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In recent years, instrumental improvements have enabled the spread of mass spectrometry-based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies., (© 2024. The Author(s).)

This study delves deeper into the impact of environmental temperature variations on the nervous system in teleost fish. Previous research has demonstrated that exposing adult zebrafish ( Danio rerio ) to 18 °C and 34 °C for 4 or 21 days induces behavioural changes compared to fish kept at a control temperature of 26 °C, suggesting alterations in the nervous system. Subsequent studies revealed that these temperature conditions also modify brain protein expression, indicating potential neurotoxic effects. The primary aim of this work was to investigate the effects of prolonged exposure (21 days) to 18 °C or 34 °C on the brain lipidomes of adult zebrafish compared to a control temperature. Analysis of the brain lipidome highlighted significant alteration in the relative abundances of specific lipid molecules at 18 °C and 34 °C, confirming distinct effects induced by both tested temperatures. Exposure to 18 °C resulted in an increase in levels of phospholipids, such as phosphatidylethanolamine, alongside a general reduction in levels of sphingolipids, including sphingomyelin. Conversely, exposure to 34 °C produced more pronounced effects, with increases in levels of phosphatidylethanolamine and those of various sphingolipids such as ceramide, gangliosides, and sphingomyelin, alongside a reduction in levels of ether phospholipids, including lysophosphatidylethanolamine ether, phosphatidylethanolamine ether, and phosphatidylglycerol ether, as well as levels of glycolipids like monogalactosyldiacylglycerol. These results, when integrated with existing proteomic and behavioural data, offer new insights into the effects of thermal variations on the nervous system in teleost fish. Specifically, our proteomic and lipidomic findings suggest that elevated temperatures may disrupt mitochondrial function, increase neuronal susceptibility to oxidative stress and cytotoxicity, alter axonal myelination, impair nerve impulse transmission, hinder synapse function and neurotransmitter release, and potentially lead to increased neuronal death. These findings are particularly relevant in the fields of cell biology, neurobiology, and ecotoxicology, especially in the context of global warming.

Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipidomic profiling has been reported as an effective approach for characterizing and differentiating brain tumors. However, since lipids can undergo non-specific enzymatic and nonenzymatic reactions due to tissue disruption, it is critical to consider the preanalytical phase of the diagnostic process (e.g., optimizing the sampling time and sampling conditions). Thus, this study assesses the ways in which the time point of sampling impacts the lipidome composition of brain tumors. Two histologically distinct brain tumors-namely, meningiomas and gliomas-were sampled using solid-phase microextraction (SPME) fibers at two time points: on-site directly after removal, and after 12 months of storage at -30 °C. The samples were analyzed via HILIC chromatography coupled with HRMS, which enabled the detection of a wide range of features, including phospholipids and sphingolipids, as well as changes in the profiles of these compounds. The samples obtained from the stored tissues tended to have elevated levels of analytes with lower m/z values. In addition, the samples obtained from the fresh and stored tissues were easily distinguished based on their lipidome compositions, regardless of the histological tumor type. Notably, while storage did not affect the possibility of differentiating meningiomas and gliomas, the biological interpretation of the obtained results were prone to bias., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Barbara Bojko reports financial support was provided by National Science Centre Poland. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chromatography-mass spectrometry-based lipidomics represents an essential tool for elucidating lipid dysfunction mechanisms and is extensively employed in investigating disease mechanisms and identifying biomarkers. However, the detection of low-abundance lipids in biological matrices, along with cumbersome operational procedures, complicates comprehensive lipidomic analyses, necessitating the development of highly sensitive, environmentally friendly, and automated methods. In this study, an online phase transition trapping-supercritical fluid extraction-chromatography-mass spectrometry (PTT-SFEC-MS/MS) method was developed and successfully applied to plasma lipidomics analysis in Type 1 diabetes (T1D) rats. The PTT strategy captured entire extracts at the column head by converting CO 2 from a supercritical state to a gaseous state, thereby preventing peak spreading, enhancing peak shape for precise quantification, and boosting sensitivity without any sample loss. This method utilized only 5 μL of plasma and accomplished sample extraction, separation, and detection within 27 min. Ultimately, 77 differential lipids were identified, including glycerophospholipids, sphingolipids, and glycerolipids, in T1D rat plasma. The results indicated that the progression of the disease might be linked to alterations in glycerophospholipid and sphingolipid metabolism. Our findings demonstrated a green, highly efficient, and automated method for the lipidomics analysis of biological samples, providing a scientific foundation for understanding the pathogenesis and diagnosis of T1D.

Study Design: A cross-sectional, age- and gender-matched study was conducted to investigate the effects of different forms of nicotine delivery on salivary lipid profiles among young adult novice smokers compared to non-smokers., Objective: To assess the effect of smoking traditional cigarettes, e-cigarettes, and heated tobacco products (HTPs) on the levels of specific sphingolipids (sphingosine, sphinganine, and sphingosine-1-phosphate), various ceramides, and lipid peroxidation products [malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)] in both unstimulated and stimulated saliva samples collected from healthy young adults who had been smoking for 1-3 years and used only 1 of the 3 nicotine delivering methods., Methods: Selection criteria included healthy young adults under 30 years old, with normal BMI and typical diet composition, and with no oral inflammatory lesions, orthodontic/dental appliances, or recent intake of medications or supplements. A total of 75 smokers and 25 non-smokers were enrolled in the study. Smokers were categorized into three groups, each comprising 25 individuals: traditional cigarette smokers, e-cigarette users, and HTPs smokers. Saliva samples were collected and analyzed for sphingolipid concentrations using ultra-high-performance liquid chromatography-tandem mass spectrometry. The concentrations of MDA and 4-HNE were measured using colorimetric and ELISA assays, respectively., Results: The average smoking intensity in the traditional cigarette group was 10 cigarettes per day. Salivary sphingolipid and ceramides concentrations were significantly lower in smokers compared to non-smokers across all nicotine delivery methods (p < 0.0001). Moreover, traditional cigarette smokers exhibited higher levels of 4-HNE and MDA in both stimulated and unstimulated saliva, compared to non-smokers (p < 0.01). In stimulated saliva, both MDA and 4-HNE in e-cigarette users, and MDA in HTPs users, showed significantly lower concentrations than their comparators in traditional cigarette smokers (p < 0.01)., Conclusion: Different nicotine delivery methods impact salivary lipid profile during the initial period of smoking habit. Reduced sphingolipids and elevated lipid peroxidation products suggest a disturbed lipid balance in the oral cavity due to enhanced oxidative stress within the salivary glands of novice smokers., (© 2024. The Author(s), under exclusive licence to British Dental Association.)

