Hydroperoxide Species Research Articles

Inhibition of Accumulation of Neutral Lipids and Their Hydroperoxide Species in Hepatocytes by Bioactive Allium sativum Extract

Our ongoing research suggests that extracts from plant-based foods inhibit the accumulation of lipid droplets (LDs) and oxidized lipid droplets (oxLDs) in liver cells. These findings suggest their potential use in the alleviation of metabolic dysfunction-associated fatty liver disease (MAFLD) and its most severe manifestation, metabolic dysfunction-associated steatohepatitis (MASH). Allium extracts (ALs: AL1–AL9) were used to assess their ability to reduce lipid droplet accumulation (LDA) and oxidized lipid droplet accumulation (oxLDA) by inhibiting neutral lipid accumulation and oxidation in LD. Among the tested Allium extracts, AL1, AL3, and AL6 demonstrated substantial inhibitory effects on the LDA. Furthermore, AL1 extract showed real-time inhibition of LDA in HepG2 cells in DMEM supplemented with oleic acid (OA) within 12 h of treatment. Our lipidomic approach was used to quantify the accumulation and inhibition of intracellular triacylglycerol (TAG) and oxidized TAG hydroperoxide [TG (OOH) n = 3] species in hepatocytes under OA and linoleic acid loading conditions. These results suggest that Allium-based foods inhibit LD accumulation by decreasing intracellular lipids and lipid hydroperoxides in the hepatocytes. The metabolomic analysis of AL1—the bioactive LDAI extract—using both LC-MS/MS and 1D-NMR [1H, 13C, and Dept (135 and 90)] approaches revealed that AL1 contains mainly carbohydrates and glucoside metabolites, including iridoid glucosides, as well as minor amino acids, organosulfur compounds, and organic acids such as the antioxidant ascorbic acid (KA2 = S13), and their derivatives, suggesting that AL1 could be a potential resource for the development of functional foods and in drug discovery targeting MAFLD/MASH and other related diseases.

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition

Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting superoxide (O2●−) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). Human MnSOD has evolved to be highly product inhibited to limit the formation of H2O2, a freely diffusible oxidant and signaling molecule. The product-inhibited complex is thought to be composed of a peroxide (O22−) or hydroperoxide (HO2−) species bound to Mn ion and formed from an unknown PCET mechanism. PCET mechanisms of proteins are typically not known due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the mechanism, we combine neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states of the enzyme to reveal the positions of all the atoms, including hydrogen, and the electronic configuration of the metal ion. The data identifies the product-inhibited complex, and a PCET mechanism of inhibition is constructed.

Synthetic Copper-(Di)oxygen Complex Generation and Reactivity Relevant to Copper Protein O2-Processing.

Synthetic copper-dioxygen complex design, generation and characterization, play a crucial role in elucidating the structure/function of copper-based metalloenzymes, including dopamine β-monooxygenase, lytic polysaccharide monooxygenases, particulate methane monooxygenase, tyrosinase, hemocyanin, and catechol oxidase. Designing suitable ligands to closely mimic the variable active sites found in these enzymes poses a challenging task for synthetic bioinorganic chemists. In this review, we have highlighted a few representative ligand systems capable of stabilizing various copper-dioxygen species such as CuII-(O2 •-)(superoxide), Cu2 II-(μ-η 1:η 1-O2 2-) (trans/cis-peroxide), Cu2 II-(μ-η 2:η 2-O2 2-)(side-on peroxide) and Cun II--OOH (hydroperoxide) species. Here, we discuss the ligand type utilized, syntheses, and spectroscopic characterization of these species. We also delineate reactivity patterns, particularly electrophilic arene hydroxylation by a side-on peroxo species which occurs via a "NIH shift" mechanism and thermodynamic-kinetic relationships among Cu2-(O2 •-)/O2 2-/-OOH moieties.

Versatile ferrous oxidation-xylenol orange assay for high-throughput screening of lipoxygenase activity.

Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as biocatalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under different conditions. This study aimed to enhance the suitability of the ferrous-oxidized xylenol orange (FOX) assay for screening LOX activity across a wide pH range with different PUFAs. The narrow linear detection range of the standard FOX assay restricts its utility in screening LOX activity. To address this, the concentration of perchloric acid in the xylenol orange reagent was adjusted. The modified assay exhibited a fivefold expansion in the linear detection range for hydroperoxides and accommodated samples with pH values ranging from 3 to 10. The assay could quantify various hydroperoxide species, indicating its applicability in assessing LOX substrate preferences. Due to sensitivity to pH, buffer types, and hydroperoxide species, the assay required calibration using the respective standard compound diluted in the same buffer as the measured sample. The use of correction factors is suggested when financial constraints limit the use of FAHP standard compounds in routine LOX substrate preference analysis. FAHP quantification by the modified FOX assay aligned well with results obtained using the commonly used conjugated diene method, while offering a quicker and broader sample pH range assessment. Thus, the modified FOX assay can be used as a reliable high-throughput screening method for determining LOX activity. KEY POINTS: • Modifying perchloric acid level in FOX reagent expands its linear detection range • The modified FOX assay is applicable for screening LOX activity in a wide pH range • The modified FOX assay effectively assesses substrate specificity of LOX.

A comprehensive experimental and kinetic modeling study of methyl tert-butyl ether combustion

A comprehensive understanding of the combustion chemistry of methyl tert‑butyl ether (MTBE) is of key importance in its application as an additive in gasoline fuels. Ignition delay times (IDTs) of MTBE/air mixtures have been measured in both a high-pressure shock tube (HPST) and in a rapid compression machine (RCM) at equivalence ratios of 0.5, 1.0, and 2.0 in air, at pressures of 10 and 30 bar over the temperature range 600 – 1350 K. Species profiles for MTBE oxidation were obtained in a jet-stirred reactor (JSR) at 1 bar, at equivalence ratios of 0.5, 1.0, and 2.0 in the temperature range 700 – 1100 K.A detailed reaction mechanism, comprising 813 species and 4319 reactions, has been developed and predicts well all of the experimental data obtained in this work and also texisitng literature data. Pressure- and temperature-dependent rate constants for the MTBE unimolecular elimination reaction producing isobutene and methanol were calculated using high-level ab-initio calculations. A sensitivity analysis reveals that this elimination reaction is important, and significantly inhibits fuel reactivity at temperatures above 1300 K. At intermediate temperatures (850 – 1300 K), the reaction MTBE + ȮH = TĊ4H8OCH3 + H2O plays a crucial role in promoting the reactivity of MTBE oxidation, whereas the reaction MTBE + ȮH = TC4H9OĊH2 + H2O is the most inhibiting reaction. At low temperatures (600 – 850 K), the isomerization reaction of TC4OCȮ2–1 ⇌ TĊ4OCO2H-2 significantly promotes the reactivity. Conversely, the reaction 2TC4OCȮ2–1 ↔ 2TC4OCȮ-1 + O2 inhibits reactivity the most. The NTC behavior in MTBE oxidation can be explained by the competition between the reactions involving the formation and consumption of cyclic ethers from TĊ4H8OCH3 radicals and the reactions associated with the formation and consumption of carbonyl hydroperoxide species.

Using Information from Public Databases to Critically Evaluate Studies Linking the Antioxidant Enzyme Selenium-Dependent Glutathione Peroxidase 2 (GPX2) to Cancer

Recent research on selenium-dependent glutathione peroxidase 2 (GPX2) tends to focus on possible roles in tumorigenesis. This is based on the idea that normally generated hydroperoxide species can damage DNA to produce mutations and react with protein sulfhydryl groups to perturb normal regulation of cancer-related pathways. GPX2 is one of many peroxidases available to control hydroperoxide levels. Altered GPX2 expression levels from normal to cancer or with cancer stages seems to be the main feature in bringing it to the attention of investigators. In this commentary, we examine this premise as a basis for cancer studies, largely by trying to place GPX2 within the larger context of antioxidant enzyme gene expression. We make use of public databases and illustrate their possible role in approaching this issue. Since use of such databases is new to us, we looked to sources in the literature to evaluate expression level data, finding general agreement with some discrepancies over the range of expression and relative expression levels among some samples. Using the database information, we critically evaluate methods used to study GPX2 in the current literature for a variety of cancers. Second, groups are now trying to compare enzymatic properties of GPX1 and GPX2 using proteins from bacterial cultures. We weigh in on these recent findings and discuss the impact on the relative GPX2 and GPX1 functions.

