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Ned A Porter

Publications and source records attributed to Ned A Porter.

At least 37 records · Page 2Linked to original sources
PubMed2004

Factors influencing the autoxidation of fatty acids: effect of olefin geometry of the nonconjugated diene.

Autoxidations of cis,cis, cis,trans, and trans,trans nonconjugated octadecadienoates and pentadecadienes were carried out in the presence of alpha-tocopherol to investigate the effect of olefin geometry on this oxidation process and provide insight into the factors that influence the autoxidation of fatty acids. We have found that as the trans character of the diene increases, the amount of O(2) trapping at the central (bis-allylic) position of the pentadienyl radical also increases. In addition, the rate constant for beta-fragmentation (k(beta) approximately 10(6) s(-1)) of the bis-allylic peroxyl radical decreased on going from the cis,cis to the trans,trans diene. We have also found that for the cis,trans nonconjugated dienes, there is a preference for trapping of the pentadienyl radical by O(2) at the transoid end, generating the cis,trans conjugated hydroperoxide as the major product.

Alkenes↗
PubMed2004

Mechanism of beta-silyl diacyl peroxide decomposition: a mild and stereoselective synthesis of beta-silyl esters.

A novel method for the formation of beta-silyl esters is presented. Mechanistic studies were carried out on the formation and decomposition of beta-silyl diacyl peroxides, showing that the decomposition pathway involves an ionic mechanism that is influenced by the presence of the beta-silyl moiety. These studies exclude a free radical decomposition pathway as evidenced by the absence of chemically induced dynamic nuclear polarization (CIDNP) during the reaction and a strong correlation of the resulting regioisomeric product distribution to sigma(+). This reaction allows for the formation of beta-silyl esters in 45-50% isolated yield with predictable regioselectivity and good to excellent diastereoselectivity. Studies demonstrate that ester products which are formed at benzylic centers have the erythro configuration, whereas ester products formed at alkyl centers have the threo configuration.

Esters↗
PubMed2004

Lysine peroxycarbamates: free radical-promoted peptide cleavage.

Strategies are reported that combine in one step a predictable chemical-based protein digestion with mass spectrometry. Lysine residue amino groups in peptides and proteins are modified by reaction with a peroxycarbonate derived from p-nitrophenol, and tert-butyl hydroperoxide. The peroxycarbonate reacts with lysine residues in peptides and proteins, and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID). Observed fragmentation of the peptides involves apparent free radical processes including Hofmann-Löffler-type rearrangements that lead to peptide chain fragmentation. Strategies for directed cleavage of peptides by free radical promoted processes are feasible, and such strategies may well simplify schemes for protein analysis.

Amino Acid Sequence↗
PubMed2004

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay.

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.

Alzheimer Disease↗
PubMed2004

Autoxidative transformation of chiral omega6 hydroxy linoleic and arachidonic acids to chiral 4-hydroxy-2E-nonenal.

Recently, we established that 13S-hydroperoxy-linoleic acid is converted to 4S-hydroperoxy-nonenal (4S-HPNE) during autoxidation, implicating hydrogen abstraction from C-8 as an initiating step [Schneider, C., et al. (2001) J. Biol. Chem. 275, 20831-20838]. On the basis of the proposed mechanism, an equivalent initiating reaction could occur from the corresponding 13S-hydroxy acid. Herein, we examined the outcome of autoxidation reactions of the omega6 hydroxy fatty acids, 13S-hydroxyoctadecadienoic acid and 15S-hydroxyeicosatetraenoic acid, as compared with reactions of the corresponding hydroperoxy substrates. Autoxidation of the hydroxy starting materials (37 degrees C, dry film) yielded 4-hydroxy-nonenal (4-HNE) as a prominent polar metabolite (and not the 4-hydroperoxide), whereas the hydroperoxide starting materials gave rise to 4-HPNE. Stereochemical analysis showed that the optical purity of the 4-hydroxy group of 4-HNE precisely matched the optical purity of the 15S- or 13S-hydroxy group of the starting fatty acid substrate (98 and 90% S, respectively). The hydroperoxide 15S-HPETE (98% 15S) gave rise to 4S-HPNE, also with retention of optical purity (98% 4S). The preservation of stereochemical configuration provides evidence that aldehyde formation does not involve participation of the hydro(pero)xy group and indicates a similar mechanism for the formation of 4-HNE and 4-HPNE during autoxidation of omega6 hydro(pero)xy fatty acids. Our results establish, moreover, that omega6 hydroxy fatty acids are potential precursors of reactive cytotoxic aldehydes in biological systems.

