Effect of Omega-3 Dosage on Cardiovascular Outcomes: An Updated Meta-Analysis and Meta-Regression of Interventional Trials.

OBJECTIVES: To quantify the effect of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on cardiovascular disease (CVD) prevention and the effect of dosage. METHODS: This study is designed as a random effects meta-analysis and meta-regression of randomized control trials with EPA/DHA supplementation. This is an update and expanded analysis of a previously published meta-analysis which covers all randomized control trials with EPA/DHA interventions and cardiovascular outcomes published before August 2019. The outcomes included are myocardial infarction (MI), coronary heart disease (CHD) events, CVD events (a composite of MI, angina, stroke, heart failure, peripheral arterial disease, sudden death, and non-scheduled cardiovascular surgical interventions), CHD mortality and fatal MI. The strength of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation framework. RESULTS: A total of 40 studies with a combined 135,267 participants were included. Supplementation was associated with reduced risk of MI (relative risk [RR], 0.87; 95% CI, 0.80 to 0.96), high certainty number needed to treat (NNT) of 272; CHD events (RR, 0.90; 95% CI, 0.84 to 0.97), high certainty NNT of 192; fatal MI (RR, 0.65; 95% CI, 0.46 to 0.91]), moderate certainty NNT = 128; and CHD mortality (RR, 0.91; 95% CI, 0.85 to 0.98), low certainty NNT = 431, but not CVD events (RR, 0.95; 95% CI, 0.90 to 1.00). The effect is dose dependent for CVD events and MI. CONCLUSION: Cardiovascular disease remains the leading cause of death worldwide. Supplementation with EPA and DHA is an effective lifestyle strategy for CVD prevention, and the protective effect probably increases with dosage.

Associations of Sedentary Behavior and Abdominal Muscle Density: The Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Sedentary behaviors (SB) may exacerbate loss of muscle mass and function, independent of physical activity levels. This study examined the associations of SB with abdominal muscle area and density, a marker of muscle quality, in adults. METHODS: A total of 1895 participants from the Multi-Ethnic Study of Atherosclerosis completed detailed health history, physical activity and SB questionnaires, computed tomography to quantify body composition, and measurements of inflammatory markers. Analyses included linear and nonlinear regression. RESULTS: The mean age and body mass index were 64.6 years and 28 kg·m-2, respectively, and 50% were women. On average, participants engaged in 28 metabolic equivalent hours·week-1 of SB. With adjustment for age, sex, race/ethnicity, physical activity, cardiovascular disease risk factors, and inflammation, multivariable regression modeling revealed a nonlinear (quadratic) relationship between SB and locomotor, stability, and total abdominal muscle density (P < .01) but not muscle area. The SB inflection point at which locomotor, stability, and total abdominal muscle density began to decrease was 38.2, 39.6, and 39.2 metabolic equivalent hours·week-1 of SB, respectively. CONCLUSIONS: SB is associated with reduced muscle density when practiced as little as 5.5 metabolic equivalent hours·day-1. These findings may have important implications for SB guidelines for targeting skeletal muscle health in older adults.

An Exploration of the Spatiotemporal and Demographic Patterns of Ebola Virus Disease Epidemic in West Africa Using Open Access Data Sources.

The purpose of this study was to investigate the utility of exploratory analytical techniques using publically available data in informing interventions in case of infectious diseases outbreaks. More exactly spatiotemporal and multivariate methods were used to characterize the dynamics of the Ebola Virus Disease (EVD) epidemic in West Africa, and propose plausible relationships with demographic/social risk factors. The analysis showed that there was significant spatial, temporal, and spatiotemporal dependence in the evolution of the disease. For the first part of the epidemic, the cases were highly clustered in a few administrative units, in the proximity of the point of origin of the outbreak, possibly offering the opportunity to stop the spread of the disease. Later in the epidemic, high clusters were observed, but only in Liberia and Sierra Leone. Although not definitely factors of risk, in the setting in which the epidemic arose, our analysis suggests infrastructure, access to and use of health services, and connectivity possibly accelerated and magnified the spread of EVD. Also, the spatial, temporal, and spatiotemporal patterns of epidemic can be clearly shown - with evident application in the early stages of management of epidemics. In particular, we found that the spatial-temporal analytic tool SaTScan may be used effectively during the evolution of an epidemic to identify areas for targeted intervention. In the case of EVD epidemic in West Africa, better data and integration local knowledge and customs may have been more useful to recognize the proper response.

