Abstract
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 N-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 (R2 > 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 × 106 cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.
Highlights
Introduction by HPLCFluorescence Detection.Biological thiols, such as cysteine (Cys) and glutathione (GSH), are ubiquitous in biological tissues and fluids (Figure 1a)
The alteration of intracellular thiol concentrations has been correlated with health conditions, such as liver disease, cancer, and cardiovascular disease [1,2,3]
The detection of intracellular thiol imbalance is important for elucidating the role of thiols in diseases
Summary
Biological thiols, such as cysteine (Cys) and glutathione (GSH), are ubiquitous in biological tissues and fluids (Figure 1a). Thiols play important roles in various intracellular reactions against oxidative stress, such as in redox equilibria. The alteration of intracellular thiol concentrations has been correlated with health conditions, such as liver disease, cancer, and cardiovascular disease [1,2,3]. Lung cancer causes a decrease in the intracellular concentrations of Cys and cysteinylglycine (CysGly), while prostate cancer leads to increased levels of Cys [4,5]. The detection of intracellular thiol imbalance is important for elucidating the role of thiols in diseases
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