Ascorbic acid deficiency and hepatic UDP-glucuronyl transferase. Qualitative and quantitative differences.

The effect of dietary ascorbate on hepatic UDP glucuronyltransferase (UDPGT) appears to be selective in that only certain isozymes of UDPGT are jeopardized. In this study, ascorbic acid deficiency produced a 68% reduction in the specific activity of hepatic UDPGT towards p-nitrophenol. Earlier studies showed a reduction in UDPGT activity towards p-aminophenol in ascorbate-deficient guinea pigs, whereas bilirubin and acetaminophen glucuronidation were unaffected. Kinetic studies suggest that p-aminophenol and p-nitrophenol are metabolized by a single isozyme in that p-nitrophenol was found to be a competitive inhibitor of p-aminophenol glucuronidation. Both qualitative and quantitative studies on partially purified UDPGT from ascorbate-deficient and ascorbate-supplemented guinea pigs were carried out to investigate the biochemical role of the vitamin. Qualitative differences were observed in UDPGT from ascorbate-deficient animals and included an increased lability to: thermal inactivation; storage at 4 degrees; and purification with UDP-glucuronic acid agarose column chromatography. Furthermore, an analysis of the microsomal membrane showed a 14% increase in membrane fluidity in ascorbate deficiency. Ascorbic acid added in vitro could not reverse the increase in fluidity observed in ascorbate-deficient microsomal membranes; however, ascorbylpalmitate, a more lipophilic form of the vitamin, was effective. Palmitic acid had no effect on membrane fluidity in microsomes from either the ascorbate-supplemented or ascorbate-deficient animals. This increase in membrane fluidity could not be explained by differences in cholesterol, total phospholipid, or phosphatidylcholine content of hepatic microsomes. Furthermore, a quantitative reduction in UDPGT partially purified from ascorbate-deficient guinea pigs was indicated by a marked reduction in protein banding at 55,000 daltons when compared to UDPGT partially purified from ascorbate-supplemented animals.

Ascorbic acid deficiency and hepatic UDP-glucuronyltransferase.

The effect of dietary ascorbic acid on hepatic microsomal UDP-glucuronyltransferase (UDPGT) activity towards p-aminophenol, bilirubin, and acetaminophen was investigated. Ascorbate deficiency produced a 33% reduction in the specific activity of UDPGT towards p-aminophenol, whereas there was no difference between microsomes from ascorbate-deficient and supplemented guinea pigs in the activity towards bilirubin and acetaminophen. This suggests that the effect of the vitamin is on a specific isozyme. This reduction was correlated with the reduced quantity of hepatic microsomal cytochrome P-450, which has been previously reported for ascorbate-deficient guinea pigs. No difference was found in the apparent affinity for the substrate, p-aminophenol, or the cofactor, UDP-glucuronic acid. Differences in microsomal UDPGT activity towards p-aminophenol occurred between the two groups with membrane-perturbing processes such as sonication and Triton X-100. Sonication and magnesium chloride were found to increase activity 329% in ascorbate-supplemented animals and 138% in the ascorbate-deficient group. The addition of ascorbate acid in vitro, or its analog d-isoascorbic acid, could protect against the detrimental effects of excess substrate by maintaining a linear enzymatic rate over a 30-min time period; there was no significant effect on the initial rate of hepatic microsomal UDPGT activity in the ascorbate-supplemented animals whereas there was a significant increase in the ascorbate-deficient group. Glutathione was as effective as ascorbic acid in protecting against the detrimental effects of excess substrate whereas cysteine and dimethyltetrapteridine were only partially effective. Ascorbyl-2-sulfate and alpha-tocopherol had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Flavin-containing monooxygenase and ascorbic acid deficiency. Qualitative and quantitative differences.

