Ethanol-ascorbate interrelationship in acute and chronic alcoholism in the guinea pig

The effects of low (200 ppm) and of high (2000 ppm) ascorbic acid, in a nutritionally adequate diet, on blood ethanol levels have been studied in permanently carotid-cannulated, ethanol-infused, unanesthetized guinea pigs. In the acute study, the postinfusion rate of ethanol decline in the blood of animals treated with ascorbic acid was significantly higher when compared with animals treated with fructose, and the rate in the two treated groups was significantly higher than in untreated controls. In the chronic study, animals were infused with sublethal doses of ethanol (30% of the total caloric intake) for 8 weeks. Blood ethanol levels monitored throughout this period showed, at 3 hr postinfusion, a lower concentration in the group on a high ascorbic acid diet. Both experimental groups receiving ethanol lost significantly more body weight in the second week of dieting; but, while the group on high ascorbic acid regained weight steadily thereafter, the group on low ascorbic acid was still 50 g below the controls at the end of the experiment. Liver, kidney, and adrenal ascorbic acid concentrations were lower in the ethanol-treated groups compared to controls. Examination of the liver revealed more fatty metamorphosis or steatosis in the low ascorbic acid group, but there was no evidence of liver fibrosis or cirrhosis. These results demonstrate the feasibility of utilizing the guinea pig for the study of the biochemical and morphological sequelae of alcoholism. They further support the contention that a diet which is nutritionally adequate may no longer be so in the presence of high ethanol intake, and that supplemental vitamin C ingestion may afford protection against ethanol toxicity.     

Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver

The production of reactive oxygen species (ROS) is considered to be a major factor in oxidative cell injury. The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against such hepatic damage. Silymarin, derived from the milk thistle plant, Silybium marianum, has been used in traditional medicine as a remedy for diseases of the liver and biliary tract. In the present study, the effect of hepatoprotective drug silymarin on body weight and biochemical parameters, particularly, antioxidant status of ethanol-exposed rats was studied and its efficacy was compared with the potent antioxidant, ascorbic acid as well as capacity of hepatic regeneration during abstention. Ethanol, at a dose of 1.6 g/kg body wt/day for 4 wks affected body weight in 16-18 week-old male albino rats (Wistar strain weighing 200-220 g). Thiobarbituric acid reactive substance (TBARS) level, superoxide dismutase (SOD), and glutathione-s-transferase (GST) activities were significantly increased, whereas GSH content, and catalase, glutathione reductase (GR) and GPx (glutathione peroxidase) activities significantly reduced, on ethanol exposure. These changes were reversed by silybin and ascorbic acid treatment. It was also observed that abstinence from ethanol might help in hepatic regeneration. Silybin showed a significant hepatoprotective activity, but activity was less than that of ascorbic acid. Furthermore, preventive measures were more effective than curative treatment.     

Kinetic study of the reaction of vitamin C with vitamin E radicals (tocopheroxyls) in solution

New stable vitamin E radicals (7-tert-butyl-5-isopropyltocopheroxyl (4), 5,7-diisopropyltocopheroxyl (5), 7-tert-butyl-5-methyltocopheroxyl (6), and 5,7-diethyltocopheroxyl (7] with two bulky alkyl substituents at ortho positions (C-5 and C-7) have been prepared, and the reaction rates of vitamin C (ascorbic acid (1) and 6-O-stearyl ascorbic acid (2] with these tocopheroxyl radicals in benzene/ethanol/water (2:1:0.1, v/v) solution have been determined spectrophotometrically, using a stopped-flow technique. The second-order rate constants, k2, obtained vary in the order of 10(3), and decrease dramatically in the order 7 greater than 6 greater than 5 greater than 4, as the size of two ortho-alkyl groups in tocopheroxyl increases. The result suggests that the effect of steric hindrance on the reaction rate is considerable. These reaction rates were compared with those of vitamin C with alpha-tocopheroxyl reported by Packer et al. (Nature 278 (1979) 737-738) and Scarpa et al. (Biochim. Biophys. Acta 801 (1984) 215-219).     

