Improved Method for the Enzymatic Microdetermination of Cholesterol in Plasma Lipoproteins1)

Summary: An improved procedure for the quantitative enzymatic determination of cholesterol in the presence of agarose is reported. The agarose samples containing cholesterol are dried in vacuo and dissolved in 6 mol/1 HC1. After neutralization, the cholesterol is determined enzymically. The reaction curve of this modified method is identical with that of the original serum method. A linear relationship is found in the range of 0.75-25.6 mmol/1 cholesterol. The coefficient of variation in the series is < 1.5 % for cholesterol concentrations> 3 mmol/1, while it is < 3 % in the lower range. The HC1 does not affect the ester bond of the cholesterol esters of the plasma samples. It is there-fore possible to determine the free and the esterified cholesterol. This modified procedure was applied to the enzymatic determination of cholesterol in the lipoprotein fractions of 15 \ capillary plasma separated by electrophoresis on agarose gel. An improved electrophoretic procedure gave an excellent separation of the lipoprotein fractions of 16 plasma samples in a single run. The lipoprotein fractions are precipitated with dextran sulfate/CaCl2, cut with a scalpel and dried in vacuo prior to dissolution with 6 mol/1 HC1. By drying the samples in vacuo, the loss of cholesterol, caused by drying them with filter paper, is avoided. The coefficients of variation in the series are as follows: 0-lipoprotein cholesterol < 2%, pre-j3-lipoprotein cholesterol 4—6%,-lipoprotein cholesterol 3—6 % for control and hyperlipidemic plasma samples. The recovery is 95-100%. The application of this procedure to the examination of 30 healthy children revealed that 7 had a cholesterol con