Synthesis of regioisomerically pure triacylglycerols containing n‐3 very long‐chain polyunsaturated fatty acids

There is growing scientific evidence that consumption of n‐3 very long‐chain polyunsaturated fatty acids (n‐3 VLC‐PUFA) helps in brain and eye development, and protects against a range of common degenerative diseases. This has provided the impetus to the scientists to develop new and renewable sources for these important fatty acids so that the food industry is able to produce and market products fortified with n‐3 VLC‐PUFA. The bioactive efficacy and stability of food products containing n‐3 VLC‐PUFA may be determined not only by the amount of n‐3 VLC‐PUFA present but also by the positional distribution of these acids within the triacylglycerol (TAG) molecules (regiopurity). Studies of the effects of positional distribution on the functionality of n‐3 VLC‐PUFA containing oils have been hampered by a general lack of pure TAG regioisomers for experimentation. This paper reviews methods that have been used for the synthesis of TAG regioisomers containing n‐3 VLC‐PUFA, with special reference to those in which one n‐3 VLC‐PUFA occurs in combination with two long‐chain saturated acids.     

Lipid changes during life cycle of marine copepod,Euchaeta japonica marukawa

All stages from egg to adult of the North Pacific copepod,Euchaeta japonica contained wax esters in their lipid stores, while triglycerides were important only in the eggs, early naupliar stages, and adults. The large lipid reserves of the eggs were wax esters and triglycerides (58% and 19% of the lipid, respectively), both of which were used rapidly during the early stages of development. Wax esters continued to decrease after triglycerides had been utilized completely for energy. The slow metabolism of lipid during starvation indicated that lipid stores in adult females may be conserved for egg production. The dominant alcohols of the wax esters of all stages were tetradecanol (24–42% of the total) and hexadecanol (25–65%). Only minor amounts of polyunsaturated alcohols were observed. There was, however, a high proportion of polyunsaturation in the wax ester fatty acids, even though octadecenoic was generally predominant (16–46% of the total wax ester fatty acids). The polyunsaturation of the wax esters fatty acids and the presence of 21∶6 hydrocarbon suggest phytoplankton in the diet of adults and in the younger stages. Cholesterol was the main sterol, but there were minor amounts of desmosterol (1–12% of the total sterols) present. The latter sterol has not been found previously in copepods, although reported from Cirripedia and Decapoda.     

How good is the evidence for a favourable effect of very long chain n‐3 polyunsaturated fatty acids on cardiovascular disease?

The evidence for beneficial effects of very long chain (VLC) n‐3 polyunsaturated fatty acids (PUFA) on cardiovascular disease (CVD) is strong because it is based on the results of adequately powered intervention trials with disease endpoints. This contrasts with the evidence for a detrimental effect of saturated fat on CVD which is weak because such studies have not been carried out. Accordingly, any set of dietary recommendations which attaches more importance to saturated fat intakes than VLC n‐3 PUFA intakes can be questioned. Despite the strength of the evidence there are practical difficulties associated with implementing the recommendation to increase intake of VLC n‐3 PUFA to 1g per day which need to be overcome. These include the acceptability of fatty fish consumption, the presence of methyl mercury in fish at the top of the food chain and declining global fish stocks.     

Lipase selectivity toward fatty acids commonly found in fish oil

The main objective of this study was to compare the fatty acid selectivity of numerous commercially available lipases toward the most ubiquitous fatty acids present in fish oils in form of their corresponding ethyl esters. Special interest was taken in their ability to separate the n‐3 long‐chain polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from the more saturated fatty acids as well as exploiting the putative discrimination between these highly valuable n‐3 PUFA. Hydrolysis of sardine oil ethyl esters in a Tris buffer solution by 12 microbial lipases is described. The results reveal that all of the lipases strongly discriminate against the n‐3 PUFA and prefer the more saturated fatty acids as substrates. Most of the lipases discriminate between EPA and DHA in favor of EPA, however, 2 bacterial lipases from Pseudomonas were observed to prefer DHA to EPA. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed reversed fatty acid selectivity when their fish oil triacylglycerol hydrolysis was studied. Thus, the n‐3 PUFA including EPA and DHA were observed to be hydrolyzed at a considerably higher rate than the more saturated fatty acids.     

Fatty Acid Specificity of Candida cylindracea Lipase

Candida cylindracea lipase (SIGMA) was tested against triglycerides (TG) and wax esters (WE) of marine origin as substrates. Under the same conditions, wax esters were hydrolysed at a lower rate than the triglycerides. The C₁₄ to C₁₈ saturated and monounsaturated fatty acids were preferentially hydrolysed whereas the longer chain monoenes (20:1 and 22:1) and particularly the polyunsaturated fatty acids (18:4,20:5 and 22:6) were resistant to the hydrolysis in triglycerides as well as in wax esters. No specificity was demonstrated for the fatty alcohols in the wax esters.     

Does fish oil prevent cardiac arrhythmias?

It is widely accepted that the very long chain n‐3 polyunsaturated fatty acids (VLC n‐3 PUFA) characteristic of marine oils protect against the development of cardiovascular disease (CVD). This idea is supported by a large body of consistent epidemiological studies showing favourable associations between intakes of VLC n‐3 PUFA and CVD risk and more importantly by several large and lengthy dietary intervention trials showing favourable effects of VLC n‐3 PUFA on the development of CVD. The effects of VLC n‐3 PUFA on sudden death and on survival from a heart attack demonstrated in these trials could be explained if they have anti‐arrhythmic effects. Recent meta‐analyses of the available dietary intervention trials suggest that neither ventricular nor atrial arrhythmias are influenced by VLC n‐3 PUFA intakes. Hence a convincing mechanism of action of VLC n‐3 PUFA on CVD has yet to emerge.     

Occurrence of Hexacosapolyenoic Acids 26:7(n‐3), 26:6(n‐3), 26:6(n‐6) and 26:5(n‐3) in Deep‐Sea Brittle Stars from Near the Kuril Islands

Significant amounts of polyunsaturated fatty acids (PUFA) with a chain length of 26 carbon atoms were detected in lipids of five deep water species of Ophiuroidea besides common fatty acids with chain lengths between 14 and 24. By means of hydrogenation, GC–MS of the methyl esters, and 4,4‐dimethyloxazoline (DMOX) derivatives of these C26 acids were characterized as 5,8,11,14,17,20,23‐hexacosaheptaenoic [26:7(n‐3)]; 8,11,14,17,20,23‐hexacosahexaenoic [26:6(n‐3)]; 5,8,11,14,17,20‐ hexacosahexaenoic [26:6(n‐6)]; and 11,14,17,20,23‐hexacosapentaenoic [26:5(n‐3)]. Concentrations of these acids varied from 0.3 to 4.5 mol% of the total FA. In all the samples investigated, the main component of C26PUFA was hexacosaheptaenoic acid 26:7(n‐3). These C26PUFA are localized mainly in polar lipids. The presence of the possible biosynthesis precursors suggests that the C26PUFA are produced by the brittle stars, and are not accumulated from food sources. This finding can also explain the presence of small amounts of the 26:7(n‐3) acid detected earlier in flesh lipids of the roughscale sole Clidoderma asperrimum, which feeds on deep water brittle stars. We suggest a possible scheme of the biosynthesis of C26 PUFA.     

