Mass Transfer Of Cholesteryl Esters Research Articles

Cholesteryl ester transfer protein and its inhibitors

Most of the cholesterol in plasma is in an esterified form that is generated in potentially cardioprotective HDLs. Cholesteryl ester transfer protein (CETP) mediates bidirectional transfers of cholesteryl esters (CEs) and triglycerides (TGs) between plasma lipoproteins. Because CE originates in HDLs and TG enters the plasma as a component of VLDLs, activity of CETP results in a net mass transfer of CE from HDLs to VLDLs and LDLs, and of TG from VLDLs to LDLs and HDLs. As inhibition of CETP activity increases the concentration of HDL-cholesterol and decreases the concentration of VLDL- and LDL-cholesterol, it has the potential to reduce atherosclerotic CVD. This has led to the development of anti-CETP neutralizing monoclonal antibodies, vaccines, and antisense oligonucleotides. Small molecule inhibitors of CETP have also been developed and four of them have been studied in large scale cardiovascular clinical outcome trials. This review describes the structure of CETP and its mechanism of action. Details of its regulation and nonlipid transporting functions are discussed, and the results of the large scale clinical outcome trials of small molecule CETP inhibitors are summarized.

The inhibition of cholesteryl ester transfer protein: a long and winding road

The presence in human plasma of a protein that promotes bidirectional transfers of neutral lipids (cholesteryl esters and triglycerides) between all lipoprotein particles was first reported in 1978 (1, 2). This protein was identified as cholesteryl ester transfer protein (CETP) and subsequently cloned in 1987 by Drayna et al. (3). CETP promotes the equilibration of cholesteryl esters and triglycerides between HDL, LDL, and triglyceride-rich lipoproteins (TRL), which include VLDL that are produced in the liver, intestinally-derived chylomicrons, and chylomicron remnants. As most of the cholesteryl esters in plasma originate in HDL in the reaction catalyzed by lecithin:cholesterol acyltransferase, and triglycerides mostly enter the plasma compartment as a component of TRL, this process of equilibration results in a net mass transfer of cholesteryl esters from potentially anti-atherogenic HDL particles to LDL and TRL, which are known to be atherogenic.

Net mass transfer of plasma cholesteryl esters and lipid transfer proteins in normolipidemic patients with peripheral vascular disease.

The role of plasma cholesteryl ester transfer and lipid transfer proteins in atherosclerosis is unclear. Recent data suggest both antiatherogenic and atherogenic properties for cholesteryl ester transfer protein (CETP). The overall effect of CETP on atherosclerosis may thus vary depending on individual lipid metabolism. To test whether lipid transfer parameters are of importance even in patients without major lipid risk factors for atherosclerosis, CETP mass and activity, net mass transfer of cholesteryl esters between endogenous lipoproteins (CET), and phospholipid transfer protein (PLTP) activity were determined in plasma from 18 normolipidemic male patients with peripheral vascular disease and 21 controls. Furthermore, lecithin: cholesterol acyltransferase (LCAT) activity was tested. The results show that CETP mass, CETP activity, and LCAT activity are not different between patients and controls. However, specific CETP activity (CETP activity/CETP mass) is lower in the patients (P < .02). On the contrary, higher CET is observed in patients' plasma (P < .001). Increased plasma PLTP activity (P = .052) is demonstrable in the patients. If the data of all subjects are combined, CET correlates positively with triglycerides ([TG], r = .45, P < .001) and with PLTP activity (r = .32, P < .05) but negatively with specific CETP activity (r = -.37 P < .05). CET and specific CETP activity remain significantly different in TG-matched patients and controls and are more strongly interrelated (r = -.71, P < .001), suggesting a higher and selective influence of lipid transfer inhibitor(s) on CET and CETP activity in the patients. CET allows the best discrimination between patients and controls in univariate and multivariate analysis. Eighty-eight percent of the subjects are correctly classified by CET as a single parameter. The results suggest that increased CET in the patients may reflect atherogenic alterations in TG metabolism and/or in lipid transfer protein activities despite normal fasting lipoprotein levels.

