13C-Breath testing in animals: theory, applications, and future directions
The carbon isotope values in the exhaled breath of an animal mirror the carbon isotope values of the metabolic fuels being oxidized. The measurement of stable carbon isotopes in carbon dioxide is called 13C-breath testing and offers a minimally invasive method to study substrate oxidation in vivo. 13C-breath testing has been broadly used to study human exercise, nutrition, and pathologies since the 1970s. Owing to reduced use of radioactive isotopes and the increased convenience and affordability of 13C-analyzers, the past decade has witnessed a sharp increase in the use of breath testing throughout comparative physiology—especially to answer questions about how and when animals oxidize particular nutrients. Here, we review the practical aspects of 13C-breath testing and identify the strengths and weaknesses of different methodological approaches including the use of natural abundance versus artificially-enriched 13C tracers. We critically compare the information that can be obtained using different experimental protocols such as diet-switching versus fuel-switching. We also discuss several factors that should be considered when designing breath testing experiments including extrinsic versus intrinsic 13C-labelling and different approaches to model nutrient oxidation. We use case studies to highlight the myriad applications of 13C-breath testing in basic and clinical human studies as well as comparative studies of fuel use, energetics, and carbon turnover in multiple vertebrate and invertebrate groups. Lastly, we call for increased and rigorous use of 13C-breath testing to explore a variety of new research areas and potentially answer long standing questions related to thermobiology, locomotion, and nutrition.