Associations between physical activity, resilience, and quality of life in people with inflammatory bowel disease.

AIM: Research has shown that moderate-to-vigorous physical activity (MVPA) is associated with higher health-related quality of life (HRQOL) in healthy individuals. Recent studies have suggested that low- to moderate-intensity physical activity can be beneficial to HRQOL in people with inflammatory bowel diseases (IBD); however, studies investigating associations between MVPA and HRQOL in this population are lacking. PURPOSE: To understand the relationships among walking, MVPA, resilience, and HRQOL in people with IBD. METHODS: People with IBD (n = 242) completed questions about physical activity, resilience and HRQOL. Pearson product-moment correlations and multiple regression analyses were used to identify associations between physical activity and HRQOL. Analysis of covariance was used to compare HRQOL over quartiles of walking and MVPA with demographic variables as covariates. RESULTS: Both walking and MVPA were independently associated with physical (ß = 0.21 and ß = 0.26, respectively; p ≤ 0.001) but not mental HRQOL (p > 0.05). Higher volumes of MVPA were significantly associated with physical HRQOL (quartile 1 40.3 ± 9.0 vs. quartile 4 47.4 ± 9.0; p < 0.001) while higher volumes of walking were associated with both physical and mental HRQOL (p ≤ 0.01). CONCLUSIONS: The findings suggest that engaging in higher volumes of MVPA above 150 min/week and walking, particularly above 60 min/week, are associated with improved HRQOL in people with IBD. Research would benefit from investigating participation in MVPA as a coping strategy, in a longitudinal manner, to determine which modes of activity may be most beneficial to people with IBD.

Mobility and Upright Posture Are Associated with Different Aspects of Cognition in Older Adults.

Objectives: Aging is associated with cognitive decline, including visuomotor and memory concerns, and with motor system changes, including gait slowing and stooped posture. We investigated the associations of visuomotor performance and episodic memory with motor system characteristics in healthy older adults. Methods: Neurologically healthy older adults (N = 160, aged 50-89) completed a battery of cognitive and motor tasks. Cognitive variables were grouped by principal components analysis (PCA) into two components: visuomotor performance and verbal episodic memory. Our primary predictor variables were two aspects of motor function: timed-up-and-go (TUG) speed and neck angle. Additional predictor variables included demographic factors (age, sex and education) and indicators of physical fitness (body mass index/BMI and grip strength). All seven predictor variables were entered stepwise into a multiple regression model for each cognitive component. Results: Poor visuomotor performance was best predicted by a combination of advanced age, high BMI and slow TUG, whereas poor verbal memory performance was best predicted by a combination of advanced age, male sex, low education and acute neck angle. Conclusions: Upright posture and mobility were associated with different cognitive processes, suggesting different underlying neural mechanisms. These results provide the first evidence for a link between postural alignment and cognitive functioning in healthy older adults. Possible causal relationships are discussed.

Availability of treatment drives decisions of genetic health professionals about disclosure of incidental findings.

Contrasting opinions exist regarding the disclosure of incidental findings detected through clinical genomic testing. This study used a discrete choice experiment to investigate genetic health professionals' preferences for the disclosure of incidental findings in an Australian paediatric setting. Four attributes of conditions relating to incidental findings were investigated: availability of prevention and treatment, chance of symptoms ever developing, age of onset and severity. Questionnaires from 59 Australian genetic health professionals were analysed. Results show that when evaluating incidental findings for disclosure, these professionals value the availability of prevention and treatment for the condition above all other characteristics included in the study. The framework of this discrete choice experiment can be used to investigate the preferences of other stakeholders such as paediatricians and parents about disclosure of incidental findings. The results of this study may be considered when assessing which categories of incidental findings are most suitable for disclosure in clinical practice.

