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Antioxidant tocopherols,

Paolo Di Mascio,

defense systems: and thiols3

Michael E Murphy, and Helmut

the role of carotenoids,


can damage DNA, and and carotenoids some potentially enzymatic prooxidants soluble explain

Reactive oxygen species proteins, carbohydrates, reactions are antioxidants radicals. The singlet ofthe

occur in tissues and and lipids. These by a system of which eliminate ability of the lipidoxygen are may indepenthe most radicals in assubstrate of nonenmembrane systems. other compounds defense. In-

and Weiss (3) in their study on the iron salt-catalyzed decomposition of H2O2 as being important in chemical, photochemical, and after electrochemical anion discovery (4). reactive species of oxygen peroxide (H2O2) as the important in the development are of a nonradical two-electron reduction of enzymology nature. state after the radical the processes. O of superoxide Biological received dismutase research by McCord on stimulus and the



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nonenzymatic scavenge free to quench properties

superoxide Fridovich Other Hydrogen became

an exceptional

molecular carotenoids,


dent of their provitamin abundant and efficient biological corbate and membranes. cellular thiols. antioxidant scavenging. also

A activity. scavengers Water-soluble Glutathione functions Thiols

Tocopherols of hydroperoxyl antioxidants is an important and is capable associated with antioxidant and antioxidant


discovery of catalase Compound I by Chance (5) and the first demonstration of H2O2 in a mammalian cell, the hepatocyte (6). Further, note. Kautsky tially (singlet made istry important molecular important ofphotooxygenation. the electronically and de Bruijn role oxygen), fundamental The excited carbonyls (7) realized quite active Foote whereas present are worthy of early the potenoxygen to the chapter species (9) Gollnick photochemon some sysdefense

for enzymatic zymatic radical proteins teractions enhance may the

be important the thiols,

to the tocopherols, cellular

of a metastable contributions


(8) and

thiols, protein

effectiveness of Nuir 1991:53:1945-2005.


singlet molecular sulihydryls,

oxygen, peroxidation lipid

oxygen radicals,


of the nonenzymatic antioxidants. tems have been reviewed in the

The enzymatic recent literature

(see Ref 1).

Carotenoids Overview

of oxidative

entails the generation of oxygen species Carotenoid they play an Carotenoids portant excited oxygen is the pigments important such biological are widely distributed in nature, where role in protecting cells and organisms. and that fl-carotene can inactivate (Fig

capable of damaging DNA, proteins, These species include the superoxide peroxide, the hydroxyl radical, and In the pattern like carotenoids, role. Oxidative ofantioxidant vitamins damage

carbohydrates, anion radical, singlet molecular

and lipids. hydrogen oxygen. compounds a prominent species

as lycopene compounds

1) are im-

electronically Singlet molecular compound and or by ie, by pho-

defense, some biological E and C, and thiols play inflicted by reactive oxygen

molecules, a process (02) is an example by photochemical of lipid

termed quenching. of such an excited reactions of biomembranes,

is generated process

or enzymatically

also referred to as oxidative stress, and reflects a shift in the prooxidant-antioxidant balance in favor of the former (1). Diverse biological processes such as inflammation, carcinogenesis, aging, radiation damage, and photobiological effects appear to involve reactive oxygen species. This field of research has provided impact in recent years in a number of fields such as biochemical pharmacology, well as pathophysiology One-electron since tron of early steps free this reduction century. may radical forms, reduction, be toxicology, and medicine. of oxygen Michaelis of general leading radiation has been out biochemistry, widely that studied one-elecformation in biological O and radical by Haber as


toexcitation or by chemiexcitation. Carotenoids may also participate in free radical reactions. For example, fl-carotene and related carotenoids decreased the rate of formation of methyl linoleate hydroperoxides (10).

(2) pointed importance

From the Institut f#{252}r Physiologische dorf, D#{252}sseldorfFRG. 2 Supported by the National Foundation


I, Universit#{228}tD#{252}sselResearch, Wash-

for Cancer

of oxygen radicals,

to intermediate are 02 and perhydroxyl recognized Am J C/in Nuir

chemistry. These their protonated HO2 and 1 94S the

forms of oxygen HO and HO. The radical HO were


ington, DC and the Deutsche Forschungsgemeinschaft, Bonn, FRG. MEM was supported by grants from the Deutscher Akademischer Austauschdienst and the Jung-Stiftung f#{252}r Wissenschaft und Forschung. 3Address reprint requests to H Sies, Institut f#{252}r Psysiologische Chemie I, Universit#{228}tD#{252}sseldorf Moorenstral3e 5, D-4000, D#{252}sseldorf!, FRG. Printed in USA. 1991 American Society for Clinical Nutrition

199 1:53:194S-200S.







xl x2



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FIG 1. Structures
of lipid-soluble antioxidants.

