Life, death and membrane bilayers
Metabolic Research Centre and Department of Biological Science, University of Wollongong, Wollongong, NSW 2522, Australia (e-mail: hulbert{at}uow.edu.au)
Accepted 24 March 2003
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Summary |
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Introduction |
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Biological membranes generally consist of bilayers of amphipathic molecules held together by non-covalent bonds. In eukaryotic cells, phospholipids are the predominant membrane lipids and consist of a hydrophilic head group to which are attached hydrophobic acyl chains. These acyl chains are either saturated, monounsaturated or polyunsaturated hydrocarbon chains that normally vary from 14 to 22 carbons in length. Fig. 1 presents the structure of two phospholipids, and Table 1 lists acyl chains commonly found in biological membranes. This commentary will concentrate on the emerging role of membrane acyl chains during the life and death of animals.
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Origins: the beginning of life and membrane bilayers |
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The three major domains of life Archaea, Bacteria and Eucarya
include two prokaryotic domains that differ markedly in the lipids
that constitute their membranes (see Itoh
et al., 2001). The Archaea inhabit the most extreme environments
on Earth and correspondingly have unusual lipids that provide a `toughness' to
their membranes (Hochacka and Somero, 2002). The core of the archaeal membrane
consists of isoprenoid chains and not the acyl chains found in Bacteria and
Eucarya. Whilst some archaeal membrane lipids are amphipathic, others are
described as bipolar lipids with two 40-carbon isoprenoid chains attached at
either end to hydrophilic glycerol head groups by ether linkages. These
tetraether lipids form `monolayer' cell membranes that are analogous to
bilayer membranes with each side covalently linked to the other. The 40-carbon
isoprenoid chains may also contain isopentane ring structures. Both the
tetraether structure of archaeal membrane lipids and the ring structures
within the isoprenoid chains provide a protective membrane `rigidity' in the
very high-temperature environments that many of the Archaea inhabit. Some
thermophilic Archaea homeostatically increase the number of ring structures
within the 40-carbon isoprenoid chains in response to higher growth
temperature (Itoh et al.,
2001
). It is of interest that the sterols, such as cholesterol,
that are membrane components in Eucarya are synthesised from isoprenoid
chains.
Lipid structure and function across three domains of life
The backbone of most membrane lipids in Archaea, Bacteria and Eucarya is
the three-carbon glycerophosphate, and the membrane lipids of Bacteria and
Eucarya are similar, consisting of fatty acyl chains ester-linked to this
three-carbon backbone. In the Bacteria, most is known about lipid metabolism
in Escherichia coli but although many bacteria follow the E.
coli paradigm others do not (Rock et
al., 1996). Bacteria do not synthesise lipids for energy storage,
thus the fundamental function of fatty acid synthesis in bacteria is the
production of membranes. The three acyl chains normally synthesised by E.
coli palmitate (16:0), palmitoleate (16:1 n-7) and vaccenate
(18:1 n-7) are incorporated into phospholipids by an acyltransferase.
Bacterial membrane bilayers contain saturates and monounsaturates but
generally lack polyunsaturates.
For normal function, membrane bilayers must be `fluid', allowing lateral
movement of membrane components. Phospholipids with two saturated acyl chains
will only be able to maintain a fluid state at high temperatures. Unsaturated
acyl chains are essential for membrane `fluidity' in the range of temperatures
typical of modern life. The fact that membrane lipids in bacteria and
eukaryotes consist of pairs of acyl chains attached to a head group is a means
of `handcuffing' an unsaturate to every saturated acyl chain, ensuring
compulsory mixing and thus bilayers that do not laterally separate into
`solid' and `fluid' patches. This does not mean that all membranes are
homogeneous in their fluidity. When grown at low temperature, E. coli
substitute 18:1 n-7 acyl chains for 16:0 acyl chains, producing phospholipids
containing two monounsaturates. This very rapid response, being evident within
30 s of lowering the temperature (Rock et
al., 1996), is termed `homeoviscous adaptation' and results in a
relatively constant membrane fluidity.
Compared with bacteria, the eukaryotes have increased the variety of lipids
that make up their membrane bilayers. Notably, they synthesise
phosphatidylcholine, a common lipid in eukaryotic membranes (see
Fig. 1), as well as
non-phospholipid membrane lipids such as sphingolipids and sterols. Compared
with prokaryotes, eukaryotes also have additional desaturase enzyme systems
capable of introducing extra double bonds into acyl chains
(Tocher et al., 1998). There
are three types of desaturases, all of which consume molecular oxygen and
electrons obtained from an electron transport chain. One type is found in the
stroma of plant plastids, a second type are associated with the endoplasmic
reticulum and chloroplast membranes of plants and thylakoid membranes of
cyanobacteria, whilst the third type are membrane-bound enzymes associated
with the endoplasmic reticulum of animal and fungal cells. These enzymes
introduce double bonds into acyl chains esterified to coenzyme A. Whilst some
invertebrate groups possess the desaturases required for de novo
production of omega-3 and omega-6 polyunsaturates, these enzymes are absent in
vertebrates and many other invertebrates. In such species, polyunsaturates are
essential dietary components, although they can elongate and further
desaturate short-chain omega-3 and omega-6 polyunsaturates. As animals are
heterotrophs and because polyunsaturates both occur in the membranes of
eukaryotic cells and are required only in small amounts, polyunsaturates will
normally be found in adequate amounts in food.
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Metabolism: the middle of life and membrane bilayers |
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Composition of metabolic rate
At a fundamental level, mitochondria maintain a substantial proton gradient
across the mitochondrial inner membrane, and this gradient is used by the
mitochondrial ATP synthase to manufacture ATP. Most of the oxygen consumed by
cells is used by the mitochondrial respiratory chain to maintain this
transmembrane proton gradient. However, even when mitochondria are not making
ATP, they still consume oxygen and pump protons. Under these conditions, the
mitochondrial proton gradient remains constant because there is a balancing
proton leak. Mitochondrial proton leak is best characterised in liver
mitochondria and is estimated to be responsible for approximately 20% of
resting oxygen consumption of mammalian hepatocytes
(Porter and Brand, 1995).
Mitochondrial proton leak is rarely mentioned in undergraduate texts.
Another significant transmembrane gradient is generated by the
Na+/K+-ATPase, ubiquitous to animal cells. The
transplasmalemmal Na+ gradient it maintains is used by a host of
other cellular activities, including ion homeostasis and regulation of
intracellular volume. It is also the immediate energy source for action
potentials in excitable cells and the active uptake of nutrients and
transcellular ion transport in some epithelial cells. Its quantitative
contribution to energy turnover varies from approximately 10% in liver to
approximately 60% in kidney and brain
(Clausen et al., 1991).
The resting oxygen consumption of the laboratory rat has been allocated
thus: 10% is non-mitochondrial, 20% is related to mitochondrial proton leak
and 70% is for mitochondrial ATP production, which is used by the
Na+/K+-ATPase (2025%), protein synthesis
(2025%), Ca2+-ATPase (5%), gluconeogenesis (7%),
myosin-ATPase (5%) and ureagenesis (2%), with other activities (including
nucleic acid synthesis) constituting 6%
(Rolfe and Brown, 1997). A
substantial amount of the energy requirements of life is associated with
membrane-linked processes. Apart from the obvious mitochondrial proton leak,
Na+/K+-ATPase and Ca2+-ATPase, some protein
synthesis is a membrane-associated activity and some non-mitochondrial oxygen
consumption is associated with membranes. For example, the desaturases are
oxygen-consuming membrane-associated enzyme complexes. Their quantitative
contribution to this non-mitochondrial oxygen consumption is currently
unknown.
The dominant influences on the resting metabolic rate of different animal
species are body size, body temperature and whether the species is ectothermic
or endothermic. With respect to body size, most studies have involved mammals,
but the findings seem generally applicable to other groups. Resting metabolic
rate of mammals varies allometrically with body mass with the exponent
approximating 0.730.75. An interesting finding has been that, although
metabolic variation is large (mass-specific metabolic rate of a mouse is about
20 times that of a cow), the relative composition of energy metabolism appears
constant with body size. For example, many processes (e.g. protein turnover,
RNA turnover and ethane exhalation) in mammals vary with the same allometric
exponent as resting metabolic rate (e.g.
