HDL Phospholipid Content and Composition as a Major Factor Determining Cholesterol Efflux Capacity From Fu5AH Cells to Human Serum
Abstract The relationships of cell cholesterol efflux to HDL phospholipid (PL) content and composition in human serum were analyzed in two groups of subjects selected on the basis of their HDL cholesterol (HDL-C) levels: a norm-HDL group (1.10 mmol/L<HDL-C<1.50 mmol/L) and a high-HDL group (HDL-C>1.75 mmol/L). In the high-HDL group, the relative fractional efflux was significantly higher than in the norm-HDL group, and in both groups, fractional efflux was correlated with a number of lipoprotein parameters, the best correlation and the only one that remained significant after multivariate analysis being with HDL phospholipid (HDL-PL). Analysis of the HDL-PL subclasses revealed that HDL in the high-HDL sera was enriched with phosphatidylethanolamine (HDL-PE) and relatively deficient in sphingomyelin (HDL-SM) compared with norm-HDL sera. Moreover, the fractional efflux values in the high-HDL group were negatively correlated with the proportion of HDL-PE (r=−.64, P<.0001) and positively correlated with the proportion of HDL-SM (r=.43, P<.01). Thus, this study provides evidence that HDL-PL concentration can be used to predict the capacity of serum to accept cellular cholesterol. Among the differences described between norm-HDL and high-HDL sera, the variability in PE to SM ratio might reflect changes in serum cholesterol acceptors that modulate the first step of reverse cholesterol transport.
- high density lipoproteins
- cholesterol efflux
- reverse cholesterol transport
- Received October 15, 1996.
- Accepted April 21, 1997.
The efflux of cholesterol from cells is an important process by which cells maintain cholesterol homeostasis and represents the first step in reverse cholesterol transport. It has been extensively documented that the movement of cholesterol between cells and the extracellular environment is mediated by a number of different serum lipoproteins (for review see References 1 and 21 2 ). In a previous investigation studying the potential of human serum to stimulate cell cholesterol efflux, we observed high correlations between efflux and a number of HDL-related parameters.3 The highest correlation observed in this study was to HDL-C concentrations; however, this later correlation explained only 50% of the variation in efflux encountered when cells were incubated with individual specimen of human serum. More recently, studies using serum from transgenic mice and rats expressing the human apoA-I gene indicated that the prime component of HDL modulating cholesterol efflux was HDL-PL.4 5 We also observed, using mouse and rat transgenic animal models, that cholesterol efflux became relatively less efficient as human apoA-I in the serum was increased. This was particularly obvious in human apoA-I transgenic (HuAITg) mice in which expression of human apoA-I increased HDL-C levels without a significant increase in efflux.4 The HuAITg rat model demonstrated that the reduced efficiency in fractional efflux in rats expressing high concentrations of human apoA-I was due to a marked decrease in the HDL-PL:apoA-I ratio in the serum.5
In the present study, we have reexamined the relationships between cell cholesterol efflux and a variety of lipid and lipoprotein parameters present in human serum, including total phospholipid and PL subclass distribution in HDL. To determine whether fractional efflux was influenced by the level of HDL in human serum, we selected for this study a group of individuals with elevated HDL concentrations compared with a group of individuals with HDL levels in a normal range.
All human serum specimens were from asymptomatic hypercholesterolemic men and were obtained from the clinical chemistry laboratory at the Hôpital Broussais in Paris, France. On the basis of the HDL-C mean values, we separated two distinct populations. The first group was comprised of 50 males presenting a normal range of HDL-C levels (1.10<HDL-C<1.50 mmol/L). The second group was represented by 37 males selected as having HDL-C concentrations >1.75 mmol/L. Blood was collected by standard venipuncture and allowed to clot for 45 to 60 minutes at room temperature. The serum was then quickly separated from the cells and frozen at −70°C in 1-mL aliquots. For the assay of efflux potential, each serum aliquot was thawed only once just before use. A serum standard was prepared using a large number of aliquots from a pool of serum samples. The serum standard was also stored at −70°C and thawed only once before use.