Generalized linear models with coarsened covariates: a practical Bayesian approach.

Coarsened covariates are a common and sometimes unavoidable phenomenon encountered in statistical modeling. Covariates are coarsened when their values or categories have been grouped. This may be done to protect privacy or to simplify data collection or analysis when researchers are not aware of their drawbacks. Analyses with coarsened covariates based on ad hoc methods can compromise the validity of inferences. One valid method for accounting for a coarsened covariate is to use a marginal likelihood derived by summing or integrating over the unknown realizations of the covariate. However, algorithms for estimation based on this approach can be tedious to program and can be computationally expensive. These are significant obstacles to their use in practice. To overcome these limitations, we show that when expressed as a Bayesian probability model, a generalized linear model with a coarsened covariate can be posed as a tractable missing data problem where the missing data are due to censoring. We also show that this model is amenable to widely available general-purpose software for simulation-based inference for Bayesian probability models, providing researchers a very practical approach for dealing with coarsened covariates.

Dietary fat and not calcium supplementation or dairy product consumption is associated with changes in anthropometrics during a randomized, placebo-controlled energy-restriction trial.

UNLABELLED: Insufficient calcium intake has been proposed to cause unbalanced energy partitioning leading to obesity. However, weight loss interventions including dietary calcium or dairy product consumption have not reported changes in lipid metabolism measured by the plasma lipidome. METHODS: The objective of this study was to determine the relationships between dairy product or supplemental calcium intake with changes in the plasma lipidome and body composition during energy restriction. A secondary objective of this study was to explore the relationships among calculated macronutrient composition of the energy restricted diet to changes in the plasma lipidome, and body composition during energy restriction. Overweight adults (n = 61) were randomized into one of three intervention groups including a deficit of 500kcal/d: 1) placebo; 2) 900 mg/d calcium supplement; and 3) 3-4 servings of dairy products/d plus a placebo supplement. Plasma fatty acid methyl esters of cholesterol ester, diacylglycerol, free fatty acids, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine and triacylglycerol were quantified by capillary gas chromatography. RESULTS: After adjustments for energy and protein (g/d) intake, there was no significant effect of treatment on changes in weight, waist circumference or body composition. Plasma lipidome did not differ among dietary treatment groups. Stepwise regression identified correlations between reported intake of monounsaturated fat (% of energy) and changes in % lean mass (r = -0.44, P < 0.01) and % body fat (r = 0.48, P < 0.001). Polyunsaturated fat intake was associated with the % change in waist circumference (r = 0.44, P < 0.01). Dietary saturated fat was not associated with any changes in anthropometrics or the plasma lipidome. CONCLUSIONS: Dairy product consumption or calcium supplementation during energy restriction over the course of 12 weeks did not affect plasma lipids. Independent of calcium and dairy product consumption, short-term energy restriction altered body composition. Reported dietary fat composition of energy restricted diets was associated with the degree of change in body composition in these overweight and obese individuals.

Enteric microbiome metabolites correlate with response to simvastatin treatment.

Although statins are widely prescribed medications, there remains considerable variability in therapeutic response. Genetics can explain only part of this variability. Metabolomics is a global biochemical approach that provides powerful tools for mapping pathways implicated in disease and in response to treatment. Metabolomics captures net interactions between genome, microbiome and the environment. In this study, we used a targeted GC-MS metabolomics platform to measure a panel of metabolites within cholesterol synthesis, dietary sterol absorption, and bile acid formation to determine metabolite signatures that may predict variation in statin LDL-C lowering efficacy. Measurements were performed in two subsets of the total study population in the Cholesterol and Pharmacogenetics (CAP) study: Full Range of Response (FR), and Good and Poor Responders (GPR) were 100 individuals randomly selected from across the entire range of LDL-C responses in CAP. GPR were 48 individuals, 24 each from the top and bottom 10% of the LDL-C response distribution matched for body mass index, race, and gender. We identified three secondary, bacterial-derived bile acids that contribute to predicting the magnitude of statin-induced LDL-C lowering in good responders. Bile acids and statins share transporters in the liver and intestine; we observed that increased plasma concentration of simvastatin positively correlates with higher levels of several secondary bile acids. Genetic analysis of these subjects identified associations between levels of seven bile acids and a single nucleotide polymorphism (SNP), rs4149056, in the gene encoding the organic anion transporter SLCO1B1. These findings, along with recently published results that the gut microbiome plays an important role in cardiovascular disease, indicate that interactions between genome, gut microbiome and environmental influences should be considered in the study and management of cardiovascular disease. Metabolic profiles could provide valuable information about treatment outcomes and could contribute to a more personalized approach to therapy.

