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OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children characterized by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease.METHODS: We performed untargeted liquid chromatography mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9,500 adults with metabolic phenotyping.RESULTS: More severe paediatric NAFLD was associated with lower levels of long chain, polyunsaturated phosphatidylcholines (PC) and triglycerides (TG). Similar trends in PC and TG chain length and saturation were seen in adults with hepatic steatosis. However, many of the specific lipids associated with NAFLD differed between children and adults. Five lipids replicated in adults (including PC(36:4)) have been directly linked to death and cardiometabolic disease, as well as indirectly via genetic variants.CONCLUSION: These findings suggest that, whilst similar pathways of lipid metabolism are perturbed in paediatric NAFLD as in cardiometabolic disease in adults, the specific lipid signature in children is different.

Peer reviewed: True, Funder: NIHR Cambridge BRC, Funder: core biochemical assay laboratory (CBAL), Funder: core metabolomic and lipidomic laboratory (CMAL), Male fertility, as manifest by the quantity and progressive motility of spermatozoa, is negatively impacted by obesity, dyslipidaemia and metabolic disease. However, the relative distribution of lipids in spermatozoa and the two compartments which supply lipids for spermatogenesis (seminal fluid and blood serum) has not been studied. We hypothesised that altered availability of lipids in blood serum and seminal fluid may affect the lipid composition and progressive motility of sperm. 60 men of age 35 years (median (range 20–45) and BMI 30.4 kg/m2 (24–36.5) under preliminary investigation for subfertility were recruited at an NHS clinic. Men provided samples of serum and semen, subject to strict acceptance criteria, for analysis of spermatozoa count and motility. Blood serum (n = 60), spermatozoa (n = 26) and seminal fluid (n = 60) were frozen for batch lipidomics analysis. Spermatozoa and seminal fluid had comparable lipid composition but showed marked differences with the serum lipidome. Spermatozoa demonstrated high abundance of ceramides, very-long-chain fatty acids (C20-22), and certain phospholipids (sphingomyelins, plasmalogens, phosphatidylethanolamines) with low abundance of phosphatidylcholines, cholesterol and triglycerides. Men with spermatozoa of low progressive motility had evidence of fewer concentration gradients for many lipid species between blood serum and spermatozoa compartments. Spermatozoa are abundant in multiple lipid species which are likely to contribute to key cellular functions. Lipid metabolism shows reduced regulation between compartments in men with spermatozoa with reduced progressive motility.

Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of a priori structural determination. In the present study, we codify the use of 31P NMR for lipidomics studies to make the technique more accessible to new users and more useful for a wider range of questions. The technique can be used in isolation (phospholipidomics) or as a part of determining lipid composition (lipidomics). We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid-containing systems. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03430-4.

Background: Male fertility, as manifest by the quantity and progressive motility of spermatozoa, is negatively impacted by obesity, dyslipidaemia and metabolic disease. Howev-er, the relative distribution of lipids in spermatozoa and the two compartments which supply lipids for spermatogenesis (seminal fluid and blood serum) has not been studied. We hypothe-sised that altered availability of lipids in blood serum and seminal fluid may affect the lipid composition and progressive motility of sperm. Methods: 60 men of age 35 years (median (range 20 - 45) and BMI 30.4 kg/m2 (24 - 36.5) under preliminary investigation for subfertility were recruited at an NHS clinic. Men provided sam-ples of serum and semen, subject to strict acceptance criteria, for analysis of spermatozoa count and motility. Blood serum (n=60), spermatozoa (n=26) and seminal fluid (n=60) were frozen for batch lipidomics analysis. Results: Spermatozoa and seminal fluid had comparable lipid composition, but showed marked differences with the serum lipidome. Spermatozoa demonstrated high abundance of ceramides, very-long-chain fatty acids (C20-22), and certain phospholipids (sphingomyelins, plasmalogens, phosphatidylethanolamines) with low abundance of phosphatidylcholines, cholesterol and tri-glycerides. Men with spermatozoa of low progressive motility had evidence of fewer concentra-tion gradients for many lipid species between blood serum and spermatozoa compartments. Conclusion: Spermatozoa are abundant in multiple lipid species which are likely to contribute to key cellular functions. Lipid metabolism shows reduced regulation between compartments in men with spermatozoa with reduced progressive motility., CLM is supported by the Diabetes UK Harry Keen Intermediate Clinical Fellowship (DUK-HKF 17/0005712) and the European Foundation for the Study of Diabetes – Novo Nordisk Foundation Future Leaders’ Award (NNF19SA058974). SF and AK acknowledge funding from the BBSRC (BB/M027252/1). This research was supported by the NIHR Cambridge BRC, the core biochemical assay laboratory (CBAL) and the core metabolomic and lipidomic laboratory (CMAL). The views expressed are those of the au-thor(s) and not necessarily those of the NIHR or the Department of Health and Social Care.

