Cholesteryl ester transfer activity in plasma measured by using solid-phase-bound high-density lipoprotein

We studied the ability of lipid-transfer factors in plasma to promote transfer, to endogenous lipoproteins, of [3H]cholesteryl ester from high-density lipoprotein (HDL) covalently bound to Sepharose 4B beads. After incubation for 2 h at 37 degrees C, 12 to 14% of the [3H]cholesteryl ester had been transferred to the lipoproteins of the plasma, in the proportions 57% to HDL and 43% to low- and very-low-density lipoproteins. This process was a function of the amount of plasma present and was stimulated by addition of partly purified lipid-transfer protein. Transfer also depended on the concentration of donor HDL but was independent of the amount of acceptor lipoprotein. This simple evaluation of cholesteryl ester transfer does not require removal of lipoproteins from the plasma before incubation.     

Cholesteryl ester transfer protein gene effect on CETP activity and plasma high-density lipoprotein in European populations. The EARS Group

BACKGROUND: Variation at the cholesteryl ester transfer protein (CETP) gene locus has been implicated in determining the levels and activity of CETP, apoAI and high-density lipoprotein (HDL) plasma concentration and the risk of developing coronary artery disease. STUDY DESIGN: The effects of two common polymorphisms of CETP, TaqIB in intron 1 and isoleucine 405 to valine (I405-->V) in exon 14, were examined in a sample of 822 men age 18-28 years from 11 countries in Europe who had participated in a study (the European Atherosclerosis Research Study II) of the offspring of myocardial infarction sufferers before the age of 55 years and age-matched control subjects. RESULTS: The frequency of the rare TaqIB allele (B2) and the rare V405 allele was 0.44 and 0.28 respectively and was the same in different regions of Europe. There was a moderate linkage disequilibrium between the two polymorphisms in all the regions (D' = +0.31, P < 0.001), explained by the preferential association between the two common alleles, B1 and I405. There was a statistically significant association of the rare alleles for both the polymorphisms with lower activity of CETP (P < 0.001), 11.2% lower for the TaqIB and 7.0% lower for the I405-->V polymorphism. The TaqIB polymorphism explained 9.1% (P < 0.001) and I405-->V explained 3.7% (P < 0.001) of the variance in CETP activity, and in combination these genotypes explained 12.0% of the variance (P < 0.001). Overall, subjects whose fathers had had an early coronary heart disease had 2.4% higher plasma CETP activity than those without such family history, which became statistically significant when adjusted for the effect of the genotypes (P = 0.015), but the significance disappeared after adjustment for the effect of lipids. There was a statistically significant effect of the TaqIB polymorphism on both plasma HDL cholesterol and apoAI level (P < 0.001), with those homozygous for the rare B2 allele having the highest level. Those individuals homozygous for the rare V405 allele had the highest HDL and apoAI levels, although these effects only reached statistical significance for HDL (P < 0.03). CONCLUSION: These results suggest that the TaqIB and I405-->V polymorphisms represent two independent functional variations in the CETP gene that may affect the activity of CETP and thus plasma levels of HDL.     

Cholesteryl ester transfer protein promotes the formation of cholesterol-rich remnant like lipoprotein particles in human plasma

BACKGROUND: Cholesteryl ester transfer protein (CETP) is suggested to be involved in the cholesterol level in remnant like lipoprotein particles (RLP), but there is no direct evidence that CETP increases cholesterol-rich RLP in plasma. METHODS: Human plasma was incubated with or without HDL containing [(3)H]-labeled cholesteryl ester ([(3)H]CE), recombinant CETP or CETP inhibitors at 37 degrees C in vitro. RESULTS: The RLP-cholesterol (RLP-C) level increased time-dependently and the amount of RLP-C increase (DeltaRLP-C) by the incubation was positively correlated with triglyceride (TG) level in plasma (r=0.597, P=0.0070). [(3)H]CE in HDL was transferred to RLP fraction under 37 degrees C incubation, and the amount of [(3)H]CE transferred to RLP correlated significantly with DeltaRLP-C in plasma (r=0.611, P=0.0156). Human recombinant CETP enhanced the RLP-C increase, while CETP inhibitor JTT-705 and anti-human CETP monoclonal antibody inhibited both the RLP-C increase and [(3)H]CE transfer to RLP. On the other hand, an inhibition of lecithin: cholesterol acyltransferase (LCAT) did not affect the RLP-C increase. In triglyceride-rich lipoproteins (TRL) fraction, JTT-705 inhibited [(3)H]CE transfer to RLP more strongly than that to non-RLP. CONCLUSIONS: CETP promotes the formation of cholesterol-rich RLP through the transfer of CE from HDL to TRL and CETP inhibitors are useful to reduce RLP-C.     

Polymorphisms in the gene coding for cholesteryl ester transfer protein are related to plasma high-density lipoprotein cholesterol and transfer protein activity

1. Cholesteryl ester transfer protein activity may have a physiological effect on high-density lipoprotein levels. 2. We examined restriction fragment length polymorphisms associated with the cholesteryl ester transfer protein gene and the apolipoprotein AI gene in a group of 60 unrelated subjects selected from an initial survey of 5000 subjects on the basis of their high-density lipoprotein levels being high or low at the extremes of the distribution. The activities of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase (phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) were also determined. Analysis by selection of those with a low high-density lipoprotein cholesterol level (less than or equal to 1.1 for males, less than or equal to 1.2 for females) gave 32 individuals with 24% B2 alleles. Selection of subjects with a high-density lipoprotein cholesterol level (less than or equal to 2 mmol/l) gave 17 with 62% B2 alleles. 3. The group with low levels of high-density lipoprotein cholesterol had higher activity of cholesteryl ester transfer protein and significantly elevated triacylglycerol levels when compared with the group with high levels of high-density lipoprotein cholesterol. 4. A further significant finding was the correlation of the MspI restriction fragment length polymorphism detected by the apolipoprotein AI gene with lecithin:cholesterol acyltransferase activity.     

心脑血管疾病患者胆固醇酯转运蛋白及某些基因缺陷

目的 分析心脑血管疾病患者和健康人血清胆固醇酯转运蛋白（CETP）水平、CETPD442G和114A突变频率和基因突变者血脂异常的特点。方法 94例心肌梗死、110例脑卒中和335名健康人,应用ELISA测定GETP浓度;合成^14C胆固醇酯标记的含载脂蛋白AI的双盘状、双层颗粒作基质测定GETP活性;并采用聚合酶链反应－限制性片断长度多态性分析CETP基因突变。结果 心梗组、脑卒中组CETP水平较健康人明显增高。心梗组、脑卒 中组和健康人D442G检出率分别为3.5%、3.6%和5％;114A检出率分别为1.2%、0.9%和1％,其中1名为健康人D442G纯合子。心梗、脑卒中患者两种CETP基因突变频率与健康人无差别。 基因突变者ETP浓度和活性仅为非基因突变者1／3,并伴有血清高密度脂蛋白胆固醇升高,甘油三酯降低。结论 心脑血管疾病患者CETP水平升高;CETP基因突变者有显著的脂蛋白异常。     

Increased high-density lipoprotein cholesterol concentration in alcoholics is related to low cholesteryl ester transfer protein activity

Cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl esters from HDL to apoB-containing lipoproteins. Since alcoholics have high HDL cholesterol and low LDL cholesterol levels, a defect in cholesteryl ester transfer could be responsible for the alcohol-induced alteration in cholesterol distribution between lipoproteins. To test this hypothesis, we compared CETP activity in plasma from 30 alcoholics without severe liver damage and 16 control subjects. Plasma CETP activity was 28% lower in the alcoholics compared with the controls (P less than 0.001), while the teetotallers among the latter had slightly higher CETP activity than those who consumed alcohol in moderation. CETP activity increased slowly after ethanol withdrawal, but did not reach the control level within the 7-day observation period. A positive correlation was observed between plasma CETP activity and the LDL cholesterol HDL cholesterol ratio (r = 0.480, P less than 0.002), whereas CETP activity showed a negative correlation with HDL cholesterol level (r = -0.467, P less than 0.001). The results indicate that defective transfer of cholesteryl esters from HDL to LDL contributes to the high HDL cholesterol levels in alcoholics.     

