Effects of gemfibrozil on very-low-density lipoprotein composition and low-density lipoprotein size in patients with hypertriglyceridemia or combined hyperlipidemia.

To examine the effects of gemfibrozil on very-low-density lipoprotein (VLDL) composition and low-density lipoprotein (LDL) size, five men with hypertriglyceridemia (HTG) alone and five men with HTG and hypercholesterolemia (combined hyperlipidemia, CHLP) were randomized for 8 weeks to Lopid SR (slow-release gemfibrozil; two 600-mg tablets once per day) or placebo in a crossover study. Drug therapy versus placebo significantly decreased plasma triglyceride (68%), and VLDL (77%), and significantly increased high-density lipoprotein cholesterol (25%); total cholesterol, apolipoprotein B and lipoprotein[a] concentrations did not change significantly. With drug, mean total apoE in plasma was 53% lower in patients with HTG and 39% lower in patients with CHLP. Gemfibrozil significantly affected VLDL composition: protein increased 26%, molar ratio of apoE to apoB reduced 48%, apoC-II increased 19%, and apoC-III decreased 9%. LDL cholesteryl ester significantly increased with drug treatment. VLDL subfractions were separated and classified as heparin binding (VLDLR, apoE rich) or nonbinding (VLDLNR-1 and VLDLNR-2, both apoE poor). All VLDL subfractions were significantly lower with drug therapy, and the differences for total VLDL and for VLDL subfractions were greater in patients with HTG. With placebo, VLDLR accounted for 41.8% of VLDL in HTG and 49.0% of VLDL in CHLP, reduced to 27.6% and 38.6%, respectively, with gemfibrozil. Taken together, these results suggest that treatment with gemfibrozil reduces plasma concentrations of VLDL and alters the apoprotein composition of VLDL in a manner that may favor LDL- and VLDL-receptor-mediated clearance of the apoE-rich VLDL subfraction, thereby reducing TG-rich particle concentrations, and possibly reducing risk for coronary heart disease.

[Inhibition of cholesterol biosynthesis in rabbit hepatocytes by 3-beta-(omega-hydroxyalkoxy)cholest-5-enes]. / Ingibirovanie biosinteza kholesterina v gepatotsitakh krolika 3beta-(omega-gidroksialkoksi)kholest-5-enami]

3 beta-(2-Hydroxyethoxy)-, 3 beta-(4'-hydroxybutoxy)-, 3 beta-(6-hydroxyhexyloxy)-, 3 beta-(9-hydroxynonyloxy)-, and 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-enes were synthesized. By means of a spin probe, the influence of the synthesized compounds on the phase transition of dimyristoylphosphatidylcholine were estimated. Time and dose dependences of the incorporation of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-ene into rabbit hepatocytes (the primary culture) were studied. 3 beta-(2-Hydroxyethoxy)- and 3 beta-(4-hydroxybutoxy)cholest-5-enes were shown to inhibit cholesterol biosynthesis from [14C]acetate in rabbit hepatocyte cultures upon a 24-hour preincubation.

Interaction of apolipoprotein[a] with apolipoproteinB-100 Cys3734 region in lipoprotein[a] is confirmed immunochemically.

Monospecific polyclonal antibodies (MPAbs) to apoB-100 regions Cys3734 and Cys4190 were isolated by affinity chromatography using the synthetic polypeptides, Q3730VPSSKLDFREIQIYKK3746 and G4182IYTREELSTMFIREVG4198, respectively, coupled to a hydrophilic resin. Molecular modeling and fluroescence labeling studies have suggested that Cys67 located in kringle type 9 (LPaK9, located between residues 3991 and 4068 of the apo[a] sequence inferred by cDNA) of the apo[a] molecule is disulfide linked to Cys3734 of apoB-100 in human lipoprotein[a] (Lp[a]). This possibility has been further explored with MPAbs. Four species of MPAbs directed to a Cys3734 region of apoB-100 (3730-3746) were isolated from goat anti-human LDL serum by a combination of synthetic peptide (Q3730VPSSKLDFREIQIYKK3746) affinity chromatography and preparative electrophoresis (electrochromatography). MPAbs to the Cys4190 region of apoB-100, a second or alternative disulfide link-site between apo[a] and apoB-100, were also isolated using a synthetic peptide (G4182IYTREELSTMFIREVG4198) affinity resin. Results of immunoassays showed that binding of these four MPAbs to Lp[a] was significantly lower than to LDL. In contrast, MPAbs to the apoB-100 region 4182-4198 which contains Cys4190, a second or alternative disulfide link-site between apo[a] and apoB-100, displayed a less significant difference in binding to Lp[a] and LDL. These results provide additional evidence that the residues 3730-3746 of apoB-100 interact significantly with apo-a- in Lp-a-, and that Cys3734 is a likely site for the disulfide bond connecting apo[a] and apoB-100.

Human Lp(a): regions in sequences of apoproteins similar to domains in signal transduction proteins.

The major apoproteins of Lp(a)--apo(a) and apo B-100--are linked by only one intermolecular disulfide bond. This linkage has been suggested to be located between apo(a) Cys4057 and apo B-100 Cys3734. Several studies, however, have suggested other noncovalent interactions between different regions of apo(a) and apo B-100. One possible mechanism for these interactions may involve the apo(a) proline-rich interkringle regions that share sequence similarities with the proline-rich regions of Src homology 3 (SH3) domain-binding proteins such as 3BP-1. SH3 and SH2 domains, and their respective ligands, proline-rich regions, and phosphotyrosine motifs, are noncatalytic segments common to signal transduction proteins. Therefore, we used sequence comparison algorithms and molecular modeling programs to identify corresponding SH3 and SH2 candidate regions as well as potential phosphotyrosine sites in the apo B-100 sequence. Six SH2 and 16 SH3 candidate regions, along with 21 potential phosphotyrosine sites, are contained in the apo B-100 sequence. In Lp(a), these regions of apo B-100 may be involved in the noncovalent, protein-protein interactions between apo(a) and apo B-100. The presence of candidate SH3 and SH2 regions in apo B-100, and potential phosphotyrosine sites in apo B-100, apo(a), and apo A-I, suggests an alternative signaling pathway unrelated to the known B/E receptor.

