Functional analysis of the chimpanzee and human apo(a) promoter sequences: identification of sequence variations responsible for elevated transcriptional activity in chimpanzee.

Lp(a) concentrations vary considerably among individuals and are primarily determined by the apo(a) gene locus. We have previously shown that mean plasma Lp(a) levels in the chimpanzee are significantly higher than those observed in humans (Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res 35, 263-270). To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5'-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5' sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5' of the apo(a) gene, present in a variable number of copies (n = 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the +93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position +94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5' sequence or the minimal promoter region -98 to +141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions -3, -2, and +8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.

Autosomal dominant type IIa hypercholesterolemia: evaluation of the respective contributions of LDLR and APOB gene defects as well as a third major group of defects.

Autosomal dominant type IIa hypercholesterolaemia (ADH) is characterised by an elevation of total plasma cholesterol associated with increased LDL particles. Numerous different molecular defects have been identified in the LDL receptor (LDLR) and few specific mutations in the apolipoprotein B (APOB) gene resulting in familial hypercholesterolaemia and familial defective apoB-100 respectively. To estimate the respective contribution of LDLR, APOB and other gene defects in this disease, we studied 33 well characterised French families diagnosed over at least three generations with ADH through the candidate gene approach. An estimation of the proportions performed with the HOMOG3R program showed that an LDLR gene defect was involved in approximately 50% of the families (P = 0.001). On the other hand, the estimated contribution of an APOB gene defect was only 15%. This low estimation of ADH due to an APOB gene defect is further strengthened by the existence of only two probands carrying the APOB (R3500Q) mutation in the sample. More importantly and surprisingly, 35% of the families in the sample were estimated to be linked to neither LDLR nor APOB genes. These data were confirmed by the exclusion of both genes through direct haplotyping in three families. Our results demonstrate that the relative contributions of LDLR and APOB gene defects to the disease are very different. Furthermore, our results also show that genetic heterogeneity is, generally, underestimated in ADH, and that at least three major groups of defects are involved. At this point, the contribution of the recently mapped FH3 gene to ADH cannot be assessed nor its importance in the group of 'non LDLR/non APOB' families.

New functional promoter polymorphism, CETP/-629, in cholesteryl ester transfer protein (CETP) gene related to CETP mass and high density lipoprotein cholesterol levels: role of Sp1/Sp3 in transcriptional regulation.

A new polymorphism located at position -629 (CETP/-629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The -629A allele was associated with lower CETP mass (P<0. 0001) and higher high density lipoprotein cholesterol (P<0.001) than the C allele in a sample of 536 control subjects from the ECTIM study. Transfection studies in HepG2 cells with a luciferase expression vector incorporating a 777-bp fragment of the CETP promoter and containing either A or C at position -629 showed significantly lower luciferase activity with the promoter fragment of the A allele (-25%, P<0.05). By gel-shift assay, DNA-protein interactions were evaluated in nuclear extracts of HepG2 cells with the use of 2 probes (A or C probe) composed of 20 bp of the promoter sequence surrounding the polymorphic site. Two specific complexes of distinct migration rate were identified with the A and the C probe. Competition with an excess of oligonucleotide containing the Sp1 consensus binding site showed that a protein(s) of the Sp transcription factor family was implicated in complex formation with the A probe but not with the C probe. Incubation with specific antibodies indicated that Sp1 and Sp3 bound specifically to the A probe. We introduced mutations in the -629-Sp1 binding site to test its functionality and to define the characteristics of transcription factor binding. We showed, by gel-shift assay, that no nuclear proteins bound to the mutated sequence. Transient transfection of HepG2 cells revealed that the expression of the mutated fragment was significantly increased compared with that of the A promoter fragment (25%, P<0.05). The mutated fragment displayed the same activity as that of the C promoter. These results indicate that Sp1 and/or Sp3 repress CETP promoter activity, whereas nuclear factors binding the C allele are without effect on promoter expression.

The promoter of human tissue factor pathway inhibitor gene: identification of potential regulatory elements.

Tissue factor pathway inhibitor is the major potent physiologic inhibitor of tissue factor-induced coagulation. Several potential binding sites for transcription factors have been described in the 750 bp of the 5' flanking region of the human tissue factor pathway inhibitor gene reported earlier. To identify elements that regulate the expression of tissue factor pathway inhibitor in endothelial, hepatocyte, and monocyte cells, the sequence of an additional 770 bp of tissue factor pathway inhibitor was determined. Comparison of this new sequence as well as that reported earlier with consensus sequences for transcription factor binding sites provided matches for GATA-2, SP1, and c-Myc sequences. Moreover, plasmids containing deletion mutants of the 5' tissue factor pathway inhibitor promoter region and the luciferase reporter gene were transfected into HepG2, ECV304, and THP1 cells. Three negative regulatory elements were localized between -548 to -390, - 390 to -75, and -1158 to -796 relative to the transcriptional start, respectively, in HepG2, ECV304 and THP-1 cells.

Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E-deficient mice.

BACKGROUND: To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: Infusion of apoE(-/-) mice fed a lipid-rich diet with rAd.RSV.TIMP-1 (1x10(11) viral particles) resulted in high hepatic expression of TIMP-1. At 2 weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 micrograms/mL (mean 14.8+/-6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 micrograms/mL (mean 4.3+/-2.1) at 4 weeks. Plasma lipid and lipoprotein levels in mice treated with rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.betaGal. However, rAd.RSV.TIMP-1-infused mice displayed a marked reduction (approximately 32%; P<0.05) in mean lesion area per section (512+/-121 micrometers(2)x10(3); n=12 sections from 4 animals) as compared with rAd.RSV.betaGal-infused mice (750+/-182 micrometers(2)x10(3); n=12 sections from 4 animals). Similarly, marked reduction in macrophage deposition as well as MMP-2, MMP-3, and MMP-13 antigens was observed. CONCLUSIONS: Histological and immunohistologic analyses of atherosclerotic lesions revealed increases in collagen, elastin, and smooth muscle alpha-actin content in mice treated with rAd.RSV.TIMP-1. These qualitative and quantitative features were the consequence of TIMP-1 infiltration from plasma to arterial intima, as immunohistochemical analyses revealed an abundance of TIMP-1 specifically in lesions of rAd.RSV. TIMP-1-treated mice.

Tissue factor pathway inhibitor is expressed by human monocyte-derived macrophages : relationship to tissue factor induction by cholesterol and oxidized LDL.

Lipid-laden macrophages express tissue factor (TF), which may activate the extrinsic coagulation pathway on rupture of the atherosclerotic plaque. Tissue factor pathway inhibitor (TFPI) is a major regulator of TF-induced coagulation. We evaluated the possibility that monocyte-derived macrophages express this protein, thereby contributing to regulation of TF activity (TFact). Equally, we investigated the effect of cholesterol and of oxidized LDL (Ox-LDL) on the expression of TFPI and TF by human monocyte-derived macrophages (HMDMs). Northern blot analysis of TFPI mRNA from cultured HMDMs revealed a single band at 4.2 kb with weak intensity; this finding was confirmed by reverse transcription-polymerase chain reaction. Gel filtration of HMDM supernatants showed the presence of an active 100-kDa form of TFPI, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions; under reducing conditions, however, the immunoblot revealed a 40-kDa form of TFPI. The TFPI in HMDM supernatants possessed heparin-binding affinity, suggesting potential interaction of TFPI with heparan sulfate proteoglycans. Stimulation of foam cell formation by incubation of macrophages for 48 hours with exogenous free cholesterol indicated that neither the biological activity nor the de novo synthesis of TFPI protein was affected. In contrast, cholesterol loading with exogenous free cholesterol induced significant upregulation of total TFact (2.6-fold: 25.0 versus 9.4 mU/mg cell protein, cholesterol-treated versus control cells; P<0. 05); such induction was not correlated with an elevation in TF antigen (8.5 versus 7.8 ng/mg cell protein, cholesterol-treated versus control cells). Similarly, cholesterol-rich Ox-LDL induced an increase in TFact (1.9-fold: 18.9 versus 10.0 mU/mg cell protein, Ox-LDL-treated versus control cells; P<0.05); by contrast, the amount of TF antigen remained unchanged (7.1 versus 7.9 ng/mg cell protein, Ox-LDL-treated versus control cells). Our data indicate that enhancement of the procoagulant activity of TF in macrophage-derived foam cells is not counterbalanced by upregulation of TFPI activity, suggesting that lesion foam cells are in a procoagulant state; they may therefore contribute to thrombus generation on plaque rupture.

