A novel GC-MS methodology to evaluate aromatase activity in human placental microsomes: a comparative study with the standard radiometric assay.

Augusto, Tiago V.; Cunha, Sara C.; Amaral, Cristina; Fernandes, José O.; da Silva, Elisiário Tavares; Roleira, Fernanda F. M.; Teixeira, Natércia; Correia-da-Silva, Georgina

Estrogens are key factors in the development of the estrogen receptor–positive (ER+) breast cancer. Estrogens, estrone (E1), and estradiol (E2) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER+ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E1 (ratio E1/AD) using isotopic analogues as internal standards. The method showed good linearity (r2 = 0.9908 for AD and 0.9944 for E1), high accuracy (more than 74% for AD and more than 66% for E1), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC50 values of the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from the radiometric assay are also compared, and similar IC50 values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs.

SEX hormones; MICROSOMES; BREAST cancer; AROMATASE; CYTOCHROME P-450; AROMATASE inhibitors; COMPARATIVE studies; ESTRADIOL

Analytical & Bioanalytical Chemistry, 2019, Vol 411, Issue 26, p7005

1618-2642

Academic Journal

10.1007/s00216-019-02076-7