Probucol is a hypolipidemic agent that causes a marked decrease in high density lipoprotein (HDL) cholesterol. To investigate the mechanism of this effect, two studies were performed in hypercholesterolemic patients who had been stabilized previously on diet and were not receiving other lipid-lowering medication. Plasma cholesteryl ester transfer protein (CETP) concentrations were measured in fasting plasma samples before and after 10 weeks of probucol therapy using a sensitive and specific radioimmunoassay. Plasma total and low density lipoprotein cholesterol concentrations decreased, whereas apolipoprotein (apo) B was unchanged. Plasma apo E concentrations increased markedly. HDL cholesterol and apo A-I decreased in all subjects. These effects of probucol were accompanied by even more striking changes in plasma CETP concentrations, which increased by a mean of 64%. In a second study of six hypercholesterolemic subjects, the time-course effects of probucol on CETP and HDL subspecies were studied. Significant increases in plasma apo E and in CETP occurred after 4 weeks, and CETP, but not apo E, increased further after 16 weeks of treatment. Concomitant and opposite changes occurred in HDL composition, with decreases in HDL cholesterol and lipoprotein containing apo A-I. The increase in plasma CETP concentrations, the decrease in HDL cholesterol, and the increase in plasma apo E concentrations observed during probucol treatment are changes consistent with a postulated increase in reverse cholesterol transport via the remnant pathway.

Apolipoprotein A-I↗

PubMedDate not supplied

Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia. Relation to cholesteryl ester transfer protein activity and other lipoprotein variables.

Cholesteryl ester transfer protein (CETP) mediates an important pathway for reverse cholesterol transport. Concentrations of CETP in fasting plasma were measured by radioimmunoassay in two different groups of hyperlipoproteinemic subjects. Plasma CETP concentrations measured by radioimmunoassay correlated closely with cholesterol ester transfer activity in normal plasma (r = 0.86). In the first group of 58 patients, plasma CETP concentrations were significantly increased, as compared with those in 79 normal subjects and in hypercholesterolemic (+26%) and combined hyperlipoproteinemic (+25%) subjects but were not altered in moderately hypertriglyceridemic subjects. Marked elevations in plasma CETP levels were documented in patients with dysbetalipoproteinemia (+68%) and severe chylomicronemia (+85%). Similar results were obtained in a second population of 50 hyperlipoproteinemic subjects. Significant correlations were found between plasma CETP levels and total cholesterol (r = 0.52), very low density lipoprotein (VLDL) cholesterol (r = 0.63), and apolipoprotein E concentration (r = 0.40). Correction of the lipoprotein phenotype by dietary means resulted in significant reductions in plasma CETP concentrations in patients with chylomicronemia and dysbetalipoproteinemia. In these subjects, plasma high density lipoprotein cholesterol concentrations increased as CETP decreased. These studies indicate that CETP levels increase in association with enhanced peripheral cholesterol transport via low density lipoprotein, beta-VLDL, or chylomicron remnants.

Adult↗

PubMedDate not supplied

Characterization of monoclonal antibodies against human low density lipoprotein.

Seven monoclonal antibodies against human low density lipoprotein (LDL) have been characterized as to their specificity and ability to interfere with the LDL pathway in cultured human fibroblasts. The immunoreactivity with LDL of two of the antibodies (2D8 and 4G3) was particularly sensitive to modification of lysine and arginine residues in LDL. Cotitration experiments indicated that the antibodies 3A8 and 3A10 may react with the same determinant and that five antibodies (5E11, 3A8, 3A10, 4G3 and 3F5) recognized determinants that were grouped in the same region of the molecule. The two other antibodies (1D1 and 2D8) reacted with determinants distant from this region. When tested in molar excess relative to LDL, Fab fragments of 5E11, 3A8, 3A10, 4G3 and 3F5 (but not 1D1 or 2D8) were capable of blocking the binding of 125I-LDL to the LDL receptor and interfering with LDL suppression of cholesterol synthesis in cultured fibroblasts. Increasing the concentration 10-fold did not change the results significantly. Based on these results we have proposed a map of the determinants as they would appear in LDL.

Antibodies, Monoclonal↗

PubMedDate not supplied

Characterization of antigenic determinants of human apolipoprotein B. Distribution on tryptic fragments of low density lipoprotein.

In the hope of obtaining useful probes to study the structure of human apolipoprotein B (apo B), we characterized monoclonal antibodies against low density lipoprotein (LDL). We examined the distribution of their corresponding antigenic determinants on tryptic fragments of LDL separated by monodimensional (SDS) or two-dimensional electrophoresis. Each antibody reacted with several different fragments even when the proteolysis was apparently complete. A peptide of 125,000 daltons was the smallest fragment recognized by all the antibodies. The antibody, 2D8, which cross-reacts with apo B-48 and 3A8 which blocks the LDL pathway both reacted with the same 43,000 dalton fragment. Two other antibodies, 3F5 and 4G3, previously shown to be close together in LDL, also appeared close together in the primary structure of apo B. A determinant present on apo B-26 (1D1) was dissociated from all others examined on fragments of less than 125,000. Similarities in the patterns of reactivities with LDL-tryptic fragments between certain monoclonal antibodies and the lectins Concanavalin A and Limax flavus agglutinin indicated the proximity of the corresponding antigenic determinants to carbohydrate moieties. Competition studies suggested that the two major carbohydrate chains of LDL do not participate in the determinants themselves.

Antibodies, Monoclonal↗

PubMedDate not supplied

Apolipoprotein B-48 and B-100 very low density lipoproteins. Comparison in dysbetalipoproteinemia (type III) and familial hypertriglyceridemia (type IV).

A protein band having the same migration as apolipoprotein (apo) B-48 was observed by SDS electrophoresis in the plasma very low density lipoprotein (VLDL) from 14 Type IV and three Type III hyperlipoproteinemic subjects and from six normal fasting subjects. The VLDL from five Type IV, three Type III, and one normal subject were separated into two subfractions, retained and nonretained, by immunoaffinity chromatography on monoclonal anti-apo B-100 Sepharose. Based on results of electrophoresis and radioimmunoassay, we have concluded that these two fractions represent apo B-48 and apo B-100 lipoproteins that we have named apo B-48 and apo B-100 VLDL. When compared to their respective apo B-100 VLDL, the apo B-48 VLDL from either Type III or Type IV was principally enriched in total lipids, in apo E, and had an electrophoretic migration similar to chylomicrons. This suggests that apo B-48 VLDL has the same origin (i.e., intestinal) in the two disorders. Both apo B-48 and apo B-100 VLDL were enriched in cholesteryl ester (CE) and depleted in triglyceride (TG) in Type III; however, both fractions were rich in TG and poor in CE in Type IV and in normal subjects. In addition, compared to their respective apo B-100 VLDL, the apo B-48 fraction was enriched in CE in Type III and in TG in Type IV. We conclude that, despite a possible similar origin for apo B-48 VLDL in Type III and in Type IV subjects, the composition of apo B-48 VLDL is variable and the CE/TG ratio is more characteristic of the type of hyperlipidemia than of the particular VLDL subfractions.

Adult↗

PubMedDate not supplied

Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apo A-I antigens.

Two series of monoclonal antibodies (MAB) directed against apolipoprotein A-I (apo A-I) reacted differentially with freshly prepared sera or plasma. MAB from Series 1 were obtained after immunization and screening with purified apo A-I, while those from Series 2 were obtained by immunization with high density lipoprotein (HDL) and screening with both HDL and apo A-I. Series 2 MAB 5F6, 3G10, and 4F7 reacted significantly better with fresh material than material stored at 4 degrees C for longer than 1 month. Conversely, Series 1 MAB 3D4, and 6B8, and Series 2 MAB 2F1 reacted more strongly with the stored preparations. Series 2 MAB 4H1 reacted equally with stored or fresh material. The inability of 3D4 and 6B8 to react with fresh sera or plasma was not due to an inhibitor in the fresh material. Preparation of HDL from serum or plasma and delipidation of this material had no effect on the above phenomena, which appears related directly to apo A-I and not to the interaction of apo A-I with lipids. The variation of immunoreactivity with storage at 4 degrees C was also shown to be unrelated to proteolysis of apo A-I. Alkaline treatment of freshly prepared sera, HDL, or apo HDL with sodium hydroxide simulated the effect of storage, allowing this material to react strongly with MAB 3D4, 6B8. As expected, there was a decrease in the reactivity of MAB 5F6 with alkali-treated fresh material compared to untreated fresh material. However, further investigation showed that alkaline treatment does not completely mimic the effect of storage. MAB 2F1, which reacts poorly with fresh material, reacted very poorly with alkali-treated fresh material, and MAB 3G10 and 4F7, which react well with fresh material, reacted even better with alkali-treated fresh material. While isoelectric focusing of apo HDL prepared from stored serum showed a redistribution of apo A-I toward more acidic isoforms compared to fresh serum, alkaline treatment of apo HDL resulted in partial cleavage of apo A-I and in the generation of isoforms more acidic than known apo A-I isomorphs. Therefore, alkaline treatment of serum is only a partial model for antigenic modification of apo A-I upon storage. These results demonstrate that in vitro conditions (storage or alkaline treatment) selectively modify certain antigenic sites of apo A-I, but not others. This phenomenon, which is probably related to deamidation, may affect apo A-I immunoassays with either monoclonal or polyclonal antibodies.

