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Analysis of pyrimidine synthesis "de Novo" intermediates in urine and dried urine filter-paper strips with HPLC-electrospray tandem mass spectrometry.

Pyrimidine nucleotides are essential for a vast number of biological processes, such as the synthesis of RNA, DNA, phospholipids, and glycogen and the sialylation and glycosylation of proteins (1). Pyrimidines are synthesized "de novo" in mammalian cells through a multistep process. The first three steps are catalyzed by CAD, [4] a trifunctional cytoplasmic enzyme cluster containing carbamylphosphate synthetase, aspartate carbamyltransferase, and dihydroorotase (Fig. 1). 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 phosphoribosyl-transferase and orotidine-5'-phosphate decarboxylase.

[FIGURE 1 OMITTED]

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 (2-4).

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 chromatographymass 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 HPLCMS/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.

Materials and Methods

CHEMICALS

[1,3-.sup.15][N.sub.2]-Uracil; [1,3-.sup.15][N.sub.2]-orotate; [1,3-.sup.15][N.sub.2]-uridine, and 2,3,3-[D.sub.3]-aspartate were obtained from Cambridge Isotope Laboratories. Carbamyl phosphate and yeast orotidine-5'phosphate pyrophosphorylase were obtained from Boehringer Mannheim GmbH. Aspartate transcarbamylase from Streptococcus faecalis and phosphoribosyl pyrophosphate were obtained from Sigma-Aldrich, and alkaline phosphatase was obtained from Roche.

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. u1 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-.sup.15][N.sub.2]-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-.sup.15][N.sub.2]-orotate, 12 mmol/L phosphoribosyl pyrophosphate, 20 mmol/L Mg[Cl.sub.2], and 50 mmol/L Tris-HCl (pH 8.0) was incubated with yeast orotidine-5'-phosphate pyrophosphorylase (2.4 g/L) at 37[degrees]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[degrees]C, the reaction mixture was deproteinized by use of a Millipore Microcon[R] 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 [micro]m] 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-.sup.15][N.sub.2]-orotate had been converted into [1,3-.sup.15][N.sub.2]-orotidine.

[D.sub.3]-N-carbamyl-aspartate was prepared by incubating a reaction mixture (2 mL) containing 13 mmol/L 2,3,3-[D.sub.3]-aspartate, 15 mmol/L carbamyl phosphate, and 50 mmol/L triethanolamine HCl (pH 7.0) with aspartate transcarbamylase (1 kU/L) at 37[degrees]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 mn. The yield of enzymatically prepared [D.sub.3]-N-carbamyl-aspartate was ~34%.

The synthesis of [1,3-.sup.15][N.sub.2]-dihydroorotate was performed with recombinant Lactococcus lactis dihydroorotate dehydrogenase and [sup.15][N.sub.2]-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 [sup.15][N.sup.2]-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[degrees]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-.sup.15][N.sub.2]-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 [micro]L of the IS and 2 [micro]L of 250 mL/L formic acid were added to 100 [micro]L of clear urine. After centrifugation (10 000g for 2 min), 50 [micro]L 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 [micro]L) was added to filter-paper strips (12 x 40 mm). The strips were dried at room temperature, and 20 [micro]L 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[degrees]C under a stream of nitrogen, and the dried sample was dissolved in 200 [micro]L of 50 mmol/L formic acid (pH 2.6) and sonicated for 5 min. After centrifugation (10 000g for 2 min), 50 [micro]L 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 [micro]m particle size] protected by a Phenomenex SecurityGuard [4 x 3.0 (i.d.) mm] [C.sub.18] 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 sputter between the HPLC column and the mass spectrometer was used to introduce the eluate at a flow of 50 [micro]L/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[degrees]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.

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 [micro]mol/L). The stable-isotope-labeled compound of each analyte was used as the I5. 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 interassay variation of the method was established by measurement of a blank urine and a urine enriched with the metabolites of interest at low (10 [micro]mol/L), medium (100 [micro]mol/L), and high (250 [micro]mol/L) concentrations. The interassay variation was established by measuring blank urines and urines enriched with the relevant metabolites (10-250 [micro]mol/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 [micro]mol/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.

Results

The specific transitions that were obtained for each compound are shown in Table 1. 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[right arrow]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.

