The natural abundance of deuterium and 18
O varies by the geographical location with 2
H ranging from 80-180 ppm and 18
O from 1900 to 2050 ppm. These isotopes are also naturally present in the body’s organic compound. When a person moves to new geographic location and drinks water of a different isotopic abundance, the isotopic enrichment of the total body water changes. This is challenging for those DLW studies where participants are moved to different location during the metabolic measurement period [34
]. For example in military or astronaut nutrition studies, participants are commonly transported to a different geographical location for their field assignment, changing the isotope abundance and causing baseline isotopic shifts [48
]. Following methods can be used to control for errors associated with administering the DLW at different geographic locations.
Correction of the baseline isotopic shifts using an equilibration period:
Introducing a period of equilibrium where subjects consume water from the new geographic location for 1-3 weeks before baseline dose administration is suggested to avoid underestimation of energy expenditure [37
]. This method has been validated [48
], but if the rate of turnover is low or if the subjects are traveling to a different location for a short period, this method may not be practical.
Correction of the baseline isotopic shifts using a control group: To account for the differences in isotopic proportions of water at a different location, it is also advisable to enroll a control group that does not receive any tracer. It is important that the control group should be involved in similar physical activity as the isotopic group for consistent isotopic elimination rate. The average change in the elimination rate of that control group is then used to estimate a baseline correction for the test group. Three studies used the control group data to correct for the error in raw isotope data [48
]. DeLany et al. tested the effect of change in food and water source on energy expenditure measurement post DLW dosage administration in soldiers during a 30-day field training [48
]. The control group did not receive any heavy water. A significant decrease in 2
H and 18
O enrichment was observed in urine samples in soldiers receiving no loading dose. These values from control soldiers were used to establish and predict the change in baseline and for calculation of the enrichment [48
]. Jones et al. also studied the energy expenditure in military troops who moved to the Arctic Circle for 10-day training [51
]. Similar to Delany et al., in this study control group showed a shift in the baseline isotopic enrichments by -4650 and -480 ppm per day for 2
H and 18
O, respectively, primarily due to low temperatures (-25 degree Celsius) and changes in food and water source. These shifts in the control group were then used to correct for the DLW measures in the experimental group [51
]. In another study of energy expenditure measurement, marines were transported to a high elevation region with temperatures of -15 to 30 degree Celsius for 11-days of high intensity training exercise. The DLW was administered to all marines except the controls and corrections were made for the shifts in the isotopic baseline for the experimental group [49
]. Therefore, using control subjects maintained the accuracy of DLW.
Correction of the baseline isotopic shifts using a combination of equilibration period and control group:
Multiple studies measuring energy expenditure during military field missions have combined equilibration period and inclusion of control group to increase accuracy of the method. Gretebeck et al. employed both these correction methods for errors due to background isotope change, where subjects were to consume water from a given source indefinitely [53
]. Eight healthy women consumed tap water enriched with 2
H and 18
O to resemble the water available on a typical space shuttle mission for 28-days. The same enriched water was used to prepare drinks and rehydrate food as well. Additionally, a control group was also included in the study that received the enriched water for 14-days for equilibration and given DLW on day 15. When the isotope disappearance rate was calculated without considering change in the background, an error of 2.9 MJ/day in TEE was observed in the 28-day enriched water consumption group [53
]. This study validated the use of an equilibration period of minimum 2 weeks to the new water source, when the isotopic enrichment of the water source for subjects is altered. The study also confirmed the validity of a control group to track changes in the isotopic background. In cases where control group is not feasible due to limited number of subjects, baseline changes can be corrected based on the final isotope ratio of the fully equilibrated baseline isotope abundance [53
]. This research group took a similar approach in another study where energy requirements were calculated for 13 healthy males during a ground based mission (control) or a space flight for 8-14 days. Levels of deuterium and 18
O in the potable water for space mission are quite different from that of ground water and therefore a modified DLW method was used to compare the two groups. The enrichment levels of potable water were predicted from the hydrogen and oxygen gases used for the fuel cells and a preload of deuterium and 18
O were given to each crewmember to raise their baseline isotope levels to that of potable water. Dosing before the flight changed the isotopic levels of the astronauts and reduced the differences between the isotopic abundances in preflight body water and drinking water in the flight [54
]. The modified combination approach has also been successfully used in a similar study where energy expenditure was measured in soldiers during a high intensity field exercise at high altitude of Mt. Rainier by obtaining a supply of the drinking water from the destination local. Soldiers drank water from the ranger station at Mt. Rainier 10-days prior to the DLW administration bringing the body of the subjects in equilibrium with the water isotope levels in the new location. The study also employed a control group to correct for the values [50
]. Similarly, the African Bush soldiers were put on a 1-week equilibration diet before high intensity field training in combination with a control group to avoid errors due to changes in the baseline body water isotopic abundances [52
]. The combination method has been the most popular method to correct for baseline shifts.
Correction of the baseline isotopic shifts using a high isotopic dose:
Corrections for baseline isotopic shifts can also be made by simply increasing the dose of the isotopes above the level of the baseline in the natural environment [37
]. A larger dose increases the signal relative to the natural abundance of deuterium and 18
O and to the random error in the isotopic measurement, thus improving the precision [53
], but costs for the labeled water also increase.
Correction of the baseline isotopic shifts using a loading dose that mimics the natural isotopic abundance ratio: 2
H and 18
O have a covariant relationship, which means any shifts in the natural abundance of both isotopes will be in the same direction and proportional. Therefore, to dramatically reduce any errors due to change in the natural isotopic abundance with change in geographic location, it is recommended to use a loading dose that mimics the ratio of natural abundances across the world [34
]. In doing so the change in baselines for the two isotopes will introduce errors into the isotope elimination rates, but those errors will cancel each other when the difference between kD
is taken while calculating TEE.
Delaying the loading dose:
Baseline isotopic errors can occur not only when a person is transported to a remote study site, but also when a person travels large distances weeks before or during the DLW period and then returns to the dosing site. For example, if a person has traveled more than 200 miles or traveled shorter distances that involve large changes in altitude two weeks before or plans to travel during the DLW period, the baseline isotopic abundance may change . To avoid errors due to changes in isotopic abundance with travel, it is recommended to delay the DLW dosing for at least one biological half-life.