The Journal of Nutrition
Methodology and Mathematical Modeling
The Doubly Labeled Water Method Produces
Highly Reproducible Longitudinal Results in
William W. Wong,3* Susan B. Roberts,4Susan B. Racette,5Sai Krupa Das,6Leanne M. Redman,6
James Rochon,7Manjushri V. Bhapkar,7Lucinda L. Clarke,4and William E. Kraus7
3USDA/Agricultural Research Service Children?s Nutrition Research Center, Baylor College of Medicine, Houston, TX;4Jean Mayer
USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA;5Washington University School of Medicine, St. Louis,
MO;6Pennington Biomedical Research Center, Baton Rouge, LA; and7Duke Clinical Research Institute, Duke University, Durham, NC
The doubly labeled water (DLW) method is considered the reference method for the measurement of energy expenditure
under free-living conditions. However, the reproducibility of the DLW method in longitudinal studies is not well
documented. This study was designed to evaluate the longitudinal reproducibility of the DLW method using 2 protocols
developed and implemented in a multicenter clinical trial—the Comprehensive Assessment of Long-term Effects of
Reducing Intake of Energy (CALERIE). To document the longitudinal reproducibility of the DLW method, 2 protocols,
1 based on repeated analysis of dose dilutions over the course of the clinical trial (dose-dilution protocol) and 1 based on
repeated but blinded analysis of randomly selected DLW studies (test-retest protocol), were carried out. The dose-dilution
protocol showed that the theoretical fractional turnover rates for2H and18O and the difference between the 2 fractional
turnover rates were reproducible to within 1% and 5%, respectively, over 4.5 y. The Bland-Altman pair-wise comparisons
of the results generated from 50 test-retest DLW studies showed that the fractional turnover rates and isotope dilution
spaces for2H and18O, and total energy expenditure, were highly reproducible over 2.4 y. Our results show that the DLW
method is reproducible in longitudinal studies and confirm the validity of this method to measure energy expenditure,
define energy intake prescriptions, and monitor adherence and body composition changes over the period of 2.5–4.4 y.
The 2 protocolscanbe adoptedbyother laboratories to document the longitudinal reproducibility of their measurementsto
ensure the long-term outcomes of interest are meaningful biologically. This trial was registered at clinicaltrials.gov as
NCT00427193.J. Nutr. 144: 777–783, 2014.
The doubly labeled water (DLW)8method was originally
developed and validated for measuring energy utilization in
small mammals (1–9). Following the validation of the DLW
method for measuring energy expenditure (EE) against indirect
calorimetry in humans (10–16), the method quickly became
the reference method for measuring habitual EE in humans,
including premature infants (17,18), newborns (19,20), children
(21), adolescents (22,23), pregnant women (24,25), lactating
women (26,27), and adults (28,29), as well as individuals with
various diseases (30–33). The DLW method is noninvasive and
does not require blood sampling. The method also has minimal
participant burden and can be used anywhere. Briefly, the DLW
method is based on the principle that the disappearance rate
of the heavier stable isotope of hydrogen (2H) reflects water
turnover rate, whereas the disappearance rate of the heavier
stable isotope of oxygen (18O) reflects both water and CO2
turnover rates. Therefore, with time, the difference between the
disappearance rates of2H and18O represent the rate of CO2
production. Based on the energy equivalent of 1 L of CO2, the
rate of CO2production can be converted to EE (16,34). The
accuracy and precision of the DLW method rely on the accuracy
and precision of the analytical instrumentation used to measure
2H and18O. Currently, isotope ratio MS is considered the best
analytical instrumentation for accurate and precise measure-
lected for the DLW method (35–38). However, the long-term
18O content in physiologic samples col-
1Funded by the National Institute on Aging grant 5U01-AG-022132-05 with
support from the USDA/Agricultural Research Service grant 6250-51000-053.
The contents of this publication do not necessarily reflect the views or policies of
the USDA or the NIH, nor does mention of trade names, commercial products, or
organizations imply endorsement.
2Author disclosures: W. W. Wong, S. B. Roberts, S. B. Racette, S. K. Das,
L. M. Redman, J. Rochon, M. V. Bhapkar, L. L. Clarke, and W. E. Kraus, no
conflicts of interest.
8Abbreviations used: CALERIE, Comprehensive Assessment of Long-term
Effects of Reducing Intake of Energy; CR, caloric restriction; DLW, doubly
labeled water; EE, energy expenditure; FM, fat mass; FFM, fat-free mass; kH,
fractional turnover rate of2H; kO, fractional turnover rate of18O; NH, isotope
dilution space of2H; NO, isotope dilution space of18O; TBW, total body water;
VCO2, carbon dioxide production rate.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
ã 2014 American Society for Nutrition.
Manuscript received November 4, 2013. Initial review completed December 2, 2013. Revision accepted January 15, 2014.
First published online February 12, 2014; doi:10.3945/jn.113.187823.
by William Wong on April 18, 2014
reproducibility of the DLW method, which is critical for longi-
tudinal studies to monitor changes in EE, energy intake, and body
composition, has not been documented.
The objective of this study was to evaluate the longitudinal
reproducibility of the DLW method based on 2 protocols: 1 for
the study dose dilutions and 1 for the test-retest reliability, which
were developed and implemented in the National Institute on
Aging?s multicenter clinical trial, the Comprehensive Assessment
of Long-term Effects of Reducing Intake of Energy (CALERIE).
