Influenza Virus Membrane Fusion Is Promoted by the Endosome-Resident Phospholipid Bis(monoacylglycero)phosphate.

The phospholipid bis(monoacylglycero)phosphate (BMP) is enriched in late endosomal and endolysosomal membranes and is believed to be involved in membrane deformation and generation of intralumenal vesicles within late endosomes. Previous studies have demonstrated that BMP promotes membrane fusion of several enveloped viruses, but a limited effect has been found on influenza virus. Here, we report the use of single-virus fusion assays to dissect BMP's effect on influenza virus fusion in greater depth. In agreement with prior reports, we found that hemifusion kinetics and efficiency were unaffected by the addition of 10-20 mol % BMP to the target membrane. However, using an assay for fusion pore formation and genome exposure, we found full fusion efficiency to be substantially enhanced by the addition of 10-20 mol % BMP to the target membrane, while the kinetics remained unaffected. By comparing BMP to other negatively charged phospholipids, we found the effect on fusion efficiency mainly attributable to headgroup charge, although we also hypothesize a role for BMP's unusual chemical structure. Our results suggest that BMP function as a permissive factor for a wider range of viruses than previously reported. We hypothesize that BMP may be a general cofactor for endosomal entry of enveloped viruses.

Lysophosphatidic acid, ceramide 1-phosphate and sphingosine 1-phosphate in peripheral blood of patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonias. Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are signaling lipids that evoke growth factor-like responses to many cells. Recent studies revealed the involvement of LPA and S1P in the pathology of IPF. In this study, we determined LPA, S1P and ceramide 1-phosphate (C1P) in peripheral blood plasma of IPF patients, and examined correlation to the vital capacity of lung (VC), an indicator of development of fibrosis. Blood plasma samples were taken from eleven patients with IPF and seven healthy volunteers. The lipids of the sample were extracted and subjected to liquid chromatography-tandem mass spectrometry for analysis. Results showed that there is a significant negative correlation between VC and plasma LPA levels, indicating that IPF patients with advanced fibrosis had higher concentration of LPA in their plasma. Average of S1P levels were significantly higher in IPF patients than those in healthy subjects. Although it is not statistically significant, a similar correlation trend that observed in LPA levels also found between VC and S1P levels. These results indicated that plasma LPA and S1P may be associated with deterioration of pulmonary function of IPF patients. J. Med. Invest. 69 : 196-203, August, 2022.

Metabolomic profiling of Dezhou donkey seminal plasma related to freezability.

Donkeys are indispensable livestock in China because they have transport function and medicinal value. With the popularization of artificial insemination on donkeys, semen cryopreservation technology has gradually become a research hotspot. Seminal plasma is a necessary medium for transporting sperm and provides energy and nutrition for sperm. Seminal plasma metabolites play an important role in the process of sperm freezing, and also have an important impact on sperm motility and fertilization rate after freezing and thawing. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to compare the metabolic characteristics of seminal plasma of high freezability (HF) and low freezability (LF) male donkeys. We identified 672 metabolites from donkey seminal plasma, of which 33 metabolites were significantly different between the two groups. Metabolites were identified and categorized according to their major chemical classes, including homogeneous non-metal compounds, nucleosides, nucleotides, and analogues, organosulphur compounds, phenylpropanoids and polyketide, organoheterocyclic compounds, organic oxygen compounds, benzenoids, organic acids and derivatives, lipids and lipid-like molecules, organooxygen compounds, alkaloids and derivatives, organic nitrogen compounds. The results showed that the contents of phosphatidylcholine, piceatannol and enkephalin in donkey semen of HF group were significantly higher than those of LF group (p < .05), while the contents of taurocholic and lysophosphatidic acid were significantly lower than those of LF group (p < .05). The different metabolites were mainly related to sperm biological pathway response and oxidative stress. These metabolites may be considered as candidate biomarkers for different fertility in jacks.

Plasma Sphingosine-1-Phosphate Levels Are Associated with Progression of Estrogen Receptor-Positive Breast Cancer.

