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Epoxyeicosatrienoic acids (EETs) are increasingly recognized as key metabolites in the arachidonic acid (AA) metabolic pathway. EETs are epoxy derivatives of AA with two chiral centers formed by cytochrome P450 (CYP) enzymes. EETs have reported biological activities as racemates; however, knowledge on specific optical isomers of EET is lacking. A main reason is the absence of practical assay to quantify EETs isomers associated with specific pathological conditions and enzymes. The reported underivatized chiral LC-MS/MS assays utilize different mobile phases and flow rates or required long run times to achieve separation of EET stereoisomers. Others incorporated a derivatization step before the separation of EETs in their assays. Therefore, the objective of this study was to develop and validate a stereoselective assay for the simultaneous quantitation of underivatized EET enantiomers using Liquid Chromatography Mass Spectrometry (LC-MS/MS) with an optimum baseline separation using binary mobile phase and gradient elution. Herein, we report the development and validation of an LC-MS/MS assay, and its application to quantify the formation of EET enantiomers mediated by human liver microsomes. Assay linearity extends over 10-600 ng/mL with r 2  > 0.99 for all EETs enantiomers. The inter-run accuracy was within ± 15 %, and precision was ≤ 15 %, and < 20 % for the LLOQ. The matrix effect for the current assay was within ≤ ±20 %, and the mean recovery for quantitative methods was 70-125 %. The assay proved to be reliable and practical for chiral analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Polyunsaturated fatty acids (PUFAs) are closely related to various physiological conditions. In several age-related diseases including Alzheimer's disease (AD) altered PUFAs metabolism has been reported. However, the mechanism behind PUFAs impairment and AD developpement remains unclear. In humans, PUFAs biosynthesis requires delta-5 desaturase (D5D), delta-6 desaturase (D6D) and elongase 2 activities; which are encoded by fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), and elongation of very-long-chain fatty acids-like 2 (ELOVL2) genes, respectively. In the present work, we aim to assess whether genetic variants in FADS1, FADS2 and ELOVL2 genes influence plasma and erythrocyte PUFA composition and AD risk. A case-control study was carried out in 113 AD patients and 161 healthy controls.Rs174556, rs174617, and rs3756963 of FADS1, FADS2, and ELOVL2 genes, respectively were genotyped using PCR-RFLP. PUFA levels were quantified using Gas Chromatography. Genotype distributions of rs174556 (FADS1) and rs3756963 (ELOVL2) were different between case and control groups. The genotype TT of rs174556 and rs3756963 single nucleotide polymorphism (SNP) increases significantly the risk of AD in our population. PUFA analysis showed higher plasma and erythrocyte arachidonic acid (AA) level in patients with AD, whereas only plasma docosahexaenoic acid (DHA) was significantly decreased in AD patients. The indexes AA/Dihomo-gamma-linolenic acid (DGLA) and C24:4n-6/Adrenic acid (AdA) were both higher in the AD group. Interestingly, patients with TT genotype of rs174556 presented higher AA level and AA/DGLA index in both plasma and erythrocyte. In addition, higher AA and AA/DGLA index were observed in erythrocyte of TT genotype ofrs3756963 carrier's patients. Along with, positive correlation between AA/DGLA index, age or Gamma-linolenic acid (GLA)/ Linoleic acid (LA) index was seen in erythrocyte and /or plasma of AD patients. After adjustment for confounding factors, the genotype TT of rs174556, erythrocyte AA and AA/DGLA index were found to be predictive risk factors for AD while plasma DHA was found associated with lower AD risk. Both rs174556 and rs3756963 influence AD risk in the Tunisian population and they are likely associated with high AA level. The combination of the two variants increases further the susceptibility to AD. We suggest that FADS1 and ELOVL2 variants could likely regulate the efficiency of AA biosynthesis which could be at the origin of inflammatory derivate., Competing Interests: Declaration of Competing Interest All authors have no conflict of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12'57' E 58°57'35″). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316-7 and RT2316-13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316-7. Percentage of EPA, DHA and the omega-6 fatty acid dihomo-γ-linolenic acid of total fatty acids of RT2316-13 was higher at the low incubation temperature. RT2316-13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316-13 a candidate for the production of lipids with different uses., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Cutaneous wound pain causes physical and psychological stress for patients with wounds. Previous studies reported that stress induces hyperalgesia and deteriorates wound healing. However, the effect of the stress response such as in hypothalamic-pituitary-adrenal (HPA) axis on local wound area is unclear. We aimed to investigate the effects of a stress response on the mechanical withdrawal threshold in the local wound area and describe the identification of a wound pain exacerbation. We topically injected adrenocorticotropic hormone (ACTH) into the granulation tissue of full-thickness cutaneous wound model rats on the fifth day postwounding and measured the mechanical withdrawal thresholds, cytochrome P450 2Bs levels and concentration of 5,6-epoxyeicosatrienoic acid in wound exudate. We found that ACTH induced mechanical hypersensitivity at 4 and 6 hours after injection (P = .004 and .021, respectively), and increased gene expression of cytochrome P450 2B12 expression (P = .046). Concentration of 5,6-EET in the wound exudate was moderately correlated with the mechanical withdrawal threshold (r = -.630). Finally, the mechanical withdrawal threshold in the 5,6-EET group was significantly lower than that in the control group at 2 hours after the injection (P = .015). We propose that 5,6-EET is one of the most promising contributors to the wound pain exacerbation. These findings could guide clinical wound and pain management., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Sarcoidosis is a systemic inflammatory multi-organ disease almost always affecting the lungs. The etiology remains unknown, but the hallmark of sarcoidosis is formation of non-caseating epithelioid cells granulomas in involved organs. In Scandinavia, > 30% of sarcoidosis patients have Löfgren's syndrome (LS), an acute disease onset mostly indicating a favorable prognosis. The impact of dysregulation of lipid mediators, which has been investigated in other inflammatory disorders, is still unknown., Methods: Using three different liquid chromatography coupled to tandem mass spectrometry targeted platforms (LC-MS/MS), we quantified a broad suite of lipid mediators including eicosanoids, sphingolipids and endocannabinoids in bronchoalveolar lavage (BAL) fluid from pulmonary sarcoidosis patients (n = 41) and healthy controls (n = 16)., Results: A total of 47 lipid mediators were consistently detected in BAL fluid of patients and controls. After false discovery rate adjustment, two products of the soluble epoxide hydrolase (sEH) enzyme, 11,12-dihydroxyeicosa-5,8,14-trienoic acid (11,12-DiHETrE, p = 4.4E-5, q = 1.2E-3, median fold change = 6.0) and its regioisomer 14,15-dihydroxyeicosa-5,8,11-trienoic acid (14,15-DiHETrE, p = 3.6E-3, q = 3.2E-2, median fold change = 1.8) increased in patients with sarcoidosis. Additional shifts were observed in sphingolipid metabolism, with a significant increase in palmitic acid-derived sphingomyelin (SM16:0, p = 1.3E-3, q = 1.7E-2, median fold change = 1.3). No associations were found between these 3 lipid mediators and LS, whereas levels of SM 16:0 and 11,12-DiHETrE associated with radiological stage (p < 0.05), and levels of 14,15-DiHETrE were associated with the BAL fluid CD4/CD8 ratio., Conclusions: These observed shifts in lipid mediators provide new insights into the pathobiology of sarcoidosis and in particular highlight the sEH pathway to be dysregulated in disease.