These carotenoids has been properties inverse ofcertain cancer,

antioxidant raised whether independent relationship


may of cancer


a possible



The decrease This

quenching in the quenching

of 02 by tocopherol order of aring, since


homologs y>

was group


to 1).

in the prevention

( 1 1 ), since

the question An tract have


#{244}-tocopherol (Table in position derivative

(3-carotene might of its provitamin fl-carotene such

have anticarcinogenic A activity (12). intake and the incidence


on a free an ester

hydroxyl or ether

6 of the chromane 6-position chemical at lower

at the


types ofcancer, has been observed

( 13,

as lung and gastrointestinal 14), and animal experiments of carotenoids in protecting

in the chromane ring eliminated the activity. A distinct reactivity ofthe tocopherol homologs with 02 occurred rates and showed the sequence a- > y- > - > fl-toin Table I , carotenoids their and contribute classes and tocopherols abilities that show and both against lipid-

revealed anticarcinogenic properties Although the role of carotenoids

( 1 5). plants against as well is esa prooxygen and to of phoof

copherol (21), As is presented an inverse correlation their concentration groups singlet soluble corbate, dition, modes ofcompounds oxygen.

the photosensitization induced by their own as in treatment of patients with photosensitivity tablished, the mechanism by which (3-carotene tective function has been shown against cancer to be capable

chlorophyll diseases may exert Singlet damage

between in plasma may two

02 quenching tissue, indicating to the ofcompounds

protection are both

remains unknown. of inducing DNA the quenching


be mutagenic
In our biological todetector recent

work, we determined by first monitoring at 1270 2), and then nm using the plotting abilities infrared diode intensity of quencher considerable compounds (Fig 02 by direct a germanium ratio ofthe

and complement the role which act as antioxidants tocopherols, ofprotection by scavenging reactions, Refs (see of enzymatic

of thiol compounds and asin the aqueous phase. In adperoxyl radicals, and 29). and thiols, demonstrate multifaceted

by a variety

chemiluminescence chemiluminescence using Stern-Volmer differences compounds in the (Table

1, 22, 28,

in the presence and absence plots (Fig 3) (19). There were rate constants of quenching (kg,

Vitamin tocopherols sponsible branes ofvitamin not =-23 taking Mmol/L 1 1 and depending E, a term that encompasses a small group of related (Fig 1), is the major lipid-soluble antioxidant refor protecting the polyunsaturated fatty acids in memagainst lipid peroxidation ranges E supplements, (23). (23), on local This diets maintains levels (30). of membranes membranes. often determines the low-density lipopro<



1). Lycopene


the greatest


ability, double that of (3-carotene. Comparison oflycopene, y-carotene, and (3-carotene revealed ofthe (3-ionone ring increased the quenching and double the two ionone (bixin), decreasing rings by carboxylate the conjugated

of the structures that the opening ability. Replacing group or ad-

(28, between with



normal values blood can

intake in adults between level of

E in US diets vitamin 1 3 IU/d

4 and average an average 23 zmol/L

22 IU/d

a methyl ester bonds (crocin),


be typical,

dition of chemical groups on the also modulated the quenching quenching properties of carotenoids energy but also The recently state, ie, the length functional on the groups.

(3-ionone ring (xanthophylls) ability, suggesting that the reside not only on the triplet double-bond also system, proposed (26). These

The vitamin susceptibility teins, agents icals,

E content of microsomal

of the conjugated

bile pigments as antioxidants

bilirubin and biliverdin were of physiological importance have been described with kq values lower tocopherols (Table has been

hepatocytes, or whole organs to damage by peroxidizing such as hydroxyl radicals, alkoxyl radicals, peroxyl radsinglet oxygen, and perhaps a number of oxygen-metal (31-34). secondary into to a chain lipids chain-propagating These agents intermediates, alkoxyl reaction and peroxyl steps. not only damage the lipids but lipid hydroperoxides, which organic radicals peroxyl without radicals, Tocopherols reacting in and peroxidation.

