Human skeletal muscle metabolic responses to 6 days of high-fat overfeeding are associated with dietary n-3PUFA content and muscle oxidative capacity.

Understanding human physiological responses to high-fat energy excess (HFEE) may help combat the development of metabolic disease. We aimed to investigate the impact of manipulating the n-3PUFA content of HFEE diets on whole-body and skeletal muscle markers of insulin sensitivity. Twenty healthy males were overfed (150% energy, 60% fat, 25% carbohydrate, 15% protein) for 6 d. One group (n = 10) received 10% of fat intake as n-3PUFA rich fish oil (HF-FO), and the other group consumed a mix of fats (HF-C). Oral glucose tolerance tests with stable isotope tracer infusions were conducted before, and following, HFEE, with muscle biopsies obtained in basal and insulin-stimulated states for measurement of membrane phospholipids, ceramides, mitochondrial enzyme activities, and PKB and AMPKα2 activity. Insulin sensitivity and glucose disposal did not change following HFEE, irrespective of group. Skeletal muscle ceramide content increased following HFEE (8.5 ± 1.2 to 12.1 ± 1.7 nmol/mg, p = .03), irrespective of group. No change in mitochondrial enzyme activity was observed following HFEE, but citrate synthase activity was inversely associated with the increase in the ceramide content (r=-0.52, p = .048). A time by group interaction was observed for PKB activity (p = .003), with increased activity following HFEE in HF-C (4.5 ± 13.0mU/mg) and decreased activity in HF-FO (-10.1 ± 20.7 mU/mg) following HFEE. Basal AMPKα2 activity increased in HF-FO (4.1 ± 0.6 to 5.3 ± 0.7mU/mg, p = .049), but did not change in HF-C (4.6 ± 0.7 to 3.8 ± 0.9mU/mg) following HFEE. We conclude that early skeletal muscle signaling responses to HFEE appear to be modified by dietary n-3PUFA content, but the potential impact on future development of metabolic disease needs exploring.

No transfer of the non-regulated mycotoxins, beauvericin and enniatins, from feeds to farmed fish reared on plant-based diets.

Concern about the risk of exposure to emerging plant-derived mycotoxins such as beauvericin and enniatins has been addressed by the European Commission who requested the European Food Safety Authority for a scientific opinion on their risk to human and animal health. The studied mycotoxins were found in feeds with enniatin B and beauvericin at average concentrations of 19.9 µg/kg and 30 µg/kg, respectively. In all cases, concentrations of all the mycotoxins analyzed were below quantification limits (<0.1 µg/kg) in fish samples (n = 82). The present work provides comprehensive and traceable data of emerging mycotoxins in plant-based aquafeeds and fish reared on the feeds, responding to increasing concerns about safety of farmed fish fed on sustainable feeds. On the basis of data reported, there was no transfer of the emerging mycotoxins, beauvericin and enniatins, from feeds to fish and so, no risk for human consumption.

Deterioration of muscle force and contractile characteristics are early pathological events in spinal and bulbar muscular atrophy mice.

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's Disease, is a late-onset X-linked progressive neuromuscular disease, which predominantly affects males. The pathological hallmarks of the disease are selective loss of spinal and bulbar motor neurons, accompanied by weakness, atrophy and fasciculations of bulbar and limb muscles. SBMA is caused by a CAG repeat expansion in the gene that encodes the androgen receptor (AR) protein. Disease manifestation is androgen dependent and results principally from a toxic gain of AR function. There are currently no effective treatments for this debilitating disease. It is important to understand the course of the disease in order to target therapeutics to key pathological stages. This is especially relevant in disorders such as SBMA, for which disease can be identified before symptom onset, through family history and genetic testing. To fully characterise the role of muscle in SBMA, we undertook a longitudinal physiological and histological characterisation of disease progression in the AR100 mouse model of SBMA. Our results show that the disease first manifests in skeletal muscle, before any motor neuron degeneration, which only occurs in late-stage disease. These findings reveal that alterations in muscle function, including reduced muscle force and changes in contractile characteristics, are early pathological events in SBMA mice and suggest that muscle-targeted therapeutics may be effective in SBMA.This article has an associated First Person interview with the first author of the paper.