Lipids, Lipoproteins, and HDL Subfractions
Lipids, apolipoproteins, and HDL subfractions were quantified using methods previously described.3 Briefly, serum total cholesterol and triglycerides were measured with enzymatic methods. HDL-C was determined enzymatically after precipitation of LDL and VLDL with PTA. Serum apoB was quantified by immunonephelometry with a BNA analyzer (Behring) using commercial polyclonal antibody (Behring). Total apoA-I was determined by electroimmunoassay using a commercial kit (Sebia). Quantitative determination of Lp A-I was performed by differential electroimmunoassay of serum apoA-I in HDL particles containing only apoA-I.6 The concentration of Lp A-I/A-II was calculated as the difference between total apoA-I and Lp A-I, both determined by electroimmunoassay, as cited above. HDL2 and HDL3 cholesterol concentrations were determined by a direct electrophoretic method in discontinuous gradient gel.7 Serum total PLs were determined with an enzymatic test kit (Biotrol) according to the manufacturer’s recommendations. In some samples, the complete quantification of lipid HDL components (free and esterified cholesterol, PL, and triglycerides) was determined on the supernatant after PTA precipitation, using enzymatic commercial kits (Biotrol).
PL Composition Analysis
The PL subclasses were determined in the HDL supernatant after PTA precipitation by high-performance liquid chromatography according to the method reported by Becart et al8 after extraction of total PLs by the method of Bligh and Dyer.9 A calibration curve was constructed using a chloroform/methanol (1:1, vol/vol) solution containing given amounts of each PL subclass and dicaproyl phosphatidylcholine (Sigma) as an internal standard. A Beckman liquid chromatographic Gold system consisting of a model 126 pump, a model 406 interface, and a Dell 466 L computer was used (Beckman). The detection was performed by a light-scattering detector, model DDL 21 (Eurosep Instruments). Briefly, PL subclasses were separated on a silica gel column (119×4 mm “Supersher” Si 60, 4 microns, Merck) using a binary gradient elution (solvent A: chloroform/methanol/ammonium hydroxide at 30% [80:19.5:0.5, by vol]; solvent B: chloroform/methanol/water/ammonium hydroxide at 30% (60:34:5.5:0.5, by vol; flow rate, 1 mL/min]]). In the light-scattering detector, the nebulization was performed at 70°C and a pressure of 2 bar. Results are expressed as both mass (milligrams per liter) and percent composition for each PL subclass.
Cholesterol Efflux Assay
The cholesterol efflux assay, which quantitates the potential of each serum specimen to remove radiolabeled cholesterol from the cell membrane, was performed as previously described.3 Briefly, individual samples of human serum diluted to 5% were incubated at 37°C with the 3[H]cholesterol-labeled Fu5AH rat hepatoma cells for 4 hours. At least three wells of cells were incubated with each serum sample. Serum samples were kept at −70°C and were then quickly thawed, thoroughly mixed, and diluted into minimum essential medium just before addition to cells. After incubation, the medium was removed from the cell monolayer, centrifuged to remove floating cells, and stored at −70°C for further analysis. At the end of the efflux period, cell monolayers were washed with PBS. The cellular lipids were extracted with isopropanol by overnight incubation at room temperature, and cellular lipid radioactivity was measured in an aliquot of the extract. The radioactivity released to the medium was expressed as the fraction of the total radioactive cholesterol present in each well. To standardize the cellular response obtained with different batches of cells and labeling medium, a standard pool of human serum was prepared and was always included in the experiment as a test serum. At least two aliquots of this pool were assayed in each efflux experiment. The fractional efflux of tested sera in different experiments was then expressed as a relative efflux by dividing the absolute efflux value by the value of the standard pool.
Quantitative variables were expressed as mean±SD. The unpaired Student’s t test was used to analyze statistical differences between the norm-HDL and high-HDL groups. Linear correlation coefficients and multiple regression were performed on a Macintosh IISI computer by using a statistical software program (Statview II, Abacus Concepts Inc). A value of P<.05 was considered statistically significant.
Biochemical Characteristics of the Serum Samples
Serum samples came from two groups of subjects selected among a population of asymptomatic hypercholesterolemic males on the basis of HDL-C concentrations. The first group of samples was obtained from individuals with normal HDL-C concentrations (norm-HDL: 1.10<HDL-C<1.50 mmol/L), and the second group was obtained from subjects with elevated HDL-C values (high-HDL: >1.75 mmol/L). Table 1⇓ presents the various serum parameters in these two groups. As expected, all of the HDL-related parameters, except Lp A-I/A-II concentrations, were significantly higher in the high-HDL group than in the norm-HDL group. Although total cholesterol was not different between the two groups, we observed an elevation of total PLs and a diminution of triglycerides, LDL-cholesterol, and apoB in the high-HDL group.
Cellular Cholesterol Efflux
The fractional efflux of radiolabeled free cholesterol from Fu5AH cells to whole serum was determined by using 5% serum and an incubation time of 4 hours. To reduce variation in the determination encountered by conducting the assays on different days and with different preparations of cells, all of the efflux data were normalized to a standard pool of human serum that was run in each separate assay. As expected, the average value for cholesterol efflux was significantly higher in the high-HDL group than in the norm-HDL group (1.30±0.15 versus 1.25±0.08, P=.042).
