An innovative method for rapid detection of urinary oxalate in goats with urolithiasis


 Background: Oxalate is a natural and abundant metabolic by-product; as a highly oxidized organic compound with powerful chelating activity, in high concentrations it can cause morbidity and mortality in both animals and humans. Elevated levels in urine (i.e., hyperoxaluria) have been found to correlate with several human diseases, especially urolithiasis or kidney stone disease (KSD)- a disease also prevalent in goats. Current methods for measuring oxalate are highly technical, cumbersome, and time-consuming, which often forces clinics to utilize expensive diagnostic laboratories. Therefore, in this study, we designed an innovative device, Oxalometer, to measure urinary oxalate in goats. Results: The results indicate that the Oxalometer performed as accurately as the standard commercially available test. The Oxalometer was able to measure higher levels of urinary oxalate in goats with urolithiasis compared to non-urolithiasis goats. The data demonstrate the accuracy and sensitivity of the Oxalometer. Conclusions: This proof-of-concept study supports the future application of the device in determining on-the-spot oxalate levels in patients and brings kidney stone prevention to point-of-care practice for ruminants.

oxalic acid, it must be excreted via urine; high levels of oxalic acid in the urine (i.e., hyperoxaluria) have been found to correlate with a number of human diseases, especially calcium-oxalate kidney stone disease (CaOx KSD), a disease also prevalent in goats (6, 7). The current oxalate test used in human medicine is relatively expensive, utilizing costly equipment performed by skilled technicians; it is timeconsuming with a longer turnaround time. Since the current test is not a point-of-care test, monitoring or treatment plans are challenging to implement for patients at the time of visit. This study developed an Oxalometer device that can measure oxalate levels in urine within two minutes. The Oxalometer exhibited similar accuracy and sensitivity as a commercially available test. Furthermore, we found that goats suffering from urolithiasis had higher levels of urinary oxalate than non-affected goats. This proof-ofconcept study supports the future application of the device in determining on-the-spot oxalate levels and brings kidney stone prevention to point-of-care practice.

