Fig. 1.

TEMPOL increased the NAD⁺/NADH ratio via ROS scavenging and the redox cycling system in vitro. (A) Schematic illustration of two pathways of NAD⁺ generation by TEMPOL. GR, glutathione reductase; NNT, nicotinamide nucleotide transhydrogenase. (B) Superoxide scavenging and simultaneous NAD⁺ production in the hypoxanthine–xanthine oxidase system. The reaction with superoxide was measured by the rate of reduction of ferricytochrome c at 550 nm. Reaction mixtures contained 120 µM hypoxanthine, 0.005 units xanthine oxidase, 24 μM cytochrome c and 1 mM NADH in 10 mM phosphate buffer (pH 7.4) with TEMPOL (0–1 mM, as indicated in the figure). (C) NAD⁺ production in the AsA–GSH redox cycle. NADH oxidation was measured at 340 nm. The reaction mixture contained 1 mM ascorbic acid (AsA), 1 mM glutathione (GSH), 1 unit of GR and 0.5 mM NADH in 10 mM phosphate buffer (pH 7.4) with TEMPOL (0–1 mM). Values are means±SD (n=4). *p<0.05 and ***p<0.005 compared with 0 mM.