Mussel Consumption as a "Food First" Approach to Improve Omega-3 Status.

Numerous United Kingdom and European Union expert panels recommend that the general adult population consumes ~250 mg of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per day through the consumption of one portion of oily fish per week. The long-chain omega-3 fatty acids EPA and DHA are only found in appreciable amounts in marine organisms. Increasing oily fish consumption conflicts with sustaining fisheries, so alternative dietary sources of EPA and DHA must be explored. Mussels are high in omega-3 polyunsaturated fatty acids (PUFAs) and a good source of essential amino acids. Therefore, we aimed to investigate the impact of introducing mussels as a protein source in the lunchtime meal three times per week for two weeks on the omega-3 status of free-living participants. Following an initial two-week monitoring period, 12 participants (eight male and four female) attended the nutrition laboratory three times per week for two weeks. Each participant received a personalised lunch constituting one-third of their typical daily calorie consumption with ~20% of the calories supplied as cooked mussels. A portion of cooked mussels from each feeding occasion was tested for total omega-3 content. The mean ± SD mussel EPA + DHA content was 518.9 ± 155.7 mg/100 g cooked weight, meaning that each participant received on average 709.2 ± 252.6 mg of EPA + DHA per meal or 304.0 ± 108.2 mg of EPA + DHA per day. Blood spot analysis revealed a significant increase in the omega-3 index (week 1 = 4.27 ± 0.81; week 4 = 5.07 ± 1.00) and whole blood EPA content during the study (%EPA week 1 = 0.70 ± 0.0.35; %EPA week 4 = 0.98 ± 0.35). Consuming mussels three times per week for two weeks as the protein source in a personalised lunchtime meal is sufficient to moderately improve the omega-3 index and whole blood DHA + EPA content in young healthy adults.

Assessment of Dietary Intake, Energy Status, and Factors Associated With RED-S in Vocational Female Ballet Students.

Elite ballet dancers are at risk of health issues associated with Relative Energy Deficiency in Sport (RED-S). This study determined the nutritional status, estimated energy status, and assessed factors related to RED-S in vocational female ballet students. Using a cross-sectional study design, we measured dietary intake (food diaries and 24 h dietary-recall) and energy expenditure (accelerometry) in vocational female ballet students (n = 20; age: 18.1 ± 1.1 years; body mass index: 19.0 ± 1.6 kg·m2; body fat: 22.8 ± 3.4%) over 7 days, including 5 weekdays (with dance training) and 2 weekend days (without scheduled dance training). Furthermore, we assessed eating behaviors, menstrual function, risk of RED-S (questionnaires), and body composition (dual x-ray absorptiometry). Energy and macronutrient intakes of vocational ballet students were similar during weekdays and weekend days (P > 0.050), whereas total energy expenditure was greater on weekdays than weekend days (P < 0.010; 95% CI: 212, 379). Energy balance was lower on weekdays (-425 ± 465 kcal·day-1) than weekend days (-6 ± 506 kcal·day-1, P = 0.015; 95% CI: -748, -92). Exercise energy expenditure was greater on weekdays (393 ± 103 kcal·day-1) than weekend days (213 ± 129 kcal·day-1; P < 0.010; 95% CI: 114, 246), but energy availability was similar between time periods (weekdays 38 ± 13 kcal·kg FFM·day-1; weekend days 44 ± 13 kcal·kg FFM·day-1; P = 0.110). Overall, 35% of participants had an energy intake <1,800 kcal·day-1, 44% had reduced energy availability (30-45 kcal·kg FFM·day-1), and 22% had low energy availability (<30 kcal·kg FFM·day-1). Menstrual dysfunctions were reported in 40% of participants; 15 and 25% reported oligomenorrhea and secondary amenorrhea, respectively; while 65% were classified at risk of RED-S (based on the Low Energy Availability in Females Questionnaire). All participants had adequate bone health (bone mineral density Z-score: 1.1 ± 0.9 SD), but 20% had <85% expected body weight. The observation of an energy deficit in vocational female ballet students was primarily attributed to an inability to plan energy intake and thereby meet higher energy requirements during ballet training weekdays. Screening for factors associated with RED-S and tailoring education programs to inform energy and nutrition requirements for health and training are recommended in elite young ballet students.

