M.J. Chapman

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 subjects 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 subjects 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)

Pravastatin Modulates Cholesteryl Ester Transfer From HDL to ApoB-Containing Lipoproteins and Lipoprotein Subspecies Profile in Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) results from genetic defects in the LDL receptor, and is characterized by a marked elevation in plasma LDL and by qualitative abnormalities in LDL particles. Because LDL particles are major acceptors of cholesteryl esters (CEs) from HDL, significant changes occur in the flux of CE through the reverse cholesterol pathway. To evaluate the effects of an HMG-CoA reductase inhibitor, pravastatin, on CE transfer from HDL to apo B-containing lipoproteins and on plasma lipoprotein subspecies profile in subjects with heterozygous FH, we investigated the transfer of HDL-CE to LDL subfractions and changes in both concentration and chemical composition of the apo B- and the apo AI-containing lipoproteins. After pravastatin treatment (40 mg/d) for a 12-week period, plasma LDL concentrations (mean +/- SD, 745.4 +/- 51.9 mg/dL) were reduced by 36% in patients with FH (n = 6). By contrast, the qualitative features of the density profile of LDL subspecies in patients with FH, in whom the intermediate (d = 1.029 to 1.039 g/mL) and dense (d = 1.039 to 1.063 g/mL) subspecies were significantly increased relative to a control group, were not modified by pravastatin. In addition, no significant effect on the chemical composition of individual LDL subfractions was observed. Furthermore, plasma HDL concentrations were not modified, although the density distribution of HDL was normalized. Indeed, the HDL density peak was shifted towards the HDL2 subfraction (ratios of HDL2 to HDL3 were 0.7 and 1.1 before and after treatment, respectively). Evaluation of plasma CE transfer protein (CETP) mass was performed with an exogenous CE transfer assay. Under these conditions, no modification of plasma CETP protein mass was induced by pravastatin administration. However, the rate of CE transfer from HDL to LDL was reduced by 24% by pravastatin (61 +/- 17 micrograms CE.h-1.mL-1 plasma; P < .0005), although intermediate and dense LDL subfractions again accounted for the majority (71%) of the total CE transferred to LDL. Thus, pravastatin induced reduction of plasma CETP activity without change in the preferential targeting of the transfer of HDL-CE towards the denser LDL subfractions. In conclusion, pravastatin reduces the elevated flux of CE from HDL to apo B-containing lipoproteins in subjects with heterozygous FH as a result of a reduction in the LDL particle acceptor concentration.

PAF-acether-degrading acetylhydrolase in plasma LDL is inactivated by copper- and cell-mediated oxidation.

In peripheral blood, native low-density lipoprotein (LDL) is a major carrier of acetylhydrolase, the enzyme that hydrolyzes the sn-2 acetate of PAF-acether, converting it to lyso PAF-acether. By controlling the level of PAF-acether, the acetylhydrolase may regulate the biologic effects of this potent inflammatory and thrombotic mediator. The biologic oxidation of LDL appears to underlie its atherogenicity. We report here that oxidative modification of LDL led to progressive loss of associated acetylhydrolase activity. Reductions of approximately 90% and 40% of acetylhydrolase activity occurred respectively in LDL oxidized for 24 hours by copper ions (2.5 mumol/L) in phosphate-buffered saline and in LDL incubated with human monocyte-like THP1 cells in Hams F-10 medium. Acetylhydrolase activity decreased as a function of the degree of LDL oxidation and was correlated with an increase in net negative charge and in the content of thiobarbituric acid-reactive substances (r = -.94 and r = -.88, respectively; P < or = .001). The acetylhydrolase of mildly oxidized LDL displayed a similar Km for PAF-acether compared with native LDL, whereas its Vmax was lower. Thus, acetylhydrolase conserved its affinity for PAF-acether, whereas a nondefined and noncompetitive inhibitor, apparently produced during oxidation, might account for the observed loss in enzymatic activity. Acetylhydrolase activity was totally recovered in LDL modified by both acetylation and malondialdehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Familial lecithin: cholesterol acyltransferase deficiency: further resolution of lipoprotein particle heterogeneity in the low density interval

Patients presenting with a familial deficiency of lecithin:cholesterol acyltransferase (LCAT) typically exhibit multiple quantitative and qualitative perturbations of apo B- and apo A-I-containing plasma lipoproteins. Marked particle heterogeneity has been detected over the low-density range (d = 1.019-1.063 g/ml), involving lipoprotein(X) (LP-X) and large molecular weight LDL (LM-LDL). We describe the chromatographic fractionation and characterization of the major particle species distributed within the low-density interval in a new French LCAT-deficient family. Detailed analyses of the plasma lipoprotein and apolipoprotein spectrum are reported. The plasma lipoproteins were enriched in unesterified cholesterol and phospholipids with markedly reduced concentrations of cholesteryl esters. By a combination of gel filtration and affinity chromatography on heparin-sepharose, the heterogeneous mixture of low-density particles was resolved into three distinct particle populations: LP-X (diameter 400 A) corresponding to LM-LDL, an apo A-I and albumin-containing particle similar to LP-X2 (diameter 300 A), and cholesteryl ester-deficient (0.9%) triglyceride-rich (58.4%) LDL containing apo B-100 (diameter 260-270 A). Use of affinity chromatography allowed separation of HDL-like particles (diameter 140-160 A) which were rich in free cholesterol (21.4%) and phospholipids (52.9%) and which were isolated in association with LP-X upon gel filtration chromatography. Ultracentrifugal density gradient analysis of plasma from the LCAT-deficient subject over a period of 3 years showed a net shift of the lipoprotein distribution in the low density range due to an increase in plasma LP-X levels. We propose that the presence of LP-X in the plasma is correlated with a progressive alteration in the renal function recently observed in this patient.

Effect of high-dose pitavastatin on glucose homeostasis in patients at elevated risk of new-onset diabetes: insights from the CAPITAIN and PREVAIL-US studies.

Abstract Aims: Statin treatment may impair glucose homeostasis and increase the risk of new-onset diabetes mellitus, although this may depend on the statin, dose and patient population. We evaluated the effects of pitavastatin 4 mg/day on glucose homeostasis in patients with metabolic syndrome in the CAPITAIN trial. Findings were validated in a subset of patients enrolled in PREVAIL-US. Methods: Participants with a well defined metabolic syndrome phenotype were recruited to CAPITAIN to reduce the influence of confounding factors. Validation and comparison datasets were selected comprising phenotypically similar subsets of individuals enrolled in PREVAIL-US and treated with pitavastatin or pravastatin, respectively. Mean change from baseline in parameters of glucose homeostasis (fasting plasma glucose [FPG], glycated hemoglobin [HbA1c], insulin, quantitative insulin-sensitivity check index [QUICKI] and homeostasis model of assessment–insulin resistance [HOMA-IR]) and plasma lipid profile were assessed at 6 months (CAPITAIN) and 3 months (PREVAIL-US) after initiating treatment. Results: In CAPITAIN (n = 12), no significant differences from baseline in HbA1c, insulin, HOMA-IR and QUICKI were observed at day 180 in patients treated with pitavastatin. A small (4%) increase in FPG from baseline to day 180 (P < 0.05), was observed. In the validation dataset (n = 9), no significant differences from baseline in glycemic parameters were observed at day 84 (all comparisons P > 0.05). Similar results were observed for pravastatin in the comparison dataset (n = 14). Conclusions: Other than a small change in FPG in the CAPITAIN study, neutral effects of pitavastatin on glucose homeostasis were observed in two cohorts of patients with metabolic syndrome, independent of its efficacy in reducing levels of atherogenic lipoproteins. The small number of patients and relatively short follow-up period represent limitations of the study. Nevertheless, these data suggest that statin-induced diabetogenesis may not represent a class effect.

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 HDL2b 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.