Nitrite as regulator of hypoxic signaling in mammalian physiology.

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

The oxidative response in the chronic constriction injury model of neuropathic pain.

BACKGROUND: In the chronic constriction injury model of rat neuropathic pain, oxidative stress as well as antioxidants superoxide dismutase and reduced glutathione (GSH) are important determinants of neuropathological and behavioral consequences. Studies of the chronic constriction injury model observed (indirect) signs of inflammation. We, therefore, investigated the level of oxidative stress and antioxidant enzymes in skeletal muscle tissue of the rat hind paw and (jugular vein) plasma at d 7 after nerve injury. MATERIALS AND METHODS: The level of reactive oxygen and nitrogen species (RONS) was determined as a measure of oxidative stress. Reduced GSH levels and the ceruloplasmin/transferrin ratio were determined as measures of overall antioxidant activity. RONS and overall antioxidant activity were measured in skeletal muscle tissue of the hind paw and jugular vein plasma. The level of RONS in muscle was determined using spin trapping combined with electron paramagnetic resonance spectroscopy. Using electron paramagnetic resonance spectroscopy, we also determined plasma levels of transferrin and ceruloplasmin. GSH levels were determined using high-performance liquid chromatography. RESULTS: In skeletal muscle tissue, the level of RONS was lower in nerve-injured hind paws than in controls. The plasma level (jugular vein) of RONS did not differ between nerve-injured and control rats. In skeletal muscle tissue, the level of GSH was higher in nerve-injured hind paws than in controls. The ceruloplasmin/transferrin ratio tended to be higher in (jugular vein) plasma of nerve-injured rats as compared to controls. CONCLUSIONS: This study shows that, at d 7 after nerve injury, oxidative stress-induced changes are present also in skeletal muscle tissue of the rat hind paw. Our findings of a decreased level of RONS in combination with an increased level of the antioxidant GSH suggest that an overshoot of antioxidant activity overrules initial oxidative stress.

Ubichromanol: a prodrug to support mitochondrial ubiquinone functions?

Ubichromanol-9 (UCa9), with a side chain consisting of nine isoprene units) is a reductive cyclization product of ubiquinone-10 (UQ10). It acts as a radical scavenging antioxidant and is about half as effective as alpha-tocopherol. Already decades ago its one-electron oxidation product, the ubichromanoxyl radical had been identified. However, nothing was known so far about the two-electron oxidation product of this antioxidant and its bioactivity. This study proves that ubichromanol can be oxidized to a ubiquinone-like compound with a hydroxyl-substituted side chain (UQ10OH), a metabolite that is naturally present in bovine liver mitochondria. The bioactivity of this ubiquinone derivative in its reduced form as substrate for mitochondrial complex III (cytochrome bc1 complex) was slightly below that of native ubiquinol, but significantly higher than that of reduced alpha-tocopheryl quinone. Since ubiquinone-like molecules (UQ10OH, UQ10) were identified as oxidation products of UCa9 during lipid peroxidation, this ubiquinone derivative could provide a possibility to combine antioxidant properties of chromanols and bioenergetic benefits of UQ10.

Skeletal muscles, heart, and lung are the main sources of oxygen radicals in old rats.

The aim of this study was to compare rat tissues with respect to their reactive oxygen and nitrogen species (RONS) generating activities as a function of age. We quantified the RONS generation in vivo in young (6 months) and in old (30 months) male Sprague-Dawley rats using the recently developed spin trap 1-hydroxy-3-carboxy-pyrrolidine, applied intravenously. This spin trap reacts with superoxide radical and peroxynitrite yielding a stable spin adduct which is detectable by means of electron paramagnetic resonance (EPR) spectroscopy in frozen tissues. In old rats RONS generation was significantly increased compared to their young counterparts in the following order: blood

Distribution of tocopheryl quinone in mitochondrial membranes and interference with ubiquinone-mediated electron transfer.

