ascorbic-acid and sorbinil

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.

Topics: Animals; Antioxidants; Ascorbic Acid; Benzene Derivatives; Glutathione; Guinea Pigs; Hepatocytes; Imidazolidines; Ketones; Lipid Peroxidation; Oxidative Stress; Rats; Reactive Oxygen Species

Using isolated rat hepatocytes we have shown that glutathione (GSH) depletion by glutathione-S-transferase (GST)-catalyzed conjugation with 1-bromoheptane or phorone was accompanied by a significant elevation in ascorbate synthesis. Glycogenolysis was also stimulated without a significant rise in glucose synthesis. Furthermore, when glycogenolysis was stimulated in control hepatocytes by increasing intracellular cAMP levels (with glucagon or dibutyryl cAMP), cellular glucose levels, but not ascorbate levels, increased. These data suggest that GSH depletion can stimulate ascorbate synthesis independently of glucose synthesis and that hepatocytes can direct glycogenolysis towards ascorbate synthesis during GSH conjugation.

Topics: Animals; Ascorbic Acid; Bucladesine; Cyclic AMP; Diamide; Dithiothreitol; Fructose; Glutathione; Glutathione Disulfide; Glycogen; Hepatocytes; Heptanes; Imidazoles; Imidazolidines; Ketones; Male; Rats; Rats, Sprague-Dawley; Uridine Diphosphate Glucose

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.

Topics: Aminopyrine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antipyrine; Ascorbic Acid; Barbital; Buthionine Sulfoximine; Cells, Cultured; Chlorobutanol; Chromatography, High Pressure Liquid; Clotrimazole; Diamide; Dose-Response Relationship, Drug; Enzyme Inhibitors; Galactosamine; Glucuronates; Glucuronic Acid; Hepatocytes; Imidazoles; Imidazolidines; Metyrapone; Models, Chemical; Preservatives, Pharmaceutical; Proadifen; Rats; Rats, Wistar; Resorcinols; Time Factors; Xenobiotics; Xylulose

The effect of long-term (2 weeks) exposure to 0-50 mM glucose and 0-1 mM sorbitol on myo-inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo-inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo-Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in > or = 30 mM glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 mM sorbinil. Ascorbic acid also prevented the reduction in myo-inositol uptake due to excess extracellular glucose, at 3 and 30 microM concentrations, but not at 300 microM. Neither sorbinil nor ascorbic acid could prevent the alterations in myo-inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo-inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo-inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.

Topics: Aldehyde Reductase; Animals; Animals, Newborn; Ascorbic Acid; Biological Transport; Cells, Cultured; Enzyme Inhibitors; Glucose; Imidazoles; Imidazolidines; Inositol; Kinetics; Phospholipids; Rats; Rats, Wistar; Schwann Cells; Sciatic Nerve

L-threose is a product of ascorbate oxidation and degradation. By virtue of its free aldehyde group it can form Schiff-bases with tissue proteins, altering their normal function. In this study, we have examined the possibility of its detoxification to L-threitol by aldose reductase in the lens. The rat lens enzyme present in fresh homogenate as well as after 100 fold purification was found to utilize L-threose with a km of 7.1 x 10(-4) M. The specificity of the reaction was affirmed by its inhibition with sorbinil and quercetin, the well known aldose reductase inhibitors. Further studies on the role of this enzyme in preventing toxicity due to degradation products of ascorbate are in progress.

