Calculation of CO 2 production in doubly-labelled water studies

Previous studies show that the doubly labeled water method is accurate for measuring energy expenditure in the adult human. To validate this method in infants, carbon dioxide production rate and energy expenditure were measured for 5 to 6 days by doubly labeled water (DLW) and periodic open circuit respiratory gas exchange (RGE) in 10 blinded studies in nine infants following abdominal surgery. Infants were maintained on consistent oral or parenteral nutrition prior to and during study. This avoided diet-related changes in baseline isotopic enrichment of body water which could theoretically contribute to significant errors in calculation of carbon dioxide production rate. For DLW, insensible water loss was assumed to be proportional to respiratory volume and body surface area, where the former was predicted from carbon dioxide production rate. Insensible water loss thus calculated averaged 18% of water turnover. Rates of carbon dioxide production measured by DLW were not significantly different from that of RGE (10.4 +/- 1.1 and 10.5 +/- 0.9 l/kg/day, mean +/- SD, respectively). Energy expenditure was calculated using respiratory quotients from dietary intake (DLW:DIET) and RGE (DLW:RGE) data. There was no significant difference between energy expenditure determined by DLW (DLW:DIET and DLW:RGE) and that measured by RGE (58.5 +/- 6.1, 56.8 +/- 6.1, and 57.3 +/- 5.1 kcal/kg/day, mean +/- SD, respectively). Rate of carbon dioxide production, DLW:diet, and DLW:RGE calculated by DLW differed from corresponding RGE values by -0.9 +/- 6.2, -1.1 +/- 6.1, and 1.6 +/- 6.2%, mean +/- SD, respectively. These findings demonstrate the validity of the doubly labeled water method for determining energy expenditure in infants without concurrent water balance studies.

Angus cattle from 2 beef cattle projects on which carbon dioxide production rate (CPR) was measured were used in this study to examine the relationships among BW, DMI, and carbon dioxide traits of beef cattle fed ad libitum on a roughage diet or a grain-based feedlot diet, and to evaluate potential proxies for DMI and feed efficiency. In both projects, the GreenFeed Emission Monitoring system, which provides multiple short-term breath measures of carbon dioxide production, was used as a tool to measure CPR. The data were from 119 Angus heifers over 15 d on a roughage diet and 326 Angus steers over 70 d on a feedlot diet. Mean (±SD) age, BW, and DMI were 372 ± 28 d, 355 ± 37 kg, and 8.1 ± 1.3 kg/d for the heifers, and 554 ± 86 d, 577 ± 69 kg, and 13.3 ± 2.0 kg/d for the steers, respectively. The corresponding mean CPR was 5760 ± 644 g/d for heifers and 8939 ± 1212 g/d for steers. Other traits studied included carbon dioxide yield (CY; CPR/DMI) and intensity (CI; CPR/BW) and 5 forms of residual carbon dioxide production (RCP), which is a measure of actual minus predicted CPR. Feed efficiency traits studied included feed conversion ratio (FCR) and residual feed intake (RFI). The relationship between CPR and DMI, and between CPR and BW was both positive and linear, for the heifers and also for the steers. For the combined heifer and steer datasets, the R2 for the relationship between CPR and BW, and between CPR and DMI was 0.82 and 0.78, respectively. The correlation between CPR and DMI (r = 0.84 for heifers; r = 0.83 for steers) was similar to that between CPR and BW (r = 0.84 for heifers; r = 0.87 for steers). Most of the carbon dioxide traits were significantly (P < 0.05) correlated with one or both feed efficiency traits. One of the RCP traits (RCPMA) was computed by maintaining metabolic BW (M) and average daily gain (A) in the formula for RFI, but substituting the DMI with CPR. The correlation (r = 0.27) between RCPMA and RFI, though significantly different from zero, was not strong enough for its use as proxy for RFI. On the other hand, a strong correlation (r = 0.73) was obtained between the CPR to gain ratio (CGR) and FCR. This indicates that, where DMI is not available, CPR could be used in its place to compute a feed efficiency trait similar to FCR, since the computation of CGR was similar to that for FCR, except that DMI was substituted with CPR in the FCR formula.

