Comprehensive detection of disorders of purine and pyrimidine metabolism by HPLC with electrospray ionization tandem mass spectrometry.
Pathways involved in 9 heritable metabolic disorders of purine metabolism and 7 heritable metabolic disorders of pyrimidine metabolism are depicted in Fig. 1 of the Data Supplement that accompanies the online version of this article at http:www.clinchem.org/content/vol52/issue6. Clinical manifestations of inherited defects of purine metabolism vary considerably, even among patients in the same family (2). The central nervous, renal, and hematologic systems are the most affected. Insoluble metabolites such as uric acid, xanthine, and 2,8-dihydroxyadenine cause urinary tract calculi and arthritis. The uric acid concentration is a useful diagnostic marker for deficiencies of phosphoribosyl pyrophosphate synthetase (PRPPS)  hypoxanthine guanine phosphoribosyl transferase (HGPRT), purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and molybdenum cofactor (2). Clinical features of pyrimidine degradation disorders are seizures and mental retardation (3). The key biochemical manifestation of all of these disorders is a change in the urinary excretion of purines or pyrimidines.
The method presented allows complete assessment of the urinary excretion of all relevant purines and pyrimidines, permitting the diagnosis of each of the disorders of purine and pyrimidine metabolism.
Materials and Methods
We purchased the following compounds from Sigma: adenine, adenosine, 2-deoxyadenosine, 2-deoxyguanosine, inosine, 2-deoxyinosine, xanthine, hypoxanthine, orotic acid, uric acid, thymine, uracil, dihydrothymine, dihydrouracil, N-carbamyl-[beta]-aminoisobutyric acid, N-carbamyl-[beta]-alanine, thymidine, uridine, pseudouracil, 5-hydroxymethyluracil, and 2-deoxyuridine. 2,8-Dihydroxyadenine was a kind gift of Ragnhild Seip (Oslo, Norway). We purchased guanosine from Acros Organics, HPLC-grade 1-propanol and formic acid from Merck, analytical-grade acetic acid from Roth, and ammonium hydroxide from Aldrich. Succinyladenosine was prepared according to the method of Jaeken and van den Berghe (4).
INTERNAL STANDARD MIXTURE
Stable-isotope-labeled reference compounds used as internal standards were purchased from Cambridge Isotope Laboratories. Unfortunately, such compounds were not available for all metabolites studied; therefore, each internal standard was selected based on its similarity to the corresponding metabolite in structure, retention time, and fragmentation pattern. To make stock solutions, we dissolved each component in 1-propanol-water (1:1 by volume).
An internal standard mixture consisting of 500 [micro]mol/L each of [8-[sup.13]C]adenine, [1'-[sup.13]C]adenosine, [1,3-[sup.15][N.sub.2]]orotic acid, [[sup.15]N]uracil, [d.sub.4]-thymine, [d.sub.6]-dihydrothymine, and 13C-labeled N-carbamyl-[beta]-alanine and 1000 [micro]mol/L d4dihydrouracil was prepared with eluent A of the HPLC method.
The control population consisted of 77 children aging from 2 months to 18 years. The urine specimens were collected in our hospital specifically for this study from completely healthy and nonsymptomatic volunteers. We investigated patient samples collected previously for laboratory tests and stored as positive controls in our hospital; the samples were from the following patients: a 6-year-old male patient with adenine phosphoribosyl transferase (APRT) deficiency; a 5-month-old male patient with HGPRT deficiency; a 1-month-old female patient with XDH deficiency; a 6-year-old male patient with PNP deficiency; an 8-month-old patient with adenosine deaminase (ADA) deficiency; a 5-year-old male patient with uridine monophosphate synthase (UMPS) deficiency; a 6-year-old male patient with adenylosuccinate lyase (ASL) deficiency; a 16-year-old male patient with thymidine phosphorylase (TP) deficiency; a 4-year-old male patient with (3-ureidopropionase (UP) deficiency; a 1-year-old patient with dihydropyrimidinase (DHP) deficiency; a 5-year-old male patient with dihydropyrimidine dehydrogenase (DPD) deficiency; and a 3-year-old male patient with molybdenum cofactor deficiency.
The sample collection for this study was approved by the ethics committee (ethics application number 071/ 2005; University Children's Hospital Heidelberg, Germany). Urine samples were stored at -20[degrees]C until analysis. Urinary creatinine concentrations were determined by the alkaline-creatinine-picrate method (5). Before analysis, urine samples were diluted to 0.5 mmol/L creatinine with eluent A and filtered with a centrifuge filter (Millipore) with a pore size of 0.1 [micro]m. We added 20 [micro]L of internal standard mixture to 180-[micro]L aliquots of urine or diluted urine and then injected 20 FxL of the prepared urine into an HPLC system with detection by electrospray ionization tandem mass spectrometry (ESI MS/MS).
HPLC-MS / MS
The HPLC system consisted of a Rheos 2000 quaternary pump and a vacuum degasser connected to a CTC pal autosampler. A reversed-phase column [Aqua C18 Minibore; 250 x 2.0 mm (i.d.); 5 [micro]m particle size; Phenomenex] protected by a guard column of the same material was used for chromatography. Column temperature was maintained at 23[degrees]C. For separation of the compounds, the following eluents were used: eluent A consisted of 0.05 mol/L acetic acid, adjusted to pH 4 with 250 g/L NH40H and adjusted again to pH 2.8 with undiluted formic acid; eluent B was a mixture of solvent A and methanol (1:1 by volume). A flow rate of 100 [micro]L/min was applied. The following linear elution gradient was used: 0-2 min, 100% A; 2-10 min, 100% A to 0% A; 10-11 min, 0% A; 11-11.5 min, 0% to 100% A. Between runs, the system was reequilibrated with 100% A for 8.5 min. HPLC ESI MS/MS analysis required 20 min.
A Quattro Ultima tandem mass spectrometer (Micromass) was used. The purines (except 2-deoxyadenosine), orotic acid, pseudouridine, uridine, 2-deoxyuridine, 5-hydroxymethyluracil, and thymidine were ionized in negative ionization mode. The pyrimidine degradation products and 2-deoxyadenosine were positively ionized. Nitrogen served as desolvation, nebulizing, and cone gas, and argon was used as collision gas. Cell pressure was 0.153 Pa. The optimum source temperature was 100[degrees]C. Compounds were analyzed in multiple-reaction-monitoring experiments, in which preselected ion pairs were entered. Ion pairs, cone voltages, and collision energies are summarized in Table 1.
The specific tandem mass spectrometric conditions for each compound were optimized by use of a 100 [micro]mol/L stock solution of each metabolite. A flow rate of 10 [micro]L/min was chosen.
Linearities and detection limits were determined by injection of urine samples enriched with different metabolite concentrations. Linearity curves for analyte peak area/internal standard peak area plotted vs standard concentration were generated. The slopes and intercepts of the linearity curves were used for analyte quantification. The detection limit was defined as the lowest concentration that gave a signal-to-noise ratio of 3.
To establish the interday variation, we analyzed a blank urine sample plus 3 urine samples enriched with low (10 [micro]mol/L), medium (50 [micro]mol/L), and high concentrations (100 [micro]mol/L) of the metabolites 10 times within 1 day. For uric acid, higher concentrations (200 and 400 [micro]mol/L) were chosen because urinary concentrations are higher. For succinyladenosine and 2,8-dihydroxyadenine, lower concentrations (7.5 and 35 [micro]mol/L and 35 and 70 [micro]mol/L, respectively) were selected. For determination of interday variation, urine samples with the same concentration as for the evaluation of the interday variation were analyzed in duplicate on 7 separate days.
We evaluated recoveries by analyzing 8 different urine samples in duplicate before and after enrichment with 25 and 75 j,mol/L of purine and pyrimidine metabolites. We determined the recoveries of succinyladenosine and 2,8-dihydroxyadenenine by measuring 4 different urine samples enriched with these analytes at 15 and 35 [micro]mol / L.
We investigated the influence of urine pH by adjusting the pH to between 3 and 9 in 4 separate urine samples. Before analysis, all 4 samples were enriched with identical concentrations of purines and pyrimidines. The resulting concentrations were compared with each other and also with the added concentrations.
To estimate reference intervals, we assayed urine samples from 77 healthy children from 2 months to 18 years of age. Age- and sex-related concentration dependence was investigated by a 5-step procedure. First, analysis of covariance (ANCOVA) was performed for each detectable metabolite as the dependent variable with sex as an independent variable and age as a covariate. Results revealed no primary effect for sex and no interaction for age by sex for any of the metabolites. Significant age effects were observed for 7 of 22 metabolites (see Table 5).
Visual partitioning of the raw data plotted by age was performed for those metabolites showing a significant result (step 2; see cutoff age in Table 5). Next (step 3) we tested the log(x + 1)-transformed distributions below and above the cutoff ages, using the algorithm published by Lahti et al. (6). The log(x + 1) transformation was used to handle the problem of log-transformation of zero values for certain metabolites. Decision for partitioning was based on R values as the ratio of the SDs of the larger by the smaller age distribution and the differences D (measured in SD units) between the age distributions at their lower ([D.sub.Lower limit]) and upper ([D.sub.upper limit]) tails. This procedure corroborated the visual age partitioning for all metabolites. In step 4, we calculated the 95% confidence intervals of the means according to the modified Cox algorithm (7). Finally (step 5), we calculated the reference intervals for all metabolites by back-transforming the logarithmic confidence intervals to the original scale. Readers should be aware that the back-transformed variables are geometric means (as estimators of the median) and the multiplicative SD (instead of the additive SD) (8). Shown in Table 5 are the means, SDs, and reference intervals. All statistical calculations were performed with the "R software environment for statistical computing" (9).
To compare chromatograms obtained for patient urine samples with those from healthy individuals, we analyzed urine samples from patients in whom a metabolic disorder had been confirmed previously. To mimic urine of patients with PRPPS superactivity, urines were enriched with hypoxanthine (100-120 mmol/mol creatinine), uric acid (>1900 mmol/mol creatinine), and xanthine (100-120 mmol/mol creatinine) (10).
The MS/MS conditions (ionization mode, ion pairs, cone voltage, and collision energy) for each metabolite are shown in Table 1. Signal intensities and retention times of the purines and pyrimidines are presented in Fig. 2 of the online Data Supplement for negatively ionized compounds and in Fig. 3 of the online Data Supplement for positively ionized metabolites. Each metabolite displayed specific ion pairs and characteristic retention times (Table 1). Optimal peak shapes were achieved for all analytes except for dihydrouracil and N-carbamyl-[beta]-alanine, which were characterized by broader and split peaks. Uracil had less response than the other metabolites.
Interferences between the metabolites in this method were overcome by HPLC separation and elution at different times. Adenosine, uridine, and thymidine produced ions that corresponded to the ion pairs of adenine, uracil, and thymine, respectively. The HPLC column separated the metabolites in the urine samples from salt, which would cause quenching of metabolite signals in the ion source of the tandem mass spectrometer.
Ion pairs for creatinine and its internal standard were also included in the method to ensure correct dilution of the sample (data not shown). This ratio was checked before quantification.
LINEARITY AND DETECTION LIMITS
The ranges of linearity and the detection limits for each metabolite and its corresponding internal standard are presented in Table 2. Eight internal standards were used for the quantification of 24 purines and pyrimidines. Several purines and pyrimidines were quantified up to 600 mmol/mol creatinine (300 [micro]mol/L). Uridine and 2-deoxyuridine were quantified up to 400 mmol/mol creatinine (200 [micro]mol/L). 2-Deoxyadenosine, 2,8-dihydroxyadenine, and N-carbamyl-(3-amino-isobutyric acid were quantifiable up to 200 mmol/mol creatinine (100 [micro]mol/L) and succinyladenosine up to 140 mmol/mol creatinine (70 [micro]mol/L). Uric acid was quantified from 200 to 1600 mmol/mol creatinine (100-800 [micro]mol/L). For sample metabolite concentrations higher than the upper limit of linearity, we diluted the sample to a concentration within the linear range and reanalyzed.
For xanthine, adenosine, 2,8-dihydroxyadenine, uracil, thymine, dihydrouracil, dihydrothymine, N-carbamyl-[beta]-amino-isobutyric acid, N-carbamyl-[beta]-alanine, uridine, and pseudouridine, the detection limit was between 1 and 10 [micro]mol/L. For all other metabolites, the detection limit was <0.2 [micro]mol/L. Patient urine samples with decreased concentrations of these metabolites (e.g., uric acid in XDH, molybdenum cofactor, or PNP deficiency) were successfully identified with this method.
The intraday variations for the various metabolites are presented in Table 3. The mean CV for urine samples with 50 [micro]mol/L added standard solution (200 [micro]mol/L for uric acid, 35 [micro]mol/L for succinyladenosine and 2,8-dihydroxyadenine) was 12%. Mean recovery was 106%. In addition to the data shown in Table 3, intraday CVs were 7%-20% for urines enriched with 10 [micro]mol/L each metabolite except for hypoxanthine, uracil, and pseudouridine, which had CVs of 31%, 28%, and 27%, respectively. The mean recovery was 104%. CVs were 6%-24% for urines enriched with 100 [micro]mol/L of each metabolite. Dihydrouracil was an exception; its CV was higher, at 42%. Dihydrouracil eluted in a split peak, making integration difficult, and showed variable background, which led to an increased CV value. Mean recovery for this concentration was 102%.
Data for the interday variation are presented in Table 3. The interday CVs were [less than or equal to]17% for urines enriched with 50 [micro]mol/L for all metabolites except uric acid (200 [micro]mol/L), succinyladenosine (30 [micro]mol/L), and 2,8-dihydroxyadenine (35 [micro]mol/L). The only higher CV was 25% for 2,8-dihydroxyadenine. Recoveries were 92%-125%. In addition, urines enriched with lower concentrations (10 [micro]mol/L) had CVs of 8%-31%. The mean recovery for these less concentrated urines was 101%. The CVs and recoveries for urine samples enriched with 100 [micro]mol/L of each metabolite were 3%-12% and 95%-109%, respectively.
