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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1819-1828

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1819-1828.)
© 1995 American Heart Association, Inc.

Articles

Triglycerides Are Major Determinants of Cholesterol Esterification/Transfer and HDL Remodeling in Human Plasma

Toru Murakami; Silvia Michelagnoli; Renato Longhi; Gemma Gianfranceschi; Franco Pazzucconi; Laura Calabresi; Cesare R. Sirtori; Guido Franceschini

From Center E. Grossi Paoletti, Institute of Pharmacological Sciences, University of Milano (T.M., S.M., G.G., F.P., L.C., C.R.S., G.F.) and Istituto di Chimica degli Ormoni, CNR (R.L.), Milano, Italy.

Correspondence to Prof Guido Franceschini, Center E. Grossi Paoletti, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milano, Italy.


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n=20) and isolated (HTG, n=20) or mixed hypertriglyceridemia (MHTG, n=20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37°C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG+MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (r=.722 and r=.642, respectively), CETR (r=.510 and r=.491, respectively), and HDL remodeling (r=.625 and r=.620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.


Key Words: HDL conversion • lecithin:cholesterol acyltransferase • atherosclerosis • hyperlipoproteinemia • cholesterol ester transfer protein


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Most prospective epidemiological studies have shown an independent inverse relation between HDL cholesterol concentration and risk of development of coronary heart disease (CHD).1 The mechanisms underlying this negative association are not fully understood, and several biological explanations have been proposed.2 Most prominent seems the function of HDL as a vehicle of cholesterol in the so-called reverse cholesterol transport,3 the process by which excess cholesterol in peripheral cells is transported to the liver for elimination from the body.

Total HDL in humans is heterogeneous, consisting of several major and minor particle subpopulations.4 Although the physiological significance of these different particles is mostly undefined, some of these particles display peculiar properties in vitro.4 Indeed, small pre–ß-migrating HDLs containing apoA-I but not apoA-II (LpA-I) show the highest capacity to retrieve cholesterol from cell membranes,5 thus providing an explanation for the lower plasma concentrations of LpA-I but not of particles containing both apoA-I and apoA-II, in subjects with premature coronary atherosclerosis.6 7 These findings can be interpreted to suggest that not all the HDL particles have the same antiatherogenic potential and that minor variations in HDL particle distribution might be associated with larger variations in the coronary risk.

The identification of factors modulating HDL particle distribution in plasma is crucial for a better understanding of the complex relation between HDL and CHD risk. The present concepts on the regulation of HDL remodeling in human plasma have been drawn primarily from experimental studies with normolipidemic plasma or reconstituted systems.8 9 10 11 12 From these studies, the following sequentially linked steps have been proposed: (1) esterification of free cholesterol occurs in small HDL3 particles by the LCAT enzyme with the formation of large HDL2a particles; (2) the transfer of newly synthesized CEs to apoB-containing lipoproteins by the CETP, in exchange for TGs, increases the lipoprotein core volume and results in larger HDL2b particles; and (3) the hydrolysis of HDL-TG and phospholipids by hepatic and lipoprotein lipases converts large HDL2b back into small HDL3 particles. Thus, the interplay of cholesterol esterification and CE transfer is essential in the remodeling of HDL structure and, in turn, may contribute to the regulation of HDL particle distribution in the single individual.13

The physiological role of LCAT in HDL remodeling and reverse cholesterol transport is best described by the biochemical/clinical features of patients with genetic defects in cholesterol esterification, ie, LCAT deficiency and fish-eye disease.14 Homozygous patients display a nearly complete absence of cholesterol esterification in plasma and hypoalphalipoproteinemia, with the accumulation of discoidal HDL precursors and small spherical HDLs.15 Although different from the described genetic conditions, the role of LCAT in the regulation of plasma HDL level/structure in normal individuals is still unclear. In normolipidemic and hyperlipidemic individuals the plasma HDL-C level correlated negatively with the CER but not with the plasma LCAT concentration as measured by a specific radioimmunoassay.16 In contrast, in two large studies on healthy individuals, the LCAT concentration when evaluated by the same radioimmunoassay, was significantly positively correlated with HDL-C and HDL3-C levels.17 18 The observation of normal lipid/lipoprotein profiles in obligate heterozygotes for LCAT deficiency19 and the lack of significant variation in plasma LCAT levels among patients with various dyslipidemias16 suggest that LCAT activity/concentration is not rate limiting in plasma cholesterol esterification and HDL remodeling.