Cholesteryl ester transfer protein in human brain

Summary The evidence that apolipoproteins are found in the cerebrospinal fluid and low-density lipoprotein receptor is found in the brain suggests that the brain may have an active lipid transport system. In plasma, cholesteryl ester transfer protein mediates the exchange and net transfer of cholesteryl ester and triglycerides among lipoproteins. Cholesteryl ester transfer activity was measured in the cerebrospinal fluid and plasma of ten neurologically normal subjects. Cholesteryl ester transfer activity was readily detectable in cerebrospinal fluid (7.4±13% cholesteryl ester was transferred per 20 μl), and this activity was completely abolished with specific antibody against the plasma cholesteryl ester transfer protein. The concentration of cholesteryl ester transfer activity in the cerebrospinal fluid was about 12% of that found in plasma, whereas the concentration of albumin in cerebrospinal fluid was only about 0.6% of that in plasma, suggesting direct synthesis of cholesteryl ester transfer protein within the brain. Cholesteryl ester transfer activity was found in conditioned medium from human neuroblastoma and neuroglioma cells and sheep choroid plexus. The data suggest that cholesteryl ester transfer protein is synthesized and secreted in the brain. This protein could play an important role in the transport and redistribution of lipids within the central nervous system. Deceased January 1991     

Cholesteryl ester transfer protein in human brain.

The evidence that apolipoproteins are found in the cerebrospinal fluid and low-density lipoprotein receptor is found in the brain suggests that the brain may have an active lipid transport system. In plasma, cholesteryl ester transfer protein mediates the exchange and net transfer of cholesteryl ester and triglycerides among lipoproteins. Cholesteryl ester transfer activity was measured in the cerebrospinal fluid and plasma of ten neurologically normal subjects. Cholesteryl ester transfer activity was readily detectable in cerebrospinal fluid (7.4 +/- 13% cholesteryl ester was transferred per 20 microliters), and this activity was completely abolished with specific antibody against the plasma cholesteryl ester transfer protein. The concentration of cholesteryl ester transfer activity in the cerebrospinal fluid was about 12% of that found in plasma, whereas the concentration of albumin in cerebrospinal fluid was only about 0.6% of that in plasma, suggesting direct synthesis of cholesteryl ester transfer protein within the brain. Cholesteryl ester transfer activity was found in conditioned medium from human neuroblastoma and neuroglioma cells and sheep choroid plexus. The data suggest that cholesteryl ester transfer protein is synthesized and secreted in the brain. This protein could play an important role in the transport and redistribution of lipids within the central nervous system.     

Absence of cholesteryl ester transfer protein-mediated cholesteryl ester mass transfer from high-density lipoprotein to low-density lipoprotein particles is a major feature of combined hyperlipidaemia

Elevated plasma cholesteryl ester transfer protein (CETP) mass is characteristic of combined hyperlipidaemia (CHL), an atherogenic dyslipidaemia characterized by increased levels of both very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) and subnormal levels of high-density lipoprotein (HDL). CETP remodels plasma lipoproteins by promoting the heteroexchange of neutral lipids. To determine the mechanism of the CETP-mediated redistribution of cholesteryl ester (CE) between plasma lipoprotein particles in CHL, we measured CE mass transfer and exchange from HDL to apoB-containing lipoproteins under physiological conditions in the plasmas of 14 CHL patients and compared the data with those in a group of normolipidaemic subjects (NLS; n=9). The rate of CE mass transfer from HDL to VLDL was significantly increased in CHL patients (24.1 ± 3.8 μg CE transferred h^?1 mL^?1 plasma) when compared with NLS (14.4±2.6 μg CE transferred h^?1 mL^?1 plasma, P=0.0001). By contrast with control subjects, no net CE mass transfer from HDL to LDL was detected in CHL patients; transfer of radiolabelled CE to LDL was, however, observed, suggesting the occurrence of CE exchange between HDL and LDL in the absence of net CE mass transfer. The LDL fraction from CHL patients displayed a significant reduction (15%; P<0.003) in its ability to accept cholesteryl ester from HDL when compared with normolipidaemic LDL. Moreover, a reduction of 10% (P<0.02) was found in the capacity of hyperlipidaemic HDL to donate cholesteryl esters to apoB-containing lipoproteins as compared with control HDL; the reduced levels (–32%) of HDL_2b particles in CHL plasmas may account for this effect. We conclude that the low affinity of hyperlipidaemic LDL particles for CETP, taken together with the elevated plasma concentrations of a qualitatively active CE acceptor, VLDL, and the low HDL levels in CHL patients, result in the absence of net CE mass transfer from HDL to LDL in Combined hyperlipidaemia.     

Heterogeneous mutations in the human lipoprotein lipase gene in patients with familial lipoprotein lipase deficiency.

The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations.     