Topp et al., 1995), which
means they constitute a constant proportion of total metabolic rate
irrespective of the metabolic intensity and body size of the mammal. Part of
the allometric variation in metabolic rate is due to variation in relative
tissue size and part is due to variation in mass-specific tissue metabolism.
Tissue metabolism varies allometrically in mammals, as does the in
vitro mass-specific sodium pump activity
(Couture and Hulbert, 1995
).
The fact that the allometric exponents for these activities are similar
indicates that sodium pump activity is a constant proportion of total cellular
metabolic activity. Similarly, the oxygen consumption of mammalian hepatocytes
varies allometrically and the proportion of total hepatocyte oxygen
consumption devoted to ATP production, mitochondrial proton leak and
non-mitochondrial processes is relatively constant irrespective of body size
(Porter and Brand, 1995
).
The difference in the resting metabolism of ectotherms and endotherms has
been analysed by the study of reptiles and mammals matched for body mass and
body temperature. The reptile species chosen are desert lizards
Amphibolurus nuchalis and A. vitticeps, with preferred body
temperatures of 37°C, thus allowing comparison of the rate of cellular
activities not complicated by the effects of temperature. Resting metabolism
is approximately 7-fold greater in the endothermic mammals (mice and rats)
compared with the ectothermic reptiles and this is associated with larger
tissues and greater mass-specific tissue metabolism in the mammals
(Brand et al., 1991). The
contribution of ATP production, mitochondrial proton leak and
non-mitochondrial processes to total oxygen consumption of hepatocytes is
similar in the reptile and mammal (Brand et
al., 1991
), whilst the proportion of tissue metabolism devoted to
sodium pump activity is also similar (Else
and Hulbert, 1987
).
`Membrane pacemaker' theory of metabolism
From both the allometric comparison of metabolism in mammals and the
ectothermendotherm comparison, the same conclusions are reached. These
are: (1) the large differences in the whole-organism metabolic rate are partly
a cellular phenomenon and (2) the relative composition of metabolism in
different species is similar. Resting metabolic rate appears to consist of
linked processes such that when one varies, all vary in unison. This suggests
that there is a single factor (or a small number of factors) that influences
all (or many) of these processes: i.e. a pacemaker for metabolism. The
importance of membrane-associated processes in determining overall metabolic
rate suggests that membranes may be the site of such a pacemaker. It has been
proposed that both the amount of membranes and their acyl composition, notably
the relative balance between monounsaturated and polyunsaturated acyl chains,
especially docosahexaenoic acid (DHA), are a pacemaker for metabolic activity
(Hulbert and Else, 1999,
2000
). Space limitations
restrict our discussion here to the compositional aspects of membranes.
The evidence supporting the `membrane pacemaker' theory is of two types: (1) the acyl composition of membrane bilayers varies in a manner similar to variations in metabolic rate and (2) variations in acyl composition of membrane bilayers influence membrane-associated processes. These influences are such that high DHA content is normally associated with increased activity of the membrane-associated processes.
Although they have the same body temperature, the acyl composition of
tissue phospholipids in the endothermic mammal had a much greater unsaturation
index (number of double bonds per 100 acyl chains) than that of the reptilian
phospholipids. The mammalian membrane bilayers had a greater content of the
omega-3 polyunsaturated DHA than did the reptilian membrane bilayer but a
lower content of the monounsaturated 18:1 n-9
(Hulbert and Else, 1989). In a
similar manner, the tissue phospholipids of small mammals have a higher
unsaturation index than those of large mammals, although the total percentage
of unsaturates does not vary with body size in mammals. The membrane bilayers
of small mammals are generally high in DHA but low in 18:1 n-9 compared with
those of large mammals. These trends in acyl composition are manifest in
phospholipids from all tissues examined except brain, which are highly
polyunsaturated in all mammals irrespective of body size
(Hulbert et al., 2002c
). The
acyl chain that shows the greatest allometric variation is DHA, which has
exponents of -0.19, -0.21, -0.34 and -0.40 in liver, kidney, heart and
skeletal muscle, respectively (Hulbert et
al., 2002c
). Recently, the same trends have also been observed in
the pectoral muscle phospholipids of birds, with DHA content having an
exponent of -0.28 (Hulbert et al.,
2002a
). These exponents are similar to the -0.27 for mass-specific
metabolic rate. DHA content represents the greatest variation in body
composition yet recorded for different-sized mammals or birds.