The influence of CETP in determining plasma HDL levels/structure is also controversial. Subjects with inherited CETP deficiency show multiple abnormalities in the HDL system, with increased plasma levels of large CE- and apoE-rich HDL particles.20 Plasma HDL-C levels were independent of CETP activity in normolipidemic subjects,21 whereas plasma CETP concentrations correlated positively with HDL-C and apoA-I levels in normolipidemic22 but not hyperlipidemic individuals.23 Whereas these discrepancies mostly reflect differences in methodology and/or patient selection, more consistent findings have been reported in individuals in whom changes in CETP activity/concentration induced by probucol24 25 or ethanol26 resulted in opposite changes in plasma HDL levels.

In the present study several parameters of the cholesterol esterification/transfer processes have been examined in parallel in both healthy subjects and patients with various forms of hyperlipidemia. The implication of cholesterol esterification/transfer in HDL remodeling was investigated by incubating total human plasmas and analyzing the changes in HDL particle size distribution by nondenaturing PAGGE. The results indicate that plasma TGs are primarily responsible for the variations in cholesterol esterification/transfer and HDL remodeling.


*    Methods
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 up arrowAbstract
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 *Methods
down arrowResults
 down arrowDiscussion
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Subjects
This study involved 60 hyperlipidemic patients referred to the E. Grossi Paoletti Lipid Clinic in Milano, Italy. Twenty normolipidemic control subjects of similar age and sex were recruited from among the Hospital and University staff. Subjects with liver/kidney disease, thyroid dysfunction, or a history of alcohol abuse and/or heavy smoking were excluded. Obese subjects (body weight >120% of ideal body weight) were also excluded. Although some of the patients presented with abnormalities in glucose tolerance, diabetes mellitus was not clearly evident in any of them. Five of the selected subjects (4 hyperlipidemic patients and 1 control subject) had suffered a myocardial infarction at least 6 months before examination; 1 patient had suffered a transient ischemic attack, but the remaining subjects had no personal history of cardiovascular disease. No subject was taking medications that had the potential of modifying lipid/lipoprotein levels; lipid-lowering therapies were stopped at least 2 months before the beginning of the study. The study protocol was approved by the Institutional Review Board, and all participating subjects were fully informed of the end points and modalities of the study.

Patients were classified into three hyperlipidemia phenotypes based on fasting lipid levels at two separate visits: (1) hypercholesterolemia (HC), LDL cholesterol (LDL-C) >130 mg/dL and TG <200 mg/dL; (2) isolated hypertriglyceridemia (HTG), LDL-C <130 mg/dL and TG >200 mg/dL; and (3) mixed hypertriglyceridemia (MHTG), LDL-C >130 mg/dL and TG >200 mg/dL. After an overnight fast, blood was collected in both empty plastic tubes and tubes containing Na2-EDTA (final concentration, 1 mg/mL). Serum and plasma were prepared by low-speed centrifugation at 4°C. Serum aliquots were added with Na2-EDTA (1 mg/mL) and solid NaBr (final concentration, 5.1 mol/L) and kept at 4°C for HDL subfraction analysis by rate-zonal ultracentrifugation.27 Plasma aliquots for enzyme activity and mass measurements were immediately frozen and stored at -80°C until assayed.

Lipid/Lipoprotein Measurements
Plasma total and free cholesterol (TC and FC) and TG levels were determined within the same day of blood collection by enzyme methodologies28 29 standardized within a World Health Organization quality control program. The CE mass was calculated as (TC-FC)x1.68. Plasma Lp(a) levels were measured by enzyme-linked immunosorbent assay.30 In individuals with plasma TG <400 mg/dL, the LDL-C level was calculated with the use of Friedewald's formula31 and corrected for the Lp(a)-C content as cLDL-C=LDL-C-(Lp[a]x0.3).30 Plasma lipoproteins were separated by sequential ultracentrifugation32 using a Beckman TL 100 ultracentrifuge equipped with a TL 100.3 rotor (Beckman Instruments). Protein concentration in the lipoprotein fractions was determined by the method of Lowry et al.33 Plasma apoA-I, apoA-II, and apoB levels were measured by immunoturbidimetry34 using a Cobas-Mira analyzer (F. Hoffmann–La Roche). Plasma glucose concentrations were determined using a Glucose HK kit (Hoffmann–La Roche) applied on the Cobas system.

