A definite relationship has been established between elevated serum cholesterol and human susceptibility to coronary heart disease (CHD), especially arteriosclerosis. Cholesterol and a ubiquitous companion, 5Alpha-cholestanol, are components of atheromatous plaques; thus, an understanding of the regulatory aspects of sterol biosynthesis is important to the control of CHD. Quantitatively, the major metabolites and excretory products of sterols in mammals are the bile acids, but the details of biosynthesis and its regulation of these acids are not completely understood.
The specific aims of this proposal are to provide comprehension of the mechanisms of regulation of the quantities of the primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), formed from cholesterol in liver, and to delineate the remaining details of their biosynthesis. Biochemical studies with new synthetic chemicals and pertinent enzymes will be augmented with improved, more sensitive analytical methods. Biochemical studies include the following: A.) Assessment through kinetic studies of the activity of new chemically modified 5Beta- and 5Alpha-sterols as competitive inhibitors of hepatic microsomal 12Alpha-hydroxylase from rats and rabbits. This enzyme controls production of a precursor of CA; when inactive; CDCA is produced. The best inhibitors will be studied in hepatocytes, and in rats to ascertain inhibition in cells and inhibition or side effects in the whole animal; B.) explore the pathway from C27 acids to C24 bile acids with each purified mitochondrial enzyme involved in Beta-oxidation, and then with hepatocytes with appropriate C27 acids as their coenzyme A derivatives; C.) assess the affects on key hepatic enzymes involved in cholesterol and bile acid biosynthesis after feeding conjugated allo (5Alpha-) bile acids to rats for 3 or 7 days; D.) reactivate efforts to separate and purify rabbit hepatic 12Alpha-steroid hydroxylase utilizing affinity chromatography with a matrix containing a modified 5Alpha-sterol from carp bile; and E.) examine selected synthetic C27 steroidal acids as potential metabolites of plant sterols. Analytical methods will be extended to provide separation and identification of molecules of interest at much lower levels particularly with glass capillary gas chromatography, mass spectrometry, micro-bore HPLC, 13C-NMR spectroscopy and other physical methods.
