International Journal of Obesity (2003) 27, 641–647 & 2003 Nature Publishing Group All rights reserved 0307-0565/03 $25.00 www.nature.com/ijo PAPER Measurement of total energy expenditure in grossly obese women: comparison of the bicarbonate– method with whole-body and free-living doubly labelled water

ER Gibney1, P Murgatroyd2, A Wright3, S Jebb3 and M Elia4*

1Department of Biochemistry, Trinity College Dublin, Dublin, Ireland; 2Wellcome Trust Clinical Research Facility, Addenbrookes Hospital, Hills Road, Cambridge, UK; 3MRC Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge, UK; and 4Institute of Human Nutrition, Southampton General Hospital, Southampton, UK

OBJECTIVE: To establish validity of the bicarbonate–urea (BU) method against direct measurements of gaseous exchange (GE) in a whole-body indirect calorimeter and to compare BU and doubly labelled water (DLW) measurements in free-living conditions in the same group of grossly obese women. DESIGN: Energy expenditure (EE) was estimated by the BU method over 24 h concurrently with whole-body indirect calorimetry and subsequently over 5 consecutive days at home concurrently with 14 day DLW. Six women, body mass index (BMI) 52.4710.4 kg/m2 (s.d.), were studied. RESULTS: Total energy expenditure (TEE) measurements by BU and GE within the metabolic chamber were not significantly different (BU ¼ 11.7971.89 MJ/day and GE ¼ 11.6471.86 MJ/day; mean difference, 0.2570.49 MJ/day, P40.05). Free-living TEE derived from BU and DLW was also similar (13.2871.86 and 13.8672.25 MJ/day, respectively; mean difference 0.1771.33 MJ/day, Po0.05). The measured physical activity level (PAL) in these very obese subjects was within the range reported in other free-living studies in less obese individuals (1.6270.14 using DLW and 1.5670.20 using BU). The BU method was well tolerated by the subjects. CONCLUSIONS: This study in grossly obese subjects, heavier than those participating in previous studies involving tracer methods, demonstrates validity of the BU against GE under controlled metabolic conditions, and the equivalence between BU and DLW under free-living conditions. The results suggest that both tracer methods are valid in this population group. This study also demonstrates the practicalities of using the BU method over 5 days, the longest application of the method so far. International Journal of Obesity (2003) 27, 641–647. doi:10.1038/sj.ijo.0802302

Keywords: energy expenditure; bicarbonate urea; indirect calorimetry; doubly labelled water; validation; free living

Introduction on inferring intake from measurements of free-living total Obesity is one of the most important public health problems energy expenditure (TEE) using tracer methods, particularly facing both developed and developing countries.1 An the doubly labelled water (DLW) method. important aspect of the study of obesity is the assessment The DLW water method is an isotopic dilution method, of energy balance and intake. However, the accurate where energy expenditure is calculated from endogenous assessment of energy intake (EI) is difficult, especially in CO2 production, which is derived by the differential obese individuals, because of considerable under-report- elimination of the isotopes 2H and 18O from the body water ing,2,3 and hence, considerable emphasis has been placed pool.4 The DLW method has been validated against both gaseous exchange (GE) (considered to be the gold standard for the estimation of energy expenditure (EE)),5–9 and energy *Correspondence: Professor M Elia, Institute of Human Nutrition, South- balance studies.10,11 Some variation has been reported ampton General Hospital, Tremona Road, Southampton SO16 6YD, UK. between the results of validation studies, which has been E-mail: [email protected] Received 18 March 2002; revised 27 January 2003; attributed to the different physiological states of the accepted 3 February 2003 individuals and errors associated with different analytical Measurement of total energy expenditure in obese women ER Gibney et al 642 methods undertaken by each laboratory.12–14 However, the rate. The two methods are thus complementary to each overall precision of estimating EE using the DLW is estimated other. to be within 73–6%.7,11,12,15–19 Therefore, the DLW method The BU method has been validated against respiratory is considered to be a valid method for estimation of free- gas exchange in both healthy lean individuals34 and living TEE, and has been applied to a range of population patients with lung cancer.35 The method has also been groups including the elderly,20 infants,18,21,22 sick22–29 applied to free-living healthy subjects,34 patients with undernourished30 and obese.15,31 lung cancer,35 meliodosis,36 HIV infection37 and chronic One validation study of particular interest is that of obstructive pulmonary (airway) disease (COPD).38 An analy- Ravussin et al,15 who compared DLW to GE in a whole-body sis of published data show that BU method underestimated metabolic chamber in a group of individuals with differing EE obtained by GE by only 0.0570.31 MJ/day, and is body mass index (BMI). The study reported an under- unrelated to the amount of body fat (r ¼ 0.05 using pooled estimation of EE by DLW that was related to fat mass data based on two studies in which total body fat ranged (r ¼À0.81, Po0.002) and percent body fat (r ¼À0.68, from 5 to 36 kg and BMI from 21 to 33 kg/m2 34,35.) However, Po0.02). The magnitude of this effect, À0.285 MJ/day for the majority of subjects in these studies were not obese and each additional 10 kg of fat, is certainly sufficient to be of span a limited range of body fat. The first element of the concern if the population is particularly obese, has wide current study, within the whole-body calorimeter, was variability in adiposity, or shows substantial changes in body designed to extend these validations to a grossly obese weight. The authors suggested that the underestimation population. could be because of larger sequestration of deuterium during The second elements is that this study tests the practicality fat synthesis in the obese subjects. However, Haggarty et al32 of the BU method under free-living conditions, provides the estimated the whole-body fatty acid and cholesterol synth- first direct comparison of the BU and DLW techniques in esis in weight-stable adults and demonstrated little evidence obese subjects, and provides long-term concurrent measure-

