CO2 scrubbing, zero gases, Keeling plots, and a mathematical approach to ameliorate the deleterious effects of ambient CO2 during 13 C breath testing in humans and animals

Abstract

¹³ C breath testing is increasingly used in physiology and ecology research because of what it reveals about the different fuels that animals oxidize to meet their energetic demands. Here I review the practice of ¹³ C breath testing in humans and other animals and describe the impact that contamination by ambient/background CO₂ in the air can have on the accuracy of ¹³ C breath measurements. I briefly discuss physical methods to avoid sample contamination as well as the Keeling plot approach that researchers have been using for the past two decades to estimate δ¹³ C from breath samples mixed with ambient CO₂ . Unfortunately, Keeling plots are not suited for ¹³ C breath testing in common situations where (1) a subject's VCO₂ is dynamic, (2) ambient [CO₂ ] may change, (3) a subject is sensitive to hypercapnia, or (4) in any flow-through indirect calorimetry system. As such, I present a mathematical solution that addresses these issues by using information about the instantaneous [CO₂ ] and the δ¹³ CO₂ of ambient air as well as the diluted breath sample to back-calculate the δ¹³ CO₂ in the CO₂ exhaled by the animal. I validate this approach by titrating a sample of ¹³ C-enriched gas into an air stream and demonstrate its ability to provide accurate values across a wide range of breath and air mixtures. This approach allows researchers to instantaneously calculate the δ¹³ C of exhaled gas of humans or other animals in real time without having to scrub ambient CO₂ or rely on estimated values.