Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:266-275
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:266-275.)
© 1999 American Heart Association, Inc.
Mass Concentration of Plasma Phospholipid Transfer Protein in Normolipidemic, Type IIa Hyperlipidemic, Type IIb Hyperlipidemic, and NonInsulin-Dependent Diabetic Subjects as Measured by a Specific ELISA
Catherine Desrumaux;
Anne Athias;
Ginette Bessède;
Bruno Vergès;
Michel Farnier;
Laurence Perségol;
Philippe Gambert;
Laurent Lagrost
From Laboratoire de Biochimie des Lipoprotéines, INSERM U498,
Université de Bourgogne (C.D., A.A., G.B., B.V., L.P., P.G., L.L.);
Service d'Endocrinologie et Diabétologie, Hôpital du Bocage
(B.V.); and Le Point Médical (M.F.), Dijon, France.
Correspondence to Laurent Lagrost, Laboratoire de Biochimie des Lipoprotéines, INSERM U498, Hôpital du BocageBP 1542, 21034 Dijon Cedex, France.
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Abstract
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AbstractMean plasma
phospholipid transfer protein (PLTP)
concentrations were measured for
the first time by using a competitive
enzyme-linked immunosorbent
assay. PLTP mass levels and phospholipid
transfer activity values,
which were significantly correlated
among normolipidemic plasma samples
(
r=0.787,
P<0.0001),
did not differ
between normolipidemic subjects (3.95±1.04
mg/L and 575±81 nmol
· mL
-1 · h
-1, respectively;
n=30),
type IIa hyperlipidemic patients (4.06±0.84 mg/L
and
571±43 nmol · mL
-1 · h
-1,
respectively;
n=36), and type IIb hyperlipidemic
patients (3.90±0.79
mg/L and 575±48 nmol ·
mL
-1 · h
-1, respectively;
n=33). No
significant correlations with plasma lipid parameters
were
observed among the various study groups. In contrast, plasma
concentrations
of the related cholesteryl ester transfer protein (CETP)
were
higher in type IIa and type IIb patients than in normolipidemic
controls,
and significant, 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 with
noninsulin-dependent
diabetes mellitus (n=50) than in normolipidemic
controls (6.76±1.93
versus 3.95±1.04 mg/L,
P<0.0001;
and 685±75
versus 575±81 nmol · mL
-1 ·
h
-1,
P<0.0001,
respectively). In contrast,
CETP levels did not differ significantly
between the 2 groups. Among
noninsulin-dependent diabetes
mellitus patients, PLTP levels were
positively correlated with
fasting glycemia and glycohemoglobin levels
(
r=0.341,
P=0.0220;
and
r=0.382,
P=0.0097, respectively) but not
with plasma lipid
parameters. It is proposed that plasma
PLTP mass levels are
related to glucose metabolism rather
than to lipid metabolism.
Key Words: cholesteryl ester transfer protein lipid transfer ELISA glucose noninsulin-dependent diabetes mellitus
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Introduction
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In vivo, plasma lipoproteins do not constitute stable
entities
but are continuously remodeled through the action of several
enzymes
and lipid transfer proteins. In particular, cholesteryl ester
transfer
protein (CETP)
1 and phospholipid transfer protein
(PLTP), 2
related proteins belonging to the lipid
transfer/lipopolysaccharide
binding protein (LT/LBP)
family,
1 can promote the exchange
of lipid species between
various plasma lipoprotein fractions.
In fact, studies over the past
few years have demonstrated that
both CETP and PLTP produce multiple
effects on lipoprotein structure
and composition. Thus, CETP promotes
the exchange of neutral
lipids, ie, CEs and triglycerides,
between plasma lipoprotein
fractions, leading to alterations in both
the neutral lipid
content and the size distribution of
lipoproteins.
2 3 PLTP
can facilitate the transfer of
phospholipids between lipoprotein
particles,
4 and it was
lately shown to transfer lipopolysaccharides,
5
unesterified cholesterol,
6 and

-tocopherol
7 as well. In addition,
PLTP
constitutes an important determinant of the size distribution
of
HDL.
3 8 9 10 11 12 Taken together, recent advances have
raised
considerable interest in elucidating the precise function
of lipid
transfer proteins in lipoprotein metabolism, and a
new
challenge of in vivo studies is to relate
pathophysiological
alterations of the plasma levels
of CETP and PLTP to atherosclerosis
susceptibility.
Mainly, 2 distinct approaches can be applied to the quantification of
lipid transfer protein levels, consisting of either evaluation of lipid
transfer activities by isotopic or net mass-transfer assays or
determination of the mass concentration of lipid transfer proteins by
specific immunoassays. Although the determination of lipid transfer
rates in plasma has proved helpful and informative, it does not
necessarily provide a reliable and specific estimate of the lipid
transfer protein mass per se, due in part to the presence of putative
modulators in total plasma. The result is that only specific
immunoassays are suitable for accurate determination of lipid transfer
protein mass in plasma samples. In 1990, the first radioimmunoassay of
human CETP, proposed by Marcel and coworkers,13 allowed
the determination of mean CETP levels in normolipidemic plasmas, and
subsequent clinical investigations with specific
immunoassays14 15 16 17 18 19 led to a significant improvement in our
knowledge of the metabolism of CETP and its
pathophysiological variations. Unlike CETP, PLTP
has been quantified only through its ability to exchange phospholipids,
and to date no specific immunoassay has been proposed to assay PLTP
mass levels in biological samples. In fact, it is noteworthy that
phospholipid exchange activity is a property that is shared by several
plasma proteins, including CETP,20 LBP,21 and
soluble CD14,21 in addition to PLTP. The latter point
suggests that only a specific immunoassay would accurately reflect the
level of PLTP in plasma, and today the lack of an adapted quantitative
tool may account, at least in part, for the paucity of information
concerning the pathophysiological relevance of
PLTP.
The present report describes the first immunoassay of human PLTP. A
competitive ELISA of PLTP was devised by using polyclonal
immunoglobulins raised against purified human PLTP. This new method was
then applied to the determination of PLTP in normolipidemic plasmas, as
well as in plasmas from type IIa hyperlipidemic, type
IIb hyperlipidemic, and noninsulin-dependent diabetes
mellitus (NIDDM) patients.
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Methods
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Study Subjects
One hundred forty-nine subjects were selected for the study,
including
30 normolipidemic subjects (15 males and 15 females; total
cholesterol
<2.50 g/L and triglycerides <1.30
g/L), 36 patients with
type IIa dyslipidemia (27 males and
9 females; total cholesterol
>2.50 g/L and
triglycerides <1.30 g/L), 33 patients with
type IIb
dyslipidemia (30 males and 3 females; total
cholesterol
>2.50 g/L and triglycerides >1.30
g/L), and 50 patients
with NIDDM (23 males and 27 females; total
cholesterol 1.02
to 3.20 g/L and triglycerides
0.45 to 4.35 g/L). Control normolipidemic
subjects were selected from
the hospital staff as healthy subjects
with normal thyroid, renal, and
hepatic functions and without
a history of
hyperlipidemia, coronary artery disease, or
diabetes.
Type IIa and type IIb patients did not present secondary
causes
of dyslipidemia, and patients with diabetes mellitus
or those
who were overweight (body mass index >30
kg/m
2) were excluded
from the type IIa and type
IIb groups. Diabetic patients suffered
from NIDDM and were treated by
either diet alone (n=8) or in
combination with oral hypoglycemic drugs
(n=42). Among the entire
population studied, neither normolipidemic
subjects nor dyslipidemic
patients received drugs known to
affect lipoprotein metabolism.
The study was approved by
the ethics committee of the Bocage
Hospital (Dijon, France), and
informed consent was obtained.
Blood Samples
Fasting blood samples were collected into EDTA-containing glass
tubes, which were placed immediately on ice. Plasma was separated by a
5-minute centrifugation at 3000g, and
aliquots were kept at -80°C until analysis.
Purification of Human Plasma PLTP
PLTP was purified from 1200 mL of citrated human plasma that was
made lipoprotein deficient by the dextran
sulfateMnCl2 precipitation procedure of
Burstein et al.22 PLTP was purified by sequential
chromatography on hydrophobic, affinity, and
anion-exchange columns as previously described.23 Only
Mono-Q fractions with high specific phospholipid transfer activity and
containing virtually only pure PLTP were selected for the study, with
the exception of the partially purified Mono-Q fractions used for plate
coating in the ELISA. For rabbit immunization and ELISA calibration,
Mono-Q fractions containing pure PLTP were further passed through an
anti-albumin immunoaffinity column to ensure removal of any
traces of human plasma albumin. Finally, before rabbit
immunization, the purified PLTP fractions were subjected to an ultimate
preparative electrophoresis step to ensure a maximal degree of purity.
In brief, purified PLTP fractions were applied to an 8%
polyacrylamide gel containing 1% SDS, and electrophoresis was
conducted in a 50 mmol/L Tris, 380 mmol/L glycine, and 0.1%
SDS, pH 8.3, buffer for 6 hours at 50 mA. After electrophoresis, the
portion of gel containing PLTP was cut off and the protein was eluted
as previously described.24 The purity of PLTP preparations
was assessed by SDS electrophoresis 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 for immunization of a New Zealand
White rabbit within 24 hours. For plate coating, a partially purified
PLTP fraction was obtained from lipoprotein-deficient fresh plasma
(d>1.21 g/mL) by a combination of phenyl-Sepharose,
heparin-Ultrogel, and anion-exchange
chromatography.23 In brief, after
anion-exchange chromatography of heparin-bound
proteins, all of the eluted fractions containing detectable
phospholipid transfer activity were pooled, and the resulting material
used for plate coating corresponded to an
500-fold increase in
specific phospholipid transfer activity compared with the starting
plasma.
