Leptospirosis with acute renal failure and paraparesis.

Leptospirosis is an important zoonosis with a worldwide distribution that is characterized by a broad spectrum of clinical manifestations ranging from inapparent infection to fulminant disease. The presentation of paraparesis in combination with acute renal failure is rare.

The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium.

PURPOSE: High levels of ascorbic acid are known to be present in the aqueous humor of many diurnal species, whereas nocturnal animals have low concentrations of the compound. The purpose of this study was to test the hypothesis that the high concentration of aqueous ascorbate in diurnal animals protects the lens against ultraviolet (UV)-induced damage to the eye. This study compares the effect of UV-B-induced DNA strand breaks on the lens epithelia of guinea pigs and rats after depletion or elevation of aqueous humor ascorbate, respectively. METHODS: Eyes of guinea pigs and rats were exposed to UV-B radiation (0.25-0.75 J/cm2 on the cornea) for 10 minutes, and DNA strand breaks in lens epithelium were measured by single-cell gel electrophoresis. Ascorbic acid concentration in the aqueous humor, lens, and lens-capsule epithelium were assayed by spectrophotometric and electrochemical methods. For depletion of aqueous humor and lens ascorbate in guinea pigs, the animals were maintained on an ascorbate-deficient diet. Aqueous ascorbic acid was elevated in the rat by intraperitoneal injections of sodium ascorbate (1 g/kg). RESULTS: The ascorbate concentration in the aqueous humor of the normal rat was approximately 3% that of the guinea pig, whereas the concentration of the compound in the lens of the normal rat was 10% that of the guinea pig. Guinea pigs fed an ascorbate-deficient diet showed a dramatic drop of more than 80% in aqueous humor ascorbate in the first week, whereas lens ascorbate decreased by approximately 25% during this time period. After a single intraperitoneal injection of sodium ascorbate in the rat, aqueous humor ascorbic acid increased nearly 30 times that in the control, whereas lens ascorbate increased by approximately 30%. The extent of DNA damage in the lens epithelium of a normal rat exposed to UV-B was significantly greater than that occurring in lenses of normal guinea pigs after exposure to the same dose of radiation. Lenses from ascorbate-deficient guinea pigs showed 50% more DNA damage than those from normal guinea pigs after UV exposure, whereas the lenses in ascorbate-injected rats exhibited significant protection against UV-induced DNA strand breaks. CONCLUSIONS: High levels of ascorbic acid in the aqueous humor had a protective effect against UV-induced DNA damage to lens epithelium. The results were consistent with the hypothesis that high ascorbic acid in diurnal animals protects the lens against the cataractogenic effect of UV radiation in sunlight.

A protective role for glutathione-dependent reduction of dehydroascorbic acid in lens epithelium.

PURPOSE: In view of the antioxidant role of ascorbic acid and the glutathione redox cycle in the lens, the authors have studied the relationship of the cycle to reduction of the oxidized product of ascorbic acid, dehydroascorbic acid (DHA), in lens epithelium. METHODS: Cultured dog lens epithelial cells and intact rabbit lenses were exposed to various concentrations of DHA in experiments performed at 20 degrees C to minimize hydrolysis of the compound (t1/2 of 5 minutes at 37 degrees C). Levels of glutathione (GSH) and oxidized glutathione (GSSG) were measured in lens cells and whole lens epithelial by electrochemical detection. RESULTS: Treatment of lens cells with 1 mM DHA for 0.5 to 3 hours in the absence of glucose (glucose is required for the reduction of GSSG through the glutathione redox cycle) produced from 60% to complete oxidation of GSH (controls contained negligible GSSG) and distinct morphologic changes (cell contraction and blebbing), as shown by scanning electron microscopy. Glucose prevented these effects and allowed nearly immediate recovery of GSH after DHA exposure in the absence of glucose. A dose-dependent response was observed for the formation of GSSG in cultured cells from 0.05 to 0.5 mM DHA in the absence of glucose. The results of experiments performed with DHA plus an inhibitor of glutathione reductase mimicked those obtained using DHA minus glucose. DHA produced a 3- to 10-fold stimulation of hexose monophosphate shunt activity in cultured lens cells and whole lenses, which was prevented by the inhibition of glutathione reductase. Treatment of whole lenses with DHA minus glucose also produced oxidation of epithelial GSH and was accompanied by the loss of lens transparency. No evidence was found for dehydroascorbate reductase activity in the lens epithelium. CONCLUSIONS: The exposure of lenses and lens epithelial cells to DHA under conditions in which the glutathione redox cycle was compromised resulted in the disappearance of GSH in the tissues and the appearance of GSSG. The reduction of DHA was shown to be linked to the glutathione redox cycle by a nonenzymatic interaction between GSH and DHA. Reduction of DHA in the lens is important because of the potential toxicity of this oxidant and/or its degradation products.

Simultaneous measurement of reduced and oxidized glutathione in human aqueous humor and cataracts by electrochemical detection.

