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Stereospecific Effects of R-Lipoic Acid on Buthionine Sulfoximine-Induced Cataract Formation in Newborn Rats
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
221, 422–429 (1996)
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Stereospecific Effects of R-Lipoic Acid on Buthionine Sulfoximine-Induced Cataract Formation in Newborn Rats Indrani Maitra,1 Elena Serbinova,* Hans J. Tritschler,† and Lester Packer*,2 *Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; and †ASTA Medica, Weismüllerstraße 45, D-60314 Frankfurt Am Main, Germany Received March 11, 1996 This study revealed a marked stereospecificity in the prevention of buthionine sulfoximine-induced cataract, and in the protection of lens antioxidants, in newborn rats by a-lipoate. R- and racemic a-lipoate decreased cataract formation from 100% (buthionine sulfoximine only) to 55% (buthionine sulfoximine + R-a-lipoic acid) and 40% (buthionine sulfoximine + rac-a-lipoic acid) (p < 0.05 compared to buthionine sulfoximine only). S-a-lipoic acid had no effect on cataract formation induced by buthionine sulfoximine. The lens antioxidants glutathione, ascorbate, and vitamin E were depleted to 45, 62, and 23% of control levels, respectively, by buthionine sulfoximine treatment, but were maintained at 84–97% of control levels when R-a-lipoic acid or rac-a-lipoic acid were administered with buthionine sulfoximine; S-a-lipoic acid administration had no protective effect on lens antioxidants. When enantiomers of a-lipoic acid were administered to animals, R-a-lipoc acid was taken up by lens and reached concentrations 2- to 7-fold greater than those of S-a-lipoic acid, with rac-a-lipoic acid reaching levels midway between the R-isomer and racemic form. Reduced lipoic acid, dihydrolipoic acid, reached the highest levels in lens of the rac-a-lipoic acid-treated animals and the lowest levels in S-a-lipoic acid-treated animals. These results indicate that the protective effects of a-lipoic acid against buthionine sulfoximine-induced cataract are probably due to its protective effects on lens antioxidants, and that the stereospecificity exhibited is due to selective uptake and reduction of R-a-lipoic acid by lens cells. © 1996 Academic Press, Inc.
By the very nature of its function the occular lens is subjected to a constant barrage of free radicals. To counteract this oxidative insult, the lens has an efficient antioxidant system in the form of enzymes and low molecular weight antioxidants. Newborn rats are unable to synthesize ascorbate, and treatment of these animals with BSO, an inhibitor of glutathione synthesis, has been used as a model of oxidative stress, with cataract formation in lens after the animals open their eyes as an easily assessed end-point (1, 2). Depletion of lens GSH appears to be the prime effect of BSO treatment, initiating a general imbalance in the antioxidant defense system. Among the various postulated functions of GSH in the lens, the one which has received most attention in the last decade is its role in protection against oxidative damage. Hence, it is likely that the mechanism for BSO-induced cataract formation is an acceleration of oxidative damage in the lens due to severe depletion of lens antioxidant defenses; this model thus serves as an ideal model system for testing protective effects of exogenous antioxidant administration against cataract formation. In our previous work (3) we observed that administration of racemic a-lipoate increased both the GSH and GSSG levels in lens tissue of BSO-treated newborn animals, as well as the levels of other antioxidants, and protected against cataract formation, but the exact mechanism of this effect was not clear. Exogenously supplied a-lipoic acid (a-lipoate), a dithiol found endogenously as covalently bound lipoamide in a-keto acid dehydrogenases and in the glycine cleavage system (4), has been shown to substitute for ascorbate in vitamin C-deficient guinea pigs (5) and to raise intracellular 1
Present address: College of St. Catherine, 601 25th Avenue S, Minneapolis, MN 55454. To whom correspondence should be addressed. Abbreviations: BSO, buthionine sulfoximine; GSH, glutathione; GSSG, glutathione disulfide.
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levels of glutathione in vitro and in vivo (6, 7). In previous work, we studied the effect of racemic a-lipoate on cataract formation in BSO-treated rats, and found that it was effective in protecting newborn animals from BSO-induced cataract formation, reducing the incidence of cataract from 100% to 45% (3). Treatment with a-lipoate maintained the antioxidant status of the cell by maintaining a higher level of ascorbate and tocopherol, possibly through recycling mechanisms, and thereby contributed to decreased GSH utilization in the tissues. a-Lipoate has an asymmetric carbon at its C6 position. For the a-ketoacid dehydrogenase complexes from Escherichia coli (9) as well as for the mammalian pyruvate dehydrogenase complex (10) it has been shown that only the R-configuration of a-lipoate is able to function as a cofactor. In E. coli, both enantiomers of lipoic acid are taken up by the cell, while only the R-form is incorporated into the pyruvate dehydrogenase complex (11). However, eukaryotic cells possess mechanisms to reduce both enantiomers (12). Dihydrolipoamide dehydrogenase, the E3 enzyme in mitochondrial a-keto acid dehydrogenases, is highly specific for R-a-lipoate, whereas glutathione reductase, which can utilize a-lipoate in addition to glutathione disulfide as a substrate, is specific for S-a-lipoate (12, 13). The stereospecificity of cellular uptake mechanisms for the two enantiomers is not known. The present investigation sought to clarify the relative effect of the enantiomers on cataract formation in lenses of BSO-induced glutathione-deficient newborn animals,. MATERIALS AND METHODS Animals Sprague-Dawley rats (female, timed pregnant) (Simonsen, Santa Clara, CA), were housed in the Research Animal Facilities and given food and water ad libitum. Newborn animals of both sexes were used. They were breast fed. All procedures were approved by the University of California, Berkeley, Animal Care and Use Committee.
