ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.     

Prolonged niacin treatment leads to increased adipose tissue PUFA synthesis and anti-inflammatory lipid and oxylipin plasma profile

Prolonged niacin treatment elicits beneficial effects on the plasma lipid and lipoprotein profile that is associated with a protective CVD risk profile. Acute niacin treatment inhibits nonesterified fatty acid release from adipocytes and stimulates prostaglandin release from skin Langerhans cells, but the acute effects diminish upon prolonged treatment, while the beneficial effects remain. To gain insight in the prolonged effects of niacin on lipid metabolism in adipocytes, we used a mouse model with a human-like lipoprotein metabolism and drug response [female APOE*3-Leiden.CETP (apoE3 Leiden cholesteryl ester transfer protein) mice] treated with and without niacin for 15 weeks. The gene expression profile of gonadal white adipose tissue (gWAT) from niacin-treated mice showed an upregulation of the “biosynthesis of unsaturated fatty acids” pathway, which was corroborated by quantitative PCR and analysis of the FA ratios in gWAT. Also, adipocytes from niacin-treated mice secreted more of the PUFA DHA ex vivo. This resulted in an increased DHA/arachidonic acid (AA) ratio in the adipocyte FA secretion profile and in plasma of niacin-treated mice. Interestingly, the DHA metabolite 19,20-dihydroxy docosapentaenoic acid (19,20-diHDPA) was increased in plasma of niacin-treated mice. Both an increased DHA/AA ratio and increased 19,20-diHDPA are indicative for an anti-inflammatory profile and may indirectly contribute to the atheroprotective lipid and lipoprotein profile associated with prolonged niacin treatment.     

Effects of supplementation with fish oil and barium selenate on performance, carcass characteristics and muscle fatty acid composition of late season lamb finished on grass-based or concentrate-based diets

The objectives of this study were to investigate the effects of fish oil supplementation on performance and muscle fatty acid composition of hill lambs finished on grass-based or concentrate-based diets, and to examine the interaction with selenium (Se) status. In September 2006, 180 entire male lambs of mixed breeds were sourced from six hill farms after weaning and finished on five dietary treatments: grazed grass (GG), grass +0.4 kg/day cereal-based concentrate (GC), grass +0.4 kg/day cereal-based concentrate enriched with fish oil (GF), ad libitum cereal-based concentrate (HC) and ad libitum fish oil-enriched concentrate (HF). Within each treatment, half of the lambs were also supplemented with barium selenate by subcutaneous injection. At the start of the trial, the proportion of lambs with a marginal (<0.76 μmol/l) or deficient (<0.38 μmol/l) plasma Se status was 0.84 and 0.39, respectively. Compared with control lambs, GG lambs treated with Se had higher (P < 0.01) plasma Se levels, whereas erythrocyte glutathione peroxidase activity was higher (P < 0.01) for Se-supplemented lambs fed diets GG and GF. However, Se supplementation had no effects on any aspect of animal performance. Fish oil increased (P < 0.05) levels of 22:5n-3 and 22:6n-3 in the Longissimus dorsi of HF lambs but otherwise had no effect on the health attributes of lamb meat. There were no significant effects of fish oil on dry matter intake, animal performance or lamb carcass characteristics. Daily carcass weight gain (CWG; P < 0.001), carcass weight (P < 0.01) and conformation score (P < 0.01) increased with increasing concentrate inputs. Lambs fed concentrate-based diets achieved a higher mean CWG (P < 0.001), dressing proportion (P < 0.001) and carcass weight (P < 0.011), and were slaughtered up to 8.3 days earlier (P < 0.05) and at 1.2 kg lower (P < 0.05) live weight than pasture-fed lambs. However, carcasses from grass-fed lambs contained lower levels of perinephric and retroperitoneal fat (P < 0.05), and had less fat over the Iliocostalis thoracis (P < 0.001) and Obliquus internus abdominis (P < 0.05). Meat from grass-fed lambs also had lower levels of 18:2n-6 and total n-6 fatty acids compared with those finished indoors. The results of this study demonstrate that fish oil supplementation has some benefits for the health attributes of meat from lambs fed concentrate-based diets but not grass-based diets. Supplementing Se-deficient lambs with barium selenate will improve Se status of lambs fed zero-concentrate diets, but has no additional benefit when lambs are already consuming their daily Se requirement from concentrates or when fish oil-enriched diets are fed.     