Effects of fish oils on ex vivo B-cell responses of obese subjects upon BCR/TLR stimulation: a pilot study.

The long-chain n-3 polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) in fish oil have immunomodulatory properties. B cells are a poorly studied target of EPA/DHA in humans. Therefore, in this pilot study, we tested how n-3 LC-PUFAs influence B-cell responses of obese humans. Obese men and women were assigned to consume four 1-g capsules per day of olive oil (OO, n=12), fish oil (FO, n=12) concentrate or high-DHA-FO concentrate (n=10) for 12 weeks in a parallel design. Relative to baseline, FO (n=9) lowered the percentage of circulating memory and plasma B cells, whereas the other supplements had no effect. There were no postintervention differences between the three supplements. Next, ex vivo B-cell cytokines were assayed after stimulation of Toll-like receptors (TLRs) and/or the B-cell receptor (BCR) to determine if the effects of n-3 LC-PUFAs were pathway-dependent. B-cell IL-10 and TNFα secretion was respectively increased with high DHA-FO (n=10), relative to baseline, with respective TLR9 and TLR9+BCR stimulation. OO (n=12) and FO (n=12) had no influence on B-cell cytokines compared to baseline, and there were no differences in postintervention cytokine levels between treatment groups. Finally, ex vivo antibody levels were assayed with FO (n=7) after TLR9+BCR stimulation. Compared to baseline, FO lowered IgM but not IgG levels accompanied by select modifications to the plasma lipidome. Altogether, the results suggest that n-3 LC-PUFAs could modulate B-cell activity in humans, which will require further testing in a larger cohort.

Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.

Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish.

Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.

Lipid Composition of Oil Extracted from Wasted Norway Lobster (Nephrops norvegicus) Heads and Comparison with Oil Extracted from Antarctic Krill (Euphasia superba).

In the UK, the Norway lobster (Nephrops norvegicus) supports its most important shellfish fishery. Nephrops are sold either whole, or as "tails-only" for the scampi trade. In the "tailing" process, the "head" (cephalothorax) is discarded as waste. A smaller crustacean species, the Antarctic krill Euphasia superba, represents an economically valuable industry, as its extractable oil is sold as a human dietary supplement. The aim of this study was to determine the amount and composition of the oil contained in discarded Nephrops heads and to compare its composition to the oil extracted from krill. Differences due to Geographical variation and seasonal patterns in the amount and composition of lipid were also noted. Results indicated that Nephrops head waste samples collected from more southern locations in Scotland (Clyde Sea area) contained higher levels of oil when compared to samples collected from northern locations in Iceland. Moreover, seasonal differences within the Clyde Sea area in Scotland were also observed, with oil extracted from Nephrops head waste peaking at around 11.5% during the summer months when larger and more mature females were caught by trawl. At this time of the year, the valuable fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) accounted for around 23% of the total fatty acid content in oil extracted from Nephrops head waste. A seasonal effect on EPA content was found, with higher levels obtained in the summer, while no trend was found in DHA percentages. Finally, oil from Nephrops head waste contained a higher proportion of EPA and DHA than krill oil but these fatty acids were more abundantly linked to the neutral lipids rather to than polar lipids. The characterization of lipid that could be extracted from Nephrops head waste should be seen as a first step for the commercial use of a valuable resource currently wasted. This approach is extremely relevant given the current limited supply of EPA and DHA and changes in the Common Fisheries Policy.

