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Nitrite as a Source of Nitric Oxide in Exercising Skeletal Muscle of Rat
stoichiometries were analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS) and chemical trapping experiments. Persulfane species have been identified include GSSxG, GSSxH, GSSxNH2, GSSxSO3H, GSSxSOH, with persulfane x values ranging from 1 to 9. The presence and distribution of each family of persulfane species within the original reaction mixtures were confirmed using Single Ion Chromatograms (SICs) to demonstrate separation on LC column and give approximate quantification by peak area. Less stable persulfane families were obtained by trapping with speciesspecific reagents. The dominance of tetrasulfanes, GSS 3H, as well as the limited distribution of persulfide amines, GSS xNH2, x = 1 to 3, suggests a common pathway of persulfane generation in this complex reaction mixture.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.210 169 1LWULWHDVD6RXUFHRI1LWULF2[LGHLQ([HUFLVLQJ 6NHOHWDO0XVFOHRI5DW
Barbora Piknova1, Ji Won Park1, Jeff Lam1, and Alan Neil Schechter1 1 NIH/NIDDK, USA Nitric oxide synthase (NOS) is the main enzyme generating nitric oxide (NO) in mammalian cells. Presence of significant quantities of functional NOS, mainly as NOS1 isoform, in skeletal muscle had been well established. We have previously shown that rat skeletal muscle also contains unusually high concentrations of nitrate, one of the members of non-enzymatic NO metabolic cycle. In the present study we hypothesize that this nitrate reservoir contributes significantly to the generation of NO in case of exercise-induced tissue hypoxia and that nitrate can be considered as one of the sources of NO needed for vasodilatation in such case. We measured nitrate and nitrite levels in Wistar rat leg tissue before and after exercise on treadmill. We found significant increase of nitrite levels accompanied by significant decrease of nitrate levels immediately after exercise. Using skeletal muscle tissue homogenate we established that xanthine oxidoreductase (XOR) is the enzyme responsible for the generation of nitrite from nitrate and that there is a low but not negligible production of NO. Taken together, these results support the hypothesis that exercise-induced functional hyperemia is, at least partially, supported by NO generated from local tissue nitrate via its reduction to NO by resident XOR.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.211 170 )RUPDWLRQDQG&KDUDFWHUL]DWLRQRI1LWUR)DWW\$FLGV LQ/'/
Mauricio Mastrogiovanni 1, Andres Trostchansky1, and Homero Rubbo1 1 Universidad de la Republica, Montevideo, Uruguay
Human LDL purified from fresh plasma of normolipidemic donors was exposed to nitrating agents including peroxynitrite and acidic nitrite. Fatty acids from LDL were analyzed by hydrolysis of triglycerides and phospholipids and the free fatty acid fraction was treated with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). Then, samples were subjected to HPLC-MS/MS MRM analysis following the loss of PTAD from the cLA-PTAD compound. Quantitation was performed comparing signal areas of samples to a calibration curve of 9,11 cLA derivatized with PTAD. NFA were determined by HPLC-MS/MS following MRM transitions with 13C and 15N internal standards in parallel with 3-nitrotyrosine, carbonyls, TBARS and lipid hydroperoxydHV /'/ ȝ0 H[SRVHG WR peroxynitrite flux (20 μM/min) or 300 μM nitrite / pH 3, 60 min, RT, showed the presence of nitro-oleic acid (m/z 326.2/46.1) and nitro-conjugated linoleic acid (m/z 324.2/46.1). Time-courses of nitro-conjugated linoleic acid formation reached picomolar levels. When looking for oxidation products and protein nitration, peroxynitrite induced greater levels of carbonyls, TBARS and 3nitrotyrosine than nitrite. These parameters positively correlated with NFA formation. Our results support the formation of NFA in LDL exposed to biologically-relevant nitrating agents. Current experiments are foccusing in demonstrate that NFA-rich LDL is anti-atherogenic through its ability to counterpart the proinflammatory actions of oxidized LDL.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.212 171 'HVLJQDQG9DOLGDWLRQRI1RYHO)OXRURJHQLF6XEVWUDWHV IRU61LWURVRJOXWDWKLRQH5HGXFWDVH Bei Sun1 and Bulent Mutus1 1 University of Windsor, Canada
Protein S-nitrosation is the covalent attachment of nitric oxide (NO) moiety to cysteine thiols and has been recognized as an important post-translational modification which affects protein function. Many studies have shown that aberrant S-nitrosation contributes to the development of pathological features, such as diabetes, cancer and neurodegeneration. Under normal conditions, the dynamic processes of nitrosation and denitrosation are regulated and controlled by cells via an array of mechanisms. One highly conserved enzyme involved in such regulation is S-nitrosoglutathione reductase (GSNOR). GSNOR is an NADH dependent, class III alcohol dehydrogenase which promotes the de-nitrosation of proteins by specifically metabolizes S-nitrosoglutathione (GSNO). In our lab, we have successfully synthesized and purified O-aminobenzoyl-GSNO and EosinGSNO as two novel fluorogenic substrates for GSNOR. When tested using recombinant GSNOR produced and purified from Ecoli, both substrates undergo fluorescence enhancement upon GSNOR-mediated alteration of the ±S=NO moiety. In addition, these substrates can be used to monitor the activity of GSNOR in vitro as well as in cultured cell systems. Supported by NSERC Discovery Grant to BM
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.213
Nitro-fatty acids (NFA) represent novel anti-inflammatory signaling mediators present in human tissue and are likely to be transported by plasma lipoproteins. Lipid and protein oxidation in low density lipoprotein (LDL) as well as apo B-100 nitration have been used as biomarkers of proatherogenic LDL. However, there are no reports about the presence of NFA in LDL that could contribute to reduce the pro-inflammatory role of oxidized-LDL. Herein we explored the presence of NFA in LDL and determine conditions that favour NFA formation to modulate LDL oxidation.
