Age-associated mitochondrial oxidative decay:
Improvement of carnitine acetyltransferase
substrate-binding affinity and activity in
brain by feeding old rats acetyl-L-
carnitine and?or R-?-lipoic acid
Jiankang Liu*†, David W. Killilea*†, and Bruce N. Ames*†‡
*Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; and†Children’s Hospital Oakland Research Institute,
Oakland, CA 94609
Contributed by Bruce N. Ames, December 28, 2001
We test whether the dysfunction with age of carnitine acetyltrans-
to decreased binding affinity for substrate and whether this
substrate, fed to old rats, restores CAT activity. The kinetics of CAT
were analyzed by using the brains of young and old rats and of old
rats supplemented for 7 weeks with the CAT substrate acetyl-L-
carnitine (ALCAR) and?or the mitochondrial antioxidant precursor
R-?-lipoic acid (LA). Old rats, compared with young rats, showed a
decrease in CAT activity and in CAT-binding affinity for both
substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to
old rats significantly restored CAT-binding affinity for ALCAR and
CoA, and CAT activity. To explore the underlying mechanism, lipid
peroxidation and total iron and copper levels were assayed; all
lipid peroxidation but did not decrease iron and copper levels. Ex
vivo oxidation of young-rat brain with Fe(II) caused loss of CAT
Fe(II) inactivated the enzyme but did not alter binding affinity.
However, in vitro treatment of CAT with the lipid peroxidation
products malondialdehyde or 4-hydroxy-nonenal caused a de-
crease in CAT-binding affinity and activity, thus mimicking age-
related change. Preincubation of CAT with ALCAR or CoA pre-
vented malondialdehyde-induced dysfunction. Thus, feeding old
rats high levels of key mitochondrial metabolites can ameliorate
oxidative damage, enzyme activity, substrate-binding affinity, and
mal metabolism (1–10). Aging is associated with a decrease in
cellular enzyme or receptor activities. Some enzyme or receptor
inactivation is due to an increase in Kmfor their substrates or
cofactors (B.N.A., J.L., and I. Elson-Schwab, unpublished work).
For example, Feuers (11) found that in female mice, the Vmaxof
mitochondrial complexes III and IV significantly decreased with
age, in parallel with a decrease of ubiquinol or cytochrome c
substrate-binding affinity. Dietary restriction, which reduces the
generation of oxidants and oxidative damage, effectively re-
versed these decreases in complex activity and substrate affinity.
On the other hand, the activity of many enzymes decreases with
age but shows no change in Km (12–14) (B.N.A., J.L., and I.
Elson-Schwab, unpublished work).
As many as one-third of mutations in a gene result in the
corresponding enzyme having a poorer binding affinity (an in-
creased Km) for its coenzyme, which in turn lowers the rate of the
reaction (15–17). When the concentration of the coenzyme is
increased by feeding the corresponding vitamin at high levels, the
enzyme activity is partially restored, and the disease phenotype is
cured or ameliorated (18). Thus, we hypothesize (18–20) that
ging appears to be due, in part, to damage caused by the
oxidants produced by mitochondria as by-products of nor-
during aging, mitochondrial oxidants deform proteins because of
by-products from lipid peroxidation. This deformation in turn
decreases the binding affinity of many enzymes for their substrates
or coenzymes. Feeding high doses of enzyme substrates or coen-
zymes can overcome the deficiencies of those enzymes with de-
creased binding affinity and restore enzyme function. Oxidative
decay is particularly acute in mitochondria (1, 4, 8). Thus, feeding
high levels of several mitochondrial substrates and vitamin precur-
sors of coenzymes might reverse some of the mitochondrial decay
of aging (5–7, 9, 18, 21, 22).
L-Carnitine is a betaine required in the mitochondria for
transporting in long chain fatty acids for ? oxidation and ATP
production, as well as for transporting out excess short and
medium chain fatty acids (23). Feeding old rats an acetyl-L-
carnitine (ALCAR)-supplemented diet restores tissue levels of
free and acyl carnitines to that found in plasma and brain tissues
of younger animals (20, 24). This diet-induced increase in
carnitine levels in older animals results in a reversion of liver and
heart mitochondria to a more youthful state, both structurally
and functionally (6, 9, 14, 25–28).
R-?-lipoic acid (LA) is a coenzyme for pyruvate dehydroge-
nase and ?-ketoglutarate dehydrogenase in mitochondria. Di-
hydrolipoic acid, the reduced form of LA, is a potent antioxidant
raise the levels of intracellular glutathione, which is critical for
neuronal function (29, 30). LA supplementation restores long-
term potentiation, a synaptic analogue of learning and memory,
in aged rodents (31) and partially restores ambulatory activity
and memory lost during aging (5, 32, 33).
