Influence of apolipoprotein composition of high density lipoprotein particles on cholesteryl ester transfer protein activity. Particles containing various proportions of apolipoproteins AI and AII

The effect of apolipoprotein (apo) composition of high density lipoproteins (HDL) on cholesteryl ester transfer protein (CETP) activity was studied by measuring the rate of radiolabeled cholesteryl esters transferred between low density lipoproteins (LDL) and HDL3 which contained various proportions of apoAI and apoAII. Ultracentrifugally isolated HDL3, which contained virtually only apoAI and apoAII in their protein moiety, were progressively enriched with apoAII upon the incubation with increasing amounts of delipidated HDL apolipoproteins. The substitution of apoAII for apoAI in HDL3 did not induce marked alteration of the lipid composition of the lipoprotein particles. The rates of cholesteryl ester exchanges with LDL in the presence of purified human CETP were significantly reduced with apoAII-enriched HDL3 as compared with non-enriched homologous particles. Consistent results were obtained by determining the rate of cholesteryl esters transferred either from LDL toward HDL3, or in the opposite direction, from HDL3 to LDL. The effect of the apoAI and apoAII content of HDL particles on CETP activity was also investigated by measuring the rate of cholesteryl esters transferred from LDL to plasma HDL3 particles which contained either only apoAI, HDL3-AI, or both apoAI and apoAII, HDL3-AIAII. HDL3-AI and HDL3-AIAII particles were isolated from human plasma by a sequential procedure which combined ultracentrifugation and anti-apoAII immunoaffinity chromatography. As observed with HDL3 artificially enriched with apoAII, cholesteryl ester transfer rates were significantly lower with plasma HDL3-AIAII than with plasma HDL3-AI particles. Kinetic analysis of the interaction of CETP with apoAII-enriched HDL3 revealed that apoAII could act as an uncompetitive inhibitor of the cholesteryl ester transfer reaction. Since the plasma levels of HDL-AI, HDL-AIAII, and HDL-AII may undergo significant physiological fluctuation, the present study suggests that HDL apoproteins may be important factors in modulating cholesteryl ester transfer rates in vivo.     

Interaction of apolipoprotein AII with the putative high-density lipoprotein receptor

There is strong evidence to indicate that binding of HDL by cells is due to recognition of apoproteins residing on the surface of the lipoprotein by the putative HDL receptor(s). Although both of the major HDL apoproteins, AI and AII, are recognized by the putative receptor, the nature of the binding interaction and the domains of the apoproteins involved are largely unknown. Previous data from this laboratory led to the proposal of a model to explain how HDL particles containing AII interacted with the HDL receptor in a different manner as compared to HDL particles which contain apoAI but not apoAII [Vadiveloo, P. K., & Fidge, N. H. (1992) Biochem. J. 284, 145-151]. The model predicted that each chain of the apoAII homodimer contained a binding domain capable of interacting with the HDL receptor. This model was tested in the current study by preparing apoAII monomers, complexing them with phospholipid, and determining the ability of these complexes to bind to putative HDL receptors in rat liver plasma membranes (RLPM) and bovine aortic endothelial cell membranes (BAECM) by ligand blotting. The data showed that these complexes were bound by HB1 and HB2 from RLPM, and to the 110-kDa HDL binding protein from BAECM, providing critical evidence to support the model. Further investigation into the binding interaction revealed that apoAII complexed with phospholipid (apoAII-PC) bound more than delipidated apoAII, which bound more than delipidated apoAII monomers. Thus, optimum binding required the presence of lipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the carboxy-terminal domain of human apolipoprotein AI in high-density-lipoprotein metabolism--a study based on deletion and substitution variants in transgenic mice

Cholesterol levels in high-density lipoprotein (HDL) of transgenic mice overexpressing human apolipoprotein AI (apoAI), a des-(190-243)-apoAI deletion mutant or an apoAI-(1-189)-apoAII-(12-77) chimera were 2.8-fold (P<0.001), 1.3-fold (P<0.05) and 2.2-fold (P<0.001) higher than in control mice, respectively. Human apolipoprotein levels in apoAI and in apoAI-(1-189)-apoAII-(12-77) transgenic mice were 5.2-fold and 3.5-fold higher than in des-(190-243)-apoAI transgenic mice, whereas their HDL cholesterol levels were 2.1-fold and 1.6-fold higher. PAGE of HDL isolated by ultracentrifugation revealed that murine HDL migrated as 9.6-nm and 7.2-nm particles. Overexpression of human apoAI and apoAI-(1-189)-apoAII-(12-77) resulted in the production of polydisperse HDL (9.6, 9.2, 8.4 and 7.2 nm) particles, whereas overexpression of des-(190-243)-apoAI primarily resulted in an increase of 7.2-nm particles. The fractional catabolic rates of human apoAI and apoAI-(1-189)-apoAII-(12-77) were very similar, whereas that of des-(190-243)-apoAI was 4.9-fold higher. The endogenous production rates of human apoAI, des-(190-243)-apoAI and apoAI-(1-189)-apoAII-(12-77) in transgenic mice were very similar. It is concluded that deletion of the carboxy-terminal domain of apoAI reduces its lipoprotein association, resulting in the production of small, phospholipid-rich HDL particles that are cleared more rapidly. Substitution of the carboxy-terminal helices of apoAI with helices of apoAII restores lipoprotein association, resulting in the production of HDL, which migrates as human HDL3 and HDL2. Although the carboxy-terminal domain of the chimera contained more than 80% of the amino acid sequence of apoAII, its HDL-distribution profile in transgenic mice was very similar to that of human apoAI. This study demonstrates the importance of the helical structure of apoAI of the carboxy-terminal domain of apoAI, rather than of its exclusive amino acid sequence, in HDL metabolism.     

Interpreting DNA mixtures

Apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) represent 80 90% of the protein content of high density lipoproteins (HDL). Previously we have identified a Finnish family with an apo A-I variant (Lys107-->0) associated with reduced plasma HDL cholesterol level and decreased lipoprotein (Lp)(AI w AII) concentration compared to unaffected family members. To determine the in vivo metabolism of apo A-I and apo A-II in the carriers of apo A-I (Lys107-->0) variant we radioiodinated normal apo A-I with 125I and apo A-II with 131I and compared the kinetic data of two heterozygous apo A-I(Lysl07-->0) patients (HDL cholesterol leves 0.31 and 0.69 mmol/l) to that of eight normolipidemic, healthy control subjects. Plasma radioactivity curves of 125I-labelled normal apo A-I of the patients demonstrated accelerated clearance of apo A-I compared to control subjects. In the two patients the fractional catabolic rates (FCR) of apo A-I were 0.347/day and 0.213/day, respectively, while the mean FCR of apo A-I of the control subjects was 0.151 +/- 0.041/day. Similarly, the plasma decay curves of the 131I-labelled apo A-II showed more rapid clearance of apo A-II in the two patients than in control subjects. The FCR of apo A-II in the two patients were 0.470/day and 0.234/day, while the mean FCR of apo A-II in control subjects was 0.154 +/- 0.029/day. The calculated production rates of apo A-I were similar in patients and in control subjects, and the production rates of apo A-II were significantly higher in patients than in control subjects. Our results show that the Lp(AI w AII) deficiency in patients with the apo A-I(Lys107-->0) is associated with increased fractional catabolic rates of normal apo A-I and apo A-II, while the production rates of these apolipoproteins are normal (apo A-I) or slightly increased (apo A-II).     

Enhanced cholesterol efflux by tyrosyl radical-oxidized high density lipoprotein is mediated by apolipoprotein AI-AII heterodimers

Myeloperoxidase secreted by phagocytes in the artery wall may be a catalyst for lipoprotein oxidation. High density lipoprotein (HDL) oxidized by peroxidase-generated tyrosyl radical has a markedly enhanced ability to deplete cultured cells of cholesterol. We have investigated the structural modifications in tyrosylated HDL responsible for this effect. Spherical reconstituted HDL (rHDL) containing the whole apolipoprotein (apo) fraction of tyrosylated HDL reproduced the ability of intact tyrosylated HDL to enhance cholesterol efflux from cholesterol-loaded human fibroblasts when reconstituted with the whole lipid fraction of either HDL or tyrosylated HDL. Free apoAI or apoAII showed no increased capacity to induce cholesterol efflux from cholesterol-loaded fibroblasts following oxidation by tyrosyl radical, either in their lipid-free forms or in rHDL. The product of oxidation of a mixture of apoAI and apoAII (1:1 molar ratio) by tyrosyl radical, however, reproduced the enhanced ability of tyrosylated HDL to induce cholesterol efflux when reconstituted with the whole lipid fraction of HDL. HDL containing only apoAI or apoAII showed no enhanced ability to promote cholesterol efflux following oxidation by tyrosyl radical, whereas HDL containing both apoAI and apoAII did. rHDL containing apoAI-apoAIImonomer and apoAI-(apoAII)2 heterodimers showed a markedly increased ability to prevent the accumulation of LDL-derived cholesterol mass by sterol-depleted fibroblasts compared with other apolipoprotein species of tyrosylated HDL. These results indicate a novel product of HDL oxidation, apoAI-apoAII heterodimers, with a markedly enhanced capacity to deplete cells of the regulatory pool of free cholesterol and total cholesterol mass. The recent observation of tyrosyl radical-oxidized LDL in vivo suggests that a similar modification of HDL would significantly enhance its ability to deplete peripheral cells of cholesterol in the first step of reverse cholesterol transport.     

