Random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsomes

The dilution of rabbit liver microsomes by soy-bean phospholipids was used as methodical approach to investigate the molecular organization of NADPH-dependent microsomal redox chain. The ultrastructural analysis of control and phospholipid diluted microsomes revealed that the incorporation of exogenous phospholipids into microsome membranes increased their surface area, as well as decreased the lateral density distribution and size of intramembrane particles. The dilution of microsome membranes by phospholipids slowed down the initial rate of cytochrome P-450 reduction by NADPH. The apparent second order rate constant of cytochrome P-450 reduction by NADPH: cytochrome P-450-reductase did not change in phospholipid-enriched microsomes. The results obtained provide strong evidence for the random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsome membranes.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.     

Distribution of NAD(P)H-dependent cytochrome P-450 mixed function oxidase system in the brush border membrane of rabbit kidney cortex.

Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome P-450 and cytochrome b5-dependent mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome P-450-dependent mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system.     

Additive effects of epinephrine and corticotropin-releasing factor (CRF) on adrenocorticotropin release in rat anterior pituitary cells

Rabbit antibody was prepared against NADPH-cytochrome c reductase of Tetrahymena microsomes. When examined by the Ouchterlony double diffusion test, anti-NADPH-cytochrome c reductase immunoglobulin formed a single precipitation line with Tetrahymena reductase but not rat liver one. The antibody inhibited the NADPH-cytochrome c reductase activity of Tetrahymena microsomes, but it did not affect either NADH-ferricyanide or NADH-cytochrome c reductase activity of Tetrahymena microsomes. The NADPH-dependent desaturation of stearoyl-CoA in Tetrahymena microsomes was inhibited by anti-reductase immunoglobuline, while the NADH-dependent desaturation was affected by neither anti-reductase nor control immunoglobuline. It was suggested that the temperature associated-alteration of NADPH-cytochrome c reductase activities would be important for regulation of microsomal NADPH-dependent desaturase activities in Tetrahymena which contains no cytochrome P-450.     

Effects of MPTP, MPP+, and paraquat on NADPH-dependent lipid peroxidation in mouse brain and lung microsomes

Both MPTP and MPP+ inhibited the NADPH-dependent microsomal LPO in mouse brain and lung. On the other hand, PQ significantly stimulated the LPO in brain microsomes in a dose-dependent manner. The herbicide, however, stimulated lung microsomal LPO only in a narrow concentration range, despite much higher P450 reductase activity in lung microsomes than that in brain microsomes. These findings suggest that the effect of PQ on microsomal LPO is different from those of the analogous neurotoxins, MPTP and MPP+, and is not uniform in brain and lung.     

Peroxide modification of membranes and isomorphic composition of cytochrome P-450 of rat liver microsomes during antioxidant deficiency]

Lipid peroxidation (LPO), physico-chemical properties of the membranes and isoformic composition of microsomal cytochrome P-450 from the rat liver were studied under conditions of antioxidant insufficiency (AOI) which was modelled by exclusion of alpha-tocopherol from the animals' ration. An insignificant accumulation of microsomal diene conjugates and schiff bases against a sharp increase of the ability to the prooxidant stimulated LPO in vitro took place. A significant decrease of membrane lipid microviscosity and a change in surface properties of microsomal membranes of rats with AOI was determined. Absence of alpha-tocopherol in the ration was accompanied by a significant change in the content of separate isoforms of cytochrome P-450 exhibited in growth of a polypeptide with m. w. 54 kDa and the lowering of proteins with m. w. 48 and 50 kDa. Less intensive quenching of tryptophan fluorescence by acrylamide was also revealed, which testified to a lower accessibility of the quencher to membrane proteins or their fluorophore sites. Modification of lipid composition and of physicochemical properties of the rat liver membrane microsomes which was observed at AOI was significantly correlated by pretreatment with the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol).     

