HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: D400010-JLR200v1 45/11/2145    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tanaka, T. Articles by Satouchi, K. Search for Related Content PubMed PubMed Citation Articles by Tanaka, T. Articles by Satouchi, K. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 45, 2145-2150, November 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate-capture molecule

Tamotsu Tanaka^1,*, Hideki Tsutsui^*, Kaoru Hirano^*, Tohru Koike, Akira Tokumura and Kiyoshi Satouchi^*

^* Department of Applied Biological Science, Fukuyama University, Higashimura, Fukuyama, 729-0292, Japan
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8551, Japan
Faculty of Pharmaceutical Sciences, University of Tokushima, 1-78-1 Shomachi, Tokushima, 770-8505, Japan

Published, JLR Papers in Press, August 16, 2004. DOI 10.1194/jlr.D400010-JLR200

¹ To whom correspondence should be addressed. e-mail: tamot{at}fubac.fukuyama-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in wound healing, embryonic development, and progression of cancer. Here, we report a procedure for the quantification of LPA by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The method is based on a characteristic mass shift with total charge change (from –2 to +1) of the phosphate species due to 1:1 complexation of LPA^2– with a dinuclear zinc (II) complex {1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex; Zn₂L³⁺} at physiological pH. The monocationic complex [LPA^2–-Zn₂L³⁺]⁺ was detected in the positive mode, in which no other signal of cation adducts of LPA^2– was observed. The detection limit of 18:1 LPA by this method was 0.1 pmol on a sample plate. The intensity ratio of [LPA^2–-Zn₂L³⁺]⁺ against an internal standard [17:0 LPA^2–-Zn₂L³⁺]⁺ increased linearly with their molar ratio. Based on the relative intensities of complex ions, we determined the amounts of LPA homologs in an egg white by this method; the results obtained were in good agreement with those by gas liquid chromatography.

This sensitive and convenient procedure for LPA-specific detection is useful for the quantification of LPA homologs occurring in biological materials.

Abbreviations: DHB, 3,5-dihydroxybenzoic acid; ESI, electrospray ionization; LPA, lysophosphatidic acid; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PC, phosphatidylcholine; THAP, 2,4,6-trihydroxy-acetophenone; Zn₂L³⁺, 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex

Supplementary key words egg white • matrix-assisted laser desorption/ionization time-of-flight mass spectrometry • serum

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Lysophosphatidic acid (LPA) is a lipid mediator that induces diverse biological responses in vitro and in vivo (1, 2). It induces platelet aggregation (3, 4), contraction of smooth muscle cells (5), and proliferation of cells (6, 7). LPA has been shown to be present in several biological fluids, such as fetal calf serum (7), rat plasma (8, 9), human plasma (10–12), and human saliva (13), at significant concentrations. Because of its growth factor-like activity, LPA is considered to play a role in the wound-healing process (13, 14). Furthermore, evidence is accumulating that LPA present in human follicular fluid (15) or hen egg white (16) plays an essential role in embryonic development or egg transport (17). Also of importance are findings that LPA accumulates at high concentrations in plasma from patients with ovarian cancer (10, 11) and that LPA in ascitic fluid from patients with ovarian cancer may act as a mitogen to ovarian carcinoma cells (18, 19). These results suggest that levels of LPA in the body fluids are of clinical importance as a potential biomarker for ovarian cancer progression (10, 11).

Usually, LPA in body fluids consists of several molecular species with different acyl (or alkyl or alkenyl) chains (1, 2). It has been reported that one LPA receptor, LPA₃, discriminates differences in the chain length and number of cis double bonds in the acyl residue of LPA as well as the type of linkage and the position of the aliphatic chain at the glycerol backbone of LPA (20). Accordingly, the potency of physiological and pathophysiological responses induced by LPA (e.g., wound-healing activity, carcinoma metastasis activity) is dependent not only on the concentration of LPA but also on the molecular species composition of LPA in the body fluids (21). To this end, a convenient and highly specific method for the quantification of molecular species of LPA would be helpful for better understanding of the physiological and pathophysiological roles of LPA and its biosynthetic route.

Several methods have been developed for the quantification of LPA (22–27). For the molecular species analysis of LPA, mass spectrometric analyses have been widely performed: GC-MS (electron impact ionization) (13, 24), fast atom bombardment MS (21), electrospray ionization (ESI) MS (11, 12, 26) and matrix-assisted laser desorption ionization (MALDI) MS (27). Among these methods, ESI MS and MALDI MS can detect lysophospholipids with high sensitivity and have the advantage of not requiring derivatization of LPA.

