Evaluation of cold atmospheric pressure plasma irradiation of water as a method of singlet oxygen generation.

We used cold atmospheric pressure plasma jet to examine in detail 1O2 generation in water. ESR with 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, a secondary amine probe, was used for the detection of 1O2. Nitroxide radical formation was detected after cold atmospheric pressure plasma jet irradiation of a 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide solution. An 1O2 scavenger/quencher inhibited the ESR signal intensity induced by cold atmospheric pressure plasma jet irradiation, but this inhibition was not 100%. As 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide reacts with oxidizing species other than 1O2, it was assumed that the signal intensity inhibited by NaN3 corresponds to only the nitroxide radical generated by 1O2. The concentration of 1O2 produced by cold atmospheric pressure plasma jet irradiation for 60 s was estimated at 8 µM. When this 1O2 generation was compared to methods of 1O2 generation like rose bengal photoirradiation and 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide) thermal decomposition, 1O2 generation was found to be, in decreasing order, rose bengal photoirradiation ≥ cold atmospheric pressure plasma jet > endoperoxide thermal decomposition. Cold atmospheric pressure plasma jet is presumed to not specifically generate 1O2, but can be used to mimic states of oxidative stress involving multiple ROS.

Method to Improve Azo-Compound (AAPH)-Induced Hemolysis of Erythrocytes for Assessing Antioxidant Activity of Lipophilic Compounds.

We examined the method of oxidative hemolysis for assessment of antioxidant activity of various compounds, especially lipophilic compounds. 2,2'-Azobis(amidinopropane) dihydrochloride (AAPH) was used as the source of free radicals for the oxidative hemolysis of horse erythrocytes. We found that absorbance at 540 nm is not appropriate for monitoring AAPH-induced hemolysis. Instead, we should use absorbance at 523 nm (an isosbestic point), because AAPH oxidizes the oxygenated hemoglobin to methemoglobin and absorbance at 540 nm does not correctly reflect the amount of released hemoglobin by AAPH-induced hemolysis. The corrected method of AAPH-induced hemolysis was applicable to assess the antioxidant activity of various hydrophilic compounds such as ascorbic acid, (-)-epicatechin, and edaravone. For the assessment of antioxidant activity of lipophilic compounds, we need appropriate dispersing agents for these lipophilic compounds. Among several agents tested, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) liposome at a concentration of 0.34 mM was found to be useful. Exogenous α-tocopherol incorporated using DMPC liposome as a dispersing agent was shown to protect erythrocytes from AAPH-induced hemolysis in a concentration-dependent manner.

Basic Investigations of Singlet Oxygen Detection Systems with ESR for the Measurement of Singlet Oxygen Quenching Activities.

Singlet oxygen (1O2) is highly oxidative and exerts strong cytotoxic effects. We tried to establish the best combination of a singlet oxygen generation system and a detection method with ESR, for measurement of the quenching activities of various substances. The photosensitizing reaction of rose bengal or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was used for the generation of 1O2, and a sterically hindered secondary amine, 2,2,6,6-tetramethyl-4-piperidone (TEMPD) or 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), was used as the 1O2 detection probe. These secondary amines were oxidized by 1O2 to form stable nitroxide radicals, which were detectable by ESR. TEMPD was found to be readily oxidized by air, causing large background signals in comparison with TEMP-OH. The ESR signal obtained by the irradiation of rose bengal with visible light in the presence of TEMP-OH consisted of two kinds of nitroxide radical overlapping. In contrast, only a single nitroxide signal was observed when TEMP-OH was reacted with 1O2 generated from EP. Therefore, the best combination should be EP as the 1O2 generator and TEMP-OH as the detection probe. When using this combination, we found that the concentrations of some organic solvents such as dimethyl sulfoxide and acetonitrile should be kept constant for reliable quantification, because the concentrations of organic solvents affect the ESR signal intensity.

Formation of reactive oxygen species by irradiation of cold atmospheric pressure plasma jet to water depends on the irradiation distance.

