Highly sensitive cholesterol assay with enzymatic cycling applied to measurement of remnant lipoprotein-cholesterol in serum.
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 (S(y/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.
Kishi
Koji
K
International Reagents Corporation, 1-1-2, Murotani, Nishi-ku, Kobe 651-2241, Japan. irckojikishi@irc-net.co.jp
Ochiai
Koji
K
Ohta
Yohsuke
Y
Uemura
Yahiro
Y
Kanatani
Kazushi
K
Nakajima
Katsuyuki
K
Nakamura
Masakazu
M
D002784:Cholesterol / Q000097:blood*; D006801:Humans; D008074:Lipoproteins / Q000097:blood*; D010088:Oxidoreductases; D012016:Reference Values; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D014280:Triglycerides / Q000097:blood*
D008074:Lipoproteins; D014280:Triglycerides; C112017:remnant-like particle cholesterol; D002784:Cholesterol; D010088:Oxidoreductases; C019884:cholesterol dehydrogenase