OPTIMIZATION OF A CLASSICAL AROMATASE-ACTIVITY ASSAY AND APPLICATION IN NORMAL, ADENOMATOUS AND MALIGNANT BREAST PARENCHYMA

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aroma tase activity in breast tissue samples. The lower activity in this tis sue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mi xture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 mi n activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatogr aphy-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, o estrone and androstenedione after incubation with non-labelled androst enedione proved that a substantial amount of the substrate is converte d into testosterone. Qualitative GC-MS steroid profiling of the incuba tion mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could hav e caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samp les and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aroma tase positivity was found in 80% of the normal samples, 56% of the ade noma samples and 48% of the carcinoma samples. Although carcinoma samp les were less often aromatase positive than normal tissue samples chi( 2) = 4.80; P < 0.050) there was no difference in absolute aromatase ac tivity. Because no less than approximately 50% of the carcinomas conta ined aromatase activity and because of the non-routine character of th e assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status. Copyright (C) 1996 Elsevier Science Ltd.