Separation of HDL Subclasses
HDL subfractions were isolated by rate-zonal ultracentrifugation in a swinging bucket rotor,27 which separates subfractions according to density, shape, and size. Two fractions, designated as HDL2 and HDL3, were collected and the TC content was measured by enzymatic methods. The Ve of the HDL2 and HDL3 peaks from the density gradient was automatically monitored and taken as an index of particle flotation rate.27

HDL particle size distribution was analyzed by nondenaturing PAGGE, using precast 4% to 30% slab gels (Pharmacia Biotec).35 Aliquots of the d<1.21 g/mL plasma fractions, which had been separated by ultracentrifugation at 100 000 rpm for 3 hours at 4°C in a Beckman TL 100.3 rotor, were applied to the gels. The gels were scanned by an LKB Ultroscan XL laser densitometer (Pharmacia Biotec) and particle sizes calculated with the LKB 2400 Gelscan XL software, with the use of thyroglobulin, apoferritin, lactate dehydrogenase, and bovine serum albumin as calibration proteins. Five HDL subpopulations were identified and classified according to the criteria of Nichols et al35 as follows: HDL2b, 9.7 to 12.9 nm; HDL2a, 8.8 to 9.7 nm; HDL3a, 8.2 to 8.8 nm; HDL3b, 7.8 to 8.2 nm; and HDL3c, 7.2 to 7.8 nm. To calculate the percentage distribution of HDL subpopulations, areas under the scanning curves were integrated by dropping vertical lines that corresponded to the subpopulation size limits; the total integrated area in the 7.2 to 12.9 nm size-interval was considered to be 100%. We then assigned each sample an HDLps36 according to the following formula:

where i is the band number (5 for HDL2b, 4 for HDL2a, 3 for HDL3a, 2 for HDL3b, and 1 for HDL3c) and Xi is the fraction of total area for that band. The HDLps combines the HDL size distribution and the concentration of each HDL subpopulation; a large score represents a particle distribution shifted toward larger sizes. The intra-assay and interassay coefficients of variation (CVs) for HDLps are 5.4% and 7.9%, respectively (n=8). To estimate plasma concentrations of each HDL subpopulation, the protein concentration of plasma HDL was multiplied with the relative HDL subpopulation areas.7 37

Determination of Cholesterol Esterification/Transfer
Cholesterol esterification was evaluated by measuring the CER (ie, the esterification in the presence of the endogenous lipoprotein substrate) in whole plasma by a radioassay method, as previously described.38 Briefly, plasma aliquots were mixed with a 5% delipidated HSA solution containing 1.5 nmol [14C]-cholesterol and incubated for 2 hours at 37°C. The reaction was stopped by placing the tubes on ice, then lipids were extracted and dried. FC and CE were separated by thin-layer chromatography, and radioactivity was counted.

The net mass transfer of CE from HDL to lower density lipoproteins was determined in fresh plasma by measuring the CETR during a 30-minute incubation period at 37°C, as previously described.24 Briefly, plasma samples were incubated at 37°C in the presence of 0.15 mol/L sodium iodoacetate as LCAT inhibitor; at progressive intervals (at 0, 10, 20, and 30 minutes) pentuplicate plasma aliquots were treated with phosphotungstic acid–MgCl2 to precipitate VLDL and LDL. The CE concentration was measured in the supernatant, and CETR was calculated as the regression line obtained by plotting HDL-CE against incubation time.24 The CE mass transfer in our CETR assay is fully dependent on CETP, being inhibited by monoclonal antibody TP-2 (kindly provided by Dr Y.L. Marcel).

Plasma LCAT and CETP concentrations were determined by an immunoradiometric assay, using anti-peptide antibodies. Peptides corresponding to residues 393 through 416 of LCAT and 458 through 476 of CETP were synthesized by the solid-phase method with a peptide synthesizer (model 431, Applied Biosystems). The purified peptides were conjugated with ovalbumin and used to immunize 3-month-old New Zealand White male rabbits (Charles River). Antibodies were purified by affinity chromatography and used in the immunoradiometric assay.39 The intra-assay and interassay CVs were 3% and 10% for LCAT and 4% and 9% for CETP, respectively.

HDL Conversion Assay
To monitor changes in HDL particle size distribution induced by the interaction of HDL with other lipoproteins, enzymes, or plasma factors,13 aliquots of fresh plasma were incubated at 37°C under N2 in a shaking water bath. At progressive intervals, plasma fractions of d<1.21 g/mL were separated by ultracentrifugation, and the HDL particle size distribution was analyzed by PAGGE as described above. Incubation resulted in a progressive accumulation of larger particles (Fig 1ADown), as indicated by the linear increase of HDLps (Fig 1BDown). For each patient, the change in HDLps ({Delta}HDLps) after 6 hours of incubation was calculated and used in statistical analyses.



