Download - Mass Concentration of Plasma Phospholipid Transfer Protein in Normolipidemic, Type IIa Hyperlipidemic, Type IIb Hyperlipidemic, and Non-Insulin-Dependent Diabetic Subjects as Measured

Mass Concentration of Plasma Phospholipid TransferProtein in Normolipidemic, Type IIa Hyperlipidemic, TypeIIb Hyperlipidemic, and Non–Insulin-Dependent Diabetic

Subjects as Measured by a Specific ELISACatherine Desrumaux, Anne Athias, Ginette Bessede, Bruno Verges, Michel Farnier,

Laurence Persegol, Philippe Gambert, Laurent Lagrost

Abstract—Mean plasma phospholipid transfer protein (PLTP) concentrations were measured for the first time by using acompetitive enzyme-linked immunosorbent assay. PLTP mass levels and phospholipid transfer activity values, whichwere significantly correlated among normolipidemic plasma samples (r50.787, P,0.0001), did not differ betweennormolipidemic subjects (3.9561.04 mg/L and 575681 nmolz mL21 z h21, respectively; n530), type IIa hyperlipidemicpatients (4.0660.84 mg/L and 571643 nmol z mL21 z h21, respectively; n536), and type IIb hyperlipidemic patients(3.9060.79 mg/L and 575648 nmol z mL21 z h21, respectively; n533). No significant correlations with plasma lipidparameters were observed among the various study groups. In contrast, plasma concentrations of the related cholesterylester transfer protein (CETP) were higher in type IIa and type IIb patients than in normolipidemic controls, andsignificant, positive correlations with total and low density lipoprotein cholesterol levels were noted. Interestingly,plasma PLTP mass concentration and plasma phospholipid transfer activity were significantly higher in patients withnon–insulin-dependent diabetes mellitus (n550) than in normolipidemic controls (6.7661.93 versus 3.9561.04 mg/L,P,0.0001; and 685675 versus 575681 nmolz mL21 z h21, P,0.0001, respectively). In contrast, CETP levels did notdiffer significantly between the 2 groups. Among non–insulin-dependent diabetes mellitus patients, PLTP levels werepositively correlated with fasting glycemia and glycohemoglobin levels (r50.341,P50.0220; andr50.382,P50.0097,respectively) but not with plasma lipid parameters. It is proposed that plasma PLTP mass levels are related to glucosemetabolism rather than to lipid metabolism.(Arterioscler Thromb Vasc Biol. 1999;19:266-275.)

Key Words: cholesteryl ester transfer proteinn lipid transfern ELISA n glucosen non–insulin-dependent diabetes mellitus

I n vivo, plasma lipoproteins do not constitute stable entitiesbut are continuously remodeled through the action of

several enzymes and lipid transfer proteins. In particular,cholesteryl ester transfer protein (CETP)1 and phospholipidtransfer protein (PLTP), 2 related proteins belonging to thelipid transfer/lipopolysaccharide binding protein (LT/LBP)family,1 can promote the exchange of lipid species betweenvarious plasma lipoprotein fractions. In fact, studies over thepast few years have demonstrated that both CETP and PLTPproduce multiple effects on lipoprotein structure and compo-sition. Thus, CETP promotes the exchange of neutral lipids,ie, CEs and triglycerides, between plasma lipoprotein frac-tions, leading to alterations in both the neutral lipid contentand the size distribution of lipoproteins.2,3 PLTP can facilitatethe transfer of phospholipids between lipoprotein particles,4

and it was lately shown to transfer lipopolysaccharides,5

unesterified cholesterol,6 anda-tocopherol7 as well. In addi-

tion, PLTP constitutes an important determinant of the sizedistribution of HDL.3,8–12 Taken together, recent advanceshave raised considerable interest in elucidating the precisefunction of lipid transfer proteins in lipoprotein metabolism,and a new challenge of in vivo studies is to relate pathophys-iological alterations of the plasma levels of CETP and PLTPto atherosclerosis susceptibility.

Mainly, 2 distinct approaches can be applied to the quan-tification of lipid transfer protein levels, consisting of eitherevaluation of lipid transfer activities by isotopic or netmass-transfer assays or determination of the mass concentra-tion of lipid transfer proteins by specific immunoassays.Although the determination of lipid transfer rates in plasmahas proved helpful and informative, it does not necessarilyprovide a reliable and specific estimate of the lipid transferprotein mass per se, due in part to the presence of putativemodulators in total plasma. The result is that only specific

Received February 11, 1998; revision accepted June 23, 1998.From Laboratoire de Biochimie des Lipoproteines, INSERM U498, Universite de Bourgogne (C.D., A.A., G.B., B.V., L.P., P.G., L.L.); Service

d’Endocrinologie et Diabetologie, Hopital du Bocage (B.V.); and Le Point Medical (M.F.), Dijon, France.Correspondence to Laurent Lagrost, Laboratoire de Biochimie des Lipoproteines, INSERM U498, Hopital du Bocage—BP 1542, 21034 Dijon Cedex,

France.© 1999 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol.is available at http://www.atvbaha.org

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immunoassays are suitable for accurate determination of lipidtransfer protein mass in plasma samples. In 1990, the firstradioimmunoassay of human CETP, proposed by Marcel andcoworkers,13 allowed the determination of mean CETP levelsin normolipidemic plasmas, and subsequent clinical investi-gations with specific immunoassays14–19 led to a significantimprovement in our knowledge of the metabolism of CETPand its pathophysiological variations. Unlike CETP, PLTPhas been quantified only through its ability to exchangephospholipids, and to date no specific immunoassay has beenproposed to assay PLTP mass levels in biological samples. Infact, it is noteworthy that phospholipid exchange activity is aproperty that is shared by several plasma proteins, includingCETP,20 LBP,21 and soluble CD14,21 in addition to PLTP.The latter point suggests that only a specific immunoassaywould accurately reflect the level of PLTP in plasma, andtoday the lack of an adapted quantitative tool may account, atleast in part, for the paucity of information concerning thepathophysiological relevance of PLTP.

