salicylates and 2-5-dihydroxybenzoic-acid

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L

Topics: Actinomycetales; Biodegradation, Environmental; Biotransformation; Carbamazepine; Carbon; Catechol 1,2-Dioxygenase; Catechols; Dioxygenases; Environmental Pollution; Gentisates; Hydroxybenzoate Ethers; Kinetics; Metabolic Networks and Pathways; Mixed Function Oxygenases; Naphthalenes; Naproxen; Salicylates

Choline salicylate (CS) as a derivative of acetylsalicylic acid is commonly used in different drug forms. In medicine, it is applied topically to inflammation of the oral cavity mucosa and in laryngology. However, this substance in the form of an ionic liquid has not been investigated enough. There are no literature studies on stability tests constituting a stage of pre-formulation research. HPLC (Nucleosil C18, 4.6 × 150 mm, 5 μm; methanol-water-acetic acid 60:40:1, 230 nm or 270 nm) and UV (276 nm) methods for the determination of CS in 2% (g/mL) aqueous solutions were developed. Under stress conditions, CS susceptibility to hydrolytic degradation in aqueous medium, hydrochloric acid, sodium hydroxide, and hydrogen peroxide, and the effect of light on the stability of CS solutions were studied with HPLC analysis. The degradation degree of CS and the purity of the solutions were also tested. Choline salicylate has been qualified as practically stable in neutral and acid media, stable in an alkaline medium, very stable in an oxidizing environment, and photolabile in solution. The HPLC-MS/MS method was used to identify 2,3- and 2,5-dihydroxybenzoic acids as degradation products of CS under the tested conditions.

Topics: Choline; Chromatography, High Pressure Liquid; Drug Combinations; Drug Stability; Gentisates; Hydrolysis; Hydroxybenzoates; Molecular Structure; Photolysis; Salicylates; Tandem Mass Spectrometry

Since its introduction, matrix-assisted laser desorption/ionization (MALDI) has been widely used for the mass analysis of biomolecules. The "soft ionization" of MALDI enables accurate mass determination of intact biomolecules. However, the ionization and desorption processes of MALDI are not adequately soft as many labile biomolecules, such as glycoconjugates containing sialic acid or the sulfate functional group, easily dissociate into fragments and sometimes, no intact molecules are observed. In this study, we compared the conventional matrix of MALDI, namely 2,5-dihydroxybenzoic acid, to various soft matrices of MALDI-specifically, 5-methoxysalicylic acid, diamond nanoparticle trilayers, HgTe nanostructures, ionic liquid, and droplets of frozen solutions-by using three labile glycoconjugates as analytes: gangliosides, heparin, and pullulan. We demonstrated that droplets of frozen solution are the softest matrices for gangliosides and heparin. In particular, droplets of frozen solution do not generate fragments for gangliosides and can be used to determine the relative abundance of various gangliosides, whereas ionic liquid 2,5-dihydroxybenzoic acid butylamine is the most suitable matrix for pullulan mass analysis. Graphical Abstract.

Topics: Gangliosides; Gentisates; Glucans; Glycoconjugates; Heparin; Hydroxybenzoate Ethers; Ionic Liquids; Nanostructures; Salicylates; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The genome of the α-proteobacterium Pseudaminobacter salicylatoxidans codes for a ferrous iron containing ring-fission dioxygenase which catalyzes the 1,2-cleavage of (substituted) salicylate(s), gentisate (2,5-dihydroxybenzoate), and 1-hydroxy-2-naphthoate. Sequence alignments suggested that the "salicylate 1,2-dioxygenase" (SDO) from this strain is homologous to gentisate 1,2-dioxygenases found in bacteria, archaea and fungi. In the present study the catalytic mechanism of the SDO and gentisate 1,2-dioxygenases in general was analyzed based on sequence alignments, mutational and previously performed crystallographic studies and mechanistic comparisons with "extradiol- dioxygenases" which cleave aromatic nuclei in the 2,3-position. Different highly conserved amino acid residues that were supposed to take part in binding and activation of the organic substrates were modified in the SDO by site-specific mutagenesis and the enzyme variants subsequently analyzed for the conversion of salicylate, gentisate and 1-hydroxy-2-naphthoate. The analysis of enzyme variants which carried exchanges in the positions Arg83, Trp104, Gly106, Gln108, Arg127, His162 and Asp174 demonstrated that Arg83 and Arg127 were indispensable for enzymatic activity. In contrast, residual activities were found for variants carrying mutations in the residues Trp104, Gly106, Gln108, His162, and Asp174 and some of these mutants still could oxidize gentisate, but lost the ability to convert salicylate. The results were used to suggest a general reaction mechanism for gentisate-1,2-dioxygenases and to assign to certain amino acid residues in the active site specific functions in the cleavage of (substituted) salicylate(s).

Topics: Alphaproteobacteria; Amino Acid Sequence; Amino Acids; Bacterial Proteins; Dioxygenases; Escherichia coli; Gene Expression; Gentisates; Kinetics; Molecular Sequence Data; Mutation; Naphthols; Oxidation-Reduction; Protein Conformation; Recombinant Proteins; Salicylates; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity

The aim of the present study was to modify and validate a high-performance liquid chromatographic (HPLC) method for determining 2,3 and 2,5 dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid in human plasma. The mobile phase was a mixture of sodium acetate/citrate (pH 2.5) 30 mM-methanol (93:7, v/v). The injection volume was 10 muL. Retention time for 2,5-DHBA, and 2,3-DHBA was 4.5 +/- 0.10 and 5.8 +/- 0.15 min, respectively. The detection and quantification limits were 10 and 40 nM for 2,3-DHBA and 8 and 20 nM for 2,5-DHBA. Linearity was evaluated in the range of 40-1600 nM for both metabolites. Inter- and intra-analysis variation coefficient was below 10%. Good recoveries of more than 99% were obtained for both metabolites using this method.

Topics: Biomarkers; Catechols; Chromatography, High Pressure Liquid; Drug Stability; Gentisates; Humans; Hydroxybenzoates; Hydroxylation; Linear Models; Oxidative Stress; Reproducibility of Results; Salicylates; Sensitivity and Specificity

The aim of this study was to evaluate the effect of 2,5-dihydroxybenzoic acid, a salicylate derived from Acetyl salicylic acid (ASA) and vitamin A (vit A) on Na(+), K(+) ATPase enzyme and GSH levels in brain of rats exposed to hyperoxia (Hyp) as oxidant protocol. Rats were treated as follow: group I (control), group II (Hyp), group III (Hyp, ASA), group IV (vit A), group V (Hyp, vit A), group VI (Hyp, vit A, ASA). Vit A was given 5 days before and during Hyp, aspirin at the end of Hyp. Na(+),K(+) ATPase and total ATPase activity was significantly increased in group V. Levels of GSH showed a significant increase in group III, besides, levels of 2,5-dihydroxybenzoic acid as salicylate in plasma were significantly increased in group II. These results elucidate differences in the biochemical response of animal towards intake of various types of antioxidant substances, with increased GSH and salicylate in hyperoxia.

Topics: Animals; Antioxidants; Brain Chemistry; Female; Free Radicals; Gentisates; Glutathione; Hyperoxia; Oxidative Stress; Rats; Rats, Sprague-Dawley; Salicylates; Sodium-Potassium-Exchanging ATPase; Vitamin A

Salicylic acid (SA) and related phenolic compounds can exert control over stomatal opening and previous work in our laboratory has shown that chronic injection of SA increases the photosynthetic rate of corn. The work reported in this paper investigated the effects of foliar applied SA, acetyl salicylic acid (ASA) and gentisic acid (GTA) on photosynthetic rates and growth of soybean (a C3 plant) and corn (a C4 plant) under greenhouse conditions. In general, the tested compounds enhanced photosynthetic rates in both soybean and corn. Stomatal conductance and transpiration were also increased. These compounds do not alter chlorophyll content. In some cases treatment with these compounds resulted in increased leaf areas and plant dry mass, however, plant height and root length were not affected.

Topics: Aspirin; Chlorophyll; Gases; Gentisates; Glycine max; Hydroxybenzoates; Molecular Structure; Phenols; Photosynthesis; Plant Leaves; Salicylates; Zea mays

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.

Topics: Animals; Cerebellum; Corpus Striatum; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Injections; Levodopa; Male; Mesencephalon; Methamphetamine; Microdialysis; Osmolar Concentration; Oxidopamine; Rats; Rats, Sprague-Dawley; Salicylates; Substantia Nigra

To elucidate the role of hydroxyl radical (.OH) species in the generation mechanism of the cochlear pathology induced by transient asphyxia and subsequent re-ventilation, the concentrations of 2,3-hydroxybenzoic acid (DHBA) and 2,5-DHBA, major products arising from the attack of .OH upon salicylate, were measured in the perilymph of the guinea pig by the high performance liquid chromatography-electrochemical/UV method. The mean value of 2, 3-DHBA concentration in the perilymph significantly increased from the pre-asphyxic level (6.4 microM) to 7.6 microM and 8.8 microM during asphyxia of 3 min duration and at 5 min after the onset of re-ventilation, respectively. The 2,5-DHBA concentration was 7.9 microM before asphyxia, and also significantly increased to 11.5 microM and 16.2 microM during and after asphyxia, respectively. These results strongly indicated that .OH was generated in the perilymph of the asphyxic and re-ventilated guinea pig cochlea, and the significance of this increased .OH in generating anoxia and re-perfusion injury is discussed with respect to iron and oxygen-derived free radicals.

Topics: Animals; Asphyxia; Chromatography, High Pressure Liquid; Electrochemistry; Gentisates; Guinea Pigs; Hydroxybenzoates; Hydroxyl Radical; Perilymph; Salicylates

The direct measurement of hydroxyl radicals in vivo is extremely difficult. Therefore, the indirect determination of hydroxyl radicals using salicylate (2-hydroxybenzoate) is widely accepted. Reverse microdialysis with glutamate led to a dose-dependent production of hydroxyl free radicals indicated by the hydroxylation adduct of salicylate, namely 2,3-dihydroxybenzoic acid. The local stimulation of hydroxyl free radical formation seems to be suitable to investigate a radical-scavenging property of potential neuroprotective drugs. In vitro experiments using the Fenton reaction may be a helpful tool to assess whether or not a substance is able to act as a radical scavenger in a cell free environment, which is easy to handle and a simple screening method before in vivo experiments were performed. In the present study we present an in vivo approach using local application of glutamate into the striatum and an in vitro screening using the Fenton reaction to induce hydroxyl radical formation. The main goal is to reliable measure hydroxyl free radicals, which are the most reactive oxygen radicals in biology and medicine.

Topics: Animals; Dose-Response Relationship, Drug; Gentisates; Glutamic Acid; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Male; Methods; Microdialysis; Rats; Rats, Wistar; Salicylates

The poor cyclooxygenase (COX) inhibitor and major aspirin metabolite salicylic acid is known to exert analgesic and anti-inflammatory effects by still unidentified mechanisms. In RAW 264.7 macrophages, lipopolysaccharide (LPS)-induced COX-2-dependent synthesis of prostaglandin E(2) (PGE(2)) was suppressed by aspirin (IC(50) of 5. 35 microM), whereas no significant inhibition was observed in the presence of sodium salicylate and the salicylate metabolite salicyluric acid at concentrations up to 100 microM. However, the salicylate metabolite gentisic acid (2,5-dihydroxybenzoic acid; 10-100 microM) and salicyl-coenzyme A (100 microM), the intermediate product in the formation of salicyluric acid from salicylic acid, significantly suppressed LPS-induced PGE(2) production. In contrast, gamma-resorcylic acid (2,6-dihydroxybenzoic acid) as well as unconjugated coenzyme A failed to affect prostanoid synthesis, implying that the para-substitution of hydroxy groups and the activated coenzyme A thioester are important for COX-2 inhibition. Using real-time RT-PCR, none of the salicylate derivatives tested were found to interfere with COX-2 expression. Overall, our results suggest that certain metabolites of salicylic acid may contribute to the pharmacological action of its parent compound by inhibiting COX-2-dependent PGE(2) formation at sites of inflammation.

Topics: Acyl Coenzyme A; Animals; Aspirin; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Gentisates; Hydroxybenzoates; Inflammation; Isoenzymes; Lipopolysaccharides; Macrophages; Mice; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salicylates; Sodium Salicylate

Aromatic carboxylic acids substituted with methoxylated groups are among the most abundant products in "alpechin", the wastes resulting from pressing olives to obtain olive oil. Degradation of o-methoxybenzoate by an stable consortium made of a gram positive bacterium, Arthrobacter oxydans, and gram negative one, Pantotea agglomerans, was shown to mineralize this compound efficiently. The concerted action of both microorganisms was needed for the two first steps in the process, namely, the conversion of o-methoxybenzoate into salycilate, and the hydroxylation of the latter to gentisate. Gentisate was further degraded by the Arthrobacter strain.

Topics: Anisoles; Arthrobacter; Biodegradation, Environmental; Gentisates; Gram-Negative Bacteria; Hydroxybenzoate Ethers; Hydroxybenzoates; Organometallic Compounds; Salicylates

Topics: Animals; Chromatography, High Pressure Liquid; Convulsants; Corpus Striatum; Gentisates; Glutarates; Hydroxybenzoates; Hydroxyl Radical; Male; Methylguanidine; Microdialysis; Rats; Rats, Wistar; Salicylates

Hydroxylation of salicylate into 2,3 and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) by human liver microsomal preparations was investigated. Kinetic studies demonstrated that salicylate was 5-hydroxylated with two apparent Km: one high-affinity Km of 606 microM and one low-affinity Km greater than 2 mM. Liver microsomes prepared from 15 human samples catalyzed the formation of 2,5-DHBA at metabolic rate of 21.7 +/- 8.5 pmol/mg/min. The formation of 2, 3-DHBA was not P-450 dependent. Formation of 2,5-DHBA was inhibited by 36 +/- 14% following preincubation of microsomes with diethyldithiocarbamate, a mechanism-based selective inhibitor of P-450 2E1. Furthermore, the efficiency of inhibition was significantly correlated with four catalytic activities specific to P-450 2E1, whereas the residual activity was correlated with three P-450 3A4 catalytic activities. Troleandomycin, a mechanism-based inhibitor selective to P-450 3A4, inhibited by 30 +/- 12% the 5-hydroxylation of salicylate, and this inhibition was significantly correlated with nifedipine oxidation, specific to P-450 3A4. The capability of seven recombinant human P-450s to hydroxylate salicylate demonstrated that P-450 2E1 and 3A4 contributed to 2, 5-DHBA formation in approximately equal proportions. The Km values of recombinant P-450 2E1 and 3A4, 280 and 513 microM, respectively, are in the same range as the high-affinity Km measured with human liver microsomes. The plasmatic metabolic ratio 2,5-DHBA/salicylate, measured 2 h after ingestion of 1 g acetylsalicylate, was increased 3-fold in 12 alcoholic patients at the beginning of their withdrawal period versus 15 control subjects. These results confirm that P-450 2E1, inducible by ethanol, is involved in the 5-hydroxylation of salicylate in humans. Furthermore, this ratio was still increased by 2-fold 1 week after ethanol withdrawal. This finding suggests that P-450 3A4, known to be also inducible by alcoholic beverages, plays an important role in this increase, because P-450 2E1 returned to normal levels in less than 3 days after ethanol withdrawal. Finally, in vivo and in vitro data demonstrated that P-450 2E1 and P-450 3A4, both inducible by alcohols, catalyzed the 5-hydroxylation of salicylate.

Topics: Alcoholism; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Gentisates; Humans; Hydroxybenzoates; Hydroxylation; Kinetics; Microsomes, Liver; Mixed Function Oxygenases; Salicylates

Administration of neurotoxic doses of methamphetamine (8 mg/kg, intraperitoneally x 4 times, at 2 hr intervals) caused a significant decrease in dopamine and 3,4-dihydroxyphenylacetic acid and an increase in 3-methoxytyramine levels in the striatum along with a decrease in serotonin and 5-hydroxyindoleacetic acid levels in the striatum and hippocampus. In addition, the methamphetamine treatment caused an increase in rat rectal temperature. Intraventricular injection of salicylate 105 min. after the last injection of methamphetamine produced an increase in 2,3- and 2,5-dihydroxybenzoic acid in the striatum and hippocampus. Moreover, the ratio of 2,3-dihydroxybenzoic acid to salicylate was significantly increased in the striatum, but not in the hippocampus. These results indicate that the hydroxyl radical may play an important role in methamphetamine-induced neurotoxicity in rat striatum and that its formation may be the result of methamphetamine-induced release of dopamine.

Topics: Animals; Body Temperature; Chromatography, High Pressure Liquid; Corpus Striatum; Dopamine; Gentisates; Hippocampus; Hydroxybenzoates; Hydroxyl Radical; Injections, Intraventricular; Male; Methamphetamine; Rats; Rats, Wistar; Salicylates; Serotonin

1. Previous studies have shown that formation of 2,3-dihydroxybenzoate (2,3-DHB) from salicylate in vivo is a sensitive and specific marker of *OH radical generation, since 2,3-DHB is formed exclusively by *OH radicals, whereas both *OH radicals and cytochrome P450 (CYP) contribute to the production of 2,5-DHB. In the present study the salicylate-hydroxylation assay was used to examine whether CYP induction by the administration of dexamethasone, phenobarbital or beta-naphthoflavone to the male rat led to oxidative stress in vivo. 2. Dexamethasone was used under conditions that induced an approximately 50-fold induction of CYP P4503A expression in liver microsomal protein. Treatment with dexamethasone caused a 17.2-fold increase in 2,3-DHB plasma concentration compared with control animals. An increase in total hydroxylated salicylate (2,3-DHB plus 2,5-DHB) of 133.5 micromol/l plasma was produced, of which--assuming that the attack by *OH in position 3 or 5 of salicylate occurs at a similar rate--10.9 micromol/l were due to *OH radical attack and 122.6 micromol/l due to metabolism by CYP. 3. Phenobarbital led to a 4.7-fold increase in 2,3-DHB plasma concentration under conditions that induced CYP P4502B and 3A. An increase in total hydroxylated salicylate of 34.3 micromol/l plasma was observed, 2.0 micromol/l due to *OH radical attack and 32.3 micromol/l due to metabolism by cytochrome P450. 4. In contrast to dexamethasone and phenobarbital, beta-naphthoflavone did not cause a significant increase in 2,3-DHB plasma concentrations. 5. SKF 525A, a mixed-function oxidase inhibitor, caused a significant reduction of mean 2,5-DHB plasma concentration by 35% (p < 0.001), whereas 2,3-DHB was not significantly reduced, indicating that in contrast to the situation after induction by dexamethasone or phenobarbital, *OH radical generation by constitutive CYP contributes only to a minor degree to total in vivo *OH radical generation. 6. This study shows for the first time, to the authors' knowledge, that induction of some (but not all) P450s is associated with the production of hydroxyl radicals in vivo.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; beta-Naphthoflavone; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dexamethasone; Enzyme Induction; Enzyme Inhibitors; Gentisates; Hydroxybenzoates; Hydroxylation; Male; Oxidative Stress; Oxidoreductases, N-Demethylating; Phenobarbital; Proadifen; Rats; Rats, Sprague-Dawley; Salicylates

Microdialysis was done on 300-400 g, awake, male rats with microdialysis probes inserted through guide cannulas into the striatum (Bregma co-ordinates A 0.5, L 2.9, D -4.0 for guide cannulas implanted 5 days previously). Rats were exposed to hyperbaric oxygen (HBO; 6 atm absolute, 5 atm gauge pressure of oxygen with carbon dioxide absorbed by soda lime). Artificial cerebrospinal fluid (CSF) containing 5 mM sodium salicylate was perfused at 1 microl/min and collected over sequential 10 min intervals with rats breathing air, then HBO, and after decompression. Times to convulsions and duration and severity of convulsions were observed and recorded. CSF samples were analyzed for 2,3- and 2,5-dihydroxybenzoic acid (DHBA), reaction products of hydroxyl radicals with salicylate, by HPLC and compared to authentic standards. Recovery of DHBAs was 48% from fluid surrounding microdialysis probes, based on in vitro tests. The average time to the first convulsion was 21 min and rats convulsed an average of 4 times during 40 min in HBO. There were no significant differences in hydroxyl radical production by this protocol during any of the 10 min collection periods in air or HBO (average in pmoles for 10 microl of all samples: 2,3-DHBA = 7.0 +/- 2.5 and 2,5-DHBA = 11.3 +/- 4.1). The failure to detect an increase in hydroxyl radicals in HBO prior to or during convulsions appears valid since each rat served as its own control.

Topics: Air; Animals; Brain Chemistry; Chromatography, High Pressure Liquid; Corpus Striatum; Electron Spin Resonance Spectroscopy; Epilepsy, Tonic-Clonic; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Hyperbaric Oxygenation; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Salicylates

The effect of protection of dopaminergic neurons by talipexole, a dopamine (DA) agonist, is investigated on a methamphetamine (MA)-induced parkinsonism model of mice (C57BL/6N). The reduction of tyrosine hydroxylase activity in the striatum 72 h after MA (5 mg/kg every 2 h, four times) treatment was attenuated by the administration of talipexole (0.25 mg/kg or 1.0 mg/kg) prior to the administration of MA. In an in vitro experiment, talipexole inhibited the adduction reaction of hydroxyl radicals to salicylate. Taken together, these data suggest that the protective effect of talipexole on DA neurons is, in part, caused by the hydroxyl radical-scavenging action of the drug.

Topics: Animals; Azepines; Central Nervous System Stimulants; Chromatography, High Pressure Liquid; Dopamine; Dopamine Agonists; Enzyme Induction; Free Radical Scavengers; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Parkinson Disease, Secondary; Receptors, Dopamine D2; Salicylates; Salicylic Acid; Tyrosine 3-Monooxygenase

To examine sera for the presence of salicylic acid and 2,3- and 2,5-dihydroxybenzoic acids (2,3- and 2,5-DHBA), in individuals not taking salicylate drugs.. Extracts of acidified serum samples were analysed by high performance liquid chromatography with electro-chemical detection. The chromatographic conditions were altered, and the retention times of the unknown compounds compared against authentic salicylic acid, 2,3-DHBA, and 2,5-DHBA. Serum samples (some spiked with salicylic acid) were incubated with salicylate hydroxylase and analyses undertaken. An extract of acidified serum was derivatised using N-methyl-N-trimethylsilyltrifluoroacetamide and the salicylic acid derivative identified by gas chromatography-mass spectrometry.. Salicylic acid, 2,3-DHBA, and 2,5-DHBA were identified as being normal constituents of serum.. Salicylic acid, 2,3-DHBA, and 2,5-DHBA possess anti-inflammatory properties. The finding that these compounds are present as normal constituents of serum, possibly arising from diet, raises important questions as to their role in the promotion of health.

Topics: Chromatography, High Pressure Liquid; Diet; Gas Chromatography-Mass Spectrometry; Gentisates; Humans; Hydroxybenzoates; Salicylates; Salicylic Acid

Free radical damage to proteins, lipids, DNA and RNA has been thought to play an important role in many diseases as well as the aging process. One free radical, the hydroxyl free radical (HFR), is extremely reactive and is difficult to measure directly. HFRs were quantified by measuring the hydroxylation products 2,3- and 2,5-dihydroxybenzoic acids (DHBAs) formed as a result of the reaction between HFR and systemically administered salicylate (SAL). DHBAs and SAL concentrations were determined using RP-HPLC with dual coulometric electrode detection. The method has limits of detection of 1 pg for the DHBAs and 100 pg for SAL (signal-to-noise ratio 3:1). A detailed interference study as well as analyte stability and linearity studies were performed. This method was used to determine basal ratios of DHBA/SAL in a variety of tissues and to study the effects of glutamatergic and dopaminergic drugs on DHBA/SAL ratios in brain region homogenates.

