Inserm, U1185, Le Kremlin-Bicêtre, F-94276, France; Fac Med Paris-Sud, Univ. Paris-Sud, Université Paris Saclay, UMR-S 1185, Le Kremlin-Bicêtre, F-94276, France; Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpital Bicêtre, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, F-94275, France.
Inserm, U1185, Le Kremlin-Bicêtre, F-94276, France; Fac Med Paris-Sud, Univ. Paris-Sud, Université Paris Saclay, UMR-S 1185, Le Kremlin-Bicêtre, F-94276, France; Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpital Bicêtre, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, F-94275, France.
J Steroid Biochem Mol Biol. 2020 Apr;198:105553. doi: 10.1016/j.jsbmb.2019.105553. Epub 2019 Nov 26.
21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative. We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35). The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids. Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient's classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles. Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.
21-羟化酶缺乏症是导致先天性肾上腺皮质增生症（CAH）最常见的酶缺陷，其特征是醛固酮和皮质醇生物合成均受损。需要密切监测氢化可的松（HC）和 9α-氟氢可的松（FDR）替代治疗，以达到最佳治疗效果。由于在儿童时期需要频繁和重复评估血浆类固醇、17-羟孕酮（17-OHP）、雄烯二酮（Δ4-A）和睾酮（TESTO），因此尿类固醇谱分析可能是一种有趣的非侵入性替代方法。我们开发并验证了一种用于测量 23-尿盐皮质激素、糖皮质激素和肾上腺雄激素的 LC-MS/MS 方法。我们研究了在单次 08 点收集的尿液斑点中进行类固醇谱分析，以区分 61 名 CAH 患者及其年龄和性别匹配的对照组的用途。根据他们的 08 点血浆 17-OHP 浓度，CAH 患者分为两组：低于（控制患者，n=26）和高于 20ng/ml（未控制患者，n=35）。定量下限和宽分析范围允许测定主要尿肾上腺皮质激素及其四氢代谢物的游离和总浓度。大多数类固醇的提取回收率高于 75%，日内精密度低于 20%。在未控制的 CAH 患者中，21-羟化酶缺陷上游的尿类固醇水平更高。在 CAH 患者中，血浆和尿 17-OHP 密切相关。与对照组相比，酶缺陷下游的类固醇在 CAH 患者中崩溃。在控制良好的患者中，这种下降比未控制的患者更明显。雄激素（Δ4-A、TESTO 和雄酮+androsterone 的总和）在未控制的 CAH 患者中积累。在控制良好的患者中，观察到血浆和尿中雄烯二酮水平之间存在强烈关系。在两个 CAH 组中，FDR 和 HC 的每日剂量和尿排泄量相似。尿 FDR 与尿钠钾比呈负相关。偏最小二乘判别分析模型允许根据尿类固醇组谱将患者分为不受影响、控制和未控制的 CAH 患者。我们的 LC-MS/MS 方法成功地在尿液中建立了类固醇谱分析，是一种根据治疗效果区分 CAH 患者的有用且非侵入性工具。
