Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum

Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of hydroxyl radicals concomitant with cAMP in carbon monoxide poisoning, because the formation of 2,3-DHBA was potentiated by the PKA inhibitor H89 and suppressed by Rp-8-Br-cAMPS, which inhibits PKA and Epac.     

Impaired phosphatidylcholine biosynthesis and ascorbic acid depletion in lung during lipopolysaccharide-induced endotoxaemia in guinea pigs

Recently there has been a moderate resurgence in the use of flax-seed in a variety of ways including bread. The scientific basis of its use is very limited. There is some claim for beneficial effects in cancer and lupus nephritis. These claims could be due to its ability to scavenge oxygen radicals. However, its antioxidant activity is not known. Recently a method has been developed to isolate secoisolariciresinol diglucoside (SDG) from defatted flax-seed in large quantity (patent pending). We investigated the ability of SDG to scavenge úOH using high pressure liquid chromatography (HPLC) method. úOH was generated by photolysis of H2O2 (1.25-10.0 \sgmaelig;moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce úOH-adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. H2O2 produced a concentration-dependent úOH as estimated by 2,3-DHBA and 2,5-DHBA. A standard curve was constructed for known concentrations of 2,3-DHBA and 2,5-DHBA against corresponding area under the peaks which then was used for measurement of 2,3-DHBA and 2,5-DHBA generated by UV irradiation of H2O2 in the presence of salicylic acid. SDG in the concentration range of 25, 50, 100, 250, 500, 750, 1000 and 2000 \sgmaelig;g/ml (36.4, 72.8, 145.6, 364.0, 728.0, 1092.0, 1456.0 and 2912.0 \sgmaelig;M respectively) produced a concentration-dependent decrease in the formation of 2,3-DHBA and 2,5-DHBA, the inhibition being 4 and 4.65% respectively with 25 \sgmaelig;g/ml (36.4 \sgmaelig;M) and 82 and 74% respectively with 2000 \sgmaelig;g/ml (2912.0 \sgmaelig;M). The decrease in úOH-adduct products was due to scavenging of úOH not and by scavenging of formed 2,3-DHBA and 2,5-DHBA. SDG prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner in the concentration range from 319.3-2554.4 \sgmaelig;M. These results suggest that SDG scavenges úOH and therefore has an antioxidant activity.     

LC/MS analysis of hydroxylation products of salicylate as an indicator of in vivo oxidative stress

Hydroxyl radical attack upon salicylate leads to the generation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and therefore can be used to assess hydroxyl radical formation both in vitro and in vivo. Evidence is presented for a highly sensitive LC/MS assay for the quantification of 2,3-DHBA. Calibration curves showed linearity within the concentration range tested (0.5-6.5 pmol/microl rat plasma) with a coefficient of determination (r2) greater than 0.99. A detection limit of less than 0.25 pmol for 2,3-DHBA has been achieved. The intra-assay and inter-assay variability were determined to be 4.1% and 12.5%, respectively. This method was evaluated for the determination of drug-induced in vivo generation of oxidative stress by means of 1,1,1-trichloroethane (TCE) a compound that is a pseudosubstrate for cytochrome P450 and is known to induce oxygen reductase activity of this enzyme(s). TCE treated rats had a 6.4-fold increase in the mean maximal plasma 2,3-DHBA concentration as compared to the saline treated rats (p = .009). The developed LC/MS assay requires minimal sample preparation and provides a rapid and sensitive method for quantification of 2,3-DHBA as a specific indicator of hydroxyl radical generation.     