DHA Supplementation Alone or in Combination with Other Nutrients Does not Modulate Cerebral Hemodynamics or Cognitive Function in Healthy Older Adults.

A number of recent trials have demonstrated positive effects of dietary supplementation with the omega-3 polyunsaturated fatty acids (n-3 PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on measures of cognitive function in healthy young and older adults. One potential mechanism by which EPA, and DHA in particular, may exert these effects is via modulation of cerebral hemodynamics. In order to investigate the effects of DHA alone or provided as one component of a multinutrient supplement (also including Gingko biloba, phosphatidylserine and vitamins B9 and B12) on measures of cerebral hemodynamics and cognitive function, 86 healthy older adults aged 50-70 years who reported subjective memory deficits were recruited to take part in a six month daily dietary supplementation trial. Relative changes in the concentration of oxygenated hemoglobin and deoxygenated hemoglobin were assessed using Near Infrared Spectroscopy (NIRS) during the performance of cognitive tasks prior to and following the intervention period. Performance on the cognitive tasks was also assessed. No effect of either active treatment was found for any of the NIRS measures or on the cognitive performance tasks, although the study was limited by a number of factors. Further work should continue to evaluate more holistic approaches to cognitive aging.

HDAC4-myogenin axis as an important marker of HD-related skeletal muscle atrophy.

Skeletal muscle remodelling and contractile dysfunction occur through both acute and chronic disease processes. These include the accumulation of insoluble aggregates of misfolded amyloid proteins that is a pathological feature of Huntington's disease (HD). While HD has been described primarily as a neurological disease, HD patients' exhibit pronounced skeletal muscle atrophy. Given that huntingtin is a ubiquitously expressed protein, skeletal muscle fibres may be at risk of a cell autonomous HD-related dysfunction. However the mechanism leading to skeletal muscle abnormalities in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that symptomatic animals developed a progressive impairment of the contractile characteristics of the hind limb muscles tibialis anterior (TA) and extensor digitorum longus (EDL), accompanied by a significant loss of motor units in the EDL. In symptomatic animals, these pronounced functional changes were accompanied by an aberrant deregulation of contractile protein transcripts and their up-stream transcriptional regulators. In addition, HD mouse models develop a significant reduction in muscle force, possibly as a result of a deterioration in energy metabolism and decreased oxidation that is accompanied by the re-expression of the HDAC4-DACH2-myogenin axis. These results show that muscle dysfunction is a key pathological feature of HD.

Nutritional quality of salmon products available from major retailers in the UK: content and composition of n-3 long-chain PUFA.

In the present study, salmon products available from UK retailers were analysed to determine the levels of n-3 long-chain PUFA (LC-PUFA), a key determinant of nutritional quality. There was a wide variation in the proportions and absolute contents of EPA and DHA in the products. Relatively high contents of 18 : 1n-9, 18 : 2n-6 and 18 : 3n-3, characteristic of vegetable oils (VO), were found in several farmed salmon products, which also had generally lower proportions of EPA and DHA. In contrast, farmed salmon products with higher levels of 16 : 0 and 22 : 1, characteristic of fish oil (FO), had higher proportions of EPA and DHA. Therefore, there was a clear correlation between the levels of VO and FO in feeds and the proportions of n-3 LC-PUFA in products. Although wild salmon products were characterised by higher proportions of n-3 LC-PUFA (20-40%) compared with farmed fish (9-26%), they contained lower total lipid contents (1-6% compared with 7-17% in farmed salmon products). As a result, farmed salmon products invariably had higher levels of n-3 LC-PUFA in absolute terms (g/100 g fillet) and, therefore, delivered a higher 'dose' of EPA and DHA per portion. Overall, despite the finite and limiting supply of FO and increasing use of VO, farmed salmon continue to be an excellent source of and delivery system for n-3 LC-PUFA to consumers.

