Fatty Acid Radicals Research Articles

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

Identification of Two Cyclooxygenase Active Site Residues, Leucine 384 and Glycine 526, That Control Carbon Ring Cyclization in Prostaglandin Biosynthesis

The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.

In vivo EPR evidence for free radical adducts of nifedipine.

Nifedipine [3,5-pyridinedicarboxylic acid, 1,4-dihydro-2, 6-dimethyl-4-(2-nitrophenyl)-dimethyl ester] is a calcium channel blocker that has been widely used as a prescription drug for patients with hypertension. After illumination by ordinary light for 24 hr, nifedipine is converted completely to its nitroso analog without further photochemical degradation. Evidence for stable, nitroxyl-like free radical generation in mice was observed 15 min after intramuscular (i.m.) or intraperitoneal (i.p.) injection of illuminated nifedipine as monitored by in vivo L-band electron paramagnetic resonance (EPR) spectrometry. This was confirmed in more detail by ex vivo measurements on excised muscle and liver tissue. The nature of these radicals was surmised by comparing the reaction of illuminated nitroso-nifedipine with polyunsaturated fatty acids. Surprisingly, identical radical spectra were detected from excised liver doped with nonilluminated nifedipine, suggesting that this drug can be enzymatically converted in vivo to its nitroso analog without the requirement for illumination. This is one of the first reports of in vivo EPR evidence for a class of unsaturated fatty acid radical conjugates resulting from the normal metabolism of a common drug. Magn Reson Med 42:691-694, 1999.

Structural Characterization of Arachidonyl Radicals Formed by Prostaglandin H Synthase-2 and Prostaglandin H Synthase-1 Reconstituted with Mangano Protoporphyrin IX

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Eugenol — an inhibitor of lipoxygenase-dependent lipid peroxidation

The effect of eugenol on enzymatic lipid peroxidation catalyzed by soybean lipoxygenase was studied in an in vitro system. Lipid peroxidation was inhibited by eugenol in a concentration-dependent manner. The half-maximal inhibition (IC 50) was found to be 380 μM eugenol. Enzyme kinetic studies showed that eugenol non-competitively inhibited lipid peroxidation by altering the maximum velocity ( V max) and without any change in Michaelis-Menten constant ( K m) values. The inhibitory mechanism implies that eugenol does not inactivate the enzyme directly but may interfere with fatty acid radical intermediates due to its hydroxy radical scavenging ability and thus play a role in inhibiting the propagation of lipid peroxidation.

Particle growth in butadiene emulsion polymerization, 4. The promoting effect of mercaptans

AbstractThe observation that butadiene emulsion polymerizations in the presence of fatty acid emulsifiers need minimal amounts of tertiary or n‐dodecanethiols to polymerize at a reasonable rate is often referred to as the promoting effect of mercaptans and is evaluated in this paper. Experimental evidence is presented which shows that fatty acid emulsifiers can actively reduce the average number of radicals per particle. In this paper it will be shown that three components are necessary for retardation of the rate of polymerization in the absence of dodecanethiol to occur: (1) Only diene monomer polymerizations show retardation. (2) Only peroxodisulfate‐initiated polymerizations show retardation. (3) Retardation of the rate of polymerization only occurs in the presence of fatty acid emulsifiers. These three components are combined for the first time in a reaction scheme which is an extension of a reaction scheme proposed by Kolthoff in 1951. Experimental evidence justifies the suggestion that reaction between a fatty acid radical and butadiene play an important role in the promoting effect.

Formation of octadecadienoate dimers by soybean lipoxygenases

AbstractThe aim of this investigation was to determine whether the regioselectivity found for lipoxygenases in the formation of fatty acid hydroperoxides from linoleic acid is reflected in the formation of dimeric products in secondary reactions involving linoleic acid, product hydroperoxide and lipoxygenase. A method was therefore developed for the separation and identification of dimers formed by fusion of two linoleic acid radicals or a linoleic acid radical and linoleate. The method includes solid‐phase extraction, preparative separation of products by thin‐layer chromatography, derivatization to the corresponding fully hydrogenated methyl esters and capillary gas chromatography (GC) coupled with electron impact mass spectrometry. We present evidence that the formation of octadecadienoate dimers, during the secondary reaction of soybean lipoxygenase‐1 or lipoxygenase‐3, is a nonenzymic process that can be envisaged by nonspecific association of intermediate fatty acid radicals (L*) that have dissociated from the enzyme. We could show that the relative amounts of different octadecadienoate dimers formed remain unaltered, regard‐less of pH and type of soybean isoenzyme used. Quantitative analysis by GC showed that under the reaction conditions used, the formation of dimers branching at the 13‐position is preferred.

Fatty acid radicals and the mechanism of soybean lipoxygenase.

Fatty acid radicals and the mechanism of soybean lipoxygenase.

Fatty acid radical formation in rats administered oxidized fatty acids: In vivo spin trapping investigation

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of α-(4-pyridyl-1-oxide)- N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-alga fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.

