Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA

Matti Jauhiainen,Vesa M Olkkonen,Minna Kärkkäinen,Christian Ehnholm,Sarah Siggins,Jari Metso,Esa Tahvanainen,Jukka Tenhunen

doi:10.1194/jlr.d300023-jlr200

Matti Jauhiainen, Vesa M Olkkonen + Show 6 more

Open Access

https://doi.org/10.1194/jlr.d300023-jlr200

Copy DOI

Abstract

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [high-activity PLTP (HA-PLTP)] and the other a low-activity (LA-PLTP) form. We present here a PLTP ELISA that allows not only for accurate measurement of PLTP concentration in plasma but also of the distribution of both LA- and HA-PLTP. To achieve similar immunoreactivity of the two PLTP forms, a denaturing sample pretreatment with 0.5% SDS was required. Distribution of LA- and HA-PLTP in plasma was assessed using size-exclusion chromatography, Heparin-Sepharose chromatography, anti-PLTP immunoaffinity chromatography, and dextran sulfate-CaCl2 precipitation. All four methods demonstrated that approximately 60% of plasma PLTP represents LA-PLTP and 40% represents HA-PLTP. According to the modified ELISA, the total serum PLTP concentration in a random Finnish population sample (n = 80) was 5.81 +/- 1.33 mg/l (mean +/- SD) (range, 2.78-10.06 mg/l) and the mean activity was 5.84 +/- 1.39 micromol/ml/h (range, 3.21-11.15 micromol/ml/h). To quantitate both forms of PLTP in sera from this sample, we combined dextran sulfate-CaCl2 precipitation with the modified PLTP ELISA. The HA-PLTP mass (mean, 1.87 +/- 0.85 mg/l) correlated significantly with serum PLTP activity, whereas that of LA-PLTP (mean, 3.94 +/- 1.4 mg/l) showed no correlation with phospholipid transfer activity.

Highlights

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [highactivity PLTP (HA-PLTP)] and the other a low-activity (LAPLTP) form
Assuming that the Western blot gives an accurate measure of PLTP mass, this result suggests that the immunoreactivity of HA-PLTP in the ELISA is underestimated compared with that of low-activity PLTP (LA-PLTP) and suggests that a denaturation step before antibody binding may improve the detectability of the protein, especially that of the active form
We report a novel, modified ELISA assay for PLTP mass determination

Summary

Introduction

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [highactivity PLTP (HA-PLTP)] and the other a low-activity (LAPLTP) form. According to the modified ELISA, the total serum PLTP concentration in a random Finnish population sample (n ‫ ؍‬80) was 5.81 ؎ 1.33 mg/l (mean ؎ SD) (range, 2.78–10.06 mg/l) and the mean activity was 5.84 ؎ 1.39 ␮mol/ml/h (range, 3.21–11.15 ␮mol/ml/h). To quantitate both forms of PLTP in sera from this sample, we combined dextran sulfate-CaCl2 precipitation with the modified PLTP ELISA. In vitro studies have identified a number of functions for phospholipid transfer protein (PLTP) in lipoprotein metabolism [1, 2]. PLTP has been shown to play a role in the secretion of apoB-containing lipoproteins [15, 17, 18]

Methods

Results

Conclusion