Fig. 2 shows some of these
allometric relationships for skeletal muscle in both mammals and birds.
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Although these relationships are for total tissue phospholipids (i.e.
pooled membrane bilayers), the same trends exist when subcellular membranes
are independently analysed. Liver mitochondrial phospholipids are more
polyunsaturated and less monounsaturated in mammals compared with
similar-sized lizards with the same body temperature
(Brand et al., 1991;
Brookes et al., 1998
). The same
difference is observed in small mammals compared with large mammals
(Porter et al., 1996
) and in
small birds compared with large birds (M. D. Brand, N. Turner, P. L. Else and
A. J. Hulbert, unpublished).
Membrane polyunsaturation and membrane-associated function
Liver mitochondrial proton leak varies allometrically with body mass in
mammals and is related to differences in membrane acyl composition, especially
DHA content (Porter et al.,
1996). Similarly, in birds, liver mitochondrial proton leak is
allometrically related to body mass and correlated with membrane acyl
composition (M. D. Brand, N. Turner, P. L. Else and A. J. Hulbert,
unpublished). Although liver mitochondrial proton leak from other ectotherms
is not as low as that from the desert lizards, it is correlated with
differences in acyl composition (Brookes et
al., 1998
; Hulbert et al.,
2002b
). When the data for mammals and ectothermic vertebrates are
combined, liver mitochondrial proton leak is positively correlated with DHA
content of mitochondrial phospholipids (r=0.66, P<0.01,
N=26) and negatively correlated with the content of the
monounsaturated 18:1 n-9 (r=-0.38, P<0.05,
N=26). The difference in proton leakiness of mitochondria from
different tissues in the rat can be similarly associated with differences in
the acyl composition of their mitochondrial phospholipids
(Rolfe et al., 1994
). Other
evidence is that proton leak of liver mitochondria from mice increased both
when membrane DHA content was increased in vivo, by feeding menhaden
oil, and in vitro, by lipid fusion
(Stillwell et al., 1997
).
In endothermic vertebrates, the molecular activity of individual
Na+/K+-ATPase units is several times that in ectothermic
vertebrates (Else et al.,
1996), and these differences in molecular activity are related to
the different membrane lipids. When delipidated, kidney and brain microsomes
from the endothermic rat (Rattus norvegicus) and ectothermic cane
toad (Bufo marinus) both show a decrease in
Na+/K+-ATPase molecular activity. Relipidation with the
original microsomal lipids restores molecular activity to normal levels;
however, when relipidated with microsomal lipids from the other species, the
molecular activity is more like that of the other species
(Else and Wu, 1999
). These
`species-crossover' results show that membrane lipid environment is a
significant factor determining Na+/K+-ATPase molecular
activity. The mechanism by which membrane lipids affect enzyme activity is
unknown; however, physical characteristics of the membrane lipids appear
important. A strong relationship between the packing of membrane lipids and
Na+/K+-ATPase molecular activity in tissues from rats
and toads has been observed (Wu et al.,
2001
). The statistical correlations of molecular activity with the
this membrane protein via its effects on the physical environment of
the membrane bilayer.
Polyunsaturated acyl chains are also associated with many other rapid
membrane-associated activities. For example, DHA content is elevated in
high-frequency skeletal muscle such as the flight muscle of hummingbirds
(Archilocus colubris) and the rattlesnake (Crotalus atrox)
tail-shaker muscle (Infante et al.,
2001). Retinal membranes have a high DHA content and this is
associated with high activity of visual system G-proteins
(Litman and Mitchell, 1996
).
Several other examples are described by Hulbert and Else
(1999
,
2000
).
