Microsomal Oxidation Research Articles

Inhibition of rat mammary microsomal oxidation of ethanol to acetaldehyde by plant polyphenols

We previously reported that the microsomal fraction from rat mammary tissue is able to oxidize ethanol to acetaldehyde, a mutagenic-carcinogenic metabolite, depending on the presence of NADPH and oxygen but not inhibited by carbon monoxide or other cytochrome P450 inhibitors. The process was strongly inhibited by diphenyleneiodonium, a known inhibitor of NADPH oxidase, and by nordihydroguaiaretic acid, an inhibitor of lipoxygenases. This led us to suggest that both enzymes could be involved. With the purpose of identifying natural compounds present in food with the ability to decrease the production of acetaldehyde in mammary tissue, in the present studies, several plant polyphenols having inhibitory effects on lipoxygenases and of antioxidant nature were tested as potential inhibitors of the rat mammary tissue microsomal pathway of ethanol oxidation. We included in the present screening study 32 polyphenols having ready availability and that were also tested against the rat mammary tissue cytosolic metabolism of ethanol to acetaldehyde. Several polyphenols were also able to inhibit the microsomal ethanol oxidation at concentrations as low was 10-50 μM. The results of these screening experiments suggest the potential of several plant polyphenols to prevent in vivo production and accumulation of acetaldehyde in mammary tissue.

What is the biological fate of mentholated cigarette smoking?

What is the biological fate of mentholated cigarette smoking?

A Baeyer-Villiger Oxidation Specifically Catalyzed by Human Flavin-Containing Monooxygenase 5

10-((4-Hydroxypiperidin-1-yl)methyl)chromeno[4,3,2-de]phthalazin-3(2H)-one (E7016), an inhibitor of poly(ADP-ribose) polymerase, is being developed for anticancer therapy. One of the major metabolites identified in preclinical animal studies was the product of an apparent oxidation and ring opening of the 4-hydroxypiperidine. In vitro, this oxidized metabolite could not be generated by incubating E7016 with animal or human liver microsomes. Further studies revealed the formation of this unique metabolite in hepatocytes. In a NAD(P)(+)-dependent manner, this metabolite was also generated by liver S9 fractions and recombinant human flavin-containing monooxygenase (FMO) 5 that was fortified with liver cytosol fractions. In animal and human liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), or a brief heat treatment at 50°C. Based on these results, the overall metabolic pathway was believed to involve a two-step oxidation process: dehydrogenation of the secondary alcohol in liver cytosol followed by an FMO5-mediated Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps were coupled via regeneration of NAD(P)(+) and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In an NAD(P)H-dependent manner, the synthetic ketone intermediate was metabolized to the same ring-opened metabolite in animal and human liver microsomes. This metabolic reaction was also inhibited by BNPP or a brief heat treatment at 50°C. Methimazole, the substrate/inhibitor of FMO1 and FMO3, did not inhibit this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the synthetic ketone intermediate in recombinant enzymes.

Fasting‐induced oxidative stress in very long chain acyl‐CoA dehydrogenase‐deficient mice

Hepatopathy and hepatomegaly as consequences of prolonged fasting or illnesses are typical clinical features of very long chain acyl-CoA dehydrogenase (VLCACD) deficiency, the most common long-chain fatty acid β-oxidation defect. Supplementation with medium-chain triglycerides (MCTs) is an important treatment measure in these defects, in order to supply sufficient energy. Little is known about the pathogenetic mechanisms leading to hepatopathy. Here, we investigated the effects of prolonged fasting and an MCT diet on liver function. Wild-type (WT) and VLCAD knockout mice were fed with either a regular long-chain triglyceride diet or an MCT diet for 5 weeks. In both groups, we determined liver and blood lipid contents under nonfasting conditions and after 24 h of fasting. Expression of genes regulating peroxisomal and microsomal oxidation pathways was analyzed by RT-PCR. In addition, glutathione peroxidase and catalase activities, as well as thiobarbituric acid reactive substances, were examined. In VLCAD knockout mice fed with a long-chain triglyceride diet, fasting is associated with excessive accumulation of liver lipids, resulting in hepatopathy and strong upregulation of peroxisomal and microsomal oxidation pathways as well as antioxidant enzyme activities and thiobarbituric acid reactive substances. These effects were even evident in nonfasted mice fed with an MCT diet, and were particularly pronounced in fasted mice fed with an MCT diet. This study strongly suggests that liver damage in fatty acid oxidation defects is attributable to oxidative stress and generation of reactive oxygen species as a result of significant fat accumulation. An MCT diet does not prevent hepatic damage during catabolism and metabolic derangement.