Granulocytic-macrophagic and macrophagic colony stimulating factors elicit colonies of mast cells in mouse bone marrow agar culture. An electron microscope study

The aim of this paper is to describe a new methodology for the separation of human high-density lipoproteins (HDL) into apolipoprotein (apo) E-poor and apo E-rich subfractions by fast protein liquid chromatography (FPLC) using a heparin affinity column. Recoveries for apolipoproteins AI, AII, CI, CII, CIII, and E were 68.9, 74.7, 71.9, 73.5, 40.0, and 55.8%, respectively. We provide suggestive evidence that apo E-rich HDL is produced from apo E-poor HDL by the displacement of apo AI by apo E. Apo E-poor HDL was the predominant fraction. The molar ratio of apo E to apo AI in apo E-poor HDL was 0.02 and 0.01 for the subjects studied while in apo E-rich HDL it was 1.86 and 1.25. The molar ratios of the C apolipoproteins to apo AI are markedly different between the subfractions.     

Adenoviral vector-mediated overexpression of serum amyloid A in apoA-I-deficient mice

Serum amyloid A (SAA) is an acute phase reactant that can become the predominant apolipoprotein of high density lipoprotein (HDL) during severe inflammatory states. However, the function of SAA is unknown. To study the ability of SAA to form HDL in the absence of apolipoprotein A-I, we expressed the mouse SAA pI 6.15 (CE/J) isoform in apolipoprotein A-I knock-out (apoA-I (-/-)) mice using a recombinant adenovirus. As a control, apoA-I (-/-) mice were injected with an adenovirus expressing human apoA-I. High level expression of plasma SAA was obtained in the absence of any endogenous acute phase SAA production. SAA expression increased plasma HDL cholesterol levels about 2-fold, but to a lesser extent than the expression of apoA-I (about 10-fold). The HDL particles isolated by density ultracentrifugation from SAA-expressing mice were heterogeneous in size and composition and rich in free cholesterol as well as apoE and apoA-IV. Of the SAA expressed in the plasma, only a small fraction (4%) was associated with HDL particles in contrast to expressed apoA-I, of which 62% was associated with HDL. We conclude that SAA is unable to substitute for apoA-I in HDL particle formation.     

Production rate determines plasma concentration of large high density lipoprotein in non-human primates

Large LpAI HDL particles, containing only apoA-I without apoA-II, are reported to be the major anti-atherogenic portion of HDL and to be increased in individuals with low risk for coronary heart disease. To determine whether the plasma concentration of large LpAI is modulated by the rate of production or catabolism of apolipoprotein A-I (apoA-I) in large LpAI, kinetic studies of large LpAI were performed in African green monkeys consuming an atherogenic diet with either high plasma HDL concentration (120 +/- 36 mg/dl, mean +/- SD, n = 3) or low plasma HDL concentration (40 +/- 13 mg/dl, n = 3). Large LpAI was isolated, without ultracentrifugation, by immunoaffinity and gel filtration and radiolabeled. After injection, the specific activity of apoA-I in large HDL, consisting of both LpAI and LpAI:AII particles, was followed. A multicompartmental model was developed for the kinetics of apoA-I in large HDL, which indicated that a portion of large HDL is distributed to a sequestered pool, outside the circulating plasma, and reenters circulating plasma approximately 3 h after injection. There was no conversion of large LpAI to smaller HDL particles or transfer of radiolabeled apoA-I to smaller HDL particles. Although the mean fractional catabolic rate was not different comparing the high and low HDL group, the mean production rate of apoA-I in large HDL was 4-fold greater in the high HDL group compared with the low HDL group. These data support the hypothesis that the plasma concentration of large HDL is controlled primarily by the rate of production of apoA-I in large HDL.     

