HDL subpopulations. It was initially characterized as a protein facilitating the transfer of phospholipids be-
tween LDL and HDL [3] or from phospholipid vesicles to HDL [4]. Later, it was shown that PLTP can induce
HDL conversion, a process which remodels a homoge- neous HDL fraction into populations of large and
small HDL particles [5,6]. The small lipid poor particles are similar to preb-HDL, the initial acceptors of mem-
brane cholesterol from peripheral cells in the reverse cholesterol transport process. Recent in vivo studies
employing adenoviral overexpression of human PLTP or human PLTP transgenic mice have shown that
PLTP has profound effects on plasma HDL. Adenovi- ral overexpression of PLTP resulted in a dramatic
decrease in HDL levels, while preb-HDL levels were substantially elevated. In human PLTP transgenic mice,
the HDL-cholesterolnon-HDL cholesterol ratio in- creased significantly. Furthermore, after the human
PLTP transgene mice were cross-bred with human apoA-I transgenics, there was an increase in HDL
phospholipids and in preb-mobile HDL [7 – 10]. The connections of plasma PLTP activity or tissue PLTP
expression levels with specific clinical states have been addressed only to a very limited extent. Increased PLTP
activity has been reported to be associated with insulin resistance in type II diabetic patients [11]. Upregulation
of PLTP expression is detected in emphysematous lungs and in cultured alveolar epithelial cells upon experimen-
tally induced hypoxia [12]. Furthermore, in a study with alcoholic patients, high alcohol consumption was
associated with increased plasma PLTP activity [13]. In a mouse model, PLTP activity was reported to be
increased by a high-cholesterol diet and decreased upon injection of lipopolysaccharide [14].
Until now most studies have assessed only the phos- pholipid transfer activity of PLTP in clinical specimens.
Measurement of plasma PLTP activity using either the endogenous [8] or exogenous [4] assays may be affected
by differences in the composition of the plasma sam- ples. Further, there may be factors other than PLTP
that facilitate phospholipid transfer in these activity assays: phospholipid exchangetransfer activity has also
been attributed to plasma lipopolysaccharide binding protein LBP [15], soluble CD14 [15], and cholesteryl
ester transfer protein CETP [16]. It is therefore crucial for our understanding of plasma phospholipid PL
transfer processes, their regulation, and their physiolog- ical implications that PLTP mass in plasma is also
determined.
In the present study we describe an enzyme-linked immunosorbent assay ELISA for the measurement of
PLTP mass, based on the use of a monoclonal capture antibody and a polyclonal detection antibody. The
ELISA was used to assay PLTP mass in plasma of 159 normal Finnish individuals. The relationships between
plasma PLTP mass, phospholipid transfer activity, and various lipoprotein parameters suggest that in healthy
individuals phospholipid transfer activity is strongly modulated by the plasma lipidlipoprotein composition.
The method provides a novel tool to elucidate the mechanisms by which PLTP regulates plasma HDL
levels and subpopulation distribution.
2. Materials and methods
2
.
1
. Standardization of the ELISA
2
.
1
.
1
. Purification of human plasma PLTP The lipoprotein-poor fraction, density \ 1.21 gml,
was isolated from fresh plasmapheresis plasma by se- quential ultracentrifugation [17]. To this fraction, apro-
tinin 50 Uml and b-mercaptoethanol 5 mM, final concentration
were added.
It was
then chro-
matographed on
a hydrophobic
Butyl-Toyopearl 650M column 5 × 20 cm essentially as described [18].
The butyl-column was equilibrated with 10 mM Tris – HCl, pH 7.4 containing 2 M NaCl and 1 mM EDTA.
The bottom fraction was recycled overnight in the column, washed with 50 mM Tris – HCl, pH 7.4 con-
taining 1 mM EDTA, and then eluted with 3 mM Tris – HCl, pH 7.4, 1 mM EDTA. The fractions con-
taining PLTP activity were combined, aprotinin and b-mercaptoethanol were added as above, and the pool
was recycled overnight through a heparin-sepharose column dimensions, 2.5 × 10 cm. After extensive
washing with TE buffer 25 mM Tris – HCl, pH 7.4, 1 mM EDTA and then with TE buffer containing 50
mM NaCl, the PLTP activity was eluted with TE buffer containing 1.0 M NaCl. The active elution fractions
were combined, dialyzed overnight against TE buffer and then again applied on a 1-ml heparin-sepharose
column Hi-Trap, Pharmacia, Uppsala, Sweden. The column was first washed with TE buffer and eluted with
a linear 60-min NaCl gradient 0 – 0.5 M. PLTP activ- ity eluted at 0.20 – 0.45 M NaCl. The active fractions
were combined and applied to a 2-ml hydroxylapatite column Bio-Rad HTP-grade, Richmond, USA equili-
brated with 1 mM Na-phosphate buffer, pH 6.8, 150 mM NaCl. PLTP activity was eluted with a linear
60-min Na-phosphate gradient 1 – 100 mM. PLTP ac- tivity eluted between 25 and 50 mM phosphate. The
active fractions were combined and stored at − 20°C. The purity of the PLTP preparations was analyzed
using homogeneous 12.5 SDS-polyacrylamide gels. The proteins were visualized by silver-staining or im-
munoblotting with a monoclonal anti-PLTP antibody. After silver staining the gel was scanned with the
BioImage System Millipore. Protein was determined by the method of Lowry et al. [19] using BSA as a
standard.
2
.
2
. Measurement of phospholipid transfer acti6ity Phospholipid transfer activity was measured using a
radiometric method [4]. Phosphatidylcholine PC lipo- somes were prepared essentially as described [4]. The
liposome preparation contained 10 mmol of egg PC, 1 mCi of [
14
C]dipalmitoyl PC, and 20 nmol of butylated hydroxytoluene. Each assay contained HDL
3
as accep- tor 250 mg protein, donor liposomes 150 nmol of
labelled liposomes, serumplasma sample 4 ml of sam- ple diluted 1:10 with TBS, 1 mM EDTA, in a final
volume of 400 ml. The assays were carried out in 1.5-ml Eppendorf tubes which were incubated at 37°C for 1.5
h. Under these conditions the assay was linear within the range of plasma phospholipid transfer activity ob-
served in the study subjects. In each series, the samples were analyzed in duplicate. Each series also contained
duplicate blank tubes without sample and duplicate control plasma samples that were stored at − 70°C
control samples did not lose PL transfer activity even after prolonged storage for 1 – 2 years at this tempera-
ture. For each new sample series, a new plasma control sample was thawed. All the other details of the assay
have been described [20]. Control samples were used to monitor the repeatability of the assay within and be-
tween the series. The intra-assay precision was 9.4 n = 11 and the interassay precision 12 n = 50.
2
.
3
. The anti-PLTP antibodies For production of the polyclonal PLTP antibody
R176, a cDNA segment encoding the C-terminal amino acid residues 425 – 493 of PLTP was expressed in Es-
cherichia coli from the pGAT-4 vector a kind gift of Dr Johan Pera¨nen, Institute of Biotechnology, Univer-
sity of Helsinki as a His
6
-glutathione-S-transferase GST fusion. The insoluble recombinant protein was
isolated by preparative SDS – PAGE after enrichment using the inclusion body purification protocol of Lin
and Cheng [21]. The protein was used for subcutaneous immunization of New Zealand White rabbits mixed
with complete first injection or incomplete subse- quent injections Freund’s adjuvant. The IgG fraction
of the antiserum was isolated using Protein A-Sep- harose CL 4B Pharmacia according to the manufac-
turer’s
instructions, dialyzed
at 4°C
against phosphate-buffered saline PBS, and stored in aliquots
at − 80°C. The monoclonal antibody, JH59, against PLTP was
produced as follows. Full-length human PLTP was produced in E. coli as a thioredoxin fusion protein
using the ThioFusion expression system Invitrogen. The predominantly insoluble protein was purified by
preparative SDS – PAGE and used for subcutaneous immunization of BALBc mice. After an intraperitoneal
booster, the mice were sacrificed and hybridomas were generated [22] using the P2-NS1-Ag4-1 myeloma fusion
partner [23]. Culture supernatants were tested for PLTP antibody by Western blotting, and the positive cultures
were cloned three times by limiting dilution. The mono- clonal hybridoma cells were passaged in Pristane-
primed BALBc mice for ascitic fluid production, and the monoclonal IgG antibody was isolated using
protein G-Sepharose CL 4B columns Pharmacia ac- cording to the manufacturer’s instructions. The IgG
was dialyzed against PBS at 4°C and stored in aliquots at − 80°C.
2
.