Relationships Between Cellular Cholesterol Efflux and Serum Parameters
Table 2⇓ presents both the univariate and multivariate analysis of the correlations obtained between cell cholesterol efflux and the measured serum lipoprotein parameters for the norm- and high-HDL populations. Univariate analysis demonstrated that a number of lipoprotein parameters significantly correlated with efflux in both groups of sera. For both groups, the highest correlation was to HDL-PL (r=.70 and r=.61 for norm-HDL and high-HDL, respectively). The positive correlation between efflux and total serum PL is a reflection of both the fact that approximately 50% of the total PL is contributed by the HDL fraction and the fact that there is a good correlation between total serum and HDL-PL levels (r=.55, P=.0001 and r=.62, P=.0001 in norm- and high-HDL groups, respectively). The comparison of the regression lines fitting HDL-PL to fractional efflux in the two groups (Fig 1⇓) shows that the slope was lower, although not significantly, in the high-HDL (0.318) group than in the norm-HDL group (0.485), suggesting a relative loss of efficiency of HDL particles for cholesterol efflux when their concentration in serum is elevated.
The correlations observed between efflux and Lp A-I and Lp A-I/A-II emphasized the differences in the HDL subclasses profile in the norm- and high-HDL groups. Indeed, efflux is correlated with Lp A-I concentrations only in the high-HDL specimens (r=.59, P<.001). On the other hand, efflux was correlated with Lp A-I/A-II in the norm-HDL group only (r=.49, P<.0003). Thus, these data indicate that the relative contribution of HDL subclasses to the efflux of cell cholesterol depends on the level of their concentrations in the serum. When the data on serum parameters and cholesterol efflux were analyzed by multiple regression (Table 2⇑), the only parameter that remained significant in both the norm-HDL and high-HDL groups was HDL-PL.
Fig 2⇓ illustrates the relationships between efflux and HDL lipid components in sera having normal HDL-C levels. Thirty-six sera in which HDL lipid values were available were examined, and the efflux was related to the concentrations of surface and core lipid components of the HDL. The surface components of the HDL particles (ie, PL and FC) demonstrated the highest correlation coefficients (r=.73 and r=.52, respectively), with much lower correlations between efflux and the lipoprotein core components (esterified cholesterol: r=.37; triglycerides: r=.36). No relationship was observed between the HDL-PL/FC ratio and efflux (r=.11).
Among the serum lipid parameters influencing fractional efflux in the norm-HDL group, we found an unexpected positive relationship with triglyceride concentrations (r=.37, P=.008), which did not exist in the high-HDL group. Since triglyceride concentrations were positively correlated with HDL-PL levels in the norm-HDL group (r=.44, P<.001) and not in the high-HDL group (r=.14), we suggest that the positive association between triglycerides and efflux in the norm-HDL group was a reflection of the influence of HDL-PL concentration on cell cholesterol efflux.
HDL-PL Composition and Relationships With Fractional Efflux
PL subclasses were separated and quantified by high-performance liquid chromatography performed on the HDL supernatant obtained after precipitation of VLDL and LDL in 37 norm-HDL sera and 34 high-HDL sera. Table 3⇓ shows the comparison of PL composition between the two groups, expressed as both concentration and percentage of each subclass. The concentrations of HDL-PC, HDL-PE, and HDL-PI were significantly higher in the high-HDL sera, whereas no difference was observed between the two groups for HDL-SM. The lack of difference in the HDL-SM mass led to a significantly lower proportion of HDL-SM in the high-HDL sera than in the norm-HDL sera. Moreover, in the high-HDL group, the proportion of PC and PE was significantly higher than in the norm-HDL group. These differences led to a significant reduction of the SM:PC ratio and a significant increase of the PE:PC ratio in the HDL of the high-HDL sera. Finally, the PE:SM ratio was twice higher in the high-HDL sera compared with the norm-HDL sera.
The relationship between HDL-PL composition and cellular cholesterol fractional efflux was analyzed on the basis of each subclass mass or relative percentage (Table 4⇓). In both the norm-HDL and the high-HDL groups, efflux was significantly positively correlated with the concentration of HDL-PC and HDL-SM. Although it did not reach statistical significance, the trend of correlation between efflux and HDL-PE concentration was opposed in the two groups, since it was positive in the norm-HDL group and negative in the high-HDL group. Finally, we observed a trend for a positive correlation between HDL-PI in the high-HDL group, which did not appeared in the norm-HDL group.