of error on DLW-derived CO2 production, with a mean ments of CO2 production by two methods with differing underestimation of À0.5% CO2 production, suggesting that metabolic dependencies from which the observations of the DLW method is unlikely to be seriously affected. No Ravussin et al15 may be further explored. study has investigated the validity of DLW in an obese group since that of Ravussin et al,15 and other studies yielding individual subject data have not supported a relation Subjects and methods 6–10 between adiposity and error in DLW-estimated TEE. Six women with a long history of morbid obesity were There is now a pressing need to re-examine Ravussin’s recruited from a hospital outpatient clinic (Table 1). Their observations and, if they are confirmed, to explore further body weight had varied by less than 2 kg in the 4 weeks prior the mechanisms by which they may occur. to the start of the study. Apart from their obesity, all subjects The bicarbonate–urea (BU) method provides an alternative were considered to be in satisfactory health. Complete to DLW for measurement of free-living CO2 production, medical history, physical examinations and routine blood from which (EE) is then calculated using appropriate measurements were performed. None of the subjects had 33,34 factors. The BU method is based on the principle of diabetes, but some had arthralgias (subject 3 had some isotopic dilution of infused labelled bicarbonate, and the symptoms of osteoarthritis) and a certain degree of exer- assumption that the specific activity of urinary urea (formed tional dyspnoea. The study took place at the MRC Dunn from CO2 in the liver) is an indicator of CO2 production Clinical Nutrition Centre, Cambridge, UK. The study was within the body. The extent of isotopic dilution depends on approved by Medical Research Council (MRC) Dunn Nutri- the rate of entry of labelled CO2, infused as bicarbonate, tion Unit and Addenbrooke’s Hospital Ethics committees. relative to the rate of endogenous CO2 production. When Informed consent was obtained in writing from the volun- CO2 production is high, the enrichment or specific activity teers before beginning the study. 14 of CO2 in urinary urea is low, and the converse. The C-BU method is relatively less expensive than the DLW and can provide estimates of EE over periods from 12–24 h to several 14C bicarbonate administration days. In contrast, the DLW typically provides an integrated The 14C bicarbonate was administered as a primed constant estimate of EE over 10–20 days in adults. Both methods infusion. The prime was given on the day prior to (GE)

estimate net CO2 production, and require conversion of CO2 measurement (day 0) and the infusion commenced. The production to EE. However, the techniques are influenced by infusion (24 Â 106 dpm/day) was administered using the different physical and metabolic processes. For example, the Graseby minipump (Graseby variable-speed driver MS26; DLW is affected by fractionation of isotopes at body surfaces Graseby Medical, Watford, UK) and a bolus urea prime (see and potential sequestration into organic substances,19 while Elia39 for calculation of dose). Details of procedures for the BU method is potentially subject to carbon fixation, inserting the subcutaneous canulla are presented else- 34 14 variable recovery of labelled CO2 through variable equilibra- where. The whole-body radiation exposure from the C tion with urea, which may not be produced at a constant was close to background daily radiation exposure.34

