Online edition:ISSN 2758-089X


Hemolysate was prepared by adding H20 (1.0 ml) to washed packed red cell (0.9 ml). Aliquot of 20μl of the hemolysate was taken to assay the concentration of hemoglobin. The rest of the hemolysate was extracted by Rose's method (isopropanol 11.0 ml + Chloroform 7.0 ml). The extract are divided into two parts, A and B. Both A (0.4 ml) and B (7.0 ml) were evaporated. The residue of A was dissolved in n-propanol (50μl) and the solution thus produced was measured for its free cholesterol concentration enzymatically. The residue B was dissolvedin 0.4 ml of Folch'schloroform : methanol (2:1, v/v), and a portion of the solution (2μl) was applied on thin layered silica gel sintered on a slender cylindrical quartz rod (chromarod : 0.9 mm in diameter, 150 mm in length, silica gel grain of 5 μm diameter). The chromarods were placed into a glass tank containing a solvent mixture of chloroform,methanol, and H20 for development. The erythrocyte membrane lipids will be separated into five components, that is free cholesterol (FC), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl choline (PC) and sphingomyelin (SM). After drying the chromarods, detection of lipid components was performed by passing them through the flame ionization detector (FID) of the Iatroscan. The electric current produced by the ionized carbon atoms in the flame was amplified electrically to be recorded as peaks appearing on a graphic paper in an autorecorder. Integrals of the peaks relevant to the individual components were calculated by a computer system to obtain the ratio of the amounts of their ionized carbon atoms, namely FC : PE : PS : PC : SM. If the values of the peak areas referring to the quantity of ionized carbon atoms produced per unit weight of purified lipid have been checked with individual standard lipid specimens preliminarily, it is able to convert the aforementioned peak area ratio into the ratio of lipid weight ratio FC : PE : PS : PC : SM. The lipid weight per 1 g of hemoglobin is also calculable, by collatingthe free cholesterol concentration (mg/dl) of hemolysate to the hemoglobin concentration (g/dl) of the hemolysate. Analytical result of normal red cell membrane lipid components (mg/g Hb) obtained in this way was as follows : FC : 4.69 ±0.57, PE : 2.94 ± 0.4, PS : 1.80 ± 0.34, PC : 2.85 ± 0.39, and SM : 2.52 ± 0.34. These estimations agreed well with those values got by the conventional chemical analysis of the red cell membrane lipid. Coefficients of variation of the analytical values of membrane lipids i. e. FC, PE, PS, PC and SM by this method were 1.5-2.9, 4.5-8.7, 5.6-12.6, 2.3-6.7, 3.0-8.7(%), respectively.

Takemoto Y