Oxidizability of atherogenic low-density lipoprotein subspecies in severe familial hypercholesterolemia: impact of long-term low-density lipoprotein apheresis.

BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathophysiology of atherosclerosis. LDL-apheresis, which involves direct removal of plasma LDL from circulating blood, is an efficient treatment of homozygous familial hypercholesterolemia (FH). METHODS: We evaluated impact of long-term LDL apheresis treatment on the atherogenicity of the major LDL subclasses (light, LDL1, and LDL2, density [d] 1.018-1.030 g/mL; intermediate, LDL3, d 1.030-1.040 g/mL, and dense LDL, LDL4 and LDL5, d 1.040-1.065 g/mL) separated by density gradient ultracentrifugation in severe FH patients. Therefore, we compared the oxidative resistance as well as the chemical and physical properties of each LDL subpopulation in the FH group with those in the corresponding LDL subfractions from normocholesterolemic control subjects. RESULTS: Both intermediate and dense LDL subfractions were significantly more resistant to copper-mediated oxidation in FH patients treated regularly by LDL-apheresis than their counterpart controls. The lag phases for LDL3, LDL4, and LDL5: 63.9+/-11.6, 55.8+/-1.2, and 47.2+/-6.5 min. in FH patients were significantly longer than those of the corresponding subfractions in normocholesterolemic controls (P <.01 for LDL3 and LDL5, P<.005 for LDL4). This protective effect was reflected in the delayed formation of biologically active lipid oxidation products such as oxysterols, lipid hydroperoxides, dienes, and dienals in the intermediate and dense LDL subfractions of FH patients. These findings may result from lower "seed" contents of lipid hydroperoxide (LOOH) detected as dienes in plasma LDL from apheresis-treated FH patients; indeed, baseline LOOH/diene contents in all 5 LDL subclasses from FH patients were significantly lower than those of the corresponding subclasses in normolipidemic subjects (P<.0005). On the other hand, the enhanced oxidative resistance of both intermediate (LDL3) and dense (LDL4 and LDL5) LDL subpopulations in FH patients could not be accounted for by any consistent modification in chemical composition or in lipophilic antioxidant content, although minor differences were observed between patients and controls in unsaturated fatty acid profile. In contrast, sphingomyelin content was enriched in FH LDL subclasses, potentially resulting in reduced penetration of the hydrophilic surface layer of LDL by oxygen radicals. CONCLUSION: We conclude that low concentrations of preformed lipid hydroperoxides and dienes, together with surface sphingomyelin enrichment, can account for the enhanced oxidative resistance of intermediate (LDL3) and atherogenic dense LDL (LDL4, LDL5) induced by long-term LDL apheresis in severe FH patients.

Familial lecithin:cholesterol acyltransferase deficiency: molecular analysis of a compound heterozygote: LCAT (Arg147 --> Trp) and LCAT (Tyr171 --> Stop).

Lecithin:cholesterol acyltransferase (LCAT) is responsible for the formation of the majority of plasma cholesteryl esters. Familial LCAT deficiency is associated with corneal opacity, anemia and proteinurea and typically results in renal failure in the 4-5th decade; this syndrome is equally characterized by the quasi-absence of plasma LCAT activity with variable enzyme mass and very low levels of plasma cholesteryl esters. In this study, we report detailed analyses of plasma lipids and lipoprotein profile in two sisters (CM and ML) presenting classical homozygous LCAT-deficiency; the younger sibling (CM) had proteinurea from an early age whereas the older sister (ML) has never exhibited renal dysfunction. We investigated the molecular defect in the 45 year-old woman (proband CM) exhibiting all clinical and biochemical features of familial LCAT deficiency: a plasma cholesterol level of 105 mg/dl, of which 95% was unesterified, an HDL-cholesterol of 6.5 mg/dl and an apo A-I level of 52 mg/dl. The proband (CM) displayed a plasma cholesterol esterification rate which corresponded to 2% of normal LCAT activity; plasma LCAT protein concentration was 0.56 microg/ml and equivalent to approximately 10% of normal LCAT mass. Analysis by single strand conformation polymorphism (SSCP) of the PCR products corresponding to exons 4 and 5 of the LCAT gene revealed a visible band shift. Sequence analyses of exons 4 + 5 revealed two separate single point mutations: a C --> T transition replacing Arg147 by Trp and a T --> G transition converting Tyr171 to a stop codon. The presence of these two point mutations was confirmed by restriction enzyme analyses: the C --> T transition abolished a MwoI site whereas the T --> G transition created an AvrII site. The Arg147 mutation was associated with a non-secreted protein. The Tyr171 mutation resulted in formation of a truncated protein lacking the catalytic site. In summary, we have identified an LCAT deficient patient corresponding to a compound heterozygote for the Arg147 --> Trp mutation and a new molecular defect involving a Tyr171 --> Stop mutation in the LCAT gene.

Plasma LDL and HDL subspecies are heterogenous in particle content of tocopherols and oxygenated and hydrocarbon carotenoids. Relevance to oxidative resistance and atherogenesis.

