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Assay of Intracellular Free and Macromolecular-bound Metabolites of 5-Fluorodeoxyuridine and 5-Fluorouracil¹

Departments of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of California, San Francisco, California 94143

Methods have been developed to assay several aspects of 5-fluoro-2'-deoxyuridine and 5-fluorouracil metabolism in tissue culture cells. These methods allow measurement of (a) intracellular levels of the covalent complex formed between 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), 5,10-methylenetetrahydrofolate, and thymidylate synthetase; (b) incorporation of drug into RNA; and (c) analysis of drug metabolites. The methods were developed using radioactively labeled drugs, but they should be adaptable to studies using nonlabeled compounds.

Sephadex G-25 chromatography or trichloroacetic acid precipitation were utilized for isolation of the macromolecular cell fraction; prior treatment of this fraction with RNase or heating at 65° for 15 min resulted in selective removal of RNA or the thymidylate synthetase complex, respectively, from the precipitable fraction. Free intracellular drug metabolites present in the acid-soluble fraction were analyzed by high-pressure liquid chromatography.

Following incubation of HTC cells with [6-³H]-5-fluoro-2'-deoxyuridine, a radioactive macromolecule was isolated and identified as a FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex. Intracellular formation of this complex was shown to be dependent on the presence of the enzyme thymidine kinase. Dissociation of the complex in vivo was first order with a t_1/2 of 6.2 hr; in contrast, a t_1/2 of 2 hr was determined for dissociation of the complex in cytosol.

Incubation of L1210 cells with [6-³H]-5-fluorouracil for 22 hr resulted in formation of 80 fmol of FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex per 10⁶ cells, as compared with 400 fmol of drug incorporated into RNA per 10⁶ cells. Intracellular FdUMP could not be detected in the acid-soluble fraction of these cells unless the cells were first heated to dissociate the complex.

¹ This study was supported by Grants CA 14266 and CA 14394, awarded by the National Cancer Institute, Department of Health, Education and Welfare, and the Cancer Research Coordinating Committee of the University of California.

² Leukemia Society of America Fellow, 1976 to 1978. Present address: Department of Pharmacology, Northwestern University Medical School, Chicago, III. 60611.

³ Recipient of a NIH Career Development Award. To whom requests for reprints should be addressed, at School of Medicine, Department of Biochemistry and Biophysics, University of California, San Francisco, Calif. 94143.

Received 3/ 8/79. Accepted 5/23/79.