Patients with primary aldosteronism (PA) have increased risk of target-organ damage, among which vascular calcification is an important indicator of cardiovascular mortality. 14, 15-Epoxyeicosatrienoic acid (14, 15-EET) has been shown to have beneficial effects in vascular remodeling. However, whether 14, 15-EET associates with vascular calcification in PA is unknown. Thus, we aimed to investigate the association between 14, 15-EET and abdominal aortic calcification (AAC) in patients with PA. Sixty-nine patients with PA and 69 controls with essential hypertension, matched for age, sex, and blood pressure, were studied. 14, 15-Dihydroxyeicosatrienoic acid (14, 15-DHET), the inactive metabolite from 14, 15-EET, was estimated to reflect serum 14, 15-EET levels. AAC was assessed by computed tomographic scanning. Compared with matched controls, the AAC prevalence was almost 1-fold higher in patients with PA (27 [39.1%] versus 14 [20.3%]; P =0.023), accompanied by significantly higher serum 14, 15-DHET levels (7.18±4.98 versus 3.50±2.07 ng/mL; P <0.001). Plasma aldosterone concentration was positively associated with 14, 15-DHET (β=0.444; P <0.001). Multivariable logistic analysis revealed that lower 14, 15-DHET was an independent risk factor for AAC in PA (odds ratio, 1.371; 95% confidence interval, 1.145-1.640; P <0.001), especially in young patients with mild hypertension and normal body mass index. In conclusion, PA patients exibited more severe AAC, accompanied by higher serum 14, 15-DHET levels. On the contrary, decreased 14, 15-EET was significantly associated with AAC prevalence in PA patients, especially in those at low cardiovascular risk., (© 2018 American Heart Association, Inc.)

The amount of Zn absorbed from Zn-biofortified wheat material has been determined using an in vivo model of Zn absorption. The erythrocyte linoleic:dihomo -γ-linolenic acid (LA:DGLA) ratio was used as a biomarker of Zn status. Two groups of chickens (n = 15) were fed different diets: a high-Zn (46.5 μg Zn g -1 ) and a low-Zn wheat-based diet (32.8 μg Zn g -1 ). Dietary Zn intakes, body weight, serum Zn, and the erythrocyte fatty acid profile were measured, and tissues were taken for gene expression analysis. Serum Zn concentrations were greater in the high Zn group (p < 0.05). Duodenal mRNA expression of various Zn transporters demonstrated expression upregulation in the birds fed a low Zn diet (n = 15, p < 0.05). The LA:DGLA ratio was higher in the birds fed the low Zn diet (p < 0.05). The higher amount of Zn in the biofortified wheat resulted in a greater Zn uptake.

Background/aims: This study aimed to explore the metabololipidome in mice upon cupping treatment., Methods: A nude mouse model mimicking the cupping treatment in humans was established by administrating four cupping sets on the back skin for 15 minutes. UPLC-MS/ MS was performed to determine the PUFA metabolome in mice skin and blood before and after cupping treatment. The significantly changed lipids were administered in macrophages to assess the production of pro-inflammatory cytokines IL-6 and TNF-α by ELISA., Results: The anti-inflammatory lipids, e.g. PGE1, 5,6-EET, 14,15-EET, 10S,17S-DiHDoHE, 17R-RvD1, RvD5 and 14S-HDoHE were significantly increased while pro-inflammatory lipids, e.g. 12-HETE and TXB2 were deceased in the skin or plasma post cupping treatment. Cupping treatment reversed the LPS-stimulated IL-6 and TNF-α expression in mouse peritoneal exudates. Moreover, 5,6-EET, PGE1 decreased the level of TNF-α, while 5,6-EET, 5,6-DHET downregulated IL-6 production in macrophages. Importantly, 14,15-EET and 14S-HDoHE inhibited both IL-6 and TNF-α induced by lipopolysaccharide (LPS). 17-RvD1, RvD5 and PGE1 significantly reduced the LPS-initiated TNF-α, while TXB2 and 12-HETE further upregulated the LPS-enhanced IL-6 and TNF-α expression in macrophages., Conclusion: Our results reveal the identities of anti-inflammatory versus pro-inflammatory metabolipidome and suggest the potential therapeutic mechanism of cupping treatment., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: Sodium metabisulfite is commonly used as preservative in foods but can oxidize to sulfite radicals initiating molecular oxidation. Ghrelin is a peptide hormone primarily produced in the stomach and has anti-inflammatory effects in many organs. This study aimed to assess endogenous omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in rat peripheral organs following sodium metabisulfite treatment and determine the possible effect of ghrelin on changes in n-6 inflammatory pathway., Methods: Male Wistar rats included in the study were allowed free access to standard rat chow. Sodium metabisulfite was given by gastric gavage and ghrelin was administered intraperitoneally for 5 weeks. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in liver, heart and kidney tissues were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in n-6 inflammatory pathway., Results: Omega-6 PUFA levels, AA/DHA and AA/EPA ratio were significantly increased in liver tissue following sodium metabisulfite treatment compared to controls. No significant change was observed in heart and kidney PUFA levels. Tissue activity of COX and PGE2 levels were also significantly increased in liver tissue of sodium metabisulfite treated rats compared to controls. Ghrelin treatment decreased n-6 PUFA levels and reduced COX and PGE2 levels in liver tissue of sodium metabisulfite treated rats., Conclusion: Current results suggest that ghrelin exerts anti-inflammatory action through modulation of n-6 PUFA levels in hepatic tissue.

Introduction: Cardioprotective effect of high density lipoprotein (HDL) is, in part, dependent on its related enzyme, paraoxonase 1 (PON1). Fatty acid composition of HDL could affect its size and structure. On the other hand, PON1 activity is directly related to the structure of HDL. This study was designed to investigate the association between serum PON1 activity and fatty acid composition of HDL in healthy men., Methods: One hundred and forty healthy men participated in this research. HDL was separated by sequential ultracentrifugation, and its fatty acid composition was analyzed by gas chromatography. PON1 activity was measured spectrophotometrically using paraxon as substrate., Results: Serum PON1 activity was directly correlated with the amount of stearic acid and dihomo-gamma-linolenic acid (DGLA). PON1/HDL-C was directly correlated with the amount of miristic acid, stearic acid, and DGLA and was inversely correlated with total amount of ω 6 fatty acids of HDL., Conclusion: The fatty acid composition of HDL could affect the activity of its associated enzyme, PON1. As dietary fats are the major determinants of serum lipids and lipoprotein composition, consuming some special dietary fatty acids may improve the activity of PON1 and thereby have beneficial effects on health.

Background: Fullerenes are molecules being investigated for a wide range of therapeutic applications. We have shown previously that certain fullerene derivatives (FDs) inhibit mast cell (MC) function in vitro, and here we examine their in vivo therapeutic effect on asthma, a disease in which MCs play a predominant role., Objective: We sought to determine whether an efficient MC-stabilizing FD (C(70)-tetraglycolate [TGA]) can inhibit asthma pathogenesis in vivo and to examine its in vivo mechanism of action., Methods: Asthma was induced in mice, and animals were treated intranasally with TGA either simultaneously with treatment or after induction of pathogenesis. The efficacy of TGA was determined through the measurement of airway inflammation, bronchoconstriction, serum IgE levels, and bronchoalveolar lavage fluid cytokine and eicosanoid levels., Results: We found that TGA-treated mice have significantly reduced airway inflammation, eosinophilia, and bronchoconstriction. The TGA treatments are effective, even when given after disease is established. Moreover, we report a novel inhibitory mechanism because TGA stimulates the production of an anti-inflammatory P-450 eicosanoid metabolites (cis-epoxyeicosatrienoic acids [EETs]) in the lung. Inhibitors of these anti-inflammatory EETs reversed TGA inhibition. In human lung MCs incubated with TGA, there was a significant upregulation of CYP1B gene expression, and TGA also reduced IgE production from B cells. Lastly, MCs incubated with EET and challenged through FcεRI had a significant blunting of mediator release compared with nontreated cells., Conclusion: The inhibitory capabilities of TGA reported here suggest that FDs might be used a platform for developing treatments for asthma., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Objective: Sex differences in cerebral ischemic injury are, in part, attributable to the differences in cerebrovascular perfusion. We determined whether the brain microvascular endothelial cells (ECs) isolated from the female brain are more resistant to ischemic injury compared with male ECs, and whether the difference is attributable to lower expression of soluble epoxide hydrolase and higher levels of vasoprotective epoxyeicosatrienoic acids (EETs). We also determined whether protection by EETs is linked to the inhibition of rho-kinase (ROCK)., Methods and Results: EC ischemic damage was measured after oxygen-glucose deprivation (OGD) using propidium iodide (PI) and cleaved caspase-3 labeling. Expression of soluble epoxide hydrolase was determined by quantitative polymerase chain reaction and immunocytochemistry, EETs levels by liquid chromatography-tandem mass spectrometry, and ROCK activity by ELISA. EC damage was higher in males compared with females, which correlated with higher soluble epoxide hydrolase mRNA, stronger immunoreactivity, and lower EETs compared with female ECs. Inhibition of soluble epoxide hydrolase abolished the sex difference in EC damage. ROCK activity was higher in male versus female ECs after OGD, and sex differences in EC damage and ROCK activity were abolished by 14,15-EET and ROCK inhibition., Conclusions: Sex differences in ischemic brain injury are, in part, attributable to differences in EET-mediated inhibition of EC ROCK activation after ischemia.

Epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH) have been associated with ischemic stroke. Salvianolic acid A (SAA) is proved to display potent cerebroprotection. However, little information is available about the link between them. This study aimed to investigate whether SAA exhibits its protective effects in rats subjected to middle cerebral artery occlusion (MCAO) through sEH and EETs. The results showed that SAA treatment ameliorated neurological deficits and reduced infarct volume. Notably, the beneficial effects of SAA were attenuated by co-administration of (14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE)), a putative selective EETs antagonist. Furthermore, SAA increased the 14,15-EET levels in the blood and brain of sham and MCAO rats. Assay for hydrolase activity showed that 1 and 3 mg/kg of SAA significantly diminished brain sEH activity of MCAO rats. A fluorescent assay in vitro indicated that SAA could inhibit recombinant human sEH activity in a concentration-dependent manner (IC(50) = 1.62 μmol/l). Immunohistochemical analysis showed that SAA at the doses of 1 and 3 mg/kg significantly decreased sEH protein expression in hippocampus CA1 region of MCAO rats. In conclusion, cerebral protection of SAA is mediated, at least in part, via inhibiting sEH to increase EETs levels.

Like arachidonic acid (AA), dihomo-γ-linolenic acid (DGLA) is a 20-carbon ω-6 polyunsaturated fatty acid and a substrate of cyclooxygenase (COX). Through free radical reactions, COX metabolizes DGLA and AA to form well-known bioactive metabolites, namely, the 1 and 2 series of prostaglandins (PGs1 and PGs2), respectively. Unlike PGs2, which are viewed as proinflammatory, PGs1 possess anti-inflammatory and anticancer activities. However, the mechanisms linking the PGs to their bioactivities are still unclear, and radicals generated in COX-DGLA have not been detected. To better understand PG biology and determine whether different reactions occur in COX-DGLA and COX-AA, we have used LC/ESR/MS with a spin trap, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN), to characterize the carbon-centered radicals formed from COX-DGLA in vitro, including cellular peroxidation. A total of five types of DGLA-derived radicals were characterized as POBN adducts: m/z 266, m/z 296, and m/z 550 (same as or similar to COX-AA) and m/z 324 and m/z 354 (exclusively from COX-DGLA). Our results suggest that C-15 oxygenation to form PGGs occurs in both COX-DGLA and COX-AA; however, C-8 oxygenation occurs exclusively in COX-DGLA. This new finding will be further investigated for its association with various bioactivities of PGs, with potential implications for inflammatory diseases., (Published by Elsevier Inc.)

The molecular bases of Alzheimer's disease (AD) remain unclear. We used a lipidomic approach to identify lipid abnormalities in the brains of subjects with AD (N = 37) compared to age-matched controls (N = 17). The analyses revealed statistically detectable elevations in levels of non-esterified monounsaturated fatty acids (MUFAs) and mead acid (20:3n-9) in mid-frontal cortex, temporal cortex and hippocampus of AD patients. Further studies showed that brain mRNAs encoding for isoforms of the rate-limiting enzyme in MUFAs biosynthesis, stearoyl-CoA desaturase (SCD-1, SCD-5a and SCD-5b), were elevated in subjects with AD. The monounsaturated/saturated fatty acid ratio ('desaturation index')--displayed a strong negative correlation with measures of cognition: the Mini Mental State Examination test (r = -0.80; P = 0.0001) and the Boston Naming test (r = -0.57; P = 0.0071). Our results reveal a previously unrecognized role for the lipogenic enzyme SCD in AD.

Alterations by perfluorinated fatty acids (PFCAs) with a chain length of 6-9 carbons in the fatty acid profile of hepatic lipids of mice were investigated. The characteristic changes caused by all the PFCAs examined were increases in the contents and proportions of oleic acid (18 : 1), palmitoleic acid (16 : 1) and 8,11,14-eicosatrienoic acid (20 : 3) in hepatic lipids. Hepatic contents of palmitic acid were also increased by the treatments with the PFCAs. These effects were almost dependent on the hepatic concentrations of PFCA molecules regardless of their carbon chain length. Perfluorooctanoic acid elevated the expressions of mRNA encoding acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, stearoyl-CoA desaturase (SCD) (SCD1 and 2), chain elongase (ELOVL5), Δ6 desaturase (Fads2), 1-acylglycerophosphocholine acyltransferase (LPCAT) (LPCAT3). The four PFCAs examined induced microsomal SCD and LPCAT in hepatic concentration-dependent manners regardless of carbon chain length. One linear regression line was confirmed between LPCAT activity and hepatic concentration of PFCA at wide range of the concentration, whereas the induction of SCD was saturable at relatively low concentration of PFCAs. These results suggest (i) that PFCAs with a chain length of 6-9 carbons change the fatty acid profile of hepatic lipids by increasing contents and proportions of 16 : 1, 18 : 1 and 20 : 3, (ii) that these alterations in fatty acid profile are caused by up-regulation of SCD, de novo fatty acid synthesis, chain elongase and Δ6 desaturase and (iii) that the mechanism underlying SCD induction is, in part, mediated through peroxisome proliferator-activated receptor α.

