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Competitive Binding Radioassay for 5-Fluorodeoxyuridine 5'-Monophosphate in Tissues

Medicine [D. S. M., T. A., H. S. S.] and Clinical Pharmacology [C. E. M., B. A. C.] Branches, Clinical Oncology Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20205

A competitive binding radioassay has been developed for 5-fluorodeoxyuridine 5'-monophosphate, based on the tight binding of this potent inhibitor to thymidylate synthetase (EC 2.1.1.45). Unbound ligand may be separated from that bound to enzyme by precipitating the intact inhibitor-enzyme complex with trichloroacetic acid. Scatchard plot analysis using a two-site model for binding yielded apparent dissociation constants of 1.2 x 10^–11 and 1.7 x 10^–10 M from a least-squares computer fit of the data. 5-Fluorodeoxyuridine 5'-monophosphate could be detected in the range of 0.02 to 2.0 pmol with no apparent interference by other substances. Assay of 5-fluorodeoxyuridine 5'-monophosphate levels in L1210 ascites tumor following 5-fluorouracil in vivo revealed peak levels occurring within the first hr with a subsequent disappearance half-life of 3.9 hr. Close agreement was found between the previously described enzyme inhibition assay and the more rapid and sensitive competitive binding method.

¹ To whom requests for reprints should be addressed, at Building 10-12N226, NIH, Bethesda, Md. 20205.

Received 8/30/78. Accepted 3/22/79.