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Abstract：
The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency\'s Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.
摘要：
乳酸/丙酮酸和β-羟基丁酸/乙酰乙酸的代谢比被认为是有价值的工具，通过估计细胞质和线粒体中的游离NAD/NADH来评估体内氧化还原细胞状态。分别。当前研究的目的是验证同时测定血浆和肝组织中四种代谢物的气相色谱质谱方法。该程序包括邻苯二胺微波辅助衍生化，然后用乙酸乙酯液-液萃取并用双(三甲基甲硅烷基)三氟乙酰胺:三甲基氯硅烷99:1进行甲硅烷基化。校准曲线呈现可接受的线性，丙酮酸的定量极限为0.001mM，β-羟基丁酸酯和乙酰乙酸酯，乳酸为0.01mM。日内和日间的准确性和精确度均符合欧洲药品管理局的指南规范。血浆或肝脏和水中三点标准代谢物加标曲线的斜率系数无显著差异，在代谢物加标样品中获得了可接受的回收率。在精确切割的肝大鼠切片中以及在挑战氧化还原状态的不同实验条件下孵育的HepG2细胞中测试了该方法的适用性。总之,经过验证的方法具有良好的灵敏度，在乳酸/丙酮酸和β-羟基丁酸/乙酸代谢物的定量中的特异性和可重复性，并且可能在体内氧化还原状态的评估中有用。