The calibration curves for dihydroorotate, orotate, orotidine, uracil, and uridine were linear up to at least 1 mmol/L ([r.sup.2] [greater than or equal to] 0.998). The calibration curve for N-C-aspartate was fitted best by a quadratic curve ([r.sup.2] = 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 [micro]mol/L for N-C-aspartate, 3 [micro]mol/L for dihydroorotate, 0.4 [micro]mol/L for orotate, 0.7 [micro]mol/L for orotidine, 3 [micro]mol/L for uridine, and 1.5 [micro]mol/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.

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.

[FIGURE 2 OMITTED]

Discussion

There is an increasing awareness that pyrimidine nucleotides synthesized via the de novo pathway play an important role in a variety of biological processes (1). In this respect, leflunomide, which is a potent inhibitor of dihydroorotate dehydrogenase, has shown promising results for the treatment of rheumatoid arthritis and other autoimmune diseases (22). Inhibitors of the pyrimidine de novo pathway might also have a profound antimalarial activity with minimal host toxicity (2, 23). The malarial parasite Plasmodium falciparum synthesizes its pyrimidine nucleotides exclusively via the de novo pathway, whereas healthy erythrocytes have no capacity for pyrimidine biosynthesis. Thus, the analysis of metabolites of the pyrimidine de novo pathway might provide more insight into the disease itself or the effectiveness of drugs targeted at enzymes of the pyrimidine de novo pathway.

Numerous methods have been published regarding the analysis of specific metabolites of the pyrimidine de novo pathway (6, 8-19), 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/M5, 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 [micro]mol/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 dihydroorotate 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.

The L. lactis dihydroorotate dehydrogenase type B was a gift from Dr. Olof Bjornberg, Molekylarbiologisk Institut, Kobenhavns Universitet (Copenhagen, Denmark). We thank Dr. Willem Kulik for critical reading of the manuscript.

References

(1.) Huang M, Graves LM. De novo synthesis of pyrimidine nucleotides; emerging interfaces with signal transduction pathways. Cell Mol Life Sci 2003;60:321-36.

(2.) Christopherson RI, Lyons SD, Wilson PK. Inhibitors of de novo nucleotide biosynthesis as drugs. Acc Chem Res 2002;35:961-71.

(3.) Ruckemann K, Fairbanks LD, Carrey EA, Hawrylowicz CM, Richards DF, Kirschbaum B, et al. Leflunomide inhibits pyrimidine de novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans. J Biol Chem 1998;273:21682-91.

(4.) Bruneau JM, Yea CM, Spinella-Jaegle S, Fudali C, Woodward K, Robson PA, et al. Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide. Biochem J 1998;336(Pt 2):299-303.

(5.) Webster DR, Becroft DMO, van Gennip AH, van Kuilenburg ABP. Hereditary orotic aciduria and other disorders of pyrimidine metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill Medical Publishing Division, 2001:2663-702.

(6.) Salerno C, Crifo C. Diagnostic value of urinary orotic acid levels: applicable separation methods. J Chromatogr B Analyt Technol Biomed Life Sci 2002;781:57-71.

(7.) van Gennip AH, Bree-glom EJ, Grift J, Degree PK, Wadman SK. Urinary purines and pyrimidines in patients with hyperammonemia of various origins. Clin Chim Acta 1980;104:227-39.

(8.) Prescott LM, Jones ME. Modified methods for the determination of carbamyl aspartate. Anal Biochem 1969;32:408-19.

(9.) Oberholzer VG, Palmer T. Increased excretion of N-carbamoyl compounds in patients with urea cycle defects. Clin Chim Acta 1976;68:73-8.

(10.) Rogers LE, Nicolaisen K. Enzymatic spectrophotometric assay for dihydroorotic acid in serum and urine. Experientia 1972;28: 1258-9.

(11.) Kesner L, Aronson FL, Silverman M, Chan PC. Determination of orotic and dihydroorotic acids in biological fluids and tissues. Clin Chem 1975;21:353-5.

(12.) Fairbanks LD, Carrey EA, Ruckemann K, Swaminathan R, Kirschbaum B, Simmonds HA. Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid. J Chromatogr B Biomed Sci Appl 1999;732:487-93.

(13.) Hayek-Ouassini M, Henseling J, Luffler M. Separation of N-carbamoyl aspartate and L-dihydroorotate from serum by high-performance liquid chromatography with fluorimetric detection. Chromatographia 2002;55:411-5.

