Humphrey S M, Garlick P B
Department of Radiological Sciences, United Medical School, Guy's Hospital, London, United Kingdom.
Am J Physiol. 1991 Jan;260(1 Pt 2):H6-12. doi: 10.1152/ajpheart.1991.260.1.H6.
Nuclear magnetic resonance (NMR) spectroscopy detects only free, unbound metabolites. We have therefore compared the free high-energy phosphate content of isolated perfused rat hearts (determined by 31P-NMR) with the total high-energy phosphates of the same hearts (determined by chemical analysis) to determine the fractions, if any, that are NMR invisible. Aerobic perfusion (40 min at 37 degrees C, Pi-free Krebs buffer) was followed by 10, 14, or 18 min total global ischemia and 30 min reperfusion (n = 6 in each group). Fully relaxed 31P-NMR spectra (40 scans using 90 degrees pulses at 15-s intervals) were collected at various times throughout the protocol, and the signal intensities of the beta-phosphate of ATP, phosphocreatine (PCr), and Pi were quantified using methylenediphosphonate as an external standard. Hearts were freeze clamped either before ischemia or at the end of reperfusion and were chemically assayed for ATP, PCr, and Pi. After 40 min of normoxia, the ATP and PCr contents determined by NMR were almost identical to the values determined by chemical analysis. However, only 39 +/- 8% of the total Pi was NMR visible. After reperfusion, after 14 or 18 min of ischemia, the proportion of NMR-visible ATP had decreased to 64 +/- 9% (P less than 0.005). After reperfusion after 18 min ischemia, the proportion of NMR-visible Pi had increased to 76 +/- 10% (P less than 0.05). In conclusion, whereas the total cellular content of PCr is always NMR visible, ischemia-reperfusion can alter the fraction of NMR-visible ATP and Pi.
核磁共振（NMR）光谱仅能检测游离的、未结合的代谢物。因此，我们比较了离体灌注大鼠心脏的游离高能磷酸盐含量（通过31P-NMR测定）与相同心脏的总高能磷酸盐含量（通过化学分析测定），以确定是否存在NMR不可见的部分。在有氧灌注（37℃下40分钟，无磷酸钾的 Krebs 缓冲液）后，进行10、14或18分钟的全心缺血，然后再灌注30分钟（每组n = 6）。在整个实验过程中的不同时间收集完全弛豫的31P-NMR光谱（使用90°脉冲，每隔15秒扫描40次），并以亚甲基二膦酸盐作为外标，对ATP的β-磷酸盐、磷酸肌酸（PCr）和Pi的信号强度进行定量分析。在缺血前或再灌注结束时将心脏冷冻钳夹，然后对ATP、PCr和Pi进行化学分析。在常氧40分钟后，通过NMR测定的ATP和PCr含量与通过化学分析测定的值几乎相同。然而，总Pi中只有39±8%是NMR可见的。在缺血14或18分钟后的再灌注后，NMR可见的ATP比例降至64±9%（P<0.005）。在缺血18分钟后的再灌注后，NMR可见的Pi比例增加到76±10%（P<0.05）。总之，虽然PCr的总细胞含量始终是NMR可见的，但缺血-再灌注可改变NMR可见的ATP和Pi的比例。
