Determination of 11β-HSD2 activity
Placental tissues (0.1–0.2g) were homogenized in ice-cold 10 mM sodium phosphate buffer (pH 7.0) containing 0.25 M sucrose. The homogenate was used immediately in the following assays. Protein concentration was determined by BCA kit (BoGuang Ltd, Shanghai) protein assay kit with BSA as standard. The 11β-HSD dehydrogenase activity was determined by measuring the rate of conversion of cortisol to cortisone(Alfaidy et al., 2002; Schoof et al., 2001). Briefly, the assay tubes were added with placental tissue homogenate (containing 20–50 mg protein), cortisol (0.5 mM) and cofactor NAD or NADP (250 mM) and then incubation at 37℃ for 30 min. Cortisol and cortisone concentration was determined by using liquid chromatography tandem mass spectrometry as described previously (Fahlbusch et al., 2013;2015) and performed by APT. Co. Ltd (Shanghai, China).