(14.) Bonham JR, Guthrie P, Downing M, Allen JC, Tanner MS, Sharrard M, et al. The allopurinol load test lacks specificity for primary urea cycle defects but may indicate unrecognized mitochondrial disease. J Inherit Metab Dis 1999;22:174-84.

(15.) McCann MT, Thompson MM, Gueron IC, Tuchman M. Quantification of orotic acid in dried filter-paper urine samples by stable isotope dilution. Clin Chem 1995;41:739-43.

(16.) Rashed MS, Jacob M, AI Amoudi M, Rahbeeni Z, Al Sayed MAD, Al Ahaidib L, et al. Rapid determination of orotic acid in urine by liquid chromatography-electrospray tandem mass spectrometry. Clin Chem 2003;49:499-501.

(17.) Ito T, van Kuilenburg ABP, Bootsma AH, Haasnoot AJ, van Cruchten AG, Wada Y, et al. Rapid screening of high-risk patients for disorders of purine and pyrimidine metabolism using HPLC electrospray tandem mass spectrometry of liquid urine or urine-soaked filter paper strips. Clin Chem 2000;46:445-52.

(18.) Salerno C, D'Eufemia P, Celli M, Finocchiaro R, Crifo C, Giardini O. Determination of urinary orotic acid and uracil by capillary zone electrophoresis. J Chromatogr B Biomed Sci Appl 1999;734:175-8.

(19.) Friedecky D, Adam T, Bartak P. Capillary electrophoresis for detection of inherited disorders of purine and pyrimidine metabolism: a selective approach. Electrophoresis 2002;23:565-71.

(20.) Nielsen FS, Andersen PS, Jensen KF. The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and [FeS] redox centers. J Biol Chem 1996;271: 29359-65.

(21.) Henry RJ. Non-protein nitrogenous constituents. Clinical chemistry, principles and techniques. New York: Hoeber Medical Division, Harper & Row, 1964:292-9.

(22.) Sanders S, Harisdangkul V. Leflunomide for the treatment of rheumatoid arthritis and autoimmunity. Am J Med Sci 2002;323: 190-3.

(23.) Gero AM, O'Sullivan WJ. Purines and pyrimidines in malarial parasites. Blood Cells 1990;16:467-84.

(24.) van Kuilenburg ABP, Dobritzsch D, Meinsma JR, Haasjes J, Waterham HR, Nowaczyk MJM, et al. Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure. Biochem J 2002;364:157-63.

ANDRE B.P. VAN KUILENBURG, [1] * HENK VAN LENTHE, [1] MONIKA LOFFLER, [2] and ALBERT H. VAN GENNIP [3]

[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.

[4] Nonstandard abbreviations: CAD, carbamylphosphate synthetase-aspartate carbamyltransferase-dihydroorotase; MS/MS, tandem mass spectrometry; IS, internal standard; N-C-aspartate, N-carbamyl-aspartate; and ESI, electrospray ionization.

* Address 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@ amc.uva.nl.

Received June 18, 2004; accepted August 26, 2004.

Previously published online at DOI: 10.1373/clinchem.2004.038869
Table 1. MS settings for each compound in the negative
ESI mode.

 Parent Daughter Cone Collision
 ion, ion, voltage, energy,
Compound Mass m/z m/z V eV

N-C-Aspartate 176 175 132 20 10

Orotidine 288 287 111 15 15

Dihydroorotate 158 157 113 20 10

Uracil 112 111 42 25 15

Orotate 156 155 111 20 10

Uridine 244 243 200 25 10

[D.sub.3]-N-C- 179 178 135 20 10
Aspartate

[1,3-.sup.15] 290 289 113 15 15
[N.sub.2]-
Orotidine

[1,3-.sup.15] 160 159 115 20 10
[N.sub.2]-
Dihydroorotate

[1,3-.sup.15] 114 113 43 25 15
[N.sub.2]-
Uracil

[1,3-.sup.15] 158 157 113 20 10
[N.sub.2]-
Orotate

[1,3-.sup.15] 246 245 201 25 10
[N.sub.2]-
Uridine

Table 2. Intraassay variation for urines
and filter-paper extracts. (a)

 Blank (b,c)