Although numerous experiments revealed an essential role of a lipid mediator, sphingosine-1-phosphate (S1P), in breast cancer (BC) progression, the clinical significance of S1P remains unclear due to the difficulty of measuring lipids in patients. The aim of this study was to determine the plasma concentration of S1P in estrogen receptor (ER)-positive BC patients, as well as to investigate its clinical significance. We further explored the possibility of a treatment strategy targeting S1P in ER-positive BC patients by examining the effect of FTY720, a functional antagonist of S1P receptors, on hormone therapy-resistant cells. Plasma S1P levels were significantly higher in patients negative for progesterone receptor (PgR) expression than in those positive for expression (p = 0.003). Plasma S1P levels were also significantly higher in patients with larger tumor size (p = 0.012), lymph node metastasis (p = 0.014), and advanced cancer stage (p = 0.003), suggesting that higher levels of plasma S1P are associated with cancer progression. FTY720 suppressed the viability of not only wildtype MCF-7 cells, but also hormone therapy-resistant MCF-7 cells. Targeting S1P signaling in ER-positive BC appears to be a possible new treatment strategy, even for hormone therapy-resistant patients.

Alterations in complex lipids in tumor tissue of patients with colorectal cancer.

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.

Potential value of circulating endothelial cells for the diagnosis and treatment of COVID-19.

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has been a formidable global challenge. As yet, there are very few drugs to treat this infection and no vaccine is currently available. It has gradually become apparant that coronavirus disease 2019 (COVID-19) is not a simple disease involving a single organ; rather, many vital organs and systems are affected. The endothelium is one target of SARS-CoV-2. Damaged endothelial cells, which break away from organs and enter the bloodstream to form circulating endothelial cells, were recently reported as putative biomarkers for COVID-19. Modulation of the expression level of sphingosine-1 phosphate via sphingosine kinase activation can control endothelial cell proliferation and apoptosis. As such, it may be possible to obtain a sensitive and specific diagnosis of the severity of COVID-19 by assessing the absolute number and the viable/apoptotic ratio of circulating endothelial cells. Furthermore, a focus on the endothelium could help to develop a strategy for COVID-19 treatment from the perspective of endothelial protection and repair.

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Endothelial damage in septic shock patients as evidenced by circulating syndecan-1, sphingosine-1-phosphate and soluble VE-cadherin: a substudy of ALBIOS.

BACKGROUND: Septic shock is characterized by breakdown of the endothelial glycocalyx and endothelial damage, contributing to fluid extravasation, organ failure and death. Albumin has shown benefit in septic shock patients. Our aims were: (1) to identify the relations between circulating levels of syndecan-1 (SYN-1), sphingosine-1-phosphate (S1P) (endothelial glycocalyx), and VE-cadherin (endothelial cell junctions), severity of the disease, and survival; (2) to evaluate the effects of albumin supplementation on endothelial dysfunction in patients with septic shock. METHODS: This was a retrospective analysis of a multicenter randomized clinical trial on albumin replacement in severe sepsis or septic shock (the Albumin Italian Outcome Sepsis Trial, ALBIOS). Concentrations of SYN-1, S1P, soluble VE-cadherin and other biomarkers were measured on days 1, 2 and 7 in 375 patients with septic shock surviving up to 7 days after randomization. RESULTS: Plasma concentrations of SYN-1 and VE-cadherin rose significantly over 7 days. SYN-1 and VE-cadherin were elevated in patients with organ failure, and S1P levels were lower. SYN-1 and VE-cadherin were independently associated with renal replacement therapy requirement during ICU stay, but only SYN-1 predicted its new occurrence. Both SYN-1 and S1P, but not VE-cadherin, predicted incident coagulation failure. Only SYN-1 independently predicted 90-day mortality. Albumin significantly reduced VE-cadherin, by 9.5% (p = 0.003) at all three time points. CONCLUSION: Circulating components of the endothelial glycocalyx and of the endothelial cell junctions provide insights into severity and progression of septic shock, with special focus on incident coagulation and renal failure. Albumin supplementation lowered circulating VE-cadherin consistently over time. CLINICAL TRIAL REGISTRATION: ALBIOS ClinicalTrials.gov number NCT00707122.

