Effect of hydrogen/air flow rates and scan rate on the flame ionisation detection response of phopholipids, and their quantitative and qualitative analysis by Iatroscan (Mark‐6s) TLC‐FID

The combined effect of scan speed, hydrogen and air flow rates on the flame ionization detection (FID) peak response of phospholipid classes has been studied to determine the optimum levels of these parameters. The phospholipid composition of different types of commercial lecithins, as well as lecithins combined with fish oils, has been analyzed by Iatroscan TLC‐FID Mark‐6s under optimized conditions. An air flow rate of 2 L/min, a hydrogen flow rate of 150–160 mL/min, and a scan speed of 30 s/rod seem to be the ideal conditions for scanning phospholipids with complete pyrolysis in the flame in the Mark‐6 model. Increasing the scan speed rapidly decreased the FID response. A hydrogen flow rate as high as 170 mL/min could be used at relatively low air flow rates (&#x003C2 L/min) and the response declined when both air flow rate and hydrogen flow rate increased simultaneously. Both linear and curvilinear relationships had highly significant correlations (p&#x003C0.01) with the sample load. Time course reactions, including the hydrolysis of phosphatidylserine using enzymes, can be successfully monitored by the Iatroscan TLC‐FID Chromarod system.

Fowl play and the price of petrel: long-living Procellariiformes have peroxidation-resistant membrane composition compared with short-living Galliformes

The membrane pacemaker hypothesis predicts that long-living species will have more peroxidation-resistant membrane lipids than shorter living species. We tested this hypothesis by comparing the fatty acid composition of heart phospholipids from long-living Procellariiformes (petrels and albatrosses) to those of shorter living Galliformes (fowl). The seabirds were obtained from by-catch of commercial fishing operations and the fowl values from published data. The 3.8-fold greater predicted longevity of the seabirds was associated with elevated content of peroxidation-resistant monounsaturates and reduced content of peroxidation-prone polyunsaturates and, consequently, a significantly reduced peroxidation index in heart membrane lipids, compared with fowl. Peroxidation-resistant membrane composition may be an important physiological trait for longevous species.

