Lipid Extraction Procedure Research Articles

Studies of Labware Contamination during Lipid Extraction in Mass Spectrometry-Based Lipidome Analysis.

In lipidomic analysis, plasticware is increasingly being used for lipid extraction and other sample processing procedures over glassware. However, a systematic investigation of the consequences of plasticware use on mass spectrometry (MS)-based lipidome analysis is lacking. In this work, we present an analytical approach for detecting and comparing solvent and labware contaminants encountered in lipidomic workflows. It is shown that the contaminant profiles varied widely between microcentrifuge tubes from different manufacturers. The most suitable polypropylene tubes tested introduced 847 labware-originating contaminant m/z's when three different manufacturing batches were tested for Folch lipid extractions. Of particular concern is that 21 primary amide and fatty acid surfactants were introduced that were identical to biological endogenous lipids, 16 of which had not been previously reported as leachables from polypropylene materials. Alternatively, the use of borosilicate glassware and PTFE-lined screw caps introduced 98 different contaminant m/z's across three manufacturing batches tested for Folch extractions. Despite the overwhelming number of labware contaminants introduced, current databases and literature only facilitated the identification of 32 contaminants. To address the dearth of publicly available contaminant information, we provide a comprehensive labware contamination repository containing high-resolution m/z values, adductation information, retention times, and MS/MS spectra. This resource should prove to be valuable for researchers in detecting and distinguishing contaminants from analytes of interest. A companion paper presents a detailed study of how labware contamination can lead to ion-suppression effects on coeluting lipids and interference in the analysis of endogenous lipids, such as those from human sera.

Prevention of Neonatal Respiratory Distress Syndrome by Antenatal Steroid Administration

Background: Exposure to antenatal steroids (A.N.S.) decreases the incidence of neonatal respiratory distress syndrome and other morbidities in premature infants. If left untreated, death may occur from progressive hypoxia and respiratory failure. In survivors, resolution begins between 2 to 4 days.Objective: To determine the efficacy of antenatal steroids in preventing respiratory distress syndrome in premature neonates. Material and Methods: A randomized trial, analytical study with a sample size of 152 was conducted in the Department of Obstetrics and Gynecology, Hayatabad Medical Complex, Peshawar. The study duration was from 16th July 2022 to 18th June 2023. The study included neonates born at 24 to 34 weeks of gestation. Neonates born at fewer than 34 (24 to 34 weeks) weeks gestation were given 3 or 4 mL of surfactant from lipids taken from calf lung lavage. The proteins were decreased from more than 10% to less than 1% of the phospholipid weight using the lipid extraction procedure. The chi-square test was used.Results: Analysis was performed for the 152 VLBW infants the infants who received antenatal corticosteroids was 37 to 45% were gestational age 2434 weeks. While infants who received surfactant decreased gradually as gestational age increased. A significant decrease in surfactant used in the late period, with a 510% reduction in each gestational group.Conclusion: This study reaffirms the value of administering antenatal steroids to premature neonates. This study showed the potential use of ante natal steroids has effectively mitigated respiratory distress syndrome. These findings underscore the significance of antenatal corticosteroid therapy in reducing respiratory morbidity in preterm neonates and emphasize its role in enhancing lung maturation and neonatal outcomes.Keywords: Antenatal corticosteroids, Preterm infants, Respiratory distress syndrome, Surfactant, Very low birth weight.

