Simultaneous Competitive Protein Binding Assay for Cortisol, Cortisone, and Prednisolone in Plasma, and Its Clinical Application

Abstract

Simultaneous measurement of cortisol, cortisone, and prednisolone is described. A dichloromethane extract of plasma is separated and purified by partition thin-layer chromatography and assayed by competitive protein binding. Using 0.2 ml of plasma, we could assay about 1 µg of these three steroids per 100 ml. We measured plasma corticosteroids in men on prednisone therapy, during ACTH stimulation, and in pregnant women at the time of delivery, and also in arterial and venous blood samples collected from the umbilical cord. Plasma concentrations of prednisolone (4.46 ± 1.07 µg/100 ml) were maximal 2 h after oral administration of prednisone (10 mg). Basal concentrations of cortisol and cortisone in these subjects were 10.50 ± 1.04 and 1.88 ± 0.69 (SD) µg/100 ml, respectively. The values decreased to less than 1 µg/100 ml 4 h after administration and returned to 12.82 ± 1.92 and 2.16 ± 0.49 µg/100 ml, respectively, by 24 h. After intramuscular injection of synthetic ACTH, values for cortisol and cortisone were maximal at 30 min, declining to pre-injection concentrations within 4 h. Plasma cortisol and cortisone values for maternal venous blood at delivery were 47.0 ± 8.5 and 7.1 ± 2.2, umbilical vein blood 4.9 ± 2.6 and 19.7 ± 2.8 and in blood from the umbilical arteries 7.3 ± 4.5 and 11.6 ± 4.9 µg/ 100 ml, respectively. Cortisol was higher (P < 0.05) in the umbilical arteries, cortisone (P < 0.01) in the umbilical vein.