Establishment of a GC-MS/MS Methodology for Comprehensive Sex Steroid Profiling in Mouse Serum

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-373
Maria E Nilsson*1, Liesbeth Vandenput2, Ã…sa Tivesten1, Anna-Karin Norlén1, Henrik Ryberg1 and Claes Ohlsson3
1Sahlgrenska University Hospital, Gothenburg, Sweden, 2University of Gothenburg, Gothenburg, Sweden, 3Sahlgrenska University Hospital, Gothenburg, Sweden
There are two major differences in sex steroid metabolism between humans and mice. (i) A substantial portion of serum sex steroids are bound with high affinity to SHBG in humans, while mice do not have SHBG. (ii) Humans, but not mice, secrete substantial amounts of C-19 androgen precursors (DHEA and Androstenedione [A]) from the adrenal gland. These differences influence the total serum levels of sex steroids and sex steroid precursors. Hence, the serum levels of sex steroids/sex steroid precursors are expected to be relatively low and difficult to determine in mice. Serum levels of sex steroids in mice have routinely been analyzed using immuno-assay based techniques, having questionable specificity in the lower range. The aim of the present study was to develop a sensitive and specific GC-MS/MS methodology for measurement of a comprehensive sex steroid profile in mice.

We established a GC-MS/MS method for the measurement of serum testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), DHEA, A, and progesterone (P). After addition of isotope-labeled standards, steroids were extracted to chlorobutane, purified on a silica-column and derivatized using pentafluorobenzyl hydroxylamine hydrochloride followed by pentafluorobenzoyl chloride. Steroids were analyzed in MRM-mode with ammonia as reagent gas.

The lower limits of quantification (LLOQ) for T, DHT, E2, E1, DHEA, A, and P were 25, 5, 2, 2, 50, 12, and 50 pg/ml, respectively. The serum levels of all these sex steroids, except T and E1, were above the LLOQ when analyzed in ≥250 µl female mouse serum (average levels: T 14, DHT 13, E2 2.4, E1 0.5, DHEA 163, A 189, and P 13912 pg/ml). E2 and E1 were undetectable in male serum but all other sex steroids were reliably measured using 100 µl of male mouse serum (average levels: T 8235, DHT 168, DHEA 149, A 207, and P 680 pg/ml). As expected, orchidectomy (orx) resulted in a dramatic reduction of T, DHT and A. In contrast, serum P was substantially increased in orx mice (+404%, p<0.01) and E2 treatment of orx mice normalized serum P, suggesting that testicle-derived T is involved in the regulation of circulating levels of adrenal-derived P via an estrogenic mechanism in male mice.

In conclusion, our developed GC-MS/MS-based methodology for the measurement of a comprehensive serum sex steroid profile in mice might be useful for the characterization of sex steroid metabolism in a variety of sex-steroid related transgenic mouse models.

Nothing to Disclose: MEN, LV, T, AKN, HR, CO

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