title: Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil.
abstract: 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 degrees 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 t1/2 of 6.2 hr; in contrast, a t1/2 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 10(6) cells, as compared with 400 fmol of drug incorporated into RNA per 10(6) 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.
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D046508:Culture Techniques; D003600:Cytosol / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000032:analysis*; D005467:Floxuridine / Q000378:metabolism*; D005468:Fluorodeoxyuridylate / Q000032:analysis*; D005472:Fluorouracil / Q000378:metabolism*; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D051381:Rats; D013937:Thymidine Kinase / Q000378:metabolism
chemical_list: D003856:Deoxyuracil Nucleotides; D005467:Floxuridine; D005468:Fluorodeoxyuridylate; D012313:RNA; D013937:Thymidine Kinase; D005472:Fluorouracil