In Vitro Polyol Production by Rat Lens Aldose Reductase: Demonstration and Inhibition, Slides of Biochemistry

Download In Vitro Polyol Production by Rat Lens Aldose Reductase: Demonstration and Inhibition and more Slides Biochemistry in PDF only on Docsity! Biochem. J. (1986) 240, 233-237 (Printed in Great Britain) Purified rat lens aldose reductase Polyol production in vitro and its inhibition by aldose reductase inhibitors Peter F. KADOR,*M Jin H. KINOSHITA,* David R. BRITTAIN,t Donald J. MIRRLEES,t Christopher M. SENNITTI and Donald STRIBLINGt *National Eye Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A., and tlmperial Chemical Industries Ltd., Alderley Park, Macclesfield, Cheshire SK1O 4TG, U.K. The production of polyols in vitro by highly purified aldose reductase (EC 1.1. 1.21) was monitored by g.l.c. In the presence ofNADPH aldose reductase reduced glucose, galactose and xylose to the respective polyols sorbitol, galactitol and xylitol. The rates of formation of these polyols closely mirrored the Km values for the substrates obtained from kinetic measurements that monitored the rate of disappearance of NADPH. No polyol production occurred in the absence of purified aldose reductase, and analysis by g.l.c. revealed only the presence of unchanged monosaccharides. Addition of the aldose reductase inhibitor sorbinil to purified rat lens aldose reductase incubated with xylose in the presence of NADPH resulted in decreased xylitol production. However, aldose reductase inhibitors produced no effect in altering the rate of Nitro Blue Tetrazolium formation from either glucose or xylose, indicating that the observed inhibition in vitro does not result from a free-radical-scavenger effect. INTRODUCTION The metabolic conversion of glucose into fructose through the sorbitol pathway was first described by Hers (1956, 1957) to occur in the seminal vesicles and placenta. In the first step of this pathway glucose was shown to be reduced to the sugar alcohol sorbitol by the enzyme aldose reductase (EC 1.1.1.21). Shortly thereafter van Heyningen (1959) observed in both animal feeding experiments and lens homogenate incubations that aldose reductase, utilizing NADPH, could convert glucose, xylose and galactose into their respective sugar alcohols sorbitol, xylitol and galactitol in the rat lens. In addition, she reported the presence of sorbitol in the human diabetic lens. These initial observations led to the development of the Polyol Hypothesis of sugar cataract development (Kinoshita, 1974), which states that the aldose reductase-initiated accumulation of polyols pro- duces a hyperosmotic effect, which results in lens fibre swelling and eventual cataract formation. This hypothesis has been verified by studies with numerous animal models and by the development of potent aldose reductase inhibitors (Kinoshita et al., 1983). Numerous studies also suggest that the aldose reductase-initiated accumulation of polyols is involved in the pathogenesis of other ocular and systemic diabetic complications (Kador et al., 1985b). This has spurred great interest in the development of potent non-toxic inhibitors of aldose reductase. Despite the overwhelming evidence for the involve- ment ofaldose reductase-initiated polyol accumulation in the pathogenesis of diabetic complications, some reports have questioned both the biochemical importance of lens polyol formation and the mechanism through which polyols are formed by aldose reductase (Crabbe, 1984; Wolff et al., 1984; Wolff & Crabbe, 1985). These questions are largely based on the inability of these investigators to demonstrate in vitro the production of polyols by purified lens aldose reductase (Crabbe, 1984). It is suggested that the spectrophotometric determination of NADPH utilization as an index of aldose reductase activity is open to question because of an artifact caused by spontaneous monosaccharide oxidation in which free radicals that can oxidize NADPH to NADP+ are generated by the formation of an enediol. By this mechanism, the monosaccharide is oxidized to an ac-dicarbonyl instead of being reduced to a polyol (Wolff et al., 1984). It is also proposed that aldose reductase inhibitors depend on an antioxidant mechanism rather than inhibition of aldose reductase as such. These reports that aldose reductase activity is an artifact have prompted us to demonstrate the NADPH- dependent production of polyols from aldose sugars in vitro by highly purified rat lens aldose reductase. Moreover, the structure-activity relationships of aldose reductase inhibitors are not consistent with a radical- scavenging mechanism, and in this paper we report the lack of activity of a selection of potent aldose reductase inhibitors in a system sensitive to superoxide dismutase. MATERIALS AND METHODS Chemicals Unless otherwise stated, all chemicals were of reagent-grade quality. (NH4)2S04 (enzyme grade) was obtained from Bethesda Research Laboratories, Rock- ville, MD, U.S.A. NADPH and NADP+ were obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN, U.S.A. Isocitrate, isocitrate