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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1388-1396

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1388-1396.)
© 1995 American Heart Association, Inc.

Articles

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

Presented in part at the 65th Scientific Sessions of the American Heart Association, New Orleans, La, and published in abstract form (Circulation. 1992;86[suppl I]:I-71.)

Laurent Lagrost; Emmanuel Florentin; Valérie Guyard-Dangremont; Anne Athias; Hassan Gandjini; Christian Lallemant; Philippe Gambert

From the Laboratoire de Biochimie des Lipoprotéines, INSERM CJF 93-10, Faculté de Médecine, Dijon, France.

Correspondence to Laurent Lagrost, Laboratoire de Biochimie Médicale, Hôpital du Bocage, 21034 Dijon, France.


*    Abstract
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 *Abstract
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Abstract 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->VLDL+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.


Key Words: plasma • lipoprotein • cholesteryl ester • triglyceride • cholesteryl ester transfer protein


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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In human plasma, the exchange of neutral lipids, ie, cholesteryl esters and triglycerides, between plasma lipoproteins is mediated by a specific factor, the CETP.1 2 The activity of CETP results in the redistribution of cholesteryl esters, synthesized within HDL by the LCAT,3 toward VLDL and LDL.4 5 Numerous clinical investigations have clearly evidenced that, in humans, increased concentrations of LDL and reduced concentrations of HDL are associated with the development of atherosclerosis.6 7 Thus, by catalyzing the transfer of cholesteryl esters from HDL toward VLDL and LDL, CETP could be regarded as a potentially atherogenic factor.8 9 That latter concept is in good agreement with the positive correlation between plasma CETP concentrations and the extent of coronary artery atherosclerosis in monkeys,10 with the increase in both the severity and progression of atherosclerotic lesions in CETP-transgenic mice as compared with control animals,11 as well as with the inverse relation between CETP activity and HDL cholesterol concentrations in normal12 13 and CETP-deficient subjects.14 15 Recent in vivo studies brought circumstantial evidence for the regulation of the CETP gene expression by environmental factors, among them the fat content of the diet.9 Complementary in vitro studies demonstrated that NEFAs with various chain length can upregulate both the expression of the CETP gene and the secretion of the protein in cultured cell models.16 17 In addition to plasma CETP concentration,18 19 a number of factors are susceptible to influence the plasma CET activity, among them the concentration and composition of donor and acceptor lipoprotein substrates.9 20 Lipoprotein lipase has been shown to enhance the CETP-mediated transfer of cholesteryl esters from HDL toward VLDL as the result of the accumulation of lipolytic products, such as NEFAs, in the lipoprotein surfaces.21 22 In vitro, oleic acid can mimic the effect of VLDL lipolysis by enhancing the binding of CETP to lipoprotein substrates and increasing the rate at which cholesteryl esters are transferred from HDL toward VLDL22 or from HDL toward LDL.23 The effect of NEFA on the CETP-mediated transfer of cholesteryl esters is dependent on the ionization of the carboxylic group22 and on the structure of the acyl chain.24 25 Therefore, results from these latter studies indicate that NEFA might increase not only the synthesis but also the activity of CETP. Earlier studies26 27 have suggested that reciprocal transfers of neutral lipids between plasma lipoprotein fractions are closely linked. However, complementary investigations have challenged this concept by indicating that the transfer of esterified cholesterol and triglycerides between lipoproteins does not involve a simple molecular exchange of one moiety for the other but is achieved by processes that are at least partially independent.28 29 30 In particular, oleic acid has been shown recently to reduce the net mass of triglycerides transferred from triglyceride-rich lipoproteins to HDL while increasing the net transfer of cholesteryl esters in the reverse direction, from HDL toward triglyceride-rich lipoproteins.30 These results suggest that NEFAs could dissociate the heteroexchange of neutral lipids between HDL and lipoproteins of lower density. However, the significance of the modulation by NEFA of lipid transfers in vivo is still uncertain and, in particular, the ability of physiological concentrations of NEFA to influence CETP activity in total plasma remains to be established.

The goals of the present study were to determine whether NEFAs in fasting human plasma can modulate CETP-mediated cholesteryl ester transfers and to investigate their effects on the reciprocal exchange of cholesteryl esters and triglycerides between HDL and apo B–containing lipoproteins.


*    Methods
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 *Methods
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Subjects
The study subjects were normolipidemic, nonsmoking healthy volunteers recruited among the biomedical laboratory personnel. Informed consent was obtained. Subjects did not take drugs other than oral contraceptives. Venous blood samples were collected in Na EDTA–containing tubes after an overnight fast and a 15-minute complete rest. Plasmas were promptly separated by low-speed centrifugation at 4°C and kept at the same temperature. All samples were analyzed within a few hours after collection.

