Abstract:
A method for assaying cortisol and cortisone using chromatography on either Paper or Sephadex LH-20 columns, followed by competitive protein binding, has been applied to umbilical cord and maternal plasma samples. In mixed cord plasma the mean cortisol concentration was 6.0 ± 0.8 µg/100 ml (n = 9) and the mean cortisone concentration was 13.5 ± 2.9 µg/100 ml (n = 9). In cord arterial plasma the mean cortisol concentration was 6.3 ± 2.9 µg/100 ml (n = 6). and the mean cortisone level was 10.1 ± 2.5 µg/100 ml (n = 6). For cord venous plasma, the mean level of cortisol was 5.6 ± 1.5 µg/100 ml (n = 6) and of cortisone was 13.5 ± 2.4 µg/100 ml (n = 6). Maternal plasma gave a mean value of cortisol of 42.3 ± 4.5 µg/100 ml (n = 6) and of cortisone of 6.2 ± 0.9 µg/100 ml (n = 6). The mean value of cortisol in mixed cord plasma of live anencephalic fetuses was 3.3 ± 1.8 µg/100 ml (n = 3) and of cortisone was 13.6 ± 3.2 µg/100 ml (n = 3).
Further assays employing a radioimmunoassay for cortisol, and competitive protein binding for cortisone have been applied to cord plasma samples of prematurely-delivered fetuses, after treatment of betamethasone to the mother (12 mg/day for one or two days). The mean value of cortisol in samples from fetuses delivered up to 24 hours after treatment was 2.6 ± 2.2 µg/100 ml (n = 4) and the mean value of cortisone was 2.2 ± 2.2 µg/100 ml (n = 14).
For those fetuses delivered over 72 hours after betamethasone treatment, mean levels of cortisol and cortisone were respectively 10.4 ± 3.2 µg/100 ml and 9.4 ± 3.4 µg/100 ml (n = 3) compared to control values without betamethasone treatment of 8.2 ± 3.5 µg/1 0 ml and 9.6 ± 3.7 µg/100 ml (n = 3) respectively.
Kinetic parameters of the human placental 11β-hydroxysteroid dehydrogenase system were assessed using a microsomal preparation in 50% glycerol buffer.
The Michaelis constant for cortisol was 1.88 x 10-6M, in the presence of NADP, and 0.56 x 10-6M, in the presence of NAD. Vmax values varied with concentration of cofactor, increasing from 0.031 to 0.076 µmoles cortisone produced per minute per mg protein with increasing concentration of NADP from 0.036 mM to 1.2 mM. The value of vmax, for an initial concentration of 1.2 mM NAD, was 0.0598.
Substrate inhibition was not evident at the level of cortisol employed (up to a cortisol concentration of 1 x 10-4M). Corticosterone, cortisone, progesterone and pregnenolone inhibited enzyme activity.
The results of this study suggest that fetal cortisol is produced by the fetal adrenal, and fetal cortisone originates primarily from the conversion of maternal cortisol to cortisone across the placenta, by the action of the 11β-hydroxysteroid dehydrogenase enzyme.