Environmental Metabolomics Using 9¹ H-NMR Spectroscopy

Environmental metabolomics is a subdiscipline of metabolomics and focuses on the study of metabolic changes in organisms in response to environmental challenges. This approach is ideal for studying multiple species within an ecosystem because it is not dependent on knowledge of an organism's genome. Unbiased measurements of an organism's metabolic composition can in principle be used to identify novel biomarker profiles and modes of action of stressors. This chapter presents protocols for the extraction of metabolites from biological samples, the measurement of metabolites using ¹H nuclear magnetic resonance (NMR) spectroscopy, and finally the analysis of the metabolic data using multivariate statistical methods. First, the preparation of biofluids (e.g., blood and urine) for NMR analysis is described together with a methanol-chloroform protocol for extracting metabolites from tissue samples. Next the NMR methods are presented, comprising a standard one-dimensional (1-D) ¹H-NMR method and a two-dimensional (2-D) ¹H-¹H J-resolved NMR experiment. The advantages and limitations of each method are discussed. Finally, two methods for analyzing the multivariate metabolic NMR data are presented. These include a traditional fingerprinting approach that comprises of a spectral preprocessing step followed by multivariate statistical analysis. Although reliable and proven, this method often produces results in terms of unidentified metabolites that are of limited value to the biologist. The second and newer method is based on metabolic profiling in which NMR spectra are deconvoluted into a list of metabolites and their concentrations. Although more biologically insightful, this latter approach can prove labor intensive.