International Journal of Obesity Measurement of total energy expenditure in obese women ER Gibney et al 643 14 Table 1 Subject characteristics full 24-h period, trapping both CO2 and total CO2 leaving the calorimeter. 7 Mean s.d. Complete urine collections were obtained on 5 consecu- tive days from 07:01 to 09:00 h and 09:01 to 07:00 h (2 and Age (y) 43.779.2 22 h collection within each 24 h period), both while in the Body weight (kg) 140.2729.3 Height (m) 1.6070.11 calorimeter and at home, for estimation of the composite Body mass index (kg/m2) 52.4710.4 mean specific activity of urea. Total body water 48.4710.1 Fat-free mass (kg) 62.2713.8 Fat mass (kg) 74.0720.8 Free-living measurements 7 Fat mass (%) 52.6 6.1 At the end of the calorimeter run (09:00 h day 2), subjects

received an oral dose of DLW (0.05 g D2O 100% enrichment and 1.5 g H18O 10% enrichment per kilogram of body Whole-body indirect calorimetry 2 weight). Subjects then refrained from eating or drinking for Continuous measurements of GE were made in all subjects. a 4-h equilibration period. A spot urine sample (B20 ml) was The calorimeter CO analysers were tested for linearity 2 obtained 4 h after dosing. The subjects then returned home throughout the working range. Infusion tests with 80% N 2 and were asked to maintain their habitual activities. Daily and 20% CO at a rate of 1.25 l/min, confirmed the accuracy 2, spot urine samples were collected at a defined time for the of the calorimeter measurements to within 71%. next 12–14 days for DLW analysis, and the remaining urine Subjects entered a whole-body calorimeter at 20:00 h on was added to the complete collections until day 5. day 0. A total of 37 h was spent in the calorimeter chamber, Owing to the size and limitations of the Graseby ending at 09:00 h on day 2. After an overnight equilibration, minipump, a home visit was made on day 3 to replace the GE measurements were recorded from 09:00 h on day 1 syringe containing the 14C bicarbonate infusion, so that the until 09:00 h on day 2. Within the calorimeter, subjects infusion could be maintained to the end of the 5 days. followed the same standardised protocol. Apart from two Weight and height were measured at the start (day 0) and 30-min exercise periods (30 min of cycling at 25 W and end (day 15) of the study. Venous blood was also taken on 30 min of stepping on and off a 20-cm block at a rate day 15 for measurement of circulating bicarbonate and urea 40/min) the subjects remained sedentary. Two subjects were concentrations, liver function tests and full blood count, unable to undertake or complete the exercise because of which were measured according to standard laboratory their physical limitations, shortness of breath (subject 6) methods. and arthritic complications (subject 3). Food was given in three isoenergetic meals (40% fat, 47% carbohydrate and 13% protein), with a food quotient of 0.857.40 Calculations

Individual diets were established for each subject at EE within the metabolic chamber was calculated from O2 41 1.35 Â predicted (BMR). Any food consumption and CO2 production using the expression of 40 and drink not eaten was weighed and recorded. Ad libitum Elia and Livesey: EE ¼ 15.818O2+5.176CO2, where O2 and decaffeinated tea and coffee was available throughout CO2 are in litres and EE is in kilojoules. the study. BMR was measured between 08:00 and 09:00 h Details of the analysis involved in the BU method, on both days 1 and 2 and the average value calculated. including the titration of hyamine hydroxide and scintilla- Subjects were lying awake but completely at rest, at 241C, tion counting of 14C bicarbonate, are given elsewhere.34

12–14 h after eating. Calculation of CO2 production from the BU method was 34 The following samples were obtained, at the times made using the equation of Elia et al: net CO2 production indicated, while the subjects were in the calorimeter: (1) (mol/day) ¼ 0.95 Â 0.85 Â dpm infused divided by the corre- Between 09:00 and 09:15 h on days 1 and 2, blood samples sponding specific activity of urea (dpm/mol).34 The energy were taken, through an airtight hatch in the calorimeter equivalent of CO2 was assumed to be 535 kJ/mol, which door, for the measurement of blood urea, bicarbonate, full approximates to the value for subjects close to nutrient blood count, and liver function tests. (2) At 09:00 h breath balance while ingesting a typical Western diet.40 Small sample for assessing specific activity of CO2 was taken. (3) correction factors to take into account changes in the Between 08:30 and 09:00 h, calorimeter air samples were amount of label in calorimeter air (between 09:00 h on day obtained for assessing specific activity of CO2.CO2 was 1 and 09:00 h on day 2) and changes in the specific activity trapped from these air samples in vials containing a weighed of the urea pool were also used as described previously.34 amount of hyamine hydroxide (B3.0 mmol/vial). Sample For DLW measurement, isotope enrichment was expressed

(3), together with the measured calorimeter air CO2 as delta per thousand, relative to the International Standard concentration, allowed calculation of the total amount of Vienna Standard Mean Ocean Water (SMOW). 2H/1H isotope 14 CO2 in the calorimeter. (4) A measured, proportional ratios were measured using an Aqua Sira mass spectrometer sample of the outgoing calorimeter air was bubbled through (Micromass, Wythenshawe, UK) with a precision of 2.6% a hyamine hydroxide/phenolphthalein mixture over the relative SMOW in duplicate measurements. For 18O/16O