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  		Figure 1. HDL remodeling in incubated human plasma. A, Nondenaturing PAGGE (4% to 30%) profiles of plasma from a normolipidemic healthy subject (left) and from a hypertriglyceridemic patient (right) before (dashed lines) and after (continuous lines) incubation at 37° for 6 hours. B, Time course of changes in HDLps during incubation of normolipidemic ({bullet}) and hypertriglyceridemic ({blacksquare}) plasma. The HDLps was calculated as in "Methods" and combines the HDL size distribution and the concentration of each HDL subclass, HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c. A large score represents a particle distribution shifted toward larger sizes. Each point represents the mean±SEM of five experiments with plasmas from different individuals.

Statistical Analyses
Results are reported as mean±SD if not otherwise stated. Logarithmic transformation was performed on individual values of skewed variables. Group differences in continuous variables were determined by two-tailed Student's t test. The significance of differences in continuous variables between more than two groups was tested by ANOVA with the Student-Newman-Keuls test for multiple comparisons. Pearson correlation coefficients were computed to assess the association between parameters. A stepwise regression analysis was performed by entering the independent variable with the highest partial correlation coefficient at each step, until no variable remained with an F value of >=4. Group differences or correlations with P<.05 were considered to be statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Lipid/Lipoprotein Parameters
Table 1Down summarizes basic characteristics of the studied subjects. Fasting plasma lipid/lipoprotein variables are reported in Table 2Down. Most of the data reflect the selection criteria. The range of TG levels for the hypertriglyceridemic population (205 to 1354 mg/dL) is characteristic of moderate to severe hypertriglyceridemia. Low HDL-C and HDL2-C levels typify patients with isolated or mixed hypertriglyceridemia; HDL3-C levels do not vary among the four studied groups. Mean plasma HDL-TG levels are elevated in all the hyperlipidemic groups, whereas only HTG patients have high plasma LDL-TG concentrations. Plasma Lp(a) levels were significantly higher in HC patients than in control subjects (NL). As expected, mean plasma apoB levels are elevated in all hyperlipidemics, but apoA-I and apoA-II concentrations do not differ significantly among the various groups. No major differences in plasma CETP and LCAT levels are observed among the four study groups; higher CETP concentrations are found in MHTG and HC patients, but in HC patients the difference when compared with NL control subjects does not reach statistical significance (P=.28).


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  		Table 1. Characteristics of the Examined Subjects


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  		Table 2. Plasma Lipid/Lipoprotein, and LCAT and CETP Levels in the Examined Subjects

HDL particle size distribution was assessed by nondenaturing PAGGE (Table 3Down). Hypertriglyceridemic patients (HTG+MHTG) have higher plasma concentrations of small HDL3b and HDL3c particles, whereas the concentration of large HDL2b particles is {approx}50% lower compared with NL control subjects. No major differences in plasma HDL subclass concentrations are found between HC patients and NL control subjects (Table 3Down). The mean HDLps is significantly lower in MHTG and HTG patients than in NL control subjects (Table 3Down). In the whole series, HDLps is correlated positively with HDL-C and HDL2-C (r=.744 and r=.811, respectively) and negatively with VLDL-TG (r=-.766) (Fig 2Down). By stepwise logistic regression, VLDL-TG, sex, LCAT, and HDL2-C, in this order, are independent and significant predictors of HDLps.


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  		Table 3. Plasma HDL in the Examined Subjects Before (Baseline) and After (Postincubation) Incubation at 37°C for 6 Hours



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  		Figure 2. Relationship between plasma VLDL-TG levels and CER, CETR, HDLps, and {Delta}HDLps in the examined subjects. The HDLps was calculated as in "Methods" and combines the HDL size distribution and the concentration of each HDL subclass, HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c. A large score represents a particle distribution shifted toward larger sizes. {Delta}HDLps represents the change in HDLps after incubation of plasma at 37° for 6 hours.

HDL subfractions were also separated by rate-zonal ultracentrifugation, and their flotation rate was evaluated by measuring the Ve from the density gradient. No difference in HDL2-Ve can be found among the various groups (Table 3Up). In contrast, slow-floating HDL3 particles are detected in both MHTG and HTG patients. A significant negative correlation between HDL3-Ve and VLDL-TG is found in the whole series (r=-.387).