A sensitive, electrochemical method was employed for the simultaneous measurement of reduced and oxidized glutathione in lens cortex, nucleus and capsule epithelia of rabbit lenses, normal human lenses and human cataracts. In addition, aqueous humor from cataract patients was also analyzed. The level of GSSG in the nucleus of human cataracts was significantly higher than that in the nucleus of normal eye bank lenses. The capsule epithelium of intracapsular extracted cataracts possessed high levels of reduced glutathione, despite the fact that much of the glutathione in the cortex and nucleus of the lenses was depleted. Levels of GSH in the aqueous humor of cataract patients were several times higher than those reported for normal aqueous humor. Electrochemical detection proved to be a useful technique for analysis of reduced and oxidized glutathione in lens and aqueous humor, especially when sample size is small, such as for capsule epithelium.

Hypertonic stress increases NaK ATPase, taurine, and myoinositol in human lens and retinal pigment epithelial cultures.

PURPOSE: Recent evidence suggests that taurine and myoinositol may serve as organic osmolytes in a number of cells, including lens and retinal pigment epithelia, but the mechanism for their increased accumulation in response to hypertonic stress is not known. To assess whether NaK ATPase contributed to the elevated levels of taurine and myoinositol in cells exposed to hypertonic media, we measured the activity of NaK ATPase, which is known to be implicated in the transport of these substances, in human lens and retinal pigment epithelia cultured in isotonic and hypertonic media. METHODS: Primary cultures of human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells were maintained in isotonic and hypertonic media for varying periods of time, and the activity of NaK ATPase and the levels of taurine and myoinositol were measured in cells cultured under two different conditions. The possible involvement of the transport enzyme in the accumulation of the two osmolytes was also investigated by inhibiting the enzyme with ouabain. RESULTS: When primary cultures of HLE and HRPE were exposed to hypertonic medium containing NaCl (600 mOsm) or cellobiose (500m Osm) for 72 hours, the concentration of taurine and myoinositol in HLE cells increased by 218% and 558% of control, respectively, in NaCl medium, whereas the corresponding increases in cellobiose medium were 147% and 439%. In HRPE cells, the increase in myoinositol levels in the two hypertonic media was more dramatic than that in taurine. Concomitant with the increase in the concentration of the osmolytes, there was an increase in NaK ATPase activity in both cell types. Although the accumulation of taurine in HLE cells in hypertonic media in a 6-hour culture was essentially prevented by 10(-8) mmol/l ouabain, myoinositol levels were affected to a lesser, but still significant, extent. In HRPE cells, which were cultured for 24 hrs in the presence of 10(-6) mmol/l ouabain, there was a more direct correlation between the inhibition of NaK ATPase and the decreased accumulation of taurine and myoinositol in the hypertonic media. CONCLUSION: Although the exact mechanism by which NaK ATPase activity increases in response to hypertonic stress remains to be established, the increased activity of the enzyme is related to the enhanced accumulation of the organic osmolytes, taurine, and myoinositol, in HLE and HRPE cells cultured in hypertonic medium.

Study of the polyol pathway and cell permeability changes in human lens and retinal pigment epithelium in tissue culture.

The polyol pathway was investigated in primary cultures of human retinal pigment epithelial (HRPE) cells and the results were compared with those in human lens epithelial (HLE) cells cultured under similar conditions. Significant levels of galactitol were formed in HRPE cells cultured for 72 hr in a medium containing 30 mmol/l D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy (TEM). Biochemical analysis revealed a significant depletion of cellular myoinositol, taurine, and a number of other free amino acids similar to those in HLE cells. These morphologic and biochemical changes observed in HRPE cells cultured in high galactose medium were inhibited or prevented by the inclusion of an aldose reductase inhibitor in the medium, further supporting the view that vacuole formation is due to the osmotic effect of polyol formation mediated by aldose reductase. The similarity of intracellular vacuole formation resulting from polyol accumulation and the biochemical changes in HRPE and HLE cells strongly suggests that a common mechanism is involved.

Exposure of rabbit lens to hyperbaric oxygen in vitro: regional effects on GSH level.

Previous studies have indicated that in vivo exposure to hyperbaric O2 may be associated with the development of nuclear cataract. In the present work, in vitro effects of hyperbaric O2 on rabbit lenses were investigated following culture of the lenses in an atmosphere of 99% O2 at pressures ranging between 1 and 100 atm. Treatment with O2 resulted in a significant decrease in the level of reduced glutathione (GSH) in the lenses even at the lower pressures studied (less than 8 atm). At 100 atm O2 the loss of GSH was 85% after a 3 hr exposure. At 8 atm O2 a significant drop in GSH concentration was shown to occur in the lens nucleus prior to loss of the tripeptide in the superficial cortex. O2-treated lenses became hazy in appearance, especially at the higher pressures, but did not become densely opaque. Pressures of N2 up to 100 atm had no effect on either lens transparency or on the concentration of GSH. Although oxidized glutathione (GSSG) was detected in the whole lens at pressures of O2 as low as 4 atm, no change in GSH level or evidence for GSSG accumulation was observed in the capsule-epithelium of the lens at pressures as high as 50 atm O2. Ninety percent of the GSSG present in lenses after exposure to 100 atm O2 could be reconverted to GSH by subsequent culture of the lenses under normal conditions. Exposure of lenses to 50 atm O2 produced a three-fold stimulation of hexose monophosphate shunt activity, equal to that which has been reported for treatment of lenses with 0.06 mM H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glutathione depletion on cation transport and metabolism in the rabbit lens.

The role of reduced glutathione (GSH) in lens membrane function was studied by depleting GSH with 1-chloro-2,4-dinitrobenzene (CDNB), a reaction catalyzed by GSH-S-transferase. Depletion of GSH in the lens epithelium by 70-90% led to a decrease in uptake and increase in efflux of 86Rb. ATP levels and Na+/K+-ATPase activity were normal while there was a slight decrease in lactate production. The results provide the first direct evidence that depletion of endogenous GSH per se does not lead to inactivation of Na+/K+-ATPase. However, lenses deficient in GSH when challenged with a normally tolerated level of H2O2 showed significant inactivation of membrane ATPase without a further increase in membrane permeability. Pretreatment with CDNB resulted in a 3-fold stimulation of the hexose monophosphate shunt activity which is attributed to the unexpected finding of a significant increase in the level of oxidized glutathione in the lens. It is concluded that deficiency of GSH causes a marked increase in membrane permeability and such lenses are susceptible to oxidative damage resulting in inactivation of the Na+/K+ pump, thus leading to ionic changes and cataract development.

The effects of X-irradiation on lens reducing systems.

Studies have been made of the effects of X-ray on various lens reducing systems, including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS) and of certain enzymes, including GSH reductase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PG). It was found that during several weeks following X-irradiation but prior to cataract formation, there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the X-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the X-rayed lens was found to decrease significantly relative to controls 1 week prior to cataract formation, and the ratio of NADPH to NADP+ in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the X-rayed lens. A corresponding decrease occurred in the activity of the HMS in X-rayed lenses as measured by culture in the presence of 1-14C-labeled glucose, G-6-PD was partially inactivated in the X-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to X-irradiation. The data indicate that X-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high-molecular-weight protein aggregates may be initiated.

Glutathione and lens epithelial function.

The relationship of the concentration of glutathione (GSH) in lens epithelium to the transport of cations in the lens was studied by decreasing the level of GSH in the epithelium and monitoring subsequent effects in the lens on the distribution of cations, the activity of Na+-K+ ATPase and the uptake and efflux of 86Rb. Oxidation of GSH in cultured rabbit lenses was accomplished by the use of 1 mM tertiary butyl hydroperoxide (TBHP), a reagent which appears to be suitable for the specific oxidation of GSH in this tissue. The concentration of GSH found in the normal lens epithelium was estimated to be 64 mum per gram wet weight or nearly six times that present in the whole lens. A decrease in the concentration of GSH in lens epithelium of 60 per cent or more leads to an increase in hydration, a shift in the distribution of Na+, K+, and Cl-, a decrease in the activity of Na+-K+ ATPase, and a decrease in the active transport, and an increase in the passive diffusion of 86Rb. In the TBHP-treated lenses there is a rapid decrease in the production of lactate, possibly as a result of the inhibition of Na+-K+ ATPase, but the effect on the level of lens ATP is delayed and less pronounced. It appears that the adverse effect on membrane permeability caused by the oxidation of GSH is partially reversed when a high level of GSH returns to the epithelium. However, the decrease in active transport of 86Rb and the inactivation of Na+-K+ ATPase are not reversed by either regeneration of GSH in the tissue or by treatment with exogenous dithiothreitol and may indicate an irreversible conformational change in the enzyme initiated by the loss of the protective effect of GSH. The data indicate that a critical level of GSH is required in the lens epithelium for the maintenance of normal cation transport.

Evidence for lens oxoprolinase, an enzyme of the gamma-glutamyl cycle.

The presence of oxoprolinase, an enzyme of the gamma-glutamyl cycle, not previously reported in the lens was demonstrated by organ-culture technique and from a study of the partially purified enzyme. The evidence for oxoprolinase in intact rabbit lens is based on the following: (1) [14C]-labeled oxoproline is utilized by the lens giving rise to labeled CO2, (2) [14C]-oxoproline is converted to glutamic acid, which is subsequently incorporated into glutathione, (3) formation of labeled glutamic acid and CO2 from [14C]-oxoproline is effectively blocked by a structural analog of the compound L-2-imidazolidone-4-carboxylic acid, a known inhibitor of oxoprolinase. The enzyme was partially purified from bovine lens capsule epithelium and certain of its properties were examined. Ocular lens was also found to contain significant amounts of oxoproline, an intermediate of the gamma-glutamyl cycle.