open-chain tetrapyrroles oxygen quenchers (27), but clearly higher than the carotenoids, of conjugated

as effective singlet than the carotenoids 1). As in the case of to the system

complexes produce can thus protect further lead


of lipid

the quenching double bonds.


by scavenging

1 96S









posomes against rapid peroxidation until it was consumed. This contrasted with our recent observation that peroxidation initiated with Fe/ADP/NADPH began already after only 15% of the vitamin rat liver antioxidant, vs different E was (37). lost This from microsomal further membranes analyses prepared of the role from of this prompted

particularly with types of prooxidants

respect to its relative (37a).


the before lation loss

4, A and B, shows
of a-tocopherol with and

the temporal
the onset ions.

of lipid There



the experiments

the free metal

was no lag period or the accumu(TBARS) in-

either the chemiluminescence of thiobarbituric acid-reactive 500 mol started FeSO4/L although

increase substances

duced by peroxidation

(Fig 4, A) indicating that lipid vitamin E content was at initial preceded induced both by chemilu100 imoI

levels. In contrast, a clear lag period minescence and TBARS accumulation

with feature lipid depletion. other

(Fig 4, B) whereas
prooxidants prooxidant starts relationship peroxidation and of this

a-tocopherol was without any apparent is that even there following is similar of human

lost as quickly as lag. The unique left before vitamin during lipoproE the

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is still a period substantial to that noted low-density

peroxidation This

(1) V

CuCl2-induced teins (34).


Other prooxidants, including ADP-chelated tants (NADPH and ascorbate), or compounds oxyl radicals (tert-buty! hydroperoxide and and the results are summarized in Table

iron with reducthat generate perAAPH) were tested site of formation,

2. The


0 5



FIG 2. Quenching of the NDPO2 generated singlet oxygen monomol emission signal at 1270 nm by lycopene. Singlet oxygen was generated chemically by using the thermodissociation ofthe endoperoxide of 3,3(l,4-naphthylidene)dipropionate (NDPO2) (19). Experiments were performed at 37 #{176}C in ethanol:chloroform:water (50:50: 1) placed in a thermostated glass cuvette. The infrared emission of O was measured after addition of 20 iL of 0.4 M NDPO2 solution. The light emission was then measured using a liquid nitrogen-cooled germanium photodiode detector (20).



Using phosphatidylcholine liposomes as a model membrane, Niki and co-workers (35, 36) found that when lipid peroxidation was initiated by the peroxyl radical-generating compound 2,2azobis(2-amidinopropane) (AAPH), vitamin E protected the li-

FIG 3. Stern-Volmer plot of lycopene, 13-carotene, and lutein. From experiments such as in Fig 2, the quenching constant (kq) was calculated according to Stern-Volmer plot (S0/S = 1 + (kq + kr) k [Q]), where S and S are the chemiluminescence intensities in the presence of the quencher, respectively, k1 is the chemical reaction rate constant, k, is the 02 decay constant in the solvent (10 ts), and [QI is the quencher concentration; because kq>>
kr, kr

was neglected.

ANTIOXIDANT TABLE 1 Singlet oxygen quenching biological compounds* Compound


SYSTEMS is initiated when peroxidation by complexes involves containing peroxyl radicals, Fe2 and

1 97S en-

peroxidation by carotenoids, tocopherols, thiols and other hanced



k$ Ms



those generated in propagation reactions (Table although the finding that vitamin E is nearly the oxyl-radical supports results tect suggest scavenging the contention that other compound that important detected antioxidant it is a major antioxidant

2). Therefore, exclusive per(4 1) (42), also our proof

in membranes factors

Carotenoids Lycopene y-Carotene Astaxanthin Canthaxanthin a-Carotene (3-Carotene Bixin Zeaxanthin Lutein

3 1 000 25 000 24 000 21 000 19 000 14 000 14000 10 000 8 000

ndII nd nd nd nd nd nd nd nd

0.5- 1.0 mol/L

membranes Ascorbic acid

before or after the oxidation of vitamin E. may be a physiologically relevant enhancer of vitamin E. For example, the chromanoxyl radical and However, although (44), has in biological of tocopherols. systems level hydrogen directly not yet transfer observed been

the effectiveness vitamin E from 0.05-0.1 0.3-0.6 mol/L mol/L phase to chemical 43 ng/retin#{224} 80 ng/retina zeaxanthin and lutein 05 tmol/L 0.3 imol/L 5-20 tmol/L using low Thiols Thiol free groups and (36, vitamin similar endogenous

it may regenerate so prolong the lag from by because ascorbate ESR in

39, 43). model

E radicals, techniques

demonstrated of the


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Cryptoxanthin Crocin Bilirubin Biliverdin Bilirubin ditaurate Tocopherols1 a-Tocopherol fi-Tocopherol -y-Tocopherol 6-Tocopherol Trolox Thiols Cysteine Glutathione Lipoate Methionine