Occurrence,function and biosynthesis of wax esters in marine organisms

Wax esters occur as a major lipid-type in at least 30 species of marine animals, distributed among 17 orders and 3 phyla. They are of limited usefulness as a chemotaxonomic character, since only in two suborders, the calanoid copepods, Calanoidei, and the toothed whales, Odontoceti, do the wax esters occur in all members so far examined. In bony fishes their occurrence in muscle correlates better with mesopelagic habitat and vertical migration patterns than with taxonomy. Homologs with 21 to 44 total carbon atoms have been reported, but the usual range for the wax esters in copepods and fish is C₃₀–C₄₂. In fishes the muscle wax esters contain predominantly one and two double bonds per molecule, while in roe lipids up to 65% of the homologs contain three or more double bonds. The component alcohols are saturated and monounsaturated, with 16∶0 and 18∶1 as the usual major constituents. The fatty acids are more diverse, but 18∶1 is most often the main component, and 16∶1 and 20∶1 are frequent major constituents; polyunsaturated acids make up 1–12% in fish muscle and whale oils and up to 45% in fish roe wax esters. Possible functions of the wax esters are for buoyancy, as energy reserves and for thermal insulation. In vitro, various tissues of marine bony fishes synthesize wax esters from long chain alcohols and fatty acids, without activation. A competing pathway for the long chain alcohols in vivo is their catabolic oxidation to the corresponding fatty acids. The key to the accumulation of wax esters is to be sought in the metabolism of the long chain alcohols, their biosynthesis and esterification vs. their catabolism. Presented at the 60th AOCS Annual Meeting, San Francisco, April 1969, as part of a Symposium on Natural Waxes.     

Has the beneficial effect of fish oil on cardiovascular disease disappeared?

Meta‐analyses of prospective cohort studies or meta‐analyses of randomised controlled trials completed before the year 2002 suggest that the very long chain polyunsaturated fatty acids (VLC n‐3 PUFA) characteristic of fish oil significantly reduce the risk of cardiovascular disease. However, randomised controlled trials and meta‐analyses of these trials published in the last decade suggest that VLC n‐3 PUFA have no effect on the risk of cardiovascular disease. While some of the more recently completed trials can be criticised as they were either uncontrolled or did not have sufficient statistical power a more important factor may be at work. By contrast with the earlier trials, the cardiovascular disease patients recruited for trials carried out in the last decade tend to be treated with statins. Demonstrating additional benefit of VLC n‐3 PUFA on cardiovascular disease over and above that conferred by statins is clearly proving problematic.     

Combined Short-Path Distillation and Solvent-Assisted Crystallization of Beef Fatty Acid Methyl Esters

Solvent‐assisted crystallization has previously been employed to remove long‐chain saturated fatty acids (≥ 18 carbons) from animal fat to improve its cold temperature biofuel properties. The same technology can be used for removing long‐chain saturated fatty acids (SFA) from animal fats for human consumption, but SFA remaining (i.e., 14:0 and 16:0) are more atherogenic than longer chain SFA. In the present study, an easy and efficient method was developed using short‐path distillation prior to solvent‐assisted crystallization for the more complete removal of SFA from beef tallow, and for the first time reports the distillation and crystallization behavior of polyunsaturated fatty acid biohydrogenation products (PUFA‐BHP). Shorter chain SFA methyl esters (i.e., 14:0 and 16:0) were efficiently removed at 90 °C, 9.3 Pa, with a rotor speed of 70 rpm and either two cycles of distillation at 90 drops/min or three cycles at 110 drops/min. Stearic acid (18:0) was then effectively removed by crystallization at ?20 °C using a sample to methanol ratio of 1:10. The remaining fraction enriched with PUFA‐BHP (i.e., rumenic acid, c9,t11‐18:2, and its precursor vaccenic acid, t11‐18:1) have potential use in disease model (i.e., cell culture and animal) studies to help further elucidate their bioactivity and mode of action, and may in the future have functional food or nutraceutical potential.     

Fatty Acid Composition of Tropical Fish Depends on Reservoir Trophic Status and Fish Feeding Habit

Eutrophication results in a deficiency of n‐3 LC‐PUFA (long‐chain polyunsaturated fatty acids) in aquatic food chains, affecting fish nutrition and physiology. The trophic transfer of FA (fatty acids) to fish species of different feeding habits was investigated in two reservoirs in southeast Brazil—the mesotrophic Ponte Nova Reservoir (PN) and the hypereutrophic Billings Reservoir (Bil). Total FA profile of stomach contents and adipose tissue, triacylglycerols (TAG), and phospholipids (PL) from liver and muscle of the omnivorous Astyanax fasciatus and the carnivorous Hoplias malabaricus were analyzed by gas chromatography. A prevalence of n‐6PUFA, as 18:2n‐6 (linoleic acid) and 20:4n‐6 (arachidonic acid, ARA) was observed in the stomach contents and in the tissues of A. fasciatus from the PN reservoir. In contrast, n‐3 LC‐PUFA, as 20:5n‐3 (eicosapentaenoic acid, EPA) was accumulated in fish tissues from Bil, resulting in higher n3/n6 and EPA/ARA ratios, compared to fish from PN. This differential FA accumulation was also observed for H. malabaricus, but differences were slightly minor, and no changes were observed in the EPA/ARA ratios between fish from both reservoirs. Regardless reservoir, FA profiles of TAG resembled that of their diet, whereas FA profiles of PL were more conservative and mainly comprised by LC‐PUFA. We conclude that reservoir trophic status affected the FA composition of food resources available to these fish species, resulting in differential allocation of n‐3 and n‐6 FA. As expected, FA profile of the investigated fish species also reflected their feeding habit and physiological demands.     

Long‐chain omega‐3 oils: Current and future supplies,food and feed applications,and stability

Increasing recognition of the health benefits of omega‐3 long‐chain (⪈C₂₀) polyunsaturated fatty acids (omega‐3 LC‐PUFA or LC omega‐3 oils) continues. But new sources are needed, with recent developments with novel land plants showing promise. The value of existing and future sources LC omega‐3 oils occurs through aquaculture, livestock and other feeding. There is also a need toenhance the stability of oils containing LC omega‐3 oils. Challenges include increasing DHA levels in land plants, increasing oxidative stability in food products and product labeling. Consumers have difficulty recognizing and differentiating long‐chain and shorter‐chain (SC, C₁₈) omega‐3 PUFA, both of which are referred to as “omega‐3” fatty acids.     

Enzymatic synthesis of steryl esters of polyunsaturated fatty acids

Steryl esters of long-chain fatty acids have water-holding properties, and polyunsaturated fatty acids (PUFA) have various physiological functions. Because steryl ester of PUFA can be expected to have both features, we attempted to synthesize steryl esters of PUFA by enzymatic methods. Among lipases used, Pseudomonas lipase was the most effective for the synthesis of cholesteryl docosahexaenoate. When a mixture of cholesterol/docosahexaenoic acid (3:1, mol/mol), 30% water, and 3000 units/g of lipase was stirred at 40°C for 24 h, the esterification extent attained 89.5%. Under the same reaction conditions, cholesterol, cholestanol, and sitosterol were also esterified efficiently with docosahexaenoic, eicosapentaenoic, arachidonic, and γ-linolenic acids.     

Fatty alcohols in capelin,herring and mackerel oils and muscle lipids: II. A comparison of fatty acids from wax esters with those of triglycerides

The fatty acids recovered from the triglycerides and wax esters of common northwest Atlantic copepods are compared with the fatty acids of wax esters recovered intact from certain fish skin and body lipid, and from commercial fish oils. The fish species, herring, capelin and mackerel, all feed on copepods, and many resemblances of the copepod lipid fatty acids to those of a previous analysis of similar copepods suggest that the basic dietary fat input for these fish may be quite constant. The two copepod fatty acid analyses differed quantitatively in triglyceride 20∶1 and 22∶1 and also in 20∶5ω3 and 22∶6ω3, confirming the primary role of the wax esters in copepods. Selectivity factors are discussed in comparing the copepod wax ester fatty acids with the fatty acids of the wax esters recovered intact from the fish lipids and oils. The basic role of copepods in supplying all types of fatty acids to fish depot fats is considered to be strongly supported by these findings.     

