Research in this laboratory focuses on the basic biochemistry and pharmacological control of lipid mediators derived from both enzymatic and nonenzymatic pathways largely employing techniques of sophisticated mass spectrometry to address critical issues. The term lipid mediators is used here in the context that many of the compounds under investigation have potent and diverse biological activities that permit cells to intercommunicate with each other. For example, following exposure to reactive oxygen species new lipid molecules are formed from endogenous lipids by covalent alteration of their chemical structure and these new molecules cause cells in a tissue to respond to the oxidant stress. A major emphasis has continued to involve the use of mass spectrometry for both qualitative and quantitative investigations in this area of lipid biochemistry. Three specific areas are the current focus of activities in this laboratory and they include:
- Eicosanoid mediator biosynthesis
- Lipid MAPS
- Reactive oxygen and lipid biochemistry
Eicosanoid mediator biosynthesis
Arachidonic acid is an abundant polyunsaturated fatty acid present in all living mammalian cells that serves important roles both as a component of esterified membrane phospholipids as well as being a precursor for a large family of biologically active metabolites termed eicosanoids.
This laboratory has been investigating the role of leukotrienes which are 5-lipoxygenase (5-LO) derived metabolites of arachidonic acid synthesized both in normal physiology as well as pathophysiology. The formation of leukotrienes is tightly regulated within cells and yet only now are we becoming aware of some of the complex interactions that are taking place that ultimately lead to the release of the primary biologically active leukotrienes, leukotriene B4 and leukotriene C4. The outline of the biochemical pathway leading from cellular phospholipids to the formation of these eicosanoids is shown in Figure 1 as well as the chemical structures of the products as well as intermediates.
: Eicosanoid biosynthetic pathways
(click for a larger image)
Current investigations in the laboratory have centered around specific biochemical events which regulate production of leukotrienes. One particular area is the reacylation of arachidonic acid liberated by cPLA2α and transfer of arachidonic acid back into cellular phospholipids. The mechanisms involved and the various enzymes engaged along the pathway have not been fully characterized and recently this pathway was found to significantly control the final quantity of leukotrienes produced, for example in the human polymorphonuclear leukocyte (1). The identity as well as characteristics of the lysoglycerophosphocholine acyltransferase that accepts arachidonoyl CoA is a major current goal of the laboratory (Figure 2). These studies bring into focus the remodeling of arachidonic acid as well as the important role that CoA ester formation plays in leukotriene production in cells.
: Remodeling of arachidonic acid into
phospholipids and link to leukotriene production
The production of leukotrienes within tissues and even within cells is a more complicated process. The intracellular location at which leukotrienes are first synthesized, namely the subcellular location where the committed biochemical intermediate leukotriene A4 (LTA4) is generated, is now known to occur at the nuclear membrane (Figure 3). This important biochemical intermediate is also a reactive conjugated triene epoxide which has a very short chemical half-life (t½ < 3 sec). In spite of the fact that LTA4 is made deep within the cell and that it is chemically reactive, it can leave the synthetic cell and enter into a donor cell where the conversion of LTA4 into either LTB4 or LTC4 is completed by a process termed transcellular biosynthesis (2,3).
: Intracellular steps and subcellular location
of enzymes of the 5-lipoxygenase cascade
A major question being addressed in the laboratory at the present time is developing means by which one can assess the efficiency of transcellular biosynthesis and to compare the amount of leukotrienes made by this cell-cell interaction mechanism compared to the total quantity of leukotrienes made within a single cell.
In order to carry out this process, the production of leukotrienes is being studied in genetically modified mice (knock out mice) that have been lethally irradiated and have undergone bone marrow transplant (Figure 4). In this case, chimeric mice are made in which certain cells have one part of the 5-LO enzymatic cascade whereas other cells complete the enzymatic cascade by having either uniquely LTA4-hydrolase or LTC4-synthase. This chimeric model will permit investigation of transcellular biosynthesis efficiency in vivo through measurement of both enzymatic and nonenzymatic LTA4 products following relevant stimulation of the animal. An important role of mass spectrometry is that not only can the entire range of 5-LO products be measured, but also cyclooxygenase products measured at the same time in a lipidomics-type approach where an entire profile of eicosanoids is measured by LC/MS/MS techniques. Recently, the use of this lipidomics approach was used in studies of stimulation of bone marrow derived neutrophils from 5-LO -/- mice (4).
: Chimeric mice made from 5-LO -/- mice lethally irradiated then rescued using bone marrow from LTA4-H (-/-) or LTC4-S (-/-) mice. In this way, LTB4 or LTC4 production must be derived by transcellular biosynthesis.
An emerging strategy of biochemical investigations has been to involve the use of global techniques to assess the nature of particular biochemical elements such as DNA, RNA, or proteins and has led to a development of terms such as genomics and proteomics. This same strategy can be applied to lipids in an emerging field termed lipidomics which would involve the determination of both the types of lipids as well as quantity of lipids present within tissues or cells. This fundamental approach to biology requires a realistic assessment of what are and are not lipid substances. The Lipid MAPS consortium has suggested a definition of lipids as hydrophobic or amphipathic biomolecules that originate either in part by condensations of carbanion thioesters and/or carbocations of isoprene units (5). Dr. Murphy's laboratory is part of the large multi-university consortium that is building important infrastructure in lipid biochemistry, including development of methods to determine glycerolipids, phospholipids, steroids, sphingolipids, as well as fatty acids and their derivatives which are present in specific cells. The first initiative has been to define those lipids present in a macrophage cell line and assess what changes both qualitatively and quantitatively occur following activation of the toll-like 4 receptor in these cells (6). Detailed information of the Lipid MAPS initiative as well as unique tools for lipid mass spectrometry can be found at the Lipid MAPS website: www.lipidmaps.org
Further advancement of mass spectrometry into the challenges of lipid biochemistry is a major focus of Lipid MAPS core research in Dr. Murphy's laboratory. One aspect of this is the development of the mass spectrometer as a powerful imaging tool to assess spacial distribution of specific lipids within tissues. Towards this end matrix assisted laser desorption time-of-flight mass spectrometry is being employed to
define regional distributions of lipids within tissues (7). An example of this work is the image of the following mouse brain in terms of the distribution of a unique glycerophospholipid containing docosahexaenoic acid, namely 16:0a/22:6-GPCho (Figure 5). This phospholipid generates a positive [M+H]+ion at m/z 844.7 and when a 10μ thick section of rat brain is put on a MALDI mass spectrometer target surface and then the laser beam rastered across the tissue, the MALDI generated ions are analyzed by a quadrupole time-of-flight mass spectrometer to generate a 4-dimensional database. The four dimensions are the XY coordinates of the laser spot, mass-to-charge ratio of detected ions and abundance of that ion. Using 100 μM steps for the MALDI raster process, a complete image of the tissue can be obtained and significant regional differences in this glycerophosphocholine lipid can be observed. This emerging technology is being developed as a robust strategy to look at the distribution of lipids in complex tissues (7).
: (A) Image of a rat brain [M+H]+ formed by the abundance of the m/z 844.7 from the phosphatidylcholine 16:0a/22:6-GPCho. Red is the most abundant and blue is low to no abundance. (B) Collisional activation of m/z 844.7 from the rat brain tissue (red zone) and corresponding product ions, including m/z184 characteristic of phosphocholine lipids.
Reactive oxygen and lipid biochemistry
A considerable body of information has been generated concerning the formation of biologically active lipids through tightly regulated enzymatic cascades such as the production of prostaglandins and leukotrienes from arachidonic acid.
However, lipids, especially lipids which are in the outer plasma membrane of cells, serve the cell as the first barrier to reactive oxygen species in that the lipids themselves can be the targets of chemical reactions if sufficiently reactive species are exposed to a cell (Figure 6). While in many cases, this leads to the elimination of a reactive chemical that could interfere with intercellular biochemistry, in some cases, biologically active products are formed from components of the plasma membrane barrier that can interact with cellular receptors or intermediary biochemistry and in this way mediate toxicity of the chemical reactive oxygen species.
: The plasma lipid bilayer as the first line of cellular defense against reactive oxygen species.
In the past, studies of the molecular basis for the toxicity of ozone led to the structural characterization of several novel products, one derived from glycerophosphocholine and others from cholesterol (Figure 7) that have significant biological activities (8,9). Mass spectrometry plays a central role in being able to structurally characterize products of reactive oxygen species, in this case ozone, with naturally occurring membrane lipids. An understanding of the reaction chemistry is currently under investigation as well as the potential role of plasmalogen phospholipids as some of the initially ozonized products. Plasmalogen phospholipids have a unique vinyl ether substituent at the sn-1 position of glycerophosphoethanolamine lipids (10).
Another reactive oxygen specie under investigation includes acrolein, a major component in cigarette smoke as well as an environmental reactive oxygen species. Unique chemical reactions with aminophospholipids have been discovered using mass spectrometry and the mechanism of action of the toxicity of acrolein appears also to involve interaction with lipid mediator biosynthesis. These studies are only possible through the use of sophisticated mass spectrometry to assess covalent modifications of naturally occurring lipid substances because of the high sensitivity and structural information content of this biophysical tool.
: Reaction of ozone with lung surfactant cholesterol to form biologically active oxysterols.
Students and postdoctoral fellows in the laboratory of Dr. Robert Murphy obtain extensive experience not only in the fundamentals of the use of sophisticated mass spectrometry, but also in the application of this analytical technology to important problems of lipid biochemistry.