Dynamic phosphometabolomic profiling of human tissues and transgenic models by ¹⁸O-assisted ³¹P NMR and mass spectrometry

Nextgeneration screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope ¹⁸O-assisted ³¹P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The ¹⁸O labeling procedure is based on the incorporation of one ¹⁸O into P _ifrom [ ¹⁸O]H ₂O with each act of ATP hydrolysis and the distribution of ¹⁸O-labeled phosphoryls among phosphatecarrying 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 ¹⁸O-labeling procedures has revealed significant differences in the dynamics of G-6-P[ ¹⁸O] (glycolysis), G-3-P[ ¹⁸O] (substrate shuttle), and G-1-P[ ¹⁸O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[ ¹⁸O], 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[ ¹⁸O] and decreased G-1-P[ ¹⁸O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit-/--deficient hearts is characterized by a higher G-6-[ ¹⁸O]P turnover rate, G-6-P levels, glycolytic capacity, γ/β-phosphoryl of GTP[ ¹⁸O] turnover, as well as β-[ ¹⁸O]ATP and β-[ ¹⁸O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus, ¹⁸O-assisted gas chromatographymass spectrometry and ³¹P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.