细胞内总游离巯基氨基酸的测定及其对不同作用机制药物的识别作用

建立了一种灵敏、特异的离线柱前衍生高效液相色谱-荧光检测法(HPLC-FLD)测定细胞内4种总的游离巯基氨基酸。以三-(2-羧乙基)膦(TCEP)为还原试剂、7-氟-2,1,3-苯唑-4-磺酸(SBD-F)为衍生试剂、N-(2-巯基丙酰基)-甘氨酸(MPG)为内标,经衍生后,在C18色谱柱上分离,12min内4种巯基氨基酸得到良好的基线分离,且其浓度与相对色谱峰面积呈良好线性关系,相关系数在0.9995～0.9998之间。结果表明,该方法具有良好的精密度、宽的线性范围及较低的定量限,适用于生物样本的测定。用此方法测定了HeLa细胞在长春新碱(25nmol/mL)及丝裂霉素(75nmol/mL)干预24h后细胞内4种巯基氨基酸,正常培养组为对照。偏最小二乘判别分析(PLS-DA)结果表明:细胞内4种巯基氨基酸能够将药物干预组与对照组之间、两个药物干预组之间很好地区分开,这种模式识别是代谢组学研究方法对药物作用机制模式识别的一种新的尝试,并希望通过这种方法找到与疾病或药物作用相关规律,用于计算机辅助药理学的研究。

Simultaneous Determination of Intracellular Free Aminotihols and the Recognition Role for Two Drugs with Different Action Mechanisms

As critical redox in organism aminothiols are involved and regulated by many physiological and pathological conditions of human diseases.Four free intracellular aminothiols have been determined by high performance liquid chromatography(HPLC) fluorescence detection with off-line pre-column derivatization,in which TCEP was selected as reduced agent,SBD-F as derivatization agent and N-(2-mercaptopropionyl)-glycine as internal standard.Four aminothiols with internal standard were separated satisfactorily in 12 min.Method validation showed that this approach has good reproducibility,wide linear range and low limit of quantification,and was suitable for biological sample analysis.Two interventioned groups by Vincristine(25 nmol/mL) and Mitomycin(75 nmol/mL) with different mechanisms for 24 h was investigated to study the recognition ability by aminothiols for drug actions.Partial least squares-discriminant analysis(PLS-DA) showed that the intracellular 4 aminothiols could be used to distinguish two anticancer drugs with different mechanisms.This study is an attempt for pattern recognition of drugs by metabolomics,and could possibly be used to find relevance between disease and drugs for computer-aided pharmacology.