Development of novel point-of-care-testing for oxalate detection in urine based on oxalate-binding ligands

Abstract

Kidney stone disease is the third most common urinary tract disease worldwide that is more prevalent in the tropics. In Thailand, the highest stone prevalence up to 17% is reported in the Northeast. The disease is highly recurrent with a recurrence rate of about 50% within 5-10 years. The most common type of kidney stones is calcium oxalate (CaOx), and an increased urinary oxalate excretion is an important risk factor. Most of kidney stone patients have a hyperoxaluria condition (urinary oxalate >40 mg/day or >0.46 mmol/day). Therefore, urinary oxalate measurement has been widely used for estimating the risk of CaOx stone formation and predicting the stone recurrence. In this study, we first aimed to develop the new method for oxalate measurement in human urine using a newly discovered compound (Cu-nat5-BPG complex) that was shown to specifically bind to oxalate in condition 80% acetonitrile aqueous solution buffered with 10 mM HEPES pH 7.0. Due to the complex matrix in urine sample, we found that the binding of oxalate to Cu-nat5-BPG complex was significantly interfered by some common urinary substances, i.e., creatinine, calcium, and magnesium. Moreover, the level of oxalate excretion in human urine is usually less than 40 mg/day, and this level was below the detection limit of the Cu-nat5-BPG complex method. We, therefore, shifted the goal to establishment of urinary oxalate measurement using oxalate oxidase (OxOx) enzyme coupled with horse radish peroxidase. We cloned and expressed the recombinant barley oxalate oxidase (rBarley-OxOx) in yeasts, Pichia pastoris. Our data showed that we successfully cloned and expressed the rBarley-OxOx in P. pastoris, and the purified recombinant proteins were confirmed to be rBarley-OxOx by mass spectrometry. However, the yield of rBarley-OxOx was relatively small, and it was not feasible to upscale for the further use. We had changed the study plan again from producing our own OxOx to buying the OxOx that commercially available. We purchased the OxOx enzyme (purified from barley seedings) from the Roche Diagnostics and established the procedure for oxalate detection in urine samples. Oxalate concentrations were measured in 191 urine samples collected from Maha Sarakham Province, Thailand. The result found that the urinary oxalate level in non-stone former (NS-group, n=122) was significantly lower than that in the kidney stone patients (KS-group, n=69). Urinary oxalate level measured by the OxOx method was positively correlated with the urinary oxalate level measured by the HPLC method. In conclusion, the present study demonstrated that the OxOx enzymatic method was a more specific and effective method for measuring urinary oxalate than the Cu-nat5-BPG complex method. Based on our findings, cloning and expressing the plant OxOx in yeasts was laboratory feasible, but it was not recommended for further commercial use mainly due to a relatively low yield.