Skip to main content
SpringerLink
Log in

Development of an Online-SPE–LC–MS/MS Method for 26 Hydroxylated Polyunsaturated Fatty Acids as Rapid Targeted Metabolomics Approach for the LOX, CYP, and Autoxidation Pathways of the Arachidonic Acid Cascade

Chromatographia volume 78, pages 415–428 (2015)Cite this article

Abstract

Hydroxylated fatty acids (OH-FAs) are formed in all branches of the arachidonic acid (AA) cascade from polyunsaturated fatty acids (PUFA). OH-FAs act as potent lipid mediators and serve as activity marker for pathways of the AA cascade, particularly the lipoxygenase branch. Current targeted metabolomics methods of the AA cascade cover several OH-FAs among other oxylipins, yet they require long runtimes and laborious sample preparation. In the present study, we developed a new rapid LC–MS method with automated sample preparation for the simultaneous quantification of 26 OH-FAs within 6.5 min. Crude biological samples are directly injected following addition of four isotopically labeled internal standards and centrifugation. The analytes are extracted from the matrix by means of online solid phase extraction on an Oasis HLB column at 3.5 mL min−1 flow rate. LC separation was carried out on a RP-18 column with fused core 1.3 µm particles. The method showed a high sensitivity with a limit of detection of 0.5–10 fmol on column and a broad linear range. Intra- and inter-batch precision and accuracy for the analytes were characterized for cell culture medium as well as human plasma and were found to be generally within 100 ± 15 %. The method was applied to the investigation of OH-FA formation in five cell lines following incubation with AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). The colon cancer cell lines HCA-7 and SW-480, as well as the fibroblast line Balb/c 3T3 showed significant formation of OH-FAs in the cell culture medium, with dominant formation of 15-HETE, 18-HEPE, 20-HDHA, and 8-HDHA from the precursor PUFAs.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

AA:

Arachidonic acid

ACN:

Acetonitrile

ALA:

Alpha-linolenic acid

CAD:

Collision activated dissociation

CE:

Collision energy

COX:

Cyclooxygenase

CXP:

Collision cell exit potential

CYP:

Cytochrome P450 monooxygenase

DHA:

Docosahexaenoic acid

DP:

Declustering potential

DPA:

Docosapentaenoic acid

DMEM:

Dulbecco’s modified Eagle’s medium

EPA:

Eicosapentaenoic acid

ESI:

Electrospray ionization

FCS:

Fetal calf serum

FWHM:

Full width at half maximal height

HAc:

Acetic acid

HDHA:

Hydroxydocosahexaenoic acid, hydroxy-DHA

5-HEDH:

5-Hydroxyeicosanoid dehydrogenase

HEPE:

Hydroxyeicosapentaenoic acid, hydroxy-EPA

HETE:

Hydroxyeicosatetraenoic acid, hydroxy-AA

HpETE:

Hydroperoxyeicosatetraenoic acid, hydroperoxy-AA

HODE:

Hydroxyoctadecadienoic acid, hydroxy-LA

HOTrE:

Hydroxyoctadecatrienoic acid, hydroxy-ALA

LA:

Linoleic acid

LC–MS:

Liquid chromatography mass spectrometry

LLOQ:

Lower limit of quantification

LOD:

Limit of detection

LOX:

Lipoxygenase

MeOH:

Methanol

OH-FA:

Hydroxy fatty acid

PUFA:

Polyunsaturated fatty acid

SPE:

Solid phase extraction

References

  1. Buczynski MW, Dumlao DS, Dennis EA (2009) J Lipid Res 50:1015–1038

    Article  CAS  Google Scholar 

  2. Fischer R, Konkel A, Mehling H, Blossey K, Gapelyuk A, Wessel N, von Schacky C, Dechend R, Muller DN, Rothe M, Luft FC, Weylandt K, Schunck WH (2014) J Lipid Res 55:1150–1164

    Article  CAS  Google Scholar 

  3. Mulugeta S, Suzuki T, Hernandez NT, Griesser M, Boeglin WE, Schneider C (2010) J Lipid Res 51:575–585

    Article  CAS  Google Scholar 

  4. Serhan CN (2014) Nature 510:92–101

    Article  CAS  Google Scholar 

  5. Weylandt KH, Chiu CY, Gomolka B, Waechter SF, Wiedenmann B (2012) Prostagladins Other Lipid Mediat 97:73–82

    Article  CAS  Google Scholar 

  6. Schuchardt JP, Schmidt S, Kressel G, Dong H, Willenberg I, Hammock BD, Hahn A, Schebb NH (2013) Prostagladins Leukot Essent Fat Acids 89:19–29

    Article  CAS  Google Scholar 

  7. Dangi B, Obeng M, Nauroth JM, Teymourlouei M, Needham M, Raman K, Arterburn LM (2009) J Biol Chem 284:14744–14759

    Article  CAS  Google Scholar 

  8. Chiu CY, Gomolka B, Dierkes C, Huang NR, Schroeder M, Purschke M, Manstein D, Dangi B, Weylandt KH (2012) Inflamm Res 61:967–976