Participants and Methods
CALERIE was a multicenter, parallel-group, randomized controlled
clinical trial conducted between 2005 and 2012 at the Jean Mayer USDA
Human Nutrition Research Center on Aging at Tufts University,
Washington University School of Medicine, and Pennington Biomedical
Research Center. Duke Clinical Research Institute served as the
coordinating center and the gas-isotope-ratio MS laboratory at Baylor
College of Medicine?s USDA/Agricultural Research Service Children?s
Nutrition Research Center served as the central DLW laboratory. The
design and conduct of the clinical trial were published elsewhere (39).
Study participants provided written informed consent. The institutional
review board for human studies at each participating institution
approved the study?s protocol.
Healthy adults were recruited for the 2-y clinical trial. Eligible
participants were between 20 and 50 y of age (men) or between 20 and
47 y of age (women), either of normalbodyweight or slightlyoverweight
(BMI of $22.0 and <28.0 kg?m22), nonsmoking, nondiabetic, and not
on any medications. Female participants had to use an acceptable form
of contraception during the clinical trial. A total of 238 participants
began the baseline evaluations, of which 218 were randomly assigned,
with a 2:1 allocation to the caloric restriction (CR) and control groups,
respectively, and were provided at least 1 follow-up evaluation (40). A
total of 191 participants provided complete follow-up data.
The DLW method was used to determine the habitual energy intake of
each study participant. Two consecutive 14-d DLW protocols were
conducted with each participant at baseline. The mean EE values from
the 2 DLW studies were used to calculate the weight maintenance energy
intake requirement at baseline and the 25% CR prescription for
participants assigned to the CR intervention. For each subsequent time
point in the study, which included 6, 12, 18, and 24 mo for CR
participants, and 12 and 24 mo for control participants, a single 14-d
DLW protocol was used.
For each DLW period, 2 baseline urine samples were collected. The
participant was then administered by mouth a mixed cocktail containing
0.1 g of2H2O at 99.98 atom percent2H and 0.16 g of 100%18O per kg
of body weight. The DLW dose was designed to minimize potential
errors introduced by the anticipated fluctuation in natural abundances of
the 2 isotopes during the CR intervention, to reduce the effect of
analytical errors on the precision of the DLW method, and to ensure
there were sufficient isotopes at the end of each 14-d DLW study period
for accurate and precise isotope ratio measurements (41–43). Six
postdose urine samples were collected: 2 at 5–6 h postdose, 2 on day
7, and 2 on day 14. Study participants were instructed to void at home in
the morning on days 7 and 14 before the postdose urine samples were
collected in the clinic. The exact time of dosing and sample collection
times were recorded.
Urine samples were transferred to 3 sets of o-ring cryovials.
Encrypted ID labels, created and printed by the coordinating center,
were affixed by site personnel to the cryovials in all follow-up DLW
studies to ensure the DLW Laboratory was unaware of the treatment
assignment and participant ID. One set of cryovials was shipped on dry
ice to the DLW Laboratory for isotope ratio measurements.
converted to H2using the zinc reduction method (36,37). The H2was
introduced via the automated sample inlet system directly into a Finnigan
2H assays, 10 mL of urine without further treatment was
instrument for hydrogen isotope ratio measurement. For
ISOPREP-18 H2O-CO2 equilibration chambers were used, in which
100 mL of urine was equilibrated with 300 mbar of CO2of known18O
content for 10 h priorto admission to the ion source of a VG instrument for
oxygen isotope ratio measurement (36). The isotope ratio measurements
were expressed in delta (d) per mil (parts per 1,000 or &) as follows:
d2H or d18Oð&Þ ¼
where RSampleand RStandardwere the2H/1H or18O/16O isotope ratios of
the sample or the laboratory working standard, respectively. The
isotopic ratios were then normalized against 2 international water
standards: Vienna-Standard Mean Ocean Water and Standard Light
Antarctic Precipitation (44). The precision (SD) for the2H assay was
1.0& for samples with natural abundance of2H and 1.8& for samples
with enriched amounts of2H (37). For18O assays, the precision was
0.21& for samples with natural abundance of
samples with enriched amounts of18O (36).
The isotope dilution space of2H (NH) and18O (NO) were calculated
18O and 0.97& for
NHor NOðmolÞ ¼
where d was the dose of2H2O or H218O in grams, Awas the amount of
laboratory water in grams used in the dose dilution, a was the amount of
2H2O or H218O in grams added to the laboratory water in the dose
dilution, Eawas the rise in d2H or d18O values in the laboratory water
after the addition of the isotopic water, and Edwas the rise in d2H or
d18O values in the urine samples at time zero obtained from the zero-
time intercepts of the2H and18O decay curves in the urine samples. The
use of dose dilution in the calculation of isotope dilution spaces was
recommended by the International Dietary Energy Consultancy Groups
to ensure accuracy of the isotope dilution calculations (45). Carbon
dioxide production rate (VCO2) was calculated from the fractional
turnover rate of2H (kH) and18O (kO) as follows (46):
_VCO2ðmol ? d21Þ ¼ 0:4812 3 ½ðkO3NOÞ2ðkH3 NHÞ?20:0246 3 rg
where rgwas the fractionated water loss, which was calculated as 1.05 3
(NOx kO2 NHx kH). The VCO2was converted to EE based on an
energy equivalent of 1 L of CO2to be 3.815/RQ + 1.2321 (16), where
RQ was the respiratory quotient provisionally estimated to be 0.86 for
all DLW measurements in this study (47).