Computational approach to characterization of human liver drug-metabolizing enzymes

Cytochromes P450 are the key enzymes for activating and inactivating many drugs; individual expression levels of CYPs may play a crucial role in drug safety and drug efficacy. Statistical comparison of biochemical profiles of 23 human liver microsomes have been used to characterize human liver samples. The profile included 12 parameters, namely activity of NADPH-cytochrome P450 reductase, cytochrome P450 content and cytochrome P450-dependent monooxygenase activities with marker substrates. Unsupervised statistical methods including cluster analysis and principal component analysis revealed with very high confidence the presence of two groups. Difference between the groups was explained by peculiarities of reductase activity and cytochrome P450 enzyme activities with 7-ethoxyresorufin, 7-methoxyresorufin, 7-methoxycoumarin, 7-benzyloxyresorufin and 7-benzyloxyquinoline. Results of biochemical assays coupled with multidimensional data analysis can be further used for targeted proteomic profiling of microsome oxidation mechanisms.

Participation of CYP2C8 and CYP3A4 in the N‐demethylation of imatinib in human hepatic microsomes

Imatinib is a clinically important inhibitor of tyrosine kinases that are dysregulated in chronic myelogenous leukaemia and gastrointestinal stromal tumours. Inter-individual variation in imatinib pharmacokinetics is extensive, and influences drug safety and efficacy. Hepatic cytochrome P450 (CYP) 3A4 has been implicated in imatinib N-demethylation, but the clearance of imatinib decreases during prolonged therapy. CYP3A phenotype correlates with imatinib clearance at the commencement of therapy, but not at steady state. The present study evaluated the possibility that multiple CYPs may contribute to imatinib oxidation in liver. Imatinib biotransformation in human liver microsomes (n= 20) and by cDNA-expressed CYPs was determined by LC-MS. Relationships between imatinib N-demethylation and other drug metabolizing CYPs were assessed. N-desmethylimatinib formation was correlated with microsomal oxidation of the CYP3A4 substrates testosterone (ρ= 0.60; P < 0.01) and midazolam (ρ= 0.46; P < 0.05), and the CYP2C8 substrate paclitaxel (ρ= 0.58; P < 0.01). cDNA-derived CYPs 2C8, 3A4, 3A5 and 3A7 supported imatinib N-demethylation, but 10 other CYPs were inactive; in kinetic studies, CYP2C8 was a high-affinity enzyme with a catalytic efficiency ∼15-fold greater than those of CYPs 3A4 and 3A5. The CYP3A inhibitors ketoconazole and troleandomycin, and the CYP2C8 inhibitors quercetin and paclitaxel decreased imatinib oxidation. From molecular modelling, the imatinib structure could be superimposed on a pharmacophore for CYP2C8 substrates. CYP2C8 and CYPs 3A contribute to imatinib N-demethylation in human liver. The involvement of CYP2C8 may account in part for the wide inter-patient variation in imatinib pharmacokinetics observed in clinical practice.

Inhibition of CYP3A-mediated oxidation in human hepatic microsomes by the dietary derived complex phenol, gallic acid.

Plant polyphenols, such as gallic acid, have been reported to have a range of biological activities including antimutagenic effects. Previously, we reported that gallic acid (3,4,5-trihydroxybenzoic acid), an agent found in wine and tea, inhibits androstenedione 6beta-hydroxylase activity (Ki 70 microm), a cytochrome P450 (CYP3A) marker in human liver microsomes. The pre-incubation of gallic acid (100 microM) with human liver microsomes in the absence of NADPH, as compared with the presence of NADPH, before assay of androstenedione 6beta-hydroxylase activity significantly increased the inhibitory effects of the gallic acid (0.03 +/- 0.03 nmol (mg microsomal protein)(-1) min(-1) compared with 0.20 +/- 0.06 nmol (mg microsomal protein) (-1) min(-1) (P < 0.05)). The antioxidant ascorbic acid and the radical scavenger glutathione prevented this observed increase in inhibition. Removal of gallic acid-derived products from the incubation completely restored CYP3A activity. In contrast, the activities of CYP1A and CYP2E, and non-CYP mediated reductive microsomal 17beta-hydroxysteroid dehydrogenase activity were refractory to inhibition by gallic acid.