The lipid based ESCRT-independent mechanism, which contributes to MVB formation, is one of the crucial procedures in exosome biogenesis. n-SMase is a key lipid metabolism enzyme in this mechanism and can induce the hydrolysis of sphingomyelins (SMs) to ceramides (Cers), thereby promoting the formation of ILVs inside MVBs. Therefore, the regulation of n-SMase can realize the alteration in exosome release. According to the fact that cancer-associated cells have a tendency to release more exosomes than healthy cells, lipid extracts in exosomes from healthy volunteers, HCC and ICC patients were analyzed by a novel pseudotargeted lipidomics method focused on sphingolipids (SLs) to explore whether cancer-related features regulate the release of exosomes through the above pathway. Multivariate analysis based on the SLs expression could distinguish three groups well indicated that the SLs expression among the three groups were different. In cancer groups, two species of critical Cers were up-regulated, denoted as Cer (d18:1&#95;16:0) and Cer (d18:1&#95;18:0), while 55 kinds of SLs were down-regulated, including 40 species of SMs, such as SM (d18:1&#95;16:0), SM (d18:1&#95;18:1) and SM (d18:1&#95;24:0). Meanwhile, several species of SM/Cer exhibited significant down-regulation. This substantial enhancement of the SMs hydrolysis to Cers process during exosome biogenesis suggested that cancer-related features may potentially promote an increase in exosome release through ESCRT-independent mechanism. Moreover, differential SLs have a capability of becoming potential biomarkers for disease diagnosis and classification with an AUC value of 0.9884 or 0.9806 for the comparison between healthy group and HCC or ICC groups, respectively. In addition, an association analysis conducted on the cell lines showed that changes in the SM/Cer contents in cells and their exosomes were negatively correlated with the levels of released exosomes, implied the regulation of exosome release levels can be achieved by modulating n-SMase and subsequent SL expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The analysis of ceramide (Cer) and sphingomyelin (SM) lipid species using liquid chromatography-tandem mass spectrometry (LC-MS/MS) continues to present challenges as their precursor mass and fragmentation can correspond to multiple molecular arrangements. To address this constraint, we developed ReTimeML, a freeware that automates the expected retention times (RTs) for Cer and SM lipid profiles from complex chromatograms. ReTimeML works on the principle that LC-MS/MS experiments have pre-determined RTs from internal standards, calibrators or quality controls used throughout the analysis. Employed as reference RTs, ReTimeML subsequently extrapolates the RTs of unknowns using its machine-learned regression library of mass-to-charge (m/z) versus RT profiles, which does not require model retraining for adaptability on different LC-MS/MS pipelines. We validated ReTimeML RT estimations for various Cer and SM structures across different biologicals, tissues and LC-MS/MS setups, exhibiting a mean variance between 0.23 and 2.43% compared to user annotations. ReTimeML also aided the disambiguation of SM identities from isobar distributions in paired serum-cerebrospinal fluid from healthy volunteers, allowing us to identify a series of non-canonical SMs associated between the two biofluids comprised of a polyunsaturated structure that confers increased stability against catabolic clearance., (© 2024. The Author(s).)

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC., Competing Interests: Declaration of competing interest M.H., R.J., and D.W. are listed as inventors on the patent EP 3514545 related to this work. All other authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sphingolipids, including sphingosine and sphinganine, are one of the major classes of lipids. They serve as constituents of cell membranes and lipid rafts and aid in the performance of cell-cell communication and adhesion. Abnormal levels of sphingolipids in the aqueous humor can indicate impaired sphingolipid metabolism and associated ocular pathologies. Sphingolipids can be extracted from the aqueous humor by the methyl-tert-butyl ether (MTBE) lipid extraction method and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). This chapter describes a modified protocol for an MTBE lipid extraction from the aqueous humor, followed by analysis with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Air pollution has been associated with the development of atherosclerosis; however, the pathophysiological mechanisms underlying pro-atherosclerotic effects of air pollution exposure remain unclear. We conducted a prospective panel study in Beijing and recruited 152 participants with four monthly visits from September 2019 to January 2020. Linear mixed-effect models were applied to estimate the associations linking short-term air pollution exposure to biomarkers relevant to ceramide metabolism, pro-inflammation (neutrophil extracellular traps formation and systemic inflammation) and pro-atherosclerotic responses (endothelial stimulation, plaque instability, coagulation activation, and elevated blood pressure). We further explored whether ceramides and inflammatory indicators could mediate the alterations in the profiles of pro-atherosclerotic responses. We found that significant increases in levels of circulating ceramides of 9.7% (95% CIs: 0.7, 19.5) to 96.9% (95% CIs: 23.1, 214.9) were associated with interquartile range increases in moving averages of ambient air pollutant metrics, including fine particulate matter (PM 2.5 ), black carbon, particles in size fractions of 100-560 nm, nitrogen dioxide, carbon monoxide and sulfur dioxide at prior up to 7 days. Higher air pollution levels were also associated with activated neutrophils (increases in citrullinated histone H3, neutrophil elastase, double-stranded DNA, and myeloperoxidase) and exacerbation of pro-atherosclerotic responses (e.g., increases in vascular endothelial growth factor, lipoprotein-associated phospholipase A2, matrix metalloproteinase-8, P-selectin, and blood pressure). Mediation analyses further showed that dysregulated ceramide metabolism and potentiated inflammation could mediate PM 2.5 -associated pro-atherosclerotic responses. Our findings extend the understanding on potential mechanisms of air pollution-associated atherosclerosis, and suggest the significance of reducing air pollution as priority in urban environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The endoplasmic reticulum (ER) plays a key role in the regulation of protein folding, lipid synthesis, calcium homeostasis, and serves as a primary site of sphingolipid biosynthesis. ER stress (ER dysfunction) participates in the development of mitochondrial dysfunction during aging. Mitochondria are in close contact with the ER through shared mitochondria associated membranes (MAM). Alteration of sphingolipids contributes to mitochondria-driven cell injury. Cardiolipin is a phospholipid that is critical to maintain enzyme activity in the electron transport chain. The aim of the current study was to characterize the changes in sphingolipids and cardiolipin in ER, MAM, and mitochondria during the progression of aging in young (3 mo.), middle (18 mo.), and aged (24 mo.) C57Bl/6 mouse hearts. ER stress increased in hearts from 18 mo. mice and mice exhibited mitochondrial dysfunction by 24 mo. Hearts were pooled to isolate ER, MAM, and subsarcolemmal mitochondria (SSM). LC-MS/MS quantification of lipid content showed that aging increased ceramide content in ER and MAM. In addition, the contents of sphingomyelin and monohexosylceramides are also increased in the ER from aged mice. Aging increased the total cardiolipin content in the ER. Aging did not alter the total cardiolipin content in mitochondria or MAM yet altered the composition of cardiolipin with aging in line with increased oxidative stress compared to young mice. These results indicate that alteration of sphingolipids can contribute to the ER stress and mitochondrial dysfunction that occurs during aging., (Published by Elsevier B.V.)

