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A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d = 1.085–1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125I by the iodine monochloride technique. 65–80% of 125I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in. the incubation mixture, and accurate with a variability of only 3–5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows

Apolipoproteins are important in the structure and metabolism of lipoproteins, and alterations in levels of apoproteins or in their interrelations occur in some forms of hyperlipemia. Pregnancy is regularly accompanied by hyperlipoproteinemia, but while data on lipoprotein lipids is available, the apopipoproteins have not been studied. To characterize the lipemia of pregnancy more completely, we studied some of the apolipoproteins in plasmas of pregnancy women. Thirty-eight normal fasting women were studied between the 18th and 39th weeks of gestation and again 23 plus or minus 17 weeks after delivery. Eight additional women were sampled every 4-6 wk during the second and third trimesters of gestation. Plasma and lipoprotein lipids were assayed by standard procedures and Apolipoprotein B (ApoB) was measured by radioimmunoassay. The interrelations of Apolipoprotein A (ApoA) in high-density lipoprotein (HDL) and of Apolipoprotein C (ApoC) in very-low-density lipoprotein (VLDL) were assessed by disc gel electrophoresis in four women during the last trimester of gestation and again 6-8 mo post partum and in four nongravid controls. Gestational triglycerides (TG) and cholesterol (Chol) were elevated in 95% of the pregnant women. TG in lipoproteins rose progressively during gestation, with VLDL-TG rising the most. Low-density lipoprotein (LDL) and HDL became enriched by TG relative to other components. Total-and VLDL-ApoB increased, while LDL-ApoB remained unchanged, resulting in a change in the density distribution of ApoB. (VLDL-ApoB X 100/total ApoB rose from 3.6% to 6.7%, P less than 0.02.) The accumulation of TG-rich LDL and the increases of VLDL-ApoB may be the result of changes in the rates of secretion or intravascular catabolism of VLDL. Which process is altered remains to be determined. The relative amounts of ApoC-II and ApoC-III in VLDL and the ApoA-I/ApoA-II ratios in HDL were unchanged in pregnancy. These results differ from those seen following high-carbohydrate diets.

Summary The effects of two apolipoproteins isolated from human very low density lipoproteins (apoLp-Glu and apoLp-Ala) on lipoprotein lipase (LPL) activity have been studied. ApoLp-Glu markedly stimulated LPL as reported by others. ApoLp-Ala isolated by techniques previously described also activated LPL at low levels. However, with further purification by hydroxylapatite chromatography all activation by apoLp-Ala was eliminated with the removal of a small contaminant immunochemically identical to apoLp-Glu. ApoLp-Ala consistently inhibits LPL when present at levels above 2% of the substrate (w/w). This inhibition was not overcome by addition of phospholipid, apoLp-Glu, or more enzyme.

These studies were designed to determine which of the apoproteins of high density lipoprotein (HDL) function as the cofactor for lipoprotein lipase (LPL). ApoLP-gln and apoLP-thr, the major HDL apoproteins, as well as apoLP-val, a minor apoprotein constituent, are inactive as cofactors even in the presence of phospholipid. ApoLP-ala, another minor constituent, is inactive alone but in the presence of phospholipid stimulates lipase activity twofold. Only apoLP-glu is able to stimulate LPL activity in the absence of phospholipid and, in the presence of phospholipid, increases activity twelvefold over baseline levels. It is possible that apoLP-glu and perhaps apoLP-ala are obligatory “co-factors” for the hydrolytic step required for normal clearing of triglyceride from the plasma.

Familial lecithin: cholesterol acyltransferase (LCAT) deficiency has recently been described as an inborn error of metabolism. The enzyme acts upon circulating lipoproteins and catalyses the transfer of fatty acid from the beta position of lecithin to the 3-β-OH group of free cholesterol. Besides characteristic changes of plasma lipid concentrations, deficiency of this enzyme is followed by changes of the physicochemical properties of the plasma lipoprotein fractions. Heterogeneity and abnormality with regard to the apolipoprotein composition are most pronounced in the low density fraction (d 1,063 to 1,21 g/ml). Three different sub-fraction can be isolated from this density class. a) lipoproteins containing only apolipoprotein B; b) lipoproteins containing apolipoprotein B and apolipoprotein C and c) lipoproteins consisting of apolipoprotein C, lipoprotein are very similar to the abnormal lipoprotein properties of the third (c) lipoprotein are very similar to the abnormal lipoprotein (LP-X) characterizing cholestasis. Since liver disease is often associated with low LCAT activity an important metabolic relationship may exist between structure of plasma lipoproteins, LCAT activity and liver function. (Coauthors: Drs. E. Gjone and J. P. Blomhoff, Oslo.)

1. 1. Lipoprotein fractions were isolated from canine serum by sequential preparative ultracentrifugation and purified. Immunochemical and quantitative chemical analyses were performed to characterize their apolipoprotein and lipid composition. 2. 2. High density (d 1.063–1.210 g/ml) lipoproteins contain most of the cholesterol and phospholipid present in canine serum and at least two immunologically distinct apolipoprotein components. Relatively more protein and phospholipid are present in these fractions than in lower density lipoproteins and the amounts of cholesterol and triglyceride are relatively less. 3. 3. Low density (d 1.006–1.063 g/ml) lipoproteins contain primarily a single apolipoprotein which is also demonstrable in very low density (d < 1.006 g/ml) lipoproteins. In addition, the very low density lipoprotein fraction contains an immunologically distinct component which may correspond with the apolipoprotein C found in human plasma. 4. 4. Immunochemical analyses also demonstrated the presence of an unusual protein component in purified very low density lipoproteins, in unpurified but not purified high density lipoproteins and in the serum sample from which lipoproteins of d