Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

Area of Science:

• Biochemistry
• Analytical Chemistry
• Cell Biology

Background:

• Biothiols, including cysteine and glutathione, are crucial for intracellular redox balance and combating oxidative stress.
• Understanding intracellular thiol levels is vital for studying cellular health and disease mechanisms.

Purpose of the Study:

• To develop and validate a high-performance liquid chromatography (HPLC) method for sensitive intracellular thiol quantification.
• To apply the developed method for analyzing thiol concentrations in human leukemia cells and assessing responses to thiol scavengers.

Main Methods:

• Thiols were derivatized using ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), a thiol-specific fluorescent reagent.
• Separation of six distinct SBD-thiols was achieved within 30 minutes using reversed-phase HPLC on an InertSustain AQ-C18 column with a citric buffer/methanol mobile phase.
• Calibration curves demonstrated excellent linearity (R² > 0.999) for all quantified thiols.

Main Results:

• The method successfully quantified intracellular thiol concentrations in human chronic myelogenous leukemia K562 cells, ranging from 5.5-153 pmol/1 × 10⁶ cells.
• The time-dependent effects of the thiol scavenger N-ethylmaleimide on intracellular thiol levels were successfully monitored.
• High linearity (R² > 0.999) was achieved for all SBD-thiols, indicating method reliability.

Conclusions:

• The developed HPLC-fluorescence method provides a robust and sensitive tool for intracellular thiol analysis.
• This technique is valuable for investigating the role of intracellular sulfur metabolism and redox homeostasis in biological systems.
• The method enables precise measurement of thiol dynamics in cellular models and under various experimental conditions.