Impact of gender on the metabolism of apolipoprotein A-I in HDL subclasses LpAI and LpAI:AII in older subjects

The behavior of apolipoprotein (apo) A-I in lipoprotein (Lp) AI and LpAI:AII was studied in 11 postmenopausal females and 11 males matched for plasma triglyceride and total cholesterol levels. Subjects consumed a baseline diet [35% fat (14% saturated, 15% monounsaturated, and 7% polyunsaturated), 15% protein, 49% carbohydrate, and 147 mg cholesterol/1000 kcal] for 6 weeks before the start of the kinetic study. At the end of the diet period, using a primed-constant infusion of [5,5,5-2H3]leucine, residence times (RT) and secretion rates (SR) of apoA-I were determined in 2 subpopulations of high-density lipoprotein (HDL) particles, LpAI and LpAI:AII. Plasma total cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations were similar in males and females. The mean plasma HDL cholesterol concentration in males (1.14 +/- 0.23 mmol/L; mean +/- SD) was lower than in females (1.42 +/- 0.18 mmol/L; P =. 0034). Similarly, the mean plasma concentration of apoA-I in males (130 +/- 21 mg/dL) was lower than that in females (150 +/- 19 mg/dL; P = .0421). The RT of apoA-I in either LpAI or LpAI:AII was similar between men and women. Despite the higher plasma apo A-I levels in female compared with male subjects, total apoA-I and apoA-I in LpAI and LpAI:AII pool sizes were similar between the two groups, attributable to the lower body weight of the female subjects. The mean SR of total apoA-I in males (8.5 +/- 2.7 mg.kg-1.d-1) was 22% lower than in females (10.9 +/- 2.3 mg.kg-1.d-1; P = .0389). The SR of both apoA-I in LpAI and LpAI:AII was lower in males than females, although the differences did not reach statistical significance. These data suggest that the difference observed in HDL cholesterol concentration between males and females is attributable to SR of apoA-I and not the catabolic rate.     

Decreased postprandial high density lipoprotein cholesterol and apolipoproteins A-I and E in normolipidemic smoking men: relations with lipid transfer proteins and LCAT activities

We have previously reported that normolipidemic smokers are lipid intolerant due to increased responses of triglyceride-rich lipoproteins (TRL) apolipoprotein B-48, triglyceride (TG), and retinyl esters to a mixed meal compared to non-smokers. To investigate whether postprandial high density lipoprotein (HDL), apolipoprotein A-I (apoA-I), apolipoprotein A-II (apoA-II), and apolipoprotein E (apoE) concentrations or lipid transfer protein activities are affected by cigarette smoking, we investigated 12 male smokers and 12 non-smokers with comparable fasting lipoprotein profile, BMI, and age. Plasma samples obtained after an overnight fast and postprandially were separated by density gradient ultracentrifugation. Postprandial apoA-I, lipoprotein AI-particles (LpA-I), HDL-cholesterol, and HDL apoE concentrations decreased in smokers, but remained unchanged in controls. Concomitantly, cholesterol and apoE concentrations increased significantly in TRL fractions in smokers. Fasting lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) activity levels, as well as esterification rates (EST) and phospholipid transfer rates were comparable between the groups. Cholesteryl ester transfer protein (CETP) activity levels were lower in the smokers. Postprandially EST increased, but CETP and PLTP activities deceased in smokers as compared to controls. We conclude, that even healthy, normolipidemic smokers have altered postprandial high density lipoprotein (HDL) cholesterol and apolipoprotein composition, as well as lipid transfer protein activities. The shift of cholesterol and apoE from HDL to the triglyceride-rich lipoprotein (TRL) fraction, together with decreased plasma apoA-I and LpA-I concentrations during alimentary lipemia may indicate impaired reverse cholesterol transport. Both the postprandial increase in TRL and the lowering of HDL may promote atherogenesis in smokers.     