Anti-PLTP Polyclonal Antibodies
Antiserum to purified human PLTP was prepared by immunization of
a 3-kg New Zealand White rabbit with 1 initial injection of 250 µg
PLTP emulsified in complete Freund's adjuvant followed by three
150-µg injections of PLTP emulsified in incomplete Freund's adjuvant
at 2-week intervals. The rabbit was bled 8 days after the last
injection, serum was recovered by low-speed
centrifugation, and the serum IgG fraction was prepared
by using a protein A column (protein ASepharose 4 Fast Flow,
Pharmacia) according to the procedure described by the manufacturer.
This experiment was performed under the framework of the Guide
for the Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH publication No. 81-23, revised
1985).
Anti-PLTP Immunoblotting
The specificity of anti-PLTP immunoglobulins was assessed by
Western blotting. To this end, plasma and purified PLTP samples were
subjected to electrophoresis in 80 to 250 g/L polyacrylamide
Phastgels under reducing conditions, and proteins were subsequently
transferred to a nitrocellulose membrane by using a Phast semidry
electrophoretic transfer system as recommended by the manufacturer
(Pharmacia). The resulting blots were blocked overnight at 4°C in
10% low-fat milk before being incubated for 1 hour at 37°C in the
presence of anti-PLTP antibodies. After being washed, nitrocellulose
membranes were incubated for 1 hour at 37°C with horseradish
peroxidaseconjugated secondary antibodies (Bio-Rad). Finally,
development was achieved by using the ECL-Western blotting detection
reagent kit from Amersham.
PLTP ELISA
A competitive ELISA of PLTP was devised according to the general
procedure previously used in our laboratory to quantify human
apoA-IV,24 apoB,25 and CETP.16
All steps of the immunoassays (pipetting, diluting, dispensing,
washing, and photometry) were carried out with a Biomek 2000
Biorobotics System (Beckman Instruments).
Plate Coating
A 100-µL volume of partially purified PLTP fraction (protein
concentration, 15 mg/L) in a 15 mmol/L
Na2CO3, 35 mmol/L
NaHCO3, and 3 mmol/L
NaN3 (pH 9.6) buffer was pipetted into each well
of a polystyrene microwell plate (Immuno 96F type I from Nunc) and
incubated overnight at 4°C. The plates were then washed 4 times with
a 150 mmol/L NaCl0.025% (vol/vol) Tween-20 washing solution and
incubated for 30 minutes at room temperature with 200 µL of a 1%
(wt/vol) human serum albumin solution containing 10 mmol/L
Na2HPO4, 5 mmol/L
NaH2PO4, and 150
mmol/L NaCl, adjusted to pH 7.2 with NaOH (albumin-phosphate
buffer).
Sample Treatment
PLTP-containing samples were diluted in the
albumin-phosphate buffer and mixed with an equal volume of
polyclonal anti-PLTP antibodies diluted in albumin-phosphate
buffer containing 1% Triton X-100 (Pierce Chemical Co). Total plasma
samples were diluted from 1:2 to 1:16 in the albumin-phosphate
buffer. The mixtures were incubated overnight at 4°C in 96 Deep-well
titer plates (Beckman). Aliquots (100 µL) were then pipetted into the
immunoplate microwells 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 Antibodies
One hundred microliters of peroxidase-conjugated anti-rabbit
antibodies (Bio-Rad) diluted in the albumin-phosphate buffer
was pipetted into each well and incubated for 1 hour at 37°C. After
completion of the incubation, the plates were washed 4 times as before,
and 100 µL of a freshly prepared 0.4 g/L
o-phenylenediamine0.68 g/L
H2O2 solution in a 6.6
mmol/L sodium phosphate3.4 mmol/L citrate buffer (pH 5.2) was
pipetted into each well. After 15 minutes at room temperature in the
dark, the reaction was stopped by addition of 50 µL of 2.5 mol/L
H2SO4. The absorbances were
read at 490 nm with a Photometry tool on the Biomek 2000 Biorobotics
station, and data were saved on a PC computer for further treatment.
Calibration
Pure PLTP (specific activity,
10 µmol ·
mg-1 · h-1) was
used to standardize the assay. The amount of PLTP in purified fractions
was determined by SDS gel capillary electrophoresis with carbonic
anhydrase as an internal standard. Capillary electrophoresis was
performed with uncoated, fused-silica capillaries (27 cmx100-µm ID)
attached to a P/ACE 2100 system that was controlled by Gold software
(Beckman Instruments). The P/ACE system 2100 was used in
reversed-polarity mode. The electrolyte buffer was a noncross-linked
gel matrix (eCAP SDS 14-200, Beckman). In brief, PLTP-containing
samples were diluted in Tris buffer (pH 6.6) containing 1% SDS, and
they were supplemented with orange G as a tracking dye and carbonic
anhydrase (0.2 g/L) as an internal standard. Electrophoresis was
conducted at 20°C at 8.10 kV, and detection was performed at 214 nm.
