Lipase specificity towards eicosapentaenoic acid and docosahexaenoic acid depends on substrate structure

The fatty acid specificity of five lipases towards eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was evaluated in the hydrolysis of fish oil, squid oil and a model system. The model system contained methyl esters of EPA, DHA and palmitic acid. All the investigated lipases discriminated against both EPA and DHA more in the model system than in the natural oils. Thus both EPA and DHA were more easily hydrolysed from a glyceride than from a methyl ester. In the model system, the lipase from Candida rugosa showed the highest discrimination against DHA, while the lipases from Pseudomonas fluorescens and Pseudomonas cepacia discriminated against EPA the most. In a glyceride, the fatty acid specificity of lipases towards EPA and DHA was affected by the positional distribution of the fatty acids and the glyceride structure due to the regiospecificity and triglyceride specificity of the lipase. In the oils, the Pseudomonas lipases also discriminated against EPA the most, while DHA was initially discriminated the most by the lipase from Thermomyces lanuginosus. However, after longer reaction times the enrichment of DHA in the glyceride fraction of the oils was greatest for the lipase from C. rugosa.     

脂肪酶二步法催化鱼油下脚料富集DHA

采用Pseudomonas sp．脂肪酶水解,Geotrichum sp．脂肪酶选择性酯化的两步法显著提高游离脂肪酸（FFA）中二十二碳六烯酸（DHA）的含量。通过筛选合适的底物醇,优化水解和选择性酯化反应条件达到富集DHA的目的。结果表明月桂醇为选择性酯化的最适底物醇。确定的最佳水解条件：4g反应底物,m（水）／m（粗鱼油）=1,1000 U Pseudomonas sp．脂肪酶,40℃,搅拌速度200r／min,反应24h;最佳酯化条件：3g反应底物,n（FFA）／n（醇）=1／2,1000 U固定化Geotrichum sp．脂肪酶,1g正己烷,30℃,200r／min搅拌,反应20h。经过水解和一次选择性酯化反应后,DHA含量从原料粗鱼油中的18．9％提纯到72．8％;经二次选择性酯化后,DHA含量上升到92％。脂肪酶水解-选择性酯化的两步法是富集DHA的有效方法。     

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.     

Effect of frozen storage on fatty acid composition of the different tissues of four scombrid and one dussumeriid species

In the current study, the effect of frozen storage at ?18°C was evaluated on fatty acid composition of different body parts (liver, muscle tissue, and viscera) of narrow‐barred Spanish mackerel (Scomberomorus commerson, Lacépède, 1800), longtail tuna (Thunnus tonggol, Bleeker, 1851), kawakawa (Euthynnus affinis, Cantor, 1849), king mackerel (Scomberomorus guttatus, Bloch & Schneider, 1801), and rainbow sardine (Dussumieria acuta, Valenciennes, 1847) caught in the Persian Gulf. Changes in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid plus docosahexaenoic acid/palmitic acid (EPA+DHA/C16), ω3 PUFA/ω6 PUFA (ω3/ω6), and polyunsaturated fatty acids/saturated fatty acids (PUFA/SFA) were investigated during a 6‐month period. A decrease in unsaturated fatty acids, particularly PUFAs (60–100%) as well as ω3/ω6, EPA+DHA/C16 (polyene index) and PUFA/SFA ratios, indicated a decrease in the nutritional values of the samples.     

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors

The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).     

固定化脂肪酶选择性水解废弃鱼油富集DHA和EPA甘油酯

以鱼油下脚料为原料．以固定化Geotrichum sp．脂肪酶为催化剂,选择性水解粗鱼油,使目标脂肪酸EPA和DHA富集于甘油骨架上,显著提高了甘油酯中EPA和DHA的质量分数。通过单因素试验和响应面分析得出最佳水解条件：2g粗鱼油,2．5g水,221．3U脂肪酶,30℃,反应11．7h。最佳条件下的水解度为42．1％。EPA和DHA质量分数分别为7．8％和41．1％。经过二次水解后,水解度提高到45．9％,EPA和DHA质量分数分别提高到8．5％和42％。与初始鱼油相比,分别提高了2倍和2．2倍。     

Low dietary fish-oil threshold for myocardial membrane n-3 PUFA enrichment independent of n-6 PUFA intake in rats

