Lipidomics is a relatively new field under the heading of systems biology. Due to its infancy, the field suffers from significant ‘growing pains’, one of which is the lack of bioinformatic analytic resources that other “-omics” fields enjoy. Here, I describe the creation and validation of the glycerophospholipid identification program VaLID. Using an in silico approach, we generated a comprehensive database containing all of the glycerophospholipids within multiple sub-classes: those containing chains of 0 to 30 carbons with up to 6 unsaturations and various linkages. Using Java, I created a web- based computer interface with a search engine and a visualization tool to access this database. In comparing results to current programs, I found that VaLID consistently contained more identity predictions than did the current gold standard LipidMAPS. Results from several tests with real datasets confirm that VaLID is more than capable as a phospholipid identification tool for use in lipidomics.

Mass spectrometry has increasingly become the method of choice for the analysis of complex biological samples, including proteins and lipids. This thesis describes the development of MS-based analytical methodologies for the analysis of complex proteomic and lipidomic samples. Chapter 3 describes the development of microfluidic proteomic reactors, in the formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex proteomic samples by combining multiple processing steps, such as rapid extraction and enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all performed in a few nanoliters effective volume, resulting in an increased protein digestion efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that were compatible with HPLC-MS/MS analysis. In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of PAF and LPC lipid species is described. In this method, lipid extracts from biological samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000 mass spectrometer, where the lipid species of interest were detected using a precursor ion scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0, C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative comparisons of other LPC lipid species of interest between different samples. The developed methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8 transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42 treatment, respectively, in comparison to controls. iii Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.

Lipidomics, a rapidly developing field of study, focuses on the classification and quantitation of lipid species. Lipidomics has emerged in the forefront of scientific research due to the key roles that lipids play in metabolism, cancer, and disease. Using mass spectrometry as a tool for analysis, understanding the role eicosanoids and sphingolipids play has advanced rapidly. Being able to observe these small molecules in vivo has led to better understanding of several lipid-driven mechanisms and identification of eicosanoid and sphingolipid biomarkers in neurodegenerative disease, cancer, sepsis, wound healing, and pre-eclampsia. In the studies herein, we developed targeted mass spectrometric methods to identify lipids of interest in inflammatory disease signaling. We further used lipidomics to identify biomarkers in a clinical trial involving patients diagnosed with pre-eclampsia, discovering that ceramide-1-phosphate, a lipid involved in the initiation of inflammation, was significantly decreased in the plasma of patients who developed pre-eclampsia. Additionally, lipidomic studies were used to elucidate the mechanism of the cPLA2α/C1P interaction, in which the binding of C1P to of cPLA2α was ablated (cPLA2α KI) resulting in resistance to sepsis and increased wound closure rate of cPLA2α KI mice. These studies show how useful lipidomics coupled with mass spectrometry can be in studying inflammatory diseases regulated by lipid signaling.

Glycosphingolipids such as α- and β-glucosylceramides (GlcCers) and α- and β- galactosylceramides (GalCers) are stereoisomers differentially synthesized by gut bacteria and their mammalian hosts in response to environmental insult. Thus, lipidomic assessment of α- and β-GlcCers and α- and β-GalCers is crucial for inferring biological functions and biomarker discovery. However, simultaneous quantification of these stereoisomeric lipids is difficult due to their virtually identical structures. Differential mobility mass spectrometry (DMS), as an orthogonal separation to high performance liquid chromatography used in electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS), can be used to separate stereoisomeric lipids. Generating LC-ESI-DMS-MS/MS methods for lipidomic analyses is exceedingly difficult demanding intensive manual optimization of DMS parameters that depend on the availability of synthetic lipid standards. Where synthetic standards do not exist, method development is not possible. To address this challenge, I developed a supervised in silico machine learning approach to accelerate method development for ion mobility-based quantification of lipid stereoisomers. I hypothesized that supervised neural network models could be used to learn the relationships between lipid structural characteristics and optimal DMS machine parameter values thereby reducing the total number of empirical experiments required to develop a DMS method and enabling users to “predict” DMS parameters for analytes that lack synthetic standards. Specifically, this thesis describes a supervised learning approach that learns the relationship between two DMS machine parameter values (separation voltage and compensation voltage) and two lipid structural features (N-Acyl chain length and degree of unsaturation). I describe here, iDMS, an algorithm that was trained on 17 lipid species, and can further simulate results of DMS manual method development and suggest optimal parameter values for 47 lipid species. This approach promises to greatly accelerate the development of assays for the detection of lipid stereoisomers in biological samples.

