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the Cell of lipids, culturing, of lipidome. intracellular investigation determination

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17.06.2018

Content:

  • the Cell of lipids, culturing, of lipidome. intracellular investigation determination
  • Host triacylglycerols shape the lipidome of intracellular trypanosomes and modulate their growth
  • Introduction
  • This analysis unexpectedly revealed a strong host lipid signature in the parasite . In this study, we have leveraged comprehensive lipid mass spectrometry to investigate the and mammalian cells was plotted to determine if the parasite lipidome could .. Mammalian cell culture and T. cruzi maintenance. To investigate this prediction, it was necessary to establish baseline lipidome .. cognate host cells were performed to determine which, if any, parasite lipid classes display .. Mammalian cell culture and T. cruzi maintenance. Early identification of disregulated PGE2 levels will allow interventions to be carried full complement of lipids (i.e., the lipidome) in cells, organs, extracellular fluids, . In lipidomics, investigations that utilize LC-MS/MS approaches lipids that elute .. Thereafter, the cells need to be cultured in the presence of the relevant.

    the Cell of lipids, culturing, of lipidome. intracellular investigation determination

    Some of the largest changes 10—fold in lipid levels arising from KLA treatment of RAW cells occurred in cholesteryl esters containing saturated and monounsaturated fatty acyl groups.

    Smaller changes were detected in cholesteryl esters containing polyunsaturated fatty acyl substituents. These results suggested that saturated and monounsaturated cholesterol esters might accumulate as a result of increases in intracellular cholesterol levels arising from TLR4 activation.

    In agreement with this hypothesis, mRNAs encoding two cholesterol esterification enzymes Soat1 and -2 increased 4—6-fold with KLA treatment. Two classes of prenols, dolichols and coenzyme Q, were increased 1. The correlation between various sterols and the genes for cholesterol biosynthesis is shown in supplemental Fig. Consistent with the observations made above, cholesterol, its precursors, and its several derivatives co-vary with the mRNA of HMG CoA reductase Hmgcr and cholesterol hydroxylase Ch25h supplemental Table 2, Cluster 2.

    Cholesterol is made through two parallel pathways from lanosterol. As a result, its profile is a composite of the two effects; desmosterol increases from 0 to 24 h, as does 7-dehydro-cholesterol supplemental Fig. However, Dhcr24 supplemental Table 2, Cluster 3 in the first branch of cholesterol biosynthesis and Dhcr27 supplemental Table 2, Cluster 1 in the second branch of cholesterol biosynthesis decrease over time increasing only at the last time point at 24 h.

    As a net result, cholesterol exhibits a slightly non-monotonic time response, although the overall response shows an increase. KLA treatment generated increases in almost every category of sphingolipid analyzed, including free sphingoid bases and their phosphates e. These increases were consistent with the induction of de novo sphingolipid biosynthesis because sphinganine and dihydroceramides are early intermediates in this pathway.

    Furthermore, the gene expression dataset revealed that mRNAs encoding two subunits of serine palmitoyltransferase Sptlc1 and Sptlc2 , the enzyme catalyzing the initial step of sphingolipid biosynthesis, were elevated in KLA-treated cells.

    These results are readily observed in the correlation and data heat maps shown in supplemental Fig. Sphingomyelins and glucosylceramides were increased with KLA treatment 1. The elevation in sphingomyelins might reflect regulatory cross-talk between cholesterol metabolism and sphingolipid metabolism as oxysterols, which were found to be elevated by KLA see above , alter sphingomyelin biosynthesis.

    The elevation in glucosylceramides was associated with an increase in the mRNA for the glycosyltransferase that makes these intermediates, UDP-glucose: After treatment with KLA, RAW cells have substantially higher levels of galactosylceramides and sulfated galactosylceramides sulfatides The increased production of sulfatides may be of biological significance as the presence of sulfated glycolipids on cells promotes their phagocytosis by macrophages 24 , and sulfatides are one of the categories of endogenous lipids that bind to the CD1d cell surface receptor.

    Two other sphingolipid categories affected by KLA treatment and which have been widely implicated in cell signaling were the sphingoid base 1-phosphates sphingosine 1-phosphate S1P and sphinganinephosphate and ceramidephosphate Cer1P. Total glycerophospholipids in KLA-treated samples remained roughly constant over the h period, whereas control samples at 12 and 24 h had less total glycerophospholipid than the KLA-treated samples at these time points.

