Glutathione reductase in normal and cataractous human lenses

Rogers, Karl; Augusteyn, Robert C.

doi:10.1016/0014-4835(78)90041-6

Cited by 71 publications

(30 citation statements)

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“…Thus, our results suggest that ascorbic acid, present in higher concentration than glucose in the healthy lens and more prone to oxidative decay, may play a major role in lens aging and cataract formation. In vivo, accelerated ascorbic acid oxidation and subsequent reactions may be secondary to a progressive malfunctioning of the protective, complex lenticular reductionoxidation system; for instance, the known decrease in glutathione reductase in aging lenses may thus eventually result in an oxidative chain reaction (12,35). Free radical formation occurs during the Maillard reaction and also can be expected to take place in the low-oxygen environment of the crystalline lens.…”

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confidence: 99%

“…The most significant of these appears to be the glutathione system in conjunction with glutathione peroxidase, superoxide dismutase, and ascorbic acid (12). In mammals, ascorbate is present in the lens in unusually high concentration, as much as [30][31][32][33][34][35] times the blood level; in man, the lens ascorbate concentration is even greater than the already high level present in the aqueous humor (13,14). However, little is known about the role of ascorbic acid in lens function and in cataractogenesis.…”

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The role of ascorbic acid in senile cataract.

Bensch¹,

Fleming²,

Lohmann³

1985

Proc. Natl. Acad. Sci. U.S.A.

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The reductone ascorbic acid, present in the crystalline lens in concentrations higher than those of glucose, is capable of undergoing nonenzymatic "browning" in the presence of lenticular proteins. We studied the nonenzymatic browning with ascorbate in model systems employing bovine serum albumin and lens crystailins. When bovine serum albumin, a-crystallin, or y-crystallin was incubated with[14C]ascorbic acid, the formation of yellow and then brown condensation products appeared to correlate with increasing protein-associated radioactivity. The fluorescence spectrum of these products was similar to that of homogenates of human cataractous lenses. We suggest that the nonenzymatic reaction of lens crystallins with ascorbic acid may contribute, at least in part, to the color changes of aging lenses and to the physical lenticular deterioration leading to senile cataract. High dietary intake of ascorbic acid did not affect the fluorescence spectrum of murine lenses; thus, we assume that the speed and extent of the lenticular browning reactions must depend on a deterioration of other factors of the multicomponent antioxidant system of the eye.Senescent cataract is a very common spontaneous eye affliction in persons beyond middle age. It becomes progressively more severe and frequent in the elderly, with more than 85% of octagenarians having some degree of lenticular opacification (1). Of particular interest to us is the most common type of senescent cataract, the so-called nuclear sclerosis, in which the central portion of the lens undergoes a gradual increase in density and opacity, usually accompanied by a yellow to dark-brown discoloration (2). What prompted us to undertake these studies was the striking similarity in the appearance of such lenses with the color changes that develop during slow spontaneous autoxidation of ascorbic acid solutions, either in the presence or the absence of proteins. We here report observations on oxidative ascorbic acid-protein interactions in vitro. Comparison of our findings with data on cataractous lenses suggests that the biochemical reactions observed by us may play a role in the aging of human lenses. MATERIALS AND METHODSAll reactions were carried out in 67 mM potassium phosphate buffer (pH 7.4). Sterile solutions of ascorbic acid (Sigma) and proteins (bovine serum albumin, bovine lens a-crystallin, and bovine lens y-crystallin) were incubated for various times up to 1 month for, fluorescence studies, both at room temperature and at 370C. Bovine serum albumin was obtained (Sigma) as Cohn fraction V, essentially fatty acid-free. Lens crystallins were a gift from T. Chiou (National Taiwan University, Institute of Biochemical Sciences, Taipei, Taiwan). Sterility of the incubation mixtures was monitored periodically by testing aliquots on nutrient agar plates. The fluorescence spectra of these incubation mixtures were obtained with an Aminco Bowman spectrophotofluorometer model 4-8106 after prolonged dialysis of the ascorbic acid-containing protein solutions against lar...

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The role of ascorbic acid in senile cataract.

Bensch¹,

Fleming²,

Lohmann³

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…The lens contains a relatively high concentration of reduced glutathione (28). A loss of glutathione in addition to a loss of glutathione reductase has been reported during cataractogenesis (29,30). This might expose the lens to higher oxidative stress and lead to increased conversion of ascorbate to its oxidative products, thus facilitating the synthesis of pentosidine.…”

Section: Resultsmentioning

confidence: 99%

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

Nagaraj

Sell

Prabhakaram

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

233

144

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Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with a-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.

