Comparison of indirect calorimetry and a new breath 13C/12C ratio method during strenuous exercise J. A. ROMIJN, E. F. COYLE, J. HIBBERT, AND R. R. WOLFE Metabolism Unit, Shriners Burns Institute, and Departments of Anesthesiology and Surgery, University of Texas Medical Branch, Galveston 77550; and Human Performance Laboratory, Department of Kinesiology and Health, The University of Texas at Austin, Austin, Texas 78712 Romijn, J. A., E. F. Coyle, J. Hibbert, and R. R. Wolfe. Comparisonof indirect calorimetry and a new breath l:C/‘*C ratio method during strenuous exercise. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E64-E71, 1992.-A new stable isotope method for the determination of substrate oxidation rates in vivo is described and compared with indirect calorimetry at rest and during high-intensity exercise (30 min at 80-85% maximal 0, uptake capacity) in six well-trained cyclists. This method uses the absolute ratios of 13C/12C in expired air, endogenous glucose, fat, and protein in addition to 0, consumption and is independent of CO, production (VCO,). Carbohydrate and fat oxidation rates at rest, calculated by both methods, were not significantly different. During exercise the breath 13C/12C ratio increased and reached a steady state after 15-20 min. Carbohy- drate oxidation rates during exercisewere 39.4 & 5.2 and 41.7 k 5.7 mg l kg-l l min-l [not significant (NS)], and fat oxidation rates were 7.3 & 1.3 and 6.9 & 1.2 mgekg-l=min-l (NS), using indirect calorimetry and the breath ratio method, respectively. We conclude that the breath 13C/12C ratio method can be used to calculatesubstrateoxidation under different conditions, such asthe basal state and exercise.In addition, the resultsobtained by this new method support the validity of the underlying as- sumption that indirect calorimetry regards %o., asa reflection of tissue CO, production, during exercise in trained subjects, even up to 80-85% maximal 0, uptake. stable isotopes; mass spectrometry; substrate oxidation PRESENTLY, TWO METHODS are available to quantify whole body substrate oxidation rates at rest and during exercise: indirect calorimetry and tracer techniques. In- direct calorimetry relies on the assumption that 0, con- sumption (Vo2) and CO2 production (VCO,), as mea- sured in expired air, reflect gas exchange at the tissue level (7). This is probably true under both conditions for 02, of which there are no large stores in the body. In contrast, VCO~ is only a reliable estimate of tissue CO2 production in the presence of a stable bicarbonate pool. However, during exercise, even of moderate intensity, bicarbonate kinetics are markedly altered (2). During exercise above the lactate threshold, a depletion of the bicarbonate pool may result in an overestimation of tis- sue CO2 production and consequently of carbohydrate oxidation (with a concomitant underestimation of fat oxidation). A second approach to quantify substrate ox- idation uses administration of 13C- or 14C-enriched sub- strates, such as glucose and amino acids (17). The oxi- dation rate is calculated from the rate of excretion of labeled CO2 and the enrichment of the substrate in the plasma. This tracer method, however, is hampered by the same potential limitations with respect to bicarbon- ate metabolism as indirect calorimetry. In addition, there may be exchange of labeled CO2 within the tri- carboxylic acid (TCA) cycle, which will result in de- creased recovery of CO2 in the breath and thus in an underestimation of substrate oxidation (19). In the ap- plication of these tracer methods, account must also be taken of changes in bicarbonate recovery between rest and exercise (2, 17, 21). To circumvent the use of VCO, and the problem of isotopic exchange in the TCA cycle, we have developed an alternative approach to calculate substrate oxidation rates at rest and during exercise. It involves the mea- surement of the absolute 13C/12C ratios in expired breath and in endogenous glucose, fat, and protein. With the measurement of these absolute 13C/12C ratios and of Vo2, the absolute rates of substrate oxidation can be calculated, independently of VCO~. This method relies on only a small difference in the absolute 13C/12C ratios of endogenous substrates. To amplify the naturally oc- curring difference between endogenous carbohydrates, protein, and fat, we used an exhaustive exercise protocol on the day before the actual measurements to deplete endogenous glycogen stores, which was followed by gly- cogen repletion with 13C-enriched cornstarch. This new breath ratio method was used to assess the validity of indirect calorimetry for estimation of carbohydrate and fat oxidation rates during high-intensity exercise. METHODS Subjects Six highly trained endurancecyclists [age 24 t 1 yr, weight 76.2 k 3.2 kg, height 1.79 t 3 m, maximal 0, uptake capacity . wo 2 max) 62 & 3 ml l kg-l. min-‘1 volunteered for this study. All subjects were healthy, asindicated by medicalhistory and phys- ical examination. They were consuminga weight-maintaining diet containing at least 250 g carbohydrates daily. To2 max had been determined several weeks before the present protocol, while the subjects cycled on a stationary ergometer (Monark 819). \io, max was determined during an incremental cycling protocol lasting 7-10 min. The study was approved by the in- stitutional review boards of the University of Texas at Galveston and Austin. Experimental Protocol The experiment was performed on two consecutive days in four subjects and on three consecutive days in two subjects (Fig. 1). The 1st day served to increase the 13C enrichment of endogenous carbohydrates by a combined glycogen depletion- repletion protocol, using IV-enriched cornstarch. To deplete glycogenin the cycling musculature and in the liver as much as possible,the subjects exercised until fatigued on the 1st day after fasting for the previous 12 h. They first cycled for 30 min at 80-85% VO, max and then for 2-3 h at 65-70% VO, m8X until blood glucoseconcentration declined below 3 mM and they began to experience leg fatigue. At this point they performed 8-12 bouts of exercise at the highest intensity they could main- tain for 2 min (70-90% \I~o, ,,,) followed by 2 min of recovery E64 0193~1849/92 $2.00 Copyright 0 1992 the American Physiological Society