Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein 19-carbon steroids before and after ACTH stimulation

Abstract

Context: A broad analysis of adrenal gland-derived 19-carbon (C19) steroids has not been reported. This is the first study that uses liquid chromatography-tandem mass spectrometry to quantify 9 C19 steroids (androgens and their precursors), estrone, and estradiol in the adrenal vein (AV) of women, before and after ACTH stimulation.

Objective: The objective of this study was to define the adrenal androgen metabolome in women before and after ACTH infusion.

Design: This was a retrospective study.

Patients: Seven women, aged 50.4 ± 5.4 years, with suspected diagnosis of an adrenal aldosterone-producing adenoma were included in the study.

Methods: AV and iliac serum samples were collected before and after administration of ACTH (15 minutes). AV samples were analyzed using for concentrations of 9 unconjugated C19 steroids, estrone, and estradiol. Dehydroepiandrosterone sulfate (DHEA-S) was quantified by radioimmunoassay.

Results: AV levels of DHEA-S were the highest among the steroids measured. The most abundant unconjugated C19 steroids in AV were 11β-hydroxyandrostenedione (11OHA), dehydroepiandrosterone (DHEA), and androstenedione (A4). ACTH significantly increased the adrenal output of 9 of the 12 steroids that were measured. ACTH increased the mean AV concentration of DHEA-S by 5-fold, DHEA by 21-fold, A4 by 7-fold, and 11OHA by 5-fold. 11β-Hydroxytestosterone and testosterone were found to be potent androgen receptor agonists when tested with an androgen-responsive cell reporter model.

Conclusion: The current study indicates that the adrenal gland secretes primarily 3 weak androgens, namely DHEA, 11OHA, and A4. Active androgens, including testosterone and 11β-hydroxytestosterone, are also produced but to a lesser degree.

Figure 1.

C₁₉ steroid effects on AR activity. MDA-kb2 cells, containing human androgen receptor and an androgen receptor-driven luciferase reporter, were treated with increasing concentrations of testosterone, 11OHT, and 11KT (A) and A4, 11OHA, and 11KA (B). After 16 hours, luciferase activity was measured in cell lysates. Data are presented as mean fold induction compared with basal controls ± SEM (n > 4 independent experiments). C, EC₅₀ for the tested C₁₉ steroids.

Figure 2.

Microarray analysis of steroidogenic enzymes involved in androgen (A) and estrogen (B) synthesis in the human adrenal glands. Dotted lines indicate that the reaction appears to be negligible in the adrenal. The enzyme transcripts present at the highest levels in the adrenal are marked in red and those with negligible expression are marked in green. The steroids that are not produced by the adrenal are marked in blue. AKR1C3, type 5 17β-hydroxysteroid dehydrogenase; CYB5, cytochrome b₅; CYP17, 17α-hydroxylase/17,20-lyase; CYP19, aromatase; HSD3B2, type 2 3β-hydroxysteroid dehydrogenase; HSD17B1, type 2 17β-hydroxysteroid dehydrogenase; HSD17B2, type 2 17β-hydroxysteroid dehydrogenase; HSD17B3, type 3 17β-hydroxysteroid dehydrogenase; SRD5A2, type 2 5α-reductase; SULT2A1, steroid sulfotransferase 2A1.