A new class of lipoxygenase inhibitor. Polyunsaturated fatty acids containing sulfur

A series of unsaturated and polyunsaturated fatty acids with a sulfur atom substituting for a methylene unit of the chain has been prepared and characterized. The syntheses were accomplished by the Wittig coupling of the ylid derived from the triphenylphosphonium salt of 9-bromononanoic acid with aldehydes containing sulfur. The newly formed double bond had predominately the natural Z geometry even when the starting aldehyde was conjugated with the sulfur atom. The sulfides 13-thia-9(Z)-octadecenoic acid ($\text{2}$), 13-thia-9(Z), 11(E)-octadecadienoic acid ($\text{5}$) and 13-thia-9(E), 11(E)-octadecadienoic acid ($\text{6}$) were readily converted into their sulfoxide derivatives by treatment with an equivalent amount of m-chloroperoxybenzoic acid. The structures of the novel compounds were confirmed by the application of ir, uv, ¹H-nmr, ¹³C-nmr, and (as methyl esters) chemical ionization mass spectrometry. Two members of this new family of fatty acids ($\text{5}$ and $\text{6}$) were found to inhibit the catalysis of the oxygenation of linoleic acid by soybean type-1 lipoxygenase. The analysis of the kinetic data for compound $\text{5}$ indicated that the type of inhibition was reversible competitive with an inhibition constant of 30 μM.     

Essential fatty acids are not required for wound healing.

Rats with essential fatty acid deficiency (EFAD) exhibit mild body growth retardation, diminished leukocyte influx in certain models of inflammation, and skin lesions characterized by ulceration, thinning and decreased pigmentation. In the present study we examined the role of EFAD in cutaneous wound healing, a process in which the inflammatory response and the macrophage play a central role. We reproduced the EFAD condition in Lewis rats (n = 35), and examined its effects in wound healing using the paired rat surgical incision model. Rats were compared with weight-matched controls, receiving standard chow diet. Skin samples harvested at days 5, 7, 14 and 21 post-wounding were evaluated for tensiometry and histology. EFAD rats exhibited all the characteristics of this condition, and the typical alteration of liver lipids. Skin samples harvested at different days post-wounding did not show difference in maximal breaking strength when compared to weight-matched controls. Histological evaluation of skin samples showed no difference in the cellular inflammatory infiltration in either EFAD rats or in weight-matched controls. Immunohistochemical studies revealed no difference in the influx of macrophages in the different groups of rats. Fatty acid supplementation of EFAD rats (n = 7), successfully reversed the EFAD state as assessed by the macroscopic skin and liver changes and liver fatty acid content, without modifying either tensile strength or cellular inflammatory infiltration. Our results suggest that EFAD does not alter the normal course of the cutaneous wound repair in rats, despite all the cutaneous alterations produced by this condition. We conclude that essential fatty acids (EFAs) are not essential for cutaneous wound repair.     

Inactivation of potato lipoxygenase by hydroperoxy acids as suicide substrates

The preincubation of potato lipoxygenase with 9(S)-hydroperoxyoctadecatrienoic acid, 15(S)-hydroperoxyeicosatetraenoic acid or 5(S)-hydroperoxyeicosatetraenoic acid which can be subjected to further lipoxygenation led to the gradual inactivation of the lipoxygenase activity, whereas 13(S)-hydroperoxy-9,13,15-octadecatrienoic acid or 15(S)-hydroperoxy-11,13,17-eicosatrienoic acid had no significant effect. The inhibitory effect of the peroxy acids was abolished by hemoglobin. Based on these observations, it is proposed that the unstable epoxide intermediates from the respective peroxy acids may be responsible for the inactivation of potato lipoxygenase. In the comparative study, it was found that 15(S)-hydroperoxyeicosatetraenoic acid possessed more effective inhibitory role than the other acids with Ki value of 250 microM.     

