Phosphonates as31P-NMR markers of extra- and intracellular space and pH in perfused rat liver

Aminomethylphosphonate and 2-aminoethylphosphonate as31P-NMR pH markers for extracellular and cytosolic spaces in the isolated perfused rat liver

“…[10][11][12]14,16 A representative 31 P-NMR spectrum of a rat liver perfused at 37°C with KH buffer in the presence of phosphonate derivatives (2 mM each) is shown in Fig. 1.…”

“…28 The NProPo intracellular concentration (0.4 AE 0.1 mmol/g liver wet weight) was small but not negligible, in agreement with the slight uptake of this phosphonate reported elsewhere. 12 MePoMe 2 distributed within the intracellular compartment at an average concentration of 3.7 AE 0.3 mmol/g liver wet weight (n = 5). In that case, it is noteworthy that MePoMe 2 resonance appearance and clearance upon perfusion and rinsing followed the same behaviour as NMePo and NProPo, two non-permeant phosphonates.…”

“…For instance, 3-aminopropylphosphonate (NProPo) is known to remain within the extracellular space, 10 whereas dimethyl methylphosphonate (Me-PoMe 2 ) is commonly used as a marker of both intraand extracellular volumes. 11,12 Following an appropriate calibration of the NMR signals, these markers were used to assess the intracellular volume. [13][14][15][16] Although some phosphonates such as MePoMe 2 do not exhibit pHtitrating properties, phosphonate-related compounds have been often thought to be good markers for reporting pH values, but to date they have been essentially used as extracellular pH probes, 10,12,15,16 whereas few intracellular applications were reported.…”

“…11,12 Following an appropriate calibration of the NMR signals, these markers were used to assess the intracellular volume. [13][14][15][16] Although some phosphonates such as MePoMe 2 do not exhibit pHtitrating properties, phosphonate-related compounds have been often thought to be good markers for reporting pH values, but to date they have been essentially used as extracellular pH probes, 10,12,15,16 whereas few intracellular applications were reported. 12 Moon and Richards have intoduced the use of inorganic phosphate (P i ) as a 31 P-NMR indicator of pH; 17 thus cytosolic pH, and in some cases of mitochondrial pH, 18,19 are currently determined from the chemical shift of P i .…”

“…We compared the behaviour of aminomethylphosphonate (NMePo) and 2-aminoethylphosphonate (NEthPo), two potential pH probes, to other well-characterized phosphonates, NProPo and Me-PoMe 2 . [10][11][12]16 NMePo and NEthPo have already been studied in Dictyostelium Discoideum amoebae, where the pinocytotic uptake of the phosphonates allowed endosomal pH determination. 21,22 NMePo and NEthPo have been shown to be good intracellular pH-probe candidates, but their uptake in the liver, which includes determination of time-dependent increase in the phosphonate signal, remained to be assessed.…”