Hydroperoxyoctadecatrienoic Acid Research Articles

Plasma metabolomic profile is near-normal in people with HIV on long-term suppressive antiretroviral therapy.

Combination antiretroviral therapy (ART) has transformed human immunodeficiency virus (HIV) infection in people with HIV (PWH). However, a chronic state of immune activation and inflammation is maintained despite achieving HIV suppression and satisfactory immunological recovery. We aimed to determine whether the plasma metabolomic profile of PWH on long-term suppressive ART and immunologically recovered approximates the normality by comparison with healthy controls with similar age and gender. We carried out a cross-sectional study in 17 PWH on long-term ART (HIV-RNA <50 copies/mL, CD4+ ≥500 cells/mm3, and CD4+/CD8+ ≥1) and 19 healthy controls with similar age and gender. Metabolomics analysis was performed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The statistical association analysis was performed by principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and Generalized Linear Models (GLM) with a gamma distribution (log-link). Significance levels (p-value) were corrected for multiple testing (q-value). PCA and PLS-DA analyses found no relevant differences between groups. Adjusted GLM showed 14 significant features (q-value<0.20), of which only three could be identified: lysophosphatidylcholine (LysoPC) (22:6) (q-value=0.148), lysophosphatidylethanolamine (LysoPE) (22:6) (q-value=0.050) and hydroperoxy-octadecatrienoic acid (HpOTrE)/dihydroperoxy-octadecatrienoic acid (DiHOTrE)/epoxy-octadecadienoic acid (EpODE) (q-value=0.136). These significant identified metabolites were directly correlated to plasma inflammatory biomarkers in PWH and negatively correlated in healthy controls. PWH on long-term ART have a metabolomic profile that is almost normal compared to healthy controls. Nevertheless, residual metabolic alterations linked to inflammatory biomarkers persist, which could favor the development of age-related comorbidities among this population.

Rice peroxygenase catalyzes lipoxygenase-dependent regiospecific epoxidation of lipid peroxides in the response to abiotic stressors

The peroxygenase pathway plays pivotal roles in plant responses to oxidative stress and other environmental stressors. Analysis of a network of co-expressed stress-regulated rice genes demonstrated that expression of OsPXG9 is negatively correlated with expression of genes involved in jasmonic acid biosynthesis. DNA sequence analysis and structure/function studies reveal that OsPXG9 is a caleosin-like peroxygenase with amphipathic α-helices that localizes to lipid droplets in rice cells. Enzymatic studies demonstrate that 12-epoxidation is slightly more favorable with 9(S)-hydroperoxyoctadecatrienoic acid than with 9(S)-hydroperoxyoctadecadienoic acid as substrate. The products of 12-epoxidation are labile, and the epoxide ring is hydrolytically cleaved into corresponding trihydroxy compounds. On the other hand, OsPXG9 catalyzed 15-epoxidation of 13(S)-hydroperoxyoctadecatrienoic acid generates a relatively stable epoxide product. Therefore, the regiospecific 12- or 15-epoxidation catalyzed by OsPXG9 strongly depends on activation of the 9- or 13- peroxygenase reaction pathways, with their respective preferred substrates. The relative abundance of products in the 9-PXG and 13-PXG pathways suggest that the 12-epoxidation involves intramolecular oxygen transfer while the 15-epoxidation can proceed via intramolecular or intermolecular oxygen transfer. Expression of OsPXG9 is up-regulated by abiotic stimuli such as drought and salt stress, but it is down-regulated by biotic stimuli such as flagellin 22 and salicylic acid. The results suggest that the primary function of OsPXG9 is to modulate the level of lipid peroxides to facilitate effective defense responses to abiotic and biotic stressors.

