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
Abstract
:1. Introduction
2. Results and Discussion
2.1. Method Optimization
2.2. Method Validation
2.3. Applicability of the Method
3. Materials and Methods
3.1. Chemical Reagents
3.2. Instrumentation
3.3. Preparation of Stock Solutions, Working Solutions, Calibrators and Quality Controls Samples
3.4. Sample Preparation
3.5. Microwave-Assisted Derivatization
3.6. Method Validation
3.6.1. Linearity of Calibration Curves and Matrix Effect
3.6.2. Accuracy and Precision
3.6.3. Recovery, Selectivity, Carry-Over and Stability of Derivatives
3.7. Method Application
3.7.1. Precision-Cut Liver Slices from Rats
3.7.2. Cultured Human Hepatic Cells
3.8. Statistical Calculations
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Madiraju, A.K.; Qiu, Y.; Perry, R.J.; Rahimi, Y.; Zhang, X.M.; Zhang, D.; Camporez, J.P.G.; Cline, G.W.; Butrico, G.M.; Kemp, B.E.; et al. Metformin Inhibits Gluconeogenesis via a Redox-Dependent Mechanism In Vivo. Nat. Med. 2018, 24, 1384–1394. [Google Scholar] [CrossRef]
- Alshawi, A.; Agius, L. Low Metformin Causes a More Oxidized Mitochondrial NADH/NAD Redox State in Hepatocytes and Inhibits Gluconeogenesis by a Redox-Independent Mechanism. J. Biol. Chem. 2019, 294, 2839–2853. [Google Scholar] [CrossRef] [Green Version]
- Mintun, M.A.; Vlassenko, A.G.; Rundle, M.M.; Raichle, M.E. Increased Lactate/Pyruvate Ratio Augments Blood Flow in Physiologically Activated Human Brain. Proc. Natl. Acad. Sci. USA 2004, 101, 659–664. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, Q.; Wang, S.Y.; Nottke, A.C.; Rocheleau, J.V.; Piston, D.W.; Goodman, R.H. Redox Sensor CtBP Mediates Hypoxia-Induced Tumor Cell Migration. Proc. Natl. Acad. Sci. USA 2006, 103, 9029–9033. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Habarou, F.; Brassier, A.; Rio, M.; Chrétien, D.; Monnot, S.; Barbier, V.; Barouki, R.; Bonnefont, J.P.; Boddaert, N.; Chadefaux-Vekemans, B.; et al. Pyruvate Carboxylase Deficiency: An Underestimated Cause of Lactic Acidosis. Mol. Genet. Metab. Rep. 2015, 2, 25–31. [Google Scholar] [CrossRef] [PubMed]
- Wu, J.; Jin, Z.; Zheng, H.; Yan, L.J. Sources and Implications of NADH/NAD+ Redox Imbalance in Diabetes and Its Complications. Diabetes Metab. Syndr. Obes. Targets Ther. 2016, 9, 145–153. [Google Scholar] [CrossRef] [Green Version]
- Levy, B.; Sadoune, L.O.; Gelot, A.M.; Bollaert, P.E.; Nabet, P.; Larcan, A. Evolution of Lactate/Pyruvate and Arterial Ketone Body Ratios in the Early Course of Catecholamine-Treated Septic Shock. Crit. Care Med. 2000, 28, 114–119. [Google Scholar] [CrossRef] [PubMed]