Functional Assays of Cholesterol Esterification and CE Transfer
Cholesterol esterification and CE net mass transfer were tested in whole plasma by substrate-dependent methods, evaluating the interaction between CETP and LCAT in plasma with the endogenous lipoprotein substrates. The initial velocities of esterification and transfer reactions were measured in such a way that no more than 5% of the substrate was used during the assay period40 and are expressed as CER and CETR, respectively. These assays were selected because they are representative of the reaction system found in plasma at steady state, being dependent on composition/concentrations of the lipoprotein substrates/products, amount and activity of LCAT and CETP, and concentrations of endogenous inhibitors.41 They do not fully reflect the physiological conditions in which CER and CETR are influenced by other variables (eg, the turnover rate of donor/acceptor lipoproteins).

The individual CER data indicate a trend toward higher rates with increasing plasma TG levels (Fig 3Down). Indeed, mean CER values are significantly elevated in MHTG (76.3±26.4 nmol/mL per hour) and HTG (91.4±20.5 nmol/mL per hour) patients compared with NL control subjects (53.1±13.6 nmol/mL per hour) or HC patients (60.0±13.1 nmol/mL per hour). In addition, CER shows a significant positive correlation with plasma TG and VLDL-TG levels in the whole series (Table 4Down, Fig 2Up). CER correlates inversely with plasma HDL-C and HDL-CE levels (Table 4Down), suggesting product inhibition of cholesterol esterification even in the presence of a CE-removing activity. CER also correlates negatively with HDLps distribution and with the plasma levels of HDL2-C and large HDL particles (HDL2b and HDL2a) (Table 4Down), indicating that the accumulation in plasma of CE-rich, large HDL inhibits further esterification of cholesterol. In contrast, a positive correlation is found between CER and the plasma levels of small, lipid-poor HDL3b and HDL3c particles (Table 4Down). CER does not correlate with plasma LDL-C or Lp(a) levels. As expected, CER is highly significantly correlated with plasma FC concentration. By contrast, no correlation is found between CER and plasma LCAT levels, indicating that enzyme concentration is not a rate-limiting factor in the reaction, with lipid and lipoprotein substrates/products being major determinants in the regulation of plasma cholesterol esterification. Superimposable correlations are found when normotriglyceridemic and hypertriglyceridemic individuals are considered separately, indicating that the same regulatory mechanisms operate in the presence of normal or elevated plasma TG levels.



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  		Figure 3. CER and CETR in NL subjects and in HC, MHTG, or HTG patients. HC, MHTG, and HTG phenotypes were identified as described in "Methods."<\/.>


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  		Table 4. Univariate Correlation Coefficients of CER, CETR, and {Delta}HDLps With Various Lipid/ Lipoprotein Parameters

CETR also increases with increasing plasma TG values (Fig 3Up), both MHTG and HTG patients show significantly elevated mean CETR values (89.5±36.2 and 114.1±45.8 nmol/mL per hour, respectively) compared with NL control subjects (57.9±15.8 nmol/mL per hour) or HC patients (60.0±22.6 nmol/mL per hour). Again, a significant positive correlation is found in the whole series between CETR and plasma TG and VLDL-TG levels (Table 4Up, Fig 2Up). However, when normotriglyceridemic and hypertriglyceridemic subjects are considered separately, CETR correlates with plasma TG and VLDL-TG in the latter (r=.315 and r=.318, respectively) but not in the former (r=-.213 and r=-.293, respectively), suggesting that the pool of TG-rich lipoproteins is rate limiting in the transfer reaction. In the whole series, CETR correlates inversely with plasma HDL-C and HDL-CE levels (Table 4Up). Again, normotriglyceridemic and hypertriglyceridemic subjects behave differently as regards this relation, with CETR strongly positively correlated with HDL-C and HDL-CE in the former (r=.460 and r=.405, respectively) but not in the latter (r=-.129 and r=-.139, respectively). A weak positive correlation is found between CETR and CETP concentration in the whole series (Table 4Up); similar correlations in separate groups (normotriglyceridemics, r=.246; hypertriglyceridemics, r=.190) do not reach the significance. Correlations between CETR and plasma levels of HDL subfractions are only marginally significant. No correlation is found between CETR and plasma LDL-C or Lp(a) levels. Finally, a positive correlation between CER and CETR is observed in the whole series (Table 4Up). This correlation is significant in hypertriglyceridemic patients (r=.366), but not in normotriglyceridemic individuals (r=.117).

Stepwise logistic regression was used to assess the independent contributions of various lipid/lipoprotein and enzyme variables to the prediction of CER and CETR. VLDL-TG is the sole independent and significant predictor of CER and CETR, explaining 75% and 65% of the variation of plasma CER and CETR, respectively.