We analyzed 8 different urine samples before and after addition of known metabolite concentrations. The mean recoveries and CVs are listed in Table 4. Recoveries ranged from 85% to 123% with the exceptions of 2-deoxyinosine, 2-deoxyadenosine, and thymidine, for which recoveries were 133%, 128%, and 128%, respectively. The CVs were 9%-24% for all metabolites except for hypoxanthine, thymine, and thymidine, which had CVs of 34%,28%, and 33%, respectively. We also determined the recoveries for lower concentrations (25 and 150 [micro]mol/L for uric acid). The mean recovery was 111%, and the mean CV was 19%.
We investigated carryover by assaying an unenriched urine sample after assaying urine samples with added metabolites. No carryover was detected.
To investigate the influence of pH, we analyzed 4 enriched urine samples with different pH values in the range of 3-9. No differences in concentrations or peak shapes were detected.
REFERENCE INTERVALS AND CONTROL RANGES
The reference intervals for all of the metabolites are listed in Table 5. For xanthine, hypoxanthine, inosine, uracil, N-carbamyl-[beta]-alanine, N-carbamyl-[beta]-aminoisobutyric acid, and pseudouridine, we found that the reference intervals were age dependent, whereas the other metabolites showed no age dependence. For 2-deoxyinosine, adenosine, 2-deoxyadenosine, guanosine, 2-deoxyguanosine, succinyladenosine, orotic acid, thymine, dihydrouracil, dihydrothymine, uridine, 2-deoxyuridine, and 5-hydroxymethyluracil, sample concentrations were below the lower limits of quantification, as were the concentrations of inosine and N-carbamyl-[beta]-aminoisobutyric acid for children older than 1 year and N-carbamyl-[beta]-alanine for children older than 4 years of age. Upper reference limits were defined by the maximum measured concentration in the first age group for inosine, N-carbamyl-[beta]-alanine, and N-carbamyl-[beta]-aminoisobutyric acid. The reference sample concentrations of these 3 metabolites were partly below the lower limits of quantification, and no mean could be calculated. 2,8-Dihydroxyadenine and thymidine were not detectable in urine from healthy persons.
SAMPLES FROM PATIENTS WITH CONFIRMED
Urine samples from patients with confirmed diagnoses [deficiencies of APRT, ADA, PNP, UMPS, ASL, TP, UP, DHP, DPD, XDH, molybdenum cofactor, and HGPRT] were analyzed. The chromatograms (Fig. 1) illustrated major differences from those of urine specimens from healthy individuals. In each case, the correct diagnosis was readily apparent. The urine sample from the patient with APRT deficiency showed a peak corresponding to 2,8-dihydroxyadenine and contained some unidentified compounds that did not interfere with the recognition and quantification of 2,8-dihydroxyadenine. There were also unidentified peaks in the chromatogram of adenosine in the urine sample from the patient with ADA deficiency. In the sample from the patient with DHP deficiency, there were unidentifiable peaks in the chromatograms of dihydrouracil and dihydrothymine.
[FIGURE 1 OMITTED]
Deficiencies in the enzymes involved in purine and pyrimidine metabolism lead to nonspecific, mostly neurologic, symptoms, e.g., mental retardation, seizures, muscular hypotonia, or urinary tract calculi. Methods are needed to identify patients with these metabolic abnormalities in the broad population of patients displaying such symptoms. Liquid chromatography-MS/MS targets specific components of complex mixtures with greater efficiency than previously available methods. The use of internal standards augments specificity.
Existing methods for the analysis of purines and pyrimidines are gas chromatography-MS, which involves time-consuming sample preparation (11,12), and reversed-phase HPLC coupled with ultraviolet detection, which requires an analytical run time of -30 min and is more susceptible to disruptive elements in the urinary matrix. A proton nuclear magnetic resonance (1H-NMR) spectrometric method can be used to measure many compounds of purine and pyrimidine metabolism (13), but it has a major disadvantage in that it fails to detect uric acid and 2,8-dihydroxyadenine, which are very useful markers for the diagnosis of APRT deficiency, XDH deficiency, molybdenum cofactor deficiency, PRPPS super-activity, and HGPRT deficiency. Capillary electrophoresis can also be used to measure many analytes, but it requires time-consuming sample preparation (14).
MS/MS is a robust clinical laboratory technique in which cleaning and maintenance procedures between analyses are reduced. The applied Z-spray reduces contamination in the tandem mass spectrometer; therefore, less cleaning is required. Higher sample throughput is also possible. A method is available that uses atmospheric pressure chemical ionization MS/MS (15) with ion-exchange purification and an evaporation step as sample preparation, making the method very time-consuming. It also does not include any pyrimidine degradation products.
