Preconditioning with hydrogen peroxide (H2O2) or ischemia in H2O2-induced cardiac dysfunction

To assess whether allantoin levels in serum and urine are influenced by exhaustive and moderate exercise and whether allantoin is a useful indicator of exercise-induced oxidative stress in humans, we made subjects perform exhaustive and moderate (100% and 40% VO₂max) cycling exercise and examined the levels of allantoin, thiobarbituric acid reactive substances (TBARS) and urate in serum and urine. Immediately after exercise at 100% VO₂max, the serum allantoin/urate ratio was significantly elevated compared with the resting levels while the serum urate levels was significantly elevated 30 min after exercise. The serum TBARS levels did not increase significantly compared with the resting levels. Urinary allantoin excretion significantly increased during 60 min of recovery after exercise, however, urinary urate excretion decreased significantly during the same period. The urinary allantoin/urate ratio also rapidly increased during 60 min of recovery after exercise. Urinary TBARS excretion decreased during the first 60 min of the recovery period and thereafter significantly increased during the latter half of the recovery period. On the contrary, after 40% VO₂max of exercise, no significant changes in the levels of urate, allantoin and TBARS in serum or urine were observed. These findings suggest that allantoin levels in serum and urine may reflect the extent of oxidative stress in vivo and that the allantoin which appeared following exercise may have originated not from urate formed as a result of exercise but from urate that previously existed in the body. Furthermore, these findings support the view that allantoin in serum and urine is a more sensitive and reliable indicator of in vivo oxidative stress than lipid peroxidation products measured as TBARS.     

The oxidation of reduced nicotinamide nucleotides by hydrogen peroxide in the presence of lactoperoxidase and thiocyanate, iodide or bromide

Lactoperoxidase (EC 1.11.1.7) catalysed the oxidation of NADH by hydrogen peroxide in the presence of either thiocyanate, iodide or bromide. In the presence of thiocyanate, net oxidation of thiocyanate occurred simultaneously with the oxidation of NADH, but in the presence of iodide or bromide, only the oxidation of NADH occurred to a significant extent. In the presence of thiocyanate or bromide, NADH was oxidized to NAD(+) but in the presence of iodide, an oxidation product with spectral and chemical properties distinct from NAD(+) was formed. Thiocyanate, iodide and bromide appeared to function in the oxidation of NADH by themselves being oxidized to products which in turn oxidized NADH, rather than by activating the enzyme. Iodine, which oxidized NADH non-enzymically, appeared to be an intermediate in the oxidation of NADH in the presence of iodide. NADPH was oxidized similarly under the same conditions. An assessment was made of the rates of these oxidation reactions, together with the rates of other lactoperoxidase-catalysed reactions, at physiological concentrations of thiocyanate, iodide and bromide. The results indicated that in milk and saliva the oxidation of thiocyanate to a bacterial inhibitor was likely to predominate over the oxidation of NADH.     

大豆过氧化物酶－过氧化氢－碘化钾体系杀菌作用

【目的】研究豆壳过氧化物酶(SBP)-H2O2-KI三元体系杀菌作用及其可能的杀菌机制。【方法】以细菌生长的浊度(OD600)为指标检测SBP-H2O2-KI体系的抑菌作用;以活细胞计数(CFU)为指标检测SBP-H2O2-KI体系的杀菌作用;以最低抑菌浓度(MIC)为指标检测亚致死剂量的SBP-H2O2-KI体系作用下连续传代细菌的敏感性变化;以物理和化学方法检测SBP-H2O2-KI体系中活性氧基团等功能基团的生成与否,以期解释SBP-H2O2-KI体系的杀菌机制。【结果】SBP-H2O2-KI体系对多种细菌有高效、快速杀菌作用,作用时间仅为几分钟。在亚致死剂量浓度下连续培养的细菌悬液对体系的耐受能力(MIC)没有显著性变化,从中也不能分离到抗性/耐性突变株。物理和化学方法检测结果表明SBP-H2O2-KI反应过程中无羟基自由基产生;化学方法检测结果表明SBP-H2O2-KI反应过程中无超氧阴离子自由基产生;而有单线态氧和单质碘的产生。【结论】根据实验结果可以推测:SBP-H2O2-KI体系的杀菌力可能主要来源于单线态氧和碘的活性中间态,而不是羟基自由基和超氧阴离子。此外,SBP-H2O2-KI体系所具备的高效、快速的杀菌作用,以及细菌对其不易产生抗性的特点,预示此体系在医疗以及植保等方面有广泛的应用前景。     

