Contents

Polyanion water activity regulation &

Preliminary notes dealing with the enthalpy-entropy compensation effect

0. Sub-transition state free energy flow chanelling may generate enthalpy entropy compensation
01. The isokinetic temperature phenomenon may give rise to stochastic structural reorganization phenomena 02. 03. Chemical affinity re-defined for stochastic chemical reactivity
The Fluctuation Theorem suggests that F (rather than F) is the chemical affinity in compensated systems

1. Brownian Motion
2. Biological Nucleation 3. Compensation effects elucidate water structure

4. Liu & Guo (2001) Review

5. Aberdeen U. (1984) Review

6. Putative Time Reversal & Compressed Time 7. Coveney (1988) Review 8. Vacuum Space and Extrathermodynamic Phenomena 9. Reverse Entropy & Vacuum Space 10. Vacuum Time 11., 12 Dehydrochlorination compensation data etc. 13 Redefining the nature of chemical reactivity 14. Soubility & Nucleation 14a. Nucleaton inhibitors ---------------------------------------------------------------------------------15. Van Wazer structural reorgnization ------------------------------------------------------------------------------16. (Heparan Sulfate & Degenerative Diseases) Mss. ex University of Aberdeen, Etc.

Manuscript POLYANION WATER ACTIVITY REGULATION A Hypothesis: Heparin/Heparan Sulphates Modulate Protein Activities by Water Activity Regulation Including Nanohole Induced Non-Equilibrium Water and Hofmeister-Like Effects Generated by Interfacial Aqueous Phases with High Ionic Strength, Multi-Element Inorganic Salt Compositions David Grant PhD MRSC* Aberdeenshire UK AB53 6SX (& University of Aberdeen) Heparan sulphate microstructure, evidently strictly regulated both temporarily and positionally during development (being responsible amongst numerous other established functions for regulating several growth factor activities [(1) cf. Bernfield et al. 1999; Hacker et al 2005] which are apparently dependent on the existence in syndecans, glypicans, agrin and other protoglycans of heparan sulphate sidechains having a great variety different linearly information-encoded sequences (dependent on the occurrence in this uronic acid (1->4) D glucosamine repeat disaccharides of sulphated iduronate, glucouronate and N acetyl, N-sulphonate or unsubstituted glucosamine 2-deoxyglucosamine, residues) which are believed to enable several types of signalling including those involving binding to basic sites

in proteins via specific information encoded sequences of sugars analogous to the pentasaccharide antithrombin-binding site heparin (Bernfield et al 1999; Lyon & Gallagher 1997) (such an information code is more complex than that of DNA to which it is obviously is however similar in principle although the heparan sulphate system apparently lacks any ability to act as a template for its self replication). It is now further suggested that binding of polysaccharides to proteins may require correct interstitial aqueous solutions (e.g. containing separate soft-ice like phases) which are now believed to contribute to the inorganic co-solute multi-ionic environments** . With heparan sulphate it seems that a system other than molecular recognition by an antithrombin (AT)-like-fine tuned electrostatic quaternary N+ protein link to individual codon sulphate mineral-like anionic patterns must allow fine structure discrimination to be established. A required high degree of selectivity by different microstructures present in different heparan sulphates seems to be the basis for their ability to select different proteins for their normal (e.g. growth factor orchestration) functions. The existence of some previously unsuspected unconventional molecular recognition system is however apparently required to facilitate this. This possibility is suggested by the work of Kreuger et al 2005) who found that most probable signalling oligosaccharides failed to discriminate between their individual FGF growth factor isoforms target binding sites when they were present as individual molecular signals (obtained from the high molecular weight by scission, separation and purification) . This finding is starkly contrary to the selectivity apparently achieved by the parent high molecular weigh proteoglycans in their in vivo environments. This situation might have arisen (as suggested by Kreuger et al) from the ability of such larger molecules (but not the smaller segments) to form additional hydrogen-bonding, Van der Waals forces and salt links (including those with inorganic ion bridges) and perhaps involving the whole heparan sulphate proteoglycan chain which may be involved. It is now proposed that the ultimate basis of heparan sulphate signalling is actually the induced water structure (e.g. this is acknowledged to be promoted in some non-specific manner by the extracellular matrix) and that this is the ultimate cause of tissue integrity. Tissue development may also require input form correct water structures associated with the longer polysaccharide segments molecules. This correct water structure for the proposed heparan sulphate signalling etc. functions could involve both hydrophilic and hydrophobic repulsive and attractive forces similar to the often long range effects which have been identified to occur in aqueous solutions between hydrophobic surfaces and hydrophobic and hydophilic mica surfaces (cf. Christenson & Claesson 2001) for which such long range effects have been identified might conceivably mimic the high anionic density + hydrophobic N-Ac region systems of heparan sulphates). These types of water structures are also believed to critically depend on the presence of sub-microscopic sized nano holes which have been in water structures (micro-bubbles) (e.g. adjacent to mica and silicate surface studies by atomic force microscopy) pointing to the possible key role of such a mechanism in the of formation of (presumable non-equilibrium, thermostatically unstable) water microstructures in the biochemical mechanism of tissue generation and its upkeep. Myelin basic protein integrity may depend on the hydrophobic effect on water structure and depend on microbubbles for its existence as it is associated with lipid induced hydrophobic waters structuring effects (Muelle et al 1999). It should be noted that the integrity of this protein has also been associated with heparan sulphate proteoglycans which enable the repair of damaged myelin sheaths, defects in which can be argued to give rise to neurological conditions such as multiple sclerosis. A scenario by which is this process becomes disrupted by a UV-vitamin D-thyroid factor dependent sulphate transporter which facilitates heparan sulphate sulphation could explain the geographical incidence of multiple sclerosis in Australia and perhaps also the possible promotion of this disease by barium intoxication (Purdey, 2004). It can also be logically suggested (e.g. Dr 2008) that protein folding must depend largely on the existence and the activities of interstitial water; The Hofmeister series can be explained by the effects of high concentrations of protein stabilisers or denaturant solutes on the surface tension of the interfacial water. This concept should also be extended it is now proposed to include the effect of nanobubbles on water structure. Since heparin and heparin-like sites in heparan sulphate are ultra-anionic, high ionic strength co-solute generating environments to those which promote the Hofmeister effect, it is also expected that the interstitial water which occurs adjacent to heparin/heparan sulphate polysaccharides could likewise

generate local Hofmeister effects which could determine how heparan sulphate attaches to and alters the conformation of target proteins. Direct evidence that such interstitial water might participates in such actions was obtained from studies of the binding of heparin to poly-L-lysine and poly-L-arginine (Grant et al. 1991) which indicated that such binding was accompanied by large changes in the overtone stretching frequencies of the water molecules attached to heparin. Related studies by these authors of the binding of inorganic ions and inorganic solids to heparin** and heparan sulphate tended to confirm that such binding depended on hydration changes (rather than electrostatic attractive forces, e.g. as had previously been believed to control this activity (cf. the Manning hypothesis). Related studies by the same authors that interstitial water and entropy changes thereof also apparently determined how a range of inorganic counterions bound to heparin. Further direct evidence for the existence of a high ionic strength environment adjacent to heparin is that crystalline akaganeite fibers (identified by X-ray diffractions) are produced at heparin surfaces (following binding of Fe2+ to heparin, its oxidation to Fe3+ Co2+ - dependent (such ions were also apparently commonly co-purified with heparin). [Formation of this fibrillar crystalline material {FeO.OH} is known to require the high ionic strength conditions]; (Williamson FB Aberdeen, personal communication of unpublished work). Biological Fluids are Seawater-Like Multielement Matrices from which Heparin and Anionic Polysaccharides Selectively Sequester the Least Abundant Solute Ions. In an internet paper (which is now no longer accessible) which discussed how water structure influences protein folding G Wilse Robinson quoted Szengt Gorgi who had earlier pointed out that humans are essentially bags of skin filled with seawater. Haraguchi, (2004) included heparin in his tabulation of topics for which a suggested that the new science of metallomics [a concept which had earlier been introduced by RJP Williams] which deals with the multi-inorganic nature of geological and biological phenomena including the seawater range of inorganic elements, which should be potentially be considered of fundamental importance to biochemistry since biological fluids are multi-inorganic ion solutions which are usually approximately similar in their multi-inorganic element compositions to seawater and other natural waters. It might further be suggested that since heparan sulphate seems to have co-evolved with multicellular animals in the sea some 109 years ago a primitive role of cell surface heparan sulphates was to act as a nutrient gatherer and buffer for bio-friendly seawater-like multi inorganic element containing salt solutions. This notion seems to be confirmed by a report from the Dietrich group of the existence of an exact mathematical relationship (Nader et al. 1983) between the amounts of tissue heparan sulphates (and other sulphated polysaccharides) and the salinities of the habitats of fifteen species of aquatic invertebrates, where habitat water might be required to directly bathe the heparan -sulphateproteoglycan-lined tissues. Anionic polysaccharides (and proteins) in animals when bathed in the multi-inorganic ion salt solution biological fluids will generate multi-inorganic ion/hydration water complexes similar to the anionic polysaccharides abundantly present in the cell walls of marine algae had been established (Wassemannn, 1949) most likely to exist in this form in vivo rather than being present, as was originally supposed, as free alginic acid Although less chemically definable than the pure polyanionc polysaccharides (alginates, carrageenans, and the polysaccharide side-chains of glycosaminoglycans etc.) but nevertheless of considerable importance to the homoeostasis of inorganic ions including carbonate, bicarbonate and Ca2+ ions in natural waters is the system of humic/fulvic polymers (a system of polymethylene, polycarbonyl, caboxylated) material which comprises the largest system of organic polymers on earth. These natural polyanions bind numerous metal ions present in seawater etc. via abundant -COO- groups, which apparently gave rise to the geological deposits of fulvate organic matter. Determination by spark source mass spectrometry (SSMS) of the multi-inorganic element contents of geological fulvates and marine alginate showed obvious qualitative similarities to the SSMS results for the multi-element contents of the animal polysaccharide heparin (Grant et al. 1987). Less information is currently available from the literature of similar studies of heparan sulphates (which are more difficult to obtain in large amounts) but studies conducted in the context of scitigraphic imaging (e.g. of tumours) has indicated that this procedure may depend upon the binding of the radionuclides to heparan sulphate proteoglycans e.g. at cell surfaces; side experiments

established that 45Ca in heparan sulphate could be replaced by a range of multivalent metal counterions in a manner consistent with heparan sulphate being normally present in vivo in the form of a multiinorganic matrix. The apparent differences in the observed biochemical/physical properties of different brands of heparin seems to at least in part have its origin in the different degrees of purification achieved by different manufacturers. It might even be suggested that such attempts at purification actually achieve inappropriate forms of heparin, at least from the requirement of biochemical if not from the standpoint of pharmacological research. What is obviously required for fundamental biochemical researches is the actual form of polyanions which are present in vivo. That the achievement of the equilibrium between the polyanion and the multi-element bathing fluids is not re-established rapidly may be deduced form the reports that the different single salt forms of heparin have different in vivo activities. The traditional view was that the multi-element character of unrefined heparin was of little scientific interest and that samples used for biochemical researches should be as free from such multi-elements as possible. [A similar hypothesis was applied to chitosan research, where the existence of multielements had been attributed to uptake from a final washing in tap water]. This idea is suggested to be incorrect and the status of the inorganic/water co-sphere around anionic polysaccharides which could potentially be part of an organometallic signalling system needs to be re-evaluated.

*This hypothesis was generated from private discussions (including with FB Williamson PhD and Professor WF Long** of the former University of Aberdeen, Marischal College Polysaccharide Laboratory, correspondence with Professor RJP Williams , Oxford University) and extensive internet literature studies made privately from Ashbank, Turriff AB53 6SX, UK as well as by use of the facilities kindly provided by Queen Mother Library Kings College and Forresterhill Medical Library, University of Aberdeen. **The majority of the published papers of this polysaccharide research group we posted by Professor WF Long at http://www/abdn.ac.uk/~bch118/publicatuions2003march.doc References arranged alphabetically Bernfield M et al 1999, Ann Rev Biochem. 68, DATA72907777; Cf. Hacker U Nybakken K Perrison N Nature Reviews Molecular Cell Biology 6 530-54- doi: 10.1038/nrm1681 Christenson HK Claesson PM 2001 Adv Colloid Interface Sci 91 (3) 391-436 Dr A 2008 Salts, interfacial water and protein conformation Biotechnol & Biotechnol Eq. 22 (1) 629-633 [Cf. Dr A Kelemen L Fbin L Taneva SG Fodor E Pli T Cupane A Cacace MG Ramsden JJ 2007 J Phys Chem 111, 5344-5350] Protein folding was traditionally viewed as an intrinsic property of the amino acid sequence in which the solvent had a secondary role; inherent hydrophilic/hydrophobic effects were believed to be directed by the amino acid sequences alone. This view has recently been challenged. Dr et al re-evaluated how proteins fold and suggested that the principal influence (or driving force) for protein folding originates from a dominant effect of water structure/activity on protein supramolecular structure/conformation. Such an influence of water structure also explains the Hofmeister or lyotropic series a phenomenon of protein denaturation characteristic of high solute (including inorganic salt) molecule concentrations. [Hofmeister salts change the hydophobic/hydrophilic properties of protein-water interfaces, kosmotropes making them more hydrobphobic while chaotropes make them more hydrophilic (these

terms were used in the context of their surface free energies). Surface tension*** of salt solutions in air had long been regarded as being related to the Hofmeister effect and indicated that this most likely arose from changes in water structure produced by the presence of high concentrations of salts (this was also suggested by overtone bands attributed to water molecule aggregates {cf., Kleeberg 1987; the ability of salt to influence the H-bonding in water is only seen in the immediate environment of the ions (Omta et al. 2003 Science 301, 347-34) which explains why high salt concentrations are needed affect the overall water structure and promote Hofmeister activities}. Grant D et al 1987 Biochem J. 244, 143-149 (Abbreviated list of multi-elements in heparin; the single ion form of heparin should also have been reported to have a list of these elements, albeit present in a much lesser amounts) Cf CPS 2000 Chemistry Preprint Archive , 2000 Oct, 94-104 available at http://preprint.chemweb.com/biochem.001002Multi element content of heparin (permanent internet file) The sample of sodium heparin chosen for evaluation by SSMS had been donated by a major pharmaceutical company presumably because of it was considered to be highly suitable for conducting academic biochemical researches. A highly purified sample using a standard industrial single salt heparin preparatory procedure was also studied by SSMS evaluation. Both heparins were found to have SSMS profiles similar to other natural polyanions (i.e. alginate or fulvate-like) multi-element matrices. These multi-element contents correlated (in log-log plots similar to those shown by Haraguchi, 2004) with those of human blood serum, human hair, marine alginates, seawater and the natural fulvates from geological deposits. A considerably body of literature was later found which supported the notion that commercial heparins are always contaminated to varying extents by such elements as Si ,Al, Cu, Zn, Mn, As, V, Ca, Sr and Ba. Cf. Harrison GE Sutton A Nature 1963 (4869) 809; Bowen HJM in Trace Element in Biochemistry, Academic Press, London, 1966; Heineman G Vogt W Biol Trace Elem Res. 2000, 75, 227-234; Alcock NM Serum versus plasma for trace metal analysis Elem Metab Man Ani Proc Int Symp 4th 1981 (Pub 1982) Eds JM Gawthorme, JMMMC Howell CL White p 678-680, Springer Verlag Berlin; Schwarz KA PNAS USA 1973. 70, 1608-1612; Bohrer D et al J Parenteral Enteral Nutrition 2005, 29 Bohrer D et al RBA 2004 36 (2) 99-103. The original report of these SSMS data was made by Moffat CF Ph D Thesis Synthesis, Characterizsation and Applications of Chemically Modified Heparins University of Aberdeen 1987 p 187-18 Grant D Tait MI Long WF Williamson FB Microstructure-dependent crystallization modulation by alginates (unpublished) Grant D Somers JA Tait MI Long WF Williamson FB Anti-calcite crystallisation activities of carrageenans Posters presented by MI Tait at the XIIIth International Seaweed Symposium Vancouver 13-18 August, 1989 Grant D et al (heparin-polypeptide interaction involvement of polymer-associated water) Biochem J 1991 277, 569-571 Grant D 2000 web.ukonline.co.uk/dgrant/dg Grant D et al (dependence on counter-ion of the degree of hydration of heparin) Biochem Soc Trans 1990, 18, 1293-1294 Haraguch H 2004 J Anal At Spectrom. 19, 5-14 Israelachvile J 1987 PNAS USA 84 4722-4724 Kleeberg H 1987 Proc Symposium in Honor of WAP Luck Marburg FRG

Kreuger J et al 2005 J Biol Chem 389, 145-150 [the oligosaccharides studied had the same charge densities, this seemed to suggest that charge density was the important factor for target protein binding not the way the charges were disposed along the oligosaccharides] Lyon M Gallagher JT Matrix Biol 1998, 17, 485-493 Cf Esko JD Lindahl U. Suggested reading list entitled Molecular diversity of heparan sulfate available at http://www.jci.org/content/full/108/2/169/DC1 Muelli H et al. 1999 Biophys J 76 (2) 1072-1079 Nader HB Medeiros MGL Paiva JF Paiva VMP Jeronimo SMB Ferreira TMPC Dietrich CP (1983) Comp Biochem Physiol. 76, 433-436 (Relationship between habitat salinity and the average total sulphated glycsoaminoglycan contents in fifteen species of Crustacea, Pelecypoda and Gastropoda) Park PW et al 2000 J Biol Chem 275 29923-29926 Purdey M (2004) Barium and multiple sclerosis (heparan sulphate mechanism) Sasisekharan R Shriver Z Venkataraman G Narayanasanu U Nat Rev Cancer 2002 2(7) 521-528 Wassermann A (1949) Cation adsorption by brown algae. The mode of occurrence of alginic acid Annals of Botany N.S. XIII (49) 79-88 Cf Black WAP Mitchell RL (1952) Trace elements in the common algae and in sea water J Mar Biol Asoc UK 30 (3) 575-584 (Alginates also bind to inorganic surfaces in a highly microstructure-discriminated manner (1); this seems also to be the case for animal polysaccharides and such binding could conceivable by utilized for sequence evaluation purposes). --------------------------------------------------------------------------------------------------------------

MANUSCRIPTS Preliminary Notes DEALING WITH THE ENTHALPY-ENTROPY COMPENSATION EFFECT Ms. 0
Consideration Of How Constancy Of A Sub-Transition State Free Energy Flow Process Arising From Channeling of Energy May Generate Exact Enthalpy Entropy Compensation so that in a set of rate constants for chemically related processes

kr (where lnkr=AexpEa/RT) Ea becomes a linear function of logA] If chemical reactions proceed via the formation of transition state complexes in which the driving force for the reaction is provided by the (Gibbs-Helmholtz) Free Energy (F)

F = H-TS
[H (enthalpy) T (absolute Temperature) x S (entropy)]

(1)

and if this driving force is exerted only at the actual part of the transition state complex which becomes changed* then for any set of chemical reactions (in which e.g. for a wide range of starting molecules which all contain the same (or a similar) sub-structure on which chemical transformation is performed) the consequence of this restriction to control the actual reacting centre by a related process over the whole set of reaction rate determining processes for which the values of

F
are (approximately) constant,
then comparison (of F, H or S) between members of a set of such selected chemical
reactions must show that such values of

F are (approximately) zero,

viz.
substituting equation (2) in equation (1) gives

F 0

(2) ;
H TS (3)

i.e. an (approximate) enthalpy ( H)-entropy ( S) compensation then exists for appropriate sets of rate constants
This simple argument shows how the poorly understood enthalpy-entropy compensation process may arise simply from a conventional free energy driving mechanism which acts only upon a restricted reaction channel in the transition state complex.

In equation (2) T (=, the Leffler isothermal temperature at which all reactions in the selected set proceed at equal rate).
(*This differs from the model of the transition state complex which was proposed by some later variations of the transition state theory which suggested that coupling of vibrations from distant parts of this molecule could contribute to energy flow) The above argument uses a single value of free energy, F , i.e. only one part of the conventional free energy driving force, ie. not, (as convention dictates) the change in free energy F which is associated with the completed reaction ; this use of partial time invariant free energy per se and not its variation time over the process of chemical change indicates that the role of time as a processor of free energy is being changed from that conceived in the clasical thermodynamics-derived expression of chemical affinity.

Ms 0-1

The Isokinetic Temperature Phenomenon May Give Rise To A General-Throughout-Chemistry Stochastic Structural Reorganization Phenomena.
It should be noted that enthalpy-entropy compensation/ isokinetic temperature occurs widely throughout chemistry (and also throughout physics) and similarly random structural reorganization of chemical substances arise under controlled pyrolysis conditions. These two sets of phenomena are now putatively directly linked together.

Stochastic Structural Reorganization The process by which during heating above a critical temperature, all types of chemical substances throughout the periodic table can under their individally appropriately (pyrolysis) temperature conditions undergo complex structural reorganizations which lead to the formation of apparently highly complex mixtures of final products. While pyrolysis of non-carbon (inorganic) systems tend to occur at lower temperature that do those for carbon centered (organic) structures a similar underlying randomization of side chains about a core structure mechanism may determine the outcomes of both kinds of pyrolysis processes (conducted e.g. under sealed tube conditions). Examples include the various poly inorganic oxy acid esters (e.g. the polysilicic acid esters and the polyphosphoric acid esters for which the process of P-O-P bond formation and redistribution in the set of [P(O)(OR)3 (P)-O-P(O)(OR)2 {(P)-O-}2P(O)(OR) {(P)-O-}3P(O) give rise to ortho, (monomer) and condensed phosphates of linear chain, crosslinked (branched) chain and ring structures via the ring/chain ratio plus the operation of the sets of reactions: monomer + middles = 2 ends; end + branch = 2 middles]. The above behavior putatively arise simply because of the occurrence of a general enthalpy-entropy compensation behavior facilitated by the exitence of a critical temperature. (In sets of chemcial reactions which exhibit enthalpy-entropy compensation there exists an isokinetic temperature at which all rates within these sets of such reactions becomes equal and the products of these sets of compensated reaction processes (especially if they are conducted in a system which leads to polymeric structures) tend to show very clearly identifiable stochastic outcomes (or for a range of progressively near isokinetic conditions a range of progressively near-stochastic outcomes arise).

MSS02

Chemical Affinity, The Driving Force of Chemical Reactivity Re-Defined for Stochastic Chemical Reactivity It should be noted that historically the original idea proposed by Bertholet and Thomsen in the early nineteenth century was that the heat produced during a reaction was a useful indicator of chemical affinity or the driving force behind chemical reactions. This could not, however, explain why endothermic reactions can occur spontaneously. Hence it was suggested (by Gibbs VantHoff and Le Chatelier) that the proper definition of the chemical reactivity (athe driving force or chemical affinity) is the maximum work which a chemical reaction can perform. Hence the Gibbs-Helmholz concept of free energy F=H-TS Work & Free Energy The classical concept of chemical reactivity, the Gibbs-Helmholtz free energy F (=H-TS) change in F as a driving force was ultimately derived from the hypothesis that reactions could occur spontaneously only if such reactions could be made to perform useful work. However, the stochastic reorganization (scrambling) reactions which came to light only after such modern analytical techniques as NMR were introduced in the twentieth century, seemed to indicate that chemical structure alteration could also arise spontaneously under conditions where the overall free energy in the reaction vessel does not change, and therefore the idea that the magnitude of potential free energy change is what always primarily causes chemical transformations to occur (and always thereby potentially performs work) seems to be flawed. What then can give a more universally valid index of chemical reactivity? (It should be n oted that the general occurrence of certain extrathermodynamic free energy relationships which were not predictable from classical thermodynamics also questions the original ideas relating to how the overall free energy change equates to chemical reactivity). Stochastic (reversible reaction quasi-equilibria) chemical reactivity during which in the overall stoichiometric process, free energy is conserved perhaps should be distinguished from the type of nonstochastic chemical reactivity where free energy change remains a valid concept as a driving force for chemical reactivity. It now seems worthwhile instead, for the elucidation of the nature stochastic processes to re-focus more on the isokinetic temperature to consider how this relates to stochastic chemical reactivity (which is putatively a central consequence of the existence of enthalpy-entropy compensation) which putatively dictates the outcome of the (free-energy change-free) stochastic processes.

This draws attention to temperature per se and the isokinetic temperature (T) in particular as a possible usely focus for deriving a more general driving force for chemical transformation. MSS03
The Fluctuation Theorem

suggests why F (and not F) may be a useful measure for the chemical reactivity driving force in compensated chemical systems.
It should be noted that S and not S appears in the Evans equation which may be written S = P/t

(where P = expSt; antilog P = P= St where t is time and P is the ratio of the probabilities that S will take a positive or a negative value (i.e. that the direction of time will be positive or negative)) Substituting Evans S in F =H-TS, F = H-TP/t From the Leffler equation (H=ST, T = H/S (where T is the isokinetic temperature))

when TT
F H-[H/S]P/t If H<<[H/S]P/t (e.g. when t is very small) then F -[H/S]P/t If F is allowed to be 0, then from the relationship F=H-TS, H/S=T applies [and further ignoring the stipulation that the Fluctuation Theorem is only strictly valid for systems away from equilibrium] the above expression for F may be simplified to

F -TP/t
i.e. the putative chemical potential under the above conditions is the product of ( temperature) x ( reverse time potential).

1st MANUSCRIPT
Note D.G. 25/4/12 Brownian Motion Reconsidered Evans Negative Time Effects New Take on Time Needs also to Reconsider the Provision of Separate Time Zones in Matter The Phase Rule Needs to be Extended to Include New Types of Phases Possibility of Separate Time Zones of Is Evans Reverse Time Flow Requires Matter Separating Boundaries i.e. The Existence of Unconventional Phases of Matter

The Fluctuation Theorem (Evans 1993) can be considered to be a further theoretical consideration of Brownian-motion (-like behavior) of microscopic particles which had been found (e.g. by A. Einstein, cf. D.V. Fenby Chem Brit 1981 17 114 ) not to obey the laws of classical thermodynamics. The Fluctuation Theorem was apparently convincingly demonstrated experimentally by G.M.Wang et al. Phys Rev Lett 89 050601 2002 (who found that the observed path of a 1micron size latex bead held in an optical tweezer system pulled through water showed that movement occurred contrary to that predicted by classical thermodynamics and that the system could be provide evidence for the existence of a reverse time flow in liquid water). The Fluctuation Theorem equates (exp. [entropy.time] to) the probability that a change of the time direction of entropy which is normally positive for large scale system will, for small systems, tend to decrease with the passage of (positive) time rather than increase, i.e. this theorem predicts that the entropy associated with small particles will tend to violate of the second law of thermodynamics to a greater extent than is the case with larger particles; it is further evident that this behavior will be of likely practical application for small scale nanoparticle engines. This includes particles the size of (e.g. common biologically relevant) chemical molecules and (reverse-time concepts) may be required to fully understand chemical transformations of such systems. It should be noted that the Fluctuation Theorem definitely suggests that the direction of time might reverse under experimentally relevant conditions: positive (normally perceived) time might becomes negative (reverse) time) as a measure of the movement of small sized entities. It is further now suggested that this time reversal must also be conditional on the existence (e.g. in fluid phases such as water) of (new types of) phase boundaries (e.g. additional those considered by the Gibbs which provide boundaries which allow defined separate zones allowing reverse time flow to co-exist in admixture with normal positive time systems. The Fluctuation Theorem and its implications for the kinetic behavior of molecular sized entities could give a more general rationalization of numerous (cf. Leffler) experimentally observed extrathermodynamic systems: (the linear free energy relationships (Hammett etc) and the related but more general which (found in comparison of exponential function e.g. rate constants k = AexpE/RT which describe the temperature variation of chemical and physical processes and which can be attributed to enthalpy-entropy compensation where a mathematical relationship such as [(allowed entropy change) (allowed enthalpy change) which cannot be predicted by the laws of classical thermodynamics] pertains). A corollary to this further prediction of the usefulness of the Fluctuation Theorem as a general working rule for the re-examination of chemical reactivity should focus on those experimental systems and states of matter which demonstrate genuine enthalpy-entropy compensation with the idea of confirming that such systems must also contain separated (reverse-) time zone (putatively novel types of) phase boundaries. In the case of liquid water, this experimental task may already have been accomplished; water is a solvent in which numerous examples of enthalpy-entropy compensation has been reported (including those which are biologically relevant) shows a kind of phase-change-like variation of near infrared spectrum combination bands near 37C (this was reported by W.A.P. Luck in in Zur Assoziation des Wassers. III Ber. Bunsengesellschaft, 1965 (7) 626-37, cf Fig 6 on p. 639 (Temperaturabhngigkeit der Wellenlgenlage des intensivsten Maximums der 1,15--H2O-Bande). In the case of the observed gas molecule reaction well defined enthapy-entropy compensation (such as is found for organic molecule dehydrochlorination reactions) where compensation has been observed the putative separate phase boundaries must be of a more subtle nature perhaps being associated with mixing imperfections (vortices etc. which show up in chemical engineering models of gas reactors).

The Nucleation of Phase Formation The mechanism of phase formation and separation of the postulated new types of phases of matter which are required to accommodate the separate zones of Evans reverse time (which putatively create defined overall enthalpy-entropy compensation) will also, it is proposed, also be subject to the same kind of general nucleation effects as the common need for seed templates for the formation of conventional crystalline phases. It should be noted that the nucleation of separate phases in water-rich gels (of anionic algal polysaccharides and collagen) formed in aqueous solutions demonstrate well-defined examples of enthalpy-entropy compensation.

2nd Manuscript

Biological Nucleation &

Enthalpy-Entropy Compensation between the apparent enthalpy and entropy change terms in the
usually employed kinetic rate constant parameters.

Some series of structurally-related but chemical distinct substances demonstrate highly credible preexponential vs. Arrhenius activation correlations. These have long been known but remain unexplained. Accurately obeyed inter-relatedness of this kind and the related Hammett equation seems somehow to breach ideas of the usefulness of traditional thermodynamic concepts such as the second law of thermodynamics (n.b. these ideas were developed during the nineteenth century for steam engines etc.) could indicate that the classical thermodynamics-based model of molecular energy transfer during chemical transformation of molecular sized entities is, at least partially, incorrect. It may be possible for some interacting chemical systems (e.g., aqueous solutions of biopolymers) to access hidden sources of energy/enthalpy; this facility may enhance biologically relevant processes. The extra energy/enthalpy may be accessed by a process which is equivalent to an ability of molecules to access interactive future state propensities e.g. by accessing currently unknown high efficiency tunneling pathways. D Grant 23/12/00-5/2/01 updated 2/12 These jotting are intended to form the basis of an Internet Discussion 1. SUMMARY Examples of enthalpy-entropy compensation which do not seem to comply with the classical model of how enthalpy and entropy should behave as a function of time crop up throughout chemistry, physics and biology. An unexplained catalogue of enthalpy-entropy compensation (1) seems to run counter to the current understanding of temperature-dependent and temperature-independent thermodynamic-laws. However the enthalpy-entropy compensation effect and the related Hammett relationship (2) has often been dismissed as likely being of a trivial theoretical importance; in any case it is a rare phenomenon which is not exhibited, even approximately, by many (or most?) chemical reactions and most likely is an artifact of experimental error. This idea is however untenable when the full range of reported instances of enthalpy-entropy compensation is taken into account.

The establishment of the origin of compensation may, it is now suggested, allow for greater insight to be obtained into the nature of chemical reactivity. It may also concern the validity of the second law of thermodynamics and perhaps even further suggest the need to formulate more accurate macroscopic system laws needed to more fully quantify how the natural one-way system of time flow applies to particles of the size of chemical molecules. It seems that the currently believed paradigm about entropy and time which is associated with the second law of thermodynamics is defective. There may be a universal tendency for enthalpy and entropy changes to be mathematically compensated during all kinds of electron redistribution process including those associated with chemical bond formation and rearrangement. This could suggest a new framework hypothesis to stimulate the attainment of novel insight into chemical reactivity in general and most especially to organic chemistry and biology in particular. This could also allow suggest possible major driving force in biology- which appears to encompass situations where the allowable rate and equilibrium states differ significant from those which describe bulk matter and for which the second law of thermodynamics as applied to macroscopic closed systems is absolutely not applicable. (It should be noted that as to the considered argument that naturally occurring biological assembly is an open system this still does not require entropy to decrease with the passage of time). It should be noted that examples of biologically relevant enthalpy entropy compensation are provided by the denaturation of proteins, the haemolysis of erythrocytes and the nucleation of polysaccharide hydrated gel formation. Such gels are sometimes held to be possible primitive analogues of biological tissues. It is now further suggested that such abnormal entropy change effects may provide and empower biological systems with morphogenic effectors. Knowledge of the ultimate mechanism of morphogenesis may also shed light on how evolution, neurological degenerative diseases and cancer etc., arise. 2. INTRODUCTION A New Paradigm for Organic Reactivity A wide range of enthalpy-entropy compensated chemical reaction processes (these being much more common than was formerly believed) include exact linear relationships with large numbers of data point which surely points to the existence of unexplored fundamental properties of common organic reaction processes. Compensated chemical reaction processes exhibit an isokinetic temperature where all reaction rates within the series become equal. This phenomenon may be why some chemical reactions are clean (e.g. single) products but multi e.g. apparently scrambled randomized reaction products arising more frequently at near isokinetic temperatures. The attention of organic chemists was directed by Leffler in 1955 (2) to 79 (or perhaps 113) types of organic reactions which displayed enthalpy-entropy correlations but only 24 classes of organic reactions could clearly be identified which lacked any enthalpy-entropy correlation. It should be noted that the latter (random enthalpy-entropy non-correlated variation) is how enthalpy and entropy (at least as it applies to bulk matter) should behave according to traditionally held views. The clearly experimentally validation of enthalpy-entropy compensation seemed, however, not to be easily explained in 1955 and this remains true today. Nevertheless the common occurrence of compensation between organic reaction kinetic data reviewed by Leffler clearly demonstrates a disallowed enthalpy - entropy compensation exists for organic reactivity but similar compensation is also known for electrical conduction, thermionic activity, heterogeneous catalysis of hydrocarbon formation in clays and compensated reactions include the biologically-relevant kinetics of the denaturation of proteins.

The correlation processes may conceivably apply to all classes of electronic redistribution reactions occurring within of atomic and molecular sized particles. Such enthalpy-entropy correlation generates an associated isokinetic temperature (where all reaction rates in the compensated series become equal and may be associated with random scrambling such as occurs with the polyphosphate and polysilicate organic acid esters). Such entropy-enthalpy smudging may be of especial relevance as a fundamental driving force of organic substance and biochemical reactivity.
Families of the often less stable systems or inorganic families of chemical substances more commonly undergo random scrambling as indicated by Van Wazer and colleagues (from 1955) to be a useful paradigm to address inorganic (polymer) structurally reorganizing systems (which as [inorganic polyphosphate] occur at all cell surfaces throughout biota (cf Kornberg).

[A related sub-class of the enthalpy-entropy chemical reactivity correlation is the basis of the Hammett sigma relationship which accords structural parameters to aromatic side chain rate constants was noted by Leffler loc. cit to apply only in those cases where the enthalpy is a linear function of the entropy but the converse is not true since the linear relationship between enthalpy and entropy is also encountered in relations to which the Hammett equation does not apply . The Hammett sigma value can also be linearly correlated to NMR chemical shifts (e.g. for phenolic OH groups cf. Dietrich et al]. A clue about the general nature of the compensation effect was also suggested by Leffler: it, at least in some instances where the reactions in question are conducted in a solvent or solvents, might arise from an temperature induced change in the structure of such solvent(s). The solvent which seems most readily able to confer compensation is liquid water. How compensation of organic reactivity by aqueous solution chemistry arises might therefore be of central relevance to biological systems. It should be noted however that compensation also is well known to occur in apparent unimolecular gas phase decompositions as well as complex radical chain propagated gas phase reactions e.g. dehydrochlorinations where both homogeneous and heterogeneous rate processes seem to obey the same compensated pre-exponential and Arrhenius activation energy relationship. This came to my attention during industrial chemical reaction model building when the existence of compensation came to light as did the inability of leaders in the field of such studies to explain this effect.

References to document l (1) Leffler J.E. J Organic Chemistry 1955, vol. 20, p. 202-1231) ). [(The enthalpy-entropy relationship and its implications for organic chemistry) a comprehensive review; n.b. this confirmation of the relevance of the compensation phenomenon appears in this a prestigious ACS peer-reviewed Journal] The Hammett equation which applies to aromatic reactivity where the entropy is variable but only in those cases where the enthalpy is a linear function of the entropy was noted to be a sub-class of a more general enthalpy-entropy relationship (2) Butler A.R. (Chem Brit 1989, Oct., 997-998) suggested that general use of the Hammett relationship (e.g. the dissociation constants for substituted benzoic acids (Ko) and for substituted phenylacetic acids Ko are found to be linearly related . Where Ko is the dissociation constant for unsubstituted benzoic acid log K vs. logK these are correlated but also (surprisingly) linear correlation can be demonstrated between ( = log K/Ko ) and logk/ko where k are rate constants for numerous reaction involving substituted benzene compounds. The latter linear Hammett correlations were, however, more-orless suspect since log-log relationships in general cam easily obtained e.g. using an entirely spurious data set which varies approximately exponentially (a plot of the twoyearly increase in the Swedish gross product between 1970 and 1980 [why choose two yearly intervals other than to deliberately select a set of suitable numbers and also surely other equally accessible datasets for two yearly changes over other decades would not be exponential and therefore would not produce a linear variation]) could be made to fit a Hammett relationship for the reaction of diazodiphenylmethane with substituted benzoic acids. This chemistry journal seems to meet the view of many scientists that it is acceptable to joke about the possible need to revise the traditional views of thermodynamic concepts.

Cf. Boon M.R. Nature 1973 243 401 discussed a suggestion by Banks B.E.C et al. ibid., 240 147, that the thermodynamic compensation rule is artifactual; cf also Harris P.S. (ibid. 401402), Banks et al (ibid. 402) and Wilson M.C. and Gawey M.C. (ibid. 402-404). [The enthalpyentropy compensation relationship was discussed by these authors for the denaturation of proteins, organic semiconductors, amorphous semiconductors, the thermal death of viruses and microorganisms as well as the heterogenous hydrocarbon formation on clays; the consensus of opinion seemed to confirm the validity of the enthalpy-entropy compensation effect. (1) (2) More recent reports of compensated enthalpy-entropy compensation behavior include Middleton J.K. et al J Virol 2002 76 1051-1061 [Thermal deactivation of Reoviral infective subparticles is mediated by the denaturation of a viral surface protein {n.b. reoviruses are nonenveloped viruses}] Several papers report compensation during food processing e.g. Beristain C.I. et al. J Food Engineering 1996 30 405-415 [Water vapour adsorption on food] Moyanici P.C. et al ibid., 2004 [Browning] and, for hydrogen adsorption on zeolites Garrone E et al. Chem Phys Lett 2008 456 68-70 Dietrich M.W. et al., Anal Chem. 1966 38 (11) 1479-1484 Van Wazer J.R. Amer Scientist 1962 50 450-472 Brown & Kornberg cf PNAS ---------------------------------------------------------------------------------------------------------------Re-typed from document printed out on 3/2/01 Biological Nucleation Is Cooperative Enthalpy-Entropy Compensation Relevant to Biology? D Grant 12/12/00 These jottings are intended to form the basis of an internet Discussion. Contents 1. Introduction 2. Hypotheses 3. Possible examples 4. Possible explanations 1. INTRODUCTION Enthalpy-entropy compensation crops up throughout chemistry, physics and biology. Since it includes rate effects (cf. Note 1) where allowable entropy changes appear to be subject to some controlling (both restricting and enhancing) influences which appear to breach the concepts expected of systems which obey the rules of classical thermodynamics, the possible influence of compensated-enthalpy-entropy on biological system seems worthy of consideration. When rate constant compensation effects (cf. Notes 1 and 2) for series of related chemical reactions were reviewed by Leffler in 1955, the origin of the compensation effects could not be identified. This remains true today. There may be a natural reluctance to openly address the scientific relevance of the compensation phenomena since to do so may seem to require a major revision of the currently accepted highly regarded status of classical thermodynamics.

Furthermore the enthalpy-entropy compensation effect had been previously dismissed by chemists as being a trivial phenomenon or an artifact arising mostly from experimental error. Furthermore many (or most?) chemical reactions series (were believed) not to exhibit the effect, even approximately. However, such dismissal of the compensation effect is felt to be unjustified when the full range of reported instances is taken into account (see Notes 2 and 3). Compensated systems are known to be induced in systems of related chemical reactions by solvent effects. Enthalpy and entropy compensations within sets of rate constants obtained from series of chemical reactions can achieve the precision of a linear relationship the slope of which gives an isokinetic temperature at which all reaction rates in the series become equal. Scrambled reaction products of chemical reactions are characteristically obtained when the reaction is conducted near the isokinetic temperature but clean products products (e.g. a high yield of a single product) tend to be formed at temperatures far removed from the isokinetic temperature (Leffler 1955). Reactions in aqueous solution seem especially prone to compensated behavior: this includes protein denaturation (which is also affected by Hofmeister lyotropic series which can be attributed to water structure effects) and such cellular interactions as haemolysis of erythrocytes (cf Good et al quoted by Brown 1971); [incidentally such haemolysis at inorganic silica particle surfaces shows a complex dependence on the size and surface properties of the silica particles as well as being markedly different for erythrocytes from patients with inflammatory diseases and well as erythrocytes from different animal species (cf. Iler 1979 and Arienzo & Bresciam 1969)]. 2. Hypotheses. It seems desirable to generate hypotheses to evaluate the chemical basis of compensated kinetics. Probing the induction of polysaccharide supramolecular structure might be a worthwhile initial task in this context since compensation effects are found for the nucleation of kappa carrageenan hydrated gel structure (see Note 4). Such gels are useful approximations to biological tissues. Since compensation between changes in entropy and enthalpy may be widely relevant to biological science and medicine the following tentative working hypothesis might be considered. HYPOTHESIS IF COOPERATIVE ENTHALPY_ENTROPY COMPENSATION IN BIOLOGY A central hypothesis is resented on the basis of the literature and other data reviewed below which suggests that biological systems possess the ability to employ cooperative interactions between enthalpy-entropy compensated systems. Water may be a particularly suitable medium for supporting or transmitting cooperative enthalpic-entropic interactive signals. In such a hypothetical co-operative system of compensated processes and increase in the enthalpy of the system would require a corresponding compensated entropy decrease (and vice versa) and if a gradual increase in enthalpy could be introduced into such a system over long periods of time it would require a corresponding decrease in the entropy of the system corresponding to increased system complexity with time. Consider a co-operatively compensated scenario: (involving the effects of surface (cf. Note 4) electrical charges (cf. Note 5) on nucleation Nucleation of phase change -> (interphase) surface formation Effect of surface charge on nucleation <- surface charge 3. POSSIBLE EXAMPLES It might be instructive to probe for compensation in the following instances:

Nucleation effects possibly responsible for the pneumonosilicotic influence of clean crystalline quartz crystals present in some mine dusts. Amorphous silicas lack this activity. The carcinogenic effect of glass fibers and asbestos. Nucleation of mitogenic activity by calcium phosphate granules present in breast tissue. Nucleation by prions of the formation of pathological prion conformation Could (compensation /nucleation) water phase surface effects also be involved in brain activity? (Could such effects be of relevance for consideration of Turing wavelength in diffusible reducing factors which are thought to be determinants of developmental biology) (cf. Schiffmann 2000)? 4. Possible Explanations of the Compensation Effect. Adequate data supports the reality that a significant proportion of rate and equilibrium data show some unified underlying thermodynamic control mechanism(s) outwith those considered by classical (especially gas reaction) kinetic theories. According to classical rate theories the pre-exponential term can be thought of as a particle collision number or a reacting bond vibration frequency. Log A (see Note 1) should have a (maximum possible) value around 13.5 sec-1; (this circumstance is in fact true for many gas phase reaction system rate constant pre-exponential values). However, in compensated systems the value of log A may (as well as being much less than this value), apparently highly illogically, also can be much greater than this value. The particle interaction model of the reaction process then requires that the compensation effect produces a systematic inhibition or enhancement of the collision process beyond any single event by an amount which is proportional to the activation energy needed for the reaction process to proceed. Blackadder & Hinshelwood (1958) had attempted to explain the compensation effect in terms of vibrational energy coupling from distant degrees of freedom in the reacting molecule. This explanation does not seem to have become accepted. Eley (1967) listed three possible explanations of the compensation effect: 1) a temperature dependent activation energy (variation), 2) a characteristic distribution of activation energies or 3) some fundamental properties of potential energy curves and free volumes of liquids. According to Lumry & Biltonen (1969), in regard to the enthalpy-entropy compensation phenomenon, It is possible that the enthalpy and entropy change during conformational changes of proteins is the single most important physical-chemical characterization of protein function *. Such compensation (or isokinetic) temperatures generally fall within the range of 23 to 47C (clustering around 20C) (Brown 1971). Kresheck, in a review of biological calorimetry commented in 1986 that the well-known compensation between enthalpy and entropy changes for aqueous solutions complicates the interpretation of these quantities except on a relative basis until a detailed molecular theory of hydration is developed Perhaps the ultimate reason for the distinction which can be made between compensated and non-compensated rate processes is to do with the wave-like rather than the particle-like nature of matter especially as it pertains to electrons (and similar entities), the structure and reactivity of hydrogen-containing substances (especially water) and hydrogen-bands and to the importance of such factors in nucleation of compensated chemical reactions. Where such wave-like propensities dominate, compensated rather than regular kinetics may ensue.

Other possibilities to be evaluated might be how the occurrence of (unsuspected) compensation in nucleation processes together with the effect of surface chemistry in providing such nucleation sites could impart apparent compensation behavior to downstream processes. 5- Are Compensation Effects ultimately due to Space Restriction at Surfaces or Point Dislocations of Nucleation Sites? Perhaps the compensation effect is peculiar to reactions at or between interfaces (including those generated by biological molecules). Since the effect seems to show up in electron release and conduction by solids (discussed below in Note 3) perhaps the effect is to do with those surfaces which have energy levels available which allow wide delocalization of electrons in two dimensional space. Values of allowable S may be decreased by being limited by the restriction effects of two dimensional space and this may particularly apply to nucleation processes (sometimes unacknowledged) which are responsible for the initiation of chemical reactions. 5.2. Evidence for Surface Effects in Enthalpy-Entropy Compensation Leffler (1955) had suggested that any series of reactions needed to proceed via the same mechanism for enthalpy-entropy compensation to be permitted. If this were true than all gas phase dehydrochlorinations which are same-compensated to those which can be demonstrated to occur at surfaces must also be rate determined by the surface processes despite apparently showing evidence to the contrary. The reason why some systems, but not others show up accurate compensation may simply be that the former cases are subject to a large extent to (often unacknowledged) surface effects (e.g. initiation or nucleation) while the latter cases are not so influenced. Are the compensation effects then a consequence of necessary critical nucleation events occurring at surfaces? Including (internal) unstable surface of hydrogen-bonded aggregated clusters such as evidently occur in water (e.g. Luck 1963, 1965). Compensated hydrogenbonded tunneling processes may be pertinent to the mechanism of coupling producing infrared continua in O-H bond array vibration in acid salts (Zndel 1969-76). Hydrated sulphonate groups (Zndel 1969, cf. Columban 1992) (a possible model for glycosaminoglycan hydration/proton conduction) and other silanol rich surfaces (e.g. in clays) may conceivably promote such effects in reactants adsorbed at HO- rich surfaces. -------------------------------------------------------------------------------------------------

Notes
Note 1 The Compensation Effect as Applicable to Rate Constants. For C (a specific chemical reaction rate constant (in the usual format) C=Aexp(-E/RT) (A is the pre-exponential factor E is the Arrhenius activation energy R is the gas constant T is the absolute temperature) When the Arrhenius rate expression Log C = logA E/2.302 RT is compared for a series of reactions by plotting log A against E, quite often a linear relationship is revealed (reviewed by Leffler 1955), giving LogA = E/(2.302RTs) + log Co + (const) (the value of the (const) is often =0;

the parameter Ts is the (isokinetic) temperature at which all reactions of the series occur at the same rate (Co). [Organic reactions apparently tend to be cleaner with regard to products from competing reactions) the further the reaction temperature is from the isokinetic temperature; the isokinetic temperature however encourages randomization from which nucleation allows phase separation as related by Leffler 1955]. The rate constant expression may be rewritten, according to the transition state complex theory of chemical reaction, in terms of entropic and enthalpic changes associated with a transition state complex formed in equilibrium amounts: C=(kT/h)exp(S*/R)exp(-H*/RT) The compensation effect thereby suggests linkage (by unknown mechanism(s)) between entropy and enthalpy changes in the equilibria which produce the activated transition state complex.

Note 2 Examples of Compensation Effects. Dehydrochlorination Rate Constants Compensation. Forty three dehydrochlorination reaction rate constant which were available from the literature (Grant 1968) gave a linear relationship between enthalpy and entropy parameters , even although some of the data were reported to be for homogeneous reactions and other data were from reactions which could be demonstrated to be heterogeneous (and even some which were possibly were probably propagated by radical chain reaction processes). Such correlation were in fact admitted as being fairly well known to classical academic chemical kineticists (such as C.N. Hinshelwood, a Nobel laureate) who were well aware of the possible importance of these phenomena to a fuller understanding of the nature of chemical reactivity but were more or less puzzled by them.
If reactions between molecules are regarded as resulting from collisions then the maximum rate is the same as the collision number. Compensated systems can however apparently exhibit apparent rate constant-suggested collision numbers which are several orders of magnitude greater than what can be deemed possible (perhaps without limit as to how much greater) on the basis of the maximum classical gas theory allowed value. This might be due e.g. to the operation of some Maxwell demon effect (e.g. an inhomogeneity of gas phase mixing) an ability to virtually access inaccessible states achieved by allowing for an increased activation energy (enthalpy) input or to some kind of anticipation by the reacting molecules of future states and perhaps. The entropy enthalpy correlation is suggestive of a virtual time flow process against that required by second law dictat.

Although dehydrochlorination reaction rate constants were compensated, apparently similar dehydrobromination reactions were not. It should be noted that seventy nine series of organic chemical reaction processes exhibiting compensation were identified by Leffer in 1955. Acid-base rate correlations and rate effects in aromatic systems (Hammett equations) provide a more limited illustration of a compensation-like effect (cf. Leffler 1955) Note 3 More General Enthalpy-Entropy Effects The compensation effect is apparently fairly general, not simply being restricted to chemical reaction rate constants, e.g. it is also applicable, as shown by Barclay & Butler in 1938, to the heats and entropies of vaporisation from solutions and pure associated liquids, and, as shown for forty six liquids by Campbell & Eley in 1967 to Etvs surface tension factors and, as reviewed by Eley in 1967, to the electrical conductivity of solids (including amino acids and proteins (possible influenced by strongly held water) , to the electrical conductivity of organic and inorganic oxides (including TiO2 , Fe2O3 and ZnO), to the thermionic emission of electrons from solid surfaces (and the transfer of ions and molecules between H2O and D2O.

It also applies to the haemolysis of erythrocytes and to the denaturation of proteins (Brown et al 1971) as well as to many heterogeneous catalytic processes including hydrocarbon formation on clays (Wilson & Galway 1973). Compensation may also contribute to balanced bond exchange randomization in polyethers, polysulphides, polysulphates, polyphosphates and organic esters of these inorganic polymers (cf. Grant 1967) etc. from which (clean) phases may however be induced to separate by nucleation. There is also compensation between the enthalpic and the entropic contributions to the second osmotic virial coefficient (often considered as a measure of the solvent power) for various (organic) polymer solvent systems (Wolf 1972). The reported occurrence of compensated effects for such a wide range of physico-chemical situations indicates that the compensation may dominate the chemical behavior and contribute to the major physico-chemical properties (activities) of such systems; (this may apply to biological systems) including by allowing interactions between compensated systems to generate a special kind of responsiveness to stimulae. Note 4 *[Compensation in Nucleation of Disorder-Order Transition in Kappa Carrageenan Compensation is apparent between enthalpy and entropy changes in the nucleation of kappa carrageenan supramolecular structure formation (disorder-order transition) in various salt (from added amounts of small anions) solutions (reported but not discussed by Austen et al 1988) induced in tetramethylammonium cation (aqueous solution) environments in which a study of the kinetics of nucleation shows a linear correlation between the values of entropy of activation and enthalpy of activation for this process, as studied by stopped flow polarometer. (Results shown in Table II of this paper suggest that the isokinetic temperature (Leffler 1055) occurs in the range 0-40C (with the most probable value being at ca 20C). These nucleation rates also follow the Hofmeister (1888)(lyotropic) series , which can be correlated to water aggregation states (as indicted e.g. by an alteration of the near infrared bands associated with water aggregates (cf. Kleeberg 1987) indicating that the supramolecular structures present in water may contribute to the compensation effect; this idea is supportive of the notion that the hydrogen-bonded aggregates present in liquid water (cf Ling and also Bernal (1965)) may be uniquely important for biology, i.e. water is not merely a relatively inert dispersion medium]. ---------------------------------------------------------------------------

2. Note 5 Effect of Electrical Fields at Surfaces Weak electromagnetic fields have been observed to alter the (nucleation of) crystallization of water and aromatic substances (Evans 1982) and are therefore likely to be of fundamental importance to the kinetics of nucleation. It would be of interest to probe for compensation effects in these systems. Electromagnetic (e.m. fields (such as those generated by nerves) have the potential to act as biological signalling and switching mechanisms; (cf., electrical charges at aqueous interphases (and associated nucleation events) can produce a discrete, reversible volume change (which can alter the volume by several orders of magnitude) by infinitisimal change in electrical potential across the polyelectrolyte gel. Similar effects are also known for such gels in acetone-water mixtures induced by small changes in temperature, solvent composition, pH or added salt (Tanaka et al 1982). The formation of aggregated water molecules with characteristic patterns of polarizability may also be subject to nucleation of structure effects which are subject to the influence by (weak) magnetic fileds and electromagnetic fields on such polarizability. Do biological systems also employ (polysaccharide protein) modulated feedback loops involving Etvs surface tension factor effects? (Cf. surfaces, as in colloidal chemistry in general, can accumulate impurities which lead to electrical double layer effects which produce Zeta potential dependent electro-osmosis/electrophoresis).

The adsorption of a layer of water plus (e.g. prebiotic) organic molecules at various silanol containing surfaces (such as clay or amorphous silica (e.g. silica sols) which was discussed by Grant et al 1992a) might have allowed restriction of rate processes to two dimensional space, thereby restricting allowable entropy change) so that complex systems such as the precursors of polysaccharides, proteins and nucleic acids were encouraged to be generated over long time intervals. Evidence in favour of the above origin of biopolymers is that quartz crystals seem to exert very strong epitaxial effects on the polymerisation of simple amino acids (Hensch 1986). The mineral-like surface of present-day glycosaminoglycans such as heparan sulphate which is evidently highly involved in growth factor and other morphogen processing in multicellualr animals , could have become evolved as a consequence of such a deriving force. Note 6 Water Surfaces and Compensation Nucleation. Water structure is considered to be the complex assembly of hydrogen-bonded water molecules apparent in the liquid state and undergoing temperature-dependent supramolecular structure transition (e.g. at 20 and 37C) which are reminiscent of phase transitions, detectable by various diffraction and vibrational spectroscopic techniques as well as by measurement of density. NIR Spectra of Water in Heparin (Na) Films and Aqueous Solutions (in Capillaries). Dehydration of Na heparin films showed a systematic alteration of the fundamental and near infrared H2O and H-O bond structure as a function of water content (Grant et al 1986). Water and aqueous solution spectra of lamellar films and water in capillaries were also extensively studied by Grant et al 1981-84 but difficulties in interpreting the spectra have delayed publication of the results. Comparison of spectra of lamellar films of aqueous solutions containing heparin with spectra recorded in capillary tubes (with the light beam normal to the tube) showed that the spectra were similar in the first overtone stretching of the OH regions but significantly different in the 5 200cm-1 band due to H-O-H bending which showed an additional, usually more intense band at lower frequency ca 5000cm-1 in the capillary tube spectra. It was tentatively concluded that the effect of the capillary surface differentially affects the combination bands containing bending vibration components owing to the higher sensitivity of such bending vibration to supramolecular structures induced by the presence of water in a capillary as opposed to a lamellar film. Previously published supramoleuclar water structure effects detected by IR (by Gans, Luck and Ford & Falk) also suggests that where bending vibrations are incorporated into the combination bands temperature dependent structural transitions are evident, however H-O bond stretching overtone bands no not show these effects so clearly.
(possible insert) **

Those aspects of water structure which might promote enthalpy-entropy compensation could be a consequence of internal (labile) surfaces generated by hydrogen-bonding (electron proton delocalization bands) which seem to show up in near infrared spectra. More conventionally the critically important reactivity and conformation folding of proteins is thought to be dependent on water interactions at hydrophobic and hydrophilic surfaces of the protein (cf. Urry 1996). Water-protein interaction has been proposed to allow retention of information under ultradilution conditions and be the explanation of homeopathic medicine. Do polysaccharides also have a biological role in (nucleating?) the formation of phases of specific water structures in, e.g., water-rich gels and even of structures in near-pure water? Are such effects also relevant to homeopathic medicine? (and be relevant to the disputed effect reported by Davenas et al. 1983 which apparently showed that homeopathic preparations contained structural information derived from IgE precursor solute no longer present (heparin present in the buffer solutions used by Davenas et al. might (Glick 1988) have been the origin of the effect this supposing that heparin molecules could adopt stable

conformations able to behave (e.g. via water arrays at such conformations) like the original IgE molecules). Polysaccharides, which most characteristically contain arrays of hydroxyl groups, are abundantly synthesized by organisms, and may have a primary role for the modulation of water structure perhaps ultimately through entropic effects. Such polysaccharides may also possess anti-nucleation abilities for the prevention of induction of inappropriate supramolecular structure as well as co-nucleation abilities which modulate and control specific nucleation events. This possibility is supported experimentally by the effect of various glycosamioglycans on the kinetics of crystallization of calcite (Grant et al 1989cf Grant et al 1992b). Surface effects on near infrared spectra of water may also be related to Etvs factors. Such spectra which are due to overtone and bending stretching combination bands show up water aggregates which are temperature and (it may be argued from experimental data) surface tension dependent (possibly being influenced by polarizability of bonds at surfaces) Added later. In the NIR spectral study of Ganz (1936) by using the third overtone water hydroxyl stretch + bending combination band in the 0.77 micron band region, the results of a study of the effect of added sucrose on the NIR water bands showed an increase in absorption with increasing sucrose concentration (0, 17.3, 32.7%) but with little change in the position of the maximum of the broad overtone band which was very similar to that of pure water. This indicates that sucrose is highly water friendly since the third overtone plus deformation combination band of water in the presence of such a disaccharide is essential the same as in pure water. Most dissolved substances have a greater effect. It is also noteworthy form the results presented in the Ganz paper that NaCl has a similar only slight shifting effect upon the band maxima of this water structure band). The use of a high concentration of trehalose trisaccharide to create a water-like environmentunder suspended animation conditions (cf. Crowe et al 1987) may be due to such an effect. The polarizability of aggregated water molecules and their abilities to exhibit greatly altered NIR overtone and combination H-O- bond related e.m. radiation absorption may be of especial importance to biological systems. Wiggins & van Ryan (1980) found evidence for a wide ranging effect of surface on water structure associated with the types of polysaccharide gels which are commonly used by biomedical researchers for gel filtration. These authors considered that the infrared spectrum of such gels demonstrated an effect of capillary exclusion which caused the formation of stretched water structure in pores which was detected by density measurement as well as by infrared absorption energy variation. The incidence of nucleation of structure by pores was not considered by these workers but this is now advanced to be a potential factor involved in the alteration of the supramolecular structure of water reported by them. Various previous workers attempting to prepare a distinct (to become disreputable) polywater (but the specific hypothesis was later withdrawn by its originator) reported similar observations but these were differently interpreted. The near infrared spectrum of water in capillaries (Grant et al. unpublished) differs most notably in the intensity of bands ascribable to water clusters form that in lamellar films such as those commonly used by spectroscopists including WAP Luck. Interpretation of such spectra were however hindered by random fluctuations in the intensity of such bands perhaps attributable to spurious unstable nucleation effects of surfaces, temperature gradients and scattering.

These results may indicate that some of the physical measurements of water in capillaries made by the polywater researchers may not have been entirely due to impurities. The existence of a long range (polarization) effect of surfaces on the infrared absorptions of water (including the results reported by Wiggins & van Ryan 1990 etc.) accords with the original ideas of Bernal et al. (reviewed by Bernal in 1985) that surfaces exert very long range forces on water structure. Insect Antifreeze Structure. The insect antifreeze protein, the structure of which was reported by Liou et al. (2000) contains a polysaccharide-like-hydroxyl group array which closely fits a planar ice crystal face structure. The rigid planarity is bolstered by the highly crosslinked secondary structure of the protein. The planar surface with five water molecule width may be the anti-ice crystal capping-off site, able to prevent ice crystal nuclei growth to a size where they do not spontaneously re-dissolve; but could the planar surface also have a water structure nucleation effect hindering ice formation?) The other biological antifreeze proteins may present similar sites as may the anticalcification sites (of a similar size) centered on NSO3- groups of heparin/heparan sulphate (cf. Grant D et al. 1989).

5.3 Involvement of Hofmeister Effects on Water Structure.

Since the involvement of compensation effects in various biological processes may depend on unknown aspects of water structure (Kresheck 1986) a fuller understanding of the compensation effect may allow greater understanding of the biochemistry of aqueous solutions of biopolymers in general. Apparent phase transitions show up in studies of the temperature dependence of the near infrared spectrum of water, being particularly noticeable bands include a water bending vibration component. This effect is found for a 37C transition in the 0.7 micron (Ganz 1936) and 1.2 micron (Luck 1965) bands bur also is evident in the fundamental region bending vibration (although perhaps apparent in the (Ford & Falk 1967) reported results this was not discussed by these authors. (It would be of interest to determine if the effect is subject to Hofmeister lyotropic effect and are compensation effects also possibly relevant for unstable internal surfaces which may occur in water and other hydrogen bonded liquids). The results of the effect of temperature on the intensity of Raman spectra of water reported by Walrafen (1964) showed in Fig. 7 a 175cm-1 hydrogen-bond stretching vibration) a scatter of results which when re-plotted was indicative of ca 20 to 40 C transition although interpreted by the authors in terms of a single bound H2O unbound H2O state

6. Self Assembly of Complex Structures Such natural build-up by self assembly of order occurs in crystallization which is subject to initiation/nucleation of structure. This is the situation where nucleation effects are most fully understood (cf. Freundlich 1928). Compensation effects are evident in the nucleation of biological structure, akin to crystallization, as reported (Austen et al 1988) in the kappa carrageenan order disorder transition as mentioned above. Compensation effects are evident in the nucleation of DNA/RNA double helix formation seem likely and may somehow be involved in gene expression and allow response to biological evolutionary pressure.

The build-up of complexity with time in biological systems, apparently violates the second law of thermodynamics as classically developed for closed systems (although open systems may naturally reduce entropy over time, they are not obliged to do so). In chemical systems in which there is a general state of compensation, however, an induced increase in enthalpy over time induces a correlated decrease in entropy over time. To invoke such compensation effects for an explanation of biological evolutionary pressures required the assumption of the existence of biological containing interactive and cooperative compensation systems. Colloidal chemistry may also provide further instances. An intermediate colloidal phase formed in the first instance, during nucleation of regular crystal structure e.g. mentioned by Nancollas et al. (1983) could be morphologicially important in directing future structure building.

Minerals have no genes but calcium carbonate and ice crystals (and perhaps also liquid water and such preparations as silica sols) display constancy of pattern and ability to change it by forming a very large number of forms. This is assumed in biology to be the exclusive function of genes but as has been argued by Lima-de-Faria (1988), genes per se may have little to do with these two basic phenomena which are functions of controlled nucleation activities in which compensational effects may have a critical role perhaps in the murky chemistry of the nucleation processes. It may not be entirely fortuitous that silica particles resemble cells in that they are capable of growing by the uptake of monomeric silicic acid demonstrating a type of morphological nucleation behavior akin to biological reproduction (briefly discussed by Grant et al 1992a) and are subject to ageing. Similar cell-like behavior has been more fully described for organic colloids produced by thermal dehydration of racemic amino acid mixtures Fox et al 1970 on lava surface (quartz) surfaces (Hench 1982). These phenomena may be examples of nucleation of less-than-crystalline order. Nucleation of crystalline order is still incompletely understood. According to Lima de Faria (1988)* the explanation of biological development needs to await the explanation of crystal growth*. Possibly biologically relevant but also not understood nucleation processes seem to occur in dendritic growth of ice crystals and tip splitting in absence of superficial tension (viscous finger instability) also discussed by Lima de Faria (1988) and similar pattern developments in concentrated polysaccharide solution phase boundaries were reported by Preston(1980). This may be an experimental probability situation where the cooperative interaction between compensated systems may promote natural creation of order. Although the ultimate molecular electronic structures responsible for the effects are not established, it its clearly experimentally established for such order building systems as crystallization and spontaneous assembling of silanol -> siloxane polymers and proteinoids as well as more generally the existence of biology, obviously this phenomenon leads spontaneously to more highly ordered systems. Nucleation phenomena in which hydration effects are involved may also contribute to the binding of ions and molecules to polysaccharides including the highly anionic heparin/heparan sulphate (cf. Grant et al. 1992c). These polysaccharides also bind to co-ions including basic amino acids in proteins by a process which is believed to be more akin to a nucleated phase change (reminiscent of carrageenans with which they have structural and physiological similarity) than to a purely electrostatic process such as that proposed in the Manning hypothesis (cf Grant et al 1992c). The importance of heparan sulphate to developmental

biology has recently been highlighted (Perrimon & Bernfield 2000). These reactions might be subject to compensation effects and research to determine this is warranted. The occurrence of compensation might also be conveniently probed in the modulation of crystallization by polysaccharides such as lambda and kappa carrageenan and alginate which were reported by Birchall & Davel (1981) to modify the crystallization habits of sodium chloride both by acting as nucleation sites and by inhibiting nucleation. In the latter instance the polysaccharides were evidently present in a different phase form from the former situation (e.g. more of a solid than solute effect being induced). Controlled nucleation studies as reported for the crystallization of calcium carbonate (calcite) as modulated by polysaccharides such as glycosaminoglycans, carrageenans and alginates (Grant et al 1989 and unpublished papers) should also be extended to probe for compensation effects.

Preston et al (1980) discussed such formation and movement of organized macroscopic structures in an otherwise convection-free solution. The origin of such self-assembly of structured patterns then might ultimately arise out of the deterministic chaotic physics of fluids (including electrical properties of surfaces); the unusual hydrogen-bonding potential of water perhaps singles it out as the most versatile fluid in this regard and why living systems can ultimately develop from complex colloidal dispersions in water through nucleation effects. Further notes Note 7 Further Suggested Experiments The special physico-chemical effects of (clusters such as Fe 2+/3+ mixed valence systems (e.g. Poganiuch et al. 1991) surfaces may induce compensation via nucleation modulation. Surfaces define and separate phases. Electro magnetic field effects of (and on) surfaces due to mixed valence iron carboxylate complexes may be implicated in causing compensation effects. Note 8 Colloidal systems high surface area systems. Biolocial colloids have even larger surfaces (than abiotic inorganic colloids) cf internal surfaces, cisternae etc. Life is to do with such surfaces, perhaps especially flexible surfaces. (Surface waves of especial importance to biology?) Especially, perhaps, the effect of these surfaces on water structure. Note 9 It is common practice to facilitate the smooth boiling of aqueous solution (e.g. when used to carry out oxidative ceriometic reactions) by employment of boiling (carborundum chips) This is an example of nucleation of phase change. Note 10 Antifreeze protein Further notes In Water in protein crystals discussed by Finney (1979) Ch2 p57 Section 2.2.3 discusses surface ordering: For proteins, suitable arrangement of hydrogen donor /acceptor sites on the molecular surface may structure the interfacial solvent sufficiently to affect both the dynamics of the molecule and diffusion processes across or close to the interface. Such effects have been invoked to explain the action of so-called antifreeze glycoproteins in Arctic and Antarctic fishes; a suitable geometric arrangement of polar sites on the (extended) protein may produce a strong affinity for the ice structure, which may thus be prevented from further growth by a protein coating on the ice front (refs given). However a somewhat different

interpretation based on the relative orientations of sugar-OH groups and H2O molecules in water and ice is that the glycoproteins inhibit ice nucleation, i.e. they favor supercooling (ref given )*). The prevention of ice formation by antifreeze proteins evidently occurs by the provision of an exact match to the ice structure. Nascent ice particles may be prevented from growing by binding to the antifreeze protein (AFP) which contains a plane surface with attached water molecules mimicking the ice structure (Liou et al. 2000). Similar beta sheets to those identified in this protein may be more generally used for water structure modulation via the provision of geometrically exact planar surfaces thus confirming the effect of restriction of dimension on (nucleation) reactions and thereby control of entropic forces. The X-ray crystal structure of the beetle Tenevria molitor AFP which is a small protein (8.d4kD, monomeric in solution, in shape a slightly flattened cylinder of 32 Angstroms in length and composed of tandem 12 residue repeats TCTxSxxCxxAx, there are also five bound water molecules regularly spaced between six carboxyl terminal loops playing a similar stabilizing role to OH groups of internal serine residues) which translates into an exceptionally stable beta helix, the most regular protein structure yet observed (e.g. most mineral surface hydroxyl array* yet observed) containing a flat, putative ice binding surface consisting of threonine-cysteine-threonine motifs. The solid protein crystals contain dimers lined by two layers of water molecules attached to the beta sheets; such ordered water molecules could participate in the mechanism of antifreeze protein ice nuclei binding (i.e. they contain a small piece of one layer thick ice plane interface). The two dimensional array of the threonine side chains makes a remarkably good march to the repeated spacing between oxygen atoms in the ice lattice on the primary prism plane 7.35 Angstroms and 4.52 Angstroms and a reasonable match to the basal plant (7,83 Angstroms and 4.52 Angstroms being highly suggestive of its ice-binding function).

Note 11 Consider Chemical Engineering Flow Patterns in Water Attempts to model chemical reactions occurring in the fluid phase require complex mixing and flow pattern detection. Some mixing systems generate vortexes which are unstable but switch position in a regular manner detectable by synchronized cinematographic evidence (cf. studies carried out in the sixties at ICI using reporter polystyrene beads aiding detection of the effects ). On a smaller scale, fluid dynamics should hardly be dismissed as of not importance biologically especially for the critical effect on water chemistry; biological systems may have resource to such systems of vibrating flows (e.g. waves on water rich surfaces?) The Couett-Taylor experiment of a fluid (usually water ) between two concentric cylinders, where the inner rotates, shows lamellar (Couett) flow at low speed changing at higher speed to Taylor vortex flow (showing periodic wave patterns) which changes at yet higher rotational speed to more complex patterns of turbulent Taylor vortices. Such experiments have been used to confirm the principles of chaotic dynamics (cf. Fiedl & Golubitsky 1992). The (deterministic) nature and their change in hydrogen bond patterns at the microscopic level of water will accompany flow-like pattern change and it might be speculated to be subject to influences allowing structure to be induced by stirring and other similar nucleating effects. Note 12 Consideration of Freuchlich Colloid & Capillary Chemistry Textbook

Work must be expended to bring liquid from the interior to the surface since the liquid state is maintained by short range attractive forces between its molecules. In a liquid the surface pressure depends on the radius of curvature but the surface tension is independent of the surface curvature (cf. Freundlich (1928). Experiments were designed to investigate such liquid internal pressure as in the experiments reported by Berthelot in 1850 (Ann de Chem et de Phys (3) 30 232) where a thick walled tube was filled as completely as possible with a liquid and its vapor and by warming the liquid is made to completely fill the tube. If we now cool the wholeif the wall of the tube were thoroughly clean, and all change air bubbles had been completely dissolved, the tube remains completely filled at low temperatures. Vibration or excessive fall of temperature will cause the liquid to resume with a snap its proper volume. (added later this is clearly a nucleation effect-the vibration will create surface waves which trigger the change in volume). The above experiment is a direct formation of stretched liquid); cf. stretched water in pores as a consequence of osmotic pressure Wiggins & van Ryan 1990 CHECK.. This is also like the DIY refitting of an (old) tubeless car tire where at a critical air pressure it suddenly snaps back into its proper position, it being inhibited from this position in this case by slight surface roughness. Note 11 Strained Water At low pH values the hydrogen moves out from the oxygen as far as 1.05 Angstroms: at high pH it is at 0.85 Angstroms; at neutral pH it is at 0.96 Angstroms; practically every surface in a cell has an ice like boundary. Water can also be considered to be a colloid with ca100 Angstrom size particles (Bernal 1966) (but considerably more mobile than ice (Picuelell 1985, Ling 1973). The symmetrical hydrogen bonded H5O2+ and H5O2(H2O)4 structures were subjected to ab initio calculations by Munitz (1985); solvation being found to convert a single minimum situation to a two minimum situation. Broken H-bond (Stillinger 1980) quantum mechanical studies suggest that hydrogen bonding in water is cooperative) or that unfulfilled H-bonds (Symons 1990) exist in water with strained bonds (Geiger et al 1979). Symons (1989) suggested that the reactivity of water was a function of the number of free HO groups and the unpaired electrons (LP water). Reactions requiring attack by H-O required free groups while nucleophilic attack required LP water. Basic solvents caused a apid decrease in H-O free detected by NIR (3000cm-1 band) added salts also affected these structures. Note 12 Hydrophobic Bonding Further discussion (by Stillinger 1980 and Sharp 1991) Nonpolar solvents which cannot form hydrogen bonds such as noble gases and hydrocarbons cannot hydrogen bond to water (also sparingly soluble) but when dissolved in water cause structure making. Note 13 4C Transition Below the 4C transition temperature liquid water contains a tridymite-like (four coordinated) water structure and a quartz-like water structure in the temperature range 4-200C and an ammonia like close packed structure between 200 and 34 0C. Pauling later proposed a dodecahedral water structure to account for similar experimental data. Cf the -45C transition is caused by redistribution of strained networks (Angell, 1980)

Note 14 Infrared data on water structure (Later note se also Symons 1989) Luck (1965, 1968) ascribed absorptions at 3725, 3700 3545, 3410 and 3355cm-1 respectively to HO groups in monomer, dimer, tetramer and polymer water; unbonded HO groups showed a distinctive absorption at 3748ccm-1.
Combine with Walrafen et al.

Raman bands at 3650 and 3250cm-1 identified fully hydrogen bonded (H2O)5 tetrahedral assemblies. Aqueous solutions of sugars showed decreased absorption at 3250cm-1 while perchlorate,which does not form hydrogen bonds, increased the absorbance at 3570cm-1 ascribed to dangling HO groups associated with de-structured water (Walrafen & Fisher 1986). Note 15 Anti-inflammatroy drugs such as hydorcortisone, aspirin, indomethacin, flufenisal and naproxen seem to contain functional groups positioned so as to fit into an ice-like lattice at the cell surface (Warner, 1973). Note 16 Recent discussion of developmental biology involving Turing wavelength and experimental work of Childs et al by Y Schiffmann (Chemweb.com Preprint Server 27 Sept 2000 14:51) (which discussed oxidation reduction potential and cAMP as Turing vectors for generation of biological polarity and form in developmental biology); however, other mechanisms might be accommodated to such hypotheses. Cf., Perrimon and Bernfield 2000) reviewed the hypothesis that heparan sulphate biochemistry may be an important morphogen control system, providing sites for the assembly of growth factors (operating via phosphorylation), a sort of highly specified smart glue but subject to modulation of its biosynthesis by oxidation-reduction potential as well as post secretion modification by oxidation reduction chemistry (especially of nitrogen based, e.g. nitric oxide metabolite effects). The modulation of planar surfaces generated adjacent to heparan sulphate helix structure which might also be of fundamental importance to entropic force regulation in assembly of biological components which modified by heparan sulphate binding will alter their water regulating effects. Perhaps the simplest morphogen model might be a form of activated water such as the hydrated electrons at (internal) water surfaces. Schiffman (2000) wrote In the literature it is often assumed, e.g., with respect to Hydra, that several Turing systems coexist and it is also assumed that maintaining the polar profile, even when the system increases in size, is important for the polarity of the final phenotype in reality there is only one Turing system. Childs system of physiological dominance , subordination and isolation are interpreted in light of the Turing wavelength P34 Traditionally there have been two great enigmas in biology the problem of epigenesis and the related problem of increasing differentiation, organization, localization, order heterogeneity and negative entropy. The fact that a system is on open one only makes the decrease in entropy possible but not obligatory Second, the problem of regulation i.e. the problem or organic purposeness, the so-called equifinality of developmental processes or the manifestation of foresight towards a goal, the goal being the creation of a normal organism- a whole-not only form a normal egg but also from a part of it, or form two fused eggs, or from pieces of hydroids or from planarians etc.

.The solution to the problem of localization (which) is simply the problem of the selfproduction of heterogeneity for homogeneity (Child 1948) could not even be conceived before Turing. This problem was a major cause of belief in vitalism. Current biological systems are likely to use highly evolved mechanism (s) which may include such entities as cAMP with a Turing inhibitor ATP but derived in the distant past from a simpler mechanism, but current biological systems may retain the ability to be influenced by primitive signaling and may be capable in principle of employing both. Current systems might also employ more than a one kind of diffusible substance for ChildTuring mechanism morphogenesis. A further example of a primitive biological mechanism, now more-or less replaced by highly evolved systems, might be the discrimination between Na+ and K+ by stretched water in pores (Wiggins & Van Ryan 1990). 7/1/01 NO and cAMP etc might be Turing morphogens working on a heparan sulphate workbench. More primitive systems might have however used more primitive morphogens (a primitive and a highly evolved mechanism may be a general feature of biological systems); modern systems might retain such abilities and have modern and old systems working as back-up. Examples of possible morphogens include hydrated electrons, superoxide anions, adducts of these with water or silicic acid or calcium salt colloid (colloidal amorphous calcium carbonate etc) internal surfaces. The thermodynamic effects of restriction on entropy trends needs to be fully addressed as regards open and closed or other restricted (e.g. the effect of surface restriction or the very occurrence of reactions at the molecular level (where particle and wave nature of matter may be inter-convertible) which was not considered as part of the framework of origins of the laws of thermodynamics as applied to self-assembly of complex systems. 8/1/01 Consider further possible Turing morphogens. The use of morphogens would come to be needed in muticellular organism. This seems to be where extracellular polysaccharides and especially heparan sulfate come into the picture. This is a highly evolved function. Such complex patterning of cellular structures would have been absent in primitive life forms where evolution of form would have been analogous to the fully crystalline states as discussed by Lma de Faria. Primitive morphogens, however, might have included: Ca2+, CO3 2-, CaCO3, oxalate, Na+ K+ Mg2+. Effect of these on water structure.

Heat alone: its effect on water structure. Heat alone its effect on phase boundaries of aqueous colloidal systems or lipid membranes at water interface. (Cf. lava lamp heat provided by light bulb creating biological-like movement of large cell-like structure.) The use of water by living organisms may be based on an ability to use water efficiently for energy transfer as well as for electrical property induction at phase boundaries. Cf. the use of a particular temperature region as e.g. 37C where there may be an additional advantage of a phase-like transition. Water may also possess an ability to generate internal temperature/structure profiles which have a degree of stability. However living organisms seem to need only liquid water at any temperature region where liquid is present. The heat/energy source may be any form. Sunlight. Oxidation reduction potential of highly charged redox system (?) Fe3+/Fe2+, oxidation of C-H compounds (e.g, in partly oxidized structures). Note 17 Limb Regeneration and Ion Currents

Cf Mackenzie (1982) who reviewed the work of Jaffe and others who demonstrated the importance of the Ca2+ ion currents. In all living systems we know of the membranes that surround the living cells maintain differential distribution of ions. (Such differential distribution of ions will produce a differential water structure equivalent to a different temperature (cf. Luck) across the membrane or phase boundary). Polarity in developmental biology may be determined by differential ion movements. In the seaweed fucus embryos light generates polarity evidently via Ca2+ gradients. (Anionic polysaccharides especially at the pericellular regions may be involved in such Ca2+ effects). *Eggs have another use for Ca2+ which takes seconds. When sperm fuses, its injects a shot of Ca2+, which releases more Ca2+ from stores within the egg. This Ca2+ releases yet more, causing a positive feedback explosion that propagates across the egg as a wave of calcium on hundred time more concentrated than normal. Jaffe et al observed the wave with the aid of a protein, aequorin, which emits light when it binds to Ca2+.* *During the contraction of the cytoplasm in cell division current enters the cell along the line where, minutes later, the furrow will appear between the two new daughter cells. In salamanders which regenerate lost limbs, the electrical current is intense at the stump of an amputated limb. The field produced by the stump currents may attract nerve axons which promote the regeneration of limbs. Can local calcium currents arrange tissue morphogeneiss? Amid the present enthusiasm for a molecular genetic nucleocentric approach to development, it must be remembered that a cell at some point receives instructions from outside The epigenetic input-that is telling a cell where and who it is and therefore which genes is would be appropriated to use is after all the crux of developmental interactions, the dynamic process through which differentiation and morphogeneiss are enacted. Many of these instructions it now appears, ride along on currents of electricity. Possible involvement of water molecules adjacent to cytochrome c oxidase (membrane bound) was discussed by Sassaroli et al (1989) from Raman vibrational spectroscopic evidence . Cytochrome c oxidase is the terminal enzyme in the electron transport chain and serves as the catalytic site for the reduction of O2 to H2O. Water molecules may take part in the proton translocation pathway. Note 18 9/1/01 Ford & Falk (1988) attributed a high scatter of apparent band maxima around the 3300cm-1 stretching (v3) vibration but not around the 2430cm-1 bending (v1) vibration in studies of HOD infrared spectra (paper discussed in original file) to condensed water droplets at the cell windows. They write The high scatter for the v3 data is probably caused by condensation of traces of water vapor on the silver chloride windows. Condensed H2O would lead to apparent broadening of the v3 band but would not affect the v1 band. Frequencies of band maxima are not affected by traces of H2O. Note 18 10/1/01 Early workers (Ganz 1936 and reference cited therein) in the fild of ir spectra of water and aqueous solutions had ready access to the NIR region and made important studies of the

influence of temperature and added substances on water structure to which the NIR spectroscopic region is especially sensitive (cf. also Symons 1989). The discontinuity in the spectrum of water at ca40C was observed in the 13000cm-1 band by Ganz (1936). Dilute (0.7N) NaClO4 showed the band but the band was shifted to higher energy (more free H2O) and as the transition to somewhat lower temperature. Conc NaClO4 5.6N, 9.24N HCl and 30% H2O2 also abolished the transition. The effect of perchlorate on the transition and the effect of the other substances mentioned is evidence that the transition is due to water structure (alteration) e.g. the way arrays of hydrogen bonds are arranged. Note 19 11/1/01 The discontinuity in the 1.1.8 micron band of water was almost considered to be an artifact by Luck (1965) being a secondary effect only as regards the use of this temperature by many homoisothermal animals due to cross over at this temperature of extinction coefficients of the various component bands at ca 37C; the amount of free HO groups as a function of temperature seems to be almost completely linear showing not discontinuities (cf Fig 4 of Luck 1963). However since the origin of the value of the extinction coefficients for various overtone bands will depend on factors related to the polarizability of water structures which could be profitably used by organisms it seems apparent that such polarizaiblity phase transition seems relevant to biochemistry. Note 19 13/1/01 Biochemistry seems to have primitive and evolved mechanism such as primitive phosphorylation and primitive functions for polysaccharides*. The highly evolved pumping selectivity of biological membranes for Na+ and K+ ions might stem from a primitive stretched water in pre selectivity discussed by Wiggins & van Ryan (1990). Such polysaccharides as heparin/heparan sulfate and Ca phosphate and silicate binding sites on these anions involving nucleation activity can be hypothesized to be involved in the mechanism of growth factor signaling. Primitive functions might be discernable in toxic elements of minerals such as asbestos, silica fibers and hydroxyapatite); cf. the calcium containing crystals associated with mammary tumors such crystallinity being an indicator of malignancy, have been suggested to be involved in promotion of the disease and via failure of cellular mechanism (which may include sulfated polysaccharides) for maintenance of the amorphous state may be part of the etiology of the disease process (cf. Grant et al. 1992b). That the Ca/P ratio of crystallites associated with mammary tumors was identical to that of milk (Plant 200) suggested that the cancer disease process was somehow triggered by aberrant milk secretion due to growth factor (e.g. IGF-1) signaling; In vitro cellular mitogenic signaling by somatomedin is required for the mitogenic effect of Ca-rich solids (cf. the somatotrophicn IGF-1 related activity which was reported by Cheung et al 1986). Calcium pyrophosphate granules present in a wide range of species (in membrane bound compartments) are maintained in an amorphous state in vivo but they crystallize in vitro, clearly indicating a role for the cellular system in maintaining the amorphous state. These intracellular granules in the snail Helix aspersa have the ability (heparin-like!) to incorporate a wide range of metal ions (Taylor et al 1990). Sulfated polysaccharides including heparin.heparan sulfate/carrageenan, xylan sulfate and polyvinyl sulfate exhibit a range of antiviral and anti-crystallization activities where the charge density seems to play an important role: thus similar levels of activity were found for heparin

and carrageenans of similar charge density. Such a charge-array effect is a mineral-like property of such sulfated polymers. The multi-ion content of heparin (Grant 2000 Chemweb preprint server) (perhaps suggesting an analogy with a geological sample of soil or natural water ( cf www.bgs.ac.uk.iugs quoted by V Ashton, Chem Brit 2001 37 (1) 33-34 reporting a conversation with J Plant)) suggests that heparan sulfate is also a multi-inorganic-ion matrix and futhermore differences between normal and virally transformed cell heparan sulfates in this aspect of the chemical nature of these polyanions, create differences in ionicity and hydration-determined cell activities (including nucleation activities). (Added later Wiggins & van Ryan (1990) studied the broad 3600cm-1 band ir spectra of water HOvibration alone or in gel (pores) and interpreted the large differences in the details of such spectra as being supportive of their hypothesis of an altered water structure). 13/1/01 Unstable (akin to deterministic chaos) patterns of cooperatively formed hydrogen bonds between water molecules where the lifetime of the individual hydrogen bond (ca 10-12 seconds)) (n.b. this kind of bond switching leads to anomalous high proton conduction in water) may additionally be subject to nucleation of structure (e.g. by magnetic and electromagnetic fields, surfaces and other weak forces with nucleating activity and biological relevance ) might be tentatively hypothesized to be important to biology and could even be a main determinant of the uniqueness of water to the biological phenomenon by allowing utilization of the interactive entropicenthalpic potentials of such systems in order to encourage the long term generation and stabilization of highly ordered non-crystalline systems. To elsewhere Evidence for the control of nucleation of mineral structure by weak magnetic fields comes from the prevention of the formation of boiler scale when the feed water is passed through a magnetic field, a treatment which may be derived from ancient Chinese tradition (Anon 1990). The mechanism of this effect (which is subject to disbelief)(might include an effect of surfaces on water structure modulation and might even be relevant to mitogenic signaling such as that produced (pathologically in synovial hyperplasia in joint disease and in breast cancer) by surfaces such a (crystalline or amorphous hydroxyapatitte surfaces of exactly the same Ca/P ratio as milk (Ashton 2001), and enhance the effects of growth factors such as IGF 1 and cf. somatotrophin). Such hydroxyapatite seemed to have a mitogenic mechanism similar to that of PDGF and may enable cells to escape their dependence on somatomedian C for DNA synthesis (Cheung et al 1988) further studies with such solids and CaCl2 and LaCl3 showed that cell surface events strongly influence mitogen responsiveness and cell densitydependent growth inhibition (Praeger & Gilchrest 1989). (Grant et al (1992) further hypothesized that anti-crystallization activity of normal heparan sulfate, but not heparan sulfate degraded by factors associated with cancer and inflammatory diseases, might have been capable of preventing such mitogenic signaling and glycosaminoglycan efficiency may be a factor in the etiology of degenerative diseases). Discussion of the possibility that growth factors present in bovine milk may account for the high incidence of breast cancer in Western society stems form epidemiological information (Ashton, 20o1) Similar dietary growth factor ingestion (principally form animal sources) together with the effects of mineralized surfaces, may be more widely relevant to the etiology of cancers and thorough epidemiological investigation is surely warranted in regard to growthfactor-like molecules which may act together with cofactor surface agents in various foods. Such mitogenic stimuli might e.g. be present in preparations where hydroxyapatite residues (e.g. from bone) are activated by the irradiation of food for sterilization.

Such relevance might even include (upper) stomach cancer. 15/1/01 Further indicators of Important Biological Nucleation. Nucleation-like induction of mitogenic activity by solid calcium phosphates at cell surfaces may be involved in the induction of breast cancer was originally discussed by Grant et al. 1992a. This possibility seems part of the model outlined by Plant (cf. Ashton 2001) where minerals of milk-like elemental composition are formed due to dietary toxins (hormone/growth factors) causing inappropriate DNA activation. Nucleation needed to produce biology from stochastic proteinoids. Nucleation events, not considered by the chemists involved, are now proposed to be the cause of a limited non-random content achieved by thermal condensation polymerization of racemic amino acids at silicate surfaces using ammonia + methane + water derived amino acid mixtures). Such non-random structure generation evidently allows such proteinoids to have enzymic activity. Condensation polymerization of phosphates, silicates and other relatively simple monomer systems has been demonstrated to occur essentially by stochastic processes (Grant 1964, 1979). The formation of multiple polymeric structures is enhanced by a further tendency of such systems to undergo stochastic ligand redistribution reactions leading to change of functionality. Only where nucleation events intervene (e.g. in the induction of crystallization of specific silicate or phosphate structures) are such systems capable of generating highly ordered systems. It seems necessary to include consideration of nucleation-like induction of structure in any rational consideration of the chemical origin of biological cells. Fox et al (1970) reviewed laboratory simulation of the chemical origins of cells. An important omission from this review is suggested to be any in depth consideration of the effect of nucleation phenomena (although this was thought to have possibly influenced the selection of L amino acids from (all20) mixed D and L amino acids formed by electrical discharge through methane, ammonia, hydrogen and water). Fox et al (1970) demonstrated that organic chemistry could generate primitive bacterial-like colloids from proteinoids formed (e.g. at geothermal vents) above 100C by thermal dehydration followed y dispersal in water. HCN processes could also five rise to heterocyclic nitrogen bases present in nucleic acid allowing formation of e.g. amino acid adenylates which allows condensation of amino acids in aqueous solution. In such a manner , it was argued, the formation of living organisms could have been envisionaged without any need to violate the second law of thermodynamics. The above argument is thought to be flawed since the occurrence of a natural increase is needed to generate even the simplest organism from the primitive proteinoid microspheres would have required an enormous generation of increased order with increasing time outwith the probability of random occurrence by classical thermodynamic influences. Only by the incorporation of nucleation events (Maxwell demons as it were) it is suggested can the essentially stochastic assembly processes outline by Fox et al be transformed into biological systems. Another analogous chemical system (which in this case is capable of forming polymeric units in aqueous solution by natural self assembly) and was found to be subject to the nucleation of sub-crystalline order in quasi-biological scenarios, are the silicic acids and derived aqueous dispersions of silica sols derived from them (Grant et al 1992b).

Nucleation filters might also be responsible for the generation of compensation between entropic and enthalpic changes in reactions occurring at surfaces and other restricted situation (as discussed above). The restriction of a natural tendency of entropy to increase with time by such influences as surface effects which may influence water structure seems somehow to offer potential insight into natural selection of form since protein structure is currently modified and controlled biologically by such water interactions. 16/1/01 Consideration of the morphogenic effect of interlinked nucleation activities. Polysaccharides may bind to other ions and molecules by a nucleation-dependent mechanism. Different physical forms of such polysaccharides, however, who different abilities to engage in such interactions and such physical forms themselves are subject to nucleation triggers for their formation. This indicates a possibility that a hierarchy of nucleation events could generate complex morphological effects, and may lie at the core of the thinking (by Bernal and Lima de Faria) linking a need for fuller understanding of the nucleation of crystallization as being a key to unravel the complexities of biological morphology generation and the emergence of biological systems in the first place. 17/1/01 It was concluded form studies of the formation of ordered structures at polymer solution interfaces where formation and movement of organized macroscopic structure (such as dendrite-like growth (cf. also refs given by Melrose et al 1990) who noted that the principles which govern a wide variety of tree patterns formed by growth under conditions beyond those of diffusion-limited aggregation are at present unclear) in dextran solution in a convection free system (Preston et al 1980) that previous ideas (cf. Nicolls & Prigogine 1977) possibly involving nucleation of non equilibrium structures allowing self-organization of complex systems nonequilibrium unexpectedly may be the source or order producing coherent behavior in space and time *Note Biological molecules seems to demonstrate both primitive and highly evolved properties. Heparin, in protection of extracellular space against insult by superoxide radicals seems to have both primitive direct non-enzymic superoxide dismutase activity as well as the highly evolved antioxidative activity dependent on the interaction of heparin/heparan sulfate with the superoxide dismutase enzyme. (The major extracellular SOD is bound to cell wall heparan sulfate). The primitive anticoagulant activity of heparin in non-enzymic dissolution of blood clots is accompanied by a complex anticoagulation mechanism e.g. that involving AT(III). The original primitive function are likely to be essential to the emergence of biological systems but are difficult to discern in modern highly evolved systems. References Addadi L Berkovitch-Yellin Z Weissbuch I van Mil J Shimon LJW Lahav M Leserowitz L (1985) Growth and dissolution of organic crystals with taylor-made inhibitors implications in stereochemistry and materials science Angew Chem Engl 24 466-484 Ashton (2001) Earthly pursuits Chem in Brit 37 (1) 33-34

(A discussion with J Plant including epidemiological evidence for pro-cancer effects of growth factors in diary products; this discussion and cited reports are evidence for the benefits of a vegan diet; cf. Bossano M (2000 the (summer issue p10)) Austen KRJ Goodall DM Norton IT (1988) Anion effects on equilibria and kinetics of the disorder-order transition of kappa carraggeenan Biopoymers 27 139-155 Barclay IM Butler JAV (1938) The entropy of solution Trans Faraday Soc 34 1445-1554 Bernal JD (1965) The structure of water and its biological implications Symp Soc Exptl Biol 19 17-32 Chem Abs 65 9242f Birchall JD Davey RJ (1981) The crystallization of sodium chloride from aqueous solution in the presence of polysaccharide J Crystal Growth 54 3233-329 Blackadder DA Hinshelwood (Sir) C (1958) The kinetics of the decomposition of the addition compounds formed by sodium bisulphite and a series of aldehydes and ketones. Part II. General Discussion of energy-entropy relations J Chem Soc 1958 2728-2734 Boon MR (1973) Thermodynamic compensation rule Nature 243 401 (cf also the immediately following letters to Nature) Brown HD (Edit.) (1971) Chemistry of the Cell interface Part B Academic Press new York Cf p70 Cheung HS Van Wyk Jj Russel WE McCarty DJ (1986) Mitogenic activity of hydroxyapatitie: requirement for somatomedin C J Cell Physiol 128 143-148 Columban P Ed (1992) Chemistry of Solid State Material Proton Conductors , Solids , Membranes and Devices Cambridge University Press Crowe JH Crowe LM Carpenter JF Aurell Wistrom C (1987) Stabilization of dry phospholipid bilayers and proteins by sugars Biochem J 242 1-10 Davenas E Beauvais F Amara J Oberbaum M Robinson B Miadonna A Tadeschi A Pomeranz B Fortner P Belon P Sainte-Luady J Poitevin B Benveniste J (1988) Human basophil degranulation triggered by very dilute antiserum against IgE Nature 333 816-818 cf Glick JL (1988) ibid 334 376 Dziewiatkowski DD (1987) Binding of calcium by proteoglycans in vitro Calcif Tissue Int 40 265-269

Edwardson JA Oakley AE Pullen RGI McArthur FK Morris CM Taylor GA Candy JM (1988) Aluminium and the pathogenesis of neurodegenerative disorders in Aluminium in Food and the Environment Ed RC Massey D taylor Royal Society of Chemistry Special Publication No 73 Finney JL (1979) The organization and function of water in protein crystals In water, a Comprehensive Treatise Vol 6 Ed F Franks Plenum New York (1979) Eley DD (1967) Energy gap and pre-exponential factor in dark conduction by organic semiconductors J Polymer Sci C (17) 73-91 Eley DD (1972) J Polym Sci A2 874 Evans GJ (1982) Influence of external fields on nucleation and crystal growth J Chem Soc Faraday Trans (1) 673-687 Field M Golubitsky M (1992) (paperback edition with corrections 1995) Symmetry in Chaos Oxford University Press ISBN 0 19 8553688 7 (Pbk) Ford TA Falk M (1968) Hydrogen bonding in water and ice Canad J Chem 46 3379-3386 Fox SW (1960) Thermal polymerization of amino acids and production of formed microparticles on lava Nature 201 336-337 Cf Fox SW Harada k ibid 201 s335-376 Fox SW Harada K Krampita G Mueller G (1970) Chemical origin of cells Chem Engineering News (June 22) 80-94 Ganz E (1936) ber dem Absorptionspectrum van wsserigen Lsungen zwischen 0.70 micron bis 0.90 micron Z Phys Chem B 33 163-178 Glick JL (1988) Only the smile is left Nature 334 378 Grant D (1967) Redistribution reactions in polymeric alkyl silicates J Inorg Nucl Chjem 26 337 and ref cited Grant 1968 ICI Central Instruments Research Laboratory Note highlighting compensation between pre-exponential and activation energy parameters of dehydrochlorination reactions and information form PJ Thomas ICI Mond Division Grant D (1978)

The rearrangement polymerization of phosphorous acid with acetic anhydride Europ Polymer J 15 1161-1165 Grant D Long WF Williamson FB (1987) Infrared spectra of heparin-cation complexes Biochem J 244 143-149 Grant D Long WF Williamosn FB (1988) Polystyrene surfaces may require structured water for effective cell adhesion Biochem Soc Trans 16 1029-1030 Grant D Long WF Wiliamson FB (1989) Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization Biochem J 259 41-45 Grant D Long WF Williamson FB (1990) The dependence on counter-cation of the degree of hydration of heparin Biochem Soc Trans 18 (6) 1283-12884 Grant D Long WF Williamson FB (1992b) A putative role for colloidal silicates in primitive evolution deduced in part from their relevance in modern pathological affliction Medical Hypotheses 38 46-48 Grant D Long WF Williamson FB (1992c) Zn2+-heparin interaction studied by potentiometric titration Biochem J 267 849-853 Grant D Long WF Somers JA Williamson FB Inhibition by carrageenan of CaCO3 (calcite) crystallization (unpublished) Grant D (2000) Web.ukonline.co.uk.dgrant Hench Cf. discussion paper by Last JA & Reiser KM (1986) Silica Biochemistry Wiley, Chichester (Ciba Foundation Symposium 121) p 180-1993 Hofmeister F (1888) Zur Lehre von der Wirkung der Salze Archiv fur Experimentelle Pathologie und Pharmakologie 24 247-2260 Krescheck GC (1988) Calorimetric methods for measurement of interactions of biomolecules with water Methods in Enzymology 127 142-150 Leffler JE (1955) The enthalpy-entropy relationship and its implications for organic chemistry J Org Chem 20 12121-1231 Liou Y-C Tocilj A Davies PL Jia Z (2000) Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein Nature 408 322-3243 Lima-de-Faria (1988) Evolution without selection. Form and function by autoevolution

Elsevier Science Publ BN (Biomedical Div) Amsterdam Ling GN (1962) A Physical Theory of the Living State Ed Negendant M & Edelmann L (1988) Scanning Microscopy Int. Chicago p 89 Luck W (1963) Beitrag zur Assoziation des flussigen Wassers. I Die temperaturabhndigheikeit der Ultrarotganded des Wassers Ber Bunsenges 67 (2) 186 (1965) II Salzeffecte ibid 69 (1) 69 (1965) III Die Temperabhndigkeit ibid., 69 (7) 626-637 (1987) Proc Symp in Honor of WAP Luck, Marburg 2-3 April; Ed Hubertus Kleeberg Springer Verlag Berlin Mackenzie D (1982) The electricity that shapes our ends New Scientist 93 (1290) 217-220 Melrose JR Hibbert DB Ball RC (199)) Interfacial velocity in electrochemical deposition and the Heckler transition Phys Rev Letters 65 (24) 3009-3012 Nancollas GH Sawada K Schutringer E (1983) Mineralization reactions involving calcium carbonate and phosphate Biomineralization and Biological Metal Accumulation EdsP Westbroek and EW de Jong Reidel Nicolls G Prigogine T (1977) Self-Organizaiton in Non-Equilibrium Systems Wiley New York (reference given by Preston et al (1980) ONeil CH Hodges GM Riddle PN Jordan PW Newman RH Flood RJ Toulson EC (1980) A find fibrous silica contaminant of flour in the high oesophagal cancer area of North-East Iran Int J Cancer 28 61-628 Pashlley KM McGuiggn PM Hinham BW Evans DF (1985) Attractive forces between uncharged hydrophilic surfaces. Direct measurement in aqueous solution Science 229 1088 Perimon N Bernfield (2000) Specificities of heparan sulphate proteoglycans in developmental processes Nature 404 728 PpoganiiuchP Lia S Papaefthymiou GC Lippard SJ (1991) A trinucelear , oxo-centred mixed-valence iron complex with unprecedented carboylate coordination [Fe3O(O2CCH3)(TACN)].2CHCl 3 (TACN is 1,3,7 triazacyclononane) J Amer Chem Soc 113 4645-4651 Preston BN Laurent TC Comper WD Checkley GJ (1980) Rapid polymer transport in concentrated solutions through the formation of ordered structures Nature 287 499-503 Praeger FC Gilchrest BA (1989)

Calcium, lanthanum, pyrophosphate, and hydroxyapatite: a comparative study in fibroblast mitogenicity Proc Soc Exper Biol Med 190 28-34 Sassaroli M Ching Y-C Dasgupta S Rousseau DL (1989) Cytochrome c oxidase : evidence for interaction of water molecules with cytochrome a Biochemistry 28 3128 3132 Schiffmann Y (2000) CPS biochem/000090002 The biochemical basis of polarity and form regulation in development and restructuring Schoolery JN Alder BJ (1955) Nuclear magnetic resonance in concentrated aqueous electrolytes J Chem Phys 23 (5) 805-811 Symons MCR (1989) Liquid water the story unfolds Chem in Brit (May 1989) 491-494, 498 Tanaka T Nishio I Sun S-T Ueno-Nishio S (1982) Collapse of gels in an electrical filed Science 218 467-469 Taylor MG Greaves GN Simkiss K (1990) Biotransformation of intracellular mineral by zinc ions in vivo and in vitro Eur J Biochem 192 7883-78 Urry D (1996) Engineers of creation Chem in Brit 32 (10) 39 (cf von Stackellberg M Muller HR (1951) Naturwiss 38 456) Freundlich H (1926) (Translated by HS Hatfield) Coloid & Capillary Chemistry Methuen & Co London Walrafen GE Raman spectral studies of water structure J Vchem Phys 40 (11) 3249-3256 Walragen GE Fischer MR (1986) Low frequency Raman scattering from water and aqueous solutions : a direct measure of hydrogen bonding Methods in Enzymology Academic 127 91113

Wiggins PM van Ryan RT (199)) Changes in ionic selectivity with changes in density of water in gels and cells Biophys J 58 585-596 Wilson MC Galway AL (1973) Compensation effect in heterogeneous catalytic reactions including hydrocarbon formation on clays Nature 243 402-404 Wolf BA (1972)

Interdependence of enthalpic and entropic contributions to the second osmotic virial coefficient J Polymer Sci A-2 10 847-856 Zndel G Murr A (1969) Hydration und hydrophobe Wechselwirkung der Ionen in Polyelectrolyten mit eniner Folgerung bezglich der biologischen Membranen Z Naturforsch 24b 375-377 Zndel G Muehlinghaus J (1971) Proton dispersive forces and continuous energy level distribution of proton in the hydrogen bonds of semiprotonated poly-L-histidine and with model substances Zeit Naturforsch 26b (6) 546-554 Zndel G (1976) Easily polarizable hydrogen bonds-their interactions with the environment- IR continuum and anomalous large proton conductivity Chapter 15 of The Hydrogen Bond. Recent Developments inThe Theory and Experiments P Schuster, G Zundel and C Sandorfy Eds North Holland Publ Co 1976 (Later addition Heparan sulfate helices may generate biologically relevant water surfaces in a similar manner to the anti-freeze protein reported by Liou et al. One may imagine that the regulation of protein shape (as in growth factor activity regulation) will include such water surface structure effects). The patterns which are observed to develop at phase boundaries formed in polysaccharide/polyether solutions/dispersions might have a related mechanism of formation if microflow patterns (e.g. of deterministic chaotic systems of changing hydrogen bonds were analogous to those of bulk flow induced patterns); A mathematical point might be the ultimate entropy decrease generator if the above argument is extended. Perhaps nucleation sites are sometimes more of the nature of points than of linear sites or two dimensional surfaces. Cf. point dislocation. Cf heparin AT(III) binding site centered at a high charge density area of the specific structured pentasaccharide. There may be a hierarchy of compensated effects where entropy is encouraged to decrease naturally, in this way sequential assembly of parts might be available to biological systems. He existence of electrical charges at surface and the effect of electro-magnetic fields on nucleation phenomena may provide mechanisms for modulation of the effects (e.g. by biological systems). TV Program Mentioning Reptile Body Temperature Reptiles use 35C. Larger individuals are able, after warming themselves in the sun (maximum body exposure position) to 35C to dive deeper and longer in the Humboldt current coastline to graze algae. If they stay too long they overcool and die.

Compensation effects with water systems (e.g. more enhanced at 37C?) possibly more restricted than simple entropy enthalpy interaction effects, might encompass restriction favoring decrease in entropy with time not simply a restriction on allowable entropy changes as required by the linearly coupled entropy-enthalpy changes. Nucleation Denaturation

The press and TV reported (21/12/00) An Edinburgh study of a mouse model of this disease (where remembered learning abilities of mice were tested) had reported that injection of an antibody to amyloid caused the dissolution of plaques and restored memory (hopefully allowing future treatment for the human disease). That dissolution of the solids from brain tissue allowed regeneration of brain function seems highly noteworthy and if substantiated may, as the press report suggested, be a breakthrough in such brain research. Are the nucleation activities of inappropriately solid surfaces a contributory factor in neurodegenerative diseases such as Alzheimers disease? And is aberrant nucleation involved in the generation of such plaques in the first place? Aluminium ions, implicated in dialysis and other dementia might have induced plaque formation since microscopic examination by NMR (Edwardson et al. 1988) indicated that aluminium silicate nuclei were present in the plaque. Toxic substance such as beryllium and aluminum salts, asbestos fiber s and denatured proteins may wreak havoc in the 600-80%w/w water-rich biological systems by affecting the supramolecular structure of water. 1-H NMR studies (starting with Shoolery & Alder (1955) indicated that beryllium and aluminum cations and phosphate anions produce large shifts in the water proton resonance. Such altered water structures may further perturb the putative binding and physiological activities of extracellular polysaccharides which, it is now suggested, may include the induction (nucleation?) of correct water structures. Heparin has a primitive property of protecting against denaturation of proteins and perhaps in reversing this process, as, e.g. in the non-enzymic fibrinolysis studies reported by Russian workers. Heparinoids (Ateroid) (containing heparan sulfate) have also been reported to improve cognitive function in AD patients by an unknown mechanism (Santinin et al.). The fibrinolytic properties of these substances may have been an impetus of the original studies; victims of strokes were also treated but in these cases the cognitive function did not improve. Perhaps the improved cognitive function in AD is due to the AD plaques being solubilized or surface modified by the heparinoid (this possibility should be checked experimentally). Linked Pathological Calcificiation and Nucleation Effects? Adverse nucleation events associated with solid calcification granules with a Ca/P ratio were reported (Ashton 2001) to be identical to that of milk; particles formed in this way may participate in the etiology of breast cancer (acting together with effects of insulin like and other growth factors as discussed by Plant 2001 in Ashton 2001); hydroxyapatitie and related crystals had previously (Cheung et al 1986) been shown to initiate mitogenic activity in fibroblasts, showing a complex dependency on somatomedin C. The formation of such mitogenic crystals might arise pathologically by a mechanism involving depletion of heparan sulfate (Grant et al. 1989a) (e.g. by nitric oxide metabolites, Grant 2000) which normally protect tissue by inhibiting nucleation of inappropriate crystal formation. Heparan sulfate, togther with glycosaminoglycans in general, including cartilage proteoglycans, seem to generate physiological phase boundaries which will also be subject to nucleation controls of such phase formation; (glycosaminoglycans as well as proteoglycans were found to behave in ion binding and in the control of calcification, as phases separate from the outside solution (Dunstone 1961, Dziewiatkowski 1987, Grant et al. 1989)). Surfaces Modulation of Water Structure Passive and Active Effects of Pores Inert surfaces may cause modulation of water structure simply by restricting solution microstructure.

Surfaces with polar groups e.g. sulfate and phosphate esters, capable of directly interacting with water may cause additional alteration of water structure The existence of pores as in polysaccharide gel filters is indicated by Wiggins & van Ryan to be sufficient to generate altered water structure under certain conditions where a solute molecule is size excluded from the pore Do ocean (surfaces) themselves or smaller water reservoirs (underground pools or even water surfaces in inclusions in rocks show similar effects? Might not such environments be good places for life to evolve?) would they show evidence of a tendency of entropy not to increase which could be due to a long term presence of such a body of water (surfaces)? 28/12/00 Surface processes may tend to enhance compensation effects between allowable entropy and enthalpy change. This is understandable since surfaces are two dimensional space, so that ordinary space time laws are constrained if consider only surface processes and entropy flow is altered as it were so that time flow is changed directionally somehow. Further restraining this sort of argument to one dimensional systems might be expected to show up further constraints on allowable entropy changes or tendencies. Thus one dimensional systems might even tend to increase its associated entropy with time. Essentially reversion the direction of local time at linear surfaces. In this way it might be seen that the linear polymer DNA is used to conserve information systems over long times. Is there evidence that linear systems might tend naturally to increase in complexity with time? The coiling up of collagen triple helices? Heparin /heparan sulfate are linear and their highly anionic character tends to maintain this (as with DNA). The use of heparan sulfate to arrange proteins might have its origin in such a tendency for linear systems to generate order. Heparan sulfate might be likened to a magic wand. Surface processes including those at surfaces of liquids tend towards entropy-enthalpy compensation such as found in the effect of temperature on surface tension expressed as the Etvs factors (surface tension multiplied by (molecular weight/density) to the power 2/3 i.e. a molecular dimension correction assuming molecular weight and spherical shape) (Campbell & Eley (1940) Trans Faraday Soc 36 854-856). References are also given in this paper to data from similar compensation effects for the formation of liquid surfaces of solution of a solute in a series of solvents and for the vapoization of pure liquids. (The Etvs temperature dependence term had been previously found (Freundlich 1922) to be smaller than expected for associated liquids, to be very small for molten salts but to be very large for some organic substances; this indicates that other apparently wider effects on the rates of chemical reactions which exhibit compensation effects between entropy and enthalpy changes may also be caused by the rates being dependent on surface activities including the surface tension effect compensations). 6/1/01 The nature of the diffusible substance capable of generating MnO2 patterns produced from added KMnO4 ascribed to reduction by metabolites may be of relevance in understanding morphogenesis in developmental biology. The reduction of KMnO4 will hardly be directly caused by cAMP (with inhibitor ATP) a proposed (Schiffmann 200) (Turing wavelength generator). (added later: cf cytochrome electron generation) Growth factors involve phosphorylation which will affect hydrophobic effects of protein surfaces and influence water structuring perhaps pointing to a primitive effect of water structuring factors in generation of morphogenic fields by the Turing mechanism. Such structuring would be likely determined by nucleation of some form of phase formation indicating a fundamental role of nucleation of such form as the basis of the Turing wavelength.

However cf cAMP signalling seems responsible for dispersed dictyostelium aggregation This, of course being a highly evolved system. What reducing agent might be responsible for permanganate (or the tetrazolium) reduction as employed by Childs? Superoxide acid? (H-superoxide anion).(water surfaces with such radical adsorbed onto them?) Ascorbate? Other reducing sugar? Nitric oxide? Other nitrogen oxides? Glutathione? (added later, electrons, oxalate) 18/1/01 The kinetics of nucleation (in the disorder to order transition) of an ordered from of kappa carrageenan studies (Austen et al 1988) These results seem in accord with the notion of a role of deterministic chaos structured hydrogen bonding arrays in water structure in the nucleation of molecular ordering of biopolymers.

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The yet-to-be-achieved rationalisation of the compensation effect could point to the how ultimate basis of water structure might be elucidated

A fairly strict entropy enthalpy compensation is found for many systems of related rate constants in organic chemistry. Cf. the review by J.E. Leffler (1955) (J Org Chem, 20 1202-1231) which showed how widespread is this phenomenon of enthalpy and entropy compensation for numerous a series of related chemical reactions. This activity can putatively be induced by changes in the structure of empty space (dissipated vacuum m energy source boundaries), some adjacent small particle surface, other molecule or the solvent) and can achieve the precision of a linear relationship which allows for a system an isokinetic temperature to be predicted at which all reactions in the series occur at the same rate. This will e.g. promote the formation of random distributions of polyethers or of phosphate esters. Studies conducted by the author in modelling chlorocarbon pyrolysis (related to studies reported in Grant D, J Appl Chem Biotechnol, 24, 49) revealed that all reported dehydrochlorination rate constants available from Chemical Abstracted journals in 1966 showed the existence of an exact correlation in pre-exponential and activation energy terms for the reported rate constants. During attempts to find wideranging principles which might pertain to scrambling reactions many inorganic of many inorganic and some organic compounds (the Van Wazer research group) had also suggested that scrambled reaction products are apparently characteristic of those reactions which occur rapidly around the isokinetic temperature while clean (e.g. single) products are to be expected for reactions conducted at temperatures far removed form this It can be rationally argued that the general existence of the enthalpy entropy compensation effect requires that a major revision of kinetic theory be attempted (and this might require a modification of some part of the current treatment of thermodynamics) and therefore a full understanding of water structure must await the attainment of such a revision in the basic science. The origin of the effect is at present not established. It is however well-known that reactions accomplished in water are frequently enthalpy-entropy compensated. It seems likely that some rigorous mathematical description might be possible which might predict the outcome of processes which are considered at present to be governed by the usual thermodynamic principles; this may especially apply to pseudo equilibria where different pathways are used for the forward and reverse reactions and are therefore such processes are not fully reversible (this might apply to the supramolecular rearrangements which occur in water rich polysaccharide gels which demonstrate a pronounced hysteresis of melting and the apparent occurrence of water memory cf. Martin Chaplin 2008 Water Structure And Science internet http://www.Isbu.ac.uk/water/ref5.html [this includes a list of references which might support the existence of a water memory effect e.g. ref. 500 (L Rey, 2004) suggests that simple salt solutions when diluted beyond the Avogadro number retain memory of the alkali metal salt); the Memory of Water article however also concludes that the apparent memory effect of water is probably due to solute and surface changes occurring during processing (for which homeopathic usage prefers glass rather than polypropylene vessels, both of these can inject ultratrace amounts of inorganic Al2O3 and TiO2 particles which like SiO2 can in principle create very high water content gels). M Chaplin believed that the main evidence against the existence of water memory, per se, however, was that hydrogen-bonded structures would be required reform their structures to allow this (despite the short. viz. picosecond lifetimes for the hydrogen bonds and milliseconds for H-O bonds in liquid water such lifetimes would not necessarily control the lifetimes of clusters which were suggested to be able to simultaneously disintegrate and reform identical structures). If water memory is real then water structure exchanges cannot be held under thermodynamic reversible equilibrium conditions which would require all water-only compositions to produce identical final distributions of aggregates and clusters. The existence of order in chaotic systems however could indicate that stable configurations even of unstable linkages undergoing interchanges in liquid water (especially the Si etc. multi-element seawater-like aqueous solution which can be argued (cf. Haraguchi 2004) to be the correct form for biochemical studies) could permit memory effects to persist for longer periods of time. This requires a chemical engineering fluid dynamic approach to water structure and the influence of true seawater or blood serum including correct amounts of the 50+ trace and ultratrace elements).

The enthalpy-entropy compensation effect had usually been dismissed as an artifact or experimental error since it was apparently not exhibited, even approximately, by many (or most?) chemical reactions. However dismissal of the compensation effect is felt to be unjustified when the full range of reported instance of this effect is taken into account (cf. Boon, MR 1973 Nature 243 401 and following letters in the journal). It is applicable, as shown by Barclay & Butler in 1938 (Trans Faraday Soc 34, 14451554) to the heats and entropies of vaporisation from solutions and pure un-associated liquids and, as shown by Campbell & Eley in 1967 (Trans Faraday Soc 36, 854-856) to the electrical conductivity of solids (including amino acids and proteins) (possibly influenced by strongly held water), to the electrical conductivity of organic and inorganic oxides (including TiO2, Fe2O3 and ZnO) to the thermionic emission of electrons from solid surfaces and the transfer of ions and molecules between H2O and D2O. It also applies to the haemolysis of erythrocytes and to the denaturation of proteins (Brown HD Ed 1971, in Chemistry of the Cell Interface, Part B, Academic Press New York, cf. p 70) as well as to many heterogeneous catalytic processes including hydrocarbon formation on clays (Wilson & Galway 1973 , Nature 243, 402-404. It applies to nucleated disorder-order transition in kappa carrageenan (which was reported but not discussed by Austin et al. in 1988 (Biopolymers 27, 139,155) the results presented by these authors suggest an isokinetic temperature around 20C); such nucleation rates also follow the Hofmeister (lyotropic) series which was correlated with water aggregation by WAP Luck (as discussed in Kleeberg H (Ed) by multiple authors 1987, in the Proceedings of a symposium in honour of WAP Luck on interactions of water in ionic and non-ionic hydrates held in Marburg FRG published by Springer Verlag) Hints as to how this rationalisation might be achieved might be gleaned from systems where randomization of chemical structure interchanges occurs {a review of such phenomena in inorganic chemistry is given by Van Wazer JR 1962 Amer Scientist 50, 450-472}. A useful model of water structure could perhaps be developed according to the randomization of structures similar to those discussed here (later work by Van Wazer et al. also established that, in addition to the occurrence of general randomisation in many systems of e.g. inorganic polymers, ring and cluster formation was also of especial importance); Van Wazer et al. believed that these phenomena could be accommodated within traditional thermodynamic principles, it is now suggested, however, that a different mathematical treatment is required. Further experimental work by the author on one of the systems where structural reorganization had been established to occur (chlorocarbon scrambling under sealed tube pyrolysis conditions; Grant D (1974; also to be the subject of a further discussion article) had suggested that the assumption of attainment of a full thermodynamic reversibility in this system is incorrect. ***A, widely applicable throughout nature, general entropy and enthalpy compensation system also applies to the surface tensions of liquids which can be considered to be limited to values which obey this relationship {Campbell H Eley DD 1940 loc cit}; this will putatively also govern the allowable surface tension changes associated with the Hofmeister effect which is dependent on water structure alteration. A clue to the origin of the enthalpy-entropy compensation process can be suggested from the circumstance that bond rearrangements may themselves require to occur extrathermodynamically and at least in part instantaneously by processes analogous to those which gives rise to the Pauli exclusion principle (and to the use of instantaneous transfer of electrons during photon uptake in light receptors of plants) [i.e. where the of time in normal Einstein spacetime becomes degraded at least intellectually]. These movements may therefore occur at rates greater than those which apply to larger bodies which obey the Einstein relativity law (with a maximum transfer rate of matter defined by the speed of light in a vacuum) and as such can be considered to be extra-spacetime and require a reconsideration of the definition of how small scale matter-time differs from large scale matter-time, essentially the former allowing extra spacetime time (e.g. future or virtual time perhaps equivalent spoilage of time by vacuum energy field) to enter into equations which govern common rate processes and are extrathermodynamic. This extra dimension of effective time is perhaps afforded most simply and can be applied in a straightforward manner to the reconsideration of many common rate processes by using the Evans Fluctuation Theorem (cf. Wang 2002 Phys Rev Lett 2002 89 050601 which confirmed that (a classical thermodynamically anomalous) Brownian motion movement of 1 latex particle though water (studied using optical tweezers towing pathway analysis methods could be described by an entropy/time

phenomenon which was exponentially and extensively determined according to the equation where P is the probability of the occurrence of reverse entropy/time logP = [Entropy].[Time] [1]

(n.b. this variation can be considered to be equivalent to a reversal of time as required for the variation of change in entropy as required by the second law of thermodynamics). which further allows the value of the entropy change S (e.g. in Gibbs free energy G=H-TS) also to become modified according to the process described equation [1] (which as confirmed by Wang et al. allows reverse entropy/time to affect processes which depend on the internal motions of particles of the size as the hypothetical chemical reaction rate kr determining transition state complexes for which equation [2] is thought to be valid). [This further suggests that the relevant scale where this reversal of time occurs will be achieved includes the process described by equation [1] and affectively suggest this could also modify the amount of the entropy change which occurs during chemical transformations of smaller particles over lesser periods of time e.g. nanomole (and perhaps over a much wider concentration ranges) of chemical molecules. e.g. 0.1nano mole of a chemical substance [(1013) molecules] over 10-13 seconds [the putative lifetime of a transition state complex kr = [kT/h. exp S*/R] .exp-H*/RT [2]

(where k and h are Boltzmann and Planks constants respectively) [cf. the conventional collision theory rate expression equivalent is kr A. exp Ea/RT [2a] ].

This is now suggested to allow extrathermodynamics or non-equilibrium thermodynamic considerations might be usefully be applied for the rationalization of the variation of rate with chemical structure (which gives rise to the Leffler relationship (equation [2b] vide infra). The ultimate origin of the extrathermodynamic relationships is here hypothesized to be the possibility of the leakage of entropy to and from the type of virtual reality which is hypothesiszed to arise in the conventional transition state theory of chemical reactivity. This may be time in the past or future in the conventional spacetime or overlap between conventional spacetime and the vacuum (e.g. where transfer of entropy to and from the vacuum is conceptually permitted to modify the observed change in entropy with time during the course of a chemical or physical transformation. If, equation [1] determines the magnitude of the entropy change S* in equation [2] over the period of time of the duration of 10-13 sec. the putative approximate bond typical vibration lifetime of the activated complex of the chemical reaction path, it may be reasonably further postulated that the alteration of the value of S*/R in [2] caused by the operation of equation[1] may permit the enthalpy-entropy compensation law to be achieved by allowing an adding or subtracting the exact amount of extra entropy generated by the process of equation [1] to achieve a fully enthalpy-entropy compensated kr for those sets of rate processes such as chemical reactions phase change, electron transport in semiconductors, etc., i.e. all processes which obey the Leffler relationship [2b] logA = Ea equation (which include numerous other temperature dependent rate processes as indicated by e.g. Liu and Guo Chem Rev 2001 101 673 and additional processes also indicated by prior literature (D. Grant unpublished)) where is the compensation temperature as defined by Leffler (J. Org Chem 1955 20 1202). The hypothesized mechanism is: S*/R in equation [2] contains an additional amount of entropy, S*p which alters the logA term in the kr= A.exp.Ea/R equation [2a] by a supplemental term (which has either a + or ve sign) S* = S*P [3]

in which the value of S* is determined by the equation: Ea = log A = S* [4]

I.e. the reversal of time (or equivalent process) allowed by equation [1] achieves a restriction of rate temperature variation to meet a presumed requirement (or law) which requires that an enthalpy-entropy compensation must be manifested. Because of the intrinsic creation of randomized behaviour (e.g. of similar compensated rates of polymer segment scrambling) of individual rate processes, this paradoxically causes an increased complexity of related chemical structure to naturally arise with the passage of time. This seems to be a related process to Prigogine dissipative structure (vide infra). This is of relevance to how biology could have developed in aqueous solutions (n.b. the medium of liquid water could be usefully probed experimentally to provide further evidence of transfer of entropy between conventional spacetime and virtual states, seems uniquely to promote compensation between enthalpy and entropy to occur in rate processes and therefore we should expect liquid water and its chemical bond vibrational system which is known to demonstrate major departure from ideal behaviour e.g. with the formation of very broad band of virtual continua). The suggested hypothetical process of adjustment of the value of the entropic log A part of log kr to increase or decrease the value of log A values may seem to require some future time transfer provision and storage of the extra potential entropy which would otherwise be required to comply with the classical thermodynamic laws which hitherto have been thought to apply fully to the quasi equilibrium processes by which the activated transition state complexes (to which equation [2] above is believed to apply) are formed as intermediates in chemical reactions. While the application of the classical thermodynamic laws to the elucidation of the mechanism of chemical reactivity appear have been largely spectacularly successful in providing a framework of enthalpy and entropy in this as well as in numerous other scientific fields, it is also possible that additional concepts are required (e.g. the present hypothesis of entropy leakage between real and virtual space may, it is suggested) to achieve an improvement of this understanding. As to whether the origin of the extra S* is actually due to a genuine reversal of time or to a process which produces an equivalent effect on S* (e.g. some other poorly understood field effect e.g. associated with vacuum energy which may overlap conventional spacetime in the highly activated reaction rate-determining transient transition state complex type of matter) requires further consideration.

4th Manuscript

Notes & Attempted Comments or Critical Resum of Liu L & Guo Q.X Chem Rev 2001 101 513-695 (Isokinetic Relationship, Isoequilibrium Relationship, and Enthalpy-Entropy Compensation) {124b references 394 papers reviewed} A linear putative enthalpy-entropy relationship is found sometimes (but perhaps should be looked for always since it is found between logAi and the Ea values (in equation (2) or between Hi* and Si* in equation (3) below (or their Meyer-Neldel effect equivalents in electronic semiconductor behavior, Barclay Butler rule for surface evaporation) widely throughout chemistry and biochemistry a list including: micellization, Langmuir adsorption, enantiomer separation, water sorption, gas chromatography, liquid chromatography, solvation thermodynamics , thermal transitions, solution extraction, conformational equilibrium, ionic hydration, dielectric relaxation, hydrogen-bonding, crystal melting , coordination chemistry, calixarene chemistry, cyclodextrin chemistry, crown ether chemistry molecular capsules, van der Walls complexes, protein chemistry, lipid chemistry, nucleic acid chemistry, antibiotics dissociation, enzyme binding and food chemistry, and oxidation, thermal decomposition thermal isomerization hydrolysis , addition reactions substitution reactions, redox reactions electron transfer, photolysis, depolymerization, proton transfer and photoisomerization, being given with references (In common with other reviews of this subject it is thought necessary first of all to diminish the possible rather wide range of reported instances of the effect since this could immediately suggest that some major revision of classical thermodynamics and rate theory might be required) by first stating that it is only likely to apply (to similar chemical substances denoted by a subscript i etc.) differing where if we have a series of values of reactions only in a single substituent in one of the reactions of closely related chemical reactions so that for a series of values of changes in enthalpy entropy and free energy which are individually related viz. Gi = Hi T Si (1),

then a linear relationship sometimes seems to exist between enthalpy and entropy changes intimated by correlations between different chemical substance log Ai and Ea (values in eqn. (2) or between activated transition
state complex formation equilibrium constant terms H*i and S*I (values in eqn. (3)
[assuming again that the following commonly used equations are valid

ki = Aiexp(-Ea/RT) k = Ai exp (-E/RT)

(2) (Arrhenius eqn.)

also

= kBT/h.exp(Si*/R).exp(-Hi*/RT)(c)1-m (3) {Transition state reaction rate theory

equation (in which kB, h and c are respectively Boltzmanns, Plancks constants and the concentration in the standard state and m is the molecularity.)}]

A further indication that compensation could be due to solvation effects was made by Lumry et al. who suggested that compensation might be a special consequence of the properties of water.

Compensation Occurring During Phase Transition Estrup et al. pointed out that a compensation effect might follow from the equality on the chemical potential of two phases at a transition.

This accorded with results obtained by Gilbert who found compensation effects during phase transitions studied for 700 organic compounds. The reviewers commented that considering the fact that in phase transitions the enthalpy change H and the entropy change S are connected via the phase-transition temperature T in the equation S = H/T, the compensation in these cases merely says that the phase-transition temperature is roughly the same among the series. Of possible significance to the elucidation of the mechanism of compensation (however, overlooked by the reviewers) is that phase change processes are generally subject to the phenomenon of nucleation. And nucleation may also be the unacknowledged trigger to allow the initiation of diverse other types of chemical reactions. [As is apparent from studies which were not reviewed it should be noted that the kinetic rate constants of nucleation of phase transition in iota and kappa carrageenan (conducted in various salt solutions) and helix coil transitions in proteins have been indicated to show enthalpy-entropy compensation]. A separate section (4.4) considered theories of isokinetic and isoequilibrium relationships. Linert et al. was noted to have considered the effect of the heat bath activating reaction rate theory and the systems which are able to accomplish vibrational-virbrational transitions where the rate of the reaction was proposed to be determined mainly by the environmental molecules and not by the interaction between the reactants themselves. Obviously this theory can explain the isokinetic effect found in those cases where a series of reactions with different reactants occur on the same solid support or in the same liquid solution. This section was summarized: any theory of the isokinetic effect must be able to explain why all the reactions in a series exhibit the same rate constant at an isokinetic T. One mechanism suggests that this is determined by the vibration frequency of the (solid) catalyst another theory suggests that it is determined by the vibration frequency of a specific chemical bond.

Protein Unfolding
The comparison of different proteins suggested that a characteristic common T (ca. 100 C) existed around which the rate of folding became the same when normalized with respect to the number of amino acids present. Hydration may, however, be the primary cause of protein folding compensation. In section 6 (Conclusions) the reviewers noted that Few concepts in chemistry have reached a state of such confusions as the enthalpy-entropy compensation, the isokinetic relationship, and the isoequilbrium relationship. Because theories are complicated with so many artifacts and misunderstandings, may scientists even warn that the exploitation of these extrathermodynamic relationships is perhaps dangerous. As shown in the present review, the compensation effect and the isokinetic (isoequilibrium) relationship are not necessarily equivalent. The compensation effect thus defined should only mean that there is a linear relationship between the logarithm of the pre-exponential factors and the activation energies, between the enthalpies and entropies of activation or between the enthalpy and entropy change of a series of similar reactions. On the other hand, the isokinetic (or isoequilibrium) effect should be defined only by the existence of a common intersection point of Arrhenius) or vant Hoff) lines describing the kinetics (or thermodynamics) of the reaction series. Statistical examination is necessary in establishing any of the three relationships, because the experimental errors can lead to an apparent correlation. .Relatively, the compensation effect can be well understood at the present stage. The misuse of the kinetic model can lead to compensation, which is of little importance.

The monotonic relationship between the binding energy and binding entropy is a kind of compensation which, however, is not generally applicable. For the often-observed large compensation effects, especially those involving solution of biomolecules, redistribution of the energy-distinguishable subspecies is most likely the physical origin. Usually two forms of redistributions play the major roles, i.e. the solvent recognition and the molar shift. On the other hand, the theory of an isokinetic (or isoequilbirum) relationship is not evident enough. Though it is established that the isokinetic (or isoequilibrium) relationship is a result of certain energy resonance between the reacting molecules and their environments, the detailed mechanism requires further investigation.
Continued Notes & Attempted Comments or Critical Resum o

Liu L & Guo Q.X Chem Rev 2001 101 513-695 (Isokinetic Relationship, Isoequilibrium Relationship, and Enthalpy-Entropy Compensation) {124b references 394 papers reviewed} Sometimes (putative) strictly linear enthalpy-entropy relationships are found (but perhaps should always be looked for since it is found between logAi and the Ea values (in equation (2) or between Hi* and Si* in equation (3) below (or their Meyer-Neldel effect equivalents in electronic semiconductor behavior, Barclay Butler rule for surface evaporation as well as) i.e. it widely shows up throughout chemistry and biochemistry. [Liu and Guo (who gave a list of references) suggested that enthalpy-entropy compensation effects occur with micellization, Langmuir adsorption, enantiomer separation, water sorption, gas chromatography, liquid chromatography, to be of relevance in the solvation thermodynamics , thermal transitions, solution extraction, conformational equilibrium, ionic hydration, dielectric relaxation, hydrogen-bonding, crystal melting , coordination chemistry, calixarene chemistry, cyclodextrin chemistry, crown ether chemistry molecular capsules, van der Walls complexation, in protein chemistry, lipid chemistry, nucleic acid chemistry, the dissociation of antibiotics as well as affecting enzyme binding, and oxidation, thermal decomposition thermal isomerization hydrolysis; it also occurred for various addition reactions, substitution reactions, redox reactions, electron transfer reactions, photolysis, depolymerization, proton transfer and photoisomerization, and was of relevance for food preparation chemistry. (In common with other reviews of this subject it is thought necessary first of all to diminish the possible rather wide range of reported instances of the effect since this could immediately suggest that some major revision of classical thermodynamics and rate theory might be required) by first stating that it is only likely to apply (to similar chemical substances denoted by a subscript i etc.) differing where if we have a series of values of reactions only in a single substituent in one of the reactions of closely related chemical reactions so that for a series of values of changes in enthalpy entropy and free energy which are individually related viz. Gi = Hi T Si (1),

then a linear relationship sometimes seems to exist between enthalpy and entropy changes intimated by correlations between different chemical substance log Ai and Ea (values in eqn. (2) or between activated transition
state complex formation equilibrium constant terms H*i and S*i (values in eqn. (3)
[assuming again that the following commonly used equations are valid

ki = Aiexp(-Ea/RT)

(2) (Arrhenius eqn.)

equation (in which kB, h and c are respectively Boltzmanns, Plancks constants and the concentration in the standard state and m is the molecularity.)}]

= kBT/h.exp(Si*/R).exp(-Hi*/RT)(c)1-m (3) {Transition state reaction rate theory

Erroneous Conclusions of Prior Authors

Include Confusion between Compensation and Isokinetic Effects.

(The latter needs to be defined solely on existence of intersection of Arrhenius curves and not assumed from the existence of enthalpy-entropy compensation). There are three separate relationships: The Compensation Effect, The Isokinetic Effect and the Isoequilibrium Effect Theories Proposed by Prior Authors Pure Mathematical Explanations. Exact Compensation is Impossible. Entropic and Energetic Aspects of Chemical Bonding --------------------------------------------------------------

Compensation and Linear Free Energy Relationships and related phenomena (cf. the Hammett equation (cf. Leffler, 1955 who argued that this was a sub-class of entropy-enthalpy compensation) have been generally accepted as being true and not artifacts (which is a common belief relating to the wider compensation phenomenon). The existence of such relationships implies that H* is a linear function of S* but to date there has been not fully clear explanation of the linear free energy relationships although Helper who formally partitioned the total free energy into internal and external enthalpies and entropies where the former represent pure substituent effects in the gas phase and the latter represent effects due to solvation and often the former Sint = 0, so that the external enthalpy and entropy compensated each other i.e. Hext =Sext. The idea that solvation effects can generate compensation effects is supported by computational chemistry where it is found that calculated enthalpy and entropy of solvation often compensate for each other. A further aspect of this effect that enthalpy-entropy compensation can be caused by two types of interactions in solution i.e. solvent reorganization and molar shift. A further indication that compensation could be due to solvation effects was made by Lumry et al. who suggested that compensation might be a special consequence of the properties of water. Numerous instances of the compensation effect have been reported for reactions conducted in aqueous solution. [The P.M. Wiggins theory of low and high density water {not considered by the reviewers) is an example of how recent advances in interpretations of vibrational spectra and careful density measurements show that the anomalous properties of liquid water can be accommodated to two type of hydrogen-bonded aggregates which cam be demonstrated to likely by present as separate enthalpy and entropy defined chemical components in this liquid system] [The idea that many reactions between highly reactive chemical substances fail to proceed in the absence of ultra-trace amounts of water is an very old one which was claimed to have been found not to be a valid concept by researches conducted in the 1960s. Since the ability to extensively dry reagents by storage in a vacuum desiccator over e.g. P2O5 is a valid technique and which would have been readily available to the nineteenth and early twentieth century chemists, the rejection of this universal catalyst activity should perhaps be revisited, as the possibility of trace contamination of many chemical reagents by very small amounts of water might, e.g., explain at least some of the currently unexplained instances of the compensation effect; apart from the provision of unspecified chemical reaction catalytic activities, water might also act as a general nucleation agent and have the effect by being able to promote phase change processes (cf. the referenced discussed by the reviewers to such an origin of the compensation effect)]. k = Ai exp (-E/RT) This surface catalyst circumstance could create the appearance of compensation.

Another theory (Benson) of how heterogeneous catalyst sites could create compensation was suggested: tighter binding at some sites will tend to create lesser entropy (lesser freedom of motion). The narrowing of the transition state hypothesis (Conner)

Molar shift phenomena can generate two components (e.g. can involve cis and trans isomers of e.g. 1,2 dichloroethane). Compensation is Observed in Heterogeneous Catalytic Reactions (The overall rate constant is expressed as a non-Arrhenius form Compensation Occurring During Phase Transition Estrup et al. pointed out that a compensation effect might follow from the equality on the chemical potential of two phases at a transition. This accorded with results obtained by Gilbert who found compensation effects during phase transitions studied for 700 organic compounds. The reviewers commented that considering the fact that in phase transitions the enthalpy change H and the entropy change S are connected via the phase-transition temperature T in the equation S = H/T, the compensation in these cases merely says that the phase-transition temperature is roughly the same among the series. Of possible significance to the elucidation of the mechanism of compensation (however, overlooked by the reviewers) is that phase change process is generally subject to the phenomenon of nucleation. And nucleation may also be the unacknowledged trigger to allow the initiation of diverse other types of chemical reactions. [As is apparent from studies which were not reviewed it should be noted that the kinetic rate constants of nucleation of phase transition in iota and kappa carrageenan (conducted in various salt solutons) and helix coil transitions in proteins have been indicated to show enthalpy-entropy compensation]. The section (4.4) considered theories of isokinetic and isoequilibrium relationships. Linert et al. considered the effect of the heat bath activating reaction rate theory and the systems which are able to accomplish vibrational-vibrational transitions where the rate of the reaction was proposed to be determined mainly by the environmental molecules and not by the interaction between the reactants themselves. Obviously this theory can explain the isokinetic effect found in the cases where a series of reactions with different reactants occur on the same solid support or in the same liquid solution. This section was summarized: any theory of the isokinetic effect must be able to explain why all the reactions in a series exhibit the same rate constant at an isokinetic T. One mechanism suggests that this is determined by the vibration frequency of the (solid) catalyst another theory suggests that it is determined by the vibration frequency of a specific chemical bond.

More on Protein Unfolding

The comparison of different proteins suggested a characteristic common T (ca. 100 C) around which the rate became the same when normalized with respect to the number of amino acids present. Hydration may be the primary cause of protein folding compensation. In section 6 (Conclusions) the reviewers noted that Few concepts in chemistry have reached a state of such confusions as the enthalpy-entropy compensation, the isokinetic relationship, and the isoequilbrium relationship. Because theories are

complicated with so many artifacts and misunderstandings, may scientists even warn that the exploitation of these extrathermodynamic relationships is perhaps dangerous. As shown in the present review, the compensation effect and the isokinetic (isoequilibrium) relationship are not necessarily equivalent. The compensation effect thus defined should only mean that there is a linear relationship between the logarithm of the pre-exponential factors and the activation energies, between the enthalpies and entropies of activation or between the enthalpy and entropy change of a series of similar reactions. On the other hand, the isokinetic (or isoequilibrium) effect should be defined only by the existence of a common intersection point of Arrhenius) or vant Hoff) lines describing the kinetics (or thermodynamics) of the reaction series. Statistical examination is necessary in establishing any of the three relationships, because the experimental errors can lead to an apparent correlation. .Relatively, the compensation effect can be well understood at the present stage. The misuse of the kinetic model can lead to compensation, which is of little importance. The monotonic relationship between the binding energy and binding entropy is a kind of compensation which, however, is not generally applicable. For the often-observed large compensation effects, especially those involving solution of biomolecules, redistribution of the energy-distinguishable subspecies is most likely the physical origin. Usually two forms of redistributions play the major roles, i.e. the solvent recognition and the molar shift. On the other hand, the theory of an isokinetic (or isoequilbirum) relationship is not evident enough. Though it is established that the isokinetic (or isoequilibrium) relationship is a result of certain energy resonance between the reacting molecules and their environments, the detailed mechanism requires further investigation.

----------------------------------------------------------------------------------

5th Manuscript
Document retype April 5 2012 (cf. scanned version of original document in http://www.scribd.com/doc/8787336/D)

Aberdeen U ms. (Grant, D. Compensation Effects)

COMPENSATION EFFECTS

NOTE

by

D. Grant

5.4.84 The Compensation Effect may be defined by the following:

log k = a -E / 4.6T

(Similar expression for equilibrium constants)

where log is the logarithm to the base 10, k is the chemical reaction rate constant, a is a constant, E is the Arrhenius activation energy , and T is the absolute temperature and where

kE + (const)

(const is often = 0).

(Similar expression from equilibrium constants)

A plot of log a vs. E gives a straight line.

The rate constant maybe re-written in terms of entropic and enthalpic changes associated with the transition state complex, according to the transition state complex theory of chemical reaction rate, viz.,

kr = (kt/h) exp ( S*/R ) exp ( - H*/RT),

(usual meaning of constants)

the pre-exponential term in the rate constant being associated with entropic changes in the formation of the activation transition state complex. A wide range of rate processes have been observed to exhibit this entropic-enthalpic interdependence; attempts to explain these interrelationships have been unsatisfactory: the effect was noted and briefly discussed by Boon (1973) , Harris (1973), Damjanovici and Vernon (1973) and rationalized in terms of special properties of surface catalysed processes by Wilson and Galway (1973). Barton and coworkers (1951) noted an apparent relationship between log A and E, Maccoll however repeated some of the work and reported different values which did not show the relationship. The problem was discussed by Blackadder and Hinshelwood (1958). The compensation effect was reviewed by Leffler (1955) who cited 81 examples from the literature. He pointed out that the relationship indicated the existence of and isokinetic temperature for a series of

reactions exhibiting the effect. It was suggested to be a general rule that any organic reaction was cleaner the further the reaction temperature was from the isokinetic temperature, hence a tendency to obtain random product distribution near the isokinetic temperature.

The compensation effect has been rationalized in terms of the occurrence of heterogeneous catalysis. This is a reasonable explanation for results obtained from hydrocarbon formation on clays and similar processes (Wilson and Galway (1973).
(but this cannot be a general explanation)

Compensation effects are also observed in the case of heats and entropies of vaporization from solutions, e.g. gases from acetone and numerous other examples, Barclay and Butler (1938); and for energy gap and pre-exponential factors in dark conduction by organic semiconductors, Eley (1967); as well as in the enthalpic and entropic contributions to the second osmotic virial coefficient , Wolf (1972). The compensation effect between dilution entropy and dilution enthalpy of polystyrene and polymethylmethacrylate have been reported by Lechner and Schulz (1973). A possibility arises that the compensation effect could be related to deviation for ideal solution behavior.

There is a tendency for reactions with higher activation energies to proceed faster than might otherwise be expected------- this might be a postulate related to the compensation effect. Alternatively put, there is a kick principle: the greater the activation energy the greater the kick which might additionally become available following dissociation of the transition state complex. Such a concept may be derived from considerations of unimolecular rate theory in

consideration of compensation-like effects, by comparison with radioactive decay or Auger effects cf., Rognisky and Rosenkewitsch (1930). A further review and discussion of the compensation effect was given by Molin et al (1965). References. -----------Boon M.R., Nature (1973) 243, 401 Harris P.S. ibid., (1973) 243, 402 Damjanovic V. and Vernon, C.A. ibid., (1973) 243, 402 Wilson, M.C. and Galwey, A.K. ibid., (1973) 243, 404 Barton D.H.R.and Howlett, K.e. J. Chem Soc 1949 165 Barton D.H.R. , and Onyon, P.F. J. Amer Chem Soc. (1950) 72 988 (edit*ch)
pr better Barton, D.H.R., Head A J. and Williams, R.J. J. Chem Soc., 1951 2039

Maccoll A. and Thomas P.J., Trans Faraday Soc. (1968) 119 Blackadder, D.A. and Hinshelwood, C. J. Chem Soc. 1958, 2728 Leffler, J.E., J. Org Chem 1955, 20, 1202 Barclay I.M. and Butler J.A.V., J. Chem Soc. 1939 1445 (edit *ch j and date) Eley D.D., J. Polym. Sci Pt C No 17 (1967) 73 Wolf, B.A. J Polym Sci Pt. A2, (1972) 847 Lechner, M.D. and Schulz, G.V. Macromol Chem (1973) 172 161

(edit* ch date)

Roginsky, S and Rosenkewitsch L., Nature 1930, 125, 347 cf., Roginsksy, S and Rosenkewitsch, L, Z Phys Chem. (1930) B 10, 47 Molin, Iu. N. Leshina T.V. and Mamaev V.P., Dok Akad Nauk SSR (1965) 163 402

6th Manuscript Putative Time Reversal & Compressed Time

Entropy changes which are possibly indicative of a direction of time reversal have been proposed to occur as a consequence of the operation of the Fluctuation Theorem which predicts that the probability that the variation of entropy with time can takes the opposite direction to that required by the second law of thermodynamics, i.e. entropy flow during a natural process can decrease as well as increase. The occurrence of virtual time reversal (the ratio of the probability that entropy will decrease to the probability that entropy is suggested to be equated to exp [entropy x time]. (1) The extensive value of exp(S) [over unit time] seems to indicate an exponentially higher probability of time reversal for smaller than for larger entities. This suggests that the value of exp (S*) which appears in chemical rate expressions (e.g. equation (2) below) is determined by (1). This circumstance provides an explanation for a hitherto inexplicable extrathermodynamic enthalpy-entropy compensation processes (e.g. in chemical reaction rate processes (cf. Leffler, and Liu and Guo) (which include the Hammett and Taft linear free energy relationships). (1) describes small size engine behaviour a ; this concept can be extended to entities involved in chemical transformations and these may include the transition state complexes which are thought to be formed between reagents and which determine the overall reaction rates according to the rate of their decay into reaction products. These entities may act as Fluctuation Theorem engines in which S is required to vary in the extra-classical thermodynamic manner i.e. S* in the usual chemical reaction rate expressions e.g. that shown in equation (2) (the process by which decay of the transition state complexes gives rise to kr (the rate constant) must always be subject to additional, hitherto not -previously-considered, counter time variation requirements demanded by equation (1)).
The expression for the rate constant in equation (2), [(the original, the simplest), activated transition theory expression, where k is the Boltzmann constant, h is the Planck constant, R is the gas constant, T is the absolute temperature and S* and H* are respectively the change in entropy and enthalpy during the establishment of an equilibrium between the starting molecule(s) and the activated transition state complex; needs also to consider the operation of equation (3) which has also been observed experimentally to be so frequently obeyed or approximated to, that some natural law seemed to be involved, which limited the allowed values of S* and H* by some hitherto puzzling extra-thermodynamic (enthalpy-entropy compensation) relationship; most often equation (3) limits the allowable values of kr for a particular type of chemical transformation carried out on sets of quite distinct chemical substances but which are chemically structure related, but also applies to numerous other physical processes which show k= A.exp.Ea/RT variation where Ea is the activation energy where log A depends on Ea similarly to equation(3)

kr = (kT/h) exp (S*/R)exp(-H*/RT) (2) S* = H* + const (3) (the value of the const. often is 0) The above argument suggests that (1) directly controls the value of S* in equation (3), i.e. the enthalpy-entropy compensation process arises directly as a corollary to the Fluctuation Theorem. The contribution of the reverse time process then can be calculated between the difference in the values of log A obtained as previously by assuming one-way time by classical statistical mechanics and the experimental observed values of log A. a Since the operation of the Fluctuation Theorem mechanism has been interpreted as meaning that small scale engines such as mitochondria can sometimes (highly counter-intuitively) be naturally enabled because of their size to run in a reverse time direction allowing their normal chemical reaction products such as carbon dioxide to also serve as reagents during periods of time reversal enabling mitochondria to regenerate the starting material used for normal time direction processes). The same argument can be applied for any other chemical transformation. This indicates the previously assumed one-way (supposed classical thermodynamics determined) experimentally observed rate expressions for the outcome of any chemical reaction must be the balance of forward and backward time rate processes which required to arise by the operation of the Fluctuation Theorem.. [N.b. the second law of thermodynamics is the only law of physics that introduces a sense of direction of time is apparently flawed and the assumption made by Classical Thermodynamics (which were originally indicated for large scale engines) that the direction of time must always be one-way and be associated with an increase in entropy. is challenged by the experimental observations associated with

Brownian motion which has been indicated however to be correctly described by the Fluctuation Theorem which seriously challenges the universality of the second law of thermodynamics.

The Fluctuation Theorem replaces the second law of thermodynamics with an exponential equation (probability of obedience to the second law depends on the exponential value of entropy multiplied by time) which allocates a simple statistical probability that entropy will either increase or decrease with the passage of time to the direct exponential function of entropy (in terms of the extensive size thereof) in such a manner as to allow the classical second law of thermodynamics to apply as before for large scale particles etc. but to be partially disallowed for small scale particles etc. where the change of entropy with time is permitted also to decrease. The above theorem introduces the idea of a natural operation of what seems to be equivalent to a timereversal phenomenon which might especially apply to the motion of small entities and shows up in e.g., Brownian motion and putatively also is encountered during the motion of chemical molecules and furthermore is of possible relevance for the fuller understanding of the mechanisms of chemical reactions. The introduction of the idea that the second law of thermodynamic should be replaced by a statistically determined equivalent law which demands a quite different entropy time variation for real small sized systems than for large systems further suggests a consideration of the notion that all aspects of time variations per se are not precisely defined for small particles but are actually the outcome of random processes. The precise definition of time can be considered to be replaced essentiality by a statistical equivalent balance between positive and negative quasi time measures. This quasi time can be considered to become factorized into different time paths accorded some arbitrary defining coordinate system which might be predicted a priori to be that provided by vacuum space.

Is entropy (S) a function of complex time flow [event pathways] alone? (i.e. is a consequence of the complex measure of time flow)
If the probability of reverse time flow (t-) relative to positive time flow (t-) varies according to (the probability of reverse time flow )= t+/t- =exp S.t (Fluctuation Theorem) and if then t = t+ - tt+/t- = exp S(t+ - t-)

and loge(t+/t-) = S(t+ - t-)

S = [loget+]/(t+ - t-) [loget-]/(t+ - t-)

Entropy arises from complex time differences in related event process determined by ([exponential-directional-time] /[net-time] ) flow as indicated by the above equations. The magnitude of entropy is then determined entirely by the above complex time flow event pathway processes involving the difference between positive and negative (complex) exponential time event pathways. Entropy arises solely as a consequence of (exponential) time event pathways when the complex nature of time is taken into account. Entropy always is established by a process which allows for both positive and negative (imaginary) time flow event pathways to occur.

Therefore entropy change must also be established by a process which allows both positive and negative time flows to occur. (Cf. this replaces the second law of thermodynamics, [entropy tends to increase with (positive) time event pathways (the magnitude of entropy must always increase with time (i.e. (with positive time [event pathway] flow)]. Compressed Time

Alternative draft of hypothesis using the concept of compressed time

The probability of time reversal for a typical transition state complex is assumed to be that given by the Evans equation where the ratio of (second law of thermodynamics defined) normal to reverse entropy flow is equated to exp. (entropy).(time). This probability ratio, which appears to create a reversal of time (seemingly equivalent to creating a separate stretched time scale within the space bounded within an engine of the size of a typical activated transition state chemical reaction rate determining complex). This process putatively thereby controls the observed (apparent) rate of chemical reaction via an equivalent process to that of an alteration of the internal motions and associated entropy and energy flow (especially the actual rates of the rotational and vibrational motions [but less so for translational motion which is determined by motions occurring external to the transition state complex?]; n.b., however, this occurs only under the conditions of the part-hypothetical short lived highly unstable transition state complex this nevertheless causes real frequency changes which alter nature of the chemical entities in regards their entropy and enthalpy characteristics during any time dependent processes (by changing the time dependencies of the energetics of the (electronic structured) chemical bond being time compressed and simultaneously chemically altered). This direct linkage can explain the apparently observed directly entropy and enthalpy compensated change which is described by Leffler equation (which is commonly perceived by macro-scale experimental observation techniques). This indicates that the time compression process changes both entropy and enthalpy in a linked manner and therefore, at least for the situation of chemical reactivity, the Evans equation may be re-written by replacing entropy with some function of (Leffler) enthalpy. The probability ratio for the occurrence of time compression indicated by the Evans equation can evidently vary widely, the reverse entropy flow ratio being apparently allowed to vary between the limits of zero to infinity, the special case arises when the probability ratio value is unity where time stops / changes direction [(t+ - t-) [(0 1) / (1 ) (being zones where t+ t-)] (all direction of motion including spin directions being required to change around). Let us suggest, therefore that there exists some typical value of this probability ratio which is perhaps that which corresponds to the typical circumstances suggested by classical thermodynamic considerations (where, of course time compression was not considered) this is logA = ca.13.5 (for a typical unimolecular rate constant kr = A.expEa/RT sec-1 where the Ea value is a fairly large fraction of the bond energy of the chemical bond strengths of the bonds being changed in the reaction process in question). Such typical rate constant A and logEa values do, however, actually represent an apparent and not the actual single direction normal time processes since some time compression/time reversal always seems to be demanded by the Evans probability theorem (cf. its experimental verification for 1 particles towed through in water over several second time intervals [this seems to be Brownian motionrelated]). If the Leffler equation linking enthalpy and entropy change is an outcome of the above hypothetical time compression processes, then time compression occurs in the transition state complexes commonly encountered under common chemical reaction conditions, e.g. chlorinated organic substance thermal dehydrochlorinations (for which all experimental rate constant data show compensated values of log A between <2 and 19.9 [2,4-dichloropentane] (and the compensated activation energies from <5 to 69.6 kcal/mole);

The suggested process of time compression may, however, be of a more complex nature that a simple linear compression. Time (in respect of matter vacuum apace interaction) could be equivalent to a potentially highly structured process which putatively also involves extradimensional space probability considerations.

-------------------------------------------------------------------------------------------------------------------------

7th Manuscript
Coveney (1988) Review
Notes from Peter V. Coveney Nature 1988 333 409-15 The second law of thermodynamics: entropy, irreversibility and dynamics This provides (a conventional) prior-to-1988 historical background which discusses attempts to reconcile the second law of thermodynamics with real systems ( i.e. how entropy time variation arises). [but this review did not mention the Leffler process at al, since to to do so it is now suggested required a complete re-think of the basic concepts deal with in classical and non-equilbrium thermodynamics]

Irreversible processes are the normal circumstances of Nature. It should be noted that Classical Thermodynamics, strictly speaking, only can be applied to fully reversible processes. These may not occur in Nature at all. The thermodynamic potentials (such as A and G (Gibbs)) exist only for equilibrium conditions). These are very special cases (and perhaps may not actually exist? This could indicate that classical thermodynamics might be a hoax and essentially in any fundamental sense must ultimately be invalid and cannot be used as a useful mathematical model on which to base chemical reactivity).
N.b. A reversible process is one in which a given system may be returned to its initial state, even after the performance of work and the exchange of heat, with no accompanying effects arising in the surroundings. All other processes are irreversible.
The distinction is sharpened by the second law of thermodynamics which asserts that, for any irreversible process, the function S, known as the entropy, must increase, whereas for a reversible process it remains constant. More particularly, the entropy of an isolated system increases monotonically until it reaches its

maximum at equilibrium - a form of words that implies a sense of the direction of time, as does the familiar statement of the second law in the form dS 0 where the equality applies only to reversible processes, more exactly this should be written dS/dt 0 which makes it clear that the positive direction of time (t) is linked to the increase in S during an irreversible process. Indeed the second law of thermodynamics is the only law of physics which introduces a
direction in time.
-----------------------------------------------------------------------------------------------------------------------------------------------(Although the energy of a system E should in principle be known from quantum mechanics which govern the behaviour of matter at the microscopic level, this can only be accurately determined for a few simple and ideal systems). -------------------------------------------------------------------------------------------------------------------------------------------------------Cf., where = 1/kT , T is the temperature, k is the Boltzmann constant and Ei is the energy of the system; for closed systems able to exchange energy but not matter with the environment, the microscopic version of the equilibrium entropy is given by

S = k ln Z + kT ( lnZ/ T)v Z is the partition function (of Equilibrium Statistical Mechanics)

Z=
i

exp

(-Ei)

But, n.b., Z cannot be rationally defined out of equilibrium.

Also non-equilibrium conditions are the normal conditions. However, the exchange of energy and matter in overall non-equilibrium system with the environment and the production of entropy (dissipation) by the system may give rise, in a non-linear system, to another hypothesis that there exists a local equilibrium which may sometimes resemble a true equilibrium condition so that those macroscopic variables which occur at equilibrium can also be applied here (at least as a working hypothesis). E.g. for an open system capable of exchanging both energy and matter with the surroundings and deS is the entropy exchanged with the surroundings and diS is the entropy produced internally within the system) Prigogine [generalized thermodynamics] non-equilibrium thermodynamics is where entropy production= P, for any k th. out-of equilibrium-process rate = J; and the (generalized) force = X, is given by P diS/dt 0 by P diS/dt 0 = JkXk 0 k [This the de Donder hypothesis further equates the generalized force with the (chemical) affinity (A)].

Fluxes Jk are further defined to depend linearly on the forces Xk

Jk = Lkk .Xk k [The Onsager hypothesis further also suggests a stronger requirement than linearity namely reciprocity, is proposed where

L kk = L kk

].

The Jk and Xk terms in the above equations then become the rates of reaction and the affinities of the species present (and possibly also the diffusive flux and chemical potential gradient if spatial inhomogeneities are relevant..) Entropy production P diS/dt (0) can itself be considered to be a potential which minimizes dissipation beyond a bifurcation point at which the original quasi reversible thermodynamic processes become unstable. However, Prigogine Dissipative Ordered States of Matter are considered to arise under conditions where minimum entropy production becomes unstable [beyond a certain value c of a parameter which measures the distance of the system from thermodynamic equilibrium with the evolution of the system beyond the point c]. {e.g. dissipative structures are associated commonly with those chemical reactions in which autocatalysis occurs; this is stated to be common in biology where the glycolytic cycle, the source of adenose triphosphate was the first confirmed example of a biological dissipative structure}. Far from equilibrium, however, where the above types of scalar potentials become invalid, a more useful equation is thought to be:

/ t( 2S) = Jk Xk
(where represents the deviations form the stationary values).
k

N.b. the right hand side of the above equation has no particular sign. The state of

minimum entropy production is no longer the state to which the system evolves. The vagueness of the second law of classical thermodynamics leaves open the question of the precise meaning of entropy out of equilibrium.
______________________________________________________________ D.G. Insert
Enhanced entropy fluxes due to the Evans Fluctuation of the direction of time may provide a rationalization of apparently anomalous aspects of chemical reactivity. Also

Do The Common Extrathermodynamic Relationships Result From The Complex Number Nature of Time? A complex number time process seems to apply to the Fluctuation Theorem which especially allows counter-to-second law entropy change to occur for
small-sizes assemblages (with a probability which depends exponentially on entropy x tim)e which futher could explain the

Entropy-enthalpy compensation

which shows up in temperature dependent rate processes throughout physics, chemistry and biology

E.g. Leffler showed in 1955 that logA vs. E correlations in k= AexpE/RT exists throughout organic chemistry and applies to small as well as for large values of A; i.e. 0.non-intuitive time processes (most easily considered as time possessing imaginary (i) plus real components)and related causation is perhaps a characteristic of all types of chemical reaction rates. According to De Donder the rate of entropy production is an important, perhaps the most important, part of the chemical affinity which promotes chemical reactivity analogous to a affinity being the application of a force which directs the flow of matter in the context of chemical transformation, but this idea may now also require also to comply with the 1993 Evans Fluctuation theorem which controversially disregards the second law of thermodynamics i.e. the requirement that, for any irreversible process, entropy must increase, but for reversible processes remain constant. The (de Donder) idea that chemical reaction rate processes are directly influenced by the rate of entropy flux along the reaction pathway coordinates suggests that the A values (which are also apparently strongly coupled to allowable values of activation energy (E)) must determine the value of k = A.exp (Ea/RT) in effect cause compensated rate constants to tend to be entropy-changedetermined. (N.b., log A and E values are generally experimentally correlated (but this phenomenon is probably counter-to-classical thermodynamicspredictions) and this becomes most obvious for the strict correlation

which exits between logA and E in numerous sets of rate constants obtained for families of related chemical reactions). Entropy change within molecules of short-lifetime must arise, according to the Fluctuation Theorem by operation of both positive and negative time entropy variation. This requires a modification of the transition state theory (assuming that time flow fluctuation processes apply, and that a virtual time reversal process critically influences the behaviour of the transient chemical rate-determining complex molecule). Irrationally large pre-exponential A factors sometimes show up experimentally in supposedly unimolecular reaction rate constants; [e.g. when written in the form kr = [units sec -1 or mole. cm-3. sec-1 the value of A can be greater (by at least x106) than the maximum possible value suggested by classical thermodynamics. This was later suggested to arise because of the existence of rate determining radial chain processes. However, alternatively, these values of A could have arisen, at least in some cases, by the operation of Fluctuation Theorem reverse time enhanced entropy flow. Rate expression enthalpy-entropy correlation may arise because of convergence of events in both real and imaginary space. While the rate of change of entropy (which is required by classical thermodynamics to be greater or equal to 0 but according to the Fluctuation Theorem time can both be greater or less than 0 [the relative probability of the reverse entropy flow being = exp. (entropy).(time)] : this is an equivalent concept to the existence of mixed positive / negative time {e.g. over the time interval of the existence of the transition rate complex (perhaps over packets of infinitesimal time intervals in complex number units with i complex number usage or somehow involving hyperactive intermediate states or random hopping into high energy levels akin to the random matrix theory)}. If the process giving rise to the A values in rate expressions is dominant, then to maintain this dominance, the allowable values Ea cannot be independent of the allowable A values in the same rate process, but must be determined by being forced by the chemical affinity pressure exerted by the process which determines the value of logA. Putatively this phenomenon, it is now suggested, is determined by causations which arise from (Fluctuation Theorem time which can be considered to be) a complex number determined event train. this complex number effect is what causes the Ea values to reflect the log A values during the time period of entropy flow reversal (which furthermore is postulated to occur in imaginary space). -------------------------------------------------------------------------------------------------------

Consider the Hypothesis that Thermodynamic and Extrathermodynamic Processes Have been Experimentally Demonstrated Both to be Equally Valid. The high status of Natural-Law-Derived Thermodynamic and partly disbelieved Empirical Rule (Paradoxes) Seemed Previously to be Incompatible. Since Classical Thermodynamics and its associated theories of chemical reactivity seem to contradict the Evans Fluctuation Theorem, it is instructive to ask if this Theorem is correct, and if so there is a wider need to revise the general assumption of the validity of the Second Law of Thermodynamics (which is thought to allow for only one directional time) The Fluctuation Theorem apparently allows for entropy decrease to occur under situations previously ruled out by Classical Thermodynamics (and seems to be equivalent to allowing two directional time variation to occur for small particles over short time intervals; this presumably applies to the situations occurring not only in transitions state complexes which have been supposed to determine chemical reaction rates, but also to the rate processes by which energy is transferred between colliding particles (this possibility suggests that revision of the kinetic theory of gases may be required).] [Overall a situation equivalent to a general enthalpy-entropy compensation process might arise, in possible agreement with empirical rules, and apply throughout physics, chemistry and biology because of the validity of the Fluctuation Theorem behavior of entropy and hence by inference the validity of the complex number measure and the concept of the general bi-direction nature of time]. The Use of a Complex Number Measure of Time however seems to allow for the thermodynamic and extrathermodynamic concepts to be reconciled (especially if reverse entropy time variation can occur in imaginary space but this can also impact in real space situations as seems to have been confirmed experimentally). DG Insert cont.
Preamble to the General Discussion of the Above

Consider (The Driving Force a for Chemical Reactivity) A Affinity (Chemical)

(Affinity) : (this is a measure [analogous to how heat is measured by temperature T] which defines the tendency for a chemical reaction to occur spontaneously)
can also be considered to be analogous to the force (like a pressure difference) under which the rate of a chemical reaction is driven (this resembles a how a fluid flow may driven through a conduit by a pressure difference which can be likened to the chemical affinity (A).

A de Donder Affinity, A = -rG (where G is Gibbs free energy (=H-TS) {(G)T,P = H TS}
Cf. the Gibbs-Helmholtz equation, G-H=T(G)/T

(where G can also be thought of as the difference in the chemical potential values of the reagents and the products of the reaction and of the starting materials).

Cf. the IUPAC definition of affinity is

A = -[(G/)P,T], being the reaction extent.

The re-arrangement of matter during a chemical reaction may be regarded as the rate of the process of the

, the extent A = dQ/d

of stoichiometric alteration of chemical concentrations;

Cf also, Prigogine gives

(where Q is the uncompensated heat of reaction) Cf. the rate of entropy increase = the product of thermodynamic force (=A/T) and the thermodynamic flow (the reaction rate) Entropy-Enthalpy Compensation in Chemical Kinetics Compensated heat (thermal energy) can be considered to drive thermally activated chemical reactions Compensated thermal energy can be regarded as

=H-TS
but when 0, H = S Compensation between allowable enthalpy and entropy flow may arise by a process suggested by the above equation which is widely applicable throughout physics, chemistry and biology. In the (pseudo equilibrium in the putative transition state complexes) for sets of rate constants for closely related reactions the expression is observed to be valid H * = S* where is the (Leffler) isothermal temperature, equivalent to T in the first two equations above) at which all rates become equal for the specific set of reactions. which characterizes the sets of related chemical reactions (kr = A exp.Ea/RT) for which log A and Ea compensation arises, indicating that (where thermal activation energy is denoted *) the values of S* and H* which become phenomenologically coupled by operation of the time variation process arising within the postulated established equilibrium molecular assembly, which in the simplest form of the transition state theory gives rise to the expression kr = (kT/h) exp (S*/R) .exp (-H*/RT)

It is now further suggested that extra-thermodynamics [e.g. for the equilibrium process ( K = exp ) between the starting materials and a transient activated transition state complex, the decay of which determines the rate of the reaction] arises by operation of the Evans Fluctuation Theorem (any entropy flow (equivalent to time) reversal must potentially arise in small scale engines such as transient transition state complexes, the internal motions of which are commonly supposed to account for chemical reactivity. An extension of this theorem now suggested is that the formation of the these rate determining complexes actually are established by equilibrium attained via a complex number dependent time variation process where the back reaction occurs in imaginary (i.e. reverse) time sets. Virtual extrathermodynamic balances are suggested to be allowed in small scale machines to be determined by equilibrium complex time (imaginary time balanced by normal time). This concept may resolve various paradoxes including that which has hitherto been associated with the transition state theory which seemed to required the establishment of a chemical equilibrium under conditions which were forbidden by the laws of classical thermodynamics.. The re-assessment of the ultimate nature of thermal energy The traditional view of the nature of thermal energy which that which arises from the interactions between colliding gas particles seems now also to require to be modified by the incorporation of Fluctuation Theorem complex number measured time interplay (as this must apply to the small entity small time interval interactions which occur during the collision processes on which traditional thinking places the origin of thermal energy. Thermal Decomposition of Chlorinated Aliphatic Hydrocarbons This extensively studied field of chemical kinetics provided a suitable database with which to probe the possible relevance of the above concepts. The role of the actual mechanism which was thought to be a critical in directing the driving force seems not to agree with how the (published data) entire set of (60+) rate constants for the overall chlorinated organic substance dehydrochlorination rates (in the form kr=Aexp Ea/RT) all fit the same linear log A vs. Ea linear through origin compensation curve which includes rates which are evidently achieved by different mechanisms (e.g. including homogeneous heterogeneous gas phase and liquid phase as well as radical chain reactions) but which were originally thought to be simple homogeneous unimolecular decomposition processes, but when subjected to in depth investigation were found to be more highly complex and variable in mechanism.. In particular chain reactions, unsuspected catalysis and autocatalytic phenomena came to light as contributors to these rate processes. When NMR was used to study rate processes the outcome was often found to be more complex than had been apparent to chemists who had relied on older analytical methods. In industrial laboratories new empirical ideas emerged especially relating to the outcome of higher-than-ambient-temperature reactions; whereas for free radical reaction processes the preceding idea was of a predominant rate-determining process, the use of electronic integration allowed the large numbers of know rate constants to be inserted into large numbers of simultaneous equations. The older ideas of reaction mechanism seemed somehow not to be valid following this treatment. [But these observations re-awakened the usefulness of the original de Donder idea of the control which might be exerted by a single type of chemical reaction driving force which was valid for particular a type of thermally achieved stoichiometric transformation].

a Historically, it was observed that exothermic reactions were much more common and more vigorous than were endothermic reactions. This seems to have led Thomsen (1854) and Berthelot (1878) to propose that the heat produced, H, in any chemical reaction should be regarded as its affinity A [Cf., The Thomsen Bertholet Principle: - chemical affinity] Gibbs, Van tHof and Le Chatelier, however, noted that endothermic reactions also occurred naturally which required a new method of defining A which was now equated with the Gibbs (or Helmholz) free energy (G) (defined by G = H-TS) (where H=E +PV) Endothermic reactions were now allowed because of the additional source of heat energy arising from entropic processes which compensated for enthalpically derived endothermicity {the additional heat

which is available from entropy multiplied by temperature follows from the definition of entropy = S as H/T }.

(Cf. also compensated energy force = heat compensation = temperature x entropy)

This compensated G, the Gibbs free energy G= H-TS and the Thomsen-Berthelot A is now seen to be valid only at absolute zero (cf., when T=0G = H in the Gibbs-Helmholtz equation G-H=T(G)/T ) Any spontaneous chemical process was also thought to need to obey the second law of thermodynamics because it must potentially yield useful work [Cf the conversion process heat work,
1st law no restriction except conservation 2nd law this conversion is restricted by the principle that no useful work may be obtained by abstracting heat from a body already at the lowest available T]

The frequent use of i e.g. in the following (Liouville and Schrdinger) equations suggests this is because the processes being described actually refers to processes where for the process under consideration is most elegantly viewed where the meter of time therein is a complex number process. End of D.G. Insert_to notes etc from Coveney review --------------- ----------------------------------------- ---------------The dynamic description of e.g. of ca 1024 particles which exist under non-equilibrium conditions may behave according to (quasi-) equilibrium conditions suggested by The Liouville equation: i /t t = Lt where = distribution function for classical systems or the density operator for quantum systems

[which represent the probability of finding the system in a given state in phase space (the space of all the coordinates q and momenta p of the particles in the system in a classical description) or in Hilbert space (the space of states, or wavefunctions, in quantum theory).]

Formally, in the Liouville spaces in which is defined, in both classical and quantum theory, satisfies the Liouville equation where L is the Liouville operator

i{H,} = i( H/ p. / q- H/ q. / p) [H,] = H x 1-1 x H

The Liouville equation is left invariant under time reversal; i.e. it can only describe reversible processes. This time-reversal symmetry is a fundamental feature of essentially all dynamical equations which is also thought to be a fundamental problem of the consideration of entropy by e.g. non-equilibrium statistical mechanics. The Gibbs expression for entropy can be written in terms of eq

Seq G = -k d eq ln eq(classical) -k Tr eq ln eq (quantal) where d is the invariant measure (volume element) of phase space , and Tr denotes the trace (the sum of the diagonal elements) of the matrix product which follows. The correspondence with equilibrium entropy follows because eq = e H/Z But this equation cannot be used for any system out of equilibrium [Cf. the Liouvillian equation shows that SG/t =-k/t di lnt = 0 such S is a constant of motion which is disallowed by the second law] For an isolated system , where L is time dependent, the solution to the Liouvillain equation for t at time t in terms of the initial condition o at time t =0 is given by

--------------------------------------------------------------------------------------------------------------------------[Boltzmanns H-theorem. The essential approximation was the replacement of the two-particle distribution
function by a product of two single-particle functions-

t = exp (-iLt)o

This is equivalnct to an assumption of molecular chaos (where particles are un-correlated before collision). Boltzmann (non-equilibrium) entropy is given by SB (t) = -kH(t)

which satisfies the inequality H/t 0. This treatment is thought to be a good approximation for the description of dilute gases which allows
mainly binary collisions but the Boltzmann argument is probably more generally ultimately invalid.]

H (t) =

d1f1logef1

f(1) f1 (The velocity distribution function f(v1) f1 (of the Liouvillian equation) has been replaced by the following equation (where is the collision cross-section, d is the element of solid angle and the prime labels (
1

, 2) are the velocities following a two body collision).

f1/t + v1f1/x1 = dd1 {f 1 f 2 - f1f2}

------------------ ----------- --------------- ----------------- ------------------ -------------- -----------The Hypothesis of Coarse Graining: seeks to describe how natural processes can be considered to produce an apparent irreversibility at the macroscopic but not at the microscopic working level) also abrogates Boltzmann conceptualizations.
[Irreversibility which shows up generally in experimental observations must be illusory at the microscopic level since classical thermodynamics has been experimentally confirmed; this seems to have been largely a successful method of dealing with natural phenomena including both classical and quantum mechanics (which is regarded as being a correct microscopic theory of dynamics). Irreversibility is observed only because we observe a coarse-grained reality: we can discern only finite, rather than infinitesimal regions of phase-space. A paradox of how macroscopic processes depend on microscopic processes are thought to be created because of our use of approximations. These are built-into our experimental methods.

But it should be noted (Prigogine et al.) irreversibility is either true on all levels or on none: it cannot emerge as if out of nothing, on going from one level to another [D.G. insert in the arguments as set out by Coveney (in the discussion of the Brussels School of irreversible thermodynamics) may (surprisingly) indicate the possible need to re-consider apparently irrational causation and e.g. this arising from the consequences of the existence in juxtaposition of spacetime of an e.g. vacuum space overlap or leakage into conventional deterministic spacetime which allows for the passage of information during particle interactions in a apparently irrational manner consequent on negative time flow being permitted.]

Brussels School Re-Description of Classical Dynamics

This hypothesizes an intellectual model in which dynamic instability (as this shows up in real
systems) is postulated to be equivalent to the kind of irreversibility which apparently breaches the second law of thermodynamics. Dynamic instability can be incorporated into a modified form of classical mechanics so as to permit an obedience of the system of the second law of thermodynamics. [Irreversibility is believed to be closely related to dynamic instability which indicates a sensitivity of the system to its initial state]. [This mathematical trick seems to allow for the incorporation of dynamic instability into a modified form of classical mechanics in order to achieve an obedience to the second law of thermodynamics]. While, if the Poincar theorem, which requires that any system will eventually return (arbitrarily close) to their initial state in the (Gibbs) phase space (even if the recurrence time very long) holds, then the variation of the system over time cannot occur by unidirectional time (entropy increase flow). Time flow is then in reality allowed to be both positive and negative. The second law of thermodynamics cannot therefore be obeyed under these conditions. [Where the second law of thermodynamics where entropy (or a function with the properties of entropy such as a Lyapounov variable or functional increases with time is ruled out classical thermodynamics should then become an inappropriate model with which to predict particle interactions over time]. The ultimate nature even of classical mechanics may actually be indeterminate and causation (the effect of time) on particles which can be indicated not to be as straightforward as originally perceived. If the system is stable, a slight change in the initial conditions will not alter its long-time evolution. For unstable systems (such as those termed K-flows in ergodic theory) neighbouring trajectories diverse exponentially in time and the Hamiltonian formalism eventually must become invalid
[Hamiltonian (total energy) H and initial conditions given by the point in phase space {q(0) , p(0) , where q denotes the coordinates and p the momenta of the particles in the system then the future (and past) evolution (or trajectory) of the system can be predicted deterministically from Hamiltons equations of motion d/dtq(t) = H/p, d/dtp(t) = -H/q ] This is equivalent to non- determinism applying to classical mechanics (i.e. erroneous assumptions have previously been made in this treatment regarding how particles interact over time. The perception or definition of time variation as a one-way process is challenged). Dynamics (as hitherto defined) seems ultimately incapable of describing the observed behaviour of particle interactions

We are obliged to take into account the fact that it is quite impossible for us to achieve absolute precision (infinite information) in measuring the initial conditions. This assertion has far reaching implications.., because the passage from infinite regions to points in (Gibbs) phase space now becomes a singular limit, the whole concept of trajectories, which has been the basis of classical mechanics for three hundred years, becomes no longer operationally meaningful for a class of sufficiently unstable dynamical systems.
The attempts to use conventional approaches to create a new modified form of classical concepts of rate processes were described.

The Liouville space formalism enables us to broaden the definition of a dynamical variable: in addition to simple multiplication by phase space functions, we can now introduce more general operators that act on the distribution function .

The Liouville operator L being the Poisson bracket of the Hamiltonian, contains derivatives with respect to both q and p. Without violating Poincars theorem we can now seek Lyapounov variables of the form

d M
t

where M is now an appropriate dynamical operator playing the role of entropy on the microscopic level. To allow M to exist a minimum amount of instability is required.

For ergodic systems whose trajectories fill the entire phase space compatible with the conservation of energy, the condition can be stated very precisely in terms of the spectral properties (the nature of the eigenvalues) of the Liouville operational term which must possess a continuous spectrum along the entire real line. This applies to the set of K-flows in which one can define an internal time operator T which as the commutation property
i [L,T] = i (LT-TL) =1 and this reflects the age of the system. An entropy operator M can then be constructed as a monotonically decreasing positive operator function of T. This equation demonstrates non-commutativity of the time operator T with the Liouvillian L. [A complete certainty in the dynamical description in terms of trajectories (through L) renders thermodynamics (through T) meaningless, whereas conversely, a fully thermodynamic description is incompatible with a compete knowledge of the dynamics ].

Non-integrable systems which begin with three body problem are of importance in physics for which the construction of the entropy operator involves the use of subdynamics. The source of the irreversibility in Boltzmanns equation is assumed to stem from nonintegrability rather then from the ergodic properties of the dynamics.
Non-unitary transformation theory: A non-unitary transformation (can be defined) through = M in terms of which the reversible unitary group Ut is related to an irreversible semigroup W*t by the relation W*t = Ut-1 (t0) This semigroup for present purposes satisfies the equality

W*tW*s = W*t+s

only for t,s 0, [equation 1]

whereas the corresponding group property UtUs = Ut+s is true for all times, t,s.

The non-unitary transformation ( definition equation above) therefore breaks the timesymmetry of the evolution originally present in the Liouvillian equation and the process becomes irreversible. The transformed distribution function (known as the physical representation p , causes the originally deterministic evolution t = Ut o to be transformed via [equation 1, above] into a stochastic Markov process:
p

t = t = Uto = W*to = W*tp0

p

allowing any convex functionals of such as

d(pt)2 or dptlnpt to satisfy an H theorem with no approximation.

---------------------------(cont.) Notes ex Coveney

The introduction of M into quantum systems It is thought that infinite but not finite quantum systems can display irreversibility (alternatively instability cannot be defined for normal quantum system in marked contrast to the situation in classical mechanics, in quantum systems, because, if follows that no M (entropy superoperator) is allowed for finite quantum systems but where the Hamiltonian has a spectrum that in a limiting sense becomes continuous from 0 to + , the superperoperator M then becomes possible.

The introduction of the entropy superoperator M causes M to be non-factorizable (viz. it cannot be written as a product of Hilbert space operators and, as a result it transforms pure states into mixtures (i.e. the system evolves irreversible; if M exists there is not longer any reason to be restricted to the notion of the wavefunction).

This resolves the measurement problem: because we have now taking into account both the macroscopic nature of the measuring apparatus and the irreversible nature of the measurement act itself. The entropy superoperator M, if allowed for infinitely large quantum systems in the thermodynamic limit, corresponding to the limit of an infinite number of particles in an infinite volume, may also permit a resolution of the measurement problem (cf. Heisenberg Uncertainty Principle).

(Cf.

according to the conventional formulation of the theory as defined by van Neumann there are two distinct types of transformation which occur to the wavefunction , (the quantity containing all the information to be known about a given system). [1] we have the unitary (reversible) deterministic evolution of pure states

, governed by the Schrdinger equation i /t = H

(H being the Hamiltonian for the system) and [2] we have the supposed reduction or collapse of the wavefunction, which occurs irreversibly and nodeterministically when a measurement is made on the system {for which no mechanism can currently be provided}. ------- ----- ----- ----- ---Coveney concluded his review by suggesting [in the section headed Ireversibility in real systems: subdynamics] that the concept of the entropy superoperator can allow progress to be made in understanding the thermodynamic behavior of large many-body systems for which the Brussel School considered that the sub-dynamics theory of irreversible processes clarified the relation between dynamics and thermodynamics and allowed kinetic equations to be derived using non-equilibrium statistic mechanics.

This theory starts off from the usual perturbation expression for the true system where L= L0 + L

L0 represents a solvable problem and L are the interactions between the units defined by Lo. The approach is based on the introduction of a complete set of correlation states (eigenstates of L0) whose hermitian projection operators P( ) commute with L0.

For the case of a dissipative system in the thermodynamic limit, we can no longer diagonalize the full Liouvilllian superoperator L , but we can show that a new complete set of non-hermitian projection operators ( ) exists, each one of which commutes with L: [( ) , L] =

( )

L-L( ) =0

If we define the components of the density operator by

() () ,
then we see that each component evolves independently

()

()

i./t
hence the term subdynamics.

A generalization of the conventional unitary quantum-mechanical transformation theory was defined that relates the projection operators () and P()

through

P() = P() -1

which as before converts the unitary description into the physical representation, where once again the evolution now shows an arrow of time
p

t = W *tp0

Coveneys Conclusions

[The ultimate description of matter may not , as is commonly assumed, be obtained from quantum-mechanics (alone) since this encounters a range of difficulties e.g., the non-exponential decay predicted for unstable particles ; general difficulties in theoretical formulation of problems involving lifetimes as well as the re-normalization programme associated with the divergence of quantum field theory and the need to use multi-photon detection apparatus for very short time-scales, and the absence of a consistent theory of measurement]. Reversible thermodynamics is also (at least partly) also an incorrect theory. This model of a completely timeless, deterministic Universe might perhaps usefully be replaced (by the Prigogine (Brussels School)) model in which static affairs are enlarged to embrace the probabilistic kinetics of processes in which of reversibility and irreversibility are accorded equal objectivity (the notions of being and becoming are thereby unified within a single conceptual framework). [Long held beliefs are challenged about the validity of classical thermodynamics (i.e. non-evolutionary thermodynamics) were apparently supported by simple, twobody problems in physics which favours exactly solvable models (e.g. those which involve periodic motion) and this usage was supported by the apparent successful usage of reversible thermodynamics in Cosmology and Particle Physics (cf. the Standard Model of the 1970s and the Inflationary Universe model and the 3K microwave black-body radiation background)]. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

8th Manuscript Vacuum Space & Extrathermodynamic Phenomena

In a perfect (classically perceived) no-particle or no-energy vacuum if the vacuum space is sufficiently large time ceases to have any reality. Time therefore is a construct of the ability of matter to exist in independent coordinates. Time cannot be realistically be perceived in the case of an isolated particle moving without reference to a frame. More complex but still simple systems cannot be conceived as being uni-directional and hence time is intrinsically bidirectional. The build-up of complexity in regard to framed boundaries restricts motion and hence creates the illusion of unidirectional time. This is the realm of practical application of heat energy and the concept of how useful work may be derived therefrom (viz. the mathematical ideas enshrined in classical thermodynamics). The possibility that a vacuum may be structured however introduces the idea that useful work (vacuum energy) may be obtained therefrom and further the possible high energy presence in a vacuum suggests further that the definition of time is less clear than in the absence of this phenomenon. It is likely that notions attempting to assess this possibility and also to more clearly define microsystems e.g. chemical reactivity restricting concepts only to those from classical thermodynamics will be incomplete or perhaps quite misleading.

Extrathermodynamic Phenomena in Chemistry The Hammett linear free-energy relationships were indicated by Leffler to be a special case of a more general enthalpy-entropy compensation process in which numerous chemical reaction rate constants k (=AexpEa) for families of chemical substances can be shown to demonstrate (an often strict) mathematical relationship between logA and the Ea (Arrhenius activation energy) terms. The equivalent terms to logA and Ea for processes in physics which vary exponentially with temperature also. Notes from review papers of relevance to a fuller understanding of the application of thermodynamics to chemistry.

Possible (Historically Identified) Extrathermodynamic Processes

include

Brownian motion,
which may breach the second law of thermodynamics

A system which appeared to undergo Brownian motion was observed by following laser tweezer- held particle trajectories (by Yang et al Phys Rev Let in 2002 to disobey the second law of thermodynamics Cf also Discussion of Brownian Motion & Calculation of Avogardros number (L) from Brownian Motion by David V Fenby in Einstein and molecular reality, Chem. Brit., 1981, 17 (3) 134-118 The study of Brownian motion- the ceaseless, irregular movements of microscopic particles suspended in a liquid-began with its observation by Brongniart and brown in 1827 and throughout the remainder of the 19th century many theories were advanced to explain the phenomenon (M.J. Nye Molecular reality. London: Macdonald, 1972). Although the correct explanation, that the motion arises from the impacts of the solvent molecules on the particles, was expressed by a few investigators in the second half of the 19th century, it was not until the studies of Gouy between 1888 and 1895 that this hypothesis attracted wide attention. Gouy regarded Brownian motion as direct and visible proof of a kinetic molecular theory and, further, pointed out that the phenomenon is contrary to the second law of thermodynamics. .. In 1905, the year in which he proposed the special theory of relativity, and the concept of radiation quanta, Einstein showed (A. Einstein , Investigation on the theory of the Brownian movement (R. Frth ed) New York: Dover. 1956) that bodies of microscopically-visible size suspended in a liquid will perform movements of such magnitude that they can be easily observed in a microscope, on account of the molecular motions of heat. In the first of his papers on this topic Einstein was not sure whether these predicted movements were identical to Brownian motion because he lacked information concerning the latter. The work of Gouy and others was soon brought to his attention , Einstein considered a solution of spherical particles of radius r very much greater than that of the solvent molecules. He calculated the diffusion coefficient D of these particles in two ways: 1. From a consideration of thermodynamic (osmotic pressure) driving force and the frictional resistance of the solvent, the latter being obtained from Stokes hydrodynamic equation, Einstein showed that D=RT/6Lr (1) ..He obtained L= 6.56 x 1023 mol-1. 2 Einstein showed that, due to the impacts of the surrounding solvent molecules, the particles would undergo irregular motions. He proved that the rectilinear displacements of the particles during some time interval followed a statistical distribution law similar to the Maxwell distribution of molecular velocities but involving D. The mean square displacement in the x-direction is given by, x2 = 2D (2)

x2

= (RT/3Lr)

(3)

 Einstein was fully cognisant of the significance of his work on Brownian motion: If the movement discussed here can actually be observed (together with the laws relating to it), then classical thermodynamics can no longer be looked upon as applicable with precision to bodies even of dimensions distinguishable in a microscope: an exact determination of actual atomic dimensions is then possible. On the other hand, [should] the prediction of this movement prove to be incorrect, a weighty
argument would be provided against the molecular-kinetic conception of heat. .).

Excerpts from an article which points out that non-equilibrium thermodynamics (i.e. irreversible processes can be a source of order within a system). Peter V Coveney The second law of thermodynamics: entropy irreversibility and dynamics Nature 1988 332 409-415 Until recently, however, it had been thought that the fundamental dynamical laws were incompatible with the objective existence of the irreversible processes that thermodynamics purports to describe. Most scientists accept the existence of irreversible processes as a brute fact of life. Being macroscopic their definition may be found in thermodynamics , which is concerned the formalization of the facts of everyday experience. A reversible process is one in which a given system may be returned to its initial state, even after the performance of work and the exchange of heat, with no accompanying effects arising in the surroundings. All other processes are irreversible; for example even if it is possible to return a system to its initial state, changes will occur in the surrounding (transfer of heat, and so on). Clearly reversible processes are very special cases. The second law The distinction is sharpened by the second law of thermodynamics, which asserts that, for any irreversible process, the function S, known as the entropy, must increase, whereas for a reversible process it remains constant. More particularly, the entropy of an isolated system increases monotonically until it reaches is maximum at equilibrium-a form of words that implies a sense of the direction of time, as does the familiar statement of the second law in the form dS 0 where the equality applies only to reversible processes; more exactly, this should be written dS/dt 0. This makes it clear that the positive direction of time (t) is linked to the increase in S during an irreversible process. Indeed the second law of thermodynamics is the only law of physics that introduces a sense of direction of time.

Mansuscript 9
Reverse Entropy & Vacuum Space

D Grant AB53 May 8 2012 Hypothesis Reverse Entropy and/or Vacuum Space: Possible Roles in Mediating Chemical Transformations The occurrence of unexplained entropy-enthalpy compensation in temperature dependent rate processes is a major scientific challenge. Explanation of this compensation seem not to be achievable by conventional ideas of causality and therefore may require some new approach to the logic of time dependence of physical phenomena which apply to small sized entities which include particles the size of chemical molecules. Two ideas are considered here. The Evans reverse entropy flow which has been experimentally verified for colloidal size particles undergoing Brownian motion and the possible role of vacuum space interactions which might behave as modulators of chemical reactivity ----------------------------------------------------------------------------------------------Insert: The physical nature of entropy D.Grant May 12 2012 Entropy might ultimately arise from how matter interacts with vacuum energy. It can be pointed out that entropy has traditionally been less well understood than is enthalpy. Previously whether the nature of entropy is subjective or objective has been seemed to require a convoluted discussions; this also applies the role of an entropy superoperator in quantum systems.

Enthalpy change is intertwined in entropy change by the phenomenon of Leffler compensation, but a possible (at present highly tentative) system highlighted by this process, may allow for a more rigorous definition of entropy. This tentative hypothesis tries to explain of the origin of this compensation effect by suggesting that the existence of vacuum energy might somehow conceivably regulate entropy flow during the course of those chemical reaction transition state complex decompositions which display the Leffler compensation phenomenon by evoking the occurrence of reverse time entropy flow [as experimentally verified by Yang et al 2002 to occur during micron bead towing though water] (putatively this is a demonstration of Brownian motion conditions where the second law of thermodynamics becomes disallowed for short timescales)]. Further extending the hypothesis from the argument that the Leffler and similar phenomenon are sufficiently general in Nature to allow (at least in principle) their extension to all chemical and physical reactivities. further suggests a new vacuum energy conceptualization which permits a new definition of entropy as an intensity factor for matter interaction between conventional space and the vacuum. (i.e. a processing (transfer) effector). Time-turnaround. The process of the time dependence of entropy (production) during chemical molecule activation (in the classical of transition state complex formation in chemical reaction types which demonstrate strong enthalpy-entropy compensation e.g. in series of rate

constants {is also a process which may however be widely replicated throughout Nature} is putatively of fundamental relevance to a fuller understanding of the properties of chemical reactivity [which is how temperature { etc.} and time affects the arrangement of matter] a variation process which possibly allows to a deeper understanding of causation in general. The hypothesis that the Leffler entropy-enthalpy compensation phenomenon (which seemed to arise because of the breach of [supposed] primary thermodynamic laws) actually ultimately arises from an adherence to some basic fundamental natural law(s) which demands an overall creation of an enthalpy-entropy correlation during the activated transition state complex decay (e.g. following chemical molecule thermal activation) is suggested to arise via a virtual processing of this virtual molecule via a virtual flow of entropy from some future (or other) virtual time coordinates. And that this arises by a similar mechanism to that by which time reversal is suggested to occur during Brownian motion [The Evans-Yang apparent experimentally verifiably time reversal process]. The scientific credibility of such time reversal processes are now considered also to permit a fundamental re-consideration of the mechanism of chemical reactivity in general. This suggests that this arises via an alteration of the entropy production during the Evans reverse entropy time period. Such a time reversal interval has, it is proposed, a profound effect on the molecule (requiring all movement including spins and vibrations to be reversed) within the particles undergoing this transformation. This process can be modeled using an analogy of a reversing (e.g. automobile) engine which needs experience power cut-out or be clutch removed from power

connection, motion slowed stopped put in reverse gear and restarted]. This is, it is now pointed can only be phenomenonologically accomplished for an engine of the size of the quasi molecule which has traditionally been proposed to exist in the theory of chemical reaction rate activated transition state complex, via the complete abolition of commonly assumed normal causality during the chemical reaction transition state quasimolecule lifetime by what it is further hypothesized, the creation of a local quasi vacuum state (causing a virtual abolition of mass and time in space occupied by the activation complex quasi molecule for the indeterminate but possibly infinitesimal (small) time interval under consideration) for which the variation of entropy interaction between vacuum energy and conventional space-time is the critical ratedetermining process which produces both the phenomenon of chemical reactivity but also the observed enthalpy-entropy correlation via a fundamental natural law which defines this process (but which also seems to require a re-definition of the physical nature entropy in terms of this relationship). Entropy arises it can therefore be suggested from the manner in which the quasi vacuum and non-vacuum states interact during the transition of individual components into normal matter from the proposed quasi vacuum state virtual matter during the competition of chemical transformation or other common equivalent transformations achieved by heat energy or other forms of energy upon matter. *(Which is now known to a more general throughout physics, chemistry and biology phenomenon than when discussed by Leffler) in which Arrhenius activation energies for separate but related

chemical reaction processes display a distinct correlation with the entropy changes in these processes.
------------------------------------------------------------------------------------------------

Document retype April 5 2012 (cf. scanned version of original document in http://www.scribd.com/doc/8787336/D)

Recap COMPENSATION EFFECTS NOTE and edit in compact spacing

by D. Grant 5.4.84

The Compensation Effect may be defined by the following: log k = a -E / 4.6T (cf. similar expression for equilibrium constants) where log is the logarithm to the base 10, k is the chemical reaction rate constant, a is a constant, E is the Arrhenius activation energy , and T is the absolute temperature and where a = kE + (const) (const is often = 0).(Similar expression from equilibrium constants) [A plot of log a vs. E gives a straight line]. The rate constant maybe re-written in terms of entropic and enthalpic changes associated with the transition state complex, according to the transition state complex theory of chemical reaction rate, viz., kr = (kt/h) exp ( S*/R ) exp ( - H*/RT), (usual meaning of constants); the pre-exponential term in the rate constant being associated with entropic changes in the formation of the activation transition state complex. A wide range of rate processes have been observed to exhibit this entropic-enthalpic interdependence. Attempts to explain these interrelationships have been unsatisfactory: the effect was noted and briefly discussed by Boon (1973) (cf. Harris (1973), Damjanovici and Vernon (1973) and rationalized in terms of special properties of surface catalyzed processes by Wilson and Galway (1973))..Barton and coworkers (1951) noted an apparent relationship between log A but E. Maccoll repeated some of the work and found different values which did not show the compensation relationship. The problem of very high logA values was discussed by Blackadder and Hinshelwood (1958). The role of enthalpy-entropy compensation in organic chemistry reviewed by Leffler (1955) who cited 81 examples of compensated rate constants. He pointed out that the relationship indicated the existence of and isokinetic temperature for a series of reactions exhibiting the effect. It was suggested to be a general rule that any organic reaction was cleaner the further the reaction temperature was from the isokinetic temperature, hence a tendency to obtain random product distribution near the isokinetic temperature. The compensation effect has been found (for some types of) heterogeneous catalysis (e.g. hydrocarbon formation on clays and similar processes (cf. Boon et al: Wilson and Galway (1973).(but this cannot be a general explanation?). Compensation effects are also observed in the case of heats and entropies of vaporization from solutions, e.g. gases from acetone and numerous other examples, Barclay and Butler (1938); and for energy gap and pre-exponential factors in dark conduction by organic semiconductors, Eley (1967); as well as in the enthalpic and entropic contributions to the second osmotic virial coefficient , Wolf (1972). The compensation effect between dilution entropy and dilution enthalpy of polystyrene and polymethylmethacrylate have been reported by Lechner and Schulz (1973). A possibility arises that the compensation effect could be related to deviation for ideal solution behavior. There is a tendency for reactions with higher activation energies to proceed faster than might otherwise be expected------- this might be a postulate related to the compensation effect. Alternatively put, there is a kick principle: the greater the activation energy the greater the kick which might additionally become available following dissociation of the transition state complex. Such a concept may be derived from considerations of unimolecular rate theory in consideration of compensation-like effects, by comparison with radioactive decay or Auger effects cf., Rognisky and Rosenkewitsch (1930). A further review and discussion of the compensation effect was given by Molin et al (1965). References. Barclay I.M. and Butler J.A.V., J. Chem Soc. 1939 1445 Barton D.H.R.and Howlett, K.e. J. Chem Soc 1949 165 Barton D.H.R. , and Onyon, P.F. J. Amer Chem Soc. (1950) 72 988; Barton, D.H.R., Head A J. and Williams, R.J. J. Chem Soc., 1951 2039, cf.Maccoll A. and Thomas P.J., Trans Faraday Soc. (1968) 119 Blackadder, D.A. and Hinshelwood, C. J. Chem Soc. 1958, 272 Boon M.R., Nature (1973) 243, 401;cf., Harris P.S. ibid., (1973) 243, 402;cf. also Damjanovic V. and Vernon, C.A. ibid., (1973) 243, 402 and Wilson, M.C. and Galwey, A.K. ibid., (1973) 243, 404 Eley D.D., J. Polym. Sci Pt C No 17 (1967) 73 Leffler, J.E., J. Org Chem 1955, 20,1202 Lechner, M.D. and Schulz, G.V. Macromol Chem (1973) 172 161 Molin, Iu. N. Leshina T.V. and Mamaev V.P., Dok Akad Nauk SSR (1965) 163 402 Roginsky, S and Rosenkewitsch L., Nature 1930, 125, 347, cf., Roginsksy, S and Rosenkewitsch, L, Z Phys Chem. (1930) B 10, 47 Wolf, B.A. J Polym Sci Pt. A2, (1972) 847 ;;

Manuscript 10
Vacuum Time Consider the hypotheses that the Leffler enthalpyentropy compensation between entropy and enthalpy is a consequence of entropy flow from virtual sources consisting either of (the vacuum) or (other time locus suggested by Evans reverse entropy).

The vacuum no-time (a may-well-be new idea) If the vacuum is without time variation, all time there is coincident with Leffler enthalpy coincidence ___________________________________________

Cf., Classical Thermodynamics/ Collision Theory ; Transition State Theory (And Modifications Thereof) of Chemical Reaction Rate Consider the Arrhenius rate expression lnk = log A Ea/RT (1) k=exp(-Ea/RT)

where k = sec-1 for first order reaction and mole cm-3 sec-1 for second order reaction A= frequency factor Ea = Arrhenius activation energy R= gas constant An apparent enthalpy-entropy correlation phenomenon became evident for the above rate process including when rate constants were interpreted in terms of statistical mechanical transition state theory which highlights

the transition state complex correlation for sets of rate constants show good correlations between S* and H*/T. A review by Leffler in 1955 (J Org Chem., 20 1202 ) suggested that rate log A vs. Ea compensated for numerous sets of rate constants which were then available for a wide range of typical classes of organic reactions. Indeed the phenomenon of compensation seemed possibly to be a general rule and not be a rare occurrence. Although experimental error undoubtedly was the origin of some of the reported instances of apparent enthalpy-entropy compensation, genuine such correlations are now believed to occur widely. This draws attention to the compensation behavior as being one of possible correlation between general enthalpy and entropy i.e. not being restricted to rate processes only.

The conventional view is that A, the frequency factor in the rate constant (which according to the collision theory of chemical rate, is the fraction of the total number of collisions which are the correct type to permit chemical reaction to occur) has a maximum value typically of ca. 1013.5 sec.-1 for the temperature and pressure conditions under which chemical reactions are usually studied. Experimental log A values were sometimes found, however, to be considerably greater than this. These values which are believed to be genuine (and not due to experimental error) were, at first thought to be wholly irrational but later were considered to arise because of a degeneracy [calculable by statistical mechanics] in the bond vibration processes which allow entropy flow within the activated complex which allowed for an increased entropy change and increased probability of the occurrence of such a change. The number of required degrees of freedom were however difficult to reconcile with those which were available. Some of the observed log A values were very considerably greater than could be explained by this hypothesis. (The values were furthermore most often found to be less than 1013.5 and these A values also showed an identical compensation dependence on experimental Ea values as the A values from the larger-than-average A value data).
[When conducted in a solvent the high A values were suggested to have possibly arisen from a cage effect which was facilitated by adjacent solvent molecules which promoted the occurrence of multiple collisions which contribute to the amplification of the thermal activation processes].

The most common form of the correlation was linear Ea = (logA) + const (and often the const was =0) the term , (can be interpreted as a temperature (the compensation temperature) at which all reactions of the series proceeded at equal rates). [The possibility of this type of iso-kinetics tends to generate over-system stochastic behavior]. {This stochastic reorganization of chemical molecules is exemplified at near ambient temperature by liquid water which when used as a solvent for chemical reactions (e.g. those of importance to biology) generates a general enthalpy-entropy compensated milieu [this is equivalent to what has been described as no-mechanism chemistry.

Leffler pointed out that the Hammett and the related more widely applicable Taft equations) are also examples (sub-types) of the general overall enthalpy-entropy compensation phenomenon. {This is a commonly used empirical organic chemistry (extrathermodynamic) empirical rule allowing systematization of ortho and para substitution rates of aromatic substances and Taft constants apply more widely and includes aliphatics}. Hammett and Taft constants furthermore vary linearly with NMR chemical shifts (these are good potentially calculable from first principle indicators of relative chemical reactivity) indicating that the shielding of nuclear spins by the electron clouds which produces the NMR chemical shifts also may give rise to the rate constant enthalpy-entropy compensation phenomena. It should be noted that entropic and enthalpic thermodynamic concepts which originally were introduced into chemistry from the field and viewpoint of mechanics and with the prime objective to consider how the transformation of chemical energy into heat could be converted (e.g. in steam energies) into work with a maximum efficiency; it was considered, that thermodynamics can be described most simply and with the greatest mathematical exactness for an (unreal) ideal system which functioned under conditions of reversibility of piston movement; this reversibly simply linked pressure and temperature and work derivation. This reversibility clause has entered physics and chemistry from this historical input. Such reversibility seems however to be a source of error (really a misunderstanding) when comparing real with ideal systems. Non-ideality is putatively intrinsically linked with the occurrence of the disallowed lack of independence between the temperature dependent and independent entropy and enthalpy terms in rate processes. Non-ideality also is engendered by how time reversal seems to be not ruled out for the equations which determine motion but time-reversal is not apparently encountered in real physical processes (at least at the scale encountered by engineers). The reversal to entropy flow on proceeding from large to small engines seems to have been experimentally demonstrated (Yang et al. 2002) over sufficiently long time intervals to now require that this phenomenon be incorporated into the mathematical models of chemical reactivity (such as the statistical mechanical transitions state activated complex formation theory). The details of how this may be accomplished could however suggest that the actual rate governing process is not as was previously believed due to conventional causality but also requires a re-assessment of allowable

causalities in which the nature of time must be modified to allow future and/or past time to co-exist with present time. Alternatively put: this process of mixing of previous, consequent and present events seems to demand some extra-spacetime information transfer system. This putatively might be afforded by the vacuum which can be accorded an event (matter, mass) mixing role under conditions where the rules which govern the event variation in conventional spacetime do not apply. This further indicates that leakage process between conventional space time and the vacuum space must be a normal phenomenon and be a controller of normal chemical and reactivity particle interactivity. -----------------------------------------------------------------------------------MR Boon etc. (several separate contributors) Nature 1973 243 401 in a discussion of whether the entropy-enthalpy compensation rule in chemical reaction rate constants is a genuine experimental fact or alternatively is an artifact of wrongful data processing drew attention to the circumstance of heterogeneous organic hydrogenation where the existence of very large range of high A values (occurring over several orders of magnitude) which showed exact correlation with Ea values countered a common belief being that experimental error is always the origin of the compensation phenomenon could be ruled out. A later critical review by Liu and Guo (Chem Rev 2001 101 673) mentioned a range of such correlated chemical reactions (and it should be noted while this review critically examines the entire field it indicates that compensation, while in some cases showed that this has been wrongly attributed, (as were some instance of identification of an isothermal temperature) most of the reported instances are probably genuine and include, phase change upon melting and the temperature-dependent behavior of semiconductors (including protein-based) and host guest solvent behavior. Enthalpy-entropy compensated reactions in aqueous solutions were seemingly rather commonly; perhaps this was because water consisted (of at least) two chemical structures components in the compensated reaction supporting fluid. (The aqueous solution compensation mechanism, it should be noted, could be of fundamental importance to e.g. protein folding and perhaps also could be implicated in the mechanism of biological evolution).

A dramatic multi-point (200,000+) curve demonstration of the enthalpyentropy correlation was later demonstrated for thermal variation of digital camera pixels dark currents.
For considering the effect of temperature on rate processes two sets of rules might however be suggested to simultaneously be extant. [One of these might be considered to be the correct one and the other is considered to be incorrect. N.b. such rules seem to underpin all basic science in that notions of space and time e.g. the second law of classical thermodynamics is thought to be of very high fundamental importance to how time is a one-way]. A problem with making the Leffler phenomenon the lead hypothesis is that classical mechanics originated statistical thermodynamics has been outstandingly successful in e.g. with statistical mechanics in allowing diverse phenomena to be quantified and compared.

[It should however also be noted that use of statistical mechanics in calculating reaction rate constants has been apparently largely on the face of its apparent spectacular success, needs to be carefully reexamined a re-examination of dehydrochlorination rate constant predictions by this method putatively fails].

The kinetics of gas phase dehydrochlorination of chlorinated organic substances were originally though by some researchers (e.g. DHR Barton) to show a definite, if an approximate, enthalpy-entropy correlation; Allan Maccoll (another prominent classical researcher in this field) had, on the other hand, after he had carefully repeated these studies, concluded that no correlation actually existed; the log A values reported for this set of rate constants seemed to cluster around an average value of ca. 1013.5 the same value which had been predicted by the number of gas molecular collisions which can achieve the thermal activation required for bond loosening. Another contribution of Allan Maccoll and colleagues was that the dehydrochlorination reaction mechanisms in both the gas phase and in a solvent were very similar and indeed also similar to the decomposition of these molecules by processes involving ionization of which occurred in a mass spectrometer. The paradigm of a electron pair type reaction mechanism seems somehow at odds with the above experimental facts but nevertheless a reaction mechanism of the conventional type was thought to have been established for this class of reaction. My re-examination of Allan Maccoll et als published results suggest that his listed values of A while mostly of values around 1013.5 actually do show a fairly strong correlation between his experimental log A and Ea values especially when an additional (through origin 0,0) point is added to the published tabulated results for which a the least squares fit of these data shows a well-defined rate constant pre-exponential value vs. Ea Arrhenius activation energy correlation ( R2 >0.9; furthermore these data also obey the same correlation curve as the numerous other lab. reported kinetic data for this and similar reactions reported in the peer-reviewed literature. [A large number of dehydrochlorination and chlorinated molecule isomerisation rate constants (originally collected by the author in an industrial laboratory) also obeyed the same well-defined linear correlation irrespective of whether the original authors believed their results had derived from homogeneous first order bimolecular free radical or entirely heterogeneous reaction mechanisms: all of these data fitted similarly onto a general correlation curve (which passes through the origin). These academic
and industrial laboratory-reported data included studies from different countries etc. and which included applied science studies of model compounds aimed at establishing the mechanism of the thermal decomposition polyvinyl chloride).

A re-analysis of my entire database dehydrochlorination set of rate constants tends to confirm the Leffler hypothesis (that the occurrence of enthalpy-entropy compensation applies to all related types of chemical reactions and by further implication also to all types of rate constants) asuggests that these results might usefully also form the basis of a new theory of chemical reactivity which could perhaps allow for a fuller understanding of the mechanism of all thermally induced chemical molecular and other energy source activation routes; it is worthwhile to especially include mechanisms using previously unexpected energy sources (vacuum energy) which might conceivably contribute to the phenomenon of chemical molecular activation. This permits a new set of thermodynamic rules to be formulated by starting off from the hypothesis that the Leffler process is a primary law of nature and that it putatively has a more relevant impact on chemical reactivity than does the second law of thermodynamics. Two Incompatible Intellectualization Processes? Two intellectually separate methods 1) classical thermodynamics and developments of this (e.g. non-equilibrium thermodynamics) and 2) what has been described as extrathermodynamic empiricalism (e.g. use of Hammett relationships etc. and possibly in need of re-interpretation are the chemical reactivities (e.g. biological evolution, chemical structure nucleation phenomena and thermal scrambling behavior which had previously were dealt with using classical thermodynamics). 2) has been generally viewed with suspicion and be of a dangerous nature as regards the integrity of science as it might contravenes the believed firm laws enshrined in 1).
If for no other reason than simply to provide an intellectual exercise, it is useful to start from the premise that the viewpoint of Leffler and other workers who have reported the existence of genuine enthalpy-entropy compensation have used experimentally sound methods. Since theory is secondary to experiment the latter must take precedence. This means that during the process of thermal activation of a chemical molecule and other physical processes which can be described by an equation of the form rate= (pre-exponential) exp. Ea/RT, the primary physical law governing all actions which lead to the Leffler equation (in which during the formation and decomposition of the transition state complex entropies occur require the introduction of pathways from extrathermodynamic entropy flows. [N.b. the traditional activated complex concept seems worth retaining as useful intellectual concept in any revision of chemical reactivity] It is instructive nevertheless to consider as a starting-off point for a re-evaluation on the basis of the assumption that all observed chemical and perhaps also physical phenomena depend somehow on a transition state complex (n.b this a virtual molecule which is formed in a non-equilibrium fashion [and therefore is at the start an extrathermodynamic concept as classical thermodynamics requires the occurrence of reversibility even although this cannot strictly be applied]). Postulate further that the fundamental driving force of the phenomena in question is the need in the said virtual state of chemical transition (where the lifetime of the activated complex is indeterminate but thought perhaps to be that of a single bond vibration e.g. 10-13 sec. (but could be much more or much less than this period) to ensure that S* is correlated with H* i.e. that the variation of entropy and enthalpy with time is fundamentally (by natural law) a linked process. Let us further suggest that the enthalpy is perhaps the dominant phenomenon in that entropy responds to enthalpy and not vice versa

Since we are considering time to be non-thermodynamic and therefore not to be directed by the second law of thermodynamics we can consider bidirectional time with equal weight to both.

A mechanism may exist during the thermal activation of matter (which increases the rate of chemical transformations) by which entropy flows with equal probability so as to either to increase or decrease the values of S* in which the excess of deficit amount of S* over or less that required to balance the Ea value is transferred to or from other virtual sources (e.g. to and from vacuum space which lies outside conventionally viewed spacetime which is bounded by directionally restricted time flow). This provides a convenient (if a highly non-conventional) model for the experimentally observed compensation phenomena which suggests that virtual space associated with the vacuum may play a dominant role in the mechanism by which Leffler compensation arises. This space may exist in an accessible form in the vacuum (cf., n.b., the general acceptance of the existence of vacuum energy is now common). An alternative viewpoint is that this virtual space exists in normal spacetime but in the past or in the future thereof thus being in a virtual form.
The experimental phenomenon of Brownian motion (or its equivalent in a latex bead towed water optical tweezers nonthermodynamically directed motion) has been, credibly rationalized, it should be noted, by use of reverse entropy flow by Yang et al (2002) by use of the time reversal Fluctuation Theorem (Evans). Another Tentative Hypothesis Wording The past time record associated with the virtuality membrane boundary between conventional spacetime and the vacuum energy system (extraspacetime) achieves entropy flow (from the store) to produce a Leffler enthalpy coincidence of proportionality with the Arrhenius activation energy. If past and future are meaningless in the context of the boundary between vacuum space and conventional spacetime (i.e. if the vacuum is without stable particulate matter, this abrogates conventional time space as conventionally regarded;

future time become equivalent, in the vacuum to past time and both can be a source of entropy flow into the real spacetime.

IVR (Intramolecular vibrational energy redistribution) Cf e.g. D. Gree et al., J. Chem Phys 1999 110 1979 which reported the eigenvalue-resolved i.r. specrum of 2-F ethanol which demonstrated the occurrenc of an IVF lifetime of 275 picosec. (the isomerisation process lifetime was 2 nanosec).
Cf Notes from Chemical Kinetics: from Molecular Structure to Chemical Reactivity by Luis G. Arnaut, Sebastio J. Formosinho and Hugh Burrows, Elsevier, 2007 Cf Introduction Berthelots understanding of the role of methematics in chemistyr the mathematicians make an incoherent block out of physical and chemical phenomena. For better of for worse, they force us to fit the results to their formulas, assuming reversibility and continuity in all sides, which, unfortunately is contradicted by a large number of chemical phenomena, in particular the law of definite proportions.

cf. Ch8.4 p 219 , Local Random Matrix Theory (LRMT)

For the transition state complex formed as a rate determing step in (unimoleuclar and other) chemicial reactions, while Slater had considered (in his traditional modified transition sate hypothesthis) that that one waits for classical motions of the modes to be in phase with each other. Chemical reaction occurs when a particular superposition of modes causes the reaction coordinate to reach the top of the reaction barrier. The anharmonicity of the bonds, however, limits this appoach because it allows energy to flow between modes. However even for the harmonic level of approximationcertain paths of motion are more facile than others. Such paths follow resonance in which a few quanta in one mode are converted into quanta of other modes, such that the total mode energy is nearly conserved
Molecules which are not high in symmetry have many localized normal modes.these supplemented by torsional modes But (following Gruebele and Wolynes) these respresent slow varying degrees of freedom which can be used to label privileged quantum states which have excitations in many modes, the so called interior states which can be occupied if the molecucle is sufficiently energized. Since these states have a rich set of possible non-linear conections this allows energy to flow in many ways. This can be viewed as sequential hopping to nearby states (e.g. those states which differ by changes on only a few quanta numbers in a few modes). These states differ from the edge states (which are considered to occur at the perphery of the state space) which have nearly all atheir energy concentrated in a single mode which restricts energy flow and which are considered to decay by a dynamical tunnelling process by which the intermediate states are never populated but facilitate hoping to energy states that are nealry resonant and which are found in the interior with very different quantum numbers.

The randomization process is allowed to occur more often for slow (higher activation enrergy) reactions where the activiated molecule has time to explore all the availbe phase space. If energy flow is slower than the reaction coordinate vibration itself, then the simple RRKM rate becomes inaccurate. Rates depleted by reaction can become repopulated via IVR and diffusion through the state space. This can becomes rate determining. Such a feature shows up in the pressure dependence of the rates because rate space diffusion is facilitated by collisons.

Manuscript 11
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The above (enthalpy-entropy compensation) plot was obtained for 1968 Chem Abs. listed chlorinated aliphatic substance dehydrochlorination rate constants k= Aexp Ea/RT ( x-axis is Ea (kcal mol and the y axis is log A) Dehydrochlorination Rate Constants: Correlation of LogA and Ea Values DG 23/4/12 The plot (shown above) of the values of log A vs Ea (Arrhenius activation energies) gas phase rate constants (k=AexpEa/RT) obtained from Chemical Abstracts in 1968 for the putative homogeneous first order reactions of the type R1R2CH-CClR3R4 R1R2C=CR3R4 +HCl suggests a strong correlation occurs between the logA and Ea values for some 42 data points (the trendline extending to the origin).

Other similar experimental data points which became available after 1968 (e.g. data obtained during modeling of polyvinylchloride thermal decomposition (Mayer Z et al 1974 [J Macromol Sci (C) Polymer Reviews) 10 (2) 263-292)] and those tabulated by Zhu & Bazzelli (2002) also fitted a similar (or the same) linear relationship. The C2 dehydrochlorination rate correlation curve did not however apply to dehydrochlorinatios of 3 single carbon compounds (although data for CHCl3 and CH2Cl2 dehydrochlorination were close to the correlation curve for C2 dechlorination the logA and Ea, the data points for CH3Cl (logA = 14.025, Ea 102.7 kcal/mol with 30+kcal/mol greater than that predicted by the C2 dehydrochlorination data).

Manuscript 12
Ms12 Key Refs. J.E. Leffler (The enthalpy-entropy relationship and its implication for organic chemistry) J Org Chem. 1955 20 1202-1231 L. Liu and Q.-X. Guo (Isokinetic relationship, isoequilbirum relationship, and enthalpy-entropy compensation) Chem Rev. 2001 101 673-695

D.G. Internet Enthalpy-Entropy Compensation Documents written in University of Aberdeen Research Laboratories and continued expansion updates from home [which developed ideas stemming from prior publications of D. Grant arising from various periods of academic and industrial research lab activities e.g. ref. (1) ] (1) Pyrolysis of chlorinated organic substances D. Grant (I.C.I. Ltd.) J Appl Chem Biotechnol. 24 49-58 This research confirmed the results from a previous paper (N.E. Aubrey and J.R. Van Wazer (Monsanto Co.) J. Amer Chem Soc. 1964 86 (20) that the pyrolysis of any fully chlorinated organic molecule produces: CCl4+ C2Cl6 + C6Cl6 (hexachlorobenzene) in amounts determined only by the Cl/C ratio of the starting substances (or substances) and a single equilibrium constant.

Whilst the Monsanto researchers had believed that in amounts of products obtained were solely determined by a reversible thermodynamic equilibrium between the above three substances, followingon studies done at I.C.I. Ltd. which I reported in ref (1) however suggested that this apparent equilibrium was not fully reversible and further that it was probably determined by extrathermodynamic processes involving critical rate determining (fluid phase) nucleation mechanisms. The nature of such nucleation processes were, however, not clear. Similar general nucleation process may apply widely to the initiation of numerous other types of chemical reactions and may be afforded by small nucleating particles for which the Evans Fluctuation Theorem (in which, for small particles the natural time direction of increased entropy which applies to large particles is reversed; the smaller the particles the more likely are Evans reverse entropy effects to be encountered. The ultimate such effect could arise from a mathematical point in space nucleating system. This Theorem putatively requires that preceding ideas of how chemical substances react and how such reactivity allows the complexity of biology to emerge, be re-assessed. I had previously worked in Van Wazers Monsanto St Louis laboratory where I studied, as a member of the same team which had included N.E. Aubrey (cf. ref (1)) and investigated some other (apparent) equilibration processes which can be achieved in the fluid phase. The realm of no-mechanism or where-mechanism-is replaced-by-reorganization-equilibria- chemistry was reviewed by J.R. Van Wazer in 1962 (ref. 1a). (1a) J.R. Van Wazer. Amer Scientist. 1962 50 450-472 My studies included polysulfides, polyselenides, polyphosphonates, polysulfates and ligand exchanges in organo tin compounds (cf. refs cited in (2) as well as preliminary investigations into the structural reorganization of organo-vanadium compounds and of the sealed-tube chlorination of organic molecules by sulfuryl chloride (these preliminary results suggested that similar (quasi) thermodynamic principles also dictated the outcome of these reactions). Numerous systems like the carbon chlorine system could be fitted to (quasi) thermodynamic-equilibrium-based-models which allowed the outcome of the observed reorganization processes to be rationalized). A similar paradigm applied also to the polysilicate esters a research into this conducted at Monsanto, Ruabon, Wales, UK (and assisted by the University of Keele) was reported in ref. (2). (2) D. Grant. J Inorg Nucl Chem. 19667 29 69-810. Another research topic from my time at the Monsanto research facility in Ruabon was the selfassembly of silica sol particles. Aspects of this process which benefited from the ability of seeds of specifically microstructures silica sol particles, surprisingly seemed to resemble biological reproduction. The overtly nucleation-determined processes of the seeding of silica sol formation were the basis of novel methods of preparing silica sols (ref. (3)) (3) Patents British Pat 1,134118 and 1143019 (Chem Abs. 70 P50947e and P98389b) . Cf. also D Grant et al. Med Hypotheses 1992 38 46-48 and ref. (4) (4) S.T. Hyde et al. Physica A 2004 339 24-33. {There is a hint (ref. (4) that the process of seeded generation of specific species of silica sols could kick start pre-biological cellular activities which might evolve using Evans reverse entropy into conventional types of biological cells}. The Hammett (and Taft ) extrathermodynamic linear free energy relationships had previously been discovered by synthetic organic chemists to gave an empirical prediction of (aromatic) chemical reactivity. Since these relationships seemed to contravene the second law of thermodynamics they have been dismissed by some chemists as being trivial and highly misleading (cf. e.g. A. E. Butler Chem Brit. 1989 997-998) Hammett and Taft factors are however closely linearly correlated with NMR (1H and 31P) chemical shift values, cf. ref (5) (5) Cf. e.g. for 1H NMR shifts of aromatic phenol OH MW Dietrich et al. Anal Chem 1966 38 11 1479-84 and 31P NMR shifts for (O)P(R)2X where R=C6H5 and CH3 and XP(R)2NP(R)2X where R = C6H5 by A. Schmidpeterand H. Brecht Angew Chem Internat Edit. 1967 6 (11) 945-946 J.E. Leffler, ref (6) (6) J.E. Leffler J. Org Chem 1955 20 1202-1231 had however earlier clearly demonstrated that Hammett equations were actually sub-class of a much more general and widely applicable-to-organic-

chemistry enthalpy-entropy correlation system. This like Hammett is extrathermodynamic meaning that it breaches the physics laws especially the 2nd law. The enthalpy entropy correlation in sets of rate constants creates to an isokinetic temperature where all reactions rate constants for different chemical molecules undergoing the same class of chemical transformation do so at the same rate as well as the general overall occurrence of compensation between pre-exponential and activation terms which tend to oppose each other tends to make reaction rates in the Hammett and related enthalpy-entropy compensated series of chemical substances to be more equal and therefore tends to increase the likelihood of stochastic processes occurring therein. My ICI Ltd. CIRL researches had included a discussion with the then scientific leaders in this field P.J. Thomas a co-worker of Allan Maccoll (UCL) who had established that the transition states formed in the pyrolysis of chlorinated organic substances involved the formation of some kind of unconventional (a partially activated carbonium ion). This idea was later extended (cf. ref (7)) (7) E. Uggerud et al (Eur J Mass Spectrom 200 6 131-134 a paper which was dedicated to the memory of Allan Maccoll) who established that alkyl group substituent reactivity effects were linearly correlated with Taft -values. This implies that enthalpy-entropy compensation is an inherent component of what allows this kind of chemical reactivity classification to arise in these additional aliphatic classes of reactions. Whilst Thomas et al. had conceded that some form of linear correlation between logA and Ea values often showed up in rate constants for series of related chemical reactions this was not the case for true homogeneous gas phase dehydrohalogenation of organic molecules for which the logA values were most often found to be 1013sec-1 (the thermal activation collision number or bond vibration rate). Data from a Chemical Abstracts survey (made in 1968 using Reading University library) in connection with my I.C.I. researches had however suggested that a clear linear correlation existed between log A and Ea for all accessed published datasets. (cf. Fig. 1, below which includes not only the homogenous but also rate constants which were though to be determined by surface effects).

Fig 1 (a plot of the gas phase dehydrochlorination rate constant data) also confirms that, for supposed homogeneous gas phase processes, values of logA tend to be correlated to Ea values and also can for some conditions which create logA pre-exponentials which are considerably in excess of the value 1013 (which according to the principal Ingold-Hinshelwood older conventional view of thermal activation should not be higher than 1013sec-1 [although Blackadder & Hinshelwood (J Chem Soc 1958) had

considered that the inclusion of the energetic coupling of in phase covalent bond vibrations from distant parts of the reacting molecule could perhaps explain this effect; however this idea seems not to have been generally accepted although a variant of it appears to have been used by Linert to explain compensated rate processes occurring in solution especially aqueous solution where hydrogen bonded aggregates allow for very large unstable vibrationally coupled continua to be generated cf. (8) a fairly comprehensive 2001 review of the enthalpy-entropy compensation phenomena]. (8) L. Lui and Q.-X. Guo Chem Rev 2001 101 673-695). The above general applicability of a single correlation curve to a class of chemical reactions irrespective of supposed mechanism usually is suggestive of thermodynamic rather than kinetic control. But the underlying control process which tends to make rate constants more similar than they rationally might be supposed to be allowed to be, is, apparently, in this case the putative extrathermodynamic (in breach of thermodynamic laws) enthalpy-entropy compensation rule which is further suggested to arise from a general type of all pervasive (perhaps from e.g. vacuum zero point energy) mechanism of nucleation which is a further manifestation of the reverse-entropy characteristics of the Evans Fluctuation Theorem which proposed that the probability of reverse time entropy change (i.e., entropy tends not to increase as required by the second law) but decreases varies exponentially with (entropy)/ (time) which are extensive properties meaning that small sized (e.g. nucleation site) entities (engines) will tend (it is now postulated) to create order naturally by processes which are equivalent to reversing the time direction of the 2nd law. The enthalpy-entropy compensation effect applies to semiconductor (traps) (for both inorganic and organic including proteins). The dark currents show individual pixel generated very large datapoint point linear curves shown in Ref (9) which are analogous to those indicated in Figs. 1 (above) and Fig. 2 (below). (9) R. Widenhorn et al. J Appl Phys. 2001 89 (12) 8179-8182) Enthapy-entropy compensation apparently controls the nucleation of helix coil (phase) transition in proteins (cf. ref (10) (and putatively also is widely applicable to the phenomenon of protein folding). (10) O.K. Vorov et al. Biophys J 2009 97 (11) 3000-3009 Fig. 2 shows the effect of inorganic counterion dependent (putative water structure generation related? enthalpy-entropy correlation in rate constant transition states) (data accessed from literature obtained during my University of Aberdeen polysaccharide group researches) enthalpy-entropy compensation and the and associated isokinetic T (= ca. 20oC) is apparent in the rates of nucleated phase change processes of marine algal biological anionic polysaccharides, [each point is for different inorganic counterion conditions]. The (enthalpy-entropy controlled supramolecular structuring) which occurs with of the marine algal polysaccharides which are of analogous structure to the mammalian anionic polysaccharides suggests that similar enthalpy-entropy controlled nucleation may putatively also control nucleation of structure therein and hence may be a driving force for animal development and evolution. The data plotted in Fig 2 are for kappa carrageenan ( points) from K.R.J. Austen et al. Biopolymers 1988 27 139-155 p 150 and Table II; the other points are for iota carrageenan I.T. Norton et al. J Chem Soc Faraday Trans 1983 79 2501-2515 Fig. 2 (n.b. 4.1859J=1cal)

Enthalpy-Entropy Correlation During Algal Polysaccharide Phase Change

The binding of counterions to heparin (mimetic of the heparan sulfate side chain of heparan sulfate proteoglycans (a prime orchestrator of growth factor activities and other determinants of animal cellular activity and achievement of morphology during tissue homeostasis, wound healing (and also putatively embryo assembly etc.) apparently achieves selectivity at least partly via a similar nucleation of supramolecular structure to that shown by the alginates illustrated in Fig.2. A simple electrostatic binding (at least for most cations with a possible exception of Mg2+) the previously held hypothesis of such binding (the Manning electrostatic mechanism) was not confirmed, instead the binding process more resemble a phase change mechanism of a similar character to that illustrated by Fig. 2. These are likely nucleated processes; such compensated nucleation processes putatively allow Evans reverse-time entropy to generate order (which in apparent breach of the classical laws of physics permits allow animal evolution to take place using reverse entropy by which animal development programs can be adapted to optimize the organisms most effective interactions with the environment. The involvement of water aggregates in the above nucleation process is suggested by the cation dependence of water cluster association with heparin. (This may conform to the Linert hypothesis further developed by Luo and Guo ref. (8) ). (Cf. also ref. 11) (11) Papers by D. Grant et al. listed in the W.F. Long and F.B. Williamson Aberdeen University group publications available at http://www.abdn.ac.uk/~bch118/publicationsmarch.doc cf., e.g., D. Grant et al. Biochem J 1992 285, 477-480; ibid., 283 243-246, 282 601-604; Seeding of crystal formation was found to be inhibited by heparin and heparin-like substances which apparently adsorbed onto seed crystal surfaces. This caused not only the blocking of crystal growth but also altered the morphologies of the inhibited crystals which were permitted to grow. Cf. ref (12, 12a) (12)

D. Grant et al Biochem J., 1989 259 41-45. This may permit endogenous heparin to protect tissues against the pro-inflammatory effects of pathologically formed crystals (e.g. at blood vessel walls, (12a) D. Grant et al. Med Hypotheses. 1992 38 49-55. Enthalpy-entropy compensated sets of equilibrium and rate constants show up throughout physics, chemistry and biology. This seems to suggest some alternative paradigm which allows for a classification of natural phenomena which challenges the system of classical thermodynamics. The similar challenge to classical thermodynamics posed by the Evans Fluctuation Theorem, Cf. Wang et al. (ref. 13) (which proposes that reverse time flow entropy increase with positive direction of time. Is an inherent property of small sized engines) is now suggested to allow a new paradigm of chemical reactivity especially as it applies to biology to be developed. (13) G.M. Wang et al. Phys Rev Lett. 2002 89 05060.

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Redefining the Nature of Chemical Reactivity.

D.G. June 2012

An Alternative Hypotheses of Causality, the Nature of Thermal Activation Chemical Reactivity and Reversibility in Chemical Equilibria ( suggested to be required by the Evans Fluctuation Theorem which suggests a need for the revision of the classical concept of chemical reactivity).

Consider the previously-described but not-previouslyunderstood extrathermodynamic enthalpy-entropy compensation phenomena where in sets of rate constants [kr = A exp Ea/RT] the observed Ea values demonstrate a direct proportionality with the observed log A values. If this enthalpy-entropy compensation process arises not from reactions which are untypical of most chemical reactions, but by a mechanism which is of a more general nature than one which is restricted only to the individual (similar process) sets of rate process and actually applies universally and includes all chemical systems, then it is obvious that the reason for a general enthalpy-entropy compensation must also be that which defines the ultimate nature of chemical reactivity. But new thinking seems to be required to re-define how present views of thermal energy and how they rate processes depend on temperature must it seems be revised to accommodate the above idea.

It is now proposed that a process of general compensation between enthalpy and entropy exists through nature (in agreement with how this has been reported to show up in numerous but restricted sets of rate processes in physics chemistry(1) and biology(2)) and that the origin of this general enthalpy-entropy compensation is a consequence of the existence of a reversal of internal motions in transition state complexes which occur by reverse entropy generation according to the Evans Fluctuation Theorem [a probability which is equal to exp. (entropy).(time)] . It is further suggested that such motion turnaround actions affect all of the operations (within the transient interaction short lived adducts [consisting of activated colliding particles or inter-reacting chemical molecules of a similar nature to the transition state complexes postulated to occur in this theory of chemical reactivity]). All thermally induced chemical reactivity must then be subject to the operation of the Evans Fluctuation Theorem [which requires that, especially for small sized entities over small time intervals, that the second law of classical thermodynamics which requires that entropy increases with time, be replaced by a more complex process, in which entropy also simultaneously decreases with time within the same molecule] which predicts a significant probability exists for an actual direct reversal of normal internal motions equivalent to an actual reversal of time. This shock it is now suggested, must apply to any kind of (albeit hitherto mostly hypothetical) adducts which are thought to be formed between colliding particles, (the traditionally considered origin of thermal energy) as well as to the

transient transition state complexes (which are commonly considered to be the origin of chemical reactivity). I.e. the Fluctuation Theorem applies widely throughout physics and chemistry and it holds with equal validity for the collision processes which creates heat and to the interaction adduct which cause molecules to react chemically. I.e. the outcome of thermal heat arising from the general motion and collision of particles and the chemical transformation of such colliding particles are both directly consequent on the Fluctuation of the direction of time within the activated complexes formed when particles collide. This new model of gas thermal heat relationship and the transition state behavior suggests that attention should be directed to what may happen during any real turn-around reversal in motion within the transient transition state complexes and during their decay which must greatly alter translations, vibrations and rotations and this per se be what produces the phenomenon of chemical reactivity).

A revision is required in the traditional idea that all enthalpy-entropy compensation process arise simply from the operation of a classical thermodynamics compatible solvent effect (as previously proposed(1), this effect is thought to especially apply to liquid water which allows generally observed biological compensation processes to arise(2)). This hypothesis can now be extended by allowing the definition of what constitutes a solvent to include (a new concept the effect on molecules of the existence of) vacuum state energy systems which is now proposed to be equivalent to a chemical solvent system (more or less an universal solvent for matter); operation of vacuum energy transfer from such a virtual solvent can be considered to allow gas state reactions to generate sets of fully compensated rate constants (e.g. those reported for chorinated aliphatic molecule pyrolysis show experimentally well-defined enthalpy-entropy compensation). Hence the proposal that the Fluctuation Theorem applies equally to organic reactions conducted in a solvent as to those gas state reactions which show well defined enthalpy-entropy compensation also seems to require a revision of the understanding of the processes by which matter and the vacuum space interact. It seems to be required to consider the possibility that vacuum energy processes can contribute to the suggested required revised nature of transition state complexes or collision adduct interactions and these must be included as being the far-reaching implications stemming from the Evans Fluctuation Theorem.

Cf. Leffler(1) attempt to explain enthalpy-entropy compensation on the basis of classical thermodynamics, must be re-interpreted.
It is now indicated that vacuum energy could overlap into transition state complexes or collision adducts and this overlap may

determine the Free Energy (Fo) relationships (expressed in the following equations) which link the standard free energy of activation change Fo with both an equilibrium constant K and the rate constant kr RTlnK = -Fo;
and H = Ho + S where is the isokinetic temperature.

RTlnkr = -Fo + RTln kT/h

(1)Cf. J.E. Leffler (The enthalpy-entropy relationship and its implication for organic chemistry) J Org Chem. 1955 20 1202-1231 (2) L. Liu and Q.-X. Guo (Isokinetic relationship, isoequilibrium relationship, and enthalpy-entropy compensation) Chem Rev. 2001 101 673-695

Manuscript 14

Ms 14

Consider the Solubility of Sparingly Soluble Phases in Natural Waters, And the Role of Nucleation, Classical Thermodynamics and Reversibility of Dissolution Processes
The ubiquitous present of humic polyanions in the aqueous and atmospheric environments prevents the attainment of thermodynamic equilibria which might otherwise determine the reactivity of sold phases. DG May 31 2012 The Supersaturation of (Sparingly Soluble) Salts exposed to the environment. Solubility is an ideal concept. It can be suggested that, from a thermodynamic viewpoint, the lack of ideality in commonly encountered natural systems prevents the use of any straightforward thermodynamic considerations for the elucidation of the behavior of such systems. (It can be added, as a corollary, that any general use of thermodynamics for the understanding of general chemical phenomena is also an ideal concept which should only be applicable under ideal, (essentially unreal) situations). This is to a large extent perhaps because natural systems are rarely pure substances. Pure substances behave differently from natural pure (ideal) chemical systems; e.g. the impurities which they contain greatly alter their reactivities. Chemical activity-determining impurities include those humic substance types which are easily transferred from the environment onto the surfaces of any inorganic phase which is exposed to naturally occurring aqueous solutions or to the atmosphere. Inorganic phase surfaces can be effectively deactivated by the build up of surfaces containing humic matter [n.b., this is the largest class of chemical substance in the biosphere]. The catalytic activity of natural biosphere-connected inorganic phases become modified by the occurrence of humic matter chemical modification of their surfaces. A further type of humic matter is formed during the thermal polymerization of a range of common roganic substances (e.g. the heterogeneous (e.g. carbonized) surface coating which can arise from the thermal decomposition of organic substances (e.g. on silicate surfaces).

A coat of a single layer of humic matter can completely deactify solid phases. However, chemical treatment of such deactivated surfaces which removes the humic matter layers can render them catalytically active. Ideal surface and solution conditions are required for classical thermodynamic solubility concepts to be valid (because the dissolution and the formation (precipitation) of solid phases is regarded as a reversible equilibrium between the solid phases and the dissolved ions or molecules which are contained in such phases). However while thermodynamic reversibility may describe the attainment of solubility at surfaces of clean phases in pure solvents (which may pertain to laboratory environments) the common occurrence of surfaceactive humic polyanions in natural waters and in the air changes the concept of solubility from a process to for which thermodynamic principles can be applied to a process which is essentially extrathermodynamic in nature and is determined to a large extent by kinetic rate determining processes. These kinetic processes may, however, be subject to extrathermodynamic enthalpy-entropy compensation restriction rules. The common presence of surface polyion coatings could also be part of the mechanism by which this compensation process is achieved The following additional hypothesis can also be suggested: where chemical reactivities are dependent on the catalytic assistance of polyioncoated surface phases act as catalysts, then the humified or mixed organic matter substance thermal decomposition products or other surface active adsorbant modified surfaces determine the kinetic parameteres of reactions which are facilitated by these surfaces. Could this then cause the rate constant expressed as kr=Aexp Ea/RT, to show a common humic matter (or related substance) generated enthalpy-entropy extrathermodynamic rule (which is seen most clearly in sets of related reactions where the activation energy Ea seems to become dependent on the log A value; the apparent correlation process may be because of specific abilities of the humic-like surface polyanion to coat and control the surface activity in such a manner to ensure that the usual thermodynamically concepts involving gas phase thermal energy and transition state complex formation are replaced by the requirement for an extrathermodynamic enthalpy-entropy polyanion-determined rate law. It should be noted that surface processes which are known to generate log A Ea compensation including surface tension related Etvs factors show entropy-enthalpy compensation (cf. Campbell & Eley) at the surfaces of liquids.

The elucidation of the behavior of sparing soluble substances* in natural waters depends on interactions between phase boundaries and natural polyanions (humic and fulvic acids) which can act as potent inhibitors of the attainment of the (thermodynamic) solubility equilibria which creates supersaturated natural poly inorganic ion solutions. The sea is the largest such example. The biological fluids which are approximately seawaterlike, include human blood serum. Other natural polyanions, present in the extracellular matrices and cell surfaces of multi-cellular animals and plants include the anionic polysaccharides, which like the humic and fulvic acids of natural water and atmosphere also contain arrays of carboxylate anionic groups (as well as more strongly ionized sulfate as with the glycosaminoglycans). These polyanions (via a process which can be considered as a smart biological inhibition of thermodynamic potential energy) can be applied both to prevent the formation of sparingly soluble inorganic phases (e.g., to prevent the deleterious formation of pathological plaques and stones) or to conceptually use the thermodynamic potential to act in the controlled manner so as to create skeletal structures. The crystallization of CaCO3 (from natural waters including biological intracelluar fluids) is thought to critically depend on the nucleation of crystallization by CaCO3 crystallites which act as seeds for the growth of larger CaCO3 crystals. The (e.g. potent ability of humic polymers to greatly inhibit this nucleating process also prevents the attainment of the (thermodynamic) equilibria which would otherwise potentially arise under the idea conditions of the presence of clean CaCO3 crystallite nuclei surfaces.
The attainment of thermodynamic reversibility between CaCO3 crystals and an aqueous solution containing Ca2+ and CO32- ions becomes impossible when the surface of CaCO3 is replaced by of another phase system consisting of the complex formed between the adsorbed polyanion and the clean crystal surface. The thermodynamic and kinetic properties of this new phase then becomes of critical relevance for a proper understanding of (apparently altered) chemical reactivity of the bulk nearCaCO3 stoichiometry phase.

*e.g. CaCO3, BaSO4 as well as the aggregation of Hg(II) sulfide in natural waters seems to be dependent on the ability of natural dissolved organic matter (humic polymers with abundant carboxylate anionic groups) inhibition of the nucleation of crystallization. 14.a Could the Notion that Nucleation Inhibitors Cause Apparent NonThermodynamic Behavior be Extended to Suggest New Thinking about General Extrathermodynamic Phenomena?

The idea that ideal thermodynamic solubility relationships become invalid under normal non-ideal situations can be applied to the reassessment of how, in chemistry, general rate processes may commonly occur via non-thermodynamic behavior (e.g. those in which a general enthalpy-entropy compensation process which is not predicted from classical thermodynamics applies) which may ultimately be caused by the non-thermodyamic control of the nucleation activity of phase boundaries such as vessel surfaces and small colloidal dust particles etc. which conceivable might behave as nucleation sites under common natural environment conditions. A complex surface assisted process, putative of the type discussed above was observed for aqueous solution phase cerium(IV) thermal decomposition to Ce(III) via oxidation of water molecules. This reaction had been previously not been observed when Ce(IV) sulfuric acid solutions were employed as volumetric reagents for the oxidative determination (e.g. of P-H bond containing substances) which had been considered to occur by straightforward (thermodynamically controlled) homogeneous solution phase biomolecular rate-determining processes. Fresh glass surfaces seemed capable of initiating chain reactions but these surfaces quickly became deactivated by insoluble reaction products allowing reaction to proceed for a short time span over which surfaceinitiated free radical chain reactions produced the observed kinetics. The idea that water impurities or surface imperfections or coatings might offer a universal catalytic surface system for gas phase dehydrochlorination reactions tabulated above is also credible. A marked log A vs. Ea linear correlation is observed for all reported experimentally determined rate constants for this class of reaction. The same curve described all rate processes irrespective if the mechanism was believed to be a homogeneous gas phase unimolecular decomposition (thermal activation by collision of gas molecules), by surface reaction or by radical chain propagated processes in the gas phase. Gas-phase supposed homogenous reactions seem also to be surface assisted. The deposition at glass or silica vessel surfaces of polymeric substances seems to alter the course of gas phase chemical reactions. This is often assumed to be a surface passification process in which new vessels require in order to be used to yield reproducible perhaps homogeneous gas phase kinetic results but may be itself have unsuspected critical impact on the course of chemical reactions.

For industrial scale organic chemistry the use of metal vessels e.g. of stainless steel, was found to produce a different type surface boundary effect which was generally much more reactive than that seen at silicate surfaces. For reactions involving chorine or hydrogen chloride the formation of surface chlorination products seems to create highly efficient free radicals production sites. These could dominate the observed reaction kinetics. Although use of methods of stopping free radical chain reactions were believed to have eliminated these reaction pathways in the decomposition of chlorinated aliphatic substances, the existence of any genuine unimoleuclar decomposition pathway for thermal decomposition (dehydrochlorination) of such molecules began to appear doubtful as suggested by the following list compiled in the 1950s at the University of Glasgow; this suggested that some at least of the alkyl chloride thermal decompositions occurred by both free radical chain (R), and unimolecular (U), decomposition mechanisms: Substance R : free radical chain reaction U: unimolecular decomposition 1,2 dichloroethane 1,1,2 trichloroethane 1,2,2,2 tetrachloroethane ethyl chloride 1,1 dichloroethane 3yBu chloride 2 chloropropane 1,2 dichloropropane 1,1,1 trichloroethane 1,1,1,2 tetrachloroethane n propyl chloride n butyl chloride 2,2/ dichlorodiphenylethane isobutyl chloride 1,4 dichlorobutane 1,1 dichloropropane 2,2 dichloropropane R R R U U U U U U U U R R R + + U U U U

R R R

+ +

Since the logA and Ea kinetic parameters from the above set of reactions as well as for all other similar dehydrochlorination reactions, obey the

same compensation curve between enthalpy and entropy (demonstrated by plotting log A vs. Ea for the different starting products) indicates that while different reaction mechanisms may be involved (and undoubtedly include some arising from rate-determining humic-wall-coats; it can be suggested that if any such processes are rate-influencing and the actual reaction mechanism consists of complex interdependent stages then each stage must show logA vis Ea compensation and this leads to the occurrence of net logA and netEa compensation in the overall expeeriemental rate process). Any series of dehydrochlorination reactions proceed by rate processes which are also all interlinked by the basic overall outcome, i.e. the overall stoichiometric equation. Which this stoichiometry may be attained by a complex multi-stage surface catalyzed process, nevertheless the overall enthalpy and entropy rule (e.g. during the creation of the perhpas several transition state complexes or the equivalent collision activation process) are all mutually compensated. This logically requires signaling back from future outcomes to achieve such a linkup This indicates that what links these reactions together is a chemical logic which is actually is an assessment of the most probable future outcome (i.e. the future formation C=C olefins from starting substances with a C-Cl bond ) [n.b the rate compensation phenomenon does, however not apply to C1 dehydrochlorinations in keeping with the idea that it is >C=C< driven]. Future events then apparently dictate the overall relatedness of the (possible several interlinking processes which generate an extrathermrodynamic law which applies to Cn>1 chloride dehydrochlorination and (similarly also to C=C isomerisations of Clcontaining olefins). Actual rate determining (perhaps heterogeneous) transition state complexes all lead to the (future) formation of >C=C< from HC-Cl (this includes chlorinated alipahtic molecule dehydrochlorination as well as chlorinated aliphatic substance isomerisation) in which the set of log A values are not restricted [as required by simple application of classical thermodynamics-based theories to a value of ca. 13.5] but vary over a wide range of values e.g. 2-20 (for kr sec-1 (apparent unimolecular rate processes) and even if the actual value is influenced by the surface polyion catalyst assisted processing) the (overall) value of Ea for any particular starting substance in the set is determined solely by the value of log A (which requires to obey the Leffler isokinetic relationship logA = Ea, [where is the isothermal temperature at which all reactions in the set occur at the same rate]). Reverse Time Theory The Fluctuation Theorem (Evans 1992, cf. Yang et al. 2002) proposed that for small entities over small time scales reverse time flow will occur to a varying extent according to the probability ratio of forward vs.

reverse time = exp. [entropy] . [time]. This time reversal was proposed to be exponentially size-related so that only forward time need be considered for larger particles such as the machines for which classical thermodynamics was designed, but for small-sized, short lifetime entities such as typical chemical molecules and transition state complexes these are now predicted to experience a real reverse time processing. This may be a critical requirement for all chemical reactivity. It is also then the ultimate origin of the enthalpy-entropy compensation phenomenon observed in sets of (chemical logic future state probability) rate constants. For individual series of chemical reactions (irrespective of the reaction mechanism) the products of such reactions can be considered to feedback virtual information (as it were from the future) into the series of their transition states so that the precise rate by which these products can be generated is achieved by a balance between the overall observed logA and Ea values. These entropy and enthalpy changes are then not independent variables but are determined by how matter interacts with virtual time in the virtual space of the transition state complex where the effect of temperature on a virtual conjoined chemical potential [the enthalpy-entropy] is determined by such a compensation process. This redefines what chemical reactivity actually is.

______________________________________________ ______________________________________________ _____________________________________________

Other Manuscripts
DG Note 27/4/12

15. Van Wazer Structural Reorganization

A review (which was originally drafted in 1975) of ideas generated from my participation in J.R. Van Wazers Structural- Reorganization-Throughout-The-Periodic-Table research program (which had been offered but not published or otherwise put to use) is now re-formatted below.

---------------------------------------------------------------------------------------SCRAMBLING REACTIONS David Grant, B.Sc., Ph.D. During the latter part of the eighteenth century, Berthollet and Proust debated the constancy of the combining proportions of elements in chemical species, (1). Berthollet recognized the importance of equilibria, regarding constant proportions as being the exception. There is a grain of truth in this, e.g., polymeric and amorphous species can exhibit variable compositions, and, while numerous compounds including those based on carbon backbones have a high probability of remaining unchanged for a long time, some substances having central atoms other than carbon at structural centers are intrinsically of lower stability than are purely organic molecular species. This applies to the liquid phase which can facilitate rapid molecular rearrangements to occur (and most especially if this process is fast with respect to the time required to separate the individual molecular species then the scrambled product is what is most commonly encountered). This scrambling, may, however, not be detected by vibrational spectroscopy or by X-ray diffraction. Numerous reaction products, previously though to be single compounds have been found to be the scrambled mixtures which, if at equilibrium will exhibit some reproducible physical properties in a manner similar to that of pure compounds. The most general form of scrambling was established by Van Wazer, (2) (ca. 1955) who extended the earlier concepts of Flory, and demonstrated that equilibria of which the following is typical, are established:
(RO)2>P(O)(OR) (neso) + -O-P(O)(OR)-O- (middles) 2 O-P(O)<(OR)2 (ends)

(further equilbria are usually measured to distinguish the middles in rings from those in chains, and the ends on long chains from those on short chains. In the phosphate-condensed phosphate systems, branch groups also take part in equilibria like : ends + branches 2 middles).
The Van Wazer method of characterization of chemical systems is useful for the elucidation of the products of synthetic organometallic chemistry where conventional organic chemical logic fails to properly identify the substances formed in any attempted stepwise synthesis of an inorganic molecular structure or a mixed inorganic-organic element (e.g. B, P, As, Si, etc.) - containing derivative.

An example of former situation arose is an attempt to synthesize an isotetraphosphate structure. While a composition which appeared to contain this structure, as suggested by some physical measurements etc. seemed to have been produced, a later re-evaluation by NMR showed that this structure, if formed initially, had completely rearranged to produce a reproducible mixture, the composition of which could be exactly predicted using the scrambling equilibrium hypothesis suggested by Van Wazer, (3). Such rationalizations were similarly useful for arriving at the correct chemical constitution of a product arising from attempts to prepare octamethyltetraminopyrophosphate (((CH3)2N)2P(O))2O) (OMPA) (cf. 3a) (the reaction product, however showed the NMR spectrum of a completely scrambled mixture of the same stoichiometry. A similarly completely scrambled mixture also apparently arose from attempts to prepared specific polysilicate esters, e.g. RO-(Si(OR)2-(OSi(OR)2)n-O-Si(OR)2-OR (where R is an organic group).

Scrambling was first recognized as an important phenomenon in tetra-coordinate Pb chemistry by Callingaert and Beatty in 1939, (4). Ethyl vs. methyl groups were found to scramble , near-randomly, on Pb and some other metal centers. Flory (5), considered that an equilbrium middles + ends middles + ends occurred between groups in polyester chains. Forbes and Anderson observed the random sorting of Cl vs. Br on C< (6) and on other centers.
Many cases, for a wide range of ligands and centers, are now known to exhibit scrambling behavior. Classification into near-random or highly non-random systems is often helpful. Many systems are near-random, e.g., halogen exchange on -V(O)< (7), -P(O)< (8 ), Ti (6 co-ord.) (9), -Hg- (2 co-ord.) (10), >Ge< (4 co-ord.) (11), >Sn< (4 co-ord.) (12), >Si< (4 co-ord.) (13), -B< (3 co-ord.) (14), [and also, e.g., RnMX4-n (R = alkyl etc., X = halogen, M = Ge, Sn, Si, etc.)]. Recent interest has been shown in CO exchange in cluster compounds, (15); different sites may exhibit markedly different exchange rates. Cf., Fe3 (CO)12 scrambling (15a). Examples of scrambling in polymer systems are: in polysiloxanes, (16); -silicates, (17); -sulphates, (18); -sulphones, (19); -sulphides, (20); -selenides, (20); -borates, (21); -phosphates, (22); -phosphonates, (23); -ethane, oxy,diphosphonates, (24); -arsenites, (25); -germthioxanes, (26); -(fluoro)arsenious methylimides, (27) and -tin sulphides, (28). Scrambling (at ambient temperature), of alkoxyl vs. Cl groups on -V(O)< is some 107 time faster than on -P(O)< but both systems are similarly random, (19). An example of scrambling on V(O)< (V(O)Cl3 / (CH3)3Si-O-Si(CH3)3) is shown in Fig. 1

Fig.1

[R=2Si/(V+2Si)]

The greater rates of scrambling on transition-metal centers may give rise to fluxional activity (rapid, often intramolecular NMR-detected scrambling) (29). The use of transition-metal compounds as catalysts for organic reactions, e.g., in hydrogenation, isomerization and polymerization, seems to be afforded by the scrambling potential at the metal, e.g., scrambling-related polymer generation arises from the -olefin monomer additions to the metalcentered scrambling centers which are the active sites of the Ziegler-Natta polymerization catalysts. In

the polymer chain propagation process the transition-metal forms metal-carbon bonds into which bonded olefinic bonds insert. This is likely to be a reversible (equilibrium) reaction, but is far displaced towards the polymer form at a polymerization temperature of ca. 70oC (30). Whether isotactic or stereospecific polymers arise during this insertion process seems to be dependent on the rate of structural reorganization of metal bound co-ligands. A further example of organic reaction catalysis at metal centers is the metathesis of olefins at W and Mo centers, which leads to an apparent scrambling of the alkyl groups about the double bond, (31). Scrambling may be catalyzed both positively or negatively, e.g., phosphorus halides scrambling is accelerated by H2O (32); for boron halides, scrambling is inhibited by bases, (33). There is probably no single type of mechanism which can explain scrambling reactions. Under scrambling conditions a mechanistic approach to the rationalization of chemical reaction products obtained at sub-scrambling temperature, so useful for organic reactions at near-ambient temperatures, is much less helpful for achieving an understanding of scrambling process. The important criteria are now knowledge of the equilibria (perhaps more accurately described as [enthalpy-entropy compensated] extrathermodynamic pseudoequilibria) which govern this behavior, (34). A general (first order approximation) principle of scrambling is that the type of equilibrium (e.g. random or non-random) is dependent on the ligands and is independent of the sites; the rate of scrambling, however, depends on the site, (35). Carbon is the slowest site. Scrambling of hydrocarbons (e.g. at 1000oC) gives CH4, C2H6, C3H8 and char (closed system). Phosphorus hydrides behave similarly (char now being red phosphorus), as do silicon hydrides, (36). The outcome of high temperature scrambling behavior of hydrocarbons is derivable from the accurate thermodynamic data which is available for these compounds, and the above scrambled corresponds to a thermodynamic equilibrium. However, at lower temperatures, reversible exchange between aliphatic and aromatic carbon-based systems, occurs. These are likely to be pseudo-equilibria, where the forward and reverse rates are not quite equal, and proceed by different mechanisms; this situation is illustrated by the scrambling of chlorocarbons in sealed-tubes, where all chlorocarbons which have been studied give CCl4, C2Cl6 and C6Cl6, with inappreciable char formation, (37). The phenomenon of aromaticity may be considered to be a double bond scrambling around the ring. Negative catalysis (Fe(CO)3 complexing sites) slow the process sufficiently for individual cyclohexatrienes to be distinguished, (38). In boranes and related compounds, a variety of BHB scrambling reactions have been reported , e.g. with B3H8 , (39),

Silicates scramble near-randomly, (17), therefore giving rise to appreciable amounts of a large number of structures. Nucleation of crystallization of a particular structure can remove it form the equilibrium, eventually converting all of the molecules present. [Bond exchange also can lead to flow in compositions beyond the gel point. In inorganic polymers such as silicates bond exchange can lead to flow whereas flow in organic polymers flow most often involves the sliding of the intact molecules over each other].

The formation of quite complicated structures in high yield in pre-biotic conditions could have been an outcome of scrambling reactions and subsequent nucleation of specific sub-types of molecules allowing the formation of specific proteinoid and poly-sugar-triphosphate molecules to be achieved in high yield.