- Debray, F.G.; Mitchell, G.A.; Allard, P.; Robinson, B.H.; Hanley, J.A.; Lambert, M. Diagnostic Accuracy of Blood Lactate-to-Pyruvate Molar Ratio in the Differential Diagnosis of Congenital Lactic Acidosis. Clin. Chem. 2007, 53, 916–921. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vassault, A.J. Lactate, Pyruvate, Acetoacetate and 3-Hydroxybutyrate. In Laboratory Guide to the Methods in Biochemical Genetics; Springer: Berlin/Heidelberg, Germany, 2008; pp. 35–51. [Google Scholar] [CrossRef]
- Williamson, D.H.; Lund, P.; Krebs, H.A. The Redox State of Free Nicotinamide-Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver. Biochem. J. 1967, 103, 514–527. [Google Scholar] [CrossRef]
- Christensen, C.E.; Karlsson, M.; Winther, J.R.; Jensen, P.R.; Lerche, M.H. Non-Invasive in-Cell Determination of Free Cytosolic [NAD+]/ [NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes. J. Biol. Chem. 2014, 289, 2344–2352. [Google Scholar] [CrossRef] [Green Version]
- Sun, F.; Dai, C.; Xie, J.; Hu, X. Biochemical Issues in Estimation of Cytosolic Free NAD/NADH Ratio. PLoS ONE 2012, 7, 31–36. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Krebs, H.A. The Redox State of Nicotinamide Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver. Adv. Enzyme Regul. 1967, 5, 409–434. [Google Scholar] [CrossRef]
- Artuch, R.; Vilaseca, M.A.; Farre, C.; Ramon, F. Determination of Lactate, Pyruvate, β-Hydroxybutyrate and Acetoacetate with a Centrifugal Analyser. Clin. Chem. Lab. Med. 1995, 33, 529–534. [Google Scholar] [CrossRef]
- Galán, A.; Hernández, J.M.; Jimenez, O. Measurement of Blood Acetoacetate and β-Hydroxybutyrate in an Automatic Analyser. J. Autom. Methods Manag. Chem. 2001, 23, 69–76. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Nuwayhid, N.F.; Johnson, G.F.; Feld, R.D. Multipoint Kinetic Method for Simultaneously Measuring the Combined Concentrations of Acetoacetate-β-Hydroxybutyrate and Lactate-Pyruvate. Clin. Chem. 1989, 35, 1526–1531. [Google Scholar] [CrossRef]
- Li, P.K.; Lee, J.T.; MacGillivray, M.H.; Schaefer, P.A.; Siegel, J.H. Direct, Fixed-Time Kinetic Assays for Beta-Hydroxybutyrate and Acetoacetate with a Centrifugal Analyzer or a Computer-Backed Spectrophotometer. Clin. Chem. 1980, 26, 1713–1717. [Google Scholar] [CrossRef] [PubMed]
- Yoon, H.-R. Simultaneous Determination of Plasma Lactate, Pyruvate, and Ketone Bodies Following Tert-Butyldimethylsilyl Derivatization Using GC-MS-SIM. Biomed. Sci. Lett. 2015, 21, 241–247. [Google Scholar] [CrossRef] [Green Version]
- Beylot, M.; Beaufrère, B.; Normand, S.; Riou, J.P.; Cohen, R.; Mornex, R. Determination of Human Ketone Body Kinetics Using Stable-Isotope Labelled Tracers. Diabetologia 1986, 29, 90–96. [Google Scholar] [CrossRef]
