Hepatic zonation of drug metabolizing enzymes. Studies on hepatocytes isolated from the periportal or perivenous region of the liver acinus

Abstract

Models of hepatic intraacinar zonation have been proposed previously; in most models, direct visualization of the acinar destruction is not possible while intact hepatocyte recovery–viability often presents a problem for subsequent metabolic studies. In the present studies, the liver is isolated in situ and perfused with Krebs–Henseleit buffer, pH 7.4. A 1.5-mL intrahepatic volume of a 7 mM digitonin solution is then injected at a flow rate of 6 mL/min for 15 s via the portal vein or via the vena cava for selective destruction of the periportal (PP) or perivenous (PV) region of the acinus. To avoid diffusion of the detergent throughout the acinus, the liver is then immediately perfused with oxygenated Hanks buffer in the direction opposite to that of digitonin injection. The preparation can then be used for histological evaluation, for studies on isolated-perfused liver, or for isolation of hepatocytes. Direct visualization of the acinar destruction can be achieved by coloring the permeabilized cells with 0.2 mM trypan blue; the liver is then fixed in situ by a 10-min perfusion with paraformaldehyde and histological evaluation is achieved by eosine staining of liver slices. Following isolation of hepatocytes by collagenase perfusion, a highly significant PV localization was found for the synthesis of glutamine, the N-demethylation of aminopyrine, and the glucuronidation of p-nitrophenol, whereas a highly significant PP zonation was found for alanine aminotransferase. By contrast, no specific acinar zonation was found for the enzymes 7-ethoxycoumarin O-deethylase and aniline p-hydroxylase. Total cytochrome P-450 was 0.42 ± 0.006 and 0.4 ± 0.03 nmol/10 6 hepatocytes in PV and PP, respectively (nonsignificant). Our data show, using histological evaluation, that selective and reproducible destruction of the liver acinus is achieved by the method used. Moreover, our findings indicate that, under unstimulated conditions, intraacinar regionalization of several enzymes involved in the biotransformation of xenobiotics is present in normal rat hepatocytes.Key words: hepatic acinar zonation, cytochrome P-450, mixed function oxidases, glucuronyl transferase, hepatocytes, digitonin.