Sugar cataracts in vitro: Implications of oxidative stress and aldose reductase I

The rat lens, when cultured in medium 199 containing 55·5 mm-glucose and 15% fetal calf serum, developed a visible equatorial cortical opacity by the 96th hour. The gerbil lens, whose aldose reductase activity was twice that of the rat lens, developed such an opacity after 24 hr. The development of opacity is thus faster in a lens with higher aldose reductase activity. The relation of aldose reductase with genesis of sugar cataracts is also evident from the observation that substitution of glucose by xylose accelerated cataract formation. Xylose is known to have lower K_m for aldose reductase than glucose. Fortification of the media with vitamin E (2·4 μm) or GSH (0·1 mm) did not prevent the cataract development. The level of lens malonaldehyde, one of the products of lipid peroxidation, was also unaffected either following culture in high sugar media or following in vivo induction of diabetes by streptozotocin. The oxidative stress, thus, does not seem to be involved in initiating the formation of sugar cataracts.     

In utero and milk-mediated effect of aldose reductase inhibitor on galactose cataracts.

Our previously reported investigations showed that cataracts could be induced in fetal lenses through the maternal feeding of galactose during pregnancy. We also reported that the lens opacity present at birth reverses completely by 30 days of age if there is no further post-natal exposure to galactose. This investigation was designed to investigate if an aldose reductase inhibitor (ARI) has any cross-placental effect in preventing galactose-induced cataracts in fetuses. We have also evaluated if there are any milk-mediated effects of galactose on cataract induction and of galactose and ARI on the maintenance or reversal of opacities induced in utero. Pregnant Sprague-Dawley rats were fed either 50% galactose or rat chow with or without the ARI, (2R,4S)-6-fluoro-2-methyl spirochroman-4,4'-imidazolidine-2',5'-dione (Eisai compound E-0722). Following parturition, pups of mothers from the different dietary groups were either fed by their own mothers or foster fed by lactating females fed either rat chow or 50% galactose with or without the ARI. Lenses of the pups were examined at desired intervals with light and scanning electron microscopes. We observed that: (a) both galactose and the ARI had a cross-placental effect on the fetal lenses in the development and inhibition of cataracts, respectively; (b) galactose had very little, if any, milk-mediated effect on either the induction of cataracts in newborn pups that were born with transparent lenses or the maintenance of cataracts induced in utero; (c) the ARI appeared to have a milk-mediated effect, which accelerates the reversal of cataract associated alterations in lenses of pups with cataracts induced in utero, leading to further reinstatement of lens transparency; and (d) the presence of ARI in the diet of rats during pregnancy and/or post-parturition provided continued protection to the lenses of pups that were exposed to a galactose diet following birth.     

Astragalin attenuates diabetic cataracts via inhibiting aldose reductase activity in rats

AIM: To investigate the aldose reductase (AR) inhibition capacity of astragalin (AST) against streptozoticin-induced diabetic cataracts (DCs) in rats. METHODS: Ex vivo investigations were conducted by treating the lens of a goat placed for 72h in artificial aqueous humor (AAH) of pH 7.8 at room temperature with cataract-causing substance (55 mmol/L of galactose) and in vivo studies were performed on rats via induction with streptozotocin. AST was administered at different dose levels and scrutinize for DC activity. RESULTS: In diabetic rats, AST improved the body weight, blood insulin, and glucose as well as the levels of galactitol in a dose-dependent way, other biochemical parameters i.e. inflammatory mediators and cytokines, and also suppress AR activity. The level of the antioxidant parameters such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) activity were also altered on a diabetic lens after the administration of the AST. CONCLUSION: AST protects against lens opacification to avoid cataracts and polyols formation, indicating that it could be used as a potential therapeutic agent for diabetes.     

