Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat

Abstract

The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatographyelectrospray ionization timeof-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.

Diethylstilbestrol decreased adrenal cholesterol and corticosterone in rats

“…The samples were prepared, and the MS analysis was carried out according to our previously reported method (Duncan et al 2009, Maeda et al 2013a. Briefly, the samples (rat blood and adrenal glands) were homogenised with acetonitrile, and the supernatants were purified using two columns.…”

“…For the identification of the steroids and conjugates in the samples, the various compounds present in the blood and adrenal gland preparations were separated using HPLC (UFLC Nexera; Shimadzu, Kyoto, Japan) and an L-column 2 (C18, 2.1!150 mm, 2 mm particle size; Chemicals Evaluation and Research Institute, Tokyo, Japan) at a flow rate of 200 ml/min (40 8C). The chemical formula for each steroid was calculated and identified via LC-TOF MS (microTOF-QII; Bruker Daltonics, Bremen, Germany) using mass error and SigmaFit, as previously reported (Maeda et al 2013a). Finally, the steroid levels were quantified with LC-MS/MS (LC: Ultimate 3000, MS: TSQ Quantum Ultra; Thermo Fisher Scientific, San Jose, CA, USA) as described in our previous report (Maeda et al 2013a).…”

“…The chemical formula for each steroid was calculated and identified via LC-TOF MS (microTOF-QII; Bruker Daltonics, Bremen, Germany) using mass error and SigmaFit, as previously reported (Maeda et al 2013a). Finally, the steroid levels were quantified with LC-MS/MS (LC: Ultimate 3000, MS: TSQ Quantum Ultra; Thermo Fisher Scientific, San Jose, CA, USA) as described in our previous report (Maeda et al 2013a).…”

“…2, see section on supplementary data given at the end of this article, and determined using MS analysis (Maeda et al 2013a). …”

“…After 1 h of treatment, the DES concentration in various organs was determined using an accurate LC-MS-based method that was previously developed by our group (Maeda et al 2013a), and the results are shown in Fig. 1.…”