Physico-chemical properties of copper-oxidized very low density lipoproteins

The aim of our study was to investigate the effect of Cu2+ catalyzed oxidation on VLDL physico-chemical properties and secondary structure of apo B-100. Incubation of very low density lipoproteins with copper ions resulted in a decrease in tryptophan and lysine residues parallel to lipid peroxidation products, conjugated dienes and TBARS. Fluorescence polarization showed an increase in the molecular order at the lipoprotein surface of VLDL, as demonstrated by the increase in Pf values of DPH. The secondary structure of apo B-100 was investigated by infrared spectroscopy. Increased order and structural changes, as observed after oxidative stress on VLDL, could be of relevance in the abnormal interactions between lipoproteins and cell membranes.     

Regulation of the assembly and secretion of very low density lipoproteins by the liver

Recent studies have suggested that there are three sites at which VLDL secretion by the liver may be controlled: (i) Newly synthesised apo-B either remains associated with the RER membrane and is degraded by the ubiquitin/proteasome system, or is translocated into the lumen and incorporated into lipid poor VLDL precursors; (ii) the lumenal apo-B is either degraded or moves on, and (iii) acquires the remaining VLDL lipids in the SER/cis-Golgi. Newly synthesised apo-B, at the cytosolic side of the RER, is stabilised and protected from degradation by the chaperone protein, hsp-70. Triacylglycerol, cholesterol ester and phospholipids have all been implicated in the translocation of apo-B and microsomal triglyceride protein plays a major role. If translocation does not occur then the apo-B is degraded. Dietary fish-oils, but not sunflower oil, inhibit movement of apo-B containing precursors from the RER and their assembly with lipids and target lumenal apo-B to degradation. This effect is reversed by inhibition of lumenal proteolysis, but not by inhibition of cytosolic proteolysis. Therefore lumenal degradation of apo-B and secretion appear to be in balance, so that if assembly of VLDL precursors is slowed, then degradation becomes predominant. If however, degradation is inhibited then VLDL assembly can proceed. These observations suggest that movement of VLDL precursors from the RER lumen to the second stage of assembly may be a further regulated step.     

Lipid metabolism in pregnancy. IV. C Apoprotein changes in very low and intermediate density lipoproteins

In vitro studies indicate that relative amounts of apolipoproteins CII and CIII in plasma may alter lipoprotein lipase mediated triglyceride removal from very low density lipoprotein. Therefore, we have determined relative amounts of these peptides in pregnant women in late gestation (mean 36 weeks) and 6 and 20 weeks postpartum. Very low density (d less than 1.006) and intermediate density (d 1.006-1.019) lipoproteins have been examined because they are affected similarly in pregnancy. Triglyceride and cholesterol in these two fractions increased 3 1/2 to 4 1/2-fold in pregnancy in keeping with previous reports. On a relative basis, apo CII was decreased and apo CIII1 and CIII2 increased in both VLDL and IDL at 36 weeks gestation. At 6 and 20 weeks postpartum relative amounts of C peptides returned to identical levels and were unaffected by lactation. It remains to be seen if in vivo triglyceride removal in pregnancy is altered by the reduction in apo CII relative to apo CIII.     

Expression of monocyte chemoattractant protein-1 in monocytes and effects of native and oxidized very low density lipoproteins

Rheumatoid neutrophilic dermatitis (RND) is a rare cutaneous finding in patients with severe rheumatoid arthritis or with high-titer rheumatoid factors. Most commonly, these lesions are erythematous papules or plaques distributed symmetrically on extensor surfaces. On histologic examination a dense dermal neutrophilic infiltrate without vasculitis is apparent. The pathogenesis of RND is unclear, and few treatments are known. With careful clinical and histologic examination, RND may be differentiated from the wide array of other cutaneous findings in rheumatoid arthritis.     

