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While maternal mental health strongly influences neurodevelopment and health in the offspring, little is known about the determinants of inter-individual variation in the mental health of mothers. Likewise, the in utero biological pathways by which variation in maternal mental health affects offspring development remain to be defined. Previous studies implicate lipids, consistent with a known influence on cognitive and emotional function, but the relevance for maternal mental health and offspring neurodevelopment is unclear. This study characterizes the placental and circulatory lipids in antenatal depression, as well as socio-emotional outcomes in the offspring. Targeted liquid chromatography-mass spectrometry covering 470 lipid species was performed on placenta from 186 women with low (n = 70) or high (n = 116) levels of antenatal depressive symptoms assessed using the Edinburgh Postnatal Depression Scale at 26 weeks’ gestation. Child socio-emotional outcomes were assessed from the Child Behavior Check List (CBCL) at 48 months. Seventeen placental lipid species showed an inverse association with antenatal EPDS scores. Specifically, lower levels of phospholipids containing LC-PUFAs: omega-3 docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and omega-6 arachidonic acid (AA) were significantly associated with depressive symptoms. Additional measurement of LC-PUFA in antenatal plasma samples at mid-gestation confirmed the reduced circulation of these specific fatty acids in mothers. Reduced concentration of the placental phospholipids also predicted poorer socio-emotional outcomes in the offspring. This study provides new insights into the role of the materno-fetal lipid cross-talk as a mechanism linking maternal mental health to that of the offspring. These findings show the potential utility of nutritional approaches among pregnant women with depressive symptoms to reduce offspring risk for later socio-emotional problems.

The de novo ceramide synthesis pathway is essential to human biology and health, but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry-specific rare functional variant, L175Q, in delta 4-desaturase, sphingolipid 1 (DEGS1), a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders and spur ongoing research of drug targets along this pathway.

There is a strong association between hepatocyte phospholipid homeostasis and non-alcoholic fatty liver disease (NAFLD). The phosphatidylcholine to phosphatidylethanolamine ratio (PC/PE) often draws special attention as genetic and dietary disruptions to this ratio can provoke steatohepatitis and other signs of NAFLD. Here we demonstrated that excessive free fatty acid (1:2 mixture of palmitic and oleic acid) alone was able to significantly lower the phosphatidylcholine to phosphatidylethanolamine ratio, along with substantial alterations to phospholipid composition in rat hepatocytes. This involved both a decrease in hepatocyte phosphatidylcholine (less prominent) and an increase in phosphatidylethanolamine, with the latter contributing more to the lowered ratio. Stable isotopic tracer phospholipidomic analysis revealed several previously unidentified changes that were triggered by excessive free fatty acid. Importantly, the enhanced cytidine diphosphate (CDP)-ethanolamine pathway activity appeared to be driven by the increased supply of preferred fatty acid substrates. By contrast, the phosphatidylethanolamine N-methyl transferase (PEMT) pathway was restricted by low endogenous methionine and consequently low S-adenosylmethionine, which resulted in a concomitant decrease in phosphatidylcholine and accumulation of phosphatidylethanolamine. Overall, our study identified several previously unreported links in the relationship between hepatocyte free fatty acid overload, phospholipid homeostasis, and the development of NAFLD.

Dysregulation of lipid homeostasis is intimately associated with defects in insulin secretion, a key feature of type 2 diabetes. Here, we explore the role of the putative lipid transporter ABCA12 in regulating insulin secretion from β-cells. Mice with β-cell-specific deletion of Abca12 display impaired glucose-stimulated insulin secretion and eventual islet inflammation and β-cell death. ABCA12's action in the pancreas is independent of changes in the abundance of two other cholesterol transporters, ABCA1 and ABCG1, or of changes in cellular cholesterol or ceramide content. Instead, loss of ABCA12 results in defects in the genesis and fusion of insulin secretory granules and increases in the abundance of lipid rafts at the cell membrane. These changes are associated with dysregulation of the small GTPase CDC42 and with decreased actin polymerisation. Our findings establish a new, pleiotropic role for ABCA12 in regulating pancreatic lipid homeostasis and insulin secretion.

Background Metabolic complications are highly prevalent in cancer survivors treated with irradiation but the underlying mechanisms remain unknown. Methods Chow or high fat-fed C57Bl/6J mice were irradiated (6Gy) before investigating the impact on whole-body or skeletal muscle metabolism and profiling their lipidomic signature. Using a transgenic mouse model (Tg:Pax7-nGFP), we isolated muscle progenitor cells (satellite cells) and characterised their metabolic functions. We recruited childhood cancer survivors, grouped them based on the use of total body irradiation during their treatment and established their lipidomic profile. Results In mice, irradiation delayed body weight gain and impaired fat pads and muscle weights. These changes were associated with impaired whole-body fat oxidation in chow-fed mice and altered ex vivo skeletal muscle fatty acid oxidation, potentially due to a reduction in oxidative fibres and reduced mitochondrial enzyme activity. Irradiation led to fasting hyperglycaemia and impaired glucose uptake in isolated skeletal muscles. Cultured satellite cells from irradiated mice showed decreased fatty acid oxidation and reduced glucose uptake, recapitulating the host metabolic phenotype. Irradiation resulted in a remodelling of lipid species in skeletal muscles, with the extensor digitorum longus muscle being particularly affected. A large number of lipid species were reduced, with several of these species showing a positive correlation with mitochondrial enzymes activity. In cancer survivors exposed to irradiation, we found a similar decrease in systemic levels of most lipid species, and lipid species that increased were positively correlated with insulin resistance (HOMA-IR). Conclusion Irradiation leads to long-term alterations in body composition, and lipid and carbohydrate metabolism in skeletal muscle, and affects muscle progenitor cells. Such changes result in persistent impairment of metabolic functions, providing a new mechanism for the increased prevalence of metabolic diseases reported in irradiated individuals. In this context, changes in the lipidomic signature in response to irradiation could be of diagnostic value.

