First use of triply labelled water analysis for energy expenditure measurements in mice

The doubly labelled water (DLW) method is widely used to determine energy expenditure. In this work, we demonstrate the addition of the third stable isotope, (17)O, to turn it into triply labelled water (TLW), using the three isotopes measurement of optical spectrometry. We performed TLW ((2)H, (18)O and(17)O) measurements for the analysis of the CO(2) production (r(CO2)) of mice on different diets for the first time. Triply highly enriched water was injected into mice, and the isotope enrichments of the distilled blood samples of one initial and two finals were measured by an off-axis integrated cavity output spectroscopy instrument. We evaluated the impact of different calculation protocols and the values of evaporative water loss fraction. We found that the dilution space and turnover rates of (17)O and (18)O were equal for the same mice group, and that values of r(CO2) calculated based on (18)O–(2)H, or on (17)O–(2)H agreed very well. This increases the reliability and redundancy of the measurements and it lowers the uncertainty in the calculated r(CO2) to 3% when taking the average of two DLW methods. However, the TLW method overestimated the r(CO2) compared to the indirect calorimetry measurements that we also performed, much more for the mice on a high-fat diet than for low-fat. We hypothesize an extra loss or exchange mechanism with a high fractionation for (2)H to explain this difference.