Generation of Cataracts Animals were divided into 8 groups and given 4 injections per day for 2 days, according to the following protocol:
Time Groups
8 a.m./4 p.m.
12 a.m./8 p.m.
Control +BSO BSO + R-LA BSO+Racemate BSO+S-LA R-LA only Racemate only S-LA only
NaCl BSO BSO BSO BSO R-LA Racemate S-LA
NaCl/NaCl:EtOH NaCl/NaCl:EtOH R-LA Racemate S-LA NaCl NaCl NaCl
Each experimental group consisted of 20–25 animals. BSO was dissolved in 0.1 M NaCl and injected at a concentration of 3 mmol/g body weight (total volume not exceeding 60 ml). a-lipoate was dissolved in ethanol: NaCl (50:50, v/v). BSO was injected subcutaneously into newborn animals (age 28–36 hours). a-lipoate (25 mg/kg b.w.) was given intraperitoneally. An equivalent volume of ethanol: NaCl and/or NaCl was given to the controls. Rats were sacrificed when they opened their eyes (14–15 days). Cataract formation was visualized with a slit-lamp microscope and the lenses were classified as opaque or clear. a-Lipoate uptake. In order to study the uptake of R-, S-, and rac-lipoate in lens, with and without BSO treatment, the same protocol as above was followed except that a total of 6 injections were given to the animals. After the last injection, the animals were sacrificed at 0, 1, 3 and 6 hrs and lenses were isolated as described below. Isolation of lenses. After decapitation of the animals the eyes were carefully dissected out from the ciliary body and the lens capsule was released from the enucleated eye globes. Each lens was weighed and placed in a microfuge tube and frozen immediately in liquid nitrogen for future analyses. 423
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Analytical Procedures Glutathione. Frozen lenses were homogenized in 5% sulfosalicylic acid, 5 mM EDTA, ice cold and bubbled with argon. After centrifugation GSH and GSSG were determined by the enzymatic recycling method (14). Ascorbate. Lenses were homogenized in ice-cold 90% methanol, 1 mM EDTA, bubbled with argon gas. Ascorbic acid and dehydroascorbic acid were measured by HPLC using electrochemical detection (15). Vitamin E. a-Tocopherol was extracted in hexane and analyzed by HPLC using electrochemical detector as described by Lang et al (16). a-Lipoate and dihydrolipoate. Frozen lenses were homogenized in 5% sulfosalicylic acid, 5 mM EDTA, ice cold and bubbled with argon. The lens homogenate was then extracted with equal volume of ethanol. After centrifugation the lens extract was analyzed by HPLC: determination of lipoic acid and dihydrolipoate was carried out on a Rainin Microsorb 3 um C18 column, 10 × 0.46 cm. The mobile phase was 55% water/25% methanol/20% acetonitrile, with 2% monochloroacetic acid. The flow rate was 1 ml/min with a 100 ul injection loop. Measurement of a-lipoate and dihydrolipoate was accomplished using an electrochemical detector with a dual Hg/Au electrode according to the method modified by Han et al (17). Chemicals. L-Buthionine-(S,R)-sulfoximine (BSO) was from Sigma Company, R-, S-, and rac-a-lipoate were from ASTA Medica (Frankfurt). HPLC grade ethanol and methanol were purchased from Fischer Scientific, Fair Lawn, NJ. All other reagents used were from Sigma Chemical Company, St. Louis, MO, or of the highest quality available.
RESULTS Cataract formation. All newborn animals treated with BSO alone developed cataracts (Table 1). None of the controls or the rats treated with (25 mg/kg body weight) enantiomers alone developed cataract. In case of BSO plus R-a-lipoate, and BSO plus rac-a-lipoate, 55% and 40%, respectively, of the animals developed cataract (Table 1). These results were significantly different from that of BSO treated group only (p<0.005). All the animals treated with S-a-lipoate and BSO developed cataract. Lens GSH and GSSG levels. GSH in the lenses of BSO-treated rats was about 55% lower than that in control lenses (Fig 1), (p < 0.0001). Treatment with BSO and R-a-lipoate or BSO and rac-a-lipoate simultaneously demonstrated a higher level of GSH (≈84–87% that of control level), significantly different from BSO treated group only (p < 0.001). Treatment with a-lipoate alone did not change the GSH level from the control level (data not shown). The efficiency of preventing the decrease in GSH levels in BSO-treated animals was about the same for R-a-lipoate and rac-alipoate. S-a-lipoate administered in conjunction with BSO was almost without effect on lenticular GSH level. Similarly, total glutathione (GSH+GSSG) level also decreases after BSO injection, from 3.24 mmoles/g lens tissue to 1.78±0.18 mmoles/g lens tissue. Treatment with R-a-lipoate and TABLE 1 Effect of Lipoic Acid Enantiomers on Cataract Formation in Newborn Animals with BSO-Induced Glutathione Deficiency Treatment
% Cataract formation
CONTROL BSO R-LA+BSO S-LA+BSO rac-LA+BSO R-LA S-LA rac-LA
0 100 55* 100 40* 0 0 0
In each experimental group n420–30. Data has been averaged from three separate experiments. * Significantly different (P < 0.005) from BSO treated group. 424
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FIG. 1. Effect of enantiomers of a-lipoic acid on glutathione levels in the lenses of newborn animals with BSO-induced glutathione deficiency. Conditions as in Methods. Filled bars, GSH. Cross-hatched bars, total glutathione (GSH + GSSG). N 4 4 for each bar. Data are given as mean ± SD. *significantly different from BSO treated group (p < 0.001).
