Methods for the analysis of oxylipins in plants

Plant oxylipins comprise a highly diverse and complex class of molecules that are derived from lipid oxidation. The initial oxidation of unsaturated fatty acids may either occur by enzymatic or chemical reactions. A large variety of oxylipin classes are generated by an array of alternative reactions further converting hydroperoxy fatty acids. The structural diversity of oxylipins is further increased by their occurrence either as free fatty acid derivatives or as esters in complex lipids. Lipid peroxidation is common to all biological systems, appearing in developmentally regulated processes and as a response to environmental changes. The oxylipins formed may perform various biological roles; some of them have signaling functions. In order to elucidate the roles of oxylipins in a given biological context, comprehensive analytical assays are available for determining the oxylipin profiles of plant tissues. This review summarizes indirect methods to estimate the general peroxidation state of a sample and more sophisticated techniques for the identification, structure determination and quantification of oxylipins.     

Simultaneous tissue profiling of eicosanoid and endocannabinoid lipid families in a rat model of osteoarthritis

We describe a novel LC method for the simultaneous and quantitative profiling of 43 oxylipins including eicosanoids, endocannabinoids, and structurally related bioactive lipids with modified acyl groups. The LC-MS/MS method uses switching at a defined time between negative and positive electrospray ionization modes to achieve optimal detection sensitivity for all the lipids. The validated method is linear over a range of 0.01–5 nmol/g (0.1–50 nmol/g for 2-arachidonoyl glycerol) with intra- and interday precision and accuracy between 1.38 and 26.76% and 85.22 and 114.3%, respectively. The method successfully quantified bioactive lipids in different tissue types in the rat, including spinal cord, dorsal root ganglia (DRGs), knee joint, brain, and plasma. Distinct regional differences in the pattern of lipid measured between tissue types were observed using principle component analysis. The method was applied to analyze tissue samples from an established preclinical rat model of osteoarthritis (OA) pain and showed that levels of 12-hydroxyeicosatetraenoic acid were significantly increased in the OA rat knee joint compared with controls, and that 15-hydroxyeicosatetraenoic acid was significantly increased in the DRGs in the model of OA compared with controls. The developed LC-MS/MS method has the potential to provide detailed pathway profiling in tissues and biofluids where the disruption of bioactive oxylipins may be involved in disease states.     

Oxylipin profile and antioxidant status of potato tubers during extended storage at room temperature

Potato tubers (cv. Bintje) (Solanum tuberosum L.) were stored under extreme conditions at 20 °C for 350 days without sprout inhibitors in order to assess whether aging- and/or senescence-related processes occurred. Under these extreme storage conditions, multiple sprouting followed by the formation of daughter tubers occurs. At the same time, an increase in respiration intensity, as evidenced by cytochrome c oxidase activity (E.C. 1.9.3.1), is observed, leading to a potential increase in reactive oxygen species (ROS) production. As polyunsaturated fatty acids are priority targets of oxidative attacks, the damage to lipids was assessed by oxylipin profiling in both free and esterified forms. Oxylipin profiling showed a predominance of linoleic acid-derived oxylipins and of 9-hydroxy and 9-hydroperoxy fatty acids in both free and esterified forms. No significant accumulation of individual oxylipin was observed 350 days after harvest. To further understand the absence of lipid breakdown products accumulation, the main enzymatic and non-enzymatic antioxidants were assessed. Antioxidant enzyme activities [superoxide dismutase (E.C. 1.15.1.1), catalase (E.C. 1.11.1.6.), ascorbate peroxidase (E.C. 1.11.1.11)] were enhanced during the advanced phase of aging. The main non-enzymatic antioxidant compound, ascorbate, decreased markedly in the early stages of storage, followed by a slower decline. Total radical scavenging activity was also maintained at the end of the storage period. Our results indicate that the enhanced aging process occurring during storage at room temperature does not seem to be associated with the changes classically encountered during leaf senescence or seed aging and that the observed degenerative processes do not surpass the protective potential of the tubers.     

Volatile and non-volatile fungal oxylipins in fungus-invertebrate interactions

Eight-carbon volatiles are characteristic of the odour profile of many filamentous fungi. They derive from enzymatic or non-enzymatic lipid oxidation and are thus termed volatile oxylipins. Collectively, non-volatile and volatile fungal oxylipins are important hormone-like factors that regulate the phenotypic status of a fungus, i.e. growth, morphological differentiation and secondary metabolite production. Given this intimate link between oxylipin formation and phenotypic change, we propose that the release of volatile oxylipins is an important means by which fungi may influence the course and outcome of interactions with animals. Such invertebrate – fungus interactions are intricate inter–kingdom relationships where either one depends on the other, or both on each other, where one is to the others benefit or detriment – eventually having even consequences on third parties and thus influencing whole foodwebs. In this review, we first highlight the connections between oxylipin formation and fungal phenotypic changes, how they affect invertebrate interactions and vice versa. We then expand this by implementing eight-carbon volatiles as infochemicals. Infochemicals are cues or signals perceived by the invertebrates' chemical senses, that are to the invertebrates' or the fungus’ benefit or detriment, through the behavioural responses they elicit. We point out, with various examples, that there is a strong analogy between fungus-invertebrate interactions mediated by fungal eight-carbon volatiles and plant-herbivore interactions mediated by six-carbon green-leaf volatiles released from wounded or stressed plants.     

Lipoxygenases – Structure and reaction mechanism

Lipid oxidation is a common metabolic reaction in all biological systems, appearing in developmentally regulated processes and as response to abiotic and biotic stresses. Products derived from lipid oxidation processes are collectively named oxylipins. Initial lipid oxidation may either occur by chemical reactions or is derived from the action of enzymes. In plants this reaction is mainly catalyzed by lipoxygenase (LOXs) enzymes and during recent years analysis of different plant LOXs revealed insights into their enzyme mechanism. This review aims at giving an overview of concepts explaining the catalytic mechanism of LOXs as well as the different regio- and stereo-specificities of these enzymes.     

Lipid peroxidation: mechanisms, inhibition, and biological effects

In the last 50 years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipids are oxidized by three distinct mechanisms; enzymatic oxidation, non-enzymatic, free radical-mediated oxidation, and non-enzymatic, non-radical oxidation. Each oxidation mechanism yields specific products. The oxidation of linoleates and cholesterol is discussed in some detail. The relative susceptibilities of lipids to oxidation depend on the reaction milieu as well as their inherent structure. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. Phospholipid hydroperoxides, for example, are reduced to the corresponding hydroxides by selenoproteins in vivo. Various kinds of antioxidants with different functions inhibit lipid peroxidation and the deleterious effects caused by the lipid peroxidation products. Furthermore, the biological role of lipid peroxidation products has recently received a great deal of attention, but its physiological significance must be demonstrated in future studies.     

Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is an autoimmune inflammatory disorder. Highly reactive oxygen free radicals are believed to be involved in the pathogenesis of the disease. In this study, RA patients were sub-grouped depending upon the presence or absence of rheumatoid factor, disease activity score and disease duration. RA Patients (120) and healthy controls (53) were evaluated for the oxidant—antioxidant status by monitoring ROS production, biomarkers of lipid peroxidation, protein oxidation and DNA damage. The level of various enzymatic and non-enzymatic antioxidants was also monitored. Correlation analysis was also performed for analysing the association between ROS and various other parameters.MethodsIntracellular ROS formation, lipid peroxidation (MDA level), protein oxidation (carbonyl level and thiol level) and DNA damage were detected in the blood of RA patients. Antioxidant status was evaluated by FRAP assay, DPPH reduction assay and enzymatic (SOD, catalase, GST, GR) and non-enzymatic (vitamin C and GSH) antioxidants.ResultsRA patients showed a higher ROS production, increased lipid peroxidation, protein oxidation and DNA damage. A significant decline in the ferric reducing ability, DPPH radical quenching ability and the levels of antioxidants has also been observed. Significant correlation has been found between ROS and various other parameters studied.ConclusionRA patients showed a marked increase in ROS formation, lipid peroxidation, protein oxidation, DNA damage and decrease in the activity of antioxidant defence system leading to oxidative stress which may contribute to tissue damage and hence to the chronicity of the disease.     

Serum Levels of Oxylipins in Achilles Tendinopathy: An Exploratory Study

Background

Linoleic acid-derived oxidation products are found in experimental pain models. However, little is known about the levels of such oxylipins in human pain. In consequence, in the present study, we have undertaken a lipidomic profiling of oxylipins in blood serum from patients with Achilles tendinopathy and controls.

Methodology/Principal findings

A total of 34 oxylipins were analysed in the serum samples. At a significance level of P<0.00147 (<0.05/34), two linoleic acid-derived oxylipins, 13-hydroxy-10E,12Z-octadecadienoic (13-HODE) and 12(13)-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) were present at significantly higher levels in the Achilles tendinopathy samples. This difference remained significant when the dataset was controlled for age, gender and body-mass index. In contrast, 0/21 of the arachidonic acid- and 0/4 of the dihomo-γ-linolenic acid, eicosapentaenoic acid or docosahenaenoic acid-derived oxylipins were higher in the patient samples at this level of significance. The area under the Receiver-Operator Characteristic (ROC) curve for 12,13-DiHOME was 0.91 (P<0.0001). Levels of four N-acylethanolamines were also analysed and found not to be significantly different between the controls and the patients at the level of P<0.0125 (<0.05/4).

Conclusions/Significance

It is concluded from this exploratory study that abnormal levels of linoleic acid-derived oxylipins are seen in blood serum from patients with Achilles tendinopathy. Given the ability of two of these, 9- and 13-HODE to activate transient receptor potential vanilloid 1, it is possible that these changes may contribute to the symptoms seen in Achilles tendinopathy.     

Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-phytodienoic acid-containing galactolipid, arabidopside E

Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (>90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7-8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.     

High Sensitivity and Wide Linearity LC-MS/MS Method for Oxylipin Quantification in Multiple Biological Samples

Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05–0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a 1,000-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.     

Adipocyte lipid storage and adipokine production are modulated by lipoxygenase-derived oxylipins generated from 18-carbon fatty acids

Generation of oxylipins (oxygenated metabolites of fatty acids) by lipoxygenases may be responsible for the beneficial effects of 20- and 22-carbon n-3 fatty acids on adipose tissue dysfunction in obesity, but the potential actions of oxylipins derived from 18-carbon fatty acids, which are generally at higher levels in the diet, are unknown. We therefore compared the effects of select lipoxygenase-derived oxylipins produced from α-linolenic acid (ALA, C18:3 n-3), linoleic acid (LA, C18:2 n-6), and arachidonic acid (AA, C20:4 n-6) on key adipocyte functions that are altered in obesity. Individual oxylipins were added to the culture medium of differentiating 3T3-L1 preadipocytes for 6 days. Lipid accumulation was subsequently determined by Oil Red O staining, while Western blotting was used to measure levels of proteins associated with lipid metabolism and characteristics of adipocyte functionality. Addition of all oxylipins at 30 nM was sufficient to significantly decrease triglyceride accumulation in lipid droplets, and higher levels completely blocked lipid production. Our results establish that lipoxygenase-derived oxylipins produced from 18-carbon PUFA differentially affect multiple adipocyte processes associated with lipid storage and adipokine production. However, these effects are not due to the oxylipins blocking adipocyte maturation and thus globally suppressing all adipocyte characteristics. Furthermore, these oxylipin species decrease the lipid content of adipocytes regardless from which precursor fatty acid or lipoxygenase they were derived. Consequently, adipocyte characteristics can be altered through the ability of oxylipins to selectively modulate levels of proteins involved in both lipid metabolism and adipokine production.     

Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine

A GC-MS-based method for the simultaneous quantification of common oxylipins along with labile and highly reactive compounds based on in situ derivatization with pentafluorobenzyl hydroxylamine to the corresponding O-2,3,4,5,6-pentafluorobenzyl oximes (PFB oximes) is presented. The approach covers oxo derivatives such as jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), certain phytoprostanes, unsaturated oxo-acids, oxo-hydroxy acids, and aldehyde fragments from the polar head of fatty acids. In the positive electron impact-MS mode, the PFB oximes display characteristic fragment ions that greatly facilitate the identification of oxylipins in complex matrices. In addition, the fluorinated derivatives allow a highly selective and low-background analysis by negative chemical ionization. Besides showing the general value of the method for the identification of a broad range of oxylipins (18 examples), we also demonstrate sensitivity, linearity, and reproducibility for the quantification of JA, OPDA, 11-oxo-9-undecenoic acid, and 13-oxo-9,11-tridecadienoic acid. The efficiency of the method is demonstrated by differential profiling of these four oxylipins in lima bean leaves after mechanical wounding and feeding by the herbivore Spodoptera littoralis. Caterpillar feeding induced several oxylipins, whereas after wounding only the level of JA increased. The rapid in situ derivatization prevents the isomerization of cis-JA to trans-JA. The resting level of JA in lima beans showed an isomer ratio of 80:20 for trans/cis-JA. After wounding, de novo synthesis of JA alters the ratio to 20:80 in favor of the cis isomer.     

Oxylipins as developmental and host-fungal communication signals

Pathogenic microbes and their hosts have acquired complex signalling mechanisms to appraise themselves of the environmental milieu in the ongoing battle for survival. Several recent studies have implicated oxylipins as a novel class of host-microbe signalling molecules. Oxylipins represent a vast and diverse family of secondary metabolites that originate from the oxidation or further conversion of polyunsaturated fatty acids. Among the microbial oxylipins, the fungal oxylipins are best characterized and function as hormone-like signals that modulate the timing and balance between asexual and sexual spore development in addition to toxin production. Coupled with other studies that implicate a role for fungal oxylipins in pathogenesis by Aspergillus and Candida spp., these results suggest that host and microbial oxylipins might interfere with the metabolism, perception or signalling processes of each other.     

Enzymatic and non-enzymatic lipid peroxidation in leaf development.

Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and (1)H nuclear magnetic resonance suggested a non-enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development.     

Lipoxygenases: occurrence, functions and catalysis

Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes. Products are hydroperoxy polyunsaturated fatty acids and metabolites derived there from collectively named oxylipins. They may either originate from chemical oxidation or are synthesized by the action of various enzymes, such as lipoxygenases (LOXes). Signalling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among the numerous products. Cloning of many LOXes and other key enzymes metabolizing oxylipins, as well as analyses by reverse genetic approaches and metabolic profiling revealed new insights on oxylipin functions, new reactions and the first hints on enzyme mechanisms. These aspects are reviewed with respect to function of specific LOX forms and on the development of new models on their substrate and product specificity.     

Conjugated linoleic acid induces lipid peroxidation in humans

Conjugated linoleic acid (CLA) is shown to have chemoprotective properties in various experimental cancer models. CLA is easily oxidised and it has been suggested that an increased lipid oxidation may contribute to the antitumorigenic effects. This report investigates the urinary levels of 8-iso-PGF(2alpha), a major isoprostane and 15-keto-dihydro-PGF(2alpha), a major metabolite of PGF(2alpha), as indicators of non-enzymatic and enzymatic lipid peroxidation after dietary supplementation of CLA in healthy human subjects for 3 months. A significant increase of both 8-iso-PGF(2alpha) and 15-keto-dihydro-PGF(2alpha) in urine was observed after 3 months of daily CLA intake (4.2 g/day) as compared to the control group (P<0.0001). Conjugated linoleic acid had no effect on the serum alpha-tocopherol levels. However, gamma-tocopherol levels in the serum increased significantly (P=0. 015) in the CLA-treated group. Thus, CLA may induce both non-enzymatic and enzymatic lipid peroxidation in vivo. Further studies of the mechanism behind, and the possible consequences of, the increased lipid peroxidation after CLA supplementation are urgently needed.     

Similarity in the distribution of F(2)-isoprostanes in the lipid subfractions of atherosclerotic plaque and in vitro oxidised low density lipoprotein

Oxidation of low density lipoprotein (LDL) in vivo is thought to play a critical role in the initiation of atherosclerosis. F(2)-isoprostanes are compounds resulting from non-enzymatic oxidation of arachidonic acid and elevated levels are present in human atherosclerotic plaque. However, little is known about the formation of F(2)-isoprostanes in plaque lesions or their distribution in lipid subclasses. Given that LDL and tissue lipid subfractions (such as phospholipids, cholesterol esters and triglycerides) all contain significant levels of arachidonic acid, the aim of this study was to examine the relative distribution of F(2)-isoprostanes in the different lipid fractions of LDL oxidised in vitro, and compare this to the distribution in atherosclerotic plaque. The results reveal that while the majority of F(2)-isoprostanes are present in the phospholipid or surface lipid fractions, the core lipids (cholesterol esters/triglycerides) contribute at least 10% of the total F(2)-isoprostanes in both LDL oxidised in vitro and human atherosclerotic plaque. The remarkably similar profiles between the oxidised LDL and advanced atherosclerotic plaque suggests oxidation in vivo, is predominantly via non-enzymatic processes directed towards the surface lipids.     

Linoleic acid derived oxylipins are elevated in kidney and liver and reduced in serum in rats given a high-protein diet

High protein (HP) diets are often used as a means to reduce obesity, but their long-term effects remain unclear. In vitro studies suggest the involvement of a subset of oxylipins in the tissue response to HP diets. To examine the role of these bioactive lipids in vivo, normal adult male Sprague Dawley rats were provided isocaloric diets with LP (low protein, 8% protein by weight), NP (normal protein, 14%) or HP (50%) diets for 2 weeks, and targeted lipidomic analysis of oxylipins in kidney (cortex and medulla), liver and serum was performed by HPLC-MS/MS. The main group of oxylipins affected by the HP diet was the oxylipins derived from linoleic acid (LA), many of which were elevated in kidney (particularly the medulla) and liver, but reduced in serum of rats provided the HP compared to NP or LP diets. A smaller proportion of other n-6 fatty acid derived oxylipins were lower in kidney and higher in liver, and none were affected in serum, by HP feeding. Few n-3 oxylipins were affected by protein level. In liver only, the oxylipin product to substrate ratios of the soluble epoxide hydrolase enzyme were higher in LP fed rats. Differences between cortex and medulla oxylipins suggest relatively higher cortex activity of 5- and 8-lipoxygenase and cytochrome P450 hydroxylase, and higher medulla cyclooxygenase and 12- and 15-lipoxygenase activity. Further studies are needed to elucidate the physiological effects of the changes in these novel oxylipins in response to short-term dietary HP.