Lysophosphatidylcholine Species Research Articles

Inverse relations of serum phosphatidylcholines and lysophosphatidylcholines with vascular damage and heart rate in patients with atherosclerosis

Background and aimsThe rapidly growing discipline of lipidomics allows the study of a wide spectrum of lipid species in body fluids and provides new insights into the pathogenesis of cardiovascular disease. We investigated serum phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) species in relation to arterial stiffness, hemodynamics, and endothelial dysfunction in symptomatic patients with atherosclerosis and in healthy controls. Methods and resultsThirty-two patients with peripheral arterial disease (age 61.7 ± 9.0 years), 52 patients with coronary artery disease (age 63.2 ± 9.2 years), and 40 apparently healthy controls (age 60.3 ± 7.1 years) were studied. Serum levels of 90 glycerophospholipids were determined with the AbsoluteIDQ™ p180 kit (BIOCRATES Life Sciences AG, Innsbruck, Austria). The technique of applanation tonometry was used for non-invasive pulse wave analysis and carotid-femoral pulse wave velocity (cf-PWV) assessment. Decreased serum levels of several individual PC and lysoPC species (e.g., PC aa C28:1, PC aa C30:0, PC aa C32:2, PC ae C30:0 and PC ae C34:2, lysoPC a C18:2) were observed for the patient groups in comparison to the healthy subjects. In addition, a considerable number of PCs and lysoPCs were inversely related to either cf-PWV, heart rate, asymmetric dimethylarginine (ADMA) or ADMA/arginine for patients with symptomatic atherosclerosis but not for the controls. ConclusionWe found altered relationships between PC and lysoPC profiles, inflammation, and arterial function in atherosclerotic patients, compared to healthy subjects.

Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner

The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-β-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.

Lysophosphatidylcholine acyltransferase 1 upregulation and concomitant phospholipid alterations in clear cell renal cell carcinoma

BackgroundThe involvement of lipid metabolism in tumourigenesis and the progression of clear cell renal cell carcinoma (ccRCC) have been reported. However, the role of phospholipid profile alterations in ccRCC has not yet been systematically explored. In the present study, we compared the phospholipid compositions between ccRCC and paired normal renal tissues.MethodsThe phospholipid compositions of paired ccRCC and normal renal tissues were evaluated using liquid chromatography tandem mass spectrometry (LC/MS/MS). To evaluate the mRNA and protein levels of lysophosphatidylcholine acyltransferase (LPCAT), which converts lysophosphatidylcholine (LPC) to phosphatidylcholine (PC), qRT-PCR, western blotting and immunohistochemistry were performed. The correlations of LPCAT1 expression with clinicopathological features and prognosis were assessed. In addition, siRNAs were used to knockdown LPCAT1 expression in ccRCC cell lines, and its effect on cell proliferation, cell cycle, migration and invasion were investigated.ResultsThe phospholipid compositions of ccRCC and normal renal tissues were significantly different. Multiple LPC species were decreased and corresponding PC species were increased in cancer tissues. The mRNA and protein levels of LPCAT1 were up-regulated in ccRCC tissues compared with normal renal tissues, and LPCAT1 expression was significantly correlated with unfavourable pathological features (higher tumour grade, higher TNM stage and larger tumour size) and overall survival. In cell line experiments, LPCAT1 knockdown depleted PCs, inhibited cell proliferation, migration and invasion and induced cell cycle arrest at the G0/G1 phase.ConclusionSelective changes in PC and LPC composition were observed in ccRCC tissues. The overexpression of LPCAT1 promotes the development and progression of ccRCC, likely through the conversion of LPC to PC.