Maternal obesity during pregnancy has immediate and long-term detrimental effects on the offspring heart. In this study, we characterized the cardiac and circulatory lipid profiles in late gestation E18.5 fetuses of diet-induced obese pregnant mice and established the changes in lipid abundance and fetal cardiac transcriptomics. We used untargeted and targeted lipidomics and transcriptomics to define changes in the serum and cardiac lipid composition and fatty acid metabolism in male and female fetuses. From these analyses we observed: (1) maternal obesity affects the maternal and fetal serum lipidome distinctly; (2) female fetal heart lipidomes are more sensitive to maternal obesity than males; (3) changes in lipid supply might contribute to early expression of lipolytic genes in mouse hearts exposed to maternal obesity. These results highlight the existence of sexually dimorphic responses of the fetal heart to the same in utero obesogenic environment and identify lipids species that might mediate programming of cardiovascular health., MRC Metabolic Diseases Unit [MRC_MC_UU_00014/4] Cambridge Home and EU Student Scholarship British Heart Foundation studentship [FS/12/64/30001] II was supported by a British Heart Foundation studentship [FS/18/56/35177]

Funder: Foundation for Liver Research, Funder: Royal Society and a CAMS-UK fellowship, The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.

Context Gestational diabetes (GDM) affects 20 million women/year worldwide and is associated with childhood obesity. Infants of affected mothers have increased adiposity from birth, which leads to obesity in later life. However, it remains unknown whether the effect of GDM upon neonatal body composition is due to hyperglycemia alone or is mediated by other pathways. Objective To investigate plasma lipid profiles in obese women according to GDM diagnosis, infant birthweight percentiles, and adiposity. Design Prospective cohort from UPBEAT trial (ISRCTN 89971375). Setting Hospital and community. Patients 867 obese pregnant women recruited in early pregnancy, assessed at 28 weeks for GDM. Offspring anthropometry was assessed at birth. Outcome (Prespecified) Neonatal birth percentile and abdominal circumference. Methods Lipidomic profiling in the fasting plasma oral glucose tolerance test sample using direct infusion mass spectrometry. Analysis included logistic/linear regression, unadjusted and adjusted for maternal age, body mass index, parity, ethnicity, UPBEAT trial arm, and fetal sex. The limit of significance was P = 0.05 for offspring anthropometry and P = 0.002 for lipidomic data. Results GDM in obese women was associated with elevated plasma concentrations of specific diglycerides [DG(32:0)] and triglycerides [TG(48:0), (50:1), (50:2)] containing fatty acids (16:0), (16:1), (18:0), and (18:1), consistent with increased de novo lipogenesis. In the whole cohort, these species were associated with birthweight percentile and neonatal abdominal circumference. Effects upon infant abdominal circumference remained significant after adjustment for maternal glycemia. Conclusions Increased de novo lipogenesis-related species in pregnant women with obesity and GDM are associated with measures of offspring adiposity and may be a target for improving lifelong health.

Hypoxic-ischemic encephalopathy (HIE) is associated with perinatal brain injury, which may lead to disability or death. As the brain is a lipid-rich organ, various lipid species can be significantly impacted by HIE and these correlate with specific changes to the lipidomic profile in the circulation. Objective: To investigate the peripheral blood lipidomic signature in dried blood spots (DBS) from newborns with HIE. Using univariate analysis, multivariate analysis and sPLS-DA modelling, we show that newborns with moderate-severe HIE (n = 46) who underwent therapeutic hypothermia (TH) displayed a robust peripheral blood lipidomic signature comprising 29 lipid species in four lipid classes; namely phosphatidylcholine (PC), lysophosphatidylcholine (LPC), triglyceride (TG) and sphingomyelin (SM) when compared with newborns with mild HIE (n = 18). In sPLS-DA modelling, the three most discriminant lipid species were TG 50:3, TG 54:5, and PC 36:5. We report a reduction in plasma TG and SM and an increase in plasma PC and LPC species during the course of TH in newborns with moderate-severe HIE, compared to a single specimen from newborns with mild HIE. These findings may guide the research in nutrition-based intervention strategies after HIE in synergy with TH to enhance neuroprotection., NIHR Cambridge Biomedical Research Centre (146281) & Biotechnology and Biological Sciences Research Council (BB/P028195/1)

Background Genetic, lifestyle, and environmental factors can lead to perturbations in circulating lipid levels and increase the risk of cardiovascular and metabolic diseases. However, how changes in individual lipid species contribute to disease risk is often unclear. Moreover, little is known about the role of lipids on cardiovascular disease in Pakistan, a population historically underrepresented in cardiovascular studies. Methods We characterised the genetic architecture of the human blood lipidome in 5662 hospital controls from the Pakistan Risk of Myocardial Infarction Study (PROMIS) and 13,814 healthy British blood donors from the INTERVAL study. We applied a candidate causal gene prioritisation tool to link the genetic variants associated with each lipid to the most likely causal genes, and Gaussian Graphical Modelling network analysis to identify and illustrate relationships between lipids and genetic loci. Results We identified 253 genetic associations with 181 lipids measured using direct infusion high-resolution mass spectrometry in PROMIS, and 502 genetic associations with 244 lipids in INTERVAL. Our analyses revealed new biological insights at genetic loci associated with cardiometabolic diseases, including novel lipid associations at the LPL, MBOAT7, LIPC, APOE-C1-C2-C4, SGPP1, and SPTLC3 loci. Conclusions Our findings, generated using a distinctive lipidomics platform in an understudied South Asian population, strengthen and expand the knowledge base of the genetic determinants of lipids and their association with cardiometabolic disease-related loci.