Selective Oxidation of Methane to Methanol over Au/H-MOR.

Selective oxidation of methane to methanol by dioxygen (O2) is an appealing route for upgrading abundant methane resource and represents one of the most challenging reactions in chemistry due to the overwhelmingly higher reactivity of the product (methanol) versus the reactant (methane). Here, we report that gold nanoparticles dispersed on mordenite efficiently catalyze the selective oxidation of methane to methanol by molecular oxygen in aqueous medium in the presence of carbon monoxide. The methanol productivity reaches 1300 μmol gcat-1 h-1 or 280 mmol gAu-1 h-1 with 75% selectivity at 150 °C, outperforming most catalysts reported under comparable conditions. Both hydroxyl radicals and hydroperoxide species participate in the activation and conversion of methane, while it is shown that the lower affinity of methanol on gold mainly accounts for higher methanol selectivity.

Hydroperoxide‐Mediated Degradation of Acetonitrile in the Lithium–Air Battery

AbstractUnderstanding and eliminating degradation of the electrolyte solution is arguably the major challenge in the development of high energy density lithium–air batteries. The use of acetonitrile provides cycle stability comparable to current state‐of‐the‐art glyme ethers and, while solvent degradation has been extensively studied, no mechanism for acetonitrile degradation has been proposed. Through the application of in situ pressure measurements and ex situ characterization to monitor the degradation of acetonitrile in the lithium–air battery, a correlation between H2O concentration within the cell and deviation from the idealized electron/oxygen ratio is revealed. Characterization of the cycled electrolyte solution identifies acetamide as the major degradation product under both cell and model conditions. A new degradation pathway is proposed that rationalizes the formation of acetamide, identifies the role of H2O in the degradation process, and confirms lithium hydroperoxide as a critical antagonistic species in lithium–air cells for the first time. These studies highlight the importance of considering the impact of atmospheric gases when exploring lithium–air cell chemistry and suggest that further exploration of the impact of hydroperoxide species on the degradation in lithium–air cells may lead to identification of more effective electrolyte solvents.

Group additivity values for entropy and heat capacities of C2–C8 alkanes, alkyl hydroperoxides, and their radicals

Group additivity values for the thermodynamic properties of oxygenated radicals are poorly determined due to the absence of high quality reference data. Here, a set of 58 group additive values (GAV) for the standard entropy and heat capacity of relevance to alkane oxidation is derived from fits to an extensive and accurate database of standard entropies (298.15 K) and heat capacities (300–3000 K) recently calculated with the “STAR-1D” formalism. The 192 species in this database represent the alkanes (RH), alkyl radicals (Ṙ), alkyl hydroperoxides (RO2H), alkyl-peroxy (RȮ2) and hydroperoxy-alkyl (Q˙OOH) radicals for all of the isomers of C2–C5 alkane fuels and a select number of isomers of C6–C9 species. The STAR-1D thermochemical data for this set of species was previously obtained from a coupling of scaled B2PLYPD3/cc-pVTZ vibrational analyses with scaled ωB97X-D/cc-pVTZ one-dimensional hindered rotor corrections. The 2σ uncertainties in the GAV results relative to the STAR-1D data set are 2.4 cal K–1 mol–1 for the entropies and at most 2.0 cal K–1 mol–1 for the heat capacities in the temperature range 500–800 K. The 2σ fitting uncertainties in the heat capacity gradually reduce at higher temperatures reaching a value of 0.8 cal K–1 mol–1 at 2000 K decreasing to only 0.5 cal K–1 mol–1 at 3000 K. The high degree of accuracy for the GAV representations is obtained through the introduction of various new group terms, together with the re-optimization of existing group terms. Among the full set of 58 GAV terms, 25 include non-next-nearest neighbor interactions (NNI) and β-corrections. The updated GAVs can be applied in the prediction of entropies and heat capacities for a wide range of hydrocarbons and hydroperoxide species and their radicals, which is important to the accurate prediction of fuel reactivity at low-temperatures in the range 600–1000 K.

Regulatory roles of ferroptosis-related non-coding RNAs and their research progress in urological malignancies.

