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EPITHELIAL LINING FLUID (ELF) AND LUNG PROTEIN NITRATION IN LUNG ALLOTRANSPLANTS

P. Jackson, T. Ohman, E. Helton, J. P. Crow, L. Viera, C.B. Alexander, S. Matalon

Birmingham DVAMC and UAB, Birmingham, AL, USA

This study investigated nitration and chlorination of ELF proteins and nitration of lung structural proteins in patients (n=29) who had undergone unilateral lung allotransplantation. We assayed nitrotyrosine (NT) by ELISA and both NT and chlorotyrosine (CT) by HPLC. Urea and nitrite concentrations were measured in bronchoalveolar lavage fluid (BALF) and serum. Assays also were done on BALF from four normal controls. We found NT by ELISA and HPLC in BALF of patients and lower, but detectable, levels in no’rmal controls. Mean per cent nitration (NT/total Tyr x 100) of BALF proteins by HPLC assay was: patients, 0.14 &0.27[SD]% and controls, 0.02 f 0.03. CT (CT/total Tyr x 100) was found only in the patients’ BALF (0.11*0.48[SD]%). Nitrite was measurable in BALF and serum of transplant patients (patients: BALF, 3.6r2.4[SD] uM; serum, 11.1~7.5). Corrected for dilution, the ELF [nitrite] was -500 pM, and the [NT) was uM based on HPLC. lmmunohistochemistry showed nitrotyrosine in some open lung biopsies. ELF of transplanted lungs is extremely rich in nitrite (nitrate) and contains both NT and CT.

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NITRATION OF y-TOCOPHEROL AND OXIDATION OF a-TOCOPHEROL BY CU,ZNSOD/H~O~/NO~-: ROLE OF NITROGEN DIOXIDE FREE RADICAL. B. Kalyanaraman, S. P. A. Goss and R. J. Singh. Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI. 53226 USA.

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (0~~) to 02 and HaOa. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of HaOa, leading to self- inactivation and formation of a potent enzyme bound-oxidant. We report that lipid peroxidation of L-o-lecithin liposomes was greatly enhanced during the SODiHzOa reaction in the presence of nitrite anion (NOa3 with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NOa- also greatly enhanced a-tocopherol (a-TH) oxidation by SOD/HsOa in saturated DLPC liposomes. The major product identified by HPLC and W-studies was a-tocopheryl quinone (a-TQ). When DLPC liposomes containing y-tocopherol (y-TI-I) were incubated with SOD/H~Oa/NO;, the major product identified was NGTH (5-NOa-y-tocopherol). Nitrone spin traps significantly inhibited the formation of a-TQ and NGTH. NO; inhibited HzOz- dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NOs- by an SOD-bound oxidant to the nitrogen dioxide radical (NOa). In this study we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NOa- is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis (FALS)-linked SOD mutants.

SPECIFIC S-NITROSOTHIOL QUANTIFICATION AS SOLUTION NITRITE AFTER VANADIUM(m) REDUCTION AND OZONE CHEMILUMINESCENT DETECTION David R. Janero and James F. Ewing* NitroMed, Inc., 12 Oak Park Drive, Bedford, MA 01730

S-Nitrosothiols (RSNOs) may represent naturally occurring nitric oxide (NO) surrogates and intermediates in NO metabolism. A facile, sensitive, and selective micromethod had been develoned and validated for RSNO quantification as mercury-displaceable NO. Brief (5 min), room temperature pretreatment of RSNO with a molar excess of aqueous HgCl, was used to liberate the NO moiety, which quantitatively converted to its stable solution end- product, nitrite. Solution nitrite was reduced back to NO with vanadium(III), and the NO was detected by gas-phase chemiluminescence after reaction with ozone in a commercial NO analyzer. A linear relationship was observed between RSNO- bound NO and assay response over a wide range (16.3-3500 pmol), and the response was quantitatively identical for equivalent amounts of nitrite and RSNO-bound NO. The method was 96% selective for nitrite vs. nitrate and evidenced negligible interference by nitrosated compounds bearing C-NO or N-NO functionalities. Lower limits of quantitative sensitivity and qualitative detection were below 50 and 20 pmol RSNO-bound NO equivalents, respectively. The intra- and inter-day coefficients of variation

were ~8%. This technique has been applied to diverse natural and synthetic S-nitrosothiols with full recovery from biological samples at levels of NO-equivalents undetectable by the popular Saville method.

*Present address: Arqule, Inc.; Waltham, MA 02154

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IRON PREVENTS EXCESSIVE NITRIC OXIDE GENERATION IN SEPTIC SHOCK MICE

A.M. Komarov, D.L. Mattson*, LT. Mak, W.B. Weglicki

The George Washington University Medical Center, Washington, DC and *Medical College of Wisconsin

We have tested the hypothesis on ‘free iron’ regulation of inducible nitric oxide synthase (iiOS) expression in murine endotoxic shock model. Mice received E. coli endotoxin (i.v.) and iron-citrate complex (s.c.). Iron and endotoxin were injected simultaneously to produce iron-dependent reduction of nitric oxide (NO) level in the blood (assessed as nitrite/nitrate and nitrosyl hemoglobin) and liver tissue of septic shock animals (detected as mononitrosyl-iron complex formed in vivo by diethyldithiocarbamate - DETC). Nitrosyl hemoglobin and DETC/FeNO complex were detected by electron paramagnetic resonance spectroscopy (BPR) at 77K. We have also found iron- dependent decrease of iNOS protein expression in the murine liver following endotoxin and iron treatment. Iron injection atIer iNOS induction (that is at 6 hours after endotoxin treatment) was without effect on the level of NO products in blood and tissue of endotoxin-treated mice The iron effect on NO output via constitutive NOS enzymes is currently under investigation. Supported by Faculty Research Enhancement Fund from GWU Medical Center (A.M.K.).

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