Induction of Hepatic Cytochrome P450s by the Herbal Medicine Sophora flavescens Extract in Rats: Impact on the Elimination of Theophylline

The roots of Sophora flavescens (Sf) have been widely used as a herbal medicine for the treatment of diarrhea, gastrointestinal hemorrhage, and eczema. Cytochrome P450 (P450) forms including CYP1A2, CYP2B, CYP2E1, and CYP3A participate in the oxidative metabolism of theophylline, which is an important bronchodilation agent with a narrow therapeutic index. To assess the interaction of Sf with theophylline, the effects of Sf extract on theophylline-metabolizing P450s and on the pharmacokinetic profile of theophylline were investigated in male Sprague-Dawley rats. Oral treatment of rats with the Sf extract caused dose-dependent increases of liver microsomal oxidation activities toward 7-ethoxyresorufin, 7-pentoxyresorufin, and nifedipine. However, nitrosodimethylamine N-demethylation activity was not affected. The ingestion of Sf extract stimulated theophylline 8-oxidation and N-demethylation activities. The increases of oxidative activities were in consensus with the elevation of the protein levels of CYP1A2, CYP2B1/2, CYP2C11, and CYP3A. Sf-treatment increased the clearance of theophylline and decreased the area under the concentration-time curve (AUC) and the area under the moment curve (AUMC). These results demonstrate that Sf reduces blood theophylline concentration through facilitating the elimination of theophylline. In patients taking Sf, possible P450 induction-induced drug interaction should be noted to decrease the risk of therapeutic failure or adverse effects resulting from the use of additional therapeutic agents.

Formulations of Valproate Alter Valproate Metabolism: A Single Oral Dose Kinetic Study

The risk for teratogenicity of valproate (VPA) increases in a dose- or concentration-dependent manner. It has been also suggested that an increased metabolic conversion of VPA to its toxic metabolites including 2-propyl-4-pentenoic acid (4-en) is involved in the mechanism of VPA toxicity at higher doses and concentrations. This study aimed to examine whether formulations of VPA alter metabolism of VPA itself. Seven healthy male volunteers received an oral dose (800 mg) of conventional and slow-release formulations of VPA on 2 separate days, consisting of 2 phases of single-dose kinetic trials. Blood sampling for determination of VPA and its monounsaturated (2-en, 3-en, and 4-en) and hydroxylated (3-OH, 4-OH, and 5-OH) metabolites by gas chromatography-mass spectrometry were performed up to 60 hours after dosing. In subjects receiving the slow-release formulation of VPA, decreased Cmax, prolonged Tmax, and reduced area under the curve of metabolites by microsomal oxidation (4-en, 4-OH, and 5-OH) were observed. In contrast, aforementioned kinetic parameters of beta-oxidative metabolites (2-en, 3-en, and 3-OH) were unchanged irrespective of VPA formulations. These results suggest that the slow-release formulation may be safer with regard to pharmacokinetic and metabolic aspects, which is characterized by decreased formation of 4-en, the most toxic metabolite, together with reduced peak concentrations of the parent compound.

Production of a reactive metabolite of troglitazone by electrochemical oxidation performed in nonaqueous medium

In order to confirm the existence of reactive metabolites by LC-MS/MS analysis, they should be modified into stable compounds, because some reactive metabolites generated by biotransformation induce drug toxicity; however, they are unstable, with very short lives, and cannot be detected in their intact forms. To overcome these problems, electrochemical oxidation of troglitazone was performed in nonaqueous medium, since such reactive compounds are stable in the absence of water. Troglitazone, an antidiabetic agent, was withdrawn from the market because of serious hepatotoxicity in some patients. It has been considered that one or more reactive metabolites are involved in hepatotoxicity, although the mechanism of the adverse reaction is unclear. Using our method of electrochemical oxidation in nonaqueous medium, we obtained a product of troglitazone derivative that may be a clue to clarify the mechanism of toxicity. The product in the reaction mixture was separated by HPLC without chemical modification and detected using UV and ESI-MS. The mass spectrum of its molecular ion showed that it was an o-quinone methide derivative of troglitazone and identified as a reactive metabolite generated by liver microsome oxidation of the drug. The product was stable over 24 h at room temperature in anhydrous acetonitrile, but it reacted with N-( tert-butoxycarbonyl)- l-cystein methylester to produce an adduct that could be identified by its m/ z value. Thus, the method of electrochemical oxidation in nonaqueous medium is considered to be useful to prepare and predict reactive metabolites of drugs that are unstable in aqueous medium or in vivo.

