- 作者：Judit Garcí ; a-Villoria ; Jose Maria Herná ; ndez-Pé ; rez ; Angela Arias ; Antonia Ribes ; aribes@clinic.ub.es
- 关键词：HPLC-ESI-MS/MS ; Cystinosis ; Cystine in granulocytes
- 刊名：Clinical Biochemistry
- 出版年：2013
- 出版时间：February, 2013
- 年：2013
- 卷：46
- 期：3
- 页码：271-274
- 全文大小：418 K
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).Method
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 ¦Ìmol/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.
Conclusion
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.