Long chain n-3 PUFA docosahexaenoic acid (DHA) is important for heart and brain function. Investigations of biologically plausible mechanisms using animal models associate cardioprotection with DHA incorporation into myocardial membranes that are largely derived from supra-physiological fish oil (FO) intake. We measured the incorporation of DHA into myocardial membranes of rats from low dietary FO intake within human dietary range and quantitatively assessed the influence of dietary n-6 PUFA. With rats fed diets containing 0.16%–5% FO, equal to 0.12%–8.7% energy (%en) as eicosapentaenoic acid (EPA) and DHA (EPA+DHA), and either 1.5%en or 7.5%en n-6 PUFA (linoleic acid) for four weeks, dietary n-6:n-3 PUFA ratios ranged from 74 to 0.3. Myocardial DHA concentration increased in a log-linear fashion with a dietary threshold of 0.019%en as EPA+DHA and half maximal dietary [EPA+DHA] equal to 0.29%en (95% CI, 0.23–0.35). Dietary linoleic acid intake did not influence myocardial DHA. Myocardial membranes are sensitive to absolute dietary intake of long chain n-3 PUFA at low %en in the rat, equivalent to a human intake of one meal of fatty fish per week or less. The dietary ratio of n-6:n-3 PUFA has no influence on long chain n-3 PUFA cellular incorporation from dietary fish oil.     

Seasonal variation in fatty acid composition of silver pomfrets,Pampus argenteus (Euphrasen), in Kuwait waters

Proximate and fatty acid composition of wild silver pomfrets, Pampus argenteus, were studied in Kuwait waters for a period of 1 year (November 2007–October 2008) to see whether there were any seasonal compositional differences between males and females. Ten adults (five males, five females) were sampled each month during (i) Pre‐spawning (March–May), (ii) Spawning (June–August), (iii) Post‐spawning (September–November), and (iv) Winter (December–February). Both sexes had significantly (P < 0.05) higher whole body moisture and lower crude protein and lipid contents in winter compared to the respective males and females sampled in other seasons. However, females had significantly higher (9.1%) lipid content during the pre‐spawning season than females in other seasons (7.0–8.2%). The most abundant fatty acid in whole body lipid in both sexes was C16 followed by C18:1n‐9, which accounted for about 31–35% and 22–24% of total lipids, respectively. Males in the pre‐spawning and spawning seasons had significantly higher total monosaturated fatty acids (MUFA) than males and females in post‐spawning and winter. Males had significantly higher total polyunsaturated fatty acids (PUFA) during post‐spawning seasons than females in pre‐spawning and winter seasons. However, there were no significant differences (P > 0.05) in total saturated fatty acids (SFA), PUFA, EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) or n‐3/n‐6 ratios between respective males and females in different seasons. Livers in males had significantly (P < 0.05) higher MUFA, SFA, PUFA, EPA and DHA than respective females in all months during the spawning season. Female gonads had significantly (P < 0.05) higher MUFA and PUFA but lower SFA content than males in different months during the spawning season. In contrast to the liver, the gonad DHA content and n‐3/n‐6 ratios in females were significantly higher than in males. The gonads from both sexes contained more than double the amount of EPA present in liver; in the case of DHA this was more than three‐fold higher in female gonads, but not in males. Thus, the presence of higher proportions of PUFA, EPA and DHA in gonads, particularly in eggs of silver pomfret, indicates their need for these fatty acids, which may be used as a guideline for dietary essential n‐3 fatty acid requirements for feed formulation of this species. A higher content of DHA in eggs also indicates the higher requirement for DHA in the broodstock diet of silver pomfret.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Individual effects of dietary EPA and DHA on the functioning of the isolated working rat heart