Ferroptosis is a new type of cell death characterized by damage to the intracellular microenvironment, which causes the accumulation of lipid hydroperoxide and reactive oxygen species to cause cytotoxicity and regulated cell death. Non-coding RNAs (ncRNAs) play an important role in gene expression at the epigenetic, transcriptional, and post-transcriptional levels through interactions with different DNAs, RNAs, or proteins. Increasing evidence has shown that ferroptosis-related ncRNAs are closely related to the occurrence and progression of several diseases, including urological malignancies. Recently, the role of ferroptosis-associated ncRNAs (long non-coding RNAs, micro RNAs, and circular RNAs) in the occurrence, drug resistance, and prognosis of urological malignancies has attracted widespread attention. However, this has not yet been addressed systematically. In this review, we discuss this issue as much as possible to expand the knowledge and understanding of urological malignancies to provide new ideas for exploring the diagnosis and treatment of urological malignancies in the future. Furthermore, we propose some challenges in the clinical application of ferroptosis-associated ncRNAs.

Systematically derived thermodynamic properties for alkane oxidation

Key combustion properties, such as ignition delay time, show strong sensitivity to the thermochemistry of the main species in the standard radical oxidation pathway at low temperatures (600–1000 K). Significant uncertainties persist in current estimates of thermodynamic properties, particularly for the larger species of relevance to common practical fuels. In this article, we use advanced computational schemes to evaluate thermodynamic properties for the fuel, fuel radical, peroxy, hydroperoxy-alkyl radical, and hydroperoxide species for a set of 17 fuels containing up to 9 carbon atoms, and with various degrees of branching in the alkane backbone. The procedure, termed STAR-1D, combines conformer sampling to find the minimum geometry, B2PLYP-D3/cc-pVTZ harmonic frequency evaluations, and ωB97X-D/cc-pVTZ one-dimensional torsional mapping. It includes two physically-based scaling routines: a frequency-dependent scaling to B2PLYP-D3/cc-pVTZ anharmonic frequencies and a scaling of one-dimensional ωB97X-D/cc-pVTZ torsional profiles to reproduce the product of the B2PLYP-D3/cc-pVTZ frequencies in the harmonic limit. Substantive comparisons with existing experimental databases, together with careful examinations of key theoretical assumptions, are used to explore the accuracy of the predictions. These computationally intensive explorations of 195 species were facilitated by automated thermochemistry software. High accuracy 0 K heats of formation from a separate study are used with the STAR-1D computations to generate NASA polynomial representations. In addition to their intrinsic value, the present results also provide a reliable database for the optimization of group additivity or machine learning schemes for scaling to larger combustion systems. Towards this end a complementary extensive conformational analysis is carried out for the medium sized species and the thermodynamic properties of the lowest energy hydrogen-bonded and non-hydrogen bonded conformers are contrasted for larger hydroperoxy-alkyl radical species.

A wide range experimental study and further development of a kinetic model describing propane oxidation

This work presents an experimental and kinetic modeling study of propane oxidation. Ignition delay times of propane were measured in a high-pressure shock tube and in rapid compression machines in the temperature range 689 – 1700 K at equivalence ratios of 0.5, 1.0 and 2.0 in ‘air’, for a wide range of pressures from 20 to 90 bar. CO and H2O mole fraction profiles for propane oxidation were measured in a shock tube behind reflected shock waves in the temperature range 1370–1840 K at equivalence ratios of 0.5, 1.0 and 2.0 and at a pressure of approximately 1.3 atm. Moreover, propane oxidation was studied using a jet-stirred reactor coupled to a synchrotron vacuum ultraviolet photoionization mass spectrometer at low temperatures in the range 565 – 690 K and at a pressure of 1 atm. This wide range of experimental datasets for propane oxidation was used to reoptimize and update our previous kinetic mechanisms, AramcoMech3.0 and NUIGMech1.1. In the current mechanism, NUIGMech1.3, the thermochemical parameters of all species relevant to low-temperature propane oxidation chemistry, including propyl-peroxyl, hydroperoxyl-propyl, hydroperoxyl-propyl-peroxyl, and carbonyl-hydroperoxide radicals, are updated based on newly calculated values at the CCSD(T)-F12/TZ-F12//B2PLYPD3/TZ///B2PLYP-D3/TZ level of theory. The improvements made in the thermochemical values and in the kinetic parameters for the low-temperature propane oxidation reactions in NUIGMech1.3 result in better model agreement with the new IDTs and speciation data, including carbon monoxide, formaldehyde, propene, acetaldehyde and various minor products such as ethylene, acetic acid, acrolein as well as various hydroperoxide and cyclic ether species.

