The metabolism of ethanol perturbs the major nucleotides of liver, the pyridine nucleotides (NAD, NADH), and the adenine nucleotides (ATP, ADP, AMP). Distributions of adenine nucleotides are also central to considerations of cellular bioenergetics, and accurate quantitative data for the subcellular distributions of enzymes are essential in analyzing metabolism in vivo. Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes yields a much faster and, in some cases, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase are illustrative. Although previously thought to be entirely cytosolic, digitonin fractionation shows that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure. The amount bound varies with the animal's nutritional state. In hepatocytes from rats that were either starved 2 days and then fed NIH stock diet ad libitum, or starved 2 days and then refed a fat-free diet 2 days, the noncytosolic activity was, respectively, 52%, 21%, or 24% of total cellular lyase. Because starvation/refeeding strongly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10 to 12 times greater than in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Adding 20 uM CoA to the digitonin fractionation medium caused all the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent,5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest that the noncytosolic ATP citrate lyase may have a special role in lipogenesis. Intracellular compartmentation and metabolic zonation can also be studied by perfusing the intact liver with digitonin, and calcium ions have a strong influence on relative patterns of enzyme release from the cytosol and mitochondria. This procedure has led to ways of isolating periportal and perivenous hepatocytes, enabling the study in vitro of metabolic zonation.
