The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.

Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

On the application of 4-hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion

Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.     

The hydroxylation of phenylalanine and tyrosine: a comparison with salicylate and tryptophan.

The hydroxylation of phenylalanine by the Fenton reaction and gamma-radiolysis yields 2-hydroxy-, 3-hydroxy-, and 4-hydroxyphenylalanine (tyrosine), while the hydroxylation of tyrosine results in 2,3- and 3,4-dihydroxyphenylalanine (dopa). Yields are determined as a function of pH and the presence or absence of oxidants. During gamma-radiolysis and the Fenton reaction the same hydroxylated products are formed. The final product distribution depends on the rate of the oxidation of the hydroxyl radical adducts (hydroxycyclohexadiene radicals) relative to the competing dimerization reactions. The pH profiles for the hydroxylations of phenylalanine and tyrosine show a maximum at pH 5.5 and a minimum around pH 8. The lack of hydroxylated products around near pH 8 is due to the rapid oxidation of dopa to melanin. The relative abilities of iron chelates (HLFe(II) and HLFe(III) to promote hydroxyl radical formation from hydrogen peroxide are nitrilotriacetate (nta) greater than ethylenediaminediacetate (edda) much greater than hydroxyethylethylenediaminetriacetate greater than citrate greater than ethylenediaminetetraacetate greater than diethylenetriaminepentaacetate greater than adenosine 5'-triphosphate greater than pyrophosphate greater than adenosine 5'-diphosphate greater than adenosine 5'-monophosphate. The high activity of iron-nta and -edda chelates is explained by postulating the formation of a ternary Fe(III)-L-dopa complex in which dopa reduces Fe(III). The hydroxylations of phenylalanine and tyrosine are similar to that of salicylate (Z. Maskos, J. D. Rush, and W. H. Koppenol, 1990, Free Radical Biol. Med. 8, 153-162) and tryptophan (preceding paper) in that oxidants augment the formation of hydroxylated products by catalyzing the dismutation of hydroxyl radical adducts to the parent compound and a stable hydroxylated product. A comparison of salicylate and the amino acids tryptophan, phenylalanine, and tyrosine clearly shows that salicylate is the best indicator of hydroxyl radical production.     

On the application of 4-hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion

Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.     

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

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

Hydroxylation of Salicylate and Phenylalanine as Assays for Hydroxyl Radicals: a Cautionary Note Visited for the Third Time

Hydroxylation of salicylate to2, 3- and2, 5-dihydroxy-benzoates (DHBs) is widely used as an index of hydroxyl radical (OH) formation in vivo and in vitro. Several recent studies indicate that peroxynitrite can lead to generation of DHBs from salicylate and it is uncertain as to whether or not OH' is involved. A similar problem may occur in the use of phenylalanine as an OH' detector. Hence formation of hydroxylation products from salicylate (or phenylalanine) may not in itself be a definitive index of OH' generation, especially in cases where such generation in physiological systems is decreased by inhibitors of nitric oxide syn-thase. Determination of salicylate (or phenylalanine) nitration products can allow distinction between peroxynitrite-dependent aromatic hydroxylation and that involving “real” OH.     

Comparison of salicylate and D-phenylalanine for detection of hydroxyl radicals in chemical and biological reactions

Summary

Hydroxylation of salicylate and D-phenylalanine was measured to test the usefulness of these compounds for hydroxyl radical (HO^?) detection in chemical and biological systems. When HO^? were produced by the photolytic decomposition of hydrogen peroxide, nearly equal amounts of 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were produced from salicylate, with catechol as a minor product. In the photolytic reaction, nearly equal concentrations of p-,m-, and o-tyrosine were formed from D-phenylalanine. When salicylate or D-phenylalanine was present with Fenton reagents or in iron(II) autoxidation systems, the relative proportions of hydroxylated products were similar to those observed after photolysis, although less total products were usually detected. In contrast, when similar experiments were conducted with isolated hepatic microsomes and perfused livers, 2,5-DHBA was the primary product from salicylate, and p-tyrosine was the major product from D-phenylalanine. Cytochrome P-450 enzymes can hydroxylate salicylate to produce 2,5-DHBA, and it is likely that phenylalanine hydroxylase produces most of the p-tyrosine detected in hepatic tissues. Thus, although both salicylate and D-phenylalanine are useful probes for hydroxyl radical formation in chemical systems, hydroxylated products formed from enzymatic reactions complicate interpretation of data from both compounds in vivo.     

