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Scavenger receptor SR-BI has been implicated in HDL-dependent atheroprotective mechanisms. We report the generation of an SR-BI conditional knockout mouse model in which SR-BI gene targeting by loxP site insertion produced a hypomorphic allele (hypomSR-BI). Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-fold elevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BI gene inactivation of the hypomorphic SR-BI allele in hepatocytes (hypomSR-BI-KO(liver)) was associated with high plasma TC concentrations, increased plasma free cholesterol/TC (FC/TC) ratio, and a lipoprotein-cholesterol profile typical of SR-BI-/- mice. Plasma TC levels were increased 2-fold in hypomSR-BI and control mice fed an atherogenic diet, whereas hypomSR-BI-KO(liver) and SR-BI-/- mice developed severe hypercholesterolemia due to accumulation of FC-rich, VLDL-sized particles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold) compared with that in controls, but to a much lower degree than in hypomSR-BI-KO(liver) (32-fold) and SR-BI-/- (48-fold) mice. The latter models did not differ in either plasma lipid levels or in the capacity of VLDL-sized lipoproteins to induce macrophage cholesterol loading. However, reduced atherosclerosis in hypomSR-BI-KO(liver) mice was associated with decreased lesional macrophage content as compared with that in SR-BI-/- mice. These data imply that, in addition to its major atheroprotective role in liver, SR-BI may exert an antiatherogenic role in extrahepatic tissues.

The plasma cholesteryl ester transfer protein (CETP) plays a key role in reverse cholesterol transport (RCT) by mediating the transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to atherogenic ApoB-containing lipoproteins, including VLDL, IDL and LDL. We describe a new polymorphism located at position −971 in the human CETP gene promoter, which corresponds to a G/A substitution at a potential Ava I restriction site. The relationship between the −971G/A polymorphism, plasma lipid parameters and plasma CETP concentration was evaluated in the Etude Cas-Temoins de l'Infarctus du Myocarde (control–myocardial infarction cases) cohort, and revealed that the −971G/A polymorphism (A allele frequency: 0.491) was significantly associated with both plasma high-density lipoprotein cholesterol (HDL-C) levels and CETP concentration ( P =0.006 and 0.009, respectively). Subjects with genotype −971GG displayed both low HDL-C levels and high plasma CETP concentration, while genotype −971AA subjects displayed the inverse relationship. Evaluation of potential interactions between the −971G/A and the −629C/A or TaqIB polymorphisms demonstrated that the −971G/A polymorphism interacts significantly with the functional −629C/A site and the TaqIB polymorphism with respect to plasma HDL-C levels ( P =0.0014 and 0.012, respectively), but does not affect plasma CETP concentration. These results clearly suggest that the interaction between the 971G/A polymorphism and either the −629C/A or the TaqIB polymorphism on plasma CETP concentration is different than that implicated in HDL-C levels. Transient transfection of HepG2 cells revealed that the −971G/A polymorphism did not modulate transcriptional activity of the human CETP gene promoter. The −971G/A promoter polymorphism therefore constitutes a non-functional marker. Furthermore, the observed effects of the −971G/A polymorphism on both plasma CETP concentration and HDL-C levels are due to functional variants in linkage disequilibrium with it. Our findings strongly suggest the existence of as yet unidentified functional polymorphisms in the CETP gene promoter that could explain the association between specific polymorphisms of the CETP gene and both plasma HDL-C and CETP concentrations.

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 theapo(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 chimpanzeeapo(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.

Abstract —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 P P P

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.

Abstract —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 P

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.

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.

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.

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 CT 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.

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.

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 (1×10 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 μg/mL (mean 14.8±6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 μg/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.βGal. However, rAd.RSV.TIMP-1–infused mice displayed a marked reduction (≈32%; P 2 ×10 3 ; n=12 sections from 4 animals) as compared with rAd.RSV.βGal-infused mice (750±182 μm 2 ×10 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 α-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.

