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Feature Articles : Jun 1, 2013 (Vol. 33, No. 11)

Deciphering the Mysteries in Lipid Biology

  • Kate Marusina, Ph.D.

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.

Sphingolipid Profiling

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.”

Eicosanoid Signatures

Mounting evidence indicates that saturated fatty acids amplify inflammation. Fatty acids can activate G-protein–coupled receptors and initiate the arachidonic acid cascade. This in turn leads to production of eicosanoids, lipid mediators of inflammation.

“Global lipid analysis is indispensable in quantitation of all products in the inflammatory cascade,” says Dr. Dennis. “One of the major achievements of the LIPID MAPS consortium is development of over 500 standards covering eight main lipid categories in plasma, as well as 150 authentic eicosanoid standards. Now we can follow production of eicosanoids in response to inflammatory stimuli and correlate the molecular profiles with physiological manifestations of inflammation.”

In the study of inflammation caused by Borrelia (an agent of Lyme disease), the team identified a number of unusual eicosanoids, including resolvins and protectins thought to help resolve inflammation.

“We are just beginning to explore ways to increase the levels of beneficial anti-inflammatory eicosanoids,” says Dr. Dennis. “By applying lipidomic tools to model systems we can follow eicosanoid fluxes in response to inflammatory stimuli. Then we overlay the effects of dietary supplements.”

While dietary fish oil, rich in omega-3 fatty acids, was long known to elicit anti-inflammatory effects, the exact mechanism of action was not understood. Using the comprehensive analysis of the whole lipid cascade, Dr. Dennis and colleagues were able to demonstrate the direct mechanistic effects of omega-3 fatty acids on the production of mediators.

“Current technological achievements in lipidomics offer incredible opportunities to understand diseases and then to find mechanisms to treat them,” explains Dr. Dennis. “Investigation of hyperalgesia, or hypersensitivity caused by previous trauma, such as a burn or mechanical injury, is an example of such lipidomic discovery.”

Peripheral injury generates eicosanoid “memory signatures” in neurons. A new discovery showed that hepoxilins, metabolites in these signatures, act on well-known receptors of calcium signaling. These findings now enable development of anti-inflammatory therapeutics targeting these receptors.

Global Triglyceride Profiles

“The field of lipidomics is opening up,” says Bruce Kristal, Ph.D., associate professor of surgery, department of neurosurgery at Brigham and Women’s Hospital/Harvard Medical School. “Development of enabling technologies provides an opportunity to examine broad lipid profiles while maintaining the concurrent ability to accurately identify and quantify each one. We can now reach deep into the lipidome in a single MS run.”

Dr. Kristal’s team applies global lipidomic profiling to probe lipidomic changes mediated by diet and biological processes. Although LIPID MAPS developed many standards for lipid identification, these remain insufficient to cover the entire structural diversity. “We often have to identify the metabolites de novo, using mass, retention time, and fragmentation pattern as inputs. Combined with biological knowledge, this gives us a pretty good idea what we are looking at,” says Dr. Kristal.

This discovery paradigm was most recently adapted to study premature infants. This high-risk population commonly suffers from GI morbidities. The ability to recognize problems with intestinal functions in early infancy would help to optimize their care. Lipids were collected from the fecal matter using an extraction technique developed specifically for this purpose.

“The fecal lipidome is very complex,” continues Dr. Kristal. “We found over 300 known endogenous lipid metabolites, as well as others that seem to be quite different from plasma species. Most of these are not yet known.” The profiling method developed is broadly applicable to biological samples where lipids play a key role. This includes the analysis of triglycerides, which is standard in medicine, but provides a rather crude disease diagnostic based on the sum total of triglycerides.

“This will change in the near future,” predicts Dr. Kristal. “We will conceivably profile all 120+ triglycerides and follow each triglyceride along with, for example, dietary changes in each individual patient.”

Sterol Analytics

Cholesterol is the most well-known member of sterol family of lipids, and it serves as a precursor of many key molecules. Sterols participate in nearly every important cellular function, but are mostly recognized for their causative effect on atherosclerotic plaque.

“We are after the undiscovered roles of sterol metabolites,” says David Russell, Ph.D., professor of molecular genetics, University of Texas Southwestern Medical Center. “Mutations in genes encoding sterol metabolic enzymes lead to serious liver and endocrine problems. And yet, until now we did not possess a robust ability to evaluate all cholesterol precursors and derivatives at once.”

Dr. Russell’s team developed a method to reproducibly detect and analyze >60 sterols in just 100 microliters of blood, plasma, or urine. “To develop this method, we first reviewed the collective scientific wisdom for sterol purification. Next, we optimized each analytical step using dozens of different sterol standards to ensure maximum recovery at each step,” continues Dr. Russell. “The initial method development was lengthy, but now we have a rapid and quantitative method to analyze a majority of sterols in human plasma.”

This analytical method was deployed to evaluate an unbiased population sample, the Dallas Heart Study. The sample consists of “typical” residents of Dallas county aged 18 to 65, who underwent multiple blood tests, diagnostic imaging, and health surveys. The team focused on 20 consistently detected sterols and created sterol profiles for over 3,000 samples. “Our approach combines sterol analytics with SNP genotyping and clinical profiles,” adds Dr. Russell. In collaboration with Merck, the Russell team is searching for correlations between sterol profiles and genetic alleles. If found, such correlations may reveal a new biomarker of disease.

Simultaneously, the team is evaluating samples from patients with clearly defined diseases, such as West Nile Virus, common influenza, and septicemia. The researchers hope that sterol profiles may provide a means to detect an infection before its clinical manifestation.