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 mo nitor treatment with cysteamine, adequate measurements of cystine concentra tions 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 cyst eine after reduction with sodium borohydride and derivatization with monobr omobimane, followed by separation with automated HPLC and fluorescence dete ction. Results: The assay was linear to 200 mu mol/L cysteine. Within-run and day- to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 mu mol/L. Added cysteine, up to 200 mu mol/L, was completely removed, and rec overy 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 reacti ons 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. (C) 1999 American Association fo r Clinical Chemistry.