The magnitude of the naturally occurring isotopic enrichment of 13 C in exhaled CO 2 is directly proportional to exercise intensity in humans Marshall D. McCue ⁎, Celeste A. Passement, Miranda Rodriguez Department of Biological Sciences, St. Mary's University, One Camino Santa Maria, San Antonio, TX 78228, United States abstract article info Article history: Received 19 June 2014 Received in revised form 26 August 2014 Accepted 28 August 2014 Available online 25 October 2014 Keywords: Breath testing Fatty acids Heart rate Isotope fractionation Lipid oxidation Metabolic rate Stable isotope Crossover effect Respiratory exchange ratio Ventilation Due to various biochemical fractionation processes during lipid synthesis, the lipid molecules in the body contain substantially lower concentrations of 13 C than the nonlipid molecules. Because of the isotopic differences between these two endogenous nutrient pools, any shift toward nonlipid fuel oxidation would be expected to in- crease in the δ 13 C of the exhaled breath. Interestingly, the possibility of whether or not an exercise-induced change actually occurs has been debated in literature for over two decades and researchers have still not reached a consensus. We measured ventilatory and metabolic variables before, during, and after exercise in forty-eight adults (n = 25 females, n = 23 males; 20.1 ± 1.9 years) assigned to either a resting treatment or one of three exercising treatments where they maintained a heart rate of 130, 150, or 170 bpm for 56 min. We found that the mean metabolic rates of the exercising groups increased 4.4-fold, 6.1-fold, and 7.7-fold above resting values, respectively. Exercise caused small increases in respiratory exchange ratios (e.g., from 0.83 ± 0.08 to 0.86 ± 0.10) indicative of increased carbohydrate oxidation, but these changes were too variable to be reliably correlated with exercise intensity. In contrast, the δ 13 C of the exhaled breath increased by 0.62 ± 0.19‰, 1.14 ± 0.29‰, and 1.79 ± 0.50‰, respectively, for the three groups and was significantly correlated with the intensity of exercise. We also show that the isotopic difference of the lipid and nonlipids of the body is similar (~ 2.7‰) even when consuming bulk diets that are isotopically distinct (N 8‰). If not corrected for, these exercise- induced changes in δ 13 C of the breath would be sufficiently large to skew the results of studies investigating the oxidative fates of exogenous nutritional supplements. © 2014 Published by Elsevier Inc. 1. Introduction 1.1. Naturally occurring differences in δ 13 C in the body The δ 13 C-values of the lipids in the bodies of organisms ranging from bacteria to humans are known to be lower than the nonlipid molecules (i.e., carbohydrates and proteins; reviewed in Hayes, 2001). The magni- tude of the isotopic depletion of the lipids ranges from approximately 1–12‰ (Hayes, 2001; Post et al., 2007; Vollaire et al., 2007; Doronin et al., 2012) and may be influenced by carbon source (Abraham et al., 1998; Cifuentes and Salata, 2001), body temperature (DeNiro and Epstein, 1977), and nutritional history (Gaye-Siessegger et al., 2004; Barnes et al., 2007) of the organism. The particular biochemical mecha- nisms responsible for this ubiquitous difference in natural abundance of 13 C are not fully understood (Gannes et al., 1998; Hayes, 2001; Gaye-Siessegger et al., 2004) yet evidently occur in both autotrophs and heterotrophs (Park and Epstein, 1961; Jacobson et al., 1970; O'Leary et al., 1992; Hayes, 2001; Chikaraishi et al., 2004). Several bio- chemical processes have been identified as potential branch points for this isotopic fractionation in animals, and the most comprehensive studies conclude that the 13 C depletion in the lipid pool results from one or more of the following processes: 1) discrimination during the conversion of 13 C-pyruvate to acetyl-CoA (DeNiro and Epstein, 1977; Monson and Hayes, 1982; Blair et al., 1985; Melzer and Schmidt, 1987), 2) discrimination of 13 C-acyl groups by carrier proteins during fatty acid elongation (Monson and Hayes, 1980, 1982), and 3) discrimi- nation of 13 C-fatty acids during esterification during synthesis of triacyl- glycerols (Monson and Hayes, 1980). The 13 C-values of exhaled CO 2 reflect the 13 C-values of the organic substrates being oxidized by an animal (Schoeller et al., 1980). Similarly, the 13 C-values of the body tissues reflect the 13 C-values of the bulk diet (DeNiro and Epstein, 1978). However, because the relative lipid content of the body, and hence the bulk δ 13 C values, can vary widely among individuals and within individuals over time (Schoeller et al., 1984), researchers measuring stable isotopes often consider the difference in δ 13 C between lipid and lean tissues to be a nuisance variable (e.g., (Cherel et al., 2009; Codron et al., 2012; Doucett et al., 1999; Focken and Becker, 1998; Logan et al., 2008; Post et al., 2007). Suggested solutions to minimize the apparent variance when measuring the δ 13 C of bulk tissues involve physically extracting or ‘washing off’ the lipids (Miller et al., 1985; Pinnegar and Polunin, 1999; Kelly, 2000; McCue, Comparative Biochemistry and Physiology, Part A 179 (2015) 164–171 ⁎ Corresponding author. Tel.: +1 201 431 8005. E-mail address: mmccue1@stmarytx.edu (M.D. McCue). http://dx.doi.org/10.1016/j.cbpa.2014.08.021 1095-6433/© 2014 Published by Elsevier Inc. 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