Abstract
Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3β-hydroxy-Δ5 structure were converted by cholesterol oxidase to 3-oxo-Δ4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.
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Björkhem, I.; Meaney, S.; Diczfalusy, U. Oxysterols in human circulation: Which role do they have? Curr. Opin. Lipidol. 2002, 13, 247–253.
Björkhem, I.; Diczfalusy, U. Oxysterols, friends, foes, or just fellow passengers? Arterioscler. Thromb. Vasc. Biol. 2002, 22, 734–742.
Axelson, M.; Mörk, B.; Sjövall, J. Occurrence of 3β-hydroxy-5-cholestenoic acid, 3β,7α-dihydroxy-5-cholestenoic acid, and 7α-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood J. Lipid Res. 1988, 29, 629–641.
Zhang, J.; Larsson, O.; Sjövall, J. 7α-Hydroxylation of 25-hydroxycholesterol and 27-hydroxycholesterol in human fibroblasts Biochim. Biophys. Acta. 1995, 1256, 353–359.
Lund, E.; Andersson, O.; Zhang, J.; Babiker, A.; Ahlborg, G.; Diczfalusy, U.; Einarsson, K.; Sjövall, J.; Björkhem, I. Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans Arterioscler. Thromb. Vasc. Biol. 1996, 16, 208–212.
Meaney, S.; Bonfield, T. L.; Hansson, M.; Babiker, A.; Kavuru, M. S.; Thomassen, M. J. Serum cholestenoic acid as a potential marker of pulmonary cholesterol homeostasis: Elevated levels in patients with pulmonary alveolar proteinosis J. Lipid Res. 2004, 45, 2354–2360.
Babiker, A.; Andersson, O.; Lindblom, D.; van der Linden, J.; Wiklund, B.; Lutjohann, D.; Diczfalusy, U.; Bjorkhem, I. Elimination of cholesterol as cholestenoic acid in human lung by sterol 27-hydroxylase: Evidence that most of this steroid in the circulation is of pulmonary origin J. Lipid Res. 1999, 40, 1417–1425.
Schroepfer, G. J. Oxysterols: Modulators of cholesterol metabolism and other processes Physiol. Rev. 2000, 80, 361–554.
Lehmann, J. M.; Kliewer, S. A.; Moore, L. B.; Smith-Oliver, T. A.; Oliver, B. B.; Su, J. L.; Sundseth, S. S.; Winegar, D. A.; Blanchard, D. E.; Spencer, T. A.; Willson, T. M. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway J. Biol. Chem. 1997, 272, 3137–3140.
Janowski, B. A.; Willy, P. J.; Devi, T. R.; Falck, J. R.; Mangelsdorf, D. J. An oxysterol signalling pathway mediated by the nuclear receptor LXRα Nature 1996, 383, 728–731.
Forman, B. M.; Ruan, B.; Chen, J.; Schroepfer, G. J.; Evans, R. M. The orphan nuclear receptor LXRα is positively and negatively regulated by distinct products of mevalonate metabolism Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10588–10593.
Janowski, B. A.; Grogan, M. J.; Jones, S. A.; Wisely, G. B.; Kliewer, S. A.; Corey, E. J.; Mangelsdorf, D. J. Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 266–271.
Song, C.; Liao, S. Cholestenoic acid is a naturally occurring ligand for liver X receptor α Endocrinology 2000, 141, 4180–4184.
Russell, D. W. Nuclear orphan receptors control cholesterol catabolism Cell. 1999, 97, 539–542.
Repa, J. J.; Mangelsdorf, D. J. The role of orphan nuclear receptors in the regulation of cholesterol homeostasis Annu. Rev. Cell Dev. Biol. 2000, 16, 459–481.
Brown, A. J.; Jessup, W. Oxysterols and atherosclerosis Atherosclerosis. 1999, 142, 1–28.
