Post-prandial tracer studies of protein and amino acid utilisation: what can they tell us about human amino acid and protein requirements?

Nitrogen balance (NB), the principal methodology used to derive recommendations for human protein and amino acid requirements, has been widely criticised, and calls for increased protein and amino acid requirement recommendations have been made, often on the basis of post-prandial amino acid tracer kinetic studies of muscle protein synthesis, or of amino acid oxidation. This narrative review considers our knowledge of the homeostatic regulation of the FFM throughout the diurnal cycle of feeding and fasting and what can and has been learnt from post-prandial amino acid tracer studies, about amino acid and protein requirements. Within the FFM, muscle mass in well fed weight-stable adults with healthy lifestyles appears fixed at a phenotypic level within a wide range of habitual protein intakes. However homoeostatic regulation occurs in response to variation in habitual protein intake, with adaptive changes in amino acid oxidation which influence the magnitude of diurnal losses and gains of body protein. Post-prandial indicator amino acid oxidation (IAAO) studies have been introduced as an alternative to NB and to the logistically complex 24 h [13C-1] amino acid balance studies, for assessment of protein and amino acid requirements. However, a detailed examination of IAAO studies shows both a lack of concern for homeostatic regulation of amino acid oxidation and  major flaws in their design and analytical interpretation, which seriously constrain their ability to provide reliable values. New ideas and a much more critical approach to existing work is needed if real progress is to be made in the area.

Post-natal muscle growth and protein turnover: a narrative review of current understanding.

A model explaining the dietary-protein-driven post-natal skeletal muscle growth and protein turnover in the rat is updated, and the mechanisms involved are described, in this narrative review. Dietary protein controls both bone length and muscle growth, which are interrelated through mechanotransduction mechanisms with muscle growth induced both from stretching subsequent to bone length growth and from internal work against gravity. This induces satellite cell activation, myogenesis and remodelling of the extracellular matrix, establishing a growth capacity for myofibre length and cross-sectional area. Protein deposition within this capacity is enabled by adequate dietary protein and other key nutrients. After briefly reviewing the experimental animal origins of the growth model, key concepts and processes important for growth are reviewed. These include the growth in number and size of the myonuclear domain, satellite cell activity during post-natal development and the autocrine/paracrine action of IGF-1. Regulatory and signalling pathways reviewed include developmental mechanotransduction, signalling through the insulin/IGF-1-PI3K-Akt and the Ras-MAPK pathways in the myofibre and during mechanotransduction of satellite cells. Likely pathways activated by maximal-intensity muscle contractions are highlighted and the regulation of the capacity for protein synthesis in terms of ribosome assembly and the translational regulation of 5-TOPmRNA classes by mTORC1 and LARP1 are discussed. Evidence for and potential mechanisms by which volume limitation of muscle growth can occur which would limit protein deposition within the myofibre are reviewed. An understanding of how muscle growth is achieved allows better nutritional management of its growth in health and disease.

The application of stable-isotope tracers to study human musculoskeletal protein turnover: a tale of bag filling and bag enlargement.

The nutritional regulation of protein and amino acid balance in human skeletal muscle carried out by the authors with Mike Rennie is reviewed in the context of a simple physiological model for the regulation of the maintenance and growth of skeletal muscle, the "Bag Theory". Beginning in London in the late 1970s the work has involved the use of stable isotopes to probe muscle protein synthesis and breakdown with two basic experimental models, primed-dose continuous tracer infusions combined with muscle biopsies and arterio-venous (A-V) studies across a limb, most often the leg, allowing both protein synthesis and breakdown as well as net balance to be measured. In this way, over a 30 year period, the way in which amino acids and insulin mediate the anabolic effect of a meal has been elaborated in great detail confirming the original concepts of bag filling within the muscle endomysial "bag", which is limited by the "bag" size unless bag enlargement occurs requiring new collagen synthesis. Finally we briefly review some new developments involving 2 H2 O labelling of muscle proteins.

Nutrition, infection and stunting: the roles of deficiencies of individual nutrients and foods, and of inflammation, as determinants of reduced linear growth of children.