Aldehydes↗
PubMed2003

Mass spectrometry of intracellular and membrane proteins using cleavable detergents.

Detergents have been used to enhance the solubility of hydrophobic biomolecules for decades. Despite the widespread use of detergents in biochemistry, the presence of these molecules often complicates further analysis by mass spectrometry. This study presents a solution to this problem by outlining a method utilizing a novel cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS). This detergent can be used to extract protein contained within the interior of the cell by disrupting cell membranes. Once the proteins are free from the cell, PPS also assists in protein solubilization by shielding the hydrophobic regions of the newly extracted protein from unfavorable interactions with water. The added advantage of PPS over conventional detergents such as sodium dodecyl sulfate or n-octylglucoside is that the detergent properties that interfere with MALDI mass spectrometry can be eliminated prior to analysis. PPS was found to improve sensitivity in MALDI analyses of both soluble proteins and membrane proteins without degrading spectral quality. The virtues of this strategy were applied to whole cell extracts. Analysis of these extracts resulted in an overall increase in both the number of peaks observed and overall signal intensity.

Bacterial Proteins↗
PubMed2003

Liquid chromatography coordination ion-spray mass spectrometry (LC-CIS-MS) of docosahexaenoate ester hydroperoxides.

Coordination ion-spray mass spectrometry (CIS-MS) is a useful tool in the detection and identification of complex mixtures of cholesterol ester and phospholipid hydroperoxides. The methyl ester, cholesterol ester, and phospholipid hydroperoxides of docosahexaenoic acid were analyzed by LC-CIS-MS and their elution orders were identified. Their corresponding alcohols were also identified. The methyl hydroperoxydocosahexaenoate (HPDHE) elution order is 14, 17, 16, 13, 20, 11, 10, 4, 7, 8 while the methyl hydroxydocosahexaenoate (HDHE) elution order is 14 > or = 17, 16, 13, 11, 10, 20, 7, 8, 4. The cholesteryl HPDHE elution order is 14, 17, 16, 13, 20, 11 > or = 10, 4, 7, 8 and the cholesteryl HDHE elution order is 17, 14, 16, 13, 11, 20, 10, 7, 8, 4. The elution order of the 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylcholine (PDPC) hydroperoxides and alcohols is 20, 16, 17, 13, 14, 10, 11, 7, 8, 4.

Cholesterol Esters↗
PubMed2003

Identification of a novel class of endoperoxides from arachidonate autoxidation.

Free radical-initiated lipid autoxidation in low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Oxidation of the lipid components of LDL leads to a complex mixture of hydroperoxides, bicyclic endoperoxides, monocyclic peroxides, and serial cyclic peroxides. The oxidation compounds and/or their decomposition products can modify protein components, which may lead to various diseases. A novel class of peroxides (termed dioxolane-isoprostanes) having a bicyclic endoperoxide moiety characteristic of the isoprostanes and a dioxolane peroxide functionality in the same molecule was identified in the product mixture formed from in vitro autoxidation of cholesteryl arachidonate. The same products are also detected in in vitro oxidized LDL. Various mass spectrometric techniques have been applied to characterize these new peroxides. The structure of these compounds has also been confirmed by independent synthesis. We reason, based on the free radical mechanism of the transformation, that only the 12- and 8-peroxyl radicals (those leading to 12-HPETE and 8-HPETE) of arachidonate can form these new peroxides. We also suggest that the formation of these peroxides provides a rationale to explain the fact that 5- and 15-series isoprostanes are formed in preference to 8- and 12-series. Furthermore, series of other isoprostanes, such as dioxolane A(2), D(2), E(2), etc., can be derived from the dioxolane-isoprostane peroxides. These findings offer further insights into the oxidation products of arachidonate and the opportunity to study their potential biological relevance.

