Jolaine M Hines1, Irina Bancos2, Cristian Bancos3, Raman D Singh1, Aditya V Avula1, William F Young2, Stefan K Grebe2,4, Ravinder J Singh5. 1. Immunochemical Core Laboratory, Mayo Clinic, Rochester, MN. 2. Department of Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN. 3. Information Technology, Mayo Clinic, Rochester, MN. 4. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. 5. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. singh.ravinder@mayo.edu.
Abstract
BACKGROUND: Steroid profiling is a promising diagnostic tool with adrenal tumors, Cushing syndrome (CS), and disorders of steroidogenesis. Our objective was to develop a multiple-steroid assay using liquid-chromatography, high-resolution, accurate-mass mass spectrometry (HRAM LC-MS) and to validate the assay in patients with various adrenal disorders. METHODS: We collected 24-h urine samples from 114 controls and 71 patients with adrenal diseases. An HRAM LC-MS method was validated for quantitative analysis of 26 steroid metabolites in hydrolyzed urine samples. Differences in steroid excretion between patients were analyzed based on Z-score deviation from control reference intervals. RESULTS: Limits of quantification were 20 ng/mL. Dilution linearity ranged from 80% to 120% with means of 93% to 110% for all but 2 analytes. Intraassay and interassay imprecision ranged from 3% to 18% for all but 1 analyte. Control women had lower excretion of androgen and glucocorticoid precursors/metabolites than men (P < 0.001), but no difference in mineralocorticoids was seen (P = 0.06). Androgens decreased with age in both sexes (P < 0.001). Compared with patients with adrenocortical adenoma (ACA), patients with adrenocortical carcinoma (ACC) had 11 steroids with increased Z scores, especially tetrahydro-11-deoxycortisol (14 vs 0.5, P < 0.001), pregnanetriol (7.5 vs -0.4, P = 0.001), and 5-pregnenetriol (5.4 vs -0.4, P = 0.01). Steroid profiling also demonstrated metabolite abnormalities consistent with enzymatic defects in congenital adrenal hyperplasia and differences in pituitary vs adrenal CS. CONCLUSIONS: Our HRAM LC-MS assay successfully quantifies 26 steroids in urine. The statistically significant differences in steroid production of ACC vs ACA, adrenal vs pituitary CS, and in congenital adrenal hyperplasia should allow for improved diagnosis of patients with these diseases.
BACKGROUND:Steroid profiling is a promising diagnostic tool with adrenal tumors, Cushing syndrome (CS), and disorders of steroidogenesis. Our objective was to develop a multiple-steroid assay using liquid-chromatography, high-resolution, accurate-mass mass spectrometry (HRAM LC-MS) and to validate the assay in patients with various adrenal disorders. METHODS: We collected 24-h urine samples from 114 controls and 71 patients with adrenal diseases. An HRAM LC-MS method was validated for quantitative analysis of 26 steroid metabolites in hydrolyzed urine samples. Differences in steroid excretion between patients were analyzed based on Z-score deviation from control reference intervals. RESULTS: Limits of quantification were 20 ng/mL. Dilution linearity ranged from 80% to 120% with means of 93% to 110% for all but 2 analytes. Intraassay and interassay imprecision ranged from 3% to 18% for all but 1 analyte. Control women had lower excretion of androgen and glucocorticoid precursors/metabolites than men (P < 0.001), but no difference in mineralocorticoids was seen (P = 0.06). Androgens decreased with age in both sexes (P < 0.001). Compared with patients with adrenocortical adenoma (ACA), patients with adrenocortical carcinoma (ACC) had 11 steroids with increased Z scores, especially tetrahydro-11-deoxycortisol (14 vs 0.5, P < 0.001), pregnanetriol (7.5 vs -0.4, P = 0.001), and 5-pregnenetriol (5.4 vs -0.4, P = 0.01). Steroid profiling also demonstrated metabolite abnormalities consistent with enzymatic defects in congenital adrenal hyperplasia and differences in pituitary vs adrenal CS. CONCLUSIONS: Our HRAM LC-MS assay successfully quantifies 26 steroids in urine. The statistically significant differences in steroid production of ACC vs ACA, adrenal vs pituitary CS, and in congenital adrenal hyperplasia should allow for improved diagnosis of patients with these diseases.
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