Limitations of acetate as a substrate for measuring cholesterol synthesis in liver.

In studies designed to measure rates of hepatic cholesterogenesis, rat liver slices were incubated with [Wlacetate in the presence of unlabeled octanoate and with [l*C]octanoate in the presence of unlabeled acetate to assess the degree of intracellular dilution and compartmentalization of the acetyl-CoA pools. It was observed that when increasing concentrations of [14C]acetate were used, the incorporation rate of this substrate into cholesterol became constant under circumstances where the specific activity of the ketone bodies continued to rise. Second, the addition of unlabeled acetate reduced the flow of radiolabeled Cp units from [Wloctanoate into cholesterol but not into ketone bodies or CO*. Third, use of the specific activity of the ketone bodies was shown to yield an appropriate correction for the flux of [14C]octanoate into cholesterol: in contrast, this correction was shown to be inappropriate in experiments in which cholesterogenesis was measured using radiolabeled acetate. Taken together, these findings indicate that [14C]acetate gives rise to a cytosolic pool of acetylCoA that is used for cholesterol synthesis but is not in isotopic equilibrium with the mitochondrial pool of acetyl-CoA used for ketogenesis. From a combination of experiments using acetate and octanoate, singly and together and alternately labeled, the apparent and absolute fluxes of CZ units through both pools were measured. Maximum rates of sterol synthesis were obtained in the presence of octanoate. In contrast, with (l*C]acetate as substrate the flux of labeled Cp units into cholesterol was only 47% of maximum while total Ce flux was 75% of that observed with octanoate. Thus, in addition to the errors associated with intracellular dilution when [14C]acetate is used, a further limitation to the use of this substrate appears to be the presence of a partially rate-limiting step in its metabolism prior to hydroxymethylglutaryl-CoA reductase.