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Biomedical subjects

S D Varma

Publications and source records attributed to S D Varma.

At least 19 recordsLinked to original sources

Oxidative damage to lens in culture: reversibility by pyruvate and ethyl pyruvate.

It is generally believed that prophylactic intake of antioxidants is beneficial in delaying the onset of some aging manifestations such as cataract. However, whether such a supplementation will also be effective if the pathophysiological process has already set in remains a largely open question. We examined this possibility with lens changes leading to cataract formation, since cataract genesis is intimately related to a continued generation of reactive oxygen species (ROS) in the aqueous humor. Since the formation of cataract is a well-defined progressive disease, starting with an early refractive change and leading to gradual enhancement of opacification, we hypothesized that even a belated start with an appropriate anti-oxidant could halt the pathological process and delay cataract maturation and vision impairment. Using lens cultures, we tested this hypothesis with pyruvate, known to be an effective and highly potent ROS scavenger. Adding pyruvate to the culture medium after lenses had sustained a 50% damage was significantly effective in preventing progress. This was apparent by better maintenance of the active rubidium transport activity in these lenses compared to controls without pyruvate treatment. Glutathione levels were also higher in the pyruvate group.

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Prevention of cataract by pyruvate in experimentally diabetic mice.

Previous studies have demonstrated that administration of pyruvate prevents cataract formation in diabetic rats. It is known that the induction of cataractous process in this case is initiated by aldose reductase (AR) catalyzed synthesis and accumulation of excessive sorbitol in the lens fibres and epithelium and their consequent osmotic hydration. Synthesis of this and other polyols is competitively inhibited by pyruvate. The objective of the present investigations was hence to determine whether pyruvate would have a similar protective effect in species where cataract formation is relatively independent of sorbitol synthesis such as in humans where the lens AR activity is extremely low, especially with glucose as a substrate. The Km of AR for glucose is known to be very high. The possible protective effect of pyruvate in the low AR models was conceived on the basis of our previous findings suggesting that it can also exert substantial antiglycating as well as antioxidant effects. The present studies have hence been conducted with mice, a species known to be low in lens AR, similar to that in humans. As stipulated, pyruvate administration has indeed been found to offer a significant protection against development of diabetic cataract in this model also. The effect correlated with the inhibition of protein glycation as well as of oxidative stress. The latter was apparent by the prevention of the loss of glutathione known to be associated with diabetes. Although there was a small but noticeable increment in the sorbitol content of the diabetic lenses, this was osmotically insignificant. Even this increase was prevented by pyruvate. The magnitude of the elevation in the contents of glycated proteins and the depression in the level of glutathione were, on the contrary, highly pronounced, suggesting a more prominent role of the latter factors. In addition, the possibility of a direct metabolic support it could offer to the tissue is also imminent by its effect on the maintenance of ATP, as shown earlier. The present studies are therefore considered more relevant to the pathogenesis of cataract in human diabetics and its possible prevention by endogenous compounds with antiglycating and antioxidant properties. Inhibition of cataract formation by pyruvate in an animal model with low lens AR, similar to that in humans, has been shown for the first time.

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Combination of glycemic and oxidative stress in lens: implications in augmentation of cataract formation in diabetes.

It is well known that the incidence of cataract is higher in diabetics as compared to non-diabetics. Its rate of maturation is also faster in the diabetics. The precise mechanism of this acceleration is not clearly understood. It is hypothesized that this could be a result of the combination of the metabolic and oxidative stress induced by glycemia itself with the age-associated increase in ambient generation of oxyradical species. In the current studies, we have investigated this possibility using the galactose cataract model. Galactosemia was induced by feeding rats a 50% galactose diet. The increased susceptibility of the glycemic lenses to physiological damage by reactive oxygen species (ROS) was studied by incubating them in Tyrode in the absence and presence of menadione. The resulting physiological damage to the lens was assessed initially in terms of its ability to maintain Na+-K+ ATPase dependent active transport of potassium ions, as represented by the uptake of rubidium ions. Subsequently, the level of ATP, indexing the general metabolic status, and the level of glutathione (GSH), indexing the status of antioxidant reserve, were also determined. The uptake of rubidium in the normal lenses incubated in the presence of the quinone was depressed to more than 50% of the controls run in the basal medium. A similar depression existed in the galactosemic lenses in comparison to the normal lenses. However, in the presence of menadione, the inhibition of the uptake was accentuated further in the case of galactosemic lenses, the uptake here being only 20% of the normal controls. Similarly, the galactosemic lenses were also more susceptible to menadione dependent decrease in ATP and GSH.