Evidence that apoB-100 of low-density lipoproteins is a novel Src-related protein kinase.

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of the src protein kinase family.

Immunoreactivity of apolipoprotein B-100 in oxidatively modified low density lipoprotein.

Thirteen monoclonal antibodies (MAbs) against apolipoprotein B-100 (apo B) were used to analyze changes in immunoreactivity of human LDL resulting from oxidation mediated by cupric ions and oxygen. Decrease in immunoreactivity of oxidized LDL was demonstrated by competitive ELISA with MAbs 5F8, BL3, Mb43, 2G8, B3, B5, and BL7 for which the epitopes are located within residues 1-1297, 4235-4355, 4027-4081, 3728-4306, 2239-2331, 1854-1878, and in the vicinity of residue 2331, respectively. Immunoreactivity of the epitope B6 (2239-2331) increased during first 4 hours of oxidation and then diminished gradually. Epitope B1 (405-539) had slightly reduced immunoreactivity during first 8 h of LDL oxidation and then its minor increase was observed. MAb 12G10, specific to the epitope within apo B thrombin-digest fragment T4 (1-1297), displayed either weak or strong binding to LDL. LDL with weak binding pattern demonstrated significant increase in immunoreactivity upon oxidation. In contrast, LDL with strong binding pattern showed little to no change. Epitopes Mb47 (3441-3569) and 8G4 (1-1297) remained unchanged in oxidized LDL. Immunoreactivity of apo B-100 epitope recognized by MAb 4C11 (residues 2377-2658) was shown to be a function of oxidation time: it increased progressively up to 16 h and was stabilized for another 24 h of LDL oxidation. This epitope may be unmasked by LDL oxidation and may provide a useful immunochemical marker to monitor the extent of LDL oxidation.

[Relative location of anthroyl derivatives of stearic acid in low density lipoproteins]. / Otnositel'noe raspolozhenie antroil'nykh proizvodnykh stearinovoi kisloty v lipoproteidakh nizkoi plotnosti.

Effective quenching constants (K'sv) of 2-, 7- and 12-(9-anthroyloxy)stearic acid (n-AS) fluorescence in LDL were determined. Spin probes I(m, n) (n = 3, 7, 10, 14) and I- anions were used as quenchers. Quenching of 2-AS and 12-AS fluorescence by I(m, n) was the more effective, the deeper spin probe nitroxyl fragment was located (the greater n was); maximal K'sv value corresponded to I(1,14). By contrast, for 7-AS the quenching by I(12,3) was the most effective. 2-AS and 12-AS spectra maxima and fluorescence polarization were similar. We concluded that the 2-AS chromophore was located deeper in LDL phospholipid monolayer than chromophore of 7-AS (as was the case for 12-AS).

[Fractionation of low density lipoproteins in NaBr density gradient]. / Fraktsionirovanie lipoproteidov nizkoi plotnosti v gradiente plotnosti NaBr.

The method of atherogenic class lipoproteins fractionation by equilibrium zonal centrifugation in SW 41 Ti rotor (density range 1,006-1,063 g/ml) was proposed. Hydrated densities, flotation coefficients and mean diameters of LDL particles from different subfractions were estimated.

[Analysis of the distribution of low density lipoprotein flotation coefficients using an analytical centrifuge equipped with absorption optics]. / Analiz raspredeleniia lipoproteinov nizkoi plotnosti po koéffitsientam flotatsii s pomoshch'iu analiticheskoi ul'tratsentrifugi, osnashchennoi absorbtsionnoi optikoi.

Procedure of polydispersity determinations of low density lipoproteins (LDL) on low concentration scale (0.04%) using analytical ultracentrifuge with absorption optics was produced. No corrections for Johnston-Ogston effect and hydrostatic compressibility effect are required. Isothermal compressibility of LDL particles was estimated to be equal to 1.9 X 10(-5) Bar-1. An equation was obtained relating the flotation coefficients of LDL from different sources with solvent density and buoyant density of their particles. It was revealed that LDL particles from individual human plasma are divided into three-four subgroups having specific flotation characteristics and particular quantities of the material in these subgroups.

[Physico-chemical properties of low density lipoproteins isolated in an equilibrium density gradient]. / Fiziko-khimicheskie svoistva podfraktsii lipoproteidov nizkoi plotnosti, poluchennykh v ravnovesnom gradiente plotnosti.

The tryptophanyls of total low density lipoproteins (LDL) (1.006-1.063 g/ml) from coronary heart disease (CHD) patients and subjects without CHD signs had different accessibility to fluorescence quenchers (I-and acrylamide). LDL were separated into subfractions in equilibrium density gradient. The coefficient of extinction , quantum yield and other spectral characteristics of LDL intrinsic fluorescence, rotational mobility of maleimide spin labels and fatty acid spin probe were different in LDL subfractions from healthy subjects. LDL subfractions with hydrated density 1.045-1.05 g/ml bound to B,E-receptors of cultured fibroblasts more effectively than did subfractions with density 1.01-1.03 g/ml. Structural differences of apo-B in the particles with different lipid to protein ratio are supposed.