A sensitive radioimmunoassay for fludrocortisone in human plasma

Abstract

Fludrocortisone has been a mainstay of therapy for orthostatic hypotension for many years. Clinical experience suggests that there exists a substantial interindividual variation in responsiveness to the drug. To assess this, we have developed an assay that permits measurement of the low concentrations of fludrocortisone found in human plasma. Fludrocortisone was detected by radioimmunoassay. A polyclonal rabbit antibody, raised against dexamethasone which cross-reacts strongly with fludrocortisone, was reacted with either standard or unknown samples in the presence of [ 125I]fludrocortisone-3-TyrNH 2 (synthesized by coupling tyrosine amide to fludrocortisone-3-oxime and iodinating with chloramine T oxidation). The ED 10, ED 50, and ED 80 were 0.34, 5.0, and 30 ng/mL of plasma, respectively. The cross reactivity with other 9-fluorinated steroids was found as follows: dexamethasone, 340%; betamethasone, 230%; and triamcinolone, 8%. To preclude an erroneous result, subjects who were pregnant or receiving any steroid medication were excluded from the study. The percent cross-reactivity with the main naturally occurring steroids was as follows: 11-desoxycortisol 3.2%, cortisol 1.1%, DOC 0.3%, pregnenolone 0.1%, corticosterone 0.06%, progesterone 0.05%, and aldosterone <0.05%. The only compound with potential for interference, because of its high level in the circulation in the early morning, was cortisol. In plasma samples obtained before administration of fludrocortisone, levels of fludrocortisone were undetectable even though the plasma cortisol ranged from 40–175 ng/mL. In preliminary studies, fludrocortisone was given orally in doses of 0.2 and 2 mg. Timed plasma samples were collected. With doses of 0.2 mg, fludrocortisone levels peaked at about 2.4 ng/mL after 1–2 h and were undetectable by 12 h. With 2 mg, the levels peaked at 17.6–24.5 ng/mL at 1 h and was undetectable by 24 h. With the 2 mg dose, plasma cortisol fell at 10 h to 71–87% (14–23 ng/mL) of baseline. This decline was greater than would have been expected from the diurnal variation in cortisol. The estimated T 1 2 for fludrocortisone was 1.6 h for the 2 mg dose and 2.4 h for the 0.3 mg dose. This new fludrocortisone assay can now be used to define the pharmacokinetics of this agent.