The aim of the current study was to test the hypothesis that maternal lipid metabolism was modulated during normal pregnancy and that these modulations are altered in gestational diabetes mellitus (GDM). We tested this hypothesis using an established mouse model of diet-induced obesity with pregnancy-associated loss of glucose tolerance and a novel lipid analysis tool, Lipid Traffic Analysis, that uses the temporal distribution of lipids to identify differences in the control of lipid metabolism through a time course. Our results suggest that the start of pregnancy is associated with several changes in lipid metabolism, including fewer variables associated with de novo lipogenesis and fewer PUFA-containing lipids in the circulation. Several of the changes in lipid metabolism in healthy pregnancies were less apparent or occurred later in dams who developed GDM. Some changes in maternal lipid metabolism in the obese-GDM group were so late as to only occur as the control dams’ systems began to switch back towards the non-pregnant state. These results demonstrate that lipid metabolism is modulated in healthy pregnancy and the timing of these changes is altered in GDM pregnancies. These findings raise important questions about how lipid metabolism contributes to changes in metabolism during healthy pregnancies. Furthermore, as alterations in the lipidome are present before the loss of glucose tolerance, they could contribute to the development of GDM mechanistically.

Maternal obesity during pregnancy has immediate and long-term detrimental effects on the offspring heart. In this study, we characterized the cardiac and circulatory lipid profiles in fetuses of diet-induced obese pregnant mice and established the changes in lipid abundance and fetal cardiac transcriptomics. We used untargeted and targeted lipidomics and transcriptomics to define changes in the serum and cardiac lipid composition and fatty acid metabolism in male and female fetuses. From these analyses we observed: (1) maternal obesity affects the maternal and fetal serum lipidome distinctly; (2) female heart lipidomes are more sensitive to maternal obesity than male fetuses; (3) changes in lipid supply might contribute to early expression of lipolytic genes in mouse hearts exposed to maternal obesity. These results highlight the existence of sexually dimorphic responses of the fetal heart to the same in utero obesogenic environment and identify lipids species that might mediate programming of cardiovascular health.

Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of structural determination a priori. In the present study, we codify the use of 31P NMR for lipidomics to make the technique more accessible to new users and more useful for a wider range of studies. We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid systems.

Background: Altered lipid metabolism in early life has been associated with subsequent weight gain and predicting this could aid in obesity prevention and risk management. Here, a lipidomic approach was used to identify circulating markers for future obesity risk in translational murine models and validate in a human infant cohort.Methods: Lipidomics was performed on the plasma of APOE*3 Leiden, Ldlr-/-.Leiden, and the wild-type C57BL/6J mice to capture candidate biomarkers predicting subsequent obesity parameters after exposure to high-fat diet. The identified candidate biomarkers were mapped onto corresponding lipid metabolism pathways and were investigated in the Cambridge Baby Growth Study. Infants' growth and adiposity were measured at 0-24 months. Capillary dried blood spots were sampled at 3 months for lipid profiling analysis.Findings: From the mouse models, cholesteryl esters were correlated with subsequent weight gain and other obesity parameters after HFD period (Spearman's r >= 0.5, FDR p values

Summary: Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

BackgroundGenetic, lifestyle, and environmental factors can lead to perturbations in circulating lipid levels and increase risk of cardiovascular and metabolic diseases. However, how changes in individual lipid species contribute to disease risk is often unclear. Moreover, little is known about the role of lipids on cardiovascular disease in Pakistan, a population historically underrepresented in cardiovascular studies.MethodsWe characterised the genetic architecture of the human blood lipidome in 5,662 hospital controls from the Pakistan Risk of Myocardial Infarction Study (PROMIS) and 13,814 healthy British blood donors from the INTERVAL study. We applied a candidate causal gene prioritisation tool to link the genetic variants associated with each lipid to the most likely causal genes, and Gaussian Graphical Modelling network analysis to identify and illustrate relationships between lipids and genetic loci.ResultsWe identified 359 genetic associations with 255 lipids measured using direct infusion high-resolution mass spectrometry in PROMIS, and 616 genetic associations with 326 lipids in INTERVAL. Our analyses revealed new biological insights at genetic loci associated with cardiometabolic diseases, including novel lipid associations at the LPL, MBOAT7, LIPC, APOE-C1-C2-C4, SGPP1, and SPTLC3 loci.ConclusionsOur findings, generated using a distinctive lipidomics platform in an understudied South Asian population, strengthen and expand the knowledge base of the genetic determinants of lipids and their association with cardiometabolic disease-related loci.

