Measurement of precipitated calcium citrate in urine by murexide-based indicator displacement assay and its clinical utility in calcium oxalate urolithiasis

Abstract

Urinary stone, mainly composed of calcium oxalate (CaOx), is a longstanding urologic condition with high prevalence and recurrence rates. Dietary habits, specifically increased consumption of high oxalate diet and low intake of citrate-containing foods, lead to hyperoxaluria and hypocitraturia that directly contribute to CaOx stone formation. Measurements of urinary oxalate and citrate are essential to estimate the risk of CaOx stone formation. Currently, high-performance liquid chromatography is used for urinary citrate measurement, but it is complicated and requires well-trained technicians. We hypothesized that instead of directly measuring citrate in urine, measurement of calcium citrate (CalCit) precipitated in urine challenging with excessive calcium would be an alternative method to distinguish CaOx stone patients from non-stone subjects. In this study, we developed a new test for measuring precipitated CalCit in supersaturated urine. Supersaturation of urine was induced by adding excessive amount of calcium chloride. CalCit was precipitated by ethanol and redissolved by hydrochloric acid. Citrate content in the redissolved CalCit was determined by an indicator-displacement assay (IDA) using murexide (Mrx) as an indicator dye. Method validation was performed. Clinical validation was subsequently conducted to evaluate whether this method had sufficient performance for diagnosing CaOx urolithiasis. We successfully established the procedure of the murexide-based indicator displacement assay (Mrx IDA) for measuring precipitated CalCit. The measurement interval of the Mrx IDA was between 0.4 and 1.4 mM. Limit of detection (LoD), lower limit of quantitation (LoQ), and upper LoQ were 0.08 mM, 0.4 mM, and 1.4 mM, respectively. Repeatability, reproducibility, and accuracy were well acceptable. For clinical validation, the test was performed in 24-hour urine samples obtained from 122 non-stone subjects and 45 CaOx stone patients. Receiver operating characteristic (ROC) analysis revealed an area under ROC curve of 0.8227 (95% CI: 0.7522-0.8931) for distinguishing CaOx stone patients from non-stone subjects. At the cutoff value of 632 µM, the test provided sensitivity, specificity, positive and negative predictive values (PPV and NPV), positive and negative likelihood ratios (LH+ and LH-), and accuracy of 84.44%, 70.49%, 74.25%, 51.35%, 92.47%, 2.41, 0.26, and 74.90%, respectively. Conclusion, we successfully developed the Mrx IDA test for measuring urinary precipitated CalCit. Method validation indicated that this newly established test was precise and accurate. Clinical accuracy testing showed high sensitivity and NPV, suggested that the Mrx IDA test was useful to rule out the CaOx urolithiasis. Therefore, the measurement of urinary precipitated CalCit developed in this study is clinically promising to be used as a screening test for CaOx urolithiasis.