Longitudinal reproducibility of the DLW method
To assess the longitudinal reproducibility of the DLW method, 2
protocols were developed and implemented in the CALERIE multicenter
Dose-dilution protocol. Two dose dilutions that spanned the range of
isotopic enrichments anticipated at 5–6 h postdose (at ;1:400 dilution)
and at 14-d postdose(at ;1:1500dilution) were preparedfrom the DLW
dose mixture used in the CALERIE clinical trial. Sufficient quantities of
the 2 dose dilutions, along with the laboratory water that was used to
prepare the dose dilutions, were stored in leak-proof containers at 5?C
for the duration of the clinical trial. Initially, the 2 dose dilutions and the
laboratory water were analyzed 10 times each for2H and18O content
each day for 10 d. The mean values were used to generate the conversion
constants to convert the monthly2H and18O measurements of the dose
dilutions and the laboratory water into the theoretical fractional
turnover rates of 0.1 for2H and 0.13 for18O. The conversion constants
were calculated as follows:
CkHor CkO¼lnðDE526hÞ ? lnðDE142dÞ
0:1 or 0:13
where CkHand CkOwere the conversion constants for
respectively; and DE5–6 hand DE14-dwere the2H and18O content of the
dose dilution above the isotopic content of the laboratory water
778 Wong et al.
by William Wong on April 18, 2014
anticipated at 5–6 h and at 14-d postdose, respectively. To monitor the
reproducibility of the
the clinical trial, monthly measurements of the 2 dose dilutions and the
laboratorywaterwere performedandthe valueswere converted tokHand
kOvalues using the respective conversion constant (CkHor CkOÞ: The
percentage difference of the kH, kO, and kO-kHvalues generated from the
monthly measurements of the 2 dose dilutions and the laboratory water
with respect to the theoretical values of 0.10, 0.13, and 0.03, respectively,
was plotted against the date of analysis to monitor the long-term
reproducibility of these measurements.
18O measurements over the course of
Test-retest protocol. All DLW studies that were performed postran-
domization from participants in both study arms were eligible for the
test-retest protocol. Baseline studies were excluded because of the require-
ment to provide baseline total energy expenditure values quickly to the
clinical sites so that the correct energy prescription could be determined.
Sample size calculations(48) indicated that a sample of at least 46 duplicate
DLW studies would be required to demonstrate that the intra-class
correlation was >0.8 with a type-I error of a = 0.05 and type-II error of
b = 0.2. Thus, 50 DLW studies, or ;8% of postrandomization DLW
studies, were included.
At periodic intervals in calendar time, DLW studies were selected for
the study. The goal was to select them when ;120 new postrandomiza-
tion DLW studies had been performed since the previous calendar point.
However, because of administrative issues, the samples were actually
selected at 4 time points when 427, 84, 116, and 10 additional samples
had accumulated. The first sampling was delayed to allow the DLW
laboratory to focus on the baseline studies. Moreover, because more
samples were found to be ineligible than expected, the sample rate was
increased toward 15% by the end of the study to meet the required study
size. Samples were selected from all new postrandomization DLW studies
using simple random sampling by the statistician at the coordinating
DLW studieswere not performed inthe control groupatmonths6 and 18,
so that stratifying by treatment group would have overrepresented studies
knowledge of treatment groups, samples that did not conform to the
standard 14-d protocol were discarded and replaced (similarly for those
lacking sufficient urine volume). Selected retest samples were labeled with
encrypted ID labels that looked identical to the regular sample ID labels.
They were printed by the coordinating center and provided to the sites,
thereby ensuring that the DLW laboratory was unaware of the group
assignment, participant ID, and protocol time point so that only the
sample collection sequence was identified. The clinical sites retrieved the
duplicate urine sample sets from their freezers, affixed the blinded labels,
and forwarded them to the DLW laboratory for analysis. When the mass
spectrometric measurements were completed, the isotopic data were
study information to the DLW laboratory to generate the DLW outcome
variables kH, kO, NH, NO, and EE.
Descriptive statistics were used to calculate the mean, SD, and range of
the participants? physical characteristics, the percentage difference from
the theoretic kH, kOand kO-kHvalues under the dose-dilution protocol,
and the mean, mean difference, and the corresponding SD and range of the
categorical variables, respectively, between the retest participants and the
nonretest participants. Paired samples t test was used to compare the test-
retest outcome variables. The Bland-Altman pair-wise comparison (49,50)
was used to evaluate the reproducibility of the test-retest results. Statistical
analyses were performed with SPSS software (SPSS).
Longitudinal reproducibility of the DLW method
Dose-dilution protocol. The longitudinal reproducibility of
the DLW method based on the dose-dilution protocol is
summarized in Figure 1. Fig. 1A illustrates the reproducibility of
the kHvalues over a period of 4.4 y and, as shown, the kHvalues
generated from the dose-dilution measurements over 4.4 y were
within 1% of the theoretic value of 0.10 for kH, with a mean
difference of 0.11 6 0.25% (mean 6 SD; range: 20.47–0.81%).
Fig. 1B illustrates the reproducibility of the kOvalues over 4.4 y
and, as shown, the kOvalues generated from the dose-dilution
with a mean difference of 0.06 6 0.35% (range: 20.76–0.76%).
The reproducibility of the difference between kO and kH is
summarized in Fig. 1C. As shown in the figure, the kO2kHvalues
were reproducible within 5% of the theoretic value of 0.03 with a
mean difference of 20.06 6 0.19% (range: 23.63–4.12%).
Test-retest protocol. The demographic and baseline physical
characteristics of the participants who were randomly selected
under the test-retest protocol and the nonretest participants are
summarized in Table 1. The follow-up DLW studies (n = 50)
dilution data over 4.4 y (n = 79) in the caloric restriction clinical trial.
Shown are the percentage difference of kH with respect to a
theoretical value of 0.10 (A); percentage difference of kOwith respect
to a theoretical value of 0.13 (B); and percentage difference of kO2kH
with respect to a theoretical value of 0.03 (C). The symbols within
each panel represent the percentage difference for each monthly
dose-dilution measurement. The solid line within each panel repre-
sents a mean difference of zero. The 2 dotted lines within each panel
represent a 1% difference around zero (A and B), and a 5% difference
around zero (C). DLW, doubly labeled water; kH, fractional turnover
rate of2H; kO, fractional turnover rate of18O.
Reproducibility of the DLW method based on the dose-
Long-Term reproducibility of the DLW method779
by William Wong on April 18, 2014
used in the test-retest protocol were obtained from 46 randomly
selected participants, because 4 participants had 2 DLW studies
that were carried out at different time points of the clinical trial.
The demographic and baseline characteristics of the 46 partic-
ipants selected for the test-retest protocol were not different
from the 172 participants who were not selected for the test-
Table 2 provides the descriptive statistics for the DLW
outcome variables obtained from the 50 DLW studies randomly
selected for the test-retest protocol. The original fractional
turnover rates for2H and18O ranged from 20.176 to 20.058 d21
and from 20.201 to 20.081 d21, respectively. The original
isotope dilution spaces for2H and18O also ranged from 27.7 to
49.7 kg and from 26.8 to 47.9 kg, respectively. With respect to
EE, the original values ranged from 1561 to 3675 kcal?d21.
Therefore, the 50 DLW studies randomly selected for the test-
retest protocol provided a wide range of these measurements to
fully evaluate the longitudinal reproducibility of the DLW
method. A paired-samples t test showed that the differences
between the original tested and retested fractional turnover rates
(kHand kO) were significant (P = 0.02). However, none of the
differences between the original tested and retested values for
NH, NO, and EE was found to be significant (P $ 0.3). The small
differences observed between the original tested and retested
values for kHand kOare considered physiologically irrelevant
because no significant difference was observed among the major
DLW outcomes (isotope dilution spaces and EE), which were
derived from these fractional turnover rates.
Figure 2 summarizes the Bland-Altman pair-wise comparisons
between the retested DLW outcome variables and the original
tested values. Fig. 2A shows that the retested kHvalues, when
compared with the original values, hada bias of0.0004 d21with
a lower and upper limit of agreement between 20.002 and
0.003 d21, respectively. With the exception of 1 data point, the
rest of the differences were within the lower and upper limit of
agreement. The comparison between the retested kOvalues and
the original values showed a bias of 0.0005 d21, with a lower
and upper limit of agreement between 20.003 and 0.004 d21,
respectively (Fig. 2B). Again, with the exception of 1 data point,
the rest of the differences all fall within the limit of agreement.
When compared with the original values, the retested NHvalues
(Fig. 2C) had a bias of 20.1 kg with a lower and upper limit of
agreement between 22.1 and 2.0 kg, respectively. The individ-
ual differences again fall within the limit of agreement, with the
exception of 1 data point. Similar results were obtained for the
retested NOvalues (Fig. 2D), with a bias of 20.1 kg and a lower
and upper limit of agreement between 21.8 and 1.7 kg, respec-
tively. For the retested EE values (Fig. 2E), a bias of 25 kcal?d21,
with a lower and upper limit of agreement between 2148 and 137
kcal?d21, respectively, was obtained. With the exception of 1 data
point, the other differences were all within the limit of agreement.
Regression analyses indicated that the differences in kHand kO
Demographic and baseline physical characteristics of participants in the CR clinical trial1
Test-retest participants (n = 46)Nonretest participants (n = 172)P value
Sex, n (%)
Race, n (%)
Ethnic group, n (%)
Hispanic or Latino
Not Hispanic or Latino
BMI status, n (%)
39.7 6 6.6 (22.9–50.6) 37.5 6 7.3 (20.7–50.8)0.06
73.5 6 9.5 (52.8–97.7)
169.7 6 9.0 (153.2–191.4)
25.5 6 1.7 (22.0–28.5)
71.3 6 9.1 (51.8–97.5)
168.4 6 8.4 (147.7–195.5)
25.1 6 1.7 (21.3–29.0)
1Values are means 6 SDs (ranges) or n (%). P values by independent samples t test for continuous variables and chi-square test for
categorical variables. CR, caloric restriction.
from the CR clinical trial under the test-retest protocol1
DLW outcome variables calculated from samples collected from human studies randomly selected
Variables OriginalRetest Difference
20.098 6 0.024 (20.176 to 20.058)
20.122 6 0.025 (20.201 to 20.081)
35.7 6 6.1 (27.7–49.7)
34.4 6 5.9 (26.8–47.9)
2242 6 407 (1561–3675)
20.098 6 0.024 (20.178 to 20.058)
20.122 6 0.026 (20.202 to 20.081)
35.8 6 6.4 (27.7–51.8)
34.5 6 6.1 (26.8–49.1)
2247 6 397 (1584–3622)
0.000 6 0.001 (20.007–0.001)
0.001 6 0.002 (20.011–0.001)
20.1 6 1.0 (20.9–7.0)
20.1 6 0.9 (21.2–5.9)
25 6 73 (2118–161)
1Values are means 6 SDs (ranges), n = 50. CR, caloric restriction; DLW, doubly labeled water; EE, energy expenditure; kH, fractional turnover rate of
2H; kO, fractional turnover rate of18O; NH, isotope dilution space of2H; NO, isotope dilution space of18O.
780Wong et al.
by William Wong on April 18, 2014
were inversely related to the mean kHand kOvalues (r2# 0.09,
P # 0.04). No relation was detected between the differences and
the mean values among the comparisons of the NH(P = 0.07), NO
(P = 0.17), and EE (P = 0.32) measurements.
