A one-step organic solvent lipid extraction method was used to separate lipids from spray-dried egg yolk. Organic solvents tested were chloroform:methanol (CM, 2:1, v:v), methyl-tert-butyl ether (MTBE), or hexane:isopropanol (HI, 3:2, v:v). The resulting defatted egg yolk powder had between 21 and 30% more protein and between 22 and 25% less lipid than the initial spray-dried egg yolk powder (p < 0.05). The solubility of the powder decreased from 20% to 4% (p < 0.05) when CM was used as the organic solvent, likely due to protein denaturation by the chloroform. Gels made from MBTE and HI-extracted protein concentrates had similar hardness (p > 0.05) and were both harder than gels made using the initial egg yolk powder (p < 0.05). MTBE gels were springier, more cohesive, and gummier (p < 0.05) with similar resistance to the initial egg yolk powder (p > 0.05). The results of this study showed that the functionality of the protein in the defatted egg yolk powder was best retained when MTBE was used as the lipid extraction solvent.

Sphingolipids, including sphingosine and sphinganine, are one of the major classes of lipids. They serve as constituents of cell membranes and lipid rafts and aid in the performance of cell-cell communication and adhesion. Abnormal levels of sphingolipids in the aqueous humor can indicate impaired sphingolipid metabolism and associated ocular pathologies. Sphingolipids can be extracted from the aqueous humor by the methyl-tert-butyl ether (MTBE) lipid extraction method and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). This chapter describes a modified protocol for an MTBE lipid extraction from the aqueous humor, followed by analysis with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS).

Fecal samples are a non-invasive and relatively accessible matrix for investigating physiological processes in resident killer whale (Orcinus orca) populations. The high lipid content of the diet (primarily salmonids) leads to lower density fecal material and slower dispersion, facilitating sample collection. As fecal discharge is relatively infrequent and the volume of sample is variable, maximizing analytical options is an important consideration. Here we present an extraction methodology to measure hormones and lipid content from the same fecal aliquot. Lipid extractions are commonly conducted using chloroform and methanol from Folch or Bligh and Dyer (B&D), while alcohol is the primary solvent for hormone extraction. We evaluated the possibility of using the methanol layer from lipid extractions to assess fecal steroid hormone levels. Folch and B&D methanol residues were assayed form metabolites of progesterone (PMs) and corticosterone (GCs), and results were compared to aliquots extracted in 70 % ethanol. Hormone concentrations measured in the methanol layer from Folch and B&D extractions were 55 % to 79 % lower than concentrations in 70 % ethanol. We developed mathematical corrections, using linear regression models fitted to Folch or B&D methanol vs 70 % ethanol hormone concentrations (p < 0.01). Fecal concentrations of PMs and GCs from methanol extractions were biologically validated and are significantly higher in confirmed pregnant females compared to non-pregnant individuals (p < 0.05). This study demonstrates that lipid extraction protocols may be used for the analysis of multiple biomarkers, maximizing the use of small-volume samples.

Lipids produced by oleaginous yeasts are considered as sustainable sources for the production of biofuels and oleochemicals. The red yeast Rhodosporidium toruloides can accumulate lipids to over 70% of its dry cell mass. To facilitate lipid extraction, a recombinant β-1,3-glucomannanase, MAN5C, has been applied to partially breakdown R. toruloides cell wall. In this study, R. toruloides NP11 was engineered for secretory expression of MAN5C to simplify the lipid extraction process. Specifically, a cassette contained a codon-optimized gene MAN5C was integrated into the genome of R. toruloides by Agrobacterium-mediated transformation. The engineered strain NP11-MAN5C was found with proper expression and secretion of active MAN5C, yet no notable compromise in terms of cell growth and lipid production. When NP11-MAN5C cell cultures were extracted with ethyl acetate without any pretreatment, 20% of total lipids were recovered, 4.3-fold higher than that of the parental strain NP11. When the cells were heat-treated followed by extraction with ethyl acetate in the presence of the culture broth supernatants, up to 93% of total lipids were recovered, confirming beneficial effects of MAN5C produced in situ. This study provides a new strategy to engineer oleaginous yeasts for more viable lipid extraction and down-stream processes.