Abstract 829: Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells

Abstract Background: Neuroblastoma (NB) is a malignancy of the neuroendocrine system affecting infants. NB is highly resistant to drug therapy, which leads to ineffective treatments and increased refractory. Current treatments have low specificity to targeted NB cells resulting in unwanted side effects. A drug delivery system that can overcome these problems is urgently needed. Incorporating drugs in optimized lipid nanoparticles such as liposomes have proven to be an effective method for drug targeting. To achieve better cellular selectivity, lipid membrane extract (LE) is derived from NB cells using a modified Dyer and Blight lipid extraction procedure. The objective of the study was to determine an optimized cell membrane lipid-extracted nanoliposomes (CLENs) for the NB cell model while considering physicochemical properties (i.e. size and molecular charge) and composition of the preparation. Methods: The neuroblastoma cell line, SK-N-DZ, was used for drug selectivity study. Expanded culture of SK-N-DZ was employed for cellular extraction purposes. The method for lipid membrane extraction was previously reported by Alharbi and Campbell, (AAPS Open 4,5(2018)). SK-N-DZ lipid extract was the main ingredient to prepare SK-N-DZ CLENs. Phase I of the study is to prepare CLENs with various compositions of DOPC, cholesterol, DPPE-PEG5000 and SK-N-DZ lipid extract. DPPE-Rhodamine was used for fluorescence and cellular uptake studies. Phase II of the study evaluated the effect of SK-N-DZ lipid extraction inclusion on drug incorporation, cellular uptake and selectivity, and cytotoxicity. Invitrogen alamarBlue HS in vitro assay was used to determine cell viability for cytotoxicity studies. SK-N-SH (brain tissue), BT-20 (breast tissue) and G401 (kidney tissue) cell lines served as negative controls. Results: The results from the cellular uptake optimization studies revealed that the optimized composition and ratio for SK-N-DZ CLENs was DOPC/Cholesterol/LE/PEG (65/10/20/5). The average particle size and value for zeta potential was 164 nm +/- 1.2 and -7.92 +/- 1.55 mV, respectively. The inclusion of SK-N-DZ lipid membrane extract (2, 10, 20 and 40 mol%) in preparation of CLENs increased cellular uptake by SK-N-DZ cells compared to corresponding control (no LE included). No difference observed in cellular uptake of preparations containing 20 and 40 mol% of LE. The additional inclusion of cholesterol (10, 20, 40 mol%) increased overall cellular uptake of preparations compared to CLENs without the additional cholesterol content. Overall, the inclusion of PEG (5, 10, and 20 mol%) in SK-N-DZ CLENs increased their uptake by the target cell population. Conclusion: In conclusion, the inclusion of SK-N-DZ lipid membrane extract in the nanoliposome preparations increased their overall cellular uptake by SK-N-DZ target cells. Additional cellular selectively and cytotoxicity studies are currently underway. Citation Format: Kevin Bumanglag, Thi Tran, Robert Campbell, Yishan Li. Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 829.

Abstract 237: Evaluation of cell membrane lipid-extracted nanoliposomes (CLENs) as a potential drug platform for targeting multiple myeloma

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy that primarily affects the elderly. MM is the second most common hematologic cancer with approximately 25,000-30,000 new cases diagnosed annually in the United States. A challenge associated with MM therapy is unwanted drug uptake by normal healthy cells. Nanoparticles provide a promising outlook to mitigate these issues, offering more selective drug uptake, while minimizing toxicity associated with treatment. The objective of this study was to develop, optimize and evaluate physicochemical and In vitro properties of MM-CLENs in comparison to conventional liposomes and relevant cell controls. Methods: RPMI-8226 MM cell line was selected cellular model for MM disease. RPMI-8226 cells were cultured and expanded In vitro for cellular extraction purposes. Lipid extraction (LE) procedures were performed to generate the RPMI-8226 LE ingredient. Other lipid ingredients used in preparation of RPMI-8226 CLENs included DOPC, cholesterol, and DPPE-Rhodamine (fluorescence indicator). Phase I included physicochemical characterization and cellular uptake studies of CLENs preparations with varying concentration of RPMI-8226-LE (0 to 40 mol%) content. Phase II began with the optimized concentration of LE determined in Phase I, along with varying concentrations of cholesterol (0 to 40 mol %) content for the nano-targeting studies in vitro. The control cell lines represent other hematological diseases, K-562-GFP (chronic myeloid leukemia), CCRF-CEM (acute lymphoblastic leukemia), and U937 (lymphoma). Results: Phase I and II physicochemical characterization and In vitro studies, revealed that the optimized preparation of RPMI-8226-CLENs consisted of 5% LE, and 5% cholesterol content. Optimal preparation consisted of DOPC: Cholesterol: LE (90:5:5) with an average values for particle size and zeta potential of 178±38 nm (n=4), and -14±4 mV (n=4), respectively. The inclusion of LE in conventional DOPC:cholesterol liposomes resulted in significantly improved uptake of the CLENs by RPMI-8226 cells in vitro (P< 0.001). However, preliminary selectivity studies suggest no significant difference in the uptake of RPMI-8226-CLENs by RPMI-8226 when compared to control cells (K-562-GFP, CCRF-CEM and U937). Conclusion: Studies involving RPMI-8226 CLENs are currently underway. Such studies include target selectivity, and influence of additional components of CLENs as a function of the microenvironment. Citation Format: Christina T. Tran, Eden Park, Pedro L. Rodriguez-Flores, Robert B. Campbell. Evaluation of cell membrane lipid-extracted nanoliposomes (CLENs) as a potential drug platform for targeting multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 237.