Lipoprotein Preparation
Lipoprotein fractions were isolated from total normolipidemic plasmas by sequential ultracentrifugation at 100 000 rpm (350 000g) in a TLA-100.2 rotor in a Beckman TL-100 ultracentrifuge. LDL was isolated as the plasma fraction 1.019<d<1.055 g/mL with one 4-hour spin at the lowest density and two 5-hour spins at the highest density. HDL3 was isolated as the plasma fraction 1.13<d<1.21 g/mL with one 5-hour spin at the lowest density and two 7-hour spins at the highest density. Lipoproteins were recovered by tube slicing and were then dialyzed overnight against a buffer of 10 mmol/L Tris, 150 mmol/L NaCl (pH 7.4) containing 5 mmol/L EDTA-Na2 and 3 mmol/L NaN3 (TBS buffer).

Purification of CETP
CETP was partially purified from citrated human plasma according to the sequential procedure previously described.31 Briefly, the ultracentrifugally isolated d>1.21 g/mL plasma proteins were fractionated successively by hydrophobic interaction chromatography on a phenyl-Sepharose CL-4B column, affinity chromatographies on Heparin-Ultrogel A4R and Blue-Trisacryl columns, and anion-exchange chromatography on a Mono-Q HR 5/5 column. Chromatographic separations were performed by using a fast protein liquid chromatography system (Pharmacia). The preparation of CETP was deficient in LCAT activity and PLTP activity.31

Radiolabeling of LDL and HDL3
HDL3 was biosynthetically labeled according to the general procedure previously described.32 A d>1.13 g/mL plasma fraction was obtained after ultracentrifugation of 20 mL total normolipidemic plasma, dialyzed against TBS, and incubated with 10 nmol [1{alpha}, 2{alpha}(n)-3H]cholesterol (specific activity, 46 Ci/mmol; Amersham) for 24 hours at 37°C in a shaking water bath. Subsequently, the 1.019<d<1.055 g/mL fraction obtained from 10 mL total normolipidemic plasma (about 15 mg LDL cholesterol) was added to the incubated mixtures. The incubation was then prolonged for a 6-hour period to allow the exchange of radiolabeled esterified cholesterol between lipoprotein substrates. That procedure presents two main advantages: (1) radiolabeled HDL3 and LDL are prepared in the same batch and (2) HDL3 is replete with unlabeled nonesterified cholesterol during the 6-hour prolongation of the incubation in the presence of isolated LDL. At the end of the incubation, the LDL and HDL3 fractions were recovered by sequential ultracentrifugation as described above. Typical labeled preparations of LDL and HDL3 obtained with this procedure had specific activities of approximately 7000 and 28 000 cpm/nmol of cholesterol, respectively. As judged by thin-layer chromatography, more than 95% of total radioactivity of both lipoprotein substrates resided in the cholesteryl ester moiety.

Isotopic Assays of Plasma CET Activity
The CET activity of a sample was measured as its capacity to promote the transfer of radiolabeled cholesteryl esters from donor to acceptor lipoprotein substrates. In native plasma, total CET activity was evaluated by measuring the rate of radiolabeled cholesteryl esters transferred either from HDL3 to endogenous apo B–containing plasma lipoproteins (CETHDL->VLDL+LDL activity) or from LDL to endogenous plasma HDL (CETLDL->HDL activity). For that purpose, a tracer dose (2.5 nmol of cholesterol) of either radiolabeled HDL3 or radiolabeled LDL was added to 25 µL of plasma in a final volume of 50 µL. Plasma LCAT activity was blocked by adding iodoacetate (final concentration, 1.5 mmol/L). In some experiments, fatty acid–free albumin (Sigma) was added (final concentration, 8 g/L). Duplicate mixtures were incubated for 3 hours at 37°C in an incubator. At the end of the incubation, the tubes were immediately placed on ice. A volume of 45 µL of the incubated mixtures was adjusted to density 1.07 g/mL Kbr in a final volume of 2 mL in Quickseal centrifugation tubes (Beckman). The tubes were then sealed and subjected to ultracentrifugation for 7 hours at 50 000 rpm (269 000g) in a 50.4 Ti rotor in an L7 ultracentrifuge (Beckman). The plasma fractions of d<1.068 g/mL (plasma apo B–containing lipoproteins) and of d>1.068 g/mL (HDL-containing plasma fraction) were recovered in a volume of about 1 mL and transferred into counting vials. A volume of 2 mL scintillation fluid (OptiScint Hisafe 3, Pharmacia) was added to each vial, and the radioactivity was assayed for 5 minutes in a Wallac 1410 liquid scintillation counter (Pharmacia). The recovery of total radioactivity in the d<1.068 and d>1.068 g/mL fractions was constantly greater than 95%. In nonincubated controls containing radiolabeled HDL3, the radioactivity recovered in the d<1.068 g/mL fraction did not exceed 4% of the total. In nonincubated controls containing radiolabeled LDL, less than 10% of the radioactivity was recovered in the d>1.068 g/mL fraction. CET activity was measured as the rate of total radiolabeled cholesteryl esters transferred from the lipoprotein tracer to the d<1.068 g/mL or the d>1.068 g/mL acceptor fraction during a 3-hour incubation at 37°C after deduction of blank values of control mixtures kept at 4°C.33 Results were expressed in percentage of radiolabeled cholesteryl esters transferred.