Phospholipase activity in human mesenteric ischaemia and infarction

Currently, diagnostic tests for mesenteric ischaemia and infarction are inadequate due to poor sensitivity and specificity. In addition, many potential markers appear too late to be clinically useful. At present, definitive diagnosis can only be made at the time of surgery, which is not ideal as surgery is often to be avoided in critically ill and elderly patients. A clinically useful, minimally invasive test is likely to decrease the currently very high mortality rate and allow monitoring of 'at risk' patients during their hospital stay. A two-dimensional electrophoresis based proteomic approach was undertaken to assess plasma protein differences between patients with surgically confirmed bowel infarction and control Intensive Care patients. The major protein differences were found to be members or variants of acute phase proteins. Serum amyloid A showed the largest difference between the two patient groups, and this protein was investigated in greater depth. An analysis was performed to compare the diagnostic ability of several commonly used indicators of critical illness and bowel infarction with serum amyloid A and phospholipase A2. Although none of the variables were ideal for clinical use, plasma phospholipase A2 activity showed the best discriminatory power, as determined by Receiver Operating Characteristic curves. From a review of the literature, phospholipase AI (PLA2) appeared to be increased in the bowel as a result of ischaemia and infarction. In one patient, matched tissues were obtained, and PLA2 activity was found to be significantly higher in infarcted bowel tissue compared to ischaemic bowel tissue. PLA2 activity was significantly greater in bowel lumen than tissue, suggesting that the protein was being released, and may enter the circulation. PLA2 activity was increased in the plasma of bowel infarction patients compared with control patients, though the difference was not significant. The phospholipase activity exhibited a number of similarities to typical phospholipase A2 proteins, but also showed a number of inconsistent characteristics. For this reason, we wished to identify the protein responsible for the increased phospholipase activity in infarcted human bowel. The PLA2 activity in human bowel could not be abolished by immunoprecipitation of the PLA2 isoforms IIA (well described in bowel) and V (a closely related isoform). To investigate these proteins, a native urea protein gel devised for snake venom phospholipase A2 was modified for use with mammalian phospholipase AI. The modified gel was used to show that the protein with phospholipase activity from infarcted gut was different from normal gut PLA2 and type IIA PLA2. A number of extensions were devised for these native gels and were found to be useful both in this investigation and for venom investigations. Protein purification was undertaken to identify the protein responsible for the increased phospholipase activity in infarcted bowel. Protein was purified from infarcted human bowel using a number of techniques that exploited unusual characteristics of the protein. The purification techniques each retained the native activity of the protein and the purification could therefore be monitored with a phospholipid hydrolysis assay at each stage. The protein identified by mass spectrometry was an excellent match for cyclophilin B, an inflammatory protein that had previously been identified in rat bowel at the mRNA level (Hasel et al, 1991, Kainer & Doris, 2000). As the purification progress had been monitored throughout with a phospholipid hydrolysis assay, cyclophilin B was an unexpected identification, as it is not known to have phospholipase activity. Cyclophilin B was removed from the highly purified samples via immunoprecipitation and this process abolished all phospholipase activity. The addition of cyclosporin A, (the pharmaceutical ligand of cyclophilin B), did not effect the phospholipase activity. Cyclophilin B protein was found in normal and infarcted human bowel using Western blotting. Cyclophilin B protein also appeared to be present in the bowel lumen and plasma of several patients with bowel infarction, but not in control patients. Immunohistochemistry confirmed the ubiquitous nature of cyclophilin B that had been reported by other groups. This project has investigated the use of two dimensional gel electrophoresis based proteomics to identify proteins present in the plasma of patients with confirmed bowel infarction and control intensive care patients. The major protein classes observed were members of the acute phase proteins, which highlights the need for pre-fractionation of plasma to identify lower abundance, disease associated proteins. A series of potential plasma markers were compared using Receiver Operating Characteristic Curves. Although no ideal marker was clear from this analysis, phospholipase activity appeared to warrant further investigation. Phospholipase activity was investigated in human infarcted bowel. Protein purification identified cyclophilin B as a bowel protein that showed unusual phospholipid hydrolysing activity. Cyclophilin B is a ubiquitous protein in intestinal cell types in both normal and infarcted tissue. There appears to be release of cyclophilin B into bowel lumen and plasma under conditions of mesenteric ischaemia and infarction.

Comparative effects of tuna oil and salmon oil on liver lipid metabolism and fatty acid concentrations in rats

The comparative effect of tuna oil (TO) and salmon oil (SO) on the plasma and liver lipid and fatty acid compositions in Sprague Dawley rats was investigated. The total triacylglycerol (TG) and total cholesterol (TC) concentrations in liver was significantly decreased in the TO group; TG level in liver was also significantly decreased in the SO group. The mRNA expression of HMG-CoA reductase in liver was significantly down-regulated in the TO and SO groups relative to the control group. The plasma TG and TC were decreased in TO, but not in SO; plasma low-density lipoprotein and very low-density lipoprotein levels in TO and SO were decreased compared with the control group. The total n-3 polyunsaturated fatty acid (PUFA) in plasma and liver phospholipids was significantly elevated in the TO and SO. Docosahexaenoic acid (22:6n-3) and eicosapentaenoic acid (20:5n-3) in tissues were significantly increased in the TO and SO, respectively. In this study, TO had a more beneficial effect on liver TC and plasma TG, TC, high-density lipoprotein in rats than SO. The likely mechanism for lowering liver and plasma cholesterol by n-3 PUFA is to suppress the mRNA expression of gene encoding HMG-CoA reductase responsible for cholesterol biosynthesis.

PRACTICAL APPLICATIONS

The beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) from fish and fish oil on human health is derived from their role in modulating membrane lipid composition and affecting metabolic and signal-transduction pathways. In the present study, we demonstrated that n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna and salmon oils can be effectively incorporated into tissue membranes. Tuna oil rich in DHA has more beneficial effect on liver total cholesterol (TC) and plasma triglyceride, TC and HDL in rats than salmon oil, which is rich in EPA. The present data could provide information for the potential application of fish oils as components of functional food, and selected for fortification with different fish oils.

