Abstract
Background
Hyperuricemia is the only biochemical index in the classification of acute gouty arthritis in American Rheumatism Association 1977 and the main basis of clinical diagnosis for most doctors. However, nearly half of the time gout occurs without hyperuricemia, especially in an acute attack，which leads to an urgent need to find a new substitute diadynamic criteria of gout. Xanthine and hypoxanthine, as precursors of uric acid, have been reported to be high in gout patients with hyperuricemia and presumed to be gout biomarkers.
Objectives
To further explore the possibility of xanthine and hypoxanthine to be gout biomarkers as substitutes for uric acid.
Methods
A reversed‐phase HPLC‐UV method was employed for simultaneous quantitative detection of uric acid (UA), xanthine (X), and hypoxanthine (HX) in gout patients’ (with and without hyperuricemia) and healthy persons’ serum.
Results
The xanthine and hypoxanthine concentrations in gout patients with hyperuricemia and without hyperuricemia are higher than in healthy persons with a P < 0.001.
Conclusions
This study supplements previous researches by confirming that xanthine and hypoxanthine are significantly elevated in gout patients’ serum especially in patients’ with normouricemia, which supported xanthine and hypoxanthine may have clinical application for the diagnosis of gout.
Keywords: diagnosis, gout, hyperuricemia, hypoxanthine, xanthine
1. INTRODUCTION
Gout is a common arthritis caused by deposition of monosodium urate (MSU) crystals within joints and is always associated with hyperuricemia.1 It occurs mostly in male populations, but is also general in older female groups.2 Hyperuricemia, defined as a serum urate level (sUA) higher than upper limit of normal range, is an important indicator of gout diagnosis.3 Surely, the normal ranges of sUA vary with age and sex: for general adult men, it is 149 ~ 416 μmol/L (2.5 ~ 7.0 mg/dL), and for adult women, it is 89 ~ 357 μmol/L (1.5 ~ 6.0 mg/dL). For people above 60 years old, the normal ranges are 250 ~ 476 μmol/L (4.2 ~ 8.0 mg/dL) for men and 190 ~ 434 μmol/L (3.5 ~ 7.0 mg/dL) for women. For children, the normal sUA is 60 ~ 90 μmol/L lower than adults, but there is no specified range value. Studies have already indicated a direct association between serum urate levels and the risk suffering from gout.4, 5 The American Rheumatism Association 1977 case definition of primary gout also regards hyperuricemia as one of twelve gout symptoms to make a diagnosis.6
However, although hyperuricemia is often used as the major diagnostic indicator in hospitals, nearly half of the time gout occurs without hyperuricemia, especially in an acute attack.7, 8 And there are four situations where normouricemia accompanies gouty arthritis: (a) The solubility of uric acid in human serum under physiological conditions at pH 7.4 and 37°C is 404 μmol/L (6.8 mg/dL), and it falls by a factor of four over a temperature range of less than 25℃, which suggests that there are parts of the body like extremities where, even at normal urate concentrations, the plasma sodium urate is chronically supersaturated and even nucleated and grew to form tophi9, 10; (b) patients with normal urate levels at the time of an acute attack due to the accompanying uric acid diuresis, which perhaps due to pain‐induced cortisol release11, 12; (c) patients with hyperuricemia caused by known factors, such as obesity, alcoholism, or diuretic use, become normouricemic when these factors are removed; and (d) patients with normal urate levels due to therapy with allopurinol or a uricosuric drug. Diagnostic and therapeutic misadventures can occur when acute gout is not considered because of these four situations which may results in months of pain suffering, the dangers inherent in massive mistaken antibiotic treatment and the unnecessary expense.
In the past decade, significant progress has been made in genomic loci associated with hyperuricemia and gout. High risk genes associated with increased serum urate concentrations involve uric acid synthesis‐related genes such as PRPSAP1,13 and renal uric acid excretion‐related genes such as ABCG2 14, 15 and SLC2A9.16 Genetic polymorphism has been proposed for the prevention and recognition of gout, but the prediction of gout by genetic variation is still limited due to insufficient knowledge of the relationship between genes and gout.