Inhibition of LPL expression in human monocyte-derived macrophages is dependent on LDL oxidation state: a key role for lysophosphatidylcholine.

The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 microg protein per milliliter) led to marked reduction in the expression of LPL activity (-62%, P<0.01) and mRNA level (-47%, P<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (-31%, P<0.05). Among the biologically active sterols formed during LDL oxidation, only 7beta-hydroxycholesterol (5 microg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 micromol/L (-34% to -53%, P<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7beta-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte-derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.

Impact of a combination of a calcium antagonist and a beta-blocker on cell- and copper-mediated oxidation of LDL and on the accumulation and efflux of cholesterol in human macrophages and murine J774 cells.

Calcium antagonists and beta-blockers may retard or inhibit atherogenesis. In the absence of data pertaining to the potential cardioprotective action of an association of such agents, we have investigated the impact of nifedipine and atenolol, alone or in combination, on the capacity of monocyte-macrophages (ex vivo) and copper ions (in vitro) to oxidize LDL and on intracellular metabolism and efflux of free and esterified forms of cholesterol in human macrophages and foam cells. At concentrations up to 100 micromol/L, atenolol had no effect on the oxidative resistance of LDL; on the contrary, nifedipine displayed a significant dose-dependent capacity to protect LDL during copper-mediated oxidation (100 micromol/L; P<.001). Using a DPPH radical generating system, nifedipine was shown to exert free radical-trapping activity (molar ratio of scavenging activity, nifedipine:alpha-tocopherol, 1:114). The addition of atenolol to nifedipine was without effect on the antioxidant activity of the calcium antagonist. In experiments in which oxidative modification was mediated by monocyte-macrophages, nifedipine but not atenolol conserved its antioxidant capacity. Furthermore, we demonstrated that association of atenolol with nifedipine did not modify the antioxidant properties of nifedipine itself. Using a human monocyte-derived macrophage culture system, nifedipine, atenolol, or a combination of the two drugs was ineffective in inhibiting foam cell formation induced by acetylated LDL or oxidized LDL. However, atenolol (100 micromol/L) increased cellular accumulation of cholesteryl ester (+17%; P<.05), whereas nifedipine (100 micromol/L) decreased total cholesterol (-37.4%; P<.05) accumulation induced by acetylated LDL in the mouse macrophage cell line J774. A combination of the two drugs neutralized these antagonistic effects. None of these results were reproduced during the oxidized LDL-induced transformation of murine J774 cells into foam cells. Furthermore, cholesterol efflux from preloaded human macrophages was equally unaffected by the addition of the drugs alone or in combination. It therefore seems unlikely that the beneficial effect of atenolol on coronary heart disease is mediated by changes in either LDL oxidizability or cholesterol metabolism in human macrophages and foam cells. Our findings with nifedipine suggest, however, that this calcium antagonist may potentially exert antiatherosclerotic properties via a reduction of the oxidative modification of LDL, thereby affecting a reduction in foam cell formation and in the pathophysiological cellular activities of oxidized lipids, rather than by inducing a direct reduction in cholesterol accumulation in human foam cells of macrophage origin.

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.

Effect of gemfibrozil on the concentration and composition of very low density and low density lipoprotein subfractions in hypertriglyceridemic patients.

