Apolipoprotein B-48 analysis by high-performance liquid chromatography in VLDL: a sensitive and rapid method.
Apolipoprotein (apo) B subspecies were separated by high-performance liquid chromatography (HPLC) to analyze Apo B-48 contents in very-low-density lipoproteins (VLDL) more easily. Apo B-100 and B-48 were eluted through two connected column of Shim-pack Diol-300 at a retention time of 24 min and 32.3 min, respectively. The molecular masses estimated by this method were approximately 600 kDa in apo B-100, 220 kDa in apo B-48. % apo B-48 in total apo B (% B-48) determined by measuring peak area of HPLC-separated apo B in a healthy subject was 76% in chylomicrons, 13% in fasting VLDL, and 20% in 2 h-postprandial VLDL. No peak of apo B-48 was detected in LDL. Recovery of Apo B determined by re-chromatography of HPLC-separated sample was 91 +/- 4.0% and 95 +/- 3.6% in Apo B-100 and apo B-48, respectively. 125I-labeled apo B in VLDL were also analyzed by both HPLC and SDS-PAGE. Percent radioactivity of apo B-48 fraction in the total apo B determined by the HPLC (17.3%) was similar to the value obtained through the measurement of radioactivity separated by the SDS-PAGE (17.6%). Coefficient of variation in % B-48 determined by the peak area was 2.5%. Percent B-48 determined by the HPLC method was significantly correlated with % B-48 by SDS-PAGE, but 6-8 times higher, which might be accounted for in part by the reported difference of chromogenicity between apo B-100 and apo B-48. % B-48 in VLDL separated from fasting plasma were 17.1 +/- 5.6% in 14 healthy subjects, and positively related to VLDL triglyceride and cholesterol concentrations. Apo B isoprotein analysis by the HPLC method is reliable and easy to perform for studying apo B metabolism in triglyceride-rich lipoproteins in physiological as well as pathological conditions.
Hidaka
H
H
Third Department of Medicine, Shiga University of Medical Science, Japan.
Kojima
H
H
Nakajima
Y
Y
Aoki
T
T
Nakamura
T
T
Kawabata
T
T
Nakano
T
T
Harano
Y
Y
Shigeta
Y
Y
D000328:Adult; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008077:Lipoproteins, LDL / Q000032:analysis / Q000097:blood; D008079:Lipoproteins, VLDL / Q000032:analysis / Q000097:blood
D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL