Effects of herbal cake-partitioned moxibustion on plasma trimethylamine, trimethylamine-N-oxide, and flavin-containing monooxygenase 3 in atherosclerotic rabbits / 针灸推拿医学（英文版）

Objective: To investigate the effects of herbal cake-partitioned moxibustion on the plasma levels of trimethylamine (TMA), trimethylamine-N-oxide (TMAO), and flavin-containing monooxygenase 3 (FMO3) in rabbits with atherosclerosis (AS), as well as to explore the possible mechanism of herbal cake-partitioned moxibustion in treating AS. Methods: After 1-week adaptive feeding, 28 male New Zealand rabbits were divided into a blank group, a model group, an antibiotic group, and a herbal cake-partitioned moxibustion group according to the random number table method, with 7 rabbits in each group. Rabbits were fed with a basic diet in the blank group, while with a basic diet plus 1％ choline in the remaining groups to prepare the AS model. Rabbits were given drinking water with broad-spectrum antibiotics in the antibiotic group, and herbal cake-partitioned moxibustion in the herbal cake-partitioned moxibustion group for 12 weeks. The atherosclerotic plaques by hematoxylin-eosin (HE) staining, the blood lipid levels, the plasma TMA and TMAO levels by liquid chromatography-mass spectrometry were detected for rabbits in each group at the end of interventions. Liver FMO3 protein expression was detected by Western blotting. Liver FMO3 mRNA expression was detected by real-time fluorescence quantitative polymerase chain reaction. Results: HE staining showed that the arterial wall was rough, the intima was significantly thickened, and more foam cells and lipid deposits were seen in rabbits of the model group. Arterial wall thickening was not obvious with a few foam cells and lipid deposits in the herbal cake-partitioned moxibustion group. Compared with the blank group, the serum levels of low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were increased (P<0.01), the plasma levels of TMA and TMAO were increased (P<0.05, P<0.01), and the expression levels of liver FMO3 protein and mRNA were all increased (P<0.05, P<0.01); while the serum high-density lipoprotein cholesterol (HDL-C) level was decreased in the model group (P<0.05). Compared with the model group, the LDL-C and TC levels were decreased (P<0.01 or P<0.05), the HDL-C levels were increased (P<0.01), the TMA and TMAO levels were decreased (P<0.05), while the protein and mRNA expression levels of FMO3 were decreased without statistical significance in the herbal cake-partitioned moxibustion group and the antibiotic group. Conclusion: Herbal cake-partitioned moxibustion can slow atherosclerotic plaque formation and regulate lipid levels in AS rabbits, and the mechanism may be related to the down-regulation of TMA and TMAO expression in the plasma.

Research advances in non-P450-mediated drug oxidative metabolism / 药学学报

The major non-P450 enzymes involved in the oxidative metabolism of drugs are:the flavincontaining monooxygenase (FMO), the monoamine oxidase (MAO), the aldehyde oxidase (AO), the xanthine oxidase (XO), the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). In recent years, the role of non-P450 enzymes in drug oxidative metabolism has garnered increasing attention. However, the contribution of non-P450 enzymes to the drug oxidative metabolism is possibly underestimated in many cases, as most metabolism studies in drug discovery and lead optimization are conducted using in vitro test systems related to P450 enzymes. In this article, these non-P450 enzymes in terms of catalyzed reaction types, common substrates, gene polymorphism and drug interaction are reviewed, and the in vitro models and factors for non-P450-mediated oxidative metabolism are summarized. Similar to P450 enzymes, non-P450 enzymes can directly catalyze the oxidation of drugs, yielding therapeutically active metabolites or toxic metabolites. These enzymes can also oxidize the toxic metabolites, generated from P450-catalyzed reaction, to nontoxic metabolites. In general, most non-P450 enzymes (such as FMO and MAO) appear to be much less inducible than P450 enzymes.

Role of FMOs in drug metabolism and development / 药学学报

The flavin-containing monooxygenase (FMOs) is recognized as an important complimentary enzyme system next to the cytochrome P450 (CYP450), which catalyzes the metabolism of many xenobiotics (nucleophilic heteroatom-containing chemicals) and several endobiotics. This article provides a comprehensive introduction of FMOs including the biological characteristics, catalytic mechanism, substrate specificity, genetic polymorphisms. The effect of FMOs on drug metabolism and individual differences and relation with diseases are also mentioned. It is valuable for the discovery of therapeutic targets and design of new drug candidate.

Plasma Concentrations of Clozapine and its Metabolites and FMO3 Variations in Korean Schizophrenic Patients

OBJECTIVE: The relationship between the total daily dose of clozapine given and the plasma concentrations of clozapine and its metabolites(N-desmethylclozapine and clozapine N-oxide) and the effect of Glu158Lys (wild-type: Glu, 'H' ; variant: Lys, 'h') and Glu308Gly(wild-type: Glu, 'D' ; variant: Lys, 'd') variation in FMO3 gene on plasma concentrations of clozapine and its metabolites was studied in schizophrenic patients. METHODS: Trough plasma concentrations of clozapine and its metabolites were measured in 34 schizophrenic patients receiving clozapine. The genetic variation of 'h' and 'd' in FMO3 were analyzed in 21 among 34 patients. RESULTS: A linear relationship between the total daily dose of clozapine given(mg/kg body weight per day) and the plasma concentrations(nM) of clozapine was revealed by regression analysis(p<0.001) in the 23 patients receiving a constant daily dose of clozapine for 8 days. The plasma molar concentration ratios of clozapine N-oxide/clozapine in 8 subjects with 'hh' or 'Hh' alleles were not different from those in 6 subjects with 'HH' alleles and the plasma molar concentration ratios in 6 subjects with 'dd' or 'Dd' alleles were not different from those in 8 subjects with 'DD' alleles. CONCLUSION: The effect of Glu158Lys and Glu308Gly variation in FMO3 gene on clozapine metabolism could not be shown.

