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Saliva and urine are the two main body fluids sampled when breast milk intake is measured with the 2H oxide dose-to-mother technique. However, these two body fluids may generate different estimates of breast milk intake due to differences in isotope enrichment. Therefore, we aimed to assess how the estimated amount of breast milk intake differs when based on saliva and urine samples and to explore whether the total energy expenditure of the mothers is related to breast milk output. We used a convenience sample of thirteen pairs of mothers and babies aged 2-4 months, who were exclusively breastfed and apparently healthy. To assess breast milk intake, we administered doubly labelled water to the mothers and collected saliva samples from them, while simultaneously collecting both saliva and urine from their babies over a 14-d period. Isotope ratio MS was used to analyse the samples for 2H and 18O enrichments. Mean breast milk intake based on saliva samples was significantly higher than that based on urine samples (854·5 v. 812·8 g/d, P = 0·029). This can be attributed to slightly higher isotope enrichments in saliva and to a poorer model fit for urine samples as indicated by a higher square root of the mean square error (14·6 v. 10·4 mg/kg, P = 0·001). Maternal energy expenditure was not correlated with breast milk output. Our study suggests that saliva sampling generates slightly higher estimates of breast milk intake and is more precise as compared with urine and that maternal energy expenditure does not influence breast milk output.

Inspired by a previous 'Sauna, sweat and science' study [Zech et al. Isot Environ Health Stud. 2015;51(3):439-447] and out of curiosity and enthusiasm for stable isotope and sauna research we aimed at answering the question 'do we sweat (isotopically) what we drink'? We, therefore, pulse-labelled five test persons in a sauna experiment with beverages that were 2 H-enriched at about +25,600 ‰. Sweat samples were collected during six sauna rounds and the hydrogen isotope composition δ 2 H sweat was determined using an isotope ratio mass spectrometer. Before pulse labelling, δ 2 H sweat - reflecting by approximation body water - ranged from -32 to -22 ‰. This is ∼35 ‰ enriched compared to usual mid-European drinking water and can be explained with hydrogen-bearing food as well as with the respiratory loss of 2 H-depleted vapour. The absence of a clearly detectable 2 H pulse in sweat after pulse labelling and δ 2 H sweat results of ≤+250 ‰ due to a fast 2 H equilibration with body water are moreover a clearly negative answer to our research question also in a short-term consideration. Given that the recovery of the tracer based on an isotope mass balance calculation is clearly below 100 %, we finally answer the question 'where did the rest of the tracer go?'

Background and objectives : There is a lack of studies investigating associations of physical activity level (PAL) and activity energy expenditure (AEE) using the doubly-labeled water (DLW) method with body composition and physical fitness in young children. Thus, we aimed to examine cross-sectional associations of PAL and AEE with body composition indices and physical fitness components in Swedish preschool children. Materials and methods: PAL was calculated as total energy expenditure measured using DLW divided by the predicted basal metabolic rate in 40 children aged 5.5 (standard deviation 0.2) years. AEE was calculated as total energy expenditure minus basal metabolic rate and the thermic effect of food, and divided by fat-free mass. Body composition was assessed using the 3-component model by combining measurements based on isotope dilution and air-displacement plethysmography. Physical fitness (muscular strength, motor fitness, and cardiorespiratory fitness) was evaluated using the PREFIT test battery. Multiple linear regression models were conducted. Results: PAL and AEE were negatively associated with body mass index, percent body fat, and fat mass index (PAL: standardized β -0.35, -0.41, and -0.45, all p < 0.036; AEE: standardized β -0.44, -0.44, and -0.47, all p < 0.006, respectively). Furthermore, PAL and AEE were positively associated with the standing long jump test (PAL: standardized β 0.37, p = 0.017; AEE: standardized β 0.38, p = 0.014). There were no statistically significant associations found regarding PAL or AEE with fat-free mass index or any other physical fitness test. Conclusions: Greater PAL and AEE at the age 5.5 were significantly associated with body fatness and improved lower-body muscular strength. Therefore, increasing physical activity, and thus energy expenditure, at young ages may be beneficial for preventing overweight/obesity. However, further studies with larger sample sizes are needed to confirm the results.

