Lipid Extracts Research Articles

Enhanced Lipidomics Analysis of Breast Cancer Cells Using Three-phase Liquid Extraction and Ultra High-performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Lipid extraction of complex biological samples is essential for high-quality data in liquid chromatography-mass spectrometry (LC-MS)-based lipidomics. This study introduces a three-phase liquid extraction (3PLE)-ultra-high-performance LC coupled with quadrupole time-of-flight tandem MS method. This method was successfully applied to lipidomics analysis of breast cancer cells, including highly metastatic MDA-MB-231 and slightly metastatic MCF7 cells. The 3PLE method employed an n-hexane/methyl tert-butyl ether/acetonitrile/water solvent system that formed one aqueous and two organic phases. Neutral and polar lipids were enriched in the upper and middle organic phases, respectively, and combined for detection, thereby reducing analysis time. Compared with the Bligh and Dyer method, 3PLE achieved higher sensitivity and detected more features, with over a 50% increase in the relative abundance of nearly 50% of the differential lipids. In total, 21 differential lipids were identified in the MDA-MB-231 group and 22 in the MCF7 group compared to normal breast epithelial cells (MCF10A). Pathway analysis suggested that lipid changes in breast cancer cells were associated with glycerophospholipid metabolism, arachidonic acid metabolism, sphingolipid metabolism, and linoleic acid metabolism. The study presents a highly efficient lipidomics method, providing a scientific foundation for understanding breast cancer pathogenesis and aiding in diagnosis.

Assessing the Effect of Minimally Invasive Lipid Extraction on Parchment Integrity by Artificial Ageing and Integrated Analytical Techniques.

Assessing the Effect of Minimally Invasive Lipid Extraction on Parchment Integrity by Artificial Ageing and Integrated Analytical Techniques.

Optimization of the marine microalgae Nannochloropsis oculata harvesting by flocculation: Effects on biomass quality and residual medium reuse for a new cycle

ABSTRACT Flocculation using natural plant-based biopolymers from Moringa oleifera seeds and Acacia mearnsii flocculant as Tanfloc® (TSG) has emerged as an efficient strategy for microalgae biomass harvesting. This study optimized the recovery process of Nannochloropsis oculata by varying flocculant concentration, sedimentation time, and culture pH. Results revealed that 1.2 mg.L−1 of TSG at pH 8.9 achieved optimal conditions, although TSG proved effective across different pH ranges. Alkaline flocculation (ALK) also enhanced microalgae harvesting, yielding a high harvesting efficiency (>96%) within 30 minutes of sedimentation. Both methods achieved a concentration factor exceeding 20. However, the use of ALK resulted in reduced lipid and carbohydrate content compared to the control condition. Carotenoid levels showed no significant difference. TSG influenced lipid extraction but not fatty acid quality or carbohydrate extraction. Importantly, the residual medium from TSG flocculation could sustain subsequent cultivation cycles without nutrient supplementation, presenting a cost-effective solution for microalgae cultivation and harvesting.

Evaluation of the pharmacological efficiency of the lipid extract from the tunic of the marine hydrobiont Halocynthia aurantium (Pallas, 1774)

It was studied effect of the lipid extract isolated from the tunic of the marine hydrobiont Halocynthia aurantium. It was evaluated its administration during hypothermia, hyperthermia, muscle load and the introduction of hexenal as well as its embryotoxic and teratogenic effects The impact of stress (vertical fixation of rats by the dorsal neck fold) was accompanied by an increase in plasma levels of total lipids, total cholesterol, cholesterol/phospholipid ratios and a decrease in total phospholipids, as well as by a change in the quantitative characteristics of classes of neutral and phospholipid. It was carried out a correction of the developed changes by a lipid extract of ascidia and a commercial reference preparation “Omega-3”. The lipid extract of H. aurantium showed a higher efficiency in restoring the lipid composition of the blood plasma under stress impact compared to the preparation “Omega-3” due to a wider range of neutral and phospholipid classes, polyunsaturated fatty acids of the n-3 and n-6 families. The tunica of ascidian can be used as a raw material for obtaining preparations with stress-protector and lipid-correcting properties.

Characterization of Extracellular Vesicles by Sulfophosphovanillin Colorimetric Assay and Raman Spectroscopy.

