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Fatty acids: branched-chain – structures, occurrence and biosynthesis



Branched-chain fatty acids are common constituents of the lipids of bacteria and animals, although
they are rarely found in the integral lipids of higher plants. Normally, the fatty acyl chain is
saturated and the branch is a methyl-group. However, unsaturated branched-chain fatty acids are
found in marine animals, and branches other than methyl may be present in microbial lipids. The
most common branched chain fatty acids are mono-methyl-branched, but di- and poly-methyl-
branched fatty acids are also known. Their main function in membranes may be to increase the
fluidity of lipids as an alternative to double bonds, which are more liable to oxidation. The following
discussion is not intended to be comprehensive.

Saturated iso- and anteiso-Methyl-Branched Fatty Acids

iso-Methyl branched fatty acids have the branch point on the penultimate carbon (one from the
end), while anteiso-methyl-branched fatty acids have the branch point on the ante-penultimate
carbon atom (two from the end) as illustrated. In the latter, the methyl group has the (S)-
configuration. The common range of fatty acids of this kind with a single branch point only in a
saturated chain are discussed in this section.
CH3 anteiso-
iso- a

Fatty acids with structures of this type and with 10 to more than 30 carbons in the acyl chain are
found in nature, but those most often encountered have 14 to 18 carbons. They are common
constituents of bacteria but are rarely found in other microorganisms. Via the food chain, they can
be found in animal tissues, especially those of marine animals and ruminants. However, they can
also be synthesised in animal tissues per se. In bacteria, their content and composition can often
be used as taxonomic markers, and in bacilli, for example, some species have fatty acids with the
iso-structure only, while others have the anteiso-structure.

These fatty acids are produced biosynthetically via the conventional mechanisms for the synthesis
of saturated fatty acids in bacteria (see the appropriate web pages), except that the nature of the
primer molecule differs.

Thus instead of acetyl-coA, 2-methylpropanyl-CoA (derived from the amino acid valine) is the
primer for the biosynthesis of iso-branched fatty acids with an even number of carbon atoms (odd-
numbered chain), while 3-methylbutyryl-CoA (derived from leucine) is the primer for iso-fatty acids
with an odd number of carbon atoms (even-numbered chain). 2-Methyl-butyryl-CoA (derived from
isoleucine) is the primer for anteiso-fatty acids to produce fatty acids with an odd number of carbon
atoms (even-numbered chain). It is more common to find iso-methyl fatty acids with an even
number of carbons in total, although the chain-length is odd-numbered, sometimes leading to
confusion in the informal nomenclatures that may be used in scientific publications (for example
13-methyl-tetradecanoate acid is sometimes abbreviated to iso-methyl-14:0 and sometimes iso-
methyl-15:0). However, because of the alternative route for iso-methyl formation and alpha-

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

oxidation processes, odd-numbered iso-methyl acids and even-numbered anteiso-methyl acids are
also found in tissues.

fatty acid
CH3 CH CO CoA iso-fatty acids
2-methylpropanyl-CoA (odd-numbered chain)

fatty acid
CH3 CH CH2 CO CoA iso-fatty acids
(even-numbered chain)

CH3 fatty acid

CH3 CH2 CH CO CoA anteiso-fatty acids
(even-numbered chain)

In animal tissues, the biosynthesis of these fatty acids de novo is normally a very minor process
and is believed to involve the same mechanism as above. However, it can occur at a significant
rate in some instances. For example, lanolin, the waxy material produced as a protective coating
for the fleece of sheep, contains a high proportion of iso and anteiso fatty acids from C10 to C34 in
chain-length (see the web page on waxes). One anteiso-branched fatty acid, 18-methyl-
eicosanoic acid, constitutes up 60% of the total fatty acids esterified directly to wool via thiol ester
bonds, and it comprises 40% or more of the same lipid in all mammalian hairs examined to date. A
significant proportion of the lipids secreted by the meibomian glands adjacent to the eye consist of
iso/anteiso-methyl-branched fatty acids. Triacylglycerols containing isovaleric (3-methylbutyric)
acid are important constituents of the blubber and melon oils of the Beluga whale, and an
alkyldiacylglycerol containing this acid occurs in rabbit harderian gland. Branched-chain acids have
also been implicated in some human disease states.

