基于肠道微生物代谢产物的人结直肠癌诊断方法研究

Establishment of a Novel Diagnostic Method for Colorectal Cancer Based on Metabolites of Intestinal Microorganism

建立气相色谱简单快速测定人粪便样品中短链脂肪酸(Short-chain fatty acids,SCFAs)的方法。粪便样品经1%HCl-75%乙醇溶液提取、高速离心,即用于GC测定。采用DB-FFAP毛细管柱(30 m×0.25 mm×0.25μm),升温程序洗脱(初始温度50℃保持1 min,以10℃/min升至190℃);气化室温度为250℃;载气(高纯氮)线速度为1.0 m L/min;分流比为50∶1,采用氢火焰离子化检测器检测。经系统方法学验证,证实本方法简单灵敏、准确可靠。采用多元统计分析方法成功区分健康志愿者和结直肠癌患者。与健康志愿者相比,结直肠癌患者粪便中乙酸、丁酸降低较为明显,提示SCFAs特别是丁酸可成为结直肠癌诊断的潜在标志物。本方法可用于结直肠癌患者和健康志愿者粪便中SCFAs的快速测定,并有望成为一种快速筛查和诊断结直肠癌的方法。

Abstract A rapid gas chromatographic （ GC ） method was established for the determination of short-chain fatty acids （ SCFAs ） in human feces. Feces samples were directly extracted by 1% HCl-75% ethanol solution, and then centrifuged at high speed for GC analysis. The chromatographic separation was performed on a DB-FFAP capillary column （ 30 m × 0 . 25 mm × 0 . 25 μm ） with a temperature program （ initial temperature at 50℃ held for 1 min, ramped to 190℃ at 10℃/min ） . The injection port temperature was 250℃ with split ratio at 50：1 . The carrier gas was high purity nitrogen with a constant linear velocity at 1. 0 mL/min. A flame ionization detector was employed to quantify SCFAs. The proposed method had been certified by systematic method validation, and used to determine feces samples. Subsequently, the health volunteer and colorectal cancer patient groups could be distinguished successfully by multivariate statistical analysis. Compared with health volunteers, the acetic acid and butyric acid of feces from colorectal cancer patients were reduced obviously, indicating that SCFAs particularly butyric acid could be considered as candidate markers for colorectal cancer diagnosis. In summary, this study provides a rapid method for the determination of SCFAs in feces form health volunteers and colorectal cancer patients. The method had a prospect for screening and diagnosing colorectal cancer rapidly.