Epidemiological data indicate that dietary tocopherols and carotenoids can exert cardioprotective effects, which may be mediated by their antioxidant actions. The oxidative modification of LDL underlies the atherogenicity of these cholesterol-rich particles. The resistance of LDL to oxidation is influenced by several endogenous factors, among which the content of tocopherols and carotenoids is prominent. Of the exogenous factors, HDL inhibits oxidation of LDL via several mechanisms. In view of the paucity of data on the distribution of diverse tocopherol and carotenoid components among the apoB- and apoA-I-containing lipoproteins of human plasma, we evaluated the quantitative and qualitative features of the LDL and HDL particle subspecies in normolipidemic subjects. The bulk of tocopherols and hydrocarbon carotenoids (lycopene, alpha- and beta-carotene) was transported in LDL (45% and 76%, respectively), in contrast to the oxygenated carotenoids (lutein/zeaxanthin, canthaxanthin, and beta-cryptoxanthin), which were equally distributed between LDL and HDL. alpha-Tocopherol content was independently associated with lipid core size (cholesteryl ester and triglyceride) in VLDL, LDL, and HDL (P < .005); by contrast, the particle content of the oxygenated carotenoids lutein/zeaxanthin and canthaxanthin was strongly related to that of phospholipids. A significant and progressive decrease in the molar content of alpha- and gamma-tocopherols was found with increase in density from light to dense LDL subspecies (LDL1 to LDL5); a similar trend was observed in HDL subspecies. Furthermore, particle contents of lutein/zeaxanthin, beta-cryptoxanthin, beta-carotene, and lycopene were markedly reduced in small, dense LDL (LDL5, d = 1.050 to 1.065 g/mL). We conclude that diminished contents in such carotenoids as well as in tocopherols could underlie not only the diminished oxidative resistance of small, dense LDL but also reduced tissue targeting of antioxidants in subjects with a dense LDL phenotype.

Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.

There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.

Plasma lipoproteins in the golden Syrian hamster (Mesocricetus auratus): heterogeneity of apoB- and apoA-I-containing particles.

We present the quantitative and qualitative characteristics of the apolipoprotein (apo) B- and apoA-I-containing lipoprotein subspecies in the plasma of male Golden Syrian hamsters. The spectrum of hamster lipoproteins of d < 1.172 g/ml was subfractionated by isopycnic density gradient ultracentrifugation. ApoB-containing subspecies were distributed up to a density of 1.074 g/ml. Hamster very low density lipoproteins (VLDL, d < 1.018 g/ml; approximately 120 mg/dl plasma) were triglyceride (TG)-rich, deficient in cholesteryl ester (CE), and highly heterogeneous in size, containing chylomicron-like particles. ApoVLDL contained proteins analogous to human apoB-100, apoB-48, and apoE. ApoB-containing subspecies with physicochemical properties typical of low density lipoproteins (LDL) were identified as a single, major size species in the density interval from 1.019 to 1.074 g/ml, particle diameter decreasing progressively with increase in density. Hamster LDL-like subspecies were distinguished from their human counterparts by a relative deficiency in core CE (< 30% by wt) and by enrichment in triglyceride. The high M(r) form of apoB was the major apolipoprotein of all LDL-like subfractions, in which apoE was detected as a minor component. Total plasma levels of LDL (d 1.019-1.074 g/ml) amounted to approximately 140 mg/dl (approximately 25% of d < 1.172 g/ml lipoproteins). The distribution of dense apoB-containing subspecies overlapped that of apoA-I-containing, high density lipoprotein-1 (HDL1)-like particles in the density interval approximately 1.039-1.074 g/ml. ApoA-I-containing subspecies with the physical and chemical characteristics of HDL were exclusively present over the density interval 1.074-1.172 g/ml. Quantitatively, these subspecies predominated in hamster plasma (approximately 270 mg/dl). Light, HDL2-like particles of d 1.065-1.103 g/ml (HDLL) were preponderant, (approximately 66% of total HDL). Marked size heterogeneity was evident, and was associated with distinct particle contents of minor apolipoproteins. Both HDLL and heavy HDL (HDLH, d 1.103-1.172 g/ml) were enriched in CE as evidenced by elevated weight ratios of CE:FC (7-9:1) and of CE:TG (up to approximately 50:1). Considered together, the core lipid contents of apoB- and apoA-I-containing lipoproteins are consistent with the hypothesis that the hamster is partially deficient in neutral lipid (CE, TG) transfer activity.

Plasma lipoproteins and apolipoproteins in the preruminant calf, Bos spp: density distribution, physicochemical properties, and the in vivo evaluation of the contribution of the liver to lipoprotein homeostasis.