Efficient and clean epoxidation of methyl oleate to epoxidized methyl oleate catalyzed by external surface of TS-1 supported molybdenum catalysts

Methyl oleate (MO) macromolecules are unable to access the microporous channel of conventional titanium silicalite-1 (TS-1) zeolite, which leads to the low catalytic activity of epoxidation of MO. Herein, the small-size TS-1 (TS-1-s) zeolite with a large surface area was synthesized successfully through steam-assisted crystallization, and then a novel blocked TS-1-s supported molybdenum (Mo/TS-1-bs) catalysts were prepared for efficient epoxidation of MO with clean H 2 O 2 to produce epoxidized methyl oleate (EMO). Various characterization results show the introduced Mo species are highly dispersed on the outer surface of TS-1-s without destroying the structure of the MFI type zeolite and the tetrahedral framework titanium (Ti 4+ ). Interestingly, a large number of Mo 6+ species exhibit certain epoxidation activity attributing to the generating of electrophilic molybdenum hydroperoxide (Mo–OOH) species via Mo species combined with H 2 O 2 , which could adsorb and react easily with MO molecules. Furthermore, the Mo species are also coordinated with Ti 4+ species through the O atom resulting in the facile attack of the Ti–O bonds by H 2 O 2 and easy to form an active intermediate. Therefore, compared with the conventional TS-1, the Mo/TS-1-bs shows higher catalytic activity in the epoxidation reaction. However, the acidity of Mo/TS-1-bs catalysts increases with increasing the Mo-loading owing to the formation of Lewis acid, which leads to the side reactions and the decrease of EMO selectivity. As a consequence, the efficient, non-corrosive and green catalytic system for epoxidation of MO was developed, and the activity improvement of Mo species and the influence of acid were also demonstrated. • The clean small-size TS-1 and Mo/TS-1-bs catalysts were prepared successfully for epoxidation of methyl oleate (MO). • Mo species can promote the MO epoxidation through generating of molybdenum hydroperoxide and coordination with Ti 4+ . • The acidity of Mo/TS-1-bs catalysts can decrease the selectivity of epoxidized methyl oleate.

Oxidative Stress Linked Organ Lipid Hydroperoxidation and Dysregulation in Mouse Model of Nonalcoholic Steatohepatitis: Revealed by Lipidomic Profiling of Liver and Kidney.

Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.

Combining trace Pt with surface silylation to boost Au/uncalcined TS‐1 catalyzed propylene epoxidation with H2 and O2

AbstractModification of bifunctional Au‐Ti catalysts for direct epoxidation of propylene with H2 and O2 is of burgeoning interest for potential commercialization requirements with desirable overall catalytic performance. Herein, we report a strategy by combining the addition of trace Pt into Au particles to enhance hydroperoxide species formation with the surface silylation to promote propylene oxide (PO) desorption. The resultant catalyst, that is, silylated Au20Pt1/uncalcined TS‐1, gives rise to significantly enhanced propylene conversion of 11%, PO selectivity of 88%, and H2 efficiency of 41%. The underlying nature of the above enhanced performance is elucidated by multiple techniques, such as high‐angle annular dark‐field scanning transmission electron microscopy, X‐ray photoelectron spectroscopy, Fourier transform infrared spectra, and theoretical calculations. The insights revealed here, the synergy of Au‐Pt‐Ti triple active sites together with the surface silylation, could guide the rational design of Au‐Ti catalysts for propylene epoxidation to boost PO production.

Pendent Relay Enhances H2O2 Selectivity during Dioxygen Reduction Mediated by Bipyridine-Based Co-N2O2 Complexes.

