Rapid Oxalate Determination in Blood and Synthetic Urine Using a Newly Developed Oxometer

Blood oxalate determination and results

Second generation glucometer strips (One-Touch, Johnson and Johnson, New Brunswick, NJ) were impregnated with 5 μL of an aqueous solution of oxidase (7.5 IU), obtained from Hordeum vulgare barley seedlings (Sigma-Aldrich, St. Louis, MO), and allowed to dry. Fresh blood (1 mL) was collected by tail bleed from a male Sprague Dawley rat, and its baseline blood glucose level was determined to be 80 mg/dL by glucometer. A 7.5 μL volume of sodium oxalate solution (ie, 0.16 mM, 0.32 mM, 0.65 mM, 1.25 mM, or 2.5 mM) was added to 12.5 μL of blood, gently mixed, and one drop placed on a glucometer strip. Standard 45-second readout in mg/dL of glucose was recorded using OneTouch^® Ultra^®2 glucometer (LifeScan, Inc; Milpitas, CA) (Fig. 2A). Relative oxalate determination was made by subtracting baseline blood glucose reading from each oxalate-spiked blood glucose reading (Fig. 2B). As each sodium oxalate sample was diluted within the blood, oxalate concentration was adjusted by calculating the absolute amount of oxalate added and regraphed for adjusted oxalate readout (Fig. 2C). A “best fit” line was calculated using a linear regression program in a TI-83 graphic calculator (Texas Instruments, Dallas, TX). As blood oxalate levels increased, measured oxalate was found to increase at a rate where the Δ reading of 0.05 mg/dL corresponded to 0.01 mg of oxalate present in the sample.

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FIG. 2.

Glucometer readings (y-axis) at 45 seconds for baseline and oxalate-spiked blood specimens with varying concentration (x-axis). (A) Double-production of hydrogen peroxide; 78±4 mg/dL glucose (gray area) marks background glucose level. (B) Relative oxalate reading without volume correction. (C) Relative oxalate reading with correction for total sample volume. Blue box delineates area of best fit line. Y-axis=oxalate concentrations.

Synthetic urine oxalate determination and results

Sodium oxalate was added to synthetic urine to create 0.6 mM and 1.25 mM oxalate solutions. Glucometer test strips impregnated with varying amounts of OxOx (ie, 15 IU, 7.5 IU, 3.8 IU, 1.9 IU, 0.95 IU, 0.0 IU) were placed within 1.5 mL polystyrene spectrophotometer cuvettes and 0.6 mL of synthetic urine/oxalate solution added. Strips were removed at 3 and 5 minutes in two separate experimental settings. The resultant color reactions were photographed, showing the graded activity levels within the vial (Fig. 3).

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FIG. 3.

Black and white photograph of blue color reaction seen within cuvettes after removal of glucometer strip. Oxalate oxidase concentration on the strips is reported in IU below each cuvette. (A) 0.6 mM oxalate solution with 5 minute strip dwell time. (B) 1.25 mM oxalate solution with 3 minute strip dwell time. Higher oxalate concentration resulted in more prominent color at a shorter time interval.

Urinary oxalate measurement

In humans, the dicarboxylic anion oxalate is the end product of liver glyoxylate and ascorbic acid metabolism and is a commonly absorbed plant product found in most diets. The majority of oxalate is eliminated through the kidney and, under predisposing conditions, may precipitate with urinary calcium to form insoluble mineral complexes and eventually kidney stones. Because the majority of kidney stones are oxalate-based, urine oxalate can be measured (normal range 0.1–0.46 mmol/day or 20–40 mg/day) to estimate stone risk and to assist with preventative dietary counseling. ⁹

Although reliable urine oxalate assays are available through a small number of commercial and private laboratories, proper sample handling and preparation are paramount for dependable results. First, urine samples must be acidified during collection and storage (pH 1–2) to limit calcium oxalate precipitation and bacterial growth. To maximize the reaction rate of the OxOx and other enzymes used in the colorimetric assay, the urine samples (once in the laboratory) must be adjusted back to pH 5–7 or risk affecting oxalate recovery. ¹⁰ Adding to the measurement complexity, urine must be mixed with freshly prepared buffers and centrifuged in a tube of activated charcoal to remove urinary proteases, ascorbic acid, and interfering divalent cations. ¹¹ Small amounts of urine are then placed into duplicate wells of a microtiter plate with controls, oxalate oxidase and dyes are added, and color changes are read by a spectrophotomer at 590 nm wavelength absorbance. ¹¹