6 000 1 100 3 200 2 300 1 200 280; 45#{216}** 270 230 160 47#{216}** 8.3 2.4 138 13

nd nd nd nd nd 3.6 0.23 2.8 1.7 nd


as intracellular enzymatic

antioxidants reactions.

by scavenging Glutathione is



23 2.3 2.3

zmol/L imol/L mol/L 0

30-100 tmol/L 3-20 imol/L 0.5-10 mmol/L 1-30 mol/L


* Except as noted, experiments with carotenoids and tocopherols were performed at 37 #{176}C in ethanol:chloroform:water (50:50:1); thiols in D20/ 50 mmol/L phosphate buffer (pD 7.4). Retinoic acid, etretinate, isotretinoin, and dihydrolipoate have no quenching ability. Esterification or ether formation at the 6-position in the chromane ring of a-tocopherol abolished the quenching ability (2 1 ). Cystine, GSSG, and the oxidation products of methionine such as methionine sulfone and methionine sulfoxide have no effect on the physical and chemical quenching of 02.

t kq values were obtained from Stern-Volmer plots (20). Data from references (19, 21, and unpublished observations). t k1 values were obtained by measuring the thiol depletion as a function of time, using the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) reaction (22). Plasma concentrations are given for carotenoids and tocopherol, typical cellular concentrations are given for thiols. Data compiled from references ( 19, 2 1, 23, 24).




II nd, not determined (usually low compared 1!Tocopherols were measured electrochemically

to kq). using reversed-phase


(25). Solvent was D20/C2H5OH (1:1) for solubility reasons. See reference 52; predominantly chemical quenching,








all influence their these differences,

reactivity with it is concluded

vitamin E and lipids. Based on that the importance of vitamin

E in protecting membranes used. The relative importance

depends on the type of prooxidant of vitamin E is diminished when

FIG 4. Time course ofchemiluminescence, thiobarbituric acid-reactive substances(TBARS) accumulation, and vitamin F loss initiated by FeSO4 (A) or CuSO4 (B). Microsomal fractions were prepared as described (38), diluted to 0.5 mg protein/mL with 0.1 mol potassium phosphate buffer/ L (pH 7.4), and bubbled with oxygen at 37 #{176}C as previously described (39). Low level chemiluminescence was measured with a single-photon counting-system as described elsewhere (38, 39). Malondialdehyde was estimated by the formation ofTBARS (40). Malondialdehyde-equivalents were calculated using = 156 mM cm. a-Tocopherol was measured electrochemically in lipid fractions using reversed-phase HPLC (25). Ferrous sulfate (A) was dissolved in nitrogen-gassed water and injected immediately to give a 500 zmol/L final concentration. Copper sulfate (B) dissolved in water was injected to give 100 Mmol/L final concentration.

1 98S TABLE 2 Loss of a-tocopherol






the onset

of chemiluminescence

in microsomes

in relation

to mechanisms Initiation

of initiation mechanism


of prooxidant*

% a-Tocopherol remaining at end of the lag before CL 3.3 31 44 51 84

Peroxyl radicals from propagation reactions Cu2-catalyzed Catalyzed by cyt P-450 (ox) Catalyzed by cyt P-450 (ox)

Exogenous peroxyl radicals


Fe2 complexes


1.5 2 1 1 1



Fe2/ADP Cyt P-450 (red) Fe2/ADP and cyt P450 (red) Fe2


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a Prooxidants: FeSO4, 500 imo1 ferrous sulfate/L; reductant; t-BOOH, 23 mol tert-butyl hydroperoxide 25 mmol 2,2azobis(2-amidinopropane)/L; CuSO4,

Fe/ADP/NADPH, 10 tM FeCl3 chelated by 1 mmol ADP/L with 0,2 mmol NADPH/L as L min; Fe/ADP/Asc, Fe/ADP as before with 0.4 mM ascorbate as reductant; AAPH, 100 zmol copper sulfate/L. From reference 37a. lag time.