Generally, cobalt-N2O2 complexes show selectivity for hydrogen peroxide during electrochemical dioxygen (O2) reduction. We recently reported a Co(III)-N2O2 complex with a 2,2'-bipyridine-based ligand backbone which showed alternative selectivity: H2O was observed as the primary reduction product from O2 (71 ± 5%) with decamethylferrocene as a chemical reductant and acetic acid as a proton donor in methanol solution. We hypothesized that the key selectivity difference in this case arises in part from increased favorability of protonation at the distal O position of the key intermediate Co(III)-hydroperoxide species. To interrogate this hypothesis, we have prepared a new Co(III) compound that contains pendent -OMe groups poised to direct protonation toward the proximal O atom of this hydroperoxo intermediate. Mechanistic studies in acetonitrile (MeCN) solution reveal two regimes are possible in the catalytic response, dependent on added acid strength and the presence of the pendent proton donor relay. In the presence of stronger acids, the activity of the complex containing pendent relays becomes O2 dependent, implying a shift to Co(III)-superoxide protonation as the rate-determining step. Interestingly, the inclusion of the relay results in primarily H2O2 production in MeCN, despite minimal difference between the standard reduction potentials of the three complexes tested. EPR spectroscopic studies indicate the formation of Co(III)-superoxide species in the presence of exogenous base, with greater O2 reactivity observed in the presence of the pendent -OMe groups.

CD36 Activation Enhances Hydrogen Peroxide Generation in Human Platelets

Cardiovascular disease remains the leading cause of death in the United States and thrombosis following atherosclerotic plaque rupture represents a significant portion of this mortality. Our lab and others have previously shown that oxidized lipids in low density lipoprotein (LDL) particles (oxLDL) serve as a “danger” signal that is recognized by CD36, a scavenger receptor of the innate immune system, found on platelets, and other vascular cells, that promotes platelet hyper-reactivity and thrombogenesis. Recently published data from our lab suggests that oxLDL-CD36 signaling in platelets involves generation of reactive oxygen species (ROS) that are important signaling pathway regulators and contribute to the sustained activation of the redox-sensitive MAPK, ERK5. This pathway is an important contributor to platelet activation and thrombosis. It is not well understood how CD36-dependent ROS generation maintains platelets under hyperlipidemic conditions in a “hypersensitive” state. Our hypothesis is that oxLDL acting through CD36 stimulates specific hydroperoxide species to modulate platelet signaling pathways. Using reverse-phase HPLC to detect 7-hydroxycoumarin (COH), the stable fluorescent product of the reaction between hydroperoxides and coumarin boronic acid (CBA), we selectively measured intracellular hydroperoxide generation in washed human platelets. Platelets showed a dose-dependent increase in COH accumulation of 70 ± 20%, 81 ± 13%, and 84 ± 16% after stimulation with oxLDL 25 μg/mL, 50 μg/mL, or 100 μg/mL, respectively (p=0.0115, p<0.01, p<0.01). Equivalent doses of native LDL produced no significant increase in COH accumulation. Platelets also showed a time-dependent increase in COH with maximal accumulation after 60 minutes of 50 μg/mL oxLDL stimulation (p<0.001). Previously published functional aggregometry data suggested that PEG-catalase, an enzyme that promotes hydrogen peroxide (H2O2) breakdown, prevents oxLDL-stimulated platelet activation and aggregation. Here we showed that pre-treatment of platelets with PEG-catalase blunted oxLDL-stimulated COH accumulation by 42 ± 8.6% (p=0.0106). In addition to H2O2, CBA reacts with peroxynitrite, but inhibition of platelet nitric oxide synthase with L-NG-Nitroarginine methyl ester (L-NAME) did not alter COH generation after oxLDL treatment when compared to oxLDL alone. Since CD36 signaling activates platelets through “non-classical” pathways, we examined how classic platelet agonists affected oxLDL-stimulated hydroperoxide generation. Treatment with collagen-related peptide (CRP) showed no significant increase in COH accumulation, but co-stimulation with oxLDL and CRP showed a 185 ± 26.4% increase in COH accumulation, as compared to oxLDL alone (p<0.0001), suggesting a synergistic effect between the oxLDL-CD36 and collagen-GPVI signaling pathways. This effect was not observed after thrombin or ADP treatment. Further studies are necessary to elucidate how oxLDL-stimulated hydroperoxide generation affects platelet signaling. Taken together our data suggests that oxLDL specifically generates H2O2 in platelets to promote a hyper-sensitive state and collagen-GPVI signaling may enhance this effect promoting thrombosis in pro-atherogenic conditions. Since mice and humans lacking CD36 do not demonstrate a bleeding diathesis and form normal thrombi, we reason that targeting CD36-mediated redox mechanisms has clinical potential to reduce the risk of atherothrombosis without compromising normal physiological hemostasis responses, unlike current anti-platelet agents in clinical use. DisclosuresNo relevant conflicts of interest to declare.

