Lipids constitute the majority of metabolites in plasma. They stand out because of their structural diversity and the sheer number of molecular species. In-depth analysis by the consortium called LIPID Metabolites And Pathways Strategy (LIPID MAPS) revealed almost 600 distinct molecular species in six main mammalian lipid categories.
“The consortium is a multi-institutional effort to identify and quantitate all of the lipid species in mammalian cells and tissues, as well as to quantitate the changes in these species under different conditions,” says Edward A. Dennis, Ph.D., distinguished professor, University of California, San Diego and director of the LIPID MAPS Consortium. Lipids’ structural diversity gives rise to enormous variations in physiological functions. Undoubtedly, many lipid metabolites could be used as diagnostic tools or therapeutic targets.
But only with the advent of advanced lipidomic technologies can some of these ideas now become possible. The very diversity of lipids presented an ultimate challenge for development of wide-scale lipid-profiling methods, until finally the analytical platforms matured enough to enable analysis of lipid molecules in great detail.
“Lipidomics is defined as detailed analysis and global characterization of the structure and function of lipids in a living system. However, even this definition may be too simplistic to describe a complex world of lipid research and analysis,” comments Robert C. Murphy, Ph.D., university distinguished professor, University of Colorado.
“Tremendous technological advances of the past two decades brought forward both the big picture of lipid metabolism and targeted analysis of specific lipids. We can now probe biosynthesis, metabolism, and functional roles of any lipid of interest.”
Dr. Murphy’s work focuses on an arachidonic acid cascade. This fatty acid is a key inflammatory intermediate and its cycle is a popular target for drug development. “The global lipid analysis of the pathway metabolites may be crucial for discovery of new drugs or identifying potential toxicities,” continues Dr. Murphy.
“COX2 inhibitors affect conversion of arachidonic acid into prostaglandins and are highly efficient in reducing inflammation. However, following the withdrawal of Vioxx from the market, development of the whole class of these products was abandoned.”
Together with colleagues from the Personalized NSAID Therapeutics Consortium, Dr. Murphy is reinvestigating the basis of toxicity. The huge complexity of prostaglandin signaling means that most COX2 inhibitors are going to affect multiple components of the pathway. Understanding the interaction of the arachidonic pathway with other biochemical networks may offer more therapeutic benefits devoid of toxicities in the future.
“Since it is not as easy to create tags to follow lipids, MS-based lipid-imaging tools are the only way to show lipid localization inside the tissues,” says Dr. Murphy. Imaging MS becomes a molecular snapshot of an individual biological system, such as lung tissue.
For this project, a new cryoembedding agent was developed to enhance diffusion of lipids from the tissue into the matrix. Increased concentrations of unsaturated phospholipids (arachidonate) found at the airways may be a protective mechanism in removing ozone. Tying this snapshot with underlying biochemistry can help to shed light on many critical biological processes.
Sphingolipids are critically important for cell structure as components of lipid bilayers and mediators of cell recognition and signaling. “Sphingolipidome” is a very large class of molecules that vary widely in structural complexity and amounts.
“This complexity is daunting,” says Alfred Merrill, Ph.D., professor and Smithgall Institute chair of molecular cell biology in the School of Biology, Georgia Institute of Technology. “We literally need a pathway map to visualize this knowledge and to focus our attention on important nodes.”
As one approach, the team developed a tool that depicts differences in gene expression or metabolite amounts in a sphingolipid metabolic pathway heatmap. Comparison of the expression profiles of normal and cancer cells directs researchers’ attention to particular sphingolipids for subsequent analysis using targeted lipid profiling and tissue imaging mass spectrometry.
“This tool also works in reverse,” continues Dr. Merrill. “If we observe changes in metabolites, we can correlate them with the expression map to generate a hypothesis of why this is happening.”
Using this approach, Dr. Merrill and his colleagues confirmed that sulfatides are elevated in ovarian cancer. In this study, the pathway heatmap used gene expression data from human ovarian epithelial cells collected by laser capture microdissection. Indeed, transcriptional profile suggested that sulfatides may be elevated in cancer cells. MALDI imaging confirmed localization of sulfatides specifically to cancer cells.
“The power of this approach is that it combines structurally rigorous and quantitative mass spectrometry methods with others that provide complimentary information,” says Dr. Merrill. “Our hope is that this will help researchers see the big picture, and maybe find new biomarkers and therapeutic interventions. We will continue to try to understand sphingolipid metabolism as part of entire biological entities, not just its individual parts, which is a truly ‘omic’ endeavor.”