Alpha-tocopherol (Toc) is an efficient lipophilic antioxidant present in all mammalian lipid membranes. This chromanol is metabolized by two different pathways: excessive dietary Toc is degraded in the liver by side chain oxidation, and Toc acting as antioxidant is partially degraded to alpha-tocopheryl quinone (TQ). The latter process and the similarity between TQ and ubiquinone (UQ) prompted us to study the distribution of TQ in rat liver mitochondrial membranes and the interference of TQ with the activity of mitochondrial and microsomal redox enzymes interacting with UQ. In view of the contradictory literature results regarding Toc, we determined the distribution of Toc, TQ, and UQ over inner and outer membranes of rat liver mitochondria. Irrespective of the preparation method, the TQ/Toc ratio tends to be higher in mitochondrial inner membranes than in outer membranes suggesting TQ formation by respiratory oxidative stress in vivo. The comparison of the catalytic activities using short-chain homologues of TQ and UQ showed decreasing selectivity in the order complex II (TQ activity not detected)>Q(o) site of complex III>Q(i) site of complex III>complex I approximately cytochrome b(5) reductase>cytochrome P-450 reductase (comparable reactivity of UQ and TQ). TQ binding to some enzymes is comparable to UQ despite low activities. These data show that TQ arising from excessive oxidative degradation of Toc can potentially interfere with mitochondrial electron transfer. On the other hand, both microsomal and mitochondrial enzymes contribute to the reduction of TQ to tocopheryl hydroquinone, which has been suggested to play an antioxidative role itself.

Dietary lipid hydroperoxides induce expression of vascular endothelial growth factor (VEGF) in human colorectal tumor cells.

Fatty acid hydroperoxides arise from unsaturated fatty acids in the presence of oxygen and elevated temperature during processing of food. Here we have studied their effects on gene expression in colorectal tumor cells using linoleic acid hydroperoxide (LOOH) as a model compound. Its addition to the medium of LT97 human adenoma cells and SW480 human carcinoma cells enhanced the production of intracellular hydrogen peroxide. Furthermore, in both cell lines, increases in VEGF mRNA and protein were observed. Unoxidized linoleic acid had little or no activity. Concomitantly, COX-2 expression was up-regulated. In the LT97 cells, the COX inhibitors SC58560 and SC58236 completely prevented the VEGF induction, suggesting that the effect was dependent on prostaglandin synthesis. In vivo prostaglandin-mediated induction of VEGF secretion is known to be essential for the growth of adenomatous polyps and their progression to carcinomas. Therefore, our results for the first time implicate dietary lipid hydroperoxide as a key risk factor in colon carcinogenesis.

Are the biological properties of kaempferol determined by its oxidation products?

Although flavonoid molecules have attracted considerable interest in recent years because of their antioxidant effect, there are considerable differences in their chemical properties. Electron paramagnetic resonance (EPR) spectroscopy was used to compare the oxidative free radical chemistry of two such molecules, kaempferol and luteolin, which have the same empirical formula but differ in the position of one OH group. Whereas the basic flavonoid structure remains intact in luteolin, structural changes occur in kaempferol after one-electron oxidation. Autoxidation of kaempferol in alkaline solution and oxidation by at pH 7 led to rapid fragmentation. In contrast, oxidation by horseradish peroxidase/hydrogen peroxide, xanthine/xanthine oxidase (X/XO) or a Fenton reaction system produced a radical whose structure appeared to be based on dimerisation of either the original or a fragment of the flavonoid. Hence, the biological properties of kaempferol are likely to be determined by the chemistry of its oxidation products.

Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?