However, in most mammalian tissues, branched-chain fatty acids of this type rarely make up more
that 1-2% of the total, and are probably derived mainly from bacteria in the intestines or from
consumption of such fatty acids in dairy products or meat from ruminant animals. Similarly fish oils
usually contain 1-2% iso- and anteiso-fatty acids of chain-length C14 to C18, which are presumed to
be derived from the marine food chain.

The free-living nematode Caenorhabditis elegans synthesises iso-methyl-tetradecanoic and

hexadecanoic acids de novo and has been shown to be absolutely dependent on these for its
growth and development.

In higher plants, 14-methylhexadecanoic occurs at a level of 0.5 to 1% in seed oils from the family
Pinaceae, where it appears to be a useful taxonomic marker. iso-/anteiso-Methyl-branched fatty
acids are major components of plant surface waxes, however (see the appropriate web pages).

Neo fatty acids, which can be considered

as having a terminal tertiary butyl group or a
with two iso-methyl groups, have been
found in certain microorganisms, algae,
plants and marine invertebrates. For
example, 13,13-dimethyltetradecanoic acid 13,13-dimethyl-tetradecanoic acid a
or ‘neopalmitic acid’ illustrated is a minor

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

component of bark and resins from conifers and other plants, and it has been found in the shell,
chitin and chitosan of a species of crab.

Saturated Mid-Chain Methyl-Branched Fatty Acids

10-R-Methyl-octadecanoic acid or a
tuberculostearic acid is a major HOOC
component of the lipids of the
tubercle bacillus and related tuberculostearic acid a
bacterial species. Indeed its
presence in bacterial cultures and sputum from patients is used in the diagnosis of tuberculosis. It
is also found in Corynebacterium and other bacterial species.

A number of fatty acids with a single methyl branch of this type have been isolated from specific
bacteria. For example, 10-methylhexadecanoic and 11-methyloctadecanoic acids are relatively
common. 12-Methylhexadecanoic acid and 14-methyloctadecanoic acid are major components of
the halotolerant bacterium Rubrobacter radiotolerans. The latter occurs in the aquatic bacterium
Rhodococcus equi also. 6- and 9-Methyltetradecanoic acids are found in lichenized fungi.
Mycobacterium phlei contains a range of methyl-branched fatty acids, including 8- and 10-
methylhexadecanoate, 9-methylheptadecanoate, 11-methylnonadecanoate, 12-methyleicosanoate,
14-methyldocosanoate and16-methyltetracosanoate. Very many different branched chain fatty
acids of this type may be found in mixed microbial populations such as those isolated from soil or
other environmental samples. Similarly sponges and some other marine organisms contain methyl-
branched fatty acids derived from microorganisms in their diet or that live in symbiosis with them.
For example, in addition to a number of iso- and anteiso-
methyl-branched fatty acids, 10-methyl-16:0, 11-methyl- a
18:0, 14-methyl-20:0, 18-methyl-24:0 and 20-methyl-
26:0 were found in the lipids of the sponge Verongia

Biosynthesis of branched chain fatty acids of this type CH2

involves methylation of oleic acid esterified as a CH3(CH2)7C CH2(CH2)7COOR
component of a phospholipid, with S-adenosyl-
methionine as the methyl donor. The resulting 10-
methyleneoctadecanoyl residue is reduced to the 10- NADPH
methyl compound with NADPH as the cofactor. A
related mechanism is in used for biosynthesis of CH3
cyclopropane fatty acids in bacteria. The intermediate CH3(CH2)7CH CH2(CH2)7COOR
10-methyleneoctadecanoic acid has been isolated from a
a Corynebacterium.

