Plasma cholesterol ester transfer protein and distribution of cell cholesterol among plasma lipoproteins in vitro in distance runners and sedentary men

Summary. Plasma cholesteryl ester transfer protein (CETP) activity and distribution of red blood cell (RBC) cholesterol among plasma lipoproteins during incubation of blood were determined in 14 distance runners and 10 sedentary men. Mean plasma CETP activity was similar in the runners (31% 10 μl^-1 18 h^-1) and the sedentary men (32% 10 μl^-1 18 h^-1). There was significantly (P<0.05) greater accumulation of cell cholesterol in the HDL fraction (runners: 0.33 mmol 1-^-1; sedentary men: 0.23 mmol l^-1) which comprised a significantly (P<0.05) larger proportion of the total amount of cell cholesterol lost to plasma (runners: 89%; sedentary men: 64%) in incubated blood from the runners. The results of this study suggest that in distance runners, high HDL concentrations are not accompanied by reduced plasma CETP levels but in conjunction with low triglyceride-rich lipoprotein levels in plasma, may promote preferential distribution of cell cholesterol into the ‘antiatherogenic’ HDL fraction.     

Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.     

Plasma protein binding of propranolol enantiomers as a major determinant of their stereoselective tissue distribution in rats

We examined whether the distribution of propranolol (PL) exhibits stereoselectivity. (+)-, (-)- or (+/-)-PL was administered by i.v. bolus injection (5 or 10 mg/kg) to rats. The concentrations of PL enantiomers in plasma (Cp) and tissues (e.g., lung, heart, brain, kidney, muscle and gastrointestinal tract) were determined at 5, 10, 30, 60 and 120 min after administration by chiral stationary-phase liquid chromatography. Plasma protein binding of PL enantiomers was evaluated by ultrafiltration. Values of tissue-to-plasma partition coefficient and tissue-to-plasma-free fraction were obtained. Cp for (+)-PL was consistently higher than that of (-)-PL in plasma. In contrast, (-)-PL showed a significantly higher distribution than (+)-PL in all tissues observed (P less than .05) at 60 min after administration of the racemate. As a result, the tissue-to-plasma partition coefficient-Cp curve was markedly different for the two enantiomers. However, because the plasma-free fraction of (+)-PL was less than that of (-)-PL, no remarkable difference was found between the concentration-dependent tissue-to-plasma-free concentration curves for the two enantiomers. In conclusion, our findings suggest that the uptake or binding of PL enantiomers to tissues is saturable and not stereoselective. Therefore, the apparent stereoselective tissue distribution of PL seems to be caused mainly by the difference in plasma protein binding of its enantiomers.     

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.     

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.     

Plasma protein binding and placental transfer of bupivacaine.

The maternal venous and umbilical venous plasma concentrations of bupivacaine were determined at delivery following epidural administration of the drug to 31 women in labor. In each case the umbilical venous plasma concentration of bupivacaine was lower than the maternal venous plasma concentration. There was no significant difference between the concentration of unbound bupivacaine in umbilical venous and maternal venous plasma at delivery. The difference in umbilical and maternal plasma concentrations of bupivacaine appears to be a consequence of greater bupivacaine binding to maternal than to fetal total plasma protein. The intersubject variation of the ratio of bupivacaine concentration in fetal plasma/bupivacaine concentration in maternal plasma was found to be related to individual variation in the extent of protein binding of bupivacaine in maternal and umbilical plasma and correlated positively with the variation of the ratio of total protein concentration in umbilical and maternal plasma.     

Inhibition of cholesteryl ester transfer protein activity by monoclonal antibody. Effects on cholesteryl ester formation and neutral lipid mass transfer in human plasma.

We have employed a neutralizing monoclonal antibody, prepared against the Mr 74,000 cholesteryl ester transfer protein (CETP), to investigate the regulation of lecithin:cholesterol acyltransferase (LCAT) activity by cholesteryl ester (CE) transfer, and also to determine which lipoproteins are substrates for LCAT in human plasma. The incubation of normolipidemic plasma led to transfer of CE from HDL to VLDL, and of triglycerides from VLDL to LDL and HDL. This net mass transfer of neutral lipids between the lipoproteins was eliminated by the monoclonal antibody. However, CE transfer inhibition had no effect on the rate of plasma cholesterol esterification in plasma incubated from 10 min to 24 h at 37 degrees C. In the absence of CE transfer, HDL and LDL exhibited cholesterol esterification activity, whereas VLDL did not. The rate of CE formation in HDL was three to four times greater than in LDL during the first hour of incubation, but CE formation in HDL decreased after 6-8 h, while that in LDL continued. Thus, (a) the Mr 74,000 CETP is responsible for all neutral lipid mass transfer in incubated human plasma, (b) the rate of CE formation in plasma is not regulated by CE transfer from HDL to other lipoproteins, and (c) HDL is the major initial substrate for LCAT; LDL assumes a more significant role only after prolonged incubation of plasma.     

Plasma creatinine as a determinant of plasma total homocysteine concentrations in the Hordaland Homocysteine Study: use of statistical modeling to determine reference limits

OBJECTIVES: We established population-based reference limits for plasma total homocysteine (tHcy) according to creatinine. DESIGN AND METHODS: In 7042 middle-aged and elderly subjects from the Hordaland Homocysteine Study, we used statistical modeling to establish nomograms for tHcy according to creatinine in the whole population and in folate-replete and healthy subgroups. RESULTS: When plotted against creatinine, tHcy 97.5th percentile almost overlapped in men and women, and, in elderly, increased up to 8 micromol/L from the 2.5th to 97.5th creatinine percentiles. Folate-replete subjects had tHcy upper limits approximately 20% below the whole population at all creatinine levels. Healthy subjects had lower creatinine, but at a given creatinine level, tHcy was the same as in the whole population. CONCLUSIONS: tHcy difference between men and women is mostly explained by creatinine. The tHcy-reducing effect of folate is independent of creatinine. In elderly people, creatinine should be taken into account when assessing tHcy levels.     

Plasma protein profiling: the diagnostic evaluation of disorders in plasma protein composition by a new immunoelectrophoretic method.

A new electroimmunoprecipitation technique is presented by means of which a great variety of antigens e.g. plasma proteins can be simultaneously and quantitatively determined with a single-step electrophoretic separation. The essential features of the new technique are: (a) subdivision of the antibody gel into gel strips containing monospecific antibodies to individual plasma proteins. (b) sample application as a "sample gel" filling a trough over the width of the immunoplate. Quantitation is based on the fact that the distance an antigen can migrate within a gel containing a defined amount of specific antibody directed against the antigen is determined by the concentration of the appropriate antigen within the sample. The area where the antigen is finally completely consumed by immunoprecipitation and antibody present in excess is sharply delineated. The applicability of the method in simultaneous quantitative determination of 15 plasma proteins is demonstrated with plasma from healthy blood donors and patients with various diseases. The advantage of the new technique as compared to commonly used clinical acetate folia electrophoresis is the high degree of specificity for the determination of a great number of individual, diagnostically meaningful plasma proteins. The advantage over common quantitative two-dimensional immunoelectrophoresis is its uncomplicated way of evaluation. The potential clinical application of the new quantitative immunoelectrophoretic technique in diagnostic screening and differential diagnosis is discussed.     

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.     

Accelerated transfer of cholesteryl esters in dyslipidemic plasma. Role of cholesteryl ester transfer protein.

Plasma cholesteryl esters, synthesized in the high density lipoproteins (HDL), may be transferred to other lipoproteins by a cholesteryl ester transfer protein (CETP). We found a twofold increase in mass transfer of cholesteryl ester from HDL to apoB-containing lipoproteins in incubated hypercholesterolemic rabbit plasma compared with control. There was a two- to fourfold increase in the activity of CETP, measured in an isotopic assay in hypercholesterolemic plasma. A CETP-like molecule was isolated in increased amounts from hypercholesterolemic plasma. Incubated plasma from four dysbetalipoproteinemic subjects also showed an increase (threefold) in cholesteryl ester mass transfer, compared with normolipidemic controls. There was a twofold increase in the activity of CETP, assayed in whole or lipoprotein-free plasma. Thus, there is increased transfer of cholesteryl esters from HDL to potentially atherogenic apoB-containing lipoproteins in dyslipidemic rabbit and human plasma. The enhanced transfer results in part from increased activity of CETP, possibly reflecting an increase in 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.     