Abstract —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 μg protein per milliliter) led to marked reduction in the expression of LPL activity (−62%, P P P P 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 (

Abstract Calcium antagonists and β-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 μmol/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 μmol/L; P P P

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 subtractions. 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.

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 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). A negative correlation was evidenced between the glucagon/C-peptide molar ratio and total or incremental TG areas (r = −.62, P < .01 and r = −.74, P < .001, respectively). No additional correlations were found in diabetic patients between any of these variables and glycemic control. Only HTG patients showed increased total and incremental PP TG areas when compared with controls, as well as associated abnormalities in lipoprotein levels and composition. They showed higher VLDL, intermediate-density lipoprotein (IDL), and apolipoprotein (apo) B levels, together with decreased HDL2. In the postprandial state, they presented higher chylomicron levels and significant TG enrichment within HDL2. Amplification of lipoprotein changes already present at fasting also occurred, mainly a significant increase in VLDL levels together with a decrease in the HDL2 cholesteryl ester (CE)TG ratio, which suggests increased net lipid transfer between these lipoproteins. These findings indicate that non-obese treated diabetic patients with only moderate fasting hypertriglyceridemia present potentially atherogenic quantitative and qualitative lipoprotein abnormalities that are better evidenced in the PP state and may contribute to their higher CHD risk.

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.

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 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.

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.

The proliferation of commercial immunoassays for the determination of apolipoproteins AI and B as parameters of risk for coronary heart disease and the discordance between the values obtained with the systems have made more urgent than ever the need for standardization of these assays. The authors report here the results of a collaborative study on the standardization of the values between the "Comité Française de Coordination des Recherches sur l'Athérosclérose et le Cholestérol (ARCOL)" and 13 Companies providing 15 different analytical systems. The results show that standardized measurements are not possible using a "consensus international standard" and that the major problem is related to the enormous variability of the analytical systems. In contrast, the present study demonstrates that six different serum pools containing increasing amounts of apolipoproteins AI and B can be used to generate a common reference curve which permits calibration of all immunoassays and standardization of results.

The chemical composition and metabolism of lipoproteins in a population of Benedictine nuns were studied after 5-month periods during which the predominant dietary fats were sunflower oil, peanut oil, palm oil, or milk fats (butter and cream). The population was divided into three groups. The control group (C) included twelve subjects selected at random by taking two subjects per age pool among those with plasma cholesterol less than 230 mg/dl. Groups H1 and H2 were selected at random in the same way, among those with plasma cholesterol greater than 230 mg/dl. Each group comprised six subjects and differed from each other in the amount of plasma cholesteryl esters, i.e., below and above the mean value of group C, for H1 and H2, respectively. Changes in the degree of saturation of the predominant fat of the diet were associated with changes in both the chemical composition of lipoproteins and their cellular metabolism studied in fibroblast cultures. No significant difference between the normocholesterolemic subjects of group C and the ''high risk'' subjects of groups H1 and H2 was found.

The binding of chlorophenoxyisobutyric (CPIB), tibric (TA) and nicotinic (NA) acids and CPIB ethyl ester (Clofibrate), TA and NA isopropyl esters (TAPE and NAPE) to human lipoproteins of low density of different classes (LDL 2 , LDL 1 and VLDL) and high density (HDL) were studied by equilibrium dialysis and Sephadex gel filtration. Clofibrate and TAPE bound strongly to lipoproteins, but their acids, CPIB and TA and also NA and NAPE, did not bind. In the same experimental conditions, Clofibrate and TAPE bound only weakly to human serum albumin (HSA) and CPIB bound to HSA with a K a of 3.3 × 10 5 M −1 for 1 site of high affinity. The Clofibrate and TAPE bound to lipoproteins did not dissociate either during dialysis or during filtration on Sephadex G 25. The binding percentage remained constant for all drug concentrations studied, and the molar ratio of bound drug rose linearly with increasing concentrations. This suggests that the interaction may be irreversible, and there is some evidence that binding may induce irreversible changes in the lipoprotein molecules. These results, and those already found in experiments made with three other drugs related to Clofibrate, lead to the proposal that in their interaction with lipoproteins, the phenyl groups are necessary and the esterification is contributory. The possible role of this interaction in the lipid-lowering effect of the drugs is discussed with special reference to their possible implication in lipoprotein synthesis within the intestinal and hepatic cells.

