PubMed

Methods Mol Biol. 2024;2816:253-263. doi: 10.1007/978-1-0716-3902-3_22.ABSTRACTLipids are compounds involved in many biologic functions including cell structure, metabolism, energy storage and are involved in signaling. A prominent lipid in these functions is cholesterol. Cholesterol also plays a part in the signaling of melanocytes, which contain melanosomes. The maturation of these melanosomes happens during melanocyte growth. The deficit of melanogenesis or melanosome maturation is associated with ocular albinism in the eye. Aberrations of melanosome maturation are also associated with pigment dispersion syndrome. Albinism and pigment dispersion manifestations are systemic. Both melanogenesis and melanocyte maturation are affected by cholesterol metabolism. Cholesterol signaling is a part of many pathways in the body, and evaluating these signals can have implications in systemic disease processes of melanogenesis and melanosome maturation, like ocular albinism and pigment dispersion. Cholesterol is carried by lipoprotein particles. Low-density lipoprotein (LDL) is usually the transport vehicle for cholesterol to reach tissues and organelles. The LDL uptake on cells often sends out a cascade of internal signaling within the cells. We describe here LDL signaling related to lipase activity changes using enzymatic methods with a kit. We describe analyses of cholesterol esters and free cholesterol with liquid chromatography and gas chromatography with or in tandem with mass spectrometry (GC-MS and LC-MS/MS). These analyses will provide insight into melanosome maturation and melanogenesis. The methods described here are applicable to all melanocytes within the body of a model mammalian organism.PMID:38977604 | DOI:10.1007/978-1-0716-3902-3_22

Methods Mol Biol. 2024;2816:241-252. doi: 10.1007/978-1-0716-3902-3_21.ABSTRACTBioactive lipids have been identified as dynamic signaling lipid mediators (LMs). These fats have the ability to activate responses and control bodily functions either directly or indirectly. Linoleic Acid (LA) and Alpha Linoleic Acid (ALA) are types of omega 3 fatty acids that possess inflammatory properties and promote resolution of inflammation either through their own actions or through their metabolites known as oxylipins. In this chapter, we provide an explanation of a method that combines chromatography with tandem mass spectroscopy (LC MS/MS) to identify and measure all the metabolites derived from LA and ALA. Additionally, we employed the described methodology to analyze human serum samples obtained before and after whole-body vibration exercise training. The results indicated an increase in some of the LA and ALA LMs that have beneficial effects in regulating the cardiovascular system.PMID:38977603 | DOI:10.1007/978-1-0716-3902-3_21

Methods Mol Biol. 2024;2816:205-222. doi: 10.1007/978-1-0716-3902-3_19.ABSTRACTThe role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).PMID:38977601 | DOI:10.1007/978-1-0716-3902-3_19

Methods Mol Biol. 2024;2816:193-204. doi: 10.1007/978-1-0716-3902-3_18.ABSTRACTWith impaired retinal ganglion cell (RGC) function and eventual RGC death, there is a heightened risk of experiencing glaucoma-induced blindness or other optic neuropathies. Poor RGC efficiency leads to limited transmission of visual signals between the retina and the brain by RGC axons. Increased focus on studying lipid messengers found in neurons such as endocannabinoids (eCBs) has importance due to their potential axonal pathway regenerative properties. 2-Arachidonoylglycerol (2-AG), a common eCB, is synthesized from an sn-1 hydrolysis reaction between diacylglycerol (DAG) and diacylglycerol lipase (DAGL). Examination of DAG production allows for future downstream analysis in relation to DAGL functionality. Here, we describe protocol guidelines for extracting RGCs from mouse retinas and subsequent mass spectrometry analysis of the DAG content present within the RGCs.PMID:38977600 | DOI:10.1007/978-1-0716-3902-3_18

Methods Mol Biol. 2024;2816:175-191. doi: 10.1007/978-1-0716-3902-3_17.ABSTRACTThe trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.PMID:38977599 | DOI:10.1007/978-1-0716-3902-3_17

