A robust high performance metabotyping strategy of C.elegans

Benjamin Blaise¹, Bénédicte Elena¹, Jean Giacomotto², Mohamed N. Triba², Laurent Ségalat², Marc-Emmanuel Dumas¹, Lyndon Emsley¹

¹Centre de Résonance Magnétique Nucléaire à Très Hauts Champs FRE 3008 and Laboratoire de Chimie, UMR 5182, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon, France.

²CGMC, UMR 5534 CNRS/Université Lyon-1 Claude Bernard, Bâtiment Mendel, 43 boulevard du 11 Novembre, 69622 Villeurbanne Cedex, France.

The nematode worm Caenorhabditis elegans was the first animal model whose genome was sequenced. The interest in this model organism is to monitor a wide range of biological process and to characterize genetic mutations responsible for human disease. However, many genetic mutations do not display any obvious morphological or behavioral phenotype under classical observations. We show that ¹H high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance spectroscopy is able to identify and quantify a large number of C. elegans metabolites. In association with biostatistics / chemometrics, we characterize metabolic fingerprints associated to genetic mutations. Here in this study, we develop, validate and apply a metabonomic protocol based on ¹H HRMAS NMR spectroscopy of intact C.elegans worms, in order to investigate the metabolic signature induced by mutations of oxidative stress enzymes. To address the reproducibility and robustness issue of C.elegans metabolic profiling using ¹H HRMAS NMR, we have tested and controlled several biological and technological factors, namely the effects of strain, age, sample preparation and ¹H HRMAS NMR analysis itself. A metabonomic analysis, i.e. the hypothesis-free interpretation of biological NMR data by multivariate statistics, is particularly suited to understand pathophysiological perturbations in C. elegans mutants. Supervised multivariate statistics reveal a remarkable discrimination between the N2 strain (WT) and mutants of oxidative stress: SOD-1 mutants sod-1(tm776), and Catalase-1 mutants ctl-1(ok1242). We identify a metabolic phenotype (metabotype) significantly associated with these mutations: a general reduction of fatty acyl resonances from triglycerides, corresponding to a compensative strategy for regulation of oxidative stress (1). On top of that we control several confounding factors that can mislead the biological interpretation such as technological (NMR stability, user effect), or statistical (bactch effect) factors. It results that ¹H HRMAS is a robust strategy to investigate subtle metabolites variations in C.elegans induced by genetic or biological processes, leading to functional genomics of model organisms at the systems biology level.

(1) Blaise, B.J. et al., Metabotyping of Caenorhabditis elegans reveals latent phenotypes. Proc Natl Acad Sci U S A 104, 19808-12 (2007).