RF21 | PSAT99 Urinary adrenal steroidome modifications by Ketoconazole and Metyrapone alter the urinary free cortisol immunoassay reliability in Cushing Syndrome

INTRODUCTION: 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 adrenal-directed medical treatment. In CS patients without medical treatment, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of interference by cross-reacting steroid metabolites. However, it is unknown how induced changes by Ketoconazole (KTZ) or Metyrapone (MTP) on the excretion of urinary steroid metabolites alter the IA cross-reactivity interference of 24h-UFC determinations and what are its clinical implications. METHODS: 24h-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, 35 urinary steroids were analyzed by GC-MS on each patient before and during treatment. RESULTS: Before treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75: 1 (95% CI 1.60 - 1.94) compared to those by GC-MS. During treatment with KTZ, the cross-reactivity interference of the 24h-UFC results by IA decreased in a dose-dependent manner (p = 0.000). Thus, in patients taking KTZ >400mg/day, 24h-UFC*IA determinations were no longer overestimated, giving similar results to those by GC-MS (factor of 0.98: 1, 95% CI, 0.83–1.20). Baseline 24h-UFC and upper limit normal (ULN) reductions were magnified (>25%) when assessed by IA vs GC-MS leading to higher falsely KTZ efficacy and misleading biochemical classification of 15% of patients. Despite dosage, in patients taking MTP the IA cross-reactivity only decreased 0.55 which resulted in persistence of overestimated 24h-UFC results (factor of 1.33: 1, 95% CI, 1.09 -1.76). When comparing MTP efficacy among IA vs GC-MS results, ULN baseline reductions were >50% different in some patients, while in others, similar reductions were found. Acceptable 24h-UFC 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). Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. 6β-Hydroxy-cortisol (6β-OH-cortisol) was the metabolite determining most of the 24h-UFC cross-reactivity variability (R2= 48.3%, p = 0.000) followed by 20α-dihydrocortisol (20α-DHF) (R2= 24.4%, p = 0.000). Each treatment induced a unique urinary steroid signature (p <0.001) and larger excretion reductions of 6β-OH-cortisol, 20α-DHF and 18-Hydroxy-cortisol in patients with KTZ elucidated the higher decrement of interference in the 24h-UFC by IA, compared to MTP treated patients (p = 0.001). CONCLUSION: KTZ and MTP alter distinctly the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment in a unique manner. 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 ULN. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m.