Enhanced Tetracycline Removal from Highly Concentrated Aqueous Media by Lipid-Free Chlorella sp. Biomass.

Microalgae are used as a lipid source for different applications, such as cosmetics and biofuel. The nonliving biomass and the byproduct from the lipid extraction procedure can efficiently remove antibiotics. This work has explored the potential use of Chlorella sp. biomasses for tetracycline (Tc) removal from highly concentrated aqueous media. Non-living biomass (NLB) is the biomass before the lipid extraction procedure, while lipid-extracted biomass (LEB) is the byproduct mentioned before. LEB removed 76.9% of Tc at 40 mg/L initial concentration and 40 mg of biomass, representing an adsorption capacity of 19.2 mg/g. Subsequently, NLB removed 68.0% of Tc at 50 mg/L and 60 mg of biomass, equivalent to 14.2 mg/g of adsorptive capacity. These results revealed an enhanced removal capacity by LEB compared with NLB and other microalgae-based materials. On the other hand, the adsorption kinetics followed the pseudo-second-order and Elovich models, suggesting chemisorption with interactions between adsorbates. The adsorption isotherms indicate a multilayer mechanism on a heterogeneous surface. Additionally, the interactions between the surface and the first layer of tetracycline are weak, and the formation of the subsequent layers is favored. The Chlorella sp. biomass after the lipid extraction process is a promising material for removing tetracycline; moreover, the use of this residue contributes to the zero-waste strategy.

Determination of free fatty acids in Antarctic krill meals based on matrix solid phase dispersion

Amino-modified mesoporous silicawas prepared by modifying mesoporous silica with 3-aminopropyltriethoxysilane and used as adsorbents in matrix solid-phase dispersion (MSPD) to analyze free fatty acids (FFAs) in krill meals for the first time. The adsorption–desorption experiments and Fourier-transform infrared spectroscopy showed amino-modified mesoporous silica with ordered mesoporous structure was successfully synthesized. The adsorption experiments including static and dynamic adsorption showed thatabsorption capacity of amino-modified mesoporous silica towards FFAs was better than that of aminated silicon microspheres at all concentrations. Under optimal extraction conditions, outstanding linearity (0.1–12000 nmol g−1), low LODs (0.05–1.25 nmol g−1), satisfactory recoveries (82.17–96.43%) and precisions (0.19–5.26%) were obtained. Moreover, the application of MSPD for FFAs analysis avoided complicated lipid extraction procedures and accomplished the homogenization, crushing, extraction and cleaning of the samples in one step. Consequently, this approach provides an alternative choice to the existing approach for analyzing FFAs in solid and semi-solid samples.

Removal of minocycline from high concentrated aqueous medium by nonliving and lipid-free Chlorella sp. biomass

This work evaluated the removal of minocycline (MC) by the nonliving Chlorella sp. biomass (NLB) and modified by a lipid extraction procedure (LEB). Both biomasses have different morphology (NLB: globular-like; LEB: flakes and blocks) and size distribution. The pH showed a significant synergistic influence on MC removal (p < 0.05). MC initial concentration (C0) and biomass dosage significantly interact, suggesting that LEB agglomeration decreased removal. NLB removed 90.8 ± 1.3% of MC and LEB 80.8 ± 1.4% at C0 = 53.89 mg/L, 50 mg of biomass and pH 10. The adsorption kinetics and isotherms suggested multilayer formation by physical and chemical adsorption on heterogeneous and macroporous surfaces. According to results, NLB as an adsorbent had an economic disadvantage because of production costs despite good removal efficiency. However, it is possible to take advantage of the biomass after removing value-added compounds (LEB) as a zero-waste strategy.