Temporal changes in human skeletal muscle and blood lipid composition with fish oil supplementation.

The aim of this study was to examine changes in the lipid profile of red blood cells and muscle tissue along with the expression of anabolic signalling proteins in human skeletal muscle. Following a 2-week control period, 10 healthy male participants consumed 5 g d(-1) of fish oil (FO) for 4 weeks. Muscle biopsies and venous blood samples were collected in the fasted state 2 weeks prior (W-2) and immediately before (W0) the initiation of FO supplementation for internal control. Muscle biopsies and venous blood samples were again obtained at week 1 (W1), 2 (W2) and 4 (W4) during FO supplementation for assessment of changes in lipid composition and expression of anabolic signalling proteins. There was no change in the composition of any lipid class between W-2 and W0 confirming control. Following FO supplementation n-3 polyunsaturated fatty acid (n-3 PUFA) muscle lipid composition was increased from W0 to W2 and continued to rise at W4. n-3 PUFA blood lipid composition was increased from W0 to W1 and remained elevated for the remaining time points. Total protein content of focal adhesion kinase (FAK) increased from W0 to W4 whereas total mechanistic target of rapamycin (mTOR) was increased from W0 at W1 with no further significant increases at W2 and W4. These data show that FO supplementation results in discordant changes in the n-3 PUFA composition of skeletal muscle compared to blood that is associated with increases in total FAK content.

Treatment with an antibody directed against Nogo-A delays disease progression in the SOD1G93A mouse model of Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder in which motor neurons in the spinal cord and motor cortex degenerate. Although the majority of ALS cases are sporadic, mutations in Cu-Zn superoxide dismutase-1 (SOD1) are causative for 10-20% of familial ALS (fALS), and recent findings show that a hexanucleotide repeat expansion in the C9ORF72 gene may account for >30% of fALS cases in Europe. SOD1(G93A) transgenic mice have a phenotype and pathology similar to human ALS. In both ALS patients and SOD1(G93A) mice, the first pathological features of disease manifest at the neuromuscular junction, where significant denervation occurs prior to motor neuron degeneration. Strategies aimed at preventing or delaying denervation may therefore be of benefit in ALS. In this study, we show that Nogo-A levels increase in muscle fibres of SOD1(G93A) mice along with the elevation of markers of neuromuscular dysfunction (CHRNA1/MUSK). Symptomatic treatment of SOD1(G93A) mice from 70 days of age with an anti-Nogo-A antibody (GSK577548) significantly improves hindlimb muscle innervation at 90 days, a late symptomatic stage of disease, resulting in increased muscle force and motor unit survival and a significant increase in motor neuron survival. However, not all aspects of this improvement in anti-Nogo-A antibody-treated SOD1(G93A) mice were maintained at end-stage disease. These results show that treatment with anti-Nogo-A antibody significantly improves neuromuscular function in the SOD1(G93A) mouse model of ALS, at least during the earlier stages of disease and suggest that pharmacological inhibition of Nogo-A may be a disease-modifying approach in ALS.

Molecular chaperone mediated late-stage neuroprotection in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1(G93A) transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1(G93A) and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1(G93A) aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.

Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

Using a fingertip whole blood sample for rapid fatty acid measurement: method validation and correlation with erythrocyte polar lipid compositions in UK subjects.