Effects of Ethanol and Polychlorinated Biphenyls in Combination on Ascorbic Acid Metabolism,Liver Drug-metabolizing Enzymes,and Lipid Metabolism in Rats

We investigated the combined effects of ethanol and polychlorinated biphenyls (PCB) on ascorbic acid metabolism, liver drug-metabolizing enzymes, and lipid metabolism in rats fed on a diet containing by 36% by energy of ethanol and 0.005% of PCB, either singly or in combination, for 49 days. Ethanol and PCB given together synergistically affected the amount of ascorbic acid excreted in the urine and the serum concentration of ascorbic acid. This synergistic effect was also observed in the activity of aniline hydroxylase in the liver. Ethanol and PCB given together seem to have had different effects on lipid metabolism. These results suggest that the effect of ethanol on the metabolism of ascorbic acid and of drugs may be enhanced by combined administration with PCB, and that the ascrobic acid deficiency and/or modification of the drug metabolism may become more serious.     

Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid

The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.     

Radical-regulating and antiviral properties of ascorbic acid and its derivatives

The ability of ascorbic acid and a number of its derivatives to suppress replication of Herpes simplex virus type I was investigated in human rhabdomyosarcoma cell line. In parallel, interaction of the test compounds with carbon- and oxygen-centered radicals formed on radiolysis of hydroxyl-containing organic compounds was studied using the steady state radiolysis method. It has been shown that 2-O-glycoside of ascorbic acid, displaying marked antiviral properties against Herpes simplex virus type I, is also capable of inhibiting fragmentation and recombination reactions of α-hydroxyl-containing carbon-centered radicals while not affecting processes involving oxygen-centered radicals.     

Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy

The formation of hydroxyl radicals in beta-glucan solutions treated with ascorbic acid and iron(II) was demonstrated by ESR spin trapping based methods. Two different spin traps were tested, namely DMPO which is commonly used to detect hydroxyl radicals, and POBN often used to detect carbon centered radicals. The experiments performed showed that the presence of iron(II) with DMPO led to low DMPO-OH adduct stability and further to DMPO dimerization. The level of hydroxyl radicals formed during the beta-glucan radical mediated degradation was evaluated using two ESR spin trapping methods based on the use POBN together with either 2% (v/v) EtOH or DMSO. The addition of ascorbic acid together with iron(II) in beta-glucan solution led to an immediate maximal production of hydroxyl radicals while the presence of ascorbic acid alone led to a progressive production of radical. Further hydroxyl radicals were found to be formed when iron(II) was added alone in beta-glucan solutions. The viscosity loss observed in the three last mentioned beta-glucan solutions were found to relate with the formation of hydroxyl radicals. These data confirm the involvement of hydroxyl radical in the beta-glucan degradation.     

Combined effect of selenium and ascorbic acid on alcohol induced hyperlipidemia in male guinea pigs

Alcoholics usually suffer from malnutrition and are especially deficient in micronutrients like vitamin C, selenium and Zn. In the present study, combined effects of selenium and ascorbic acid on alcohol-induced hyperlipidemia were studied in guinea pigs. Four groups of male guinea pigs were maintained for 45 days as follows: control (1 mg ascorbate (AA)/100 g body mass/day), ethanol (900 mg ethanol/100 g body mass + 1 mg AA/100 g body mass/day), selenium+ascorbic acid [(25 mg AA + 0.05 mg Se)/100 g body mass/day], ethanol+selenium+ascorbic acid [(25 mg AA + 0.05 mg Se + 900 mg ethanol)/100 g body mass/day]. Co-administration of selenium and ascorbic acid along with alcohol reduced the concentration of all lipids, as also evidenced from the decreased activities of hydroxymethylglutaryl-CoA reductase and enhanced activities of plasma lecithin cholesterol acyl transferase and lipoprotein lipase. Concentrations of bile acids were increased. We conclude that the supplementation of Se and ascorbic acid reduced alcohol induced hyperlipidemia, by decreased synthesis and increased catabolism.     