 Mean (SD),
Compound [micro]mol/L CV, %

Urines
 N-C-Aspartate 2.61 (0.11) 4
 Dihydroorotate ND (e)
 Orotate 2.17 (0.05) 2
 Orotidine 2.66 (0.03) 1.3
 Uridine 0.68 (0.10) 14
 Uracil 7.9 (0.6) 8
Filter-paper extracts
 N-C-Aspartate 2.45 (0.11) 5
 Dihydroorotate ND
 Orotate 2.18 (0.06) 3
 Orotidine 2.7 (0.2) 6
 Uridine 0.7 (0.2) 31
 Uracil 7 (2) 28

 Low (b,d)

 Mean (SD),
Compound [micro]mol/L CV, %

Urines
 N-C-Aspartate 11.8 (0.2) 1.3
 Dihydroorotate 9.9 (0.5) 5
 Orotate 11.94 (0.14) 1.2
 Orotidine 12.2 (0.6) 5
 Uridine 10.5 (0.6) 5
 Uracil 18.2 (0.9) 5
Filter-paper extracts
 N-C-Aspartate 10.9 (0.3) 3
 Dihydroorotate 9.7 (0.9) 9
 Orotate 11.9 (0.2) 2
 Orotidine 12.2 (0.3) 2
 Uridine 11.0 (0.8) 7
 Uracil 18.1 (0.8) 4

 Medium (b,d)

 Mean (SD),
Compound [micro]mol/L CV, %

Urines
 N-C-Aspartate 95 (2) 2
 Dihydroorotate 102 (5) 5
 Orotate 101 (2) 2
 Orotidine 99 (2) 2
 Uridine 103 (3) 3
 Uracil 110 (3) 3
Filter-paper extracts
 N-C-Aspartate 89.8 (1.5) 2
 Dihydroorotate 96 (3) 4
 Orotate 100 (2) 2
 Orotidine 101 (3) 3
 Uridine 104 (3) 3
 Uracil 110 (4) 3

 High (b,d)

 Mean (SD),
Compound [micro]mol/L CV, %

Urines
 N-C-Aspartate 233 (8) 3
 Dihydroorotate 251 (10) 4
 Orotate 254 (6) 3
 Orotidine 255 (3) 1.3
 Uridine 260 (9) 3
 Uracil 267 (11) 4
Filter-paper extracts
 N-C-Aspartate 226 (5) 2
 Dihydroorotate 236 (16) 7
 Orotate 252 (4) 2
 Orotidine 251 (7) 3
 Uridine 257 (4) 2
 Uracil 257 (11) 4

(a) n = 5 for each method at each concentration.

(b) The same urine was used for both
direct injection and the filter-paper procedure.

(c) Urine without supplementation.

(d) Urine supplemented with low (10 mol/L), medium
(100 mol/L), or high (250 mol/L) concentrations
of relevant compounds.

(e) ND, not detected.

Table 3. Interassay variation for urines
and filter-paper extracts. (a)

 Blank (b,c)

Compound Mean (SD), CV, %
 mol/L
Urines
 N-C-Aspartate 2.5 (0.3) 11
 Dihydroorotate ND (e)
 Orotate 2.20 (0.12) 6
 Orotidine 2.8 (0.2) 6
 Uridine 0.5 (0.2) 33
 Uracil 7.9 (0.5) 6
Filter-paper extracts
 N-C-Aspartate 2.5 (0.3) 12
 Dihydroorotate ND
 Orotate 2.22 (0.11) 5
 Orotidine 2.77 (0.14) 5
 Uridine 0.52 (0.10) 20
 Uracil 7.36 (0.08) 1.1

 Low (b,d)

Compound Mean (SD), CV, %
 mol/L

Urines
 N-C-Aspartate 11.7 (0.6) 5
 Dihydroorotate 10.2 (0.4) 4
 Orotate 12.0 (0.2) 2
 Orotidine 12.4 (0.4) 3
 Uridine 10.0 (0.6) 6
 Uracil 18.0 (1.0) 6
Filter-paper extracts
 N-C-Aspartate 12.1 (1.1) 9
 Dihydroorotate 9.7 (0.4) 4
 Orotate 11.9 (0.3) 3
 Orotidine 12.3 (0.4) 4
 Uridine 9.9 (0.3) 3
 Uracil 17.4 (1.0) 6

 Medium (b,d)