Funder: Joint Programming Initiative A healthy diet for a healthy life; doi: http://dx.doi.org/10.13039/100013279, Introduction: Blood-based sample collection is a challenge, and dried blood spots (DBS) represent an attractive alternative. However, for DBSs to be an alternative to venous blood it is important that these samples are able to deliver comparable associations with clinical outcomes. To explore this we looked to see if lipid profile data could be used to predict the concentration of triglyceride, HDL, LDL and total cholesterol in DBSs using markers identified in plasma. Objectives: To determine if DBSs can be used as an alternative to venous blood in both research and clinical settings, and to determine if machine learning could predict ‘clinical lipid’ concentration from lipid profile data. Methods: Lipid profiles were generated from plasma (n = 777) and DBS (n = 835) samples. Random forest was applied to identify and validate panels of lipid markers in plasma, which were translated into the DBS cohort to provide robust measures of the four ‘clinical lipids’. Results: In plasma samples panels of lipid markers were identified that could predict the concentration of the ‘clinical lipids’ with correlations between estimated and measured triglyceride, HDL, LDL and total cholesterol of 0.920, 0.743, 0.580 and 0.424 respectively. When translated into DBS samples, correlations of 0.836, 0.591, 0.561 and 0.569 were achieved for triglyceride, HDL, LDL and total cholesterol. Conclusion: DBSs represent an alternative to venous blood, however further work is required to improve the combined lipidomics and machine learning approach to develop it for use in health monitoring.

Typical lipidomics methods incorporate a liquid&ndash, liquid extraction with LC&ndash, MS quantitation, however, the classic sample extraction methods are not high-throughput and do not perform well at extracting the full range of lipids especially, the relatively polar species (e.g., acyl-carnitines and glycosphingolipids). In this manuscript, we present a novel sample extraction protocol, which produces a single phase supernatant suitable for high-throughput applications that offers greater performance in extracting lipids across the full spectrum of species. We applied this lipidomics pipeline to a ruminant fat dose&ndash, response study to initially compare and validate the different extraction protocols but also to investigate complex lipid biomarkers of ruminant fat intake (adjoining onto simple odd chain fatty acid correlations). We have found 100 lipids species with a strong correlation with ruminant fat intake. This novel sample extraction along with the LC&ndash, MS pipeline have shown to be sensitive, robust and hugely informative (&gt, 450 lipids species semi-quantified): with a sample preparation throughput of over 100 tissue samples per day and an estimated ~1000 biological fluid samples per day. Thus, this work facilitating both the epidemiological involvement of ruminant fat, research into odd chain lipids and also streamlining the field of lipidomics (both by sample preparation methods and data presentation).

Dried blood spots (DBS) and dried milk spots (DMS) represent convenient matrices for collecting and storing human samples. However, the use of these sample types for researching lipid metabolism remains relatively poorly explored, and especially the efficiency of lipid extraction is unclear. A visual inspection of punched DBSs after standard extraction suggests that the samples remain largely intact. DMSs comprise a dense aggregate of milk fat globules on one side of the card, suggesting that the lipid fraction may be physically inaccessible. This led us to the hypotheses that decoagulating may facilitate lipid extraction from both DBSs and DMSs. We tested decoagulation using a mixture of strong chaeotropes (guanidine and thiourea) in both DBS and DMS in the context of high throughput lipidomics (96/384w plate). Extraction of lipids from DMSs was tested with established extractions and one novel solvent mixture in a high throughput format. We found that exposure of DBSs to chaeotropes facilitated collection of the lipid fraction but was ineffective for DMSs. The lipid fraction of DMSs was best isolated without water, using a mixture of xylene/methanol/isopropanol (1:2:4). We conclude that decoagulation is essential for efficient extraction of lipids from DBSs and that a non-aqueous procedure using a spectrum of solvents is the best procedure for extracting lipids from DMSs. These methods represent convenient steps that are compatible with the sample structure and type, and with high throughput lipidomics.HighlightsThe efficiency of lipid extractions on dried milk and dried blood spots was testedThe number of lipid variables and the total signal strength were used as objective measuresDecoagulation of dried blood spots improved extraction efficiencyA mixture of xylene, methanol and isopropanol isolates the lipid fraction best from DMSsAn aqueous extraction using dichloromethane was the most efficient method for isolating lipids from DBSs

Background & aimsGenome-wide association studies in adults have identified variants in HSD17B13 and MARC1 as protective against NAFLD. It is not known if they are similarly protective in children and, more generally, whether the peri-portal inflammation of pediatric NAFLD and lobular inflammation seen in adults share common genetic influences. Therefore, we aimed to: establish if these variants are associated with NAFLD in children, and to investigate the function of these variants in hepatic metabolism using metabolomics.Methods960 children (590 with NAFLD, 394 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MARC1. Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of samples. In silico tools were used to model the effect of rs2642438G>A (p.Ala165Thr) on MARC1.Resultsrs72613567T>TA in HSD17B13 was associated with lower odds of NAFLD diagnosis (OR 0.7 (95%CI 0.6-0.9) and lower grade of portal inflammation (PA in MARC1 was associated with lower grade of hepatic steatosis (P=0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective allele, whereas MARC1 levels were not affected by genotype. Both variants showed downregulation of hepatic fibrotic pathways, upregulation of retinol metabolism and perturbation of phospholipid species. Modelling suggests that p.Ala165Thr would disrupt the stability and metal-binding of MARC1.ConclusionsThere are shared genetic mechanisms between pediatric and adult NAFLD, despite their differences in histology. MARC1 and HSD17B13 are involved in phospholipid metabolism and suppress fibrosis in NAFLD.

