Elsevier

Clinica Chimica Acta

Volume 333, Issue 1, 1 July 2003, Pages 59-67
Clinica Chimica Acta

A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method

https://doi.org/10.1016/S0009-8981(03)00165-7Get rights and content

Abstract

Background: Several methods for measuring concentrations of lysophosphatidic acid (LPA), a lipid mediator, have been reported to date. However, these methods are not routinely used because most of them require specialized instrument and a complicated protocol. Methods: We developed a novel LPA assay using enzymatic cycling. LPA in a sample is hydrolyzed with lysophospholipase to glycerol-3-phosphate, followed by enzymatic cycling using glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase. Amplified concentrations of hydrogen peroxides, a product of the enzymatic cycling, are then colorimetrically measured. Results: This method was specific for LPA, being insensitive to the presence of phosphatidic acid or lysophosphatidylcholine. The within-run and between-run CVs were 1.31–1.32% and 0.73–1.03%, respectively. The recoveries of exogenous LPA added to plasma were 100.3–101.6%. In males, LPA concentrations (mean±S.D.) of human serum and EDTA-plasma were 0.41±0.14 and 0.08±0.02 μmol/l, respectively. In females, they were 0.41±0.12 and 0.09±0.02 μmol/l, respectively. Conclusions: This novel colorimetric assay for determination of LPA using enzymatic cycling is simple and highly sensitive. It can be used with an automatic analyzer. It may also be useful for further studies of the biological functions of LPA as well as clinical applications in various disorders.

Introduction

Lysophosphatidic acid (1-acyl-2-hydroxy-sn-glycerol-3-phosphate or 1-hydroxy-2-acyl-sn-glycerol-3-phosphate; LPA), the simplest phospholipid, is involved in various biological responses. LPA has been shown to elicit cell proliferation [1], [2], [3], [4], platelet aggregation [5], smooth muscle contraction [6], tumor cell invasion [7], neurotransmitter release [8], [9], chemotaxis [10], stress fiber formation [11], [12], dedifferentiation of vascular smooth muscle cells [13], cellular motility [14], matrix metalloproteinase activation [15] and activation of peroxisome proliferator-activated receptor gamma known as an intracellular receptor [16]. These observations suggest that LPA plays an important physiological role in multiple cellular processes. LPA seems to evoke these multiple reactions through LPA receptors, which are G protein-coupled receptors belonging to the endothelial cell differentiation (EDG) family [17], [18], [19].

LPA has been found in several biological fluids including serum [20], plasma [21], ascites in patients suffering from ovarian cancer [22], aqueous humor and lacrimal gland fluid of the rabbit eye [23], follicular fluid [24] and saliva [25]. It has also been reported that the plasma LPA concentrations of patients with gynecologic cancers such as ovarian cancer are significantly higher compared with that of controls [21]. In addition to ovarian cancer, increased serum LPA concentrations were found in patients with multiple myeloma [26].

It has been reported that LPA in blood is produced by lysophospholipase D [27]. Recently lysophospholipase D was purified from fetal bovine serum [28] and human plasma [29]. Interestingly and surprisingly, lysophospholipase D was identified with autotaxin, which is known as a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants [30].

Various methods for measurement of LPA concentrations have been reported to date. For example, there is the analysis of fatty acid methyl esters by gas chromatography following lipid extraction and thin layer chromatography [21], [26], [27], a radioenzymatic assay [31], a stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry assay [32] and an enzyme-linked fluorometric assay [33]. However, most of these LPA assay protocols are not routinely used for diagnosis because they require specialized instrument and a complicated protocol such as lipid extraction and LPA isolation from other lipids in plasma.

We developed a novel colorimetric assay for the measurement of LPA using enzymatic cycling. Enzymatic cycling is a method for amplifying the sensitivity of enzymatic assays and leads to successful determination of very low LPA concentrations in blood. Some assays using enzymatic cycling have been examined for determination of different substances [34], [35], [36]. In this paper, we report a simple, specific and sensitive method to measure LPA concentrations in plasma.

Section snippets

Reagents

Lysophospholipase (EC 3.1.1.5), glycerol-3-phosphate oxidase (G3PO; EC 1.1.3.21) and 3 α-hydroxysteroid dehydrogenase (HSD; EC 1.1.1.50) were from Asahi Kasei (Tokyo, Japan). Peroxidase (EC 1.11.1.7) was from Toyobo (Osaka, Japan). Glycerol-3-phosphate dehydrogenase (G3PDH; EC 1.1.1.8) was from Roche Diagnostics (Mannheim, Germany). N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dihydrate (TOOS) was from Dojindo Laboratories (Kumamoto, Japan), 4-aminoantipyrine from Kishida

Standard curve

LPA calibration fluids having six different LPA concentrations (0, 0.625, 1.25, 2.5, 5.0, 10 μmol/l) were prepared by serially diluting 10 μmol/l 1-oleoyl-LPA with saline containing 0.1% TritonX-100. The absorbance changes per minute caused by these calibration fluids yielded a slightly non-linear standard curve in relation between LPA concentration and absorbance/min (Fig. 2).

Time course

The time courses of the reactions in calibration fluids (Fig. 3A) and in two different human fresh frozen plasma (Fig.

Discussion

In order to more easily and more sensitively determine low concentrations of LPA in plasma, we developed a novel colorimetric assay for LPA utilizing enzymatic cycling. In this LPA assay, hydrogen peroxides amplified by enzymatic cycling reactions are colorimetrically measured with TOOS and 4-aminoantipyrine in the presence of peroxidase. The background assay, which measures pre-existing metabolic intermediates of the LPA assay in plasma, was carried out simultaneously. Its procedure allows

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