Isotope dilution spaces of mice injected simultaneously with deuterium, tritium and oxygen-18

The isotope dilution technique for measuring total body water (TBW), and th e doubly labelled water (DLW) method for measuring energy expenditure, are both sensitive to small variations in the ratio of the hydrogen to oxygen-1 8 dilution space. Since the dilution space ratio varies between individuals , there has been much recent debate over what causes this variability (i.e. physiological differences between individuals or analytical error in the i sotope determinations), and thus which values (individual or a population m ean dilution space ratio) should be employed for TBW and DLW calculations. To distinguish between physiological and analytical variability, we injecte d 15 non-reproductive and 12 lactating mice (Mus musculus, outbred MF1) sim ultaneously with deuterium, tritium and oxygen-18, The two hydrogen labels were administered and analysed independently, therefore we expected a stron g correlation between dilution space ratios based on deuterium and tritium if most of the variation in dilution spaces was physiological, but only a w eak correlation if most of the variation was analytical. Dilution spaces we re significantly influenced by reproductive status. Dilution spaces express ed as a percentage of body mass averaged 15.7 % greater in lactating mice t han in non-reproductive mice. In addition, the hydrogen tracer employed had a significant effect (deuterium spaces were 2.0 % larger than tritium spac es). Deuterium and tritium dilution spaces, expressed as a percentage of bo dy mass, were highly correlated. Dilution space ratios ranged from 0.952 to 1.146 when using deuterium, and from 0.930 to 1.103 when using tritium, Di lution space ratios based on deuterium and tritium were also highly correla ted. Comparison of standard deviations of the dilution space ratio based on deuterium in vivo and in vitro indicated that only 4.5 % of the variation in the dilution space ratios observed in the mice could be accounted for by analytical variation in the deuterium and oxygen-18 analyses. Although our results include data which were outside the limits previously regarded as biologically possible, the correlations that we detected strongly suggest t hat variation in the observed dilution space ratio was mostly physiological rather than analytical.