Procedure
Total body water (TBW) was measured on two consecutive days using stable, nonradioactive, nontoxic isotopes. For the first measurement of TBW, the participant was given an oral dose of an isotope of oxygen (O18), in the form of water (H
2
18
O). The subject consumed 1.5 g/kg body weight of 4% H
2
18
O via a syringe to minimise residue. The dose consumed was recorded to the nearest 10 mg. A single urine sample was obtained before the dose and subsequent urine samples were collected 4–6 h postdose and then once every 24 h (approximately) for the following 15 days. The subject recorded the time each urine sample was collected.
At the same time the following day, TBW was again measured using an isotope of hydrogen (
2H) in the form of water (
2H
2O, deuterium oxide). The subject consumed via a syringe 0.05 g/kg body weight of 99.9% deuterium oxide mixed into a sample of 50 ml tap water plus a xanthan gum fluid thickener (EasyThick, Flavour Creations). Xanthan gum was chosen for the purpose of illustrating our novel stable isotope method because it is a commonly used fluid thickener, and it was one of thickening agents tested by Sharpe et al. [
3]. Other thickening agents may be substituted for labeling with
2H, and the
2H could be added to sample fluids other than tap water (e.g., juice, cordial, milk, infant formula). The solution was thickened according to the manufacturer’s instructions to produce a solution thickened to full (pudding) thick. The dose was recorded to the nearest 10 mg. A single urine sample was obtained before the dose and 4–6 h postdose. The daily urine samples collected for
18O analysis were also used for
2H analysis. All urine samples were frozen until analysis. It was not necessary to place predose restrictions on the participant with respect to food or drink consumption on either of the testing days, as this method yields an average result from the collection of multiple urine samples over a 2-week period. In addition, the isotopic enrichment of each postdose sample is analysed with respect to the predose urine sample; this effectively serves as a control. Therefore, unlike methods that measure the rate of appearance of isotopes in bodily fluid, this method is not affected by short-term differences in gastric emptying or intestinal absorption rates in relation to prior food or beverage consumption.
To determine each dilution space and measure TBW, the enrichment of 2H and 18O in the predose and postdose urine samples, the dose administered, and the local tap water were assessed via isotope ratio mass spectrometry (IRMS) (PDZ Europa, UK).
To measure the enrichment of
2H, 0.5 ml of each urine sample was pipetted into a 12-ml vacuutainer. A catalyst, approximately 1 mg of platinum on alumina powder (Sigma Aldrich) contained in a 0.5-ml vial (Hewlett Packard Pty), was introduced into the vacuutainer while ensuring no direct contact of the catalyst and the urine. Each vacuutainer was evacuated for a period of 5 min before being filled with 99% hydrogen gas. The vaccutainers were then kept at room temperature for 3 days to allow the
2H in the sample to achieve equilibrium with the hydrogen gas [
4]. All reference waters were prepared at the same time and in the same manner as the urine samples.
Similarly, to measure the enrichment of 18O, 0.5 ml of each urine sample were pipetted into a 12-ml vacuutainer. The vacuutainers were then evacuated for 5 minutes, and 5% carbon dioxide, 95% nitrogen gas was introduced into the vacuutainer. The samples were then kept at room temperature for 24 h to facilitate the equilibrium between the 18O in the sample and the gas. Again, reference waters were prepared at the same time and in the same manner as the urine samples.
The enrichment of both the 2H and 18O samples were measured in duplicate with the results being expressed in delta units as ‰ (per mil) relative to standard mean ocean water (SMOW).
The theoretical instantaneous enrichments of body water, which could be obtained by the administered dose and defined as the intercept obtained by back-extrapolation of the semilogarithmic plot of observed enrichment against time, is given by a rearrangement of the equation of Halliday and Miller [
5] as
$$ E_{\rm{D}} = \frac{{T_{\rm{D}} \cdot A_{\rm{D}} \,\cdot\,F_{\rm{D}}}}{{a_{\rm{D}}}}\frac{{\left( {E_{\rm{aD -
}}E_{\rm{tD}}} \right)}}{{1.04 \cdot {\text{TBW}}}} $$
for deuterium, and
$$ E_{\rm{O }} = \frac{{T_{\rm{O}} \cdot A_{\rm{O}}\,\cdot
\,F_{\rm{O}}}}{{a_{\rm{O}}}}\frac{{\left( {E_{\rm{aO -
}}E_{\rm{tO}}} \right)}}{{1.01 \cdot {\text{TBW}}}} $$
for
18O. In these equations,
A is the mass of the isotope administered to the subject (in g),
a is the mass of the dose retained and diluted in
T litres of tap water for IRMS analysis, with the subscripts D for deuterium and O for
18O, respectively.
E
aD and
E
aO are the enrichments of deuterium and
18O in the dilute dose, and
E
tD and
E
tO are those of the tap water. The factors of 1.04 and 1.01 are correction factors to relate isotope dilution space to total body water (TBW) [
6], and are introduced to account for exchange with sites of nonaqueous species. Finally, we have explicitly included in this equation bioavailability factors
F
D and
F
O. For pure water these would be taken as unity (i.e.,
F
O = 1); however, if it is the case that the thickening agent reduces the degree to which water can be absorbed, then
F
D would be found to be less than 1.
F
D can be calculated by combining the two equations:
$$ F_{\rm{D}} =
1.035\frac{{E_{\rm{D}}a_{\rm{D}}A_{\rm{O}}T_{\rm{O}}\left(
{E_{\rm{aO - }}E_{\rm{tO}}}
\right)}}{{E_{\rm{O}}a_{\rm{D}}A_{\rm{D}}T_{\rm{D}}\left(
{E_{\rm{aD - }}E_{\rm{tD}}} \right)}} $$