Neuroprotection by the endogenous cannabinoid anandamide and arvanil against in vivo excitotoxicity in the rat: role of vanilloid receptors and lipoxygenases.

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.

Exogenous anandamide protects rat brain against acute neuronal injury in vivo.

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Neuroprotection by Delta9-tetrahydrocannabinol, the main active compound in marijuana, against ouabain-induced in vivo excitotoxicity.

Excitotoxicity is a paradigm used to explain the biochemical events in both acute neuronal damage and in slowly progressive, neurodegenerative diseases. Here, we show in a longitudinal magnetic resonance imaging study that Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the main active compound in marijuana, reduces neuronal injury in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain to elicit excitotoxicity. In the acute phase Delta(9)-THC reduced the volume of cytotoxic edema by 22%. After 7 d, 36% less neuronal damage was observed in treated rats compared with control animals. Coadministration of the CB(1) cannabinoid receptor antagonist SR141716 prevented the neuroprotective actions of Delta(9)-THC, indicating that Delta(9)-THC afforded protection to neurons via the CB(1) receptor. In Delta(9)-THC-treated rats the volume of astrogliotic tissue was 36% smaller. The CB(1) receptor antagonist did not block this effect. These results provide evidence that the cannabinoid system can serve to protect the brain against neurodegeneration.

Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation.

Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme. The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin. EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding. Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation. Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature. Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.

Fatty acid hydroperoxide lyase: a plant cytochrome p450 enzyme involved in wound healing and pest resistance.

Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The first step in the lipoxygenase pathway is the reaction of linoleic or linolenic acids with molecular oxygen, catalyzed by the enzyme lipoxygenase. The hydroperoxy fatty acids thus formed are highly reactive and dangerous for the plant and therefore further metabolized by other enzymes such as allene oxide synthase, hydroperoxide lyase, peroxygenase, or divinyl ether synthase. Recently, these enzymes have been characterized as a special class of cytochrome P450 enzymes. Hydroperoxide lyases cleave the lipoxygenase products, resulting in the formation of omega-oxo acids and volatile C6- and C9-aldehydes and -alcohols. These compounds are major contributors to the characteristic "fresh green" odor of fruit and vegetables. They are widely used as food flavors, for example, to restore the freshness of food after sterilization processes. The low abundance of these compounds in nature and the high demand make it necessary to synthesize them on a large scale. Lipoxygenase and hydroperoxide lyase are suitable biocatalysts for the production of "natural" food flavors. In contrast to lipoxygenase, which has been extensively studied, little is yet known about hydroperoxide lyase. Hydroperoxide lyases from different organisms have been isolated, and a few genes have been published lately. However, the structure and reaction mechanism of this enzyme are still unclear. The identification of this enzyme as a cytochrome P450 sheds new light on its structure and possible reaction mechanism, whereas recombinant expression brings a biocatalytic application into sight.

Oxygenation of (3Z)-alkenals to 4-hydroxy-(2E)-alkenals in plant extracts: a nonenzymatic process.

There is large interest in 4-hydroxy-(2E)-alkenals because of their cytotoxicity in mammals. However, the biosynthetic pathway for these compounds has not been elucidated yet. In plants, 4-hydroxy-(2E)-alkenals were supposed to be derived by the subsequent actions of lipoxygenase and a peroxygenase on (3Z)-alkenals. The presence of 9-hydroxy-12-oxo-(10E)-dodecenoic acid (9-hydroxy-traumatin) in incubations of 12-oxo-(9Z)-dodecenoic acid (traumatin) in the absence of lipoxygenase or peroxygenase, has prompted us to reinvestigate its mode of formation. We show here that in vitro 9-hydroxy-traumatin, 4-hydroxy-(2E)-hexenal and 4-hydroxy-(2E)-nonenal, are formed in a nonenzymatic process. Furthermore, a novel product derived from traumatin was observed and identified as 11-hydroxy-12-oxo-(9Z)-dodecenoic acid. The results obtained here strongly suggest that the 4-hydroxy-(2E)-alkenals, observed in crude extracts of plants, are mainly due to autoxidation of (3Z)-hexenal, (3Z)-nonenal and traumatin. This may have implications for the in vivo existence and previously proposed physiological significance of these products in plants.

Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death.

Oxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2-induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2-induced apoptosis. On the other hand, lentil lipoxygenase products 9- and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.

Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics.

Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases.

Formation of a new class of oxylipins from N-acyl(ethanol)amines by the lipoxygenase pathway.

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both alpha-N-linolenoylethanolamine and gamma-N-linolenoylethanolamine (18:3), as well as alpha-N-linolenoylamine and gamma-N-linolenoylamine were converted into their (13S)-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of alpha-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z)-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)-hydroperoxy-alpha-N-linolenoylethanolamine and the (13S)-hydroperoxide of linoleic acid and its ethanolamine derivative. alpha-Ketols and gamma-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S)-hydroperoxy-alpha-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.

Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase.

Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at -80 degrees C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and alpha-linolenic acid (alpha-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 microM a typical Michaelis-Menten curve was found with a K(m) of 77 microM. At higher alpha-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mumol alpha-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from alpha-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred.

Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase.

Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been reported to contain a hydroperoxide lyase specific for 13-hydroperoxy linoleic and linolenic acid only. However, in addition to 13-hydroperoxide lyase activity we found substantial 9-hydroperoxide lyase activity in alfalfa seedlings as well. The specific activity for 9-hydroperoxy fatty acids was about 50% of the activity for the 13-isomers. Furthermore, alfalfa seedlings contain a 3Z:2E-enal isomerase that converts the 3Z-enal products to their 2E-enal isoforms.

Anandamide hydrolysis by human cells in culture and brain.

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet, AnNH inactivation has not been studied in humans. Here, a human brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 +/- 0.2 microM and 800 +/- 75 pmol.min-1.mg of protein-1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 degreesC, respectively, and the activation energy of the reaction was 43.5 +/- 4.5 kJ.mol-1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Ki values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 +/- 0.9 microM) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 +/- 0.02 microM and 30 +/- 3 pmol.min-1.mg of protein-1 (CHP100 cells) and 0.13 +/- 0.01 microM and 140 +/- 15 pmol.min-1.mg of protein-1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.

With anandamide as substrate plant 5-lipoxygenases behave like 11-lipoxygenases.

Anandamide, an endogenous ligand for cannabinoid receptors CB1 and CB2, was incubated with purified 5-lipoxygenases from barley and tomato. This yielded 11S-hydroperoxy-5,8,12,14-eicosatetraenoylethanolamide (11S-HPANA) as major product (about 70%). This is in contrast with the dioxygenation of arachidonic acid, where 5S-HPETE is the major product. This observation implies that the regiospecificity of the dioxygenation, catalyzed by nonmammalian 5-lipoxygenases, is altered by a modification at the carboxylic end of the substrate. Soybean 15-lipoxygenase forms 15S-HPANA (95%) and 11S-HPANA (5%), and in the second dioxygenation 5,15-diHPANA (45%) and 8,15-diHPANA (55%) are formed. Apparently, the regiospecificity of the soybean 15-lipoxygenase reaction is only slightly affected using anandamide as substrate.

Oxidation of dilinoleoyl phosphatidylcholine by lipoxygenase 1 from soybeans.

Soybean lipoxygenase-1 is able to oxidize dilinoleoyl phosphatidylcholine at pH 7.5 and 10. The reaction could be followed spectrophotometrically from the increase of the absorbance at 234 nm. An intermediate product and a final product were detected. In the intermediate product only one of the linoleoyl chains (either sn1 or sn2) was oxidized. In the final product, both linoleic acid units were converted into hydroperoxides. Apparently, oxidation of one of the linoleoyl chains leads to a disruption of the structure of the mixed bilayer disk, making the remaining fatty acid unit more accessible to the action of the enzyme. The specificity of lipoxygenase-1 when acting on phospholipids is not affected by pH. The exclusive production of 13-hydroperoxyoctadecadienoic acid derivatives of dilinoleoyl phosphatidylcholine at pH 7.5 and 10 may result from the blockage of the carboxylic end of the fatty acid.

The effect of hydroxylation of linoleoyl amides on their cannabinomimetic properties.

As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH2 influences their binding abilities to the CB-1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid [3H]CP 55.940 from the CB-1 receptor (Ki > 1 microM). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.

Dioxygenation of N-linoleoyl amides by soybean lipoxygenase-1.

Anandamide, a novel neurotransmitter, has been reported to be dioxygenated by brain lipoxygenase [1,11]. Anandamides constitute a new class of neuroregulatory fatty acid amides. However, little is known about the enzymatic dioxygenation of these lipids. Therefore, we have tested several members of the neuroactive fatty acid amide class containing a 1Z,4Z-pentadiene system whether they could be dioxygenated by soybean lipoxygenase-1, which is a model enzyme for mammalian lipoxygenases. In this study it was found that lipoxygenase-1 converts N-linoleoylethanolamide (ODNHEtOH), N-linoleoylamide (ODNH2), N-linoleoylmethylamide (ODNHMe) and N,N-linoleoyldimethylamide (ODN(Me)2 into 13-(S)-hydroperoxy-9Z,11E-octadeca-9,11-dienoyl amides derivatives. The apparent Km values for ODNHEtOH (23.6 +/- 3.7 microM), ODNH2 (8.60 +/- 0.65 microM) and linioleic acid (OD: 8.85 +/- 0.74 microM) are not significantly different. The k(cat) for ODNH2 (32.4 +/- 1.2 s(-1)) is twice as small as compared to the turnover numbers of the other substrates, viz. ODNHEtOH (61.6 +/- 5.0 s(-1)) and OD (54.4 +/- 2.0 s(-1). The results suggest that N-linoleoyl ethanolamide and N-linoleoyl amide can be readily converted by lipoxygenases in vivo.

Ozone stress modulates amine oxidase and lipoxygenase expression in lentil (Lens culinaris) seedlings.

The effect of ozone stress on polyamine metabolism and membrane lipid peroxidation in lentil seedlings through the amine oxidase and lipoxygenase activity and expression has been investigated. Ozone is shown to control the expression of these enzymes at the transcriptional level, down-regulating the amine oxidase gene and up-regulating the lipoxygenase gene. The decrease of amine oxidase activity correlated with the increase of putrescine concentration in the ozone-treated plantlets, whereas the increase of lipoxygenase activity was paralleled by enhanced membrane lipid peroxidation. Finally, polyamines are shown to inhibit lipoxygenase activity in lentils.

Lipoxygenase is irreversibly inactivated by the hydroperoxides formed from the enynoic analogues of linoleic acid.

Triple bond analogues of natural fatty acids irreversibly inactivate lipoxygenase during their enzymatic conversion [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538-10545]. To gain insight into the mechanism of the irreversible inactivation of soybean lipoxygenase-1, we studied the enzymatic conversion of two linoleic acid analogues, 9(Z)-octadec-9-en-12-ynoic acid (9-ODEYA) and 12(Z)-octadec-12-en-9-ynoic acid (12-ODEYA). During the inactivation process, Fe(III)-lipoxygenase converts 9-ODEYA into three products, i.e. 11-oxooctadec-9-en-12-ynoic acid, racemic 9-hydroxy-10(E)-octadec-10-en-12-ynoic acid, and racemic 9-hydroperoxy-10(E)-octadec-10-en-12-ynoic acid. Fe(II)-lipoxygenase does not convert the inhibitor and is not inactivated by 9-ODEYA. Fe(III)-lipoxygenase converts 12-ODEYA into 13-hydroperoxy-11(Z)-octadec-11-en-9-ynoic acid (34/66 R/S), 13-hydroperoxy11(E)-octadec-11-en-9-ynoic acid (36/64 R/S), 11-hydroperoxyoctadec-12-en-9-ynoic acid (11-HP-12-ODEYA, enantiomeric composition of 33/67), and 11-oxooctadec-12-en-9-ynoic acid (11-oxo-12-ODEYA) during the inactivation process. Also, Fe(II)-lipoxygenase is inactivated by 12-ODEYA. It converts the inhibitor into the same products as Fe(III)-lipoxygenase does, but two additional products are formed, viz. 13-oxo-11(E)-octadec-11-en-9-ynoic acid and 13-oxo-11(Z)-octadec-11-en-9-ynoic acid. The purified reaction products were tested for their lipoxygenase inhibitory activities. The oxo compounds, formed in the reaction of 9-ODEYA and 12-ODEYA, do not inhibit Fe(II)- or Fe(III)-lipoxygenase. The 9- and 13-hydroperoxide products that are formed from 9-ODEYA and 12-ODEYA, respectively, oxidize Fe(II)-lipoxygenase to its Fe(III) state and are weak lipoxygenase inhibitors. 11-HP-12-ODEYA is, however, the most powerful inhibitor and is able to oxidize Fe(II)-lipoxygenase to Fe(III)-lipoxygenase. 11-HP-12-ODEYA is converted into 11-oxo-12-ODEYA by Fe(III)-lipoxygenase. We propose a mechanism for the latter reaction in which Fe(III)-lipoxygenase abstracts the bisallylic hydrogen H-11 from 11-HP-12-ODEYA, yielding a hydroperoxyl radical which is subsequently cleaved into 11-oxo-ODEYA and a hydroxyl radical which may inactivate the enzyme.

Chemical and quantum mechanical studies of the free radical C-C bond formation in the lipoxygenase-catalyzed dimerisation of octodeca-9,12-diynoic acid.

Triple bond analogues of poly-unsaturated fatty acids are well-known inactivators of lipoxygenases. In an earlier study we proposed that, since 11-oxo-octadeca-9,12-diynoic acid (11-oxo-ODYA) is the only oxygenated product formed during the irreversible inactivation of soybean lipoxygenase-1, the inactivation should proceed via a C11 centered octadeca-9,12-diynoic acid radical (ODYA radical). In the present study we investigated the lipoxygenase-catalysed formation of the ODYA radical. In the reaction of lipoxygenase with ODYA in the absence of dioxygen and in the presence of 13(S)-hydroperoxy-octadeca-9Z, 11E-dienoic acid (13-HPOD), free ODYA radicals were formed which resulted in the formation of three dimeric ODYA products in which one ODYA moiety is linked via its C9 (12%), C11 (72%) or C13 (16%) to the C11 methylene of the other ODYA moiety. With the ab initio Hartree-Fock method, using the 2,5-heptadiynyl radical as a model compound, the electron spin in the ODYA radical was calculated to be located for 12.0, 75.0 and 12.0% on carbon atoms C9, C11 and C13 of the ODYA radical, respectively. The ODYA-ODYA dimer formation could thus be explained on the basis of the electron spin distribution in the ODYA radical. The dimer formation, i. e. reaction of an ODYA radical with an ODYA molecule was compared with the reaction of the ODYA radical with dioxygen. On the basis of this comparison it is concluded that a) the ODYA dimer formation occurs at the carbon atom with the highest electron spin population; b) ODYA dimer formation is predominantly a kinetically determined process; c) the electron spin distribution in the ODYA radical can be used to predict the composition of the dimer mixture; and d) the regiospecific oxygen addition in the formation of 11-oxo-ODYA is enzymatically controlled.