European Journal of Clinical Nutrition (2007) 61, 1250–1255
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ORIGINAL ARTICLE
Implications of the variability in time to isotopic
equilibrium in the deuterium dilution technique
RC Colley, NM Byrne and AP Hills
School of Human Movement Studies, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane,
Queensland, Australia
Objective: To investigate the variability in isotopic equilibrium time under field conditions, and the impact of this variability on
estimates of total body water (TBW) and body composition.
Design and Setting: Following collection of a fasting baseline urine sample, 10 women and 10 men were dosed with deuterium
oxide (0.05 g/kg body weight). Urine samples were collected every hour for 8 h. The samples were analysed using isotope ratio
mass spectrometry. Time to equilibration was determined using three commonly employed data analysis approaches.
Results: Isotopic equilibrium was reached by 50, 80 and 100% of participants at 4, 6 and 8 h, respectively. The mean group
equilibration determined using the three different plateau determination methods were 4.871.5, 3.870.8 and 4.971.4 h.
Isotopic enrichment, TBW, and percent body fat estimates differed between early (3–5 h), but not later sampling times (5–8 h).
Conclusion: Although the three different plateau determination approaches resulted in differences in equilibration time, all
suggest that sampling at 6 h or later will decrease the likelihood of error in body composition estimates resultant from
incomplete isotopic equilibration in a small proportion of individuals.
European Journal of Clinical Nutrition (2007) 61, 1250–1255; doi:10.1038/sj.ejcn.1602653; published online 7 February 2007
Keywords: total body water; body composition; urine; sampling time
Introduction
Deuterium dilution measurements of total body water (TBW)
and subsequent estimates of body composition are increasingly common in health-related research. The technique is
commonly used in field studies, where many of the stringent
conditions achieved in controlled laboratory settings are
unrealistic to maintain. Administration of a known dose of
isotope (2H) is given to an individual after collection of a
baseline sample. Although multiple post-dose samples can
be collected, more commonly a single post-dose sample is
provided at a time when isotopic enrichment has been
Correspondence: RC Colley, Institute of Health and Biomedical Innovation,
Queensland University of Technology, Brisbane, Queensland, Australia.
E-mail: [email protected]
Guarantor: RC Colley.
Contributors: RCC was responsible for the data collection, study design,
manuscript writing, data analysis, data interpretation and statistical analysis.
NMB initiated the study and was largely involved in the study design, data
analysis, data interpretation, statistical analysis and manuscript editing. APH
oversaw the initiation and design of the study and manuscript editing.
Received 23 January 2006; revised 29 November 2006; accepted 12
December 2006; published online 7 February 2007
reached within the body fluid. Sampling mediums include
blood, saliva, urine and, more recently, measures of expired
air (Smith et al., 2002). The timing of the post-dose sample
collection is not the same for each sampling medium as the
time to reach isotopic equilibrium is longer when urine is the
sampling medium. Furthermore, as time to equilibration is
highly variable between individuals (Schoeller et al., 1980), a
practical concern in body composition research is identifying the test protocol, which will ensure that isotopic
equilibrium is likely to be achieved in all individuals.
Early research investigating isotope dilution and body
water suggested that the equilibrium time of deuterium in
urine is between 1.5 and 3 h (Schloerb et al. 1950; Mendez
et al. 1970), however more recent research tends to suggest
sampling times closer to 6 h are required (Schoeller et al.,
1980, 1982). Blanc et al. (2002) found that a post-dose
sampling time of 3 h was insufficient to reach an isotopic
plateau in 21% of participants. In 1989, Wong et al. (1989)
reported that equilibrium was not attained in 100% of
participants at 6 h, suggesting that later time points needed
to be investigated. Van Marken Lichtenbelt et al. (1994)
suggested that it may take up to 10 h for deuterium to
equilibrate completely with body water; a conclusion based
Variability in isotopic equilibrium
RC Colley et al
1251
on a comparison with underwater weighing. Recent studies
have reported post-dose urine sampling at 5 h (Rush et al.,
2003; Bauer et al., 2005) or between 4 and 6 h (Puiman et al.,
2004). It is unclear whether such protocol variance compromises the accuracy of the deuterium dilution technique
(Lukaski and Johnson, 1985; Speakman, 1997; Wong, 2003).
