BACKGROUND: Cystinosis is a rare autosomal recessive disorder characterized by an accumulation of intralysosomal cystine due to a defect in cystine transport across the lysosomal membrane. This disorder can be treated specifically using high doses of cysteamine. Accurate measurement of intracellular cystine content is necessary for the diagnosis and monitoring of treatment with cysteamine. Here we describe a new method to measure intracellular cystine. It relies on a liquid chromatography-tandem mass spectrometry assay. We compare this novel method with the cystine-binding protein assay. METHOD: Cells were isolated and lysed in the presence of N-ethylmaleimide to avoid interference from cysteine. After deproteinization, addition of stable isotope d6 cystine and butylation, cystine was measured using an API 3000 MSMS. RESULTS: The cystine assay was linear to at least 50 micromol/L. Within-run and between-run coefficients of variation were 2.9% and 5.7% respectively. CONCLUSION: It is possible to measure very low concentrations of intracellular cystine with liquid chromatography-tandem mass spectrometry. The results obtained with this novel method correlate very well with those obtained using the cystine-binding protein assay.
BACKGROUND:Cystinosis is a rare autosomal recessive disorder characterized by an accumulation of intralysosomal cystine due to a defect in cystine transport across the lysosomal membrane. This disorder can be treated specifically using high doses of cysteamine. Accurate measurement of intracellular cystine content is necessary for the diagnosis and monitoring of treatment with cysteamine. Here we describe a new method to measure intracellular cystine. It relies on a liquid chromatography-tandem mass spectrometry assay. We compare this novel method with the cystine-binding protein assay. METHOD: Cells were isolated and lysed in the presence of N-ethylmaleimide to avoid interference from cysteine. After deproteinization, addition of stable isotope d6 cystine and butylation, cystine was measured using an API 3000 MSMS. RESULTS: The cystine assay was linear to at least 50 micromol/L. Within-run and between-run coefficients of variation were 2.9% and 5.7% respectively. CONCLUSION: It is possible to measure very low concentrations of intracellular cystine with liquid chromatography-tandem mass spectrometry. The results obtained with this novel method correlate very well with those obtained using the cystine-binding protein assay.
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Authors: Virginie Janssens; Héloïse P Gaide Chevronnay; Sandrine Marie; Marie-Françoise Vincent; Patrick Van Der Smissen; Nathalie Nevo; Seppo Vainio; Rikke Nielsen; Erik I Christensen; François Jouret; Corinne Antignac; Christophe E Pierreux; Pierre J Courtoy Journal: J Am Soc Nephrol Date: 2019-09-23 Impact factor: 10.121
Authors: Martijn J Wilmer; Joost P Schoeber; Lambertus P van den Heuvel; Elena N Levtchenko Journal: Pediatr Nephrol Date: 2010-08-24 Impact factor: 3.714
Authors: Mohamed A Elmonem; Samuel H Makar; Lambertus van den Heuvel; Hanan Abdelaziz; Safaa M Abdelrahman; Xavier Bossuyt; Mirian C Janssen; Elisabeth Am Cornelissen; Dirk J Lefeber; Leo Ab Joosten; Marwa M Nabhan; Fanny O Arcolino; Fayza A Hassan; Héloïse P Gaide Chevronnay; Neveen A Soliman; Elena Levtchenko Journal: Orphanet J Rare Dis Date: 2014-11-19 Impact factor: 4.123