Deuterium Equilibrium Time in Saliva of Newborn Infants

Abstract

There is an increasing interest about the use of stable isotopes for body composition analysis in pediatrics. To ensure the success of total body water analysis by the deuterium dilution method, it is fundamental to determine the equilibrium time (plateau) of deuterium in the body fluid studied.

Objectives: 

We report here the equilibration time of deuterium oxide in the saliva of newborns after oral intake of the isotope.

Methods: 

Twenty healthy term newborn infants, 10 males and 10 females, were analyzed. Saliva was collected from each newborn before the oral administration of a 100 mg/kg dose of deuterium oxide (baseline sample) and then at 1-hour intervals for 5 hours after administration. Deuterium enrichment of saliva was determined by isotope ratio mass spectrometry according to the recommendations of the International Atomic Energy Agency.

Results: 

The plateau time of deuterium in saliva occurred 3 hours after oral administration of the stable isotope.

Conclusion: 

These data are essential for further studies on the body composition of newborn infants. To the best of our knowledge, this is the first study regarding the equilibration time of deuterium in the saliva of term newborns.

There is increasing interest about the use of stable isotopes for body composition analysis in pediatrics, a fundamental step in the study of nutritional and metabolic status ^(1,2). The understanding that fat deposits practically contain no water and that water represents about 73.2% of fat-free mass in adults and 80% of fat-free mass in newborns has stimulated investigations using total body water (TBW) as the basis for the calculation of body composition ^(1,3,4). The principal method for measuring TBW is the use of a stable isotope dilution and deuterium is the stable isotope most frequently utilized ^(5,6).

Specifically in pediatrics, the use of stable isotopes has the advantage of being safe and of allowing the use of small amounts of different biological specimens such as plasma, expired air, saliva, and urine ⁽⁷⁾. The standard approaches to the measurement of TBW include overnight equilibration and the intercept and plateau methods ⁽⁶⁾. Overnight equilibration seems to be the least optimal approach for fieldwork because it requires correcting for changing water balance during the long overnight period of equilibration. The other 2 approaches do not have this limitation, but they strongly depend on a correct estimate of isotope equilibration time ⁽⁸⁾.

The plateau method is usually applied to adults and infants in fieldwork ^(9,10) because it only requires 1 sample after equilibration. During the initial period after a dose is administered, deuterium concentration changes until it acquires a relatively stable value, which is defined as the plateau. The appearance of the plateau indicates that equilibration of the tracer has been reached within the body ⁽⁸⁾. The deuterium steady state has been reported to be approximately 3 hours for adults ⁽¹⁾ and 4.85 ± 3 hours for infants ranging in age from 6 to 11 months ⁽⁸⁾. To the best of our knowledge, there are no reports in the literature about the equilibrium time of deuterium in the saliva of newborn infants.

The current study presents the plateau time for deuterium in the saliva of healthy term newborn infants.

METHODS

Twenty adequate for gestational age term newborn infants with less than 72 hours after birth, 10 males and 10 females, were randomly selected at the University Hospital, School of Medicine of Ribeirão Preto, University of São Paulo. To be included in the study, infants had to meet the following criteria: gestational age of more than 37 weeks, being adequate for gestational age, absence of congenital abnormalities, absence of any conditions or clinical events requiring oxygen, ventilatory support, corticosteroids, inotropic agents, or any care that may compromise nutrition, and ability to breast feed.

Gestational age was determined by ultrasound at the first trimester. Adequacy for gestational age was determined using the curve proposed by Alexander et al ⁽¹¹⁾.

After obtaining written maternal consent, an oral dose of 100 mg/kg (roughly 1 mL/kg of a 10% deuterium oxide stock solution) was carefully weighed using a scale with a sensitivity of 0.001 g (Toledo AB204 scale, SP, Brazil). The solution was prepared at the University Hospital pharmacy by the gravimetric dilution of a 99.8% deuterium oxide solution (Cambridge Isotopes Laboratories, Andover, MA) with filtered tap water under sterile conditions. The deuterium solution was added to an empty syringe, which was weighed with the solution using the same scale. The volume was carefully offered to the infant by mouth and, after ingestion, the syringe was weighed again. The difference between weights was considered to be the amount of deuterium ingested. If any amount of the dose was lost during the procedure the experiment was terminated at this point. A 2-mL sample of the 10% deuterium stock solution was saved routinely for analysis.

Saliva samples (0.5–0.7 mL) were collected at baseline before deuterium administration, and 1, 2, 3, 4, and 5 hours after its administration. Samples were obtained using a standard butterfly catheter infusion set, 21 gauge, without the needle. A 10-mL syringe was used to maintain continuous suction and the saliva collected was stored until analysis at −20°C in freezing tubes with caps and rubber rings (Sarstedt-Numbrecht, Germany) protected with a Parafilm strip (Menasha,WI). The procedure took less than 15 minutes and was not considered for the time after dosing.

The deuterium enrichment of the samples was measured by isotope ratio mass spectrometry. Saliva samples (300 μL aliquots) were analyzed in triplicate. Samples were equilibrated with 100% ultrapure hydrogen injected into each tube by an automated system (Gilson Autosampler), and equilibrium was obtained by platinum catalysis (Thermoquest platinum catalyst rods, ThermoFinniganMAT, Bremen, Germany) under conditions of controlled temperature (25°C) ⁽¹²⁾. Hydrogen gas was subsequently analyzed with a continuous flow isotope ratio mass spectrometer (Hydra System/ANCA 20-20, Europa Scientific, Cheshire, UK) at the Mass Spectrometry Laboratory of the Medical School of Ribeirão Preto, University of São Paulo. Tap water, internal standards, and diluted dose were analyzed within each sample batch.

