| Abstract: | Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles. |
