Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography–tandem mass spectrometry
2014
Yurek, David | Yu, Lan | Schrementi, James | Bell, Michael G. | McGee, James | Kowala, Mark | Kuo, Ming-shang | Wang, Jian
Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography–tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z′ value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
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