DIMETHYL METHYLPHOSPHONATE (DMMP) - A P-31 NUCLEAR-MAGNETIC-RESONANCESPECTROSCOPIC PROBE OF INTRACELLULAR VOLUME IN MAMMALIAN-CELL CULTURES

Dimethyl methylphosphonate (DMMP), when added to a suspension of eryth rocytes, has been reported to have a lower frequency chemical shift in side of cells than outside. This work further investigates the same ph enomenon in hollow-fiber bioreactor cultures of six mammalian cell lin es and describes the application of DMMP as a measure of intra- versus extracellular volumes in mammalian cell cultures. No toxic effects of the DMMP were observed at the concentrations used here. The dependenc e of the shift of intracellular DMMP on intracellular protein content was shown to be similar for cultured mammalian and red blood cells. Al so consistent with shifts in erythrocytes, an increase in the intracel lular protein concentration due to a reduction in cultured cell volume increased the magnitude of the shift to lower frequency. Longitudinal relaxation (T1) values for intra- and extracellular DMMP were measure d so that partially saturated DMMP peaks in P-31 NMR spectra of mammal ian cell cultures can be corrected to give the relative volumes of the intra- and extracellular compartments; this information provides a re lative measure of culture growth. Intracellular volume measured by thi s method can also be used to quantify intracellular metabolites such a s ATP during the growth of the culture. To explore the mechanism behin d the intracellular shift, we have also addressed the three possible c ontributions to the chemical shift of DMMP: hydrogen-bonding interacti ons, magnetic susceptibility, and ionic strength. Data is presented wh ich eliminates the latter two mechanisms and strongly supports the hyp othesis that the observed intracellular shift is due to a reduction in hydrogen bonding between water and DMMP in the cytoplasm.