The Effect of α-Tocopherol on the Nitration of γ-Tocopherol by Peroxynitrite

It has been proposed (S. Christenet al. Proc. Natl. Acad. Sci. USA94, 3217–3222, 1997) that although α-tocopherol (α-TH) is an efficient antioxidant, the presence of γ-tocopherol (γ-TH) may be required to scavenge peroxynitrite-derived reactive nitrogen species. To investigate the reactions between α-TH, γ-TH, and peroxynitrite, endogenous levels of both α-TH and γ-TH were monitored when low-density lipoprotein was oxidized in the presence of the peroxynitrite generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1). SIN-1 oxidized α-TH while γ-TH levels remained constant. The sparing of γ-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing α-TH and γ-TH were incubated with either SIN-1 or peroxynitrite. Our data show that α-TH inhibits peroxynitrite-mediated γ-TH nitration, i.e., 5-NO2-γ-tocopherol formation. The rate constants for the reactions between both α-TH and γ-TH with peroxynitrite suggest that the sparing of γ-TH by α-TH does not occur by competitive scavenging, but may be due to the formation of a transient γ-TH intermediate. Nitration of γ-TH becomes significant only after α-TH levels have been depleted. We conclude α-TH alone is sufficient to remove any peroxynitrite-derived reactive nitrogen species, as the presence of α-TH attenuates nitration of both γ-TH and tyrosine. The present results also indicate that a bolus addition of peroxynitrite or SIN-1 to liposomes containing γ-TH forms 5-NO2-γ-tocopherol in similar yields. This is in contrast to their reaction profile with tyrosine in aqueous solution. Under these conditions, SIN-1 does not form nitrotyrosine at detectable yields.