ABSTRACT
We have developed a new assay for cortisone (E) in serum, saliva, and urine involving Celite chromatography followed by RIA with 125I-labeled E and scintillation proximity assay. The chromatography step separates cortisol (F) from E, and in combination with their RIAs, permits assessment of the status of the F-E shuttle. We report the results of basal, postcorticotropin (ACTH), and postdexamethasone E and F concentrations and their circadian fluctuations in the serum, saliva, and urine of healthy volunteers. The serum and urine F/E ratios were increased in patients with ectopic ACTH secretion, whereas in adrenal adenoma and Cushing disease only the urinary ratio was increased. In chronic renal insufficiency this ratio was increased in serum (23.5 +/- 3.9) but diminished in saliva (0.38 +/- 0.11), and in apparent mineralocorticoid excess the ratios were high in serum (44.3 +/- 9.3) and urine (5.35 +/- 0.85) compared with those of healthy subjects (serum 9.8 +/- 3.5, urine 0.52 +/- 0.29, saliva 0.52 +/- 0.29).
Subject(s)
Cortisone/analysis , Hydrocortisone/metabolism , Radioimmunoassay , 11-beta-Hydroxysteroid Dehydrogenases , Adrenal Gland Diseases/metabolism , Adrenocorticotropic Hormone/metabolism , Adult , Chromatography , Circadian Rhythm , Cortisone/blood , Cortisone/immunology , Cortisone/metabolism , Cortisone/urine , Diatomaceous Earth , Female , Humans , Hydrocortisone/immunology , Hydroxysteroid Dehydrogenases/deficiency , Immune Sera/immunology , Kidney Failure, Chronic/metabolism , Male , Middle Aged , Mineralocorticoids/metabolism , Radioimmunoassay/methods , Reference Values , Reproducibility of Results , Saliva/chemistry , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Chromatography/methods , Hydrocortisone/urine , Radioimmunoassay , Adult , Chromatography, High Pressure Liquid , Diatomaceous Earth , False Positive Reactions , Female , Gas Chromatography-Mass Spectrometry , Humans , Male , Quality Control , Radioimmunoassay/statistics & numerical data , Reference Values , Sensitivity and Specificity , Sex Characteristics , Steroids/urineABSTRACT
We have studied the photosensitized oxidation, via singlet oxygen production, of histidine (His) and tryptophan (Trp) in the serum of porphyria patients and in the serum of healthy volunteers before or after addition of hematoporphyrin. It was observed that free plasma His and Trp are good probes of singlet oxygen production in the blood under visible light irradiation. However, Trp, which is mostly bound to serum albumin, is much less susceptible to photooxidation than His, which remains free in the plasma. These results must not be ignored in systemic effects of porphyrias and in photochemotherapy with hematoporphyrin derivatives.