Urinary lysophopholipids are increased in diabetic patients with nephropathy

Diabetic nephropathy (DN) is a major cause of chronic kidney disease that frequently leads to end stage renal failure. Lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are lysophospholipid mediators shown to accumulate in kidney and to promote renal inflammation and tubulo-interstitial fibrosis in diabetic rodent models. Here we assessed whether LPA and LPC were associated to the development of nephropathy in diabetic human patients. Several molecular species of LPA and LPC were quantified by LC/MS–MS in urine and plasma from type 2 diabetic patients with (cases; n=41) or without (controls, n=41) nephropathy symptoms (micro/macro-albuminuria and eGFR<60ml/min/1.73m2). Cases and controls were matched for sex, age and diabetes duration. Six species were detected in urine for both LPA and LPC, LPA16:0, LPA20:4, LPC16:0, LPC18:0, LPC18:1, and LPC18:2 that were significantly more concentrated in cases than in controls. Total LPC and LPA (sum of detected species) were significantly and exclusively associated with albuminuria (P<0.0001 and P=0.0009 respectively) and were significantly higher in the 3rd when compared to the 1st albuminuria tertile in cases. Plasma lysophospholipids showed a different species profile urine and their concentrations were not different between cases and controls. In conclusion, urine concentration of lysophospholipids increases in diabetic patients with DN as the likely result of their co-excretion with albumin combined with possible local production by kidney. Because LPA and LPC are known to promote renal inflammation and tubulo-interstitial fibrosis, their increased production in DN could participate to the development of kidney damage associated with diabetes.

Role of prefrontal cortical calcium-independent phospholipase A2 in antinociceptive effect of the norepinephrine reuptake inhibitor antidepresssant maprotiline

The prefrontal cortex is essential for executive functions such as decision-making and planning. There is also accumulating evidence that it is important for the modulation of pain. In this study, we investigated a possible role of prefrontal cortical calcium-independent phospholipase A2 (iPLA2) in antinociception induced by the norepinephrine reuptake inhibitor (NRI) and tetracyclic (tricyclic) antidepressant, maprotiline. Intraperitoneal injections of maprotiline increased iPLA2 mRNA and protein expression in the prefrontal cortex. This treatment also reduced grooming responses to von-Frey hair stimulation of the face after facial carrageenan injection, indicating decreased sensitivity to pain. The antinociceptive effect of maprotiline was abrogated by iPLA2 antisense oligonucleotide injection to the prefrontal cortex, indicating a role of this enzyme in antinociception. In contrast, injection of iPLA2 antisense oligonucleotide to the somatosensory cortex did not reduce the antinociceptive effect of maprotiline. Lipidomic analysis of the prefrontal cortex showed decrease in phosphatidylcholine species, but increase in lysophosphatidylcholine species, indicating increased PLA2 activity, and release of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after maprotiline treatment. Differences in sphingomyelin/ceramide were also detected. These changes were not observed in maprotiline-treated mice that received iPLA2 antisense oligonucleotide to the prefrontal cortex. Metabolites of DHA and EPA may help to strengthen a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate a role of prefrontal cortical iPLA2 and its enzymatic products in the antinociceptive effect of maprotiline.

Systemic saturated lysophosphatidylcholine is associated with hepatic function in patients with liver cirrhosis

Serum lysophosphatidylcholine (LPC) is described to decline in patients with chronic liver diseases. Here it was evaluated which of the LPC species are associated with liver function. LPC species were quantified by direct flow-injection electrospray ionization tandem mass spectrometry in serum of 45 patients with mainly alcoholic liver cirrhosis. Saturated LPC is 52.1 (31.7–110.0) μmol/l in serum of patients with CHILD-PUGH score C (decompensated liver cirrhosis) and significantly lower compared to patients with well-compensated disease (CHILD-PUGH score A) with 114.1 (12.3–401.4) μmol/l. Mono- and polyunsaturated LPC are not changed in these groups. Saturated LPC is negatively correlated with the model for end-stage liver disease score, bilirubin and galectin-3 and positively with Quick prothrombin time. Ascites and varices are complications of liver cirrhosis. Saturated LPC does, however, not correlate with hepatic venous pressure gradient, ascites volume and variceal size. Unexpectedly, saturated LPC measured in serum of 42 patients declines from 88.4 (27.8–177.5) μmol/l to 72.4 (27.6–141.8) μmol/l shortly after transjugular intrahepatic portosystemic shunt implantation. Hepatic vein saturated LPC (82.3 (12.4–161.7) μmol/l) is higher than portal vein levels (78.8 (10.1–161.0) μmol/l) suggesting hepatic release of this lipid species. Current data demonstrate that systemic saturated LPC species are reduced in patients with decompensated liver cirrhosis and associated with mortality risk.

Mitochondrial Reactive Oxygen Species Mediate Lysophosphatidylcholine-Induced Endothelial Cell Activation.

Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is the mechanism underlying hyperlipidemia-induced EC activation. Here, we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis. Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0, and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout mice during early atherosclerosis. Using electron spin resonance and flow cytometry, we found that LPC 16:0, 18:0, and 18:1 induced mtROS in primary human aortic ECs, independently of the activities of nicotinamide adenine dinucleotide phosphate oxidase. Mechanistically, using confocal microscopy and Seahorse XF mitochondrial analyzer, we showed that LPC induced mtROS via unique calcium entry-mediated increase of proton leak and mitochondrial O2 reduction. In addition, we found that mtROS contributed to LPC-induced EC activation by regulating nuclear binding of activator protein-1 and inducing intercellular adhesion molecule-1 gene expression in vitro. Furthermore, we showed that mtROS inhibitor MitoTEMPO suppressed EC activation and aortic monocyte recruitment in apolipoprotein E knockout mice using intravital microscopy and flow cytometry methods. ATP synthesis-uncoupled, but proton leak-coupled, mtROS increase mediates LPC-induced EC activation during early atherosclerosis. These results indicate that mitochondrial antioxidants are promising therapies for vascular inflammation and cardiovascular diseases.

Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Mortality prediction in patients with severe septic shock: a pilot study using a target metabolomics approach.

Septic shock remains a major problem in Intensive Care Unit, with high lethality and high-risk second lines treatments. In this preliminary retrospective investigation we examined plasma metabolome and clinical features in a subset of 20 patients with severe septic shock (SOFA score >8), enrolled in the multicenter Albumin Italian Outcome Sepsis study (ALBIOS, NCT00707122). Our purpose was to evaluate the changes of circulating metabolites in relation to mortality as a pilot study to be extended in a larger cohort. Patients were analyzed according to their 28-days and 90-days mortality. Metabolites were measured using a targeted mass spectrometry-based quantitative metabolomic approach that included acylcarnitines, aminoacids, biogenic amines, glycerophospholipids, sphingolipids, and sugars. Data-mining techniques were applied to evaluate the association of metabolites with mortality. Low unsaturated long-chain phosphatidylcholines and lysophosphatidylcholines species were associated with long-term survival (90-days) together with circulating kynurenine. Moreover, a decrease of these glycerophospholipids was associated to the event at 28-days and 90-days in combination with clinical variables such as cardiovascular SOFA score (28-day mortality model) or renal replacement therapy (90-day mortality model). Early changes in the plasma levels of both lipid species and kynurenine associated with mortality have potential implications for early intervention and discovering new target therapy.

Abstract B06: TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism

Abstract Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease characterized by multi-organ proliferation of TSC2-deficient cells with aberrant activation of the mechanistic/mammalian Target of Rapamycin Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Up to 80% of women with TSC develop lymphangioleiomyomatosis (LAM), which consists of diffuse proliferation of TSC2-deficient smooth muscle-like cells with progressive cystic destruction of the lung. LAM can also occur as a sporadic disorder of women. A downstream effector of mTORC1, the Sterol Regulatory Element-Binding Protein (SREBP), regulates the transcription of de novo fatty acid synthesis enzymes in TSC2-deficient cells. The impact of loss of the TSC proteins on the lipidome and how complex lipid species are affected by rapamycin and its analogs are unknown. Here, we report the first systematic study of the TSC lipidome, revealing unexpected findings. Using mass spectrometry, we profiled 131 complex lipid species discovered elevated levels (p&amp;lt;0.05) of 4 lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, C20:4) in the plasma of LAM patients compared with healthy control women. LPC are a class of bioactive lipids generated by phospholipase A (PLA) activity. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro pre-clinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid, including the LPC precursors phosphatidylcholines (PC) and sphingolipids, and neutral lipid species, including triacylglycerols and cholesterol esters. To determine whether TSC2-deficient cells generate LPC using de novo synthesized PC, TSC2-deficient and TSC2-expressing cells were labeled with deuterated choline (choline chloride 1,1,2,2-d4) for 6 hours, and lipids isolated to measure choline incorporation into two saturated LPC (C16:0 and C18:0) and two parent PC (C32:0 and C34:0) species. Incorporation of deuterated choline into PC and LPC was greater than 2 fold higher in TSC2-deficient cells compared to TSC2-expressing controls (p&amp;lt;0.05). Surprisingly, treatment with rapamycin, the mTOR catalytic inhibitor Torin1, or downregulation of the mTORC1-regulated lipogenic transcription factor SREBP (Sterol Regulatory Element-Binding Protein) did not suppress LPC in TSC2-deficient cells, while they suppressed other phospholipid species. Finally, pharmacologic and genetic approaches revealed that inhibition of distinct isoforms of PLA2 may decrease the proliferation of TSC2-deficient but not TSC2-expressing cells. Collectively, these results demonstrate for the first time that TSC2-deficient cells have enhanced choline phospholipid metabolism, revealing a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies. Citation Format: Carmen Priolo, Stéphane J. H. Ricoult, Damir Khabibullin, Harilaos Filippakis, Jane Yu, Brendan Manning, Clary Clish, Elizabeth P. Henske. TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B06.

