Characterisation of Hepatocyte Sub‐populations Generated by Centrifugal Elutriation

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Abstract
Protocols for the fractionation of isolated [rat] hepatocytes into 8 subpopulations using centrifugal elutriation are described. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre-perfused with 1 of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P-450 content and response to induction by 3-methylcholanthrene and phenobarbitone are reported in addition to the distribution of lactate dehydrogenase, glucose-6-phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the subpopulations. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of 8 factors studied, the distributions of 6 were consistent with the subpopulations being derived from different acinar zones. Two major discrepancies were noted, however: the distribution of pyruvate kinase activity and the response of the subpopulations to phenobarbitone. While a metabolic heterogeneity was revealed in the subpopulations generated, further characterization is required to determine whether acinar zone separation has occurred and, if so, to what extent.