The enrichment of n−3 polyunsaturated fatty acids using aminopropyl solid phase extraction columns

A rapid, simple and reliable method is described for the preparation of concentrates of methyl or ethyl esters of n−3 polyunsaturated fatty acids by solid phase extraction using aminopropyl bonded silica columns. After applying mixtures of fatty acid esters in hexane, saturated and monounsaturated fatty acid esters are preferentially eluted with hexane whereas polyunsaturated fatty acids (PUFA) can subsequently be eluted with dichloromethane. Concentrates containing 80–90% n−3 PUFA can thus be obtained using fish oil fatty acids esters as a starting material.     

n‐3 PUFA Esterified to Glycerol or as Ethyl Esters Reduce Non‐Fasting Plasma Triacylglycerol in Subjects with Hypertriglyceridemia: A Randomized Trial

To date, treatment of hypertriglyceridemia with long‐chain n‐3 polyunsaturated fatty acids (n‐3 PUFA) has been investigated solely in fasting and postprandial subjects. However, non‐fasting triacylglycerols are more strongly associated with risk of cardiovascular disease. The objective of this study was to investigate the effect of long‐chain n‐3 PUFA on non‐fasting triacylglycerol levels and to compare the effects of n‐3 PUFA formulated as acylglycerol (AG‐PUFA) or ethyl esters (EE‐PUFA). The study was a double‐blinded randomized placebo‐controlled interventional trial, and included 120 subjects with non‐fasting plasma triacylglycerol levels of 1.7–5.65 mmol/L (150–500 mg/dL). The participants received approximately 3 g/day of AG‐PUFA, EE‐PUFA, or placebo for a period of eight weeks. The levels of non‐fasting plasma triacylglycerols decreased 28 % in the AG‐PUFA group and 22 % in the EE‐PUFA group (P < 0.001 vs. placebo), with no significant difference between the two groups. The triacylglycerol lowering effect was evident after four weeks, and was inversely correlated with the omega‐3 index (EPA + DHA content in erythrocyte membranes). The omega‐3 index increased 63.2 % in the AG‐PUFA group and 58.5 % in the EE‐PUFA group (P < 0.001). Overall, the heart rate in the AG‐PUFA group decreased by three beats per minute (P = 0.045). High‐density lipoprotein (HDL) cholesterol increased in the AG‐PUFA group (P < 0.001). Neither total nor non‐HDL cholesterol changed in any group. Lipoprotein‐associated phospholipase A2 (LpPLA2) decreased in the EE‐PUFA group (P = 0.001). No serious adverse events were observed. Supplementation with long‐chain n‐3 PUFA lowered non‐fasting triacylglycerol levels, suggestive of a reduction in cardiovascular risk. Regardless of the different effects on heart rate, HDL, and LpPLA2 that were observed, compared to placebo, AG‐PUFA, and EE‐PUFA are equally effective in reducing non‐fasting triacylglycerol levels.     

Molecular Finger Printing of Nutra-Coconut Oil with Improved Health Protective Phytoceuticals and its Efficacy as Frying Medium

Coconut oil is rich in medium chain triglycerides but lacks polyunsaturated fatty acids (PUFA) and bio‐active phytoceuticals. In the present work nutra‐coconut oil was prepared by blending coconut oil and flaxseed oil (70:30) and adding 3000 ppm of flaxseed cake concentrate using ethanol, methanol and 20 % aqueous ethanol. The concentrate prepared from flaxseed was from ethanol as it gave maximum yield. The different bio‐active molecules in flaxseed concentrate observed are polyphenols (39.04 %), tocopherols (4.37 %), ferulic acid (0.17 mg g^?1), p‐coumaric acid (2.24 mg g^?1), chlorogenic acid (16.11 mg g^?1), gallic acid (8.58 mg g^?1), sinapic acid (0.64 mg g^?1) and secoisolariresinol (30.13 mg g^?1). The nutra‐coconut oil was found to have polyphenols (2.86 %), tocopherols (442.96 ppm) and antiradical activity (94 %). The PUFA content was found to increase in nutra‐coconut oil significantly (p < 0.05) (2–22 %). The FT‐IR spectra of nutra‐coconut oil revealed that the peak at 3009 and 1651 cm^?1 was associated with the presence of unsaturated fatty acids. There was no significant (p > 0.05) difference observed in sensory attributes of snack food fried using coconut oil and nutra‐coconut oil indicating that the later could be used as a frying medium and useful for food processing industries.     

