P. S. Devamanoharan

PREVENTION OF SELENITE CATARACT BY VITAMIN C

Studies have been conducted to determine the efficacy of vitamin C in the prevention of cataracts induced by selenite. Administration of the latter to rat pups results in the development of advanced cataracts within 5 days. Treatment with ascorbate had a significant preventive effect. The observations indicate that selenite cataract is due to an oxidative stress to the lens. In addition, the findings are in conformity with our view that ascorbate functions as an anticataractogenic substance.

PREVENTION OF INTRACELLULAR OXIDATIVE STRESS TO LENS BY PYRUVATE AND ITS ESTER

Pyruvate is a well-known scavenger of hydrogen peroxide (H2O2). In addition, it scavenges superoxide radical (O2.-). However, evidence on its intracellular antioxidant function is meager at present. Hence, we have examined the effectiveness of this metabolite and its ethyl ester against intracellular oxidative damage to the lens under organ culture. Menadione, a redoxcycling quinone, was used to generate the reactive oxygen species (ROS). It was found to inhibit lens metabolism as evidenced by a decrease of ATP. Additionally, tissue oxidation was apparent by loss of glutathione (GSH), and increase in the level of oxidized glutathione (GSSG), coupled with increase of the urea soluble proteins (water insoluble). The overall physiological damage was apparent by the inhibition of the Na+-K+-ATPase dependent cation pump, as evidenced by a decreased rubidium transport. These deleterious effects were attenuated by pyruvate and ethyl-pyruvate. The later was found to be more effective.

Radio-isotopic determination of hydrogen peroxide in aqueous humor and urine

The concentrations of hydrogen peroxide in the aqueous humor and urine of several animal species and humans have been determined. The determinations are based on peroxide-dependent decarboxylation of I-[14C]-alpha-ketoglutaric acid and measurement of the resulting 14CO2 by quantitating the radioactive disintegration. The levels of H2O2 in most animals varied between 5.0 and 41 microM for aqueous, and 115 and 187 microM for urine. The levels of peroxide in the urine of steer, cat and baboon were lower and fell out of the above range. In the aqueous of humans with cataracts, the levels ranged from 33 to 324 microM, the overall average being 189 +/- 88 microM. The source of such high levels in the aqueous of cataract patients is currently being studied.

HYDROGEN PEROXIDE IN HUMAN BLOOD

Blood and plasma of humans and rats were analyzed for hydrogen peroxide. The samples were analyzed after deproteinization with trichloroacetic acid, immediately after they were withdrawn from human volunteers or rats. A radio-isotopic technique based on peroxide-dependent decarboxylation of 1-14C-alpha-ketoacids and consequent liberation of 14CO2 was used. The results demonstrate the presence of micromolar levels of H2O2, both, in the plasma as well as in the whole blood. The values in the whole blood were substantially greater than the plasma. This was true for rats as well as humans. The presence of such significant quantities of H2O2 in the blood have been demonstrated for the first time. The investigation, therefore, opens a newer avenue of research on diseases purported to be related to the generation of oxygen radicals in vivo.

Prevention of oxidative damage to rat lens by pyruvate in vitro: Possible attenuation in vivo

Studies have been conducted to assess the possible preventive effect of pyruvate against lens protein oxidation and consequent denaturation and insolubilization. Rat lens organ culture system was used for these studies. The content of water insoluble proteins (urea soluble) increased if the lenses were cultured in medium containing hydrogen peroxide. Incorporation of pyruvate in the medium prevented such insolubilization. The insolubilization was associated primarily with loss of gamma crystallin fraction of the soluble proteins. PAGE analysis demonstrated that insolubilization is related to -S-S- bond formation which was preventable by pyruvate. Since pyruvate is a normal tissue metabolite the findings are considered pathophysiologically significant against cataract formation. This was apparent by the prevention of selenite cataract in vivo by intraperitoneal administration of pyruvate.

Excretion of Hydrogen Peroxide in Human Urine

The excretion of hydrogen peroxide in human urine has been demonstrated for the first time. This was accomplished by a new radioactive method developed on the basis of decarboxylation of alpha-ketoglutaric acid by H2O2. Urine samples were incubated with alpha ketoglutarate pulsed with iwts 1-14C-analogue, and CO2 formed by decarboxylation was determined by radioactivity measurements. Blanks were prepared by pre-incubation of the samples with catalase. Both male and female subjects were studied. On an average the concentration of H2O2 in urine was approximately 100 +/- 60 microM (10(-4) M). Peroxide excretion was found to be unexpectedly high and might thus be useful for clinical diagnosis and therapy in diseases purported to be related to oxidative stress.

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.

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.

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.

Photoinduction of cataracts in rat lens in vitro. Preventive effect of pyruvate

Intact rat lenses were incubated in riboflavin-containing Tyrode solution or medium-199, generating photochemically active species of oxygen and the oxidative stress measured in terms of the decrease in active accumulation of rubidium, and the fall in the levels of glutathione and ATP. Addition of pyruvate to the medium prevented the tissue against oxidative damage as evidenced by a greater accumulation of rubidium and higher levels of glutathione and ATP. Pyruvate was thus found to be effective against the toxicity of oxygen derivatives, particularly the hydrogen peroxide. In dark experiments also, conducted in glucose-free medium, the uptake of rubidium was substantially greater in the presence of pyruvate. The levels of ATP were also higher. These results, therefore, suggest that this ketoacid is beneficial to the tissue through its ability to decompose H2O2 as well through providing a metabolic support. The development of in vitro cataract under the photochemical effects of riboflavin and oxygen was also effectively thwarted by pyruvate. The results are thus potentially useful from the point of view of developing pyruvate and similar compounds as effective anticataract agents.