Objective— High-density lipoprotein (HDL) lipid composition and function may better reflect cardiovascular risk than HDL cholesterol concentration. This study characterized the relationships between HDL composition, metabolism, and function in metabolic syndrome (MetS) patients and how changes in composition after weight loss (WL) and exercise treatments are related to function. Approach and Results— Plasma samples from MetS patients (n=95) and healthy individuals (n=40) were used in this study. Subsets of the MetS group underwent 12 weeks of no treatment (n=17), WL (n=19), or WL plus exercise (WLEX; n=17). HDL was isolated using density-gradient ultracentrifugation. The HDL lipidome was analyzed by mass spectrometry, and particle size determined by nuclear magnetic resonance. Cholesteryl ester transfer protein activity and ex vivo HDL cholesterol efflux capacity (CEC) were assessed. The HDL lipidome in the MetS patients was substantially different from that in healthy individuals, mean particle size was smaller, and CEC was lower. Several HDL phospholipid and sphingolipid species were associated with HDL diameter and CEC. The HDL lipidome and particle size were modified toward the healthy individuals after WL and WLEX treatments, with greater effects observed in the latter group. Cholesteryl ester transfer protein activity was reduced after WL and WLEX, and CEC was improved after WLEX. Conclusions— WLEX treatment in MetS patients normalizes the HDL lipidome and particle size profile and enhances CEC. HDL lipids associated with diminished CEC may represent novel biomarkers for early prediction of HDL dysfunction and disease risk and may represent potential therapeutic targets for future HDL therapies. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00163943

Lipids are known to influence tumour growth, inflammation and chemoresistance. However, the association of circulating lipids with the clinical outcome of metastatic castration-resistant prostate cancer (CRPC) is unknown. We investigated associations between the plasma lipidome and clinical outcome in CRPC. Lipidomic profiling by liquid chromatography-tandem mass spectrometry was performed on plasma samples from a Phase 1 discovery cohort of 96 CRPC patients. Results were validated in an independent Phase 2 cohort of 63 CRPC patients. Unsupervised analysis of lipidomic profiles (323 lipid species) classified the Phase 1 cohort into two patient subgroups with significant survival differences (HR 2.31, 95% CI 1.44-3.68, p = 0.0005). The levels of 46 lipids were individually prognostic and were predominantly sphingolipids with higher levels associated with poor prognosis. A prognostic three-lipid signature was derived (ceramide d18:1/24:1, sphingomyelin d18:2/16:0, phosphatidylcholine 16:0/16:0) and was also associated with shorter survival in the Phase 2 cohort (HR 4.8, 95% CI 2.06-11.1, p = 0.0003). The signature was an independent prognostic factor when modelled with clinicopathological factors or metabolic characteristics. The association of plasma lipids with CRPC prognosis suggests a possible role of these lipids in disease progression. Further research is required to determine if therapeutic modulation of the levels of these lipids by targeting their metabolic pathways may improve patient outcome.

Chronic immune activation persists despite antiretroviral therapy (ART) in HIV+ individuals and underpins an increased risk of age-related co-morbidities. We assessed the Frailty Index in older HIV+ Australian men on ART. Immunometabolic markers on monocytes and T cells were analyzed using flow cytometry, plasma innate immune activation markers by ELISA, and lipidomic profiling by mass spectrometry. The study population consisted of 80 HIV + men with a median age of 59 (IQR, 56–65), and most had an undetectable viral load (92%). 24% were frail, and 76% were non-frail. Frailty was associated with elevated Glucose transporter-1 (Glut1) expression on the total monocytes (p = 0.04), increased plasma levels of innate immune activation marker sCD163 (OR, 4.8; CI 1.4–15.9, p = 0.01), phosphatidylethanolamine PE(36:3) (OR, 5.1; CI 1.7–15.5, p = 0.004) and triacylglycerol TG(16:1_18:1_18:1) (OR, 3.4; CI 1.3–9.2, p = 0.02), but decreased expression of GM3 ganglioside, GM3(d18:1/18:0) (OR, 0.1; CI 0.0–0.6, p = 0.01) and monohexosylceramide HexCerd(d18:1/22:0) (OR, 0.1; CI 0.0–0.5, p = 0.004). There is a strong inverse correlation between quality of life and the concentration of PE(36:3) (ρ = − 0.33, p = 0.004) and PE(36:4) (ρ = − 0.37, p = 0.001). These data suggest that frailty is associated with increased innate immune activation and abnormal lipidomic profile. These markers should be investigated in larger, longitudinal studies to determine their potential as biomarkers for frailty., Highlights • Frailty in older HIV + men is associated with heightened monocyte glycolytic metabolism and innate immune activation. • Lipid classes including phosphatidylethanolamine, GM3 ganglioside and monohexosylceramide are dysregulated in frailty. • Reduced quality of life in aging HIV + men is associated with monocyte glycolytic metabolism and plasma lipid dysregulation. Despite the achievement of long-term survival, people living with HIV have higher rates of age-related comorbidities and frailty (low physiological reserve) compared to their HIV-negative counterparts, for reasons that are currently unknown. We found that immune cells (monocytes) from frail individuals may be incapable of using glucose efficiently, leading to immune dysfunction. Furthermore, we identified a group of lipids in the blood that is associated with frailty. These previously unrecognized factors might underpin age-related comorbidities including frailty in the aging HIV + population. This knowledge may improve our understanding, and management of the health conditions in people aging with HIV.

Aim: to determine lipid species that change in response to a change in dairy consumption. In addition, to investigate whether dairy associated lipid species are correlated with changes in measures of vascular structure and function. Methods: A 12-mo randomised controlled trial was conducted to determine the effect of increased consumption of fruit, vegetables and dairy, compared to usual diet, on measures of vascular structure and function in adults with type 1 and type 2 diabetes (n = 108). This paper comprises post-hoc analyses investigating the relationship between dairy intake, serum lipid species and vascular health. Central and peripheral blood pressure, carotid femoral pulse wave velocity, augmentation index, serum lipid species and dietary intake were measured at baseline and 3-mo. Common carotid artery intima media thickness was measured at baseline and 12-mo. Results: serum lipid species [lysophosphatidylcholine (LPC) 14:0, LPC 15:0, LPC 16:1, phosphatidylcholine (PC) 29:0 PC 30:0, PC 31:0 and cholesterol ester (CE) 14:0] were associated with the change in full fat dairy consumption (rho 0.19-0.25; p < 0.05). The 3-mo change in some lipids was positively associated with the 3-mo change in central systolic [LPC 14:0 (rho 0.30; p = 0.007), PC 30:0 (rho 0.28; p = 0.010)] and diastolic blood pressure [LPC 14:0 (rho 0.32; p = 0.004), LPC 15:0 (rho 0.23; p = 0.04), LPC 16:1 (rho 0.23; p = 0.035), PC 29:0 (rho 0.28; p = 0.01), PC 30:0 (rho 0.36; p = 0.001), PC 31:0 (rho 0.30; p = 0.007)] and 12-mo change in common carotid artery intimal medial thickness [CE 14:0 (rho 0.22; p = 0.02)]. Pulse wave velocity and augmentation index were unrelated to dairy and lipid species Conclusion: an increase in dairy associated lipids appears to be associated with an increase in blood pressure and common carotid intimal medial thickness Refereed/Peer-reviewed

Background Differential plasma concentrations of circulating lipid species are associated with pathogenesis of type 2 diabetes (T2D). Whether the wide inter-individual variability in the plasma lipidome contributes to the genetic basis of T2D is unknown. Here, we investigated the potential overlap in the genetic basis of the plasma lipidome and T2D-related traits. Results We used plasma lipidomic data (1202 pedigreed individuals, 319 lipid species representing 23 lipid classes) from San Antonio Family Heart Study in Mexican Americans. Bivariate trait analyses were used to estimate the genetic and environmental correlation of all lipid species with three T2D-related traits: risk of T2D, presence of prediabetes and homeostatic model of assessment – insulin resistance. We found that 44 lipid species were significantly genetically correlated with one or more of the three T2D-related traits. Majority of these lipid species belonged to the diacylglycerol (DAG, 17 species) and triacylglycerol (TAG, 17 species) classes. Six lipid species (all belonging to the triacylglycerol class and containing palmitate at the first position) were significantly genetically correlated with all the T2D-related traits. Conclusions Our results imply that: a) not all plasma lipid species are genetically informative for T2D pathogenesis; b) the DAG and TAG lipid classes partially share genetic basis of T2D; and c) 1-palmitate containing TAGs may provide additional insights into the genetic basis of T2D. Electronic supplementary material The online version of this article (doi:10.1186/s12863-017-0515-5) contains supplementary material, which is available to authorized users.

Background Asian subjects are at increased cardio-metabolic risk at comparatively lower body mass index (BMI) compared with white subjects. Sympathetic nervous system activation and dyslipidemia, both characteristics of increased adiposity, appear to be related. We therefore analyzed the association of muscle sympathetic nerve activity (MSNA) with the plasma lipidomic profile in young adult Asian and white subjects. Methods Blood samples were collected from 101 participants of either Asian or white background (age, 18 to 30 years; BMI, 28.1 ± 5.9 kg/m2). Lipids were extracted from plasma and analyzed using electrospray ionization-tandem mass spectrometry. MSNA was quantified using microneurography. The association of MSNA and obesity with lipid species was examined using linear regression analysis. Results The plasma concentrations of total dihydroceramide, ceramide, GM3 ganglioside, lysoalkylphosphatidylcholine, alkenylphosphatidylethanolamine, and lysophosphatidylinositol were elevated in the Asian subjects relative to the white subjects. After adjustment for confounders, diacylglycerols and triacylglycerols, cholesterol esters, phosphatidylinositols, phosphatidylethanolamines, and phosphatidylglycerols bore significant associations with MSNA but only in the Asian subjects. These associations remained significant after further adjustment for the participants' degree of insulin resistance and appeared not to be related to differences in diet macronutrient content between groups. Conclusions The lipidomic profile differs between Asian and white subjects. There exists a strong relationship between certain lipid species and MSNA. The association is stronger in Asian subjects, despite their lower BMI. This study demonstrates an association between circulating lipids and central sympathetic outflow. Whether the stronger association between the lipid profile and sympathetic activation underpins the apparent greater risk posed by increased adiposity in Asian individuals merits further attention.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

The de novo ceramide synthesis pathway is essential to human biology and health, but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry-specific rare functional variant, L175Q, in delta 4-desaturase, sphingolipid 1 (DEGS1), a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders and spur ongoing research of drug targets along this pathway.

Dysregulation of lipid homeostasis is a precipitating event in the pathogenesis and progression of hepatosteatosis and metabolic syndrome. These conditions are highly prevalent in developed societies and currently have limited options for diagnostic and therapeutic intervention. Here, using a proteomic and lipidomic-wide systems genetic approach, we interrogated lipid regulatory networks in 107 genetically distinct mouse strains to reveal key insights into the control and network structure of mammalian lipid metabolism. These include the identification of plasma lipid signatures that predict pathological lipid abundance in the liver of mice and humans, defining subcellular localization and functionality of lipid-related proteins, and revealing functional protein and genetic variants that are predicted to modulate lipid abundance. Trans-omic analyses using these datasets facilitated the identification and validation of PSMD9 as a previously unknown lipid regulatory protein. Collectively, our study serves as a rich resource for probing mammalian lipid metabolism and provides opportunities for the discovery of therapeutic agents and biomarkers in the setting of hepatic lipotoxicity. The integration of liver and plasma quantitative lipidomic and proteomic data from 107 distinct mouse strains provides important insights into regulators of mammalian lipid metabolism.

