QUANTIFICATION OF 5-FLUOROURACIL INCORPORATION INTO RNA OF HUMAN AND MURINE TUMORS AS MEASURED WITH A SENSITIVE GAS-CHROMATOGRAPHY MASS-SPECTROMETRY ASSAY

5-Fluorouracil (5FU) can exert its cytotoxic activity by either inhibi tion of thymidylate synthase or incorporation into RNA. The extent and importance of the latter in tumors of patients are not clear, due to the lack of sensitive and reproducible methods. RNA from 5FU-treated h uman WiDr colon tumor cells was isolated and [C-14]5FU incorporation i nto RNA was measured by traditional scintillation counting while that of nonradiolabeled 5FU was measured with the present, new method. For the latter purpose, isolated RNA was incubated with RNase, alkaline ph osphatase, and uridine phosphorylase, resulting in a complete degradat ion of RNA, nucleotides, and nucleoside to 5FU. 5FU was then measured with gas chromatography coupled to mass spectrometry. For both methods RNA incorporation was 0.4 pmol/h/mu g RNA at 25 mu M 5FU while a simi lar time (up to 4 h) and concentration dependence (25 to 50 mu M) were found. Reproducibility of the assay was more than 95%. In a murine co lon tumor 5FU incorporation into RNA reached a peak of 10 pmol/mu g RN A at 2 h after administration of the maximum tolerated dose of 80 mg 5 FU/kg, which was retained until at least 72 h at 2.5 pmol/mu g. In tum ors from patients treated with 500 mg 5FU/m(2) incorporation into RNA after 24 h amounted to 1.0-1.5 pmol/mu g; RNA. In conclusion, a novel approach, combining different sensitive and reproducible techniques, w as established to measure 5FU incorporation into RNA in clinical tumor specimens enabling determination of its clinical relevance. (C) 1995 Academic Press, Inc.