Measurement of oxalate in urine is important for the diagnosis of primary hyperoxaluria (McKusick 259900) and the secondary forms produced by excessive intake or abnormal intestinal absorption of oxalate (1). Determination of glycolic acid is essential for the diagnosis of primary hyperoxaluria type 1. Finally, to estimate the risk of stone formation in calcium oxalate urolithiasis and nephrocalcinosis, simultaneous determination not only of calcium but also of citrate (a potent inhibitor of calcium oxalate and calcium phosphate crystallization) and other constituents (electrolytes, phosphate and sulfate) is required to calculate urinary calcium saturation (2). Ion-chromatography HPLC (3)(4) and specific enzymatic assays (5)(6) are available only in specialized laboratories. In addition, preservation and storage of liquid samples may influence the stability of oxalate and glycolate (7). Use of urinary filter spots is a practical alternative for the collection and safe transport of samples to be analyzed for many metabolic disorders. To evaluate the age-related changes of oxalate, glycolate, citrate, and sulfate in a pediatric population, we developed an automated ion-chromatography system for the simultaneous measurement of these anions in urine and established their reference values for liquid urine samples as well as for dried urine on filter paper. We studied 20 individuals from each age group (<6 months, 7 months–2 years, 3–7 years, 8–16 years, and >16 years), all without renal or metabolic disease, all on a routine food intake at the time of study. The urines were preserved with 6 mol/L HCl at pH 1–2 to prevent nonenzymatic conversion of ascorbate to oxalate. Filter paper strips (3 × 5 cm filter paper backing, cat. no. 165-0921, Bio-Rad) were dipped into urine to 1 cm below the upper edge. Excess urine was wiped off, and the filter was left to dry at room temperature (18–20 °C). The filter strip was stored for up to 2 weeks before analysis at room temperature. Urinary filter paper spots were then cut into small pieces, shaken in 3 mL of water for 15 min at room temperature, and sonicated with an Aerograph Ultrasonic Cleaner (Branson Instruments) for 5 min at room temperature. The extracts were filtered through an Ultrafree-MC filter with a 10-kDa cutoff (Millipore) at 2000g for 10 min at room temperature. The clear supernatant was analyzed by HPLC. System 1 (used to separate oxalate, glycolate, citrate, and sulfate from chloride, nitrate, and phosphate) consisted of a Series 4000i gradient ion chromatography system (Dionex, Sunnyvale, CA). The background conductivity was minimized using the anion suppressor unit ASRS-I (Dionex) at 30 μS (microSiemens). Separation was performed on the IonPack AS11 analytical column (4 × 250 mm; Dionex) connected to the IonPack AG11 guard column (5 × 50 mm; Dionex), using a linear gradient of A: 100 mL of 50 mmol/L sodium hydroxide in 900 mL water; and B: 50 mmol/L sodium hydroxide, at a flow of 2.0 mL/min. The first 4 min was an isocratic run with solvent A; the gradient was run from 4 to 12 min. System 2 was essentially the same as system 1 except that a CarboPack PA1 column (4 × 250 mm; Dionex) was used and isocratic separation was performed with 14 mmol/L sodium tetraborate at a flow of 1.5 mL/min. For the recovery calculation, microliter amounts of dissolved oxalate (1.0 mmol/L), glycolate (1.0 mmol/L), citrate (3.0 mmol/L), and sulfate (6.0 mmol/L) were added to urine, reflecting concentrations in the physiological range or slightly above. Results for liquid samples and samples stored on filter paper were compared by paired t-test. These comparisons were considered significantly different if P ≤0.05. The Friedman test was used to evaluate the age dependency of values. Statistical analyses were performed using WinSTAT 3.1 (Kalmina). The urinary HPLC chromatograms (system 1) of the standard mixture, nondiseased urine, and urine from a pediatric patient with primary hyperoxaluria type 1 are shown in Fig. 1 , A-C. In this system, contaminative fluoride may interfere with glycolate; therefore, all samples with increased glycolate concentrations needed to be reanalyzed in system 2 (Fig. 1 , D-F). However, in all samples analyzed, there was no contamination with fluoride. Thus, system 1 can be used for screening all urinary samples to differentiate between primary hyperoxaluria type 1 and other hyperoxalurias. Stability of oxalate and glycolate is one of the most important factors and may be