When the doubly labeled water (DLW) method is used to measure total daily energy expenditure (TDEE), isotope measurements are typically performed using isotope ratio mass spectrometry (IRMS). New technologies, such as off-axis integrated cavity output spectroscopy (OA-ICOS) provide comparable isotopic measurements of standard waters and human urine samples, but the accuracy of carbon dioxide production (V̇co 2 ) determined with OA-ICOS has not been demonstrated. We compared simultaneous measurement V̇co 2 obtained using whole-room indirect calorimetry (IC) with DLW-based measurements from IRMS and OA-ICOS. Seventeen subjects (10 female; 22 to 63 yr) were studied for 7 consecutive days in the IC. Subjects consumed a dose of 0.25 g H 2 18 O (98% APE) and 0.14 g 2 H 2 O (99.8% APE) per kilogram of total body water, and urine samples were obtained on days 1 and 8 to measure average daily V̇co 2 using OA-ICOS and IRMS. V̇co 2 was calculated using both the plateau and intercept methods. There were no differences in V̇co 2 measured by OA-ICOS or IRMS compared with IC when the plateau method was used. When the intercept method was used, V̇co 2 using OA-ICOS did not differ from IC, but V̇co 2 measured using IRMS was significantly lower than IC. Accuracy (~1-5%), precision (~8%), intraclass correlation coefficients ( R = 0.87-90), and root mean squared error (30-40 liters/day) of V̇co 2 measured by OA-ICOS and IRMS were similar. Both OA-ICOS and IRMS produced measurements of V̇co 2 with comparable accuracy and precision compared with IC.

Purpose: To compare the degree to which four accelerometer metrics-total activity counts per day (TAC per day), steps per day (steps per day), physical activity energy expenditure (PAEE) (kcal·kg·d), and moderate- to vigorous-intensity physical activity (MVPA) (min·d)-were correlated with PAEE measured by doubly labeled water (DLW). Additionally, accelerometer metrics based on vertical axis counts and triaxial counts were compared., Methods: This analysis included 684 women and 611 men age 43 to 83 yr. Participants wore the Actigraph GT3X on the hip for 7 d twice during the study and the average of the two measurements was used. Each participant also completed one DLW measurement, with a subset having a repeat. PAEE was estimated by subtracting resting metabolic rate and the thermic effect of food from total daily energy expenditure estimated by DLW. Partial Spearman correlations were used to estimate associations between PAEE and each accelerometer metric., Results: Correlations between the accelerometer metrics and DLW-determined PAEE were higher for triaxial counts than vertical axis counts. After adjusting for weight, age, accelerometer wear time, and fat free mass, the correlation between TAC per day based on triaxial counts and DLW-determined PAEE was 0.44 in women and 0.41 in men. Correlations for steps per day and accelerometer-estimated PAEE with DLW-determined PAEE were similar. After adjustment for within-person variation in DLW-determined PAEE, the correlations for TAC per day increased to 0.61 and 0.49, respectively. Correlations between MVPA and DLW-determined PAEE were lower, particularly for modified bouts of ≥10 min., Conclusions: Accelerometer measures that represent total activity volume, including TAC per day, steps per day, and PAEE, were more highly correlated with DLW-determined PAEE than MVPA using traditional thresholds and should be considered by researchers seeking to reduce accelerometer data to a single metric.