3-Ketocholanoic Acid Is the Major in Vitro Human Hepatic Microsomal Metabolite of Lithocholic Acid

3alpha-Hydroxy-5 beta-cholan-24-oic (lithocholic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified, and metabolite formation was quantified using a liquid chromatography/mass spectrometry-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation: 3-oxo-5 beta-cholan-24-oic (3-ketocholanoic) acid, 3 alpha,6 alpha-dihydroxy-5 beta-cholan-24-oic (hyodeoxycholic) acid, 3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic (ursodeoxycholic) acid, 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic (murideoxycholic) acid, and 3 alpha-hydroxy-6-oxo-5 beta-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent K(m) and V(max) values of 22 muM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination.

Rat cytochromes P450 oxidize 3-aminobenzanthrone, a human metabolite of the carcinogenic environmental pollutant 3-nitrobenzanthrone

3-Aminobenzanthrone (3-ABA) is a human metabolite of carcinogenic 3-nitrobenzanthrone (3-NBA), which occurs in diesel exhaust and air pollution. Understanding which cytochrome P450 (CYP) enzymes are involved in metabolic activation and/or detoxication of this toxicant is important in the assessment of an individual's susceptibility to this substance. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 3-ABA and to examine the metabolites formed during such an oxidation. The metabolites formed by CYPs in rat hepatic microsomes were separated by high performance liquid chromatography (HPLC). 3-ABA is oxidized by these enzymes to three metabolites, which were separated by HPLC as distinguish product peaks. Using co-chromatography with synthetic standards, two of them were identified to be oxidative metabolites of 3-ABA, N-hydroxy-3-ABA and 3-NBA. The structure of another 3-ABA metabolite remains to be characterized. To define the role of rat hepatic CYP enzymes in metabolism of 3-ABA, we investigated the modulation of its oxidation using different inducers of CYPs for treatment of rats to enrich the liver microsomes with individual CYPs. Based on these studies, we attribute most of 3-ABA oxidation in rat hepatic microsomes to CYP2B, followed by CYP1A, although a role of other hepatic CYPs cannot be ruled out. Inhibition of 3-ABA oxidation by selective inhibitors of individual CYPs, supported this finding.

In Vitro Metabolism Study of Combretastatin A-4 in Rat and Human Liver Microsomes

The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6' of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.

Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased; however, the mechanisms involved in the pathogenesis of NAFLD have not been thoroughly investigated in humans. In this study, we evaluated the expression of fatty acid metabolism-related genes in NAFLD. Real-time RT-PCR was performed using liver biopsy samples from 12 NAFLD patients. The target genes studied were: acetyl-CoA carboxylase (ACC) 1, ACC2, and fatty acid synthase (FAS) for the evaluation of de novo fatty acid synthesis; carnitine palmitoyltransferase 1a (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), and long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase alpha (HADHalpha) for beta-oxidation in the mitochondria; peroxisome proliferator-activated receptor- (PPAR-) alpha and cytochrome P450 2E1 (CYP2E1) for oxidation in peroxisomes and microsomes (endoplasmic reticulum) respectively; and diacylglycerol O-acyltransferase 1 (DGAT1), PPAR-gamma, and hormone sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, expression of ACC1 and ACC2, but not FAS was increased, indicating that de novo fatty acid synthesis is enhanced in NAFLD. In contrast, expression of CTP1a, a rate-limiting enzyme, was remarkably decreased, indicating that beta-oxidation in the mitochondria was decreased, although the expression of LCAD and HADHalpha was increased. Expression of PPAR-alpha was increased, whereas that of CYP2E1 was reduced. The expression of DGAT1, PPAR-gamma, and HSL was enhanced. These data suggest that in NAFLD, increased de novo synthesis and decreased beta-oxidation in the mitochondria lead to accumulation of fatty acids in hepatocytes, although the extent of oxidation in peroxisomes and microsomes remains unclear.

