New method for determining cystine in leukocytes and fibroblasts.
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.
de Graaf-Hess
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Laboratory of Paediatrics and Neurology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Trijbels
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Blom
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D000328:Adult; D001894:Borohydrides; D001643:Bridged Bicyclo Compounds; D002457:Cell Extracts / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003543:Cysteamine / Q000627:therapeutic use; D003553:Cystine / Q000032:analysis* / Q000097:blood; D003554:Cystinosis / Q000175:diagnosis / Q000188:drug therapy; D005033:Ethylmaleimide; D005347:Fibroblasts / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008875:Middle Aged; D015203:Reproducibility of Results
D001894:Borohydrides; D001643:Bridged Bicyclo Compounds; D002457:Cell Extracts; D003553:Cystine; D003543:Cysteamine; C025364:sodium borohydride; D005033:Ethylmaleimide; C028381:monobromobimane