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Lipoic Acid Inhibits Oxidative Stress
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Journal of Exercise Physiologyonline
(JEPonline)
Volume 11 Number 1 February 2008
Managing Editor
Tommy Boone, Ph.D.
Editor-in-Chief
Jon Linderman, Ph.D.
Review Board
Todd Astorino, Ph.D.
Julien Baker, Ph.D.
Tommy Boone, Ph.D.
Lance Dalleck, Ph.D.
Dan Drury, DPE.
Hermann Engels, Ph.D.
Eric Goulet, Ph.D..
Robert Gotshall, Ph.D.
Len Kravitz, Ph.D.
James Laskin, Ph.D.
Melissa Knight-Maloney,
Ph.D.
Derek Marks, Ph.D.
Cristine Mermier, Ph.D.
Daryl Parker, Ph.D.
Robert Robergs, Ph.D.
Brent Ruby, Ph.D.
Jason Siegler, Ph.D.
Greg Tardie, Ph.D.
Lesley White, Ph.D.
Chantal Vella, Ph.D.
Thomas Walker, Ph.D.
Ben Zhou, Ph.D.
Official Research Journal
of The American Society of
Exercise Physiologists
(ASEP)
ISSN 1097-9751
Nutrition and Exercise
DIETARY SUPPLEMENTATION WITH LIPOIC ACID
INHIBITS EXERCISE-INDUCED OXIDATIVE STRESS
ANGELA PENDLETON1, SUNAM GURUNG1, SHAWN STOVER1 .
1Department
of Biology and Environmental Science, Davis & Elkins
College, Elkins, West Virginia, USA
ABSTRACT
Pendleton A, Gurung S, Stover S. Dietary Supplementation With
Lipoic Acid Inhibits Exercise-Induced Oxidative Stress. JEPonline
2008;11(1):53-59. Acute anaerobic exercise can promote oxidation of
lipids. It was hypothesized that exercise-induced lipid peroxidation
would be significantly reduced in fast-twitch skeletal muscle by 12 weeks
of dietary supplementation with lipoic acid, and that lipid peroxidation
would be reduced even further by combining 12 weeks of
supplementation with 12 weeks of high intensity sprint training. Twentyfour mice were randomly divided into three groups: control, lipoic acid
supplemented (LA), and supplemented/sprint trained (LA+Ex). LA and
LA+Ex mice were fed lipoic acid (150 mg/kg body weight) two days a
week for 12 weeks. LA+Ex mice participated in a high intensity training
program consisting of treadmill running two days a week for 12 weeks.
After the diet and exercise training regimen, experimental and control
mice engaged in six consecutive 30 s sprints on a rodent treadmill at a
pace of 30 m/min, with a 1 min recovery interval between each sprint.
Concentrations of malondialdehyde (MDA), a product of lipid
peroxidation, were determined spectrophotometrically in fast- and slowtwitch muscles. In mice receiving 12 weeks of dietary supplementation
with lipoic acid, MDA concentration was unchanged in the slow-twitch
soleus. However, MDA concentration was dramatically reduced in the
fast-twitch extensor digitorum longus (EDL), relative to control, indicating
a significant decrease in lipid peroxidation. Mice that had been both
sprint trained and supplemented for 12 weeks also expressed a
significant decrease in EDL MDA concentration, relative to control. The
effects of the two treatments do not appear to be additive, as there was
no significant difference found between LA and LA+Ex groups.
Key Words: Antioxidant, Peroxidation, Malondialdehyde
Lipoic Acid Inhibits Oxidative Stress
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INTRODUCTION
The generation of reactive oxygen species (ROS) such as singlet oxygen, superoxide radical, and
hydroxyl radical occurs as a consequence of normal cellular metabolism (1). ROS-related molecular
damage includes DNA strand breaks and single base modifications (2), oxidation of amino acid side
chains and fragmentation of polypeptides (3), and the degradation of polyunsaturated fatty acids and
phospholipids by lipid peroxidation (4). Processing of ROS is carried out by the body’s endogenous
antioxidant defense system, which includes enzymatic activity of superoxide dismutase (SOD),
glutathione peroxidase (GPx), and glutathione reductase (GR), in conjunction with exogenous
antioxidants consumed through diet (1). Oxidative stress may be defined as a condition in which the
cellular production of ROS exceeds the body’s physiological capacity to render it inactive (4).
The increase in oxygen uptake during aerobic exercise is accompanied by an elevation in ROS.
Acute aerobic exercise generates ROS by creating a disturbance in electron transport that leads to
excessive leakage of superoxide radicals (4). However, long-term endurance training effectively
reduces the damage associated with increased oxygen uptake by enhancing the body’s antioxidant
defenses. It has been demonstrated that GPx (5), GR (5), and SOD (6) activities increase in
response to endurance training.
Acute anaerobic exercise can promote oxidation of lipids. In rats, a single one-minute sprint at 45
m/min elevates lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS) in skeletal
muscle, indicating significant lipid peroxidation (7). In mice, six 30 s sprints at a pace of 30 m/min
significantly increase the concentration of malondialdehyde (MDA), a lipid peroxidation marker, in
skeletal muscle (8). In humans, six 150 m sprints significantly increase plasma levels of MDA (9).
Furthermore, previous studies indicate that sprint training can attenuate the effects of oxidative
stress. In rats, activities of GPx and GR increase significantly in cardiac and skeletal muscle
following sprint training (10). In mice, sprint training reduces lipid peroxidation in skeletal muscle, as
indicated by a decrease in MDA concentration (8). In humans, sprint training produces a decrease in
plasma concentrations of MDA and protein carbonyls, both biomarkers of oxidative stress, when
compared to untrained subjects (11).
In human tissues, endogenous -lipoic acid (LA) can be found in trace amounts in -ketoacid
dehydrogenase, -ketogutarate dehydrogenase, and pyruvate dehydrogenase complexes (12).
Exogenous LA is taken up by a variety of cells and reduced by NADH- or NADPH-dependent
enzymes to dihydrolipoate, or DHLA (13). Both LA and DHLA exhibit antioxidant activity by chelating
transition metals such as iron, copper, and mercury (14). Furthermore, the reduced and oxidized
forms of lipoic acid are able to scavenge a broad array of reactive oxygen and nitrogen species,
including hydrogen peroxide, hydroxyl radical, and nitric oxide radical (15). Finally, DHLA is a strong
reducing agent and is able to regenerate several major physiological antioxidants, including vitamin
C, vitamin E, and glutathione (13).
Glutathione plays a major role in defending tissues against oxidative stress (16). The availability of
cysteine, a precursor for glutathione synthesis, is a critical determinant of cellular glutathione levels
(17). DHLA provides cysteine by reducing cystine, which is abundant in extracellular space (18).
While reducing cystine to cysteine, DHLA is oxidized to LA, which is taken up by cells and reduced
back to DHLA. Thus, DHLA, a potent reducing agent, can be continuously regenerated (17).
Several lines of evidence demonstrate the effectiveness of LA supplementation in reducing lipid
peroxidation. Manda et al. (19) reported a significant decrease in the generation of TBARS in Xirradiated mouse cerebellar tissue following treatment with LA. Baydas et al. (20) demonstrated an
Lipoic Acid Inhibits Oxidative Stress
55
LA-mediated protective effect against lipid peroxidation in the glial cells of diabetic rats. Finally, a
study by Sundaram and Panneerselvan (21) reported that administration of LA in conjunction with
carnitine, a mitochondrial metabolite, produces a significant decrease in skeletal muscle lipid
peroxidation in aged rats.
The present study investigated the hypotheses that 1) exercise-induced lipid peroxidation can be
significantly reduced in fast-twitch skeletal muscle by long-term dietary supplementation with LA, and
2) exercise-induced lipid peroxidation in fast-twitch skeletal muscle can be reduced even further by
combining long-term LA supplementation with long-term, high intensity sprint training.
METHODS
Animals
The Institutional Animal Care and Use Committee of Davis & Elkins College approved this study.
Twenty-four male albino ICR (CD-1®) mice (Harlan, Indianapolis, IN), 5-7 weeks old when the study
began, were housed individually in ventilated cages (Maxi-Miser Positive Individual Ventilation
System, Thoren Caging Systems, Inc., Hazelton, PA). The caging system was located in a room
maintained at 18-24°C with 12:12 light-dark cycles. The 24 mice were randomly divided into three
groups: control (n=8), lipoic acid supplemented (LA, n=10), and lipoic acid supplemented/sprint
trained (LA+Ex, n=6). Initially, each group had eight mice. However, two mice in the LA+Ex group
refused to run on the treadmill during the first week of training and were transferred to the LA group.
