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Methodology for Profiling the Steroid Metabolome in Animal Tissues Using Ultraperformance Liquid Chromatography-Electrospray-Time-of-Flight Mass Spectrometry Anel M. Flores-Valverde and Elizabeth M. Hill* Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Falmer, Brighton, U.K. BN1 9QG The advent of mass spectrometry-based metabolite profiling techniques should allow investigations into the behavior and regulation of many of the low abundant signaling molecules present in the animal metabolome such as hormones and so aid investigations into the mechanisms of endocrine system function and disorders. Examples of their potential applications include endocrinerelated diseases such as some cancers, as well as endocrine disruption in wildlife and humans caused by some environmental contaminants. In the present study, a method was developed to profile a variety of vertebrate steroids and their conjugates in fish tissues using solid phase extraction (SPE) prior to ultraperformance liquid chromatography linked to electrospray-time-of-flight mass spectrometry (UPLC-TOF MS). Analysis of tissue extracts spiked with steroids revealed that most analytes could be detected at subnanogram per gram concentrations in liver or testes, but ovarian extracts caused ion supression of estrogens. A comparison of the steroid metabolome of the ovaries and testes using partial least-squares discrimination analysis (PLS-DA) revealed that the androgens 11ketotestosterone, 11-hydroxyandrostenedione, androstenedione,andtwounidentifiedcortisol-typeglucocorticoids were discriminatory steroid markers in testes extracts. In contrast, cortisol was the predominant glucocorticoid marker in ovary extracts, and cortisone was abundant in extracts of both testes and ovaries. A number of other unidentified nonsteroidal metabolites were also differentially expressed between the testes and ovarian tissues. These studies reveal that metabolite profiling using UPLC-TOF MS is a promising approach to investigate steroid homeostasis in animal tissues. Metabolomics is the study of the changes in the metabolite profiles within organisms which may occur, for instance, as a result of normal physiological processes, from onset of disease, or from exposure to environmental toxins or other stressors.1,2 Electrospray ionization mass spectrometry (ESI-MS) is increas* To whom correspondence should be addressed. Phone: +44 1273 678382. Fax: 44 1273 877586. E-mail:
[email protected]. (1) Kaddurah-Daouk, R.; Kristal, B. S.; Weinshilboum, R. M. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 653–683. (2) Viant, M. R. Mar. Ecol: Prog. Ser. 2007, 332, 301–306. 10.1021/ac8014966 CCC: $40.75 2008 American Chemical Society Published on Web 10/24/2008
ingly being used for profiling metabolites commonly ranging from 50-2000 amu, as it enables detection of many biologically important molecules at the subnanogram level.3,4 However a common problem of ESI-MS technology is the observed ion suppression of less abundant or less ionizable metabolites by other components in the sample.5 Therefore one of the challenges of using this technology for metabolomics is to develop analytical approaches to enable profiling of less abundant molecules, for example, steroids which are often present in tissues or body fluids at 99% D atom), were purchased from Cambridge Isotope Laboratories Inc. (MA), while estradiol 2,4,16,16-d4 (E2-d4, 95-97% D atom) and progesterone-2,2,4,6,6,17R,21,21,21-d9 (P-d9, 98% D atom) were purchased from CDN isotopes (Quebec, Canada). All other standards and reagents chemicals were purchased from Steroloids Inc., Newport, RI and Sigma-Aldrich Company Ltd., Dorset, U.K. All solvents were high-performance liquid chromatography grade, purchased from Rathburn Chemicals Ltd., Walkerburn, Scotland, U.K. DSC-NH2 SPE cartridges (100 mg) were supplied from Supelco, Sigma-Aldrich Ltd., Dorset, U.K. StrataX-AW SPE cartridges (33 µm sized particles, 200 mg, 6 mL) were purchased from Phenomenex Ltd., Macclesfield, Cheshire, U.K. (14) Peters, R. E. M.; Courtenay, S. C.; Cagampan, S.; Hewitt, M. L.; MacLatchy, D. L. Aquat. Toxicol. 2007, 85, 154–166. (15) Lee, S. H.; Woo, H. M.; Jung, B. H.; Lee, J. G.; Kwon, O. S.; Pyo, H. S.; Choi, M. H.; Chung, B. C. Anal. Chem. 2007, 79, 6102–6110. (16) Lutz, U.; Lutz, R. W.; Lutz, W. K. Anal. Chem. 2006, 78, 4564–4571. (17) Wang, Y. Q.; Griffiths, W. J. Neurochem. Int. 2008, 52, 506–521. (18) Gibson, R.; Smith, M. D.; Spary, C. J.; Tyler, C. R.; Hill, E. M. Environ. Sci. Technol. 2005, 39, 2461–2471. (19) Wilson, I. D.; Plumb, R.; Granger, J.; Major, H.; Williams, R.; Lenz, E. A. J. Chromatogr., B 2005, 817, 67–76.
