Comparison of Formation of D2/E2-Isoprostanes and F2-Isoprostanesin Vitroandin Vivo— Effects of Oxygen Tension and Glutathione

The isoprostanes (IsoPs) are bioactive prostaglandin-like compounds derived from the free-radical-catalyzed peroxidation of arachidonic acidin vitroandin vivo.IsoPs possessing either an F-type prostane ring (F2-IsoPs) or D/E-type prostane rings (D2/E2-IsoPs) are formed depending on whether IsoP endoperoxide intermediates undergo reduction or isomerization, respectively. Little, however, is known regarding factors influencing the formation of various classes of IsoPs, particularly D2/E2-IsoPs. Thus, studies were undertaken to examine the formation of D2/E2-IsoPs in relation to F2-Isops bothin vitroandin vivo.In peroxidizing rat liver microsomes, the formation of D2/E2-IsoPs increased in a time- and oxygen-dependent manner and correlated with F2-IsoP generation and loss of precursor arachidonic acid, although the absolute amount of D2/E2-IsoPs formed exceeded by over 5-fold the levels of F2-IsoPs formed. Surprisingly, however, in liver tissue from rats exposed to an oxidant stress, levels of F2-IsoPs were up to 10-fold greater than those of D2/E2-IsoPs, suggesting that an endogenous process causes IsoP endoperoxide reductionin vivo.Addition of glutathione (GSH) to peroxidizing microsomes at concentrations from 0.01 to 5 mM increased the formation of F2-IsoPs at the expense of D2/E2-IsoPs. Boiling of microsomes did not alter the effect of GSH. Formation of D2/E2-IsoPs in liver tissuein vivowas greatly enhanced compared to F2-IsoPs in rats depleted of GSH. Thus, GSH modulates the formation of different classes of IsoPsin vitroandin vivo.Other thiols, including β-mercaptoethanol, dithiothreitol, and cysteine, were able to substitute for GSH. These studies indicate that GSH promotes F2-IsoP formation and diminishes D2/E2-IsoP levelsin vitroandin vivoby causing reduction of IsoP endoperoxides.