Phosphorus-31 nuclear magnetic resonance spectroscopic observation of the intracellular transformations of oncostatic cyclophosphamide metabolites

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Abstract
31P NMR spectroscopy was used to directly monitor, for the first time, the intracellular chemistry of the ultimate active metabolite of cyclophosphamide, namely, phosphoramide mustard. These NMR studies utilized a human histiocytic lymphoma cell line (U937), embedded in agarose gel threads, and perfused with medium containing synthetically derived metabolites (4-hydroxycyclophosphamide (2), aldophosphamide (3), and phosphoramide mustard (4)). Metabolites 2 or 3 or both readily crossed the cell membrane; in contrast, the membrane was relatively impermeable to 4. Intracellular concentrations of 4 could, therefore, be attributed primarily to the intracellular fragmentation of 3. Signals suggestive of either carboxyphosphamide or 4-ketophosphamide were not detected. Spectral data were used to calculate a rate constant of (5.4 .+-. 0.3) .times. 10-3 min-1 for the intracellular disappearance of 4 at 23.degree. C. The intracellular pH was determined to be 7.1 from the chemical shift of the internal inorganic phosphate signal.