In situ detection and imaging of lysophospholipids in zebrafish using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

In this paper, a matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) (MALDI-FTICR-MS) imaging method was developed to rapid and in situ detect the spatial distribution of lysophospholipids (LPLs) in zebrafish. The combination of MALDI with ultrahigh-resolution FTICR-MS achieves the MS imaging of LPLs with a mass resolution up to 50 000, which allows accurate identification and clear spatial visualization of LPLs in complex biological tissues. A series of lysophosphatidylcholines (LPCs) was detected using positive ion detection mode, and their concentration differences and spatial distributions were clearly visualized in different parts of zebrafish tissue. The method is rapid, simple, and efficient, being a desirable way to understand the spatial distribution of LPLs in biosome.

Development and validation of a QTrap method for sensitive quantification of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.

"Modeling-Prediction" Strategy for Deep Profiling of Lysophosphatidic Acids by Liquid Chromatography-Mass Spectrometry: Exploration Biomarkers of Breast Cancer.

Lysophosphatidic acids (LPAs) are important bioactive phospholipids consisting of various species involved in a wide array of physiological and pathological processes. However, LPAs were rarely identified in untargeted lipidomics studies because of the incompatibility with analytical methods. Moreover, in targeted studies, the coverages of LPAs remained unsatisfactorily low due to the limitation of reference standards. Herein, a "modeling-prediction" workflow for deep profiling of LPAs by liquid chromatography-mass spectrometry was developed. Multiple linear regression models of qualitative and quantitative parameters were established according to features of fatty acyl tails of the commercial standards to predict the corresponding parameters for unknown LPAs. Then 72 multiple reaction monitoring (MRM) transitions were monitored simultaneously and species of LPA 14:0, LPA 16:1, LPA 18:3, LPA 20:3 and LPA 20:5 were firstly characterized and quantified in plasma. Finally, the workflow was applied to explore the changes of LPAs in plasma of breast cancer patients compared with healthy volunteers. Multi-LPAs indexes with strong diagnostic ability for breast cancer were identified successfully using Student's t- test, orthogona partial least-squares discrimination analysis (OPLS-DA) and logistic regression- receiver operating characteristic (ROC) curve analysis. The proposed workflow with high sensitivity, high accuracy, high coverage and reliable identification would be a powerful complement to untargeted lipidomics and shed a light on the analysis of other lipids.

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis.

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

Sphingosine Kinase 1 is Associated With Immune Cell-Related Gene Expressions in Human Breast Cancer.

BACKGROUND: Although previous experiments have implicated sphingosine-1-phosphate (S1P) as a links between immune reactions and cancer progression, the exact mechanism of this interaction has not comprehensively studied in clinical human samples. This study sought to evaluate the S1P regulation by sphingosine kinase 1 (SPHK1), an S1P-producing enzyme, in the immunity/immuno-reactivity of clinical human breast cancer surgical specimens. METHODS: S1P levels were examined in tumor, peritumoral, and normal human breast samples using mass spectrometry. Genomics Data Commons data portal of The Cancer Genome Atlas cohort was used to assess the expression of S1P-related and immune-related genes. RESULTS: S1P levels were significantly higher in tumor samples compared to peritumoral (P < 0.05) or normal human breast samples (P < 0.001). SPHK1 gene expression was elevated in tumoral samples compared to normal breast samples (P < 0.01). Furthermore, the elevated expression of SPHK1 in breast cancer tissue was associated with an increased expression of the different kinds of immune-related genes, such as CD68, CD163, CD4, and FOXP3 (forkhead box P3), in HER2-negative breast cancer. Network analysis showed the central role of SPHK1 in the interaction of S1P signaling and expression of immune cell-related proteins. CONCLUSIONS: We demonstrated that S1P is mainly produced by tumor tissue, rather than peritumoral tissue, in breast cancer patients. Our data revealed the involvement of S1P signaling in the regulation of immune-related genes, suggesting the links between S1P and complicated immune-cancer interactions in breast cancer patients.

The Unique Phospholipidome of the Enteric Pathogen Campylobacter jejuni: Lysophosholipids Are Required for Motility at Low Oxygen Availability.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.

Dually Crosslinked Supramolecular Hydrogel for Cancer Biomarker Sensing.

