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Supplement: Waltham International Symposium

Lipoic Acid as an Antioxidant in Mature Thoroughbred Geldings: A Preliminary Study

Carey A. Williams³, Rhonda M. Hoffman, David S. Kronfeld, Tanja M. Hess, Korinn E. Saker^* and Pat A. Harris

Virginia Polytechnic Institute and State University, Blacksburg, VA, ^* Virginia–Maryland Regional College of Veterinary Medicine, Blacksburg, VA and Equine Studies Group, Waltham Centre for Pet Nutrition, Leicestershire, UK

³To whom correspondence should be addressed. E-mail: cawilli4{at}vt.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
-Lipoic acid (LA) has demonstrated antioxidant effects in humans and laboratory animals. The objective of this study was to determine whether the effects of LA are similar in horses. Five Thoroughbred geldings were supplemented with 10 mg/kg/d DL--lipoic acid in a molasses and sweet feed carrier and five received only the carrier as a placebo (CON). Blood samples were obtained at baseline (0 d), after 7 and 14 d of supplementation, and 48 h postsupplementation (16 d). Blood fractions of red and white blood cells (RBC and WBC, respectively) and plasma were analyzed for glutathione (GSH), glutathione peroxidase (GPx) and total plasma lipid hydroperoxides (LPO). An experienced veterinarian observed no adverse clinical effects. Plasma LPO baselines differed between groups (P = 0.002). When covariates were used, there was a decrease over time in the LA group (P = 0.015) and concentrations were lower in the LA group than in the CON group at 7 and 14 d (P = 0.022 and P = 0.0002, respectively). At baseline, GSH concentration was 69 ± 7 in WBC and 115 ± 13 mmol/mg protein in the RBC, with no differences resulting from either time or treatment. The GPx activity was 47 ± 4 and 26 ± 5 U/g protein at baseline WBC and RBC, respectively, with a lower concentration in the LA group’s WBC at 7 (P = 0.019) and 14 d (P = 0.013). The results show that 10 mg/kg LA had no evident adverse effects, and moderately reduced the oxidative stress of horses allowed light activity. These findings encourage studying of LA in horses subjected to strenuous exercise.

KEY WORDS: • oxidative stress • horse • lipid hydroperoxides • glutathione • dihydrolipoate

-Lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have received widespread attention as antioxidants with both preventative and therapeutic uses in humans and laboratory animals (1). They have potential value in production and companion animals during periods of oxidative stress. -Lipoic acid is an eight-carbon structure that contains a disulfide bond as part of a dithiolane ring with a five-carbon tail. It is a cofactor in the conversion of pyruvate to acetyl CoA as part of the pyruvate dehydrogenase complex. Both LA and DHLA also protect the integrity of cell membranes by interacting with antioxidants—glutathione (GSH) and vitamins E and C (1). In vitro, intracellular GSH was positively correlated to the concentration of LA, which may play a role with cysteine (2,3).

Lipoic acid is both water- and fat-soluble, and thus may regenerate water- and fat-soluble antioxidants, such as vitamin C and vitamin E. Rats consuming a vitamin E–deficient diet with added LA had no symptoms of vitamin E deficiency, unlike the rats on a vitamin E–deficient diet alone (4). The low -tocopherol (-TOC) concentrations in the tissues of LA-supplemented rats suggests that LA eliminates symptoms of vitamin E deficiency by sparing -TOC.

Another function of LA and DHLA is to scavenge free radicals. Both LA and DHLA eliminate hydroxyl radicals and hypochlorous acid with a potency comparable to that of glutathione and N-acetylcysteine (5,6). However, only DHLA eliminated superoxide, peroxyl and ascorbyl radicals. Both LA and DHLA did not interact directly with chromanoxyl radicals of vitamin E; however, DHLA did reduce ascorbyl radicals and showed a DHLA/ascorbic acid–mediated reduction of chromanoxyl radicals (7,8). These studies demonstrate the importance of LA as a protective compound during oxidative metabolism.