- Pacenti, M.; Dugheri, S.; Traldi, P.; Degli Esposti, F.; Perchiazzi, N.; Franchi, E.; Calamante, M.; Kikic, I.; Alessi, P.; Bonacchi, A.; et al. New Automated and High-Throughput Quantitative Analysis of Urinary Ketones by Multifiber Exchange-Solid Phase Microextraction Coupled to Fast Gas Chromatography/Negative Chemical-Electron Ionization/Mass Spectrometry. J. Autom. Methods Manag. Chem. 2010, 2010, 1–13. [Google Scholar] [CrossRef] [Green Version]
- Rocchiccioli, F.; Leroux, J.P.; Cartier, P. Quantitation of 2-ketoacids in Biological Fluids by Gas Chromatography Chemical Ionization Mass Spectrometry of O-trimethylsily-quinoxalinol Derivatives. Biol. Mass Spectrom. 1981, 8, 160–164. [Google Scholar] [CrossRef]
- Carragher, F.M.; Bonham, J.R.; Smith, J.M. Pitfalls in the Measurement of Some Intermediary Metabolites. Ann. Clin. Biochem. 2003, 40, 313–320. [Google Scholar] [CrossRef]
- Payne, B. Pitfalls in the Measurement of Some Intermediary Metabolites: Stabilization of Lactate and Pyruvate. Ann. Clin. Biochem. 2004, 41, 83. [Google Scholar]
- Woolf, L.I.; Hasinoff, C.; Perry, A. Estimation of Branched-Chain α-Keto Acids in Blood by Gas Chromatography. J. Chromatogr. B Biomed. Sci. Appl. 1982, 231, 237–245. [Google Scholar] [CrossRef]
- Pailla, K.; Blonde-Cynober, F.; Aussel, C.; De Bandt, J.-P.; Cynober, L. Branched-Chain Keto-Acids and Pyruvate in Blood: Measurement by HPLC with Fluorimetric Detection and Changes in Older Subjects. Clin. Chem. 2000, 46, 848–853. [Google Scholar] [CrossRef] [Green Version]
- Casals, G.; Marcos, J.; Pozo, O.J.; Alcaraz, J.; Martínez de Osaba, M.J.; Jiménez, W. Microwave-Assisted Derivatization: Application to Steroid Profiling by Gas Chromatography/Mass Spectrometry. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2014, 960, 8–13. [Google Scholar] [CrossRef] [PubMed]
- Fernández-Galán, E.; Massana, N.; Parra-Robert, M.; Hidalgo, S.; Casals, G.; Esteve, J.; Jiménez, W. Validation of a Routine Gas Chromatography Mass Spectrometry Method for 2-Hydroxyglutarate Quantification in Human Serum as a Screening Tool for Detection of Idh Mutations. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2018, 1083, 28–34. [Google Scholar] [CrossRef]
- Miles, J.M.; Schwenk, W.F.; McClean, K.L.; Haymond, M.W. Determination of Ketone Body Turnover in Vivo with Stable Isotopes, Utilizing Gas Chromatography/Mass Spectrometry. Anal. Biochem. 1984, 141, 110–115. [Google Scholar] [CrossRef]
- European Medicines Agency. European Medicines Agency, Guideline on Bioanalytical Method Validation; European Medicines Agency: Amsterdam, The Netherlands, 2011. [Google Scholar]
- Goodman, R.P.; Markhard, A.L.; Shah, H.; Sharma, R.; Skinner, O.S.; Clish, C.B.; Deik, A.; Patgiri, A.; Hsu, Y.-H.H.; Masia, R.; et al. Hepatic NADH Reductive Stress Underlies Common Variation in Metabolic Traits. Nature 2020, 583, 122–126. [Google Scholar] [CrossRef] [PubMed]