Standard analysis methods of the deuterium dilution
technique include the plateau method, the intercept method
and overnight equilibration. The plateau method is often
applied in fieldwork because it only requires one sample after
dosing. After dose administration, deuterium enrichment in
the body water pools is highly variable until it reaches a
relatively stable value, which is defined as the plateau.
Isotopic equilibrium of urine takes longer than in other body
fluids because of water previously stored in the bladder.
Collecting the urine too early can result in samples
misrepresenting the true enrichment level at a particular
point in time (Blanc et al., 2002). The accuracy of the
deuterium dilution technique, therefore, relies heavily on
ensuring that the collection of the post-dose sample occurs
after full isotopic equilibration has taken place. Several
different plateau determination methods exist, and this lack
of consistency has made comparison between studies
challenging.
A plateau or equilibrium has been defined as the point
when the difference in isotopic enrichment between two
consecutive time points becomes less than 3% (Fjeld et al.,
1988); however, a more conservative value of o2% has more
frequently been suggested and is considered an acceptable
limit for analytic considerations (Salazar et al., 1994;
Schoeller et al., 2000; Blanc et al., 2002). Jankowski et al.
(2004) defined a reference value as the average of three
samples taken during the fourth hour post-dosing and
suggested that equilibrium occurred when the enrichment
became p2% of the reference value. In contrast, other
researchers use the 6 h enrichment value as a reference
(Schoeller et al., 1980, 1982; Wong et al., 1989).
The objective of this study was therefore to investigate the
variability in equilibrium time under field conditions, and
the impact of this variability on estimates of TBW and body
composition. By tracking deuterium enrichment from postdose urine samples collected on an hourly basis for 8 h, the
present study determined the differences incurred when
sampling occurs earlier or later than a standard time point of
6 h. It was hypothesised that a greater relative measurement
error in TBW and body composition estimates would result
from sampling o6 h when compared to sampling X6 h.
voiding dysfunctions were indicated. Descriptive characteristics of the participants are presented in Table 1. The study
protocol was approved by the Queensland University of
Technology Human Research Ethics Committee.
Dosing and sample collection protocol
Measurements of TBW were made using the stable, nonradioactive, non-toxic isotope deuterium oxide, in the form
of water (2H2O). Participants provided a baseline urine
sample before consuming an oral bolus equating to 0.05 g/
kg body weight of 2H2O. The pre-dosing sample was used to
correct for background 2H2O concentration values in the
post-dose sample. The container and straw were weighed
following oral ingestion of the dose to account for any
leftover dose in the container. This was then subtracted from
the weight of the container, straw, and dose to give the exact
amount of dose ingested. A single urine sample was provided
every hour for 8 h post-dose during which participants
undertook a sedentary workday. Participants were instructed
to collect each sample as close to the hour as possible
(75 min) and to not void in between the hourly sample
collections. Participants were fasted at the time of dosing;
however, no restrictions on dietary or fluid intake were
imposed on the participants during the equilibrium period
to ensure typical field conditions.
Deuterium enrichment measurements
The enrichment of the local tap water, the dose given and
the 2H2O in the pre-dose and post-dose samples were
measured using isotope ratio mass spectrometry (Hydra,
PDZ Europa, Crew, UK). Each urine sample (500 ml) was
pipetted into 10 ml exetainers (Labco Ltd., Califon, NJ, USA)
for analysis. A catalyst, approximately 1 mg of platinum on
alumina powder (Sigma Aldrich Inc., St Louis, MO, USA)
contained in a 0.5 ml vial (Chromacol Ltd., Herts, UK), was
introduced into the exetainer ensuring no direct contact
with the urine. Each sample was evacuated for 5 min before
being filled with 99% hydrogen gas for no more than 2 s. The
samples were left at room temperature for a minimum of
72 h to equilibrate the deuterium in the urine sample with
the hydrogen gas above it. All reference waters were prepared
at the same time and in the same manner. Isotopic
enrichments were expressed as the difference per unit
volume (%O) from standard mean ocean water . All assays
Table 1
Methods
Participants
Twenty healthy adults (10 males, 10 females) participated in
the study. Participants were not selected based on any age,
activity level or body composition criteria but were excluded
from the study if they were pregnant or lactating. No renal or
Physical characteristics of the participants (Mean7s.d.)
Variable
N
Age (years)
Height (m)
Weight (kg)
Body Mass Index (kg/m2)
Males
Females
10
31.6710.3
1.870.1
77.979.9
25.073.3
10
31.379.6
1.770.1
65.9712.6
22.173.0
European Journal of Clinical Nutrition
Variability in isotopic equilibrium
RC Colley et al
1252
were performed in duplicate with repeat assays in our
laboratory demonstrating coefficients of variance (CV) of
o2.0% at low enrichment levels and o1% for higher values.
Calculations and formulae
The dilution space was calculated (Eq. 1) by subtracting the
pre-dose urine sample enrichment from that of the post-dose
sample.
ð1Þ
N ¼ TA=a ðEa Et Þ=ðEs Ep Þ
N is the dilution space in grams, A the isotope given in
grams, a a portion of the dose (in grams) retained for mass
spectrometer analysis, T the amount of tap water (ml), in
which a is diluted before analysis, and Ea, Et, Ep and Es are the
isotopic enrichments (in units) of the portion of dose, the
tap water used, the pre-dose sample of physiological fluid
(i.e. urine) and the post-dose sample of physiological fluid,
respectively. The calculation was carried out with the eight
different post-dose (Es) result.
Total body water (l) was subsequently calculated using
Eq. 2:
TBW ¼ ðN=1:04Þ=1000
ð2Þ
The division of N by 1.04 corrects for the exchange of
deuterium with non-aqueous hydrogen. On the basis of
assumption that the hydration of fat free mass (FFM) is 0.73
for adults (Wong, 2003), FFM (kg) was calculated using Eq. 3.
Percent body fat (%) was subsequently derived from FFM and
body weight (W) using Eq. 4:
FFM ¼ TBW=0:73
ð3Þ
and standard deviations (s.d.) were used as summary
statistics. Differences in TBW and percent body fat over the
8-h period were assessed using repeated measures analysis
of variance (ANOVA). Where significant differences were
identified (Po0.05), post-hoc analyses using a Bonferroni
adjustment for multiple comparisons were employed. In a
sub-sample (N ¼ 9) dietary intake was recorded; in this group
correlations between the plateau time and fluid intake before
hour 6 and fluid intake before the time of plateau were
investigated.
Results
The anthropometric data of the 20 participants is presented
in Table 1. All participants were able to provide a urine
sample at each of the eight post-dose time points. Figure 1
depicts the proportion of participants who reached isotopic
equilibration at all time points using the three different
plateau determination techniques described in the methods.
Isotopic equilibrium occurred in the whole group, for all
three methods, by hour 8. Using the most commonly cited
method (1), the proportion of participants equilibrated at
hour 4 was 50%. This proportion increased to 80% by hour 6
and 100% by hour 8.
Using the three plateau determination procedures described in the methods, the mean time to equilibration was
assessed. Method 1 (consecutive values o2%) resulted in a
mean equilibration time of 4.871.5 h, method 2 (consecutive values o3%) had a mean equilibration time of
3.870.8 h and method 3 (o2% of hour 6) resulted in mean
equilibration time of 4.971.4 h. Methods 1 and 2 resulted in
% Body Fat ¼ ððW FFMÞ=WÞ100
ð4Þ
Data analysis
On the basis of methods most widely discussed in the
literature, the following three methods of assessing plateau
were evaluated in the present study:
(1) Plateau defined as the earliest time point when consecutive enrichment values become o2% different from
the previous hour.
(2) Plateau defined as the earliest time point when consecutive enrichment values become o3% different from
the previous hour.