The aim of this study was to evaluate the effects of dietary pure eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the physiology of the heart in normoxic conditions and during postischemic reperfusion. These effects were compared with those of dietary n-6 polyunsaturated fatty acids (PUFA). Rats were fed a diet containing either sunflower seed oil (75 g x kg(-1), SSO group), or a mixture of EPA (20:5 n-3) ethyl ester and SSO (10:90, EPA group), or a mixture of DHA (22:6 n-3) ethyl ester and SSO (10:90, DHA group), or a mixture of EPA + DHA ethyl esters and SSO (4.2:5.8:90, e+D group) for 6 weeks. The hearts were then perfused according to the working mode. The perfusion was maintained either in normoxic conditions or stopped for 17 min (global zero-flow ischemia) and restored for 33 min (reperfusion). The aortic and coronary flows, aortic developed pressure, and electrocardiogram were continuously monitored. When rats were fed a diet containing either EPA and (or) DHA, the n-6/n-3 PUFA ratio of cardiac phospholipids decreased. The proportion of arachidonic acid was reduced more with DHA than dietary EPA. In the EPA group, the percentage of DHA was lower than in the DHA group, but the percentage of EPA and docosapentaenoic acid (22:5 n-3) was higher. These changes in membrane fatty acid composition altered the cardiac function. In normoxic conditions, the coronary flow was higher in the SSO group than in the DHA and EPA groups. The heart rate was lower in the DHA and e+D groups than in the EPA and SSO groups. The aortic flow, cardiac output, and aortic developed pressure were not affected. During postischemic reperfusion, the recovery of aortic flow, coronary flow, and aortic developed pressure was similar in the four groups. A slightly improved recovery of cardiac function was noticed in the EPA group, but the difference was not significant. Feeding rats 5% fish oil + 5% SSO instead of 10% SSO for 8 weeks increased the incorporation of EPA in cardiac phospholipids and favored the recovery (+120%) of aortic flow during postischemic reperfusion. In conclusion, the beneficial effect of dietary fish oil on the recovery of cardiac pump activity during reperfusion was not observed with DHA or EPA alone. It appears to be positively related to the accumulation of EPA in membrane phospholipids. The dietary conditions favouring EPA accumulation remain to be determined.     

Change in fatty acid composition of serum lipids in obese females after short-term weight-reducing regimen with the addition of n-3 long chain polyunsaturated fatty acids in comparison to controls

Short-term weight-reducing regimens were shown to influence fatty acid composition of serum lipids unfavorably. Adding long chain n-3 polyunsaturated fatty acids (n-3 LC PUFA) to a low-calorie diet (LCD) could avoid these changes. The aim of this study was to examine the effect of a short-term in-patient weight-reducing regimen including LCD with yogurt enriched by low doses of n-3 PUFA (n-3 LCD). The enriched yogurt contained 790 mg of fish oil, predominantly eicosapentaenoic (20:5n-3; EPA) and docosahexaenoic (22:6n-3; DHA). Forty obese women were randomly assigned to the group consuming LCD and joghurt either with or without n-3 enrichment. Following the 3-week diet in the n-3 LCD group a significantly higher increase in the proportion of n-3 LC PUFA (sum of n-3 FA, EPA and DHA) in serum lipids was confirmed. In phospholipids (PL) a significant difference in the sum of n-6 fatty acids was found, a decrease in the n-3 LCD group and an increase in LCD group. Significantly higher increase in the PL palmitate (16:0) was shown in the LCD group. The results suggest that low doses of n-3 fatty acid enrichment can help to avoid unfavorable changes in fatty acid composition in serum lipids after a short-term weight-reducing regimen.     

Grouping Newly Isolated Docosahexaenoic Acid-Producing Thraustochytrids Based on Their Polyunsaturated Fatty Acid Profiles and Comparative Analysis of 18S rRNA Genes

Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.     

N-3 polyunsaturated fatty acid consumption produces neurobiological effects associated with prevention of depression in rats after the forced swimming test

Epidemiological data and clinical trials suggest that n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have preventive and therapeutic effects on depression; however, the underlying mechanism remains elusive. The present study aimed to examine the behavioral effects and antidepressant mechanism of n-3 PUFA using a forced swimming test. Eleven-week-old male Sprague-Dawley rats were fed an American Institute of Nutrition-93M diet containing 0%, 0.5% or 1% EPA and DHA relative to the total energy intake in their diet for 12 weeks (n=8 per group). Total dietary intake, body weight and hippocampus weights were not significantly different among groups. The groups administered 0.5% and 1% EPA+DHA diets had significantly higher levels of n-3 PUFA in their brain phospholipids compared to those in the control group. The immobility time was significantly decreased and the climbing time was significantly increased in the 0.5% and 1% EPA+DHA groups compared with those in the 0% EPA+DHA group. Plasma serotonin concentration and hippocampus c-AMP response element binding protein (CREB) expression were significantly increased in the 0.5% and 1% EPA+DHA groups compared with those in the 0% EPA+DHA group. Conversely, interleukin (IL)-6 expression was significantly reduced in the 0.5% and 1% EPA+DHA groups compared with that in the 0% EPA+DHA group. However, there were no dose-dependent effects of n-3 PUFA and no significant differences in expressions of IL-1β, tumor necrosis factor-α, brain-derived neurotrophic factor or phosphorylated CREB. In conclusion, long-term intake of EPA+DHA induced antidepressant-like effects in rats and overexpression of CREB via decreased IL-6 expression.     

Docosahexaenoic acid is selectively enriched in plasma phospholipids during pregnancy in Trinidadian women--results of a pilot study

The fetal demand for docosahexaenoic acid (DHA) has to be satisfied by the mother. We determined the fatty acids in maternal plasma non-esterified fatty acid (NEFA), triacylglycerol (TAG) and phosphatidylcholine (PC), in a cross-sectional study of non-pregnant (n = 10), pregnant (n = 19), and postpartum (n = 9) women. There were lipid class-dependent differences in plasma polyunsaturated fatty acid (PUFA) concentrations between groups. During pregnancy, DHA was most highly enriched in PC, about 230%, with more modest enrichment for linoleic acid (LA) and arachidonic acid (AA), and no enrichment of alpha-linolenic acid (alpha-LNA). There was relative enrichment of LA, AA and alpha-LNA in TAG, but not of DHA. There was no specific enrichment of any PUFA in the NEFA pool. These data accord with the suggestion that the enrichment of alpha-LNA in TAG and of DHA in phospholipids reflects hepatic regulation of n-3 PUFA metabolism which potentially enhances the delivery of DHA to the placenta.     

Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenoic acid on breastmilk fatty acid composition

We investigated whether supplementation with arachidonic acid (20:4 omega 6; AA), or a combination of AA and docosahexaenoic acid (22:6 omega 3; DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition. Ten women were daily supplemented with 300 mg AA, eight with 300 mg AA, 110 mg eicosapentaenoic acid (20:5 omega 3; EPA) and 400 mg DHA, for one week and eight women served as unsupplemented controls. Milk samples were collected on days 0, 1 and 7. The fatty acid composition of the milk was analyzed by capillary gas chromatography with flame ionisation detection. Supplementation with AA alone had no effect on breastmilk AA, but tended to reduce EPA and DHA levels. Administration of a combination of AA, EPA and DHA tended to increase both milk AA and long chain PUFA (LCPUFA)omega 3 content. A larger simultaneous increase of milk AA, DHA and EPA than observed in the present study can probably be accomplished by the use of a combination of a lower LCPUFA omega 6/LCPUFA omega 3 ratio and higher AA, EPA and DHA dosages.     

Docosahexaenoic acid, but not eicosapentaenoic acid, lowers ambulatory blood pressure and shortens interval QT in spontaneously hypertensive rats in vivo

This study was designed to evaluate the effects of individual dietary long-chain n-3 polyunsaturated fatty acids (LCPUFA) on hypertension and cardiac consecutive disorders in spontaneously hypertensive rats (SHR) as compared to Wistar-Kyoto rats (WKY). Rats were fed for 2 months an eicosapentaenoic (EPA)- or docosahexaenoic acid (DHA)-rich diet (240 mg/day) or an n-3 PUFA-free diet. Male SHR (n=6), implanted with cardiovascular telemetry devices, were housed in individual cages for continuous measurements of cardiovascular parameters (blood pressure (BP) and heart rate (HR)) during either activity or rest periods, ECG were recorded during the quiet period. The n-6 PUFA upstream of arachidonic acid was affected in SHR tissues. The cardiac phospholipid fatty acid profile was significantly affected by dietary DHA supply, and EPA in a very lower extent, since DHA only was incorporated in the membranes instead of n-6 PUFAs. Endothelium n-6 PUFA content increased in all SHR groups. Compared to WKY, linoleic acid content decreased in both studied tissues. Cardiac noradrenalin decreased while the adrenal catecholamine stores decreased in SHR as compared to WKY. Both n-3 PUFA supply induced a decrease of adrenal catecholamine stores. Nevertheless after 6 weeks, DHA but not EPA induced a lowering-blood pressure effect and shortened the QT interval in SHR, most probably through its tissue enrichment and a specific effect on adrenergic function. Dietary DHA supply retards blood pressure development and has cardioprotective effect. These findings, showing the cardioprotective effects of DHA in living animals, were obtained in SHR, but may relate to essential hypertension in humans.     