Topics: Animals; Benzoates; Cerebral Cortex; Chromatography, High Pressure Liquid; Corpus Striatum; Dizocilpine Maleate; Drug Stability; Excitatory Amino Acid Antagonists; Free Radicals; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Kidney; Liver; Male; N-Methylaspartate; Rats; Rats, Sprague-Dawley; Salicylates; Sensitivity and Specificity

Topics: Animals; Brain Chemistry; Chromatography, High Pressure Liquid; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Liver; Male; Microdialysis; Myocardium; Rats; Rats, Wistar; Salicylates

The effect of pargyline, a monoamine oxidase inhibitor, on the generation of hydroxyl free radicals (.OH) was investigated using cardiac microdialysis. Salicylic acid in Ringer's solution (0.5 nmol x microL(-1) x min(-1)) was infused directly through a microdialysis probe to detect the generation of .OH as reflected by the formation of dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rats. When pargyline (100 nmol x microL(-1) x min(-1)) was infused in rat heart, the level of norepinephrine (NE) gradually increased in a time-dependent manner and an increase of DHBA was also observed. When NE was administered to the pargyline pretreated animals, a marked elevation in the levels of 2,3- and 2,5-DHBA formation was obtained, as compared to the group treated with NE only, showing a positive linear correlation between NE and .OH formation trapped as 2,3-DHBA (R2 = 0.981) or 2,5-DHBA (R2 = 0.984) in the dialysate. NE clearly produced an increase in .OH formation. These results indicate that accumulation of NE in the extracellular fluid elicited by pargyline can be auto-oxidized, which in turn, leads (possibly by an indirect mechanism) to the formation of cytotoxic .OH free radicals.

Topics: Animals; Dose-Response Relationship, Drug; Gentisates; Hydroxybenzoates; Hydroxyl Radical; In Vitro Techniques; Male; Microdialysis; Monoamine Oxidase Inhibitors; Myocardium; Norepinephrine; Pargyline; Rats; Rats, Wistar; Salicylates; Salicylic Acid

Free-radicals are reported to cause the tissue-damage associated with some toxins and diseases, yet there is no suitable method for routine in-vivo monitoring of these species. This paper introduces an in-vivo microdialysis technique in which the hydroxyl radical reacts with salicylate to generate dihydroxybenzoic acids (DHBA) which are measured by HPLC with electrochemical detection. When pargyline, a monoamine oxidase inhibitor, was infused into rat brain, the levels of DHBA increased markedly. When noradrenaline was administered to animals pre-treated with pargyline, DHBA levels increased markedly compared with the group treated with noradrenaline only. When the heart was subjected to 15-min regional ischaemia by occlusion of the left anterior descending coronary artery, levels of DHBA in heart dialysate were unchanged. Electrical stimulation of the stellate ganglion resulted in marked elevation of levels of DHBA the myocardial dialysate. Infusion of Fe2+ into rat liver resulted in increased formation of DHBA. When the intestine was rendered ischaemic for 10, 20 and 30 min, the highest DHBA level was obtained after 10-min ischaemia and the lowest after 30 min. These results confirm that free-radical production might make a major contribution at certain stages in the progression of the injury.

Topics: Animals; Brain; Chromatography, High Pressure Liquid; Electric Stimulation; Gentisates; Hydrogen Peroxide; Hydroxybenzoates; Hydroxyl Radical; Iron; Ischemia; Jejunum; Liver; Male; Microdialysis; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Myocardium; Norepinephrine; Pargyline; Rats; Rats, Wistar; Reperfusion Injury; Salicylates; Superoxides; Sympathetic Nervous System

Coenzyme Q10 is an essential cofactor of the electron transport chain and is an antioxidant. We examined the effects of oral feeding with coenzyme Q10 in young animals on brain concentrations. Feeding with coenzyme Q10 at a dose of 200 mg/kg for 1-2 months in young rats resulted in significant increases in liver concentrations, however, there was no significant increase in brain concentrations of either reduced- or total coenzyme Q10 levels. Nevertheless there was a reduction in malonate-induced increases in 2,5 dihydroxybenzoic acid to salicylate, consistent with an antioxidant effect. In other studies we found that oral administration of coenzyme Q10 significantly reduced increased concentrations of lactate in the occipital cortex of Huntington's disease patients. These findings suggest that coenzyme Q10 might be useful in treating neurodegenerative diseases.

Topics: Administration, Oral; Animals; Antioxidants; Brain; Coenzymes; Corpus Striatum; Electron Transport; Gentisates; Humans; Hydroxybenzoates; Injections; Liver; Male; Malonates; Neurodegenerative Diseases; Neuroprotective Agents; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Salicylates; Salicylic Acid; Ubiquinone

The healing-promotion property of saliva has been observed in the past, but its underlying mechanism has never been elucidated. We hypothesized a mechanism based on salivary proteins binding to redox active metal ions, rendering them nonactive in their capacity for free radical production.. Examination of this mechanism was conducted by comparing the redox activity of protein-rich saliva with protein-poor saliva. We also examined the redox activity mediated by these 2 kinds of saliva following the in vitro addition of iron, copper, and manganese. Saliva samples were analyzed for their redox activity by measuring the ascorbate-driven and saliva (diluted 1:2)-mediated conversion of salicylate to its 2,3- and 2.5-dihydroxybenzoates and catechol metabolites.. The concentrations of salicylate metabolites formed by protein-rich saliva were significantly lower by 45% (P < .05), 66% (P < .01), and 54% (P < .05), respectively, when compared with those formed by protein-poor saliva. The capacity of saliva in suppressing redox activity was found to be inversely related to the concentrations of iron and copper added (but not manganese), but correlated well with the protein content. When the highest concentrations of iron (15 mumol/L) and copper (10 mumol/L) were added to protein-rich saliva, the concentrations of salicylate metabolites produced were only 0.3% to 1% of those of non-saliva-containing controls (P < .01). However, when these concentrations of iron and copper were added to protein-poor saliva, significantly higher values of redox activity were detected, and the concentrations of the salicylate derivatives produced were 2.1% to 8.1% of those of non-saliva-containing controls (P < .01). In contrast, when the lowest concentrations of iron (2 mumol/L) and copper (0.1 mumol/L) were added, 2.8 to 4 times lower concentrations of salicylate derivatives were produced (P < .01).. These results substantiate our hypothesis that saliva has a profound capacity for reducing redox activity rendered by transition metal ions, correlating well with its protein content.

Topics: Ancitabine; Animals; Antimetabolites, Antineoplastic; Antioxidants; Ascorbic Acid; Catechols; Copper; Free Radicals; Gentisates; Hydroxybenzoates; Iron; Iron Chelating Agents; Male; Manganese; Metals; Oxidation-Reduction; Parasympathomimetics; Parotid Gland; Pilocarpine; Protein Binding; Rats; Rats, Wistar; Salicylates; Saliva; Salivary Proteins and Peptides; Wound Healing

Although calcium (Ca2+) is important in cardiac dysfunction and has also been reported as a source of oxidative toxicity, the connection between Ca2+ overload and oxygen free radicals in the myocardium is not clear. We have investigated whether Ca2+ overload generates hydroxyl free radicals in rat ventricle. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl free radical reacts with salicylate. Ringer's solution containing salicylic acid (0.5 nmol microL-1 min-1) was infused through a microdialysis probe in the region of the left anterior descending coronary artery of the rat ventricle. A positive linear correlation was obtained between Ca2+ and hydroxyl free radical formation trapped as 2,3-DHBA (r2 = 0.976) and 2,5-DHBA (r2 = 0.982) in the myocardial dialysate. The administration of ouabain (1 mg kg-1, i.v.), a Ca2+ elevator, into the femoral vein significantly increased the level of 2,3- and 2,5-DHBA. These results indicate that Ca2+ overload generates hydroxyl free radicals in rat heart.

Topics: Animals; Calcium; Cardiotonic Agents; Dose-Response Relationship, Drug; Gentisates; Heart; Heart Ventricles; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Male; Microdialysis; Mixed Function Oxygenases; Myocardium; Ouabain; Rats; Rats, Wistar; Salicylates; Salicylic Acid

Using the microdialysis technique and laser-Doppler flowmetry, we performed simultaneous measurement of salicylate hydroxylation and glutamate release along with local CBF in the ischemic penumbral cortex of rat brain subjected to normothermic transient middle cerebral artery (MCA) occlusion. Cortical CBF fell to 24 +/- 11% (mean +/- SD) during ischemia and recovered to 84 +/- 16% during reperfusion. Extracellular glutamate levels increased by 6.5-fold above baseline 10 min following MCA occlusion but subsequently returned to near baseline levels in spite of the persistent ischemia. Increase in 2,3- and 2,5-dihydroxybenzoic acid (DHBA) concentrations in the microdialysis perfusate was confirmed during both ischemia and reperfusion phase. Although the temporal profile and amount of salicylate hydroxylation were heterogeneous among individual animals, integrated 2,3-DHBA concentrations during reperfusion were correlated positively with integrated glutamate concentrations during ischemia and negatively with mean postischemic CBF. These relationships suggest a possible association of the enhanced production of 2,3-DHBA during reperfusion with larger amounts of intraischemic glutamate release and lower levels of post-ischemic CBF.

Topics: Acid-Base Imbalance; Analysis of Variance; Animals; Cerebral Cortex; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Free Radical Scavengers; Gentisates; Glutamic Acid; Hydroxybenzoates; Hydroxylation; Iron Chelating Agents; Ischemic Attack, Transient; Laser-Doppler Flowmetry; Male; Rats; Rats, Sprague-Dawley; Regression Analysis; Salicylates; Salicylic Acid

Bioactivation of phenytoin and related teratogens by peroxidases such as prostaglandin H synthase (PHS) may initiate hydroxyl radical (.OH) formation that is teratogenic. Salicylate is hydroxylated by .OH at the third and fifth carbon atoms, forming 2,3- and 2,5-dihydroxybenzoic acids (DHBA). In vivo salicylate metabolism produces only the 2,5-isomer, so 2,3-DHBA formation may reflect .OH production. In the present study, we validated the salicylate assay using the known .OH generator paraquat and evaluated .OH production by phenytoin. Female CD-1 mice were treated with paraquat (30 mg/kg, intraperitoneally) given 30 min after acetylsalicylic acid (ASA) (200 mg/kg, intraperitoneally). Blood was collected at 5, 15, and 30 min and 1 and 2 hr after paraquat, and plasma was analyzed for DHBA isomers and glucuronide conjugates by high performance liquid chromatography with electrochemical detection. Paraquat increased 2,3-DHBA formation 19.2-fold, with substantial inter-individual variability in the time of maximal formation (p = 0.0001). The 2,3-DHBA glucuronide conjugates in vivo and in hepatic microsomal studies amounted to approximately 11% and 0.43%, respectively, of total 2,3-DHBA equivalents. To investigate putative .OH production initiated via PHS-catalyzed phenytoin bioactivation, ASA was given 30 min before phenytoin (65 or 100 mg/kg, intraperitoneally), resulting in respective 7.6-fold (p = 0.02) and 14.2-fold (p = 0.003) increases in phenytoin-initiated maximal 2,3-DHBA formation. Maximal 2,3-DHBA formation was 2.1-fold higher when ASA was administered after rather than before the same dose (65 mg/kg) of phenytoin (p = 0.03), indicating ASA inhibition of PHS-catalyzed phenytoin bioactivation. Urinary analysis was much less sensitive, and the 2,5-isomer reflected enzymatic rather than .OH-mediated hydroxylation. The paraquat studies demonstrate the importance of timing in accurately quantifying 2,3-DHBA formation and suggest that glucuronidation does not interfere. The substantial, dose-dependent initiation of 2,3-DHBA formation by phenytoin, and its inhibition by ASA, provide the first in vivo evidence that PHS-dependent .OH formation could contribute to the molecular mechanism of phenytoin teratogenesis.

Topics: Animals; Biotransformation; Female; Gentisates; Glucuronidase; Hydrolysis; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Mice; Paraquat; Phenytoin; Salicylates; Salicylic Acid; Teratogens

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C18 column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3-4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2-20 micrograms/ml and linearity was observed from 0.1-200 micrograms/ml and 5-2000 micrograms/ml for SA in plasma and urine, respectively. The method was validated to 0.2 microgram/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.

