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Biomedical subjects

Ned A Porter

Publications and source records attributed to Ned A Porter.

At least 19 recordsLinked to original sources
PubMed2006

In vivo and in vitro lipid peroxidation of arachidonate esters: the effect of fish oil omega-3 lipids on product distribution.

The effect of lipid composition on the distribution of free radical oxidation products derived from arachidonic acid (20:4) esters has been studied in vitro and in vivo. Pro-inflammatory prostaglandin (PG) F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo and in vitro by the free radical-catalyzed peroxidation of arachidonic acid. Controlled free radical oxidation of mixtures of fatty acid esters in vitro showed that the formation of IsoPs from arachidonate is dramatically influenced by the presence of other fatty acid esters in the reaction mixture. Thus, three lipid mixtures containing the same arachidonate concentration but different amounts of other fatty esters (16:0; 18:1; 18:2; 20:5, and 22:6) were oxidized, and the product yields were determined by GC and LC/MS/MS analysis. The yield of F2-IsoP formed after 1 h of oxidation was 18% (based on arachidonate consumed) for mixtures containing arachidonate as the only oxidizable PUFA, but yields of these biologically active compounds dropped to 6% in polyunsaturated fatty acid (PUFA) mixtures typical of those found in tissues of fish oil-fed animals. F2-IsoP levels were also monitored in the livers of mice on diets supplemented with eicosapentaenoic acid (C20:5 omega-3; EPA), the PUFA most abundant in fish oil. While the level of arachidonic acid present in livers was not significantly different from that in control animals, levels of IsoPs in the liver were reduced in the EPA-fed mice compared to those in controls under conditions of oxidative stress (60 +/- 25% reduction, n = 5) or at baseline (48 +/- 14% reduction, n = 5). These results suggest that dietary omega-3 PUFAs may influence the formation of bio-active peroxidation products derived from omega-6 PUFAs by channeling the free radical pathway away from the F2-IsoPs.

Arachidonic Acid↗
PubMed2006

Urinary prostaglandin F2alpha is generated from the isoprostane pathway and not the cyclooxygenase in humans.

Prostaglandins (PGs) derived from the enzymatic oxidation of arachidonic acid by the cyclooxygenases (COXs) are potent lipid mediators involved in human physiology and pathophysiology. Structurally similar compounds, the isoprostanes (IsoPs), are generated from the free radical-catalyzed oxidation of arachidonic acid independent of COX. IsoPs exhibit significant bioactivity and play a role in the pathogenesis of diseases associated with oxidant injury. As one of the major PGs, prostaglandin F(2alpha) (PGF(2alpha)) is present in human urine in significant concentrations and is presumed to be derived from COX activity. We determined, however, that levels of putative PGF(2alpha) in urine cannot be suppressed by nonsteroidal anti-inflammatory agents, suggesting that it is generated via another mechanism(s). An important difference between COX-derived PGF(2alpha) and the IsoPs is that the former is an optically pure compound, whereas IsoPs are racemic. Utilizing a rodent model of oxidative stress, we now show that significant amounts of compounds identical in all respects to PGF(2alpha) and its enantiomer, ent-PGF(2alpha), are formed in equal amounts esterified in tissue phospholipids, suggesting that these compounds are derived via the IsoP pathway. Further, employing liquid chromatography/mass spectrometry, the vast majority of putative PGF(2alpha) in human urine is derived from the free radical-initiated peroxidation of arachidonate independent of COX and is composed of PGF(2alpha) and its enantiomer, although the latter compound is approximately 2-fold more abundant. Thus, quantification of urinary PGF(2alpha) actually reflects oxidative stress status as opposed to COX activity. Indeed, levels of this compound are elevated in urine from cigarette smokers and in humans with hypercholesterolemia, two conditions associated with oxidant stress. The elucidation that urinary PGF(2alpha) in humans is derived from the IsoP pathway has implications regarding PG formation and inhibition in vivo.

Animals↗
PubMed2006

Visible light excitation of CdSe nanocrystals triggers the release of coumarin from cinnamate surface ligands.

The photochemical properties of organic ligands on the surface of nanocrystalline CdSe particles were examined. A number of thiols carrying a substituted cinnamate tail was synthesized. In solution, these cinnamate compounds undergo light-induced (374 nm) E-Z isomerization, followed by a nonphotolytic lactonization to give highly fluorescent coumarin. The cinnamate-thiols were successfully exchanged onto the CdSe nanocrystal, and the photochemical behavior of these conjugates was studied. Upon aerobic photolysis at 374 nm, the surface cinnamates released coumarin accompanied by rapid nanocrystal degradation. This degradation was not observed under similar anaerobic conditions or when the organic ligands did not contain the cinnamate group. Surprisingly, very similar results were obtained upon irradiation at visible wavelengths at which the cinnamate has no absorption. With the aid of UV-visible absorption spectroscopy, fluorescence spectroscopy, and electrochemistry, a unified theory for both the increased photoinstability of the nanocrystal as well as the coumarin release was proposed. It involves cinnamate radical anions on the CdSe surface, formed upon electron transfer from the excited nanocrystal to the surface cinnamate, undergoing E-Z isomerization. Practically, this results in the remarkable ability to release coumarin from nanocrystal ligands simply by exciting the nanocrystal with visible light. This new photorelease protocol not only aids in the understanding of fundamental nanocrystal-ligand interactions but may also offer new opportunities in the areas of drug delivery and imaging.

Journal Article↗
PubMed2006

N-Terminal amino acid side-chain cleavage of chemically modified peptides in the gas phase: a mass spectrometry technique for N-terminus identification.

Although genome databases have become the key for proteomic analyses, de novo sequencing remains essential for the study of organisms whose genomes have not been completed. In addition, post-translational modifications present a challenge in database searching. Recognition of the b or y-ion series in a peptide MS/MS spectrum as well as identification of the b1 - and yn-1 -ions can facilitate de novo analyses. Therefore, it is valuable to identify either amino-acid terminus. In previous work, we have demonstrated that peptides modified at the epsilon-amino group of lysine as a t-butyl peroxycarbamate derivative undergo free radical promoted peptide backbone fragmentation under low-energy collision-induced dissociation (CID) conditions. Here we explore the chemistry of the N-terminal amino group modified as a t-butyl peroxycarbamate. The conversion of N-terminal amines to peroxycarbamates of simple amino acids and peptides was studied with aryl t-butyl peroxycarbonates. ESI-MS/MS analysis of the peroxycarbamate adducts gave evidence of a product ion corresponding to the neutral loss of the N-terminal side chain (R), thus identifying this residue. Further fragmentation (MS3) of product ions formed by N-terminal residue side-chain loss (-R) exhibited an m/z shift of the b-ions equal to the neutral loss of R, therefore labeling the b-ion series. The study was extended to the analysis of a protein tryptic digest where the SALSA algorithm was used to identify spectra containing these neutral losses. The method for N-terminus identification presented here has the potential for improvement of de novo analyses as well as in constraining peptide mass mapping database searches.

Amines↗
PubMed2006

Peroxyl radical clocks.

A series of peroxyl radical clocks has been developed and calibrated based on the competition between the unimolecular beta-fragmentation (k(beta)) of a peroxyl radical and its bimolecular reaction with a hydrogen atom donor (k(H)). These clocks are based on either methyl linoleate or allylbenzene and were calibrated directly with alpha-tocopherol or methyl linoleate, which have well-established rate constants for reaction with peroxyl radicals (k(H-tocopherol) = 3.5 x 10(6) M(-1) s(-1), k(H-linoleate) = 62 M(-1) s(-1)). This peroxyl radical clock methodology has been successfully applied to determine inhibition and propagation rate constants ranging from 10(0) to 10(7) M(-1) s(-1).

Calibration↗
PubMed2006

Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid.

