Fatty Acyl Chains Research Articles

Oxidative lipidomics to elucidate the non-volatile derivatives of four typical triglycerides in vegetable oils under simulated frying conditions

Vegetable oils with different saturations have varied composition of triglycerides (TGs) and produce different non-volatile derivatives during oxidation. Precise characterization of the non-volatile derivatives of TGs is essential for understanding the degradation of TGs and the production pattern of non-volatile derivatives. Oxidative lipidomics was combined with collision-induced dissociation and electron-activated dissociation to elucidate the precise structures of non-volatile derivatives produced under simulated frying conditions by 1,3-dipalmitoyl-2-oleoylglycerol (POP), triolein (OOO), trilinolein (LLL), and trilinolenin (LnLnLn). The results indicate that the unsaturated fatty acyl chains at the sn-2 position were more susceptible to oxidation compared with those at the sn-1/3 position. Species of non-volatile derivatives included epoxy-, hydroperoxy-, hydroxy-, and oxo-TGs, as well as degradation products. The potential reaction pathways of TGs and their non-volatile derivatives were also proposed. This study elucidated oxidative degradation mechanisms of the four typical TGs and provided a theoretical basis for changes of vegetable oils during frying.

Role of biomembrane phospholipid fatty acyl chains in membrane protein function.

Role of biomembrane phospholipid fatty acyl chains in membrane protein function.

Targeted Lipidomics Analysis of Adipose and Skeletal Muscle Tissues by Multiple Reaction Monitoring Profiling.

Lipid homeostasis is critical for maintaining normal cellular functions including membrane structural integrity, cell metabolism, and signal transduction. Adipose tissue and skeletal muscle are two major tissues involved in lipid metabolism. Adipose tissue can store excessive lipids in the form of triacylglyceride(TG), which can be hydrolyzed to release free fatty acids (FFAs)under insufficient nutrition states. In the highly energy-demanding skeletal muscle, lipids serve as oxidative substrates for energy production but can cause muscle dysfunction when overloaded. Lipids undergo fascinating cycles of biogenesis and degradation depending on physiological demands, while dysregulation of lipid metabolism has been increasingly recognized as a hallmark of diseases such as obesity and insulin resistance. Thus, it is important to understand the diversity and dynamics of lipid composition in adipose tissue and skeletal muscle. Here, we describe the use of multiple reaction monitoring profiling, based on lipid class and fatty acyl chain specific fragmentation, to explore various classes of lipids in skeletal muscle and adipose tissues. We provide a detailed method for exploratory analysis of acylcarnitine (AC), ceramide (Cer), cholesteryl ester (CE), diacylglyceride (DG), FFA, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and TG. Characterization of lipid composition within adipose tissue and skeletal muscle under different physiological situations will provide biomarkers and therapeutic targets for obesity-related diseases.

Structural elucidation and relative quantification of sodium- and potassium-cationized phosphatidylcholine regioisomers directly from tissue using electron induced dissociation

Comprehensive structural characterization of phosphatidylcholines (PCs) is essential to understanding their biological functions and roles in metabolism. Electron induced dissociation (EID) of protonated PCs directly generated from biological tissues has previously been shown to provide in-depth structural information on the lipid headgroup, regiosiomerism of fatty acyl tails and double bond positions. Although phosphatidylcholine ions formed via alkali metal cationization (i.e., [M + Na]+ and [M + K]+) are commonly generated during matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry experiments, the gas-phase ion chemistry behavior of EID on sodium- and potassium-cationized phosphatidylcholine ion types has not been studied for ions generated directly from tissue. Herein, we demonstrate EID on [M + Na]+ and [M + K]+ ion types in a MALDI imaging mass spectrometry workflow for lipid structural characterization. Briefly, near-complete structural information can be obtained upon EID of sodium- and potassium-cationized PCs, including diagnostic fragmentation of the lipid headgroup as well as identification of fatty acyl chain positions and double bond position. EID of cationized lipids generates sn-specific glycerol backbone cleavages as well as a favorable combined loss of sn-2 fatty acid with choline over sn-1, allowing for facile differentiation and relative quantification of PC regioisomers. Moreover, relative quantification of sn-positional isomers from biological tissue reveals that the relative percentages of sodium- and potassium-cationized sn-positional isomers varies significantly in different regions of rat brain tissue.

