Doubly-labeled water

Doubly-labeled water

Doubly-labeled water is water in which both the hydrogen and the oxygen has been partly or completely replaced for tracking purposes (i.e., "labeled") with an uncommon isotope of these elements.

In theory, radioactive forms of hydrogen and oxygen could be used for such labeling. In practice, for both practical and safety reasons, almost all "doubly-labeled water" tests use water labeled with the heavy, non-radioactive forms of the elements deuterium and oxygen-18 (O-18 or 18O).

In particular, the term "doubly-labeled water test" refers to a particular type of test of metabolic rate, in which average metabolic rate of an organism is measured over a period of time. This is done by administering a dose of doubly-labeled water, and then tracking the loss of deuterium and O-18 in the subject, over time, through the use of regular sampling of heavy isotope concentrations in the body water (by sampling saliva, urine, or blood).

Mechanism of the test

The test essentially measures the subject's carbon dioxide production over the interval. The key concept which allows this, is that oxygen in body water (including the marker dose of O-18) equilibrates with the body's bicarbonate and dissolved carbon dioxide pool (through the action of the enzyme carbonic anhydrase), and is therefore lost from the body, in carbon dioxide. "Additionally" the oxygen in water is lost through body moisture loss (mostly urine). However, since deuterium (as a second label and marker for body water) is only lost in moisture, the deuterium change in body water over time can be used to "subtract out" (or mathematically compensate) for the loss of tracer O-18 by the water-loss route. This leaves only the remaining net loss of O-18 in carbon dioxide, and thus an estimate for total carbon dioxide production. Once this is known, the total metabolic rate may be estimated from simplifying assumptions, regarding the ratio of oxygen used in metabolism (and therefore heat generated), to carbon dioxide eliminated (see respiratory quotient).

Practical isotope administration

Doubly-labeled water may be administered by injection, or orally (the usual route in humans). Since the isotopes will be diluted in body water, there is no need to administer them in a state of high isotopic purity, no need to employ water in which all or even most atoms are heavy atoms, or even to begin with water which is doubly-labeled. In practice, doses of doubly-labeled water for metabolic work are prepared by simply mixing a dose of deuterium oxide (heavy water) (90 to 99%) with a second dose of H218O, which is water which has been separately enriched with O-18 (though usually not to a high level, since this is expensive and unnecessary for this use), but otherwise contains normal hydrogen. The mixed water sample then contains both types of heavy atoms, in a far higher degree than normal water, and is now doubly-labeled. The free interchange of hydrogens between water molecules (via normal ionization) in liquid water ensures that the pools of oxygen and hydrogen in any sample of water will be separately equilibrated in a short time with any added heavy isotope(s).


The doubly-labeled water test is particularly useful for measuring average metabolic rate over relatively long periods of time (a few days or weeks), in subjects for which other types of direct or indirect calorimetric measurements of metabolic rate would be difficult or impossible. For example, the technique can measure the metabolism of animals in their wild state (deer on a hillside or birds foraging for their young), with the technique problems being related mainly to how to administer the dose of isotope, and collect several samples of body water at later times to check for differential isotope dilution.

Administration of doubly-labeled water to animals in the wild has been successfully accomplished in many ways. Animals such as snakes may be captured and simply injected. Insect-eating birds may have their body water labeled by offering them caterpillars which have previously been injected with the heavy isotopes, and so on. In both animals and humans, the test is made more accurate if a single determination of respiratory quotient has been made for the organism eating the standard diet at the time of measurement, since this value changes relatively little (and more slowly) compared with the much larger metabolic rate changes related to normal movement and activity.

Because the heavy hydrogen and oxygen isotopes used in the standard doubly-labeled water test are non-radioactive, and also non-toxic in the doses used (see heavy water), the doubly-labeled water measurement of mean metabolic rate has been used extensively in human volunteers, and even in infants PMID 3104873 and pregnant women PMID 1858878.

Additional references (see also reference biographies)

*Review: PMID 3142975
*History and theory: PMID 9771875
*See also: PMID 14576749

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