Age Related Changes in NAD+ Metabolism Briefly, 10 ml of sample was added to 10 ml of Metmyoglobin and 150 ml of Chromogen. The reaction was initiated by adding 40 ml of hydrogen peroxide working solution. The samples were incubated on a shaker for 5 min at room temperature and the absorbance was read at 405 nm using the Model 680XR microplate reader (BioRad, Hercules). The absorbance values obtained for the samples were compared with a standard curve obtained using Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2- Figure 4. Increased DNA damage was reported in the rat liver, heart, kidney, and lung with age. (A) DNA damage was determined by measuring the flourescence of phosphorylated H2AX at Serine139 using the Fluostar Optima Fluorometer (NY, USA). All values are means 6 S.E from tissue obtained from eight different rats for each age group. Significance *p,0.01 compared to 3 month old rats. (B) Immunodetection of phosphor- H2AX-Serine139 in the liver, heart, kidney, and lung from 3 month, 12 month and 24 month old rats. Phosphor-H2AX-Serine139 (green) and DAPI (blue). Higher immunoreactivity for phosphor-H2AX-Serine139 was observed in 12 and 24 month old rats compared to 3-month old rats. doi:10.1371/journal.pone.0019194.g004 PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e19194