Stable isotope technique to assess body composition · IAEA International Atomic Energy Agency...
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IAEA International Atomic Energy Agency Stable isotope technique to assess body composition Christine Slater, PhD Nutrition Specialist IAEA [email protected]
Transcript of Stable isotope technique to assess body composition · IAEA International Atomic Energy Agency...
IAEA International Atomic Energy Agency
Stable isotope technique to
assess body composition
Christine Slater, PhD
Nutrition Specialist IAEA
IAEA
Body composition
• The main components of the body are:
• Water
• Protein
• Fat
• Mineral matter
http://www.jawon.com/reng/res/body-composition.html
The relative amounts of these can change with age,
ethnicity and nutritional status
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Fat
FFM
2C
Total Body Water (TBW)
• The body is mainly composed of water
• At birth the body is 70-75% water
• The adult body contains 50-60% water,
decreasing to less than 40% in obese adults
• In adults FFM contains ~73% water
• Water is found exclusively within the Fat Free
Mass (FFM)
• Measure TBW
• Calculate FFM (TBW / hydration factor)
• Body fat = Body weight - FFM
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TBW by isotope dilution
• Total body water includes both intracellular and
extracellular water
• When a person takes a drink of labelled water, the
label mixed with the body water within a few hours
• This is called isotope dilution
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Back to basics!
• Atoms are composed of protons,
neutrons and electrons
• Protons and neutrons form the
nucleus of an atom
In a neutral atom the number protons = number of electrons
Neutrons are the glue that stops the nucleus falling apart
The mass of an electron is 0.00055 Daltons
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What is an isotope?
• Isotopes of an element have
the same the number of
protons in the nucleus
(atomic number)
but
• different atomic mass
(the sum of number of
protons + number of neutrons)
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Isotopes of hydrogen
• Hydrogen has 3 isotopes
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Isotopes of oxygen
• Oxygen has 3 isotopes
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Natural abundance
• The natural abundance is the concentration of a
stable isotope present in the background (baseline)
sample
• The natural abundance of 2H in water is ~0.015%
• In normal water 15 out of 100,000 H atoms will be 2H
• The natural abundance of 18O in water is ~0.20%
• Baseline samples must be collected in any study
using stable isotope techniques
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Enrichment
• Enrichment is the concentration of a stable
isotope in a sample after the background
has been subtracted
• The target enrichment depends on the
method that will be used to analyse the
sample
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Total body water by isotope dilution
• Deuterium (2H), tritium (3H) and 18O have all been used
to measure total body water (TBW)
• 2H is the method of choice because of the radiation
hazard associated with the use of 3H, and the relative
expense of using 18O
(H218O is ~40 x more expensive than 2H2O)
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• Total body water can be measured by deuterium
dilution
• Deuterium (2H or D) is a stable isotope of hydrogen
• Deuterium oxide is water labelled with 2H
O
H H
O
D D
water deuterium oxide
2H2O or D2O (99.8 atom % D)
Deuterium oxide
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Procedure: equilibration technique
• After collection of a
baseline sample, a
known quantity of
D2O is consumed
• The D2O mixes with
body water within a
few hours
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Appearance of 2H in body water
Illustration: Christine Slater data
from Les Bluck
• In healthy participants, enrichment of deuterium in body water reaches a
“plateau” after 2-3 hours in saliva
• The plateau enrichment lasts for several hours
• In saliva, an early overshoot occurs where the enrichment is above the
“plateau” as the D2O has not fully mixed with ICF
0
20
40
60
80
100
120
140
160
0 2 4 6 8
Time post dose (hours)
% p
late
au
en
rich
me
nt
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Procedure: equilibration technique
• Two post-dose samples are
collected at the plateau
enrichment
• TBW can be sampled as
saliva, urine or plasma:
depends on the method of
analysis available
Deuterium enrichment in saliva
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Analysis: IRMS
Isotope ratio mass spectrometry: requires high-tech laboratory,
with experienced personnel, very accurate and precise, can
be used for analysis urine, plasma, saliva or human milk
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Analysis: FTIR
Fourier Transform Infrared Spectrometry: cheaper to buy,
easier to use and to maintain, but not as sensitive as
IRMS, requires 10 times as much D2O.
Cannot be used for urine samples
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Equilibration time
• Equilibration takes longer in urine than in saliva
• Equilibration takes longer in adults than children
Healthy adult
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12
Time post-dose (hours)
En
rich
men
t (p
pm
excess 2
H)
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Equilibration in urine
Healthy adult
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12
Time post-dose (hours)
En
rich
men
t (p
pm
excess 2
H)
Elderly adult
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Time post-dose (hours)E
nri
ch
men
t (p
pm
excess 2
H)
• Equilibration takes longer in the elderly
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Safety of deuterium oxide
• There is no radiation hazard associated with deuterium oxide.
