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

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 C breath testing in humans and other animals and describe the impact that contamination by ambient/background CO in the air can have on the accuracy of 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 δ C from breath samples mixed with ambient CO . Unfortunately, Keeling plots are not suited for C breath testing in common situations where (1) a subject's VCO is dynamic, (2) ambient [CO ] 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 [CO ] and the δ CO of ambient air as well as the diluted breath sample to back-calculate the δ CO in the CO exhaled by the animal. I validate this approach by titrating a sample of 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 δ C of exhaled gas of humans or other animals in real time without having to scrub ambient CO or rely on estimated values.