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General Clinical Chemistry |
1 Academic Medical Center, University of Amsterdam, Emma Children’s Hospital and Departments of Clinical Chemistry, Amsterdam, The Netherlands.
2 Philipps-University, Institute for Physiological Chemistry, Marburg, Germany.
3 Academic Hospital Maastricht, Departments of Clinical Genetics and Clinical Chemistry, Maastricht, The Netherlands.
aAddress correspondence to this author at: Academic Medical Center, Laboratory Genetic Metabolic Diseases, F0-224, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Fax 31-206962596; e-mail a.b.vankuilenburg{at}amc.uva.nl.
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Abstract |
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Methods: Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. 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 de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4–3 µmol/L. The intra- and interassay variation for urine with added compounds was 1.2–5% for urines and 2–9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97–106% for urine samples and 97–115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls.
Conclusion: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.
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Introduction |
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). Glutamine, ATP, and carbon dioxide are converted by CAD into dihydroorotate. Subsequently, the oxidation of dihydroorotate to orotate occurs at the mitochondrial inner membrane by dihydroorotate dehydrogenase, with ubiquinone being the electron acceptor. The final two steps in this pathway, leading to the synthesis of UMP, occur again in the cytoplasm and are catalyzed by the bifunctional enzyme UMP synthase, which contains orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase.
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In addition to de novo synthesis, pyrimidine nucleotides can also be synthesized via salvage of the pyrimidine nucleosides uridine and cytidine. The relative contribution of the de novo and the salvage pathways to the maintenance of the nucleotide pools, however, varies in different cells and tissues. Proliferating cells usually require a functional pyrimidine de novo pathway to sustain their increased demand for nucleotides. Several inhibitors have therefore been developed against enzymes of the pyrimidine de novo pathway, such as dihydroorotate dehydrogenase, which show potent antiproliferative effects in tumor cells or proliferating T lymphocytes(2)(3)(4).
Pathologic conditions such as a deficiency of UMP synthase or a urea-cycle defect can lead to altered excretion of metabolites of the pyrimidine de novo pathway. Patients with a deficiency of UMP synthetase excrete excessive amounts of orotate in their urine(5)(6). In contrast, patients suffering from a urea-cycle disorder can, in addition to orotate, also excrete highly increased amounts of orotidine, uridine, and uracil(5)(7). Several methods have been described for the detection of some of the metabolites of the pyrimidine de novo pathway, using colorimetry(8), thin-layer chromatography(9), enzymatic spectrophotometric assays(10), liquid-liquid chromatography(11), HPLC(6)(12)(13), gas chromatography-mass spectrometry(14)(15), HPLC–tandem mass spectrometry (HPLC-MS/MS)(16)(17), and capillary electrophoresis(18)(19). However, these procedures are usually laborious, requiring extensive manipulations, and/or they do not detect all metabolites. We therefore have developed a rapid and sensitive method, using HPLC-MS/MS, that allows the detection of all pyrimidine de novo metabolites from urine or urine-soaked filter paper strips within a single analytical run of 14 min. The usefulness of the method was demonstrated by the analysis of urine samples from patients with urea-cycle defects and patients with a deficiency of the pyrimidine degradation pathway.
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Materials and Methods |
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patient and control populations
The control population consisted of 155 patients admitted to our hospital with clinical and biochemical findings not indicative of inborn errors in the urea cycle or purine and pyrimidine metabolism. In addition, four patients with a disorder of the urea cycle and two patients with a dihydropyrimidine dehydrogenase deficiency were investigated. Samples were obtained according to the "Code for proper use of human tissue" as formulated by the Dutch Federation of Medical Scientific Societies.