Methods Mol Biol. 2024;2816:151-159. doi: 10.1007/978-1-0716-3902-3_15.ABSTRACTDeveloping robust analytical techniques is a vital phase to facilitate understanding the roles and impacts of various omic profilings in cellular functions. The comprehensive analysis of various biological molecules within a biological system requires a precise sample preparation technique. Solid-Phase Extraction (SPE) has proven to be an indispensable method in lipidomic analysis, providing an uncomplicated and user-friendly technique for extraction and purification of lipid components from complex biological matrices. Of all the factors influencing the reliability and success of SPE, column or adsorbent materials, flow rate, and storage conditions are paramount in terms of their significance. In this chapter, we will discuss in detail the SPE steps for lipidomic analysis in biofluid samples (serum and plasma) and muscle tissues.PMID:38977597 | DOI:10.1007/978-1-0716-3902-3_15

Methods Mol Biol. 2024;2816:139-144. doi: 10.1007/978-1-0716-3902-3_13.ABSTRACTPhosphatidic acid (PA) is a key signaling lipid that plays a crucial role in regulating various cellular processes. Studies have shown that azobenzene-containing PA analogues can be used as an all-chemical strategy for light-mediated control of PA signaling. These photoswitchable lipids offer a solution to the limitations of traditional bulk dosing methods by allowing for light- and shape-dependent interactions with protein effectors and lipid-metabolizing enzymes. This chapter describes how to synthesize AzoPA and dAzoPA.PMID:38977595 | DOI:10.1007/978-1-0716-3902-3_13

Methods Mol Biol. 2024;2816:129-138. doi: 10.1007/978-1-0716-3902-3_12.ABSTRACTPhospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s).PMID:38977594 | DOI:10.1007/978-1-0716-3902-3_12

Methods Mol Biol. 2024;2816:87-100. doi: 10.1007/978-1-0716-3902-3_9.ABSTRACTLaparotomy (EL) is one of the most common procedures performed among surgical specialties. Previous research demonstrates that surgery is associated with an increased inflammatory response. Low psoas muscle mass and quality markers are associated with increased mortality rates after emergency laparotomy. Analysis of lipid mediators in serum and muscle by using liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has proven to be a sensitive and precise technique. In this chapter, we describe an LC-MS/MS protocol for the profiling and quantification of signaling lipids formed from Eicosapentaenoic Acid (EPA) and Eicosatetranoic acid (ETA) by 5, 12, or 15 lipoxynases. This protocol has been developed for and validated in serum and muscle samples in a mouse model of surgical stress caused by laparotomy.PMID:38977591 | DOI:10.1007/978-1-0716-3902-3_9

Methods Mol Biol. 2024;2816:53-67. doi: 10.1007/978-1-0716-3902-3_6.ABSTRACTThis chapter conducts an in-depth exploration of the impact of musculoskeletal (MSK) disorders and injuries, with a specific emphasis on their consequences within the older population demographic. It underscores the escalating demand for innovative interventions in MSK tissue engineering. The chapter also highlights the fundamental role played by lipid signaling mediators (LSMs) in tissue regeneration, with relevance to bone and muscle recovery. Remarkably, Prostaglandin E2 (PGE2) emerges as a central orchestrator in these regenerative processes. Furthermore, the chapter investigates the complex interplay between bone and muscle tissues, explaining the important influence exerted by LSMs on their growth and differentiation. The targeted modulation of LSM pathways holds substantial promise as a beneficial way for addressing muscle disorders. In addition to these conceptual understandings, the chapter provides a comprehensive overview of methodologies employed in the identification of LSMs, with a specific focus on the Liquid Chromatography-Mass Spectrometry (LC-MS). Furthermore, it introduces a detailed LC MS/MS-based protocol tailored for the detection of PGE2, serving as an invaluable resource for researchers immersed in this dynamic field of study.PMID:38977588 | DOI:10.1007/978-1-0716-3902-3_6

Methods Mol Biol. 2024;2816:35-40. doi: 10.1007/978-1-0716-3902-3_4.ABSTRACTSphingolipids, 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).PMID:38977586 | DOI:10.1007/978-1-0716-3902-3_4

Methods Mol Biol. 2024;2816:25-33. doi: 10.1007/978-1-0716-3902-3_3.ABSTRACTInterconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.PMID:38977585 | DOI:10.1007/978-1-0716-3902-3_3