Introduction The identification of metabolomic dysregulation appears promising for the prediction of type 1 diabetes and may also reveal metabolic pathways leading to beta-cell destruction. Recent studies indicate that regulation of multiple phospholipids precede the presence of autoantigens in the development of type 1 diabetes. Objectives We hypothesize that lipid biomarkers in plasma from children with recent onset type 1 diabetes will reflect their remaining beta-cell function and predict future changes in beta-cell function. Methods We performed targeted lipidomic profiling by electrospray ionization tandem mass spectrometry to acquire comparative measures of 354 lipid species covering 25 lipid classes and subclasses in plasma samples from 123 patients

Background Inflammatory bowel disease (IBD), which encompasses ulcerative colitis (UC) and Crohn's disease (CD), is believed to be caused by abnormal host immune responses to the intestinal microbiome. However, the precise etiology of IBD remains unknown. Lipid metabolism and signaling are suggested to play important roles in inflammation with significant implications for IBD. In this study, we aimed to characterize lipidomic profiles in IBD with comparison between healthy controls, UC, and CD. Methods Patients with IBD (n = 40, UC: 16 and CD: 24) and age- and gender-matched healthy volunteers (n = 84) were recruited. Plasma lipid profiles containing 333 lipid species were measured using electrospray ionization-tandem mass spectrometry. Results A total of 86 individual lipid species were significantly changed in CD compared with controls (78 decreased while 8 increased), with the majority belonging to the ether lipids including the alkylphospholipids (alkylphosphatidylcholine and alkylphosphatidylethanolamine) and plasmalogens (alkenylphosphatidylcholine and alkenylphosphatidylethanolamine). Of these 86 lipid species, 33 remained significantly and negatively associated with CD after adjusting for age, sex, waist circumference, current smoking, and diastolic blood pressure in logistic regression. In contrast, only 5 lipid species significantly differed between UC and controls. Conclusions We demonstrate that a number of ether lipids (alkylphospholipid and plasmalogens) are significantly and negatively associated with CD. These alterations of lipid profiles particularly plasmalogens may contribute to the pathogenesis of IBD.

Lipidomic approaches are now widely used to investigate the relationship between lipid metabolism, health and disease. Large-scale lipidomics studies typically aim to quantify hundreds to thousands of lipid molecular species in a large number of samples. Consequently, high throughput methodology that can efficiently extract a wide range of lipids from biological samples is required. Current methods often rely on extraction in chloroform:methanol with or without two phase partitioning or other solvents, which are often incompatible with liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). Here, we present a fast, simple extraction method that is suitable for high throughput LC ESI-MS/MS. Plasma (10 μL) was mixed with 100 μL 1-butanol:methanol (1:1 v/v) containing internal standards resulting in efficient extraction of all major lipid classes (including sterols, glycerolipids, glycerophospholipids and sphingolipids). Lipids were quantified using positive-ion mode LC ESI-MS/MS. The method showed high recovery (&gt, 90%) and reproducibility (%CV &lt, 20%). It showed a strong correlation of all lipid measures with an established chloroform:methanol extraction method (R2 = 0.976). This method uses non-halogenated solvents, requires no drying or reconstitution steps and is suitable for large-scale LC ESI-MS/MS-based lipidomic analyses in research and clinical laboratories.

Background— Cardiovascular disease (CVD) is the most common cause of death in the United States and is associated with a high economic burden. Prevention of CVD focuses on controlling or improving the lipid profile of patients at risk. The human lipidome is made up of thousands of ubiquitous lipid species. By studying biologically simple canonical lipid species, we investigated whether the lipidome is genetically redundant and whether its genetic influences can provide clinically relevant clues of CVD risk. Methods and Results— We performed a genetic study of the human lipidome in 1212 individuals from 42 extended Mexican American families. High-throughput mass spectrometry enabled rapid capture of precise lipidomic profiles, providing 319 unique species. Using variance component–based heritability analyses and bivariate trait analyses, we detected significant genetic influences on each lipid assayed. Median heritability of the plasma lipid species was 0.37. Hierarchical clustering based on complex genetic correlation patterns identified 12 genetic clusters that characterized the plasma lipidome. These genetic clusters were differentially but consistently associated with risk factors of CVD, including central obesity, obesity, type 2 diabetes mellitus, raised serum triglycerides, and metabolic syndrome. Also, these clusters consistently predicted occurrence of cardiovascular deaths during follow-up. Conclusions— The human plasma lipidome is heritable. Shared genetic influences reduce the dimensionality of the human lipidome into clusters that are associated with risk factors of CVD.

Summary High-throughput targeted lipid profiling with liquid chromatography-mass spectrometry (LC-MS) has been used extensively to identify associations between plasma lipid species and disease states. Such methods, used to characterize larger clinical cohorts, often suffer from an inability to differentiate isomeric forms of glycerophospholipids that are typically reported as the sum fatty acid carbons and double bonds. Here we report a chromatography gradient coupled with a detailed characterization of the human plasma lipidome to provide improved resolution and identification of 636 lipid species, including previously unreported species, in a 15-min analysis. We have utilized this method on a subset of the Australian Diabetes, Obesity, and Lifestyle Study and have detailed associations of plasma lipid species with anthropometric and blood glucose measures. These results highlight the importance and power of high-throughput lipidomics coupled with a detailed characterization of the lipidome to better understand lipid biology in a population setting.