Bioavailable Flavonoids: Cytochrome P450-Mediated Metabolism of Methoxyflavones

Methoxylated flavones were recently shown to be promising cancer chemopreventive agents. Their high metabolic stability compared with the hydroxylated analogs was shown in our laboratory using the human hepatic S9 fraction with cofactors for glucuronidation, sulfation, and oxidation. In the present study, the resistance of methoxylated flavones toward oxidative metabolism was investigated with human liver microsomes and recombinant cytochrome P450 (P450) isoforms. Among 15 methoxylated flavones investigated, the two partially methylated compounds, tectochrysin and kaempferide, were among the most susceptible to microsomal oxidation (Cl(int) 283 and 82 ml/min/kg). Of the fully methylated compounds, 5,7-dimethoxyflavone and 5-methoxyflavone were the most stable (Cl(int) 13 and 18 ml/min/kg, respectively), whereas 4'-methoxyflavone, 3'-methoxyflavone, 5,4'-dimethoxyflavone, and 7,3'-dimethoxyflavone were the least stable (Cl(int) 161, 140, 119, and 92 ml/min/kg, respectively), emphasizing the importance of the positions of the methoxy substituents in the flavone ring system. Among the five P450 isoforms tested, CYP1A1 showed the highest rate of metabolism of fully methylated compounds, followed by CYP1A2 and CYP3A4. CYP2C9 and CYP2D6 gave minimal disappearance of the parent compound. Finally, in incubations with hepatic S9 fraction with cofactors for oxidation and both conjugation reactions, partially methylated flavones, as expected, were much less metabolically stable than fully methylated flavones, confirming that oxidative demethylation is the rate-limiting metabolic reaction for fully methylated flavones only. In summary, the rate of oxidative metabolism of methoxylated flavones, mainly involving CYP1A1 and CYP1A2, varied widely, even between compounds with very similar structures.

In Vitro Rat Hepatic Metabolism of n-Alkanes: Nonane, Decane, and Tetradecane

Jet propellant 8 (JP-8) jet fuel is a complex mixture of aromatic and aliphatic hydrocarbons. The aim of this study was to determine in vitro metabolic rate constants for semivolatile n-alkanes, nonane (C9), decane (C10), and tetradecane (C14), by rat liver microsomal oxidation. The metabolism was assessed by measuring the disappearance of parent compound by gas chromatography. Various concentrations of n-alkanes were incubated with liver microsomes from adult male F-344 rats. Nonlinear kinetic constants for nonane and decane were V(max) (nmol/mg protein/min) = 7.26 +/- 0.20 and 2.80 +/- 0.35, respectively, and K(M) (micro M) = 294.83 +/- 68.67 and 398.70 +/- 42.70, respectively. Metabolic capacity as assessed by intrinsic clearance (V(max)/K(M)) was approximately four-fold higher for nonane (0.03 +/- 0.005) than for decane (0.007 +/- 0.001). There was no appreciable metabolism of tetradecane even with higher microsomal protein concentration and longer incubation time. These results show a negative correlation between metabolic clearance and chain length of n-alkanes. These metabolic rate constants will be used to update existing physiologically based pharmacokinetic (PBPK) models for nonane and decane as part of developing a PBPK model for JP-8.

Impaired Microsomal Oxidation of the Atypical Antipsychotic Agent Clozapine in Hepatic Steatosis

Hepatic lipid infiltration (steatosis) is a complication of the metabolic syndrome and can progress to nonalcoholic steatohepatitis and severe liver injury. Microsomal cytochrome P450 (P450) drug oxidases are down-regulated in experimental steatosis. In this study we evaluated the separate and combined effects of lipid accumulation and P450 down-regulation on the microsomal oxidation of the antipsychotic agent clozapine (CLZ), the use of which is associated with an increased incidence of the metabolic syndrome. Several important drug oxidizing P450s were down-regulated, and the formation of N-desmethyl-CLZ (norCLZ) and CLZ N-oxide was decreased in microsomal fractions from orotic acid-induced early steatotic rat liver. Inclusion of lipids extracted from steatotic, but not control, liver decreased the free concentration of CLZ in microsomes and suppressed norCLZ formation; CLZ N-oxidation was unchanged. Triglycerides increased in steatotic liver to 15-fold of control, whereas increases in the monounsaturated oleic acid to 10-fold of control and total polyunsaturated and saturated fatty acids to 4- and 5-fold of control also occurred. Addition of triglycerides containing esterified omega-6 and omega-3 fatty acids inhibited the microsomal formation of norCLZ but not that of CLZ N-oxide; triglycerides esterified with unsaturated and monounsaturated fatty acids were inactive. Thus, drug oxidation may be suppressed in steatosis by P450 down-regulation and the accumulation of polyunsaturated fatty esters. In contrast, the activity of the flavin-containing monooxygenase that mediates CLZ N-oxidation was unimpaired. Lipid deposition in livers of patients with the metabolic syndrome may necessitate dosage adjustments for toxic drugs, including CLZ.