The in vivo role of the liver in lipoprotein homeostasis in the preruminant calf, a functional monogastric, has been evaluated. To this end, the hydrodynamic and physicochemical properties, density distribution, apolipoprotein content, and flow rates of the various lipoprotein particle species were determined in the hepatic afferent (portal vein and hepatic artery) and efferent (hepatic vein) vessels in fasting, 3-week-old male preruminant calves. Plasma lipoprotein profiles were established by physicochemical analyses of a series of subfractions isolated by isopycnic density gradient ultracentrifugation. Triglyceride-rich very low density lipoproteins (VLDL) (d less than 1.018 g/ml) were minor plasma constituents (approximately 1% or less of total d less than 1.180 g/ml lipoproteins). The major apolipoproteins of VLDL were apoB-like species, while the complement of minor components included bovine apoA-I and apoC-like peptides. Particles with diameters (193-207 A) typical of low density lipoproteins (LDL) were present over the density interval 1.026-1.076 g/ml; however, only LDL of d 1.026-1.046 g/ml were present as a unique and homogeneous size subspecies, containing the two apoB-like species as major protein components in addition to elevated cholesteryl ester contents. LDL represented approximately 10% of total d less than 1.180 g/ml lipoproteins in fasting plasma from all three hepatic vessels. Overlap in the density distribution of particles with the diameters of LDL and of high density lipoproteins (HDL) occurred in the density range from 1.046 to 1.076 g/ml; these HDL particles were 130-150 A in diameter. HDL were the major plasma particles (approximately 90% of total d less than 1.180 g/ml substances) and presented as two distinct populations which we have termed light (HDLL) and heavy (HDLH) HDL. Light HDL (d 1.060-1.091 g/ml) ranged in size from 120 to 140 A, and were distinguished by their high cholesteryl ester (29-33%) and low triglyceride (1-3%) contents; apoA-I was the principal apolipoprotein. Small amounts of apolipoproteins with Mr less than 60,000, including apoC-like peptides, were also present. Heavy HDL (d 1.091-1.180 g/ml) accounted for almost half (47%) of total calf HDL, and like HDLL, were also enriched in cholesteryl ester and apoA-I; they ranged in size from 93 to 120 A. The protein moiety of HDLH was distinct in its possession of an apoA-IV-like protein (Mr 42,000). Blood flow rates were determined by electromagnetic flowmetry, thereby permitting determination of net lipoprotein balance across the liver. VLDL were efficiently removed during passage through the liver (net uptake 1.06 mg/min per kg body weight).(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity.

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics of cysteine ethyl ester in rat.

1. The pharmacokinetics of the mucolytic compound 35S-cysteine ethyl ester in rat show that it is completely absorbed after oral administration, with a bioavailability of 0.59. 2. By autoradiography, tissue distribution of 35S-cysteine ethyl ester showed a high affinity for the lung, different to that observed with 35S-cysteine. 3. Unchanged cysteine ethyl ester, cysteine and inorganic sulphates were present in the lung. 4. After oral or i.v. administration of 35S-cysteine ethyl ester the total radioactivity excreted in the urine amounted to 16% dose and 40% was excreted in the faeces, following biliary excretion. It was metabolized to inorganic sulphate, cysteine and taurine, which were excreted in the urine.

Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation.

The molecular basis of the heterogeneity of plasma low density lipoproteins (LDL, d 1.024-1.050 g/ml) was evaluated in 40 normolipidemic male subjects following fractionation by isopycnic density gradient ultracentrifugation into eight major subspecies. The mass profile of our subjects' LDL uniformly displayed single symmetric or asymmetric peaks as a function of density; the peak occurred most frequently (20 subjects) in subfraction 7 (d 1.0297-1.0327 g/ml). Several physicochemical properties (hydrodynamic behavior, electrophoretic mobility, chemical composition, size and particle heterogeneity, and apolipoprotein heterogeneity) of the LDL subfractions were examined. Hydrodynamic analyses revealed unimodal distributions and distinct peak Sf degree rates in individual subfractions. Such behavior correlated well with particle size and heterogeneity data, in which LDL subspecies were typically resolved as unique narrow bands by gradient gel electrophoresis. Subspecies with average densities of 1.024 to 1.0409 g/ml ranged from 229 to 214 A in particle diameter. LDL protein content increased in parallel with density while the proportion of triglyceride diminished; cholesteryl esters predominated, accounting for approximately 40% or more by weight. Distinct differences in net electric charge were demonstrated by electrophoresis in agarose gel, the subspecies with average density of 1.0314 g/ml displaying the lowest net negative charge. ApoB-100 was the major apoprotein in all subspecies, and constituted the unique protein component over the density interval 1.0271-1.0393 g/ml. ApoE and apo[a] were detected at densities less than 1.0271 and greater than 1.0393 g/ml. While apoE was evenly distributed within these two regions, representing up to 2% of apoLDL, the distribution of apo[a] was skewed towards the denser region, in which it amounted to 3-7% of apoLDL. ApoC-III was detectable as a trace component at densities greater than 1.0358 g/ml. Calculation of the number of molecules of each chemical component per LDL subspecies showed the presence of one copy of apoB-100 per particle, in association with decreasing amounts of cholesteryl ester, free cholesterol, and phospholipid. These data indicate that a similar overall molecular organization and structure is maintained in a unimodal distribution of LDL particle subspecies over the density range approximately 1.02 to 1.05 g/ml. In sum, our data may be interpreted to suggest that microheterogeneity in the physicochemical properties of human LDL subspecies reflects dissimilarities in their origins, intravascular metabolism, tissular fate, and possibly in their atherogenicity.