Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity. Screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity.
Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of the naturally occurring pyrimidines, uracil and thymine, and the fluoropyrimidine anticancer drug, 5-fluorouracil (FUra) to 5,6-dihydropyrimidines. Previous studies have demonstrated that cancer patients who are DPD deficient exhibit severe toxicity (including death) following treatment with FUra. To date, the direct measurement of DPD enzyme activity has been the only reliable method to identify DPD deficient cancer patients. We now report a semi-automated radioassay for measuring DPD activity in human peripheral lymphocytes. Following incubation of lymphocyte cytosol (at a fixed protein concentration of 200 micrograms) with [6-14C]FUra at timepoints ranging from 0 to 30 min, samples are ethanol precipitated, filtered and analyzed by HPLC. Determination of radioactivity is accomplished using an in-line flow scintillation analyzer with automatic quantitation of peaks. This method provides the first specific assay for DPD enzyme activity which is rapid, reproducible and sensitive enough to be used in the routine screening of cancer patients for DPD deficiency prior to treatment with FUra.
Johnson
M R
MR
Department of Pharmacology and Toxicology, University of Alabama at Birmingham 35294, USA.
Yan
J
J
Shao
L
L
Albin
N
N
Diasio
R B
RB
D000964:Antimetabolites, Antineoplastic / Q000009:adverse effects*; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil / Q000009:adverse effects*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D010088:Oxidoreductases / Q000097:blood* / Q000172:deficiency; D011854:Radiochemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000964:Antimetabolites, Antineoplastic; D002250:Carbon Radioisotopes; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil