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-lipoic acid
1
* Linus Pauling Institute and
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA; and
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
1Correspondence: Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331, USA. E-mail: tory.hagen{at}orst.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol. A significant age-related increase (P =
0.05) in steady-state levels of oxidative DNA damage was observed, as
monitored by 8-oxo-2'-deoxyguanosine levels. To investigate whether
dietary supplementation with (R)--lipoic acid (LA)
was effective at reducing oxidative stress, young and old rats were fed
an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death.
Cardiac myocytes from old, LA-supplemented rats exhibited a markedly
lower rate of oxidant production that was no longer significantly
different from that in cells from unsupplemented, young rats. Lipoic
acid supplementation also restored myocardial ascorbic acid levels and
reduced oxidative DNA damage. Our data indicate that the aging rat
heart is under increased mitochondrial-induced oxidative stress, which
is significantly attenuated by lipoic acid supplementation.—Suh,
J. H., Shigeno, E. T., Morrow, J. D., Cox, B., Rocha,
A. E., Frei, B., Hagen, T. M. Oxidative stress in the aging
rat heart is reversed by dietary supplementation with
(R)--lipoic acid.
Key Words: aging • cardiac myocytes • oxidative stress • lipoic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the U.S. Because of the enormous
suffering and health care burden that cardiac dysfunction causes, much
effort has gone into understanding the mechanisms leading to
age-related myocardial decline. It has been difficult, however, to
separate the effects of aging per se from those of
age-associated diseases (atherosclerosis, diabetes, hypertension) on
cardiac performance. Thus, the relative contribution of ‘aging’ to
myocardial dysfunction is not well defined.
Even though the mechanisms leading to alterations in cardiac
performance are not well understood, there is reason to suspect
increased oxidative stress to significantly contribute to myocardial
dysfunction with age. It is generally agreed that isolated
mitochondrial preparations from old compared to young hearts produce
more reactive oxygen species (ROS), reflecting an age-related decline
in coupling of electron transport to ATP production. These changes in
mitochondria may lead to the reported increase in superoxide and
hydrogen peroxide production in mitochondria prepared from old vs.
young rats (2
3
4)
. Thus, it is conceivable that dietary
interventions with antioxidants, which could augment endogenous
antioxidant compounds to either prevent the formation or quench the
higher levels of oxidants, could provide an effective means to improve
or maintain myocardial function with age.
(R)--lipoic acid (LA) is a thiol compound found naturally
in plants and animals (4)
. Lipoamide dehydrogenases, found
only in mitochondria, reduce free LA to dihydrolipoic acid, which is a
potent antioxidant. Thus, LA supplementation may increase cellular and
mitochondrial antioxidant status, thereby effectively attenuating any
putative increase in oxidative stress with age (5)
.
Aside from acting as a potent antioxidant in its own right, LA
increases or maintains levels of other low molecular weight
antioxidants such as ubiquinone, glutathione (GSH), and ascorbic acid.
LA may exert these effects by ‘sparing’ or reducing, ubiquinone
(6)
, GSH, and vitamin C (7)
or, in the case
of GSH, by increasing the cellular uptake of cysteine (8)
,
which is the rate-limiting substrate for GSH biosynthesis.
The purpose of the present study was to 1) examine the age-related changes to myocardial oxidant production, low molecular weight antioxidant status, and indices of oxidative damage, and 2) determine whether dietary supplementation of lipoic acid could improve those indices of oxidative stress. Overall, our results show that the aging rat myocardium exhibits increased oxidant production, significantly lower ascorbic acid levels, and a marked increase in steady-state levels of oxidative DNA damage. LA supplementation significantly reverses the age-related decline in myocardial ascorbic acid content, and lowers the rate of oxidant production and the steady-state levels of oxidative DNA damage. Our results thus indicate that dietary supplementation with lipoic acid may be an effective means to lower increased myocardial oxidative stress with age.
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MATERIALS AND METHODS |
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-aminoethyl ether) N,N,N',N'-tetraacetic acid), heparin (sodium
salt), rhodamine 123, dithiothreitol, L-ascorbic acid, meta-phosphoric
acid, and 8-oxo-dG standard (Sigma, St. Louis, Mo.);
2',7'-dichlorofluoroscein diacetate (DCFH) (Molecular Probes, Eugene,
Oreg.); collagenase, type 2 (Worthington, Lakewood, N.J.); nuclease P1,
alkaline phosphatase, sodium iodide, proteinase K, and RNAse A (Roche
Pharmaceuticals, Indianapolis, Ind.). All other reagents were of
reagent grade or better.
Animals
Rats (Fischer 344, virgin male, outbred albino), both young
(2–5 months) and old (24–28 months; National Institute of Aging
animal colonies), were acclimatized in the Oregon State University
animal facilities for at least 1 wk prior to experimentation. Animals
were placed on an AIN-93M standard diet; some rats were given an
AIN-93M diet supplemented with 0.2% (w/w) (R)--lipoic
acid (Asta Medica, Germany) for 2 wk prior to death. Water was given
ad libitum throughout.
In experiments examining tissue antioxidant and oxidative DNA damage levels, rats were anesthetized with diethyl ether and a midline incision was made in the abdomen. Animals were killed by cutting through the diaphragm, followed by severing of the superior vena cava. Hearts were quickly removed and cut into small pieces; the pieces were placed individually in cryotubes and snap frozen in liquid nitrogen.
Cardiac myocyte isolation
For analysis of cellular oxygen consumption, average
mitochondrial membrane potential, and oxidant production, the heart was
dispersed into single cells by perfusion with 1% collagenase
(9)
. Typically, the isolation procedure yielded
5.0–7.5 x 106 calcium-tolerant ventricular
cells per heart, which exhibited typical rod-shaped appearance and
morphological striations. Cell viability, as measured by trypan blue
exclusion, was typically between 60 and 80%. These values agree with
those cited in the literature (10)
.
It was necessary to modify the above procedure in order to isolate cells from old rats. Old rats had to be injected with twice the amount of heparin to remove all blood from the heart. The amount of collagenase perfused through the heart was also increased to 1.2% (w/v) and the perfusion flow rate was raised from 2.8 to 7.0 ml/min. These changes were necessary because of the increased fibrotic nature of the heart in old vs. young rats. Even with these modifications, cell yield and viability were more variable than that for young rats: 3.0–7.0 x 106 cells isolated per heart from old rats, with viability ranging from 45 to 80%. Because of the possibility that low cell viability and yield would result in data that do not accurately represent the in vivo situation, no isolated cell preparations were used that had an initial viability below 70% and a yield of <5.0 x 106 cells per heart.
DCFH measurement
Formation of oxidants in isolated cardiac myocytes was
determined by assaying the fluorescence of 2',7'-dichlorofluorescein
(DCF), the oxidation product of DCFH. Duplicate samples were routinely
monitored. Fluorescence was measured with a Hitachi F-2500 fluorescent
spectrophotometer (Hitachi Instruments, Tokyo, Japan) using standard
fluorescein filters and Hitachi-supplied software. To determine whether
age-related changes to cellular oxygen consumption affected apparent
changes in oxidant production, we measured cellular oxygen consumption
and expressed data as the fluorescence change/mol O2
consumed/106 cells.
Ascorbic acid analysis
Ascorbate was measured essentially as in ref 11
.
Briefly, tissue homogenates were mixed with an equal volume of
meta-phosphoric acid (10% w/v) containing 1 mmol/l of the
metal chelator diethylenetriaminepentaacetic acid (DTPA) and
centrifuged to remove the precipitated proteins. Ascorbic acid was
separated by high-performance liquid chromatography (HPLC)
(11)
and detected at an applied potential of +0.6 V by a
LC 4B (Bioanalytical Systems, Inc., West Lafayette, Ind.)
electrochemical detector.
Vitamin E analysis
Myocardial vitamin E levels were determined as in ref
12
. Briefly, myocardial tissue (50 mg) was homogenized in
10 mM phosphate-buffered saline containing 1 mmol DTPA. 50 µl of
the homogenate was extracted in hexane:methanol (5:1), and the hexane
phase was collected and dried under a constant stream of nitrogen. The
sample was reconstituted with methanol and analyzed by HPLC with
electrochemical detection at an applied potential of +0.5 V by a LC 4B
electrochemical detector (12)
.
Oxidative DNA damage
Analysis of oxidative damage to nuclear DNA was measured by the
method of Helbock et al. (13)
. DNA was extracted using the
chaotropic sodium iodide method by a DNA Extractor WB kit (Wako
BioProducts, Richmond, Va.). DNA hydrolysates were analyzed by HPLC
with electrochemical coulometric detection, as described
(13)
.
Oxidative lipid damage
Analysis of oxidative damage to lipids was quantified by
measuring F2- isoprostanes in lipids using a
highly precise and accurate assay employing gas chromatography/mass
spectrometry (14)
.
Statistical analysis
Statistical significance was determined by the unpaired
Student’s t test, using Stat-View statistical software.
Results are expressed as the mean ± SE. A
P value of less than 0.05 was considered significant.
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RESULTS |
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). These results suggest an age-associated decrease in the basal
metabolic rate of the cardiac myocytes.
|
To examine how the decline in mitochondrial function affected cellular
oxidant production, the rates of DCFH oxidation in freshly isolated
cardiac myocytes from old and young rats were determined. In cardiac
myocytes isolated from old rats, total cellular oxidant production was
125% higher than in myocytes from young rats (Fig. 2
). This increase in oxidant production was even more pronounced when
normalized to the rates of oxygen consumption. Thus, the rate of DCFH
fluorescence per mmol O2 consumed was threefold higher in
cardiac myocytes from old rats compared to young rats. These results
strongly suggest that, with age, cardiac myocytes are under increased
oxidative stress, possibly due to a decline in mitochondrial function.
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To investigate the effects of this age-associated increase in oxidative
stress on cellular antioxidant capacities, ascorbate and -tocopherol
levels in the freshly isolated hearts from old and young rats were
measured. Consistent with the data on oxidant production (Fig. 2)
, we
observed a 57% decrease in the tissue ascorbate concentration of old
rats (1.40±0.87 nmol/mg protein) compared to young rats (3.08±1.10
pmol/mg tissue (P=0.02; Table 1
). In contrast, no age-related decrease in vitamin E levels was observed
(0.97±0.29 and 1.16±0.62 nmol/mg protein in hearts from young and
old). This result suggests that there is a differential consumption of
water-soluble vs. lipid-soluble antioxidants in vivo due to
aging.
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To further explore this notion of different levels of oxidative stress
with aging in the aqueous and lipid compartments, we examined oxidative
damage to DNA, as assessed by the levels of 8-oxo-dG, and lipids, as
assessed by levels of F2-isoprostanes. Consistent with the
pattern of antioxidant depletion with age, we found a twofold increase
in oxidative DNA damage in the heart of old rats
(4.00±4.80-oxo-dG/105 dG bases) compared to young rats
(2.01±0.25 8-oxo-dG/105 dG bases
(P=0.002; Fig. 4
). In contrast, the levels of
F2-isoprostanes in hearts from old rats (3.74±0.41 ng/g
wet tissue) were not significantly different from those in young rats
(3.47±0.02 ng/g wet tissue). These results suggest that in the heart,
macromolecules in an aqueous environment may be more susceptible to
damage due to age-associated increase in oxidative stress than membrane
lipids.
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In light of the above evidence of increased oxidative stress in the
aging heart, we examined whether supplementation with
(R)--lipoic acid can reverse the decline in mitochondrial
function and reduce oxidative stress. To this end, Fischer 344 rats of
varying ages (young: 2–4 months; old: 24–28 months) were fed an
AIN-93M diet with or without 0.2% (w/w) (R)--lipoic acid
for 2 wk. Results show a significant decrease in the cellular oxidant
production in the old LA-supplemented vs. old unsupplemented animals
(Fig. 3
). In agreement with these data, we also found a significant twofold
improvement in ascorbate levels in the hearts from old LA-treated vs.
untreated animals (2.90±0.36 vs. 1.4±2.9 nmol/mg protein,
respectively; P=0.03). In fact, cardiac ascorbate levels in
LA-fed old rats were not different from those in young rats (2.90±0.72
nmol/mg protein; Fig. 4
). However, a similar increase in tissue ascorbate levels was not
achieved by LA feeding to young rats. Furthermore, there was no effect
of LA treatment on the levels of -tocopherol.
|
To investigate whether the decreased ROS production and increased
ascorbate levels in LA-treated old rats translated into a decrease in
oxidative damage to DNA and lipids, 8-oxo-dG and
F2-isoprostane levels were measured. Indeed, we found a
30% decrease in cardiac 8-oxo-dG levels in the supplemented old rats
(2.84±0.39 8-oxo-dG/105 dG bases) compared to
their age-matched controls (4.00±0.40
8-oxo-dG/105 dG bases (P=0.05;
Fig. 5
). In the young animals, LA supplementation did not alter the
steady-state levels of 8-oxo-dG in the heart. As expected, treatment
with LA had no effect on the steady-state levels of
F2-isoprostanes in young or old rats. Thus, by decreasing
the rate of oxidant production and increasing the level of antioxidant
protection in the aqueous phase, LA treatment caused a marked reversal
in steady-state levels of 8-oxo-dG in old rats to levels found in the
young rats.
