Zheng Xiaojiao, Qiu Yunping, Zhong Wei, Baxter Sarah, Su Mingming, Li Qiong, Xie Guoxiang, Ore Brandon M, Qiao Shanlei, Spencer Melanie D, Zeisel Steven H, Zhou Zhanxiang, Zhao Aihua, Jia Wei
Center for Translational Medicine, and Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
Metabolomics. 2013 Aug 1;9(4):818-827. doi: 10.1007/s11306-013-0500-6.
Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography mass spectrometry (GC-MS) analysis. A one-step derivatization using 100 µL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations (RSDs) of all targeted compounds showed good intra- and inter-day (within 7 days) precision (< 10%), and good stability (< 20%) within 4 days at room temperature (23-25 °C), or 7 days when stored at -20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistics analysis of the concentrations of these target metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.
涉及肠道微生物群的肥胖和代谢紊乱研究需要可靠的方法来精确测量短链脂肪酸（SCFAs）和支链氨基酸（BCAAs）的浓度。在此，我们报告了一种快速方法，该方法使用氯甲酸丙酯（PCF）衍生化，然后进行气相色谱-质谱（GC-MS）分析，同时测定生物样品中的SCFAs和BCAAs。在pH值为8的水、丙醇和吡啶（v/v/v = 8:3:2）反应体系中，使用100 µL PCF进行一步衍生化可提供最佳衍生化效率。衍生化产物的最佳提取效率通过用己烷进行两步提取来实现。该方法在广泛的浓度范围内表现出良好的衍生化效率和回收率，每种化合物的检测限较低。所有目标化合物的相对标准偏差（RSDs）在日内和日间（7天内）均显示出良好的精密度（< 10%），在室温（23 - 25°C）下4天内或在 - 20°C储存7天时具有良好的稳定性（< 20%）。我们应用该方法测量了给予不同饮食的大鼠粪便样本中的SCFA和BCAA水平。对这些目标代谢物浓度的单变量和多变量统计分析能够区分饮食中含有乙醇干预和不同油脂的三组。该方法还成功用于测定正常人粪便、血浆和尿液中的SCFA和BCAA，提供了这些代谢物浓度的重要基线信息。这项新颖的代谢谱研究在转化研究方面具有巨大潜力。