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Tissue Extraction. Sexually mature roach (Rutilus rutilus) were supplied by Jon Wall Fisheries (Melton Mowbray, Leics, U.K.) in November 2005 and acclimatized for 1 week prior to experimental studies. Details of fish and their holding conditions are given in Supporting Information, Table S-1. Ovaries (2.0-2.6 g), testes (0.8-1.1 g), and liver (0.1-0.2 g) were extracted with two volumes of methanol (i.e., 2 mL g-1) using an Microson XL2000 ultrasonic probe (Misonix Farmingdale) (gonads 18 W × 2 min, liver 6 W × 1 min). Samples were centrifuged (3000g, 20 min), the supernatant collected, and the remaining pellets were extracted again with two volumes of methanol. The supernatants of each sample were combined, reduced under vacuum to 1 mL, and diluted with sodium acetate buffer (50 mM, pH 7.0) so that the final methanol concentration was 0.5) were analyzed using orthogonal PLS (OPLS) to select data that was only due to class separation. The most discriminative variables (retention time, RT, × m/z data) between male and female roach were then visualized using an “S”-plot which is a scatter plot of covariance and correlation loading profiles resulting from the OPLS model (see Wicklund et al.20 for further explanation). Significant metabolite markers were ranked according to their p-value. However because the number of false positives (type 1 errors) in the statistical analysis of the data were likely to be high due to the abundance of variables being tested,21 then an estimate of the false discovery rate (FDR) was made using the q-value to assign a significance to each metabolite marker. The q-values were calculated from the p-values of markers in the whole data set according to Storey et al.22 using q-value software obtained from http://genomics.princeton.edu/storeylab/qvalue/index.html. Metabolites with q-values e0.05 were deemed to be significantly different between the sex classes. The m/z signals of significant discriminatory markers were noted at between 400-1000 counts per second (cps) in the mass spectra of the samples to avoid inaccuracies in mass measurement due to TOF dead time in the mass detector. The observed masses were used to calculate theoretical masses of candidate molecules with the elemental composition tool (version 4.00) of the MassLynx software. Once obtained, this information was compared with the RT and accurate mass of standard compounds. For further identification of the androgen markers, some samples were combined and fractionated by UPLC at 0.5 min intervals using the same LC conditions as previously described above. Androgens in the fractions were analyzed by GC/MS. GC/MS Analysis of Marker Steroids. Steroids were derivatized to their O-methyloxime (MO)-trimethylsilyl ethers (TMS) prior to GC/MS. For the preparation of MO-TMS derivatives, fractions containing the markers were dried under vacuum and incubated for 30 min at 80 °C in 50 µL of methoxyamine hydrochloride in pyridine 2% (w/w). The reagent was evaporated under nitrogen, and the residue was dissolved in 80 µL of 1:1 pyridine and BSTFA, (1% TMCS). After heating at 65 °C for 20 min, the sample volume was reduced to 3. Steroids were extracted using Strata-X-AW and DSC-NH2 cartridges and analyzed using UPLC-ESITOF MS metabolite profiling methods and quantified using external calibration. b Steroids were extracted using Strata-X-AW and DSC-NH2 cartridges and analyzed using UPLC-ESI-TOF MS metabolite profiling methods and quantified using internal calibration.
or DSC-NH2 SPE cartridges (see Table S-2 in the Supporting Information) which suggested that there was little ion suppression of steroids in this matrix. The detection of steroids recovered from testes extracts was generally poorer in comparison to the SPE recovery from standard solutions but less so where an internal standard was used for quantification, which suggested that losses in signal of many of the steroids was due to ion suppression of the testes extract. None of the spiked (hydroxy)estrogens were detected in ovary extracts despite recoveries from standard solutions of >74% on Strata-X-AW or DSC-NH2 SPE, indicating a severe matrix effect of ovary extracts. Nevertheless a range of steroids could be detected in extracts of liver and testes tissue, as well as androgens and glucocorticoids in extracts of ovarian tissue, at estimated LODs of low nanogram per gram concentrations. Concentrations of free and conjugated sex steroids in gonads of juvenile fish have been reported as between 0.25-16 ng g-1,25 which indicates that the level of detection reported in this work would be sufficient to detect endogenous steroids present in fish liver or testes tissues. These results also suggest that for metabolite profiling it is necessary to assess both which analytes can and which cannot be detected by the analytical methodology used, in order to avoid making false conclusions on the presence of metabolites in the tissue sample. Comparision of Steroid Profiles in Tissues from Male and Female Fish. UPLC-TOF MS profiling of gonad and liver extracts from eight female and eight male fish revealed detection (25) Labadie, P.; Budzinski, H. Chemosphere 2006, 64, 1274–1286.
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of endogenous steroids in all the tissues despite high signals of other components of the metabolome present in the samples. Representative ion chromatograms of the SPE fractions from the tissue extracts are given in parts A-C of Figure 2, together with the base peak ion chromatogram (BPI) of all the other components in the sample. Steroids were identified by retention time and exact mass data (m/z accuracy of