Because application of cold atmospheric pressure plasma jet (CAPPJ) to biological samples have taken large attentions, it is important to examine the effects of various CAPPJ parameters on the generation of reactive species. Here, we investigated the generation of reactive species in water by CAPPJ irradiation by changing the following parameters: irradiation time, sample volume, and irradiation distance between the sample surface and plasma jet tip. We measured 1) change in the ESR signal intensity of 4-hydroxy-2,2,6,6-tetrametylpeperidine-1-oxyl (Tempol), 2) spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3) Fricke dosimeter reaction, and 4) hydrogen peroxide (H2O2) formation induced by CAPPJ irradiation. By the experiment of volume dependency, it is suggested that the reactive species detected in water are formed largely in the plasma gas phase. The reduction of ESR signal intensity of Tempol and the formation of DMPO-OH were strongly dependent on irradiation distance, but the relationship between H2O2 generation and distance was weak. The formation of species that oxidize Fe2+ to Fe3+ was shown by the Fricke dosimeter reaction, and reactions at irradiation distances longer than 3 cm were mainly attributable to H2O2. It may be possible to apply different reactive species to the samples by changing the CAPPJ irradiation distance.

Trapping of fatty acid allyl radicals generated in lipoxygenase reactions in biological fluids by nitroxyl radical.

Nitroxyl radicals can trap fatty acid allyl radicals on ferric-lipoxygenases at lower oxygen content, which are an intermediate in the lipoxygenase reaction. In the present study, we examined whether nitroxyl radical-trapping of fatty acid allyl radicals on the enzyme proceeds in biological fluids with abundant antioxidants. The fatty acid allyl radical-nitroxyl radical adducts were quantified by HPLC with electrochemical detection (HPLC-ECD); the adducts in eluate degraded into nitroxyl radical by passing through heating coil at 100 degrees C, and then nitroxyl radical was detected by electrochemical detector. Soybean 15-lipoxygenase and nitroxyl radical (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl, CmDeltaP) were mixed with rat serum prepared from fresh venous blood, and the solution was stood at 37 degrees C for 1 h. One volume of the solution was mixed with 5 vols of cold acetonitrile. After centrifugation, the supernatant was subjected to HPLC-ECD. Arachidonate allyl radical-CmDeltaP adducts as well as linoleate allyl radical-CmDeltaP adducts were detected in the solution, and the content of these adducts remarkably increased in the presence of phospholipase A(2). It is proved for the first time that nitroxyl radical traps fatty acid allyl radicals generated in the lipoxygenase reaction in biological fluid without competition from endogenous antioxidants.

Feedback activation of ferrous 5-lipoxygenase during leukotriene synthesis by coexisting linoleic acid.

Ferrous lipoxygenases seem to be activated through a feedback control mechanism via FA hydroperoxides generated from PUFAs by partially existing ferric lipoxygenases. However, during leukotriene synthesis, feedback activation of ferrous 5-lipoxygenase in the presence of arachidonic acid (AA) was not observed. In the present study, we examined the feedback activation of ferrous 5-lipoxygenase in the 5-lipoxygenase/AA system in the presence of linoleic aicd (LA), which is a predominant component of membrane phospholipids. When potato 5-lipoxygenase was incubated with AA and LA in the presence of nitroxyl radical, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP), one-electron redox cycle reaction between ferric and ferrous 5-lipoxygenase was detected. For each revolution of the cycle, one molecule of PUFA and one molecule of its hydroperoxide were converted into PUFA-allyl radical-CmDeltaP adduct ([PUFA-H].-CmDeltaP) and PUFA-epoxyallyl radical-CmDeltaP adduct ([PUFA-H+O].-CmDeltaP), respectively. The ratios, [AA-H].-CmDeltaP/[LA-H].-CmDeltaP and [AA-H+O].-CmDeltaP/[LA-H+O].-CmDeltaP, were estimated to be 1.7 and 0.13, respectively. These facts indicate that ferrous 5-lipoxygenase is activated through feedback control in the presence of LA, and that resulting ferric 5-lipoxygenase catalyzes the stoichiometric synthesis of leukotrienes from AA. In conclusion, the biosynthesis of leukotrienes is remarkably efficient.

Regulation of S-thiolation and S-nitrosylation in the thiol/nitric oxide system by radical scavengers.