Background and Aims: Despite current best care, patients with peripheral artery disease (PAD) remain at high risk of myocardial infarction and biomarkers to more accurately assess cardiovascular risk are needed. This study assessed the relationship between the serum lipidome and incident myocardial infarction in a cohort of PAD patients. Methods: 265 PAD patients were followed up for a median of 23 months, during which 18 people suffered a myocardial infarction. Fasting serum concentrations of 332 lipid species were measured via mass spectrometry and their association with incident myocardial infarction was assessed via Cox regression. Secondary analyses investigated prognostic potential of specific lipid species. Results: Total serum concentrations of alkyl-phosphatidylcholine and alkenylphospatidylcholine (plasmalogen) lipids were inversely associated with incident myocardial infarction after adjusting for multiple testing (hazards ratio (95% confidence intervals): 0.43 (0.24-0.74); p=0.032; and 0.28 (0.14-0.56), p=0.010, respectively). Specifically, 10 alkenylphosphatidylcholine species and 6 alkyl-phosphatidylcholine species were negatively associated with incident myocardial infarction after adjusting for traditional risk factors and correcting for multiple testing (hazards ratios ranging from 0.07-0.51, p

Context: Postprandial dysmetabolism in type 2 diabetes (T2D) is exacerbated by prolonged sitting and may trigger inflammation and oxidative stress. It is unknown what impact countermeasures to prolonged sitting have on the postprandial lipidome. Objective: In this study, we investigated the effects of regular interruptions to sitting, compared with prolonged sitting, on the postprandial plasma lipidome. Design: Randomized crossover experimental trial. Setting: Participants underwent three 7-hour conditions: uninterrupted sitting (SIT); light-intensity walking interruptions (LW); and simple resistance activity interruptions (SRA). Participants and Samples: Baseline (fasting) and 7-hour (postprandial) plasma samples from 21 inactive overweight/obese adults with T2D were analyzed for 338 lipid species using mass spectrometry. Main Outcome Measures: Using mixed model analysis (controlling for baseline outcome variable, gender, body mass index, and condition order), the percentage change in lipid species (baseline to 7 hours) was compared between conditions with Benjamini–Hochberg correction. Results: Thirty-seven lipids were different between conditions (P < 0.05). Compared with SIT, postprandial elevations in diacylglycerols, triacylglycerols, and phosphatidylethanolamines were attenuated in LW and SRA. Plasmalogens and lysoalkylphosphatidylcholines were reduced in SIT, compared with attenuated reductions or elevations in LW and SRA. Phosphatidylserines were elevated with LW, compared with reductions in SIT and SRA. Conclusion: Compared with SIT, LW and SRA were associated with reductions in lipids associated with inflammation; increased concentrations of lipids associated with antioxidant capacity; and differential changes in species associated with platelet activation. Acutely interrupting prolonged sitting time may impart beneficial effects on the postprandial plasma lipidome of adults with T2D. Evidence on longer-term intervention is needed.

Plasma lipidome is now increasingly recognized as a potentially important marker of chronic diseases, but the exact extent of its contribution to the interindividual phenotypic variability in family studies is unknown. Here, we used the rich data from the ongoing San Antonio Family Heart Study (SAFHS) and developed a novel statistical approach to quantify the independent and additive value of the plasma lipidome in explaining metabolic syndrome (MS) variability in Mexican American families recruited in the SAFHS. Our analytical approach included two preprocessing steps: principal components analysis of the high-resolution plasma lipidomics data and construction of a subject-subject lipidomic similarity matrix. We then used the Sequential Oligogenic Linkage Analysis Routines software to model the complex family relationships, lipidomic similarities, and other important covariates in a variance components framework. Our results suggested that even after accounting for the shared genetic influences, indicators of lipemic status (total serum cholesterol, TGs, and HDL cholesterol), and obesity, the plasma lipidome independently explained 22% of variability in the homeostatic model of assessment-insulin resistance trait and 16% to 22% variability in glucose, insulin, and waist circumference. Our results demonstrate that plasma lipidomic studies can additively contribute to an understanding of the interindividual variability in MS.

Aims The optimal management of asymptomatic subjects at intermediate risk of coronary artery disease (CAD) is often uncertain. Re-stratification to a high- or low-risk category may enable optimization of preventative strategies. Coronary computed tomographic angiography (CCTA) enables a minimally invasive assessment of coronary artery plaque quantity and composition. Non-calcified plaque by CCTA is lipid-rich and more vulnerable to rupture and resultant acute coronary syndromes. The purpose of this study was to determine whether novel approaches to CAD risk stratification, such as plasma lipid profiling, may predict non-calcified plaque burden in intermediate risk subjects. Methods and results CCTA and several markers of CAD (including plasma lipid profiling, carotid intima-media thickness, aortic pulse wave velocity, and high-sensitivity C-reactive protein) were prospectively performed in 100 asymptomatic patients at intermediate CAD risk according to the Framingham risk score. Segment stenosis scores (SSS) were calculated to evaluate the burden of total, calcified, and non-calcified coronary artery plaque. Non-calcified plaque was observed in 66 subjects and 158 (11%) of 1425 coronary artery segments. Eighteen lipid species demonstrated significant associations with non-calcified plaque burden, but not with total plaque or calcified plaque burden. No other marker of CAD was found to predict coronary artery plaque burden. Conclusions Plasma lipidomic analysis can predict the burden of non-calcified coronary plaque in asymptomatic subjects at intermediate risk of CAD. Re-stratification of these patients by plasma lipid profiling may enable more appropriate and effective primary prevention management strategies.