    Article  CAS  Google Scholar 

  9. Porter NA, Lehman LS, Weber BA, Smith KJ (1981) J Am Chem Soc 103:6447–6455

    Article  CAS  Google Scholar 

  10. Yin H, Brooks JD, Gao L, Porter NA, Morrow JD (2007) J Biol Chem 282:29890–29901

    Article  CAS  Google Scholar 

  11. Konkel A, Schunck WH (2011) Biochim Biophys Acta 1814:210–222

    Article  CAS  Google Scholar 

  12. Inceoglu B, Wagner K, Schebb NH, Morisseau C, Jinks SL, Ulu A, Hegedus C, Rose T, Brosnan R, Hammock BD (2011) Proc Natl Acad Sci USA 108:5093–5097

    Article  CAS  Google Scholar 

  13. Miyata N, Roman RJ (2005) J Smooth Muscle Res 41:175–193

    Article  Google Scholar 

  14. Bylund J, Kunz T, Valmsen K, Oliw EH (1998) J Pharmacol Exp Ther 284:51–60

    CAS  Google Scholar 

  15. Buckley CD, Gilroy DW, Serhan CN (2014) Immunity 40:315–327

    Article  CAS  Google Scholar 

  16. Bento AF, Claudino RF, Dutra RC, Marcon R, Calixto JB (2011) J Immunol 187:1957–1969

    Article  CAS  Google Scholar 

  17. Gonzalez-Periz A, Planaguma A, Gronert K, Miquel R, Lopez-Parra M, Titos E, Horrillo R, Ferre N, Deulofeu R, Arroyo V, Rodes J, Claria J (2006) Faseb J 20:2537–2539

    Article  CAS  Google Scholar 

  18. Lima-Garcia JF, Dutra RC, da Silva KABS, Motta EM, Campos MM, Calixto JB (2011) Br J Pharmacol 164:278–293

    Article  CAS  Google Scholar 

  19. Neuhofer A, Zeyda M, Mascher D, Itariu BK, Murano I, Leitner L, Hochbrugger EE, Fraisl P, Cinti S, Serhan CN, Stulnig TM (2013) Diabetes 62:1945–1956

    Article  CAS  Google Scholar 

  20. Wilson R, Lyall K (2002) Lipids 37:917–924

    Article  CAS  Google Scholar 

  21. Tsikas D, Zoerner AA (2014) J Chromatogr B 964:79–88

    Article  CAS  Google Scholar 

  22. Dumlao DS, Buczynski MW, Norris PC, Harkewicz R, Dennis EA (2011) Biochim Biophys Acta 1811:724–736

    Article  CAS  Google Scholar 

  23. Gomolka B, Siegert E, Blossey K, Schunck WH, Rothe M, Weylandt KH (2011) Prostagladins Other Lipid Mediat 94:81–87

    Article  CAS  Google Scholar 

  24. Inceoglu B, Wagner KM, Yang J, Bettaieb A, Schebb NH, Hwang SH, Morisseau C, Haj FG, Hammock BD (2012) Proc Natl Acad Sci USA 109:11390–11395

    Article  CAS  Google Scholar 

  25. Yang J, Schmelzer K, Georgi K, Hammock BD (2009) Anal Chem 81:8085–8093

    Article  CAS  Google Scholar 

  26. Strassburg K, Huijbrechts AM, Kortekaas KA, Lindeman JH, Pedersen TL, Dane A, Berger R, Brenkman A, Hankemeier T, van Duynhoven J, Kalkhoven E, Newman JW, Vreeken RJ (2012) Anal Bioanal Chem 404:1413–1426

    Article  CAS  Google Scholar 

  27. Willenberg I, Elsner L, Steinberg P, Schebb NH (2015) Food Chem 166:537–543

    Article  CAS  Google Scholar 

  28. Schebb NH, Huby M, Morisseau C, Hwang SH, Hammock BD (2011) Anal Bioanal Chem 400:1359–1366

    Article  CAS  Google Scholar 

  29. Schebb NH, Flores I, Kurobe T, Franze B, Ranganathan A, Hammock BD, Teh SJ (2011) Aquat Toxicol 105:448–454

    Article  CAS  Google Scholar 

  30. Schebb NH, Inceoglu B, Rose T, Wagner K, Hammock BD (2011) Anal Methods 3:420–428

    Article  CAS  Google Scholar 

  31. Hsieh Y, Duncan CJG, Brisson JM (2007) Anal Chem 79:5668–5673

    Article  CAS  Google Scholar 

  32. Bollinger JG, Thompson W, Lai Y, Oslund RC, Hallstrand TS, Sadilek M, Turecek F, Gelb MH (2010) Anal Chem 82:6790–6796

