This study investigated the effects of docosahexaenoic acid (DHA) supplementation on delayed onset muscle soreness (DOMS), physical function, and inflammation following eccentric exercise-induced muscle damage in physically trained male and female adults (training ≥ 5d/wk). Thirty-eight participants (12 Control, 26 DHA) completed a 12-week double-blind, placebo-controlled matched-pair trial. The Control group received high-oleic acid tablets. The DHA group received 715 mg/d of microencapsulated DHA tablets. Participants performed eccentric cycling at weeks 0 and 12, with assessments conducted pre-exercise, 0-h, 24-h, and 48-h post-exercise. The primary outcomes were DOMS (visual analogue scale) and the Omega-3 Index (O3I) (estimated by finger-stick dry blood spot). Secondary outcomes included neuromuscular function and inflammatory cytokines. O3I was not different between groups at week 0 but was elevated in the DHA group (∆2.43

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin gene (HTT). While classically considered a disease of grey matter, recent imaging data have revealed presymptomatic abnormalities in white matter (WM) tracts. Here, we report lipid changes in glycerophospholipids and sphingolipids from enriched myelin extracts of three WM tracts (internal capsule (IC), dorsomedial prefrontal cortex (dmPFC), corpus callosum (CC)) of HD and control donors. We found no difference in total lipid concentration between HD and control myelin. However, changes were observed at the lipid class level for the CC and dmPFC, with a reduction in the proportion of Hexosylceramides in HD myelin. When lipids were examined at a species level, there was a shift towards shorter glycerophospholipid fatty acid chain length in HD for all three regions, most notably in phosphatidylethanolamine species. This coincides with previous data showing a reduction in fatty acyl chain lengths in sphingolipid species in the caudate of the same donor cohort and suggests a widespread impact of HD on fatty acid metabolism in the brain.

Exercise triggers a proportional inflammatory response that is crucial for muscle fiber repair and adaptation. However, excessive or chronic inflammation can delay recovery and increase injury risk. Biomarkers such as creatine kinase and interleukins are commonly used to assess muscle damage and inflammation, respectively, but they have limitations in specificity and sensitivity. However, lipid mediators that signal all inflammatory events with distinct roles in pro-inflammatory, anti-inflammatory, and pro-resolving phases could provide a more complete understanding of the inflammatory response to exercise. In this review, we discuss the limitations of current biomarkers and the potential of lipid mediators to theoretically offer more precise insights into the inflammatory processes following exercise. Further research is needed to establish standardized protocols for measuring lipid mediators and to understand their temporal dynamics in relation to inflammation and recovery. This knowledge could lead to improved strategies for monitoring and enhancing recovery in athletes.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide valuable insights into the metabolome of complex biological systems such as organ tissues and cells. However, obtaining metabolite data at single-cell spatial resolutions presents a few technological challenges. Generally, spatial resolution is defined by the increment the sample stage moves between laser ablation spots. Stage movements less than the diameter of the focused laser beam (i.e., oversampling) can improve spatial resolution; however, such oversampling conditions result in a reduction in sensitivity. To overcome this, we combine an oversampling approach with laser postionization (MALDI-2), which allows for both higher spatial resolution and improved analyte ionization efficiencies. This approach provides significant enhancements to sensitivity for various metabolite classes (e.g., amino acids, purines, carbohydrates etc.), with mass spectral intensities from 6 to 8 μm pixel sizes (from a laser spot size of ∼13 μm) being commensurate with or higher than those obtained by conventional MALDI at 20 μm pixel sizes for many different metabolites. This technique has been used to map the distribution of metabolites throughout mouse spinal cord tissue to observe how metabolite localizations change throughout specific anatomical regions, such as those distributed to the somatosensory area of the dorsal horn, white matter, gray matter, and ventral horn. Furthermore, this method is utilized for single-cell metabolomics of human iPSC-derived astrocytes at 10 μm pixel sizes whereby many different metabolites, including nucleotides, were detected from individual cells while providing insight into cellular localizations.

