A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification

Abstract

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [14C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [14C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanol-extracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.—Saulnier-Blache, J. S., A. Girard, M-F. Simon, M. Lafontan, and P. Valet. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification. J. Lipid Res. 2000. 41: 1947–1951. The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [14C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [14C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanol-extracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.—Saulnier-Blache, J. S., A. Girard, M-F. Simon, M. Lafontan, and P. Valet. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification. J. Lipid Res. 2000. 41: 1947–1951. Lysophosphatidic acid (LPA; 1-acyl-2-hydroxy-sn-glycero-3-phosphate) is a key intermediate in glycerolipid synthesis, and a bioactive phospholipid (1Gaits F. Fourcade O. Le Balle F. Gueguen G. Gaige B. Gassama-Diagne A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis.FEBS Lett. 1997; 410: 54-58Google Scholar, 2Jalink K. Hordijk P.L. Moolenaar W.H. Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator.Biochim. Biophys. Acta. 1994; 1198: 185-196Google Scholar). It controls a wide variety of cellular responses (mitogenesis, cytoskeletal rearrangements, cell adhesion, ion transport, and apoptosis) through the activation of specific G protein-coupled receptors (3Goetzl E. An S. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate.FASEB J. 1998; 12: 1589-1598Google Scholar, 4Chun J. Contos J.J. Munroe D. A growing family of receptor genes for lysophosphatidic acid (LPA) and other lysophospholipids (LPs).Cell Biochem. Biophys. 1999; 30: 213-242Google Scholar). LPA has been detected in various biological fluids including blood (5Xu Y. Shen Z. Wiper D.W. Wu M. Morton R. Elson P. Kennedy A. Belinson J. Markman M. Casey G. Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers.J. Am. Med. Assoc. 1998; 280: 719-723Google Scholar, 6Tigyi G. Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochomocytoma cells.J. Biol. Chem. 1992; 267: 21360-21367Google Scholar, 7Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets.Biochem. J. 1993; 291: 677-680Google Scholar, 8Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta.Am. J. Physiol. 1994; 267: C204-C210Google Scholar, 9Sasagawa T. Suzuki K. Shiota T. Kondo T. Okita M. The significance of plasma lysophospholipids in patients with renal failure on hemodialysis.J. Nutr. Sci. Vitaminol. 1998; 44: 809-818Google Scholar), aqueous humors (10Liliom K. Guan Z. Tseng J-L. Desiderio D.M. Tigyi G. Watsky M.A. Growth factor-like phospholipids generated after corneal injury.Am. J. Physiol. 1998; 274: C1065-C1074Google Scholar), and adipose tissue microdialysates (11Valet P. Pagès C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes.J. Clin. Invest. 1998; 101: 1431-1438Google Scholar). Abnormally high concentrations of LPA have been found in ascites and plasma from patients with ovarian cancer (12Xu Y. Gaudette D. Boynton J. Frankel A. Fang X. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Holub B. Mills G. Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients.Clin. Cancer Res. 1995; 1: 1223-1232Google Scholar, 13Westermann A.M. Havik E. Postma F.R. Beijnen J.H. Dalesio O. Moolenar M.W. Rodenhuis S. Malignant effusions contain lysophosphatidic acid (LPA)-like activity.Ann. Oncol. 1998; 9: 437-442Google Scholar). Several cell types have been shown to produce LPA in vitro: platelets (14Mauco G. Chap H. Simon M-F. Douste-Blazy L. Phosphatidic and lysophosphatidic acid production in phospholipase C- and thrombin-treated platelets. Possible involvement of a platelet lipase.Biochimie. 1978; 60: 653-661Google Scholar, 15Gerrard J.M. Robinson P. Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin.Biochim. Biophys. Acta. 1989; 1001: 282-285Google Scholar), adipocytes (11Valet P. Pagès C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes.J. Clin. Invest. 1998; 101: 1431-1438Google Scholar), and ovarian cancer cells (16Shen Z. Belinson J. Morton R. Yan X. Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells.Gynecol. Oncol. 1998; 71: 364-368Google Scholar). LPA has also been shown to be produced from oxidized low density lipoprotein (LDL) (17Siess W. Zangl K. Essler M. Bauer M. Brandl R. Corrinth C. Bittman R. Tigyi G. Aepfelbacher M. Lysophosphatidic acid mediates the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions.Proc. Natl. Acad. Sci. USA. 1999; 96: 6931-6936Google Scholar). Several possible metabolic pathways could be involved in cellular production of LPA: phospholipase A2-dependent deacylation of phosphatidic acid (PA) (18Fourcade O. Simon M.F. Viodé C. Rugani N. Leballe F. Ragab A. Fournié B. Sarda L. Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.Cell. 1995; 80: 919-927Google Scholar), lysophospholipase D-dependent hydrolysis of lysophosphatidylcholine (19Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma.Biochim. Biophys. Acta. 1986; 875: 31-38Google Scholar, 20Tokomura A. Miyake M. Nishioka Y. Yamano S. Aono T. Fukuzawa K. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients.Biol. Reprod. 1999; 61: 195-199Google Scholar), acyltransferase-dependent acylation of monoacylglycerol phosphate (21Haldar D. Vancura A. Glycerophosphate acyltransferase from liver.Methods Enzymol. 1992; 209: 64-72Google Scholar), or acyldihydroxyacetone phosphate reductase-dependent reduction of acyldihydroxyacetone phosphate (22Dunlop M.E. Larkins R.G. Pancreatic islets synthesize phospholipids de novo from glucose via acyl-dihydroxyacetone phosphate.Biochem. Biophys. Res. Commun. 1985; 132: 467-473Google Scholar). To understand the precise role of LPA in the development of particular pathologies such as cancer or obesity, and to clarify the metabolic origin of LPA produced by cells, precise and sensitive detection of LPA is necessary. In the present work, a recombinant LPA acyltransferase (LPAAT) was used to develop a simple and highly sensitive radioenzymatic assay to quantify LPA in various biological fluids. The rat 1-acyl-sn-glycerol-3-phosphate acyltransferase cDNA encoding an enzyme behaving as an LPAAT was previously cloned in bacterial expression vector pTrcHis (23Kume K. Shimizu T. cDNA cloning and expression of murine 1-acyl-sn-glycerol-3-phosphate acyltransferase.Biochem. Biophys. Res. Commun. 1997; 237: 663-666Google Scholar). Fifty microliters of DH5α-competent cell suspension (Life Technologies, Gaithersburg, MD) was transformed with 0.5 μg of pTrcHis-AGPAT plasmid (generous gift from K. Kume, Department of Biochemistry and Molecular Biology, University of Tokyo, Japan) and grown in Luria broth (LB) medium containing ampicillin (50 μg/ml). When the A600 reached approximately 0.6, bacteria were cultured for 3 h in the presence of 1 mM isopropyl-β-d-thiogalactopyranoside. Bacteria were collected by centrifugation at 3,000 g for 10 min, and the pellet was disrupted by sonication in 0.2 M Tris-HCl (pH 7.4). After centrifugation at 10,000 g for 20 min, the supernatant was recovered and further centrifuged at 100,000 g (microsomes) for 90 min. The yield of a typical preparation is 6 to 7 mg of microsomal protein per liter of culture. The resulting pellet was resuspended in the same buffer to a final protein concentration of 1 μg/μl and was used for radioenzymatic detection of LPA. Kinetic analysis showed that the activity of transformation of LPA into PA of a typical microsome preparation varied from 0.8 to 1 nmol/min per mg of protein. Na+ salt 1-oleoyl-sn-glycero-3-phosphate (LPA) (from Sigma, St. Louis, MO) was solubilized in phosphate-buffered saline (PBS) containing 1% lipid-free albumin (from ICN, Costa Mesa, CA). 