Among the 50 DLW studies chosen for the test-retest protocol,
2 studies were found to provide outcomes significantly different from
the original values. Repeated MS analyses on the samples yielded
the same outcomes. Despite these findings, the results presented in
Fig. 2 show that all the biases between the repeated measurements
and the original measurements were clustering around zero. Ex-
cluding these 2 studies from the comparisons yielded tighter
limits of agreement (kH: 20.001–0.001 d21; kO: 20.001–0.001 d21;
biases closer to zero (kH: 20.0003 d21; kO: 0.0003 d21; NH: 0.03 kg;
NO: 0.04 kg; EE: 0.6 kcal?d21), as anticipated. The significant
relation observed between the differences and the mean values for
kHand kOalso disappeared (kH: P = 0.64; kO: P = 0.99) after
excluding these 2 studies from the comparisons.
Because the DLW studies were randomly selected for the test-
retest protocol, the time elapsed since the original measurements
varied between 12 d and 2.5 y, with 68% being repeated within
1 y and 32% being repeated between 1 and 2.5 y. An independent
samples t test found no differences in the biases among the DLW
outcome variables between measurements repeated within 1 y
and those repeated after 1 y (P $ 0.1).
Applications of the DLW method in the CALERIE clinical
CR prescription. In the CALERIE clinical trial, 2 consecutive
14-d DLW studies were carried out at baseline to determine the
EE of each study participant and to establish the CR prescrip-
tion for those randomly assigned to the CR intervention.
Because these participants were healthy and were not taking
part in any dietary or physical activity programs to lose weight,
the mean EE measurements derived from these 2 consecutive
DLW studies were assumed to equal their ad libitum energy
intakes. The pre-intervention energy intakes of 10 participants,
5 males and 5 females, who were assigned to the CR inter-
vention in the CALERIE clinical trial with best adherence are
summarized in Table 3. The 25% CR prescriptions were cal-
culated as 75% of ad libitum energy intake as determined by
the DLW method.
Adherence monitoring. Adherence measures were used to deter-
mine the degree of CR actually achieved. Adherence was character-
%CR ¼ 100½12ðEIP=EIALÞ?;
where EIPrepresents mean daily energy intake over the period of
interest and EIALrepresents the ad libitum daily energy intake
before the start of the intervention. Ad libitum energy intake was
characterized by the mean of 2 consecutive measures of EE
performed at baseline using the DLW method. Based on the
relation, EI = EE + DES, where EE was the mean daily energy
expenditure during the period of interest and DES was the change
DLW method based on outcomes
collected from 50 human studies
randomly selected from the caloric
restriction clinical trial for the test-
retest protocol over 2.5 y (n = 50).
Shown are the Bland-Altman pair-
wise comparison between the
retested kH values and the origi-
nally calculated kH values (A);
Bland-Altman pair-wise compari-
son between the retested kO
values and the originally calculated
kO values (B); Bland-Altman pair-
wise comparison between the
retested NHvalues and the origi-
nally calculated NH values (C);
Reproducibility of the
Bland-Altman pair-wise comparison between the retested NOvalues and the originally calculated NOvalues (D); and Bland-Altman pair-wise
comparison between the retested EE values and the originally calculated EE values (E). The solid line within each panel represents zero
difference. The dotted line within each panel represents the bias or mean difference between the retest and original values. The 2 dashed lines
within each panel represent the 95% CIs of the bias. The symbols within each panel represent the individual difference between the retest and
the original values. DLW, doubly labeled water; EE, energy expenditure; kH, fractional turnover rate of2H; kO, fractional turnover rate of18O; NH,
isotope dilution space of2H; NO, isotope dilution space of18O.
clinical trial among study participants who were randomly
assigned to the caloric restriction intervention1
Applications of the DLW method in the CALERIE
Nutrition applicationsMean values
Adherence monitoring (baseline to 6 mo)
Body compositional changes (baseline to 6 mo)
NOat baseline, kg
TBW at baseline, kg
FFM at baseline, kg
FM at baseline, kg
2467 6 443 (1883–3225)
1803 6 325 (1400–2346)
1803 6 325 (1400–2346)
1696 6 341 (1254–2393)
2363 6 126 (2614 to 2219)
2467 6 443 (1883–3225)
31 6 5 (25–38)
36.2 6 7.8 (26.1–48.0)
35.8 6 7.7 (25.9–47.6)
49.1 6 10.6 (35.4–65.2)
26.5 6 5.9 (19.0–34.4)
20.8 6 1.1 (22.5–0.8)
21.0 6 1.5 (23.3–1.0)
28.6 6 3.4 (215.2 to 24.6)
1Values are means 6 SDs (ranges), n = 10. CALERIE, Comprehensive Assessment of
Long-term Effects of Reducing Intake of Energy; CR, caloric restriction; DLW, doubly
labeled water; EIAL,ad libitum energy intake; EIp, energy intake over the period of
interest; FM, fat mass; FFM, fat-free mass; NO, isotope dilution space of18O; TBW, total
body water; %CR, percentage of CR achieved; DES, change in body energy stores; DFM,
change in FM; DFFM, change in FFM; DNO, change in the isotope dilution space of18O.
Long-Term reproducibility of the DLW method781
by William Wong on April 18, 2014
in body energy stores during the period of interest. For intervals
between 2 DLW measures, EE was computed as the mean of the
EEestimates acrossthe2 timepoints.For intervalsspanningmore
weighted by the duration of the interval, was applied. DES was
estimated by calculating the change in energy stores (measured by
dual energy x-ray absorptiometry) from the beginning to the end
of the interval. DES was calculated using standard coefficients for
changes in fat mass (FM) (FM: 9300 kcal?kg21) and fat-free mass
(FFM) (FFM: 1100 kcal?kg21). The EE, EIP, DES, and %CR for
10 participants who were assigned to the CR intervention over a
6-mo period are summarized in Table 3.