BSO or rac-a-lipoate and BSO prevented the decrease in the total glutathione level almost to the same extent. Lens ascorbate levels. Administration of BSO led to a decrease in total lens ascorbate levels to 62% of control values (Fig 2). 93% and 90% of control values of lens ascorbate were found after treatment with BSO + R-a-lipoate or BSO + rac-a-lipoate, respectively, significantly different from the group treated with BSO only (p < 0.001). The level of ascorbate in lenses from animals treated with S-a-lipoate plus BSO was 68% of the control level, not significantly different from that in animals treated with BSO only. Lens vitamin E levels. Vitamin E levels in lenses dropped significantly after BSO administration,
FIG. 2. Effect of enantiomers of a-lipoic acid on ascorbate levels in the lenses of newborn animals with BSO-induced glutathione deficiency. Conditions as in Methods. Filled bars, ascorbate. Open bars, total ascorbate (ascorbate + dehydroascorbate). N46 for each bar. Data are given as mean ± SD. aSignificantly different from BSO-treated group (p < 0.001). b Significant from BSO-treated group. Other conditions as in Fig 1. 425
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from 6.0 ± 3.3 nmol/g tissue in control animals to 1.4 ± 0.6 nmol/g tissue in BSO-treated animals (23% of the control level) (p < 0.0001). Administration of BSO + R-a-lipoate maintained vitamin E levels at 97% of control levels (5.8±0.9 nmol/g tissue) and administration of BSO + rac-a-lipoate maintained it at 88% of control value (Fig 3), both significantly different (p < 0.005) from the group treated with BSO only. Lens vitamin E levels in animals treated with BSO + S-a-lipoate were not significantly different from those in animals treated with BSO only. Timecourse of uptake and reduction of lipoic acid by lens in animals. In control animals, increased lens levels of a-lipoate and dihydrolipoate were observed within one hour of injection of a-lipoate (Fig 4) and at this time uptake of R-a-lipoate or rac-a-lipoate was almost the same. But after three hours there was 3 times as much a-lipoate in the lenses of R-a-lipoate treated animals as in the animals treated with racemate mixture. In S-a-lipoate treated animals uptake of a-lipoate was very low compared to the other isomers. Dihydrolipoate formation was highest in the rac-alipoate treated animals. No a-lipoate or dihydrolipoate was detected after 6 hours for any treatment (Fig 4). In BSO-treated animals, the uptake of a-lipoate was much more rapid, though the same stereospecificity was present—the uptake of the R-form was highest and S-form was lowest (Fig. 4). DISCUSSION The present investigation reveals a high degree of stereospecificity for protection by exogenous lipoate against the BSO-induced formation of lens cataract in the newborn rat. The R-enantiomer and racemic mixture were highly protective, whereas the S-enantiomer was almost completely ineffective. We suggest that these marked differences in protective effect are probably due to differences in uptake and reduction of R-, rac- and S-a-lipoate. These results are consistent with several previous observations: (i) the existence of two pathways of reduction of a-lipoate with differing stereospecificities: NADH-dependent mitochondrial lipoamide dehydrogenase reduction specific for the R-enantiomer and the NADPH-dependent glutathione reductase reduction specific for the S-enantiomer (12, 13); (ii) dihydrolipoate can participate in reducing other antioxidants in the antioxidant network, including vitamin C (from dehydroascorbate or the semi-ascorbyl radical(18)), and glutathione (from glutathione disulfide(19).); (iii) the observation in other studies that isolated the rat lenses in culture induced to form opacity (cataracts) by high glucose exposure (a
FIG. 3. Effect of enantiomers of a-lipoic acid on vitamin E levels in the lenses of newborn animals with BSO-induced glutathione deficiency. Conditions as in Methods. N 4 4 for each bar. Data are given as mean ± SD. *Values significantly diData are given as mean ± SD.fferent from BSO treated group (p < 0.005) 426
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FIG. 4. Timecourse of uptake of and reduction of enantiomers of a-lipoic acid by lens in control animals and in animals treated with BSO. Conditions as in Methods. N 4 4–6 for each point. Data are given as mean ± SD. Closed symbols and solid lines: a-lipoic acid. Open symbols and dashed lines: dihydrolipoic acid. Circles: animals treated with R-a-lipoic acid. Triangles: animals treated with S-a-lipoic acid. Squares: animals treated with rac-a-lipoic acid.