Methods Mol Biol. 2024;2816:13-24. doi: 10.1007/978-1-0716-3902-3_2.ABSTRACTZebrafish (Danio rerio) has emerged as a pivotal model organism in vertebrate development research over several decades. Beyond its contributions to developmental biology, zebrafish have increasingly played a crucial role in the field of lipidomics. Lipidomics, a comprehensive analysis of lipids within biological systems, offers profound insights into lipid metabolism and signaling pathways. This chapter explores the zebrafish's unique attributes that make it an ideal candidate for lipidomics studies. With a genome sharing numerous genetic similarities with humans, zebrafish serve as a powerful model for dissecting lipid metabolism and unraveling the complexities of lipid mediator-related diseases. In this chapter, we delve into specific protocols tailored for utilizing zebrafish in lipidomics research and similar investigations. Through a comprehensive exploration of zebrafish as a model organism, this chapter aims to provide researchers with valuable insights and methodologies for advancing lipidomics studies using zebrafish.PMID:38977584 | DOI:10.1007/978-1-0716-3902-3_2

Int Microbiol. 2024 Jul 8. doi: 10.1007/s10123-024-00556-0. Online ahead of print.ABSTRACTThis study explored the extracellular metabolomic responses of three different Salmonella enterica serotype Typhimurium (S. Typhimurium) strains-ATCC 13311 (STy1), NCCP 16964 (STy4), and NCCP 16958 (STy8)-cultured at refrigeration temperatures. The objective was to identify the survival mechanisms of S. Typhimurium under cold stress by analyzing variations in their metabolomic profiles. Qualitative and quantitative assessments identified significant metabolite alterations on day 6, marking a critical inflection point. Key metabolites such as trehalose, proline, glycerol, and tryptophan were notably upregulated in response to cold stress. Through multivariate analyses, the strains were distinguished using three metabolites-4-aminobutyrate, ethanol, and uridine-as potential biomarkers, underscoring distinct metabolic responses to refrigeration. Specifically, STy1 exhibited unique adaptive capabilities through enhanced metabolism of betaine and 4-aminobutyrate. These findings highlight the variability in adaptive strategies among S. Typhimurium strains, suggesting that certain strains may possess more robust metabolic pathways for enhancing survival in refrigerated conditions.PMID:38977514 | DOI:10.1007/s10123-024-00556-0

Life Sci. 2024 Jul 6:122891. doi: 10.1016/j.lfs.2024.122891. Online ahead of print.ABSTRACTThere is a growing body of evidence suggesting that the composition of intestinal flora plays a significant role in regulating lipid metabolism. 2', 3', 5'-tri-O-acetyl-N6-(3-hydroxyphenyl) adenosine (IMMH007) is a new candidate compound for regulating blood cholesterol and other lipids. In this study, we conducted metagenomic and metabolomic analyses on samples from high-fat diet-fed (HFD) hamsters treated with IMMH007. Our findings revealed that IMM-H007 reversed the imbalance of gut microbiota caused by a high-fat diet. Additionally, it activated adiponectin receptor and pantothenate and CoA biosynthesis pathway-related genes, which are known to regulate lipid and glucose metabolism. Furthermore, IMM-H007 promotes cholesterol metabolism by reducing the abundance of genes and species associated with 7α-dehydroxylation and bile salt hydrolase (BSH). Metabolomics and pharmacological studies have shown that IMM-H007 effectively improved glucose and lipid metabolism disorders caused by HFD, reduced the aggregation of secondary bile acids (SBAs), significantly increased the content of hyodeoxycholic acid (HDCA), and also activated the expression of VDR in the small intestine. As a result, there was a reduction in the leakage of diamine oxidase (DAO) into the bloodstream in hamsters, accompanied by an upregulation of ZO-1 expression in the small intestine. The results suggested that IMM-H007 regulated glucose and lipid metabolism, promoted cholesterol metabolism through activating the expression of VDR, inhibiting inflammatory and improving the permeability of the intestinal barrier. Thus, our study provides new understanding of how IMM-H007 interacts with intestinal function, microbiota, and relevant targets, shedding light on its mechanism of action.PMID:38977060 | DOI:10.1016/j.lfs.2024.122891