The present report describes the first immunoassay ofhuman PLTP. A competitive ELISA of PLTP was devised byusing polyclonal immunoglobulins raised against purifiedhuman PLTP. This new method was then applied to thedetermination of PLTP in normolipidemic plasmas, as well asin plasmas from type IIa hyperlipidemic, type IIb hyperlip-idemic, and non–insulin-dependent diabetes mellitus(NIDDM) patients.

MethodsStudy SubjectsOne hundred forty-nine subjects were selected for the study, includ-ing 30 normolipidemic subjects (15 males and 15 females; total

cholesterol,2.50 g/L and triglycerides,1.30 g/L), 36 patients withtype IIa dyslipidemia (27 males and 9 females; total cholesterol.2.50 g/L and triglycerides,1.30 g/L), 33 patients with type IIbdyslipidemia (30 males and 3 females; total cholesterol.2.50 g/Land triglycerides.1.30 g/L), and 50 patients with NIDDM (23males and 27 females; total cholesterol 1.02 to 3.20 g/L andtriglycerides 0.45 to 4.35 g/L). Control normolipidemic subjectswere selected from the hospital staff as healthy subjects with normalthyroid, renal, and hepatic functions and without a history ofhyperlipidemia, coronary artery disease, or diabetes. Type IIa andtype IIb patients did not present secondary causes of dyslipidemia,and patients with diabetes mellitus or those who were overweight

Figure 1. Volume-dependent (A) and time-dependent (B) trans-fer of phospholipids in normolipidemic plasma. Phospholipidtransfer activity was measured in an endogenous lipoprotein-independent assay as the rate of transfer of radiolabeled phos-phatidylcholine from [14C]DPPC liposomes toward isolated HDL(see Methods).

Figure 2. SDS–polyacrylamide gel electrophoresis of purifiedPLTP. One-microliter volumes of b-mercaptoethanol–treated,purified PLTP (lower profile), purified PLTP without reducingagent (intermediate profile), and low-molecular-weight calibra-tion kit standards (Pharmacia; upper profile) were subjected toelectrophoresis in Pharmacia 8% to 25% Phastgel. At the endof electrophoretic migration, the gel was stained with Coomas-sie brilliant blue R-250 and analyzed on a Bio-Rad GS-670imaging densitometer.

Figure 3. Capillary gel electrophoretic profile of purified PLTP inthe presence of carbonic anhydrase as an internal standard (seeMethods).

Desrumaux et al February 1999 267

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(body mass index.30 kg/m2) were excluded from the type IIa andtype IIb groups. Diabetic patients suffered from NIDDM and weretreated by either diet alone (n58) or in combination with oralhypoglycemic drugs (n542). Among the entire population studied,neither normolipidemic subjects nor dyslipidemic patients receiveddrugs known to affect lipoprotein metabolism. The study wasapproved by the ethics committee of the Bocage Hospital (Dijon,France), and informed consent was obtained.

Blood SamplesFasting blood samples were collected into EDTA-containing glasstubes, which were placed immediately on ice. Plasma was separatedby a 5-minute centrifugation at 3000g, and aliquots were kept at280°C until analysis.

Purification of Human Plasma PLTPPLTP was purified from 1200 mL of citrated human plasma that wasmade lipoprotein deficient by the dextran sulfate–MnCl2 precipita-

tion procedure of Burstein et al.22 PLTP was purified by sequentialchromatography on hydrophobic, affinity, and anion-exchange col-umns as previously described.23 Only Mono-Q fractions with highspecific phospholipid transfer activity and containing virtually onlypure PLTP were selected for the study, with the exception of thepartially purified Mono-Q fractions used for plate coating in theELISA. For rabbit immunization and ELISA calibration, Mono-Qfractions containing pure PLTP were further passed through ananti-albumin immunoaffinity column to ensure removal of any tracesof human plasma albumin. Finally, before rabbit immunization, thepurified PLTP fractions were subjected to an ultimate preparativeelectrophoresis step to ensure a maximal degree of purity. In brief,purified PLTP fractions were applied to an 8% polyacrylamide gelcontaining 1% SDS, and electrophoresis was conducted in a50 mmol/L Tris, 380 mmol/L glycine, and 0.1% SDS, pH 8.3, bufferfor 6 hours at 50 mA. After electrophoresis, the portion of gelcontaining PLTP was cut off and the protein was eluted as previouslydescribed.24 The purity of PLTP preparations was assessed by SDSelectrophoresis in 80 to 250 g/L polyacrylamide gradient gels(Phastsystem, Pharmacia) and by SDS gel capillary electrophoresis,as indicated. The purified protein was concentrated and used forimmunization of a New Zealand White rabbit within 24 hours. Forplate coating, a partially purified PLTP fraction was obtained fromlipoprotein-deficient fresh plasma (d.1.21 g/mL) by a combinationof phenyl-Sepharose, heparin-Ultrogel, and anion-exchange chroma-tography.23 In brief, after anion-exchange chromatography ofheparin-bound proteins, all of the eluted fractions containing detect-able phospholipid transfer activity were pooled, and the resultingmaterial used for plate coating corresponded to an'500-foldincrease in specific phospholipid transfer activity compared with thestarting plasma.

