Abstract
IntroductionTwenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment. However, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of steroid metabolites’ cross-reaction. It is still uncertain how ketoconazole (KTZ)- and metyrapone (MTP)-induced changes on the urinary steroid metabolites can alter the 24h-UFC*IA determinations’ reliability.Methods24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ, and 39 during MTP) from 34 CS patients. In addition, urinary steroidome was analyzed by GC-MS on each patient before and during treatment.ResultsBefore treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75 (95% CI 1.60–1.94) compared to those by GC-MS. However, during KTZ treatment, 24h-UFC*IA results were similar (0.98:1) to those by GC-MS (95% CI, 0.83–1.20). In patients taking MTP, IA bias only decreased 0.55, resulting in persistence of an overestimation factor of 1.33:1 (95% CI, 1.09–1.76). High method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Upper limit normal (ULN) reductions in patients taking KTZ were 27% larger when using 24h-UFC*IA compared to 24h-UFC*GC-MS, which resulted in higher false efficacy and misleading biochemical classification of 15% of patients. Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. Larger urinary excretion reductions of 6β-hydroxy-cortisol, 20α-dihydrocortisol, and 18-hydroxy-cortisol in patients with KTZ elucidated the higher 24h-UFC*IA bias decrement compared to MTP-treated patients.ConclusionKTZ and MTP alter the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of reference ranges and ULN.
Highlights
Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment
We demonstrated that 86% of the extent of the 24h-UFC*IA cross-reactivity interference was explained by the abundancy variation of 22 steroid metabolites and that the different degrees of 24h-UFC*IA bias found in patients with KTZ vs. MTP were caused by distinct reductions of urinary metabolites like 6b-OH-cortisol
Different degrees of cross-reaction interference on 24h-UFC*IA determinations before treatment and during KTZ or MTP make IA less suitable for cortisol evaluation
Summary
Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment. 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of steroid metabolites’ cross-reaction. It is still uncertain how ketoconazole (KTZ)- and metyrapone (MTP)-induced changes on the urinary steroid metabolites can alter the 24h-UFC*IA determinations’ reliability. Steroidogenesis inhibitors (SEI) are the drugs most used in CS, reducing cortisol biosynthesis by inhibiting enzymes of the adrenal steroidogenic pathway. While KTZ blocks multiple steps of the adrenal steroidogenic pathway through the inhibition of numerous cytochrome p450 enzymes [5], MTP is a more specific inhibitor of the 11b-hydroxylase enzyme, which blocks 18-hydroxylase to a minor extent [6]
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