t Not a significant Chemiluminescence

initiator in the prooxidant system. (CL) began without any measurable

the most





as a substrate

for several





on exper-

transferases, peroxidases, mitigate the deleterious protection of biological is an interesting aspect thiol prevents is enzymatically been attributed

and other enzymes that prevent or effects ofoxygen free radicals (45). The membranes against lipid peroxidation of its function, since this water-soluble This protection on vitamin E; it has peroxidase and/or

imental conditions, dihydrolipoate (46, 47) and other thiols(39) can also protect membranes nonenzymatically, preventing lipid peroxidation and sparing tocopherol. This suggested that the thiols tioxidant tathione oxidation Glutathione associated role, should and with and sparing (GSH) that membrane the proteins along with (48, may of protein 49). were both able to slow also have thiols an anby gluof perprotection

damage in a lipid environment. mediated and is dependent to oxidant neutralizing, lipid

be considered

the prevention

of tocopherols or dihydrolipoate

the loss of protein thiols from microsomes during lipid peroxidation (Fig 5). This effect paralleled the ability ofthese thiols to
.-i 0


delay the onset of chemiluminescence and (Fig 5) and slow the loss of tocopherol (47). Other thiols have varying abilities to protect membranes (results not shown), in contrast to previous reports, can which lipid indicate that glutathione However, is the it was only noted thiol that that the prevent peroxidation.



C U,
0 ..-i

buffer composition in the experiments greatly affected this result, since when Tris chloride was used to replace the usual potassium phosphate buffer, the relative specificity for GSH increased. If GSH enzymatically regenerates tocopherol from its oneelectron idation tioxidant transfer oxidation product, then and protection ofprotein the prevention thiols would of lipid peroxbe secondary an-




effects. The direct demonstration from GSH to tocopheryl radicals

of a one-electron by ESR is rendered However, by properties

Time (mm)
FIG 5. Effect ofthiols on the time course ofchemiluminescence and protein sulffiydryl loss initiated by Fe/ADP and NADPH. Microsomal fractions were incubated as described in Figure 4 except that FeCI3 (12 mol/L) chelated with ADP(l mM) was added and peroxidation initiated with NADPH (0.2 mM). Concentrations ofglutathione (GSH) and dihydrolipoate (DHL) were chosen to provide similar lag periods. Measurement of protein thiols was modified from Jocelyn (50), using = 14.2 mM cm for the product of 2,2-dinitro-5,5-dithiodibenzoic acid (DTNB) reduction.

difficult by the low tocopherol content an enzymatic mechanism was supported

ofmembranes. indirectly

such as a substrate specificity for GSH (40, 5 1). The specificity for tocopherol isomers was analyzed by preparing microsomes with equal amounts of a- and 6-tocopherol. The relative loss of the two isomers and the addition the protection 3). Thus, of enzymatic were roughly parallel during lipid peroxidation, ofGSH or dihydrolipoate did not greatly favor of either tocopherol isomer compared exists with controls the (Table specificity to support hypothesis

no isomer


ANTIOXIDANT TABLE 3 Effect of thiols



1 99S

on the onset

of chemiluminescence Lag before CLt mm

and the relative

rate of a- vs. -tocopherol

loss during


lipid peroxidation Ratio of a-TH to #{244}-TH loss


Rate nmo/

of a-TH mg 3.8 2.9 1.4

loss min

Rate nmo/.

of #{244}-TH loss mg 3.4 2.2 1.5

min 1.12 1.37 0.91 to provide similar

Control Glutathione Dihydrolipoate


(0.5 mmol/L) (1 mmol/L) observations

17 28 30 (Scholich

2 4f 5 et al). Concentrations

0.3 0.2 0.2 and dihydrolipoate

0.3 0.2t 0.3

0.09 0.16 0.09

Data are unpublished different different

of glutathione

were chosen

lag periods.

t CL, chemiluminescence.

f Significantly Significantly

from control value (P < 0.05). from 0.5 mmol/L glutathione value




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Portions of this work were done in cooperation with Thomas PA Devasagayam, P Stephan Kaiser, Astrid Scheschonka, and Heiner Scholich. We thank Ursula Rabe for excellent technical assistance.