Secondary Fish-Odor Syndrome Can be Acquired by Nitric Oxide-mediated Impairment of Flavin-containing Monooxygenase in Hepatitis B Virus-Infected Patients

Primary fish-odor syndrome (FOS) is a genetic disorder caused by defective flavin-containing monooxygenase 3 gene (FMO3) with deficient N-oxidation of trimethylamine (TMA), causing trimethylaminuria (TMAU). By contrast, secondary FOS can be acquired by decreased FMO activities in patients with chronic liver diseases, but the underlying mechanisms are unknown. In the present study, we examined plasma NOx concentrations and viral DNA contents as well as in vivo FMO activities and their correlations in chronic viral hepatitis (CVH) patients. Plasma concentration of NOx was significantly increased by 2.1 fold (56.2+/-26.5 vs. 26.6+/-5.4micrometer, p< 0.01), and it was positively correlated with plasma hepatitis B virus (HBV) DNA contents (r2=0.2838, p=0.0107). Furthermore, the elevated plasma NOx values were inversely and significantly correlated with in vivo FMO activities detected by ranitidine-challenged test (8.3% vs. 20.0%, r2=0.2109, p=0.0315). TMA N-oxidation activities determined in CVH patients without challenge test were also significantly low (73.6% vs. 95.7%, p< 0.05). In conclusion, these results suggested that secondary FOS could be acquired by the endogenously elevated NO in patients with CVH.

Advance in flavin-containing monooxygenase 3(FMO3):structure/function and genetic polymorphisms / 中国药理学通报

Flavin-containing monooxygenase 3 (FMO3) is an important hepatic microsomal enzyme that oxidizes a number of drugs,xenobiotics and other chemicals.Many variants in the gene encoding FMO3 have been identified,some of which result in altered enzymatic activity,consenquently,altered substrate metabolism.Studies also implicate individual and ethnic differences in FMO3.Thus,it is anticipated that knowledge regarding functionally-relevant FMO3 genetic variability will become increasingly important for drug development and patient therapeutic choices.

Alteration of Substrate Specificity by Common Variants, E158K/E308G and V257M, in Human Hepatic Drug-metabolizing Enzyme, Flavin-containing Monooxygenase 3

Our earlier studies found a significant correlation between the activities of ranitidine N-oxidation catalyzed by hepatic flavin-containing monooxygenase (FMO) and the presence of mutations in exon 4 (E158K) and exon 7 (E308G) of the FMO3 gene in Korean volunteers. However, caffeine N-1 demethylation (which is also partially catalyzed by FMO) was not significantly correlated with these FMO3 mutations. In this study, we examined another common mutation (V257M) in exon 6 of FMO3 gene. The V257M variant, which is caused by a point mutation (G769A), was commonly observed (13.21% allele frequency) in our subjects (n=159). This point mutation causes a substitution of Val257 to Met257, with transformation of the secondary structure. The presence of this mutant allele correlated significantly with a reduction in caffeine N-1-demethylating activity, but was not correlated with the activity of N-oxidation of ranitidine. In a family study, the low FMO activity observed in a person heterozygous for a nonsense mutation in exon 4 (G148X) and heterozygous for missense mutation in exon 6 (V257M) of FMO3 was attributed to the mutations. Our results suggest that various point mutations in the coding regions of FMO3 may influence FMO3 activity according to the probe substrates of varying chemical structure that correlate with each mutation on the FMO3 gene.

Thiobenzamide S-oxidation in perfused rat liver: Ex vivo determination of hepatic flavin-containing monooxygenase activity

An ex vivo assay determining the flavin-containing monooxygenase (FMO) activity in perfused rat liver has been developed by assessing the rate of thiobenzamide S-oxide (TBSO) formation from the infused thiobenzamide (TB). The hepatotoxicity by TB or TBSO was not a critical factor for maintaining the FMO activity for up to 50 min. The FMO activity expressed in nmoles TBSO produced/g liver/min was the same for the recycling and non-recycling perfusion. This implies that reduction of the oxidized TBSO back to the parent compound (TB) is negligible. Hydrolysis of the collected perfusates with either beta-glucuronidase or arylsulfatase did not increase the TBSO level and thus, TBSO does not appear to undergo conjugation either to glucuronide or sulfate esters. Thus, measuring the rate of TB S-oxidation in the isolated perfused liver with 1 mM TB for 50 min provides a useful tool for evaluation of the hepatic FMO activity in the absence of hepatic necrosis and without the interferences caused by further conjugation or back reduction of the TBSO to the parent TB.