model of diabetic cataractogenesis) show virtually complete stereospecificity for protection by the R as compared to the S form of lipoate(20). Several steps may be important for lipoate to be effective in protection of the lens against oxidatively-induced cataract. It must be taken up by lens cells. Pathways of reduction are also important, because dihydrolipoate is a potent reductant which can regenerate other antioxidants. In control rats, lens levels of R-a-lipoate were approximately 7-fold greater than those of S-a-lipoate three hours after intraperitoneal injection (Fig. 4); in BSO-treated rats the difference was approximately two-fold (Fig. 4). In both cases the racemic mixture was midway between the two enantiomers. Since R-a-lipoate is the naturally occurring enantiomer of a-lipoate in the body, a selective transport process of R-a-lipoate may already exist in the cells which might account for the greater degree of accumulation of R-a-lipoate in the lenses, and these differences in uptake probably accounted for part of the differences in protection. The levels of dihydrolipoate in lens do not follow the same pattern as lipoate. In both control and BSO-treated animals, the racemic mixture produced the highest levels, followed by the Renantiomer. Lipoate has been shown to be reduced to dihydrolipoate in all cells and tissues thus far 427
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examined: in the perfused rat liver (21), and isolated largendorff rat heart (13), isolated hepatocytes (22),, erythrocytes and leukocytes (13), keratinocytes (23) and fibroblasts (24), and in mitochondria (25),. There are two pathways for the reduction of exogenously supplied lipoate, one via NADH dependent lipoamide dehydrogenase of mitochondria, which is highly specific for the Renantiomer, and the other via NADPH-dependent glutathione reductase in the cytosol and mitochondrial matrix, which is slightly more specific for the S-enantiomer (12, 13). Our results are consistent with the operation of both pathways at near Vmax when lipoate is administered at the concentration used in this study. Thus, the racemate, which contains both the R- and the S- forms of lipoate, can utilize both pathways and produces the greatest concentration of dihydrolipoate. This may be occurring in the lens itself or in other tissues, which then release dihydrolipoate which is carried to the lens and taken up. If lipoate were administered in lower concentrations (as would be the case for chronic, as opposed to acute treatment), the pathways of reduction would likely not be operating at Vmax, and it would be predicted that in this case the R-enantiomer would produce the higher concentration of dihydrolipoate. Our results indicate that the effects of dihydrolipoate on other antioxidants may be especially important. We observed a restoration of glutathione, ascorbate, and vitamin E levels after treatment with R-a-lipoate and the racemic mixture (Figs 1, 2 and 3, respectively), as well as protection against cataract (Table 1), whereas S-a-lipoate did not provide any protection for other antioxidants, nor did it protect against cataract. These results correspond with the much higher levels of dihydrolipoate in the R-a-lipoate and rac-a-lipoate lenses, and support the idea tha dihydrolipoate is exerting its protective effects at least partially by restoring other antioxidants to near-normal levels. These results confirm and extend results found in our previous study, in which we found that administration of R,S lipoate largely prevented the losses of GSH and GSSG in the lens of BSO-treated newborn animals. In addition, R, S-lipoate protected against the loss of ascorbate and vitamin E and protected against cataract formation. In isolated rat lenses, similar stereospecific effects to those seen in this study were found, with R- and rac-a-lipoate protecting against glucoseinduced cataract formation and S-a-lipoate being completely ineffective. Cataract formation is a result of chemical modification of proteins; protein SH groups become oxidized (26, 27). It is possible that a-lipoate prevents the formation of cataract by maintaining a supply of thiols (in the form of dihydrolipoate). The greater protective effect of the racemic mixture compared to the R-enantiomer against BSO-induced cataract (Table 1), may be due to the fact that it produced a greater concentration of dihydrolipoate. In summary, R- and rac-a-lipoate maintain antioxidants mechanism in the lenses of newborn animals and protect against BSO-induced cataract formation. Since the S-form was completely ineffective, our results may be explained by preferential uptake of the R-enantiomer together with the presence of a high activity of the NADH-dependent pathway for lipoate reduction present in mitochondria with perhaps some contribution by NADPH pathways for reduction of the Senantiomer in the R, S-lipoate system. Together, these two pathways contribute to produce high levels of dihydrolipate when the R-enantiomer or the racemic mixture is administered in a single, acute dose. REFERENCES 1. Calvin, H. I., Medvedovsky, C., and Worgul, B. V. (1986) Science 233, 553–555. 2. Meister, A. (1989) in Glutathione: Chemical, Biochemical, and Medical Aspects (D. Dolphin, R. Poulson, O. Avramovic, Eds.) pp. 367–373, Wiley, New York. 3. Maitra, I., Serbinova, E., Tritschler, H., and Packer, L. (1995) Free Rad. Biol. Med. 18, 823–829. 4. Patel, M. S., and Smith, R. L. (1994) in The evolution of antioxidants in modern medicine (K. Schmidt, A. T. Diplock, and H. Ulrich, Eds.), pp. 65–77, Hippocrates Verlag, Stuttgart. 5. Rosenberg, H. R., and Culik, R. (1959) Arch. Biochem. Biophys. 80, 86–93. 6. Han, D., Tritschler, H. J., and Packer, L. (1995) Bioch. Biophys. Res. 207, 258–264. 428
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Busse, E., Zimmer, G., Schopohl, B., and Kornhuber, B. (1992) Arzneimittel-Forschung 42, 829–831. Packer, L., Witt, E. H., and Tritschler, H. J. (1995) Free Radic. Biol. Med. 19, 227–250. Yang, Y.-S., and Frey, P. A. (1989) Arch. Biochem. Biophys. 268, 465–474. Koplin, R., Ulrich, H., and Bisswanger, H. (1991) in Biochemistry and Physiology of Thiamine Diphosphate Enzymes, pp. 242–250, VCH Weinheim. Oehring, R., and Bisswanger, H. (1992) Biol. Chem. Hoppe-Seyler 373, 333–335. Pick, U., et al. (1995) Biochem. Biophys. Res. Comms. 206, 724–730. Haramaki, N., Han, D., Handelman, G. J., and Packer, L. (1996) Free Rad. Biol. Med. submitted. Anderson, M. E. (1985) Methods Enzymol. 113, 548–552. Dhariwal, K. R., Hartzell, W. O., and Levine, M. (1991) Am. J. Clin. Nutr. 54, 712–716. Lang, J. K., Gohil, K., and Packer, L. (1986) Anal. Biochem. 157, 106–116. Han, D., Handelman, G. J., and Packer, L. (1995) Meth. Enzymol. 251, 315–325. Kagan, V. E., et al. (1992) Biochem. Pharmacol. 44, 1637–1649. Jocelyn, P. C. (1967) Eur. J. Biochem. 2, 327–331. Kilic, F., Handelman, G. J., Serbinova, E., Packer, L., and Trevithick, J. R., Biochem. Mol. Biol. Int., in press. Peinado, J., Sies, H., and Akerboom, T. P. M. (1989) Arch. Biochem. Biophys. 273, 389–395. Müller, L., and Menzel, H. (1990) Biochim. Biophys. Acta 1052, 386–391. Podda, M., Han, D., Koh, B., Fuchs, J., and Packer, L. (1994) Clin. Res. 42, 41a. Handelman, G. J., Han, D., Tritschler, H., and Packer, L. (1994) Biochem. Pharmacol. 47, 1725–1730. Armstrong, M., and Webb, M. (1966) Biochem. J. 103, 913–922. Harding, J. J., Crabbe, M. J. C., and Davson, H., Eds., (1984) Vol. 2, Academic Press, New York. Kikugawa, K., Kato, T., Beppu, M., and Hayasaka, A. (1991) Biochem. Biophys. Acta 1096, 108–114.