PLTP mass concentration was determined by comparing the area of the
PLTP peak to the area of the peak obtained with a known amount of
carbonic anhydrase. Finally, an ELISA primary standard curve
constructed from a set of dilutions of purified PLTP was used to
determine PLTP levels in a pool of frozen, normolipidemic human plasmas
that constituted a secondary standard. Routinely, 8 dilutions (PLTP
concentrations from 0.0275 to 3.52 mg/L) were used to construct a
secondary calibration curve for each immunotitration plate. Standard
curves were fitted to the data points by using data analysis
software (Immunofit EIA/RIA data analysis software, Beckman).
Four dilutions of each sample were assayed, and the PLTP concentration
was calculated as the mean of the 4 results.
Isolation of Lipoproteins
HDLs were isolated as the 1.07<d<1.21 g/mL fraction
of normolipidemic, fresh, and citrated human plasma at a speed of
55 000 rpm (223 000g) in a 70-Ti rotor on an L7
ultracentrifuge (Beckman) by two 20-hour spins at the lower
density and one 30-hour spin at the higher density. The HDL fraction
was finally washed with one 8-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 isolated
lipoproteins were dialyzed overnight against a 10 mmol/L Tris,
150 mmol/L NaCl, 1 mmol/L tetrasodium-EDTA, and 3 mmol/L
NaN3, pH 7.4, buffer.
CETP ELISA
CETP concentration in total plasma samples was measured by a
competitive ELISA on a Biomek 2000 laboratory automatic workstation
(Beckman) as previously described.16 CETP mass
concentration values were determined from a calibration curve obtained
with a frozen plasma standard, and they were calculated by using data
analysis software (Immunofit EIA/RIA data analysis
software, Beckman). Four dilutions of each sample were assayed, and the
CETP concentration was calculated by averaging the 4 results.
Phospholipid Transfer Activity Assay
Plasma phospholipid transfer activity was determined as the
capacity of a plasma sample to induce the transfer of radiolabeled
dipalmitoyl phosphatidylcholine ([14C]DPPC)
from [14C]DPPC liposomes to an excess of
isolated HDL. In brief, liposomes (110 nmol phosphatidylcholine) were
incubated at 37°C with isolated HDL (250 µg protein) in the
presence of total plasma and iodoacetate (1.5 mmol/L) in a final
volume of 400 µL. Phospholipid liposomes and apoB-containing
lipoproteins were subsequently precipitated, and radioactivity was
assayed in supernatants. Phospholipid transfer activity was calculated
as the amount of total radiolabeled phospholipids transferred from
liposomes to HDL after deduction of blank values that were obtained by
incubating liposomes and HDL at 37°C for 90 minutes in the absence of
plasma. Phospholipid transfer after a 90-minute incubation increased
gradually as the amount of added normolipidemic plasma was increased
from 0 to 20 µL (Figure 1A
). When 10 µL of plasma was added,
the phospholipid transfer assay was linear over a 2-hour period (Figure 1B
). Throughout the study, phospholipid transfer activity
measurements among various plasma samples were conducted by using
10-µL plasma aliquots that were incubated for 90 minutes at 37°C.
The assay was proved to be independent of the phospholipid exchange
activity catalyzed by CETP.4 23 26 27 28

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Figure 1. Volume-dependent (A) and time-dependent (B)
transfer of phospholipids in normolipidemic plasma. Phospholipid
transfer activity was measured in an endogenous
lipoprotein-independent assay as the rate of transfer of radiolabeled
phosphatidylcholine from [14C]DPPC liposomes toward
isolated HDL (see Methods).
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CETP Activity Assay
Plasma CETP activity was determined as the capacity of a plasma
sample to promote the transfer of radiolabeled CEs
([3H]CE) from [3H]CE
HDL3 to an excess of isolated LDL. Radiolabeled
HDL3 (0.8 µg cholesterol) was
incubated for 18 hours at 37°C with isolated LDL (150 µg protein)
in the presence of 5 µL of total plasma and iodoacetate (1.5
mmol/L) in a final volume of 150 µL. Donor and acceptor particles
were subsequently separated by ultracentrifugation, and
CETP activity was calculated as the percentage of total radiolabeled
CEs transferred from HDL3 to LDL after deduction
of nonincubated control values.
Other Analyses
All chemical assays were performed on a Cobas-Fara centrifugal
analyzer (Roche). Total cholesterol concentration
was measured by an enzymatic method using a Boehringer Mannheim
reagent. HDL cholesterol was measured after selective
precipitation of apoB-containing lipoproteins with Boehringer
phosphotungstic acid/MgCl2 reagent, as
recommended by the manufacturer. LDL cholesterol
concentration was calculated using the Friedewald
formula.29 Triglycerides were assayed by an
enzymatic method using Roche reagents. Glycemia, ie, plasma glucose
level, was determined by an enzymatic method. Glycohemoglobin (HbA1c)
was determined by high-performance liquid
chromatography on a Diamat analyzer (Bio-Rad).
Plasma C-peptide was measured by radioimmunoassay (Mallinckrodt
Medical). Plasma insulin was measured by radioimmunoassay (CIS Bio
International).