Carnitine acetyltransferase (CAT) (EC 188.8.131.52) catalyses the
reversible conversion of acetyl-CoA and carnitine to acetylcar-
nitine and CoA. CAT’s essential functions are to regenerate
CoA, which allows peroxisomal ?-oxidation to proceed, and to
facilitate transport of acetyl moieties to mitochondria for oxi-
dation (34). More than 70% of CAT is located in the mitochon-
(34–36). An age-associated decrease of CAT activity has been
reported in rat soleus, diaphragm, and heart (37, 38) and in brain
and muscles in vitamin E-deficient rats (39), although Moret et
Abbreviations: ALCAR, acetyl-L-carnitine; CAT, carnitine acetyltransferase; HNE, 4-
hydroxynonenal; LA, R-?-lipoic acid; MDA, malondialdehyde; TBS, Tris-buffered saline.
‡To whom reprint requests should be addressed at: Children’s Hospital Oakland Research
Institute 5700 Martin Luther King, Jr., Way, Oakland, CA 94609. E-mail: bnames@
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
February 19, 2002 ?
vol. 99 ?
al. (40) did not find altered CAT activity in the brain of
Long–Evans rats with age in healthy animals over a moderate
age range. CAT activity was found to decrease in Alzheimer’s
patient brain microvessels and cerebellum (41, 42), although
there are contrary findings (43). CAT activity was also found to
decrease in fatal ataxic encephalopathy (44), mitochondrial
encephalomyopathy (45), and severe peripheral vascular disease
(46). Sulfhydryl reactive agents cause a decrease in CAT-binding
affinity for substrates (increase in Km) and in CAT activity, and
the addition of CAT substrates or antioxidants such as mercap-
toethanol prevents or partially reverses CAT inhibition (47). No
study has attempted to examine the age-associated changes in
CAT substrate-binding affinity and the effects of substrates or
antioxidant treatments on the Kmof CAT.
The present study was designed to test the Kmhypothesis by
assaying the kinetics of CAT in the brains of young and old rats,
and old rats fed ALCAR and?or LA. The mechanism for the
change in kinetics was also explored.
Materials and Methods
Materials. ALCAR (hydrochloride salt) was a gift of Sigma Tau
(Pomezia, Italy) and LA (the natural R-isomer), of ASTA
Medica (Frankfurt?Main, Germany). All other reagents were
reagent grade or the highest quality available from Sigma, unless
Animals. Fischer 344 male rats were obtained from the National
Institute on Aging. Control animals were fed an AIN93M diet
from Dyets (Bethlehem, PA) and MilliQ (Millipore) water (pH
5.2). The rats in the experimental groups were fed either 0.5%
ALCAR in their drinking water, 0.2% LA in AIN93M diet, or
both, for 7 weeks. The young rats were 4.5 months, and the old
than 7 weeks older when they were killed with ether anesthesia.
The brains were removed, immediately put into liquid nitrogen,
and stored in a ?80°C freezer until analysis.
Kinetic Analysis. Because there are no brain regional differences
in CAT activity as well as Kmin rats (40) or in rabbits (36), we
used the whole brain. Brain tissue was homogenized with 50 mM
Tris-buffered saline (pH 7.5) containing 2 mM EDTA, 5 mM
MgCl2, 0.8 mM DTT, 1 ?M protease inhibitor mixture, and 0.25
mM phenylmethylsulfonyl fluoride (freshly made in acetone and
added to the homogenizing tube before homogenization). Ho-
mogenates were then sonicated on ice for 3 ? 10 s and
centrifuged at 3,500 ? g for 5 min to obtain the mitochondrial
and microsomal portion containing more than 90% of the
enzyme (36). The CAT activity was assayed immediately after
the centrifugation as described (48, 49). The assay medium
contained about 0.5 mg of protein?ml brain homogenate super-
natant, 50 mM Tris, 2 mM EDTA, 25 mM malate, 0.25 mM
NAD, 12.5 ?g?ml of rotenone, 12.5 ?g?ml of malate dehydro-
genase, 50 ?g?ml of citrate synthase, and 0.04% Triton-100. The
kinetics were determined over a range of ALCAR concentra-
tions from 0.015 to 5 mM with a constant concentration of 1.25
mg?ml of CoA (Km for ALCAR) or over a range of CoA
concentrations from 6.25 to 400 ?M with a constant concentra-
tion of 2 mM acetyl-L-carnitine (Kmfor CoA). The results were
plotted with the double-reciprocal plot of reciprocal rate 1?v
against reciprocal substrate concentration 1?S. Results were also
calculated by the direct linear plot with the equation of Vmax?
v ? (v?S)Km(50).
