Article

Liu, J., Killilea, D. & Ames, B. N. 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-alpha-lipoic acid. Proc. Natl Acad. Sci. USA 99, 1876-1881

Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2002; 99(4):1876-81. DOI: 10.1073/pnas.261709098
Source: PubMed

ABSTRACT

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due 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-alpha-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 increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with 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 decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.

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Available from: David W Killilea, Jan 18, 2015
Age-associated mitochondrial oxidative decay:
Improvement of carnitine acetyltransferase
substrate-binding affinity and activity in
brain by feeding old rats acetyl-
L-
carnitine andor
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-
ferase (CAT), a key mitochondrial enzyme for fuel utilization, is due
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) andor 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
increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited
lipid peroxidation but did not decrease iron and copper levels. Ex
vivo oxidation of young-rat brain with Fe(II) caused loss of CAT
activity and binding affinity. In vitro oxidation of purified CAT with
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
mitochondrial dysfunction.
A
ging appears to be due, in part, to damage caused by the
oxidants produced by mitochondria as by-products of nor-
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 K
m
for 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 V
max
of
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 K
m
(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 K
m
) 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
during aging, mitochondrial oxidants deform proteins because of
direct oxidation, changes in membranes, and adduction of aldehyde
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
that can recycle other antioxidants, such as vitamins C and E, and
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 2.3.1.7) 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-
drial matrix, and it appears to be present in all mammalian tissues
(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@
uclink4.berkeley.edu.
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.
1876–1881
PNAS
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al. (40) did not find altered CAT activity in the brain of
LongEvans rats with age in healthy animals over a moderate
age range. CAT activity was found to decrease in Alzheimers
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 K
m
) 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 K
m
of CAT.
The present study was designed to test the K
m
hypothesis by
assaying the kinetics of CAT in the brains of young and old rats,
and old rats fed ALCAR andor 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 (FrankfurtMain, Germany). All other reagents were
reagent grade or the highest quality available from Sigma, unless
otherwise indicated.
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
ones were 24.5 months at the start of experiment; they were more
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 K
m
in 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
MgCl
2
, 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 proteinml brain homogenate super-
natant, 50 mM Tris, 2 mM EDTA, 25 mM malate, 0.25 mM
NAD, 12.5
gml of rotenone, 12.5
gml of malate dehydro-
genase, 50
gml 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
mgml of CoA (K
m
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 (K
m
for CoA). The results were
plotted with the double-reciprocal plot of reciprocal rate 1v
against reciprocal substrate concentration 1S. Results were also
calculated by the direct linear plot with the equation of V
max
v (vS)K
m
(50).
Ex Vivo
Oxidation of Rat-Brain Homogenate. The young-rat-brain
homogenate was incubated with FeSO
4
for 15 min at 37°C. The
kinetics were assayed as described above, and the oxidation was
assayed by measuring malondialdehyde (MDA) with a gas
chromatographymass 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
gml) was incubated with PBS and various concentrations of
FeSO
4
with or without metal chelatorsantioxidants at room
temperature for 15 min. CAT activity was assayed immediately
by using the method described above (CAT at 0.036
gml of
protein).
MDA- and 4-Hydroxynonenal (HNE)-Induced Inactivation and
K
m
Change of Purified CAT
in Vitro
. MDA was prepared by derivatization
of 1,1,3,3-tetramethoxypropane with 0.01 M HCl and stored 36
weeks at 4°C. The concentration of MDA was determined at 245
nm by using an extinction coefficient of 13,700M (52). HNE was
obtained from Calbiochem, and its concentration was determined
at 224 nm by using an extinction coefficient of 13,750M (53).
Similar to the above incubation, CAT (0.36
gml) was incubated
in PBS or Tris-buffered saline (TBS) with MDA or HNE at room
temperature. The CAT kinetics were assayed immediately as above
(CAT at 0.036
gml 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 chromatographymass spectrometric method to measure
the level of MDA (51, 52).
Results
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 V
max
and apparent K
m
are shown
in Fig. 1 C and D. Compared with young rats, old rats showed a
moderate decrease in enzyme activity (V
max
) (14%, Fig. 1C) and
an increase in K
m
[160% of K
m
for ALCAR and 180% of K
m
for
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 (K
m
for ALCAR, P 0.019; K
m
for 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) (110 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 FeSO
4
, 134 1.6 pmolmg of protein). As expected,
metal chelators such as EDTA at 1 mM (MDA 48.6 0.6
pmolmg) 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) (0.11 mM) induced a concentration-dependent
inactivation of CAT with a 50% inactivation at a concentration
of 95
Mat37°C for 15 min (0.5 unitsml). 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). FeSO
4
at 200
M concentration caused 80%
inactivation of CAT. As expected, metal chelators EDTA and
Liu et al. PNAS
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deferoxamine, at 1 mM concentration, protected the enzyme
from inactivation by 70 and 94% respectively, as did the sulfhy-
dryl antioxidants (all at 1 mM), reduced glutathione (23%), DTT
(32%), and dihydrolipoic acid (35%), although they were less
effective. Catalase (1 mgml) 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,