Methods
Animal recruitment and sample collection.
Owners of goats admitted to the University of Florida Large Animal Hospital were asked if they would participate. The animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida. Clinical examination upon hospitalization consisted of a complete physical examination and diagnostic imaging procedures. Transabdominal ultrasonography was performed with a 3.5 MHz sector probe to con rm bladder enlargement and a diagnosis of obstructive urolithiasis. The urine samples were collected from goats with obstructive urolithiasis (n = 12) and goats without urinary tract disorders (n = 15) either by free catch or ultrasound-guided cystocentesis as presented in Table 1.
Uroliths obtained as part of the treatment in some goats or necropsy examination of all goats presenting for obstructive urolithiasis were collected. Recovered uroliths were submitted to the Minnesota Urolith Center in St. Paul, Minnesota, where uroliths were screened by optical crystallography and infrared spectroscopic for con rmation of urolith composition. Design and Stereolithographic Apparatus (SLA) 3D printing of the oxalometer ltration cartridge.
The oxalometer ltration cartridge was designed using Trimble Sketchup 3D design software. The column design interfaced a slanted grate with the sidewall of a standard 1.5 mL polystyrene disposable 1 cm path length cuvette (ThermoFisher, USA). In essence, the design employed the cohesive natural properties of water and adhesion to the cuvette surface to uniformly and consistently draw uid through the lter. 3D printing was achieved using the Form 1 + SLA 3D Printer (FormLabs, USA), which uses a blue light laser (305 nm) to fuse resin layers to form a water-tight object. STL les from Sketchup were imported into the proprietary PreForm software. Positioning and support structures were automatically optimized by the software before printing, and the same settings were used on each following print. The build surface (150 mm x 150 mm) was capable of printing 29 ltration columns simultaneously after optimization. Clear Resin (#GPCL02) was lled into the reservoir tank, and 3D printing was initiated at a print resolution of 200 microns. Completed prints were scraped off the build stage, rinsed in isopropyl alcohol by repeatedly dunking for 2 minutes, and submerged for 5 minutes. The prints were air dried for 5 minutes and cured afterward using a long wavelength UV light for 30 minutes. Cured prints had the supports trimmed off using end-cut pliers. Columns were treated with Elmer's spray glue, and glass ber (Whatman GF-D) lter paper was adhered to the inside bottom face of the columns. 150 mg/mL of 100 mesh activated charcoal in water were mixed, and 1 mL was pipetted into each column and allowed to drip dry to create the nal ltration column (Fig. 1).
Succinate buffer was prepared by mixing 26.8 mL of ddH 2 O, 5 mL of 500 mmol/L of succinic acid (pH 3.8), 0.6 mL of 100 mmol/L of p-Dimethylaminobenzaldehyde (DMAB), and 0.6 mL of 100 mmol/L of 3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH). 4 mL of 0.2 U/mL enzyme mixture containing oxalate oxidase and horseradish peroxidase (Trinity Biotech, USA) was added to the succinate buffer. The mixture was vortexed, and 500 µL was pipetted into empty cuvettes, then capped and stored in a -80°C freezer until use. Cuvettes were thawed for approximately 15 minutes, caps were removed, and 50 µL of urine was added to each oxalometer ltration charcoal cartridge. Cuvettes were centrifuged (1000 rcf, 30 seconds), then lter cartridges were removed, and cuvettes were re-capped; cuvettes were incubated at 37 0 C for 15 minutes. Immediately after, the results were read at 590 nm on the Tecan M200 Pro Spectrophotometer with a cuvette adapter. All samples were measured in duplicate, and the experiment was repeated three times.
Determining urinary oxalate using a commercial kit.
The colorimetric detection system was used to measure urinary oxalate per the manufacturer's instructions (Oxalate kit, 591-C, Trinity Biotech, USA). Brie y, oxalate reagents A, B, and the sample diluent were suspended with 10 mL, 2 mL, and 100 mL, respectively. Standard concentrations of 0.357 mM oxalate and 0.5 mM oxalate were prepared using potassium oxalate in normal simulated urine (Carolina, # 695955). For each sample, a 1:1 ratio of urine: sample diluent was utilized and stabilized at a pH between 5 and 7 prior to being transferred to a charcoal tube. Each sample was vortexed, then centrifuged (1500xg, 5 minutes); 100 µL of sample Reagent A was added to each well, followed by 5 µL of standards, sample urines, or water (negative control), to respective wells. 10 µL of Reagent B was added to each well and incubated (room temperature, 5 minutes), then absorbance at 590 nm was read on the Tecan M200 Pro Spectrophotometer. All samples were measured in duplicate, and the experiment was repeated three times.
Statistical analysis.
GraphPad Prism 6 software was used for all statistical analyses. Differences between the two groups (urolithiasis v non-urolithiasis goats) were determined by an unpaired two-tailed parametric Mann-Whitney test, and the median was reported. Statistical signi cance was considered if p < 0.05 in all analyses.

Results
Evaluating the accuracy of the Oxalometer.
The Oxalometer has a few components that could compromise the accuracy of the device. For example, the ltration charcoal cartridge may impede the ow of the liquid sample by gravity, or the ow rate could affect the colorimetric reaction in the cuvette. Therefore, it is imperative that we determined the reliability and accuracy of the Oxalometer against a commercially available standard test (Trinity oxalate kit). Simulated urine (Item # 695955, Carolina Biological, USA) was spiked with de ned concentrations of oxalate (0.25, 0.5, 1.0 uM). Absorbance for each concentration was determined using the Oxalometer and Trinity oxalate kit. As presented in Fig. 2 Measuring goat urinary oxalate using the Oxalometer.
As demonstrated, the Oxalometer performed as well as the commercial standard with known oxalate concentrations in simulated urine. To further evaluate the sensitivity and accuracy of the Oxalometer, clinical urinary samples were utilized. Urinary oxalate levels in goats with obstructive urolithiasis were compared to urinary oxalate levels in non-urolithiasis goats. Using the Trinity kit, measurement of urinary oxalate in goats with urolithiasis was signi cantly elevated above that of non-urolithiasis goats ( Fig. 3A and 3B; mean absorbance in non-urolithiasis goats: 0.551 ± 0.040, urolithiasis goats: 0.797 ± 0.068, p = 0.0144, mean concentration in non-urolithiasis goats: 5.762 ± 0.563, urolithiasis goats: 9.228 ± 0.954, p = 0.0144 ). Next, we sought to determine the accuracy of the oxalometer with goat urines. As presented in Fig. 3C, there was a signi cant difference between the urinary oxalate in non-urolithiasis and urolithiasis goats (mean absorbance in non-urolithiasis goats: 0.050 ± 0.008, urolithiasis goats: 0.126 ± 0.018, p = 0.0109). The mean concentration was 4.254 µM ± 0.772 for non-urolithiasis goats and 10.554µM ± 1.401 for urolithiasis goats, p = 0.0028 (Fig. 3D). The result indicates that the Oxalometer could accurately measure urinary oxalate in clinical samples.