References (1) -Cf. A Short History of Chemistry , J.R. Partington, Macmilllan, London, 1957, p. 153 (2) Phosphorus and Its Compounds Vol I, J.R. Van Wazer, Interscience, New York, 1959; C.F. Callis , J. R Van Wazer, J.N. Schoolery and W.A. Anderson, J Amer. Chem. Soc., (1957),79, 2719; J.R. Van Wazer, C.F. Callis and J.N. Schoolery, (1955), 77, 4945 (3) E. Schwarzman and J.R. Van Wazer, J. Amer. Chem. Soc., (1960), 82, 6009 (3a) cf., e.g., J.R. Van Wazer, Amer. Scientist, ref. (36) (4) G. Caligingaert and H.A. Beatty, J. Amer. Chem. Soc. (1939), 61, 2748 G. Calingaert, H.A. Beatty and H.R. Neal, ibid., (1939), 61, 2755 G. Calingaert and H Soros, ibid., (1939), 61, 2758 G. Calingaert, H.A. Beatty and H. Soroos, ibid. (1940), 62, 1099 (5) P.J. Flory, J. Amer Chem. Soc., (1942), 64, 2205 (6) G.S. Forbes and H.H. Anderson, J. Amer. Chem. Soc., (1944), 66, 931 (7) R.J.H. Clark and P.D. Mitchell, J.Chem. Soc. Dalton, 1972, 2429 (8) L.C.D. Groenwege and J.H. Payne Jr., J. Amer Chem. Soc., (1959), 81, 6357 (9) P.A.W. Dean and D.F. Evans, J. Chem. Soc., A, 1970, 2569

(10) M. D. Rausch and J.R. Van Wazer. Inorg. Chem. (1964), 3,761 Cf. J.C. Lockart, Chem. Rev., (1965), 65, 131 (11) G.M. Burch and J.R.Van Wazer, J. Chem. Soc. A. 1966, 586 cf. Inorg. Chem., 1964, 3, 268 (12) J.J. Burke and P.C. Lauterbur, J. Amer. Chem. Soc. (1961), 83, 326; G.S. Forbes and H.H. Anderson, ibid., (1945), 67, 1911; (1944), 66, 931, G. Calingaert, H. Soroos and V. Hnizda, ibid., (1940), 62, 1107 D. Grant and J.R. Van Wazer, J. Organometal. Chem., (1965), 4, 229 (13) K. Moedritzer and J.R. Van Wazer Inorg. Chem. (1964), 3, 268 and refs. cited; cf J.R. Van Wazer and K. Moedritzer, J. Inorg. Nucl. Chem. (1964), 24, 73 (14) H.K Hofmeister and J.R. Van Wazer, J Inorg. Nucl. Chem., (1964), 26, 1209 M.F. Lappert, M.R. Litzow et al., J. Chem Soc.(A) 1971, 383; (15) L. Milone, S. Aime, E.W. Randall and E Rosenberg J.C.S. Chem. Commun., 1975, 452 T.J. Marks and G.W. Grynkewich, J. Organometallic Chem., (1975), 91, C9-12,

F.A. Cotton, D.L. Hunter and P. Lahuerti, Inorg. Chem., (1975), 14, 511. (15a) B.F.G. Johnson, J.C.S. Chem. Commun., 1976, 703 (16) K. Moedritzer and J. R. Van Wazer, J. Amer. Chem. Soc. (1964), 86, 802 (17) D. Grant, J. Inorg. Nucl. Chem., (1967), 29, 69 R.O. Gould, B.M. Lowe and N.A. MacGilp, J.C.S. Chem. Commun., 1974, 720 (18) J.R. Van Wazer, D. Grant and C.H. Dungan, J. Amer. Chem. Soc., (1965), 87, 3333 (19) Unpublished work of D. Grant and J.R. Van Wazer (20) J.R. Van Wazer, D. Grant, J. Amer. Chem. Soc., (1964), 86, 3012 (21) H.K. Hofmeister and J.R. Van Wazer, J Inorg. Nucl. Chem., (1964), 26, 1201 (22) J.R. Van Wazer, C.F. Callis, J.N. Shoolery and R.C. Jones, J. A Amer. Chem. Soc., (1956), 78, 5709 and 5715 C.F. Callis, J.R. Van Wazer, J.N. Shoolery and W.A. Anderson, J. Amer. Chem. Soc. (1957), 79, 2719 D.P. Ames, S. Ohashi, C.F. Callis and J.R. Van Wazer ibid., (1959), 81, 6350 M. M. Crutchfield, C.F. Callis, R.R. Irani and G.C. Roth, Inorg. Chem., (1962), 1, 813 L.C.D. Groenweghe, J.H. Payne and J.R. Van Wazer, J. Amer. Chem. Soc., (1960), 82, 5305 E. Schwarzmann and J.R. Van Wazer, ibid., (1961), 83, 365 D.R. Cooper and J.A. Semlyen, Polymer, (1972), 13, 414 J.R. Van Wazer and S. Norval, J. Amer. Chem. Soc., (1966), 88, 4415 L.C.D. Groenweghe and J.H. Payne Jr., J. Amer. Chem. Soc., (1959), 81, 6357 (23) D. Grant, J.R. Van Wazer and C.H. Dungan , J. Polymer Sci., (1967),A-1,5, 57 (24) Unpublished work of D. Grant (Glasgow University manuscript in preparation [(added later: eventually published in Eur. Polym. J. (1979), 15, 1161)] (25) J.R. Van Wazer, K. Moedritzer and D.W. Matula, J. Amer. Chem. Soc., (1964), 86, 807 (26) K. Moedritzer and J.R. Van Wazer, J. Amer. Chem. Soc., (1968), 90, 1520 (27) M.D. Rausch, J.R. Van Wazer and K. Moedritzer, J. Amer. Chem. Soc., (1964), 86, 814 (28) K. Moedritzer and J.R. Van Wazer, Inorg. Chem., (1964), 3, 943 (29) F.A. Cotton, Chem. Brit., (1968), 4, 345 Cf. S. Cradock, E.A.V. Ebsworth, H. Moretto and D.W.H. Rankin, J.C.S. Dalton, 1975, 390; A.J. Campbell, C.A. Fyfe and E. Maslowsky Jr., Chem Commun., 1971, 1032; P.C. Angus and

S.R. Stobart, J.C.S. Dalton, 1973, 2374 (30) Cf. D. Grant , J Polymer Sci., Polymer Letters , (1975), 13,1 (31) Cf. N. Calderon and R.N. Hinrichs, Chemtech., (1974), 4, 627 E.L. Muetterties and M.A. Busch, J.C.S. Chem. Commun., 1974, 754 and refs. cited; A.J. Amass, Br. Polymer J. (1972), 4, 327

(32) A.D. Jordan and R.G. Cavell, Inorg. Chem., (1972), 11, 564 (33) B. Benton-Jones, M.E.A. Davidson, J.S. Hartman, J.J. Klassen and J.M. Miller, J.C.S. Dalton, 1972, 2603 Cf. M.J. Bula, J.S. Hartman and C.V. Raman, ibid., 1974, 725 (34) D.W. Matula, L.C.D. Groenweghe and J.R. Van Wazer, J Chem. Phys. (1964), 41, 3105 R.M. Levy and J.R. Van Wazer, ibid., (1966), 45, 1824 L.C.D. Groenweghe, J.R. Van Wazer and A.W. Dickenson, Anal. Chem., (1964), 36, 303 J.R. Van Wazer and K. Moedritzer, Angew. Chem. Internat. Edit., (1966), 5, 341 K. Moedritzer, Inorg. Chim. Acta, 1970, 4, 613 J.R. Van Wazer and L.C.D. Groenweghe, Nuclear Magnetic Resonance in Chemistry, B. Pesce, Ed., (1965), 283 J.R. Van Wazer Inorganic Polymer Chemistry J. Macromol. Sci., 1967, A1, 29 J.C. Lockart, Chem. Rev., (1965), 65, 131
Note added to ms. later: the Van Wazer scrambling phenomena are likely to be afforded by extrathermodynamic pseudoequilibria associated with enthalpy-entropy compensation phenomena, the general occurrence of which throughout biology, chemistry and physics putatively requires a re-think of classical thermodynamics, an e.g. involves application of reverse time and vacuum energy concepts ]

(35) J.R. Van Wazer, Proc. Conf. Coord. Chem., 8th, Vienna, 1964, Springer-Verlag, Vienna, Ed. V. Gutman, p.162 (36) J.R. Van Wazer, Amer. Scientist, (1962) , 50, 450 (37) N.E. Aubrey and J.R. Van Wazer, J. Amer Chem Soc., (1964), 86, 4380 D. Grant, J. Appl. Chem. Biotechnol., (1974), 24, 49 (38) R. Victor, R. Ben-Shoshan and S. Sarel, Chem. Commun., 1970, 1680 (39) H. Beall and C.H. Bushweller, Chem. Rev., (1973), 73, 465 cf, E.L. Muetterties, E.L. Hoel, C.G. Salentine and M.F. Hawthorne, Inorg. Chem., (1975), 14, 950 Further References, Scrambling Centre Indicated. J.R. Van Wazer et al, J. Inorg. Nucl. Chem. (1964), 26, 1209 (boron); Inorg. Chem., (1964), 3, 139 (arsenic); J. Amer. Chem. Soc., (1964), 86, 811; Inorg. Chem., (1964), 3, 280 (phosphorus); Ibid., (1965), 4, 1294 (silicon) (silicon, germanium) J. Inorg. Nucl. Chem., (1964), 26, 737 (silicon) J. Organometal. Chem., (1968), 12, 69 (silicon)

Ibid., (1975), 85, 41 ([silicon] phosphorus) Inorg. Chim. Acta. (1967), 1, 407; Ibid., (1967), 1, (1967), 152 (silicon, germanium) (cf. K. Moedritzer ibid. (1971), 5, 547; (1974), 10, 163 (silicon, germanium K.M. Abraham and J.R. Van Wazer, J. Inorg. Nucl. Chem., (1975), 37, 541 (silicon, germanium) E. Fluck, J.R. Van Wazer and L.C.D. Groenweghe, J. Amer. Chem. Soc., (1959), 81, 6363 (phosphorus) J. Inorg. Nucl. Chem., (1967), 29,1571 (germanium); Ibid. (1964), 26, 737; Ibid., (1967), 29, 1851 (silicon) Inorg. Chem., (1965), 4, 1294 (review) J. Chem. Phys. (1964), 41, 3122 (several elements) J.G. Reiss and S.C. Pace, Inorg. Chim. Acta, (1974), 9, 61 (silicon) M.W. Grant and R.H. Prince, J. Chem. Soc., (A), 1969, 1138 (silicon)(germanium) F. Glocking, S.R. Stobart and J.J. Sweeney, J.C.S. Dalton, 1973, 2029 (mercury); A.G. Lee and G.M. Sheldrick, ibid., (A), 1969, 1055 (thallium); J.A.S. Howell and K.C. Moss, ibid., (A), 1971, 2483 (tantalum); R. Davis, M.N.S. Hill, C.E. Holloway, B.F.G. Johnson and K.H. Al-Obaidi, ibid., (A), 1971, 994 (molybdenum and tungsten); H. Hagnauer, G.C. Stocco and R.S. Tobias, J. Organometal. Chem., (1972), 46, 179 (gold); C.E. Holloway, J. Coord. Chem., 1971,1, 253 (tantalum); J. Evans, B.F.G. Johnson, J. Lewis and J.R. Norton, J.C.S. Chem, Commun., 1973, 807 (rhodium); J.F. Nixon, B. Wilkins and D.A. Clement, J.C.S. Dalton, 1974, 1993 (rhodium); J. Evans, B.F.G. Johnson, J. Lewis and R.Watt, ibid., 1974, 2368 (rhodium); M. Green and G.J. Parker, ibid., 1974, 333 (rhodium and iridium); A.J.P. Domingos, B.F.G. Johnson and J. Lewis, ibid., 1974, 145 (ruthenium); T.H. Whitesides and R.A. Budnik, J.C.S. Chem. Commun., 1974, 302 (ruthenium); F. Calderazzo, M. Pasquali and T. Salvatori, J.C.S. Dalton, 1974, 1102 (uranium); R.J. Cross and N.H. Tennent, ibid., 1974, 1444 (platinium); T. Mole, Organometal. Reactions, (1970),1,1, (review, aluminum); N.S. Ham and T. Mole, Progr. Nuclear Magnetic Resonance Spectrosocpy, Ed. J.W. Feeney and L.H. Sutcliff, Pergamon Press, London, (1969), 4, 91 (review); F.A. Cotton, Accounts Chem. Res., (1968), 1, 251 (review). (Examples of scrambling on carbon); K. Moedritzer and J.R. Van Wazer, J. Org. Chem., (1965), 30, 3920 (polyoxymethylenes); Ibid., (1965), 30, 3925 (acetals and orthoformates) J.T. Bursey , M.M. Bursey and D.G.I. Kingston, Chem. Rev., (1973), 73, 191 (intramolecular hydrogen transfer on carbon during mass spectrometery).
Added Later Following my postgraduate research (which had been funded by an Albright & Wilson Mfg. U.K. Studentship and supervised by D.S. Payne at the University of Glasgow, Scotland, U.K, (earning Ph.D. in 1962 for a thesis A Study of Phosphites [cf. D. Grant et al., J. Inorg. Nucl. Chem., 1964, 26, 1985 and ibid., 26, 2103) and a study of the use of Ce(IV) for oxidative analytical chemistry [Anal. Chim. Acta, 1961, 25, 422 ]), I had the honor to work as a Postdoctoral Fellow with John R Van Wazer in his (Monsanto Co, St Louis) research group (which conducted fundamental researches into the phenomenon of stochastic structural reorganization; this processes, which had been found to determine the chemical constitution of condensed phosphates (and further seemed to offer insight into the in biological use of condensed phosphates for energy transfer and as a basic determinant of the structure and activity of nucleic

acids), also seemed to allow for a fuller understanding of the thermal stability of numerous kinds of substances (including inorganic, organometallic and organic polymers). I later contributed to I.S.R. U.K. research activities (which ceased in 1975) [cf., e.g. E.W. Duck et al. Eur. Polymer J., 1974, 10, 77; ibid., 481; ibid., 1979, 15, 625) and, The Pertinence Of The Scrambling Behaviour of Ligands on Transition-Metal Centres to Ziegler-Natta Catalyst Activities (J. Polymer Sci., Polymer Lett., 1975, 13, 1 ) which suggested that the polymerization of olefins (as well as the related process of olefin metathesis) occurred by a process akin to Van Wazer structural reorganization at the catalytic center monomer adducts].

Mss. ex University of Aberdeen

Archival Literature Surveys from personal files retained from Marischal College Polysaccharide Research Group
D. Grant, Ph.D., Turriff AB53, Scotland, UK

Contents

A Survey Conducted by Nancy E

Woodhead

B Survey Conducted by David Grant

A A Perspective Suggested by Peer-Reviewed Literature a of How Glycosaminoglycans May Mediate Cellular Activities Including Proliferation and Transformation

----------------------------------

A topic which is believed to be of public interest was the subject of a presentation made in 1982 (to the W.F. Long, F.B. Williamson Polysaccharide Research Group at Marischal College Aberdeen) by Nancy E. Woodhead. This document contains an edited transcript of my shorthand notes made at the time to which I now append an update of the peer-reviewed literature in this field and also an edited version of a hypothesis

(Ascorbate and Nitric Oxide in Redox Control of Heparan Sulphate) which has originally posted on the internet in 2000 on a site which is no longer active. ===================================================================== Glycosaminoglycans and Cellular Transformation Prior to 1982 literature reports in this field were classified by Nancy Woodhead as follows: 1. 2. 3. 4. 5. Experiments directly relating glycosaminoglycans (GAGs) to cell growth control and Transformation. Alterations in GAG contents of cultured cells after transformation. Alterations of GAG contents of mammalian tumors compared to normal tissue; [this provides more material than is available from cells in culture]. Changes in complex composition of certain cell surface GAGs; [especially of HS)] Searches for a specific changes in function of cell surface GAGs; [possible functional change due to regional alterations in GAG structure].

1. Includes reports of experiments relating GAGs to cell growth control and cellular transformation; starting from 1932: Year Author 1932 Zakrezewski 1957 Sister M. Lippman Reported that heparin suppresses growth of normal embryonic tissue and Jensen sarcoma tissue. Reported experimental evidence that heparin can act as a mitotic inhibitor. [This was an in vivo study of the effect of how administration of subcutaneous heparin affected the measured size of Ehrlich Ascites tumors in rats. It was shown that heparin, under the conditions studied, produced a 40-50% regression of tumors].

1960 Ozzello et al.

Umbilical cord extracted hyaluronate (HA) or chondroitin sulfate (CS) promoted the growth of human mammary carcinoma cells in culture. Any of these substance taken singly promoted tumor growth. Heparin prevented mitosis. Evidence for intracellular action of heparin. Heparin also caused formation of microvili on cell surfaces. (Is this an abnormal effect of heparin or is at an apparent normal intracellular function of heparin-like molecules?) It was apparent that the phenomenon was probably not a unique property of EDTA (a high affinity Ca2+ chelator) which had been known to produce microvilli formation. This outcome may simply be caused by the removal of Ca2+ from the cell surface. (Heparin-like molecules may have this function in vivo).

1964 Costachel et al.

1966 Takeuchi

CS enhanced of tumor growth. Hydrocortisone inhibited the growth of tumors, but this inhibition was prevented by the presence of CS. Heparin CS and HA : maintained cells in culture after they degenerated in normal culture medium. Some effect of CS and HA on cell surfaces was suggested to

1974 Takeuchi et al.

increase their potential for affecting cell growth. 1975 Olin et al. ? Salt effects ? (and related sources of salt e.g. heparin etc.) can apparently [link to this document lost] can exert a concentration-dependent control effect on tumor cell growth. Large doses inhibited epithelial cell growth but low cell surface doses accelerated growth. E.g. 50-100g/ml level (at) cell surface inhibited cell growth while 0.5g/ml promoted growth. Summary of the pre-1975 findings 1. Polyanions can enhance tumor growth by protecting cell surface antigenic sites. 2. The production of new connective tissue giving GAGs is favorable to cancer cell growth More GAGs (if present) they can be chosen for cancers to (allow cells to stick?) 3. Effect of heparin on nucleoproteins. Evidence shows that heparin interacts with proteins in the nucleus (and thereby affects DNA) and so (affects) protein synthesis. 4. Heparin removes Ca from cell surfaces. Cellular proliferation might be affected by this mechanism. Studies which report an Alteration in GAG content of cultured cells after transformation 1966 Ishimoto et al. Avian sarcoma virus in chicken embryo fibroblasts led to (i) 5x increase in HA synthesized, (ii) extracellular HA increases. 1973 Satoh et al. Herpes type II or SV40 virus transforming virus in hamster embryo fibroblasts led to (i) increased HA (ii) HS proportion of total GAGs

1977

Hopwood and Dorfman SV40 in human skin fibroblasts led to (i) increased HA; increased HS, decreased dermatan sulfate (DS). (ii) HA acid synthesis is inversely proportional to cell density in normal but not in transformed cells. (i) Primary and permanent cell lines (Associated) increased CS and increased HS in permanent cell lines (ii) (Associated) increased DS and increased HS in primary cell lines. These results were similar for transformed (cell lines) (these are equivalent to permanent?) Rous sarcoma virus (RSV) transformed chondrocytes, showed: (i) less cell surface GAGs; (ii) HS (was) shed into the cell culture medium. (1) Chemical transformation of liver parenchymal cell clones led to: Increased CS; production of (usually less) HS.

1978 Dietrich et al.

1979 Mikuni-Takagali and Toole i 1980 Ninomaya et al.

Suggested reason/(consequences) for these changes: (1) CS acts as stimulant for cell division, with HS and DS having different roles such as recognition and adhesiveness. (2) HS may act as a negative control element of growth. (3) Loss of activity of GAG degrading enzymes or increase in GAG synthesizing enzymes: not a direct effect of transformation. 1978 Chiarugi et al. Assessed Mammalian Tumors 100 cases of human cancer studied: normal and neoplastic GAG content compared: (i) All neoplastic tissue showed changed GAG contents. (ii) Malignant tissue showed larger changes than non invasive tumors (iii) Most common effects - increase in CS or increase in HA or decrease in HA or CS. Often an increase (occurs in) total GAG

(1) Increase in HA and/or CS is a characteristic abnormality of GAGs in cancerous tissue. (2) Increase in total GAG in tumor tissue gives overall change in negative charge associated with the tissue. ------Studies Reporting Changes in Chemical Structure of Cell Surface GAG Following Cellular Transformation 3TS cell and SV40 transformed cells (i) (ii) 1978 Nakamura et al. HA and CS not changed. HS from transformed cells elutes at lower ionic strength from anion exchange column.

1975 Underhill and Keller

AH130 Ascites hepatoma Cell-associated GAG 93% HS (extracellular) fluid 58% HS, 26% HA, 16% CS Cell associated (i) HS less sulfated. (ii) HS (is) highly heterogeneous (as indicated by electrophoresis).

1978 Winterbourne and Mora 3TS/SV3TS cells (i) HS elutes from anion exchanger at lower anionic strength. 35 3 (ii) S/ H ratio lower. (But) no change in overall turnover rate of HS. 1979 Chandreschan and Davidson Normal human breast cell line (compared with) human breast carcinoma cell lines.

Cancer cell lines (i) Mainly CS and HS. (i) Heterogeneous HS. (ii) Size Charge (and) NAc/N-SO3 altered.

1980 Keller et al.

3TS/SV3T3

(Following transformation) (i) HS charge density is lower. (ii) 8% decrease (occurs) in O-sulfate 1981 Winterbourne and Mora 3TS/SV3TS (i) (ii) (iii) (iv) HS charge-density is lower. O-sulfate containing disaccharides of HNO2 degraded HS; the same. O-sulfate-containing oligosaccharides of HNO2 degraded HS show lower 6-O sulfation. Overall sulfation a decrease occurs in 6 (-O-sulfate).

CONCLUSION(S TO BE) DRAWN FROM THE ABOVE EXPERIMENTS 1. 2. 3. 4. Change in sulfate incorporation into GAGs may reflect change in substrate PAPS pool sizes. T antigen* (SV40 early gene product) may be responsible for change in HS structure. Ca2+ binding of the cell surface may be altered by lower HS charge. Changes in HS structure may lead to altered binding to cell surface protein e.g. fibronectin (this is of interest since fibronectin is not found on many transformed cells).

* Binds heparin therefore could possibly change the structure of HS on the cell surface HS was found only on cells expressing T antigen. -------

Specific Change in Properties of Cell Surface GAGs 1982 Fransson et al. (i) (ii) (iii) (comparing) 3T3/8V3TS and PyY 3TS cells HS (is) heterogeneous HS has a lower change density (in transformed cells) HS iduronate/glucuronate bearing N-sulfate segments (are less common in transformed cells).

Increased heterogeneity is (also) associated with reduction in self-aggregation properties. 1982 1983 Culp and Dorfman Highly N-sulfated HS sequences (bind) most efficiently to fibronectin affinity columns.

Stamatoglou and Keller 3TS/8V3TS HS elution from fibronectin (or) collagen. No difference between normal and transformed cells (when using physiol.) NaCl Only heparin will displace HS from collagen (but) heparin and DS can displace HS from fibronectin.

1982 Castellot et al.

Smooth muscle cell growth is inhibited by heparin like substances. Endothelial cells exposed to heparinoids are released from growth inhibition.

Heparitinase released from cells or platelets may cause stimulation of mitosis by degrading heparin like compounds. If you get damage to an endothelial surface to a blood vessel then the platelets will adhere to this surface (but only if they) release heparinase and so stimulate cell growth. The release of thrombin also will occur under these conditions. It should be noted that: Cancer (n.b. is the) uncontrolled growth of cells. Transformation - (is the) event leading to cancerous growth of cells. Metastasis (is the critical-for-cancer-mortality) loss of cells from tumor surface to form subsidiary tumors elsewhere. N.b. Inflammatory conditions - (are those conditions where) CS and HS also tend to increase N.b. also: GAGs structures become altered before or during cellular transformation. But Validity of Considertion of Fibronectin as a Driving Force ? Could such evidence point in the wrong direction? The important events in cellular transformation may be associated (most specifically) with the GAGs. {Note added later. The current paradigm is that (usually multiple) mutation(s) of DNA produce cellular transformation and neoplasia. Whilst this mechanism is undoubtedly the primary cause of cancer the follow-on effect on the glycosylation system is putatively also an additional critical part of the etiologies of these diseases; especially the transformation-associated alteration of extracellular proteoglycan glycosylation regulatory functions afforded by HS may actually cause the greatest damage to the organism by allowing uncontrolled cellular growth angiogeneiss and metastasis to occur}. N.b. The HS information encoded processing system is now thought to be a major epigenetic driver. Chiarugi et al. had noted in 1974 (Biochim Biophys Acta. 345 283-293) that The effect of N-sulfated polyanions on tissue growth in vivo and in vitro has been recognized since 1932 (this was intimated by Zakrezewki loc. cit } But (at what stage) in the transformation process are the HS or other GAGs related to the change in events involved ? The initial important change must be there - the insertion of (some) new genetic interaction. Perhaps a clue can be gained from the circumstances where Ca is chelated out (cf. thrombin, antithrombin and the heparin effect) Mammalian GAGs - can be anti-inflammatory -- can be anti-cancer.

(Cf.) A consideration which might be of relevance is how cross-linked carrageenan beads can (behave) like proteoglycan complexes; (they are set in cells but they are not actually in the cells).

a Adapted from notes made during a lecture/discussion literature survey of how GAGs (especially heparan sulfate (HS)) participated in the etiology of cellular transformation and neoplasia presented to the Marishcal College (Aberdeen) Polysaccharide Research Group* on 18 November 1982 by Nancy E. Woodhead (then a graduate student member of the W.F. Long and F.B. Williamson Polysaccharide Research Group) written down in a University of Aberdeen Lab. Notebook 16/3/82-26/11/82 [Page heading Nancys Talk]

retained by D Grant AB53 UK.

Cf also NE Woodhead et al., IRCS Medical Science : Biochemistry ; Cancer; Cell and Molecular Biology; Connective Tissue, Skin and Bone ; Pathology [IRCS Med. Sci., 14 427428 (1986) (Heparan sulfates from fibroblasts exhibiting a temperature-dependent transformed growth trait). (This was a comparative study of normal vs. chemicallytransformed cells which exhibited a transformed growth trait at 37oC. The transformed cells reverted to a normal growth pattern at a lower temperature. The observed changes in heparan sulfates may have mediated this phenotype change.
These results remain of topical interest.

*[The hypothesis that cellular proliferation is, at least in part, determined by the binding of Ca2+ ions to cell surface glycosaminogycans was, for about thirty years, a principal research
focus for a polysaccharide research group headed by W.F. Long and F.B. Williamson at the University of Aberdeen. Barr L. et al. reported in 2006 (FASEB J. 20 E963-E975) that a Ca2+ dependent pathway (involving the completion of the glycosaminogycan protein linkage tetrasaccharide formation) controls chondroitin and heparan sulfate proteoglycan biosynthesis].

The Addendum (vide infra) lists a fairly random selection of more recent similar topic peer-reviewed literature

References Citations given in the 1982 lecture by N.E. Woodhead are listed below in the order in

which they appear in my notes (which were taken at the time); these are also listed in the The Heparan Sulphates of Control, Virally-Transformed and ChemicallyTransformed Fibroblasts Nancy Elizabeth Woodhead Ph.D. Thesis University of Aberdeen, , 1985. It should be noted that this thesis also reported for the first time on how a chemical carcinogen (N-methyl-N-nitro-N-nitrosoguandine)transformation of cells gave rise to similar diminution of HS sulphation to that observed with viral transformation or as is commonly found in HS extracted from cancer tissue. A previous Aberdeen University Ph.D. Thesis by H.H.K Watson (1980) had also dealt with related researches.
Additional references to those discussed in the 1982 lecture and which further support the hypothesis that the etiology of neoplasia could depend on alteration of GAG composition following cellular transformation which were Chapter 1.8 [Glycosaminoglycans in Cancer] cited of the above Thesis are: Kuroda et al. (1974) [Rat tumors] Cancer Res. 34 308-312;

Kupchella et al. (1981) [Rat tumors ] ibid., 41 419-424 Kojima at al. (1975) Horai et al. (1981) [Human hepatic tumor] ibid., 502-547 [Human lung tumor] Cancer. 48 2016-2021

Knudsen et al. (1984) [Murine tumors synthesized 20x the amounts of GAGs in vivo/in vitro] J Cell Biochem. 25 183-196 David and Van den Berg (1983) [Transformed mouse epithelial cells] J Biol Chem. 258 7338-7344; Eur J Biochem. 1989

178 609-617 Robinson et al. (1984) ibid., 253 668-793 Ohkuboka (1983) Cancer Res. 43 2712-7 Additional Related-Topic Older References Mitotic Gelation (Water Structure?) Cf. Chiarugi V.P. et al. (1974) Biochim Biophys Acta. 345 283-293: Heparin has been found to prevent mitotic gelation ; (a periodic release of free heparin occurs in synchrony with the cell cycle); Augusti-Tocco G. and Chiarugi V.P. (1976) Cell Differentiation 5 (3)161-170 (Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell culture) the switch from the round to the neuron-like cells can be obtained by a simple change of the culture conditions, which causes an increase of cell adhesion this is accompanied by an increasing ability of cells to retain heparan sulfate. Control of Cellular Activities by Cell Surface GAG Selective Binding of Ca2+ Cf. Long W.F. and Williamson W.F. loc. cit. and Cf. Vannucchi S. et al. Biochem J. 1978 170 185-187. Table 4 of this article indicates that the relative Ca2+ binding capacity of commercial GAGs was found to be HA 1.00, heparin 2.76, HS 2.00, CS(A) 1.60, CS(B) 1.67 and CS(C) 1.80. Role of Ascorbate as a Heparan Sulfation Control Agent Watson and Edward (1980) Biochem Soc Trans. 8 134-136 (Cf. Edward and Oliver (1983) [Ascorbate boosts HS sulfation] ibid., 11 383; ibid. 12 304 {this was confirmed by Kao et al. (1990) Exp Mol Pathol. 1990 53 1-10}and may be part of the mechanism by which ascorbate demonstrates anti-cancer activity as indicated, e.g. by Cameron and Pauling (1985) PNAS 75 4538-4542 cf. ibid., 79 3685-3689}) -------------------------------------------------------------------------------------------------------------------------------------------------Esko J.D. Rostand K.S. Weinke J.L.(1988) (Tumor formation dependent on proteoglycan biosynthesis) Science. 241 1092-1096; cf. Barr L. et al., (2006) 20 E963-E975 (Evidence of calcium-dependent pathways in the regulation of human 1,3glucuronosyltransferase-1 (GlcAT-I) gene expression : a key enzyme in proteoglycan synthesis) FASEB J. [These GAG chains are important regulators in a wide range of biological events, such as matrix deposition, intracellular signaling, morphogenesis, cell migration normal and tumor cell growth] Fedarko N.S. Ishihara M and Conrad H.E. (1989) (Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan) J Cell Physiol. 139 287-294 PMID 2715188 (Additional related references accessed by Marion Ross a later member of the Aberdeen University polysaccharide group)) Zimina N.P. et al. (1987) Biokhimia. 52 (5) 856-861 [All types of sulfated GAGs in actively proliferating tissues (except

regenerating tissues) have a reduced degree of sulfation)

Kosir M.A. and Culp L.A. (1988) Surg Forum Med. 39 424-426 Matuoka K et al. (1984) Cell Structure and Function 9 357-367 [1. HS plays a particular function in contact regulation of cell proliferation. 3. Transformation-related changes in the structure of HS molecules do not much affect the function of HS. 3. The cellular transformation, however, is accompanied by alteration in the growth regulating system sensitive to extracellular HS. Heparin inhibited growth in both normal and transformed cells]

Cf. also Long WF and Williamson FB (1979)

(Glycosaminoglycans, calcium ions and control of cell proliferation)

IRCS Journal Med Sci. 7 429-434; Cf. also Med Hypoth. 11 285-308 and ibid., 13 385-394 Most of the relevant Aberdeen University Polysaccharide Research Group publications including those of

N.E. Woodhead (up to 2003) which had been undertaken in part to advance the above hypothesis were listed by Professor W.F. Long at
web. abdn.ac.uk/~bch118/publications2003march.doc NE Woodhead et al. also conducted in vivo and in vitro studies relating to the Long-Williamson GAG divalent metal ion animal cellular control hypothesis (cf., Biochem J. 1986 237 281-284)

HS, the ubiquitous component cell surfaces and extracellular matrices of animals, may affect transformation of cells and also be a controller of their proliferation, at least in part, via HS-mediated control of the activities of (Ca2+ and Zn2+) metal ion signaling activities.
Zakrezewki Z. (1933) Z Krebsforsch. 36 513-521 Lippman M. (1957) Cancer Res. 17, 11-14 Ozzello L. et al. (1960) ibid., 20, 600-605 Costachel O et al. (1964) Exptl Cell Res. 34 542-547 Takeuchi J. (1966) ibid., 26, 797-802 Takeuchi J. et al. (1976) ibid., 36, 2133-2139 Olin (source not found) Obrink B. et al. (1975) Conn Tiss Res. 3 187-193 Ishimoto N. et al. (1966) J Biol Chem. 241 2052-2057 Satoh C. et al. (1973) Proc Natl Acad Sci USA. 70 54-63 Hopwood J.J. and Dorfman A. (1977) J Biol Chem. 252 4771-4785 Dietrich C.P. and Armelin H.A. (1978) Biochem Biophys Res Commun. 84 794-801; Cf. Dietrich C.P. and DeOca H .M. ibid., 80 805-812; Mikuni-Takagaki Y. and Toole B.P. (1979) J Biol Chem. 254 8409-8415 Chiarugi V.P. et al. (1978) Cancer Res. 38 4717-4721 Underhill C.B. and Keller J.M. (1975) Biochem Biophys Res Commun. 63 448-454; cf., J Cell Physiol. 89 53-64 Nakamura N. et al. (1978) Biochim Biophys Acta. 538 445-457 Winterbourne D.J. and Mora P.T. (1978) J Biol Chem. 253 5109-5120 (1981) ibid. 256 4310-4320 Keller L. et al. (1980) Biochemistry. 19 2529-2536 Fransson L.-. and Havsmark B. (1982) Carbohydr Res. 110 135-144 Stamatoglou S.C. and Keller J.M. (1982) J Cell Biol. 96 1820-1823 Castellot J.J. et al. (1982) J Biol Chem. 257 11256-11260 ADDENDUM

Selected Later References

which further extend knowledge of the (heparin/HS involvement in animal cell proliferation and transformation) research topic

Zhou H. et al. (M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression) PloS ONE. 2011 6 (6) e21106 Chao B.H. et al. (Clinical use of the low-molecular-weight heparins in cancer patients: focus on the improved patient outcomes) Thrombosis. 2011 2011:530183; PMID 22084664 Borsing L. et al. (Sulfated hexasaccharides attenuate metastasis by inhibition of P-selectin and heparanase) Neoplasia. 2011 13 (5) 445-452 Casu B. et al. (Heparin-derived HS mimics that modulate inflammation and cancer) Matrix Biol. 2010 29 (16) 442-452 Raman K and Kuberan B (Chemical tumor biology of HS proteoglycan) Curr Chem Biol. 2010 4 (10) 20-31 [Heparan sulfate is a profound target for developing novel cancer therapeutics because modifying HS chains would affect Hpa, Hsulf-1, Hsulf-2, H-sulf-2, and 3-OST {these are key enzymes involved in HS biosynthesis and catabolism} activity in tumor cells, which in turn would affect angiogenesis, growth factor signal over amplification, and tumor growth, invasion and metastasis] Barash U. et al. (Proteoglycans in health and disease: new concepts for heparanase function in tumor progression and metastasis) FEBS J. 2010 277 (19) 3890-3903 Lee Y.D. et al. (Antiangiogenic activity of orally absorbable heparin derivatives in different types of cancer cells) Pharm Res. 2009 26 (12) 2667-76; PMID 19830530; Zacharski L.R. and Lee A.Y. (Heparin as an anticancer theraapeutic) Expert Opin Investgated Drugs. 2008 17 (7) 1029-1037 Pumphrey CY et al. (Neoglycans, carbodiimide-modified glycosaminoglycans: a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis) Cancer Res. 2002 62 (13) 3722-3728 Engelberg H. Cancer. 1999 85 (23) 257-272 [Heparin is a potential anti-cancer drug] Cf. e.g. Goldberg I.D. Ann N Y Acad Sci 1986 463 289-291 Coombe D.R. and Kett W.C. (Heparan sulfate-protein interactions: therapeutic potential through structure-function insights) Cell Mol Life Sci. 2005 62 410-424 [Cf. also the later review by Casu et al. loc. cit.) is of especially relevance as it outlines why GAGs and particularly heparin/heparin -HS-like structures are now attracting considerable interest as a source of new therapeutics for the treatment of infectious diseases, inflammation and allergic diseases and cancers; attempts to provide a critical review of the historical development emphasises the complexity of the biochemistry of HS (which is confirmed to be a major master system which inter alia controls embryo development wound healing, hemostasis and the immune system) and points out that in vitro

studies may not reproduce the in vivo conditions especially as regards the present of metal cation cofactors and pH both of which can have profound effects on the microstructure of HS chains on which outcome of the interactions with the target proteins depend]. ANGIOGENESIS Ritchie J.P. et al., (SST0001, a chemically modified heparin, inhibits myeloma growth and angiogeneis via disruption of the heparanase/syndecan-1 axis) Clin Cancer Res. 2011 17 (6) 1382-1393 Logie J.J. et al. (Glucocorticoid-mediated inhibition of angiogenic changes in human endothelial cells is not caused by reductions in cell proliferation or migration) PloS ONE. 2010 5 (12) e14476; [Anti-angiogenic actions of glucocorticoids may be in part mediated by induction of thrombospondin-1 (TSP-1); this in turn implicates a key role of HS in this process as cf. Feitsma K. et al. who had indicated in J Biol Chem. 2000 275 (13) 9396-9402 (Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. The microstructure of the HS cell surface binding sites for thrombospondin-1(TSP-1) (which are responsible for TST-1 endocytosis) contains a trisulfated 2-Osulfated iduronic acid-N-sulfated 6-O-sulfated glucosamine disaccharide unit which is distinct from the HS structure which is required for e.g. basic fibroblast growth factor binding]. Jakobsson L. et al., (Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis) Developmental Cell. 2006 10 625-634
[Cf. Medical News Today (www.medicalnewstoday.com/articles/43115.php (New discovery about role of sugar in cell communication,,, A research team from Uppsala Univeristy has uncovered an entirely new mechanism for how communication between cells

is regulated. By functioning like glue, a certain type of sugar in the body can make cell communication more effective and stimulate the generation of new blood vessels. The discovery paves the way for the development of drugs for cancer and rheumatism, for example)]
Linhardt R.J. (Heparin-induced cancer cell death) Chem Biol. 2004 11 (4) 420-422 Blackhall F.H. et al. (Binding of endostatin to endothelial HS shows a differential requirement for specific sulfates) Biochem J. 2003 375 (1) 131-139 [Endostatin is believed to inhibit angiogenesis (and hence tumorigeneis) by a mechanism which appears to involve the binding of endostatin to HS and for which 6-O sulfates played a dominant role in site selectivity]; Folkman J. (Angiogeneiss inhibition and tumor regression caused by heparin or a heparin fragment in the presence of cortisol) Science. 1983 221 719-725; Cf. Crum R. et al., (A new class of steroids inhibits angiogenesis in the presence of heparin or HS fragments) Ibid., 1985, 230, 1375-1378 Cf. Schachtschabel D.O. and Sluke G. (Effect of cortisol on glycosaminoglycan synthesis and growth of diploid, human fibroblasts (WI-38) in relation to in vitro aging) Z Gerontol. 1984 17 (3) 141-149 PMID 6475191 [Contrary to the medium the pattern of the cell surface GAGs was changed by 1.4x10-7M cortisol with an increase in HA synthesis and a decrease in that of sulfated GAGs; this effect of cortisol is equivalent to a counter aging influence]

HEPARIN AFFIN REGULATORY PEPTIDE (HARP) [Pleiotrophin] Vacherot F. et al., (Involvement of heparin affin regulatory peptide in human prostate cancer) Prostate. 1999 38 126-136 Cf. Heroult M et al. (HARP binds to VEGF and inhibits VEGF-induced angiogenesis) Oncogene. 2004 23 1745-1753 Lee T-Y. Folkman J. and Javaherian K. (HSPG (heparan sulfate proteoglycan)-binding peptide corresponding to the exon 6a-encoded domain of VEGF (vascular endothelial growth factor [VEGF]) inhibits tumor growth by blocking angiogenesis in murine model) PloS ONE. 2010 5 (4) e9945; Cf. Chen J.-L et al. (Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor (VEGF), angiogenesis and metastasis of arthotopic implantation of human gastric cancer} World J Gastroenterol. 2007 13 (3) 457-461 [N-desulfated heparin inhibits tumor metastasis and angiogeneis via an inhibition of the expression of VEGF (Possible Therapeutic Potential of (Non-Animal-Sourced) HEPARINOIDS (HEPARIN-LIKE SULFATED POLYSACCHARIDE) Zaslau S et al., Amer J Surg. 2006 192 (5) 640-643 (Pentosan polysulfate (Elmiron): In vitro effects on prostate cancer cells regarding cell growth and vascular endothelial growth factor production); [This heparinoid is a sulfated beech wood xylan also known as SP54] Cf. Noda H et al. (Antitumor activity of polysaccharides and lipids from marine algae) Nippon Suisan Gakkaishi 1989 55 (7) 1265-1271 EXT (Heparan sulfate related) TUMOR SUPPRESSOR Kitagawa H. et al. (The tumor suppressor EXT-like gene EXTL2 encodes a key enzyme for the chain initiation of heparan sulfate) J Biol Chem. 1999 274 (20) 13933-13937; Lind T. et al. (The putative tumor suppressors EXT1 and EXT2 are glycosyltrqansferases required for the biosynthesis of heparan sulfate) J Biol Chem 1998 273 (41) 26265-26268 Rahmoune H. et al. Biochem Soc Trans. 1996 24 (3) 355S [While the usual effect of cellular transformation is a lower production of heparan sulfate of decreased degree of sulfation, cellular transformation can also be accompanied by an augmentation of heparan sulfate biosynthesis (with increasing cell surface as well as culture medium released heparan sulfate) relative to normal cells)] Cf. Biochem J. 1998 273 33 21111-21114 [A MCF-7 tumor cell HS microstructure which binds laminin-1 (implicated in tumor-host adhesion) was identified (IdoA(2-O-SO3-)-GlcNSO3-(6-O-SO3-)]5[IdoA(2-O-SO3-)-AManR(6S-O-SO3-was generated using a
hydrazinolysis/deaminative procedure which cleaves deacetylated N-acetylglucosaminic bonds).

Liuzzo J.P. and Moscatelli D. (Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and HS: Down modulation of FGF receptors by phorbol ester) Blood. 1996 87 (1) 245-255

HEPARAN SULFATE AT CELL NUCLEUS Buczek-Thomas J.A. et al. (Inhibition of histone acetyltransferase by glycosaminoglycans) J Cell Biochem. 2008 105 (1) 108-120 Hsia E. et al. (Nuclear localization of basic fibroblast growth factor is mediated by HS proteoglycans through protein kinase C signaling) J Cell Biochem. 2003 88 (6) 1214-1225 Richardson T.P. et al. (Regulation of HS protoglycan nulcear localization by fibronectin) J Cell Sci. 2001 114 (9) 1613-1623 HEPARANASE (Effect at Nucleus) Purushothaman A et al., (Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive phenotype J Biol Chem. 2011 286 (35) 30377-30383 Yang Y. et al. (Heparanase enhances local and systemic osteolysis in multiple myeloma by upregulating the expression and secretion of RANKL) Cancer Res. 2010 70 (21) 8329-8338 Chen L. and Sanderson R.D. (Heparanase regulates levels of syndecan-1{HS proteoglycan} in the nucleus) PloS ONE. 2009 4 (3) e4947 [Although HS function within the nucleus is not well understood there is emerging evidence that it may act to repress transcriptional activity] Mani K et al. (Tumor attenuation by 2(hydroxynapthyl)--D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products) Glycobiology. 2004 14 (5) 387-397
[N.b. the HS oligomers which were found to signal to the nucleus in these studies showed anhydroMan end groups. These are formed during (nitric oxide ascorbate Cu/Zn facilitated) nitrosative cleavage i.e. the (partly) non-enzymic cleavage of HS (to give putative hormone-like HS fragments) from un-substituted GlcNH2 groups in HS pre-primed for nitrosative cleavage. (While such structures are known to occur in HS although the mechanism of their insertion e.g. during primarly biosynthesis details are still unknown but putatively may aberrantly be augmented as a result of non-enzymic de-N sulfonation during acidosis or redox metal ion dyshomeosasis of precursor GlcN-SO3- groups. Or perhaps by other mechanisms which putatively contribute to the aetiologies of neoplasia and other degenerative diseases}]

HEPARANASE - GROWTH FATOR PHOSPHORYLATIION Cohen-Kaplan V. et al., (Heparanase augments epidermal growth factor receptor phosphorylation: correlation with head and neck tumor progression) Cancer Res. 2008 68 (24) 10077-85 HEPARANASE -METASTASIS Cohen E. et al. (Heparanase is overexpressed in lung cancer and correlates inversely with patient survival) Cancer. 2008 113 (5) 1004-1011; Faye C. et al.

(Molecular interplay between endostatin, integrins and heparan sulfate) J Biol Chem. 2009 284 (33) 22029-22040 Liu D et al. (Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis) PNAS USA 2002 99 (2) 568-573 [Specific different HS microstructure mixtures generated in vivo by exogenous heparinase apparently signal for opposite growth effects on tumor cells was suggested by the observation of the effect of difference between heparinases I and III when injected into mice with B16BL6 melanoma; while the HS oligomer mixture arising from heparinase III digestion caused tumour cell inhibition that produced by heparinase I digestion caused tumor cell growth to be enhanced] Vlodavsky I and Friedman Y. (Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis) J Clin Invest. 2001 108 (3) 341-347 CELLULAR IMMUNE ANTI-TUMOR RESPONSE Dziarski R. (Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin) J Immunol. 1989 143 (1) 356-365 INORGANIC CALCIUM ION : COFACTOR IN HEPARAN SULFATE ACTION Ca2+ etc. chelation, heparan sulfate/chondroitin sulfate ratio METASTASIS Cf. Tmr J et al. (Modulation of heparan-sulfate/chondroitin-sulfate ratio by glycosaminoglycan biosynthesis inhibitors affects liver metastatic potential of tumor cells) Int J Cancer. 1995 62 755-761 [Ethane-1-hydroxyl-1-1-diphosphonate [EHDP] a well tolerated pharmaceutical (and a Ca2+ binding ligand) and other (different mechanism) GAG biosynthesis inhibitors, were apparently able to diminish tumor metastasis (by putatively Ca2+ dependent) inhibition of heparan sulfate biosynthesis. It should be noted that EHDP and related bisphosphonates osteoporosis therapeutic use also has been indicated to produce an extended life expectance by an unknown mechanism; it should also be noted that bisphosphonates per se have been reported to demonstrate anti-metastatic effects in e.g. in prostate cancer cf. e.g. Montaque R et al., Eur Urol. 2004 46 (3) 389-401] Kan M. et al. J Biol Chem. 1996 271 26143-26148 [Divalent metal ions are essential cofactors for basic fibroblast growth factor assembly] for correct interaction between heparan sulfate and L-selectin as well as 2 and 3 integrins and for annexinV assembly on cell surfaces]; Valencia-Snchez A. et al. Mol Androl. 1995 7 (1,2) 57 [Heparan sulfate Ca2+ flux control during capacitation and modulation of acrosome reaction by heparin] Takeuchi Y. et al. J Biol Chem. 1990 265 (23) 13661-13668 [Extracellular Ca2+ concentration regulates the distribution and transport of heparan sulfate proteglycans and heparan fragments in a rat parathyroid cell line] Vandewalle B. et al. J Cancer Res Clin Oncol. 1994 120 (7) 389 Ca2+ enhanced HS proteoglycan activity which modulate tumor cell growth Hayashi M. and Yamada K.M. J Biol Chem. 1982 257 5263-5267 [Divalent cations are required for heparin binding to fibronectin] Boehm T. et al. (Zinc binding of endostatin is essential for its anti-angiogenic activity)

Biochem Biophys Res Commun. 1990 252 (1) 190-194;

The roles of Ca2+ and other metal ions in the inorganic biochemistry of heparan sulfate
allows for a system of Ca2+ Zn2+, Cun+ etc. activity regulation (and perturbation of this toxic Mn+ metal ions) could be relevant inter alia to a fuller understanding of how GAGs contribute to animal tissue homeostasis and non-specific immune protection and wound healing.

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Ascorbate and Nitric Oxide In Redox Control Of Heparan Sulphate Structure of Relevance to Cancer
http://www.google.com/search? q=cache:7kSouJC8zHUJ:web.ukonline.co.uk/dgrant/dg4/+/dgrant/dg4/&amp;hl=en&amp;ct=clnk&amp;cd=1&amp;ie=UTF-"Hepa ran Redox Hypothesis." from home-based continuation of a literature survey following laboratory studies of heparin/heparan sulphate (with WF Long and FB Williamson Department of Molecular and Cell Biology University of Aberdeen)

CONTENTS 1. Summary 2. Introduction 3. ACTIVE NITROGEN Derivatives in Chemistry and Biology 3.1 Physiological utilisation of nitric oxide 3a Active nitrogen derivatives as promoters of degenerative diseases 3.2 Various potentially biologically active nitrogen derivatives 3.3 Nitrogen compounds as aqueous system denaturants 3.4 Nitrogen-centred free-radicals 3.5 Metal-catalysed effects in active nitrogen formation? 3.5.1 Iron and copper 3.5.2 Vanadium and titanium 3.5.3 Nickel and cobalt 3.5.4 Molybdenum 3.6 Peroxynitrite 3.7 Cysteine-tyrosine active nitrogen traps? 4. ASCORBATE Redox metal effects 5. Summary of HEPARIN/HEPARAN SULPHATE BIOCHEMISTRY 5a Activation of heparin/heparan sulphate from nitrous acid cleavage by removal of protective groups from glucosamine nitrogens 5b Evidence for the relevance of heparin/heparan sulphate biochemistry to cancer. Cancer-related metal ion binding by heparan sulphates

5c Effects of oxygen, glucose, toxins (including toxic metals) & ascorbate on heparan sulphate biosynthesis 6. Conclusions 7. References 8. Appendix a. Transition metal ion catalysis of reactive nitrogen damage b. Heparin/heparan sulphate brain development and function Neurodegenerative diseases including Alzheimer's disease (AD) Introduction Heparin/heparan sulphate in AD Zinc in AD Cognitive function in AD effect of Ateroid Neurite outgrowth Lack of correlation between extracted brain heparan suphate and AD Transferrin receptor function and heparan sulphate Fenton reaction free-radical damage in AD etc c. Supramolecular structure of heparin/heparan sulphate and binding potential d. Silicates and heparin/heparan sulphate 9. Heparin/heparan sulphate involvement in prion diseases 10. Water binding to heparin/heparan sulphate <BR>Hofmeister effects on water structure Modulation of crystallisation by heparin/heparan sulphate 11. Established laboratory use of nitrous acid for heparin/heparan sulphate structure evaluation 12. Binding of pathogens to heparan sulphate 13. Acknowledgements

1. SUMMARY The prevention of deaminative cleavage of heparan sulphate by Vitamin C and its derivatives is postulated to be critically important to health by blocking dysfunction which may occur in heparan sulphate biochemistry under iron overload, pathogen induced acidosis and thiocyanate intoxication. Critically required heparan sulphate biosynthesis is suggested to be sensitive to cellular redox status involving ascorbate and nitric oxide. 2. INTRODUCTION Linus Pauling promoted the use of Vitamin C as an anticancer, antiviral and procognition agent but lacked a convincing mechanism by which to account for such wide benefits to health (additional to antioxidant activity and promotion of collagen hydroxylation-dependent increased extracellular matrix resistance to tumour metastasis). It is becoming apparent that many tissue protective roles depend on heparin/heparan sulphate biochemistry, including highly microstructural-dependent growth factor signalling (cf Perrimon & Bernfield 2000) which might be abrogated by nitrous acid derived from nitric oxide and this could underpin the epidemiological and other evidence (cf Numerous authors, 1991) for an anti-cancer role of ascorbate (which is a highly efficient anti-nitrous acid agent). Although nitrous acid deaminative cleavage has been employed for decades and as a diagnostic test for heparan sulphates (and pathological nitric oxide related dysfunction of heparan sulphate biochemistry and possible metal ion catalysis of this has been evident for some time (Grant 1992) more recent reports (Vilar et al 1997; Ghael et al 1997) confirm that deaminative cleavage of heparan sulphate is of likely importance to pathology.

Ascorbate anti-nitrite activity is most likely due to the rapid chemical reduction of nitrous acid to nitric oxide in agreement with the results of Tsao (1991) who showed that both D- and L- ascorbate had similar anti-tumour effectiveness in an animal model, indicating a pure-chemical (e.g. redox control) not an 'essentially biochemical' mechanism; ascorbate nevertheless is indicated to have further cancer-related biochemical effects. Dehydroascorbate, diketogulonic acid and copper ascorbate apparently display individually different anti-tumour activities; dehydroascorbate may directly inhibit cellular mitosis (Edgar 1970) perhaps involving control of cellular redox potential which also influence the biosynthesis of heparan sulphate of altered sulphation and chain length. Copper ascorbate perhaps sensitises some tumour cells for subsequent destruction. Ascorbate has been reported to directly increase the biosynthesis of heparan sulphate chains of higher degrees of sulphation (Kao et al 1990, cf Malemud et al 1978) this may be related to cellular redox control effects; apoliprotein E is also reported (Paka et al 1999) to boost the biosynthesis of more highly sulphated complex microstructured anticoagulant heparan sulphate chains in endothelial cells. Lipolysis, recently reported (Anderson et al 1999) to be modulated by nitric oxide in human adipose tissue is known to be influenced by the heparin/heparan system, further potentially linking ascorbate, heparan sulphate, lipid and nitric oxide biochemistry and redox chemistry. 3. ACTIVE NITROGEN DERIVATIVES IN CHEMISTRY AND BIOLOGY 3.1 Physiological Utilisation of Nitric Oxide Nitric oxide homeostasis in blood is believed to be controlled by binding to iron in erythrocyte haemoglobin. Ascorbate is, however well understood to provide protection against the formation of carcinogenic nitrosamines produced from dietary nitrite and is also likely to be involved more widely in the physiological control of the nitric oxide air oxidation product nitrous acid. Arginine-derived nitric oxide, produced by nitric oxide synthases upon stimulation by agents like calcium ions, calmodulin, cytokines and tumour necrosis factor, is utilised physiologically as a chemical messenger (for guanylate cyclase) potentiating endothelial relaxation (which is reported by Kouretas et al 1998 to be influenced in immune responses by non-anticoagulant heparin via inhibitioin of nitric oxide synthase by a guanine nucleotide regulatory protein); nitric oxide is also involved in mitochondrial activity, gene expression, apoptosis and haemostasis, and is a neurotransmitter with putative involvement in learning (Hawkins et al 1998) and lipid metabolism (Anderson et al 1999).

3a Nitrogen-Containing Carcinogens

The majority of compounds known to be carcinogenic seem to be N containing, and include aromatic amines, alkyl nitrosoamides, aryl dialkyl triazines, carbamates, azo and acridine dyes, nitrosamines and pyrrolizidine alkaloids. Some types of nitrogen compounds demonstrate both pro- and anti-cancer effects (cf Ferguson 1975). Although nitrate ester and hydrazine derivatives have long been known to generate (therapeutically useful) cardiovascular effects, now known to be due to nitric oxide formation (Servent et al 1989), nitrogen oxide chemistry was traditionally thought to mainly impinge on biology in terms of noxious byproducts of hydrocarbon and tobacco combustion. Active nitrogen intermediates are implicated in the aetiology of rheumatoid diseases where urinary secreted nitrate exceeds ingested nitrate in amounts correlated with the severity of the disease and inhibitors of inducible nitric oxide synthase are show promising therapeutic activity against such diseases (Cochran 1994, Stichentoch & Frollich 1998). That active nitrogen intermediates derived from nitric oxide produced by autoimmune effects are likely implicated in the degradation of cartilage components in these diseases concurs with the putative use of glycosaminoglycan-selective colorimetric assay for improved diagnosis of rheumatoid diseases (Kery et al 1992) and with the apparent therapeutic success of dietary fish oil (an active component, docosahexaneoic acid, inhibits macrophage production of nitric oxide/nitrous acid (Jeyarajah et al 1999). Glucosamine or glucosamine sulphate dietary supplements are in common use to combat osteoarthritis (cf Theodosakis et al 1997) by apparently enhancing the biosynthesis of heparan sulphate (McCarty 1995).
Nitrous acid produced from nitrites present as food preservatives is a known inducer of stomach cancer (cf Cameron & Pauling 1979) and the above discussion of the role of nitrous acid in the aetiology arthritic diseases can logically be extended to carcinogenesis, in keeping with the notion that biochemistry seems highly centred on the unique properties of organic nitrogen compounds of which nitric oxide /nitrous acid is a part, capable of reacting with various organic nitrogens in proteins, nucleic acid bases as well as in amino sugars such as those in glycosaminoglycans. A complex nitrogen oxidation-reduction chemistry exists with nine stable oxidation states including molybdenum-dependent microbiochemistry influenced by a wide range of redox metal (iron, copper, titanium, vanadium, chromium, perhaps nickel) and acid/base(zinc) catalysis. In the deaminative cleavage of glucosamines, unstable intermediate diazonium derivatives are believed to be formed (as is general from primary amines) and may be accompanied by other unstable intermediates such as intrinsically unstable nitrite esters (cf Barton et al 1977). Nitrous acid itself is believed to equilibrate with more reactive N2O3 but other species may also be involved in similar equilibria Nitric oxide is readily oxidized to nitrous acid which, however, can be efficiently reduced back to nitric oxide by ascorbate. Nitrous acid reacts with amines, e.g. with polysaccharide glucosamines to cause deaminative cleavage of the polymer chains. Aromatic and aliphatic primary amines are affected differently by nitrous acid, the former produces diazonium, the latter nitrogen gas. Secondary amines produce (often carcinogenic)

nitrosamines. Nitrite is commonly assessed by the Greiss reaction. 3.2 Various potentially biologically active nitrogen derivatives In addition to nitric oxide and its metabolites, other potentially damaging active nitrogen species include nitrogen-centred free-radicals, various nitrogen oxides, peroxides (especially peroxynitrite, Beckman 1990), oxyacids, hydrides, halides, pseudohalides and such species as cyanides, cyanates, isocyanates, thiocyanates, isothiocyanates and nitrenes. Amine derivatives should also be included (cf hydroxylamines have similar DNA mutagen effect to nitrous acid cf Mahler &amp; Cordes 1966 and their formation was considered to explain the carcinogenicity of aromatic amines) and hydrazine, azides and small ring heterocycles (e.g. azirines, aziridines and oxaziridines); redox metal complexes of the above nitrogen species as well as other long-known entities - activated molecular nitrogen and polymeric adducts of hydrogen atoms and nitric oxide, sulphurcontaining Fremy's salt (cf Emeleus & Anderson 1946) and thionitrite. Polyamines should also be included, as their formation may be linked with the promotion of stomach cancer by Helicobacter pylori (cf Xu et al 1998; Linsalata et al 1998). An indication of potential biological reactivity of suspected active nitrogen chemical species might be revealed by their nitrogen nmr chemical shifts; damaging species conceivably having the most downfield shifted resonances (cf Mason 1983). 3.3 Nitrogen Compounds as Aqueous System Denaturants Numerous nitrogen compounds can be considered as aqueous solution denaturants, relevant to biological damage. Urea and guanidinium have this property shared with amines, such metal complexes as cuprammonium, ammonium salts, nitrosylated derivatives, cyclic amino oxides (Johnson et al 1966) and thiocyanate. 3.4 Nitrogen-Centred Free Radicals Chloramines (formed by immune response myeloperoxidase hydrogen peroxide and chloride generated hypochlorite) are reported (Hawkins & Davies 1998) to yield potentially damaging nitrogen-centred free-radicals (especially from lysine) which are repairable by reduction by ascorbate. Such repair could be a component of ascorbate tissue protection under autoimmune disease promotion conditions. Nitric oxide (NO) is a stable free-radical. The chemistry of NO , however, includes redox shifts to NO+ (nitrosium) and NO- (nitroxyl). 3.4.1 R-S-H Adducts of NO Cellular Redox Status, NO and Ascorbate Controlling Effect NO homostasis may involve linked oxidation-reduction of S-H groups including those in redox metal complexes (cf Stamler et al 1992); involvement of glutathione perhaps in concert with ascorbate might be anticipated. Synergy between various such species may be relevant to the tissue damage mechanisms. The effect of exogenous metals on the stability of such NO adducts may be relevant to the deleterious effects of iron overload (cf Singh et al 1996). Redox input into altered heparan sulphate microstructure at both the biosynthetic stage and post synthetic stage is believed to be of high importance for normal and disease and therapeutic-related control of cellular activity. 3.4.2 NO as an Antioxidant It has long been known that nitric oxide is an efficient antioxidant, e.g., radical chain reactions were detectable by stopping them by its injection(Hinshelwood 1957)and similar broad antioxidant function is also believed to be relevant to biology (Kanner et al 1991).

3.5 Metal Catalysis (Redox and Acid/Base Effects) in Active Nitrogen Chemistry

Significance of NO deaminative cleavage of heparan sulphate at physiological pH 3.5.1 Iron and Copper Vilar et al (1997) reported the deaminative cleavage at physiological pH of aceylated glucosamines by nitric oxide gas in the presence of phosphate buffer but not in the presence of imidazole buffer. The former situation is often affected by trace transition metal catalysts, especially by iron and copper, absent from the latter situation. Further experiments are required to test this possibility. Previously it had been though that acid pH <5 was required for deaminative cleavage. The details of the nitric oxide derivatives (perhaps involving effects of thiol (and metal) adducts) which are responsible for endothelial relaxation(Stamler et al 1992)) may also require further experimental work. Factors which influence the reactivity of nitric oxide metabolites include thiocyanate (Fan & Tannenbaum 1973) may also be a function of trace redox metal catalysts (Chalis et al 1978; Park & Aust 1998).
Redox metal complexes formed with the active nitrogen intermediates also serve as antibiotics by binding and deactivating pathogen redox centres but nitric oxide is scavenged by binding to iron in haemoglobin without deactivating it; immonium oxide formation has been proposed to account for the catalase-like activity of haem nitroxides and related compounds (Mehlhorn &amp; Swanin 1992). 3.5.2 Vanadiuim, Titanium (& Chromium) Redox metal activity analogous to Fenton-iron hydroxyl radical production which is observed for V(IV), Cr(III), Ti(III) analogously to the well-known Fe(II) reaction with hydrogen peroxide as discussed by Bodini & Sawer (1976) may also generate highly reactive nitrogen-centred free-radicals (such as from Cr(II) perchlorate studied by Wells & Salam (1968); Cr(III), however, which is highly reducing, would normally be expected to be rapidly oxidised by dissolved oxygen , e.g., in the stomach. The occurrence of redox processes involving protons such as TiO2+ 2H+ = e = Ti3+ + H2O indicate that Ti(III) and probably V(III) to be potentially more strongly reducing under neutral than pH 1 conditions where they may more readily stimulate active nitrogen formation. 3.5.2.a Possibly relevant V biochemistry

Although V(III) is usually stable in aqueous solution only at pH <3, it may, however, be stabilised at high pH values by ligands such as, e.g. may occur in tunicate blood. The ligand contained in fungal chlorooxidase shows V bound by three non-solvent oxygens, a histidine and a N3 azide group (Messerschmidt & Wever 1996) and the V(IV) in amavadin has NO and CO2 - binding groups (S,S)-2,2(hydroxyimino)dipropionic acid (Garner et al 1999). Bromo/iodoperoxidases containing V(V) in their active centres occur in brown and red seaweed and lichen. Horse muscle contains vanadyl ATP which mimics insulin effects. Some isopolyvanadates demonstrate heparin-like inhibition of inositol (1,4,5)triphosphate receptors but with low specificity (Potter & Nahorskji 1992). A vanadium-containing ascorbate complex was indicated to be the oubain-like factor in human urine (Kramer et al 1998). 3.5.3 Nickel (& Cobalt) Is the redox chemistry of Ni altered by biological ligands a possible risk factor for heparan sulphate deaminative cleavage? A reported Ni(II) induced alteration in EGF and beta TGF mediated growth control during malignant transformation of human kidney epithelial cells(Mollerup et al; 1996) could conceivably have a deaminatively cleaved heparan sulphate link e.g. via active nitrogen generated from redox processes facilitated by biological ligand stabilized Ni(III). 3.6 Peroxynitrite Peroxynitrite formed from nitrous acid and superoxide anion is believed to be an important reactive nitrogen species responsible for the carcinogenic DNA damage apparently previously ascribed to the effects of hydroxyl radicals (Beckman et al 1990) which may explain various carcinogenic effects of particulates including that of iron-rich varieties of asbestos (Park & Aust 1997). Peroxynitrite is also implicated in the aetiology of various neurodegenerative diseases (Beasl 1997; Cookson & Shaw 1999; Love 1999)including (familial) motor neuron disease (Rosen et al 1993; Calder 1995)this providing an explanation for the pivotal involvement of genetically defective superoxide dismutaseI. Key polysaccharide heparan sulphates implicated in antioxidant defense (cf Adachi et al refs)are also likely to be subject to peroxynitrite damage as well as to deaminative cleavage by other active nitrogen species.

