Body composition , energy expenditure and physical activity Maximizing precision and accuracy of the doubly labeled water method via optimal sampling protocol , calculation choices , and incorporation of 17 O measurements

Background/Objectives The doubly labeled water (DLW) method is the gold standard methodology for determination of free-living, total daily energy expenditure (TEE). However, there is no single accepted approach for either the sampling protocols (daily vs. two-point, in which samples are collected after dosing and at the end of the measurement period) or the calculations used in the determination of the rate of carbon dioxide production (rCO2) and TEE. Moreover, fluctuations in natural background abundances introduce error in the calculation of rCO2 and TEE. The advent of new technologies makes feasible the possibility of including additional isotope measures (O) to account for background variation, which may improve accuracy. Subjects/Methods Sixteen subjects were studied for 7 consecutive days in a whole-room indirect calorimeter (IC) with concurrent measurement of TEE by DLW. Daily urine samples were obtained and isotope ratios were determined using offaxis integrated cavity output spectroscopy (OA-ICOS). Results We determined the best combination of approaches for estimating dilution spaces and elimination rates and calculated average daily volume of carbon dioxide production (VCO2) using six different published equations. Using this best combination, multi-point fitting of isotope elimination rates using the daily urine samples substantially improved the average precision (4.5% vs. 6.0%) and accuracy (−0.5% vs. −3.0%) compared with the two-point method. This improvement may partly reflect the less variable day-to-day chamber measurements of energy expenditure. Utilizing O measurements to correct for errors due to background isotope fluctuations provided additional but minor improvements in precision (4.2% vs. 4.5%) and accuracy (0.2% vs. 0.5%). Conclusions This work shows that optimizing sampling and calculation protocols can improve the accuracy and precision of DLW measurements. The gold standard for measuring total daily energy expenditure (TEE) in free-living individuals is the doubly labeled water (DLW) method. The general principle of the method is that, after consuming a dose of water labeled with the “heavy” stable isotopes of H and O, the elimination rates of these isotopes can be measured in samples of urine, saliva, or blood [1, 2]. A baseline sample is obtained prior to dosing, which is assumed to represent the background * John R. Speakman j.speakman@abdn.ac. uk 1 Kairos Aerospace, Mountain View, CA, USA 2 Division of Geriatric Medicine, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 3 Geriatric Research, Education, and Clinical Center, VA Eastern Colorado Health Care System, Denver, CO, USA 4 Division of Endocrinology, Metabolism, and Diabetes, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 5 Institute of Biological and Environmental Sciences, Aberdeen University, Aberdeen, UK 6 State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 12 34 56 78 90 () ;,: 12 34 56 78 90 (); ,: isotope levels. One or two samples are obtained shortly after dosing (typically within 4–6 h), which are assumed to represent the point at which the labeled isotopes have fully equilibrated with the body water pools. Samples are then obtained at one or more points after dosing (typically within 1–21 days) and used to determine the rate of elimination of oxygen (kO) and hydrogen (kD), which are then used to determine the average daily rate of carbon dioxide production (rCO2 in mole/day) over the measurement period. rCO2 is then converted to the average daily volume of CO2 production (VCO2, L/day), which is used to estimate TEE, using an assumed respiratory quotient (typically 0.85) and the equation of Weir [3]. There are a number of assumptions built into these equations, and the impacts they have on the determination of rCO2 have been previously considered (reviewed in Speakman [2]). Some of these issues were conclusively resolved, such as the use of a two-pool model (i.e., separate pools for the loss of O and H) to estimate the dilution spaces for large animals including humans and the use of population rather than individual estimates of the dilution space ratio when calculating the size of the total body water (TBW) pool [2, 4, 5]. However, while there has been some discussion in the literature of other issues such as estimating TBW using the plateau or intercept methods [6, 7], and whether to use one or the average of the two post-dose samples in this estimate, these issues have not been resolved and continue to vary from laboratory to laboratory. Furthermore, although many laboratories use only two samples (post-dose and end of study) to determine kO and kD, it is conceivable that using multiple samples (e.g., daily) to determine kO and kD may improve the accuracy and precision of the DLW method [8–10]. In addition, there have been several different equations published to estimate rCO2. Which combination of approaches to estimate TBW (plateau vs. intercept), kO and kD (twopoint model vs. exponential decay), and equations to determine rCO2 produces the greatest accuracy and precision is not known. Moreover, the existence of multiple protocols and calculation types potentially compromises comparisons between studies. Another factor that impacts the accuracy and precision of the DLW technique is that H and O occur naturally and their background levels fluctuate over time, primarily due to variations in sources of water and food in the diet [11]. To accurately determine kD and kO, the sample enrichments must be expressed as enrichments above background. The most common practice is to obtain a sample prior to dosing and assume that the background levels remain constant over the measurement period [9, 12]. However, if the natural (background) abundance changes substantially during the measurement period, this can result in errors in determining rCO2 and TEE [12]. It has been suggested that background fluctuations of O and H during the course of a typical DLW experiment might be reconstructed from changes in the levels of O [13–15]. Measurements of natural abundance variations of O by mass spectrometry are, however, technically challenging. The emergence of laser spectroscopy approaches to isotope determination in water has made measurement of O much more feasible [16, 17]. Indeed, we have previously shown using off-axis integrated cavity output spectroscopy (OA-ICOS) that background fluctuations of O are highly correlated with background fluctuations of both O and H and suggested that measurements of O could be used to measure and correct for the background isotope fluctuations of O and H in body water [13]. However, this approach has one substantial limitation, namely, that the O stable isotope is enriched along with the O stable isotope in all commercially available water enriched in O, and therefore O in dosed subjects is not at background abundance. Nevertheless, there are mathematical approaches that may overcome this limitation. The purpose of the analysis presented here is to determine the combination of approaches to calculate TBW, kO and kD, and equations to estimate rCO2, which produces the best accuracy and precision of the final, calculated daily VCO2. To achieve this, we used data from our recently completed OA-ICOS validation study using near continuous measurements from whole-room indirect calorimetry (IC) with additional measurements of O by OAICOS [18]. In addition, we explored the potential to use these OA-ICOS O measurements to improve the accuracy and precision of the DLW method by correcting for background enrichment fluctuations.