Human low density lipoproteins (LDL) in combination with cholesterol or cholesteryl linoleate as precursors for progesterone synthesis of human placenta in organ culture.

After preincubation of term placental tissue in organ culture for 24 h, progesterone synthesis is 2-3 fold lower than without preincubation. By adding human male serum proteins (MW less than 12,400), we obtained 2-3.5 fold lower tissue levels of progesterone. Serum proteins with high molecular weight (MW greater than 12,400) are postulated to facilitate progesterone release by binding free medium progesterone. In test series without preincubation, there are no significant (p less than 0.05) differences in progesterone formation in the presence of cholesterol (C), cholesteryl linoleate (CL), and LDL. In test series with preincubation, LDL causes a twofold increase in medium progesterone with C (0.1 and 1 mM) and CL (0.1 mM) in the presence of the low molecular weight serum protein (MW less than 12,400) solution. A decrease of 50% was obtained by 1 mM CL with/and without LDL. In culture medium containing high molecular weight serum proteins (MW greater than 12,400), 0.1 and 1 mM C and CL induce a twofold increase in progesterone production without any significant (p less than 0.05) differences between the single values. No further stimulation could be observed by LDL because there was sufficient LDL for maximal progesterone formation. In conclusion, LDL enhances the utilization of cholesterol and cholesteryl linoleate for progesterone production in term placenta. A lipoprotein cholesterol receptor is suspected.

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.