Improved Quantitative Approach for Monitorization of Gangliosides Structural Diversity in Fungal Cell Factories by LC-MS/MS

Gangliosides are glycolipids occurring in higher animals, with a sphingoid core in the form of ceramide, bound to a glycan moiety including several units of sialic acid. Gangliosides are involved in important (patho)-physiological processes as components of cell membranes in humans, which has led to intensive study and interest in production strategies. Their structural variability depends on the combination of a sphingoid base, a fatty acyl chain, and an attached oligosaccharide. The combinatorial diversity differs and grows exponentially in synthetic biology approaches, e.g., use of microbial cell factories. A specific analytical platform accounting for this complexity is not available to date. However, quantification of the intermediates of the whole biosynthetic route is needed to boost projects on biotechnological ganglioside production. In this study, a fast high-throughput quantitative LC-MS/MS methodology was developed to cover analysis of gangliosides, with a wider structural perspective adapted to fungal organisms. This work was achieved using metabolically engineered strains that further allowed to test detection in biological complex matrixes. Ganglioside backbones—hitherto uncharacterized—with the five most common fungal sphingoid bases and both simple and hydroxylated fatty acids were subjected to characterization. The addition of glycans to the polar head was also successfully monitored with up to 4 units—corresponding to GD3 which bears two sialic acid units and furthermore represents the common precursor for the whole ganglio-series. This platform represents an improved methodology to study the biochemical diversity associated to gangliosides for natural and metabolically engineered biosynthetic pathways.

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

Lipid desaturation regulates the balance between self-renewal and differentiation in mouse blastocyst-derived stem cells

Stem cells are defined by their ability to self-renew and differentiate, both shown in multiple studies to be regulated by metabolic processes. To decipher metabolic signatures of self-renewal in blastocyst-derived stem cells, we compared early differentiating embryonic stem cells (ESCs) and their extra-embryonic counterparts, trophoblast (T)SCs to their self-renewing counterparts. A metabolomics analysis pointed to the desaturation of fatty acyl chains as a metabolic signature of differentiating blastocyst-derived SCs via the upregulation of delta-6 desaturase (D6D; FADS2) and delta-5 desaturase (D5D; FADS1), key enzymes in the biosynthesis of polyunsaturated fatty acids (PUFAs). The inhibition of D6D or D5D by specific inhibitors or SiRNA retained stemness in ESCs and TSCs, and attenuated endoplasmic reticulum (ER) stress-related apoptosis. D6D inhibition in ESCs upregulated stearoyl-CoA desaturase-1 (Scd1), essential to maintain ER homeostasis. In TSCs, however, D6D inhibition downregulated Scd1. TSCs show higher Scd1 mRNA expression and high levels of monounsaturated fatty acyl chain products in comparison to ESCs. The addition of oleic acid, the product of Scd1 (essential for ESCs), to culture medium, was detrimental to TSCs. Interestingly, TSCs express a high molecular mass variant of Scd1 protein, hardly expressed by ESCs. Taken together, our data suggest that lipid desaturation is a metabolic regulator of the balance between differentiation and self-renewal of ESCs and TSCs. They point to lipid polydesaturation as a driver of differentiation in both cell types. Monounsaturated fatty acids (MUFAs), essential for ESCs are detrimental to TSCs.

Structure Analysis of Unsaturated Polymyxin E Components Based on High-Performance Liquid Chromatography - Quadrupole/ Time of Flight Tandem Mass Spectrometry and Photochemical Reaction

Background: Polymyxin E (PME), which is a complex of cationic cyclic lipodecapeptides, is used to treat multidrug-resistant gram-negative bacterial infections. Besides the main components PME1 and PME2, polymyxin containing unsaturated fatty acyl (FA) group with lower contents is hardly to determine the structure without chromatographic preparations and NMR. Introduction: The peptide sequences of PME components has been carried out based on high performance liquid chromatography-quadrupole / time-of-flight mass spectrometry (HPLC-Q/TOF-MS). However, the components with double bond on the FA, such as 2’, 3’-dehydro PME1, were difficult to be determined or easily misjudged by MS/MS. The transformation of such unsaturated components to be epoxidized components or di-hydroxylated components can promote the acquisition of more fragment ions in the MS/MS, so as to assist in judging the position of double bonds on FA. Methods: In this paper, the PME mixtures were dissolved in an equal proportion of 20% ACN aqueous solution and 2-acetylpyridine. The above PME solution was transferred to a quartz cuvette and irradiated with the ultraviolet lamp at 254 nm for 8h. The dehydro PME components were converted to be epoxy PMEs and dihydroxy PMEs. A fragmentation pathway of epoxidized components or di-hydroxylated components based on Q/TOF-MS/MS was proposed for the first time. Results: According to the characteristic ions of epoxidized components and di-hydroxylated components, 2’, 3’-epoxy PME1/E2 and 2’, 3’-dihydroxy PME1/E2 were confirmed. It can be inferred that the double bond is located at the 2’, 3’-position of FA. Conclusion: The structure of unsaturated PME component with double bond on the FA is elucidated by HPLC-Q/TOF-MS combined with photochemical reaction. This strategy is applicable to other lipopeptides containing unsaturated FA chain.