Metformin and thiazolidinediones (TZDs) are believed to exert their antidiabetic effects via different mechanisms. As evidence suggests that both impair cell respiration in vitro, this study compared their effects on mitochondrial functions. The activity of complex I of the respiratory chain, which is known to be affected by metformin, was measured in tissue homogenates that contained disrupted mitochondria. In homogenates of skeletal muscle, metformin and TZDs reduced the activity of complex I (30 mmol/l metformin, -15 +/- 2%; 100 micromol/l rosiglitazone, -54 +/- 7; and 100 micromol/l pioglitazone, -12 +/- 4; P < 0.05 each). Inhibition of complex I was confirmed by reduced state 3 respiration of isolated mitochondria consuming glutamate + malate as substrates for complex I (30 mmol/l metformin, -77 +/- 1%; 100 micromol/l rosiglitazone, -24 +/- 4; and 100 micromol/l pioglitazone, -18 +/- 5; P < 0.05 each), whereas respiration with succinate feeding into complex II was unaffected. In line with inhibition of complex I, 24-h exposure of isolated rat soleus muscle to metformin or TZDs reduced cell respiration and increased anaerobic glycolysis (glucose oxidation: 270 micromol/l metformin, -30 +/- 9%; 9 micromol/l rosiglitazone, -25 +/- 8; and 9 micromol/l pioglitazone, -45 +/- 3; lactate release: 270 micromol/l metformin, +84 +/- 12; 9 micromol/l rosiglitazone, +38 +/- 6; and 9 micromol/l pioglitazone, +64 +/- 11; P < 0.05 each). As both metformin and TZDs inhibit complex I activity and cell respiration in vitro, similar mitochondrial actions could contribute to their antidiabetic effects.

Mechanisms of vasodilatation induced by nitrite instillation in intestinal lumen: possible role of hemoglobin.

It has been shown that nitrite can be reduced to nitric oxide (NO) in intestine and a number of other tissues and released into the blood to form nitrosylhemoglobin (NO-Hb), existing in an equilibrium with S-nitrosohemoglobin. The latter has been suggested to be an NO transporter to distant organs. The aim of this study was to define the pathway of nitrite reduction to form NO in intestinal wall and to estimate whether this pathway has an effect on peripheral circulation. We have shown that in rat intestine at pH 7.0 70% of nitrite is converted to NO in mitochondria. At pH 6.0, nonenzymatic nitrite reduction becomes as efficient as the mitochondrial pathway. To prove whether the NO formed from nitrite in intestine can induce vasodilatation, sodium nitrite was instilled into intestinal lumen and the concentration of NO formed and diffused into the blood was followed by measuring of NO-Hb complex formation. We found that the concentration of NO-Hb gradually increases with the increase of nitrite concentration in intestinal lumen. However, it was not always accompanied by a decrease in systemic blood pressure. Blood pressure dropped down only after NO-Hb reached a threshold concentration of approximately 10 microM. These data show that NO-Hb cannot provide enough NO for vasodilatation if the concentration of NO bound to Hb is < 10 microM. The exact mechanism underlying vasodilatation observed when the concentration of NO-bound Hb was > 10 microM is, however, not clear yet and requires further studies.

Intracellular generation of reactive oxygen species by mitochondria.

Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H(2)O(2) from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc(1) complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H(2)O(2) via superoxide radicals.

The protection of bioenergetic functions in mitochondria by new synthetic chromanols.

alpha-Tocopherol is the most important lipophilic antioxidant of the chromanol type protecting biomembranes from lipid peroxidation (LPO). Therefore, alpha-tocopherol and its derivatives are frequently used in the therapy or prevention of oxygen radical-derived diseases. In the present study, novel chromanol-type antioxidants (twin-chromanol, cis- and trans-oxachromanol) as well as the well-known short-chain analogue of alpha-tocopherol, pentamethyl-chromanol, were tested for their antioxidative potency in rat heart mitochondria (RHM). Our experiments revealed that the bioenergetic parameters of mitochondria were not deteriorated in the presence of chromanols (up to 50 nmol/mg protein). Exposure of RHM to cumene hydroperoxide and Fe2+ (final concentrations 50 microM each), inducing LPO, significantly affected their bioenergetic parameters which were determined in the presence of glutamate and malate (substrates of mitochondrial complex I). Alterations of the bioenergetic parameters were partially prevented in a concentration-dependent manner by preincubating RHM with antioxidants before adding the radical-generating system. In the lower concentration range, twin-chromanol turned out to be more efficient than pentamethyl-chromanol, both being far more protective than cis- and trans-oxachromanol. Measurement of protein-bound SH groups and thiobarbituric acid-reactive substances revealed that this protective effect was due to their antioxidative action. Furthermore, HPLC measurements of alpha-tocopherol and alpha-tocopheryl quinone in rat liver mitochondria demonstrated an alpha-tocopherol-sparing effect of twin-chromanol. In conclusion, new chromanol-type antioxidants, especially twin-chromanol, were able to improve bioenergetic and biochemical parameters of mitochondria exposed to oxidative stress.

Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals.

Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO (t1/2=ca. 45 s), and even over DEPMPO (t1/2=ca. 14 min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by 1H and 13C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO (t1/2=ca. 8 min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.

Very stable superoxide radical adducts of 5-ethoxycarbonyl- 3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives.

Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free radical formation in biological systems. Superoxide spin adducts with half-lives up to 20 min at physiological pH have recently been reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major improvement over DMPO (t(1/2) ca. 45 s), and even DEPMPO (t(1/2) ca. 14 min). In this study, an additional methyl group was introduced into position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic properties of the investigated spin traps. The structure of all compounds was confirmed by (1)H and (13)C-NMR with full signal assignment. In comparison with EMPO (t(1/2) ca. 8 min) or DEPMPO (t(1/2) ca. 14 min), the superoxide adducts of all novel spin traps were considerably higher (t(1/2) ca. 12-55 min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g. derived from methanol or linoleic acid hydroperoxide) were also detected.

Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure-activity relationship.

Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure-activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O(2)(*-) (5,5-dimethyl-1-pyrroline-N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3',4',5-tetrahydroxystilbene (IC(50): 2.69microM; k(9): 443000M(-1)s(-1)), 3,4,4',5-tetrahydroxystilbene (IC(50): 41.5microM; k(9): 882000M(-1)s(-1)) and 3,3',4,4',5,5'-hexahydroxystilbene (IC(50): 5.02microM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds 1 and 3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.

Suppression of tumour-promoting factors in fat-induced colon carcinogenesis by the antioxidants caroverine and ubiquinone.

Fatty acid hydroperoxides are produced from unsaturated fatty acids in the presence of oxygen at elevated temperatures during food processing. Their effects on gene expression in colorectal tumour cells were studied using linoleic acid hydroperoxide (LOOH) as a model compound. Addition of LOOH to the medium of LT97 adenoma and SW480 carcinoma cells enhanced the production of hydrogen peroxide. Both cell lines were observed to increase VEGF factors based on mRNA. High consumption of dietary fat promotes colon carcinogenesis in the long-term. While this effect is well known, the underlying mechanisms are not understood. An approach was made starting from the assumption that LOOH is present in dietary fats as a result of heating. LOOH undergoes homolytic cleavage in the presence of iron. Various radicals are formed on mixing LT97 or SW480 cells with LOOH. The expression of tumour-promoting factors was inhibited by caroverine and ubiquinone, which may be justified as active chemopreventive agents.

Release and hemodynamic influence of nitro-glycerine-derived nitric oxide in endotoxemic rats.

BACKGROUND AND AIM: Nitric oxide released from nitro-glycerine (NG) has been considered to improve the microcirculation. Septic conditions are, however, associated with excessive formation of nitric oxide (NO), which is formed from l-arginine by the inducible NO synthase (iNOS) activity. Since the characteristics and influence of NG-derived NO in sepsis remains unclear, the major aims of the present study were to quantify the release and to determine the effects of NO formed from NG on systemic blood pressure under endotoxemic conditions. MATERIAL AND METHODS: Four hours following endotoxin challenge (10 mg/kg intraperitoneally), rats received an infusion of NG (0.5 or 5.0 micromol/kg/h) over 45 min. We determined the changes in blood pressure and the NO concentrations generated in brain, heart, intestine, kidney, liver, and lung by means of NO trapping and EPR technique. RESULTS: NG infusion in control rats and endotoxin challenge decreased systemic blood pressure to the same extent. However, in rats subjected to endotoxin challenge NG infusion did not affect the blood pressure. The endotoxin-induced increase in tissue NO concentrations were found to be 15-folds higher than tissue levels of NO following NG infusion. CONCLUSION: Our results suggest that under endotoxic shock conditions in rats NG may not additionally affect the systemic blood pressure. This may relate to the excessive tissue NO levels induced by endotoxin that are not further increased by NG.

