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Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry
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. Howe...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055858/ https://www.ncbi.nlm.nih.gov/pubmed/29517154 http://dx.doi.org/10.1002/bmc.4238 |
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author | Jamalpoor, Amer Sparidans, Rolf W. Pou Casellas, Carla Rood, Johannes J.M. Joshi, Mansi Masereeuw, Rosalinde Janssen, Manoe J. |
author_facet | Jamalpoor, Amer Sparidans, Rolf W. Pou Casellas, Carla Rood, Johannes J.M. Joshi, Mansi Masereeuw, Rosalinde Janssen, Manoe J. |
author_sort | Jamalpoor, Amer |
collection | PubMed |
description | 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 μmol/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. |
format | Online Article Text |
id | pubmed-6055858 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-60558582018-07-30 Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry Jamalpoor, Amer Sparidans, Rolf W. Pou Casellas, Carla Rood, Johannes J.M. Joshi, Mansi Masereeuw, Rosalinde Janssen, Manoe J. Biomed Chromatogr Research Articles 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 μmol/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. John Wiley and Sons Inc. 2018-04-15 2018-08 /pmc/articles/PMC6055858/ /pubmed/29517154 http://dx.doi.org/10.1002/bmc.4238 Text en © 2018 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Research Articles Jamalpoor, Amer Sparidans, Rolf W. Pou Casellas, Carla Rood, Johannes J.M. Joshi, Mansi Masereeuw, Rosalinde Janssen, Manoe J. Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
title | Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
title_full | Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
title_fullStr | Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
title_full_unstemmed | Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
title_short | Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
title_sort | quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055858/ https://www.ncbi.nlm.nih.gov/pubmed/29517154 http://dx.doi.org/10.1002/bmc.4238 |
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