Age Related Changes in NAD+ Metabolism Animals Female wistar rats were used and experiments were designed to target the following age groups: 3 months old (equivalent to a young human adult aged 20 years), 12 month old (equivalent to a middle-aged human aged 40 years) and 24 month old (equivalent to an aged human greater than 80 years) [37]. The animals were individually housed in an environmentally controlled room under a 12:12 hour light/dark cycle at 23uC and were given commercial rat chow and water ad libitum. All experiments were performed according to the Animal Ethics Committee of the University of Sydney. Anaesthesia was induced with a mixture of O2, NO2, and 5% halothane, and then maintained by an intraperitoneal injection of sodium pentobarbitone (60 mg kg21). Transcardial perfusion was performed using 0.1 M DPBS, pH 7.4. Whole heart, lung, liver and kidney, were removed, washed with DPBS and used immediately for a variety of biochemical and histochemical procedures. Assay of o- and m- tyrosine for the Measurement of Protein Carbonyl Formation The assay for o- and m- tyrosine was performed as previously described [38]. Briefly, calibration standards of phenylalanine (313 pmol and 50 nmol) and o- and m- tyrosine (313 fmol to 50 pmol) were used for quantification. Labelled [13C6]-phenylal- anine was used as an internal standard and was added to all samples and standards. The liver, heart, kidney and lung were Figure 1. Increased formation of oxidatively modified proteins in the liver, heart, kidney, and lung with age. Increased levels of (A) o- tyrosine and (B) m- tyrosine were reported in the brain after 12 months of age compared to 3 month old rats. 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.g001 PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e19194