• The maximum enrichment of 2H in body water is ~0.1% or 1000 mg/kg (ppm) when a 30 g dose of deuterium oxide is given to an adult
• Deuterium oxide had been used in studies involving humans for over 50 years. No harmful effects have been observed in mammals below 15% enrichment of body water
References
Jones PJH & Leatherdale ST (1991) Clin Sci 80, 277-280
Kotetzko B et al (1997) Eur J Pediatr 156 [suppl 1], S12-S17
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Principle of isotope dilution
• A known amount of tracer is added to a pool
(volume) of unknown size
• The tracer is allowed to mix freely with the pool,
and the pool is sampled
• The amount of tracer in the sample is measured
• The size of the pool can be calculated
C1V1 = C2V2
V2 = C1V1/C2
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Assumptions of the technique
1. The deuterium oxide is distributed only in body water
2. The deuterium oxide is equally distributed in all body water compartments (e.g. saliva, urine, plasma, sweat, human milk)
3. The rate of equilibration of deuterium oxide is rapid
4. Neither deuterium oxide nor body water is lost during the equilibration time
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1. The deuterium oxide is distributed only
in body water
False
• 2H can be sequestered into organic compounds in the body (mainly proteins)
• 2H exchanges with “active” H atoms (-NH2, -COOH, -OH) of amino acids
• 2H can be lost from the body water pool during synthesis of proteins and fatty acids
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Non-aqueous exchange
• Dilution space of 2H (VD) is ~4% higher than TBW
TBW (kg) = VD/1.04
• 1.04 is the non-aqueous exchange factor for 2H
• There is less non-aqueous exchange of 18O
• The non-aqueous exchange factor for 18O is 1.01
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2. The deuterium oxide is equally
distributed in all body water compartments
True, true for water in the body (liquid water), but not for water leaving the body as water vapour
• Deuterium oxide (2H2O) is not chemically identical to water
• When D2O mixes with body water, three isotopic forms are found
99.8001% 0.1998% 0.0001%
1H2O 1H2HO 2H2O
H2O HDO D2O
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2. The deuterium oxide is equally
distributed in all body water compartments
• The bond between 2H and O is slightly shorter than the bond between 1H and O
• The energy of the bond between 2H and O is slightly greater than the energy of the bond between 1H and O
• This can lead to isotopic fractionation when water undergoes a chemical or physical change
• Isotopic fractionation of water occurs when water liquid becomes water vapour
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Fractionation factor
• The isotope fractionation factor between liquid
water and water vapour at 25°C is 0.941
• This means that the concentration of
deuterium in water vapour is 94.1% of the
deuterium concentration in the liquid water
from which it evaporated
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Effect of fractionation if 100 μL of condensation is clinging to
the lid of a sample vial containing 4 mL saliva, which
originally contained 1000 mg/kg 2H
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The effect of fractionation is more pronounced when
the volume of saliva is small
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Why is fractionation important?
• Water vapour that condenses on the caps of bottles used for storing doses, body water specimens and calibration standards contains less deuterium than the bulk of the liquid
• Therefore dose bottles should be inverted to mix the contents before opening and body water specimens should be centrifuged before analysis
• Do not leave bottles open to the atmosphere, especially in warm humid climates
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Fractionation
• The effect of increased loss of water as
water vapour, which contain less 2H
than body water, is to concentrate the 2H2O left behind
• This can lead to an underestimation of
TBW and therefore an overestimation of
body fat
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Isotopic fractionation: summary
• There is very little isotopic fractionation of water within the
body. Plasma, urine, sweat and human milk show little
fractionation
• Water leaving the body as water vapour (in breath and
evaporation from the skin) contains less deuterium than
body water
• It is important to avoid physical activity during the
equilibration period to avoid loss of water as water vapour
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3. The rate of equilibration of
deuterium oxide is rapid
True
• Depends on your definition of rapid. In healthy participants, equilibration in saliva and plasma is usually achieved after 2-3 hours. It takes longer to achieve equilibration in urine
but
• The time required for equilibration tends to be longer in subjects with slow water turnover (e.g. the elderly or very ill)
• In these people, the plateau enrichment in saliva is reached after about 4-5 hours
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4. Neither D2O nor body water is lost
during the equilibration time
• Losses in urine can be
minimised by limiting fluid
intake
• Losses in sweat and breath
can be minimised by limiting
physical activity during the
equilibration period
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Important Issues: Hydration of FFM
• In healthy subjects the water content varies by <2% from day to day
• Hydration of fat free mass is assumed to be 73.2% in adults
• Use Lohman (1992) or Foman (1982) hydration factors in children
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Hydration of lean tissue in children
(% water content of fat-free body)
Age (years) Male Female
1 79.0 78.8
1-2 78.6 78.5
3-5 77.8 78.3
5-6 77.0 78.0
7-8 76.8 77.6
9-10 76.2 77.0
11-12 75.4 76.6
13-14 74.7 75.5
15-16 74.2 75.0
17-20 73.8 74.5
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Hydration of lean tissue in infants (% water content of fat-free body)
Age (months) Male Female
Birth 80.6 80.6
1 80.5 80.5
2 80.3 80.2
3 80.0 79.9
4 79.9 79.7
5 79.7 79.5
6 79.6 79.4
9 79.3 79.0
12 79.0 78.8
18 78.5 78.4
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Important Issues: Hydration of FFM
• The assumption that the hydration of FFM is 73.2 % may not be valid in situations where patients have expanded extracellular water e.g. during pregnancy and in children with oedema (SAM)
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Procedure for measuring TBW by
deuterium dilution with analysis of
saliva samples by FTIR
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Doses for children
Preparation of the doses is a critical step in the
method, which depends on knowing the exact weight
of D2O consumed by the child.