Fish oil replacement in starter, grow-out, and finishing feeds for farmed aquatic animals

As aquaculture production continues to grow, there will be an increased use of lipid resources (oils and fats) alternative to fish oil for feed production. The potential for the use of these alternatives varies depending on the feeds in which they are included according to the production phase of the animals to which they are being fed. In starter feeds, where rapid growth, high survival, and normal development are critical priorities, there will remain a need for the use of lipid resources high in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Fish in this starter phase have a critical requirement for the n-3 LC-PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and fish oils remain the only cost-effective source of these nutrients in the volumes required. However, the greatest demand for lipids is in those diets for the grow-out phase. Most studies on alternative lipid use with animals in this part of the production phase show positive outcomes, in that there are few studies where all the added fish oil cannot be replaced. There are some species, however, where potential replacement levels are suggested to be more conservative, and a general substitution level in this production phase of 75% has been suggested. One of the key effects noted across the grow-out phase is that all alternatives affect the flesh fatty acid characteristics by reducing the level of n-3 LC-PUFA. This issue has provoked the concept of finisher diets, whereby a high n-3 LC-PUFA content diet is fed in order to restore the desired meat fatty acid profiles. Studies examining this concept have found that the tissue triacylglycerol fatty acids were greatly modified and responded in a simple dilution process to the added oil fatty acid composition, whereas the fatty acids of tissue phospholipids were less influenced by dietary fatty acid makeup.

Immobilized artificial membrane chromatography : quantitative structure-retention relationships of structurally diverse drugs

The chromatographic capacity factors (log k‘) for 32 structurally diverse drugs were determined by high performance liquid chromatography (HPLC) on a stationary phase composed of phospholipids, the so-called immobilized artificial membrane (IAM). In addition, quantitative structure-retention relationships (QSRR) were developed in order to explain the dependence of retention on the chemical structure of the neutral, acidic, and basic drugs considered in this study. The obtained retention data were modeled by means of multiple regression analysis (MLR) and partial least squares (PLS) techniques. The structures of the compounds under study were characterized by means of calculated physicochemical properties and several nonempirical descriptors. For the carboxylic compounds included in the analysis, the obtained results suggest that the IAM-retention is governed by hydrophobicity factors followed by electronic effects due to polarizability in second place. Further, from the analysis of the results obtained of two developed quantitative structure-permeability studies for 20 miscellaneous carboxylic compounds, it may be concluded that the balance between polarizability and hydrophobic effects is not the same toward IAM phases and biological membranes. These results suggest that the IAM phases could not be a suitable model in assessing the acid-membrane interactions. However, it is not possible to generalize this observation, and further work in this area needs to be done to obtain a full understanding of the partitioning of carboxylic compounds in biological membranes. For the non-carboxylic compounds included in the analysis, this work shows that the hydrophobic factors are of prime importance for the IAM-retention of these compounds, while the specific polar interactions, such as electron pair donor−acceptor interactions and electrostatic interactions, are also involved, but they are not dominant.

Metabolic rate and membrane fatty acid composition in birds : a comparison between long-living parrots and short-living fowl

Both basal metabolic rate (BMR) and maximum lifespan potential (MLSP) vary with body size in mammals and birds and it has been suggested that these are mediated through size-related variation in membrane fatty acid composition. Whereas the physical properties of membrane fatty acids affect the activity of membrane proteins and, indirectly, an animal's BMR, it is the susceptibility of those fatty acids to peroxidation which influence MLSP. Although there is a correlation between body size and MLSP, there is considerable MLSP variation independent of body size. For example, among bird families, Galliformes (fowl) are relatively short-living and Psittaciformes (parrots) are unusually long-living, with some parrot species reaching maximum lifespans of more than 100 years. We determined BMR and tissue phospholipid fatty acid composition in seven tissues from three species of parrots with an average MLSP of 27 years and from two species of quails with an average MLSP of 5. 5 years. We also characterised mitochondrial phospholipids in two of these tissues. Neither BMR nor membrane susceptibility to peroxidation corresponded with differences in MLSP among the birds we measured. We did find that (1) all birds had lower n-3 polyunsaturated fatty acid content in mitochondrial membranes compared to those of the corresponding tissue, and that (2) irrespective of reliance on flight for locomotion, both pectoral and leg muscle had an almost identical membrane fatty acid composition in all birds.