Cholesteryl Ester and Triglyceride Mass Transfer Assay
The mass transfer of cholesteryl esters and triglycerides between HDL and apo B–containing lipoproteins was measured during the long-term incubation of total plasma at 37°C in the presence of an LCAT inhibitor.34 For that purpose, 100 µL aliquots of total plasma were supplemented with iodoacetate (final concentration, 1.5 mmol/L) and incubated at 37°C for 24 hours. At the end of incubation, apo B–containing lipoproteins were selectively precipitated with a phosphotungstic acid/MgCl2 reagent (Boehringer Mannheim) as recommended by the manufacturer. Unesterified cholesterol, total cholesterol, and triglyceride concentrations present in the supernatant were measured, and the mass of esterified cholesterol and triglycerides transferred were calculated as compared with control samples maintained at 4°C.

CETP ELISA
CETP concentration in total human plasma was measured by using a competitive ELISA on a Biomek 1000 Biorobotic System (Beckman Instruments).35 Calibration was carried out by comparison with a standard curve obtained with a frozen plasma standard.35 Calculation of CETP concentrations was realized by using a data analysis software (Immunofit EIA/RIA Data Analysis Software, Beckman). The CETP concentration value for each plasma sample was calculated by averaging quadruplicate determinations. Intra-assay and interassay coefficients of variation were 4% and 6%, respectively.

Specific CET Activity
CETHDL->VLDL+LDL and CETLDL->HDL specific activities were calculated as the ratio of plasma CET activity to plasma CETP mass concentration and expressed in percentage of total radiolabeled cholesteryl esters transferred per microgram of CETP.

Precipitation of the Total Plasma Lipoprotein Fraction
Lipoproteins were removed from total human plasma by using a single-step adsorption method based on the ability of FSD (SERVA Feinbiochemica) to remove large particles, such as lipid emulsions and lipoprotein particles, from aqueous media.36 37 38 Briefly, total plasmas were treated for 10 minutes at room temperature with various concentrations of FSD according to the general procedure previously described.38 At the end of the incubation, precipitates were removed by low-speed centrifugation, and remaining amounts of NEFA, cholesterol, apo B, apo A-I, and albumin were assayed in the supernatants.

Other Analytical Methods
All chemical assays were performed on a Cobas-Fara Centrifugal Analyzer (Roche). Total cholesterol, unesterified cholesterol, and triglyceride concentrations were measured by enzymatic methods using Boehringer reagents. HDL cholesterol was determined after precipitation of apo B–containing lipoproteins with phosphotungstic acid/MgCl2 as described above. Concentrations of plasma apo B, apo AI, and apo AII were determined by immunoturbidimetry39 with anti-apo B, anti-apo AI, and anti-apo AII antibodies purchased from Behring. Apo B and apo AI standards were purchased from Behringwerke AG. Apo AII standard was purchased from Immuno AG. NEFA concentrations were determined by using both a commercially available enzymatic kit (Wako Pure Chemical Industries) and a capillary gas chromatography procedure.40 By use of the statistical method of Bland and Altman,41 a good agreement was observed between enzymatic and gas chromatography plasma NEFA determinations. Albumin concentrations were measured by using the A-Gent Albumin Test from Abbott.

Statistical Analysis
Student's t test was used to determine the significance of the difference between data means. Coefficients of correlation were calculated by using linear regression analysis. Multiple regression analysis was used to determine significant contributions of triglycerides, HDL cholesterol, VLDL+LDL cholesterol, and NEFA to the prediction of CET activity in human plasma.


