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Noninvasive Spectroscopic Analysis of Fluoropyrimidine Metabolism in Cultured Tumor Cells¹

Departments of Pharmaceutical Chemistry [M. K., R. H. S., T. L. J.] and Radiology [T. L. J.] and Cancer Research Institute [C. B.], University of California, San Francisco, California 94143

Nuclear magnetic resonance spectroscopy is a technique that may be used noninvasively to follow the intracellular metabolism of fluorinated antimetabolites such as 5-fluorouracil (FUra) and 5-fluorouridine. Intracellular ¹⁹F spectral peaks are assigned by comparison with the pH-dependent chemical shifts measured for eight commercially available fluoropyrimidine metabolites as well as by comparison with the literature recorded values of five known catabolites of FUra. Five murine and human tumor cell lines (N₁S₁, Sarcoma 180, L1210, HL-60, and MiaPaCa) were exposed in vitro for 24 h to cytostatic doses of FUra or 5-fluorouridine. Treated cells were harvested and analyzed immediately or following a subsequent incubation under either nutrient-rich or nutrient-poor conditions. A major narrow component peak at 4.6–4.9 ppm was observed in all cell samples analyzed immediately after treatment. This peak was identified as intracellular FUra nucleotides, and its T₁ value was approximately 800 ms. No fluoropyrimidine catabolites were detectable in any of the treated cell lines. Free FUra could be measured in cells only after subsequent incubation under nutrient-poor conditions, and this was associated with a decline in the prominent FUra nucleotide peak. In treated cells chased with drug-free media containing 1 µM thymidine, spectra revealed a broad component signal underlying and downfield from the narrow nucleotide-containing peak. By biochemically fractionating treated cells into an acid-soluble fraction and phenol-purified cytoplasmic and nuclear RNA extracts, we were able to completely separate the nucleotide peak from the broad component signal resulting from FUra incorporation into RNA. Thymidine produced a marked enhancement of this ¹⁹F signal into both cytoplasmic and nuclear RNA without affecting the nucleotide signal from the acid-soluble fraction. The present ability of nuclear magnetic resonance to monitor the metabolic channeling of fluoropyrimidines in intact tumor cells suggests that future spectroscopic imaging of patients treated with fluorinated antimetabolites may provide clinically important information about tumor biochemistry and drug sensitivity.

¹ Supported in part by grants CA-36769, CA-36773, and CA-27343 from the National Cancer Institute and CH-235B and NP-437 from the American Cancer Society.

² To whom requests for reprints should be addressed.

Received 8/14/85. Revised 12/30/85. Accepted 1/ 2/86.