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Technical Briefs |
1 Department of Laboratory Medicine and Pathology and2 Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, MN
aaddress correspondence to this author at: Mayo Clinic, Division of Nephrology and Hypertension, 200 First Street SW, Rochester, MN 55905; fax 507-266-9315, e-mail Lieske.John{at}mayo.edu
Precise measurement of plasma oxalate is difficult because the concentration in healthy humans is fairly low (1–3 µmol/L) (1)(2). A colorimetric enzymatic assay that uses oxalate oxidase is commonly used for oxalate detection (2)(3). This assay is fairly straightforward for detecting urinary oxalate, which occurs at concentrations in the millimolar range. Plasma oxalate, however, occurs at concentrations in the micromolar range, and signals generated by oxidase degradation are difficult to detect. Our reference laboratory previously used an oxalate oxidase–based assay that required enzyme immobilization on a nylon coil and uses an HPLC with spectrophotometric detection (1). This method has several disadvantages, including limited automation potential, and requires subjective estimation of peak size. We therefore took advantage of the enhanced sensitivity of currently available spectrophotometers (absorbance measured down to a sensitivity of 0.0001) to develop a plasma assay that uses soluble oxalate oxidase (1)(2)(3)(4).
The reagents used were as follows: oxalate reagents A and B and the Oxalate Urine Control Elevated, purchased from Trinity Biotech; hydrochloric acid (0.01 and 12 mol/L), sodium hydroxide (10 mol/L), potassium citrate monohydrate (crystalline; formula weight 324.22), EDTA disodium salt dihydrate (crystalline powder; formula weight 372.24), citric acid monohydrate (granular; formula weight 210.14), and sodium nitrite (crystalline; formula weight 69.00) from Fisher; and oxalic acid dihydrate (formula weight 126.07) and Triton from Sigma. Disposable polystyrene semimicro (1.5 mL) cuvettes were purchased through Fisher. Absorbance was measured with the Beckman Coulter DU800 UV/Visible Spectrophotometer.
Stock citrate buffer (0.33 mol/L) was prepared by dissolving 30.5 g of potassium citrate, 50 g of citric acid, and 2 g of EDTA disodium salt in 1 L of distilled, deionized H2O, with a mean (SD) resulting pH of 3.3 (0.2). Working citrate buffer (0.066 mol/L) was prepared from the stock and filtered through a 0.2 µm, 47-mm nylon filter just before use.
Oxalate stock solution (1.0 mmol/L) was prepared by dissolving oxalic acid dihydrate in 0.01 mol/L HCl. Five working calibrators (50.0, 10.0, 5.0, 2.5, and 1.0 µmol/L) were prepared from the stock and also diluted in 0.01 mol/L HCl. Oxalate Urine Control Elevated was reconstituted and diluted 1:50 and 1:500 in 0.01 mol/L HCl to prepare high and low controls, respectively. Control solutions were divided among single-use vials and frozen at –70 °C.
Blood from healthy volunteers was drawn in a 10-mL sodium heparin tube and immediately placed on wet ice. Within 1 h, samples were centrifuged at 4 °C to isolate plasma, which was adjusted within the next hour to a pH range of 2.3–2.7 with 
10 µL of concentrated (12 mol/L) HCl per 1.0 mL of plasma. Once acidified, samples were deproteinized by centrifugation at 1000g at 20 °C for 1.5–2 h with an Amicon Ultra-4 filter. Sample filtrates (500 µL), controls, calibrators, and an HCl blank were next treated with sodium nitrite (30 µL of 5 mmol/L sodium nitrite in working citrate buffer) to convert sample ascorbate to dehydroascorbate.
Proper sample processing and acidification are essential (2)(5). Ascorbate converts to oxalate nonenzymatically at a pH >4.0; the higher the pH the more rapid the conversion (1). It is therefore necessary to maintain a low pH during sample processing. Deproteinization during subsequent steps is also required to prevent ascorbate precipitation, which can produce overwhelming turbidity, precluding spectrophotometric analysis, and trap unpredictable quantities of oxalate (5). Finally, recovery through the filters was found to be dependent on maintaining the pH in the desired range (2.3–2.7) (1).
Acidified, deproteinized, nitrite-treated plasma filtrates (100 µL), controls, calibrators, and HCl blanks were combined with 500 µL of Trinity Biotech oxalate reagent A [3.2 mmol/L 3-(dimethylamino)benzoic acid, 0.22 mmol/L 3-methyl-2-benzothiazolinone hydrazone along with buffer (pH 3.1) and nonreactive ingredients and stabilizers] in a cuvette, and the absorbance (0.0400) was measured at 590 nm (A readings). Trinity Biotech oxalate reagent B [40 µL, containing 3000 U/L oxalate oxidase (barley) and 100 000 U/L peroxidase (horseradish)] was added, and the absorbance at 590 nm (approximate range, 0.0400–0.1500) was measured again (B readings). The A readings were subtracted from the B readings for each unknown, calibrator, control, and HCl blank. The subtracted HCl blank was then subtracted from each unknown, calibrator, and control. A calibration curve was generated to determine unknown values.