Some such methyl branched fatty acids occur in a few disparate tissues in the animal kingdom.
Perhaps the best known is the uropygial (preen) gland of birds that secretes a waxy material that
serves to waterproof the feathers. The precise composition of this varies from species to species,
but all are characterized by high concentrations of branched-chain fatty acids (and alcohols).
Usually the branch is a methyl group, but ethyl and propyl branches are also known. The positions
of these and the chain-lengths of the various components cover a wide range, but for the
monomethyl fatty acids, the branch-points are most often in positions 2 to 6. Di-, tri- and
tetramethyl-branched fatty acids are also present. A common pattern is to find series such as 2,4-,
2,6-, 2,8- and 4,6-dimethyl, and so forth, with 2,4,6-, 2,4,8- and 2,6,8-trimethyl, and 2,4,6,8-
tetramethyl fatty acids. In some species these can comprise 90% of the total fatty acids. However,
the preen gland of the barn owl contains 3-methyl- and 3,5-, 3,7-, 3,9-, 3,11-, 3,13-, and 3,15-
dimethyl-branched fatty acids.

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

As an example, the composition of the fatty acids in the

uropygial gland of the fulmar is listed in Table 1. Much
remains to be learned of the mechanism of Table 1. Branched-chain components in
biosynthesis of these fatty acids, but it appears that a the preen gland of the fulmar (wt% of the
high proportion at least is produced by a conventional
type of fatty acid synthase that utilizes methylmalonyl- Position Chain-length Amount (%)
CoA to insert the methyl group as opposed to malonyl-
CoA per se. 2- C8 0.4
3- C7 to C12 53.3
Ruminant fats also contain high proportions of 4- C7 to C12 22.6
branched-chain components, especially when they are 6- C10 to C12 4.0
fed carbohydrate-rich diets, when up to 9% of the 2,4-/2,6- C8 to C10 6.5
subcutaneous fat can comprise such fatty acids. 3,7- C9 to C11 8.3
Relatively high proportions of propionic acid (as 4,6-/4,8- C10 0.4
opposed to acetic and butyric) are produced by the
Jacob,J. and Zeman,A. Z. Naturforsch.,
rumen microorganisms, and this is in turn converted to 26b, 33 (1971).
methylmalonyl-CoA, which is incorporated into fatty
acids by the fatty acid synthase. A consequence of this mechanism is that the methyl groups are
all in the even-numbered positions, and are distributed randomly in fatty acids of varying chain-
lengths. In fact more than 120 different mono-, di- and tri-methyl fatty acids (and some ethyl-
branched components) have been identified in ruminant fats.

A further interesting example is Vernix caseosa, the waxy skin secretion that covers newborn
babies. In addition to a high proportion of iso-/anteiso-methyl-branched fatty aids, this contains
approximately 10% of components from C11 to C18 in chain-length with methyl groups in the even-
numbered positions from 2 to 12, which are once more presumably synthesised using
methylmalonate as a substrate.

Non-isoprenoid dimethyl-branched fatty acids are frequently reported from bacteria. For example,
4,9-dimethyl-10:0, 4,10- and 4,11-dimethyl-12:0, and 4,13-dimethyl-14:0 acids, with 2,13- and
2,12-dimethyl-14:0 acids were identified in a halophilic Bacillus sp. Multi-branched fatty acids with
the methyl branches in positions 2-, 4-, 6- and 8- are present in certain Mycobacteria. Dimethyl
fatty acids are occasionally reported from sponges, where they are presumed to be derived from
bacteria in the food chain or that are symbiotic, e.g. 9,13- and 10,13-dimethyl-14:0, 8,10-dimethyl-
16:0 and 3,13-dimethyl-14:0.

Dimethyl, dibasic acids such as 14,15-dimethyltriacontanedioic acid are sometimes found in plant
waxes and in bacteria.

Isoprenoid Fatty Acids

A number of isoprenoid fatty acids occur naturally in animal tissues that are derived from the
metabolism of phytol (3,7,11,15-tetramethylhexadec-trans-2-en-1-ol), the aliphatic alcohol moiety
of chlorophyll. These range from 2,6-dimethylheptanoic to 5,9,13,17-tetramethyloctadecanoic
acids, but those encountered most often are 3,7,11,15-tetramethylhexadecanoic (phytanic) and
2,6,10,14-tetramethyl-pentadecanoic (pristanic) acids.

