Age Related Changes in NAD+ Metabolism Figure 2. Increased oxidative damage to lipids in selected brain regions with age. (A) Liver, (B) heart, (C) kidney, and (D) lung were analysed by Western blotting using anti-4HNE 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. (E) Immunodetection of 4-HNE in the liver, heart, kidney, and lung from 3 month, 12 month and 24 month old rats. 4-HNE (green) and DAPI (blue). Higher immunoreactivity for 4-HNE was observed in 12 and 24 month old rats compared to 3-month old rats. doi:10.1371/journal.pone.0019194.g002 placed in tapered glass vial. The protein was then allowed to precipitate (,18 h, 220uC) following the addition of 10 volumes of acetone/HCl (100:1, v:v). The precipitate was centrifuged (14,000 g, 30 min, ambient temperature) and the supernatant discarded. Internal standard (10 ml, 1 mg/ml [13C6]-phenylala- nine) containing trace quantities of [13C6]-tyrosine, was added to all samples and standards, and dried under reduced pressure (,1 Torr, 1–2 h). Afterwards, gas phase acid hydrolysis was performed at 150uC for 1 h. Samples and standards were then derivatised using a three-step derivatisation approach, using heptafluorobutyric anhydride (HFBA), pentafluorobenzylbromide (PFBBr) and N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA), vacuum dried for 10 min, and reconstituted in 50 ml ethyl acetate. Each standard and sample (1 ml) was injected into the GC/MS using pressure pulse splitless loading (6890N gas chromatograph interfaced to 5973 mass selective detector; Agilent technologies, Ryde, Australia), with methane as the ECNI reagent gas. Chromatography was performed on a fused silica capillary column (HP-5MS; 30 m60.25 mm i.d.60.25 mm, film thickness), with helium carrier gas and using the following chromatographic parameters: inlet temperature 250uC, transfer line temperature 280uC, programmed oven temperature gradient with initial temperature of 150uC for 3 min, then a temperature ramp of 30uC/min to 290 and a final time of 3 min at 290uC. Final o- and m- tyrosine levels were expressed relative to phenylalanine levels. Western Blots for the Detection of 4-Hydroxynonenal, Poly(ADP)-ribose, Sirt1, and Total/Acetylated p53 Protein Expression The whole liver, heart, kidney and lung were homogenised in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4); IGEPAL 1%; 0.25% Na-deoxycholate; 1 mM EDTA, 150 mM NaCl; 1 mg/ml each of aprotinin, leupeptin and pepstatin; 1 mM Na3VO4; and 1 mM NaF. After 1 h, the homogenate was obtained by centrifuging at 14,000 g, for 30 min at 4uC. Protein concentration was determined using the Bradford protein assay. Equal amounts of protein extract (30 mg) were dissolved in Laemmli sample buffer (Hercules, CA, USA), boiled for 5 min, electrophoresed on 8–12% (v/v) polyacrylamide SDS-PAGE gels (BioRad, Hercules, CA, USA), and electrotransferred onto PVDF membranes (BioRad, Her- cules, CA, USA). Membranes were blocked with 5% non-fat milk dissolved in TBS for 1 h and incubated with primary antibody overnight at 4uC. The primary antibodies used are described in Table 1. After incubation with primary antibodies, membranes were washed in TBS-Tween-20 and incubated with HRP conjugated secondary antibodies (Sigma, Castle Hill, Australia) for 1 h at room temperature. After further washing in TBS-Tween-20, the membranes were incubated with an ECL plus reagent (RPN2132, Amersham) and protein bands were visualised on X-ray films. The bands were quantified by ImageJ software, and normalised to b-actin, which served as an internal control. Total Antioxidant Capacity Assay The total antioxidant capacity in aging liver, heart, kidney and lung was evaluated by Trolox equivalent antioxidant capacity assay using a standard antioxidant assay kit (Cayman, Michigan, USA). Briefly, the tissue was homogenised on ice in a buffer containing 5 mM potassium phosphate; 0.9% NaCl; and 0.1% glucose, pH 7.4. The samples were centrifuged at 10,000 g for 15 min at 4uC, and the supernatant was collected for the assay. Figure 3. Total antioxidant capacity significantly declined in the rat liver, heart, kidney, and lung with age. 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. doi:10.1371/journal.pone.0019194.g003 PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e19194