Urinary Free Cortisol: Interference in the Determination

Introduction: The excretion of urinary free cortisol (UFC) is a sensitive indicator of hypercortisolism. However, the low concentration of UFC compared with its total urinary metabolites and the molecular similarity between them, make a reliable UFC measurement difficult. A typical example is the increase in the measurement of UFC secondary to an increase in 6 beta-hydroxycortisol (6 beta-OHF), an unconjugated metabolite of cortisol. Since 6 beta-OHF excretion may increase up to 60 more by the administration of drugs whose metabolism is also carried out by p450 cytochrome, exists the possibility of interference with importance for the diagnosis. This problem has largely been overcome, by submitting the urine sample to a previous extraction with an organic solvent. Nevertheless, some immunoassay methods dispense the extraction step and choose to indicate reference values obtained without extraction. Aim: (1) To evaluate the influence of dichloromethane extraction in UFC levels; (2) To evaluate the interference of 6 beta-OHF in the measurement of UFC using five immunoassays; (3) To confirm the absence of 6 beta-OHF in the organic phase after extraction. Methods: The UFC concentration was determined by immunoassay, before (D) and after (E) dichloromethane extraction, in patient's urine samples and in one urine sample after addition of known amounts of 6 beta-OHF. The presence of 6 beta-OHF was evaluated by gas chromatography analysis. Results: (1) The difference between UFC results before and after extraction showed a variation between -7 and 878%; (2) The UFC levels in samples without exogenous 6 beta-OHF, showed a variation of 56-90 mu g/24 h (E) and 71-671 mu g/24 h (D). The range of UFC values after the addition of increasing amounts of 6 beta-OHF was 53-231 mu g/24 h (E) and 71-2842 mu g/24 h (D); (3) After extraction, gas chromatography analysis only showed the presence of 6 beta-OHF in the aqueous phase. Conclusion: It was demonstrated the existence of variable degrees of interference induced by the presence of 6 beta-OHF in CLU measurement by immunoassay. Each laboratory should confirm the need to submit the urine samples to a previous extraction with an organic solvent.