Arachidonic Acid↗
PubMed2003

Theoretical calculations of carbon-oxygen bond dissociation enthalpies of peroxyl radicals formed in the autoxidation of lipids.

Theoretical calculations were carried out to provide a framework for understanding the free radical oxidation of unsaturated lipids. The carbon[bond]hydrogen bond dissociation enthalpies (BDEs) of organic model compounds and oxidizable lipids (R[bond]H) and the carbon[bond]oxygen bond dissociation enthalpies of peroxyl radical intermediates (R[bond]OO*) have been calculated. The carbon[bond]hydrogen BDEs correlate with the rate constant for propagation of free radical autoxidation, and the carbon[bond]oxygen BDEs of peroxyl radicals correlate with rate constants for beta-fragmentation of these intermediates. Oxygen addition to intermediate carbon radicals apparently occurs preferentially at centers having the highest spin density. The calculated spin distribution therefore provides guidance about the partitioning of oxygen to delocalized carbon radicals. Where the C[bond]H BDEs are a function of the extent of conjugation in the parent lipid and the stability of the carbon radical derived therefrom, C[bond]OO* BDEs are also affected by hyperconjugation. This gives way to different rates of beta-fragmentation of peroxyl radicals formed from oxygen addition at different sites along the same delocalized radical. We have also studied by both theory and experiment the propensity for benzylic radicals to undergo oxygen addition at their ortho and para carbons which, combined, possess an equivalent unpaired electron spin density as the benzylic position itself. We find that the intermediate peroxyl radicals in these cases have negative C[bond]OO* BDEs and, thus, have rate constants for beta-fragmentation that exceed the diffusion-controlled limit for the reaction of a carbon-centered radical with oxygen.

Arachidonic Acid↗
PubMed2003

Formation of prostaglandins E2 and D2 via the isoprostane pathway: a mechanism for the generation of bioactive prostaglandins independent of cyclooxygenase.

It has heretofore been assumed that the cyclooxygenases (COXs) are solely responsible for peostaglandin (PG) synthesis in vivo. An important structural feature of PGH2 formed by COX is the trans-configuration of side chains relative to the prostane ring. Previously, we reported that a series of PG-like compounds termed isoprostanes (IsoPs) are formed in vivo in humans from the free radical-catalyzed peroxidation of arachidonate independent of COX. A major difference between these compounds and PGs is that IsoPs are formed from endoperoxide intermediates, the vast majority of which contain side chains that are cis relative to the prostane ring. In addition, unlike the formation of eicosanoids from COX, IsoPs are formed as racemic mixtures because they are generated nonenzymatically. IsoPs containing E- and D-type prostane rings (E2/D2-IsoPs) are one class of IsoPs formed, and we have reported previously that one of the major IsoPs generated is 15-E2t-IsoP (8-iso-PGE2). Unlike PGE2, 15-E2t-IsoP is significantly more unstable in buffered solutions in vitro and undergoes epimerization to PGE2. Analogously, the D-ring IsoP (15-D2c-IsoP) would be predicted to rearrange to PGD2. We now report that compounds identical in all respects to PGE2 and PGD2 and their respective enantiomers are generated in vivo via the IsoP pathway, presumably by epimerization of racemic 15-E2t-IsoP and 15-D2c-IsoP, respectively. Racemic PGE2 and PGD2 were present esterified in phospholipids derived from liver tissue from rats exposed to oxidant stress at levels of 24 +/- 16 and 37 +/- 12 ng/g of tissue, respectively. In addition, racemic PGs, particularly PGD2, were present unesterified in urine from normal animals and humans and represented up to 10% of the total PG detected. Levels of racemic PGD2 increased 35-fold after treatment of rats with carbon tetrachloride to induce oxidant stress. In this setting, PGD2 and its enantiomer generated by the IsoP pathway represented approximately 30% of the total PGD2 present in urine. These findings strongly support the contention that a second pathway exists for the formation of bioactive PGs in vivo that is independent of COX.

