TY - JOUR
T1 - Cyanide-Nitroprusside Colorimetric Assay
T2 - A Rapid Colorimetric Screen for Urinary Cystine
AU - Brady, Clayton T.
AU - Giesen, Callen D.
AU - Voskoboev, Nikolay
AU - Chirackal, Robin S.
AU - Gavrilov, Dimitar K.
AU - Flanagan, Ryan M.
AU - Lieske, John C.
N1 - Publisher Copyright:
© 2017 American Association for Clinical Chemistry.
PY - 2017/7/1
Y1 - 2017/7/1
N2 - 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.
AB - 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.
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U2 - 10.1373/jalm.2016.022582
DO - 10.1373/jalm.2016.022582
M3 - Article
C2 - 33636959
AN - SCOPUS:85102160157
SN - 2576-9456
VL - 2
SP - 55
EP - 64
JO - Journal of Applied Laboratory Medicine
JF - Journal of Applied Laboratory Medicine
IS - 1
ER -