Phosphonates as 31P-NMR markers of extra- and intracellular space and pH in perfused rat liver.

K Bruynseels, N Gillis, P Van Hecke, F Vanstapel

Index: NMR Biomed. 10(6) , 263-70, (1997)

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Abstract

We evaluated phosphonates (Po) as markers of the extra- and intracellular space in perfused rat liver. (i) In- and outwash behaviour of phenylphosphonate (PhePo), 3-amino-propylphosphonate (NProPo) and methyl phosphonate (MePo) was compared with that of creatine phosphate (CrP), a marker of the extracellular space, and of dimethyl methylphosphonate (MePoMe2), a marker of the total water-accessible space. In- and outwash of CrP was accurately predicted by the time constant (approximately 12 s) for the in- and outwash of inulin, a standard marker of the extracellular space. MePoMe2 rapidly distributed over the total liver volume (about three times the CrP accessible space). PhePo, NProPo and MePo washed rapidly into the extracellular space with CrP, and then steadily spilled over into the MePoMe2-accessible space. Upon outwash, Po signals rapidly declined in phase with that of CrP. Residual Po (PhePo >> NProPo approximately equal to MePo) reflected the amount internalized during prolonged (60 min) inwash. Proportional amounts of residual Po were found in extracts of livers harvested after outwash of perfusate and extracellular markers. Consistent with exclusion from the cells, CrP went undetected in these extracts. (ii) The resonance frequency of residual PhePo after outwash of the extracellular fraction corresponded with the pH reported by cytosolic P1 and responded to transient changes of the intracellular pH, induced by perfusion with and withdrawal of 20 mM NH4Cl. (iii) MePoMe2 homogeneously distributed over perfusate, parenchyma and bile, consistent with unrestricted permeability. Other Po were transported transcellularly and excreted in bile. CrP was virtually excluded from the bile, attesting to a minimal role for 'bulk-phase pinocytotic' transcellular transport, or for 'paracellular' leakage. In summary, charged Po can be used as extracellular markers in liver, provided experimental conditions are adjusted to minimize their internalization. Some Po (e.g. PhePo) can reach intracellular concentrations which suffice for the compound to act as a reporter molecule of the cytosolic pH.