Convenient chromatographic prepurification step before measurement of urinary cortisol by radioimmunoassay

We applied various prepurification protocols (extraction with different solvents, liquid/solid separation on bonded silica media, Celite, and Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the results with the concentrations measured by a HPLC method validated with reference to isotope dilution gas chromatography–mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minicolumn (Waters™ Sep-Pak Vac RC) in combination with dichloromethane extraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication. We tested numerous steroids for interference and found that free polar cortisol derivatives (hydroxylated or hydrogenated) could only partially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.

A correlative study between glucocorticoid receptor levels in human mononuclear leukocytes and biochemical data in Cushing's disease

We determined glucocorticoid receptors in human monouclear leukocytes in 9 patients with Cushing's disease, in order to correlate them with laboratory data. Receptors were measured by a whole-cell assay method, after incubation with [3H]-dexamethasone in the presence or absence of excess unlabelled hormone. In Cushing's disease, there were 4425 ± 364 sites/cell (N = 9), similar to in the controls: 4473 ± 476 (N = 10); average Kd was 2.42 ± 0.52 nmol/l (N = 3) similar to in the controls: 2.0 ± 0.20 nmol/l (N = 3). In Cushing's patients we found significant negative correlations between basal glucocorticoid receptors and: 1) morning blood cortisol (r = −0.67, P< 0.05), and 2) 17-ketogenic steroids after 2 mg of dexamethasone (r = −0.85, P< 0.01). No correlations were observed with afternoon blood cortisol, free urinary cortisol, basal and post-8-mg dexamethasone 17-ketogenic steroids, TRH-TSH area, urinary calcium. plasma glucose, or systolic blood pressure. Conclusions: In Cushing's disease, a subtle receptor down-regulation may exist, as suggested by the inverse relationship between glucocorticoid receptors and morning blood cortisol. Secondly, the relationship between basal receptors and 17-ketogenic steroids after 2 mg of dexamethasone suggests that glucocorticoid receptors in human mononuclear leukocytes could reflect the sensitivity of the nervous system-pituitary-adrenal axis to dexamethasone inhibition.

URINARY EXCRETION OF FREE CORTISOL IN IMPAIRED RENAL FUNCTION

ABSTRACT Total cortisol and free, non protein-bound cortisol in plasma and urinary excretion of unconjugated free cortisol were measured during iv infusion of cortisol at varying dose rates in eight patients with impaired renal function. The results showed that free urinary cortisol decreased with decreased glomerular filtration rate (GFR), also compared to free cortisol level in plasma. An increase in free cortisol in plasma had no influence on GFR. It is concluded that determination of free urinary cortisol, otherwise useful in diagnosing Cushing's syndrome, may be of less value in patients with impaired renal function.

URINARY EXCRETION OF FREE CORTISOL IN HYPERCALCAEMIA

ABSTRACT Human and animal studies have uniformly demonstrated increased hypothalamic-pituitary-adrenocortical (HPA) activity during acute hypercalcaemia. The HPA-activity during chronic hypercalcaemia was investigated by means of free urinary cortisol excretion. No difference in HPA activity could be demonstrated between a hyperparathyroid hypercalcaemic and a normocalcaemic group of patients. Based on these results it is suggested that during chronic hypercalcaemia, the HPA feed back mechanism overcomes the influence of hypercalcaemia on the HPA-axis.

FREE URINARY CORTISOL AS A REPRESENTATIVE STEROID OF THE URINARY PORTER-SILBER-CHROMOGENS

ABSTRACT In this investigation an attempt was made to find a compound based on urinary steroids which characterise the mean cortisol metabolites. Free urinary cortisol turned out to be useful for this purpose. Thus a positive correlation can be demonstrated under basal conditions as well as after standardized loading tests with metyrapone and β1-24-tetracosactide2). With regard to the lens-effect described in a previous paper and the mechanisms leading to this effect it was considered whether the determination of free urinary cortisol could be used as a sensitive estimation of enhanced adrenal activity, providing this rise in activity led to an increase of the biologically active plasma cortisol fraction. Further studies of free urinary cortisol in pathological conditions other than Cushing's syndrome as well as calculations of ratios of urinary C-17-OHCS and plasma cortisol are necessary, however, in order to evaluate the diagnostic possibilities in use of free urinary cortisol as a parameter.