Lipidomic analysis of variation in response to simvastatin in the Cholesterol and Pharmacogenetics Study.

Statins are commonly used for reducing cardiovascular disease risk but therapeutic benefit and reductions in levels of low-density lipoprotein cholesterol (LDL-C) vary among individuals. Other effects, including reductions in C-reactive protein (CRP), also contribute to treatment response. Metabolomics provides powerful tools to map pathways implicated in variation in response to statin treatment. This could lead to mechanistic hypotheses that provide insight into the underlying basis for individual variation in drug response. Using a targeted lipidomics platform, we defined lipid changes in blood samples from the upper and lower tails of the LDL-C response distribution in the Cholesterol and Pharmacogenetics study. Metabolic changes in responders are more comprehensive than those seen in non-responders. Baseline cholesterol ester and phospholipid metabolites correlated with LDL-C response to treatment. CRP response to therapy correlated with baseline plasmalogens, lipids involved in inflammation. There was no overlap of lipids whose changes correlated with LDL-C or CRP responses to simvastatin suggesting that distinct metabolic pathways govern statin effects on these two biomarkers. Metabolic signatures could provide insights about variability in response and mechanisms of action of statins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-010-0207-x) contains supplementary material, which is available to authorized users.

Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis.

Macrophages show endoplasmic reticulum (ER) stress when exposed to lipotoxic signals associated with atherosclerosis, although the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. Here we show that mitigation of ER stress with a chemical chaperone results in marked protection against lipotoxic death in macrophages and prevents macrophage fatty acid-binding protein-4 (aP2) expression. Using genetic and chemical models, we show that aP2 is the predominant regulator of lipid-induced macrophage ER stress. The absence of lipid chaperones incites an increase in the production of phospholipids rich in monounsaturated fatty acids and bioactive lipids that render macrophages resistant to lipid-induced ER stress. Furthermore, the impact of aP2 on macrophage lipid metabolism and the ER stress response is mediated by upregulation of key lipogenic enzymes by the liver X receptor. Our results demonstrate the central role for lipid chaperones in regulating ER homeostasis in macrophages in atherosclerosis and show that ER responses can be modified, genetically or chemically, to protect the organism against the deleterious effects of hyperlipidemia.

The plasma lipidomic signature of nonalcoholic steatohepatitis.

UNLABELLED: Specific alterations in hepatic lipid composition characterize the spectrum of nonalcoholic fatty liver disease (NAFLD), which extends from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). However, the plasma lipidome of NAFLD and whether NASH has a distinct plasma lipidomic signature are unknown. A comprehensive analysis of plasma lipids and eicosanoid metabolites quantified by mass spectrometry was performed in NAFL (n = 25) and NASH (n = 50) subjects and compared with lean normal controls (n = 50). The key findings include significantly increased total plasma monounsaturated fatty acids driven by palmitoleic (16:1 n7) and oleic (18:1 n9) acids content (P < 0.01 for both acids in both NAFL and NASH). The levels of palmitoleic acid, oleic acid, and palmitoleic acid to palmitic acid (16:0) ratio were significantly increased in NAFLD across multiple lipid classes. Linoleic acid (8:2n6) was decreased (P < 0.05), with a concomitant increase in gamma-linolenic (18:3n6) and dihomo gamma-linolenic (20:3n6) acids in both NAFL and NASH (P < 0.001 for most lipid classes). The docosahexanoic acid (22:6 n3) to docosapentenoic acid (22:5n3) ratio was significantly decreased within phosphatidylcholine (PC), and phosphatidylethanolamine (PE) pools, which was most marked in NASH subjects (P < 0.01 for PC and P < 0.001 for PE). The total plasmalogen levels were significantly decreased in NASH compared with controls (P < 0.05). A stepwise increase in lipoxygenase (LOX) metabolites 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 8-HETE, and 15-HETE characterized progression from normal to NAFL to NASH. The level of 11-HETE, a nonenzymatic oxidation product of arachidonic (20:4) acid, was significantly increased in NASH only. CONCLUSIONS: Although increased lipogenesis, desaturases, and LOX activities characterize NAFL and NASH, impaired peroxisomal polyunsaturated fatty acid (PUFA) metabolism and nonenzymatic oxidation is associated with progression to NASH.

