Stable Isotope Technique to Assess Body Composition

Stable isotope technique to assess body composition

Christine Slater, PhD Nutrition Specialist IAEA

IAEA International Atomic Energy Agency 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

IAEA Total Body Water (TBW)

2C • The body is mainly composed of water

Fat • 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

FFM • Water is found exclusively within the Fat Free Mass (FFM) • Measure TBW • Calculate FFM (TBW / hydration factor) • Body fat = Body weight - FFM

IAEA 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 IAEA 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

IAEA The mass of an electron is 0.00055 Daltons 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)

IAEA Isotopes of hydrogen

• Hydrogen has 3 isotopes

IAEA Isotopes of oxygen

• Oxygen has 3 isotopes

IAEA 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 IAEA 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

IAEA 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 18 2 (H2 O is ~40 x more expensive than H2O)

IAEA Deuterium oxide

• 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 O

H H D D

water deuterium oxide

2 H2O or D2O (99.8 atom % D)

IAEA 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

IAEA Appearance of 2H in body water

160

140

120

100

60 % plateau enrichment plateau% 40

0 Illustration: Christine Slater data 0 2 4 6 8 from Les Bluck Time post dose (hours)

• 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 IAEA Procedure: equilibration technique

• Two post-dose samples are

collected at the plateau Deuterium enrichment in saliva enrichment • TBW can be sampled as saliva, urine or plasma: depends on the method of analysis available

IAEA 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

IAEA 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

IAEA Equilibration time

• Equilibration takes longer in urine than in saliva • Equilibration takes longer in adults than children

140

120

H) 2 100

40 Enrichment (ppm excess Enrichment (ppm excess 20 Healthy adult

0 0 2 4 6 8 10 12 Time post-dose (hours)

IAEA Equilibration in urine

• Equilibration takes longer in the elderly

140 70

120 60

2 2 100 50

80 40

60 30

40 20

Enrichment (ppm excess Enrichment (ppm excess Enrichment (ppm excess Enrichment (ppm excess 20 Healthy adult 10 Elderly adult

0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time post-dose (hours) Time post-dose (hours)

IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA Non-aqueous exchange

2 • Dilution space of H (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

IAEA 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

2 • Deuterium oxide ( H2O) is not chemically identical to water

• When D2O mixes with body water, three isotopic forms are found

99.8001% 0.1998% 0.0001%

1 1 2 2 H2O H HO H2O H2O HDO D2O

IAEA IAEA 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

IAEA 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

IAEA 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

IAEA The effect of fractionation is more pronounced when the volume of saliva is small

IAEA 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

IAEA Fractionation

• The effect of increased loss of water as water vapour, which contain less 2H than body water, is to concentrate the 2 H2O left behind • This can lead to an underestimation of TBW and therefore an overestimation of body fat

IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA 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)

IAEA Procedure for measuring TBW by deuterium dilution with analysis of saliva samples by FTIR

IAEA 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.

IAEA 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

IAEA Recommended doses for IRMS

Category Body weight Dose 99 atom %

range (kg) 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. IAEA Recommended doses for FTIR

Doses should be weighed in a food preparation area, not in a laboratory.

Body weight Weight of D2O (kg) required (g) <10 3

10-20 6

.. 20-30 10

30-50 20

>50 30

IAEA

Dose Consumption

IAEA Flow Chart of procedure (children> 2y)

Ask the child to empty bladder

Weigh child in light clothing (to 0.1 kg)

Collect baseline Sample

Invert dose bottle to ensure contents are fully mixed 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. 3 h saliva Wait for 3-4 hours for dose to equilibrate with body water 4 h urine Collect first post - dose sample

Wait for a further 0.5 - 1 hour

Collect second post - dose sample

IAEA 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 IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA 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

IAEA IAEA Human Health Series (12 & 13)

IAEA http://humanhealth.iaea.org Coming soon: eLearning !

IAEA http://humanhealth.iaea.org