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Technical Briefs |
1 Analysis Center, Institute of Biomedicine, Tsinghua University, Beijing, China;2 XieHe Hospital, Beijing, China; 3 Capital Institute of Pediatrics, Beijing, China;
aaddress correspondence to this author at: Analysis Center, Institute of Biomedicine Tsinghua University, Beijing 100084, People’s Republic of China; fax 86-10-62781688, e-mail luoga{at}mail.tsinghua.edu.cn
Hyperuricemia, a serum urate concentration >0.45 mmol/L (7.0 mg/dL) in men and 0.36 mmol/L (6.0 mg/dL) in women, is the biochemical hallmark of gout (1)(2)(3), but many individuals with life-long hyperuricemia do not develop gouty arthritis (4). Conversely, serum urate concentrations may be within reference values in some patients with acute gout, particularly during the early phases of the disorder. We hypothesized that gout patients may have a different profile of purine precursors than do asymptomatic people with hyperuricemia and that concentrations of these compounds may reflect disorders of purine metabolism. To our knowledge, purine metabolites have not been studied and compared in gout and asymptomatic hyperuricemia.
With established methods for measuring purine compounds in blood and urine (5)(6)(7)(8)(9), retention time is not always adequate for identification of every peak because urine and blood usually contain many interfering compounds. Quantification by tandem mass spectrometry (MS/MS) (10)(11) may require an internal standard, which often is hard to obtain.
In this study of 10 purine compounds in the blood of gout patients and hyperuricemia patients with no gout symptoms, we used HPLC for serum sample separation, MS/MS for peak identification, and ultraviolet (UV) detection for quantification.
Purines were purchased from Fluka and Sigma. The HPLC-MS experiment was performed on an Agilent 1100 series liquid chromatograph with a mass spectrometric detector trap. Separation was carried out on a SupelcosilTM LC-18-DB column [25 cm x 4.6 mm (i.d.); 5-µm film thickness]. The column temperature was maintained at 25 °C. The mobile phases were as follows: 10 mmol/L ammonium acetate, adjusted to pH 6.5 with glacial acetic acid (eluant A), and methanol (eluant B). The elution gradient was as follows (flow rate, 1 mL/min): 0 to 10 min, 100% A; 10 to 16 min, 100% A to 92% A; 16 to 30 min, 92% A to 80% A; 30 to 45 min, 80% A to 0% A; 45 to 50 min, 0% A to 100% A; 50 to 60 min, equilibration with 100% A. All gradient steps were linear, and the total analysis time, including equilibration, was 60 min. The column eluate was monitored at 254 nm. A splitter was used between the HPLC column and the mass spectrometer, and 100–200 µL/min of eluate was introduced into the mass spectrometer. Negative electrospray ionization mode was used. Nitrogen was used as both nebulizing and collision gas, and capillary voltage was maintained at 3.5 kV. Other MS conditions were as follows: nebulizer, 25.0 psi; dry gas, 8.0 L/min; dry temperature, 325 °C; target mass, 200 m/z; compound stability, 60%; trap drive level, 50%; ICC target, 20 000. Auto-MS/MS was performed on molecular ions of all peaks to obtain their fragment information. Compound peaks were identified by their retention times and auto-MS/MS. Identification results for the molecular ions and the most abundant ions of 10 compounds are listed in Table 1
of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue9/. Quantification was based on UV detection at 254 nm.
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Blood samples were obtained from 49 untreated gout patients, 12 treated gout patients, 20 patients with untreated asymptomatic hyperuricemia, 13 patients with treated asymptomatic hyperuricemia, and 35 healthy controls. All patients and controls were males with no renal dysfunction. All treated patients were receiving 0.1 g/day allopurinol. All blood samples were centrifuged to obtain serum in the hospital and sent to our laboratory, where they were analyzed within 2 h or stored at –80 °C. Before analysis, 100 µL of serum was mixed with 500 µL of methanol for deproteinization, centrifuged at 14 800g for 10 min, dried with gentle nitrogen at 50 °C, and then mixed with 100 µL of water for HPLC-UV-MS/MS analysis.
The limits of detection and the calibration equations for 10 compounds are listed in Table 2 of the online Data Supplement. We determined the intraassay variation of the method (see Table 3 of the online Data Supplement) by measuring a serum sample 7 times and a serum sample enriched with synthetic compounds at low (100 µg/L), medium (500 µg/L), and high (5 mg/L) concentrations; we determined the interassay variation (see Table 4 of the online Data Supplement) by measuring a blank serum sample and serum samples enriched with synthetic compounds (500 µg/L) for 7 days. Recovery (see Table 5 of the online Data Supplement) was calculated based on measurements of 7 serum samples before and after enrichment with known concentrations of synthetic purines (100 µg/L, 500 µg/L, and 5 mg/L).
Retention times were used to identify each peak. To avoid problems caused by interference from other impurities with similar retention times and by retention time variations of the compounds under investigation, we used auto-MS/MS to identify each peak. The separation of pure purine compounds added to blank serum is shown in Fig. 1
. The results of HPLC separations of sera from gout patients and hyperuricemia patients are shown in Figs. 1 and 2, respectively, of the online Data Supplement. A buffer pH of 6.5 was chosen for the separation because the MS response of purine compounds decreased rapidly at a buffer pH >7, but retention times were too long at a buffer pH <6.5.
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The calibration curves for each compound were linear up to at least 50 mg/L (r2 >0.999). Except for uric acid, concentrations of purine metabolites in patients’ serum samples were in the linear part of the calibration curve. When necessary, samples were diluted to concentrations within the linear part of the curve and reanalyzed.
We used UV quantification for these analytes because the detection limits for this method were as low as those for MS with multiple reaction monitoring. The absorbance maxima for most purine compounds were between 245 and 260 nm (12); we therefore chose 254 nm as the detection wavelength.
Intraassay CVs were 1.0%–11% for nonenriched serum samples, 0.9%–9.2% for sera enriched with low concentrations, 0.6%–6.2% for sera enriched with medium concentrations, and 0.8%–2.3% for sera enriched with high concentrations. Interassay CVs were 3.7%–25% for nonenriched sera and 4.4%–19% for enriched sera. The recoveries were 84.5%–97% (CVs, 1.7%–7.1%) for sera enriched with 100 µg/L, 88%–97.4% (CVs, 1.2%–6.2%) for sera enriched with 500 µg/L, and 91%–98% (CVs, 0.9%–3.1%) for sera enriched with 5 mg/L.
The concentrations of purines in patients and controls are listed in Table 1
and in Table 6 of the online Data Supplement. Both gout patients and patients with asymptomatic hyperuricemia had higher uric acid, xanthine, and hypoxanthine concentrations than the healthy controls (P <0.01, Student t-test). Hypoxanthine and xanthine are both precursors of uric acid in purine metabolism; therefore, high uric acid concentrations affect the metabolism of xanthine and hypoxanthine, increasing their serum concentrations. Uric acid concentrations were similar in the 2 patient groups and therefore cannot be regarded as a single target marker for distinguishing between these 2 types of patients. Hypoxanthine and xanthine differed significantly, however, as did the ratios of hypoxanthine and xanthine to uric acid. Serum concentrations [mean (SD)] in 5 acute gout patients with no medical treatment were markedly high for hypoxanthine [80.7 (27.0) µmol/L] and xanthine [10.1 (1.6) µmol/L]. In 8 gout patients who reported frequently drinking and eating food high in purines, serum concentrations were 58.2 (18.5) µmol/L for hypoxanthine, 11.1 (3.4) µmol/L for xanthine, and 486 (81) µmol/L for uric acid, indicating that serum hypoxanthine and xanthine concentrations were also affected by intake of purine-containing foods. The concentrations in allopurinol-treated patients were similar to those in untreated patients, but both treated and untreated gout patients had lower uric acid and higher hypoxanthine and xanthine concentrations than did allopurinol-treated hyperuricemic patients, The differences in hypoxanthine and xanthine concentrations in gout patients compared with patients with asymptomatic hyperuricemia were not as large in treated as in untreated patients. Allopurinol-treated patients had lower uric acid, xanthine, and hypoxanthine concentrations, as expected from the action of allopurinol on purine metabolism. Other nucleosides and deoxynucleosides also differed to some degree in the 2 patient groups, although not as markedly as hypoxanthine and xanthine. Hypoxanthine and xanthine can thus be regarded as marker compounds for gout diagnosis.
The HPLC-UV-MS/MS method we describe is specific, simple, and inexpensive, requiring only a small volume of serum (100 µL) and easy sample preparation. Use of auto-MS/MS avoided errors in peak identification.
Acknowledgments
This study was supported by the "973" National Key Foundational Research Project of China (No. 2001CB510306).
References
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