The effect of gemfibrozil treatment on very low (VLDL) and low (LDL) density lipoprotein subfractions has been investigated in 9 moderate hypertriglyceridemic (HTG) patients (triglyceride (TG) levels 237-426 mg/dl). Three VLDL subfractions, VLDL1 (Sf 175-400), VLDL2 (Sf 100-175) and VLDL3 (Sf 20-100) and 4 LDL subspecies, LDL1 (1.023-1.028 g/ml), LDL2 (1.029-1.037 g/ml), LDL3 (1.038-1.049 g/ml) and LDL4 (1.050-1.062 g/ml) were prepared by density gradient ultracentrifugation. High density lipoprotein (HDL) chemical composition and lipolytic activities after heparin injection were also determined. Gemfibrozil induced a net decrease in VLDL1 and VLDL2 concentrations (P < 0.01). Cholesteryl ester (CE) weight percent was significantly reduced in both VLDL1 and VLDL2 subfractions. VLDL3 concentration and composition were not changed by the treatment. Lipoprotein lipase (LPL) activity was reduced in HTG patients (P < 0.05). After treatment, LPL activity increased (P < 0.05) but remained lower than in control, normotriglyceridemic (NTG) subjects. Furthermore, gemfibrozil increased HDL cholesterol (P < 0.05) and normalised the elevated HDL2 and HDL3 TG content. The increase of LDL cholesterol, often observed after gemfibrozil treatment, was due to a rise in the LDL2 subfraction (1.029 < d < 1.037 g/ml), the main fraction present in control subjects. However, despite the significant decrease of total plasma TG (329 mg/dl vs. 174 mg/dl), LDL3 subfraction (1.038 < d < 1.049 g/ml) was not reduced.

Postprandial amplification of lipoprotein abnormalities in controlled type II diabetic subjects: relationship to postprandial lipemia and C-peptide/glucagon levels.

Lipoprotein abnormalities, mainly high very-low-density lipoprotein (VLDL) and low high-density lipoprotein (HDL) levels, increase the risk of coronary heart disease (CHD) in type II diabetic patients. To investigate the relationship between these lipoprotein abnormalities and the postprandial (PP) lipid-clearing capacity, triglyceride (TG) and hormonal levels were determined hourly up to the 4th hour after a mixed meal containing 32.5 g lipids/m2 body surface in 14 treated non-obese type II diabetic patients with adequate nutritional and glycemic control (hemoglobin A1C [HbA1C] < 7%) and in 12 healthy subjects matched for age, sex, and body mass index (BMI). Mean cholesterol levels did not differ between patients and controls, with fasting TG moderately increased in diabetics (140 +/- 70 v 66 +/- 34 mg/dL, P < .01). Whereas fasting TG levels in patients showed a continuous distribution from 55 to 250 mg/dL, postprandial TG clearly identified two different subgroups. A "high-responder" or hypertriglyceridemic subgroup (HTG) showed PP TG levels significantly higher than control levels (290 +/- 62 v 106 +/- 41 mg/dL, P < .001), with higher fasting TG as well (181 +/- 52, P < .01), whereas both fasting and PP TG levels were not different from control levels in the normotriglyceridemic (NTG) diabetic subgroup. The magnitude of the PP triglyceridemic area showed a negative correlation with HDL2 cholesterol (r = .66, P < .001) and a positive correlation with PP HDL2 TG enrichment (r = .80, P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic and phenotypic heterogeneity in familial lecithin: cholesterol acyltransferase (LCAT) deficiency. Six newly identified defective alleles further contribute to the structural heterogeneity in this disease.