*    Results
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 up arrowMethods
 *Results
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Relations Between CETP Mass Concentrations and Lipoprotein Parameters in Normolipidemic Human Plasma
Plasma CETP concentrations were determined among a population of 27 normolipidemic subjects (plasma cholesterol range, 121 to 234 mg/dL; plasma triglyceride range, 27 to 84 mg/dL; plasma NEFA range, 0.07 to 0.73 mmol/L) by using a competitive ELISA as described under "Methods." Plasma CETP levels (0.28±0.06 mg/dL; range, 0.19 to 0.42 mg/dL) were significantly and positively correlated only with the level of NEFA (r=.50; P=.008) and the VLDL+LDL:HDL cholesterol ratio (r=.39; P=.043). In contrast, no significant relations were observed between CETP and other lipid or apolipoprotein components.

Correlation of Plasma NEFA Concentration With CET Activity
CETP activity was evaluated by measuring the transfer of tritiated cholesteryl esters from a tracer dose of exogenous radiolabeled lipoproteins toward endogenous plasma lipoprotein fractions (see "Methods"). As shown in Fig 1Down, the addition of increasing concentrations of purified CETP to total normolipidemic plasmas resulted in CET assays that gave linear response and permitted the measurement of plasma CETP activity over a wide range of values.



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  		Figure 1. Line graphs showing the relations between purified CETP amounts added to total normolipidemic plasmas and CET activity values. CETHDL->VLDL+LDL activity (top) was determined as the percentage of radiolabeled cholesteryl esters transferred from a tracer dose of exogenous HDL3 toward the plasma d<1.068 g/mL fraction. CETLDL->HDL activity (bottom) was determined as the percentage of radiolabeled cholesteryl esters transferred from a tracer dose of exogenous LDL toward the plasma d>1.068 g/mL fraction (see "Methods").

Among a population of 37 normolipidemic subjects (plasma total cholesterol range, 121 to 240 mg/dL; plasma triglyceride range, 27 to 142 mg/dL; plasma NEFA range, 0.07 to 1.02 mmol/L), the rate of transfer of cholesteryl esters from radiolabeled HDL3 to the d<1.068 g/mL fraction correlated positively with total NEFA concentrations (r=.46, P<.005) (Fig 2Down), while no significant relation was observed with plasma albumin levels (results not shown).



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  		Figure 2. Scatterplot showing the correlation of CET toward VLDL+LDL with NEFA concentrations in plasmas from 37 normolipidemic subjects. CETHDL->VLDL+LDL activity was determined as the percentage of radiolabeled cholesteryl esters transferred from a tracer dose of exogenous HDL3 toward the plasma d<1.068 g/mL fraction (see "Methods").

To investigate further the effect of NEFA on the bidirectional transfer of cholesteryl esters between HDL and VLDL+LDL plasma fractions, CET activity was measured in 27 normolipidemic plasmas (total cholesterol range, 121 to 234 mg/dL; plasma triglyceride range, 27 to 84 mg/dL) in two ways, either from a tracer dose of radiolabeled HDL3 toward plasma apo B–containing lipoproteins (CETHDL->VLDL+LDL activity) or from a tracer dose of LDL toward plasma HDL (CETLDL->HDL activity). As shown in Table 1Down, CETHDL->VLDL+LDL activity correlated negatively with HDL cholesterol and esterified cholesterol:triglyceride ratio in HDL and correlated positively with VLDL+LDL cholesterol, VLDL+LDL:HDL cholesterol ratio, plasma triglyceride, and NEFA concentrations. Conversely, CETLDL->HDL activity correlated negatively with VLDL+LDL cholesterol, VLDL+LDL:HDL cholesterol ratio, and triglyceride levels and positively with HDL cholesterol levels. In contrast to CETHDL->VLDL+LDL activity, CETLDL->HDL activity did not correlate significantly with NEFA concentrations (Table 1Down). Multiple regression analysis revealed that plasma triglyceride, HDL cholesterol, VLDL+LDL cholesterol, and NEFA concentrations, when combined in a four-variable model, accounted for 75% of the variability in CETHDL->VLDL+LDL activity, with only NEFA reaching a significant level (P=.025), and for 68% of the variability in CETLDL->HDL activity, with both HDL cholesterol and VLDL+LDL cholesterol levels reaching significant levels (P=.033 and P=.010, respectively). Correlations between plasma NEFA concentration and specific CET activities calculated as the ratio of plasma CET activity to plasma CETP mass concentration did not reach the significance level.


