A method of direct measurement for the enzymatic determination of cholesteryl esters

Abstract

A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC. Free cholesterol (FC) has an important role as a component of cell membranes and a starting material for bile acid synthesis. However, cholesteryl ester (CE) is inactive when it is stored. In the progression of arteriosclerosis, CE accumulates in macrophages and smooth muscle cells and leads to the formation of foam cells. Determination of CE in cells or various tissues is of great importance in the fundamental research into atherosclerosis and the development of anti-atherosclerotic drugs.Several methods of enzymatic determination for FC and total cholesterol (TC) have been published (1-5). Measurement of TC has measured FC resulting from the decomposition of CE by cholesterol esterase, and FC was contained in the native sample. To measure CE, individual TC and FC are measured, and FC is then subtracted from TC (indirect assay). Measuring CE by indirect assay is difficult in a sample solution with a low ratio of CE to FC.This report describes a direct assay of CE by enzymatic determination without measuring TC and FC. In the first step, FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide is decomposed into water and oxygen by catalase. At the second step, CE is measured by enzymatic determination using a colorimetric method or a fluorometric method. CE is hydrolyzed to FC by cholesterol esterase. FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Thus, the enzymatic method for assaying FC is based on the measurement of hydrogen peroxide by way of peroxidase-coupled oxidation of hydrogen-sensitive probes. The hydrogen peroxide reacts with 4-aminoantipyrine or Amplex Red in the presence of peroxide to form a pigment or fluorescent products. The enzymatic reactions involved in the assay are as follows:CE content in human plasma, rat liver, and cultured cells was measured using this method, and its usefulness was evaluated.

A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

“…We determined that there was no need to specifi cally inactivate the catalase before the enzymatic cholesterol quantifi cation, which is dependent upon hydrogen peroxide generation. A prior study also used catalase treatment before measurement of cholesterol esters by an enzymatic assay , and these investigators also did not need to inactivate the catalase ( 10 ). The reason that catalase does not need to be inactivated is most likely due to catalase's instability at low concentra- …”

Berberine promotes the development of atherosclerosis and foam cell formation by inducing scavenger receptor A expression in macrophage

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Semisynthesis of novel monacolin J derivatives: hypocholesterolemic and neuroprotective activities

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