A method for calculating the distribution of pH in tissues and a new source of pH error from the 31P-NMR spectrum

The true distribution of the pH in tissues can be determined from the in vivo 31P-nuclear magnetic resonance (NMR) spectrum by converting the parts per million (PPM) axis of the pH responsive resonance to pH using the Henderson-Hasselbalch equation. In addition, the intensity axis of the resonance must be divided by the derivative of the Henderson-Hasselbalch equation to correct for the nonlinear relationship between pH and PPM. This nonlinear relationship causes the apparent center of the resonance in PPM to be dependent not only on the center of the pH distribution but also on its width and distance from the pKa, where Ka is the association constant. Therefore, the pH determined from uncorrected spectra may be in significant error, particularly if the pH distribution is distant from the pKa and is broad. The method was applied to the isolated perfused Morris hepatoma 5123C to determine the distribution of intracellular pH (pHi) using resonances from two intracellular compounds. The two resonances did not report the same pHi unless the spectral data were properly corrected. The method should be of interest to anyone interested in pHi.