preparation of internal standards
1,3-15N2-Orotidine was prepared enzymatically by the concerted action of orotidine-5'-phosphate pyrophosphorylase and alkaline phosphatase. A reaction mixture (4 mL) consisting of 5 mmol/L 1,3-15N2 orotate, 12 mmol/L phosphoribosyl pyrophosphate, 20 mmol/L MgCl2, and 50 mmol/L Tris-HCl (pH 8.0) was incubated with yeast orotidine-5'-phosphate pyrophosphorylase (2.4 g/L) at 37 °C in the dark for 4 h. Subsequently, alkaline phosphatase was added to the reaction mixture to a final concentration of 0.9 g/L (2700 kU/L). After an incubation period of 2 h at 37 °C, the reaction mixture was deproteinized by use of a Millipore Microcon® YM-10 centrifugal filter unit. The clear supernatant was analyzed by reversed-phase HPLC at ambient temperature on a Phenomenex Aqua analytical column [250 x 4.6 mm (i.d.); particle size, 5 µm] with a gradient from 100% solvent A (50 mmol/L formic acid, pH 2.6) to 60% solvent B [methanol–50 mmol/L formic acid (pH 2.6); 1:1 by volume] in 6 min, at a flow rate of 1 mL/min. Detection of the various products at a wavelength of 266 nm showed that 51% of the substrate 1,3-15N2-orotate had been converted into 1,3-15N2-orotidine.
D3-N-carbamyl-aspartate was prepared by incubating a reaction mixture (2 mL) containing 13 mmol/L 2,3,3-D3-aspartate, 15 mmol/L carbamyl phosphate, and 50 mmol/L triethanolamine HCl (pH 7.0) with aspartate transcarbamylase (1 kU/L) at 37 °C for 2 h. The reaction mixture was deproteinized by use of a Microcon YM-10 centrifugal filter unit. Separation of D3-N-carbamyl-aspartate from 2,3,3-D3-aspartate was performed isocratically [50 mmol/L formic acid (pH 2.6), at a flow rate of 1 mL/min] by reversed-phase HPLC at ambient temperature, as described above, with online ultraviolet detection at 210 nm. The yield of enzymatically prepared D3-N-carbamyl-aspartate was 
34%.
The synthesis of 1,3-15N2-dihydroorotate was performed with recombinant Lactococcus lactis dihydroorotate dehydrogenase and 15N2-orotate(20). Dihydroorotate dehydrogenase was added, to a final concentration of 20 mg/L, to a reaction mixture (75 mL) containing 1.15 mmol/L 15N2-orotate sodium salt, 3.5 mmol/L NADH, 1 mmol/L dithiothreitol, and 100 mmol/L Tris-HCl (pH 6). After 30 min at 30 °C, the reaction mixture was deproteinized by centrifugation using a Microcon YM-3 centrifugal filter unit and lyophilized. Analysis by HPLC-MS/MS, as described below, showed a yield of 1,3-15N2-dihydroorotate of 95%.
Analysis of the enzymatically synthesized internal standards by HPLC-MS/MS, as described below, did not reveal the presence of any interferences. These crude internal standards were therefore used directly to prepare the internal standard (IS) mixture without further purification. The IS mixture was prepared in water containing 1 mmol/L each of stable-isotope-labeled N-carbamyl-aspartate (N-C-aspartate), dihydroorotate, orotate, orotidine, uridine, and uracil.
sample preparation
Urine samples were centrifuged at 10 000g for 5 min to remove debris, and 10 µL of the IS and 2 µL of 250 mL/L formic acid were added to 100 µL of clear urine. After centrifugation (10 000g for 2 min), 50 µL of this urine was injected into the HPLC-MS/MS system. Urinary creatinine concentrations were determined by the conventional alkaline-picrate method(21).
Urine (200 µL) was added to filter-paper strips (12 x 40 mm). The strips were dried at room temperature, and 20 µL of the IS was deposited on the center of each strip. After drying, the strip was placed in a 2-mL Eppendorf tube and extracted with 1.5 mL of 750 mL/L methanol by sonification for 10 min. The extract was dried at 40 °C under a stream of nitrogen, and the dried sample was dissolved in 200 µL of 50 mmol/L formic acid (pH 2.6) and sonicated for 5 min. After centrifugation (10 000g for 2 min), 50 µL of the clear extract was injected into the HPLC-MS/MS system. The remaining extract could be used to measure the creatinine concentrations by the conventional alkaline-picrate method(21).
hplc-electrospray ionization ms/ms
The metabolites of interest were separated at ambient temperature on a Phenomenex Aqua analytical column [250 x 4.6 (i.d.) mm; 5 µm particle size] protected by a Phenomenex SecurityGuard [4 x 3.0 (i.d.) mm] C18 ODS column. Solvent A consisted of 50 mmol/L formic acid (pH 2.6) and solvent B consisted of a mixture of 50 mmol/L formic acid (pH 2.6) and methanol (1:1 by volume). Elution was performed by use of a linear gradient, at a flow rate of 1 mL/min, as follows: 0–6 min, 100% solvent A to 60% solvent B; 6–6.1 min, 60% solvent B to 100% solvent B; 6.1–9 min, 100% solvent B; 9–9.1 min, 100% solvent B to 100% solvent A; 9.1–14 min, equilibration with 100% solvent A.