Blocking cardiac ATP-sensitive K+ channels reduces hydroxyl radicals caused by potassium chloride-induced depolarization in the rat myocardium

The current study examined whether opening of the ATP-sensitive K(+) (K(ATP)) channel can induce hydroxyl free radical (OH) generation, as detected by increases in nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels in the rat myocardium. When KCl (4-140mM) was administered to rat myocardium through microdialysis probe, the level of 2,3-DHBA increased gradually in a potassium ion concentration ([K(+)](o))-dependent manner. The [K(+)](o) for half-maximal effect of the level of 2,3-DHBA production (ED(50)) was 67.9microM. The maximum attainable concentration of the level of 2,3-DHBA (E(max)) was 0.171microM. Induction of glibenclamide (10microM) decreased OH formation. The half-maximal inhibitory effect (IC(50)) for glibenclamide against the [K(+)](o) (70mM)-evoked increase in 2,3-DHBA was 9.2microM. 5-Hydroxydecanoate (5-HD, 100microM), another K(ATP) channel antagonist, also decreased [K(+)](o)-induced OH formation. The IC(50) for 5-HD against the [K(+)](o) (70mM)-evoked increase in 2,3-DHBA was 107.2microM. The heart was subjected to myocardial ischemia for 15min by occlusion of left anterior descending coronary artery (LAD). When the heart was reperfused, the normal elevation of 2,3-DHBA in the heart dialysate was not observed in animals pretreated with glibenclamide (10microM) or 5-HD (100microM). These results suggest that opening of cardiac K(ATP) channels by depolarization evokes OH generation.     

Dopaminergic denervation enhances susceptibility to hydroxyl radicals in rat neostriatum

To determine if greater amounts of hydroxyl radical (*OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134microg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5mg/kg i.p.) was administered 30min before vehicle, L-DOPA (60mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60mg/kg i.p.); and this was followed 30min later (and 15 min before termination) by the spin trap, salicylic acid (8micromoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses *OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) *OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed *OH.     

Increased generation of hydroxyl radicals in the rat hypertrophied myocardium: in vivo study by microdialysis

A comparative study of the generation of hydroxyl radicals (OH*) in the hypertrophic myocardium of SHR-SP rats (n = 8) and in the myocardium of WKY (n = 5) and Wistar (n = 12) rats was performed using the microdialysis technique. The experiments were carried out on anesthetized open-chest male rats (ketamine intraperitoneally, 10 mg/kg) with artificial ventilation. The amount of OH* produced was estimated by high-performance liquid chromatography with electrochemical detection using as a marker 2,3-dihydroxybenzoic acid (2,3-DHBA), a product of the reaction of the hydroxyl radical with salicylic acid added to the perfusate. The quantity of 2,3-DHBA in the dialysate was estimated by the external standard method and expressed in percent of the 2,3-DHBA concentration in the perfusion fluid. The mean baseline value of 2,3-DHBA in dialysate samples in SHR-SP rats (157 +/- 22%, n = 8) was significantly higher than in Wistar (90 +/- 15%, n = 12, p = 0.0001) and Wistar-Kyoto rats (106 +/- 12%, n = 5, p = 0.005). The basal 2,3-DHBA level in SHR-SP rats was positively correlated (r = 0.831, n = 7, p < 0.05) with the degree of hypertrophy of the left ventricle expressed as the ratio of the left ventricle weight to the body weight. The data presented demonstrate that the hypertrophy of the left ventricle in SHR-SP rats is accompanied by the elevation of the level of free oxygen radicals.     

Enzymatic determination of itoic acid, a Bacillus subtilis siderophore, and 2,3-dihydroxybenzoic acid.

A specific enzymatic method to determine the amounts of itoic acid, a Bacillus subtilis siderophore, and 2,3-dihydroxybenzoic acid (2,3-DHBA) was devised. A sample was incubated first with hippurate hydrolase and then with 2,3-DHBA-3,4-dioxygenase. Itoic acid was estimated from the increase in A374. The incubation with the first enzyme was omitted for the determination of 2,3-DHBA.     