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DISCUSSION |
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. However, a recent study
by Hansford and co-workers suggested that this increase may be
attributed to an artifact stemming from assay conditions due to
excessively high levels of substrate (succinate) used
(15)
. In addition, Hagen and co-workers have found that
selective loss of defective mitochondria during the isolation process
can further complicate the interpretation of results from studies using
isolated mitochondria (16)
. However, we have avoided these
potential problems here by examining the age-associated changes in
metabolic function and antioxidant status in intact cardiac myocytes
isolated from young and old animals. In the present study, we observed an apparent increase in DCF fluorescence in myocytes from old when compared to young animals. This increase in oxidant levels may be attributed to at least three possibilities: 1) increased oxidant production mainly from decaying mitochondria, 2) decline in cellular antioxidant status with no increase in oxidant flux, and 3) both an increase in oxidant production and a decline in antioxidant status. The present work cannot distinguish between these possibilities. However, it is notable that we observed a significant decline in myocardial ascorbate levels and oxygen consumption with age. Lower oxygen consumption would seemingly be a compensatory mechanism to also lower mitochondrial oxidant flux. Along with loss of ascorbate, our results would suggest that the decline in antioxidant status may be a significant contributing factor in the apparent age-related increase in myocardial oxidative stress.
In contrast to the age-related decrease in the levels of ascorbate and
an increase in the levels of 8-oxo-dG, the levels of -tocopherol and
F2-isoprostanes did not change with age. Because these
moieties are located primarily in lipophilic environments, these data
suggest that the effects of endogenous oxidants may be confined to the
aqueous milieu of the heart. In support of this notion, we had
previously found that F2-isoprostane formation is reduced
by -tocopherol (17)
. Ascorbate however, does not affect
the formation of F2-isoprostanes, at least in
vitro in microsomes (18)
.
One factor contributing to such compartmentalization may be due to the
presence of redox-active transition metal ions, which can catalyze the
conversion of hydrogen peroxide and superoxide into the more highly
reactive hydroxyl radical via Fenton chemistry. Previous in
vitro experiments by Lodge and co-workers show that dihydrolipoic
acid can inhibit copper-induced LDL oxidation by direct chelation of
free copper ions (19)
. In addition, LA appears to inhibit
iron-induced ascorbate oxidation, possibly by reducing redox active
iron (J. H. Suh et al., unpublished observation). We are currently
examining the nature of the interaction between transition metals and
LA. Thus, in addition to its antioxidant effect, dihydrolipoic acid may
protect against lipid peroxidation by chelating free metal ions
in vivo.
Ascorbate may be consumed more rapidly than -tocopherol in
vivo. First, the reduction potential of ascorbate (280 mV) is much
lower than that of -tocopherol (500 mV) (21)
. Thus,
ascorbate is a more effective antioxidant capable of inhibiting lipid
peroxidation against a number of different oxidant species
(11)
. Its low reduction potential also allows it to
regenerate -tocopherol whereas the reverse is not possible
(20)
. Second, the concentration of ascorbate is two- to
threefold higher than -tocopherol; thus, ascorbate may spare
-tocopherol from oxidation.
In contrast to a previous study showing an increase in urinary
F2-isoprostanes with age, our results show that myocardial
levels of F2-isoprostanes did not increase
(22)
. Myocardial F2-isoprostanes may not
accumulate due to rapid repair and/or clearance, whereas plasma or
urinary F2-isoprostanes would be reflective of released
F2-isoprostanes from all tissues and renal clearance.
