Impact of 2H and 18O Pool Size Determinations on the Calculation of Total Energy Expenditure

Measurement of total energy expenditure using [²H,¹⁸O]water requires both accurate and precise determination of the rates of disappearance of ²H and ¹⁸O from body water over time and determination of the ²H and ¹⁸O pool sizes. However, the impact of the isotopic determination of body water upon the determination of energy expenditure is often overlooked. For measurement of total body water per se, the delay after administration before sampling body fluids becomes important, and saliva sampling can be used to resolve the timing of early samples for body water determination. For energy expenditure measurement per se, linear regression can be used to define the initial dilution. Because the hydrogen tracer dilutes into a pool significantly larger than body water pool per se due to the presence of labile hydrogens, a correction to the isotope pool size must be applied. The theoretical calculations of the exchangeable hydrogen pool presented here suggest that the hydrogen pool size is <3% greater than the body water pool and data are provided to support this idea. Finally, the two approaches used to define the body water pool space contribution to the calculation of energy expenditure using ²H₂¹⁸O are reviewed. Using a pool size based upon the average of the two pool spaces limits the effect of pool size error in the calculation of energy expenditure.     

Effect of bolus fluid intake on energy expenditure values as determined by the doubly labeled water method.

The doubly labeled water (DLW, 2H(2)18O) method is a highly accurate method for measuring energy expenditure (EE). A possible source of error is bolus fluid intake before body water sampling. If there is bolus fluid intake immediately before body water sampling, the saliva may reflect the ingested water disproportionately, because the ingested water may not have had time to mix fully with the body water pool. To ascertain the magnitude of this problem, EE was measured over a 5-day period by the DLW method. Six subjects were dosed with 2H2(18)O. After the reference salivas for the two-point determination were obtained, subjects drank water (700-1,000 ml), and serial saliva samples were collected for the next 3 h. Expressing the postbolus saliva enrichments as a percentage of the prebolus value, we found 1) a minimum in the saliva isotopic enrichments were reached at approximately 30 min with the minimum for 2H (95.48 +/- 0.43%) being significantly lower than the minimum for 18O (97.55 +/- 0.44, P less than 0.05) and 2) EE values calculated using the postbolus isotopic enrichments are appreciably higher (19.9 +/- 7.5%) than the prebolus reference values. In conclusion, it is not advisable to collect saliva samples for DLW measurements within approximately 1 h of bolus fluid intake.     

Measurement of energy expenditure in humans by doubly labeled water method

The utility of the doubly labeled water method for the determination of energy expenditure and water output was investigated in humans. Approximately 10 g of 18O and 0.5 g of 2H as water was orally administered to four healthy adults. Total body water was determined from the isotope dilution, and the ensuing 18O and 2H disappearance rates from body water were determined for 13 days by mass spectrometric isotope ratio analysis of the urinary water. During this period, subjects were maintained on a measured diet to determine energy and water intake. The energy expenditure from the doubly labeled water method differed from dietary intake plus change in body composition by an average of 2%, with a coefficient of variation of 6%. The water outputs determined by the two methods differed by 1%, with a coefficient of variation of 7%. The doubly labeled water method is noninvasive, and the subjects could maintain their daily activities without restriction.     

Evaluation of dietary assessment instruments against doubly labeled water, a biomarker of habitual energy intake

Epidemiological studies of diet and disease rely on the accurate determination of dietary intake and subsequent estimates of nutrient exposure. Although methodically developed and tested, the instruments most often used to collect self-reported intake data are subject to error. It had been assumed that this error was only random in nature; however, an increasing body of literature suggests that systematic error in the reporting of true dietary intake exists as well. Here, we review studies in which dietary intake by self report was determined while energy expenditure was simultaneously measured using the doubly labeled water (DLW) method. In seeking to establish the relative accuracy of each instrument to capture true habitual energy intake, we conclude that none of the self-reported intake instruments demonstrates greater accuracy against DLW. Instead, it is evident that the physical and psychological characteristics of study participants play a significant role in the underreporting bias observed in these studies. Further research is needed to identify underreporters and to determine how to account for this bias in studies of diet and health.     

A rapid analytical technique for the determination of energy expenditure by the doubly labelled water method

The doubly labelled water method involves the administration of water enriched in 2H and 18O followed by determination of the turnover rates of these isotopes. Since 18O is eliminated from the body as both CO2 and water, while 2H leaves only as water, the difference between the two turnover rates provides a measure of CO2 production and hence energy expenditure. Isotopic analysis by conventional stable isotope ratio analysis (SIRA) is labour intensive and time consuming, as it requires off-line conversion of water samples to gases (H2 and CO2) followed by sequential analysis for each of the two isotopes using the mass spectrometer. Lack of suitable automated instrumentation with the ability to process large numbers of samples has prevented routine application of the method. We describe here an automated technique in which body water samples (urine, saliva, breath water or milk) are analysed simultaneously for 2H and 18O. The single bench system comprises two mass spectrometer analysers, one for measuring 2H from H2 gas, the other for measuring 18O from the water vapour (masses 18, 20). Both analysers share a common heated inlet system into which microlitre quantities of the body fluids are injected from an autosampler (102 samples). The water vapour flows both directly to one analyser for 18O measurement and into a uranium reduction furnace for conversion to H2, prior to 2H measurement by the second analyser. Both analysers also share vacuum and electronic components, enabling savings in both space and cost. In this paper we present results illustrating performance characteristics and procedures for routine application to human subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple Laboratory Comparison of the Doubly Labeled Water Technique*

A double-blind study was conducted to determine between-laboratory variability in the doubly labeled water method for measurement of total energy expenditure in humans, and to compare the accuracy and precision of three widely-used procedures for calculating rates of carbon dioxide production from the original isotope data. Eighteen laboratories from five countries participated in the study. All laboratories were provided with five water standards containing varying amounts of ²H and ¹⁸O, and in addition 11 laboratories were provided with urine and dose specimens from one (six laboratories) or two (five laboratories) healthy elderly subjects of normal height and weight undergoing a calorimetric validation of the doubly labeled water method. The data from the five water standards were analyzed to predict between-laboratory variability in the doubly labeled water technique in all laboratories. In addition, data from the subjects were analyzed using the “slope-intercept”, “2-point” and “modified” methods of calculation. The results confirm that the doubly labeled water method can be an accurate technique for the measurement of energy expenditure in adult human subjects in some laboratories. However, there was substantial between-laboratory variability in the results and some laboratories returned physiologically impossible results. There was no significant effect of calculation procedure on the accuracy of the technique in this limited comparison, although the slope-intercept procedure appeared to be more susceptible to analytical error than the other procedures. The isotope standards analyzed by participants in this study will be made available to other investigators on request.     

Replacing sweetened caloric beverages with drinking water is associated with lower energy intake

Objective: Reduced intake of sweetened caloric beverages (SCBs) is recommended to lower total energy intake. Replacing SCBs with non‐caloric diet beverages does not automatically lower energy intake, however. Compensatory increases in other food or beverages reportedly negate benefits of diet beverages. The purpose of this study was to evaluate drinking water as an alternative to SCBs. Research Methods and Procedures: Secondary analysis of data from the Stanford A TO Z intervention evaluated change in beverage pattern and total energy intake in 118 overweight women (25 to 50 years) who regularly consumed SCBs (>12 ounces/d) at baseline. At baseline and 2, 6, and 12 months, mean daily beverage intake (SCBs, drinking water, non‐caloric diet beverages, and nutritious caloric beverages), food composition (macronutrient, water, and fiber content), and total energy intake were estimated using three 24‐hour diet recalls. Beverage intake was expressed in relative terms (percentage of beverages). Results: In fixed effects models that controlled for total beverage intake, non‐caloric and nutritious caloric beverage intake (percentage of beverages), food composition, and energy expenditure [metabolic equivalent (MET)], replacing SCBs with drinking water was associated with significant decreases in total energy intake that were sustained over time. The caloric deficit attributable to replacing SCBs with water was not negated by compensatory increases in other food or beverages. Replacing all SCBs with drinking water was associated with a predicted mean decrease in total energy of 200 kcal/d over 12 months. Discussion: The results suggest that replacing SCBs with drinking water can help lower total energy intake in overweight consumers of SCBs motivated to diet.     

