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Clinical Chemistry 48: 737-741, 2002;
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2002;48:737-741.)
© 2002 American Association for Clinical Chemistry, Inc.

Highly Sensitive Cholesterol Assay with Enzymatic Cycling Applied to Measurement of Remnant Lipoprotein-Cholesterol in Serum

Koji Kishi1a, Koji Ochiai1, Yohsuke Ohta1, Yahiro Uemura1, Kazushi Kanatani2, Katsuyuki Nakajima2 and Masakazu Nakamura3

1 International Reagents Corporation, 1-1-2, Murotani, Nishi-ku, Kobe 651-2241, Japan.

2 Japan Immunoresearch Laboratories Co., Ltd., 351-1, Nishiyokete-cho, Takasaki 370-0021, Japan.

3 Osaka Medical Center for Cancer and Cardiovascular Disease, 1-3-3, Nakamichi, Nishinari-ku, Osaka 537-8511, Japan.

aAuthor for correspondence. Fax 81-78-992-1082; e-mail irckojikishi{at}irc-net.co.jp.

Background: Remnant lipoprotein-cholesterol (RLP-C) concentrations in sera of healthy individuals are very low (0.080–0.437 mmol/L), making conventional cholesterol methods poorly suited to this purpose. We have developed a highly sensitive cholesterol assay (CD method) and applied it to the RLP-C assay.

Methods: The CD shuttled cholesterol reversibly between reduced and oxidized forms in the presence of thio-NAD and NADH. The production rate of thio-NADH correlated with the cholesterol concentration and was measured by the absorbance at 404/500 nm. This CD method was combined with an immunoaffinity separation procedure with specific monoclonal antibodies to apolipoprotein (apo) A1 and apo B-100 and used for RLP-C assay. Results were compared with a RLP-C method that uses cholesterol oxidase, peroxidase, and chromogenic substrate.

Results: The CD method could detect 0.10 x 10-3 mmol/L cholesterol and was at least 5 times more sensitive than the conventional enzymatic method. Within- and between-day imprecision (as CVs) of the RLP-C assay with the CD method was <4%. Regression analysis of RLP-C assays with the new (y) and conventional (x) cholesterol methods yielded: y = 1.02x - 0.008 mmol/L (Sy|x = 0.0065 mmol/L; r = 0.997; n = 297).

Conclusions: Serum RLP-C can be measured by the CD method. The CD method may be useful for other assays that require sensitive cholesterol measurements in biological materials.




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