It is well accepted that n-3 long-chain PUFA intake is positively associated with a range of health benefits. However, while benefits have been clearly shown, especially for CVD, the mechanisms for prevention/benefit are less understood. Analysis of plasma and erythrocyte phospholipids (PL) have been used to measure the status of the highly unsaturated fatty acids (HUFA), especially EPA (20 : 5n-3) and DHA (22 : 6n-3), although the time and complexity of the process places limitations on the sample numbers analysed. An assay has been developed using whole blood, collected by finger prick, and stored on absorbant paper, subjected to direct methylation and fatty acids quantified by automated GC. Tests on fatty acid stability show that blood samples are stable when stored at - 20°C for 1 month although some loss of HUFA was seen at 4°C. A total of fifty-one patients, including twenty-seven who consumed no fatty acid supplements, provided a blood sample for analysis. Concentrations of all major fatty acids were measured in erythrocyte PL and whole blood. The major HUFA, including EPA, DHA and arachidonic acid (ARA; 20 : 4n-6), as well as the ARA:EPA ratio and the percentage n-3 HUFA/total HUFA all showed good correlations, between erythrocyte PL and whole blood. Values of r2 ranged from 0.48 for ARA to 0.95 for the percentage of n-3 HUFA/total HUFA. This assay provides a non-invasive, rapid and reliable method of HUFA quantification with the percentage of n-3 HUFA value providing a potential blood biomarker for large-scale nutritional trials.

Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: cloning, functional characterisation, and tissue distribution of an Elovl4 elongase.

The elongases of very long-chain fatty acids (Elovl) account for the rate-limiting condensation step of the elongation process in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovl family, Elovl4, has been regarded as a critical enzyme in vertebrates in the production of the so-called very long-chain fatty acids (VLC-FA), a group of compounds that has been scarcely explored in fish. Here we report on the cloning of a novel Elovl4-like elongase from Atlantic salmon (Salmo salar). The salmon Elovl4 cDNA codes for a putative protein containing 306 amino acids. Heterologous expression in yeast demonstrated that salmon Elovl4 efficiently elongated saturated FAs up to 36:0, with 24:0 and 26:0 appearing as preferred substrates. Additionally, salmon Elovl4 effectively converted C20 and C22 polyunsaturated fatty acids to elongated polyenoic products up to C36. Tissue distribution showed that Elovl4 mRNA transcripts are abundant in eye, brain and testes, suggesting that, as described in mammals, these tissues are important metabolic sites for the biosynthesis of VLC-FA. Our results are discussed in comparison with the functional analyses observed in Elovl4 proteins from other vertebrates, and also other Elovl proteins investigated previously in Atlantic salmon.

Influence of the dietary protein:lipid ratio and fish oil substitution on fatty acid composition and metabolism of Atlantic salmon (Salmo salar) reared at high water temperatures.

A factorial, two-way, experimental design was used for this 10-week nutritional trial, aiming to elucidate the interactive effects of decreasing dietary protein:lipid level and substitution of fish oil (FO) with rapeseed oil (RO) on tissue fatty acid (FA) composition and metabolism of large Atlantic salmon (Salmo salar L.) reared at high water temperatures (sub-optimal, summer temperatures: 11·6°C). The six experimental diets were isoenergetic and formulated to include either FO or RO (60 % of the added oil) at three dietary protein:lipid levels, specifically (1) 350 g/kg protein and 350 g/kg lipid, (2) 330 g/kg protein and 360 g/kg lipid, (3) 290 g/kg protein and 380 g/kg lipid. Final weight, specific growth rate and thermal growth coefficient were positively affected by the dietary RO inclusion at the expense of FO, while no significant effects were seen on growth due to the decreasing protein level. The oil source had a significant effect on muscle and liver FA composition. However, the changes in muscle and liver FA indicate selective utilisation or retention of individual FA and moderate reductions in tissue EPA and DHA. Pyloric caeca phospholipid FA composition was significantly affected by the two factors and, in some cases, significant interactions were also revealed. Liver and red muscle ß-oxidation capacities were significantly increased due to RO inclusion, while an interactive effect of protein level and oil source was shown for white muscle ß-oxidation capacity. The results could explain, at least partially, the better performance that was shown for the RO groups and the enhanced protein-sparing effect.

Vertebrate fatty acyl desaturase with Δ4 activity.

Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 182n - 6 to produce 183n - 6 that is elongated to 203n - 6 followed by Δ5 desaturation. Synthesis of EPA from 183n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 225n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 225n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above.