Formation of hydroxanthommatin-derived radical in the oxidation of 3-hydroxykynurenine.

Using ESR, a radical (g = 2.004) was detected in the reaction mixture of 3-hydroxykynurenine (3-HKY), H2O2, and horseradish peroxidase. The radical was stable and was detected even after 5 h. On HPLC analysis of the reaction mixture, two radical peaks (Peak-1 and Peak-2) were detected using ESR. The ESR spectra of Peak-1 and Peak-2 radicals were the same and identical with that of the original radical in the reaction mixture. The retention times of Peak-1 and Peak-2 corresponded to those of authentic xanthommatin (XA) and hydroxanthommatin (Hydro-XA), respectively, XA being formed in the oxidation of 3-HKY by potassium ferricyanide and Hydro-XA being formed in the reduction of XA by sodium metabisulfite. The absorbance spectra of Peak-1 and Peak-2 were nearly identical with those of authentic XA and Hydro-XA. The absorbance spectrum of Peak-2 changed from that of Hydro-XA to that of XA, indicating that Hydro-XA auto-oxidized to XA in the air. The ESR signal intensity of the Peak-2 radical developed in accordance with the progress of this auto-oxidation of Hydro-XA to XA. It was supposed that the Peak-2 radical was generated in the auto-oxidation of Hydro-XA after its elution from the HPLC column. Thus, the radical seemed to be the one-electron oxidized form of Hydro-XA. The Peak-1 radical appeared to be the true retention of the radical on the column and to be eluted with a much larger amount of XA. The separation of the radical from XA was impossible on the column. Hemoglobin (Hb) or hematin also induced the same radical in the reaction mixture of 3-KHY, H2O2, and Hb or hematin.

Oxidation of 3-hydroxykynurenine catalyzed by methemoglobin with hydrogen peroxide.

Methemoglobin (metHb) with H2O2 catalyzed the oxidation of 3-hydroxykynurenine (3-HKY) in the reaction mixture of metHb, 3-HKY, and H2O2. The spectrophotometric experiments suggest the following mechanism for the 3-HKY oxidation by metHb with H2O2. MetHb first reacts with H2O2 to form the ferryl complex of Hb. This species then oxidizes 3-HKY, while it returns to metHb. 3-HKY was more reactive with the ferryl complex than glutathione but less reactive than ascorbic acid. Scavengers of the hydroxyl radical, dimethyl sulfoxide and ethanol, scarcely inhibited the 3-HKY oxidation by metHb with H2O2. Desferrioxamine, a metal chelator, hardly suppressed the 3-HKY oxidation. These results indicate that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H2O2.

Superoxide dismutase enhances the toxicity of 3-hydroxyanthranilic acid to bacteria.

Cu,Zn.superoxide dismutase (SOD) enhanced the toxicity of 3-hydroxyanthranilic acid (3-HAT) to Salmonella typhimurium strain TA 102, evaluated as ability to form colonies. MnSOD showed the same effect. Inactivated Cu.ZnSOD had no effect. SODs accelerated the oxidation of 3-HAT, but inactivated Cu.ZnSOD caused little acceleration. It is proposed that the acceleration of 3-HAT oxidation leads to the enhancement of the 3-HAT toxicity. Catalase protected the bacteria from the toxicity of 3-HAT enhanced by Cu,ZnSOD, indicating that hydrogen peroxide generated in the oxidation of 3-HAT is involved in the toxicity. SODs accelerate the oxidation of 3-HAT and generate more hydrogen peroxide, that causes the enhancement of the 3-HAT toxicity to the bacteria. However, hydrogen peroxide alone was not so toxic. Hydrogen peroxide with 3-HAT was more toxic to the bacteria.

Superoxide dismutases enhance the rate of autoxidation of 3-hydroxyanthranilic acid.

The autoxidation of 3-hydroxyanthranilate to cinnabarinate at 37 degrees C and at pH 7.4 is hastened by superoxide dismutase (SOD). The Cu,Zn-containing enzyme from bovine erythrocytes and the Mn-containing enzyme from Escherichia coli were equally effective in this regard; whereas the H2O2-inactivated Cu,Zn enzyme was ineffective. Catalase appears to augment the effect of superoxide dismutase, because it prevents the bleaching of cinnabarinate by H2O2. It follows that O2-, which is a product of the autoxidation, slows the net autoxidation by engaging in back reactions and that SOD increases the rate of autoxidation by removal of O2- and thus by prevention of these back reactions.

The effects of caffeic acid and its related catechols on hydroxyl radical formation by 3-hydroxyanthranilic acid, ferric chloride, and hydrogen peroxide.

The effect of caffeic acid on hydroxyl radical formation through a reaction, which contained 0.22 M carbonate buffer (pH 7.4), 0.22 mM 3-hydroxyanthranilic acid, 87 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2.9 mM hydrogen peroxide, and 14 microM FeCl3, was investigated. The addition of 30 microM caffeic acid resulted in the decrease of hydroxyl radical formation in the reaction mixture. Chlorogenic acid, 3,4-dihydroxy-phenylalanine noradrenaline, gallic acid, dopamine, epicatechin, and D-(+)-catechin also suppressed the hydroxyl radical formation. In regard to the positional isomers of benzenediol, o-benzenediol inhibited the hydroxyl radical formation, but m and p-benzenediol did not. The inhibitory effect of the hydroxyl radical formation seems to be due to the chelation of iron ions by the catechols. Supporting evidence includes the diminished effect of catechols in the presence of EDTA (a potent iron ion chelator) and the observation of a visible band at 450 nm caused by the interaction between caffeic acid and iron ions. Additionally, the visible band (506 nm) was observed in the solution of o-benzenediol and ferric chloride but not in the solution of m- or p-benzenediol and ferric chloride. Thus compounds with adjacent hydroxyl groups on aromatic rings might inhibit hydroxyl radical formation.

