• What is assessed?
• How is the measurement conducted?
• When is this method used?
• How are estimates of body composition derived?
• How is the measurement conducted?
• Populations
• Further considerations
• Resources required
• Instrument library
• References

Hydrometry or total body water (TBW) by isotope dilution is a common method for the assessment of body composition at the molecular level.

The method is based on the principle that water is distributed in all parts of the body except body fat. Water is found exclusively within the fat free mass (FFM), which is approximately 73.2% water in adults. Water is the largest component of the human body and includes both intracellular fluid and extracellular fluid. At birth, the body contains approximately 80% water, but as the body matures, this proportion decreases to 50–60% in lean adults and to less than 40% in obese adults.

With measurement of TBW by isotope dilution, the amount of FFM can be estimated using hydration factors. Body fat mass (FM) is the difference between body weight and FFM.

Total body water (TBW) assessment is based on the principle of isotope dilution. Enrichment of the body water pool (see Figure 1) following a bolus dose of deuterium oxide (²H₂O) allows the isotope dilution space to be calculated using the following equations:

F₁N₁=F₂N₂

N₂=F₁N₁/F₂

F₁ is the deuterium enrichment (concentration) of the dose

F₂ is the deuterium enrichment (concentration) of the distribution space

N₁ is the size of the deuterium dose

N₂ is the size of the distribution space

N.B F₁N₁ = F₂N₂ is the isotope version of the principle of C₁V₁ = C₂V₂, where C is concentration and V is volume.

All that remains is for corrections due to non-aqueous exchange of the isotope to be made. That is, corrections for the loss of isotope outside of the TBW into fats and proteins predominantly.

TBW can theoretically be measured using water labelled with either of the stable isotopes deuterium (²H₂O) or oxygen-18 (H₂¹⁸O) or the radio-active isotope tritium (³H₂O). This is sometimes referred to as the ‘tracer’. Stable isotopes are preferred over radio-active isotopes due to participant acceptance and minimal risk; water labelled with deuterium is far more economical than ¹⁸O.

A known bolus dose of deuterium oxide is given orally to the participant. This mixes with the body water pool (see Figure 1). Biological fluids such as usually, plasma, saliva or urine are sampled.

Two methods to determine total body water

There are two methods used to determine TBW: 1) the intercept method and 2) the equilibration/plateau method.

1. The intercept method usually utilises urine sampling and can take between 7-14 days. Briefly the natural logarithm of the elimination of tracer from the body water is plotted against time and the intercept gives the tracer dilution at the time of dosing.
2. The equilibration/plateau method utilises saliva sampling and is a much shorter protocol taking up to 5 hours.

Key instructions for participants

Intercept method

1. A baseline urine sample of at least 1 mL is collected prior to dosing. This is a vital sample as it allows the baseline isotopic enrichment of the body water pool to be determined.
2. Participant is weighed in minimal clothing.
3. A weighed dose (0.07 g/kg BW) of isotope is ingested; this is usually through a straw to avoid spillage. Approximately 50 mL of drinking water is added to the dose bottle and the participant drinks this.
4. Post-dose urine samples (minimum 1 mL) are taken over a sampling period of 1-14 days. For children this sampling period may be shortened.
5. Urine samples should not be the first void after waking, and ideally at a similar time each day.
6. Enrichment of the saliva samples are measured using either Isotope Ratio Mass Spectrometry or Fourier Transform Infrared (FTIR) Spectrometry. FTIR cannot be used for urine samples.

Equilibration/plateau method

1. A baseline saliva sample of approximately 1 mL is collected prior to dosing. This is a vital sample as it allows the baseline isotopic enrichment of the body water pool to be determined.
2. Participant is weighed in minimal clothing.
3. A weighed dose (0.07 g/kg BW) of isotope is ingested; this is usually through a straw to avoid spillage. Add approximately 50 mL of drinking water to the dose bottle and have the participant drink this.
4. Post-dose saliva samples (1 mL) are taken at 3, 4 and 5 hours for adults and 2, 3, 4 and 5 hours for children.
5. Participants should avoid drinking during the equilibration period, but if this is not possible, the volume of all fluids consumed should be recorded.
6. Enrichment of the saliva samples are measured using either Isotope Ratio Mass Spectrometry or Fourier Transform Infrared (FTIR) Spectrometry. If FTIR is to be used, the isotope dose must be increased accordingly.

N.B Participants should not drink at least 30-min before a saliva sample is taken.

