TPPS4 and TMPyP. The generation of LOOH was threefold higher in the presence of the TMPyP that exhibits higher affinity to the acidic membrane. The photodamage in GUVs analyzed by optical microscopy showed faster vesicle fragmentation in the presence of TMPyP. Controls experiments with D 2 O, azide and sorbic acid suggested preferential damage promoted by porphyrin triplet species rather than singlet oxygen. The results also show that affinity of the porphyrin to the membrane is crucial to determine the degree and the mechanism of lipid damage. Supported by UFABC, Fapesp and CNPq 30 Singlet Oxygen in Intact Heart Mitochondria Decreases Supercomplexes Junhwan Kim 1 , Kwangwon Lee 1 , and Charles Hoppel 1 1 Case Western Reserve University Singlet oxygen is produced by absorption of red light by the phthalocyanine dye Pc 4. Previous studies showed that irradiation of mitochondria pre-incubated with Pc 4 caused damage to mitochondrial structure and function by generating singletoxygen. The major mitochondrial functional damage was characterized as inhibition of state-3 respiration and uncoupling. However, detailed mechanisms for this dysfunction were not understood. Cardiolipin is a unique phospholipid localized primarily in the mitochondrial inner membrane and its oxidation produces functional damage to the mitochondrial electron transport chain system. We hypothesized that the oxidation of cardiolipin is responsible for the mitochondrial damage following exposure to activated Pc 4. To test this hypothesis, we performed a time course study to examine inactivation of mitochondrial respiration and cardiolipin oxidation caused by Pc 4-generated singlet oxygen. As previously reported, 10 min of irradiation significantly decreased mitochondrial respiration. At this time point, by HPLC- MS, we did not observe any change in either the content or composition of cardiolipin or any other phospholipid. Instead, we found decreased content of supercomplexes (monomer complex I/ dimer complex III/ complex IV). These results indicate that the damage initiated by singlet oxygen in mitochondria decreases supercomplexes leading to respiratory depression and suggest that oxidation of cardiolipin is not a factor. 31 Jaundice Is Associated with Oxidative Stress in Patients with Plasmodium Vivax Malaria Emerson Silva Lima 1 , Camila Fabbri 1 , Rita de Cássia Mascarenhas Netto 2 , and Marcus V.G. Lacerda 3 1 Federal University of Amazonas, 2 Uberlandia Federal University, 3 Tropical Medicine Foundation Dr. Heitor Vieira Dourado, Brazil The present study aimed elucidates the lipid peroxidation and variations of antioxidant enzymes profile in patients infected with Plasmodium vivax that developed jaundice in the course of the disease and also check the influence of high concentration of bilirubin. Were studied P. vivax malaria patients with jaundice, without jaundice and healthy individuals with no history of malaria. All patients had their antioxidant enzymes activities and lipid peroxidation marker malondialdehyde measured on day 1 and day 14 (after the beginning of therapy and malaria free). The malondialdehyde levels (μM) and activity of celuroplasmin(U/L) and glutathione reductase(U/L) were increased in plasma of patients with P. vivax with jaundice (2.3±0.97; 77.3±35.93; 89.9±38.00) compared to control group (1.5±0.29; 38.6±13.5; 47.0±7.0), on day 1, respectively. Differently, the activity of thioredoxin reductase (μmol/min) was decreased compared to the control group (6.63±2.02 versus 12.1±2.0). The enzymes glutathione reductase, thioredoxin reductase, thiols and malondialdehyde also showed significant difference in patients P. vivax malaria with jaundice (89.9±38.00; 6.63±2.02; 303.9±84.32; 2.3±0.97) when compared with patients P. vivax without jaundice (63.9±12.6; 8.88±2.18; 240.82±93.79; 1.67±0.64) on day 1, respectively. There was an increase of lipid peroxidation and significant changes in profile of antioxidant enzymes in patients who develop severe malaria and presented jaundice when compared with that developed mild form of the disease. The high concentrations of bilirubin may be a signaling of oxidative process and contributing to malaria pathogenesis. Financial support: MCT/CNPq/INCT Redoxoma, FAPEAM 32 In Vivo Detection of Myeloperoxidase Activity by LC/MS/MS Analysis of 2-Chloro-Ethidium, a Novel Product Formed from the Reaction of Hydroethidine with Hypochlorous Acid and Chloramines Gus J Maghzal 1 , Katie Cergol 1 , Cacang Suarna 1 , Darren Newington 1 , Anthony J Kettle 2 , Richard J Payne 1 , and Roland Stocker 1,3 1 University of Sydney, Australia 2 University of Otago, New Zealand 3 Victor Chang Cardiac Research Institute, Australia Hypochlorous acid (HOCl) is a strong oxidant produced by myeloperoxidase (MPO) expressed in neutrophils (PMN). HOCl can damage biomolecules and deplete antioxidants either directly or through secondary reactions via chloramines, and it is thought to contribute to atherosclerosis and related vascular diseases via multiple mechanisms including inhibition of reverse cholesterol transport by HDL and foam cell formation by LDL. Indeed, HOCl- modified LDL and HDL are present in human atherosclerotic plaques. In vivo analysis of HOCl/MPO activity is commonly assessed by the accumulation of 3-chloro-tyrosine (3-Cl-Tyr). A potential caveat with 3-Cl-Tyr as a biomarker for MPO activity is its low yield of formation. We set out to establish a method for the detection of HOCl in cells and in vivo using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Here, we demonstrate that HOCl reacts with HE with a rate constant of ∼1.5 x 10 5 M -1 s -1 to form several products, including 2-chloro- ethidium (2-Cl-E + ), as determined by MS and NMR. We also show that formation of 2-Cl-E + is not radical-mediated. Using LC/MS/MS, we detected 2-Cl-E + in: (i) the reaction of HE with either reagent HOCl, MPO-derived HOCl, or chloramines of taurine and glycine; (ii) HE-loaded human PMN after stimulation with phorbol myristate acetate; and (iii) in mouse arteries in vivo 2-14 days after placement of a non-occlusive cuff as a model of local arterial inflammation and after HE injection. Interestingly, and in contrast to the situation with 2-Cl-E + , there was no significant difference in 3-Cl-Tyr levels between cuffed and sham- operated arteries, despite large infiltration of PMNs in the adventitia of the injured tissue. We propose 2-Cl-E + as a novel surrogate for HOCl/MPO activity in vitro and in vivo. SFRBM 2012 S24 doi:10.1016/j.freeradbiomed.2012.10.056 doi:10.1016/j.freeradbiomed.2012.10.057 doi:10.1016/j.freeradbiomed.2012.10.058 doi:10.1016/j.freeradbiomed.2012.10.059