Accumulation of apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with plasma cholesteryl ester transfer protein deficiency.

This study characterized the plasma lipoproteins of familial hyperalphalipoproteinemic patients with or without deficiency of cholesteryl ester transfer protein (CETP) activity. The subjects with CETP deficiency have increased levels of apolipoprotein (apo) E. The increased concentration of apo E in these subjects was correlated to the appearance of apo E-rich high density lipoproteins (HDL). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed that these lipoproteins contained predominantly the apo E (82%) and little amount of apo A-I (18%). These apo E-rich HDL displayed a much higher affinity than human LDL in binding to LDL receptors on human fibroblasts. Furthermore, 3.5 times fewer apo E-rich HDL than LDL were required to saturate the receptors on fibroblasts. These data indicated that the apo E-rich HDL in CETP-deficient human subjects contained multiple copies of apo E and bound to the LDL receptor through multiple interactions. The apo E-rich HDL, with similar properties as cholesterol-induced apo E HDLc, were not detectable in normal human subjects or in hyperalphalipoproteinemic subjects with normal CETP activity. The apo E-containing HDL in the latter subjects were smaller and contained only small amounts of apo E (14%). The difference in apo E-containing HDL in these subjects suggests a correlation between CETP level and the appearance of apo E-rich HDL.     

Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.     

A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins.

Plasma HDL are a negative risk factor for atherosclerosis. Cholesteryl ester transfer protein (CETP; 476 amino acids) transfers cholesteryl ester from HDL to other lipoproteins. Subjects with homozygous CETP deficiency caused by a gene splicing defect have markedly elevated HDL; however, heterozygotes have only mild increases in HDL. We describe two probands with a CETP missense mutation (442 D:G). Although heterozygous, they have threefold increases in HDL concentration and markedly decreased plasma CETP mass and activity, suggesting that the mutation has dominant effects on CETP and HDL in vivo. Cellular expression of mutant cDNA results in secretion of only 30% of wild type CETP activity. Moreover, coexpression of wild type and mutant cDNAs leads to inhibition of wild type secretion and activity. The dominant effects of the CETP missense mutation during cellular expression probably explains why the probands have markedly increased HDL in the heterozygous state, and suggests that the active molecular species of CETP may be multimeric.     

Contribution of glycaemic control, endogenous lipoproteins and cholesteryl ester transfer protein to accelerated cholesteryl ester transfer in IDDM

Abstract In an earlier study we demonstrated that the transfer of cholesteryl ester (CET) estimated as the net mass of CE lost from HDL to the apoB-containing lipoproteins (VLDL + LDL) during incubation of plasma is accelerated in normolipidaemic patients with insulin-dependent diabetes mellitus (IDDM). Recombination experiments with isolated lipoprotein fractions employing this same mass transfer assay indicated that this disturbance resulted from dysfunction of VLDL and not from changes in the activity of CE transfer protein (CETP). In this study, we sought first to determine whether CET estimated with an isotopic method that measures the transfer of radiolabelled CE from exogenous HDL from non-diabetic controls to endogenous VLDL + LDL was also increased in IDDM and, if so, the extent to which this disturbance was affected by glycaemic control, VLDL and CETP. As observed with the mass transfer assay, the rate of transfer of the HDL-CE label to VLDL + LDL was also significantly accelerated in IDDM plasma (IDDM: k = 0·256±0·07; control: k = 0·092±0·05; mean±SD; P < 0·001). Fasting glucose and fructosamine correlated with both isotopic transfer (k) (r= 0·54, P= 0·009; r= 0·57, P= 0·005, respectively) and the mass of CE transferred at 2 h (r= 0·55, P= 0·006; r= 0·59, P= 0·004, respectively). Recombination experiments revealed that isotopic CET was accelerated when: (a) IDDM VLDL were combined with controls HDL and d > 1·21 fractions; and (b) IDDM d > 1·21 plasma fractions containing CETP were combined with controls VLDL + LDL and HDL. While CETP concentrations in a subset of the study group were higher in the diabetic than in the non-diabetic controls, the difference was not statistically significant (IDDM 2·25±0·97 vs. control 1·58±0·58 μg ml^-1; mean±SD; P<0·1). These findings indicate that dysfunction of VLDL and increased CETP concentrations both contribute to the pathological acceleration of isotopic transfer in IDDM plasma and that the magnitude of this proatherogenic defect correlates closely with glycaemic control.     

Delayed catabolism of high density lipoprotein apolipoproteins A-I and A-II in human cholesteryl ester transfer protein deficiency.

Deficiency of the cholesteryl ester transfer protein (CETP) in humans is characterized by markedly elevated plasma concentrations of HDL cholesterol and apoA-I. To assess the metabolism of HDL apolipoproteins in CETP deficiency, in vivo apolipoprotein kinetic studies were performed using endogenous and exogenous labeling techniques in two unrelated homozygotes with CETP deficiency, one heterozygote, and four control subjects. All study subjects were administered 13C6-labeled phenylalanine by primed constant infusion for up to 16 h. The fractional synthetic rates (FSRs) of apoA-I in two homozygotes with CETP deficiency (0.135, 0.134/d) were found to be significantly lower than those in controls (0.196 +/- 0.041/d, P < 0.01). Delayed apoA-I catabolism was confirmed by an exogenous radiotracer study in one CETP-deficient homozygote, in whom the fractional catabolic rate of 125I-apoA-I was 0.139/d (normal 0.216 +/- 0.018/d). The FSRs of apoA-II were also significantly lower in the homozygous CETP-deficient subjects (0.104, 0.112/d) than in the controls (0.170 +/- 0.023/d, P < 0.01). The production rates of apoA-I and apoA-II were normal in both homozygous CETP-deficient subjects. The turnover of apoA-I and apoA-II was substantially slower in both HDL2 and HDL3 in the CETP-deficient homozygotes than in controls. The kinetics of apoA-I and apoA-II in the CETP-deficient heterozygote were not different from those in controls. These data establish that homozygous CETP deficiency causes markedly delayed catabolism of apoA-I and apoA-II without affecting the production rates of these apolipoproteins.     

Mechanisms of enhanced cholesteryl ester transfer from high density lipoproteins to apolipoprotein B-containing lipoproteins during alimentary lipemia.

In vitro lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins as a result of lipolysis-induced alterations in lipoprotein lipids that lead to increased binding of CETP. To determine if there are similar changes during alimentary lipemia, we measured the transfer of cholesteryl esters from HDL to apo B-containing lipoproteins in incubated fasting and postprandial plasma. In seven normolipidemic subjects there was 2-3-fold stimulation of cholesteryl ester transfer in alimentary lipemic plasma. Cholesteryl ester transfer was stimulated when either the d less than 1.063-or d greater than 1.063-g/ml fraction of lipemic plasma was recombined with its complementary fraction of fasting plasma. To determine the distribution of CETP, plasma was fractionated by agarose chromatography and CETP activity was measured in column fractions in a standardized assay. In fasting plasma, most of the CETP was in smaller HDL, and a variable fraction was nonlipoprotein bound. During lipemia there was increased binding of CETP to larger phospholipid-enriched HDL and in two subjects an increase in CETP in apo B-containing lipoproteins. The total CETP activity of fractions of lipemic plasma was increased 1.1-1.7-fold compared with fasting plasma. Lipemic CETP activity was also increased when measured in lipoprotein-free fractions after dissociation of CETP from the lipoproteins. When purified CETP was incubated with phospholipid-enriched HDL isolated from alimentary lipemic or phospholipid vesicle-treated plasma, there was increased binding of CETP to the phospholipid-enriched HDL compared with fasting HDL, with a parallel stimulation in CETP activity. Thus, the pronounced stimulation of cholesteryl ester transfer during alimentary lipemia is due to (a) an increased mass of triglyceride-rich acceptor lipoproteins, (b) a redistribution of CETP, especially increased binding to larger phospholipid-enriched HDL, and (c) an increase in total activity of CETP, perhaps due to an increased CETP mass.     

Variation at the cholesteryl ester transfer protein gene in relation to plasma high density lipoproteins cholesterol levels and carotid intima-media thickness

BACKGROUND: Cholesteryl ester transfer protein (CETP) plays a major role in lipoprotein metabolism. We have screened the CETP gene for mutations and polymorphisms regulating high density lipoproteins cholesterol (HDL-C) levels and the development of atherosclerosis, and found some polymorphisms (I405V and R451Q) to have minor effects. DESIGN: The purpose of this study was to investigate the combined effect of the several polymorphisms of the CETP gene so far found on HDL-C levels and carotid intima-media thickness (IMT), and, in addition, to study whether the recently found functional polymorphism in the promoter region of the CETP gene (C to A, - 629 relative to the first transcribed nucleotide) explains the previous associations due to linkage disequilibrium. The genotypes were determined in a population sample of 481 men and women. RESULTS: There were no significant differences in plasma CETP activity or carotid IMT between the genotypes of the promoter polymorphism. The women with the CC genotype of the promoter polymorphism had the lowest HDL-C levels (P < 0.001), but no such difference was seen in men. Detected polymorphisms of the CETP gene explained about 8% of the variation in HDL-C in women and about 7 and 10% of the variation in carotid IMT in women and men, respectively. The associations of the promoter, I405V and R451Q-A373P polymorphisms with HDL-C and carotid IMT seemed to be independent of each other. The associations with IMT were independent of total HDL-C levels, suggesting that HDL subfractions may have more effect on IMT. CONCLUSION: The CETP gene locus was found to be polymorphic and its polymorphisms explained a reasonable proportion of the variation in the degree of carotid atherosclerosis.     

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.     

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.     

胆固醇酯转运蛋白活性测定在心脑血管疾病监测中的意义

目的探讨胆固醇酯转运蛋白(CETP)活性与心脑血管疾病的关系。方法应用体外合成14C标记的高密度脂蛋白(HDL)样颗粒,测定了45例冠心病患者、26例脑卒中患者和40例健康人CETP活性。结果冠心病患者、脑卒中患者CETP活性分别为(17.6±5.4)%、(15.2±3.8)%,而健康人为(12.7±2.0)%,两组患者与健康人比较差异均有统计学意义(P<0.01)。结论CETP与动脉粥样硬化的发生、发展密切相关。     

Probucol increases cholesteryl ester transfer protein activity in hypercholesterolaemic patients

Probucol, a widely used lipid lowering drug, reduces both low- and high-density (LDL and HDL) lipoprotein levels and can induce a regression of tissue lipid deposits in both animals and man. The suggested mechanism(s) involve the prevention of LDL oxidative modifications and, possibly, an improvement in the reverse cholesteryl ester transport system. Probucol administration to 10 hypercholesterolaemic patients increased the activity of the cholesteryl ester transfer protein (CETP) by 50%. The rise of CETP activity was significantly related with the plasma steady-state drug levels (r = 0.51, P less than 0.005), thus suggesting that probucol may directly stimulate CEPT synthesis and/or release. Furthermore, CETP activity was inversely related with HDL-cholesterol levels, both in the whole series of 10 patients (r = -0.56, P less than 0.001) and, more so, in the single individuals (r between -0.77 and -0.97), thus suggesting that the reduction of plasma HDL-cholesterol levels is a direct consequence of CETP stimulation. These findings support the hypothesis that an improvement in the reverse cholesteryl ester transport is a major mechanism of probucol and that this may explain the drug induced plasma lipoprotein changes.     

Monoclonal antibody inhibition of cholesteryl ester transfer protein activity in the rabbit. Effects on lipoprotein composition and high density lipoprotein cholesteryl ester metabolism.

Cholesteryl ester transfer protein (CETP) promotes in vitro transfer of cholesteryl ester (CE) and triglyceride (TG) between lipoproteins. We studied the function of CETP in vivo in rabbit lipoprotein metabolism using a neutralizing monoclonal antibody (MAb, TP1) to CETP. Rabbits were injected with TP1 (n = 8), or irrelevant MAb or saline (control, n = 8), resulting in an initial 71% inhibition of CETP, which fell to 45% after 48 h. HDL CE rose in the inhibited animals, reaching levels that doubled initial and control values at 48 h (P less than 0.001). HDL TG fell reciprocally, but HDL protein did not change, suggesting a CE for TG exchange. VLDL CE/TG decreased. Rabbits were also given [3H]cholesteryl ether HDL (a CE analogue). CETP inhibition delayed the initial clearance of radioactivity from HDL (control 6.8 vs. TP1 4.1 pools/d) and plasma (7.8 vs. 5.2 pools/d). We conclude that CETP plays a quantitatively important role in HDL CE catabolism in the rabbit, promoting the exchange of TG for CE and the clearance of CE from plasma.     

Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.

Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.     

