Skip to main content
SpringerLink
Log in

Qualitative and quantitative analysis of lipoxygenase products in bovine corneal epithelium by liquid chromatography-mass spectrometry with an ion trap

  • Published:
Lipids

Abstract

Electrospray ionization ion trap mass spectra of 5-, 12-, and 15-hydroperoxyeicosatetraenoic (HPETE), hydroxyeicosatetraenoic (HETE), and ketoeicosatetraenoic (KETE) acids were recorded. The HPETE were partly dehydrated to the corresponding KETE in the heated capillary of the mass spectrometer. 12-HPETE and 15-HPETE were also converted to KETE by collision-induced dissociation (CID) in the ion trap, whereas CID of 5-HPETE yielded little formation of 5-KETE. Subcellular fractions of bovine corneal epithelium were incubated with arachidonic acid (AA) and the metabolites were analyzed. 15-HETE and 12-HETE were consistently formed, whereas significant accumulation of HPETE and KETE was not detected. Biosynthesis of 12- and 15-HETE was quantified with octadeuterated 12-HETE and 15-HETE as internal standards. The average biosynthesis of 15-HETE and 12-HETE from 30μM AA by the cytosol was 38±8 and below 3 ng/mg protein/30 min, respectively, which increased to 78±21 and 10±4 ng/mg protein/30 min in the presence of 1 mM free Ca2+. The microsomal biosynthesis was unaffected by Ca2+. The microsomes metabolized AA to 15-HETE as the main metabolite at a low protein concentration (0.3 mg/mL), whereas 12-HETE and 15-HETE were formed in a 2∶1 ratio at a combined rate of 0.7±0.2 μg/mg protein/30 min at a high protein concentration (1.8 mg/mL). The level of 12-HETE in corneal epithelial cells was 50±13 pg/mg tissue, whereas the endogenous amount of 15-HETE was low or undetectable (<3 pg/mg tissue). Incubation of corneas for 20 min at 37°C before processing selectively increased the amounts of 12-HETE in the epithelium fourfold to ∼0.2 ng/mg tissue. We conclude that 12-HETE is the main endogenously formed lipoxygenase product of bovine corneal epithelium.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AA:

arachidonic acid

CID:

collision-induced dissociation

GC:

gas chromatography

HETE:

hydroxyeicosatetraenoic acid

HPETE:

hydroperoxyeicosatetraenoic acid

HPLC:

high-performance liquid chromatography

KETe:

ketoeicosatetraenoic acid

LC:

liquid chromatography

LOX:

lipoxygenase

MS:

mass spectrometry

PBS:

phosphate buffered saline

RP:

reversed phase

RT-PCR:

reverse transcription-polymerase chain reaction

UV:

ultraviolet

References

  1. Kühn, H. (1999) Lipoxygenases, inProstaglandins, Leukotrienes and Other Eicosanoids (Marks, F., and Fürstenberger, G., eds.), pp. 109–141, Wiley-VCH, Weinheim.

    Google Scholar 

  2. Brash, A.R. (1999) Lipoxygenases: Occurrence, Functions, Catalysis, and Acquisition of Substrate,J. Biol. Chem. 274, 23679–23682.

    Article  PubMed  CAS  Google Scholar 

  3. Funk, C.D. (1993) Molecular Biology in the Eicosanoid Field,Prog. Nucleic Acid Res. Mol. Biol. 45, 67–98.

    PubMed  CAS  Google Scholar 

  4. Funk, C.D. (1996) The Molecular Biology of Mammalian Lipoxygenases and the Quest for Eicosanoid Functions Using Lipoxygenase-Deficient Mice,Biochim. Biophys. Acta 1304, 65–84.

    PubMed  Google Scholar 

  5. Prigge, S.T., Boyington, J.C., Gaffney, B.J., and Amzell, L.M. (1996) Structure Conservation in Lipoxygenases: Structural Analysis of Soybean Lipoxygenase-1 and Modeling of Human Lipoxygenases,Proteins 24, 275–291.

    Article  PubMed  CAS  Google Scholar 

  6. MacMillan, D.K., and Murphy, R.C. (1995) Analysis of Lipid Hydroperoxides and Long-Chain Conjugated Keto Acids by Negative Ion Electrospray Mass Spectrometry,J. Am. Soc. Mass Spectrom. 6, 1190–1201.

    Article  CAS  Google Scholar 

  7. Hall, L.M., and Murphy, R.C. (1998) Electrospray Mass Spectrometric Analysis of 5-Hydroperoxy and 5-Hydroxyeicosatetraenoic Acids Generated by Lipid Peroxidation of Red Blood Cell Ghost Phospholipids,J. Am. Soc. Mass. Spectrom. 9, 527–532.

    Article  PubMed  CAS  Google Scholar 

  8. Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F., and Whitehouse, C.M. (1989) Electrospray Ionization for Mass Spectrometry of Large Biomolecules,Science 246, 64–71.

