Inhibition of lipid peroxidation

Lipid peroxy radicals (ROO-) were detected by electron spin resonance (ESR) at low temperature after formation by addition of H₂O₂ into a suspension of mice lymphocites. If lymphocytes were treated with selenomethionine (Se-Met) prior to addition of H₂O₂, ROO-formation was inhibited in a fashion that was dependent on Se-Met concentration. Formation of ROO- in the spleen of mice was induced by⁶⁰Co irradiation. Animals that were supplemented with Na₂SeO₃ prior to irradiation exhibited a lower ROO-concentration than that of nontreated animals. Based on our experiments, we have concluded that Se has an oxygen-free radical scavenging effect. This should be a protective effect against lipid peroxy radical cellular attack.     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

Multiple reactions in vanadyl-V(IV) oxidation by H2O2

Oxidation of vanadyl sulfate by H₂O₂ involves multiple reactions at neutral pH conditions. The primary reaction was found to be oxidation of V(IV) to V(V) using 0.5 equivalent of H₂O₂, based on the loss of blue color and the visible spectrum. The loss of V(IV) and formation V(V) compounds were confirmed by ESR and⁵¹V-NMR spectra, respectively. In the presence of excess H₂O₂ (more than two equivalents), the V(V) was converted into diperoxovanadate, the major end-product of these reactions, identified by changes in absorbance in ultraviolet region and by the specific chemical shift in NMR spectrum. The stoichiometric studies on the H₂O₂ consumed in this reaction support the occurrence of reactions of two-electron oxidation followed by complexing two molecules of H₂O₂. Addition of a variety of compounds—Tris, ethanol, mannitol, benzoate, formate (hydroxyl radical quenching), histidine, imidazole (singlet oxygen quenching), and citrate—stimulated a secondary reaction of oxygen-consumption that also used V(IV) as the reducing source. This reaction requires concomitant oxidation of vanadyl by H₂O₂, favoured at low H₂O₂:V(IV) ratio. Another secondary reaction of oxygen release was found to occur during vanadyl oxidation by H₂O₂ in acidic medium in which the end-product was not diperoxovanadate but appears to be a mixture of VO ₃ ⁺ (–546 ppm), VO³⁺ (–531 ppm) and VO ₂ ⁺ (–512 ppm), as shown by the⁵¹V-NMR spectrum. This reaction also occurred in phosphate-buffered medium but only on second addition of vanadyl. The compounds that stimulated the oxygen-consumption reaction were found to inhibit the oxygen-release reaction. A combination of these reactions occur depending on the proportion of the reactants (vanadyl and H₂O₂), the pH of the medium and the presence of some compounds that affect the secondary reactions.     

H2 oxidation,O2 uptake and CO2 fixation in hydrogen treated soils

In many legume nodules, the H₂ produced as a byproduct of N₂ fixation diffuses out of the nodule and is consumed by the soil. To study the fate of this H₂ in soil, a H₂ treatment system was developed that provided a 300 cm³ sample of a soil:silica sand (2:1) mixture with a H₂ exposure rate (147 nmol H₂ cm^–3hr^–1) similar to that calculated exist in soils located within 1–4 cm of nodules (30–254 nmol H₂ cm^–3hr^–1). After 3 weeks of H₂ pretreatment, the treated soils had a K_m and V_max for H₂ uptake (1028 ppm and 836 nmol cm^–3 hr^–1, respectively) much greater than that of control, air-treated soil (40.2 ppm and 4.35 nmol cm^–3 hr^–1, respectively). In the H₂ treated soils, O₂, CO₂ and H₂ exchange rates were measured simultaneously in the presence of various pH₂. With increasing pH₂, a 5-fold increase was observed in O₂ uptake, and CO₂ evolution declined such that net CO₂ fixation was observed in treatments of 680 ppm H₂ or more. At the H₂ exposure rate used to pretreat the soil, 60% of the electrons from H₂ were passed to O₂, and 40% were used to support CO₂ fixation. The effect of H₂ on the energy and C metabolism of soil may account for the well-known effect of legumes in promoting soil C deposition.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide

Among various metal ions of physiological interest, Cu²⁺ is uniquely capable of catalyzing the oxidation of NADH by H₂O₂. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (1-methyl imidazole, 2-methyl imidazole, andN-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine,L-histidine methyl ester, andL-carnosine), however, inhibit the Cu²⁺-catalyzed peroxidation of NADH. Other chelating agents such as EDTA andL-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H₂O₂ utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD⁺. D₂O, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding¹O₂ as a mediator. Similarly, OH· is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H₂O₂.     