Statistical Analysis
ELISA curves were constructed by polynomial regression
analysis. Sigmoidal competitive curves were linearized by
logit-log transformation. Coefficients of correlation were calculated
by linear regression analysis. Multiple regression
analysis was used to determine the contribution of age and
diabetic state to the rise in PLTP mass concentration in the diabetic
subpopulation. Data means were compared by using a 1-way ANOVA.
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Results
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Purification and Characterization of Human PLTP
PLTP was purified from fresh human plasma by using a sequential
procedure
involving dextran sulfate/MnCl
2
precipitation followed by chromatographic
and
electrophoretic steps (see Methods). As shown in Figure
2

, purified PLTP appeared as a single homogeneous band after
electrophoresis
in denaturing polyacrylamide gradient gels,
with an apparent
molecular weight of 56 kDa. In good agreement with
previous
studies,
7 the apparent molecular weight of pure
PLTP rose to
70 kDa when preincubated with ß-mercaptoethanol before
denaturing
polyacrylamide gradient gel electrophoresis (Figure 2

). Further
analysis of PLTP preparations by the
high-resolution capillary
electrophoresis technique confirmed
the presence of a single
protein peak (Figure 3

). Complementary
experiments revealed
that the mean isoelectric point of purified PLTP
(pI 5.0) was
identical to the pI value previously reported by Tollefson
and
coworkers.
4 Purified PLTP preparations
presented elevated specific
phospholipid transfer activity,
determined as the rate of transfer
of radiolabeled phosphatidylcholine
from [
14C]DPPC-liposome
donors toward isolated
HDL acceptors.

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Figure 2. SDSpolyacrylamide gel electrophoresis of
purified PLTP. One-microliter volumes of ß-mercaptoethanoltreated,
purified PLTP (lower profile), purified PLTP without reducing agent
(intermediate profile), and low-molecular-weight calibration kit
standards (Pharmacia; upper profile) were subjected to electrophoresis
in Pharmacia 8% to 25% Phastgel. At the end of electrophoretic
migration, the gel was stained with Coomassie brilliant blue R-250 and
analyzed on a Bio-Rad GS-670 imaging densitometer.
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Figure 3. Capillary gel electrophoretic profile of purified
PLTP in the presence of carbonic anhydrase as an internal standard (see
Methods).
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Production of a Specific Anti-PLTP Antiserum and
Development of an ELISA
Specific anti-PLTP IgGs were prepared from the serum of the rabbit
successively injected with pure PLTP over a 2-month period (see
Methods). As shown in Figure 4
, anti-PLTP immunoglobulins
(concentration range, 0 to 100 µg/µL) were found to inhibit plasma
phospholipid transfer activity in a concentration-dependent manner.
When 5 distinct plasma samples were supplemented with high
concentrations of anti-PLTP immunoglobulins (150 µg/µL), the
maximal inhibition of phospholipid transfer activity ranged between
70% and 87%. Under the same experimental conditions, the CE transfer
activity of the related CETP remained unchanged (Figure 4
). As
shown in Figure 5
, a single 70-kDa band was detected by Western
blotting of either plasma or purified PLTP samples under reducing
conditions. Anti-PLTP immunoglobulins were used to establish a
competitive ELISA (see Methods). As shown in Figure 6
, a typical
ELISA displacement curve was obtained with purified PLTP with a 0.1 to
10 µg/mL working concentration range. In contrast, no displacement
curves were observed when purified CETP or albumin solutions
were used (Figure 6
). When displacement curves were obtained by
using various PLTP-containing fractions with distinct degrees of
purity, the logit-log lines were parallel, indicating that the affinity
of polyclonal anti-PLTP IgG was unaffected by the presence of other
protein components in the mixture to be assayed (Figure 7
). In
addition, parallel logit-log lines were obtained with serial dilutions
of normolipidemic or hyperlipidemic plasmas, indicating
that under the experimental conditions used, plasma lipid levels did
not alter the immunoaffinity of immunoglobulins for PLTP (Figure 8
).

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Figure 4. Effect of anti-PLTP polyclonal immunoglobulins on
phospholipid transfer and CE transfer activities in human plasma. For
phospholipid transfer activity measurements, various amounts of control
( ) or anti-PLTP ( ) rabbit IgGs were added
to 10 µL of human plasma. The mixtures were preincubated for 16 hours
at 4°C in a final volume of 200 µL and centrifuged for 30
minutes at 10 000 rpm. The resulting supernatants were then incubated
at 37°C for 90 minutes in the presence of [14C]DPPC
liposomes (110 nmol phosphatidylcholine), isolated HDL (250 µg
protein), and iodoacetate (1.5 mmol/L) in a final volume of 400
µL. For CE transfer activity determinations, 5 µL of plasma was
preincubated for 16 hours at 4°C with the indicated amounts of
nonimmune ( ) or immune ( ) rabbit IgG. The
mixtures were then centrifuged, and the supernatants were
incubated at 37°C for 18 hours with [3H]CE
HDL3 (0.8 µg cholesterol), isolated LDL (150
µg protein), and iodoacetate (1.5 mmol/L) in a final volume of
150 µL. At the end of the incubations, donor and acceptor lipoprotein
fractions were separated, and transfer activity values were calculated
as the percentage of radioactivity transferred from the donor to the
acceptor fraction after deduction of blank values. Data are expressed
as percentages of remaining lipid transfer activity compared with
controls incubated without immunoglobulin supplementation (phospholipid
transfer activity, 692 nmol · mL-1 ·
h-1). Each point represents the mean of duplicate
determinations.