    Interestingly, significant changes in the phosphatidic acid and phosphatidylinositol classes were observed after 8—24 h of KLA stimulation. This trend was especially noticeable for the saturated and monounsaturated species in that the The phosphatidylinositols exhibited a similar trend toward larger -fold increases for saturated species, whereas certain polyunsaturated species such as These results demonstrate that lipid second messengers and key metabolic intermediates i.

    We present herein an integrated view of lipid metabolism and remodeling that includes cross-talk between the six lipid categories discussed above as illustrated in the comprehensive overview of the major mammalian lipid categories and their interconnections shown in Fig.

    Pathways to remodeling begin with the fatty acyl-CoA species. Careful measurements of acyl-CoAs after treatment with KLA show that although saturated CoA levels with the most chain lengths increase upon treatment, all the unsaturated CoA levels are decreased.

    Also, transcript levels of many desaturase genes are decreased in macrophages upon KLA treatment and are presumed to lead to reduced enzymatic levels for production of unsaturated acyl-CoAs. The temporal changes in the different lipid categories also support the remodeling of fatty acyls between glycerolipids, glycerophospholipids, sphingolipids, and sterol esters. The increased levels of palmitoyl CoA result in increased de novo synthesis of sphingolipids; the transcript level of serine palmitoyl transferase, the rate-limiting enzyme for the de novo synthesis of sphingolipids, is significantly elevated from the 4-h time point onward and peaks at the h time point.

    The concomitant increase in the biosynthetic products desmosterol and lanosterol via acetyl CoA points to the involvement of sterol synthesis as well. Members of the statin class of drugs inhibit cholesterol biosynthesis by blocking the conversion of HMG-CoA into mevalonic acid. We reasoned that use of this selective inhibitor together with KLA would provide insight into how perturbations in one lipid biosynthetic pathway reverberate through the lipidome. As expected, compactin blocked the KLA-stimulated increases in desmosterol Fig.

    These relative changes are also visible in the data heat map for the combined data shown in supplemental Fig. In the large cluster, desmosterol and 24,epoxy-cholesterol are adjacent to each other, as are cholesterol and hydroxycholesterol. Although these four lipids are in the same cluster, the two subgroups are far apart. Compactin treatment similarly slowed the increase in coenzyme Q that occurred with or without KLA stimulation, but in these experiments, levels of dolichol were unchanged under all conditions.

    The legend is as follows: Activation of LXR by 24,epoxy-cholesterol is known to enhance the expression of genes encoding Abca1 and Abcg1, transporters that mediate cholesterol efflux, and to inhibit the expression of inflammatory response genes An unexpected observation made in the compactin experiments was that inhibition of sterol biosynthesis caused an increase in the levels of two eicosanoids, PGD 2 and PGE 2 , in response to KLA Fig.

    This is also evident from the high correlation among these genes and lipids shown in supplemental Fig. All four are in the same cluster. Studies in bone marrow-derived macrophages excluded roles for the LXRs in this apparent cross-talk between the sterol and eicosanoid pathways, suggesting the existence of a novel regulatory pathway involving transcription and signaling.

    Changes in multiple lipid molecular species of glycerophospholipids and cholesteryl esters were also observed as illustrated in Fig. Using the protocols developed for mass spectrometric identification of lipids, we identified several novel lipids associated with macrophage cells. Several adrenic acids and elongated prostaglandins were found Analysis of the phospholipid content of macrophage cell types i.

    RAW, resident peritoneal macrophages, bone marrow-derived macrophages, and foam cells has revealed dozens of rare or previously uncharacterized species of phospholipids, including phosphatidylthreonines, ether-linked phosphatidylinositols, phosphatidylserines, and glycerophosphatidic acids, as well as phosphatidylcholines and phosphatidylethanolamines containing very long polyunsaturated fatty acids In preliminary studies, we have observed that KLA increased the 1-deoxy-sphingoid bases as well as their N -acyl-metabolites.

    Analysis of macrophage sterols revealed a novel role for a previously described lipid of unknown function. The oxysterol hydroxycholesterol was initially described in the literature in 31 , 32 , and despite hundreds of studies in which this lipid was studied, a biological function remained undefined.