In situ demonstration of the activity of 3 beta-hydroxysteroid dehydrogenase on steroid hormone secreting cells within the paraaortic lymph nodes of golden hamster.

The in situ activity of 3 beta-hydroxysteroid dehydrogenase was detected in clustered cells which showed steroid-producing morphology, within the capsule of the paraaortic lymph node. In light and electron microscopic studies, the positive reaction products were detected on intracapsular cell clusters. This result indicates that these unique cells may have a steroid secreting function within the lymph nodes.

Enhancement by catechols of hydroxyl-radical formation in the presence of ferric ions and hydrogen peroxide.

The effect of caffeic acid, a kind of catechol, on the Fenton reaction was examined by using the ESR spin trapping technique. Caffeic acid enhanced the formation of hydroxyl radicals in the reaction mixture, which contained caffeic acid, hydrogen peroxide, ferric chloride, EDTA, and potassium phosphate buffer. Chlorogenic acid, which is an ester of caffeic acid with quinic acid, also stimulated the formation of the hydroxyl radicals. Quinic acid did not stimulate the reaction, suggesting that the catechol moiety in chlorogenic acid is essential to the enhancement of the hydroxyl-radical formation. Indeed, other catechols and related compounds such as pyrocatechol, gallic acid, dopamine, and noradrenaline effectively stimulated the formation of the hydroxyl radicals. The above results confirm the idea that the catechol moiety is essential to the enhancement. Ferulic acid, 4-hydroxy-3-methoxybenzoic acid, and salicylic acid had no effect on the formation of the hydroxyl radicals. The results indicate that the enhancement by the catechols of the formation of hydroxyl radicals is diminished if a methyl ester is formed at the position of the hydroxyl group of the catechol. In the absence of iron chelators such as EDTA, DETAPAC, desferrioxamine, citrate, and ADP, formation of hydroxyl radicals was not detected, suggesting that chelators are essential to the reaction. The enhancement of the formation of hydroxyl radicals is presumably due to the reduction of ferric ions by the catechols. Thus, the catechols may exert deleterious effects on biological systems if chelators such as EDTA, DETAPAC, desferrioxamine, citrate, and ADP are present.

Separation of polyunsaturated fatty acid radicals by high-performance liquid chromatography with electron spin resonance and ultraviolet detection.

In the reaction of soybean lipoxygenase (EC 1.13.11.12) with polyunsaturated fatty acids such as linoleic, linolenic and arachidonic acids, some radical species were detected using the electron spin resonance (ESR) spin-trapping technique. The radical species derived from the three polyunsaturated fatty acids were not distinguishable because the ESR spectra of the spin adducts of nitrosobenzene with their three radical species showed no difference in their hyperfine splittings. To overcome this defect of the spin-trapping technique, these spin-adducts were separated by employing high-performance liquid chromatography (HPLC) combined with ESR spectroscopy. The spin adducts were eluted from a C18 reversed-phase column in the order linolenic acid, linoleic acid and arachidonic acid. The half-lives of the spin adducts separated by HPLC-ESR were determined as linoleic acid 600 min, linolenic acid 360 min and arachidonic acid 160 min. The use of an ultraviolet detector together with the HPLC-ESR technique resulted in a 500-fold increase in sensitivity in the detection of the radical species.

Suppression of the DTH reaction in mice by means of moxibustion at electro-permeable points.

The effects of moxibustion stimulation on picryl chloride-induced cutaneous contact hypersensitivity, a delayed type of hypersensitivity (DTH) reaction, were investigated in mice. Three electro-permeable points (B-20, LI-15, S-36) were selected as moxibustion points. Moxibustion at point B-20 (spleen associated point) significantly suppressed the DTH reaction. Adoptive transfer of spleen T cells from moxibustion-stimulated donors resulted in an increased suppression of the DTH reaction in the recipients. These studies suggest that suppression of the DTH reaction with moxibustion may be related to cellular immunity, especially to the induction of the suppressive activity of T cells in the spleen.

Detection of nucleic acid-derived radicals formed by alloxan.

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.

Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen.

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.

An electron microscopic study of the acupuncture or moxibustion stimulated regional skin and lymph node in experimental animals.

Ultrastructures of the electro-acupuncture or moxibustion stimulated skin and regional lymph nodes in experimental animals were examined by scanning and transmission electron microscopy. In the scanning electron microscopy, small numbers of infiltrating erythrocytes and lymphocytes were found in the vicinity of the penetrated acupuncture needle in the acupuncture-stimulated skin, whereas moxibustion-treated instances revealed a large number of immunocyte infiltrations in the region. Transmission electron microscopically revealed that immunocytes consisted of lymphocytes, monocytes and some granulocytes and mast cells. In addition, the moxibustion-stimulated regional lymph nodes increased in weight after the stimulation and induced numerous immunocyte influx through afferent lymphatics. The acupuncture-stimulated nodes, however, revealed no remarkable change in the weight and morphology. A functional significance of the acupuncture and moxibustion-stimulated skin and regional nodes was discussed in reference to previous immunohistological descriptions of immune-reacted lymph node of the same animals.