[Lipoproteins associated with lipoprotein B in human serum low density lipoproteins]. / Lipoprotéines associeés aux lipoprotéines B dans les lipoprotéines de basse densité de serum humain.

Small amounts of lipoprotein C and lipoprotein D could be observed in low density lipoproteins (1.030-1.055 g/ml), using electroimmunomigration and polyacrylamide gel electrophoresis. Lipoprotein structures containing several apolipoproteins such as lipoprotein (B+C) or (B+D) were not detected in these low density lipoproteins. Lipoproteins C and D could not be separated from lipoprotein B by using gel filtration and affinity chromatography on heparin-Sepharose. Apolipoproteins C-III and D measured by electroimmunoassay are 3.2 +/- 1.2% and 1.15 +/- 0.6%, respectively, of the proteins found in the density range 1.030-1.055 g/ml, so there is, therefore, about 1 mol of apolipoprotein C-III and 0.1 mol of apolipoprotein D per mol of apolipoprotein B.

Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma.

High density lipoproteins (HDL) consist of a mixture of chemically and functionally distinct families of particles defined by their characteristic apolipoprotein (Apo) composition. The two major lipoprotein families are lipoprotein A-I (LP-A-I) and lipoprotein A-I:A-II (LP-A-I:A-II). This study describes the isolation of a third minor HDL family of particles referred to as lipoprotein A-II (LP-A-II) because it lacks ApoA-I and contains ApoA-II as its main or sole apolipoprotein constituent. Because ApoA-II is an integral protein constituent of three distinct lipoprotein families (LP-A-I:A-II, LP-A-II: B:C:D:E and LP-A-II), LP-A-II particles were isolated from whole plasma by sequential immunoaffinity chromatography on immunosorbers with antisera to ApoA-II, ApoB and ApoA-I, respectively. In normolipidemic subjects, the concentration of LP-A-II particles, based on ApoA-II content, is 4-18 mg/dl accounting for 5-20% of the total ApoA-II not associated with ApoB-containing lipoproteins. The lipid composition of LP-A-II particles is characterized by low percentage of triglycerides and cholesterol esters and a high percentage of phospholipids in comparison with lipid composition of LP-A-I and LP-A-II: A-II. The major part of LP-A-II particles contain ApoA-II as the sole apolipoprotein constituent; however, small subsets of LP-A-II particles may also contain ApoD and other minor apolipoproteins. The lipid/protein ratio of LP-A-II is higher than those of LP-A-I and LP-A-I:A-II. In homozygous ApoA-I and ApoA-I/ApoC-III deficiencies, LP-A-II particles are the only ApoA-containing high density lipoprotein with levels found to be within the same range (7-13 mg/dl) as those of normolipidemic subjects. However, in contrast to normal LP-A-II, their lipid composition is characterized by higher percentages of triglycerides and cholesterol esters and a lower percentage of phospholipids and their apolipoprotein composition by the presence of ApoC-peptides and ApoE in addition to ApoA-II and ApoD. These results show that LP-A-II particles are a minor HDL family and suggest that, in the absence of ApoA-I-containing lipoproteins, they become an efficient acceptor/donor of ApoC-peptides and ApoE required for a normal metabolism of triglyceride-rich lipoproteins. Their other possible functional roles in lipid transport remain to be established in future experiments.

Monoclonal antibodies to human apolipoprotein A-I: characterization and application as structural probes for apolipoprotein A-I and high density lipoprotein.

Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.

Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo.

We describe an ELISA technique able to recognize malondialdehyde-modified low-density lipoproteins (LDL). For this purpose we produced antibodies to malondialdehyde-LDL, specific for the malondialdehyde modification of LDL; these antibodies recognized essentially malondialdehyde-LDL. Coating ELISA plates with the antibodies to malondialdehyde-LDL and using peroxidase-labelled antibodies to LDL, which reveal only apolipoprotein B, we obtained an accurate method of detecting malondialdehyde-modified apolipoprotein B. Preliminary studies demonstrated that this method allows the detection of lipoproteins containing malondialdehyde-modified apolipoprotein B in the serum of patients with cardiovascular diseases.