The relative proportion of each PL subclass in the norm-HDL group had no influence on the fractional efflux values, since none of the correlations studied were significant. By contrast, in the high-HDL group we found that the two PL subclasses that differed from the norm-HDL group, percent SM and percent PE, exhibited significant correlations with fractional efflux. The fractional efflux values were positively related to the percent SM (r=.43, P<.01) and negatively related to the percent PE (r=−.64, P<.0001). Overall, in this group, the variability of efflux values was strongly and negatively associated to the variability of the PE:SM ratio (r=−.64, P<.0001) (Fig 3⇓).
As an extension of a previous study on HDL metabolic properties in whole serum,3 4 5 we have addressed, in the present report, the role of HDL-PL concentration and composition as a marker for cell cholesterol efflux to human serum. Thus, we have compared the lipoprotein profile, the PL pattern, and the fractional efflux of cellular cholesterol of two groups of human sera presenting either elevated or normal HDL levels.
Despite their recognized physiological and structural importance, PLs have not been extensively investigated as mediators for reverse cholesterol transport or as markers for coronary artery disease. The routine determination of PLs in this study was performed using an enzymatic kit method that is relatively simple, although it does not measure minor subspecies like phosphatidylserine, PI, and PE. Our results demonstrated that HDL-PL level best reflects the capacity of human serum to release radiolabeled cholesterol from Fu5AH cells. In both norm- and high-HDL groups, the correlation between fractional efflux and HDL-PL was much stronger than with any other measured lipoprotein parameter (Table 2⇑). This result corroborates our recent studies showing that in the serum of rats transgenic for human apoA-I, PL content was the prime component of HDL modulating cholesterol efflux.5 The predominant role of PLs on the release of cholesterol from cells can be attributed to their specific amphiphilic properties, which facilitate the capture of membrane free cholesterol by extracellular acceptors. It has been shown in vitro that the PL depletion of HDL particles by phospholipase A2 or hepatic lipase treatment,10 11 and more recently by mild copper oxidation,12 reduces cell free cholesterol efflux. Also, enrichment of lipoproteins with PL enhances their capacity to remove cholesterol from cells, as evidenced when PL was added to whole serum from rats5 or humans.13 Although the PL:FC ratio on the acceptor particles has been shown to be critical in determining the bidirectional flux of cholesterol that occurs when cells are incubated with lipoproteins, we did not find a direct influence of this ratio on the values of fractional efflux in this study. As previously demonstrated by Johnson et al,10 the impact of the PL:FC ratio rather influences the influx of lipoprotein free cholesterol into the cells and would more directly affect the intracellular content of cholesterol.
Although the correlation between cholesterol efflux and HDL-PL was highly significant in both the norm- and the high-HDL groups (Fig 1⇑), the data indicate that the efflux efficiency of the mixture of HDL particles present in the high-HDL group is somewhat lower than in serum from individuals with a normal range of HDL concentrations. It can be estimated that it requires from 40% to 50% more HDL-PL in the serum obtained from the high-HDL group to produce a change in fractional efflux equivalent to that obtained with the sera from norm-HDL individuals.
The analysis of HDL-PL subclass composition demonstrates differences between the two groups of serum specimens, which might account for the difference in efflux efficiency. First, as expected from the increased concentration in total HDL-PL in the high-HDL group, the concentrations of HDL-PC, HDL-PE, and HDL-PI were higher than in the norm-HDL group, whereas the concentrations in HDL-SM were not different between the two groups. This result led to a significant reduction in the relative proportion of HDL-SM in the high-HDL group compared with the norm-HDL group. The importance of SM in modulating cellular cholesterol efflux clearly appeared from this study, since HDL-SM concentration correlated with fractional efflux in both groups. Moreover, when the results obtained in both groups were pooled and divided according to the median value of HDL-SM, the average fractional efflux value for the samples below the median was significantly lower than for the samples above the HDL-SM median (1.22±0.12 versus 1.32±0.10, P<.001). By contrast, no significant differences were observed in average efflux values when the whole population was separated on the basis of the HDL-PC, HDL-PE, or HDL-PI concentration median values.
