Atherosclerosis 151 2000 451 – 461
Quantification of human plasma phospholipid transfer protein PLTP: relationship between PLTP mass and phospholipid
transfer activity
Jarkko Huuskonen, Minna Ekstro¨m, Esa Tahvanainen, Anu Vainio, Jari Metso, Pirkko Pussinen, Christian Ehnholm, Vesa M. Olkkonen, Matti Jauhiainen
Department of Biochemistry, National Public Health Institute, Mannerheimintie
166
, FIN-
00300
Helsinki, Finland Received 4 June 1999; received in revised form 20 September 1999; accepted 8 October 1999
Abstract
A sensitive sandwich-type enzyme-linked immunosorbent assay ELISA for human plasma phospholipid transfer protein PLTP has been developed using a monoclonal capture antibody and a polyclonal detection antibody. The ELISA allows for the
accurate quantification of PLTP in the range of 25 – 250 ng PLTPassay. Using the ELISA, the mean plasma PLTP concentration in a Finnish population sample n = 159 was determined to be 15.6 9 5.1 mgl, the values ranging from 2.30 to 33.4 mgl. PLTP
mass correlated positively with HDL-cholesterol r = 0.36, P B 0.001, apoA-I r = 0.37, P B 0.001, apoA-II r = 0.20, P B 0.05, LpA-I r = 0.26, P = 0.001 and LpA-IA-II particles r = 0.34, P B 0.001, and negatively with body mass index BMI
r = − 0.28, P B 0.001 and serum triacylglycerol TG concentration r = − 0.34, P B 0.001. PLTP mass did not correlate with phospholipid transfer activity as measured with a radiometric assay. The specific activity of PLTP, i.e. phospholipid transfer
activity divided by PLTP mass, correlated positively with plasma TG concentration r = 0.568, P B 0.001, BMI r = 0.45, P B 0.001, apoB r = 0.45, P B 0.001, total cholesterol r = 0.42, P B 0.001, LDL-cholesterol r = 0.34, P B 0.001 and age
r = 0.36, P B 0.001, and negatively with HDL-cholesterol r = − 0.33, P B 0.001, LpA-I r = − 0.21, P B 0.01 as well as LpA-IA-II particles r = − 0.32, P B 0.001. When both PLTP mass and phospholipid transfer activity were adjusted for plasma
TG concentration, a significant positive correlation was revealed partial correlation, r = 0.31, P B 0.001. The results suggest that PLTP mass and phospholipid transfer activity are strongly modulated by plasma lipoprotein composition: PLTP mass correlates
positively with parameters reflecting plasma high density lipoprotein HDL levels, but the protein appears to be most active in subjects displaying high TG concentration. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
High density lipoprotein metabolism; Plasma lipids; Specific activity; Triacylglycerol www.elsevier.comlocateatherosclerosis
1. Introduction
The observation that high density lipoproteins HDL have powerful antiatherogenic properties has
stimulated research into this lipoprotein fraction [1]. The mechanism of the protection is unknown but may
be due to the involvement of HDL in the process of reverse cholesterol transport RCT, whereby choles-
terol is transported from extrahepatic tissues to the liver for excretion from the body. The circulating HDL
are heterogeneous, comprising a number of subpopula- tions of particles of distinct size, charge, composition
and function. There is evidence that some HDL sub- populations are better than others in protecting against
atherosclerosis [1]. HDL are continuously remodelled in plasma through the action of enzymes and lipid transfer
proteins [2].
The human plasma phospholipid transfer protein PLTP plays an important role in the regulation of
plasma HDL levels and governs the distribution of
Abbre6iations
:
BMI, body mass index; CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; HL, hepatic lipase;
mAb, monoclonal antibody; PL, phospholipid; PLTP, phospholipid transfer protein; SDS – PAGE, sodium dodecyl sulfate polyacrylamide
gel electrophoresis; TG, triacylglycerol. Corresponding author. Tel.: + 358-9-47448467; fax: + 358-9-
47448281. E-mail address
:
matti.jauhiainenktl.fi M. Jauhiainen 0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 1 5 0 9 9 0 0 4 2 9 - 3
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
