Metabolic changes in Japanese medaka (Oryzias latipes) during embryogenesis and hypoxia as determined by in vivo ^3^1P NMR [An article from: Comparative Biochemistry and Physiology, Part C]

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Description:
In vivo ^3^1P nuclear magnetic resonance spectroscopy (NMR) was used to determine phosphometabolite changes in medaka (Oryzias latipes) during embryogenesis and hypoxia. NMR data were acquired using a flow-through NMR tube perfusion system designed to both deliver oxygenated water to embryos and accommodate a hypoxic challenge. Measurements of embryogenesis at 12- and 24-h intervals throughout 8 days of development (n=3 per time point, 900 embryos per replicate) and during acute hypoxia (n=6, 900 embryos at Iwamatsu stage 37 per replicate) were performed via NMR, and replicate samples (n=4, 250 embryos each) were flash frozen for HPLC analysis. The hypoxic challenge experiment consisted of data acquisition with recirculating water (pre-hypoxic control period; 1 h), without recirculating water (hypoxic challenge; 1 h), then again with recirculating water (recovery period; 1.3 h). Concentrations of ATP, phosphocreatine (PCr), orthophosphate (P"i), phosphomonoesters (PME), phosphodiesters (PDE), and intracellular pH (pH"i) were determined by NMR, and ATP, ADP, AMP, GTP, GDP, and PCr were also determined via HPLC. During embryogenesis, [ATP] and [PCr] as determined by HPLC increased from 1-day post fertilization (DPF) levels of 0.93+/-0.08 and 2.48+/-0.21 @mmol/mg (dry tissue), respectively, to 7.24+/-0.77 and 15.66+/-1.08 @mmol/mg, respectively, by day 8. [ATP] and [PCr] measured by both NMR and HPLC fluctuated over 1-3 DPF, then increased significantly (p