International Journal of Obesity Measurement of total energy expenditure in obese women ER Gibney et al 644 42 ratio, samples were equilibrated with CO2. Samples (3 ml) Results were equilibrated with 13 ml CO2 at 400 mbar for 6 h in an Subject characteristics Isoprep system (Micromass, Wythenshawe, UK), measured All subjects were grossly obese. Mean age was 43.779.2 y, 7 7 2 relative to tank CO2, corrected for isotopic interferences height 1.60 0.11 m and BMI 52.4 10.4 kg/m (Table 1). according to Hoffman42 and expressed relative to SMOW. Weight change within the calorimeter was 0.3570.7 kg and The precision of the sample measurements was 0.4% rel during the free-living element was 0.7770.8 kg. The weight SMOW. change in the calorimeter was not significantly related to the

Estimates of CO2 production by the DLW method were energy balance (r ¼ 0.23), and was probably largely because obtained using the multipoint method with fractionation of changes in fluid balance. corrections as described by Coward.5,17 This procedure also The subjects tolerated the infusion well, and the mini- uses observed values for isotope distribution spaces rather pump did not appear to interfere with normal daily than normalised values.43 It has been suggested that the ratio activitiesFincluding work and household activities. One of the isotope distribution spaces may be increased in obese subject, subject 4, found the pump uncomfortable and it was subjects and the significance of this in DLW calculations has removed on day 4 at her request. The subcutaneous infusion been discussed by Coward et al.12 Average estimates of the did not cause any local inflammation in any of the subjects. internal precision of the estimates was 4.3572.44%, calcu- Subjects had satisfactory full blood count (FBC), urea, lated according to Ritz et al.19 creatinine and liver function tests (haemoglobin 13.070.6 Body composition was estimated using TBW measure- 109/l; haematocrit 37.176.5 1/1 Â 100, white cell count ments, with the assumed water content of FFM to be 0.73.44 6.873.9 109/l; plasma urea 4.0570.9 mmol/l; plasma Bicar- Physical activity levels (PAL) were calculated as the ratio TEE/ bonate 25.472.0 mmol/l; plasma albumin 33.574.9 g/l; BMR. plasma bilirubin 9.876.1 mmol/l).

Statistical methods EE in whole-body calorimetry 7 Results are expressed as mean s.d. Results obtained by BU vs Mean CO2 production by the calorimeter method and BU GE and BU vs DLW were compared using Student’s paired t- method was 22.0273.11 and 21.7773.48 mol/day, respec- tests. tively (Table 2). Mean TEE within the whole-body chamber Sample size calculations for examining possible differences was 11.7871.66 and 11.6471.86 MJ/day with the calori- in EE between DLW and BU methods in free-living meter and BU method, respectively (Table 2). This produced circumstances were carried out as follows. Using the data a group mean difference of 0.2570.49 MJ/day45 between the of Ravussin et al15 to establish a regression equation between two methods; however, this was not significant (paired the discrepancy in EE (DLWÀGE) and body fat (r ¼ 0.812), it sample t-test, P40.28). Mean PAL within the calorimeter was was possible to calculate that the extent to which DLW 1.3570.06 (range 1.27–1.42). This variability in PAL was underestimated EE obtained by GE was 0.93 MJ/day for partly because of the inability of two individuals to complete individuals with 50 kg fat, and 1.52 MJ/day for individuals the exercise periods, because of acute arthritis and shortness with 75 kg fat. These were associated with prediction of breath (see individual PAL values in Table 3). There was no standard deviations (s.d. prediction) of 0.54 and 0.59 MJ/ significant relation between the difference in TEE between day, respectively.45 Data from previous BU studies involving the two methods (BU–GE) and body fat expressed as subjects with 5–36 kg fat show that BU method provides kilograms or percentage, either in this study alone unbiased estimates of TEE that do not change with increas- (r2 ¼ 0.035 and 0004, respectively) or when data from this ing body fat.34,35 Using the above information, sample size study were combined with results from previous studies calculations were made for obese individuals with 50 and (r2 ¼ 0.009 and o0.001, respectively).34,35 75 kg fat using SPSS SamplePower2. To detect a difference Not all subjects consumed all the food that was provided. between DLW and BU of 0.86 MJ/day (0.93–0.05 MJ/day) at Hence the mean energy balance during whole-body calori- 50 kg with B80% power and significance of Po0.05 (two- meter measurements was slightly negative (À0.9571.36 MJ/ tailed), a sample size of only six would be required. To detect day). Energy intake in subjects 2 and 4 was less than EE by a difference of 1.58 (1.63–0.05) MJ/day at 75 kg, with the more than 2 MJ/day (Table 3). This may have decreased BMR same power and significance, a sample size of only four below habitual levels, but it does not alter the comparison of would be required. These calculations assume that the BU total EE measured by GE and the BU method. method does not begin to produce biased estimates of EE above 36 kg body fat (tested in the present paper) and that any discrepancies obtained between BU and GE are un- Free-living EE measurements

correlated with the discrepancies obtained between DLW and Using the DLW method, the mean estimated CO2 produc- GE. If these were correlated, the sample size needed to detect tion was found to be 25.9374.21 mol/day, corresponding to hypothesised differences between DLW and BU methods an EE of 13.8672.25 MJ/day (Table 2). Measured space ratios, 5,12 would be reduced even further. used in the calculation of CO2 for DLW method, gave a

International Journal of Obesity Measurement of total energy expenditure in obese women ER Gibney et al 645

Table 2 Comparison of net CO2 production and total energy expenditure (TEE) (a) over 24 h between the bicarbonate–urea method (BU) and indirect calorimetry, and (b) free-living between the BU method and the doubly labelled water (DLW) method

Subject In whole-body calorimeter Free living

Calorimeter BU CO2 production BU as DLW BU CO2 production BU as % of calorimeter (%) % of DLW (%) b a CO2 24 h EE CO2 24 h EE CO2 24 h EE CO2 24 h EE (mol/day) (MJ/day) (mol/day) (MJ/day) (mol/day) (MJ/day) (mol/day) (MJ/day)

1 20.69 11.07 20.35 10.88 99.08 23.19 12.40 23.38 12.51 100.82 2 26.10 13.96 27.10 14.50 103.83 33.88 18.11 30.71 16.42 90.64 3 20.33 10.87 19.46 10.41 95.72 24.39 13.04 26.96 14.42 110.54 4 25.18 13.47 24.93 13.34 99.00 25.50 13.63 23.81 12.74 93.37 5 21.90 11.72 20.74 11.09 94.70 26.56 14.20 23.40 12.51 88.10 6 17.89 9.57 18.05 9.66 100.89 22.06 11.79 20.94 11.19 94.92

Mean 22.02 11.78 21.77 11.64 98.87 25.93 13.86 24.87 13.29 96.40 s.d. 3.11 1.66 3.48 1.86 3.35 4.21 2.25 3.45 1.84 8.16 a F EE calculated using equation of Elia and Livesey; EE (KJ/min) ¼ 15.818O2+5.176CO2 see Table 3. bResults obtained using the Coward method of analysis of doubly labelled water.

Table 3 Gaseous exchange, (RQ), basal metabolic rate (BMR), energy expenditure (EE), physical activity level (PAL), energy intake (EI) and energy balance (EB) during 24-h whole-body measurements in a calorimeter

Subject Basal state (BMR) 24-h indirect calorimetry

O2 (ml/min) CO2 (ml/min) RQ BMR (MJ/day) O2 (ml/min) CO2 (ml/min) RQ EE (MJ/day) PAL EI (MJ/day) EB (MJ/24 h)

1 305.90 236.10 0.77 7.93 389.70 322.20 0.83 11.28 1.29 11.00 À0.28 2 369.00 279.40 0.76 10.49 505.60 404.60 0.80 14.53 1.39 12.41 À2.12 3 251.80 213.60 0.85 7.33 345.60 303.70 0.88 10.14 1.38 11.05 +0.91 4 348.80 280.50 0.80 9.43 475.10 392.20 0.83 13.75 1.37 11.00 À2.75 5 290.20 238.70 0.82 8.39 410.80 340.90 0.83 11.89 1.42 10.60 À0.40 6 276.00 221.40 0.80 7.94 349.70 278.6 0.80 10.04 1.27 9.842 À0.19

Mean 306.95 244.95 0.80 8.82 412.75 340.37 0.83 11.94 1.35 10.98 À0.95 s.d. 44.46 28.65 0.03 1.22 65.62 49.60 0.03 1.86 0.06 0.84 1.36

mean ratio of 1.0370.02 and the rate constants for 18O and comparison with GE in a whole-body calorimeter. The BU 2H were found to be 0.103170.014 and 0.078170.012/day, method predicted TEE to be 99.172.5% of that obtained by 43,13 respectively, similar to previous studies. CO2 production indirect calorimetry, which is similar to the value of results from the free-living bicarbonate period are displayed 102.173.4% obtained in lung cancer patients35 and 7 34 in Table 2. A mean production of 24.8773.45 mol CO2 was 100 5% in healthy lean men. This study also provides calculated compared to 25.9374.21 using the DLW method the first comparison between the BU and DLW methods (a and calculations based on Coward et al.12 This gave a mean difference of À3.478.01%, Table 2). It illustrates the 7 difference of 1.06 2.19 mol CO2 between the two groups. practicality of infusing subcutaneous bicarbonate in free- The corresponding values for total EE were 13.2971.84 MJ living conditions for longer periods than previously at- and 13.8672.25 MJ/day for BU and DLW, respectively. The tempted (5 days). As in previous studies,34,36–38 the pump mean difference between the two methods (0.1771.33 (MJ/ was well tolerated and did not cause local inflammatory day)) was not significant (paired sample t-test, P40.2). PAL reaction. Although one individual reported mild irritation, (TEE/BMR) calculated using measured BMR (GE) and esti- no individual reported that the procedure prevented normal mates of TEE using the DLW and BU method were 1.6270.14 daily activities. and 1.5670.20, respectively. Each method used to measure EE carries some inherent

errors. The precision of CO2 measurements in a whole-body chamber is better than 1.0%, and that of the BU method up Discussion to about 2.5%,20 which are at best sufficient to account for This study provides evidence for the validity of BU about half of the variance in the difference between the two method in a group of grossly obese individuals through methods (s.d. 3.8%). The remaining variance may then be

International Journal of Obesity Measurement of total energy expenditure in obese women ER Gibney et al 646 attributed to biological factors influencing the two methods % body fat (8.2–31.0%), or BMI (19.4–30.5 kg/m2), but still in different ways. they do not support the Ravussin et al15 report of progressive In free-living conditions, though the BU and DLW underestimation of EE by the DLW as body fat increases. methods did not yield significantly different results, the In summary, this study confirms the validity of the BU standard deviation of the difference between them was 8%, method against measurements of EE by direct measurements much larger than for the BU–GE comparison. Assuming that of GE in a whole-body indirect calorimeter, and demon- analytical variability of the BU method is B2.5% and that of strates the practical application of using the BU method over the DLW method is approximately 4%,20 the pooled 5 days, the longest application of this technique so far. In measurement precision errors amount to 4.7%. Thus, a addition, it is the first study to demonstrate correspondence substantial part of the variability may be of biological origin. between the BU and DLW methods for measuring TEE in Identification and calculation of the biological variance free-living conditions. In so doing, it counters previous between the two methods in this study is difficult. One of reports suggesting a large systematic underestimation of EE the difficulties in comparing the methods is that free-living DLW in grossly obese individuals. measurements using the BU method were taken over 5 days, whereas DLW measurements were taken over 12–14 days. It is therefore possible that there may have been some real differences in EE between the two periods. The lack of Acknowledgements significant difference between free-living measurements We thank G Jennings, WA Coward and staff at the former made using the two tracer methods can be interpreted in MRC Dunn Nutrition Unit for their help with this study. two ways. One is that a difference did exist but was not demonstrated because of a type II error (small sample size), and the other is that the measurements were indeed References comparable with no systematic difference between them. It 1 WHO. Obesity: preventing and managing the global epidemic. Report can be shown, using the assumptions stated in the Statistical of WHO Consultation in Obesity WHO: Geneva; 1998. pp 1–276. Methods section, that with 75 kg fat, a sample size of 6 would 2 Schoeller DA, Bandini LG, Dietz WH. Inaccuracies in self reported be sufficient to detect a significant (P 0.05) difference of intake identified by comparison with the doubly labelled water o method. Can J Physiol 1990; 68: 941–949. 0.96 MJ/day with 80% power. This is considerably less than 3 Black AE, Goldberg GA, Jebb SA, Livingstone MBE, Cole TJ, the 1.63 MJ/day underestimation suggested by the study of Prentice AM. Critical evaluation of energy intake data using Ravussin et al.15 This implies that DLW does not under- fundamental principles of energy physiology: 2. Evaluating the Am J Clin Nutr estimate total EE in free-living conditions to the extent results of published surveys. 1991; 45: 583–599. 15 4 Murgatroyd PR, Shetty PS, Prentice AM. Techniques for the suggested by Ravussin et al, especially since the mean DLW measurement of human energy expenditure: a practical guide. Int EE was 0.59 MJ/day greater than that obtained by the BU J Obes Relat Metab Disord 1993; 17: 549–568. method, not lower as might be expected from the Ravussin 5 Coward WA, Cole TJ. The doubly labelled water method for the data. It is possible that Rauvssin’s finding is gender-specific, measurement of energy expenditure in humans: risks and benefits. In: RG Whitehead, A Prentice (eds). New techniques in since our study was carried out in women and that of nutritional research. Academic Press: London; pp 139–176. Ravussin et al in men. An analysis of published studies in 6 Roberts SB, Coward WA, Schlingenseipe KH, Nohria V, Lucas A. which DLW has been validated against GE6–10 do not support Comparison of the doubly labeled water method with indirect this, but is limited by the small number of women studied calorimetry and a nutrient balance study for simultaneous determination of energy expenditure, water intake, metabolisable (31 individual validations; 27 M, four F). energy intake in preterm infants. Am J Clin Nutr 1986; 44: Data from previously published studies can also be used to 315–322. assess if the difference in EE (DLW/GE Â 100) is related to fat 7 Schoeller DA, Webb P. Five day comparison of the doubly labeled water method with respiratory gas exchange. Am J Clin Nut 1984; mass. Using individual subject results from three studies in 7,9,15 40: 153–158. which body fat was reported, the relation was found to 8 Westerterp KR, Bronus F, Saris WHM, Hoor FT. Comparison of be weak (r2 ¼ 0.10, gradient 0.51%EE/kg; n ¼ 27), not sig- doubly labelled water with respirometry at low and high activity nificant, and positive rather than negative, implying that, if levels. Am J Physiol 1988; 65: 53–56. 9 Schoeller DA, Kushner RF, Jones PJH. Validation of doubly labeled anything, an increase in body fat tends to be associated with water for measuring energy expenditure during parenteral an overestimation rather than an underestimation of EE. nutrition. Am J Clin Nutr 1986; 44: 291–298. Similar overall trends were obtained when the results were 10 Seale JL, Rumpler WV, Conway JM, Miles CW. Comparison of examined using analysis of covariance (this showed no doubly labeled water, intake-balance, and direct and indirect calorimetry methods for measuring energy expenditure. Am J Clin study–fat interaction, r ¼ 0.25; gradient 0.45%EE/kg fat; 2 Nutr 1990; 52: 66–71. n ¼ 27) and when each study was analysed separately. In 11 Seale JL, Rumpler WV. Comparison of energy expenditure addition, the relations of % difference in EE with % body fat measurements by diet records, energy intake balance, doubly (r2 ¼ 0.04, gradient 0.25% EE/kg fat; n ¼ 27) and BMI labeled water and room calorimetry. Eur J Clin Nutr 1997; 51: 856–863. (r2 ¼ 0.082, gradient 1.130%EE/kgfat; n ¼ 27) were also weak, 12 Coward WA, Ritz P, Cole TJ. Revision of calculations in the doubly not significant, and positive. These results were derived from labeled water method for measurement of energy expenditure in subjects with a limited range of body fat (range 5.8–24.9 kg), humans. Am J Physiol 1994; 267: E805–E807.