Objective Waist circumference (WC), the clinical marker of central obesity, is gaining popularity as a screening tool for type 2 diabetes (T2D). While there is epidemiologic evidence favoring the WC-T2D association, its biological substantiation is generally weak. Our objective was to determine the independent association of plasma lipid repertoire with WC. Methods Samples and data from the San Antonio Family Heart Study of 1208 Mexican Americans from 42 extended families were used. Association of plasma lipidomic profiles with the cross-sectionally assessed WC was determined. Plasma lipidomic profiling entailed liquid chromatography with mass spectrometry. Statistical analyses included multivariable polygenic regression models and bivariate trait analyses using the SOLAR software. Results After adjusting for age and sex interactions, body mass index, homeostasis model of assessment—insulin resistance, total cholesterol, triglycerides, high density lipoproteins and use of lipid lowering drugs, dihydroceramides as a class were associated with WC. Dihydroceramide species 18:0, 20:0, 22:0, and 24:1 were significantly associated and genetically correlated with WC. Two sphingomyelin species (31:1 and 41:1) were also associated with WC. Conclusions Plasma dihydroceramide levels independently associate with WC. Thus, high resolution plasma lipidomic studies can provide further credence to the biological underpinnings of the association of WC with T2D.

The study of the metabolism of docosapentaenoic acid (DPA, 22:5n � 3) in humans has been limited by the unavailability of pure DPA and the fact that DPA is found in combination with eicosapentaenoic acid (EPA, 20:5n � 3) and docosahexaenoic acid (DHA, 22:6n � 3) in natural products. In this double blind cross over study, pure DPA and EPA were incorporated in meals served to healthy female volunteers. Mass spectrometric methods were used to study the chylomicron lipidomics. Plasma chylomicronemia was significantly reduced after the meal containing DPA compared with the meal containing EPA or olive oil only. Both EPA and DPA were incorporated into chylomicron TAGs, while there was less incorporation into chylomicron phospholipids. Lipidomic analysis of the chylomicron TAGs revealed the dynamic nature of chylomicron TAGs. The main TAG species that EPA and DPA were incorporated into were EPA/18:1/18:1, DPA/18:1/16:0 and DPA/18:1/18:1. There was very limited conversion of DPA and EPA to DHA and there were no increases in EPA levels during the 5 h postprandial period after the DPA meal. In conclusion, EPA and DPA showed different metabolic fates, and DPA hindered the digestion, ingestion or incorporation into chylomicrons of the olive oil present in the meal.

The sphingolipids sphingosine-1-phosphate (S1P) and ceramide are important bioactive lipids with many cellular effects. Intracellular ceramide accumulation causes insulin resistance, but sphingosine kinase 1 (SphK1) prevents ceramide accumulation, in part, by promoting its metabolism into S1P. Despite this, the role of SphK1 in regulating insulin action has been largely overlooked. Transgenic (Tg) mice that overexpress SphK1 were fed a standard chow or high-fat diet (HFD) for 6 weeks before undergoing several metabolic analyses. SphK1 Tg mice fed an HFD displayed increased SphK activity in skeletal muscle, which was associated with an attenuated intramuscular ceramide accumulation compared with wild-type (WT) littermates. This was associated with a concomitant reduction in the phosphorylation of c-jun amino-terminal kinase, a serine threonine kinase associated with insulin resistance. Accordingly, skeletal muscle and whole-body insulin sensitivity were improved in SphK1 Tg, compared with WT mice, when fed an HFD. We have identified that the enzyme SphK1 is an important regulator of lipid partitioning and insulin action in skeletal muscle under conditions of increased lipid supply. Refereed/Peer-reviewed

Introduction Rheumatoid arthritis (RA) is linked to increased cardiovascular morbidity and mortality, not completely explained by traditional risk factors. Importantly, the increased risk occurs despite lower levels of total and low-density lipoprotein cholesterol. Whilst systemic inflammation may be a factor, it is possible that changes in individual lipid species contribute to the increased cardiovascular risk.

Adipocytes package incoming fatty acids into triglycerides and other glycerolipids, with only a fraction spilling into a parallel biosynthetic pathway that produces sphingolipids. Herein, we demonstrate that subcutaneous adipose tissue of type 2 diabetics contains considerably more sphingolipids than non-diabetic, BMI-matched counterparts. Whole-body and adipose tissue-specific inhibition/deletion of serine palmitoyltransferase (Sptlc), the first enzyme in the sphingolipid biosynthesis cascade, in mice markedly altered adipose morphology and metabolism, particularly in subcutaneous adipose tissue. The reduction in adipose sphingolipids increased brown and beige/brite adipocyte numbers, mitochondrial activity, and insulin sensitivity. The manipulation also increased numbers of anti-inflammatory M2 macrophages in the adipose bed and induced secretion of insulin-sensitizing adipokines. By comparison, deletion of serine palmitoyltransferase from macrophages had no discernible effects on metabolic homeostasis or adipose function. These data indicate that newly synthesized adipocyte sphingolipids are nutrient signals that drive changes in the adipose phenotype to influence whole-body energy expenditure and nutrient metabolism.

Although skeletal muscle insulin resistance has been associated with activation of c-Jun N-terminal kinase (JNK), whether increased JNK activity causes insulin resistance in this organ is not clear. In this study we examined the metabolic consequences of isolated JNK phosphorylation in muscle tissue.Plasmids containing genes encoding a wild-type JNK1 (WT-JNK) or a JNK1/JNKK2 fusion protein (rendering JNK constitutively active; CA-Jnk) were electroporated into one tibialis anterior (TA) muscle of C57Bl/6 mice, with the contralateral TA injected with an empty vector (CON) to serve as a within-animal control.Overproduction of WT-JNK resulted in a modest (~25%) increase in phosphorylation (Thr(183)/Tyr(185)) of JNK, but no differences were observed in Ser(307) phosphorylation of insulin receptor substrate 1 (IRS-1) or total IRS-1 protein, nor in insulin-stimulated glucose clearance into the TA muscle when comparing WT-JNK with CON. By contrast, overexpression of CA-Jnk, which markedly increased the phosphorylation of CA-JNK, also increased serine phosphorylation of IRS-1, markedly decreased total IRS-1 protein, and decreased insulin-stimulated phosphorylation of the insulin receptor (Tyr(1361)) and phosphorylation of Akt at (Ser(473) and Thr(308)) compared with CON. Moreover, overexpression of CA-Jnk decreased insulin-stimulated glucose clearance into the TA muscle compared with CON and these effects were observed without changes in intramuscular lipid species.Constitutive activation of JNK in skeletal muscle impairs insulin signalling at the level of IRS-1 and Akt, a process which results in the disruption of normal glucose clearance into the muscle.

