Extent Of Oxidative Modification Research Articles

MORPHOLOGICAL RESEARCH ON FREE-RESIDUE OXIDATION PROCESSES IN CASES OF DECIDUAL CELLS OF PLACENTA IN CHORIOAMNIONOTIS AND BASAL DECIDUITIS COMBINED WITH IRON-DEFICIENCY ANEMIA IN THE PREGNANT

Background. The oxidative modification of proteins is lately pivotal to pathologists and it is a new way of research on different pathological conditions, as well as the diagnostics of inflammation processes in placenta.Objective. The study was aimed at the research of nitro peroxides and establishing the specific features of oxidative modification of proteins in inflammation of placenta with iron deficient anaemia in the pregnant.Methods. Сhemiluminescent and histochemical technique (with bromphenol blue on ‘acidic’ and ‘basic’ proteins according to Mikel Calvo) was applied.Results. The intensity of nitro peroxides glow in chorioamnionitis and basal deciduitis increased in comparison with the samples of physiological and iron deficient anaemia gestation. At the same time in chorioamnionitis the glow intensity is higher than in basal deciduitis.Due to the results of immune histochemical technique held while analysing the samples, together with chorioamnionitis and basal deciduitis the R/B increases and in basal deciduitis the rate, is probably, higher, than in chorioamnionitis. At the same time, the extent of oxidative modification of proteins in cases of inflammation with iron deficient anaemia in the pregnant is on the average higher than with no iron deficient anaemia in the pregnant.Conclusions. High level of nitro peroxides in placentae basal plate in secundines inflammation, the increase in R/B rate, in other words the prevalence of ‘acidic’ proteins over ‘basic’ ones, is evidenced due to the increase of the intensity of oxidative modification processes of proteins in cases of deciduitis.

Exploring Oxidative Reactions in Hemoglobin Variants Using Mass Spectrometry: Lessons for Engineering Oxidatively Stable Oxygen Therapeutics.

Worldwide demand has driven the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) as potential acellular oxygen therapeutics. HBOCs have the potential to provide an oxygen bridge to patients and minimize current problems associated with supply and storage of donated blood. However, to date, safety and efficacy issues have hampered the approval of viable HBOCs in the United States. These previous efforts have underscored the need for a better molecular understanding of toxicity to design safe and oxidatively stable HBOCs. Recent Advances: High-resolution accurate mass (HRAM) mass spectrometry (MS) has recently become a versatile tool in characterizing oxidative post-translational modifications that occur in Hb. When integrated with other analytical techniques, HRAM data have been invaluable in providing mechanistic insight into the extent of oxidative modification by quantifying oxidation in amino acids near the reactive heme or at specific "oxidative hotspots." In addition to providing a deeper understanding of Hb oxidative toxicity, HRAM MS studies are currently being used toward developing suitable HBOCs using a "two-prong" strategy that involves (i) understanding the mechanism of Hb toxicity by evaluating mutant Hbs identified in patients with hemoglobinopathies and (ii) utilizing this information toward designing against (or for) these reactions in acellular oxygen therapeutics that will result in oxidatively stable protein. Future HRAM studies are aimed at fully characterizing engineered candidate HBOCs to determine the most oxidatively stable protein while retaining oxygen carrying function in vivo. Antioxid. Redox Signal. 26, 777-793.

Di-n-butyl phthalate (DnBP) and diisobutyl phthalate (DiBP) metabolism in a human volunteer after single oral doses

An individual (male, 36years, 87kg) ingested two separate doses of di-n-butyl phthalate (DnBP) and diisobutyl phthalate (DiBP) at a rate of ~60μg/kg. Key monoester and oxidized metabolites were identified and quantified in urine continuously collected until 48h post-dose. For both DnBP and DiBP, the majority of the dose was excreted in the first 24h (92.2% of DnBP, 90.3% of DiBP), while only <1% of the dose was excreted in urine on day 2. In each case, the simple monoesters were the major metabolites (MnBP, 84%; MiBP, 71%). For DnBP, ~8% was excreted as various side chain oxidized metabolites. For DiBP, approximately 20% was excreted mainly as the oxidized side chain metabolite 2OH-MiBP, indicating that the extent of oxidative modification is around 2.5 times higher for DiBP than for DnBP. All DnBP and DiBP metabolites reached peak concentrations between 2 and 4h post-exposure, followed by a monotonic decline. For DnBP metabolites, the elimination halftime of MnBP was 2.6h; longer elimination halftimes were estimated for the oxidized metabolites (2.9-6.9h). For DiBP metabolites, MiBP had the shortest halftime (3.9h), and the oxidized metabolites had somewhat longer halftimes (4.1 and 4.2h). Together with the simple monoesters, secondary oxidized metabolites are additional and valuable biomarkers of phthalate exposure. This study provides basic human metabolism and toxicokinetic data for two phthalates that have to be considered human reproductive toxicants and that have been shown to be omnipresent in humans.

