Systematic re-evaluation of the long-used standard protocol of urease-dependent metabolome sample preparation

In the urinary metabolomics for finding biomarkers in urine, owing to high concentrations of urea, for chromatography-based metabolomic analysis, urea needed to be degraded by urease. This urease pretreatment has been the key step of sample preparation for standard urinary metabolomics until today even for mass spectrometry-based analysis. The urease pretreatment involving incubation of urine with urease contradicts the concept of metabolome sampling, which should immediately arrest metabolic reactions to prevent alterations of a metabolite profile. Nonetheless, the impact of urease pretreatment has not been clearly elucidated yet. We found that activities of urease and endogenous urinary enzymes and metabolite contaminants from the urease preparations introduce artefacts into metabolite profiles, thus leading to misinterpretation.

在旨在筛选尿液生物标志物的尿液代谢组学研究中，由于尿液尿素浓度极高，在基于色谱的代谢组学分析中，需通过脲酶（urease）对尿素进行降解。时至今日，该脲酶预处理步骤仍是标准化尿液代谢组学样本制备的核心环节，即便针对基于质谱的分析亦不例外。然而，将尿液与脲酶共孵育的预处理流程，与代谢组采样的核心原则相悖：代谢组采样应即刻终止代谢反应，以避免代谢物谱发生改变。尽管如此，脲酶预处理对代谢组分析的影响迄今尚未得到明确阐明。本研究发现，脲酶活性、尿液内源酶以及脲酶制剂中携带的代谢物污染物，会向代谢物谱中引入人为假象（artefacts），进而导致解读偏差。