3.7 Are Clusters of Aromatic- and Sulphur-Containing Amino Acids in Proteins Active Nitrogen Traps? A postulated antioxidant function of conserved clusters of tyrosines, cysteines and methionines in proteins (Grant et al 1989a) might be logically extended to include anti-active nitrogen function (in addition to the previously known nitrene reactivity) this according both with these groupings being potential indirect scavengers of peroxynitrite by deactivating hydroxyl radical precursors as well as likely acting as direct scavengers since nitro tyrosine is observed on reaction with peroxynitrite as are analogous derivatives from chloramines (Hawkins & Davies 1998). The formation of hydroxyl radicals by ascorbate, oxygen and Cu(II) led to free radical activated adducts producing randomisation around aromatic-cysteine S-S bond systems including insulin and oxytoxin. Cysteine-tyrosine systems have been postuated to be involved in cysteine directed signal transduction (crosstalk) of growth factors in gene expression(Nakashima 1996) inappropriate perturbation of which by active nitrogens might be relevant to the aetiology of cancer but normal involvement in nitric oxide in such signalling might be anticipated.

4. Ascorbate As with the biochemical role of heparan sulphates it is commonly stated that the biological function of ascorbic acid is not yet established. A role in redox control seems likely directly and indirectly inputting into heparan sulphate biochemistry. Ascorbate is believed to be a key aqueous phase extracellular antioxidant; this function is extended now to it performing key anti-active nitrogen protective functions. The antioxidant function is supposed synergistic with uric acid and with lipid-soluble tocopherol and glutathione; ascorbate is likely to provide aqueous phase extracellular antioxidant and antiactive nitrogen protection augmented by related intracellular activities of ascorbate sulphates and perhaps other ascorbate derivatives which may be later identified. An established role of ascorbate is in the in vivo utilisation of ascorbate is in dopamine norepinephrine conversion.
Adrenal glands and eye lens have a high ascorbate concentration. In studies directed to probe the possibility of damaging effects of high ascorbate dietary supplementation; the consensus of opinion seems to be that damage may easily be demonstrated in vitro but less easily in vivo. Minetti et al (1992) found no evidence for hydroxyl radical formation in esr studies of ascorbate free-radicals in moderately iron overloaded plasma although this had been previously observed. Phosphate and other ligands for iron may catalyse the iron redox damage in presence of oxygen, cf, ascorbate, Cu(II) and oxygen promoted hydroxyl radical formation in vitro, scrambling disulphide bonds in insulin and oxytocin (Inoue & Hirobe 1987). Podore et al (1998) discussed cellular DNA damage initiated by ascorbate which might, after all, have been of a protective nature (cf discussion by Levine, Salonen et al 1998). Older work identified pro-cancer effects of ascorbate (Fukushima et al 1983,1987). Other possibly detrimental effects of excess ascorbate dietary supplementation lie in the increased risk of calculi formation from oxalate crystals (ascorbate metabolite) and increased ascorbate promoted collagen crosslinking which might (in some instances adversely) affect arterial walls (Dwyor 2000) and perhaps also reduce the permeability of Bruch's membrane causing visual damage. Vitamin C dietary supplementation releases iron from insolubles such as oxalates and carbohydrate complexes (cf Sharma & Marthur 1995). Ascorbate deficiency likely impairs the correct biosynthesis of the extracellular matrix components collagen and glycosaminoglycans including heparan sulphate (cf Kao et al 1990); syndecan-1 mediated differential collagen interaction may be affected (cf Sanderson et al 1994). Ascorbic acid has standard oxidation reduction potential of 0.058V pH7 and has pKa = 4.2 (C3-OH); a di-anion is also formed by dissociation of C2-OH.

An X-ray crystallographic structure (Hughes 1973) of the Tl salt showed delocalisation of electrons between lactone ring carbons 1-3, the anionic charge being shared between O(1) and O(3) atoms. NMR studies show apparently various co-existing forms of ascorbate including the possibility of the formation of oligomers and polymers (Matusch 1977). Further simple to perform NMR studies are warranted. Ascorbate is irreversibly transformed at higheer pH values into ketogulonic acid and by browing reactions. Ascorbate sulphate esters, e.g., 2-O-sulphate, may have sulphotransfaerase properties and their oxidation reduction properties are likely to be relevant to tissue protection. <P>Evidence of anti-active nitrogen protection by ascorbate is afforded by the improvement of endothelial function by reversal of the oxidation of nitric oxide by free radicals in patients with chronic heart failure (Hornig et al 1998) and the likely role in anti-nitrite/nitrosamine carcinogenesis (e.g. Kirchner and Hopkins 1991). The activity of ascorbate derivates such as ascorbalaminic acid present in plants (Couchman et al 1972) may also be relevant to protection by dietary 'ascorbate' against excess nitrous acid in the stomach. The oxidation of ascorbate by dissolved oxygen is iron and copper catalysed(cf e.g. Minetti et al 1992) proceeding via the formation of ascorbyl and hydroxyl free radicals. Redox recycling by ferrioxidase activity in caeruloplasmin may contribute to iron overload dependent rapid oxidation of ascorbate in plasma. Copper-centred enzymic activity is also responsible for the ascorbyl oxidase activity of plants. Hydroxyl radicals generated from ascorbate peroxide and Cu(II) rearrange substituents around aromatic containing groups attached to disulphide bonds in peptides such as insulin (Inoue & Hirobe 1987) and cause microstructural alteration (perhaps to Nsulphonate groups) in heparin detectable by abrupt reduction in the anti-Factor Xa activity (a similar reaction with iron results in less selective alteration (Liu & Perlin 1994). Ascorbate may protect against damaging oxidative activity by higher oxidation states of chromium (Connett & Wetterhahn 1983) and perhaps vanadium. Older work on the degradative activity of ascorbate is likely to have been influenced by the ability of trace redox metals (usually present unless special measures are taken to eliminate them) to generate hydroxyl radicals (as in the degradation of gastric and salivary mucins and hyaluronic acid etc in cartilage, vitreous humour and pneumonococci capsular polysaccharides reported by Roberson(1941) by ascorbate + hydrogen peroxide in phosphate buffer). The formation of ascorbyl radicals/ascorbate degradation was found to be catalysed in a dose-dependent manner by Cu+, Cu2+, Fe2+, Mn2+, Fe3+ as well as by Zn2+ but not by Na+, K+ ,Ca2+ or Mg2+. Transition metals are normally strongly protein-bound in serum with the possible exception of Cu2+. Metal ions may regulate (Satoh &amp; Sakagaimi 1997) and be regulated by the biological activity of ascorbate (e.g. the iron homeostasis mechanism in lens epithelial cells which includes ascorbate-boosted de novo synthesis and likely ascorbate-assisted iron binding to ferritin (Goralska et al 1998)). Vanadyl ascorbate complexes may be involved in ATPase regulation (possibly relevant to human health) being detected in human urine (Kramer et al 1998). Maillard reactions, including threose ascorbate decomposition produts of abnormal ascorbate metabolism, may have a role in lens caractognesis (galactose exposed lenses showed enhanced crystallin-bound Schiff base-linked ascorbate degradation products, (Saxena et al 1996)). Cellular ascorbate activity (e.g. in erythrocytes) may mimic or augment that of NADH and glutathione having a role in the maintenance of oxidation-reduction potential. High dose ascorbate therapy is believed to counter infection by augmenting such NAD(P)H high energy electron provision, e.g., for providing the respiratory burst of phagocytes (Cathcart 1991).

Oxalate

Ascorbate eventually metabolises to oxalate which may crystallise under defective anti-mineralization inhibitor conditions (cf Singh 1993).

5. SUMMARY OF HEPARIN/HEPARAN SULPHATE BIOCHEMISTRY The most highly evolved glycosaminoglycans seem to be the heparan sulphates(mainly from the surfaces of most animal cells and extracellular matrix) and heparin (from mast cells and leukocytes but stored in endothelial cells (Hiebert & McDuffie 1989). Heparan sulphates, probably obligatory for multicellular animal organisms, are broadly similarly constituted to heparin but have characteristic differences (Gallagher et al 1985). The assembly of heparan sulphates has recently been indicated to be more complicated than previously thought, requiring multiple N-deacetylase/N-sulphotransferase (NDST) isoenzymes (Aikawa et al 1999). Precise sequence-dependent heparan sulphate microstructure is likely a fundamental component of much growth factor signalling (Krufka et al 1996) and heparan sulphate proteoglycans (HSPGs) are fundamental to the distribution and processing of extracellular signals that pattern fields of cells in developmental biology. The HSPGs syndecans and glypicans are associated with cell surfaces and perlecan (and agrin) with basement membranes(Perrimon & Bernfield 2000). Perlecan core protein synthesis and sulphation of heparan sulphate side chains were reduced by Pb2+ suggesting that this toxin inhibits endothelial cell proliferation by lowered response to bFGF (Fujiwara &amp; Kaji 1999). Nitrous acid perturbation of heparan sulphate dependent growth factor signalling in mucosal replendishment has been suggested(Ascensio et al 1995)to promote stomach cancer.
Although many biological processes (e.g. as listed by Belford et al 1992 who gave prior references)are known to have potential inputs from heparin/heparan sulphate biochemistry, it is still usually stated that the main biochemical role of these substances has yet to be established. Apart from growth factors small ions are bound to heparin/heparan sulphate which may function in part as a conveyor belt for their distribution and this function may be influenced by a form of hydration dependent phase change behaviour discussed below. The original function of glycosaminoglycans may have been for provision of a correct water and ionic biological environment similar to the believed function of polysaccharides secreted by bacteria, the extracellular polysaccharides of algae and even the polyanionic humic acid which confer biofriendly hydration and mutinutrient reservoir properties on soils. Evolutionary pressure in development of multicellular organisms would require more complex functions such as the provision of anchoring sites (a 'workbench' or processor function) for a variety of effectors required to orchestrate such complex processes as haematopoiesis (cf Gupta et al 1998) and anticoagulant /coagulant activities including antithrombin (III) binding and processing (cf Rosenberg & De Agnostini 1992; Bourin &

Lindahl 1993); their distant ancestral precursors evidently resembled bacterial capsular polysaccharides (Cf [GlcUAGlcNAc]n which can be structurally manipulated in vitro to yield heparin analogues (Lormeau et al 1992). An unusually evolved flexibility of iduoronate rings may facilitate adoption of multiple environmentally directed conformations by heparin, heparan sulphate and dermatan sulphate (e.g. Sanderson et al 1995; Grant et al 1991). A similar evolution of increased functional complexity is also evident in the extracellular polysaccharides of plants (McNeil et al 1984). It is this structural complexity of the highly evolved heparan sulphate structures, dependent on highly specific polysaccharide microstructures (cf Perrimon & Bernfield 2000) requiring correctly arranged substituted glucosamine sugars, which renders them especially susceptible to damage by nitrous acid generated by the cellular immune response to pathogens which bound to cell surface heparans sulphate chains. Formation of complexes between heparin/heparan sulphate and proteins is often critical for stabilising protein conformation and aiding correct functionality, such complexes are formed by polysaccharide binding to groups of basic amino acids which include highly consderved consensus sequences such as the Trp-Ser-Pro-Trp sequence bounded by basic amino acids (Guo et al 1992). Consensus sequences for glycosaminoglycan recognition were determined as X-BB-X-B-X and X-BBB-XX-B-X where B is a basic residue and X is a hydrophobic residue (Cardin & Weintraub 1989). Faham et al reported the first crystal structure of a protein-GAG complex(for bFGFheparin); computer simulation of its receptor docking (Bitomsky &Wade 1999) can lead to insight into such complex formation, perhaps improved by including solvation effects. It is evident that nitrous acid or other active nitrant attack on vulnerable amides may affect both parts of the binding sites. Heparin/heparan sulphate proteoglycans are believed to be involved in haemostasis, fibrinolysis, capillary permeability, matrix assembly, potentiation of mitogenic, chemotactic, neurotrophoc and angiogenic activities, inter and intracellular communication, cellular recognition, immunological signalling, adhesion, differentiation, proliferation, wound healing, apoptosis, gene expression, capacitation, morphogenesis, embryogenesis, nerve and brain development and function (neurite outgrowth, synaptic function, myelination) the provision of links between extra and intracellular space, phosphatidyl inositide anchoring and cytoskeleton function, lipid processing, glomerular filtration and antioxidant protection, involving both the intact polymers and, at least in some instances, fragments. (Oligosaccharides formed during normal heparin/heparan sulphate turnover have, however, likely specific intracellular functions following endocytosis as well as protecting the blood and urinary systems against damage by crystal formation and inhibiting viral, bacterial and prion infection). It may be no coincidence that nitric oxide is also implicated in similar functions and therefore active nitrogen processing of heparin/heparan sulphate may be a normal part of such biological activity. The presence of tightly bound Cu2+ in heparin and heparan sulphate but not in other GAGs (Rej et al 1990) may conceivably influence such 'active nitrant' processing. Nitric oxide synthase activity is reported (Kouretas et al 1998) to be modulated by non-anticoagulant heparin (of possible therapeutic value) via a guanine nucleotide regulatory protein mechanism . Nitric oxide production by bovine endothelial cells has been correlated with shear stress (Korenaga et al 1992); heparan sulphate is also thought to be involved in flow sensing (e.g. by polymer coil conformation alteration caused change in Ca2+ and Na+ balance, studied by Siegel et al 1998). The biosynthesis of endothelial heparan sulphate proteoglycans was reported to be greatly influenced by flow, although no feedback effect of polymer fragments could be detected (Grimm et al 1988).

5a.

ACTIVATION OF HEPARAN SULPHATE FOR NITROUS ACID CLEAVAGE BY REMOVAL OF PROTECTIVE GROUPS FROM GLUCOSAMINE NITROGENS Initial proton-catalysed removal of the sulphonate groups, or reaction of acyl groups with hydrazine, yields glucosamines which are much more highly reactive towards nitrous acid than are the acetyl or sulphonate substituted glucosamines of native heparan sulphate. The stability of the N-sulphonate groups(studied with heparin) towards(nitrous) acid catalysed hydrolysis is affected by counterion binding by a mechanism which is believed to involve hydration changes and phase boundary effects. Alternative explanations(due to Manning)in terms of a critical charge density, above and below which outer or inner ion-binding occurs, was not substantiated by detailed analysis; additional catalysis of such effects may occur by iron, copper and perhaps vanadyl ions. A tightening or loosening of the hydration sheath is engendered by cation-dependent binding with alteration in the accessibility of the N-sulphonate groups and susceptibility towards nitrous acid cleavage. The standard procedure uses replacement of counterions by pyridinium ion to render the molecule more readily de-Nsulphonated (an example of synergy between nitrogen-based reagents in the degradation of heparin). This counterion alteration effect evidently induces the formation of a less hydrated form of the heparin molecule. These situations may mimic pro-disease scenarios related to heparan sulphate damage. Unsubsituted glucosamines in heparin/heparan sulphate (which will undergo immediate nitrous acid scission) were originally believed to be absent from tissue heparan sulphates or to be extraction artifacts, since the currently accepted mechanism of the biosynthesis of heparin/heparan sulphate requires that all glucosamine amino groups must be either N-acetylated or N-sulphonated (Kjellen et al 1992); such a rigid requirement seems, however, to be in disagreement with the recent reports of the presence of unsubstituted glucosamines, immediately deaminatively cleavable by nitrous acid at pH 4.6, in heparan sulphate present in endothelial cells although absent from heparan sulphate similarly obtained from CHO cells (Norgard-Sumnichi & Varki 1995, cf Van den Born et al 1995).

The presence of unsubstituted glucosamines in vivo may be evidence for the occurrence of pathological postsynthetic modification. The free amino groups in pharmaceutical heparin detectable from nitrophenylation, included both amino acid end groups and glucosamines (Arai et al 1993). Older work (Linker &amp; Hovingh 1973) reported unexpectedly larger amounts of unsubstituted glucosamines in heparan sulphate from amyloid liver but not from healthy tissues which may indicate that catalysis of the de-N-sulphonation by e.g. quaternary nitrogen counterions or trace transition metals occurred in the acidic proteolysis extracts or even a disease-related elevation of free glucosamine residues in heparan sulphate biosynthesis. Further studies of heparan sulphate alteration by active nitrogen (including possible iron and copper catalysis) in conjunction with effects of other metal ions, notably zinc, aluminium as well as silicates are warranted in this context. Heparan sulphates with sulphate-rich segments, which inhibit inappropriate arterial well cellular proliferation, were diminished in arteriosclerosis patients. Oligosaccharides produced by nitrous acid deaminative cleavage of heparan sulphate at pH 3.9 showed marked antiproliferative activity against smooth muscle cells (of relevance to the prevention of arteriosclerosis) but this property was absent from oligosaccharides produced by cleavage of heparan sulphate conducted at pH 1.5 (Schmidt et al 1992). Extended sequences of 7-8 N-sulphonated disaccharides, in which a proportion of the iduronic residues are sulphated at C-2, appeared to be required for cell adhesion and migration (Walker et al 1996). The following cancer-related situations (cf Ferguson 1975) may also conceivably involve heparan sulphaterelated signalling processes: conversion of carcinogenic aromatic amines to N-sulphonated derivates (possibly mimicking heparin/heparan sulphate structures) was apparently required to induce cancer and various nonalkylating antileukaemic drugs possess a potency apparently related to the possession of O-N-O triangulation sites reminiscent of N-sulphonate centred binding in heparin/heparan sulphate.

5b.EVIDENCE FOR THE RELEVANCE OF HEPARIN/HEPARAN SULPHATE BIOCHEMISTRY TO THE AETIOLOGY OF CANCER

The effect of heparin and related compounds on cell and tissue growth has been recognised since 1932. Release of heparin/heparan sulphate from cells was found to be in synchrony with the cell cycle (Chiarugi et al 1974). Heparin and heparinoids have demonstrated substantial antitumour effects in animal models (Zacharski 1988) and are likely to have similar effects in humans with potential therapeutic benefit (Engleberg 1999). Plant xylan derived heparinoids have demonstrated anti-tumour activities (Wessel et al 1998). Some anti-cancer drugs while supposed to act by deactively crosslinking of DNA may (or in addition) block proliferation-related cell surface sites as appears to be the case for the inhibition of cell proliferation by Suramin(Nakajima et al 1991)a nonpolysaccharide heparinoid (Grunicke 1991). At least two members of the EXT family of 'tumour suppressors' were found to encode D-glucosyl and N-acetylglucuronyl transferases required for the chain elongation of heparan sulphate (Lin & Lindahl et al 1998). The HSPG syndecan-1 is reduced in cervical cancer where excessive expression supressed the growth of the cancer cells (Nakanishi et al 1999). Heparin (but much less so de-N-sulphonated heparin) was indicated to have multiple potential immuno-enhancing effects, including stimulation of IL-1 mediated activities inducing increased cytotoxic responses against histocompatible tumours in mice (Dziarski 1989, 1992). Heparin/heparan sulphate binding growth factors appear to be involved in cellular physiology of relevance to cancer. Heparin affinity regulated peptide (HARP) was associated with epithelial cells in prostate cancer but not with those of normal or benign hyperplasia and may be involved in the regulation of prostate tumour cell proliferation (Vacherit et al 1999). Heparin selectively inhibits cell cycle phases in endothelial cell proliferation (Kimura et al 1992) and regulates protein tyrosine phosphorylation in vascular smooth muscle cells(Mishra & Castellot 1999). Heparan sulphate-dependent internalisation and catabolism likely controls the in vivo availability of bFGF (Colin et al 1999) and bFGF alters the intracellular distribution of the GAG degradation products (Tumova et al 1999). Many pathogens are known to initially interact with the host via a heparan sulphate linkage (David 1998); this includes organisms which are known to promote cancer (especially noteworthy is Helicobacter pylori which may perturb b-FGF heparan sulphate signalling putatively linked to stomach cancer by Ascensio et al (1995) in agreement with more virulent strains demonstrating increased heparan sulphate binding ability (Chmiela et al 1995)). Ascorbyl radicals were increased with increasing H pylori acitivity in these tissues (Drake et al 1996) in accord with for an antioxidant/nitrant anti-cancer role of ascorbate in countering

immunologically produced anti-bacterial responses which if inadequete would lead to inappropriate degradation of heparan sulphates. Cancer cells often show a large difference from normal cells in their surface heparan sulphates both in amount and in microstructure, most characteristically by a decrease in amount and decrease in charge density of the surface bound heparan sulphates. This is likely to be a consequence of altered biosynthesis as well as augmented degradation both by enzymic and non-enzymic (including oxidant/nitrant) routes. In the cell culture model of cancer, an alteration in heparan sulphate biosynthesis is found in both cellular transformations produced by tumour viruses and by nitrosoguanidine derivatives (cf Woodhead et al 1987). More recent studies identify types of HSPG putatively involved (e.g., syndecan-1 studied by Nakanishi in 1999). Oncogenic transformation may also be accompanied by biosynthesis of heparan sulphates of increased charge density (Rahmoune et al 1996). High grade glioma derived cells expressed markedly increased amounts of hyaluronic acid and heparan sulphate (Steck et al 1989). The cancer inducer phorbol ester triggers HSPG-dependent tumour cell adhesion (Timar et al 1996). Heparan sulphate moieties, and not the core protein, of a phosphatidyl inositol anchored proteoglycan, initiated the mouse melanoma cell adhesion to fibronectin (Drake et al 1992). The relative virulence and metastatic potential of cancer cell lines often correlates with their secreted heparanase acitivites (Nakajima et al 1986-1992). Katz et al(1996) showed that inhibition by heparin of lung colonization by in vitro virally transformed 3T3 cells rendered highly tumorigenic by in vitro passage, was achieved by the inhibition of the transformed cell heparanse activity. The degradation of basement membrane by prostate tumour heparanse is believed to be a key factor in the malignancy and potentially may be countered by inhibition of the GAG-ase (Kosir et al 1999). Basement membrane heparan sulphates studies of implanted Lewis lung carcinomas showed that more highly metastatic cancer cells possessed greater basement membrane organization, this being perhaps asttributable to the effects of accompanying heparan sulphates with greater overall sulphation (Nakanisha et al 1992). Heparin/heparan sulphates provide binding sites for the major extracellular superoxide dismutase (cf Adachi et al) and were indicated to possess intrinsic antioxidant effects (Grant et al 1987b) demonstrating some intrinsic superoxide dismutase activity (Grant et al 1988) and also binding trace transition metal(e.g. iron) ions thus countering potential free-radical and peroxynitrite DNA damage (Ross et al 1992).

5c CANCER-RELATED METAL ION BINDING BY HEPARAN SULPHATES Heparan sulphate biochemistry seems to be implicated in various cation transportations of relevance to cancer.

Transferrin glycosylation is believed to include heparan sulphate side chains (Fransson et al 1984)and tumour cells may have more heparan sulphate directed iron entry. Anti-tumour chelation therapy may include interruption of such iron transport. Trace bound redox metals may contribute to the superoxide dismutase activity of heparin although activity was found to remain after attempting their removal and sequestration (Grant et al 1988). Altered heparan sulphate in tumour cells allows radioctive tracer tumour imaging (gallium was believed to mimic iron binding; (cf transferrin binding sites are believed to have heparan sulphate side chains). Gallium ions stick selectively to tumour and inflammatory lesions via binding to heparan sulphates((Kojima et al 1983; Table I of this article reports that heparan sulphate exhibited much higher binding than heparin) cf also Hama et al 1983 and 1984) endocytosis and degradation of gallium-heparan sulphate was indicated to occur in lysosomes. Therapeutic utilisation of gallium nitrate for the normalization of hypocalcemia of malignancy is reported (Warrell et al 1991) superior to other treatments such as ethane 1-hydroxyl,1,1-diphosphonate (a blocker of CaCO3 crystal nucleation of similar activity to pyrophosphate or heparan sulphate Grant et al 1989b). A complex calciumsensitive heparan sulphate cell engulfment has been demonstrated in parathyroid cells (Takeuchi, Yanagishita et al 1990) and similar mechanisms may also apply to other cell types. Formation of crystals capable of surface enrichment of bound redox-metals capable of initiating free radical cascades as has been hypothesised for asbsetos-linked cancerigenesis may also apply to other pathological crystals. Heparin/heparan sulphate has a role in the prevention of such crystallisation but N-sulphonate depeleted heparin was found to be much less effective (Grant et al 1989b). Calcium-rich crystals may also generate mitogenic signals directly, pathological calcification may therefore be both a cause and an effect of malignancy (cf discussion of relevant literature by Grant et al 1992b). In summary, metal ion homeostsis is likely subect to alteration by disease processes which affect and are affected by heparan sulphate microstructures.

5e. EFFECTS OF OXYGEN, GLUCOSE, TOXINS & ASCORBATE ON HEPARAN SULPHATE MICROSTRUCTURE Cell culture experiments have indicated that heparan sulphate biosynthesis may be sensitive to cellular oxidation-reduction status but the details vary between cell types. Heparan sulphates from bovine arterial endothelial cells showed dependence of heparan sulphate biosynthetic patterns on oxygen pressure, hypoxia inducing reduction in chain length and decrease in sulphation (Karlinsky et al 1992). Chlorate is used routinely in vitro cell cultures to decrease sulphate incorporation into heparan sulphate, monensin having similar effects (Sampaio et al 1992). Ascorbate, however, specifically boosted the biosynthesis of heparan sulphate in fibroblast cell culture and increased sulphate incorporation into the polymer (Kao et al 1992); a mechanism involving redox status seems indicated. Ascorbate thus may both potentially promote biosynthesis as well as provide protection against inappropriate post-secretion damage to heparan sulphate by active oxygen/nitrogen. (Priior work had demonstrated that ascorbate increased collagen and glycosaminglycan synthesis).

5ei Effect of glucose on heparan sulphate biosynthesis

In a model of diabetic nephropathy glucose augmentation in mouse glimerular epithelial culture differentially reduced the biosynthesis of various heparan sulphate proteoglycans (Morano et al 1999)(this effect likely impairs glomerular filtration). 5eii Effect of toxins on heparan sulphate biosynthesis Toxins (e.g. lead (Kaji et al 1991) cadmium (Cardenas et al 1992) and endotoxin (Dietrich et al 1996) reduced heparan sulphate biosynthesis. There may be an additional, normal, age-related diminution of heparan sulphate as a proportion of the total glycosaminoglycans at arterial walls where a linear decrease with age has been reported (Murata &amp; Yokoyama 1989). The effects age, excess glucose, toxins and ascorbate deficiency(through dietary insufficiency or in smokers) may impair heparan sulphate function.

6. Research into intervention of carcinogenesis and other degenerative diseases using the above model might reasonably consider heparan suphate replacement therapy via administration of preformed heparins, semisynthetic heparinoids or dietarty supplementa of ascorbate, glucosamine or glucosamine sulphate or provision of additional protection by other ascorbate derivatives, improved inducible nitric oxide synthase inhibitors used in conjunction with redox metal chelation therapy. (Perhaps inhibitors of inducible nitric oxide synthases studied for rheumatic disease therapy e.g. dietary components, warrant investigation as anti-cancer agents). The hypothesis of a central role of redox status for cellular activity control in which heparan sulphate microstructure is involved offers a new framework for research.
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APPENDIX 8.a. TRANSITION METAL CATALYSIS OF REACTIVE NITROGEN DAMAGE (further notes) 8a-1. Iron & Copper Catalysis of Nitrosylation & Deaminative Cleavage. 8a-2. Nitrous Acid Reduction to Nitric Oxide by Ascorbate. Effect of Redox Metals. 8a-3. Anti-Cancer Effects of Nitrite. 8a-4. Deaminative Cleavage of Heparin/Heparan Sulphate at Physiological pH. 8a-5. Thiocyanate Effects. 8a-6. Phosphate Effects. 8a-7. Multi-Ion Binding Properties of Heparin/Heparan Sulphate. 8.a-7-1. Pathological Species May Seek Nutrients from Heparin/Heparan Sulphate. 8.a-7-2. Spark Source Mass Spectrometry of Heparin. 8.a-7-2-1. Ppm Elements Before and After Cation Exchange. Possible Pathologically-Relevant Redox Metals.

8.a-7-2-2 Titanium. 8.a-7-2-3. Chromium. 8.a-7-2-4. Vanadium. 8.a-7-2-5. Manganese. 8.a-7-2-6. Molybdenum. 8.a-7-2-7. Silver. 8.a-7-2-8. Lanthanum, Neodymium, Zirconium & Yttrium. 8.a-7-2-9. Cerium & Antimony. 8.a-7-2-10. Mercury & Cadmium. 8.a-7-2-11. Aluminium. 8.a-7-3. Nickel Effects. 8.a-7-4. Anions in Heparin. 8.a-7-4-1. Phosphorus 8.a-7-4-2. Silicon. 8.a-7-4-3 Boron. 8.a-7-5. The Binding of Aluminium & Fluoride to Heparin 8.a-7-6. Importance of Nucleation Factors & Lipids in Induction of Polysaccharide Supramolecular Structures. 8.a-7-6-1. Hydrophobic Polyanion Binding Conditions. 8b. Heparan Sulphate, Brain Development, Function & Neurodegenerative Diseases Including Alzheimer's Disease (AD). 8b-1. Introduction. 8b-2. Heparin/Heparan Sulphate in AD. 8.b-2-1. Learning & Heparin/Heparan Sulphate. 8.b-2-2. Zinc in AD. 8.b-2-3. Cognitive Function in AD. Therapeutic Use of Heparin/Heparan Sulphate. 8.b-2-4. Neurite Outgrowth Stimulation by Heparin/Heparan Sulphate. 8.b-2-4-1 Possible Heparin/Heparan Sulphate-Dependent Growth Factor Effects in AD. 8.b-2-5. Extracted Brain Heparan Sulphate - Lack of Correlation with AD. 8.b-2-6. Possible Transferrin Receptor Function. Aluminium & Heparin/Heparan Sulphate in AD. 8.b-2-7. Fenton Reaction/Free-Radical Damage in Neurodegenerative Diseases. 8c. Supramolecular Structure of Heparin/Heparan Sulphate PGs and their Binding Potential. 8c-1 Silicates in GAGs. 9. Involvement of Heparin/Heparan Sulphate in Modulation of Prion Protein Activity. 10. Water Binding to Heparin/Heparan Sulphate. 10a. Hofmeister Effects on Water Structure. 11. Modulation of Crystallization by Heparin/Heparan Sulphate. 12. Established Laboratory Use of Nitrous Acid for Heparin/Heparan Sulphate Analysis. 13. Binding of Pathogens to Heparan Sulphate. 14. References. 15. Acknowledgements. 8a-1. Iron & Copper Catalysis of Nitrosylation & Deaminative Cleavage. Dietary nitrite is traditionally thought to lead to carcinogenic nitrosamine formation; iron-nitroso complexes (imparting a characteristic red colouring to cured meat) potentially enhance nitrosamine formation under iron overload conditions e.g. exacerbate by chronic (autoimmune) nitric oxide production which may contribute to the aetiology of various degenerative diseases including cancer, arthritis and Alzheimer's disease in which inappropriate nitrous acid deaminative cleavage of heparin/heparan sulphate polysaccharides, (important mediators of immunological signalling, haemostasis, cardiovascular function and wound healing) may be involved. Iron may catalyse such deaminative cleavage both by pre-priming heparan sulphate via de-Nsulphonation (unpublished observations of the author) and by directly catalysing the nitrous acid depolymerizaton process. Available iron, indicated by serum ferritin, may increase naturally with age in the absence of specific observable disease (Jarrett et al 1989) according with a redox-metal centred reactive oxidant (or nitrant) hypothesis of ageing.

The depletion of heparan sulphate in atherosclerotic arteries increased linearly with age (plot of data in Table 4 of Murata & Yokoyama (1989)) this effect may be related to an iron dependent heparan sulphate degradation in atherosclerosis. In vitro experimental evidence for iron and copper salt catalysis of nitrosoamine formation, although not widely researched, was provided by the studies of Challis et al (1978). Evidence for possible transition metal-related reactive nitrogen damage to health is :an increased incidence in men than pre-menstrual but not post-menstrual women of upper stomach cancer supposed to be promoted by nitrosamines (McColl 1999); an iron-catalysed nitric oxide/nitrous acid DNA damage implicated (Park & Aust 1998) in the mutagenicity of iron-rich asbestos and an iron-overload correlated mortality of Eastern Finnish men (from serum ferritin epidemiological studies reported by Salonen et al 1992). A possibly related ability of heparin to inhibit free-radical reactions initiated by Fe(II) was found (Ross et al 1992) to be dependent on the integrity of heparin, damage produced to heparin by nitrous acid eliminating this protective effect. 8a-2. Nitrous Acid Reduction to Nitric Oxide by Ascorbate. Effect of Redox Metals. Ascorbic acid influences the availability of nitrous acid by efficiently reducing it (back) to nitric oxide. Iron and copper ions impair this anti-nitrite protection by catalysing the reaction of ascorbic acid/ascorbate with molecular oxygen (to give dehydroascorbic acid). 8a-3 Anti-Cancer Effects of Nitrite. Nitrite, supplemented by that produced by (molybdenum enzyme-dependent) reduction of dietary nitrate, can also have an anti-cancer effect (Benjamin 1997) probably by killing cancer-inducing bacteria. The balance of beneficial and damaging influences of nitrite/nitrous acid is suggested to be a function of the activation of spurious redox metals capable of enhancing nitrosylation and deaminative cleavage of heparin/heparan sulphate, balanced by the availablilty of reductants, principally ascorbate for blocking these reactions and the effectiveness and availability of redox metals in the oxidative depletion of ascorbate. 8a-4. Deaminative Cleavage of Heparin/Heparan Sulphate at Physiological pH. Nitric oxide, previously not thought capable of producing cleavage of N-sulphonated or N-acetylated heparin/heparan sulphate at physiological pH, is now established from the work of Vilar et al (1997) to undergo cleavage by nitric oxide gas in vitro, under conditions of phosphate, but not imidazole buffering. The effect of trace iron and copper impurities normally present in phosphate buffers (Nader & Krishnamoorthy 1983, cf Singh et al 1996) is surely relevant to this situation. Further investigation is required. 8a-5. Thiocyanate Effects Thiocyanate, augmented in the saliva and serum of smokers (Boyland & Walker 1974; Zhang et al 1997) is a potent catalyst of nitrosylation (McColl et al 1997) facilitating the formation of nitrosium cations, and possibly additionally complexing with and enhancing the deaminative catalytic characteristics of thiocyanate metal complexes. The effect of thiocyante on the deaminative cleavage of heparin/heparan sulphate requires investigation. Thiocyanate-promoted denaturation of biological hydrogen-bonds may allow deep penetration of associated redox metal complexes such as those of copper, chromium, vanadium and iron (the bright red iron (III) thiocyanate complex is well-known sensitive laboratory test for ferric ions) should be considered as potential catalysts of heparan sulphate cleavage by active nitrants. 8a-6. Phosphate Effects. Phosphate containing ligands may increase damaging activity of redox metals both by oxidative and nitrant routes. Phosphate, polyphosphates and phosphate ester-liganded redox metals are likely to both enhance nitrosylation reactions and also to increase the depletion of ascorbate by oxidation processes. The form and availability of more active forms of liganded phosphate may be critical to such catalytic processes. Experimental studies are needed to assess catalysis of such reactions as well as the relevance of perhaps related stochastic scrambling, long known to be a characteristic of abiological phosphate/polyphosphate and related systems (Van Wazer 1959); applied to DNA this results in mutagenic effects, as yet is unevaluated by biochemists). 8a-7.

MULTI-ION BINDING PROPERTIES OF HEPARIN/HEPARAN SULPHATE. The more highly charged segments of heparan sulphate (sometimes as heparin-like segments) may provide multi-trace-metal binding sites which provide antioxidant protection as well as nutrient ion reservoirs. 8a-7-1. Pathological Species May Seek Nutrients from Heparin/Heparan Sulphate. Inappropriate active nitrogen depolymerization may provide pathological organisms with the otherwise growth-limiting nutrients. The large number of pathological organisms which become attached to heparan sulphate may do so in part to gain such nutrient advantage. 8a-7-2. Spark Source Mass Spectrometry of Heparin. Numerous transition metals assayed by spark source mass spectroscopy in ('sodium') heparin from mixed mammalian sources with a very high trace metal loading (the trace metals are presumably present in the original animal polysaccharide as well as being picked up during processing e.g. from tap water (a major drug company provided for academic study a presumably non-commercial high-ash heparin (far outwith the allowable specification believed to be a common preparation 'problem'; EDTA is apparently used to diminish the unacceptably high toxic ash contents of some preparations and residual EDTA poses an contaminant problem in pharmaceutical heparin)) in addition to the presence of major cations reported by Grant et al (1987a) which were (ppm before/after cation exchange resin percolation):- nickel(170/5), molybdenum(7/0?), chromium (30/1); the sample also contained vanadium; manganese, however, was below the detection limit of 1ppm (in ppm before/after Tl+ replacement on a cation exchange column). 8.a-7-2-1. Ppm Elements Before (x) and After (y) Cation Exchange. (listed thus (x/y*)) Cations Iron & Copper Caesium & Rubidium iron(1100/10*), Cs 9/0*, copper(730/,5*) Rb 3/0.2* Lead, Arsenic & Tin Pb 16/4*, As 15/1*, Sn 5/0.3* Possible Pathologically-Relevant Redox Metals. 8.a-7-2-2. Titatnium Titanium showed up relatively abundantly in heparin at (>390/4*). If present in a polymerized oxybridged Ti oxy/hydroxy species it would be less damaging than if, e.g. liganded by halide. 8.a-7-2-3. Chromium Cr in heparin was (30/1*). Part of the anti-cancer role of ascorbate might be to counter pro-oxidant effects as in the chemical reduction of Cr(VI) which is likely to be responsible for ascorbate protection against chromate induced cancer (Connett & Wetterhahn 1983). 8.a-7-2-4. Vanadium Vanadium, found in lesser amounts than the above elements in mammalian heparin (3/?*), could partly originate from fungal sources where it is abundant, forms similar medium stability complexes with thiocyanate to thiocyanate-iron(III) (but iron(II) interacts more weakly)(Bahta et al 1997) but may sometime pose a 'vanadium overload' threat. 8.a-7-2-5. Manganese 8.a-7-2-6. Molybdenum Molybdenum dependent enzymic action is involved in the oxidation-reduction biology of nitrogen compounds. Mo in heparin was (7/?*). 8.a-7-2-7. Silver Silver occurs in mammalian heparin. Ag (4/0.5*), and may be similarly involved in thiocyanate-related pathology. 8.a-7-2-8. Lanthanum, Neodymium, Zirconium & Yttrium.

La 7/1*,W 5/0*, Nd 5/0*, Zr 5/0.3*,Y 3/0* were present in significant amounts in the studied heparin. 8.a-7-2-9. Cerium and Antimony Cation exchange replacement of cations by Tl+ appeared to scarcely remove Ce (7/3.5*) and Sb (2/1.7*); it must be assumed therefore that these elements occur in anionic or form unusually strongly bound adducts with heparin or are artifacts of the analytical procedure. Inappropriate release of such stored metals may stimulate inappropriate, pro-disease, redox activity. 8.a-7-2-10. Mercury and Cadmium Mass spectral analysis shows that although mercury is below the detection limit in the above mammalian animal heparin, both mercury and cadmium have been reported (Muzzarelli 1983) to be present in chitosan evidently via marine pollution; intoxication from this source and dental fillings have been listed in Internet discussions of mercury toxicity to be a major causative factor in AD. Further work is required to fully epidemiologically quantify such trace metal loading of heparin/heparan sulphate and other mammalian GAGs and to determine possible pathological links. 8.a-7-2-11. Aluminium The sample was not assayed for Al because of the use of an aluminium powder sampling procedure. Further work is required to assess Al contents of high ash and normal pharmaceutical heparin. 8a-7-3. Nickel Effects. Nickel metal provokes hypersensitivity in some persons. Nickel ions normally do not demonstrate well-defined aqueous solution redox behaviour, but heterocyclic N-containing ligands have been observed to stabilise otherwise difficult-to-produce Ni(III) (Lindol, 1975) which may redox cycle with Ni(II) leading to the formation of active-oxygen and active-nitrogen species. As Ni is employed in mammalian enzyme centres, relevant Ni-dependent heparan sulphate interaction might be anticipated. Further studies are required to test this possibility. Ni in heparin was relatively abundant (170/?*). 8a-7-4. Anions in Heparin. Major, likely anionic 'contaminants' detected by mass spectrometry in the above heparin sample were also surprisingly largely removable by cation exchange resin treatment, including 2000ppm chloride, 130 ppm bromide, 10 ppm iodide and surprisingly 890 ppm fluoride (removable to 4ppm upon Tl+ exchange, therefore likely to be attached to heparin-bound cations such as Al3+). 19-F NMR studies might be of value in establishing the mode of binding of such fluorine to heparin. Strong binding of heparin to iodide, chloride and sulphate has previously been described in a Patent claim (Fo-We, 1962). In a 1980 review Jaques stated that heparin exhibits "strong binding of counterions of sodium, potassium, ammonium, quaternary ammonium radicals, and co-ions such as sulfate, phosphate and acetate". Sulphate anions in heparin were found not to be removed by dialysis (Simon 1982) as would be expected if present as a consequence e.g. of de-N-sulphonation; unpublished observations of the author confirmed that a proportion of the 35S labelled sulphate in heparin may behave anomalously on gel filtration separation in keeping with the formation of a bound sulphate anion-heparin complex. 8.a-7-4-1. Phosphorus The above analysis also showed the presence of 440ppm phosphorus (which was reduced to 30ppm on cation exchange resin treatment), likely present as phosphate ((1348ppm as PO43- or other P(V) oxyspecies derivatives) in heparin. Additional interest of the high phosphorus content of heparin is related to a likely influence of such phosphate and related species in enhancing damaging metal redox behaviour. Condensed phosphates if bound to heparin/heparan sulphate would be expected to provide strong metal ion binding sites. Calcium in the studied heparin was 30000ppm which was reduced to 30 ppm upon passage through the cation exchange resin column in agreement with the chemical environment of heparin-bound phosphorus being possibly present as a calcium P(V)oxy-species-heparin complex. Ir spectra of a heparin solution peptized hydroxyapatite (obtained in studies of the inhibition of crystallization of this substance by heparin) confirmed the presence oxy-phosphate attached to the heparin chains in such a way as to significantly alter the heparin ir asymmetric S-O vibration absorbance bands (Grant et al 1992, unpublished). 8.a-7-4-2.

Silicon The mass spectral results of heparin discussed above also showed 5900ppm Si, 100ppm being retained bound to the heparin after passage through a cation exchange resin. Clearly a large part of the silicon is likely to be present as silicate and be bridged to heparin by cations such as Ca2+ and Al3+ . Schwarz (1973) found that polyuronides, including GAGs, contained strongly linked (molybdateinactive) silicate, (e.g. as a crosslinking silicic acid derivative, 29-Si NMR would be usefully employed to determine the environment of Si in such complexes); this author reported that heparan sulphate had 427ppm Si which could not be removed with 8M urea. Grant et al (1992f) presented experimental evidence for the importance of multi-phase phenomena in various cation (including calcium)-heparin interaction as an altenative explanation to the Manning hypothesis for the existence of critical binding behaviour. Such a phase change mechanism may explain why inorganic anions are often irreversibly bound to the highly anionic polysaccharides (of additional likely relevance to understanding the mechanism of DNA and RNA cation binding where a similar situation pertains) and may further be of relevance to the possible gating of ion, including proton, conduction by such polymers as well, perhaps being relevant to understanding of the apparently unrelated phenomena such as the physical chemistry of the formation of pathological amyloid (heparan sulphate containing) deposits in which metal ion loading may have some causative participation (cf, Ca2+ but not other M2+ except non-physiological Cd2+ mediated the association of human serum amyloid P component with heparan and dermatan sulphate (Hamazaki 1987)). A mechanism (Long & Williamson 1979) of control of cellular proliferation by heparin/heparan sulphate interactions with Ca2+ might be extended to include the co-binding of phosphate and silicate to Ca2+ (perturbable by such cations as Al3+?) to heparin/heparan sulphate and be of further possible relevance to the involvement of GAGs in calcification. 8.a.-7-4-3. Boron. The mass spectrometric results showed that the starting heparin contained <25ppm B, likely as borates or related anions, reducible to <1ppm after cation exchange. 8.a-7-5. The Binding of Aluminium & Fluoride to Heparin. A previous indication by Kojima et al (1983) that Al3+ binds strongly to heparin/heparan sulphate was confirmed by Grant et al (unpublished results obtained using potentiometric and osmotic methods). Both heparin/heparan sulphate biochemistry and (more controversially) the effect of aluminium intoxication, have been implicated in neurodegenerative diseases such as AD (e.g. Lehmann 1992). Fluoride intoxication at high levels is reported to alter the GAG content of bones (Susheela & Jahr; Priince & Navia) which might include the biological effects of Al3+-polysaccharide binding (perhaps causing perturbation of Ca2+-heparan sulphate binding?). Biologically-relevant effects of Al3+ plus F- include adenylate cyclase activation by F- (involving trace Al3+ or Be2+ co-catalysis cf, e.g, Martin, 1988). 8.a-7-6. Importance of Nucleation Factors & Lipids in Induction of Polysaccharide Supramolecular Structures. Further ideas of relevance of binding of anions to heparin/heparan sulphate might be an involvement of these polysaccharides in various currently poorly-understood physiological nucleation phenomena and disease-related alteration in such activity due to erroneous supramolecular structural distorting effects. Since Ca/Mg pyrophosphate granules are believed to nucleate calcification (e.g. Taylor et al 1990) such pyrophosphate granules attached to glycosaminoglycans may have similar properties. Nucleation initiating catalysts may control specific glycosaminoglycan phase change; iron impurities are believed to be initiators of carrageenan supramolecular structure formation (Williamson FB, Aberdeen, personal communication). 8.a-7-6-1. Hydrophobic Polyanion Binding Conditions. Physiological phase changes in the pericellular environment clearly involve the physical chemistry of lipids. Whereas under normal physiological saline conditions, metal ions are often only relatively weakly bound by mono and polysaccharides, in non-aqueous media including pericellular lipid-rich environments, large conformational alterations are found upon cation sugar interaction, suggesting that this is unusually strong in character (cf Tajmir-Riahi 1989), The nmr and infrared spectra of various heparin metal ion complexes provide some insight into details of polyanion-cation-water interaction. 13-C nmr spectra are influenced by a time averaging of fast cation exchange processes exemplified by a dominant effect of Na in mixed Na/Ca heparin and by a surprisingly masked effect of paramagnetic

ions although an apparent specific binding of paramagnetic Cu(II) shows up more clearly in 1-H nmr spectra (Rej et al 1990). 205-Tl nmr shows a relatively uninformative distorted single broad absorption indicating a poorly resolved distribution of cation environments. Solid samples studied by ir provide evidence of cation-specific interaction with clear dependence of the importance of cation-dependent water binding under these conditions. Binding of heparin to basic polypeptides in hydrophobic media show the importance of glue-like activities of water molecules associated with the cation/polyanion complex (Grant et al 1992). 8b HEPARAN SULPHATE, BRAIN DEVELOPMENT, FUNCTION & NEURODEGENERATIVE DISEASES INCLUDING ALZHEIMER'S DISEASE (AD). 8b.-1 Introduction. Heparan sulphate proteoglycans, nitric oxide (Salvemini et al 1998), and perhaps redox systems which will include Zn2+ effects (perturbed by Al3+ ?) ions are implicated in the pathogenesis of AD and related diseases as well as in normal memory function. A chronic inflammatory state of the brain may occur in AD and might explain why patients on antiinflammatory drug therapy for rheumatoid arthritis appear less susceptible to AD (McGeer et al 1990) agreeing with a likely involvement of immunologically produced nitric oxide/nitrous acid /redox metal heparan sulphate damage in AD. Inappropriate modulation of heparan sulphate by nitric oxide metabolites influenced by trace metal ions (perhaps including inappropriate initiation of polyanion phase change as discussed in the previous section) might instigate neurological malfunction and contribute e.g. to the formation of amyloid deposits in various amyloidoses which have the common non-covalently linked basement membrane heparan sulphate PG component (Narindrasorasak et al 1991). Neutralization of inappropriately formed nitric oxide generated nitrous acid by ascorbate reduction or by sacrificial deaminative cleavage of glucosamines may provide essential protection against such effects. Iron (iron ions are especially potent potential redox and phase change damaging agents) is reported to be selectively accumulated with aluminium in neurofibrillar tangles (NFT) of AD (Perl et al 1992). Membrane spanning heparan sulphate proteoglycans are thought likely to be important regulators of activity-dependent modulation of neuronal connectivity (Laun et al 1999) perhaps linking cytoskeletal elements with the pericellular environment. Nitrous acid also can potentially disrupt neurotransmitter monoamine oxidase activity directly deaminately cleaving dopamine and its metabolites and cause DNA cross-linking (Kirchner & Hopkins 1991). Liver amyloid was reported to show a very high level of de-N-sulphonated heparan sulphate chains absent from other heparan sulphates (Linker & Hovingh 1977) allowing uncontrolled nitrous acid cleavage. 8b-2. Heparin/Heparan Sulphate in AD. Fukuchi et al 1998 recently reviewed the involvement of heparin/heparan sulphate (in beta-amyloid protein, neurite plaque, NFT tau protein fibril formation and ApoE4 lipid processing) in AD but did not deal with the effects of nitric oxide and its metabolites in this disease which is now suggested (as a main theme of these notes) to damage heparan sulphate molecules under conditions pertaining to such diseases. 8.b-2-1. Learning and Heparin/Heparan Sulphates. Nitric oxide neurotransmitter activity may be involved in learning (Hawkins et al 1998, cf also Breen 1992, Fukuchi et al 1998). N-CAM glycoprotein function in synaptic connectivity was found to be heparin-dependent (Bullock & Rose 1992) (Cole et al 1986) with a possible memory function release of heparan sulphate (Sugara & Der 1994); injection of heparin into the adult rat hippocampus induced seizures (Mudher et al 1998); the heparan sulphate proteoglycan syndecan-3 was implicated in neuron extracellular matrix interaction in the cellular mechanism responsible for synaptic plasticity, associated with cortactin src type and fyn tyrosine kinases, playing a crucial role in long term potentiation (LTP) (enzymic cleavage of heparan sulphate as well as the addition of heparin prevented development of LTP in rat hippocampal cells studied by Laun et al, 1999); heparan sulphate may also provide resevoirs for growth factors, zinc and neurotransmitters. 8.b-2-2. Zinc in AD. Zinc is a potential modulator of heparin/heparan sulphate activity. Zinc is an intracellular messenger causing protein kinase C (PKC) to translocate to polymerised actin components in the cytoskeleton (Zalewski et al 1990).

A neurological function in sustained cellular responses involves PKC is long-term potentiation of synaptic transmission in the hippocampus (Ackers et al 1986); this activity is also heparan sulphate dependent (Laun et al 1999). Phosphorylation of cytoskeletal elements modulatable by heparin/heparan suphate may be implicated in short-term memory. Involvement of zinc APP-heparan sulphate interactions (Multhaup 1994) in AD therapy (Masters et al 1993) suggest possible modulation of APP activity by heparin/zinc. Zinc supplementation could apparently impair congnitive function in AD patients, restorable on cessation of the zinc supplementation (cf Smith et al 1994). The reported anti-heparinase activity of Zn2+ (cf Patent Spec RO96508) needs to be investigated further, but indicates a possible role of Zn2+ in the modulation of heparin/heparan sulphate signalling via alteration of its degradation rate. Are there linkages between Zn2+ - heparin/heparan, Zn2+- ascorbate, Zn2+- finger linked biochemistry Zn in SOD (Cu,Zn SOD), and effects of Zn2+ on NO biochemistry? Zinc disturbance in AD may include a function of specialized Zn2+-GAG binding (Grant et al 1992f). Zn2+ may counter a neurological protection afforded by heparin (considered to be against proteolytic cleavage of APP, the integral trans-membrane protein that is released from cells in culture following proteolytic cleavage) but nitric oxide related mechanisms might also be relevant. Selective Zn2+ binding to heparin (e.g. Woodhead et al 1986), an unusual proton binding effect produced uniquely by Zn2+ with heparin (Grant et al 1992f) (and perhaps a role in proton conduction) may have relevance for zinc-dependent heparan sulphate processing. Zn2+ catalysis of de-N-sulphonation of heparin by co-binding of metals and the role of Zn fingers in proteins in modulation of gene expression may be part of overall Zn2+ controlled mechanisms which include, at some stage, heparan sulphate-linked redox ascorbate and nitric oxide biochemistry and nitric oxide modulation of metal-protein binding (cf Draper et al 1996). Zn2+ at 50 nM (the effect saturated at 70 micro M) promoted heparin binding to amyloid precursor protein APP and abolished a protective effect of heparin against its proteolytic cleavage. Zinc dietary supplementation was reported to impair cognitive function in AD patients (Masters et al (1993). Heparan sulphate was separated from other GAGs by its greater readiness to form Zn2+ cross- linked phospholipid precipitates (Wu & Cohen 1984). Parrish & Fair (1981) reported that Zn2+ was bound to heparin but less readily by chondroitin 4 and 6 sulphates, dermatan sulphate or hydaluronic acid. NMR studies by Whitfield & Sarkar (1991) suggested that Zn2+-carboxyl interactions controlled the conformation of the iduronate rings of heparin monosaccharides and Li+ ions appear similarly to affect iduronate conformations in heparin preparations (Grant et al 1991; perhaps these observations are relevant to heparan sulphate mechanisms in neurological physiology. It might further be hypothesized that similar effects might include nucleation of phase change related to cycloskeletal elements involved neurological function and in memory function, further modulatable by nitric oxide. 8.b-2-3 Cognitive Function in AD. Therapeutic Use of Heparin/Heparan Sulphate A heparan sulphate containing GAG preparation ('Ateroid') has been reported by Ferrero et al (1989) to improve cognitive function in AD patients. A mechanism which might be relevant to such effects by blocking by exogenous heparin of heparan sulphate beta amyloid peptide interaction was suggested by Leveugle et al (1994). Vascular malfunction involving heparin/heparan sulphate and nitric oxide is also possibly involved the aetiology of neurodegenerative diseases. Heparin induced endothelial cell cytoskeletal reorganization was suggested to be a possible mechanism for vascular relaxation (Mandil et al 1995) and nexin-2 an isoform of the AD amyloid beta protein precursor secreted in large quantities by platelets upon vascular injury may be potentiated by heparin/heparan sulphate. Glia-derived nexin-1 binds to heparin (Rovelle et al 1992). The nmr spectrum of heparin was perturbed by Gd3+ binding in a less selective manner than by Cu2+ (Rej et al 1990). Cu in heparin may contribute both to its SOD and angiogenic activity (Raju et al 1982). 8.b-2-4. Neurite Outgrowth Stimulation by Heparin/Heparan Sulphate. Neurite outgrowth is stimulated by heparin (e.g. Sedden et al 1994) and heparan sulphate (Dow et al 1992). A heparin-binding domain in APP is also thought to be involved in regulation of neurite

outgrowth (Smith et al 1994) in which there is a likely autocrine role of heparan sulphate proteoglycans (Dow & Riolelle 1992) perhaps via a thrombospondin route (O'Rouke et al 1992). 8b-2-4-1. Possible Heparin/Heparan Sulphate-Dependent Growth Factor Effects in AD. Since the brain has long been recognized as a plentiful source of mitogenic acitivity and is a rich source of FGFs (Gimenea-Gallego et al 1985) subject to modulation by heparan sulphate (or by exogenous heparin), heparan sulphate proteoglycans are likely also to control bFGF activity in developing brain (Hondermark et al 1992) and be of relevance to neurodegenerative disease as is implied by the usefulness of a bFGF binding assay (Peiry et al 1992) to study the incidence of heparan sulphate in plaques in AD and other neurodegenerative diseases. Regulation of nerve growth factor (NGF) (is likely to be heparan sulphate-dependent (Damon et al 1992). Cellular therapy to achieve NGF replacement has been proposed for AD therapy (Hawkins, press report 2000). NGF was reported to change the invasive properties of neuro-ectoderm melanoma cells, possibly dependent on heparanase activity (Maschetti et al 1999); such activity, as discussed above, may also be Zn2+-dependent. 8.b-2-5. Extracted Brain Heparan Sulphate - Lack of Correlation with AD. Although brain heparan sulphates differed from those of other organs (Lindahl et al 1995), little difference was detected between the sugar composition and partial sequences of heparan sulphates from AD and control brains, indicating that the amount and primary microstructure of heparan sulphates may not contribute directly to AD; however the complexity of heparan sulphate biochemistry, involving the possibility of multiple cation and anion binding as well as multi-phase behaviour, does not rule out some future establishment of a direct heparan sulphate (supramolecular) structural link with neurodegnerative disease pocesses. It is suggested that, in addition to the polysaccharide microstructure, the inorganic content of the normal and AD heparan sulphates needs to be examined by e.g. spark source mass spectroscopy. Of particular interest is aluminium and silicon contents since there are putative links of these with the disease. (Cf silicic acid has been suggested to exert a protective role in aluminium neurotoxicity cf Birchall & Chappell 1989). 8.b-2-6. Possible Transferrin Receptor Function, Aluminium & Heparin/Heparan Sulphate in AD. Transferrin receptors which contain heparan sulphate side chains (Fransson et al, cf Gallagher et al 1986 and Hu & Reogoeczi 1992) are 67-Ga3+ markers for brain aluminium transport and coincide with the areas of the brain vulnerable to AD (although unaltered in abundance in AD) (Edwardson et al 1990). 67-Ga3+ binding which was much more strongly bound to heparan sulphate than to other GAGs (Kojima et al 1983) likely mimicking effects of Al3+. Al3+ is also implicated in dialysis dementia and Guam disease. Al3+ may inhibit calcium-mediated proteolysis of cytoskeletal proteins and induces NFT to form complexes thought to be via phosphate ester binding (Nixon et al, 1990). Cis-aconitate is thought be a likely pathological carrier of Al in AD and Al-citrate backs up transferrin and ferritin binding (Lehmann 1992). 8.b-2-7. Fenton Reaction /Free-Radical Damage in Neurodegenerative Diseases. Complex interactions involving heparin/heparan sulphate are seen to be involved directly and indirectly in control of both redox balance, antioxidant and other enzymic activity. Redox balance of which glutathione, ascorbate, nitric oxide and multiple antioxidant activity is part, is suggested as a theme of these notes to be a dominant factor in heparin/heparan sulphate biochemistry, pro-disease, abnormal heparan sulphate arising from its perturbation which include abnormal iron, aluminium and silicon loading which could adversely affect the pro-antioxidant activity and proteolytic modulatory actions of heparin/heparan sulphate. Al3+ directly deactivates SOD (Shainkin-Kestenbaum et al 1989) and brain proteases (Nixon et al 1990) and perhaps other relevant Ca2+, Cu(II) and Zn2+ dependent mechanisms. In AD, Down's syndrome, amylotrophic lateral sclerosis, Parkinson's and Guam dementias (Leveugle et al 1994), affected neurons apparently over-synthesise lactoferrin (involved in iron and aluminium transport). Antioxidant SOD genetic insufficiency has been identified in the familial form of amylotropic lateral sclerosis (Rosen et al 1993, cf Calder et al 1995), in agreement with an augmented superoxide initiated

damage involving reactive nitrogen species including peroxynitrite (Cookson & Shaw 1999, cf Love 1999 who reviewed oxidative stress in brain ischemia). Iron, phosphate (and aluminium) intoxication effects, however, may also be implicated in the above disease processes. As discussed in section 3.5 above, reactive oxygen free-radicals generated by iron-Fenton reactions are likely to damage GAGs. Further evidence of this is provided by Nagasawa et al 1992 who observed such damage to hyaluronic acid and heparin/heparan sulphate in less sulphated regions. Another example is the Cu(I) hydroxyl radical formation damage to hyaluronic acid in metallosis of the eye (Sterk et al 1985). 8c. Supramoleular Structure of Heparin/Heparan Sulphate PGs and their Binding Potential. Ca2+ (Liang et al 1982, Dais et al 1987) and Zn2+ binding the heparin may be critically dependent on the cooperative effects of supramolecular structure and arrayed N-sulphonate groups (Grant et al 1992c,f) to which crosslinking by small amounts of e.g. Al3+ could lead to disruption of the requirements for protein folding and binding. Nitric oxide damage to heparan sulphates in neurodegenerative disease may enhance such inappropriate binding activities. 8c-1. Silicates in GAGs. Silicates have been found to be associated with aluminium in AD plaques (Edwardson et al 1990). Although aluminium and silicate involvement in AD may be related to Zn2+ or Ca2+ homeostasis, silicon is an essential nutrient although the details of its biochemistry are still obscure and (inorganic) silicates are believed to occur in normal association with glycoaminoglycans (Iler,1979). It is conceivable that silicate acquisition may be a normal function of GAGs. The effect, if any, of Al3+ on this is worthy of study. Differences in the amounts of bound silicates and phosphates by glycosaminoglycans from different species organs and diseases and work-up procedures might have contributed to difficulties in comparing metal ion binding and other activities of such GAG preparations. 9. Involvement of Heparin/Heparan Sulphate in Modulation of Prion Protein Activity. Prion proteins have a heparin/heparan sulphate perspective (by differentially binding to heparin-like molecules, the synthesis and metabolic fate of prion proteins in scrapie infected cells is inhibited by reversing their phenotype back to normal (Gabizone et al 1993)). Part of the normal cellular function of prion proteins may be conceivably of relevance to memory function as they are evidently implicated in neurodegenerative diseases, principally scrapie but also Kuru, Creutzfeld-Jacob and Gerstmann-Straussler-Scheinkler diseases (Prusnier 1995). The antiscrapie effect of heparin and related molecules was established by workers (Diringer & Ehlers 1991) who do not, however, agree with the prion hypothesis (Diringer 1985). A further factor which does not seem to have been considered is the effect of non-physiological metal intoxication of heparin/heparan sulphate from the perspective of prion protein biochemistry. 10. WATER BINDING TO HEPARIN/HEPARAN SULPHATE. The number of water molecules, strongly bound to sulphate half-ester groups of heparin was found to the highly dependent on the counterions present, averaging six for sodium, three for potassium and one for calcium. A quasi phase change mechanism of counter ion binding to heparin/heparan sulphate may have relevance to switchable ion and proton pumping. Commercial sulphonated polymers find use as proton conductors, such activity being dependent on cation-dependent water binding to the arrays of sulphonate groups (Colomban 1992) which is analogous to a similar effect of counterions on the stoichiometry of water molecule binding to sulphate half-ester groups in highly sulphated heparin-like molecules. Hydration changes may also be relevant to the coil conformation changes thought to be responsible for signalling by sodium/calcium - heparan sulphate chains in the proposed mechanism of sensing of blood flow in vivo (Siegel et al 1998) and perhaps other servo-control mechanisms, evidently employing potentially involving heparan sulphate conformation -dependent effects, such as control of calcium/heparin binding sites in parathyroid cells (Takeuchi et al 1990).

