Advertisement

Quantitation of lanosterol and its major metabolite FF-MAS in an inhibition assay of CYP51 by azoles with atmospheric pressure photoionization based LC-MS/MS

  • Eva R. Trösken
  • Ellen Straube
  • Werner K. Lutz
  • Wolfgang VölkelEmail author
  • Christopher Patten
Articles

Abstract

Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14α-demethylase (CYP51) is an enzyme inhibited by azoles. Only few data have been reported showing their inhibitory potency since an assay in an in vitro system is not available so far. In the present work an inhibition assay using human recombinant CYP51, coexpressed with human P450 oxido-reductase by the baculovirus/insect cell expression system, and LC-MS/MS as analytical method is described. Atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were used with a triple quadrupole mass spectrometer to compare quantitation of lanosterol (substrate) and 4,4-dimethyl-5α-cholesta-8,14,24-triene-3β-ol (FF-MAS) (product of CYP51) with d6-2,2,3,4,4,6-cholesterol (d6-cholesterol) as internal standard. Optimization of analytical parameters resulted in a LC-APPI-MS/MS method with a LOQ of 10 pg on column for FF-MAS. The sensitivity of the method (LOD 0.5 ng/ml) makes it possible to analyze supernatants of inhibition experiments after precipitation of proteins by isopropanol without any sample enrichment. The coefficient of variation of the analytical method was <20% (n=5) for FF-MAS, lanosterol and d6-cholesterol. The external calibration curve was linear from 1 to 10,000 ng/ml with R2≥0.999 and an accuracy of 94–115%. Compared with APCI, APPI provides a ten- to 500-fold increase in sensitivity for the analytes in this study. IC50 values of epoxiconazole and miconazole—two widely used azole fungicides used in agriculture and in human medicine, respectively—were 1.95 µM and 0.057 µM.