The presence of lecithin:cholesterol acyltransferase (LCAT) deficiency in six probands from five families originating from four different countries was confirmed by the absence or near absence of LCAT activity. Also, other invariate symptoms of LCAT deficiency, a significant increase of unesterified cholesterol in plasma lipoproteins and the reduction of plasma HDL-cholesterol to levels below one-tenth of normal, were present in all probands. In the probands from two families, no mass was detectable, while in others reduced amounts of LCAT mass indicated the presence of a functionally inactive protein. Sequence analysis identified homozygous missense or nonsense mutations in four probands. Two probands from one family both were found to be compound heterozygotes for a missense mutation and for a single base insertion causing a reading frame-shift. Subsequent family analyses were carried out using mutagenic primers for carrier identification. LCAT activity and LCAT mass in 23 genotypic heterozygotes were approximately half normal and clearly distinct from those of 20 unaffected family members. In the homozygous patients no obvious relationship between residual LCAT activity and the clinical phenotype was seen. The observation that the molecular defects in LCAT deficiency are dispersed in different regions of the enzyme suggests the existence of several functionally important structural domains in this enzyme.

A multicenter comparison of the effects of simvastatin and fenofibrate therapy in severe primary hypercholesterolemia, with particular emphasis on lipoproteins defined by their apolipoprotein composition.

This multicenter, double-blind, randomized study was designed to compare the effects of simvastatin (20 mg/d and 40 mg/d) and fenofibrate (400 mg/d) on plasma lipids, lipoproteins, apolipoproteins (apo), and lipoprotein particles defined by their apo composition (Lp A-I, Lp A-II:A-I, Lp E:B, Lp C-III:B) in primary hypercholesterolemia. After 6 and 10 weeks of therapy, both drugs lowered plasma cholesterol, low-density lipoprotein (LDL) cholesterol, and apo B. The effect on LDL and apo B was significantly more pronounced for simvastatin (P = .01). Simvastatin increased Lp A-I, but did not change Lp A-II:A-I, while fenofibrate decreased Lp A-I and increased Lp A-II:A-I. Lp E:B and Lp C-III:B were decreased with both drugs, but fenofibrate was significantly more effective in reducing these particles than simvastatin. This study demonstrates that both drugs have beneficial effects on the parameters positively or negatively correlated with the atherosclerotic risk, with simvastatin being more effective in reducing some of them. These results suggest that the drugs led to different structural modifications of the lipoproteins, which would not be revealed by examination of lipoprotein density classes. These differences are probably related to the different mechanisms of action of the agents.

Lipoprotein particle analysis comparing simvastatin and fenofibrate.

This study compares the effects of fenofibrate and simvastatin in primary hypercholesterolemia, with particular regard to lipoprotein particles, as defined by their apolipoprotein composition: LpAI, LpAII: AI, LpE:B, LpCIII:B. This was a double-blind study in which patients were randomized to 2 groups, one receiving simvastatin 20 mg once daily and the other receiving fenofibrate 200 mg b.i.d., if their total cholesterol and their LDL cholesterol remained above 7.60 mmol/l (300 mg/dl) and 4.95 mmol/l (195 mg/dl) after a 4-week placebo period. Simvastatin dosage was doubled at the end of 6 weeks of therapy if the LDL-cholesterol level remained above 3.55 mmol/l (140 mg/dl). Analyses were done after 6 and 10 weeks of therapy. Apolipoprotein AI was increased significantly only at week 10 with fenofibrate (+7.4%). Simvastatin had a more pronounced effect than fenofibrate on apolipoprotein B. There was a significant difference between drugs at weeks 6 and 10. No change was observed in the LpAII:AI level with simvastatin, whereas fenofibrate increased these particles quite significantly (+13.9 and +22.3%). The drugs had opposite effects on LpAI (+2.5 and +5.6% with simvastatin; -12.8 and -15.1% with fenofibrate). LP E:B (-33.0 and -40.8% with simvastatin; -53.8 and -52.2% with fenofibrate) and LpCIII:B (-23.8 and -31.8% with simvastatin; -35.1 and -43.5% with fenofibrate) were decreased by both drugs, but fenofibrate was significantly more effective in reducing these particles than simvastatin at week 6. This study suggests that both drugs led to different structural modifications of the lipoproteins, which would not be revealed by total apolipoprotein analysis. These differences are probably related to the mechanisms of action of these drugs.