Correlation between adult diabetic cataracts and red blood cell aldose reductase levels

PURPOSE: To investigate the correlation between adult diabetic cataracts and levels of aldose reductase (AR) in red blood cells (RBCs). METHODS: The study involved 337 eyes of 337 patients with diabetes. The extent and severity of lens opacity was assessed according to the Lens Opacities Classification System III (LOCS III). The AR levels within RBCs were determined with an ELISA. The relationship between the AR level in RBCs and the prevalence of nuclear cataract, cortical cataract, and posterior subcapsular cataract in patients with diabetes was examined. RESULTS: There were no significant alterations in AR level in RBCs in patients with a diabetes duration of < or = 10 years and patients < 60 years of age. In each subgroup, a higher amount of AR levels in RBCs significantly correlated with the prevalence of posterior subcapsular cataracts. A significant association between cortical cataract and AR level in RBCs was also seen in a subgroup of patients younger than 60 years. CONCLUSIONS: AR emerges as an important factor affecting the onset of posterior subcapsular cataracts at the early stages of diabetes mellitus. This raises the possibility that AR inhibitors could play a useful role in treatment of adult diabetic cataract through its inhibition of AR activities.     

Relative abundance of aldose reductase mRNA in rat lens undergoing development of osmotic cataracts

Aldose reductase (AR) messenger RNA concentration was determined in normal rat lens and in lens from rats fed a 50% galactose diet over a period of 20 days. The AR mRNA was detected by using a previously described AR cDNA clone. The relative concentration of the AR mRNA was estimated by cpm of 35S-UTP labeled antisense RNA hybridized to dot-blots prepared from cytosols isolated from single lens, decapsulated lens (cortex) and its respective capsule (epithelia). The results demonstrated that the concentration of the AR mRNA in the epithelium doubled over the 20 day period. Correspondingly, an increase in the concentration of the DNA was also observed, suggesting that the increase in epithelial cytosolic mRNA might be partially due to the increase in the number of epithelial cells occurring in lens undergoing cataractogenesis. The increase in AR mRNA in the epithelia was gradual, and it doubled by day 12 on galactose, while the increase in DNA was rapid and reached an optimum level by about day 4. By day 4 the cortex AR mRNA concentration increased, then rapidly decreased to insignificant levels by day 20. Changes in AR mRNA and in DNA following a high influx of galactose in the lens might suggest a heightened gene response to changes in the cellular environment for the lens epithelium.     

Rat lens aldose reductase: rapid purification and comparison with human placental aldose reductase

Aldose reductase (alditol:NADP oxidoreductase EC .1.1.1.21), an enzyme in the sorbitol pathway which has been implicated in the pathogenesis of diabetic complications, has been purified from rat lens (RLAR) by affinity chromatography with Amicon Matrex Gel Orange A and its properties have been compared to those of purified human placental aldose reductase (HPAR). The RLAR appears to be closely associated with alpha- and beta-crystallin and has a higher affinity for the dye Matrex column than HPAR. The purified enzyme, obtained upon elution from the column, appears as a closely-spaced doublet of approximately 38 K MW on SDS-PAGE which does not immunologically cross-react with antibodies raised against the single 38 K MW HPAR. Antibodies raised against RLAR however do cross-react with HPAR. Kinetic studies indicated both enzymes to have a greater apparent affinity for aliphatic and aromatic aldehydes than for aldose sugars. Compared to DL-glyceraldehyde, RLAR displayed an 80-fold greater affinity for p-nitrobenzaldehyde and a 1000-fold decreased affinity for D-glucose while HPAR displayed a 15-fold greater affinity for p-nitrobenzaldehyde and 600-fold less affinity for D-glucose. Both enzymes displayed only trace activity with 200 mM L-gulonic acid.     

