Apolipoproteins as the basis for heterogeneity in high‐density lipoprotein2 and high‐density lipoprotein3

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Abstract
A method is described for the isoelectric focusing (IEF) of [human serum] lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide 2-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analyzed by 2-dimensional IEF-SDS [sodium dodecyl sulfate] electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed 8 different fractions to be collected which have the same pI [isoelectric point] in either lipoprotein class. The apolipoprotein composition of each isolated band was analyzed by electroimmunoassays for apolipoproteins AI, AII, CI, CII, CIII, D and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, aopCIII and apoD. ApoCII and apoCIII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3. In addition, subtle differences in the distribution of apolipoproteins in HDL2 and HDL3 indicate the occurrence of some different lipoprotein species in these 2 subclasses of HDL.

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