4,8,12-Trimethyltridecanoic acid is especially common in fish and other marine organisms.

Phytanic acid is formed in animal tissues by oxidation of phytol to phytenic acid (only encountered
in tissues under artificial feeding conditions), followed by reduction. The shorter chain isoprenoid
fatty acids are formed from this by sequential α- and/or β-oxidation reactions. In natural phytanic
acid, each of the methyl groups would be expected to have the D-configuration, but in that
prepared via chemical hydrogenation of phytol, the 3-methyl group is racemic (D,L).

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

Normally, these fatty acids occur at low levels

only in tissues, with the concentrations being a
highest in herbivores. For example, phytanic
acid is found at levels of up to 1% normally in HOOC
milk fat and adipose tissue from cows.
However, much higher concentrations can 3,7,11,15-tetramethylhexadecanoic (phytanic) acid
occur on occasion. For example, up to 20% of
the fatty acids in the triacylglycerols of bovine
plasma can consist of this acid, because the
methyl-branch in position 3 of the chain inhibits
the action of the enzyme lipoprotein lipase that 2,6,10,14-tetramethylpentadecanoic (pristanic) acid
clears triacylglycerols from plasma.

In humans, several inborn metabolic errors with

one or more deficiencies in the degradation of HOOC
phytanic and pristanic acids have been 4,8,12-trimethyltridecanoic acid
described that lead to an accumulation of these a
acids in tissues and body fluids. There are
various clinical expressions of these disorders, some of which can be fatal, the best known of
which is Refsum’s disease. Because of the presence of the 3-methyl group, degradation of
phytanic acid by β-oxidation is impossible. Rather, phytanic acid is oxidized by α-oxidation in
peroxisomes, yielding pristanic acid, which can then be subjected to three cycles of β-oxidation
with 4,8-dimethylnonanoyl-CoA as the end product; this is transported to mitochondria where full
oxidation occurs. In Refsum’s disease, defects in one or other steps in the α-oxidation system, but
mainly in the enzyme phytanoyl-CoA 2-hydroxylase, lead to the clinical symptoms.

Isoprenoid branched-chain fatty acids, such as phytanic and pristanic acids, appear to be signalling
molecules that function by regulating the expression of those genes that affect the catabolism of
lipids in animal tissues. They are transported to the nucleus by binding to a liver-type fatty acid
binding protein, and they exert their effects by binding to the α subtype of the peroxisome
proliferator-activated receptors (PPAR), which induces the transcription of enzymes involved in
fatty acid degradation by β- and ω-oxidation. In a sense, they are regulators of their own
degradation. Phytanic acid is also a regulator of aspects of glucose and retinoic acid metabolism.
Similarly, the retinoic acids, isoprenoid acids derived from vitamin A, are potent regulators of genes
involved in cell growth and differentiation via distinct transport proteins and nuclear receptors.

Unsaturated Methyl-Branched Fatty Acids

Monounsaturated methyl branched-chain fatty acids have been detected in bacteria and marine
animals. Often, the branch is in the iso/anteiso-position, but it can also be more central in the
aliphatic chain. For example, one of the first acids of this type to be described was 7-methyl-7-
hexadecenoic acid from lipids of the ocean sunfish (Mola mola), while 7-methyl-6- and 7-methyl-8-
hexadecenoic acids were later found in sponges. Similar fatty acids with iso-/anteiso-methyl groups
detected in related marine organisms include 13-methyltetradec-4-enoic, 14-methylhexadec-6-
enoic, 14-methylpentadec-6-enoic and 17-methyloctadec-8-enoic acids, and many others. It is
possible that the primary origin of these fatty acids is in bacteria, since many comparable fatty
acids have been found in bacteria, for example in Bacillus cereus, B. megaterium and
Desulfovibrio desulfuricans.

Many different demospongic acids, i.e. with bis-methylene-interrupted double bonds (usually in the
5,9-positions), have been found with iso- and anteiso-methyl branches (see our webpages on
polyunsaturated fatty acids). In addition, several related fatty acids have been described with the
methyl group in more central positions, e.g. 17-methyl-5,9-24:2, 21-methyl-5,9-26:2 and 22-
methyl-5,9-28:2. Unusual multibranched polyunsaturated and very-long-chain fatty acids have

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

been located in slime moulds and freshwater sponges from Israel, including
enoic acid from seven different species of myxomycetes.