Main sources of errors

1. Failure to administer an accurately measured dose.
2. Contamination of the samples by the dose.
3. Dilution of saliva samples by drinking immediately before collection.

Figure 1 Estimating TBW by deuterium dilution. At baseline, the body water pool naturally contains a small amount of deuterium. After a known bolus dose of deuterium oxide is given orally to the participant, this mixes with and enriches the body water pool.
Source: International Atomic Energy Agency (2010).

TBW can be measured in the field using the deuterium oxide dilution technique. An advantage of this technique is that it can be used to assess longitudinal changes in body composition before and after an intervention. In an ideal situation, this method should be used alongside other body composition measures as it is limited to estimating fat mass (FM) and fat free mass (FFM) (2-component (2C) model). Error rates have been estimated to be 1% for TBW and 0.5% for FFM.

TBW by isotope dilution is also used to derive the criterion method for overall body composition, the 4-component model together with other body composition methods (bone mass from whole body DEXA scan and body volume/density by hydrostatic underwater weighing or air displacement plethysmography).

The first step is to calculate TBW:

1. a is the amount of oral dosing solution, in grams, administered to the subject
2. W is the amount of deionised tap water used to dilute the enriched isotope dose, in grams
3. a is the amount of enriched isotope dose, in grams
4. Ea is enrichment of the diluted dose a in W
5. Ew is the enrichment of the tap water diluent
6. Es is the mean enrichment of saliva samples at 3, 4 and 5 hours for the plateau method or the zero time intercept for the back extrapolation method
7. Ep is the enrichment of the pre dose sample
8. Division by 1.041 accounts for non-aqueous exchange

Once TBW has been calculated, FFM is simply TBW divided by the hydration factor:

FFM = TBW / hydration factor

FM = Weight - FFM

In adults, the hydration factor is assumed to be 73.2%. The actual values range from 67.4 to 77.5%. These variations can result in considerable error when calculating total body fat. For studies involving infants and/or children the hydration factor varies with age and sex.

An overview of the characteristics of hydrometry is outlined in Table 1.

Strengths

1. Error rates have been estimated to be 1% for TBW and 0.5% for FFM.
2. Stable isotopes, like deuterium are safe to be used in children and in pregnancy.

Limitations

1. Cost of the equipment, analysis and isotope:
   1. For the plateau method ~£150 per participant
   2. Back extrapolation method ~£200 per participant
2. Sample preparation of the isotopes prior to administration might be tedious.
3. This method assumes that the isotope is distributed only in water and equally distributed in all anatomical water compartments.

Table 1 Characteristics of the hydrometry method.

Considerations relating to the use of hydrometry in specific populations are described in Table 2.

Table 2 Anthropometry by hydrometry in different populations.

1. Water turnover is slower in the elderly, unless they are taking diuretics, in pregnant women, and in patients with expanded extracellular water volume (such as malnourished children with oedema). Therefore, a longer equilibration time should be allowed for these participants
2. It is preferable to use the back extrapolation procedure with urine sampling to assess TBW in infants. It is advisable to seek the help of an expert if you are planning to assess TBW in infants, as special precautions must be taken to ensure that the dose is consumed correctly.
3. The adult hydration factor of 0.732 is not appropriate for use in children and infants. For example; in a 5-6 years old boy hydration of FFM is 77% and in a 3-month old baby girl, FFM is 79.9%. Hydration factors for children and infants are available from Lohman (1992) and Fomon et al. (1982).
4. Corresponding data for prematurely born infants are lacking. Thus, until more information becomes available, any assessment of body fat in premature infants should use three or four component models of body composition.
5. There is presently no consensus on the most appropriate hydration coefficients for different stages of pregnancy. Therefore, the deuterium dilution technique is not recommended for a two component model assessment of body composition in women in the second and third trimesters of pregnancy.
6. A readymade batch dose of deuterium oxide can be useful when dosing a series of participants.
7. A sample of each batch dose should be frozen as a reference.

Refer to section: practical considerations for objective anthropometry

1. Deuterium oxide (99.8 or 99.9 at % 2H)
2. Fridge for storage of isotope
3. Sterile dose bottles
4. 2 µm filters
5. Sterile 20 mL syringes
6. Electronic balance weighing scales (2dp)
7. Freezer (–20°C) for storing samples
8. FTIR or isotope ratio mass spectrometers
9. Detailed written instructions for participants including a record sheet to record date and time of sample collection
10. Trained operators
11. Collaboration with a research group experienced in this technique is strongly recommended.

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