The beneficial effects of dietary supplementation of
(R)--lipoic acid toward ameliorating the age-related
changes in cardiac myocytes may be attributed to its ability to act as
an antioxidant as well as its role as in modulating metabolism. Along
with the reduction in oxidant production, (R)--lipoic
acid supplementation restores myocardial ascorbate levels from old
rats. Previous work by Lykkesfeldt and co-workers have reported that
there is no change in hepatic ascorbate synthesis with age
(23)
, which suggests that (R)--lipoic acid
may restore myocardial ascorbate levels by stimulating hexose
transporter activity and/or by direct regeneration of dehydroascorbate.
Furthermore, LA can indirectly increase GSH levels by increasing
cysteine uptake, which is a rate-limiting step for GSH biosynthesis.
This increase in thiol antioxidant levels can in turn enhance the rate
of ascorbate recycling.
Dietary supplementation of LA also leads to significantly lower
steady-state levels of 8-oxo-dG in hearts from old animals. This
reduction suggests that the age-related accumulation of 8-oxo-dG
in vivo may be due to increased oxidative insult rather than
decreased repair capacity. In support of this, a recent study by
Souza-Pinto and co-workers has reported that there is an age-dependent
increase in 8-oxo-dG glycosylate/AP lyase activity in rat mitochondria
with age (24)
.
The causes for this age-related decline in myocardial mitochondrial
function are not completely understood. One possibility is that with
age there may be a loss of essential cofactors such as LA, which may
limit optimal mitochondrial performance. Supplementation with LA may
replenish needed cofactors for -keto acid dehydrogenases (pyruvate
oxidoreductase and 2-oxo-glutarate oxidoreductase) used in pyruvate and
fatty acid metabolism. It has been shown that the proportion of the
active form of pyruvate oxidoreductase declines with age
(25)
, possibly due to modification of the lipoamide moiety
of the E2 subunit (26)
. Thus, feeding old rats LA may
reverse the age-associated decline in LA-dependent oxidoreductase
activity. This may also explain why we only observed a beneficial
effect of LA only in old and not in young animals.
Other necessary cofactors necessary for mitochondrial function, such as
carnitine and cardiolipin, also decline with age (27
, 28)
.
Carnitine loss, for example, can limit the transport of fatty acids
into mitochondria for ß-oxidation, which is the major source for ATP
synthesis. In addition, decline in cardiolipin has been shown to
decrease substrate transport in isolated mitochondrial preparations and
lower cytochrome c oxidase activity.
One possible physiological consequence of these decreased cofactors
would be loss of ATP production, which may lead to cardiac stiffness.
To maintain myocardial function, a constant supply of ATP is required
and small reserves are maintained. This suggests that when energy
supply is interrupted (ischemia) or impaired (aging), ATP levels
decline rapidly. Like systolic contraction, diastolic relaxation also
requires high levels of ATP, because ATP acts as an allosteric effector
to disassociate actin from myosin (29)
. Thus, any
decrement in mitochondrial ATP synthesis affects cardiac stiffness
appreciably. A decline in ATP synthesis also compromises
Ca2+ reuptake into the sarcoplasmic reticulum
from the cytosol, again affecting myocardial relaxation (30
, 31)
. The Na+/Ca2+
transporter is also energy dependent, and a decline in myocardial ATP
levels would thus slow cardiac relaxation by decreasing the rate of
Ca2+ removal from the cytosol (30
, 31)
. It is notable that a general attribute of myocardial aging
is a prolonged cytosolic calcium transient and slower myocardial
relaxation rate (32
, 33)
.
The exact physiological consequences associated with these cellular changes remains to be elucidated. Although these changes may not affect the heart’s function under normal conditions, it is possible that a loss in bioenergetic capacity along with antioxidant protection may severely limit the hearts ability to respond to physical stress. More studies are needed to carefully correlate the limitation brought about by age-associated changes in the mitochondrial function.
In conclusion, our present findings suggest that
(R)--lipoic acid supplementation may be a safe and
effective means of improving systemic decline in over all metabolic
function and also increase protection against both endogenous and
external production of ROS. However, long-term feeding studies with LA
are needed to determine whether benefits of LA seen in old animals can
be sustained over time.
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ACKNOWLEDGMENTS |
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Received for publication April 7, 2000.
Revision received August 11, 2000.
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45% more
oxidants, as measured by DCFH fluorescence, than cells from young (3
months) rats. B) The rate of oxidant production is even
more marked when oxidant production is normalized to the cellular rate
of O2 consumption. Results are expressed as the mean ± SE of 3 experiments.
