Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-I and lipid components

Journal Article · Tue Nov 01 04:00:00 EST 1977 · J. Lipid Res.; (United States)

OSTI ID:7076116

Foreman, J R; Karlin, J B; Edelstein, C; Juhn, D J; Rubenstein, A H; Scanu, A M

A method has been developed for separating major serum lipoproteins from 1 ml or less of human serum by isopycnic density gradient ultracentrifugation. The serum, in a step gradient, was spun for 48 hr at 38,000 rpm at 10/sup 0/C and, in each fraction, apolipoproteins B and A-I were quantified by radioimmunoassays. Markers for lipid distribution were (4-/sup 14/C)cholesterol and (U-/sup 14/C)lecithin, each incubated with serum at 20/sup 0/C for 75 min prior to ultracentrifugation. In control sera, three main fractions, very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins were clearly separated from a bottom fraction. Their flotational, electrophoretic, and chemical properties agreed with those reported for lipoproteins separated by conventional ultracentrifugation. Both apo B and apo A-I were fully recovered. Essentially all of the apo B was found in VLDL (9.3 +- 3.5%) and LDL (87 +- 4.6%); of the apo A-I, 81.0 +- 5.7% was in HDL and the remainder (17.0 +- 5.8%) was in the bottom fraction. Peak activities of (/sup 14/C)cholesterol coincided with the peak of apo B in both LDL and VLDL, and with the peak of apo A-I in HDL. Results with the radiolabeled cholesterol agreed with those from chemical analyses. Labeled lecithin was not an accurate marker of phospholipid distribution because a significant amount of the lecithin was converted into its lyso derivative. The mechanism was not established; it appeared unrelated to activities of lecithin-cholesterol acyl transferase or a Ca/sup 2 +/-dependent phospholipase. Our method also proved successful in the separation of the serum lipoproteins of the few patients with dyslipoproteinemia (abetalipoproteinemia and familial hypercholesterolemia) who were examined. The applicability to all dyslipoproteinemias was not assessed. The single-spin method could be useful in clinical studies as a complement to other established techniques.

• Research Organization: Univ. of Chicago
• OSTI ID: 7076116
• Journal Information: J. Lipid Res.; (United States), Journal Name: J. Lipid Res.; (United States) Vol. 18:6; ISSN JLPRA
• Country of Publication: United States
• Language: English

Related Subjects

550000* -- Biomedical Sciences
Basic Studies
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BLOOD SERUM
CENTRIFUGATION
CHOLESTEROL
ESTERS
FRACTIONATION
HYDROXY COMPOUNDS
IMMUNOLOGY
LECITHINS
LIPIDS
LIPOPROTEINS
MAMMALS
MAN
MEDICINE
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PATIENTS
PHOSPHOLIPIDS
PRIMATES
PROTEINS
RADIOIMMUNOLOGY
SEPARATION PROCESSES
STEROIDS
STEROLS
ULTRACENTRIFUGATION
VERTEBRATES