Eicosapentaenoic acid (EPA, C20:5, omega-3) is the most abundant polyunsaturated fatty acid (PUFA) in fish oil. Recent studies suggest that the beneficial effects of fish oil are due, in part, to the generation of various free radical-generated non-enzymatic bioactive oxidation products from omega-3 PUFAs, although the specific molecular species responsible for these effects have not been identified. Our research group has previously reported that pro-inflammatory prostaglandin F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether F2-IsoP-like compounds (F3-IsoPs) are formed from the oxidation of EPA in vivo. Oxidation of EPA in vitro yielded a series of compounds that were structurally established to be F3-IsoPs using a number of chemical and mass spectrometric approaches. The amounts formed were extremely large (up to 8.7 + 1.0 microg/mg EPA) and greater than levels of F2-IsoPs generated from arachidonic acid. We then examined the formation of F3-IsoPs in vivo in mice. Levels of F3-IsoPs in tissues such as heart are virtually undetectable at baseline, but supplementation of animals with EPA markedly increases quantities up to 27.4 + 5.6 ng/g of heart. Interestingly, EPA supplementation also markedly reduced levels of pro-inflammatory arachidonate-derived F2-IsoPs by up to 64% (p < 0.05). Our studies provide the first evidence that identify F3-IsoPs as novel oxidation products of EPA that are generated in vivo. Further understanding of the biological consequences of F3-IsoP formation may provide valuable insights into the cardioprotective mechanism of EPA.

Animals↗
PubMed2006

Molecular dynamics simulations of arachidonic acid complexes with COX-1 and COX-2: insights into equilibrium behavior.

The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors.

Arachidonic Acid↗
PubMed2006

Molecular dynamics simulations of arachidonic acid-derived pentadienyl radical intermediate complexes with COX-1 and COX-2: insights into oxygenation regio- and stereoselectivity.

The two cyclooxygenase enzymes, COX-1 and COX-2, are responsible for the committed step in prostaglandin biosynthesis and are the targets of the nonsteroidal antiinflammatory drugs aspirin and ibuprofen and the COX-2 selective inhibitors, Celebrex, Vioxx, and Bextra. The enzymes are remarkable in that they catalyze two dioxygenations and two cyclizations of the native substrate, arachidonic acid, with near absolute regio- and stereoselectivity. Several theories have been advanced to explain the nature of enzymatic control over this series of reactions, including suggestions of steric shielding and oxygen channeling. As proposed here, selective radical trapping and spin localization in the substrate-derived pentadienyl radical intermediate can also be envisioned. Herein we describe the results of explicit, 10 ns molecular dynamics simulations of both COX-1 and COX-2 with the substrate-derived pentadienyl radical intermediate bound in the active site. The enzymes' influence on the conformation of the pentadienyl radical was investigated, along with the accessible space above and below the radical plane and the width of several channels to the active site that could function as access routes for molecular oxygen. Additional simulations demonstrated the extent of molecular oxygen mobility within the active site. The results suggest that spin localization is unlikely to play a role in enzymatic control of this reaction. Instead, a combination of oxygen channeling, steric shielding, and selective radical trapping appears to be responsible. This work adds a dynamic perspective to the strong foundation of static structural data available for these enzymes.

Arachidonic Acid↗
PubMed2005

Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods.

Isoprostanes are isomers of prostaglandins that are generated from free radical-initiated autoxidation of arachidonic acid. Quantification of F(2)-isoprostanes is regarded as the "gold standard" to assess oxidative stress in various human diseases. There are 32 possible racemic isoprostane isomers that exist as four sets of regioisomers. Each regioisomer is composed of eight diastereomers. We report liquid chromatographic/mass spectrometric methods to separate and identify F(2)-isoprostane stereoisomers. These methods have been applied to the analysis of F(2)-isoprostanes derived from tissues of rats exposed to an oxidative stress and are useful to assess the relative formation of various regioisomers and stereoisomers generated in vitro and in vivo. The delineation of the more abundant isomers formed will allow for studies to examine the biological relevance of selected compounds in vivo.

Animals↗
PubMed2005

Lipid-soluble 3-pyridinol antioxidants spare alpha-tocopherol and do not efficiently mediate peroxidation of cholesterol esters in human low-density lipoprotein.

Because alpha-tocopherol (alpha-TOH) mediates the peroxidation of cholesterol-esterified lipids in human low-density lipoprotein (LDL) in vitro and has displayed disappointing results against the onset and development of atherosclerosis, it may not be appropriate for use as a therapeutic in the prevention and/or treatment of the disease. Herein are described the experimental results demonstrating that 3-pyridinols spare alpha-TOH, do not efficiently mediate lipid peroxidation, and protect lipoprotein tryptophan residues in human LDL.