Assessment of lipid profile in fat body and eggs of Rhipicephalus microplus engorged females exposed to (E)-cinnamaldehyde and α-bisabolol, potential acaricide compounds

In the present study, the lipid profile from the fat body and eggs of Rhipicephalus microplus was evaluated after exposure of engorged females to (E)-cinnamaldehyde and α-bisabolol, substances which have acaricide potential according to the literature. Engorged females collected from artificially infested cattle were immersed in a concentration of 10.0 mg/mL of each substance. Dissection of the female fat bodies was performed at different times (72 h and 120 h), for subsequent lipid extraction. In addition, on the fifth day of oviposition, were collected 50.0 ml50.0 mL aliquots of the egg mass of each treatment to perform the same lipid extraction procedure. To assess the lipid profiles, the samples were submitted to the thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GCMS) analysis. Furthermore, an in silico analysis was performed using PASS online® software to predict the possible molecular targets of (E)-cinnamaldehyde and α-bisabolol. As result, the main lipids identified from the fat body were triacylglycerides, fatty acids, and cholesterol, whereas, triacylglycerides (TAG), fatty acids (FA), and cholesterol (CHO) and cholesterol esters (CHOE), were identified in the eggs. The results also showed a significant increase (p < 0.05) of CHO in the fat body in the group exposed to (E)-cinnamaldehyde at 72 h (0.12 μg/fat body) and 120 h (0.46 μg/fat body), in the eggs from females treated with this same substance, there was a significant reduction (p < 0.05) in the amount of CHO (0.21 μg), compared to the water control group (0.45 μg). In the GCMS technique, 5 chemical classes were found, and variations were observed between these substances, mainly hydrocarbons and steroids, in the different groups, and (E)-cinnamaldehyde promoted the greatest changes. From the predictions of the in silico study, 38 and 20 targets were selected, respectively, which are mainly related to alterations in lipid metabolism, immune system and nervous system. This study provides the first report of changes in lipid metabolism of R. microplus exposed to (E)-cinnamaldehyde and α-bisabolol, as well as presenting possible activity on the molecular targets of these substances, expanding knowledge for the potential use of these compounds in the development of botanical acaricides.

Comprehensive lipid profiling of Microchloropsis gaditana by liquid chromatography - (tandem) mass spectrometry: Bead milling and extraction solvent effects

Liquid chromatography - mass spectrometry (LC-MS) experiments were used to qualitatively and semi-quantitatively monitor the impact of bead milling and the influence of the nature of the extraction solvents, Bligh and Dyer (B&D) versus n-heptane, on the composition of the structural lipids extracted from the microalgae Microchloropsis gaditana (M. gaditana).Our data reveal that lipid extraction with n-heptane as the solvent becomes as efficient as the standard Bligh and Dyer method when a milling step is included before the extraction. Indeed, lipid extraction yields amount to ~1% (n-heptane on intact cells), ~5% (B&D on intact cells), ~5% (n-heptane on milled cells) and ~6% (B&D on milled cells). In all the extracts, using liquid chromatography separation combined to accurate mass measurements and tandem mass spectrometry experiments, we identified a huge number of lipids belonging to different structural lipid families, including LPC (lyso-phosphatidylcholine), PC (phosphatidylcholine), DGTS (diacylglyceryl-N-trimethylhomoserine), MGTS (monoacylglyceryl-N-trimethylhomoserine), DGDG (digalactosyldiacylglycerol), DGMG (digalactosylmonoacylglycerol), MGDG (monogalactosyldiacylglycerol), PG (phosphatidylglycerol), PI (phosphatidylinositol), SQDG (sulfoquinovosyldiacylglycerol), SGMG (sulfoquinovosylmonoacylglycerol) and free fatty acids (FFA). We further demonstrated that the milling step induces strong modifications in the lipid composition due to extensive lipid degradation into free fatty acids - probably by enzymatic processes, even when the milled cells are conserved at −20 °C.From a method development perspective, this work represents to the best of our knowledge the most complete structural analysis investigation of lipids extracted from intact and milled microalgae, including the use of a sector diagram representation that could be relevant for all lipidomic investigations. One of the main outcomes of the study is the confirmation that liquid chromatography - tandem mass spectrometry (LC-MS/MS) experiments are essential when lipid extraction procedures from any microalgae biomass have to be optimized. The analytical protocol developed in the present study certainly deserves to be extended to other microalgae.

