Isolation of high density lipoproteins from rat intestinal epithelial cells.
Previous studies have defined forms of high density lipoproteins (HDL) in rat mesenteric lymph, suggesting that they have a secretory origin. This study describes the isolation and characterization of intestinal intracellular HDL. Two preparations were made as follows: (a) Rat enterocytes were isolated and a Golgi organelle fraction was prepared. (b) Cell homogenates were subjected to nitrogen cavitation and a cytoplasmic fraction was prepared. Lipoproteins were isolated from both preparations by sequential ultracentrifugation. When the HDL fraction (1.07-1.21 g/ml) was subjected to isopyknic density gradient ultracentrifugation, a peak of apoproteins A-I and B (apoA-I and apoB, respectively) was found at a density of 1.11-1.14 g/ml. Electron microscopy of the fraction showed spherical particles ranging in size from 6 to 13 nm. Immunoelectrophoresis revealed a precipitin arc in the alpha region against apoA-I which extended into the pre-beta region where a precipitin arc against apoB was also seen. ApoB antisera depleted the pre-beta particles whereas the alpha migrating particles remained. Lipid analysis of the whole HDL fraction revealed phospholipid, cholesteryl ester, and triglyceride as the major lipids. [3H]leucine was then administered into the duodenum and a radiolabeled intracellular HDL fraction was isolated. The newly synthesized apoproteins of the HDL fraction, as determined by gel electrophoresis, were apoB, apoA-I, and apolipoprotein A-IV (ApoA-IV). Immunoprecipitation of the apoB particles revealed apoA-I and apoA-IV in the supernatant. These data demonstrate that there are at least two intracellular intestinal forms of HDL particles, one of which contains apoB. The other particle contains apoA-I and apoA-IV, has alpha mobility, is spherical, and resembles a particle found in the lymph.
D000818:Animals; D001053:Apolipoproteins / Q000032:analysis; D001054:Apolipoproteins A / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002914:Chylomicrons / Q000032:analysis; D003593:Cytoplasm / Q000648:ultrastructure; D004847:Epithelial Cells; D004848:Epithelium / Q000032:analysis / Q000648:ultrastructure; D007122:Immunoelectrophoresis; D007422:Intestines / Q000032:analysis* / Q000166:cytology / Q000648:ultrastructure; D007930:Leucine / Q000378:metabolism; D008055:Lipids / Q000032:analysis; D008075:Lipoproteins, HDL / Q000302:isolation & purification*; D008196:Lymph / Q000166:cytology; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D001053:Apolipoproteins; D001054:Apolipoproteins A; D002914:Chylomicrons; D008055:Lipids; D008075:Lipoproteins, HDL; D014316:Tritium; D007930:Leucine