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“…The reasons for this difference is not clear at present. However, Rogers and Augusteyn [ 31 ] have suggested that a small amount of GSH-R activity could maintain normal GSH levels even in cataractous lenses. It is also possible that this difference could be due to differences in the strains of rats used.…”

Section: Pv Rao and Ks Bhat Discussionmentioning

confidence: 99%

Influence Dietary Riboflavin Deficiency on Lenticular Glutathione Redox Cycle, Lipid Peroxidation, and Free Radical Scavengers in the Rat

Rao

Bhat

1989

J. Clin. Biochem. Nutr.

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(first set of animals) or 0.05 M sodium phosphate buffer, pH 7.2 (second set of animals) using a Potter/Elvehj am glass homogenizer. A 7.5% homogenate was prepared from two lenses (third set of animals) in 5% ice-cold TCA. Aliquots of J. Clin. Biochem. Nutr. INFLUENCE OF RIBOFLAVINDEFICIENCY ON RAT LENS 197 the lens homogenate (Tris-EFTA buffer) were analysed for GSH and GSSG measured fluorimetrically [11] in a Hitachi 650-lOS spectrofluorimeter and for total and non-protein sulfhydryl groups with Ellman's reagent [12]. Lipid peroxide level was estimated (in terms of malondialdehyde (MICA)) in the phosphate buffer by the thiobarbituric acid (TBA) reaction [13] and ascorbic acid in 5,000>< g supernatant of 5% TCA homogenate, using orthophosphoric acid, ferric chloride and aa '-dipyridyl [14]. The remaining homogenate (Tris-EFTA buffer) was centrifuged at 20.000>< g in a Sorvall RCSB super-speed centrifuge at 4°C for 30 min. The 20,000>< g supernatant was used for the assay of glutathione redox cycle enzymes. Glucose 6-phosphate dehydrogenase (G6PDH) was assayed spectrophotometrically as described by Selvaraj, and Bhat [15]. The reaction mixture contained in 1.2 ml: 50,u mol glycylglycine buffer (pH 8.7), 12 ,u mol KCl, 12 ,u mol MgC12, 3 ,u mol nicotinamide, 61u mol glucose 6-phosphate, and 75 1a l lens tissue supernatant. The reaction was started by adding 0.12 ,u mol NADP+. GSH-R was assayed by the method of Bayoumi, and osalki [16] as described elsewhere [9]. Glutathioneperoxidase (GSH-PX) was assayed as described by Hochstein and Utley [17] with the following modification: The reaction mixture contained in 1 ml: 50 ,u mol phosphate buffer (pH 7.2), 2.5 ,u mol GSH, 0.5 ,u mol NaN3, 0.3 ,u mol EFTA, 0.1 ,u mol NADPH, 0.5 IU GSH-R, and 75 ,u 1 supernatant (enzyme preparation, 1 : 5 dilution). The reaction mixture was incubated at 37°C for 10 min and the reaction started by the addition of 0.251a mol tert-butyl hydroperoxide. Catalase in the 750 X g supernatant fraction of the homogenate prepared in phosphate buffer was assayed polarographically as described by Goldstein [18] with a polarograph (YSI, Model 5301) equipped with an oxygen monitor (model 53) and a recorder (LKB 2210). The activity of superoxide dismutase (SOIL) in supernatant fractions of the lens homogenate (phosphate buffer) obtained by centrifugation at 35,000>< g and 0-4°C for 25 min was determined spectrophotometrically by measuring the inhibition of autooxidation of pyrogallol [19]. Protein concentration was measured by the method of Lowry et al. [20]. Statistical analysis. The data was analysed by one way analysis of variance. The mean differences were also further tested by Duncan's multiple range test. RESULTSThis study spans three different animal experiments in every one of which riboflavin-deficient rats showed a severe growth retardation, hair loss, and skin lesions, all of which are characteristic features of riboflavin deficiency.Ocular examination revealed severe corneal vascularization in riboflavindeficient rats (30%) as comp...

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Glutathione reductase in normal and cataractous human lenses

Cited by 71 publications

References 5 publications

The role of ascorbic acid in senile cataract.

The role of ascorbic acid in senile cataract.

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

Influence Dietary Riboflavin Deficiency on Lenticular Glutathione Redox Cycle, Lipid Peroxidation, and Free Radical Scavengers in the Rat

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