Measurement of steroid synthesis in zona glomerulosa cells by liquid chromatography-electrospray ionization-mass spectrometry: inhibition by nitric oxide.
A liquid chromatography-electrospray ionization-mass spectrometry method was developed to simultaneously determine the concentrations of aldosterone, corticosterone, cortisol, deoxycorticosterone, pregnenolone, and progesterone in bovine adrenal zona glomerulosa (ZG) cells. Steroids were extracted by liquid-liquid extraction, separated on a reverse-phase C18 column, ionized by electrospray, and detected by single-quadrupole mass spectrometry in a positive ion mode. All steroids formed sodium adducts at high abundance. Factors affecting the formation and signal of sodium adducts were investigated. The limits of detection (S/N=3) using selected ion monitoring are 2 pg for these steroids and 10 pg for pregnenolone. DETA NONOate, a nitric oxide donor, inhibited the basal, angiotensin-II-stimulated, and 25-hydroxycholesterol-stimulated syntheses of these steroids in ZG cells in a concentration-dependent manner. The technique demonstrates the ability to determine the individual steroid in each enzymatic step of aldosterone synthesis and the activity of steroidogenic enzymes in adrenal ZG cells.
Nithipatikom
Kasem
K
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Holmes
Blythe B
BB
Isbell
Marilyn A
MA
Hanke
Craig J
CJ
Gomez-Sanchez
Celso E
CE
Campbell
William B
WB
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003903:Deuterium; D009569:Nitric Oxide / Q000378:metabolism*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013256:Steroids / Q000096:biosynthesis*; D015384:Zona Glomerulosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D013256:Steroids; D000804:Angiotensin II; D009569:Nitric Oxide; D003903:Deuterium