Rapid Assay of Hypothalamic Aromatase Using High Performance Liquid Chromatography

Abstract

A rapid assay for hypothalamic aromatase has been developed using HPLC. Each assay tube contained rat anterior hypothalamus homogenized in Na phosphate buffer (pH 7.0), NADPH regenerating system, unlabelled estradiol and estrone, and 10 μCi of ³H-androstenedione. After agitation at 37°C for 3 hours, the reaction mixture was extracted with CH₂Cl₂, partitioned between 90% methanol and hexane, and the methanol layer chromatographed on a Waters 10μ C-18 Radial-PAK column using acetonitrile/water (70:30). The estrogens were collected together and rechromatographed with THF/water (35:65). The estrone peak was collected, and the ³H-estrone produced was determined by liquid scintillation. The estrone collected from the second chromatographic step was found to be at least 95% pure by methylation and rechromatography. Using the HPLC procedure, enzymatic ³H-estrone formation was found to be linear in a time and protein dependant manner.