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Does N-acetylcysteine act as an intracellular cysteine precursor in human erythrocytes?

J.E. Raftos,1 S. Whillier1 and P.W. Kuchel,2 1Department of Biological Sciences, Macquarie University, NSW 2109, Australia and 2School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia.

In sickle cell disease haemoglobin S oxidises at a rate of 6% per day releasing reactive oxygen species that reduce the concentration of the important antioxidant glutathione (GSH). Oral administration of N-acetylcysteine (NAC) has been shown to increase the GSH concentrations in sickle red blood cells (RBCs). RBCs with higher GSH concentrations are less likely to become irreversibly sickled and patients with low populations of irreversibly sickled RBCs have fewer vaso-occlusive episodes (Pace et al., 2003). GSH is synthesized from glutamate, cysteine and glycine and in RBCs the rate of synthesis is controlled by feedback inhibition of GSH. When RBCs are depleted of GSH, the supply of cysteine from the plasma becomes rate limiting. It has been assumed that on oral administration NAC enters the RBCs where it is deacetylated to release the cysteine required for GSH production. The aim of this study was to determine whether this was in fact the mode of action of NAC.

In vitro, both extracellular cysteine and NAC can provide a source of cysteine for GSH production in RBCs that have been depleted of 80% of their initial GSH concentration by incubation with 1-chloro-2,4-dinitrobenzene. However, while 1.0 mM NAC was required for the maximum rate of GSH synthesis cysteine at only 60 μM supported synthesis at the same rate (Raftos et al., 2007). To determine why NAC was a relatively inefficient cellular cysteine precursor, we measured the kinetics of the uptake and deacetylation of NAC.

GSH depleted RBCs were incubated in a range of extracellular NAC concentrations up to 10 mM and NAC uptake was measured as the initial rate of increase in total RBC thiols. Uptake did not approach saturation at 10 mM extracellular NAC and the first order rate constant was 2.04 ± 0.07 min-1. When the experiment was repeated with 50 μM DNDS or 5 μM DIDS, NAC uptake was inhibited by 58% indicating that the majority of the NAC enters by low affinity transport via the anion exchange protein. This is not surprising as NAC carries a negative charge at plasma pH. The Km and Vmax for deacetyaltion of NAC measured using 1H NMR in concentrated RBC haemolysates were 1.49 ± 0.16 mM and 1.61 ± 0.018 μmol L-1 min-1, respectively (Raftos et al., 2007). Oral NAC at the dosage sufficient to increase GSH concentration in the RBCs of sickle cell patients produces a plasma concentration of only 10 μM while free cysteine increases by 50 μM (Burgunder et al., 1989). At this concentration the measured rates of uptake and deacetylation of NAC are too slow to supply intracellar cysteine rapidly enough to maintain GSH synthesis. We conclude that NAC acts in the plasma by breaking thiol bonds to replace bound cysteine. The freed plasma cysteine can then enter the RBCs to support GSH synthesis.

Burgunder JM, Varriale A & Lauterburger BH. (1989) European Journal of Clinical Pharmacology, 36: 127-31.

Pace BS, Shartava A, Pack-Mabien A, Mulekur M, Ardia A & Goodman SR. (2003) American Journal of Hematology, 73 26-32.

Raftos JE, Whillier S, Chapman BE & Kuchel PW. (2007) International Journal of Biochemistry and Cell Biology, 39: 1698-1706.