Short-term Effects of Ethanol Intoxication on Ascorbic Acid and Lipid Metabolism and on Drug-metabolizing Enzymes in Liver of Rats

The effects of one-time ethanol intoxication on ascorbic acid and lipid metabolism and on drug-metabolizing enzymes in liver of rats were investigated. Male Donryu rats that had been fed semi-purified feed were given 5 g/kg ethanol solution (25%, w/v) via a stomach tube and killed 16 h after intubation. The amount of ascorbic acid excreted in the urine after ethanol administration increased, but renal and adrenal concentrations of ascorbic acid decreased. The serum levels of total cholesterol, high-density-lipoprotein cholesterol, triglycerides, phospholipids, and non-esterified fatty acids were elevated in rats given ethanol, but hepatic level of total lipids, cholesterol, triglycerides, phospholipids were not. The hepatic concentrations of cytochrome P-450 and cytochrome b₅ did not increase, but this large dose of ethanol increased the activities of aminopyrine N-demethylase and cytochrome c reductase.

These results indicated that the single dose of ethanol affected the ascorbic acid and lipid metabolism of rats, and induced drug-metabolizing enzymes in their liver.     

Dietary ascorbic acid deficiency in guinea pigs: No effect on ethanol preference,spiroperidol binding,or monoamine oxidase activity

Ascorbic acid levels are commonly reported to be decreased in alcoholics. Although this deficiency could be due to dietary factors, there is evidence that ascorbic acid may be involved in the metabolism and acute effects of ethanol, possibly related to the pathogenesis of alcoholism. Therefore, we examined ethanol preference in guinea pigs receiving an ascorbate deficient $\text{vs}$ a normal diet. Brain and spleen ascorbic acid levels were dramatically decreased, but ethanol preference was not altered by the acute dietary deficiency of this vitamin. In addition, an acute stressor (cold water swim), alone or in combination with ascorbate deficiency, had no effect on ethanol preference. At termination of the experiment, two measures of brain aminergic function (MAO activity and ³H-spiroperidol binding), purportedly altered by ethanol or ascorbic acid or both, were not associated with tissue ascorbate levels.     

Hyaluronan degradation by copper(II) chloride and ascorbate: rotational viscometric, EPR spin-trapping, and MALDI-TOF mass spectrometric investigations

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl(2) present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.     

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.     

Pyrazole and 4-methylpyrazole inhibit oxidation of ethanol and dimethyl sulfoxide by hydroxyl radicals generated from ascorbate, xanthine oxidase, and rat liver microsomes

Pyrazole and 4-methylpyrazole, which are potent inhibitors of alcohol dehydrogenase, inhibited the oxidation of ethanol and of dimethyl sulfoxide by two model hydroxyl radical-generating systems. The systems used were the iron-catalyzed oxidation of ascorbic acid and the coupled oxidation of xanthine by xanthine oxidase. Pyrazole and 4-methylpyrazole were more effective inhibitors at lower substrate concentrations than at higher substrate concentrations; the oxidation of ethanol was inhibited to a greater extent than the oxidation of dimethyl sulfoxide. These results are consistent with competition between pyrazole or 4-methylpyrazole with the substrates for the generated hydroxyl radicals. Pyrazole and 4-methylpyrazole appear to be equally effective in reacting with hydroxyl radicals. An approximate rate constant of about 8 × 10⁹m^?1 s^?1 was calculated from the inhibition curves, indicating that pyrazole and 4-methylpyrazole are potent scavengers of the hydroxyl radical. Previous studies have implicated a role for hydroxyl radicals in the microsomal pathway of ethanol oxidation. In the presence of azide (to inhibit catalase), pyrazole and 4-methylpyrazole inhibited the NADPH-dependent microsomal oxidation of ethanol, as well as several other hydroxyl radical-scavenging agents. This inhibition by pyrazole and by 4-methylpyrazole may reflect a mechanism involving competition for hydroxyl radicals generated by the microsomes. However, the kinetics of inhibition by pyrazole were mixed, not competitive, and pyrazole and 4-methylpyrazole also inhibited aminopyrine demethylase activity. Pyrazole has been shown by others to interact with cytochrome P-450. It is suggested that pyrazole and 4-methylpyrazole affect microsomal oxidation of ethanol via effects on the mixed-function oxidase system and via competition for the generated hydroxyl radicals. In view of these results, low concentrations of pyrazole and 4-methylpyrazole should be used in studies on pathways of alcohol metabolism, and caution should be made in interpreting the actions of these compounds when used at high concentrations.     