Characterization of PtAOS1 Promoter and Three Novel Interacting Proteins Responding to Drought in Poncirus trifoliata

Jasmonic acid (JA) plays a crucial role in various biological processes including development, signal transduction and stress response. Allene oxide synthase (AOS) catalyzing (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) to an unstable allene oxide is involved in the first step of JA biosynthesis. Here, we isolated the PtAOS1 gene and its promoter from trifoliate orange (Poncirus trifoliata). PtAOS1 contains a putative chloroplast targeting sequence in N-terminal and shows relative to pistachio (Pistacia vera) AOS. A number of stress-, light- and hormone-related cis-elements were found in the PtAOS1 promoter which may be responsible for the up-regulation of PtAOS1 under drought and JA treatments. Transient expression in tobacco (Nicotiana benthamiana) demonstrated that the P−532 (−532 to +1) fragment conferring drive activity was a core region in the PtAOS1 promoter. Using yeast one-hybrid, three novel proteins, PtDUF886, PtDUF1685 and PtRAP2.4, binding to P−532 were identified. The dual luciferase assay in tobacco illustrated that all three transcription factors could enhance PtAOS1 promoter activity. Genes PtDUF1685 and PtRAP2.4 shared an expression pattern which was induced significantly by drought stress. These findings should be available evidence for trifoliate orange responding to drought through JA modulation.

15-Lipoxygenase metabolites of α-linolenic acid, [13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome.

The ratio of ω-6 to ω-3 polyunsaturated fatty acids (PUFAs) appears to be critical in the regulation of various pathophysiological processes and to maintain cellular homeostasis. While a high proportion of dietary intake of ω-6 PUFAs is associated with various inflammatory disorders, higher intake of ω-3 PUFAs is known to offer protection. It is now well established that beneficial effects of ω-3 PUFAs are mediated in part by their oxygenated metabolites mainly via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. However, the down-stream signaling pathways that are involved in these anti-inflammatory effects of ω-3 PUFAs have not been elucidated. The present study evaluates the effects of 15-LOX metabolites of α-linolenic acid (ALA, ω-3 PUFA) on lipopolysaccharide (LPS) induced inflammation in RAW 264.7 cells and peritoneal macrophages. Further, the effect of these metabolites on the survival of BALB/c mice in LPS mediated septic shock and also polymicrobial sepsis in Cecal Ligation and Puncture (CLP) mouse model was studied. These studies reveal the anti-inflammatory effects of 13-(S)-hydroperoxyoctadecatrienoic acid [13-(S)-HPOTrE] and 13-(S)-hydroxyoctadecatrienoic acid [13-(S)-HOTrE] by inactivating NLRP3 inflammasome complex through the PPAR-γ pathway. Additionally, both metabolites also deactivated autophagy and induced apoptosis. In mediating all these effects 13-(S)-HPOTrE was more potent than 13-(S)-HOTrE.

Tomato lipoxygenase D involved in the biosynthesis of jasmonic acid and tolerance to abiotic and biotic stress in tomato

TomloxD is a lipoxygenase from Lycopersicon esculentum Mill, which has lipoxygenase activity. The expression of TomloxD can be stimulated by wounding, pathogen infection, jasmonate, and systemin. To investigate the function of TomloxD, transgenic tomato plants silencing tomloxD gene was produced. The suppression of TomloxD expression led to a marked reduction in the levels of lipoxygenase activity and endogenous jasmonic acid content, which suggested TomloxD can catalyze α-linolenic acids to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT), and the 13-HPOT is metabolized further to synthesize jasmonic acid. Real-time RT-PCR revealed that the expression of defense genes LeHSP90, LePR1 and LePR6 was also less in the transformants than in the wild-type tomato plant. Resistance assay showed that the suppression of TomloxD in transgenic tomato plants reduced thermotolerance of tomato and increased its susceptibility to Cladosporium fulvum. Collectively, the data presented here suggest that the TomloxD plays a role as a component of the octadecanoid defense-signaling pathway and involved in the generation of endogenous jasmonic acid, and in turn regulates the expression of plant defense genes and resistance to high temperature and pathogen attack.