A splitter between the HPLC column and the mass spectrometer was used to introduce the eluate at a flow of 50 µL/min into the mass spectrometer. The eluate from 4.2 to 8 min was introduced into the mass spectrometer by use of an electrically operated valve. A Waters Micromass Quattro II tandem mass spectrometer was used in the negative electrospray ionization (ESI) mode, and nitrogen was used as the nebulizing gas. Argon was used as the collision gas, and the cell pressure was 0.25 Pa. The source temperature was set at 80 °C, and the capillary voltage was maintained at 3.5 kV. Multiple-reaction monitoring was used to detect the metabolites by the specific m/z transition of precursor ion to fragment. The transitions, cone voltages, and collision energies established for each compound are listed in Table 1
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validation
The mass spectrometer was optimized for each metabolite by use of various settings as the compounds were eluted from the HPLC column. The linearity and detection limits for each compound were established by injection of calibration mixtures with different concentrations (0, 5, 100, 250, and 1000 µmol/L). The stable-isotope-labeled compound of each analyte was used as the IS. The concentration of each analyte was determined by use of the slope and intercept of the calibration curve that was obtained from a least-squares regression for the analyte/IS peak-area ratio vs the concentration of the analyte in the calibration mixture.
The intraassay variation of the method was established by measurement of a blank urine and a urine enriched with the metabolites of interest at low (10 µmol/L), medium (100 µmol/L), and high (250 µmol/L) concentrations. The interassay variation was established by measuring blank urines and urines enriched with the relevant metabolites (10–250 µmol/L) during a period of 3 weeks. The recovery of the method was established by measuring five different urines before and after enrichment with 10, 100, and 250 µmol/L of the relevant metabolites. The extraction efficiency of creatinine was obtained by comparing the creatinine concentrations in urine samples with those of the filter-paper extracts of the same urines (10 different urines with creatinine concentrations of 4.8–11.6 mmol/L).
To compensate for losses that might occur during preparation of the samples and loss of sensitivity attributable to quenching of the signal by coeluting compounds, the IS mixture was added to the samples.
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Results |
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. The development of a flow injection procedure was not possible because of interference of some of the compounds of interest with the detection of others. For example, the detection of uracil using the m/z 111
42 transition was hampered by interference from orotate. Apparently, orotate is partly degraded during the process of ionization to uracil. To circumvent the interference and suppression of the signals by salts, the samples were introduced into the mass spectrometer via reversed-phase HPLC. Fig. 2
shows the multiple-reaction-monitoring signals and the unambiguous identification of each compound present in the calibration mixture.
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The calibration curves for dihydroorotate, orotate, orotidine, uracil, and uridine were linear up to at least 1 mmol/L (r2 
0.998). The calibration curve for N-C-aspartate was fitted best by a quadratic curve (r2 = 0.998). To establish the detection limits of the mass spectrometer for the various compounds, no special precautions were taken, such as cleaning of the high-voltage lens and sample cone. Under these conditions, the detection limits (defined as a signal-to-noise ratio of 3) were 1 µmol/L for N-C-aspartate, 3 µmol/L for dihydroorotate, 0.4 µmol/L for orotate, 0.7 µmol/L for orotidine, 3 µmol/L for uridine, and 1.5 µmol/L for uracil.
The mean (SD) extraction efficiency for creatinine from filter-paper strips was 71 (5)% (n = 10). The intra- and interassay variations (CVs) of the procedure to detect the various compounds in urine and in filter-paper extracts are shown in Tables 2
and 3
, respectively. For urine with added compounds, the intra- and interassay variation was 1.2–5% for liquid urines and 2–9% for filter-paper extracts of the urines. No indication of degradation of the various metabolites was observed when we tested urine-soaked filter-paper strips that had been stored at room temperature for 3 weeks. The recovery data for the various metabolites are summarized in Table 4
. Recoveries of the added metabolites were 97–106% for urine samples and 97–115% for filter-paper extracts of the urines.