Topics: Chromatography, High Pressure Liquid; Gentisates; Glucuronates; Hippurates; Humans; Hydroxybenzoates; Reproducibility of Results; Salicylates; Salicylic Acid; Sensitivity and Specificity

Garlic has been reported to provide protection against hypercholesterolemic atherosclerosis and ischemia-reperfusion-induced arrhythmias and infarction. Oxygen free radicals (OFRs) have been implicated as causative factors in these diseases and antioxidants have been shown to be effective against these conditions. The effectiveness of garlic in these disease states could be due to its ability to scavenge OFRs. However, the OFR-scavenging activity of garlic is not known. Also it is not known if its activity is affected by cooking. We therefore investigated, using high pressure liquid chromatography, the ability of garlic extract (heated or unheated) to scavenge exogenously generated hydroxyl radical (.OH). .OH was generated by photolysis of H2O2 (1.2-10 mumoles/ml) with ultraviolet (UV) light and was trapped with salicylic acid (500 nmoles/ml). H2O2 produced .OH in a concentration-dependent manner as estimated by .OH adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. Garlic extract (5-100 microliters/ml) produced an inhibition (30-100%) of 2,3-DHBA and 2,5-DHBA generated by photolysis of H2O2 (5.00 pmoles/ml) in a concentration-dependent manner. Its activity is reduced by 10% approximately when heated to 100 degrees C for 20, 40 or 60 min. The extent of reduction in activity was similar for the three heating periods. Garlic extract prevented the .OH-induced formation of malondialdehyde in the rabbit liver homogenate in a concentration-dependent manner. It alone did not affect the MDA levels in the absence of .OH. These results indicate that garlic extract is a powerful scavenger of .OH and that heating reduces its activity slightly.

Topics: Animals; Chromatography, High Pressure Liquid; Free Radical Scavengers; Garlic; Gentisates; Hot Temperature; Hydroxybenzoates; Hydroxyl Radical; Lipid Peroxidation; Liver; Malondialdehyde; Photolysis; Plant Extracts; Plants, Medicinal; Rabbits; Salicylates; Salicylic Acid; Sensitivity and Specificity; Superoxides; Ultraviolet Rays

Apolipoprotein E (Apo-E) is linked to the pathogenesis of Alzheimer's disease. Apo-E deficient mice have increased lipid peroxidation in plasma. In the present study we examined two markers of oxidative stress in brains of Apo-E deficient mice. The ratios of 2,3 and 2,5 dihydroxybenzoic acid (DHBA)/salicylate, an index of hydroxyl radical generation, were unchanged except for an increase in 2.5-DHBA/salicylate in the cerebellum. 3-Nitroxyrosine is a marker for nitration of proteins produced by peroxynitrite. Concentrations of 3-nitrotyrosine were significantly increased in the cerebral cortex, hippocampus, brainstem and cerebellum of Apo-E deficient mice. These results suggest the Apo-E may modulate oxidative stress produced by peroxynitrite.

Topics: Animals; Apolipoproteins E; Biomarkers; Brain Chemistry; Free Radical Scavengers; Free Radicals; Gentisates; Hydroxybenzoates; Mice; Mice, Inbred C57BL; Oxidative Stress; Salicylates; Salicylic Acid; Tyrosine

Most of the procedures currently performed by capillary zone electrophoresis (CZE) with laser induced fluorescence detection requires prior derivatization. This increases cost, the turn-around-time and chances of extraneous contaminations. CZE with laser induced resonance energy transfer is demonstrated as a viable alternative for detecting non-fluorescent compounds with no prior derivatization. The feasibility of this approach is demonstrated by separating and directly detecting salicylic acid (2,4-dihydroxybenzoic acid), gentisic acid (o-methoxybenzoic acid), salicyluric acid (o-hydroxyhippuric acid) and 4-aminosalicylic acid in urine. The detection of salicylate in serum is also shown. The method is highly sensitive with detection limits in the 1.10(-7) M range. Importantly it requires no prior preconcentration or sample preparation and can be used with complex sample matrices such as serum and urine.

Topics: Aminosalicylic Acid; Aspirin; Benzoates; Chemistry, Clinical; Electrophoresis, Capillary; Energy Transfer; Gentisates; Hippurates; Humans; Hydroxybenzoates; Lasers; Linear Models; Salicylates; Sensitivity and Specificity

Identification of a case of acute salicylate intoxication using 300 MHz 1H NMR spectroscopy of a urine sample is reported. It has been achieved by using a combination of a one-dimensional experiment with water presaturation and a two-dimensional homonuclear J-resolved experiment. By these means, lysine and the three major metabolites of acetylsalicylic acid have been assigned in the crude urine. The results are compared with those obtained at 600 MHz and with classical biochemical methods. The use of this method for routine diagnosis in biological analysis is discussed.

Topics: Adolescent; Aspirin; Female; Gentisates; Hippurates; Humans; Hydroxybenzoates; Magnetic Resonance Spectroscopy; Poisoning; Salicylates; Salicylic Acid

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Aspirin; Cerebrovascular Disorders; Cyclooxygenase Inhibitors; Female; Free Radical Scavengers; Gentisates; Humans; Hydroxybenzoates; Male; Neuroprotective Agents; Platelet Aggregation Inhibitors; Salicylates; Salicylic Acid

Results from various in vitro experiments have indicated that excitotoxicity and oxidative stress are two interrelated major mechanisms in causing neuronal damage in brain disorders such as cerebral ischemia/reperfusion. Thus, we have conducted experiments to investigate whether in the striatum of anesthetized rats the elevated brain extracellular concentrations of glutamate could increase the formation of hydroxyl radical. Elevation of glutamate levels and trapping of hydroxyl radical were accomplished by perfusing Ringer solutions containing both glutamate and salicylic acid through microdialysis probes implanted in rat striatum. The formation of hydroxyl radical was quantitated as the increased amounts of 2,3 and 2,5 dihydroxybenzoic acid (DHBA) which were the hydroxylative products of salicylic acid. Eluted microdialysates were directly injected onto high performance liquid chromatography (HPLC) with electrochemical detector via an on-line automatic injector. This method was authenticated by in vitro studies employing Fenton-type hydroxyl radicals generation system. Our results indicated that elevated glutamate concentrations (15 mM, 1.5 mM, and 150 microM glutamate in perfusing solutions) would significantly increased both the concentrations of 2,3 and 2,5 DHBA. In conclusion, we have obtained direct evidence showing that the elevated glutamate concentrations in brain extracellular space would increase the formation of hydroxyl radical, and these results implied that oxidative stress occurring in brain disorders might be induced by excitotoxicity.

Topics: Anesthesia; Animals; Chromatography, High Pressure Liquid; Corpus Striatum; Extracellular Space; Gentisates; Glutamic Acid; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Kinetics; Male; Rats; Rats, Sprague-Dawley; Salicylates; Salicylic Acid

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 degrees C), hypothermic (30 degrees C), or hyperthermic (39 degrees C) but was held at 37 degrees 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Body Temperature; Corpus Striatum; Dialysis; Free Radicals; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Ischemic Attack, Transient; Kinetics; Male; Rats; Rats, Wistar; Reperfusion; Salicylates; Salicylic Acid

The in vivo measurement of highly reactive free radicals, such as hydroxyl radical (.OH), in humans is very difficult if not impossible. Specific markers are currently under investigation (amino acid hydroxylatin, protein, DNA adducts, and aromatic probes). They are based on the ability of .OH to attack aromatic molecules to produce hydroxylated compounds that can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives, i.e., 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA). The measurement of 2,3-DHBA, following oral administration of salicylate or its acetylated form (aspirin), has been proposed for assessment of in vivo oxidative stress. In this work, a sensitive method for the detection of in vivo .OH generation is presented. The methodology employs a high-pressure liquid chromatography with electrochemical detection for the identification and quantification of the hydroxylation products from the reaction of .OH with salicylate. A detection limit of less than 0.1 pmol for the hydroxylation products has been achieved with electrochemical detector responses which were linear over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA and dihydroxylated derivatives/salicylic acid ratios following the administration of 1000 mg aspirin in 20 healthy subjects. In the same individuals, plasma levels of thiobarbituric acid reactants (TBARs), a major index of lipid peroxidation, were also measured and correlation with hydroxylated products was sought. The plasma level of TBARs was positively correlated with the 2,5-DHBA/salicylic acid ratio, but not with the absolute plasma level of 2,3-DHBA.

Topics: Adult; Aspirin; Calibration; Chromatography, High Pressure Liquid; Female; Free Radical Scavengers; Gentisates; Humans; Hydroxybenzoates; Hydroxyl Radical; Male; Oxidative Stress; Reproducibility of Results; Salicylates; Salicylic Acid; Thiobarbituric Acid Reactive Substances

We examined in vivo monitoring of hydroxyl radical (.OH) generation during myocardial ischemia for 30 min by occluding the left anterior descending (LAD) in dog heart using a microdialysis technique. The hydroxyl radical reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acids (DHBA) which can be measured electrochemically in picomole quantity by a high pressure liquid chromatography-electrochemical (HPLC-EC) procedure. When the premature ventricular contraction (PVC) occurred at almost 25 sec intervals, marked elevation of the levels of 2,3- and 2,5-DHBA was observed in the heart dialysate of 30-min ischemia. This study demonstrated the generation of .OH in the canine heart subjected to 30-min ischemic insult.

Topics: Animals; Chromatography, High Pressure Liquid; Dogs; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Microchemistry; Microdialysis; Myocardial Contraction; Myocardial Ischemia; Salicylates; Salicylic Acid

The hydroxyl radical (.OH) is a highly reactive oxygen free radical that has been implicated as a cause of lung injury following exposure to silica and silicates. Despite evidence that silica generates .OH in vitro, there has been no previous demonstration of in vivo production of .OH after exposure to nonfibrous mineral oxide dusts. We tested the hypothesis that instillation of silica into rat lungs is associated with greater .OH production and acute lung inflammation in vivo relative to the instillation of a less toxic nonsilicate particle, titanium dioxide. The production of .OH in the lungs following dust instillation was measured using sodium salicylate as an .OH trap. Seven days after dust exposure, the rats were given intraperitoneal salicylate, the lungs isolated, and salicylate hydroxylation products (2,3- and 2,5-dihydroxybenzoic acid), reflecting .OH, were measured. There was significantly more 2,3-dihydroxybenzoic acid in silica-exposed lungs compared with lungs instilled with titanium dioxide. In addition, the instillation of silica into rat lungs in vivo was associated with a greater acute inflammatory response. We conclude that following in vivo exposure, silica stimulates greater .OH production relative to the less toxic particle, titanium dioxide. These differences in .OH generation correspond to disparities in acute lung inflammation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Dust; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Lung; Lung Injury; Organ Size; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Salicylates; Salicylic Acid; Silicon Dioxide; Silicosis; Titanium

Rhodococcus erythropolis strain S1 uses the gentisate pathway to metabolize salicylate and m-hydroxybenzoate and the protocatechuate pathway to degrade p-hydroxybenzoate. m-Hydroxybenzoate 6-hydroxylase was induced by growth on m-hydroxybenzoate or gentisate, and salicylate 5-hydroxylase only by growth on salicylate. p-Hydroxybenzoate 3-hydroxylase could be induced only by growth on p-hydroxybenzoate. m-Hydroxybenzoate or p-hydroxybenzoate could repress the induction of salicylate 5-hydroxylase. Maleylpyruvate isomerase in the gentisate pathway did not require reduced glutathione.

Topics: cis-trans-Isomerases; Gentisates; Glutathione; Hydroxybenzoates; Isomerases; Kinetics; Mixed Function Oxygenases; Oxygen Consumption; Parabens; Rhodococcus; Salicylates; Salicylic Acid

The difficulty in direct detection of oxygen-derived free radicals (OFR) in the intact kidney has left uncertain the role of OFR in renal hypoperfusion injury. Salicylate hydroxylation was used as a sensitive method of estimating the extent of production of highly reactive hydroxyl radicals in renal ischaemia-reperfusion injury in the intact rat kidney perfused with recirculating cell-free medium. The reaction products were detected and quantified by HPLC with electrochemical detection. Hydroxyl radicals were detected as 2,5-dihydroxybenzoic acid (2,5-DHBA). Ischaemia for 15 min followed by reperfusion for 15 min caused more than a twofold increase in 2,5-DHBA concentration (to 2279 +/- 225 pg/g tissue weight) compared to controls (933 +/- 103, P < 0.001). Addition of 15 mM dimethylthiourea (DMTU) before induction of ischaemia prevented this increase. Induction of hypoxia for 15 min with continued perfusion (as a model of low-flow ischaemia) had no significant effect on hydroxyl radical formation. We conclude that significant quantities of hydroxyl radicals form in the absence of circulating leucocytes during reperfusion following ischaemia, but not during hypoxia in the perfused rat kidney.

