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 #x3BC;Ci of3H-androstenedione. After agitation at 37#xB0;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 10#x3BC; 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 the3H-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, enzymatic3H-estrone formation was found to be linear in a time and protein dependant manner.
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