Original Article

Ann Clin Biochem 2000; 37: 355±359

Effect of urine storage on urinary uric acid concentrations Jun-ichi Yamakita, Tetsuya Yamamoto, Yuji Moriwaki, Sumio Takahashi, Zenta Tsutsumi and Toshikazu Hada From the Third Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 663-8501, Japan

Accurate determination of serum and urinary uric acid concentrations is essential for the diagnosis and classi®cation of gout according to uric acid metabolism derangement. Urine and/or serum samples are often kept at either 4 C or 3 20 C until assayed, when a large number of samples are handled simultaneously. Our preliminary study indicated a signi®cant decrease in urinary uric acid concentration after preservation, regardless of the storage temperature. Uric acid crystals were often observed in these cases which showed a marked decrease in urinary uric acid concentration after storage. In the present study, we sought the factor(s) that might cause this decrease in urinary uric acid concentration, as well as measures to overcome the problem. High urinary uric acid concentration and low pH proved to play major roles in the decrease in urinary uric acid concentration after storage. In contrast, dilution of the urine samples before storage resulted in no signi®cant change in urinary uric acid concentration. Based on these results, we recommend diluting urine before storage for determination of uric acid concentration and avoiding underestimation. SUMMARY.

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INTRODUCTION Diagnosis of gout and its derangement of uric acid metabolism is usually based on the measurement of serum uric acid concentration, uric acid clearance, and 24-h urinary uric acid excretion. Based on these values, gout patients are classi®ed as overproducers of uric acid, who excrete over 1000 mg/day of urinary uric acid,1 or underexcreters of uric acid, whose fractional clearance of uric acid (uric acid clearance/ creatinine clearance2 100) is below 4%.2 It is standard practice to administer xanthine oxidase inhibitor (allopurinol) to overproducers and uricosuric agents to underexcreters. For this purpose, accurate measurements of serum and urinary uric acid concentrations are important. However, in the process of treating large amounts of urine and serum, the samples are often kept at either 4 C or 3 20 C until analysis. We incidentally found a discrepancy in urinary uric acid concentrations between fresh and preserved samples during the analysis of uric acid excretion in 78 patients with gout (see Fig. 1); urinary uric acid concentration was

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Correspondence: Dr Yuji Moriwaki. E-mail: [email protected]

signi®cantly decreased in some preserved samples. Therefore, the present study was conducted to clarify the factor(s) causing this

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FIGURE 1. Correlation of uric acid concentrations between fresh urine samples and preserved ones (stored at 4 C and warmed by incubation at 37 C) of 78 patients with gout. A linear regression line (r=1) was observed in most cases, but in some others the dots deviated from the line.

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discrepancy and to propose solutions for the problem. SUBJECTS AND METHODS Eighty-six healthy men took part in the study, after informed consent was obtained. Spot urine was collected in a plastic tube, and the samples were divided into three groups. In one group, urinary uric acid concentration was measured immediately after voiding, while in the remaining two groups, urine was kept at either 4 C or 3 20 C overnight. The stored urine samples were incubated in 37 C or 60 C containers for 10 min before measurement, and then determination of urinary uric acid concentration was performed at room temperature by the uricase method3 using a commercially available kit (Uric acid B-test Wako, Wako Pure Chemical Industries, Osaka, Japan). The degree of imprecision (percentage coef®cient of variation; %CV) with this kit is below 1´5% according to the manufacturer’s instruction. Values are expressed as the mean6 SD. Observed differences between groups were assessed by Student’s t-test. A stepwise regression analysis was used to assess the association between the decrease in urinary uric acid concentration after storage and other measured variables. P values below 0´05 were considered statistically signi®cant.

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RESULTS Correlation of uric acid concentrations between fresh urine samples and preserved ones The relationship of uric acid concentrations from fresh urine samples and preserved ones (stored at 4 C and warmed by incubation at 37 C) was analysed in 78 patients with gout. As shown in Fig. 1, a linear regression line (r6 1) was observed in most cases, but there were discrepancies in urinary uric acid concentrations between fresh and preserved samples in some cases. Urinary uric acid concentration was signi®cantly decreased in these preserved samples. A similar tendency was observed in samples from 86 healthy subjects (see Fig. 2).

