Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase

Yuzo Kayamori, Hiroyuki Hatsuyama, Tadayoshi Tsujioka, Masato Nasu, Yoshiaki Katayama

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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 β-NADH from β-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 β-NADH from the reaction of β-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. Conclusion: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.

Original languageEnglish
Pages (from-to)2158-2163
Number of pages6
JournalClinical Chemistry
Volume45
Issue number12
Publication statusPublished - Dec 17 1999

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Cholesterol
NAD
Sterol Esterase
Serum
hydrazine
Cholesterol Oxidase
Endpoint Determination
Chromogenics
cholesterol dehydrogenase
Cholesterol Esters
Automation
Reducing Agents
Bilirubin
Peroxidase
Ascorbic Acid
Assays
Hemoglobins
Recovery

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Kayamori, Y., Hatsuyama, H., Tsujioka, T., Nasu, M., & Katayama, Y. (1999). Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase. Clinical Chemistry, 45(12), 2158-2163.

Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase. / Kayamori, Yuzo; Hatsuyama, Hiroyuki; Tsujioka, Tadayoshi; Nasu, Masato; Katayama, Yoshiaki.

In: Clinical Chemistry, Vol. 45, No. 12, 17.12.1999, p. 2158-2163.

Research output: Contribution to journalArticle

Kayamori, Y, Hatsuyama, H, Tsujioka, T, Nasu, M & Katayama, Y 1999, 'Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase', Clinical Chemistry, vol. 45, no. 12, pp. 2158-2163.
Kayamori Y, Hatsuyama H, Tsujioka T, Nasu M, Katayama Y. Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase. Clinical Chemistry. 1999 Dec 17;45(12):2158-2163.
Kayamori, Yuzo ; Hatsuyama, Hiroyuki ; Tsujioka, Tadayoshi ; Nasu, Masato ; Katayama, Yoshiaki. / Endpoint colorimetric method for assaying total cholesterol in serum with cholesterol dehydrogenase. In: Clinical Chemistry. 1999 ; Vol. 45, No. 12. pp. 2158-2163.
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abstract = "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 β-NADH from β-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 β-NADH from the reaction of β-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. Conclusion: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.",
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