Detailed characterization of extracellular vesicles (EVs) is crucial for their application in medical diagnostics. However, the complexity of their chemical composition and the heterogeneity of EV populations make their characterization challenging. Here we describe two analytical procedures that can help overcome this challenge. Small EVs were isolated from conditioned cell culture media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Raman spectroscopy was used to assess the overall composition of the isolated samples and lipids extracted from them. Sulfophosphovanillin (SPV) colorimetric assay was used to quantify the contents of lipid. Six samples of EVs were characterized. The lipid contents measured using SPV assay was in reasonable agreement with the quantitative estimates based on the particle size and concentration measured using NTA. The most peaks observed in the Raman spectra could be attributed to either proteins or lipids, and their origins was confirmed by lipid extraction. The protein-to-lipid ratio was estimated based on the Raman spectra. The experiential procedures described in this study will help to overcome the challenge of quick and highly informative characterization of the EVs.

Environmental impact assessment in microalgal lipid production: carbon footprint and net emissions

This study focuses on analyzing the role of microalgae in mitigating climate change through CO2 capture and lipid production, which can be used in the development of energy products and commercially relevant products. The objective was to evaluate the net CO2 emissions in three lipid production scenarios from microalgae, in addition to analyzing the impact of various biomass pretreatment technologies before lipid extraction. For this purpose, only the emissions directly generated by energy consumption in the process and the amount of CO2 that can be captured by microalgae cultivation were considered. In all three scenarios, CO2 capture during biomass cultivation and emissions associated with maintaining the process in operation were evaluated. The results show that the emissions generated by energy consumption were 3.56 kg-CO2/kg-oil, 2.19 kg-CO2/kg-oil, and 2.36 kg-CO2/kg-oil in scenarios 1, 2, and 3, respectively, while the CO2 emissions captured in microalgae cultivation were three to four times higher, ultimately resulting in negative emissions in all scenarios. The efficiency of CO2 capture per kilogram of oil produced varies between scenarios, suggesting that the choice of technology and process conditions significantly influence the overall environmental impact. This analysis identifies areas of opportunity to improve microalgal biomass utilization processes, highlighting the stages of the process with the greatest impact on the carbon footprint and enabling the comparison of different technologies on the same basis.

Ultrasound-assisted extraction vs. microwave-assisted extraction for sustainable development goals: Selecting the ideal lipid extraction and fatty acid profile

The objective of this study was to compare the efficiency of ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) methods for extracting lipids from the green microalga Chlorococcum novae-angliae. This study specifically focused on the fatty acid profiles of the extracted lipids, using ethanol as the solvent and wet biomass as the starting material. Ultrasound-assisted extraction yielded a maximum of 0.026 ± 0.001 g lipid/g wet biomass at a biomass ratio of 1:25 for 2 min with a 1-second cycle at 180 W and 20 kHz, which was 21% higher than that of microwave-assisted extraction conducted at a ratio of 1:30 for 2 min at 300 W and 35 °C. Ultrasound-assisted extraction enhanced saturated fatty acids (SFAs), which were 1.5 times higher than to microwave-assisted extraction, while microwave-assisted extraction significantly increased polyunsaturated fatty acids (PUFAs) by 4.4 times. The findings suggest that ultrasound-assisted extraction is more suitable for applications requiring high SFA content, such as in the fuel industry, whereas microwave-assisted extraction is preferable for sectors focused on fatty acid quality, such as food and health. This comparative analysis contributes to the literature by highlighting the impact of extraction methods on fatty acid profiles and supports sustainable development goals (SDGs), particularly SDG 12, by promoting environmentally friendly extraction techniques.

Mechanistic insights into lipid extraction from wet microalgae using hydrophobic deep eutectic solvents under hydrothermal conditions

Hydrophobic deep eutectic solvents (HDESs) were first employed under hydrothermal conditions to form a two-phase system for instantaneous and efficient lipid extraction from wet microalgae, preventing lipid degradation and avoiding the use of toxic solvents. Ten HDESs were assessed for their efficiency and selectivity in lipid extraction. Among them, OCT-DEC, composed of octanoic acid and decanoic acid, achieved the highest extraction efficiency at 95.42 %, while also preventing the co-extraction of proteins and reducing sugars. Molecular dynamics simulations revealed that lipid extraction efficiency is primarily influenced by lipid-HDES interactions, which drive lipid diffusion, and the interfacial properties of the two-phase hydrothermal system, which resist lipid diffusion. The superior performance of OCT-DEC is attributed to its high hydrogen bond density and short bond lifetimes, which, combined with its structural stability, synergistically reduce resistance to lipid diffusion at the interface. Additionally, OCT-DEC significantly enhances the thermal stability of unsaturated fatty acids, such as C20:5, effectively preventing the degradation of high-value lipids and highlighting its potential for hydrothermal extraction of these valuable compounds.

