Measurement Of Cystine In Granulocytes Using Liquid Chromatography-Tandem Mass Spectrometry In Iranian Children

Background and aim: Cystinosis is a rare autosomal recessive disorder characterized by an accumulation of intra lysosomal cystine due to a defect in cystine transport across the lysosomal membrane. This results in cystine accumulation and crystallization in the cells causing widespread tissue damage. This disorder can be treated specifically using high doses of cysteamine. Although the pathogenesis is not completely elucidated, research in the field of cystinosis has been spurred in the late 1990s by the identification of the CTNS gene, which encodes for cystinosin, a lysosomal cystine carri .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 In the 1960s, Schneider et al. showed for the first time increased intracellular cystine levels in granular fractions of leukocytes from patients, identifying cystinosis as a cystine storage disease that was associated with Fanconi syndrome. Cysteamine (Cystagon) depletes lysosomal cystine by a disulfide exchange reaction, resulting in the equimolar generation of a cysteine-cysteamine molecule and a molecule of cysteine . Both compounds can exit lysosomes via system c transporters, bypassing the defective cystinosin pathway . The efficacy of cysteamine can be monitored in clinical practice by measuring intracellular cystine levels in polymorphonuclear (PMN) leukocytes and is considered to be a reflection of tissue cystine content. Over the years, several methods have been developed to measure intracellular cystine levels for the diagnosis of cystinosis and for monitoring cysteamine treatment. Currently, HPLC and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) are the most widely used. LC-MS/MS is the most sensitive method (allows detection of as little as 0.02 μmol cystine/l, compared with 0.15 μmol cystine/l using HPLC) and has recently been developed for cystine determination in granulocytes Materials And Method: In order to measure final concentration of cystine in nanomoles of half cystine by milligrams of proteins it is necessary to measure the amountof cystine in lyzed RBCs and the protein content of cell derbis both analysis are equally important 7-10 ml venous blood in ACD or heparin or EDTA or monitoring treatment sample should be collected 6 hr after cystamine treatment was started Whole blood is conserved at room temperture until sample is prepared PMN cells are prepared whitin 24 hr according to guidelines from group cystine in white blood cells isolation of PMNs increases the sensitivity for detection of heterozygotes after simple prepration and leukocyte isolation Then 50ul of sample is injected into LC_MS/chromatographic seperation is achieved on an agilent 1100 system using a C18 ODB column the column elute is injected directly in to Applied Bio system API 13000 MS/MS This method allows to quantify amount of cystine in lower than 0.25 nmol ½ cystine/mg protein in normal subjects 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.Results: We analysed leukocyte cysteine level of 308 patients( 146 Females and 162 Males) with mean age 8. 5 yr (range 0.01 – 71 yr) reffered to our laboratory between 2015-2017|. The results were shown in the table. The mean cystin level of leukocyted was 0.64 (range 0- 6.54). Which shows a frequency of about 50% for patients with cysteine leve l> 0.4 Conclusion: Since efficacy of cysteamine can be monitored in clinical practice by measuring intracellular cystine levels in polymorphonuclear (PMN) leukocytes and is considered to be a reflection of tissue cystine content we recommend this method for diagnosing of the disease and follow up of patients receiving Cystamine to evaluate the efficacy of treatment