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Variation of the dilution space ratio (Nd/No) between deuterium ((2)H) and oxygen-18 ((18)O) impacts the calculation of total energy expenditure (TEE) by doubly labeled water (DLW). Our aim was to examine the physiological and methodological sources of variation of Nd/No in humans. We analyzed data from 2,297 humans (0.25-89 yr old). This included the variables Nd/No, total body water, TEE, body mass index (BMI), and percent body fat (%fat). To differentiate between physiologic and methodologic sources of variation, the urine samples from 54 subjects were divided and blinded and analyzed separately, and repeated DLW dosing was performed in an additional 55 participants after 6 mo. Sex, BMI, and %fat did not significantly affect Nd/No, for which the interindividual SD was 0.017. The measurement error from the duplicate urine sample sets was 0.010, and intraindividual SD of Nd/No in repeats experiments was 0.013. An additional SD of 0.008 was contributed by calibration of the DLW dose water. The variation of measured Nd/No in humans was distributed within a small range and measurement error accounted for 68% of this variation. There was no evidence that Nd/No differed with respect to sex, BMI, and age between 1 and 80 yr, and thus use of a constant value is suggested to minimize the effect of stable isotope analysis error on calculation of TEE in the DLW studies in humans. Based on a review of 103 publications, the average dilution space ratio is 1.036 for individuals between 1 and 80 yr of age., (Copyright © 2016 the American Physiological Society.)

Background/objectives: The method of choice for measuring total energy expenditure in free-living individuals is the doubly labeled water (DLW) method. This experiment examined the behavior of natural background isotope abundance fluctuations within and between individuals over time to assess possible methods of accounting for variations in the background isotope abundances to potentially improve the precision of the DLW measurement., Subjects/methods: In this work, we measured natural background variations in (2)H, (18)O and (17)O in water from urine samples collected from 40 human subjects who resided in the same geographical area. Each subject provided a urine sample for 30 consecutive days. Isotopic abundances in the samples were measured using Off-Axis Integrated Cavity Output Spectroscopy., Results: Autocorrelation analyses demonstrated that the background isotopes in a given individual were not temporally correlated over the time scales of typical DLW studies. Using samples obtained from different individuals on the same calendar day, cross-correlation analyses demonstrated that the background variations of different individuals were not correlated in time. However, the measured ratios of the three isotopes (2)H, (18)O and (17)O were highly correlated (R(2)=0.89-0.96)., Conclusions: Although neither specific timing of DLW water studies nor intraindividual comparisons were found to be avenues for reducing the impact of background isotope abundance fluctuations on DLW studies, strong inter-isotope correlations within an individual confirm that use of a dosing ratio of 8‰:1‰ (0.6 p.p.m.: p.p.m.) optimizes DLW precision. Theoretical implications for the possible use of (17)O measurements within a DLW study require further study.

Isotopic determination of total energy expenditure (TEE) by the doubly labeled water (DLW) method may be affected by urine retention in the elderly. The isotopic enrichments in urine and plasma sampled simultaneously 4 h post-DLW dose were compared in a subset of 281 subjects [139 women, 142 men, 75 +/- 3 (SD) yr] of the 3,075 participants in the Health, Aging, and Body Composition study. Based on analytic precisions, a +/- 2% urine-plasma difference was set as the cut-off value. Ten percent of the population presented a difference lower than -2%, suggesting a delay in urine isotopic equilibration. This -13 +/- 10% urine-plasma difference was not linked to analytic errors, illnesses, the sampling time, or the time and quantity of water intake, suggesting that urine retention may be the main factor. The consequences are an 18 +/- 13 and 21 +/- 16% overestimation of the total body water and the TEE, respectively. Unexpectedly, 21% of the population presented a urine-plasma difference higher than +/- 2% that resulted, however, in a nonsignificant TEE underestimation of -3 +/- 5%. In conclusion, the delayed isotopic equilibration observed in urine reduces the accuracy of the DLW method in the elderly. It is recommended, when blood sampling is impossible, to adopt the intercept method with urine sampling 24 h postdose.