Dietary Supplementation
All mice received and consumed approximately six grams of food daily (Fortified Diet for Rats and
Mice, Kaytee Products, Inc., Chilton, WI). -lipoic acid (Sigma, St. Louis, MO) was mixed with
powdered food and fed to LA and LA+Ex mice at a dose of 150 mg/kg body weight two days a week
for 12 weeks. All mice had free access to water.
Table 1. The mouse training schedule.
Training
Number of
Speed
Incline (°)
Week
Sprints
(m/min)
Exercise Protocols
LA+Ex mice participated in a high intensity training
program consisting of treadmill running two days a week
1-3
3
24
5
4
4
24
5
for 12 weeks. Each session included three to six 30 s
sprints at a pace of 24-30 m/min on a 5-15° incline (Table
5-6
4
27
10
1), with a 1 min recovery interval between each sprint.
7-8
5
27
10
LA and control mice were not involved in the training
9
5
30
15
process. At the end of the training period, trained and
10-12
6
30
15
untrained mice engaged in six consecutive 30 s sprints
on a rodent treadmill at a pace of 30 m/min (15° incline), with a 1 min recovery interval between each
sprint. An electrified grid (0.1 mA) at the rear of treadmill was used sparingly as motivation to run. All
exercise procedures were carried out between 8:00 and 9:00 a.m.
Assessment of Lipid Peroxidation
Mice were sacrificed by cervical dislocation. Sacrifice of exercised animals was carried out
immediately after the final 1 min recovery interval. Soleus and extensor digitorum longus (EDL)
muscles were extracted from hind limbs, rinsed with cold distilled water, homogenized in 20 mM TrisHCl buffer (pH 7.4), and reacted with N-methyl-2-phenylindole (R1) at 45°C. R1 combines with MDA
to generate a stable chromophore with maximal absorbance at 586 nm (Colorimetric Assay for Lipid
Peroxidation, Oxford Biomedical Research, Oxford, MI). To account for background absorbance,
matched negative controls containing Tris-HCl buffer in place of tissue homogenate were prepared.
Experimental samples and controls were analyzed spectrophotometrically at 586 nm. MDA
Lipoic Acid Inhibits Oxidative Stress
56
concentrations were calculated from the absorbance values using the following formula: [MDA] =
([A586 – b]/a) x df, where [MDA] is the M concentration of MDA in the sample, A586 is the net
absorbance of the sample at 586 nm, a is the slope of the MDA standard curve (MDA standards were
included in the assay kit), b is the intercept of the standard curve, and df is the dilution factor of the
sample.
Statistical Analyses
A one-way analysis of variance (ANOVA) was used to determine variability between group means.
Two-sample t tests were used to compare specific groups in the one-way ANOVA. All t tests were
two-tailed, and an alpha level of p<0.05 was regarded as statistically significant. Data are expressed
as mean ± standard deviation.
RESULTS
In mice receiving 12 weeks of dietary supplementation with lipoic acid, MDA concentration was
unchanged in the slow-twitch soleus. However, MDA concentration was dramatically reduced in the
fast-twitch EDL
,
indicating a significant
decrease in lipid peroxidation (Figure 1). Furthermore, mice that had been both sprint trained and
LA-supplemented for 12 weeks also expressed a significant decrease in EDL MDA concentration
(Figure 1). There was no significant difference between the LA
and LA+Ex groups.
20
18
16
MDA ( M)
14
* *
LA
12
10
LA+Ex
8
Control
6
4
2
0
Soleus
EDL
Tris buffer
Figure 1. Results of lipoic acid supplementation and sprint training. Values represent mean ± standard deviation;
*Significantly different from control (p<0.05); Samples containing only the homogenization buffer were analyzed to control
for nonspecific absorbance.
DISCUSSION
In the present study, experimental and control mice engaged in an acute anaerobic exercise session,
consisting of six consecutive 30 s sprints on a rodent treadmill at a pace of 30 m/min (15° incline),
with a 1 min recovery interval between each sprint. It has been previously demonstrated that this
protocol effectively induces lipid peroxidation in fast-twitch skeletal muscle, as indicated by a
significant increase in MDA concentration in the EDL (8). However, the mechanism by which acute
anaerobic exercise leads to oxidative stress remains unclear. It has been suggested that glycolytic
power peaks very early during high intensity exercise and is followed by significant mitochondrial ATP
synthesis (22,23). In addition to the contribution of aerobic metabolism, and the subsequent
Lipoic Acid Inhibits Oxidative Stress
57
disturbance in electron transport, the elevation in ROS generated by sprint exercise may also be
affected by ischemia/reperfusion. The sudden uptake of oxygen following a sprint may react with
accumulated metabolic intermediates to generate elevated levels of ROS (4,24).