Fatty acid oxidation products (‘green odour’) released from perennial ryegrass following biotic and abiotic stress, potentially have antimicrobial properties against the rumen microbiota resulting in decreased biohydrogenation

In this experiment, we investigated the effect of 'green odour' products typical of those released from fresh forage postabiotic and biotic stresses on the rumen microbiota and lipid metabolism. Hydroperoxyoctadecatrienoic acid (HP), a combination of salicylic and jasmonic acid (T), and a combination of both (HPT) were incubated in vitro in the presence of freeze-dried ground silage and rumen fluid, under rumen-like conditions. 16S rRNA (16S cDNA) HaeIII-based terminal restriction fragment length polymorphism-based (T-RFLP) dendrograms, canonical analysis of principal coordinates graphs, peak number and Shanon-Weiner diversity indices show that HP, T and HPT likely had antimicrobial effects on the microbiota compared to control incubations. Following 6 h of in vitro incubation, 15.3% of 18:3n-3 and 4.4% of 18:2n-6 was biohydrogenated in control incubations, compared with 1.3, 9.4 and 8.3% of 18:3n-3 for HP, T and HPT treatments, respectively, with negligible 18:2n-6 biohydrogenation seen. T-RFLP peaks lost due to application of HP, T and HPT likely belonged to as yet uncultured bacteria within numerous genera. Hydroperoxyoctadecatrienoic acid, T and HPT released due to plant stress potentially have an antimicrobial effect on the rumen microbiota, which may explain the decreased biohydrogenation in vitro. These data suggest that these volatile chemicals may be responsible for the higher summer n-3 content of bovine milk.

Overexpression of the Tomato 13-Lipoxygenase Gene TomloxD Increases Generation of Endogenous Jasmonic Acid and Resistance to Cladosporium fulvum and High Temperature

Expression of the tomato gene encoding 13-lipoxygenase,TomloxD, is stimulated by wounding, pathogen infection, jasmonate, and systemin, but its role during growth and development of tomato (Lycopersicon Spp.) remains unclear. To assess the physiological role of TomloxD, we produced transgenic tomato plants with greatly increased TomloxD content using sense constructs under the control of the CaMV 35S promoter. Overexpression of TomloxD in transgenic tomatoes led to a marked increase in the levels of lipoxygenase activity and content of endogenous jasmonic acid (JA), which suggested that TomloxD can use α-linolenic acid as a substrate to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT); the 13-HPOT produced appears to be metabolized further to synthesize JA. Real-time RT-PCR revealed that the expression levels of defense genes LeHSP90, LePR1, LePR6 and LeZAT in the transformants were higher than those in non-transformed plants. Assay for resistance to pathogenic fungus and high temperature stresses suggested that transgenic plants harboring TomloxD were more tolerant to Cladosporium fulvum and high temperature stress than non-transformed tomato plants. The data presented here indicate clearly that TomloxD is involved in endogenous JA synthesis and tolerance to biotic and abiotic stress. The tomloxD gene has potential applications in engineering cropping plants that are resistant to biotic and/or abiotic stress factors.

Use of isotope mass probes for metabolic analysis of the jasmonate biosynthetic pathway

In order to quantitatively study the jasmonate biosynthetic pathway, we chemically synthesized a pair of isotope mass probes and established a labeling protocol. The pair of mass probes used in our work were ω-bromoacetonylpyridinium bromide (BPB) and d(5)-ω-bromoacetonylpyridinium bromide (d(5)-BPB), which contain carboxylic acid reactive groups, isotopically labeled groups and permanent positive charges. High performance liquid chromatography (HPLC) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-QTOF-MS) were used for the detection of labeled standard mixtures and plant samples. In comparison to negative mode electrospray ionization detection of unlabeled analytes, the ESI signal of reverse charge labeled compounds was shown to improve by 20- to 80-fold. Accurate relative quantification was achieved as no isotopic effects of the different isotope labeled phytohormones during RP/SCX mixed-mode liquid chromatographic separation were observed. A data analysis method was established for analyzing metabolic pathways using our labeling strategy. We then applied our method and examined the jasmonate biosynthetic pathway of rice under salt stress and the premature senescence mutant. Here we found that under salt stress conditions, rice showed up-regulation in (13S)-hydroperoxyoctadecatrienoic acid (HOPT), cis-(+)-12-oxophytodienoic acid (OPDA), 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8) and jasmonoyl-valine (JA-Val) levels, while α-linolenic acid (LA) and jasmonic acid (JA) showed down-regulation, and three components (HPOT, OPC-8 and JA-Val) were accumulated. The premature senescence mutant showed up-regulation in all major components of the jasmonate biosynthetic pathway with the exception of LA, and an accumulation of HPOT, OPC-6 and JA-Val. This study demonstrates that our chemical stable isotope labeling strategy can be used as a powerful tool for metabolic pathway analysis of phytohormones in plants.

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12 S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13 S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.

Lipoxygenase from Banana Leaf: Purification and Characterization of an Enzyme That Catalyzes Linoleic Acid Oxygenation at the 9-Position

The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.

COX and friends

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.

Regulation of oxylipin synthesis

Two very common groups of oxylipins formed in plants involve the conversion of fatty acid hydroperoxides, such as hydroperoxy-octadecatrienoic acid, into further metabolites by allene oxide synthase and hydroperoxide lyase. Both of these oxylipin branch pathways appear to be ubiquitous or nearly so in plants, but the relative activities of these two branches vary among plant species. In most plants examined, including Arabidopsis, product formation from either of these pathways is minimal until elicited by wounding or some other means, upon which products from both pathways, such as jasmonic acid and C(6) aldehydes and alcohols, can increase by orders of magnitude. In some plant species such as Artemisia and Jasminum spp. oxylipin product formation is heavily skewed towards allene oxide synthase products. Others such as watermelon (Citrullus lanatus) produce 10-fold higher amounts or more of hydroperoxide lyase than allene oxide synthase products. Arabidopsis and tobacco are intermediate between these extremes. Artemisia and Jasminum are also unusual in that they do not require wounding or other types of induction for high oxylipin product formation. Release of non-esterified fatty acids appears to be correlated with oxylipin formation, but phospholipase A(2) appears not to be involved with oxylipin production, at least in the case of Artemisia leaves.

Overexpression of a cytoplasm-localized allene oxide synthase promotes the wound-induced accumulation of jasmonic acid in transgenic tobacco.

Jasmonic acid (JA) is involved in regulating the expression of certain plant defense genes and response to various stresses. JA biosynthesis is hypothesized to occur both in chloroplasts and the cytoplasm. In order to test whether or not a cytosol-localized allene oxide synthase (AOS) can promote JA biosynthesis, transgenic tobacco plants containing a flax AOS cDNA without a chloroplast transit sequence under the control of a tetracycline-inducible promoter were generated. Induction of the flax AOS gene in transgenic plants with chlor-tetracycline (Tc) led to the expression of the flax AOS mRNA and protein, which resulted in high level of metabolism of 13(S)-hydroperoxyoctadecatrienoic acid (13(S)-HPOT) and formation of 12-oxo-phytodienoic acid (12-O-PDA). Subcellular fractionation demonstrated that the flax AOS protein and activity were associated with the cytosol. Overexpression of the flax AOS in induced transgenic plants did not increase JA levels in healthy, undamaged leaf tissues. However, in wounded tissues overexpressing a flax AOS, levels of JA and the transcript of a pathogenesis-related gene (PR-1) dramatically increased when compared to those not expressing the flax AOS. Analysis of the release of wound-induced C6 volatiles showed that the level of (Z)-3-hexen-1-ol decreased about 30% due to overexpression of the cytoplasm-localized AOS, while (Z)-3-hexenal and (Z)-3-hexenyl acetate appeared not to be significantly altered. The data indicate that cytoplasmic AOS responds to wounding by increasing the levels of the wound-induced JA which in turn directly or indirectly enhances the expression of plant defense genes.

Some Biochemical Aspects of the Protective Effect of Trimetazidine on Rat Cardiomyocytes During Hypoxia and Reoxygenation

This study was undertaken to evaluate the direct cardioprotective effect of trimetazidine (TMZ), an anti-anginal drug devoid of haemodynamic action, on isolated myocytes. Cultured rat ventricular myocytes were treated with the drug 16 h and 1 h before the experiments. The drug-treated cells and control cells were placed in a substrate free medium and submitted in a specially designed device to either normoxia (N4), or hypoxia (150 min. H2.5, or 240 min, H4), or 150 min hypoxia followed by 90 min reoxygenation (HR). The treatment of the cells with TMZ (5 × 10-4 M) resulted in a significant decrease of lactate dehydrogenase (LDH) leakage (-58% in H2.5, -36% in H4 and -37% in HR). The LDH release provoked by oxidizing agents, H2O2 and 13-s-HpOTrE (13(S)-hydroperoxyoctadecatrienoic acid) during post-hypoxic reoxygenation was also lowered by TMZ. However, this effect reflected the beneficial action of TMZ during hypoxia since the drug was not efficient in altering the LDH leakage induced by the oxidizing agents in normal conditions. Moreover, the hypoxia-induced decreased of ATP content was not affected by TMZ, and resynthesis of ATP during substrate-free reoxygenation was similar in TMZ-treated and control cells. The respiration parameters have been studied in rat heart mitochondria isolated from control and TMZ-treated rats, in the presence or absence of TMZ in the respiration medium (10-4 M). The main result was a rapid and potent inhibition of palmitoylcarnitine oxidation, when TMZ was added to the respiration medium. The chronic treatment only resulted in a slight alternation of pyruvate oxidation. In conclusion, a pre-treatment of ventricular myocytes with TMZ resulted in an increased cell resistance to hypoxic stress, as evidenced by LDH leakage. This cytoprotective effect to TMZ should not be mediated through an antioxidant activity, but could be related to a modification of lipid metabolism.

Enzymatic formation of 9,16-dihydro(pero)xyoctadecatrienoic acid isomers from α-linolenic acid

Incubation of α-linolenic acid with soybean lipoxygenase at pH 6.5 led to formation of conjugated triene oxidation products exhibiting maximum uv absorption at 267 nm, which were converted into four 9,16-dihydroxyoctadecatrienoic acid isomers. In the precursorsubstrate study, it seems that 9,16-dihydroxy acid isomers are derived from the doubly oxygenated products and the epoxide intermediate, which are both produced from hydrogen removal at C-14 of 9( S)-hydroperoxyoctadecatrienoic acid. Optimum pH and K m values for soybean lipoxygenase-1-catalyzed conversion of 9( S)-hydroperoxyoctadecatrienoic acid into the conjugated triene products were 8.5 and 80 μm, respectively.

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 K i value of 250 μM.

The possible role of 9(S)-hydroperoxyoctadecatrienoic acid as a suicide substrate of soybean lipoxygenase

While incubation of soybean lipoxygenase with α-linolenic acid resulted in the gradual decrease of lipoxygenase activity, the incubation with linoleic acid had no change. The inactivation of soybean lipoxygenase during incubation with α-linolenic acid was markedly observed at pH 6.5, but not at pH 9.0. Among the lipoxygenation products of α-linolenic acid, only 9(S)-hydroperoxyoctadecatrienoic acid caused the inactivation of lipoxygenase. 9(S)-Hydroxyoctadecatrienoic acid, 13(S)-hydroperoxyoctadecatrienoic acid or 9,16-dihydroperoxy conjugated trienoic acid was without effect. Accordingly, it is suggested that the epoxide intermediate, one conversion product of 9(S)-hydroperoxyoctadecatrienoic acid, might be involved in the direct inactivation of lipoxygenase.