Effects of an exogenous ketone ester using multi-omics in skeletal muscle of aging C57BL/6J male mice.

Exogenous ketone ester supplementation provides a means to increase circulating ketone concentrations without the dietary challenges imposed by ketogenic diets. Our group has shown that oral R,S-1,3, butanediol diacetoacetate (BD-AcAc2) consumption results in body weight loss or maintenance with moderate increases in circulating ketones. We have previously shown a diet consisting of 25% BD-AcAc2 can maintain lean body mass (LBM) and induce fat mass (FM) loss in young, healthy male mice, but the underlying mechanisms are still unknown. Therefore, the purpose of this study was to determine if a diet consisting of 25% BD-AcAc2 (ketone ester, KE) would alter body composition, transcriptional regulation, the proteome, and the lipidome of skeletal muscle in aged mice. We hypothesized that the KE group would remain weight stable with improvements in body composition compared to controls, resulting in a healthy aging phenotype. Male C57BL/6J mice (n = 16) were purchased from Jackson Laboratories at 72 weeks of age. After 1 week of acclimation, mice were weighed and randomly assigned to one of two groups (n = 8 per group): control (CON) or KE. A significant group by time interaction was observed for body weight (P < 0.001), with KE fed mice weighing significantly less than CON. FM increased over time in the control group but was unchanged in the KE group. Furthermore, LBM was not different between CON and KE mice despite KE mice weighing less than CON mice. Transcriptional analysis of skeletal muscle identified 6 genes that were significantly higher and 21 genes that were significantly lower in the KE group compared to CON. Lipidomic analysis of skeletal muscle identified no differences between groups for any lipid species, except for fatty acyl chains in triacylglycerol which was 46% lower in the KE group. Proteomics analysis identified 44 proteins that were different between groups, of which 11 were lower and 33 were higher in the KE group compared to CON. In conclusion, 72-week-old male mice consuming the exogenous KE, BD-AcAc2, had lower age-related gains in body weight and FM compared to CON mice. Furthermore, transcriptional and proteomics data suggest a signature in skeletal muscle of KE-treated mice consistent with markers of improved skeletal muscle regeneration, improved electron transport chain utilization, and increased insulin sensitivity.

Diversity-Oriented Synthesis of a Molecular Library of Immunomodulatory α-Galactosylceramides with Fluorous-Tag-Assisted Purification and Evaluation of Their Bioactivities in Regard to IL-2 Secretion.

Structural variants of α-galactosylceramide (α-GalCer) that stimulate invariant natural killer T (iNKT) cells constitute an emerging class of immunomodulatory agents in development for numerous biological applications. Variations in lipid chain length and/or fatty acids in these glycoceramides selectively trigger specific pro-inflammatory responses. Studies that would link a specific function to a structurally distinct α-GalCer rely heavily on the availability of homogeneous and pure materials. To address this need, we report herein a general route to the diversification of the ceramide portion of α-GalCer glycolipids. Our convergent synthesis commences from common building blocks and relies on the Julia-Kocienski olefination as a key step. A cleavable fluorous tag is introduced at the non-reducing end of the sugar that facilitates quick purification of products by standard fluorous solid-phase extraction. The strategy enabled the rapid generation of a focused library of 61 α-GalCer analogs by efficiently assembling various lipids and fatty acids. Furthermore, when compared against parent α-GalCer in murine cells, many of these glycolipid variants were found to have iNKT cell stimulating activity similar to or greater than KRN7000. ELISA assaying indicated that glycolipids carrying short fatty N-acyl chains (1fc and 1ga), an unsubstituted (1fh and 1fi) or CF3-substituted phenyl ring at the lipid tail, and a flexible, shorter fatty acyl chain with an aromatic ring (1ge, 1gf, and 1gg) strongly affected the activation of iNKT cells by the glycolipid-loaded antigen-presenting molecule, CD1d. This indicates that the method may benefit the design of structural modifications to potent iNKT cell-binding glycolipids.

