Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Increased NADPH-dependent chemiluminescence by microsomes after chronic ethanol consumption

The generation of reactive oxygen intermediates by microsomes from ethanol-fed rats and pair-fed controls was determined by assaying for NADPH-dependent chemiluminescence. In the absence or presence of added ferric complexes, microsomal light emission was elevated several-fold after chronic ethanol consumption. Iron complexes such as ferric-citrate or ferric-ATP stimulated, while ferric-EDTA, inhibited microsomal chemiluminescence. Freeze-thawing the microsomes to elevate their content of lipid hydroperoxides resulted in large increases in chemiluminescence; under all conditions, the light emission remained several-fold higher with microsomes from the ethanol-fed rats. Chemiluminescence was not sensitive to superoxide dismutase, catalase, or the hydroxyl radical scavenging agent, dimethyl sulfoxide, but was inhibited by antioxidants and by glutathione. Replacing air with a mixture of 50% nitrogen-50% air or 50% carbon monoxide-50% air had no effect on chemiluminescence by microsomes from the pair-fed controls. However, the chemiluminescent response by microsomes from the ethanol-fed rats was inhibited about 50% by the nitrogen mixture, and was further inhibited (about 75% of values found with 100% air, and 50% of values found with 50% nitrogen-50% air) with the carbon monoxide mixture. The sensitivity to carbon monoxide suggests the possibility that the alcohol-inducible cytochrome P-450 isozyme may contribute, in part, to the elevated light emission produced by microsomes from the ethanol-fed rats. The increase in chemiluminescence by microsomes after chronic ethanol consumption appears to reflect an elevated level of lipid hydroperoxides as well as an increased rate of generation of reactive oxygen species.     

Increased NADH-dependent production of reactive oxygen intermediates by microsomes after chronic ethanol consumption: comparisons with NADPH.

Microsomes from chronic ethanol-fed rats were previously shown to catalyze the NADPH-dependent production of reactive oxygen intermediates at elevated rates compared to controls. Recent studies have shown that NADH can also serve as a reductant and promote the production of oxygen radicals by microsomes. The current study evaluated the influence of chronic ethanol consumption on NADH-dependent microsomal production of reactive oxygen intermediates, and compared the results with NADH to those of NADPH. Microsomal oxidation of chemical scavengers, taken as a reflection of the production of hydroxyl radical (.OH)-like species was increased about 50% with NADH as cofactor and about 100% with NADPH after chronic ethanol consumption. The potent inhibition of the production of .OH-like species by catalase suggests a precursor role for H2O2 in .OH production. Rates of NADH- and NADPH-dependent H2O2 production were increased by about 50 and 70%, respectively, after chronic ethanol consumption. A close correlation between rates of H2O2 production and generation of .OH-like species was observed for both NADH and NADPH, and increased rates of H2O2 production appear to play an important role in the elevated generation of .OH-like species after chronic ethanol treatment. Microsomal lipid peroxidation was elevated about 60% with NADH, and 120% with NADPH, after ethanol feeding. With both types of microsomal preparations, the characteristics of the NADH-dependent reactions were similar to the NADPH-dependent reactions, e.g., sensitivity to antioxidants and free radical scavengers and catalytic effectiveness of ferric complexes. However, rates with NADPH exceeded the NADH-dependent rates by 50 to 100%, and the increased production of reactive oxygen intermediates by microsomes after ethanol treatment was greater with NADPH (about twofold) than with NADH (about 50%). Oxidation of ethanol results in an increase in hepatic NADH levels and interaction of NADH, iron, and microsomes can produce potent oxidants capable of initiating lipid peroxidation and oxidizing .OH scavengers. These acute metabolic interactions produced by ethanol-derived NADH are increased, not attenuated, in microsomes from chronic ethanol-fed rats, and it is possible that such increases in NADH (and NADPH)-dependent production of reactive oxygen species play a role in the development of oxidative stress in the liver as a consequence of ethanol treatment.     

