Comparison of Enzymatic, Colorimetric and Gas-Chromatographic Analyses in the Assay of Cholesterol

Comparisons of several enzymatic methods, colorimetric methods and the gas chromatographic method for the determination of cholesterol were undertaken. Three kinds of enzymatic method consisting of the respective cholesterol oxidase (EC 1. 1. 3. 6) isolated from microorganisms (Nocardia erythropolis and Brevibacterium sterolium) or fungus (Schizophyllum commune) were applied to the assay of serum cholesterol. In the obtained data among these enzyme methods, there was no difference dependent on the variation of origin of enzyme. Substrate specificity of these enzymes was also investigated by comparing relative intensity of coloration to authentic cholesterol or analogues. Enzymatic responses of these methods to authentic sitosterol or stigmasterol were estimated as about 60-70% of that to cholesterol. In the enzymatic method, concomitant presence of neutral sterols with cholesterol in foods also disturbed the determination of true cholesterol concentration. A enzymatic and two colorimetric (Liebermann-Burchard and Kiliani) methods was compared to make clear the specificity for sterols (lanosterol, campesterol, sitosterol, stigmasterol, fucosterol, desmosterol and coprosterol). The enzymatic method gave lower values to various sterols measured as cholesterol than the above colorimetric methods. The gas chromatographic procedure was the most suitable method for the determination of net cholesterol concentration in foods containing several neutral sterols.