Bioresour Technol. 2024 Jul 6:131078. doi: 10.1016/j.biortech.2024.131078. Online ahead of print.ABSTRACTVitamin D (VD) production-based microalgae biosynthesis presents various benefits including sustainability, fast expansion, and the capacity to generate substantial quantities. However, this approach suffers from serious challenges that require effective cultivation methods and extraction processes. Indeed, further researches are of significant interest to understand the biosynthesis pathways, enhance the processes, and ensure its viability. In this context, the present review focuses on an in-depth understanding of the chemistry of VD and its analogues and provides a comprehensive explanation of the biosynthesis pathways, precursors, and production methods. In addition, this work discusses the state of the art reflecting the recent advances researches and the global market of microalgae as a potential source of VD. In sum, this paper demonstrates that microalgae can efficiently biosynthesize various forms of VD, presenting a sustainable alternative for VD production.PMID:38977035 | DOI:10.1016/j.biortech.2024.131078

Anal Chem. 2024 Jul 8. doi: 10.1021/acs.analchem.4c01706. Online ahead of print.ABSTRACTDiscovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.PMID:38976774 | DOI:10.1021/acs.analchem.4c01706

Proc Natl Acad Sci U S A. 2024 Jul 16;121(29):e2313851121. doi: 10.1073/pnas.2313851121. Epub 2024 Jul 8.ABSTRACTMass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.PMID:38976734 | DOI:10.1073/pnas.2313851121

Adv Sci (Weinh). 2024 Jul 8:e2402025. doi: 10.1002/advs.202402025. Online ahead of print.ABSTRACTAs a significant infectious disease in livestock, porcine reproductive and respiratory syndrome (PRRS) imposes substantial economic losses on the swine industry. Identification of diagnostic markers and therapeutic targets has been a focal challenge in PPRS prevention and control. By integrating metabolomic and lipidomic serum analyses of clinical pig cohorts through a machine learning approach with in vivo and in vitro infection models, lysophosphatidic acid (LPA) is discovered as a serum metabolic biomarker for PRRS virus (PRRSV) clinical diagnosis. PRRSV promoted LPA synthesis by upregulating the autotaxin expression, which causes innate immunosuppression by dampening the retinoic acid-inducible gene I (RIG-I) and type I interferon responses, leading to enhanced virus replication. Targeting LPA demonstrated protection against virus infection and associated disease outcomes in infected pigs, indicating that LPA is a novel antiviral target against PRRSV. This study lays a foundation for clinical prevention and control of PRRSV infections.PMID:38976572 | DOI:10.1002/advs.202402025

J Ocul Pharmacol Ther. 2024 Jul 8. doi: 10.1089/jop.2024.0067. Online ahead of print.ABSTRACTIntroduction: The lens's metabolic demands are met through a continuous circulation of aqueous humor, encompassing a spectrum of components such as organic and inorganic ions, carbohydrates, glutathione, urea, amino acids, proteins, oxygen, carbon dioxide, and water. Metabolomics is a pivotal tool, offering an initial insight into the complexities of integrated metabolism. In this investigative study, we systematically scrutinize the composition of intraocular fluid in individuals afflicted with cataracts. Methods: The investigation involved a comprehensive analysis of aqueous humor samples from a cohort comprising 192 patients. These individuals were stratified by utilizing the SPONCS classification system, delineating distinct groups characterized by the hardness of cataracts. The analytical approach employed targeted quantitative metabolite analysis using HILIC-based liquid chromatography coupled with high-resolution mass spectrometric detection. The metabolomics data analysis was performed with MetaboAnalyst 5.0. Results: The results of the enrichment analysis have facilitated the inference that the discerned disparities among groups arise from disruptions in taurine and hypotaurine metabolism, variations in tryptophan metabolism, and modifications in mitochondrial beta-oxidation of short-chain saturated fatty acids and pyrimidine metabolism. Conclusion: A decline in taurine concentration precipitates diminished glutathione activity, prompting an elevated requirement for NAD+ and instigating tryptophan metabolism along the kynurenine pathway. Activation of this pathway is additionally prompted by interferon-gamma and UV radiation, leading to the induction of IDO. Concurrently, heightened mitochondrial beta-oxidation signifies a distinctive scenario in translocating fatty acids into the mitochondria, enhancing energy production.PMID:38976556 | DOI:10.1089/jop.2024.0067