Lipoic acid has been shown to have effects during exercise stress, aging and metabolic disease. Khanna et al. (9) compared rested and exercised rats supplemented with or without LA. The LA supplemented rats, both rested and exercised, had a higher GSH concentration, and a lower lipid peroxidation level measured by thiobarbituric acid reactive substances compared to those of nonsupplemented rats. During aging, mitochondria use oxygen inefficiently in ATP synthesis. Old rats supplemented with LA had a higher mitochondrial membrane potential, ambulatory activity, GSH and ascorbate concentration; furthermore, the malondialdehyde concentration was five times higher in the nonsupplemented rats (10). Lipoic acid was also used in humans to test its effectiveness on relieving diabetic symptoms (11). Three different doses of LA were compared to a placebo over a period of 19 d. After 5 d patients dosed with 1200, 600 or 100 mg LA had a greater decrease in the severity of their symptoms than patients given a placebo. Other human tests also show positive results when tested under different diseased states or illness conditions, including Alzheimer-type dementia (12).

Oxidative stress is evident in horses undergoing exhaustive and endurance exercise (13–15); this warrants the testing of antioxidant supplements. The objective of this study was to evaluate the safety and antioxidant potential of LA in horses undergoing light voluntary pasture exercise.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Ten mature Thoroughbred geldings, 7 to 16 y of age, weighing 626.8 ± 8.3 kg, were used in this study. The protocol was approved by the institutional animal care and use committee and performed at the Virginia Tech Middleburg Agricultural Research and Extension Center.

The geldings were maintained on 30 acres of mixed grass/clover pasture during the month of January with ad libitum access to orchard grass/alfalfa hay. Five of the geldings were randomly assigned to the treatment group (LA) and received 10 mg/kg body weight DL--lipoic acid (Sigma Chemical, St. Louis, MO) mixed into a carrier (70 g of molasses and sweet feed) offered by hand once/d at 1300 h for 14 d. The other five geldings were assigned to the control group (CON) and received the carrier as a placebo at the same time as the LA supplement.

Blood samples were collected by venous puncture in 10 mL sodium heparin–containing Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) before supplementation (0 d), after 7 and 14 d of supplementation, and 48 h after termination of the supplement (16 d). Blood samples were taken at 1300 h and immediately processed into red blood cell, white blood cell and plasma aliquots.

For assays using erythrocyte lysate, 500 µL of whole blood was transferred into a microcentrifuge tube and centrifuged at 2500 x g for 5 min at 4°C. The plasma was removed and discarded from the sample. Erythrocytes were resuspended in 1 mL ice-cold deionized water, then frozen at -80°C until analysis. For the determinations using white blood cells, the buffy coat, located at the interface of the red cell pellet and the plasma was removed after centrifugation of whole blood at 2500 x g for 5 min at 4°C, and transferred to a tube containing 10 mL of lysis buffer (0.15 M NH₄Cl, 0.01 M NaHCO, 0.03 M EDTA free acid). White blood cells were washed once in the lysis buffer to lyse the red blood cells, and then washed twice in Hank’s balanced salt solution (HBSS; Life Technologies, Carlsbad, CA). After the last wash, 1 mL HBSS was added to the pellet and mixed thoroughly, after which 0.5 mL was transferred into microtubes and frozen at -80°C until sample analysis. Plasma aliquots were prepared by centrifuging the Vacutainer tubes at 2500 x g for 5 min at 4°C, then transferring the plasma to microtubes, which were frozen at -80°C until sample analysis.

Red blood cell lysate and white blood cells were analyzed for total glutathione (Oxis Health Products, Portland, OR, Biotech GSH-420, kit no. 51023; interassay CV 7.0%, intra-assay CV 5.6%) and glutathione peroxidase [GPx; Oxis Health Products, Biotech GPx-340, kit no. 51017; interassay CV 4.2%, intra-assay CV 5.0%) using an OxyScan Automated Oxidative Stress Analyzer (Oxis Health Products)]. Total plasma lipid hydroperoxides (LPO; Oxis Health Products, Biotech LPO-560, kit no. 21025) were also analyzed using a spectrophotometer (interassay CV 3.0%, intra-assay CV 4.6%).