Antioxidants↗
PubMed2005

Combination detergent/MALDI matrix: functional cleavable detergents for mass spectrometry.

This study reports the synthesis of the first functional cleavable detergent designed specifically for applications in mass spectrometry. Upon cleavage, two inert compounds and the MALDI matrix are formed, eliminating sources of potential interference originating from traditional cleavable detergents. Analysis of peptides demonstrates that MALDI matrix generated in situ results in MALDI spectra equivalent to those prepared using established protocols. Analysis of the membrane protein diacylglycerol kinase was accomplished using the combination detergent/MALDI matrix. Applications of the functional cleavable detergents to the profiling of whole cell lysates results in increased signal-to-noise ratios of many ions and the detection of additional proteins previously not observed.

Acrylates↗
PubMed2005

Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo.

Isoprostanes (IsoPs) are isomers of prostaglandins that are generated from the free radical-initiated peroxidation of arachidonic acid (C20.4 omega-6). IsoPs exert potent bioactivity and are regarded as the "gold standard" to assess oxidative stress in various human diseases. Analogously, autoxidation of docosahexaenoic acid (DHA, C22.6 omega-3) generates an array of IsoP-like compounds that are termed neuroprostanes (NPs). A major class of NPs identified in vitro and in vivo contains F-type prostane rings and are know as F4-NPs. A number of different F4-NP regioisomers are formed from the peroxidation of DHA. Among the eight possible regioisomeric groups, we hypothesize that 4- and 20-series NPs are generated in greater amounts than other classes because the precursors that lead to regioisomers other than those of the 4- and 20-series can be further oxidized to form novel dioxolane-IsoP-like compounds, analogous to those generated from arachidonate. Various mass spectrometric approaches, including electron capture atmospheric pressure chemical ionization mass spectrometry, were utilized to analyze NPs formed in vitro and in vivo based on their characteristic fragmentation in the gas phase. Experimental results were consistent with our hypothesis that 4- and 20-series NP regioisomers are preferentially generated. The discovery of regioselectivity in the formation of NPs will allow studies of the biological activities of NPs to focus on the more abundantly generated compounds to determine their role in modulating the pathophysiological consequences of DHA oxidation and oxidant stress.

Animals↗
PubMed2005

Nonacid cleavable detergents applied to MALDI mass spectrometry profiling of whole cells.

Although cleavable detergents were first synthesized a number of years ago, they have only recently been successfully applied to problems involving biological molecules. Recent reports have demonstrated that these compounds are useful for applications involving both 2D PAGE and mass spectrometry. However, most cleavable surfactants have utilized acid-labile functional groups to affect cleavage. In applications where extreme pH is required, acid cleavable detergents have limited usefulness. We report the synthesis of fluoride cleavable silane compounds and photolabile cinnamate esters as cleavable detergents having alternative cleavage chemistries than previously reported cleavable detergents. These compounds were applied to whole cell analysis using MALDI mass spectrometry, and it was demonstrated that their use results in an increase in the number of proteins analyzed by increasing protein solubility.

Cell Line, Tumor↗
PubMed2005

Synthesis of dihydroperoxides of linoleic and linolenic acids and studies on their transformation to 4-hydroperoxynonenal.

