Separation of three compositionally distinct subclasses of rat high density lipoproteins by heparin-affinity chromatography.

The composition of 3 subclasses of plasma high density lipoproteins (HDL) separated by heparin-affinity chromatography was characterized. Plasma was obtained from Fischer-344 adult male rats fed a semi-purified diet containing 1% cholesterol. HDL particles were isolated by ultracentrifugation and agarose column chromatography. The purified HDL fraction was applied to a column (1.0 X 28 cm) packed with heparin-Sepharose CL-6B and eluted at 4 degrees C with 5 mM Tris buffer (pH 7.4) with varying concentrations of NaCl. The first peak (P1) eluted with 50 mM NaCl and 25 mM MnCl2 was albumin; the second peak (P2) eluted at 70 mM NaCl accounted for 78% of total plasma HDL-cholesterol (HDL-C) and 82% of total HDL protein. The particles of this HDL subclass measured 113 A in diameter and were devoid of apolipoprotein (apo) E, but high in apo A-I. The third peak (P3) eluted with 290 mM NaCl represented 4.3% of total HDL-C and 6.0% of total HDL protein, and contained apo E (25% of its protein). The average size of the particles was 126 A. The last peak (P4) eluted at 0.6 M NaCl accounted for 18% of total HDL-C and 12% of HDL protein. The particles of P4 were considerably larger in size (156 A) relative to those of P2 and P3, and rich in apo E (73% of its protein) with relatively low concentrations of apo A-I and C. Based on the compositional characteristics and sizes of the particles, the HDL subclasses of P2, P3 and P4 were designated as HDL2 with no apo E, HDL2 with moderate apo E, and HDL1 (or HDLc), respectively. The above results provide evidence for the existence of 3 compositionally distinct subclasses of plasma HDL in the rat, which may differ with regard to their roles in the transport and metabolism of cholesterol.