The gold standard for diagnosis isaccurate identification of MSU crystals, but the difficulty for patients to accept the pain of joint puncture, inexperience with aspirating joints, time pressures during clinical visits, and inaccessibility or inconvenience of compensated polarized light microscopy greatly limit its clinical application.17 Thus, physicians usually diagnose gout based on clinical impressions instead. Other diagnostic methods such as X‐ray radiography, dual source CT(DSCT), magnetic resonance imaging(MRI), high‐frequency ultrasound also have the shortages of inability to assess early soft tissue changes, low positive rate, and high price.18, 19, 20, 21 Under the abovementioned circumstances, there is a pressing need for a simple, accurate，rapid, and economical method in clinical practice for the diagnosis of gout, especially a biochemical detection method for replacement of uric acid.
The direct cause of gout is that high concentration of urate in the blood subsequently deposits in the joint that triggers inflammation. The reason for excessive accumulation of urate depends on the balance between dietary intake, synthesis, and the rate of excretion (include renal and gut excretion) .22, 23, 24 Figure 1 shows the production and excretion mechanism of urate in humans.25 It can be seen that urate is the final product of purine metabolism in vivo, and there is a close relationship between xanthine, hypoxanthine, and uric acid. Xanthine and hypoxanthine are both the precursor metabolites of uric acid in the purine metabolic pathway and surveys such as that conducted by Zhao et al(2005) have shown that xanthine and hypoxanthine concentrations increase in gout patients whose uric acid level is shown to be abnormal high.26, 27, 28 However, researchers have not treated this phenomenon in much detail that cases with high uric acid level were discussed only, while those with normouricemia were not involved, and which is essential for using xanthine and hypoxanthine as diadynamic criteria replacements for uric acid. This study made up for the gap in the previous researches by quantifying the levels of xanthine and hypoxanthine in gout patients with normouricemia at the same time as those with hyperuricemia. And it is concluded via analyzing 201 clinical samples that the levels of xanthine and hypoxanthine in gout patients whether with hyperuricemia or not are higher than those in healthy people, which confirms that xanthine and hypoxanthine can be used as more accurate diagnostic indexes of gout instead of uric acid.
Figure 1.
Purine metabolic pathway. Purine bases derived from tissue Nucleic acids are reused through the salvage pathway. The enzyme hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) salvages hypoxanthine to inosine monophosphate (IMP) and guanine to guanosine monophosphate (GMP). In a similar salvage pathway, adenine phosphoribosyl transferase (APRT) converts adenine to adenosine monophosphate (AMP). A small portion of urate overproduction owing to HGPRT deficiency and phosphoribosyl pyrophosphate (PRPP) synthetase (PRPPS) superactivity. Futhermore, the net degradation of ATP result in the accumulation of adenosine diphosphate (ADP) and adenosine monophosphate (AMP), which can be rapidly degraded to uric acid. Xanthine oxidase (XO) is the enzyme that catalyzes the oxidation of hypoxanthine to xanthine, and xanthine to uric acid
2. MATERIALS AND METHODS
2.1. Chemicals
Standard of uric acid sodium salt was purchased from Sigma‐Aldrich, Co., and both xanthine and hypoxanthine (purity>98%) were from Yuannuo Tiancheng Technology Co., Ltd. (Chengdu China). Perchloric acid (GR) was obtained from Macklin Biochemical Co., Ltd. (Shanghai China); ammonium acetate (purity>98%) and methanol (HPLC grade) were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China); dextran 70 and activated charcoal were from Macklin Biochemical Co., Ltd.; and acetic acid (AR) glacial and sodium hydroxide (AR) were from Guangdong Chemical Reagent Engineering‐technological Research and Development Center. Ultra‐pure water of HPLC grade (18.25ΜΩ) was used for preparation of all the solutions and for HPLC analysis.
2.2. Stock and working standard solutions preparation
The stock solutions of uric acid, xanthine, and hypoxanthine were prepared in sodium hydroxide solution (0.1 mol/L), accelerated dissolution by ultrasound. All stock solutions were stored at −20℃ and were thawed at room temperature to prepare working standard solutions. Table 1 shows the concentrations of stock and working standard solutions.
Table 1.