Lpcat3 deficiency promotes palmitic acid-induced 3T3-L1 mature adipocyte inflammation through enhanced ROS generation.

Phosphatidylcholines (PCs) are major phospholipids in the mammalian cell membrane. Structural remodeling of PCs is associated with many biological processes. Lysophosphatidylcholine acyltransferase 3 (Lpcat3), which catalyzes the incorporation of polyunsaturated fatty acyl chains into the sn-2 site of PCs, plays an important role in maintaining plasma membrane fluidity. Adipose tissue is one of the main distribution organs of Lpcat3, while the relationship between Lpcat3 and adipose tissue dysfunction during overexpansion remains unknown. In this study, we reveal that both polyunsaturated PC content and Lpcat3 expression are increased in abdominal adipose tissues of high-fat diet-fed mice when compared with chow-diet-fed mice, indicating that Lpcat3 is involved in adipose tissue overexpansion and dysfunction. Our experiments in 3T3-L1 adipocytes show that inhibition of Lpcat3 does not change triglyceride accumulation but increases palmitic acid-induced inflammation and lipolysis. Conversely, Lpcat3 overexpression exhibits anti-inflammatory and anti-lipolytic effects. Furthermore, mechanistic studies demonstrate that Lpcat3 deficiency promotes reactive oxygen species (ROS) generation by increasing NOX enzyme activity by facilitating the translocation of NOX4 to lipid rafts, thereby aggregating 3T3-L1 adipocyte inflammation induced by palmitic acid. Moreover, overexpression of Lpcat3 exhibits the opposite effects. These findings suggest that Lpcat3 protects adipocytes from inflammation during adipose tissue overexpansion by reducing ROS generation. In conclusion, our study demonstrates that Lpcat3 deficiency promotes palmitic acid-induced inflammation in 3T3-L1 adipocytes by enhancing ROS generation.

Effects of non-toxic doses of various food additives on the structure of mammalian retina: Investigation by mid-infrared spectroscopy

Potential health effects of food additives have captured the interest of both public and scientific community because of the controversy regarding their side effects, and in particular their possible role in vision impairment. In this study we analyzed the effects of ingestion of non-toxic doses of tartrazine, carmoisine, sodium benzoate and their mixture on rat’s retinae using mid-infrared spectroscopy after 45 days and 90 days of ingestion. The resulted infrared spectra were statistically evaluated to clarify the relationship between the ingestion periods of the food additives and their effects on retinal structure. The obtained spectra after ingestion of food additives either separately or their mixture were different than the control spectra. Regarding the lipid moiety of retinal membranes, there are changes in lateral packing of the fatty acyl chains while; lipid chain length and branching were different in food additives groups than the control ones. Several modifications in proteins secondary structure were accompanying food additives ingestion, more specifically the detection of β-turns. The effect was obvious in the mixture group where, retinal proteins structure is dominant by β-sheet. Multivariate analysis indicates that there is a reverse relationship between the biological effects of tartrazine, carmoisine and the food additives mixture and the ingestion periods. Paradoxically, this is in contrast to the effects of sodium benzoate. The present study indicates that there is a positive, confirmed relationship between the ingestion of food additives and the changes in both lipids and proteins of the retina, this will affect its function and consequently, vision.

A look into DGAT1 through the EM lenses

With the advent of modern detectors and robust structure solution pipeline, cryogenic electron microscopy has recently proved to be game changer in structural biology. Membrane proteins are challenging targets for structural biologists. This minireview focuses a membrane embedded triglyceride synthesizing machine, DGAT1. Decades of research had built the foundational knowledge on this enzyme's activity. However, recently solved cryo-EM structures of this enzyme, in apo and bound form, has provided critical mechanistic insights. The flipping of the catalytic histidine is critical of enzyme catalysis. The structures explain why the enzyme has preference to long fatty acyl chains over the short forms.

LPCAT4 Knockdown Alters Barrier Integrity and Cellular Bioenergetics in Human Urothelium.

