State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Anal Chem. 2022 Jul 12;94(27):9880-9887. doi: 10.1021/acs.analchem.2c01816. Epub 2022 Jun 27.
Urine sample storage after collection at ultra-low-temperature (e.g., -80 °C) is normally required for comparative metabolome analysis of many samples, and therefore, freeze-thaw cycles (FTCs) are unavoidable. However, the reported effects of FTCs on the urine metabolome are controversial. Moreover, there is no report on the study of how urine FTCs affect biomarker discovery. Herein, we present our study of the FTC effects on the urine metabolome and biomarker discovery using a high-coverage quantitative metabolomics platform. Our study involved two centers located in Hangzhou, China, and Edmonton, Canada, to perform metabolome analysis of two separate cohorts of urine samples. The same workflow of sample preparation and dansylation isotope labeling LC-MS was used for in-depth analysis of the amine/phenol submetabolome. The analysis of 320 samples from the Hangzhou cohort consisting of 80 healthy subjects with each urine being subjected to four FTCs resulted in relative quantification of 3682 metabolites with 3307 identified or mass-matched. The analysis of 176 samples from the Edmonton cohort of 44 subjects with four FTCs quantified 3516 metabolites with 3166 identified or mass-matched. Multivariate and univariate analyses indicated that significant variations (fold change ≥ 1.5 with -value ≤ 0.05) from FTCs were only observed in a very small fraction of the metabolites (<0.3%). Moreover, various metabolites did not show a consistent pattern of concentration changes from one to four FTCs, allowing the use of two separate cohorts of samples to remove these randomly changed metabolites. Three metabolite biomarkers for separating males and females were discovered, and FTC did not influence their discovery.
尿液样本采集后通常需要在超低温（例如-80°C）下储存，以便对许多样本进行比较代谢组分析，因此不可避免地会经历冻融循环（FTC）。然而，关于 FTC 对尿液代谢组的影响的报道存在争议。此外，尚无关于 FTC 如何影响生物标志物发现的研究报告。在此，我们使用高覆盖定量代谢组学平台研究了 FTC 对尿液代谢组和生物标志物发现的影响。我们的研究涉及位于中国杭州和加拿大埃德蒙顿的两个中心，对两个独立的尿液样本队列进行代谢组分析。对杭州队列的 320 个样本（包括 80 名健康个体，每个尿液样本进行了 4 次 FTC）进行了相同的样品制备和 Dansyl 同位素标记 LC-MS 工作流程，以对胺/酚亚代谢组进行深入分析。结果共相对定量了 3682 种代谢物，鉴定或质量匹配的代谢物有 3307 种。对埃德蒙顿队列的 176 个样本（包括 44 名个体，每个尿液样本进行了 4 次 FTC）进行分析，定量了 3516 种代谢物，鉴定或质量匹配的代谢物有 3166 种。多变量和单变量分析表明，只有一小部分代谢物（<0.3%）的 FTC 会产生明显变化（倍数变化≥1.5，-值≤0.05）。此外，各种代谢物从一次 FTC 到四次 FTC 的浓度变化没有一致的模式，允许使用两个独立的样本队列来去除这些随机变化的代谢物。发现了三个用于区分男性和女性的代谢物生物标志物，FTC 并未影响其发现。
