A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method
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
References (40)
- et al.
Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins
Cell
(1989) - et al.
Lysophosphatidic acid induction of transforming growth factors alpha and beta: modulation of proliferation and differentiation in cultured human keratinocytes and mouse skin
Exp. Cell Res.
(1995) - et al.
Induction of in vitro tumor cell invasion of cellular monolayers by lysophosphatidic acid or phospholipase D
Biochem. Biophys. Res. Commun.
(1993) - et al.
Neurotransmitter release from lysophosphatidic acid stimulated PC12 cells: involvement of lysophosphatidic acid receptors
Biochem. Biophys. Res. Commun.
(1993) - et al.
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors
Cell
(1992) - et al.
Phosphatidic acid and lysophosphatidic acid induce haptotactic migration of human monocytes
J. Biol. Chem.
(1995) - et al.
Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid
J. Biol. Chem.
(1998) - et al.
Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid
J. Biol. Chem.
(1999) - et al.
Lysophosphatidic acid, a growth factor-like lipid, in the saliva
J. Lipid Res.
(2002) - et al.
Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase
J. Biol. Chem.
(2002)
Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein
J. Biol. Chem.
A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification
J. Lipid Res.
Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry
Anal. Biochem.
Serum lysophosphatidic acid is produced through diverse phospholipase pathways
J. Biol. Chem.
Optimizing enzymatic cycling assays: spectrophotometric determination of low levels of pyruvate and l-lactate
Anal. Biochem.
A continuous spectrophotometric method based on enzymatic cycling for determining l-glutamate
Anal. Biochem.
An enzymatic assay for lysophosphatidylcholine concentration in human serum and plasma
Clin. Biochem.
Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 phenochromocytoma cells
J. Biol. Chem.
Mitogenic action of lysophosphatidic acid and phosphatidic acid on fibroblasts. Dependence on acyl-chain length and inhibition by suramin
Biochem. J.
Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta
Am. J. Physiol.
Cited by (55)
Preclinical detection of lysophosphatidic acid: A new window for ovarian cancer diagnostics
2022, TalantaCitation Excerpt :Although this method required prior lipid extraction through the Bligh and Dyer method, excellent linearity could be obtained in a very low concentration range of 0.1–10 nM which clearly illustrated the ultra-high sensitivity of the method. In a subsequent work, Kishimoto et al. [114] reported an enzymatic cycling based colorimetric assay for detecting LPA in plasma with the characteristics of simple operation and high sensitivity. The dihydroxyacetone phosphate generated by glycerol-3-phosphate in the enzymatic reaction could resynthesize glycerol-3-phosphate in the presence of NADH, whereby the enzymatic circulation could be guaranteed.
Different origins of lysophospholipid mediators between coronary and peripheral arteries in acute coronary syndrome
2017, Journal of Lipid ResearchChallenges in accurate quantitation of lysophosphatidic acids in human biofluids
2014, Journal of Lipid ResearchMethods for quantifying lysophosphatidic acid in body fluids: A review
2014, Analytical BiochemistryCitation Excerpt :It is known that acyl chains located at the sn-2 position of lysophospholipids are unstable, spontaneously migrating to the sn-1 position [42,43]. In addition, it is important to recognize that total serum LPA levels increase dramatically during incubation [44,45]. Therefore, appropriate sample collection, preparation, and storage are important for obtaining reliable measurements.
Increased lysophosphatidic acid levels in culprit coronary arteries ofpatients with acute coronary syndrome
2013, AtherosclerosisCitation Excerpt :We measure plasma levels of LPA and LPC, and serum levels of autotaxin (ATX), soluble CD40 ligand (sCD40L), hs-CRP, and Lp-PLA2. Plasma LPA and LPC concentrations were enzymatically determined as described [14–16]. In brief, LPA was hydrolyzed with lysophospholipase to glycerol 3-phosphate, followed by enzymatic cycling using glycerol 3-phosphate oxidase and glycerol 3-phosphate dehydrogenase.
Autotaxin in liver fibrosis
2012, Clinica Chimica ActaCitation Excerpt :Lysophosphatidic acid (1- or 2-acyl-lysophosphatidic acid; LPA) elicits a wide variety of biological responses including cell migration, neurogenesis, angiogenesis, smooth-muscle contraction, platelet aggregation, and wound healing [1,2]. Because it is present in blood at a concentration of ~ 0.1 μmol/L [3], ie, close to the concentration that exerts various effects on cells in vitro [1,2], LPA is regarded as a circulatory paracrine mediator. A major portion of LPA in blood is generated by lysophospholipase D (lysoPLD) from lysophospholipids, mainly lysophosphatidylcholine [4].