Human liver microsomes enzyme kinetics

Table 9.2 Summary of enzyme kinetic parameters and inhibitor potencies for 11 human CYP activities in pooled human liver microsomes [117]. 

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

Cytochrome P-450 from rat or human liver microsome preparations is inactivated when incubated anaerobically with carbon tetrachloride in the presence of NADPH and an oxygen-scavenging system (Manno et al. 1988 1992). Inactivation involved destruction of the heme tetrapyrrolic structure, and followed pseudo first-order kinetics with fast and slow half lives of 4.0 and 29.8 minutes. When compared with rat liver microsomes, the human preparations were 6-7 times faster at metabolizing carbon tetrachloride, and only about one- eighth as susceptible to suicide inactivation (about 1 enzyme molecule lost for every 196 carbon tetrachloride molecules metabolized). 

Chiba et al. [163] studied the oxidative metabolism of omeprazole in 14 human liver microsomes in relation to the 4 -hydroxylation capacity of S-mephenytoin. The formation of 5-hydroxyomeprazole and omeprazole sulfone from omeprazole exhibited a biphasic kinetic behavior, indicating that at least two distinct enzymes are involved in either of the metabolic pathways of omeprazole. These findings suggest that S-mephenytoin 4 -hydroxylase is an enzyme primarily responsible for the 5-hydroxylation of omeprazole and further metabolism of omeprazole sulfone, but not for the sulfoxidation of omeprazole in human liver microsomes. 

A second type of nonhyperbolic saturation kinetics became apparent during studies on the metabolism of naproxen to desmethylnaproxen (32). Studies with human liver microsomes showed that naproxen metabolism has biphasic kinetics and is activated by dapsone (T. Tracy, unpublished results). The unactivated data shows what appears to be a typical concentration profile for metabolism by at least two different enzymes. However, a similar biphasic profile was obtained with expressed enzyme (25). This biphasic kinetic profile is observed with the two-substrate model when V/rn2 > Eml and Kml Km2. The appropriate equation for the two-site model when [S] < Kml is... 

Human liver microsomes and recombinant human CYP enzymes are the preferred test systems because kinetic measurements are not confounded by cellular uptake and other metabolic processes present in hepatocytes and liver slices. 

Patten CJ, Thomas PE, Guy RL, et al. Cytochrome P450 enzymes involved in acetaminophen activation by rat and human liver microsomes and their kinetics. Chem Res Toxicol 1993 6 511-518. 

Table 3 summarizes typical incubation conditions and kinetic constants of marker substrate reactions of Human P450 enzymes in a pool of human liver microsomes (Mandan 2002). 

The sulfation of phenol and the glucuronidation of its hydroquinone metabolite were measured in human liver cytosols and microsomes, respectively. The rate of phenol sulfation varied between 0.31 and 0.92 nmol/mg protein/min this is slightly higher than the rate for mice (0.46) and lower than that for rats (1.20). The rate of hydroquinone glucuronidation was between 0.10 and 0.28 mnol/mg protein/min, slightly higher than that for rats (0.08) and lower than that for mice (0.22). These enzyme-kinetic data were subsequently used to simulate phenol metabolism in mice, rats and humans in vivo, using a com-partmental pharmacokinetic model with benzene as phenol precursor (Seaton et al., 1995). 

Wen Z, Tallman MN, Ali SY, et al. UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for etoposide glucuronidation in human liver and intestinal microsomes structural characterization of phenolic and alcoholic glu-curonides of etoposide and estimation of enzyme kinetics. Drug Metab Dispos 2007 35(3) 371-380. 

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