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. 2021 Apr 30;22(9):4752.
doi: 10.3390/ijms22094752.

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

Robin Wijngaard  1 , Meritxell Perramón  1 , Marina Parra-Robert  1 , Susana Hidalgo  1 , Gina Butrico  2 , Manuel Morales-Ruiz  1   3   4 , Muling Zeng  5 , Eudald Casals  5 , Wladimiro Jiménez  1   3 , Guillermo Fernández-Varo  1   3 , Gerald I Shulman  2 , Gary W Cline  2 , Gregori Casals  1   4
Affiliations

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

Robin Wijngaard et al. Int J Mol Sci. .

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.

Keywords: GC-MS; ketone bodies; microwave-assisted derivatization; nicotinamide adenine dinucleotide; redox state.

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Conflict of interest statement

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of derivatization yields of pyruvate and lactate after quinoxalinol deritvatitzation step performed using microwave or thermal block at different incubation times (mean of three plasma pools). RF: response factor.
Figure 2
Figure 2
(a) Mass chromatograms of a rat liver sample for lactate (m/z 117) and pyruvate (m/z 217) (tube 1). The concentrations were 1.23 mM and 0.18 mM, for lactate and pyruvate, respectively. Note that an additional peak can be observed at 3.7 min, derived from the elution of β-hydroxybutyrate, which was stable against the MAD quinoxalinol derivatization step. (b) Mass chromatograms of a rat liver sample for β-hydroxybutyrate (m/z 191) and acetoacetate (m/z 231) (tube 2). The concentrations were 0.56 mM and 0.031 mM, for β-hydroxybutyrate and acetoacetate, respectively.
Figure 3
Figure 3
(a) Ratios of lactate/pyruvate and β-hydroxybutyrate/acetoacetate in precision-cut liver slices incubated under control conditions (control), exposed to ethanol (17 mM) or H2O2 (1.5 mM). (b) Ratios of lactate/pyruvate in human hepatic cells (HepG2) cultivated under normal conditions (control), stimulated with 2 mM H2O2 (H2O2) and stimulated with 2 mM H2O2 and treated with CeO2NPs (5 µg/mL) (H2O2 + CeO2NPs).
Figure 4
Figure 4
(a) Schema of the assay procedure for the determination of lactate and pyruvate (Tube 1) and β-hydroxybutyrate and acetoacetate (Tube 2) in plasma samples. (b) Schema of the assay procedure for determination of lactate and pyruvate (Tube 1) and β-hydroxybutyrate and acetoacetate (Tube 2) in liver samples. 5-SAS: 5-sulfosalicylic acid; BSTFA: bis(trimethylsilyl)trifluoroacetamide; HCl: hydrochloric acid; IS: internal standard; OPD: o-phenylenediamine.
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