    Article  CAS  Google Scholar 

  33. Masoodi M, Mir AA, Petasis NA, Serhan CN, Nicolaou A (2008) Rapid Commun Mass Spectrom 22:75–83

    Article  CAS  Google Scholar 

  34. Miller TM, Donnelly MK, Crago EA, Roman DM, Sherwood PR, Horowitz MB, Poloyac SM (2009) J Chromatogr B 877:3991–4000

    Article  CAS  Google Scholar 

  35. Harkewicz R, Fahy E, Andreyev A, Dennis EA (2007) J Biol Chem 282:2899–2910

    Article  CAS  Google Scholar 

  36. Masoodi M, Eiden M, Koulman A, Spaner D, Volmer DA (2010) Anal Chem 82:8176–8185

    Article  CAS  Google Scholar 

  37. Schebb NH, Ostermann AI, Yang J, Hammock BD, Hahn A, Schuchardt JP (2014) Prostagladins Other Lipid Mediat. doi:10.1016/j.prostaglandins.2014.05.002

    Google Scholar 

  38. Norris PC, Dennis EA (2012) Proc Natl Acad Sci USA 109:8517–8522

    Article  CAS  Google Scholar 

  39. Norris PC, Reichart D, Dumlao DS, Glass CK, Dennis EA (2011) J Leukoc Biol 90:563–574

    Article  CAS  Google Scholar 

  40. Glasgow WC, Eling TE (1990) Mol Pharmacol 38:503–510

    CAS  Google Scholar 

  41. Little JM, Kurkela M, Sonka J, Jantti S, Ketola R, Bratton S, Finel M, Radominska-Pandya A (2004) J Lipid Res 45:1694–1703

    Article  CAS  Google Scholar 

  42. Turgeon D, Chouinard S, Belanger P, Picard S, Labbe JF, Borgeat P, Belanger A (2003) J Lipid Res 44:1182–1191

    Article  CAS  Google Scholar 

  43. Grant GE, Rokach J, Powell WS (2009) Prostagladins Other Lipid Mediat 89:98–104

    Article  CAS  Google Scholar 

  44. Green FA (1990) Lipids 25:618–623

    Article  CAS  Google Scholar 

  45. Powell WS, Gravel S, Gravelle F (1995) J Lipid Res 36:2590–2598

    CAS  Google Scholar 

  46. Sharma RA, Gescher A, Plastaras JP, Leuratti C, Singh R, Gallacher-Horley B, Offord E, Marnett LJ, Steward WP, Plummer SM (2001) Carcinogenesis 22:1557–1560

    Article  CAS  Google Scholar 

  47. Sheng H, Shao J, Kirkland SC, Isakson P, Coffey RJ, Morrow J, Beauchamp RD, DuBois RN (1997) J Clin Invest 99:2254–2259

    Article  CAS  Google Scholar 

  48. Yoshinaga M, Buchanan FG, DuBois RN (2004) Prostagladins Other Lipid Mediat 73:111–122

    Article  CAS  Google Scholar 

  49. Bednar W, Holzmann K, Marian B (2007) Food Chem Toxicol 45:508–514

    Article  CAS  Google Scholar 

  50. Melstrom LG, Bentrem DJ, Salabat MR, Kennedy TJ, Ding XZ, Strouch M, Rao SM, Witt RC, Ternent CA, Talamonti MS, Bell RH, Adrian TA (2008) Clin Cancer Res 14:6525–6530

    Article  CAS  Google Scholar 

  51. Tavolari S, Bonafe M, Marini M, Ferreri C, Bartolini G, Brighenti E, Manara S, Tomasi V, Laufer S, Guarnieri T (2008) Carcinogenesis 29:371–380

    Article  CAS  Google Scholar 

  52. Lepage C, Liagre B, Cook-Moreau J, Pinon A, Beneytout JL (2010) Int J Oncol 36:1183–1191

    CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by a Kekulé Ph.D. fellowship of the Fonds der Chemischen Industrie to IW, and a grant of the German Research Foundation (DFG) (WE 2908) to KWH as well as a Marie Curie Career Integration Grant (CIG 293536) of the European Union and a Grant of the German Research Foundation (SCHE 1801) to NHS.

Author information

Authors and Affiliations

  1. Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany

    A. I. Ostermann, I. Willenberg & N. H. Schebb

  2. Department of Hepatology, Gastroenterology and Metabolism, Rudolf-Virchow-Hospital, Charité University Medicine, Berlin, Germany

    K. H. Weylandt

  3. Lipid Clinic, Experimental and Clinical Research Center (ECRC), Max-Delbrück-Center for Molecular Medicine, Charité University Medicine, Berlin, Germany

    K. H. Weylandt

  4. Institute of Food Chemistry, University of Wuppertal, Wuppertal, Germany

    N. H. Schebb

Authors
  1. A. I. Ostermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. Willenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. H. Weylandt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. H. Schebb
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to N. H. Schebb.

Additional information

Published in the topical collection Recent Developments in Clinical Omics with guest editors Martin Giera and Manfred Wuhrer.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 58 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ostermann, A.I., Willenberg, I., Weylandt, K.H. et al. Development of an Online-SPE–LC–MS/MS Method for 26 Hydroxylated Polyunsaturated Fatty Acids as Rapid Targeted Metabolomics Approach for the LOX, CYP, and Autoxidation Pathways of the Arachidonic Acid Cascade. Chromatographia 78, 415–428 (2015). https://doi.org/10.1007/s10337-014-2768-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10337-014-2768-8

Keywords

search

Navigation