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.     

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.     

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.     

A novel green enzymatic synthetic route of 2, 3-dihydroxybenzoic acid from glucose and CO2 fixation

The enzymatic carbon fixation is a promising approach to deal with greenhouse gas emission and is usually accompanied with energy consumption during the reduction of CO₂. As a very important route, the carboxylation can convert CO₂ to organic carbon without extra requirement of reduction power and is hoped as a greener solution, especially for some non-bulk chemicals, such as medical intermediates. Here, a concept-proof trail of green enzymatic process of conversing both of CO₂ and benzene to produce 2,3-dihydroxybenzoic acid (2,3-DHBA), which is the intermediate for fine chemicals, is introduced with O₂ from air and glucose. The results showed that the conversion catechol by 2, 3-dihydroxybenzoic acid decarboxylase (2,3-DHBD) alone was around 30 %, with an overall conversion from phenol of 2.4 %, which was limited by the in-situ production of catechol. This trail contributed a green enzymatic route for the production of 2,3-DHBA.     

Biosynthesis of salicylic acid in fungus elicited Catharanthus roseus cells

Feeding experiments using [1-¹³C]-d-glucose to Catharanthus roseus (L.) G.Don cell suspension cultures followed by elicitation with Pythium aphanidermatum extract were performed in order to study the salicylic acid (SA) biosynthetic pathway and that of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a comparison. A strongly labeled C-7 and a symmetrical partitioning of the label between C-2 and C-6 would occur if SA was synthesized from phenylalanine. In case of the isochorismate pathway, a relatively lower incorporation at C-7 and a non-symmetrical incorporation at C-2 and C-6 would be obtained. Relatively, high- and non-symmetrical enrichment ratios at C-2 and C-6, and a lower enrichment ratio at C-7 were observed in both SA and 2,3-DHBA detected by ¹³C NMR inverse gated spectrometry leading to the conclusion that the isochorismate pathway is responsible for the biosynthesis of both compounds. However, different enrichment ratios of the labeled carbons in SA and 2,3-DHBA indicate the use of different isochorismate pools, which means that their biosynthesis is separated in time and/or space.     

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.     

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.     

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.     

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.     

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.     

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.     

Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation

We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (*OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer's solution through the microdialysis probe at a rate of 1 microl/min. Sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused directly through a microdialysis probe to detect the generation of *OH. Induction of [K(+)](o) (70 mM) or tyramine (1 mM), significantly increased the formation of *OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, significantly decreased the K(+) depolarization-induced *OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 microM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to L-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K(+) depolarization-induced *OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat 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 level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 microM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced *OH generation by inhibiting LDL oxidation in the heart.     

Biodegradation kinetics of peptone and 2,6-dihydroxybenzoic acid by acclimated dual microbial culture

This study evaluated the kinetics of simultaneous biodegradation of peptone mixture and 2,6-dihydroxybenzoic acid (2,6-DHBA) by an acclimated dual microbial culture under aerobic conditions. A laboratory-scale sequencing batch reactor was sustained at steady-state with peptone mixture feeding. During the study period, peptone mixture feeding was continuously supplemented with 2,6-DHBA. Related experimental data were derived from three sets of parallel batch reactors, the first fed with the peptone mixture, the second with 2,6-DHBA and the third one with the two substrates, after acclimation of microbial culture and simultaneous biodegradation of both organics. A mechanistic model was developed for this purpose including the necessary model components and process kinetics for the model calibration of relevant experimental data. Model evaluation provided all biodegradation characteristics and kinetics for both peptone mixture and 2,6-DHBA. It also supported the development of a dual microbial community through acclimation, with the selective growth of a second group of microorganisms specifically capable of metabolizing 2,6-DHBA as an organic carbon source.     

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.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

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.     

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.     

Sensitive determination of aspirin and its metabolites in plasma by LC-UV using on-line solid-phase extraction with methylcellulose-immobilized anion-exchange restricted access media

We describe a sensitive determination of aspirin (ASA) and its three metabolites (salicylic acid [SA], 2,3-dihydroxybenzoic acid [2,3-DHBA], and 2,5-dihydroxybenzoic acid [gentisic acid (GA)]) in rat plasma. Analysis was carried out by on-line solid-phase extraction (SPE) using a methylcellulose-immobilized-strong anion-exchanger (MC-SAX), followed by liquid chromatography (LC) coupled with UV detection. The lower limits of quantitation for ASA and SA were 60 ng/mL in 100 microL of plasma, respectively. This method was validated with respect to intra- and inter-day precision, accuracy, and linearity up to concentrations of 20,000 ng/mL for ASA, SA, 2,3-DHBA and gentisic acid, respectively. The method was successfully applied to an analysis of the pharmacokinetics of ASA and SA in rats.     

Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCE) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats

Hydroxyl free radicals react with salicylate to form 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Utilizing the technique of high pressure liquid chromatography with electrochemical detection (LCED), it is possible to detect DHBAs at the level of femtomoles. Since salicylate is relatively non-toxic, we have administered it as a trapping agent in a first attempt to examine the use of the LCED method as a sensitive measure of in vivo OH production. Utilizing adriamycin administration as a model to induce oxygen free radical tissue damage, we found that the level of DHBAs present in drug treated rats versus controls was increased 100-fold in heart and muscle, 30-fold in lung, and 3- and 4-fold in brain and blood, respectively. These first observations support the theory that adriamycin induced OH in tissue and indicates that the LCED method may prove to be useful to measure oxygen free radical production in vivo.