AbstractIn recent years there has been a significant interest in the development of innovative lipidomics techniques capable of resolving lipid isomers. To date, methods applied to resolving sn‐isomers have resolved only a limited number of species. We report a workflow based on ozone‐induced dissociation for untargeted characterisation of hundreds of sn‐resolved glycerophospholipid isomers from biological extracts in under 20 min, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn‐isomer pairs identified as compared to previous reports and reveals that sn‐isomer populations are tightly regulated and significantly different between cell lines. The sensitivity of this method and potential for de novo molecular discovery is further demonstrated by the identification of unexpected lipids containing ultra‐long monounsaturated acyl chains at the sn‐1 position.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few treatment options and poor survivability. In this work we sought to characterise metabolic adaptions to gemcitabine (GEMC)-based chemotherapy exposure to discover new therapeutic targets for improving treatment efficacy. We show that GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2 cells through increased activity and expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). We also discovered alternate fatty acid desaturase 2 (FADS2) activity in Panc1 cells, which led to the production of sapienic acid (FA 16:1n-10, cis) from palmitic acid (FA 16:0). Knockdown of key lipid synthesis enzymes sensitised cells to GEMC treatment, with FAS (both cell lines), SCD1 (MiaPaCa2 only) and SCD1+FADS2 (Panc1) knockdown showing the greatest reduction in cell growth when combined with GEMC treatment. In Panc1 cells, either desaturase upregulated its activity when the alternate was knocked down, necessitating the need for dual desaturase knockdown in this cell line. PDAC cells attenuated to grow in combination GEMC/paclitaxel (CombAT) also displayed enhanced de novo lipogenesis; however, combination chemotherapy significantly downregulated FADS2 expression and activity in Panc1 CombAT cells rendering them more sensitive to SCD1 knockdown. We conclude that co-targeting lipid synthesis in PDAC could be a viable strategy for improving the efficacy of both GEMC monotherapy and combination GEMC/PTX therapy. ### Competing Interest Statement The authors have declared no competing interest.

Yeast is a poikilothermic organism and adapts its lipid composition to the environmental temperature to maintain membrane physical properties. Studies addressing temperature-dependent adaptation of the lipidome have described changes in the phospholipid composition at the level of sum composition (e.g. PC 32:1) and molecular composition (e.g. PC 16:0_16:1). However, there is little information at the level of positional isomers (e.g. PC 16:0/16:1 versus PC 16:1/16:0). Here, we used collision- and ozone-induced dissociation (CID/OzID) mass spectrometry to investigate homeoviscous adaptation of PC, PE and PS to determine the phospholipid acyl chains at the sn-1 and sn-2 position. Our data establish the sn-molecular species composition of PC, PE and PS in the lipidome of yeast cultured at different temperatures.

The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon-carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon-carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.

We report a workflow based on ozone-induced dissociation for untargeted characterization of hundreds of sn- resolved glycerophospholipid isomers from biological extracts in under 20 minutes, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn- isomer pairs identified compared to previous reports, reveals that sn -isomer populations are tightly regulated and significantly different between cell lines, and enables identification of rare lipids containing ultra-long chain monounsaturated acyl chains.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) of metabolites can reveal how metabolism is altered throughout heterogeneous tissues. Here negative ion mode MALDI-MSI has been coupled with laser post-ionisation (MALDI-2) and applied to the MSI of low molecular weight (LMW) metabolites (<m/z 600) to investigate the benefits MALDI-2 offers for spatial metabolomics in terms of metabolite coverage and sensitivity. When applied to mouse kidney tissue MALDI-2 provided almost double the number of on-tissue specific mass features compared to conventional MALDI. MALDI-2 also resulted in not only the increased detection sensitivity for multiple metabolite species but also permitted the imaging of LMW metabolites (e.g. uridine) that were not detected using conventional MALDI-MSI. When compared against ∼140 publically available kidney datasets submitted through the METASPACE analysis platform using the same N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) matrix, MALDI-2 provided 34 unique metabolite m/z features that were not consistently annotated previously. To further evaluate the usefulness of this MALDI-2 approach to metabolite imaging, MALDI-2 was applied to the imaging of mouse liver tissue containing a metastasised breast cancer at a pixel size of 20 μm. Using a co-localisation analysis, MALDI-2 detected six tumour-specific metabolites that were not detected using conventional MALDI, as well as providing an up to 20-fold increase in signal intensities for many others (e.g., glutamate). This work provides one of the first reports of MALDI-2 applied to metabolite imaging and demonstrates the dramatic improvements in sensitivity and metabolite coverage it provides.