(3) Plateau defined as the first time point at which the
enrichment value differs from the enrichment level at
hour 6 by o2%.
Statistical analyses were carried out using SPSS Version
12.0.01, 2003 (SPSS Inc., Chicago, IL, USA). As all outcome
variables were determined to be normally distributed, means
European Journal of Clinical Nutrition
Proportion of Participants
Reaching Isotopic Equilibrium (%)
120
Method 1
Method 2
Method 3
100
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
Time (hrs)
Figure 1 The proportion of participants reaching isotopic equilibration at each hour using three different methods of plateau
determination. Method 1: Plateau defined as the earliest time point
when consecutive enrichment values become o2% different from
the previous hour. Method 2: Plateau defined as the earliest timepoint when consecutive enrichment values become o3% different
from the previous hour. Method 3: Plateau defined as the earliest
time point, which is o2% different from the enrichment level at
hour 6.
Variability in isotopic equilibrium
RC Colley et al
1253
Table 2 TBW and percent body fat across the 8-hourly time-points
(Mean7s.d.)
8
Equilibration Time (hrs)
7
Hour
Hour
Hour
Hour
Hour
Hour
Hour
Hour
6
5
1
2
3
4
5
6
7
8
TBW (litres)
Percent body fat (%)
50.8714.5
37.477.3
38.476.9
39.376.8
39.976.7
40.376.2
40.576.5
40.776.5
0.9733.7
28.579.4
26.577.6
24.877.8
23.677.6
22.677.6
22.277.6
22.077.8
Abbreviation: TBW; total body water.
All time-points include n ¼ 20.
4
Table 3
Summary of post-hoc analysis for TBW during 8-h period
Hour 3
Hour 4
Hour 5
Hour 6
Hour 7
Hour 8
NS
NS
*
*
*
*
*
*
*
NS
*
*
*
NS
NS
*
*
*
NS
NS
NS
3
Method 2
Method 3
Figure 2 Boxplot depicting the mean, s.d., range, and outliers in
isotopic equilibration using the three different methods. Method 1:
Plateau defined as the earliest time point when consecutive
enrichment values become o2% different from the previous hour.
Method 2: Plateau defined as the earliest time-point when
consecutive enrichment values become o3% different from the
previous hour. Method 3: Plateau defined as the earliest time point,
which is o2% different from the enrichment level at hour 6.
significantly different equilibration times (P ¼ 0.004), as did
methods 2 and 3 (P ¼ 0.006). Method 1 equilibration times
were not different to method 3. The mean equilibration
time, s.d., range and outliers of each method are depicted in
Figure 2.
Means and s.d. for TBW and percent body fat are presented
in Table 2. A significant effect of time was found in isotopic
enrichment (F1.26 ¼ 12.38, Po0.001), TBW (F1.05 ¼ 9.52,
Po0.005) and percent body fat (F1.05 ¼ 8.64, Po0.007) across
the eight hours. Table 3 displays the results of the post-hoc
analysis for TBW. Hours 3 and 4 were significantly different
to each other (Po0.000) and both were significantly
different to hours 5, 6, 7 and 8. There were no significant
differences between any combination of hours 5, 6, 7 and 8.
Similar results were found for isotopic enrichment and
percent body fat data (data not shown). Figure 3 demonstrates that sampling earlier (2–6 h) resulted in greater
difference in TBW relative to hour 6 (reference time point),
whereas sampling later resulted in an overestimation of TBW
relative to hour 6. The difference resultant from sampling
later was less than that for sampling earlier.
Fluid and dietary intake data were available from nine of
the participants. The total mean cumulative fluid intake over
the course of the 8 h protocol period was 16927799 ml
(range: 250–2600), equating to an average intake of approximately 200 ml/h.
Hour
Hour
Hour
Hour
Hour
Hour
2
3
4
5
6
7
Abbreviation: TBW; total body weight.
*Indicates a statistically significant difference (Po0.05).
NS indicates no statistically significant difference (P40.05).