玉米叶表皮短细胞发育过程的形态特征及栓质细胞作用研究

采用大田试验,直接撕表皮或对叶片进行固定处理,结合单染、复染、荧光染色等多种细胞学显色方法,利用光学显微镜、荧光显微镜和扫描电子显微镜系统观察玉米叶表皮短细胞的发生时期、发育过程、分布规律以及形态结构特征,研究K⁺和H₂O₂在栓质细胞中的分布变化与表皮其它细胞中K⁺和H₂O₂的分布及气孔器开关的关系,为进一步挖掘短细胞的新功能提供细胞学依据。结果表明：（1）短细胞是同步发生在玉米多叶位新表皮组织形成过程中,所有植株从第7新生叶,大部分第6叶,极少数第5叶的基部同时开始发生短细胞,之后新生的高位叶也均发生短细胞,并随着叶位的升高叶片各部位短细胞密度均增大,所有植株的1~4叶（因不再生长）均无短细胞出现。（2）初期发育的叶表皮细胞进行不对称分裂,生成相互交替的长、短细胞,有的短表皮细胞横（垂直叶脉）分裂,形成栓质细胞和硅质细胞对;栓质细胞基部与叶肉细胞相邻,硅质细胞嵌在栓质细胞和表皮细胞间偏上。（3）有短细胞发生的叶片,宏观背面发亮且覆有蜡质层,微观表皮细胞的着色特性发生了变化;栓质细胞为面包形柱状细胞,硅质细胞为哑铃形扁细胞。（4）气孔器张开时,栓质细胞中没有K⁺和H₂O₂的积累;气孔器关闭时,栓质细胞中积累了大量的K⁺和H₂O₂,且栓质细胞中K⁺和H₂O₂的积累始终与副卫细胞中K⁺和H₂O₂的积累变化一致,而硅质细胞和长细胞没有K⁺和H₂O₂的积累。该研究确定了玉米叶表皮短细胞发生的时期;展示了其发育过程的形态学变化特征;发现栓质细胞中K⁺和H₂O₂的积累随气孔器开关呈周期性变化,且与副卫细胞中K⁺和H₂O₂的积累变化保持一致。     

Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands

Hydrogen peroxide (H₂O₂)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C. Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca²⁺ ions were not required but Ca²⁺ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H₂O₂ concentrations but only extent of aggregation at optimal H₂O₂ concentrations. Ba²⁺, Sr²⁺, Cd²⁺, Mn²⁺ and Ni²⁺ ions (1 mM) and Zn²⁺, Pb²⁺ and Hg²⁺ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the prostaglandin H₂/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane A₂, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein kinase C modulator, Zn²⁺ ions, inhibited by different mechanisms. The results indicate direct action of H₂O₂ at the prostaglandin H₂/thromboxane A₂ receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products. The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Pre-treatment with H<Subscript>2</Subscript>O<Subscript>2</Subscript> induces salt tolerance in Barley seedlings

Barley seedlings were pre-treated with 1 and 5 μM H₂O₂ for 2 d and then supplied with water or 150 mM NaCl for 4 and 7 d. Exogenous H₂O₂ alone had no effect on the proline, malondialdehyde (MDA) and H₂O₂ contents, decreased catalase (CAT) activity and had no effect on peroxidase (POX) activity. Three new superoxide dismutase (SOD) isoenzymes appeared in the leaves as a result of 1 μM H₂O₂ treatment. NaCl enhanced CAT and POX activity. SOD activity and isoenzyme patterns were changed due to H₂O₂ pre-treatment, NaCl stress and leaf ageing. In pre-treated seedlings the rate of ¹⁴CO₂ fixation was higher and MDA, H₂O₂ and proline contents were lower in comparison to the seedlings subjected directly to NaCl stress. Cl⁻ content in the leaves 4 and 7 d after NaCl supply increased considerably, but less in pre-treated plants. It was suggested that H₂O₂ metabolism is involved as a signal in the processes of barley salt tolerance.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

15N2 incorporation and acetylene reduction by Azospirillum isolated from rice roots and soils

Summary Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by¹⁵N₂ incorporation and C₂H₂ reduction. C₂H₂ reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in¹⁵N₂ incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N₂-fixing ability and that plant genotype is of importance for optimal associations.     

Scavenging of free radicals in gas-phase mainstream cigarette smoke by immobilized catalase at filter level

Catalase is well known as capable of inducing the decomposition of H₂O₂. In this study, a kind of immobilized catalase (entrapped in cross-linked chitosan beads) was dispersed in conventional acetate filter as an antioxidant additive. Quantitative estimation of the free radicals in mainstream cigarette smoke (MCS) was performed to address the effect of this modified filter. It was found that the levels of PBN adduct and NO^?/NO₂^? associated with the gas-phase mainstream cigarette smoke (GPCS) were efficiently decreased by ～40% through catalase filtering. Besides, the modified filter was found to lower the MCS-induced adverse biological effects including lipid peroxidation and mutagenicity. This was proved to be substantially attributed to the catalase-dependent breakdown of NO^?, which was stimulated by some of peroxides (most probably being H₂O₂), the dismutation products of tar particulate matters (TPM). These results highlighted a promising approach to reduce the smoking-associated health risks to passive smokers. Moreover, the mechanisms of catalase filtering may be helpful for the development of appropriate immobilized enzyme systems to be applied for reducing health risks associated with gaseous pollutants.     

Deconvolution of the three components of the photoacoustic signal by curve fitting and the relationship of CO2 uptake to proton fluxes

The photoacoustic response of the photosynthetic apparatus to a short light pulse consists of three components: heat evolution, O₂ evolution and CO₂ uptake. Recent attempts of deconvoluting the individual components by curve-fitting by means of model curves [Kolbowski et al. (1990) Photosynth Res 25: 309–316] suffered from the fact that the model curve for CO₂ uptake changed its curve shape with CO₂ concentration. Here, it is shown that good fits can be obtained if a stretching factor is incorporated into the fitting routine which adjusts the shape of the uptake model curve. The relationship between CO₂ uptake und H⁺ transport across the thylakoid membrane was investigated by experiments in different CO₂ concentrations from 0 to 7%. It was found that under limiting conditions (7% CO₂) the flux ratio CO₂: O₂ was close to 4. This was compared with the value expected from the stoichiometries of the linear electron transport chain.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

Abstract

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the ‘ECoScreen’ in healthy calves (body weight 63–98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system ‘ECoCheck’. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138–624 nmol l^?1 EBC or 0.10–0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC–H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC–pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC–pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

H2O2 Sensors of Lungs and Blood Vessels and Their Role in the Antioxidant Defense of the Body

This paper considers the composition and function of sensory systems monitoring H₂O₂ level by the lung neuroepithelial cells and carotid bodies. These systems are localized in the plasma membrane of the corresponding cells and are composed of O ² -generating NADPH-oxidase and an H₂O₂-activated K⁺ channel. This complex structure of the H₂O₂ sensors is probably due to their function in antioxidant defense. By means of these sensors, an increase in the H₂O₂ level in lung or blood results in a decrease in lung ventilation and constriction of blood vessels. This action lowers the O₂ flux to the tissues and, hence, intracellular [O₂]. The [O₂] decrease, in turn, inhibits intracellular generation of reactive oxygen species. The possible roles of such systems under normal conditions (e.g., the effect of O ² in air) and in some pathologies (e.g., pneumonia) is discussed.     

Generation of hydrogen peroxide by brown adipose tissue mitochondria

This is the first report on the generation of H₂O₂ by brown adipose tissue mitochondria. Flavin dehydrogenase-linked substrates like succinate, glycerol-1-phosphate, and fatty acyl CoA were good substrates for the reaction, while NAD⁺-linked substrates were less effective. In cold-acclimated animals the activity showed a substantial increase (2.5-fold). TheK _m andV _max of the reaction were considerably lower than those of the respective dehydrogenase. Metal ions, particularly Cu²⁺ and Fe²⁺ were potent inhibitors of the reaction. Nucleoside diphosphates, which were inhibitors by themselves, potentiated the inhibitory action of Fe²⁺ ions. In most of the properties, the H₂O₂ generator of brown adipose tissue mitochondria resembled that of liver mitochondria.