Lipoprotein composition influences cholesteryl ester transfer in alcohol abusers

Alcohol use is known to increase high-density lipoprotein (HDL) cholesterol, which is at least in part mediated by the alcohol-induced reduction in plasma cholesteryl ester transfer protein (CETP) activity and mass. We have shown that the high plasma HDL concentration reduces the CETP-mediated net mass transfer of cholesteryl esters from HDL to very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), or even reverses the direction of transfer in plasma incubations. Therefore, we studied the effect of lipoprotein composition on lipid net mass transfers in 14 male alcohol abusers and nine male control subjects by incubating plasma for up to 2 h. The cholesteryl ester net mass transfer in the alcohol abusers was mainly predicted by the VLDL and LDL lipid composition in multiple linear regression, while the HDL composition was the main factor in the controls. The observed difference in the effect of the lipoprotein composition on cholesteryl ester net mass transfer support our previous finding in rabbits that CETP binding to lipoproteins may differ during ethanol oxidation. The results suggest that ethanol oxidation induces alterations which may affect the binding of CETP to lipoproteins.

Differential Interaction of the Human Cholesteryl Ester Transfer Protein with Plasma High Density Lipoproteins (HDLs) from Humans, Control Mice, and Transgenic Mice to Human HDL Apolipoproteins

Plasma high density lipoproteins (HDLs) from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoprotein A-II (HuAIITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with the human cholesteryl ester transfer protein (CETP) was studied. Whereas cholesteryl ester transfer rates were gradually enhanced by the addition of moderate amounts of HDL from the different sources, striking differences appeared when HDL levels kept increasing beyond a maximal transfer value. Indeed, while a plateau value corresponding to maximal CETP activity was maintained when raising the concentration of HuAITg HDL and HuAIAIITg HDL, inhibitions could be observed with the highest levels of human, control mouse, and HuAIITg mouse HDL. The concentration-dependent inhibition of CETP activity could be reproduced by the addition of delipidated HDL apolipoproteins from control mice, but it was abolished by a 1-h preheating treatment at 56 degrees C. In contrast, no significant inhibition of CETP activity was observed with the delipidated protein moiety of HuAITg HDL, and cholesteryl ester transfer rates remained unchanged before and after a 1-h, 56 degrees C preheating step. Finally, the CETP-mediated transfer of radiolabeled cholesteryl esters from human low density lipoprotein to human HDL was significantly higher in the presence of lipoprotein-deficient plasma from HuAITg mice than in the presence of lipoprotein-deficient plasma from control mice. Interestingly, cholesteryl ester transfer rates measured with both control and HuAITg lipoprotein-deficient plasmas became remarkably similar following a 1-h, 56 degrees C preheating treatment. It is concluded that human, control mouse, and HuAIITg mouse HDL contain a heat-labile lipid transfer inhibitory activity that is absent from HDL of HuAITg and HuAIAIITg mice. Alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitory activities.

Reduced cholesteryl ester transfer in plasma of patients with lipoprotein lipase deficiency.

The net mass transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to the apolipoprotein (apo) B-containing lipoproteins, very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in plasma (cholesteryl ester transfer (CET)) from three patients lacking lipoprotein lipase (LpL) activity was significantly lower (P < 0.001) than in plasma from fasting control subjects with comparable triglyceride levels. Chylomicrons isolated from LpL-deficient fasting plasma showed the same low level of CET activity as observed in the intact plasma when combined with HDL and cholesteryl ester transfer protein (CETP)-containing d 1.063 g/ml bottom fractions from control subjects. Preincubation of chylomicrons and large triglyceride-rich lipoproteins (Sf > 400) from LpL-deficient plasma with milk LpL, however, stimulated the capacity to engage in CET 4- to 5-fold to the same level as chylomicrons and VLDL from control subjects after a fat load. Consistent with these measurements of CET activity in plasma, chylomicrons obtained from the LpL-deficient subjects after a 14-h fast had higher TG/CE ratios than chylomicrons from controls 3 h after ingesting a fat load (LpL-deficient 26.3 +/- 9.0 vs. controls 6.9 +/- 2.1; mean +/- SD). The mass of CETP did not differ in LpL-deficient and control subjects (LpL-deficient 1.03 +/- 0.22 micrograms/ml vs. controls 1.58 +/- 0.58 micrograms/ml). These studies are consistent with earlier in vitro studies showing that the actions of lipoprotein lipase and its lipolytic products are essential, for maximal cholesteryl ester transfer protein activity.

