Establishing a GC-MS/MS method to quantify redox markers in NDUFS4 knockout mice

Abstract

Through normal metabolic reactions, electron carriers such as nicotinamide adenine dinucleotide reduced and flavin adenine dinucleotide reduced (NADH and FADH2) are formed through various metabolic pathways which drive the mitochondrial electron transport chain in the mitochondrial matrix towards adenosine triphosphate (ATP) synthesis. NADH and FADH2 donates electrons to complex I and II of the electron transport chain (ETC) respectively, regenerating oxidized NAD+ and FAD+ levels which would ultimately be reduced again. Leigh syndrome (LS) is a phenotype characterized by mutations of the NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4) gene, causing complex I deficiency and with that, disturbance of the NAD+/NADH redox pair. Targeting reduced and oxidized constituents (redox metabolites) could reveal whether a patient has a redox (NAD+/NADH) imbalance and if possible treatment options could restore the balance. As such, diagnosing patients with redox imbalance requires accurate and reproducible quantification of redox metabolites. Due to the reactive nature of NAD(P)H and NAD(P), reliable quantification of these species remains difficult, which prompts researchers to rather quantification redox metabolites such as pyruvate and lactate to investigate redox state. Also, to confirm hypothesis generating studies such as untargeted metabolomics investigating redox status, a method that is able to quantify metabolites linked to redox metabolism more accurately is required to confirm the hypothesis of previous untargeted studies. As such, a gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to accurately quantify 24 redox metabolites, aiding treatment studies focusing on restoring redox balance. The newly developed method was applied to NDUFS4 KO and WT mouse liver, brain, and heart tissue to reveal changes in redox metabolites as a result of NAD/NADH redox imbalance. Multiple perturbations in redox markers were revealed throughout the liver brain and heart tissue, with some of the perturbations only occurring in certain tissue types. The heart exhibited the most perturbations in redox metabolites and redox ratios, followed by the liver and then the brain. As a whole, it seemed that the liver released accumulated redox metabolites into the circulation, while the heart and brain import the accumulated intermediates of metabolism to restore redox balance to ensure that energy homeostasis is maintained.