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A novel chiral stationary phase HPLC-MS/MS method to discriminate between enzymatic oxidation and auto-oxidation of phosphatidylcholine

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Abstract

To elucidate the role of enzymatic lipid peroxidation in disease pathogenesis and in food deterioration, we recently achieved stereoselective analysis of phosphatidylcholine hydroperoxide (PCOOH) possessing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE) using HPLC-MS/MS with a CHIRALPAK OP (+) column. Because enzymatic oxidation progresses concurrently with auto-oxidation, we need to distinguish them further. Here, we attempted such an analysis. First, we used lipoxygenase, linoleic acid, and lysophosphatidylcholine (LPC) to synthesize the enzymatic oxidation product 13(S)-9Z,11E-HPODE PC, and the auto-oxidation products 13(RS)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC, which were used as standards to test the ability of various columns to separate the enzymatic oxidation product from auto-oxidation products. Separation was achieved by connecting in series two columns with different properties: CHIRALPAK OP (+) and CHIRALPAK IB-3. The CHIRALPAK OP (+) column separated 13(R)-9Z,11E-HPODE PC and 13(S)-9Z,11E-HPODE PC, whereas CHIRALPAK IB-3 enabled separation of 13(S)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC. The results for the analysis of both enzymatically oxidized and auto-oxidized lecithin (an important phospholipid mixture in vivo and in food) indicate that our method would be useful for distinguishing enzymatic oxidation and auto-oxidation reactions. Such information will be invaluable for elucidating the involvement of PCOOH in disease pathogenesis and in food deterioration.

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Abbreviations

16:0 LPC:

1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine

16:0/18:2 PC:

1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine

C18:

octadecylsilane

CD3OD:

methanol-d4

CSP:

chiral stationary phase

DCC:

dicyclohexylcarbodiimide

DMAP:

4-dimethylaminopyridine

HPETE:

hydroperoxyeicosatetraenoic acid

HPODE:

hydroperoxyoctadecadienoic acid

LA:

linoleic acid

LOX:

lipoxygenase

LPC:

lysophosphatidylcholine

MxP:

2-methoxypropene

NMR:

nuclear magnetic resonance

PC:

phosphatidylcholine

PCOOH:

phosphatidylcholine hydroperoxide

PPTS:

pyridinium p-toluenesulfonate

SRM:

selected reaction monitoring.

References

  1. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med. 1996;20(5):707–27.

    Article  CAS  Google Scholar 

  2. Kinoshita M, Oikawa S, Hayasaka K, Sekikawa A, Nagashima T, Toyota T, et al. Age-related increases in plasma phosphatidylcholine hydroperoxide concentrations in control subjects and patients with hyperlipidemia. Clin Chem. 2000;46:822–8.

    CAS  Google Scholar 

  3. Glass CK, Witztum JL. Atherosclerosis the road ahead. Cell. 2001;104(4):503–16.

    Article  CAS  Google Scholar 

  4. Bochkov VN, Oskolkova OV, Birukov KG, Levonen AL, Binder CJ, Stöckl J. Generation and biological activities of oxidized phospholipids. Antioxid Redox Signal. 2010;12(8):1009–59.

    Article  CAS  Google Scholar 

  5. Ito J, Nakagawa K, Kato S, Miyazawa T, Kimura F, Miyazawa T. The combination of maternal and offspring high-fat diets causes marked oxidative stress and development of metabolic syndrome in mouse offspring. Life Sci. 2016;151:70–5.

    Article  CAS  Google Scholar 

  6. Xu W, Takahashi Y, Sakashita T, Iwasaki T, Hattori H, Yoshimoto T. Low density lipoprotein receptor-related protein is required for macrophage-mediated oxidation of low density lipoprotein by 12/15-lipoxygenase. J Biol Chem. 2001;276(39):36454–9.

    Article  CAS  Google Scholar 

  7. Funk CD, Cyrus T. 12/15-lipoxygenase, oxidative modification of LDL and atherogenesis. Trends Cardiovasc Med. 2001;11(3–4):116–24.

    CAS  Google Scholar 

  8. Kühn H, Borchert A. Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes. Free Radic Biol Med. 2002;33(2):154–72.

    Article  Google Scholar 

  9. Ito J, Nakagawa K, Kato S, Hirokawa T, Kuwahara S, Nagai T, et al. Direct separation of the diastereomers of phosphatidylcholine hydroperoxide bearing 13-hydroperoxy-9Z,11E-octadecadienoic acid using chiral stationary phase high-performance liquid chromatography. J Chromatogr A. 2015;1386:53–61.

