Use of Diethyl(2-methylpyrrolidin-2-yl)phosphonate as a Highly Sensitive Extra- and Intracellular 31P NMR pH Indicator in Isolated Organs

Abstract

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.Because of the dependence of the chemical shift of phosphates on pH (1-3), 31 P NMR spectroscopy has progressively become the standard method for the measurement of intracellular pH (pH i ) 1 in biological systems, mostly using inorganic orthophosphate (P i ) as a naturally occurring pH probe (4 -6). Although this technique was first applied to measure pH in a variety of biological fluids, the development of pulsed Fourier transform NMR and wide-bore supraconducting magnets has soon allowed the noninvasive study of cell cultures and isolated perfused organs under physiologic or pathologic conditions. In particular, the use of 31 P NMR has considerably increased our understanding of the dynamics of pH i changes during ischemia-induced acidosis in the heart (7-9) and liver (10, 11). Although P i resonance has been successfully used to describe the main pH i -regulatory systems in these organs (12, 13), more subtle trans-sarcolemmal proton movements that could occur in different pathologies may escape investigation if they are related to extra-and intracellular pH values different by less than 0.2-0.3 pH units (4). In addition to this relative lack of resolution, P i levels vary with cell metabolism, and the chemical shift of P i has been demonstrated to be affected by ionic strength (4 -6).The search for improved exogenous 31 P NMR pH indicators as alternatives to P i yielded a variety of alkyl-and aminoalkylphosphonic acids having their resonance peak distinct from that of phosphorylated metabolites, and an NMR sensitivity ⌬␦ ab (defined as the mean difference between the chemical shifts of the protonated ␦ a and the unprotonated ␦ b forms) in the range of P i (i.e. 2-3 ppm). There have been a number of studies on the in v...

Ion-Trapping, Microsomal Binding, and Unbound Drug Distribution in the Hepatic Retention of Basic Drugs

“…A comparison of the predicted iontrapping and the experimental ratios obtained by data fitting showed good similarity (Table 3). It is recognized that the predicted ion-trapping is only an approximation as a range of pHs have been reported for intracellular pH (7.19 -7.29) (Le Couteur et al, 1993;Burns et al, 1999;Pietri et al, 2001), mitochondrial pH (6.7-7.0) (Soboll et al, 1980), and lysosomal pH (4 -5) (MacIntyre and Cutler, 1988;Myers et al, 1995;Proost et al, 1997) as well as differing lysosomal fractional volumes (0.68 -1%) (Rhoades and Pflanzer, 1996).…”

“…Monensin is routinely used in the cattle and a Vesicular to cytosolic concentration ratio ϭ (1 ϩ 10 pKa Ϫ pHv)/(1 ϩ 10 pKa Ϫ pHi) (Myers et al, 1995), where pHi Ϸ 7.27 is the assumed cytosolic pH (Le Couteur et al, 1993), pHv Ϸ 4.4 is the assumed lysosomal pH (Daniel et al, 2001) and pHv Ϸ 6.67 is the assumed mitochondrial pH in the fasted state (Pietri et al, 2001).…”

Functional in vivo EPR Spectroscopy and Imaging Using Nitroxide and Trityl Radicals

“…However, pH assessment using 31 P-NMR and inorganic phosphate (P i ) has its own limitations which are rarely discussed, including the lack of resolution (about 0.2-0.3 pH units and even less at lower pH), the fact that P i concentrations vary with metabolism and ischemia, and the chemical shift dependence on ionic strength. 93,94 Because of these problems, exogenous pH probes are being designed for NMR spectroscopy to improve detection of myocardial acidosis 93 and extracellular pH in tumors. 95,96 Upon application of exogenous probes, low field EPR spectroscopy has higher sensitivity compared with NMR for the same probe concentration, and reasonable depth of penetration in living tissues.…”

In vivo Spectroscopy and Imaging of Nitroxide Probes

“…1 mM, respectively). Because of these problems, exogenous pH probes are being designed for NMR spectroscopy to improve detection of myocardial acidosis (Pietri et al, 2001) and extracellular pH in tumors (Gillies et al, 1994;2004). Upon application of exogenous probes, low-field EPR spectroscopy has higher sensitivity compared with NMR for the same probe concentration, and reasonable depth of penetration in living tissues.…”

“…In vivo pH assessment using 31 P-NMR and inorganic phosphate, Pi, suffers from low intrinsic NMR sensitivity and lack of functional resolution (about 0.2-0.3 pH units) (Gillies et al, 1982;Pietri et al, 2001). It is assumed that the chemical shift of endogenous Pi reflects intracellular pHi because of the generally higher fraction of intracellular volume, and 2-3-fold higher intracellular Pi concentrations over extracellular ones (2-3 mM and ca.…”