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Dynamic Regulation of Alpha-Tocopherol Secretion from Hepatocytes by the Alpha-Tocopherol Transfer Protein
transplantation but damage occurs during the preservation episode. Our laboratory has demonstrated that cold storage leads to increased mitochondrial superoxide and mitochondrial damage, and that addition of mitochondrial targeted anti-oxidant Mitoquinone (MitoQ) to the preservation solutions blunted this injury. To better translate the rodent study, we used a porcine kidney cold storage model to determine whether MitoQ blunted damage. Using High Resolution Respirometry (HRR) and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex respiration following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. To further dissect the mechanism of injury following cold storage, we tested the hypothesis that cold preservation disrupts mitochondrial homeostasis which contributes to altered mitochondrial function. Results showed that cold preservation induced tubular damage and cell death in proximal tubules, but not within the distal nephron following 24 hours of cold storage. Interestingly, we observed increased mitochondrial complex proteins (biogenesis) in the distal nephrons. Using HRR and fresh kidney tissues we assessed mitochondrial function at 4°C and showed that all respiratory complexes were severely compromised after cold storage. When cold stored biopsies were ° allowed to warm to 37 C complex II/III remained decreased, while other complex activities increased. Together, results suggested that cold storage induces oxidative stress leading to renal damage with proximal tubules being vulnerable to cold ischemia induced renal damage and cell death, whereas the distal tubules cope with this cold ischemic stress by triggering mitochondrial biogenesis. Furthermore, MitoQ decreased oxidative stress, tubular damage and cell death by decreasing tyrosine nitration and improving mitochondrial function during cold storage. Therefore, further studies using mitochondrial targeted antioxidants should be considered as an integral part of organ preservation solution prior to transplantation. This work is supported by NIH grant R43 DK089659.
doi:10.1016/j.freeradbiomed.2012.10.268
263 Melatonin Prevents Hypochlorous Acid Mediated Cyanocobalamin Destruction and Cyanogen Chloride Generation 1
Dhiman Maitra , Rasha M. Abdulridha1, Ibrahim Abdulhamid2, Ghassan M. Saed1, and Husam M. Abu-soud1 1 2 Wayne State University, Children's Hospital of Michigan Hypochlorous acid (HOCl) is a potent cytotoxic oxidant generated by the enzyme myeloperoxidase (MPO). MPO is known to play a role in various pathological conditions through its ability to oxidatively modify several biomolecules. Vitamin B12 (VB12) is a water soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. Deficiency of VB12 has been known to be associated with cardiovascular and neurodegenerative conditions, among many others. Cyanocobalamin (Cobl) is a common pharmacological form of VB12. Recently, we have shown that interaction of Cobl with HOCl leads to corrin ring destruction and generation of toxic molecular products such as cyanogen chloride (CNCl). Detailed stopped-flow kinetic and mass-spectrometric studies revealed that HOCl-mediated Cobl destruction occurs through a mechanism that involves Į-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and subsequent cleavage of the phosphonucleotide moiety. Here, we show that melatonin prevents HOCl-mediated Cobl chlorination, corrin destruction and CNCl formation, using a combination of UV-Vis
spectrophotometry, HPLC analysis, and colorimetric CNCl assay. We attribute this protective role of melatonin to its ability to directly scavenge HOCl. Collectively, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can also exert its protective effect by inhibiting HOCl-mediated Cobl destruction and subsequent CNCl generation.
doi:10.1016/j.freeradbiomed.2012.10.269
264 Dynamic Regulation of Alpha-Tocopherol Secretion from Hepatocytes by the Alpha-Tocopherol Transfer Protein 1
Stacey Chung1, Jeffrey Atkinson2, and Danny Manor 1 2 Case Western Reserve University, Brock University Vitamin E, a neutral plant-derived lipid, is an essential nutrient for all vertebrates, which scavenges free radicals in biological membranes thereby preventing oxidative stress. Of the eight naturally-occurring forms of vitamin E, Į-tocopherol is the most biologically active form. This discrimination is achieved by the selective retention of hepatic Į-tocopherol transfer protein (ĮTTP), and degradation of all other vitamin E isoforms by CYP4F2. In the liver, Į-TTP resides in lysosomes and facilitates the secretion of endocytosed Į-tocopherol to the circulation. At the intracellular level, Į-TTP transfers lysosomal Į-tocopherol to transport vesicles, which subsequently transports Į-tocopherol to the plasma membrane, from where the vitamin is released into circulation for uptake by extrahepatic target tissues. We aim to elucidate how Į-toc status changes the intracellular localization of Į-TTP and the molecular mechanism by which Į-TTP regulates Įtoc egress from hepatocytes. Using live-cell immunofluorescence, we found that localization of Į-TTP in hepatocytes is dynamic: in the absence of Į-tocopherol, the protein is found in a punctate perinuclear pattern but it rapidly disperses into a diffuse cytosolic appearance upon treatment with vitamin E. These findings suggest that dynamic trans-localization of Į-TTP plays an important role in the protein’s function, indicating a dynamic and homeostatic regulation of Į-tocopherol egress from hepatocytes.