• IRMS analysis: 0.05 g D2O per kg body weight.
• FTIR analysis: 0.5 g D2O per kg body weight.
• It is much easier to make the doses in batches,
based on the mean body weight expected.
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Dose preparation
• The dose must be accurately weighed (to at least 0.01 g)
• Doses can be prepared in advance and stored in a leak proof, screw cap bottle in a fridge or freezer until required
• Do not store doses (very highly enriched water, 99.8 atom % D) in the same fridge or freezer as saliva samples (low enrichment, <0.1 atom % 2H)
Leak proof, screw cap,
autoclaveable polypropylene
bottles suitable for storing
D2O doses
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Recommended doses for IRMS
Category Body weight
range (kg)
Dose 99 atom %
D2O (g)
Adult >70 6
Adolescent 35-70 3
Child 10-35 1
Infant <10 0.3
• Batches should be made from a single bottle of D2O, as it
is necessary to analyse a dilution of the dose with the
urine samples.
• A 1 in 10 dilution of the highly enriched (99.8 atom %)
D2O is prepared in a large glass bottle and mixed.
• This is then dispensed into leak-proof bottles.
• Doses must be accurately weighed by trained personnel.
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Recommended doses for FTIR
Doses should be weighed in a food preparation area,
not in a laboratory.
..
Body weight
(kg)
Weight of D2O
required (g)
<10 3
10-20 6
20-30 10
30-50 20
>50 30
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Dose Consumption
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Flow Chart of procedure (children> 2y)
Wait for a further 0.5 - 1 hour
Wait for 3-4 hours for dose to equilibrate with body water
Invert dose bottle to ensure contents are fully mixed
Ask the child to empty bladder
Weigh child in light clothing (to 0.1 kg)
Collect baseline
Sample
Participant drinks the dose Add 50 mL drinking water to the bottle and ask the participant to consume this through the same straw.
Add another 50mL and repeat.
Collect first post - dose
sample
Collect second post - dose sample
3 h saliva
4 h urine
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Measuring body weight
• An accurate measure of body weight is required as body fat is calculated by difference from body weight
• This is particularly important in longitudinal studies
• The scales must be placed on a level surface
• The scales must be calibrated regularly
• The participant should be weighed in underwear or a light gown of known weight
Illustration: Mauro E. Valencia
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Procedure for saliva sampling
• At least 2 mL saliva is required, preferably 4 mL to keep some in reserve in case a repeat analysis is needed
• Ensure that the child has not consumed any food or drink for at least 30 minutes before sampling saliva
• Saliva is collected by asking the participant to chew on a cotton wool ball until it is sodden
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Saliva sampling vials
• Examples of suitable vials for storing saliva or urine
samples: cryovials with screw caps
• Vials must have a good seal to prevent loss of
sample and ingress of moisture from the atmosphere
during storage
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Notes on saliva sampling
• Wear a new pair of gloves for each saliva sample
• Do not touch the dose bottle until after the baseline saliva sample has been collected
• All equipment must be completely dry before use
• Do not re-use disposable plastic syringes or sample vials
• Ensure that caps are tightly closed to prevent loss of sample and cross-contamination between samples
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Storage of samples
• Samples can be stored in a cool box until transfer back to the lab
• Store the samples in a freezer (-20ºC) until analysis
• Samples can be stored in a fridge if analysis is to take place within a week
• Do not store samples in the same fridge or freezer as doses. Doses contain more than 1000 times as much deuterium as saliva or urine samples
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Avoid cross-contamination!
• Avoid cross-contamination at all costs
• Between doses and saliva samples. Do not store
in the same fridge/freezer
• Between the dose and baseline saliva samples.
Do not open the dose bottle until after the
baseline samples have been collected
• Between pre-dose and post-dose saliva samples.
Use screw cap vials with a good seal and wrap in
separate zip-lock bags
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Food and Drink Intake
• If it is not possible to fast during the
equilibration period (children and lactating
women), a small meal may be given 1 hour
after the dose was consumed
• Keep a note of the volume of fluids consumed.
This volume should be subtracted from the
calculated TBW