Topics: Animals; Chromatography, High Pressure Liquid; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Hypoxia; Ischemia; Kidney; Male; Rats; Rats, Wistar; Reperfusion; Salicylates

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 mumol) 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.

Topics: Animals; Biogenic Monoamines; Brain; Corpus Striatum; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Injections, Intraperitoneal; Injections, Intraventricular; Methamphetamine; Rats; Rats, Sprague-Dawley; Salicylates; Salicylic Acid

It has been suggested that salicylate (SA) hydroxylation can be used to detect hydroxyl radical formation in vivo. Here we describe a rapid and sensitive HPLC method using ultraviolet absorbance (UV) and electrochemical detection (EC) to detect SA (UV), its hydroxylated adducts 2,3- and 2,5-dihydroxybenzoic acids (DHBA) and catechol in combination with catechol- and indoleamines and related metabolites (EC) in one isocratic run. These compounds were measured in acidified cerebrospinal fluid (CSF) and perchlorate extracts of striatal tissues of untreated and SA-loaded rats (300 mg/kg SA, i.p.). Peaks were identified by comparing retention times of samples and standards, by adding standards to biological samples, by voltamograms, and by comparing chromatograms of manganese (Mn2+)-injected striata of SA-loaded rats with several control conditions. Six hours after unilateral injection of 0.4 mumol Mn2+ into striatum, 2,3-DHBA and 2,5-DHBA levels in striatum were respectively 4- and 7-fold increased as compared to non-injected (contralateral) striata, suggesting in vivo hydroxyl radical formation. In addition, dopamine and serotonin levels were depleted in Mn(2+)-injected striata by 46% and 64%, respectively. In CSF of Mn(2+)-injected rats, DHBA/SA ratios were not significantly changed as compared to those of control rats. In conclusion, the described technique can be applied to study in vivo hydroxyl radical formation in direct relation with dopaminergic and serotonergic neurotransmitter changes during neurotoxic processes.

Topics: Animals; Biogenic Amines; Catechols; Chromatography, High Pressure Liquid; Electrochemistry; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Indoles; Magnesium; Male; Manganese Poisoning; Neostriatum; Rats; Rats, Wistar; Reference Standards; Salicylates; Salicylic Acid; Spectrophotometry, Ultraviolet

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 mumoles/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 micrograms) 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 micrograms/ml was 32.36% and 43.2% respectively while with 36.0 micrograms/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.

Topics: Animals; Antioxidants; Chromatography, High Pressure Liquid; Disulfides; Free Radical Scavengers; Garlic; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Lipid Peroxidation; Liver; Malondialdehyde; Plants, Medicinal; Rabbits; Reactive Oxygen Species; Salicylates; Salicylic Acid; Sulfinic Acids; Tablets

To provide more experimental evidence for the proposed role of oxygen free radicals in red blood cell (RBC) damage in beta-thalassemia, hydroxyl radical generation was studied in thalassemic (Th) vs. normal (N) RBC. .OH fluxes were quantified by the conversion of salicylic acid (SA) into its hydroxylated products, 2,3- and 2,5-dihydroxybenzoic acids (DHBA) and catechol, assayed with HPLC coupled to electrochemical detection. No significant difference in spontaneous .OH generation between N-RBC and Th-RBC was found. Ascorbic acid (0.5-3.0 mM) induced many-fold increases in SA hydroxylation in a dose-dependent manner in both types of cells. In the presence of ascorbate (1.0 mM), the SA hydroxylated products were determined in Th-RBC vs. N-RBC as follows (nmol/ml): 2,5-DHBA, 1.45 +/- 0.06 vs. 1.81 +/- 0.05 (p = 0.001); 2,3-DHBA, 1.89 +/- 0.21 vs. 1.15 +/- 0.08 (p = 0.008) and catechol, 0.87 +/- 0.13 vs. 0.38 +/- 0.05 (p = 0.006). The results showed significant increase in the total SA hydroxylation in Th-RBC as compared to N-RBC with a tendency to form 2,3-DHBA and catechol at the expanse of 2,5-DHBA. The excessive .OH generation in Th-RBC is attributed to the abnormally high content of redox active iron in the cytosolic and/or membrane compartments of these cells.

Topics: beta-Thalassemia; Catechols; Erythrocytes; Gentisates; Humans; Hydroxybenzoates; Hydroxyl Radical; In Vitro Techniques; Iron; Oxidation-Reduction; Salicylates; Salicylic Acid

Topics: Animals; Bromocriptine; Corpus Striatum; Dopamine; Free Radical Scavengers; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Iron Chelating Agents; Kinetics; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Salicylates; Salicylic Acid

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 mumoles) 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.

Topics: Antioxidants; Benzocycloheptenes; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Food Preservatives; Free Radical Scavengers; Gentisates; Hydrogen Peroxide; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Photolysis; Salicylates; Salicylic Acid; Ultraviolet Rays

The ability of caffeine to potentiate the analgesic effect of aspirin was studied in the pain-induced functional impairment model in the rat. Female Wistar rats received an intra-articular injection of 30% uric acid in the right hind limb, inducing its dysfunction. Once the dysfunction was complete, animals received aspirin oral doses of 0, 0.55, 0.98, and 1.74 mmol/kg with and without 0.17 mmol/kg of caffeine, and the recovery of functionality over time was considered as an expression of analgesia. Blood samples were drawn simultaneously with hind limb functionality determinations, and plasma concentrations of aspirin, salicylic acid, and gentisic acid were measured by high-performance liquid chromatography. Aspirin induced a dose-dependent analgesic effect. Caffeine alone was ineffective. However, caffeine significantly increased the analgesic effect of aspirin at all doses, without modifying aspirin, salicylic acid, or gentisic acid plasma levels. It is concluded that caffeine potentiates the analgesic effect of aspirin by a pharmacodynamic, but not by a pharmacokinetic mechanism.

Topics: Animals; Aspirin; Caffeine; Drug Synergism; Female; Gentisates; Hindlimb; Hydroxybenzoates; Pain; Rats; Rats, Wistar; Salicylates; Salicylic Acid

There has been controversy concerning the products formed by a Fenton reaction. We determined the hydroxyl radical (.OH) generated in a Fenton reaction system with no iron chelator using micellar electrokinetic capillary chromatography (MECC). The hydroxyl radical generated in this Fenton system attacked salicylic acid to produce major products of 2,3- and 2,5-dihydroxybenzoic acid (DHB), 2,3-DHB being prominent. Hydroxyl radical scavengers, such as mannitol, ethanol, thiourea and a ferric chelator, Desferal, significantly diminished the peaks for DHBs, showing production of .OH. We compared the MECC method with the electron paramagnetic resonance (EPR) spin trapping technique. The quantity of DHBs obtained by MECC increased dose-dependently up to 1 microM Fe2+ at a fixed concentration of H2O2, whereas that of the spin adduct by EPR showed a bell-shaped curve. This quantitation of .OH adducts by MECC supports the proposal that the oxidizing species formed by a Fenton reaction with no chelator is .OH. The EPR spin trapping method appears to be erroneous, particularly when iron is present at a higher concentration than hydrogen peroxide. The application of this method to the paraquat effect in vitro is demonstrated, and the possibility for analysis of .OH in vivo is also discussed.

Topics: Chromatography; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Iron; Micelles; Salicylates; Salicylic Acid

Systemic administration of salicylate (SA) to rats (100 mg kg-1 i.p. ) was used as an in vivo trap of hydroxyl radicals (.OH). In the brain SA reacts with hydroxyl radicals to form the stable adducts 2, 3- and 2,5 dihydroxybenzoic acid (DHBAs) which can thus be taken as an index of .OH formation. The DHBAs were recovered by intracerebral microdialysis in hippocampus or striatum and quantified by high pressure liquid chromatography (HPLC) with electrochemical detection. There were no peaks corresponding to 2,5-DHBA or 2,3-DHBA in the chromatograms from rats not receiving SA. A basal level of 2,5-DHBA was seen in the dialysates from all animals given SA whereas 2, 3-DHBA was not detected. In one group of rats generation of free oxygen radicals was induced in the striatum by adding Fe2+ and ascorbate to the perfusion fluid to test the sensitivity of the system. Addition of Fe2+ ascorbate to the perfusion fluid induced a significant 7-fold increase in 2,5-DHBA that gradually returned to baseline after removal of Fe2+/ascorbate. In two other groups the microdialysis probes were implanted in either the striatum or the hippocampus and the animals were subjected to 20 min of four-vessel occlusion + hypotension (4-VOH). Significant reductions in 2,5-DHBA were detected during ischaemia followed by significant increases of 5-fold and 3-fold in the striatum and hippocampus, respectively, beginning immediately upon reperfusion and lasting for the remainder of the observation period (160 min).

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Male; Microdialysis; Monoamine Oxidase; Rats; Rats, Wistar; Reperfusion; Salicylates; Salicylic Acid

1. We applied in vivo microdialysis technique to examine the effect of Fe2+, Fe3+, Cu2+ and Zn2+ on free radical-generation of rat liver. The hydroxyl free radical (.OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA) which can be measured electrochemically in picomole quantity by HPLC-EC procedure. 2. The relative rates of recovery of 2,3- and 2,5-DHBA at 1 microliter/min in vitro were an average of 10.1 +/- 0.8% and 10.5 +/- 9%, respectively. 3. When the metal ion infused through the dialysis probe, Fe2+ but not Fe3+, Cu2+ and Zn2+ caused an increase in the formation of DHBA of rat liver. 4. After the Ringer solution containing 10 mumole/kg was injected into the penile vein, the levels of 2,3- and 2,5-DHBA were increased about 60% and 40%, respectively. 5. These results indicate that non-enzymatic oxidation in the extracellular fluid may play a key role in Fe2+ generation of -OH in liver. Free radical formation processes may contribute to in vivo free radical formation induced by Fe2+.

Topics: Animals; Extracellular Space; Ferrous Compounds; Free Radicals; Gentisates; Hydroxybenzoates; Hydroxylation; Iron Chelating Agents; Liver; Metals; Microdialysis; Perfusion; Rats; Rats, Wistar; Salicylates; Salicylic Acid

In the passive upright position, arterial and venous pressure in human feet increases capillary pressure, which leads to the filtration of fluid from the circulating plasma into the tissues of the feet. Loss of fluid concentrates both red cells and plasma so that the hematocrit and plasma protein concentration of venous blood leaving the feet greatly exceed their mean values in the circulation. To study this phenomenon in animals, the authors used beagle dogs maintained in an upright position and compared the results to those maintained in a prone position. In the passive upright position, red cell count, hematocrit, and total plasma protein concentrations were significantly increased. Therefore, we have determined the level of hydroxyl free radical formed as assessed by salicylate hydroxylation in the saphenous vein of dogs in an upright and normal position. Hydroxyl free radical formation was significantly higher in the veins of dogs in an upright position than in those in a prone position. The upright position model, which causes increases in hematocrit red cell count and total plasma protein in the saphenous vein, seems suitable for the study of drugs interfering in the pathophysiology of venous stasis. Indeed, the hematocrit increase of red cell count and total plasma protein in the saphenous vein of the beagle is similar to modifications observed in venous blood leaving the human foot after sitting. Moreover, the increase of .OH, concomitant with these modifications, may explain certain pathological observations, for instance, edema. The data clearly suggest that macromolecular extravasculation may be due to tissue injury caused by oxygen free radical formation in the blood vessels.