Body compositional changes. Isotope dilution has been long
considered one of thereference methods forthe measurements of
body composition. It has been well documented that FFM in
healthy adults has a hydration of 73% (51). Knowing the NO
from the DLW protocol, total body water (TBW) can be
calculated using the equation TBW = NO/1.01 because the NOis
assumed to overestimate TBW by 1% (51). Therefore, FFM can
be calculated from TBW using the equation FFM = TBW/0.73.
FM is simply the difference between body weight and FFM. The
changes in body composition (body weight, FFM, FM) among
10 participants who were assigned to the CR group over a 6-mo
period are summarized in Table 3.
Our results represent the first study to document the longitudinal
reproducibility of the DLW method.
The DLW method is considered the reference method for EE
measurements under free-living conditions because it is noninvasive,
nonrestrictive with minimal participant burden, and has no known
adverse effects. The other advantage of the DLW method is that it
can be implemented almost anywhere and the samples can be
shipped back to the analytical laboratory. Because both2H and18O
are nonradioactive stable isotopes, they do not decay or emit
harmful radiation and therefore can be kept for a long time under
proper conditions to support longitudinal studies. As shown in
The results also demonstrated that the isotope ratio measurements
reproducibility of the DLW method was further supported by the
results obtained from the blinded test-retest protocol (Fig. 2),
showing that the results were highly reproducible up to 2.5 y.
One previous study examined the reliability of the DLW
method in 5 participants (52). However, that study was not
blinded and the DLW protocol was repeated on the same
participants after a 3-d break. Therefore, although that study
could be used to evaluate the reliability of the DLW method
within participants, it could not be used to evaluate the
longitudinal reproducibility of the DLW method.
Unfortunately, the DLW method is not widely used in cross-
sectional or longitudinal studies because the method is expensive
and requires specialized instrumentation such as isotope ratio
MS to measure the stable isotopes. Therefore, other dietary
assessment methods such as 24-h dietary recalls and FFQs often
are employed in surveys and longitudinal studies. However,
these less-expensive methods are known to have large measure-
ment errors, particularly among children, different ethnic
groups, and overweight or obese participants (53–56).
Because the reproducibility results were obtained using isotope
ratio MS, the results might not be applicable to DLW studies
carried out using other instrumentation such as cavity ring-down
spectroscopy (57–60) or Fourier transform infrared spectroscopy
(61,62). The long-term reproducibility of the DLW method using
these other instruments will need to be documented.
Our results demonstrate that the DLWoutcome variables are
highly reproducible longitudinally. Therefore, other laboratories
can use these 2 protocols to document the long-term reproduc-
ibility of their measurements to ensure the biologic significance
of the long-term outcomes of interest.
All authors were involved in the research design to evaluate the
longitudinal reproducibility of the DLW method. S.B. Roberts
and S.K.D. were the principal investigators at the Jean Mayer
USDA Human Nutrition Research Center on Aging, Tufts
University. S.B. Racette was the principal co-investigator at
Washington University School of Medicine. L.M.R. was the
principal coinvestigator at the Pennington Biomedical Research
Center. J.R. and W.E.K. were the principal investigators at the Duke
Clinical Research Institute, Duke University. M.V.B. was the study
database manager at the coordinating center. W.W.W. was the
principal investigator of the DLW Laboratory at the USDA/
Agricultural Research Service Children?s Nutrition Research
Center, Baylor College of Medicine. L.L.C. performed all the
MS measurements in the DLW Laboratory. W.W.W. and M.V.B.
performed the statistical analysis. W.W.W. wrote the initial draft of
the manuscript and had primary responsibility for the final content.
All authors read and approved the final manuscript.
1. Lee JS, Lifson N. Measurement of total energy and material balance in
rats by means of doubly labeled water. Am J Physiol. 1960;199:238–42.
Lifson N, Gordon GB, McClintock R. Measurement of total carbon
dioxide production by means of D2O18. J Appl Physiol. 1955;7:704–10.
Lifson N, Lee JS. Estimation of material balance of totally fasted rats by
doubly labeled water. Am J Physiol. 1961;200:85–8.
Lifson N, McClintock R. Theory of use of the turnover rates of body water
for measuring energy and material balance. J Theor Biol. 1966;12:46–74.
Little WS, Lifson N. Validation study of D218O method for determi-
nation of CO2output of the eastern chipmunk (Tamais striatus). Comp
Biochem Physiol Comp Physiol. 1975;50:55–6.
McClintock R, Lifson N. CO2output and energy balance of hereditary
obese mice. Am J Physiol. 1957;189:463–9.
McClintock R, Lifson N. Applicability of the D2O18method to the
measurement of the total carbon dioxide output of obese mice. J Biol
McClintock R, Lifson N. Determination of the total carbon dioxide
outputs of rats by the D2O18method. Am J Physiol. 1958;192:76–8.
McClintock R, Lifson N. CO2output of mice measured by D2O18under
conditions of isotope re-entry into the body. Am J Physiol. 1958;195:
10. Klein PD, James WP, Wong WW, Irving CS, Murgatroyd PR, Cabrera
M, Dallosso HM, Klein ER, Nichols BL. Calorimetric validation of the
doubly-labelled water method for determination of energy expenditure
in man. Hum Nutr Clin Nutr. 1984;38:95–106.
11. Schoeller DA, Webb P. Five-day comparison of the doubly labeled water
method with respiratory gas exchange. Am J Clin Nutr. 1984;40:153–8.
