| المستخلص: |
Background: Measuring actual free cortisol in the plasma is a more precise way to assess adrenocortical function compared to total cortisol or to the free cortisol estimated by calculation methods. Equilibrium dialysis and ultrafiltration are considered as the gold standard methods used for direct quantification of free plasma cortisol. The aim of the study was to develop, optimize and validate an equilibrium dialysis method that allows us to separate free cortisol from bound cortisol in human plasma. Subsequently, this would allow us to investigate if cortisol turnover in the free pool is faster than in the total pool. Methods: Various self-assembled equilibrium dialysis cells were used to optimize the most appropriate equilibrium dialysis technique to separate free cortisol from bound. Equilibrium was tested with tritiated steroid and with colorimetric dye. Cortisol ELISA was used to measure cortisol concentrations in the samples and dialysate. Intra-assay coefficient of variation was calculated for equilibrium dialysis method experiments. Inter-assay coefficient of variation was calculated for equilibrium dialysis method experiments that were done more than once. A commercially available ultrafiltration device was used to validate our equilibrium dialysis method. Results: Data has shown that dialysis equilibrium can occur before 16 hours, at 6 hours. It also has shown that various in-house equilibrium dialysis techniques produced somewhat inconsistent results with high inter-assay coefficients of variation (>15 %). An alternative direct method of measuring free cortisol, the ultrafiltration method and the calculated free cortisol methods produced more consistent results than the equilibrium dialysis method developed here. Conclusions: The equilibrium dialysis method developed here was not a very precise method of measuring free cortisol as it tended to produce inconsistent results.
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