The total body water (TBW) in humans can be subdivided in different water pools. Major pools are the extracellular fluid (ECF) and the intracellular fluid, where the ECF is subdivided into plasma and interstitial fluid. In urological practice questions remain on the impact of the interstitium on the pathophysiology of different urological diseases. A typical example is the pathogenesis of edema and its connection with nocturnal polyuria. In patients with spinal cord lesions for example, a high incidence of nocturnal polyuria linked with a high incidence of edema. However, the exact mechanism explaining both observations is not known. Probably, the interstitium plays a crucial role in the origin and maintenance of this edema, but this hypotheses has never been studied. 

Aim of this research was evaluating the relevance of the potential use of an isotopic marker and bio-impedance assessment (BIA), to observe human’s TBW and the size and function of the interstitium for its further use in urological research.

Nineteen healthy individuals were recruited in this prospective trial combining ingestion of deuterium oxide (2H2O) enriched water en BIA. Blood and saliva samples were obtained every 10 minutes after ingestion of 2H2O enriched water, urine was collected once before the isotopic labelling and once 95 min after labeling. This specific volume of 2H2O corresponded with 0.25% of the TBW of the person, estimated using the Watson formula. 

BIA (Inbody S10) was done every 5 minutes. Posture changes (standing to lying and lying to standing) were done to assess their impact on fluid distribution between body compartments. The 2H abundance (a2H) of the water in blood, saliva and urine was determined using cavity ring down laser spectroscopy after extraction of the water by cry-distillation. 

Subsequently, TBW was measured in 6 different manners. First, TBW was calculated based on a2H in the sampled water pools (blood, saliva and urine), after the following data-processing: the pool size was assessed using a mass balance of the dilution of the deuterium tracer by the pool as

n_P  = n_LW∙(a^2 H_LW-a^2 H_LP)/(a^2 H_LP-a^2 H_T0 )

With nP and nLW being the amount (mol) of the water of the pool to be assessed and the labeled water (i.e. 330 mL ingested), respectively, and a2HLW, a2HLP, and a2HT0 being the a2H of the labeled water, of the sampled pool and of the pool before ingestion of the labeled water, respectively. To estimate TBW for the a2H saliva and blood samples, calculations were done using the last data point. However, some precautions should be taken, as it was clear that equilibrium was not yet attained at the end of the experiment.

Secondly, TBW based on BIA measurement were calculated by the device during standing and lying position. Lastly, TBW was calculated based on the Watson formula (men: TBW = 2.447 L – (0.09516 L∙ years -1 * Age) + ( 0.1074 L∙cm -1 * Height) + (0,3362 L∙kg -1* mass); women: TBW = -2.097 L + (0.1069 L ∙ years -1 * Height) + (0.2466 L ∙ cm -1  * Weight).

Wilcoxon signed-ranked test was used to assess differences of BIA in different postures. Mann Whitney U test was used to observe gender differences. A p-value of <0.05 was considered statistically significant. The study was approved by the hospital’s ethical board.

The uptake of 2H2O in blood and saliva changed over time. In saliva, a first peak could be visualized 10 minutes after ingestion due to the maintain of ingested 2H2O in the mouth. After attaining this maximal a2H of the saliva, a dilution takes place through interaction of the saliva pool with a slow exchangeable pool.

During the first period (from uptake of the isotopic label till reaching of the maximum a2H in blood) a first order kinetic, representing the exchange of water in the gastronomic track to the blood, was assumed. An exchange rate could be determined using a correlation analyses on following equation.     
a^2 H_(t,S)= a^2 H_(T0,S)+a^2 H_(max,S) (1-e^((-k∙t) ))

With a2Ht,S, a2HT0,S, a2Hmax,S, being the a2H of the serum at specific time, at start point T0 and at the point of maximal isotopic enrichment respectively. While clear individual differences could be noted, gender had a big influence on the exchange rate, with a significantly slower exchange for female than for males (Mann Whitney U test, p = 0.041, male group median 0.052 min -1; female 0.038 min -1). After achieving this maximal a2H, a dilution of the a2H in the blood was observed. 

The TBW determined by this last urine sample slightly exceeded the Watson TBW (median female 104% (88% - 112%), median male 104% (IQR 84 – 124%)).

BIA measurements showed that trunk and leg impedance decreased significantly with posture changes, which visualizes the increase of water volume in both trunk and leg, linked with the change of position from lying down to standing up.
TBW was measured and calculated in 6 different manners (Table 1).

The results of this pilot study demonstrate the potential value of an isotopic marker and BIA for further urological research, as the kinetics of exchange between the intravascular, slow and fast exchangeable pool and the impact of different body positions could be determined. The method to observe a2H in different samples and to calculate the TBW based on this results was developed. However, for further research, a longer time frame (+- 4 hours) for the protocol is necessary. 

TBW was calculated in 6 different manners. Small differences between the methods were observed, although they were all appropriate to measure TBW in this young and healthy group. However, the Watson formula has some limitations as it does not include body composition and thus will not differ between patients with the same mass and length but with a discrepancy in fat or muscle constitution. Moreover, for older, obese or comorbid patients the Watson formula is not feasible, as it does not take into account edema, fluid storage problems or intake of (anti)diuretic medication. For this ‘non-standard’ population, TBW assessed by a deuterium dilution experiment or BIA have more potential, as they take into account aforementioned comorbidities.

The volume, spread and change of the interstitial fluid could not be visualized using BIA and ingestion of 2H2O  in healthy volunteers. However, it was obvious that ingestion of isotopically labelled water is informative for many questions concerning the absorption and storage of ingested water. BIA at the other hand, seems a good tool to observe difference in fluid contain of different body parts and the impact of posture changes. Future research in patients with fluid storage problems is necessary to observe their disabled water handling and to potentially visualize the effect in the interstitium. Moreover this research should contain renal function and edema measurement to see the impact of different positions on diuresis and edema.

In this prospective pilot study both methods to observe fluid distributions in patients were tested and found to be appropriate to observe TBW in healthy volunteers and to visualize the effect of posture changes on the fluid contain of different body parts. However, the interstitium could not yet be visualized. Further research in e.g. patients with edema will most certainly provide more insights into the functioning of the interstitium and the possible link with edema and diuresis.