[Determination of lipoprotein Lp(a). A semi-automatic immuno-turbidimetric method]. / Dosage de la lipoprotéine Lp(a). Une méthode immuno-turbidimétrique semi-automatique.

A sensitive, accurate, semiautomatic immunoturbidimetric assay for determining Lp(a) levels using a Hitachi 705 apparatus is described. A polyclonal antibody against anti-Lp(a) produced by Immuno-france is used. Studies proved that this test is reliable and that results are well correlated with those obtained by immunonephelometry.

[The efficacy and tolerance of simvastatin and fenofibrate in primary hypercholesterolemia]. / Efficacité et tolérance de la simvastatine et du fénofibrate dans l'hypercholestérolémie primaire.

The effects of simvastatin and fenofibrate on blood lipids and the side-effects of these two drugs were compared in a double-blind trial involving 184 adults with primary hypercholesterolemia (total cholesterol levels: 3.87 +/- 1.02 g/l in the simvastatin group, 3.78 +/- 0.79 g/l in the fenofibrate group; N.S.). During a 10-week period, the patients received either fenofibrate 200 mg twice daily or simvastatin 20 mg once daily with doubling of the dosage at week 6 if the LDL-cholesterol level remained above 1.40 g/l. Simvastatin significantly (P less than 0.01) reduced total cholesterol by -29.9 percent, LDL-cholesterol by -35.4 percent, apoprotein B by -27.3 percent and triglycerides by -16.7 percent. Fenofibrate significantly (P less than 0.01) reduced total cholesterol by -19.2 percent, LDL-cholesterol by -22.3 percent apoprotein B by 13.9 percent and triglycerides by 28.9 percent. Reduction of the first 3 parameters was significantly (P less than 0.01) greater with simvastatin and reduction of triglycerides significantly greater with fenofibrate. Apoprotein A1 levels were increased by fenofibrate (+ 7.4 percent) but not by simvastatin. Side-effects occurred with a frequency of 6 percent under simvastatin and 9 percent under fenofibrate.

Plasma lipids, lipoproteins and apolipoproteins in two kindreds of hypobetalipoproteinemia.

Plasma lipids and apolipoproteins were quantified in two kindreds of hypobetalipoproteinemia. All affected members were asymptomatic but showed a decrease of 75% in apolipoprotein B and of 69% in LDL-cholesterol. There were no major changes in apo A-I and A-II but all affected family members had reduced levels of apo C-II (by 58%) and C-III (by 59%) without significant decrease in apo C-I and no specific decrease of apo C-III1. Apolipoprotein E is decreased in SDS-PAGE. The plasma level and phenotype of Lp(a) are not affected by HBL, suggesting that a catabolic rather than a synthetic mechanism is responsible for the disease. As shown by density gradient ultracentrifugation, HDL2 particles that contain essentially apolipoprotein A-I, cholesterol and phospholipids represent in affected subjects the major part of HDL. Due to the net reduction of apolipoprotein B-containing particles (VLDL and LDL) as acceptors of lipids in HBL, there is an accumulation of large particles rich in cholesteryl esters.

Fluidity changes and chemical composition of lipoproteins in type IIa hyperlipoproteinemia.

The chemical composition and the physical properties of lipoproteins (VLDL, LDL and HDL) were studied in two groups of patients: 14 healthy normolipidemic subjects and 15 type IIa familial hypercholesterolemic patients. The steady-state fluorescence anisotropy rs was estimated in lipoproteins by the fluorescence depolarization of two fluorescent probes: the DPH (1,6-diphenyl-1,3,5-hexatriene) and the TMA-DPH (1,4-trimethylammonium phenyl-6-1,3,5-hexatriene). A structured order parameter S was calculated from the DPH fluorescence anisotropy. The flow activation energies were calculated for LDL and HDL from both groups from the Arrhenius plots (log r DPH versus 1/T). By using TNBS (trinitrobenzene sulfonic acid) as a distance control quencher, the two probes were located in the outer shell of LDL. In HDL, TMA-DPH remained at the surface of the particles, while DPH was more deeply embedded in the lipid core. There was no difference in the physico-chemical properties of VLDL between the two groups studied. DPH fluorescence anisotropies were significantly increased in LDL and HDL from the hypercholesterolemic group compared to the control particles (P less than 0.05 and P less than 0.01, respectively). In LDL this modification of the fluorescence anisotropy can be related to a change in the lipid composition of particles. LDL from hypercholesterolemic patients contained significantly less triacylglycerol (P less than 0.01) and more cholesteryl ester (N.S.). Their cholesteryl ester to triacylglycerol ratio was significantly higher. In HDL, there was no difference in chemical composition between the two groups. The increase in DPH fluorescence anisotropy can be related to the presence of smaller particles in HDL from HC group. No difference was noted in the TMA-DPH fluorescence anisotropy at 37 degrees C in the LDL from the two groups. In contrast, TMA-DPH fluorescence anisotropy in HDL from hypercholesterolemic group was significantly higher than in control HDL. The flow activation energy of DPH was also significantly higher in both LDL and HDL from the hypercholesterolemic group than in control group particles. In both LDL and HDL from the control group, DPH fluorescence anisotropy was negatively correlated with TG/protein and TG/PL ratios and positively correlated with the CE/TG ratio. No correlation was observed between lipid composition and DPH fluorescence anisotropy values in hypercholesterolemic particles. The modification in fluidity parameters, especially the increase in the flow activation energies in LDL and HDL from hypercholesterolemic patients, could lead to a restriction of cholesterol movements in these particles. From a physiological point of view, this could represent a loss of functional capacity.

Comparative study of the precipitation of low density lipoproteins by aortic proteodermatan sulphate and heparin.

The aortic proteoglycans and heparin were shown to form insoluble complexes with human low density lipoproteins (LDL). The effect of temperature, polyethylene glycol and ionic strength on the formation of complexes between porcine aortic proteodermatan sulphate (PDS) and LDL has been studied by laser nephelometry and comparisons made with heparin LDL complexes. Turbidity was a nonlinear function of the quantity of LDL precipitated by PDS. The turbidity of aggregates was constant at temperatures between 2 degrees C and 30 degrees C but increased with temperature above 30 degrees C up to 50 degrees C. The formation of insoluble complexes decreased rapidly with increasing NaCl concentration. Polyethylene glycol enhanced the turbidity at 20 degrees C but not at 37 degrees C. It also increased the resistance of complexes to dissociation by increasing ionic strength. The turbidity of heparin--LDL complexes was linearly correlated with the quantity of precipitated LDL. The heparin-LDL aggregates were less sensitive to modification of temperature and ionic strength than the PDS-LDL aggregates. These results suggest that ionic interactions are weaker in PDS-LDL complexes than in the heparin-LDL complexes. Non-coulombic interactions and/or temperature dependent conformational changes may be involved in the stabilization of supramolecular PDS-LDL aggregates. No such interactions or changes appear to be involved in complex formation between heparin and LDL.

Efficacy and safety of simvastatin (alone or in association with cholestyramine). A 1-year study in 66 patients with type II hyperlipoproteinaemia.

The effects and safety of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, were investigated alone or in association with cholestyramine in 66 patients with hypercholesterolaemia, in a 1-year study. In type IIa hypercholesterolaemia (41 patients), the association was more effective than simvastatin used alone in lowering total cholesterol (37% vs 29%) and LDL-cholesterol (45% vs 37%). In type IIb hypercholesterolaemia (23 patients), the association simvastatin-cholestyramine did not appear more effective than simvastatin used alone. The decrease of apoprotein B was parallel to the LDL-cholesterol decrease. Apoprotein A1 did not change significantly. The long-term safety of simvastatin was good. No lens opacity was noted. The most serious side-effect in our study was myolysis which occurred in two patients with a marked increase in creatine phosphokinase.