Adenosine Triphosphate↗

Cataracts in experimentally diabetic mouse: morphological and apoptotic changes.

AIM: The objective of these investigations was to extend our earlier study on the induction of cataracts in diabetic mice, a low aldose reductase (AR) animal model at morphological level. Previous studies were done primarily at biochemical level. METHODS: Diabetes was induced by intraperitoneal administration of streptozotocin. The lenses isolated after the establishment of diabetes were then subjected to histologic and electron microscopic studies. RESULTS: Morphological alterations were characterized by shrinkage, elongation and lobulization of the nuclei of the epithelial cells. This was associated with chromatin condensation and its margination. Similar structural aberrations were also observed in a significant number of the subepithelial fibre cells representing defect in fibre maturation. More interestingly, unlike that in other common animal models of diabetic cataract, such abnormally nucleated cells were also found to be prevalent in the posterior subcapsular region, a finding common in human diabetics also. CONCLUSION: The present studies further affirm the suitability of the mouse model for a study of cataractogenesis induced by diabetes. Because of the findings reported herein, as well as the known biochemical similarity between the lenses of the mice and humans in respect of AR deficiency, contrary to the rat model where it is very high, use of this species is considered more useful towards understanding the basic aetiology as well as for evaluating the efficacy of various referred nutritional and metabolic antioxidants against such cataracts.

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Protective effect of ascorbate against oxidative stress in the mouse lens.

The purpose of this study was to determine if high ascorbate of the human aqueous protects the lens against oxidative stress. Previous studies with the rat lens have been inconclusive because of its fortification with aldose reductase (AR), an important antioxidant. The human lens is deficient in this activity. These studies were hence done with the mouse lens, a species deficient in this enzyme. The reactive oxygen species (ROS)-induced physiological damage to the tissue was assessed in organ culture, by measuring its ability to actively transport 86Rb(+) ions, in the absence and presence of ascorbate. In addition, the status of tissue metabolism and its antioxidant reserve were assessed by quantitating ATP and glutathione (GSH). As expected, ROS decreased the membrane transport activity as well as the levels of ATP and GSH. Ascorbate minimized these toxic effects substantially. The presence of high ascorbate, therefore, appears highly beneficial in protecting the lens against oxidative damage and cataract formation, despite a deficiency of AR. The findings therefore appear to be significant from the point of view of using this nutrient for delaying the onset of cataract development in human beings, therapeutically as well as nutritionally.

Adenosine Triphosphate↗

Effect of alpha-ketoglutarate against selenite cataract formation.

We have previously shown that pyruvate protects against reactive oxygen species (ROS) induced damage to lens in vitro. It has also a significant effect against cataract development. Its effectiveness has been ascribed to the presence of alpha-keto-carboxylate group in the molecule, acting as a scavenger of ROS. Hence, it was felt desirable to determine if other alpha-keto-acids could have similar effects. These studies have hence been conducted with alpha-ketoglutarate (alpha-KG), a compound with greater stability and without any known significant effect on the glycolysis. Its effectiveness has been assessed by monitoring cataract development in rat pups given sodium selenite. A large percentage of such animals (about 80%) developed nuclear opacity 7-8 days after its administration. In animals treated with alpha-ketoglutarate, the incidence of cataracts was only 23%. The agent therefore has a very substantial anticataractogenic effect, as apparent by direct slit lamp examination followed by photography, as well as by examination of the isolated lenses through transillumination. The significance of the ophthalmologic findings was apparent also by better physiological maintenance of the tissue, reflected by higher levels of ATP and GSH. In view of these in vivo beneficial effects, studies are in progress to identify the biochemical and metabolic sites of its action. Whether the effectiveness is related only to its action as a ROS scavenger or it could be contributed also by some metabolic effects independent of ROS remains to be determined.

Adenosine Triphosphate↗

Morphogenetic and apoptotic changes in diabetic cataract: prevention by pyruvate.

