Apolipoprotein B determination in the dissolved precipitate obtained after precipitation of LDL with polyvinylsulphate. An alternative method for the determination of LDL apolipoprotein B without using ultracentrifugation.
Using a commercially available test for LDL cholesterol (Boehringer Mannheim), a method was developed for determination of LDL apolipoprotein B without using ultracentrifugation. The infranatant obtained by precipitation of serum with polyvinylsulphate was redissolved in a saline citrate solution and incubated with phospholipase A2, phospholipase C, phospholipase D or triglyceride lipase, respectively. When the saline-citrate redissolved precipitate was monitored by electron microscopy, it appeared as a fibriform network. The additional incubation with phospholipase A2, C, D, or triglyceride lipase resulted in a molecularization of LDL particles. By electron microscopy these particles could not be distinguished from LDL particles isolated by ultracentrifugation. In radial immunodiffusion tests, the additional incubation of the redissolved precipitate with phospholipase C or phospholipase D resulted in a total loss of the slight immunoreactivity observed before phospholipase incubation. However, additional incubation with triglyceride lipase resulted in a significant increase in immunoreactivity. Only the additional incubation of the redissolved precipitate with phospholipase A2 resulted in an immunoprecipitation reaction comparable to that with LDL particles isolated by ultracentrifugation. Using a resolubilized and phospholipase A2-incubated precipitate of a pool serum as apolipoprotein B standard, a good correlation was obtained between apolipoprotein B values measured in this dissolved precipitate and those measured in the d greater than 1.006 kg/l fraction isolated by ultracentrifugation (r = 0.95; y = 0.95x + 0.018; n = 44).
D001055:Apolipoproteins B / Q000032:analysis*; D003902:Detergents; D005615:Freezing; D007202:Indicators and Reagents; D008049:Lipase; D008077:Lipoproteins, LDL / Q000032:analysis*; D008854:Microscopy, Electron; D010740:Phospholipases; D011145:Polyvinyls; D014461:Ultracentrifugation
D001055:Apolipoproteins B; D003902:Detergents; D007202:Indicators and Reagents; D008077:Lipoproteins, LDL; D011145:Polyvinyls; C019050:polyvinyl sulfate; D010740:Phospholipases; D008049:Lipase