Laboratory of Metabolomics, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 5, 779 00, Olomouc, Czech Republic; Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, I.P. Pavlova 6, 775 20, Olomouc, Czech Republic.
Department of Biochemistry, Faculty of Science, Palacký University Olomouc, Šlechtitelů 27, 783 71, Olomouc, Czech Republic.
J Chromatogr A. 2019 Nov 8;1605:360355. doi: 10.1016/j.chroma.2019.07.009. Epub 2019 Jul 10.
Urea, as an end product of protein metabolism and an abundant polar compound, significantly complicates the metabolomic analysis of urine by GC-MS. We developed a sample preparation method removing urea from urine samples prior the GC-MS analysis. The method based on urease immobilized on magnetic microparticles was compared with the others that are conventionally used (liquid-liquid extraction, free urease protocol), and samples without any treatment. To study the impact of sample preparation approaches on the quality of analytical data, we employed comprehensive metabolomic analysis (using both GC-MS and LC-MS/MS platforms) of standard material based on human urine. Multivariate statistical analysis has shown that immobilized urease treatment provides similar results to a free urease approach. However, significant alterations in the profiles of metabolites were observed in the samples without any treatment and after the extraction. Compared to other approaches that were tested, the immobilization of urease on microparticles reduces both the number of artifacts and the variability of the metabolites (average CV of extraction 19.7%, no treatment 11.4%, free urease 5.0%, and immobilized urease 2.5%). The method that was developed was applied in a GC-MS metabolomic experiment of glutaric aciduria type I, where both known diagnostically important biomarkers and unknowns, as the most discriminating compounds, were found.
尿素作为蛋白质代谢的终产物和丰富的极性化合物，极大地增加了 GC-MS 对尿液代谢组学分析的复杂性。我们开发了一种在 GC-MS 分析前从尿液样本中去除尿素的样品制备方法。该方法基于固定在磁性微球上的脲酶，与传统方法（液液萃取、游离脲酶方案）和未经任何处理的样品进行了比较。为了研究样品制备方法对分析数据质量的影响，我们采用基于人尿液的标准物质进行了全面的代谢组学分析（使用 GC-MS 和 LC-MS/MS 平台）。多变量统计分析表明，固定化脲酶处理与游离脲酶方法提供了相似的结果。然而，在未经任何处理和提取后的样品中观察到代谢物图谱发生了显著变化。与测试的其他方法相比，脲酶在微球上的固定化减少了伪影的数量和代谢物的可变性（提取的平均 CV 为 19.7%，未经处理为 11.4%，游离脲酶为 5.0%，固定化脲酶为 2.5%）。所开发的方法应用于 I 型戊二酸血症的 GC-MS 代谢组学实验中，在其中发现了已知具有诊断重要性的生物标志物和未知物，作为最具区分性的化合物。
