Analysis of the distribution of cholesterol in the intact cell

We have used the enzyme cholesterol oxidase, which catalyzes the oxidation of cholesterol to cholest-4-en-3-one, to examine the distribution of cholesterol in cultured fibroblasts, Chinese hamster ovary cells, and isolated rat liver hepatocytes. While the plasma membrane normally was not attacked by cholesterol oxidase, we found that treating cells with low ionic strength buffer and glutaraldehyde rendered their cholesterol highly susceptible to oxidation. Most of the cholesterol was oxidized in all three cell types: 94% in fibroblasts, 92% in Chinese hamster ovary cells, and 80% in hepatocytes. Given that the enzyme had access only to the outer surface of the cells and cholesterol can move rapidly across the fixed plasma membrane, these values are taken to reflect the fraction of cellular cholesterol present in the plasma membrane. Additional experiments confirmed this interpretation. Fibroblasts were labeled with [3H]cholesterol by brief exposure to exogenous radiolabel and incubated with [14C]mevalonic acid to label cholesterol biosynthetically. Cholesterol oxidase attacked at least 97% of the exogenous label but as little as 10% of the biosynthetically labeled cholesterol. These data suggest that the cholesterol oxidase did not reach the intracellular pool and that cholesterol in the plasma membrane is not in rapid equilibrium with internal membranes. A study of the transfer of cholesterol to plasma from cells labeled biosynthetically with [3H]cholesterol and exogenously with [14C]cholesterol confirmed the different subcellular distribution of the two labels. These studies demonstrate that an unexpectedly high proportion of cell cholesterol is associated with plasma membranes and that this cholesterol pool can be rapidly and selectively labeled and oxidized. These features make cholesterol a useful specific marker for the plasma membrane.