- Casals, E.; Zeng, M.; Parra-Robert, M.; Fernández-Varo, G.; Morales-Ruiz, M.; Jiménez, W.; Puntes, V.; Casals, G. Cerium Oxide Nanoparticles: Advances in Biodistribution, Toxicity, and Preclinical Exploration. Small 2020, 16, 1907322. [Google Scholar] [CrossRef] [PubMed]
- Parra-Robert, M.; Casals, E.; Massana, N.; Zeng, M.; Perramón, M.; Fernández-Varo, G.; Morales-Ruiz, M.; Puntes, V.; Jiménez, W.; Casals, G. Beyond the Scavenging of Reactive Oxygen Species (ROS): Direct Effect of Cerium Oxide Nanoparticles in Reducing Fatty Acids Content in an In Vitro Model of Hepatocellular Steatosis. Biomolecules 2019, 9, 425. [Google Scholar] [CrossRef] [Green Version]
Metabolite | Derivative | Selected Ion (m/z) | Retention Time (min) |
---|---|---|---|
Lactate | 2-TMS | 117 | 2.9 |
l-lactate-3-13C | 2-TMS | 118 | 2.9 |
Pyruvate | TMS-quinoxalinol | 217 | 5.1 |
Pyruvate-1-13C | TMS-quinoxalinol | 218 | 5.1 |
β-hydroxybutyrate | 2-TMS | 191 | 3.7 |
β-Hydroxybutyrate-d4 | 2-TMS | 195 | 3.7 |
Acetoacetate | 2-TMS | 231 | 4.2 |
Lactate | Pyruvate | β-Hydroxybutyrate | Acetoacetate | ||||||
---|---|---|---|---|---|---|---|---|---|
Standard | mM | A (%) | P (%) | A (%) | P (%) | A (%) | P (%) | A (%) | P (%) |
Std 1 | 0.001 | - | - | 93.3 | 12.4 | 100.0 | 0.0 | 120.0 | 22.0 |
Std 2 | 0.01 | 101.7 | 14.7 | 110.3 | 4.6 | 99.7 | 6.0 | 86.7 | 15.7 |
Std 3 | 0.025 | 98.9 | 12.9 | 100.9 | 9.9 | 98.5 | 8.5 | 92.1 | 7.5 |
Std 4 | 0.1 | 99.4 | 6.3 | 101.7 | 9.2 | 95.7 | 1.8 | 97.0 | 9.4 |
Std 5 | 0.25 | 105.3 | 8.0 | 99.8 | 6.2 | 92.7 | 11.1 | 102.8 | 5.6 |
Std 6 | 1 | 98.4 | 3.5 | 100.0 | 0.6 | 93.9 | 10.0 | 99.7 | 0.5 |
Std 7 | 5 | 99.6 | 1.4 | - | - | 101.8 | 1.4 | - | - |
Human Plasma | Rat Liver | ||||||
---|---|---|---|---|---|---|---|
Added (mM) | Detected (mM) | Expected (mM) | Recovery (%) | Detected (mM) | Expected (mM) | Recovery (%) | |
Lactate | - | 0.37 | - | - | 0.66 | ||
0.1 | 0.41 | 0.47 | 87.2 | 0.69 | 0.76 | 90.8 | |
0.4 | 0.79 | 0.77 | 102.6 | 1.10 | 1.06 | 103.8 | |
Pyruvate | - | 0.13 | - | - | 0.13 | ||
0.1 | 0.22 | 0.23 | 95.7 | 0.23 | 0.23 | 100.0 | |
0.4 | 0.53 | 0.53 | 100.0 | 0.57 | 0.53 | 107.5 | |
β-hydroxybutyrate | - | 0.12 | - | - | 0.17 | ||
0.1 | 0.20 | 0.22 | 90.0 | 0.24 | 0.22 | 88.9 | |
0.4 | 0.53 | 0.52 | 101.9 | 0.53 | 0.52 | 98.2 | |
Acetoacetate | - | 0.07 | - | - | 0.07 | ||
0.1 | 0.16 | 0.17 | 94.1 | 0.15 | 0.17 | 88.2 | |
0.4 | 0.53 | 0.47 | 112.8 | 0.41 | 0.47 | 87.2 |
Intra-Day (n = 3) | Inter-Day (n = 3) | ||||
---|---|---|---|---|---|
mM | A (%) | P (%) | A (%) | P (%) | |
Lactate | |||||
QC2 | 0.05 | 106.3 | 5.0 | 98.5 | 15.3 |
QC3 | 0.5 | 101.2 | 9.1 | 105.1 | 8.4 |
QC4 | 2.5 | - | - | 95.9 | 3.0 |
Pyruvate | |||||