Ascorbic acid deficiency causes qualitative and quantitative differences in the guinea pig hepatic flavin-containing monooxygenase (FMO). Kinetic studies with purified FMO indicated no significant change in the apparent Km of dimethylaniline or NADPH in ascorbate-supplemented or -deficient animals. Following purification of ascorbate-deficient guinea pig FMO by DEAE-cellulose and blue agarose chromatography, exogenous FAD was required for 15% of the FMO microsomal activity recovered. In contrast, only 5% of the total microsomal enzyme recovered from ascorbate-supplemented animals required exogenous FAD. Furthermore, there was an enhanced sensitivity to time-dependent nonlinearity with the purified ascorbate-deficient guinea pig FMO. The degree of time-dependent nonlinearity was related to the concentration of substrate. Also, purified ascorbate-supplemented guinea pig FMO was stable for 4 weeks at -20 degrees, whereas the ascorbate-deficient enzyme was inactivated. A decrease in the quantity of ascorbate-deficient guinea pig FMO compared to ascorbate-supplemented was indicated by a marked reduction in total FMO activity recovered from blue agarose chromatography and reduced protein staining intensity with SDS-PAGE at 56,000 daltons.

Ascorbic acid and elevated SGOT levels after an acute dose of ethanol in the guinea pig.

Male guinea pigs were maintained on a vitamin C-deficient chow diet and supplemented with either 0.05 or 2.0 mg of ascorbic acid/ml drinking water for 3 weeks prior to receiving an intraperitoneal injection of 4.0 g of ethanol/kg body weight. The following biochemical parameters were measured prior to, and hourly for 12 hours after, ethanol administration: serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvate transaminase (SGPT), serum triglycerides, and blood ethanol clearance. The animals were killed 12 hours after ethanol administration and liver weight to body weight ratios and hepatic ascorbic acid concentrations determined. Acute ethanol administration resulted in a 12-fold increase in SGOT levels in animals with hepatic ascorbic acid concentrations at or below 16 mg/100 g of liver. A marked reduction, 60%, in this increase was observed in animals that had concentrations of hepatic ascorbic acid above 16 mg/100 g of liver. No effect of hepatic ascorbic acid concentration was observed on elevated levels of SGPT, serum triglycerides, or blood ethanol clearance.

Effect of ascorbic acid on the consequences of acute alcohol consumption in humans.

This study examines the influence of ascorbic acid pretreatment on ethanol clearance, toxicity, and behavioral impairment after an acute dose of ethanol in humans. Ascorbic acid or a placebo was given to 20 healthy male subjects for 2 weeks before ethanol consumption. The dose of ethanol was 0.95 gm/kg body weight and was consumed during a 2 1/2-hour period. Thirty minutes after ethanol consumption, motor coordination and intellectual function were assessed by Goldberg's "Finger-Finger" and "Serial Sevens" tests. In addition, color discrimination was measured with the use of the Farnsworth-Munsell 100 Hue Color Test. Hourly blood samples were taken for 10 hours after ethanol consumption to measure serum triglyceride levels, blood lactate/pyruvate ratios, and serum enzymes. Blood ethanol clearance was also determined. Ethanol consumption elevated serum triglyceride levels and blood lactate/pyruvate ratios and impaired performance of the behavioral tests but did not alter serum enzyme levels. Ascorbic acid pretreatment resulted in significant enhancement in blood ethanol clearance and an increase in serum triglyceride levels after ethanol consumption in half of the subjects. Ascorbic acid pretreatment also resulted in improved motor coordination and color discrimination after ethanol consumption in half of the subjects. Ascorbic acid pretreatment did not influence elevated blood lactate/pyruvate ratios or impaired intellectual function.

Modulation of the flavin-containing monooxygenase in guinea pigs by ascorbic acid and food restriction.

