Recommendation Method for the Determination of Total Cholesterol in Serum

Committee on Biochemical Constituents and Their Analytical Reagents, Japan Society of Clinical Chemistry

Abstract

A proposed recommendation method for the determination of total cholesterol (TC) using comparative method for evaluation in serum is described. This proposed method is a full enzymatic cholesterol assay system (CE-CD-UV method) combining cholesterol dehydrogenase (CD, EC number not assigned) from Nocardia species and cholesterol esterase (CE, EC 3. 2. 1. 13) from Pseudomonas species. Cholesterol esters in serum are converted to free cholester-ol by hydrolysis with CE in the first stage of the assay. The free cholesterol is then quantita-tively oxidized by CD to LΔ-4-cholesten-3-one re-sulting in the production of a stoichiometric amount of reduced P-nicotinamide-adenine-di-nucleotide (β-NADH). The P-NADH is measured spectrophotometrically to quantitate total serum cholesterol. The performance of the proposed method is described below. The average withinay and day-to-day precisions expressed as coefficient of variation were 0.37 to 0.78% and 0.42 to 0.49%, respectively. The assay was linear from 0 mg/dl to approximately 720 mg/dl (18.6 mmol/l) total serum cholesterol. Reduced substances (bilirubin, etc.) did not interfere. The correlation between the TC values determined by the project facilities using the proposed method (y) and the values determined by the Abell-Levy-Brodie-Kendall method (χ)(Osaka Medical Center for Cancer & Cardiovascular Disease: a member of U. S. National Cholesterol Reference Method Laboratory Network) was coincidence. The equation was best fit as y= 1.004x+0.450, 7=0.999 and n=12. In addition, an assay for TC using CD coupled with alkaline saponification (the alkaline saponification method) was investigated to confirm that the hydrolysis of CE was complete or not. The results of the proposed method (y) showed a linear correlation with the alkaline saponification method (x). The equation was best fit as y=1.003x-2.849, r=0.993 and n=25. In conclusion, it is recommended that this proposed method is a comparative method for total serum cholesterol determination in serum.