    Activated RAW macrophages were found to induce the cholesterol hydroxylase gene and the synthesis of hydroxycholesterol, which in turn was shown to regulate the production of immunoglobulin A by B cells of the adaptive immune system Novel N -acyl-phosphatidylserine molecular species were detected in the phospholipids of mouse RAW cells N -Acyl-phosphatidylserines may be the precursor of N -acylserine, a signaling lipid present in bovine brain, and it is possible that phospholipase D could cleave N -acyl-phosphatidylserine to generate N -acyl serine.

    A living cell represents a highly integrated unit in which many thousands of lipid gene products must interact to ensure homeostasis in cell division, metabolism, and responsiveness. The switch from a reductionist to a constructionist approach in the scientific study of cells must begin with the identification of the many molecules that make up the cell.

    Recent studies along these lines have produced complete gene, mRNA, and protein parts lists, and here we add to these building blocks by reporting initial efforts to identify the many thousands of lipid molecular species that make up the mammalian macrophage. This number represents an underestimate as undoubtedly, not all lipids were extracted from the RAW cells by the organic solvents used and not all that were resolved were quantified by our analytical methods, so there will certainly be additional novel lipids present.

    Indeed, in the course of these studies, we discovered many lipid species not previously reported in macrophages including dihomoprostaglandins, ether-linked triglycerides, ether-linked phosphatidylinositol, ether-linked phosphatidylserine, phosphatidylthreonine, N -acyl phosphatidylserine, and deoxyceramides data not shown.

    The changes in lipid levels reported here were detected in multiple independent experiments, suggesting that each has a biological meaning.

    In some cases, such as for eicosanoids, the consequences for macrophage function and inflammation are well defined, whereas for a majority of the other lipid changes, determining the effects and physiological significance will require future investigations. We have presented herein a comprehensive picture of the dynamics of lipid metabolism in the functioning of a mammalian cell using a systems biology approach see supplemental Fig. This is the first quantitative approach toward the complete characterization of the lipidome of a mammalian cell and its regulation during immunological stimulation and pharmacological inhibition in a model for disease processes.

    VanNieuwenhze Indiana University , Dr. White University of California, Irvine , Dr. Allegood, Aaron Armando, Michelle D. Armstrong, Robert Byrnes, Christopher A.

    Lincoln, and Rebecca Shaner for technical assistance and Masada Disenhouse for administrative assistance. W Russell and B. You'll be in good company. Journal of Lipid Research.

    Alex Brown d , 1 , Stephen B. Milne d , David S. Myers d , Christopher K. Merrill Jr f , 1 , M. Murphy g , 1 , Christian R. Raetz h , 1 , Teresa A. Garrett h , Ziqiang Guan h , Andrea C. Ryan h , David W. Russell i , 1 , Jeffrey G. McDonald i , Bonne M. Thompson i , Walter A. Previous Section Next Section. Fatty acids, Eicosanoids, and Fatty Acyl-CoA Eicosanoids and fatty acids were detected and quantitated by mass spectrometric methods Glycerolipids Glycerolipids triacylglycerols, diacylglycerols, and monoether diacylglycerols were detected and quantitated by mass spectrometric methods Glycerophospholipids Glycerophospholipids excluding cardiolipins were detected and quantitated by mass spectrometric methods Sphingolipids Sphingolipids were detected and quantitated by mass spectrometric methods Coenzyme Qs and Dolichols Coenzyme Qs and dolichols were detected and quantitated by mass spectrometric methods Sterols and Cholesteryl Esters Sterols were detected and quantitated by mass spectrometric methods Fatty Acids and Fatty Acyls Activation of phospholipase A 2 to release arachidonic acid for subsequent metabolism is a hallmark of inflammation.

    Sterols and Prenols Over a h period of stimulation, intracellular cholesterol levels doubled in treated cells, and the amounts of two intermediates in the cholesterol biosynthetic pathway, lanosterol and desmosterol, increased by 8- and 4-fold, respectively Fig. Sphingolipids KLA treatment generated increases in almost every category of sphingolipid analyzed, including free sphingoid bases and their phosphates e. Lipid Remodeling and Pharmacological Perturbation of Activated Macrophages by a Statin We present herein an integrated view of lipid metabolism and remodeling that includes cross-talk between the six lipid categories discussed above as illustrated in the comprehensive overview of the major mammalian lipid categories and their interconnections shown in Fig.