A comprehensive comparison of four methods for extracting lipids from Arabidopsis tissues

BackgroundThe plant lipidome is highly complex, and the composition of lipids in different tissues as well as their specific functions in plant development, growth and stress responses have yet to be fully elucidated. To do this, efficient lipid extraction protocols which deliver target compounds in solution at concentrations adequate for subsequent detection, quantitation and analysis through spectroscopic methods are required. To date, numerous methods are used to extract lipids from plant tissues. However, a comprehensive analysis of the efficiency and reproducibility of these methods to extract multiple lipid classes from diverse tissues of a plant has not been undertaken.ResultsIn this study, we report the comparison of four different lipid extraction procedures in order to determine the most effective lipid extraction protocol to extract lipids from different tissues of the model plant Arabidopsis thaliana.ConclusionWhile particular methods were best suited to extract different lipid classes from diverse Arabidopsis tissues, overall a single-step extraction method with a 24 h extraction period, which uses a mixture of chloroform, isopropanol, methanol and water, was the most efficient, reproducible and the least labor-intensive to extract a broad range of lipids for untargeted lipidomic analysis of Arabidopsis tissues. This method extracted a broad range of lipids from leaves, stems, siliques, roots, seeds, seedlings and flowers of Arabidopsis. In addition, appropriate methods for targeted lipid analysis of specific lipids from particular Arabidopsis tissues were also identified.

Evaluation of the quantity and the carbon isotopic composition of amino acids by using diverse sample residues after lipid extraction.

Compound-specific stable isotope analysis (CSIA) becomes a critical tool in the fields of ecology and biogeochemistry to trace basal carbon sources such as amino acids (AAs) and fatty acids (FAs) in natural environments. However, in many studies, either AAs or FAs have been analyzed due to restricted sample amounts. The aim of this study is to report a single-sample preparation method by identifying AAs using lipid-free residues derived from a typical FA analytic procedure. The capability of the lipid-free residues was evaluated by determining AA quantities and carbon isotopic compositions (δ13C) across diverse sample matrices, including soil, suspended particulate organic matter (SPM), fish tissue, and cultured cyanobacteria (N = 3). Our result showed that the use of lipid-free residues after a typical lipid extraction procedure in most samples did not significantly reduce the AA quantities, relative to the conventional AA method (done by using whole samples), except in case of samples of soil. Moreover, δ13C compositions of the most AAs (from essential to nonessential AAs) in all samples were not significantly different, with an average isotope difference of < 2.1‰ between the two methods. Overall results indicate that the residual parts after extracting lipid fraction are useful for reliable AA quantification and individual isotopic information. Ultimately, one sample preparation for determining FA and AA data enables us to characterize one (or multiple) basal carbon source(s) within rare samples despite their limited abundance. Graphical abstract.

Comparison of different lipid extraction procedures applied to three microalgal species

The increase in the world’s energy demand has contributed to the emergence of new sustainable energy sources, such as microalgae, with their great potential to provide biofuels and other high value co-products for the food and health’s markets. However, current biorefinery methodologies are either too complex to extract the targeted components such as high-value products, or require solvents with toxicity for humans and the environment. This work aims to evaluate different lipid extraction approaches applied to three microalgal species: Chlorella zofingiensis, Phaeodactylum tricornutum, and Arthrospira platensis, while employing less toxic and more economical solvents for the lipids extraction. Experimental results showed a promising outcome to tune current biorefinery methodologies, enhancing product yield as well as decreasing potential hazards.

Stable isotope analyses reveal unique trophic role of reef manta rays (Mobula alfredi) at a remote coral reef.