胆固醇酯转运蛋白的提纯和鉴定

目的建立一种新的分离纯化人血清胆固醇酯转运蛋白（ＣＥＴＰ）的方法,为制备ＣＥＴＰ单克隆抗体和ＣＥＴＰ浓度测定提供物质基础。方法制备无脂血清,用ＢｕｔｙｌＳｅｐｈａｒｏｓｅ４ＦＦ、ＣＭＳｅｐｈａｒｏｓｅＦＦ和ＳｅｐｈａｃｒｙｌＳ２００三步柱层析分离、纯化ＣＥＴＰ。结果纯化ＣＥＴＰ经ＳＤＳＰＡＧＥ电泳鉴定呈单一区带,分子量６５０００,蛋白质含量０３２５ｍｇ／ｍｌ,与国外和作者研制ＣＥＴＰ单抗的免疫斑点试验呈特异性反应,与纯化脂蛋白（ａ）、载脂蛋白（Ａｐｏ）Ａ１、ＡｐｏＢ、ＩｇＧ、ＩｇＡ、ＩｇＥ、Ｃ３、Ｃ４抗体无免疫反应。结论经三步柱层析获得了纯化ＣＥＴＰ,得率约２５％,活性每小时３０ｎｍｏｌ／μｇ。     

新生儿血清胆固醇酯转运蛋白浓度及其与LDL组成的关系

目的分析新生儿血清胆固醇酯转运蛋白(CETP)浓度与LDL组成的关系,以及对血脂、脂蛋白水平的影响。方法采用ELISA法测定40例脐带血CETP浓度,并与80例健康成人比较;用序列超速离心法分离LDL后再用常规方法分析其中TG、CE含量。结果新生儿CETP呈偏态分布、中位数0.20 mg/L,范围(0.11~0.49)mg/L,均值(0.25±0.11)mg/L,明显低于成人(2.19±2.04)mg/L(P<0.0001);男女间无显著差异[(0.26±0.10)mg/L vs(0.22±0.16)mg/L,P>0.05]。新生儿血脂、脂蛋白浓度均低于成人,LDL-CE/LDL-TG比值也明显低于成人(1.5 vs 4.7)。结论新生儿血清CETP浓度仅是成人的九分之一,低CETP水平可能是新生儿LDL-CE/LDL-TG比值低的主要原因。     

Accelerated cholesteryl ester transfer in patients with insulin-dependent diabetes mellitus

Abnormalities in cholesteryl ester transfer (CET) may play a role in the development of diabetic arterial vascular complications. To assess this important step systematically in reverse cholesterol transport, we have studied 20 treated, clinically stable, normolipidaemic patients. Contrary to the impairment in CET described previously in NIDDM, the mass of CE transferred from HDL to VLDL + LDL was significantly greater in IDDM patients than in controls at 1,2, and 4 h (P less than 0.001). When the d less than 1.063 plasma fractions from IDDM subjects were combined with controls d less than 1.063 fractions, an accelerated CET response was observed which was identical to that found in intact IDDM plasma. This finding, which indicates that this disturbance in CET was associated with the acceptor lipoproteins, was confirmed when we found that it was reproduced by the addition of IDDM VLDL and not LDL to control d greater than 1.063 fractions. Changes observed in lipoprotein core lipid composition were consistent with accelerated CET occurring in IDDM in vivo: the TG/CE core lipid ratio was decreased in VLDL from six subjects (diabetic 9.5 +/- 0.8 vs control 12.9 +/- 3.4; P less than 0.1) and increased in their HDL (diabetic 0.55 +/- 0.11 vs control 0.42 +/- 0.04; P less than 0.025). No correlation was demonstrable between estimates of diabetic control (glycoalbumin, fasting glucose) and CET. These data indicate that CET may be abnormally increased in normolipidaemic IDDM patients. A defect of this type may be atherogenic because it increases the number of lipoprotein particles in plasma which resemble cholesteryl ester-enriched chylomicron and VLDL remnants but whose normal receptor-mediated catabolism may be altered.     

慢性肾衰透析患者胆固醇酯转运蛋白及血脂变化

本文检测77例慢性肾衰血透患者(HD)的胆固醇酯转运蛋白(CETP)和血脂,结果显示HD患者较对照组的CETP、TC、HDL-C、apoAI明显降低,TG明显升高.按TG分组,高TG组和正常TG组HD患者CETP仍显著低于对照组,但高TG组HD较正常TG组HD患者TC、apoB明显升高,apoAI、HDL-C下降.CETP和血脂异常与HD患者高发心、脑血管疾病密切相关,其发病机理需进一步探讨.