Modulation of the flavin-containing monooxygenase (FMO) by varying the ascorbic acid and food intake was investigated. Hepatic activity of the FMO in ascorbic acid-deficient guinea pigs fed a restricted amount of diet which resulted in a 10-15% body weight loss, was 17% of that in animals fed restricted amounts of the adequate diet. FMO hepatic activity in ascorbic acid-supplemented guinea pigs on a food-restricted regimen was 176% of that found in animals fed the adequate diet ad libitum. This increase in activity was not related to stress. Alteration in the activity of this important drug-metabolizing enzyme system by a combination of ascorbic acid deficiency and reduced food intake could potentially alter the rate of metabolism of a great variety of pharmaceutical drugs and environmental chemicals.

Ascorbic acid chronic alcohol consumption in the guinea pig.

Protection against the toxic effects of chronic alcohol consumption was observed in male guinea pigs maintained on a high-ascorbic-acid diet (vitamin C-deficient chow plus 2.0 mg ascorbic acid/ml drinking water) as compared to animals on a low-ascorbic-acid diet (vitamin C-deficient chow and from 0.025 to 0.050 mg ascorbic acid/ml drinking water). Alcohol was orally administered to the guinea pigs at a dose of 2.5 g/kg for up to 14 weeks. Levels of serum aspartate aminotransferase and serum alanine aminotransferase were significantly elevated in animals on the low-ascorbic-acid diet that received alcohol, 120 and 250%, respectively. In contrast, in animals on the high-ascorbic-acid diet that received alcohol, levels of alanine aminotransferase were not significantly elevated and levels of aspartate aminotransferase were elevated 50%. In addition, some of the animals on the low-ascorbic-acid diet that received alcohol for 12 to 14 weeks developed hepatic steatosis and necrosis, whereas none of the animals on the high-ascorbic-acid diet that received alcohol for the same length of time manifested these changes.

Ascorbic acid deficiency and the flavin-containing monooxygenase.

Activity of the flavin-containing monooxygenase (FMO) was reduced significantly in ascorbic acid deficient guinea pigs. Reduction in oxidation of dimethylaniline (DMA) and of thiobenzamide was associated with a decrease in the activity of the FMO. In both ascorbate supplemented and deficient guinea pig hepatic 12,000 g supernatant fractions, SKF-525A and n-octylamine did not inhibit DMA N-oxidation. Phenobarbital pretreatment did not increase the rate of N-oxidation of DMA. In addition, hepatic supernatant fractions thermally treated at 50 degree were unable to N-oxidize DMA, but 80% of the cytochrome P-450 activity was retained. Also, N-oxidation of DMA was reduced by 53% at pH 7.0, while oxidation of cytochrome P-450 specific substrates was inhibited by only 19%. Kinetic studies of DMA N-oxidation indicate no significant change in the apparent Km in ascorbate supplemented or deficient animals. The in vitro addition of ascorbic acid had no effect on the activity of the FMO. The toxicological implications of the reduction in FMO activity in ascorbic acid deficiency are discussed.

Effect of ascorbate on covalent binding of benzene and phenol metabolites to isolated tissue preparations.

[14C]Phenol and [14C]benzene are metabolized in the presence of NADPH and hepatic microsomes isolated from phenobarbital- or benzene-pretreated or untreated guinea pigs to intermediates capable of covalently binding to microsomal protein. When 1 mM ascorbate was included in the incubation mixture containing benzene as the substrate, covalent binding was inhibited by 55%. Increasing the ascorbate concentration to 5 mM inhibited binding by only an additional 17%. In contrast, when phenol was used as the substrate, 1 mM ascorbate inhibited binding by 95%. When DT-diaphorase was included in the incubation mixture containing benzene as the substrate, binding was inhibited by only 18%. This degree of inhibition is in contrast to 70% inhibition with phenol. These results indicate that different metabolites are responsible for a portion of the covalent binding depending upon the substrate employed. GSH inhibited covalent binding greater than 95% with either substrate. The metabolism of phenol to hydroquinone was unaffected by the addition of ascorbate or GSH. The metabolism of benzene to phenol was unaffected by the addition of GSH; however, the addition of ascorbate decreased the formation of phenol by 35%. Tissue ascorbate could be modulated by placing guinea pigs on different dietary intakes of ascorbate. Bone marrow ascorbate concentrations could be modulated 10-fold without any significant change in the GSH concentrations. Bone marrow isolated from guinea pigs on different dietary intakes of ascorbate were incubated with H2O2 and phenol. Bone marrow with low ascorbate concentrations displayed 4-fold more covalent binding of phenol equivalents than those with high ascorbate concentrations. This is an example of how the dietary intake of ascorbate can result in a differential response to a potentially toxic event in vitro.