1,3-Bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc (II) complex (Zn₂L³⁺; Phos-tag^®) is a dinuclear zinc (II) complex acting as a phosphate-capture molecule (28) (Fig. 1). Takeda et al. (29) have reported that phosphorylated peptides, adenosine monophosphate, acetylphosphate, and LPA can be detected by MALDI-time-of-flight (TOF) MS as a monocationic complex with Zn₂L³⁺. In the current study, we report a simple and sensitive procedure for the quantitative analysis of LPA homologs in biological samples by MALDI-TOF MS using this novel phosphate-capture molecule.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Structure of the lysophosphatidic acid–1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (18:1 LPA2–-68Zn2L3+)+.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Syntheses of LPAs
First, 1-alkenyl (alkyl)-2-acyl-PC was synthesized by the condensation of 1-alkenyl (alkyl)-2-lyso-PC with anhydride of oleic acid (18:1), linoleic acid (18:2), or arachidonic acid (20:4) as described previously (31). The synthesized PC (1.6 µmol) was suspended in 0.6 ml of 0.2 M sodium acetate buffer (pH 5.8) containing 0.1 M CaCl₂, and the suspension was mixed with 0.4 ml of diethyl ether. The reaction was started by adding 0.4 ml of the active fraction of phospholipase D from cabbage. The reaction mixture was stirred vigorously at room temperature for 2 h. After evaporation of the ether phase under a stream of N₂ gas, phospholipids including 1-alkenyl (alkyl)-2-acyl-phosphatidic acid (PA) were extracted by the method of Bligh and Dyer (32) from the reaction mixture acidified to pH 2 with 2 M HCl. The phospholipids were then dissolved in 3.8 ml of chloroform-methanol-2.5 M HCl (1:2:0.8, v/v/v) to hydrolyze the 1-alkenyl-2-acyl-PA to 1-lyso-2-acyl-PA. After vigorous shaking for 15 min, phospholipids were extracted by the method of Bligh and Dyer (32) from the reaction mixture and subjected to TLC using chloroform-methanol-28% ammonia hydroxide (65:35:6, v/v/v) for the purification of 1-lyso-2-acyl-PA. The yield of 1-lyso-2-acyl-PA was 20% of that of 1-alkenyl (alkyl)-2-acyl-PC. Because the acyl residue at the sn-2 position of the glycerol backbone of lysophospholipid is liable to migrate to the sn-1 position, the 2-acyl LPA was analyzed by MALDI-TOF MS within 24 h after its preparation. 1,2-Dipalmitoyl-PC, 1,2-dimargarinoyl-PC, and 1,2-dioleoyl-PC were synthesized by condensation of L--glycerophosphorylcholine with anhydride of palmitic acid, margaric acid, and oleic acid, respectively, as described previously (33). The synthesized PCs were treated with phospholipase A₂ as described (34). The resulting 1-acyl-2-lyso-PC was hydrolyzed by phospholipase D from cabbage, and the product was purified by TLC as described above. The yield of 1-acyl-2-lyso-PA was 20% of the starting material, 1,2-diacyl-PC. The content of lipid phosphorus in LPA was determined by the method of Bartlett (35).

Extraction and purification of LPA from biological fluids
LPA in calf serum was extracted and purified by the method reported by Nakane et al. (16) with minor modifications. Three nanomoles of 17:0 LPA, 7.5 ml of chloroform, 15 ml of methanol, and 4.2 ml of distilled water were added to 2 ml of calf serum in a glass tube. After vigorous shaking, 7.5 ml of chloroform, 7.5 ml of distilled water, 0.05 ml of 14% ammonia hydroxide, and 0.7 g of KCl were added for phase separation. After centrifugation, the lower phase was discarded and the upper phase was washed with 15 ml of chloroform again. The upper phase was acidified to pH 2 with concentrated HCl and washed twice with 15 ml of chloroform. The lower phases were combined and evaporated to dryness. The residue was dissolved in a small volume of chloroform-methanol (2:1, v/v) and subjected to TLC using chloroform-methanol-28% ammonia hydroxide (65:35:6, v/v/v). The silica gel containing LPA was scraped off the plate, and LPA was extracted by the method of Bligh and Dyer (32) under acidic conditions (adjusting the pH of the upper phase to 2 with HCl). Similar methods for the preparation of LPA were used with 24 g of hen egg white. In this experiment, 17:0 LPA (161 nmol) as an internal standard was added to hen egg white before lipid extraction. The extraction was conducted with 4 g of egg white per glass tube. One-tenth of the LPA fraction obtained from 24 g of hen egg white was subjected to MALDI-TOF MS. The remaining nine-tenths of the LPA fraction of egg white was used for GC analysis. The yield of the egg white LPA prepared by our method was 25%.

