Mass Spectrometry-based Shotgun Lipidomics Research Articles

An altered plasma lipidome-phenome network characterizes heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a multifactorial, multisystemic syndrome that involves alterations in lipid metabolism. This study aimed to test whether distinct plasma lipid profiles or lipid entities or both are associated with clinical and functional echocardiographic parameters in HFpEF. We examined the human plasma lipidome in HFpEF patients (n=18) with left ventricular ejection fraction ≥50% and N-terminal pro-brain natriuretic peptide (NT-proBNP) >125pg/mL and control subjects (n=12) using mass spectrometry-based shotgun lipidomics. The cohort included 8 women and 22 men with average age of 67.8±8.6 SD. The control and disease groups were not significantly different with respect to age, body mass index, systolic and diastolic blood pressure, and waist-to-hip ratio. The disease group experienced more fatigue (P<0.001), had more often coronary artery disease (P=0.04), and received more medications (beta-blockers, P<0.001). The disease group had significantly different levels of HFpEF-relevant parameters, including NT-proBNP (P<0.001), left ventricular mass index (P=0.005), left atrial volume index (P=0.001), and left ventricular filling index (P<0.001), and lower left ventricular end-diastolic diameter (P=0.014), with no difference in left ventricular ejection fraction. Significant differences in lipid profiles between HFpEF patients and controls could not be detected, including no significant differences in abundance of circulating lipids binned by carbon chain length or by double bonds, nor at the level of individual lipid species. However, there was a striking correlation between selected lipids with smoking status that was independent of disease status, as well as between specific lipids and hyperlipidaemia [with corresponding significance of either false discovery rate (FDR) <0.1 or FDR<0.01]. In an exploratory network analysis of correlations, we observed significantly stronger correlations within the HFpEF group between individual lipids from the cholesterol ester and phosphatidylcholine (PC) classes and clinical/echocardiographic parameters such as left atrial volume index, left ventricular end-diastolic diameters, and heart rate (FDR<0.1). In contrast, the control group showed significantly stronger negative correlations (FDR<0.1) between individual species from the PC and sphingomyelin classes and left ventricular mass index or systolic blood pressure. We did not find significant direct associations between plasma lipidomic parameters and HFpEF and therefore could not conclude that any specific lipids are biomarkers of HFpEF. The validation in larger cohort is needed to confidently conclude the absence of first-order associations.

Quantifying yeast lipidomics by high-performance thin-layer chromatography (HPTLC) and comparison to mass spectrometry-based shotgun lipidomics.

Lipidomic analysis in diverse biological settings has become a frequent tool to increase our understanding of the processes of life. Cellular lipids play important roles not only as being the main components of cellular membranes, but also in the regulation of cell homeostasis as lipid signaling molecules. Yeast has been harnessed for biomedical research based on its good conservation of genetics and fundamental cell organisation principles and molecular pathways. Further application in so-called humanised yeast models have been developed which take advantage of yeast as providing the basics of a living cell with full control over heterologous expression. Here we present evidence that high-performance thin-layer chromatography (HPTLC) represents an effective alternative to replace cost intensive mass spectrometry-based lipidomic analyses. We provide statistical comparison of identical samples by both methods, which support the use of HPTLC for quantitative analysis of the main yeast lipid classes.

Cationic amphiphilic drugs induce accumulation of cytolytic lysoglycerophospholipids in the lysosomes of cancer cells and block their recycling into common membrane glycerophospholipids.