Free radical generation and coupled thiol oxidation by lactoperoxidase/SCN-/H2O2.

The lactoperoxidase-catalyzed oxidation of glutathione (GSH) and thiocyanate (SCN-) was studied. Oxidation of SCN- was recorded by ultraviolet spectroscopy and by electron spin resonance (ESR). Consumption of GSH was measured by amperometric titration. One or two moles of GSH was oxidized per mole of H2O2 added, depending on the reaction conditions. Omission of SCN- prevented the oxidation of GSH. The oxidation of GSH required only catalytic amounts of SCN-, which was therefore recycled. Iodide (I-) could replace SCN-, while chloride or bromide were ineffective. The apparent Michaelis constant for SCN- was 17 microM. Oxidation of SCN- gave rise to two reactive intermediates, one stable and one unstable. The stable intermediate (-OSC. = N-(?)) decayed by a second-order reaction with a rate constant of 1.1 M-1 s-1. The decay of the unstable radical was very fast. The data (a) explain the short- and long-term antibacterial effects of lactoperoxidase-halide-H2O2 system, (b) point to possible deleterious effects due to glutathione depletion, (c) are of relevance for free radical diseases involving sulphur-centered free radicals, and (d) support previous observations on lipid peroxidation/halogenation in biological membranes, liposomes, and unsaturated fatty acids.     

Salicylic acid-induced inactivation of creatine kinase in the presence of lactoperoxidase and H2O2

To clarify one mechanism of aspirin-induced gastric mucosal damage, inactivation of creatine kinase (CK) by salicylic acid that is easily produced from aspirin in vivo was examined in the presence of lactoperoxidase (LPO) and H2O2 (LPO-H2O2). Salicylic acid inactivated CK (rabbit muscle) during its interaction with LPO-H2O2. CK activity in gastric mucosal homogenate decreased dependent on the concentration of salicylic acid in the presence of LPO-H2O2. Oxygen radical scavengers did not prevent the inactivation of CK. Direct detection of free radicals of salicylic acid by electron spin resonance was unsuccessful. However, glutathionyl radicals were formed during the interaction of salicylic acid with LPO-H2O2 in the presence of reduced glutathione and 5,5-dimethyl-1-pyrroline oxide as a spin trap agent. Among salicylic acid-related drugs, salsalate, but not aspirin and ethenzamide, inactivated CK, indicating the phenolic hydroxyl group is oxidized by LPO-H2O2. During oxidation of salicylic acid by LPO-H2O2, the sulfhydryl group in CK markedly decreased, and salicylic acid bound to CK. These results indicate that CK was inactivated through loss of the sulfhydryl group and binding of salicylic acid.     

ELF electromagnetic fields increase hydrogen peroxide (H2O2)-induced mutations in pTN89 plasmids

We have examined the mutational effects of hydrogen peroxide (H(2)O(2)) in the presence and absence of an extremely low-frequency magnetic field (ELFMF), using pTN89 plasmids. Mutations were detected in the supF gene carried by these plasmids in Escherichia coli. The plasmids were either treated with H(2)O(2) (1microM) alone at 37 degrees C for 4h, or were exposed to an ELFMF (60Hz, 5millitesla (mT)) simultaneously with H(2)O(2) treatment. The mutation frequency was 2.28 x 10(-4) for H(2)O(2) treatment alone, and 5.81 x 10(-4) for ELFMF exposure with H(2)O(2) treatment. We did not observe any mutations using treatment with ELFMF exposure alone. This indicates that the ELFMF may potentiate H(2)O(2)-induced mutation. Sequence analysis of the supF mutant plasmids revealed that base substitutions, G: C-->A :T transitions and G:C-->T:A transversions were dominant in both treatment groups, and there was no difference in the mutation spectrum or the hotspots between the groups. Therefore, ELFMFs may interact and potentiate the damage induced by H(2)O(2), resulting in an increase in the number of mutations.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Enhanced gellan gum production by hydrogen peroxide (H2O2) induced oxidative stresses in Sphingomonas paucimobilis

In this study, the effect of H₂O₂-induced oxidative stress on gellan gum production and cell growth were investigated. Gellan gum production was improved and cell growth was inhibited by H₂O₂. A multiple H₂O₂ stresses with different concentrations were developed to optimize gellan gum production. A maximal gellan gum yield (22.52 g/L), which was 35.58 % higher than the control, was observed with 2, 2, 3, 4 mmol/L H₂O₂ added at 6, 12, 18, 24 h, respectively. Moreover, UDP-glucose pyrophosphorylase activity and glucosyltransferase activity were increased with H₂O₂ stresses. This new strategy of multiple H₂O₂-induced oxidative stresses would be further applied to gellan gum production in future study.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation.

Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H2O2. When low levels of thiocyanate (SCN-) were used in the GO system, H2O2 was detected and lactoperoxidase (LP) was inactivated when SCN- was depleted. With 10-fold higher SCN-, LP remained active and H2O2 was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H2O2 in a system employing low SCN-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H2O2 was not detected during the course of the experiment. At high SCN- levels, relatively high concentrations of oxidation product were produced immediately, with H2O2 undetectable. The results suggest that the final product of the LP reaction depends on the method of H2O2 generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37 degrees C.     

Irreversible inactivation of lactoperoxidase in the course of iodide oxidation

In the course of lactoperoxidase-catalysed I- oxidation, which is a model for the initial step of thyroid hormone biosynthesis, irreversible enzyme inactivation can occur if free molecular iodine (I2) or other oxidized iodine species accumulate. Evidence is presented that the breakdown of the catalytic activity is the result of the iodination of the peroxidase-apoprotein. This kind of enzyme inactivation, which can be prevented by iodine acceptors' such as thyroglobulin or high concentrations of I-, may well play a role in the regulation of the synthesis of thyroid hormones in vivo.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

The degradation of cytochrome c by hydrogen peroxide

Cytochrome c is degraded by a large excess of hydrogen peroxide, leading to opening of the heme porphyrin ring and loss of the Soret absorption bands. The kinetic parameters of this reaction have been determined, and it is shown that a small concentration of oxygen is liberated at the same rate as degradation. Low-level chemiluminescence and release of a hydroxylating species also accompany heme destruction. It is proposed that heme iron activates hydrogen peroxide to a more powerful oxidant, perhaps the hydroxyl radical, which remains bound to the heme iron and initiates attack on the porphyrin ring. Chemiluminescence appears to result from a side reaction involving singlet oxygen attack on the alpha-methene bridge, yielding a dioxetane. The in vivo degradation of cytochrome c by excess hydrogen peroxide may interfere with respiration, accelerate aging, and enhance the metabolism of carcinogens.     

Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.     

Effect of lactoperoxidase on the antimicrobial effectiveness of the thiocyanate hydrogen peroxide combination in a quantitative suspension test

Background  

The positive antimicrobial effects of increasing concentrations of thiocyanate (SCN-) and H₂O₂ on the human peroxidase defence system are well known. However, little is known about the quantitative efficacy of the human peroxidase thiocyanate H₂O₂ system regarding Streptococcus mutans and sanguinis, as well as Candida albicans. The aim of this study was to evaluate the effect of the enzyme lactoperoxidase on the bactericidal and fungicidal effectiveness of a thiocyanate-H₂O₂ combination above the physiological saliva level. To evaluate the optimal effectiveness curve, the exposure times were restricted to 1, 3, 5, and 15 min.     