Urothelium is a transitional, stratified epithelium that lines the lower urinary tract, providing a tight barrier to urine whilst retaining the capacity to stretch and rapidly resolve damage. The role of glycerophospholipids in urothelial barrier function is largely unknown, despite their importance in membrane structural integrity, protein complex assembly, and the master regulatory role of PPARγ in urothelial differentiation. We performed lipidomic and transcriptomic characterisation of urothelial differentiation, revealing a metabolic switch signature from fatty acid synthesis to lipid remodelling, including 5-fold upregulation of LPCAT4. LPCAT4 knockdown urothelial cultures exhibited an impaired proliferation rate but developed elevated trans-epithelial electrical resistances upon differentiation, associated with a reduced and delayed capacity to restitute barrier function after wounding. Specific reduction in 18:1 PC fatty acyl chains upon knockdown was consistent with LPCAT4 specificity, but was unlikely to elicit broad barrier function changes. However, transcriptomic analysis of LPCAT4 knockdown supported an LPC-induced reduction in DAG availability, predicted to limit PKC activity, and TSPO abundance, predicted to limit endogenous ATP. These phenotypes were confirmed by PKC and TSPO inhibition. Together, these data suggest an integral role for lipid mediators in urothelial barrier function and highlight the strength of combined lipidomic and transcriptomic analyses for characterising tissue homeostasis.

Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed invitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing invivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.

Investigating sphingolipids as biomarkers for the outcomes of acute ischemic stroke patients receiving endovascular treatment

Long-chain ceramides are associated with the mechanisms and clinical outcomes of acute ischemic stroke (AIS). This study aimed to investigate the plasma ceramides and sphingosine-1-phosphate in AIS patients undergoing endovascular thrombectomy (EVT) and their associations with outcomes. Plasma samples were collected from 75 AIS patients who underwent EVT before (T1), immediately after (T2), and 24h after (T3) the procedures and 19 controls that were matched with age, sex, and co-morbidities. The levels of ceramides with different fatty acyl chain lengths and sphingosine-1-phosphate were measured by UHPLC-ESI-MS/MS. A poor outcome was defined as a modified Rankin Scale score of 3-6at 3 months after stroke. The plasma levels of long-chain ceramides Cer (d18:1/16:0) at all three time points, Cer (d18:1/18:0) at T1 and T3, and Cer (d18:1/20:0) at T1 and very-long-chain ceramide Cer (d18:1/24:1) at T1 were significantly higher in AIS patients than those in the controls. In contrast, the plasma levels of sphingosine-1-phosphate in AIS patients were significantly lower than those in the controls at all three time points. Among the AIS patients, 34 (45.3%) had poor functional outcomes at 3 months poststroke. Multivariable analysis showed that higher levels of Cer (d18:1/16:0) and Cer (d18:1/18:0) at all three time points, Cer (d18:1/20:0) at T1 and T2, and Cer (d18:1/24:0) at T2 remained significantly associated with poor functional outcomes after adjustment for potential confounding factors. Plasma ceramides were elevated early in AIS patients with acute large artery occlusion. Furthermore, Cer (d18:1/16:0) and Cer (d18:1/18:0) could be early prognostic indicators for AIS patients undergoing EVT.

Novel Ambient Oxidation Trends in Fingerprint Aging Discovered by Kendrick Mass Defect Analysis.

A Kendrick mass defect (KMD) plot is an efficient way to disperse complex high-resolution mass spectral data in a visually informative two-dimensional format which allows for the rapid assignment of compound classes that differ by heteroatom content and/or unsaturation. Fingerprint lipid oxidation has the potential to be used to estimate the time since deposition of a fingerprint, but the mass spectra become extremely complex as the lipids degrade. We apply KMD plot analysis for the first time to sebaceous fingerprints aged for 0–7 days to characterize lipid degradation processes analyzed by MALDI-MS. In addition to the ambient ozonolysis of fingerprint lipids previously reported, we observed unique spectral features associated with epoxides and medium chain fatty acid degradation products that are correlated with fingerprint age. We propose an ambient epoxidation mechanism via a peroxyl radical intermediate and the prevalence of omega-10 fatty acyl chains in fingerprint lipids to explain the features observed by the KMD plot analysis. Our hypotheses are supported by an aging experiment performed in a sparse ozone condition and on-surface Paternò–Büchi reaction. A comprehensive understanding of fingerprint degradation processes, afforded by the KMD plots, provides crucial insights for considering which ions to monitor and which to avoid, when creating a robust model for time since deposition of fingerprints.

Novel Aza-Paternò-Büchi Reaction Allows Pinpointing Carbon-Carbon Double Bonds in Unsaturated Lipids by Higher Collisional Dissociation.