Three HPLC ESI MS/MS methods have been described for which sample preparation is simple (16-18), but to date, our method is the only one that provides quantification of purines, pyrimidines, and pyrimidine degradation products and requires minimum sample preparation while enabling complete analysis of relevant purine and pyrimidine metabolites in 1 analytical run. An additional advantage over previous methods is the inclusion of uric acid and 2,8-dihydroxyadenine because hyper- and hypouricemia are important in establishing diagnoses of 5 different disorders of purine metabolism (2).
Because of its polar endcapping, the Aqua HPLC column used for separation of metabolites retains basic compounds more effectively than conventional [C.sub.18] columns. The combination of positive and negative ionization enables analysis of purines and pyrimidines in 1 analytical run. Uracil and thymine are also negatively ionizable (16), but in our experience the signal intensities are much higher with positively charged parent ions. No interferences of purines and pyrimidines were encountered, whereas with a previously reported method (16), dihydropyrimidines and N-carbamyl compounds could not be measured without affecting the measurement of other metabolites of the purine and pyrimidine pathways. We also did not observe any interference of 5,6-dihydrouridine with 5,6-dihydrouracil, as described previously (19). With our method, no spontaneous fragmentation of 5,6-dihydrouridine in the ion source was identified (see Fig. 4 in the online Data Supplement).
In our method, the HPLC column was necessary to separate the metabolites from unidentifiable peaks. Every metabolite was analyzed with its own specific ion pair, only adenosine, uridine, and thymidine also showed the same ion pairs as adenine, uracil, and thymine because of spontaneous fragmentation in the ion source.
The linearity ranges apply for concentrations within the pertinent reference intervals as well as pathologic concentration ranges of 2-600 mmol/mol creatinine for purines and 10-600 mmol/mol creatinine for pyrimidines. Dihydrouracil is quantifiable at 100-600 mmol/ mol creatinine. The lower quantification limit of 100 mmol/mol creatinine is acceptable because concentrations in patients with DHP deficiency are usually 150-630 mmol/mol creatinine (10). The method also permits the detection of decreases in uric acid and pseudouridine concentrations.
Succinyl-5-amino-4-imidazole carboxamide riboside (SAICAR) and succinyladenosine are the 2 metabolites associated with adenylosuccinate lyase deficiency (20). Succinyladenosine was readily detectable with this method, but SAICAR was not ionizable or became instable in the ion source. SAICAR can be detected by the modified Bratton-Marshall test (21). Succinyladenosine concentrations are higher in the milder form of the disease (20).
5-Amino-4-imidazolecarboxamide ribosiduria is detectable by a combination of the modified Bratton-Marshall test and our new approach. Succinyladenosine is also excreted in concentrations that are increased but lower than those found in ASL-deficient patients (22).
In conclusion, our method allows rapid, specific, and reliable screening for defects in the purine and pyrimidine pathways. This method can be used to correctly diagnose deficiencies of APRT, ADA, PNP, UMPS, ASL, TP, DPD, DHP, UP, XDH, molybdenum cofactor, PRPPS, and HGPRT.
We thank Dr. B. Assmann (University Children's Hospital Dusseldorf, Germany), Dr. G. Kutschke (University Children's Hospital Mainz, Germany), and Dr. G. Seidlitz (University Children's Hospital Greifswald, Germany) for sending samples from patients with confirmed disorders of purine and pyrimidine metabolism. We also thank A. Anninos for technical assistance. Finally, we thank the editors and reviewers for their comments and fruitful discussions.
Received August 12, 2005; accepted March 24, 2006.
Previously published online at DOI: 10.1373/clinchem.2005.058842
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SUSEN HARTMANN,  * JURGEN G. OKUN,  CHRISTIANE SCHMIDT,  CLAUS-DIETER LANGHANS,  SVEN F. GARBADE,  PETER BURGARD,  DOROTHEA HAAS,  John OLIVER SASS  WILLIAM L. NYHAN  and GEORG F. HOFFMANN 
 Division of Metabolic Diseases, Department of General Pediatrics, University Children's Hospital Heidelberg, Heidelberg, Germany.
 Laboratory of Metabolism, Department of General Pediatrics and Adolescent Medicine, University Children's Hospital Freiburg, Freiburg, Germany.
 University of California, San Diego, La Jolla, CA.
* Address correspondence to this author at: Division of Metabolic Diseases, Department of General Pediatrics, University Children's Hospital Heidelberg, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany. Fax 49-6221-564069; e-mail Susen.Hartmann@med.uni-heidelberg.de.
 Nonstandard abbreviations: PRPPS, phosphoribosyl pyrophosphate synthetase; HGPRT, hypoxanthine guanine phosphoribosyl transferase; PNP, purine nucleoside phosphorylase; XDH, xanthine dehydrogenase; APRT, adenine phosphoribosyl transferase; ADA, adenosine deaminase; UMPS, uridine monophosphate synthase; ASL, adenylosuccinate lyase; TP, thymidine phosphorylase; UP, [beta]-ureidopropionase; DHP, dihydropyrimidinase; DPD, dihydropyrimidine dehydrogenase; ESI MS/MS, electrospray ionization tandem mass spectrometry; and SAICAR, succinyl-5-amino-4-imidazole carboxamide riboside.
Table 1. Summary of ionization mode, ion pairs, cone voltages, and collision energies. Ionization Parent Daughter Metabolite mode ion, m/z ion, m/z Purines Adenine - 134 107 Xanthine - 151 108 Hypoxanthine - 135 92 Inosine - 267 135 2-Deoxyinosine - 251 135 Adenosine - 312 134 2-Deoxyadenosine + 252 136 Guanosine - 282 150 2-Deoxyguanosine - 266 150 Uric acid - 168 124 2,8-Dihydroxyadenine - 166 123 Succinyladenosine - 382 134 Pyrimidines Orotic acid - 155 111 Uracil + 113 96 Thymine + 127 110 Dihydrouracil + 115 73 Dihydrothymine + 129 69 N-Carbamyl-[beta]-alanine + 133 115 N-Carbamyl-[beta]- + 147 86 aminoisobutyric acid Pseudouridine - 243 153 Uridine - 243 200 2-Deoxyuridine - 227 184 5-Hydroxymethyluracil - 141 123 Thymidine - 241 151 Cone Collision Metabolite voltage, V energy, eV Purines Adenine 40 20 Xanthine 40 20 Hypoxanthine 40 20 Inosine 40 20 2-Deoxyinosine 40 20 Adenosine 40 15 2-Deoxyadenosine 35 24 Guanosine 40 16 2-Deoxyguanosine 40 16 Uric acid 40 20 2,8-Dihydroxyadenine 40 20 Succinyladenosine 35 30 Pyrimidines Orotic acid 40 20 Uracil 35 15 Thymine 35 15 Dihydrouracil 35 15 Dihydrothymine 35 15 N-Carbamyl-[beta]-alanine 35 10 N-Carbamyl-[beta]- 35 15 aminoisobutyric acid Pseudouridine 35 10 Uridine 35 8 2-Deoxyuridine 35 10 5-Hydroxymethyluracil 35 13 Thymidine 35 10 Table 2. Summary of corresponding internal standards, range of linearity, and detection limits. Linearity, mmol/mol Metabolite Internal standard creatinine Purines Adenine [8-[sup.13]C Adenine 2-600 Xanthine [8-[sup.13]C Adenine 2-600 Hypoxanthine [8-[sup.13]C Adenine 4-600 Inosine [8-[sup.13]C Adenine 2-600 2-Deoxyinosine [8-[sup.13]C Adenine 2-600 Adenosine [1'-[sup.13]C Adenosine 2-600 2-Deoxyadenosine [1'-[sup.13]C Adenosine 2-200 Guanosine [1'-[sup.13]C Adenosine 2-600 2-Deoxyguanosine [1'-[sup.13]C Adenosine 2-600 Uric acid [8-[sup.13]C Adenine 200-1600 2,8-Dihydroxyadenine [8-[sup.13]C Adenine 2-200 Succinyladenosine [8-[sup.13]C Adenine 10-140 Pyrimidines Orotic acid [1,3-[sup.15] [N.sub.2] 2-600 Orotic acid Uracil [sup.15][N.sub.2] Uracil 5-600 Thymine [d.sub.4]-Thymine 10-600 Dihydrouracil [d.sub.4]-Dihydrouracil 100-600 Dihydrothymine [d.sub.6]-Dihydrothymine 10-600 N-Carbamyl-[beta]-alanine [sup.13]C -N-Carbamyl- 10-600 [beta]-alanine N-Carbamyl-[beta]- [sup.13]C -N-Carbamyl- 10-200 aminoisobutyric acid [beta]-alanine Pseudouridine [1'-[sup.13]C Adenosine 10-600 Uridine [1'-[sup.13]C Adenosine 10-400 2-Deoxyuridine [1'-[sup.13]C Adenosine 2-400 5-Hydroxymethyluracil [sup.15][N.sub.2] Uracil 10-600 Thymidine [1'-[sup.13]C Adenosine 10-600 Detection limit, Metabolite r mol/L Purines Adenine 0.98 0.1 Xanthine 0.99 2 Hypoxanthine 0.99 0.02 Inosine 0.97 0.1 2-Deoxyinosine 0.99 0.02 Adenosine 0.99 2 2-Deoxyadenosine 0.93 0.02 Guanosine 0.99 0.02 2-Deoxyguanosine 0.99 0.2 Uric acid 0.97 0.2 2,8-Dihydroxyadenine 0.98 1 Succinyladenosine 0.98 0.2 Pyrimidines Orotic acid 0.95 0.2 Uracil 0.99 5 Thymine 0.96 5 Dihydrouracil 0.98 10 Dihydrothymine 0.98 5 N-Carbamyl-[beta]-alanine 0.99 5 N-Carbamyl-[beta]- 0.92 5 aminoisobutyric acid Pseudouridine 0.96 1 Uridine 0.98 2 2-Deoxyuridine 0.96 0.2 5-Hydroxymethyluracil 0.98 0.1 Thymidine 0.93 0.2 Table 3. Intra- and interday variations for urines with metabolites added at known concentration. (a) Intraday variation (n = 10) Mean Added measured concentration, concentration, Metabolite [micro]mol/L [micro]mol/L Purines Adenine 50 49 Xanthine 50 56 Hypoxanthine 50 46 Inosine 50 57 2-Deoxyinosine 50 50 Adenosine 50 52 2-Deoxyadenosine 50 52 Guanosine 50 62 2-Deoxyguanosine 50 58 Uric acid 200 245 2,8-Dihydroxyadenine 35 30 Succinyladenosine 35 32 Pyrimidines Orotic acid 50 50 Uracil 50 54 Thymine 50 44 Dihydrouracil 50 49 Dihydrothymine 50 58 N-Carbamyl-[beta]- 50 52 alanine N-Carbamyl-[beta]- 50 65 aminoisobutyric acid Pseudouridine 50 50 Uridine 50 60 2-Deoxyuridine 50 49 5-Hydroxymethyluracil 50 56 Thymidine 50 51 Mean, (b) CV, Metabolite % % Purines Adenine 97 8 Xanthine 112 9 Hypoxanthine 95 10 Inosine 114 8 2-Deoxyinosine 100 8 Adenosine 105 13 2-Deoxyadenosine 104 8 Guanosine 123 16 2-Deoxyguanosine 116 19 Uric acid 122 18 2,8-Dihydroxyadenine 88 19 Succinyladenosine 91 17 Pyrimidines Orotic acid 100 24 Uracil 108 10 Thymine 89 11 Dihydrouracil 97 17 Dihydrothymine 118 7 N-Carbamyl-[beta]- 104 8 alanine N-Carbamyl-[beta]- 131 8 aminoisobutyric acid Pseudouridine 100 14 Uridine 120 14 2-Deoxyuridine 98 14 5-Hydroxymethyluracil 112 10 Thymidine 103 8 Interday variation (n = 7) Mean Added measured concentration, concentration, Metabolite [micro]mol/L [micro]mol/L Purines Adenine 50 54 Xanthine 50 57 Hypoxanthine 50 52 Inosine 50 60 2-Deoxyinosine 50 55 Adenosine 50 57 2-Deoxyadenosine 50 59 Guanosine 50 60 2-Deoxyguanosine 50 62 Uric acid 200 246 2,8-Dihydroxyadenine 35 40 Succinyladenosine 30 31 Pyrimidines Orotic acid 50 54 Uracil 50 47 Thymine 50 50 Dihydrouracil 50 52 Dihydrothymine 50 55 N-Carbamyl-[beta]- 50 50 alanine N-Carbamyl-[beta]- 50 57 aminoisobutyric acid Pseudouridine 50 46 Uridine 50 55 2-Deoxyuridine 50 50 5-Hydroxymethyluracil 50 50 Thymidine 50 54 Mean, (b) CV, Metabolite % % Purines Adenine 108 7 Xanthine 114 11 Hypoxanthine 104 16 Inosine 120 11 2-Deoxyinosine 110 9 Adenosine 115 13 2-Deoxyadenosine 118 14 Guanosine 120 9 2-Deoxyguanosine 125 17 Uric acid 123 6 2,8-Dihydroxyadenine 113 25 Succinyladenosine 102 13 Pyrimidines Orotic acid 107 12 Uracil 94 13 Thymine 100 17 Dihydrouracil 104 11 Dihydrothymine 110 15 N-Carbamyl-[beta]- 101 7 alanine N-Carbamyl-[beta]- 113 10 aminoisobutyric acid Pseudouridine 92 9 Uridine 109 10 2-Deoxyuridine 99 9 5-Hydroxymethyluracil 100 8 Thymidine 109 8 (a) The blank sample was subtracted from the enriched urines and is not presented in the table. (b) Measured concentration as a percentage of the added concentration. Table 4. Mean recoveries and CVs for urine samples (n = 8). (a) Added Recovery concentration, Mean, Metabolite [micro]mol/L [micro]mol/L Purines Adenine 75 78 Xanthine 75 86 Hypoxanthine 75 81 Inosine 75 64 2-Deoxyinosine 75 100 Adenosine 75 68 2-Deoxyadenosine 75 96 Guanosine 75 76 2-Deoxyguanosine 75 90 Uric acid 300 278 2,8-Dihydroxyadenine 35 38 Succinyladenosine 15 18 Pyrimidines Orotic acid 75 72 Uracil 75 68 Thymine 75 67 Dihydrouracil 75 80 Dihydrothymine 75 68 N-Carbamyl-[beta]-alanine 75 79 N-Carbamyl-[beta]- 75 69 aminoisobutyric acid Pseudouridine 75 82 Uridine 75 90 2-Deoxyuridine 75 83 5-Hydroxymethyluracil 75 65 Thymidine 75 96 Recovery Metabolite Mean, % CV, % Purines Adenine 104 9 