Inhibition of aldose reductase from human retina

Aldose reductase was prepared from a pool of 21 male and 16 female human retinas by ammonium sulphate fractionation (40-75% saturation) and chromatography on DEAE-Sephacel and Matrex-OA. The overall purification was 132-fold with 50% recovery of enzyme activity. The concentrations of the aldose reductase inhibitors Sorbinil, Statil and M79175 required to give 50% inhibition (IC50 value) of enzyme activity with the model substrate 4-nitrobenzaldehyde (4NB) were 3.4 microM, 2.3 microM and 0.22 microM respectively. This indicated that M79175 was the most effective inhibitor tested of aldose reductase with 4NB in vitro. These inhibitors were more effective when tested against aldose reductase activity with glucose, the substrate which might play a role in the pathogenesis of diabetic complications. Sorbinil gave an IC50 (glucose) of 0.40 microM; M79175 and Statil were more effective. At an inhibitor concentration of 0.1 microM the %-inhibitions observed were: Sorbinil 20% M79175 55%, Statil 76%. Thus Statil was the most potent compound tested against human retinal enzyme using the more physiological substrate in vitro. This report provides the first direct evidence that human retinal aldose reductase is susceptible to inhibition by compounds designed for chemotherapy of diabetic complications, and indicates that the concentrations of inhibitor required for a substantial block of activity in vitro are lower than those attained in plasma in man.     

Intrinsic inhibition of aldose reductase.

The development of aldose reductase inhibitors for the treatment of diabetic complications, such as cataract and retinopathy, has been of intense interest in the pharmaceutical community for the last 20 years. To date, aldose reductase inhibitors have been synthetically developed from leads obtained from in vitro screening studies. Recently, we have observed that mammalian tissues contain intrinsic inhibitors of aldose reductase, which may be used as potential drugs for treating diabetic complications with potentially less side effects than synthetic aldose reductase inhibitors. Intrinsic inhibitor(s) of aldose reductase have been observed in the methanolic extracts from rat and human kidneys and bovine lenses that were subjected to a number of chromatographic techniques, including counter current chromatography, flash chromatography, gel filtration and high pressure liquid chromatography. This inhibition results from a direct interaction between the inhibitor and enzyme. The intrinsic inhibitor, present in the lipophilic fraction of human kidney and bovine lens extracts, can easily penetrate into the lens to inhibit sugar alcohol formation. Intraperitoneal injection of partially purified bovine lens extract inhibited lens polyol formation in young rats fed 50% galactose diet.     

Topical aldose reductase inhibitor formulations for effective lens drug delivery in a rat model for sugar cataracts.

PURPOSE: In the topical delivery of drugs to the lens, drug retention on the eye surface is considered to be important because increased retention on the ocular surface should lead to increased ocular absorption of a drug through the cornea into the aqueous humor and subsequently the lens. The aim of this study was to investigate whether increasing the viscosity of a topical aldose reductase inhibitor suspension increases the lenticular bioavailability of the inhibitor and whether such a formulation can arrest sugar cataract formation. METHODS: Five topical suspensions of 3% 2-methylsorbinil (2-MS) were prepared using (1) hydroxypropyl methylcellulose (HPMC, 0.5% w/v), (2) xanthan gum (0.5% w/v), (3) gellan gum (0.5% w/v), (4) carbopol (0.25% w/v), and (5) carbopol (0.25% w/v)--hydroxypropyl methylcellulose (HPMC) (0.25% w/v). Viscosity measurements were conducted with a viscometer. Lenticular levels of 2-MS were determined in the lenses from young Sprague Dawley rats receiving 1 drop of selected topical suspension twice-daily for 7 days. The efficacy of the suspensions to arrest sugar cataract formation was evaluated by administering the suspensions for 21 days to similar rats fed a diet containing 50% galactose. Lens changes were examined by portable slit lamp following mydriasis. RESULTS: Lenticular levels of 2-MS was highest in rats administered suspensions containing 0.25% carbopol + 0.25% HPMC as vehicles followed by 0.5% gellan gum, 0.5% HPMC, 0.25% carbopol, and 0.5% xanthan gum. All untreated rats fed a 50% galactose diet developed hypermature cataracts within 15 days; however, none of the topical treated rats demonstrated cortical vacuole formation after 21 days of galactose feeding. CONCLUSIONS: In the suspensions examined, no direct relationship between the lenticular drug levels of 2-MS and either viscosity or pH of the vehicles were observed. The observed arrest of sugar cataract formation indicated that therapeutically adequate lenticular levels of 2-MS were provided by all topical suspensions.     