The regulation of linear growth by nutritional and inflammatory influences is examined in terms of growth-plate endochondral ossification, in order to better understand stunted growth in children. Linear growth is controlled by complex genetic, physiological, and nutrient-sensitive endocrine/paracrine/autocrine mediated molecular signalling mechanisms, possibly including sleep adequacy through its influence on growth hormone secretion. Inflammation, which accompanies most infections and environmental enteric dysfunction, inhibits endochondral ossification through the action of mediators including proinflammatory cytokines, the activin A-follistatin system, glucocorticoids and fibroblast growth factor 21 (FGF21). In animal models linear growth is particularly sensitive to dietary protein as well as Zn intake, which act through insulin, insulin-like growth factor-1 (IGF-1) and its binding proteins, triiodothyronine, amino acids and Zn2+ to stimulate growth-plate protein and proteoglycan synthesis and cell cycle progression, actions which are blocked by corticosteroids and inflammatory cytokines. Observational human studies indicate stunting to be associated with nutritionally poor, mainly plant-based diets. Intervention studies provide some support for deficiencies of energy, protein, Zn and iodine and for multiple micronutrient deficiencies, at least during pregnancy. Of the animal-source foods, only milk has been specifically and repeatedly shown to exert an important influence on linear growth in both undernourished and well-nourished children. However, inflammation, caused by infections, environmental enteric dysfunction, which may be widespread in the absence of clean water, adequate sanitation and hygiene (WASH), and endogenous inflammation associated with excess adiposity, in each case contributes to stunting, and may explain why nutritional interventions are often unsuccessful. Current interventions to reduce stunting are targeting WASH as well as nutrition.

Sex differences in the composition of weight gain and loss in overweight and obese adults.

Sex differences in the ratio of fat mass (FM):fat-free mass (FFM) during weight change should differentially affect the extent of weight change during energy imbalance in men and women. In the present study, we determined FM and FFM contents by dual-energy X-ray absorptiometry and calculated the P-ratios (protein energy/total energy) of excess weight and weight loss during a randomised controlled trial of four commercial weight loss regimens. Overweight and obese women (n 210) and men (n 77) were studied at baseline and at 2 and 6 months during weight loss on four dietary regimens: Dr Atkins' New Diet Revolution; The Slim-Fast Plan; Weight-Watchers programme; Rosemary Conley's Diet and Fitness Plan. At baseline, the percentage of FFM (%FFM) and P-ratios of excess weight were 40 % and 0·071 for men and 27 % and 0·039 for women. At 2 months, men had lost twice as much weight as women and three times more FFM than women, indicating higher FFM content and P-ratios of weight loss for men, 0·052, than for women, 0·029, with no dietary effects. Between 2 and 6 months, the rate at which weight was lost decreased and the %FFM of weight loss decreased to similar low levels in men (7 %) and women (5 %): i.e. P-ratios of 0·009 and 0·006, respectively, with no dietary effects. Thus, for men compared with women, there were greater FFM content and P-ratios of weight change, which could partly, but not completely, explain their greater weight loss at 2 months. However, protein-conserving adaptations occur with increasing weight loss and over time, more extensively in men, eventually eliminating any sex difference in the composition of weight loss.

Energy balance and obesity: a UK perspective on the gluttony v. sloth debate.

Obesity in the UK was assumed to have developed against a population decline in physical activity, with health messages focused on diet and exercise prevention strategies. Doubly-labelled water (DLW) studies of energy expenditure have indicated the alternative scenario that the increased obesity prevalence reflects excessive food energy intake with physical activity levels unchanged. This analysis is questionable, deriving in part from a weakness of the DLW methodology in identifying changing physical activity levels within populations of increasing body weight. This has resulted in an underestimation of the reduction in physical activity in the overweight and obese, as revealed by direct studies of such behaviour. Furthermore, a close examination of food energy supply, household food purchases and individual food energy consumption since 1955, in relation to likely estimates of current intakes indicated by simple modelling of predicted energy expenditure, identifies: (a) food energy supply as markedly overestimating energy intakes; (b) individual food energy consumption as markedly underestimating energy intakes; and (c) household food purchase data as the closest match to predicted current food energy intakes. Energy intakes indicated by this latter method have fallen by between 20 to 30%, suggesting comparable falls in physical activity. Although unequivocal evidence for a matching UK trend in falling physical activity is limited, as is evidence that obesity follows reductions in physical activity, such a link has been recently suggested in a large prospective study in adolescents. Thus, for the UK, obesity has developed within a 'move less-eat somewhat less but still too much' scenario. A focus on both diet and exercise should remain the appropriate public health policy.