Topics: Animals; Blood Proteins; Body Fluids; Dogs; Edema; Erythrocyte Count; Free Radicals; Gentisates; Hematocrit; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Male; Models, Biological; Posture; Salicylates; Salicylic Acid; Saphenous Vein

Oxygen free radicals have been implicated as a causal factor in posttraumatic neuronal cell loss following cerebral ischemia and head injury. The conversion of salicylate to dihydroxybenzoic acid (DHBA) in vivo was employed to study the formation of hydroxyl radical (.OH) following central nervous system (CNS) injury. Bilateral carotid occlusion (BCO) in gerbils and concussive head trauma in mice were selected as models of brain injury. The lipid peroxidation inhibitor, tirilazad mesylate (U-74006F), was tested for its ability to attenuate hydroxyl radical formation in these models. In addition, U-74006F was studied as a scavenger of hydroxyl radical in an in vitro assay based on the Fenton reaction. For in vivo experimentation, hydroxyl radical formation was expressed as the ratio of DHBA to salicylate (DHBA/SAL) measured in brain. In the BCO model, hydroxyl radical formation increased in whole brain with 10 min of occlusion followed by 1 min of reperfusion. DHBA/SAL was also found to increase in the mouse head injury model at 1 h postinjury. In both models, U-74006F (1 or 10 mg/kg) blocked the increase in DHBA/SAL following injury. In vitro, reaction of U-74006F with hydroxyl radical gave a product with a mol wt that was 16 greater than U-74006F, indicative of hydroxyl radical scavenging. We speculate that U-74006F may function by blocking oxyradical-dependent cell damage, and thereby maintaining free iron (which catalyzes hydroxyl radical formation) concentrations at normal levels.

Topics: Animals; Brain Injuries; Brain Ischemia; Free Radical Scavengers; Gentisates; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Lipid Peroxides; Male; Mice; Pregnatrienes; Salicylates; Salicylic Acid

The transport of aspirin (ASP) and its metabolites (salicylic acid (SA), salicyluric acid (SAU), gentisic acid (GA) and gentisuric acid (GAU)) through human erythrocyte membrane was investigated. ASP permeated rapidly into the erythrocytes and the concentration dwindled gradually after the maximum concentration was attained almost within one minute. It was suggested that SA is released from the erythrocytes after ASP transported into the erythrocytes is hydrolyzed in them. In both an inward and outward direction, the transport rates of SA and GA were rapid, while those of SAU and GAU were lower by conjugating glycine. It was suggested that GAU remains for a long time in a living body. The rate of transport of GA and GAU were markedly obstructed by the band 3 protein inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate, although the transport rates of SA and SAU were obstructed only slightly. It was suggested that the transport of GA and GAU are mediated through band 3 protein.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Aspirin; Chemical Phenomena; Chemistry, Physical; Culture Media; Cytosol; Erythrocyte Membrane; Gentisates; Hippurates; Humans; Hydrolysis; Hydroxybenzoates; In Vitro Techniques; Salicylates; Salicylic Acid

The salicylate trapping method was used to investigate the changes in hydroxyl radical (.OH) levels in the selectively vulnerable hippocampus compared to the cerebral cortex of gerbils subjected to a 10 min period of near complete forebrain ischemia. Salicylate-derived 2,5-dihydroxybenzoic acid (2,5-DHBA) was measured in sham-operated animals and at 1, 5, and 15 min of reperfusion. A basal level of 2,5-DHBA was also seen in non-ischemic gerbil brain, both in the hippocampus and cortex. The hippocampal basal level was 160% higher than in the cortex (P < .01). Treatment with the cytochrome P450 inhibitor SKF-525A (50 mg/kg s.c. 30 min before measurement) did not affect this basal level in either hippocampus or cortex, which argues against a contribution of metabolic salicylate hydroxylation as its source. In contrast, pretreatment with the arachidonic acid cyclo-oxygenase inhibitor ibuprofen (20 mg/kg s.c.) decreased (-68.8%) the level of salicylate hydroxylation in the hippocampus, but not the cortex. In animals subjected to 10 min of forebrain ischemia, a selective increase in 2,5-DHBA was observed in the hippocampus at 1 min of reperfusion which subsided by 5 min. No increase in salicylate hydroxylation was apparent in the cortex within the same time frame. The increase in .OH in the hippocampus at 1 min of reperfusion was accompanied by a significant decrease (-15.7%; P < .03) in the hippocampal levels of vitamin E. No loss of vitamin E was observed in the cortex at the same time.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Brain Ischemia; Cerebral Cortex; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Free Radicals; Gentisates; Gerbillinae; Hippocampus; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Ibuprofen; Lipid Peroxides; Male; Proadifen; Reperfusion; Salicylates; Salicylic Acid; Vitamin E

Merocyanine 540 (MC540) is a photosensitizing dye of potential use in the purging of cancer cells from autologous bone marrow explants. Treatment of marrow with MC540, followed by illumination with visible light, selectively kills neoplastic cells while sparing a sufficient number of stem cells to allow marrow engraftment. The photodynamic action of MC540 is thought to be mediated by reactive oxygen species, particularly singlet oxygen. We have previously shown that salicylic acid (SA) scavenges MC540-generated singlet oxygen. In this work, we sought to abrogate MC540-mediated cell killing of murine L1210 and human K562 leukemia cells with salicylate. Paradoxically, the presence of salicylate during illumination in the presence of MC540 appreciably enhanced cell killing. Enhancement was dependent on salicylate concentration in the range 0.1 to 10 mM, with 1.0 mM SA potentiating the MC540-mediated reduction in survival of L1210 and K562 cells by factors of 2.7 and 1.9, respectively. Neither preincubation with SA followed by washing prior to illumination nor addition of SA following illumination altered MC540-mediated cell killing, indicating that potentiation was dependent on the presence of SA during illumination. Illumination in the presence of salicylate alone did not diminish cell viability. In addition to SA, a number of structurally related compounds including dihydroxybenzoic acids, aspirin, and sodium benzoate also enhanced MC540-mediated cell killing. Potentiation of leukemic cell killing by salicylate could provide a basis for enhancing the clinical efficacy of MC540-mediated phototherapy.

Topics: Animals; Cell Survival; Drug Synergism; Gentisates; Humans; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Leukemia; Leukemia L1210; Oxygen; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Salicylates; Salicylic Acid

Isolated rat brain microvessels have been utilized to examine whether they produce hydroxyl free radicals if they are subjected to a 10- to 20-min anoxia period followed by a 40-min reoxygenation period. Hydroxyl free radical flux was assessed utilizing salicylate as a trap. The 2,3- and 2,5-dihydroxybenzoic acids (DHBA) products as well as salicylate in the microvessels were quantitated utilizing high-pressure liquid chromatography (HPLC) with electrochemical and fluorescence detection. The results show that a period of anoxia followed by reoxygenation resulted in an enhanced formation of DHBA compared to the normoxic control microvessels. Addition of superoxide dismutase (SOD) and catalase to the microvessels undergoing anoxia decreased the amount of hydroxyl free radicals trapped, suggesting that superoxide and hydrogen peroxide were produced and excreted from the endothelial cell surfaces and then unless quenched reentered the cells to form hydroxyl free radicals within. The amount of 2,5-DHBA formed closely correlated with the amount of 2,3-DHBA formed, indicating that either product can be used to assess hydroxyl free radical flux in brain microvessels.

Topics: Animals; Brain; Catalase; Chromatography, High Pressure Liquid; Free Radicals; Gentisates; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Male; Microcirculation; Oxygen; Rats; Rats, Sprague-Dawley; Salicylates; Salicylic Acid; Superoxides

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.

Topics: Animals; Antioxidants; Calcium; Free Radical Scavengers; Gentisates; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; In Vitro Techniques; Male; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid

To elucidate the pathophysiological role of the hydroxyl radical (.OH) during the postischemic reperfusion of the heart, we measured the .OH product in the coronary effluent from isolated perfused rat heart during a 30-minute reperfusion period after various ischemic intervals of 5, 10, 15, 20, 30, and 60 minutes. Salicylic acid was used as the probe for .OH, and its derivative, 2,5-dihydroxybenzoic acid (2,5-DHBA), was quantified using high-performance liquid chromatography with ultraviolet detection. 2,5-DHBA was negligible in the effluent from nonischemic hearts, but a significant amount was detected from the hearts rendered ischemic for 10 minutes or longer. The peak of 2,5-DHBA was seen within 90 seconds after the onset of reperfusion in every group. The accumulated amount of 2,5-DHBA was maximal in the group with 15-minute ischemia (6.73 +/- 1.04 nmol/g wet heart wt after 30 minutes of reperfusion); it decreased as the ischemic time was prolonged and was 2.38 +/- 0.84 nmol/g wet wt after 30 minutes of reperfusion in the group with 60-minute ischemia. In the model of 15-minute ischemia/30-minute reperfusion, there was no correlation between the accumulated amount of 2,5-DHBA and functional recovery (+/- dP/dt, heart rate, and coronary flow), lactate dehydrogenase release, and morphological damage. Although treatment with 0.5 mM deferoxamine, an iron chelator, significantly decreased 2,5-DHBA (from 6.73 +/- 1.04 to 2.29 +/- 0.80 nmol/g wet wt after 30 minutes of reperfusion, p less than 0.01), it failed to reduce the postischemic myocardial injury in the group with 15-minute ischemia. The results suggest that .OH production is influenced by the preceding ischemic interval and that .OH does not exert an immediate direct effect on postischemic damage during early reperfusion in the isolated perfused rat heart, although a possibility remains that the small portion of .OH trapped by salicylic acid may not be intimately associated with myocardial injury.

Topics: Animals; Coronary Disease; Deferoxamine; Free Radicals; Gentisates; Heart; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Male; Myocardial Reperfusion Injury; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid; Time Factors

In vivo metabolism of salicylic acid produces two main hydroxylated derivatives (2,5- and 2,3-dihydroxybenzoic acid). The former can be produced by enzymatic pathways through the cytochrome P-450 system, while the latter is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-dihydroxybenzoate, following oral administration of salicylate in its acetylated form (aspirin), has been proposed for assessment of oxidative stress. In this article we report plasma levels of 2,3- and 2,5-dihydroxybenzoates following the administration of 1 g aspirin and plasma levels of thiobarbituric acid-reactive material (TBARM) in well-controlled diabetic patients and in healthy subjects. 2,3-Dihydroxybenzoate levels were significantly higher (23%) in diabetic patients than in controls (63.4 +/- 20.1 versus 49.0 +/- 6.8 nM; p < .05). On the other hand, TBARM values were not significantly different between groups. These results suggest that the method is useful to reveal in vivo oxidative stress independently from the peroxidation of lipids, and they support the hypothesis that oxygen radicals are involved in the pathogenesis of chronic complications of diabetes.

Topics: Adult; Aspirin; Diabetes Mellitus, Type 1; Female; Gentisates; Humans; Hydroxybenzoates; Hydroxylation; Male; Oxidation-Reduction; Reactive Oxygen Species; Salicylates; Thiobarbiturates

A method using liquid-liquid extraction has been developed for the isolation of acetylsalicylic acid and its metabolites, salicylic, gentisic or possibly salicyluric acids, from whole blood, isolated erythrocytes and plasma. Methylene chloride proved to be the best of the organic solvents tested. For whole blood and isolated erythrocytes it was necessary to carry out haemolysis prior to their extraction. The high-performance liquid chromatographic conditions for the quantitation of acetylsalicylic acid and its metabolites from samples of whole blood, erythrocytes and whole plasma were optimized. Separation was performed using reversed-phase chromatography on Separon SGX C18 and ultraviolet detection at 236 nm. A mixture of methanol-water (80:100, v/v) was the mobile phase, acidified with perchloric acid to pH 2.5.

Topics: Animals; Aspirin; Cells, Cultured; Chromatography, High Pressure Liquid; Erythrocytes; Gentisates; Hippurates; Hydroxybenzoates; Rabbits; Reproducibility of Results; Salicylates; Salicylic Acid; Spectrophotometry, Ultraviolet

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.

Topics: Biodegradation, Environmental; Gentisates; Hydroxybenzoates; Mixed Function Oxygenases; NADP; Naphthalenes; Oxygen Consumption; Rhodococcus; Salicylates; Salicylic Acid; Soil Microbiology; Species Specificity

Ischemia of rat intestine was induced in vivo by occlusion of the superior mesenteric artery (SMA) for 15 min. Sodium salicylate, 100 mg/kg, given IP, 30 min prior to the ischemic event served as a specific trap for hydroxyl radicals. Portions of the bowel were sequentially isolated and removed--2 min prior to ischemia, 2 min prior to declamping of the SMA, and 10 min following reperfusion. The bowel segments were homogenized in 3% TCA. The homogenate was centrifuged and filtrated through a 0.22 mu filter. The hydroxylation products of salicylate, dihydroxybenzoic acid (DHBA) derivatives, were isolated, identified, and quantified by HPLC coupled with electrochemical detection (ECD). The level of 2,5-DHBA (M +/- SE, ng/g tissue) in the preischemic bowel (N = 21) was 241.8 +/- 10.0. In the ischemic specimen the level of 2,5-DHBA increased significantly to 313.3 +/- 15.5 (p = 0.0129), and remained unchanged in the reperfusion period (322.8 +/- 15.5). The histological examination correlated well with these levels: mild villi damage in the ischemic period with no further exacerbation during the reperfusion period. This study in an in vivo animal model of intestinal ischemia-reperfusion provides direct evidence for the involvement of free radicals during the ischemic insult.