Susceptibility of aldehyde and aldose reductases of human tissues to aldose reductase inhibitors

The effect of aldose reductase inhibitors such as sorbinil, alrestatin, and quercitrin has been studied on the aldose reductase purified from human brain and lens, and aldehyde reductase I purified from human liver, and aldehyde reductase II purified from human brain, liver, and red cells. None of the aldose reductase inhibitors have been found to be specific for aldose reductase. Fifty micromolar sorbinil besides inhibiting aldose reductase, completely inhibits aldehyde reductase II from the brain, liver and red cells. Similarly, alrestatin and quercitrin also are potent inhibitors of aldehyde reductase I and aldehyde reductase II.     

Hexokinase, glucose-6-phosphatase dehydrogenase and aldose reductase in human fetal lenses.

The lens HK, G6PD, AR activity and its relationship with fetal age was determined. There is a positive correlation between the age of fetus and the activity (IU/mg pro.) of HK and G6PD (r = 0.8069, 0.8204, P < 0.01) and a negative correlation between the age of fetus and activity of AR (r = -0.810 1, 0.05 > P > 0.01).     

Correlation between erythrocyte aldose reductase level and human diabetic retinopathy

AIM: To examine the relation between aldose reductase (AR) and the development and progression of diabetic retinopathy by comparing the erythrocyte AR levels with the prevalence of diabetic retinopathy in NIDDM patients. METHODS: A clinic based cross sectional study was used. 611 NIDDM patients and 73 controls were enrolled. Erythrocyte AR levels were determined by ELISA. These AR levels were then correlated with patient age, duration of diabetes, and HbA(1c) levels. AR levels were also correlated with the prevalence of diabetic retinopathy in the entire NIDDM patient group and in three subgroups formed by separating the NIDDM patients by their duration of diabetes. The prevalence of diabetic retinopathy significantly increased with increased erythrocyte AR levels in patients with duration of diabetes of less than 10 years. A similar, but non-significant correlation between the prevalence of retinopathy and increased erythrocyte AR levels was observed in patients with diabetes duration of 10-20 and >/=20 years. RESULTS: The prevalence of diabetic retinopathy increased with increased erythrocyte AR levels in NIDDM patients with a duration of diabetes of less than 10 years. CONCLUSION: It was suggested that the inhibition of AR in patients with early NIDDM might be beneficial in reducing the development of diabetic retinopathy.     

Efficacy of Alrestatin, an aldose reductase inhibitor, in human diabetic and nondiabetic lenses.

Immediately after cataract extraction, lenses from diabetic and nondiabetic patients were collected, classified, and assayed or incubated in high-glucose medium. The distribution of cataract types within the diabetic and nondiabetic groups was almost identical. The aldose reductase (AR) inhibitor AY22,284 (Alrestatin) was as effective in blocking sorbitol formation in diabetic as in nondiabetic lenses. While there was no difference in the level of intralenticular glucose, the diabetic lens produced significantly more sorbitol than did the nondiabetic lens. Also, the activity of polyol dehydrogenase (PD) was much lower in the diabetic population. The diabetic lenses swelled slightly more (P <.2) than nondiabetic lenses in high glucose media, and AY22,284 was effective in reducing the swelling of diabetic lenses in 35.5 mM glucose medium. While these results are preliminary, they suggest that diabetes, in some way, may confer on the human lens an increased susceptibility to osmotic stress via the sorbitol pathway. It is also reassuring to note that an AR inhibitor is no less effective in blocking the more active AR in the diabetic than in the nondiabetic lens. The therapeutic implications of this are discussed.     

