Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and β-Hydroxybutyrate/Acetoacetate in Biological Samples

International Journal of Molecular Sciences

卷:22

GaryW. Cline¹  Gina Butrico¹  GeraldI. Shulman¹  Eudald Casals²  Muling Zeng²  Guillermo Fernández-Varo³  Robin Wijngaard³  Susana Hidalgo³  Meritxell Perramón³  Manuel Morales-Ruiz³  Marina Parra-Robert³  Gregori Casals³  Wladimiro Jiménez³ 

[1] Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06510, USA;

[2] School of Biotechnology and Health Sciences, Wuyi University, 99 Yingbing Middle Rd., Jiangmen 529020, China;

[3] Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Carrer de Villarroel 170, 08036 Barcelona, Spain;

关键词: redox state;    GC-MS;    microwave-assisted derivatization;    nicotinamide adenine dinucleotide;    ketone bodies;   

DOI  :  10.3390/ijms22094752

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown