Use of diethyl(2-methylpyrrolidin-2-yl)phosphonate as a highly sensitive extra- and intracellular P-31 NMR pH indicator in isolated organs - Direct NMR evidence of acidic compartments in the ischemic and reperfused rat liver

The novel phosphorylated pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosph onate (DEPMPH) was evaluated as a P-31 NMR probe of the pH changes associat ed with ischemia/reperfusion of rat isolated hearts and livers. In vitro ti tration curves indicated that DEPMPH exhibited a 4-fold larger amplitude of chemical shift variation than inorganic phosphate yielding an enhanced NMR sensitivity in the pH range of 5.0-7.5 that allowed us to assess pH variat ions of less than 0.1 pH units. At the non-toxic concentration of 5 mM, DEP MPH distributed into external and cytosolic compartments in both normoxic o rgans, as assessed by the appearance of two resonance peaks. An additional peak was observed in normoxic and ischemic livers, assigned to DEPMPH in ac idic vesicles (pH 5.3-5.6). During severe myocardial ischemia, a third peak corresponding to DEPMPH located in ventricular and atrial cavities appeare d (pH 6.9). Mass spectrometry and NMR analyses of perchloric extracts showe d that no significant metabolism of DEPMPH occurred in the ischemic liver. Reperfusion with plain buffer resulted in a rapid washout of DEPMPH from bo th organs. It was concluded that the highly pH-sensitive DEPMPH could be of great interest in noninvasive ex vivo studies of pH gradients that may be involved in many pathological processes.