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Total
Body Water Measurements
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Metabolic Solutions offers project
design assistance and a mass spectrometry service to
help researchers determine total body water (body composition)
using deuterium oxide (D2O) and Oxygen 18
stable isotope methods.
Total body water (body composition) has been measured
using stable isotopes of oxygen (Oxygen 18) and deuterium
(deuterium oxide, D2O) for more than 40 years. It is
a very useful and highly popular technique to estimate
lean body mass. The principle is based on the theory
that water is distributed in all parts of the body except
body fat. Most researchers use deuterium oxide as the
tracer because of its low cost. The principal drawback
of using hydrogen isotopes is that the water space is
overestimated because of exchange with protein and other
body constituents. The distribution of oxygen 18 (18O)
more accurately reflects the true volume distribution
of water, but the oxygen 18 (18O)isotope is significantly
more expensive than deuterated water.
The protocols for determining Total Body Water (body
composition) using Deuterium Oxide
(D2O) and Oxygen 18 (18O)are
provided below:
Typical Protocol for Total
Body Water (Body Composition) with Deuterium Oxide (D2O)
- Test subject should undergo an overnight fast
or be fasted for 2 hours following a small breakfast.
- Obtain a "pre" baseline plasma, saliva
or urine sample before administration of isotope.
It would be preferable to collect about 1 ml fluid,
although smaller samples can be analyzed. If blood
is collected, extract the plasma and place in a
plastic test tube and freeze at -20°C. A 1 ml
aliquot of urine or saliva can be stored in a plastic
test tube at -20°C.
- Weigh out the heavy water to the nearest 0.1 mg.
A typical dose of isotope (99.9% enriched in Deuterium)
is:
D (gm D2O) = 0.15 gm/kg Body Weight
- Administer the heavy water orally or intravenously
(use IV grade only deuterium oxide). If the dose
is given orally, rinse the cup with 50-100 ml of
unlabeled water. Record the exact weight of isotope
administered.
- Wait 3-6 hours for complete equilibration, at
which time a second sample should be collected.
Sample Measurements
Three deuterium measurements are necessary to determine
body composition. Send an aliquot of (a) pre-dose
sample, (b) post-dose sample and (c) undiluted isotope
dose to Metabolic Solutions. Samples should be shipped
frozen (preferable with dry ice) by express overnight
service. Note: If several subjects use the same bottle
of isotope, only one sample of the dose is required.
Deuterium Oxide Analysis
Principle
The resulting HD gas is measured with an isotope ratio
mass spectrometer. The methodology is described by
Scrimgeour, C.M., Rollo, M.M., Mudambo, M.K.T., Handley,
L.L. and Prosser, S.J. (1993) 'A simplified method
for deuterium/hydrogen isotope ratio measurements
on water samples of biological origin' Biological
Mass Spectrometry, 22: 383-387.
Instrumentation
Europa Hydra continuous flow isotope ratio mass spectrometer,
for measuring deuterium and 18O in aqueous samples
following equilibration with a gas phase.
General Technique
The following steps are performed in triplicate for
the analysis of D2O in fluids:
|
| Step |
Action |
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1
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0.3 ml of fluid sample is placed in a 10cc Exetainer
tube. |
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2
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0.2 ml disposable glass insert containing ~
4mg of platinum or alumina powder is added to
the Exetainer tube. |
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3
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Exetainer tube is sealed and evacuated with
a vacuum line. |
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4
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Exetainer tube is filled with hydrogen gas.
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5
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Samples are allowed to equilibrate for seven
(7) days at room temperature.
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6
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Samples are analyzed by IRMS within 2 weeks
of equilibration. |
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Mass Spectrometry
The mass spectrometer measures the sample gas against
a lab reference gas (hydrogen gas generated from tap
water). The ratio of mass 3 (HD) to mass 2 (H2) is measured
by the mass spectrometer. During a run, the instrument
measures reference and standard gases that have been
placed throughout the samples in order to assure precision
and accuracy. At the completion of the run, results
are drift corrected, under control of the instrument
software.
Reporting Results
The results of the isotope ratio analysis are reported
as a delta relative to a reference gas. The delta is
measured in parts per thousand, expressed as (‰).
The delta between sample and reference gas is defined
as:
Delta D = [(Ratio of Sample - Ratio of Reference) /
(Ratio of Reference)] x 1000
The International Atomic Energy Agency (IAEA) in Vienna,
Austria has recommended that all deuterium measurements
to be expressed relative to the Vienna Standard Mean Ocean
Water (V-SMOW). Each day of analysis, a V-SMOW sample
is run against the working reference gas. Results are
reported relative to the working reference gas, versus
V-SMOW and as atom percent excess (APE).
Correction Factors
The results of the IRMS instrument are corrected for
a small contribution from H3+ ions generated within the
mass spectrometer. The H3+ correction is applied with
the software. The analyst minimizes the H3+ correction
during the tuning of the instrument.
Accuracy
The IAEA standards comprising about 500 and 1000 ‰
D relative to V-SMOW were analyzed using the hydrogen
equilibration method. The values obtained are shown
below:
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| Sample
|
Expected Enrichment (‰)
|
95 % Confidence Interval |
Actual Measurement (‰) |
| 302A |
508.4 |
505.5 - 511.3 |
507.2 |
| 302B |
996 |
987 - 1004 |
996.3 |
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Precision
The precision of the methodology for biological samples
at 1000 ‰ is ± 2%. The percent coefficient
of variation is typically 0.75% daily and varies no more
than 2% with international standards run throughout the
year.
Quality Control
The analyses are supported by three quality control
standards. Quality control charts are used to continually
monitor the analytical method. Good laboratory practices
(GLPs) are followed with complete documentation.
Calculation of Total Body Water
(Body Composition) with Deuterium Oxide Measurements
The delta deuterium values for the pre-dose dpre)
and post-dose samples (dpost) are determined.
The deuterium dose is diluted with tap water.
The amount of dose diluted and water used is recorded.
The deuterium content of the tap water (dtap)
and diluted dose (ddose) are measured.
Total Body Water Calculations
Total body water (TBW) in moles is calculated from the
dilution of heavy isotope using the equation:
TBW (moles) = ((ddose - dtap)
/ (dpost - dpre)) x (WA / 18.02a)
where W = Amount of water (grams) used to dilute the
dose, A = Amount of dose (grams) administered to subject,
a = amount of dose (grams) diluted for analysis.
To convert TBW to kilograms:
TBW (kg) = TBW (moles) x 18.02 / 1000 g/kg
It has been experimentally determined that deuterium
oxide overestimates total body water by 4%. Some
deuterium can bind to acidic amino acids of body protein
or other non-exchangeable sites. Therefore, to
correct for the non-exchange of deuterium in the body,
the total body water measurement is divided by 1.04.
Corrected TBW (kg) = TBW (kg) / 1.04
Lean Body Mass Lean body mass (LBM) is calculated
from the formula:
LBM (kg) = corr TBW (kg) / 0.72
where 0.72 is the correction for the distribution
of water in lean body mass.
Fat Mass
Fat Mass is then determined as the difference between
body weight and lean body mass.
Fat Mass (kg) = Body Weight (kg) - LBM (kg)
Typical Protocol for Total Body Water (Body Composition) with Oxygen-18 Water
This technique is very popular because it is easy
to administer, noninvasive, accurate, and can be used
in free living subjects. The following is a sample
protocol:
- Test subject should undergo an overnight fast
or be fasted for 2 hours following a small breakfast.
- Obtain a "pre" baseline plasma sample
before administration of isotope. It would be preferable
to collect about 1 to 2 ml serum, although small
samples can be analyzed. Extract the serum and place
in a plastic test tube and freeze.
- Weigh out the heavy water to the nearest 0.1 mg.
To calculate the correct dose of isotope (10% enriched
in Oxygen-18), solve for D:
D (gm H218O) = (subject wt (kg)/70
kg) x 38 gm
38 gm H218O (10% enriched) is
the correct dose for a 70 kg adult.
- Administer the heavy water orally or intravenously.
Record the exact weight of isotope administered.
- Wait 4 hours for complete equilibration, at which
time the second sample should be collected.
Sample Measurements
Three oxygen-18 measurements are necessary to determine
body composition. Send an aliquot of (a) pre-dose
sample, (b) post-dose sample and (c) undiluted isotope
dose to Metabolic Solutions. Samples should be shipped
frozen (preferable with dry ice) by express overnight
service. Note: If several subjects use the same bottle
of isotope, only one sample of the dose is required.
Oxygen-18 Analysis in Fluids
Principle
The 18O content of aqueous fluid is measured
by equilibration of the fluid with CO2 of
known 18O content. The following reaction occurs:
H218O + C16O2
--> H216O + C16O18O
The resulting CO2 gas can be isolated and
purified for isotope ratio measurement. The methodology
is described by Wong, Lee and Klein, Am. J. Clin. Nutr.
45: 905-913, 1987.
Instrumentation
A Europa Scientific Tracermass isotope ratio mass
spectrometer (IRMS) instrument is used for the analysis.
The system uses a continuous-flow sample preparation module,
Automated Breath Carbon Analyzer (ABCA). The continuous-flow
preparation system converts the sample matrix into pure
gas (typically CO2 and N2) in a
helium carrier. Known reference materials are analyzed
in an identical manner before and after batches of samples.
General Technique
The following steps are used to analyze 18O
in fluids: |
| Step |
Action |
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1
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Inject a known amount of reference CO2
gas into a 10 cc Exetainer tube with a plastic
syringe. |
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2
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Inject a known amount of aqueous fluid
into the Exetainer tube with a plastic syringe.
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3
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Equilibrate samples in a temperature controlled
shaker bath for a set time period depending
on the volume of aqueous fluid. |
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4
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Prepare reference samples using identical
procedure. |
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5
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Analyze head space gas directly in Exetainer
tubes with Europa Tracermass, ABCA module
and Gilson autosampler. |
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CO2 Equilibration Time
The following table shows the amount of reference CO2
gas added to various amounts of water sample and the
equilibration time: |
| Aqueous Size |
CO2 Gas Added |
Equilibration Time |
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500 µl
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0.5 ml
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3 hrs
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250 µl
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0.5 ml
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4hrs
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25 µl
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0.5 ml
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96 hrs
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10 µl
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0.5 ml
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96 hrs
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Reporting Results
The results of the isotope ratio analysis are reported
as a delta relative to a reference gas. The delta
is measured in parts per thousand, expressed as (‰).
The International Atomic Energy Agency (IAEA) in Vienna,
Austria has suggested that all 18O measurements be expressed
relative to the Vienna Standard Mean Ocean Water (V-SMOW).
Metabolic Solutions reports results relative to V-SMOW
standard, our reference CO2 gas and as atom
percent excess (APE).
Correction Factors
There will be some isotopic fractionation between the
water and the added carbon dioxide gas. However,
using our water to CO2 ratio, this fractionation error
will be less than 0.01 ‰. The results are not corrected
since this fractionation error is below the accuracy and
precision of the method.
Accuracy
The IAEA standards comprising about 250 and 500 ‰ 18O
relative to V-SMOW were analyzed using the water/CO2
equilibration method. The values obtained are
shown below: |
Sample
|
Expected Enrichment (‰)
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95% Confidence Interval
|
Actual Measurement (‰)
|
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304A
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251.7
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249.2 - 254.2
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252.4 ± 0.2
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304B
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502.5
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498.9 - 506.1
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502.4 ± 0.3
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Precision
The precision of the methodology for water samples at natural abundance ± 0.10
‰ and for biological samples at 250 ‰ the precision is
± 0.20 ‰.
Quality Control
Two or three standards are run daily to insure quality assurance of
the analyses. Quality control charts are used to evaluate the analytical
method daily. Good laboratory practices (GLPs) are followed with complete
documentation.
Calculation of
Total Body Water (Body Composition) with Oxygen-18
Measurements
The delta oxygen-18 values for the pre-dose (dpre) and post-dose
(dpost) are determined. The oxygen-18
dose is diluted with tap water. The amount of dose
diluted and water used is recorded. The deuterium
content of the tap water ((dtap) and diluted
dose (ddose) are measured.
Total Body Water Calculations
Total
body Water (TBW) in moles is calculated from the dilution of heavy
isotope using the equation:
TBW (moles) = ((ddose
- dtap)
/ (dpost - dpre))
x (WA / 18.02a)
where W =
Amount of water (grams) used to dilute the dose, A = amount of dose
(grams) administered to subject, a = amount of dose (grams) diluted for
analysis.
To
convert TBW to kilograms:
TBW (kg) = TBW (moles) x 18.02 / 1000
g/kg
It
has been experimentally determined that oxygen-18 overestimates
total body water by 1%. Some oxygen-18 can bind to acidic amino
acids of body protein or other non-exchangeable sites. Therefore,
to correct for the non-exchange of oxygen-18 in the body, the total body
water measurement is divided by 1.01:
Corrected TBW (kg) = TBW (kg) / 1.01
Lean Body Mass
Lean
body mass (LBM) is calculated from the formula:
LBM (kg) = corr TBW (kg) / 0.72 where 0.72 is the correction
for the distribution of water in lean body mass.
Fat Mass
Fat
Mass is then determined as the difference between body weight and lean
body mass.
Fat Mass (kg) = Body Weight (kg) - LBM (kg)
Published Total Body Water (Body Composition) Studies Analyzed By Metabolic
Solutions
1. Andrews, F.M., Nadeau, J.A., Saabye, L., Saxton, A.M. Measurement of
total body water in horses using deuterium oxide dilution. Am J Vet Res
58(10):1060-1064, 1997.
"Deuterium oxide appears safe and efficacious
for determining total body water content in horses and may be helpful for determining
changes in total body water content during exercise and disease."
2. Armstrong, L.E., Kenefick, R.W., Castellani, J.W., Riebe, D., Kavouras, S.A.,
Kuznicki, J.T., Maresh, C.M., Bioimpedance spectroscopy technique: intra-, extracellular,
and total body water., Medicine & Science in Sports and Excerise, 29(12):1657-1663,
1997.
"The purpose of this study was to test the validity of a multiple frequency
bioimpedance spectroscopy (BIS) technique that estimates extracellular fluid volume
(ECV), intracellular fluid volume (ICV), and total body water (TBW)."
3. Cincotta, A.H., MacEachern, T.A., Meier, A.H.
Bromocriptine redirects metabolism and prevents seasonal onset of obese
hyperinsulinemic state in Syrian hamsters. Am. J. Physiol.
264(27):E285-E293, 1993.
"Metabolic and hormonal effects of
bromocriptine were studied in seasonally obese female Syrian
hamsters."
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Nashua, NH 03063
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