Introduction: Whole-body energy expenditure measurements with indirect calorimetry provide the respiratory exchange ratio (RER), a valuable index of the balance between carbohydrate and fat oxidation. However, RER alone cannot distinguish the specific sources of oxidized substrates, such as dietary vs. endogenous carbohydrate and fat, and may not fully capture dynamic changes in substrate utilization across different physiological conditions or interventions. Here, we determined the precision and detection limits of a new method that combines indirect calorimetry with stable-isotope tracers of glucose or palmitate in a controlled metabolic enclosure (ME). Material and method: An indirect calorimetry system (Promethion Gas Analyzer 3m2, Flow generator 250, Sable Systems International, North Las Vegas, NV, USA) with an integrated laser-based stable-isotope gas analyzer and connected to an ME (Photopod, Weatherpod, Cincinnati, OH, USA, 1.2m x 1.2m x 2m, DxWxH, 2880 liters), was stationed in a controlled environment with stable ambient temperature (22°C) and air circulation. In a preliminary study, a participant sat in the ME, breathing normally, for fasted, pre-prandial, and post-prandial states. Non-physical activities (e.g., reading or using a laptop) were allowed. Exhaled air dilution factors of 13CO 2 enrichments coming from an ingested 1-13C-glucose (0.2, 1 or 2 g) or U-13C-palmitate (0.25 or 0.5 g) enriched standard test meal (ENSURE plus, Abbott: 350 kcal, 16 g protein, 20 g CHO, 11 g fat) were determined and optimized by measuring continuously alternating CO 2 , O 2 , concentrations and δ-13C in ambient and ME air (1 sample/sec). In every cycle (6 min), the mean ± SD of resp. 60 and 30 samples from ME air and ambient air were determined. Enrichments in exhaled air were determined mathematically. Statistics: Graphpad Prism 10, Precision comparison of 10 cycles with unpaired T-test. Correlation with Pearson. Results: Lowering the airflow in the ME from 240 to 100 l/min reduced the dilution of exhaled air by ambient air in the ME (ambient air fraction: 240: 0.48 vs. 100: 0.31). It did not affect the ME temperature when occupied (24°C), but caused a delay in stable readings (time to 95% of ME CO 2 concentration baseline plateau: 240: 8.5 min versus 100: 27 min). Less dilution increased the precision of the δ-13C in the ME air (SD240: 0.58‰ vs. SD100: 0.43‰, p< 0.0002) and natural abundance δ-13CO 2 in exhaled air (30 min baseline measurement SD240: 0.88‰ vs. SD100: 0.17‰) and therefore increased the sensitivity to detect enrichment of exhaled air (LOD = 3 x SD, LOD240: 2.6‰ vs. LOD100: 0.51‰). Increasing amounts of 1-13C-glucose in the test meal were linear with the exhaled 13CO 2 enrichment (AUC over 5 h, Pearson r = 1, p< 0.0009). U-13C-palmitate intake resulted in a more delayed 13CO 2 curve (1-13C-glucose Tmax enrichment: 220 min vs. U-13C-palmitate 400 min). Conclusion: Achieving precise 13CO 2 enrichment in exhaled air through a room calorimetry approach and stable-isotope-enriched test meals offers a new, advanced, and non-invasive method for assessing changes in macronutrient substrate oxidation. This technique can be applied in studies involving different physiological conditions or interventions. Funding source: USDA/ARS 6026-10700-001-000D, NIH/NIGMS P20-GM109096 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Have et al. (2026) studied this question.