New assays for the enzymatic conversion of cholesterol to pregnenolone

Cholesterol side chain cleavage is determined by means of separation of (26-C14)-cholesterol and its radioactively labeled side chain (1-C14)k-isocaproic acid. Alumina minicolumn assay (AMCA): adsorption of cholesterol from an aqueous phase by aluminium oxide, while isocaproic acid can percolate through the column. In modification of a previously described technique (1), cholesterol is quantitatively eluted by ethanol. Filter assay (FA): retention of cholesterol by a membrane filter (pore size less than or equal to 0.1 um) while isocaproic acid can pass the filter. Two-phase scintillation assay (TPSA): pH-dependent partition of isocaproic acid between an organic scintillation mixture and an aqueous phase. The TPSA can be applied for all enzymatic reaction in which the polarity of the radioactive residue which is split off depends on pH values or when the total charge of a polar molecule is changed to an apolar state by cleaving one non-radioactive group (e.g. steroid sulfates) and vice versa. The criteria or reliability of the test systems are described. Bovine adrenal mitochondria were incubated and the side chain cleavage of (26-C14)-cholesterol was studied by the new tests systems and compared to the conversion rates of (4-C14)-cholesterol to its metabolites are determined by thin layer chromatography. A good agreement of all tests was found.