1-O-Hexadecyl-sn-glycero-3-phosphate (alkyl-LPA) was obtained from 1-O-hexadecyl-2-acetyl-sn-glycerol-3-phosphocholine (platelet-activating factor; PAF) (from Sigma). PAF was dispersed by sonication in a 50 mM Tris buffer (pH 7.4) containing 5 mM Ca2+, and incubated first with pig pancreas phospholipase A2 (from Sigma) for 2 h followed by a 30-min treatment with Streptomyces chromofuscus phospholipase D (from Sigma). Lipids were extracted by the Bligh and Dyer procedure (24Bligh E. Dyer W. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) under acid conditions. After evaporation alkyl-LPA was solubilized in ethanol. The real concentration of the stock solutions of LPA and alkyl-LPA was determined by phosphorus measurement (25Böttcher C. Gent C.V. Priest C. A rapid and sensitive submicrophosphorus determination.Anal. Chim. Acta. 1961; 24: 203-204Google Scholar). 1-Oleoyl-LPA or lipids contained in serum or plasma were extracted with 1 volume of 1-butanol. After vigorous shaking and centrifugation (5 min at 3,000 g), the upper butanol phase was collected and evaporated under nitrogen at 50°C. This procedure allowed 85% to 90% extraction of LPA. Dry lipid extract was resuspended in 200 μl of reaction medium (1 μl of [14C]oleoyl-CoA [specific radioactivity (RAS), 55 mCi/mmol; New England Nuclear, Boston, MA], 20 μl of 200 mM Tris (pH 7.5), 10 μl of recombinant LPAAT, 8 μl of 500 μM sodium orthovanadate, and 161 μl of H2O containing Tween 20 at 1 mg/ml) and incubated for 120 min at 20°C. The mixture was vortexed every 15 min. The reaction was stopped by addition of 400 μl of CHCl3–methanol–12 M HCl 40:40:0.26 followed by vigorous shaking and a 10-min centrifugation at 3,000 g. The lower CHCl3 phase was evaporated under nitrogen, resuspended in 20 μl of CHCl3–methanol 1:1, and spotted on a silica gel 60 thin-layer chromatography (TLC) glass plate (Merck, Rahway, NJ), and then separated with CHCl3–methanol–NH4OH–H2O 65:25:0.9:3 as a solvent. Alternatively, a two-dimensional TLC using CHCl3–methanol–NH4OH–H2O 65:25:0.9:3 as the solvent of the first migration, and CHCl3–methanol–acetic acid–H2O 40:20:5:0.5 as the solvent of the second migration, was performed. The plate was autoradiographed overnight to localize 14C-labeled lipids. Identification of radioactive spots was performed by comigration with cold lipids visualized under iodine vapors. [14C]PA spots were then scraped and counted with 3 ml of scintillation cocktail. The following formula was used to convert radioactivity to picomoles: dpm = 1/(2.22 × RAS) pmol, RAS being the specific radioactivity of [14C]oleoyl-CoA. Fetal calf sera were from BioWhittaker Europe (Verviers, Belgium; lot 6SB0011), GIBCO Life Technologies (Rockville, MD; lot 4001585K), and Boehringer Mannheim France (Meylan, France; lot 210463). Donor calf sera were from BioWhittaker Europe (lot 6M1955) and GIBCO Life Technologies (lot 3016082D). Human plasma was collected in EDTA with the consent of the subjects and was approved by the Ethics Committee of the hospital. Mouse plasma was collected from FVB mice after carotid section. Collection was performed in tubes containing 50 μl of a 2% solution of EDTA, disodium salt. The tubes were centrifuged for 5 min at 1,500 g and LPA was measured in the platelet-poor plasma. Animals were handled in accordance with the principles and guidelines established by the National Institutes of Medical Research (INSERM). The ability of LPAAT to acylate LPA to form PA was tested after semipurification of the enzyme from rat LPAAT cDNA-transformed Escherichia coli. For that, 1-oleoyl-LPA or butanol-extracted lipids from serum [known to contain a significant amount of LPA (6Tigyi G. Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochomocytoma cells.J. Biol. Chem. 1992; 267: 21360-21367Google Scholar, 7Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets.Biochem. J. 1993; 291: 677-680Google Scholar, 8Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta.Am. J. Physiol. 1994; 267: C204-C210Google Scholar, 9Sasagawa T. Suzuki K. Shiota T. Kondo T. Okita M. The significance of plasma lysophospholipids in patients with renal failure on hemodialysis.J. Nutr. Sci. Vitaminol. 1998; 44: 809-818Google Scholar)] were incubated in the presence of LPAAT-transformed E. coli microsomes and [14C]oleoyl-CoA. To identify the products of the reaction, they were separated by two-dimensional TLC (Fig. 1). In the absence of LPA (Fig. 1, cont), no [14C]PA was detected. A radioactive was found at the (Fig. 1, It to of [14C]oleoyl-CoA extracted with 1-butanol. spots (Fig. 1, upper were also The lower with cold acid was as was also in the absence of microsomes from a of [14C]oleoyl-CoA. The upper with and and was as 14C-labeled lipids resulting from the presence of in E. coli and with LPAAT in In the presence of LPA or from a with cold PA was [14C]PA was as the of the of into LPA. This the presence of LPAAT in E. coli A but was also 2% of [14C]PA was further the of LPA species found in biological fluids and are a are alkyl-LPA T. S. S. K. Y. A. D. of lysophosphatidic acid and in rat and of biological Biophys. Acta. 1999; Scholar). acylation of alkyl-LPA was was from PAF and and to LPAAT and [14C]oleoyl-CoA. shown in or alkyl-LPA to the of [14C]PA was from PAF or The of the radioenzymatic assay was then For that, of 1-oleoyl-LPA were incubated in the presence of LPAAT and [14C]oleoyl-CoA. This to the of an amount of [14C]PA (Fig. of the radioactivity into that LPA acylation into PA was complete and linear to from 0 to 200 pmol, with a detection of 0.2 pmol (Fig. The radioenzymatic assay was used to quantify LPA in bovine sera of various shown in 1, the concentration of LPA measured in 20 μl of serum varied from to The concentration of LPA in calf sera was to the concentration of LPA found in calf present at lower concentrations in significant concentrations of LPA (from to were also found in human and mouse platelet-poor plasma of LPA in bovine of present in μl of or calf serum were extracted with and incubated in the presence of LPAAT and [14C]oleoyl-CoA as in and The products of the reaction were separated by TLC and autoradiographed as in and of [14C]PA was scraped and The radioactivity was into and the concentration of LPA was to of in a of LPA in human and mouse of 8 50 present in 200 μl of human or mouse platelet-poor plasma were extracted with and incubated in the presence of LPAAT and [14C]oleoyl-CoA as in and The products of the reaction were separated by TLC and autoradiographed as in and of [14C]PA was scraped and The radioactivity was into and the concentration of LPA was to of are in in a Lipids present in μl of or calf serum were extracted with and incubated in the presence of LPAAT and [14C]oleoyl-CoA as in and The products of the reaction were separated by TLC and autoradiographed as in and of [14C]PA was scraped and The radioactivity was into and the concentration of LPA was to of Lipids present in 200 μl of human or mouse platelet-poor plasma were extracted with and incubated in the presence of LPAAT and [14C]oleoyl-CoA as in and The products of the reaction were separated by TLC and autoradiographed as in and of [14C]PA was scraped and The radioactivity was into and the concentration of LPA was to of are in Several have previously been used to LPA. chromatography of acids from LPA J.M. Robinson P. Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin.Biochim. Biophys. Acta. 1989; 1001: 282-285Google Scholar). This is an assay of the acid of LPA. method is not sensitive and high of LPA. In not of the presence of For that is to hydrolysis and to T. S. S. K. Y. A. D. of lysophosphatidic acid and in rat and of biological Biophys. Acta. 1999; Scholar). The presence of LPA in a biological also be by using This of assay of the of an on a cellular to be by such as platelet (17Siess W. Zangl K. Essler M. Bauer M. Brandl R. Corrinth C. Bittman R. Tigyi G. Aepfelbacher M. Lysophosphatidic acid mediates the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions.Proc. Natl. Acad. Sci. USA. 1999; 96: 6931-6936Google Scholar), in Xenopus oocytes (10Liliom K. Guan Z. Tseng J-L. Desiderio D.M. Tigyi G. Watsky M.A. Growth factor-like phospholipids generated after corneal injury.Am. J. Physiol. 