Ex Vivo Oxidation of Rat-Brain Homogenate. The young-rat-brain
homogenate was incubated with FeSO4for 15 min at 37°C. The
kinetics were assayed as described above, and the oxidation was
assayed by measuring malondialdehyde (MDA) with a gas
chromatography–mass spectrometric method (51, 52).
In Vitro Oxidation of Purified CAT with Iron. CAT (purified from
pigeon breast muscle) was obtained from Sigma. The enzyme
was diluted with PBS and used immediately. The enzyme (0.36
?g?ml) was incubated with PBS and various concentrations of
FeSO4 with or without metal chelators?antioxidants at room
temperature for 15 min. CAT activity was assayed immediately
by using the method described above (CAT at 0.036 ?g?ml of
MDA- and 4-Hydroxynonenal (HNE)-Induced Inactivation and Km
of 1,1,3,3-tetramethoxypropane with 0.01 M HCl and stored 3–6
weeks at 4°C. The concentration of MDA was determined at 245
nm by using an extinction coefficient of 13,700?M (52). HNE was
obtained from Calbiochem, and its concentration was determined
at 224 nm by using an extinction coefficient of 13,750?M (53).
Similar to the above incubation, CAT (0.36 ?g?ml) was incubated
in PBS or Tris-buffered saline (TBS) with MDA or HNE at room
(CAT at 0.036 ?g?ml of protein).
Total Metal Assay with Inductively Coupled Plasma Spectrometry
(ICP). Six metals (iron, copper, calcium, magnesium, manganese,
and zinc) were analyzed by using ICP with modification of a
reported method (54, 55).
Lipid Peroxidation Assay. Lipid peroxidation was assayed by using
a gas chromatography–mass spectrometric method to measure
the level of MDA (51, 52).
CAT Kinetics. The double-reciprocal plots of CAT reaction veloc-
ity versus ALCAR or CoA concentration in rat brain are shown
in Fig. 1 A and B. The values of Vmaxand apparent Kmare shown
in Fig. 1 C and D. Compared with young rats, old rats showed a
moderate decrease in enzyme activity (Vmax) (14%, Fig. 1C) and
an increase in Km[160% of Kmfor ALCAR and 180% of Kmfor
CoA (Fig. 1D)], suggesting a decrease in substrate-binding
affinity. Supplementation with ALCAR in old rats significantly
increased the binding affinity for ALCAR. Supplementation
with LA showed a small increase in binding affinity that was not
statistically significant. The combination of ALCAR and LA
significantly elevated enzyme activity and binding affinity for
both substrates (Kmfor ALCAR, P ? 0.019; Kmfor CoA, P ?
0.018). The combination also significantly increased CAT activ-
ity (P ? 0.04).
Ex Vivo Oxidation of Brain Homogenate from Young Rats. Incubation
of Fe(II) (1–10 mM) with brain homogenate from young rats for
15 min induced a concentration-dependent inactivation of CAT.
The inactivation was accompanied by a significant decrease in
substrate-binding affinity (Fig. 2A), similar to that associated
with aging (Fig. 1). Incubation of rat-brain homogenate with
Fe(II) induced a marked increase in membrane lipid peroxida-
tion, as shown by the level of MDA (control, 15.2 ? 0.2; addition
of 0.2 mM FeSO4, 134 ? 1.6 pmol?mg of protein). As expected,
metal chelators such as EDTA at 1 mM (MDA 48.6 ? 0.6
pmol?mg) and deferoxamine at 1 mM (MDA 8.5 ? 0.2 pmol?
mg) protected lipid membranes from peroxidation.
In Vitro Oxidation of Purified CAT. Incubation of purified enzyme
with Fe(II) (0.1–1 mM) induced a concentration-dependent
inactivation of CAT with a 50% inactivation at a concentration
of 95 ?M at 37°C for 15 min (0.5 units?ml). Unlike ex vivo
oxidation of rat-brain homogenate, this oxidation induced a
decrease in enzyme activity but not in the substrate-binding
affinity (Fig. 2B). FeSO4at 200 ?M concentration caused 80%
inactivation of CAT. As expected, metal chelators EDTA and
Liu et al. PNAS ?
