Measurement of small high density lipoprotein subclass by an improved immunoblotting technique

Abstract

An improved method of immunoblotting of plasma onto agarose gel matrix containing antiapolipoprotein A-I is described. Fresh plasma samples were subjected to gradient polyacrylamide gel electrophoresis (4-25%) followed by electrotransfer onto agarose gel layer containing antiapolipoprotein A-I. This method was compared with immunoblotting onto nitrocellulose where the transfer onto agarose gel matrix has been shown to be more convenient, quantitative, and can be kept permanently. Plasma apolipoprotein A-I was found to be distributed among regions of varying molecular weights ranging from 43,000 to 800,000. A small size fraction of molecular weight range of 43,000-50,000 (small HDL) was found in normolipidemic and hyperlipidemic subjects. The proportion of the latter fraction varied considerably among subjects (range: 0.0-32%), being lower in normolipidemic subjects (mean +/- SEM: 11.6 +/- 1.4%), and higher in hyperlipidemic subjects (mean +/- SEM: 23.7%/- 1.7%, P < 0.001). Physiological increase in the level of the small HDL was observed in normolipidemic subjects 4 h after fat ingestion (difference: 5.0%, P < 0.001); moreover, the level was higher in normolipidemic subjects who consumed moderate amounts of alcohol (mean +/- SEM: 17.9 +/- 1.2%, P < 0.001) compared with normolipidemic subjects who do not drink alcohol at all.