Validation of fluorouracil metabolite analysis in excised tumor - Lability of anabolites

Rapid excision and freezing of tissue commonly is assumed to preserve the molecular composition of the tissue just prior to its removal from the host. We examined the lability of radiolabeled 5-fluorouracil (FUra) and its anabolites during excision and freeze-clamping in a rat tumor model. Acid-soluble metabolites were identified by HPLC. Two rats, each bearing multiple, subcutaneously-implanted colon tumors, were treated with eniluracil (an inactivator of dihydropyrimidine dehydrogenase) to prevent catabolism of FUra and then injected intravenously with [H-3]FUra. After 2 hr, tumors were harvested sequentially and segmented. The tumor pieces were kept at room temperature for various times up to 4 min prior to freezing. These specimens showed a decrease (P < 0.01) in labeled nucleoside triphosphate content of 13 +/- 2%/min and commensurate increases (P < 0.005) in labeled nucleoside monophosphates and nucleosides with increasing time-to-freeze. The amounts of labeled macromolecules, nucleoside diphosphates, and FUra each remained approximately constant. The study indicates that substantial errors may occur in measured tissue concentrations of pyrimidine nucleosides and nucleotides due to lability during tissue excision and freeze-clamping. Such errors can be corrected using data of the type obtained in this study. BIOCHEM PHARMACOL 60;7:963-967, 2000. (C) 2000 Elsevier Science Inc.