GC-MS and GC-MS/MS of pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether (PFB-TMS) derivatives of hydroxylated long-chain fatty acids including arachidonic acid metabolites, the eicosanoids, in the electron-capture negative-ion chemical ionization (ECNICI) mode are the most sensitive and accurate approaches to quantify carboxyl groups-containing compounds in complex biological fluids such as plasma and urine. Under ECNICI conditions, PFB-TMS derivatives of eicosanoids ionize to form very few ions, with the carboxylates [M-PFB] - being typically the most intense. Less intense ions may be additionally formed by consecutive neutral loss (NL) of trimethylsilanol (TMSOH, 90Da) groups ([M-PFB-(TMSOH) n ] - ). By using [1,1- 18 O 2 ]- and [1,ω- 18 O 2 ]-eicosanoids, we studied ion processes following collisionally activated dissociation (CAD) of the precursor ions [M-PFB] - . We found that CAD resulted in formation of product ions due to NL of a TMS 18 OH (92Da) group in monocarboxylic and of a PFB 18 OH (200Da) group in dicarboxylic eicosanoids. TMS 18 OH NL implies an intra-molecular transfer of the TMS group from hydroxyl groups to their carboxylate anions [M-PFB] - . From a mechanistic point of view, this rearrangement may explain formation of unique product ions in GC-MS/MS of eicosanoids under ECNICI conditions. From the quantitative point of view, quantification by GC-MS/MS of product ions due to [M-PFB-(TMSOH) n ] - and [M-PFB-TMS 18 OH-(TMSOH) n-1 ] - would reveal incorrect data, if [1,1- 18 O 2 ]-eicosanoids are used as internal standards and if no correction for the 18 O-loss is performed. In 18 O-labelled dicarboxylic eicosanoids, such as the major urinary metabolite (MUM) of E prostaglandins, i.e., [1,ω- 18 O 2 ]-PGE-MUM), no TMS ester/TMS ether rearrangement was observed. Yet, 18 O-loss occurred upon CAD of [M-PFB] - due to NL of PFB 18 OH (200Da). In both cases the extent of 18 O-loss needs to be determined and considered for accurate quantification of monocarboxylic acids such as 8-isoprostaglandin F 2α (8-iso-PGF 2α ) and dicarboxylic eicosanoids such as PGE-MUM., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

The doubly labeled water (DLW) method is used to measure free-living energy expenditure in humans. Inherent to this technique is the assumption that natural abundances of stable isotopes (2)H and (18)O in body water remain constant over the course of the measurement period and after elimination of the loading dose of DLW will return to the same predose level. To determine variability in the natural abundances of (2)H and (18)O in humans living in a region with seasonal shifts in rain patterns and sources of drinking water, over the course of 12 mo we collected weekly urine samples from four individuals living in southwest Nigeria as well as samples of their drinking water. From ongoing regional studies of hypertension, obesity, and energy expenditure, we estimated average water turnover rate, urine volumes, and sodium and potassium excretion. Results suggest that (2)H and (18)O in urine, mean concentrations of urinary sodium and potassium, urine volume, and total body turnover differed significantly from dry to rainy season. Additionally, seasonal weather variables (mean monthly maximum temperatures, total monthly rainfall, and minimum relative humidity) were all significantly associated with natural abundances in urine. No seasonal difference was observed in drinking water samples. Findings suggest that natural abundances in urine may not remain constant as assumed, and studies incorporating DLW measurements across the transition of seasons should interpret results with caution unless appropriate doses of the tracers are used., (Copyright © 2015 the American Physiological Society.)

Rationale: Total Body Water (TBW) and Total Energy Expenditure (TEE) are routinely measured in free-living conditions by the (2)H2(18)O method. Isotope eliminations can be measured from spot urine samples by HTC-EA IRMS, but only after cumbersome cryogenic distillation to extract water. Distillation may, however, be replaced by charcoal treatment and filtration. This study tested (1) the effect of sample treatments (filtration versus distillation) on the isotope ratios, (2) the effect of different ways of normalization that respect or not the principle of identical treatment of the sample and references, and (3) the impact on the biological outcomes., Methods: Two filters (PES membrane; 10 kDa) accepting volumes of urine samples (V500: 0.5 mL versus V6: 3.0 mL) were tested. In-house water standards and in-house urine standards were prepared and normalized against the international scale to calibrate the urine samples. The δ(2)H and δ(18)O values from water in the urine were measured by HTC-EA IRMS., Results: Filtered urine normalized with water standards showed a bias in the δ(2)H values that was corrected when calibration was performed with urine standards. At a δ(2)H value of 1101.4‰, the accuracy increased from -11.9 to -0.2 δ‰ (V500) and from -3.8 to 0.4 δ‰ (V6). The TBW errors were greatest with V500 and water calibration (1.20%) and lowest with V6 and urine calibration (0.34%; preparation-by-calibration interaction p = 0.027). For the δ(18)O values the accuracy of enrichments and TBW were not affected whatever preparations and normalization were used. The average TEE was not affected but the variability increased from 0.6 to 2.7% versus cryogenic distillation., Conclusions: Cryogenic distillation remains the gold standard for small sample size experiments where small changes in TEE are to be detected. Filtration offers an alternative for large-scale experiments. When the body composition is derived from (2)H2O dilution, it is strongly recommended that urine standards should be used to eliminate the effect of filtration., (Copyright © 2015 John Wiley & Sons, Ltd.)

The stable isotopes of hydrogen (δ(2)H) and oxygen (δ(18)O) in human urine are measured during studies of total energy expenditure by the doubly labeled water method, measurement of total body water, and measurement of insulin resistance by glucose disposal among other applications. An ultrasensitive laser absorption spectrometer based on off-axis integrated cavity output spectroscopy was demonstrated for simple and inexpensive measurement of stable isotopes in natural isotopic abundance and isotopically enriched human urine. Preparation of urine for analysis was simple and rapid (approximately 25 samples per hour), requiring no decolorizing or distillation steps. Analysis schemes were demonstrated to address sample-to-sample memory while still allowing analysis of 45 natural or 30 enriched urine samples per day. The instrument was linear over a wide range of water isotopes (δ(2)H = -454 to +1702 ‰ and δ(18)O = -58.3 to +265 ‰). Measurements of human urine were precise to better than 0.65 ‰ 1σ for δ(2)H and 0.09 ‰ 1σ for δ(18)O for natural urines, 1.1 ‰ 1σ for δ(2)H and 0.13 ‰ 1σ for δ(18)O for low enriched urines, and 1.0 ‰ 1σ for δ(2)H and 0.08 ‰ 1σ for δ(18)O for high enriched urines. Furthermore, the accuracy of the isotope measurements of human urines was verified to better than ±0.81 ‰ in δ(2)H and ±0.13 ‰ in δ(18)O (average deviation) against three independent isotope-ratio mass spectrometry laboratories. The ability to immediately and inexpensively measure the stable isotopes of water in human urine is expected to increase the number and variety of experiments which can be undertaken.

During nutritional interventions, the ingestion of d(31)-palmitic acid and H(2)(18)O allows the assessment of dietary fatty acid oxidation from cumulative (2)H recovery in urine and the estimation of the total body water pool (TBW) from (18)O dilution. Continuous-flow isotope ratio mass spectrometry (CF-IRMS) coupled to either equilibration or high-temperature conversion (HTC) techniques permits (2)H- and (18)O-enrichment measurements in biological fluids. Thus it was of great interest to compare these methods applied to the determination of dietary fatty acid oxidation. The linearity, accuracy and correlation between CF-equilibration and CF-HTC were first checked using (2)H- and (18)O-enriched water and urine samples. Urine samples from 14 subjects were then measured with both methods. The (2)H and (18)O raw data were normalised against calibration lines. The final aim was to study the impact of the normalised raw results on physiological data (i.e. TBW and d(31)-palmitate recovery). No significant difference was observed between the (18)O- and (2)H-enrichment measurements depending on the analytical method used. The TBW volumes calculated from the (18)O enrichments measured either with CF-equilibration or CF-HTC were not significantly different: respectively, 45.1 ± 1.0 L or 45.7 ± 1.0 L (mean ± sem, p = 0.09). The palmitic acid oxidation results obtained from the (2)H-enrichment measurements and the TBW from CF-equilibration vs. CF-HTC were not significantly different (p ≥ 0.26): with δ(2)H values of, respectively, 16.2 ± 1.6% vs. 16.2 ± 1.1% at 8 h, 18.7 ± 2.0% vs. 17.6 ± 1.3% at 12 h and 21.7 ± 1.9% vs. 21.5 ± 1.3% at 3 days post-dose (mean ± sem). Thus, even if CF-HTC was preferred because it was more practical to carry out, both methods allow the study of dietary lipid oxidation in man and generate similar results., (Copyright © 2011 John Wiley & Sons, Ltd.)

The doubly labeled water method provides an objective and accurate measure of total energy expenditure in free-living subjects and is considered the gold-standard method for this measurement. Its use, however, is limited by the need to employ isotope ratio mass spectrometry (IRMS) to obtain the high-precision isotopic abundance analyses needed to optimize the dose of expensive (18) O-labeled water. Recently, cavity-ring down spectroscopy (CRDS) instruments have become commercially available and may serve as a less expensive alternative to IRMS. We compared the precision and accuracy of CRDS with those of IRMS for the measurement of total energy expenditure from urine specimens in 14 human subjects. The relative accuracy and precision (SD) for total body water was 0.5 ± 1% and for total energy expenditure was 0.5 ± 6%. The CRDS instrument displayed a memory between successive specimens of 5% for (18) O and 9% for (2) H. The memory necessitated carefully ordering of specimens to reduce isotopic disparity, performance of several injections of each specimen to condition the analyzer, and use of a mathematical memory correction on subsequent injections. These limited the specimen throughput to about one urine specimen per hour. CRDS provided accuracy and precision for isotope abundance measurements of urine that were comparable with those of IRMS. The memory problems were easily recognized by our experienced laboratory staff, but future efforts should be aimed at reducing the memory of the CRDS so that it would be less likely to result in poor reproducibility in laboratories using doubly labeled water for the first time., (Copyright © 2010 John Wiley & Sons, Ltd.)

The assessment of food intake is essential for the development of dietetic interventions. Accuracy is low when intake is assessed by questionnaires, the under-reporting of food intake being frequent. Most such studies, however, were performed in developed countries and there is little data about the older population of developing nations. This study aimed to verify the total energy expenditure (TEE) of independent older Brazilians living in an urban area, through the doubly labelled water (DLW) method and to compare it with the reported energy intake obtained through the application of a food frequency questionnaire (FFQ). Initially, 100 volunteers aged from 60 to 75 years had their body composition determined by dual-energy X-ray absorptiometry (DEXA). Five volunteers of each quartile of body fat percentage had their energy expenditure determined by DLW. The mean age of the subjects included in this phase of the study was 66.4 +/- 3.5 years, and ten of the subjects were men. The mean TEE was 2565 +/- 614 and 2154 +/- 339 kcal.day(-1) for men and women, respectively. The Physical Activity Level (PAL) was 1.58 +/- 0.31 and 1.52 +/- 0.22, respectively. Under-reporting of food intake was highly prevalent, with a mean percentage of reported intake in relation to measured TEE of -17.7%. Thus, under-reporting of food intake is highly prevalent among Brazilian independent older persons. The DLW method is an important tool in nutritional studies and its use is to be recommended in developing countries., (Copyright 2010 John Wiley & Sons, Ltd.)

The doubly labelled water method is valuable for measuring energy expenditure in humans. It usually involves blood or urine sampling, which might be difficult in neonates and children with cerebral palsy or other disabilities. We therefore aimed to validate a method making use of saliva samples analyzed by automated thermal conversion elemental analyzer in combination with isotope ratio mass spectrometry (TC-EA/IRMS). The subjects received labelled water orally and urine and saliva samples were collected and analyzed. Deuterium as well as oxygen18 was measured in one single run using a peak jump method. Excellent linearity was found for measurement of enrichments of deuterium (R2 = 0.9999) and oxygen18 (R2 = 0.9999). The intra-assay precision and the inter-assay precision of the measurement of two standards were good for both deuterium and oxygen18. The variation between urine and saliva samples was small (4.83% for deuterium and 2.33% for oxygen18 n = 40). Saliva sampling is to be preferred, therefore, as it can be easily collected and is non-invasive. Moreover, its time of production is almost exactly known. The TC-EA/IRMS method is a good alternative to the more laborious off-line IRMS measurements., (Copyright 2009 John Wiley & Sons, Ltd.)

The stable oxygen and hydrogen isotope compositions of organic samples are increasingly being used to investigate patterns of animal migration. Relatively few studies have applied these techniques to modern humans, despite a variety of potential forensic applications. We analyzed drinking water and food at two geographic locations, East Greenbush, New York (USA) and Fairbanks, Alaska (USA), with different delta(18)O and deltaD values for precipitation and tap water. Foods varied widely in measured delta(18)O and deltaD values, but not systematically by purchase location. We measured delta(18)O and deltaD values of scalp hair from five residents at each location. We used a mixing model to establish the proportion of oxygen and hydrogen in head hair derived from drinking water ( approximately 27% and approximately 36%, respectively). Finally, we analyzed the delta(18)O and deltaD values of facial hair and urine from a subject who traveled from Fairbanks to East Greenbush, on to the UK and back to Fairbanks. Urine delta(18)O and deltaD values responded immediately and strongly to travel-related change in drinking water, and were well described by a single-pool turnover model. Beard hair delta(18)O values tracked changes in urine delta(18)O closely, and oscillated between the values for the resident populations in both locations. In contrast, beard hair deltaD values did not track changes in urine deltaD as well, and retained a signature of the traveler's permanent residence. Our findings show that the delta(18)O and deltaD values of urine and facial hair (specifically delta(18)O) can provide a record of the geographical movements of humans., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Background: Strategies for the prevention and treatment of childhood obesity require a better understanding of the relation between the pattern of free-living physical activity and total energy expenditure (TEE)., Objective: We assessed the relations between TEE and physical activity level (PAL) during engagement in different intensities of physical activity., Design: We used a cross-sectional study of 104 children (median age: 5.4 y) in Scotland. TEE was measured with use of doubly labeled water (DLW), and resting energy expenditure was predicted to determine PAL. Time spent sedentary and in light-intensity activity and in moderate- and vigorous-intensity physical activity (MVPA) was assessed by accelerometry concurrent with DLW measurements. Correlation and regression were used to assess the relations between measures of sedentary behavior, intensities of activity, and PAL as the dependent variable., Results: Time spent sedentary was negatively correlated with PAL (r = -0.33, P < 0.01), and time spent in light-intensity activity was positively correlated with PAL (r = 0.31, P < 0.01). In multiple regression analyses, both time spent sedentary and in light-intensity activities were significantly associated with PAL. Time spent in MVPA was not associated with PAL; engagement in MVPA was limited in this sample (median: 3% of waking hours; range: 0-14%). PAL was significantly higher in boys than in girls., Conclusion: In this sample and setting, PAL was not influenced by engagement in MVPA but was influenced by time spent sedentary and in light-intensity activities. This study suggests that in young children, MVPA could make only a minor contribution to free-living TEE and PAL.

Background: Concerns of a decrease in physical activity levels (PALs) of children and a concurrent increase in childhood obesity exist worldwide. The exact relation between these two parameters however has as yet to be fully defined in children., Objective: This study examined the relation in 47 children, aged 5-10.5 y (mean age 8.4+/-0.9 y) between habitual physical activity, minutes spent in moderate, vigorous and hard intensity activity and body composition parameters., Design: Total energy expenditure (TEE) was calculated using the doubly labelled water technique and basal metabolic rate (BMR) was predicted from Schofield's equations. PAL was determined by PAL=TEE/BMR. Time spent in moderate, vigorous and hard intensity activity was determined by accelerometry, using the Tritrac-R3D. Body fatness and body mass index (BMI) were used as the two measures of body composition., Results: Body fat and BMI were significantly inversely correlated with PAL (r=-0.43, P=0.002 and r=-0.45, P=0.001). Times spent in vigorous activity and hard activity were significantly correlated to percentage body fat (r=-0.44, P=0.004 and r=-0.39, P=0.014), but not BMI. Children who were in the top tertiles for both vigorous activity and hard activity had significantly lower body fat percentages than those in the middle and lowest tertiles. Moderate intensity activity was not correlated with measures of body composition., Conclusions: As well as showing a significant relation between PAL and body composition, these data intimate that there may be a threshold of intensity of physical activity that is influential on body fatness. In light of world trends showing increasing childhood obesity, this study supports the need to further investigate the importance of physical activity for children.

Purposes: To validate the energy expenditure estimated from The Minnesota Leisure Time Physical Activity Questionnaire (MLTPAQ) with total energy expenditure (TEE) measured by doubly labeled water (DLW), and to present and examine the validity of an extended version of the MLTPAQ with additional questions about inactivity during leisure time (eMLTPAQ), in a sample of Swedish 15-yr-old adolescents., Methods: Thirty-five 15-yr-old adolescents were interviewed using the eMLTPAQ. In addition to anthropometry, indirect calorimetry was measured to assess basal metabolic rate, and TEE was assessed by the DLW method over a 14-d period., Results: Energy expenditure calculated from MLTPAQ correlated well with TEEDLW (r=0.49, P<0.01), and the correlation increased when including questions about inactivity (r=0.73, P<0.01). However, eMLTPAQ underestimated TEE in 34 of the 35 students, with a mean difference between the methods of 2.8 MJ.d(-1) (95% limits of agreement: -0.1 to 5.6 MJ.d(-1)), which mainly was explained by a relative high intensity in the time which remained unreported., Conclusion: eMLTPAQ is valid in ranking adolescents energy expenditure and in describing patterns of leisure time physical activities.

We have developed an assay for determining the 18O enrichment of water in biological fluids. Urine, plasma, or whole blood is reacted with phosphorous pentachloride to yield phosphoric acid. Derivatization of phosphoric acid with diazomethane generates trimethyl phosphate. The enrichment of trimethyl phosphate is nearly four times that of water and is assayed using gas chromatography-mass spectrometry (electron impact ionization). Yang et al. (1998, Anal. Biochem. 258, 315-321) assayed the 2H enrichment of body water after exchange with acetone, by gas chromatography-mass spectrometry. The combination of our 18O method and the 2H method of Yang et al. allows one to measure energy expenditure via "doubly labeled" water (2H(2)O + H(2)18O), using small samples of body fluids. These techniques were used to measure energy expenditure in mice, in which the 18O enrichment of body water can be monitored down to 0.025%.

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.

The doubly labeled water method for measuring total energy expenditure is subject to error from natural variations in the background 2H and 18O in body water. There is disagreement as to whether the variations in background abundances of the two stable isotopes covary and what relative doses of 2H and 18O minimize the impact of variation on the precision of the method. We have performed two studies to investigate the amount and covariance of the background variations. These were a study of urine collected weekly from eight subjects who remained in the Madison, WI locale for 6 wk and frequent urine samples from 14 subjects during round-trip travel to a locale > or = 500 miles from Madison, WI. Background variation in excess of analytical error was detected in six of the eight nontravelers, and covariance was demonstrated in four subjects. Background variation was detected in all 14 travelers, and covariance was demonstrated in 11 subjects. The median slopes of the regression lines of delta2H vs. delta18O were 6 and 7, respectively. Modeling indicated that 2H and 18O doses yielding a 6:1 ratio of final enrichments should minimize this error introduced to the doubly labeled water method.