Peroxynitrite-Dependent Aromatic Hydroxylation and Nitration of Salicylate and Phenylalanine. Is Hydroxyl Radical Involved?

There is considerable dispute about whether the hydroxylating ability of peroxynitrite (ONOO^-)-derived species involves hydroxyl radicals (OH*). This was investigated by using salicylate and phenylalanine, attack of OH* upon which leads to the formation of 2, 3- and 2, 5-dihydroxybenzoates, and o-, m- and p-tyrosines respectively. On addition of ONOO- to salicylate, characteristic products of hydroxylation (and nitration) were observed in decreasing amounts with rise in pH, although added products of hydroxylation of salicylate were not recovered quantitatively at pH 8.5, suggesting further oxidation of these products and underestimation of hydroxylation at alkaline pH. Hydroxylation products decreased in the presence of several OH* scavengers, especially formate, to extents similar to those obtained when hydroxylation was achieved by a mixture of iron salts, H₂O₂ and ascorbate. However, OH* scavengers also inhibited formation of salicylate nitration products. Ortho, p- and m-tyrosines as well as nitration products were also observed when ONOO^- was added to phenylalanine. The amounts of these products again decreased at high pH and were decreased by addition of OH* scavengers. We conclude that although comparison with Fenton systems suggests OH* formation, simple homolytic fission of peroxynitrous acid (ONOOH) to OH* and NO₂ would not explain why OH* scavengers inhibit formation of nitration products.     

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA²⁻) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o- phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA²⁻ in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ' ex vivo' showed TA²⁻ to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe²⁺ + H₂O₂) indicate a mechanism whereby TA²⁻ forms a primary complex with Fe²⁺ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.     

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.     

Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species

We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (*OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite ((-)OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and superoxide (O2*-) by using HPF or APF alone, but (-)OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluorescein, and for the first time we could visualize (-)OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and (-)OCl in many biological and chemical applications.     

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.     

Hydroxylation of aromatic compounds as indices of hydroxyl radical production: A cautionary note revisited

While setting up an intracerebral microdialysis system to estimate the extent of oxidative stress induced by the neurotoxin, N-methylphenylpyridinium ion (MPP⁺), we encountered a problem in the use of hydroxybenzoic acids as traps of hydroxyl radicals. Using either 2-hydroxybenzoate (salicylate) or 4-hydroxybenzoate as trapping agents, we observed a nonspecific, that is, nontissue derived, production of hydroxyl radicals as measured by the hydroxylation products, 2,3- and 2,5-dihydroxybenzoate from 2-hydroxybenzoate and 3,4-dihydroxybenzoate from 4-hydroxybenzoate. This production of dihydroxybenzoates was 10 times that expected due to the administration of MPP⁺, thus making it impossible to interpret our results. Careful investigation of the various components of the microdialysis system indicated that contact of the microdialysate with metal surfaces resulted in dihydroxybenzoic acid formation. These results should serve as a reminder to perform stringent tests of the experimental system prior to experiments with biological tissues to evaluate the contribution of hydroxyl radical production from nonbiological sources. Therefore, along with the possibility of enzymatic production of dihydroxybenzoates, artefactual production by components of the experimental apparatus must be considered before assuming that one is measuring hydroxyl radical production by a biological system.     

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

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

HPLC and LC-MS studies of hydroxylation of phenylalanine as an assay for hydroxyl radicals generated from Udenfriend's reagent

An HPLC assay method and an LC-MS method were used to study the Udenfriend reaction and its variations by using phenylalanine as the hydroxylation substrate. The results indicate that (1). citric acid can replace EDTA as the promoter for the production of hydroxyl radicals in the Undenfriend reaction, albeit in a somewhat less efficient way, (2). dihydroxylation of the hydroxylation substrate, phenylalanine, readily occurs with the Udenfriend systems (with either EDTA or citric acid), and (3). a novel oxidative degradation pathway may exist for o-tyrosine. It is cautioned that dihydroxylation needs to be accounted for when interpreting hydroxylation results in HPLC-based HO(z.rad;) assay systems with phenylalanine as the substrate.     