Beneficial effects of high intensity sprint training have also been established by previous research.
Lipid peroxidation, as indicated by MDA concentration, is significantly decreased in the fast-twitch
EDL after 12 weeks of sprint training (8). The mechanism behind the peroxidation reduction is
unclear. It is possible that the beneficial effect is due to an increased capacity for oxidative
phosphorylation. Previous studies have confirmed increased mitochondrial mass in response to
conditions of oxidative stress (25,26). Furthermore, an increase in reduced glutathione (GSH) may
contribute to the training adaptation. Endurance training has been shown to increase GSH content in
hind limb muscles of dogs (27) and rats (6). High intensity sprint training may produce a similar
effect.
GSH plays a prominent role in the cellular defense against oxidative stress by scavenging ROS, both
directly and as a substrate for GPx (16). Conversion of GSH to GSSG, the oxidized form of the
molecule, is catalyzed by GPx during the reductive detoxification of hydrogen peroxide (28). GSH is
regenerated from GSSG by activity of GR (29). Although some epithelial cells have the capacity to
take up intact GSH (30), most cells rely on de novo synthesis to maintain cellular stores. GSH is
synthesized by a two-step process involving the enzymes, -glutamate-cysteine ligase (GCL) and
glutathione synthetase (GS). GCL catalyzes the intracellular reaction that results in attachment of a
sulfhydryl-containing cysteine residue to a glutamate residue. Activity of GS adds a glycine residue to
complete the tripeptide (16). Normally, ROS are broken down rapidly by defensive mechanisms.
Toxic effects of oxidative stress are only observed when the rate of ROS formation exceeds the rate
of inactivation.
Previous studies have demonstrated that LA can effectively inhibit the adverse effects of oxidative
stress, both in vitro and in vivo. It has been reported that LA protects cultured neurons against
glutamate-induced cytotoxicity by regenerating GSH levels (31).
Furthermore, it has been
demonstrated that, in old rats, a diet supplemented with LA improves mitochondrial function,
increases metabolic rate, and decreases lipid peroxidation by inhibiting age-related loss of GSH (32).
In the present study, it was hypothesized that lipid peroxidation would be significantly reduced in the
EDL by 12 weeks of dietary supplementation with LA, and that lipid peroxidation in the EDL would be
reduced even further by combining 12 weeks of LA supplementation with 12 weeks of high intensity
sprint training. Although the first hypothesis was supported by data, there was no significant
difference found between the LA and LA+Ex groups, in terms of lipid peroxidation (Figure 1). It is
possible that there is a ceiling on the amount of GSH that can be regenerated. A sprint trainingand/or LA-induced upregulation of GSH will have to be quantified to determine if this is true.
CONCLUSIONS
In summary, results of the present study are consistent with previous work that demonstrates a
decrease in lipid peroxidation in response to long-term, high intensity sprint training (8). Furthermore,
results indicate that long-term dietary supplementation with LA can also reduce the impact of
oxidative stress by preventing excessive lipid peroxidation. However, the effects of the two
treatments do not appear to be additive, as there was no significant difference found between LA and
LA+Ex groups.
Lipoic Acid Inhibits Oxidative Stress
58
ACKNOWLEDGEMENTS
Research equipment was obtained through grants from the West Virginia Experimental Program to
Stimulate Competitive Research (WVEPSCoR) and the West Virginia IDeA Network of Biomedical
Research Excellence (WV-INBRE). The project described was supported by the National Center for
Research Resources (NCRR), a component of the National Institutes of Health (NIH). Contents of
this publication are solely the responsibility of the authors and do not necessarily represent the official
views of the NCRR or NIH. Support was provided by a subcontract from the West Virginia University
Research Corporation under an NIH Prime Agreement through the Marshall University Research
Corporation.
Address for correspondence: Shawn Stover, Ph.D., Department of Biology and Environmental
Science, Davis & Elkins College, 100 Campus Drive, Elkins, WV 26241. Phone: (304) 637-1275;
Email: [email protected].
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