Objectives:Regulators of circadian rhythm, including melatonin, influence fundamental biological processes. Measuring the melatonin metabolite 6-sulfatoxymelatonin in urine can estimate melatonin production. 6-sulfatoxymelatonin is mainly analyzed by immunoassays, but these methods are hampered by cross-reactivity and poor reproducibility when used to analyze small molecules. Therefore, we validated a high-throughput liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify 6-sulfatoxymelatonin in urine. We evaluated age-dependent 24-h excretion of 6-sulfatoxymelatonin into urine and the biological variation of urinary excretion in healthy individuals. Methods:The online solid phase extraction method combined with LC-MS/MS was validated according to international guidelines, and used to measure the excretion of 6-sulfatoxymelatonin into urine of 240 healthy individuals. Biological variation of 6-sulfatoxymelatonin excretion was examined in 10 healthy individuals. Results:Urinary 6-sulfatoxymelatonin results were well within the validation criteria (interassay coefficient of variation: <5.4%, quantification limit: 0.2 nmol/L). There was an age-related decrease in 6-sulfatoxymelatonin excretion into 24-h urine [F(5, 234)=13.9; p<0.001]. Within-subject variation of 6-sulfatoxymelatonin was 39.2% in day urine, 15.1% in night urine, and 12.2% in 24-h urine. Between-subject variation was 39.1% in day urine, 37.9% in night urine, and 36.8% in 24-h urine. Conclusions:This MS-based method enables straightforward, reproducible, and sensitive quantification of 6-sulfatoxymelatonin in urine. Urinary 6-sulfatoxymelatonin levels decreased with age. Biological variation of 6-sulfatoxymelatonin excretion into urine was high between subjects and lower within subjects, indicating that repeated measurements of 6-sulfatoxymelatonin in 24-h urine are needed in future studies.

译文

目的:昼夜节律的调节剂,包括褪黑激素,影响基本的生物学过程。测量尿液中的褪黑素代谢物6-硫酸根氧基褪黑素可以估计褪黑激素的产生。主要通过免疫测定法分析6-硫酸氧褪黑素,但这些方法在用于分析小分子时受到交叉反应性和重现性差的困扰。因此,我们验证了串联质谱法(LC-MS / MS)进行的高通量液相色谱法定量尿液中的6-磺酰氧基褪黑素。我们评估了健康个体中6-巯基氧基褪黑激素随年龄的24小时排泄和尿液排泄的生物学变化。
方法:按照国际指南对在线固相萃取结合LC-MS / MS方法进行了验证,并用于测定240名健康人尿液中6-磺酰氧基褪黑素的排泄量。在10名健康个体中检查了6-硫氧嘧啶褪黑素排泄的生物学变化。
结果:尿6-磺酰氧基褪黑激素的结果完全符合验证标准(测定间变异系数:<5.4%,定量极限:0.2nmol / L)。与24小时尿液中的6-巯基氧基褪黑激素的排泄量存在年龄相关性的降低[F(5,234)= 13.9; p <0.001]。受试者体内6-硫酸氧褪黑激素的日间变异为39.2%,夜间尿为15.1%,24小时尿为12.2%。受试者之间的差异为白天尿液为39.1%,夜间尿液为37.9%,24小时尿液为36.8%。
结论:这种基于质谱的方法可对尿液中的6-磺酰氧基褪黑素进行直接,可重现和灵敏的定量。尿中的6-硫酸酯化褪黑激素水平随着年龄的增长而降低。受试者之间尿中6-巯基氧基褪黑激素排泄的生物学变化较高,而受试者中则较低,这表明在未来的研究中需要对24小时尿液中的6-巯基氧基褪黑激素进行重复测量。

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