Lin, Y. Y.; Welch, M.; Liebermann, S. The detection of 20(S)-hydroxycholesterol in extracts of rat brains and human placenta by a gas chromatograph/mass spectrometry technique J. Steroid Biochem. Mol. Biol. 2003, 85, 57–61.
Yao, Z. X.; Brown, R. C.; Teper, G.; Greeson, J.; Papadopoulos, V. 22-Hydroxycholesterol protects neuronal cells from β-amyloid-induced cytotoxicity by binding to β-amyloid peptide J. Neurochem. 2002, 83, 1110–1119.
Axelson, M.; Larsson, O.; Zhang, J.; Shoda, J.; Sjövall, J. Structural specificity in the suppression of HMG-CoA reductase in human fibroblasts by intermediates in bile acid biosynthesis J. Lipid Res. 1994, 36, 290–298.
Bjorkhem, I.; Reihner, E.; Angelin, B.; Ewerth, S.; Akerlund, J. E.; Einarsson, K. On the possible use of the serum level of 7α-hydroxycholesterol as a marker for increased activity of the cholesterol 7α-hydroxylase J. Lipid Res. 1987, 28, 889–894.
Axelson, M.; Aly, A.; Sjövall, J. Levels of 7α-hydroxy-4-cholesten-3-one in plasma reflects rates of bile acid synthesis in man FEBS Lett. 1988, 239, 324–328.
Axelson, M.; Björkhem, I.; Reihner, E.; Einarsson, K. Plasma level of 7α-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7α-hydroxylase in man FEBS Lett. 1991, 284, 216–218.
Björkhem, I.; Diczfalusy, U.; Lütjohann, D. Removal of cholesterol from extrahepatic sources by oxidative mechanisms Curr. Opin. Lipidol. 1999, 10, 161–165.
Lund, E. G.; Guileyaedo, J. M.; Russel, D. W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7238–7234.
Björkhem, I.; Andersson, U.; Ellis, E.; Alvelius, G.; Ellegard, L.; Diczfalusy, U.; Sjövall, J.; Einarsson, C. From brain to bile. Evidence that conjugation and ω-hydroxylation are important for elimination of 24S-hydroxycholesterol (cerebrosterol) in humans J. Biol. Chem. 2001, 276, 37004–37010.
Bodin, K.; Bretillon, L.; Aden, Y.; Bertilsson, L.; Broome, U.; Einarsson, C.; Diczfalusy, U. Antiepileptic drugs increase plasma levels of 4β-hydroxycholesterol in humans: Evidence for involvement of cytochrome P450 3A4 J. Biol. Chem. 2001, 276, 38685–38689.
Wentworth, P.; McDunn, J. E.; Wentworth, A. D.; Takeuchi, C.; Nieva, J.; Jones, T.; Bauttista, C.; Ruedi, J. M.; Gutierrez, A.; Janda, K. A.; Babior, B. M.; Eschenmoser, A.; Lerner, R. A. Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation Science. 2002, 298, 2195–2199.
Wentworth, P.; Nieva, J.; Takeuchi, C.; Galve, R.; Wentworth, A. D.; Dilley, R. B.; DeLaria, G. A.; Saven, A.; Babior, B. M.; Janda, K. D.; Eschenmoser, A.; Lerner, R. A. Evidence for ozone formation in human atherosclerotic arteries Science. 2003, 302, 1053–1056.
Zhang, Q.; Powers, E. T.; Nieva, J.; Huff, M. E.; Dendle, M. A.; Bieschke, J.; Glabe, C. G.; Eschenmoser, A.; Wentworth, P.; Lerner, R. A.; Kelly, J. W. Metabolite-initiated protein misfolding may trigger Alzheimer’s disease Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4752–4757.
Pulfer, M. K.; Murphy, R. C. Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant J. Biol. Chem. 2004, 279, 26331–26338.
