Breath water-based doubly labelled water method for the noninvasive determination of CO₂ production and energy expenditure in mice

We explored a novel doubly labelled water (DLW) method based on breath water (BW-DLW) in mice to determine whole body CO₂ production and energy expenditure noninvasively. The BW-DLW method was compared to the DLW based on blood plasma. Mice (<i>n</i> = 11, 43.5 ± 4.6 g body mass (BM)) were administered orally a single bolus of doubly labelled water (1.2 g H₂¹⁸O kg BM⁻¹ and 0.4 g ²H₂O kg BM⁻¹, 99 atom% (AP) ¹⁸O or ²H). To sample breath water, the mice were placed into a respiration vessel. The exhaled water vapour was condensed in a cold-trap. The isotope enrichments of breath water were compared with plasma samples. The ²H/¹H and ¹⁸O/¹⁶O isotope ratios were measured by means of isotope ratio mass spectrometry. The CO₂ production (RCO₂) was calculated from the ²H and ¹⁸O enrichments in breath water and plasma over 5 days. The isotope enrichments of breath water vs. plasma were correlated (R² = 0.89 for ²H and 0.95 for ¹⁸O) linearly. The RCO₂ determined based on breath water and plasma was not different (113.2 ± 12.7 vs. 111.4 ± 11.0 mmol d^–1), respectively. In conclusion, the novel BW-DLW method is appropriate to obtain reliable estimates of RCO₂ avoiding blood sampling.