Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Salisburylaan 133, B-9820, Merelbeke, Belgium.
Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Salisburylaan 133, B-9820, Merelbeke, Belgium; Institute for Global Food Security, School of Biological Sciences, Queen's University, University Road, Belfast, United Kingdom.
Anal Chim Acta. 2020 Apr 29;1108:79-88. doi: 10.1016/j.aca.2020.02.046. Epub 2020 Feb 26.
Faecal metabolomics markedly emerged in clinical as well as analytical chemistry through the unveiling of aberrations in metabolic signatures as reflection of variance in gut (patho)physiology and beyond. Logistic hurdles, however, hinder the analysis of stool samples immediately following collection, inferring the need of biobanking. Yet, the optimum way of storing stool material remains to be determined, in order to conserve an accurate snapshot of the metabolome and circumvent artifacts regarding the disease and parameter(s) under observation. To address this problem, this study scrutinised the impact of freeze-thaw cycling, storage duration, temperature and aerobicity, thereby using ultra-high performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS)-based polar metabolomics and lipidomics methodologies for faecal metabolomics. Both targeted (n > 400) and untargeted approaches were implemented to assess storage effects on individual chemical classes of metabolites as well as the faecal fingerprint. In general, recommendations are that intact stool samples should be divided into aliquots, lyophilised and stored at -80 °C for a period no longer than 18 weeks, and avoiding any freeze-thawing. The first preservation week exerted the most decisive impact regarding storage temperature, i.e. 12.1% and 6.4% of the polar metabolome experienced a shift at -20 °C and at -80 °C, respectively, whereas 8.6% and 7.9% was observed to be changed significantly for the lipidome. In addition, aside from the negligible impact of aerobicity, the polar metabolome appeared to be more dependent on the storage conditions applied compared to the lipidome, which emerged as the more stable fraction when assessing the storage duration for 25 weeks. If the interest would greatly align with particular chemical classes, such as branched-chain amino acids or short-chain fatty acids, specific storage duration recommendations are reported. The provided insights on the stability of the faecal metabolome may contribute to a more reasoned design of experiments in biomarker detection or pathway elucidation within the field of faecal metabolomics.
粪便代谢组学通过揭示代谢特征的异常，作为肠道（病理）生理学及其他方面变化的反映，在临床和分析化学领域中显著出现。然而，逻辑障碍阻碍了对采集后立即进行的粪便样本的分析，这暗示了生物库的必要性。然而，为了准确地保留代谢组的快照并避免有关疾病和观察参数的人为因素，仍然需要确定存储粪便样本的最佳方法。为了解决这个问题，本研究通过使用基于超高效液相色谱-高分辨率质谱（UPLC-HRMS）的极性代谢组学和脂质组学方法，研究了冻融循环、储存时间、温度和需氧性对粪便代谢组学的影响。本研究同时采用靶向（n＞400）和非靶向方法来评估储存对个体代谢物化学类别的影响以及粪便指纹图谱。一般来说，建议将完整的粪便样本分成等分，冻干并储存在-80°C 下，时间不超过 18 周，并避免任何冻融。在储存温度方面，第一个保存周的影响最为显著，即极性代谢组的 12.1%和 6.4%分别在-20°C 和-80°C 下发生了转移，而脂质组的 8.6%和 7.9%观察到显著变化。此外，除了需氧性的影响可以忽略不计之外，与脂质组相比，极性代谢组似乎更依赖于所应用的储存条件，在评估 25 周的储存时间时，脂质组表现出更稳定的特征。如果特别关注某些化学类别的情况，例如支链氨基酸或短链脂肪酸，可以提供特定的储存时间建议。本研究关于粪便代谢组稳定性的见解可能有助于在粪便代谢组学领域中更合理地设计生物标志物检测或途径阐明实验。
