| New method for determining cystine in leukocytes and fibroblasts. | |
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MedLine Citation:
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PMID: 10585356 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/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 degrees 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. |
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Authors:
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A de Graaf-Hess; F Trijbels; H Blom |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Clinical chemistry Volume: 45 ISSN: 0009-9147 ISO Abbreviation: Clin. Chem. Publication Date: 1999 Dec |
Date Detail:
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Created Date: 1999-12-17 Completed Date: 1999-12-17 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 9421549 Medline TA: Clin Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 2224-8 Citation Subset: IM |
Affiliation:
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Laboratory of Paediatrics and Neurology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Adult Bicyclo Compounds Borohydrides Cell Extracts / chemistry Chromatography, High Pressure Liquid / methods* Cysteamine / therapeutic use Cystine / analysis*, blood Cystinosis / diagnosis, drug therapy Ethylmaleimide Fibroblasts / metabolism* Fluorometry Humans Leukocytes / metabolism* Middle Aged Reproducibility of Results |
| Chemical | |
Reg. No./Substance:
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0/Bicyclo Compounds; 0/Borohydrides; 0/Cell Extracts; 128-53-0/Ethylmaleimide; 16940-66-2/sodium borohydride; 56-89-3/Cystine; 60-23-1/Cysteamine; 71418-44-5/monobromobimane |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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