    Article  CAS  Google Scholar 

  10. Kato S, Nakagawa K, Suzuki Y, Asai A, Nagao M, Nagashima K, et al. Liquid chromatography-tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation. Anal Biochem. 2015;471:51–60.

    Article  CAS  Google Scholar 

  11. Ibusuki D, Nakagawa K, Asai A, Oikawa S, Masuda Y, Suzuki T, et al. Preparation of pure lipid hydroperoxides. J Lipid Res. 2008;49(12):2668–77.

    Article  CAS  Google Scholar 

  12. Kato S, Nakagawa K, Suzuki Y, Suzuki K, Mizuochi S, Miyazawa T. Preparation of 13 or 9-hydroperoxy-9Z,11E (9E,11E) or 10E,12Z (10E,12E)-octadecadienoic phosphatidylcholine hydroperoxide. J Oleo Sci. 2014;63(5):431–7.

    Article  CAS  Google Scholar 

  13. Ito J, Mizuochi S, Nakagawa K, Kato S, Miyazawa T. Tandem mass spectrometry analysis of linoleic and arachidonic acid hydroperoxides via promotion of alkali metal adduct formation. Anal Chem. 2015;87(9):4980–7.

    Article  CAS  Google Scholar 

  14. Schneider C, Yu Z, Boeglin WE, Zheng Y, Brash AR. Enantiomeric separation of hydroxy and hydroperoxy eicosanoids by chiral column chromatography. Methods Enzymol. 2007;433:145–57.

    Article  CAS  Google Scholar 

  15. Garscha U, Nilsson T, Oliw EH. Enantiomeric separation and analysis of unsaturated hydroperoxy fatty acids by chiral column chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;872(1–2):90–8.

    Article  CAS  Google Scholar 

  16. Younes AA, Mangelings D, Vander Heyden Y. Chiral separations in normal phase liquid chromatography: enantioselectivity of recently commercialized polysaccharide-based selectors. Part I: enantioselectivity under generic screening conditions. J Pharm Biomed Anal. 2011;55(3):414–23.

    Article  CAS  Google Scholar 

  17. Geryk R, Kalíková K, Vozka J, Plecitá D, Schmid MG, Tesařová E. Enantioselective potential of chiral stationary phases based on immobilized polysaccharides in reversed phase mode. J Chromatogr A. 2014;1363:155–61.

    Article  CAS  Google Scholar 

  18. Homann J, Lehmann C, Kahnt AS, Steinhilber D, Parnham MJ, Geisslinger G, et al. Chiral chromatography-tandem mass spectrometry applied to the determination of pro-resolving lipid mediators. J Chromatogr A. 2014;1360:150–63.

    Article  CAS  Google Scholar 

  19. Song Y, Jing W, Yang F, Shi Z, Yao M, Yan R, et al. Simultaneously enantiospecific determination of (+)-trans-khellactone, (+/−)-praeruptorin A, (+/-)-praeruptorin B, (+)-praeruptorin E, and their metabolites, (+/−)-cis-khellactone, in rat plasma using online solid phase extraction-chiral LC-MS/MS. J Pharm Biomed Anal. 2014;88:269–77.

    Article  CAS  Google Scholar 

  20. van Willem N, Mabel CT. Update on vegetable lecithin and phospholipid technologies. Lipid Sci Technol. 2008;110:472–86.

    Article  Google Scholar 

  21. Cui L, Decker EA. Phospholipids in foods: prooxidants or antioxidants? J Sci Food Agric. 2016;96(1):18–31.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) Grant Number 15H04497.

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Authors and Affiliations

  1. Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan

    Junya Ito, Kiyotaka Nakagawa, Shunji Kato & Teruo Miyazawa

  2. Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan

    Takafumi Hirokawa & Shigefumi Kuwahara

  3. Tsukishima Foods Industry Co., Ltd., Tokyo, 134-8520, Japan

    Toshiharu Nagai

  4. New Industry Creation Hatchery Center (NICHe), Tohoku University, Sendai, 980-8579, Japan

    Teruo Miyazawa

Authors
  1. Junya Ito
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  2. Kiyotaka Nakagawa
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  3. Shunji Kato
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  4. Takafumi Hirokawa
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  5. Shigefumi Kuwahara
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  6. Toshiharu Nagai
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  7. Teruo Miyazawa
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Correspondence to Kiyotaka Nakagawa.

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Ito, J., Nakagawa, K., Kato, S. et al. A novel chiral stationary phase HPLC-MS/MS method to discriminate between enzymatic oxidation and auto-oxidation of phosphatidylcholine. Anal Bioanal Chem 408, 7785–7793 (2016). https://doi.org/10.1007/s00216-016-9882-4

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  • DOI: https://doi.org/10.1007/s00216-016-9882-4

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