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Stereospecific Effects of R-Lipoic Acid on Buthionine Sulfoximine-Induced Cataract Formation in Newborn Rats Indrani Maitra,1 Elena Serbinova,* Hans J. Tritschler,† and Lester Packer*,2 *Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; and †ASTA Medica, Weismüllerstraße 45, D-60314 Frankfurt Am Main, Germany Received March 11, 1996 This study revealed a marked stereospecificity in the prevention of buthionine sulfoximine-induced cataract, and in the protection of lens antioxidants, in newborn rats by a-lipoate. R- and racemic a-lipoate decreased cataract formation from 100% (buthionine sulfoximine only) to 55% (buthionine sulfoximine + R-a-lipoic acid) and 40% (buthionine sulfoximine + rac-a-lipoic acid) (p < 0.05 compared to buthionine sulfoximine only). S-a-lipoic acid had no effect on cataract formation induced by buthionine sulfoximine. The lens antioxidants glutathione, ascorbate, and vitamin E were depleted to 45, 62, and 23% of control levels, respectively, by buthionine sulfoximine treatment, but were maintained at 84–97% of control levels when R-a-lipoic acid or rac-a-lipoic acid were administered with buthionine sulfoximine; S-a-lipoic acid administration had no protective effect on lens antioxidants. When enantiomers of a-lipoic acid were administered to animals, R-a-lipoc acid was taken up by lens and reached concentrations 2- to 7-fold greater than those of S-a-lipoic acid, with rac-a-lipoic acid reaching levels midway between the R-isomer and racemic form. Reduced lipoic acid, dihydrolipoic acid, reached the highest levels in lens of the rac-a-lipoic acid-treated animals and the lowest levels in S-a-lipoic acid-treated animals. These results indicate that the protective effects of a-lipoic acid against buthionine sulfoximine-induced cataract are probably due to its protective effects on lens antioxidants, and that the stereospecificity exhibited is due to selective uptake and reduction of R-a-lipoic acid by lens cells. © 1996 Academic Press, Inc.
By the very nature of its function the occular lens is subjected to a constant barrage of free radicals. To counteract this oxidative insult, the lens has an efficient antioxidant system in the form of enzymes and low molecular weight antioxidants. Newborn rats are unable to synthesize ascorbate, and treatment of these animals with BSO, an inhibitor of glutathione synthesis, has been used as a model of oxidative stress, with cataract formation in lens after the animals open their eyes as an easily assessed end-point (1, 2). Depletion of lens GSH appears to be the prime effect of BSO treatment, initiating a general imbalance in the antioxidant defense system. Among the various postulated functions of GSH in the lens, the one which has received most attention in the last decade is its role in protection against oxidative damage. Hence, it is likely that the mechanism for BSO-induced cataract formation is an acceleration of oxidative damage in the lens due to severe depletion of lens antioxidant defenses; this model thus serves as an ideal model system for testing protective effects of exogenous antioxidant administration against cataract formation. In our previous work (3) we observed that administration of racemic a-lipoate increased both the GSH and GSSG levels in lens tissue of BSO-treated newborn animals, as well as the levels of other antioxidants, and protected against cataract formation, but the exact mechanism of this effect was not clear. Exogenously supplied a-lipoic acid (a-lipoate), a dithiol found endogenously as covalently bound lipoamide in a-keto acid dehydrogenases and in the glycine cleavage system (4), has been shown to substitute for ascorbate in vitamin C-deficient guinea pigs (5) and to raise intracellular 1
Present address: College of St. Catherine, 601 25th Avenue S, Minneapolis, MN 55454. To whom correspondence should be addressed. Abbreviations: BSO, buthionine sulfoximine; GSH, glutathione; GSSG, glutathione disulfide.
