April 1, 2008 (Vol. 28, No. 7)

Susan Aldridge, Ph.D.

HPLC, Mass Spec, and Nuclear Magnetic Resonance Are Among Elucidating Technologies
Metabolomics is the newest of the omics approaches and perhaps the one that is the closest reflection of what is actually taking place in the living cell. Researchers are looking to this platform, which draws on many different technologies, for new insights into biochemical pathways and hope it will thereby yield products such as biomarkers and new drug targets. Delegates at “metabomeeting 2008,” which will take place later this month in Lyon, France, will present a range of specific contributions and approaches to metabolomics research.

John P. Shockcor, Ph.D., director of metabolic profiling business development at Waters, says that small molecule profiling (as metabolomics is sometimes known) is about four simple questions: what has gone up, what has gone down, what is missing, and what is new?

Waters’ HPLC/UPLC and mass spectrometry technologies can help answer these questions by providing information about the composition of complex molecular mixtures. Thereafter, it is the role of the biochemist to integrate this with proteomics and genomic data to understand the context of these changes that may, or may not lead to the identification of a useful biomarker.

Dr. Shockcor will present Waters’ recent work on ion mobility mass spectrometry—a technology that is the basis of the company’s Synapt high-definition MS (HDMS) system. Ion mobility refers to the diffusion of an ion through a gas under the influence of an electric field. The rate of this diffusion depends upon the charge on the ion and its rotational cross-sectional area—the latter being more relevant to metabolomic work.

The Waters system places an ion mobility separation stage just prior to the mass analyzer, and when combined with UPLC, a type of 2-D separation is achieved. The technology allows fine separation of ions with similar masses, and the output gives an accurate mass compared to the nominal mass, which is the more usual output of MS techniques.

Ion mobility with MS is a tool that Waters has pioneered, according to Dr. Shockcor, and other companies are now following its lead. “Waters will continue to develop ion-mobility mass spectrometry beyond the first generation and will focus upon the utility of accurate mass for analysis of complex mixtures,” he adds.

Synapt HDMS is already being used for a wide range of small molecule studies. Dr. Shockcor will discuss its application to lipidomics at the meeting. “So many of the chronic diseases that plague us, heart disease and diabetes, for instance, are linked to the regulation and absorption of lipids,” he explains. “There is a massive subset of tens of thousands of lipids that are extremely challenging to analyze in metabolomic studies. We are developing tools for looking at these in experiments that have not previously been possible.”

Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) is another key technology for metabolomics, and Norbert Lutz, Ph.D., research professor at the Faculty of Medicine of the Université de la Méditerranée, will present some cutting-edge applications.

“Metabolomics is, broadly speaking, the comprehensive study of metabolism in biological tissue,” he says. Such studies are not focused on individual metabolic questions but cover a vast range of metabolic events. Nor is metabolomics restricted to a specific analytical method or statistical tool.

“Metabolomics starts with measuring a large number of metabolite concentrations without selecting a specific pathway or event,” he explains. “It is of interest because it deals with biochemical events that are directly linked to the performance of cells, therefore providing more direct insight into the functioning and malfunctioning of cells, tissues, or organs than genomics or proteomics.”

Studying a relatively large number of molecules through this approach can reveal the interdependence of biochemical processes and pathways and represents the complexity of cellular and tissue activity better than the more traditional approach of looking at only a few molecules. These are the important goals of biomedical research, he adds, and “it is hoped that metabolomics can lead to better diagnostics and the discovery of novel forms of therapy.”

At “metabomeeting,” Dr. Lutz will present some findings from his integrated metabolomic NMR spectroscopy studies. NMR allows the simultaneous determination of the nature and amount of a number of metabolites in intact tissue, cell suspensions, perfused cells, and tissue extracts (the latter giving the highest resolution).

NMR on biopsy samples is a more recent development. Dr Lutz’ studies involve the integration of NMR spectroscopy with findings from other technologies and sources such as MRI, microscopy, enzyme activity measurements, and clinical examination. In the future, metabolomics should, ideally, be combined with genomics and proteomics data to give a more comprehensive picture.