The concentrations of the mixed stock and working standard solutions
| Uric acid (μmol/L) | Xanthine (μmol/L) | Hypoxanthine (μmol/L) | |
|---|---|---|---|
| Mixed stock standard solution | 11 783.89 | 3155.61 | 3526.56 |
| 1 | 1178.39 | 210.37 | 117.55 |
| 2 | 589.19 | 105.19 | 58.78 |
| 3 | 294.60 | 52.59 | 29.39 |
| Mixed working standard solution | |||
| 4 | 147.30 | 26.30 | 14.69 |
| 5 | 117.84 | 4.21 | 4.70 |
| 6 | 58.92 | 2.10 | 2.35 |
| 7 | 29.46 | 1.05 | 1.18 |
2.3. Serum samples
Serum samples from gout patients (n = 101) and healthy people (n = 100) were collected from biochemical testing center of West China Hospital，Sichuan University. In order to avoid gender‐related errors, all samples are from men. The serum samples were stored at 4℃ in the hospital for no more than 1 week and then stored at −20℃ in our laboratory.
2.4. Blank serum preparation
In order to avoid the interference of endogenous uric acid, xanthine, and hypoxanthine, the blank serum was prepared with dextran‐coated activated charcoal as published previously.29 After washed twice, 1 g activated charcoal was added to 20 mL ultra‐pure water containing 0.1 g dextran 70, adjusted to pH 4.2, and stirred at room temperature for 24 hours. Thereafter, the mixture was centrifuged, and the sedimentary dextran‐coated activated charcoal mixed with 15 mL serum, stirred at 37°C for 2 hours, filtered to eliminate the bacterium and then stored at −20°C.
2.5. Sample treatment
The blank serum samples were treated firstly by mixing 200 μL of serum with 40 μL of 0.0125 mol/L NaOH to reach the same pH and volume as QC samples. Then, the proteins in the serum sample were precipitated by addition of 400 μL of 20% perchloric acid solution. After vortex for 2 minutes, the serum samples were centrifuged at 13 400 g for 10 minutes. The clear supernatant was injected for HPLC analysis directly. QC samples for standard curves and quality control were prepared through a similar process. Two hundred microliters of the blank serum was mixed with 40 μL of mixed working standard solutions of different concentrations and precipitated by 400 μL of 20% perchloric acid, after that the same steps were taken as described before.
2.6. HPLC conditions
Twenty microliters of the pretreated samples was injected into the HPLC system, which consisted of an Agilent Technologies (USA) 1100 liquid chromatograph comprising a G1310A pump, a 7725i manual injection valve with a 20 μL quantitative loop, a Shimadzu CTO‐6A column oven, and a G1314F 1260 VWD detector. The compounds were separated on a Gem, ODS (5 μ,150*4.6mm) column at 30℃, and the detection wavelength was 250 nm. The mobile phase was prepared from 10 mmol/L ammonium acetate in ultra‐pure water, adjusted to pH 4.0 with glacial acetic acid,filtered through a 0.45‐μm filter and then degassed by ultrasound. The flow rate of mobile phase was set at 1 mL/min, and the total run time under these conditions was ~40 minutes.
Statistics.
The Student t test was performed on the obtained data to compare between groups. And P < 0.05 was considered significant.
3. RESULTS
3.1. Selectivity
The HPLC system provided good separation among the UA, HX, and X, and each of the three metabolites had a good peak shape. The retention time (t R) and resolution (R) of the three metabolites are shown in the Table 2.
Table 2.
The retention time (t R), theoretical plate number (N), tailing factor (T), and resolution (R) of the three metabolites for selectivity validation
| Concentration (μmol/L) | t R(min) | N | T | R | |
|---|---|---|---|---|---|
| Uric acid | 832.79 | 7.120 | 9911 | 0.85 | 3.40 |
| Hypoxanthine | 117.55 | 8.300 | 4103 | 0.80 | 2.93 |
| Xanthine | 105.19 | 9.573 | 4392 | 0.99 | 2.32 |
3.2. Standard curve and linear range
A series of QC samples used to calculate standard curves were processed by mixed working standard solution 1‐7 and analyzed as described in “Sample Treatment,” and all operations were repeated in three batches. Linear regression was performed on peak area against concentrations of UA, HX, and X (converted into serum concentrations) to obtain regression equations and correlation coefficients (r). The regression equations and correlation coefficients (r) are listed in Table 3. All the r values were>0.99, and the range of concentrations were suitable for analyzing all serum samples.
Table 3.
Regression equations, correlation coefficients (r), and linear ranges of the three metabolites
| Duplicates | Regression equation | r 2 | Correlation coefficients (r) | Linear range（μmol/L） | |
|---|---|---|---|---|---|
| Uric acid | 1 | y = 1.2517x‐15.646 | 0.9994 | 0.9997 | 29.46‐1178.39 |
| 2 | y = 1.083x + 7.5557 | 0.9998 | 0.9999 | ||
| 3 | y = 1.0875x + 3.1064 | 0.9997 | 0.99985 | ||
| Hypoxanthine | 1 | y = 7.3465x‐15.327 | 0.9994 | 0.9997 | 1.18‐117.55 |
| 2 | y = 6.5207x‐3.4843 | 0.9992 | 0.9996 | ||
| 3 | y = 6.4965x‐5.1062 | 0.9993 | 0.99965 | ||
| Xanthine | 1 | y = 0.9992x‐0.8982 | 0.9984 | 0.9992 | 1.05‐210.37 |
| 2 | y = 0.9065x + 1.3086 | 0.9997 | 0.99985 | ||
| 3 | y = 0.911x + 0.8091 | 0.9997 | 0.99985 |
3.3. Lower limit of quantitation
QC samples with minimum concentration of linear range were analyzed for five duplicate samples to validated lower limit of quantitation (LLOQ). The LLOQ recoveries of the three metabolites were all >85% and <120%, and the RSD of duplicates did not exceed 10% (Table 4).
Table 4.
Precision and accuracy validation
| Metabolites | Concentration (μmol/L) |
Within‐day (n = 5) DAY1 |
Within‐day (n = 5) DAY2 |
Within‐day (n = 5) DAY3 |
Between‐day (n = 3) |
|||
|---|---|---|---|---|---|---|---|---|
| Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | RSD (%) | ||
| Uric acid | 29.46 | 86.75 | 2.29 | 104.08 | 2.13 | 97.56 | 3.43 | 8.09 |
| 58.92 | 92.47 | 1.33 | 100.63 | 5.86 | 94.63 | 2.81 | 5.23 | |
| 147.30 | 95.86 | 3.41 | 101.12 | 2.79 | 95.95 | 2.42 | 3.74 | |
| 552.37 | 96.75 | 2.83 | 96.24 | 4.33 | 102.32 | 2.39 | 4.18 | |
| Hypoxanthine | 1.18 | 110.60 | 6.74 | 114.94 | 2.10 | 88.91 | 4.79 | 12.13 |
| 2.35 | 96.87 | 1.63 | 103.36 | 3.02 | 87.24 | 2.06 | 7.48 | |
| 14.69 | 95.58 | 2.90 | 103.49 | 2.60 | 96.57 | 2.24 | 4.40 | |
| 88.16 | 96.06 | 2.69 | 95.91 | 4.15 | 100.57 | 3.22 | 3.90 | |
| Xanthine | 1.05 | 102.73 | 4.98 | 103.76 | 3.13 | 86.88 | 4.23 | 9.05 |
| 2.10 | 93.52 | 1.79 | 99.08 | 4.78 | 88.41 | 0.00 | 5.60 | |
| 13.15 | 95.83 | 3.19 | 103.02 | 3.01 | 96.96 | 2.89 | 4.35 | |
| 78.89 | 96.00 | 2.64 | 95.98 | 4.05 | 100.91 | 3.15 | 3.95 | |
3.4. Precision and accuracy
QC samples of lower limit of quantitation (LLOQ), low (L), medium (M), and high (H) concentrations were prepared in five duplicates and determined in three analytical batches on different dates. Accompanying standard curve was made every day. Data on the precision and accuracy of the HPLC method for measurement of UA, HX, and X are given in Table 5. Recovery rates were within the range of 85%‐115% and 80%‐120% at LLOQ for all three metabolites. Within‐day and between‐day RSD were both below 15% (Table 4).
Table 5.
