In this appendix, some additional preliminary in vivo NMR results are presented. In paticular two experiments are presented which typify the types of results obtained in the study of phosphate energetics during anoxia and recovery and are meant to be a supplement to the dual spectroscopy NMR results presented in chapter six. The first of these experiments was one of the first demonstrations of the double-resonated coil developed in chapter three and was performed before the development of the unidirectional breathing technique. In this experiment ``normoxia'' periods were effected by manually ventilating the animal with several 15-25 cc ``breaths'' of room air in between NMR acquisitions, and the ``recovery'' period was effected by ventilating the animal as above every minute or so for approximately ten minutes. NMR acquisition parameters were: SW = + 3000 Hz, # points = 4096, # scans = 256, TR = 2.3 seconds, total acquisition time about 10 minutes, and ³¹P 90° PW = 25 ms.

Figure F.1 shows ³¹P NMR spectra collected during an initial ``normoxia'' period (A,B) followed by successive acquisitions during an anoxic period during which the animal was not ventilated (C,D,E,F,G,H) and finally an acquisition after approximately 10 minutes of ``recovery'' ventilation (I). Spectra have been processed with 15 Hz line-broadening and zero- and first-order phase correction. Figure F.2 shows the relative levels of PCr and b-ATP extracted from the NMR spectral data as % of those in spectrum (A). During anoxia, PCr stores clearly become depleted though b-ATP levels remain relatively constant. The inconsistency in the levels of PCr available in normoxic experiments (A,B) is most likely due to the ineffectualness of the intermittent tidal breathing protocol employed and should be well addressed by the unidirectional technique.

Scans were:

A.) Normoxia

B.) Normoixia

C.) 20 Minutes Anoxia

D.) 45 Minutes Anoxia

E.) 65 Minutes Anoxia

F.) 85 Minutes Anoxia

G.) 105 Minutes Anoxia

H.) 145 Minutes Anoxia

I.) After 10 Minutes Recovery

Note that PCr peak becomes substantially diminished during anoxia, but b-ATP peak remains relatively constant in amplitude. Upon recovery, the PCr peak begins to reappear.

The second experiment included here was performed using the same double-tuned coil but involved continuous ventilation using the unidirectional technique. Normoxia consisted of ventilation with a mixture of 97% O ₂ and 3% CO₂ while anoxia consisted of ventilation with 97% N₂ and 3% CO₂. Recovery was effected by switching the ventilation gas back to the 97% O₂ mixture. NMR acquisition parameters were:

SW = + 3000 Hz, # points = 4096, # scans = 256, TR = 2.3 seconds, total acquisition time about 10 minutes, and ³¹ P 90° PW = 25 ms. Spectra are presented in Figure F.3 and have been processed with 15 Hz line-broadening and zero- and first-order phase correction. Figure F.4 shows the relative levels of PCr and b-ATP extracted from spectra. Again b-ATP remains available even though a marked decrease in PCr is evident during anoxia. Here, after 20 minutes of recovery, the PCr peak is again evident and is fully recovered perhaps illustrating the increased effectiveness of the unidirectional ventilation technique over tidal ventilation.

Scans were:

A.) Normoxia

B.) 20 Minutes Anoxia

C.) 40 Minutes Anoxia

D.) 65 Minutes Anoxia

E.) 80 Minutes Anoxia

F.) 95 Minutes Anoxia

G.) 145 Minutes Anoxia

H.) 3 Hours Anoxia

I.) 15 Minutes Recovery

After 3 Hours Anoxia

J.) 35 Minutes Recovery

K.) 50 Minutes Recovery

Note that PCr peak becomes substantially diminished during anoxia, but b-ATP peak remains relatively constant in amplitude. Upon recovery, the PCr peak reappears fully to normoxic levels.

Figure F.3: Successive ³¹P NMR Scans from In vivo Turtle Heart with Unidirectional Breathing.