Studies on Polyacrylamide-gel Block Electrophoresis of Serum Lipoprotein, with Special Reference to Recovery Rate of Cholesterol, Triglyceride and Phospholipid from Each Block

Abstract

Authors investigated the recovery rate of cholesterol (TC), triglyceride (TG) and phospholipid (PL) from each gel-block contained lipoprotein after electrophoresis. Method: Authors separated in several lipoprotein fraction from the 4ml to 5ml of serum by Wada's method (Clinical Chemistry 19: 235-239, 1973). Electric current was 8mA in 130-140 volts. The period of phoresis was 6-8hrs. Each fraction was cutted and each gel-block was weighed and it was crushed to pieces (2-3mm²) through pushout-type-crusher acompaning wire netting. The lipoprotein fraction was extracted by stirring saline with crushed piecies at 4sC for 20-24hrs. After extraction, the saline contained the crushed pieces was centrifuged in 27500g for 30min. at 4sC, then the supernatant was concentrated by riofilyzing in polyethylenglycol 4000. The concentrated solution was diluted to the original serum volume again. TC, TG, PL, β-lipoprotein (β-Lp) and α₁-lipoprotein (α₁-Lp) in each extract-solution were estimated. The β-Lp was measured by turbidity method with dextran sulphate (β-Lp I) and immunodiffusion method (β-Lp II). The α₁-Lp was measured by immunodiffusion method. Results: I) Recovery rate of β-Lp by β-Lp II in Sudan black B stained block was better than in unstained block. It is difficult to explain the cause of this result for the present.2) Recovery rate of β-Lp by β-Lp I in each block was poor regardless of staining in all cases. It was concluded that electric charge on surface of β-Lp-molecule was changed by phoresis with POLYACRYLAMIDE-GEL. 3) Recovery rate of alubumin was almost perfect. It was supposed that protein in serum was not degenerated by phoresis. Therefore, the decrease of basic charge on β-Lp by electrophoresis was resulted from degeneration of lipid part in β-Lp or caused by degeneration of lipid part with Sudan Black B staining. 4) A patient suffered from primary biliary livercirrhosis had a few α₁-Lp but her serum contained much more PL, TC, TG, and β-Lp than in normal subjects, especially the concentration of TG in pre-β-Lp was higher than in normal subjects. It was concluded from above results that there were apoprotein-C-deficiency in serum of patients (P. B. C.) which was not able to convert from pre-β-Lp to β-Lp by lipoprotein lipase. 5) The percentage of recovery of each lipid fractions were as follows;