Greenhouse gases (GHGs) emissions due to increasing energy demand have raised the need to identify effective solutions to produce clean and renewable energy. Biotechnologies are an effective platform to attain green transition objectives, especially when synergically integrated to promote health and environmental protection. In this context, microalgae-based biotechnologies are considered among the most effective tools for treating gaseous effluents and simultaneously capturing carbon sources for further biomass valorisation. The production of biodiesel is regarded as a promising avenue for harnessing value from residual algal biomass. Nonetheless, the existing techniques for extracting lipids still face certain limitations, primarily centred around the cost-effectiveness of the process.This study is dedicated to developing and optimising an innovative and cost-efficient technique for extracting lipids from algal biomass produced during gaseous emissions treatment based on algal-bacterial biotechnology. This integrated treatment technology combines a bio-scrubber for degrading gaseous contaminants and a photobioreactor for capturing the produced CO2 within valuable algal biomass. The cultivated biomass is then processed with the process newly designed to extract lipids simultaneously transesterificated in fatty acid methyl esters (FAME) via In Situ Transesterification (IST) with a Kumagawa-type extractor. The results of this study demonstrated the potential application of the optimised method to overcome the gap to green transition. Energy production was obtained from residuals produced during the necessary treatment of gaseous emissions. Using hexane-methanol (v/v = 19:1) mixture in the presence KOH in Kumagawa extractor lipids were extracted with extraction yield higher than 12% and converted in fatty acid methyl esters. The process showed the enhanced extraction of lipids converted in bio-sourced fuels with circular economy approach, broadening the applicability of biotechnologies as sustainable tools for energy source diversification.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Urine analysis has remained a fundamental and widely used method in clinical diagnostics for over a century. With its minimal invasive nature and comprehensive range of analytes, urine has established itself as a clinical diagnostic tool for various disorders, including renal, urological, metabolic, and endocrine diseases. Furthermore, urine\'s unique attributes make it an attractive matrix for biomarker discovery, as well as in assessing the metabolic and physiological states of patients and healthy individuals alike. However, limitations in our knowledge of average values and sources of urinary lipids decrease the wider clinical application of urinary lipidomics. In this context, untargeted lipidomics analysis relies heavily on the extraction and analysis of lipids in biological samples. Nevertheless, this type of analysis presents challenges in lipid identification due to the diverse nature of lipids. Therefore, proper sample treatment before analysis is crucial to obtain robust and reproducible lipidomic profiles. To address this gap, we conducted a comparative study of a urine pool sample collected from twenty healthy volunteers using four different lipid extraction methods: one biphasic and three monophasic protocols. The extracted lipids were then analyzed using UHPLC-MS and MS/MS, and the semi-quantification of all the accurately annotated lipid species was performed for each extraction method.

The extraction of lipids by the Folch method from the muscles of all the fish studied led to statistically significant differences in the values of δ15N. At the same time, lipid extraction led to a statistically significant increase in δ13C in pike and roach, and to a statistically insignificant decrease in δ13C in perch and bream. Thus, lipid extraction cannot serve as a universal method of sample preparation for the analysis of the isotopic composition of carbon (13C/12C) and nitrogen (15N/14N) in fish muscles. The differences between the δ13C values in the samples before and after lipid extraction were statistically investigated by different models. It is shown that mathematical correction method models can be used, but the results are depending on the fish types.

One of the most recent additions to the family of two-dimensional (2D) materials, graphitic C3N3 (g-C3N3), has been considered a viable contender for biomedical applications, although its potential toxicity remains elusive. We perform all-atom molecular dynamics simulations to decipher the interactions between model lipid membranes and g-C3N3 as a first step toward exploring the cytotoxicity induced at the nanoscale. We show that g-C3N3 can easily insert into the cellular membranes following a multistage mechanism consisting of simultaneous desolvation of the 2D material along with enrichment of nanomaterial-lipid interactions. Free energy calculations indicate that g-C3N3 is more stable in a membrane-bound state compared to an aqueous solution; however, the insertion of the material does not disturb the structural integrity of lipid membranes. After being inserted into a membrane, g-C3N3 is unlikely to be released into the cellular environment and is incapable of extracting lipid molecules from the membrane. The nature of interaction between the 2D material and membranes is found to be independent of the nanomaterial size. Also, the performance of g-C3N3 toward biomolecular delivery is shown to be significantly improved compared to the state-of-the-art 2D materials graphene and hexagonal boron nitride (h-BN). It is revealed that, the affinity of g-C3N3 toward lipid membranes is weaker compared to the nanotoxic graphene and h-BN, while being marginally higher than h2D-C2N, which in turn, increases the biocompatibility of the material, thereby brightening its future as a noncytotoxic material for forthcoming biomedical applications.

Lipids play many important physiological roles in mammalian reproduction, being essential for the acquisition of oocyte competence and post-fertilization embryonic development. Lipid profiling in samples of minute size, such as oocytes, is challenging but has been achieved by mass spectrometry technologies such as multiple reaction monitoring (MRM) profiling. With the goals of further simplifying sample workflow and investigating the influence of pre-analytical conditions, we have evaluated how different extraction methods and transportation of lipid extracts in vacuum and at room temperature impacted the lipid profile of bovine oocytes. Using a comprehensive method, 316 MRMs associated with lipids of 10 different classes were screened in oocyte lipid extracts prepared by 2 extraction methods (one-step methanol addition or Bligh and Dyer) and transporting them in dry ice or at room temperature inside vacuum packages. No changes in the multivariate analysis (PCA) were noticeable due to transportation temperature, while lipid profiles were more affected by the lipid extraction protocol. Sample extraction using pure methanol favored the detection of phospholipids uniformly, while Bligh and Dyer favored the detection of neutral intracellular lipids. Triacylglycerol lipids and free fatty acids yielded decreased abundances when samples were transported at room temperature. We conclude that if samples are submitted to the same lipid extraction protocol and same transportation batch at room temperature coupled with vacuum conditions it is possible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.