Lipidomics is of increasing interest in studies of biological systems. However, high-throughput data collection and processing remains non-trivial, making assessment of phenotypes difficult. We describe a platform for surveying the lipid fraction for a range of tissues. These techniques are demonstrated on a set of seven different tissues (serum, brain, heart, kidney, adipose, liver, and vastus lateralis muscle) from post-weaning mouse dams that were either obese (> 12 g fat mass) or lean (

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children and adults characterized by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease. We hypothesized that change in the concentration of lipid species in pediatric NAFLD would mimic the alterations known to be associated with CVD in adults (and be largely reflective of insulin resistance). Here, we performed untargeted liquid chromatography mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9,500 adults with metabolic phenotyping. Phosphatidylcholine (PC) and triglyceride (TG) desaturation and chain length were inversely associated with histological severity of paediatric NAFLD. Nine lipids species (lyso-PC, PC, and TG classes) were also associated with hepatic steatosis and insulin resistance in an independent cohort of adults. Five of the 9 lipids replicated in the adults cohort (including PC(36:4)) have been directly linked to death and cardiometabolic disease in adults, as well as indirectly via genetic variants that influence the concentration of these species. Together, these findings suggest that lipid metabolism is altered in paediatric NAFLD in a similar way as in cardiometabolic disease in adults and it is therefore critical to alleviate insulin resistance in these children to mitigate their long-term morbidity.

To understand the interaction between diet and health, biomarkers that accurately reflect consumption of foods of perceived health relevance are needed. The aim of this investigation was to use direct infusion-mass spectrometry (DI-MS) lipidomics to determine the effects of fish oil supplementation on lipid profiles of human adipose tissue. Adipose tissue samples from an n-3 polyunsaturated fatty acid (PUFA) supplementation study (n = 66) were analyzed to compare the pattern following supplementation equivalent to zero or four portions of oily fish per week. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were incorporated into highly unsaturated (≥5 double bonds) triglycerides (TGs), phosphocholines, and phosphoethanolamines as well as being detected directly as the nonesterified fatty acid forms. Multivariate statistics demonstrated that phospholipids were the most accurate and sensitive lipids for the assessing EPA and DHA incorporation into adipose tissue. Potential confounding factors (adiposity, age, and sex of the subject) were also considered in the analysis, and adiposity was also associated with an increase in highly unsaturated TGs as a result of incorporation of the n-6 PUFA arachidonic acid. DI-MS provides a high-throughput analysis of fatty acid status that can monitor oily fish consumption, suitable for use in cohort studies.

International audience; At high altitude oxygen delivery to the tissues is impaired leading to oxygen insufficiency (hypoxia). Acclimatisation requires adjustment to tissue metabolism, the details of which remain incompletely understood. Here, metabolic responses to progressive environmental hypoxia were assessed through metabolomic and lipidomic profiling of human plasma taken from 198 human participants before and during an ascent to Everest Base Camp (5,300 m). Aqueous and lipid fractions of plasma were separated and analysed using proton (1 H)-nuclear magnetic resonance spectroscopy and direct infusion mass spectrometry, respectively. Bayesian robust hierarchical regression revealed decreasing isoleucine with ascent alongside increasing lactate and decreasing glucose, which may point towards increased glycolytic rate. Changes in the lipid profile with ascent included a decrease in triglycerides (48-50 carbons) associated with de novo lipogenesis, alongside increases in circulating levels of the most abundant free fatty acids (palmitic, linoleic and oleic acids). Together, this may be indicative of fat store mobilisation. This study provides the first broad metabolomic account of progressive exposure to environmental hypobaric hypoxia in healthy humans. Decreased isoleucine is of particular interest as a potential contributor to muscle catabolism observed with exposure to hypoxia at altitude. Substantial changes in lipid metabolism may represent important metabolic responses to sub-acute exposure to environmental hypoxia. Reduced cellular oxygen availability (hypoxia) is a feature of many disease states. Oxygen delivery may be globally impaired by diseases of the heart or lung, or by anaemia (in which the concentration of oxygen-carrying haemoglobin is reduced); or regionally or locally impaired by macrovascular and microvascular disease respectively 1,2. Diffusion of oxygen in the lungs and/or tissues may be impaired through accumulation of parenchymal fluid and/

