抄録
The purpose of the present study was to compare the several current methods for quantitation of high density lipoprotein cholesterol (HDL-C). HDL was fractionated by ultracentrifugation or precipitation procedures utilizing heparin-MnCl2 (heparin-Mn), reagents of which were prepared by ourselves, heparin-CaCl2 (heparin-Ca; N company's kit), phosphotungustate-MgCl2 (PT-Mg; F company's kit) or dextran sulfate-MgCl2 (Dex-Mg; NK company's kit). Cholesterol concentrations in HDL separated by ultracentrifugation and by precipitation procedure using heparin-Mn Cl2 were measured by o-phthalaldehyde (OPA) method. Cholesterol concentrations in HDL separated by other precipitation procedures were analyzed by enzymatic method utilizing the kit from N company. HDL-C (Mean±SD) measured by ultracentrifugation procedure, heparin-Mn procedure, heparin-Ca procedure, PT-Mg procedure and Dex-Mg procedure were 48.1±12.6, 45.6±11.0, 29.6±13.2, 24.1±11.5 and 24.4±11.2 mg/100ml, respectively. There was no significant difference between HDL-C value obtained by ultracentrifugation procedure and that by heparin-Mn procedure. However, HDL-C values obtained by other three precipitation procedures utilizing the kits were significantly lower than the value by ultracentrifugation procedure. Correlation coefficients among 5 methods were statistically significant. The data were analyzed separately in two groups; the first group in which HDL-C by ultracentrifugation procedure was equal to and higher than 40mg/100ml and the second group in which HDL-C was lower than 40mg/100ml. In both groups, mean values of HDL-C determined by three kits were significantly lower than that obtained by ultracentrifugation. In order to investigate the effects of cholesterol measurement on HDL-C values obtained by precipitation procedure, HDL-C of 22 patients were measured by heparin-Mn and heparin-Ca procedure. Cholesterol in supernatant after precipitation were analyzed by OPA method and enzymatic method utilizing N company's kit. HDL-C measured by heparin-Mn procedure with cholesterol measurement by OPA, heparin-Mn procedure with cholesterol measurement by enzymatic method, heparin-Mn procedure (W company's kit) and heparin-Ca procedure (N company's kit) with cholesterol measurement by enzymatic method were 44.7±9.5, 42.5±13.1, 30.3±12.2 and 28.1±10.9mg/100ml, respectively. There was no difference between the HDL-C values obtained by heparin-Mn procedure with cholesterol measurement by OPA and enzymatic method. The HDL-C values measured by heparin-Mn and heparin-Ca procedure (kit) with cholesterol measured by enzymatic method were significantly lower than HDL-C measured by heparin-Mn procedure with cholesterol measurement by OPA method. Therefore, we could not decide apparently different effects of OPA method and enzymatic method for cholesterol determination in HDL fractionated by different precipitation procedures. Factors which could affect HDL fractionation by precipitation procedure such as polyanion concentration, divalent cation concentration, pH of solution for precipitation, pH of solution after mixing plasma with precipitation solution and ratio of solution volume to plasma volume, were investigated. All these factors were within optimum range. However, the ratio of solution volume to plasma volume might be one of the problems. The presence of Apo A-I in the precipitates has been reported. We studied the occurrence of Apo A-I in the precipitates obtained by precipitation procedure utilizing anti-Apo A-I serum. There were precipitin lines observed between precipitates and anti-Apo A-I serum by double immunodiffusion method. The further studies are necessary to quantitate Apo A-I in the precipitates obtained by different precipitation procedures. Furthermore, sometimes, background staining or turbidity of reagent blank f