12. SealeJL, Conway JM, CanaryJJ.Seven-dayvalidationofdoublylabeledwater
method using indirect room calorimetry. J Appl Physiol. 1993;74:402–9.
13. Seale JL, Rumpler WV. Comparison of energy expenditure measure-
ments by diet records, energy intake balance, doubly labeled water and
room calorimetry. Eur J Clin Nutr. 1997;51:856–63.
14. Seale JL, Rumpler WV, Conway JM, Miles CW. Comparison of doubly
labeled water, intake-balance, and direct- and indirect-calorimetry
methods for measuring energy expenditure in adult men. Am J Clin
15. Coward WA, Prentice AM, Murgatroyd PR, Davies HL, Cole TJ, Saylor
KE, Glodberg GR, Halliday D, Macnamara JP. Measurement of CO2and
782Wong et al.
by William Wong on April 18, 2014
water production rates in man using2H,18O-labelled H2O: comparisons
between calorimeter and isotope values. In: van Es AJ, editor. Clinical
nutrition and metabolic research. Munich: Karger; 1984. p. 169–77.
16. Ravussin E, Harper IT, Rising R, Bogardus C. Energy expenditure by
doubly labeled water: validation in lean and obese subjects. Am J
17. Jensen CL, Butte NF, Wong WW, Moon JK. Determining energy
expenditure in preterm infants: comparison of
indirect calorimetry. Am J Physiol. 1992;263:R685–92.
18. Roberts SB, Coward WA, Schlossman SF, Schlingensseipen KH, Nohria
V, Lucas A. Comparison of the doubly labeled water (2H2-18O) method
with indirect calorimetry and a nutrient-balance study for simultaneous
determination of energy expenditure, water intake, and metabolizable
energy intake in preterm infants. Am J Clin Nutr. 1986;44:315–22.
19. Roberts SB, Coward WA, Ewing G, Savage J, Cole TJ, Lucas A. Effect of
weaning on accuracy of doubly labeled water method in infants. Am J
20. Butte NF, Wong WW, Ferlic L, Smith EO, Klein PD, Garza C. Energy
expenditure and deposition of breast-fed and formula-fed infants during
early infancy. Pediatr Res. 1990;28:631–40.
21. Treuth MS, Butte NF, Wong WW. Effects of familial predisposition to
obesity on energy expenditure in multiethnic prepubertal girls. Am J
Clin Nutr. 2000;71:893–900.
22. Wong WW, Butte NF, Ellis KJ, Hergenroeder AC, Hill RB, Stuff JE,
Smith EO. Pubertal African-American girls expended less energy at rest
and during physical activity than Caucasian girls. J Clin Endocrinol
23. Bandini LG, Schoeller DA, Dietz WH. Energy expenditure in obese and
nonobese adolescents. Pediatr Res. 1990;27:198–203.
24. Butte NF, Wong WW, Treuth MS, Ellis KJ, O’Brian SE. Energy
requirements during pregnancy based on total energy expenditure and
energy deposition. Am J Clin Nutr. 2004;79:1078–87.
25. Kopp-Hoolihan LE, Van LMD, Wong WW, King JC. Longitudinal
assessment of energy balance in well-nourished, pregnant women. Am J
Clin Nutr. 1999;69:697–704.
26. Villalpando SF, Butte NF, Wong WW, Flores-Huerta S, Hernandez-
Beltran MJ, Smith EO, Garza C. Lactation performance of rural
Mesoamerindians. Eur J Clin Nutr. 1992;46:337–48.
27. Barbosa L, Butte NF, Villalpando S, Wong WW, Smith EO. Maternal
energy balance and lactation performance of Mesoamerindians as a
function of body mass index. Am J Clin Nutr. 1997;66:575–83.
28. Ma ˆsse LC, Fulton JE, Watson KL, Mahar MT, Meyers MC, Wong WW.
Influence of body composition on physical activity validation studies
using doubly labeled water. J Appl Physiol. 2004;96:1357–64.
29. Schulz LO, Schoeller DA. A compilation of total daily energy expenditures
and body weights in healthy adults. Am J Clin Nutr. 1994;60:676–81.
30. Schoeller DA, Levitsky LL, Bandini LG, Dietz WW, Walezak A. Energy
expenditure and body composition in Prader-Willi syndrome. Metab-
31. Casper RC, Schoeller DA, Kushner R, Hnilicka J, Gold ST. Total daily
energy expenditure and activity level in anorexia nervosa. Am J Clin
32. Motil KJ, Schultz RJ, Wong WW, Glaze DG. Increased energy expenditure
associated with repetitive involuntary movement does not contribute to
growth failure in girls with Rett syndrome. J Pediatr. 1998;132:228–33.
33. Delikanaki-Skaribas E, Trail M, Wong WW, Lai EC. Daily energy
expenditure, physical activity, and weight loss in Parkinson’s disease
patients. Mov Disord. 2009;24:667–71.
34. Weir JB. New methods for calculating metabolic rate with special
reference to protein metabolism. J Physiol. 1949;109:1–9.
35. Wong WW, Klein PD. A review of techniques for the preparation of
biological samples for mass-spectrometric measurements of hydrogen-2/
hydrogen-1 and oxygen-18/oxygen-16 isotope ratios. Mass Spectrom
36. Wong WW, Lee LS, Klein PD. Deuterium and oxygen-18 measurements
on microliter samples of urine, plasma, saliva, and human milk. Am J
Clin Nutr. 1987;45:905–13.
37. Wong WW, Clarke LL, Llaurador M, Klein PD. A new zinc product for
the reduction of water in physiological fluids to hydrogen gas for2H/1H
isotope ratio measurements. Eur J Clin Nutr. 1992;46:69–71.
