The Journal of Nutrition

Methodology and Mathematical Modeling

A Hydrogen Gas-Water Equilibration Method

Produces Accurate and Precise Stable
Hydrogen Isotope Ratio Measurements
in Nutrition Studies1,2
William W. Wong* and Lucinda L. Clarke

Department of Pediatrics, USDA/Agricultural Research Service ChildrenÕs Nutrition Research Center, Baylor College of Medicine,
Houston, TX

Downloaded from jn.nutrition.org at UNIVERSITY OF NORTH DAKOTA on June 13, 2015

Abstract
Stable hydrogen isotope methodology is used in nutrition studies to measure growth, breast milk intake, and energy
requirement. Isotope ratio MS is the best instrumentation to measure the stable hydrogen isotope ratios in physiological
fluids. Conventional methods to convert physiological fluids to hydrogen gas (H2) for mass spectrometric analysis are labor
intensive, require special reagent, and involve memory effect and potential isotope fractionation. The objective of this
study was to determine the accuracy and precision of a platinum catalyzed H2-water equilibration method for stable
hydrogen isotope ratio measurements. Time to reach isotopic equilibrium, day-to-day and week-to-week reproducibility,
accuracy, and precision of stable hydrogen isotope ratio measurements by the H2-water equilibration method were
assessed using a Thermo DELTA V Advantage continuous-flow isotope ratio mass spectrometer. It took 3 h to reach
isotopic equilibrium. The day-to-day and week-to-week measurements on water and urine samples with natural
abundance and enriched levels of deuterium were highly reproducible. The method was accurate to within 2.8 o/oo and
reproducible to within 4.0 o/oo based on analysis of international references. All the outcome variables, whether in urine
samples collected in 10 doubly labeled water studies or plasma samples collected in 26 body water studies, did not differ
from those obtained using the reference zinc reduction method. The method produced highly accurate estimation on ad
libitum energy intakes, body composition, and water turnover rates. The method greatly reduces the analytical cost and
could easily be adopted by laboratories equipped with a continuous-flow isotope ratio mass spectrometer. J. Nutr. 142:
2057–2062, 2012.

Introduction
The stable hydrogen isotope method has been used in nutrition
studies to measure growth (1), breast milk intake (2,3), lipid
metabolism (4), and free-living energy expenditure (EE) (5–9).
With obesity reaching epidemic proportions in the United States,
the use of the stable hydrogen isotope method to measure growth
and EE under free-living conditions is of particular importance,
because the tracer, deuterium (2H)3 oxide, is nonradioactive and
1
Supported by the NIH National Center for Research Resources Shared
Instrumentation Grant (1S10RR026764-01) and USDA/Agricultural Research
Service grant no. 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.
2
Author disclosures: W. W. Wong and L. L. Clarke, no conflicts of interest.
3
Abbreviations used: DLW, doubly labeled water; EE, energy expenditure; H2,
hydrogen gas; 2H, deuterium; GISP, Greenland Ice Sheet Precipitation; IAEA,
International Atomic Energy Agency; kH, fractional turnover rate of deuterium;
NH, isotope dilution space of deuterium; NH:NO, ratio between the isotope
dilution spaces of deuterium and oxygen-18.
* To whom correspondence should be addressed. E-mail: wwong@bcm.edu.
ã 2012 American Society for Nutrition.
Manuscript received August 3, 2012. Initial review completed August 16, 2012. Revision accepted August 27, 2012.
First published online September 26, 2012; doi:10.3945/jn.112.167957.
the heavier isotope, 2H, is present in body tissues and in foods
and beverages that are being consumed by humans every day.
Therefore, the stable hydrogen isotope method can be used in
all age groups, including premature infants, newborns, toddlers,
children, and pregnant and lactating women with no known
adverse effects.
Isotope ratio MS is considered to be the best instrumentation
for the accurate and precise measurements of 2H in physiological
samples (10,11). However, stable hydrogen isotope ratio mea-
surement is considered most difficult, because the process to
reduce water in the physiological fluids to hydrogen gas (H2) for
isotope ratio measurement is labor intensive, involves a poten-
tial huge isotope fractionation effect, and requires correction for
memory effect.
In the current study, we sought to determine whether a
platinum catalyzed H2-water equilibration method, without
the requirement to reduce the physiological fluid to H2, could
produce accurate and reproducible stable hydrogen isotope
ratio measurements to support nutrition studies using the stable
hydrogen isotope methodology.
2057
Methods
Mass spectrometer system
A Thermo Delta V Advantage continuous-flow isotope ratio mass
spectrometer system equipped with a Finnigan GasBench II (Thermo
Electron North America) was used to assess the accuracy and reproduc-
ibility of the platinum catalyzed H2-water equilibration method for stable
hydrogen isotope ratio measurements. Briefly, the mass spectrometer
system was set to H mode with high voltage at 3 kV and the magnet at
1579 steps. The reference H2 (Air Liquide Healthcare) was set at 449
kPa and the carrier gas, helium (at 99.999%, Air Liquide Healthcare)
was set at 414 kPa. All stable hydrogen isotope ratio measurements are
expressed in d per mil unit (o/oo) vs. the international reference materials,
Standard Mean Ocean Water (SMOW), and Standard Light Antarctic
Precipitation (SLAP) (12,13) as follows:

2
d2 HSMOW=SLAP ð&Þ ¼ ð H=1 HÞsample =ð H=1 HÞreference 21Þ3103 ;
2
where (2H/1H)sample and (2H/1H)reference represent the 2H:1H ratio of the
sample or the reference H2, respectively. A change in 1 d2HSMOW/SLAP
unit is equivalent to a change of 65 mmol/L in 2H content.
Sample preparation procedure
After uncapping a 12-mL Exetainer (Labco International), ;5 mg of
activated charcoal (Fisher Scientific) and ;200 mg of copper powder
(Fisher Scientific) were introduced into the Exetanier followed with a
platinum catalytic rod (ThermoScientific). The activated charcoal and
copper powder were added to remove any potential contaminants in
the samples that might poison the catalyst. After putting 0.2 mL of
sample into the Exetainer, the Exetainer was recapped and placed into
the GasBench II and flushed with 2% H2 in helium at 483 kPa
(M0001710-P-44 at 99.999% H2 and 99.996% helium, Air Liquide
Healthcare) for 7 min. The sample was allowed to equilibrate with the
H2 at room temperature. At the end of the equilibration, an aliquot of
the H2 in the Exetainer was injected into the Thermo Delta V Advantage
mass spectrometer system for stable hydrogen isotope ratio measurement
against the reference H2.
Equilibration time
The time to reach isotopic equilibrium was assessed by allowing 18
Exetainers containing a water sample with a d2HSMOW/SLAP value of +963%
to equilibrate between 0.7 and 8.8 h. At the end of each equilibration period,
an aliquot of the H2 in the Exetainers was injected into the mass
spectrometer system for stable hydrogen isotope ratio measurement.
Day-to-day and week-to-week reproducibility
Two water samples, one at natural abundance (d2HSMOW/SLAP = 214.9 o/oo)
and one at an enriched level of 2H (d2HSMOW/SLAP = +1096.8 o/oo) as well as
2 urine samples, one at natural abundance (d2HSMOW/SLAP = 237.9 o/oo)
and one at an enriched level of 2H (d2HSMOW/SLAP = +1077.3 o/oo), were
analyzed once per day for 8 d and once per week for 5 wk.
Accuracy and precision of stable hydrogen isotope ratio
measurements
The accuracy and precision of the H2-water equilibration method for
stable hydrogen isotope ratio measurements were assessed using 3
international reference materials: Greenland Ice Sheet Precipitation (GISP)
(12,13), International Atomic Energy Agency (IAEA) 302A, and IAEA
302B (14). Three separate aliquots of the GISP were analyzed and each
analysis represented 10 measurements. The IAEA reference materials were
analyzed during a period of 4 d and each analysis again represented 10
measurements.
Accuracy of the H2-water equilibration method in doubly
labeled water and body water studies
To further assess the accuracy of the platinum catalyzed H2-water
equilibration method, urine samples collected from 10 doubly labeled
water (DLW) studies with EE values ranging between 1943 and 3006
kcal/d were analyzed using the platinum catalyzed H2-water equili-
bration method following the stable hydrogen isotope ratio measurements
2058 Wong and Clarke
using the reference zinc reduction method (15) and the stable oxygen
isotope ratio measurements using the reference H2O-CO2 equilibra-
tion method (11). The DLW outcome parameters: fractional turnover
rate of 2H (kH), isotope dilution space of 2H (NH), ratio between the
isotope dilution spaces of 2H and oxygen-18 (NH:NO), and EE derived
from the stable hydrogen isotope ratio measurements using the H2-
water equilibration method, were compared with those obtained using
the reference methods (11,15). To determine whether the equilibration
method was able to accurately measure the stable hydrogen isotope content
in serum samples, serum samples collected in 26 body water studies with
NH ranging between 46 and 86 kg were analyzed. The NH obtained from
the equilibration method was compared with those obtained using the
reference zinc reduction method (15).
Nutrition applications of the H2-water equilibration method
Ad libitum energy intake. In a clinical trial (Comprehensive Assess-
ment of Long-term Effects of Reducing Intake of Energy) to determine
the effect of energy restriction on human physiology, the ad libitum
energy intake of 221 normal-weight study participants was determined
using the DLW method. In the DLW method, after the collection of 2
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baseline urine samples, each participant ingested 1.7 g/kg body weight
of DLW. Two urine samples were collected 4–5 h postdose. Four more
urine samples, 2 on each day, were collected 7 and 14 d postdose. Because
these participants were weight stable, the ad libitum energy intake was
equal to EE. To demonstrate the accuracy and precision of the H2-water
equilibration method to estimate ad libitum energy intake, urine samples
collected from 10 study participants in this clinical trial were analyzed
using the traditional zinc reduction method and again using the H2-
water equilibration method. The ad libitum energy intakes were then
compared between the 2 analytical methods.
Body fat measurements. In a companion animal study to look at the
effect of diet on body composition, the 2H dilution spaces of house cats
(Felis catus) at various stages of obesity were measured by the 2H oxide
dilution method. After collection of a baseline serum sample, each cat
was given orally 60 mg/kg body weight of 2H oxide. Another serum
sample was collected 3 h postdose. The serum samples collected from
10 cats were analyzed for 2H content using both the traditional zinc
reduction method and the H2-water equilibration method. Total body
water was calculated by dividing the 2H dilution spaces by 1.04,
because 2H dilution is known to overestimate total body water by 4%.
Lean body mass was calculated by dividing total body water by 0.73,
the hydration constant of lean body mass in mature cats. Body fat was
calculated as the difference between body weight and lean body mass.
The body fat obtained in these 10 studies was compared between the 2
analytical methods.
Water turnover rates. Water turnover rates can be estimated using the
2
H dilution method. The information can be used to estimate breast milk
production in lactating women or fluid requirement under challenging
field conditions. After collection of a baseline urine sample, the study
participants ingested 100 mg/kg body weight of 2H oxide. Six more
postdose urine samples were collected on d 1, 7, and 14. The urine samples
from 10 soldiers in a training field study were analyzed using the traditional
zinc reduction method and again using the H2-water equilibration method.
The 2H dilution spaces were calculated as described in the body fat
measurements. The fractional turnover rates of 2H were calculated from
the monoexponential disappearing curves of 2H over time in the urine
samples. Water turnover rates were calculated as the product between
the 2H dilution spaces and the fractional turnover rates. The water
turnover rates were compared between the 2 analytical methods.
All human and animal protocols described in this manuscript have
been approved by the Institutional Review Board for Human and Animal
Studies at the study institutions (Baylor College of Medicine, Pennington
Biomedical Research Institute, and Nestle Purina PetCare).
Statistical analysis
The Bland and Altman pairwise comparison analysis (16) was used to
evaluate the accuracy of the platinum catalyzed H2-water equilibration
method for the measurements of kH, NH, NH:NO, and/or EE against
those generated using the reference zinc reduction method (11,15). The be: 20.00013 d21 and the lower and upper limits of agreement to
paired samples t test was used to compare the results obtained in the studies be 20.00156 and 0.00129 d21, respectively. For NH (Fig. 1B), the
to measure ad libitum energy intake, body fat, and water turnover rate. An bias was 20.01 kg and the lower and upper limits of agreement
a value of 0.05 was used in all statistical analyses and all values in the text were 20.39 and 0.38 kg, respectively. The NH:NO ratio (Fig.
are mean 6 SD.
1C) also was very similar between the 2 methods (bias: 20.0004;
lower and upper limits of agreement: 20.0096 and 0.0088,
respectively). Measurements of EE (Fig. 1D) also were highly
Results reproducible between the 2 methods (bias: 27.1 kcal/d; lower and
upper limits of agreement: 2169.5 and 155.4 kcal/d, respectively).
It took ~3 h for the stable hydrogen isotopes to reach isotopic
As shown by the P values in each figure, no significant relationship
equilibrium at room temperature. The stable hydrogen isotope
was detected between the differences and the mean values.
ratios remained stable for up to at least 9 h.
The accuracy of the equilibration method to measure the stable
The day-to-day and week-to-week measurements of a water
hydrogen isotope content in serum samples is demonstrated in
material at natural abundance of 2H (d2HSMOW/SLAP = 214.9
o Figure 2. As shown in the figure, the NH values measured by the
/oo) were reproducible to within 1.5 and 1.4 o/oo, respectively.
equilibration method were only 0.16 kg (bias) higher than that
For a water material at the enriched level of 2H (d2HSMOW/SLAP =
obtained using the zinc reduction method, with lower and upper
+1096.8 o/oo), the day-to-day and week-to-week measurements
limits of agreement at 20.67 and 0.99 kg, respectively.
were reproducible to within 4.6 and 4.6 o/oo, respectively.
Table 2 summarizes the comparison of ad libitum energy
For a urine material at natural abundance of 2H (d2HSMOW/

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o intakes, body fat, and water turnover rates between the 2 analytical
SLAP = 237.9 /oo), the day-to-day and week-to-week measure-
methods. As shown in the table, ad libitum energy intakes were
ments were reproducible to within 1.5 and 1.7 o/oo, respectively.
accurate to 8 6 81 kcal/d between the 2 methods. Body fat was
At the enriched level of 2H (d2HSMOW/SLAP = +1,077.3 o/oo), the
accurate to 0.01 6 0.01 kg between the 2 methods. Water turnover
day-to-day and week-to-week measurements were reproducible
rates were not different (0.00 6 0.01 kg/d) between the 2 methods.
to within 3.8 and 4.1 o/oo, respectively.
Essentially, the nutritional outcome values measured using the H2-
Table 1 summarized the accuracy and precision of the stable
water equilibration method were not significantly different from
hydrogen isotope ratio measurements of the equilibration method.
those measured using the reference zinc reduction method. In fact,
Three aliquots of GISP were analyzed and measured. The d2HSMOW/
o the ad libitum energy intakes, body fat, and water turnover rates
SLAP values for GISP were accurate to +0.7 /oo compared with
were highly correlated (r = 0.98) between the 2 analytical methods.
the accepted d2HSMOW/SLAP value for GISP (12,13). The overall
precision of the GISP measurement was determined to be 1.5 o/oo.
With the IAEA 302A reference material at a d2HSMOW/SLAP value
Discussion
of +506.2 o/oo, the measured d2HSMOW/SLAP values over a period
of 4 d were accurate to 21.4 o/oo and reproducible to within 4.0 o/oo. Our results showed that the platinum catalyzed H2-water equil-
With the IAEA 302B reference material at a d2HSMOW/SLAP ibration method, using a small sample size of 200 mL, produced
value of +992.3 o/oo, the measured d2HSMOW/SLAP values over highly accurate and reproducible stable hydrogen isotope ratios in
a period of 4 d were accurate to +2.8 o/oo and reproducible to natural waters, international reference materials, and physiolog-
within 2.7 o/oo. ical fluids at both natural abundance and enriched levels of 2H
Figure 1 compared the kH, NH, NH:NO, and EE measurements commonly encountered in the DLW and body water studies using
by the H2-water equilibration method with those obtained using the stable hydrogen isotope methodology. The method is very
the reference zinc reduction method. The kH values were found to straightforward, because it has minimal sample pretreatment and
be similar between the 2 methods (Fig. 1A), with bias calculated to does not require the reduction of water samples into H2 prior to

TABLE 1 Accuracy and precision of stable hydrogen isotope ratio measurements by the platinum
catalyzed H2-water equilibration method using international water reference materials1

References Accepted d2H value Measured d2H value2 Accuracy2 Mean accuracy Overall precision3
o
/oo
GISP 2189.7 (2190.2 to 2189.1) 2189.7 6 1.3 20.0 +0.7 1.5
2190.1 6 1.7 20.4
2188.8 6 1.5 +0.9
IAEA 302A 506.2 (504.0–508.4) 504.8 6 2.4 21.4 21.4 4.0
507.9 6 3.4 +1.7
503.0 6 4.4 23.2
503.5 6 5.4 22.7
IAEA 302B 992.3 (985.6–999.0) 991.6 6 2.4 20.7 +2.8 2.7
995.4 6 3.0 +3.1
998.7 6 3.2 +6.4
994.8 6 2.1 +2.5
1
Values are mean 6 SD or accepted values (95%CI), n = 10. GISP, Greenland Ice Sheet Precipitation; 2H, deuterium; IAEA 302A,
International Atomic Energy Agency 2H enriched reference water material 302A; IAEA 302B, International Atomic Energy Agency 2H enriched
reference water material 302B.
2
Accuracy (o/oo) = measured d2H value (o/oo) – accepted d2H value (o/oo).
qffiffiffiffiffiffiffiffiffi
2
3
Overall precision (o/oo) = +ðSDÞ
n , where SD is the internal precision or SD of each set of measurements and n is the total number of sets of
2
d H values used in the evaluation.

Stable hydrogen isotope ratios by equilibration 2059

 information on ad libitum energy intakes, body fat, and water
turnover rates. In fact, several NIH-funded projects are using the
DLW method to evaluate the accuracy and precision of various
instruments for gathering energy intakes in free-living subjects as
well as to determine the energy requirements in toddlers. The
DLW method also has been used to monitor study compliance
in participants undergoing a weight-loss program or to look at
changes in thermogenesis with diets. The H2-water equilibration
method will definitely facilitate the sample processing in these
studies.
The H2-water equilibration method for stable hydrogen
isotope ratio measurements was first described by Horita (17,18)
with a precision of 1.5 o/oo on natural waters. The method was
subsequently adapted by Coplen (19), showing a precision of 1.3
o
/oo on natural water samples. He reported the use of copper
granules to prevent the sulfide in the water samples to poison the
platinum catalyst. Coplen (20) subsequently tested the H2-water
equilibration method on stable hydrogen isotope ratio measure-

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ments on urine samples collected from 2 diabetic patients. The
d2HSMOW/SLAP values of the urine samples ranged between 251
and +890 o/oo. Compared to the reference zinc reduction method,
the authors reported that the accuracy varied between 2211
and +71 o/oo. The authors attributed the huge variation in
accuracy to potential incomplete reduction of the water to H2
or the inherent poor precision of the zinc reduction method.
This is a possibility, because their zinc reduction procedure put
the urine samples in direct contact with the zinc reagent, thus
providing an opportunity for compounds in the urine samples
to poison the zinc reagent. However, when the sample is not
allowed to come in contact with the zinc reagent, as we described
in our zinc reduction procedure (15), we have not observed such
an effect.
Scrimgeour (21) subsequently tested the H2-equilibration
method on 3 tap water, 3 urine samples, and 3 plasma samples
at natural abundance of 2H as well as urine samples collected
in one body water study and urine samples collected in 3 DLW
studies. Using a sample size of 400 mL, the results were highly
reproducible but took 3 d to reach isotopic equilibrium. In the
DLW studies, the kH values were found to be similar to those
obtained using the zinc reduction method. The study lacked

FIGURE 1 Bland and Altman pairwise comparison of the DLW

outcome variables: kH (A), NH (B), NH:NO (C), and EE (D) generated
from urine samples collected in 10 DLW human studies by the
platinum catalyzed H2-water equilibration method and the reference
zinc reduction method. The y-axis represents the differences between
the 2 measurements. The x-axis represents the means between the 2
measurements. The solid lines represent the bias and the dashed lines
represent the lower and upper limits of agreement. The P value in
each figure represents the significant value by linear regression
analysis between the differences and the mean values. DLW, doubly
labeled water; EE, energy expenditure; H2, hydrogen gas; kH,
fractional turnover rate of deuterium; NH, isotope dilution space of FIGURE 2 Bland and Altman pairwise comparison of the isotope
deuterium; NH:NO, ratio between the isotope dilution spaces of deuterium dilution spaces of 2H generated from serum samples collected in 26
and oxygen-18. body water studies in common house cats between the platinum
catalyzed H2-water equilibration method and the reference zinc
reduction method. The y-axis represents the differences between
the 2 measurements. The x-axis represents the means between the
mass spectrometric analysis. The equilibration method also does 2 measurements. The solid lines represent the bias and the dashed
not require correction for memory effect commonly associated lines represent the lower and upper limits of agreement. The
with the in-line reduction method. P value represents the significant value by linear regression analysis
As shown in Table 2, the H2-water equilibration method can between the differences and the mean values. H2, hydrogen gas; 2H,
be used in many nutrition studies, including studies requiring deuterium.

