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.     

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.     

Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with electrochemical detection.

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.     

Antioxidant activity of allicin,an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 g) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 g/ml was 32.36% and 43.2% respectively while with 36.0 g/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.     

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.     

Hydroxyl Radical Formation in Hyperglycemic Rats During Middle Cerebral Artery Occlusion/Reperfusion

Preexisting hyperglycemia is associated with enhanced reperfusion injury in the postischemic rat brain. The goal of this study was to evaluate whether the hyperglycemic exacerbation of brain injury is associated with enhanced generation of hydroxyl radicals in rats subjected to middle cerebral artery occlusion (2 h), followed by reperfusion (2 h). Magnetic resonance images revealed the exacerbation of focal brain injury in hyperglycemic rats. The salicylate trapping method was used in conjunction with microdialysis to continuously estimate hydroxyl radical production by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) during ischemia/reperfusion. In normoglycemic rats, from a mean baseline level of 130 nmol/l, 2,3-DHBA levels surged to peak levels of 194 nmol/l 45 min into ischemia and to 197 nmol/l 15–30 min into the reperfusion period, returning to baseline by 2 h into reperfusion. A similar temporal profile was observed in hyperglycemic rats, except that absolute 2,3-DHBA levels were higher (165 nmol/l at baseline, 317 nmol/l peak during ischemia, 333 nmol/l peak during reperfusion), and levels remained significantly high (p < .05) throughout the reperfusion period. These results suggest that hydroxyl radical is an important contributor to the exacerbation of neuronal and cerebrovascular injury after focal ischemia/reperfusion in hyperglycemic rats.     

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.     

Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.     

Determination of salicylate hydroxylation products as an in vivo oxidative stress marker

The in vivo measurement of highly reactive free radicals, such as the z.rad OH radical, is very difficult. New specific markers, which are based on the ability of z.rad OH to attack the benzene rings of aromatic molecules, are currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). The latter acid can be also produced by enzymatic pathways through the cytochrome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2, 3-DHBA, following oral administration of the drug acetyl salicylate, could be proposed for assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from the reaction of z. rad OH with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system. A detection limit of < 1 pmol for the hydroxylation products has been achieved with linear response over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following administration of 1 g acetyl salicylate in 20 healthy subjects.     

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.     

Evaluation of hydroxyl radical-scavenging property of purpurogallin using high pressure liquid chromatography

Purpurogallin (PPG) has been used as an additive to edible and non-edible oils or fats to retard oxidation. Its antioxidant mechanism is not known. We investigated the ability of PPG to scavenge exogenously generated hydroxyl radicals (·OH) using a sensitive high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles) with UV light and was trapped with salicylic acid (500 nmoles). Salicylic acid is hydroxylated to produce ·OH adduct products 2,3-and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced concentration-dependent ·OH as estimated by generation of 2,3- and 2,5-DHBA. PPG (100, 200, 300, 400, 500 and 600 nmoles) produced concentration-dependent decreases in ·OH adduct products (approximately 70% inhibition with 600 nmoles of PPG). It did not affect the peak of standard 2,3- and 2,5-DHBA indicating that the decrease in the adduct product generated by H₂O₂ is due to scavenging of ·OH. These results indicate that photolysis of H₂O₂ by UV light produces ·OH and that PPG scavenges ·OH.     

Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury

During postischemic reperfusion, free radicals are produced and have deleterious effects in isolated rat hearts. We investigated whether melatonin (MEL) reduces the production of hydroxyl radical (*OH) in the effluent and aids in recovery of left ventricular (LV) function. Hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. Salicylic acid (SAL) was used as the probe for *OH, and its derivatives 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were quantified using HPLC. In addition, thiobarbituric acid reactive substances (TBARS) in the myocardium was measured. Plateaus in the measurement of 2,5- and 2,3-DHBA were seen from 3 to 8 min after reperfusion in each group. The group that received 100 microM MEL+ SAL had significantly reduced amounts of 2,5- and 2,3-DHBA by multiple folds, compared to the SAL group. TBARS was significantly decreased in the 100 microM MEL group (1.20+/-0.36 vs 1.85+/-0.10 micromol/g of drug-free group, p<0.001). More importantly, the 100 microM MEL group significantly recovered in LV function (LV developed pressure, +dp/dt, and -dp/dt; 63.0%, 60.3%, and 59.4% in the 100 microM MEL group; 30.2%, 29.7%, and 31.5% in the drug-free group, respectively; p<0.05). Duration of ventricular tachycardia or ventricular fibrillation significantly decreased in the 100 microM MEL group (100 microM MEL, 159+/-67 sec; drug-free, 1244+/-233 sec; p<0.05). As a result of scavenging *OH and reducing the extent of lipid peroxidation, MEL is an effective agent for protection against postischemic reperfusion injury.     

Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats

To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-induced arthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 μl of emulsion containing bovine type II collagen in complete Freund's adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting; high tumour necrosis factor-α (TNF-α) plasma levels; and synovial neutrophil accumulation. Treatment with HYA and C4S, starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase, decreased plasma TNF-α levels, and limited synovial neutrophil infiltration. These data confirm that erosive destruction of the joint cartilage in CIA is due at least in part to free radicals released by activated neutrophils and produced by other biochemical pathways. The beneficial effects obtained with the treatment suggest that HYA and C4S could be considered natural endogenous macromolecules to limit erosive damage in CIA or as a useful tool with which to study the involvement of free radicals in rheumatoid arthritis.     

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.     

Estimating Hydroxyl Radical Content in Rat Brain Using Systemic and Intraventricular Salicylate: Impact of Methamphetamine

Abstract: Free radicals have been implicated in the etiology of many neurodegenerative conditions. Yet, because these species are highly reactive and thus short-lived it has been difficult to test these hypotheses. We adapted a method in which hydroxyl radicals are trapped by salicylate in vivo, resulting in the stable and quantifiable products, 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. After systemic (100 mg/kg i.p.) or intraventricular (4 µmol) administration of salicylate, the amount of DHBA in striatal tissue correlated with tissue levels of salicylate. After systemic salicylate, the ratio of total DHBA to salicylate in neostriatum was at least 10-fold higher than that observed after central salicylate. In addition, systemic salicylate resulted in considerably higher concentrations of 2,3- and 2,5-DHBA in plasma than in brain. Therefore, a large portion of the DHBA present in brain after systemic salicylate may have been formed in the periphery. A neurotoxic regimen of methamphetamine increased the concentration of DHBA in neostriatum after either central or systemic administration of salicylate. The increase in 2,3-DHBA after the central administration of salicylate was significant at 2 h, but not at 4 h, after the last dose of methamphetamine. These results suggest that (1) when assessing specific events in brain, it is preferable to administer salicylate centrally, and (2) neurotoxic doses of methamphetamine increase the hydroxyl radical content in brain in a time-dependent manner.     

Detection of Free Radical Activity During Transient Global Ischemia and Recirculation: Effects of Intraischemic Brain Temperature Modulation

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two-vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3- and 2,5-DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3- and 2,5-DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3- and 2,5-DHBA levels increased by 2.5- and 2.8-fold, respectively. Levels of 2,3-DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5-DHBA levels persisted for 2 h. The increases in 2,3- and 2,5-DHBA levels observed following hyperthermic ischemia were significantly higher (3.8- and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical-induced injury in the development of ischemic damage.     

Time course of production of hydroxyl free radical after subarachnoid hemorrhage in dogs

Vasospasm after subarachnoid hemorrhage (SAH) is associated with lipid peroxidation. However, lipid peroxides increase in a delayed fashion after SAH and may be a byproduct of but not a cause of vasospasm. This study correlated vasospasm with hydroxyl free radical and lipid peroxide levels. 24 dogs had baseline cerebral angiography and induction of SAH by 2 injections of blood into the cisterna magna at baseline and 2 days later. Angiography was repeated 4, 7, 10, 14 or 21 days after the first injection (n = 4 per group) and a microdialysis catheter was inserted into the premedullary cistern. Control dogs (n = 4) underwent angiography and microdialysis but not SAH. Salicylic acid, 100 mg/kg, was administered intravenously, and microdialysis fluid was collected and analyzed by high pressure liquid chromatography for 2,3- and 2,5-dihydroxybenzoic acids (DHBA). Malondialdehyde was measured in subarachnoid clot removed from the prepontine cistern and in the basilar artery itself at the time of euthanasia. Significant vasospasm developed 4 to 14 days after SAH. Malondialdehyde levels were significantly elevated in the basilar artery and subarachnoid clot 4 days after SAH (p < 0.0001, ANOVA) but not at other times. 2,5-DHBA levels were significantly greater than control at 4 to 14 days and they peaked at 4 days (p < 0.05, ANOVA). 2,3-DHBA was significantly increased at 4 days after SAH (p < 0.05, ANOVA). There were significant correlations between basilar artery malondialdehyde levels and vasospasm and cerebrospinal fluid 2,5-DHBA levels and vasospasm. These results suggest the presence of hydroxyl free radical after SAH and demonstrate a correlation between such production, as measured by trapping with salicylate, and the early phase of vasospasm. The correlation with vasospasm implicates free radicals and lipid peroxidation in this phase of vasospasm.     

Brain Hydroxyl Radical Generation in Acute Experimental Head Injury

Abstract: The time course and intensity of brain hydroxyl radical (^?OH) generation were examined in male CF-1 mice during the first hour after moderate or severe concussive head injury. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2,3- and/or 2,5-dihydroxybenzoic acid (DHBA) in brain 15 min after salicylate administration was used as an index of ^?OH formation. In mice injured with a concussion of moderate severity as defined by the 1-h posttraumatic neurologic recovery (grip score), a 60% increase in 2,5-DHBA formation was observed by 1 min after injury compared with that observed in uninjured mice. The peak in DHBA formation occurred at 15 min after injury (+67.5%; p < 0.02, compared with uninjured). At 30 min, the increase in DHBA lost significance, indicating that the posttraumatic increase in brain ^?OH formation is a transient phenomenon. In severely injured mice, the peak increase in DHBA (both 2,3- and 2,5-) was observed at 30 min after injury, but also fell off thereafter as with the moderate injury severity. Preinjury dosing of the mice with SKF-525A (50 mg/kg i.p.), an inhibitor of microsomal drug oxidations, did not blunt the posttraumatic increase in salicylate-derived 2,5-DHBA, thus showing that it is not due to increased metabolic hydroxylation. Neither injury nor SKF-525A administration affected the DHBA plasma levels. However, saline perfusion of the injured mice to remove the intravascular blood before brain removal eliminated the injury-induced increase in 2,5-DHBA, but did not affect the baseline levels seen in uninjured mice. This implies that the source of the increased DHBA in the injured mice is the microvasculature, probably the endothelium. The administration of the 21-aminosteroid lipid antioxidant, tirilazad mesylate, which possesses ^?OH scavenging properties, also attenuated the posttraumatic increase in DHBA, further supporting that it reflects an increase in ^?OH radical formation. These results are the first direct demonstration of the occurrence and time course of increased ^?OH production in injured brain.     

Hydroxyl radical-scavenging property of indomethacin

The ability of indomethacin to scavenge hydroxyl radical (.OH) using high pressure liquid chromatography (HPLC) was investigated. .OH radical was generated by photolysis of H₂O₂ (1.5–10 mmoles/L) with UV light and was trapped with salicyclic acid (500 nmoles). H₂O₂ produced .OH in a concentration-dependent manner as estimated by .OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). Indomethacin in increasing concentrations (5–600 moles/L) produced increasing inhibition of generation of 2,3-DHBA (7–67%) and of 2,5-DHBA (7–77%). The results indicate that indomethacin scavenges .OH in a concentration-dependent manner.     

The AraC-like transcriptional regulator DhbR is required for maximum expression of the 2,3-dihydroxybenzoic acid biosynthesis genes in Brucella abortus 2308 in response to iron deprivation

Phenotypic evaluation of isogenic mutants derived from Brucella abortus 2308 indicates that the AlcR homolog DhbR (2,3-dihydroxybenzoic acid [2,3-DHBA] biosynthesis regulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.