Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase.
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 beta-NADH from beta-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 beta-NADH from the reaction of beta-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; S(y|x) = 0.117 mmol/L; n = 50.


CONCLUSIONS
This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.
Kayamori
Y
Y
Department of Clinical Chemistry, National Cardiovascular Center Hospital 5-7-1, Fujishirodai, Suita, Osaka 5658565, Japan. ykayamor@hsp.ncvc.og.jp
Hatsuyama
H
H
Tsujioka
T
T
Nasu
M
M
Katayama
Y
Y
D002784:Cholesterol / Q000097:blood* / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D006834:Hydrazines; D009243:NAD; C029424:hydrazine; D002784:Cholesterol; D010088:Oxidoreductases; C019884:cholesterol dehydrogenase