Gas chromatography-mass spectrometry assay of the (18)o enrichment of water as trimethyl phosphate

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%.     

Milk intake and energy expenditure of free-ranging northern fur seal, Callorhinus ursinus, pups

Milk ingested by mammalian offspring, coupled with offspring's utilization of this energetic investment, influences survival and growth. A number of studies have examined milk intake in otariids, but few have examined milk intake over the entire lactation period, and none has independently measured energy expenditure concurrent with milk intake. We concurrently examined milk intake, field metabolic rate (FMR), and body composition of 41 pups over the entire lactation interval in 1995 and 1996 on St. Paul Island, Alaska. One hundred two metabolic measurements were obtained with isotope dilution methods. Mean milk intake did not differ annually but increased with age and mass, ranging from 3,400+/-239 to 6,780+/-449 (+/-SE) mL per suckling bout. Milk energy consumption did not vary with age on a mass-specific basis. No differences were detected in milk volume consumed by male and female pups, either absolutely or on a mass-specific basis. Mass-specific FMR peaked during molting, was lowest postmolt, and did not vary by sex. Pups in 1995 had lower FMR than pups in 1996 and were also fatter. Mean milk energy utilized for maintenance metabolism decreased over time from 77% to 43% in 1995 and remained at 71% in 1996. Pup body mass was negatively correlated with the percentage of total body water and positively correlated with the percentage of total body lipid (TBL). Pups increased the percentage of TBL from 16% to 37%. Northern fur seal pups increased energy intake over lactation, while concurrent changes in body composition and pelage condition resulted in mass-specific metabolic savings after the molt.     

Sensitivity of methods for calculating energy expenditure by use of doubly labeled water

Attempts to estimate human energy expenditure by use of doubly labeled water have produced three methods currently used for calculating carbon dioxide production from isotope disappearance data: 1) the two-point method, 2) the regression method, and 3) the integration method. An ideal data set was used to determine the error produced in the calculated energy expenditure for each method when specific variables were perturbed. The analysis indicates that some of the calculation methods are more susceptible to perturbations in certain variables than others. Results from an experiment on one adult human subject are used to illustrate the potential for error in actual data. Samples of second void urine, 24-h urine, and breath collected every other day for 21 days are used to calculate the average daily energy expenditure by three calculation methods. The difference between calculated energy expenditure and metabolizable energy on a weight-maintenance diet is used to estimate the error associated with the doubly labeled water method.     

Approaches for quantifying energy intake and %calorie restriction during calorie restriction interventions in humans: the multicenter CALERIE study

Calorie restriction (CR) is a component of most weight loss interventions and a potential strategy to slow aging. Accurate determination of energy intake and %CR is critical when interpreting the results of CR interventions; this is most accurately achieved using the doubly labeled water method to quantify total energy expenditure (TEE). However, the costs and analytical requirements of this method preclude its repeated use in many clinical trials. Our aims were to determine 1) the optimal TEE assessment time points for quantifying average energy intake and %CR during long-term CR interventions and 2) the optimal approach for quantifying short-term changes in body energy stores to determine energy intake and %CR during 2-wk DLW periods. Adults randomized to a CR intervention in the multicenter CALERIE study underwent measurements of TEE by doubly labeled water and body composition at baseline and months 1, 3, and 6. Average %CR achieved during the intervention was 24.9 ± 8.7%, which was computed using an approach that included four TEE assessment time points (i.e., TEE(baseline, months 1, 3, and 6)) plus the 6-mo change in body composition. Approaches that included fewer TEE assessments yielded %CR values of 23.4 ± 9.0 (TEE(baseline,) months 3 and 6), 25.0 ± 8.7 (TEE(baseline,) months 1 and 6), and 20.9 ± 7.1% (TEE(baseline, month 6)); the latter approach differed significantly from approach 1 (P < 0.001). TEE declined 9.6 ± 9.9% within 2-4 wk of CR beginning and then stabilized. Regression of daily home weights provided the most reliable estimate of short-term change in energy stores. In summary, optimal quantification of energy intake and %CR during weight loss necessitates a TEE measurement within the first month of CR to capture the rapid reduction in TEE.     

Doubly labeled water measurement of human energy expenditure during strenuous exercise

The energy expenditures (EE) of 23 adult male Marines were measured during a strenuous 11-day cold-weather field exercise at 2,200- to 2,550-m elevation by both doubly labeled water (2H2 18O, DLW) and intake balance methods. The DLW EE calculations were corrected for changes in baseline isotopic abundances in a control group that did not receive 2H2 18O. Intake balance EE was estimated from the change in body energy stores and food intake. Body energy-store changes were calculated from anthropometric [-1,574 +/- 144 (SE) kcal/day] and isotope dilution (-1,872 +/- 293 kcal/day) measurements made before and after the field exercise. The subjects kept daily logbook records of ration consumption (3,132 +/- 165 kcal/day). Mean DLW EE (4,919 +/- 190 kcal/day) did not differ significantly from intake balance EE estimated from food intake and either anthropometric (4,705 +/- 181 kcal/day) or isotope dilution (5,004 +/- 240 kcal/day) estimates of the change in body energy stores. The DLW method can be used with at least the same degree of confidence as the intake balance method to measure the EE of active free-living humans.     

Variation in energy metabolism and water flux of free-ranging male lace monitors,Varanus varius (Squamata: Varanidae)

The energy and water used by Varanus varius correlated with changes in weather, activity, and possibly the availability of prey. In summer, CO(2) production and water influx rates were high (0.147 mL CO(2) g(-1) h(-1) and 23.6 mL H(2)O kg(-1) d(-1)) but substantially lower during autumn (0.053 mL CO(2) g(-1) h(-1) and 9.1 mL H(2)O kg(-1) d( -1)) and winter (0.016 mL CO(2) g(-1) h(-1) and 2.4 mL H(2)O kg(-1) d(-1)), increasing again in spring (0.052 mL CO(2) g(-1) h(-1) and 7.9 mL H(2)O kg(-1) d(-1)). The summer-winter difference represented more than a ninefold reduction in energy expenditure and water flux. However, individual V. varius could manipulate their energy and water requirements by up to sixfold during the summer period by regulating activity. Although we found no adaptive benefits of increased or decreased level of activity, we did find that larger animals moved more frequently and over greater distances than smaller animals. We hypothesise that V. varius regulates its activity on the basis of the trade-off between energy expenditure through activity and energy acquisition through foraging.     

Constraints on energy intake in fish: the link between diet composition, energy metabolism, and energy intake in rainbow trout

The hypothesis was tested that fish fed to satiation with iso-energetic diets differing in macronutrient composition will have different digestible energy intakes (DEI) but similar total heat production. Four iso-energetic diets (2 × 2 factorial design) were formulated having a contrast in i) the ratio of protein to energy (P/E): high (H(P/E)) vs. low (L(P/E)) and ii) the type of non-protein energy (NPE) source: fat vs. carbohydrate which were iso-energetically exchanged. Triplicate groups (35 fish/tank) of rainbow trout were hand-fed each diet twice daily to satiation for 6 weeks under non-limiting water oxygen conditions. Feed intake (FI), DEI (kJ kg(-0.8) d(-1)) and growth (g kg(-0.8) d(-1)) of trout were affected by the interaction between P/E ratio and NPE source of the diet (P<0.05). Regardless of dietary P/E ratio, the inclusion of carbohydrate compared to fat as main NPE source reduced DEI and growth of trout by ~20%. The diet-induced differences in FI and DEI show that trout did not compensate for the dietary differences in digestible energy or digestible protein contents. Further, changes in body fat store and plasma glucose did not seem to exert a homeostatic feedback control on DEI. Independent of the diet composition, heat production of trout did not differ (P>0.05). Our data suggest that the control of DEI in trout might be a function of heat production, which in turn might reflect a physiological limit related with oxidative metabolism.     