February 19, 2002 ?
vol. 99 ?
no. 4 ?
deferoxamine, at 1 mM concentration, protected the enzyme
from inactivation by 70 and 94% respectively, as did the sulfhy-
(32%), and dihydrolipoic acid (35%), although they were less
effective. Catalase (1 mg?ml) also showed protection (87%),
suggesting that enzyme inactivation is mediated by Fenton
chemistry. The substrates of the enzyme protected against
Fe(II)-induced inactivation: ALCAR at 1 mM, 92%; L-carnitine
at 1 mM, 36%; CoA at 0.6 mM, 91%; and acetyl-CoA at 0.6 mM,
In Vivo Lipid Peroxidation Levels in Rat Brain.Compared with young
product of lipid peroxidation. Old rats fed LA or LA plus
ALCAR had significantly lowered levels of brain MDA (Fig. 3).
Effect of MDA and HNE on the Kmof Purified CAT.Fig.4AandBshow
the effects of MDA and HNE in PBS on reciprocal plots of CAT
for the substrate ALCAR. Both MDA and HNE caused a
concentration-dependent inactivation of CAT accompanied by
an increase in Kmwhen incubated in PBS (Fig. 4) or in TBS (data
not shown). MDA was a more powerful inhibitor than HNE. In
PBS incubation, MDA at 25, 50, and 100 ?M inhibited CAT
activity to 69, 54, and 30% and increased the Kmfor ALCAR to
135, 152, and 259%, whereas HNE at 0.5, 0.75, 1.0, and 2.0 mM
inhibited CAT activity to 96, 88, 79, and 60% and increased the
Km for ALCAR to 135, 164, and 269%). The concentration
was calculated by using Student’s t test between young and old groups (*, P ? 0.05,**, P ? 0.01) and by using one-way ANOVA with Dunnett’s multiple
comparison test between old and other treated groups (#, P ? 0.05).
Double-reciprocal plots of reaction velocity versus substrate ALCAR (A) or CoA (B) concentrations in rat brain. (C) Vmax; (D) apparent Kmfor ALCAR and
brain with and without 5 mM Fe(II) (A) and for purified CAT (from pigeon
breast muscle) with and without 0.1 mM Fe(II) (B). Significant difference was
calculated with Student’s t test (**, P ? 0.01).
www.pnas.org?cgi?doi?10.1073?pnas.261709098Liu et al.
required for 50% inhibition of CAT activity by MDA is 45 ?M
in PBS solution and 400 ?M in TBS solution. Preincubation with
substrates, ALCAR, L-carnitine, CoA, and acetyl-CoA pro-
tected MDA-induced CAT inactivation and increase in Km
Total Metal Content in Rat Brain. Compared with young rats, old
old, 81.6 ? 2.2 ng?mg of dry tissue; P ? 0.001) and copper
(young, 10.3 ? 0.2, and old, 17.4 ? 0.6 ng?mg of dry tissue; P ?
0.001) in the brain; no changes in Ca, Mg, Zn, and Mn were
found (data not shown). Supplementing with ALCAR and?or
LA did not cause a significant decrease in the levels of total iron
(old ? ALCAR, 85.1 ? 4.2; old ? LA, 80 ? 1.7; and old ?
ALCAR ? LA, 76.3 ? 4.5 ng?mg of dry tissue) or total copper
(data not shown).
Old-rat brain is shown to have a moderate age-associated
decrease in CAT activity and a marked decrease in binding
affinity for the substrates ALCAR (young 100 ?M, old 150 ?M;
Fig. 1D) and CoA. Feeding old rats ALCAR for 7 weeks, which
elevates the level of free and acyl carnitines in blood and brain
to a level of about 100 ?M (20), significantly restored this
age-associated decrease in binding affinity for ALCAR; the
activity and its binding affinity for the substrates ALCAR and
CoA to the levels observed in young rats. CAT has two separate
binding sites: one for CoA?acetyl-CoA involving the sulfhydryl
group of a cysteine residue and a second for L-carnitine?
ALCAR (56). Feeding old rats LA significantly enhanced the
effect of ALCAR, although LA alone had only a small effect on
CAT activity and substrate-binding affinity.
Although extrapolation from in vitro to in vivo results should
be viewed with caution, we suggest two plausible mechanisms
that could account for the age-associated loss of binding affinity
and activity: (i) adduction to the protein of aldehyde products of
lipid peroxidation, or (ii) oxidation of the protein either directly
from lipid peroxidation, increases with age in parallel with a
decrease in CAT activity and binding affinity for substrates (Fig.