Systemic administration of d-amphetamine induces long-lasting oxidative stress in the rat striatum

The long-term effect of d-amphetamine (AMPH) on the induction of oxidative stress was examined in vivo in the rat brain. In this study, 2,3-dihydroxybenzoic acid (2,3-DHBA) and malonaldehyde (MDA) were used as the index of the hydroxyl radical and lipid peroxidation, respectively. The levels of 2,3-DHBA, MDA and dopamine (DA) in striatal homogenates were examined 7 days following injection of a single large dose of AMPH (7.5 mg/kg, i.p.) in rats pretreated with desipramine (10 mg/kg, i.p.), an agent that inhibits the metabolism of AMPH. Our results showed that 2,3-DHBA and MDA levels were significantly increased by AMPH, whereas DA and its metabolites, DOPAC and HVA were depleted in the striatum. Pretreatment with the glutamate NMDA receptor subtype antagonist MK-801 (1 mg/kg, i.p.) attenuated the increases of 2,3-DHBA and MDA, and provided partial protection against the long-lasting loss of DA produced by AMPH. Overall, the results demonstrate that AMPH could induce sustained production of free radical and oxidative damage, and lead to DA terminal degeneration in the striatum of the rat.     

Role of Reactive Oxygen Species in the Sensitivity of Rat Hypertrophied Myocardium to Ischemia

The relationship between hydroxyl radical (OH*) generation in the zone of ischemia/reperfusion and the size of infarction formed was investigated in 18-22-week-old anaesthetized male SHRSP and Wistar rats using a myocardial microdialysis technique. The marker of OH* generation, 2,3-dihydroxybenzoic acid (2,3-DHBA), was analyzed in dialyzates by high performance liquid chromatography with electrochemical detection. Myocardial ischemia was induced by ligation of the descending branch of the left main coronary artery for 30 min. The mean value of basal 2,3-DHBA level in the dialyzate samples from SHRSP (243 +/- 21 pg for 30 min) was significantly higher than that from Wistar rats (91 +/- 4 pg for 30 min, p < 0.0002); it positively correlated with left ventricular hypertrophy (r = 0.806; p < 0.05). During reperfusion total 2,3-DHBA output was 1.8-fold higher in SHRSP than in Wistar rats (659 +/- 60 pg versus 364 +/- 66 pg for 60 min, respectively, p < 0.0002). At the same time, 2,3-DHBA increase above the basal level was the same in Wistar and SHRSP rats (181 +/- 25 and 172 +/- 36 pg for 60 min, respectively). The infarct size in SHRSP (45.4 +/- 4.3%) was significantly higher (p < 0.05) than in Wistar rats (32.8 +/- 3.3%). There was a significant positive correlation between basal level of 2,3-DHBA and total reperfusion 2,3-DHBA content in SHRSP (r = 0.752; p < 0.05). Thus, data obtained clearly indicate that the hypertrophied myocardium of SHRSP was less tolerant to ischemia/reperfusion than that of Wistar rats due to chronically increased OH* production and enhanced total OH* output during reperfusion. Greater myocardial damage in SHRSP than in Wistar rats following the equal increase in OH* production above the basal level suggests the existence of deficit of the antioxidant defense in the hypertrophied myocardium.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Effects of α-Phenyl-tert-Butylnitrone and Selegiline on Hydroxyl Free Radicals in Rat Striatum Produced by Local Application of Glutamate

Abstract: The hydroxyl radical is a very reactive oxygen species that damages biomolecules in the brain and in other tissues. The possible pharmacological intervention to prevent hydroxyl radical formation was studied in vivo using the microdialysis technique in brains of nonanesthetized rats. Hydroxyl radicals form stable adducts [mainly 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA)] via an aromatic hydroxylation reaction with salicylic acid. 2,3-DHBA was separated and quantified by HPLC and electrochemical detection. Microdialysis probes were implanted into the striatum 1 day before measurement of levels of hydroxyl radicals. The next day, the probes were first perfused for 120 min with a modified Ringer's solution containing 5 m M salicylic acid, to obtain stable baselines. Afterward, the perfusion solution was switched to another solution that in addition contained 50 m M glutamate, to stimulate radical formation. Twenty minutes later, α-phenyl- tert -butylnitrone (PBN; 100 mg/kg), selegiline (10 mg/kg), or saline was administered intraperitoneally. The glutamate perfusion produced marked two- to 2.5-fold increases in 2,3-DHBA content. Treatment with PBN significantly antagonized the rise of 2,3-DHBA level, indicating that PBN is a direct radical scavenger not only in vitro but also in vivo. Acute treatment with selegiline failed to reduce significantly the glutamate-induced radical formation. The acute experiments presented here do not support the suggestion that the neuroprotective effects of selegiline described in the literature are due to a potential hydroxyl radical scavenging property of the drug.     