Keywords

High Performance Liquid Chromatography Azole Collision Induce Dissociation Lanosterol Triple Quadrupole Mass Spectrometer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Andersen, H. R.; Vinggaard, A. M.; Rasmussen, T. H.; Gjermandsen, I. M.; Bonefeld-Jorgensen, E. C. Effects of Currently Used Pesticides in Assays for Estrogenicity, Androgenicity, and Aromatase Activity in Vitro. Toxicol. Appl. Pharmacol. 2002, 179, 1–12.CrossRefGoogle Scholar
  2. 2.
    Lepesheva, G. I.; Virus, C.; Waterman, M. R. Conservation in the CYP51 Family. Role of the B′ Helix/BC Loop and Helices F and G in Enzymatic Function. Biochemistry 2003, 42, 9091–9101.CrossRefGoogle Scholar
  3. 3.
    Pfaller, M. A.; Riley, J.; Koerner, T. Effects of Terconazole and Other Azole Antifungal Agents on the Sterol and Carbohydrate Composition of Candida albicans. Diagnos. Microbiol. Infect. Dis. 1990, 13, 31–35.CrossRefGoogle Scholar
  4. 4.
    Majdic, G.; Parvinen, M.; Bellamine, A.; Harwood, H. J., Jr.; Ku, W. W.; Waterman, M. R.; Rozman, D. Lanosterol 14α-Demethylase (CYP51), NADPH-Cytochrome P450 Reductase and Squalene Synthase in Spermatogenesis: Late Spermatids of the Rat Express Proteins Needed to Synthesize Follicular Fluid Meiosis Activating Sterol. J. Endocrinol. 2000, 166, 463–474.CrossRefGoogle Scholar
  5. 5.
    Cotman, M.; Jezek, D.; Fon Tacer, K.; Frangez, R.; Rozman, D. A Functional Cytochrome P450 Lanosterol 14α-Demethylase CYP51 Enzyme in the Acrosome: Transport Through the Golgi and Synthesis of Meiosis Activating Sterols. >Endocrinology 2004, 145, 1419–1426.CrossRefGoogle Scholar
  6. 6.
    Zarn, J. A.; Bruschweiler, B. J.; Schlatter, J. R. Azole Fungicides Affect Mammalian Steroidogenesis by Inhibiting Sterol 14α-Demethylase and Aromatase. Environ. Health Perspect. 2003, 111, 255–262.CrossRefGoogle Scholar
  7. 7.
    Bellamine, A.; Mangla, A. T.; Nes, W. D.; Waterman, M. R. Characterization and Catalytic Properties of the Sterol 14α-Demethylase from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8937–8942.CrossRefGoogle Scholar
  8. 8.
    Lamb, D. C.; Fowler, K.; Kieser, T.; Manning, N.; Podust, L. M.; Waterman, M. R.; Kelly, D. E.; Kelly, S. L. Sterol 14α-Demethylase Activity in Streptomyces coelicolor A3(2) is Associated with an Unusual Member of the CYP51 Gene Family. Biochem. J. 2002, 364, 555–562.CrossRefGoogle Scholar
  9. 9.
    Baltsen, M.; Byskov, A. G. Quantitation of Meiosis Activating Sterols in Human Follicular Fluid Using HPLC and Photodiode Array Detection. Biomed. Chromatogr. 1999, 13, 382–388.CrossRefGoogle Scholar
  10. 10.
    Razzazi-Fazeli, E.; Kleineisen, S.; Luf, W. Determination of Cholesterol Oxides in Processed Food Using High-Performance Liquid Chromatography-Mass Spectrometry with Atmospheric Pressure Chemical Ionization. J. Chromatogr. A 2000, 896, 321–334.CrossRefGoogle Scholar
  11. 11.
    Nitahara, Y.; Aoyama, Y.; Horiuchi, T.; Noshiro, M.; Yoshida, Y. Purification and Characterization of Rat Sterol 14-Demethylase P450 (CYP51) Expressed in Escherichia coli. J. Biochem. 1999, 126, 927–933.Google Scholar
  12. 12.
    Penman, B. W.; Crespi, C. L. Analysis of Human Lymphoblast Mutation Assays by Using Historical Negative Control Data Bases. Environ. Mol. Mutagen. 1987, 10, 35–60.CrossRefGoogle Scholar
  13. 13.
    Stromstedt, M.; Rozman, D.; Waterman, M. R. The Ubiquitously Expressed Human CYP51 Encodes Lanosterol 14α-Demethylase, a Cytochrome P450 Whose Expression is Regulated by Oxysterols. Arch. Biochem. Biophys. 1996, 329, 73–81.CrossRefGoogle Scholar
  14. 14.
    Hashimoto, F.; Hayashi, H. Identification of Intermediates after Inhibition of Cholesterol Synthesis by Aminotriazole Treatment in Vivo. Biochim. Biophys. Acta 1991, 1086(115), 124.Google Scholar
  15. 15.
    Shyadehi, A. Z.; Lamb, D. C.; Kelly, S. L.; Kelly, D. E.; Schunck, W. H.; Wright, J. N.; Corina, D.; Akhtar, M. The Mechanism of the Acyl-Carbon Bond Cleavage Reaction Catalyzed by Recombinant Sterol 14α-Demethylase of Candida albicans (Other Names are: Lanosterol 14α-Demethylase, P-45014DM, and CYP51). J. Biol. Chem. 1996, 271, 12445–12450.CrossRefGoogle Scholar
  16. 16.
    Desbrow, C.; Routledge, E. J.; Brighty, G. C.; Sumpter, J. P.; Waldock, M. Identification of Estrogenic Chemicals in STW Effluent 1. Chemical Fractionation and In Vitro Biological Screening. Environ. Sci. Technol. 1998, 32, 1549–1558.CrossRefGoogle Scholar
  17. 17.
    Singh, G.; Gutierrez, A.; Xu, K.; Blair, I. A. Liquid Chromatography/Electron Capture Atmospheric Pressure Chemical Ionization/Mass Spectrometry: Analysis of Pentafluorobenzyl Derivatives of Biomolecules and Drugs in the Attomole Range. Anal. Chem. 2000, 72, 3007–3013.CrossRefGoogle Scholar
  18. 18.
    Higashi, T.; Takido, N.; Yamauchi, A.; Shimada, K. Electron-Capturing Derivatization of Neutral Steroids for Increasing Sensitivity in Liquid Chromatography/Negative Atmospheric Pressure Chemical Ionization-Mass Spectrometry. Anal. Sci. 2002, 18, 1301–1307.CrossRefGoogle Scholar
  19. 19.
    Greig, M.; Bolanos, B.; Quenzer, T.; Bylund, J. M. R. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Using Atmospheric Pressure Photoionization for High-Resolution Analyses of Corticosteroids. Rapid Commun. Mass Spectrom. 2003, 17, 2763–2768.CrossRefGoogle Scholar
  20. 20.
    Robb, D. B.; Covey, T. R.; Bruins, A. P. Atmospheric Pressure Photoionization: An Ionization Method for Liquid Chromatography-Mass Spectrometry. Anal. Chem. 2000, 72, 3653–3659.CrossRefGoogle Scholar
  21. 21.
    Byskov, A. G.; Andersen, C. Y.; Nordholm, L.; Thogersen, H.; Xia, G.; Wassmann, O.; Andersen, J. V.; Guddal, E.; Roed, T. Chemical Structure of Sterols that Activate Oocyte Meiosis. Nature 1995, 374, 559–562.CrossRefGoogle Scholar
  22. 22.
    Nassar, A. E.; Varshney, N.; Getek, T.; Cheng, L. Quantitative Analysis of Hydrocortisone in Human Urine Using a High-Performance Liquid Chromatographic-Tandem Mass Spectrometric-Atmospheric-Pressure Chemical Ionization Method. J. Chromatogr. Sci. 2001, 39, 59–64.Google Scholar
  23. 23.
    Peng, S. X.; Barbone, A. G.; Ritchie, D. M. High-Throughput Cytochrome P450 Inhibition Assays by Ultrafast Gradient Liquid Chromatography with Tandem Mass Spectrometry Using Monolithic Columns. Rapid Commun. Mass Spectrom. 2003, 17, 509–518.CrossRefGoogle Scholar
  24. 24.
    Headley, J. V.; Peru, K. M.; Verma, B.; Robarts, R. D. Mass Spectrometric Determination of Ergosterol in a Prairie Natural Wetland. J. Chromatogr. A. 2002, 958, 149–156.CrossRefGoogle Scholar
  25. 25.
    Joos, P. E.; Van Ryckeghem, M. Liquid Chromatography-Tandem Mass Spectrometry of Some Anabolic Steroids. Anal. Chem. 1999, 71, 4701–4710.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2004

Authors and Affiliations

  • Eva R. Trösken
    • 1
  • Ellen Straube
    • 1
  • Werner K. Lutz
    • 1
  • Wolfgang Völkel
    • 1
    Email author
  • Christopher Patten
    • 2
  1. 1.Department of ToxicologyUniversity of WürzburgWürzburgGermany
  2. 2.BD-GentestBD Biosciences Discovery LabwareWoburnUSA

Personalised recommendations