Cyanide-Nitroprusside Colorimetric Assay: A Rapid Colorimetric Screen for Urinary Cystine

Abstract

Background Cystinuria is an autosomal recessive disorder resulting in poor proximal tubule reabsorption of cystine in the nephron, increasing the risk of cystine stone formation. A fast, inexpensive assay to screen for urinary cystine is needed because cystine stones are difficult to noninvasively differentiate from more common calcium-containing ones. Tandem mass spectrometry (MS/MS) is sensitive and specific but is labor-intensive and costly. Alternatively, a colorimetric assay is fast and cost-effective; however, creatinine interference is an issue. Methods A published cyanide-nitroprusside colorimetric assay was modified for a high-throughput microplate format. Creatinine interference was reduced using 0.1 mol/L PBS and a standard reaction time of 60 s and was further corrected using a formula derived from the slope of multiple creatinine standard curves. Results The limit of blank was determined to be 2.6 mg/L, the limit of detection 11.9 mg/L, and the limit of quantitation 15.3 mg/L. The analytic measurement range was established as 15.3–100 mg/L cystine. Intraassay and interassay CV was calculated to be 9.6% and 8.0%, respectively, for a high-level cystine concentration (83.6 mg/L). Low-level cystine (36.4 mg/L) intraassay and interassay CV was determined to be 18.1% and 17.6%, respectively. Passing–Bablok regression analysis of colorimetric vs LC-MS/MS results revealed a slope of 1.10 and y intercept of −7.14 mg/L, with an overall bias of 2% by Bland–Altman plot analysis. Conclusions We analytically validated a rapid colorimetric assay suitable to quantify urinary cystine. The effect of thiol drugs on this assay remains to be determined.

Chemical Modification of Tiopronin for Dual Management of Cystinuria and Associated Bacterial Infections

“…The standard sodium nitroprusside assay (Figure a) quantifies cystine solubility in urine samples. , During the study, it was found that as the pH increased from 5.5 to 7.5, cystine solubility in a healthy patient urine sample increased in the presence of both tiopronin and tiopronin-NO (Figure b–d). This indicates that thiol exchange occurs more quickly at pH 7.5 and reaches equilibrium at 48 h since alkali treatments break down cystine stones .…”