title: Dynamic phosphometabolomic profiling of human tissues and transgenic models by 18O-assisted ³¹P NMR and mass spectrometry.
abstract: Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope (18)O-assisted (31)P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The (18)O labeling procedure is based on the incorporation of one (18)O into P(i) from [(18)O]H(2)O with each act of ATP hydrolysis and the distribution of (18)O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded (18)O-labeling procedures has revealed significant differences in the dynamics of G-6-P[(18)O] (glycolysis), G-3-P[(18)O] (substrate shuttle), and G-1-P[(18)O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[(18)O], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1-/-), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[(18)O] and decreased G-1-P[(18)O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit-/--deficient hearts is characterized by a higher G-6-[(18)O]P turnover rate, G-6-P levels, glycolytic capacity, γ/β-phosphoryl of GTP[(18)O] turnover, as well as β-[(18)O]ATP and β-[(18)O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus, (18)O-assisted gas chromatography-mass spectrometry and (31)P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.
authors.affiliation: Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Emirhan
authors.initials: E
authors.lastname: Nemutlu
authors.forename: Song
authors.initials: S
authors.lastname: Zhang
authors.forename: Anu
authors.initials: A
authors.lastname: Gupta
authors.forename: Nenad O
authors.initials: NO
authors.lastname: Juranic
authors.forename: Slobodan I
authors.initials: SI
authors.lastname: Macura
authors.forename: Andre
authors.initials: A
authors.lastname: Terzic
authors.forename: Arshad
authors.initials: A
authors.lastname: Jahangir
authors.forename: Petras
authors.initials: P
authors.lastname: Dzeja
mesh_terms: D000263:Adenylate Kinase / Q000172:deficiency; D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism; D006325:Heart Atria / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010103:Oxygen Isotopes; D010759:Phosphorus Isotopes; D010766:Phosphorylation; D051381:Rats; D013312:Stress, Physiological
chemical_list: D010103:Oxygen Isotopes; D010759:Phosphorus Isotopes; D003402:Creatine Kinase; D000263:Adenylate Kinase