高效液相色谱法检测细胞能量代谢物质的方法建立与应用

建立高效液相色谱法(HPLC)测定细胞中ATP、ADP和AMP含量的方法,以比较高糖培养的MRC-5细胞和D-半乳糖诱导的拟衰老MRC-5细胞能量代谢水平。体外培养的细胞经高氯酸裂解后,用反向HPLC法检测两组细胞ATP、ADP和AMP的含量。HPLC系统采用Ecosil C18反相柱(4.6*250mm 5μm pH 2～8),流动相为15mM磷酸二氢钾缓冲液和10%甲醇,流速0.5mL/min,紫外检测波长为254nm;检测温度为25℃,进样量20μL。以标准曲线法计算ATP、ADP和AMP含量,并计算腺苷酸能荷(EC)。结果显示拟衰老细胞的ATP含量及EC较对照组显著降低,而AMP含量明显升高,差异具有统计学意义(p<0.05),但两组之间的ADP含量没有明显差别。结果表明高效液相色谱法可检测出不同状态细胞能量物质的差异,其具有操作简便、快速、重现性好等特点,可以作为线粒体能量代谢功能的评估方法之一。

Establishment of High -performance Liquid Chromatography for Energy Metabolic Materials Assay and Its Applications

The objective of this study is to establish a simple and rapid HPLC method for measurements of ATP,ADP and AMP content in cells,and compare the energy metabolism level between high-glucose-treated and D-galactose-treated MRC-5 cells.Cells were collected and homogenized in 0.1mol/L perchloric acid solution,and then the content of ATP,ADP and AMP was detected by HPLC analysis.HPLC system included a C18 reverse phase hydrophilic column(4.6*250 mm,5 μm,pH2~8,Ecosil).Detections were operated using 50 mM monopotassium phosphate and 10% methyl alcohol as mobile phase with a flow rate of 0.5 ml/min and wavelength of 254 nm.For HPLC analysis,20 μl of standards or samples were injected.Concentrations of ATP,ADP and AMP were quantified according to the standard curve and energy charge(EC) was calculated.Significant reduction was observed in ATP contents and EC while AMP content was highly elevated in the D-galactose-treated cells(p<0.05).There was no significant difference in ADP content between high-glucose-treated and D-galactose-treated.The data indicated that HPLC is a simple,rapid and accurate method for detection of ATP,AMP and ADP in different phase of the cultured cells.It can be used as a method of assess function of mitochondrial energy metabolism.