The purity of the LPA fraction prepared from egg white was assessed by TLC with chloroform-methanol-20% ammonia hydroxide (60:35:6, v/v/v) as a solvent system that separates lysophosphatidylinositol from LPA. We could not detect lysophosphatidylinositol in this LPA fraction.

Analysis of LPA by TOF MS
LPA was dissolved in 0.1 ml of 10 mM Tris-borate buffer (pH 8.0). Five microliters of ⁶⁸Zn₂L³⁺ solution (0.1–1.0 mM) was mixed with 10 µl of the LPA solution, and 0.5 µl of the mixture was spotted on a sample plate. Immediately, 0.5 µl of THAP solution (10 mg/ml in acetonitrile) was layered on the mixture as a matrix solution. The solution of matrix/analyte on the sample plate was dried for a few minutes. The matrix/analyte cocrystal that formed was subjected to MALDI-TOF MS. In the case of MALDI-TOF MS without the use of ⁶⁸Zn₂L³⁺, LPA was dissolved in 0.1 ml of chloroform-methanol (2:1, v/v). One microliter of LPA solution was mixed with 9 µl of DHB solution in the same solvent (10 mg/ml) as an alternative matrix. One microliter of the mixed solution was spotted on a sample plate. The solution of matrix/analyte on the sample plate was dried for a few minutes. The matrix/analyte cocrystal that formed was subjected to MALDI-TOF MS. In both procedures, MALDI-TOF mass spectra were acquired on a Voyager DE STR (Applied Biosystems, Framingham, MA) in the positive mode. The wavelength of the nitrogen-emitting laser, the pressure in the ion chamber, and the accelerating voltage were 337 nm, 3.7 x 10^–7 Torr, and 20 kV, respectively. The detection was conducted in the reflector mode. The low mass gate was set at 400 Da. To enhance the reproducibility, 256 single shots from the laser were averaged for each mass spectrum. For GC analysis of LPA, the fatty acyl moiety of LPA was converted to fatty acid methylesters using 5% methanolic HCl, and the resultant fatty acid methylester was analyzed by GC equipped with a capillary column coated with 0.25 µm film of polar CBP 20, as described previously (34).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Detection of [LPA^2–-⁶⁸Zn₂L³⁺]⁺ by MALDI-TOF MS
Figure 2A shows a MALDI-TOF mass spectrum of ⁶⁸Zn₂L³⁺ adducts of 18:1 LPA^2–. An intense ion peak assigned to [18:1 LPA^2–-⁶⁸Zn₂L³⁺]⁺ was detected at m/z 1,023.3, but neither the protonated molecule of 18:1 LPA^2– nor the molecular ion of its cation adduct was detected in the mass spectrum, as previously reported by Takeda et al. (29). The detection limit of 18:1 LPA appears to be 0.1 pmol on a sample plate (Fig. 2B). When 18:1 LPA was analyzed in the absence of ⁶⁸Zn₂L³⁺ in the positive mode using DHB as a matrix, two ions of the cation adduct of 18:1 LPA^2– were detected at m/z 459.2 [LPA^2–-2H⁺-Na⁺]⁺ and m/z 481.2 [LPA^2–-H⁺-2Na⁺]⁺ in the mass spectrum (Fig. 2C), as reported previously (27). In this case, more than 10 pmol of 18:1 LPA on a sample plate was required for its detection under our analytical conditions. Although a single ion assigned to be [LPA^2–-H⁺]^– was detected in the negative mode, more than 50 pmol of 18:1 LPA on a sample plate was required for its detection (data not shown). Detection of LPA as forms of multiple adducts is a problem for qualitative and quantitative analysis of LPA in biological samples, in which LPA comprises several molecular species with different acyl chain lengths. In this context, sensitive detection of LPA as a single form of [LPA^2–-⁶⁸Zn₂L³⁺]⁺ is advantageous for molecular species analysis of LPA in biological fluid. Detection of LPA as [LPA^2–-⁶⁸Zn₂L³⁺]⁺ has another advantage: low background noise in a mass range over m/z 900, in which ions of the ⁶⁸Zn₂L³⁺ adduct of LPA^2– are detected.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Positive ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of the 68Zn2L3+ adduct of 1-18:1 LPA2– (A and B) and the proton-sodium adduct of LPA2– (C). A and B: The LPA2–-68Zn2L3+ solution (0.5 µl) was applied to the sample plate followed by the same volume of 2,4,6-trihydroxy-acetophenone (THAP) solution (10 mg/ml in acetonitrile). The amounts of 1-18:1 LPA2– and 68Zn2L3+ on the sample plate were 10 pmol and 17 pmol (A) and 0.1 pmol and 1.7 pmol (B), respectively. C: 1-18:1 LPA in a mixture of chloroform-methanol (2:1, v/v) was mixed with 9 µl of 3,5-dihydroxybenzoic acid solution (10 mg/ml in the same solvent). One microliter of the mixed solution was spotted on a sample plate. The amount of 1-18:1 LPA on the sample plate was 500 pmol.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Detection efficiencies of various molecular species of LPA. Different amounts (0.25–5 nmol) of 1-16:0 LPA (A), 1-18:1 LPA (B), 2-18:2 LPA (C), or 2-20:4 LPA (D) were mixed with a fixed amount of 17:0 LPA (5 nmol) in 0.1 ml of 10 mM Tris-borate buffer. Ten microliters of the mixed LPA solution was mixed with 5 µl of 0.2 mM 68Zn2L3+ solution. The mixed solution (0.5 µl) was applied to a sample plate and mixed with 0.5 µl of 10 mg/ml THAP solution in acetonitrile. The amount of 17:0 LPA on the sample plate was 17 pmol. Values are the means ± SD of three independent experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Quantitative analysis of LPA homologs in hen egg white. The LPA fraction was prepared from hen egg white (24 g) that was mixed with 17:0 LPA (161 nmol) as an internal standard before lipid extraction. One-tenth of the LPA fraction was dissolved in 0.1 ml of 10 mM Tris-borate buffer, and 10 µl of the LPA solution was mixed with 5 µl of 1 mM 68Zn2L3+ solution. The solution of LPA2–-68Zn2L3+ was applied to a sample plate as a 0.5 µl droplet and mixed with 0.5 µl of 10 mg/ml THAP solution in acetonitrile. MALDI-TOF MS was conducted in the positive mode.