Global trends in marine nutraceuticals

An expanding body of scientific research indicates that the marine environment is emerging as a unique resource of functional food ingredients with health-promoting properties. Significant attention has been paid to exploration of potential nutraceuticals and pharmaceuticals derived from the ocean. Marine-based  nutraceuticals are gaining attention due to their unique features, which are absent in terrestrial-based resources. A number of new marine nutraceutical products have been introduced in the nutraceuticals and functional foods markets. The main sources and products of primary interest for marine nutraceuticals and ingredients include omega-3 fish/algal oil, phospholipids, micro/macro algal nutrition supplements, fish proteins and peptides, hydrolysates, shellfish chitin, fish collagen, and mineral supplements.

A short-term n-3 DPA supplementation study in humans

Purpose Despite the detailed knowledge of the absorption and incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into plasma lipids and red blood cells (RBC) in humans, very little is known about docosapentaenoic acid (DPA, 22:5 n-3). The aim of this study was to investigate the uptake and incorporation of pure DPA and EPA into human plasma and RBC lipids.

Methods Ten female participants received 8 g of pure DPA or pure EPA in randomized crossover double-blinded manner over a 7-day period. The placebo treatment was olive oil. Blood samples were collected at days zero, four and seven, following which the plasma and RBC were separated and used for the analysis of fatty acids.

Results Supplementation with DPA significantly increased the proportions of DPA in the plasma phospholipids (PL) (by twofold) and triacylglycerol (TAG) fractions (by 2.3-fold, day 4). DPA supplementation also significantly increased the proportions of EPA in TAG (by 3.1-fold, day 4) and cholesterol ester (CE) fractions (by 2.0-fold, day 7) and of DHA in TAG fraction (by 3.1-fold, day 4). DPA proportions in RBC PL did not change following supplementation. Supplementation with EPA significantly increased the proportion of EPA in the plasma CE and PL fractions, (both by 2.7-fold, day 4 and day 7) and in the RBC PL (by 1.9-fold, day 4 and day 7). EPA supplementation did not alter the proportions of DPA or DHA in any lipid fraction. These results showed that within day 4 of supplementation, DPA and EPA demonstrated different and specific incorporation patterns.

Conclusion The results of this short-term study suggest that DPA may act as a reservoir of the major long-chain n-3 fatty acids (LC n-3 PUFA) in humans.

Up-regulated Desaturase and Elongase Gene Expression Promoted Accumulation of Polyunsaturated Fatty Acid (PUFA) but Not Long-Chain PUFA in Lates calcarifer, a Tropical Euryhaline Fish, Fed a Stearidonic Acid- and γ-Linoleic Acid-Enriched Diet