HDL Remodeling in Plasma
The PAGGE profile of HDL from normotriglyceridemic individuals is characterized by two major components, migrating in the HDL2b and HDL3a size intervals (Fig 1AUp, left). With increasing plasma TG levels, the contribution of HDL2b and HDL3a decreases progressively and a component migrating in the HDL3b size interval becomes predominant (Fig 1AUp, right). Incubation dramatically alters the particle size distribution of HDL: In all subjects there is a marked reduction in the HDL3a and HDL3b components together with the appearance of large HDL2a-2b species (Fig 1AUp). The particle size of the remaining HDL3a-3b particles does not change. Larger HDL2 particles accumulate in hypertriglyceridemic versus normotriglyceridemic (9.93±0.10 versus 9.59±0.06 nm, respectively) plasmas (Fig 1AUp). The contribution of small HDL3c particles to the whole HDL profile also increases after incubation, more so in hypertriglyceridemic patients (Fig 1AUp). The changes in HDL particle distribution in individual plasmas were evaluated by calculating {Delta}HDLps, which provides an index of changes in both size and amount of HDL subclasses.36

The mean {Delta}HDLps value is significantly different among the various groups, being twofold to threefold higher in MHTG and HTG patients versus NL control subjects (Table 3Up, Fig 4Down), ie, consistent with a more pronounced accumulation of larger HDL particles in patients with elevated plasma TG levels. The increase in HDLps after incubation is statistically significant in MHTG and HTG patients but not in NL and HC individuals. {Delta}HDLps is strongly positively correlated with plasma TG and VLDL-TG levels (Fig 2Up). {Delta}HDLps is also positively correlated with both CER and CETR, thus suggesting that cholesterol esterification and transfer may contribute to the enlargement of HDL particles. However, no significant correlation is found with plasma CETP and LCAT levels. The concentration of HDL particles in native plasma is also variably correlated with {Delta}HDLps, the formation of large HDL2 being related directly to the content of small HDL3a and HDL3b particles and inversely to that of large HDL2b. A highly significant negative correlation is also found between {Delta}HDLps and baseline HDLps values (Table 4Up).



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  		Figure 4. Individual changes in HDLps after incubation of plasma at 37°C for 6 hours in NL and in HC, MHTG, and HTG groups. HC, MHTG, and HTG phenotypes were identified as described in "Methods." HDLps was calculated as in "Methods" and combines the HDL size distribution and the concentration of each HDL subclass, HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c. A large score represents a particle distribution shifted toward larger sizes.

The independent contribution of the various parameters to the {Delta}HDLps was assessed by stepwise logistic regression. Baseline VLDL-TG and HDLps, in this order, are independent and significant predictors of {Delta}HDLps, accounting for 84% of the variation of {Delta}HDLps. This result is consistent with a relation in which the availability of an adequate pool of "acceptor" particles and the concentration/structure of HDL substrates/products determine the remodeling of HDL.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrates that in plasmas from hypertriglyceridemic subjects cholesterol esterification and net CE transfer out of HDL are higher than in NL or HC individuals. In the present in vitro system, the conversion of small HDL into large, lipid-rich HDL is also faster in hypertriglyceridemic than normotriglyceridemic plasmas. Correlation analyses indicate that a direct relation exists between fasting triglycerides and cholesterol esterification, net CE transfer, and HDL remodeling. Plasma LCAT and CETP concentrations are not rate-limiting in the esterification/transfer reactions and do not contribute significantly to HDL remodeling.

The efflux of cholesterol from peripheral cells is the first step in reverse cholesterol transport, the process responsible for the removal of excess cholesterol from peripheral tissues and transport to the liver for excretion.4 The extent of cholesterol efflux from cells is dependent on the presence of specific acceptors in the extracellular space5 and in the maintenance of an FC gradient between cell membranes and plasma.42 43 The esterification of cholesterol within HDL and the CETP-mediated transfer of newly formed CE out of HDL primarily contribute to the FC gradient.42 LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles are continuously converted in plasma through the interaction with other lipoproteins, transfer proteins, and lipolytic enzymes.13 Particles with a high capacity for cell cholesterol uptake are generated through this process.9 10 44 Therefore, an efficient cholesterol esterification/transfer and HDL remodeling would improve reverse cholesterol transport and decrease the risk for cardiovascular disease. Investigation of factors determining cholesterol esterification/transfer and HDL remodeling in human plasma is necessary to understand the significance of these pathways and to identify potential targets for intervention.