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levels of glutathione in vitro and in vivo (6, 7). In previous work, we studied the effect of racemic a-lipoate on cataract formation in BSO-treated rats, and found that it was effective in protecting newborn animals from BSO-induced cataract formation, reducing the incidence of cataract from 100% to 45% (3). Treatment with a-lipoate maintained the antioxidant status of the cell by maintaining a higher level of ascorbate and tocopherol, possibly through recycling mechanisms, and thereby contributed to decreased GSH utilization in the tissues. a-Lipoate has an asymmetric carbon at its C6 position. For the a-ketoacid dehydrogenase complexes from Escherichia coli (9) as well as for the mammalian pyruvate dehydrogenase complex (10) it has been shown that only the R-configuration of a-lipoate is able to function as a cofactor. In E. coli, both enantiomers of lipoic acid are taken up by the cell, while only the R-form is incorporated into the pyruvate dehydrogenase complex (11). However, eukaryotic cells possess mechanisms to reduce both enantiomers (12). Dihydrolipoamide dehydrogenase, the E3 enzyme in mitochondrial a-keto acid dehydrogenases, is highly specific for R-a-lipoate, whereas glutathione reductase, which can utilize a-lipoate in addition to glutathione disulfide as a substrate, is specific for S-a-lipoate (12, 13). The stereospecificity of cellular uptake mechanisms for the two enantiomers is not known. The present investigation sought to clarify the relative effect of the enantiomers on cataract formation in lenses of BSO-induced glutathione-deficient newborn animals,. MATERIALS AND METHODS Animals Sprague-Dawley rats (female, timed pregnant) (Simonsen, Santa Clara, CA), were housed in the Research Animal Facilities and given food and water ad libitum. Newborn animals of both sexes were used. They were breast fed. All procedures were approved by the University of California, Berkeley, Animal Care and Use Committee.
Generation of Cataracts Animals were divided into 8 groups and given 4 injections per day for 2 days, according to the following protocol:
Time Groups
8 a.m./4 p.m.
12 a.m./8 p.m.
Control +BSO BSO + R-LA BSO+Racemate BSO+S-LA R-LA only Racemate only S-LA only
NaCl BSO BSO BSO BSO R-LA Racemate S-LA
NaCl/NaCl:EtOH NaCl/NaCl:EtOH R-LA Racemate S-LA NaCl NaCl NaCl
Each experimental group consisted of 20–25 animals. BSO was dissolved in 0.1 M NaCl and injected at a concentration of 3 mmol/g body weight (total volume not exceeding 60 ml). a-lipoate was dissolved in ethanol: NaCl (50:50, v/v). BSO was injected subcutaneously into newborn animals (age 28–36 hours). a-lipoate (25 mg/kg b.w.) was given intraperitoneally. An equivalent volume of ethanol: NaCl and/or NaCl was given to the controls. Rats were sacrificed when they opened their eyes (14–15 days). Cataract formation was visualized with a slit-lamp microscope and the lenses were classified as opaque or clear. a-Lipoate uptake. In order to study the uptake of R-, S-, and rac-lipoate in lens, with and without BSO treatment, the same protocol as above was followed except that a total of 6 injections were given to the animals. After the last injection, the animals were sacrificed at 0, 1, 3 and 6 hrs and lenses were isolated as described below. Isolation of lenses. After decapitation of the animals the eyes were carefully dissected out from the ciliary body and the lens capsule was released from the enucleated eye globes. Each lens was weighed and placed in a microfuge tube and frozen immediately in liquid nitrogen for future analyses. 423
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Analytical Procedures Glutathione. Frozen lenses were homogenized in 5% sulfosalicylic acid, 5 mM EDTA, ice cold and bubbled with argon. After centrifugation GSH and GSSG were determined by the enzymatic recycling method (14). Ascorbate. Lenses were homogenized in ice-cold 90% methanol, 1 mM EDTA, bubbled with argon gas. Ascorbic acid and dehydroascorbic acid were measured by HPLC using electrochemical detection (15). Vitamin E. a-Tocopherol was extracted in hexane and analyzed by HPLC using electrochemical detector as described by Lang et al (16). a-Lipoate and dihydrolipoate. Frozen lenses were homogenized in 5% sulfosalicylic acid, 5 mM EDTA, ice cold and bubbled with argon. The lens homogenate was then extracted with equal volume of ethanol. After centrifugation the lens extract was analyzed by HPLC: determination of lipoic acid and dihydrolipoate was carried out on a Rainin Microsorb 3 um C18 column, 10 × 0.46 cm. The mobile phase was 55% water/25% methanol/20% acetonitrile, with 2% monochloroacetic acid. The flow rate was 1 ml/min with a 100 ul injection loop. Measurement of a-lipoate and dihydrolipoate was accomplished using an electrochemical detector with a dual Hg/Au electrode according to the method modified by Han et al (17). Chemicals. L-Buthionine-(S,R)-sulfoximine (BSO) was from Sigma Company, R-, S-, and rac-a-lipoate were from ASTA Medica (Frankfurt). HPLC grade ethanol and methanol were purchased from Fischer Scientific, Fair Lawn, NJ. All other reagents used were from Sigma Chemical Company, St. Louis, MO, or of the highest quality available.