Stable isotope analyses provide the means to examine the trophic role of animals in complex food webs. Here, we used stable isotope analyses to characterize the feeding ecology of reef manta rays (Mobula alfredi) at a remote coral reef in the Western Indian Ocean. Muscle samples of M. alfredi were collected from D'Arros Island and St. Joseph Atoll, Republic of Seychelles, in November 2016 and 2017. Prior to analysis, lipid and urea extraction procedures were tested on freeze-dried muscle tissue in order to standardize sample treatment protocols for M. alfredi. The lipid extraction procedure was effective at removing both lipids and urea from samples and should be used in future studies of the trophic ecology of this species. The isotopic signatures of nitrogen (δ15N) and carbon (δ13C) for M. alfredi differed by year, but did not vary by sex or life stage, suggesting that all individuals occupy the same trophic niche at this coral reef. Furthermore, the isotopic signatures for M. alfredi differed to those for co-occurring planktivorous fish species also sampled at D'Arros Island and St. Joseph Atoll, suggesting that the ecological niche of M. alfredi is unique. Pelagic zooplankton were the main contributor (45%) to the diet of M. alfredi, combined with emergent zooplankton (38%) and mesopelagic prey items (17%). Given the extent of movement that would be required to undertake this foraging strategy, individual M. alfredi are implicated as important vectors of nutrient supply around and to the coral reefs surrounding D'Arros Island and St. Joseph Atoll, particularly where substantial site fidelity is displayed by these large elasmobranchs.

Enhanced isolation of lipids from microalgal biomass with high water content for biodiesel production

In present study, lipids were extracted from unbroken microalga Chlorella vulgaris with high water content (50% microalgal solution) through three-phase partitioning (TPP). The method was found to extract around 15.9% of total lipid transformable to methyl esters (LTMEs) from unbroken microalgal cells which is two times of Bligh and Dyer method. We investigated the effects of various parameters on TPP performance and were optimised through response surface methodology. The results indicated that incubation duration, temperature and extraction time were positively correlated with LTME extraction efficiency. The optimum temperature was 60 °C, incubation duration was 120 min, extraction time was 60 min, ratio of solvent to DKP was 1:1. The FAME yield was calculated as 12.05% and major fatty acids together accounted for 71.33% which indicated the great potential of the proposed lipid extraction procedure for microalga-based biodiesel production.

Comprehensive and quantitative analysis of white and brown adipose tissue by shotgun lipidomics

ObjectiveShotgun lipidomics enables an extensive analysis of lipids from tissues and fluids. Each specimen requires appropriate extraction and processing procedures to ensure good coverage and reproducible quantification of the lipidome. Adipose tissue (AT) has become a research focus with regard to its involvement in obesity-related pathologies. However, the quantification of the AT lipidome is particularly challenging due to the predominance of triacylglycerides, which elicit high ion suppression of the remaining lipid classes. MethodsWe present a new and validated method for shotgun lipidomics of AT, which tailors the lipid extraction procedure to the target specimen and features high reproducibility with a linear dynamic range of at least 4 orders of magnitude for all lipid classes. ResultsUtilizing this method, we observed tissue-specific and diet-related differences in three AT types (brown, gonadal, inguinal subcutaneous) from lean and obese mice. Brown AT exhibited a distinct lipidomic profile with the greatest lipid class diversity and responded to high-fat diet by altering its lipid composition, which shifted towards that of white AT. Moreover, diet-induced obesity promoted an overall remodeling of the lipidome, where all three AT types featured a significant increase in longer and more unsaturated triacylglyceride and phospholipid species. ConclusionsThe here presented method facilitates reproducible systematic lipidomic profiling of AT and could be integrated with further –omics approaches used in (pre-) clinical research, in order to advance the understanding of the molecular metabolic dynamics involved in the pathogenesis of obesity-associated disorders.

Biomolecular Component Analysis of Phospholipids Composition in Live HeLa Cells.

The alteration of the phospholipid composition within the cell, in particular the ratio between saturated and unsaturated fatty acids, can serve as an important biomarker to prognosis of the disease progression (e.g., fatty-liver disease, prostate cancer, or neurodegenerative disorders). Major techniques for lipid analysis in biological samples require a lipid extraction procedure that is not compatible with live cell studies. To address this challenge, we apply microRaman-Biomolecular Component Analysis (BCA) for comparative analysis of phospholipid composition and sensing the saturation degree of fatty acid lipid chain in live HeLa cells and lipids extracted from HeLa cells. After processing raw Raman data, acquired in lipid droplets (LDs) free cytoplasmic area, LDs and extracted lipids with BCA, the lipid component was isolated. Despite the similarity in general profiles of processed Raman spectra acquired in live cells and extracted lipids, some clear differences that reflect diversity in their phospholipids composition were revealed. Furthermore, using the direct relation between the number of double bonds in the fatty acid chain and the intensity ratio of the corresponding Raman bands, the saturation degree of fatty acids was estimated.