A plateau was defined as the earliest time point when consecutive enrichment values became <2% different from the previous hour, as proposed by Wong et al ⁽¹³⁾. The study and the information protocol were approved by the Hospital Ethics Committee and written informed consent was obtained from the parents of each infant.

The Statistical Package for the Social Sciences (SPSS) version 12.0 (SPSS Inc, Chicago, IL) was used for data analysis. Values are expressed as mean ± SD and were compared by ANOVA and the paired Student t test.

RESULTS

The 20 infants studied were born with a gestational age of 39.0 ± 1.1 weeks, birth weight of 3185 ± 422 g, and length of 49.2 ± 1.5 cm. All were adequate for gestational age. Deuterium was offered 31.0 ± 15.1 hours after birth.

No difference between sexes was found regarding deuterium content of the saliva expressed as delta-SMOW for each time point, and therefore, male and female infants were considered as a single group for analysis.

Figure 1 presents the percent differences between consecutive values during the 5-hour observation period. On the basis of the 2% variation, it can be noted that equilibrium was achieved after 3 hours and was maintained until the 5th hour.

FIG. 1: Differences between means, expressed as percentage, during the 5-hour observation period. Consecutive values were used to calculate the differences.

Applying the data of the deuterium equilibrium curve in saliva to the equation developed by Ducatti et al ⁽¹⁴⁾ studying the recycling of carbon-13 in mammalian and bird tissues, it was possible to obtain theoretically the exact equilibrium time:

δ²H(t) = 705.95 − 737.1 e^−2.6156t. The calculated steady state was 2.7 ± 0.4 hours.

Approximately, the same result was obtained using the model of Wolfe and Chinkes ⁽¹⁵⁾:

which resulted in an equilibrium time of 2.6 hours.

DISCUSSION

The present study defined that a plateau, the steady state of the ingested deuterium in the saliva of normal term newborns, occurred after 3 hours. A plateau was defined as the earliest time point when consecutive enrichment values became <2% different from the previous hour, as proposed by Wong et al ⁽¹³⁾. Fjeld et al ⁽¹⁰⁾, studying deuterium equilibrium time in the urine of pediatric patients, considered a plateau to have been reached when the variation was less than 3%. Applying mathematical models to our data indicated that a plateau should be reached after 2.7 hours. Similar to the results obtained by Wong et al ⁽¹³⁾, there was no sex difference regarding the equilibrium time.

To avoid errors related to insufficient isotope ingestion ^(16,17), we used a dose of 100 mg/kg, more than sufficient for adequate tissue saturation. In addition, care was taken to guarantee that the correct dose would be offered and fully ingested. For this purpose, a high precision scale was used to measure the dose of deuterium offered, which was administered to the infant in a slow and careful manner to prevent any loss of the deuterium volume offered.

It should be remembered that the equilibration period of 3 hours for adults is obtained under fasting conditions. This is an important issue for newborn infants because they were breast-fed on demand, usually every 2 or 3 hours. In the present study, in agreement with the study by Salazar et al ⁽⁸⁾, infants received the dose after breast-feeding, and were breast-fed on demand throughout the sampling period. There may have been a delay in equilibration because of the ingestion of breast milk, but this could not be ethically avoided, and represents the true field condition. Although the volume ingested during breast-feeding was not measured, the equilibrium of deuterium in saliva was established after 3 hours. However, the fact that isotopic equilibrium is achieved within 3 hours does not mean that lack of adjustment for fluid intake leads to the correct value for body water ⁽¹⁸⁾. During the first days of life, milk intake is likely to be low, especially in breast-fed infants and should not be an issue. Nevertheless, if body water were 2 L in a 3.5-kg baby, even a 50-mL fluid intake, uncorrected for, would lead to an error of 2.5% in body water. For example, older infants should have their intake estimated by test weighing, with the value subtracted from final body water as a correction. An alternative to recording fluid intake in older babies is to use the back extrapolation method ⁽¹⁸⁾, although the plateau method is preferable in neonates.

The steady state in saliva was found to be higher in older infants. Studying 3 infants aged 2 to 3 months, Infante et al ⁽¹⁹⁾ found a plateau after 6 to 8 hours. Salazar et al ⁽⁸⁾, studying children during the first year of life, detected a plateau after 4.85 hours.

The practical consequence of establishing equilibration time in an infant with a nonfasting regime is to determine when it is necessary to obtain the first saliva sample after the administration of the deuterium dose in order to obtain valid results for TBW. The use of shorter times for homogeneous isotope distribution before equilibrium may explain, for instance, the disagreements in the initial validations of the measurement of milk intake by the deuterium dilution technique compared to other methods ⁽²⁰⁾. Because in adults the equilibrium time has been defined as 3 hours, several investigators assume that children, because of a faster water balance, should obtain equilibrium earlier, within about 2 hours ^(19–21).

Although Wong et al ⁽¹³⁾ did not find differences in the equilibrium time of deuterium in saliva and urine of adult patients, the study of equilibrium time of deuterium in urine is far more complicated than in saliva in pediatrics. There is always the possibility of contamination of the collected specimen with residual urine. In newborns, it is particularly difficult to avoid contamination and it is unethical to introduce a bladder catheter to collect a urine specimen from a healthy newborn.

We believe that the use of saliva samples is far more appropriate for the study of body composition in the newborn population. The ideal time to sample saliva after deuterium ingestion in newborns is 3 hours. These data are essential for further studies on the body composition of newborn infants. To the best of our knowledge, this is the first study regarding the equilibration time of deuterium in the saliva of term newborns.