31P NMR quantitation of phosphorus metabolites in rat heart and skeletal muscle in vivo.
The aim of this study was to examine two methods of 31P NMR quantitation of phosphocreatine (PCr), ATP, and P(i) in rat heart and skeletal muscle in vivo. The first method employed an external standard of phenylphosphonic acid (PPA; 10 mM), and the second method used an enzymatic measurement of tissue ATP equated to the area under the betaATP peak. With the use of the external standard, the concentrations of ATP, PCr, and P(i) in the rat heart were 4.48 +/- 0.33, 9.21 +/- 0.65, and 2.25 +/- 0.16 micromol/g wet wt, respectively. With the use of the internal ATP standard, measured on the same tissue, the contents (means +/- SE) were 4.78 +/- 0.19, 9.83 +/- 0.18, and 2.51 +/- 0.33 micromol/g wet wt, respectively (n = 7). In skeletal muscle, ATP, PCr, and P(i) were 6.09 +/- 0.19, 23.44 +/- 0.88, and 1.81 +/- 0.18 micromol/g wet wt using the PPA standard and 6.03 +/- 0.19, 23.30 +/- 1.30, and 1.82 +/- 0.19 micromol/g wet wt using the internal ATP standard (n = 6). There was no significant difference for each metabolite as measured by the two methods of quantification in heart or skeletal muscle. The results validate the use of an external reference positioned symmetrically above the coil and imply that each has similar NMR sensitivities (similar signal amplitude per mole of 31P between PPA and tissue phosphorus compounds). We conclude that PCr, ATP, and P(i) are nearly 100% visible in the normoxic heart and nonworking skeletal muscle given the errors of measurement.