4
. The sandwich ELISA for PLTP mass determination
The monoclonal antibody JH59 was diluted in 0.1 M carbonate buffer, pH 9.6 to a final concentration of
2.50 mgml, and 200 ml of the solution was added to the wells of Nunc Cert. Maxisorb 96-well plates. The plates
were coated overnight at 4°C, and then washed six times with 350 ml PBS, 0.05 Tween 20 using the
Labsystems Helsinki, Finland Multiwash apparatus. Unspecific binding was blocked by incubating 300 ml of
PBS, 0.05 Tween 20, 1 bovine serum albumin BSA; Sigma A3803 in the wells for 1 h at 37°C, followed by
six washes with PBS, 0.05 Tween 20 see above. The secondary standard plasma of a normolipidemic male
subject and the plasma specimens to be analyzed were diluted 110, 130, and 190 in PBS, 0.1 Tween 20,
and duplicate samples of each dilution 200 ml were applied in the coated wells. The plates were then incu-
bated for 1 h at 37°C and washed six times as above. The detection antibody, polyclonal rabbit anti-PLTP
R176 IgG, was diluted with PBS, 0.05 Tween 20, 1 BSA to 10.85 mgml, and incubated in the wells 200
mlwell for 1 h at 37°C, followed by six washes with PBS, 0.05 Tween. The bound detection antibody was
visualized using HRP-coupled goat anti-rabbit IgG Bio-Rad 72734 diluted 110 000 in PBS, 0.05 Tween
20, 0.5 BSA, and incubated 200 mlwell for 1 h at 37°C. The plates were finally washed six times with
PBS, 0.05 Tween, and the color reaction with the substrate solution 10 mg o-phelylenediamine, Sigma
P8287, 10 ml H
2
O
2
per 25 ml of 25 mM citric acid, 50 mM Na
2
HPO
4
; 200 mlwell was developed at room temperature in the dark for 4 min, stopped by adding
50 ml of 3 M H
2
SO
4
per well, and the absorbances at 490 nm were recorded using a Wallac Victor 2, 1420
Multilabel Counter. For standardization, a freshly purified human plasma PLTP primary standard see
above was diluted in PBS, 0.1 Tween to obtain PLTP 30, 60, 81, 122, 162, 194, and 243 ng PLTPwell, and
analyzed in parallel with the secondary standard 200 ml applied per well. The secondary standard was stored in
small aliquots at − 80°C, and a fresh aliquot was thawed for each ELISA experiment. A secondary stan-
dard dilution series was present in duplicate on every single ELISA plate.
2
.
5
. Other methods Total cholesterol and HDL cholesterol after precipi-
tation of b-lipoproteins with dextran sulphate-magne- sium
chloride were
determined enzymatically
CHOD-PAP method
using the
reagents from
Boehringer Mannheim, Mannheim, Germany [24]. Tri- acylglycerol concentration was determined with the
fully enzymatic method of Wahlefeld [25]. Apolipo- proteins apo A-I, A-II and B were quantified by
immunoturbidometry [26]. LDL cholesterol was calcu- lated according to the formula of Friedewald [27].
Concentrations of LpA-I and LpA-IA-II particles were measured by rocket immunoelectrophoresis using
commercial reagents Sebia, Paris, France. In Western blot analysis, human plasma samples 1 ml of 1:10
diluted plasma per well were subjected to SDS – PAGE analysis on 12.5 homogeneous polyacrylamide gels
under reducing conditions, whereafter the proteins were transferred onto nitrocellulose sheets. After anti-PLTP
antibody treatment, the blots were visualized using the Enhanced
ChemiLuminescence detection
system Amersham.
2
.
6
. The study population The study subjects, n = 159, were from a large
Finnish cross-sectional study FINRISK [28]. They represented men and women in four age groups from
25 to 65 years in 10-year intervals. The participants of the FINRISK study were randomly selected from the
inhabitants of North Karelia, the Kuopio province, and Southwestern Finland, thus closely representing the
Finnish population. Among the study subjects, 86.2 were moderate drinkers, 8.8 drank so much they felt
intoxicated at least once per week and 5 did not use alcohol. Of the subjects, 51 were regular tobacco
users, 15 were occasional smokers, and 34 had never smoked. Among women, 12.3 used oral contra-
ceptives, 1.4 used intrauterine devices delivering hor- mones, 19.2 used estrogen for menopause symptoms,
and 1.4 were gravid. Diabetes type 1 or 2 had been diagnosed in 2.5 of the participants. Blood samples
from the study subjects were collected in non-fasting state. All participants have given informed consent and
the study has been approved by an ethics committee.
2
.
7
. Statistical analyses Statistical testing was performed using the Statistical
Package for Social Sciences SPSS version 7.5 SPSS, Chicago, USA. The dependence of serum PLTP mass
or phospholipid transfer activity on gender was tested by analysis of variance ANOVA. The correlation of
PLTP mass, phospholipid transfer activity and specific activity with age, body mass index BMI, serum
cholesterol, triacylglycerol, HDL cholesterol, apoB, apoA-I, apoA-II, apoA-I in LpA-I or LpA-IA-II
particles, and serum gamma glutamyl transferase S- GT was analyzed with unadjusted values.
3. Results