In this study Probucol transport and the effect of the drug on lipoprotein composition in 9 cases of type IIa hypercholesterolemia were investigated. Probucol lowered plasma cholesterol by 20%, without affecting triglycerides. HDL cholesterol was decreased and a slight reduction in LDL cholesterol was noted. This was due to a reduction in the number of circulating lipoprotein particles, without modification in the lipid/protein ratio, mainly cholesterol/protein ratio. Probucol was almost entirely removed by lipoproteins; 75% of the drug was found in LDL, the remainder being equally distributed in VLDL and HDL. There was no correlation between the serum Probucol level, or the amount of Probucol bound to lipoproteins, and the decrease in serum or lipoprotein cholesterol. However, there was a significant increase of the EC/TC (esterified cholesterol/total cholesterol) ratio in VLDL and LDL, but not in HDL.

G P 45.699 is a chemical hypolipidemic agent related to Clofibrate. The present study, achieved in man during a clinical assay, shows that the drug, after its intestinal absorption, is carried in blood by serum albumin, and also by low density lipoproteins, chiefly density 1.006–1.063 lipoproteins. In 4 normal subjects, the drug is found to be bound to albumin for 93–98%; to low density lipoproteins for only 2–6%. But when molecular weights are considered, the mole ratio of the drug per mole of albumin is less than 1, whereas per mole of lipoprotein it reaches 13 and even 25. Similar results were obtained in 4 hyperlipidemic subjects. This ability of low density lipoproteins to transport G P 45.699 in vivo was confirmed in vitro by the ability of purified β lipoproteins to bind the drug in saline. The presence of the drug on low density lipoproteins allows a new hypothesis concerning the hypolipidemic action of G P 45.699 and of related chemical compounds, Clofibrate being the best known. According to this hypothesis, the drug when binding with peptide B (apo-β-lipoprotein) within the mucosal cell, would reduce its ability to bind and transport triglycerides and cholesterol. A direct interaction between the drug and lipoproteins would fit better with its effects on lipidemia. Its interaction with albumin being responsible for some other effects, such an interference with several antivitamine-K anticoagulants.