Extraction recovery validation
| Metabolites | Concentration (μmol/L) | Extraction recovery (%) | RSD (%) |
|---|---|---|---|
| Uric acid | 58.92 | 96.54 | 1.55 |
| 147.30 | 89.11 | 1.96 | |
| 552.37 | 83.34 | 7.38 | |
| Hypoxanthine | 2.35 | 75.61 | 6.13 |
| 14.69 | 90.70 | 3.11 | |
| 88.16 | 80.64 | 6.99 | |
| Xanthine | 2.10 | 79.51 | 8.00 |
| 13.15 | 90.19 | 2.42 | |
| 78.89 | 80.80 | 7.00 |
3.5. Extraction recovery
QC samples of low (L), medium (M), and high (H) concentrations were pretreated in five duplicates as described in “Sample Treatment.” The preparation of control samples was the same as QC samples, except that the mixed working standard solutions were added into supernatants after the removal of the protein instead of blank serum. Extraction recoveries were calculated based on the peak area ratio of two batches of samples and were between 70% and 97%. Since all the RSD were no more than 10% (Table 5), the method was stable and feasible for analysis of the three metabolites.
3.6. Stability
To avoid degradation of the compounds, the samples were stored at −20℃ after collected from the hospital, thawed at room temperature before pretreated, and cooled with ice‐water bath until injected. The ice‐water bath stability for 24 hours, repeated freeze‐thaw stability between −20℃ and RT after three times and long‐term freezing stability at −20℃ for 100 days were validated with QC samples of low (L), medium (M), and high (H) concentrations in five duplicates, and the RSD of recovery rates were all within 15% and 20% near LLOQ (Table 6).
Table 6.
Ice‐water bath, repeated freeze‐thaw, and long‐term freezing stability validation
| Metabolites |
Concentration (μmol/L) |
Ice‐water bath | Freeze‐thaw three times | Long‐term freezing | |||
|---|---|---|---|---|---|---|---|
| Recovery (%) | RSD（%） | Recovery (%) | RSD（%） | Recovery (%) | RSD（%） | ||
| Uric acid | 58.92 | 97.31 | 2.39 | 101.20 | 2.38 | 89.67 | 1.50 |
| 147.30 | 98.24 | 0.59 | 110.29 | 6.00 | 102.18 | 2.67 | |
| 552.37 | 99.94 | 3.16 | 107.32 | 5.29 | 97.20 | 1.81 | |
| Hypoxanthine | 2.35 | 87.65 | 2.48 | 103.98 | 1.85 | 96.76 | 1.94 |
| 14.69 | 105.07 | 4.67 | 99.62 | 4.26 | 95.72 | 4.10 | |
| 88.16 | 102.81 | 3.80 | 97.09 | 4.77 | 89.81 | 1.88 | |
| Xanthine | 2.10 | 103.61 | 1.37 | 109.60 | 2.40 | 101.40 | 2.22 |
| 13.15 | 105.20 | 4.93 | 102.15 | 4.77 | 94.02 | 2.98 | |
| 78.89 | 102.79 | 3.74 | 101.77 | 4.72 | 90.77 | 2.11 | |
3.7. The Student t test
The gout patients, gout patients with hyperuricemia, gout patients with no hyperuricemia, and healthy persons were grouped from the hospital's diagnosis results, and our test results of these groups were compared with the Student t test (Table 7). The urate concentration in gout patients with hyperuricemia is significantly higher than in gout patients with no hyperuricemia and healthy persons with a P < 0.001, and there were significant differences between the xanthine and hypoxanthine levels of gout patients, gout patients with hyperuricemia, gout patients with no hyperuricemia, and healthy persons with a P < 0.001 and also between the level of xanthine of gout patients with hyperuricemia and with no hyperuricemia with a P < 0.01.
Table 7.