Discussion
Oxalate is a natural by-product of metabolism that cannot be broken down by mammals instead of excreted through urination. Hyperoxaluria can result in kidney stone disease (KSD) or urolithiasis. Often clinics are forced to utilize expensive diagnostic laboratories to measure urine oxalate because current methods are highly technical and cumbersome. The standard of care oxalate methods (Trinity Biotech, MilliporeSigma) which the diagnostic laboratories often use, involve expensive instruments, such as incubators, mixers, spectrophotometers, and can take up to three hours to complete and additional days to obtain the result. Therefore, in this study, we designed a rapid and innovative device called Oxalometer as an alternative to measuring urinary oxalate. We rst validated the measurements of urinary oxalate levels in simulated urine spiked with de ned concentrations of oxalate. Both the standard commercial kit and the Oxalometer exhibited a direct relationship between oxalate concentrations and absorbance; speci cally, as the oxalate concentration increased, the absorbance also increased. Interestingly, the Rsquared of the Oxalometer device was higher than that of the commercial assay, suggesting that Oxalometer has a better t for the data. Furthermore, using clinical goat urines, the Oxalometer reproduced the same results as the commercial assay, suggesting that our new device can accurately and consistently detect urinary oxalate levels. Health Study II (n = 101,824 younger women) reported that higher dietary oxalate was associated with increased stone risk in men and older women (16). When researchers focused on those with a recurrent stone disease in this cohort (~ 6,000 individuals), stone formation risk signi cantly rose with increasing urine calcium and oxalate excretion (17). Of these two factors, Siener et al. demonstrated that increased oxalate excretion, not calcium, was the major urinary risk factor for relapse during follow-up with recurrent calcium oxalate stones (18). This nding was further corroborated by in-vivo and in-vitro studies that showed small increases in urinary oxalate could have a confounding effect on the factors which in uence nucleation, crystal growth, and aggregation of calcium oxalate in urine (19).
Increased awareness of oxalate-based diseases and the prevalence of urolithiasis in animals and humans have catalyzed a need for simple, rapid assays that provide temporal-based monitoring of oxalate levels in urine and/or blood. Current methods for determining oxalate, however, are relatively complex, labor-intensive, and require specialized equipment in the laboratory setting. In general, a spectrophotometer is needed to measure the intensity of indamine dye at 590 nm as an end-product of oxalate, 3-methyl-2-benzothiazolinone hydrazone (MBTH), and 3-dimethylaminobenzoic acid (DMAB) catalyzed by oxalate oxidase and peroxidase (20)(21)(22). A modi ed HPLC spectrophotometric detector can be used to improve sensitivity (23), while ion chromatography can be used to determine oxalate and multiple other ion concentrations (24, 25). The current oxalate test is relatively expensive and performed by skilled technicians using costly equipment. With turnaround times between 3-7 days, it is not a pointof-care test, resulting in inpatient treatment plans that are di cult to prescribe and implement. While testing for oxalate would seem an essential standard of care in stone disease, urinary oxalate testing is not performed routinely.

Conclusions
In this study, we developed Oxalometer device to determine the level of urinary oxalate accurately. The critical feature of the device is the simplicity in manufacturing and application and its convenience and timeliness to run in-clinic or at home. Routine monitoring would enable animal owners and veterinarians to become aware of the animal's oxalate levels and to take measurements quickly to prevent or reduce the possible recurrence of kidney stones or other oxalate-related diseases through changes in diet and uid intake. One major drawback of the study is the small sample size. Therefore, additional studies with larger cohorts will be needed to validate the results furthers.   Figure 1 Oxalometer assembly schematic: Using 3D printing, the Oxalometer was designed with a urine ltration unit coupled with an enzyme-dye preloaded cuvette to allow for a simple and quick urinalysis. The Oxalometer ltration cartridge was developed using the Trimble Sketchup 3D design software, while the column design consisted of a standard 1.5 mL polystyrene disposable 1cm path length cuvette interfaced with a slanted grate. Glass, the ber lter paper was adhered to the inside bottom face of the columns, and mesh activated charcoal was added into each column to create the ltration column. This device employs the properties of cohesion and adhesion to the cuvette surface to homogenously draw uid through the lter.