Anti-PLTP Polyclonal AntibodiesAntiserum to purified human PLTP was prepared by immunizationof a 3-kg New Zealand White rabbit with 1 initial injection of 250mg

Figure 4. Effect of anti-PLTP polyclonal immunoglobulins onphospholipid transfer and CE transfer activities in humanplasma. For phospholipid transfer activity measurements, vari-ous amounts of control (E) or anti-PLTP (F) rabbit IgGs wereadded to 10 mL of human plasma. The mixtures were preincu-bated for 16 hours at 4°C in a final volume of 200 mL and cen-trifuged for 30 minutes at 10 000 rpm. The resulting superna-tants were then incubated at 37°C for 90 minutes in thepresence of [14C]DPPC liposomes (110 nmol phosphatidylcho-line), isolated HDL (250 mg protein), and iodoacetate(1.5 mmol/L) in a final volume of 400 mL. For CE transfer activitydeterminations, 5 mL of plasma was preincubated for 16 hoursat 4°C with the indicated amounts of nonimmune (M) or immune(f) rabbit IgG. The mixtures were then centrifuged, and thesupernatants were incubated at 37°C for 18 hours with [3H]CEHDL3 (0.8 mg cholesterol), isolated LDL (150 mg protein), andiodoacetate (1.5 mmol/L) in a final volume of 150 mL. At the endof the incubations, donor and acceptor lipoprotein fractionswere separated, and transfer activity values were calculated asthe percentage of radioactivity transferred from the donor to theacceptor fraction after deduction of blank values. Data areexpressed as percentages of remaining lipid transfer activitycompared with controls incubated without immunoglobulin sup-plementation (phospholipid transfer activity, 692 nmol z mL21 zh21). Each point represents the mean of duplicatedeterminations.

Figure 5. Anti-PLTP Western blot anal-ysis of various protein fractions. Immu-nolocalization of PLTP was performedafter electrophoresis in 80 to 250 g/Lpolyacrylamide gradient gels asdescribed under Methods. Lane 1, low-molecular-weight calibration kit stan-dards after Coomassie brilliant bluestaining of gradient gels. Lane 2, immu-

noblot of Pharmacia low-molecular-weight calibration kit stan-dards. Lane 3, immunoblot of 1 mL diluted (1/5) normolipidemichuman plasma. Lane 4, immunoblot of a heparin-bound, par-tially purified PLTP fraction (25 ng protein).

Figure 6. ELISA displacement curves obtained with PLTP (F),CETP (E), and human serum albumin (M). Values are mean ofduplicates. On the abscissa is plotted the amount of protein permicrowell.

Figure 7. Logit-log representation of displacement curvesobtained with a plasma standard (E) and with different proteinfractions during the PLTP purification procedure: phenyl-Sepharose fraction (f), heparin-Sepharose fraction (M), andpure Mono-Q fraction (F). On the abscissa is plotted theamount of total protein per microwell. Specific activities of typi-cal phenyl-Sepharose, heparin-Sepharose, and Mono-Q frac-tions were '100-fold, 400-fold, and 1000-fold, respectively,higher than in total plasma.23

268 PLTP Mass Concentration in Human Plasma



PLTP emulsified in complete Freund’s adjuvant followed by three150-mg injections of PLTP emulsified in incomplete Freund’sadjuvant at 2-week intervals. The rabbit was bled 8 days after the lastinjection, serum was recovered by low-speed centrifugation, and theserum IgG fraction was prepared by using a protein A column(protein A–Sepharose 4 Fast Flow, Pharmacia) according to theprocedure described by the manufacturer. This experiment wasperformed under the framework of theGuide for the Care and Useof Laboratory Animalspublished by the US National Institutes ofHealth (NIH publication No. 81-23, revised 1985).

Anti-PLTP ImmunoblottingThe specificity of anti-PLTP immunoglobulins was assessed byWestern blotting. To this end, plasma and purified PLTP sampleswere subjected to electrophoresis in 80 to 250 g/L polyacrylamidePhastgels under reducing conditions, and proteins were subsequentlytransferred to a nitrocellulose membrane by using a Phast semidryelectrophoretic transfer system as recommended by the manufacturer(Pharmacia). The resulting blots were blocked overnight at 4°C in10% low-fat milk before being incubated for 1 hour at 37°C in thepresence of anti-PLTP antibodies. After being washed, nitrocellulosemembranes were incubated for 1 hour at 37°C with horseradishperoxidase–conjugated secondary antibodies (Bio-Rad). Finally, de-velopment was achieved by using the ECL-Western blotting detec-tion reagent kit from Amersham.

PLTP ELISAA competitive ELISA of PLTP was devised according to the generalprocedure previously used in our laboratory to quantify humanapoA-IV,24 apoB,25 and CETP.16 All steps of the immunoassays(pipetting, diluting, dispensing, washing, and photometry) werecarried out with a Biomek 2000 Biorobotics System (BeckmanInstruments).

Plate CoatingA 100-mL volume of partially purified PLTP fraction (proteinconcentration, 15 mg/L) in a 15 mmol/L Na2CO3, 35 mmol/LNaHCO3, and 3 mmol/L NaN3 (pH 9.6) buffer was pipetted into eachwell of a polystyrene microwell plate (Immuno 96F type I fromNunc) and incubated overnight at 4°C. The plates were then washed4 times with a 150 mmol/L NaCl–0.025% (vol/vol) Tween-20washing solution and incubated for 30 minutes at room temperaturewith 200 mL of a 1% (wt/vol) human serum albumin solutioncontaining 10 mmol/L Na2HPO4, 5 mmol/L NaH2PO4, and150 mmol/L NaCl, adjusted to pH 7.2 with NaOH (albumin-phosphate buffer).

