"The Nuts and Bolts of NMR-Based Metabolomics" (Powerpoint presentation: File size 4.6 MB)
Presented at the NIEHS Center for Environmental Health Sciences 5th Annual Conference, Napa, California, August 2003.

Currently, high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are the principal analytical platforms for metabolomic experiments. Both techniques enable a relatively rapid and simultaneous measurement of potentially hundreds of endogenous metabolites in tissue extracts and biofluids. Furthermore, due to the vast quantity of multi-dimensional data that can be generated, both approaches rely extensively upon statistical bioinformatics for the appropriate management and analysis of the datasets.

This presentation will describe the NMR-based metabolomic approach, including technical details pertaining to sample preparation, NMR analysis, and a brief introduction to multivariate data processing. It will highlight several advantages of the NMR approach such as minimal sample preparation, flexible sample formats, high-throughput analyses, and unbiased detection of low molecular weight organic metabolites. The principal disadvantage of relatively limited sensitivity, compared to MS methods, will also be discussed.

The results from several on-going experiments will be used to illustrate key points, with an emphasis on nutritional studies. These will include: (1) an investigation of withering syndrome in red abalone (Haliotis rufescens), which is a chronic muscle wasting disease that has impacted the abalone aquaculture industry in California. (2) A preliminary metabolomic study of plasma samples from Peruvian children, both before and after fortification with Fe and Zn. (3) An investigation of the metabolic changes occurring during development of Japanese medaka fish embryos (Oryzias latipes). This will highlight methods for summarizing and visualizing the similarities and differences between the metabolic profiles of stage-specific embryos, and will illustrate the concept of a 'developmental trajectory' through multi-dimensional metabolic space.

Physical, chemical and biological stressors can induce adverse biochemical effects within marine invertebrates, including alterations in gene expression, protein concentrations, and metabolic status. Whereas transcriptomics and proteomics have recently emerged as powerful tools for assessing the effects of stressors in the aquatic environment, the concept of metabolomics has yet to receive attention. This state-of-the-art approach involves the quantitative measurement of the time-dependent metabolic response of living organisms to various stimuli. High-resolution nuclear magnetic resonance (NMR) spectroscopy is particularly appropriate for investigating metabolic status, since potentially hundreds of endogenous metabolites can be quantified rapidly in tissues or biofluids, with minimal sample preparation.

Here we describe our preliminary study of withering syndrome in red abalone (Haliotis rufescens) using NMR-based metabolomics. These marine molluscs are an important aquaculture species along the West Coast of the United States. Recently, both wild and farmed abalone have been impacted by a fatal muscle withering syndrome (WS). Although this disease is of bacterial origin, recent studies suggest that the presence of the pathogen in combination with elevated seawater temperatures can synergistically stimulate the pathogenesis of WS. Before investigating the potential roles of environmental stressors in WS development, we first need to identify novel metabolic biomarkers of disease progression.

One-dimensional ¹H NMR spectra (at 500 MHz) of hemolymph and perchloric acid extracts of foot muscle and digestive gland tissue, from both healthy and diseased abalone have been recorded. Several metabolites have been identified and subsequently confirmed by two-dimensional NMR methods, including homonuclear correlation spectroscopy (COSY) and heteronuclear single quantum correlation spectroscopy (HSQC). Principal component analyses have revealed several differences in metabolite concentrations between healthy and diseased animals. These include decreased levels of valine, alanine, carnitine, glycine, tyrosine, phenylalanine and glycogen, and elevated levels of lactate and homarine in withered abalone. The identification of biomarkers will provide a powerful tool for characterizing several aspects of WS, including the effects of environmental stressors and the efficacy of feed-based antibiotic treatments that are currently under development. Additionally, this study confirms that NMR-based metabolomics is effective at revealing novel biomarkers in diseased marine invertebrates.