Recent advances in single-cell genomics and transcriptomics technologies have transformed our understanding of cellular heterogeneity in growth, development, ageing, and disease; however, methods for single-cell lipidomics have comparatively lagged behind in development. We have developed a method for the detection and quantification of a wide range of phosphatidylcholine and sphingomyelin species from single cells that combines fluorescence-assisted cell sorting with automated chip-based nanoESI and shotgun lipidomics. We show herein that our method is capable of quantifying more than 50 different phosphatidylcholine and sphingomyelin species from single cells and can easily distinguish between cells of different lineages or cells treated with exogenous fatty acids. Moreover, our method can detect more subtle differences in the lipidome between cell lines of the same cancer type. Our approach can be run in parallel with other single-cell technologies to deliver near-complete, high-throughput multi-omics data on cells with a similar phenotype and has the capacity to significantly advance our current knowledge on cellular heterogeneity.

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography–mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM.

K. GreenK. NewellS. DownieP. StapleyJ. SteeleT. MitchellJ. SampsonP. ElseH. GroellerB. MeyerM. BrownD. McGheeX.F. Huang

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole–linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18–104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

The caudate, a region of targeted neurodegeneration in Huntington's disease, has a shift in the relative abundance of sphingolipid species influenced by fatty acyl chain length. Long-chain species are favoured over very-long-chain species in multiple classes. Modifications to ceramide synthases may underlie these shifts. Huntington's disease is a devastating neurodegenerative disorder that onsets in late adulthood as progressive and terminal cognitive, psychiatric and motor deficits. The disease is genetic, triggered by a CAG repeat (polyQ) expansion mutation in the Huntingtin gene and resultant huntingtin protein. Although the mutant huntingtin protein is ubiquitously expressed, the striatum degenerates early and consistently in the disease. The polyQ mutation at the N-terminus of the huntingtin protein alters its natural interactions with neural phospholipids in vitro, suggesting that the specific lipid composition of brain regions could influence their vulnerability to interference by mutant huntingtin; however, this has not yet been demonstrated in vivo. Sphingolipids are critical cell signalling molecules, second messengers and membrane components. Despite evidence of sphingolipid disturbance in Huntington's mouse and cell models, there is limited knowledge of how these lipids are affected in human brain tissue. Using post-mortem brain tissue from five brain regions implicated in Huntington's disease (control n = 13, Huntington's n = 13), this study aimed to identify where and how sphingolipid species are affected in the brain of clinically advanced Huntington's cases. Sphingolipids were extracted from the tissue and analysed using targeted mass spectrometry analysis; proteins were analysed by western blot. The caudate, putamen and cerebellum had distinct sphingolipid changes in Huntington's brain whilst the white and grey frontal cortex were spared. The caudate of Huntington's patients had a shifted sphingolipid profile, favouring long (C13-C21) over very-long-chain (C22-C26) ceramides, sphingomyelins and lactosylceramides. Ceramide synthase 1, which synthesizes the long-chain sphingolipids, had a reduced expression in Huntington's caudate, correlating positively with a younger age at death and a longer CAG repeat length of the Huntington's patients. The expression of ceramide synthase 2, which synthesizes very-long-chain sphingolipids, was not different in Huntington's brain. However, there was evidence of possible post-translational modifications in the Huntington's patients only. Post-translational modifications to ceramide synthase 2 may be driving the distinctive sphingolipid profile shifts of the caudate in advanced Huntington's disease. This shift in the sphingolipid profile is also found in the most severely affected brain regions of several other neurodegenerative conditions and may be an important feature of region-specific cell dysfunction in neurodegenerative disease.