A new approach to establishing dietary energy reference values.

PURPOSE OF REVIEW: To describe the methodology behind the new UK Dietary Recommendations for Energy. RECENT FINDINGS: Large interindividual variation in discretionary activity prevents prediction of energy expenditure, [total energy expenditure (TEE)], as a function of physical activity levels (PALs), [e.g. TEE = PAL × basal metabolic rate (BMR)] with the previously assumed accuracy. An alternative simplified approach derives PAL values in terms of population median and values for those exhibiting less or more activity (25th and 75th centiles) from suitable reference doubly labelled water (DLW) data. DLW data sets were used to identify PAL values: for adults, from two large randomized studies of urban populations (n = 929), and for children (aged 1-3, 3 to less than 10 and 10-18 years), from all published studies reporting mean values (n = 170 studies, 2502 patients) from which PAL values were reported or could be derived. Values for TEE were then derived from anthropometric determinants of BMR and PAL, and prescriptive estimated average requirement, (EAR) values were calculated on the basis of healthy body weights: BMI = 22.5 kg/m for adults and reference growth data for children. Energy costs of tissue energy deposition were modelled as a simple 1% increase in PAL values for children within the three age groups. SUMMARY: The model for calculation of the EAR is simplified and widely applicable in most population settings.

Knowledge gained from studies of leucine consumption in animals and humans.

Leucine's wide-ranging metabolic influences have made it subject to special interest. It is abundant in the diet, especially in some milk and cereal proteins, in part due to its allocation of 6 codons in the genetic code, and individual dietary intakes range up to >250 mg · kg(-1) · d(-1). It influences many cell functions by various mechanisms, which include allosteric activation of enzymes, enabling ATP generation and insulin secretion from the pancreatic islet cell, and activation of signaling pathways. It is a mediator of the anabolic drive of dietary amino acids, stimulating anabolic hormone secretion and directly signaling protein deposition and growth through the stimulation of protein synthesis and restraint of proteolysis. Its signaling may involve the mammalian target of rapamycin complex and rapamycin-insensitive pathways responding to a leucine "transceptor," which combines leucine cellular transport, fueled by the intracellular-extracellular glutamine gradient, and a signaling response to changes in ionic and water balance and cell volume. In animal studies, dietary leucine supplementation has reversed many of the adverse influences of a high-fat diet, consistent with a benefit for healthy weight maintenance in humans for which evidence is accumulating. The implications for safety of leucine-supplemented diets are discussed in terms of adversely lowering valine and isoleucine concentrations and inducing hyperammonemia through overloading peripheral glutamine synthetic pathways. Finally, the apparently high human leucine requirement is explained in terms of both an adaptive metabolic demand model of requirements and the design and analysis of human studies, which may overestimate values.

Identifying recommended dietary allowances for protein and amino acids: a critique of the 2007 WHO/FAO/UNU report.

The WHO/FAO/UNU (2007) report examines dietary protein and amino acid requirements for all age groups, protein requirements during pregnancy, lactation and catch-up growth in children, the implications of these requirements for developing countries and protein quality evaluation. Requirements were defined as the minimum dietary intake which satisfies the metabolic demand and achieves nitrogen equilibrium and maintenance of the body protein mass, plus the needs for growth in children and pregnancy and lactation in healthy women. Insufficient evidence was identified to enable recommendations for specific health outcomes. A meta analysis of nitrogen balance studies identifies protein requirements for adults 10 % higher than previous values with no influence of gender or age, consistent with a subsequently published comprehensive study. A new factorial model for infants and children, validated on the basis of the adequacy of breast milk protein intakes and involving a lower maintenance requirement value, no provision for saltatory growth and new estimates of protein deposition identifies lower protein requirements than in previous reports. Higher values for adult amino acid requirements, derived from a re-evaluation of nitrogen balance studies and new stable isotope studies, identify some cereal-based diets as being inadequate for lysine. The main outstanding issues relate to the biological implausibility of the very low efficiencies of protein utilisation used in the factorial models for protein requirements for all population groups especially pregnancy when requirements may be overestimated. Also considerable uncertainty remains about the design and interpretation of most of the studies used to identify amino acid requirement values.