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Effect of storage on the urinary uric acid concentrations The mean spot urinary uric acid concentrations were 3´606 1´81 mmol/L in fresh samples, 2´946 1´23 mmol/L in ones stored at 4 C and warmed by incubation at 37 C, 3´286 1´05 mmol/L in ones stored at 4 C and warmed by incubation at 60 C (see Fig. 3a),

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Ann Clin Biochem 2000: 37

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FIGURE 2. Correlation of uric acid concentrations between fresh urine samples and preserved ones (stored at 4 C and warmed by incubation at 37 C) of 86 healthy subjects. As with Fig. 1, a linear regression line (r=1) was observed in most cases, but in some others the dots deviated from the line.

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3´376 1´62 mmol/L in ones stored at 3 20 C and warmed by incubation at 37 C, and 3´316 1´53 mmol/L in ones stored at 3 20 C and warmed by incubation at 60 C (see Fig. 3b). Urinary uric acid concentration was found to be signi®cantly decreased after preservation, regardless of storage and incubation temperatures (P< 0´01). Needle-like crystals were often observed in cases that showed a marked decrease in urinary uric acid concentration after storage (see Fig. 4). Infrared absorption spectrophotometry demonstrated that the crystals were almost entirely (98%) composed of uric acid.

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Comparison of various biochemical parameters between groups with marked and slight decreases in uric acid concentration after storage In 20 cases (group A), a marked decrease in urinary uric acid concentration (> 0´59 mmol/ L; 5´586 1´61 mmol/L in fresh urine and 2´746 1´66 mmol/L in 4 C-stored urine, P< 0´0001) was observed, while in the remaining 66 cases (group B) the decrease was only slight (< 0´59 mmol/L). To determine the causative factors involved in this difference, several biochemical components, such as uric acid concentration, pH and electrolytes, were compared between the two groups, using fresh samples. As shown in Table 1, the urinary concentrations of uric acid, urea nitrogen,

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Effect of storage on urinary uric acid concentrations

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FIGURE 3. Effect of storage on urinary uric acid concentrations. (a) Samples stored at 4 C: lane 1=fresh samples; lane 2=incubation at 37 C; lane 3=incubation at 60 C. (b) Samples stored at 3 20 C: lane 1=fresh samples; lane 2=incubation at 37 C; lane 3=incubation at 60 C. A signi®cant decrease in urinary uric acid concentrations was observed in the stored samples, irrespective of the storage and incubation temperatures.

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protein, glucose and creatinine, as well as urine osmolarity, were signi®cantly higher in group A than in group B (5´586 0´35 mmol/L versus 3´086 0´15 mmol/L, P< 0´01; 0´356 0´02 mol/L versus 0´286 0´01 mol/L, P< 0´05; 746 7 mg/L versus 526 2 mg/L, P< 0´01; 0´536 0´09 mmol/L versus 0´286 0´02 mmol/L, P< 0´01; 13´6 6 0´83 mmol/L versus 10´66 0´58 mmol/L, P< 0´01; 819´26 36´3 mOsm/kg H2O versus 700´46 25´8 mOsm/kg H2O, respectively, P< 0´05). Urine pH was signi®cantly lower in group A than in

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group B (5´46 0´1 versus 5´86 0´1, P< 0´05). Urinary concentrations of sodium, potassium, chloride, calcium, phosphate and magnesium did not differ between the two groups (data not shown). To further clarify which factor(s) contributed most strongly to the decrease in urinary uric acid concentration after storage, a stepwise regression analysis was performed. Consequently, urinary uric acid concentration immediately after voiding, urine osmolarity and pH were found to make the strongest contributors to a decrease in urinary

TABLE 1. Biochemicalparametersbetween groups with marked (< 0´59 mmol/L; group A) and slight (< 0´59 mmol/L; group B) decreases in urinary uric acid concentration after storage Group A (n=20) Uric acid in fresh urine (mmol/L) Urine pH Urea nitrogen (mol/L) Protein (mg/L) Glucose (mmol/L) Osmolarity (mOsm/kg H2O) Creatinine (mmol/L)

5´586 5´46 0´356 746 0´536 819´26 13´66

0´35 0´1 0´02 7 0´09 36´3 0´83

Group B (n=66) 3´086 5´86 0´286 526 0´286 700´46 10´66

0´15 0´1 0´01 2 0´02 25´8 0´58

P value

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