    Novel Lipids Using the protocols developed for mass spectrometric identification of lipids, we identified several novel lipids associated with macrophage cells. Conclusion A living cell represents a highly integrated unit in which many thousands of lipid gene products must interact to ensure homeostasis in cell division, metabolism, and responsiveness.

    CrossRef Medline Google Scholar. Laboratory Automation 12 , — Cell 25 , 57 — Related articles Papers of the Week: A Mouse Macrophage Lipidome J. View this article with LENS. Prev Next Table of Contents.

    This Article First Published on October 5, doi: Services Email this article to a friend Alert me when this article is cited Alert me if a correction is posted Alert me when eletters are published Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Download to citation manager Request Permissions.

    Responses Submit a Letter to the Editor. Google Scholar Articles by Dennis, E. Articles by Subramaniam, S. C2C12 mouse skeletal myoblast and HFF human foreskin fibroblasts. Host cell infection was established with T. The major lipid subclasses eluting at different retention times min are indicated above the chromatogram. The chromatographic profiles for T.

    A total of lipid molecular species were confidently identified after manual curation of LipidSearch-assigned spectra, with coverage of 30 different lipid subclasses, across all samples S1 and S2 Tables; Supporting Information: Over half of the lipids identified belong to the glycerophospholipid lipid GP category unique molecular species , with most of this diversity associated with diacyl glycerophosphocholine PC or diacyl- and ether-linked glycerophosphatidylethanolamine PE subclasses and unique species, respectively.

    Next, the proportion of each of the major lipid classes within the lipidomes of T. To this end, the relative abundance of each major lipid class identified in T. Apart from the increased TG content in infected fibroblasts as compared to the uninfected cell Fig 2 , HFF , the lipid class distribution of mammalian host cells remained largely unchanged after 48 hours of T.

    The lipid class relative abundance profiles of isolated T. This finding suggests that, at least for the phase of the intracellular T. Notably, this lipid identity includes a relatively enriched TG pool in T. The lipidomic signatures of the extracellular trypomastigotes diverge from their amastigote counterparts suggestive of developmental regulation of lipid class ratios in this parasite [ 30 ].

    In addition, the lipidome profiles of T. These observations indicate that while the T. To compare parasite and host lipidomic signatures in more depth, analyses of the detailed lipid species distributions within each of the major lipid subclasses described above were undertaken.

    Two-dimensional principal component analyses PCA were performed to identify overall trends in the data Fig 3. When total lipidomes were compared across samples, T. To determine if this relationship might be driven by a sub-compartment of the total lipidome, additional PCA plots were generated for individual lipid subclasses. A more complex relationship between host and parasite lipid signatures emerged in these analyses Fig 3B and 3C ; S2 Fig. Within the TG class, tight clustering between T.

    Still other lipid classes showed no distinct trend S2 Fig. Combined, these data suggest that the steady state lipidome of T. The first two principle components are plotted PC1 and PC2 with proportion of variance for each component shown in parenthesis. With several enzymatic activities absent in mammals, T.

    An example is linoleic acid C Conversely, trypanosomes have been reported to have lower levels of oleic acid and palmitic acid than human cells [ 33 , 35 ]. We therefore predict that lipids produced endogenously by T.

    With these criteria in mind, a deeper analysis of the parasite and host lipidome data was conducted in which detailed comparisons of the FA moiety composition of each lipid subclass of T. Because of the differences between parasite and mammalian cell FA elongase and desaturase enzymatic machineries mentioned above, emphasis was given to the comparison of long-chain and very long-chain polyunsaturated FA LC-PUFA and VLC-PUFA, respectively moiety distributions of each of the major lipid subclasses of T.

    In most cases, this approach identified T. Also, the parasite-derived PE and PI pools exhibited much higher abundance of ether-bound moieties 1- 0- alkyl and 1 alkenyl, represented as Taken together, these data suggest that T. In contrast to these observations, the glycerolipid GL pools of T. This led us to hypothesize that T. To obtain direct biochemical evidence of FA scavenging by T. This protocol resulted in a striking fold increase in the C Lipidomic analysis of uninfected HFF uninfected , T.