Effect of adiposity on plasma lipid transfer protein activities: a possible link between insulin resistance and high density lipoprotein metabolism

Abstract. The mechanisms responsible for the decreased high density lipoprotein (HDL) cholesterol levels associated with obesity and insulin resistance are not well understood. Lecithin: cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) are key factors in the esterification of cholesterol in HDL and the subsequent transfer of cholesteryl ester towards apolipoprotein B-containing lipoproteins. Phospholipid transfer protein (PLTP) may be involved in the regulation of HDL particle size. We therefore measured the activities of LCAT, CETP and PLTP using exogenous substrate assays, as well as lipids, lipoproteins, insulin and C-peptide in fasting plasma from eight healthy obese men (body mass index >27 kg m^-2) and 24 non-obese subjects. The obese men had lower levels of HDL cholesterol (P<0·05) and higher levels of plasma triglycerides (P<0·05), insulin (P<0·05) and C-peptide (P<0·01), as compared to the quartile of subjects with the lowest body mass index (BMI <22·4 kg m^-2). CETP and PLTP activities were elevated in the obese men by 35% (P<0·01) and by 15% (P<0·05), respectively. LCAT activity was comparable among the quartiles. Linear regression analysis showed that CETP activity was positively correlated with body mass index (P<0·02), fasting blood glucose (P7lt;0·05) and plasma C-peptide (P<0·05). PLTP activity was positively related to body mass index (P<0·01), waist to hip circumference ratio (P<0·001), as well as to fasting blood glucose (P<0·05) and plasma C-peptide (P<0·05) It is concluded that the activities of CETP and PLTP are influenced by adiposity and possibly by insulin resistance. Elevated lipid transfer protein activities may provide a mechanism that contributes to alterations in HDL in insulin resistant states.     

Beta-adrenoceptor blockade and plasma lipoproteins. Comparison of the effects of propranolol and pindolol on plasma lipoproteins including high-density lipoprotein subfractions

Beta-Adrenoceptor blocker therapy is known to cause disturbances of the lipoprotein profile. The long-term effects of beta-adrenoceptor blockers and the influence of intrinsic sympathomimetic activity (ISA) has not been clearly defined. We measured serum lipoproteins during chronic beta-adrenoceptor blockade in patients with stable angina pectoris treated with propranolol (without ISA) (n = 21) or pindolol (with ISA) (n = 19). No significant changes occurred in the lipoprotein profile of the patients taking pindolol. In those taking propranolol, very low density lipoprotein (VLDL) increased at 52 weeks (P less than 0.05) and total high density lipoprotein (HDL) decreased at 26 weeks (P less than 0.01) and at 52 weeks (P less than 0.05). However, HDL2 rose significantly at 52 weeks (P less than 0.05). There was a corresponding increase in HDL2/HDL3 ratio. We conclude that pindolol is less likely to exert a harmful effect on plasma lipoproteins than beta-adrenoceptor blockers without ISA.     

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.     

The passage of apoproteins from plasma lipoproteins into the lipoproteins of peripheral lymph in man.

1. The transport of apoprotein B from the lipoprotein of plasma into the lipoproteins of lymph draining the foot has been studied in four men with type III hyperlipoproteinaemia. 2. Three subjects were given autologous 125I-labelled very-low-density lipoprotein (VLDL) and 131I-labelled low-density lipoprotein (LDL) by intravenous injection; the fourth was given autologous 125I-labelled VLDL and 131I-labelled intermediate-density lipoprotein (IDL) plus LDL. 3. The 125I/131I ratios in serum and lymph apoprotein B, and the 125I and 131I specific radioactivities of apoprotein B in VLDL, IDL and LDL from serum and lymph, indicate that apoprotein B in the circulating VLDL can reach peripherallymph without the intermediacy of circulating LDL.