Lysosomes are acidic organelles responsible for lipid catabolism, and their functions can be disrupted by cationic amphiphilic drugs that neutralize lumenal pH and thereby inhibit most lysosomal hydrolases. These drugs can also induce lysosomal membrane permeabilization and cancer cell death, but the underlying mechanism remains elusive. Here, we uncover that the cationic amphiphilic drugs induce a substantial accumulation of cytolytic lysoglycerophospholipids within the lysosomes of cancer cells, and thereby prevent the recycling of lysoglycerophospholipids to produce common membrane glycerophospholipids. Using quantitative mass spectrometry-based shotgun lipidomics, we demonstrate that structurally diverse cationic amphiphilic drugs, along with other types of lysosomal pH-neutralizing reagents, elevate the amounts of lysoglycerophospholipids in MCF7 breast carcinoma cells. Lysoglycerophospholipids constitute ∼11 mol% of total glycerophospholipids in lysosomes purified from MCF7 cells, compared with ∼1 mol% in the cell lysates. Treatment with cationic amphiphilic drug siramesine further elevates the lysosomal lysoglycerophospholipid content to ∼24 mol% of total glycerophospholipids. Exogenously added traceable lysophosphatidylcholine is rapidly acylated to form diacylphosphatidylcholine, but siramesine treatment sequesters the lysophosphatidylcholine in the lysosomes and prevents it from undergoing acylation. These findings shed light on the unexplored role of lysosomes in the recycling of lysoglycerophospholipids and uncover the mechanism of action of promising anticancer agents.

Lysosomal Changes in Mitosis.

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Maternal Metals/Metalloid Blood Levels Are Associated With Lipidomic Profiles Among Pregnant Women in Puerto Rico.

Background/Aim: The association between heavy metal exposure and adverse birth outcomes is well-established. However, there is a paucity of research identifying biomarker profiles that may improve the early detection of heavy metal-induced adverse birth outcomes. Because lipids are abundant in our body and associated with important signaling pathways, we assessed associations between maternal metals/metalloid blood levels with lipidomic profiles among 83 pregnant women in the Puerto Rico PROTECT birth cohort.Methods: We measured 10 metals/metalloid blood levels during 24–28 weeks of pregnancy. Prenatal plasma lipidomic profiles were identified by liquid chromatography–mass spectrometry-based shotgun lipidomics. We derived sums for each lipid class and sums for each lipid sub-class (saturated, monounsaturated, polyunsaturated), which were then regressed on metals/metalloid. False discovery rate (FDR) adjusted p-values (q-values) were used to account for multiple comparisons.Results: A total of 587 unique lipids from 19 lipid classes were profiled. When controlling for multiple comparisons, we observed that maternal exposure to manganese and zinc were negatively associated with plasmenyl-phosphatidylethanolamine (PLPE), particularly those containing polyunsaturated fatty acid (PUFA) chains. In contrast to manganese and zinc, arsenic and mercury were positively associated with PLPE and plasmenyl-phosphatidylcholine (PLPC).Conclusion: Certain metals were significantly associated with lipids that are responsible for the biophysical properties of the cell membrane and antioxidant defense in lipid peroxidation. This study highlighted lipid-metal associations and we anticipate that this study will open up new avenues for developing diagnostic tools.

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P) from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As2O3) could reduce the level of S1P in human gastric cancer cell line MGC-803, indicating that As2O3 may inhibit the activity of SphK1. In this study, the effect of As2O3 on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used to evaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomics method (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). The results revealed that As2O3 did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However, As2O3 increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS and PA in the MGC-803 cells. The outcomes suggested that As2O3 may enhance the activity of SphK1 by increasing the levels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggested that the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P) from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As2O3) could reduce the level of S1P in human gastric cancer cell line MGC-803, indicating that As2O3 may inhibit the activity of SphK1. In this study, the effect of As2O3 on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used to evaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomics method (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). The results revealed that As2O3 did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However, As2O3 increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS and PA in the MGC-803 cells. The outcomes suggested that As2O3 may enhance the activity of SphK1 by increasing the levels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggested that the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.

A Lipidomics Atlas of Selected Sphingolipids in Multiple Mouse Nervous System Regions.

Many lipids, including sphingolipids, are essential components of the nervous system. Sphingolipids play critical roles in maintaining the membrane structure and integrity and in cell signaling. We used a multi-dimensional mass spectrometry-based shotgun lipidomics platform to selectively analyze the lipid species profiles of ceramide, sphingomyelin, cerebroside, and sulfatide; these four classes of sphingolipids are found in the central nervous system (CNS) (the cerebrum, brain stem, and spinal cord) and peripheral nervous system (PNS) (the sciatic nerve) tissues of young adult wild-type mice. Our results revealed that the lipid species profiles of the four sphingolipid classes in the different nervous tissues were highly distinct. In addition, the mRNA expression of sphingolipid metabolism genes—including the ceramidase synthases that specifically acylate the N-acyl chain of ceramide species and sphingomyelinases that cleave sphingomyelins generating ceramides—were analyzed in the mouse cerebrum and spinal cord tissue in order to better understand the sphingolipid profile differences observed between these nervous tissues. We found that the distinct profiles of the determined sphingolipids were consistent with the high selectivity of ceramide synthases and provided a potential mechanism to explain region-specific CNS ceramide and sphingomyelin levels. In conclusion, we portray for the first time a lipidomics atlas of select sphingolipids in multiple nervous system regions and believe that this type of knowledge could be very useful for better understanding the role of this lipid category in the nervous system.