    Article  PubMed  CAS  Google Scholar 

  9. Deterding, L.J., Curtis, J.F., and Tomer, K.B. (1992) Tandem Mass Spectrometry Identification of Eicosanoids: Leukotrienes and Hydroxyeicosatetraenoic Acids,Biol. Mass. Spectrom. 21, 597–609.

    Article  CAS  Google Scholar 

  10. Wheelan, P., Zirrolli, J.A., and Murphy, R.C. (1993) Low-Energy Fast Atom Bombardment Tandem Mass Spectrometry of Monohydroxy Substituted Unsaturated Fatty Acids,Biol. Mass Spectrom. 22, 465–473.

    Article  PubMed  CAS  Google Scholar 

  11. Schneider, C., Schreier, P., and Herderich, M. (1997) Analysis of Lipoxygenase-Derived Fatty Acid Hydroperoxides by Electrospray Ionization Tandem Mass Spectrometry,Lipids 32, 331–336.

    Article  PubMed  CAS  Google Scholar 

  12. Bylund, J., Erickson, J., and Oliw, E.H. (1998) Analysis of Cytochrome P450 Metabolites of Arachidonic and Linoleic Acids by Liquid Chromatography-Mass Spectrometry with Ion Trap MS2,Anal. Biochem. 265, 55–68.

    Article  PubMed  CAS  Google Scholar 

  13. Oliw, E.H., Su, C., Skogstrom, T., and Benthin, G. (1998) Analysis of Novel Hydroperoxides and Other Metabolites of Oleic, Linoleic, and Linolenic Acids by Liquid Chromatography-Mass Spectrometry with Ion Trap MSn,Lipids 33, 843–852.

    Article  PubMed  CAS  Google Scholar 

  14. Williams, R.N., Delamere, N.A., and Paterson, C.A. (1985) Generation of Lipoxygenase Products in the Avascular Tissues of the Eye,Exp. Eye Res. 41, 733–738.

    Article  PubMed  CAS  Google Scholar 

  15. Hurst, J.S., Balazy, M., Bazan, H.E., and Bazan, N.G. (1991) The Epithelium, Endothelium, and Stroma of the Rabbit Cornea Generate 12(S)-Hydroxyeicosatetraenoic Acid as the Main Lipoxygenase Metabolite in Response to Injury,J. Biol. Chem. 266, 6726–6730.

    PubMed  CAS  Google Scholar 

  16. Oliw, E.H. (1993) Biosynthesis of 12(S)-Hydroxyeicosatetraenoic Acid by Bovine Corneal Epithelium,Acta Physiol. Scand. 147, 117–121.

    PubMed  CAS  Google Scholar 

  17. Liminga, M., Fagerholm, P., and Oliw, E.H. (1994) Lipoxygenases in Corneal Epithelia of Man and Cynomolgus Monkey,Exp. Eye Res. 59, 313–321.

    Article  PubMed  CAS  Google Scholar 

  18. Liminga, M., Hörnsten, L., Sprecher, H.W., and Oliw, E.H. (1994) Arachidonate 15-Lipoxygenase in Human Corneal Epithelium and 12- and 15-Lipoxygenases in Bovine Corneal Epithelium: Comparison with Other Bovine 12-Lipoxygenases,Biochim. Biophys. Acta 1210, 288–296.

    PubMed  CAS  Google Scholar 

  19. Liminga, M., and Oliw, E.H. (1999) cDNA Cloning of 15-Lipoxygenase Type 2 and 12-Lipoxygenases of Bovine Corneal Epithelium,Biochim. Biophys. Acta 1437, 124–135.

    PubMed  CAS  Google Scholar 

  20. Bazan, H.E., Birkle, D.L., Beuerman, R., and Bazan, N.G. (1985) Cryogenic Lesion Alters the Metabolism of Arachidonic Acid in Rabbit Cornea Layers,Invest. Ophthamol. Vis. Sci. 26, 474–480.

    CAS  Google Scholar 

  21. Offord, E.A., Sharif, N.A., Mace, K., Tromvoukis, Y., Spillare, E.A., Avanti, O., Howe, W.E., and Pfeifer, A.M. (1999) Immortalized Human Corneal Epithelial Cells for Ocular Toxicity and Inflammation Studies,Invest. Ophthalmol. Vis. Sci. 40, 1091–1101.

    PubMed  CAS  Google Scholar 

  22. Conners, M.S., Stoltz, R.A., and Schwartzman, M.L. (1996) Chiral Analysis of 12-Hydroxyeicosatetraenoic Acid Formed by Calf Corneal Epithelial Microsomes,J. Ocul. Pharm. Ther. 12, 19–26.

    Article  CAS  Google Scholar 

  23. Vafeas, C., Mieyal, P.A., Urbano, F., Falck, J.R., Chauhan, K., Berman, M., and Schwartzman, M.L. (1998) Hypoxia Stimulates the Synthesis of Cytochrome P450-Derived Inflammatory Eicosanoids in Rabbit Corneal Epithelium,J. Pharmacol. Exp. Ther. 287, 903–909.