The cytotoxic aldehydes 4-hydroxynonenal, 4-hydroperoxynonenal (4-HPNE), and 4-oxononenal are formed during lipid peroxidation via oxidative transformation of the hydroxy or hydroperoxy precursor fatty acids, respectively. The mechanism of the carbon chain cleavage reaction leading to the aldehyde fragments is not known, but Hock-cleavage of a suitable dihydroperoxide derivative was implicated to account for the fragmentation [Schneider, C., Tallman, K.A., Porter, N.A., and Brash, A.R. (2001) Two Distinct Pathways of Formation of 4-Hydroxynonenal. Mechanisms of Nonenzymatic Transformation of the 9- and 13-Hydroperoxides of Linoleic Acid to 4-Hydroxyalkenals, J. Biol. Chem. 275, 20831-20838]. Both 8,13- and 10,13-dihydroperoxyoctadecadienoic acids (diHPODE) could serve as precursors in a Hock-cleavage leading to 4-HPNE via two different pathways. Here, we synthesized diastereomeric 9,12-, 10,12-, and 10,13-diHPODE using singlet oxidation of linoleic acid. 8,13-Dihydroperoxyoctadecatrienoic acid was synthesized by vitamin E-controlled autoxidation of gamma-linolenic acid followed by reaction with soybean lipoxygenase. The transformation of these potential precursors to 4-HPNE was studied under conditions of autoxidation, hematin-, and acid-catalysis. In contrast to 9- or 13-HPODE, neither of the dihydroperoxides formed 4-HPNE on autoxidation (lipid film, 37 degrees C), regardless of whether the free acid or the methyl ester derivative was used. Acid treatment of 10,13-diHPODE led to the expected formation of 4-HPNE as a significant product, in accord with a Hock-type cleavage reaction. We conclude that, although the suppression of 4-H(P)NE formation from monohydroperoxides by alpha-tocopherol indicates peroxyl radical reactions in the major route of carbon chain cleavage, the dihydroperoxides previously implicated are not intermediates in the autoxidative transformation of monohydroperoxy fatty acids to 4-HPNE and related aldehydes.

Aldehydes↗
PubMed2005

Peroxidation of polyunsaturated fatty acid methyl esters catalyzed by N-methyl benzohydroxamic acid: a new and convenient method for selective synthesis of hydroperoxides and alcohols.

A new catalytic system, based on N-methyl benzohydroxamic acid (NMBHA), was developed for the selective synthesis of lipid hydroperoxides and alcohols by aerobic oxidation of lipid precursors. The reactions were carried out under mild conditions with air at atmospheric pressure and 37 degrees C. The products were isolated in much better yields (50-60%) as compared to previously reported procedures. NMBHA can be recovered at the end of the reaction and recycled. The putative reactive intermediate, a nitroxyl radical, readily abstracts a hydrogen atom from polyunsaturated lipid precursors such as linoleate esters, and NMBHA itself is an excellent hydrogen atom donor, thus conferring high selectivity to oxidations catalyzed by this reagent.

Alcohols↗
PubMed2004

Synthesis and reactivity of some 6-substituted-2,4-dimethyl-3-pyridinols, a novel class of chain-breaking antioxidants.

The synthesis and study of a series of 6-substituted-2,4-dimethyl-3-pyridinols having interesting antioxidant properties is reported. The general synthetic strategy leading to the compounds involved a low-temperature aryl bromide-to-alcohol conversion as the last step. 2,4-dimethyl-3-pyridinol (1a), 2,4,6-trimethyl-3-pyridinol (1b), and 2,4-dimethyl-6-(dimethylamino)-3-pyridinol (1d) were thus prepared from the corresponding 3-bromopyridine precursor. The methoxy derivative 2,4-dimethyl-6-(methoxy)-3-pyridinol (1c) was also prepared by an alternate route via a Baeyer-Villiger reaction on the substituted benzaldehyde precursor. Novel bicyclic pyridinols 2 and 3 required prior construction of the ring structure. Thus, 2 was prepared by the use of a 6-step intramolecular Friedel-Crafts strategy, and 3 required an 11-step sequence with a thermolytic intramolecular inverse-demand Diels-Alder reaction between a pyrimidine ring and an alkyne as the key step. Basicities of the pyridinols approached physiological pH with increasing electron density in the ring. Pyridinols 1a-d were found to be indefinitely stable to air oxidation while 2 and 3 decomposed upon extended exposure to the atmosphere. The reactivities of the pyridinols toward chain-carrying peroxyl radicals in homogeneous organic solution were examined by studying the kinetics of radical-initiated styrene autoxidations under controlled conditions. These experiments revealed that some of the newly synthesized pyridinols are the most effective phenolic chain-breaking antioxidants reported to date.

Antioxidants↗
PubMed2004

Identification and analysis of products formed from phospholipids in the free radical oxidation of human low density lipoproteins.

Phospholipids reside in the surface layer of LDLs and constitute approximately 20-25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of alpha-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while alpha-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while alpha-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products. These results suggest that alpha-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core.

Alcohols↗