International Journal of Obesity Measurement of total energy expenditure in obese women ER Gibney et al 647 13 Goran MI, Poehlman ET, Nair KS, Danforth E. Effect of gender, 30 Casper RC, Schoeller DA, Kushner R, Hnilicka J, Trainer Gold S. body composition and equilibration time on the 2H to 18O Total daily energy expenditure and activity level in anorexia dilution space ratio. Am J Physiol 1992; 263: E1119–E1124. nervosa. Am J Clin Nutr 1991; 52: 1143–1150. 14 Goran MI, Poehlman ET, Danforth E. Experimental reliability of 31 Bandini LG, Schoeller DA, Dietz WH. Energy expenditure in the doubly labeled water technique. Am J Physiol 1994; 266: obese and nonobese adolescents. Pediatr Res 1990; 27: 198–203. E510–E515. 32 Haggarty P, Shetty P, Thangam S, Kumar S, Kurpad K, Ashton J, 15 Ravussin E, Harper IT, Rising R, Clifdon B. Energy expenditure by Milne E, Earl C. Free and esterified fatty acid and cholesterol doubly labeled water: validation in lean and obese subjects. Am J synthesis in adult males and its effect on the doubly labelled Physiol 1991; 261: E402–E409. water method. Br J Nutr 2000; 83: 227–234. 16 Roberts SB, Dietz W, Sharp T, Dallal GE, Hill JO. Multiple 33 Elia M. Energy equivalents of Co2 and their importance in laboratory comparison of the doubly labelled water technique. assessing energy expenditure when using tracer techniques. Am J Obes Res 1995; 3(Suppl 1): 3–13. Physiol 1991; 260: E75–E88. 2 18 17 Coward WA. The doubly labelled water ( H2 O) methodFprin- 34 Elia M, Jones MG, Jennings G, Poppitt SD, Fuller NJ, Murgatroyd ciples and practice. Proc Nutr Soc 1988; 47: 209–218. PR, Jebb SA. Estimating energy expenditure from specific activity 14 18 Jones PJH, Winthrop AL, Schoeller DA, Swyer PR, Smith J, Filler of urine urea during lengthy subcutaneous NaH CO3 infusion. RM, Heim T. Validation of doubly labeled water for Am J Physiol 1995; 269: E172–E182. assessing energy expenditure in infants. Pediatr Res 1987; 21: 35 Gibney E, Elia M, Jebb SA, Murgatroyd P, Jennings G. Total energy 242–246. expenditure in patients with small cell lung cancer: results of a 19 Ritz P, Cole TJ, Couet C, Coward WA. Precision of DLW energy validated study using the bicarbonate urea method. Metabolism expenditure measurements: contribution of natural abundance 1997; 46: 1412–1417. variations. Am J Physiol 1996; 270: E164–E169. 36 Paton NI, Angus B, Chaowagul W, Simpson AJ, Supputtamongkol 20 Elia M, Ritz P, Stubbs RJ. Total energy expenditure in the elderly. Y, Elia M, Calder G, Milne E, White NJ, Griffin GE. Protein and Eur J Clin Nutr 2000; 54(Suppl 3): S92–S103. energy metabolism in chronic bacterial infection: studies in 21 Jensen CL, Butte NF, Wong WW, Moon JK. Determining melioidosis. Clin Sci 2001; 100: 101–110. 