RESULTS Cataract formation. All newborn animals treated with BSO alone developed cataracts (Table 1). None of the controls or the rats treated with (25 mg/kg body weight) enantiomers alone developed cataract. In case of BSO plus R-a-lipoate, and BSO plus rac-a-lipoate, 55% and 40%, respectively, of the animals developed cataract (Table 1). These results were significantly different from that of BSO treated group only (p<0.005). All the animals treated with S-a-lipoate and BSO developed cataract. Lens GSH and GSSG levels. GSH in the lenses of BSO-treated rats was about 55% lower than that in control lenses (Fig 1), (p < 0.0001). Treatment with BSO and R-a-lipoate or BSO and rac-a-lipoate simultaneously demonstrated a higher level of GSH (≈84–87% that of control level), significantly different from BSO treated group only (p < 0.001). Treatment with a-lipoate alone did not change the GSH level from the control level (data not shown). The efficiency of preventing the decrease in GSH levels in BSO-treated animals was about the same for R-a-lipoate and rac-alipoate. S-a-lipoate administered in conjunction with BSO was almost without effect on lenticular GSH level. Similarly, total glutathione (GSH+GSSG) level also decreases after BSO injection, from 3.24 mmoles/g lens tissue to 1.78±0.18 mmoles/g lens tissue. Treatment with R-a-lipoate and TABLE 1 Effect of Lipoic Acid Enantiomers on Cataract Formation in Newborn Animals with BSO-Induced Glutathione Deficiency Treatment
% Cataract formation
CONTROL BSO R-LA+BSO S-LA+BSO rac-LA+BSO R-LA S-LA rac-LA
0 100 55* 100 40* 0 0 0
In each experimental group n420–30. Data has been averaged from three separate experiments. * Significantly different (P < 0.005) from BSO treated group. 424
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FIG. 1. Effect of enantiomers of a-lipoic acid on glutathione levels in the lenses of newborn animals with BSO-induced glutathione deficiency. Conditions as in Methods. Filled bars, GSH. Cross-hatched bars, total glutathione (GSH + GSSG). N 4 4 for each bar. Data are given as mean ± SD. *significantly different from BSO treated group (p < 0.001).
BSO or rac-a-lipoate and BSO prevented the decrease in the total glutathione level almost to the same extent. Lens ascorbate levels. Administration of BSO led to a decrease in total lens ascorbate levels to 62% of control values (Fig 2). 93% and 90% of control values of lens ascorbate were found after treatment with BSO + R-a-lipoate or BSO + rac-a-lipoate, respectively, significantly different from the group treated with BSO only (p < 0.001). The level of ascorbate in lenses from animals treated with S-a-lipoate plus BSO was 68% of the control level, not significantly different from that in animals treated with BSO only. Lens vitamin E levels. Vitamin E levels in lenses dropped significantly after BSO administration,
FIG. 2. Effect of enantiomers of a-lipoic acid on ascorbate levels in the lenses of newborn animals with BSO-induced glutathione deficiency. Conditions as in Methods. Filled bars, ascorbate. Open bars, total ascorbate (ascorbate + dehydroascorbate). N46 for each bar. Data are given as mean ± SD. aSignificantly different from BSO-treated group (p < 0.001). b Significant from BSO-treated group. Other conditions as in Fig 1. 425
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from 6.0 ± 3.3 nmol/g tissue in control animals to 1.4 ± 0.6 nmol/g tissue in BSO-treated animals (23% of the control level) (p < 0.0001). Administration of BSO + R-a-lipoate maintained vitamin E levels at 97% of control levels (5.8±0.9 nmol/g tissue) and administration of BSO + rac-a-lipoate maintained it at 88% of control value (Fig 3), both significantly different (p < 0.005) from the group treated with BSO only. Lens vitamin E levels in animals treated with BSO + S-a-lipoate were not significantly different from those in animals treated with BSO only. Timecourse of uptake and reduction of lipoic acid by lens in animals. In control animals, increased lens levels of a-lipoate and dihydrolipoate were observed within one hour of injection of a-lipoate (Fig 4) and at this time uptake of R-a-lipoate or rac-a-lipoate was almost the same. But after three hours there was 3 times as much a-lipoate in the lenses of R-a-lipoate treated animals as in the animals treated with racemate mixture. In S-a-lipoate treated animals uptake of a-lipoate was very low compared to the other isomers. Dihydrolipoate formation was highest in the rac-alipoate treated animals. No a-lipoate or dihydrolipoate was detected after 6 hours for any treatment (Fig 4). In BSO-treated animals, the uptake of a-lipoate was much more rapid, though the same stereospecificity was present—the uptake of the R-form was highest and S-form was lowest (Fig. 4). DISCUSSION The present investigation reveals a high degree of stereospecificity for protection by exogenous lipoate against the BSO-induced formation of lens cataract in the newborn rat. The R-enantiomer and racemic mixture were highly protective, whereas the S-enantiomer was almost completely ineffective. We suggest that these marked differences in protective effect are probably due to differences in uptake and reduction of R-, rac- and S-a-lipoate. These results are consistent with several previous observations: (i) the existence of two pathways of reduction of a-lipoate with differing stereospecificities: NADH-dependent mitochondrial lipoamide dehydrogenase reduction specific for the R-enantiomer and the NADPH-dependent glutathione reductase reduction specific for the S-enantiomer (12, 13); (ii) dihydrolipoate can participate in reducing other antioxidants in the antioxidant network, including vitamin C (from dehydroascorbate or the semi-ascorbyl radical(18)), and glutathione (from glutathione disulfide(19).); (iii) the observation in other studies that isolated the rat lenses in culture induced to form opacity (cataracts) by high glucose exposure (a
FIG. 3. Effect of enantiomers of a-lipoic acid on vitamin E levels in the lenses of newborn animals with BSO-induced glutathione deficiency. Conditions as in Methods. N 4 4 for each bar. Data are given as mean ± SD. *Values significantly diData are given as mean ± SD.fferent from BSO treated group (p < 0.005) 426
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FIG. 4. Timecourse of uptake of and reduction of enantiomers of a-lipoic acid by lens in control animals and in animals treated with BSO. Conditions as in Methods. N 4 4–6 for each point. Data are given as mean ± SD. Closed symbols and solid lines: a-lipoic acid. Open symbols and dashed lines: dihydrolipoic acid. Circles: animals treated with R-a-lipoic acid. Triangles: animals treated with S-a-lipoic acid. Squares: animals treated with rac-a-lipoic acid.