Integrated multi‐omics analysis supports role of lysophosphatidylcholine and related glycerophospholipids in theLotus japonicus–Glomus intraradicesmycorrhizal symbiosis

Interaction of plant roots with arbuscular mycorrhizal fungi (AMF) is a complex trait resulting in cooperative interactions among the two symbionts including bidirectional exchange of resources. To study arbuscular mycorrhizal symbiosis (AMS) trait variation in the model plant Lotus japonicus, we performed an integrated multi-omics analysis with a focus on plant and fungal phospholipid (PL) metabolism and biological significance of lysophosphatidylcholine (LPC). Our results support the role of LPC as a bioactive compound eliciting cellular and molecular response mechanisms in Lotus. Evidence is provided for large interspecific chemical diversity of LPC species among mycorrhizae with related AMF species. Lipid, gene expression and elemental profiling emphasize the Lotus-Glomus intraradices interaction as distinct from other arbuscular mycorrhizal (AM) interactions. In G. intraradices, genes involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs were enhanced, while in Lotus, FA synthesis genes were up-regulated during AMS. Furthermore, FAS protein localization to mitochondria suggests FA biosynthesis and elongation may also occur in AMF. Our results suggest the existence of interspecific partitioning of PL resources for generation of LPC and novel candidate bioactive PLs in the Lotus-G. intraradices symbiosis. Moreover, the data advocate research with phylogenetically diverse Glomeromycota species for a broader understanding of the molecular underpinnings of AMS.

Alterations of plasma glycerophospholipid and sphingolipid species in male alcohol-dependent patients

BackgroundAlcohol abuse is a major risk factor for somatic and neuropsychiatric diseases. Despite their potential clinical importance, little is known about the alterations of plasma glycerophospholipid (GPL) and sphingolipid (SPL) species associated with alcohol abuse. MethodsPlasma GPL and SPL species were quantified using electrospray ionization tandem mass spectrometry in samples from 23 male alcohol-dependent patients before and after detoxification, as well as from 20 healthy male controls. ResultsA comparison of alcohol-dependent patients with controls revealed higher phosphatidylcholine (PC; P-value=0.008) and phosphatidylinositol (PI; P-value=0.001) concentrations in patients before detoxification, and higher PI (P-value=0.001) and phosphatidylethanolamine (PE)-based plasmalogen (PE P; P-value=0.003) concentrations after detoxification. Lysophosphatidylcholines (LPC) were increased by acute intoxication (P-value=0.002). Sphingomyelin (SM) concentration increased during detoxification (P-value=0.011). The concentration of SM 23:0 was lower in patients (P-value=2.79×10−5), and the concentrations of ceramide Cer d18:1/16:0 and Cer d18:1/18:0 were higher in patients (P-value=2.45×10−5 and 3.73×10−5). Activity of lysosomal acid sphingomyelinase (ASM) in patients correlated positively with the concentrations of eight LPC species, while activity of secreted ASM was inversely correlated with several PE, PI and PC species, and positively correlated with the molar ratio of PC to SM (Pearson's r=−0.432; P-value=0.039). ConclusionPlasma concentrations of numerous GPL and SPL species were altered in alcohol-dependent patients. These molecules might serve as potential biomarkers to improve the diagnosis of patients and to indicate health risks associated with alcohol abuse. Our study further indicates that there are strong interactions between plasma GPL concentrations and SPL metabolism.

Regulation of hepatic lipase activity by sphingomyelin in plasma lipoproteins

Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content.

Tuberous sclerosis complex 2 loss increases lysophosphatidylcholine synthesis in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease affecting women. LAM is caused by mutations in the tuberous sclerosis complex (TSC) genes. The TSC protein complex inhibits the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), which is a master regulator of cellular metabolism. Using mass spectrometry-based lipid profiling, we analyzed plasma from patients with LAM and discovered elevated levels of four lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, and C20:4) compared with those in healthy control women. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro preclinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid and neutral lipid species. Treatment with rapamycin or torin1 or down-regulation of sterol regulatory element-binding protein (SREBP), a lipogenic transcription factor, did not suppress LPC in TSC2-deficient cells. Inhibition of distinct isoforms of phospholipase A2 decreased the proliferation of TSC2-deficient cells. Collectively, these results demonstrate that TSC2-deficient cells have enhanced choline phospholipid metabolism and reveal a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies.

Identification of isobaric lyso-phosphatidylcholines in lipid extracts of gilthead sea bream (Sparus aurata) fillets by hydrophilic interaction liquid chromatography coupled to high-resolution Fourier-transform mass spectrometry.

The numerous and varied biological roles of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) have fueled a great demand for technologies that enable rapid, in-depth structural examination of these lipids in foodstuffs. Here, we describe the capabilities of a newly configured combination of high-efficiency liquid chromatography and high-resolution/accuracy Fourier-transform mass spectrometry with electrospray ionization (LC-ESI-FTMS), designed for lipidomics applications that require the identification of PC in their lyso forms. The devised strategy, involving a separation by hydrophilic interaction liquid chromatography (HILIC) on spherical, fused-core ultrapure silica particles (2.7μm) of a narrow-bore column (2.1mm i.d.), enabled the identification of as many as 71 LPC species in the lipid extracts of gilthead sea bream (Sparus aurata) fillets. In this way, LPC as proton (43) and sodium (28) adducts, i.e., [M + H](+) and [M + Na](+) ions (with M representing the zwitterionic form), were identified. In several cases, the extremely high (sub-ppm) mass accuracy and the high chromatographic efficiency available with the adopted instrumentation enabled the distinction of isobaric and closely eluting LPC species. Informative tandem mass spectra, based on high-energy collision induced dissociation (HCD), were also obtained, thus distinguishing regioisomeric LPC species (i.e., LPC differing only for the location of the residual side chain on the glycerol backbone) and between proton and sodium adducts. Graphical Abstract Extracted Ion Current chromatogram (XIC) obtained for the m/z value 568.339, showing the presence of two regioisomeric Lysophosphatidylcholines. The corresponding high collisional energy tandem MS spectra, obtained using a HCD cell, are shown as insets.

Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy

BackgroundPre-symptomatic hematopoietic stem cell transplantation is essential to achieve best possible outcomes for patients with the childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD). We describe a high-throughput method for measurement of C20–C26 lysophosphatidylcholines (LPCs) and biochemical diagnosis of X-ALD using the same dried blood spots (DBS) routinely used for newborn screening. MethodsLPCs are extracted from 3-mm DBS punch with methanol containing an isotopically labeled LPC as internal standard. This extract is transferred to a 96-well plate, evaporated and then reconstituted in mobile phase for flow injection analysis tandem mass spectrometry (FIA–MS/MS) in selected reaction monitoring mode for measurement of four different LPCs (C20, C22, C24, C26) and the internal standard (d4-C26-LPC). Analysis time is 1.5min per sample. ResultsThe mean CVs from the intra- and inter-assay experiments for LPCs were 6.3–15.1% for C20-LPC, 4.4–18.6% for C22-LPC and 4.5–14.3% for C24-LPC. Limits of detection were determined for C20-LPC (LOD=0.03μg/mL), C22-LPC (0.03μg/mL), C24-LPC (0.03μg/mL) and C26-LPC (0.01μg/mL). Reference ranges were established from DBS of 130 newborns and 20 adults. Samples of patients with X-ALD (n=16), peroxisomal biogenesis disorders (n=8), and X-ALD carriers (n=12) were analyzed blindly and all were correctly identified. ConclusionAnalysis of LPC species by FIA-MS/MS is a fast, simple and reliable method to screen for X-ALD and other peroxisomal disorders in DBS. To maximize specificity, abnormal results can be verified by a 2nd tier assay using LC–MS/MS.

Abstract 11678: Mitochondrial Reactive Oxygen Species Mediate Lysophosphatidylcholine-Induced Endothelial Cell Activation

Background: Lysophosphatidylcholines (LPCs) are a class of pro-inflammatory lipids that play important roles in atherogenesis. LPC activates endothelial cells (ECs) to upregulate adhesion molecules and chemokines, which is the initiation step of atherogenesis. However, the mechanisms underlying LPC-triggered EC activation are not fully understood. Previously considered as the toxic by-products of cellular metabolism, mitochondrial ROS (mtROS) are recently found to directly contribute to both the innate and adaptive immune responses. Here we tested a novel hypothesis that mtROS serve as signaling mediators for LPC-induced EC activation. Methods and Results: Using electron spin resonance and flow cytometry with fluorescence probe MitoSOX, we found that several LPC species including LPC 16:0, 18:0, and 18:1 induced mtROS in human primary aortic ECs (HAECs). Mechanistically, our analysis using mitochondrial calcium inhibitor and Seahorse XF96 mitochondrial function analyzer showed that LPC induced mtROS via increasing mitochondrial calcium entry which resulted in mitochondrial proton leakage. In addition, we found that mtROS scavenger MitoTEMPO abolished LPC-induced EC activation by downregulating Intercellular adhesion molecule 1 (ICAM-1) and Vascular cell adhesion protein 1 (VCAM-1) in HAECs. Moreover, our analysis with transcription factor profiling screening showed that MitoTEMPO acts by blocking LPC-induced nuclear translocation of pro-inflammatory transcription factor activator protein-1 (AP-1). Conclusions: Our results indicate that mtROS are responsible for LPC-induced EC activation and that mtROS may serve as a novel therapeutic target for vascular inflammation.

Alterations of Plasma Lysophosphatidylcholine Species in Obesity and Weight Loss

BackgroundObesity and related diseases of the metabolic syndrome contribute to the major health problems in industrialized countries. Alterations in the metabolism of lipid classes and lipid species may significantly be involved in these metabolic overload diseases. However, little is known about specific lipid species in this syndrome and existing data are contradictive.MethodsIn this study, we quantified plasma lipid species by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in obese subjects before and after 3 month weight loss as well as in a control group.ResultsThe comparison of obese subjects with control subjects before weight loss revealed significantly lower lysophosphatidylcholine (LPC) concentrations in obesity. LPC concentrations did not significantly increase during the observed period in the weight loss group. Analysis of LPC species revealed a decrease of most species in obesity and negative correlations with C-reactive protein (CRP) and body mass index (BMI). Correlating BMI ratio before and after weight loss with the ratio of total LPC and individual LPC species revealed significant negative relationships of LPC ratios with BMI ratio.ConclusionsOur findings contribute to the contradictive discussion of the role of LPC in obesity and related chronic inflammation strongly supporting pre-existing data in the literature that show a decrease of LPC species in plasma of obese and a potentially anti-inflammatory role in these subjects.

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi.

Background Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.Methodology/Principal findingsHere, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.Conclusions/SignificanceTaken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.

Improved fatty acid analysis of conjugated linoleic acid rich egg yolk triacylglycerols and phospholipid species.

Reports from chicken conjugated linoleic acid (CLA) feeding trials are limited to yolk total fatty acid composition, which consistently described increased saturated fatty acids and decreased monounsaturated fatty acids. However, information on CLA triacylglycerol (TAG) and phospholipid (PL) species is limited. This study determined the fatty acid composition of total lipids in CLA-rich egg yolk produced with CLA-rich soy oil, relative to control yolks using gas chromatography with flame ionization detection (GC-FID), determined TAG and PL fatty acid compositions by thin-layer chromatography-GC-FID (TLC-GC-FID), identified intact PL and TAG species by TLC-matrix-assisted laser desorption/ionization mass spectrometry (TLC-MALDI-MS), and determined the composition of TAG and PL species in CLA and control yolks by direct flow infusion electrospray ionization MS (DFI ESI-MS). In total, 2 lyso-phosphatidyl choline (LPC) species, 1 sphingomyelin species, 17 phosphatidyl choline species, 19 TAG species, and 9 phosphatidyl ethanolamine species were identified. Fifty percent of CLA was found in TAG, occurring predominantly in C52:5 and C52:4 TAG species. CLA-rich yolks contained significantly more LPC than did control eggs. Comprehensive lipid profiling may provide insight on relationships between lipid composition and the functional properties of CLA-rich eggs.