Topics: Animals; Disease Models, Animal; Free Radicals; Gentisates; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Intestines; Male; Rats; Reperfusion Injury; Salicylates; Salicylic Acid

The formation of hydroxyl radical in the post-ischemic reperfused heart was measured with high performance liquid chromatography and ultraviolet detection using salicylic acid. Hydroxyl radicals react with salicylic acid yielding 2,3- and 2,5-dihydroxybenzoic acid, which can be separated by the liquid chromatography. Isolated rat hearts were perfused with 1 mM salicylic acid and were subjected to 30 mins of global ischemia followed by aerobic or anaerobic reperfusion at 37 degrees C. The effluent from the hearts was collected at various intervals, extracted with ether, and injected into the high performance liquid chromatography unit. 2,5-dihydroxybenzoic acid was present only after aerobic reperfusion and was not detected before ischemia. The liquid chromatography peak of 2,3-dihydroxybenzoic acid was too small for quantitation. The concentration of 2,5-dihydroxybenzoic acid was the highest within 300 s of reperfusion. 2,5-dihydroxybenzoic acid was not detected in the ischemic hearts during anaerobic reperfusion. In ischemic hearts perfused with mannitol, the amount of 2,5-dihydroxybenzoic acid after reperfusion was reduced. These data suggest that hydroxyl radicals are produced in the post-ischemic reperfused heart and that the present method is useful and reliable for the measurement of hydroxyl radicals in the heart.

Topics: Animals; Chromatography, High Pressure Liquid; Gentisates; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; In Vitro Techniques; Ischemia; Male; Myocardial Reperfusion; Oxygen; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid

Experimental evidence suggests that the oxidative metabolites 2,3- and 2,5-dihydroxybenzoic acid (DIOH) may be responsible for the nephrotoxicity of salicylic acid (SAL). In the present study, enzymuria in conjunction with glucose (GLU) and protein (PRO) excretion were used as endpoints to compare the relative nephrotoxicity of SAL with 2,3- and 2,5-DIOH. In addition, the effect of age on enzymuria and GLU and PRO excretion following treatment with SAL or 2,3- and 2,5-DIOH was investigated because the elderly are at greater risk for SAL-induced nephrotoxicity. Three and 12-month male Fischer 344 rats were administered either no treatment, vehicle, SAL, 2,3-DIOH, or 2,5-DIOH at 500 mg/kg p.o. in 5 ml/kg corn oil/DMSO (5:1). Effects of these treatments on functional integrity of renal tissue was assessed from 0--72 h after dosing by measurement of urinary creatinine, GLU, and PRO, as well as excretion of proximal and distal tubular renal enzymes. Enzymes measured as indicators of proximal tubular damage were N-acetyl-beta-glucosaminidase (NAG), gamma glutamyltransferase (GGT), alanine aminotransferase (ALT), and alkaline phosphatase (AP), while urinary lactate dehydrogenase (LD) and aspartate aminotransferase (AST) were measured as indicators of distal tubular damage. In comparison to 3-month vehicle-treated rats, 2,3- and 2,5-DIOH caused a significant increase between 0-8 h in excretion of urinary GLU and activities of AST, NAG, and LD, with peak effects occurring between 4-8 h. Toxic effects of either metabolite were not evident beyond 24 h, and toxicity of 2,5-DIOH was significantly greater in comparison to 2,3-DIOH. SAL treatment resulted in similar effects on enzymuria as well as GLU and PRO excretion, but peak effects did not occur until 16-24 h, and often persisted until 72 h after dosing. Maximal enzymuria in response to SAL treatment was significantly greater in 12- vs. 3-month rats for AST, NAG, and LD. In response to 2,3-DIOH treatment, the maximal response was significantly greater in 12- vs. 3-month rats for LD and AST, and for NAG in response to 2,5-DIOH treatment. The results of this study suggest that both 2,3- and 2,5-DIOH are nephrotoxic metabolites of SAL, but implicate 2,5-DIOH as the more potent nephrotoxic metabolite. The relative lack of an age effect for 2,3- and 2,5-DIOH vs. SAL supports the hypothesis [2] that age-related differences in biotransformation of SAL, and not increased tissue sensitivity to 2,3- or 2,5-DIOH, contribute t

Topics: Acetylglucosaminidase; Administration, Oral; Aging; Alanine Transaminase; Alkaline Phosphatase; Animals; Gentisates; Glycosuria; Hydroxybenzoates; Kidney; Male; Rats; Rats, Inbred F344; Salicylates; Salicylic Acid

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.

Topics: Animals; Cytochrome P-450 Enzyme System; Free Radical Scavengers; Gentisates; Hydroxides; Hydroxybenzoates; Hydroxylation; Lipid Bilayers; Microsomes, Liver; Rabbits; Rats; Salicylates; Salicylic Acid; Substrate Specificity

Administration of salicylates during prodromal viral illness has been associated with the development of Reye's syndrome (RS). We studied salicylate biotransformation in RS patients and compared it with those on chronic salicylate therapy for juvenile rheumatoid arthritis (JRA). Urine of RS patients contained significantly more salicylic acid and less gentisic acid than that of JRA patients while the conjugated metabolites were not different between the two groups. These results suggest decreased salicylate microsomal oxidation in RS. The role of altered salicylate metabolism in the pathogenesis of RS is unclear.

Topics: Arthritis, Juvenile; Biotransformation; Gentisates; Humans; Hydroxybenzoates; Oxidation-Reduction; Reye Syndrome; Salicylates; Salicylic Acid

The yield of 2,3- and 2,5-dihydroxybenzoates (dHB's) from the reaction of .OH radicals with salicylate (SA) ions has been measured as a function of pH and in the presence of oxidants. Under steady-state radiolysis conditions, the production of these products occurs via the reactions .OH + SA----HO-SA. (radical adduct) HO-SA. H+.OH+----2-carboxyphenoxyl radical (SA.) + H2O HO-SA. + SA.----2,3-/2,5-dHB + SA The addition of the oxidants O2, Fe3+ edta, or Fe(CN)63- increases the relative yield of 2,5-dHB/2,3-dHB from about 0.2 to 1. A model to account for this effect is presented. Steady-state radiolyses of 3- and 4-hydroxybenzoate give dihydroxybenzoate products consistent with the phenol group being an ortho-para director in the electrophilic attack of the hydroxyl radical on the aromatic ring. A comparison of product distributions from the reaction of ferrous edta with hydrogen peroxide using salicylate as a scavenger strongly suggests that the same hydroxyl radical adducts are formed as in the radiation experiments.

Topics: Azides; Chemical Phenomena; Chemistry; Edetic Acid; Free Radicals; Gamma Rays; Gentisates; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxybenzoates; Hydroxylation; Iron; Kinetics; Oxidation-Reduction; Salicylates

The simultaneous determination of salicylic acid in binary and/or ternary mixtures and its two main urinary metabolites is proposed. Mixtures of salicylic, salicyluric and gentisic acids are resolved by synchronous spectrofluorimetry, in combination with first-derivative measurements. The urine is extracted with diethyl ether in acid medium. Salicylic and salicyluric acids are re-extracted into glycine-sodium hydroxide buffer solution of pH 11.6 and determined at that pH, and salicylic and gentisic acids are re-extracted into boric acid-sodium hydroxide buffer solution of pH 8.5 and determined at pH 6.

Topics: Gentisates; Hippurates; Humans; Hydrogen-Ion Concentration; Hydroxybenzoates; Salicylates; Salicylic Acid; Spectrometry, Fluorescence

Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.

Topics: Amitrole; Azides; Chromatography, High Pressure Liquid; Cyanides; Gentisates; Glutathione; Humans; Hydroxybenzoates; Hydroxylation; Neutrophils; Peroxidase; Salicylates; Salicylic Acid; Superoxide Dismutase; Tetradecanoylphorbol Acetate

Organophosphorus (OP) pesticides are considered to be environmental contaminants, and chronic exposure to low levels through the diet may affect drug action. To study this possible interaction, ethyl parathion was administered by intubation to female rats for 35 consecutive days at a dose of 0.05 or 0.2 mg/kg of body weight per day. At 7, 21 and 35 days after parathion was initiated, rats were administered a single dose of 20 mg/kg sodium salicylate intraperitoneally. Total salicylates, salicylic acid (SA), salicyluric acid (SU) and gentisic acid (GA) were determined in urine. At 7 days, parathion treatment slowed the excretion of total salicylates. This effect was more evident at longer treatment times. Total excretion of SA was increased at the expense of GA at 7 days. However, this effect was reversed at 21 and 35 days. Excretion of SU was drastically diminished after 21 days of treatment with parathion. The results suggest that subchronic oral administration of parathion to female rats changes the excretion kinetics of sodium salicylate through combined effects on renal excretion mechanisms and biotransformation processes. Thus, exposure to low concentrations of environmental contaminants may produce important changes in drug action.

Topics: Animals; Drug Interactions; Female; Gentisates; Hippurates; Hydroxybenzoates; Kinetics; Parathion; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid; Sodium Salicylate; Time Factors

It has been postulated that oxygen radicals may play a role in the pathogenesis of inflammatory bowel disease. If so, then a drug like 5-aminosalicylate (5-ASA), which is used to treat such diseases, might work by interacting with oxygen-derived species. We found that activated mononuclear cells and activated granulocytes, as well as the products of the Fenton reaction, transformed [14C]5-ASA to a number of metabolites, among which we have characterized salicylate and gentisate. We also found that the lethal effect on cultured Chinese hamster ovary cells of adding either superoxide radical or hydrogen peroxide, components of the respiratory burst of activated white blood cells, was diminished by the addition of 100 micrograms/ml (0.65 mM) of 5-ASA. Thus, we have demonstrated that 5-ASA was oxidized by the oxidative burst of white blood cells and that 5-ASA protected cells from damage by oxygen-derived species, two findings which may offer an explanation for the role of 5-ASA in the treatment of inflammatory bowel disease.

Topics: Aminosalicylic Acids; Animals; Cell Line; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Female; Free Radicals; Gentisates; Humans; Hydrogen Peroxide; Hydroxybenzoates; Leukocytes; Lymphocyte Activation; Mesalamine; Ovary; Oxidation-Reduction; Salicylates; Salicylic Acid; Superoxides

The profile of urinary salicylate metabolites was determined after the oral administration of acetylsalicylic acid (ASA) to CCl4-cirrhotic rats, CCl4-cirrhotic rats treated with colchicine for 1 month, and control groups. The following enzymatic activities were determined: liver and plasma ASA-esterase, liver UDP-glucuronyltransferase, and liver aniline hydroxylase. The time-course of plasma concentration of salicylates in similar groups was followed after the intraperitoneal administration of salicylic acid (SA) or gentisic acid (GA). The cirrhotic animals showed a lack of urinary glucuronates and an increase in urinary gentisic and salicylic acids. The activities of plasma and liver ASA-esterases were increased significantly in cirrhosis, whereas aniline hydroxylase was reduced and UDP-glucuronyltransferase remained unchanged. The plasma half-lives of salicylates were reduced in the cirrhotic animals regardless of the administered parent compound. Colchicine treatment reversed almost completely the alterations. The heterogeneity of liver metabolic dysfunctions present in chronic liver disease was demonstrated. It is emphasized that the pharmacokinetic alterations produced by liver damage are the result of a complex set of factors involving changes in the hepatic circulation, protein binding, and the existence of other routes of elimination.

