Catabolism of aromatic acids in Trichosporon cutaneum.

Trichosporon cutaneum readily metabolized protocatechuate, homoprotocatechuate, and gentisate, but lacked ring fission dioxygenases for these compounds. Benzoic, salicylic, 2,3-dihydroxybenzoic, and gentisic acids were converted into beta-ketoadipic acid before entry into the Krebs cycle. Benzoic acid gave rise successively to 4-hydroxybenzoic acid, protocatechuic acid, and hydroxyquinol (1,3,4-trihydroxybenzene), which underwent ring fission to maleylacetic acid. Salicylate and 2,3-dihydroxybenzoate were both initially metabolized to give catechol. 2,3-Dihydroxybenzoate was the substrate for a specific nonoxidative decarboxylase induced by salicylate, although 2,3-dihydroxybenzoate was not a catabolite of salicylate. Gentisate was metabolized to maleylacetic acid and was also readily attacked by salicylate hydroxylase at each stage of a partial purification procedure. Phenylacetic acid was degraded through 3-hydroxyphenylacetic, homogentisic, and maleylacetoacetic acids to acetoacetic and fumaric acids. All the reactions of these catabolic sequences were catalyzed by cell extracts, supplemented with reduced pyridine nucleotide coenzymes where necessary, except for the hydroxylations of benzoic and phenylacetic acids which were demonstrated with cell suspensions and isotopically labeled substrates.     

The hydroxylation of the salicylate anion by a Fenton reaction and T-radiolysis: a consideration of the respective mechanisms

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.     

Determination of hydroxyl free radical formation in human platelets using high-performance liquid chromatography with electrochemical detection

The formation of the hydroxyl free radical (HFR) can be quantified indirectly, by measuring two products of the hydroxylation of salicylic acid, 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB). In this study, we used reversed-phase high-performance liquid chromatography with electrochemical (coulometric) detection to measure 2,3- and 2,5-DHB levels in human platelets. The limits of detection of the method were 10 and 5 fmol on column for 2,3-DHB and 2,5-DHB, respectively. We tested the technique by measuring increases in dihydroxybenzoate levels after exposure of platelets to experimentally induced oxidative stress. Then, we measured platelet levels of 2,3- and 2,5-DHB in patients with Parkinson’s disease, under therapy with l-DOPA, and in normal subjects. We also measured platelet concentrations of l-DOPA and its major metabolite, 3-O-methyldopa (3-OMD). Parkinsonian patients showed increased levels of both 2,3- and 2,5-DHB. Platelet levels of 2,3-DHB were positively correlated with platelet levels of l-DOPA and 3-OMD. The technique we describe proved simple and extremely sensitive and may represent a useful tool for the study of oxidative stress in humans.     

Plasmid-borne Tn5 insertion mutation resulting in accumulation of gentisate from salicylate.

Plasmid-borne Tn5 insertion mutants of a Pseudomonas species which accumulated 2,5-dihydroxybenzoate (gentisate) following growth on 2-hydroxybenzoate (salicylate) were obtained from a pool of mutants that were unable to grow on naphthalene. One such mutant was characterized further. The ability of this mutant to oxidize gentisate was 100-fold less than the ability of a Nah+ Sal+ strain harboring the unmutagenized plasmid, although both strains oxidized and grew on salicylate. These bacteria were presumably able to metabolize salicylate via catechol, since they possessed an inducible, plasmid-encoded catechol 2,3-dioxygenase. Our results suggest that there is an alternate, plasmid-encoded route of salicylate degradation via gentisate and that some plasmid-associated relationship between this pathway and naphthalene oxidation exists.     

Separation of dihydroxybenzoates,indicators of in-vivo hydroxyl free radical formation,in the presence of transmitter amines and some metabolites in rodent brain,using high-performance liquid chromatography with electrochemical detection

Based on a detailed study of retention parameters, reversed-phase ion-pair chromatographic methods were developed for the simultaneous determination of dihydrocybenzoates, indicators of in-vivo hydroxyl free radical formation, transmitter amines and some metabolites to facilitate neurochemical investigations in rodent brain. Coupling of the separation methods with electrochemical detection and the use of short-chain perfluorinated carboxylic acids for ion-pairing, allowed for a fast and sensitive determination of salicylate-derived 2,3- and 2,5-dihydroxybenzoic acids and the major electroactive, hydroxylated aromatic compounds present in brain samples. Detection limits for the dihydroxybenzoates (signal-to-noise ratio = 2) were 18–22 fmol injected on the column. Basal levels of 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate in the striatum of mice treated with salicylate were 72±13 and 94±11 ng/g wet tissue, respectively.     

Use of salicylate as a probe for .OH formation in isolated ischemic rat hearts

Salicylic acid was used as a probe for .OH formed during reperfusion of the ischemic myocardium. .OH adds to the phenolic ring of salicylate to yield dihydroxybenzoic acid species. The two principal dihydroxybenzoic acids formed are the 2,3- and 2,5-derivatives and can be isolated and quantitated using HPLC combined with electrochemical detection. In these experiments, dihydroxybenzoic acids were detectable in the f molar range. Rat hearts were perfused in the Langendorff mode with Krebs-Henseleit buffer containing 100 microM salicylate. Following 20 min of global ischemia a 173% increase in tissue content of 2,5-dihydroxybenzoic acid was detected after 2.5 min of reperfusion. The duration of ischemia did not significantly affect tissue content of 2,5-dihydroxybenzoic acid peaked at 250 to 300% of control within 2.5 min of reperfusion. The inclusion of 100 microM salicylate in the perfusion buffer had no effect on myocardial function during the duration of the experiments. The results indicate that salicylate can be used as a very sensitive probe for .OH in the isolated ischemic heart.     

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

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

Plasmid-borne Tn5 insertion mutation resulting in accumulation of gentisate from salicylate

Plasmid-borne Tn5 insertion mutants of a Pseudomonas species which accumulated 2,5-dihydroxybenzoate (gentisate) following growth on 2-hydroxybenzoate (salicylate) were obtained from a pool of mutants that were unable to grow on naphthalene. One such mutant was characterized further. The ability of this mutant to oxidize gentisate was 100-fold less than the ability of a Nah+ Sal+ strain harboring the unmutagenized plasmid, although both strains oxidized and grew on salicylate. These bacteria were presumably able to metabolize salicylate via catechol, since they possessed an inducible, plasmid-encoded catechol 2,3-dioxygenase. Our results suggest that there is an alternate, plasmid-encoded route of salicylate degradation via gentisate and that some plasmid-associated relationship between this pathway and naphthalene oxidation exists.     

Production of hydroxyl free radical by brain tissues in hyperglycemic rats subjected to transient forebrain ischemia

Preischemic hyperglycemia is known to aggravate brain damage resulting from transient ischemia. In the present study, we explored whether this aggravation is preceded by an enhanced formation of reactive oxygen species (ROS) during the early reperfusion period. To that end, normo- and hyperglycemic rats were subjected to 15 min of forebrain ischemia and allowed recovery periods of 5, 15, and 60 min. Sodium salicylate was injected intraperitoneally in a dose of 100 mg/kg, and tissues were sampled during recirculation to allow analyses of salicylic acid (SA) and its hydroxylation products, 2,3- and 2,5-dihydroxybenzoate (DHBA). Tissue sampled from thalamus and caudoputamen in normoglycemic animals failed to show an increase in 2,3- or 2,5-DHBA after 5 and 15 min of recirculation. However, such an increase was observed in the neocortex after 60 min of recirculation, with a suggested increase in the hippocampus as well. Hyperglycemia had three effects. First, it increased 2,5-DHBA in the thalamus and caudoputamen to values exceeding normoglycemic ones after 15 min of recirculation. Second, it increased basal values of 2,5- and total DHBA in the neocortex. Third, it increased the 60-min values for 2,5- and total DHBA in the hippocampus. These results hint that, at least in part, hyperglycemia may aggravate damage by enhancing basal- and ischemia-triggered production of ROS.     

Catabolism of tryptophan, anthranilate, and 2,3-dihydroxybenzoate in Trichosporon cutaneum.

Trichosporon cutaneum degraded L-tryptophan by a reaction sequence that included L-kynurenine, anthranilate, 2,3-dihydroxybenzoate, catechol, and beta-ketoadipate as catabolites. All of the enzymes of the sequence were induced by both L-tryptophan and salicylate, and those for oxidizing kynurenine and its catabolites were induced by anthranilate but not by benzoate; induction was not coordinate. Molecular weights of 66,100 and 36,500 were determined, respectively, for purified 2,3-dihydroxybenzoate decarboxylase and its single subunit. Substrates for this enzyme were restricted to benzoic acids substituted with hydroxyl groups at C-2 and C-3; no added coenzyme was required for activity. Partially purified anthranilate hydroxylase (deaminating) catalyzed the incorporation of one atom of 18O, derived from either 18O2 or H2(18)O, into 2,3-dihydroxybenzoic acid.     

Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients.

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.     

比色法测定Fenton反应产生的羟自由基

Fenton反应产生的羟自由基能与水杨酸生成羟基化产物2,3－二羟基苯甲酸,用比色法测定其含量能间接测定羟自由基的生成量．通过对测定条件的研究,得到最佳的测定方案．可作为一种简便的筛选羟自由基清除剂的方法     

Commentary Salicylate Trapping of -OH as a Tool for Studying Post-Ischemic Oxidative Injury in the Isolated Rat Heart

The use of salicylate as a chemical trap for -OH represents a simple and convenient alternative to the use of spin trapping techniques to study oxidative injury in isolated perfused organs. In these systems, salicylate is included in the perfusion buffer at concentrations ranging from 0.1 to 2mM depending on the detection apparatus employed. In our studies, we have used a coulometric detector, which has a theoretical efficiency of 100% as compared to 1-5% for the standard glassy carbon electrode. We have been able to generate reproducible results by inclusion of only 100 μM salicylate, a concentration demonstrated not to affect pre- or post-ischemic cardiac function. In initial studies, we observed an increase in perfusate 2,5-dihydroxybenzoic acid consistent with an early post-ischemic burst of -OH, not unlike that reported using spin trapping techniques. Since then we and others have used this technique to examine possible relationships between -OH formation and treatments that alter post-ischemic cardiac functional recovery. For example, preischemic loading of hearts with copper results in increases in postischemic dysfunction and LDH release that were associated with an increase in 2,5-dihydroxybenzoate and by inference, -OH formation. Alternatively, we have reported that the nitroxide spin label, TEMPO, reputed to be a superoxide dismutase mimetic, decreased post-ischemic arrhythmias and 2,5-dihydroxybenzoate formation. Most recently, we have observed that preischemic loading of hearts with zinc-bis-histidinate results in improved post-ischemic cardiac function and decreased LDH release; changes that were associated with decreased 2,5-dihydroxybenzoate formation. These studies indicate that under certain conditions, salicylate is a valuable alternative to spin trapping techniques to probe the role of -OH in cardiac oxidative injury, particularly when applied to the isolated perfused heart preparation.     

Involvement of Oxygen Free Radicals in Ischaemia-Reperfusion Injury to Murine Turnours: Role of Nitric Oxide

Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO*). in vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH*) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO*) and normal skin. Significantly greater OH* generation followed I/R of murine adenocarcinoma CaNT tumours (low NO* production) compared to round cell sarcoma SaS tumours (high NO* production) and normal skin. These data suggest that tumour production of NO* confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH* generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO*. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.     

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

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

Measurement of loosely-bound iron in brain regions using redox cycling and salicylate.

A sensitive iron assay was developed for measuring non-heme and loosely bound iron in regions of rat brain. The method is based on the salicylate trapping of hydroxyl radicals generated from ascorbate-driven redox cycling of Fe3+-EDTA. This assay has high sensitivity (about 20 nM) because of amplification obtained with redox-cycling and fluorescent detection of the salicylate hydroxylation product, 2,5-dihydroxybenzoate. The assay detects iron as Fe2+ and Fe3+ combined. Values of non-heme and loosely bound iron are given for three areas of cortex, caudate, hippocampus, thalamus and brainstem of the rat brain.     

Ischemic preconditioning decreases the reperfusion-related formation of hydroxyl radicals in a rabbit model of regional myocardial ischemia and reperfusion: The role of KATP channels

The objective of this study was to assess the effects of ischemic preconditioning (IP) on hydroxyl free radical production in an in vivo rabbit model of regional ischemia and reperfusion. Another goal was to determine whether K_ATP channels are involved in these effects.