Ascorbic acid and alcohol oxidation.

Methanol and ethanol were rapidly metabolized to formaldehyde and acetaldehyde in the presence of ascorbate, 1,10-phenanthroline and either guinea pig hepatic 100,000 g supernatant or 12,000 g pellet fractions. The specific activity of methanol oxidation was 1720 nmoles formaldehyde formed/min/mg protein in the 100,000 g fraction and 790 in the 12,000 g pellet fraction. The specific activity of ethanol oxidation was 1590 nmoles acetaldehyde formed/min/mg protein in the 100,000 g fraction and 820 in the 12,000 g pellet fraction. The activity was enzymatic in that it was linear with time, proportional to protein concentration, and sensitive to temperature. Catalase appeared to be the enzymatic component responsible for the oxidation. In this ascorbate-dependent alcohol oxidation system, oxygen was consumed and H2O2 was formed. When purified catalase and ascorbate were used, complex I was detected and methanol was oxidized.

DT-diaphorase and peroxidase influence the covalent binding of the metabolites of phenol, the major metabolite of benzene.

The role of various enzymes and biological molecules on the activation and deactivation of the metabolites of phenol was investigated in vitro. Phenol, the major metabolite of benzene, is metabolized to hydroquinone and catechol. Activation of these metabolites and deactivation of their oxidized forms was assessed by the amount of covalent binding to microsomal protein. [14C]Phenol and NADPH were incubated with hepatic microsomes isolated from phenobarbital-pretreated guinea pigs, and 2.33 nmoles of hydroquinone and 0.12 nmole of catechol were formed per minute per milligram of microsomal protein. Covalent binding of the metabolites to microsomal protein incubated with microsomes isolated from guinea pigs pretreated with phenobarbital was 252 pmoles bound/min/mg; with microsomes from untreated guinea pigs, covalent binding was 146 pmoles bound/min/mg. Covalent binding was inhibited greater than 90% with the addition of N-octylamine, ascorbate, or GSH. The addition of superoxide dismutase inhibited covalent binding with microsomes isolated from phenobarbital-pretreated guinea pigs 35% but did not inhibit it with microsomes isolated from untreated animals. Partially purified guinea pig hepatic DT-diaphorase [NAD(P)H (quinone acceptor) oxidoreductase, EC 1.6.99.2] inhibited covalent binding 70%. This effect was reversed in the presence of dicumarol, a specific inhibitor of DT-diaphorase. DT-diaphorase present in the 10(5) X g supernatant fraction was also active in inhibiting covalent binding but only after the removal of endogenous reduced glutathione. This effect could also be reversed by dicumarol. The addition of diaphorase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) partially purified from Clostridium kluyveri inhibited covalent binding 86%. The addition of hydrogen peroxide and horseradish peroxidase (peroxidase, EC 1.11.17) or myeloperoxidase(s) increased covalent binding 30-fold and 6-fold, respectively. Ascorbate decreased this binding greater than 95%. These results indicate that hydroquinone, catechol, and phenol as well as their oxidized forms can be activated or deactivated by several of the above model systems. These systems may play a role in the myelotoxicity of benzene by modulating covalent binding.

Hepatic bromobenzene epoxidation and binding: prevention by ascorbyl palmitate.