Functionalized Hydroperoxide Formation from the Reaction of Methacrolein-Oxide, an Isoprene-Derived Criegee Intermediate, with Formic Acid: Experiment and Theory.

Methacrolein oxide (MACR-oxide) is a four-carbon, resonance-stabilized Criegee intermediate produced from isoprene ozonolysis, yet its reactivity is not well understood. This study identifies the functionalized hydroperoxide species, 1-hydroperoxy-2-methylallyl formate (HPMAF), generated from the reaction of MACR-oxide with formic acid using multiplexed photoionization mass spectrometry (MPIMS, 298 K = 25 °C, 10 torr = 13.3 hPa). Electronic structure calculations indicate the reaction proceeds via an energetically favorable 1,4-addition mechanism. The formation of HPMAF is observed by the rapid appearance of a fragment ion at m/z 99, consistent with the proposed mechanism and characteristic loss of HO2 upon photoionization of functional hydroperoxides. The identification of HPMAF is confirmed by comparison of the appearance energy of the fragment ion with theoretical predictions of its photoionization threshold. The results are compared to analogous studies on the reaction of formic acid with methyl vinyl ketone oxide (MVK-oxide), the other four-carbon Criegee intermediate in isoprene ozonolysis.

Ferric Heme Superoxide Reductive Transformations to Ferric Heme (Hydro)Peroxide Species: Spectroscopic Characterization and Thermodynamic Implications for H-Atom Transfer (HAT).

A new end-on low-spin ferric heme peroxide, [(PIm )FeIII -(O22- )]- (PIm -P), and subsequently formed hydroperoxide species, [(PIm )FeIII -(OOH)] (PIm -HP) are generated utilizing the iron-porphyrinate PIm with its tethered axial base imidazolyl group. Measured thermodynamic parameters, the ferric heme superoxide [(PIm )FeIII -(O2⋅- )] (PIm -S) reduction potential (E°') and the PIm -HP pKa value, lead to the finding of the OO-H bond-dissociation free energy (BDFE) of PIm -HP as 69.5 kcal mol-1 using a thermodynamic square scheme and Bordwell relationship. The results are validated by the observed oxidizing ability of PIm -S via hydrogen-atom transfer (HAT) compared to that of the F8 superoxide complex, [(F8 )FeIII -(O2.- )] (S) (F8 =tetrakis(2,6-difluorophenyl)porphyrinate, without an internally appended axial base imidazolyl), as determined from reactivity comparison of superoxide complexes PIm -S and S with the hydroxylamine (O-H) substrates TEMPO-H and ABNO-H.

Ferric Heme Superoxide Reductive Transformations to Ferric Heme (Hydro)Peroxide Species: Spectroscopic Characterization and Thermodynamic Implications for H‐Atom Transfer (HAT)

AbstractA new end‐on low‐spin ferric heme peroxide, [(PIm)FeIII−(O22−)]− (PIm‐P), and subsequently formed hydroperoxide species, [(PIm)FeIII−(OOH)] (PIm‐HP) are generated utilizing the iron‐porphyrinate PIm with its tethered axial base imidazolyl group. Measured thermodynamic parameters, the ferric heme superoxide [(PIm)FeIII−(O2⋅−)] (PIm‐S) reduction potential (E°′) and the PIm‐HP pKa value, lead to the finding of the OO−H bond‐dissociation free energy (BDFE) of PIm‐HP as 69.5 kcal mol−1 using a thermodynamic square scheme and Bordwell relationship. The results are validated by the observed oxidizing ability of PIm‐S via hydrogen‐atom transfer (HAT) compared to that of the F8 superoxide complex, [(F8)FeIII−(O2.−)] (S) (F8=tetrakis(2,6‐difluorophenyl)porphyrinate, without an internally appended axial base imidazolyl), as determined from reactivity comparison of superoxide complexes PIm‐S and S with the hydroxylamine (O‐H) substrates TEMPO‐H and ABNO‐H.