Xanthine 115 18 Hypoxanthine 108 34 Inosine 85 18 2-Deoxyinosine 133 17 Adenosine 91 11 2-Deoxyadenosine 128 10 Guanosine 101 18 2-Deoxyguanosine 120 16 Uric acid 93 14 2,8-Dihydroxyadenine 109 16 Succinyladenosine 123 9 Pyrimidines Orotic acid 91 11 Uracil 91 11 Thymine 89 28 Dihydrouracil 107 17 Dihydrothymine 91 15 N-Carbamyl-[beta]-alanine 105 15 N-Carbamyl-[beta]- 92 24 aminoisobutyric acid Pseudouridine 109 21 Uridine 120 20 2-Deoxyuridine 111 15 5-Hydroxymethyluracil 87 23 Thymidine 128 33 (a) Blank samples were subtracted from enriched urines and are not presented in the table. Table 5. Reference intervals for purines and pyrimidines by age based on 95% confidence intervals of the mean (n = 77). Age, Metabolite Controls, n years Purines Adenine 77 0-18 Xanthine 17 [less than or equal to] 1 60 >1 Hypoxanthine 17 [less than or equal to] 1 60 >1 Inosine 17 [less than or equal to] 1 59 >1 2-Deoxyinosine 77 0-18 Adenosine 77 0-18 2-Deoxyadenosine 77 0-18 Guanosine 77 0-18 2-Deoxyguanosine 77 0-18 Uric acid 76 0-18 2,8-Dihydroxyadenine 77 0-18 Succinyladenosine 77 0-18 Pyrimidines Orotic acid 77 0-18 Uracil 21 [less than or equal to] 4 48 >4 Thymine 77 0-18 Dihydrouracil 77 0-18 Dihydrothymine 77 0-18 N-Carbamyl-[beta]- 25 [less than or equal to] 4 alanine 51 >4 N-Carbamyl-[beta]- 17 [less than or equal to] 1 aminoisobutyric acid 60 >1 Pseudouridine 17 [less than or equal to] 1 60 >1 Uridine 77 0-18 2-Deoxyuridine 77 0-18 5-Hydroxymethyluracil 77 0-18 Thymidine 77 0-18 Reference interval, Metabolite Mean (a) SD (a) mmol/mol creatinine Purines Adenine 8.54 0.19 8-9 Xanthine 13.85 1.09 10-34 9.29 0.74 9-14 Hypoxanthine 10.94 1.34 7-34 7.7 0.95 7-13 Inosine -(b) <6 (c) -(d) <2 (e) 2-Deoxyinosine -(d) <2 (e) Adenosine -(d) <2 (e) 2-Deoxyadenosine -(d) <2 (e) Guanosine -(d) <2 (e) 2-Deoxyguanosine -(d) <2 (e) Uric acid 443.4 0.95 443-695 2,8-Dihydroxyadenine ND (f) Succinyladenosine -(d) <10 (e) Pyrimidines Orotic acid -(d) <2 (e) Uracil 10.65 0.81 8-20 6.04 0.90 6-10 Thymine -(d) <10 (e) Dihydrouracil -(d) <100 (e) Dihydrothymine -(d) <10 (e) N-Carbamyl-[beta]- -(b) <19 (c) alanine -(d) <10 (e) N-Carbamyl-[beta]- -(b) <17 (c) aminoisobutyric acid -(d) <10 (e) Pseudouridine 83.85 0.70 64-145 21.91 0.71 21-31 Uridine -(d) <10 (e) 2-Deoxyuridine -(d) <2 (e) 5-Hydroxymethyluracil -(d) <10 (e) Thymidine ND (a) Geometric mean as an estimator of the mean and multiplicative SD back-transformed from logarithmic parameters. For detailed explanation, see the text. (b) Calculation of the mean was not possible; concentrations of most reference samples were below the lower limit of quantification. (c) Upper limit of the reference interval was defined by the maximum measured concentration. (d) Calculation of the mean was not possible; concentrations of all reference samples were below the lower limit of quantification. (e) Upper limit of the reference interval was defined by the lower limit of quantification. (f) ND, not detectable.
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|Title Annotation:||Endocrinology and Metabolism|
|Author:||Hartmann, Susen; Okun, Jurgen G.; Schmidt, Christiane; Langhans, Claus-Dieter; Garbade, Sven F.; Bur|
|Date:||Jun 1, 2006|
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