Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, 6229 HX Maastricht, The Netherlands.
Department of Biochemistry, CARIM Cardiovascular Research Institute Maastricht, Maastricht University, 6229 HX Maastricht, The Netherlands.
J Proteome Res. 2021 Nov 5;20(11):5079-5087. doi: 10.1021/acs.jproteome.1c00617. Epub 2021 Sep 29.
While substantial efforts have been made to optimize and standardize fecal metabolomics for studies in adults, the development of a standard protocol to analyze infant feces is, however, still lagging behind. Here, we present the development of a hands-on and robust protocol for proton H NMR spectroscopy of infant feces. The influence of extraction solvent, dilution ratio, homogenization method, filtration, and duration of centrifugation on the biochemical composition of infant feces was carefully evaluated using visual inspection of H NMR spectra in combination with multivariate statistical modeling. The optimal metabolomics protocol was subsequently applied on feces from seven infants collected at 8 weeks, 4, and 9 months of age. Interindividual variation was exceeding the variation induced by different fecal sample preparation methods, except for filtration. We recommend extracting fecal samples using water with a dilution ratio of 1:5 feces-to-water to homogenize using bead beating and to remove particulates using centrifugation. Samples collected from infants aged 8 weeks and 4 months showed elevated concentrations of milk oligosaccharide derivatives and lactic acid, whereas short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) were higher in the 9 month samples. The established protocol enables hands-on and robust analyses of the infant gut metabolome. The wide-ranging application of this protocol will facilitate interlaboratory comparison of infants' metabolic profiles and finally aid in a better understanding of infant gut health.
尽管已经做出了大量努力来优化和规范成人粪便代谢组学研究，但婴儿粪便分析的标准方案的发展仍然滞后。在这里，我们提出了一种用于婴儿粪便质子 H NMR 光谱分析的实用且稳健的方案。通过结合 H NMR 光谱的直观检查和多变量统计建模，仔细评估了提取溶剂、稀释比、均化方法、过滤和离心时间对婴儿粪便生化成分的影响。随后将优化的代谢组学方案应用于 8 周、4 个月和 9 个月大的 7 名婴儿的粪便样本。个体间的变异超过了不同粪便样本制备方法引起的变异，除了过滤。我们建议使用粪便与水的 1:5 稀释比提取粪便样本，使用珠磨均质化，并使用离心去除颗粒物质。从 8 周和 4 个月大的婴儿采集的样本中，乳寡糖衍生物和乳酸的浓度升高，而 9 个月大的样本中短链脂肪酸（SCFA）和支链氨基酸（BCAA）的浓度更高。所建立的方案能够对婴儿肠道代谢组进行实用且稳健的分析。该方案的广泛应用将促进婴儿代谢谱的实验室间比较，并最终有助于更好地了解婴儿肠道健康。