Mean (SD) concentrations of purine metabolites in samples from patients with gout and healthy persons
| Metabolites |
Gout patients (n = 101) |
Gout patients with hyperuricemia (n = 25) |
Gout patients with no hyperuricemia (n = 76) |
Healthy persons (n = 100) |
|---|---|---|---|---|
| Uric acid μmol/L | 504.3752 (186.19166) | 734.31 (157.19670)a, b | 428.74 (122.04322) | 471.67 (84.05432) |
| Xanthine μmol/L | 10.67 (8.49921)a | 7.92 (6.92121)a | 11.57 (8.81167)a | 3.71 (1.01997) |
| Hypoxanthine μmol/L | 70.48 (49.80081)a | 81.3422 (61.26823)a | 66.91 (45.31427)a | 20.43 (4.73386) |
μmol/L conversion in mg/dL
1 µmol/L = 0.016811 mg/dL
1 µmol/L = 0.015211 mg/dL
1 µmol/L = 0.013611 mg/dL
Compared with healthy persons P < 0.001
Compared with gout patients with no hyperuricemia P < 0.001
4. DISCUSSION
Hyperuricemia is the only biochemical index in the classification of acute gouty arthritis in ACR 1977 and the main basis of clinical diagnosis for most doctors.6 However, uric acid level is normal in almost half of the time of gout attack, especially in an acute attack，which causes misdiagnosis, leading to unnecessary health, economic, and time losses.7, 8 Thus, it is very important to find an accurate and simple biochemical indicator that can replace the uric acid.
Hypoxanthine and xanthine, the metabolic precursors of uric acid, are presumably the best alternative indicators for uric acid26 and were determined with urate in serum by a strictly validated method established in this study, and the Student t test was performed on the comparison between four groups of gout patients, gout patients with hyperuricemia, gout patients without hyperuricemia, and healthy persons. The result shows that there are 76 cases with serum uric acid level within a normal range in the 101 gout cases and there was no significant difference between the average serum uric acid of gout patients and healthy persons. It is noteworthy that we did not collect patient samples based on uric acid levels (with or without hyperuricemia), but just blindly took the 101 samples from patients diagnosed as gout. A total of 76 cases of them have a normal uric acid level is an objective phenomenon. Two reasons may explain this phenomenon. One is that most of these patients were in an acute attack, urate precipitation, renal feedback excretion, or stress response leading to the reduction of concentration in blood. The other is that these patients had taken drugs for lowering urate. Another important finding is that the concentrations of xanthine and hypoxanthine in gout patients (with or without hyperuricemia) are higher than those in healthy people, which approves there was a significant positive correlation between xanthine and hypoxanthine, and gout, and these two compounds can be used as marker metabolites and can even replace uric acid. Besides, the xanthine concentration in gout patients with hyperuricemia was significantly lower than in gout patients with no hyperuricemia and the reason is still to be done in the near future.
Gout can be generally divided into three categories according to its pathogenesis as follows： low excretion type with blocked uric acid excretion (90%), high excretion type due to overproduction of uric acid (10%), and mixed type with a combination of two.23, 24 A possible explanation for the high serum concentrations of xanthine and hypoxanthine in patients with gout is that excessive synthesis or obstruction of downstream pathway. However, one small flaw of this study is that, due to practical constraints, we cannot guarantee all these three kind of samples were involved in our subjects. Further studies, which take this into account, need to be undertaken by a means of classifying gout patients based on the ratio of uric acid clearance to creatinine clearance, monitoring xanthine and hypoxanthine levels in different types of gout patients, and comprehensively verifying the relationship between the two compounds and gout.
5. CONCLUSIONS
With a simple, rapid, and sensitive HPLC‐UV method, this study has determined hypoxanthine, xanthine, and uric acid in gout patients and healthy people, and the analytical‐contrast results of this investigation show that xanthine and hypoxanthine in gout patients (with or without hyperuricemia) are higher than healthy people. These findings confirms previous researches and contributes more comprehensive evidence that suggests xanthine and hypoxanthine can be used as alternative indicators of uric acid for the diagnosis of gout in clinical practice, even if the serum uric acid value is normal, and also has offered a framework for the further exploration of application of these two compounds in clinical diagnosis of gout.
ACKNOWLEDGMENTS
The authors are very grateful to the Ministry of Education of the People's Republic of China for its financial support to 2017 Innovative Entrepreneurship training Program for College students (Project number: 201710610275).
Wang Y, Deng M, Deng B, Ye L, Fei X, Huang Z. Study on the diagnosis of gout with xanthine and hypoxanthine. J Clin Lab Anal. 2019;33:e22868 10.1002/jcla.22868
Wang, Deng, Ye, and Fei contributed equally to these work
Contributor Information
Liming Ye, Email: yeliminglaoshi@126.com.
Xiaofan Fei, Email: fxiaofan@yahoo.com.cn.
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