Genetic variants in FADS1 and ELOVL2 increase level of arachidonic acid and the risk of Alzheimer's disease in the Tunisian population.

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.

Antarctic thraustochytrids: Producers of long-chain omega-3 polyunsaturated fatty acids.

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.

Topically injected adrenocorticotropic hormone induces mechanical hypersensitivity on a full-thickness cutaneous wound model in rats.

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.

Soluble epoxide hydrolase derived lipid mediators are elevated in bronchoalveolar lavage fluid from patients with sarcoidosis: a cross-sectional study.

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.

Downregulated Serum 14, 15-Epoxyeicosatrienoic Acid Is Associated With Abdominal Aortic Calcification in Patients With Primary Aldosteronism.

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 (ß=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.

Anti- Versus Pro-Inflammatory Metabololipidome Upon Cupping Treatment.

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.

Linoleic Acid:Dihomo-γ-Linolenic Acid Ratio Predicts the Efficacy of Zn-Biofortified Wheat in Chicken (Gallus gallus).

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.

Induction of omega 6 inflammatory pathway by sodium metabisulfite in rat liver and its attenuation by ghrelin.

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.

Serum paraoxonase 1 activity is associated with fatty acid composition of high density lipoprotein.

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.

Protection of salvianolic acid A on rat brain from ischemic damage via soluble epoxide hydrolase inhibition.

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.

Epoxyeicosatrienoic acids are involved in the C(70) fullerene derivative-induced control of allergic asthma.

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.

Soluble epoxide hydrolase: sex differences and role in endothelial cell survival.

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.

Elevated stearoyl-CoA desaturase in brains of patients with Alzheimer's disease.

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.

Effects of perfluorinated fatty acids with different carbon chain length on fatty acid profiles of hepatic lipids in mice.

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.

Characterization of free radicals formed from COX-catalyzed DGLA peroxidation.

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.

[The role of modification of fatty acid composition of erythrocyte lipids in pathogenesis of arterial hypertension].

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.

Development of a high throughput cell-based assay for soluble epoxide hydrolase using BacMam technology.

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.

Changes in fatty acid profiles in testis and spermatozoa of red deer exposed to metal pollution.

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.

Nutrition for the eye: different susceptibility of the retina and the lacrimal gland to dietary omega-6 and omega-3 polyunsaturated fatty acid incorporation.

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.

Direct and simultaneous profiling of epoxyeicosatrienoic acid enantiomers by capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection.

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.