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  		Table 1. Correlation Between Plasma CETP Activities and Plasma Lipid Parameters in 27 Normolipidemic Subjects

Distribution of NEFA in Normolipidemic Human Plasma
To evaluate the relative proportions of lipoprotein-bound and albumin-bound NEFA, lipoproteins were removed from total human plasma by using FSD, as described under "Methods." As shown in Fig 3Down, precipitated amounts of total plasma cholesterol, apo B, and apo AI increased gradually while raising the final concentration of FSD. When present at a concentration of 2.5 mg/mL, FSD removed the totality of plasma lipoprotein components, whereas only 5% of plasma albumin was coprecipitated (Fig 3Down). Interestingly, specific removal of plasma lipoproteins was accompanied by an approximately 20% reduction in plasma NEFA concentration (Fig 3Down). NEFA content of the plasma lipoprotein fraction was determined in 14 fasting plasmas with NEFA levels ranging from 0.16 to 1.07 mmol/L. A significant, positive correlation was observed between total plasma NEFA concentrations and NEFA amounts in the plasma lipoprotein fraction (Fig 4Down). In good agreement with previous data of Shafrir,42 parameters of linear regression analysis indicated that approximately 20% of plasma NEFA localized in the lipoprotein fraction, independent of NEFA levels in total plasma (Fig 4Down).



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  		Figure 3. Line graph showing the concentration-dependent precipitation of plasma components by FSD. One pool of normolipidemic plasmas was treated with increasing concentrations of FSD as described under "Methods." Remaining amounts of apo B, apo AI, total cholesterol, NEFA, and albumin were measured in the supernatants, and differences between concentrations in native and FSD-treated plasma samples were calculated. Values are expressed as percentage of the total plasma content and are mean±SD of triplicate determinations.



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  		Figure 4. Scatterplot showing the correlation between NEFA concentrations in total plasma and in the plasma lipoprotein fraction. NEFA levels were measured in 14 normolipidemic plasmas before and after precipitation of total plasma lipoproteins in the presence of FSD (2.5 mg/mL) (see "Methods"). The concentration of NEFA in the plasma lipoprotein fraction was calculated as the difference between NEFA levels in native and FSD-treated plasma samples.

Among a subset of 10 plasmas (cholesterol range, 145 to 240 mg/dL; triglyceride range, 49 to 142 mg/dL; NEFA range, 0.16 to 1.02 mmol/L; albumin range, 4.4 to 5.7 mg/dL), a positive correlation between the amount of NEFA bound to the plasma lipoprotein fraction and CETHDL->VLDL+LDL activity was observed (r=.65; P<.05).

Effect of Albumin on Plasma CET Activity
Albumin, the major carrier of NEFA in human plasma, is able to remove NEFA from plasma lipoprotein substrates when added in excess.42 In the present study, we made use of the high binding capacity of albumin for NEFA to confirm further that plasma NEFAs were implicated in the modulation of the CET reaction. For that purpose, we investigated the effect of the supplementation of plasmas with fatty acid–free albumin (final concentration, 8 g/L) on the rate of CET between the d<1.068 g/mL and d>1.068 g/mL plasma fractions. As assessed by using denaturing polyacrylamide gradient gel electrophoresis and specific anti-apolipoprotein immunoassays, and as indicated by the supplier (Sigma), the purified albumin fraction used in the present study was at least 99% pure (results not shown). The addition of albumin induced a significant reduction in CETHDL->VLDL+LDL activity (18.3±5.5% versus 9.8±3.1%; n=27; P<.0001). In contrast, CETLDL->HDL activity was not significantly modified in the presence of albumin (22.3±4.5% versus 23.3±5.1%; n=27; NS). As shown in Fig 5Down, CETHDL->VLDL+LDL activity did not correlate anymore with HDL cholesterol levels in the presence of albumin. In contrast, a significant positive correlation of CETHDL->VLDL+LDL activity with VLDL+LDL cholesterol levels subsisted and was even more pronounced after albumin supplementation (Fig 5Down). Whereas CETLDL->HDL activity correlated positively with HDL cholesterol levels (r=.447; P<.05) and negatively with VLDL+LDL cholesterol levels (r=-.483; P<.05), the addition of albumin to the incubation mixture abolished the significant correlations (r=.176, NS, and r=.239, NS, respectively).



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  		Figure 5. Scatterplots showing the correlations between CET toward VLDL+LDL and cholesterol concentrations in plasmas from 27 normolipidemic subjects. CETHDL->VLDL+LDL activity was determined as the percentage of radiolabeled cholesteryl esters transferred from a tracer dose of exogenous HDL3 toward the d<1.068 g/mL fraction of total plasma samples, which were supplemented (right) or not (left) with fatty acid–free albumin (final concentration, 8 g/L).