Sample TreatmentPLTP-containing samples were diluted in the albumin-phosphatebuffer and mixed with an equal volume of polyclonal anti-PLTPantibodies diluted in albumin-phosphate buffer containing 1% TritonX-100 (Pierce Chemical Co). Total plasma samples were dilutedfrom 1:2 to 1:16 in the albumin-phosphate buffer. The mixtures wereincubated overnight at 4°C in 96 Deep-well titer plates (Beckman).Aliquots (100mL) were then pipetted into the immunoplate micro-wells and incubated for 3 hours at 37°C. At the end of the incubation,the plates were washed 4 times with the Tween-20 solution.

Detection of Bound Anti-PLTP AntibodiesOne hundred microliters of peroxidase-conjugated anti-rabbit anti-bodies (Bio-Rad) diluted in the albumin-phosphate buffer waspipetted into each well and incubated for 1 hour at 37°C. Aftercompletion of the incubation, the plates were washed 4 times asbefore, and 100mL of a freshly prepared 0.4 g/Lo-phenylenedi-amine–0.68 g/L H2O2 solution in a 6.6 mmol/L sodium phosphate–3.4 mmol/L citrate buffer (pH 5.2) was pipetted into each well. After15 minutes at room temperature in the dark, the reaction was stoppedby addition of 50mL of 2.5 mol/L H2SO4. The absorbances were readat 490 nm with a Photometry tool on the Biomek 2000 Bioroboticsstation, and data were saved on a PC computer for further treatment.

CalibrationPure PLTP (specific activity,'10 mmol z mg21 z h21) was used tostandardize the assay. The amount of PLTP in purified fractions wasdetermined by SDS gel capillary electrophoresis with carbonicanhydrase as an internal standard. Capillary electrophoresis wasperformed with uncoated, fused-silica capillaries (27 cm3100-mmID) attached to a P/ACE 2100 system that was controlled by Goldsoftware (Beckman Instruments). The P/ACE system 2100 was usedin reversed-polarity mode. The electrolyte buffer was a non–cross-linked gel matrix (eCAP SDS 14-200, Beckman). In brief, PLTP-containing samples were diluted in Tris buffer (pH 6.6) containing1% SDS, and they were supplemented with orange G as a tracking

TABLE 1. Correlation of CETP and PLTP Mass ConcentrationWith Plasma Parameters in Normolipidemic Subjects (n530)

PLTP CETP

PLTP 0.041

CETP 0.041

Total cholesterol 0.148 0.396*

Triglycerides 0.139 0.176

LDL cholesterol 0.140 0.362*

HDL cholesterol 0.014 0.120

Glycemia 0.041 0.066

*P,0.05.

Figure 8. Logit-log representation of displacement curvesobtained with normolipidemic and various hyperlipidemicplasma samples. Total cholesterol and triglyceride concentra-tions were 3.07 and 1.44 g/L (E), 2.10 and 3.67 g/L (M), 3.16and 5.22 g/L (F), 2.76 and 0.61 g/L (‚), and 2.00 and 0.79 g/L(Œ), respectively.

Figure 9. Correlation of plasma PLTP mass with plasma phos-pholipid transfer activity among 30 normolipidemic healthy sub-jects. PLTP mass was determined by ELISA, and phospholipidtransfer activity was determined by an isotopic transfer assay asdescribed under Methods.




dye and carbonic anhydrase (0.2 g/L) as an internal standard.Electrophoresis was conducted at 20°C at 8.10 kV, and detection wasperformed at 214 nm. PLTP mass concentration was determined bycomparing the area of the PLTP peak to the area of the peak obtainedwith a known amount of carbonic anhydrase. Finally, an ELISAprimary standard curve constructed from a set of dilutions of purifiedPLTP was used to determine PLTP levels in a pool of frozen,normolipidemic human plasmas that constituted a secondary stan-dard. Routinely, 8 dilutions (PLTP concentrations from 0.0275 to3.52 mg/L) were used to construct a secondary calibration curve foreach immunotitration plate. Standard curves were fitted to the datapoints by using data analysis software (Immunofit EIA/RIA dataanalysis software, Beckman). Four dilutions of each sample wereassayed, and the PLTP concentration was calculated as the mean ofthe 4 results.

Isolation of LipoproteinsHDLs were isolated as the 1.07,d,1.21 g/mL fraction of normo-lipidemic, fresh, and citrated human plasma at a speed of 55 000 rpm(223 000g) in a 70-Ti rotor on an L7 ultracentrifuge (Beckman) bytwo 20-hour spins at the lower density and one 30-hour spin at thehigher density. The HDL fraction was finally washed with one8-hour spin at the density of 1.21 g/mL at a speed of 90 000 rpm(561 000g) in an NVT-90 rotor on an XL-90 ultracentrifuge.

Densities were adjusted by addition of solid KBr. The isolatedlipoproteins were dialyzed overnight against a 10 mmol/L Tris,150 mmol/L NaCl, 1 mmol/L tetrasodium-EDTA, and 3 mmol/LNaN3, pH 7.4, buffer.

CETP ELISACETP concentration in total plasma samples was measured by acompetitive ELISA on a Biomek 2000 laboratory automatic work-station (Beckman) as previously described.16 CETP mass concentra-tion values were determined from a calibration curve obtained witha frozen plasma standard, and they were calculated by using dataanalysis software (Immunofit EIA/RIA data analysis software, Beck-man). Four dilutions of each sample were assayed, and the CETPconcentration was calculated by averaging the 4 results.