Topics: Aldehyde Reductase; Animals; Ascorbic Acid; Crystallins; Enzyme Inhibitors; Glycosylation; Imidazoles; Imidazolidines; Kinetics; Lens, Crystalline; NADP; Oxidation-Reduction; Quercetin; Rats; Substrate Specificity; Tetroses

Sorbitol formation in rat lenses incubated with high levels of glucose was related to activation of aldose reductase (AR). The hyperglycaemia-activated aldose reductase was inhibited by alpha-lipoic (thioctic) acid, O-phenanthroline and aldose reductase inhibitors (ARIs) including Zeopolastat (ZPLS), Sorbinil (SBN) and AL-1576. This study also examined ARIs for the ability to chelate metal ions. We found that ARIs suppress copper-dependent ascorbate oxidation, lipid peroxidation and hydrogen peroxide production in erythrocytes. ARIs also increased partition of copper ions into noctanol, which indicates formation of lipophilic complexes. Our data support the hypothesis that transition metals may be involved in activation of the polyol (aldose reductase) pathway. Also, ARIs function as metal-chelating antioxidants that may contribute to their therapeutic role for diabetic complications.

Topics: 1-Octanol; Aldehyde Reductase; Animals; Ascorbic Acid; Chelating Agents; Copper; Enzyme Activation; Enzyme Inhibitors; Erythrocytes; Fluorenes; Glucose; Humans; Hydantoins; Hydrogen Peroxide; Hyperglycemia; Imidazoles; Imidazolidines; In Vitro Techniques; Ions; Lens, Crystalline; Lipid Peroxidation; NADP; Octanols; Oxidation-Reduction; Phenanthrolines; Rats; Thioctic Acid

Recent work from our laboratory revealed a correlation between the degree of protein pigmentation in human cataractous lens and the advanced Maillard reaction as reflected by pentosidine formation. Although the data suggested a role for ascorbate in pentosidine formation in senile cataractous lenses, elevated pentosidine levels in diabetic cataracts suggested that glucosylation may be involved directly in pentosidine biosynthesis. To clarify this issue, we quantified pentosidine in lenses from rats with experimental galactosemia with and without aldose reductase inhibitor treatment. At 12 months, pentosidine-like fluorescence (335/385 nm) was three to six times higher (P < 0.0001) in water soluble and insoluble crystallins of galactosemic compared with nongalactosemic rats. Actual pentosidine levels increased shortly after onset of galactosemia. Contents in water-insoluble crystallins were 6.32 +/- 2.2 and 1.40 +/- 0.66 pmol/mg protein in galactosemic and control lenses, respectively (P < 0.001). Fluorescence and pentosidine were suppressed to almost control levels upon treatment with sorbinil. Incubation experiments showed that pentosidine could form slowly from galactose, but much more rapidly from ascorbate and its oxidation products. Its formation could be inhibited partly by both reduced and oxidized glutathione or epsilon-aminocaproic acid. The requirement of oxygen for pentosidine formation suggests that oxidative stress associated with glutathione depletion and ascorbate oxidation are plausible mechanisms for rapid pentosidine formation upon onset of galactosemia. In contrast, Maillard reaction by glycoxidation products may account for the sustained increase in pentosidine. Both these events may be linked to the newly recognized pseudohypoxic state of cells exposed to high sugar concentrations.

Topics: Aldehyde Reductase; Animals; Arginine; Ascorbic Acid; Diet; Female; Galactitol; Galactose; Galactosemias; Glutathione; Imidazoles; Imidazolidines; Lens, Crystalline; Lysine; Maillard Reaction; Rats; Rats, Sprague-Dawley; Time Factors

Transition metal-catalysed oxidations have been implicated in the complications of diabetes. We report here that some experimental inhibitors of the enzyme aldose reductase (implicated in diabetes mellitus via its ability to catalyse glucose reduction to sorbitol) are also potent inhibitors of transition metal-catalysed ascorbate oxidation. The inhibition appears to be dependent upon the presence of a spirohydantoin group. It is conceivable that the copper- and iron-binding capacity of these compounds may contribute to some of their observed biological effects and may provide a starting point for a new generation of experimental drugs for the treatment of diabetes mellitus.

Topics: Aldehyde Reductase; Antioxidants; Ascorbic Acid; Copper; Diabetes Mellitus; Dose-Response Relationship, Drug; Fluorenes; Humans; Hydantoins; Imidazoles; Imidazolidines; Iron; Oxidation-Reduction; Structure-Activity Relationship