Dzeletovic, S.; Breuer, O.; Lund, E.; Diczfalusy, U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry Anal. Biochem. 1995, 225, 73–80.
Breuer, O.; Björkhem, I. Simultaneous quantification of several cholesterol autoxidation and monohydroxylation products by isotope-dilution mass spectrometry Steroids. 1990, 55, 185–192.
Zhang, Z.; Li, D.; Blanchard, D. E.; Lear, S. R.; Erickson, S. K.; Spencer, T. A. Key regulatory oxysterols in liver: analysis as Δ4-3-ketone derivatives by HPLC and response to physiological perturbations J. Lipid Res. 2001, 42, 649–658.
Ogishima, T.; Okuda, K. An improved method for assay of cholesterol 7-α-hydroxylase activity Anal. Biochem. 1986, 158, 228–232.
Griffiths,W. J.; Liu,S.; Alvelius,G.; Sjövall,J. Derivatives Revisited: Analysis of Neutral Steroids by ESMS. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, June, 2002.
Griffiths, W. J.; Alvelius, G.; Liu, S.; Hornshaw, M.; Sjövall, J. Analysis of Oxysterols in Brain. Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics; Nashville, TN, May, 2004.
Wang, Y.; Alvelius, G.; Liu, S.; Bodin, K.; Hornshaw, M. ; Sjövall, J.; Griffiths, W. J. Steroidomics of Brain. Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics; San Antonio, TX, June, 2005.
Griffiths, W. J.; Liu, S.; Alvelius, G.; Sjövall, J. Derivatization for the characterization of neutral oxosteroids by electrospray and matrix-assisted laser desorption/ionization tandem mass spectrometry: The Girard P derivative Rapid Commun. Mass Spectrom. 2003, 17, 924–935.
Griffiths, W. J.; Alvelius, G.; Liu, S.; Sjövall, J. High-energy collision-induced dissociation of oxosteroids derivatized to Girard hydrazones Eur. J. Mass Spectrom. 2004, 10, 63–88.
Shackleton, C. H. L.; Chuang, H.; Kim, J.; de la Torre, X.; Segura, J. Electrospray mass spectrometry of testosterone esters: Potential for use in doping control Steroids. 1997, 62, 523–529.
Dharmasiri,K. A. N.; Huang,Z.-H.; Watson,J. T. Derivatives for detection of anabolic ketosteroids by positive electrospray mass spectrometry. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics; San Francisco, CA, May, 1993.
Lai, C. C.; Tsai, C. H.; Tsai, F. J.; Lee, C. C.; Lin, W. D. Rapid monitoring assay of congenital adrenal hyperplasia with microbore high-performance liquid chromatography/electrospray ionization tandem mass spectrometry from dried blood spots Rapid Commun. Mass Spectrom. 2001, 15, 2145–2151.
Lai, C. C.; Tsai, C. H.; Tsai, F. J.; Wu, J. Y.; Lin, W. D.; Lee, C. C. Monitoring of congenital adrenal hyperplasia by microbore HPLC-electrospray ionization tandem mass spectrometry of dried blood spots Clin. Chem. 2002, 48, 354–356.
Lai, C. C.; Tsai, C. N.; Tsai, F. J.; Wu, J. Y.; Lin, W. D.; Lee, C. C. Rapid screening assay of congenital adrenal hyperplasia by measuring 17 α-hydroxyprogesterone with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry from dried blood spots J. Clin. Lab. Anal. 2002, 16, 20–25.
Higashi, T.; Shimada, K. Derivatization of neutral steroids to enhance their detection characteristics in liquid chromatography-mass spectrometry Anal. Bioanal. Chem. 2004, 378, 875–882.
Shoda, J.; Axelson, M.; Sjövall, J. Synthesis of potential C27-intermediates in bile acid biosynthesis and their deuterium-labeled analogs Steroids 1993, 58, 119–125.
