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Technical Briefs |
1 Academic Medical Center, University of Amsterdam, Emma Children’s Hospital and Department of Clinical Chemistry, PO Box 22700, 1100 DE Amsterdam, The Netherlands
aaddress correspondence to this author at: Academic Medical Center, Laboratory for Genetic Metabolic Diseases, F0-224, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; fax 31-206962596, e-mail a.b.vanKuilenburg{at}amc.uva.nl
In humans, the pathway for the catabolism of uracil and thymine consists of three consecutive steps. Dihydropyrimidine dehydrogenase catalyzes the reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. The second step is catalyzed by dihydropyrimidinase and consists of reversible hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid, respectively. Finally, ß-ureidopropionase catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, respectively, ammonia, and CO2.
Patients with a defect in one of the enzymes of the pyrimidine degradation pathway can be diagnosed by an aberrant excretion profile of the pyrimidine bases and their degradation products in urine (1). For example, in patients with a complete deficiency of dihydropyrimidinase, highly increased concentrations of 5,6-dihydrouracil and 5,6-dihydrothymine and moderately increased concentrations of uracil and thymine can be detected in urine. It has also been suggested that the 5,6-dihydrouracil/uracil ratio in plasma of patients with cancer is a prognostic indicator for the toxicity of 5-fluorouracil-based chemotherapy (2). In addition, increased concentrations of 5,6-dihydrouridine, a naturally occurring component in prokaryote and eukaryote tRNA, have been found in the urine of cancer patients.
Recently we developed a screening procedure for defects in the pyrimidine degradation pathway that combines reversed-phase HPLC with electrospray ionization tandem mass spectrometry (1). Surprisingly, the analysis of 5,6-dihydrouridine in urine with the chromatographic and MS conditions as described for the pyrimidine bases and their degradation products showed that the presence of this compound interferes with the detection of 5,6-dihydrouracil. An intense multiple-reaction monitoring signal for the transition m/z 115
73 was observed for 5,6-dihydrouridine, a transition previously selected for the detection of 5,6-dihydrouracil (see Fig. 1, A and B
). In the ion source, 5,6-dihydrouridine ([M + H]+ = m/z 247) is partly degraded to m/z 115, which is identical to the [M + H]+ ion of dihydrouracil. Because 5,6-dihydrouridine coelutes with dihydrouracil, it contributes substantially to the transition peak attributed to dihydrouracil. The interference of 5,6-dihydrouridine could be eliminated by use of the transition m/z 132115 for the detection of dihydrouracil (see Fig. 1, C and D
). The selected parent ion [M + NH4]+ at m/z 132 cannot be produced by 5,6-dihydrouridine; thus, it no longer forms a source of peak contamination. The transition for the internal standard ([13C4,15N2]-dihydrouracil) was adjusted accordingly.
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The detection limits (defined as a signal-to-noise ratio of 3) for 5,6-dihydrouracil were 2–5 µmol/L and 0.3 µmol/L for urine and plasma, respectively. Table 1
shows the intraassay (within-day) variation and recovery of 5,6-dihydrouracil in urine and plasma. At the detection limit, we observed a relatively large variation for the reproducibility and recovery of 5,6-dihydrouracil in urine and plasma. However, at higher 5,6-dihydrouracil concentrations that were comparable to those found in patients with a dihydropyrimidinase deficiency, we obtained excellent reproducibility and recoveries for urine, urine-soaked filter paper strips, and plasma. Reference values were established for 5,6-dihydrouracil in urine for four different age groups: 0–2 years (n = 45), mean (SD), 7.2 (6.0) µmol/mmol of creatinine (range, 2.0–31 µmol/mmol of creatinine); 2–6 years (n = 25), 3.0 (1.5) µmol/mmol of creatinine (range, 1.0–6.0 µmol/mmol of creatinine); 6–10 years (n = 12), 3.8 (3.3) µmol/mmol of creatinine (range, 1.0–11.0 µmol/mmol of creatinine); >10 years (n = 23), 2.0 (1.9) µmol/mmol of creatinine (range, 1.0–10.0 µmol/mmol of creatinine). In plasma, a low concentration of 5,6-dihydrouracil was detected with a mean concentration of 1.3 (0.6) µmol/L (n = 10). Comparable values for 5,6-dihydrouracil have been found in plasma and urine by conventional reversed-phase HPLC (3)(4).
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In urine from newborns, the mean concentration of 5,6-dihydrouridine is 
40.4 (5.5) µmol/mmol of creatinine (5). Thus, the urinary concentrations of 5,6-dihydrouridine are sixfold higher than the 5,6-dihydrouracil concentrations. Nevertheless, the specific transition of m/z 132115 for 5,6-dihydrouracil allows the identification of all pyrimidine bases and their degradation products within one analytical run of 15 min.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Svobaite, I. Solassol, F. Pinguet, L. Ivanauskas, J. Bres, and F. M. M. Bressolle HPLC with UV or Mass Spectrometric Detection for Quantifying Endogenous Uracil and Dihydrouracil in Human Plasma Clin. Chem., September 1, 2008; 54(9): 1463 - 1472. [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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