Data were summarized as means ± SE. Baseline values for the two groups were compared by Student’s t-test and, when different, were subtracted from values at later times (this assumes a covariance of 1). The effects of time, treatment and time by treatment interaction were evaluated on the raw data or increments by analysis of variance with repeated measures using the general linear models procedures of SAS (16). Significance was inferred at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
An experienced veterinarian observed no adverse clinical signs, including gastrointestinal distress, in the group supplemented with LA. Total plasma LPO (Fig. 1) was 9.32 ± 0.78 µM for CON and 13.56 ± 0.45 µM for LA at baseline (P = 0.0016). The concentrations decreased over time in the LA group (P = 0.015) and were lower in the LA group than in the CON group at 7 and 14 d (P = 0.022 and P = 0.0002, respectively). There was also an effect of treatment and time by treatment interaction (P = 0.031). The area under the LPO curve was found to be greater (P = 0.004) for the LA group (96.9 ± 15.7 µM d^-1) than for the CON group (28.2 ± 7.1 µM d^-1). At baseline, the GSH concentration was 69 ± 7 and 115 ± 13 mmol/mg protein in the white and red blood cells, respectively, and was not significantly different between groups. Red blood cell GSH tended to increase with time (P = 0.074) and white blood cell GSH increased with time (P = 0.017) and tended to increase with treatment (P = 0.071). There were no significant interactions between time and treatment for either red blood cell or white blood cell GSH. The GPx activity in the white blood cells (Fig. 2) was 50.6 ± 1.6 for CON and 43.6 ± 033 mmol/mg protein for LA at baseline (P = 0.041). The change in concentration was higher in the LA group at 14 (P = 0.019) and 16 d (P = 0.013) than that in the CON group. The red blood cell GPx showed no differences for time or treatment.

View larger version (19K): [in this window] [in a new window]   FIGURE 1 Total plasma lipid hydroperoxides (LPO) for horses receiving the control supplement (CON; n = 5) or lipoic acid–containing supplement (LA; n = 5). The LPO baselines are subtracted from 7-, 14- and 16-d samples assuming a covariance of 1. Results for ANOVA found time (P = 0.015), treatment and time by treatment interaction (P = 0.031) significant. Data are shown as means ± SE.

View larger version (20K): [in this window] [in a new window]   FIGURE 2 White blood cell (WBC) glutathione peroxidase (GPx) for horses receiving the control supplement (CON; n = 5) or lipoic acid–containing supplement (LA; n = 5). The white blood cell GPx baselines are subtracted from 7-, 14- and 16-d samples assuming a covariance of 1. There were significant effects of time (P < 0.001), and treatment (P = 0.033) and a significant time by treatment interaction (P = 0.031). Data are shown as means ± SE.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The dose of 10 mg/kg body weight was chosen by referring to LA’s effectiveness in other species. It is effective in the rat at a dose of 10 to 100 mg/kg or about 40 to 200 mg/kg^0.75 (9,10) and in humans at a dose of 600 to 1200 mg (about 8.5 to 17 mg/kg) or about 25 to 50 mg/kg^0.75 (11). It caused gastrointestinal distress, however, at the high dosage (11). Our hypothesis was that a dose of 10 mg/kg or about 50 mg/kg^0.75 would diminish oxidative stress in horses after being supplemented for 14 d, which is the average duration studied in other species, without causing any clinical signs of toxicity.

In humans LA was used in 260 non-insulin-dependent diabetic patients to determine whether it relieved symptoms of diabetic neuropathy including pain, burning, paralysis and numbness (11). Three different doses of LA were compared to a placebo over a period of 19 d. Patients were tested and assigned a total symptom score by a physician three times during this period for improvements in neuropathic symptoms caused by the diabetes. After 5 d, patients dosed with 1200, 600 and 100 mg LA had a greater decrease (P < 0.01) in total symptom score than with the placebo, and after 12 and 19 d the decrease was found in the 1200 and 600 mg dosed patients.

Total plasma LPO may be regarded as a cumulative index of antioxidant status. Even after the 7 d of dosing with LA there was a further decrease in LPO indicating a lower oxidative stress level or higher antioxidant status. By 48 h after the termination of the LA supplement plasma LPO in the LA was not different from that in the CON group. Other studies using malondialdehyde or thiobarbituric acid-reactive substances (indicators of lipid peroxidation) found that after intense, exhaustive exercise lipid peroxidation was lower in the liver (P < 0.05), muscle (P < 0.05) and heart (P < 0.001) tissue of rats supplemented with 100 mg/kg LA for 3 wk (9). In aged rats malondialdehyde was five times higher than that in young rats; however, with LA supplementation these levels were significantly reduced in the old rats (10).

