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1
Academic Medical Center, University of Amsterdam, Emma Children’s Hospital, and the Department of Clinical Chemistry, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
2
Department of Pediatrics, Nagoya City University Medical
School, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
a Author for correspondence. Fax 31-20-6962596;
Background: Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism.
Methods: We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N-carbamyl-ß-aminoisobutyric acid, and N-carbamyl-ß-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards.
Results: All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4–4 µmol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3–12% for liquid urines and 1.0–10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3–11% (100–200 µmol/L). Recoveries were 89–99% at 100–200 µmol/L and 95–106% at 1 mmol/L in liquid urines, and 93–103% at 100–200 µmol/L and 100–106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects.
Conclusions: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.
The following articles in journals at HighWire Press have cited this article:
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  A. B.P. van Kuilenburg, H. van Lenthe, L. Zoetekouw, and W. Kulik HPLC-Electrospray Tandem Mass Spectrometry for Rapid Determination of Dihydropyrimidine Dehydrogenase Activity Clin. Chem., March 1, 2007; 53(3): 528 - 530. [Abstract] [Full Text] [PDF]
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S. Hartmann, J. G. Okun, C. Schmidt, C.-D. Langhans, S. F. Garbade, P. Burgard, D. Haas, J. O. Sass, W. L. Nyhan, and G. F. Hoffmann Comprehensive Detection of Disorders of Purine and Pyrimidine Metabolism by HPLC with Electrospray Ionization Tandem Mass Spectrometry Clin. Chem., June 1, 2006; 52(6): 1127 - 1137. [Abstract] [Full Text] [PDF]
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 A. B.P. van Kuilenburg, R. Meinsma, E. Beke, B. Assmann, A. Ribes, I. Lorente, R. Busch, E. Mayatepek, N. G.G.M. Abeling, A. van Cruchten, et al. {beta}-Ureidopropionase deficiency: an inborn error of pyrimidine degradation associated with neurological abnormalities Hum. Mol. Genet., November 15, 2004; 13(22): 2793 - 2801. [Abstract] [Full Text] [PDF]
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A. B.P. van Kuilenburg, H. van Lenthe, A. van Cruchten, and W. Kulik Quantification of 5,6-Dihydrouracil by HPLC-Electrospray Tandem Mass Spectrometry Clin. Chem., January 1, 2004; 50(1): 236 - 238. [Full Text] [PDF]
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 A. B. P. van Kuilenburg, R. Meinsma, B. A. Zonnenberg, L. Zoetekouw, F. Baas, K. Matsuda, N. Tamaki, and A. H. van Gennip Dihydropyrimidinase Deficiency and Severe 5-Fluorouracil Toxicity Clin. Cancer Res., October 1, 2003; 9(12): 4363 - 4367. [Abstract] [Full Text] [PDF]
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J. J. Pitt, M. Eggington, and S. G. Kahler Comprehensive Screening of Urine Samples for Inborn Errors of Metabolism by Electrospray Tandem Mass Spectrometry Clin. Chem., November 1, 2002; 48(11): 1970 - 1980. [Abstract] [Full Text] [PDF]
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