View larger version (30K): [in this window] [in a new window]   Fig. 5. MALDI-TOF mass spectrum of LPAs extracted from 2 ml of calf serum. The LPA fraction was prepared from 2 ml of calf serum that was mixed with 17:0 LPA (3 nmol) as an internal standard before lipid extraction. The TLC-purified LPA fraction was dissolved in 0.1 ml of 10 mM Tris-borate buffer. Ten microliters of the LPA solution was mixed with 5 µl of 0.1 mM 68Zn2L3+ solution. The solution of LPA2–-68Zn2L3+ was then applied to a sample plate (0.5 µl droplet) and mixed with 0.5 µl of 10 mg/ml THAP solution in acetonitrile. MALDI-TOF MS was conducted in the positive mode.

In the analysis of the mixture of LPA homologs, relative intensities of ions of LPA homologs did not vary much with the laser target point of the crystal. This observation suggests that homogeneous matrix/analyte cocrystal is formed under our procedure. As a matrix, THAP rather than DHB is better for the detection of [LPA^2–-⁶⁸Zn₂L³⁺]⁺. Possibly, DHB decreases the pH of the sample solution and thus prevents the formation of [LPA^2–-⁶⁸Zn₂L³⁺]⁺ (29).

Because MALDI-TOF MS cannot detect fragment ions from the parent ion, information on the structure of LPA, such as the position of the acyl group at the glycerol backbone, is limited. The detailed structural analysis can be achieved using the ESI tandem MS system (11).

MALDI-TOF MS is used by many scientists for structural analysis of biological materials and is becoming commonly available not only for analyses of proteins but also for lipids, such as triglyceride, phospholipids, and glycosphingolipid (36, 37). The sensitive and convenient procedure for LPA-specific detection is useful for the quantification of molecular species of LPA that occur in small volumes of biological materials.

Manuscript received June 14, 2004 and in revised form August 3, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

1. Tokumura, A. 1995. A family of phospholipid autocoids: occurrence, metabolism and bioactions. Prog. Lipid Res. 34: 151–184.[CrossRef][Medline]

2. Moolenaar, W. H. 1999. Bioactive lysophospholipids and their G protein-coupled receptors. Exp. Cell Res. 253: 230–238.[CrossRef][Medline]

3. Schumacher, K. A., H. G. Classen, and M. Spath. 1979. Platelet aggregation evoked in vitro and in vivo by phosphatidic acids and lysoderivatives: identity with substances in aged serum (DAS). Thromb. Haemostasis. 42: 631–640.[Medline]

4. Gerrard, J. M., S. E. Kindom, D. A. Peterson, J. Peller, K. E. Krantz, and J. G. White. 1979. Lysophosphatidic acids. Influence on platelet aggregation and intracellular calcium flux. Am. J. Pathol. 96: 423–438.[Abstract]

5. Tokumura, A., K. Fukuzawa, and H. Tsukatani. 1982. Contractile actions of lysophosphatidic acids with a chemically-defined fatty acyl group on longitudinal muscle from guinea-pig ileum. J. Pharm. Pharmacol. 34: 514–516.[Medline]

6. van Corven, E. J., A. Groenink, K. Jalink, T. Eichholtz, and W. H. Moolenaar. 1989. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell. 59: 45–54.[CrossRef][Medline]

7. Tokumura, A., M. Iimori, Y. Nishioka, M. Kitahara, M. Sakashita, and S. Tanaka. 1994. Lysophospatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta. Am. J. Physiol. 267: C204–C210.

8. Tokumura, A., K. Harada, K. Fukuzawa, and H. Tsukatani. 1986. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma. Biochim. Biophys. Acta. 875: 31–38.[Medline]

9. Tokumura, A., Y. Nishioka, O. Yoshimoto, J. Shinomiya, and K. Fukuzawa. 1999. Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma. Biochim. Biophys. Acta. 1437: 235–245.[Medline]

10. Xu, Y., Z. Shen, D. W. Wiper, M. Wu, R. E. Morton, P. Elson, A. W. Kennedy, J. Belinson, M. Markman, and G. Casey. 1998. Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. J. Am. Med. Assoc. 280: 719–723.[Abstract/Free Full Text]

11. Xiao, Y., Y. Chen, A. W. Kennedy, J. Belinson, and Y. Xu. 2000. Evaluation of plasma lysophospholipids for diagnostic significance using electrospray ionization mass spectrometry (ESI-MS) analyses. Ann. N. Y. Acad. Sci. 905: 242–259.[Abstract/Free Full Text]

12. Baker, D. L., E. S. Umstot, D. M. Desiderio, and G. J. Tigyi. 2000. Quantitative analysis of lysophosphatidic acid in human blood fractions. Ann. N. Y. Acad. Sci. 905: 267–269.[Free Full Text]

13. Sugiura, T., S. Nakane, S. Kishimoto, K. Waku, Y. Yoshioka, and A. Tokumura. 2002. Lysophosphatidic acid, a growth factor-like lipid in the saliva. J. Lipid Res. 43: 2049–2054.[Abstract/Free Full Text]

14. Balazs, L., J. Okolicany, M. Ferrebee, B. Tolley, and G. Tigyi. 2001. Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280: R466–R472.[Abstract/Free Full Text]

15. Tokumura, A., M. Miyake, Y. Nishioka, S. Yamano, T. Aono, and K. Fukuzawa. 1999. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients. Biol. Reprod. 61: 195–199.[Abstract/Free Full Text]

16. Nakane, S., A. Tokumura, K. Waku, and T. Sugiura. 2001. Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: distinct molecular species compositions. Lipids. 36: 413–419.[Medline]

17. Tokumura, A. 2002. Physiological and pathophysiological roles of lysophosphatidic acids produced by secretory lysophospholipase D in body fluids. Biochim. Biophys. Acta. 1582: 18–25.[Medline]

18. Xu, Y., X. Fang, G. Casey, and G. B. Mills. 1995. Lysophospholipids activate ovarian and breast cancer cells. Biochem. J. 309: 933–940.