Phospholipid Transfer Activity AssayPlasma phospholipid transfer activity was determined as the capacityof a plasma sample to induce the transfer of radiolabeled dipalmitoylphosphatidylcholine ([14C]DPPC) from [14C]DPPC liposomes to anexcess of isolated HDL. In brief, liposomes (110 nmol phosphati-dylcholine) were incubated at 37°C with isolated HDL (250mgprotein) in the presence of total plasma and iodoacetate (1.5 mmol/L)in a final volume of 400mL. Phospholipid liposomes and apoB-containing lipoproteins were subsequently precipitated, and radioac-tivity was assayed in supernatants. Phospholipid transfer activity wascalculated as the amount of total radiolabeled phospholipids trans-ferred from liposomes to HDL after deduction of blank values thatwere obtained by incubating liposomes and HDL at 37°C for 90minutes in the absence of plasma. Phospholipid transfer after a90-minute incubation increased gradually as the amount of addednormolipidemic plasma was increased from 0 to 20mL (Figure 1A).When 10mL of plasma was added, the phospholipid transfer assaywas linear over a 2-hour period (Figure 1B). Throughout the study,phospholipid transfer activity measurements among various plasmasamples were conducted by using 10-mL plasma aliquots that were

TABLE 2. Characteristics of the Study Subjects

Normolipidemic Type IIa Type IIb NIDDM

Age, y 38611 42616 49611* 60612†

Sex, M/F 15/15 27/9 30/3 23/27

BMI, kg/m2 23.262.9 23.363.1 25.862.3 31.366.8†

Total cholesterol, g/L 1.7960.34 2.9660.64† 3.1360.49† 2.1360.42‡

Triglycerides, g/L 0.7760.17 0.8960.25 1.9160.55† 1.6560.90†

HDL cholesterol, g/L 0.5860.14 0.5560.18 0.4460.11* 0.4660.11*

LDL cholesterol, g/L 1.0560.31 2.2760.72† 2.3160.50† 1.6960.41†

Glycemia, g/L 0.8660.07 0.8860.08 0.9360.12 1.8860.71†

HbA1c, % ND ND ND 8.7262.22

Insulinemia, mU/L ND ND ND 9.3468.19

C-peptide, mg/L ND ND ND 3.3962.70

PLTP mass, mg/L 3.9561.04 4.0660.84 3.9060.79 6.7661.93†

Phospholipid transfer activity,nmol z mL21 z h21

575681 571643 575648 685675†

CETP mass, mg/L 2.6760.55 2.9960.63 3.2360.73‡ 2.3460.67

BMI indicates body mass index; ND, not determined.*P,0.001, †P,0.0001, ‡P,0.05 vs normolipidimic subjects.

TABLE 3. Correlation of CETP and PLTP Mass ConcentrationWith Plasma Parameters in Type IIa Patients (n536)

PLTP CETP

PLTP 20.292

CETP 20.292 z z z

Total cholesterol 20.274 0.423*


LDL cholesterol 20.316 0.397*


Glycemia 0.006 20.115

*P,0.02.

TABLE 4. Correlation of CETP and PLTP Mass ConcentrationWith Plasma Parameters in Type IIb Patients (n533)

PLTP CETP

PLTP 20.15

CETP 20.15 z z z

Total cholesterol 0.104 0.259


LDL cholesterol 0.079 0.306


Glycemia 0.076 20.336




incubated for 90 minutes at 37°C. The assay was proved to beindependent of the phospholipid exchange activity catalyzed byCETP.4,23,26–28

CETP Activity AssayPlasma CETP activity was determined as the capacity of a plasmasample to promote the transfer of radiolabeled CEs ([3H]CE) from[3H]CE HDL3 to an excess of isolated LDL. Radiolabeled HDL3 (0.8mg cholesterol) was incubated for 18 hours at 37°C with isolatedLDL (150 mg protein) in the presence of 5mL of total plasma andiodoacetate (1.5 mmol/L) in a final volume of 150mL. Donor andacceptor particles were subsequently separated by ultracentrifuga-tion, and CETP activity was calculated as the percentage of totalradiolabeled CEs transferred from HDL3 to LDL after deduction ofnonincubated control values.

Other AnalysesAll chemical assays were performed on a Cobas-Fara centrifugalanalyzer (Roche). Total cholesterol concentration was measured byan enzymatic method using a Boehringer Mannheim reagent. HDLcholesterol was measured after selective precipitation of apoB-containing lipoproteins with Boehringer phosphotungstic acid/MgCl2

reagent, as recommended by the manufacturer. LDL cholesterolconcentration was calculated using the Friedewald formula.29 Tri-glycerides were assayed by an enzymatic method using Rochereagents. Glycemia, ie, plasma glucose level, was determined by anenzymatic method. Glycohemoglobin (HbA1c) was determined byhigh-performance liquid chromatography on a Diamat analyzer(Bio-Rad). Plasma C-peptide was measured by radioimmunoassay(Mallinckrodt Medical). Plasma insulin was measured by radioim-munoassay (CIS Bio International).

Statistical AnalysisELISA curves were constructed by polynomial regression analysis.Sigmoidal competitive curves were linearized by logit-log transfor-mation. Coefficients of correlation were calculated by linear regres-sion analysis. Multiple regression analysis was used to determine thecontribution of age and diabetic state to the rise in PLTP massconcentration in the diabetic subpopulation. Data means were com-pared by using a 1-way ANOVA.