Antibodies, Monoclonal↗

PubMedDate not supplied

Mapping of human apolipoprotein B antigenic determinants.

A minimum of 16 epitopes which provide a group of topographical markers to study the conformation of apolipoprotein (apo) B have been mapped in relation to elements of the sequence of apo B-100. Six of these epitopes are identified by monoclonal antibodies (Mabs) directed against low density lipoprotein (LDL) apo B, while at least 10 others react with Mabs obtained by immunization with delipidated and solubilized apo B. Five epitopes which are also expressed on apo B-48 have been assigned to the thrombolytic fragment T4 on the N-terminal side of apo B-100. None of these five epitopes requires the presence of lipids for its expression, suggesting that the conformation of the T4 region of apo B is more dependent on peptide-chain interactions than on peptide-lipid interactions. Four distinct epitopes have been assigned to the median thrombolytic fragment T3 of apo B-100, all of which require the presence of lipids for their expression; those epitopes closer to the C-terminus of T3 require specific interaction with cholesteryl esters. The same lipid dependence also characterizes a cluster of epitopes mapped to the N-terminal region of fragment T2. The epitopes that are close to the T2/T3 cleavage site and depend on the presence of cholesteryl esters for their expression are also those that react with the Mabs that inhibit the binding of LDL to its receptor. Therefore this region, which in addition contains two sequences with structural homology to the apo E receptor binding domain, probably constitutes a physiologically important receptor binding site for apo B. Finally, four other distinct epitopes which do not require the presence of lipids for their expression have been mapped on T2. In conclusion, the present report presents evidence that the immunochemical analogy of apo B-48 and apo B-100 is on the N-terminal half of apo B-100, whereas the apo B receptor binding domain is localized on the C-terminal half of apo B-100 close to the T2/T3 cleavage site.

Antibodies, Monoclonal↗

PubMedDate not supplied

Expression of apolipoprotein B epitopes in lipoproteins. Relationship to conformation and function.

The immunochemical properties of apolipoprotein (apo) B have been studied in very low density lipoprotein (VLDL)1 (Sf 100 to 400), VLDL2 (Sf 60 to 100), VLDL3 (Sf 20 to 60), different intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) subfractions isolated from patients with type IV hypertriglyceridemia. In these lipoproteins, we characterized the association of apo B with other apolipoproteins and the expression and immunoreactivity of several apo B epitopes close to the apo B receptor binding sites (3F5, 4G3, 3A8, and 5E11) and of other epitopes located on the apo B100-B48 common region (1D1 and 2D8). Immunoprecipitation showed that the proportion of lipoprotein particles expressing each apo B epitope increased from VLDL1 to LDL2; this was more apparent with 3A8 and 5E11 than with 3F5. The VLDL that were negative for apo E epitopes (60% or more of the total) were enriched in apo C. The lipoprotein particles containing apo E and/or apo C-III decreased progressively from VLDL1 (30% and 85%, respectively) to LDL2 (10% and 25%, respectively). Similar observations were made for apo C-I and apo D, demonstrating that apolipoprotein heterogeneity is greatest in the lightest lipoproteins. By competitive radioimmunoassay, the epitope for 4G3 was equally immunoreactive in each lipoprotein subclass, and the affinity constant (Ka) of 4G3 for different lipoproteins showed little variation. In contrast, both immunoreactivity and Ka of 3A8 and 5E11 increased progressively and significantly with the increasing density of the lipoprotein subclasses. This phenomenon is correlated with the increasing binding affinity of apo B in these lipoprotein subclasses to the LDL receptor of fibroblasts. We conclude that, as the apo B-containing lipoproteins become smaller, the conformation of specific regions of apo B is modified: in the receptor binding domain, the conformation of epitope 4G3, which is mapped between residues 2980 and 3080, remains constant, while that of 3A8 and 5E11 (residues 3441 to 3568) changes progressively. We propose the theory that the change in conformation in the domain spanning residues 3441 and 3568 allows the maximum expression of epitopes 3A8 and 5E11 and of the receptor binding site.

Adult↗