Differences of energy intake and energy expenditure of elite Taekwondo players receiving summer vs. winter intensive training

[Purpose]

This study aimed to evaluate the energy expenditure and energy intake as an experiment of energy balance of elite Taekwondo players receiving summer vs. winter intensive training.

[Methods]

The summer training group (STG, n = 15) and the winter training group (WTG, n = 18) wore an accelerometer for the measurement of energy expenditure and maintained a daily dietary record for measurement of energy intake, for seven consecutive days during summer or winter intensive training.

[Results]

The total energy expenditure (TEE) (834.1 kcal, p < .001), the total counts (1,867 counts, p = .038), and the energy expenditure during moderate (384.6 kcal, p < .001) and vigorous activity (351.8 kcal, p < .001) were significantly lower in the STG than in the WTG. On the other hand, the macronutrient intake showed that intake of energy (902.7 kcal, p < .001), carbohydrates (82.6g, p < .001), and protein (93.9g, p < .001) in the STG were significantly lower than those of the WTG. When comparing TEE and total energy intake, the STG consumed 902.7 kcal less as energy than the WTG, while the STG performed 834.1 kcal more as TEE than that of the WTG.

[Conclusion]

In conclusion, both of the summer and winter intensive training are not proper for energy balance condition. Therefore, to promote the optimal energy balance during the summer or winter intensive training period, a training and sports nutrition program based on the amount of energy expenditure and energy intake for Taekwondo players should be considered.     

Milk intake and carbon dioxide production of piglets determined with the doubly labelled water technique

The present study was undertaken to study different methodological aspects of quantifying CO2 production and milk intake of suckling piglets using the doubly labelled water (DLW) technique. In total, 37 piglets were enriched intraperitoneally with DLW to study equilibration time of 18O (n = 3), to validate the estimation of milk intake and CO2 production (n = 10) of piglets fed milk replacer and to quantify milk intake and CO2 production of piglets nursed ordinarily by sows (n = 24). Enrichment of 18O in expired air was analysed without any sample preparation, whereas enrichment of 18O in serum was analysed after a minimum step of sample preparation, which included pipetting of the sample, blowing gaseous CO2 into the vial for 3 s and equilibrating for 24 h. The 18O enrichment of CO2 in expired air was constant within 30-40 min of intraperitoneal injection, suggesting that DLW was equilibrated within the body water by that time. For piglets fed milk replacer, the estimation of the daily CO2 production by the DLW method (64.0 ± 2.7 l CO2/day) was in agreement with that obtained by respiration trials (64.7 ± 1.8 l CO2/day). Furthermore, the intake of milk replacer (891 ± 63 g/day) determined by deuterium oxide (D2O) dilution was similar in magnitude to that found by weighing the milk disappearance (910 ± 58 g/day). The milk intake of piglets fed milk replacer was comparable with that of sucking piglets, but sucking piglets had a remarkably higher CO2 production than artificially reared piglets, which likely was caused by a higher intake of milk solids and a higher activity level. For sucking piglets, the daily CO2 production increased curvilinearly with increasing live weight (LW) in kg: piglet CO2 production (l/day) = 25.75 × LW - 1.01 × LW2. In conclusion, 18O equilibrates fast within the body water pool when administered intraperitoneally, and the accuracy of assessing milk intake and rate of CO2 production using the DLW technique is promising. Assessment of excess enrichment of 18O in serum proved to be robust. Finally, the CO2 production of piglets fed milk replacer differs considerably from that of sucking piglets.     

The effect of lysine intake on energy partitioning and utilization in growing pigs

An experiment utilizing 12 castrated male pigs within a body weight range of 23 - 147 kg was conducted to ascertain whether the alteration of protein quality by varying the level of lysine intake is influencing total energy retention, heat production and therewith efficiency of energy utilization for growth. The animals were allotted to two treatments of a constant medium (11.5 g/d) or high lysine intake (13.5 g/d) level on the basis of an isonitrogenous diet at an energy intake level of 1.3 MJ ME/kg BW0.75. Representing a tool for determining body composition, at target body weights of 35, 55, 80, 115 and 145 kg measurements of deuterium dilution space were undertaken. Protein and lipid accretion were calculated by difference, assuming accretion to contain 23.8 and 39.0 kJ/g, respectively. The results show a significant effect (p < 0.05) between treatment groups for the values of energy retained in protein, thus ensuring the intended alteration by protein quality. Furthermore total energy retention, heat production (difference between ME intake and energy retention) and therewith energy utilization demonstrate independence from the composition of body weight (BW) gain. These observations confirm earlier results, but however, seem to be in contrast to the supposition of a constant efficiency for protein (kp) and fat (kf) accretion, respectively. This may be attributed to a variable kp, in fact to a smaller kp at minor values for protein accretion due to an increased whole body protein turnover. Lacking evidence from experimental data for advantages in using constant values for kp and kf to determine the accurate energy requirement for growth, a uniform value for the efficiency of total energy retention seems to be more adequate.     

Effects of endurance exercise on metabolic water production and plasma volume

Six endurance-trained and heat-acclimatized adult males ran for 1 h (or until exhaustion) at room temperature (23.8 degrees C) on three occasions. The work loads approximated 37, 56, and 74% of the subjects' aerobic capacities. Venous blood samples were drawn, and urine was collected before and immediately after each exercise bout. Metabolic cost was partitioned by energy substrate, and metabolic water production was quantified from urinary nitrogen, oxygen, and carbon dioxide production. Total body water loss was recorded as the decrease in body weight during the exercise. All subjects completed 1 h of exercise at the two lower exercise intensities but, due to exhaustion, averaged only 35.5 min at the highest work intensity. There were no significant changes in plasma volume after the exercise bouts. Metabolic water production increased with increasing work intensity as did the fraction of total caloric expenditure derived from carbohydrate metabolism. Plasma protein content significantly increased at all levels of exercise intensity. Metabolic water production alone would be of minimal help in plasma volume maintenance and thermoregulation during endurance exercise.     

Use of the doubly labeled water technique in humans during heavy sustained exercise

We measured energy expenditure with the doubly labeled water technique during heavy sustained exercise in the Tour de France, a bicycle race lasting more than 3 wk. Four subjects were observed for consecutive intervals of 7, 8, and 7 days. Each interval started with an oral isotope dose to reach an excess isotope level of 200 ppm 18O and 130 ppm 2H. The biological half-lives of the isotopes were between 2.25 and 3.80 days. Energy expenditure was compared with simultaneous measurements of energy intake, and body mass and body composition did not change significantly. The doubly labeled water technique gave higher values for energy expenditure than the food record technique. The discrepancy showed a systematic increment from the first to the third interval, being 12.9 +/- 7.9, 21.4 +/- 9.8, and 35.3 +/- 4.4% of the energy expenditure calculated from dietary intake, respectively. Possible explanations for the discrepancy are discussed. The subjects reached an average daily metabolic rate of 3.4-3.9 or 4.3-5.3 times basal metabolic rate based on the food record technique and the doubly labeled water technique, respectively. Thus, when measured with the same technique, the energetic ceiling for performance in humans is comparable with that of animals like birds.     

Divergence of melanocortin pathways in the control of food intake and energy expenditure

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.