Quantification of Intracellular Thiols by HPLC-Fluorescence Detection
Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-04-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/8/2365 |
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| author | Hiroki Yamamoto Takuya Fujiwara Takashi Funatsu Makoto Tsunoda |
| author_facet | Hiroki Yamamoto Takuya Fujiwara Takashi Funatsu Makoto Tsunoda |
| author_sort | Hiroki Yamamoto |
| collection | DOAJ |
| description | Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and <i>N</i>-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R<sup>2</sup> > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 10<sup>6</sup> cells. The time-dependent effect of a thiol scavenger, viz. <i>N</i>-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism. |
| first_indexed | 2024-03-10T12:11:53Z |
| format | Article |
| id | doaj.art-c8f6b979ee8c4b99afb9533cf2164cc6 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T12:11:53Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-c8f6b979ee8c4b99afb9533cf2164cc62023-11-21T16:09:49ZengMDPI AGMolecules1420-30492021-04-01268236510.3390/molecules26082365Quantification of Intracellular Thiols by HPLC-Fluorescence DetectionHiroki Yamamoto0Takuya Fujiwara1Takashi Funatsu2Makoto Tsunoda3Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 1130033, JapanGraduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 1130033, JapanGraduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 1130033, JapanGraduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 1130033, JapanBiothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and <i>N</i>-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R<sup>2</sup> > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 10<sup>6</sup> cells. The time-dependent effect of a thiol scavenger, viz. <i>N</i>-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.https://www.mdpi.com/1420-3049/26/8/2365cysteineglutathionederivatizationoxidative stress |
| spellingShingle | Hiroki Yamamoto Takuya Fujiwara Takashi Funatsu Makoto Tsunoda Quantification of Intracellular Thiols by HPLC-Fluorescence Detection Molecules cysteine glutathione derivatization oxidative stress |
| title | Quantification of Intracellular Thiols by HPLC-Fluorescence Detection |
| title_full | Quantification of Intracellular Thiols by HPLC-Fluorescence Detection |
| title_fullStr | Quantification of Intracellular Thiols by HPLC-Fluorescence Detection |
| title_full_unstemmed | Quantification of Intracellular Thiols by HPLC-Fluorescence Detection |
| title_short | Quantification of Intracellular Thiols by HPLC-Fluorescence Detection |
| title_sort | quantification of intracellular thiols by hplc fluorescence detection |
| topic | cysteine glutathione derivatization oxidative stress |
| url | https://www.mdpi.com/1420-3049/26/8/2365 |
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