Re-evaluation of the structure of low density lipoproteins

Low density lipoprotein (LDL) is an established atherogenic factor. Much effort has therefore been devoted to elucidation of its structure, yielding the generally accepted model according to which the neutral lipids (cholesterol ester and triglycerides) form a lipid core emulsified by phospholipids, cholesterol and the amphipathic Apolipoprotein B. Yet, the detailed structure of LDL is not clear. The present work was carried out with the aim of re-evaluating the LDL structure using the minimal number of assumptions: in view of the previously noted surface deficit (lack of sufficient PL and cholesterol to cover the surface of the lipid core) we have assumed that polar head groups are not covered by apo B. Other than that, we have 'allowed' Apo B to penetrate into the PL monolayers and the lipidic core and to pertrude into the solution (be elevated above the PL head group level). We have also 'allowed' neutral lipid penetration into the monolayer and variation of the thickness of the phospholipid monolayers within reasonable boundaries. Based on the established values of relevant constants (molecular weights and volumes, densities and surface areas) we have computed the radius of the particle, the penetration of Apo B into lipidic milieus and the fraction of the surface area covered by Apo B as functions of the LDL composition, the monolayer thickness and the 'elevation' of Apo B above this monolayer. These computations show that at least 40% of the LDL surface must be covered by protein and that the protein penetrates, on the average, only about a half of the PL monolayer. Thus it is not very likely to penetrate into the lipid core. These general features are preserved in the smaller LDL particles of hypertriglyceridemic patients. Assuming that no PL head group is covered by Apo B, the previously described immobilization of 20% of the phospholipids is likely to result from the interaction of Apo B with neighboring PL. According to our computations this can be regarded consistent with the previously proposed arrangement of the apo B as a '3-4 domain structure' or a long string configuration but inconsistent with 'one domain' or 'twenty domain' structures.     

Effects of low dose oral contraceptives on very low density and low density lipoprotein metabolism

Oral contraceptives (OC) raise plasma triglyceride and VLDL levels, which may be of concern, since some conditions characterized by elevated triglycerides are associated with atherosclerosis. To identify the responsible mechanism, we studied 11 healthy premenopausal women, 5 of whom were taking OC containing 0.035 mg ethinyl estradiol, and 6 of whom were not. Their rates of VLDL and LDL metabolism were measured by endogenously labeling apoB, the protein component of VLDL and LDL, by an intravenous infusion of deuterated leucine. OC use had the greatest effect on the large, triglyceride-rich VLDL subfraction (Sf 60-400), increasing plasma levels threefold and production rates fivefold (P < 0.05). Among OC users, small VLDL (Sf 20-60) levels were 2.2 times higher, and production rates were 3.4-fold higher (P < 0.05). The fractional catabolic rates of large and small VLDL were similar among OC users and nonusers. LDL levels and metabolic rates were not significantly different between the two groups. Thus, contemporary low dose OC substantially raise VLDL levels by increasing the production rate of large, triglyceride-rich VLDL, and not by slowing VLDL catabolism. Since VLDL catabolism is not impaired, we speculate that the hypertriglyceridemia induced by OC may be less atherogenic than that of hypertriglyceridemia resulting from impaired lipolysis. This may explain why long-term OC use does not appear to promote atherosclerosis.     

Transient triglyceridemia in healthy normolipidemic men increases cellular processing of large very low density lipoproteins by fibroblasts in vitro

Exaggerated and prolonged postprandial triglyceridemia is a characteristic of patients with precocious coronary heart disease. Although large very low density lipoprotein (VLDL) particles accumulate during alimentary lipemia, the biological properties of the postprandial VLDL remain unknown. In the present study, an intravenous infusion of a chylomicron-like emulsion was given to healthy normolipidemic men to examine the effects of transient triglyceridemia in vivo on compositional and cell biological characteristics of VLDL. The postinfusion large(Svedberg flotation rate (Sf) (60-400) VLDL was found to have increased capacity to inhibit low density lipoprotein (LDL) binding to the LDL-receptor and a greater ability to suppress the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity of cultured fibroblasts compared to VLDL isolated from fasting plasma. These alterations in cellular interactions were accompanied by increases in the number of apolipoprotein (apo) E, C-I, and C-III molecules per large VLDL particle and loss of apoC-II, compositional changes similar to those observed after an oral fat load. The increase in number of apoE molecules per large VLDL particle correlated positively and significantly with the increase in the capacity of large VLDL to inhibit LDL binding to the LDL receptor (r = 0.76, P = 0.01, n = 10). In contrast, the composition of the small (Sf 20-60) VLDL particles did not change significantly, nor was the LDL receptor-mediated processing of these particles altered consistently. These observations indicate that large VLDL particles that accumulate during alimentary lipemia undergo compositional changes that render them more prone to cellular binding and uptake.     

Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 +/- 5.9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.     

Diurnal heterogeneity in structure and function of low density lipoproteins of normolipidemic males

Structural changes in low density lipoproteins (LDL) have been shown to alter their metabolism and atherogenic potential. We investigated the diurnal changes in size and composition of LDL in seven healthy, non-obese, normolipidemic male volunteers consuming a standard diet (14.5% protein, 31.9% fat, 53.6% carbohydrate and 383 mg cholesterol/day) and continuing their daily routine. The food was divided into three meals and three snacks, and blood samples were obtained at 7 AM (after 12 h fasting), noon, 8 PM, midnight and 3 AM. LDL were isolated by both sequential and density gradient ultracentrifugation (d = 1.019 - 1.050 g/ml), and analyzed for lipids, apolipoproteins, size, and affinity to LDL receptors. Diurnal LDL preparations differ from fasting LDL in both chemical and physical parameters. The former get richer in triglyceride (TG/cholesterol weight ratio 0.23 vs. 0.16), larger in diameter (21.2 +/- 0.2 vs. 22.4 +/- 0.1 nm), and enriched in a more buoyant fraction (74.0 +/- 4.6 vs. 41.9 +/- 3.8% of LDL cholesterol in d = 1.019 - 1.035 g/ml). These structural changes in LDL were associated with enhanced affinity to LDL receptors in both human skin fibroblasts and HepG2 cells, as demonstrated by competition experiments with fasting human 125I-LDL. The observed diurnal heterogeneity in both the structure and the function of LDL may be attributed to the absorptive state as it did not occur during prolonged fasting. These diurnal changes may be important for better understanding LDL metabolism in vivo and for the elucidation of the atherogenic process.     

Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation

Given the multiple impairments in host defense that occur during HIV infection, patients with AIDS are at risk for a variety of pleural infections and neoplasms. Of infectious causes, bacterial parapneumonic effusions and empyemas and tuberculous pleurisy occur more frequently than effusions caused by P. carinii. In each case, therapy is directed at eradication of the causative organisms. In the setting of systemic Kaposi's sarcoma, pleural involvement is common, although diagnosis is difficult and therapeutic options are limited. Pleural effusions caused by non-Hodgkin's lymphoma often occur in the setting of pulmonary parenchymal disease and can be diagnosed cytologically. The recently described entity of primary effusion lymphoma occurs in the absence of solid-organ involvement. The development of a spontaneous pneumothorax in a HIV-infected individual should prompt a search for P. carinii infection. Although these pneumothoraces often recur and are difficult to manage, recent series suggest that surgical approaches to bronchopleural fistulas are reasonable in selected patients.     

Effect of metal cations on the copper induced peroxidation of the low density lipoproteins

The effect of metal cations on copper-catalyzed lipid peroxidation (LPO) of low density lipoproteins (LDL) was examined. The presence of metal cations in the incubation media containing LDL (0.8 mg protein/ml) and CuSO4 (0-80 microM) influenced on LPO of LDL as evident by the measurement of TBARS. With the concentrations of CuSO4 less than 10 microM, the metal cations caused an increase in LDL peroxidation. Zn2+ appeared to be the most effective inductor, Mn2+ was less effective, and the influence of Ca2+ and Mg2+ was insignificant. With greater CuSO4 concentrations Mg2+ showed no effect on TBARS formation in LDL while the addition of other nontransition metal cations to the incubation mixture led to the inhibition of LDL peroxidation. The capacity for inhibition decreased in the row Mn2+ > Zn2+ > Ca2+ > Mg2+. The possible mechanism explaining these results may be in the competition of metal ions for copper binding sites on LDL. Our results allow to suggest the existence of two types of copper binding sites on LDL, tight-binding sites which are non-effective in LPO and effective weak-binding sites.     