The hearts of anesthetized and mechanically ventilated New Zealand White rabbits were exposed through a left thoracotomy. After IV salicylate (100?mg/kg) administration, all animals underwent a 30-min stabilization period followed by 40?min of regional ischemia and 2?h of reperfusion. In the IP group, IP was elicited by 5?min of ischemia followed by 10?min of reperfusion (prior to the 40-min ischemia period). Glibenclamide, a K_ATP channel blocker, was administered prior to the preconditioning stimulus. Infarct size was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We quantified the hydroxyl-mediated conversion of salicylate to its 2,3 and 2,5-dihydroxybenzoate derivatives during reperfusion by high performance liquid chromatography coupled with electro-chemical detection.

IP was evidenced by reduced infarct size compared to control animals: 22% vs. 58%, respectively. Glibenclamide inhibited this cardioprotective effect and infarct size was 53%. IP limited the increase in 2,3 and 2,5-dihydroxybenzoic acid to 24.3 and 23.8% above baseline, respectively. Glibenclamide abrogated this effect and the increase in 2,3 and 2,5-dihydroxybenzoic acid was 94.3 and 85% above baseline levels, respectively, similar to the increase in the control group. We demonstrated that IP decreased the formation of hydroxyl radicals during reperfusion. The fact that glibenclamide inhibited this effect, indicates that K_ATP channels play a key role in this cardioprotective effect of IP.     

Ischemic preconditioning decreases the reperfusion-related formation of hydroxyl radicals in a rabbit model of regional myocardial ischemia and reperfusion: the role of K(ATP) channels

The objective of this study was to assess the effects of ischemic preconditioning (IP) on hydroxyl free radical production in an in vivo rabbit model of regional ischemia and reperfusion. Another goal was to determine whether K_ATP channels are involved in these effects.

The hearts of anesthetized and mechanically ventilated New Zealand White rabbits were exposed through a left thoracotomy. After IV salicylate (100 mg/kg) administration, all animals underwent a 30-min stabilization period followed by 40 min of regional ischemia and 2 h of reperfusion. In the IP group, IP was elicited by 5 min of ischemia followed by 10 min of reperfusion (prior to the 40-min ischemia period). Glibenclamide, a K_ATP channel blocker, was administered prior to the preconditioning stimulus. Infarct size was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We quantified the hydroxyl-mediated conversion of salicylate to its 2,3 and 2,5-dihydroxybenzoate derivatives during reperfusion by high performance liquid chromatography coupled with electro-chemical detection.

IP was evidenced by reduced infarct size compared to control animals: 22% vs. 58%, respectively. Glibenclamide inhibited this cardioprotective effect and infarct size was 53%. IP limited the increase in 2,3 and 2,5-dihydroxybenzoic acid to 24.3 and 23.8% above baseline, respectively. Glibenclamide abrogated this effect and the increase in 2,3 and 2,5-dihydroxybenzoic acid was 94.3 and 85% above baseline levels, respectively, similar to the increase in the control group. We demonstrated that IP decreased the formation of hydroxyl radicals during reperfusion. The fact that glibenclamide inhibited this effect, indicates that K_ATP channels play a key role in this cardioprotective effect of IP.     

Biological Reactions of Peroxynitrite: Evidence for an Alternative Pathway of Salicylate Hydroxylation

Salicylate hydroxylation has often been used as an assay of hydroxyl radical production in vivo. We have examined here if hydroxylation of salicylate might also occur by its reaction with peroxynitrite. To test this hypothesis, we exposed salicylate to various concentrations of peroxynitrite, in vitro. We observed the hydroxylation of salicylate at 37°C by peroxynitrite at pH 6, 7 and 7.5, where the primary products had similar retention times on HPLC to 2,3- and 2,5-dihydroxy-benzoic acid. The product yields were pH dependent with maximal amounts formed at pH 6. Furthermore, the relative concentration of 2,3- to 2,5-dihydroxyben-zoic acid increased with decreasing pH. Nitration of salicylate was also observed and both nitration and hydroxylation reaction products were confirmed independently by mass spectrometry. The spin trap N-t-butyl-a-phenylnitrone (PBN), with or without dimethyl sulfoxide (DMSO), was incapable of trapping the peroxynitrite decomposition intermediates. Moreover, free radical adducts of the type PBN/'CH₃ and PBN/ 'OH were susceptible to destruction by peroxynitrite (pH 7, 0.1 M phosphate buffer). These results suggest direct peroxynitrite hydroxylation of salicylate and that the presence of hydroxyl radicals is not a prerequisite for hydroxylation reactions.     

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

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