We tested the hypothesis that both postnatal feeding and conditions in utero affect lipid metabolism in infants. Infants who experienced restrictive growth conditions in utero and others exposed to maternal hyperglycaemia were compared to a control group with respect to feeding mode. Dried blood spots were collected from a pilot subset of infant participants of the Cambridge Baby Growth Study at 3mo. Groups: (a) a normal gestation (control, n = 40), (b) small for gestational age (SGA, n = 34) and (c) whose mothers developed hyperglycaemia (n = 59). These groups were further stratified by feeding mode; breastfed, formula-fed or received a mixed intake. Their phospholipid, glyceride and sterol fractions were profiled using direct infusion mass spectrometry. Statistical tests were used to identify molecular species that indicated differences in lipid metabolism. The abundance of several phospholipids identified by multivariate analysis, PC(34:1), PC(34:2) and PC-O(34:1), was 30–100% higher across all experimental groups. SM(39:1) was around half as abundant in in utero groups among breastfed infants only. The evidence from this pilot study shows that phospholipid metabolism is modulated by both conditions in utero and postnatal feeding in a cohort of 133 Caucasian infants, three months post partum.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) can progress from simple steatosis (i.e., nonalcoholic fatty liver [NAFL]) to nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. Currently, the driver for this progression is not fully understood; in particular, it is not known how NAFLD and its early progression affects the distribution of lipids in the liver, producing lipotoxicity and inflammation. In this study, we used dietary and genetic mouse models of NAFL and NASH and translated the results to humans by correlating the spatial distribution of lipids in liver tissue with disease progression using advanced mass spectrometry imaging technology. We identified several lipids with distinct zonal distributions in control and NAFL samples and observed partial to complete loss of lipid zonation in NASH. In addition, we found increased hepatic expression of genes associated with remodeling the phospholipid membrane, release of arachidonic acid (AA) from the membrane, and production of eicosanoid species that promote inflammation and cell injury. The results of our immunohistochemistry analyses suggest that the zonal location of remodeling enzyme LPCAT2 plays a role in the change in spatial distribution for AA-containing lipids. This results in a cycle of AA-enrichment in pericentral hepatocytes, membrane release of AA, and generation of proinflammatory eicosanoids and may account for increased oxidative damage in pericentral regions in NASH. CONCLUSION: NAFLD is associated not only with lipid enrichment, but also with zonal changes of specific lipids and their associated metabolic pathways. This may play a role in the heterogeneous development of NAFLD. (Hepatology 2017;65:1165-1180).

[Image: see text] Direct infusion high-resolution mass spectrometry (DIHRMS) is a novel, high-throughput approach to rapidly and accurately profile hundreds of lipids in human serum without prior chromatography, facilitating in-depth lipid phenotyping for large epidemiological studies to reveal the detailed associations of individual lipids with coronary heart disease (CHD) risk factors. Intact lipid profiling by DIHRMS was performed on 5662 serum samples from healthy participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). We developed a novel semi-targeted peak-picking algorithm to detect mass-to-charge ratios in positive and negative ionization modes. We analyzed lipid partial correlations, assessed the association of lipid principal components with established CHD risk factors and genetic variants, and examined differences between lipids for a common genetic polymorphism. The DIHRMS method provided information on 360 lipids (including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids), with a median coefficient of variation of 11.6% (range: 5.4–51.9). The lipids were highly correlated and exhibited a range of associations with clinical chemistry biomarkers and lifestyle factors. This platform can provide many novel insights into the effects of physiology and lifestyle on lipid metabolism, genetic determinants of lipids, and the relationship between individual lipids and CHD risk factors.

At high altitude oxygen delivery to the tissues is impaired leading to oxygen insufficiency (hypoxia). Acclimatisation requires adjustment to tissue metabolism, the details of which remain incompletely understood. Here, metabolic responses to progressive environmental hypoxia were assessed through metabolomic and lipidomic profiling of human plasma taken from 198 human participants before and during an ascent to Everest Base Camp (5,300 m). Aqueous and lipid fractions of plasma were separated and analysed using proton (1H)-nuclear magnetic resonance spectroscopy and direct infusion mass spectrometry, respectively. Bayesian robust hierarchical regression revealed decreasing isoleucine with ascent alongside increasing lactate and decreasing glucose, which may point towards increased glycolytic rate. Changes in the lipid profile with ascent included a decrease in triglycerides (48–50 carbons) associated with de novo lipogenesis, alongside increases in circulating levels of the most abundant free fatty acids (palmitic, linoleic and oleic acids). Together, this may be indicative of fat store mobilisation. This study provides the first broad metabolomic account of progressive exposure to environmental hypobaric hypoxia in healthy humans. Decreased isoleucine is of particular interest as a potential contributor to muscle catabolism observed with exposure to hypoxia at altitude. Substantial changes in lipid metabolism may represent important metabolic responses to sub-acute exposure to environmental hypoxia.

