​METABOLOMICS : GOING BEYOND THE LIMITS OF CLASSICAL APPROACHES

Understanding how diet, drugs, toxic compounds, or disease disrupt metabolism remains a major challenge. Until now, metabolic studies have largely relied on targeted isotopic methods, often radioactive, which consist of tracking a specific precursor within a given metabolic pathway, an approach that is “effective, but limited…," points out Annelaure Damont (SPI/DMTS), first author of the study, who continues : “…because it assumes prior knowledge of which biological pathways are affected. Yet the effects of a treatment or a pathology can be multiple, unexpected, and sometimes far removed from the initial target."

The ambition of this work was to broaden the ability to observe metabolism globally and without prior assumptions by labeling all carbon-containing molecules in an organism, thereby generating an isotopic map of metabolism. This represented an extraordinary challenge, which the authors addressed thanks to a conceptually simple but technically demanding idea: newborn mice were fed for six weeks with a mixture of ¹³C-labeled spirulina and ¹³C-labeled wheat flour, corresponding to a diet whose carbon content was enriched to 97.2% in ¹³C, a stable and non-radioactive isotope of carbon. While ¹³C-labeled spirulina is commercially available from a few fine-chemicals suppliers, the production of ¹³C-labeled wheat proved far more complex and required the expertise of the Phytotec platform, specialized in plant cultivation under controlled atmospheric conditions (see box).

The ¹³C-fed animals developed normally, and their urine was collected daily. Three control mice were treated in the same way but received a non-enriched diet. Metabolomic analyses and isotopic tracing were performed on urine samples by high-resolution mass spectrometry (SPI/DMTS, see box). Comparative analysis of the ¹²C and ¹³C samples enabled the unambiguous identification of 238 metabolites, whose ¹³C-labeling profiles were subsequently monitored over a 39-day period. The authors observed that numerous metabolites reached labeling levels exceeding 90% in ¹³C.

​Protocol for in vivo 13C labelling. © Damont et al., Metabolomics, 2025

​​ Circos representation of the 13C content of the 128 ​metabolites identified at 4, 15 and 39 days​

By demonstrating not only that mice can tolerate a ¹³C-labeled diet for nearly six weeks, this study also reveals the dynamics of a large fraction of the metabolome in a reliable and robust manner. It provides valuable information that comprehensively covers metabolic pathways, in contrast to targeted approaches, and represents an unprecedented methodological achievement.

POTENTIAL IMPACTS IN MEDICINE AND TOXICOLOGY​
Nutrition, toxicology, and pharmacology: the potential fields of application of such an approach are numerous. Ultimately, the use of fluids or tissues fully labeled with ¹³C could make it possible to detect metabolic disorders more precisely and quantitatively from a simple blood, urine, or biopsy sample taken from a living organism. It would offer the opportunity to simultaneously identify both the expected effects of a drug and its unwanted side effects, as well as to characterize the overall impact of a diet or an environmental toxin.
This work would not have been possible without the close collaboration between researchers from BIAM and DMTS (Joliot). All the results obtained highlight, if proof were still needed, the importance of fundamental research, without which applied research could not exist. By demonstrating that a whole-body isotopic labeling is both feasible and exploitable, the scientists provide their community with a tool that makes it possible to envision new applications for a better understanding of living systems.

Joliot contacts :  Annelaure Damont (annelaure.damont@cea.fr) ; Éric Ezan (eric.ezan@cea.fr) ; François Fenaille (francois.fenaille@cea.fr)