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Deprecated: Implicit conversion from float 265.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Biomed+Chromatogr 2018 ; 32 (8): ä Nephropedia Template TP
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Quantification of cystine in human renal proximal tubule cells using liquid chromatography?tandem mass spectrometry #MMPMID29517154
Jamalpoor A; Sparidans RW; Pou Casellas C; Rood JJ; Joshi M; Masereeuw R; Janssen MJ
Biomed Chromatogr 2018[Aug]; 32 (8): ä PMID29517154show ga
Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body, causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all of the symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high?performance liquid chromatography?tandem mass spectrometry (HPLC?MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N?ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium?labeled cystine?D4, as the internal standard. The assay developed demonstrated linearity to at least 20??mol/L with a good precision. Accuracies were between 97.3 and 102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared with cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells.