Determination of thymidine phosphorylase activity in leukemic blast cells by a high-performance liquid-chromatographic assay

A high-performance liquid chromatographic (HPLC) method has been developed for measuring thymidine and thymine levels, so these could be used to deter mine thymidine phosphorylase (TP) activity. The method described has the se lectivity to separate the analytes from an enzymatic reaction mixture witho ut mutual disturbance. Correlation of peak areas of thymine, thymidine, and 2-thio-6-azauridine with amounts in standards and spiked matrices was line ar in the concentration range 0.04-2 mu g mL(-1) for 2-thio-6-azauridine, 0 .08-3 mu g mL(-1) for thymine, and 0.02-2.5 mu g mL(-1) for thymidine. Intr a-and interday precision were measured by analyzing the retention times of replicate standards or spiked matrices within one (intra assay, n = 8) and eight consecutive days (inter assay, n = 8) over the concentration ranges 0 .1-0.8 mu g mL(-1) for thymine, 0.02-0.4 mu g mL(-1) for thymidine, and 0.1 -0.5 mu g mL(-1) for 2-thio-6-azauridine. Recovery was monitored by use of 2-thio-6-azauridine as internal standard. The precision, accuracy, and spee d of the method were excellent. The smallest detectable (LOD) and quantifia ble (LOQ) quantities were, respectively, 1.3 ng mL(-1) and 3.4 ng mL(-1) fo r thymine, 2.68 ng mL(-1) and 7.46 ng mL(-1) for thymidine, and 1.7 ng mL(- 1) and 4.92 ng mL(-1) for 2-thio-6-azauridine. At 25 degrees C the TP reaction was linear for at least 45 min. At 37 degre es C the reaction occurred much faster and was linear for 15 min only. We d ecided, therefore, to quantify thymidine and thymine after an incubation ti me of 30 min at 25 degrees C. The Michaelis constant (KM = 59 nmol) and max imum enzymatic reaction velocity (v(max) = 1.626 nmol min(-1) x 10(6) cells ) for TP were determined by extrapolation of the regression line (y = 32.53 x + 90.73; r = 0.992) to the x and y axes, respectively. Enzymatic activity was calculated from: Activity (units x 10(10) cells) = (A thymidine 30 min A thymidine 0 min) x 0.00021 where A is the peak area from the chromatogram.