Fluorometric Determination of Corticosteroids in Human Blood; Comparison of Results with the Silber-Porter Method

Abstract

A method for determining corticosteroid concentrations in 1 ml samples of serum is described. The initial purification depends upon successive extractions with 2,2,4-trimethylpentane and chloroform. The chloroform extract is subjected to a single alkaline wash and the steroid is extracted directly into the final fluorescence reagent (75% ethanolic H₂SO₄) v/v). The influence of varying concentrations of sulfuric acid and ethanolic sulfuric acid upon the development of fluorescence has been explored in order to ascertain an optimum reaction with cortisol. Recoveries of cortisol from plasma are virtually complete (95.5–105.4%). The proportionate standard deviation of duplicate analyses varied from 0.163 at concentrations in the range of 9.7 μg/100 ml to 0.018 at a mean concentration of 76.3 μg/100 ml. The activation and fluorescence spectra of normal plasma extracts consistently paralleled those observed with crystalline cortisol and corticosterone. The nonsteroid background fluorescence extracted from plasma of patients with untreated Addison's disease was equivalent to less than the intensity produced by 0.04 μg of cortisol. The results of the fluorescence method have been compared to those of the Silber-Porter method in a series of plasma samples obtained from patients during ACTH infusions, after infusions of cortisol, following operations, and from obstetrical patients. The results of the 2 methods are in close agreement in the samples obtained in each of these clinical circumstances. The fluorescence inducing corticosteroid concentration in human cord blood, however, was uniformly less than was the 17-hydroxycorticosteroid concentration. This finding is in agreement with that reported by Ulstrom and his co-workers. They observed the presence of a phenylhydrazine reacting substance in cord blood which rendered the results of the Silber-Porter method an unreliable index of corticosteroid concentrations in this circumstance.