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Figure 5. Anti-PLTP Western blot analysis
of various protein fractions. Immunolocalization of PLTP was performed
after electrophoresis in 80 to 250 g/L polyacrylamide gradient
gels as described under Methods. Lane 1, low-molecular-weight
calibration kit standards after Coomassie brilliant blue staining of
gradient gels. Lane 2, immu-noblot of Pharmacia
low-molecular-weight calibration kit standards. Lane 3,
immunoblot of 1 µL diluted (1/5) normolipidemic human
plasma. Lane 4, immunoblot of a heparin-bound, partially
purified PLTP fraction (25 ng protein).
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Intra-assay and interassay coefficients of variation were evaluated by
analyzing the same plasma sample 5 times in the same microwell plate on
the same day and on 5 consecutive days, respectively. The values of the
intra-assay and interassay coefficients of variation were 5.7% and
7.8%, respectively.
Determination of PLTP and CETP Mass Concentrations in Plasma From
Normolipidemic Subjects
PLTP mass concentration was assayed among plasmas from 30
normolipidemic subjects (15 males, 15 females). The mean plasma
concentration of PLTP was 3.95±1.04 mg/L (range, 1.98 to 5.71), with
identical levels in males and females. The mean plasma phospholipid
transfer activity among the normolipidemic population was 575±81
nmol · mL-1 ·
h-1. As shown in Figure 9
, plasma PLTP
mass levels were correlated positively and significantly with
phospholipid transfer activity values measured as the transfer of
radiolabeled phosphatidylcholine from [14C]DPPC
liposomes toward exogenous HDL (r=0.787,
P<0.0001). Among the same normolipidemic subpopulation, the
mean plasma concentration of CETP was 2.67±0.55 mg/L, with slightly
higher levels in females than in males (2.73±0.67 mg/L and 2.61±0.41
mg/L, respectively). Plasma CETP concentration was positively
correlated with total and LDL cholesterol levels
(r=0.40, P=0.0301; and r=0.36,
P=0.0495, respectively). In contrast, plasma PLTP levels
were not significantly correlated with any of the plasma lipid
parameters determined among the normolipidemic
subpopulation (Table 1
).

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Figure 9. Correlation of plasma PLTP mass with plasma
phospholipid transfer activity among 30 normolipidemic healthy
subjects. PLTP mass was determined by ELISA, and phospholipid transfer
activity was determined by an isotopic transfer assay as described
under Methods.
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Table 1. Correlation of CETP and PLTP Mass Concentration With
Plasma Parameters in Normolipidemic Subjects (n=30)
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Determination of PLTP and CETP Mass Concentrations in Plasma From
Patients With Type IIa Hyperlipidemia
As shown in Table 2
, a marked and significant rise in LDL
cholesterol levels constituted the main abnormality of type
IIa hyperlipidemic patients (n=36), accounting for the
significantly higher total cholesterol levels compared with
normolipidemic controls (P<0.0001), whereas
triglyceride and HDL cholesterol levels in type
IIa and normolipidemic populations were similar. No significant
differences in PLTP mass and phospholipid transfer activity levels were
observed between normolipidemic and type IIa groups (Table 2
).
As observed with the normolipidemic subpopulation, plasma PLTP levels
were not significantly correlated with any of the plasma lipid
parameters among type IIa patients (Table 3
). In
contrast, regression analysis revealed a positive correlation
of CETP mass levels with both total and LDL cholesterol
levels among type IIa patients (r=0.423,
P=0.0102; and r=0.397, P=0.0166,
respectively).
Determination of PLTP and CETP Mass Concentrations in Plasma From
Patients With Type IIb Hyperlipidemia
Type IIb hyperlipidemic patients presented
significantly higher levels of total cholesterol, LDL
cholesterol, and triglycerides
(P<0.0001 in all cases) compared with normolipidemic
controls (Table 2
). In contrast, HDL cholesterol
levels were significantly lower in type IIb patients than in
normolipidemic subjects (P=0.0004; Table 2
). CETP
mass concentration was significantly higher in type IIb patients than
in normolipidemic controls. No significant differences in mean PLTP
mass and phospholipid transfer activity levels were observed between
normolipidemic and type IIb groups (Table 2
). Neither CETP nor
PLTP mass levels were significantly correlated with any of the plasma
lipid parameters among the type IIb
hyperlipidemic subpopulation (Table 4
).
Nevertheless, in agreement with data observed in the normolipidemic and
type IIa subpopulations, CETP mass levels tended to be positively
correlated with plasma LDL cholesterol levels in the type
IIb population (r=0.306, P=0.0836).