10a HOFMEISTER EFFECTS ON WATER STRUCTURE The Hofmeister series ranks the relative protein denaturation abilities of aqueous salt solutions. Water aggregate structures present in various metal salt solutions, deducible from near infrared spectroscopy, also varied with the Hofmeister series (Luck 1969) indicating that the ionic content of aqueous solutions affects the aggregation state of hydrogen bonded water molecules thought to be composed, at physiological temperature, of several hundred H2O units but salt ions 'melt' these aggregates according to their Hofmeister activities. Flickering clusters of such labile hydrogen-bonded arrays may also behave in a determinisitic chaotic manner, e.g. being influenced by phase boundary conditions which although poorly understood, may be relevant to biology (which usually restricts such considerations to hydrophobicity indices) and even to the mechanism of memory storage and retrieval. Heparinized surfaces may create bio-friendly non-denaturing surfaces by providing correct hydrophilicity/hydrophobicity in addition to providing anticoagulant protection as well as binding sites for antioxidants and growth factors. Heparin/heparan sulphate, as well as other biological polyanions, can be considered to provide Hofmeister-series activity modulation through Hofmeister-effectempowered-hydration. This is thought to be related to the established morphological role of heparin/heparan sulphate and related polysaccharides in calcification and to a hypothetical role in e.g. cytoskeletal controls. The entropic driven properties of quasi phase change behaviour (such consideration are also perhaps relevant to the reversible binding and diffusion of bFGF on HSPGs of basement membranes (Dowd et al 1999) and evently an unconventional discussion of water structure as an information carrier in organisms (Schwabl 1994) are possibly related the the unusual thermodynamic properties of water-rich gels perhaps underlying the evolution of sulphated polysaccharides for flexible ligand binding, the basis of developmental biology (Comper 1994). Another example of such gel effect is the intestinal mucous coat with an unstirred water layer (Smithson et al 1981). 11. MODULATION OF CRYSTALLIZATION BY HEPARIN/HEPARAN SULPHATE. A biological function of glycosaminoglycans and related polysaccharides in calcification has been well established. A similar morphological role is possible with other biological mineral and protein structures. This includes cytoskeletal element dependent fundamental cell activity such as of the mitotic spindle (Roussel et al 1990) and perhaps also involves the nucleus (Bhavanandan & Davidson 1975). Seeded calcite crystallisation studies demonstrated that lower molecular weight or de-N-sulphonated heparins (such as might be produced as a result of nitric oxide/nitrous acid reaction) were much less effective calcification inhibitors than intact highly sulphated polymers (Grant et al 1989b). A pro-disease effect of pathological in vitro crystallisation and its prevention by heparin-like molecules has been discussed by Grant et al (1992b). The ability of structurally sound heparan sulphates, but not pathologically altered heparan sulphates, both to inhibit crystallisation and to act as seeds for the induction of correct protein folding could be related phenomena. Rate determining processes during crystallization may be dependent on relative hydration energies in transition states and related cation and hydration-dependent polysaccharides may provide enthalpic driven proximity and flexibility for ligand binding requirements of multicellular organisms, which is at the basis of developmental biology (Comper 1994) and presently hypothesized to the an important fundement of the pathology of degenerative diseases and autoimmune processes. Foreign particles (such as asbestos fibres) or abnormal solids (e.g. amyloid fibres) may provoke immunological reaction including nitric oxide/nitrite formation unless adequately deactivated by suitable biopolymers including heparan sulphate which may restore an acceptably inert structure. 12. ESTABLISHED LABORATORY USE OF NITROUS ACID FOR HEPARIN/HEPARAN SULPHATE ANALYSIS Nitrous acid has long been known to be a useful organic chemical reagent, employed by synthetic organic chemists to allow accurately defined molecular transformations to be achieved, e.g. yielding alcohols from aliphatic primary amines, diazo compounds from aromatic primary amines and nitrosoamines from secondary amines. Prior to the current awareness of nitric oxide and its metabolites

as being highly pertinent to biochemistry and medicine, the high specificity of the nitrous acid deaminative cleavage of heparan sulphates encouraged chemists to employ this reaction to confirm the presence or absence of heparin/heparan sulphates in biological samples and to further characterise the fragments obtained by gel filtration etc. The reaction is normally employed to allow scission of the N-sulphonated glucosamines leaving the Nacetyl glucosamines intact. Hydrazine treatment, however, selectively de-N-acetylates which when followed by nitrous acid treatment at pH 4, provides further heparan sulphate fragments for analysis. 13 BINDING OF PATHOGENS TO HEPARAN SULPHATE Such binding is common for many pathogens allowing therapeutic intervention to be achieved by heparin and heparinoids by blocking the heparin binding sites and by other mechanisms including inhibition of reverse transcriptases. Helicobacter pylori (among the many heparan sulphate binding organisms) has a marked pro-cancer effect, enhanced in cyclotoxin associated antigen CagA strains. Exposure of gastric epithelial cells to H pylori induced activation of the transcription factor protein 1 and activation of the proto-oncogenes c fos and c jun. This might be a crucial step for the induction of neoplasia (Meyer-Ter-Veh et al 2000). Heparin/heparan sulphate biochemistry might also be implicated in H pylori induction of cancer e.g. through perturbation of growth factors e.g. by the displacement of FGFs from heparan sulphate PG sites by bacterial heparan sulphate binding peptides (Ascencio et al 1995). Anti-proliferative effects of heparin on c fos and c jun in arterial walls appeared to be indirect perhaps via alteration of other cell cycle events (CA 120 124557c). 14. REFERENCES. Vide infra). 15. ACKNOWLEDEMENTS Grateful thanks are due to Professor KEL McColl and Ms J Grant (Glasgow), Drs Roger Worthington, Frank Williamson and Bill Long (Aberdeen) as well as to the Carnegie Foundation for the Universities of Scotland, for assistance. Spark source mass spectrometric results were kindly provided by Dr Bacon (Macaulay Inst Aberdeen) originally at the instigation of Colin Moffat (Aberdeen). * Mass spectroscopic results are given thus : (parts per million by weight (ppm) in untreated heparin/ppm in heparin after cation exchange on a Tl+ loaded Amberlite IR 120 column).
References to DG uk 2000 web postings on heparan sulfate

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more potent than heparin as potentiators of angiostatic steroids Heparin Cu biaffinity chromatography refs Shively JE Conrad HE (1976) Stoicheometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycans Biochemistry 15 3932-3942 cf ibid 9(1) 33-43 Different N oxides cleave N-sulphonated and N unsubstituted glucosamines Siczkowski M Clarke D Gordon MY (1992) Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulphate Blood 80(4) 912-919 Siegel G Malmsten M Lindman B (1998) Colloids and surfaces A: Physiological and Engineering Aspects 138 345 Flow sensing at the endothelium-blood interface (A disfunction of this sensor which involves heparan sulphate conformation alteration by sodium calcium balance can lead to organ insufficiency, hypertension and arteriosclerosis) Simon K (1982) Naturwiss Runds 35(11) 452-453 Commercial heparin contains 12% of inorganic sulphate which cannot be dialysed out Singh PP (1993) Ascorbic acid is an abettor in calcium urolithiasis Experimental Study Scanning Micros 1993 (3)1041 CA 120 188838c Singh RJ Hogg N Joseph J Kalyanaraman B (1996) J Biol Chem 271(31) 18596 Mechanism of nitric oxide release from S-nitrosothiols R-S-NO stable at 37C pH7.4 (only) in the presence of transition metal ion chelators Simon KH (1982) Heparin: Alte Indikationen, neuer Wirkungsmechanismus Naturwiss Rundschau 35(11) 452-455 Commercial heparin contains 12% inorganic sulphate (non-dialysable) Singh RJ Hogg N Joseph J Kalyanaraman B (1996) J Biol Chem 271 18596-18603 Sinnarajah S et al (1999) Heparin modulates single channel kinetics of reconstituted AMPA receptors from rat brain Synapse (NY) 31(3) 203-209 CA 130 277241b Smith DH et al (1994) A heparin binding domain in amyloid protein precursor of Alzheimer's disease is involved in regulation of neurite outgrowth J Neurosci 14(4) 2117 CA 121 6093w Smithson KW et al (1981) Intestinal diffusion barrier: unstirred water layer on mucous coat surface Science 214 1241-1244 Sommer A Rifkin DB (1989) Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan J Cell Physiol 138 215-229 Complete protection of bFGF by heparin is achieved at ratios of bFGF/heparin of 10 or where hydrophobic complexes formed are insoluble Spear P Shieh MT Herold BC Dunn DW Koshy TI (1992) Adv Exp Med Biol 313 (Heparin related polysaccharides) 341 CA 118 166066f Review Binding of Herpes simplex to cell surface heparan sulphate Stamler JS Singel DJ Loscalzo J (1992) Biochemistry of nitric oxide and its redox-activated forms Science 258 1898-1901 Stanley MJ Liebersbach BF Liu W Anhalt DJ Sanderson RD (1995) Heparan sulphate mediated cell aggregation. Syndecan-1 and 4 mediate intracellular adhesion following their transfection in to human B lymphoid cells J Biol Chem 270(10) 5077-83 CA122 204833w Steck PA Moser RP Bruner JM Liang L Freidman AN Hwang T-L Yung AWK (1989) Altered expression and distribution of heparan sulphate proteoglycans in human gliomas Cancer Research 49(8) 2096-2103 Stephen CC et al (1998) Role for heparin-binding growth factors in glucose-induced vascular disfunction Diabetes 47(11) 1771 CA 130 64586f (Heparinoid) suramin a heparin binding of inhibitor blocks glucose induced vascular disfunction Sterk H Braun M Schmut O Feichtinger H (1985) Investigation of the hyaluronic acid-copper complex by NMR spectroscopy Carbohydr Res 145 1-11 Cu(I) depolymerization of hyaluronic acid by free radicals/redox cycling Stichentoth DO Frolich JC (1998) Nitric oxide and inflammatory joint diseases Br J Rheumatol 37 246257 CA 129 52689h Sugahara K Yokumura Y Yamashina I (1989) Engelbreth-Holm-Swam mouse tumours produce undersulfated heparan sulphate and oversulphated galactosamineglycans Biochem Biophys Res Commun 162(1) 189 CA 111 112937c Sugiura M Dow KE (1994) Activity dependent regulation of neuronal synthesis and release of neurite promoting heparan sulphate proteoglycans Dev Biol 164(1) 102 CA 121 222603e Sushella AK Kharb P (1990) Aortic calcification in chronic fluoride poisoning biochemical and electron microscopic evidence Exp Mol Pathol 53(1) 72-80 CA 113 206391p Aortas ex rabbits admin 10mg NaF/kg 17-24mo Incr GAG but decr DeS possibly assoc with calcification;

cf Frege J Graef C Dominolk GW (1990) The orientation of glycosaminoglycans in osteocyte capsules of fluoride treated rats Fluoride 23(1) 27-30 CA 113 22557r Oriented microstructure of ECM in ostiotic capsules may be due to F reduction in spatial orientation of GAGs cf Susheela AK Jha M (1981) Effects of fluoride on glycosaminoglycan of cancellous and cortical bone of rabbits Experientia 37(10) 1097 CA 95 215959s Rabbit 10 mg NaF/kg 6mo showed likely both enhanced sulphation and GAG formation in cortial and cancellous bones; Prince & Navia (1983) J Nutr 113(8) 1576 CA 99 134944b Fluoritic bones (>5000ppm F) had 3x ChS and 2x DeS Taetle R Honeysett JM Bergeron R (1989) J Natl Cancer Inst 81 1229 Iron chelation therapy Tajima S et al (1995) J Biochem (Tokyo) 117(2) 353 CA 122 230439 Heparin is multifunctional - cell proliferation + modulator of collagen I Takeuchi Y Sakaguchi K Yanagishita M Aurbach GD Hascall VC (1990) Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line J Biol Chem 265 (23) 13661 CA 113 112891z Takeuchi Y Yanagishita M Hascall VC (1992) Recycling transferrin receptor and heparan sulfate proteoglycans in rat parathyroid cell line J Biol Chem 267 (21) 14685 CA 117 128638r Tajmir-Riahi H-A (1989) D-glucose adducts with zinc-group metal ions Synthesis and spectroscopic and structural characterization of Zn(II) Cd(II) and Hg(II) complexes with D-glucose and the effects of metal-ion binding on the sugar anomeric structures Carbohydr Res 190 29-37 Zinc group: sugar major interaction in solid and nonaqueous but not in aqueous solution Tanahari T et al (1995) Exp Cell Res 258(1) 233 CA 122 30727n FGF-1 may have different potency according to the quantity and type of cell surface heparan sulphate present Tekolte H et al (1994) Disaccharide composition of heparan sulphates: brain, nervous tissue storage organelles, kidney and lung J Neurochem 62(3) 1126 CA 120 187842n Terada M Yoshida T Sakamoto H Hattori Y Iida S Yamada Y Yokota J Katoh O Hirohashi S Sugimura T (1991) Origims of Human Cancer. A Comprehensive Review Cold Spring Harbor Laboratory Press 675-683 Heparin-binding growth factor genes hst-1 and hst-2/FGF6 Growth factor mechanism of oncogensis involving deregulation of normal hst-1 protein (no requirement of structural change in this gene for NIH-3T3 cell transformation by transfection) Teranishi K Poston RS Reitz BA Robbins RC (1999) Effect of low molecular weight heparin on graft vascular disease in the rat carotid allograft model Transplant Proc 131(1/2) 103-105 CA 131 27696s Low mol wt heparin prolonged graft survival time possibly via NO mechanism since NO synthase inhibition reversed the heparin effect Theodosakis J Adderly B Fox B (1997) The Arthritis Cure Century Books ISBN 07126 7813 1 Thornton J Court M Palmer A (2000) The Sunday Telegraph (London) April 16 p 21 FOCUS STOP TAKING THE TABLETS Vitamins are good for you...arent'they Press release on possible health risks of high ascorbate dietary supplement Timar J et al (1994) Anticancer Res 14(3A) 1227 CA 1227582n PMA induces HSPG change with respect to tumour cell adhesion Timpl R (1989) Structure and biological activity of basement membrane proteins Eur J Biochem 180 487-502 Tsao CS (1991) Am J Clin Nutr 54 1274S Tsen G et al (1995) Agrin is a heparan sulphate proteoglycan J Biol Chem 270 (7)3392 CA 122 128959z Agrin is prominent in embryonic chick brain neural development Turnbull JE Fernig DE Ke Y Wilkinson MC Gallagher JT (1992) Identification of basic fibroblast growth factor binding sequences in fibroblast heparan sulphate J Biol Chem 267(15) 10337-10341 CA 117 40872b (Primary role for continuous GlcN-sulphonated sequences) Turnbull JE Gallagher JT (1990) Biochem J 265 715 CA 130 306854m Molecular organization of heparan sulphate from human skin fibroblasts Turnbull JE Hopwood JJ Gallagher JT (1999) A strategy for rapid sequencing of heparan sulphate and heparin saccharides Proc Natl Acad Sci USA 96(6) 2698-2703 CA 131 16059k Nitrous acid followed by enzyme cleaved end-tagged fluorescent reading frame + PAGE allows direct reading of sequences Tumova S et al (1999) bFGF does not prevent heparan sulfate catabolism in intact cells, but it alters the distribution of the GAG degradation products Biochem J 337(3) 471 GAG accompanies bFGF to nucleus Tye RJ (1989) Review paper Industrial and non-food uses for carrageenan Carbohydrate Polymers 10 259-280 cf p 268 Carrageenan interacts very strongly with transition metals and very effectively coats and stabilized iron-based pigments at 62% solids Tyrrell DJ Page CP (1996) Haemostassologie (Stutt.) 16(1)21 CA125 48167m Review - heparin as anti-inflammatory drug

Ueda S Hayashi T Namiki M (1986) Effect of ascorbic acid on lipid auto-oxidation in a model food system Agric Biol Chem 50 (1) 1-7 (Ascorbyl free radicals) Ushino S (1996) Jpn J Cancer Res 87(1)68 HB-EGF p91 (transcription factor) activation, induction of plasminogen activator and tubulin morphogenesis in human microvascular endothelial cells Vacherit F et al (1999) Involvement of heparin affinity regulated peptide in human prostate cancer Prostate (NY) 38(2) 126 CA 1130 309881d Valencia-Sanchez A et al (1995) Mol Androl 7(1,2)57 CA 122 282178a Heparin controls acrosome reaction, also Ca flux for capacitation Van den Born J et al (CHECK) J Biol Chem 270 (52) 31303 Van den Born J et al (1995) Diabetologia 38(2) 161 CA 236801a Glomerular HSPG proteinurea Van den Born J et al (1996) J Biol Chem 271(37) 22802 CA 125 272673j NAc domains Ab HSPGs disease cf E coli capsular polysaccharide [GlcUA-GlcNAc]n Van der Voorst R et al (1999) Heparan sulphate modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase cMet J Biol Chem 274(10) 6499 CA 130 336182h Van Wazer JR (1958) Phosphorus and its Compounds Vol I Interscience New York Van Wazer (1962) Structural re-organization through ligand exchange American Scientist 50 450-472 Venktaraman G et al (1996) PNAS USA 93 (2) 845 bFGF dimerization - heparan sulphate Vilar RE Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997) Nitric oxide degradation of heparin and heparan sulphate (both nitrous acid and nitric oxide degrade heparin; buffer dependence of nitric oxide degradation at physiol pH suggests unidentified catalyst or inhibitor) Biochem J 324 473-497 Vlodavsky T (CHECK) et al (1995) Invasion Metastasis 14 (1-6) 290 cf Kaatz BZ (1995) ibid 276 Vlodavsky I Michaeli IR Mohzen M Bar-Shavit R Catane R Elere HPT (1992) Adv Exp Med Biol 315 (heparin and related polysaccharides) 317 CA 117 225598k Walker A Gallagher J (1996) Biochem J 317(3) 871 CA 125 188757d Structural domains of heparan sulphate for specific recognition of the C terminal heparin binding domain of human plasma fibronectin (HEP II) Warrell RP Jr Murphy WK Schulman P O'Dwer PJC Helller G (1991) J Clin Oncol 9(8) 1467-75 Randomized-double blind gallium nitrate compared with etridronate for acute control of cancer related hypercalcaemia Warren C Manley (1984) Measurement of heparan sulphate in normal human serum by laser nephelometry Clin Chim Acta 137(3) 355 CA 100 188260v Heparan sulphate in blood serum of healthy humans was 1.6mg/L Wanake Shinker A (1994) Heparin binding growth factors in neural development and injury Kenkyu No Shinpo 38(6) 896 CA 122 130206v Pleiptrophin and midkine synthesized in neurons, mainly localized to the cellular suface, may function as ECM molecules in an autocrine and/or paracrine fashion, upregulated in injury Weksler BB (1990) Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostacyclin H synthase and prostacyclin synthase J Cell Physiol 142 514-522 Wells CF Salam MA (1968) The kinetics of the reaction of chromium(II) with hydrazine, hydroxylamine and hydrazoid acid in perchlorate media: the formation of halegeno- and sulphatocomplexes of chromium(II) J Chem Soc (A) 1568-1575 Cr(II) causes formation of N-centred free radicals and Fenton like chemistry Wei W et al (1999) CA 130 309799h Overexpression of nitric oxide synthase I related to lymph node metastasis in non small cell lung carcinoma Wessel HP et al (1998) Carbohydr Mimics 417-431 Ed Y Chapleux Wiley VCH Verlag GmbH Weinheim Germany CA 130 182657c From GAGs to heparinoid mimetics with antiproliferative activity (Review with 37 refs on the preparation of heparinoid mimetics as anti-tumour and smooth muscle cell inhibitors) Willenbourg DO Parish CR (1988) Inhibition of allergic encephalomyelitis in rats by treatment with sulfated polysaccharides J Immunol 140 3401-3405 Whitfield DM Sarkar B (1992) Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation Biopolymers 32 597-619 Whitfield DM Sarkar B (1992) Metal binding to heparin monosaccharides: D-glucoasamine-6-sulphate, D-glucuronic acid, and L-iduronic acid J Inorg Biochemistry 41 157-170 Williamson FB (1989) personal communication Williamson MF Long WF Williamson FB (1992) The effect of heparin on the UV absorption properties of Fe(II) and Fe(III) Biochem Soc Trans 20(4) 360s

Winterbourne DJ Mora PT (1981) Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulphate with reduced degree of sulfation J Biol Chem 256(9) 4310-4320 Wong SF Halliwell B Richmond R Skowroneck WR (1981) The role of superoxide and hydroxyl radicals in the degradaton of hyaluronic acid induced by metal ions and by ascorbic acid J Inorg Biochem 14(2)127-34 Ascorbic-induced depolymerization is inhibited by desferrioxamine, catalase and scavengers of hydroxyl radical Woodhead NE Long WF Williamson FB (1986) Heparan sulphates from fibroblasts exhibiting a temperature-dependent transformed growth trait IRCS Med Sci 14 427-428 cf Biochem Soc Trans 12(2) 300 CA 100 189661p (chemical transformation affected by N-methyl-N'-nitro-N-nitroguanidine) Woodhead NE Long WF Williamson FB (1986) Binding of zinc ions to heparin Biochem J 237 281284 Wrenchall LE Cerra FB Singh RK Platt JL (1995) Heparan sulfate initiates signals in murine macrophages leading to divergent biological outcomes J Immunol 154(2) 871-80 CA 122 79080v Heparan sulphate modulates cellular immunological macrophages with cytokine signalling PGE2, IL6 activation of tyrosine kinase, nuclear factor kappa B, protein kinase C Wright TC Jr Castellot JJ Petitou M Lormeau J-C Choay J Karnovsky MJ (1989) Structural determinants of heparin's growth inhibitory activity Interdependence of oligosaccharide size and charge J Biol Chem 264(3) 1534-1542 2-O sulphate not required for antiproliferative activity Wu Van-Yu Cohen MP (1984) A competitive binding assay for measurement of heparan sulphate in tissue digests Anal Biochem 139 218-223 WuDunn D Spear PG (1989) Initial interaction of Herpes Simplex virus with cells is binding to heparan sulphate J Virol 63(1) 52-58 Wunde A et al (1995) Thromb Res 78(2)139 CA 122 255935p Anti-atheroma effect of heparin Xu G Yuan M-B Zhong X-J Fu S-Ji Tang S-L Zhao L Sun Y-P Jian P (1998) Huaren Xiaohua Zashi 6(2) 125-127 CA 130 294939c Polyamines increase with Helicobactor pylori infection in stomach (Perhaps some nitrous acid degradation of relevance to cancer?) Yanagishita M (1992) Metabolism of plasma membrane associated heparan sulphate proteoglycans Adv Exp Med Biol 313 (Heparin related polysaccharides) 113 Yanagishita M (1992) Glycosylphosphatidylinositiol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotoic and intracellular degradative pathways J Biol Chem 267(14) 9505-9511 Yanagishita M Hascall VC (1992) Cell surface heparan sulfate proteoglycans J Biol Chem 267(14) 9451-9454 Yang L Yang Y-C (1995) Heparin inhibits the expression of IL11 and granulocyte-macrophage colonystimulating factor in primate bone marrow stromal fibroblasts through mRNA destabilization Blood 86(6) 2526 CA 123 246454z Mechanism for regulating cytokines via heparin induced mRNA degradation Yoshizumi M Kourembanas S Temizer DH Cambria RP Quertermous T Lee M-E (1992) Tumor necrosis factor increases transcription of the heparin-binding epidermal growth factor-like growth factor gene in vascular endothelial cells J Biol Chem 267(14) 9467-9469 Zalewski PD et al (1990) Synergy between zinc and phorbol ester in translocation of protein kinase CV to cytoskeleton FEBS Lett 273(1-2) 131-4 4 Zhang G Li B Fan J Feng S (1997) Determination of trace thiocyanate in body fluids by a kinetic fluorimetric method Talanta 44 1141-1147 Zhang L Keuchi Y Lu J Rosenberg RD (1999) Anticoagulant heparan sulphate precursor structure in F9 embryonal carcinoma cells J Biol Chem 274(9) 5681 CA 120 323265n Demonstrates for the first time the processing assembly mechanism of specific sequences in heparan sulphate Structure differences around potential 3-O sulphate acceptors in the active and inactive anticoagulant versions of heparan sulphate Zou S Magura CE Hurley WL (1992) Heparin-binding properties of lactoferrin and lysozyme Comp Biochem Physiol 103B(4) 889-895 Binding of biotinylated heparin to lactoferrin and lysozyme in presence of Na, Ca, Cu, Zn, Fe(II) and Fe(III) showed complex buffer, pH and especially cation dependent behaviour. Heparin binding to lactoferrin unaffected by less than 10 microM Zn or Cu but was enhanced at Cu of 100 microM and inhibited above 500 microM and was inhibited at all Zn > 10 microM; Fe(III) was inhibitory at all concentrations but Fe(II) enhanced binding slightly at 10 microM. May be of significance for effect of e.g. iron overload on antimicrobial properties of studied proteins or modulation of heparin/ heparan.

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The Putative Multi-Inorganic Metallomic Element Reservoir Associated with Heparin/Heparan Sulphate Proteoglycans may Provide Essential Inorganic Cofactors Required for Nitrosative Signaling and Discriminated Protein Binding David Grant Ph.D. (Turriff, U.K.) (and University of Aberdeen*) Summary Highly sulphated animal polysaccharides, in analogy with marine alginates and natural water and soil humic polymers, are predicted to exist as multi-metal counterion adducts in vivo. Those metal ions (e.g. Ca, Zn and Cu) which have consistently been reported to be relevant to heparin/heparan sulphate signaling have also been found to consistently co-purify with heparin. It can be further suggested that the wide range of other inorganic elements which commonly occur in heparin may also be required for similar functions.

Introduction
The emerging science of biological metallomicsa, which has been hitherto been of interest for the provision of those metal ions which are essentially required to create the active metal sites in proteins, could also apply to the provision of the putatively essential inorganic cofactors required for heparin/heparan sulphate [H/HS] signaling processes.
a

The ubiquitous presence of trace and ultratrace elements in biological matrices which include found by mass spectrometric analysis has led H. Haraguchi to propose that many elements, which formerly were classified as being nonphysiological, should be now included within a revised definition of metallomics (the branch of science originated by R.J.P. Williams to the study of some 20 physiological metal ions concerning, inter alia, the connectivity between geological and biological matrices). A range of pharmaceutical heparins (mast cell derived highly purified anionic polysaccharide mixtures which are widely used as a blood anticoagulant) which had been purified to different extents during manufacture seem are good examples of metallomic matrices. This included highly multi-element substitued sodium heparin as well as a purified forms of lithium heparin (with reduced of toxic element contents to more fully conform with Pharmacopoeia requirements) suggests that the retained multi-inorganic element contents in heparin could have arisen by their sequestration in vivo from extracellular multi-element containing bathing fluids. This sequestration process is, however, evidently more complex than a simple counterion condensation since both cations and anions as well as colloidal inorganic particles can apparently be simultaneously sequestered.

Systemic dysfunction of inorganic-ion-determined supramolecular structures in heparin HS glycocalyxes could be relevant to the fuller understanding of the etiology of degenerative diseases such as cancer and atherosclerosis 1a ; this could, e.g., explain why intact H/HS molecules, their fractions or inorganic adduct polymers, oligomers and their mimetics apparently can both inhibit (H. Engelberg 1) as well as promote (cf., M.O. Ouida et al., 1) tumour growth and similarly both inhibit or promote a range of other degenerative diseases as well as the virulence of pathological organisms. HS-related molecules also seem similarly able both to promote as well as to inhibit prion dysfunction diseases. H/HS biochemistry has been implicated in the mechanism of antioxidant protection. This may include a non-enzymic mechanism of pacification of redox active metal ions (including of Cu and Fe ions and aggregates) by their incorporation into sparingly soluble metal ion H/HS complexes (including those containing amyloid fibrils) 1a-3 (additional to the more commonly acknowledged role of HS in potentiating the enzymic superoxide dismutase and selenoprotein antioxidative systems). A similar redox metal ion pacification mechanism may also contribute to how H/HS exerts control over normal and pathological calcification (D. Grant et al. 1) and proteinaceous plaque formation. Metal Ion Assisted H/HS Signaling Nitrosative cleavage of H/HS is believed to constitute a major signaling pathway involving oligosaccharide messengers the formation of which depends on redox cycling by Cu2+ , + apparently assisted by Zn2+ ions1 Examples of another, perhaps more general, type of non-redox-metal ion-determined-H/HS-signaling include inter alia, the absolute requirements for divalent cations (e.g. Ca2+) for the facilitation of basic fibroblast growth factor receptor assembly (cf., M. Kan et al. 1) the inhibition of inositol trisphosphate Ca2+ ion release by H (cf., T.D. Hill et al.1a4) and the binding of specifically unsaturated sugar H dimer to membrane Na+/Ca2+ ion exchanger allows bacterial heparanase to inhibit this activity (S.K. Shinjo et al.,
1

)).

Could HS be a Biological Clock?

HS protoglycans (in concert with Na+, K+, Ca2+, Mg2+ other metal ions and nitric oxide) may empower temporal control systems which include those directing cellular assembly during embryogenesis as well as the controlled alterations of HS microstructure which influences the course of the normal ageing processes in individual tissues and putatively also in the entire animal organism. Human ageing was suggested by results tabulated by K. Murata & Y. Yokoyama 1 to be accompanied by a relative diminution of the relative amounts HS at arterial surfaces; E. Feyzi et al.1a-5, in related groundbreaking studies, found a linear agerelated alteration of HS microstructure (this effect of age on HS [or vice versa] seems to classify HS aas being unique amongst biopolymers). Aged-HS could be ultimately responsible for the age-related increase in atheroma at vascular surfaces; if this phenomenon is found also to apply to HS in other tissues it would suggest that altered HS activities produced by an age-determined alteration of microstructures could act as some kind of long-time-span biological clock which can ultimately determine lifespan. A related short time span HS clock may also influence the cell cycle; cf., the short time span biologicalclockrelated-HS-microstructural alteration which has been associated with the absorption of light (cf. circadian rhythm) by the pineal gland1a-6. Metal ion binding properties of HS can be rationally suggested to participate in the suggested HS instrument or clock-like actions, perhaps by a related mechanism to that by which vascular surface HS metal ion binding alteration of conformation may act as a servo feedback sensor for blood flow regulation (experimental evidence for which has been presented by A. Siegel et al. 1)

A Putative Heparan Sulphate Inorganic Ion Environmental Interface

A mechanism by which various environmental inputs (both chemical and physical) can positively or negatively impart into HS activity include direct effects of diet on nitric oxide biochemistry and metal ion-dependent nitrosative postsynthetic alteration of HS. This could ultimately account for how diet controls health. Cf., 1a-7 which lists how some known dietary factors have been reported to effect change

in HS structured.
.
b

Evidently allowing H-mimetic drugs such as pentosan polysulphate (PPS) to provide an effective therapeutic intervention in prion diseases, this was first reported by Diringer & Ehlers1 and confirmed by later workers; H/HS and PPS is also highly effective against intractable viruses such as HIV-1 and II (cf., e.g. E. De Clercq TiPs. 1990, 11, 198-205; similar results were obtained by Aberdeen polysaccharide researchers for an algal PPS-like polysaccharide).
c

An alternative explanation of the blood vessel wall change in HS could be suggested to a servo-feedback response to counter the age-related diminution of fibrinolytic activity, increase of serum ferritin (Jarrett et al.l )or tendency for formation of hydroxyapatite crystallites at blood vessel surfaces which could conceivably

signal for altered HS microstructure generation to combat these potential threats (this process is suggested by the results published by F.T. Borges et al. 8).
d

A similar list can be suggested for effect of diet on nitric oxide biochemistry but the details of how this fully impinges on HS biochemistry are less clear apart from the utilization of nitrosative scission for generation of HS oligomers used as inter and intracellular signaling molecules.

HS microstructure can, it seems, be induced to change directly in response to such molecules as glucose and ascorbate, the presence of pathological organisms, inorganic surfaces (e.g. calcium oxalate crystal surfaces which can apparently also specific crystal surface which promote HS microstructures which inhibit the growth of such crystals (cf. F.T. Borges et al.,8 ; these studies suggest a possible similar scenario with hydoxyapatite). This scenario seems to point to a general sensitivity of HS microstructure towards a wide variety of extracellular and environmental signals. C.f., while ascorbate, retinoic acid, and other beneficial fatty acid types, apparently induce beneficial HS structural alterations, excess blood glucose, oxidized lipids (and other inappropriate lipids?), virus infections etc. 1a-7, and toxic ions (Pb2+, Cd2+ and Hg(II)) and high concentrations of F- (cf. Table II) negatively perturb HS microstructure and associated activity.

How Highly Specific Sugar Sequence Signals Are Achieved ?

The well-established antithrombin-binding pentasaccharide sequence in H is now known to be the principal mechanism by which H promotes blood anticoagulation. Various other HS microstructures had been proposed to similarly select specific proteins and thereby modulate their activities e.g. C. Vanpouille et al. 1 recently reported that Cyclophilin A binds via rare sites containing 3-O-sulphated, N-unsubstituted glucosamine groups in H/HS and e.g. differently structured H fragments differentially inhibit the binding of lipoproteinase to 2macroglobulin (A. Larnkjaer et al. 1) However, J. Kreuger et al.,1 failed to confirm the specific HS sugar sequence signaling hypothesis.

Fragments of HS (Kreuger et al. 1) were studied in a highly purified form in vitro. These had been selected from in vivo information which had been predicted such epitopes on their own to be capable of selective binding of FGF4, FGF7 and FGF8b. A possible explanation of these findings is that in vivo HS discrimination involves additional hydrogen bonding or Van der Walls effects which the low molecular weight epitope lack. The failure of these studies of purified oligosaccharide epitopes could also suggest that multi-inorganic ions co-spheres might have provided some essential inorganic cofactor for a discriminated HS epitope binding suggesting that e.g. a quaternary HS-FGF-FGFR-divalent metal ion complex might have been required to allow the in vivo discrimination.

Inorganic components of H/HS could be an essential part of the signaling and regulatory mechanism. Examples of a growing literature database on this topic are collected in Table I. Table II lists instances where other effects of inorganic ions on HS. Zn2+ ions known to be absolutely required to achieve HS endostatin (collagen XVIII) binding (S. Ricard-Blum et al.1). When such inorganic cofactors are removed during purification the target protein fails to bind to HS but binding can be restored by the addition of Zn2+. Basic fibroblast growth factor receptor assembly seems similarly to depend on the presence of divalent cations (M. Kan et al 9b-3-1 and redox metal ions are required (K. Ding et al. a-9) for nitric oxide-dependent H/HS oligomer generation. (Such inorganic ion inputs can also be suggested to include: core protein proteolytic shedding actions of Zn-dependent metalloproteinases (induced by cellular stress, mechanical, heat shock and hyperosmolality1) induction or seeding of H/HS or H/HS-protein supramolecular structure or aggregate formation; effects of inorganic cofactors in DNA expression, m-RNA production or activity and the modulation of the intracellular Golgi apparatus assembly process and putatively also the roles of metal ion for the modulation of enzymic post-synthetic HS processing by heparanase (D. Lui et al. 1), and other GAG structure processing enzyme activites1).

Table I:

Some Examples of Reported of Metal Ion Dependent HS Signaling

HS potentiation of Ca2+ signaling (The original hypothesis that HS was centrally involved in inorganic biochemistry) W.F.Long & F.B.Williamson, IRCS J. Med. Sci. 1979, 7, 429-434 Bivalent cations (Ca2+, Mg2+, Mn2+) Are required for HS modulation of Basic fibroblast factor receptor dimerisation; M. Kan et al., J. Biol. Chem., 1996, 271, 26143-26148; G. Siegel, et al., Cardiovasc. Res. 2003, 58, 696-705. (This apparently also requires the antihrombin binding (H pentasaccharide epitope) in HS W.L. McKeehan et al. ibid., 1999, 31, 21511-21514 which is especially sensitive to the effect of counterions as revealed

by comparison of the shift induced by counterions in the NMR signals between the antithrombin binding site and regular disaccharide repeat units in H. (Reported by D. Grant et al., Cell Biol. Intern. Rep. 1987, 11, 220)
Similar HS protein interactions, which had been previously reported which were discussed by Kan et al. loc. cit. include Bivalent cations required for correct HS interaction with 2 and 3 integrins, Platelet/endothelial cell adhesion molecule-1 (PECAM)-1) Cadhedrin and L-selectin; Cf., L-selectin in nonlymphoid endothelial cells; K.E. Norgard-Sumnich et al., Science. 1993; 261: 480-483. Ca2+ is also required for HS mediated Annexin V assembly on cell surfaces I. Capilla et al. Structure (Camb.) 2001; 9: 67-64, cf., A Lewit-Bentley et al. Eur J Biochem 1992; 210: 73-77 Bivalent cations Zn2+ etc. are required for Endostatin binding to H and HS S. Ricard-Blum et al. J Biol Chem. 2004: 275: 33688-33696); Ca2+ is required for Serum amyloid P binding to HS H Hamakai, J Biol Chem. 1987; 262 ; 1456-1460); Ca2+ enhances Serum amyloid P aggregation inhibition by H and HS E.H Nielson et al. APIMS. 1994; 102: 420) Divalent cations are required for H binding to Fibronectin M. Hayashi & K.M. Yamada, J Biol Chem. 1982; 257: 5263-5267) Ca2+ is required for extracellular Fibrillin-1 binding to HS proteoglycans K. Tidemann et al., J Biol Chem. 2001; 276: 36035-36042) Extracellular Ca2+ concentration regulates the distribution and transport of HS proteoglycans in a rat parathyroid cell line Y. Takeuchi et al. J. Biol. Chem. 1990, 265 (23) 13661-13668 Ca2+ is required for aggregation of Syndecan-1 transfected cells, mediated by HS, M.J. Stanley et al., J. Biol. Chem. 1995; 270: 5077-5083 Zn2+ and Ca2+ affect the neutralizing ability of Histidine rich glycoprotein by H Y. Kazama & T. Koide, Thrombosis Haemostasis, 1992; 67: 50; Cf. also A.L. Jones et al. J Biol Chem., 2004, 279, (29) 30114-30122) Zn2+ promotes the association of HS with biotyinylated H-kininogen T. Renne, J. Biol. Chem. 2000; 275: 33688-33696) Zn2+ Cu(II) Amyloid protein precursor (evidence for a Zn modulated H superfamily) A.J. Bush et al., J. Biol. Chem. 1994, 269 (43), 26618 forms bridges in oligomeric complexes in Prion protein - HS and GAG complexes R. Gonzales-Iglesias et al., J Mol Biol., 2002: 319: 527-540) Potentiation by a Cu(II) tripeptiede (GHK) complex of glycosaminoglycan (and collagen,metalloporoteinase elaastin and protease expression) Simeon et al. J. Invest. Dermatol., 2000, 115 (6) 962-968

Cu(II)

Cu2+/+ and Zn2+ potentiate mitrosative scission of H/HS at GlcNH2 sites

Cf. e.g., F. Cheng et al. J. Neurochem., 2006, 98, 1445-1457

Fe2+and Cu2+ ions apparently catalyse HS-GlcNSO3- HS-GlcNH2 i.e. prime HS for nitrosative cleavage D. Grant et al. unpublished observations Mn2+ Potentiation of the inhibition of soluble guanylate cyclase by HS and other sulphated polyanions M.A. Liebel et al., Biophys. Res. Commun., 1982, 104, 957-964

Possible Clue as to why Si occurs in H/HS-to Act as a multi-elelment conveyor and sorter
SiO2-based chromatographic packing medium+heparin for chromatography of inorganic ions (where both cations and anions can be

separated on a single column)

T. Takeuchi et al. Analusis. 1998, 26, 61-64

Table II Some Examples of Reports of the Effects of Inorganic Ions on HS Biosynthesis

Direct influence on HS biosynthesis

Extracellular Ca2+ concentration Y. Takeuchi et al., J Biol Chem,1990, 265, 13661-13668

Mn2+ and Ca2+

a GlcNAc transferase I can use both Mn2+ and Ca2+ but a-GlcNAc transferase II can use only Mn2+ TA Fritz et al., J Biol Chem. 1994, 269, 28809-28814;

Mn2+
AZ Kalea et al., Biometals. 2006; 19: 535-416 Mg2+ P Jaya PA Kurup, J Biosci. 1986; 10: 487-493; Ca2+ Pb2+ Y Fujiwara T Kaji, J Health Sci. 2002; 48: 460-466; Toxicology, 1999; 133: 159-169 Cd2+ A Cardenas et al., Toxicology, 1992; 76: 219-231;

Hg(II), Cd2+, Mn2+, Ni(II), Pb2+

DM Templeton Proc Trace Element Health Disease IUPAC Symp .1990 p 209-219;

FK Pawalowska-Goral et al., Fluoride. 1998; 31: 193-202 SO42PA Dawson & D .Markovich, Pflugers Arch-Eur J Physiol,. 2002; 444: 353-359 Ca Oxalate Crystal Surfaces (a specific crystal structure which can signal for altered HS biosynthesis) Cf. F.T. Borges et al. Kidney Int. 2005, 68, 1630-1642

Possible (Bone) Apatite-Related Effects

Si probably as SiO2 (suggested input into HS biosynthesis) M.F.McCarty Med. Hypoth. 1997, 49, 177-179 Possible HS/bone related role of Sr2+ for augmentaing and modulating GAG biosynthsis Y. Henrotin et al., J Bone Miner Res. 2001, 16, 299-308 E.R. Wise et al., Chem. Mater. 2007, 19, 5055-5057 have recently confirmed that bone calcification seems highly dependent on surface sulphated polysaccharides as the organic mineral

interface in bone is predominantly polysaccharide, putatively HS.

Hypothesis that Heparan Sulphate Influences Metal Ion Activities

W.F. Long & F.B. Williamson had suggested in 1979, 1a, on the basis of personal experimentation coupled with an extensive a literature review, that HS evolved by replacing most of the glucuronic aicd anionic sites with more flexible iduronic acid sites to flexibly bind Ca2+ ions with sufficient physiologically relevant affinity to enable HS to control the multiple signaling activities of this second messenger ion, which includes a major influence on cellular proliferation. (This research group also showed that the conformation of H and its biological activity could be modified following the sequestration of Ca2+ 1b by H; earlier studies by Gould et al.1 had shown, by X-ray crystallography, that interlinking between metal ions uronic acid groups, water molecules and sugar oxygens was functionally relevant to the supramolecular structure formation and related activities of of cell wall polysaccharides; Ca2+ as well as Na+ seemed to facilitate the formation of polysaccharidemetal ion egg-box aggregates (cf., G.I. Grant et al.1 and S.H. Seale et al.. 1 ). A possibly related HS selfassembly process identified by L-A Fransson & Havsamark1 which might also conceivably have depended on a similar interlinking effect of metal ions sited between HS proteoglycans). In plants, anionic pectin oligosaccharides seem to modulate Ca2+-dependent signaling processes 1a-8 analogously to the suggested (W.F. Long & F.B. Williamson 1) role of HS.

Physiological Occurrence of Nitrite Cleavage of Heparin/Heparan Sulphate

Vilar et al had also reported1e the unexpected ability of nitrite to deaminatively cleave H under physiologically relevant conditions (it occurring when the reaction was performed in the presence of phosphate buffer but not when an imidazole buffer was used; the critical factor here seems to be that the former buffer ubiquitously contains trace amounts of redox metal ions). Hitherto it had been thought essential that highly acidic non-physiological conditions were essentially required to achieve nitrosative scission of H/HS, which was known further to require the presence of unsubstituted glucosamine NH2 groups in the H/HS chains, which were also further believed not to exist naturally in H/HS under in vivo conditions.

Later work found that these assumptions were incorrect 1f . It has now become fairly well established that a major HS signaling process occurs by the controlled insertion into HS of unsubstituted glucosamine groups primed for nitrosative cleavage under slightly acidic conditions in response to biochemical stimulae; these are apparently coupled to servo feedback coupled microstructural alteration of HS. Such H+ and/or redox-metal-ion-dependent non-enzymic transformations apparently also directly primes for nitrosative cleavage by promoting the (heparan)-N-SO3NH2reaction 1g,h . Redox metal ions (as well as Ca2+ and Zn2+) present in the metallomic profile of H/HS could, it is now proposed, facilitate nitrosative scission of HS which is now believed to major animal protein control and signaling system which generates hormone-like HS oligomer messenger molecules which engage in such tissue protective and other functions, a mechanism which is, however, vulnerable to pathological perturbation by toxic metal ions.

Early Studies of Metal Ion Effects Nitrosative Processing

A traditional, highly effective, method of identifying HS in a mixture of polysaccharides is by reaction with nitrous acid which is highly specific for H and HS, being widely used in the laboratory to show their presence in proteoglycan mixtures. This was originally thought to be an entirely non-physiological action. Nitrite/nitrous acid was also known to promote stomach cancer, an effect which had been attributed to a pro-cancer mutagenic action on DNA. That nitrite/nitrous acid was such a highly specific target reagent for a major HS animal cell surface receptor might also , however, have alerted researchers to the possibility that some perturbation of HS biochemistry might have accounted for such pathological actions of nitrous acid, but this only became a credible hypothesis following the later, unexpected, discovery that nitric oxide is actually a major in vivo messenger molecule in animal physiology. N.b., nitrite is a primary metabolite of nitric oxide. It was further discovered was that if nitric oxide formation and metabolism became unbalanced, appreciable pathological in vivo protein tyrosine nitration always ensues and such nitration is a hallmark of many, or indeed of all, degenerative diseases 1a-8. Prior to the discovery of the normal physiological roles of nitric oxide, B. C. Challis et al. had

established that various inorganic catalysts (e.g. copper, iron, silver iodide and thiocyanate ions, the latter being augmented in the serum of cigarette users) catalyze nitrosative reactions. A corollary to this scenario is that anthropgenic inputs from such inorganic redox ions could induce inappropriatly perturb nitric oxide-dependent HS signaling processes. A reversal of inappropriate nitrite formation is however achievable by the reaction of nitrite with ascorbate which efficiently reconverts it back to nitric oxide. Another role for ascorbate in nitrosative HS signaling has also been identified, this being for the reduction of Cu2+ to Cu+ needed to enable the recycling of nitric oxide held at HS-core-protein cysteine stores1a-9.

Antioxidant & Antinitrant Activity (Related to Modulation of Superoxide Dismutase by H/HS) and Sequestration and Deactivation of Redox Active Iron, Copper & Silver Ions, Etc. by Heparin/Heparan Sulphate)
The following articles discuss this topic: H/HS-Superoxide Dismutase T. Adachi & S.L. Marklund J. Biol. Chem. 1989, 264 (15) 8557 J. Biochem. (Tokyo) 1995, 117 (3) 586-590 Biol. Pharm. Bull. 1998, 21 (10) 1090; Chem. Abs. 130. 64588h

------------------------------------------------------------------------------------------------------------------Cf. also, D. Grant et al., Biochem. Soc. Trans., 1996, 24, 194s (Heparins as essential antioxidants) Ibid., 1988, 16, 1030-1031 (Effect of heparin on dismutation of superoxide anion)
D. Grant et al., Med. Hypoth. 1987, 23, 67-71 (Review article) D. Grant et al. had found the absence of NMR detected paramagnetic effects expected for high levels of Fe and Cu in some H samples; this was rationalized as due to occurrence of these elements within the H supramolecular structure in a separate nonsolution phase attached to H. Cf., G. Mackintosh, Ph.D. Thesis Heparin-Iron Interaction and its Possible Relevance to Antioxidant Activity University of Aberdeen 1995

[This study involved in vitro and in vivo investigations into possible inflammatory actions of Fe(II) ions which are putatively inhibited by binding of such ions to H/HS or to H-mimetics such as pentosan polysulphate]
Cf. also M.A. Ross, M.Sc. Thesis, Heparin as an Antioxidant, University of Aberdeen 1992 M.A. Ross et al. Biochem J. 1992, 287, 849-853 Cf., R. Albertini et al., Int. J. Mol. Med. 2000, 6, 129-136; FEBS Lett., 1995, 377, 240-242 F.B. Williamson et al. (unpublished, personal communication) showed that Fe(II) heparin undergoes auto-oxidation to generate Fe(III) (O.OH) akagaenite crystals detectable by their characteristic X-ray diffraction pattern. Other realted unpublished studies have indicated that iron ions can promote pathological calcification and fibrin aggregate formation which is upon ingestion by macrophages and resultant inappropriate cytokine induction, promotes degenerative disease processes .

(Related solid phase, antioxidant function indicated by removal of potentially damaging redox iron ions by polysaccharides was reported by Merce et al.2a and Sipos et al. 2b for anionic polysaccharides) [P. Sipos, O. Berkesi, E. Tombacz, T.G. St Pierre & J. Webb, J Inorg. Biochem. 203; 95: 55-63 A.L. Merce, L.C. Marques Carrera, L.K. Santos Romanholi & M.A. Lobo Reico, J Inorg Biochem., 2002; 89: 212-218] D.J.R. Jarrett et al. J. Clin. Exp. Gerontol., 1989, 11, (3-4) 145-154 Ageing as a cause of raised serum feritin in the absence of disease J.T. Salonen et al. (Circulation, 1992, 86, (3) 803-811) found an epidemiological correlation between mortality in Eastern Finnish men with appropriately corrected serum ferritin values. This study supported a possible role of redox active iron ions in degenerative cardiovascular dysfunction. This can be further suggested to include a redox iron related alteration of HS signaling.

Cf. also T. Aoyagi et al., Biol. Pharm. Bull. 1994, 17 (2) 340, Chem. Abs., 120, 294925b

Age dependent decreases in fibrinolytic enzyme activity in serum of healthy subjects.

M.A. Liebel & A..A. White, Biochem. Biophys. Res Commun. 1982, 104 (3) 957; Chem. Abs., 138749q Reportd that soluble guanylate cyclase was inhibited by H in a Mn2+ dependent manner. Sulpated algal polysaccharides demonstrate an H-like modulatory activiey for this process in which the effectiveness of carrageenans were >>.

Z. Liu and A.S. Perlin had reported1c that Cu2+ causes selective free-radical degradation of H and in a related study by R.N. Rej et al 1d, found that trace amounts (ca. 1/185 mol ratio with respect to H) together with the generation of reactive oxygen free-radicals (from H2O2, plus ascorbate) which reduced the anti-FactorXa activity of H without causing any detectable alteration in its NMR spectrum. Notes
b

L. Kjellin & U. Lindahl (Annu. Rev. Biochem. , 1991, 60, 443-465 in a review of the structure and function of proteoglycans listed these as: provision of mechanical support, negative charge, regulation of cell migration and aggregation, roles in the development and stabilization of synaptic structures, endothelial regeneration, stabilization of basement membranes, modulation of collagen fibrilogenesis including the transparency of the cornea, regulation of cell growth, urinary trypsin inhibition, provision of a filtration barrier, role in morphogeneis, link to cytoskeleton and ECM, mediation of adhesion and morphogensis, assembly of matrix phosphatidylinositol linkage, reservoir for basic fibroblast growth factor, regulation of blood coagulation, mediation of transferrin functions, and uptake of antigen presentation. The initial step in biosynthesis is the formation of the protein linkage region GlcAGal-Gal-Xyl-Ser by transfer of xylose from the UDP-xylose to specific Ser residues in the core protein. The same linkage region occurrs with HS and chondroitin sulphate had been highly conserved from flies to humans. Bernfield et al. ibid., 1999, 68, 729-77 also provided an extensive review updating similar studies of HS systems (the most common members being now known to be glycoylphosphoinositide-linked glypicans and transmembrane syndecans) which provide the most abundant receptor system at adherent animal cell surfaces for tissue morphogenesis and wound repair as well as for host defense and energy metabolism) via interactions with the extracellular matrix, e.g. to control growth factors.
c

The possible benefits of ascorbate therapy for the treatment of cancer and viral infections was discussed by Linus Pauling. Such effects can now be rationally ascribed to the augmentation of HS protection afforded by ascorbate therapy (cf. D Grant, internet discussions posted in 2000 at web.ukonline.co.uk/dgrant/dg4/ [similar addresses with dg2/ /dg5/ and dg8/]

Heparin/Heparan Sulphate Biochemistry is Relevant to Multiple Sclerosis Research (Could the Defective Nitrosative Cleavage which Occurs in Nieman-Pick Disease alaso be Relevant to Cancer and other Degenerative Diseases?)
Defective nitric oxide-dependent deaminative cleavage of HS (and pathological nitration) has been reported to be associated with Niemann-Pick disease (K. Mani et al. 1a-13-2 )(a condition where an HSdependent recycling of cholesterol is defective but this becomes less severe in presence of adequate ascorbate which is believed to increase defective nitric oxide availability, e.g. via the redox metal ion catalytic process for nitric oxide cycling). A related dependence of aberrant nitric oxide metabolite HS-related tissue damage on insufficiency of ascorbate is probably also centrally relevant to the etiology of cancer in a redox-related HS scenarioc (an idea supported by the observation that ascorbate in cell culture experimental observations to greatly

increases the degree sulphation of fibroblast HS (evidently via the modulation of the primary biosynthesis1a-7). The etiologies of some forms of cancer and other degenerative diseases1a could also centrally involve perturbation of polysaccharide protein cross-linking (especially that facilitated by Ca2+) involving an altered modulation of the activities of growth factors involved in wound healing and which are putatively are perturbed in all degenerative diseases. An possible example of this is multiple sclerosis which from epidemiological and geographical/geological evidence is suggested to be at least in part, promoted by a chronic dietary, environmental/workplace intoxication by toxic metal ions ( e.g. Ba2+ 2). which putatively perturb the HS (multivalent metal ion) determined growth factors/growth factor receptor assembly activities which are required for myelin sheath renewal.
The electrophoresis of H in a Ba2+ containing buffer solution while providing an effective method of demonstrating the complexity of the mixture also suggests how in vivo signaling or its disruption could depend on interactions of H with alkaline earth cations. This may be relevant for the mechanism of fibroblast growth factor (FGF) signaling thought normally to depend on a quaternary complex of HS-FGF -(Ca or Mg)-fibroblast growth factor receptor (FGFR)9b-3 and how replacement of Ca or Mg with other metal ions might account for disruption FGF-FGFR signaling in demyelinating degenerative diseases which have been suggested might have been triggered by the chronic exposure to inappropriate amounts of Cu, Zn, Pb, Al, Ba or Mn ions.

Evidence for the Co-Occurrence of a Seawater Range of Inorganic Elements in Heparin

In agreement with the hypothesis that multicellular animals first evolved in a marine environments, the full 60+ inorganic ion compositions of animal (especially extracellular) biological fluids can be shown to mimic that of seawater4. This phenomenon may by why the total amounts of HS and other glycosaminoglycans occurring in the tissues of in fifteen species of aquatic including marine invertebrates demonstrated an exact exponential mathematical relationship with the degree of salinity of their habitats5. This finding could add support to the notion that the sequestration of inorganic elements from natural growth media might have been the primary reason for the evolution of HS during the early stages of the evolution of multicellular animals (HS appeared at the same time as this1a-1,2) in the sea, a phenomenon which could still be of relevance to the full understanding of the modus operandi of these polysaccharides in present day organisms). A further confirmation of this notion is that H, [n.b. this is a highly purified, protein free, animal mast cell- derived polysaccharide] apparently always clearly demonstrates, (e.g. when studied by mass

spectrometric techniques cf. Table II) a (skewed but) characteristic seawater/extracellular biological fluid like multi-inorganic element fingerprint similar to that of seawater bathed marine polysaccharide matrices, suggesting that H and the structurally closely related HS (a likely difference, however, is that the protein cores of HS can also bind additional metal ions) simultaneously sequester the full range of inorganic ions and particles which can naturally occur in their normal bathing solutions. Numerous reports6a confirm that a variety of metal ions, including highly charged multivalent ultratrace elements, bind at often modest but physiologically relevant (and rather similar) affinities to HS under in vitro and in vivo conditions (such investigations include those relating to scintigraphic visualisation of tumours and a arange of multivalent metal ions used for the histochemical staining of tissues6a).

Anions are also known to bind to H. These include SO42- which commonly contains ca. 12% S in this form. Multi elements occur at the greatest abundance in the traditional form of unfractionated Na H but they lso occur albeit in reduce amounts in Lithium H (Cf. Table III). Such (claimed) single counterion forms of H apparently should more correctly be described as modified multi-element matrices. Interference from Inorganic Constituents of Heparin During Blood Analysis This circumstance confirms older observations 3a that, if commercial H is employed as an anticoagulant for blood inorganic element determinations, account needs to be taken of the potential interference for the accurate determination of the inorganic elements in blood samples; the inorganic contaminants present in H clearly especially affect the accurate determination of those elements occurring in ultratrace amounts in biological samples.

Variation in Degree of Purification of Heparin from Inorganic Components

It is suggested that possible variation in purification procedures between commercial operations can also gives rise to previously reported but hitherto inexplicable irreproducibility in the biological activities

of commercial H samples between different brands 3b-10, 7. For fundamental biochemical researches the in vivo forms of H and HS are obviously required; these are most likely the natural multi-element forms. This hypothesis, needs, however, to be more fully established.

Binding of Anionic Polysacchrides to Seed Crystals

A major additional potential role as part of this signaling and information processing of HS is that the binding to colloidal inorganic particles such as crystal seeds can occur in a functionally relevant way; the crystal surfaces of calcium oxalate appear to induce by biofeedback an alteration in the kidney biosynthesis of HS to augment the specific anti-Ca oxalate crystallization abilities8. Similar specific interactions between crystal surfaces and alginates seem to be highly specific as was the case for a range of protein-free alginates polysaccharides of defined microstructures which adsorbed on inorganic seed crystals and caused inhibition of crystallisation; the relative degree of inhibition showed a mathematical relationship between the crystal growth rate constants (which could be accurately determined) and the detailed information encoded known microstructures of these polysachcarides 9. Essentially this is an in vitro demonstration of how information in polysaccharides with complex microstructure can be directly read. Calcium salt crystallization rates are similarly dependent on H/HS microstructure (n.b. the similar binding of HS fragments to sparingly soluble calcium salt seeds is thought to be relevant to the mechanism of control of both urinary and cardiovascular calcification9b and also to the age-associated human cardiovascular dysfunction in which (programmed?) changes in blood vessel wall HS microstructures are believed to contribute to1a-5. By an extension of this hypothesis, both normal ageing and degenerative diseases in animals could have an ultimate link to age-related changes in HS sugar sequences which may include an age-dependent alteration in the suggested inorganic cofactor effects). A. Lima de Faria suggested10 that the seeded crystallization of specific complex crystalline forms of CaCO3 obviously without any role for DNA suggests that at its most fundamental level biological reproduction also does uniquely require DNA-based genes10; reproduction of specific kinds of CaCO3 particles by templating can be suggested to be a related process to DNA replication. A corollary to this idea is that information held in specific (e.g. anionic) sugar sequences in H/HS could potentially be modulated by inorganic surfaces or membranes to which the inorganic elements which also occur (albeit mostly in very small amounts) in biological fluids and tissues as well as in seawater4. (These will include single ions, ion pairs and aggregates which are potential crystal growth nuclei (n.b. physiological solutions are supersaturated as regards the formation of solid phases of sparingly soluble Ca salts e.g. CaCO3 (calcite), apatite etc. Apatites characteristically are, like H, non-stoicheiometric salts contain a range of isomorphously substituted inorganic ions.

Colloidal complexes of hydroxyapatite - H can be prepared in the laboratory 1h. Such entities putatively have biological significance.

The Antithrombin Binding Pentasaccharide Epitope of Heparin

Perhaps the most fully understood epitope in H/HS is the pentsaccharide antithrombin-binding (PSA) site which occurs most characteristically in mast cell H and in some HSs. Pharmaceutical H, which has been used for many years as a blood anticoagulant, is now available in a variety of salt and molecular weight forms which includes the single PSA epitope, an artificially produced PSA molecule as well as unfractionated H (Na heparin) from porcine mucosa (but also from bovine lung) which derives a major part of its anticoagulant activity from the PSA epitope content. Further evidence for the role of metal ions in H-protein interaction is that in vitro studies of the kinetics of the interaction of PSA-H with proteins involved in blood anticoagulation markedly different kinetic rate constants in NaCl, KCl or LiCl solutions of the same ionic strength9c ; NMR resonances of the central trisulphated moiety in PSA-H were also especially sensitive to the presence of a range of inorganic ions which systematically displaced them to a much greater extent than was the case for the major disaccharide repeat unit resonances of H9b-2. Although pharmaceutical H is essentially protein-and nucleic acid free, it is not a single stoicheiometric molecule, but a library of at least 120 chemically distinct molecular species (which can, it is also now suggested, also be classified as a metallomic multielement, organometallic array).

The Association of Metallomic Multi-Element Arrays with Anionic Polysaccharides

Spark source mass spectrometry (SSMS) and inductively coupled plasma mass spectrometry (ICP-MS) multi-element analytical data for several H samples from different manufacturers suggest that H always contains a seaweed-like inorganic element content (recent ICP-MS reports of blood sampling tube H-

associated contaminants suggested3b-10 the co-occurrence with H of the full seawater range of some 80 elements 4). Such single ion enriched Na and Li H (e.g. by use of ion exchange resin column technology as used in medical grade H preparation) as well as native H appears to be greatly enriched in those elements which occur in the least amounts in natural waters, a situation which is reminiscent of the cell wall polysaccharides of plants1a-10 where a similar range of inorganic ions as are believed to contribute to cell wall control over Ca2+ second messenger actions via the borate crosslink polysaccharide oligosaccharide generator1a-10.

The occurrence in marine algal tissues of major cell wall polysaccharides in multi-counterion salt forms rather than, as originally thought, free acid forms, was established by A. Wasserman3 and confirmed by later workers including W.A.P. Black & R.L. Mitchell3a. It is now thought that marine algal polysaccharides contain most or all of the inorganic elements (60+ in number) which occur in seawater3b from which anionic polysaccharide ligands apparently are able to simultaneously bind a wide range of inorganic counterions and particles by a mechanism which apparently enables these polymeric ligand systems to become selectively enriched in those elements which are least abundant in their natural bathing solutions. H. Haraguchi4 suggested that for updating of the metallomic concept, many elements which had hitherto been classified as being non-physiological must be included, furthermore it has now become evident that such elements occur both in seawater and human blood serum (these solutions demonstrate a degree of inter-relatedness in their inorganic element metallomic compositions 4). The non-physiological elements also show up in H cf. Table III, indicating that a possible physiological role of such element might be to modulate the biochemical activities of H/HS. Table III compares the metallomic profiles of alginate, heparin and human hair, a common used tissue employed for detection of metal ion status of humans and for providing evidence of metal ion intoxication. Comparison of the multi-element compositions of the earths crust with seawater and H

show much less correlation (data not shown). Table IIIa lists results reported for metal ion impurities present in chitosan which have been attributed to uptake from a final washing with tap water13a. Polysaccharide-rich biomasses of land plants, brown and red marine algae, as well as mollusc shells, bone, mast cell H and H-like segments of HS may constitute a related system of natural (salinity induced) metallomic matrices which seems also to be related to the inorganic element contents of human hair15 (which is currently the most well researched multi-inorganic element containing matrix) and chitosan13a ( cf. Table IIIa). Human scalp hair (e.g., 15 ; except for Zn, which is augmented in hair) is compared with the data shown in Table I for Na and Tl heparin. This correlation is best for the least perturbed samples, from schoolboys 15. Table III

Comparison of the Elemental Contents (mg/kg) of Kelp, Heparin & Human Scalp Hair
Baltic Seaweed3a-4 Commercial Kelp3a-1 Na+ Heparin
Element (D after ion exchange) (commercially produced)

Li Heparin

3b-9 3b-10 (A:before ion exchange) (E after ion exchange)

Tl+ Heparine,3b-9 (a)

Human Scalp Hair15 (s) (b)

(c)

(academic lab product)

Si Mg Mn Ca Sr Ba Na K Rb

4300-6000 410-420 1200-5400 2180-2210 372-381 460-510 2000-7000

1642 2130 1235 19040 749 12.8 12800 0.05 trace 2.93 trace 6.3 896 35 12.2 0.06 15.9 193 0.12 0.01 trace 0.04 0.33 0.19 35.2 0.14 trace 1.4 2110 5.3 194 624 36800 326.5 trace

A D 5900 116 1300 ? [1-65](d) ? 30000 171 65 2 140 1 principal 2000 23 3 0.1

E1 E2 126 1170 (30) 260 (0) ? <50 125 0.04 1 0.3 14 143 336 42 22 <0.002 0.1

Cs 0.047-1.08 Tl Cr 1.8 Cu <10-20 Fe 280-320 Ni 9.2-13.2 Co Sn Mo <0.027 Al 120-140 Ti As 327 Zr 10.7 Ce Ag Nd W La Zn 250-310 Pb Ga Sb Cd P V 0.6-0.9 B I 130-160 Cl F Br 140-160

9 0.01 <0.002 8 0.2 0.0008 30 trace 730 16 7 10 1100 5 <4 24 <170 trace 1.3 0.6 <80 trace 5 trace 7 trace ? 14 16 <390 0.2 (0) 2 <15 ? 0.08 0.3 5 ? 0.3 7 ? (0) 3.5 4 0.002 0.003 0.01 5 0.04 0.008 0.1 5 0.03 7 0.005 0.004 0.003 <80 16 33 49 16 20 2 (1) 440 0.8 0.2 0.4 0.005 0.003 0.003 3 ? 0.003 0.3 0.12 0.1 61 77 435 ? 16 (0) 30 0.7 2 3

0.07

B C A/C 100 510(s) 11.6 10 28.0(a) 59 3.6 28 (0) 6 (s) 30 450 (a) 67 11 1.4 1.2(a) 54 1 0.16-1.6(b) ca.90+ 9 90 40 20.4(s) 98 0.2 0.051(s) 59 0.15 (s) 20 0.1 principal 1 0.99(a) 30.3 <5 22.1(a) 33 70 10 19.0(a) 58 24 10 1.49(a) ca.100 ca.125 2 0.67(a) ca.100 0.3 0.24 21 <1 0.43(a) 16 96 ? 4 0.79(a) <493 <126 1 0.274(s) <55 <167 0.21(s) 24 0.093(s) 75 0.5 0.034(s) 118 1 0.039(s) 128 <1 0.037(s) 135 1 0.045(s) 156 7 150(a) <0.5 185(s) <0.43 4 7.1(a) 2.2 1 3.4(s) 5.9 1.7 1.5 ca.30 <1 1 <1 1000 4 0.9 195(a) 169(s) 1.22(a) 1.41(s) 2.9(s) <580(s) 4.46(s) 2.3 2.6 2.5 <17.7 3.4 >10 29

3 <25 10 1.3 5600 890 130

Notes: Data for Na H (which had been recommended as being suitable as a standard reference material to be distributed amongst a number of academic institutions by a major manufacturer, which was further purified by extensive dialysis) before ion exchange and Tl He after ion exchange are taken from ref. 3b-9. Data for Na and Li H after ion exchange are taken from ref. 3b-10.
Further Background to the Studies of Metal Ion Binding and the Multi-Element Content of H Commercial H, readily available as a highly purified, protein-free polysaccharide mixture, avidly sequesters both cations and anions in a relatively indiscriminate manner and therefore potentially can host a full repertoire of inorganic ions6, 6a (both from ex vivo contamination13a and from uptake from the wide range of small amounts of inorganic elements which occur in biological fluids4). Although it is possible that the toxic ions in H are bound in some less toxic form, their occurrence in H used for dialysis and parentereal nutrition solutions is a topic of current concern3b-7,8. Although LB Jaques in his excellent 1978 review6a had emphasised the unique physiological relevance of the sequestration of both positively and negatively charged inorganic ions by H, at the present time there seems to be lack of awareness or interest in the wider relevance of this phenomenon. An analyses of a carefully selected typical porcine sodium heparin by spark source mass spectrometry (SSMS) revealed the presence of some 38 inorganic elements in amounts equal to or greater than 1ppm. A larger range of inorganic elements remained in purified H studied by ICP-MS (in the context of commercial H anticoagulant employment in blood collection tubes); the ICP-MS results agree surprisingly closely with those previously reported for both native and ion exchange purified H previously studied by SSMS. C.F. Moffat Synthesis, characterisation and applications of chemically modified heparins, Ph.D. Thesis University of Aberdeen, 1987.