Plasma LCAT and CETP levels were measured by specific immunoassays in a series of patients with various forms of hyperlipidemia. No significant variation in plasma LCAT levels was observed among the various hyperlipidemias, confirming earlier findings by Albers et al.16 In contrast, the CER was significantly higher in hypertriglyceridemic than normotriglyceridemic plasma. Correlation analyses provide a number of possible explanations for the higher CER in hypertriglyceridemic plasma. (1) Increased CER may be consequent to the HDL particle distribution shift toward small, dense HDL, with high affinity and reactivity for LCAT. In vitro studies with reconstituted HDL or cell-derived lipoproteins demonstrate that LCAT interacts preferentially with small, CE-poor HDL3 particles,45 46 whereas, large, lipid-rich HDL2 may have an inhibitory effect on LCAT activity.47 The CER in VLDL-LDL–depleted plasma is, furthermore, significantly higher in hypertriglyceridemic subjects and is positively correlated with plasma TG levels.48 (2) Lipolysis reduces HDL reactivity with LCAT, primarily by altering the lipid composition of the HDL outer shell, ie, by decreasing the phospholipid/FC ratio49 and increasing the content of inhibitory fatty acids.50 Hypertriglyceridemic VLDLs are generally resistant to lipolysis by lipoprotein lipase,51 and the reduced lipolytic efficiency may prevent the accumulation of inhibitory molecules on the HDL surface. (3) Cholesterol esterification in hypertriglyceridemic plasma can be facilitated by the accelerated transfer of newly synthesized CE to apoB-containing lipoproteins. LCAT and CETP are found in the same HDL subpopulation,52 and there is agreement that a close integration exists between the cholesterol esterification and transfer reactions.53 Indeed, a positive correlation was found between CER and CETR, especially in hypertriglyceridemic patients in whom a more efficient transport of CE out of HDL can relieve the product inhibition on the LCAT reaction.

The plasma CETP concentration tended to be higher in subjects with elevated cholesterol, supporting previous data in different cohorts of hyperlipidemic patients23 and consistent with the hypothesis that CETP is actively expressed in response to an excessive flux of cholesterol to peripheral tissues.54 Despite a faster CETR, hypertriglyceridemic patients had normal plasma CETP levels. Moreover, the plasma CETP concentration did not contribute to the prediction of CE transfer in the whole series of examined subjects. These findings suggest that in the normal population the CETP concentration is not rate limiting in the transfer reaction. It would be different in the case of subjects with extreme CETP deficiencies55 or of those treated with drugs that drastically affect plasma CETP levels.25 56 In the present series of subjects with widely different plasma lipid/lipoprotein levels, the net CE mass transfer was primarily determined by the relative pools of substrates (ie, the proportion of neutral lipids, TG, and CE) in acceptor and donor lipoproteins. This is consistent with in vitro experiments showing that the addition of increasing amounts of VLDL to normal plasma enhances the net mass transfer of CE out of HDL.57 More recently, a marked heterogeneity has been described among VLDL-LDL subspecies as acceptors of HDL-CE.58 59 60 In particular, the capacities of buoyant, TG-rich VLDL and LDL to accept HDL-CE were significantly greater than that of denser CE-rich species. Therefore, abnormalities in the distribution of VLDL-LDL subfractions in hypertriglyceridemic plasma, characterized by the prevalence of large VLDL and small LDL particles,61 62 also likely contribute to the increased CETR. Although no differentiation was made between net CE transfer to VLDL or LDL in the present study, it appears that the elevated acceptor capacity of large VLDL particles can overcome the negative effect of small LDL on CE transfer,59 60 since no correlation was found between CETR and LDL concentration or composition. Experiments with reconstituted systems clearly demonstrated that the efficiency of the transfer reaction is also dependent on various properties of lipoprotein substrates (eg, charge characteristics,63 apolipoprotein composition, and fatty acid content64 ), all affecting the binding of CETP to lipoproteins. These same studies showed that the binding capacity of lipoproteins for CETP far exceeds CETP concentrations in plasma,63 ie, consistent with the present lack of correlation between CETP concentration and CETR. It is possible that the increased CETR in hypertriglyceridemic plasma is partly due to a facilitated interaction between CETP and hypertriglyceridemic lipoproteins. This hypothesis is currently being investigated in our laboratory. Several other factors are known to modulate net CE transfer in humans. CETP activity and mass are higher in obese subjects and in smokers65 66 and lower in heavy drinkers.26 To avoid such confounding influences, only nonsmoking subjects with normal body weight and drinking pattern were recruited for the present study.