dehydrogenase, 0.3 M-ZnSO4 and 0.3 M-Ba(OH)2 were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Matrex Gel Orange A was obtained from Amicon Corp., Danvers, MA, U.S.A. NO-Bis(trimethylsilyl)trifluoroacetamide containing 1% chlorotrimethylsilane was purchased from Regis Chemical Co., Morton Grove, IL, U.S.A. The SE 30 Chromosorb g.l.c. column was obtained from Supelco, Bellefonte, PA, U.S.A. Nitro Blue Tetrazolium (grade III crystalline) was purchased from Sigma Chemical Co., Poole, Dorset, U.K. $ To whom correspondence should be addressed. Vol. 240 233 P. F. Kador and others Purification of rat lens aldose reductase Aldose reductase was purified from rat lens by affinity chromatography as previously described but with a modified phosphate buffer, namely 20 mM-sodium phos- phate buffer, pH 7.5, containing 7 mM-2-mercapto- ethanol, 0.5 mM-EDTA and 10% (v/v) glycerol (Herrmann et al., 1983; Shiono et al., 1986). Batches of 800 lenses were homogenized in 600 ml of buffer, centri- fuged at 12000 g and fractionated with (NH4)2SO4. The precipitate of the 40-70o/ -saturation fraction was collected by centrifugation at 12000 g, dialysed against the same buffer and applied to a 2.5 cm x 45 cm Bio-Rad column containing Amicon Matrex Gel Orange A. The column was washed with 450 ml of buffer, and the the enzyme was eluted with buffer containing 0.1 mM- NADPH. Fractions containing aldose reductase activity were concentrated with an Amicon column concentrator containing a YM-10 membrane. The enzyme had an approximate specific activity of 3.5 units (,umol/min)/mg of protein as determined with glyceraldehyde and appeared as a single band of Mr approx. 38000 on SDS/polyacrylamide-gel electrophoresis (Fig. 1). Incubation in the presence of an NADPH-generating system Sealed 7 ml glass vials containing purified aldose reductase (approx. 1.05 units), 10 mM-aldose sugar, 10 mM-NADPH, 1 mM-NADP+, 1 mM-DL-isocitrate, 2 mM-MgCl2, isocitrate dehydrogenase (13.8 units) and 0.1 M-sodium phosphate buffer, pH 7.8, in 2 ml final volume were placed in a 37 °C incubator. After overnight incubation (14 h) the solution was treated with 1 ml each of 0.3 M-Ba(OH)2 and 0.3 M-ZnSO4. The supernatant obtained by centrifugation was freeze-dried and treated in pyridine with bis(trimethylsilyl)trifluoro- acetamide containing 1% chlorotrimethylsilane. Incubation in the absence of an NADPH-generating system Sealed 7 ml glass vials containing in a total 1 ml volume purified aldose reductase (approx. 0.5 unit), 10 mM-xylose, 10 mM-NADPH and 0.1 M-sodium phos- phate buffer, pH 6.2, were placed in a 37 °C incubator. After overnight incubation (14 h) the solution was treated with 0.5 ml each of 0.3 M-Ba(OH)2 and 0.3 M- ZnSO4 and then processed as above. G.l.c. G.l.c. with an SE 30 Chromosorb glass column at 150 °C and helium flow under 0.4 MPa (58 lbf/in2) pressure was conducted on a Varian model 3700 gas chromatograph equipped with a Vista CDS-401 data system. The retention times of the aldose sugars and their corresponding polyols were determined through chro- matography of appropriate standards. Evaluation of aldose reductase inhibitors against generated free radicals The reduction of Nitro Blue Tetrazolium to formazan by aldopentoses was monitored spectrophotometrically in an adaption of the method of Kashimura et al. (1982). The reaction mixture consisted of 0.15 mM-Nitro Blue Tetrazolium and 0.1 mM-EDTA (dipotassium salt) in 3 ml of 0.05 M-sodium bicarbonate buffer, pH 10.2, and an aldopentose, generally D(+)-xylose (10 mg/ml). The rate of formazan production was monitored spectro- lo-, x Mr~ 92.5 66.24 45 31 21.5 14.4 1 2 Fig. 1. SDS/polyacrylamide-gel electrophoresis of purified rat lens aldose reductase For experimental details see the text. Lane 1, Mr markers; lane 2, rat lens aldose reductase. photometrically at 560 nm with a Pye-Unicam SP.8200 instrument. RESULTS In order to demonstrate the production of polyols in vitro by rat lens aldose reductase, the highly purified enzyme was initially added to a sealed system capable of generating NADPH from NADP+. This system consisted of isocitrate dehydrogenase and its substrate isocitric acid in phosphate buffer at the pH optimum of isocitrate dehydrogenase (pH 7.8) and containing MgCl2. Over- night (14 h) incubation at 37 °C of aldose reductase with 10 mM-xylose, -galactose or -glucose and 10mM-NADPH in this sealed system resulted in the production of the corresponding polyol xylitol, galactitol or sorbitol (Scheme 1). G.l.c. analysis indicated nearly 1000% conversion of xylose into xylitol, 450 conversion of galactose into galactitol and 250 conversion of glucose into sorbitol (Fig. 2). Only the starting aldose and corresponding polyol product peaks could be detected in the chromatograms. No conversion of xylose into xylitol was observed in the absence of aldose reductase compared with 19.7 + 0.3 Itmol in the complete incubation mixture. Omitting the NADP+ from the reaction mixture did not decrease xylitol production 1986 234