Obesity is characterised by increased storage of fatty acids in an expanded adipose tissue mass and in peripheral tissues such as the skeletal muscle and liver, where it is associated with the development of insulin resistance. Insulin resistance also develops in the central nervous system with high-fat feeding. The capacity for hypothalamic cells to accumulate/store lipids, and the effects of obesity remain undefined. The aims of this study were (1) to examine hypothalamic lipid content in mice with increased dietary fat intake and in obese ob/ob mice fed a low-fat diet, and (2) to determine whether endurance exercise training could reduce hypothalamic lipid accumulation in high-fat fed mice. Male C57BL/6 mice were fed a low- (LFD) or high-fat diet (HFD) for 12 weeks; ob/ob mice were maintained on a chow diet. HFD-exercise (HFD-ex) mice underwent 12 weeks of high-fat feeding with 6 weeks of treadmill exercise training (increasing from 30 to 70 min day(-1)). Hypothalamic lipids were assessed by unbiased mass spectrometry. The HFD increased body mass and hepatic lipid accumulation, and induced glucose intolerance, while the HFD-ex mice had reduced body weight and improved glucose tolerance. A total of 335 lipid molecular species were identified and quantified. Lipids known to induce insulin resistance, including ceramide (22%↑), diacylglycerol (25%↑), lysophosphatidylcholine (17%↑), cholesterol esters (60%↑) and dihexosylceramide (33%↑), were increased in the hypothalamus of HFD vs. LFD mice. Hypothalamic lipids were unaltered with exercise training and in the ob/ob mice, suggesting that obesity per se does not alter hypothalamic lipids. Overall, hypothalamic lipid accumulation is regulated by dietary lipid content and is refractory to change with endurance exercise training.

The early mechanisms regulating progression towards beta cell failure in type 1 diabetes (T1D) are poorly understood, but it is generally acknowledged that genetic and environmental components are involved. The metabolomic phenotype is sensitive to minor variations in both, and accordingly reflects changes that may lead to the development of T1D. We used two different extraction methods in combination with both liquid- and gas chromatographic techniques coupled to mass spectrometry to profile the metabolites in a transgenic non-diabetes prone C57BL/6 mouse expressing CD154 under the control of the rat insulin promoter (RIP) crossed into the immuno-deficient recombination-activating gene (RAG) knockout (−/−) C57BL/6 mouse, resembling the early stages of human T1D. We hypothesized that alterations in the metabolomic phenotype would characterize the early pathogenesis of T1D, thus metabolomic profiling could provide new insight to the development of T1D. Comparison of the metabolome of the RIP CD154 × RAG−/− mice to RAG−/− mice and C57BL/6 mice revealed alterations of >100 different lipids and metabolites in serum. Low lysophosphatidylcholine levels, accumulation of ceramides as well as methionine deficits were detected in the pre-type 1 diabetic mice. Additionally higher lysophosphatidylinositol levels and low phosphatidylglycerol levels where novel findings in the pre-type 1 diabetic mice. These observations suggest that metabolomic disturbances precede the onset of T1D. Electronic supplementary material The online version of this article (doi:10.1007/s11306-015-0889-1) contains supplementary material, which is available to authorized users.

The ability of the fatty acid composition of dietary phosphatidylcholine (PC) to affect hepatic lipid levels was investigated in C57BL/6 mice (n=8-10 per group) by feeding: (1) a high-fat semi-purified diet (HF), (2) HF diet supplemented with 1.25 wt% soy PC (SPC), (3) HF with 1.25 wt% hydrogenated soy PC (SPCH), (4) HF with 1.25 wt% egg PC (EPC), and (5) HF with 1.25 wt% hydrogenated egg PC (EPCH). The polyunsaturated fatty acid content (C18:2+C18:3+C20:4) of soy, egg and hydrogenated PC was 70%, 20% and 0%, respectively. Total liver lipid was significantly lower in SPCH and EPCH vs. HF (8.7 ± 0.1 and 8.5 ± 0.5 vs. 11.8 ± 0.6g/100, P

Summary Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo , via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.

Using Shannon's entropy, we quantified the plasticity of the human plasma lipidome (defined as the extent to which the lipidome departs froma homeostatic equilibrium). Our objective was to test whe...

Although the association between obesity and hypertension is well-known, the underlying mechanism remains elusive. Previously, we have shown that 3 week fat feeding in rabbits produces greater visceral adiposity, hypertension, tachycardia and elevated renal sympathetic nerve activity (RSNA) compared to rabbits on a normal diet. Because hyperinsulinaemia, hyperleptinemia, and dyslipidaemia are independent cardiovascular risk factors associated with hypertension we compared plasma insulin, leptin, and lipid profiles in male New Zealand White rabbits fed a normal fat diet (NFD 4.3% fat, n = 11) or high fat diet (HFD 13.4% fat, n = 13) at days 1, 2, 3 and weeks 1, 2, 3 of the diet. Plasma concentrations of diacylglyceride (DG), triacylglyceride (TG), ceramide and cholesteryl esters (CE) were obtained after analysis by liquid chromatography mass spectrometry. Plasma insulin and glucose increased within the first 3 days of the diet in HFD rabbits (P < 0.05) and remained elevated at week 1 (P < 0.05). Blood pressure and heart rate (HR) followed a similar pattern. By contrast, in both groups, plasma leptin levels remained unchanged during the first few days (P > 0.05), increasing by week 3 in fat fed animals alone (P < 0.05). Concentrations of total DG, TG, CE, and Ceramide at week 3 did not differ between groups (P > 0.05). Our data show plasma insulin increases rapidly following consumption of a HFD and suggests that it may play a role in the rapid rise of blood pressure. Dyslipidaemia does not appear to contribute to the hypertension in this animal model.