The concentration of angiotensins I and II in blood from the pulmonary artery and left ventricle of man

The concentrations of angiotensin I (AI) and II (AII) were determined by radioimmunoassay in blood from the main pulmonary artery (MPA) and left ventricule (LV) of ten subjects with rheumatic valvular heart disease. The levels of AI were consistently higher in MPA plasma (21.8+/-2.4 pmol/1) than in LV plasma (14.7+/-2.0 pmol/1), paired t, P less than 0.001. The levels of AII were consistently lower in MPA plasma (21.8+/-4.7 pmol/1) than in LV plasma (33.8+/-7.2 pmol/1), paired t, P less than 0.001. The AII antiserum cross-reacted with three metabolites of the hormone, [des-Asp1]angiotensin II, [des-(Asp1, Arg2)angiotensin II [des-(Asp1, Arg2 Val,3]angiotensin II. To characterize the nature of circulating AII immunoreactive material, paper chromatography was used to separate AII from its immunoreactive metabolites. The results showed that 84-100% of the AII immunoreactive material from both MPA and LV plasma chromatographed with the mobility of authentic angiotensin II. The mean pulmonary conversion of endogenous AI was 33+/-4.8% and the net extraction of AII by peripheral tissues was 33+/-4.1%.     

Increases in dietary cholesterol and fat raise levels of apoprotein E-containing lipoproteins in the plasma of man

Previous studies from this laboratory have determined that diets containing the usual amounts of fat to which are added 750-1500 mg/day cholesterol elevate the plasma cholesterol concentration by variable amounts, depending upon the ratio of polyunsaturated to saturated fatty acids (P/S ratio) of the diet. Diets with P/S ratios of 0.25-0.4 are accompanied by elevations of low density lipoprotein (LDL) cholesterol, whereas diets with a P/S ratio of 2.5 produce no significant changes in cholesterol levels. On the low P/S ratio diets, the structure, composition, and interaction with cultured fibroblasts of LDL are not significantly changed. Plasma high density lipoprotein (HDL) cholesterol levels remain constant, but HDL2 increase relative to HDL3. In the present study, not only dietary cholesterol but also total dietary fat was altered. Six normal young men were fed a basal diet consisting of 18% protein, 51% carbohydrate, and 30% fat, containing 250 mg/day cholesterol. After 2 weeks, an experimental diet consisting of 18% protein, 42% carbohydrate, and 39% fat, containing 1760 mg/day cholesterol, was fed for 4 weeks. The P/S ratios of both diets were about 0.4. Plasma samples were taken twice during each dietary period from 12- to 14-h-fasted subjects and analyzed for their contents of lipoprotein lipids. Plasma levels of LDL and HDL cholesterol increased by 30 and 13 mg/dl, respectively; total and very low density lipoprotein (VLDL) triglyceride concentrations were unaltered. The plasma concentrations of apoproteins (apo) B, E. and A-I, but not A-II, were elevated. Plasma samples also were studied by zonal ultracentrifugation, gel permeation column chromatography, and Pevikon electrophoresis. Although on zonal ultracentrifugation the total concentrations of LDL were increased, the flotation properties and chemical compositions of LDL were not changed. By contrast, HDL2 and HDL3L concentrations increased, and HDL2 became enriched with cholesteryl esters. On gel permeation chromatography, with the subjects on the basal diet, plasma cholesterol eluted in two peaks, corresponding to LDL and HDL. The sizes of the peaks increased on the experimental diet. ApoE eluted in two peaks: one at the leading edge of LDL (corresponding to VLDL or IDL) and the other in the area between LDL and HDL, corresponding to HDLC. On the experimental diet, the apoE peak between LDL and HDL increased. On Pevikon electrophoresis apoE migrated between the LDL and HDL bands. This apoE peak was increased on the experimental diet. These findings suggest that increasing the concentrations of both dietary cholesterol and total fat can increase the levels of plasma LDL, HDL2, and HDLC in fasting normal subjects. Thus, the concentrations of some putatively atherogenic as well as antiatherogenic lipoproteins increased in plasma, and the apparent paradox between the epidemiological and metabolic behaviors of some lipoproteins remains. Clearly, more work is needed to resolve the roles of various lipoproteins in plasma in atherosclerosis.     