The first of Dr. Lutz’ studies concerns a form of mental retardation called Rett syndrome, for which a transgenic mouse model exists. Here, metabolic fingerprints derived from brain NMR indicated reduced growth and altered activity and volume regulation of brain cells, which have been correlated with diminished brain size and energy uptake. Integrating these findings is now helping elucidate the molecular mechanisms of Rett syndrome.

In the second study, Dr. Lutz and his colleagues uncovered metabolic indicators of resistance to anticancer treatment in thymic lymphoma cells. The resistant cells exhibit altered glucose and glutamine metabolism compared to drug-sensitive cells.
The metabolomic findings were correlated with reduced loss of specific enzymes from mitochondria. Also, the resistant cells had more efficient phospholipid turnover. Animal experiments showed that resistant cells develop tumors more readily than sensitive cells. Therefore, treatment resistance and tendency to develop tumors may be linked through the same metabolic abnormalities.

Martial Piotto, Ph.D., NMR application manager of Bruker Biospin, is also scheduled to speak at the meeting on the metabolic analysis of human brain biopsies in a medical environment using HRMAS (high-resolution magic angle spinning) NMR.

HRMAS can be considered a hybrid between solid-state NMR and classical-solution NMR that is suited to examining complex tissue samples.

The bioinformatics aspect of metabolomics will be presented by Irena Spasic, Ph.D., of the Manchester Centre for Integrative Systems Biology (MCISB) at the University of Manchester. She says that small changes in the activities of individual enzymes can lead to large changes in metabolite concentrations.

This observation is backed by a wealth of experimental and mathematical evidence and means that metabolites are amplified relative to changes in the transcriptome, proteome, or gross phenotype. Therefore, metabolomics, being downstream of transcriptomics and proteomics, lends itself to biological analysis more readily, and the number of metabolites is more tractable. “These facts establish metabolomics as a valuable tool for the purposes of functional genomics, biomarker development, and systems biology,” Dr. Spasic says.

As with the other omics, metabolomics can only be of value if dedicated and effective data-management systems are in place. At “metabomeeting,” Dr. Spasic will present MeMo, a data model she developed to support effective and efficient computational analysis of large volumes of experimental data generated through the use of high-throughput metabolomics technologies.

The development of MeMo arose from the need for storage and analysis of the GC-MS data generated for the metabolic footprints of the genome-wide collection of yeast mutants. MeMo was implemented as a relational database to support the storage, management, and dissemination of large amounts of curated metabolomic data and related metadata.

In order to make room for incoming methods, instruments, and data resources and to support ongoing standardization efforts, MeMo was designed to be modular and easily extensible. These properties, in addition to the MeMo metadata description module, make the systems portable between the omics domains, according to Dr. Spasic.

MeMo has continued to evolve to reflect the information needs at MCISB, which include storing and analyzing various types of omics data in an integrated fashion. The metadata module of MeMo is used to store the core metadata common to various experiments performed. The experimental data produced is mainly stored in a MeMo database.

Since the introduction of MeMo, Dr. Spasic’s team has used Pierre, a model-driven, user-interface development tool, to provide a user interface to MeMo, including a web interface, a stand-alone application, a command-line service, and a web service. Apart from providing access to domain experts, the MeMo interface provides programmatic access to the data through the use of web services. “This is particularly important in the context of systems biology, whose integrative nature poses a typical problem of dealing with data from disparate sources,” she says.

“We are now well placed to start putting together an experimentally verified functioning model of the human metabolic network. This is the grand challenge of metabolic systems biology.”

Finally, Alexander Amberg Ph.D., head of metabonomics, drug safety evaluation at sanofi-aventis, will talk about the application of metabolomics in preclinical drug-safety evaluation within the PredTox consortium. This is a major initiative between the European Commission, industry, and academia with the goal of improving drug safety, where metabolomics can make a contribution to ADME studies.

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