In what at first appears to be a contradiction of the membrane pacemaker
theory, many aquatic organisms, especially fish, have highly polyunsaturated
membranes. This is associated with adaptation to cold environments and the
effect of low temperatures slowing physiological processes. It is also
associated with increased metabolic activity. Fish mitochondrial membranes
have a higher DHA content than those of mammals and are leakier to protons
(Brookes et al., 1998). In
fish, cold acclimation generally involves an increase in both monounsaturate
and polyunsaturate content (especially DHA) of membrane bilayers, and these
changes result in altered activity of membrane-associated proteins
(Hazel, 1995
). For example,
Na+/K+-ATPase molecular activity is increased by cold
acclimation in trout (Oncorhynchus mykiss;
Raynard and Cossins, 1991
),
and mitochondrial lipids from cold-acclimated goldfish (Carassius
auratus) exhibit a greater reactivation of delipidated mitochondrial
enzyme than do those from warm-acclimated fish
(Hazel, 1972
). Lee
(1991
) has proposed that the
effects of membrane lipids on membrane proteins are not related to membrane
fluidity. Similarly, Hazel
(1995
) has suggested that the
increase in DHA is not directly related to homeoviscous adaptation, as the
increase in monounsaturates is adequate for maintaining membrane fluidity. He
suggested that the elevated DHA may have some other function.
Polyunsaturated DHA: synthesis, structure and function in eukaryotic
membranes
Recent studies have highlighted the physical properties that DHA imparts to
membranes. Molecular dynamic simulations suggest that there are hundreds of
conformations likely for DHA in membrane bilayers
(Feller et al., 2002). Several
of these conformations have the methyl end of the molecule located at the
outer edge of the bilayer rather than in the middle of the membrane bilayer,
as is normally shown in static diagrams (see
Fig. 1). These simulations
present an image of DHA thrashing about in the hydrocarbon core of the
membrane bilayer. Such molecular movement of DHA in a membrane bilayer
suggests that it will likely speed up, in a relatively non-specific manner,
many processes catalysed by membrane proteins.
The synthesis of DHA from short-chain omega-3 polyunsaturates involves
peroxisomes and is thus different to the synthesis of other membrane acyl
chains (Sprecher, 2000).
Regulation of the acyl composition of membrane bilayers is both via
the elongase and desaturase enzyme systems as well as via the
constant deacylationreacylation processes of membrane remodelling. In
rat hepatocytes, only four molecular species (16:0/18:1, 16:0/18:2, 16:0/22:6
and 18:1/18:2) of phospholipids are synthesised de novo and all other
molecular species are made by the deacylationreacylation of these four
molecular species (Schmid et al.,
1995
). Whilst it is not completely clear what particular membrane
property is being regulated, the observation that an acyltransferase enzyme is
influenced by its surrounding membrane lipid environment (Fyrst et al., 1996)
suggests that membrane acyl composition may be regulated at this level.
Changes in acyl composition of membrane bilayers can be very rapid, occurring
within minutes in cultured cells
(Chakravarthy et al.,
1986
).
The unsaturation of membrane bilayers varies predictably between different tissues but is not very responsive to diet. Omega-6 and omega-3 polyunsaturates are essential components of the diet for higher animals. If polyunsaturates are not present in the diet, the systems involved in regulation of membrane acyl composition will result in greater amounts of both 18:1 n-9 and 20:3 n-9 (an unusual polyunsaturate that higher animals can synthesise de novo) in phospholipids. The latter acyl chain is indicative of dietary polyunsaturated fatty acid (PUFA) deficiency, as it is normally not observed in tissue phospholipids. In some animals, gut organisms may also be a significant source of PUFA synthesis. The homeostatic regulation of membrane bilayer composition relative to diet, as well as the tissue specificity, is illustrated in Fig. 3.
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The increase in metabolic rate associated with the evolution of endothermy
is associated with elevated DHA in membranes and this may have resulted from
co-opting the same enzymatic processes used by the ectothermic vertebrates
during homeoviscous adaptation to cold
(Hulbert and Else, 1999). It
is intriguing to speculate that the differences in metabolic rates of mammals
associated with food habits may be due to a distinctive acyl composition of
particular foods. For example, are the low metabolic rates of naked mole rats
(Heterocephalus glaber) and termite eaters due to a diet severely
deficient in polyunsaturates? Future investigations into such questions will
need to take into account the role of gut microrganisms, but the membrane
pacemaker theory of metabolism provides a framework for investigating such
mechanistic explanations. For example, Pan and Storlien
(1993
) have shown that the
acyl composition of the diet significantly alters the metabolic rate of
rats.
