Improvement of the determination method of lecithin cholesterol acyltransferase activity.

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  • レシチンコレステロールアシルトランスフェラーゼ活性測定法の改良
  • レシチン コレステロール アシル トランスフェラーゼ カッセイ ソクテイホウ

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Description

Lecithin cholesterol acyltransferase(LCAT) catalyzes esterification of free cholesterol (FC) in blood, and the enzyme activity has been utilized for the diagnostic purpose of the hepatic parenchymal disturbances. However, currently used simple Nagasaki-Akanuma's method (N-A method) of the LCAT activity determination in practical clinical laboratory has low sensitivity as compared with the method with radio isotopes or gas chromatography and thus the application of the N-A method has been limited. In the present study, the N-A method has been throughly re-examined to overcome any shortcomings, and as the result, a simple and sensitive method of determination for LCAT activity has been developed. FC, decrease as the reaction proceeds, is used in the original N-A method, and the determination limit of FC is approx. 25μg. In practice, when EDTA treated plasma of rats was used for the determination of LCAT, the final FC level near the end point, after the incubation at 37°C for 2h, was about 19μg, which was lower than determination limit. Linearity of the LCAT activity with time was not obtained until 30min after starting the enzymatic reaction. Thus, colorization of the reaction system of FC was taken into consideration to increase the sensitivity. As coloring agents, p-chlorophenol, N, N-diethlaniline, and nitrocatechol have been examined. It was found that p-chlorophenol was more sensitive and more stable than the conventionally used phenol. LCAT activity did not change linearly up to 30min after starting the enzymatic reaction, and become linear between 30 to 180min. In order to avoid such delay in the enzyme activity, solubilization of the substrate, dipalmitoyl lecithin, was attempted. Since, LCTA activity was completely inhibited by p-hydroxymerucuribenzoate, a known inhibitor of LCAT, only LCAT activity was determined in the present method. Relative standard deviation of the present method was about 6% and the sensitivity of LCAT assay with p-chlorophenol was approx. doubled. LCAT activity should be determined at the linear region of reaction time course, that is, during 30120min and 30180min, with satisfactory sensitivity.

Journal

  • BUNSEKI KAGAKU

    BUNSEKI KAGAKU 34 (3), T21-T25, 1985

    The Japan Society for Analytical Chemistry

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