A delayed-addition enzyme immunoassay for the relative cholesteryl ester transfer protein mass in patients with deficient plasma cholesteryl ester transfer activity

The biochemical basis for the apparent deficiency of cholesteryl ester (CE) transfer activity was investigated in two unrelated subjects with markedly elevated high density lipoprotein-cholesterol (Atherosclerosis 1988; 70:7-12). Essentially no CE or triglyceride transfer activity was detected in the patients' plasma, utilizing four different lipid transfer assays. Using polyclonal antibodies raised against human plasma cholesteryl ester transfer protein (CETP), a delayed-addition enzyme immunoassay was developed to determine plasma CETP mass. CETP could not be detected with this assay in the plasma of the two subjects with transfer activity deficiency, indicating that the CE transfer activity deficiency in these subjects is due to the absence of plasma CETP. In addition, three hyperalphalipoproteinemic subjects with a partial deficiency of CE transfer activity had a reduced level of CETP mass. There was a good correlation between plasma CETP activity and mass levels. The principles of this immunoassay may be applicable to measure the mass levels of other proteins with catalytic activities.     

Cholesteryl ester transfer protein inhibitors: challenges and perspectives

Introduction: Cholesteryl ester transfer protein (CETP) inhibitors substantially increase the concentration of high-density lipoprotein cholesterol (HDL-C), which may have a possible beneficial effect for cardiovascular disease risk reduction.

Areas covered: Current data regarding the effects of CETP inhibitors on cardiovascular disease risk and possible mechanisms for their effects and safety are presented in this review.

Expert commentary: The first CETP inhibitor, torcetrapib, was discontinued because of increased off-target adverse effects (increased serum aldosterone and blood pressure levels). The development program of dalcetrapib and evacetrapib, which were not associated with increased blood pressure, was terminated due to futility (insufficient efficacy) concerning cardiovascular outcomes. Although the failure of torcetrapib has been attributed to specific off-target effects, there are some common characteristics between CETP inhibitors pointing to the possibility that certain adverse effects may be class-specific. The newer CETP inhibitors anacetrapib and TA-8995 have promising effects on lipid profile and metabolism (increase of HDL-C and reduction of both low-density lipoprotein cholesterol and lipoprotein (a) levels), but their cardiovascular effects and safety profile have not yet been confirmed in large outcome trials.     

Accelerated cholesteryl ester transfer in plasma of patients with hypercholesterolemia.

To discern the mechanism(s) that underlie abnormal cholesteryl ester transfer (CET) in patients with hypercholesterolemia, we have studied this dysfunctional step in reverse cholesterol transport in 13 subjects with genetically heterogeneous forms of hypercholesterolemia (HC). In all HC patients, the mass of CE transferred in whole plasma from HDL to VLDL and LDL increased rapidly initially and was significantly greater than in controls at 1, 2, and 4 h (P less than 0.005). To further characterize this disturbance, we performed a series of recombination experiments. Combining HC d less than 1.063 containing acceptor VLDL + LDL with the d greater than 1.063 fraction from controls containing donor HDL + CE-transfer protein (CETP) and not the converse combination showed the same characteristics of accelerated CET noted with intact HC plasma, indicating that abnormal transfer was associated with the HC acceptor lipoproteins. When HC VLDL and its subfractions and LDL were isolated separately and then combined with control d greater than 1.063 fractions, accelerated CET was only associated with VLDL1. Consistent with an acceleration of the neutral lipid transfer reaction occurring between HDL and VLDL1 in HC in vivo, we found that the triglyceride/CE ratio was decreased in HC VLDL1 (P less than 0.001), and increased in HDL (P less than 0.25). CETP mass was significantly increased in HC plasma (HC 2.3 +/- 4 micrograms/ml vs. control 1.3 +/- 0.3 micrograms/ml; mean +/- SD; P less than 0.025). This series of observations demonstrate that CET is accelerated in the plasma of HC patients, and this disturbance results from dysfunction of the VLDL1 subfraction rather than an elevation of CETP levels. Since an abnormality of this type in vivo can lead to the accumulation of potentially atherogenic CE-enriched apoB-containing lipoproteins in plasma, it may be an additional previously unrecognized factor that increases cardiovascular risk in HC patients.     

Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels

It has been proposed that the plasma phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids and cholesterol from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). To evaluate the in vivo role of PLTP in lipoprotein metabolism, we used homologous recombination in embryonic stem cells and produced mice with no PLTP gene expression. Analysis of plasma of F2 homozygous PLTP-/- mice showed complete loss of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and partial loss of free cholesterol transfer activities. Moreover, the in vivo transfer of [3H]phosphatidylcholine ether from very-low-density proteins (VLDL) to HDL was abolished in PLTP-/- mice. On a chow diet, PLTP-/- mice showed marked decreases in HDL phospholipid (60%), cholesterol (65%), and apo AI (85%), but no significant change in non-HDL lipid or apo B levels, compared with wild-type littermates. On a high-fat diet, HDL levels were similarly decreased, but there was also an increase in VLDL and LDL phospholipids (210%), free cholesterol (60%), and cholesteryl ester (40%) without change in apo B levels, suggesting accumulation of surface components of TRL. Vesicular lipoproteins were shown by negative-stain electron microscopy of the free cholesterol- and phospholipid-enriched IDL/LDL fraction. Thus, PLTP is the major factor facilitating transfer of VLDL phospholipid into HDL. Reduced plasma PLTP activity causes markedly decreased HDL lipid and apoprotein, demonstrating the importance of transfer of surface components of TRL in the maintenance of HDL levels. Vesicular lipoproteins accumulating in PLTP-/- mice on a high-fat diet could influence the development of atherosclerosis.     

Cholesteryl ester transfer protein gene polymorphisms are associated with carotid atherosclerosis in men

BACKGROUND: The cholesteryl ester transfer protein (CETP) is involved in the reverse cholesterol transport and is therefore a candidate gene for atherosclerosis. DESIGN: The prevalences of the I405V and the R451Q polymorphisms were studied in a population sample of 515 men and women. Genotypes were determined by PCR and carotid atherosclerosis by ultrasonography as the mean intima-media thickness (IMT) of the carotid arteries. RESULTS: The Q451 allele was associated with significantly lower intima media thickness in men (P = 0.001). The Q451 allele was, in our earlier study, associated with high plasma CETP activity in men. The VV405 genotype was associated with lower plasma CETP activity compared with the II405 genotype (P < 0.01 for the difference). In the general linear model general factorial procedure the interaction between alcohol consumption and the I405V genotype on IMT was significant (P = 0.013) in men, and when the interaction term was taken into the model the I405V genotype also significantly affected IMT (P = 0.008). The VV405 genotype seems to be most harmful for men with the highest alcohol consumption. CONCLUSIONS: We describe two polymorphisms of the CETP gene associated with intima media thickness in men. A significant interaction was found between alcohol consumption and the I405V genotype on IMT.     

Decreased affinity of low density lipoprotein (LDL) particles for LDL receptors in patients with cholesteryl ester transfer protein deficiency

Abstract. We have reported that the disorder of lipoprotein metabolism in hyperalphalipoproteinae-mic patients with a deficiency of cholesteryl ester transfer protein (CETP) is characterized by the poly-disperse low density lipoprotein (LDL) particles and the accumulation of cholesteryl ester (CE) in high density lipoprotein (HDL) particles, forming cholesterol-induced HDL (HDLc)-like particles. In the present study we have investigated the interaction of these abnormal LDL with LDL receptors of normal human fibroblasts. Since the ultracentrifugally separated LDL fraction (1.019 < d < 1.063 gmL^-1) from the CETP-deficient patients contained HDLc-like particles, these particles were removed by anti-apolipoprotein (apo) A-I immunoaffinity column chromatography. The lipoproteins eluted in the unbound fraction of this column did not contain apo A-I, so this fraction was considered to be authentic LDL. The authentic LDL of the patients were deficient in CE and rich in triglycerides and apo B. The authentic LDL itself showed polydispersity, ranging in size from 23 nm to 30 nm. The affinity of these abnormal LDL particles for LDL receptors was analysed by a competitive assay in which cold LDL from the patients or control compete with ¹²⁵I-labelled LDL for fibroblast LDL receptors. The concentration of LDL particles at which 50% of ¹²⁵I-labelled normal LDL was replaced was two to three times higher for the patients than for the normal control. Therefore, the affinity of patient LDL was thought to be reduced compared to that of control LDL. These results demonstrate that CETP may play an important role in making LDL particles homogeneous and rich in CE. This modulation of LDL by CETP may enhance the affinity of LDL for LDL receptors to deliver cholesterol to peripheral tissues.