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J. Biol. Chem., Vol. 276, Issue 3, 1750-1758, January 19, 2001

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Use of Diethyl(2-methylpyrrolidin-2-yl)phosphonate as a Highly Sensitive Extra- and Intracellular ³¹P NMR pH Indicator in Isolated Organs
DIRECT NMR EVIDENCE OF ACIDIC COMPARTMENTS IN THE ISCHEMIC AND REPERFUSED RAT LIVER^*

Sylvia Pietri^§, Sophie Martel, Marcel Culcasi, Marie-Christine Delmas-Beauvieux^¶, Paul Canioni^¶, and Jean-Louis Gallis^¶

From the  Structure et Réactivité des Espèces Paramagnétiques, CNRS-UMR 6517 Universités d'Aix-Marseille I et III, F-13397 Marseille Cedex 20 and the ^¶ Résonance Magnétique des Systèmes Biologiques, CNRS-UMR 5536, Université Victor Segalen Bordeaux 2, F-33076 Bordeaux Cedex, France

The novel phosphorylated pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH) was evaluated as a ³¹P NMR probe of the pH changes associated with ischemia/reperfusion of rat isolated hearts and livers. In vitro titration 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 variations of less than 0.1 pH units. At the non-toxic concentration of 5 mM, DEPMPH distributed into external and cytosolic compartments in both normoxic organs, as assessed by the appearance of two resonance peaks. An additional peak was observed in normoxic and ischemic livers, assigned to DEPMPH in acidic vesicles (pH 5.3-5.6). During severe myocardial ischemia, a third peak corresponding to DEPMPH located in ventricular and atrial cavities appeared (pH 6.9). Mass spectrometry and NMR analyses of perchloric extracts showed that no significant metabolism of DEPMPH occurred in the ischemic liver. Reperfusion with plain buffer resulted in a rapid washout of DEPMPH from both 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.

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