Changes in high density lipoprotein subfraction lipids during neutral lipid transfer in healthy subjects and in patients with insulin‐dependent diabetes mellitus

While it is known that the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to the apo B-containing lipoproteins is increased in patients with diabetes, the extent to which the various lipoprotein fractions engage in neutral lipid exchange and the magnitude to which triglyceride (TG) is translocated is not known. To examine in greater detail neutral lipid net mass transfer in diabetes, the HDL subfractions and the apo B-containing lipoproteins were separated, and the net mass transfer of CE and TG was compared to that of control subjects. In both groups, bidirectional transfer of CE from HDL3 to very low density lipoprotein (VLDL) + low density lipoprotein (LDL) and of TG from VLDL + LDL to HDL3, took place, but this process was significantly greater (P < .01) in insulin-dependent diabetes mellitus (IDDM). In contrast, CE and TG accumulated in HDL2 to a similar degree in normal and IDDM subjects. In recombination experiments with each of the apo B-containing lipoproteins, IDDM VLDL had a greater capacity to facilitate the exchange of core lipids from both IDDM and control HDL3: on the other hand, LDL from IDDM and control subjects both donated TG and CE to HDL2 and affected little change in HDL3. These findings indicate that all the major plasma fractions normally participate in the trafficking of CE and TG among the lipoproteins during neutral lipid transfer and show that the principal perturbation in cholesteryl ester transfer in IDDM involves altered interaction between VLDL and the HDL3 subfraction.

Evidence for Nonesterified Fatty Acids as Modulators of Neutral Lipid Transfers in Normolipidemic Human Plasma

The relations between the level of plasma nonesterified fatty acid (NEFA) and both the mass concentration and activity of the cholesteryl ester transfer protein (CETP) were studied in fasted normolipidemic subjects. Plasma NEFA correlated positively with both CETP mass concentration (r = .50; P < .01) and the transfer of cholesteryl ester from HDL toward plasma VLDL+LDL (CETHDL-->VLDL+LDL activity) (r = .46; P < .05) but not with the transfer of cholesteryl ester from LDL toward plasma HDL (CETLDL-->HDL activity) (r = .05; NS). The high binding capacity of albumin for NEFA was used to investigate whether lipoprotein-bound NEFAs were implicated in the modulation of the cholesteryl ester transfer reaction. As compared with nonsupplemented controls, the addition of an excess of fatty acid-free albumin (8 g/L) to total normolipidemic plasmas reduced CETHDL-->VLDL+LDL activity (18.3 +/- 5.5% versus 9.8 +/- 3.1%; P < .0001) but not CETLDL-->HDL activity (22.3 +/- 4.5% versus 23.3 +/- 5.1%; NS). Moreover, CETHDL-->VLD+LDL and CETLDL-->HDL activities correlated negatively when measured in native plasma (r = -.45; P < .05) but positively when measured in albumin-supplemented plasma (r = .40; P < .05). In long-term incubation experiments, lipoprotein-bound NEFA increased the net mass transfer of cholesteryl esters from HDL toward VLDL+LDL but reduced the net mass transfer of triglycerides in the opposite direction, from VLDL+LDL toward HDL. Taken together, data of the present study brought strong and concordant arguments in favor of a dual effect of plasma NEFA in modulating both the mass and the activity of CETP in vivo.

Increased net mass transfer of cholesteryl esters and phospholipid transfer protein activity but normal cholesteryl ester transfer protein mass and activity in patients with peripheral vascular disease

Increased net mass transfer of cholesteryl esters and phospholipid transfer protein activity but normal cholesteryl ester transfer protein mass and activity in patients with peripheral vascular disease

Preferential cholesteryl ester acceptors among the LDL subspecies of subjects with familial hypercholesterolemia.

Elevated cholesteryl ester transfer protein-mediated transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) may contribute to the atherogenicity of LDL in subjects with familial hypercholesterolemia (FH). To identify the major CE acceptors among LDL subspecies, we investigated the qualitative and quantitative features of CE transfer and exchange to LDL on incubation of plasma under physiological conditions. LDL subspecies were fractionated by density-gradient ultra-centrifugation. Both mass transfer and exchange of HDL CE to and with very-low-density lipoprotein plus intermediate-density lipoprotein and LDL were linear for the first 6 hours of incubation. Thereafter mass transfer ceased, but exchange continued at a comparable rate. The rate of CE mass transfer to apolipoprotein B-containing lipoproteins was significantly enhanced in heterozygous FH subjects compared with normolipidemic individuals (91.6 +/- 28.2 versus 52.9 +/- 19.6 micrograms CE/h per milliliter plasma, FH versus normal subjects, P < .02). In FH subjects the predominant LDL subspecies (LDL 3 and 4, d = 1.029 to 1.050 g/mL) accounted for 59.7 +/- 9.2% of the total CE transferred to LDL from HDL. By contrast, expression of CE mass transfer relative to the mass of each lipoprotein acceptor showed the triglyceride (TG)-rich (10.7% to 17.3%), light LDL subspecies (LDL 1 and 2, d = 1.019 to 1.029 g/mL) to represent the preferential CE acceptors (LDL 1 and 2, 94.8 to 136.5 micrograms CE/mg LDL mass; LDL 3 through 5 [d = 1.029 to 1.063 g/mL], 47.1 to 64.1 micrograms CE/mg LDL mass).(ABSTRACT TRUNCATED AT 250 WORDS)

