Figure 4.

The antioxidant effects of ketones (KB) are mediated by NADH oxidation and not by an increase in reduced glutathione. (A) Acutely dissociated neurons were incubated with monochlorobimane (MCB), a fluorescent marker of reduced glutathione. The decrease in MCB fluorescence induced by 10 μM glutamate alone (GLU; n = 7) for 10 min was prevented by ketones (GLU + KB; n = 8; p < 0.01). The administration of 10 mM diamide, a specific thiol oxidant, for 10 min temporarily decreased MCB fluorescence as well (diamide; n = 10 mM; p = 0.01) but ketones did not have any effect under these conditions (diamide + KB; n = 5). Moreover, ketones did not affect baseline levels of MCB (KB; n = 5). (B) Glutamate (n = 7) increased the NAD(P)H signal relative to controls (n = 6), suggesting that the observed decrease in glutathione levels was secondary to increased production of reactive oxygen species (ROS). Ketones blocked the effect of glutamate on NAD(P)H, confirming that they decrease ROS rather an increase glutathione (n = 6). Differences were statistically significant (p = 0.01 at 20 min and 0.04 at 25 min). The effects of 1 mM potassium cyanide (KCN) are also presented for comparative purposes. Exposure to KCN for 10 min significantly increased NAD(P)H fluorescence almost to baseline levels. Horizontal bars indicate 10 min treatment periods. * p < 0.05; ** p < 0.01; *** p < 0.001.