Profiles of volatile compounds in milk containing fish oil analyzed by HS‐SPME‐GC/MS

Long‐chain polyunsaturated fatty acids (LC‐PUFA) have various positive biological effects. Fish oil represents a major source of LC‐PUFA; therefore it is extensively used to enrich food products as, for example, infant formulae, dairy products and fruit juices. However, in the presence of oxygen and metals, LC‐PUFA readily degrade, producing off‐flavors and decreasing the nutritional value of the product. The deterioration of sensory properties (taste and odor) can be easily perceived by the consumer, due to the formation of volatile compounds that are formed by decomposition of lipid hydroperoxides, also known as primary oxidation products. In this study, we used the headspace solid‐phase microextraction‐gas chromatography/mass spectrometry technique (HS‐SPME‐GC/MS) to characterize and quantify volatile compounds in a food matrix supplemented with fish oil. We demonstrated that the HS‐SPME‐GC/MS method is a valuable tool to monitor lipid oxidation at early stages. We identified t‐2‐hexenal and c‐4‐heptenal as possible oxidation markers during the storage of milk enriched with 5% of cod oil.     

Lipids of freshwater dolphinSotalia fluviatilis: Comparison of odontocete bioacoustic lipids and habitat

The melon and jaw lipids of the freshwater dolphinSotalia fluviatilis are composed mainly of isovaleroyl wax esters and diisovaleroyl triglycerides. The blubber fat contains only a trace of wax ester and is mostly tri- (long chain) and monoisovaleroyl triglycerides. Detailed gas liquid chromatographic analyses of the intact wax esters and triglycerides and of the derived fatty acids and fatty alcohols indicate common compositional patterns in the wax esters and triglycerides of the respective head lipids. Both odd and even long chain (C₁₂−C₁₆) iso-structures are prominent in the melon and jaw lipids, but only higher odd chain length iso-acids are major components in the blubber.Sotalia fluviatilis (family Delphinidae) andInia geoffrensis (family Platanistidae) share the same freshwater habitat in the upper Amazon River, and both utilize echolocation to navigate and to find food. Comparison of their respective bioacoustical lipid compositions shows distinctive types of head fats,Sotalia being rich in iso-5∶0 andIndia lacking iso-5∶0. This indicates that isovaleric acid per se has no obligatory role in dolphin echolocation.     

Composition of wax esters,triglycerides and diacyl glyceryl ethers in the jaw and blubber fats of the Amazon River dolphin (Inia geoffrensis)

The lower jaw fat of the Amazon River dolphinInia geoffrensis contains 52.8% wax ester, 44.7% triglyceride and 2.5% diacyl glyceryl ether, while its dorsal blubber fat is >98% triglyceride. Examination of the intact lipids, the derived fatty acids and the derived fatty alcohols by gas chromatography reveals that the blubber triglycerides show characteristics of freshwater fish fats, but the jaw fat lipids have several distinctive features. Jaw fat wax esters, triglycerides and diacyl glyceryl ethers are all rich in C₁₀, C₁₂ and C₁₄ fatty acids and contain no polyunsaturated acids. The fatty alcohols in the wax esters are over 90% saturated. The major carbon numbers in the jaw fat triglycerides (C₃₈–C₄₆) are considerably lower than those of the blubber triglycerides (C₄₈–C₅₄). The possible adaptation of the jaw lipids for use in the underwater echolocation process of this dolphin is discussed.