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

13C 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 13C 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 13C 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 δ13C from breath samples mixed with ambient CO2. Unfortunately, Keeling plots are not suited for 13C 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 δ13CO2 of ambient air as well as the diluted breath sample to back‐calculate the δ13CO2 in the CO2 exhaled by the animal. I validate this approach by titrating a sample of 13C‐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 δ13C of exhaled gas of humans or other animals in real time without having to scrub ambient CO2 or rely on estimated values. [ABSTRACT FROM AUTHOR]