2 18 energy expenditure in preterm infants: comparison of H2 O 37 Paton NIJ, Elia M, Jebb SA, Jennings G, Macallan DC, Griffin GE. method and indirect calorimetry. Am J Physiol 1992; 263: Total energy expenditure and physical activity measured with the R685–R692. bicarbonate urea method in patients with human immunodefi- 22 Jones PJH, Winthrop AL, Schoeller DA, Filler RB, Sawyer PR, ciency virus infection. Clin Sci 1996; 91: 241–245. Smith J, Heim T. Evaluation of doubly labeled water for 38 Tsang NIS, Chung MI, Elia M, Hui E, Lum CM, Luk JKH, Jones measuring energy expenditure during changing nutrition. Am J MG, Woo J. Total daily energy expenditure in wasted chronic Clin Nutr 1988; 47: 799–804. obstructive pulmonary disease patients. Eur J Clin Nutr 2002; 56: 23 Bandini LG, Schoeller DA, Fukagawa NK, Wykes LJ, Dietz WH. 1–5. Body composition and energy expenditure in adolescents with 39 Elia M. Estimation of short term energy expenditure by the cerebral palsy or myelodysplasia. Pediatr Res 1991; 29: 70–77. labeled bicarbonate method. In: RG Whitehead, A Prentice (eds). 24 Chong R, Jung RT, Rennie MJ, Scrimgeour CM. Energy expendi- New techniques in nutritional research. Academic Press: New York; ture in lean and obese diabetic patients using the doubly labelled 1991. pp 207–227. water method. Diabetic Med 1993; 10: 729–735. 40 Elia M, Livesey G. Energy expenditure and fuel selection in 25 Chong RKK, Jung RT, Scrimgeour CM, Rennie MJ, Paterson CR. bioloigical systems: the theory and practice of calculations based Energy expenditure and body composition in growth hormone on indirect calorimetry and tracer methods. In: Simipoulos AP deficient adults on exogenous growth hormone. Clin Endocrinol (ed). Metabolic control of eating, energy expenditure and 1994; 40: 103–110. of obesity, World Review of Nutrition & Dietetics: Basel, Karger; 26 Goran MI, Peters EJ, Herndon DN, Wolfe RR. Total energy 1992 pp 68–131. expenditure in burned children using the doubly labelled water 41 Schofield WN, Schofield C, James WPT. Basal metabolic rate. technique. Am J Physiol 1990; 259: E576–E585. Human nutr. Clin Nutr 1985; 39: 1–96. 27 Koea JB, Wolfe RR, Shaw JHF. Total energy expenditure during 42 Hoffman C. Isotopic standards for carbon and and total parenteral nutrition: ambulatory patients at home versus correction factors for mass-spectrometric analysis of carbon patients with sepsis in surgical intensive care. Surgery 1995; 118: dioxide. Geochim Cosmochin Acta 1957; 12: 133–149. 54–62. 43 Schoeller DA, vanSanten E, Peterson DW, Dietz W, Jaspan J, Klein 28 Schoeller DA, Levitsky LL, Bandini LG, Dietz WW, Walczak. PD. Total body water measurements in humans with 18O and 2H Energy expenditure and body composition in Prader Willi labeled water. Am J Clin Nutr 1980; 33: 2686–2693. syndrome. Metabolism 1988; 37: 115–120. 44 Elia M. Body composition analysis: an evaluation of 2 component 29 Taggart DP, McMillan DC, Preston C, Richardson R, Burns RJG, models, multicomponent models and bedside techniques. Clin Wheatley DJ. Effects of cardiac surgery and intraoperative Nutr 1992; 11: 1–13. hypothermia on energy expenditure as measured by the doubly 45 Altman DG, Bland JM. Measurement in medicine: the analysis of labelled water. Br J Surg 1991; 78: 237–241. method comparison studies. Statistician 1983; 32: 307–317.

International Journal of Obesity