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 Vmax and Km from extended experiments, by m...

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Bibliographic Details
Published in:Xenobiotica 2002-10, Vol.32 (10), p.895-906
Main Authors: Bousquet-Melou, A., Laffont, C. M., Laroute, V., Toutain, P.-L.
Format: Article
Language:English
Subjects:
Animals
Anti-Inflammatory Agents
Anti-Inflammatory Agents - pharmacology
Area Under Curve
Biological and medical sciences
Biological material (laboratory animals, preparation of biological tracers, etc.)
Cell Survival
Cells, Cultured
Fundamental and applied biological sciences. Psychology
General aspects
Hepatocytes
Hepatocytes - drug effects
Hepatocytes - metabolism
Hydrocortisone
Hydrocortisone - pharmacology
Kinetics
Life Sciences
Male
Models, Chemical
Pharmaceutical sciences
Pharmacology
Rats
Rats, Wistar
Time Factors
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Summary: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 Vmax and Km from extended experiments, by modelling Vmax as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. Vmax and Km 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 Vmax decrease with culture time from estimates of time parameters, t1/2 or t50. In our system, this Vmax 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.
ISSN:0049-8254
1366-5928
DOI:10.1080/00498250210163261