Lysophosphatidic acid (LPA) as the biomarker of early stage ovarian cancer is essentially difficult to detect due to lack of target spots. A dually crosslinked supramolecular hydrogel (DCSH) was developed to achieve sensing of LPA, which acts as a competitive guest molecule triggering the responsive crosslinking of the DCSH. Through this strategy, the surface plasmon resonance combined with optical waveguide spectroscopy could be used to quantitatively detect LPA with a responsive range covering physiological conditions (in pure form as well as mimicking LPA plasma solution) with high selectivity and sensitivity. LPA efficiently immerses into the host molecule ß-cyclodextrin (ß-CD) up to a 1:2 ratio by the competitive interaction mechanism, confirmed by one-dimensional nuclear overhauser effect spectroscopy (1D NOESY), high-resolution mass spectrometry (HRMS), isothermal titration calorimetry (ITC), and computational simulation. Our method opens a new strategy to detect biomarkers without target spots and provides a platform for surface plasmon resonance (SPR)-based sensors measuring small molecules.

The imbalance in the aortic ceramide/sphingosine-1-phosphate rheostat in ovariectomized rats and the preventive effect of estrogen.

BACKGROUND: The prevalence of hypertension in young women is lower than that in age-matched men while the prevalence of hypertension in women is significantly increased after the age of 50 (menopause) and is greater than that in men. It is already known that sphingosine-1-phosphate (S1P) and ceramide regulate vascular tone with opposing effects. This study aimed to explore the effects of ovariectomy and estrogen supplementation on the ceramide/S1P rheostat of the aorta in rats, and to explore a potential mechanism for perimenopausal hypertension and a brand-new target for menopausal hormone therapy to protect vessels. METHODS: In total, 30 female adult SD rats were randomly divided into three groups: The sham operation group (SHAM), ovariectomy group (OVX) and ovariectomy plus estrogen group (OVX + E). After 4 weeks of treatment, the blood pressure (BP) of the rats was monitored by a noninvasive system; the sphingolipid content (e.g., ceramide and S1P) was detected by liquid chromatography-mass spectrometry (LC-MS); the expression of the key enzymes involved in ceramide anabolism and catabolism was measured by real-time fluorescence quantitative polymerase chain reaction (qPCR); and the expression of key enzymes and proteins in the sphingosine kinase 1/2 (SphK1/2)-S1P-S1P receptor 1/2/3 (S1P1/2/3) signaling pathway was detected by qPCR and western blotting. RESULTS: In the OVX group compared with the SHAM group, the systolic BP (SBP), diastolic BP (DBP) and pulse pressure (PP) increased significantly, especially the SBP and PP (P < 0.001). For aortic ceramide metabolism, the mRNA level of key enzymes involved in anabolism and catabolism decreased in parallel 2-3 times, while the contents of total ceramide and certain long-chain subtypes increased significantly (P < 0.05). As for the S1P signaling pathway, SphK1/2, the key enzymes involved in S1P synthesis, decreased significantly, and the content of S1P decreased accordingly (P < 0.01). The S1P receptors showed various trends: S1P1 was significantly down-regulated, S1P2 was significantly up-regulated, and S1P3 showed no significant difference. No significant difference existed between the SHAM and OVX + E groups for most of the above parameters (P > 0.05). CONCLUSIONS: Ovariectomy resulted in the imbalance of the aortic ceramide/S1P rheostat in rats, which may be a potential mechanism underlying the increase in SBP and PP among perimenopausal women. Besides, the ceramide/S1P rheostat may be a novel mechanism by which estrogen protects vessels.

31P NMR Quantification of Phospholipids and Lysophospholipids in Food Emulsions.

For food emulsions containing enzymatically modified egg yolk, the conventional Folch extraction does not fully recover the polar lysophospholipids. This can be overcome by repeated methanol extractions. After solvent evaporation, the extracted (lyso)phospholipids are solubilized into mixed micelles with cholate as a detergent. The solubilized (lyso)phospholipids can be accurately quantified by 31P NMR with recoveries ranging between 96% and 108%. Detection at a high (16.4 T) relative to a mainstream (9.4 T) magnetic field strength did not offer a significant advantage since the slow molecular tumbling of the mixed micelles increased line widths. This was due to field-strength-dependent chemical shift anisotropy relaxation. Method precision is similar at 9.4 and 16.4 T, with within-laboratory reproducibilities of 7-22% and 12-25%, respectively. The method can be implemented as a routine analytical procedure at 9.4 T (400 MHz NMR spectrometer), and the limits of detection and quantification are adequate for the verification of the standard of identity of a mayonnaise prepared with enzymatically modified egg yolk.

