Ketoconazole- and Metyrapone-Induced Reductions on Urinary Steroid Metabolites Alter the Urinary Free Cortisol Immunoassay Reliability in Cushing Syndrome

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.

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders

“…Finally, although the urine of the patients treated with steroidogenic inhibitors did not show changes in 11βHSD2 activity, a reduction in CYP3A4 activity was observed. Although the causal effect of each drug on CYP3A4 activity cannot be concluded from this study due to the small number of patients and concomitant medications, the results show that patients treated with steroidogenesis inhibitors may present with significant changes in their excretion of free steroid metabolites, as previously observed in a total steroid urine profile [39]. Although the clinical relevance of monitoring these changes in cortisol metabolism should be further assessed, the results point to a considerable impact on the follow-up of these patients if urine cortisol measurements are performed using immunoassay and support the use of mass spectrometry measurements for a proper and comprehensive evaluation of the effects.…”

Medical Therapy of Endogenous Cushing’s Syndrome with Steroidogenesis Inhibitors: Treatment Rationale, Available Drugs, and Therapeutic Effects

“…Furthermore, a recent publication reported that ketoconazole and metyrapone may alter the urinary excretion of steroid metabolites, thereby leading to a high risk of overestimating their biochemical impact [44]. With respect to LNSC a high intra patient variation of almost 50% is has been reported [43].…”

“…Nevertheless, repetitive blood sampling over a day may allow for serum cortisol day curves, which provide a robust estimate of the endogenous cortisol secretion capacity over 24 h. 24h-UFC is also widely used in the monitoring of steroidogenesis inhibitors [40], but a high intra-patient coefficient of variation has to be considered [42,43]. Furthermore, a recent publication reported that ketoconazole and metyrapone may alter the urinary excretion of steroid metabolites, thereby leading to a high risk of overestimating their biochemical impact [44]. With respect to LNSC, a high intra-patient variation of almost 50 % has been reported [43].…”

Pitfalls in the Diagnosis and Management of Hypercortisolism (Cushing Syndrome) in Humans; A Review of the Laboratory Medicine Perspective

“…Exposure to excess glucocorticoids can result in complications, such as diabetes mellitus and dyslipidaemia. Assessment of the metabolome using ultra-high-performance liquid chromatography-tandem mass spectrometry has been shown to discriminate hypercortisolaemic patient specimens versus eucocortisolaemic [ 144 ]. It is possible that this type of technique, including molecular (see below), may have value in borderline cases where neither clinical nor biochemical findings can confirm or refute the diagnosis of hypercortisolaemia, but it has yet to be used outside a research setting.…”

“…However, these agents result in structurally similar metabolites to cortisol, such as 11-deoxycortisol, which risks over treatment of spurious hypercortisolism or missing hypoadrenalism due to over treatment [ 25 , 26 ]. It is therefore vital to assess cortisol with an assay free from interference in the presence of steroidogenesis inhibition, and the method of choice would be LC-MS/MS methods validated to be free from interference [ 25 , 144 ]. However, protocols involving immunoassay with urine and saliva have been reported [ 149 ].…”