4A). We also show that MDA and HNE, another lipid peroxi-
dation product, decrease the Vmaxand binding affinity of CAT
in vitro, whereas a direct oxidant, i.e., iron, does not. Lipid
peroxidation may be due in part to age-associated increases in
iron and copper levels. In agreement with this are our results
from the ex vivo oxidation of young-rat brain with Fe(II), in
which CAT Kmand activity change in the same way as during
aging. Aldehyde products from lipid peroxidation of membranes
have been shown to react with both amino and sulfhydryl groups
in protein (57), thus potentially inactivating them (53, 58). The
level of MDA needed to inhibit CAT in vitro is consistent with
the MDA level observed in vivo. The level of MDA required for
50% inhibition of CAT is 45 ?M, which is not too far from its
concentration in brain (20 pmol?mg of protein, i.e., about 4 ?M
in tissue) (Fig. 3). The MDA concentration in mitochondria is
likely to be much higher. A fraction of MDA is bound to proteins
(59). Most in vitro studies used a 10–10,000 ?M range of MDA
to show it toxic or mutagenic (57). MDA and HNE are only two
of the many known active aldehydes formed from lipid peroxi-
dation, many of which may contribute to enzyme inactivation.
Lowering aldehydes from lipid peroxidation does not seem to
be the sole explanation for the effects of ALCAR and LA on
improving CAT function and Km. Although the combination of
ALCAR and LA lowered MDA levels and restored CAT
function, the results with the individual compounds indicate a
more complex model. In vivo, LA significantly lowered MDA
levels, whereas ALCAR did not, yet ALCAR significantly
restored CAT function, whereas LA did not. Extrapolating from
in vitro experiments to in vivo conclusions, however, depends on
physiological concentration and time, as both mitochondria and
protein turn over, and a definitive conclusion as to mechanism
is not yet possible. In vitro enzyme inactivation by aldehydes and
the protective effect of the substrate ALCAR (Fig. 4C) are likely
to explain the in vivo decrease with age of CAT-binding affinity
and Vmaxand their reversal by feeding ALCAR. Then why does
LA not appreciably improve CAT function by itself, although it
lowers the level of aldehydes? Our data show that LA enhanced
the effect of ALCAR on Km, especially for CoA, therefore LA
may increase binding affinity and enzyme function to a small
extent. LA is synergistic or additive with ALCAR in a number
of studies (Figs. 1 and 2). Thus, the most likely mechanism for
our observations appears to be the interaction of aldehydes from
lipid peroxidation with CAT and a protective effect of the
substrate ALCAR, with the additional beneficial effect of LA’s
contribution in lowering mitochondrial lipid peroxidation.
The observed improvement of CAT activity and binding
affinity by ALCAR and LA may depend on protein and mito-
chondrial turnover. Damaged proteins and mitochondria are
turned over by proteasomal and lysosomal degradation, respec-
tively (60). Oxidative damage, especially lipid peroxidation, may
be responsible for some forms of proteasome dysfunction in the
central nervous system, by blocking either substrate binding or
by mixed-function oxidation reaction has been suggested
in protein turnover and aging (62). Enzymes with aldehyde-
inactivated SH groups can be reactivated by excess reduced
glutathione and cysteine (57). It is possible LA may play a role
in reactivating CAT and in preventing proteasomes from oxi-
MDA was more potent than HNE in affecting CAT kinetics.
This may be because: (i) 4-hydroxyalkenals are highly specific
reagents for SH groups, although they may also modify lysine,
LA groups. Significant difference was calculated by using Student’s t test
between young and old groups (**, P ? 0.01), and by using one-way ANOVA
with Dunnett’s multiple comparison test between old and other treated
groups (#, P ? 0.05).
MDA levels in the rat brain measured with a gas chromatography–
Liu et al.PNAS ?
February 19, 2002 ?
vol. 99 ?
no. 4 ?
histidine, serine, and tyrosine; and (ii) MDA can readily modify
proteins under physiological conditions, although it is less reac-
tive with free amino acids. MDA reacts primarily with lysine
crosslinks (57). The effect of MDA on the activity and Kmof
CAT was reduced greatly in TBS, presumably due to the relative
stable covalent binding of MDA to the amino group of Tris. The
effect of HNE, unlike that of MDA, on the activity and Kmof
CAT was not greatly affected by TBS. In experiments with
malondialdehyde, there are two possible complications whose
importance has not yet been clarified: (i) when MDA is prepared
from bis-acetal, small amounts of ?-ethoxy or ?-methoxy acro-
lein, highly reactive aldehydes, are unavoidably formed during
acid hydrolysis (63), and a variety of similar 2-alkenals are
formed during lipid peroxidation including HNE; and (ii) MDA
in solution forms reactive aldol type condensation products
including dimers and trimers (64), and these condensation
products may also modify proteins (57).