Lipidomics-based assays coupled with computational approaches can identify novel phospholipase A2 inhibitors.

Phospholipase A2 (PLA2) enzymes play a major role in many diseases including the inflammatory cascade and specific potent small molecule inhibitors could be useful in studying their physiological role as well as for the development of drugs. In order to discover novel small molecule inhibitor platforms for members of the PLA2 superfamily of enzymes, we have applied computational approaches to determine the binding mode of potent inhibitors specific for particular PLA2s to the screening of chemical libraries. This has including the U.S. National Institutes of Health (NIH) National Cancer Institute (NCI) Diversity Set V and the ChemBridge commercial compound libraries. We have then subjected identified inhibitor structures to recently developed lipidomics based screening assays to determine the XI(50) and specificity of the identified compounds for specific PLA2s. Herein we review this approach and report the identity of initial hits for both the Group IVA cytosolic PLA2 and the Group VIA calcium-independent PLA2 that are worthy of further structural modification to develop novel platforms for inhibitor development.

Promotion of plasmalogen biosynthesis reverse lipid changes in a Barth Syndrome cell model.

In Barth syndrome (BTHS) mutations in tafazzin leads to changes in both the quantities and the molecular species of cardiolipin (CL), which are the hallmarks of BTHS. Contrary to the well-established alterations in CL associated with BTHS; recently a marked decrease in the plasmalogen levels in Barth specimens has been identified. To restore the plasmalogen levels, the present study reports the effect of promotion of plasmalogen biosynthesis on the lipidome of lymphoblasts derived from Barth patients as well as on cell viability, mitochondria biogenesis, and mitochondrial membrane potential. High resolution 31P NMR phospholipidomic analysis showed an increase in the levels of plasmenylethanolamine (the major plasmalogen in lymphoblasts), which reached values comparable to the control and a compensatory decrease in the levels of its diacyl-PE counterpart. Importantly, 31P NMR showed a significant increase in the levels of CL, while not altering the levels of monolysocardiolipin. Mass spectrometry measurements showed that the promotion of plasmalogen biosynthesis did not change the molecular species profile of targeted phospholipids. In addition, promotion of plasmalogen biosynthesis did not impact on cellular viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential. Overall, the results showed the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and highlight the potential beneficial effect of a diet supplemented with plasmalogen precursors to BTHS patients.

Analysis of Phospholipids, Lysophospholipids, and Their Linked Fatty Acyl Chains in Yellow Lupin Seeds (Lupinus luteus L.) by Liquid Chromatography and Tandem Mass Spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS) was used to characterize the phospholipidome of yellow lupin (Lupinus luteus) seeds. Phosphatidylcholines (PC) were the most abundant species (41 ± 6%), which were followed by lyso-forms LPC (30 ± 11%), phosphatidylethanolamines (PE, 13 ± 4%), phosphatidylglycerols (PG, 5.1 ± 1.7%), phosphatidic acids (PA, 4.9 ± 1.8%), phosphatidylinositols (PI, 4.7 ± 1.1%), and LPE (1.2 ± 0.5%). The occurrence of both isomeric forms of several LPC and LPE was inferred by a well-defined fragmentation pattern observed in negative ion mode. An unprecedented characterization of more than 200 polar lipids including 52 PC, 42 PE, 42 PA, 35 PG, 16 LPC, 13 LPE, and 10 PI, is reported. The most abundant fatty acids (FA) as esterified acyl chains in PL were 18:1 (oleic), 18:2 (linoleic), 16:0 (palmitic), and 18:3 (linolenic) with relatively high contents of long fatty acyl chains such as 22:0 (behenic), 24:0 (lignoceric), 20:1 (gondoic), and 22:1 (erucic). Their occurrence was confirmed by reversed-phase (RP) LC-ESI-FTMS analysis of a chemically hydrolyzed sample extract in acid conditions at 100 °C for 45 min.