Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography.
A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by liquid scintillation counting. The assay was linear with time and protein concentration. The minimum amount of radiolabeled products which could be determined proved to be 12 pmol using a purified stock solution of [2-(14)C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a dihydropyrimidinase deficiency.
Van Kuilenburg
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AB
Academic Medical Center, Laboratory Genetic Metabolic Diseases, Amsterdam, The Netherlands.
Van Lenthe
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H
Van Gennip
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AH
D000581:Amidohydrolases / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D015203:Reproducibility of Results; D012588:Scintillation Counting; D012680:Sensitivity and Specificity
D002250:Carbon Radioisotopes; D000581:Amidohydrolases; C020047:dihydropyrimidinase