Branched-chain fatty acids are uncommon in plants, but small amounts of 16-methyl-cis-9-
octadecenoic and 16-methyl-cis-9,cis-12-octadecadienoic acids have been found in wood and
seeds of certain Gymnosperm species.

Mycolic and Related Fatty Acids

The mycolic acids, major components of the Mycobacteria and related species, are β-hydroxy-α-
alkyl branched structures of high molecular weight (60 to 88 carbons or more). Depending on
species, these can contain a variety of functional groups, including double bonds of both the cis-
and trans-configuration (but when the latter they also possess an adjacent methyl branch) and
cyclopropane rings, which can be of the cis- and trans-configuration. In addition, they can contain,
methoxy-, epoxy- and keto groups, which are also adjacent to a methyl branch normally. Some
representative structures are illustrated below.

alpha'-mycolic acid
from M. smegmatis

alpha-mycolic acids CH2(CH2)20CH3

from M. smegmatis


from M. tuberculosis

epoxymycolic acid CH2(CH2)20CH3

from M. fortuitum

ketomycolic acid

from M. tuberculosis (x+y+z = 49 or 51)


Structural analysis of such fatty acids is much more difficult than with conventional fatty acids. The
first step usually involves pyrolysis to yield an alpha-branched fatty acid and a meroaldehyde,
which can be analysed separately.

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

These two components are each synthesised by

different enzyme systems in the microorganisms, a
H Mero O
before they are condensed to form a typical mycolic Mero O
acid. H
Alpha O
The mycolic acids are key structural components of H Alpha
the membranes of Mycobacteria, where they appear OH
OH a
to confer distinctive properties, including a low
permeability to hydrophobic compounds, resistance
to dehydration, and the capacity to survive the hostile environment of the macrophage. The β-
hydroxyl group is especially important in that it is believed to modulate both the phase transition
temperature and the molecular packing within the membrane. The cell envelope of Mycobacterium
tuberculosis, for example, has a distinctive lipid composition that is associated with its
pathogenicity in tuberculosis infections. Thus, lipid mycolates are important as structural
components of the cell wall. Also, there is a thick layer of lipid on the outer part of the cell, which
protects the tubercle bacillus from the host’s immune system. Mycolic acids are the major
constituents of this layer as components of distinctive lipids, including an arabinogalactanmycolate
covalently linked to the cell wall peptidoglycan via a phosphodiester bond, phenolphthiocerol and
phthiocerol dimycocerosates, and trehalose esters, which include sulfatides and di- to
polyacyltrehaloses. The cyclopropane rings in mycolic acids contribute to the structural integrity of
the cell wall complex and are protective against oxidative stress. Experimentally induced changes
to the structures of the mycolic acids lead to loss of virulence.

Many details of the biosynthesis of mycolic acids have yet to be determined experimentally.
However, it is believed that in M. tuberculosis a fatty acid synthetase I provides C20-S-coenzyme A
to a fatty acid synthetase II system (see our web pages on the biosynthesis of saturated fatty
acids). Cis double bonds are introduced at two locations on a growing meroacid chain to yield
three different forms of cis,cis-diunsaturated fatty acyl intermediates, which can then be converted
to methyl, cyclopropane, methoxy- and keto-meroacids. Finally, the mature meroacids and a C26-S-
acyl carrier protein enter into a Claisen-type condensation catalysed by a polyketide synthase to
yield the mycolic acids.

Mycobacteria also contain multi-methyl-branched fatty acids (C14 to C32), often with the methyl
branches in positions 2, 4, 6 and 8, and sometimes with hydroxyl groups in position 3, together
with long-chain (C36 to C47) fatty acids, termed mycobacteric acids, which are structurally related
to mycolic acids and contain cyclopropyl rings, double bonds (trans and cis) and/or oxygenated

a mycobacteric acid from M. brumae



a phthioceranic acid from M. tuberculosis a

Indeed, the latter are formed from the mycolic acids by a cleavage between carbons 3 and 4 by a
Baeyer-Villiger-like reaction. Mycobacteric acids are present only in the triacylglycerol fraction
where they are esterified to one of the three hydroxyl groups of glycerol.