A series of studies focusing on the LA interaction with GSH performed by Han et al. (2,3) stimulated our interest in this same interaction in red and white blood cells. In vitro, human T-lymphocyte cells, bathed in various concentrations of LA, increased the intracellular level of GSH; this increase had a positive correlation to the concentration of LA added to the medium (3). When 100 µM LA was added, GSH increased 45.8% over that of the control group after 24 h, and DHLA and GSH levels were positively correlated (r = 0.57, P < 0.01). This result suggests that DHLA is a contributing factor to the GSH increase after the addition of LA.

In our study, however, the GSH concentration might have changed transiently in response to LA then reverted to normal values after 24 h when the next blood sample was taken. Lipoic acid is rapidly converted to DHLA once inside the cell; it is then transported out of the cell and used as an antioxidant (1). Dihydrolipoic acid is very reactive and rapidly used so the effects on GSH may not be long lasting. Further studies are planned to see whether the same dose of 10 mg/kg LA would have antioxidant effects on antioxidant status and oxidative stress from immediately after supplementation up to 48 h postsupplementation.

In our study, the decrease in white blood cell GPx activity in the CON group, and the slightly less marked decrease in the LA group may be attributable to the weather conditions. During wk 1 of the study (mid-January) the temperature was mild with sunny conditions, into wk 2 of the study, for the last two sample times, temperatures dropped to near freezing and precipitation increased to freezing rain/snow. It is possible that the weather played a role in the level of oxidative stress in these geldings, which are housed outside 24 h/d. This environmental stress would increase activity level and oxygen consumption, thus increasing reactive oxygen species and oxidative stress, and might explain why the LA group had less of a change in the GPx activity compared to that of the CON group.

Other studies have used GPx as well as other antioxidant enzymes to measure the effects of oxidative stress. Superoxide dismutase, catalase and GPx, along with a few nonenzymatic antioxidants, were used to determine whether LA decreased lipid peroxidation and increased antioxidant status in the mitochondria of aged rats (17). They found that with LA supplementation there was a lower level of lipid peroxidation and increased level of antioxidant action in the mitochondria, as determined by enzymatic and nonenzymatic antioxidants. In horses, Ji et al. (15) found that superoxide dismutase, catalase, glutathione reductase and GPx were numerically increased with an exercise protocol of 12 min of trotting at 4.5 m/s on a grade of 11%; however, these values were not significant. They also found that, after supplementing with vitamin E before exercise, these oxidative stress measures were also unchanged. This could possibly be because these horses were conditioned to the protocol and the amount of work was not great enough to induce oxidative stress.

Implications

The supplementation of -lipoic acid has been shown previously to increase antioxidant status and decrease oxidative stress in the horse and in other species. It favors regeneration of antioxidants including vitamin E and C, and glutathione, and thus decreases the need for their supplementation. The present findings encourage testing of lipoic acid supplementation in horses subjected to oxidative stress, for example, during strenuous exercise and prolonged transport.

	ACKNOWLEDGMENTS

 
The technical assistance and encouragement of the staff at the Virginia Tech MARE Center is gratefully acknowledged.

	FOOTNOTES

 
¹ Presented as part of the Waltham International Symposium: Pet Nutrition Coming of Age held in Vancouver, Canada, August 6–7, 2001. This symposium and the publication of symposium proceedings were sponsored by the Waltham Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris, University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl L. Keen, University of California, Davis, and D’Ann Finley, University of California, Davis.

² Supported by the late Paul Mellon, the John Lee Pratt Fellowship Program in Animal Nutrition and the Waltham Centre for Equine Nutrition and Care, Leicestershire, UK.