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Echinoderms are of broad interest for abundant bioactive lipids. The comprehensive lipid profiles in eight echinoderm species were obtained by UPLC-Triple TOF-MS/MS with characterization and semi-quantitative analysis of 961 lipid molecular species in 14 subclasses of 4 classes. Phospholipids (38.78-76.83%) and glycerolipids (6.85-42.82%) were the main classes in all investigated echinoderm species, with abundant ether phospholipids, whereas the proportion of sphingolipids was higher in sea cucumbers. Two sulfated lipid subclasses were detected in echinoderms for the first time; sterol sulfate was rich in sea cucumbers, whereas sulfoquinovosyldiacylglycerol existed in the sea star and sea urchins. Furthermore, PC(18:1/24:2), PE(16:0/14:0), and TAG(50:1e) could be used as lipid markers to distinguish eight echinoderm species. In this study, the differentiation of eight echinoderms was achieved by lipidomics and revealed the uniqueness of the natural biochemical fingerprints of echinoderms. The findings will help evaluate the nutritional value in the future.

In our research on sphingolipids from marine invertebrates, a mixture of phytoceramides was isolated from the sponge Monanchora clathrata (Western Australia). Total ceramide, ceramide molecular species (obtained by RP-HPLC, high-performance liquid chromatography on reversed-phase column) and their sphingoid/fatty acid components were analyzed by NMR (nuclear magnetic resonance) spectroscopy and mass spectrometry. Sixteen new ( 1b , 3a , 3c , 3d , 3f , 3g , 5c , 5d , 5f , 5g , 6b - g ) and twelve known ( 2b , 2e , 2f , 3b , 3e , 4a - c , 4e , 4f , 5b , 5e ) compounds were shown to contain phytosphingosine-type backbones i -t17:0 ( 1 ), n -t17:0 ( 2 ), i -t18:0 ( 3 ), n -t18:0 ( 4 ), i -t19:0 ( 5 ), or ai -t19:0 ( 6 ), N -acylated with saturated (2 R )-2-hydroxy C 21 ( a ), C 22 ( b ), C 23 ( c ), i- C 23 ( d ), C 24 ( e ), C 25 ( f ), or C 26 ( g ) acids. The used combination of the instrumental and chemical methods permitted the more detailed investigation of the sponge ceramides than previously reported. It was found that the cytotoxic effect of crambescidin 359 (alkaloid from M. clathrata ) and cisplatin decreased after pre-incubation of MDA-MB-231 and HL-60 cells with the investigated phytoceramides. In an in vitro paraquat model of Parkinson's disease, the phytoceramides decreased the neurodegenerative effect and ROS (reactive oxygen species) formation induced by paraquat in neuroblastoma cells. In general, the preliminary treatment (for 24 or 48 h) of the cells with the phytoceramides of M. clathrata was necessary for their cytoprotective functions, otherwise the additive damaging effect of these sphingolipids and cytotoxic compounds (crambescidin 359, cisplatin or paraquat) was observed.

Extraction protocols and liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) methods for the measurement of four lipid categories, namely glycerophospholipids, glycerolipids, sphingolipids and sterol lipids in human plasma, are described here. Step-by-step instructions are provided for the liquid-liquid extraction methods, including solution preparation and the non-endogenous lipid internal standards used. All instrumental conditions, chromatography columns and solutions required for the LC-MS and LC-MS/MS methods are also provided in detail., (© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

In late November 2019, Prof. Lina M. Obeid passed away from cancer, a disease she spent her life researching and studying its intricate molecular underpinnings. Along with her husband, Prof. Yusuf A. Hannun, Obeid laid down the foundations of sphingolipid biochemistry and oversaw its remarkable evolution over the years. Lipids are a class of macromolecules that are primarily associated with cellular architecture. In fact, lipids constitute the perimeter of the cell in such a way that without them, there cannot be cells. Hence, much of the early research on lipids identified the function of this class of biological molecules as merely structural. Nevertheless, unlike proteins, carbohydrates, and nucleic acids, lipids are elaborately diverse as they are not made up of monomers in polymeric forms. This diversity in structure is clearly mirrored by functional pleiotropy. In this chapter, we focus on a major subset of lipids, sphingolipids, and explore their historic rise from merely inert structural components of plasma membranes to lively and necessary signaling molecules that transmit various signals and control many cellular processes. We will emphasize the works of Lina Obeid since she was an integral pillar of the sphingolipid research world., (Copyright © 2023. Published by Elsevier Inc.)

Pichia pastoris is an expression platform widely used for foreign protein expression, while it is unknown how the global lipid profiles changed during the cultivation process, which is crucial for fermentation optimization and chassis design. Therefore, this study aimed to reveal the diverse lipid profiles of P. pastoris controlled by constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase gene and to unravel their change in the lag, logarithmic, stationary, and death phases, using ultra-performance liquid chromatography/nano-electrospray ionization-tandem mass spectrometry. Two hundred forty lipid species across 11 lipid classes were detected, including various glycerolipids, glycerophospholipids, and sphingolipids. Pichia cells displayed high diversity and variability of lipids in lipid profile, relative intensity, phosphatidylinositol/phosphatidylserine ratio, fatty acid chain length, and unsaturation degree. Notably, increase of unsaturated triacylglycerol level was accompanied by rise of malondialdehyde level under oxidative stress. The increased ceramide with long fatty acid chain could be a key feature at death phase. This work deepened our understanding of the physiology of P. pastoris during cultivation and provided valuable information for further improvement of the P. pastoris expression system.