Bioactive Lipids in Dunaliella salina: Implications for Functional Foods and Health.

Dunaliella salina is a green microalga extensively explored for β-carotene production, while knowledge of its lipid composition is still limited and poorly investigated. Among lipids, polar lipids have been highlighted as bioactive phytochemicals with health-promoting properties. This research aimed to provide an in-depth lipidome profiling of D. salina using liquid and gas chromatography coupled with mass spectrometry. The lipid content was 6.8%, including phospholipids, glycolipids, betaine lipids, sphingolipids, triglycerides, diglycerides, and pigments. Among the total esterified fatty acids, 13.6% were 18:3 omega-3 and 14.7% were 18:1 omega-9. The lipid extract of D. salina showed anti-inflammatory activity by inhibiting cyclooxygenase-2 activity at 100 µg/mL, dose-dependent antioxidant scavenging activity, and antidiabetic activity by inhibiting α-glucosidase activity at 25 and 125 µg/mL. In conclusion, the lipid extract of D. salina has the potential to be used as a functional food ingredient or in the nutraceutical and cosmeceutical industries.

Radical-directed dissociation mass spectrometry for differentiation and relative quantitation of isomeric ether-linked phosphatidylcholines

BackgroundEther-linked phosphatidylcholines (PCs) include both plasmanyl and plasmenyl PCs, which contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. Profiling and quantifying ether PCs with accurate structural information is challenging because of the common presence of isomeric and isobaric species in a lipidome. ResultsIn the present study, radical directed dissociation (RDD) from collision-induced dissociation (CID) of the bicarbonate anion adduct of ether PCs has been investigated to differentiate and relatively quantify ether PCs. Alkyl- and alkenyl- PCs give diagnostic characteristic fragment patterns that enable their confident identification and isomer differentiation. Additionally, the sn-position specific product ions have proven effective for relative quantitation among isomers in ether PCs and their isobaric PC species. Using this methodology, we successfully identified a total of 30 PC-O species, 21 PC-P species at the chain composition level, and 22 species of isobaric PC at the sn-position level in the human plasma lipid extract. The quantitative analysis revealed that ether PCs with a 20:4 fatty acyl chain are relatively more abundant in human plasma. Finally, the profile of ether PCs in type 2 diabetic (T2D) groups compared to normal control groups revealed a significant decrease in PC-O 18:1/20:5. We also found it is the PC species containing a 17-carbon fatty acyl chain, rather than their isobaric ether PCs, that shows a decreasing trend in the T2D groups. Significanceether-linked PCs are firstly investigated by RDD mass spectrometry.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

Elevated Porcupine Disrupts Lipid Metabolism and Promotes Inflammatory Response in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) presents a high incidence globally and is a major cause of cirrhosis and hepatocellular carcinoma, lacking of efficient interventions. Patients with MASLD exhibit exceeded serum levels of palmitic acid (PA). However, the association between PA and MASLD remains obscure. Gene expression omnibus dataset analysis, western blotting, mRNA-sequencing, RT-qPCR, a click chemistry-immunoprecipitation-immunofluorescence system, ELISA, lipid extraction and UHPLC-MS/MS analysis, CyTOF mass cytometry, gene knockdown via lentivirus-mediated shRNA, and high-fat methionine and choline-deficient diet-fed WT and db/db mice models were used to reveal the expression and functions of Porcupine in MASLD development both invitro and invivo. Our findings show that PA, as a crucial substrate for protein palmitoylation, induced the expression of palmitoyltransferase Porcupine in a time-dependent manner. This induction was closely associated with dysregulated lipid metabolism and stimulated inflammatory response observed invitro. Porcupine protein levels were significantly increased in liver tissues from both MASLD mice models, which was predominantly localised in lipid droplet-rich hepatocytes. Pharmacological inhibition of Porcupine by Wnt974 markedly ameliorated the aberrant lipid accumulation and inflammatory response in mouse livers. Furthermore, increased Porcupine positively correlated with CD36 at protein levels, and its inhibition or knockdown decreased CD36 protein levels via mechanisms irrelevant to transcriptional regulation, but primarily dependent on protein palmitoylation. The current study reveals that PA-induced Porcupine disrupts lipid metabolism and promotes inflammatory response during MASLD development, which can be ameliorated by the Porcupine inhibitor Wnt974. Therefore, Porcupine may be a potential pharmacological target for the treatment of MASLD.

A lipidomic approach towards identifying the effects of fragrance hydroperoxides on keratinocytes.

Limonene and linalool are used in cosmetic products for their floral scents, but their oxidation products are strong contact allergens whose mechanisms of action are still not fully understood. The effects of limonene hydroperoxide (Lim-2-OOH) and linalool hydroperoxides (Lin-6/7-OOH) on the lipid profile of a human keratinocyte cell line (HaCaT) were evaluated. 2,4-Dinitrofluorobenzene (DNFB) was also included. Lim-2-OOH and Lin-6/7-OOH were synthesised according to previous methods. HaCaT cells were treated with allergens (10 μM) for 24 h and the cellular lipid extracts were analysed by C18 liquid chromatography with tandem mass spectrometry (LC-MS/MS). Data analysis was performed using Lipostar software. Statistical analysis was carried out using Metaboanalyst and R software. All three sensitisers used caused significant changes in the lipidome of HaCaT cells in a similar trend. There was an upregulation in several plasmanyl/plasmenyl phospholipids (O-/P-phosphatidylcholines [PC] and O-/P-phosphatidylethanolamines [PE]), sphingolipids (HexCer) and triacylglycerol lipid species, and a decrease in some polyunsaturated fatty acids-containing phospholipid (PE and PC) species suggesting oxidative stress and inflammation. This study is the first to evaluate the plasticity of the HaCaT cell lipidome in response to allylic hydroperoxide allergens Lim-2-OOH and Lin-6/7-OOH, together with the experimental contact allergen DNFB. These allergens are able to upregulate and downregulate certain lipid classes to a varying degree.

Mathematical lipid correction of δ13C and effect of lipid extraction on δ15N of European eel (Anguilla anguilla) muscle: Lipid correction of δ13C in European eels.

Carbon (δ13C) and nitrogen (δ15N) stable isotope analysis is a powerful tool to investigate diverse questions in fish ecology, such as their trophic position or migration strategies. These questions appear particularly important to protect endangered European eel. However, elevated lipid content in eel muscle can bias δ13C values, as lipids are 13C-depleted compared to proteins and carbohydrates. We measured δ13C and δ15N values of bulk and lipid-free samples of eel muscle. Lipid-free samples were obtained after the extraction of lipids with cyclohexane. Lipid-corrected δ13C values, using five different mathematical equations based on bulk δ13C values, were compared to lipid-free δ13C values. We also evaluated the effect of lipid extraction on δ15N values. The analyses were based on linear regression performed on 333 individuals captured in nine lagoons and four rivers. Independently to the capture site or habitat (river or lagoon), the predicted lipid-corrected δ13C values were highly consistent with the measured lipid-free δ13C values (R2 > 0.90). The application of specific equations for each habitat or capture site only slightly increases these R2 (1.5% or less). The lipid extraction treatment significantly decreased by 0.2‰ the δ15N values compared to bulk samples. Given the excellent prediction of mathematical equations and the small decrease of δ15N values after lipids extraction, we propose to use mathematical correction to estimate δ13C values of eel muscle. As the habitats or sites did not strongly influence the results, the coefficients from our study can be applied to other studies on European eel.

Nannochloropsis oceanica Lipid Extract Moderates UVB-Irradiated Psoriatic Keratinocytes: Impact on Protein Expression and Protein Adducts.

Psoriasis is characterized by excessive exfoliation of the epidermal layer due to enhanced pro-inflammatory signaling and hyperproliferation of keratinocytes, further modulated by UV-based anti-psoriatic treatments. Consequently, this study aimed to evaluate the impact of a lipid extract derived from the microalgae Nannochloropsis oceanica on the proteomic alterations induced by lipid derivatives in non-irradiated and UVB-irradiated keratinocytes from psoriatic skin lesions compared to keratinocytes from healthy individuals. The findings revealed that the microalgae extract diminished the viability of psoriatic keratinocytes without affecting the viability of these cells following UVB exposure. Notably, the microalgae extract led to an increased level of 4-HNE-protein adducts in non-irradiated cells and a reduction in 4-hydroxynonenal (4-HNE)-protein and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2)-protein adducts in UVB-exposed keratinocytes from psoriasis patients. In healthy skin cells, the extract decreased the UV-induced elevation of 4-HNE-protein and 15d-PGJ2-protein adducts. The antioxidant/anti-inflammatory attributes of the lipid extract from the Nannochloropsis oceanica suggest its potential as a protective agent for keratinocytes in healthy skin against UVB radiation's detrimental effects. Moreover, it could offer therapeutic benefits to skin cells afflicted with psoriatic lesions by mitigating their proliferation and inflammatory responses during UV radiation treatment.

Impaired Suppression of Plasma Lipid Extraction and its Partitioning Away from Muscle by Insulin in Humans with Obesity.

Humans with obesity and insulin resistance exhibit lipid accumulation in skeletal muscle, but the underlying biological mechanisms responsible for the accumulation of lipid in the muscle of these individuals remain unknown. We investigated how plasma insulin modulates the extraction of circulating triglycerides (TGs) and non-esterified fatty acids (NEFAs) from ingested and endogenous origin in the muscle of lean, insulin-sensitive humans (Lean-IS) and contrasted these responses to those in humans with obesity and insulin resistance (Obese-IR). The studies were performed in a postprandial state associated with steady-state plasma TG concentrations. The arterio-venous blood sampling technique was employed to determine the extraction of circulating lipids across the forearm muscle before and after insulin infusion. We distinguished kinetics of TGs and NEFAs from ingested origin from those from endogenous origin across muscle by incorporating stable isotope-labeled triolein in the ingested fat. Insulin infusion rapidly suppressed the extraction of plasma TGs from endogenous, but not ingested, origin in the muscle of the Lean-IS, but this response was absent in the muscle of the Obese-IR. Furthermore, in the muscle of the Lean-IS, insulin infusion decreased the extraction of circulating NEFAs from both ingested and endogenous origin; however, this response was absent for NEFAs from ingested origin in the muscle of the Obese-IR subjects. Partitioning of circulating lipids away from the skeletal muscle when plasma insulin increases during the postprandial period is impaired in humans with obesity and insulin resistance.

Facial Amphiphile-Modified Lipids Highly Sensitize Liposomes toward Secretory Phospholipase A2.

Upregulated secretory phospholipase A2 (sPLA2) in tumors has been proposed as a stimulus to trigger drug release from liposomes for therapeutic effects. However, the current strategy for developing sPLA2-responsive liposomes merely considering substrate preference suffers from limited membrane disruptive effects induced by enzymatic hydrolysis and safety issues resulting from the overuse of sPLA2-preferred lipids. Here, a membrane-destabilizing mechanism based on enzymatic extraction and the transition of facial amphiphiles (FAs) within lipid membranes was introduced. Enzymatic degradation of FA-modified lipids, a process involving substrate extraction of lipids from membranes and cleavage of sn-2 ester bonds by sPLA2, rotation, and interface settling of detached FAs, caused tremendous efflux of payloads from liposomes, termed the SECRIS effect. In the presence of sPLA2, oxaliplatin (L-OHP) loaded liposomes containing FA-modified lipids showed enhanced drug release, comparable in vitro cytotoxicity, and excellent in vivo antitumor efficacy and reduced adverse syndromes in Colo205-bearing mice compared to conventional sPLA2-labile formulations. The discovery of the SECRIS effect creates a new pathway to engineer liposome platforms for the treatment of sPLA2-positive tumors.

Detailed lipid investigation of edible seaweeds by photochemical derivatization and untargeted lipidomics.

Seaweeds are macrophytic algae that have been gaining interest as alternative healthy foods, renewable drug sources, and climate change mitigation agents. In terms of their nutritional value, seaweeds are renowned for their high content of biologically active polyunsaturated fatty acids. However, little is known about the regiochemistry-the geometry and position of carbon-carbon double bonds-of free and conjugated fatty acids in seaweeds. In the present work, a detailed characterization of the seaweed lipidome was achieved based on untargeted HRMS-based analysis and lipid derivatization with a photochemical aza-Paternò-Büchi reaction. A triple-data processing strategy was carried out to achieve high structural detail on the seaweed lipidome, i.e., (i) a first data processing workflow with all samples for aligning peak and statistical analysis that led to the definition of lipid sum compositions (e.g., phosphatidylglycerol (PG) 34:1), (ii) a second data processing workflow in which the samples of each seaweed were processed separately to annotate molecular lipids with known fatty acyl isomerism (e.g., PG 16:0_18:1), and (iii) the annotation of lipid regioisomers following MS/MS annotation of the lipid derivatives obtained following the aza-Paternò-Büchi reaction(e.g., PG 16:0_18:1 ω-9). Once the platform was set up, the lipid extracts from 8 seaweed species from different seaweed families were characterized, describing over 900 different lipid species, and information on the regiochemistry of carbon-carbon double bonds uncovered unknown peculiarities of seaweeds belonging to different families. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of seaweeds.

Nosema ceranae infection reduces the fat body lipid reserves in the honeybee Apis mellifera

Nosema ceranae is an intestinal parasite frequently found in Apis mellifera colonies. This parasite belongs to Microsporidia, a group of obligate intracellular parasites known to be strongly dependent on their host for energy and resources. Previous studies have shown that N. ceranae could alter several metabolic pathways, including those involved in the nutrient storage. To explore the impact of N. ceranae on the fat body reserves, newly emerged summer bees were experimentally infected, and we measured (1) the lipid percentage of the abdominal fat body at 2-, 7- and 14-days post-inoculation (p.i.) using diethyl ether lipid extraction, (2) the triglyceride and protein concentrations by spectrophotometric assay methods, and (3) the amount of intracellular lipid droplets in trophocytes at 14- and 21-days p.i. using Nile Red staining. Comparing the three methods used to evaluate lipid stores, our data revealed that Nile Red staining seemed to be the simplest, fastest and reliable method. Our results first revealed that the percentage of fat body lipids significantly decreased in infected bees at D14 p.i. The protein stores did not seem to be affected by the infection, while triglyceride concentration was reduced by 30% and lipid droplet amount by 50% at D14 p.i. Finally, a similar decrease in lipid droplet reserves in response to N. ceranae infection was observed in bees collected in fall.

Mobile Phase Contaminants Affect Neutral Lipid Analysis in LC-MS-Based Lipidomics Studies.

Lipidomics is a well-established field, enabled by modern liquid chromatography mass spectrometry (LC-MS) technology, rapidly generating large amounts of data. Lipid extracts derived from biological samples are complex, and most spectral features in LC-MS lipidomics data sets remain unidentified. In-depth analyses of commercial triacylglycerol, diacylglycerol, and cholesterol ester standards revealed the expected ammoniated and sodiated ions as well as five additional unidentified higher mass peaks with relatively high intensities. The identities and origin of these unknown peaks were investigated by modifying the chromatographic mobile-phase components and LC-MS source parameters. Tandem MS (MS/MS) of each unknown adduct peak yielded no lipid structural information, producing only an intense ion of the adducted species. The unknown adducts were identified as low-mass contaminants originating from methanol and isopropanol in the mobile phase. Each contaminant was determined to be an alkylated amine species using their monoisotopic masses to calculate molecular formulas. Analysis of bovine liver extract identified 33 neutral lipids with an additional 73 alkyl amine adducts. Analysis of LC-MS-grade methanol and isopropanol from different vendors revealed substantial alkylated amine contamination in one out of three different brands that were tested. Substituting solvents for ones with lower levels of alkyl amine contamination increased lipid annotations by 36.5% or 27.4%, depending on the vendor, and resulted in >2.5-fold increases in peak area for neutral lipid species without affecting polar lipid analysis. These findings demonstrate the importance of solvent selection and disclosure for lipidomics protocols and highlight some of the major challenges when comparing data between experiments.