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Clinical Chemistry 45: 2158-2163, 1999;
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(Clinical Chemistry. 1999;45:2158-2163.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Endpoint Colorimetric Method for Assaying Total Cholesterol in Serum with Cholesterol Dehydrogenase

Yuzo Kayamoria, Hiroyuki Hatsuyama, Tadayoshi Tsujioka, Masato Nasu and Yoshiaki Katayama

Department of Clinical Chemistry, National Cardiovascular Center Hospital 5-7-1, Fujishirodai, Suita, Osaka 5658565, Japan.
a Author for correspondence. Fax 81-6-6833-9865; e-mail ykayamor{at}hsp.ncvc.go.jp

Background: Various methods are available to measure serum cholesterol concentrations. Of these, the cholesterol ester hydrolase (CEH)-cholesterol oxidase-peroxidase chromogenic method is widely used. However, this method has the disadvantage of interference by reducing substances. We developed and evaluated an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method.

Methods: Cholesterol esters are first hydrolyzed to free cholesterol by CEH. The free cholesterol is then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of ß-NADH from ß-NAD+. At equilibrium, the CDH reaction gives incomplete conversion of cholesterol to cholest-4-ene-3-one. To overcome this disadvantage, we added hydrazine monohydrate to the reaction mixture to remove cholest-4-ene-3-one, which allowed the reaction to proceed to completion and gave stoichiometric production of ß-NADH from the reaction of ß-NAD+ with cholesterol.

Results: We tested whether the amount of cholesterol added was equivalent to the absorbance change of NADH at 340 nm with six aqueous samples. Recoveries were 97.1–100.3%. The reaction was linear up to 20.28 mmol/L. The mean within-day (n = 20) and between-day (n = 10) imprecision (CV) was 0.29–0.43% and 0.22–0.61%, respectively. No interference by bilirubin, hemoglobin, ascorbic acid, and other reducing agents was observed. The equation obtained in comparison with the modified Abell-Levy-Brodie-Kendall method was: y = 0.992x - 0.0058 mmol/L; r = 0.997; Sy|x = 0.117 mmol/L; n = 50.

Conclusion: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.






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Copyright © 1999 by the American Association for Clinical Chemistry.