Dynamic high-resolution 1H and 31P NMR spectroscopy and 1H T2 measurements in postmortem rabbit muscles using slow magic angle spinning

Postmortem changes in rabbit muscle tissue with different glycogen status (normal vs low) were followed continuously from 13 min postmortem until 8 h postmortem and again 20 h postmortem using simultaneous magic angle spinning (1)H and (31)P NMR spectroscopy together with measurement of the transverse relaxation time, T(2), of the muscle water. The (1)H metabolite spectra were measured using the phase-altered spinning sidebands (PASS) technique at a spinning rate of 40 Hz. pH values calculated from the (31)P NMR spectra using the chemical shifts of the C-6 line of histidine in the (1)H spectra and the chemical shifts of inorganic phosphate in the (31)P spectra confirmed the different muscle glycogen status in the tissues. High-resolution (1)H spectra obtained from the PASS technique revealed the presence of a new resonance line at approximately 6.8 ppm during the postmortem period, which were absent in muscles with low muscle glycogen content. This new resonance line may originate from the aminoprotons in creatine, and its appearance may be a result of a pH effect on the exchange rate between the amino and the water protons and thereby the NMR visibility. Alternatively, the new resonance line may originate from the aromatic protons in tyrosine, and its appearance may be a result of a pH-induced protein unfolding exposing hydrophobic amino acid residues to the aqueous environment. Further studies are needed to evaluate these hypotheses. Finally, distributed analysis of the water T(2) relaxation data revealed three relaxation populations and an increase in the population believed to reflect extramyofibrillar water through the postmortem period. This increase was significantly reduced (p < 0.0001) in samples from animals with low muscle glycogen content, indicating that the pH is controlling the extent of postmortem expulsion of water from myofibrillar structures. The significance of the postmortem increase in the amount extramyofibrillar water on the water-holding capacity was verified by centrifugation, which showed a reduced centrifugation loss in muscles with low preslaughter glycogen status (0.9 vs 1.9%, p = 0.07).