Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR Spectroscopy G. A. Nagana Gowda 1,2 *, Lauren Abell 2 , Chi Fung Lee 2 , Rong Tian 2 and Daniel Raftery 1,2 * 1 Northwest Metabolomics Research Center and 2 Mitochondria and Metabolism Center Anesthesiology and Pain Medicine, University of Washington, USA Emails: ngowda@uw.edu and draftery@uw.edu 1
Profiling Redox and Energy Coenzymes in Tissue and Blood using NMR Spectroscopy Graphical Abstract Coenzymes Antioxidants 350 20 700 18 300 Metabolites 16 600 Whole Blood 14 250 500 Cardiac Tissue 12 200 400 10 Liver Tissue 8 150 300 Brain Tissue 6 200 Skeletal muscle tissue 100 4 100 Kidney Tissue 2 50 0 0 3-… Glutami… 0 NADH NADPH NADP+ NAD+ AMP Betaine Serine Acetate Mannose Creatine Leucine Lysine Glycerol Threonine Valine Glycine Proline Alanine Glutamine ADP ATP GSSG Coenzymes GSSG GSSG NAD + GSH AMP GSH ADP NADPH Antioxidants ATP NADH NADP + 8.60 8.55 8.50 8.45 ppm 2
Abstract: Coenzymes of redox reactions and cellular energy mediate biochemical reactions fundamental to the functioning of all living cells. To date, no simple method exists to profile these coenzymes in biological samples such as blood and different types of tissue in a single step. Conventional methods that measure them individually carry high risk of errors outweighing their biological variations. We show here that coenzymes including antioxidants and a vast pool of other metabolites can be profiled quantitatively using a simple 1 H NMR experiment, simultaneously, in tissue and whole blood. Coenzymes of redox reactions including oxidized/reduced nicotinamide adenine dinucleotide (NAD + and NADH) and (NADP + nicotinamide adenine dinucleotide phosphate and NADPH); the coenzymes of energy including adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and the antioxidants including the sum of oxidized and reduced glutathione (GSSG and GSH) can be measured with essentially no additional effort along with the nearly 70 metabolites that were shown to be quantitated in serum/plasma, recently. Considering that coenzymes and antioxidants represent a sensitive measure of cellular functions in health and numerous diseases, the simple NMR spectroscopy method described here for profiling tissue and blood potentially opens a new avenue for applications in the metabolomics field. Keywords: Redox and energy coenzymes; antioxidants; 1 H NMR; quantitation 3
Introduction NAD + , NADH, NADP + and NADPH mediate biochemical reactions fundamental to the • functioning of living cells • ATP along with ADP and AMP, represent the cellular energy currency. • No simple method exists currently to measure all quantitatively and simultaneously Conventional analysis method: Enzymatic Assay: Challenges : • Separate protocol required for each coenzyme. • Interference from sample matrix • Finite linear range 4
Can you Analyze by Mass Spectrometry? Introduction O O OH NH 2 OH NH 2 P + O O N O P O O N NAD + MW=664.4 O MW=665.4 NADH O OH OH NH 2 O OH OH NH 2 N N N H N Yes, but… P O O N N H O P O OH O N N OH O OH OH O OH OH Challenges O OH NH 2 OH NH 2 + O P O N P O O O N O NADPH • Ion suppression NADP + O MW=744.4 O OH OH NH 2 OH OH NH 2 MW= 745.4 N N N N H H P O O O P O • Interference due to unit mass N N N N OH OH O O difference OH O OH O P OH O P OH O OH OH ATP NH 2 • Interference due to similar N O N O O H HO P P P O O O chromatographic retention time N N OH OH OH O NH 2 OH OH N N • In-source fragmentation NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 O H HO P O N N N N N N N N N N N N N N N N O O O O O O O O O O O O O O OH H H H H H H H O HO HO HO HO HO HO HO P P P P P P P O O O O O O O P P P P P P P O O O O O O O N N N N N N N N N N N N N N OH OH OH OH OH OH OH OH OH OH OH OH OH OH O O O O O O O AMP ADP OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH 5
Can you Analyze by NMR Spectroscopy? Introduction O O OH NH 2 OH NH 2 P + O O N O P O NAD + O N O NADH O OH OH NH 2 O OH OH NH 2 Challenges: N N N H N P O O N H N O P O OH O N N OH O • Complexity OH OH O OH OH O OH NH 2 OH NH 2 + O P O N P • O O Similar structures O N NADP + O NADPH O O OH OH NH 2 OH OH NH 2 N N • Low concentration N N H H P O O O P O N N N N OH OH O O • Instability OH O OH O P OH O P OH O OH - Enzyme activity OH ATP NH 2 N - Oxidation O N O O H HO P P P O O O N N OH OH OH O NH 2 OH OH N N NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 O H HO P O N N N N N N N N N N N N N N N N O O O O O O O O O O O O O O OH H H H H H H H O HO HO HO HO HO HO HO P P P P P P P O O O O O O O P P P P P P P O O O O O O O N N N N N N N N N N N N N N OH OH OH OH OH OH OH OH OH OH OH OH OH OH O O O O O O O AMP ADP OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH 6
Results and Discussion Coenzymes in Tissue Extracts O O OH NH 2 OH NH 2 + O P O N O P O O N NAD + O NADH O OH OH NH 2 O OH Skeletal Muscle OH NH 2 N N N N H O P O N H N O P O OH O N N OH O OH OH O OH OH O OH NH 2 OH NH 2 + O P O N Liver O P O N O O NADPH NADP + O O OH OH NH 2 OH OH NH 2 N N N N H H O P O O P O N N N N OH OH O O Brain OH O OH O O P OH O P OH OH OH NH 2 ATP Kidney N O N O O H HO P P P O O O N N OH OH OH O NH 2 OH OH N NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 N O Heart H N N N N N N N P HO O N N N N N N N O O O O O O O O O O O O O O N N H H H H H H H OH HO HO HO HO HO HO HO P P P P P P P O O O O O O O P P P P P P P O O O O O O O O N N N N N N N N N N N N N N OH OH OH OH OH OH OH OH OH OH OH OH OH OH O O O O O O O AMP ADP OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH 7
Results and Discussion Founding work for coenzymes analysis Anal Chem. 2015 Jan 6;87(1):706-15 8
Results and Discussion Founding work for coenzymes analysis • Nearly 70 metabolites identified and quantitated in human blood • ~35% were newly identified • Unprecedented for 1D NMR. Anal Chem. 2015 Jan 6;87(1):706-15 9
Results and Discussion 9 8 7 6 5 4 3 2 1 ppm NAD + ATP/ ADP ADP/ AMP Mouse Liver Tissue Extract AMP NADH/NADPH NADH NAD + NAD + ATP NADP + NAD + NADP + NADP + NADPH NADP + NADH NAD + NADP + NADPH 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 ppm Redox and Energy Coenzymes NAD + AMP ADP NADPH ATP NADH NADP + 8.60 8.55 8.50 8.45 ppm 10
Results and Discussion: Coenzymes in Different Tissues O O OH NH 2 NAD + OH NH 2 O P O ADP N AMP + O P O O N ATP O NADPH NAD + O NADP + OH OH NADH NH 2 NADH O OH OH NH 2 Liver N N N N H O P O ATP H N N P O O OH N O N OH O OH OH OH OH Skeletal Muscle O O OH NH 2 NH 2 OH + O P O O P O N N O NADPH NADP + O O O OH OH OH OH NH 2 NH 2 Brain N N N N H H O P O O P O N N N N OH O OH O OH O OH O O P OH O P OH OH Kidney NH 2 OH N O N O O H HO P P P O O O ATP N N OH OH OH O ATP NH 2 N OH OH N O Heart H NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 P HO O N N OH N N N N N N N O N N N N N N N O O O O O O O O O O O O O O H H H H H H H HO HO HO HO HO HO HO P P P P P P P O O O O O O O P P P P P P P O O O O O O O N N N N N N N N N N N N N N 8.60 8.55 8.50 8.45 ppm OH OH OH OH OH OH OH OH OH OH OH OH OH OH AMP O O O O O O O OH OH ADP OH OH OH OH OH OH OH OH OH OH OH OH OH OH 11
Results and Discussion Sensitivity of coenzymes to tissue harvesting/extraction AMP The same mouse ADP NAD + heart tissue extracted ATP under different NADH NADP + NADPH MeOH-Water (d ) conditions MeOH-CHCl 3 , in vivo (c ) (b ) MeOH-CHCl 3 , perfusion ATP (a) MeOH-CHCl 3 8.60 8.55 8.50 8.45 ppm 12
Results and Discussion Simultaneous Analysis of Coenzymes Mouse hearts 500 n=6 400 300 ng/mg tissue ATP NAD + 200 NADH NADPH AMP NADP + 100 0 8.60 8.55 8.50 8.45 ppm 13
Results and Discussion Coenzymes from ~5mg tissue Mouse hearts 500 n=6 * NAD + 400 NADH 5 mg Tissue 300 NAD + ng/mg tissue NADPH * NADH * 200 20 mg Tissue NAD + 100 NADH NADPH NADP + * 0 80 mg Tissue 8.48 8.46 8.44 8.42 ppm 14
Results and Discussion Coenzymes and Antioxidants in Whole Blood • Blood Metabolomics: Serum or Plasma • Whole blood: serum/plasma + Cells -Nearly 50 % of blood volume is cells and 99% of cells are RBC. • Whole Blood Metabolomics: -Provides access to wider pool of metabolites with no additional efforts. 15
Coenzymes and Antioxidants in Blood Results and Discussion 16
Results and Discussion Coenzymes in Whole Blood extract ATP ADP NAD + NADPH AMP NADP + NADH Whole Blood Formic acid Blood Plasma ppm 8.60 8.55 8.50 8.45 17
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