H2O2-mediated cross-linking between lactoperoxidase and myoglobin: elucidation of protein-protein radical transfer reactions

The H(2)O(2)-dependent reaction of lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse myoglobin (HoMb) produces LPO-Mb cross-linked species, in addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb) dimer and LPO(HoMb)(2) trimer. Dityrosine cross-links are shown by their fluorescence to be present in the oligomeric products. Addition of H(2)O(2) to myoglobin (Mb), followed by catalase to quench excess H(2)O(2) before the addition of LPO, still yields LPO cross-linked products. LPO oligomerization therefore requires radical transfer from Mb to LPO. In contrast to native LPO, recombinant LPO undergoes little self-dimerization in the absence of Mb but occurs normally in its presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical electron paramagnetic resonance signal located primarily on LPO, confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or Trp-14 did not. However, because DBNBS-trapped LPO radicals were observed with all the mutants, DBNBS traps LPO radicals other than those involved in protein oligomerization. The results clearly establish that radical transfer occurs from Mb to LPO and suggest that intermolecularly transferred radicals may reside on residues other than those that are generated by intramolecular reactions.     

Metabolism of hydrogen peroxide in isolated hepatocytes: Relative contributions of catalase and glutathione peroxidase in decomposition of endogenously generated H2O2

The compartmentation of hydrogen peroxide catabolism was studied in isolated hepatocytes. Hydrogen peroxide generation in the peroxisomal compartment was stimulated by addition of glycolate and in the endoplasmic reticular compartment (cytosolic compartment) by ethylmorphine. The rate of catabolism by catalase was estimated from the concentration of methanol required to decrease the steady-state concentration of catalase Compound I to the half-maximal value. The rate of catabolism by glutathione peroxidase was assessed in a semiquantitative manner by the rate of GSSG efflux. The relationship of GSSG efflux to catalase-dependent metabolism of H₂O₂ in the presence of increasing concentrations of glycolate was sigmoidal. This indicates that the function of glutathione peroxidase is small relative to that of catalase at low rates of H₂O₂ production in the peroxisomal fraction, but that the contribution of the former system increases as the peroxisomal H₂O₂ production rate is enhanced, and suggests that the accumulation of a steady-state concentration of H₂O₂ in the nanomolar range in the peroxisomes is sufficient to allow diffusion of H₂O₂ into the cytosol. Following pretreatment of animals with aminotriazole to inhibit catalase, glycolate caused GSSG release at rates nearly double those in control cells. This indicates that even incomplete inhibition of catalase in cells can result in enhanced release of H₂O₂ into the cytosol and demonstrates the relationship of GSSG release to H₂O₂ production under these conditions. An estimate of the rate of H₂O₂ diffusion to catalase during ethylmorphine metabolism was made from the steady-state level of Compound I and measured formate concentrations. This rate increased threefold as the rate of GSH loss increased from 1 to 2 nmol/10⁶ cells per min, indicating that as the rate of H₂O₂ production in the endoplasmic reticulum becomes maximally stimulated in the presence of ethylmorphine, the rate of H₂O₂ metabolism by catalase becomes larger. A comparison of ethylmorphine-stimulated rates of GSSG efflux from cells of control and aminotriazole-treated rats shows that, unlike experiments with glycolate, no difference in the rate of efflux is observed. These results support the conclusion that in hepatocytes catalase has a relatively minor role in catabolism of H₂O₂ at low rates of H₂O₂ generation in the endoplasmic reticulum, but that the catalase function increases as the rate of H₂O₂ production is enhanced.