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).