Unique properties of NADPH- and NADH-dependent metabolism of p-nitroanisole catalyzed by cytochrome P-450 isozyme 2 in pulmonary and hepatic microsomal preparations from rabbits

Approximately 90% of the NADPH- and NADH-dependent O-demethylation of p-nitroanisole (PNA) in the hepatic microsomal fraction from phenobarbital (PB)-treated rabbits and in the pulmonary microsomal fraction from untreated rabbits is catalyzed by the same isozyme of cytochrome P-450. This isozyme of cytochrome P-450 catalyzes less than 60% of this reaction in the hepatic microsomal fraction from untreated rabbits. Antibodies to NADPH-cytochrome P-450 reductase inhibit NADPH-dependent metabolism of p-nitroanisole by about 90% but have no effect on NADH-dependent metabolism. Hepatic NADPH-dependent metabolism of pNA and reduction of cytochrome c are inhibited to the same extent with varying amounts of antibodies to NADPH cytochrome P-450 reductase. The same relationship between inhibition of monooxygenase and reductase activities is observed for the hepatic and pulmonary metabolism of benzphetamine and 7-ethoxycoumarin. In contrast, the relationship between inhibition of the pulmonary NADPH-dependent metabolism of pNA and reductase activity is biphasic; at 75% inhibition of reductase activity, metabolism of pNA is inhibited by less than 25%. For NADH-dependent metabolism of pNA, our results indicate that both electrons are transferred to cytochrome P-450 from cytochrome b5.     

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.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Electron paramagnetic resonance decay constant and oxidative stresses in liver microsomes of the selenium-deficient rat

The free radical-reducing activity and the membrane fluidity of liver microsomes from selenium-deficient (SeD) rats were examined by means of electron paramagnetic resonance (EPR) spin label method using nitroxyl-labeled stearic acids. Our findings show that the membrane fluidity and lipid peroxidation levels in SeD rat liver microsome were relatively unchanged compared with normal rat. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity. The nitroxyl spin probes are substrates for reduction-relating cytochrome P-450. Previous in vivo studies suggested that the total liver free radical reduction activity in SeD rat was decreased. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity, and the reduction rate of nitroxyl radical existing at shallow depth in membrane was increased. Selenium-deficient rats experienced an increase in hydrogen peroxide (H2O2) due to a pronounced loss of glutathione peroxidase (GSH-Px) activity. This masked the overall reduction rate of the nitroxyl spin probe by reoxidation of the hydroxylamine form. Although the SeD condition caused induction of liver cytochrome P-450 and chronic increased H2O2, this did not result in oxidative liver damage. An increased level of glutathione in SeD liver was also evident, likely due to the absence of GSH-Px activity. Using the EPR spin label method, we have shown that SeD causes complicated redox changes in the liver, notably, alterations in the levels of cytochrome P-450 and GSH-Px systems.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.     

Role of the microsomal ethanol-oxidizing system in regulating linoleoyl CoA desaturase activity in long-term ethanol loading

Long-term ethanol load resulted in a decrease of the rat liver linoleyl desaturase activity and the activation of MEOS accompanied by an increase in the activity of NADPH-dependent chain and the initial steps of NADH-dependent chain of microsomal electron transport, indicating electron transfer from NADH to cytochrome P-450. It is suggested that, when the main potential of NADH- and NADPH-dependent chains is transferred to microsomal ethanol oxidation, insufficient electron supply for linoleyl-CoA desaturase decreases the activity of this process.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Irreversible binding of DOPA and dopamine metabolites to protein by rat liver microsomes.

Rat liver microsomes catalyze NADPH-dependent irreversible binding of metabolites of DOPA and DOPAmine to microsomal protein and to BSA. Binding is inhibited by cysteine and the singlet oxygen quencher 1,4-diaza-bicyclo(2.2.2)octane. Irreversible binding to BSA is also catalyzed by mushroom tyrosinase, xanthine oxidase, and NADPH-cytochrome c reductase. The results suggest that in the microsomal system the participation of the hemoprotein, cytochrome P-450, is not an absolute requirement for the irreversible binding of metabolites of DOPA and DOPAmine to proteins.     

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.     

Stabilizing effect of hydroxybenzimidazole and its derivatives on biological membranes during activation of lipid peroxidation

Inhibition of lipid peroxidation (LPO) by oxybenzimidazole (OBI) and its derivatives--alkyloxybenzimidazole (AOBI) and alkylethoxybenzimidazole (AEBI) was studied in liver microsomes and brain synaptosomes. It has been shown that both OBI and AOBI strongly inhibit LPO in microsomes and not synaptosomes. AEBI failed to inhibit LPO in microsomes. AOBI is more potent than OBI both in ascorbate- and NADPH-dependent LPO of microsomes. An antioxidant effect of both compounds is more marked in ascorbate-dependent LPO. The investigation of the possible use of AOBI for the protection of liver membranes in various pathological conditions associated with LPO activation seems promising.