A High-Throughput Method (ASE-GC/MS/MS/MRM) for Quantification of Multiple Hydrocarbon Compounds in Marine Environmental Samples

AbstractA high-throughput method was applied in small and complex environmental samples for targeting multiple organic fractions (polar, nonpolar). The analytical method consists of a single-step lipid extraction and purification procedure (accelerated solvent extraction [ASE] system) coupled with a single-run step using gas chromatography with tandem mass spectrometry, operated in multiple reaction monitoring mode (GC/MS/MS/MRM). Successful application of this method is summarized for multiple chemical groups (aliphatics, terpanes, steranes, triaromatic steroids, polycyclic aromatic hydrocarbons, and oxidation products).

Effects of lipid extraction and different collagen extraction methods on archaeological fish bones and its implications for fish bone diagenesis

Gelatin extracted from archaeological fish bones typically exhibits relatively high C/N ratios, presumed to be caused by contamination with lipids or humic substances. The effects of lipid extraction and different collagen extraction methods applied has been studied on modern fish bones but has never been studied systematically on archaeological specimens because of taphonomic and experimental reasons. In this study, the effects of lipid extraction and order of NaOH treatment in collagen extraction method on carbon and nitrogen stable isotope analysis of archaeological fish bone (n = 30) excavated from the Hamanaka 2 site, Hokkaido, Japan (approximately 8th BC–10th AD) is investigated. Gelatin extracted from the same fish bone subsamples with or without lipid extraction procedure indicates neither significant nor systematic differences in carbon and nitrogen stable isotope ratios, elemental concentrations, C/N ratios, and yield. However, reproducibility of stable isotope ratios and elemental concentrations decrease in gelatin extracted from poorly-preserved cod bones (<3.5% yield). Gelatin extracted from archaeological fish bones may contain humic contaminants, and its effect becomes greater in gelatin with lower extraction yield. Although there is no significant change in the stable isotope and C/N ratios, change in atomic concentration of carbon and nitrogen suggests that the purity of extracted gelatin increases when NaOH treatment is applied after decalcification. Because this is only a study from one archaeological site, further case studies that evaluate lipids and diagenesis in fish bones are required.

Reproducibility of Phosphoinositide Lipid Extraction &amp; Presentation in Bioanalytical Assays

Phosphatidylinositol 4‐phosphate (PI4P) produced by Phosphatidylinositol 4‐Phosphate kinases is an abundant Golgi phosphoinositide functioning in lipid exchange and vesicle transport within mammalian cells. Researchers wanting to quantify PI4P use the Bligh and Dyer method for extracting and separating phospholipids in cells and biological tissues. This common lipid extraction procedure is known to have inherent variation. Using a human promyelocytic leukemia cell line and an ELISA that is sensitive and specific for PI4P, we examine sources of variation in bioassays including sample (HL‐60 cells), the extraction procedure, and lipid detection. We determined a PI4P Mass ELISA has low coefficient of variation (&lt;10%) if the extracted sample volume is held constant and samples are prepped and lipids presented with standardized procedures. Thus, the organic solvent extraction process is the major source of variation in typical Bligh‐Dyer preparations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy-Based Analysis of Lipid Components in Serum/Plasma of Patients with Duchenne Muscular Dystrophy (DMD).

Proton nuclear magnetic resonance spectroscopy (1H NMR) is a useful tool for the analysis of lipid components in biofluids such as serum/plasma. Such tool is applied for the analysis of lipid components in serum/plasma of patients with Duchenne muscular dystrophy (DMD). The practical approach of sampling, storage, lipid extraction procedure, sample preparation before performing the 1H NMR spectroscopy experiments is presented. All the experimental parameters of NMR spectroscopy are also described. Details of the assignments of lipid components (qualitative analysis) and quantification of particular lipid components (quantitative analysis) are explained.