Fractionation and characterization of rat hepatocytes isolated from ethanol-induced fatty liver.

A procedure for fractionation of hepatocytes according to density is presented. Hepatocytes were prepared from rats fed an ethanol-containing liquid diet, a liquid diet without ethanol, or a chow diet. The hepatocytes were fractionated in a Metrizamide density gradient and the cell fractions obtained were characterized with respect to protein synthesis, fatty acid oxidation, and esterification and ATP content. From ethanol-fed rats, two fractions of hepatocytes were obtained, one with a density of approximately 1.04 gm. per ml. and one with a density of ca. 1.08 gm. per ml. Cells from the lighter fraction contained 3 times more triacylglycerol than cells from the heavier fraction. Fifty per cent of the cells from the lighter fraction contained large (greater than 5 micron) lipid droplets as compared to 6% in the heavier fraction. The average yield from a 170-gm. rat of lipid-rich and lipid-poor cells was about 65 x 10(6) and 170 x 10(6) cells, respectively. Electron micrographs of the liver before perfusion and of the isolated and washed cell fraction revealed intact ultrastructure. Fractionation of hepatocyte suspensions from rats fed a high fat liquid diet without ethanol or a chow diet normally yielded two cell fractions with densities of ca. 1.08 and 1.10 gm. per ml. and 1.10 and 1.12 gm. per ml., respectively. The metabolic capacity of the cell fractions was unaltered by the fractionation procedure as judged by the ATP content, the rate of palmitate oxidation and esterification, and the rate of protein synthesis.