Catabolism of Deoxycytidine in Human Peripheral Blood Mononuclear Cells and Its Interference with the Determination of in Situ Thymidylate Synthase Activity

Abstract

Resting and stimulated human peripheral blood lymphocytes and monocyte-derived macrophages were incubated with tritiated deoxycytidine labeled at the 5-position. Release of tritiated water into the medium was thereupon detected utilizing its lack of binding to active charcoal, which is an established technique to measure in situ thymidylate synthase activity. It was found that tritiated dihydrouracil, a deoxycytidine catabolite, was formed during incubation with tritiated deoxycytidine. Like water, dihydrouracil does not bind to active charcoal, and its presence in the cell medium can result in an overestimation of the in situ thymidylate synthase activity. The catabolism of dCyd was highest in macrophages where 25% of the added dCyd (0.5 μM, 0.5 nmol/million cells) had been converted to dihydrouracil after 30 min, and 90% after 12 h. The in situ thymidylate synthase activity was found to be the highest in stimulated lymphocytes. If the interference of dihydrouracil had not been considered, the activity in macrophages would have been greatly overestimated and would have appeared to be higher than that of stimulated lymphocytes.