Transfer and reoxidation of reducing equivalents as the rate-limiting steps in the oxidation of ethanol by liver cells isolated from fed and fasted rats

A study was made of factors regulating the oxidation of ethanol in liver cells isolated from fed and fasted rats. The rate of ethanol oxidation was greater in liver cells from fed rats than from fasted rats. Inhibitors of the malate-aspartate shuttle decreased the rate of ethanol oxidation, suggesting that this shuttle contributes to the reoxidation of cytosolic NADH produced during the oxidation of ethanol. The greater inhibition of ethanol oxidation by antimycin than by rotenone suggests that the α-glycerophosphate shuttle also plays an important role in transporting reducing equivalents. The components of the malate-aspartate and α-glycerophosphate shuttles stimulated ethanol oxidation to a greater extent in liver cells from fasted rats than those from fed rats, suggesting that in the fasted state, ethanol oxidation is regulated by the intracellular concentrations of substrate shuttle components which transfer reducing equivalents into the mitochondria. Therefore, uncoupling agents, which stimulate oxygen consumption, do not stimulate ethanol oxidation, and concentrations of antimycin which depress oxygen uptake are much less effective in decreasing ethanol oxidation. By contrast, in liver cells from fed rats, the rate of ethanol oxidation was increased by uncoupling agents. Such stimulation was not observed when cells were prepared in the absence of albumin, probably due to leakage of shuttle substrates which leads to abnormally low intracellular levels. Indeed, when the shuttle substrates were added back to these preparations, uncouplers were effective in stimulating the rate of ethanol oxidation beyond the stimulation produced by the shuttle substrates alone. Thus, under conditions of sufficient intracellular levels of the intermediates of the substrate shuttles, ethanol oxidation is regulated by the capacity of the mitochondrial respiratory chain to reoxidize reducing equivalents generated by the alcohol dehydrogenase reaction.     

Effects of Oxidized Cooking Oil and α-Lipoic Acid on Liver Antioxidants: Enzyme Activities and Lipid Peroxidation in Rats Fed a High Fat Diet

The effects of administration of oxidized rapeseed oil and α-lipoic acid on activities of hepatic antioxidant enzymes and lipid peroxidation were studied in laboratory rats. There was an increase of the activities of superoxide dismutase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase in rats fed a high fat diet to which 10% oxidized oil was added. Administration of α-lipoic acid resulted in a decrease of the activities of these enzymes. Addition of oxidized oil also resulted in increased production of oxygen radicals, evidenced by elevated malondialdehyde production. Such effect was counteracted by administration of α-lipoic acid.     

Diabetes impairs the enzymatic disposal of 4-hydroxynonenal in rat liver

This study assesses whether the HNE accumulation we formerly observed in liver microsomes and mitochondria of BB/Wor diabetic rats depends on an increased rate of lipoperoxidation or on impairment of enzymatic removal. There are three main HNE metabolizing enzymes: glutathione-S-transferase (GST), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). In this study we show that GST and ALDH activities are reduced in liver microsomes and mitochondria of diabetic rats; in contrast, ADH activity remains unchanged. The role of each enzyme in HNE removal was evaluated by using enzymatic inhibitors. The roles of both GST and ALDH were markedly reduced in diabetic rats, while ADH-mediated consumption was significantly increased. However, the higher level of lipohydroperoxides in diabetic liver indicated more marked lipoperoxidation. We therefore think that HNE accumulation in diabetic liver may depend on both mechanisms: increased lipoperoxidation and decreased enzymatic removal. We suggest that glycoxidation and/or hyperglycemic pseudohypoxia may be involved in the enzymatic impairment observed. Moreover, since HNE exerts toxic effects on enzymes, HNE accumulation, deficiency of HNE removal, and production of reactive oxygen species can generate vicious circles able to amplify the damage.     

Effect of vitamin E from by-products of cotton oil processing on the glutathione peroxidase system in rats

The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione peroxidase from liver, skeletal muscles and erythrocytes of rats fed a vitamin E-deficient, or supplemented, diet were studied. Vitamin E was added in the diet either as a pure pharmacy form of alpha-tocopherol or as a tocopherol mixture derived from oil wastes. The deficiency of vitamin E caused an increase in the activity of the above mentioned enzymes. Both alpha-tocopherol and the tocopherol mixture were found to influence the glutathione peroxidase system. The dose-dependent response of the glutathione peroxidase system was revealed. Possible mechanisms of the changes in the antioxidizing enzymes induced by vitamin E are discussed.     

Effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity in rats

In order to find out the effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity, male rats fed with ethanol 1.6g/kg body weight per day for four weeks were studied. Besides a drastic reduction in body and testis weight, there was decrease in ascorbic acid, reduced glutathione and activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase in the testicular tissue of the treated animals. Simultaneously, there was increase in lipid peroxidation and glutathione S-transferase activity. Activities of 3 beta-hydroxy steroid dehydrogenase and 17 beta-hydroxy steroid dehydrogenase were also found decreased in the treated animals. The results indicate that chronic ethanol administration resulted in increase in oxidative stress and decrease in the activities of steroidogenic enzymes in the rat testes.     

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.     

Rate-limiting factors in the oxidation of ethanol by isolated rat liver cells.

The oxidation of ethanol by isolated liver cells from starved rats is limited by the rate of removal of reducing equivalents generated in the cytosol by alcohol dehydrogenase. Evidence is presented suggesting that, in these cells, transfer of reducing equivalents from the cytosol to the mitochondria is regulated by the intracellular concentrations of the intermediates of the malate-aspartate and glycerol 3-phosphate cycles, as well as by flux through the respiratory chain. In liver cells isolated from fed rats, the availability of substrate increased the cell content of intermediates of the hydrogen-transfer cycles, and enhanced ethanol uptake. Under these conditions, ethanol consumption is limited by the availability of ADP for oxidative phosphorylation.     

Protective effect of dietary squalene supplementation on mitochondrial function in liver of aged rats

Mitochondria are an important intracellular source and target of reactive oxygen species. The life span of a species is thought to be determined, in part, by the rate of mitochondrial damage inflicted by oxygen free radicals during the course of normal cellular metabolism. In the present study, we have investigated the protective effect of squalene supplementation for 15 days and 30 days on energy status and antioxidant defense system in liver mitochondria of 18 young and 18 aged rats. The dietary supplementation of 2% squalene significantly minimized aging associated alterations in mitochondrial energy status by maintaining the activities of TCA cycle enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) at higher level in the liver mitochondria of aged rats compared with unsupplemented controls. It exerted an antioxidant effect by inhibiting mitochondrial lipid peroxidation (malondialdehyde) in liver of young and aged rats. Supplementation with squalene also maintained the mitochondrial antioxidant defense system at higher rate by increasing the level of reduced glutathione and the activities of glutathione-dependent antioxidant enzymes (glutathione peroxidase and glutathione-S-transferase) and antiperoxidative enzymes (superoxide dismutase and catalase) in the liver of young and aged rats. The results of this study provide evidence that dietary supplementation with squalene can improve liver mitochondrial function during aging and minimize the age-associated disorders in which reactive oxygen species are a major cause.     

Effect of glutamate on the control of fatty-acid synthesis in white adipose tissue of the rat. Inhibition of pyruvate dehydrogenase.

Glutamate (5mM) inhibited glucose conversion to fatty acids by approximately one-third in adipocytes from fed rats. This inhibition was significantly less in the pressence of pyruvate or 2-oxoglutarate. After incubation of adipose tissue from fed rats with glucose and insulin, pyruvate dehydrogenase activity was 180 plus or minus 17 mU/g wet weight. Addition of glutamine to the incubation medium decreased this activity significantly (118 plus or minus 14 mU/g wet weight). This inhibition by glutamate was also diminished when 2-oxoglutarate or pyruvate were present. Glutamate added to homohentates of adipose tissue had no effect on the activation of pyruvate dehydrogenase by Mg-2+. However, glutamate inhibited the active form of the enzyme and enhanced the rate of inactivation of the enzyme complex by ATP and Mg-2+. Aminooxyacetate, a transaminase inhibitor, did not reverse the effects of glutamate on pyruvate dehydrogenase nor fatty acid synthesis.     

Mechanism of the inhibition of catalase by ascorbate. Roles of active oxygen species, copper and semidehydroascorbate

