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Clinical Chemistry 21: 1605-1608, 1975;
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Clinical Chemistry, Vol 21, 1605-1608, Copyright © 1975 by the American Association for Clinical Chemistry

Fluorometric Enzymatic Determination of Total Cholesterol in Serum

Hua-shan Huang 1, Jui-chang W. Kuan 1, and George G. Guilbault 1

1 Department of Chemistry, University of New Orleans, New Orleans, La. 70122

We describe a fluorometric enzymatic method for determining total serum cholesterol, based on hydrolysis of cholesterol esters to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol formed, as well as that initially present, is then oxidized by cholesterol oxidase (EC 1.1.3.6) to cholest-4-en-3-one with simultaneous production of hydrogen peroxide. The latter catalytically oxidizes homovanillic acid in the presence of peroxidase (EC 1.11.1.7) to form the highly fluorescent 2,2'-dihydroxy-3,3'-dimethoxy-biphenyl-5,5'-diacetic acid. A calibration curve is constructed from data on a series of standard cholesterol solutions vs. the corresponding fluorescence change (Dgrf/5 min). This curve is linear up to 4.0 g of total serum cholesterol per liter of serum. The method is specific, precise, accurate, rapid, and simple, and results correlate well with those obtained by both the Liebermann-Burchard procedure and the colorimetric enzymatic method (correlation coefficients, 0.984 and 0.981, respectively)


Key Words: enzymatic methods

Submitted on June 2, 1975
Accepted on July 10, 1975






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Copyright © 1975 by the American Association for Clinical Chemistry.