In native plasmas, regression analysis revealed the existence of a significant negative correlation between CETHDL->VLDL+LDL activity and CETLDL->HDL activity (r=-.45, P<.05). By contrast, when CETs were measured in plasma supplemented with albumin, we observed no more a negative but a significant positive correlation between the transfers in both directions (r=.40, P<.05).

Net Mass Transfers of Cholesteryl Esters and Triglycerides Between HDL and VLDL+LDL Plasma Fractions
To investigate whether nonesterified fatty acids could influence not only the velocity of CETs but also the capacity of lipoprotein substrates to accommodate or donate neutral lipids, total plasmas, containing an LCAT inhibitor (iodoacetate, 1.5 mmol/L), were incubated at 37°C for 24 hours in the presence or in the absence of albumin. As shown in Table 2Down, incubation of plasmas in the absence of albumin significantly decreased the esterified cholesterol content of HDL but increased its triglyceride content (variations compared with plasmas kept at 4°C: -4.5±1.4 mg/dL, P<.001, and +14.4±4.7 mg/dL, P<.001, respectively). The depletion in HDL esterified cholesterol induced by the incubation of total plasma was significantly lower in the presence of albumin than in nonsupplemented samples (Table 2Down). Conversely, the increase in HDL triglycerides was significantly higher in the presence of albumin than in nonsupplemented samples (Table 2Down).


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  		Table 2. Effect of Albumin on the HDL Esterified Cholesterol and Triglyceride Contents After Incubation of Total Normolipidemic Plasmas (n=27)


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
During the past few years, NEFAs have been implicated at different stages of the metabolism of plasma lipoproteins, including the clearance of triglyceride-rich lipoproteins,43 the esterification of plasma cholesterol by LCAT,44 and the triglyceride hydrolysis by lipoprotein lipase.45 In addition, several in vitro studies revealed that NEFA can regulate both the secretion of CETP by the CaCo216 and HepG217 cell lines and its interaction with lipoprotein substrates.22 23 24 46 However, the physiological significance of such a modulation by NEFA of CETP-mediated neutral lipid transfers in total human plasma remains uncertain. In particular, since most of plasma NEFAs are normally bound to albumin,42 their putative role in facilitating in vivo the interaction of CETP with plasma lipoprotein substrates has been placed in doubt. However, it is noteworthy that a small but significant fraction of NEFA is normally transported in vivo as a component of plasma lipoproteins.42 It is tempting therefore to speculate that small amounts of lipoprotein-bound NEFA may play a significant role in regulating cholesteryl ester transfers in vivo. That important issue was addressed in the present study, and we conclude that both exchange and net mass transfer of neutral lipids in normolipidemic human plasma are significantly influenced by NEFA.

In the normolipidemic population studied, we searched first for relations between plasma CETP mass concentration, as determined by using a specific ELISA,35 and plasma lipid parameters. Plasma CETP levels were positively and significantly correlated only with NEFA levels and the VLDL+LDL:HDL cholesterol ratio, whereas no significant relations were observed with other plasma lipoprotein components. The observation of a positive correlation between plasma NEFA and CETP mass concentrations, together with the previously described ability of fatty acids to enhance in vitro the secretion of CETP by cultured cells,16 17 suggest that NEFA might upregulate the synthesis and/or secretion of CETP in vivo. Furthermore, the positive relations between NEFA and CETP levels might account, at least in part, for recent observations of Moulin and coworkers,47 who reported increased plasma CETP concentrations in dyslipidemic patients with nephrotic syndrome, a pathology shown to be associated with abnormally elevated NEFA content of plasma lipoproteins.42