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Reference values established for the various compounds in urine for four different age groups showed a gradual decrease in the concentrations of the metabolites per mmol of creatinine with age (Table 5
). In all patients with a deficiency of ornithine transcarbamylase or argininosuccinase, increased concentrations of N-C-aspartate and orotate were present in the urine (Table 6
). In patient 3 and 4, moderately increased concentrations of dihydroorotate were also observed. Orotidine was highly increased in patient 2, whereas it was within the reference values in a different urine sample that was obtained 1 year later. However, in that urine sample from patient 2, highly increased concentrations of uridine and uracil were detected (Table 6
). All three patients with an ornithine transcarbamylase deficiency had highly increased concentrations of uracil. In contrast, a uracil concentration within reference values was observed for the patient with an argininosuccinase deficiency. This phenomenon has been observed before and can be explained by the high excretion of other nitrogen-containing metabolites in patients with an argininosuccinase deficiency(5)(7). In patients suffering from a dihydropyrimidine dehydrogenase deficiency, the concentrations of the pyrimidine de novo metabolites were within reference values and only highly increased concentrations of uracil were present in the urine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous methods have been published regarding the analysis of specific metabolites of the pyrimidine de novo pathway(6)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), but no procedure has been available that allows the detection of all metabolites within a single run. In this study, we demonstrate that with HPLC-MS/MS, all pyrimidine de novo metabolites and their degradation products could be measured within a single analytical run of 14 min with lower limits of detection of 0.4–3 µmol/L. Because stable-isotope-labeled standards of N-C-aspartate, dihydroorotate, and orotidine were not commercially available, these compounds were synthesized enzymatically from stable-isotope-labeled substrates. The use of stable-isotope-labeled IS enabled correction of the signals for quenching by coeluting compounds, giving high recoveries of 97–115%. The reproducibility of our method is demonstrated by the low intra- and interassay variation (1.2–9%).
The concentrations of the pyrimidine de novo metabolites in urine, normalized for creatinine, gradually decreased with age. A conceivable explanation for this phenomenon might be the increase in creatinine excretion in older individuals attributable to the increase in muscle mass. Data regarding the urinary concentrations of pyrimidine de novo metabolites are scant. Nevertheless, the concentrations of orotate in urine measured in our method are in perfect agreement with those obtained by others(15)(16).
The accumulation of carbamyl phosphate, which is associated with inherited defects of the urea cycle, stimulates the pyrimidine de novo pathway, leading to increased production of orotate. In patients with a defect in one of the enzymes of the urea cycle, the rate of pyrimidine excretion is dependent on the ammonia concentrations in plasma(7). In our study, all four patients with a defect in either ornithine transcarbamylase or argininosuccinase presented with highly increased concentrations of orotate and N-C-aspartate. In addition, some of these patients also had highly increased concentrations of orotidine, uridine, and uracil, which are in line with results obtained by others(7)(9). Only two patients had moderately increased concentrations of dihydroorotate, which might be attributable to the fact that dihydroorotase favors the reverse reaction(12). Patients with a dihydropyrimidine dehydrogenase deficiency had concentrations of the pyrimidine de novo metabolites that were within reference values. Dihydropyrimidine dehydrogenase is involved the first and rate-limiting step in the degradation of the pyrimidine bases uracil and thymine, and patients with a deficiency of this enzyme excrete large amounts of these pyrimidine bases in their urine(24).
In conclusion, the analysis of urine by HPLC-ESI MS/MS, as described in this study, allows the rapid diagnosis of inborn errors affecting the pyrimidine de novo pathway. Furthermore, we demonstrated that the results obtained with dried urine filter-paper strips were comparable to those obtained with urine. Because the collection of liquid urine, especially from neonates, might be difficult and the shipment of frozen urine is expensive, the use of filter-paper strips offers the advantage of easy collection, transport, and storage of the urine samples.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  M. E. Brosnan and J. T. Brosnan Orotic Acid Excretion and Arginine Metabolism J. Nutr., June 1, 2007; 137(6): 1656S - 1661S. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, carbamylphosphate synthetase; 
, aspartate transcarbamylase; 
, dihydroorotase; 
, dihydroorotate dehydrogenase; 
, orotate phosphoribosyltransferase; 
, orotidine 5'-monophosphate decarboxylase; 
, orotidine 5'-monophosphate phosphohydrolase; 
, pyrimidine 5'-nucleotidase; 
, uridine kinase;
, uridine phosphorylase.