Oxidative Modification of Von Willebrand Factor Inhibits Its Cleavage by ADAMTS13

During inflammation, activated neutrophils go through the oxidative burst, releasing various oxidants, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions. HOCl preferentially oxidizes cysteine and methionine residues to cysteine sulfenic acid and methionine sulfoxide, respectively, at rates ~100 times faster than it oxidizes tyrosine, another commonly oxidized amino acid. HOCl can also oxidize tyrosine to chlorotyrosine. Of great interest in this regard is the fact that the ADAMTS13 cleavage site in VWF, the Tyr1605–Met1606 peptide bond, contains two residues that are potential targets for myeloperoxidase-mediated oxidation. Given previous studies from our laboratory that VWF cleavage by ADAMTS13 is inhibited by oxidants, we hypothesized that neutrophil oxidants might oxidize either or both of these two amino acid residues and thereby potentially inhibit ADAMTS13-mediated cleavage. We tested our hypothesis using a peptide substrate for ADAMTS13 based on the VWF A2 sequence Leu1591–Arg1668. We incubated the VWF A2 peptide either without HOCl or with 25 or 75 μM HOCl, followed by quenching the oxidant with free methionine. The peptides were then incubated with purified recombinant ADAMTS13 and the reaction sampled every 15 min for one hour. We analyzed the cleavage reaction in two ways:by electrophoretic separation on a Tricine gel and densitometric quantification of the cleavage product, andby liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) to determine the location and extent of oxidative modification and quantity of the cleavage product.We found that, after exposure to 75 μM HOCl, the A2 peptide contained methionine sulfoxide at position 1606 in 99% of the molecules in the sample, whereas only 0.3% contained both chlorotyrosine at position 1605 and methionine sulfoxide at 1606. The rate of substrate cleavage by ADAMTS13 was markedly reduced with oxidation, as measured by both assays, with the rate for the peptide treated with 75 μM HOCl being only 20% of that of the non-oxidized peptide. Taken together, these findings suggest that oxidants released by activated neutrophils during inflammation have a prothrombotic effect, mediated at least in part by inhibition of VWF cleavage by ADAMTS13.

Effects of polymerization on the oxygen carrying and redox properties of diaspirin cross-linked hemoglobin

Human hemoglobin site specifically cross-linked with bis(3,5-dibromosalicyl)fumarate results in a low oxygen affinity hemoglobin-based red cell substitute (α-DBBF). Polymerization of α-DBBF by bis(maleoylglycylamide) polyethylene glycol (BMAA-PEG) yields poly α-DBBF which offers the added benefits of reduced renal clearance and increased retention in the vascular circulation. Oxygen equilibrium curves for poly α-DBBF are slightly left-shifted (higher O 2 affinity) compared to those of α-DBBF; with a diminished cooperativity and a reduced Bohr effect. In rapid mixing experiments (oxygen dissociation and carbon monoxide binding), poly α-DBBF exhibits a several fold increase in the overall rate of deoxygenation and carbon monoxide binding kinetics over its cross-linked counterpart. The rate of nitric oxide binding to the oxidized form of poly α-DBBF shows little or no change compared to the intramolecularly cross-linked derivative. The reduction of cyanomet poly α-DBBF by dithionite is several fold faster than that of HbA 0 and α-DBBF whereas the slow subsequent cyanide dissociation from the ferrous iron remained unchanged among all proteins. The propensity of poly α-DBBF for auto-oxidation is slightly enhanced over α-DBBF whereas the extent of oxidative modification by hydrogen peroxide is very similar. Polymerization appears to selectively modify ligand interactions and redox kinetics of the tetrameric cross-linked form which reflects a possibly more open heme pocket. The data suggests that changes in oxygenation properties of hemoglobin brought about by a given modification are not necessarily predictive of other functional changes.

The extent of oxidative modification of LDLs is critical in determining their effects on platelet function

The extent of oxidative modification of LDLs is critical in determining their effects on platelet function

Enhanced LDL oxidation by murine macrophage foam cells and their failure to secrete nitric oxide

Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFNy and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68–99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFNy/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFNγ/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation.

Homocysteine induces iron-catalyzed lipid peroxidation of low-density lipoprotein that is prevented by alpha-tocopherol.

Homocystinuria is an inborn error of methionine metabolism that is characterized by the premature development of arteriosclerosis. As one of the major factors in the pathogenesis of arteriosclerosis, modification of low-density lipoprotein (LDL) has received widespread attention by many investigators. In this study, to elucidate the relationship between elevated homocysteine levels and premature arteriosclerosis, we investigated the role of homocysteine in the iron-catalyzed oxidative modification of LDL. When LDL isolated from a healthy subject was incubated with homocysteine and ferric ion, a gradual decrease of polyunsaturated fatty acids (PUFA), formation of thiobarbituric acid-reactive substances (TBARS) and fluorescent substances, and the fragmentation of apoprotein B (apoB) were observed. The extent of oxidative modification was dependent on the concentration of homocysteine. Modification of LDL was suppressed until the remaining alpha-tocopherol concentration reached a critical level. When the alpha-tocopherol content of LDL was increased by 2.6-fold, both the formation of TBARS and the fragmentation of apoB were suppressed. These results suggest that homocysteine might promote iron-catalyzed oxidation of LDL and imply its role for the development of premature arteriosclerosis.