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. 2013 Jan;153(1):63-71.
doi: 10.1093/jb/mvs121. Epub 2012 Oct 10.

Accurate Determination of Tissue Steroid Hormones, Precursors and Conjugates in Adult Male Rat

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Free PMC article

Accurate Determination of Tissue Steroid Hormones, Precursors and Conjugates in Adult Male Rat

Naoyuki Maeda et al. J Biochem. .
Free PMC article

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 chromatography-electrospray ionization time-of-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.

Figures

Fig. 1
Fig. 1
Preparation of lipophilic steroids and hydrophilic conjugate forms of the steroids from various organs for MS analysis.
Fig. 2
Fig. 2
Critical steps for separation of lipophilic steroids and hydrophilic conjugate forms of the steroids and removing various contaminants from the extracted preparations of organs.
Fig. 3
Fig. 3
Identification and determination of steroids by the SigmaFit algorithm and LC-TOF MS analysis. The chromatograms of the preparations from the testis by TOF MS analysis developed in this study (A) and theoretical abundances of testosterone containing an isotope (B). A δ-value was calculated by the program ‘sigmaFit algorithm’ after comparing the two chromatograms A and B. The δ-value between 0 and 1.0 shows higher identification. In this case, small δ-value was observed at 0.0118 and the mass error was only 2.0 mDa showing that TS in the sample was identified. The identification of other steroids was performed by the same procedure using sigmaFit algorithm. Identically, TS precursors were identified from testis. Product-ion spectrums were obtained for TS by MS/MS analysis (C). SRM of TS was used for quantitative analysis as described in Materials and Methods section (D).

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