Superoxide, hydroxyl and fatty acid radical scavenging by aminosalicylates: Direct evaluation with electron spin resonance spectroscopy

Reactive oxygen radicals such as Superoxide and hydroxyl radicals, as well as intermediate unsaturated fatty acid radicals, have been proposed as playing an important role in various diseases including inflammatory bowel disease (IBD). In this study we evaluated radical scavenger properties of aminosalicylates used in the therapy of IBD using spin trapped electron spin resonance spectroscopy. 5-Aminosalicylic acid (5-ASA), 4-aminosalicylic acid and olsalazine had Superoxide radical scavenger properties ( ic 50 = 0.4, 0.4 and 1.0 mM , respectively). 5-ASA and benzalazine also inhibited hydroxyl radicals ( ic 50 = 6.5 mM ). Fatty acid radicals were not inhibited by aminosalicylates. Our results support the hypothesis that therapeutically active compounds may be oxygen radical scavengers and that fatty acid radical scavenging has to be performed by drugs other than aminosalicylates.

Perhydroxyl radical (HOO.) initiated lipid peroxidation. The role of fatty acid hydroperoxides

It is demonstrated that the perhydroxyl radical (HOO., the conjugate acid of superoxide (O2-], initiates fatty acid peroxidation (a model for biological lipid peroxidation) by two parallel pathways: fatty acid hydroperoxide (LOOH)-independent and LOOH-dependent. Previous workers (Gebicki, J. M., and Bielski, B. H. J. (1981) J. Am. Chem. Soc. 103, 7020-7025) demonstrated that HOO., generated by pulse radiolysis, initiates peroxidation in ethanol/water fatty acid dispersions by abstraction of the bis-allylic hydrogen atom from a polyunsaturated fatty acid. Addition of O2 to the fatty acid radicals forms peroxyl radicals (LOO.s), the chain-propagating species of lipid peroxidation. In this work it is demonstrated that HOO., generated either chemically (KO2) or enzymatically (xanthine oxidase), is a good initiator of fatty acid peroxidation in linoleic acid ethanol/water dispersions; O2- serves only as the source of HOO., and HOO. initiation can be observed at physiologically relevant pH values. In contrast to the previous results, the initiating effectiveness of HOO. is related directly to the initial concentrations of LOOHs in the lipids to be peroxidized. This defines a LOOH-dependent mechanism for fatty acid peroxidation initiation by HOO., which parallels the previously established LOOH-independent pathway. Since the LOOH-dependent pathway is much more facile than the LOOH-independent pathway, LOOH is the kinetically preferred site of HOO. attack in these systems. Experiments comparing HOO./LOOH-dependent fatty acid peroxidation with transition metal- and peroxyl radical-initiated peroxidation rule out the participation of the latter two species as initiators, which defines the HOO./LOOH initiation system as mechanistically unique. LOOH product studies are consistent with either a direct or indirect hydrogen atom transfer between LOOH and HOO. to yield LOO.s, which propagate peroxidation. The LOOH-dependent pathway of HOO.-initiated fatty acid peroxidation may be relevant to mechanisms of lipid peroxidation initiation in vivo.

Cerebral function and preservation during cardiac arrest.

Neurologic impairment remains a serious consequence of cardiac arrest. While current investigations are difficult to compare due to their lack of standardization, our understanding of the pathophysiology of CNS ischemia has been greatly increased. Ion fluxes, especially K and Ca, may contribute to injury by initiating a cascade of events culminating in free fatty acid, prostaglandin, and free radical formation, with their related pathogenetic potential. Treatment measures currently consist of CPR (although disagreement exists as to which form of CPR), standard supportive measures, and attention to intracranial pressure control. There is some experimental evidence to support the use of calcium channel-blockers, phenytoin, prostaglandin inhibitors, and free-radical scavengers or inhibitors; however, no human trials have been performed. Steroids and barbiturates have been investigated in human trials and do not appear to be efficacious in ameliorating CNS injury after cardiac arrest.

Separation of polyunsaturated fatty acid radicals by high-performance liquid chromatography with electron spin resonance and ultraviolet detection

In the reaction of soybean lipoxygenase (EC 1.13.11.12) with polyunsaturated fatty acids such as linoleic, linolenic and arachidonic acids, some radical species were detected using the electron spin resonance (ESR) spin-trapping technique. The radical species derived from the three polyunsaturated fatty acids were not distinguishable because the ESR spectra of the spin adducts of nitrosobenzene with their three radical species showed no difference in their hyperfine splittings. To overcome this defect of the spin-trapping technique, these spin-adducts were separated by employing high-performance liquid chromatography (HPLC) combined with ESR spectroscopy. The spin adducts were eluted from a C 18 reversed-phase column in the order linolenic acid, linoleic acid and arachidonic acid. The half-lives of the spin adducts separated by HPLC-ESR were determined as linoleic acid 600 min, linolenic acid 360 min and arachidonic acid 160 min. The use of an ultraviolet detector together with the HPLC-ESR technique resulted in a 500-fold increase in sensitivity in the detection of the radical species.

