N-acetyl-l-cysteine (NAC), a well-known antioxidant, has been successfully used as adjuvant therapy for late-stage childhood cerebral adrenoleukodystrophy (c-ALD); however, the mechanisms of NAC action are poorly understood. Previous research indicates that NAC serves as a precursor to l-cysteine (Cys), the rate-limiting substrate in the biosynthesis of glutathione (GSH), a potent, endogenous antioxidant. We hypothesized that NAC acts by liberating protein-bound Cys in plasma in an NAC concentration-dependent manner, which increases unbound Cys available for GSH biosynthesis. Human plasma was incubated for 1 h with varying, clinically relevant concentrations of NAC (0-1000 μg/mL). The effect of this interaction over time was evaluated by incubating plasma for 5-90 min with 100 μg/mL NAC. Unbound and bound Cys and NAC were separated by ultrafiltration, and concentrations were measured using high-performance liquid chromatography-mass spectrometry. Significant increases in unbound Cys were observed with increasing NAC concentrations. Also, Cys plasma protein binding decreased from 85% (10 μg/mL NAC) to approximately 0% (1000 μg/mL). Total endogenous Cys was 66% unbound at 5 min after incubation. These results demonstrate that NAC liberates endogenous, protein-bound Cys in human plasma at clinically relevant NAC concentrations. A greater understanding of NAC actions will aid in the optimization of NAC therapy including its use in c-ALD.
- HPLC (high-performance/pressure liquid chromatography)
- Protein binding