Lipids perform multiple biological functions and reflect the physiology and pathology of cells, tissues, and organs. Here, we sought to understand lipid content in relation to tumor pathology by characterizing phospholipids and sphingolipids in the orthotopic mouse glioma using MALDI MS imaging (MSI) and LC-MS/MS. Unsupervised clustering analysis of the MALDI-MSI data segmented the coronal tumoral brain section into 10 histopathologically salient regions. Heterogeneous decrease of the common saturated phosphatidylcholines (PCs) in the tumor was accompanied by the increase of analogous PCs with one or two additional fatty acyl double bonds and increased lyso-PCs. Polyunsaturated fatty acyl-PCs and ether PCs highlighted the striatal tumor margins, whereas the distributions of other PCs differentiated the cortical and striatal tumor parenchyma. We detected a reduction of SM d18:1/18:0 and the heterogeneous mild increase of SM d18:1/16:0 in the tumor, whereas ceramides accumulated only in a small patch deep in the tumoral parenchyma. LC-MS/MS analyses of phospholipids and sphingolipids complemented the MALDI-MSI observation, providing a snapshot of these lipids in the tumor. Finally, the proposed mechanisms responsible for the tumoral lipid changes were contrasted with our interrogation of gene expression in human glioma. Together, these lipidomic results unveil the aberrant and heterogeneous lipid metabolism in mouse glioma where multiple lipid-associated signaling pathways underline the tumor features, promote the survival, growth, proliferation, and invasion of different tumor cell populations, and implicate the management strategy of a multiple-target approach for glioma and related brain malignancies., Competing Interests: Conflict of Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes / Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Background: Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in <5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination., Objectives: We sought to identify circulatory and skin lipid biomarkers associated with EH and EV., Methods: Stratum corneum and plasma samples from 15 subjects with AD and a history of EH, 13 age- and gender-matched subjects with AD and without EH history, and 13 healthy nonatopic (NA) controls were analyzed by liquid chromatography tandem mass spectrometry for sphingolipid content. Sphingosine-1-phosphate (S1P) and ceramide levels were validated in plasma samples from the Atopic Dermatitis Vaccinia Network/Atopic Dermatitis Research Network repository (12 NA, 12 AD, 23 EH) and plasma from 7 subjects with EV and 7 matched subjects with AD. S1P lyase was downregulated in human primary keratinocytes to evaluate its effect on herpes simplex virus 1 (HSV-1) replication in vitro., Results: The stratum corneum of patients with EH demonstrated significantly higher levels of free sphingoid bases than those in patients who were NA, indicating enhanced sphingolipid turnover in keratinocytes (P < .05). Plasma from 2 independent cohorts of patients with EH had a significantly increased S1P/ceramide ratio in subjects with EH versus those with AD and or who were NA (P < .01). The S1P level in plasma from subjects with EV was twice the level in plasma from subjects with AD (mean = 1,533 vs 732 pmol/mL; P < .001). Downregulation of S1P lyase expression with silencing RNA led to an increased S1P level and doubled HSV-1 titer in keratinocytes., Conclusions: Our data point to long-term abnormalities in the S1P signaling system as a biomarker for previous disseminated viral diseases and a potential treatment target in recurring infections., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Sphingolipids are essential building blocks of eukaryotic membranes and important signaling molecules that are regulated tightly in response to environmental and physiological inputs. While their biosynthetic pathway has been well-described, the mechanisms that facilitate the perception of sphingolipid levels at the plasma membrane remain to be uncovered. In Saccharomyces cerevisiae, the Nce102 protein has been proposed to function as a sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that the production of Nce102 increases following sphingolipid biosynthesis inhibition and that Nce102 is internalized when excess sphingolipid precursors are supplied. This finding suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. The physiological role of Nce102 in the regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of hydroxylated complex sphingolipids in response to heat stress in the nce102 Δ deletion mutant. We also demonstrate that Nce102 behaves analogously in the widespread human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species. IMPORTANCE Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately. The primary site of both stress sensing and adaptation is the plasma membrane. We identified the yeast protein Nce102 as a marker of local sphingolipid levels and fluidity in the plasma membrane. Nce102 is an important structural and functional component of the membrane compartment Can1 (MCC), a plasma membrane microdomain stabilized by a large cytosolic hemitubular protein scaffold, the eisosome. The MCC/eisosomes are widely conserved among fungi and unicellular algae. To determine if Nce102 carries out similar functions in other organisms, we analyzed the human fungal pathogen Candida albicans and found that Nce102 responds to sphingolipid levels also in this organism, which has potential applications for the development of novel therapeutic approaches. The presented study represents a valuable model for how organisms regulate plasma membrane sphingolipids.

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

Sphingolipids are a class of lipids with high structural diversity and biological pleiotropy. Mounting evidence supports a role for sphingolipids in regulating pathophysiology of cardiometabolic diseases, and they have been proposed as potential cardiometabolic biomarkers. Current methods for quantifying sphingolipids require laborious pretreatment and relatively large sample volumes, and cover limited species, hindering their application in epidemiological studies. Herein, we applied a time-, labor-, and sample-saving protocol simply using methanol for plasma sphingolipid extraction. It was compared with classical liquid-liquid extraction methods and showed significant advantages in terms of simplicity, sphingolipid coverage, and sample volume. By coupling the protocol with liquid chromatography using a wide-span mobile phase polarity parameter and tandem mass spectrometry operated in dynamic multiple reaction monitoring mode, 37 sphingolipids from 8 classes (sphingoid base, sphingoid base phosphate, ceramide-1-phosphate, lactosylceramide, hexosylceramide, sphingomyelin, ceramide, and dihydroceramide) were quantified within 16 min, using only 10 μL of human plasma. The current method showed good performance in terms of linearity (R 2  > 0.99), intra- and interbatch accuracy (70-123%) and precision (RSD < 12%), matrix effect (91-121%), recovery (96-101%), analyte chemical stability (deviation < 19%), and carryover (< 16%). We successfully applied this method to quantify 33 detectable sphingolipids from 579 plasma samples of an epidemiological study within 10 days. The quantified sphingolipid concentrations were comparable with previous studies. Positive associations of ceramide C22:0/C24:0 and their precursors with homeostasis model assessment of insulin resistance suggested that the synthesis of the ceramides might be involved in insulin resistance. This novel method constitutes a simple and rapid approach to quantify circulating sphingolipids for epidemiological studies using targeted lipidomic analysis, which will help elucidate the sphingolipid-regulated pathways underlying cardiometabolic diseases., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Phospholipids (PLs) and sphingolipids (SLs) perform critical structural and biological functions in cells. The structure of these lipids, including the stereospecificity and double-bond position of fatty acyl (FA) chains, is critical in decoding lipid biology. In this study, we presented a simple in-source fragmentation (ISF) MALDI/TOF mass spectrometry method that affords complete structural characterization of PL and SL molecules. We analyzed several representative unsaturated lipid species including phosphatidylcholine (PC), plasmalogen PC (pPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin (CL), sphingomyelin (SM), and ceramide (Cer). Fragment ions reflecting the FA chains at sn-1 and sn-2 position, and those characteristics of the head groups of different PL classes, are readily identified. Specific fragment ions from cleavages of the C-C bond immediately adjacent to the cis C=C double-bond position(s) of FA chains and the trans C=C double bond of the sphingosine constituents allow precise localization of double bonds. The identities of the exemplary product ions from vinylic, allylic, and double-bond cleavages were also verified by LIFT-TOF/TOF. Identification of individual PL species in the lipid mixture was also carried out with ISF-MALDI/TOF. Together, this approach provides a simple yet effective method for structural characterization of PLs and SLs without the additional modification on the instrument hardware, and serves as a simple tool for the identification of lipids., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Sphingolipids (SLs) are structural components of the lipid bilayer regulating cell functions. In biological fluids, their distribution is sex-specific and is at variance in aging and many disorders. The aim of this study is to identify SL species associated with the decelerated aging of centenarians. SLs, extracted from serum of adults (Ad, 35-37 years old), aged (Ag, 75-77 years old) and centenarian (C, 105-107 years old) women were analyzed by LC-MS/MS in combination with mRNA levels in peripheral blood mononuclear cells (PBMCs) of SL biosynthetic enzymes. Results indicated in Ag and C vs. Ad a comparable ceramides (Cers) increase, whereas dihydroceramide (dhCer) decreased in C vs. Ad. Hexosylceramides (HexCer) species, specifically HexCer 16:0, 22:0 and 24:1 acyl chains, increased in C vs. Ag representing a specific trait of C. Sphingosine (Sph), dihydrosphingosine (dhSph), sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), increased both in Ag and C vs. Ad, with higher levels in Ag, indicating a SL fine-tuning associated with a reduced physiological decline in C. mRNA levels of enzymes involved in ceramide de novo biosynthesis increased in Ag whereas enzymes involved in sphingomyelin (SM) degradation increased in C. Collectively, results suggest that Ag produce Cers by de novo synthesis whereas C activate a protective mechanism degrading SMs to Cers converting it into glycosphingolipids.