Peroxynitrite scavenging by different antioxidants. Part I: convenient assay.

A convenient "tube" assay to quantify relative antioxidant activities in aqueous solutions has been developed. Peroxynitrite was employed as a biologically relevant source of radicals with Pyrogallol Red as a detecting molecule. A variety of compounds have been examined, namely polyphenols, uric acid, glutathione, and ascorbic acid. Competition kinetics were observed for the majority of examined compounds, except thymol and ascorbic acid. Pyrogallol Red was fully protected by ascorbic acid against the bleaching by peroxynitrite until its total consumption. The deviation from competition kinetics in the case of thymol was due to the formation of radicals from thymol and their subsequent reaction with Pyrogallol Red. Quercetin was the most efficient scavenger of free radicals. The measurements of relative antioxidant activities using Pyrogallol Red and other detecting molecules, such as gallocyanine and carminic acid, were in fair agreement. The assay was successfully used for a screening of antioxidant activity of plant extracts of unknown composition.     

Effect of ascorbic acid on prevention of hypercholesterolemia induced atherosclerosis

The notion that oxidation of lipids and propagation of free radicals may contribute to the pathogenesis of atherosclerosis is supported by a large body of evidence. To circumvent the damage caused by oxygen free radicals, antioxidants are needed which provide the much needed neutralization of free radical by allowing the pairing of electrons. In this study we have investigated the effect of ascorbic acid, a water soluble antioxidant on the development of hypercholesterolemia induced atherosclerosis in rabbits. Rabbits were made hypercholesterolemic and atherosclerotic by feeding 100 mg cholesterol/day. Different doses of ascorbic acid were administered to these rabbits. Low dose of ascorbic acid (0.5 mg/100 g body weight/day) did not have any significant effect on the percent of total area covered by atherosclerotic plaque. However, ascorbic acid when fed at a higher dose (15 mg/100 g body weight/day) was highly effective in reducing the atherogenecity. With this dose the percent of total surface area covered by atherosclerotic plaque was significantly less (p < 0.001). This suggests that use of ascorbic acid may have great promise in the prevention of hypercholesterolemia induced atherosclerosis.     

The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.     

Anaerobic respiration of superoxide dismutase-deficientSaccharomyces cerevisiae under oxidative stress

The ethanol productivity of superoxide dismutase (SOD)-deficient mutants ofSaccharomyces cerevisiae was examined under the oxidative stress by Paraquat. It was observed that MnSOD-deficient mutant ofS. cerevisiae had higher ethanol productivity than wild type or CuZnSOD-deficient yeast both in aerobic and in anaerobic culture condition. Pyruvate dehydrogenase activity decreased by 35% and alcohol dehydrogenase activity increased by 32% were observed in MnSOD-deficient yeast grown aerobically. When generating oxygen radicals by Paraquat, the ethanol productivity was increased by 40% in CuZnSOD-deficient or wild strain, resulting from increased activity of alcohol dehydrogenase and decreased activity of pyruvate dehydrogenase. However, the addition of ascorbic acid with Paraquat returned the enzyme activities at the level of control. These results imply that SOD-deficiency in yeast strains may cause the metabolic flux to shift into anaerobic ethanol fermentation in order to avoid their oxidative damages by Paraquat.     