Difference in TBW Relative to Hour 6 (%)
Method 1
4.0
2.0
0.0
– 2.0
– 4.0
– 6.0
– 8.0
– 10.0
– 12.0
– 14.0
– 16.0
2
3
4
5
6
7
8
Time (hrs)
Figure 3 Underestimation and overestimation in TBW resulting
from early versus later sampling. Error bars represent s.d.
Discussion
It is important to establish that the present study did not aim
to determine a single ‘optimal’ urine sampling time. Rather,
this study investigated the between-individual variance in
time to isotopic equilibrium in urine; a finding that has
important implications on how the technique is administered. We accept that many individuals will reach isotopic
equilibrium in as early as 3–4 h and that most will do so in
6 h. In fact, 85% of participants in the present study reached
European Journal of Clinical Nutrition
Variability in isotopic equilibrium
RC Colley et al
1254
equilibrium by 6 h. Those who did not equilibrate in 6 hours
were free from ailments (oedema, ascites, pleural effusion,
shock) which have been previously suggested as causes of
prolonged equilibration time (McMurrey et al., 1958). The
findings of the present study are consistent with previous
research (Schoeller et al., 1980; Wong et al., 1989), which has
suggested that a minimum of 6 h is required for isotopic
equilibrium to be achieved in urine. However, we have also
shown that equilibrium was achieved in 100% of participants at 8 h. This finding is not novel as others have
previously found that 4–6 h is inadequate for full equilibrium, suggesting that an overnight equilibrium of 10 h is
preferable (van Marken Lichtenbelt et al., 1994).
The plateau method requires that the post-dose sample is
collected in a window of time where full isotopic equilibrium
within the bladder has been reached. Equilibrium time in
urine varies greatly between individuals, therefore making it
impossible to predict an optimal single time point for sample
collection. If the single post-dose urine sample is collected
too early, the non-equilibrium enrichment value may
impose significant error in TBW and body composition
estimates. Errors in data reporting may occur because of
difficulty in determining which individuals, if any, will have
prolonged equilibration times. In addition, it is unrealistic in
field settings to collect multiple urine samples and create
individual equilibrium curves as was done in the present
study. Therefore, the recommended window to collect the
single post-dose sample becomes a critical component of the
deuterium dilution technique. On the basis of available
literature, three common methods of plateau determination
were evaluated in the present study to give a broad sense of
how the chosen methodology can alter the resultant
equilibrium time. The present study found that the method
of plateau determination resulted in significantly different
equilibration times. This result is indicative of the inconsistency in the literature relating to plateau determination.
Undoubtedly, this can only lead to further confusion for
researchers administering the technique. Figure 1 demonstrates that regardless of which plateau determination
method is used, the chance of reaching isotopic equilibrium
in 100% of participants is increased when using a later postdose sampling time.
Confusion may arise if the incorrect assumption is made
that the deuterium dilution technique can be administered
using the same sampling time protocol for different
sampling mediums. The evidence in support of a 3 h
equilibrium time is well established in serum and breath
(Schoeller et al., 1980, 1982; Wong et al., 1988). However,
equilibration time is longer when urine is the sampling
medium, primarily because it takes longer for the isotope to
fully mix with the fluid in the bladder (Schoeller et al., 1980).
Wong et al. (1989) demonstrated that 100% equilibration
had not been achieved at 6 h in a group of 10 lactating
women. It was suggested that pooling of water in the bladder
may have contributed to unequal distribution of the isotope.
van Marken Lichtenbelt et al. (1994) compared TBW
European Journal of Clinical Nutrition
estimates from urine samples collected at 4, 6 and 10 h
post-dose. Incomplete mixing of deuterium in the bladder
was evident in the samples collected at 4 and 6 h, but not at
10 h. Fluid retention can occur in some individuals,
particularly the elderly, which also affects isotope mixing
(Blanc et al., 2002). The present study is in agreement with
these previous studies in the suggestion that most, but not
all, individuals will reach equilibrium at 6 h.