doi:10.1016/j.freeradbiomed.2012.10.270
265 Peroxyl Scavenging Capacity of Carotenoid Extracts from Brazilian Native Fruits 1
Lilian Regina Barros Mariutti , Eliseu Rodrigues1, and Adriana Zerlotti Mercadante1 1 UNICAMP, Brazil Brazil has a wide variety of native, wild and not well-known fruits and no information about the antioxidant capacity of their lipophilic compounds, such as the carotenoids, is available. Mana-cubiu (Solanum sessiflorum) is a fruit native from the Amazonian region and widely distributed across the humid equatorial regions of Brazil, Peru and Colombia. It has 5-6 cm in diameter and the comestible fraction represents approximately 91% of total fresh weight (9% of peel). Golden spoon (Byrsonima crassifolia) is a tropical tree native from the North and Northest regions of Brazil, which is also found in Mato Grosso and Minas Gerais, and in some countries of Central America and Caribbean. The spherical fruit (1-2 cm diameter) is appreciated by the local population due to its typical rancid cheese-like aroma. Both fruits have been used by the local population in the treatment of gastrointestinal and gynecological inflammations, skin infections and snake bites. In
SFRBM 2012
S111
doi:10.1016/j.freeradbiomed.2012.10.268
263 Melatonin Prevents Hypochlorous Acid Mediated Cyanocobalamin Destruction and Cyanogen Chloride Generation 1
Dhiman Maitra , Rasha M. Abdulridha1, Ibrahim Abdulhamid2, Ghassan M. Saed1, and Husam M. Abu-soud1 1 2 Wayne State University, Children's Hospital of Michigan Hypochlorous acid (HOCl) is a potent cytotoxic oxidant generated by the enzyme myeloperoxidase (MPO). MPO is known to play a role in various pathological conditions through its ability to oxidatively modify several biomolecules. Vitamin B12 (VB12) is a water soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. Deficiency of VB12 has been known to be associated with cardiovascular and neurodegenerative conditions, among many others. Cyanocobalamin (Cobl) is a common pharmacological form of VB12. Recently, we have shown that interaction of Cobl with HOCl leads to corrin ring destruction and generation of toxic molecular products such as cyanogen chloride (CNCl). Detailed stopped-flow kinetic and mass-spectrometric studies revealed that HOCl-mediated Cobl destruction occurs through a mechanism that involves Į-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and subsequent cleavage of the phosphonucleotide moiety. Here, we show that melatonin prevents HOCl-mediated Cobl chlorination, corrin destruction and CNCl formation, using a combination of UV-Vis
spectrophotometry, HPLC analysis, and colorimetric CNCl assay. We attribute this protective role of melatonin to its ability to directly scavenge HOCl. Collectively, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can also exert its protective effect by inhibiting HOCl-mediated Cobl destruction and subsequent CNCl generation.
doi:10.1016/j.freeradbiomed.2012.10.269
264 Dynamic Regulation of Alpha-Tocopherol Secretion from Hepatocytes by the Alpha-Tocopherol Transfer Protein 1
Stacey Chung1, Jeffrey Atkinson2, and Danny Manor 1 2 Case Western Reserve University, Brock University Vitamin E, a neutral plant-derived lipid, is an essential nutrient for all vertebrates, which scavenges free radicals in biological membranes thereby preventing oxidative stress. Of the eight naturally-occurring forms of vitamin E, Į-tocopherol is the most biologically active form. This discrimination is achieved by the selective retention of hepatic Į-tocopherol transfer protein (ĮTTP), and degradation of all other vitamin E isoforms by CYP4F2. In the liver, Į-TTP resides in lysosomes and facilitates the secretion of endocytosed Į-tocopherol to the circulation. At the intracellular level, Į-TTP transfers lysosomal Į-tocopherol to transport vesicles, which subsequently transports Į-tocopherol to the plasma membrane, from where the vitamin is released into circulation for uptake by extrahepatic target tissues. We aim to elucidate how Į-toc status changes the intracellular localization of Į-TTP and the molecular mechanism by which Į-TTP regulates Įtoc egress from hepatocytes. Using live-cell immunofluorescence, we found that localization of Į-TTP in hepatocytes is dynamic: in the absence of Į-tocopherol, the protein is found in a punctate perinuclear pattern but it rapidly disperses into a diffuse cytosolic appearance upon treatment with vitamin E. These findings suggest that dynamic trans-localization of Į-TTP plays an important role in the protein’s function, indicating a dynamic and homeostatic regulation of Į-tocopherol egress from hepatocytes.
doi:10.1016/j.freeradbiomed.2012.10.270
265 Peroxyl Scavenging Capacity of Carotenoid Extracts from Brazilian Native Fruits 1
Lilian Regina Barros Mariutti , Eliseu Rodrigues1, and Adriana Zerlotti Mercadante1 1 UNICAMP, Brazil Brazil has a wide variety of native, wild and not well-known fruits and no information about the antioxidant capacity of their lipophilic compounds, such as the carotenoids, is available. Mana-cubiu (Solanum sessiflorum) is a fruit native from the Amazonian region and widely distributed across the humid equatorial regions of Brazil, Peru and Colombia. It has 5-6 cm in diameter and the comestible fraction represents approximately 91% of total fresh weight (9% of peel). Golden spoon (Byrsonima crassifolia) is a tropical tree native from the North and Northest regions of Brazil, which is also found in Mato Grosso and Minas Gerais, and in some countries of Central America and Caribbean. The spherical fruit (1-2 cm diameter) is appreciated by the local population due to its typical rancid cheese-like aroma. Both fruits have been used by the local population in the treatment of gastrointestinal and gynecological inflammations, skin infections and snake bites. In
SFRBM 2012
S111