Wu Lauren D, Ruhaak L Renee, Lebrilla Carlito B
Department of Chemistry, University of California, Davis, CA, 95616, USA.
Department of Translational Molecular Pathology, UT MD Anderson Cancer Center, 6767 Bertner Avenue, Houston, TX, 77030, USA.
Methods Mol Biol. 2017;1503:121-129. doi: 10.1007/978-1-4939-6493-2_10.
Human milk oligosaccharides (HMOs) are a highly abundant constituent in human milk, and its protective and prebiotic properties have attracted considerable attention. HMOs have been shown to directly and indirectly benefit the overall health of the infant due to a number of functions including serving as a beneficial food for gut bacteria, block to pathogens, and aiding in brain development. Researchers are currently exploring whether these structures may act as possible disease and nutrition biomarkers. Because of this, rapid-throughput methods are desired to investigate biological activity in large patient sets. We have optimized a rapid-throughput protocol to analyze human milk oligosaccharides using micro-volumes of human breast milk for nutritional biomarkers. This method may additionally be applied to other biological fluid substrates such as plasma, urine, and feces. The protocol involves lipid separation via centrifugation, protein precipitation using ethanol, alditol reduction with sodium borohydride, and a final solid-phase extraction purification step using graphitized carbon cartridges. Samples are analyzed using HPLC-Chip/TOF-MS and data filtered on Agilent MassHunter using an in-house library. Individual structural identification is matched against a previously developed HMO library using accurate mass and retention time. Using this method will allow in-depth characterization and profiling of HMOs in large patient sets, and will ease the process of discovering significant nutritional biomarkers in human milk.
人乳寡糖（HMOs）是人乳中含量极为丰富的成分，其保护和益生元特性已引起了广泛关注。由于具有多种功能，包括作为肠道细菌的有益食物、阻挡病原体以及助力大脑发育等，HMOs已被证明能直接和间接地有益于婴儿的整体健康。研究人员目前正在探索这些结构是否可能作为潜在的疾病和营养生物标志物。因此，需要高通量方法来研究大量患者群体中的生物活性。我们已经优化了一种高通量方案，使用微量人乳来分析人乳寡糖以寻找营养生物标志物。该方法还可应用于其他生物流体样本，如血浆、尿液和粪便。该方案包括通过离心分离脂质、用乙醇沉淀蛋白质、用硼氢化钠进行糖醇还原，以及最后使用石墨化碳柱进行固相萃取纯化步骤。使用高效液相色谱-芯片/飞行时间质谱仪（HPLC-Chip/TOF-MS）对样本进行分析，并使用内部数据库在安捷伦MassHunter软件上对数据进行过滤。通过精确质量和保留时间，将个体结构鉴定与先前开发的HMO数据库进行比对。使用这种方法将能够对大量患者群体中的HMOs进行深入表征和分析，并将简化在人乳中发现重要营养生物标志物的过程。
