测定HepG2细胞内^13C标记氨基酸及代谢物同位素丰度的方法

Method for determining isotope abundance of C-labeled amino acids and metabolites in HepG2 cells

目的利用稳定同位素和气相色谱-质谱(gas chromatography-mass spectrometer,GC-MS)衍生化法检测四君子汤(Sijunzi Decoction,SJZD)与丝裂霉素C(mitomycin C,MMC)联用对HepG2细胞内丙氨酸代谢的影响,探讨不同给药组与空白组HepG2细胞内丙氨酸代谢的差异及其意义。方法 HepG2细胞分4组(空白组、SJZD组、MMC组和SJZD与MMC联用组),分别给^13C标记丙氨酸(^13C-labeled alanine,^13C-Ala)培养12 h,回收细胞并加入体积分数为80%的甲醇溶液超声破碎后,离心取上清液,氮吹仪吹干,用体积分数为1%三甲基氯硅烷(trimethylchlorosilane,TMCS)的N-甲基-N-(三甲基甲硅烷基)三氟乙酰胺(N-methyl-N-(trimethylsilyl) trifluoroacetamide,MSTFA)衍生化,利用GC-MS检测并分析检测结果。结果 ^13C-Ala同位素丰度在10.00%~98.00%内均可以被准确测定,所建立的方法具有良好的准确度和精密度,用该方法对10组供试品进行测定,并根据EI图谱的离子碎片信息对^13C-Ala同位素丰度进行计算,统计分析结果显示,空白组与SJZD组无显著性差异(P>0.05),空白组与MMC组有极显著性差异(P<0.01),联用组与MMC组有极显著性差异(P<0.01);采用相对峰面积(Area%)比较各组之间的差异,所得结果与采用同位素丰度进行计算的结果一致。^13C-Ala下游产物乳酸的代谢采用同位素丰度进行计算,结果显示:各组之间无显著性差异。但Area%结果显示:SJZD组与空白组比较无显著性差异(P>0.05),MMC组与空白组有极显著性差异(P<0.01),联用组与MMC组相比有极显著性差异(P<0.01)。结论 SJZD单独作用对丙氨酸及其代谢产物乳酸的影响不大,与MMC联用给药后对MMC的干扰有明显的抑制作用,所得结果与前期靶向代谢组学的结论相一致,本方法可作为HepG2细胞内氨基酸更为精确的测定方法,具有良好的应用前景。

Objective Gas chromatography-mass spectrometry(GC-MS) derivatization was used to detect the difference of Alanine metabolism in HepG2 cells by Sijunzi Decoction(SJZD) combined with mitomycin C(MMC).The HepG2 cells in the drug-administered group and the blank group were investigated.The difference in the metabolism of Alanine and its significance were explored.Methods HepG2 cells were simultaneously cultured with ^13C-LabeLed Alanine(^13C-Ala),SJZD,and MMC for 12 hours.The cells were recovered and sonicated with 80% methanol.The upper layer of liquid nitrogen was dried and subjected to MSTFA derivatization,which was detected and analyzed by GC-MS.Results The Alanine ^13C isotope abundance can be accurately determined within 10%-98%.The method had the good accuracy and tightness.The established samples were used to determine 10 groups of samples,and the ions according to the EI spectrum were determined.The ^13C-Ala isotope abundance was calculated by the fragmentation in formation.The statistical analysis showed that there was no significant difference between the control group and the SJZD group(P>0.05).There was a significant difference between the control group and the MMC group(P<0.01).There was a significant difference between the two groups(P<0.01).The relative peak area(Area%) was used to compare the differences between the groups.The results were consistent with those calculated by isotopic abundance.The metabolism of lactic acid in the downstream product of ^13C-Ala was calculated by isotope abundance.The results showed that there was no significant difference between the groups,but the Area% results showed that there was no significant difference between the SJZD group and the control group(P>0.05).There was a significant difference between the group and the control group(P<0.01),and there was a significant difference between the control group and the MMC group(P<0.01).Conclusion The results obtained are consistent with the conclusions of the previous targeted metabolomics:SJZD alone has little effect on the Alanine and its metabolite lactic acid,and it can significantly inhibit the interference of MMC after administration with MMC.The method can be used as a more accurate method for determining amino acids in HepG2 cells,and has a good application prospect.