    PubMed  CAS  Google Scholar 

  24. Oliw, E.H., and Sprecher, H. (1989) Metabolism of Polyunsaturated Fatty Acids by an (n−6)-Lipoxygenase Associated with Human Ejaculates,Biochim. Biophys. Acta 1002, 283–291.

    PubMed  CAS  Google Scholar 

  25. Reddanna, P., Whelan, J., Maddipati, K.R., and Reddy, C.C. (1990) Purification of Arachidonate 5-Lipoxygenase from Potato Tubers,Methods Enzymol. 187, 268–277.

    Article  PubMed  CAS  Google Scholar 

  26. Becker, H.-D., Björk, A., and Adler, E. (1980) Quinone Dehydrogenation. Oxidation of Benzylic Alcohols with 2,3-Dichloro-5,6-dicyanobenzoquinone,J. Org. Chem. 45, 1596–1600.

    Article  CAS  Google Scholar 

  27. Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,Anal. Biochem. 7, 248–254.

    Article  Google Scholar 

  28. Jakobsson, P.-J., Odlander, B., Steinhilber, D., Rosén, A., and Claesson, H.-E. (1991) Human B Lymphocytes Possess 5-Lipoxygenase Activity and Convert Arachidonic Acid to Leukotriene B4.Biochem. Biophys. Res. Commun. 178, 302–308.

    Article  PubMed  CAS  Google Scholar 

  29. Maas, R.L., and Brash, A.R. (1983) Evidence for a Lipoxygenase Mechanism in the Biosynthesis of Epoxide and Dihydroxy Leukotrienes from 15(S)-Hydroperoxyeicosatetraenoic Acid by Human Platelets and Porcine Leukocytes,Proc. Natl. Acad. Sci. USA 80, 2484–2888.

    Article  Google Scholar 

  30. Brinckmann, R., Schnurr, K., Heydeck, D., Roesenbach, T., Kolde, G., and Kühn, H. (1998) Membrane Translocation of 15-Lipoxygenase in Hematopoietic Cells Is Calcium-Dependent and Activates the Oxygenase Activity of the Enzyme,Blood 91, 64–74.

    PubMed  CAS  Google Scholar 

  31. Kilty, I., Logan, A., and Vickers, P.J. (1999) Differential Characteristics of Human 15-Lipoxygenase Isozymes and a Novel Splice Variant of 15S-Lipoxygenase,Eur. J. Biochem. 266, 83–93.

    Article  PubMed  CAS  Google Scholar 

  32. Oliw, E.H. (1990) Biosynthesis of 20-Hydroxyeicosatetraenoic Acid (20-HETE) and 12 (S)-HETE by Renal Cortical Microsomes of the Cynomolgus Monkey,Eicosanoids 3, 161–164.

    PubMed  CAS  Google Scholar 

  33. Takahashi, Y., Ueda, N., and Yamamoto, S. (1988) Two Immunologically and Catalytically Distinct Arachidonate 12-Lipoxygenases of Bovine Platelets and Leukocytes,Arch. Biochem. Biophys. 266, 613–621.

    Article  PubMed  CAS  Google Scholar 

  34. Hada, T., Ueda, N., Takahashi, Y., and Yamamoto, S. (1991) Catalytic Properties of Human Platelet 12-Lipoxygenase as Compared with the Enzymes of Other Origins,Biochim. Biophys. Acta 1083, 89–93.

    PubMed  CAS  Google Scholar 

  35. Qiao, N., Takahashi, Y., Takamatsu, H., and Yoshimoto, T. (1999) Leukotriene A Synthase Activity of Purified Mouse Skin Arachidonate 8-Lipoxygenase Expressed inEscherichia coli, Biochim. Biophys. Acta 1438, 131–139.

    PubMed  CAS  Google Scholar 

  36. Dieter, P. (1999) The Generation of Free Arachidonic Acid, inProstaglandins, Leukotrines and Other Eicosanoids (Marks, F., and Fürstenberger, G., eds.), pp. 47–59, Wiley-VCH, Weinheim.

    Google Scholar 

  37. Fruteau de Laclos, B., Braquet, P., and Borgeat, P. (1984) Characteristics of Leukotriene (LT) and Hydroxy Eicosatetraenoic Acid (HETE) Synthesis in Human Leukocytesin vitro: Effect of Arachidonic Acid Concentration,Prostaglandins Leukotrienes Med. 13, 47–52.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dept. of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, P.O. Box 591, SE-751 24, Uppsala, Sweden

    Maria Liminga & Ernst Oliw

Authors
  1. Maria Liminga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ernst Oliw
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Liminga.

About this article

Cite this article

Liminga, M., Oliw, E. Qualitative and quantitative analysis of lipoxygenase products in bovine corneal epithelium by liquid chromatography-mass spectrometry with an ion trap. Lipids 35, 225–232 (2000). https://doi.org/10.1007/BF02664773

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02664773

Keywords

Search

Navigation