19. Xu, Y., D. C. Gaudette, J. D. Boynton, A. Frankel, X. J. Fang, A. Sharma, J. Hurteau, G. Casey, A. Goodbody, A. Mellors, B. J. Holub, and G. B. Mills. 1995. Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients. Clin. Cancer Res. 1: 1223–1232.[Abstract]

20. Bandoh, K., J. Aoki, A. Taira, M. Tsujimoto, H. Arai, and K. Inoue. 2000. Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species: structure-activity relationship of cloned LPA receptors. FEBS Lett. 478: 159–165.[CrossRef][Medline]

21. Tokumura, A., J. Sinomiya, S. Kishimoto, T. Tanaka, K. Kogure, T. Sugiura, K. Satouchi, K. Waku, and K. Fukuzawa. 2002. Human platelets respond differentially to lysophosphatidic acids having highly unsaturated fatty acyl group and alkyl ether-linked lysophosphatidic acids. Biochem. J. 365: 617–628.[Medline]

22. Aoki, J., A. Taira, Y. Takanezawa, Y. Kishi, K. Hama, T. Kishimoto, K. Mizuno, K. Saku, R. Taguchi, and H. Arai. 2002. Serum lysophosphatidic acid is produced through diverse phospholipase pathways. J. Biol. Chem. 277: 48737–48744.[Abstract/Free Full Text]

23. Saulnier-Blache, J. S., A. Girard, M. F. Simon, M. Lafontan, and P. Valet. 2000. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification. J. Lipid Res. 41: 1947–1951.[Abstract/Free Full Text]

24. Tokumura, A., K. Harada, Y. Yoshioka, H. Tsukatani, and Y. Handa. 1984. Analyses of lysophosphatidic acids by gas chromatography mass spectrometry without hydrolytic pretreatment. Biomed. Mass Spectrom. 11: 167–171.[CrossRef][Medline]

25. Holland, W. L., E. C. Stauter, and B. J. Stith. 2003. Quantification of phosphatidic acid and lysophosphatidic acid by HPLC with evaporative light-scattering detection. J. Lipid Res. 44: 854–858.[Abstract/Free Full Text]

26. Yoon, H-R., H. Kim, and S-H. Cho. 2003. Quantitative analysis of acyl-lysophosphatidic acid in plasma using negative ionization tandem mass spectrometry. J. Chromatogr. 788: 85–92.[CrossRef]

27. Petkovic, M., J. Schiller, J. Muller, M. Muller, K. Arnold, and J. Arnhold. 2001. The signal-to-noise ratio as the measure for the quanification of lysophospholipids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Analyst. 126: 1042–1050.[CrossRef][Medline]

28. Kinoshita, E., M. Takahashi, H. Takeda, M. Shiro, and T. Koike. 2004. Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc (II) complex. Dalton Transactions. 8: 1189–1193.

29. Takeda, H., A. Kawasaki, M. Takahashi, A. Yamada, and T. Koike. 2003. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule. Rapid Commun. Mass Spectrom. 17: 2075–2081.[CrossRef][Medline]

30. Davidson, F. M., and C. Long. 1958. The structure of the naturally occurring phosphoglycerides. IV. Action of cabbage-leaf phospholipase D on ovolecithin and related substances. Biochem. J. 69: 458–466.[Medline]

31. Satouchi, K., M. Sakaguchi, M. Shirakawa, K. Hirano, and T. Tanaka. 1994. Lysophosphatidylcholine from white muscle of bonito Euthynnus pelamis (Linnaeus): involvement of phospholipase A1 activity for its production. Biochim. Biophys. Acta. 1214: 303–308.[Medline]

32. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911–917.

33. Satouchi, K., and K. Saito. 1976. Studies on trimethylsilyl derivatives of 1-alkyl-2-acylglycerols by gas-liquid chromatography mass spectrometry. Biomed. Mass Spectrom. 3: 122–126.[CrossRef][Medline]

34. Tanaka, T., K. Ikita, T. Ashida, Y. Motoyama, Y. Yamaguchi, and K. Satouchi. 1996. Effects of growth temperature on the fatty acid composition of the free-living nematode Caenorhabditis elegans. Lipids. 31: 1173–1178.[CrossRef][Medline]

35. Bartlett, G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466–468.[Free Full Text]

36. Schiller, J., J. Arnhold, S. Benard, M. Muller, S. Reichl, and K. Arnold. 1999. Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: a methodological approach. Anal. Biochem. 264: 46–56.

37. Murphy, R. C., J. Fiedler, and J. Hevko. 2001. Analysis of nonvolatile lipids by mass spectrometry. Chem. Rev. 101: 479–526.[CrossRef][Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, and T. Koike Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins Mol. Cell. Proteomics, April 1, 2006; 5(4): 749 - 757. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: D400010-JLR200v1 45/11/2145    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tanaka, T. Articles by Satouchi, K. Search for Related Content PubMed PubMed Citation Articles by Tanaka, T. Articles by Satouchi, K. Social Bookmarking                      What's this?