Ubiquinol and the papaverine derivative caroverine prevent the expression of tumour- promoting factors in adenoma and carcinoma colon cancer cells induced by dietary fat.

High consumption of dietary fat promotes colon carcinogenesis. While this effect is well known the underlying mechanism is not understood. Fatty acid hydroperoxides (LOOH) arise from unsaturated fatty acids in the presence of oxygen and elevated temperature during food processing. An approach was made starting from the assumption that LOOH are present in dietary fats as a result of boiling. LOOH undergoes homolytic cleavage in the presence of iron. We studied their effects on gene expression in colorectal tumour cells using linoleic acid hydroperoxide (LOOH) as model compound. Addition to the medium of LT97 adenoma and SW480 carcinoma cells enhanced the production of hydrogen peroxide. Both cell lines were observed to increase VEGF and COX-II expression based on mRNA. Expression of VEGF was inhibited by caroverine and ubiquinon.

Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species.

The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO(-) and O(2)(*)(-), as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.

Various intracellular compartments cooperate in the release of nitric oxide from glycerol trinitrate in liver.

1. Glycerol trinitrate (GTN) has been used in therapy for more than 100 years. Biological effects of GTN are due to the release of the biomediator nitric oxide (NO). However, the mechanism by which GTN provides NO, in particular in liver, is still unknown. In this study, we provide experimental evidence showing that cytoplasm, endoplasmic reticulum, and mitochondria are required for the release of NO from GTN in the liver. 2. NO and nitrite (NO(2)(-)) were determined using low-temperature electron paramagnetic resonance and the Griess reaction, respectively. 3. The first step of GTN biotransformation is the release of NO(2)(-). This step is performed in cytoplasm and catalyzed by glutathione-S-transferase. The second step is the rate-limiting step where NO(2)(-) is slowly reduced to NO. This is mainly catalyzed by cytochrome P-450. The second phase can be significantly enhanced by decreasing the pH value, a situation which occurs during ischemia. At high NADPH concentrations exceeding physiological values, cytochrome P-450 catalyzes GTN biotransformation without the involvement of cytoplasmic glutathione-S-transferase. 4. In conclusion, our data show that NO(2)(-) derived from the first step of biotransformation of GTN in the liver is the precursor of NO but not a product of NO degradation; consequently, NO(2)(-) levels are not likely to be a marker of NO release from GTN as earlier suggested.

ESR analysis of spin adducts of alkoxyl and lipid-derived radicals with the spin trap Trazon.

Detection of oxygen-centered radicals was performed using the spin trap 1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ene-N-oxide (Trazon), a bicyclic nitrone spin trap that is easily synthesized from the corresponding amine via hydrogen peroxide mediated oxidation in the presence of the catalyst, sodium tungstate. Compared to monocyclic spin traps such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), the ESR spectra of Trazon spin adducts provide additional structural information due to long-range hyperfine splitting constants and also due to the fact that different stereoisomers can be distinguished. This is especially helpful for the detection of lipid-derived alkoxyl radicals which can be identified according to their characteristic hyperfine splitting pattern. Due to the relatively high stability of the Trazon spin adducts with lipid alkoxyl radicals, which were formed from peroxidizing linoleic acid, ESR experiments could be performed using a stationary system, whereas a slow-flow system is recommended for DMPO. A series of structurally different alkoxyl radical adducts were synthesized by iron-catalyzed nucleophilic addition of the respective alcohol to the spin trap Trazon and the spectra were analyzed by computer simulation. Both the molecular weight of the alcohol and the position of the alcoholic hydroxyl group were of significant influence on the ESR spectra. Two stereochemically different spin adducts were formed in a ratio typical of the alcohol used, thus allowing structural classification of the alkoxyl radical trapped.