Effect of frozen storage on fatty acid composition and changes in lipid content of Scomberomorus commersoni and Carcharhinus dussumieri

Investigated were the changes in fatty acid composition, oxidation and enzymatic deterioration of lipids in frozen (−30°C) fish fillets from the Persian Gulf. The narrow barred Spanish mackerel ( Scomberomorus commersoni ) and white cheek shark ( Carcharhinus dussumieri ) were tested with storage times of 0, 1, 2, 3, 4, 5 and 6 months at −18°C. Statistical results showed that the major fatty acids among the saturated and monounsaturated fatty acids of each fish species were palmitic (C16:0) and oleic (C18:1n-9) acids, respectively. Both linoleic acid (C18:2n-6) and arachidonic acid (AA) (C20:4n-6) were predominant in total n-6 polyunsaturated fatty acids in both mackerel and shark. The EPA (eicosapentaenoic acid; C20:5 n-3) and DHA (docosahexaenoic acid; C22:6 n-3) acids were the major fatty acids among total n-3 acids in both fishes. During frozen storage, the PUFA (40.1 and 23.94%), n-3 (48 and 42.83%), ω 3/ ω 6 (41.36 and 50%), PUFA/SFA (56 and 42.23%) and EPA + DHA/C16 (55.55 and 46.66%) contents decreased in S. commersoni and C. dussumieri , respectively. Also peroxide, thiobarbituric acid (TBA) and free fatty acid (FFA) values significantly increased (P < 0.01) with the time of storage.     

DHA and EPA reverse cystic fibrosis-related FA abnormalities by suppressing FA desaturase expression and activity

Patients and models of cystic fibrosis (CF) exhibit consistent abnormalities of polyunsaturated fatty acid composition, including decreased linoleate (LA) and docosahexaenoate (DHA) and variably increased arachidonate (AA), related in part to increased expression and activity of fatty acid desaturases. These abnormalities and the consequent CF-related pathologic manifestations can be reversed in CF mouse models by dietary supplementation with DHA. However, the mechanism is unknown. This study investigates this mechanism by measuring the effect of exogenous DHA and eicosapentaenoate (EPA) supplementation on fatty acid composition and metabolism, as well as on metabolic enzyme expression, in a cell culture model of CF. We found that both DHA and EPA suppress the expression and activity of Δ5- and Δ6-desaturases, leading to decreased flux through the n-3 and n-6 PUFA metabolic pathways and decreased production of AA. The findings also uncover other metabolic abnormalities, including increased fatty acid uptake and markedly increased retroconversion of DHA to EPA, in CF cells. These results indicate that the fatty acid abnormalities of CF are related to intrinsic alterations of PUFA metabolism and that they may be reversed by supplementation with DHA and EPA.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases

This work deals with the production of structured triacylglycerols (STAG) with caprylic acid (CA) located in positions 1 and 3 of the molecule of glycerol and docosahexaenoic acid (DHA) in position 2, by acidolysis of tuna oil and CA, catalyzed by several lipases. To this end several lipases and immobilization supports were tested with the aim of avoiding the acyl-migration observed in previous works. The determination of the best catalyst (i.e. the lipase and the immobilization support as a whole) was carried out by experiments of acidolysis of cod liver oil and CA in a bath reactor. The best results were obtained with the lipases from Rhizopus oryzae (Lipase D) and Rhizopus delemar (Lipase Rd), immobilized on Accurel MP1000 (a microporous polypropylene) with a lipase/support ratio 1:1.5 (w/w). The activity of these immobilized lipases was stable for a minimum of 5 days in the operational conditions (up to 40 °C).Lipase Rd was selected for the next step in which it was immobilized on Acurrel MP1000 to obtain STAG enriched in DHA by acidolysis of tuna oil (20% DHA) with CA. The experiments were carried out by recirculating the reaction mixture through an immobilized lipase packed bed reactor at different substrate/hexane ratios, as well as in absence of solvent. In the latter case, STAG with 51% CA and 13% DHA were obtained at 73 h. This result indicates that with this catalyst an acceptable reaction rate was attained in absence of solvent. A structural analysis by the pancreatic lipase method carried out to STAG with 45% CA and 16% DHA indicated that 91% of the CA incorporated is located in positions 1 and 3, and that 51% of the DHA is located in position 2 (MLM structure). This position is also rich in palmitic, eicosapentaenoic and oleic acids.After the acidolysis reaction a mixture of STAG and free fatty acids was obtained. The recovery of STAG from this reaction mixture is difficult because of the high content of free fatty acids. A separation method based on the neutralization of the free fatty acids with a KOH hydroalcoholic solution has been developed. By this procedure pure (100%) STAG were obtained with a recovery yield of 80%.