Oxyradicals under UV-b stress and their quenching by antioxidants

Formation of oxyradicals under UV-B stress was investigated using cucumber cotyledons. UV-B radiation induced production of free radicals which were analyzed by ESR spectroscopy. Evidence was obtained for the formation of superoxide and hydroxyl radicals in the tissues by comparing PBN-adducts formed with radicals obtained by chemical autooxidation of KO2 and Fenton's reaction. Addition of superoxide dismutase (SOD) to the reaction mixture partially reduced the intensity of signals confirming the production of superoxide radical as well as hydroxyl radicals. These radicals were quenched in vitro by the natural antioxidants alpha-tocopherol, ascorbic acid and benzoquinone. Changes in the level of antioxidants were also monitored under UV-B stress. The endogenous level of ascorbic acid was enhanced and alpha-tocopherol level was reduced in the tissue after exposure to UV-B radiation. The present report happens to be the first direct evidence obtained for the formation of superoxide and hydroxyl radicals in plant tissues exposed to UV-B radiation.     

Additive effect of alpha-tocopherol and ascorbic acid in combating ethanol-induced hepatic fibrosis

Abstract

Objective

To investigate the efficacy of combined administration of alpha-tocopherol (AT) and ascorbic acid (AA) in reducing ethanol-induced hepatotoxicity.

Methods

Rats were maintained for 90 days and grouped as follows: I – control rats, II – ethanol, III – alpha-tocopherol, IV – ethanol + alpha-tocopherol, V – AA, VI – ethanol + ascorbic acid, VII – alpha-tocopherol + ascorbic acid, VIII – ethanol + alpha-tocopherol + ascorbic acid. At the end of the experimental period, markers of hepatic function, oxidative stress, and the expression of markers of inflammation and fibrosis were assayed.

Results

The markers of hepatic function, lipid peroxidation products, protein carbonyls, and the expression of nuclear factor kappa B, tumor necrosis factor alpha, transforming growth factor beta 1, cytochrome P4502E1, and collagen Type I were elevated after ethanol administration. All these parameters were reduced in the ethanol group administered AT and AA in combination. The activities of antioxidant enzymes which were reduced by ethanol administration were enhanced on combined administration of AT and AA. The reduction in hepatic fibrosis was almost 20% more in AT and AA co-administered group compared with AT and AA alone treated groups.

Discussion

Combined administration of fat soluble AT and water soluble AA was beneficial against ethanol-induced hepatotoxicity. This may be due to their different subcellular localizations.     

Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102

An assay for the ability of antioxidants to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 cells was developed. Protection against hydrogen-peroxide-induced mutagenicity was observed for quercetin, caffeic acid, ascorbic acid and dimethyl sulfoxide (used as a solvent for water-insoluble antioxidants). No protective effect was observed for green tea extract (weakly pro-oxidative), catechin, rutin, sinigrin, ferulic acid and alpha-tocopherol. Mutagenicity caused by tert-butyl hydroperoxide (tBOOH) was prevented most effectively by quercetin and ascorbic acid, whereas weaker effects were observed for green tea extract and for rutin, and no effect being observed for the other antioxidants tested. The results for hydrogen peroxide indicate iron chelation to be the most important protective mechanism. Radical scavenging appeared to be effective only with dimethyl sulfoxide and ascorbic acid, which are effective scavengers of hydroxyl radicals and were used here in high concentrations. It is proposed that the hydrogen-peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA. Protection against mutagenicity caused by tert-butyl hydroperoxide appears to occur mainly through the scavenging of alkoxyl and possibly of alkyl radicals.