I172 1

TURNOVER-DEPENDENT CONSUMPTION OF NITRIC OXIDE BY 15LIPOXYGENASE: MODULATION OF NITRIC OXIQE SIGNALLING AND 15-LIPOXYGENASE ACTIVITY. V.B. O’Donnell, K.B. Taylor, S. Parthasarathy, H. Kuhn, V.M. Darley-Usmar and B.A. Freeman,

Dept of Anesthesiology, Biochemistry and Molecular Genetics, and the UAB Center for Free Radical Biology, University of Alabama at Birmingham, AL 35233.

The reactions of nitric oxide (*NO) during inflammatory conditions must be revealed in order to gain insight into the metabolic fates of *NO in viva In a cell line expressing physiological levels of 15- lipoxygenase (LOX), rates of linoleate-stimulated *NO consumption, 7-fold higher than rates of *NO production by activated rodent macrophages, were seen. This *NO consumption did not occur in l3- galactosidase-transfected controls, indicating that cellular 15-LOX has the potential to antagonize *NO signalling. Inhibition of IS- lipoxygenases by *NO was previously proposed to result from iron- nitrosyl complex formation between *NO and the active site ferrous iron. However, at biological *NO concentrations, no reaction with either native (Bred), or ferric lipoxygenase to form nitrosyl complexes was found. Rather, *NO consumption resulted from reaction with an enzyme-substrate Intermediate formed during the dioxygenase cycle, most probably the enzyme-bound lipid peroxyl radical, BredLOO*. During *NO consumption, partial reversible inhibition of 13(S)HPODE generation occurred, but lipid product profile was unchanged. Kinetic modelling of diene conjugation and *NO consumption rates revealed a rapid reaction of *NO with BredLOO*. Since co-generation of *NO and lS-lipoxygenase products occurs in inflammatory conditions such as atherosclerosis, interactions between these pathways will result in modulation of both *NO bioavailability and extents of production of lipid signalling mediators.

I 174 I I 1

THE EFFECT OF INTRAUTERINE HYPOXIA ON EXCRETION OF NITRITES AND NITRATES IN NEWBORN URINE Or&ha Tl., Andreem AA, Arutjwym A.V. D.O.Ott lnrtitute Obst&.bCyn.

The effect P

oal of the present investigation was to evaluate the o acute hypoxia or asphyxia on nitric oxide formation in

newborn organisms. A group of 42 full-term newborn children were observed. 20 of them have acute intrauterine hypoxia and 22 were healthy. Nitrite and nitrate level in urine were evaluated by Griess reaction after incubation of the sample with bacterial nitratereductase. Thts index was used to characterize nitric oxide forrnation in organism of the newborn. It was revealed that in newborn &i&en with acute hypoxia and sharp clinical brain circulation disorders the production of nitric oxide both in daytime and night was increased. At the same time normal circadian rhythm of nitric oxide production has been preserved. Thus nitric oxide can play an important role in genesis of brain circulation disorders and damage of neurons.

NITRATION OF UNSATURATED FATTY ACIDS BY NITRIC OXIDE-DERIVED REACTIVE NITROGEN SPECIES.

V.B. O’Donnell, A. Bloodsworth, J.P. Eiserich, P.H. Chumley, M.J. Jablonsky, N.R. Krishna, M. Kirk, S. Barnes V.M. Darley-Usmar, and B.A. Freeman.

Dept of Anesthesiology, Biochemistry and Molecular Genetics, and the UAB Center for Free Radical Biology, University of Alabama at Birmingham, AL 35233.

Nitric oxide (*NO)-derived reactive nitrogen species are potent nitrosating/nitrating agents formed during inflammation. Using HPLC coupled with UV detection, mass spectrometry ([14N], [15N]) and [15N]NMR nitrated products of peroxynitrite (ONOO-) reaction with linoleic acid (nitrolinoleate, LNO2), were detected. Nitration of cholesteryl linoleate in low density lipoprotein (LDL) with ONOO- also yielded LNO2, and analysis of lipids isolated from atherosclerotic plaque showed a cholesterol-containing species with mass/charge (m/z) and retention time on HPLC identical to LNO2 seen in ONOO--treated, but not control native LDL. Several other nitration/nitrosation strategies also yielded nitrated linoleate derivatives. These include, reaction of linoleic acid with either nitrogen dioxide (*N02) or nitronium tetrafluoroborate (N02BF4) yielding LNO2, and reaction of 13(S)hydroperoxyoctadecedienoic acid (HPODE) with either acidified nitrite (HONO) or isobutyl nitrite yielding a species (LON02) with a nitro functional group. We suggest this is a nitroepoxylinoleic acid formed via: (i) disso-ciation of the initial product LOON0 to LO* and *NO,, (ii) rearrangement of LO* to an epoxyallylic radical L(O)* and (iii) recombination to give L(O)NO,. Since unsaturated lipids of membranes and lipoproteins are targets for reactive oxygen and nitrogen species under both normal and acidic conditions (e.g. phagolysosome), these reactions lend insight into mechanisms for the formation of novel nitrogen-containing lipids in viva.

I 175 LACK OF TYROSINE NITRATION BY PEROXYNITRITE GENERATED IN SITU FROM NITRIC OXIDE AND SUPEROXIDE

. . . &D&&&I and Bemd Mayer

Institut fit Pharmakologie und Toxikologie, Karl-Franzens- UniversitHt Graz, Universithtsplatz 2, A-8010 Graz, Austria

Tyrosine nitration of proteins was suggested as a marker for peroxynitrite-mediated tissue injury. The nitration reaction has been extensively studied by bolus addition of peroxynitrite, an experimental approach hardly reflecting ~RJ&Q situations in which peroxynitrite is thought to be formed continuously from *NO and 02’-. Here we measured the nitration of free tyrosine by *NO and 02’- generated at defined rates from spermine NONOate and the xantbine oxidase reaction, respectively, and compared the results with nitration by authentic peroxynitrite. Bolus addition of peroxynittite to tyrosine (1 mM each) yielded 36.77 f 1.67 ph4 3- nitrotyrosine, corresponding to a recovery of -4 8, whereas ‘NO and w- generated at about equal rates from spermine NONOate and xantbine oxidaselhypoxanthine, respectively, were much less efficient (recovery -0.07 46 of total product flow). Nitration was most efficient with the ‘NO donor alone (-0.3 8 recovery). This ‘NO-triggered nitration had a pH optimum of 8.2, increased prog- ressively with increasing tyrosine concentrations, ‘and was not enhanced by NaHC03 (up to 20 mM), indicating that it was mediated by ‘NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from ‘NO and 0~‘~ as a mediator of tyrosine nitration igy&~.

OXYGEN ’ 9 8