No / Ultraviolet radiation (UVR) in sunlight has deleterious effects on skin, while behavioural changes have resulted in people gaining more sun exposure. The clinical impact includes a year-on-year increase in skin cancer incidence, and topical sunscreens alone provide an inadequate measure to combat overexposure to UVR. Novel methods of photoprotection are being targeted as additional measures, with growing interest in the potential for systemic photoprotection through naturally sourced nutrients. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are promising candidates, showing potential to protect the skin from UVR injury through a range of mechanisms. In this review, we discuss the biological actions of n-3 PUFA in the context of skin protection from acute and chronic UVR overexposure and describe how emerging new technologies such as nutrigenomics and lipidomics assist our understanding of the contribution of such nutrients to skin health.

No / Acute exposure of human skin to the ultraviolet radiation (UVR) in sunlight results in the sunburn response. This is mediated in part by pro-inflammatory eicosanoids and other bioactive lipids, which are in turn produced via mechanisms including UVR-induction of oxidative stress, cell signalling and gene expression. Sunburn is a self-limiting inflammation offering a convenient and accessible system for the study of human cutaneous lipid metabolism. Recent lipidomic applications have revealed that a wider diversity of eicosanoids may be involved in the sunburn response than previously appreciated. This article reviews the effects of UVR on cutaneous lipids and examines the contribution of bioactive lipid mediators in the development of sunburn. Since human skin is an active site of polyunsaturated fatty acid (PUFA) metabolism, and these macronutrients can influence the production of eicosanoids/bioactive lipids, as well as modulate cell signalling, gene expression and oxidative stress, the application of PUFA as potential photoprotective agents is also considered.

No / Because of confounding factors, the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on type 1 diabetes remain to be clarified. We therefore evaluated whether fat-1 transgenic mice, a well-controlled experimental model endogenously synthesizing n-3 PUFA, were protected against streptozotocin (STZ)-induced diabetes. We then aimed to elucidate the in vivo response at the pancreatic level. Beta-Cell destruction was produced by multiple low-doses STZ (MLD-STZ). Blood glucose level, plasma insulin level, and plasma lipid analysis were then performed. Pancreatic mRNA expression of cytokines, the monocyte chemoattractant protein, and GLUT2 were evaluated as well as pancreas nuclear factor (NF)-kB p65 and inhibitor of kB (IkB) protein expression. Insulin and cleaved caspase-3 immunostaining and lipidomic analysis were performed in the pancreas. STZ-induced fat-1 mice did not develop hyperglycemia compared with wild-type mice, and Beta-cell destruction was prevented as evidenced by lack of histological pancreatic damage or reduced insulin level. The prevention of Beta-cell destruction was associated with no proinflammatory cytokine induction (tumor necrosis factor-alpha, interleukin-1Beta, inducible nitric oxide synthase) in the pancreas, a decreased NF-kB, and increased IkB pancreatic protein expression. In the fat-1-treated mice, proinflammatory arachidonic-derived mediators as prostaglandin E2 and 12-hydroxyeicosatetraenoic acid were decreased and the anti-inflammatory lipoxin A4 was detected. Moreover, the 18-hydroxyeicosapentaenoic acid, precursor of the anti-inflammatory resolvin E1, was highly increased. Collectively, these findings indicate that fat-1 mice were protected against MLD-STZ-induced diabetes and pointed out for the first time in vivo the beneficial effects of n-3 PUFA at the pancreatic level, on each step of the development of the pathology-inflammation, Beta-cell damage-through cytokine response and lipid mediator production.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24993号 / 医博第5027号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 岩田 想, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

