Agilent Measurement Journal - Issue 5 - 2008 - (Page 71)

A After many years behind the scenes, lipids are making a spectacular comeback by being thrust into the limelight of biomedicine. This renewed excitement in lipids has been sparked by a series of convergent discoveries in the ﬁelds of biochemistry, cell biology and receptor pharmacology. Each of these discoveries is revealing the various roles played by lipid messengers and their receptors in health and disease. Most of these discoveries have been fueled by progress in techniques, such as mass spectrometry and atomic-force microscopy, which have opened experimental opportunities that were virtually unthinkable just ten years ago. Indeed, we are at a point today where a major goal is within our reach: proﬁling large-scale changes in lipid composition or determining the topographical distribution of individual lipid species in just a few cells. The ability to proﬁle the lipid composition of biological samples is important in disease diagnosis and drug discovery. However, constraints imposed both by the topological localization and small quantities of most biologically active lipids now demand the development of novel analytical approaches, and in particular, liquid chromatography mass spectrometry (LC/MS) techniques for high-sensitivity lipidomics. The research work described in this article was performed in collaboration with the Department of Pharmacology, University of California Irvine (UCI). It was supported by an Agilent Technologies Foundation grant. One of the challenges, for example, involves lipid metabolites of low abundance — which include many bioactive lipid messengers. These metabolites may remain undetected using the MS imaging technique due to suppression by more abundant constituents (e.g., phospholipids). Also, absolute analyte quantiﬁcation is often difﬁcult due to the lack of appropriate internal standardization. Consequently, the main analytical challenge facing MS imaging is its current lack of sensitivity and accurate quantiﬁcation, both of which are important to the application of this technology to the understanding of neurological and psychiatric diseases. We have begun to address these challenges working at the forefront of measurement technologies through Agilent’s Program for University Research and with ﬁnancial support from the Agilent Technologies Foundation. As described in the following paragraphs, the bioanalytical solution that we developed involves the use of laser microdissection (LMD), in combination with microﬂuidics LC coupled to MS, for highspatial resolution and high-sensitivity quantiﬁcation of trace amounts of lipid molecules in brain microstructures. LC/MS system: Enabling lipidomic analyses of brain macrostructures The hippocampus is a brain structure that plays a central role in memory and spatial navigation. Hippocampal degeneration is a key element in the pathogenesis of Alzheimer’s disease. For the analysis of lipids in the hippocampus, a general workﬂow consists of slicing a frozen rat brain with a cryostat and then manually punching out hippocampal subregions (Figure 1a). The punches are homogenized in a small volume of cold methanol (0.3 ml) containing appropriate internal standards. The lipids are extracted using a mixture of chloroform and water (2:1; vol:vol). The organic layer is then collected and analyzed by LC/MS. In the example provided in Figure 1b, we separated multiple lipid classes using a small-particle-size column (1.8 µm). This allowed both high-throughput and high-resolution analysis. Novel analytical approaches to lipidomic analysis In biological tissues, many bioactive lipids are present only in trace amounts. As a result, their measurement represents a major analytical challenge. Furthermore, most analytical approaches do not take into account the topological distribution of lipids in tissues and therefore often miss critical information. High-spatial resolution analyses of lipid metabolites would enable scientists to fully characterize the state and functionality of biological tissues. Topographical analysis by MS imaging, as detailed in the sidebar, has shown great promise in this area. However, the technique presents some limitations that must be addressed. Agilent Measurement Journal 71

Table of Contents Feed for the Digital Edition of Agilent Measurement Journal - Issue 5 - 2008

Agilent Measurement Journal Issue 5 Finding a New Path when Assumptions Break Down Contents Testing MIPI D-PHY Protocols Oscilloscope Firmware Update DNA Replication Joining the LTE/SAE Trial Initiative Testing Proteins Praise for DC Power Analyzer Scripting Features for AMDS Testing Hybrid PCBA Systems Improving the Gene Expression System Workflow Turning a “Good Enough” Test Strategy into One That’s Reliable, Repeatable and Traceable Understanding the Effects of Limited-Bandwidth Channels on Digital Data Signals Introducing the 3GPP LTE Downlink Validating the 26 Validating the Physical and Protocol Layers in DDR Memory Interfaces Understanding Total Jitter Measurements of Low Probabilities Using Behavioral-Model Simulation to Accurately Predict First-Order PLL Performance Creating Synchronous High-Frequency Sampling across Multiple Digitizers Overcoming the Challenges of Testing FlexRay Networks Understanding Operating Life and Repeatability of Electromechanical Switches and their Effect on Total Cost of Ownership Implementing Micro and Nano LC/MS Techniques for High Sensitivity Lipidomics Analysis

Agilent Measurement Journal - Issue 5 - 2008

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