Linoleic acid intake and susceptibility of very-low-density and low density lipoproteins to oxidation in men

Lipoprotein peroxidation is thought to play an important role in atherogenesis. In the Kuopio Atherosclerosis Prevention Study (KAPS) the intake of fat and fatty acids, the oxidation susceptibility of the plasma very-low-density + low-density lipoprotein (VLDL+LDL) fraction (by induction with copper or hemin and hydrogen peroxide), and concentrations of plasma antioxidants, serum lipids, and lipoproteins were measured in 393 men. In the multivariate-regression model dietary linoleic acid was the most important determinant of the maximal oxidation velocity for the hemin assay (standardized regression coefficient = 0.294, P<0.0001). In the copper assay the association of dietary linoleic acid and maximal oxidation velocity was second in order of strength (standardized regression coefficient = 0.324, P< 0.0001). We conclude that high linoleic acid intake is associated with increased oxidation susceptibility of atherogenic lipoproteins in men.     

Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins

Lipoprotein peroxidation, especially the modification of apolipoprotein B-100, has been implicated to play an important role in the pathogenesis of atherosclerosis. However, there have been few detailed insights into the chemical mechanism of derivatization of apolipoproteins during oxidation. In the present study, we provide evidence that the formation of the toxic pollutant acrolein (CH2=CH-CHO) and its conjugate with lysine residues is involved in the oxidative modification of human low density lipoprotein (LDL). Upon incubation with LDL, acrolein preferentially reacted with lysine residues. To determine the structure of acrolein-lysine adduct in protein, the reaction of acrolein with a lysine derivative was carried out. Employing Nalpha-acetyllysine, we detected a single product, which was identified to be a novel acrolein-lysine adduct, Nalpha-acetyl-Nepsilon-(3-formyl-3,4-dehydropiperidino )lysine. The acid hydrolysis of the adduct led to the derivative that was detectable with amino acid analysis. It was revealed that, upon in vitro incubation of LDL with acrolein, the lysine residues that had disappeared were partially recovered by Nepsilon-(3-formyl-3, 4-dehydropiperidino)lysine. In addition, we found that the same derivative was detected in the oxidatively modified LDL with Cu2+ and that the adduct formation was correlated with LDL peroxidation assessed by the consumption of alpha-tocopherol and cholesteryl ester and the concomitant formation of cholesteryl ester hydroperoxide. Enzyme-linked immunosorbent assay that measures free acrolein revealed that a considerable amount of acrolein was released from the Cu2+-oxidized LDL. Furthermore, metal-catalyzed oxidation of arachidonate was associated with the formation of acrolein, indicating that polyunsaturated fatty acids including arachidonate represent potential sources of acrolein generated during the peroxidation of LDL. These results indicate that acrolein is not just a pollutant but also a lipid peroxidation product that could be ubiquitously generated in biological systems.     

Effect of L-carnitine treatment on very low density lipoprotein kinetics in the hyperlipidemic rabbit

This study examined the hypolipidemic effect of 4 weeks of L-carnitine treatment (170 mg/kg b.w./day) in New Zealand White rabbits fed a high fat diet (5% corn oil/0.5% cholesterol). Specifically, [3H] glycerol and [125I] very low density lipoprotein (VLDL) turnover studies were conducted to examine the effect of treatment on VLDL kinetics. The masses of plasma VLDL-triglycerides (VLDL-TG) and VLDL-apoprotein B (VLDL-apoB) were significantly increased by the high-fat diet. Four weeks of treatment with L-carnitine significantly reduced these masses. Kinetic analysis indicated that fat feeding reduced the fractional catabolic rates (FCRs) of VLDL-TG and VLDL-apoB relative to chow-fed controls. The transport of these VLDL components was not altered by the diet. L-carnitine treatment had no effect on the FCRs of VLDL-TG and VLDL-apoB or on the transport of VLDL-apoB. Yet, treatment significantly lowered the transport of VLDL-TG. These data indicate that the lipid-lowering effect of L-carnitine in this animal model was due, in part, to a decrease in the transport and not due to an alteration in the fractional catabolic rate of VLDL-TG.     