Mazindol Attenuates the 3,4-Methylenedioxymethamphetamine-Induced Formation of Hydroxyl Radicals and Long-Term Depletion of Serotonin in the Striatum

The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine. The MDMA-induced increase in the extracellular concentration of both dopamine and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine-dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long-term depletion of brain 5-HT content.     

L-DOPA does not enhance hydroxyl radical formation in the nigrostriatal dopamine system of rats with a unilateral 6-hydroxydopamine lesion

The debate about the toxicity of L-DOPA to dopaminergic neurons has not been resolved. Even though enzymatic and nonenzymatic metabolism of L-DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L-DOPA generates an oxidant stress in vivo. This study determined whether acute or repeated administration of L-DOPA caused in vivo production of hydroxyl radicals in striatum and other brain regions in rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal projections. Salicylate trapping combined with in vivo microdialysis provided measurements of extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA) in striatum following L-DOPA administration systemically (100 mg/kg, i.p.) or by intrastriatal perfusion (1 mM, via the microdialysis probe). Tissue concentrations of 2,3-DHBA and salicylate were also measured in striatum, ventral midbrain, and cerebellum following repeated administration of L-DOPA (50 mg/kg, i.p., once daily for 16 days). In each instance, treatment with L-DOPA did not increase 2,3-DHBA concentrations, regardless of the nigrostriatal dopamine system's integrity. When added to the microdialysis perfusion medium, L-DOPA resulted in a significant decrease in the striatal extracellular concentration of 2,3-DHBA. These results suggest that administration of L-DOPA, even at high doses, does not induce hydroxyl radical formation in vivo and under some conditions may actually diminish hydroxyl radical activity. Furthermore, prior damage to the nigrostriatal dopamine system does not appear to predispose surviving dopaminergic neurons to increased hydroxyl radical formation following L-DOPA administration. Unlike L-DOPA, systemic administration of methamphetamine (10 mg/kg, s.c.) produced a significant increase in the concentration of 2,3-DHBA in striatal dialysate, suggesting that increased formation of hydroxyl radicals may contribute to methamphetamine neurotoxicity.     

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Protective effect of histidine on iron (II)-induced hydroxyl radical generation in rat hearts.

We investigated the efficacy of histidine on iron (II)-induced hydroxyl radical (.OH) generation in extracellular fluid of the rat myocardium using a flexibly mounted microdialysis technique (O system). Rats were anesthetized and a microdialysis probe was implanted in the left ventricular, followed by infusion of sodium salicylate in Ringer's solution (0.5 nmol/microL/min) to detect the generation .OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). Iron (II) clearly produced a concentration-dependent increase in .OH formation. A positive linear correlation between iron (II) and the formation of 2,3-DHBA (R2 = 0.987) was observed. However, histidine (25 mM) was infused through a microdialysis probe; iron (II) failed to increase the 2,3-DHBA formation obtained. To examine the effect of histidine on ischemia-reperfusion of the myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the heart dialysate. When corresponding experiments were performed with histidine (25 mM)-pretreated animals, histidine prevented the ischemia-reperfusion induced .OH generation trapped as 2,3-DHBA. These results indicate that histidine protects the myocardium against ischemia-reperfusion damage by .OH generation.     

The salicylate trapping method: is oxidation of salicylic acid solution oxygen and time dependent and metal catalysed?

For a microdialytic trapping method we systematically investigated changes in concentrations of 2,5-dihydroxy-benzoic acid (2,5-DHBA) and 2,3-dihydroxy-benzoic acid (2,3-DHBA) in freshly prepared solutions of salicylic acid (SA). The solvent was 0.9% saline exposed to different atmospheric concentrations of oxygen (0, 21, and 100%). The solutions were treated by freezing-thawing and an ultrasonic bath in presence and absence of aluminium foil. Without aluminium the concentrations of 2,5-DHBA and 2,3-DHBA kept constant over an observed period of 160 min on different levels from below 20 ng/ml to about 100 ng/ml. In presence of aluminium the concentrations increased to maximum 307 ng/ml after 160 min. Ultrasonic irradiation amplified this effect to maximum 341 ng/ml. HPLC/ECD processing and quantitative analysis of dihydroxy-benzoic acids (DHBAs) in microdialysis may be artificially influenced by varying oxygen environment and metal catalysis.     

Glutamate Release and Free Radical Production Following Brain Injury: Effects of Posttraumatic Hypothermia

Abstract: Posttraumatic hypothermia reduces the extent of neuronal damage in remote cortical and subcortical structures following traumatic brain injury (TBI). We evaluated whether excessive extracellular release of glutamate and generation of hydroxyl radicals are associated with remote traumatic injury, and whether posttraumatic hypothermia modulates these processes. Lateral fluid percussion was used to induce TBI in rats. The salicylate-trapping method was used in conjunction with microdialysis and HPLC to detect hydroxyl radicals by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA). Extracellular glutamate was measured from the same samples. Following trauma, brain temperature was maintained for 3 h at either 37 or 30°C. Sham-trauma animals were treated in an identical manner. In the normothermic group, TBI induced significant elevations in 2,3-DHBA (3.3-fold, p < 0.01), 2,5-DHBA (2.5-fold, p < 0.01), and glutamate (2.8-fold, p < 0.01) compared with controls. The levels of 2,3-DHBA and glutamate remained high for approximately 1 h after trauma, whereas levels of 2,5-DHBA remained high for the entire sampling period (4 h). Linear regression analysis revealed a significant positive correlation between integrated 2,3-DHBA and glutamate concentrations ( p < 0.05). Posttraumatic hypothermia resulted in suppression of both 2,3- and 2,5-DHBA elevations and glutamate release. The present data indicate that TBI is followed by prompt increases in both glutamate release and hydroxyl radical production from cortical regions adjacent to the impact site. The magnitude of glutamate release is correlated with the extent of the hydroxyl radical adduct, raising the possibility that the two responses are associated. Posttraumatic hypothermia blunts both responses, suggesting a mechanism by which hypothermia confers protection following TBI.     

High-resolution analysis of catechol-type siderophores using polyamide thin layer chromatography

The iron-deficient culture supernatant of a soil bacterial strain identified as Erwinia sp. was analyzed using a new high-resolution polyamide thin layer chromatography (TLC) and a silica TLC. The results showed both TLC methods were very effective for separating simple catechol compounds such as 2,3-dihydroxybenzoic acid (2,3-DHBA) and catechol. However, in the analysis of more complicated catechol compounds or true catechol-type siderophores (conjugates of 2,3-DHBA and amino acids), the polyamide TLC had the higher resolution. Polyamide TLC analysis showed that strain S1 produced three distinct catechol-type siderophores.     

Protective effect of histidine on potassium chloride depolarization enhances 1-methyl-4-phenylpyridinium ion-induced hydroxyl radical generation in the rat striatum

The present study examined the antioxidant effect of histidine on extracellular potassium ion concentration, [K+]o-induced depolarization enhances 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical (*OH) generation in the rat striatum. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/M microl/min) was infused through a microdialysis probe to detect the generation of *OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of [K+]o (20, 70 and 140 mM) significantly increased the level of 2,3-DHBA by the action of MPP+ (5 mM) in a concentration-dependent manner. However, histidine (25 mM) reduced the [K+]o-induced *OH formation. Although the level of MPP+-induced dopamine (DA) and 2,3-DHBA formation after [K+]o (70 mM) treatment increased, [K+]o failed to increase either the level of MPP+-induced DA and 2,3-DHBA in the reserpinized group. When iron (II) was administered to [K+]o (70 mM)-pretreated rats, iron (II) clearly produced a dose-dependent increase in the level of 2,3-DHBA, as compared with MPP+-only treated rats. However, in the presence of histidine (25 mM), the effect of [K+]o was abolished. These results indicated that histidine may reduce the [K+]o-induced depolarization enhanced *OH formation by the action of MPP+ in the rat striatum.