Rationale Lipoproteins belong to the most commonly measured clinical biochemical parameters. Lipidomics is an orthogonal approach and aims to profile the individual lipid molecules that jointly form the lipoprotein particles. However, in the first step of the extraction of lipid molecules from serum, an organic solvent is used leading to dissociation of the lipoproteins. Thus far it has been impossible to combine lipidomics and lipoprotein analysis in one analytical system. Methods Human plasma was diluted in phosphate‐buffered saline (PBS) and injected onto a Superose 6 PC 3.2 column with PBS as a mobile phase to separate lipoproteins. The eluent was led to a Syrris FLLEX module, which also received CHCl3/MeOH (3:1). The two phases were mixed and subsequently separated using a Teflon membrane in an especially designed pressurized flow chamber. The organic phase was led to a standard electrospray source of an Orbitrap mass spectrometer. Results Size‐exclusion chromatography (SEC) has been commonly applied to separate lipoproteins and is considered a practical alternative to ultracentrifugation. Through the on‐line liquid‐liquid extraction method it becomes possible to obtained detailed mass spectra of lipids across different lipoprotein fractions. The extracted ion chromatograms of specific lipid signals showed their distribution against the size of lipoprotein particles. Conclusions The application of on‐line liquid‐liquid extraction allows for the continuous electrospray‐based mass spectral analysis of SEC eluent, providing the detailed lipid composition of lipoprotein particles separated by size. This approach provides new possibilities for the study of the biochemistry of lipoproteins. © 2015 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

INTRODUCTION: Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools. OBJECTIVES: We have developed massPix-an R package for analysing and interpreting data from MSI of lipids in tissue. METHODS: massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries. RESULTS: Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering. CONCLUSION: massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

With the increase in incidence of type 1 diabetes (T1DM), there is an urgent need to understand the early molecular and metabolic alterations that accompany the autoimmune disease. This is not least because in murine models early intervention can prevent the development of disease. We have applied a liquid chromatography (LC-) and gas chromatography (GC-) mass spectrometry (MS) metabolomics and lipidomics analysis of blood plasma and pancreas tissue to follow the progression of disease in three models related to autoimmune diabetes: the nonobese diabetic (NOD) mouse, susceptible to the development of autoimmune diabetes, and the NOD-E (transgenic NOD mice that express the I-E heterodimer of the major histocompatibility complex II) and NOD-severe combined immunodeficiency (SCID) mouse strains, two models protected from the development of diabetes. All three analyses highlighted the metabolic differences between the NOD-SCID mouse and the other two strains, regardless of diabetic status indicating that NOD-SCID mice are poor controls for metabolic changes in NOD mice. By comparing NOD and NOD-E mice, we show the development of T1DM in NOD mice is associated with changes in lipid, purine, and tryptophan metabolism, including an increase in kynurenic acid and a decrease in lysophospholipids, metabolites previously associated with inflammation.

We tested the hypothesis that the lipid composition of infant formula is consistent between manufacturers, countries and target demographic. We developed techniques to profile the lipid and glyceride fraction of milk and formula in a high throughput fashion. Formula from principal brands in the UK (2017&ndash, 2019, bovine-, caprine-, soya-based), the Netherlands (2018, bovine-based) and South Africa (2018, bovine-based) were profiled along with fresh British animal and soya milk and skimmed milk powder. We found that the lipid and glyceride composition of infant formula differed by region, manufacturer and date of manufacture. The formulations within some brands, aimed at different target age ranges, differed considerably where others were similar across the range. Soya lecithin and milk lipids had characteristic phospholipid profiles. Particular sources of fat, such as coconut oil, were also easy to distinguish. Docosahexaenoic acid is typically found in triglycerides rather than phospholipids in formula. The variety by region, manufacturer, date of manufacture and sub-type for target demographics lead to an array of lipid profiles in formula. This makes it impossible to predict its molecular profile. Without detailed profile of the formula fed to infants, it is difficult to characterise the relationship between infant nutrition and their growth and development.

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

OBJECTIVE To investigate the relationship between early second trimester serum lipidomic variation and maternal glycemic traits at 28 weeks and to identify predictive lipid biomarkers for gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS Prospective study of 817 pregnant women (discovery cohort, n = 200; validation cohort, n = 617) who provided an early second trimester serum sample and underwent an oral glucose tolerance test (OGTT) at 28 weeks. In the discovery cohort, lipids were measured using direct infusion mass spectrometry and correlated with OGTT results. Variable importance in projection (VIP) scores were used to identify candidate lipid biomarkers. Candidate biomarkers were measured in the validation cohort using liquid chromatography–mass spectrometry and tested for associations with OGTT results and GDM status. RESULTS Early second trimester lipidomic variation was associated with 1-h postload glucose levels but not with fasting plasma glucose levels. Of the 13 lipid species identified by VIP scores, 10 had nominally significant associations with postload glucose levels. In the validation cohort, 5 of these 10 lipids had significant associations with postload glucose levels that were independent of maternal age and BMI, i.e., TG(51.1), TG(48:1), PC(32:1), PCae(40:3), and PCae(40:4). All except the last were also associated with maternal GDM status. Together, these four lipid biomarkers had moderate ability to predict GDM (area under curve [AUC] = 0.71 ± 0.04, P = 4.85 × 10−7) and improved the prediction of GDM by age and BMI alone from AUC 0.69 to AUC 0.74. CONCLUSIONS Specific early second trimester lipid biomarkers can predict maternal GDM status independent of maternal age and BMI, potentially enhancing risk factor–based screening.

Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high-resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high-speed reversed-phase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome. Copyright © 2009 John Wiley & Sons, Ltd.

Early life exposures and metabolic programming are associated with later disease risk. In particular lipid metabolism is thought to play a key role in the development of the metabolic syndrome and insulin resistance in later life. Investigative studies of metabolic programming are limited by the ethics and practicalities of sample collection in small infants. Dried blood spots on filter paper, derived from heel pricks are considered as the most suitable option for this age group. We validated a novel lipid profiling method, based on high resolution mass spectrometry to successfully determine the lipid composition of infants using dried blood spots. The spotting and air drying of blood on paper has noticeable effects on many of the lipids, leading to lipid oxidation and hydrolysis, which demand careful interpretation of the obtained data. We compared the lipid profiles from plasma or whole blood samples and the results from dried blood spots to determine if these revealed the same inter-subject differences. The results from dried blood spots were no less reproducible than other lipid profiling methods which required comparatively larger sample volumes. Therefore, lipid profiles obtained from dried blood spots can be successfully used to monitor infancy lipid metabolism and we show significant differences in the lipid metabolism of infants at age 3 versus 12 months. Electronic supplementary material The online version of this article (doi:10.1007/s11306-014-0628-z) contains supplementary material, which is available to authorized users.

In the present work we describe the development of an analytical technique for simultaneous profiling of over 100 biochemically related lipid mediators in biological samples. A multistep procedure was implemented to extract eicosanoids and other bioactive lipids from the biological matrix, chromatographically separate them using fast reversed-phase liquid chromatography, tentatively identify new candidate eicosanoids through a matching process of retention times, isotope distribution patterns, and high-resolution orbitrap MS/MS fragmentation patterns, and subsequently quantify tentative candidates by means of analytical reference standards. Key new aspects of this profiling technique included the classification of bioactive lipids into 12 groups according to their calculated exact masses and the development of optimized liquid chromatographic conditions for these groups to achieve sufficient separation of the numerous isobaric and isomeric species, many of which exhibited virtually identical collision-induced dissociation behavior. Importantly, no analytical standards were required at this screening stage of the assay, and tentative identifications were achieved by matching results to selected reference species from each of the groups. The analytical figures of merit for the orbitrap assay such as linear dynamic range, limit of detection, limit of quantitation, and precision demonstrated that the performance of the assay was very similar to that of a quadrupole linear ion trap assay, which was used for validation purposes. The method allowed us to examine eicosanoid profiles within the signaling cascade in chronic lymphocytic leukemia (CLL) cells under basal conditions and following arachidonic acid stimulation. The preliminary screening based on high-resolution tandem mass spectrometry data along with isotope pattern and retention time matching revealed the presence of 15 bioactive lipids, belonging to a range of prostaglandin, leukotriene, and hydroxy and epoxy fatty acid lipid mediators produced by CLL cells.

Introduction Links between early life exposures and later health outcomes may, in part, be due to nutritional programming in infancy. This hypothesis is supported by observed long-term benefits associated with breastfeeding, such as better cognitive development in childhood, and lower risks of obesity and high blood pressure in later life. However, the possible underlying mechanisms are expected to be complex and may be difficult to disentangle due to the lack of understanding of the metabolic processes that differentiate breastfed infants compared to those receiving just formula feed. Objective Our aim was to investigate the relationships between infant feeding and the lipid profiles and to validate specific lipids in separate datasets so that a small set of lipids can be used as nutritional biomarkers. Method We utilized a direct infusion high-resolution mass spectrometry method to analyse the lipid profiles of 3.2 mm dried blood spot samples collected at age 3 months from the Cambridge Baby Growth Study (CBGS-1), which formed the discovery cohort. For validation two sample sets were profiled: Cambridge Baby Growth Study (CBGS-2) and Pregnancy Outcome Prediction Study (POPS). Lipidomic profiles were compared between infant groups who were either exclusively breastfed, exclusively formula-fed or mixed-fed at various levels. Data analysis included supervised Random Forest method with combined classification and regression mode. Selection of lipids was based on an iterative backward elimination procedure without compromising the class error in the classification mode. Conclusion From this study, we were able to identify and validate three lipids: PC(35:2), SM(36:2) and SM(39:1) that can be used collectively as biomarkers for infant nutrition during early development. These biomarkers can be used to determine whether young infants (3–6 months) are breast-fed or receive formula milk. Electronic supplementary material The online version of this article (doi:10.1007/s11306-017-1166-2) contains supplementary material, which is available to authorized users.