5055 Background: Biomarker studies of metastatic castration-resistant prostate cancer (CRPC) have mainly focused on changes in the cancer, however, the host environment and its interactions with cancer is increasingly important, especially given the increasing association of PC outcomes and obesity. We sought associations between the plasma lipidome and clinical outcome in CRPC. Methods: Plasma samples were obtained from a Phase 1 discovery cohort of 96 CRPC patients before and after the first cycle of docetaxel chemotherapy. Lipidomic profiling of the plasma samples was performed by liquid chromatography and electrospray ionisation-tandem mass spectrometry. Results were subsequently assessed in a Phase 2 validation cohort of 63 CRPC patients. Results: Lipidomic profiling detected 323 lipid species in plasma samples from the Phase 1 cohort. Patients could be classified into two subgroups with significant survival differences according to their baseline lipidomic profiles (median overall survival 13.7 vs 21.7 months; HR 2.31, 95% CI 1.44-3.68, p = 0.0005). The baseline levels of 46 lipids were individually prognostic (p < 0.01) and predominantly sphingolipids. A prognostic three-lipid signature was derived (ceramide (d18:1/24:1), sphingomyelin (d18:2/16:0), phosphatidylcholine (16:0/16:0)) (11.7 versus 21.7 months, p = 0.00001; HR 2.94, 95% CI 1.80-4.81, p = 0.00002). The signature was associated with shorter overall survival in the Phase 2 cohort (HR 4.78, 95% CI 2.06-11.1, p = 0.0003), and was an independent prognostic factor when modeled with clinicopathological factors and metabolic characteristics (BMI, cholesterol and triacylglycerol). The AUC of ROC analysis of 12 month survival for a clinicopathological model (AUC 0.70, 95% CI 0.54-0.87, p = 0.03) was enhanced by the addition of the 3-lipid signature (AUC 0.73, 95% CI 0.57-0.89, p = 0.01). Conclusions: Our study is the first to comprehensively profile the plasma lipidome of men with CRPC using cutting-edge lipidomic profiling technology, to identify a plasma lipid signature that can reproducibly predict outcome in CRPC and can improve on clinicopathological models. Therapeutic modulation of the levels of these lipids by targeting their metabolic pathways may improve patient outcome.

Background— Currently, the relationship between circulating lipids and abdominal aortic aneurysm (AAA) is unclear. We conducted a lipidomic analysis to identify serum lipids associated with AAA presence. Secondary analyses assessed the ability of models incorporating lipidomic features to improve stratification of patient groups with and without AAA beyond traditional risk factors. Methods and Results— Serum lipids were profiled via liquid chromatography tandem mass spectrometry analysis of serum from 161 patients with AAA and 168 controls with peripheral artery disease. Binary logistic regression was used to identify AAA-associated lipids. Classification models were created based on a combination of (1) traditional risk factors only or (2) lipidomic features and traditional risk factors. Model performance was assessed using receiver operator characteristic curves. Three diacylglycerols and 7 triacylglycerols were associated with AAA. Combining lipidomic features with traditional risk factors significantly improved stratification of AAA and peripheral artery disease groups when compared with traditional risk factors alone (mean area under the receiver operator characteristic curve [95% confidence interval], 0.760 [0.756–0.763] and 0.719 [0.716–0.723], respectively; P Conclusions— A group of linoleic acid containing triacylglycerols and diacylglycerols were significantly associated with AAA presence. Inclusion of lipidomic features in multivariate analyses significantly improved prediction of AAA presence when compared with traditional risk factors alone.

BackgroundThe increased risk of coronary artery disease in human immunodeficiency virus (HIV) positive patients is collectively contributed to by the human immunodeficiency virus and antiretroviral-associated dyslipidaemia. In this study, we investigate the characterisation of the plasma lipid profiles of treated HIV patients and the relationship of 316 plasma lipid species across multiple lipid classes with the risk of future cardiovascular events in HIV-positive patients.MethodsIn a retrospective case-control study, we analysed plasma lipid profiles of 113 subjects. Cases (n = 23) were HIV-positive individuals with a stored blood sample available 12 months prior to their diagnosis of coronary artery disease (CAD). They were age and sex matched to HIV-positive individuals without a diagnosis of CAD (n = 45) and with healthy HIV-negative volunteers (n = 45).ResultsAssociation of plasma lipid species and classes with HIV infection and cardiovascular risk in HIV were determined. In multiple logistic regression, we identified 83 lipids species and 7 lipid classes significantly associated with HIV infection and a further identified 74 lipid species and 8 lipid classes significantly associated with future cardiovascular events in HIV-positive subjects. Risk prediction models incorporating lipid species attained an area under the receiver operator characteristic curve (AUC) of 0.78 (0.775, 0.785)) and outperformed all other tested markers and risk scores in the identification of HIV-positive subjects with increased risk of cardiovascular events.ConclusionsOur results demonstrate that HIV-positive patients have significant differences in their plasma lipid profiles compared with healthy HIV-negative controls and that numerous lipid species were significantly associated with elevated cardiovascular risk. This suggests a potential novel application for plasma lipids in cardiovascular risk screening of HIV-positive patients.

We have performed plasma lipid profiling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 μl of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identified statistically significant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was significantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk.