Myeloperoxidase oxidation states involved in myeloperoxidase-oxidase oxidation of thiols.

The human red-blood-cell glyoxalase system was modified by incubation with high concentrations of glucose in vitro. Red-blood-cell suspensions (50%, v/v) were incubated with 5 mM- and 25 mM-glucose to model normal and hyperglycaemic glucose metabolism. There was an increase in the flux of methylglyoxal metabolized to D-lactic acid via the glyoxalase pathway with high glucose concentration. The increase was approximately proportional to initial glucose concentration over the range studied (5-100 mM). The activities of glyoxalase I and glyoxalase II were not significantly changed, but the concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, and the percentage of glucotriose metabolized via the glyoxalase pathway, were significantly increased. The increase in the flux of intermediates metabolized via the glyoxalase pathway during periodic hyperglycaemia may be a biochemical factor involved in the development of chronic clinical complications associated with diabetes mellitus.     

Intracellular oxidation of hydroethidine: Compartmentalization and cytotoxicity of oxidation products

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.     

Microbial oxidation of cumene

Summary A total of 1229 cultures, including 230 actinomycetes, 508 other bacteria, 12 fungi and 479 yeasts were screened for their ability to oxidize the isopropyl side chain of 2-phenyl propane (cumene). Four strains of actinomycetes and six strains of bacteria but no yeasts were found positive in converting 2-phenyl propane to its oxygenated products. Eight strains oxidized cumene through the alkyl side chain producing 2-phenyl-1-propanol. TwoBacillus strains oxidized cumene to an oxygenated product.Pseudomonas oleovorans NRRL B-3429 exhibited the highest alkyl side chain oxidation activity. The optimum reaction conditions for strain B-3429 are: 25 °C, pH 6.5 and 48 h of reaction. Octane-grown cells of strain B-3429 produced higher product yields (about 7.2-fold) than the glucose-grown cells. Prolonged incubation resulted in an increase in 2-phenyl-1-propionic acid production at the expense of 2-phenyl-1-propanol. The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.1%. Reaction in the presence of methanol favored the accumulation of 2-phenyl-1-propionic acid and also increased the total yield. (The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 14.9%.) Structures of the reaction products were confirmed by GC/MS and GC/IR analyses. Products contained 92% R(–) isomer.     

Peroxidase oxidation of phenols

Partially purified preparations of horseradish peroxidase were able to catalyze the effective transformation of such phenol compounds as phenol, o-chlorophenol, 2,4,6-trichlorophenol, pentachlorophenol (giving rise to the formation of polymer products insoluble in water), resorcinol, and thymol (giving rise to the formation of low-molecular-weight products). The following conditions were found to be optimal for peroxidase oxidation and provide the maximum extent of elimination of phenol compounds: temperature, 15-25 and 25-30 degrees C for phenol and chlorophenol compounds, respectively; molar ratio H2O2/phenol, 1:1; and transformation time, 1-3 h. Although effective transformation was observed within a broad range of pH, the efficiency of the process slightly increased at a pH from 6.0 to 7.5. It was suggested to carry out multiple peroxidase oxidations of phenols using partially purified peroxidase enclosed in a dialysis membrane bag placed into a solution of a phenol compound containing hydrogen peroxide.     

Enzymatic oxidation of L-homocysteine

Homocyst(e)ine, a normal metabolite, accumulates in certain inborn errors of sulfur amino acid metabolism. Since many amino acids are converted by enzymatic oxidation and by transamination to the corresponding alpha-keto acid analogs and related products, which may exert inhibitory effects on metabolism, and because the alpha-keto acid analog of homocysteine has not yet been prepared, the enzymatic oxidation of homocysteine was investigated with the aim of obtaining alpha-keto-gamma-mercaptobutyric acid. Oxidation of DL-homocysteine by L-amino acid oxidase led to formation of at least seven products that react with 2,4-dinitrophenylhydrazine; of these, five were identified: alpha-keto-gamma-mercaptobutyrate, the mono and diketo analogs of homolanthionine, and the mono and diketo analogs of homocystine. In addition, one product was tentatively identified as alpha-ketomercaptobutyric acid gamma-thiolactone. In the course of this work alpha-keto-gamma-mercaptobutyrate was found to be a substrate of lactate dehydrogenase. L-Homocysteine and its alpha-keto acid analog were shown to be substrates of glutamate dehydrogenase and kidney glutamine transaminase. DL-Homocysteine reacts readily with alpha-keto acids to form stable hemithioketals, which were found to be substrates of L- and D-amino acid oxidases. A scheme is presented which integrates some of the complexities involved in the oxidation metabolism of homocyst(e)ine. The significance of these findings is considered in relation to the toxicity of homocysteine, which accumulates in certain pathological states.     