Assaying for hydroxyl radicals: hydroxylated terephthalate is a superior fluorescence marker than hydroxylated benzoate.

Generation of hydroxyl radicals in terephthalate (benzene-1,4-dicarboxylic acid) solution yields fluorescent 2-hydroxy-terephthalate. The reaction product is stable for hours and can readily be assessed using standard fluorimeters. The efficiency, i.e. the relative increase of fluorescence per *OH radical, is about three times higher than that of the formation of salicylate (2-hydroxy-benzoate) from benzoic acid and approximately hundred-fold higher than that of the hydroxylation of phenylalanine. As the terephthalate molecule is symmetric with respect to ring-hydroxylation, only one isomer is formed; hence, mechanistic interpretation of the hydroxylation reaction is facilitated. The scavenging rate constant of terephthalate for *OH yielding the hydroxycyclohexadienyl adduct as first intermediate is close to the diffusion controlled limit (k = 3.3 x 10(9) M(-1) s(-1)). Therefore, competition of the detector molecule with biomolecules being present under physiological conditions is expected to be efficient. The assay can be used to detect 'free' *OH radicals produced by the radiolysis of water as well as 'hydroxyl analogous species' that have been suggested to arise from the interaction of complex-bound reduced metal with either oxygen or hydrogen peroxide, e.g. from Fenton reactions. Based on calibration with radiolytically generated hydroxyl radicals the detection limit of the method is estimated to be around 50 nmol/dm3. Terephthalate is classified non-toxic and hence may also prove useful for microdialysis and continuous flow experiments as observation of fluorescence is 'non-destructive' and the reporter substance does not necessarily have to be subjected to HPLC.     

Novel method for in vivo hydroxyl radical measurement by microdialysis in fetal sheep brain in utero.

Hydroxyl radical (.OH) is a reactive oxygen species produced during severe hypoxia, asphyxia, or ischemia that can cause cell death resulting in brain damage. Generation of .OH may occur in the fetal brain during asphyxia in utero. The very short half-life of .OH requires use of trapping agents such as salicylic acid or phenylalanine for detection, but their hydroxylated derivatives are either unstable, produced endogenously, or difficult to measure in the small volume of microdialysis samples. In the present study, we used terephthalic acid (TA), hydroxylation of which yields a stable and highly fluorometric isomer (excitation, 326 nm; emission, 432 nm). In vitro studies using .OH generated by the Fenton reaction showed that hydroxylated TA formed quickly (<10 s), was resistant to bleaching (<5% change in fluorescence), and permitted detection of <0.5 pmol .OH. In vivo studies were performed in fetal sheep using microdialysis probes implanted into the parasagittal cortex. The probe was perfused at 2 mul/min with artificial cerebrospinal fluid containing 5 mM TA, and samples were collected every 30 min. Fluorescence measured in 10 mul of dialysate was significantly greater than in the efflux from probes perfused without TA. High-performance liquid chromotography analysis showed that the fluorescence in dialysis samples was entirely due to hydroxylation of TA. Thus this study shows that it is possible to use TA as a trapping agent for detecting low concentrations of .OH both in vitro and in vivo and that low concentrations of .OH are present in fetal brain tissue and fluctuate with time.     

