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Using the dilution principle for total body water (TBW)

This is an excerpt from ACSM's Body Composition Assessment With Web Resource by American College of Sports Medicine,Timothy Lohman & Laurie Milliken.

By Robert M. Blew, MS; Luis B. Sardinha, PhD; and Laurie A. Milliken, PhD, FACSM


Water is the most abundant component in the human body comprising about 60% of body mass in the reference man. Because it is mostly found in the fat-free body in a relatively constant amount, assessment of body water has been of interest as a method of body composition assessment for almost 100 years. Unlike the other molecular body components, the water component consists of a single molecular species (H2O), which simplifies the task of its measurement. Water's characteristic as a singular molecular species offers itself to the use of the dilution principle, which in its simplest form, states that the volume of the component is equal to the amount of isotope added to the component divided by the concentration of the isotope in that component.


In 1915, the dilution principle was first used in the study of human body composition when the use of a red dye to measure the plasma volume was extrapolated. The investigators verified that the concentration of the dye after mixing was not constant because it “disappeared” from blood plasma. Using a mathematical approach, a reasonable estimate was made to calculate the volume of plasma in which the dye was first diluted. Following this investigation and using the same principle, tracer material was injected intravenously and allowed to reach a uniform distribution, and from the dilution achieved at equilibrium, the constituents of the body were measured. Both radioactive and stable isotopes were thus used to measure the potassium and sodium of the body.


Tritiated water was first described by Pace et al. as an isotope for measuring TBW. The main advantage of using tritium (3H), the radioactive isotope of hydrogen, is that it is readily available and easily assayed by scintillation counting. On the other hand, a large amount of tritiated water must be administered to obtain adequate precision, eliminating its use in cases where the use of radionuclides is restricted.


Currently, deuterium (2H) and oxygen-18 (18O), which correspond to nonradioactive stable isotopes, are the most commonly used isotopes for the measurement of TBW. Oxygen-18 has the advantage that its dilution space more closely approximates TBW, but it can be adequately measured only by isotope ratio mass spectrometry, and the cost of 18O-labeled water is about 15 times more than that of deuterium. Thus, deuterium is the most frequently used isotope to estimate TBW because it is a stable isotope and easy to obtain and has lower costs than tritium or oxygen-18 with no radioactivity exposure. Deuterium can be measured by infrared spectrometry but preferably by mass spectrometry. Greater technical errors have been found using the infrared approach.


When using isotope dilution, particularly deuterated water, two body fluid samples from urine, blood, or saliva are collected: one just before administration of the deuterium dose to determine the natural background levels and the second after allowing enough time for penetration of the isotope. If the amount of isotope is known and the baseline and equilibration concentrations are measured, the volume in which the isotope has been diluted can be calculated.


There are four basic assumptions that are inherent in any isotope dilution technique.

  1. The isotope is distributed only in the exchangeable pool. None of the commonly used isotopes are distributed only in water. But tracer exchanges with nonaqueous molecules are minimal, and consequently, the volume of distribution or dilution space of the isotope can be determined, albeit slightly greater than the water pool. Deuterium exchange with nonaqueous molecules is estimated at 4.2% in human adults.
  2. The isotope is equally distributed within the pool. Isotopic tracers are identical to body water, except for differences in molecular weight, which can lead to isotopic fractionation. Isotopic fractionation corresponds to the process that accounts for the relative abundances of isotopes and consequent redistribution of isotopes within the body. Samples collected from plasma, urine, and sweat do not show fractionation, whereas samples from water vapor do.
  3. Isotope equilibration is achieved relatively rapidly. The equilibration time corresponds to the point where all body fluid compartments have the same proportion of the isotope. The rate of equilibration as a function of the route of deuterium oxide administration has been investigated by Schloerb et al., who observed that equilibration was reached 2 h after intravenous administration and 3 h after subcutaneous or oral administration. Wong et al. verified that the time to equilibration was approximately 3 h regardless of whether plasma, breath carbon dioxide, breath water, saliva, or urine was sampled. Schoeller et al. detected less TBW at 3 h than at 4 h after oral isotope administration. Considering these findings, equilibration time for TBW was set at 4 h, with the exception of patients with expanded extracellular water compartments, where 5 h equilibration time is required. Considering TBW assessment, urine has demonstrated a low isotope enrichment relative to venous plasma water. Still, it is important to consider voids after tracer administration. Three voids are recommended after the dose when urine is used as the biological sample.
  4. The tracer is not metabolized during the equilibration time. Body water is in a constant state of flux. In temperate climates, the average fractional turnover rate in adults is 8% to 10% each day. This turnover comprises inputs of water from beverages, food, metabolic water produced during the oxidation of fuels, and water exchange with atmospheric moisture. The inputs are balanced by an output of water in the form of urine, sweat, breath water, or transdermal evaporation. This constant turnover has led to two approaches when assessing TBW: the plateau method and the back-extrapolation, or slope-intercept, method. For body composition research, the plateau method is the usual approach. Deuterated water is administered, samples are collected for 3 to 5 h, and TBW is calculated from the samples collected before and after the enrichment has reached a plateau, or a constant value.