RESUMEN
Lactic acid and pyruvic acid are important metabolites in the tricarboxylic acid cycle that can reflect the cytoplasmic redox state and mitochondrial respiratory chain function. The combination of these acids is considered as a screening index for mitochondrial disorders. Due to their biological effects, a derivatization method was developed to simultaneously detect pyruvic acid and lactic acid in tissue and cell culture media using gas chromatography. In this work, the combined derivatization method with methoxyamine hydrochloride and isobutyl chloroformate was first proposed. To improve the efficiency of derivatization, in situ derivatization-ultrasound-assisted emulsification microextraction (USAEME) was used in this study. After optimizing the volume of reagents and reaction times, good linearity values were obtained from 50 to 1000 µmol/L and 1 to 100 µmol/L for lactic acid and pyruvic acid, respectively. The limits of detection (LODs) were 0.12 µmol/L for lactic acid and 0.29 µmol/L for pyruvic acid. The recoveries of the two analytes were between 93.60 and 102.80%, and the precisions were less than 6.20%. This method was successfully applied to quantify pyruvic acid and lactic acid in the animal and cellular hypoxia models which provided an auxiliary means for the diagnosis of mitochondrial diseases. Graphical abstract á .
Asunto(s)
Cromatografía de Gases/métodos , Medios de Cultivo/metabolismo , Ácido Láctico/metabolismo , Enfermedades Mitocondriales/diagnóstico , Ácido Pirúvico/metabolismo , Sonicación/métodos , Animales , Células Cultivadas , Ciclo del Ácido Cítrico , Emulsiones , Límite de Detección , Ratones , Enfermedades Mitocondriales/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Lactic acid represents an important metabolite that reflects mitochondria function and may further serve as energy source for cancer cells. In light of this physiological and pathological significance, we developed a novel and sensitive gas chromatography method to detect lactic acid in cell culture media. Here, ethyl chloroformate was selected as derivative reagent and the derivatization process was further optimized in terms of number of reagents and reaction time as well as extraction reagents. Under optimal conditions, good linearity was achieved in the tested calibration range. The limit of detection (LOD) was determined to be 0.67⯵mol/L, the recovery rates were 99.6%-106% and the precision rate RSD was <5.49%. Furthermore, this method has been applied to quantify the secretion of lactic acid in cells exposed to mono2ethylhexyl phthalate at different doses and in cancer cells over time. Taken in concert, our method proved to be both sensitive and reliable and may be applied for studies on mitochondrial function and cell glycolysis conditions.