ResultsPurification and Characterization of Human PLTPPLTP was purified from fresh human plasma by using asequential procedure involving dextran sulfate/MnCl2 precip-itation followed by chromatographic and electrophoreticsteps (see Methods). As shown in Figure 2, purified PLTPappeared as a single homogeneous band after electrophoresisin denaturing polyacrylamide gradient gels, with an apparentmolecular weight of 56 kDa. In good agreement with previ-

ous studies,7 the apparent molecular weight of pure PLTProse to 70 kDa when preincubated withb-mercaptoethanolbefore denaturing polyacrylamide gradient gel electrophore-sis (Figure 2). Further analysis of PLTP preparations by thehigh-resolution capillary electrophoresis technique confirmedthe presence of a single protein peak (Figure 3). Complemen-tary experiments revealed that the mean isoelectric point ofpurified PLTP (pI 5.0) was identical to the pI value previ-ously reported by Tollefson and coworkers.4 Purified PLTPpreparations presented elevated specific phospholipid transferactivity, determined as the rate of transfer of radiolabeledphosphatidylcholine from [14C]DPPC-liposome donors to-ward isolated HDL acceptors.

Production of a Specific Anti-PLTP Antiserumand Development of an ELISASpecific anti-PLTP IgGs were prepared from the serum of therabbit successively injected with pure PLTP over a 2-monthperiod (see Methods). As shown in Figure 4, anti-PLTPimmunoglobulins (concentration range, 0 to 100mg/mL) werefound to inhibit plasma phospholipid transfer activity in aconcentration-dependent manner. When 5 distinct plasmasamples were supplemented with high concentrations ofanti-PLTP immunoglobulins (150mg/mL), the maximal inhi-bition of phospholipid transfer activity ranged between 70%and 87%. Under the same experimental conditions, the CEtransfer activity of the related CETP remained unchanged(Figure 4). As shown in Figure 5, a single 70-kDa band wasdetected by Western blotting of either plasma or purifiedPLTP samples under reducing conditions. Anti-PLTP immu-noglobulins were used to establish a competitive ELISA (seeMethods). As shown in Figure 6, a typical ELISA displace-ment curve was obtained with purified PLTP with a 0.1 to 10mg/mL working concentration range. In contrast, no displace-ment curves were observed when purified CETP or albuminsolutions were used (Figure 6). When displacement curveswere obtained by using various PLTP-containing fractionswith distinct degrees of purity, the logit-log lines wereparallel, indicating that the affinity of polyclonal anti-PLTP

TABLE 5. Correlation of CETP and PLTP Mass ConcentrationWith Plasma Parameters in NIDDM Patients (n550)

PLTP CETP

PLTP 0.199

CETP 0.199

Total cholesterol 0.116 0.159


LDL cholesterol 0.123 0.201


Glycemia 0.341* 20.022

HbA1c 0.382† 20.183

C peptide 0.036 0.162

Insulinemia 20.074 0.135

*P,0.05, †P,0.01.

Figure 10. Correlation of PLTP mass concentration with glyce-mia (upper panel) and HbA1c (lower panel) in NIDDM patients(n550). PLTP mass, glycemia, and the proportion of HbA1cwere determined as described under Methods.




IgG was unaffected by the presence of other protein compo-nents in the mixture to be assayed (Figure 7). In addition,parallel logit-log lines were obtained with serial dilutions ofnormolipidemic or hyperlipidemic plasmas, indicating thatunder the experimental conditions used, plasma lipid levelsdid not alter the immunoaffinity of immunoglobulins forPLTP (Figure 8).

Intra-assay and interassay coefficients of variation wereevaluated by analyzing the same plasma sample 5 times in thesame microwell plate on the same day and on 5 consecutivedays, respectively. The values of the intra-assay and interas-say coefficients of variation were 5.7% and 7.8%,respectively.

Determination of PLTP and CETP MassConcentrations in Plasma FromNormolipidemic SubjectsPLTP mass concentration was assayed among plasmas from30 normolipidemic subjects (15 males, 15 females). Themean plasma concentration of PLTP was 3.9561.04 mg/L(range, 1.98 to 5.71), with identical levels in males andfemales. The mean plasma phospholipid transfer activityamong the normolipidemic population was 575681 nmolzmL21 z h21. As shown in Figure 9, plasma PLTP mass levelswere correlated positively and significantly with phospho-lipid transfer activity values measured as the transfer ofradiolabeled phosphatidylcholine from [14C]DPPC liposomestoward exogenous HDL (r50.787, P,0.0001). Among thesame normolipidemic subpopulation, the mean plasma con-centration of CETP was 2.6760.55 mg/L, with slightlyhigher levels in females than in males (2.7360.67 mg/L and2.6160.41 mg/L, respectively). Plasma CETP concentrationwas positively correlated with total and LDL cholesterollevels (r50.40,P50.0301; andr50.36, P50.0495, respec-tively). In contrast, plasma PLTP levels were not significantlycorrelated with any of the plasma lipid parameters determinedamong the normolipidemic subpopulation (Table 1).

Determination of PLTP and CETP MassConcentrations in Plasma From Patients WithType IIa HyperlipidemiaAs shown in Table 2, a marked and significant rise in LDLcholesterol levels constituted the main abnormality of type IIahyperlipidemic patients (n536), accounting for the signifi-cantly higher total cholesterol levels compared with normo-lipidemic controls (P,0.0001), whereas triglyceride andHDL cholesterol levels in type IIa and normolipidemicpopulations were similar. No significant differences in PLTPmass and phospholipid transfer activity levels were observedbetween normolipidemic and type IIa groups (Table 2). Asobserved with the normolipidemic subpopulation, plasmaPLTP levels were not significantly correlated with any of theplasma lipid parameters among type IIa patients (Table 3). Incontrast, regression analysis revealed a positive correlation ofCETP mass levels with both total and LDL cholesterol levelsamong type IIa patients (r50.423,P50.0102; andr50.397,P50.0166, respectively).

