Food Sci. Biotechnol. 20(4): 1133-1136 (2011) DOI 10.1007/s10068-011-0154-y

RESEARCH NOTE

Effects of α-Lipoic Acid Supplementation on Malondialdehyde Contents and Superoxide Dismutase in Rat Skeletal Muscles Wan-Jae Kim, Jun-Yong Kang, Dae-Keun Kwon, Young-Ju Song, and Kwang-Ho Lee

Received: 16 December 2010 / Revised: 25 May 2011 / Accepted: 27 May 2011 / Published Online: 31 August 2011 © KoSFoST and Springer 2011

Abstract This study investigated the effects of α-lipoic acid (ALA) supplementation on the malondialdehyde (MDA) contents and superoxide dismutase (SOD) protein expression in rat skeletal muscles under non-exercising condition. Fourteen Sprague-Dawley male rats at the age of 6 weeks were randomly divided into 2 groups CS, (normal diet group, n=7) and AS (0.5% ALA supplemented diet group, n=7), and keep non-exercising condition for 4 weeks. The muscle MDA concentration of the AS group was significantly lower than that of the CS group in the both soleus and extensor digitorum longus muscles. The expressions of Cu/Zn-SOD and Mn-SOD proteins in the soleus muscle were significantly higher in the AS group than that in the CS group. From these results, ALA supplementation under non-exercising condition had a role in increasing of antioxidant enzymes in skeletal muscle types of rats. Keywords: α-lipoic acid, malondialdehyde, superoxide dismutase, skeletal muscle

Introduction Increase of reactive oxygen species (ROS) production is crucial to the remodeling that occurs in skeletal muscle in response to both exercise training and prolonged periods of disuse (1). It is well documented that exercising muscle Wan-Jae Kim, Kwang-Ho Lee ( ) Department of Biotechnology, Konkuk University, Chungju, Chungbuk 380-701, Korea Tel: +82-43-840-3613; Fax: +82-43-840-3613 E-mail: [email protected] Jun-Yong Kang, Dae-Keun Kwon, Young-Ju Song Laboratory of Sports Nutrition, Sunmoon University, Asan, Chungnam 336-708, Korea

produces ROS such as superoxide anions and hydroxyl radical (2). Even at rest, low levels of ROS and markers of oxidative stress are detectable in animal tissues (3,4). Most previous researches mainly reported ROS effect on skeletal muscle under exercising condition (1,2,5,6). But it is also necessary for considering about ROS accumulation in muscles under non-exercising condition, such as daily life. However, to protect against the potentially damaging effects of ROS, cells possess several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). α-Lipoic acid (ALA) acts as a potent antioxidant by inhibiting the process of lipid peroxidation and revitalizing antioxidants in the brain, heart, liver, and skeletal muscles of young rats (7,8). Chae et al. (9) reported that ALA supplementation combined with aerobic treadmill exercise decreased malondialdehyde (MDA) content and increased SOD and GPx activity in the soleus and red gastrocnemius muscles in rats. Moreover, many researchers reported that ALA supplementation on peripheral organs significantly increased antioxidant enzymes such as SOD, CAT, GPx, and reduced oxidative stress in various tissues by alleviating lipid peroxidation through scavenging free radicals (10-12). However, although most of the studies focused on ALA supplementation combined with exercising condition, it has not yet been clearly demonstrated that effect of ALA treatment on antioxidant enzyme expression at different skeletal muscle types. Skeletal muscles mainly consist of red (slow twitch) and white (fast twitch) muscle fibers. Especially, skeletal muscles have lower levels of antioxidants than other tissues (13) and thus ROS-induced oxidative damage may have a more serious impact. It appears that ALA may have a protective role against ROSinduced oxidative damage in skeletal muscles. Therefore, we investigated the anti-oxidant effects of ALA supplementation on types of skeletal muscle of rats

1134

under non-exercising condition by estimation of MDA contents and SOD protein expression.

