Separation and characterization of hepatocytes from immature and adult rats into distinct subpopulations by centrifugal elutriation.

Hepatocytes were isolated from immature and adult rat liver by retrograde perfusion with calcium free buffer, followed by enzymic digestion, and separated into subpopulations by centrifugal elutriation. Several subpopulations with increasing cell diameters were distinguished. The smaller cells were attributed to the periportal area, the larger ones to the perivenous (centrilobular) region. Profiles of total cytochrome P-450 concentration, benzphetamine N-demethylation and ethoxyresorufin O-deethylation, NADPH-cytochrome c-reductase, glucose-6-phosphatase and glutamate-pyruvate-transaminase activities were determined in all subpopulations. With adult hepatocytes an increasing cytochrome P-450 concentration with increasing cell diameter (increasing from periportal to perivenous hepatocytes) could be observed, paralleled by increasing activities of benzphetamine N-demethylation and ethoxyresorufin O-deethylation activities. While NADPH-cytochrome c-reductase did not show a distinct zonation, glucose-6-phosphatase and glutamate-pyruvate-transaminase revealed increasing activities with increasing cell diameter. Immature hepatocytes (rats aged 11-15 days) were smaller, and more fragile. They could not be isolated with the same enzyme solution as adult hepatocytes and they did not show any zonation of cytochrome P-450 concentration, although the zonation of benzphetamine N-demethylation and ethoxyresorufin O-deethylation was almost fully developed. For NADPH-cytochrome c-reductase a zonation with higher activities in the perivenous cells could be demonstrated, in contrast to the lack of zonation in adult rats. Glucose-6-phosphatase activity showed a decline with increasing cell diameter in immature hepatocytes, whereas glutamate-pyruvate-transaminase activity did not show any zonation. In rats aged 20 days the zonation of these parameters in liver was in between that of younger and older animals. Zonation of the liver lobule develops postnatally with individual patterns for the different parameters.