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³¹P Nuclear Magnetic Resonance Study of a Human Colon Adenocarcinoma Cultured Cell Line¹

Laboratoire de Biologie Physicochimique, Institut de Chimie Biologique, Université d'Aix-Marseille, Place Victor Hugo 13003 Marseille ( France)

³¹P nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the energy metabolism in a human colon adenocarcinoma cell line (HT 29). NMR spectra were recorded at 80.9 MHz on approximately 2.5 x 10⁸ cells continuously perfused with culture medium within a 20-mm NMR sample tube.

Typical NMR spectra display a series of well-resolved resonances assigned to nucleoside triphosphates (mainly adenosine 5'-triphosphate), uridine diphosphohexose derivatives (uridine 5'-diphosphate-N-acetylglucosamine, uridine 5'-diphosphate-N-acetylgalactosamine, uridine 5'-diphosphate-glucose), intra- and extracellular inorganic phosphate, and phosphomonoesters (mainly phosphorylcholine and glucose 6-phosphate). Measurement of phosphorylated metabolite concentrations from the intensity of NMR signals is in good agreement with the results provided by conventional biochemical assays.

³¹P NMR allows to follow noninvasively the effect of anoxia on HT 29 cells. The results indicate that the cells are able to maintain about 60% of their initial nucleoside triphosphate level after 2 h of anaerobic perfusion. Cells accumulate inorganic phosphate during anoxia and the intracellular-extracellular pH gradient increases from 0.5 in well-oxygenated cells to more than 1 pH unit under anoxic conditions. The value of intracellular pH of well-oxygenated HT 29 cells is 7.1.

The effect of glucose starvation upon energy metabolism has also been examined in real time by NMR: a rapid decline of adenosine 5'-triphosphate down to 10% of the initial value is observed over a period of 2 h. In contrast, the level in uridine diphosphohexoses reaches a new steady state value representing 60% of the initial one. Refeeding the cells with 25 mM glucose leads to a dramatic drop of internal pH reflecting the activation of the glycolytic pathway.

¹ This work was supported by the Centre National de la Recherche Scientifique (UA 202) and grants from the Ministère de la Recherche et de la Technologie (GBM 83-M-0802, 84-M-0804 and 85-M-0564), the CNAMTS (Contrat de Recherche Externe 1983–1986), the Ministère de l'Education, and the Fondation pour la Recherche Médicale.

² To whome requests for reprints should be addressed.

Received 12/ 4/84. Revised 1/21/86. Accepted 4/17/86.