Age Related Changes in NAD+ Metabolism Figure 5. Increased Poly(ADP-ribose) activity in the brain with age. (A) PARP activity was determined in aging tissue using a spectrophotometric assay. 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) Western blotting for poly(ADP-ribose) in (i) liver, (ii) heart, (iii) kidney, and (iv) lung with aging using anti-Poly(ADP- ribose) (10H) antibody. The blots shown are representative tracings of an experiment done eight times. Graphs are mean 6 S.E from tissue obtained from eight different rats for each age group. Each bar of the quantification graph represents the corresponding band for each age group. Significance *p,0.01 compared to 3 month old rats. (C) Immunodetection for poly(ADP-ribose) in the liver, heart, kidney and lung from 3 month, 12 month and 24 month old rats. Poly(ADP-ribose) (green) and DAPI (blue). Higher immunoreactivity for poly(ADP-ribose) was observed in 12 and 24 month old rats compared to 3-month old rats. doi:10.1371/journal.pone.0019194.g005 carboxylic acid), a permeable cell derivative of vitamin E commonly employed as an antioxidant. The results were expressed as mmol Trolox/L/mg protein. DNA Damage Assay Since H2AX phosphorylation at serine 139 correlates very closely with each double-stranded DNA break, phosphor-H2AX is a sensitive marker for DNA damage [39]. We measured phosophorylation of H2AX at serine 139 using a DNA damage assay kit (Active Motif, Carlsbad, California, USA). Briefly, the liver, heart, kidney and lung was suspended on ice in a buffer containing 1 mM PMSF and pelleted at 10,000 g for 4 min at 4uC. Afterwards 200 ml of warm SDS lysis buffer containing 1 mg/ml each of aprotinin, leupeptin and pepstatin and 1 mM PMSF were then added and incubated for 10 min on ice. All samples were sonicated to generate 100 - to 1000-bp DNA fragments. 5 mg/ml of anti-phospho-H2AX-ser139 was added to the lysate with cold DPBS and allowed to incubate overnight at 4uC. The primary antibody was removed by washing the lysate 3 times with 200 ml DPBS. Alexis 488 goat anti-rabbit IgG secondary antibody (1:1000) was added to the lysate and incubated for 1 hour at room temperature. The secondary antibody was removed by washing the lysate twice with 200 ml DPBS. The fluorescence was read using Fluostar Optima Fluorometer (NY, USA). Filter excitation and emission was set at 485 nm and 520 wavelengths respectively. Isolation and Extraction of Nuclei for PARP and Sirt1 deacetylase Activity Assays Aliquots of homogenate from the liver, heart, kidney and lung (without protease inhibitors) were spun through 4 ml of 30% sucrose solution containing 10 mM Tris HCl (pH 7.4); 10 mM NaCl; and 3 mM MgCl2 at 1,300 g for 10 min at 4uC. The remaining pellet was washed with cold 10 mM Tris-HCl (pH 7.4) containing 10 mM NaCl. The nuclei was later suspended in 50– 100 ml extraction buffer containing 50 mM Hepes KOH (pH 7.4); 420 mM NaCl; 0.5 mM EDTA; 0.1 mM EGTA; and glycerol 10%, sonicated for 30 s, and allowed to stand on ice for 30 min. After centrifugation at 10,000 g for 10 min, an aliquot of the supernatant was used to determine protein concentration using the Bradford protein assay. PARP Activity Assay PARP activity was measured in nuclear extracts from the liver, heart, kidney and lung of young, middle-aged, and aged rats, using a new operational protocol relying on the chemical quantification of NAD+ modified from Putt et al. [40]. The final reaction mixture contained 10 mM MgCl2, Triton X-100 (1%), and 20 mM NAD+ in 50 mM Tris buffer, pH 8.1. The plate was then incubated for 1 hour and the amount of NAD+ consumed was measured by the NAD(H) microcycling assay using the Model 680XR microplate reader (BioRad, Hercules). The results were expressed as NAD+ consumed/h/mg protein. Sirt1 deacetylase Activity Sirt1 deacetylase activity was evaluated in nuclear extracts from the liver, heart, kidney and lung of young, middle-aged, and aged rats, using the Cyclex SIRT1/Sir2 Deacetylase Flourometric Assay Kit (CycLex, Nagano, Japan). The final reaction mixture (100 ml) contained 50 mM Tris-HCl (pH 8.8), 4 mM MgCl2, 0.5 mM DTT, 0.25 mA/ml Lysyl endopeptidase, 1 mM Trichos- tatin A, 200 mM NAD+, and 5 ml of nuclear sample. The samples were mixed well and incubated for 10 min at room temperature and the fluorescence intensity (ex. 340 nm, em. 460 nm) was measured every 30 s for a total of 60 min immediately after the addition of fluorosubstrate peptide (20 mM final concentration) using Fluostar Optima Fluorometer (NY, USA) and normalised by the protein content. The results are reported as relative fluorescence/mg of protein (AU). Measurement of Intracellular NAD+/NADH Levels + Total intracellular NAD (NADH+NAD ) concentration was measured spectrophotometrically using the thiazolyl blue micro- cycling assay established by Bernofsky and Swan (1973) adapted for 96 well plate format by Grant and Kapoor (1998) [41,42]. Briefly, each assay contained 100 mM bicine, pH 7.8; 0.42 mM MTT, and 1.66 mM PMS. For NAD+ measurement, 20 ml of ADH in 0.15% ethanol was added to the reaction mixture. The amount of NAD and NADH were measured as the change in absorbance at 590 nm at 37uC for 10 minutes with a Model 680XR microplate reader (BioRad, Hercules). The ratio of NAD+/NADH was calculated based on results of NAD+ and NADH concentrations. Isolation of Mitochondria All procedures were carried out at 0–4uC. Briefly, the liver, heart, kidney and lung were excised, washed with DPBS, and treated with proteinase K (1 mg/ml) for 30 s, washed with buffer A (250 mM mannitol; 0.5 mM EGTA; 5 mM Hepes; and fatty acid free bovine serum albumin 0.1%, pH 7.4 at 4uC). The samples homogenised with buffer A using a Teflon pestle. The homogenate was centrifuged twice at 600 g for 5 min at 4uC. The supernatants were mixed and centrifuged at 10,300 g for 10 min at 4uC. The mitochondrial pellets were suspended in 0.5 ml buffer A and transferred to ultracentrifuge tubes containing 1.4 ml buffer B (225 mM mannitol; 1 mM EGTA; 25 mM Hepes; and fatty acid free bovine serum albumin (BSA) 0.1%, pH 7.4 at 4uC) and 0.6 ml Percoll. The mixture was centrifuged at 105,000 g for 30 min at 4uC. The pure mitochondrial fraction was purified by washing twice with buffer A at 10,300 g for 10 min at 4uC to remove the Percoll and frozen to 280uC. An aliquot was used to determine the mitochondrial protein content using the Bradford protein assay. Determination of Mitochondrial Complexes I, II, III, and IV activities To determine the complex I activity, submitochondrial fractions (0.6 mg/ml) were incubated for 3 min in buffer solution PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e19194