Ligand-blotting visualization of the LDL receptor in plasma membranes and in two classes of coated vesicles from adrenocortical cells.

Two populations of coated vesicles, different in size, have been isolated from the bovine adrenal cortex. The enrichment of the LDL receptor from the plasma membrane to the large coated vesicles and then to the small ones was evidenced by ligand-blotting ELISA assays. The LDL receptor has been characterized as a 130-kDa proteic component which retains the binding specificity and structural features in plasma membranes as well as in the two classes of coated vesicles.

[Human apolipoprotein E: polymorphism and binding domain of the receptors]. / Apolipoproteine E humaine: polymorphisme et domaine de fixation aux récepteurs.

This paper summarizes present knowledge of the LDL receptor-binding domain of apolipoprotein E, with special emphasis on the influence of apolipoprotein polymorphism on the interaction with apo B/E receptors.

[Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum]. / Présence et isolement de deux lipoprotéines immunologiquement apparentées aux apo A I dans le sérum humain.

Very high density lipoproteins d : 1.23--1.25 g/ml (VHDL2) have been isolated from human serum by preparative ultracentrifugation. They contain 80 per cent proteins and 20 per cent lipids. Lipids are mainly phospholipids (80 per cent). The proportion of lysolecithin (50 per cent) is higher than that of lecithin (40 per cent). The quantity of cholesterol is low, the free cholesterol: total cholesterol ratio is 0.35. VHDL2 consisted principally in lipoprotein D and two lipoproteins immunologically apparented to apolipoprotein A I, called LP A I1 and LP A I2. The LP A I1 has a molecular weight slightly higher and a hydrated density lower than that of LP AI2. Our experiments suggest that LP A I1 exists in the serum before ultracentrifugation while LP A I 2 comes from HDL degradation during ultracentrifugation. The immunological heterogeneity of apo A I forming different protein-lipid complexes is discussed.

[A new protein from human plasma lipoproteins: isolation and partial characterization of apolipoprotein G]. / Une nouvelle protéine des lipoprotéines du sérum humain: isolement et caractérisation partielle d'une apolipoprotéine G.

A new apolipoprotein has been identified in VHDL1 and in HDL. This protein is immunologically distinct from already isolated apoproteins. It was isolated by column chromatography on hydroxylapatite. In polyacrylamide gel electrophoresis, its mobility is very close to that of apo D. The amino acid composition differs from those of the well characterized polypeptides of the human plasma lipoproteins. It contains glucosamine. The apparent molecular weight is 72 000 +/- 2 000 in the presence and absence of reducing agent. According to the ABCDEF nomenclature, this protein can be named apolipoprotein G (apo G). It is present in a lipoprotein distinct from the lipoproteins A and D among the VHDL1 : this new lipoprotein can be named lipoprotein G (LPG).

[Preparation and seric form of rabbit C reactive protein]. / Préparation et forme sérique de la protéine réactive C de lapin.

The preparation of rabbit C-reactive protein (CRP) involves a single step affinity chromatography. This preparation takes advantage of the calcium-dependent affinity of CRP for an agarose gel bearing 2-aminoethanol dihydrogen-phosphate as a ligand. A prior chromatography on agarose gel without the ligand allows the uptake of the serum amyloid P-component (SAP). The CRP prepared according to this method is able to form precipitating complexes in agarose with rabbit lipoproteins. The specificity of these interactions is studied. It is demonstrated that CRP-High Density Lipoproteins (HLD) association produces a second precipitation arc when the pure CRP is revealed by a specific antiserum in agarose. Moreover, CRP in the serum is shown to be in the bound form only, and the binding involves Low Density Lipoproteins (LDL) exclusively.

[Inhibition by the high density lipoprotein HDL2 and HDL3 of DNA and sterol biosynthesis in human lymphocytes stimulated with concanavalin A]. / Inhibition par les lipoprotéines de haute densité HDL2 et HDL3 de la synthèse du DNA et des stérols des lymphocytes humains stimulés par la concanavaline A.