Studies have been conducted to ascertain the preventive effect of pyruvate against diabetes induced damage to DNA and associated morphogenetic changes in the mouse lens. Such changes were characterized by DNA nicks as well as by gross morphological changes in the nuclei, evident respectively by TUNEL and Hoechst staining procedures. Morphogenetic changes were also apparent by abnormal diferentiation of the germinal epithelial cells and errors in their migratory pathway. These changes were prevented by simultaneous administration of pyruvate to the diabetic animals. The preventive effect of this agent is attributable to its property of scavenging oxy-radicals generated by high levels of the sugars.

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Ophthalmoscopic and morphogenetic changes in rat lens induced by galactose: attenuation by pyruvate.

BACKGROUND: Investigations have been conducted on the potential of pyruvate, a normal tissue metabolite, in the prevention of cataract formation. METHODS: Cataract was induced by maintaining young rats on a diet containing 30% galactose. The progress of cataract was monitored by visual inspection and ophthalmoscopic and slit-lamp examinations. The protective effect of pyruvate was assessed by incorporating it in the galactose diet and drinking water. The progress of cataract was substantially thwarted by this dietary regimen. RESULTS: Substantial morphogenetic changes in the lenses of the galactosaemic animals, which by themselves can offer obstruction to light penetration through the lens and scattering, were significantly attenuated. These changes were ascertained by histological detection of errors in cellular differentiation and their migration in unwanted areas. CONCLUSIONS: As per previous studies, the pyruvate effect is attributed to its direct effect on the biochemistry of lens related to the inhibition of oxidative stress, as well as to its effect on tissue physiology related to the lifelong process of organogenesis, characteristic of this tissue.

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Oxidative damage to mouse lens in culture. Protective effect of pyruvate.

Studies have been conducted to examine the feasibility of preventing oxyradical-dependent oxidative stress to mouse lens in culture, using pyruvate as an antioxidant. The extent of oxidative damage to the tissue was assessed by measurement of the status of Na(+)-K(+) ATPase dependent active transport of rubidium 86Rb(+). The tissue levels of adenosine triphosphate (ATP), glutathione (GSH), malonaldehyde (MDA) and catalase were also determined. While the measurement of 86Rb(+) uptake provides an assessment of the integrity of the primary active transport system, measurement of the other components reflects the status of intracellular oxidative stress. ATP measurement also reflected on the overall status of metabolic integrity. Incubation of the lens with xanthine (XA)/xanthine oxidase (XO) system had an adverse effect on all these parameters. Incorporation of pyruvate was strikingly protective. The protective effect of pyruvate is apparently due to its ability to scavenge ROS generated in the medium with the possibility of its action on tissue metabolism as well. The findings are hence considered useful for further studies on the prevention of oxidative stress to tissues by exogenous supplementation with pyruvate, specially the human lens where the biochemistry of its antioxidant mechanisms is similar to the mouse lens, contrary to the rat lens.

Adenosine Triphosphate↗

Establishment of mouse as an animal model for study of diabetic cataracts: biochemical studies.

AIM: The primary aim of this study was to understand the pathogenesis of diabetic cataracts at biochemical level in an animal model where lens aldose reductase (AR) activity is low, similar to that in the human lens. METHODS: Mouse, which is known to have low lens AR, was selected for these studies. Diabetes was induced by intraperitoneal administration of streptozotocin. Biochemical changes in the lens were monitored in freshly isolated lenses with standard chromatographic, enzymatic and culture experiments described in the section on methods. RESULTS: The present studies provide evidence of significant biochemical changes associated with such cataract formation despite very low levels of aldose reductase. The level of glycated proteins increased to 9 mg/100 mg of total water-soluble lens protein in the diabetic lenses, as compared with the normal lenses where it was only about 1.3 mg/100 mg of total protein. Glutathione (GSH), the major antioxidant in the lens, decreased from 2.35 micro mol/g in the normal lenses to about 1.17 micro mol/g in the diabetic lenses. Malonadehyde, a product of lipid peroxidation, increased from 50 micro mol/100 g in the normal to 70 micro mol/100 g in the diabetic lens. The level of adenosine triphosphate (ATP), an indicator of the overall metabolic status of the tissue, also decreased from 962 +/- 154 nmol/g in the normal to 487 +/- 130 nmol/g in the diabetic lenses. The function of the Na+-K+ ATPase was also adversely affected in diabetes, as indicated by the ability of the lens to accumulate (86)rubidium ions against its concentration gradient. The transport activity, expressed as CL/CM, was 24 in the normal lens, whereas it was only 12 in the diabetic lens. The level of sorbitol in the diabetic lens was only in the micromolar region. Hence, it was considered osmotically insignificant. CONCLUSION: Overall, the results suggest that induction of cataracts in diabetes can be related to multiple biochemical effects such as oxidative stress and glycation. Sorbitol accumulation in low aldose reductase situations, being minor, could, however, act synergistically with other factors.