The evaluation of double bond positions in fatty acyl chains has always been of great concern given their significance in the chemical and biochemical role of lipids. Despite being the foremost technique for lipidomics, it is difficult in practice to obtain identification beyond the fatty acyl level by the sole high-resolution mass spectrometry. Paternò–Büchi reactions of fatty acids (FAs) with ketones have been successfully proposed for pinpointing double bonds in FAs in combination with the collision-induced fragmentation technique. In the present paper, an aza-Paternò–Büchi (aPB) reaction of lipids with 6-azauracil (6-AU) was proposed for the first time for the determination of carbon–carbon double bonds in fatty acyl chains using higher collisional dissociation in the negative ion mode. The method was optimized using free FA and phospholipid analytical standards and compared to the standard Paternò–Büchi reaction with acetone. The introduction of the 6-AU moiety allowed enhancing the ionization efficiency of the FA precursor and diagnostic product ions, thanks to the presence of ionizable sites on the derivatizing agent. Moreover, the aPB derivatization allowed the obtention of deprotonated ions of phosphatidylcholines, thanks to an intramolecular methyl transfer from the phosphocholine polar heads during ionization. The workflow was finally applied for pinpointing carbon–carbon double bonds in 77 polar lipids from an yeast (Saccharomyces cerevisiae) extract.

Voltage-Controlled Divergent Cascade of Electrochemical Reactions for Characterization of Lipids at Multiple Isomer Levels Using Mass Spectrometry.

Cascading divergent reactions in a single system is highly desirable for their intrinsic efficiency and potential to achieve multilevel structural characterization of complex biomolecules. In this work, two electrochemical reactions, interfacial electro-epoxidation and cobalt anodic corrosion, are divergently cascaded in nanoelectrospray (nESI) and can be switched at different voltages. We applied these reactions to lipid identification at multiple isomer levels using mass spectrometry (MS), which remains a great challenge in structural lipidomics. The divergent cascade reactions in situ derivatize lipids to produce epoxidized lipids and cobalt-adducted lipids at different voltages. These lipids are then fragmented upon low-energy collision-induced dissociation (CID), generating diagnostic fragments to indicate C═C locations and sn-positions that cannot be achieved by the low-energy CID of native lipids. We have demonstrated that lipid structural isomers show significantly different profiles in the analysis of healthy and cancerous mouse prostate samples using this strategy. The application of divergent cascade reactions in lipid identification allows the four-in-one analysis of lipid headgroups, fatty acyl chains, C═C locations, and sn-positions simply by tuning the nESI voltages within a single experiment. This feature as well as its low sample consumption, no need for an extra apparatus, and quantitative analysis capability show its great potential in lipidomics.

Occult infection with hepatitis B virus PreS variants synergistically promotes hepatocellular carcinoma development in a high-fat diet context by generating abnormal ceramides

BackgroundSome occult hepatitis B virus (HBV) infections are resulted from PreS mutations that reduce secretion of envelope protein (HBsAg). We investigated the ceramide amounts and species in hepatocytes infected with PreS variants that were isolated from HBsAg-seronegative patients with hepatocellular carcinoma (HCC) and the ceramide effects on autochthonous HCC development in murine models.MethodsHBV PreS/S regions from 35 HBsAg-seronegative HCC patients were sequenced. Hepatocyte cell lines and male C57BL/6J mouse livers were transfected with two PreS variant representatives. The ceramides with variated lengths of fatty acyl chains were quantified. Tumour development was examined in the HBV-transfected mice fed different diet types.ResultsIn HBsAg-seronegative HCC patients, nonneoplastic liver tissues harboured HBsAg and replication-competent HBV. The most frequently detected PreS/S variants carried mutations of altered amino acid properties in HBsAg compared with an isolate from one HBsAg-seronegative HCC patient. Hepatocyte infection with PreS variants caused HBsAg retention within the endoplasmic reticulum and generated more amounts of ceramides with C16:0 ceramide elevated the highest. Saturated fatty acids aggravated the PreS variant-infected hepatocytes to generate abnormal amounts and species of ceramides, which with HBV proteins synergistically activated NLRP3 inflammasome in liver inflammatory macrophages. Liver tumours were only detected in HBV-transfected mice fed high-fat diet, with higher tumour loads in the PreS variant-transfected, associated with abnormal ceramide generation.ConclusionsHBV PreS mutations which altered amino acid properties of envelope proteins inhibited HBsAg secretion. Hepatocyte infection with PreS variants generated abnormal ceramides which with HBV proteins coactivated NLRP3 inflammasome in liver macrophages to promote autochthonous HCC development.