2.2. TEE and Body Composition Measurement with DLW

TEE was measured for 7 days using the DLW method. Upon arrival at the kindergarten on day 0, a urine sample was acquired for the measurement of baseline ²H and ¹⁸O enrichment before the DLW dose. Each participant drank DLW containing a premixed dose of approximately 0.12 g/kg estimated total body water (TBW) of ²H₂O (99.8 at.%, Taiyo Nippon Sanso, Tokyo, Japan) and 2.5 g/kg estimated TBW of H₂¹⁸O (10.0 at.%, Taiyo Nippon Sanso) [15]. Urine samples were collected in the morning and evening of the next day (day 1, Dec. 9th) as well as day 8 (Dec. 16th). The urine samples were stored frozen at −15 °C for later analysis by isotope ratio mass spectrometry (IRMS, Hydra 20-20, Sercon Ltd., Crewe, UK) at Fukuoka University Research Institute for Physical Activity (FUIPA). Detailed analysis using IRMS was conducted as previously described [15,16].

The dilution space of ¹⁸O and ²H (No and Nd, respectively) was determined by dividing the dose of the administered tracer by the intercept method using two day 1 samples and two day 8 samples. All samples collected on day 8 exceeded 8‰ from baseline. The Nd/No values of the present study (1.032 ± 0.015, range 1.007–1.055) were similar to those of previous studies [17,18]. Thus, total body water (TBW) (g) was calculated as the average of the value obtained with Nd divided by 1.041 and No divided by 1.007. TBW (mol) was obtained as TBW (g)/18.02. The elimination rate of ¹⁸O and ²H (ko and kd, respectively) was determined, and the carbon dioxide production rate (rCO2) (mol/day) was calculated as 0.4554 × TBW (mol) × (1.007ko − 1.041kd), with the assumption that isotope fractionation applies only to breath water using Equation (A6) by Schoeller et al. [19], with the revised dilution space constant provided by Racette et al. [17]. The rCO₂ (L/d) was obtained as 22.4 × rCO2 (mol/day). We assumed that the respiratory quotient (RQ) was 0.87 [20], and TEE was calculated using the modified Weir’s equation [21] as follows: TDEE (kcal/d) = 1.1rCO2 + 3.9rCO2/RQ. The quality checklist is described in International Atomic Energy Agency (IAEA) documents [22]. FFM was calculated using TBW with the age-dependent hydration factor of children [23]. Fat mass (FM) and percent fat (%fat) were calculated using FFM and body weight.

The predicted BMR was calculated using the equation of the Recommended Dietary Allowances for Japanese [24] as in previous studies [16], which systematically reviewed the previous BMR measurements in the Japanese population and established equations with age categories from infants to older adults. The physical activity level (PAL) was calculated as TDEE divided by the BMR predicted by the Japanese equation [25,26]. For further analysis, we also calculated BMR from the Schofield equation [27,28].