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Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate.

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.

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Diabetes-induced biochemical changes in rat lens: attenuation of cataractogenesis by pyruvate.

AIM: Studies have been conducted to determine the effect of pyruvate administration on the biochemistry of rat lens and the status of its transparency as affected by diabetic conditions. METHODS: Sprague-Dawley rats were rendered diabetic by intravenous (i.v.) injection of streptozotocin (55 mg/kg body weight (b.w.)) and treated with sodium pyruvate (2%) in drinking water. The levels of glucose, fructose, sorbitol, ATP, GSH, MDA as well as glycated proteins in the lenses were determined at various intervals after the onset of diabetes and the values compared with untreated diabetic controls. The progress of cataract formation and associated histological changes in the tissue were also monitored. RESULTS: Studies show that the pyruvate treatment decreased the extent of several biochemical changes known to be associated with cataract formation, such as the elevation in the levels of glycated proteins, sorbitol, lipid peroxidation (MDA) and inhibition of the cation pump. The progress of cataract was also significantly delayed. CONCLUSION: Exogenous administration of this compound hence was found to exert an overall protective effect against cataract formation induced by the diabetic conditions.

Adenosine Triphosphate↗

Fructose induced deactivation of antioxidant enzymes: preventive effect of pyruvate.

Glycation initiated changes in tissue proteins, which are triggered by the Schiff base formation between the sugar carbonyl and the protein -NH2, have been suggested to play an important role in the development of diabetes-related pathological changes such as the formation of cataracts. While the initial reaction takes place by the interaction of >C=O of the parent sugars with the -NH2 of proteins, reactive oxygen species (ROS) dependent generation of more reactive dicarbonyl derivatives from the oxidation of sugars also plays a significant role in these changes, altering the structural as well as functional properties of proteins. The purpose of this study was to examine whether the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), catalase and superoxide dismutase (SOD) could be affected by the high levels of fructose prevalent in diabetic lenses. Incubation of the enzymes with this sugar led to a significant loss of their activities. GAPDH was inactivated within a day. This was followed by the inactivation of catalase (3-4 days) and SOD (6 days). The loss of the activities was prevented significantly by incorporation of pyruvate in the incubation mixture. The protective effect is ascribable to its ability to competitively inhibit glycation as well as to its ROS scavenging activity. Hence, it could play a significant role in the maintenance of lens physiology and cataract prevention.

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Attenuation of sugar cataract by ethyl pyruvate.

Studies describe an attenuation of sugar cataract formation by topical administration of ethyl pyruvate. Cataract formation was induced by feeding young rats a 30% galactose diet. Mature cataracts appeared in about thirty days. Instillation of the eye drops containing 5% ethyl pyruvate decelerated the process significantly. Biochemically, the effect was reflected by lowering in the contents of dulcitol and glycated proteins. The ATP levels were also higher in comparison to the placebo treated group. The effects are hence attributable to the effect of pyruvate in inhibiting dulcitol synthesis and protein glycation, in addition to its antioxidant properties and metabolic support. The use of esterified pyruvate instead of the unesterified pyruvate was preferred because of its greater penetration through the cornea and consequently a higher concentration attained in the aqueous humor.

Adenosine Triphosphate↗

Non-enzymatic glycation of lens proteins and haemoglobin-inhibition by pyruvate: an in-vivo study.