Compound Mean (SD), CV, %
 mol/L

Urines
 N-C-Aspartate 97 (2) 2
 Dihydroorotate 104 (8) 5
 Orotate 103 (2) 2
 Orotidine 103 (2) 2
 Uridine 102 (4) 4
 Uracil 112 (4) 3
Filter-paper extracts
 N-C-Aspartate 99 (6) 6
 Dihydroorotate 101 (7) 6
 Orotate 101.3 (0.4) 0.4
 Orotidine 104 (0.3) 3
 Uridine 100 (2) 2
 Uracil 107.0 (1.1) 1

 High (b,d)

Compound Mean (SD), CV, %
 mol/L
Urines
 N-C-Aspartate 236 (5) 2
 Dihydroorotate 252 (14) 6
 Orotate 252 (6) 2
 Orotidine 258 (4) 2
 Uridine 254 (15) 6
 Uracil 268 (8) 3
Filter-paper extracts
 N-C-Aspartate 238 (18) 7
 Dihydroorotate 256 (17) 7
 Orotate 252 (6) 2
 Orotidine 255 (7) 3
 Uridine 251 (6) 2
 Uracil 266 (9) 3

(a) n = 5 for each method at each concentration.

(b) The same urine was used for both direct injection
and the filter-paper procedure.

(c) Urine without supplementation.

(d) Urine supplemented with low (10 mol/L), medium
(100 mol/L), or high (250 mol/L) concentrations of
relevant compounds.

(e) ND, not detected.

Table 4. Accuracy of measurement in
urines and filter-paper extracts. (a)

 Concentration
 in blank
 (0 mol/L), (b)
Compound [micro]mol/L
 Enriched urines
 N-C-Aspartate 3.1 (1.4-5.8)
 Dihydroorotate ND (e)
 Orotate 3.4 (2.1-5.9)
 Orotidine 3.7 (2.2-5.8)
 Uridine 2.1 (0.6-5.2)
 Uracil 24 (8.0-50)
Enriched filter-
paper extracts
 N-C-Aspartate 3.1 (1.4-5.8)
 Dihydroorotate ND
 Orotate 3.3 (2.1-5.7)
 Orotidine 3.8 (2.6-6.2)
 Uridine 2.0 (0.6-4.9)
 Uracil 21 (7.5-44)

 10 [micro]mol/L (c)

 Recovery,
Compound (d) % CV, %
 Enriched urines
 N-C-Aspartate 99 (7) 7
 Dihydroorotate 100 (4) 4
 Orotate 100 (3) 3
 Orotidine 97 (5) 5
 Uridine 102 (15) 14
 Uracil 106 (17) 16
Enriched filter-
paper extracts
 N-C-Aspartate 97 (11) 11
 Dihydroorotate 99 (5) 5
 Orotate 97 (3) 3
 Orotidine 98 (3) 4
 Uridine 100 (8) 8
 Uracil 115 (23) 20

 100 [micro]mol/L (c)

 Recovery,
Compound (d) % CV, %
 Enriched urines
 N-C-Aspartate 102 (5) 5
 Dihydroorotate 101 (6) 6
 Orotate 99 (3) 3
 Orotidine 98 (2) 2
 Uridine 103 (5) 5
 Uracil 98 (3) 3
Enriched filter-
paper extracts
 N-C-Aspartate 100 (6) 6
 Dihydroorotate 100 (6) 6
 Orotate 97 (2) 2
 Orotidine 99 (3) 3
 Uridine 103 (3) 3
 Uracil 102 (8) 8

 250 [micro]mol/L (c)

 Recovery,
Compound (d) % CV, %
 Enriched urines
 N-C-Aspartate 106 (9) 9
 Dihydroorotate 102 (5) 5
 Orotate 104 (3) 3
 Orotidine 102 (5) 5
 Uridine 105 (7) 7
 Uracil 103 (6) 6
Enriched filter-
paper extracts
 N-C-Aspartate 104 (9) 9
 Dihydroorotate 96 (2) 2
 Orotate 103 (5) 5
 Orotidine 102 (4) 4
 Uridine 104 (9) 9
 Uracil 103 (8) 8

(a) n = 5 samples for each procedure at each
concentration. The same enriched urines were
used for direct injection and for the
filter-paper procedure.

(b) Mean (range) of endogenous concentration of
compounds in urines (n = 5) used for enrichment.

(c) Concentration added to urines.

(d) Mean (SD).

(e) ND, not detected.

Table 5. Concentrations of the pyrimidine de novo
metabolites in urine from controls.