Animals↗
PubMed2003

Mechanisms for the formation of isoprostane endoperoxides from arachidonic acid. "Dioxetane" intermediate versus beta-fragmentation of peroxyl radicals.

The isoprostanes are a class of autoxidation products generated from arachidonic acid (or its esters) by a free radical initiated process. The potent biological activity of these compounds has been attracting intense research interest since they were detected in humans as well as animal models in the early 1990s. The measurement of these compounds has been regarded as one of the most useful non-invasive biomarkers for oxidative stress status. Two mechanisms for the formation of these compounds have been proposed. In the first mechanism, a peroxyl radical undergoes successive 5-exo cyclizations analogous to the enzymatic mechanism proposed for prostaglandin biosynthesis. The second mechanism starts with a 4-exo cyclization of a peroxyl radical leading to an intermediate dioxetane, a mechanism that has also been proposed for prostaglandin biosynthesis as well as for the formation of 4-hydroxy nonenal (HNE). Autoxidation of cholesteryl-15-HpETE under free radical conditions provides Type IV isoprostanes. The "dioxetane" mechanism for isoprostane generation from 15-HpETE requires that optically pure products are formed from an optically pure reactant, whereas an alternate mechanism for the process involving beta-fragmentation of the 15-peroxyl would give racemic isoprostane products. We have carried out a test of the mechanism based upon these stereochemical requirements. The results of analysis of the product mixture derived from autoxidation of optically pure Ch-15-HpETE by atmospheric pressure chemical ionization-mass spectrometry coupled with chiral high performance liquid chromatography indicate that the major isoprostane diastereomers are formed as a racemic mixture. These experimental results are consistent with a mechanism for isoprostane formation involving beta-fragmentation of the 15-peroxyl radical followed by re-addition of oxygen to form the 11-HPETE peroxyl, and they exclude a mechanism proceeding through the formation of a dioxetane intermediate.

Arachidonic Acid↗
PubMed2003

Role of hyperconjugation in determining carbon-oxygen bond dissociation enthalpies in alkylperoxyl radicals.

[reaction: see text] Theoretical calculations of carbon-oxygen bond dissociation enthalpies in substituted methylperoxyl radicals (YCH(2)OO*) reveal that bond strengths are not governed solely by the stability of YCH2* radicals but are largely affected by hyperconjugation when Y is electron-donating or conjugating. In many cases, this hyperconjugative effect is greater than stabilization of the methyl radical by Y. All electron-withdrawing Y exert small destabilizing effects via inductive withdrawal of electrons from the polarized C-OO* bond.

Free Radicals↗
PubMed2003

Specificity of the ferrous oxidation of xylenol orange assay: analysis of autoxidation products of cholesteryl arachidonate.

Autoxidation of polyunsaturated fatty acids and esters leads to a complex mixture containing hydroperoxides and cyclic peroxides. The oxidation mixture of cholesteryl arachidonate, which has been characterized by a variety of mass spectrometry techniques, was subject to analysis by conventional thiobarbituric acid-reactive substance (TBARS) and ferrous oxidation in xylenol orange (FOX) assays. Our results indicate that the FOX assay is not specific for hydroperoxides. Cyclic peroxides, such as monocyclic peroxides and serial-cyclic peroxides, give a positive FOX response even after triphenylphosphine reduction. We suggest that bicyclic endoperoxides are the major TBARS active compounds present in cholesteryl arachidonate oxidation mixtures. These compounds give a positive FOX assay before reaction with triphenylphosphine but negative TBARS and FOX assays after this reaction. Caution should be exercised when the FOX assay is used to analyze highly oxidized lipids, especially arachidonyl-containing lipids.

Arachidonic Acids↗
PubMed2003

Antioxidants significantly affect the formation of different classes of isoprostanes and neuroprostanes in rat cerebral synaptosomes.