Prediction of total propofol clearance based on enzyme activities in microsomes from human kidney and liver

Propofol is commonly used for anesthesia and sedation in intensive care units. Approximately 53% of injected propofol is excreted in the urine as the glucuronide and 38% as hydroxylated metabolites. Liver, kidneys and intestine are suspected as clearance tissues. We investigated the contribution of the liver and kidneys to propofol metabolism in humans using an in vitro-in vivo scale up approach. Renal tissue was obtained from five patients who received nephrectomies. Each renal hydroxylation and glucuronidation enzymatic activities in microsomal fractions from patients were performed discretely and their estimation based on the decrease of propofol concentration. Hepatic hydroxylation and glucuronidation activities were also performed separately using human liver microsomes. This estimation is based on the decrease of propofol concentration, assuming that the contribution of hydroxylation activity without NADPH-generating system and glucuronidation activity without UDPGA in each microsomal fraction are negligible. Both renal and hepatic clearances were estimated assuming a well-stirred model. Enzymatic activity of propofol oxidation in renal microsomes was negligible. Although glucuronidation activity in microsomes from kidneys was comparable to that from liver, the hepatic intrinsic clearance predicted from in vitro study was higher than that in kidneys due to the larger tissue volume and higher protein concentration. However, glucuronidation clearance in kidney is relatively similar to that in liver because of blood flow limitation of clearance in both tissues. The high degree of hydroxylation activity in liver microsomes is consistent with the blood flow-limited hepatic clearance of propofol. Although the activity of propofol glucuronidation in liver is higher, glucuronidation in kidney may be a substantial contributor.

Inhibition of Hemoglobin- and Iron-Promoted Oxidation in Fish Microsomes by Natural Phenolics

Natural phenolic antioxidants have been tested in hake (Merluccious merluccious) microsomes as inhibitors of lipid oxidation promoted by fish muscle prooxidants: hemoglobin (Hb), enzymatic NADH-iron and nonenzymatic ascorbate-iron. The phenolics selected were as follows: (a) a grape phenolic extract (OW), (b) a fraction (IV) with isolated grape procyanidins with a medium-low degree of polymerization and galloylation percentage, (c) hydroxytyrosol obtained from olive oil byproducts, and (d) a synthetic phenolic antioxidant, propyl gallate. All compounds delayed lipid oxidation activated by Hb, enzymatic NADH-iron, and nonenzymatic ascorbate-iron, excluding hydroxytyrosol that was not an effective antioxidant on oxidation promoted by nonenzymatic iron. The relative antioxidant efficiency was independent of the prooxidant system, IV > propyl gallate > OW > hydroxytyrosol, and showed a positive correlation with their incorporation into microsomes (p < 0.05). The reducing capacity or ability for donating electrons and the chelating properties may also contribute to the antioxidant activity of phenolics, although these factors were less decisive than their affinity for incorporating into the microsomes. Conversely, the inhibition of Hb oxidation by phenolics and their polarity did not seem to play an important role on antioxidant mechanism. These results stressed the importance of incorporating the exogenous antioxidants into the membranes where are located key substances for fish lipid oxidation (highly unsaturated phospholipids, iron-reducing enzymes, and endogenous alpha-tocopherol).

The detoxication peculiarities by microsomal oxidation enzymes and glutathione dependent enzymes under pseudotuberculosis at children

There was evaluated detoxication function state by the antipyrine test and glutathione dependent enzymes activity among the children with combined form of middle-degree pseudotuberculosis of sharp smooth tendency. Liver disorder was proven by the clin-ical and biochemical approaches. Our results show that it is desirable to allow detoxication biochemical processes peculiarities un-der Yersinia pseudotuberculosis infection while determining disease prognosis.