Control materials, calibrators, and a plasma pool were analyzed for intraassay precision (Table 1
). Plasma was collected as described earlier, acidified, and pooled. This pool was separated into 10 aliquots and deproteinized. After nitrite treatment, each of the 10 aliquots was run in duplicate (total n = 20). Two concentrations of control material were analyzed (n = 20) over a 2-week period for interassay precision. Review of the data suggested that values as low as 1.0 µmol/L can be reliably detected with a CV 
20%. Patient samples (n = 3) along with the 50 µmol/L calibrator were serially diluted (undiluted to 1:32 dilution) with nitrite-treated 0.01 mol/L HCl and assayed; the results demonstrated that the method is linear from 1 to 50 µmol/L (y = 0.9635x + 0.0918 µmol/L; r2 = 0.9975; see the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue12/).
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Previous studies suggested that 15% of labeled oxalate is lost when the deproteinizing step is performed at pH 2.3–2.7 (1). Therefore, before performing the assay, we added increasing amounts of the 50 µmol/L oxalate calibrator to a normal plasma sample and pooled plasma samples (n = 3). The mean recovery of the added oxalate was 84%.
Oxalate oxidase is a specific enzyme for oxalate. There have been many interference studies done in the past that have looked at 38 compounds, such as glyoxalate, glycolate, lactate, and pyridoxine (6)(7)(8)(9)(10). These compounds caused no interference. Ascorbic acid was found to be an inhibitor in concentrations >125 µmol/L (6).
We performed stability studies for acidified plasma and acidified deproteinized filtrate (Table 1
) because plasma must be acidified immediately to avoid the nonenzymatic conversion of ascorbate to oxalate (1). Acidified, deproteinized, filtered samples (n = 4) were stable when stored frozen for 28 days. Acidified plasma samples (n = 3) were stable for at least 14 days when stored frozen (–20 °C). Because of the presence of denatured proteins, however, plasma that is acidified and frozen before deproteinization is extremely viscous when thawed, making further analysis difficult. Deproteinization of acidified samples before storage is therefore advisable.
Healthy volunteers (n = 102; 47 males and 55 females) were recruited through the Mayo Department of Laboratory Medicine and Pathology Quality Assurance Office. Mean donor age was 43 years (range, 22–76 years). Individuals with a history of kidney disease, nephrolithiasis, Crohn disease, gastric bypass or other gastrointestinal resection, or primary hyperoxaluria were excluded. Donors were requested to fast overnight and avoid vitamin C supplements for 24 h before the collection. For this adult population, the upper reference limit (95th percentile) of <1.8 µmol/L was established, which agrees with previous studies (1)(2). No variation with age or sex was apparent (Fig. 1
).
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Oxalate is a small, 2-carbon organic acid found in most plant tissues. It is thought that only 10% of ingested oxalate is absorbed. Approximately one third of the oxalate in urine is from absorbed oxalate, and approximately two thirds is from oxalate synthesized by the liver (11). Oxalate intake has been reported to vary from 70 to 930 mg/day in a typical Western diet (12). Because humans have no enzymes to metabolize oxalate, it must be eliminated from the body via the kidneys (13).
Plasma oxalate concentrations are increased in patients with primary hyperoxaluria (14)(15)(16)(17), an autosomal recessive disorder of glyoxalate metabolism characterized by excessive production and urinary excretion of oxalate resulting from defects in specific liver enzymes (alanine:glyoxalate transferase in type 1 and glycolate reductase in type 2). Accurate determination of plasma oxalate can be an important diagnostic test, particularly in young children, for whom collection of 24-h urine samples can be difficult, or in patients presenting in renal failure. In the latter group, a rapid and accurate diagnosis is particularly important because systemic oxalosis in primary hyperoxaluria patients causes them to do poorly on standard dialysis (18)(19)(20). Frequent determinations of oxalate and aggressive dialysis are necessary until kidney and/or liver transplantation can be performed (14). Plasma oxalate concentrations are increased in all patients with end-stage renal failure, regardless of the cause, although not to the extent seen in primary hyperoxaluria (21). Individuals with diverse gastrointestinal conditions that cause fat malabsorption often overabsorb oxalate from their diet (22)(23)(24)(25), a condition termed enteric hyperoxaluria. Relatively little is known regarding the range of plasma oxalate concentrations observed in the enteric hyperoxaluric patient group, although oxalosis has been observed in some patients, associated with end-stage renal failure (23)(26). Therefore, accurate determination of plasma oxalate concentrations could be valuable for these enteric hyperoxaluric patients as well.
In conclusion, we describe a new, rapid, reliable spectrophotometric plasma oxalate assay that is less labor-intensive and technically demanding than our previously used assay. The Beckman Coulter DU800 spectrophotometer allows for enhanced sensitivity, down to 1 µmol/L, with a larger linear calibration curve and measuring range. The enhanced sensitivity of this method enables routine detection at the upper end of the reference interval with improved differentiation between results within and outside the reference interval.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. Li and K. E. McMartin Strain differences in urinary factors that promote calcium oxalate crystal formation in the kidneys of ethylene glycol-treated rats Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1080 - F1087. [Abstract] [Full Text] [PDF]
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, males (n = 47); •, females (n = 55).