Determination of PLTP and CETP Mass Concentrations in Plasma From
Patients With NIDDM
NIDDM patients (n=50) presented significantly higher
levels of total cholesterol, LDL cholesterol,
and triglycerides (P=0.0324,
P<0.0001, and P<0.0001, respectively) and
significantly lower levels of HDL cholesterol
(P=0.0005) than in normolipidemic subjects (Table 2
).
In addition, fasting glycemia was markedly and significantly higher in
diabetics than in nondiabetics (Table 2
). Plasma PLTP mass and
phospholipid transfer activity levels were significantly higher in
diabetics than in controls, whereas CETP mass levels did not vary
significantly between the 2 groups (Table 2
). Phospholipid
transfer activity levels, but not PLTP mass levels, were lower in
diabetics treated with a combination of hypoglycemic drugs and diet
than in diabetics treated with diet alone (phospholipid transfer
activity, 676±76 versus 750±55 nmol ·
mL-1 · h-1,
respectively, P=0.02; PLTP concentration, 6.75±1.80 versus
7.03±2.15 mg/L, respectively, NS). Whereas no significant
relationships between lipid transfer protein levels and plasma lipid
parameters were noted among the diabetic subpopulation,
PLTP but not CETP was correlated positively and significantly with both
fasting glycemia (r=0.341, P=0.0220) and HbA1c
(r=0.382, P=0.0097) levels (Table 5
and
Figure 10
). Because diabetic patients tended to be older than
normolipidemic controls, multiple regression analysis was used
to determine the contribution of age and the diabetic state to the
prediction of PLTP mass and phospholipid transfer activity levels. From
this analysis it was found that diabetic/nondiabetic state, but
not age, contributed significantly to the rise in both PLTP mass levels
(P<0.0001) and phospholipid transfer activity levels
(P=0.0009) in the diabetic subpopulation.

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|
Figure 10. Correlation of PLTP mass concentration with
glycemia (upper panel) and HbA1c (lower panel) in NIDDM patients
(n=50). PLTP mass, glycemia, and the proportion of HbA1c were
determined as described under Methods.
|
|
 |
Discussion
|
|---|
The importance of the roles of CETP and PLTP in lipoprotein
metabolism
has been suggested by a number of in vitro
studies (for a review
see Reference 2
2 ), and recent observations in
animal models
indicated that human PLTP expression can markedly
influence
HDL metabolism.
30 31 32 These
observations gave rise to a great
interest in evaluating the
pathophysiological variations of
CETP and PLTP
levels in vivo. To this end, a basic requirement
is to devise specific
immunoassays allowing the quantification
of CETP and PLTP mass levels
in biological samples from various
human populations. The present
report describes the first immunoassay
of human plasma PLTP that was
applied to the determination of
PLTP levels in plasma from 30
normolipidemic subjects, 36 type
IIa hyperlipidemic
patients, 33 type IIb hyperlipidemic patients,
and 50
NIDDM patients. The parallel determination of CETP levels
among the
same subpopulations allowed a comparative analysis
of the
variability of lipid transfer protein levels in humans.
A specific
polyclonal antiserum was raised in rabbit against
pure PLTP that was
isolated from normolipidemic human plasma,
and pure PLTP fractions
exhibited the same characteristics as
those previously described by
others. Hence, PLTP preparations
were able to transfer
phosphatidylcholine from liposomes toward
isolated HDL, 1 feature that
is not shared by human CETP.
4 23 26 27 28 In addition, the
present work confirmed that the mean
apparent molecular weight of
pure PLTP, as determined by denaturing
polyacrylamide gradient
gel electrophoresis, is 70 kDa under
reducing conditions, but 56 kDa in
the absence of a reducing
agent.
7 Moreover, anti-PLTP
polyclonal antibodies were able
to remove most of the phospholipid
transfer activity in total
human plasma while CE transfer activity
remained unaffected.
Polyclonal anti-PLTP antibodies were used to
establish a competitive
ELISA that proved to be a specific, sensitive,
and accurate
assay for PLTP concentration in plasma samples from
normolipidemic
and dyslipidemic subjects. No
cross-reactivity against other
plasma proteins, including the related
CETP, was noted, and
plasma PLTP mass concentrations were correlated
significantly
with phospholipid transfer rates as measured by an
endogenous
lipoprotein-independent assay.