QC1 | 0.005 | - | - | 102.7 | 14.1 |
QC2 | 0.05 | 90.8 | 10.3 | 103.1 | 6.4 |
QC3 | 0.5 | 91.4 | 8.5 | 101.6 | 8.2 |
β-hydroxybutyrate | |||||
QC1 | 0.005 | - | - | 91.3 | 6.7 |
QC2 | 0.05 | 90.3 | 11.5 | 96.4 | 10.9 |
QC3 | 0.5 | 93.8 | 11.2 | 91.0 | 9.6 |
QC4 | 2.5 | - | - | 101.3 | 11.9 |
Acetoacetate | |||||
QC1 | 0.005 | - | - | 86.7 | 15.0 |
QC2 | 0.05 | 87.7 | 12.2 | 95.5 | 8.6 |
QC3 | 0.5 | 85.0 | 13.8 | 109.0 | 9.0 |
Intra-Day (n = 3) | Inter-Day (n = 3) | |||
---|---|---|---|---|
Mean (mM) | P (%) | Mean (mM) | P (%) | |
Lactate | ||||
Plasma | 1.3875 | 6.5 | 1.8025 | 6.2 |
Liver | 0.9253 | 1.8 | 1.3778 | 10.1 |
Pyruvate | ||||
Plasma | 0.1319 | 7.9 | 0.1459 | 8.9 |
Liver | 0.0502 | 14.9 | 0.1984 | 7.2 |
β-hydroxybutyrate | ||||
Plasma | 0.0605 | 4.4 | 0.0663 | 8.6 |
Liver | 0.0752 | 4.6 | 0.5991 | 10.1 |
Acetoacetate | ||||
Plasma | 0.0107 | 2.9 | 0.0115 | 10.5 |
Liver | 0.0047 | 6.2 | 0.0310 | 21.9 |
Mean (mM) | Accuracy (%) | Precision (%) | ||
---|---|---|---|---|
24 h | 96 h | |||
Lactate | ||||
QC2 | 0.05 | 95.4 | 90.3 | 5.1 |
QC3 | 0.5 | 95.7 | 96.1 | 2.4 |
QC4 | 2.5 | 94.0 | 102.0 | 4.2 |
Pyruvate | ||||
QC1 | 0.005 | 108.1 | 114.5 | 6.8 |
QC2 | 0.05 | 95.5 | 106.8 | 5.7 |
QC3 | 0.5 | 101.6 | 110.8 | 5.6 |
β-hydroxybutyrate | ||||
QC1 | 0.005 | 100.0 | 98.0 | 1.2 |
QC2 | 0.05 | 96.8 | 97.3 | 1.8 |
QC3 | 0.5 | 102.4 | 104.4 | 2.1 |
QC4 | 2.5 | 86.0 | 86.4 | 8.8 |
Acetoacetate | ||||
QC1 | 0.005 | 100.0 | 106.1 | 3.4 |
QC2 | 0.05 | 93.0 | 104.5 | 5.8 |
QC3 | 0.5 | 96.2 | 98.7 | 2.0 |
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Wijngaard, R.; Perramón, M.; Parra-Robert, M.; Hidalgo, S.; Butrico, G.; Morales-Ruiz, M.; Zeng, M.; Casals, E.; Jiménez, W.; Fernández-Varo, G.; et al. 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. Int. J. Mol. Sci. 2021, 22, 4752. https://doi.org/10.3390/ijms22094752
Wijngaard R, Perramón M, Parra-Robert M, Hidalgo S, Butrico G, Morales-Ruiz M, Zeng M, Casals E, Jiménez W, Fernández-Varo G, et al. 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. 2021; 22(9):4752. https://doi.org/10.3390/ijms22094752
Chicago/Turabian StyleWijngaard, Robin, Meritxell Perramón, Marina Parra-Robert, Susana Hidalgo, Gina Butrico, Manuel Morales-Ruiz, Muling Zeng, Eudald Casals, Wladimiro Jiménez, Guillermo Fernández-Varo, and et al. 2021. "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 22, no. 9: 4752. https://doi.org/10.3390/ijms22094752
APA StyleWijngaard, R., Perramón, M., Parra-Robert, M., Hidalgo, S., Butrico, G., Morales-Ruiz, M., Zeng, M., Casals, E., Jiménez, W., Fernández-Varo, G., Shulman, G. I., Cline, G. W., & Casals, G. (2021). 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, 22(9), 4752. https://doi.org/10.3390/ijms22094752