38. Wong WW, Clarke LL. A hydrogen gas-water equilibration method
produces accurate and precise stable hydrogen isotope ratio measure-
ments in nutrition studies. J Nutr. 2012;142:2057–62.
2H218O method and
39. Rochon J, Bales CW, Ravussin E, Redman LM, Holloszy JO, Racette SB,
Roberts SB, Das SK, Romashkan S, Galan KM, et al. Design and conduct of
the CALERIE study: comprehensive assessment of the long-term effects of
reducing intake of energy. J Gerontol A Biol Sci Med Sci. 2011;66:97–108.
40. Stewart TM, Bhapkar M, Das S, Galan K, Martin CK, McAdams L,
Piepeer C, Redman L, Roberts S, Stein RI, et al. Comprehensive
Assessment of Long-term Effects of Reducing Intake of Energy Phase 2
(CALERIE Phase 2) screening and recruitment: methods and results.
Contemp Clin Trials. 2013;34:10–20.
41. Schoeller DA. Energy expenditure from doubly labeled water: some fun-
damental considerations in humans. Am J Clin Nutr. 1983;38:999–1005.
42. Horvitz MA, Schoeller DA. Natural abundance deuterium and 18-
oxygen effects on the precision of the doubly labeled water method. Am
J Physiol Endocrinol Metab. 2001;280:E965–72.
43. Trabulsi J, Troiano RP, Subar AF, Sharbaugh C, Kipnis V, Schatzkin A,
Schoeller DA. Precision of the doubly labeled water method in a large-
scale application: evaluation of a streamlined-dosing protocol in the
Observing Protein and Energy Nutrition (OPEN) study. Eur J Clin Nutr.
44. Gonfintini R. Standards for stable isotope measurements in natural
compounds. Nature. 1978;271:534–6.
45. Prentice AM, editor. The doubly-labelled water method for measuring
energy expenditure—technical recommendations for use in humans.
Vienna, Austria: International Atomic Energy Agency; 1990.
46. Schoeller DA. Measurement of energy expenditure in free-living humans
by using doubly labeled water. J Nutr. 1988;118:1278–89.
47. Black AE, Prentice AM, Coward WA. Use of food quotients to predict
respiratory quotients for the doubly-labelled water method of measur-
ing energy expenditure. Hum Nutr Clin Nutr. 1986;40:381–91.
48. Donner A, Eliasziw M. Sample size requirements for reliability studies.
Stat Med. 1987;6:441–8.
49. Altman DG, Bland JM. Measurement in medicine: the analysis of
method comparison studies. Statistician. 1983;32:307–17.
50. Bland JM, Altman DG. Statistical methods for assessing agreement
between two methods of clinical measurement. Lancet. 1986;1:307–10.
51. Wang Z, Deurenberg P, Wang W, Pietrobelli A, Baumgartner RN,
Heymsfield SB. Hydration of fat-free body mass: review and critique of
a classic body-composition constant. Am J Clin Nutr. 1999;69:833–41.
52. Goran MI, Poehlman ET, Danforth E Jr. Experimental reliability of the
doubly labeled water technique. Am J Physiol. 1994;266:E510–5.
53. Schoeller DA, Bandini LG, Dietz WH. Inaccuracies in self-reported
intake identified by comparison with the doubly labelled water method.
Can J Physiol Pharmacol. 1990;68:941–9.
54. Schoeller DA, Thomas D, Archer E, Heymsfield SB, Blair SN, Goran
MI, Hill JO, Atkinson RL, Corkey BE, Foreyt J, et al. Self-report-based
estimates of energy intake offer an inadequate basis for scientific
conclusions. Am J Clin Nutr. 2013;97:1413–5.
55. Bandini LG, Schoeller DA,Cyr HN,DietzWH.Validityof reported energy
intake in obese and nonobese adolescents. Am J Clin Nutr. 1990;52:421–5.
56. Bandini LG, Must A, Cyr H, Anderson SE, Spadano JL, Dietz WH.
Longitudinal changes in the accuracy of reported energy intake in girls
10–15 y of age. Am J Clin Nutr. 2003;78:480–4.
57. Meier C, Knoche M, Merz R, Weise SM. Stable isotopes in river waters
in the Tajik Pamirs: regional and temporal characteristics. Isotopes
Environ Health Stud. 2013;49:542–54.
58. Tremoy G, Vimeux F, Cattani O, Mayaki S, Souley I, Favreau G.
Measurements of water vapor isotope ratios with wavelength-scanned
cavity ring-down spectroscopy technology: new insights and important
caveats for deuterium excess measurements in tropical areas in
comparison with isotope-ratio mass spectrometry. Rapid Commun
Mass Spectrom. 2011;25:3469–80.
59. Brand WA, Geilmann H, Crosson ER, Rella CW. Cavity ring-down
spectroscopy versus high-temperature conversion isotope ratio mass spec-
alcohol/water mixtures. Rapid Commun Mass Spectrom. 2009;23:1879–84.
60. Thorsen T, Shriver T, Racine N, Richman BA, Schoeller DA. Doubly
labeled water analysis using cavity ring-down spectroscopy. Rapid
Commun Mass Spectrom. 2011;25:3–8.
61. Jennings G, Bluck L, Wright A, Elia M. The use of infrared spectropho-
tometry for measuring body water spaces. Clin Chem. 1999;45:1077–81.
62. Ferrier L, Robert P, Dumon H, Martin L, Nguyen P. Evaluation of body
composition in dogs by isotopic dilution using a low-cost technique,
Fourier-transform infrared spectroscopy. J Nutr. 2002;132:1725S–7S.
Long-Term reproducibility of the DLW method783
by William Wong on April 18, 2014