31P nuclear magnetic resonance study of a human colon adenocarcinoma cultured cell line

F. Desmoulin, Jean-Philippe Galons, P. Canioni

Research output: Contribution to journalArticle

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Abstract

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 diphosphohexose 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.

Original languageEnglish (US)
Pages (from-to)3768-3774
Number of pages7
JournalCancer Research
Volume46
Issue number8
StatePublished - 1986
Externally publishedYes

Fingerprint

Cultured Cells
Colon
Adenocarcinoma
Magnetic Resonance Spectroscopy
Cell Line
HT29 Cells
Nucleosides
Energy Metabolism
Uridine Diphosphate N-Acetylgalactosamine
Phosphates
Uridine Diphosphate N-Acetylglucosamine
Uridine Diphosphate Glucose
Glucose
Glucose-6-Phosphate
Proton-Motive Force
Phosphorylcholine
Uridine
Starvation
Culture Media
Perfusion

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

31P nuclear magnetic resonance study of a human colon adenocarcinoma cultured cell line. / Desmoulin, F.; Galons, Jean-Philippe; Canioni, P.

In: Cancer Research, Vol. 46, No. 8, 1986, p. 3768-3774.

Research output: Contribution to journalArticle

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N2 - 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 diphosphohexose 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.

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