Rabe T, Rabe D, Bierwirth A M, Runnebaum B
Steroids. 1981 May;37(5):555-71. doi: 10.1016/s0039-128x(81)90381-0.
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.
胆固醇侧链裂解是通过分离（26-C14）-胆固醇及其放射性标记的侧链（1-C14）-异己酸来确定的。氧化铝微柱分析法（AMCA）：氧化铝从水相中吸附胆固醇，而异己酸可渗透过柱子。在对先前描述的技术（1）进行改进时，胆固醇用乙醇定量洗脱。过滤分析法（FA）：膜过滤器（孔径小于或等于0.1μm）保留胆固醇，而异己酸可通过过滤器。两相闪烁分析法（TPSA）：异己酸在有机闪烁混合物和水相之间的pH依赖性分配。TPSA可应用于所有酶促反应，其中裂解掉的放射性残基的极性取决于pH值，或者当通过裂解一个非放射性基团（如类固醇硫酸盐）使极性分子的总电荷变为非极性状态时，反之亦然。描述了测试系统的标准或可靠性。孵育牛肾上腺线粒体，通过新的测试系统研究（26-C14）-胆固醇的侧链裂解，并与通过薄层色谱法测定的（4-C14）-胆固醇向其代谢物的转化率进行比较。发现所有测试结果吻合良好。