    We find that OCFA were unequally distributed across lipid classes in the host and parasite lipidomes, with C Thus, in conjunction with lipidomic profiling data, metabolic labeling studies provide further evidence that T. Furthermore, our results implicate host TG metabolism as a critical factor influencing FA incorporation by T. We sought to determine the impact of decreased access to host FA through the TG pool on amastigote growth. Using a flow cytometry-based method to follow amastigote proliferation using CFSE-labeled parasites [ 22 ] we find that T.

    These combined lipidomic, metabolic labeling, and proliferation data suggest that T. However, amastigotes are still able to proliferate in host cells lacking the capacity for TG synthesis via the major DGAT-dependent pathway [ 37 ].

    This strategy may be more energetically favorable and offer a level of flexibility that can facilitate pathogen survival under changing environmental conditions. Here, we demonstrate that, despite the predicted capacity for de novo FA synthesis by the kinetoplastid protozoan parasite, Trypanosoma cruzi [ 31 ], the obligate intracellular amastigote stages of this parasite readily incorporate long-chain fatty acids LCFA , acquired from mammalian host cell glycerolipid GL pools, into their own lipid storage and synthesis pathways.

    Our findings expose a biochemical and functional link between parasite and host lipid metabolism and demonstrate the potential or T. The unbiased, quantitative lipidomics approach adopted in this study, which focused on FA moiety compositions in different parasite and host cell lipid subclasses, was instrumental in revealing the hybrid nature of the T.

    A key element of our study design was the generation of parallel comprehensive lipidomic datasets for T. A deep analysis of the FA moiety distribution within each lipid subclass was conducted with extensive manual curation to facilitate the high confidence assignment of host- and parasite-like lipid signatures.

    Overall, we find that T. First, the proportion of several major lipid classes identified in T. Such conservation of class-specific lipid moiety distribution in T. However, unlike the parasite signatures noted above, the T. In fact, the FA moiety profiles for these lipid subclasses were nearly identical to their specific host cell counterparts, suggesting that these lipids were acquired by the parasites from their mammalian host cells.

    This conclusion is supported by metabolic labeling studies in which incorporation of exogenous FA into amastigote neutral lipids and phospholipids involves flux through host TG pools. Exogenous provision of either odd-chain FA C However, trafficking of these exogenous FA tracers into host-resident T. Furthermore, as the proliferative capacity of T. Together, our observations support a model in which intracellular T. Scavenged lipids may serve as a source of FA during the proliferative phase of the T.

    In contrast, exogenous FA were not detectably incorporated into T. Since the bulk of PE in T. The lack of flux of exogenous FA into parasite glycosomes would also explain the relatively low labeling of the T.

    While more detailed metabolic flux analyses are required to fully appreciate the contribution of scavenged FA to the biology of intracellular T. This modular synthesis involves 3 elongases ELO 1—3 , which convert C4: Although ELO are highly expressed in host cell resident T. Based on our current finding that T. In addition to generating FA de novo , T. The relative reliance of T. Our data strongly suggest that T. As lipid droplets are highly dynamic organelles that function as critical hubs for FA trafficking in cells with key roles in cellular lipid and energy metabolism [ 49 — 51 ], it is not surprising that host LD are frequently targeted by intracellular pathogens.

    LD accumulation is a common cellular response to pathogen infection [ 52 — 55 ], which can occur in response to increased oxidative stress [ 56 , 57 ] or paracrine signals [ 58 ]. With high rates of FA flux, host cell LD represent a readily accessible source of FA for a number of intracellular pathogens, such as Chlamydia trachomatis , Mycobacterium tuberculosis , and M. Our demonstration of a biochemical interaction between T.

    During acute Chagas disease, inflammatory macrophages typically exhibit increased formation of LD enriched with arachidonic acid AA , which is a precursor for the synthesis of proinflammatory eicosanoids such as prostaglandin E 2 [ 61 ]. Moreover, these LD have been shown to contain eicosanoid-forming enzymes cyclooxygenases and lipoxygenases that are upregulated during T. Despite the importance of LD for the storage of AA in inflammatory macrophages and other leukocytes [ 62 , 63 ], we were unable to detect appreciable levels of this FA in the TG pools of either infected or mock-infected host cells, or T.

    These differences likely reflect the substantial variation in lipid droplet composition and function between cell types, and even within a homogenous cell population under different environmental conditions [ 64 ].