High density lipoprotein particle size restriction in apolipoprotein A-I(Milano) transgenic mice

Human carriers of apolipoprotein A-I(Milano) (Arg173 --> Cys substitution in apolipoprotein A-I) are characterized by an HDL deficiency in which small, dense HDL accumulate in plasma. Because affected individuals are heterozygous for this mutation, the full impact of apolipoprotein A-I(Milano) (apoA-I(Milano)) on HDL-cholesterol metabolism is unknown. In this study, apoA-I(Milano) transgenic mice were used to evaluate the extent of apoA-I(Milano) dimerization and HDL particle size restriction in the absence of wild-type apoA-I. Murine apoA-I knockout mice were utilized to express apoA-I(Milano) and human apoA-II in the presence of wild-type, human apoA-I (apoA-IMilano/A-Iwt/A-II) and in its absence (apoA-IMilano/A-II). Plasma HDL-cholesterol concentrations were similar (30 mg/dl) in both lines of apoA-I(Milano) transgenic mice. In the apoA-IMilano/A-Iwt/A-II phenotype, 14% of the apoA-I(Milano) formed homodimers and 33% formed heterodimers with apoA-II. ApoA-I(Milano) homodimers increased by 71% in the apoA-IMilano/A-II transgenics and was associated with an abundance of small, 7.6-nm HDL3-sized particles compared to the 9.5, 8.3, and 7.6-nm-sized particles in apoA-IMilano/A-Iwt/A-II mice. The unesterified cholesterol/cholesteryl ester mole ratio of HDL was elevated by 45% in apoA-IMilano/A-Iwt/A-II mice and by 90% in apoA-IMilano/A-II transgenics compared to wild-type (human apoA-I/A-II). Both apoA-I(Milano) transgenics possessed normal levels of plasma LCAT activity, but endogenous cholesterol esterification rates were reduced by 50% compared to controls. Thus, HDL particle size restriction was not the result of impaired LCAT activation; rather, dimerization of apoA-I(Milano) limited the esterification of cholesterol on endogenous HDL. In the absence of wild-type apoA-I, the more extensive dimerization of apoA-I(Milano) severely limited cholesteryl ester accumulation on plasma HDL accounting for the abundance of small, 7.6-nm HDL3 particles in apoA-IMilano/A-II mice.     

Retinoic acid enhances fibrinolytic activity in-vivo by enhancing tissue type plasminogen activator (t-PA) activity and inhibits venous thrombosis

BACKGROUND: Simvastatin and pravastatin are both competitive inhibitors of the rate limiting enzyme for cholesterol biosynthesis (HMG CoA) reductase, but data from individual clinical trials suggest significant differences in potency for cholesterol reduction between the two drugs. AIM: To assess any differences in efficacy and safety between simvastatin and pravastatin in a direct, comparative study. METHODS: A double-blind, double-dummy, randomised study design was used, involving 48 patients with primary hypercholesterolaemia. Following a 6 week placebo baseline period, patients were randomly allocated to treatment with either simvastatin or pravastatin, commencing at a dose of 10 mg daily. The dose levels were titrated up to the recommended maximum effective dose of 40 mg daily at 6 weekly intervals if LDL cholesterol levels remained > or = 3.4 mmol/L. After 18 weeks of therapy, all patients were transferred to simvastatin therapy for a further 6 weeks, continuing at their week 18 dose level. Patients complied with a standard lipid lowering diet (containing < 30% of energy as total fat) throughout the study period. RESULTS: Over the 18 week direct comparison of the two drugs, there was a significant difference (p < 0.001) in response between simvastatin and pravastatin for reduction in levels of total cholesterol (32% vs 21% respectively), LDL cholesterol (38% vs 27%) and apolipoprotein B levels (34% vs 23%). No significant difference in drug effect was seen for the small reduction in levels of apolipoprotein AI (5% vs 6% respectively), nor for the increased levels of apolipoprotein AII (14% vs 11%) and HDL cholesterol (11% vs 7%). Lp(a) levels remained unchanged. When pravastatin was replaced with simvastatin for the final 6 weeks of the study in the 23 patients initially randomised to pravastatin, there were further reductions (p < 0.01) in total and LDL cholesterol, and apolipoprotein B. These results establish the advantage of simvastatin over pravastatin in terms of efficacy, for the treatment of primary hypercholesterolaemia.     

Polymorphism in the promoter region of the apolipoprotein AI gene associated with differences in apolipoprotein AI levels: the European Atherosclerosis Research Study