50%.
In Vivo
Lipid Peroxidation Levels in Rat Brain. Compared with young
rats, old rats showed a significant increase in brain MDA, a major
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
K
m
of Purified CAT. Fig. 4 A and B show
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 K
m
when 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 K
m
for 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
K
m
for ALCAR to 135, 164, and 269%). The concentration
Fig. 1. Double-reciprocal plots of reaction velocity versus substrate ALCAR (A) or CoA (B) concentrations in rat brain. (C) V
max
;(D) apparent K
m
for ALCAR and
CoA. All values are mean SE of 10 animals for young and old groups, 5 for the LA group, and 6 for the ALCAR and ALCAR plus LA groups. Significant difference
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).
Fig. 2. The kinetic parameters (V
max
and K
m
for ALCAR) of CAT for young-rat
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).
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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 K
m
(Fig. 4C).
Total Metal Content in Rat Brain. Compared with young rats, old
rats had a significant increase in total iron (young, 66.4 1.8, and
old, 81.6 2.2 ngmg of dry tissue; P 0.001) and copper
(young, 10.3 0.2, and old, 17.4 0.6 ngmg 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 andor
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 ngmg of dry tissue) or total copper
(data not shown).
Discussion
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
combination of ALCAR and LA significantly restored both CAT
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 CoAacetyl-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
by oxidants or by metal-catalyzed oxidation. We propose that the
adduction mechanism is more likely. In vivo, brain MDA, derived
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 V
max
and 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 K
m
and 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 pmolmg 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 1010,000
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 K
m
. 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 V
max
and 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 K
m
, 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 LAs
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
protein modification (61). Inactivation of key metabolic enzymes
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-
dative modification.
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,
Fig. 3. MDA levels in the rat brain measured with a gas chromatography
mass spectrometric assay. The values are mean of seven animals for the young
and old groups, three for the LA group, and ve for the ALCAR and ALCAR plus
LA groups. Signicant difference was calculated by using Students t test
between young and old groups (
**
, P 0.01), and by using one-way ANOVA
with Dunnetts multiple comparison test between old and other treated
groups (#, P 0.05).
Liu et al. PNAS
February 19, 2002
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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
residues and can then form more stable intra- and intermolecular
crosslinks (57). The effect of MDA on the activity and K
m
of
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 K
m
of
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)
in parallel to the increase in iron and copper (see Results). Ex vivo
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
Fig. 4. Concentration-dependent effects of MDA (A) and HNE (B) in PBS on reciprocal plots of CAT for substrate ALCAR. Different concentrations of MDA or
HNE were incubated with 0.36
gml of CAT enzyme for1hatroom temperature in PBS. The kinetics were assayed with an assay mixture containing 0.036
gml
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.
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alter the binding affinity. This increase of iron is consistent with
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 andor LA did not signifi-
cantly effect transition metal accumulation in these short-term
studies, the possibility of chelating the labile or ‘‘free’’ transition
metals in the brain and consequently inhibiting oxidative damage
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 andor
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 HealthNational Institute on
Aging postdoctoral training grant (5 T32 AG0026602) to D.W.K.
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1881
BIOCHEMISTRY
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    • "For example, mitochondrial biogenesis increases in muscle cells upon exercise (Holloszy, 1967). Conversely, research has shown that as mammals age, there is a general decline in both mitochondrial mass and function (Yan and Sohal, 1998; Liu et al., 2002; Chistiakov et al., 2014). There is a wide range of clinical conditions that result from mitochondrial dysfunction, including muscular disorders, cardiomyopathy, diabetes, cancer, deafness, lactic acidosis, and skeletal myopathy (Vafai and Mootha, 2012). "
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    • "Damage to mitochondria is primarily caused by ROS generated by the mitochondria themselves [11] [12], in particular by complexes I and III of the electron respiratory chain [13]. Direct damage to mitochondrial proteins decreases their affinity for substrates or coenzymes and, thereby, decreases their function [14]. ROS represented the mechanism of mitochondrial dysfunction during inflammation . "
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    • "Given that mitochondria-centered dysfunction was an essential feature of unloading, we developed a nutrient formula designed to synergistically promote mitochondrial biogenesis, improve mitochondrial dysfunction, and balance oxidative stress using compounds whose mitochondrial beneficial effects have been well documented by our laboratory and other laboratories. For example, LA plus ALCAR, cofactors located within mitochondria, have been shown to improve mitochondrial metabolism and integrity by both enhancing acetyltransferase substrate-binding affinity and activity and ameliorating oxidative stress [40] [42]. HT, a natural polyphenol compound from olive oil, activates PGC-1α-mediated mitochondrial biogenesis in retinal pigment epithelial cells, adipocytes, and muscles [24] [25] [43]; CoQ 10 , a mitochondrial electron transporter, promotes mitochondrial function when applied to Goto–Kakizaki rats [26]. "
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