摘要 : In vivo P-31 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 perfusio... 展开 In vivo P-31 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 mu mol/mg (dry tissue), respectively, to 7.24 +/- 0.77 and 15.66 +/- 1.08 mu mol/mg, respectively, by day 8. [ATP] and [PCr] measured by both NMR and HPLC fluctuated over 1-3 DPF, then increased significantly (p < 0.05) over 3-8 DPF, while [PME] and [PDE] decreased (p < 0.05) throughout embryogenesis. NMR and HPLC measurements revealed 1-3, 4-5, and 6-8 DPF as periods of embryogenesis significantly different from each other (p < 0.05), and representing important transitions in metabolism and growth. During hypoxic challenge, [ATP] and [PCr] declined (p < 0.05), [PME] and [PDE] decreased slightly, and [Pi] increased (p < 0.05). All phosphometabolites returned to pre-hypoxia concentrations during recovery. The pH(i) decreased (p < 0.05) from 7.10 +/- 0.03 to 6.94 +/- 0.03 as a result of hypoxia, and failed to return to pre-hypoxic levels within the 1.3-h recovery phase. Results demonstrate the utility of in vivo P-31 NMR to detect significant alterations in phosphorylated nucleotides and phosphometabolites at specific developmental stages during medaka development and that late-stage medaka utilize PCr to generate ATP under hypoxic conditions. (c) 2005 Elsevier Inc. All rights reserved. 收起