Effect of insulin on aldose reductase activity in the lens of rats with streptozotocin-induced diabetes

Sprague-Dawley rats (5 weeks old) were injected intravenously with 65mg of streptozotocin (STZ)/kg body weight. The diabetic rats were injected subcutaneously with insulin chronically or acutely and biochemical parameters in the lens were determined. Increases in aldose reductase (AR) activity and contents of sobitol, glucose and fructose were found in the STZ-diabetic rats, compared with normal control rats. When the STZ-diabetic rats were injected with 10 units of monotard insulin on each of 7 successive days, AR activity tended to decrease, and contents of sorbitol and fructose significantly decreased, but the glucose content did not significantly decrease, compared with STZ-diabetic rats given no insulin. There was no decrease in AR activity in the STZ-diabetic rats 3 hours after an injection of 10 units of actrapid insulin. These results demonstrated that the chronic administration of monotard insulin may decrease AR activity and sorbitol content in the lens of STZ-diabetic rats without lowering glucose levels.     

The inhibition of bovine lens aldose reductase by Clinoril, its absorption into the human red cell and its effect on human red cell aldose reductase activity

The protein aldose reductase has been implicated in cataract in diabetes and galactosaemia. Recently it has been suggested that a number of non-steroidal anti-inflammatory agents have inhibitory activity against aldose reductase activity, and therefore might be used to prevent diabetic complications including cataract. Steady state kinetic experiments show that Clinoril (Sulindac sulphoxide) acts as a non-competitive inhibitor of NADPH oxidation with purified bovine lens aldose reductase, with an action that may involve binding to more than one site on the protein. As a preliminary to studying the effect on human lens and cataract, a double-masked, placebo-controlled study using random allocation into parallel groups was conducted on 20 volunteers to determine the penetration of Clinoril (Sulindac) and its metabolites into normal human red cells, and the effect of the drug on red cell NADPH-oxidising activity. It was found that while Clinoril, the sulphoxide form of the drug, and its metabolites the sulphone and the sulphide could be detected in the appropriate plasma samples (up to 36 micrograms of the sulphone/ml of plasma), very little could be detected in the red cells. There was no significant effect on red cell NADPH-oxidising activity.     

Polymorphisms of the aldose reductase gene and susceptibility to retinopathy in type 1 diabetes mellitus

PURPOSE: Aldose reductase (ALR2) is the first and rate-limiting enzyme of the polyol pathway and is involved in the pathogenesis of diabetic retinopathy. Polymorphisms of the ALR2 gene are associated with susceptibility to diabetic retinopathy in Chinese and Japanese patients with type 2 diabetes. There are no reports investigating these polymorphisms in white patients with type 1 diabetes from either Western Europe or North America. A CA dinucleotide repeat polymorphism (5'ALR2; located at -2100 bp) as well as a novel C(106)T polymorphism was investigated in 229 white patients with type 1 diabetes, with or without retinopathy. METHODS: The DNA was typed for these polymorphisms using conventional polymerase chain reaction techniques. RESULTS: There was a highly significant increase in the frequency of the Z-2 5'ALR2 allele and Z-2/X (where X is not Z+2) genotype in patients with diabetic retinopathy (n = 159) compared with those without who had diabetes of 20 years' duration (uncomplicated, n = 70; chi(2) = 17.0, P < 0.0001). There was a similar decrease in the Z+2/Y genotype (where Y is not Z-2; chi(2) = 30.1, P < 0.000,001) in the patients with retinopathy compared with the uncomplicated diabetes group. The C/Z-2 C(-106)T/5' ALR2 haplotype was found in 33.3% of the patients with retinopathy and 8.7% of the patients with uncomplicated diabetes. CONCLUSIONS: These results confirm previous studies in other populations and in type 2 diabetes showing that polymorphisms in the promoter region of the ALR2 gene are associated with susceptibility to diabetic retinopathy.