Further multimethyl branched fatty acids found in Mycobacteria include mycoceranic (2,4,6-
trimethyloctacosanoic), mycolipenic (2,4,6-trimethyl-trans-2-tetracosenoic) and mycocerosic

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Fatty acids: branched-chain – structures, occurrence and biosynthesis

(2,4,6,8-tetramethyl-dotriacontanoic (C32)) acids. The phthioceranic acids are hepta- or octamethyl

fatty acids, some of which are also hydroxylated (hydroxyphthioceranic acid).

Recommended Reading

o Adida, A. and Spener, F. Intracellular lipid binding proteins and nuclear receptors involved in branched-
chain fatty acid signaling. Prostaglandins, Leukotrienes Essential Fatty Acids, 67, 91-98 (2002).
o Barry,C.E., Lee,R.E., Mdluli,K., Sampson,A.E., Schroeder,B.G., Slayden,R.A. and Yuan,Y. Mycolic
acids: structure, biosynthesis and physiological functions. Prog. Lipid Res., 37, 143-179 (1998).
o Body, D.R. Branched-chain fatty acids. In: 'Handbook of Chromatography. Vol. I. Lipids', pp. 241-275
(edited by H.K. Mangold, CRC Press, Boca Raton) (1984).
o Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis
(4 edition), 446 pages (Oily Press, Bridgwater, U.K.) (2010).
o Dembitsky, V.M. Natural neo acids and neo alkanes: their analogs and derivatives. Lipids, 41, 309-340
o Jacob, J. Bird waxes. In: 'Chemistry and Biochemistry of Natural Waxes’, pp. 93-146 (ed. P.E.
Kolattukudy, Elsevier, Amsterdam) (1976).
o Jones, L.N. and Rivett, D.E. The role of 18-methyleicosanoic acid in the structure and formation of
mammalian hair fibres. Micron, 28, 469-485 (1997).
o Kaneda, T. iso-Fatty and anteiso-fatty acids in bacteria - biosynthesis, function, and taxonomic
significance. Microbiol. Rev., 55, 288-302 (1991).
o Kniazeva, M., Crawford, Q.T., Seiber, M., Wang, C.Y. and Han, M. Monomethyl branched-chain fatty
acids play an essential role in Caenorhabditis elegans development. PLOS Biol., 2, 1446-1459 (2004).
o Mukherji, M., Schofield, C.J., Wierzbicki, A.S., Jansen, G.A., Wanders, R.A.J. and Lloyd, M.D. The
chemical biology of branched-chain lipid metabolism. Prog. Lipid Res., 42, 359-376 (2003).
o Nicolaides, N., Apon, J.M.B. and Wong, D.H. Further studies of the saturated methyl branched fatty
acids of Vernix caseosa lipid. Lipids, 11, 781-790 (1976).
o Rafidinarivo, E., Lanéelle, M.-A., Montrozier, H., Valero-Guillén, P., Astola, J., Luquin, M., Promé, J.-C.
and Daffé, M. Trafficking pathways of mycolic acids: structures, origin, mechanism of formation, and
storage form of mycobacteric acids. J. Lipid Res., 50, 477-490 (2009).
o Takayama, K., Wang, C. and Besra, G.S. Pathway to synthesis and processing of mycolic acids in
Mycobacterium tuberculosis. Clin. Microbiol. Rev., 18, 81-101 (2005).
o van den Brink, D.M. and Wanders, R.J.A. Phytanic acid: production from phytol, its breakdown and role
in human disease. Cell. Mol. Life Sci., 63, 1752-1765 (2006).

William W. Christie
Scottish Crop Research Institute (and Mylnefield Lipid Analysis), Invergowrie,
Dundee (DD2 5DA), Scotland
Last updated: June 28 , 2010

W.W. Christie © lipidlibrary.aocs.org 8