The effect associated with the substitution of adenine (A) for guanidine (G) in the promoter region of the apolipoprotein AI gene (-75 bp) with plasma apo AI and high-density lipoprotein (HDL) levels was investigated in the European Atherosclerosis Research Study (EARS). This is a study of healthy offspring (cases) of fathers who had suffered premature myocardial infarction (MI) before age 55 years (n = 565) and age- and sex-matched controls (n = 1,078) from 12 European countries, divided into 5 regions based on geography and language. The frequency of the polymorphism was not significantly different among the regions and the relative frequency of the rare A allele was similar in cases and controls (0.159 vs. 0.142) combining data from all regions. Individuals with one or more A allele had significantly higher plasma apo AI levels (P < 0.05) than individuals homozygous for the G allele. This effect was consistent in all regions. The data were analyzed separately in males and females. In females, those with one or more A allele had significantly higher apo AI levels (P = 0.05) than individuals homozygous for the G allele, and this raising effect of the A allele was greater in cases than controls for both apo AI (5.23% vs. 1.56%) and HDL (4.48% vs. 1.89%). In males, the A allele was associated with higher levels of apo AI and HDL, but the effect was much smaller and the differences did not reach statistical significance. In the females, where the effect of the A allele was strongest, the effect on apo AI associated with genotype was evident in non-smokers, and individuals with one or two A alleles had 3.6% higher apo AI and 3.14% higher HDL levels than individuals homozygous for the G allele. However, in the female smokers the raising effect of the A allele was greatly reduced (0.56%). Thus genetic variation in the promoter region of the apo AI gene is associated with differences in apo AI and HDL levels in healthy individuals throughout Europe, but the effect is modulated by gender, environmental factors such as smoking, and a family history of MI.     

Unravelling high density lipoprotein-apolipoprotein metabolism in human mutants and animal models

Apolipoprotein A-I plays an essential structural and functional role in HDL metabolism and apolipoprotein A-II has important effects on HDL metabolism and function. Kinetic studies in humans have established that variation in plasma HDL-cholesterol and apolipoprotein A-I concentrations is primarily determined by variation in the rate of apolipoprotein A-I catabolism. In contrast, plasma apolipoprotein A-II levels are primarily determined by the rate of apolipoprotein A-II production. Genetic factors play an important role in modulating the plasma levels of HDL-cholesterol and apolipoproteins A-I and A-II. Studies in humans have established that mutations in genes encoding enzymes that esterify cholesterol (lecithin : cholesterol acyltransferase), transfer cholesterol (cholesteryl ester transfer protein) and hydrolyze lipids (hepatic lipase, lipoprotein lipase) regulate HDL-cholesterol and apolipoprotein A-I levels by modifying the lipid content (and therefore the size) of HDL particles. Recent studies in transgenic and knockout animals have confirmed the key role of HDL lipid-modifying proteins in HDL, apolipoprotein A-I and apolipoprotein A-II metabolism and have expanded our understanding of the role of lipid modification in determining plasma concentrations of HDL-cholesterol and apolipoprotein A-I, as well as the potential functional roles of apolipoprotein A-II.     

Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5-10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non-carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n=178) or asymptomatic (n=99) non-carriers. The percentage of subjects with prothrombin levels above cut-off values of 1.15u/ml or 1.30u/ml was significantly higher in carriers of the prothrombin variant as compared to non-carriers, regardless of a previous thrombosis. However, among non-carriers the percentage of those with prothrombin levels above cut-off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non-carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.     

Analysis of particle size and lipid composition as determinants of the metabolic clearance of human high density lipoproteins in a rabbit model

Hypertriglyceridemia is commonly associated with triglyceride (TG) enrichment of high density lipoprotein (HDL) and reduction in HDL cholesterol and apolipoprotein A-I levels. We have recently reported that lipolytic modification of TG-rich HDL, which reduces particle size, enhances its clearance from the circulation. In the present study, we examined the role of particle size and lipid composition in determining the metabolic clearance of human HDL, in the absence of substantial in vivo modification of the particle by hepatic lipase. The rabbit, which has a very low hepatic lipase activity, was used for this purpose. Plasma fractions d < 1.21 g/ml were first isolated by ultracentrifugation from fasting humans with normal (NTG, n=6, mean plasma TG concentration=1.26+/-0.21 (SEM) mmol/l) or elevated plasma TG levels (HTG, n=5, TG=4.49+/-0.65 mmol/l). Small and large HDL particles were separated by gel filtration chromatography and were labeled with either 125I or (131)I. Large HDL were cleared more rapidly than small HDL in 10 out of 11 studies (P=0.006). There was, however, no difference in the fractional catabolic rate (FCR) of large HDL isolated from NTG versus from HTG subjects or in the FCR of small HDL from NTG versus HTG individuals. There was also no correlation between the TG content of HDL and its FCR. In summary, large, lipid-rich human high density lipoproteins (HDL) are cleared more rapidly than small human HDL in rabbits. These results, combined with our previous observation, also support the hypothesis that triglyceride enrichment of HDL, in the absence of substantial lipolytic modification, is not sufficient to enhance its clearance from the circulation.     