The viral lifecycle is critically dependent upon host lipids. Enveloped viral entry requires fusion between viral and cellular membranes. Once an infection has occurred, viruses may rely on host lipids for replication and egress. Upon exit, enveloped viruses derive their lipid bilayer from host membranes during the budding process. Furthermore, host lipid metabolism and signaling are often hijacked to facilitate viral replication. We employed an untargeted HILIC-IM-MS lipidomics approach and identified host lipid species that were significantly altered during vesicular stomatitis virus (VSV) infection. Many glycerophospholipid and sphingolipid species were modified, and ontological enrichment analysis suggested that the alterations to the lipid profile change host membrane properties. Lysophosphatidylcholine (LPC), which can contribute to membrane curvature and serve as a signaling molecule, was depleted during infection, while several ceramide sphingolipids were augmented during infection. Ceramide and sphingomyelin lipids were also enriched in viral particles, indicating that sphingolipid metabolism is important during VSV infection.

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 μm lateral resolution allows for the analysis of Arabidopsis thaliana ( A. thaliana ) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

Traumatic brain injury (TBI) causes neuroinflammation and neurodegeneration leading to various pathological complications such as motor and sensory (visual) deficits, cognitive impairment, and depression. N-3 polyunsaturated fatty acid (n-3 PUFA) containing lipids are known to be anti-inflammatory, whereas the sphingolipid, ceramide (Cer), is an inducer of neuroinflammation and degeneration. Using Fat1 + -transgenic mice that contain elevated levels of systemic n-3 PUFA, we tested whether they are resistant to mild TBI-mediated sensory-motor and emotional deficits by subjecting Fat1-transgenic mice and their WT littermates to focal cranial air blast (50 psi) or sham blast (0 psi, control). We observed that visual function in WT mice was reduced significantly following TBI but not in Fat1 + -blast animals. We also found Fat1 + -blast mice were resistant to the decline in motor functions, depression, and fear-producing effects of blast, as well as the reduction in the area of oculomotor nucleus and increase in activated microglia in the optic tract in brain sections seen following blast in WT mice. Lipid and gene expression analyses confirmed an elevated level of the n-3 PUFA eicosapentaenoic acid (EPA) in the plasma and brain, blocking of TBI-mediated increase of Cer in the brain, and decrease in TBI-mediated induction of Cer biosynthetic and inflammatory gene expression in the brain of the Fat1 + mice. Our results demonstrate that suppression of ceramide biosynthesis and inflammatory factors in Fat1 + -transgenic mice is associated with significant protection against the visual, motor, and emotional deficits caused by mild TBI. This study suggests that n-3 PUFA (especially, EPA) has a promising therapeutic role in preventing neurodegeneration after TBI., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Drug resistance is responsible for the failure of many available anticancer drugs. Several studies have demonstrated the association between the alteration in sphingolipids (SPLs) and the development of drug resistance. To investigate the association between SPLs metabolism and doxorubicin (dox)-resistance in MCF-7 cells, a comparative sphingolipidomics analysis between dox-sensitive (parental) and -resistant MCF-7 cell lines along with validation by gene expression analysis were conducted. A total of 31 SPLs representing 5 subcategories were identified. The data obtained revealed that SPLs were clustered into two groups differentiating parental from dox-resistant cells. Eight SPLs were significantly altered in response to dox-resistance including SM (d18:1/16), SM (d18:1/24:2), SM (d18:1/24:0), SM (d18:1/20:0), SM (d18:1/23:1), HexCer (d18:1/24:0), SM (d18:1/15:0), DHSM (d18:0/20:0). The current study is the first to conclusively ascertain the potential involvement of dysregulated SPLs in dox-resistance in MCF-7 cells. SPLs metabolism in dox-resistant MCF-7 cells is oriented toward the downregulation of ceramides (Cer) and the concomitant increase in sphingomyelin (SM). Gene expression analysis has revealed that dox-resistant cells tend to escape from the Cer-related apoptosis by the activation of SM-Cer and GluCer-LacCer-ganglioside pathways. The enzymes that were correlated to the alteration in SPLs metabolism of dox-resistant MCF-7 cells and significantly altered in gene expression can represent potential targets that can represent a winning strategy for the future development of promising anticancer drugs., Competing Interests: The authors have declared that no competing interests exist.

The reason behind the high inter-individual variability in response to SARS-CoV-2 infection and patient's outcome is poorly understood. The present study targets the sphingolipid profile of twenty-four healthy controls and fifty-nine COVID-19 patients with different disease severity. Sera were analyzed by untargeted and targeted mass spectrometry and ELISA. Results indicated a progressive increase in dihydrosphingosine, dihydroceramides, ceramides, sphingosine, and a decrease in sphingosine-1-phosphate. These changes are associated with a serine palmitoyltransferase long chain base subunit 1 (SPTLC1) increase in relation to COVID-19 severity. Severe patients showed a decrease in sphingomyelins and a high level of acid sphingomyelinase (aSMase) that influences monosialodihexosyl ganglioside (GM3) C16:0 levels. Critical patients are characterized by high levels of dihydrosphingosine and dihydroceramide but not of glycosphingolipids. In severe and critical patients, unbalanced lipid metabolism induces lipid raft remodeling, leads to cell apoptosis and immunoescape, suggesting active sphingolipid participation in viral infection. Furthermore, results indicated that the sphingolipid and glycosphingolipid metabolic rewiring promoted by aSMase and GM3 is age-dependent but also characteristic of severe and critical patients influencing prognosis and increasing viral load. AUCs calculated from ROC curves indicated ceramides C16:0, C18:0, C24:1, sphingosine and SPTLC1 as putative biomarkers of disease evolution.

There is an unmet need to develop analytical strategies that not only characterize the lipid composition of the viral envelope but also do so on a time scale that would allow for high-throughput analysis. With that in mind, we report the use of atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) combined with lithium adduct consolidation to profile total lipid extracts rapidly and confidently from enveloped viruses. The use of AP-MALDI reduced the dependency of using a dedicated MALDI mass spectrometer and allowed for interfacing the MALDI source to a mass spectrometer with the desired features, which included high mass resolving power (>100000) and tandem mass spectrometry. AP-MALDI combined with an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone spiked with lithium salt, resulted in a robust and high-throughput lipid detection platform, specifically geared to sphingolipid detection. Application of the developed workflow included the structural characterization of prominent sphingolipids and detection of over 130 lipid structures from Influenza A virions. Overall, we demonstrate a high-throughput workflow for the detection and structural characterization of total lipid extracts from enveloped viruses using AP-MALDI HRMS and lithium adduct consolidation.

Objective: Lipidomic changes were causally linked to metabolic diseases, but the scenario for colorectal cancer (CRC) is less clear. We investigated the CRC lipidome for putative tumor-specific alterations through analysis of 3 independent retrospective patient cohorts from 2 clinical centers, to derive a clinically useful signature., Design: Quantitative comprehensive lipidomic analysis was performed using direct infusion electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS) and high-resolution mass spectrometry (HR-MS) on matched nondiseased mucosa and tumor tissue in a discovery cohort (n = 106). Results were validated in 2 independent cohorts (n = 28, and n = 20), associated with genomic and clinical data, and lipidomic data from a genetic mouse tumor model (Apc 1638N )., Results: Significant differences were found between tumor and normal tissue for glycero-, glycerophospho-, and sphingolipids in the discovery cohort. Comparison to the validation collectives unveiled that glycerophospholipids showed high interpatient variation and were strongly affected by preanalytical conditions, whereas glycero- and sphingolipids appeared more robust. Signatures of sphingomyelin and triacylglycerol (TG) species significantly differentiated cancerous from nondiseased tissue in both validation studies. Moreover, lipogenic enzymes were significantly up-regulated in CRC, and FASN gene expression was prognostically detrimental. The TG profile was significantly associated with postoperative disease-free survival and lymphovascular invasion, and was essentially conserved in murine digestive cancer, but not associated with microsatellite status, KRAS or BRAF mutations, or T-cell infiltration., Conclusion: Analysis of the CRC lipidome revealed a robust TG-species signature with prognostic potential. A better understanding of the cancer-associated glycerolipid and sphingolipid metabolism may lead to novel therapeutic strategies., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Accumulating evidence indicates alterations in lipid metabolism and lipid composition in neoplastic tissue. Earlier nuclear magnetic resonance studies showed that the contents of major lipid groups, such as triacylglycerols, phospholipids and cholesterol, are changed in colon cancer tissue., Methods: In this study, a more detailed analysis of lipids in cancer and tumor adjacent tissues from colorectal cancer patients, using liquid chromatography-mass spectrometry, allowed for comparison of 199 different lipids between cancer tissue and tumor adjacent tissue using principal component analysis., Results: Significant differences were found in 67 lipid compounds between the two types of tissue; many of these lipid compounds are bioactive lipids such as ceramides, lysophospholipids or sterols and can influence the development of cancer. Additionally, increased levels of phospholipids and sphingolipids were present, which are major components of the cell membrane, and increases in these lipids can lead to changes in cell membrane properties., Conclusions: This study showed that many complex lipids are significantly increased or decreased in colon cancer tissue, reflecting significant alterations in lipid metabolism. This knowledge can be used for the selection of potential molecular targets of novel anticancer strategies based on the modulation of lipid metabolism and the composition of the cell membrane in colorectal cancer cells., (© 2021. The Author(s).)