2060 Wong and Clarke

 TABLE 2 Accuracy and precision of the platinum catalyzed H2-water equilibration method in a caloric
restriction study in normal-weight adults, in an obesity study on Felis catus, and in a combat field study
against the reference zinc reduction method1
Nutrition Values by reference
applications zinc reduction method
Ad libitum energy intake, kcal/d 2309 6 4474
Body fat, kg 1.71 6 0.84
Water turnover rate, kg/d 4.0 6 0.6
1
Values are expressed as mean 6 SD, n = 10. H2, hydrogen gas.
2
Accuracy = values by H2-water equilibration method – values by reference zinc reduction method.
3
Precision = SD of the differences between the 2 methods.
4
P values level by paired samples t test.
the evaluation of the equilibration method on the day-to-day
and week-to-week reproducibility of the measurements as well as
the accuracy of the measurements against international reference
materials. The study also did not evaluate the equilibration
method with a wide range of body water or EE values as we
performed in our study. The long equilibration time reported
possibly could be due to the limited surface contact between the
H2, the catalyst and the sample, because the catalyst (platinum-on-
alumina) was placed inside a glass vial (32 mm 3 5 mm o.d.) in
this study. Our study showed that it took only 3 h to reach isotopic
equilibrium using a smaller sample size of 200 mL.
The platinum catalyzed H2-water equilibration method has
several advantages over the conventional methods. The classical
method to use uranium turnings to reduce water into H2 requires
the use of a dedicated vacuum line and a mercury Topler pump to
transfer the H2 into a sample bulb, because H2 is not condensable
at liquid nitrogen temperature. The procedure obviously has
memory issue and is labor intensive. The development of an
in-line uranium reduction method coupled with an isotope ratio
mass spectrometer (22) eliminated the requirement of a separate
sample preparation vacuum line but has huge memory effect. In
both cases, replacement of the uranium turnings required shutting
down the vacuum line or the sample inlet.
The zinc reduction method to convert water or water in
physiological fluids to H2 has no memory effect but is labor intensive
and required a special zinc reagent to ensure complete reduction
(11,15).
With the development of the continuous-flow isotope ratio
MS, manufacturers have been able to include an in-line chromium
reactor to reduce water into H2 for stable hydrogen isotope ratio
measurements. The method is well accepted by most users,
because it requires a very small amount of sample and is
relatively quick. The method, however, involves some memory
effect and the standard chromium reactor is good for only
~200 injections. Replacement of the chromium reactor also
requires cooling down the reactor and shutting down the inlet
system. The samples also need to be pretreated with activated
charcoal followed by the removal of the activated charcoal
from the treated sample through a 0.2-mm filter. The filtration
process requires at least 1–5 mL of sample to avoid possible
contamination from moisture adhered to the barrel of the syringe,
plunger, and filter.
In conclusion, our manuscript represents the most detailed
evaluation of a platinum catalyzed H2-water equilibration method
for stable hydrogen isotope ratio measurements and its potential
application in many nutrition studies. The method is simple,
highly reproducible, accurate, and without memory effect. There-
fore, any laboratories equipped with a continuous-flow isotope
ratio mass spectrometer system should be able to adopt the method
Values by H2-water
equilibration method Accuracy2 Precision3 P4
2317 6 438 8 81 0.77
1.72 6 0.85 0.01 0.01 0.21
4.0 6 0.6 0.0 0.01 0.77
to support their nutrition studies using the stable hydrogen
isotope methodology.
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Acknowledgments
L.L.C. and W.W.W. designed research; L.L.C. conducted
research; and W.W.W. wrote the manuscript and analyzed data.
Both authors read and approved the final manuscript.
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