Menahem Fainaru

Radioimmunoassay of human high density lipoprotein apoprotein A-I

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 equal to 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 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-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: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.

Apoprotein content of plasma lipoproteins of the rat separated by gel chromatography or ultracentrifugation.

Abstract Rat lipoproteins were separated from each other and from unbound apoproteins by chromatography on 10% agarose gel. Fresh rat serum yielded two main lipoprotein peaks: Peak I corresponded mainly to very low density lipoproteins (VLDL) and peak II corresponded to high density lipoproteins (HDL). Recovery of total cholesterol and apolipoprotein A-I (A-I) in the two peaks was similar when fresh serum or lipoproteins of d d d > 1.21 g/ml was applied to the same column. Comparison of lipoproteins isolated by gel chromatography with those obtained by conventional flotation ultracentrifugation showed that ∼30% of ARP was lost from VLDL and ∼60% was lost from HDL during ultracentrifugation. The ratio of ARP to A-I fell progressively across the second chromatographic peak, indicating heterogeneity of HDL apoproteins as a function of particle size.

The effects of prednisone therapy on plasma lipoproteins and apolipoproteins: A prospective study☆

The effect of prednisone therapy on plasma lipoproteins and apolipoproteins A-I, A-II, and E levels was studied prospectively in a heterogeneous group of six male and six female subjects. All patients were in a good general condition. The patients had normal hepatocellular, renal, and thyroid functions. During the first month of therapy, the following changes were noted: Plasma triglyceride (TG) levels increased slightly in female patients only. In the entire group, plasma cholesterol level increased (17.3% of initial value, P less than 0.01). Plasma high-density lipoprotein cholesterol (HDL-C) level increased by 68% (P less than 0.001), while plasma low-density lipoprotein cholesterol (LDL-C) level increased by only 10.9% (not significant), resulting in an increased ratio of cholesterol in the two (P less than 0.01). No change in levels of plasma apolipoproteins A-I, A-II, and E was evident. The ratio of HDL-C to plasma apolipoprotein A-I increased (P less than 0.01), indicating an increased lipid to protein ratio for this lipoprotein. Most of these changes were already apparent and significant 48 hours after initiation of treatment and persisted throughout the follow-up period (up to 18 months in some patients). Our results show that in patients with no major metabolic abnormality, prednisone induces significant changes of the lipoprotein system, especially in HDL.

Radioimmunoassay of apolipoprotein A-I of rat serum

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.

Evidence that a separate particle containing B-apoprotein is present in high-density lipoproteins from perfused rat liver.

High-density lipoproteins (HDL) (1.075 less than d less than 1.175) from perfusates of rat liver, unlike those of blood plasma, contain protein with the properties of B-apolipoprotein. This protein remains near the origin upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, has beta electrophoretic mobility in agarose gel, is insoluble in tetramethylurea, and precipitates with antisera to the B-apoprotein isolated from low-density lipoprotein. B-apolipoprotein in HDL from perfusates binds to concanavalin-A Sepharose and can thus be separated from the characteristic HDL, the chemical and physical properties of which are otherwise preserved. These observations suggest that in addition to the discoidal lipoproteins, another particle that contains B-apoprotein exists in HDL of perfusates.

Isolation and Characterization of Lipid-Protein Complexes Present in Commercial Albumin Preparations

Most commercially available albumin preparations examined by us contain phospholipids, cholesterol and apolipoprotein impurities. As these albumin preparations are frequently used in large amounts in systems involving lipoprotein metabolism these impurities may reach remarkable levels to introduce exogenous effects in these studies. We have studied in detail tow bovine albumin preparations differing in their content of these contaminants. Using preparative ultracentrifugation, we have isolated from both albumins a lipid protein complex at a buoyant density of d = 1.063-1.21 g/ml with a chemical composition resembling plasma high density lipoproteins. This complex when further characterized proved also to have a similar apoprotein composition to bovine plasma high density lipoproteins. Electron microscopic study of this complex revealed discoidal particles closely resembling nascent high density lipoproteins recovered from rat liver or lymph. The similarity of these lipid-protein complexes to high density lipoproteins, accounts for some reported effects caused by commercially available albumin preparations on cholesterol excretion from cells in tissue culture and their ability to act as acceptors for surface remnants released upon VLDL catabolism in vitro.