In vivo, after their absorption by the intestine, the hypolipidaemic drugs related to Clofibrate are for the major part transported in the circulating blood, bound to serum albumin. But a recent work demonstrated that one of them, phenoxybenzyl heptanoic acid (GP 45.699 Geigy) was for about 5% bound to circulating low-density lipoproteins specially of D 1.019 to 1.063 (beta-lipoproteins). Here the interaction of GP 45.699 and two other drugs related to Clofibrate (SU 13.437 Ciba and XD 448 Sandoz) was studied in vitro against the proteins of man, rabbit, rat and hamster serum. The following results were collected: 1. (1) GP 45.699 is bound in vitro by human beta and alpha-lipoproteins, albumin, but not by IgG. 2. (2) Seventy-nine to more than 1000 molecules of the drug can be bound by one molecule of beta-lipoprotein, depending on the concentration of the drug in the medium. 3. (3) During the binding of the drug, beta-lipoprotein looses a lipid fraction containing free cholesterol and phospholipids. As a result of this molecular alteration appears first a slowering of the electrophoretic mobility which is well seen when about 100 molecules are bound. Later, when more molecules are bound, the beta-lipoprotein precipitates. In those experiments the drug precipitates with the B peptide (apobeta-lipoprotein) with which it is bound and a fraction containing free cholesterol and phospholipids stays in solution. 4. (4) The molar ratios (GP/beta-Lp) found in vitro with low concentrations of drug are close to the ratios found in vivo with similar concentrations of drug in the circulating blood. 5. (5) The interaction with the beta-lipoproteins is seen as well in the presence of whole serum or of concentrated albumin than when the lipoproteins are in purified form. 6. (6) Alpha-lipoproteins and albumin bind the GP in different proportions and do not precipitate even with highly concentrated drug solutions. 7. (7) Very similar interactions are seen when rat, hamster and rabbit serum is used instead of human serum; or when SU 13.437 or XD 448 are used instead of GP 45.699. So, in vitro, the interaction of hypolipidaemic drugs related to Clofibrate and serum lipoproteins was seen with all drugs and lipoproteins tested and looks like a general phenomenon. This supports that such an interaction may be responsible for the hypolipidaemic action of these drugs which is still open to discussion. In this hypothesis, the following sequence of events is expected: Interaction of the drug with the B peptid within the intestinal and hepatic cells → synthesis of lipoproteins containing the drug and less free cholesterol and phospholipids than normal lipoproteins → altered lipoproteins which can solubilize less neutral lipids than normal lipoproteins → impairment of fat transport → reduced exogenous and endogenous lipaemia, reduced chylomicra, lipomicra and VLDL in the circulating blood. If this hypothesis is true, an in vitro screening of the ability of drugs to interact with lipoproteins may be useful to look for new hypolipidaemic drugs.

Oral administration of large doses of vitamin A permits one to study pos-prandial lipemia. Thanks to a new fluorescimetric and automatic method, estimation of vitamin A is now much easier than formerly. The normal results of the oral vitamin A tolerance test are evaluated with this new method and compared with those given by the previous method.

A possible mechanism of action of probucol on low density lipoprotein uptake was examined in 7 type Ila hypercholesterolemic subjects. Probucol administration effectively lowered plasma cholesterol. Both apo B-associated cholesterol and HDL cholesterol were decreased but a great inter-patient variability was noted. Plasma triglycerides were unchanged and phospholipids decreased. The composition of the isolated LDL was unaffected. The LDL displaceable activity of reference [ 125 I]LDL measured by competition assays in control fibroblasts, was increased in 3 subjects, decreased in 1 subject and unchanged in the other 3. No correlation was found between the change in apo B-associated cholesterol and the change in the in vitro catabolism of LDL of treated patients. The results did not allow a simple mechanism of action to be ascribed to the drug, but questioned the origin of the hypercholesterolemia.

Human LDL were bound and internalized by cultured human fibroblasts. When an antilipoprotein IgA kappa from a case of myeloma with hyperlipidemia and xanthomatosis was introduced into the system, LDL uptake dropped by 50% but LDL binding to fibroblasts was unchanged. In the same system, IgG and IgA controls were inactive.

LDL interact with fibroblasts through specific membrane sites. This reaction is the first step of a mechanism which leads to the regulation of intracellular cholesterol synthesis. When LDL form a complex with an IgA from a myeloma serum with mixed hyperlipidemia and xanthomatosis, they no longer function as a regulator, and the result is excess production of free intracellular cholesterol. No excess cholesterol production is observed when myeloma IgA is replaced by control IgG and IgA.

The effect of treatment with simvastatin, a new HMG-CoA reductase inhibitor, has been investigated in 27 patients with primary hypercholesterolaemia. It produced a significant decrease of cholesterol and phospholipids in plasma, LDL and apolipoprotein B-containing lipoproteins. Plasma apolipoproteins B, C-III and E were also significantly lowered. The concentration of lipoprotein particles recognized by monoclonal antibodies (BL3, BL5 and BL7), associated with atherosclerotic disease, was also lowered by the treatment. Lipoproteins LpA-II:A-I were not changed, while LpA-I, which has been suggested to be the protective fraction of the apo A-I-containing lipoproteins, was slightly and inconsistently increased.