 Age, years

Compound,
 mol/mmol 0-2 2-6
creatinine (n = 52) (n = 43)
N-C-Aspartate
 Mean (SD) 1.2 (1.0) 0.7 (0.5)
 Range ND (a) to 3.8 ND to 3.0
Dihydroorotate
 Mean (SD) 0.01 (0.07) 0.01 (0.06)
 Range ND to 0.5 ND to 0.4
Orotate
 Mean (SD) 1.5 (1.2) 1.3 (0.8)
 Range ND to 6.2 0.1-3.8
Orotidine
 Mean (SD) 2.3 (1.0) 1.3 (0.8)
 Range ND to 4.8 ND to 4.2
Uridine
 Mean (SD) 0.6 (0.9) 0.4 (0.5)
 Range ND to 3.5 ND to 2.2
Uracil
 Mean (SD) 9.4 (7.9) 9.0 (4.3)
 Range ND to 29.6 2.9-22.7
Pyr total (b)
 Mean (SD) 15.0 (8.8) 12.7 (5.1)
 Range 0.7-39.7 5.8-27.1

 Age, years

Compound,
 mol/mmol 6-10 >10
creatinine (n = 20) (n = 40)
N-C-Aspartate
 Mean (SD) 0.6 (0.4) 0.4 (0.3)
 Range 0.2-1.9 ND to 1.6
Dihydroorotate
 Mean (SD) 0.02 (0.09) 0.01 (0.08)
 Range ND to 0.4 ND to 0.5
Orotate
 Mean (SD) 1.1 (0.8) 0.7 (0.5)
 Range 0.2-3.7 0.1-2.3
Orotidine
 Mean (SD) 0.9 (0.4) 0.8 (0.4)
 Range 0.4-2.3 ND to 1.8
Uridine
 Mean (SD) 0.1 (0.2) 0.3 (0.5)
 Range ND to 1.0 ND to 2.4
Uracil
 Mean (SD) 6.9 (4.3) 5.3 (4.3)
 Range 2.6-20.5 1.0-25
Pyr total (b)
 Mean (SD) 9.7 (4.9) 7.6 (4.3)
 Range 5.4-22.9 3.2-26

(a) ND, not detectable.

(b) Pyr total = N-C-aspartate + dihydroorotate + orotate
+ orotidine + uridine uracil.

Table 6. Pyrimidine de novo metabolites in urine of
patients with urea-cycle and pyrimidine degradation
defects.

 Patients Enzyme defect

 1 Ornithine transcarbamylase
 2 Ornithine transcarbamylase
 3 Ornithine transcarbamylase
 4 Argininosuccinase
 5 Dihydropyrimidine dehydrogenase
 6 Dihydropyrimidine dehydrogenase
 7 Dihydropyrimidine dehydrogenase
Controls
(n = 155)
 Mean (SD)
 Range

 Pyrimidine de novo metabolites,
 [micro]mol/mmol creatinine

 Patients N-C-Aspartate Dihydroorotate

 1 15 NDa
 2 34 0.6
 21 ND
 3 53 3.8
 4 36 4.7
 5 0.7 ND
 6 0.8 ND
 7 1.1 ND
Controls
(n = 155)
 Mean (SD) 0.8 (0.7) 0.01 (0.07)
 Range ND to 3.8 ND to 0.5

 Pyrimidine de novo metabolites,
 [micro]mol/mmol creatinine

 Patients Orotate Orotidine

 1 155 3
 2 823 102
 263 2
 3 235 3.2
 4 330 4.1
 5 0.8 0.7
 6 0.8 0.9
 7 1.1 3.4
Controls
(n = 155)
 Mean (SD) 1.2 (0.9) 1.4 (1.0)
 Range ND to 6.2 ND to 4.8

 Pyrimidine de novo metabolites,
 [micro]mol/mmol creatinine

 Patients Uridine Uracil

 1 2.6 288
 2 1.5 45
 46 231
 3 3.3 85
 4 5.1 5.6
 5 0.3 272
 6 0.4 333
 7 1.8 725
Controls
(n = 155)
 Mean (SD) 0.4 (0.7) 7.9 (6.0)
 Range ND to 3.5 ND to 30

(a) ND, not detected.
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Title Annotation:General Clinical Chemistry
Author:van Kuilenburg, Andre B.P.; van Lenthe, Henk; Loffler, Monika; van Gennip, Albert H.
Publication:Clinical Chemistry
Date:Nov 1, 2004
Words:5655
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