The limited activity of Δ6 fatty acid desaturase (FAD6) on α-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids in marine fish alters the long-chain (≥C₂₀) polyunsaturated fatty acid (LC-PUFA) concentration in fish muscle and liver when vegetable oils replace fish oil (FO) in aquafeeds. Echium oil (EO), rich in stearidonic acid (SDA, 18:4n-3) and γ-linoleic acid (GLA, 18:3n-6), may enhance the biosynthesis of n-3 and n-6 LC-PUFA by bypassing the rate-limiting FAD6 step. Nutritional and environmental modulation of the mechanisms in LC-PUFA biosynthesis was examined in barramundi, Lates calcarifer, a tropical euryhaline fish. Juveniles were maintained in either freshwater or seawater and fed different dietary LC-PUFA precursors present in EO or rapeseed oil (RO) and compared with FO. After 8 weeks, growth of fish fed EO was slower compared to the FO and RO treatments. Irrespective of salinity, expression of the FAD6 and elongase was up-regulated in fish fed EO and RO diets, but did not lead to significant accumulation of LC-PUFA in the neutral lipid of fish tissues as occurred in the FO treatment. However, significant concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), but not docosahexaenoic acid (DHA, 22:6n-3), appeared in liver and, to a lesser extent, in muscle of fish fed EO with marked increases in the phospholipid fraction. Fish in the EO treatment had higher EPA and ARA in their liver phospholipids than fish fed FO. Endogenous conversion of dietary precursors into neutral lipid LC-PUFA appears to be limited by factors other than the initial rate-limiting step. In contrast, phospholipid LC-PUFA had higher biosynthesis, or selective retention, in barramundi fed EO rather than RO.

Greater effect of diet than exercise training on the fatty acid profile of rat skeletal muscle

We determined the interaction of diet and exercise-training intensity on membrane phospholipid fatty acid (FA) composition in skeletal muscle from 36 female Sprague-Dawley rats. Animals were randomly divided into one of two dietary conditions: high-carbohydrate (64.0% carbohydrate by energy, n = 18) or high fat (78.1% fat by energy, n = 18). Rats in each diet condition were then allocated to one of three subgroups: control, which performed no exercise training; low-intensity (8 m/min) treadmill run training; or high-intensity (28 m/min) run training. All exercise-trained rats ran 1,000 m/session, 4 days/wk for 8 wk and were killed 48 h after the last training bout. Membrane phospholipids were extracted, and FA composition was determined in the red and white vastus lateralis muscles, Diet exerted a major influence on phospholipid FA composition, with the high-fat diet being associated with a significantly (P < 0.01) elevated ratio of n-6/n-3 FA for both red (2.7-3.2 vs. 1.0-1.1) and white vastus lateralis muscle (2.5-2.9 vs. 1.2). In contrast, alterations in FA composition as a result of either exercise-training protocol were only minor in comparison. We conclude that, under the present experimental conditions, a change in the macronutrient content of the diet was a more potent modulator of skeletal muscle membrane phospholipid FA composition compared with either low- or high-intensity treadmill exercise training.

Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA

Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds. © 2014 Zhou, Callahan, Shrestha, Liu, Petrie and Singh.

Dietary repletion with ω3 fatty acid or with COX inhibition reverses cognitive effects in F3 ω3 fatty-acid-deficient mice

Dietary deficiency of ω3 fatty acid during development leads to impaired cognitive function. However, the effects of multiple generations of ω3 fatty-acid deficiency on cognitive impairment remain unclear. In addition, we sought to test the hypothesis that the cognitive impairments of ω3 fatty-acid-deficient mice are mediated through the arachidonic acid-cyclooxygenase (COX) pathway. To address these issues, C57BL/6J mice were bred for 3 generations and fed diets either deficient (DEF) or sufficient (SUF) in ω3 fatty acids. At postnatal day 21, the F3 offspring remained on the dam's diet or were switched to the opposite diet, creating 4 groups. In addition, 2 groups that remained on the dam's diet were treated with a COX inhibitor. At 19 wk of age, spatial-recognition memory was tested on a Y-maze. Results showed that 16 wk of SUF diet reversed the cognitive impairment of F3 DEF mice. However, 16 wk of ω3 fatty-acid-deficient diet impaired the cognitive performance of the F3 SUF mice, which did not differ from that of the F3 DEF mice. These findings suggest that the cognitive deficits after multigenerational maintenance on ω3 fatty-acid-deficient diet are not any greater than are those after deficiency during a single generation. In addition, treatment with a COX inhibitor prevented spatial-recognition deficits in F3 DEF mice. Therefore, cognitive impairment due to dietary ω3 fatty-acid deficiency appears to be mediated by the arachidonic acid-COX pathway and can be prevented by 16 wk of dietary repletion with ω3 fatty acids or COX inhibition.