Method for determination of intracellular sodium in perfused cancer cells by 23Na nuclear magnetic resonance spectroscopy.
Changes of intracellular sodium concentrations are often an indication of disease or malfunction. In this work, shift reagent-aided 23Na NMR spectroscopic determination of intracellular sodium was adapted to measurements with perfused cells embedded in agarose gel threads. Ehrlich ascites tumor cells (EHR2) and their multidrug-resistant counterparts (EHR2/DNR+) were immobilized and perfused until the metabolic steady state had been reached as shown by 31P NMR spectroscopy. Subsequent addition of 5 mM dysprosium(III) bis(tripolyphosphate) to the perfusion medium caused a separation of extracellular and intracellular 23Na NMR signals, making quantification of the intracellular sodium possible. The dysprosium shift reagent was apparently nontoxic to the cells, as shown by the unchanged level of ATP and other intracellular phosphates. NMR visibility of the intracellular sodium was determined in suspensions of EHR2 and EHR2/DNR+ cells by treatment with digitonin; the increase of intensity of the extracellular sodium resonance observed after the digitonin treatment corresponded well (97 +/- 3%) to the sum of intracellular and extracellular sodium observed with intact cells prior to the digitonin treatment. The resistant EHR2/DNR+ cells contained a moderately higher intracellular sodium level than the wild-type EHR2 cells, 1.02 +/- 0.10 and 0.77 +/- 0.07 mumol Na/mg protein, respectively. Closely similar levels of intracellular sodium were found by flame photometry. Thus, 23Na NMR offers a reliable method for noninvasive quantification of intracellular sodium in perfused cancer cells.
Hansen
L L
LL
Department of Organic Chemistry, Royal Danish School of Pharmacy, Copenhagen.
Rasmussen
J
J
Friche
E
E
Jaroszewski
J W
JW
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000737:chemistry*; D003630:Daunorubicin / Q000494:pharmacology; D004351:Drug Resistance; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D010477:Perfusion; D012964:Sodium / Q000032:analysis*; D014407:Tumor Cells, Cultured
D012964:Sodium; D003630:Daunorubicin