model of diabetic cataractogenesis) show virtually complete stereospecificity for protection by the R as compared to the S form of lipoate(20). Several steps may be important for lipoate to be effective in protection of the lens against oxidatively-induced cataract. It must be taken up by lens cells. Pathways of reduction are also important, because dihydrolipoate is a potent reductant which can regenerate other antioxidants. In control rats, lens levels of R-a-lipoate were approximately 7-fold greater than those of S-a-lipoate three hours after intraperitoneal injection (Fig. 4); in BSO-treated rats the difference was approximately two-fold (Fig. 4). In both cases the racemic mixture was midway between the two enantiomers. Since R-a-lipoate is the naturally occurring enantiomer of a-lipoate in the body, a selective transport process of R-a-lipoate may already exist in the cells which might account for the greater degree of accumulation of R-a-lipoate in the lenses, and these differences in uptake probably accounted for part of the differences in protection. The levels of dihydrolipoate in lens do not follow the same pattern as lipoate. In both control and BSO-treated animals, the racemic mixture produced the highest levels, followed by the Renantiomer. Lipoate has been shown to be reduced to dihydrolipoate in all cells and tissues thus far 427
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examined: in the perfused rat liver (21), and isolated largendorff rat heart (13), isolated hepatocytes (22),, erythrocytes and leukocytes (13), keratinocytes (23) and fibroblasts (24), and in mitochondria (25),. There are two pathways for the reduction of exogenously supplied lipoate, one via NADH dependent lipoamide dehydrogenase of mitochondria, which is highly specific for the Renantiomer, and the other via NADPH-dependent glutathione reductase in the cytosol and mitochondrial matrix, which is slightly more specific for the S-enantiomer (12, 13). Our results are consistent with the operation of both pathways at near Vmax when lipoate is administered at the concentration used in this study. Thus, the racemate, which contains both the R- and the S- forms of lipoate, can utilize both pathways and produces the greatest concentration of dihydrolipoate. This may be occurring in the lens itself or in other tissues, which then release dihydrolipoate which is carried to the lens and taken up. If lipoate were administered in lower concentrations (as would be the case for chronic, as opposed to acute treatment), the pathways of reduction would likely not be operating at Vmax, and it would be predicted that in this case the R-enantiomer would produce the higher concentration of dihydrolipoate. Our results indicate that the effects of dihydrolipoate on other antioxidants may be especially important. We observed a restoration of glutathione, ascorbate, and vitamin E levels after treatment with R-a-lipoate and the racemic mixture (Figs 1, 2 and 3, respectively), as well as protection against cataract (Table 1), whereas S-a-lipoate did not provide any protection for other antioxidants, nor did it protect against cataract. These results correspond with the much higher levels of dihydrolipoate in the R-a-lipoate and rac-a-lipoate lenses, and support the idea tha dihydrolipoate is exerting its protective effects at least partially by restoring other antioxidants to near-normal levels. These results confirm and extend results found in our previous study, in which we found that administration of R,S lipoate largely prevented the losses of GSH and GSSG in the lens of BSO-treated newborn animals. In addition, R, S-lipoate protected against the loss of ascorbate and vitamin E and protected against cataract formation. In isolated rat lenses, similar stereospecific effects to those seen in this study were found, with R- and rac-a-lipoate protecting against glucoseinduced cataract formation and S-a-lipoate being completely ineffective. Cataract formation is a result of chemical modification of proteins; protein SH groups become oxidized (26, 27). It is possible that a-lipoate prevents the formation of cataract by maintaining a supply of thiols (in the form of dihydrolipoate). The greater protective effect of the racemic mixture compared to the R-enantiomer against BSO-induced cataract (Table 1), may be due to the fact that it produced a greater concentration of dihydrolipoate. In summary, R- and rac-a-lipoate maintain antioxidants mechanism in the lenses of newborn animals and protect against BSO-induced cataract formation. Since the S-form was completely ineffective, our results may be explained by preferential uptake of the R-enantiomer together with the presence of a high activity of the NADH-dependent pathway for lipoate reduction present in mitochondria with perhaps some contribution by NADPH pathways for reduction of the Senantiomer in the R, S-lipoate system. Together, these two pathways contribute to produce high levels of dihydrolipate when the R-enantiomer or the racemic mixture is administered in a single, acute dose. REFERENCES 1. Calvin, H. I., Medvedovsky, C., and Worgul, B. V. (1986) Science 233, 553–555. 2. Meister, A. (1989) in Glutathione: Chemical, Biochemical, and Medical Aspects (D. Dolphin, R. Poulson, O. Avramovic, Eds.) pp. 367–373, Wiley, New York. 3. Maitra, I., Serbinova, E., Tritschler, H., and Packer, L. (1995) Free Rad. Biol. Med. 18, 823–829. 4. Patel, M. S., and Smith, R. L. (1994) in The evolution of antioxidants in modern medicine (K. Schmidt, A. T. Diplock, and H. Ulrich, Eds.), pp. 65–77, Hippocrates Verlag, Stuttgart. 5. Rosenberg, H. R., and Culik, R. (1959) Arch. Biochem. Biophys. 80, 86–93. 6. Han, D., Tritschler, H. J., and Packer, L. (1995) Bioch. Biophys. Res. 207, 258–264. 428
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