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Death: the end of life and membrane bilayers |
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A link between body size, metabolic rate and lifespan was first suggested
about a century ago and was elaborated later into the `rate of living theory'
of aging. In the 1950s, it was given a molecular basis with the `free radical
theory' of aging (Harman,
1956). Taking antioxidant defences into account, it has evolved
over the past 50 years into an `oxidative stress theory'. This theory has as
its basis the rate of aerobic metabolism and is currently the most popular
theory of aging. However, it has a number of problems yet to be resolved. For
example, among mammals, humans and bats are long-lived for their size yet have
a typically mammalian level of metabolism
(Austad and Fischer, 1991
).
Similarly, birds have metabolic rates slightly higher than mammals but, on
average, have lifespans more than twice as long as similar-sized mammals
(Holmes and Austad, 1995
).
The most favoured current view of death from old age is that it is the
result of accumulated damage from reactive oxygen species (ROS) that are an
inevitable byproduct of mitochondrial oxygen consumption. Proteins,
carbohydrates, nucleic acids and lipids are all targets of such oxidative
damage. In lipids, it is the carbon atoms between the C=C units
found in polyunsaturated acyl chains that are most susceptible to oxidative
attack (Halliwell and Gutteridge,
1999). Saturated and monounsaturated acyl chains lack such carbon
atoms. The long-chain polyunsaturates in membrane bilayers of mitochondria,
however, are very susceptible to damage, both chemically and also because of
their location close to the site of ROS production. Membrane lipid
peroxidation is an autocatalytic chain reaction, and many of its products,
including hydroxynonenal (from omega-6 PUFA) and hydroxyhexanal (from omega-3
PUFA), are themselves very potent damagers of proteins
(Halliwell and Gutteridge,
1999
).
The low level of phospholipid unsaturation in large mammal species has been
related to decreased lipid peroxidation and lipoperoxidative tissue damage and
has been suggested to be an adaptation to their long MLSPs (Pamplona et al.,
1998,
2000
). Although both mammals
and birds show allometric variation in membrane acyl composition, there are
differences between these two groups of endotherms. Birds have a lower
unsaturation index in their muscle phospholipids than do mammals
(Fig. 2A), which is related to
a higher ratio of n-6 PUFA to n-3 PUFA (Hulbert et al., 2002). It has been
proposed that this lower unsaturation in birds is related to their longer
lifespan compared with similar-sized mammals
(Pamplona et al., 1996
). The
proposal that the MLSP difference between birds and mammals is related to a
lower rate of mitochondrial ROS production in birds
(Ku and Sohal, 1993
) has
recently been questioned (St Pierre et
al., 2002
). If the acyl composition of membrane bilayers is
involved in explaining the association of MLSP with body size, as well as the
difference between birds and mammals, then we might expect the same
relationship for both birds and mammals. Unsaturation index of muscle
phospholipids differs between birds and mammals
(Fig. 2A), but when the
peroxidizability index of muscle phospholipids is plotted against MLSP
(Fig. 4), birds and mammals
(including humans) all appear to follow the one relationship. Similar
relationships exist for other tissues.
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Physiological treatments that extend lifespan can also give insight into
the mechanisms underlying aging. Calorie restriction is the only physiological
treatment known to extend lifespan in a wide range of animals
(Sohal and Weindruch, 1996).
During calorie restriction, metabolic rate is not reduced but there is a
substantial decrease in lipid peroxidation in rats. This is not attributable
to changes in membrane vitamin E content but is associated with changes in
membrane acyl composition of both mitochondria and microsomes, resulting in a
decreased susceptibility of these membrane bilayers to lipid peroxidation
(Laganiere and Yu, 1987
).
Calorie restriction also modifies acyl composition of muscle membranes
(Cefalu et al., 2000
), as well
as both phosphatidylcholine and phosphatidylethanolamine in liver
(Jeon et al., 2001
), such as
to decrease their ability to undergo lipid peroxidation.
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Conclusions |
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