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.

中文翻译：

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通过LC-MS / MS监测尿液的类固醇成分，以监测经典的21-羟化酶缺乏症。

21-羟化酶缺乏症是与先天性肾上腺皮质增生（CAH）相关的最常见的酶缺陷，其特征是醛固酮和皮质醇的生物合成均受到损害。需要对氢化可的松（HC）和9α-氟氢化可的松（FDR）替代疗法进行密切的临床和生物学监测，以实现最佳治疗效果。由于在儿童期需要对血浆类固醇，17-羟基孕酮（17-OHP），雄烯二酮（Δ4-A）和睾丸激素（TESTO）进行频繁且反复的重新评估，因此尿类固醇分析可能是一种有趣的非侵入性替代方法。我们开发并验证了用于测量23种尿盐皮质激素，糖皮质激素和肾上腺雄激素的LC-MS / MS方法。研究人员在上午8点00分采集的单点尿液中研究了类固醇谱分析的有用性，以区分61名CAH患者及其年龄和性别匹配的对照。根据他们的08h00血浆中17-OHP浓度将CAH患者分为两组：低于（对照患者，n = 26）和高于20 ng / mL（未对照患者，n = 35）。定量的下限和宽的分析范围允许分析主要尿肾上腺皮质激素及其四氢代谢产物的游离浓度和总浓度。大多数类固醇的提取回收率均高于75％，测定内精度低于20％。在不受控制的CAH患者中，21-羟化酶缺陷上游的尿类固醇水平较高。在CAH患者中，血浆和尿液17-OHP密切相关。与对照相比，在CAH患者中，酶缺陷下游的类固醇塌陷。受控制的患者比不受控制的患者更为明显。在不受控制的CAH患者中积累了雄激素（Δ4-A，TESTO和依托胆醇酮+雄甾酮的总和）。在血浆和雄烯二酮的尿液水平之间观察到强烈的关系。在两个CAH组中，FDR和HC的日剂量和尿排泄量相似。尿FDR与尿中钠钾比成反比。偏最小二乘判别分析模型允许根据尿类固醇组学特征对未受影响，受控制和不受控制的CAH患者进行分类。