The measurement, esterification and biological significance of serum 7 - hydroxycholestrol

Since bile acids are major excretion products of cholesterol in the body, changes in rates of production will affect the turnover rate of cholesterol in the body. 7 a - hydroxycholesterol is the product of the rate-limiting reaction in the biosynthesis of bile acids and it has been suggested that its concentration in serum is a good parameter for establishing disorders involving the metabolic conversion of 7 ahydroxycholesterol towards bile acids. The objectives of the project were to validate the methodology towards measurement of 7a-hydroxycholesterol in serum - to investigate serum levels of this analyte in human and rat sera and to develop a methodology based on discoidal complexes for esterification studies of 7a-hydroxycholesterol. The method involved the extraction of lipids from serum using the Folch method, hydrolysis of the sample to yield total amounts present, enzymatic conversion of 7a-hydroxycholesterol to its enone derivative which was then detected and measured at 240 nm using reverse phase HPLC. A significant amount of 7a-hydroxycholesteroI was found to be esterified, which resulted in studies being carried out on (a) its possible esterification in the liver by acyl coenzyme A : cholesterol-O-acyltransferase, or (b) in the plasma by lecithin : cholesterol acyltransferase, which was facilitated by the synthesis of discoidal complexes, containing 7a-hydroxycholesterol, by the sodium cholate dialysis method and which resembled native high density lipoproteins. Electron microscopy was carried out to examine the physical structures of these complexes. The biological significance of serum 7a-hydroxycholesterol will be discussed in the thesis