Lipid rafts, membrane microdomains enriched with (glyco)sphingolipids, cholesterol, and select proteins, act as cellular signalosomes. Various methods have been used to separate lipid rafts from bulk (non-raft) membranes, but most often, non-ionic detergent Triton X-100 has been used in their isolation. However, Triton X-100 is a reported disruptor of lipid rafts. Histological evidence confirmed raft disruption by Triton X-100, but remarkably revealed raft stability to treatment with a related polyethylene oxide detergent, Brij O20. We report isolation of detergent-resistant membranes from mouse brain using Brij O20 and its use to determine the distribution of major mammalian brain gangliosides, GM1, GD1a, GD1b and GT1b. A different distribution of gangliosides-classically used as a raft marker-was discovered using Brij O20 versus Triton X-100. Immunohistochemistry and imaging mass spectrometry confirm the results. Use of Brij O20 results in a distinctive membrane distribution of gangliosides that is not all lipid raft associated, but depends on the ganglioside structure. This is the first report of a significant proportion of gangliosides outside raft domains. We also determined the distribution of proteins functionally related to neuroplasticity and known to be affected by ganglioside environment, glutamate receptor subunit 2, amyloid precursor protein and neuroplastin and report the lipid raft populations of these proteins in mouse brain tissue. This work will enable more accurate lipid raft analysis with respect to glycosphingolipid and membrane protein composition and lead to improved resolution of lipid-protein interactions within biological membranes., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Yeast is a suitable model system to analyze the mechanism of lifespan. In this study, to identify novel factors involved in chronological lifespan, we isolated a mutant with a long chronological lifespan and found a missense mutation in the sur2+ gene, which encodes a homolog of Saccharomyces cerevisiae sphingolipid C4-hydroxylase in fission yeast. Characterization of the mutant revealed that loss of sur2 function resulted in an extended chronological lifespan. The effect of caloric restriction, a well-known signal for extending lifespan, is thought to be dependent on the sur2+ gene., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human milk lipids are an important energy source and essential nutrients for the growth and development of infants. The UPLC/Q-TOF-MS was used to qualitatively and quantitatively analyze human milk lipids. Totally, 411 species of lipids were identified, in which the content of OPL was generally higher than that of OPO; SM (75.38 mg/L, 40.39%), PE (51.12 mg/L, 27.39%) and PC (40.10 mg/L, 21.49%) had the highest contents among polar lipids, mainly including SM42:2:2 (22.24 mg/L), PE36:2 (C18:0-C18:2, 21.39 mg/L) and PC36:2 (C18:0-C18:2, 19.80 mg/L). In human milk, TAG56:7 (137.14 mg/L), TAG56:8 (59.49 mg/L), TAG58:8 (65.90 mg/L) and TAG58:9 (49.99 mg/L) were the main sources of AA and DHA; PE was an important source of AA and DHA in polar lipids; and linoleic acyl in glycerides and phospholipids had higher contents than other polyunsaturated fatty acyls. These results provided the scientific basis for the simulation of human milk at molecular level., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acid (AC), neutral (NC) and alkaline ceramidase 3 (ACER3) are the most ubiquitous ceramidases and their therapeutic interest as targets in cancer diseases has been well sustained. This supports the importance of discovering potent and specific inhibitors for further use in combination therapies. Although several ceramidase inhibitors have been reported, most of them target AC and a few focus on NC. In contrast, well characterized ACER3 inhibitors are lacking. Here we report on the synthesis and screening of two series of 1-deoxy(dihydro)ceramide analogs on the three enzymes. Activity was determined using fluorogenic substrates in recombinant human NC (rhNC) and both lysates and intact cells enriched in each enzyme. None of the molecules elicited a remarkable AC inhibitory activity in either experimental setup, while using rhNC, several compounds of both series were active as non-competitive inhibitors with K i values between 1 and 5 μM. However, a dramatic loss of potency occurred in NC-enriched cell lysates and no activity was elicited in intact cells. Interestingly, several compounds of Series 2 inhibited ACER3 dose-dependently in both cell lysates and intact cells with IC 50 's around 20 μM. In agreement with their activity in live cells, they provoked a significant increase in the amounts of ceramides. Overall, this study identifies highly selective ACER3 activity blockers in intact cells, opening the door to further medicinal chemistry efforts aimed at developing more potent and specific compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Sphingolipid biosynthesis occurs in both the endoplasmic reticulum (ER) and the Golgi apparatus. Ceramide synthesized in the ER is transported to the Golgi and incorporated into complex sphingolipids. Here, we present a step-by-step protocol to analyze sphingolipid metabolism in budding yeast. Ceramide and inositolphosphorylceramide (IPC) are classes of sphingolipids present in yeast and are metabolically labeled with radioactive precursors. This protocol for metabolic labeling can be used to investigate ceramide transport in an in vivo environment. For complete details on the use and execution of this protocol, please refer to Ikeda et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

Fusarium verticillioides causes multiple diseases of Zea mays (maize) including ear and seedling rots, contaminates seeds and seed products worldwide with toxic chemicals called fumonisins. The role of fumonisins in disease is unclear because, although they are not required for ear rot, they are required for seedling diseases. Disease symptoms may be due to the ability of fumonisins to inhibit ceramide synthase activity, the expected cause of lipids (fatty acids, oxylipins, and sphingolipids) alteration in infected plants. In this study, we explored the impact of fumonisins on fatty acid, oxylipin, and sphingolipid levels in planta and how these changes affect F. verticillioides growth in maize. The identity and levels of principal fatty acids, oxylipins, and over 50 sphingolipids were evaluated by chromatography followed by mass spectrometry in maize infected with an F. verticillioides fumonisin-producing wild-type strain and a fumonisin-deficient mutant, after different periods of growth. Plant hormones associated with defense responses, i.e., salicylic and jasmonic acid, were also evaluated. We suggest that fumonisins produced by F. verticillioides alter maize lipid metabolism, which help switch fungal growth from a relatively harmless endophyte to a destructive necrotroph.

While previous studies have characterized the fatty acids and global lipid families of the chicken egg yolk, there have been no publications characterizing the individual lipids in these lipid families. Such an in-depth characterization of egg yolk lipids is essential to define the potential benefits of egg yolk consumption for the supply of structural and anti-inflammatory lipids. Historically, the major focus has been on the cholesterol content of eggs and the potential negative health benefits of this lipid, while ignoring the essential roles of cholesterol in membranes and as a precursor to other essential sterols. A detailed analysis of egg yolk lipids, using high-resolution mass spectrometric analyses and tandem mass spectrometry to characterize the fatty acid substituents of complex structural lipids, was used to generate the first in-depth characterization of individual lipids within lipid families. Egg yolks were isolated from commercial eggs (Full Circle Market) and lipids extracted with methyl-t-butylether before analyses via high-resolution mass spectrometry. This analytical platform demonstrates that chicken egg yolks provide a rich nutritional source of complex structural lipids required for lipid homeostasis. These include dominant glycerophosphocholines (GPC) (34:2 and 36:2), plasmalogen GPC (34:1, 36:1), glycerophosphoethanolamines (GPE) 38:4 and 36:2), plasmalogen GPE (36:2 and 34:1), glycerophosphoserines (36:2 and 38:4), glycerophosphoinositols (38:4), glycerophosphoglycerols (36:2), N-acylphosphatidylethanolamines (NAPE) (56:6), plasmalogen NAPE (54:4 and 56:6), sphingomyelins (16:0), ceramides (22:0 and 24:0), cyclic phosphatidic acids (16:0 and 18:0), monoacylglycerols (18:1 and 18:2), diacylglycerols (36:3 and 36:2), and triacylglycerols (52:3). Our data indicate that the egg yolk is a rich source of structural and energy-rich lipids. In addition, the structural lipids possess ω-3 and ω-6 fatty acids that are essential precursors of endogenous anti-inflammatory lipid mediators. These data indicate that eggs are a valuable nutritional addition to the diets of individuals that do not have cholesterol issues., (Copyright © 2020. Published by Elsevier Inc.)

