粪便短链脂肪酸的测定方法优化及联合补充纳豆红曲对短链脂肪酸水平的影响

Optimization of the determination method of fecal short-chain fatty acids and the effect of combined supplementation of natto and red yeast rice on the level of short-chain fatty acids

目的建立液相色谱串联质谱(LC-MS/MS)方法测定粪便短链脂肪酸(SCFAs),并检测经纳豆红曲干预后小鼠粪便中SCFAs的水平。方法利用2-硝基苯肼盐酸盐(2-NPH·HCl)对粪便样本进行衍生,上清液萃取吹干后初始流动相复溶上机。将41只7周龄~8周龄ApoE-/-雄性小鼠随机分成4组:低脂饲料组(LFD)、高脂高胆固醇饲料组(HFD)、低脂饲料+0.3 g/kg纳豆红曲组(LFD+NR)和高脂高胆固醇饲料+0.3 g/kg纳豆红曲组(HFD+NR)。干预12周后检测粪便SCFAs水平和空腹血糖水平。结果目标待测物在LC-MS/MS下分离良好。所有待测物线性相关系数在0.997以上,回收率均在80.95%~119.45%,精密度均在0.2%~5.4%,满足方法学要求。联合补充纳豆红曲12周增加LFD饲养的ApoE-/-小鼠粪便丙酸和异丁酸水平。粪便中乙酸与空腹血糖水平成负相关,辛酸与空腹血糖水平成正相关。结论通过LC-MS/MS测定粪便SCFAs水平的方法准确度、灵敏度高,定量准确。粪便SCFAs含量与空腹血糖水平具有相关性,提示纳豆红曲可能通过影响粪便SCFAs含量影响血糖水平,需进一步探索机制。

Objective This paper aims to establish a liquid chromatography tandem mass spectrometry(LC-MS/MS)method for the determination of short chain fatty acids(SCFAs)in feces and investigate the effect of combined supplementation of natto and red yeast rice on the level of fecal SCFAs in mice.Methods The stool samples were derived with 2-nitrophenyl hydrazine hydrochloride(2-NPH·HCl),the supernatant was then extracted,dried,redissolved with the initial mobile phase and analyzed on a LC-MS/MS.Forty-one ApoE-/-male mice aged 7 to 8 weeks were randomly allocated into four groups:low-fat diet group(LFD),high-fat and high-cholesterol diet group(HFD),low-fat diet+0.3 g/kg natto and red yeast rice group(LFD+NR),and high-fat and high-cholesterol diet+0.3 g/kg natto and red yeast rice group(HFD+NR).After 12 weeks of supplementation,concentrations of fecal SCFAs and fasting blood glucose were measured.Results The target substances SCFAs and VFAs were well separated under LC-MS/MS.The linear correlation coefficient of all the substances was above 0.997,the recovery rates was between 80.95%and 119.45%,and the precision was between 0.2%and 5.4%.All of these parameters meet the methodological requirements.Combined supplementation with natto and red yeast rice for 12 weeks resulted in higher levels of propionic acid and isobutyric acid in the feces of ApoE-/-mice fed with LFD.The level of acetic acid in stool was inversely correlated with fasting blood glucose concentrations,while the level of octanoic acid was positively correlated with fasting blood glucose concentrations.Conclusion The method of determining fecal SCFAs levels by LC-MS/MS has the advantages of high accuracy,quantitative accuracy and simple operation.The levels of fecal SCFAs were correlated with fasting blood glucose concentrations,suggesting that combined supplementation with natto and red yeast rice may alter blood glucose concentrations via fecal SCFAs levels,which requires further research on underlying mechanisms.