Lipid peroxidation has been implicated in the pathogenesis of a number of diseases, including neurodegenerative disorders. Evidence that antioxidants can affect the clinical course of neurodegenerative diseases is limited. In the present study, we examined the ability of five common antioxidants or antioxidant combinations, alpha-tocopherol, gamma-tocopherol, ascorbic acid, GSH ethyl ester, and a combination of ascorbate and alpha-tocopherol, to modulate lipid peroxidation in peroxidizing rat cerebral synaptosomes, a well-characterized model of oxidant injury. In these studies, we quantified isoprostanes (IsoPs) derived from arachidonic acid as an index of whole tissue oxidation and neuroprostanes (NeuroPs) formed from docosahexaenoic acid as a marker of selective neuronal peroxidation. We report that these various antioxidants displayed markedly different capacities to inhibit IsoP and NeuroP formation with the most potent effects on IsoPs observed for ascorbate, GSH ethyl ester, and the alpha-tocopherol-ascorbate combination. alpha-Tocopherol was slightly less potent and gamma-tocopherol significantly less effective. The concentration-response relationships were significantly different for NeuroP formation with the antioxidants being significantly less potent than for IsoP generation. In particular, alpha-tocopherol did not inhibit NeuroP formation at concentrations up to 100 microM. We also determined that tocopherols, in particular alpha-tocopherol, act in vitro as reducing agents to convert IsoP and NeuroP endoperoxides to reduced F-ring compounds, a finding we have observed previously in vivo in brain. These studies are of importance because they have further defined the role of antioxidants to modulate the formation of lipid peroxidation products in peroxidizing brain tissue. In addition, they suggest that alpha-tocopherol may not be a particularly effective agent to inhibit oxidant stress in the terminal compartment of neurons in the central nervous system.

Animals↗
PubMed2003

Analysis of unsaturated compounds by Ag+ coordination ionspray mass spectrometry: studies of the formation of the Ag+/lipid complex.

Coordination ionspray mass spectrometry (CIS-MS) is a useful tool in the detection and identification of cholesterol ester and phospholipid hydroperoxides and diacyl peroxides. Extensive studies of a series of cholesterol esters using CIS-MS revealed the following: (1) Cholesterol esters with equal number of double bonds as the internal standard showed a linear relative response in the mass spectrometer while compounds with non-equal numbers of double bonds gave a nonlinear relative response. (2) Complex adducts containing cholesterol ester, silver ion, AgF, AgBF(4), and 2-propanoxide form when silver is in molar excess of cholesterol esters, reducing the [M + Ag](+) signal. (3) In a mixture of cholesterol esters where silver is limiting, Ch22:6 and Ch20:4 bind to silver at the expense of Ch18:2 and have a higher signal in the mass spectrometer. (4) In a mixture of cholesterol esters where silver concentration is twofold greater than total cholesterol ester concentration, Ch22:6 and Ch20:4 form large complex adducts more frequently than Ch18:2 and have a lower signal in the mass spectrometer.

Cations, Monovalent↗
PubMed2002

Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension.

Free radicals have been implicated in the pathogenesis of an increasing number of diseases. Lipids, which undergo peroxidation, are major targets of free radical attack. We report the discovery of a pathway of lipid peroxidation that forms a series of isomers in vivo that are characterized by a substituted tetrahydrofuran ring structure, termed isofurans (IsoFs). We have proposed two distinct pathways by which IsoFs can be formed based on 18O2 and H2 18O labeling studies. Measurement of F2-isoprostanes (IsoPs), prostaglandin F2-like compounds formed nonenzymatically as products of lipid peroxidation, is considered one of the most reliable approaches for assessing oxidative stress status in vivo. However, one limitation with this approach is that the formation of IsoPs becomes limited at high oxygen tension. In contrast, the formation of IsoFs becomes increasingly favored as oxygen tension increases. IsoFs are present at readily detectable levels in normal fluids and tissues, and levels increase dramatically in CCl4-treated rats, an animal model of oxidant injury. The ratio of IsoFs to IsoPs in major organs varies according to normal steady-state tissue oxygenation. In addition, IsoFs show a marked increase early in the course of hyperoxia-induced lung injury, whereas IsoPs do not significantly increase. We propose that combined measurement of IsoFs and IsoPs should provide a more reliable index of oxidant stress severity than quantification of either alone because of the opposing modulation of the two pathways by oxygen tension, which can vary widely in different organs and disease states.

Animals↗