The mechanism responsible for the increased CETR in hypertriglyceridemic plasma has been previously investigated by Mann et al67 in a small series of hypertriglyceridemic patients. Similar to the present findings, net CE transfer was elevated in HTG patients, despite a normal CETP activity. However, correlation analyses showed that CE transfer is determined by plasma VLDL levels in normolipidemic control subjects and by CETP "mass" in hypertriglyceridemic patients. In contrast to the present study, net CE transfer was determined over a longer incubation period (6 hours) and therefore does not reflect the initial velocity of the transfer reaction (CETR).40 Moreover, the substrate-independent CETP activity, taken as a surrogate of CETP concentration, can be affected by the presence of CETP inhibitors in plasma.41 The present findings also disagree with a recent study that showed an increased net CE transfer, primarily to large LDL1 species, in patients with familial hypercholesterolemia (FH).59 We were able to make a definite diagnosis of heterozygous FH in only four of our HC patients; indeed, these had significantly higher CETR compared with the other HC patients (92.2±28.8 versus 54.3±17.5 nmol/mL per hour). It is likely that variations in the distribution of LDL subfractions with different acceptor capacities for HDL-CE59 60 among hypercholesterolemic patients68 account for the discrepancies between the results from these two studies.

The role of LCAT, CETP, and the various lipoproteins in modulating HDL remodeling was examined in whole plasma from individuals with widely different plasma lipid/lipoprotein levels. Determination of HDL remodeling in incubated plasma reflects only partially the physiological processes, since the role of lipolytic enzymes is not taken into account in such assays. Incubations of postheparin plasma are currently being performed in our laboratory to evaluate the relative contribution of lipolytic enzymes to HDL remodeling in hyperlipidemic plasma. In the absence of lipolytic enzymes small HDL are clearly converted into larger particles.11 This HDL conversion proceeds at a faster rate in hypertriglyceridemic than in normotriglyceridemic plasma. Since such TG-rich, large HDL2 particles are the preferred substrate for hepatic lipase,69 whose plasma content/activity is generally normal in hypertriglyceridemic plasma,70 one would predict that the whole HDL interconversion cycle (ie, the conversion of small HDL3 into large, lipid-rich HDL2 and then back into lipid-poor small HDL313 ) proceeds at a faster rate in hypertriglyceridemia. Indeed, the steady state content of small HDL3 is significantly higher in hypertriglyceridemic than in normotriglyceridemic individuals and is directly correlated to the extent of hypertriglyceridemia. Certain particle subspecies of small HDL3 may be equivalent to pre-ß-migrating HDL5 and, indeed, the concentration of pre-ß-HDL has been reported to be increased in hyperlipidemia.71 Thus, the accelerated HDL remodeling in hypertriglyceridemic plasma would result in the accumulation of HDL particles with a good capacity for cell cholesterol uptake.5 On the other hand, the accelerated HDL interconversion would result in an enhanced HDL-CE turnover, with low steady state plasma HDL-cholesterol levels as typically found in HTG patients. The dissociation of apoA-I from lipoprotein particles could possibly increase,72 resulting in a faster removal, primarily through the kidney,73 and in lower plasma steady state apoA-I levels. This was not found to be the case since no differences in plasma apoA-I levels were detected among the various hyperlipidemic groups, thus suggesting that no direct relation exists between HDL remodeling and apoA-I turnover in human plasma.

Taken together, the results of this study demonstrate that three major pathways in reverse cholesterol transport (ie, cholesterol esterification, CE transfer, and HDL remodeling) occur at a faster rate in hypertriglyceridemic than in normotriglyceridemic plasma. The impact of this accelerated transport on cardiovascular risk then critically depends on the individual capacity to remove apoB-containing lipoproteins through the liver.


*    Selected Abbreviations and Acronyms
 
C = cholesterol (when combined, eg, HDL-C)
CE = cholesteryl ester
CER = cholesterol esterification rate
CETP = cholesteryl ester transfer protein
CETR = CE transfer rate
HC = patient group with hypercholesterolemia
HDLps = HDL particle score
HTG = patient group with isolated hypertriglyceridemia
LCAT = lecithin:cholesterol acyltransferase
MHTG = patient group with mixed hypertriglyceridemia
NL = normolipidemic healthy study subjects
PAGGE = polyacrylamide gradient gel electrophoresis
TG = triglyceride
Ve = elution volume


*    Acknowledgments
 
Dr Murakami was supported by a Fellowship of the Fondazione Emilio Trabucchi, Milano, Italy. This work was supported in part by the Regione Lombardia (Progetto di Ricerca Finalizzato 1.4.5.6.).

Received April 28, 1995; accepted September 8, 1995.


*    References
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