Amino acid scoring patterns for protein quality assessment.

The 1985 FAO/WHO/UNU protein report defined reference amino acid patterns for infants based on breast milk and for preschool children, schoolchildren and adults from age specific estimates of dietary indispensible amino acid requirements divided by the safe protein requirement for each age group. This report argued that the protein quality of a diet should be estimated from its digestibility adjusted by its amino acid score calculated from its limiting amino acid in comparison with the reference amino acid pattern. Subsequently a joint FAO/WHO expert consultation on protein quality evaluation (1991) endorsed this protein digestibility-corrected score approach. However it rejected the adult scoring pattern identified in the 1985 report arguing that the amino acid values for this pattern were too low. As an interim measure it suggested that the scoring pattern for preschool children should be used for all age groups apart from infants. The recent WHO/FAO/UNU (2007) report endorsed the 1985 report in recommending the amino acid content of breast milk as the best estimate of infant amino acid requirements. However it was only able to identify reliable requirement values for adults and adopted a factorial approach to derivation of age-related scoring patterns. This utilized the adult pattern for maintenance, and the pattern of human tissue protein for growth. Thus scoring patterns were derived for children aged 0·5, 1-2, 3-10, 11-14, 15-18 years and for adults. The total dietary amino acid requirements calculated for these age groups were divided by the mean protein requirement to give the scoring pattern which should be used to adjust digestible intakes to identify the available protein in specific diets. However because the adult values were determined in subjects at protein intakes much higher than the mean minimum protein requirement, i.e. at 1 g/kg/d rather than 0·66 g/kg/d, the pattern is likely to include higher values than the minimum requirement and should therefore be referenced against the safe allowance.

Interactions between Growth of Muscle and Stature: Mechanisms Involved and Their Nutritional Sensitivity to Dietary Protein: The Protein-Stat Revisited.

Childhood growth and its sensitivity to dietary protein is reviewed within a Protein-Stat model of growth regulation. The coordination of growth of muscle and stature is a combination of genetic programming, and of two-way mechanical interactions involving the mechanotransduction of muscle growth through stretching by bone length growth, the core Protein-Stat feature, and the strengthening of bone through muscle contraction via the mechanostat. Thus, growth in bone length is the initiating event and this is always observed. Endocrine and cellular mechanisms of growth in stature are reviewed in terms of the growth hormone-insulin like growth factor-1 (GH-IGF-1) and thyroid axes and the sex hormones, which together mediate endochondral ossification in the growth plate and bone lengthening. Cellular mechanisms of muscle growth during development are then reviewed identifying (a) the difficulties posed by the need to maintain its ultrastructure during myofibre hypertrophy within the extracellular matrix and the concept of muscle as concentric "bags" allowing growth to be conceived as bag enlargement and filling, (b) the cellular and molecular mechanisms involved in the mechanotransduction of satellite and mesenchymal stromal cells, to enable both connective tissue remodelling and provision of new myonuclei to aid myofibre hypertrophy and (c) the implications of myofibre hypertrophy for protein turnover within the myonuclear domain. Experimental data from rodent and avian animal models illustrate likely changes in DNA domain size and protein turnover during developmental and stretch-induced muscle growth and between different muscle fibre types. Growth of muscle in male rats during adulthood suggests that "bag enlargement" is achieved mainly through the action of mesenchymal stromal cells. Current understanding of the nutritional regulation of protein deposition in muscle, deriving from experimental studies in animals and human adults, is reviewed, identifying regulation by amino acids, insulin and myofibre volume changes acting to increase both ribosomal capacity and efficiency of muscle protein synthesis via the mechanistic target of rapamycin complex 1 (mTORC1) and the phenomenon of a "bag-full" inhibitory signal has been identified in human skeletal muscle. The final section deals with the nutritional sensitivity of growth of muscle and stature to dietary protein in children. Growth in length/height as a function of dietary protein intake is described in the context of the breastfed child as the normative growth model, and the "Early Protein Hypothesis" linking high protein intakes in infancy to later adiposity. The extensive paediatric studies on serum IGF-1 and child growth are reviewed but their clinical relevance is of limited value for understanding growth regulation; a role in energy metabolism and homeostasis, acting with insulin to mediate adiposity, is probably more important. Information on the influence of dietary protein on muscle mass per se as opposed to lean body mass is limited but suggests that increased protein intake in children is unable to promote muscle growth in excess of that linked to genotypic growth in length/height. One possible exception is milk protein intake, which cohort and cross-cultural studies suggest can increase height and associated muscle growth, although such effects have yet to be demonstrated by randomised controlled trials.