Th ICP-MS results show the occurrence in H of ultratrace amounts of ((Au))((Be))(Bi)((Dy))(Gd)Ge Hf Ho (Ir) Li Lu Nb (Os) Pd Pr Pt Re Rh Ru Sc Se (Sm) Ta (Tb) Te Th Tm U Y and Yb (including in single counterion enriched, purified forms of H_ Data in parenthesis and double parenthesis are progressively less well established than those without parenthesis3b-10. Comparison of the data for multi-inorganic elements present when H or K2EDTA were used as the anticoagulant in blood collection tubes suggested that on the basis of anonic equivalents purified H contains a higher inorganic element profile than K2EDTA, especially for Pb, Fe, Al Na Ca Mg Ag Cd Cu I Si Sm and V which appeared to occur in purified H in amounts some 100 times greater whereas the amounts of La Mn Eu Mo B Ti Pt Lu and Hf were present in similar amount in both K2EDTA and purified H. Much larger amounts of Rb and Cs occurred in the K2EDTA than in the purified H 3b-10. Table I shows the multi-element contents of a range of H and of alginate-based biomasses 3a. The inorganic elements of a Baltic seaweed3a-4 seem to include a large anthropomorphic component while the commercial kelp inorganic element values suggest an oceanic seawater origin of these elements the distribution of which also shows greater similarity than does the Baltic data to the native Na H data of Table I which further suggests that the inorganic components of the H samples listed in Table I are derived from a natural source. [The results shown in Table III also suggest, however that Rb, Cu, Ni and Sn bind more strongly to the combined NSO3- OSO3- ,C(O)O- anionic system of heparin than to single C(O)O- anionic groups system of kelp alginate, the most likely ligand in that matrix].
G.E. Harrison & A. Sutton3b-2 had also, many years ago, reported the Ca, Sr and Ba contents of five commercial H samples; these data appear also to be approximately related to the contents of these elements in a range of other (polysaccharide containing) matrices reported by HJM Bowen14. This author, in 1966 had listed the then known reported information on the multi-element contents of H (./g) as: Mn 3.6, Ca 30-2900, Sr 5-92 Ba 2.5-12, Cu 0.4 and Zn 28 3b-1.

(a) ref. 15; (s) ref. 15a; (b) other internet listings; (c) ref. 15b ; (d) ref. 15d.
e

This (highly toxic counterion form) was selected for in vitro 205Tl NMR (which showed rapid alongchain site interchange). Tl seems to occur in natural heparin in similar amounts to that found in kelp but when present in such matrices may not act as a high risk human intoxicant 3b-9 but further specific studies are required to confirm this hypothesis.

Table IIIa
Comparison of the elemental content (mg/kg) data from ref. 13 of chitosan from crab shells (obtained by using a final wash with demineralised water which avoids the uptake of metal ions from tap water, a source of suspected but not documented contamination)

Element Fe Ni Cu Cr V Mn Pb Ag Hg Cd

Pure Kelp Na heparin Tl Heparin Chitosan 898 35 6.3 trace 5.3 high 0.14 0.04 1.9 trace 1100 170 730 30 3 <1 16 4 <1 <1 10 10 5 1 <1 <1 4 0.5 <1 1.5 high 2.03 1.03 0.36 0.12 0.09 0.59 0.02 0.025 0.22

The controlled uptake and release of inorganic ions from H-like polysaccharides could, it is proposed, be part of a wider servocontrol feedback system. The presence of multiple elements in naturally occurring salts as well as chemical reagents (including K2EDTA and gels employed for blood clot phase separation 3b-10) is a common phenomenon. (Such naturally occurring salts include apart from polysaccharides, proteins such as those reposnsible for the multi-elemnts in hair and nails, the apatie mineral of bone (as well as naturally occurring apatites), polyphosphates (which occur ubiquitously at cell surfaces throughout biota) and soil and natural water humic salts (which include a fraction termed fulvate). It is entirely in keeping with the general occurrence of multielement in salts found in nature (unless these have been subjected to chemical purification by crystallization and recrystallization processes) that the ultra salt-like highly sulphated polysaccharides H and HS which are the extreme forms of such biologically utilized salt systems exist as multiple inorganic ion arrays and that inorganic ions can influence or control the heparanome. The HS inorganic array may, however be subject to pathological or anthropogenoic skewing. In general the metallome can influence the supramolecular structure of polysaccharides and thereby its biological activities.

The Metallome-Heparanome
The inorganic components of H and putatively HS may also normally find use in animal tissues to link specific parts of H/HS to proteins as part of a cooperative inorganic ion/H/HS manner, is suggestive of a metallomic-heparanome nature of the inorganic biochemistry of polysaccharides. A possible function of small colloidal particles containing e.g. Ca, Sr, Ba, P, Si, Fe, V etc. in H/HS could be to provide links similar to the borate crosslinks in plant polysaccharides (in a similar manner to how hyaluronan 2a and chitosan act as a ligand for Fe-rich particles2b). Currently there is fairly strong evidence that single Ca, Mg, Sr, Mn, Zn and Cu ions have been reported to be required to allow a number of specific protein HS microstructures to interlink (cf. Table I). Exogenously administered H (which it is now pointed out also includes multi-inorganic elements) is also reported to induce an increased secretion of H-like HS fragments from the endothelium11, the mechanism of which may at least perhaps partly be due to (a now established) Cu (redox-ascorbate)/Zn dependent nitric oxide/ HS oligosaccharide generation12 process as well as the effects of such inorganic ions on the Golgi apparatus exemplified by the effect of the presence of Ca2+ (perhaps Sr2+), Mg2+ and Mn2+ in cell culture experiments which boost HS biosynthesis (toxic elements, which can

also occur in heparin (e.g., Pb2+ ,Cd2+ , Hg(II) and F- (cf. Table II) were found in similar experiments to inhibit this).

The notion that H and hair multielements represents a systematic perturbation of the amounts of inorganic elements present in pure seawater also applies to exchange of inorganic ions between two types of polymer substituted sulphate or sulphonate ion exchanger systems and applies to the exchange of multielement containing heparin dispersions with a single counterion substituted polystyrene polysulphonate ion exchange resin column (a routine method of purifying heparin). The binding and redistribution of the inorganic elements by an analogous natural polyuronic acid ion exchange system may also be an important determinant of the observed distribution of inorganic elements in seawater (which contains ion exchanger organic matter as ca. 2g /ml polyuronate-like humic acid containing humicC(O)O- anionic groups which are analogous to such groups in animal and plant anionic polysaccharides). Such a structured humate system in seawater evidently is part of a system of natural anionic inorganic ion homeostasis agents (a possible mechanism by which this occurs in seawater (or seawater-like biological fluids in primitive and perhaps also in more highly evolved animal organisms) is that such polyanonic molecules act as highly efficient inhibitors of crystallization enabling them to promote the sort of long-term supersaturated solutions which exist both in seawater and biological fluids (as clearly shown by studies of the comparative rates of seeded crystallization of e.g. CaCO3 and BaSO4 in the presence of such anionic materials (i.e., humic and fulvic acids from marine and land humified organic matter as well as the more accurately molecular structure defined polyanionic polysaccharides). Ca2+ is the most abundant multi-valent ion present in the proposed series of materials and is also is most easily exchanged (viz. the exchange of Tl+ for Ca2+ occurred in heparin with an efficiency of 103 while Ce(III/IV) was exchanged by Tl+ (the least efficient exchange) occurred at an efficiency of 0.5 (cf. Table III)). The residual Ca in each of the samples defines the purity or the degree of ion exchanged purification. This gives a simple, but apparently also a scientifically robust, method for classifying various commercial heparins which have been purified to different extents starting from a similar multi-element containing crude tissue extract giving rise, following extensive purification (during which the least strongy held countercations such as Mn and Mg are lost) to the sort of heparin Na heparin listed in Table I (arising from natural buffer systems supporting multi-element solutions like biological fluids or uncontaminated seawater). It should be noted that the same method of classifying index does not seem to apply to algal polysaccharides affected by highly polluted waters but it does apply to such substances obtained from organisms grown in natural seawaters). Requirement for Nucleation ( e.g. by SiO 2 ) of Supramolecular Structures Involving Polysacchcaride Salts ? The high Si content in both human hair and H and the well-known association of Si with glycosaminoglycans 3b-6 is in accord with the likely importance of this element for mammalian biochemistry. Although Si is known to be an essential animal nutrient and be critically involved with glycosaminoglycans, especially with H/HS, the mechanisms of binding and physiological action are unknown. Si could be the key element linking geological and biological metallomics, suggesting the need for a wider geoglyco-metallome conceptualisation by which the inorganic metallome influences the heparanome, operating in a manner receptive to inorganic matrices via cooperative interactions involving ultra-anionic polysaccharide side chains for which an absolute requirement for specific metal ions e.g. Ca, Zn and Cu have already been established for several such processes. An analogous role for Si might be anticipated The association of metal ions and inorganic moieties with anionic polysaccharides seemed more to

resemble a phase change process than e.g. an electrostatic controlled counterion condensation process (the Aberdeen polysaccharide laboratory studies of metal ion binding to H reported by Grant et al. 6b-2-2 suggests this hypothesis). Such a phase change would be subject, according to the general principles governing the formation of phases in physical chemistry, should occur via a nucleation-seed-sensitive mechanism (e.g, facilitated by suitable inorganic or organometallic nanoparticles) . The actual chemical nature of the putative nucleation seed particles in use by biological systems are currently unknown. A possible silica particle role in this function, however, seems credible. Sicontaining particles (putatively amorphous SiO2 sols similar to those discussed by D. Grant et al. 6b-2-2) seem always to be a characteristic component of anionic polysaccharides (K.A. Schwarz 6). Their presence in such an association to fulfill a nucleation of phase change role might account at least in part for the fact that Si is an essential nutrient for animals and other species.

Similarity Between H/HS and Commercial Ionomers HS proteoglycans (e.g. glypicans, syndecans, agrin and collagen XVIII) must exist in vivo in mixed salt forms similarly to H. Their functions may, like man-made ionomers based on arrays of SO3- groups be further jointly on the inorganic counterions which induce a functionally important associated water cluster system. It had been previously noted that the amounts of HS and other sulphated polysaccharides present in fifteen species of aquatic animal organisms increased with the habitat salinity5. This was thought to be a requirement to inhibit high ionic strength induced tissue disaggregation, but the organism may require the correct inorganic seawater constituents including similar amounts of Na+ and polysaccharideSO3- to form the correct membrane heparan sulphate water cluster system. Apart from the principal ions needed to form the polysaccharide - SO3- Na+ (H2O)6 array system both natural as well as man-made ionomer functions may require a form of doping by seawater ultratrace inorganic components.

The results presented for what seems to be a unrefined Na H (Table III) suggest that an apparent systematic enrichment of trace and ultratrace multi- inorganic elements from some seawater(-like) animal tissue bathing fluid or perhaps brine used during manufacture (which requires further research to confirm the relative importance of each route) similar to the enrichment of seawater elements by marine algal polysaccharides.

References
1 Proteoglycans containing heparan sulphate (HS) (e.g. the syndecans with transmembrane core proteins and the glypicans with phosphidylinositol membrane anchors) contain highly anionic, variously sulphated linear uronic acid-glucosamine-disaccharide-based polysaccharide side chains which engage a wide variety of biochemical functions 1. Supramolecularly structured H/HS proteoglycan-inorganicglycocalyxes can be predicted to beformed at all adherent animal cell surfaces and extracellular matrices. Such organometallic matrices are suggested to potentiate H/HS participatation inter alia, in blood anticoagulation, antioxidant protection, protection against pathogen insult, energy metabolism including the modulation of lipoprotein lipase activities as well as complex cellular activities such as embryogenesis, wound healing and immune surveillance.

The extremely wide range of different possible sugar conformations and arrangements of sugar-O and glucosamine N-acetate and N-sulphate groups which facilitate inter- and intra-cellular signaling and control system in multicellular animals 1 seem also to constitute major metal ion interaction sites which may further participate, together with the nitric oxide stores which apparently occur at conserved cysteines in the HS core proteins, to enable facile nitrosative scission of HS chains by the recently discovered metal ion dependent mechanism..

Nitrosative signaling catalyzed by metal ions

K. Mani et al., Glycobiology, 2006, 16 (8) 711-718 and refs.cited

K. Mani et al.. J. Biol Chem., 2007, 282, 21934-21940

R. Cappai et al., J. Biol. Chem. 2005, 289; 13913-13920; K. Mani et al., ibid., 2004, 279, 12918-12923; K. Mani et al., J. Biol. Chem. 2003, 278, 38956-38965 K. Ding et al., ibid., 2002, 277, 33353-33360, K. Mani et al., Glycobiology, 2000, 10 (6) 577-586

Cf. Cf.

M.Belting et al., J. Biol.Chem., 2003, 278 (47) 47181-47189 F. Cheng et al., ibid., 2002, 277, (46) 44431- 44439 D.Grant, Chemistry Preprint Archive 2000 Oct 94-104

This system of nitrosative signaling catalyzed by metal ions putatively constitutes a major inorganic ion assisted H/HS information processing and control system in animals which seems to have been highly conserved throughout animal evolution (cf. 1a-1,2) and which could have enabled environmental inputs to influence the course of such evolution. This system might have arisen from an earlier system where HS evolved to mimic the natural humic (poly carboxylic acid) polyanionic buffer systems of natural waters. This notion is in keeping with the report that concentration of dispersed or soluble HS in blood (cf., W. Manley et al., 1) is similar to that of the poly-carboxylated poly-counterion salt containing natural (humic) polymers in natural waters including the sea where they putatively contribute to inorganic ion homeostasis (including by the potent inhibition of crystallization of sparingly soluble salts such as CaCO3). H/HS: General References
M. Bernfield, et al., Ann. Rev. Biochem., 1999, 68, 729-777 cf., Turnbull et al., Trends Cell Biol., 2001, 11, 75-82; cf., P.W. Park

et al., J. Biol. Chem., 2000, 275, 29923-29926; M. Lyon and J.T. Gallagher, Matrix Biol., 1998, 17, 485-493;
N Perrimon M Bernfield Nature. 2000; 404: 725-728 JD Esko and U Lindahl have listed in a J Chem Invest. Supplement) A Suggested reading list entitled Molecular diversity of heparan sulfate (available at http://www.jci.org/content/full/108/2/169/DC1) Cf. also Editorial in The New England Journal of Medicine. 2001, 344; 673-675

and B. Mullow, An. Acad. Bras. Cienc. 2005 ,77 (4) 651-664 Example of an Absolute Requirement for Bivalent Metal Ions for HS Signaling
M Kan et al., J. Biol. Chem., 1996, 271, 26143-26148

(Rare HS motifs facilitate selective binding of target proteins) C. Vanpouille (with M. Lyon and D.G. Fernig) et al., J. Biol Chem. 2007, 282 (33) 24416-24429

(Structure of heparin fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to 2macroglobulin-receptor/low density lipoprotein receptor related protein by heparin fragments) A. Larnkjaer et al., Biochem J. , 1995, 307 (1) 205; Chem. Abs. 122, 285358v
Heparanase I generated HS polyanonic sequences by scission at highly sulphated regions which promoted tumour growth while heparnase III generated HS sequences which inhibited tumour growth via scission at less sulphated regions. The modus operandi is believed to be HS microstructure-dependent modulation of basic fibroblast growth factor activity. (M.-O Ouida et al., Biochem. Biophys. Res. Commun. 2007,

(Difficulty with a DNA-analogy signaling system model for HS)

Cf., J. Kreuger, P. Jemeth, E. Sanders-Lindberg, L.Eliahu, D. Ron, C. Salmivirta and U. Lindahl, Biochem J. 2005; 389, 145-150 J. Kreuger et al., J Cell. Biol. 2006, 34 (3) 461-469 HS may act as a biosensor for flow control via biofeedback at blood vessel walls via conformation determined by bound Na/Ca S. Siegel et. al.. Colloids & Surfaces A: Physiol. & Energy Aspects, 1998, 138, -351 (Overpurification could have removed the necessary inorganic cofactor to achieve HS-protein interaction?) It was found that that separated-out HS microstructures shared their binding sites growth factor variants, which had been predicted to discriminate between these specific HS microstructures. It can be suggested that the actual in vivo discrimination process must require cofactors (putatively multi-inorganic element containing) suggesting the use of a more fuzzy logic, analogue recognition system rather than an exact digital type of organic core polysaccharide microstructure electrostatic recognition similar to the hydrogen-bonding etc. method with DNA. The function of a polymer rather than oligomer-associated metallomic array might be worth establishing experimentally in this context. (This idea is suggested by published as well as unpublished concepts suggested from Aberdeen University polysaccharide laboratory studies). Cf., S. Ricard-Blum et al. J. Biol Chem. 2004, 279, 2927-2936 S.H. Gould et al. (with D.A Rees) J. Chem. Soc. Perkin Trans. II, 1975, 237-242 G.T. Grant et al., (with D.A. Rees) FEBS Lett. 1973, 32 (1) 195-198 R. Seale, E.R Morris and D.A. Rees, Carbohydr. Res. 1982, 110, 101-112 L.-A. Fransson & B. Havsmark, Carbohydr. Res. 1982, 105, 215-225 Cf. L.-A. Fransson, ibid., 1982, 110, 127-133; Eur. J. Biochem., 1981, 120, 251-255

(H-like molecules bind differentially to prion proteins and change their metabolic fate)
R. Gabizone et al., J Cell. Physiol. 1993, 157, (23) 317; Chem. Abs. 123, 249546j

Scrapie infection of neuroblastoma cells precludes nitric oxide production when the cells are challenged with lipopolysaccharides (Lindegren et al. J. Neurosci. Res. 2003, 71, 291-299) suggests need for relevant studies of the extent of S-nitrosylation of HS glypican-1 core protein and formation of nitrosative cleavage marker (anhydro mannosecontaining degradative products) in healthy and diseases brain tissue (K. Mani et al., Glycobiology, 2004, 14, 599-607 [The HS-specific epitope 10E4 is nitric oxide sensitive and partly inaccessible in glypican-1 HS][; it was noted that glypican-1 core protein nitric oxide recycling for nitrosative cleavage is dependent on the Cu2+,Cu+ system and Cu deficiency has been demonstrated in brain tissues of humans afflicted with neurodegenerative diseases). Cf .also (The cellular prion protein binds copper in vivo) D.R. Brown et al., Nature, 1997, 390, 684-686 (PrPC may regulate some aspects of copper metabolism and Cu,Zn superoxide dismutase (SOD) activity by influencing Cu incorporation into the molecule)
D.R. Brown & R.Besinger, Biochem. J.,1998, 334, 423-429 C. Treiber, A. Simons & A. Multhaup, Biochemistry, 2006, 45, 6674-6680 (Mn ions and less severely Cu ions can re-fold normal prions to the pathological misfolded form;

these authors used a yeast cellular vector). N.G. Rainov et al. (with K. Doh-ura) Expert Opin. Biol. Therapy, 2007, 7 (5) 714

Roles of Metal Ions in Bacterial Heparanase A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilisation of a loop forming one wall of the substrate-binding site (from studies of heparinase II form Pedobacter heparinus (Flavobacterium) and its complex with HSrelated disaccharide (D. Shaya et al., J Biol. Chem. 2006, 261, (22) 15525-15535); The Ca binding site of bacterial heparanase I is essential for its activity) (D. Liu et al., J. Biol. Chem. 1999, 274, 4089-4095). The addition of zinc acetate has been indicated to prevent loss due to heparinase activity during heparin extraction from animal tissues (G. Mihailescu & K.J. Nitelea, Rom. Pat. RO 76, 508 Chem Abs., 99, 93718x). Role of H/HS Disaccharides Obtained by Bacterial heparinase in Control of Cytosolic Ca2+ by Inhibiton of Na+/Ca2+ Exchanger K.Shinko et al. (with C.P. Dietrich and H.B. Nader (Heparin and heparan sulfate disaccharides bind to the exchagner inhibitor peptide region of Na+/Ca2+ exchanger and reduce the cytosolic calcium of smooth muscle cell lines requiremnt of C4-C6 unsaturation and 1->4 glycosidic linkage for activity, J. Biol. Chem. 2002, 277, (50) 48227-48233

1a (Review of Metal Ions in H/HS Signaling)

W.F. Long & F.B. Williamson, IRCS J. Med. Sci (Library Compendium) 1979, 7, 429-434; A more recent article which includes a review of inorganic ion effects on HS signaling was given by D.R. Coombe & W.C. Kett Cellular & Molecular Life Sciences, 2005, 62 (4) 410-4249d (H and Blood Coagulation) M.C. Bourin & U. Lindahl, Biochem. J. 1993, 289, 313

(H and Cancer)
H Engelberg, Cancer (New York) 1999, 85 (23), 257-272. S.M. Smorenburg & C.J. Van Norden Pharmacol. Rev. 2001, 53 (1) 93-105 [This study suggested that low molecular weight heparin might have therapeutically verifiable anti-cancer therapeutic value but the results from unfrationated heparin were less certain. The lack of awareness of variation in the inorganic cofactors present in Hs some of which (e.g. Ga3+ and Pt complexes) are known to inhibit tumour growth and which differ in amounts present between batches of H cf.Table III might have contributed to the difficult of evaluating these results) K. Ono et al., Br J Cancer, 2002, 86, 1803-1812 R.J. Linhardt (Heparin-induced cancer cell death) Chemistry & Biology, 2004, 11, 420-422 Cf. Vandewalle et al., (Ca2+ regulation of HS proteoglycans in breast cancer cells) J. Cancer Res. Clin. Oncol., 1994, 120 (7) 389 Chem Abs.121, 105487j and

(H and Atherosclerosis) Cf. K. Murata & Y. Yokoyama Atherosclerosis H. Engelberg, Pharmacol. Rev. 1984, 35, (2) 91-104 Cf. H. Engelberg Circulation, 1961,23, 573-577 (Cf. role of natural serum H) Cf. D. Grant, W.F. Long & F.B. Williamson (Degenerative and inflammatory diseases may result from defects in antimineralization mechanisms afforded by glycosaminoglycans) Med. Hypoth. 1992, 38, 49-55, Cf. Biochem J.,1989, 259, 41-45

(H and Dementia)
H. Engleberg, Dement. Geriat. Cogn. Disord., 2004, 18, 3-4 ----------------------------------------------------------------------------------------------

H/HS and Prion Dysfunction Diseases HS has been indicated to determine the metabolic fate of prions which also seem to bind and can be misfolded by bound metal ions (for which Mnn+ ions cf. C. Treiber et al.1, seem to be of especial relevance for the possible augmentation of prion misfolding under the conditions of Cu depletion and Mn intoxication. Cf. also the putative antioxidant roles of Cu ions in prion biochemistry discussed by D.R. Brown et al. 1). The H/HS-mimetic pentosan polysulfate (PPS) which can apparently prevent or inhibit misfolded prion diseases may do so, at least in part, by influencing the putative redox metal ion-

HS-prion-nitric oxide signaling system, the efficiency of which could influence how prions are normally prevented from misfolding in vivo.
1a -1 H.B. Nader, T.M.P.C. Ferreira, L. Toma, S.F. Chavanto, C.P. Dietrich, B. Casu and G. Torri, Carbohydr Res. 1988; 236,165-180 1a -2 H.B. Nader, H.K. Takahasi, J.A. Guimaraes and C.P. Dietrich, Int. J. Biol. Macromols. 1981; 356-360 1a-3 D. Grant, W.F. Long and F.B. Williamson, Med. Hypoth. 1987, 23, 67-73; cf. ibid.29, 245-253 1a-4 D. Grant, W.F. Long and F.B. Williamson Biochem J. 1989; 259; 41-45; cf. also Med Hypoth. 38, 49-55 T.D. Hill et al., Biochem Biophys. Res. Commun., 1987, 149 (3) 897-901 1a-5 E. Feyzi, T. Saldeen, E. Larsson, U. Lindahl & M. Salmivirta, J Biol Chem., 1998; 273: 13395-13398 1a-6 B. Kuberan et al. (with R.D. Rosenberg) J. Biol. Chem. 2004, 279, 5053-5054 (Light induced 3-O-sulfotransferase expression alters pineal HS fine structure-a surprising link to circadian rhythm) (i.e. HS-structural-alteration by light). 1a-7 Putatively beneficial effects on HS biosynthesis are produced by: Ascorbic acid (which stimulates GAGs (especially HS production) in cultured fibroblasts [J Kao et al., Exp. Mol. Pathol. 1990, 53 (1) 1-10 (confirming the earlier reports of M. Edward and R.F. Oliver, Biochem. Soc. Trans. 1983, 11, 383; ibid., 12, 304).

Retinoic acid and cAMP up-regulation of 3-O-sulfotransferase-1 led to a dramatic augmentation of the anticoagulant form of HS in F9 embryonal carcinoma cells [L. Zhang et al., (with R.D. Rosenberg) J. Biol. Chem., 2003, 273, 27998-28003]; Retionoids increased HS synthesis in human skin fibroblasts [M. Edward, Br. J Dermatol., 1995, 133, 223-230]
Binding of Anticoagulant H (plus a necessary compound Y cofactor) to endothelial cells was found to stimulates the biosynthesis of antithrombotically active HS proteoglycan, [M.A.S. Pinal et al., Braz. J. Med. Biol. Res. 1994, 27p, 2191-2195 (Cf., Antithrombotic agents had previously been reported to stimulate the biosynthesis of HS having an increased degree of sulphation [M.A.S. Pinhal et al. Thromb.. Res. 1994, 74 (2) 143]) Apolipoprotein E containing HDLP also stimulates endothelial production of HS rich in H-like domains [L. Paka et al, J. Biol. Chem. 1999, 274 (6) 4816-4823]. Calcium oxalate crystals upregulates the formation of HS anti-calcium oxalate crystallization inhibitors [ F.T. Borges et al., Kidney Int. 2005, 68, 1630-1642] Cf. Oxidized LDL regulates the synthesis of monkey aortic smooth muscle cell proteoglycans (including HS) that have enhanced native LDL binding properties [M.Y. Chang et al., J. Biol. Chem. 275 (7) 4766-4773 Hyperbaric Oxygen apparently also caused an overall increase in glycosaminglycan synthesis in wound fibroblasts [G.P. Roberts & K.Y. Harding, Br. J. Dermatol. 1994, 131 (5) 630-633]; Mechanical stress has been reported to regulate syndecan-4 HS expression in vascular smooth muscle cells, Thyroid hormones were reported (Y. Tshiba et al. ) to caused an increase of protoglycan synthesis in fibroblasts in culture.

(Further work is required to establish the precise effect of such hormones on HS microstructure)

However (albumin bound) Nonesterified fatty acids (NEFA) reduced the degree of sulphation of HS (this apparently cuase an increased permeability to albumin); in smooth muscle cells NEFA-albumin increased expression of the genes for core proteins of HS mediated by peroxisome proliferator-activated receptor gamma; the matrix produced by cells treated with NEFA-albumin had a highly affinity for LDLs [G. Camejo et al., Curr. Atheroscler. Rep. 1999, 1 (2) 142-149] Homocysteine also diminished anticoagulant HS expression in cultured porcine aortic endothelial cells [M. Nishinage et al., J. Clin Invest. 1993, 92, 1381-1386] and high Glucose adversely modifies HS biosynthesis by mouse glomerular epithelial cells [S. Morano et al., Diabetes/Metab. Res. & Rev. 1999, 15 (1) 13-20] ; Cf. also, Lysolecithin may also adversely alter subendothelial HS proteoglycan production [P. Sivaram et al., J. Biol Chem. 1995, 270,(30) 29760-29765]; Human-rIL1-b and TNF suppressed the formation of H-like compounds on cultured porcine aortic endothelial cells [M. Kobayashi et al., J. Cell. Physiol., 1990, 144, 383-390] Infection with Herpes simplex virus type I in vitro also inhibited proteoglycan synthesis in human endothelial cells, [R. Kanner et al., Am. J. Respir. Cell. Mol. Biol. 1990, 2, 423-431] and Antimicrotubular agents (colchicine and vinbalstine) inhibited glycosminglycan synthesis by embryonic chick tibia and chondrocytes in culture [H.W. Jansen & P Bornstein Biochim. Biophys. Acta 1974, 362, 150-159]; Amyloid fibrils apparently stimulated the biosynthesis of glycosaminoglycans in cell cultures in which hyaluronan was increase but HS was decreased [M.J. Palmoski & K.D. Brandt, Biochem. 1975, 148 (1) 145-147] [L.Li & E.L. Chaikof, Arterioscler. Thromb. Vasc. Biol., 2002, 22 51-68. H/HS, alginates and sulphated marine anionic polysaccharides e.g. carageenenans and soluble humic acids (fulvic acids) act as potent modulators of the crystallization of sparingly soluble e.g. CaCO3 and BaSO4 and hence influence the amounts of Ca2+, Ba2+, CO3- and SO4- present in solution

The naturally occurring amount of HS and fulvic acid to achieve such buffering activity seems to correspond with that reported [1.6mg/L] for HS to occur in vivo by W.C. Manley et al., Clin. Chim. Acta. 1984, 137, (3) 315 1a-8 H. Ischiropoulos, Arch. Biochem. Biophys., 198, 356, 1-11 Cf. H. Tohgi et al. Ann. Neurol.,1999, 46, 129-131 (Remarkable increase in cerebrospinal fluid 3-nitrotyrosine in patients with sporadic amylotrophic lateral sclerosis) 1a-9 Cf., K Mani F Cheng B Havsmark, M Jonsson, M Belting & L-A Fransson, J. Biol. Chem. 2003: 278: 38956-38965

1a-10 F. Baluska, J. Samaj, P. Wojraszek, D. Volkman and D Menzel, Plant Physiol. 2003: 133: 482-491 E . Melotto & J.M. Labovitch, R. Bras. Fisiol. Bras. Veg. 1994; 6: 75-82
M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Ann Rev Biochem. 1984; 6: 75-82 cf., T. Matsunaga & T Ishii, Anal. Sci. 2004.; 20: 1389-1393 [In plants pectin-like oligosaccharides were originally supposed to engage in hormone-like activities but are now thought to influence Ca2+ second messenger actions] 1a-11 D. Lagunov & G. Warren, Arch. Biochem. Biophys., 1962, 99, 396-400; J.E.Shivley & H.E. Conrad, Biochemistry, 1970, 8 (1) 33-43

1a-12

Cf. C. Leteux et al., J. Biol Chem.,276 (16) 12539-12545

[Scrapie antigen indicated co- occurrence with HS with GlcNH2, unsulphated motif: this is the site which is targeted by NO metabolites to create HS oligosaccharides]

1a-13

H. Diringer & B. Ehlers, J. Gen Virol. 1991, 72, 457 [Initial communication dealing with the inhibition of scrapie by sulphated polysaccharides]

1a-13-1 Communication from Graham Steel, Glasgow (2007) (including Professor Ian Bone Report) The H-mimetic PPS has a stimulatory effect of proteoglycan synthesis and a potent suppressor of angiogeneis and tumour growth. In a pivotal study by Doh-ura (2004) PPS was confirmed to suppress CJD attributable to misfolded PrP. The drug was directly infused into the brain using Tg7 mice expressing hamster than endogenous mouse PrP). Later work on some 20+ human patients worldwide corroborated the effect in humans.

1a-13-2 PPS-like molecules produced by normal stress-related cleavage of HS may act as cofactors for clearance of misfolded prions from cells.
Cf. K. Mani et al., J. Biol. Chem. 2007, 282, (30) 21934-21944 (In Niemann-Pick fibroblasts where nitrosative degradative processing of HS is defective carboxylated proteins were abundant When glypican-1 was silenced in normal fibroblasts the level of such proteins increased suggesting that the deaminative cleavage of HS is involved in the clearance of such abnormal including misfolded proteins) 1a-14 B.C. Challis et al., IARC Sci. Publ., 1978, 19 (Environ Aspects N-Nitroso Compd.) 127-142; Chem. Abs. 89, 196583x Cf. E. Boyland & S.A. Walker, Nature, 1974, 601-602; T.Y. Fan & S.R. Tannenbaum, J. Agr. Food Chem. 1973, 21(2) 237-240 B.C. Challis & J.R. Outram JCS Chem. Commun. 1978, (16) 707-708 (article highlighting silver catalysis of nitrosative

processes)
1b J. Boyd, F.B. Williamson and J Gettins, J. Mol..Biol., 1980, 137, 175-190 Cf. D Grant WF Long FB Williamson, Biochem J. 1987; 244: 143-149 (The infrared spectra of different H salts show individual counterion input into H structure as detected by vibrational

spectroscopy in solution and solid phases which are different from a purely electrostatic effects)
and D. Grant et al., Cell Biology International Reports, 11 (3) 220 [(Cation interaction with heparin at antithrombin-binding sites differ from that occurring elsewhere in the polymer) It was noted that especially larger shifts in NMR absorptions due to metal ion interaction were observed close to the hypersulphated region which occurs in the centre of this epitope)] 1c 1d 1e Z. Liu & A.S. Perlin, Carbohydr. Res. , 1994, 255, 183-191 R.N. Rej et al. ibid., 1990, 207, 143-152 R.E. Vilar et al., Biochem. J. 1997, 324, 473-497

1f 1g 1h 2 2a 2b 3

Van den Born J. et al. J. Biol Chem., 270 (52) 31303 F. Cheng et al., J Neurochem 2006 98,1445-1457; Mani et al., J Biol Chem 2003 278 38956-38965 and refs.cited D. Grant et al. unpublished observations Marischal College, University of Aberdeen M. Purdey, Med Hypotheses, 2004, 54, 278-306 A.L. Merce et al., J. Inorg Biochem. 2002, 89, 212-218 P. Sipos et al., ibid., 2003, 5,55-63 (Spheridal iron(III) oxyhydroxide nanopartilces sterically stabilised by chitosan in aqueous solution)

A. Wassermann, Ann. Bot. 1949 NS 13 (49) 79-88; Cf. W.A.P. Black & R.L. Mitchell. J. Mar. Biol. Assoc. U.K .,1952, 30 (3) 575-584 Cf. brief mention by F. Mo et al., Carbohydr. Res., 1985, 145, 13-24 (cf. also refs. cited herein ). 3a-1 E.g., The presence of inorganic elements in ALGINATE have been reported by Ascophylum nodosum polysacchride-based ion exchanger kelp elements are tabulated by several internet sites, e.g. http://www/alginure.co.uk/ascophylum-nodosum.html 3a-2 for Ecklonia maxima at http://www.gairesearch.co.za/kelp.html. 3a-3 Cf., also T.A. David et al., Appl. Biochem. Biotechnol.,2003, 110, 75-909 Su 3a-4 K Truus et al., Proc. Eston. Acad. Sci., Chem., 2001, 50, 95-103 for apparently anthropogenically polluted seaweed from the Baltic). Data from this paper are listed in Table I which also lists similar multi-element analysis (which was apparently provided by the Norwegian Seaweed Research Institute) of less polluted polysaccharide from . 3b-1 The presence of inorganic elements in HEPARIN have been reported by H.J.M. Bowen in Trace Elements in Biochemistry, Academic Press, London, 1966, cf., p.63 noted that since heparin contains appreciable amounts of Ba, Ca, Cu , Mn, Sr and Zn; (where serious interference problems for estimation of normal blood were caused by Mn in heparin as an anticoagulant in the solutions to be analysed); 3b-2 3b-3 3b-4 3b-5 Symp. (for Ca, Sr, Ba) by G.E. Harrison & A. Sutton, Nature, 1963, (4869), 809 Cf. M.E. Lyon, Clin. Biochem. 1995; 28: 79 The presence of paramagnetic metals in natural heparin can also affect NMR resonances cf. G. Gatti et al., Macromoleucles,1979, 12 (5) 1001-1007 (for V in H ) G. Heineman & W. Vogt, Biol. Trace Elem. Res. 2000, 75, 227-234 (for Cr, Mn in H) N.W. Alcock Serum versus plasma for trace metal analysis, Elem. Metab. Man, Anim. Proc. Int. 4th, 1981 (Pub 1982). Eds. J.M. Gawthorme, J.M.M.M.C. Howell, C.L. White, p. 6578-680; Springer, Berlin; Chem. Abs., 96, 213646 3b-6 (for Si in H/HS) K.A. Schwarz., Proc. Natl. Acad. Sci. USA, 1973; 70: 1608-16124

Cf. RK Iler The Chemistry of Silica ,Wiley, New York, 1979

and D. Grant, W.F. Long and F.B. Williamson, Med Hypoth. 1992; 38: 49-55 3b-7 3b-8 3b-9 added Nd, W, La, Pb, Ga, Sb, Cd, B and I in Na H and Mg, K, Rb, Cs, Cr, Fe, Ni, Co, Sn, Mo, Ti, Zr,Ce, Ag, Nd,W, La, Pb,Ga, Sb, Cd, P, B, I, Cl, F and Br in Tl H (D. Grant, Chemistry Preprint Archive, 2000 Oct., 94-104; available at http://preprint.chemweb.com/biochem/0010002; cf. also a preliminary summary of these data was given by D., Grant, et al., Biochem J.,1987, 244: 143-149) and more completely for Na and Tl Hs in C.F. Moffat, Ph.D. Thesis University of Aberdeen, 1987, briefly discussed the the 205Tl NMR spectrum of Tl H (on p.144) and the possible lack of toxicity of 8 ppm Tl in H is also discussed on p. 189. The relevance of larger amounts of possibly toxic or therapeutic elements (such as Ga which has demonstrated antitumours activities) are worth pursuing. 3b-10 MS for web.alsglobal.se/hem2005/pdf/blood_collection_tubes_eng.pdf (2005) tabulated multi-element data obtained by ICP(for As in H) D. Bohrer et al., J Parenteral Enteral Nutrition, 2005; 29 (1) 1-7 (for Al in H) D. Bohrer et al., RBAC. 2004; 36(2) 99-103 The presence of 38 seawater multielements in H was confirmed in Na and Tl H of the elements listed at a-f (except Al during the spark source mass spectrosocpic (SSMS) analysis of Mg, K, Rb, Cs, Tl, Fe, Ni, Co, Sn, Mo,Ti, Zr,Ce, Ag,

types of H commonly present as additives in blood assay tubes which were extracted with 0.01M HNO3 . This study also included the possible inorganic element inputs from empty tubes and needles employed for blood sampling which serve also as blanks for correction of the H analyses for background contamination; the data also reported ICP-MS data substances commonly added to blood collection tubes (EDTA and a phase boundary enhancing agent which is rich Si, Al and in lanthanides etc). The data for extracts from six types of heparinized blood collection tubes confirm the seawater-like pattern of inorganic cofactors present in H previously identified by the author. This data can be extracted from the tabulated information (which included the mainly H-derived SO3- group inorganic S contents of extracts, as well as those of the principal counterion for the employed single counterion-enriched forms of H.

4
5

H. Haraguchi, J. Anal. At. Spectrom., 2004, 19, 5-14

H.B. Nader, M.G.L. Medeiros, J.F. Paiva, V.M.P. Paiva, S.M.B. Jeronimo, T.M.P.C. Ferreira & C.P. Dietrich Comp. Biochem. Physiol., 1983; 76: 433-436 This study established that an exact mathematical relationship exists between the bathing habitat salt concentrations {in fifteen species of Crustacea, Pelecypoda and Gastropoda} and the average total sulphated glycosaminoglycans (GAGs) present in these organisms increased from 125 to 5265 g/g dry weight with increased habitat salinity. This suggests that differing salinity induces evolution/biosynthesis of the exactly balancing amounts of GAGs SO3- groups required to titrate against Na+ (the results of ref.1h suggest that the sum of the SO3-(H2O)n groups present i.e. the total GAG-(O)-SO3- + GAG- (N)-SO3- rather than the GAGC(O)Ocontents correlated with how individual GAG types were induced by different habitats with a range of different natural saline, principally NaCl habitats (i.e. seawater and brackish seawater etc.)

The work of S. Kodima et al., ( e.g. Eur J. Nucl. Med., 1983, 8, 52-59, cf., Y. Hama et al., ibid., 1984, 9, 51-56 who by in vivo and in vitro multivalent ion binding studies e.g. of to 45Ca labelled HS (relating to the scintigraphic observation of tumours via 67 Ga binding which were indicated to occur at HS sites) support the notion that HS will occur in vivo as multi-inorganic element substituted systems.

Mast cells in vivo histochemicaly stain with ruthenium red by binding to heparin in the mast cell granules (E. Phil, Histochem. Cell. Biol., 1970, 22, 302-315) Cf., also: he formation of the adduct 99Tc-H enabled the bio-distribution of H to be achieved in the rat N. Vigneron et al., J. Biophys. Med. Nucl. 1984, 8 (2-3) 192; Cf., M. Decousus et al., ibid., 1984, 8 (2-3) 180; cf., 99m Tc-H studies by Mosberk et al., Nuclearmedizin (Stuttgart), 1981, 18, 343; Chem. Abs., 96, 30896w; 111 InCl3-H for visualisation of thrombosed arteries, Chem Abs., 97, P168866w; cf. related 169Yb and 161Yb H binding studies Chem. Abs., 98, 194186g; 167Tm has strong affinity for tumour tissues especially to HS and other glycosaminoglycans: A. Ando et al., Eur. J. Nucl. Med. Mol. Imaging, 1983, 8 (10), 440-446 Eu(III)-H complex formation enables H to be spectrophotometrically assayed:

M. Risk, Spectroscop. Lett., 1995, 28, 1235 (Log10Kassoc. for Eu3+ binding to H was reported as 5.079; cf., the log10Kassoc.value for La3+ was found by Rabenstein et al. to be 4.5 6a Reviews of the binding of inorganic elements include:
LB Jaques, Science. 1978; 206: 528-533; and in Ions in Polymers (Based on a symposium by the Division of Polymer Chemistry at the 176th Meeting of the American Chemical Society, Miami Beach Fla., Sept 11-15, 1978) Amer. Chem. Soc. Adv. Chem. Ser. 187 (Pub 1980) p. 350-360

These perceptive articles remain highly relevant. J.R. Dunstone, Biochem. J., 1962, 85, 336-351 and J.E. Scott, Ch. 12, p 171-187 in The Chemical Physiology of Mucopolysaccharides, Ed., G. Quintarelli, Churchill, London, 1968
The formation of adducts with a range of inorganic salts (e.g. iodides) with heparin was discussed in a Patent (Fo-We Forschung und Verweltungs-Anstalt [Therapeutically active product ] Brit. Pat. 890,622, 1959) The Binding of Inorganic Sulphate to H

K. Simon Naturwiss. Rundschau 1982, 35 (11) 452-453 discussed the occurrence of ca. 12% of total S in H in the form bound inorganic SO42J.R. Helbert & M.A. Marini, Biochem. Biophys. Acta 1964, 83,122-134 Studied the binding of inorganic sulphate anions to H 6b-1 K.A. Schwarz, Proc. Natl. Acad. Sci., USA, 1973, 70, 1608-1612

(Bound form of silicon in glycosaminoglycans and polyuronides)

6b-2 A list of published studies from the former Aberdeen polysaccharide group (WF Long FB Williamson et al.) which includes reports of studies by a variety of physical chemistry methods of metal ion and polyamine binding to heparin and related roles of hydration and phase separation, was posted on the internet by W.F. Long at http://www.abdn.ac.uk/~bch118/publications 2003march.doc 6b-2-1 Articles which deal with the binding of metal ions by heparin include: D. Grant et al., Biochem. J., 1991, 275, 193-197; ibid., 1992, 287, 849-853; ibid., 1992, 282, 601-604; ibid., 1992, 283, 243246; ibid., 1992, 285, 477-480; Biochem. Soc. Trans., 1991, 18, 1281-1282; ibid., 18, 1282-1283; ibid., 1992, 20, 361S; ibid., 1991,; ibid., 1991, 19, 390S; ibid, 1996, 24, 203S; ibid., 1996, 24, 204S; D.L. Rabenstein et al., Carbohydr. Res., 1995, 278, 239-256 L. Herwats, et al., Nov. J. Chem. 1977, 1, 173-176 6b-2-2 D. Grant et al., Med Hypoth. 1992, 38, 46-48 (A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflictions). Silicate anions in aqueous solution above a threshold concentration (ca. 50 ppm) undergo self-association which is likely to be enhanced at H/HS surfaces in keeping with the strong association of polyuronide with inorganic silicon in biological tissues where they are believed to contribute to the mechanism of nucleation of bone formation. There is also some evidence that insufficiency of dietary Si is associated with augmented oxidative damage to tissue (soluble silicate in drinking water has been negatively correlated with the incidence of degenerative diseases such as atherosclerosis (cf., M. F. McCarty, Med Hypoth.,1997, 49, 177-179 ). Both HS and redox metal dyshomeostasis may, however, be to be implicated in this effect of Si where a putative role of silicate enhanced redox metal ion sequestration and pacification can be suggested. A variety of types of SiO2 sols and gels have been form many years available from the chemical industry; during research into the self assembly of such materials it became evident to the author that a form of nucleation behaviour which mimicked biological activity was apparent. A hypothesis of silicic acid generated seeding/Darwinian evolution is rationally derivable from these observations. This type of structural seeding could continue to be of importance in present day organisms especially in how HS supramolecular inorganic composite material structures are nucleated. 6b-2-3 Some Aberdeen Polysaccharide Group Studies These had confirmed the results of previous workers (as well as data from industrial use of sulphonated organic polymer membranes) that H contains a similar system of interacting -SO3- - associated water cluster structures (the details of which vary between individual metal counterions, but the greatest degree of hydration, 6H2O/-SO3-, is found for Na+ counterions and the least for quaternary N+ counterions (D. Grant et al. Biochem. Soc. Trans. 1990, 18, 1293-1294; ibid., 1983, 11, 96; ibid., 1984; 12: 302. The heparin-associated water clusters showed a systematic change in their near infrared water cluster absorptions upon poly-Llysine or poly-L-arginine binding. (Glue-like surface clusters of H-metal ion associated of water structure (cf. 9d ) become more obvious in a hydrophobic medium. (D. Grant et al., 1991, Biochem J. 1991; 277: 569-572). Such types of interactions are reminiscent of the reversible adsorptions thought to involve both the protein core and polysaccharide side chains of cartilage proteoglycans with chromatographic media9d which have been described as non-specific). New research is required to search for ion conducting properties of GAG hydration superstructures which can be predicted to exist since this is how man-made organic polymer-SO3-(H2O)n membrane proton conductors are employed in commercial use, their mechanism of action is believed to depend on the properties of such water clusters (cf., e.g., James et al., J Materials Sci. 2000; 35: 5111-5119); these man-made sulphonated polymers also find use in commercial fuel cell membranes which show improved thermal stability when the membrane contains additional SiO2 particles (Adjemian et al., J Electrochem Soc. 2002, 149; A256-261) suggesting a related role for the presence of Si (e.g., as SiO2 particles) in GAGs (Schwarz, 1973, loc. cit., cf. Grant et al. Med Hypoth. 1992; 38: 36-48). Further hints of how interaction between SiO2 and GAGs, especially, H/HS, may be physiologically relevant comes from the report by Takeuchi et al . (Analysis. 1998; 28: 61-64) that a heparinised SiO2 chromatographic medium separated both cations and anions on the same column. Si(OH)4 perhaps forms crosslinks analogous to B(OH)3 in plant rhamnogalacturonan II. Anionic Polysaccharides Resemble Humic Polymers as Multi-Element Matrices
In vitro studies of H indicates an ability, which it seems to share with other anionic polysacharides such as the alginate-rich biomass of kelp, to simultaneously sequester a wide range non-physiological (and therefore potentially harmful) ions from the full range of trace and ultratrace ions etc. which occur in natural waters and biological fluids including blood serum this property is also shared with soil humic acids. The perturbation of NMR

resonance due to the presence of abundant Mn and Fe in humic acids for a long time prevented the true chemical nature of these polymers to be determined; a commonly held view was that they were aromatic compounds related to lignin; D. Grant, Nature, 1977, 270, 709-710 suggested a procedure by which the paramagnetic ions could be removed by placing sparingly soluble detergent formulated pyrophosphate granules in solutions of humic substances. This caused a slow removal of the paramgnetic ions into the solid chelator and a gradual narrowing of the solution phase NMR peaks. In the case of H containing 1100ppm, Fe and 730 ppm Cu there seemed to be no detectable paramagnetc broadening. H evidently can coordinate these metals suffiencltly strongly to prevent NMR resonance perturbation. This is indirect evidence that H can behave as an antioxidant and antinitrant (by blocking of the promotion of reactive free radical formation Cu and Fe).

Review articles are in preparation by. D Grant reporting on published articles which suggest that a range of cytokines, dietary and physical factors greatly influence heparan sulphate biosynthesis.

J. Folkman, Science. 1983, 221, 719-725 cf. p722-3 Cf. also J. Folkman & M Klagsburn, Science, 1987, 235, 442-447 J. Folkman et al. Nature, 1989, 339, 58-61 (steroid induced H-related anti-angiogenesis effects differed between Hs from different manufacturers) J.-M. Herbert & J.-P. Maffand, J. Cell. Physiol., 1989, 138, 424-432 (similar HS oligosaccharides generated non-enzymically from H from different manufacturers differed markedly in their antiproliferative properties for vascular endothelia and smooth muscle cells)

8.

F.T. Borges et al., Kidney Int. 2005, 68, 1630-1642

A communication entitled Microstructure-dependent modulation of crystallization by alginates (by D. Grant, M.I. Tait, W.F. Long & F.B. Williamson) was presented by M.I. Tait at the International Seaweed Symposium, Vancouver British Columbia, Canada in 1989. This reported that the rate constants of BaSO4 crystal growth on seed crystals (obtained in a similar manner to that described by Grant et al., Biochem J. 1989; 259: 41-45) for a range of alginates (detailed by J. Boyd, Enzymic approach to the structure and function of alginic acid, Ph.D. Thesis, University College of North Wales, 1975, cf., J. Boyd & J.R. Turvey, Carbohydr. Res. 1978, 66, 187-194 and A.J. Currie in The structure and biosynthesis of alginic acid, Ph.D. Thesis University College of North Wales, 1983) were dependent on the microstructure of M (mannuronate) and G (guluronate) environments rather than degree of polymerisation; individual alginates: (1) SS/DJ , mainly a random structure with some preponderance of polyguluronate units; (2) F387 , an industrial product obtained from Ascophyllum nodosum also studied by Grasdalen et al. (cf. Carbohydr. Res. 1981; 89;: 179-11) which contained proportions of M =0.58, G =0.42 of which were in diads with MM = 0.40, GG = 0.24, MG and GM = 0.18, triad MMM = 0.08, MMG = 0.08, GMM = 0.08, GMG = 0.10, GGG = 0.18, GGM = 0.06, MGG = 0.06 and MGM = 0.12}; (3) was polymannuronic acid from fruiting bodies of Ascophyllum nodosum; (4) was poly M blocks (5) was poly alternating blocks and (6) was poly G blocks; (4)-(6 ) were obtained from commercial samples by enzymic degradation followed by fractionation according to the methods of Haug et al. (Acta. Chem. Scand. 1967, 21; 691-704). Second order rate constants were for (1)-(6) respectively 0, 55, 55, 54, 62 and 81 for the primary and 59.6, 18.8, 15.2, 14.6. 6.0 and 36.6 for the (principal) secondary reaction processes. The relative potency of the alginates was further correlated with infrared spectrocopic variation in the 630-1190cm-1 region which was indicative of the microstructural differences. This is a simple illustration of how different microstructures in complex polysaccharides such as alginates which (analogously to HS) potentially contain complex information encoding sequences can control complex chemical activities, is that the alginateinhibited seeded crystallization of BaSO4 which occurs by consecutive second order rate process, shows a systematic variation of the observed rate constants as a function of the alginate microstructure (described in terms of guluronate and mannuronate blockiness). 9b -2 D. Grant W.F. Long & F.B. Williamson, Thrombosis Research, 1986; Supplement VI, 92; Cell Biol. Int. Reports, 1987, 11, 220 9b-3 D. Grant W.F. Long & F.B. Williamson, Biochem J., 1991, 277, 569-571 9b-3-1 M. Kan, F. Wang, M. Kan, B. To, J.L. Gabriel & W.L. McKeehan, J Biol Chem. 1996; 271: 26143-26148 (Although the interaction of H with FGF was not affected by divalent cations, the presence of such cations especially Ca2+ or Mg2+ 9b-3-1 is reported absolutely to be required for high affinity (Kd= 10nM) binding of H to FGFR). 9c 9d L.C .Pedersen & M. Jorgensen, Biochem J. 1983; 211; 91-97 L.J.J. Hronowski & T.P., Anastassiades, Biochem. Biophys. Res. Commun. 1990; 167; 81-88; Anal. Biochem. 1990, 191 (1) 50-57

10 A. Lima de Faria Evolution without selection : form and function by autoevolution Elsevier Science Publishers B.V. (Biomedical diviison)Amsterdam, & New York 1988 11 M.A.S Pinal et al., Braz J. Med Biol Res. 1994, 27p, 2191-2195 H.B. Nader , V. Buonassisi, P. Colburn & C.P. Dietrich, Cell. Physiol. 1989; 140: 305-310

12 R. Cappai, F. Cheng, G.D. Ciccotosto, B.E. Needham, C.L. Masters, G.Multhaup. L.-A. Fransson and K. Mani, J Biol. Chem. 2005; 280: 13913; cf., K Mani et al., ibid., 2004; 279, 1291812923; ibid., 2003, 278: 38956-38965;
13 (D Grant suggested a similar mechanism of redox metal ion and ascorbate nitric oxide metaoblite dependent HS

Oligosacchride signalling in preliminary draft discussion articles entitled Ascorbate and Nitric Oxide in Redox Control of Heparan Sulphate and Ascorbate & Cancer originally posted on web.ukonline.co.uk/dgrant/dg4/- etc are now available as cached files from Yahoo (but not now Google) using the search terms /dgrant/dg4- and /dgrant/dg5- (with the references at /dgrant/dg8/-) cf., also /dgrant/dg2- and /dgrant/dg5- )
13a R.A.A. Muzzarelli, Polymer Sci. Technol. 1983; 23: 359-374 (which describes the preparation of heparin-like derivatives of chitosan which are evidetly highly contaminated with iron H.J.M. Bowen, J. Mar. Bio. Assoc. 1956; 35: 451-458

14

15 a V.N Senofonte and S Caroli, J. Trace Elem. Med. Biol., 2000, 14, 6-13 b I. Rodushkin and M.D. Axelsson, Sci Total Environ., 2000, 250, 83-100; c I. Rodushkin and M.D. Axelsson, ibid., 2000, 262, 21-36; [cf. also http://www.analytica.se/hem2001/human/research.asp; cf., D.A. Bass et al., Altern. Med. Rev., 2001, 6, 472-481 and A.R. Bleise et al. Analytical Qualtity Control Services, (Hair Elements Reference Sheet), International Atomic Energy Agency, A-1400,Vienna, Austria RAA 16 A. Kornberg (with M.R.W. Brown) PNAS USA, 2004, 46, 16095 17 J.M. Somers, M.L. Tait, W..F Long and F.B, Williamson, Hydrobiologica, 1990; 204/205: 491-497

*Compiled post a fixed-term research study with the polysaccharide groups at Marischal College, University of Aberdeen, U.K. the management of which (FB Williamson and WF Long) are thanked
for their support. This remains the most recent institutional affiliation of the author whose current address for communication, Ashbank Cottage 2a High Street, New Deer, Turriff, Scotland, UK, AB53 6SX) Professor RJP Williams of Oxford University is also warmly thanked for conducting a long correspondence offering advice and criticism of the subject matter of this communication.

Concluding Remarks
Polysaccharides apart from consisting of complex information-containing sequences of differently sulphated polysaccharides (the heparanome) also co-exist with an array of inorganic ions and particles including alkali metal and alkaline earth counterions present in biological fluids as well as trace and ultratrace elements also present in such fluids. The complex patterns of sulphated anionic sugar HS proteoglycans, perhaps partly as flexible metallo-polysaccharide complexes can logically be proposed to be of sufficient functional complexity to fulfill the sought after role of a nongenetic biochemical information handing and storage system suggested to be required for the post-human genome era The inforamtion-encoded H/HS system is that it is known to be involved in a systemic signaling and could explain why the human geneome is much smaller than expected.

It is suggested that the modulation of the activity of heparan sulphate (HS) sequences by metal ions could act as an amplification loop to extend the information processing abilities of otherwise inadequate, limited size genomes may contribute to the development and perhaps also the evolution ofcomplex animal organisms

Heparan sulphate associated multi-inorganic ion arrays are now suggested to promote the formation of functionally relevant heparan sulphate supramolecular structures, which, further subject to the efficiency of probably required nucleation agents, and perturbation by pathological and anthropgenic factors, are suggested to be highly relevant to the etiologies of those degenerative diseases in which heparan sulphate biochemistry has been implicated.
Sequence patterns in HS arise via servo feedback controlled alterations of the sequential utilization of the enzymic modifications during their Gogli apparatus assembly as well as via related postsynthetic modifications which also include an important non-enzymic (environment-linked) putatively metallomic input. .

The single counterion diluted forms of heparin exhibit skewed inorganic ion distributions relative to the less purified form a distribution which evidently can vary between different manufacturers, depending on the details of the purification procedures used which is a source of potential often ignored variation in the activities of H used to provide a convenient molecular structural model library system for biochemical investigations of HS activities.

Purified oligosaccharide epitopes are expected to lack the natural multi-inorganic ions normally associated with the intact polysaccharide molecules and which, can be suggested to be essential cofactors to allow the necessary discrimination to accomplish developmental regulation. H and HS are now known able to bind and modulate the activities of such a wide range of proteins, many of which are known to require inorganic cofactors, that this circumstance can be putatively be extrapolated to suggest that H/HS is involved, in addition to embryogenesis and ageing, also, in virtually all physiological and pathological processes; this further could suggest that the complex inorganic multi-element metallomic biochemistry of H/HS could contriubte to the mechanism of evolutionary change in animal genetics, probably by servo control feedback processes enabling environmental factors to influence genetic factors via the intermediatry alteration of HS signals which are known to inteconnect extracelular spce with the geneome.
Heparin, marine alginate polysaccharides and other natural polyanionc systems (e.g. humic acids) can simultaneously sequester many of the inorganic ions which occur in natural bathing solutions (e.g. seawater and blood serum) by mechanisms which tend to favour the preferential uptake of the least abundant elements present. Such inorganic reservoirs may provide cofactors for heparan sulphate-metal-ion-assisted protein attachment and facilitate hormone oligosaccharide generation by nitrosative cleavage.

On the other hand highly purified oligosaccharides or low molecular weight heparin may be depleted of the multi-inorganic ions normally associated with the intact polysaccharide/proteglycan molecules.
Si, Al, As, Pb, Sr and the rare earth elements which have been tentatively implicated as having normal roles in animal biochemistry but by unknown mechanisms, might then conceivably act to modulate of heparan sulphate activities both in normal and also under pathological, e.g., anthropogenically altered metal ion presence might also contribute to animal plasma membrane hydration and water activities and also

modulate normal and pathological calcification and by binding unliganded redox metals such as Fe3+ which will confer antioxidant and antinitrant protection). Altered heparan sulpahte biosynthesis seems to occur in response to c changes in Ca2+Mg2+ and Mn2+ ionic concnetrations as well as those of ascorbate, glucose and a wide range of other dietary factors (those which promote health tend to boost the biosynthesis of heparan sulphates of increased its degree of sulphation whilst those which are detrimental diminish this). Heparin provides a convenient model for the study of the heparanome 3 which includes signalling functions of oligosacharides generated by polysaccharides (perhaps more especially those with the most highly substituted with anionic groups). Although much purer (e.g., Li-enriched or low molecular weight) forms of heparin are now available, possibly unacceptably high levels of Al and As still occur in commercial heparins. The animal anionic polysaccharide heparin simultaneously binds a wide range of those elements which occur in seawater and in the anionic pectic polysaccharides of plant walls. It may not be entirely coincidental that a similar range of inorganic elements may occur in all major anionic polysaccharide systems of animals, land plants and marine algae.

The system of plant anionic polysaccharide, pectin generated oligosaccharide dependent Ca2+ second messenger actions10 could serve as a provisional hypothetical model for heparan sulphate signalling in animals which is known, at least in part, to require Ca2+ and Zn2+ as well as Cu+/Cu2+( for nitric oxide/(ascorbate) dependent oligosaccharide generation)3a.

(Fig. 1d.) as their occurrence in unpolluted alginate, carageenan etc., and calcified skeletal matrices which (perhaps forming crosslinks analogous to borate in plant rhamnogalacturonan II) 4 (e.g. with Na, K Mg Ca and Sr and range of other counterions
(H.B. Nader, V. Buonasisi, P. Colburn, C.P. Dietrich, Cell Physiol. 1989; 140: 305-310)

Cf. M. Purdey, Med. Hypoth., 2004, 62, 746-754;

conceivably being required to modulate the primary digital information-encoded heparan sulphate -core-held information.