Process considerations of continuous fat modification with an immobilized lipase

AbstractLipases will catalyze the incorporation of new fatty acids into the triglyceride component of a fat (acidolysis) or can rearrange/redistribute the existing fatty acid radicals in the fat's triglycerides (transesterification). Both processes can dramatically change the physical characteristics of the fat. Immobilization of the lipase allows for the use of the catalyst in a continuous column operation. Examples illustrating the commercial utility of such immobilized enzymes are presented. Process configurations, scale‐up issues and catalyst lifetime/productivity are discussed.

ChemInform Abstract: Physico‐Chemical Studies on Calcium Soaps

Calcium soaps were prepared by the metathesis of the corresponding potassium soap with an aqueous solution of calcium acetate. The IR results showed that the fatty acids exist in a dimeric structure as a result of hydrogen bonding between the carboxyl groups of two acid molecules, whereas calcium palmitate has an ionic character. The X-ray analysis indicated that the zig-zag chains of the fatty acid radical constituent of the soap molecules extend straightforward on both sides of each basal plane and that the molecular axes of the soaps are slightly inclined to the basal plane. The thermal decomposition of calcium palmitate was found to be kinetically of zero order and the energy of activation for the decomposition reaction was in the region of 3 KJ/mole.

Inverse Relationship of Oxygen Concentration and Fatty Acid Radical Leakage during Lipoxygenase Catalysis Implications for Lipoxygenase Functiona

Annals of the New York Academy of SciencesVolume 524, Issue 1 p. 385-387 Inverse Relationship of Oxygen Concentration and Fatty Acid Radical Leakage during Lipoxygenase Catalysis Implications for Lipoxygenase Functiona R. KRUPINSKI-OLSEN, R. KRUPINSKI-OLSEN Smith Kline & French Laboratories Swedeland, Pennsylvania 19479Search for more papers by this authorL-C. YUAN, L-C. YUAN Smith Kline & French Laboratories Swedeland, Pennsylvania 19479Search for more papers by this authorC. KEMAL, C. KEMAL Smith Kline & French Laboratories Swedeland, Pennsylvania 19479 Send correspondence to: C. Kemal, Chevron Chemical Company, Richmond, California 94804–0010.Search for more papers by this author R. KRUPINSKI-OLSEN, R. KRUPINSKI-OLSEN Smith Kline & French Laboratories Swedeland, Pennsylvania 19479Search for more papers by this authorL-C. YUAN, L-C. YUAN Smith Kline & French Laboratories Swedeland, Pennsylvania 19479Search for more papers by this authorC. KEMAL, C. KEMAL Smith Kline & French Laboratories Swedeland, Pennsylvania 19479 Send correspondence to: C. Kemal, Chevron Chemical Company, Richmond, California 94804–0010.Search for more papers by this author First published: April 1988 https://doi.org/10.1111/j.1749-6632.1988.tb38562.xCitations: 1 a This work was supported in part by NIH Grant No. GM 37286 to C. Kemal. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume524, Issue1Biology of the LeukotrienesApril 1988Pages 385-387 RelatedInformation

Relaxational properties and structure of compositions based on an epoxy resin modified with monoepoxide esters of fatty acids

A relationship has been found between the position of dielectric loss peaks and the structure of epoxy polymers modified with monoepoxide esters, i.e. with products of epoxy oligomer esterification with fatty acids. It is shown that phase separation accounts for a second peak appearing in epoxy-epoxide ester polymers in the region for α relation. Parameters of dipole-segmental processes have been determined for structural elements of the studied systems. It was found that a lengthening of the fatty acid radical in the additive molecule leads to increased segmental mobility and to a fall in the glass-transition temperature.

Physico‐chemical studies on calcium soaps

AbstractCalcium soaps were prepared by the metathesis of the corresponding potassium soap with an aqueous solution of calcium acetate. The IR results showed that the fatty acids exist in a dimeric structure as a result of hydrogen bonding between the carboxyl groups of two acid molecules, whereas calcium palmitate has an ionic character. The X‐ray analysis indicated that the zig‐zag chains of the fatty acid radical constituent of the soap molecules extend straightforward on both sides of each basal plane and that the molecular axes of the soaps are slightly inclined to the basal plane. The thermal decomposition of calcium palmitate was found to be kinetically of zero order and the energy of activation for the decomposition reaction was in the region of 3 KJ/mole.

Phospholipids from industrial cottonseed meal

The qualitative and quantitative compositions of the phospholipids isolated from industrial cottonseed meal have been studied. The meal contains a mixture of phospholipids that may be a source for the production of individual phospholipids. The total and position distributions of the fatty acid radicals in individual phospholipids have been determined.

Short-Lived Radicals Produced in the Course of the Reactions of 13-L-Hydroperoxylinoleic Acid with Ferrous Ions Detected by Spin Trapping and Continuous Flow ESR Spectroscopy

Abstract Not only stable spin adducts of unsaturated fatty acid radicals but also unstable ones which had been too short-lived to be observed by usual ESR measurements, could be measured by continuous flow ESR spectroscopy combined with the spin trapping method in a 13-L-hydroperoxylinoleic acid-Fe2+ reaction system.