⁴ Abbreviations used: -TOC, -tocopherol; CON, control supplemented group; DHLA, dihydrolipoic acid; GSH, glutathione; GPx, glutathione peroxidase; HBSS, Hank’s balanced salt solution; LA, lipoic acid; LPO, lipid hydroperoxides.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Packer, L., Witt, E. H. & Tritschler, H. J. (1995) Alpha-lipoic acid as a biological antioxidant. Free Radic. Biol. Med. 19:227-250.[Medline]

2. Han, D., Handelman, G., Marcocci, L., Sen, C. K., Roy, S., Kobuchi, H., Tritschler, H. J., Flohe, L. & Packer, L. (1997) Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization. BioFactors 6:321-338.[Medline]

3. Han, D., Tritschler, H. J. & Packer, L. (1995) -Lipoic acid increases intracellular glutathione in a human T-lymphocyte Jurkat cell line. Biochem. Biophys. Res. Commun. 207:258-264.[Medline]

4. Podda, M., Tritschler, H. J., Ulrich, H. & Packer, L. (1994) -Lipoic acid supplementation prevents symptoms of vitamin E deficiency. Biochem. Biophys. Res. Commun. 204:98-104.[Medline]

5. Biewenga, G. P., de Jong, J. & Bast, A. (1994) Lipoic acid favors thiolsulfinate formation after hypochlorous acid scavenging: a study with lipoic acid derivatives. Arch. Biochem. Biophys. 312:114-120.[Medline]

6. Haenen, G.R.M.M. & Bast, A. (1991) Scavenging of hypochlorous acid by lipoic acid. Biochem. Pharmacol. 42:2244-2246.[Medline]

7. Kagan, V. E., Shvedova, A., Sebinova, E., Khan, S., Swanson, C., Powell, R. & Packer, L. (1992) Dihydrolipoic acid: a universal antioxidant both in the membrane and the aqueous phase. Biochem. Pharmacol. 44:1637-1649.[Medline]

8. Suzuki, Y. J., Tsuchiya, M. & Packer, L. (1991) Thioctic acid and dihydrolipoic acid are novel antioxidants which interact with reactive oxygen species. J. Free Radic. Res. Commun. 15:255-263.

9. Khanna, S., Atalay, M., Laaksonen, D. E., Gul, M., Roy, S. & Sen, C. K. (1999) -Lipoic acid supplementation: tissue glutathione homeostasis at rest and after exercise. J. Appl. Physiol. 86:1191-1196.[Abstract/Free Full Text]

10. Hagen, T. M., Ingersoll, R. T., Lykkesfeldt, J., Liu, J., Wehr, C. M., Vinarsky, V., Bartholomew, J. C. & Ames, B. N. (1999) (R)--Lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate. FASEB J 13:411-418.[Abstract/Free Full Text]

11. Ziegler, D., Hanefeld, M., Ruhnau, K. J., Meibner, H. P., Lobisch, M., Schutte, K. & Gries, F. A. (1995) Treatment of symptomatic diabetic peripheral neuropathy with the anti-oxidant -lipoic acid. Diabetologia 38:1425-1433.[Medline]

12. Hager, K., Marahrens, A., Kenklies, M., Riederer, P. & Munch, G. (2001) Alpha-lipoic acid as a new treatment option for Alzheimer type dementia. Arch. Gerontol. Geriatrics 32:275-282.[Medline]

13. Hargreaves, B. J., Kronfeld, D. S., Waldron, J. E., Lopes, M. A., Gay, L. A., Saker, K. E., Cooper, W. L., Sklan, D. J. & Harris, P. A. (2001) Oxidative status of endurance horses. Equine Nutr. Physiol. Soc. Proc. 17:280-281.

14. Ishida, N., Hobo, S., Takahashi, T., Nanbo, Y., Sato, F., Hasegawa, T. & Mukoyama, H. (1999) Chronological changes in superoxide-scavenging ability and lipid peroxide concentration of equine serum due to stress from exercise and transport. Equine Vet. J. Suppl. 30:430-433.[Medline]

15. Ji, L. L., Dillon, D. A., Bump, K. D. & Lawrence, L. M. (1990) Antioxidant enzyme response to exercise in equine erythrocytes. Equine Vet. Sci. 10:380-383.

16. SAS. (1999) SAS/STAT User’s Guide (release 8 0) 1999 SAS Institute Cary, NC.

17. Arivazhagan, P., Ramanathan, K. & Panneerselvam, C. (2001) Effect of DL--lipoic acid on the status of lipid peroxidation and antioxidants in mitochondria of aged rats. J. Nutr. Biochem. 12:2-6.[Medline]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Williams, C. A. Articles by Harris, P. A. Search for Related Content PubMed PubMed Citation Articles by Williams, C. A. Articles by Harris, P. A.