Lipoproteins HDL2 and HDL3 inhibit DNA synthesis and sterol synthesis in human Con A-stimulated lymphocytes cultured in a medium supplemented with 20 per cent lipoprotein deficient serum. On the basis of the amount of proteins added, HDL2 is more efficient on DNA and sterol synthesis than HDL3 and less efficient than LDL. However, on the basis of the amount of cholesterol added, the inhibition of sterol synthesis induced by these three lipoproteins is not significantly different. At all concentrations of these three lipoproteins, the inhibition of sterol synthesis is higher than the inhibition of DNA synthesis.

[Two-dimensional immunoelectrophoresis of spontaneous dissociation of low density lipoproteins]. / Etude par immunoélectrophorèse bidimensionnelle de la dissociation spontanée des lipoprotéines de basse densité

Plasma-lipoproteins isolated between d 1.020 and d 1.055 g/ml were partially delipidated with ethyl ether at 4 degrees C. This treatment induces a transformation of the lipoproteins which is evolutive during several days. Bidimensional immunoelectrophoresis reveals the lipoproteins dissociation and the appearance of 4 immunologically different fractions. The time dependent formation of these subunits is slowed down by EDTA and less efficiently by antioxydants. Once started, the dissociation can be accelerated by heating at 37 degrees C or by UV exposition. Another lipopeptide is more easily revealed by anti VLDL antiserum. It can be shown in the native and in the partially or completely delipidated LDL. Its presence does not depend on lipoprotein dissociation.

[Dissociation of human serum low density lipoproteins in several immunologically distinct subunits. Isolation and characterization of a B-III subunit]. / Dissociation of human serum low density lipoproteins in several immunologically distinct subunits. Isolation and characterization of a B-III subunit.

Low density lipoproteins (d = 1.030-1.055) were partially delipidated with ethyl-ether (LDLe). These LDLe exhibit a spontaneous dissociation several days after delipidation. Four different immunoprecipitation complexes (B-I to B-IV) are observed when using two dimensional immunoelectrophoresis against anti LDLe immunserum. The various subunits of LDLe have different behaviour upon electrophoresis. On disc gel electrophoresis containing urea three bands can be seen; all are phospholipoproteins. The apolipoprotein moiety of LDL and LDLe have the same apparent molecular weight around 550 000. With time several subunits appear in LDLe, the majority of them have a molecular weight around 370 000, 260 000 and 125 000. One of the components from dissociated LDLe containing the immunodeterminant B-III, has been separated by chromatography on heparin-agarose. This LDLe-B-III has the same phospholipid/protein ratio as total LDLe and a protein moiety with an apparent molecular weight of 110 000. This part of apolipoprotein B has no affinity for heparin. Immunocompetition studies by the ELISA technique indicated that sialic acid, one of terminal residues of LDL glycoprotein, is involved in the immunological recognition of LDL and of its derivatives by anti LDL and anti LDLe antisera.

Redistribution of apolipoproteins C removed from human very low density lipoprotein during in vitro lipolysis by lipoprotein lipase.

In order to localize the labelled apolipoproteins C removed from Very Low Density Lipoproteins (VLDL) during lipolysis by lipoprotein lipase, we used human VLDL labelled with 125I-labelled apolipoproteins C and employed density gradient ultracentrifugation to analyze lipolytic products. Triacylglycerol hydrolysis occurs when albumin is present even in the absence of serum or High Density Lipoproteins (HDL). In this case, apolipoproteins C were found to be located in several fractions, in different density regions. When either HDL or serum were present in the incubation medium, the removed apolipoproteins C were recovered in only one main fraction in the high density region (1.10 g/ml). Incubations in the presence of either HDL2 or HDL3 gave quite similar results.

Association between coagulation factors VII and X with triglyceride rich lipoproteins.