Many urologists avoid the need to measure urinary oxalate and simply encourage their calcium oxalate stone formers to avoid foods high in oxalate. This seemingly easy recommendation can overwhelm persons already on multiple other dietary restrictions—eg, patients with diabetes, hypertension. In addition, oxalate is a known constituent in a number of “healthy” vegetable and plant-based foods, and its bioavailability can vary depending on food preparation and other meal constituents. ¹² Empowering a patient to self-monitor oxalate would lower confusion and allow a more tailored dietary approach. In the preventative diabetic literature, there is good evidence to support the use of glucose monitoring and patient self-involvement, ¹³ and we speculate that a similar behavior would be observed for a urinary oxometer.

Systemic oxalate measurements

As incredible as it sounds, measuring systemic oxalate is even more challenging than urine. More than 40 articles have been published during the last 80 years describing a variety of sample preparation, storage, and analysis methods for whole blood, serum, and plasma. ^14,15 To appreciate the simplicity of our oxometer, a brief review of systemic oxalate measurements is appropriate. First, whole blood, serum, and plasma oxalate levels occur at concentrations 100-fold less than urine (1–3 μmol range), so detection itself can be difficult. Even as recent as the 1980s, the reported normal range of plasma oxalate varied anywhere from 1 μmol/L to 200 μmol/L, depending on the laboratory. ^16,17

Explanations for these differences start just after the blood draw; several authors describe increased sample oxalate (oxalalogenesis) from the breakdown of glyoxalate or ascorbate if blood is allowed to stand more than a few minutes or if the samples are frozen and stored. ^15,18 Moreover, oxalate stored in an acidified environment can bind to plasma proteins, resulting in up to a 70% oxalate underestimation for some ultrafiltration assay protocols. ¹⁹ Oxalate may also be lost during multiple sample preparatory centrifugations, acidifications, deproteinizations, and nitrite-treated filtrations. ⁹ Like urine, blood may also contain substances that can affect OxOx enzyme activity, so samples need to be processed through ion-exchange resins and/or activated charcoal before enzyme exposure. ^10,20

To address these concerns, [¹⁴C] isotope-labeled oxalate internal standard techniques were introduced. ^14,20 Unfortunately, this labeling process was impractical and limited in humans because of long intravenous isotope infusions (up to 6 hours duration), need for multiple blood collections (sometimes up to 100 mL of blood), inaccurate measurements of radioactive urinary oxalate, and the potential radiation exposure to patients and laboratory and healthcare workers. ²¹ The oxometer outlined in this article would circumvent many, if not all, of these issues.

Limitations and future directions

Whole blood and urine oxalate measurements are obviously complex, so we have exploited existing, well-tested technologies for patient-centered monitoring. Real-time measurements avoid collection and storage problems, but limitations do exist. Our artificial urine contained oxalate concentrations in the mmol range, similar to what is seen clinically. Our experiments, however, do not take into account interfering substances and proteases that are found in normal human urine, nor do they involve any type of grading outside of a visual scale. We envision that a “color chart” could accompany the test kit, similar to the one provided for dipstick urinalysis. More testing and validation by traditional oxalate assays, ion chromatography, and use of actual human urine is under way.

As far as whole blood, the “double-production” of H₂O₂ by GOx and OxOx on glucometer strips is novel and has the potential to be a screening test for a variety of oxalate-related diseases. The oxalate concentration tested in our experimental blood samples was supraphysiologic, so the ability of our oxometer to measure significantly lower oxalate levels remains to be determined. Newer electrochemical blood glucometers that measure electron transfer between the solution and electrode may be more sensitive than the second-generation glucometers used in this preliminary study. An electrochemical strip of this nature would need commercial production and ideally contain only OxOx (Patent Pending #024164). Despite its shortcomings, the simple oxometer described here, if validated in future clinical trials, could usher in a new era of oxalate measurement, management, and research, paving the way for large-scale screening and preventative investigations across a variety of oxalate-related disease states.