From a cost–benefit perspective, the argument to sample at
6 h or later is valid if no negative effects are imposed by this
recommendation. Given that deuterium dilution is the
criterion measure of TBW, validation of the correct sampling
time in the traditional sense has not been possible (Speakman,
1997). No significant differences were found when TBW
values derived from deuterium dilution were compared with
underwater weighing (van Marken Lichtenbelt et al., 1994)
as well as bioelectrical impedance (Isenring et al., 2004). In
place of a validation measure, reference time points have
been established. Jankowski et al. (2004) suggested a
reference steady-state enrichment value at 4 h, however 6 h
has more frequently been used (Schoeller et al., 1980, 1982;
Wong et al., 1989). For the purposes of this discussion, hour 6
will be considered as the reference time point. A practical
issue thus becomes deciding what range of time to instruct
individuals to collect the post-dose sample. Figure 3 demonstrates that sampling earlier (2–6 h) results in an underestimation of TBW, which is less than the potential
overestimation of TBW which results from sampling later
(6–8 h). Significant differences between consecutive hours of
TBW and percentage body fat estimates were evident
amongst early time points (3–5 h) and none existed between
any combination of hours 5, 6, 7 and 8. Therefore, sampling
later than 6 h not only increases the likelihood that all
individuals will have reached equilibrium, but it appears as
though there is also less clinically significant error imposed
on body composition estimates.
Several studies using the deuterium dilution technique
have strictly controlled diet and fluid intake both before and
after dosing (Halliday and Miller, 1977; Schoeller et al., 1980,
1982; Lukaski and Johnson, 1985; Wong et al., 1988; Racette
et al., 1994; Blanc et al., 2002; Raman et al., 2004), wheres
others have acknowledged the practical limitations this
imposes in clinical or field settings (Salazar et al., 1994;
Isenring et al., 2004; Puiman et al., 2004). The present study
was purposely designed with no restrictions placed on
dietary and fluid intake during the equilibrium period. Like
Salazar et al. (1994), we aimed to investigate the technique
under the same conditions that it is presently being used in
body composition research. Nonetheless, dietary intake
during the equilibrium period can have an effect on the
time it takes equilibrium to be reached. It has been suggested
that large boluses (2–3% of the body water pool size) of fluid
can disturb isotopic enrichment (Drews and Stein, 1992).
Therefore, dietary and fluid intake is commonly recorded
during the equilibrium period (Schoeller, 1996) for later
corrections. Although seldom reported, the influence of fluid
Variability in isotopic equilibrium
RC Colley et al
1255
intake seems to be of concern only when large boluses of
fluid are taken in the hour before the sample collection. A
dietary and fluid record in the present study enabled us to
confirm that no large boluses of fluid were consumed during
the later hours of the equilibrium period. However, equilibrium is generally reached earlier in the fed state, primarily
because of increased intestinal transport and absorption
through the intestine wall (van Marken Lichtenbelt et al.,
1994). Given that the present study’s participants were not
fasting, the equilibrium times found were likely earlier than
they would have been under fasted conditions. This
strengthens the argument for later sampling as it suggests
that an individual not consuming much food during the
equilibrium period could potentially have a delayed isotopic
equilibrium due to slowed intestinal transport. A separate
study, with a larger sample size, is required to elucidate more
definitely the true effect of dietary and fluid intake on time
to isotopic equilibrium.
The most important contribution of the present study is
the provision of practical advice regarding the appropriate
timing of urine sample collection when the deuterium
dilution technique is used under field conditions to estimate
body composition. Given the unpredictable nature of fluid
shifts coming in and out of the bladder, the time to isotopic
equilibrium is highly variable. This was evident in a group of
healthy adults and could potentially be more pronounced in
special populations. Confidence that isotopic equilibrium
has been reached in the majority, if not all, participants is
greatly increased if the single post-dose urine sample is
obtained at 6 h, or preferably beyond 6 h and up to 8 h.
Acknowledgements
The authors thank Connie Wishart for her laboratory
assistance, Scott Wearing for his contribution to data
analysis and Ryan van Asten for his assistance in the early
phases of data collection.
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