S80
SFRBM 2015
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.210 169 1LWULWHDVD6RXUFHRI1LWULF2[LGHLQ([HUFLVLQJ 6NHOHWDO0XVFOHRI5DW
Barbora Piknova1, Ji Won Park1, Jeff Lam1, and Alan Neil Schechter1 1 NIH/NIDDK, USA Nitric oxide synthase (NOS) is the main enzyme generating nitric oxide (NO) in mammalian cells. Presence of significant quantities of functional NOS, mainly as NOS1 isoform, in skeletal muscle had been well established. We have previously shown that rat skeletal muscle also contains unusually high concentrations of nitrate, one of the members of non-enzymatic NO metabolic cycle. In the present study we hypothesize that this nitrate reservoir contributes significantly to the generation of NO in case of exercise-induced tissue hypoxia and that nitrate can be considered as one of the sources of NO needed for vasodilatation in such case. We measured nitrate and nitrite levels in Wistar rat leg tissue before and after exercise on treadmill. We found significant increase of nitrite levels accompanied by significant decrease of nitrate levels immediately after exercise. Using skeletal muscle tissue homogenate we established that xanthine oxidoreductase (XOR) is the enzyme responsible for the generation of nitrite from nitrate and that there is a low but not negligible production of NO. Taken together, these results support the hypothesis that exercise-induced functional hyperemia is, at least partially, supported by NO generated from local tissue nitrate via its reduction to NO by resident XOR.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.211 170 )RUPDWLRQDQG&KDUDFWHUL]DWLRQRI1LWUR)DWW\$FLGV LQ/'/
Mauricio Mastrogiovanni 1, Andres Trostchansky1, and Homero Rubbo1 1 Universidad de la Republica, Montevideo, Uruguay
Human LDL purified from fresh plasma of normolipidemic donors was exposed to nitrating agents including peroxynitrite and acidic nitrite. Fatty acids from LDL were analyzed by hydrolysis of triglycerides and phospholipids and the free fatty acid fraction was treated with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). Then, samples were subjected to HPLC-MS/MS MRM analysis following the loss of PTAD from the cLA-PTAD compound. Quantitation was performed comparing signal areas of samples to a calibration curve of 9,11 cLA derivatized with PTAD. NFA were determined by HPLC-MS/MS following MRM transitions with 13C and 15N internal standards in parallel with 3-nitrotyrosine, carbonyls, TBARS and lipid hydroperoxydHV /'/ ȝ0 H[SRVHG WR peroxynitrite flux (20 μM/min) or 300 μM nitrite / pH 3, 60 min, RT, showed the presence of nitro-oleic acid (m/z 326.2/46.1) and nitro-conjugated linoleic acid (m/z 324.2/46.1). Time-courses of nitro-conjugated linoleic acid formation reached picomolar levels. When looking for oxidation products and protein nitration, peroxynitrite induced greater levels of carbonyls, TBARS and 3nitrotyrosine than nitrite. These parameters positively correlated with NFA formation. Our results support the formation of NFA in LDL exposed to biologically-relevant nitrating agents. Current experiments are foccusing in demonstrate that NFA-rich LDL is anti-atherogenic through its ability to counterpart the proinflammatory actions of oxidized LDL.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.212 171 'HVLJQDQG9DOLGDWLRQRI1RYHO)OXRURJHQLF6XEVWUDWHV IRU61LWURVRJOXWDWKLRQH5HGXFWDVH Bei Sun1 and Bulent Mutus1 1 University of Windsor, Canada
Protein S-nitrosation is the covalent attachment of nitric oxide (NO) moiety to cysteine thiols and has been recognized as an important post-translational modification which affects protein function. Many studies have shown that aberrant S-nitrosation contributes to the development of pathological features, such as diabetes, cancer and neurodegeneration. Under normal conditions, the dynamic processes of nitrosation and denitrosation are regulated and controlled by cells via an array of mechanisms. One highly conserved enzyme involved in such regulation is S-nitrosoglutathione reductase (GSNOR). GSNOR is an NADH dependent, class III alcohol dehydrogenase which promotes the de-nitrosation of proteins by specifically metabolizes S-nitrosoglutathione (GSNO). In our lab, we have successfully synthesized and purified O-aminobenzoyl-GSNO and EosinGSNO as two novel fluorogenic substrates for GSNOR. When tested using recombinant GSNOR produced and purified from Ecoli, both substrates undergo fluorescence enhancement upon GSNOR-mediated alteration of the ±S=NO moiety. In addition, these substrates can be used to monitor the activity of GSNOR in vitro as well as in cultured cell systems. Supported by NSERC Discovery Grant to BM
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.213
Nitro-fatty acids (NFA) represent novel anti-inflammatory signaling mediators present in human tissue and are likely to be transported by plasma lipoproteins. Lipid and protein oxidation in low density lipoprotein (LDL) as well as apo B-100 nitration have been used as biomarkers of proatherogenic LDL. However, there are no reports about the presence of NFA in LDL that could contribute to reduce the pro-inflammatory role of oxidized-LDL. Herein we explored the presence of NFA in LDL and determine conditions that favour NFA formation to modulate LDL oxidation.
S80
SFRBM 2015