Another feature of high-HDL sera was a significant increase in HDL-PE, both in concentration and in relative proportion, which was inversely associated with fractional efflux. We have no explanation for the enrichment with PE nor for the relative deficiency in HDL-SM in the sera containing elevated concentrations of HDL. However, both the positive and negative relationships with efflux are consistent with the known differences in affinity of these PLs for cholesterol. Indeed, it has been reported that PE has the lowest affinity for cholesterol of any PL tested14 and, as an expected consequence, the lowest ability to deplete tissue-culture cells or PL vesicles of cholesterol.15 16 17 By contrast, it also has been demonstrated that SM is a very efficient solvent for cholesterol18 and has a higher affinity toward cholesterol in small unilamellar vesicles than other PLs.19 Recently, Zhao et al20 have shown that addition of increasing amounts of SM in Lp A-I reconstituted particles increased their ability to accept cellular cholesterol, whereas no difference was observed when PC content was changed. It has been proposed that SM may create packing defects on the surface of the acceptors, thus creating spaces for incoming cholesterol molecules.20
It is likely that the variations in PL subclass composition in the high-HDL sera are related to shifts in HDL subfraction distribution. For example, HDL2 subfraction, which is almost doubled in the high-HDL sera, has been reported to be enriched with PE compared with HDL3 subfraction.21 Moreover, studies investigating the properties of PL-protein complexes, have shown that PE is a preferential substrate for phospholipolysis activity of hepatic lipase.22 Since it is established that in hyperalphalipoproteinemia the accumulation of HDL2 is attributed, at least in part, to low hepatic lipase activity,23 the enrichment with PE in the HDL of high-HDL subjects might result from an increase of slowly catabolized HDL2.
We also observed that Lp A-I concentration, but not Lp A-I/A-II, was increased in the high-HDL group and was significantly correlated with efflux, whereas in the norm-HDL group, Lp A-I/A-II, but not Lp A-I, was correlated with efflux. The critical impact of Lp A-I and LpA-I/A-II subfractions on cholesterol efflux to whole serum has been discussed recently by Syvänne et al.24 In their study, it is shown that, within sera of controls and diabetic patients having HDL concentrations in a normal and low range, only Lp A-I/A-II is positively correlated to cell cholesterol efflux. Although it has been demonstrated that both particles can function as acceptors of cholesterol,25 26 it is now apparent that Lp A-I and Lp A-I/A-II represent heterogeneous populations of particles,27 and variations in serum concentration among individuals might result in differences in efflux capacity.28 Whether this structural heterogeneity is related to PL composition is not known. Thus, the link between HDL subclass distribution and PL subclass composition is not yet established and deserves to be carefully investigated to clarify how the PL composition differs among HDL particles and to what extent this influences the first steps of reverse cholesterol transport.
Another interesting result reported by Syvänne et al24 was the positive association between PL transfer protein activity and cholesterol efflux to whole serum. In our study, we have shown positive relationships between triglycerides, HDL-PL, and cholesterol efflux, which suggests that hypertriglyceridemia, at least to a moderate extent, might be able to produce an increase in HDL-PL content, leading to an enhancement of cholesterol efflux. This hypothesis fits with a role of PL transfer protein of indirectly stimulating cellular cholesterol efflux by modulating the exchange of PLs between triglyceride-rich lipoproteins and HDL.29
In conclusion, this study provides evidence that HDL-PL concentration is a simple and easily determined parameter that can be used to predict the capacity of serum to remove cellular cholesterol. This observation is of particular interest with regard to clinical studies that have shown that HDL-PL is decreased in patients with coronary artery disease.30 31 32 In addition, it has been shown that the severity of coronary artery disease is more strongly correlated with the decrease in HDL-PL than in HDL-cholesterol.32 33 The relationship between clinical assessment of atherosclerosis and reverse cholesterol transport requires a more careful investigation, taking into account the importance of HDL-PL. The second aspect emphasized in this study is that as it was previously shown in the mouse and rat models transgenic for human apoA-I, human serum with high HDL concentrations is not as efficient for cholesterol efflux as might be predicted on the basis of results obtained in the norm-HDL group. Among the differences that we described between the two groups, the variability in SM and PE might reflect changes in the distribution of the circulating cholesterol acceptors that modulate the first steps of reverse cholesterol transport.
Selected Abbreviations and Acronyms
|PTA||=||phosphotungstic acid/MgCl2 reagent|
This work was supported in part by a research grant from Fournier Laboratories, Dijon, France; by a North Atlantic Treaty Organization collaboration research grant No. 930317 (V. Atger); by program project grant HL22633 (G. Rothblat); by a Minority Investigator Research Supplement (MIRS) grant; by a Mentored Research Scientist Development Award HL03522 (M. de la Llera Moya); and by an Allegheny-Singer Research Institute Grant 95-045-3MCP. We wish to thank Dr A. Simon and the PCVMETRA Group (Groupe de Prévention Cardiovasculaire en Médecine du Travail, Boulogne, France) for providing the serum samples.
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