Clinical Chemistry
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Clinical Chemistry 45: 2224-2228, 1999;
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(Clinical Chemistry. 1999;45:2224-2228.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

New Method for Determining Cystine in Leukocytes and Fibroblasts

Adriana de Graaf-Hess1, Frans Trijbels1 and Henk Bloma,1

1 Laboratory of Paediatrics and Neurology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
a Author for correspondence. Fax 31-243618900; e-mail H.Blom{at}ckslkn.azn.nl

Background: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required.

Methods: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection.

Results: The assay was linear to 200 µmol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 µmol/L. Added cysteine, up to 200 µmol/L, was completely removed, and recovery of added cystine was 69–86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 °C

Conclusions: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.




The following articles in journals at HighWire Press have cited this article:


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E. Levtchenko, A. de Graaf-Hess, M. Wilmer, L. van den Heuvel, L. Monnens, and H. Blom
Altered status of glutathione and its metabolites in cystinotic cells
Nephrol. Dial. Transplant., September 1, 2005; 20(9): 1828 - 1832.
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 Clin. Chem.Home page
E. Levtchenko, A. de Graaf-Hess, M. Wilmer, L. van den Heuvel, L. Monnens, and H. Blom
Comparison of Cystine Determination in Mixed Leukocytes vs Polymorphonuclear Leukocytes for Diagnosis of Cystinosis and Monitoring of Cysteamine Therapy
Clin. Chem., September 1, 2004; 50(9): 1686 - 1688.
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J. M. Geelen, L. A. H. Monnens, and E. N. Levtchenko
Follow-up and treatment of adults with cystinosis in the Netherlands
Nephrol. Dial. Transplant., October 1, 2002; 17(10): 1766 - 1770.
[Abstract] [Full Text] [PDF]


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