Lipidex chromatography in the radioimmunoassay of serum and urinary cortisol

Abstract

A highly specific method for the determination of cortisol in human serum and urine is described. The sample is first extracted with diethyl ether/ethyl acetate (1 : 1, by vol.), then chromatographed on a highly lipophilic derivative of Sephadex (hydroxyalkoxypropyl Sephadex, Lipidex) in light petroleum/ chloroform (1 : 1, by vol.), and finally cortisol is measured by radioimmunoassay using a cortisol-21-BSA antiserum. Bound and unbound radioactivities are separated using dextran-coated charcoal technique. The 8 a.m. values (mean ± S.D.) of cortisol among 11 young females and 16 young males were 152 ± 32 ng/ml (range 111–235) and 185 ± 32 ng/ml (103–232), respectively. The respective values at 4 p.m. were 84 ± 29 ng/ml (26–117) and 84 ± 36 ng/ml (32–172). The importance of Lipidex chromatography was demonstrated with assays of serum samples from children with congenital adrenal hyperplasia; without chromatography cortisol values were 6 times those with chromatography. Specific cortisol assays from pregnancy serum also necessitated Lipidex chromatography. Among 11 young men and 9 young women the mean daily urinary cortisol excretion was 56 ± 26 μg (32–109) and 60 ± 24 μg (39–109), respectively. Specific urinary cortisol determination could not be achieved without Lipidex chromatography.