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Methodological aspects
Application of the doubly labelled method for the meas- urement of energy expenditure includes design of a pro- tocol, preparation of the isotope dose, sample collection, sample analysis, and calculation of energy expenditure from the results obtained (Table 2). The most critical step is sample analysis, ideally performed in a dedicated labo- ratory with specific sample preparation systems and an isotope ratio mass spectrometer (Speakman 1997). One can set up a laboratory or send samples away to get them analyzed. There are commercial laboratories and some university laboratories performing analyses of external samples.
With regard to the protocol, the optimal observation interval for the measurement of energy expenditure with doubly labelled water is one to three times the biological half-life of the isotopes (Lifson and McClintock 1966). The biological half-life of 18O ranges from 3 days in young children or extremely active adult subjects to about 10 days in very sedentary adult and old subjects (West- erterp 1999). In addition, measuring energy expenditure under daily living conditions with weekly cycles of phys- ical activity routines, one often measures over multiples of a week. Thus, a typical observation interval is 1 week in children and endurance athletes, 2 weeks in adult sub- jects, and 3 weeks in the elderly.
With regard to the isotope dose, body water volume of the subject mainly determines the isotope dose. Larger subjects require more isotopes, as do men compared to similar mass women, because of their larger body water containing fat-free mass. The ultimate determinant of the dose is the final body water enrichment of the isotopes at the end of the observation interval, in combination with the variation in background enrichment and the analysis precision of body water samples. With the current sam- ple analysis precision with isotope ratio mass spectrom- etry, subjects are dosed with at least 1.8 g water/kg body water of water with 10% 18O atoms and 0.12 g water/kg body water of water with 99% 2H atoms (International Atomic Energy Agency 2009). Thus, the initial back- ground enrichment of body water for 18O of 2000 ppm
is increased with 180 ppm and the initial background enrichment of body water for 2H of 150 ppm is increased with 120 ppm, or slightly higher values. The doubly labelled water method is considered safe for subjects of all ages, with no indications of health effects of the two isotopes at these concentrations (Leatherdale and Jones 1991).