Antioxidant potential of anaerobic human plasma: role of serum albumin and thiols as scavengers of carbon radicals

Extracellular fluids contain low-molecular-weight antioxidants that are actively involved in the defense against reactive oxygen species. The antioxidant activity of these compounds is largely due to their ability to trap oxygen radicals. Less known is the ability of extracellular antioxidants to scavenge carbon-centered free radicals (C-radicals). These radicals can be involved in the damage under hypoxic/anoxic conditions as well as in ischemia/reperfusion injury. We studied the reactivity of some plasma antioxidants toward a water-soluble C-radical generated by the azocompound 2,2'-azobis(2-amidinopropane) hydrochloride (AAP) under anaerobic conditions. The AAP C-radical in plasma was trapped by the spin trap 3,5-dibromo-4-nitrosobenzene-sulfonic acid (DBNBS) and produced a DBNBS radical. The scavenging properties of urate, cysteine, glutathione, natural amino acids, and serum albumin were assessed by the inhibition of the intensity of DBNBS radical. The antioxidant activity of ascorbate and that of vitamin E was measured directly by the formation of their free radicals. Urate, vitamin E and non-SH amino acids were ineffective and ascorbate was a poor scavenger of AAP C-radical. At variance, cysteine and glutathione (0.1-1.0 mM) were effective scavengers of AAP C-radicals and, importantly, protected plasma ascorbate from oxidation under both aerobic or anaerobic conditions. Our data show that ascorbate in aerobic plasma can reduce vitamin E radical and the oxidized ascorbate may be recycled by a thiol antioxidant cycle. Low-molecular-weight antioxidants accounted only partially for plasma scavenging activity of C-radicals. Plasma strongly reduced the intensity of DBNBS radical and, after dialysis, its activity was reduced by approximately 10%. Serum albumin showed an antioxidant activity comparable to dialyzed plasma. Also the cysteine residue of serum albumin was an efficient scavenger of C-radicals as shown by approximately 20% decrease in the protein scavenging activity after thiol alkylation. These results suggest that elevation in the concentration of total reduced thiols in plasma may improve its antioxidant activity under hypoxic/anoxic conditions. This may be particularly useful since other important antioxidant mechanisms such as urate, ascorbate, and vitamin E appear to be inefficient.