AIM: Previous studies have demonstrated that pyruvate can prevent protein glycation and oxidative stress under in-vitro conditions. The aim of this study was to examine the in-vivo effectiveness of this metabolite against glycation of lens crystallins and haemoglobin in galactosemic rats. METHODS: Sprague-Dawley rats were maintained on a 30% or 50% galactose-containing diet in the absence or presence of 2% or 5% pyruvate in food and water, respectively. The animals were killed subsequently and the extent of glycation of lens crystallins and haemoglobin was determined using an affinity column chromatograpic technique. RESULTS: Maintenance of rats on the high galactose diet resulted in a significant increase in glycation of both the proteins. The increase was faster and more substantial in the animals maintained on the 50% galactose diet than that in the animals fed a 30% galactose diet. The increase in the latter was also very significant. Supplementation with pyruvate inhibited the process. CONCLUSION: The inhibition is attributable to a competitive binding of pyruvate to the protein NH2 groups as well as to the antioxidant effect of the compound. The studies therefore suggest that this and other alpha-keto-acids may be physiologically useful in minimizing glycation and oxidative stress induced tissue pathology by the hyperglycaemic conditions, such as diabetes and galactosemia. The results are also considered pharmacologically significant.

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Attenuation of galactose-induced cataract by pyruvate.

Data in the present paper demonstrate a significant inhibition in the progress of sugar cataract formation by systemic administration of pyruvate. The formation of the cataract was induced by feeding young rats a diet containing 30% galactose. All animals fed this diet developed nuclear lens opacity by the end of 30 days. This was delayed if the diet and water contained, in addition, 2% sodium pyruvate. The incidence of cataract in the latter group was 0% at day 30 and only 25% at day 55. Physiologically, the inhibition was associated with the prevention of lens membrane damage as reflected by its ability to maintain transport of rubidium ions against a concentration gradient; decreased tissue hydration as indexed by the lens wet weight; inhibition of protein glycation, and higher levels of ATP. Since pyruvate, being a normal tissue metabolite, is likely to be non-toxic, the findings are considered useful for further pharmacological studies with this and other similar metabolites, relevant to protection against various secondary complications of diabetes and galactosemia.

Adenosine Triphosphate↗

Oxidative stress to rat lens in vitro: protection by taurine.

The concentration of taurine is high in the lens. However, its function therein remains unknown. Studies from other tissues suggest that in addition to several other modes of action, it acts as an antioxidant. We therefore hypothesize that taurine may be a part of the antioxidant defense mechanisms involved in protecting the lens against oxidative stress and consequent cataract formation. In these studies, the protective effect of taurine was examined using lens culture system with menadione as an oxidant. Inclusion of this compound in the incubation medium was found to have several adverse effects on the lens, such as a decrease in its ability to accumulate rubidium against a concentration gradient and fall in the levels of glutathione, ATP and an increase in water insoluble proteins. All these deleterious effects were attenuated significantly by addition of physiological amounts of taurine to the menadione-containing medium.

Adenosine Triphosphate↗

Fructose induced deactivation of glucose-6-phosphate dehydrogenase activity and its prevention by pyruvate: implications in cataract prevention.

Glucose-6-phosphate dehydrogenase (G6PDH) is an important lens enzyme diverting about 14% of the tissue glucose to the hexose monophosphate shunt pathway. The main function of such a pronounced activity of the enzyme is to support reductive biosyntheses, as well as to maintain a reducing environment in the tissue so as to prevent oxy-radical induced damage and consequent cataract formation. Sugars are one of the well-known cataractogenic agents. Several reports suggest that the cataractogenic effect of the sugars in diabetes as well as in normal aging is initiated by the glycation of the proteins including the enzymes and subsequent formation of more complex and biologically inactive or harmful structures. In a diabetic lens the concentration of fructose exceeds significantly the concentration of glucose, suggesting that the contribution of fructosylation may be greater than that of glucosylation. These studies were undertaken to examine further the possibility that in addition to glycation, generation of oxygen free radicals by fructose and consequent oxidative modifications in certain enzymes may be an important participant in the cataractogenic process. This hypothesis was tested by using G6PDH. The enzyme was incubated with various levels of fructose (0-20mM) and its activity determined as a function of time. This led to a significant loss of its activity, which was prevented by superoxide dismutase, catalase, mannitol and myoinositol. Most interestingly, pyruvate at levels between 0.2 and 1.0 mM also offered substantial protection. Hence, the results, while elucidating further the mechanism of enzyme deactivation by sugars such as fructose, also demonstrate the possibility of therapeutic prevention of cataracts by pyruvate and other such keto acids, in diabetes and other disabilities involving oxygen free radicals in the pathogenetic process.

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