The association between the concentration of different plasma lipoproteins and plasma factor VII (F VII) was analysed by isolating plasma very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) lipoproteins and assessing their in vitro interaction with F VII by immunoenzyme assay using peroxidase labelled anti-factor VII immunoglobulins to determine whether F VII coagulant activity is prognostic for cardiovascular mortality. F VII bound to triglyceride rich lipoproteins, the fixation being stronger on chylomicrons and VLDL fractions than on LDL fractions. In our experiments HDL did not bind to F VII. The fixation of coagulation factor X (FX) tested by the same method is comparable with that of F VII. The nature of this fixation seemed to arise from hydrophobic interaction as calcium was not necessary and the use of Tween 20 inhibited the interaction. The binding of factors VII and X was increased when lipids were previously treated by phospholipase C and the interaction seemed to be completely dependent on the lipid part of the lipoproteins. Hyrophobic fixation is a possible mechanism of interaction of plasma lipoproteins and F VII and X, and it may be of importance in the covariance of triglyceride concentrations and the activity of vitamin K dependent coagulation factors.

Electroimmunoassay of human serum lipoproteins containing Apo A-I by monoclonal antibodies.

Three monoclonal antibodies to human serum apolipoprotein (Apo) A-I (4A12, 4B11 and 2G11) were produced by Sanofi. They were directed to three distinct epitopes of the Apo A-I molecule, present on the surface of the lipoprotein particles. They were able to precipitate individually some lipoprotein units from the serum. Only the mixture of the three monoclonal antibodies could allow a precipitation of all Apo A-I containing particles. An electroimmunoassay using this oligoclonal mixture was assessed to standardize the Apo A-I measurement in serum. Results agreed well with those obtained by electroimmunoassays using polyclonal antisera. Moreover no pretreatment of serum samples with dissociating agents or detergents was required. Therefore, this specific, rapid (7-8 h), precise (within- and between-assay precisions were 4.25 and 3.1%, respectively) immunoassay is routinely available for apolipoprotein A-I measurement in serum.

Plasma lipoproteins in infantile visceral leishmaniasis: deficiency of apolipoproteins A-I and A-II.

This study describes the plasma lipoprotein system of young children with visceral Leishmaniasis (Kala-azar disease). In addition to the presence of amastigote forms in the sternal aspirates of bone marrow, the patients exhibited fever, anemia, hepatosplenomegaly, various degrees of pancytopenia and a slight liver cytolysis. Patients had normal total cholesterol levels and increased triglyceride levels in the plasma. The concentrations of HDL and LDL were 30% and 50% of these reported for normolipemic subjects, respectively. In contrast, there was a three-fold increase in the concentration of VLDL. The ratio of free to total cholesterol was high; this was further substantiated by electron microscopy of HDL showing the presence of disc-like particles. Quantitative determination of apolipoproteins revealed a three- and seven-fold decrease of apolipoproteins (Apo) A-I and A-II, respectively, whereas Apo B levels were within the normal range. The presence of LP-A-II particles was demonstrated by two-dimensional immunoelectrophoresis in most of the patients' plasma during the acute phase of disease.

[Structure and metabolism of lipoproteins]. / Structure et métabolisme des lipoprotéines.

Lipoproteins constitute in plasma a dynamic system allowing lipid transport. For this purpose, apolipoproteins play a very important part. They control and regulate lipid transfer between lipoproteins themselves and among cells, from their hepatic as intestinal synthesis sites to their hepatic as peripheral degradation sites. Enzymatic systems and specific receptors are involved to operate this metabolic pathway.

[Immunological assay of apolipoprotein A-I]. / Dosage immunologique de l'apolipoprotéine A-I.

Since lipid free apo A-I and lipid rich apo A-I (HDL, LP A, LP A-I) have different accessible antigenic sites, the electroimmunoassay ("rocket") does not allow the determination of the apo A-I concentration in both lipoproteins and serum when lipid free apo A-I are taken as controls. The results are found more significant when an isolated HDL or serum are used as reference. A comparison is made with the results (height of the pics) obtained for the same sample with different antibodies anti apo A-I, checked with the same HDL control, great differences two to one is observed. It is only explained by the various composition of antibodies directed against different apo A-I antigenic sites. Differences obtained in the levels of apo A-I in HDL2 and in HDL3 by immunoprecipitation show that the more lipoproteins are lipid rich the less their apo A-I are reached by antibodies.