    In summary, the application of a comparative lipidomics approach successfully distinguished parasite- and host-specific lipidomic signatures, providing evidence that T. Chan School of Public Health [ 37 ]. Cell culture reagents were purchased from Gibco. To establish intracellular T. Cells were then rinsed twice with PBS to remove extracellular parasites and fresh DMEM-2 medium added to flasks and incubated for a further 46 h.

    Parallel cultures of mock-infected mammalian cell monolayers were also established. The isolation of the i ntra c ellular a mastigotes ICA form of T. Briefly, infected monolayers were washed extensively with PBS, detached from the flask using mild trypsinization Gibco, 0. Of this infected cell suspension, 0. The lysate was then passed over a 4 ml DEAE-Sephacel Sigma column pre-washed with 10 column volumes cytosolic buffer , and cytosolic buffer was added such that three 10 ml flow-through fractions could be collected.

    Isolated amastigote from each fraction were enumerated by haemocytometer and fractions containing T. Membranes were blocked for 1 h at ambient temperature with shaking in 1: All secondary antibodies were incubated for 30 m at room temperature at the following conditions AlexaFluor goat anti-mouse 1: All antibodies were diluted with a 1: All solvents used were of HPLC grade or higher.

    The lipid extraction protocol was modified from [ 42 ]. Samples were vortexed vigorously for 5 min, and centrifuged for 10 min at 1, g at room temperature. After centrifugation, the supernatants were transferred to new Pyrex tubes, and the pellets were dried under N 2 stream. After centrifugation, the lower organic and upper aqueous phases were separated into fresh Pyrex tubes.

    Lipids were fractionated by reverse-phase chromatography over a 46 min gradient mobile phase A: The mass spectrometer acquisition settings were as follows for both positive and negative ionization mode: All data were analyzed using LipidSearch Software Version 4. Only those classes for which standards were detected in each run were considered for analysis. Each pie chart is the average of at least 3 biological replicates.

    Neutral lipid TLC were conducted using a protocol modified from [ 71 ]. The TLC chamber was equilibrated with hexane: The identity of different lipid classes on the TLC plate was inferred by comparing their migration pattern to that of commercial standards run on the same plate.

    Phospholipid TLC were conducted using a protocol modified from [ 72 ]. The TLC chamber was equilibrated with acetone: Once developed, TLC plates were dried, covered with mylar, and exposed to a storage phosphorimager screen overnight. After imaging, TLC plates were exposed to iodine vapor to visualize equal loading. The web-based server MetaboAnalyst 3.

    Bar and pie charts were generated using GraphPad Prism 7, version 7. After staining, trypomastigotes were washed and re-suspended in DMEM-2 and allowed to infect host cells as described. Immediately prior to acquisition amastigotes were pelleted at 4, g for 10 min and resuspended in a 0. Amastigotes were identified based on size and DAPI staining.

    Proliferation modeling based on signal intensity from undivided parasites collected at 18 hpi were generated using FlowJo Tree Star proliferation software. Greater than 10, events in the final amastigote gate were acquired for each sample. Methylated FA were recovered by dichloromethane: Data are from a representative experiment. Summary of the lipid species detected in at least one biological replicate of each sample.

    Averages from all lipid classes are shown. This work was supported by the National Institutes of Health https: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. Published online Dec Author information Article notes Copyright and License information Disclaimer.

    Received Jun 5; Accepted Dec 8. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

    This article has been cited by other articles in PMC. Detailed supplemental methods with corresponding references are included in file. Negative ion mode base peak chromatograms of T. Principle component analysis of host and parasite lipidomes at the lipid species level. Trends in host and parasite FA composition varies between lipid classes. Immunoblot confirmation of ectopically expressed DGAT2.

    Detection of lipid species across all samples. Summary of lipid class breakdown in T. Total lipidome fatty acyl composition by average area percent.

    Host triacylglycerols shape the lipidome of intracellular trypanosomes and modulate their growth

    High-throughput lipidomics has great potential when investigating the identification of lipid species (reviewed in [7–9]). .. phospholipids in sperm cells is required for the intra- and intercellular membrane fusion events in. Studies investigating the lipid profiles of cells following MWCNT exposure particles measured with DLS in dispersion and cell culture media determined .. amount of intracellular ROS in the cells which confirms the ability of. Despite the central role of lipids in mammalian cell function, there has been To account for biological variability of cells grown in culture and to enhance . for each analyte was determined relative to a known amount of internal .. might accumulate as a result of increases in intracellular cholesterol levels.

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