Alteration in plasma testosterone levels in male mice lacking soluble epoxide hydrolase.

Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH(2)-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism.

Effects of sample handling and storage on quantitative lipid analysis in human serum.

There is sparse information about specific storage and handling protocols that minimize analytical error and variability in samples evaluated by targeted metabolomics. Variance components that affect quantitative lipid analysis in a set of human serum samples were determined. The effects of freeze-thaw, extraction state, storage temperature, and freeze-thaw prior to density-based lipoprotein fractionation were quantified. The quantification of high abundance metabolites, representing the biologically relevant lipid species in humans, was highly repeatable (with coefficients of variation as low as 0.01 and 0.02) and largely unaffected by 1-3 freeze-thaw cycles (with 0-8% of metabolites affected in each lipid class). Extraction state had effects on total lipid class amounts, including decreased diacylglycerol and increased phosphatidylethanolamine in thawed compared with frozen samples. The effects of storage temperature over 1 week were minimal, with 0-4% of metabolites affected by storage at 4 degrees C, -20 degrees C, or -80 degrees C in most lipid classes, and 19% of metabolites in diacylglycerol affected by storage at -20 degrees C. Freezing prior to lipoprotein fractionation by density ultracentrifugation decreased HDL free cholesterol by 37% and VLDL free fatty acid by 36%, and increased LDL cholesterol ester by 35% compared with fresh samples. These findings suggest that density-based fractionation should preferably be undertaken in fresh serum samples because up to 37% variability in HDL and LDL cholesterol could result from a single freeze-thaw cycle. Conversely, quantitative lipid analysis within unfractionated serum is minimally affected even with repeated freeze-thaw cycles.

Lipid metabolism predicts changes in body composition during energy restriction in overweight humans.

Dietary weight loss regimens could be more effective by selectively targeting adipose while sparing lean mass (LM) if predictive information about individuals' lipid metabolic responses to an intervention were available. The objective of this study was to examine the relationships among changes in 4 anthropometric outcomes, weight, waist circumference (WC), percent body fat (BF), and percent LM, and comprehensive circulating lipid metabolites in response to energy reduction in overweight participants. This was a cohort study (n = 46) from a larger multi-center (n = 105) weight loss trial. We used stepwise regression to examine relationships among baseline plasma fatty acids of 7 lipid classes, biochemical metabolites, and diet to explain the variance of 4 anthropometric outcomes after intervention. No predictor variables explained the variance in the percent change in body weight. The circulating concentration of FFA 18:1(n-9) at baseline explained 31% of the variance in percent change of WC, with adjustment for energy intake at 12 wk. Circulating concentrations of phosphatidylcholine 18:0 and FFA 18:1(n-9) at baseline together explained 33% of the variance in percent LM change. The circulating concentration of phosphatidylcholine 18:0 at baseline explained 23% of the variance in the change in percent BF. This study determined relationships among comprehensive and quantitative measurements of complex lipid metabolites and metabolic outcomes as changes in body composition. Measurements of plasma circulating metabolites explained 20-30% of the variance in changes in body composition after a weight loss intervention. Thus, circulating lipids reflect lipid metabolism in relation to changes in body composition.

Assessing individual metabolic responsiveness to a lipid challenge using a targeted metabolomic approach.