Epoxyeicosatrienoic acids (EETs) play important protective functions in cardiovascular and renal systems. Under physiological conditions, EETs are quickly converted by the soluble epoxide hydrolase (sEH) to diols which do not have the beneficiary roles. Inhibition of sEH with small molecules to increase the concentration of EETs therefore provides an attractive therapeutic strategy for cardiovascular diseases. We describe here the development of a high throughput cell-based assay to measure sEH activity and screen small molecular compounds as sEH inhibitors. This assay is based on the technology of fluorescence polarization (FP), utilizing a Cy3B labeled 14,15-DHET ligand and a rabbit anti-14,15-DHET antibody. With the optimized assay, we measured the cellular sEH activity of several cell lines expressing endogenous sEH as well as sEH BacMam transduced HEK-293 cells. The inhibitory effect of several known sEH inhibitors was evaluated in sEH BacMam transduced HEK-293 cells. Our data show that there is good agreement of pIC(50) values obtained between the FP format and a commercially available ELISA kit. To our knowledge, this is the first report of a high throughput cell-based assay for screening sEH inhibitors.

Lowered sperm quality associated with reduced superoxide dismutase activity in testis and spermatozoa has been observed in red deer from a mined area in South-central Spain. Here we present fatty acid profiles for testis and spermatozoa of deer from this mined area (n=29) and a control area (n=33). Despite elevated Pb in liver and bone of red deer from this area, concentrations in testis and sperm were not significantly higher than in control areas; however, Cu in testis was lower in mined areas. Testis from mined areas also contained higher percentages of linoleic acid (18:2n-6) and dihomo-gamma-linolenic acid (20:3n-6), but lower arachidonic acid (20:4n-6). The percentage of 20:4n-6 was also lower in spermatozoa of deer from the mined area. Copper levels in testis correlated positively with the percentage of 20:4n-6. The imbalance in Cu homeostasis caused by metal pollution may have caused the observed effects on deer sperm., (Copyright 2010 Elsevier Inc. All rights reserved.)

We used liquid chromatography for analysis of fatty acids (FA) in lipids of erythrocytes of patients with hypertensive disease (HD) with normo- (group 1) and hyperlipidemia (group 2). Abnormalities of FA composition of erythrocyte lipids were revealed in both groups. In group 1 we found deficit of polyenic acids of omega-6 family, accumulation of Mead acid - prostanoid precursor with pronounced vasoconstrictor and pro inflammatory properties. In group 2 we noted more profound rearrangement of lipid matrix of erythrocyte membrane manifested as deficiency of omega-3 polyenic acids, accumulation of palmitinic and arachidonic acids. Preponderance of saturated FA in erythrocytes and deficiency of polyenic acids might evidence for pathology of their ligand-receptor transport into the cell. Blockade of active FA transport initiates formation of HD, promotes accumulation of atherogenic fractions of lipoproteins in blood. These results evidence for important pathogenetic role of FA in development of hypertension.

The purpose of this study was to compare the susceptibility of the retina and the exorbital lacrimal gland to dietary supplies of long-chain omega-3 (omega3) and omega-6 (omega6) polyunsaturated fatty acids (LC-PUFAs). Male Wistar rats were fed a 5% lipid diet containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% gamma-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA or (4) a balanced diet deprived of EPA, DHA and GLA for 3 months. Lipids were extracted from plasma phospholipids, retina and exorbital lacrimal gland, and fatty acid composition was determined by gas chromatography. Dietary supplementation with EPA and DHA increased omega3 PUFA levels in plasma phospholipids as well as in the retina and the exorbital lacrimal gland. By contrast, GLA supplementation favored omega6 PUFA incorporation, and particularly the incorporation of the end-chain omega6 product, docosapentaenoic acid (DPA), into all tissues. Supplementation with EPA, DHA and GLA increased the levels of DHA, EPA and dihomo-GLA (dGLA), whereas arachidonic acid (AA) was unchanged and DPA decreased in the retina and the lacrimal gland. The ability of both tissues to incorporate PUFAs from blood was evaluated. The results showed that the retina was more selective than the lacrimal gland for EPA. In spite of the different susceptibility of the retina and the lacrimal gland to dietary PUFAs, these results suggest that the concomitant use of dietary omega3 and omega6 PUFAs may be useful in modulating inflammation in both tissues., (Copyright 2009 S. Karger AG, Basel.)

Despite first evidence for the cytochrome P450-mediated enantioselective biosynthesis and activity of cis-epoxyeicosatrienoic acids (EETs), as yet little is known about the stereospecifity of EET generation and physiology, because the existing chiral methods are time consuming, labor intensive, and not sensitive enough. We present a method for highly sensitive, direct, and simultaneous chiral analysis of all eight EET enantiomers consisting of (i) solid-phase extraction, (ii) reversed-phase high-performance liquid chromatographic purification followed by (iii) consecutive regio- and enantiomeric separation of the four underivatized EET regioisomers within one chromatographic run employing capillary tandem column chiral-phase liquid chromatography with (iv) reliable dual online photodiode array and gentle electrospray ionization tandem mass spectrometric identification and quantitation of the eluting optical antipodes. This one-step, simple, expeditious, and highly sensitive measurement allows profiling of all eight EET enantiomers at once, thus avoiding substance loss and enabling high sample throughput. Limits of quantification in the low picogram range were achieved by the use of capillary columns with typical high quantitative sensitivity instead of conventional columns with low chromatographic signal intensity employed by previous methods. Application to tissue homogenates demonstrated the suitability of this approach for routine and reliable "enantioprofiling" of free endogenous EETs, i.e., EETs not esterified into cellular membrane phospholipids, typically occurring at very low concentrations. The technique can readily be employed for preparative purification of enantiomers in the microgram range using large-inner-diameter columns.

Fish oil, a rich source of omega-3 fatty acids, has never been used as the sole source of lipid in clinical practice for fear of development of essential fatty acid deficiency, as it lacks the believed requisite levels of linoleic acid, an omega-6 fatty acid. The objectives of this study were to establish biochemical standards for fish oil as the sole fat and to test the hypothesis that fish oil contains adequate amounts of omega-6 fatty acids to prevent essential fatty acid deficiency. Forty mice were divided into 2 groups that were either pair fed or allowed to eat ad libitum. In each group, 4 subgroups of 5 mice were fed 1%, 5%, and 10% fish oil diets by weight or a control soybean diet for 9 weeks. Blood was collected at 4 time points, and fatty acid analysis was performed. Food intake and weight status were monitored. All groups but the pair-fed 1% fish oil group gained weight, and the 5% fish oil group showed the highest caloric efficiency in both pair-fed and ad libitum groups. Fatty acid profiles for the 1% fish oil group displayed clear essential fatty acid deficiency, 5% fish oil appeared marginal, and 10% and soybean oil diets were found to prevent essential fatty acid deficiency. Fish oil enhances growth through higher caloric efficiency. We established a total omega-6 fatty acid requirement of between 0.30% and 0.56% of dietary energy, approximately half of the conventionally believed 1% as linoleic acid. This can presumably be attributed to the fact that fish oil contains not only a small amount of linoleic acid, but also arachidonic acid, which has greater efficiency to meet omega-6 fatty acid requirements.

Background: It has been suggested that atopic dermatitis (AD) is associated with impaired delta-6 desaturase activity and the subsequent altered composition of n-6 essential fatty acids (EFAs)., Objective: To investigate whether n-6 EFA deficiency accounts for AD by affecting transepidermal water loss or the immune response., Methods: Serum levels of n-6 EFAs were measured using gas chromatography-mass spectrometry in a well-defined group of 35 children with AD (IgE level >150 U/mL); 35 age-matched children with allergic rhinitis, asthma, or both (IgE level >150 U/mL); and 31 nonatopic controls (IgE level <100 U/mL). Skin barrier function was evaluated by measuring transepidermal water loss and severity of AD by computing the Scoring Atopic Dermatitis (SCORAD) index., Results: Atopic children had higher levels of linoleic acid (LA) and lower levels of its metabolites. Furthermore, gamma-linolenic acid to LA and dihommo-gamma-linolenic acid to LA ratios were significantly reduced in atopic patients. Transepidermal water loss and the SCORAD index were negatively correlated with serum levels of LA metabolites. There was no correlation between the SCORAD index and IgE level (P = .51) or between n-6 EFA concentrations and IgE level (P > .10)., Conclusions: Deficits in n-6 EFAs were correlated with the severity of AD by affecting skin barrier function and cutaneous inflammation. The link between impaired n-6 EFA metabolism and IgE level could not be defined.

There is a relationship between the fatty acid profile in skeletal muscle phospholipids and peripheral resistance to insulin in adults, but similar data have not been reported in infancy and childhood. The objective of this study was to investigate the fatty acid composition of skeletal muscle and adipose tissue across the paediatric age range. The fatty acid profile of skeletal muscle phospholipids and adipose tissue triacylglycerols was analysed in ninety-three healthy Spanish infants and children distributed into four groups: group 1 (0 to <2 years, n 10); group 2 (2 to <5 years, n 41); group 3 (5 to <10 years, n 24); group 4 (10 to 15 years, n 18). In skeletal muscle phospholipids, oleic acid (18: 1n-9cis) content decreased significantly whereas that of linoleic (18: 2n-6) acid increased significantly with age (P for trend <0.01). In adipose tissue, the contents of triacylglycerol and linoleic acid increased significantly across the paediatric age range (P for trend <0.01), whereas dihomo-gamma-linolenic (20: 3n-6) and arachidonic (20: 4n-6) showed significant differences between groups. The variations in fatty acid composition observed with age indicated an imbalance in dietary n-3/n-6 long-chain PUFA.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

In this study, we examined the effects of sesamin and vegetable oil on the concentrations of polyunsaturated fatty acid (PUFA) and lipids (triacylglycerol, free cholesterol, and phospholipid), and beta-oxidation enzyme activities in the rat liver. Rats were fed a diet containing 5% (low-fat diet) or 20% (high-fat diet) salad oil (rapeseed oil: soybean oil, 7:3) with or without sesamin (0.5% w/w) for 4 wk. As a result, the concentrations of linoleic acid (LA, n-6), alpha-linolenic acid (ALA, n-3), and total PUFA in the liver increased significantly as the result of the high-fat diet. In the high-fat diet groups, sesamin administration decreased the concentrations of LA, ALA, and total PUFA to almost the same level as the low-fat diet group, while it increased the concentrations of dihomo-gamma-linolenic acid (DGLA, n-6) and arachidonic acid (AA, n-6). The activities of carnitine acyltransferase and acyl-CoA dehydrogenase in liver mitochondria were enhanced by the intake of the high-fat diet, and were further enhanced by the administration of sesamin. Peroxisomal acyl-CoA oxidase activity was also enhanced by sesamin, while it was not affected by the dietary fat level. These results suggest that sesamin suppressed the increase of hepatic PUFA concentration caused by feeding the high-fat diet through enhancing the enzyme activities of fatty acid beta-oxidation and PUFA metabolism from LA and ALA.

Human trophoblast cells are known to release a range of arachidonic acid metabolites into culture medium, including cyclo-oxygenase, lipoxygenase and epoxygenase products. In this study we investigated the effects of dibutyryl cyclic AMP (db cAMP) on arachidonic acid metabolism in human first trimester trophoblast cells, and also determined the distribution of metabolites between intracellular and extracellular compartments. db cAMP increased intracellular levels of radioactivity within 2 min, and extracellular levels of radioactivity were increased after 30 min. These changes were reflected in increased levels of arachidonic acid metabolites in both compartments, indicating that arachidonic acid was metabolised. db cAMP increased intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) within 2 min of addition to cultured cells. No changes were detected after 5-10 min, but substantial changes were found 30 min after the addition of db cAMP. The dihydroxyeicosatrienoic acid (DiHETrE) breakdown products also increased with similar kinetics. In contrast, levels of 14,15-EpETrE increased after 5-10 min.

Each of the four regioisomers of epoxyeicosatrienoic acids (EETs) is a candidate for being an endothelial-dependent hyperpolarizing factor (EDHF). One regioisomer, 14,15-EET, stereospecifically blocks cyclooxygenases from converting arachidonic acid to prostaglandins and stereospecifically binds to cellular receptors. Both stereospecific actions emphasize the need to establish the tissue availability of the 14,15-EET enantiomers. The present work describes a method to quantitate picogram amounts of 14,15-EET enantiomers by capillary electrophoresis. The 14,15-EET enantiomers were baseline resolved (R = 1.3) using unsubstituted beta-cyclodextrin and 32% acetonitrile (v/v). When absorption at 194 nm was monitored using a photodiode array detector, 8 and 1 pg of underivatized 14,15-EET were readily quantitated and detected, respectively. Capillary electrophoresis accurately assessed chiral excesses up to 97:3 for either 14,15-EET enantiomer. Moreover, capillary electrophoresis with a photodiode array detector was sufficiently sensitive to detect and measure 14,15-EET enantiomers from murine liver. Thus, unlike chiral-phase high-performance liquid chromatography, capillary electrophoresis can be used to directly assess the chirality of trace amounts of underivatized eicosanoids.

Long-chain PUFA play an important role in early human neurodevelopment. Significant inverse correlations were reported between values of trans isomeric and long-chain PUFA in plasma lipids of preterm infants and children aged 1-15 yr as well as in venous cord blood lipids of full-term infants. Here we report FA compositional data of cord blood vessel wall lipids in 308 healthy, full-term infants (gestational age: 39.7 +/- 1.2 wk, birth weight: 3528 +/- 429 g, mean +/- SD). The median (interquartile range) of the sum of 18-carbon trans FA was 0.22 (0.13) % w/w in umbilical artery and 0.16 (0.10) % w/w in umbilical vein lipids. Nonparametric correlation analysis showed significant inverse correlations between the sum of 18-carbon trans FA and both arachidonic acid and DHA in artery (r = -0.38, P < 0.01, and r = -0.20, P < 0.01) and vein (r = -0.36, P < 0.01, and -0.17, P < 0.01) wall lipids. In addition, the sum of 18-carbon trans FA was significantly positively correlated to Mead acid, a general indicator of EFA deficiency, in both artery (r = +0.35, P < 0.01) and vein (r = +0.31, P< 0.01) wall lipids. The present results obtained in a large group of full-term infants suggest that maternal trans FA intake is inversely associated with long-chain PUFA status of the infant at birth.

The objective of this report was to determine whether vaccenic acid (t11-18:1) is converted efficiently to conjugated linoleic acid (c9,t11-18:2, CLA) in rats via the delta 9-desaturase reaction and, if so, whether vaccenic acid could substitute for CLA as an anticancer agent. In Study 1, rats were fed 1%, 2%, or 3% vaccenic acid in their diet, and tissue levels of CLA and CLA metabolites were determined in liver and mammary gland. In general, concentrations of CLA and CLA metabolites increased proportionately with an increase in vaccenic acid intake, at least up to the 2% dose level. Beyond this dose, there was clearly a plateauing effect. Thus vaccenic acid concentration increased from an undetectable level in the control to 78.5 nmol/mg lipid in the liver of rats fed a 2% vaccenic acid diet. This was accompanied by an increase in CLA from 2.3 to 33.6 nmol/mg lipid. These changes were also mirrored in the mammary gland, where increases in vaccenic acid (from 27.5 to 163.2 nmol/mg lipid) and CLA (from 17.8 to 108.9 nmol/mg lipid) were similarly observed. Vaccenic acid at 2% produced a CLA concentration in the mammary gland that was historically associated with a positive response in tumor inhibition based on our past experience. This provided the basis for selecting 2% vaccenic acid in Study 2, which was designed to evaluate its efficacy in blocking the development of premalignant lesions in the rat mammary gland. In this experiment, formation of histologically identifiable pathology due to intraductal proliferation of terminal end bud cells of mammary epithelium was used as the end point of analysis at 6 wk after carcinogen administration. Treatment with vaccenic acid reduced the total number of these premalignant lesions by approximately 50%. We hypothesize that the anticancer response to vaccenic acid is likely to be mediated by its endogenous conversion to CLA via delta 9-desaturase.

Long-chain polyunsaturated fatty acids are important components of human milk that seem to influence infant development. After oral administration of U-13C-labeled linoleic acid to a lactating woman the recovery of tracer in milk linoleic acid was 6.4%, whereas tracer recovery in dihomo-gamma-linolenic acid (DGLA) was 0.3% and in arachidonic acid (AA), <0.01%. Some 14.9% of linoleic acid intake was converted to breath-CO2. In combination with data on dietary intake and quantitative milk output, we estimate that 23% of milk linoleic acid, 7% of milk dihomo-gamma-linolenic acids and 0.5% of milk arachidonic acid were contributed by direct endogenous conversion and transfer from dietary linoleic acid. These findings lead us to conclude that long-chain polyunsaturated fatty acids for human milk synthesis are produced in the body, but either a relatively low percentage is contributed under these dietary conditions, or there are large body pools for intermediate storage of these fatty acids that are utilized for secretion into milk.

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11, 12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C(18) microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.

A modification of the human monooxygenase system have been previously associated with the sudden infant death syndrome (SIDS): the hepatic CYP2C content was markedly enhanced and resulted from an activation of CYP2C gene transcription. To determine the possible consequence of the up-regulation of CYP2C in SIDS, we examined the metabolism of arachidonic acid (AA) an endogenous substrate of CYP2C involved in the physiologic regulation of vascular tone. The overall AA metabolism was extremely low during the fetal period and rose after birth to generate 14,15 epoxyeicosatrienoic acid (EET), 11,12 EET and the sum of 5,6 dihydroxyeicosatrienoic acid (diHETE)+omega/omega-1 hydroxy AA. In SIDS, the accumulation of CYP2C proteins was associated with a significant increase in the formation of 14,15 and 11,12 diHETE, which were shown to be supported by individually expressed CYP2C8 and 2C9 and HETE1 (presumably 15 HETE). This increase was markedly inhibited by addition of sulfaphenazole, a selective inhibitor of CYP2C9. So, we propose that the higher CYP2C content in SIDS stimulates the production of EETs and diHETEs and might have severe pathologic consequences in children.

The corneal epithelium metabolizes arachidonic acid by a cytochrome P450-(CYP) mediated pathway to 12(R)hydroxy-5,8,10,14-eicosatrienoic acid [12(R)-HETE] and 12(R)hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE]. Both metabolites possess potent inflammatory properties with 12(R)-HETrE being a powerful angiogenic factor and assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea, in vivo. We developed an in vitro model of corneal organ culture to characterize the biochemical and molecular events involved in the increased synthesis of these metabolites. These cultured corneas exhibit epithelial cytochrome P450 CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis as indicated by chiral analysis and by the ability of CYP enzyme inhibitors to repress their synthesis. Hypoxia greatly and selectively stimulated the synthesis of 12(R)-HETE (7-fold over control normoxic conditions) and 12(R)-HETrE. The bacterial endotoxin, lipopolysaccharide, also increased the synthesis of these eicosanoids, substantiating the notion that this activity may function as an inflammatory pathway. These metabolites were detected in the culture medium by gas chromatography/mass spectroscopy (GC/MS) analysis and their levels significantly increased in hypoxia-treated corneas, further indicating their endogenous formation in response to injury. This in vitro model provides an excellent preparation for studying factors regulating the synthesis of these inflammatory eicosanoids and for isolating, identifying and characterizing the CYP protein responsible for their synthesis.

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

Abnormal fetal and infant growth have increasingly been correlated with adult onset cardiovascular disease. To date, there is little known about the lipid fatty acid profiles in infant cardiovascular tissue. Therefore, we analysed total lipid fatty acids from thoracic and abdominal aorta intima and media from 24 normally grown sudden infant death syndrome cases. Aorta from small for gestational age (n = 2), failure to thrive from birth (n = 3), and premature (n = 1) infants were also examined. Dihomo-gamma-linolenic acid (C20:3n-6) and oleic acid (C18:1n-9) concentrations were significantly lower in the thoracic than in the abdominal aorta. Similar dietary related differences were found in the subgroup (n = 15) of infants fed on formula milks. Both abdominal and thoracic intimal arachidonic (C20:4n-6) to dihomo-gamma-linolenic acid ratios were greater in the infants with retarded growth after birth than in their normally grown counterparts. Growth restriction in infancy might disrupt the normal accretion of vascular endothelial polyunsaturated fatty acids.

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-gamma-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E2 and F2 alpha). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50-4800 ng g-1 of PGE2 and 6-30 ng g-1 of PGF2 alpha). Several micro-organisms also produced prostaglandins in the culture medium (2.2-137.6 micrograms 1-1 for PGE2 and 0.4-7.8 micrograms 1-1 for PGF2 alpha).

Objective: To test the null-hypothesis that no age difference in adipose tissue fatty acid composition exists independent of dietary fat intake., Design: A cross-sectional survey of coronary heart disease risk factors, the Scottish Heart Health Study, provided needle biopsy adipose tissue fatty acid data and food frequency-derived dietary data., Setting: Twenty-two Scottish Districts between 1984 and 1986., Subjects: A total of 10,359 men and women aged 40-59 y were randomly recruited in sex and five-year age bands from GP lists. A sub-set of 2308 men and 2049 women (42%) provided satisfactory adipose tissue and dietary data., Main Outcome and Measures: Multiple regression analysis (adjusting for dietary fats, body mass index and smoking, with and without menopause status for women) of the relationship between individual fatty acids in adipose tissue and age, and between age and the ratio of linoleic acid (C18:2, n-6) to gamma-linolenic acid (C18:3, n-6) as an indicator of delta-6 desaturase activity., Results: Sex-consistent changes with age occurred for linoleate (adjusted regression slope +/- s.e. for men -0.299 +/- 0.1339 and for women -0.504 +/- 0.1731) and gamma-linolenate (adjusted regression slope +/- s.e. for men -0.141 +/- 0.0341 and for women -0.154 +/- 0.0469) both P < 0.0001. These changes gave rise to a significant increase (P < or = 0.005) in the C18:2, n-6 to C18:3, n-6 ratio with age). Dihomo-gamma-linolenic acid (C20:3, n-6) and docosahexa- plus docosapentaenoic acids (C22:5 + C22:6, n-3) also increased significantly with age (P < or = 0.01). For the latter, the adjusted regression slopes were far greater for women (0.596 +/- 0.0575) than men (0.131 +/- 0.0417)., Conclusions: The results show that ageing does influence adipose tissue fatty acid composition independent of diet. The sex differences may partially be due to inadequate adjustment for changes in sex hormone status in males with ageing. Using the current indicator, a decline in the rate limiting step of beta-6 desaturation appeared to occur with age, and was greater in women than in men. These results may indicate that an increase in dietary gamma-linolenic acid (C18:3, n-6) is necessary with age to offset the relative imbalance between PUFA levels which appears to occur. However, any direct health benefit regarding the common diseases of ageing from such a strategy still remain to be clarified.

The effects of gamma-linolenic acid-rich borage oil (BO), in combination with different marine oils, namely an eicosapentaenoic acid (EPA) rich oil (MO) or a DHA-rich oil (TO), on tissue fatty acid composition and prostaglandin production were investigated in turbot, a species which lacks appreciable delta5 fatty acyl desaturase activity. The juvenile turbot grew well on the experimental diets and there were no significant differences in final weights between dietary treatments. Irrespective of the marine oil component, both the BO-containing diets increased tissue phospholipid levels of 18:2n-6 and 18:3n-6, and their respective elongation products, 20:2n-6 and 20:3n-6, compared to fish fed a control diet containing a standard Northern hemisphere fish oil. Both the BO-containing diets increased the production of 1-series prostaglandins (PG), this being observed across all tissues investigated with PGF and especially PGE. The BO/MO diet also reduced 20:4n-6 in tissue phospholipids without affecting 20:5n-3, whereas the BO/TO combination decreased 20:5n-3 but increased 20:4n-6. The production of 2-series and 3-series PGs was also altered by the dietary treatments but the changes were less dependent upon the tissue levels of their respective precursor fatty acids, 20:4n-6 and 20:5n-3. The BO-containing diets had very significant effects on gross fatty acid compositions of the phospholipids including increased proportions of saturated fatty acids and n-6 polyunsaturated fatty acids (PUFA) and decreased proportions of monounsaturated fatty acids and n-3 PUFA. Overall, this study shows that eicosanoid production in turbot tissues can be influenced by dietary fatty acids, not only by changes in the absolute and relative levels of specific eicosanoid precursor PUFA in tissue phospholipids, but also by general effects on membrane composition, structure and function induced by gross fatty acid compositional changes.

A fluoroimmunoassay (FIA) for 14,15-epoxyeicosatrienoic acid (14,15-EET) and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), cytochrome P450 epoxygenase products of arachidonic acid, was developed using fluorescence polarization. 14-15-EET was hydrolyzed and analyzed as 14,15-DHET. 14,15-DHET was conjugated to thyroglobulin and a specific antibody was raised in rabbits. Both [3H8]14,15-DHET in radioimmunoassay or fluorescein-labeled 14,15-DHET (14, 15-DHET*) in FIA bound to this antibody and were competitively displaced by 14,15-DHET. The binding activity and cross-reactivity of 14,15-DHET antibody were also studied by RIA compared to FIA. The antibody cross-reacted < or = 1% with 11,12-DHET and 14,15-EET and < 0.1% with other regioisomeric DHETs and arachidonic acid metabolites. The detection limit of 14,15-DHET was 2 pg/0.6 ml by FIA. Using this method, we found that A23187 stimulated the production of 14,15-EET by endothelial cells by angiotensin II stimulated 14,15-EET release from zona glomerulosa cells. The production of 14,15-EET in these samples was confirmed by gas chromatography/mass spectrometry. These studies demonstrate a sensitive and specific FIA for 14,15-EET and 14,15-DHET and that agonists stimulate the release of these eicosanoids in two cell types, bovine coronary artery endothelial cells and bovine zona glomerulosa cells.

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ratio. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-14C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.

Cytochrome P450 (P450) monooxygenases catalyze the epoxidation of arachidonic acid to form epoxyeicosatrienoic acids, which modulate bronchial smooth muscle tone and airway transepithelial ion transport. We recently described a new human P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3). Northern analysis of lung RNA using CYP2J cDNA probes demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed in the lung. Immunoblotting of microsomal fractions prepared from human and rat lungs using a polyclonal antibody raised against recombinant human CYP2J2 revealed a single 56-kDa band confirming abundant pulmonary CYP2J2 and CYP2J3 protein expression. Immunohistochemical analysis of formalin-fixed paraffin-embedded human and rat lung sections using the anti-human CYP2J2 IgG and avidin/biotin/peroxidase detection showed that CYP2J proteins were primarily expressed in ciliated epithelial cells lining the airway. Prominent staining was also noted in nonciliated airway epithelial cells, bronchial and pulmonary vascular smooth muscle cells, pulmonary vascular endothelium, and alveolar macrophages, whereas less intense staining was noted in alveolar epithelial cells. Endogenous epoxyeicosatrienoic acids were detected in both human and rat lung using gas chromatography/mass spectrometry, thus providing direct evidence for the in vivo human and rat pulmonary P450 metabolism of arachidonic acid. Based on these data, we conclude that CYP2J2 and CYP2J3 are abundant pulmonary arachidonic acid epoxygenases and that CYP2J products, the epoxyeicosatrienoic acids, are endogenous constituents of human and rat lung. In addition to known effects on airway smooth muscle tone and transepithelial electrolyte transport, the localization of CYP2J proteins to vascular smooth muscle and endothelium suggests that epoxyeicosatrienoic acids may also be involved in the modulation of pulmonary vascular tone.

The effect of hormone replacement therapy (HRT) on platelet size was examined in 38 post-menopausal women before and at end of a 6 week period of HRT. Subjects were treated with cyclical L-norgestrel 75 mg daily from days 17 to 28 of a 28-day cycle combined with continuous conjugated equine oestrogens 0.625 mg daily. Platelet-rich plasma was obtained to measure platelet indices by Coulter analysis. Plasma measurements of luteinizing hormone, follicle stimulating hormone and 17 beta-oestradiol were measured by immunoassays. Platelet membrane fatty acids were measured by gas-liquid chromatography. A significant reduction in platelet membrane linoleic acid, di-homo-gamma-linoleic acid and arachidonic acid of 8.1%, 14.3% and 17.8%, respectively was noted after 6 weeks of HRT (p < 0.01). There was an increase in the platelet count (not significant) and platelet volume (MPV) (P < 0.05) after 6 weeks of hormone therapy. HRT appears to increase mean platelet volume which may indicate an increase in platelet reactivity. There was no correlation between the changes in mean platelet volume and membrane fatty acid changes in platelets after such therapy.

Chondrocytes are imbedded in an avascular, highly charged extracellular matrix which could form a barrier to the transfer of dietary essential fatty acids (EFA) to chondrocytes. A study was designed to assess the composition of immature and mature joint cartilage with respect to essential and nonessential fatty acids relevant to EFA deficiency. Cartilage and muscle samples were obtained from human fetus, infant and adult cadavers, and from fetal and mature sheep. Lipid extracts were prepared and the fatty acid composition determined. In human and sheep joint cartilage, linoleic acid (LA; 18:2n-6) content was lower, and n-9 eicosatrienoic acid (ETrA; 20:3n-9) and arachidonic acid (AA; 20:4n-6) were higher in fetuses compared to mature subjects. An intermediate pattern was seen in infant cartilage. n-3 Fatty acids tended to be higher in fetal than in mature cartilage in humans and in sheep. In human muscle (and in other noncartilaginous comparison tissues), similar differences between fetuses and adults were seen in LA and AA, but not in ETrA. In fetal sheep muscle, very low LA, reduced AA and raised ETrA levels compared to mature sheep muscle were seen. However, although the pattern is characteristic of EFA deficiency, the abundance of n-6 EFA in liver and spleen of human fetuses and of n-3 EFA in liver and spleen of fetal sheep suggests that placental transfer of EFA is not likely to be limiting. During fetal development, the metabolism of fatty acids is distinctive and differs between the species. ETrA appears to be a readily measurable component of some tissues at certain stages of development when its presence in tissues does not indicate EFA deficiency.

Facile methods for the detection of intact hepoxilins, monohydroxy-epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway, are unavailable because (i) an absence in these compounds of an appropriate chromophore for sensitive detection by uv exists, (ii) these compounds are sensitive to the acidic workup leading to varying degrees of decomposition, and (iii) they decompose to the derivatization procedures required for their analysis by gas chromatography mass spectrometry. Herein we apply a method which introduces a fluorescent ester chromophore to the carboxylic group of the hepoxilins under conditions which do not require acidification leading to stabilization of the derivative which is extracted into an organic solvent in situ. This procedure quantitatively derivatizes hepoxilins in a biological sample, permitting the detection of hepoxilins after a TLC purification with a limit of 50 pg/sample. This method permits the profiling of 12-HETE, hepoxilins A3 and B3, as well as the corresponding epoxide hydrolase products, trioxilins A3 and B3, in a biological sample by reverse-phase HPLC with fluorescent detection. We also report on the fluorescent and mass spectral properties of these derivatives using a liquid chromatography mass spectrometry LCMS interface with thermospray ionization.

We examined the effect of dietary alpha-linolenic acid (ALA) on the indices of lipid and coagulation status and on the fatty acid composition of serum and peripheral blood mononuclear cell (PBMNC) lipids in ten healthy men (age 21-37 yr) who consumed all their meals at the Western Human Nutrition Research Center for 126 d. There was a stabilization period of 14 d at the start when all 10 subjects consumed the basal diet (BD) containing 23.4 energy percent (en%) fat and two intervention periods of 56 d each. During the first intervention period, 5 subjects consumed the BD containing 23.4 en% fat, and 5 subjects consumed a diet providing 6.3% calories from alpha-linolenic acid [flaxseed oil (FSO) diet containing 28.8 en% fat]. Diets were crossed over between the two groups during the second intervention period. Feeding the FSO diet did not significantly alter serum triglycerides, cholesterol, high-density lipoproteins, low-density lipoproteins, apoprotein A-I and apoprotein B when compared to the corresponding values in the subjects fed the BD, nor was there any effect of the FSO diet on the bleeding time, prothrombin time and partial prothrombin time for these subjects. Feeding the ALA-containing diet did cause a significant increase in ALA concentration in serum (P < 0.001) and PBMNC lipids (P < 0.05). It also caused a significant increase (P < 0.05) in the eicosapentaenoic and docosapentaenoic acid contents of PBMNC lipids, and a decrease (P < 0.01) in linoleic and eicosatrienoic acid contents of serum lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [14C]arachidonic acid was purified on a Sep-Pak C18 cartridge and derivatized with diazomethane, O-methylhydroxylamine and dimethyl-isopropylsilylimidazole. The recovery of total 14C-radioactivity was 93.1 +/- 7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [14C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.

Peroxidation of cellular membrane lipids has been implicated in a wide variety of acute and chronic pathologies. We have developed a method for quantifying lipid peroxidation products using gas chromatography-mass spectrometry (GC-MS) in order to help elucidate the role of lipid peroxidation in such disorders. The method involves analysis of the methyl ester, trimethylsilyl ether derivatives of various hydroxyeicosatetraenoic acids (HETEs). 16-Hydroxy-9,12,14-heneicosatrienoic acid was synthesized for use as an internal standard. The assay involves the following steps: The internal standard is added to the sample and cellular lipids are extracted and trans-esterified. Next, any hydroperoxides are reduced with triphenylphosphine and the samples are subjected to two steps of solid phase extraction. The samples are then hydrogenated and the trimethylsilyl ether derivative of the hydroxyls formed. The derivatized HETEs are analyzed by electron impact GC-MS. 12-HETE, 11-HETE, 9-HETE, and 8-HETE are assayed by monitoring ions at m/z 301, 287, 259, and 271, respectively. Standard curves were constructed for each HETE and were linear over the range 1 to 250 ng; correlation coefficients were typically greater than 0.99. The assay has been applied to the study of autoxidation of lipids in both in vitro and in vivo systems.

SK-N-SH neuroblastoma cells grown under standard culture conditions contain significant amounts of Mead acid (20:3 omega 9) in phospholipids, indicating essential fatty acid (EFA) deficiency. The amount of esterified 20:3 omega 9 was augmented by growth in a chemically defined EFA-free medium, whereas its presence could be virtually eliminated by supplementation of the culture medium with either arachidonic (20:4 omega 6; AA), eicosapentaenoic (20:5 omega 3; EPA), or linolenic (18:3 omega 3) acids. Substitution of Mead acid for omega 6 fatty acids, particularly evident in phosphatidylinositol (PI), indicates a compensatory replacement of omega 9 for omega 6 fatty acids during EFA deficiency. Studies evaluating [3H]scopolamine binding to the M3 muscarinic acetylcholine receptors (mAChRs) present in these neurotumor cells as well as effects of carbachol on phosphoinositide turnover and intracellular Ca2+ mobilization, indicate that the biosubstitution of 20:4 omega 6 with 20:3 omega 9 does not detectably impair these measures of signal transduction. Stimulation of mAChRs with carbachol increased the cellular mass of diacylglycerol (DAG) approximately 60%. On the basis of distinctive fatty acid "signatures" of each of the phospholipid classes, it is concluded that the DAG initially released following muscarinic stimulation is derived from phosphoinositide breakdown. After several minutes, however, a significant amount of DAG comes from phosphatidylcholine (PC) as well. In contrast to DAG, the composition of phosphatidate (PA) following receptor stimulation closely resembles that of the phosphoinositides, even at the later time points examined. These results support a selective phosphorylation of DAG arising from the stimulated breakdown of phosphoinositides, favoring the conservation of the 1-stearoyl, 2-arachidonoyl (or 20:3 omega 9) moiety.