To determine further whether NEFA can affect not only the mass but also the activity of CETP, we chose to assay CET activity in total, native plasma. In previous studies, combinations of radiolabeled (donor particles) and unlabeled (acceptor particles) lipoprotein substrates have been used widely to evaluate the lipid transfer rates in plasma.9 20 As they have been performed either with total plasma, delipidated plasma, or with isolated plasma fractions, plasma lipid transfer assays are somewhat different, and all of them have their limitations.9 20 In vitro studies have demonstrated that CET activity is dependent on many factors including, in addition to CETP mass,18 19 the amount,48 49 the apoprotein composition,32 50 51 52 and the lipid composition50 53 54 of lipoprotein substrates (see Reference 99 for a review). Since in the present study CETP activity was measured in freshly collected native plasmas without prior treatment, values reflect the activity of the CETP as modulated by the endogenous plasma factors. That latter procedure led to results that are in good agreement with previously reported data. Indeed, the negative correlation we observed between the rate of CET toward VLDL+LDL and both HDL cholesterol levels and esterified cholesterol:triglyceride ratio in HDL confirmed the results from previous studies,55 56 57 58 which have shown that high levels of plasma CETP activity in vivo cause lower HDL cholesterol levels. Positive correlations with VLDL+LDL cholesterol concentrations, VLDL+LDL:HDL cholesterol ratio, and triglyceride concentrations observed in the present study confirmed the direct relationship that has recently been described in normolipidemic subjects between plasma VLDL concentrations and net CET from HDL to apo B–containing lipoproteins.58 While plasma CET activity has been measured usually in only one direction, it is noteworthy that CETP is actually an exchange protein that promotes the transfer of cholesteryl esters in several directions.9 20 The diversity in CETs is important to consider since a recent study in dyslipoproteinemic patients has shown that increased transfer of cholesteryl esters toward plasma VLDL+LDL can be associated with decreased transfer toward HDL.59 Thus, in the present study, plasma CET activity was evaluated not only by measuring the transfer of radiolabeled cholesteryl esters from HDL toward VLDL+LDL (CETHDL->VLDL+LDL activity) but also from LDL toward HDL (CETLDL->HDL activity). Globally, the relations between plasma lipid parameters and CETLDL->HDL activity were in contrast with those observed with CETHDL->VLDL+LDL activity. Indeed, CETLDL->HDL activity correlated positively with HDL cholesterol but negatively with plasma triglycerides, VLDL+LDL cholesterol, and VLDL+LDL:HDL cholesterol ratio. It must be noted that the fraction of radioactivity transferred may be underestimated in the CETLDL->HDL assay due to undetected transfers from LDL toward VLDL. In particular, because of the negative correlation between VLDL and HDL concentrations in plasma, a reduced transfer from LDL toward HDL might be associated with an increased transfer from LDL toward VLDL. That latter point might account in part for the significant correlations of CETLDL->HDL activity with HDL cholesterol, plasma triglycerides, VLDL+LDL cholesterol, and VLDL+LDL:HDL cholesterol ratio.

One of the novel findings from the present study is the significant positive correlation of plasma NEFA concentration with CETHDL->VLDL+LDL activity. In contrast, no significant correlation with CETLDL->HDL activity was observed, indicating again that the choice of both donor and acceptor lipoprotein substrates constitutes one source of variation in CETP activity measurement. Plasma NEFA concentration did not correlate significantly with transfer activities expressed relative to CETP mass, ie, specific CET activities, indicating that (1) as expected, CETP mass constitutes one major determinant of plasma neutral lipid transfers and (2) when CETP mass is taken into account, NEFAs do not constitute the single, most important modulators of the transfer reaction. In fact, it is well known that CETP activity can be regulated by many other plasma factors,9 and the lack of significance of the correlation of NEFA levels with specific transfer activities does not rule out the potential role of NEFA in modulating CETP activity. Depletion of NEFA content of lipoproteins in the presence of fatty acid–free albumin sustains that latter view. Indeed, since in vivo albumin binds the major part of plasma NEFA,42 60 it is likely to play a determinant role in modulating the interactions between NEFA and CETP in plasma. In agreement with observations of Shafrir,42 we demonstrated that, despite the fact that plasma NEFAs are mainly bound to albumin, a small fraction is normally carried by lipoprotein particles. To confirm that this latter fraction is available for interaction with CETP, we studied the effect of the supplementation of plasma with fatty acid–free albumin, an experimental protocol that has been shown previously to remove NEFA from plasma lipoprotein particles.42 When total plasmas were supplemented with albumin (20% increase in plasma albumin content), a significant reduction in CETHDL->VLDL+LDL activity was observed. Under identical experimental conditions, we reported that the positive relation between plasma CETP mass and CETP activity was improved in the presence of albumin, indicating further that plasma NEFA can modulate the activity of CETP.35 Taken together, these observations brought strong functional arguments in favor of the role of lipoprotein-bound NEFA in the modulation of CET activity in normolipidemic plasma. The effect of albumin supplementation is more likely to relate to its ability to bind NEFA rather than to a putative, direct effect on the transfer reaction. Indeed, as demonstrated in previous studies,22 23 24 albumin supplementation abolished the ability of NEFA to modulate the CETP-mediated exchange of cholesteryl esters between isolated lipoprotein substrates. On the contrary, in the absence of addition of NEFA, albumin alone did not affect the CETP-mediated CET reaction.24 Since the inhibition of CET by LTIP would concern mainly VLDL-LDL exchange,61 resulting in a concomitantly greater exchange between VLDL and HDL, one can speculate that NEFAs might also affect LTIP activity in addition to their ability to modulate directly the interaction of CETP with lipoprotein substrates. Whether that mechanism might account in part for differences in the relations between plasma NEFA concentrations and either CETHDL->VLDL+LDL or CETLDL->HDL activities remains to be established.