Polyunsaturated fatty acids are potent neuroprotectors

Results reported in this work suggest a potential therapeutic value of polyunsaturated fatty acids for cerebral pathologies as previously proposed by others for cardiac diseases. We show that the polyunsaturated fatty acid linolenic acid prevents neuronal death in an animal model of transient global ischemia even when administered after the insult. Linolenic acid also protects animals treated with kainate against seizures and hippocampal lesions. The same effects have been observed in an in vitro model of seizure-like activity using glutamatergic neurons and they have been shown to be associated with blockade of glutamatergic transmission by low concentrations of distinct polyunsaturated fatty acids. Our data suggest that the opening of background K(+) channels, like TREK-1 and TRAAK, which are activated by arachidonic acid and other polyunsaturated fatty acids such as docosahexaenoic acid and linolenic acid, is a significant factor in this neuroprotective effect. These channels are abundant in the brain where they are located both pre- and post-synaptically, and are insensitive to saturated fatty acids, which offer no neuroprotection.     

Activity of soybean lipoxygenase isoforms against esterified fatty acids indicates functional specificity

In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present. Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning. In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coil. The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and alpha-linolenic acid, and the reaction products with the substrates linoleic acid, alpha-linolenic acid, arachidonic acid, gamma-linolenic acid, and the triacylglycerol trilinolein. All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid. The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols. Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf. Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed.     

The action of sialidases on substrates containing O-acetylsialic acids

O-Acetyl substitution of sialic acids in glycoconjugates reduces the rate of action of sialidases on these substrates. A plasma glycoprotein fraction and an erythrocyte ganglioside containing 4-O-acetylsialic acids were isolated and characterized from equine blood, and a sialyllactose preparation with Neu5,9Ac2 was purified from rat urine. Using the novel substrates II3Neu4Ac5Gc-LacCer and II3Neu5,9Ac2-Lac the influence of individual mono-O-acetylated sialic acids on bacterial and viral sialidases could be clearly shown. This extends and clarifies observations with glycoproteins containing mixtures of mono-, di- and higher O-acetylated sialic acids with substitution at the hydroxyls on carbons 4, 7, 8 and 9. A 4-O-acetyl substitution in sialic acids blocks the action of bacterial sialidases for substrates containing these derivatives, while viral enzymes show low but significant activity, reflected in Km and Vmax values. A small reduction in bacterial sialidase activity was observed for II3Neu5,9Ac2-Lac relative to II3Neu5Ac-Lac in agreement with kinetic analysis. Newcastle disease virus sialidase showed a 50% reduction in hydrolysis rate for the 9-O-acetylated substrate and ten-fold reductions of both Km and Vmax values.     

RNA-DNA hybrids containing damaged DNA are substrates for RNase H

During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H. Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal. The rates and sites of digestion by E. coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined. The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex. There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs. Cleavage is partially redirected to the position adjacent to the damaged sites. The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex. These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand.     

Requirement of monohydroperoxy fatty acids for the oxygenation of 15LS-HETE by reticulocyte lipoxygenase

The lipoxygenase from reticulocytes oxygenates 15LS-HETE to 8-hydroperoxy-15-hydroxy-5,9,11,13-eicosatetraenoic acid and 5-hydroperoxy-15-hydroxy-6,8,11,13-eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15LS-HETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15LS-HETE.     

The effect of bio-converted polyunsaturated fatty acids on the oxidation of TAG containing highly unsaturated fatty acids

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.     

Oxygenation of trans polyunsaturated fatty acids by lipoxygenase reveals steric features of the catalytic mechanism

Lipoxygenase, a nonheme iron dioxygenase, catalyzes the oxygenation of 1,4-diene units in polyunsaturated fatty acids, forming conjugated diene hydroperoxides as the primary products. The naturally occurring all-Z geometry for the olefins in the polyunsaturated fatty acid has long been thought to be a substrate requirement for the enzyme. A rigorous test of this hypothesis using the two isomeric (9E,12Z)- and (9Z,12E)-9,12-octadecadienoic acids was carried out. Both isomeric substrates were found to be catalytically oxygenated by soybean lipoxygenase 1 at a significant fraction of the rate of the reaction of the natural substrate, linoleic acid. Product determinations revealed that a thermodynamically unfavorable E to Z isomerization at the 9,10-position occurred when (9E,12Z)-9,12-octadecadienoic acid was converted into the 13-hydroperoxide by lipoxygenase 1. Determination of the stereochemistry at the oxygenated position in the products indicated that a comparable isomerization at the 12,13-position did not occur when the 9Z,12E isomer was employed. The distribution of products obtained from oxygenation at the 9-position supported the hypothesis that the enzyme catalyzes the reaction in one of two substrate orientations, conventional and head to tail reversed. The observations can be understood on the basis of the steric demands on intermediates in the proposed mechanism of action as well as by catalysis by the active-site iron atom.     

Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific?oxygenated signaling compounds.     

Binding to mitochondrial proteins of lecithins containing known fatty acids

Lecithins from livers of normal rats and rats deficient in essential fatty acids were separated into fractions containing mainly 1, 2, 3 or 4 double bonds. The fractions thus produced were dispersed in aqueous buffer by ultrasonic irradiation and their rates of binding to mitochondria, whose lipids had been extracted with aqueous acetone, were measured. The kinetics of this reaction can be explained by an initial Brownian coagulation between lecithin and protein particles followed by a rate limiting interaction of lecithin molecules with the mitochondrial protein. A rate equation for these two steps was derived containing three parameters which represent respectively the rate constant for the coagulation step, the surface area of the mitochondria and the rate constant of the second step. The experimental data was fitted to this equation. The effect of variation in fatty acid composition was most marked on the value of the rate constant of the second step, the interaction of lecithin with mitochondrial protein. Lecithins containing arachidonic acid interacted significantly more slowly than those containing 5, 8, 11-eicosatrienoic acid or mixtures of oleic and linoleic acids. The significance of this difference is discussed in relation to the structural function of the essential fatty acids.     

Determination of cis,cis-methylene interrupted polyunsaturated fatty acids in aqueous solutions by lipoxygenase chemiluminescence

The chemiluminescent reaction of luminol during lipoxygenase-catalyzed oxygenations was studied with the purpose of developing a specific luminometric assay for cis,cis-1,4-pentadiene fatty acids directly in aqueous solutions. The addition of picomole levels of either linoleic or arachidonic acids to reaction systems containing 0.04 mM luminol and 40 micrograms/ml of purified soybean lipoxygenase-1 gave light emission curves with a single sharp maximum. Under these conditions the peak heights were linearly dependent on the fatty acid concentration and the detection limit for both of the fatty acids was 2 pmol with a signal to noise ratio of 2. For maximum reproducibility of the assays a procedure for the proper quantitation of the enzyme was developed. The fact that the assay proved to be relatively interference-free was ascribed to the high molar enzyme/substrate ratio (above 1).     

Hypothalamic responses to long-chain fatty acids are nutritionally regulated

Central administration of the long-chain fatty acid oleic acid inhibits food intake and glucose production in rats. Here we examined whether short term changes in nutrient availability can modulate these metabolic and behavioral effects of oleic acid. Rats were divided in three groups receiving a highly palatable energy-dense diet at increasing daily caloric levels (below, similar, or above the average of rats fed standard chow). Following 3 days on the assigned diet regimen, rats were tested for acute biological responses to the infusion of oleic acid in the third cerebral ventricle. Three days of overfeeding virtually obliterated the metabolic and anorectic effects of the central administration of oleic acid. Furthermore, the infusion of oleic acid in the third cerebral ventricle failed to decrease the expression of neuropeptide Y in the hypothalamus and of glucose-6-phosphatase in the liver following short term overfeeding. The lack of hypothalamic responses to oleic acid following short term overfeeding is likely to contribute to the rapid onset of weight gain and hepatic insulin resistance in this animal model.