Differences in lipoprotein lipid concentration and composition modify the plasma distribution of cyclosporine

PURPOSE: The purpose of this study was to define the relationship between lipoprotein (LP) lipid concentration and composition and the distribution of cyclosporine (CSA) in human plasma. METHODS: 3H-CSA LP distribution was determined in normolipidemic human plasma that had been separated into different LP and lipoprotein-deficient plasma (LPDP) fractions by either affinity chromatography coupled with ultracentrifugation, density gradient ultracentrifugation or fast protein liquid chromatography. 3H-CSA LP distribution (at a concentration of 1000 ng/ml) was also determined in patient plasma samples with defined dyslipidemias. Furthermore, 3H-CSA LP distribution was determined in patient plasma samples of varying LP lipid concentrations. Following incubation, the plasma samples were separated into their LP and LPDP fractions by sequential phosphotungistic acid precipitation in the dyslipidemia studies and by density gradient ultracentrifugation in the specific lipid profile studies and assayed for CSA by radioactivity. Total plasma and lipoprotein cholesterol (TC), triglyceride (TG) and protein (TP) concentrations in each sample were determined by enzymatic assays. RESULTS: When the LP distribution of CSA was determined using three different LP separation techniques, the percent of CSA recovered in the LP-rich fraction was greater than 90% and the LP binding profiles were similar with most of the drug bound to plasma high-density (HDL) and low-density (LDL) lipoproteins. When 3H-CSA was incubated in dyslipidemic human plasma or specific patient plasma of varying LP lipid concentrations the following relationships were observed. As the very low-density (VLDL) and LDL cholesterol and triglyceride concentrations increased, the percent of CSA recovered within the VLDL and LDL fractions increased. The percent of CSA recovered within the HDL fraction significantly decreased as HDL triglyceride concentrations increased. The percent of CSA recovered in the LPDP fraction remained constant except in hypercholesterolemic/hypertriglyceridemic plasma where the percent of CSA recovered decreased. Furthermore, increases in VLDL and HDL TG/TC ratio resulted in a greater percentage of CSA recovered in VLDL but less in HDL. CONCLUSIONS: These findings suggest that changes in the total and plasma LP lipid concentration and composition influence the LP binding of CSA and may explain differences in the pharmacological activity and toxicity of CSA when administered to patients with different lipid profiles.     

Lipid, apoprotein and fibrinogen levels in the blood of male patients following myocardial infarct and the effect of diet on these parameters in ischemic heart disease

The authors have found significantly higher the levels of two not routinely examined risk factors, fibrinogen and lipoprotein (a) in 28 male patients after myocardial infarction than the corresponding data of the PROCAM-study and in the case of fibrinogen than in 23 healthy blood donors. A positive correlation was observed between the LDL-cholesterol and total cholesterol, the LDL-cholesterol and the main apoprotein of LDL, the Apo B level, and between the HDL-cholesterol and the main apoprotein of HDL, the Apo AI. During a 3 week long treatment in the Cardiac Rehabilitation Department the effect of low cholesterol, high unsaturated fatty acid content diet on the lipid, apolipoprotein and fibrinogen levels of male patients suffering from coronary heart disease with cholesterol level higher than 5.2 mmol/l was studied. Significantly decreased the total cholesterol (from 6.21 +/- 0.96 mmol/l to 5.87 +/- 0.98 mmol/l, -5.5%), the LDL-cholesterol (from 3.87 +/- 1.02 mmol/l to 3.61 +/- 0.96 mmol/l, -6.7%), the HDL-cholesterol (from 1.16 +/- 0.39 mmol/l to 1.04 +/- 0.28 mmol/l, -10.3%), the main apoprotein of HDL, the Apo AI (from 1.47 +/- 0.23 g/l to 1.33 +/- 0.29 g/l, -9.5%) and the main apoprotein of LDL, the Apo B level (from 1.59 +/- 0.43 g/l to 1.46 +/- 0.50 g/l, -8.1%). The change of fibrinogen lipoprotein (a) level was not significant. According to the earlier observation of the authors and the data of the literature, the effect of low cholesterol diet on the change of HDL cholesterol was not favourable. The investigation of apolipoprotein levels failed to get closer to the understanding of its mechanism.