Dimethyl methylphosphonate (DMMP): a 31P nuclear magnetic resonance spectroscopic probe of intracellular volume in mammalian cell cultures.
Dimethyl methylphosphonate (DMMP), when added to a suspension of erythrocytes, has been reported to have a lower frequency chemical shift inside of cells than outside. This work further investigates the same phenomenon in hollow-fiber bioreactor cultures of six mammalian cell lines 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 dependence of the shift of intracellular DMMP on intracellular protein content was shown to be similar for cultured mammalian and red blood cells. Also consistent with shifts in erythrocytes, an increase in the intracellular 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 measured so that partially saturated DMMP peaks in 31P NMR spectra of mammalian cell cultures can be corrected to give the relative volumes of the intra- and extracellular compartments; this information provides a relative measure of culture growth. Intracellular volume measured by this method can also be used to quantify intracellular metabolites such as ATP during the growth of the culture. To explore the mechanism behind the intracellular shift, we have also addressed the three possible contributions to the chemical shift of DMMP: hydrogen-bonding interactions, magnetic susceptibility, and ionic strength. Data is presented which eliminates the latter two mechanisms and strongly supports the hypothesis that the observed intracellular shift is due to a reduction in hydrogen bonding between water and DMMP in the cytoplasm.
Barry
J A
JA
Department of Biochemistry, University of Arizona Health Sciences Center, Tucson 85724.
McGovern
K A
KA
Lien
Y H
YH
Ashmore
B
B
Gillies
R J
RJ
D000818:Animals; D016466:CHO Cells; D002477:Cells / Q000737:chemistry / Q000648:ultrastructure*; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry / Q000648:ultrastructure; D005910:Glioma; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009943:Organophosphorus Compounds; D051381:Rats; D012997:Solvents; D014407:Tumor Cells, Cultured
D009943:Organophosphorus Compounds; D012997:Solvents; C031116:dimethyl methylphosphonate