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.
The novel phosphorylated pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH) was evaluated as a (31)P 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.
Pietri
S
S
Structure et Réactivité des Espèces Paramagnétiques, CNRS-UMR 6517 Universités d'Aix-Marseille I et III, F-13397 Marseille Cedex 20, France. pietri@srepir1.univ-mrs.fr
Martel
S
S
Culcasi
M
M
Delmas-Beauvieux
M C
MC
Canioni
P
P
Gallis
J L
JL
D000818:Animals; D003331:Coronary Vessels / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007511:Ischemia; D008099:Liver / Q000098:blood supply / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D009206:Myocardium / Q000737:chemistry*; D063065:Organophosphonates; D010759:Phosphorus Isotopes; D011759:Pyrrolidines / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D015424:Reperfusion
D063065:Organophosphonates; D010759:Phosphorus Isotopes; D011759:Pyrrolidines; C113107:diethyl(2-methyl-2-pyrrolidinyl)phosphonate