Microbial oxidation of ebastine

The microbial oxidation of ebastine to carebastine was investigated. Among the 15 micro-organisms examined, only theCunninghamella strains showed the desired biotransformation.Cunninghamella blakesleeana oxidised the substrate within 7 days, via the intermediates alcohol and aldehyde, mainly to carebastine, the corresponding carboxylic acid. Optimisation of the culture conditions increased the yield from initially 10% up to a reproducible 40%. For the synthesis of carebastine a substrate concentration of 200 mg/l, a starting pH of 5.0 and the addition of 1% poly(vinyl alcohol) is favourable. The results achieved in experiments with shaking flasks are transferable to the fermentation scale and yielded 270 mg carebastine in a 3-l fermentation of 600 mg ebastine. The progress of the reaction was detected by TLC and HPLC, the products were identified by mass spectrometry and NMR.     

Photochemical oxidation of tyrosinase

Dye sensitized photo-oxidation inactivates tyrosinases isolated from Neurospora and Agaricus. The rate of inactivation is enhanced by cyanide and is dependent on pH.     

Myeloperoxidase-mediated oxidation of methionine

The myeloperoxidase-mediated oxidation of methionine was studied using a purified canine myeloperoxidase preparation. The system required the simultaneous presence of myeloperoxidase, H₂O₂, and a halide anion. When 0.1 mM H₂O₂ was used, the system has a pH optimum of approximately pH 5–5.5. Bromide and iodide were much more effective than chloride in the myeloperoxidase-mediated oxidation of methionine. Horseradish peroxidase was unable to oxidize methionine whether chloride or iodide was used. In contrast, lactoperoxidase oxidized methionine in the presence of iodide but not chloride. Based on studies of (1) the effect of various inhibitors and singlet oxygen quenchers, as well as (2) the effect of D₂O on the oxidation of methionine, by the myeloperoxidase system, OCl^?, or methylene blue, it was shown that the oxidation of methionine by the myeloperoxidase system was not mediated by OCl^? or ¹O₂. The mechanism of the myeloperoxidase-mediated oxidation of methionine remains unclear. However, it may be one mechanism by which the myeloperoxidase system damage microorganisms.     

Mechanisms of DNA oxidation

Oxidative damage of DNA caused by a variety of chemical and physical agents appears to be linked to cancer. However, it is becoming increasingly clear that endogenous generation of oxidants, such as hydroxyl radical and peroxynitrite, lead to oxidation of DNA, and this may cause cancer in individuals where no obvious exposure to chemical or physical agents known to be carcinogenic has occurred. The mechanisms for generation of these two oxidants in living organisms will be discussed and their reactivities with DNA to produce oxidized products (e.g., 8-oxo-dG) will be presented with special emphasis on the individual characteristics of the generation and reactivity of each oxidant.     

Radical-induced oxidation of trans-resveratrol

trans-Resveratrol (RVT) (3,5,4'-trihydroxystilbene), a polyphenolic constituent of red wine, is thought to be beneficial in reducing the incidence of cardiovascular diseases, partly via its antioxidant properties. However, the mechanism of action by which trans-resveratrol displays its antioxidant effect has not been totally unravelled. This study aimed at establishing a comprehensive scheme of the reaction mechanisms of the direct scavenging of HO(*) and O(2)(*-) radicals generated by water gamma radiolysis. Aerated aqueous solutions of trans-RVT (from 10 to 100μmolL(-1)) were irradiated with increasing radiation doses (from 25 to 400Gy) and further analyzed by UV-visible absorption spectrophotometry for detection of trans-RVT oxidation products. Separation and quantification of RVT and its four oxidation products previously identified by mass spectrometry, i.e., piceatannol (PCT), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,5-dihydroxybenzaldehyde (3,5-DHB) and para-hydroxybenzaldehyde (PHB), were performed by HPLC/UV-visible spectrophotometry. Determination of the radiolytic yields of trans-RVT consumption and oxidation product formation has allowed us to establish balance between trans-RVT disappearance and the sum of oxidation products formation. Under our conditions, O(2)(-) radicals seemed to poorly initiate oxidation of trans-RVT, whereas the latter, whatever its initial concentration, quantitatively reacted with HO() radicals, via a dismutation mechanism. Two reaction pathways involving HO()-induced trans-RVT primary radicals have been proposed to explain the formation of the oxidation end-products of trans-RVT.