Measurement of BMR and TEE

BMR was measured by indirect calorimetry (Quark BMR; COSMED, Rome, Italy). Before BMR measurements, the subjects were allowed to ingest only water for 12 h. The test was performed between 08:30 and 10:00. The Quark BMR measures the amount of oxygen consumed and carbon dioxide exhaled, and calculates the BMR using the modified Weir Eq. [33]. Before measurement, the procedure was explained to the subjects, who had comfortably rested on a bed for 30 min. A steady state was achieved for more than 5 min by the Quark BMR after 10–15 min of breathing while the subjects lay awake in the supine position.

TEE of each patient was measured over 13–15 days by the DLW method. The patients were instructed to behave as usual during the study period. The DLW protocol details are given elsewhere [34]. In brief, on the morning of day 1 (visit 1), after the collection of background blood and urine samples, subjects took an oral dose of 0.1 g ²H₂O and 2.0 g H₂¹⁸O (Taiyo Nippon Sanso, Tokyo, Japan) per kilogram of estimated total body water. Post-dose blood sample was collected at only 4 h while post-dose urine samples were collected at 2, 3, and 4 h. Further samples were collected on the mornings of days 13–15 at visit 2 (once for blood and twice for urine at a 1-h interval). In the collected samples, the representative TEE value was calculated by determining the average TEEs obtained from both samples at background, 4 h post-ingestion, and at visit 2 after emptying the urinary bladder. Isotope analyses of the blood and urine samples were performed at ESTech Kyoto (Kyoto, Japan) in duplicate using an isotope-ratio mass spectrometer (Hydra 20–20 Stable Isotope Mass Spectrometer; Sercon, Crewe, UK). The DLW method provides an average TEE value per day during the period of assessment. The PAL was calculated using the following equation: PAL =  (TEE estimated by DLW method)/ (BMR measured by indirect calorimetry) [35].