Modelling the loss of metabolic capacities of cultured hepatocytes: application to measurement of Michaelis-Menten kinetic parameters in in vitro systems.
1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V(max) and K(m) from extended experiments, by modelling V(max) as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V(max) and K(m) were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V(max) decrease with culture time from estimates of time parameters, t(1/2) or t(50). In our system, this V(max) decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments.
Bousquet-Melou
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Unité Associée INRA de Physiopathologie et Toxicologie Expérimentales, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse, France. a.bousquet-melou@envt.fr
Laffont
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Laroute
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Toutain
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D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D019540:Area Under Curve; D002470:Cell Survival; D002478:Cells, Cultured; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D006854:Hydrocortisone / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008956:Models, Chemical; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D000893:Anti-Inflammatory Agents; D006854:Hydrocortisone