Determination of PLTP and CETP MassConcentrations in Plasma From Patients WithType IIb HyperlipidemiaType IIb hyperlipidemic patients presented significantlyhigher levels of total cholesterol, LDL cholesterol, and

triglycerides (P,0.0001 in all cases) compared with normo-lipidemic controls (Table 2). In contrast, HDL cholesterollevels were significantly lower in type IIb patients than innormolipidemic subjects (P50.0004; Table 2). CETP massconcentration was significantly higher in type IIb patientsthan in normolipidemic controls. No significant differences inmean PLTP mass and phospholipid transfer activity levelswere observed between normolipidemic and type IIb groups(Table 2). Neither CETP nor PLTP mass levels were signif-icantly correlated with any of the plasma lipid parametersamong the type IIb hyperlipidemic subpopulation (Table 4).Nevertheless, in agreement with data observed in the normo-lipidemic and type IIa subpopulations, CETP mass levelstended to be positively correlated with plasma LDL choles-terol levels in the type IIb population (r50.306,P50.0836).

Determination of PLTP and CETP MassConcentrations in Plasma From PatientsWith NIDDMNIDDM patients (n550) presented significantly higher levelsof total cholesterol, LDL cholesterol, and triglycerides(P50.0324, P,0.0001, andP,0.0001, respectively) andsignificantly lower levels of HDL cholesterol (P50.0005)than in normolipidemic subjects (Table 2). In addition,fasting glycemia was markedly and significantly higher indiabetics than in nondiabetics (Table 2). Plasma PLTP massand phospholipid transfer activity levels were significantlyhigher in diabetics than in controls, whereas CETP masslevels did not vary significantly between the 2 groups (Table2). Phospholipid transfer activity levels, but not PLTP masslevels, were lower in diabetics treated with a combination ofhypoglycemic drugs and diet than in diabetics treated withdiet alone (phospholipid transfer activity, 676676 versus750655 nmol z mL21 z h21, respectively,P50.02; PLTPconcentration, 6.7561.80 versus 7.0362.15 mg/L, respec-tively, NS). Whereas no significant relationships betweenlipid transfer protein levels and plasma lipid parameters werenoted among the diabetic subpopulation, PLTP but not CETPwas correlated positively and significantly with both fastingglycemia (r50.341, P50.0220) and HbA1c (r50.382,P50.0097) levels (Table 5 and Figure 10). Because diabeticpatients tended to be older than normolipidemic controls,multiple regression analysis was used to determine thecontribution of age and the diabetic state to the prediction ofPLTP mass and phospholipid transfer activity levels. Fromthis analysis it was found that diabetic/nondiabetic state, butnot age, contributed significantly to the rise in both PLTPmass levels (P,0.0001) and phospholipid transfer activitylevels (P50.0009) in the diabetic subpopulation.

DiscussionThe importance of the roles of CETP and PLTP in lipoproteinmetabolism has been suggested by a number of in vitrostudies (for a review see Reference 2), and recent observa-tions in animal models indicated that human PLTP expressioncan markedly influence HDL metabolism.30–32 These obser-vations gave rise to a great interest in evaluating the patho-physiological variations of CETP and PLTP levels in vivo. Tothis end, a basic requirement is to devise specific immuno-assays allowing the quantification of CETP and PLTP masslevels in biological samples from various human populations.




The present report describes the first immunoassay of humanplasma PLTP that was applied to the determination of PLTPlevels in plasma from 30 normolipidemic subjects, 36 type IIahyperlipidemic patients, 33 type IIb hyperlipidemic patients,and 50 NIDDM patients. The parallel determination of CETPlevels among the same subpopulations allowed a comparativeanalysis of the variability of lipid transfer protein levels inhumans. A specific polyclonal antiserum was raised in rabbitagainst pure PLTP that was isolated from normolipidemichuman plasma, and pure PLTP fractions exhibited the samecharacteristics as those previously described by others.Hence, PLTP preparations were able to transfer phosphati-dylcholine from liposomes toward isolated HDL, 1 featurethat is not shared by human CETP.4,23,26–28In addition, thepresent work confirmed that the mean apparent molecularweight of pure PLTP, as determined by denaturing polyacryl-amide gradient gel electrophoresis, is 70 kDa under reducingconditions, but 56 kDa in the absence of a reducing agent.7

Moreover, anti-PLTP polyclonal antibodies were able toremove most of the phospholipid transfer activity in totalhuman plasma while CE transfer activity remained unaf-fected. Polyclonal anti-PLTP antibodies were used to estab-lish a competitive ELISA that proved to be a specific,sensitive, and accurate assay for PLTP concentration inplasma samples from normolipidemic and dyslipidemic sub-jects. No cross-reactivity against other plasma proteins, in-cluding the related CETP, was noted, and plasma PLTP massconcentrations were correlated significantly with phospho-lipid transfer rates as measured by an endogenouslipoprotein-independent assay.

When PLTP was assayed in total plasma from normolipi-demic subjects, the mean concentration was 3.9561.04 mg/L,with no difference between men and women. The meanplasma PLTP level was in the same range as that of othermembers of the LT/LBP family,13,33,34 and in the samenormolipidemic group, the mean plasma CETP concentrationwas 2.6760.55 mg/L. Whereas CETP mass levels werecorrelated positively and significantly with total and LDLcholesterol levels, PLTP mass levels were not correlatedsignificantly with any of the plasma lipid parameters mea-sured. In support of recent in vivo studies,35 the presentobservations suggest that plasma LDL cholesterol levelsmight constitute a key determinant of plasma CETP levels,possibly through upregulation ofCETPgene expression.36–38

In contrast, parameters other than LDL cholesterol mightconstitute the major determinants of PLTP expression. Nosignificant relationship between plasma CETP and PLTPmass levels were noted, and overall observations in normo-lipidemic subjects indicate that plasma CETP and PLTPexpression would be differentially regulated. The latter viewis supported by several recent observations: (1) Plasma PLTPactivity but not plasma CETP activity is affected by thesaturated versustrans-unsaturated fatty acid content of thediet.39 (2) Opposite tendencies in diet-induced variations inCETP and PLTP activities have been reported among variousinbred rabbit strains.40 (3) Alcohol withdrawal in alcoholicpatients produces different effects on plasma CETP andPLTP activities.41

Another point of the present study was the first determi-nation of PLTP levels in plasmas from type IIa and type IIbdyslipidemic patients. In fact, the PLTP concentration was

remarkably similar in normolipidemic, type IIa, and type IIbsubpopulations despite marked abnormalities in the plasmalipid levels of the dyslipidemic groups. Again, these obser-vations sustain the hypothesis for the lack of a direct linkbetween PLTP and plasma lipid levels. In good agreementwith previous observations,42,43 CETP mass levels weresignificantly higher in type IIb patients than in normolipi-demic controls, and a similar tendency was observed for typeIIa patients.

Finally, a specific ELISA was applied to the determinationof plasma PLTP levels in another pathological state associ-ated with dyslipidemia, ie, NIDDM. This part of the studywas hastened by recent reports addressing alterations inplasma phospholipid transfer activity in diabetic patients.However, the data are controversial, with either no alter-ation44 or a significant decrease45 in plasma phospholipidtransfer activity being reported in NIDDM, as assessed bydistinct isotopic activity assays. In addition, circumstantialevidence in favor of increased PLTP-mediated conversion ofHDL3 to HDL2 in plasma from hypertriglyceridemic NIDDMpatients compared with normolipidemic controls has recentlybeen reported.46 In the present study, we found a marked andsignificant increase in PLTP mass levels in plasmas fromNIDDM patients compared with normolipidemic controls.Again, as described above in normolipidemic subjects as wellas in type IIa and type IIb patients, no significant correlationof PLTP levels with lipid parameters was observed inNIDDM patients. Because homologies between plasma lipidabnormalities were noted in NIDDM, type IIa, and type IIbpatients, it is unlikely that the significant increase in plasmaPLTP concentrations in NIDDM is related to the dyslipid-emic state per se. In fact, analysis of additional plasmaparameters revealed a significant, positive correlation be-tween fasting glycemia and PLTP levels among the diabeticsubpopulation, whereas no significant relationship betweenPLTP mass and insulin levels was found. Together with thepositive correlation between HbA1c levels and PLTP levels,the results indicate that plasma glucose might be a putativedeterminant of plasma PLTP levels, and the significantincrease in plasma glucose in NIDDM could account for theconcomitant increase in PLTP mass. Interestingly, 1 recentstudy reported that isotopic transfer of phospholipids in obesemen is positively related to both body mass index and fastingblood glucose concentration.47 Because we did not observe asignificant relationship between body mass index and PLTPmass levels among diabetics, we postulate that plasma glu-cose rather than body mass index would determine PLTPlevels in NIDDM patients. In fact, increased PLTP levels inthe diabetic subpopulation of the present study might actuallybe related to the insulin resistance that is associated withlong-lasting, elevated levels of plasma glucose rather than arapid response to transiently elevated plasma glucose levels.Indeed, decreased plasma phospholipid transfer activity wasrecently observed in healthy men under acute hyperglycemia-induced hyperinsulinemia, and a significant negative correla-tion between plasma phospholipid transfer rates and insulinsensitivity was reported.48 We propose that the latter pointmight also apply to the NIDDM population and that theincreased PLTP levels in these patients would be part of theinsulin resistance syndrome. In contrast to PLTP, CETP massconcentrations in normolipidemic and NIDDM groups did




not differ significantly. The latter point was in good agree-ment with previous studies that reported normal CETP masslevels in NIDDM patients despite elevated plasma CE trans-fer rates.49,50

In conclusion, the PLTP ELISA described in the presentreport constitutes the first tool for the measurement of PLTPmass concentration in plasma from normolipidemic as well asdyslipidemic subjects. Whereas PLTP mass levels, unlikeCETP mass levels, did not vary significantly in type IIa andtype IIb dyslipidemic groups compared with normolipidemicsubjects, a highly significant rise was observed in NIDDMpatients. Whether PLTP mass concentration is linked toglucose metabolism rather than to lipid metabolism deservesfurther attention.

AcknowledgmentsThe study was supported by grants from the Universite de Bour-gogne, the Conseil Regional de Bourgogne, the Institut National dela Sante et de la Recherche Medicale, and Parke-Davis (to C.D.). Thetechnical assistance of Dominique DeBaudus, Elisabeth Niot, andLiliane Princep is greatly acknowledged.

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Perségol, Philippe Gambert and Laurent LagrostCatherine Desrumaux, Anne Athias, Ginette Bessède, Bruno Vergès, Michel Farnier, Laurence

as Measured by a Specific ELISAInsulin-Dependent Diabetic Subjects−Hyperlipidemic, Type IIb Hyperlipidemic, and Non

Mass Concentration of Plasma Phospholipid Transfer Protein in Normolipidemic, Type IIa

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