A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies. Identification of preferential cholesteryl ester acceptors in human plasma.

To date, several methods have been developed to determine the activity of plasma lipid transfer proteins. These methods have largely involved the addition of the transfer protein in question to labeled substrates, followed by prolonged incubation (4 to 18 hours) and subsequent evaluation of the radioactivity transferred to precipitated low-density lipoprotein (LDL). While adequate for determining the activity of cholesteryl ester transfer protein (CETP), these methods generally do not take into account the composition or levels of lipoproteins present within a given individual plasma because pools of high-density lipoprotein (HDL) are labeled and used for the transfer experiments. Both the direction and the extent of lipid transfer are dependent on the composition and relative abundance of both donor and acceptor particles as well as the activity of the lipid transfer protein(s). Here we describe a new method for the determination of the capacity of plasma samples to facilitate cholesteryl ester transfer from HDL to LDL and very-low-density lipoprotein (VLDL), a method that has several advantages. First, the subject's HDL is labeled and used for transfer. Second, the labeled HDL, in a quantity equivalent to 1% of the plasma HDL mass, is added to the subject's plasma, and therefore the relative abundance of both donor and acceptor particles is preserved at their physiological levels. Third, both cholesteryl ester mass and radioactivity are determined, allowing the net mass transfer of cholesteryl ester and cholesteryl ester exchange to be quantified separately.(ABSTRACT TRUNCATED AT 250 WORDS)

ROLE OF NON‐ESTERIFIED FATTY ACIDS IN REGULATING PLASMA CHOLESTEROL TRANSPORT

1. The cholesteryl ester transfer protein (CETP) promotes an equimolar exchange of cholesteryl esters between the high density lipoproteins (HDL) and low density lipoproteins (LDL) in human plasma. 2. Sodium oleate converts this CETP-mediated process of exchange into one of net mass transfer of cholesteryl esters from HDL to LDL. 3. Thus, conditions which increase the concentration of non-esterified fatty acids in plasma may favour the redistribution of cholesterol from the non-atherogenic HDL to the atherogenic LDL fraction.

Net mass transfer of cholesteryl esters from low density lipoproteins to high density lipoproteins in plasma from normolipidemic subjects.

Net mass transfer of lipids was measured in plasma from fasted, normolipidemic subjects. The plasma was incubated, and lipoproteins were subsequently separated by polyanion precipitation or density gradient ultracentrifugation. Total cholesterol, unesterified cholesterol, and triglycerides were measured in the isolated lipoprotein fractions. The rate of cholesterol esterification was measured simultaneously. All plasma samples showed an increase in high density lipoprotein (HDL) cholesteryl esters during 1-hour incubations. In most cases, this increase was higher than the cholesteryl ester formation in total plasma due to cholesterol esterification. Therefore, we concluded that a net mass transfer of cholesteryl esters occurred from the very low plus low density lipoprotein (VLDL + LDL) fractions to HDL in plasma from most of the subjects studied. Transfer of triglycerides occurred from VLDL + LDL to HDL in plasma from all subjects. The cholesteryl ester transfer (measured after 1 hour) is not related to the activity of cholesteryl ester transfer protein. Inhibition of cholesterol esterification did not change the direction of cholesteryl ester or triglyceride transfer. Ultracentrifugal separation of plasma lipoproteins revealed that both HDL and VLDL are acceptors of cholesteryl esters and that VLDL donates triglycerides to both LDL and HDL. Removal of VLDL from plasma by ultracentrifugation did not affect the cholesteryl ester transfer from LDL to HDL. We conclude that LDL may act as a donor of cholesteryl esters during incubation of normolipidemic plasma.