Uptake of hypertriglyceridemic very low density lipoproteins and their remnants by HepG2 cells: the role of lipoprotein lipase, hepatic triglyceride lipase, and cell surface proteoglycans

Hypertriglyceridemic very low density lipoproteins (HTG-VLDL, S(f) 60-400) are not taken up by HepG2 cells. However, addition of bovine milk lipoprotein lipase (LPL) at physiological concentrations markedly stimulates uptake. In the present study, we determined whether: a) LPL catalytic activity is required for uptake, b) LPL functions as a ligand, and c) cell surface hepatic triglyceride lipase (HL) and/or proteoglycans are involved. Incubation of HepG2 cells with HTG-VLDL plus LPL (8 ng/ml) increased cellular cholesteryl ester (CE) 3.5-fold and triglyceride (TG) 6-fold. Heat-inactivation of LPL abolished the effect. Addition of tetrahydrolipstatin (THL, an LPL active-site inhibitor) to HTG-VLDL + LPL, inhibited the cellular increase in both CE and TG by greater than 90%. Co-incubation of HTG-VLDL + LPL with heparin, heparinase, or heparitinase, blocked CE accumulation by 70%, 48%, and 95%, respectively, but had no effect on the increase in cellular TG. Pre-treatment of cells with 1 mM 4-methylumbelliferyl-beta-D-xyloside, (beta-xyloside) to reduce cell surface proteoglycans inhibited the increase in CE induced by HTG-VLDL + LPL by 78%. HTG-VLDL remnants, prepared in vitro and isolated free of LPL activity, stimulated HepG2 cell CE 2.8-fold in the absence of added LPL, a process inhibited with THL by 66%. Addition of LPL (8 ng/ml) to remnants did not further enhance CE accumulation. HepG2 cell HL activity, released by heparin, was inhibited 95% by THL. The amount of HL activity and immunoreactive mass, released by heparin, was reduced 50-60% in beta-xyloside-treated cells. These results indicate that physiological concentrations of LPL promote HepG2 cell uptake of HTG-VLDL primarily due to remnant formation and that LPL does not play a major role as a ligand. HL activity and cell surface proteoglycans significantly enhance the subsequent uptake of VLDL remnants.     

A composite regulatory element in the first intron of the estrogen-responsive very low density apolipoprotein II gene

Over 12 years, from 1984 to 1995, we conducted a prospective study of overall and malaria specific mortality among three rural populations in the Sahel, savanna and forest areas of Senegal. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality in each of the studied populations. After the emergence of chloroquine resistance, the risk of malaria death among children 0-9 years old in the three populations was multiplied by 2.1, 2.5 and 5.5, respectively. This is the first study to document malaria mortality at the community level in Africa before and after the emergence of chloroquine resistance. Findings suggest that the spread of chloroquine resistance has had a dramatic impact on the level of malaria mortality in most epidemiological contexts in tropical Africa.     

Susceptibility to in vitro lipid peroxidation of low density lipoproteins and erythrocyte membranes from liver cirrhotic patients

All retinoic acid receptors (RAR alpha, beta, gamma) have two isoforms, whose function is unknown. We now show that at least for RAR gamma, the isoforms are differentially distributed in the embryo. RAR gamma 1 and RAR gamma 2 are detected in the head region, whereas RAR gamma 2 is the sole isoform expressed in the tail region. Specifically, it is expressed in the chordoneural hinge, a region of the tailbud that has organizing properties. Treatment with high doses of retinoic acid (RA) reduces expression in this region. The results are discussed in terms of the known teratogenic effects of RA in the tail region.