Bromobenzene undergoes metabolic activation via 2,3- and 3,4-epoxidation catalyzed by the hepatic cytochrome P-450 mixed-function oxidase system. Its reactive metabolites, especially bromobenzene 3,4-oxide, presumably lead to severe centrolobular necrosis. A study of relative rate of binding of 14C-bromobenzene metabolites to hepatic microsomal protein indicated a significant difference in the rate of binding of the bromobenzene 3,4-oxide compared to its positional isomer, bromobenzene 2,3-oxide. However, the rate of bromobenzene metabolism indicated no significant difference in the formation of products o-bromophenol and p-bromophenol. A search for protective agents revealed that 6,7-dimethyl-5,6,7,8-tetrahydropterine and ascorbyl palmitate were very effective in protecting against macromolecular adduct formation at a concentration of 1 mM-in fact, at least a twofold increase in protection compared to the known protective agents such as glutathione or cysteine. Furthermore, 6,7-dimethyl-5,6,7,8-tetrahydropterine and ascorbyl palmitate inhibited the metabolism of bromobenzene over 90% at a concentration of 2.5 mM.

Bromobenzene epoxidation leading to binding on macromolecular protein sites.

Bromobenzene is metabolized via the hepatic mixed-function oxygenase system to reactive intermediates; i.e., 2,3- and 3,4-bromobenzene epoxides. These metabolites presumably bind to tissue macromolecules evoking cytotoxicity. However, the specific sites of macromolecular proteins are not known and this was investigated using microsomal protein and hemoglobin. The results indicate that 2,3- and 3,4-epoxide bind to macromolecules at different rates. The 3,4-epoxide is more reactive, binding covalently to microsomal protein at the site of its synthesis, whereas bromobenzene 2,3-epoxide is more stable, leaving the microsomal protein compartment and binding covalently to soluble protein, i.e., the hemoglobin beta chain. Structural analysis with fingerprint mapping of the hemoglobin beta chain after pretreatment with cysteine and histidine blocking agents such as p-nitrophenacyl bromide, 2-bromoethylamine hydrobromide and diethylprocarbonate indicated that the 2,3-epoxide preferentially binds to the cysteinyl residue, whereas the 3,4-epoxide binds to the histidinyl residue. This difference in binding of the 2,3- and 3,4-bromobenzene epoxides may be an important factor in determining the degree of their cytotoxicity.

Interactions of glucose-6-phosphate dehydrogenase deficiency with drug acetylation and hydroxylation reactions.

We hypothesize that the bimodal distribution of hemolytic response by G6PG-deficient individuals to particular drugs such as sulfones may be due to the genetically determined acetylation rate of those drugs. Since metabolism, e.g., hydroxylation, may be required for these drugs to become hemolytic, genetically and environmentally determined variation in hydroxylation of a drug may also contribute to this variability in hemolytic response. To test the possibilities that acetylation and hydroxylation alter the hemolytic potential of these drugs, we incubated G6PG-deficient and normal red cells with mouse liver microsomes at two states of hydroxylase activity (uninduced and induced), an NADPH-generating system, and acetylated or unacetylated drug. We then measured GSH depletion in the cells as an indicator of prelytic cell damage. We found that in the presence of induced (high hydroxylase activity) microsomes, thiazolsulfone (Promizole) or DDS in unacetylated form caused the highest level of GSH depletion in G6PD-deficient red cells. Acetylation protected against GSH depletion. The specificity of the hydroxylation reaction in producing marked GSH depletion was shown by the protective effect of a specific hydroxylation inhibitor. Our results indicate that G6PD-deficient, genetically slow acetylators, having high microsomal activity, would be most susceptible to Promizole- or DDS-induced hemolysis, compared to other metabolic phenotypes. In addition, the bimodality in hemolytic response to Promizole probably corresponds to the bimodal distribution of acetylator phenotype in the population.