Dietary protein and bone health: towards a synthesised view.

The present paper reviews published literature on the relationship between dietary protein and bone health. It will include arguments both for and against the anabolic and catabolic effects of dietary protein on bone health. Adequate protein intake provides the amino acids used in building and maintaining bone tissue, as well as stimulating the action of insulin-like growth factor 1, which in turn promotes bone growth and increases calcium absorption. However, the metabolism of dietary sulphur amino acids, mainly from animal protein, can lead to increased physiological acidity, which may be detrimental for bone health in the long term. Similarly, cereal foods contain dietary phytate, which in turn contains phosphate. It is known that phosphate consumption can also lead to increased physiological acidity. Therefore, cereal products may produce as much acid as do animal proteins that contain sulphur amino acids. The overall effect of dietary protein on physiological acidity, and its consequent impact on bone health, is extremely complex and somewhat controversial. The consensus is now moving towards a synthesised approach. Particularly, how anabolic and catabolic mechanisms interact; as well as how the context of the whole diet and the type of protein consumed is important.

Estimates of daily net endogenous acid production in the elderly UK population: analysis of the National Diet and Nutrition Survey (NDNS) of British adults aged 65 years and over.

Dietary intake has been shown to influence acid-base balance in human subjects under tightly controlled conditions. However, the net effect of food groups on alkali/acid loading in population groups is unclear. The aims of the present study were to: (1) quantify estimates of daily net endogenous acid production (NEAP) (mEq/d) in a representative group of British elderly aged 65 years and older; (2) compare and characterise NEAP by specific nutrients and food groups likely to influence dietary acid loading; (3) determine whether geographical location influenced NEAP. The National Diet and Nutrition Survey dataset, consisting of a 4 d weighed record and anthropometric data, was used to estimate dietary acidity. Dietary under-reporters were excluded by analysing only subjects with energy intakes >/= 1.2 x BMR. NEAP was estimated as the dietary potential renal acid load+organic acid excretion, the latter as a multiple of estimated body surface area. NEAP was lower in women compared with men (P < 0.001), and lower than values reported in a Swedish elderly cohort. Lower dietary acidity was significantly associated with higher consumption of fruit and potatoes and lower consumption of meat, bread and eggs (P < 0.02 to P < 0.001). Lower intakes of fish and cheese were associated with lower NEAP in men only (P < 0.01 to P < 0.001). There were regional differences for NEAP, with higher intakes in Scotland/Northern regions compared with Central/South-Western and London/South-Eastern regions (P = 0.01). These data provide an insight into the acid-generating potential of the diet in the British elderly population, which may have important consequences in this vulnerable group.

Effect of varying the ratio of n-6 to n-3 fatty acids by increasing the dietary intake of alpha-linolenic acid, eicosapentaenoic and docosahexaenoic acid, or both on fibrinogen and clotting factors VII and XII in persons aged 45-70 y: the OPTILIP study.

BACKGROUND: Elevated fibrinogen, activated factor XII (FXIIa), and factor VII coagulant activity (FVIIc) are associated with higher risk of fatal ischemic heart disease. This study tested the hypothesis that lowering the dietary ratio of n-6 to n-3 polyunsaturated fatty acids (n-6:n-3) would modify these risk factors in older men and women. OBJECTIVE: The objective of the study was to measure fasting hemostatic risk factors and postprandial changes in activated FVII (FVIIa) concentrations after a 6-mo alteration in dietary n-6:n-3. DESIGN: In a randomized, parallel design in 258 subjects aged 45-70 y, we compared 4 diets providing 6% of energy as polyunsaturated fatty acids at an n-6:n-3 between 5:1 and 3:1 with a control diet that had an n-6:n-3 of 10:1. The diets were enriched in alpha-linolenic acid, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, or both. RESULTS: Fasting and 3-h plasma triacylglycerol concentrations were 11.1% and 7.2% lower with the diet that had an n-6:n-3 of approximately 3:1 and that was enriched with EPA and DHA than with the other diets. Fasting fibrinogen, FXIIa, FVIIc, FVIIa, and FVII antigen and postprandial FVIIa were not influenced by the diets. Avoiding foods high in fat the day before measurement decreased FVIIc and FVIIa by 8% and 19.2%, respectively. A test meal containing 50 g fat resulted in a mean 47% (95% CI: 42%, 52%) increase in FVIIa 6 h later, but the response did not differ by n-6:n-3. CONCLUSION: Decreasing the n-6:n-3 to approximately 3:1 by increasing the intake of EPA and DHA lowers fasting and postprandial plasma triacylglycerol concentrations in older persons but does not influence hemostatic risk factors.