Huntington's disease (HD) is a genetic, neurodegenerative illness that onsets in late adulthood as a series of progressive and terminal cognitive, motor, and psychiatric deficits. The disease is caused by a polyQ mutation in the Huntingtin gene (HTT), producing a polyglutamine expansion in the Huntingtin protein (HTT). HTT interacts with phospholipids in vitro; however, its interactions are changed when the protein is mutated in HD. Emerging evidence suggests that the susceptibility of brain regions to pathological stimuli is influenced by lipid composition. This study aimed to identify where and how phospholipids are changed in human HD brain tissue. Phospholipids were extracted using a modified MTBE method from the post-mortem brain of 13 advanced-stage HD patients and 13 age- and sex-matched controls. Targeted precursor ion scanning mass spectrometry was used to detect phospholipid species. In the white cortex of HD patients, there was a significantly lower abundance of phosphatidylcholine (PC) and phosphatidylserine (PS), but no difference in phosphatidylethanolamine (PE). In HD putamen, ester-linked 22:6 was lower in all phospholipid classes promoting a decrease in the relative abundance of ester polyunsaturated fatty acids in PE. No differences in phospholipid composition were identified in the caudate, grey cortex or cerebellum. Ether-linked PE fatty acids appear protected in the HD brain, as no changes were identified. The nature of phospholipid alterations in the HD brain is dependent on the lipid (subclass, species, and bond type) and the location.

Male and female Plasmodium falciparum gametocytes are the parasite lifecycle stage responsible for transmission of malaria from the human host to the mosquito vector. Not only are gametocytes able to survive in radically different host environments, but they are also precursors for male and female gametes that reproduce sexually soon after ingestion by the mosquito. Here, we investigate the sex-specific lipid metabolism of gametocytes within their host red blood cell. Comparison of the male and female lipidome identifies cholesteryl esters and dihydrosphingomyelin enrichment in female gametocytes. Chemical inhibition of each of these lipid types in mature gametocytes suggests dihydrosphingomyelin synthesis but not cholesteryl ester synthesis is important for gametocyte viability. Genetic disruption of each of the two sphingomyelin synthase genes points towards sphingomyelin synthesis contributing to gametocytogenesis. This study shows that gametocytes are distinct from asexual stages, and that the lipid composition is also vastly different between male and female gametocytes, reflecting the different cellular roles these stages play. Taken together, our results highlight the sex-specific nature of gametocyte lipid metabolism, which has the potential to be targeted to block malaria transmission. This article has an associated First Person interview with the first author of the paper.

The rapid increase in lipidomic data has triggered a community-based movement to develop guidelines and minimum requirements for generating, reporting and publishing lipidomic data. The creation of a dynamic checklist summarizing key details of lipidomic analyses using a common language has the potential to harmonize the field by improving both traceability and reproducibility.

Purpose: This study explored whether the non-polar lipids in the human tear fluid lipidome show diurnal variation with and without contact lens wear. It also addressed the relationship between changes in ocular comfort during the day with the level of non-polar lipids. Methods: Tear samples were collected in the morning and evening with and without contact lenses using fine glass capillary tubes and were analysed by chip-based nano-electrospray ionization tandem mass spectrometric techniques. Tear levels of cholesteryl esters (CE), wax esters (WE) and triacylglycerides (TAG) were quantified. Results: TAG 48:0, 52:0 and WE 26:0/16:0, and 27:0/17:0 increased from morning to evening. TAG 52:2, WE 21:0/16:0, 21:0/18:1 and 28:0/18:1 decreased during the day when no lenses were worn. CE 21:0 was the only non-polar lipid that increased from morning to evening in contact lens wear. WE 21:0/16:0 and 27:0/17:0 were lower in the morning in contact lens wear compared to no lens wear (p <= 0.05). The level of non-polar lipids did not correlate with ocular comfort at the end of the day. Conclusion: Even though the level of some of non-polar lipid species changed from morning to evening the total level of major tear non-polar lipids remained unchanged during the day with and without contact lens wear. The effect of change in the quantity and structure of lipid species on tear stability and ocular comfort warrants more investigation.