title: Isolation of nascent high-density lipoprotein from rat liver perfusates by immunoaffinity chromatography: effects of oleic acid infusion.
abstract: Immunoaffinity chromatography on a column of rabbit IgG anti-rat apolipoprotein (apo) A-I covalently bonded to agarose was used to isolate nascent high-density lipoprotein (nHDL) from recirculated perfusates of rat livers. After passage through the affinity column, the bound material was eluted with sodium thiocyanate and analyzed for apolipoproteins and lipids. The protein content was 52% and the lipid composition was 37% triglyceride, 40% phospholipid, and 23% cholesterol. Apolipoproteins E and A-I each comprised approximately one third of the total, and very little apo B was detectable as judged by SDS-PAGE analysis. The affinity-isolated particles were therefore similar in composition to the major apo A-I:apo E-rich subfraction of nHDL isolated by ultracentrifugation in earlier work. It is concluded that the apo E in this class of nHDL (containing both apo E and apo A-I) is present in the secreted particle and is not a consequence of a loss of apo E from very-low-density lipoprotein (VLDL) during ultracentrifugation. The high triglyceride content in the virtual absence of apo B confirms and extends previous analyses and reinforces the conclusion that nHDL particles are enriched in triglyceride compared to plasma HDL. The inclusion of 4% albumin in the perfusion medium did not significantly change the total triglyceride output of 115 micrograms/g liver/h, but it decreased the triglyceride output isolated by anti-apo A-I affinity chromatography from 3.2 to 0.48 micrograms/g liver/h. The addition of oleic acid complexed to albumin increased the total triglyceride output by 70% and that associated with the immunoaffinity column increased from 0.48 to 2.7 micrograms/g liver/h.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia 19129.
authors.forename: J B
authors.initials: JB
authors.lastname: Marsh
authors.forename: M R
authors.initials: MR
authors.lastname: Diffenderfer
mesh_terms: D000818:Animals; D001053:Apolipoproteins / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D007158:Immunologic Techniques; D008075:Lipoproteins, HDL / Q000302:isolation & purification*; D008099:Liver / Q000737:chemistry*; D008297:Male; D019301:Oleic Acid; D009829:Oleic Acids / Q000008:administration & dosage; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014280:Triglycerides / Q000032:analysis
chemical_list: D001053:Apolipoproteins; D008075:Lipoproteins, HDL; D009829:Oleic Acids; D014280:Triglycerides; D012710:Serum Albumin, Bovine; D019301:Oleic Acid