All cells are delimited by membranes that protect the cell from the surrounding environment. In eukaryotic cells the same principle applies at subcellular level where membranes delimit functional cell organelles. The membrane structure, properties and function are defined in part by their lipid composition. Lipidomics is the large‐scale study of pathways and networks of cellular lipids in biological systems. It involves the identification and quantitation of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites. Lipidomics has been greatly facilitated by recent advances in ionization technology and mass spectrometric capabilities which have simplified the sample processing prior to analysis, giving rise to shotgun lipidomics. Shotgun lipidomics is fast, highly sensitive, and can identify hundreds of lipids missed by other methods. However, Glycosphingolipids are an important lipid family that was out of the scope of shotgun lipidomics due to the lack of suitable analytical tools. The aim of my thesis was two‐fold. The first aim was the establishment of Glycosphingolipid identification and quantification by shotgun approach. This allowed us to perform lipidomic studies with unprecedented comprehensiveness (~300 lipid species from 15 different lipid classes) from low sample amounts and with minimal sample processing. The second was the application of this technology in studies of the role of lipids in several processes like vesicular carrier formation, cell polarization, protein delivery to the plasma membrane and viral budding. This work resulted in several findings. We found that there is sorting of sphingolipids and sterols into plasma membrane targeted vesicular carriers in budding yeast. When kidney cells change from a mesenchymal to an epithelial morphology there is a profound remodeling of their lipidome, with the synthesis of longer, more saturated, more hydroxylated, and more glycosylated sphingolipids. When these sphingolipids and sterols are depleted in epithelial cells, the apical transport in epithelial cells is impaired. These data strongly support the idea that lipid rafts play an important role in sorting and delivery of lipid and protein cargo to the plasma membrane. Finally, we found that the envelopes of vesicular stomatitis virus and Semliki forest virus assert little specificity in the incorporation of lipids from the plasma membrane. This weak specificity seems to be related to a combination of virus lipid bilayer asymmetry and curvature.

All cells are delimited by membranes that protect the cell from the surrounding environment. In eukaryotic cells the same principle applies at subcellular level where membranes delimit functional cell organelles. The membrane structure, properties and function are defined in part by their lipid composition. Lipidomics is the large‐scale study of pathways and networks of cellular lipids in biological systems. It involves the identification and quantitation of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites. Lipidomics has been greatly facilitated by recent advances in ionization technology and mass spectrometric capabilities which have simplified the sample processing prior to analysis, giving rise to shotgun lipidomics. Shotgun lipidomics is fast, highly sensitive, and can identify hundreds of lipids missed by other methods. However, Glycosphingolipids are an important lipid family that was out of the scope of shotgun lipidomics due to the lack of suitable analytical tools. The aim of my thesis was two‐fold. The first aim was the establishment of Glycosphingolipid identification and quantification by shotgun approach. This allowed us to perform lipidomic studies with unprecedented comprehensiveness (~300 lipid species from 15 different lipid classes) from low sample amounts and with minimal sample processing. The second was the application of this technology in studies of the role of lipids in several processes like vesicular carrier formation, cell polarization, protein delivery to the plasma membrane and viral budding. This work resulted in several findings. We found that there is sorting of sphingolipids and sterols into plasma membrane targeted vesicular carriers in budding yeast. When kidney cells change from a mesenchymal to an epithelial morphology there is a profound remodeling of their lipidome, with the synthesis of longer, more saturated, more hydroxylated, and more glycosylated sphingolipids. When these sphingolipids and sterols are depleted in epithelial cells, the apical transport in epithelial cells is impaired. These data strongly support the idea that lipid rafts play an important role in sorting and delivery of lipid and protein cargo to the plasma membrane. Finally, we found that the envelopes of vesicular stomatitis virus and Semliki forest virus assert little specificity in the incorporation of lipids from the plasma membrane. This weak specificity seems to be related to a combination of virus lipid bilayer asymmetry and curvature.