18. Decuyper-Debergh D, Piette J, Laurent C, Van de Vorst A. Cytotoxic and genotoxic effects of extracellular generated singlet oxygen in human lymphocytes in vitro. Mutat Res l989;225:l 1-4. 19. Di Mascio P. Kaiser 5, Sies H. Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch Biochem Biophys

References 20. 1. Sies H. Biochemistry

1986;25:l058-7 1.

of oxidative




Int Ed EngI

Di Mascio P. Sies H. Quantification of singlet oxygen generated thermolysis of 3,3-(l,4-naphthylidene) dipropionate. Monomol dimol photoemission and the effects of l,4-diazabicyclo[2.2.2]octane.
J Am Chem Soc 1989;! 1 1:2909-14.

by and

2. Michaelis L. Currents in biochemical research, New York: Interscience, 1946:207. 3. Haber F, Weiss J. The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Lond A l934;l47:332-35l. 4, McCord J, Fridovich I. Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein). J Biol Chem l969;244:6049-55. 5. Chance B. An intermediate compound in the catalase-hydrogen peroxide reaction. Acts Chem Scand l947;l:236-67. 6. Sies H, Chance B. The steady-state level of catalase compound I in isolated hemoglobin-free perfused rat liver. FEBS Lett 1970;! 1: 172-6. 7. Kautsky H, de Bruijn H. How oxygen cancels out the photoluminescence offluorescent systems: formation ofactive diffusable oxygen molecules by stabilization. Naturwissenschaften 193 l;19:l043. 8. Foote CS. Photosensitized oxygenations and the role of singlet oxygen. Acc Chem Res l968;l:104-l 10. 9. Gollnick K. Type II photooxygenation reactions in solution. Adv Photochem l968;6: 1-22. 10. Terao J. Antioxidant activity of fl-carotene-related carotenoids in solution. Lipids l989;24:659-6 1. I I , Ames BN. Dietary carcinogens and anticarcinogens. Science
1983;22 I:1256-64.


Kaiser 5, Di Mascio scavenging ofsinglet


P. Murphy ME, Sies H. Physical molecular oxygen by tocopherols.

and chemical Arch Biochem

1990;277: 101-8.






Moron MM, De Pierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochem Biophys Acta 1979:582:67-78. MachIm U. Use and safety of elevated dosages of vitamin F in adults, In: Walter P. Brubacher G, St#{228}helin H, eds. Elevated dosages of vitamins. Toronto: Hans Huber Publishers, 1989:56-68. Lehman J, Rao DD, Canary JJ, Judd JJ. Vitamin F and relationships among tocopherols is human plasma, platelets, lymphocytes, and red blood cells. Am J Clin Nutr l988;47:470-4. Murphy ME, KehrerJP, Simultaneous measurement of tocopherols and tocopheryl quinone in tissue fractions using high-performance liquid chromatography with redox-cycling electrochemical detection. J Chromatogr Biomed AppI 1987:421:71-82. Stocker R, Yamamoto Y, McDonagh AF, Glaser AN, Ames BN. Bilirubin is an antioxidant ofpossible physiological importance. Science 1987;235: 1043-6.


12. Pung A, Rundhaug E, Yoshizawa, Bertram J. (3-Carotene and canthaxanthin inhibit chemicallyand physically-induced neoplastic transformation in lOTl/2 cells. Carcinogenesis 1988;9: 1533-9. 13. Peto R, Doll R, Buckley JD, Sporn MB. Can dietary beta-carotene materially reduce human cancer rates? Nature (Lond) 198 l;290:
20 1-8.

28. 29.

McDonagh AF. Evidence for singlet oxygen quenching by biliverdin IX-a dimethyl ester and its relevance to bilirubin photo-oxidation. Biochem Biophys Res Commun 1972;48:408-l4. Horwitt MK. The promotion of vitamin E. J Nutr l986;l 16:
137 1-7.

Burton GW, Ingold KU. fl-Carotene: tioxidant. Science I 984:224:569-73.

an unusual

type of lipid an-

14. Connett JE, Kuller LH, Kjelsberg MO, Polk BE, et al. Relationship between carotenoids and cancer. Cancer 1989;64: 126-34. 15. Mathews-Roth MM. Carotenoids and cancer prevention-experimental and epidemiological studies. Pure AppI Chem l985;57:7l722. 16. Di Mascio P. Wefers H, Do-Thi HP, Lafleur MVM, Sies H. Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-strand breaks. Biochim BiophysActa l989;l007:lSl-7. 17. Di Mascio P. Menck CFM, Nigro RG, Sarasin A, Sies H. Singlet molecular oxygen induced mutagenicity in a mammalian SV4Obased shuttle vector. Photochem Photobiol l990;Sl:17-20.