In support of a role of lipoprotein-bound NEFA in modulating CETs in total native plasma, correlations observed between CETP activity and lipoprotein concentrations were mostly suppressed in the presence of an excess of albumin. Indeed, after albumin supplementation, CETLDL->HDL activity no longer correlated with plasma HDL and VLDL+LDL cholesterol levels. Similarly, the correlation of CETHDL->VLDL+LDL activity with plasma HDL cholesterol levels was suppressed. Only a significant positive correlation of CETHDL->VLDL+LDL activity with plasma VLDL+LDL cholesterol levels was still apparent in the presence of albumin. These results indicate that lipoprotein-bound NEFA may account in part for the significant correlations between plasma CETP activity and the concentration of various lipoprotein fractions. However, in accordance with previous studies,2 48 49 58 the VLDL+LDL concentration remains a strong, independent determinant of plasma neutral lipid transfers, even when the modulating effect of lipoprotein-bound NEFA is cancelled.

Since under certain circumstances CETP can catalyze the rapid exchange of cholesteryl esters between lipoprotein particles without promoting any net mass transfer,62 we sought after the ability of plasma NEFA to alter the CETP-mediated mass redistribution of cholesteryl esters and triglycerides between plasma lipoproteins. To this end, HDL cholesteryl ester and triglyceride masses were measured in incubated plasmas, supplemented or not with albumin. Long-term incubation of native, nonsupplemented plasmas induced the transfer of cholesteryl esters to VLDL+LDL with a reciprocal transfer of triglycerides to HDL. This observation is consistent with the existence of an interdependence between cholesteryl ester and triglyceride transfer in plasma. However, results obtained after incubation of plasmas in the presence of albumin compared with plasmas without supplementation indicated that NEFA increased the mass of cholesteryl esters transferred from HDL to apo B–containing lipoproteins but, simultaneously, decreased the mass of triglycerides transferred from apo B–containing lipoproteins to HDL. These observations are consistent with a recent study30 that demonstrated that NEFA can dissociate the CETP-mediated heteroexchange of cholesteryl esters and triacylglycerol between HDL and triacylglycerol-rich lipoproteins. Our data indicated further that such an effect of NEFA may occur in native plasma.

Taken all together, the results of the present study suggest that plasma NEFA can modulate neutral lipid transfers through a dual effect on both the level and the activity of CETP. By increasing CETP mass concentrations and dissociating both the homoexchange of cholesteryl esters and the heteroexchange of cholesteryl esters and triglycerides between HDL and VLDL+LDL fractions, NEFAs globally favor the redistribution of neutral lipids toward plasma apo B–containing lipoproteins. The control of neutral lipid transfers by plasma NEFA could have some important implications, in particular under physiopathological conditions associated with abnormally low plasma albumin concentrations and high NEFA content of plasma lipoproteins, ie, analbuminemia63 and nephrotic syndrome.42 In support of that latter view, increased ratios of plasma NEFA to albumin have also been observed in patients with coronary heart disease.64 Whether these observations relate at least in part to the ability of NEFA to enhance the CETP-mediated transfer of cholesteryl esters from the "anti-atherogenic" HDL to the "atherogenic" VLDL and LDL fractions remains to be clarified.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CETHDL->VLDL+LDL = cholesteryl ester transfer from HDL toward VLDL+LDL
CETLDL->HDL = cholesteryl ester transfer from LDL toward HDL
CETP = cholesteryl ester transfer protein
ELISA = enzyme-linked immunosorbent assay
FSD = fumed silicon dioxide
LCAT = lecithin:cholesterol acyltransferase
NEFA = nonesterified fatty acid
LTIP = lipid transfer inhibitor protein
PLTP = phospholipid transfer protein


*    Acknowledgments
 
This investigation was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Fondation pour la Recherche Médicale. The technical assistance of Liliane Princep is greatly acknowledged.

Received March 7, 1995; accepted June 26, 1995.


*    References
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