Effects of altering the ratio of dietary n-6 to n-3 fatty acids on insulin sensitivity, lipoprotein size, and postprandial lipemia in men and postmenopausal women aged 45-70 y: the OPTILIP Study.

BACKGROUND: Insulin resistance is associated with elevated plasma triacylglycerol, low HDL concentrations, elevated postprandial lipemia, and a predominance of small, dense LDLs (sdLDLs). It has been hypothesized that the dietary ratio of n-6 to n-3 (n-6:n-3) polyunsaturated fatty acids (PUFAs) may have favorable effects on these risk factors by increasing insulin sensitivity. OBJECTIVE: The objective was to measure changes in insulin sensitivity, lipoprotein size, and postprandial lipemia after a 6-mo alteration in n-6:n-3. DESIGN: In a randomized, parallel design in 258 subjects aged 45-70 y, we compared 4 diets providing 6% of energy as PUFAs with an n-6:n-3 between 5:1 and 3:1 with a control diet that had an n-6:n-3 of 10:1. The diets were enriched in alpha-linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), or both. Insulin sensitivity was assessed with the homeostatic model assessment of insulin resistance and the revised quantitative insulin sensitivity test. RESULTS: Dietary intervention did not influence insulin sensitivity or postprandial lipase activities. Fasting and postprandial triacylglycerol concentrations were lower, and the proportion of sdLDLs decreased (by 12.7%; 95% CI: -22.9%, 2.4%), with an n-6:n-3 of approximately 3:1, which was achieved by the addition of long-chain n-3 PUFAs (EPA and DHA). CONCLUSIONS: Decreasing the n-6:n-3 does not influence insulin sensitivity or lipase activities in older subjects. The reduction in plasma triacylglycerol after an increased intake of n-3 long-chain PUFAs results in favorable changes in LDL size.

Influence of alpha-linolenic acid and fish-oil on markers of cardiovascular risk in subjects with an atherogenic lipoprotein phenotype.

We tested the hypothesis that dietary alpha-linolenic acid (ALA) can exert effects on markers of cardiovascular risk similar to that produced by its longer chain counterparts in fish-oil. A dietary intervention study was undertaken to examine the effects of an ALA-enriched diet in 57 men expressing an atherogenic lipoprotein phenotype (ALP). Subjects were randomly assigned to one of three diets enriched either with flaxseed oil (FXO: high ALA, n = 21), sunflower oil (SO: high linoleic acid, n = 17), or SO with fish-oil (SOF n = 19) for 12 weeks, resulting in dietary intake ratios of n-6:n-3 PUFA of 0.5, 27.9 and 5.2, respectively. The relative abundance of ALA and EPA in erythrocyte membranes increased on the FXO diet (p < 0.001), whereas both EPA and DHA increased after fish-oil (p < 0.001). There were significant decreases in total plasma cholesterol within (FXO -12.3%, p = 0.001; SOF -7.6%, p = 0.014; SO -7.3%, p = 0.033) and between diets (p = 0.019), and decreases within diets after 12 weeks for HDL cholesterol on flaxseed oil (FXO -10%, p=0.009), plasma TG (-23%, p < 0.001) and small, dense LDL (-22% p = 0.003) in fish-oil. Membrane DHA levels were inversely associated with the changes in plasma TG ( p= 0.001) and small, dense LDL (p<0.05) after fish-oil. In conclusion, fish-oil produced predictable changes in plasma lipids and small, dense LDL (sdLDL) that were not reproduced by the ALA-enriched diet. Membrane DHA levels appeared to be an important determinant of these fish-oil-induced effects.