The development of assessment techniques with immediate clinical applicability is a priority for resolving the growing epidemic in metabolic disease. Many imbalances in diet-dependent metabolism are not detectable in the fasted state. Resolving the high inter-individual variability in response to diet requires the development of techniques that can detect metabolic dysfunction at the level of the individual. The intra- and inter-individual variation in lipid metabolism in response to a standardized test meal was determined. Following an overnight fast on three different days, three healthy subjects consumed a test meal containing 40% of their daily calories. Plasma samples were collected at fasting, and 1, 3, 6, and 8 h after the test meal. Plasma fatty acid (FA) concentrations within separated lipid classes and lipoprotein fractions were measured at each time point. The intra-individual variation within each subject across three days was lower than the inter-individual differences among the three subjects for over 50% of metabolites in the triacylglycerol (TG), FA, and phosphatidylcholine (PC) lipid classes at 6 h, and for 25-50% of metabolites across lipid classes at 0, 1, 3, and 8 h. The consistency of response within individuals was visualized by principal component analysis (PCA) and confirmed by ANOVA. Three representative metabolites that discriminated among the three individuals in the apolipoprotein B (ApoB) fraction, TG16:1n7, TG18:2n6, and PC18:3n3, are discussed in detail. The postprandial responses of individuals were unique within metabolites that were individual discriminators (ID) of metabolic phenotype. This study shows that the targeted metabolomic measurement of individual metabolic phenotype in response to a specially formulated lipid challenge is possible even without lead-in periods, dietary and lifestyle control, or intervention over a 3-month period in healthy free-living individuals.

Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.

Dysregulation of lipid metabolism in individual tissues leads to systemic disruption of insulin action and glucose metabolism. Utilizing quantitative lipidomic analyses and mice deficient in adipose tissue lipid chaperones aP2 and mal1, we explored how metabolic alterations in adipose tissue are linked to whole-body metabolism through lipid signals. A robust increase in de novo lipogenesis rendered the adipose tissue of these mice resistant to the deleterious effects of dietary lipid exposure. Systemic lipid profiling also led to identification of C16:1n7-palmitoleate as an adipose tissue-derived lipid hormone that strongly stimulates muscle insulin action and suppresses hepatosteatosis. Our data reveal a lipid-mediated endocrine network and demonstrate that adipose tissue uses lipokines such as C16:1n7-palmitoleate to communicate with distant organs and regulate systemic metabolic homeostasis.

A lipidomic analysis of nonalcoholic fatty liver disease.

UNLABELLED: The spectrum of nonalcoholic fatty liver disease (NAFLD) includes a nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). The specific types and amounts of lipids that accumulate in NAFLD are not fully defined. The free fatty acid (FFA), diacylglycerol (DAG), triacylglycerol (TAG), free cholesterol (FC), cholesterol ester, and phospholipid contents in normal livers were quantified and compared to those of NAFL and NASH, and the distribution of fatty acids within these classes was compared across these groups. Hepatic lipids were quantified by capillary gas chromatography. The mean (nmol/g of tissue) DAG (normal/NAFL/NASH: 1922 versus 4947 versus 3304) and TAG (13,609 versus 128,585 versus 104,036) increased significantly in NAFLD, but FFA remained unaltered (5533 versus 5929 versus 6115). There was a stepwise increase in the mean TAG/DAG ratio from normal livers to NAFL to NASH (7 versus 26 versus 31, P < 0.001). There was also a similar stepwise increment in hepatic FC (7539 versus 10,383 versus 12,863, P < 0.05 for NASH). The total phosphatidylcholine (PC) decreased in both NAFL and NASH. The FC/PC ratio increased progressively (0.34 versus 0.69 versus 0.71, P < 0.008 for both). Although the levels for linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) remained unaltered, there was a decrease in arachidonic acid (20:4n-6) in FFA, TAG, and PC (P < 0.05 for all) in NASH. Eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3) were decreased in TAG in NASH. The n-6:n-3 FFA ratio increased in NASH (P < 0.05). CONCLUSIONS: NAFLD is associated with numerous changes in the lipid composition of the liver. The potential implications are discussed.