affected by ascorbate, pH, temperature, and time of storage. We investigated the recovery of liquid samples compared with the dried urine on filter paper stored for 1 week and 2 weeks at room temperature in two different urine pools (controls and patient with primary hyperoxaluria type 1). Concentrations of urinary oxalate on the filter spot were higher (P <0.005) when stored for 1 week and almost identical after 2 weeks compared with values in the control liquid sample measured immediately after collection (Table 1). No significant differences were found between the liquid sample, the dried spot after 1 week, and the dried spot after 2 weeks for glycolate, citrate, and sulfate. In the sample from a patient with the primary hyperoxaluria type 1, values for oxalate and glycolate were much higher than the controls measured in the liquid sample, the dried spot after 1 week, and the dried spot after 2 weeks (Table 1). The values of oxalate were unaffected by storage, whereas glycolate decreased after 1 week (P <0.001) and 2 weeks (P <0.001). Collection of urine on filter paper was more critical for glycolate than for oxalate when present in higher concentrations. The values for citrate were similar to those in the control urine and were only slightly lower after 2 weeks (P <0.05; Table 1). Sulfate was higher in pathological urine than in controls, and collection on filter paper produced lower values than in liquid urine (P <0.005). There was no difference in values between filter papers stored at room temperature or at 4 °C (data not shown). Comparison of oxalate, glycolate, citrate, and sulfate in liquid samples and dried spots after 1 and 2 weeks1 and reference values in pediatric population. Means ± SD (n = 6), expressed as mmol/mol creatinine for oxalate, glycolate, and citrate and as mol/mol creatinine for sulfate, are shown. Significantly different from liquid sample, P <0.005. Significantly different from liquid sample, P <0.001. Significantly different from liquid sample, P <0.05. Comparison of oxalate, glycolate, citrate, and sulfate in liquid samples and dried spots after 1 and 2 weeks1 and reference values in pediatric population. Means ± SD (n = 6), expressed as mmol/mol creatinine for oxalate, glycolate, and citrate and as mol/mol creatinine for sulfate, are shown. Significantly different from liquid sample, P <0.005. Significantly different from liquid sample, P <0.001. Significantly different from liquid sample, P <0.05. Chromatographic separation of oxalate (5), glycolate (1), citrate (7), and sulfate (4) from chloride (2), nitrate (3), and phosphate (6) in urine filter paper spot by ion chromatography, using two different systems. (A and D) Standard mixture, 100 μmol/L; (B and E) nondiseased urine; (C and F) urine from patient with primary hyperoxaluria type 1. Recovery experiments were performed with control urine. One aliquot with the four ions added was spotted on filter paper, stored for 1 week at room temperature, and analyzed in triplicate. Mean recoveries were 98% ± 8.1% for oxalate, 87% ± 12.3% for glycolate, 83% ± 2.3% for citrate, and 96% ± 7.0% for sulfate. These findings suggest that urine dried on filter paper can be stored for more than 1 week without diagnostically significant changes in concentrations of oxalate, glycolate, citrate, and sulfate. The molar ratios of urinary oxalate, glycolate, and citrate vs creatinine were found to be age-dependent. This is in agreement with data published previously by other methods (8)(9)(10)(11). The ratios were highest in infants, particularly below the age of 2 years (Table 1). For oxalate and citrate, the highest values were observed in infants below 6 months of age. In children over 2 years of age, values for oxalate, glycolate, citrate, and sulfate decreased. The Friedman test showed an age-dependent distribution of values for all four analytes. In conclusion, our improved method allows the simultaneous determination of oxalate, glycolate, citrate, and sulfate in liquid urine and urine filter paper spots, enabling differential diagnosis of oxaluria. Urine filter papers can be stored up to 1 week without diagnostically significant changes in concentrations of the above compounds. The greatest advantage of this new method is that the samples can be mailed in envelopes, speeding delivery and reducing shipping costs.
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Blau et al. (1998) studied this question.
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