Topics: Aniline Hydroxylase; Animals; Aspirin; Carbon Tetrachloride; Carboxylic Ester Hydrolases; Colchicine; Gentisates; Glucuronates; Glucuronosyltransferase; Hydroxybenzoates; Kinetics; Liver; Liver Cirrhosis, Experimental; Male; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid

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 induces OH in tissue and indicates that the LCED method may prove to be useful to measure oxygen free radical production in vivo.

Topics: Animals; Chromatography, High Pressure Liquid; Doxorubicin; Electrochemistry; Female; Free Radicals; Gentisates; Heart; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Rats; Rats, Inbred F344; Salicylates; Salicylic Acid

Attack by .OH radicals, generated by a Fenton system, upon salicylate produces 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate as major products and catechol as a minor product. H.p.l.c. separation combined with electrochemical detection was used to identify and quantify 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate in human plasma and synovial fluid. We propose that conversion of salicylate into 2,3-dihydroxybenzoate, or of other aromatic compounds into specific hydroxylated products, may be a useful assay for .OH formation in the human body.

Topics: Adult; Chromatography, High Pressure Liquid; Electrochemistry; Free Radicals; Gentisates; Hippurates; Humans; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Male; Salicylates; Salicylic Acid; Synovial Fluid

Mitochondria are known to contain a P-450 like system similar to that found in microsomes. Since previous in vivo studies from this laboratory have suggested that renal mitochondria may metabolize salicylate (SAL) to a reactive intermediate capable of protein binding, the ability of isolated kidney and liver mitochondria to activate salicylate was investigated. Renal mitochondria were 4 times more active than liver in converting SAL to a reactive intermediate and metabolized approx. 1% of the SAL to 2,3-dihydroxybenzoic acid, the catechol analogue of SAL. The formation of 2,3-dihydroxybenzoate (2,3-DHBA) and the amount of radiolabel bound to mitochondrial protein was decreased in the presence of SKF 525-A; however, excess unlabeled metabolite had no effect on binding. These data indicate that kidney mitochondria activate SAL via a cytochrome P-450 like system, but suggest that the binding species is not 2,3-DHBA itself. Oxidation of SAL and covalent binding of radiolabel, however, were also observed after the addition of ferrous iron and ascorbic acid to a model system containing [14C]SAL and bovine serum albumin. Mannitol decreased SAL oxidation and covalent binding, suggesting radical formation may represent a non-enzymatic mechanism for SAL activation.

Topics: Animals; Ascorbic Acid; Chromatography, Thin Layer; Ferrous Compounds; Gentisates; Hydroxybenzoates; In Vitro Techniques; Kidney; Male; Mannitol; Mitochondria, Liver; Proadifen; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid

Topics: Gentisates; Hippurates; Humans; Hydroxybenzoates; Salicylates; Statistics as Topic; Synovial Fluid

Indirect measurement of salicylphenolic glucuronide (SPG) has suggested that the formation of this metabolite from therapeutic doses of salicyclic acid (SA) is capacity-limited in humans. A direct high performance liquid chromatographic (HPLC) assay for SPG in human urine is described. SPG was prepared by a published method and purified by HPLC. On treatment with beta-glucuronidase, SPG yielded the expected amount of SA. Spectroscopic data, melting point, and optical rotation of the glucuronide and/or its triacetyl dimethyl ester derivative were consistent with the proposed structure. SPG was assayed using a 5-micron C18 column (temperature 55 degrees C) and fluorescence detection. A nonlinear gradient mobile phase at a flow rate of 2 ml/min was used, beginning with 100% 0.1 M pH 2.1 phosphate buffer and finishing with 84% buffer, 16% acetonitrile. Total run time was 25 min. Urine (10 microliter) was injected directly on the column, and quantitation was performed using urine standards. Within-run precision for SPG ranged from 1.2% at 150 mg/L to 2.4% at 5 mg/L. The limit of detection was less than 1 mg/L. A pilot study in two volunteers, each receiving a single 500-mg dose of sodium salicylate, was carried out to validate the usefulness of the assay.

Topics: Adult; Chromatography, High Pressure Liquid; Gentisates; Glucuronates; Hippurates; Humans; Hydrogen-Ion Concentration; Hydroxybenzoates; Kinetics; Magnetic Resonance Spectroscopy; Salicylates; Salicylic Acid

In vitro salicylates /aspirin, salicylic acid, salicylamide and gentisic acid/ inhibited formation of 12-lipoxygenase products in intact human washed platelets which were stimulated with thrombin or arachidonic acid. Salicylates did not affect 12-lipoxygenase activity in platelet lysates. Ex vivo aspirin or salicylamide at a dose of 1 g given orally to healthy volunteers potentiated formation of 12-lipoxygenase products in washed platelets. It is concluded that the effect of salicylates on 12-lipoxygenase pathway is independent from their influence on cyclooxygenase activity in platelets and aspirin cannot be considered as a selective inhibitor of platelet cyclooxygenase.

Topics: Adult; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Aspirin; Blood Platelets; Chromatography, High Pressure Liquid; Gentisates; Humans; Hydroxybenzoates; In Vitro Techniques; Lipoxygenase Inhibitors; Salicylamides; Salicylates; Salicylic Acid

Rats and mice were fed a diet containing aspirin at levels of 0, 0.3, 0.6 and 1.2% for 1 and 4 weeks. Haemorrhagic death and/or haemorrhagic anaemia occurred in rats in a dose-dependent manner. Prothrombin and kaolin-activated partial thromboplastin time indices were also decreased depending on the daily doses. However, no conspicuous haemorrhagic signs were found in mice given aspirin. These results suggest marked differences in haemorrhagic effects of aspirin between rats and mice. From results of supplementary experiments with two metabolites of aspirin, salicylic acid and gentisic acid, and from the fact of close relationship between hepatic concentration of salicylic acid and haemorrhagic effects of aspirin, it is inferred that salicylic acid may be a precursor for the active metabolite(s) to cause haemorrhage. The mechanism of species differences of aspirin is discussed.

Topics: Animals; Aspirin; Dose-Response Relationship, Drug; Gastrointestinal Hemorrhage; Gentisates; Hydroxybenzoates; Kaolin; Liver; Male; Mice; Mice, Inbred ICR; Organ Size; Partial Thromboplastin Time; Prothrombin Time; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid; Species Specificity; Time Factors

Equilibration of salicylic, salicyluric and gentisic acids between plasma and synovial fluid (SF) was measured in 36 patients receiving chronic salicylate therapy and from whom SF was required for diagnostic purposes. Gentisic and salicyluric acids equilibrated completely, while SF salicylic acid concentration was less than that in plasma. The presence of significant gentisic acid concentrations in SF could contribute to the therapeutic response to chronic salicylate therapy, since its antiinflammatory effect is even greater than that of acetylsalicylic acid.

Topics: Adult; Aged; Arthritis; Aspirin; Female; Gentisates; Hippurates; Humans; Hydroxybenzoates; Male; Middle Aged; Salicylates; Salicylic Acid; Synovial Fluid

The in vitro effects of ascorbic acid, acetaminophen, salicylic acid, and gentisic acid on home blood-glucose-measurement systems were studied. Whole blood in the normoglycemic range was spiked with quantities of each study drug at low, moderate, and high therapeutic concentrations. The glucose concentration of the blood was measured using Chemstrip bG, Dextrostix, and Visidex II home blood-glucose-monitoring systems. Serum samples were also measured by two automated systems, a glucose-oxidase method and a hexokinase method. Mean values were compared within the same glucose-measurement system to determine the extent of drug interference, and to values obtained by other systems to determine the reliability among systems. Changes between the control and treatment glucose mean concentrations of 20% or more were considered clinically important. Drug interference was observed with all three home blood-glucose-measurement systems and was drug-concentration dependent. Both automated systems were associated with drug interference at the highest concentration of salicylic acid, and the hexokinase method was influenced at the highest concentration of gentisic acid. No clinically important differences were observed between the automated systems; however, differences were observed between the automated and home blood-glucose-monitoring systems. Salicylic acid, acetaminophen, and ascorbic acid interfere with home blood-glucose-measurement systems. Switching between home blood-glucose-measurement systems could result in a poor assessment of blood-glucose values. In general, the values determined by home and automated systems should not be compared clinically.

Topics: Acetaminophen; Ascorbic Acid; Blood Glucose; Drug Interactions; Gentisates; Glucose Oxidase; Humans; Hydroxybenzoates; Indicators and Reagents; Monitoring, Physiologic; Reagent Kits, Diagnostic; Salicylates; Salicylic Acid

The inadequacy of single, simple arithmetic corrections for salicylate interference in the widely employed Glynn and Kendal technique for plasma paracetamol assay is highlighted by reference to an actual case of combined salicylate/paracetamol intoxication in an infant. Attention is drawn for the first time to the not insubstantial contribution to such interference made by even the minor metabolites of salicylate. The conclusion is reached that is necessary, particularly in the assessment of paracetamol toxicity, to determine the presence of salicylate, and when present, to employ a specific method for the estimation of paracetamol.

Topics: Acetaminophen; Aspirin; Biotransformation; Chromatography, High Pressure Liquid; Gentisates; Humans; Hydroxybenzoates; Infant; Male; Salicylates; Salicylic Acid

An improved method has been developed for the determination of acetylsalicylic acid, salicylic acid, gentisic acid, and salicyluric acid in plasma and urine of rabbits and man. Samples are extracted with dichloromethane containing mephenytoin as an internal standard, the solvent is evaporated under reduced pressure, the residue reconstituted and analyzed by high-performance liquid chromatography. Extraction efficiencies, linearity and assay precision were determined. This method has been applied to human bioavailability studies and the data are presented.

Topics: Animals; Aspirin; Biological Availability; Chromatography, High Pressure Liquid; Gentisates; Hippurates; Humans; Hydroxybenzoates; Kinetics; Rabbits; Rats; Salicylates; Salicylic Acid; Species Specificity

Salicylate availability from salsalate (SSA) and aspirin (ASA) was examined in six rheumatoid arthritis patients in a multiple-dose double-blind crossover study. Doses contained equimolar amounts of salicylic acid. After initial ASA treatment to achieve therapeutic salicylate levels (150 to 300 micrograms/ml) the patients received equimolar doses of SSA or ASA. When steady state was achieved patients were hospitalized, and blood and urine specimens were obtained during three dosing intervals and during the washout period that followed. Thereafter, patients were placed on the alternate medication for at least a week and the in-hospital pattern was repeated. Despite insignificant differences in absorption of the formulations, as measured by urinary salicylate recovery, the plasma salicylic acid AUC was lower after SSA. Evidence indicates that this apparent lower availability of salicylate from SSA is due to incomplete hydrolysis to salicylic acid, the unhydrolyzed SSA being excreted mainly as glucuronide conjugates.

Topics: Arthritis, Rheumatoid; Aspirin; Biological Availability; Chromatography, High Pressure Liquid; Double-Blind Method; Drug Evaluation; Female; Gentisates; Humans; Hydroxybenzoates; Male; Salicylates; Salicylic Acid

Salicylic acid, beta-resorcylic acid and gentisic acid were acetylated and then reacted with N-hydroxysuccinimide according to the DDC procedure to give the corresponding activated esters. The reaction of these N-hydroxysuccinimidic esters with human serum albumin and bovine gamma globulin yielded modified proteins containing hydroxybenzoyl residues linked to amino groups, the mode of linkage being of the acid amide type.

Topics: Acetylation; Animals; Antigens; Cattle; gamma-Globulins; Gentisates; Humans; Hydroxybenzoates; Phenols; Salicylates; Salicylic Acid; Serum Albumin; Succinimides

Topics: Animals; Aspirin; Fever; Gentisates; Salicylates; Salicylic Acid

Topics: Aspirin; Gentisates; Hippurates; Salicylates; Uric Acid

Topics: Blood Pressure; Blood Pressure Determination; Gentisates; Pressure; Respiration; Salicylates

Topics: Anthelmintics; Autonomic Nervous System; Gentisates; Research; Salicylates

Topics: Aminosalicylic Acid; Anesthesia; Anesthesiology; Animals; Bile; Chloralose; Cholagogues and Choleretics; Diuresis; Diuretics; Dogs; Gentisates; Salicylates; Salicylic Acid; Salts; Sodium

Topics: Antipyretics; Antipyrine; Aspirin; Gentisates; Salicylates