and salinomycin, which are produced by Streptomyces, are widely used as additives in broiler feed and cattle feed. However, at a certain level, these antibiotics are toxic. For the quality control of feed and premix it is thus very important to monitor the concentrations of these antibiotics. At present, colorimetric methods are widely used for this purpose, but they are rather time-consuming. The present paper describes the application of colorimetric methods to flow injection analysis (FIA) of these antibiotics. The established method facilitates the rapid analysis of feed and premix for the quality checking.
An in-vitro study was conducted to investigate the metabolism of nefiracetam in human liver microsomes and to identify the enzymes responsible for the metabolism. Nefiracetam was hydroxylated by human liver microsomes to 5-hydroxynefiracetam (5-OHN). Eadie-Hofstee plots for the formation of 5-OHN suggested substrate activation. The kinetic parameters, apparent Km, Vmax, and Hill coefficient, for the formation of 5-OHN by pooled human liver microsomes were 4012 microM, 2.66 nmol min(-1) (mg protein)(-1), and 1.65, respectively. The formation of 5-OHN was significantly correlated with cytochrome P450 (CYP)3A4-mediated testosterone 6beta-hydroxylase activity and dextromethorphan N-demethylase activity. The 5-OHN formation was inhibited (94%) by antibody to human CYP3A4/5. The 5-OHN formation was also inhibited by the CYP3A4 inhibitors ketoconazole and troleandomycin, but not significantly inhibited by several other P450 inhibitors. The microsomes containing cDNA-expressed CYP3A4 formed 5-OHN with sigmoidal kinetics. CYP3A5-containing microsomes did not form 5-OHN. These results indicated that CYP3A, most likely CYP3A4, was the major isozyme responsible for the formation of 5-OHN in human liver microsomes. CYP1A2 and CYP2C19 microsomes were also capable of forming 5-OHN. However, the contribution of CYP1A2 was considered to be relatively minor compared with that of CYP3A4, and the contribution of CYP2C19 was assumed to be negligible, based on the result of the immunoinhibition study and taking into account both the turnover rate by each isozyme and the relative abundance of each isozyme in human liver. We conclude that on average the formation of 5-OHN, the major metabolite of nefiracetam, is principally mediated by CYP3A4 with a relatively minor contribution by CYP1A2.
1. In vitro studies identified the hepatic cytochrome P450 (CYP) enzyme(s) involved in the major metabolism of ecabapide in human. 2. Ecabapide mainly underwent N-dealkylation to form M1 and 6-hydroxylation of the benzamide moiety to form M6. 3. The rates of formation of the major metabolites M1 and M6 were significantly correlated with CYP3A-selective testosterone 6beta-hydroxylase activities in 14 different human liver microsomes. The formation of both metabolites was markedly decreased by ketoconazole, miconazole or troleandomycin (TAO), CYP3A-selective inhibitors, and also was inhibited by anti-CYP3A antibodies. 4. These results strongly indicate that CYP3A is the predominant isozyme responsible for the major metabolism of ecabapide in human liver microsomes. 5. Marginal inhibition of the formation of M1 and M6 by nifedipine, a substrate of CYP3A with a Ki > 100 microM, suggested that nifedipne has a limited potential to inhibit the major metabolic pathways of ecabapide.
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