Sphingolipids, including ceramide (Cer) and glucosylceramide (GlcCer), have the characteristic structural units called sphingoid bases, and are constituents of cell and vacuole membranes. Plant sphingolipids bear highly diverse base structures and the base composition differs depending on the plant species. It is thought that the composition of sphingolipid classes and sphingoid bases is related to membrane fractions. However, there is little information about differences in sphingolipids among plant cultivars and the changes occurring in sphingolipids during food processing. This study investigated sphingolipids in sake rice (saka-mai) cultivars grown for sake (rice wine), and the changes in sphingolipids during polishing and brewing. In six brown rice samples, there were no large differences of the base composition among Cer or GlcCer of cultivars, whereas there were differences in their sphingolipid contents. When compared to brown rice, highly polished rice contained lower levels of sphingolipids, especially Cer. For three rice brans from different polishing steps, the Cer content was higher in the outer bran than in the inner bran. Sake and sake lees (sake-kasu) were produced by three different starter cultures (shubo preparations: the mixture of koji rice as an enzyme cocktail containing amylases, sake yeast, and adding rice as a carbohydrate source). The Cer/GlcCer ratio in sake and sake lees depended on the starter culture; Cer and GlcCer in sake lees possessed a fungi-specific base, 9-methyl-trans-4,trans-8- sphingadienine. In addition, sake lees had a higher Cer/GlcCer ratio when compared to highly polished rice as a sake source. These results suggest that the sphingolipid content of brown rice differs depending on the rice cultivar; further, the sphingolipids and the sphingolipid composition in sake and sake lees are affected by fungal sphingolipids and self-digestion during brewing.

Sphingolipids have diverse structural and bioactive functions that play important roles in many key biological processes. Factors such as low relative abundance, varied structures, and a dynamic concentration range provide a difficult analytical challenge for sphingolipid detection. To further improve mass-spectrometry-based sphingolipid analysis, lithium adduct consolidation was implemented to decrease spectral complexity and combine signal intensities, leading to increased specificity and sensitivity. We report the use of lithium hydroxide as a base in a routine hydrolysis procedure in order to effectively remove common ionization suppressants (such as glycolipids and glycerophospholipids) and introduce a source of lithium into the sample. In conjunction, an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone (THAP) is used to facilitate lithium adduct consolidation during the MALDI process. The result is a robust and high-throughput sphingolipid detection scheme, particularly of low-abundance ceramides. Application of our developed workflow includes the detection of differentially expressed liver sphingolipid profiles from a high-fat-induced obesity mouse model. We also demonstrate the method's effectiveness in detecting various sphingolipids in brain and plasma matrices. These results were corroborated with data from UHPLC HR MS/MS and MALDI FT-ICR, verifying the efficacy of the method application. Overall, we demonstrate a high-throughput workflow for sphingolipid analysis in various biological matrices by the use of MALDI TOF and lithium adduct consolidation.

As biomolecules, sphingolipids represent a broad spectrum of structures ranging from simple long chain bases to complex glycosphingolipids. While several different mass spectrometry based approaches have been proven to be useful in qualitative and quantitative analysis of sphingolipids, we find that electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring (MRM) mode using a triple quadrupole instrument, coupled to high-performance liquid chromatography (HPLC), is the most suitable approach for the analysis. In this chapter, we describe the method in a step-by-step manner towards the targeted analysis of sphingolipids in fungi. With optimized HPLC separation and instrument settings, this MRM approach affords detection of many sphingolipid species simultaneously with good sensitivity.

Mass spectrometry has increasingly been used as a tool to complement studies of sphingolipid metabolism and biological functions in plants and other eukaryotes. Mass spectrometry is now essential for comprehensive sphingolipid analytical profiling because of the huge diversity of sphingolipid classes and molecular species in eukaryotes, particularly in plants. This structural diversity arises from large differences in polar head group glycosylation as well as carbon-chain lengths of fatty acids and desaturation and hydroxylation patterns of fatty acids and long-chain bases that together comprise the ceramide hydrophobic backbone of glycosphingolipids. The standard methods for liquid chromatography-mass spectrometry (LC-MS)-based analyses of Arabidopsis thaliana leaf sphingolipids profile >200 molecular species of four sphingolipid classes and free long-chain bases and their phosphorylated forms. While these methods have proven valuable for A. thaliana based sphingolipid research, we have recently adapted them for use with ultraperformance liquid chromatography separations of molecular species and to profile aberrant sphingolipid forms in pollen, transgenic lines, and mutants. This chapter provides updates to standard methods for LC-MS profiling of A. thaliana sphingolipids to expand the utility of mass spectrometry for plant sphingolipid research.

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB 4 , 6t-LTB 4 , LTC 4 , LTB 5 , and 6t-LTB 5 . The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The main objective of this work was to develop an analytical method that can be used in a dairy manufacturing facility for the quantitation of phospholipids in dairy products. Total lipids from a dairy matrix were obtained first by Folch extraction. The total lipid extract was then applied to a silica gel-based solid-phase extraction column, and triglycerides and other nonpolar lipids were separated from the phospholipids and sphingolipids. Quantitation was performed by hydrophilic interaction HPLC coupled to evaporative light-scattering detection using a quaternary separation method. The method was validated using a commercial whey protein phospholipid concentrate and was used to analyze phospholipid and sphingolipid composition in buttermilk, whey protein concentrate, whey protein phospholipid concentrate, and several other dairy ingredients. This method was sensitive and reproducible and can be used in the dairy industry as a research tool to develop new value-added dairy phospholipid products, then later as a standard protocol for quality assurance analysis of current and future products., (© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Podbielska et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Sphingolipids constitute a heterogeneous lipid category that is involved in many key cellular functions. For high-throughput analyses of sphingolipids, tandem mass spectrometry (MS/MS) is the method of choice, offering sufficient sensitivity, structural information, and quantitative precision for detecting hundreds to thousands of species simultaneously. While glycerolipids and phospholipids are predominantly non-hydroxylated, sphingolipids are typically dihydroxylated. However, species containing one or three hydroxylation sites can be detected frequently. This variability in the number of hydroxylation sites on the sphingolipid long-chain base and the fatty acyl moiety produces many more isobaric species and fragments than for other lipid categories. Due to this complexity, the automated annotation of sphingolipid species is challenging, and incorrect annotations are common. In this study, we present an extension of the Lipid Data Analyzer (LDA) "decision rule set" concept that considers the structural characteristics that are specific for this lipid category. To address the challenges inherent to automated annotation of sphingolipid structures from MS/MS data, we first developed decision rule sets using spectra from authentic standards and then tested the applicability on biological samples including murine brain and human plasma. A benchmark test based on the murine brain samples revealed a highly improved annotation quality as measured by sensitivity and reliability. The results of this benchmark test combined with the easy extensibility of the software to other (sphingo)lipid classes and the capability to detect and correctly annotate novel sphingolipid species make LDA broadly applicable to automated sphingolipid analysis, especially in high-throughput settings.