The enzyme dysfunction induced by lipid peroxidation prod-
ucts such as MDA and HNE, rather than being specific for CAT,
may be a common mechanism of age-associated dysfunction of
enzymes with amino and sulfhydryl groups at or near their active
sites. We have shown that HNE also causes a decrease in
pyruvate dehydrogenase (PDH)-binding affinity for pyruvate
(data not shown), confirming a study on the loss of activity of
PDH by HNE (58). MDA also causes a loss of PDH activity and
a decrease in binding affinity for pyruvate (data not shown).
The brain tissue of old rats showed a significant increase in
iron and copper accumulation, which can cause oxidative dam-
age by catalyzing oxidant generation and lipid peroxidation. It
should be emphasized, however, that we have assayed total iron,
not free redox active iron. MDA accumulates with age (Fig. 3B)
oxidation of young-rat brain with Fe(II) induced similar reduc-
tion of enzyme activity and binding affinity; in vitro oxidation of
purified CAT with Fe(II) inactivated the enzyme but did not
of CAT. (C). The protection of substrate ALCAR, L-carnitine, CoA, and acetyl-CoA on reciprocal plots of CAT for substrate ALCAR (0.5 mM), L-carnitine (0.25 mM),
CoA (25 ?M), and acetyl-CoA (50 ?M). The MDA used was 50 ?M with 1-h incubation. The substrates were added before MDA.
Concentration-dependent effects of MDA (A) and HNE (B) in PBS on reciprocal plots of CAT for substrate ALCAR. Different concentrations of MDA or
www.pnas.org?cgi?doi?10.1073?pnas.261709098Liu et al.
alter the binding affinity. This increase of iron is consistent with Download full-text
previous studies in liver and brain using the atomic absorption
technique (J.L., J.-Y. Park, Q. Jiang, L. Youngman, H. Atamna,
and B.N.A., unpublished work) and spectrophotometric mea-
surements (65). Although ALCAR and?or LA did not signifi-
cantly effect transition metal accumulation in these short-term
studies, the possibility of chelating the labile or ‘‘free’’ transition
cannot be excluded, as the accumulation of metals and oxidative
damage is a lifelong process. LA, in addition to its oxidant-
scavenging effect, is an efficient chelator of copper (66) and iron
(67) that reduces the catalytic activity of transition metals in
oxidant generation reactions. A higher dose of LA (0.5%) did in
fact reduce iron in old-rat brain (J. H. Suh and T. M. Hagen,
personal communication). Although iron and copper accumu-
lation with age remains plausible as a cause of the increased lipid
peroxides, further studies are warranted.
This study, as well as others on the effects of ALCAR and?or
LA on cognition (33) and mitochondrial functions (5, 6, 68), and
studies (B.N.A., J.L., and I. Elson-Schwab, unpublished work) of
the age-associated decrease in binding affinity of other brain-
and memory-related enzymes and receptors suggest that a
decrease in enzyme-binding affinity by oxidative damage is an
important contributor to age-associated memory decline, which
may be ameliorated by feeding high doses of mitochondrial
enzyme substrates and antioxidants.
We thank E. Roitman for technical assistance and Jack Kirsch, Larry
Marnett, and John Nides for critical comments. This work was supported
by grants to B.N.A. from the Ellison Foundation, the National Institute
on Aging, the Wheeler Fund of the Dean of Biology, and the National
Institute of Environmental Health Sciences Center (Grant P30-
ES01896), and by a National Institutes of Health?National Institute on
Aging postdoctoral training grant (5 T32 AG00266–02) to D.W.K.
1. Harman, D. (1972) J. Am. Geriatr. Soc. 20, 145–147.
2. Harman, D. (1981) Proc. Natl. Acad. Sci. USA 78, 7124–7128.
3. Ames, B. N., Shigenaga, M. K. & Hagen, T. M. (1993) Proc. Natl. Acad. Sci.
USA 90, 7915–7922.
4. Shigenaga, M. K., Hagen, T. M. & Ames, B. N. (1994) Proc. Natl. Acad. Sci.
USA 91, 10771–10778.
5. Hagen, T. M., Ingersoll, R. T., Lykkesfeldt, J., Liu, J., Wehr, C. M., Vinarsky,
V., Bartholomew, J. C. & Ames, A. B. (1999) FASEB J. 13, 411–418.
6. Hagen, T. M., Ingersoll, R. T., Wehr, C. M., Lykkesfeldt, J., Vinarsky, V.,
Bartholomew, J. C., Song, M. H. & Ames, B. N. (1998) Proc. Natl. Acad. Sci.
USA 95, 9562–9566.
7. Hagen, T. M., Vinarsky, V., Wehr, C. M. & Ames, B. N. (2000) Antioxid. Redox.
Signal 2, 473–483.
8. Hagen, T. M., Yowe, D. L., Bartholomew, J. C., Wehr, C. M., Do, K. L., Park,
J. Y. & Ames, B. N. (1997) Proc. Natl. Acad. Sci. USA 94, 3064–3069.
9. Hagen, T. M., Wehr, C. M. & Ames, B. N. (1998) Ann. N.Y. Acad. Sci. 854,
10. Beckman, K. B. & Ames, B. N. (1998) Physiol. Rev. 78, 547–581.
11. Feuers, R. J. (1998) Ann. N.Y. Acad. Sci. 854, 192–201.
12. Paradies, G. & Ruggiero, F. M. (1990) Biochim. Biophys. Acta. 1016, 207–212.
13. Paradies, G. & Ruggiero, F. M. (1991) Arch. Biochem. Biophys. 284, 332–337.
14. Paradies, G., Ruggiero, F. M. & Dinoi, P. (1992) Int. J. Biochem. 24, 783–787.
15. Cox, T. C., Bottomley, S. S., Wiley, J. S., Bawden, M. J., Matthews, C. S. & May,
B. K. (1994) N. Engl. J. Med. 330, 675–679.
16. Fenton, W. A. & Rosenberg, L. E. (1995) in The Metabolic and Molecular Bases
of Inherited Disease, ed. Scriver, C. (McGraw–Hill, New York), Vol. II, pp.
17. Mudd, S. H., Skovby, F., Levy, H. L., Pettigrew, K. D., Wilcken, B., Pyeritz,
R. E., Andria, G., Boers, G. H., Bromberg, I. L., Cerone, R., et al. (1985) Am. J.
Hum. Genet. 37, 1–31.
18. Ames, B. N., Elson-Schwab, I. & Silver, E. (2002) Am. J. Clin. Nutr., in press.
19. Ames, B. N. (1998) Toxicol. Lett. 102–103, 5–18.
20. Liu, J., Atamna, H., Kuratsune, H. & Ames, B. N. (2002) Ann. N.Y. Acad. Sci.
959, in press.
21. Lykkesfeldt, J., Hagen, T. M., Vinarsky, V. & Ames, B. N. (1998) FASEB J. 12,
22. Suh, J. H., Shigeno, E. T., Morrow, J. D., Cox, B., Rocha, A. E., Frei, B. &
Hagen, T. M. (2001) FASEB J. 15, 700–706.
23. Rebouche, C. J. (1992) FASEB J. 6, 3379–3386.
24. Maccari, F., Arseni, A., Chiodi, P., Ramacci, M. T. & Angelucci, L. (1990) Exp.
Gerontol. 25, 127–134.
25. Paradies, G., Petrosillo, G., Gadaleta, M. N. & Ruggiero, F. M. (1999) FEBS
Lett. 454, 207–209.
26. Paradies, G., Petrosillo, G. & Ruggiero, F. M. (1997) Biochim. Biophys. Acta.
27. Paradies, G., Ruggiero, F. M., Petrosillo, G., Gadaleta, M. N. & Quagliariello,
E. (1994) FEBS Lett. 350, 213–215.
28. Paradies, G., Ruggiero, F. M., Petrosillo, G., Gadaleta, M. N. & Quagliariello,
E. (1994) Ann. N.Y. Acad. Sci. 717, 233–243.
29. Packer, L., Tritschler, H. J. & Wessel, K. (1997) Free Radical Biol. Med. 22,
30. Packer, L., Roy, S. & Sen, C. K. (1997) Adv. Pharmacol. 38, 79–101.
31. McGahon, B. M., Martin, D. S., Horrobin, D. F. & Lynch, M. A. (1999)
Neurobiol. Aging 20, 655–664.
32. Hagen, T. M., Liu, J., Lykkesfeldt, J., Wehr, C. M., Ingersoll, R. T., Vinarsky,
V., Bartholomew, J. C. & Ames, B. N. (2002) Proc. Natl. Acad. Sci. USA 99,
33. Liu, J., Head, E., Gharib, A. M., Yuan, W., Ingersoll, R. T., Hagen, T. M.,
Cotman, C. W. & Ames, B. N. (2002) Proc. Natl. Acad. Sci. USA 99, 2356–2361.
34. Zammit, V. A. (1999) Prog. Lipid Res. 38, 199–224.
35. Bieber, L. L. (1988) Annu. Rev. Biochem. 57, 261–283.
36. McCaman, R. E., McCaman, M. W. & Stafford, M. L. (1966)J. Biol. Chem.241,
37. Hansford, R. G. (1978) Biochem. J. 170, 285–295.
38. Hansford, R. G. & Castro, F. (1982) Mech. Ageing Dev. 19, 191–200.
39. Sung, S. C., Sandberg, P. R. & McGeer, E. G. (1978) Neurochem. Res. 3,
40. Moret, C., Pastrie, I. & Briley, M. (1990) Neurobiol. Aging 11, 57–59.
41. Kalaria, R. N. & Harik, S. I. (1992) Ann. Neurol. 32, 583–586.
42. Makar, T. K., Cooper, A. J., Tofel-Grehl, B., Thaler, H. T. & Blass, J. P. (1995)
Neurochem. Res. 20, 705–711.
43. Maurer, I., Zierz, S. & Moller, H. J. (1998) Alzheimer. Dis. Assoc. Disord. 12,
44. DiDonato, S., Rimoldi, M., Moise, A., Bertagnoglio, B. & Uziel, G. (1979)
Neurology 29, 1578–1583.
45. Melegh, B., Seress, L., Bedekovics, T., Kispal, G., Sumegi, B., Trombitas, K.
& Mehes, K. (1999) J. Inherit. Metab. Dis. 22, 827–838.
46. Brevetti, G., Angelini, C., Rosa, M., Carrozzo, R., Perna, S., Corsi, M.,
Matarazzo, A. & Marcialis, A. (1991) Circulation 84, 1490–1495.
47. Fritz, I. B. & Schultz, S. K. (1965) J. Biol. Chem. 240, 2188–2192.
48. Chase, J. F. A. (1969) Methods Enzymol. 13, 387–393.
49. Chase, J. F. A. & Tubbs, P. K. (1966) Biochem. J. 99, 32–40.
50. Cornish-Bowden, A. & Wharton, C. W. (1988) Enzyme Kinetics (IRL, Oxford).
51. Liu, J., Yeo, H. C., Doniger, S. J. & Ames, B. N. (1997) Anal. Biochem. 245,
52. Yeo, H. C., Liu, J., Helbock, H. J. & Ames, B. N. (1999) Methods Enzymol. 300,
53. Humphries, K. M., Yoo, Y. & Szweda, L. I. (1998) Biochemistry 37, 552–557.
54. Killilea, D. W., Armstrong, G. & Ames, B. N. (2001) Free Radical Biol. Med 31,
55. Verbanac, D., Milin, C., Domitrovic, R., Giacometti, J., Pantovic, R. & Ciganj,
Z. (1997) Biol. Trace Elem. Res. 57, 91–96.
56. Alhomida, A. S. (1996) Biochem Mol. Biol. Int. 39, 923–931.
57. Esterbauer, H., Schaur, R. J. & Zollner, H. (1991) Free Radical Biol. Med. 11,
58. Humphries, K. M. & Szweda, L. I. (1998) Biochemistry 37, 15835–15841.
59. Yeo, H. C., Helbock, H. J., Chyu, D. W. & Ames, B. N. (1994) Anal. Biochem.
60. Terman, A. (2001) Redox. Rep. 6, 15–26.
61. Ding, Q. & Keller, J. N. (2001) Free Radical Biol. Med. 31, 574–584.
62. Fucci, L., Oliver, C. N., Coon, M. J. & Stadtman, E. R. (1983) Proc. Natl. Acad.
Sci. USA 80, 1521–1525.
63. Marnett, L. J. & Tuttle, M. A. (1980) Cancer Res. 40, 276–282.
64. Golding, B. T., Patel, N. & Watson, W. P. (1989) J. Chem. Soc. Perkin Trans.
65. Cook, C. I. & Yu, B. P. (1998) Mech. Ageing Dev. 102, 1–13.
66. Ou, P., Tritschler, H. J. & Wolff, S. P. (1995) Biochem. Pharmacol. 50, 123–126.
67. Persson, H. L., Svensson, A. I. & Brunk, U. T. (2001) Redox Report 6,
68. Paradies, G., Ruggiero, F. M., Gadaleta, M. N. & Quagliariello, E. (1992)
Biochim. Biophys. Acta 1103, 324–326.
Liu et al. PNAS ?
February 19, 2002 ?
vol. 99 ?
no. 4 ?