Aryl hydroxylation: A selective mechanism for the herbicides, diclofop-methyl and clofop-isohutyl, in gramineous species

Esters of substituted phenoxy-phenoxy propionic acid constitute a new class of herbicides that are effective against gramineous weed and crop species. Slight changes in chemical structure alter drastically the spectrum of weeds controlled by this class of herbicides. Wheat (Triticum aestivum L.) is resistant to diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy] propanoate) (DM) and clofop-isobutyl (iso-butyl 2-[4-(4′-phenoxy)phenoxy] propanoate) (CI), oat (Avena sativa L.) and wild oat (Avena fatua L.) are susceptible to DM but resistant to CI, and corn (Zea mays L.) is susceptible to both compounds. The antagonism of IAA-induced elongation in the coleoptile straight growth test was determined to measure biological activity of the herbicides. The basis for the differential responses by gramineous species was related to the metabolism and deioxication of the herbicides in coleoptiles. Growth of wheat coleaptiles was relatively unaffected by both compounds, oat coleoptile growth was inhibited by DM but not by CI. but corn coleoptile growth was inhibited equally by both compounds. Coleoptiles and excised shoots of the three species rapidly hydrolyzed both compounds to their respective acids (diclofop, clofop). The acids were conjugated to a water-soluble ester conjugate or they were hydroxylated in the chlorine-substituted phenyl ring and conjugated as a phenolic conjugate. Aryl hydroxylation is a detoxication mechanism in resistant plants. Plants resistant to DM or CI contained low concentrations of the parent ester and the free or bound (ester conjugate) acid and a high concentration of free or bound (phenolic conjugate) aryl hydroxylated acid in coleoptile and shoot tissues, Differential responses by the three gramineous species to DM and CI axe due apparently to differences in their detoxication mechanism. The enzyme for aryl hydroxylation in oat appears to have a higher affinity for the 4-chloro- than for the 2,4-dichloro-substituted moiety. Therefore, oat hydroxylated clofop rapidly and was tolerant to CI but the limited ability of oat to hydroxylate diclofop resulted in oat being extremely susceptible to DM.     

The hydroxylation of tryptophan.

Products of the chemical hydroxylation of tryptophan by Fenton and Udenfriend reactions are similar to those obtained by ionizing radiation. When tryptophan is exposed to either of these systems, a mixture of four hydroxytryptophans, oxindole-3-alanine, and N-formylkynurenine is formed. This observation indicates that the hydroxyl radical attacks the aromatic nucleus as well as the 2 and 3 positions of the pyrrole ring. During gamma-radiolysis of nitrous oxide-saturated tryptophan solution and in the absence of oxygen or ferric edta, the hydroxyl radical adduct (or hydroxycyclohexadienyl radical) of tryptophan undergoes dimerization and polymerization, which results in a yellow product with maximal absorbance at 425 nm. In the presence of ferric edta, or in a Fenton system, the hydroxyl radical adduct disproportionates, and hydroxylated derivatives are formed. The yields of the hydroxytryptophans are proportional to the concentration of ferric edta to a limiting yield of 54% of the theoretical yield, which is taken to be one hydroxylated product per two hydroxyl radicals. Under these conditions, 4-, 5-, 6-, and 7-hydroxy-derivatives of tryptophan are found in the proportion 4:2:2:3, respectively. The presence of dioxygen during gamma-radiolysis increases the yield of N-formylkynurenine, but does not affect the total yield of hydroxytryptophans. Similarly, tryptophan subjected to the Udenfriend reaction yields 4-, 5-, 6-, and 7-hydroxytryptophan and N-formylkynurenine in approximately equal amounts.     

Dimerization of hydroxylated species of m-aminophenol by cytochrome c with hydrogen peroxide

The cytochrome c and hydrogen peroxide-dependent oxidation of m-aminophenol was investigated by electrochemistry and spectrophotometry. The results indicated that the hydroxylated species of m-aminophenol have at least two conjugated substituted groups on the ring system (most possibly, its oxidized form 2-hydroxy-4-iminoquinone), and that the degradation of cytochrome c by hydrogen peroxide can also be prevented in the presence of m-aminophenol. The hydroxyl radical scavengers, mannitol and sodium benzoate, almost completely eliminate the hydroxylation of m-aminophenol. But oxo-heme species scavenger, uric acid, does not inhibit the hydroxylation. Combining the results of mass spectrum, nuclear magnetic resonance and element analysis with that of spectrophotometry, electrochemistry and chemical scavengers, it is suggested that cytochrome c may act as a peroxidase, which facilitates the hydroxylation and subsequent dimerization of m-aminophenol.