When PLTP was assayed in total plasma from normolipidemic subjects, the
mean concentration was 3.95±1.04 mg/L, with no difference between men
and women. The mean plasma PLTP level was in the same range as that of
other members of the LT/LBP family,13 33 34 and in the
same normolipidemic group, the mean plasma CETP concentration was
2.67±0.55 mg/L. Whereas CETP mass levels were correlated positively
and significantly with total and LDL cholesterol levels,
PLTP mass levels were not correlated significantly with any of the
plasma lipid parameters measured. In support of recent in
vivo studies,35 the present observations suggest that
plasma LDL cholesterol levels might constitute a key
determinant of plasma CETP levels, possibly through upregulation of
CETP gene expression.36 37 38 In contrast,
parameters other than LDL cholesterol might
constitute the major determinants of PLTP expression. No significant
relationship between plasma CETP and PLTP mass levels were noted, and
overall observations in normolipidemic subjects indicate that plasma
CETP and PLTP expression would be differentially regulated. The latter
view is supported by several recent observations: (1) Plasma PLTP
activity but not plasma CETP activity is affected by the saturated
versus trans-unsaturated fatty acid content of the
diet.39 (2) Opposite tendencies in diet-induced
variations in CETP and PLTP activities have been reported among various
inbred rabbit strains.40 (3) Alcohol withdrawal in
alcoholic patients produces different effects on plasma CETP and PLTP
activities.41
Another point of the present study was the first determination of
PLTP levels in plasmas from type IIa and type IIb
dyslipidemic patients. In fact, the PLTP concentration was
remarkably similar in normolipidemic, type IIa, and type IIb
subpopulations despite marked abnormalities in the plasma lipid levels
of the dyslipidemic groups. Again, these observations
sustain the hypothesis for the lack of a direct link between PLTP and
plasma lipid levels. In good agreement with previous
observations,42 43 CETP mass levels were significantly
higher in type IIb patients than in normolipidemic controls, and a
similar tendency was observed for type IIa patients.
Finally, a specific ELISA was applied to the determination of plasma
PLTP levels in another pathological state associated with
dyslipidemia, ie, NIDDM. This part of the study was
hastened by recent reports addressing alterations in plasma
phospholipid transfer activity in diabetic patients. However, the data
are controversial, with either no alteration44 or a
significant decrease45 in plasma phospholipid transfer
activity being reported in NIDDM, as assessed by distinct isotopic
activity assays. In addition, circumstantial evidence in favor of
increased PLTP-mediated conversion of HDL3 to
HDL2 in plasma from
hypertriglyceridemic NIDDM patients
compared with normolipidemic controls has recently been
reported.46 In the present study, we found a marked
and significant increase in PLTP mass levels in plasmas from NIDDM
patients compared with normolipidemic controls. Again, as described
above in normolipidemic subjects as well as in type IIa and type IIb
patients, no significant correlation of PLTP levels with lipid
parameters was observed in NIDDM patients. Because
homologies between plasma lipid abnormalities were noted in NIDDM, type
IIa, and type IIb patients, it is unlikely that the significant
increase in plasma PLTP concentrations in NIDDM is related to the
dyslipidemic state per se. In fact, analysis of
additional plasma parameters revealed a significant,
positive correlation between fasting glycemia and PLTP levels among the
diabetic subpopulation, whereas no significant relationship between
PLTP mass and insulin levels was found. Together with the positive
correlation between HbA1c levels and PLTP levels, the results indicate
that plasma glucose might be a putative determinant of plasma PLTP
levels, and the significant increase in plasma glucose in NIDDM could
account for the concomitant increase in PLTP mass. Interestingly, 1
recent study reported that isotopic transfer of phospholipids in obese
men is positively related to both body mass index and fasting blood
glucose concentration.47 Because we did not observe a
significant relationship between body mass index and PLTP mass levels
among diabetics, we postulate that plasma glucose rather than body mass
index would determine PLTP levels in NIDDM patients. In fact, increased
PLTP levels in the diabetic subpopulation of the present study
might actually be related to the insulin resistance that is associated
with long-lasting, elevated levels of plasma glucose rather than a
rapid response to transiently elevated plasma glucose levels. Indeed,
decreased plasma phospholipid transfer activity was recently observed
in healthy men under acute hyperglycemia-induced
hyperinsulinemia, and a significant negative
correlation between plasma phospholipid transfer rates and insulin
sensitivity was reported.48 We propose that the latter
point might also apply to the NIDDM population and that the increased
PLTP levels in these patients would be part of the insulin resistance
syndrome. In contrast to PLTP, CETP mass concentrations in
normolipidemic and NIDDM groups did not differ significantly. The
latter point was in good agreement with previous studies that reported
normal CETP mass levels in NIDDM patients despite elevated plasma CE
transfer rates.49 50
In conclusion, the PLTP ELISA described in the present report
constitutes the first tool for the measurement of PLTP mass
concentration in plasma from normolipidemic as well as
dyslipidemic subjects. Whereas PLTP mass levels, unlike
CETP mass levels, did not vary significantly in type IIa and type IIb
dyslipidemic groups compared with normolipidemic subjects,
a highly significant rise was observed in NIDDM patients. Whether PLTP
mass concentration is linked to glucose metabolism rather
than to lipid metabolism deserves further attention.
 |
Acknowledgments
|
|---|
The study was supported by grants from the Université
de
Bourgogne, the Conseil Régional de Bourgogne, the
Institut
National de la Santé et de la Recherche Médicale,
and
Parke-Davis (to C.D.). The technical assistance of Dominique
DeBaudus,
Elisabeth Niot, and Liliane Princep is greatly
acknowledged.
Received February 11, 1998;
accepted June 23, 1998.
 |
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