A novel sn-1,3 specific lipase from Janibacter sp. as catalysts for the high-yield synthesis of long-medium-long type structured triacylglycerols

Our study offers a novel sn-1,3 specific lipase MAJ1 from marine member Janibacter sp. strain HTCC2649 for preparing long-medium-long (LML) type structured triacylglycerols (TAGs). Firstly, the resin ECR1030 was selected as a suitable support for the immobilization of lipase MAJ1. An efficient synthesis of LML-type structured TAGs by the immobilized lipase MAJ1-catalyzed interesterification of methyl palmitate and tricaprylin was studied in a solvent-free system. The reaction conditions, including substrate molar ratio, temperature and enzyme loading, were optimized. Under the optimum conditions (immobilized lipase MAJ1 of 45 U/g, substrate molar ratio of 4:1, temperature of 35 °C, reaction time of 24 h), the structured TAGs with double long chains (DLCST) were obtained in a yield of 44.3 mol%. Secondly, multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) was employed to quantify each TAG positional isomer in DLCST. The content of 1,3-dipalmitoyl-2-capryloyl-sn-glycerol in DLCST was 97.6% determined by the MDMS-SL technology.

Dendrobium officinale Regulates Fatty Acid Metabolism to Ameliorate Liver Lipid Accumulation in NAFLD Mice.

Dendrobium officinale (DOF) is a traditional Chinese edible and officinal plant. Ultrafine DOF powder (DOFP) can regulate lipids and histopathology in the liver, but the underlying mechanisms of hepatic fatty acid (FA) metabolism, which is generally correlated with the development of nonalcoholic fatty liver disease (NAFLD), remain unclear. The purpose of the present study was to investigate whether DOFP treatment alters hepatic FA metabolism in NAFLD mice by using multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) and analyse the underlying mechanisms. A 3-week DOFP treatment prevented lipid deposition and improved hepatic histopathology in NAFLD mice after withdrawal from the high-sucrose, high-fat (HSHF) diet, and it decreased triglyceride and FA content in the liver. Furthermore, the C16 : 0/C14 : 0 and C18 : 1/18 : 0 ratios in FAs were significantly decreased in the DOFP treatment group, and the C20 : 4/C20 : 3 and C22 : 4/C22 : 3 ratios were increased, and saturated FA was inhibited. Additionally, DOFP treatment significantly increased the content of two FA β-oxidation-related proteins (carnitine palmitoyltransferase 1-α and acyl-coenzyme A oxidase 1). It also decreased the content of a FA synthesis-related protein (fatty acid synthase), a FA desaturation-related protein (stearoyl-coenzyme A desaturase-1), and a FA uptake-related protein (fatty acid transport protein 2). Moreover, DOFP treatment improved dysregulated levels of major phospholipids in the livers of model mice. The results of this study confirm that DOFP treatment in NAFLD mice has liver recovery effects by regulating FA metabolism.

Sphingolipids of plant pathogenic fungi

Glycosphingolipids in filamentous fungi are significant components of the plasma membrane and are vital for different cellular processes, such as growth, morphological transition or signal transduction. Fungal growth inhibitors targeting glycosylinositolphosphoceramide (GIPCs) biosynthesis or antifungal compounds binding to GIPCs present in membranes could present a safe way of preventing fungal growth on crops since GIPCs are not present in mammalian cells. Mass spectrometry-based shotgun lipidomics was used to analyze sphingolipids of 11 fungal strains isolated from plant material. Molecular species with inositol ceramides containing zero to five carbohydrates were identified. Differences in the amount of individual molecular species were influenced by the taxonomic affiliation. All tested strains exhibited a relatively high content (more than 40 mol.%) of GIPCs with three and more saccharides attached to the polar head. It could be assumed that the sphingolipid profiles of the tested plant pathogens would be an adaptation mechanism to antifungal plant defensins.

Adverse Effects of Refeeding on the Plasma Lipidome in Young Individuals With Anorexia Nervosa?

Refeeding is the cornerstone of anorexia nervosa (AN) treatment, but little is known regarding the optimal pace and dietary composition or possible adverse effects of current clinical practices. Plasma lipids may be a moderating factor underlying unfavorable refeeding effects in AN, such as an abnormal central body fat distribution. The objective of this study was to analyze the plasma lipidome in the acutely underweight state of AN before and after refeeding. Using high-throughput quantitative mass spectrometry-based shotgun lipidomics, we measured 13 lipid classes and 204 lipid species or subspecies in the plasma of young female patients with acute AN, before (n= 39) and after (n= 23) short-term weight restoration during an intensive inpatient refeeding program (median body mass index [BMI] increase= 26.4%), in comparison to those in healthy control participants (n= 37). Before inpatient treatment, patients with AN exhibited increased concentrations of cholesterol and several other lipid classes. After refeeding, multiple lipid classes including cholesterol and ceramides, as well as certain ceramide species previously associated with obesity or overfeeding, showed increased concentrations, and a pattern of shorter and more saturated triacylgycerides emerged. A machine learning model trained to predict BMI based on the lipidomic profiles revealed a sizable overprediction in patients with AN after weight restoration. The results point toward a profound lipid dysregulation with similarities to obesity and other features of the metabolic syndrome after short-term weight restoration. Thus, this study provides evidence for possible short-term adverse effects of current refeeding practices on the metabolic state and should inspire more research on nutritional interventions in AN.

Lipid Metabolism and Lipidomics Applications in Cancer Research.

Lipids are the critical components of cellular and plasma membrane, which constitute an impermeable barrier of cellular compartments, and play important roles on numerous cellular processes including cell growth, proliferation, differentiation, and signaling. Alterations in lipid metabolism have been implicated in the development and progression of cancers. However, unlike other biomolecules, the diversity in the structures and characteristics of lipid species results in the limited understanding of their metabolic alterations in cancers. Lipidomics is an emerging discipline that studies lipids in a large scale based on analytical chemistry principles and technological tools. Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) uses direct infusion to avoid difficulties from alterations in concentration, chromatographic anomalies, and ion-pairing alterations to improve resolution and achieve rapid and accurate qualitative and quantitative analysis. In this chapter, lipids and lipid metabolism relevant to cancer research are introduced, followed by a brief description of MDMS-SL and other shotgun lipidomics techniques and some applications for cancer research.

Analysis of monohexosyl alkyl (alkenyl)-acyl glycerol in brain samples by shotgun lipidomics

Lipid species possess very different structures, leading to their very diversified cellular functions in biological systems. Lipidomics represents a powerful technology for deep analysis of hundreds to thousands of intact lipid molecular species. In the current study, a cluster of unknown ion peaks was displayed when we profiled cerebroside species in rat spinal cord samples by neutral loss scan of 162 Da in the positive ion mode using a multi-dimensional mass spectrometry-based shotgun lipidomics strategy. In order to identify the structural identities of these unknown ion peaks, MS2 and MS3 analyses of these ions were performed by high mass resolution mass spectrometry. Extensive lines of evidence allowed us to identify that these unknown ion peaks were monohexosyl alkyl-acyl glycerol (HAAG) species, including their sn-positional isomers and alkyl-acyl compositional isomers. We also applied the developed method to identify and quantify HAAG species present in a variety of mouse nerve tissues. We believe that the first kind of lipidomics study on HAAG species present in mammalian nerve tissue samples provided the foundation for future biological research in this unknown area.

Hypertensive Rats Treated Chronically With Nω-Nitro-L-Arginine Methyl Ester (L-NAME) Induced Disorder of Hepatic Fatty Acid Metabolism and Intestinal Pathophysiology.

Nω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) biosynthesis, results in hypertension and liver injury. This study aimed at investigating the changes of liver lipometabonomics and exploring the underlying mechanisms of liver injury in the L-NAME-treated rats. The male Sprague-Dawley (SD) rats were treated with L-NAME (40 mg/kg, p.o.) for 8 weeks. After that, the liver, aorta, fecal, and serum were collected for analysis. The results showed that L-NAME induced hypertension and disordered the endothelial nitric oxide synthase (eNOS)-NO pathway in the treated rats. L-NAME could also increase the levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate transaminase (AST). The multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) analysis showed that L-NAME could induce significant changes of the total hepatic lipids and most hepatic triglycerides, as well as fatty acid (FA). A positive correlation was found between the blood pressure and TAG. Immunofluorescence and Western-Blot experiments indicated that the L-NAME treatment significantly influenced some FA β-oxidation, desaturation, and synthesis-related proteins. The increase of intestinal inflammation, decrease of microcirculation and tight junction proteins, as well as alterations of microbial communities were observed in the L-NAME induced hypertensive rats, as well as alterations of microbial communities were notable correlation to TAG and FA species. This study demonstrated that the L-NAME-induced hypertensive rats exhibiting liver injury were the joint action of hepatic abnormal fatty acid metabolism and microcirculation disorder. Furthermore, the gut microflora, as well as the changes of FA β-oxidation (ACOX, CPT1α), desaturation (SCD-1), and synthesis (FAS) may be the potential mechanisms for abnormal fatty acid metabolism.

Sensitive analysis of fatty acid esters of hydroxy fatty acids in biological lipid extracts by shotgun lipidomics after one-step derivatization.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of biological/biomedical conditions, and facilitate the understanding of these lipid species in the patho(physio)logical process.

Sustained maternal smoking-associated changes in the physico-chemical properties of fetal RBC membranes might serve as early markers for vascular comorbidities

Maternal smoking-induced congenital heart and microvascular defects are closely associated with the impaired functioning of the in-utero feto-placental circulation system. Current groundbreaking facts revealed intimate crosstalk between circulating red blood cells (RBCs) and the vascular endothelium. Thus, RBCs have become the protagonists under varied pathological and adverse pro-oxidative cellular stress conditions. We isolated and screened fetal RBCs from the arterial cord blood of neonates, born to non-smoking (RBC-NS) and smoking mothers (RBC-S), assuming that parameters of fetal RBCs are blueprints of conditions experienced in-utero. Using atomic force microscopy and mass spectrometry-based shotgun lipidomics in the RBC-S population we revealed induced membrane stiffness, loss in intrinsic plastic activities and several abnormalities in their membrane-lipid composition, that could consequently result in perturbed hemodynamic flow movements. Altogether, these features are indicative of the outcome of neonatal microvascular complications and suggest unavailability for the potential rescue mechanism in cases of vascular endothelium impairment due to altered membrane integrity and rheological properties.

Ceramide subclasses identified as novel lipid biomarker elevated in women with polycystic ovary syndrome: a pilot study employing shotgun lipidomics

This study aimed to identify potential lipid biomarkers in women with polycystic ovary syndrome (PCOS) and determine their predictive value for PCOS. Eighteen women with PCOS and 17 healthy controls were enrolled. A multi-dimensional mass spectrometry-based shotgun lipidomics approach was employed to analyze serum lipid profiles. Shotgun lipidomics revealed that the concentrations of ceramide (Cer) and phosphatidylcholine (PC) were higher (PC: 831.6 ± 217.4 vs. 605.2 ± 164.2 μmol/l; Cer: 3,387.6 ± 829.9 vs. 2,552.2 ± 679.4 nmol/l, respectively), whereas that of lysophosphatidylcholine was lower, in PCOS women than in healthy controls (82.02 ± 39.49 vs. 133.62 ± 65.36 μmol/l, respectively). Receiver operating characteristic analysis showed that the combination of Cer (OH_N16:0/N18:0) and Cer (N22:0) had the greatest discriminatory power to differentiate between women with and without PCOS (area under the curve: 0.889, 95% confidence interval: 0.784–0.994). These results indicate that the combination of Cer (OH_N16:0/N18:0) and Cer (N22:0) may represent a novel lipid predictor of PCOS.

Strategies to Improve/Eliminate the Limitations in Shotgun Lipidomics.

Direct infusion-based shotgun lipidomics is one of the most powerful and useful tools in comprehensive analysis of lipid species from lipid extracts of various biological samples with high accuracy/precision. However, despite many advantages, the classical shotgun lipidomics suffers some general dogmas of limitations, such as ion suppression, ambiguous identification of isobaric/isomeric lipid species, and ion source-generated artifacts, restraining the applications in analysis of low-abundance lipid species, particularly those less ionizable or isomers that yield almost identical fragmentation patterns. This article reviews the strategies (such as modifier addition, prefractionation, chemical derivatization, charge feature utilization) that have been employed to improve/eliminate these limitations in modern shotgun lipidomics approaches (e.g., high mass resolution mass spectrometry-based and multidimensional mass spectrometry-based shotgun lipidomics). Therefore, with the enhancement of these strategies for shotgun lipidomics, comprehensive analysis of lipid species including isomeric/isobaric species is achieved in a more accurate and effective manner, greatly substantiating the aberrant lipid metabolism, signaling trafficking, and homeostasis under pathological conditions.

PO-264 Target Mitochondria Metabolism and Energy Expenditure in Muscle Atrophy, Sarcopenia and Cachexia

Objective Muscle atrophy is a common and clinically important outcome of many diseases and hospitalization bed rest. Mechanical unloading of skeletal muscles may result in a rapid loss of muscle mass and mitochondria dysfunction. And the recovery from disuse muscle atrophy is usually complete in young healthy adults but is delayed and often incomplete in older patients. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. To this end, we employed multiple approaches to address the role of mitochondrial function and metabolism in muscle atrophy and sarcopenia. And we are also engaged to develop novel mitochondria-targeted antioxidant peptides to mitigate this mitochondria-related functional impairment by ROS and improve the recovery from muscle atrophy and cachexia.&#x0D; Methods Tail Suspension Hind Limb Unloading for adult mice (6-month-old) and aged mice (22-24month old). Cardiometabolic Phenotyping：Measurement of fat and lean mass (g) is accomplished using a LF90II TD-NMR (Bruker, Madison, WI). Locomotor activity measurements are obtained using a Promethion Mouse Multiplexed Metabolic System(Sable Systems International, Las Vegas, NV).Mitochondrial Respiration :Respirometry assays were conducted using an Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria), Mitochondria H2O2 emission was measured with Amplex Red reagent which reacts with H2O2 to produce the stable fluorescent compound resorufin. Resorufin fluorescence was monitored using a Fluorescence. A continuous, spectrophotometric assay was utilized to measure mitochondrial calcium retention capacity (CRC) within soleus fiber bundles. RNA Sequencing and Informatics, KEGG Pathway and Upstream Regulator Analysis, Real-Time Quantitative-PCR. Multidimensional mass spectrometry-based shotgun lipidomics was employed to measure and characterize the lipid patterns inmouse soleus muscle. Metabolomics Acylcarnitines A panel of acylcarnitines was quantitated by LC/ MS/MS (Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer). Amino Acids. Quantitation of amino acids was achieved using LC/MS/MS (Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer).&#x0D; Results Old mice have impaired early recovery of soleus mass following unloading induced muscle atrophy. And mitochondrial function and metabolism does not Improve during early recovery in aged Mice. Divergent metabolomic response was found between adult and aged Mice during unloading and recovery. However, transcriptomic response to unloading and reloading is similar in adult and aged Mice.&#x0D; Conclusions Here, we report that aged mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass after disuse muscle atrophy. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid β-oxidation and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. Our findings also showed cellular metabolic changes during muscle atrophy and sarcopenia may induce higher production of oxygen radicals that play a significant role in the progression of age-related sarcopenia. These findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse. We are engaged to develop novel mitochondria-targeted antioxidant peptides to mitigate this mitochondria-related functional impairment by ROS and improve the recovery from muscle atrophy and cachexia. Co-administration of other targeted compounds was also promising to improve recovery from muscle atrophy and cachexia.&#x0D;