Improvement of the cystine measurement in granulocytes by liquid chromatograhy-tandem mass spectrometry.
BACKGROUND
Cystinosis is an autosomal recessive genetic disorder due to mutations in CTNS gene, which causes a defect of cystinosin, impairing the transport of free cystine out of lysosomes and causing irreversible damage to various organs, particularly the kidney. The diagnosis of Cystinosis is carried out by measuring the cystine content in leucocytes. Accurate quantification of cystine is of capital importance not only for the diagnosis, but also for monitoring the effectiveness of cysteamine treatment. Here we describe an improvement to measure cystine in granulocytes using high-pressure-liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS).


METHODS
Granulocytes were isolated from heparinized blood by isopycnic centrifugation. After lysis and deproteinization, the concentration of di-butylated cystine was measured by HPLC-ESI-MS/MS, using deuterium labelled, d6-cystine, as internal standard.


RESULTS
The assay was linear to at least 50 μmol/L with a good precision. Within-day and between-day coefficients of variation were about 6%. The recovery was higher than 98%. Control values were clearly distinguishable from pathological levels, even if patients were under treatment. A good correlation was observed with cystine binding protein (CBP) assay, one of the most sensitive and specific methods.


CONCLUSIONS
This method results in good analytical performance, and is useful for diagnosis and follow up of Cystinosis. This method offers several advantages over the CBP assay: it is less expensive, easier, quicker and it does not require radioactivity. In addition, when comparing with the HPLC-ESI-MS/MS method previously described by Chabli et al. 2006, our assay exhibits more sensitivity and is faster.
Sección de Errores Congénitos del Metabolismo-IBC, Servicio de Bioquímica y Genética Molecular, Hospital Clínic. CIBER de enfermedades raras (CIBERER), IDIBAPS, Barcelona, Spain.
Judit
J
García-Villoria
Jose Maria
JM
Hernández-Pérez
Angela
A
Arias
Antonia
A
Ribes
D016022:Case-Control Studies; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003543:Cysteamine / Q000627:therapeutic use; D003553:Cystine / Q000097:blood*; D003554:Cystinosis / Q000175:diagnosis / Q000188:drug therapy; D006098:Granulocytes / Q000737:chemistry*; D006801:Humans; D007223:Infant; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D003553:Cystine; D003543:Cysteamine