Brooks, C. J. W.; Cole, W. J.; Lawrie, T. D. V.; MacLachlan, J.; Borthwick, J. H.; Barrett, G. M. Selective reactions in the analytical characterization of steroids by gas chromatography-mass spectrometry J. Steroid Biochem. 1983, 19, 189–210.
MacLachlan, J.; Wotherspoon, A. T. L.; Ansell, R. O.; Brooks, C. J. W. Cholesterol oxidase: Sources, physical properties, and analytical applications J. Steroid Biochem. Mol. Biol. 2000, 72, 169–195.
Liu, S.; Sjövall, J.; Griffiths, W. J. Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes Rapid Commun. Mass Spectrom. 2000, 14, 390–400.
Liu, S.; Sjövall, J.; Griffiths, W. J. Neurosteroids in rat brain: Extraction, isolation, and analysis by capillary column liquid chromatography-electrospray mass spectrometry Anal. Chem. 2003, 75, 5835–5846.
Wheeler, O. H. The Girard reagents J. Chem. Educ. 1968, 45, 435–437.
Williams, T. M.; Kind, A. J.; Houghton, E.; Hill, D. W. Electrospray collision induced dissociation of testosterone and testosterone hydroxy analogs J. Mass Spectrom. 1999, 34, 206–216.
Griffiths, W. J. Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids Mass Spectrom. Rev. 2003, 22, 81–152.
Lütjohann, D.; Brzezinka, A.; Barth, E.; Abramowski, D.; Staufenbiel, M.; von Bergmann, K.; Beyreuther, K.; Multhaup, G.; Bayer, T. A. Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain J. Lipid Res. 2002, 43, 1078–1085.
Clifton, P. M.; Noakes, M.; Sullivan, D.; Erichsen, N.; Ross, D.; Annison, G.; Fassoulakis, A.; Cehun, M.; Nestel, P. Cholesterol-lowering effects of plant sterol esters differ in milk, yoghurt, bread, and cereal. Eur. J. Clin. Nutr. 2004, 58, 503–509.
Fahy, D. M.; O’Callaghan, Y. C.; O’Brien, N. M. Phytosterols: Lack of cytotoxicity but interference with β-carotene uptake in Caco-2 cells in culture Food Addit. Contam. 2004, 21, 42–51.
Dhar, A. K.; Teng, J. I.; Smith, L. L. Biosynthesis of cholest-5-ene-3β, 24-diol (cerebrosterol) by bovine cortical microsomes J. Neucochem. 1973, 21, 51–60.
Meaney, S.; Babiker, A.; Lütjohann, D.; Diczfalusy, U.; Axelson, M.; Björkhem, I. On the origin of cholestenoic acids in human circulation Steroids 2003, 68, 595–601.
Zhang, J.; Akwa, Y.; El-Etr, M.; Baulieu, E. E.; Sjövall, J. Metabolism of 27-, 25-, and 24-hydroxycholesterol in rat glial cells and neurons Biochem. J. 1997, 322, 175–184.
Zhang, J.; Akwa, Y.; Baulieu, E. E.; Sjövall, J. 7α-Hydroxylation of 27-hydroxycholesterol in rat brain microsomes C. R, Acad. Sci. III. 1995, 318, 345–349.
Mast, N.; Norcross, R.; Andersson, U.; Shou, M.; Nakayama, K.; Bjorkhem, I.; Pikuleva, I. A. Broad substrate specificity of human cytochrome P450 46A1 which initiates cholesterol degradation in the brain Biochemistry 2003, 42, 14284–14292.
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Published online January 25, 2006
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Griffiths, W.J., Wang, Y., Alvelius, G. et al. Analysis of oxysterols by electrospray tandem mass spectrometry. The official journal of The American Society for Mass Spectrometry 17, 341–362 (2006). https://doi.org/10.1016/j.jasms.2005.10.012
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DOI: https://doi.org/10.1016/j.jasms.2005.10.012