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.

若年性角膜輪を伴う高HDL&lt;sub&gt;2&lt;/sub&gt;-コレステロール血症の臨床的・生化学的検討

We recently discovered two homozygous cases of hyper-HDL2-cholesterolemia associated with premature corneal opacities. Patient 1 is a 55-year-old man, whose serum HDL-cholesterol levels were much elevated to -140mg/dl, while serum total cholesterol was only slightly increased and serum triglyceride moderately reduced. Serum levels of apolipoprotein (apo) A-I and E were also elevated. Patient 2 is a 58-year-old man whose serum HDL-cholesterol was also much increased to 130-169mg/dl. The lipid and lipoprotein patterns of these patients were closely similar by ultracentrifugal analysis and the increase of HDL-cholesterol was attributed solely to the enrichment of cholesteryl ester (CE) in HDL2 fraction (1.063<d<1.125g/ml). The trait was considered to be inherited in the family of Patient 2. In postheparin plasma, LPL activities were increased in these patients, whereas H-TGL showed reduced activities. In order to elucidate the mechanism of accumulation of CE in HDL2, we examined CE net transfer rate according to the modified method of Fielding et al. In control subjects there was a net mass transfer of CE from HDL to VLDL and LDL after a 1hr's incubation with DTNB at 37°C, while in both cases there was a net transfer of CE in the opposite direction leading to the accumulation of CE in HDL.We also examined the effect of probucol on the lipoprotein and apoprotein levels of these patients. Probucol reduced serum levels of HDL-cholesterol, apo A-I and apo E parallel to the reduction of serum total cholesterol. The decrease of HDL-cholesterol was largely due to the reduction of cholesterol in HDL2 fraction. Examination of patients' sera by HPLC disclosed that large HDL2 particles became smaller to almost normal sizes after the treatment with probucol and that the change was proved to be reversible after stopping administration of the drug.We also examined CE net transfer rate after treatment with probucol, and found that the reversion of CE net transfer was normalized in both cases after the therapy. We therefore conclude that abnormalities in CE net transfer might be involved in the pathogenesis of hyper-HDL2-cholesterolemia.

Comparison of very-low-density lipoproteins isolated from rat liver perfusate, rat serum and human plasma as acceptors for cholesteryl ester transfer

Using two methods, we have compared the ability of three types of very-low-density lipoprotein (VLDL), namely, rat hepatic perfusate VLDL, rat serum VLDL and human plasma VLDL, to accept cholesteryl esters from human plasma high-density lipoprotein (HDL). Both net mass and isotopic transfers of cholesteryl ester were evaluated. The VLDL concentration in the incubation media was adjusted on the basis of equivalent amounts of either cholesteryl ester, apolipoprotein B or triacylglycerol. In each case, the net mass transfer of cholesteryl ester was found to be greatest to rat hepatic VLDL, lower to rat serum VLDL and lowest to human plasma VLDL. Similar results were obtained with the isotopic transfer method when the VLDL concentration was adjusted on the basis of cholesteryl ester or apolipoprotein B. However, when VLDL were matched on the basis of triacylglycerol concentration, the isotopic transfer rate into human plasma VLDL was twice as high as into rat hepatic VLDL which presumably reflects the decreased number of hepatic VLDL particles by this criterion. In conclusion under fasted conditions (chylomicron excluded), nascent VLDL have been shown to be the best cholesteryl ester acceptor compared to their plasma counterpart.

Lipoprotein lipase enhances the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins.

These studies were undertaken to examine the effects of lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) on the transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL). Human or rat VLDL was incubated with human HDL in the presence of either partially purified CETP, bovine milk LPL or CETP plus LPL. CETP stimulated both isotopic and mass transfer of cholesteryl esters from HDL into VLDL. LPL caused only slight stimulation of cholesteryl ester transfer. However, when CETP and LPL were both present, the transfer of cholesteryl esters from HDL into VLDL remnants was enhanced 2- to 8-fold, compared to the effects of CETP alone. The synergistic effects of CETP and LPL on cholesteryl ester transfer were more pronounced at higher VLDL/HDL ratios and increased with increasing amounts of CETP. In time course studies the stimulation of cholesteryl ester transfer activity occurred during active triglyceride hydrolysis. When lipolysis was inhibited by incubating LPL with either 1 M NaCl or 2 mM diethylparanitrophenyl phosphate, the synergism of CETP and LPL was reduced or abolished, and LPL alone did not stimulate cholesteryl ester transfer. These experiments show that LPL enhances the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. This property of LPL is related to lipolysis.

