Measurement of cystine in granulocytes using liquid chromatography-tandem mass spectrometry.
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.


METHODS
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.


CONCLUSIONS
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.
Chabli
A
A
AP-HP, Hôpital Necker-Enfants Malades, Service de Biochimie B, Université Paris V, 75015 Paris, France.
Aupetit
J
J
Raehm
M
M
Ricquier
D
D
Chadefaux-Vekemans
B
B
D002853:Chromatography, Liquid / Q000379:methods*; D003553:Cystine / Q000032:analysis*; D003554:Cystinosis / Q000175:diagnosis; D029968:Escherichia coli Proteins / Q000378:metabolism; D006098:Granulocytes / Q000737:chemistry*; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods*
D029968:Escherichia coli Proteins; D003553:Cystine