Liquid chromatography–tandem mass spectrometry analysis of human adrenal vein corticosteroids before and after adrenocorticotropic hormone stimulation

Abstract

Context Although steroid hormones produced by the adrenal gland play critical roles in human physiology, a detailed quantitative analysis of the steroid products has not been reported. The current study uses a single methodology (liquid chromatography-tandem mass spectrometry, LC-MS/MS) to quantify ten corticosteroids in adrenal vein (AV) samples pre and post adrenocorticotropic hormone (ACTH) stimulation. Design/methods Three men and six women with a diagnosis of an adrenal aldosterone-producing adenoma (APA) were included in the study. Serum was collected from the iliac vein (IV) and the adrenal vein (AV) contralateral to the diseased adrenal. Samples were collected, before and after administration of ACTH. LC-MS/MS was then used to quantify serum concentrations of unconjugated corticosteroids and their precursors. Results Prior to ACTH stimulation the four most abundant steroids in AV were cortisol (90%), cortisone (4%), corticosterone (3%) and 11-deoxycortisol (0.8%). Post ACTH administration, cortisol remained the major adrenal product (79%), however, corticosterone became the second most abundantly produced adrenal steroid (11%) followed by pregnenolone (2.5%) and 17α-hydroxypregnenolone (2%). ACTH significantly increased the absolute adrenal output of all ten corticosteroids measured (P<0.05). The four largest post ACTH increases were pregnenolone (300-fold), progesterone (199-fold), 17α-hydroxypregnenolone (187-fold) and deoxycorticosterone (82-fold). Conclusion Using LC-MS/MS we successfully measured 10 corticosteroids in peripheral and adrenal vein serum samples under pre and post ACTH stimulation. This study demonstrates the primary adrenal steroid products and their response to ACTH.

Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method

“…Except in a few studies [22,23], aldosterone was always quantified without any derivatization step, as we report it here. The tendency is for analyzing aldosterone like DHEAS in the negative mode because of a higher signal-tonoise ratio [7][8][9][10][11][12]16,[24][25][26][27].…”

Regulation of Human 3β-Hydroxysteroid Dehydrogenase Type 2 by Adrenal Corticosteroids and Product-Feedback by Androstenedione in Human Adrenarche

“…Relative to purified, solubilized 3bHSD2, cortisol is a 2.5-fold better inhibitor of the liposome-bound enzyme, although even the liposome-bound K i of 217 mM greatly exceeds the concentration of intra-adrenal cortisol (Dickerman et al, 1984;Nakamura et al, 2012). The inhibition mode of cortisol for both purified 3bHSD2 preparations is uncompetitive, unlike the competitive mode found in 3bHSD2-tranfected COS-7 cells (Topor et al, 2011).…”

“…Cortisone may also competitively inhibit adrenal 3bHSD2, but much higher concentrations compared with androstenedione are required based on the measured K i values. The liposome-bound 3bHSD2 K i value for cortisone (34 mM) is considerably higher than reported human adrenal levels (Dickerman et al, 1984;Nakamura et al, 2012). Based on the liposomal 3bHSD2 K i 5 217 mM, cortisol appears to be a very low-affinity, uncompetitive inhibitor of 3bHSD2 that requires the enzyme to be bound to lipid membranes for inhibition even at high, nonphysiologic levels (Dickerman et al, 1984;Nakamura et al, 2012).…”

Regulation of human 3-beta-hydroxysteroid dehydrogenase type-2 (3βHSD2) by molecular chaperones and the mitochondrial environment affects steroidogenesis

“…112 The R195P-1 mutation shifts the high-affinity, competitive 113 inhibition of wild-type 3b-HSD1 by trilostane to the much lower 114 affinity, noncompetitive inhibition profile similar to that of 3b-115 HSD2 containing Pro195. The P195R-2 mutation of 3bHSD2 shifts 116 the low-affinity, noncompetitive inhibition profile of wild-type 117 3b-HSD2 by trilostane to a high affinity, competitive inhibition 118 profile similar to that of 3b-HSD1 containing Arg195. The predicted roles of key residues that are non-identical in 3b-120 HSD1 and 3b-HSD2 are being tested by the creation of chimeric 121 mutants of each isoenzyme using site-directed mutagenesis.…”

“…We 701 determined the kinetics of inhibition of 3bHSD2 by cortisol, 702 cortisone and androstenedione and found that cortisol is a very low 703 affinity, uncompetitive inhibitor of solubilized 3bHSD2 (K i = 542 704 mM). Cortisol is a 2.5-fold better inhibitor of the liposome-bound 705 enzyme, although even the liposome-bound K i of cortisol greatly 706 exceeds the concentration of intra-adrenal cortisol[117,118].707 Cortisone is a 1.6-fold more effective competitive inhibitor of 708 liposomal than solubilized 3bHSD2 and is a much more effective 709 inhibitor of 3bHSD2 than cortisol, having 10-fold and 6.4-fold710 lower K i values than does cortisol for the solubilized and liposomal 711 enzymes, respectively. On the other hand,…”