Nitric oxide (NO) is a possible agent, which induces crosslinking among molecules containing sulfhydryl groups. However, the S-thiolation is essentially accompanied by S-nitrosylation. In the present study, we evaluated radical scavengers as a regulator for S-thiolation and S-nitrosylation by NO released from NO-generator, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (P-NONOate). When glutathione was incubated with P-NONOate in 4% (vol/vol) O(2)-saturated buffer solution (pH 7.4) in the presence of nitrone spin-trapping agent, 5,5'-dimethyl-1-pyroline-N-oxide (DMPO), the prevention of S-thiolation and the promotion of S-nitrosylation were observed. The DMPO adduct was identified to be thiyl radical-DMPO adduct via ESR study. In contrast, nitroxyl radical, radical scavenger against oxygen-centered radicals, promoted the S-thiolation but prevented S-nitrosylation. Nitronyl nitroxide, radical scavenger against nitric oxide, can convert nitric oxide into nitrogen dioxide in the O(2)-independent manner. In the presence of nitronyl nitroxide in the thiol/P-NONOate system, S-thiolation was remarkably enhanced up to 60% (mol/mol) of sulfhydryl groups. However, nitronyl nitroxide at enough content (>or=1.0 mM) almost completely prevented S-nitrosylation, whereas nitronyl nitroxide at comparatively lower content (0.5 mM) contrarily enhanced the S-nitrosylation. Based on these facts, it appeared to be possible to consequently regulate S-thiolation and S-nitrosylation through controlling the thiyl radical chain reaction by radical scavengers.

Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content.

Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM alpha-linolenic acid, 1.0 muM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA-H+O(2)].-CmDeltaP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C(2)H(5)-CH(CmDeltaP)-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C(2)H(5)-.CH-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity.

Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content.

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical-lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)-MS/MS (tandem MS), four E,Z-linoleate allyl radical-CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical-CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content.

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition.

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).

Identification and characterization of three Drosophila melanogaster glucuronyltransferases responsible for the synthesis of the conserved glycosaminoglycan-protein linkage region of proteoglycans. Two novel homologs exhibit broad specificity toward oligosaccharides from proteoglycans, glycoproteins, and glycosphingolipids.

The Drosophila melanogaster genome contains three putative glucuronyltransferases homologous to human GlcAT-I and GlcAT-P. These enzymes are predicted to be beta1,3-glucuronyltransferases involved in the synthesis of the glycosaminoglycan (GAG)-protein linkage region of proteoglycans and the HNK-1 carbohydrate epitope of glycoproteins, respectively. The genes encode active enzymes, which we have designated DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII (where BS stands for "broad specificity"). Protein A-tagged truncated soluble forms of all three enzymes efficiently transfer GlcUA from UDP-GlcUA to the linkage region trisaccharide Galbeta1-3Galbeta1-4Xyl. Strikingly, DmGlcAT-I has specificity for Galbeta1-3Galbeta1-4Xyl, whereas DmGlcAT-BSI and DmGlcAT-BSII act on a wide array of substrates with non-reducing terminal beta1,3- and beta1,4-linked Gal residues. Their highest activities are obtained with asialoorosomucoid with a terminal Galbeta1-4GlcNAc sequence, indicating their possible involvement in the synthesis of the HNK-1 epitope in addition to the GAG-protein linkage region. Galbeta1-3GlcNAc and Galbeta1-3GalNAc, disaccharide structures widely found in N- and O-glycans of glycoproteins and glycolipids, also serve as acceptors for DmGlcAT-BSI and -BSII. Transcripts of all three enzymes are ubiquitously expressed throughout the developmental stages and in adult tissues of Drosophila. Thus, all three glucuronyltransferases are likely involved in the synthesis of the GAG-protein linkage region in Drosophila, and DmGlcAT-BSI and -BSII appear to be involved in various GlcUA transfer reactions for the synthesis of proteoglycans, glycoproteins, and glycolipids. This activity distinguishes these glucuronyltransferases from their mammalian homologs GlcAT-P and GlcAT-D (or -S). Sequence alignment of the Drosophila glucuronyltransferases with homologs in human, rat, and Caenorhabditis elegans demonstrates the conservation of a majority of the critical amino acid residues in the active sites of the three Drosophila enzymes.