Isolation and characterization of human plasma lipid transfer proteins.

A highly purified protein that facilitates the exchange and net mass transfer of cholesteryl ester (CE), and triacylglycerol (TG), and the transfer of phosphatidylcholine (PC), between plasma lipoproteins was isolated from the d-1.21-1.25 g/ml plasma fraction. Transfer activities showed similar distributions through ultracentrifugation, phenyl-Sepharose, DEAE-Sepharose, CM-cellulose, and hydroxyapatite chromatography. The lipid transfer protein appears to be an acidic protein with an apparent molecular weight of 64,000 +/- 1600 (n = 4) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent molecular weight of 65,000 by gel filtration chromatography, and has a pI of 5.0 +/- 0.2 (n = 5) by both analytical isoelectricfocusing and chromatofocusing. The purified transfer protein facilitated the net mass transfer of CE from high density lipoprotein (HDL) or low density lipoprotein (LDL) to very low density lipoprotein (VLDL) and the net mass transfer of TG from VLDL to LDL or HDL. Chromatography of lipid transfer protein fractions on a heparin-Sepharose column yielded two separate fractions with PC transfer activity. The first fraction did not bind to heparin column, was relatively resistant to elevated temperature (at 58 degrees C, only 5% activity was lost in 1 hour) and eluted with the CE and TG transfer activities which were also temperature-resistant. The second PC transfer activity bound to the heparin column and was temperature-sensitive (at 58 degrees C, 90% activity was lost in 1 hour). Addition of the temperature-resistant lipid transfer fraction (LTP-1) and purified lecithin cholesterol acyltransferase (LCAT) to whole plasma stimulated the endogenous plasma cholesterol esterification rate by approximately 50%, whereas addition of either LTP-1 or LCAT only slightly enhanced the esterification rate. The transfer of CE, TG, and PC was mediated by a temperature-resistant plasma protein or proteins with very similar properties. Plasma also contained a distinct lipid transfer protein which was temperature-sensitive and facilitated the transfer of PC, but not CE or TG.

High density lipoprotein subfractions isolated by heparin-Sepharose affinity chromatography and their role in cholesteryl ester transfer to very low density lipoproteins.

Normal human plasma HDL was applied to a column of heparin-Sepharose in the presence of MnCl(2) and three fractions were obtained by stepwise elution with increasing NaCl concentrations: a non-retained fraction (NR, 78% of protein) and two retained fractions (R(1) and R(2), 18 and 2.5% of protein, respectively). Both unesterified and esterified cholesterol increased from NR to R(1) to R(2) but the increment was more pronounced for unesterified cholesterol. ApoA-II to apoA-I ratio was-lower in R(1) compared to NR but R(1) contained more apoC than NR. ApoE increased from NR to R(1) to R(2) (0.07, 0.4, and 14% of protein in each fraction, respectively) while apoB was found only in R(2). Agarose gel electrophoresis and immunoadsorbers for apoB and apoE showed that R(2) consisted of two major lipoprotein populations, one containing apoB and some apoE and the other containing apoE and no apoB. Cholesteryl ester transfer between each HDL subfraction and VLDL in the presence of partially purified cholesterol ester transfer protein was studied. NR and R(1) gave the highest initial rates of transfer for labeled cholesteryl ester which were corroborated by significant mass transfer of cholesteryl esters. From these results, we concluded that there is no connection between cholesteryl ester transfer and apoE. On the other hand, transfer from R(2) to VLDL followed different kinetics with a high zero hour transfer but with subsequently lower rates when compared to NR and R(1). The cholesteryl ester transfer activity in R(2) was mainly due to the presence of apoE-containing lipoproteins whereas those containing apoB had minimal transfer activity. However, because this transfer of label was not translated into significant mass transfer of cholesteryl ester to VLDL, the apoE-containing lipoproteins appear involved mainly in the equilibration of cholesteryl esters.-Marcel, Y. L., C. Vézina, D. Emond, R. B. Verdery, and R. W. Milne. High density lipoprotein subfractions isolated by heparin-Sepharose affinity chromatography and their role in cholesteryl ester transfer to very low density lipoproteins.