Materials and Methods Experimental animals and diets Fourteen, 6-week-old Sprague-Dawley rats weighing 230-250 g (BW) were purchased from SamtacoBio Korea (Hwaseong, Korea). The rats were divided into 2 groups (CS: normal diet with sedentary group, n=7, AS: ALA supplemented diet with sedentary group, n=7), given free access to tap water and diet for 4 weeks, and housed in groups of 2/cage under controlled temperature (23±1oC) and relative humidity (50±5%). The light/dark cycle was automatically controlled (alternation 12 h periods). All studies were approved by Animal Studies Committee of Sunmoon University. The composition of the experimental diet was based on the AIN-76 standard diet. ALA (5 g/kg diet) is supplied to AS group instead of corn oil amount of CS group. At the end of the experimental period, the rats were sacrificed by anesthesia, and each muscle was collected. All samples were stored at −70oC until analyzed. Biochemical analysis MDA levels in soleus muscle and extensor digitorum longus (EDL) were determined colorimetrically using thiobarbituric acid according to the method of Buege and Aust (14). Briefly, 1 g of muscle tissue was homogenized with physiological saline and then centrifuged at 8,000×g for 5 min. the supernatant was added to thiobarbituric acid (TBA)-HCl reagent. The mixture was then placed in a boiling water bath for 15 min. On cooling, the protein precipitate was removed by centrifuging at 10,000×g for 5 min, and the absorbance of the clear supernatant fraction was read at 535 nm. MDA values were calculated using molar extinction coefficient, and expressed as µmol/g weight. Western blot analysis Soleus and EDL muscles were homogenized on ice with a polytron homogenizer in 19 volumes of 20 mmol/L Tris-HCl buffer (pH 7.5) containing 5 mmol/L EDTA, 2 mmol/L phenylmethysulfonyl fluoride (PMSF), 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10 µg/mL pepstatin A (Sigma-Aldrich, St. Louis, MO, USA). The homogenates were centrifuged at 1,200×g for 10 min and then the supernatant was collected and recentrifuged at 10,000×g for 10 min. The supernatants were used to detect Cu/Zn-SOD and Mn-SOD protein expression. Protein concentrations were determined by the use of a Bradford reagent from Bio-Rad, with bovine serum albumin as the standard. All protein extraction procedures were conducted at 4oC. Each aliquot of tissue extract containing 40 (Cu/Zn-SOD) or 20 µg (Mn-SOD) of

Kim et al.

protein was mixed with an equal volume of Laemmli buffer, heated at 100oC in heating block for 5 min, and subjected to SDS-PAGE along with a mixture of molecular weight standards from Bio-Rad (Hercules, CA, USA). After electrophoresis, the protein was transferred to a (PVDF) membrane in a Mini Transfer-Blot Electrophoretic Transfer cell (Bio-Rad). After treating with blocking buffer [phosphate-buffered saline (PBS) containing 10% skim milk] for 90 min at room temperature, the membranes were incubated with primary polyclonal antibodies for 2 h at room temperature. These included anti-SOD1 and antiSOD2 (Santa Cruz Technology, Santa Cruz, CA, USA). The antibodies were raised in goat against the rat isoforms of the above proteins. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibodies, which were anti-goat IgG and anti-rabbit IgG (Santa Cruz Technology), as appropriate, for 1 h at room temperature. The target proteins were detected by an enhanced chemiluminscent kit (GE, Piscataway, NJ, USA). The films were photographed and the protein bands of interest were quantified with the band analyzer software (Bio-Rad). Statistical analysis All data were analyzed using Statistical Package for the Social Sciences Version 11.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean± standard error (SE), and values were analyzed by the independent samples t-test. Significance was defined as α= 0.05.

Results and Discussion As shown in Fig. 1, the MDA contents of the AS group were significantly lower than CS group in soleus and EDL muscles (p<0.05; Fig. 1A and p<0.01; Fig. 1B, respectively). This result is in accordance with the earlier studies stating that the supplementation of ALA decreased the level of TBA reactive substances in the muscle tissues (9,15). Khanna et al. (15) suggested that although ALA supplementation did not play a protective role against oxidative damage in the vastus lateralis muscle, there was a protective effect against oxidative lipid damage in the red gastrocnemius with the intake of 150 mg/kg BW for 8 weeks. Chae et al. (9). reported that the reduction of MDA levels prominently appeared in the type I and type IIa muscles, such as soleus and red gastrocnemius, with the intake of 100 mg/kg BW of ALA for 8 weeks. The types of skeletal muscle are classified into red and white. Red skeletal muscle has the characteristics of highly dense capillary networks and mitochondria, and a relatively higher blood flow than white muscle. This leads to increased oxygen consumption and promotes oxidative damage (17). Because the type II fibers have a relatively

Effects of α-Lipoic Acid on Skeletal Muscles

1135

Fig. 1. Effect of ALA supplementation on the MDA contents of different muscle types (A, soleus muscle; B, extensor digitorum longus muscle). CS, control group; AS, α-lipoic acid supplemented group. *Significantly different between CS and AS groups; Values are mean±SE; *p<0.05 and **p<0.01 are compared with control.

Fig. 2. Effect of ALA supplementation on Cu/Zn-SOD protein expression in different muscle types (A, soleus muscle; B, extensor digitorum longus muscle). CS, control group; AS, α-lipoic acid supplemented group. *Significantly different between CS and AS groups; Values are mean±SE (% of control); *p<0.05 are compared with control.

Fig. 3. Effect of ALA supplementation on Mn-SOD protein expression in different muscle types (A, soleus muscle; B, extensor digitorum longus muscle). CS, control group; AS, α-lipoic acid supplemented group. *Significantly different between CS and AS groups; Values are means±SE (% of control); **p<0.01 are compared with control.

lower oxygen rate than the type I fibers, type II fibers generation lower ROS and are more positively related to MDA contents (18). In this study, our data showed that the

MDA level, according to 78 mg/kg BW of ALA supplementation, was significantly lower in the AS group than the CS in the soleus and EDL muscles.

1136

Figure 2 and 3 represents the expressions of Cu/Zn-SOD and Mn-SOD proteins, respectively. The expressions of Cu/Zn-SOD and Mn-SOD proteins of the AS group was significantly higher than that of the CS group in the soleus muscle (p<0.05; Fig. 2A and p<0.01; Fig. 3A). However, there was no significant difference in the EDL muscle (Fig. 2B, 3B). Many studies reported that ALA supplementation plays a protective role by reducing associated oxidative damage to lipids and improves antioxidant enzymes (11,12), Chea et al. (9) reported that ALA treatment increased SOD levels with the intake of 100 mg/kg BW of ALA for 8 weeks, and it may have reduced lipid peroxidation in the soleus muscle. Khanna et al. (15) also reported that intake of 150 mg/kg BW of ALA for 8 weeks decreased lipid peroxidation in the gastrocnemius muscle by increasing antioxidant enzymes, such as CAT and GPx. These kinds of antioxidant enzymes protect SOD against inactivation by H2O2. Reciprocally, SOD protects CAT and GPx against superoxide anions (18). Previous studies supported our result that SOD levels were increased in red muscle fibers by ALA administration, but not in the white muscle fibers (9,15). In conclusion, ALA supplementation under non-exercising condition in rats during 4 weeks may have a protective role against oxidation in skeletal muscles by decreasing MDA levels and increasing SOD protein expression that is dependent on fiber types of skeletal muscle in rats. These data may contribute the potential usage of ALA in the protection of muscle aging and atrophy by accumulation of oxidants during daily life. Acknowledgments This work was supported by Konkuk University in 2010.

References 1. Powers SK, Duarte J, Kavazis AN, Talbert EE. Reactive oxygen species are signaling molecules for skeletal muscle adaptation. Exp. Physiol. 95: 1-9 (2010)

Kim et al. 2. Diaz PT, She ZW, Davis WB, Storey KB. Quantification of salicylate by the in vitro diaphram: Evidence for hydroxyl radical production during fatigue. J. App. Physiol. 75: 540-545 (1993) 3. Chance B, Sies B, Boveries A. Hydroperoxide metabolism in mammalian organs. Physiolol. Rev. 59: 527-605 (1979) 4. Floyd RA. Measurment of oxidative stress in vivo. pp. 89-103. In: The Oxygen Paradox. CLEUP University Press, Padova, Italy (1995) 5. Delliaux S, Brerro-Saby C, Steinberg JG, Jammes Y. Reactive oxygen species activate the group IV muscle afferents in resting and exercising muscle in rats. Pflüg. Arch. 459: 143-150 (2009) 6. Niess AM, Simon P. Response and adaptation of skeletal muscle to exercise-the role of reactive oxygen species. Bioscience 1: 48264838 (2007) 7. Packer L, Kraemer K, Rimbach G. Molecular aspects of lipoic acid in the prevention of diabetes complication. Nutrition 17: 888-895 (2001) 8. Savitha S, Tamilselvan J, Anusuyadevi M, Panneerelvam C. Oxidative stress on mitochondrial antioxidant defense system in the aging process. Role of DL-α-lipoic acid and L-carnitine. Int. J. Clin. Chem. 355: 173-180 (2005) 9. Chae CH, Shin CH, Kim HT. The combination of α-lipoic acid supplementation and aerobic exercise inhibits lipid peroxidation in rat skeletal muscles. Nutr. Res. 28: 399-405 (2008) 10. Coombes JS, Power SK, Rowell B, Hamilton KL, Dodd SL, Shanely A, Sen CK, Packer L. Effects of vitamin E and α-lipoic acid on skeletal muscle contractile properties. J. App. Physiol. 90: 1424-1430 (2001) 11. Stevens MJ, Obrosova I, Cao X, Van HC, Greene DA. Effects of DL-α-lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy. Diabetes 49: 1006-1015 (2000) 12. Martins VD, Manfredini V, Peralba MCR, Benfato MS. α-Lipoic acid modifies oxidative stress parameters in sickle cell trait subjects and sickle cell patients. Clin. Nutr. 28: 192-197 (2009) 13. Valberg S, Häggendal J, Lindholm A. Blood chemistry and skeletal muscle metabolic responses to exercise in horses with recurrent exertional rhabdomyolysis. Equine Vet. J. 25: 17-22 (1993) 14. Buege JA, Aust SD. Microsomal lipid peroxidation. Method. Enzymol. 52: 302-310 (1978) 15. Khanna S, Atalay M, Laaksonen DE, Gul M, Roy S, Sen CK. αLipoic acid supplementation: Tissue glutathione homeostasis at rest and after exercise. J. App. Physiol. 86: 1191-1196 (1999) 16. Sen CK, Roy S, Han D, Packer L. Regulation of cellular thiols in human lymphocytes by α-lipoic acid: A flow cytometric analysis. Free Radical Bio. Med. 22: 1241-1257 (1997) 17. Sacheck JM, Blumberg J. Role of vitamine E and oxidative stress in the exercise. Nutrition 17: 809-814 (2001) 18. Selvakumar E, Prahalathan C, Mythili Y, Varalakshmi P. Protective effect of DL-α-lipoic acid in cyclophosphamide induced oxidative injury in rat testis. Reprod. Toxicol. 19: 163-167 (2004)