1998; 274: C1065-C1074Google Scholar), or cell (11Valet P. Pagès C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes.J. Clin. Invest. 1998; 101: 1431-1438Google Scholar). bioactive phospholipids other LPA are also to biological be specific for LPA quantification. LPA also be detected after cellular phospholipid with and of by TLC T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets.Biochem. J. 1993; 291: 677-680Google Scholar, P. Pagès C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes.J. Clin. Invest. 1998; 101: 1431-1438Google Scholar, O. Simon M.F. Viodé C. Rugani N. Leballe F. Ragab A. Fournié B. Sarda L. Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.Cell. 1995; 80: 919-927Google Scholar). method is to cultured cells, and is not LPAAT, also as 1-acyl-sn-glycerol-3-phosphate acyltransferase catalyzes LPA into a key in glycerolipid LPAAT has been to be highly specific for LPA and not by LPA acid S. K. Y. S. from rat acyltransferase and Enzymol. 71: Scholar). the high of LPAAT enzyme an in the development of a assay for LPA quantification. using a LPAAT produced in E. coli (23Kume K. Shimizu T. cDNA cloning and expression of murine 1-acyl-sn-glycerol-3-phosphate acyltransferase.Biochem. Biophys. Res. Commun. 1997; 237: 663-666Google Scholar) have a simple, and highly sensitive radioenzymatic assay to quantify LPA in various biological fluids. using assay is possible to quantify LPA in butanol-extracted lipids and TLC of LPA. It is also that assay of and present at a lower concentration alkyl-LPA has been to be in platelet M.F. Chap H. Douste-Blazy L. Human platelet by acid and a of phospholipid Biophys. Res. Commun. Scholar). assay not and the amount of alkyl-LPA present in a biological be For that the biological be with a such as phospholipase to LPA to radioenzymatic assay is also highly It is possible to 0.5 pmol of LPA. This high of LPA concentration from low of biological It for possible to quantify LPA in 200 μl of plasma or μl of are with blood in human and This high allowed to quantify as low a concentration as LPA in human and mouse plasma. It is to that, by using that the concentration of LPA in human plasma was (5Xu Y. Shen Z. Wiper D.W. Wu M. Morton R. Elson P. Kennedy A. Belinson J. Markman M. Casey G. Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers.J. Am. Med. Assoc. 1998; 280: 719-723Google Scholar). the method is the concentration determined in bovine sera are in with previously (6Tigyi G. Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochomocytoma cells.J. Biol. Chem. 1992; 267: 21360-21367Google Scholar, 7Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets.Biochem. J. 1993; 291: 677-680Google Scholar, 8Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta.Am. J. Physiol. 1994; 267: C204-C210Google Scholar, 9Sasagawa T. Suzuki K. Shiota T. Kondo T. Okita M. The significance of plasma lysophospholipids in patients with renal failure on hemodialysis.J. Nutr. Sci. Vitaminol. 1998; 44: 809-818Google Scholar). is that calf sera contain a concentration of LPA calf the precise of to be to be into when cell In radioenzymatic assay represents a and highly sensitive method for LPA quantification. of and of method could be in or in or LPA. of for with pTrcHis-AGPAT also for and for This work was by from the National de de the Cancer the de and the phosphate acyltransferase chromatography Luria broth low density lipoprotein lysophosphatidic acid, 1-oleoyl-sn-glycero-3-phosphate lysophosphatidic acid acyltransferase phosphatidic acid 1-O-hexadecyl-2-acetyl-sn-glycerol-3-phosphocholine phosphate-buffered saline specific