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.