30. Gey KF, Brubacher vitamins in relation


GB, St#{228}helin HB. Plasma levels of antioxidant to ischemic heart disease and cancer. Am J Clin M, Rabe U, Sies H. Evaluation in microsomal lipid peroxidation of a-tocopherol as detected by

1987;45: 1368-77.

31. Cadenas E, Ginsberg antioxidant activity 32.

low-level chemiluminescence. Biochem J 1984:223:755-9. Reed DJ, Fariss MW, Pascoe GA. Mechanisms ofchemical toxicity and cellular protective systems. Fund AppI Toxicol l986;6:59l-7.

33. Pascoe GA, Reed DJ. Vitamin E protection against chemical-induced cell injury. II. Evidence for a threshold effect ofcellular a-tocopherol in prevention of adriamycin toxicity. Arch Biochem Biophys
l987;256: 159-66.




ET 42.

AL Burton GW and Ingold KU. Vitamin F: application ofthe principles of physical organic chemistry to the exploration ofits structure and function. Acc Chem Res l986;19:l94-20l. Tappel AL. Will antioxidant nutrients slow the aging processes? Geriatrics l968;23( lO):97-l05. Lambelet P, Saucy F and LOliger J. Chemical evidence for interactions between vitamins E and C. Experientia l985;41:l384-8. Sies H. On the biochemistry of the thiol group. The role of glutathione. Naturwissenschaften 1989;76:57-64. Bast A and Haenen GRMM. Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation. Biochem Biophys Acts 1988;963:558-6 1. Scholich H, Murphy ME and Sies H. Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on a-tocopherol. Biochim Biophys Acts l989;lOOl:256-6l. Wayner DDM, Burton GW, Ingold KU and Locke S. Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. FEBS
Leus l985;l87:33-7.

34. Quehenberger 0, J#{252}rgens G, Zadravec 5, Esterbauer H. Oxidation of human low density lipoprotein initiated by copper(II)chloride. In: Taylor K. Ward J. von Sonntag C., et al., eds.). Oxygen radicals in biology and medicine. Basic life sciences. Vol. 49. New York: Plenum Press, 1988:387-90. 35. Yamamoto Y, Haga 5, Niki E, Kamiya Y. Oxidation of lipids. V. Oxidation of methyl linoleate in aqueous dispersions. Bull Chem
Soc Jpn l984;59:l260-l264.

43. 44. 45, 46.

Niki F, Kawakami A, Yamamoto Y, Kamiya Y. Oxidation of lipids. VIII. Synergistic inhibition of oxidation of phosphatidylcholine liposome in aqueous dispersion by vitamin E and vitamin C. Bull Chem Soc Jpn 1985:58:1971-5. 37. Murphy ME. Scholich H, Wefers H, Sies H. Alpha-tocopherol in microsomal lipid peroxidation. Ann NY Acad Sci l989;570:480-6. 37a.Scheschonka A, Murphy ME, Sies H. Temporal relationships between the loss of vitamin F, protein sulfhydryls and lipid peroxidation in microsomes challenged with different prooxidants. Chem Biol Interact I 990:74:233-52. 38. Cadenas E and Sies H. Low level chemiluminescence of liver microsomal fractions initiated by tert-butyl hydroperoxide. Eur J Biochem 1982; 124:349-56. 39. Wefers H and Sies H. The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin F. EurJ Biochem l988;l74:353-7. 40. Haenen GRMN and Bast A. Protection against lipid peroxidation by a microsomal glutathione-dependent labile factor. FEBS Lest 1983; 159:24-8. 41. Burton GW, Joyce A, Ingold KU. Is vitamin F the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes? Arch Biochem Biophys l983;22 1:281-90.




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Frei B, Stocker R, and Ames BN. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Nat! Acad Sci USA 1988;85:9748-52. 50. Jocelyn PC. Spectrophotometric assay of thiols. Methods Enzymol 1987; 143:44-67. 51. Burk RF. Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation. Biochim Biophys Acta l983;757: 2 1-8. 52. Devasagayam TPA, Sundquist AR, Di Mascio P, Kaiser S, Sies H. Activity of thiols as singlet molecular oxygen quenchers. J Photochem Photobiol B (in press).