Abstract
A simple, selective, and sensitive method utilizing tritium ((3)H) release from (3)H-deoxyuridine 5'-monophosphate (dUMP) substrate for accurate and precise determination of the low basal thymidylate synthase activity (TSA) in normal healthy peripheral blood mononuclear cells (PBMCs) was developed and validated. The method is based on the removal of the remaining substrate after the TSA reaction by absorption onto activated carbon and measurement of the supernatant fluid by liquid scintillation counting. The method background was substantially decreased by using lyophilized substrate and optimized binding conditions of remaining substrate onto carbon after TSA reaction. The concentration of cofactor N (5),N (10) methylene-(6R,S)-tetrahydrofolate was increased to obtain maximal TSA. Method sensitivity was further increased by omission of ethylenediaminetetraacetic acid from the reaction mix and by using longer reaction times. The validation parameters included specificity, linearity, sensitivity, precision, and stability. The lower limit of quantification was 25 μg PBMC cytosolic lysate, which released 1.4 pmol (3)H/h. TSA was stable in PBMC pellets stored for 6 months at -80 °C. The applicability of the method was demonstrated by the successful determination of TSA in PBMC cytosolic lysates from ten healthy volunteers with and without the specific TSA inhibitor FdUMP.