Effect of dietary fish oil on selected inflammatory markers in pigs

The present study tested a hypothesis that dietary fish oil (eicosapentaenoic acid+docosahexaenoic acid) in a commonly achievable dose ameliorates a systemic inflammation in pigs. Two groups of pigs of 16 animals each were fed a diet with either 2.5% of fish oil (F) or a control diet with 2.5% of palm oil (P). After 70 days of fattening, eight F and eight P pigs were challenged (F⁺; P⁺) i.v. by lipopolysaccharide. After 3 h, all pigs were sacrificed and blood, liver and visceral adipose tissue (VAT) samples were taken. No significant effect (P>0.05) of dietary oil on the feed intake and daily weight gain was found out. Less neutrophils (16.8% v. 28.8%; P<0.05) were found in the F⁺-leukocytes of the peripheral blood; F⁺ pigs had lower (P<0.05) percentage of the swine leukocyte antigen-D-related CD163⁺ (SLA-DR⁺ CD163⁺) macrophages in the VAT (15.4% v. 21.8%) and lower expression of the SLA-DR-CD163⁺ surface molecules of the VAT macrophages. No difference (P>0.05) between F⁺ and P⁺ pigs in the peroxisome proliferator-activated receptor γ, GPR120, Adipor1 and Adipor2 (adiponectin receptor) gene expression, respectively, was established; plasma adiponectin was the same (21.1 ng/ml) in F⁺ and P⁺ pigs. In comparison with the P⁺ pigs, increased expression of the lipopolysaccharide-binding protein (LBP) gene and intercellular adhesion molecule 1 (ICAM1) gene was found out in the liver of the F⁺ pigs; expression of the tumor necrosis factor α (TNFα) gene was higher in the liver but lower in the VAT of the F⁺ pigs (P<0.05). The F⁺ pigs had higher (P<0.05) plasma concentration of both anti-inflammatory cytokine interleukin-4 (0.46 v. 0.04 ng/ml) and pro-inflammatory TNF-α (13.41 v. 7.72 ng/ml). It was concluded that dietary fish oil at the tested amount had a negligible effect on expression of the evaluated receptor genes and plasma adiponectin, and had an ambiguous effect on expression of cytokine genes and plasma cytokine levels.     

EPA,not DHA,prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor β1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.     

Regulation of inflammatory and lipid metabolism genes by eicosapentaenoic acid-rich oil

Omega-3-PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are associated with prevention of various aspects of metabolic syndrome. In the present studies, the effects of oil rich in EPA on gene expression and activation of nuclear receptors was examined and compared with other ω3-PUFAs. The EPA-rich oil (EO) altered the expression of FA metabolism genes in THP-1 cells, including stearoyl CoA desaturase (SCD) and FA desaturase-1 and -2 (FASDS1 and -2). Other ω3-PUFAs resulted in a similar gene expression response for a subset of genes involved in lipid metabolism and inflammation. In reporter assays, EO activated human peroxisome proliferator-activated receptor α (PPARα) and PPARβ/γ with minimal effects on PPARγ, liver X receptor, retinoid X receptor, farnesoid X receptor, and retinoid acid receptor γ (RARγ); these effects were similar to that observed for purified EPA. When serum from a 6 week clinical intervention with dietary supplements containing olive oil (control), DHA, or two levels of EPA were applied to THP-1 cells, the expression of SCD and FADS2 decreased in the cells treated with serum from the ω3-PUFA-supplemented individuals. Taken together, these studies indicate regulation of gene expression by EO that is consistent with treating aspects of dyslipidemia and inflammation.     

In rat hepatocytes, myristic acid occurs through lipogenesis, palmitic acid shortening and lauric acid elongation

The origin of myristic acid in mammalian cells and the regulation of its endogenous cellular low concentration are not known. Another intriguing question is the potential metabolic properties of endogenous myristic acid as compared with exogenous myristic acid. In the present paper, we hypothesised and demonstrated that, in liver cells, in addition to the usual fatty acid synthase (FAS) pathway that produces predominantly palmitic acid and minor amounts of myristic acid, part of endogenous cellular myristic acid also comes from a shortening of palmitic acid, likely by peroxisomal β-oxidation and from lauric acid by elongation. From a nutritional point of view, C16:0 is universally found in natural fats and its shortening to myristic acid could contribute to a non-negligible source of this fatty acid (FA) in the organism. Then, we measured the distribution of endogenously synthesised myristic acid in lipid species and compared it with that of exogenous myristic acid. Our results do not support the hypothesis of different metabolic fates of endogenous and exogenous myristic acid and suggest that whatever the origin of myristic acid, its cellular concentration and lipid distribution are highly regulated.     

Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans

Newly synthesized triglyceride (TG) may exit the liver immediately as VLDL-TG or be stored and secreted after a delay. We quantified the contributions from plasma NEFA, diet, and de novo lipogenesis (DNL) to VLDL-TG via immediate and delayed pathways in five lean, normolipidemic subjects; six obese, hypertriglyceridemic (HPTG) nondiabetics; and six obese, HPTG diabetics. Intravenous [(2)H(31)]palmitate and [1-(13)C(1)] acetate and oral [(2)H(35)]stearate were administered for 30 h preceding an overnight fast. [1,2,3,4-(13)C(4)]palmitate was infused during the subsequent 12 h fast. Contributions from plasma NEFA via the immediate pathway were 64 +/- 15, 33 +/- 6, and 58 +/- 2% in control, HPTG, and diabetic HPTG, respectively. Delayed pool fractional contributions were as follows: dietary FA, 2.0 +/- 0.9, 2.5 +/- 1, and 12 +/- 2%; DNL, 3 +/- 0.3, 14 +/- 3, and 13 +/- 4%; delayed NEFA, 15 +/- 4, 20 +/- 4, and 30 +/- 3%. VLDL-TG production rates and absolute input rates from the delayed pool were significantly higher in HPTG and diabetic HPTG than in controls. In conclusion, we provide direct kinetic evidence for a hepatic TG storage pool in humans and document its metabolic sources. The turnover time and sources of this pool differ in diabetic HPTG and nondiabetic HPTG, with potential therapeutic implications.     

Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.     

Increased arachidonic acid concentration in the brain of Flinders Sensitive Line rats, an animal model of depression

Depression may be associated with impaired membrane PUFA composition, especially decreased n-3 PUFA. This assumption has not been tested at the level of brain tissue. Moreover, most studies were confounded by dietary variability. We examined the FA composition of selected brain areas in an animal model of depression, the Flinders Sensitive Line (FSL) rat, and compared the findings with those in controls fed identical diets. In all brain regions studied, the concentration of arachidonic acid (AA) was significantly higher in the FSL rats: in the hypothalamus by 21%, in the nucleus accumbens by 24%, in the prefrontal cortex by 31%, and in the striatum by 23%. No significant differences were observed for n-3 PUFA or for the saturated and monounsaturated FAs. Our results confirm the existence of altered brain PUFA composition in an animal model of depression. The finding of increased AA, an n-6 PUFA, rather than decreased n-3 PUFA, emphasizes the importance of both PUFA families in the pathophysiological processes underlying depression. The FSL rat is a useful tool for further elucidation of the FA disturbances in depression.     

The synthesis and accumulation of stearidonic acid in transgenic plants: a novel source of 'heart-healthy' omega-3 fatty acids

Dietary omega-3 polyunsaturated fatty acids have a proven role in reducing the risk of cardiovascular disease and precursor disease states such as metabolic syndrome. Although most studies have focussed on the predominant omega-3 fatty acids found in fish oils (eicosapentaenoic acid and docosahexaenoic acid), recent evidence suggests similar health benefits from their common precursor, stearidonic acid. Stearidonic acid is a Δ6-unsaturated C18 omega-3 fatty acid present in a few plant species (mainly the Boraginaceae and Primulaceae ) reflecting the general absence of Δ6-desaturation from higher plants. Using a Δ6-desaturase from Primula vialii , we generated transgenic Arabidopsis and linseed lines accumulating stearidonic acid in their seed lipids. Significantly, the P. vialii Δ6-desaturase specifically only utilises α-linolenic acid as a substrate, resulting in the accumulation of stearidonic acid but not omega-6 γ-linolenic acid. Detailed lipid analysis revealed the accumulation of stearidonic acid in neutral lipids such as triacylglycerol but an absence from the acyl-CoA pool. In the case of linseed, the achieved levels of stearidonic acid (13.4% of triacylglycerols) are very similar to those found in the sole natural commercial plant source ( Echium spp.) or transgenic soybean oil. However, both those latter oils contain γ-linolenic acid, which is not normally present in fish oils and considered undesirable for heart-healthy applications. By contrast, the stearidonic acid-enriched linseed oil is essentially devoid of this fatty acid. Moreover, the overall omega-3/omega-6 ratio for this modified linseed oil is also significantly higher. Thus, this nutritionally enhanced linseed oil may have superior health-beneficial properties.     

Epigenome-wide analysis of neonatal CD4+ T-cell DNA methylation sites potentially affected by maternal fish oil supplementation

Supplementation of fish oil rich in omega-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy has been shown to confer favorable health outcomes in the offspring. In a randomized controlled trial, we have previously shown that n-3 PUFA supplementation in pregnancy was associated with modified immune responses and some markers of immune maturation. However, the molecular mechanisms underlying these heritable effects are unclear. To determine whether the biological effects of maternal n-3 PUFA supplementation are mediated through DNA methylation, we analyzed CD4⁺ T-cells purified from cryo-banked cord blood samples from a previously conducted clinical trial. Of the 80 mother-infant pairs that completed the initial trial, cord blood samples of 70 neonates were available for genome-wide DNA methylation profiling. Comparison of purified total CD4⁺ T-cell DNA methylation profiles between the supplement and control groups did not reveal any statistically significant differences in CpG methylation, at the single-CpG or regional level. Effect sizes among top-ranked probes were lower than 5% and did not warrant further validation. Tests for association between methylation levels and key n-3 PUFA parameters, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or total n-3 PUFAs were suggestive of dose-dependent effects, but these did not reach genome-wide significance. Our analysis of the microarray data did not suggest strong modifying effects of in utero n-3 PUFA exposure on CD4⁺ T-cell methylation profiles, and no probes on the array met our criteria for further validation. Other epigenetic mechanisms may be more relevant mediators of functional effects induced by n-3 PUFA in early life.     

Linoleic acid biosynthesis in the pea aphid,Acyrthosiphon pisum (Harris)

The pea aphid Acyrthosiphon pisum (Harris) incorporated [1-¹⁴C]acetate into a phospholipid dienoic fatty acid in a time-dependent manner. In 2-h incubations, the incorporation of radioactivity into the 18:2 fraction was minimal, whereas after 45 h 18:2 was the major fatty acid labeled. Ozonolysis of the isolated dienoic fatty acid methyl ester followed by radio-gas-liquid chromatography showed that radioactivity was associated with fragments containing carbons 1–9 and 13–18. These data established the location of the double bonds in the 9,12 positions and indicated that the entire molecule was labeled from [1-¹⁴C]acetate. Tetracycline-treated aphids synthesized linoleic acid in the same proportions as untreated controls. Scanning electron microscopy showed that over 50% of the treated insects had greatly reduced numbers of intracellular symbiotes or lacked them or most of the existing symbiotes had an abnormal appearance. Therefore, we conclude that intracellular symbiotes are not involved in the biosynthesis of linoleic acid in the pea aphid.     

Epigenome-wide analysis of neonatal CD4+ T-cell DNA methylation sites potentially affected by maternal fish oil supplementation

Supplementation of fish oil rich in omega-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy has been shown to confer favorable health outcomes in the offspring. In a randomized controlled trial, we have previously shown that n-3 PUFA supplementation in pregnancy was associated with modified immune responses and some markers of immune maturation. However, the molecular mechanisms underlying these heritable effects are unclear. To determine whether the biological effects of maternal n-3 PUFA supplementation are mediated through DNA methylation, we analyzed CD4⁺ T-cells purified from cryo-banked cord blood samples from a previously conducted clinical trial. Of the 80 mother-infant pairs that completed the initial trial, cord blood samples of 70 neonates were available for genome-wide DNA methylation profiling. Comparison of purified total CD4⁺ T-cell DNA methylation profiles between the supplement and control groups did not reveal any statistically significant differences in CpG methylation, at the single-CpG or regional level. Effect sizes among top-ranked probes were lower than 5% and did not warrant further validation. Tests for association between methylation levels and key n-3 PUFA parameters, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or total n-3 PUFAs were suggestive of dose-dependent effects, but these did not reach genome-wide significance. Our analysis of the microarray data did not suggest strong modifying effects of in utero n-3 PUFA exposure on CD4⁺ T-cell methylation profiles, and no probes on the array met our criteria for further validation. Other epigenetic mechanisms may be more relevant mediators of functional effects induced by n-3 PUFA in early life.     

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 μl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 μl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 ± 1.1 pmol, which was in complete agreement with the observed X¯₀ = 15.0 ± 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.