Ascorbate reversibly inhibits catalase, and this inhibition is enhanced and rendered irreversible by the prior addition of copper(II)-bishistidine. In the absence of copper, the inhibition was prevented and reversed by ethanol, but not by superoxide dismutase, benzoate, mannitol, thiourea, desferrioxamine, or DETAPAC. In the presence of the copper complex mannitol, benzoate, and superoxide dismutase still had no effect, but thiourea, desferrioxamine, DETAPAC, or additional histidine decreased the extent of inactivation to that seen in the absence of copper. In the presence of copper, ethanol protected at [ascorbate] less than 1 mM, but was ineffective at [ascorbate] greater than 2 mM, even in the absence of oxygen. Although in the absence of copper, complete removal of oxygen provided full protection against inactivation by ascorbate, this protection was not seen if the catalase was briefly preincubated with H2O2 prior to flushing with nitrogen, or if copper was present. In fact, if copper was present, inactivation was enhanced by the removal of oxygen. Increasing the concentration of oxygen from ambient to 100% slowed the inactivation, whether or not copper was present. It is concluded that the initial reversible inactivation involves reaction with H2O2 to form compound I, followed by one electron reduction of compound I to compound II. In the presence of added copper, the initial (reversible) inactivation allows H2O2 to accumulate sufficiently to permit irreversible inactivation. Since in the presence of copper oxygen is not required, and neither the reversible nor the irreversible inactivation was prevented by conventional scavengers of active forms of oxygen, the inactivation is likely mediated by semidehydroascorbate, and/or it may involve site-specific generation of the damaging intermediates.     

Paradoxical effect of sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5′-diphosphoglucuronic add or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.     

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.     

Stimulation of the pentose phosphate pathway and glutathione levels by dehydroascorbate, the oxidized form of vitamin C.

Ascorbic acid, or vitamin C, generally functions as an antioxidant by directly reacting with reactive oxygen intermediates and has a vital role in defenses against oxidative stress. However, ascorbic acid also has pro-oxidant properties and may cause apoptosis of lymphoid and myeloid cells. The present study shows that dehydroascorbate, the oxidized form of vitamin C, stimulates the antioxidant defenses of cells, preferentially importing dehydroascorbate over ascorbate. While 200-800 microM vitamin C caused apoptosis of Jurkat and H9 human T lymphocytes, pretreatment with 200-1000 microM dehydroascorbate stimulated activity of pentose phosphate pathway enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase, elevated intracellular glutathione levels, and inhibited H(2)O(2)-induced changes in mitochondrial transmembrane potential and cell death. A 3. 3-fold maximal glutathione elevation was observed after 48 h stimulation with 800 microM dehydroascorbate. In itself, dehydroascorbate did not affect cytosolic or mitochondrial reactive oxygen intermediate levels as monitored by flow cytometry using oxidation-sensitive fluorescent probes. The data reveal a novel mechanism for increasing glutathione levels through stimulation of the pentose phosphate pathway and identify dehydroascorbate as an antioxidant for cells susceptible to the pro-oxidant and proapoptotic properties of vitamin C.     

Irreversible inactivation of calcium-dependent proteinases from rat liver by biological disulfides

The effect of low-molecular-mass biological disulfides and their related reduced compounds on the activity of two calcium-dependent neutral proteinases (calpains) from rat liver has been investigated. L-Cystine and L-cystamine bring about the inactivation of both enzymes, while the related reduced compounds L-cysteine and L-cysteamine are without effect. Calpain II is more sensitive to the inactivating effect of glutathione disulfide in comparison with calpain I. The inactivation rates of both calpains depend on the concentration of glutathione disulfide. Reduced glutathione, added at physiological concentration (5 mM), neither affects the proteinase activities nor protects the enzymes from the inactivating effect of glutathione disulfide. The enzymes inactivated by biological disulfides cannot be restored by a large excess of a reducing thiolic compound (dithiothreitol). It is suggested that calcium-dependent proteinases might be inactivated also in vivo by enhanced level of glutathione disulfide.     

Association of a reactive intermediate derived from 1′,6‐dihydroxy metabolite with benzbromarone‐induced hepatotoxicity

Treatment with benzbromarone can be associated with liver injury, but the detailed mechanism remains unknown. Our recent studies demonstrated that benzbromarone was metabolized to 1′,6‐dihydroxybenzbromarone and followed by formation of reactive intermediates that were trapped by glutathione, suggesting that the reactive intermediates may be responsible for the liver injury. The aim of this study was to clarify whether the reactive intermediates derived from 1′,6‐dihydroxybenzbromarone is a risk factor of liver injury in mice. An incubation study using mouse liver microsomes showed that the rates of formation of 1′,6‐dihydroxybenzbromarone from benzbromarone were increased by pretreatment with dexamethasone. Levels of a hepatic glutathione adduct derived from 1′,6‐dihydroxybenzbromarone were increased by pretreatment with dexamethasone. Furthermore, plasma alanine amino transferase activities were increased in mice treated with benzbromarone after pretreatment with dexamethasone. The results suggest that the reactive intermediate derived from 1′,6‐dihydroxybenzbromarone may be associated with liver injury.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.