The doubly labeled water (DLW) method has become widely used in studies of energy expenditure and body composition. Researchers differ in the analytical methods used to calculate the dilution spaces for deuterium and oxygen-18. Some determine dilution spaces using isotope enrichments extrapolated to the instant of dosing with DLW (slope-intercept method), but others use measured enrichments from body water samples obtained 3-10 h after dosing (plateau method). These differences limit the comparability of analyses across labs. I derive a simple mathematical approach for recalculating reported dilution spaces to any time point post dosing, using reported dilution spaces and rates of isotope depletion. Simulated data are used to examine the effects of different dilution space protocols. Recalculating dilution spaces enables researchers to determine outcome variables of interest (e.g., total body water, energy expenditure, and water throughput) from different labs under a unified protocol for determining dilution spaces, and improves comparisons among studies. Differences between dilution space protocols can lead to substantial differences in outcome variables of interest in DLW studies. When comparing results of DLW studies that employ different dilution space protocols, dilution spaces should be recalculated for a common time point, and outcome variables recalculated as needed, prior to comparison across studies.

Eddy covariance serves as one the most effective techniques for long-term monitoring of ecosystem fluxes, however long-term data integrations rely on complete timeseries, meaning that any gaps due to missing data must be reliably filled. To date, many gap-filling approaches have been proposed and extensively evaluated for mature and/or less actively managed ecosystems. Random forest regression (RFR) has been shown to be stable and perform better in these systems than alternative approaches, particularly when filling longer gaps. However, the performance of RFR gap filling remains less certain in more challenging ecosystems, e.g., actively managed agri-ecosystems and following recent land-use change due to management disturbances, ecosystems with relatively low fluxes due to low signal to noise ratios, or for trace gases other than carbon dioxide (e.g., methane).In an extension to earlier work on gap filling global carbon dioxide, water, and energy fluxes, we assess the RFR approach for gap filling methane fluxes globally. We then investigate a range of gap-filling methodologies for carbon dioxide, water, energy, and methane fluxes in challenging ecosystems, including European managed pastures, Southeast Asian converted peatlands, and North American drylands.Our findings indicate that RFR is a competent alternative to existing research standard gap-filling algorithms. The marginal distribution sampling (MDS) is still suggested for filling short (< 12 days) gaps in carbon dioxide fluxes, but RFR is better for filling longer (> 30 days) gaps in carbon dioxide fluxes and also for gap filling other fluxes (e.g. sensible heat, latent energy and methane). In addition, using RFR with globally available reanalysis environmental drivers is effective when measured drivers are unavailable. Crucially, RFR was able to reliably fill cumulative fluxes for gaps > 3 moths and, unlike other common approaches, key environment-flux responses were preserved in the gap-filled data.

Unpublished results obtained by J. K. Hardy (1939) indicate that, under conditions in which carbon dioxide was allowed to accumulate in the atmosphere as a result of the activity of apples when enclosed in gas-tight containers in the absence of oxygen, there was no evidence, over a short period, of a fall in the rate of carbon dioxide production with increasing concentrations of carbon dioxide in the atmosphere, and hence in the tissues. Previous results (Kidd & West, I937) had shown that in air the rate of carbon dioxide production of apples during their post-climacteric phase is progressively depressed with rising concentration of carbon dioxide in the atmosphere, and hence in the tissues. If aerobic carbon dioxide production is depressed by the presence of carbon dioxide but anaerobic carbon dioxide production is not depressed, the fact must be of interest in connexion with the intermediate theories of metabolism of the respiratory process. Experiments have been conducted to check Hardy's results by a method in which the fruits were exposed to a relatively constant pressure of carbon dioxide over a longer period. The method used for the estimation of carbon dioxide production by apples in the presence of carbon dioxide i-n air has already been described (Kidd & West, I933). The experiments were carried out at io0C. Three samples of Bramley's Seedling apples were obtained on 29 September I939. In the case of two of these samples (nos. i and 2) the fruit was pre-climacteric, and in the other (no. 3) post-climacteric. The respiratory history of these three samples is shown in Fig. i (top). The first sample (no. i) was exposed, at the outset, to an atmosphere of pure nitrogen, and it can be seen, by comparing the results with those of the second sample (no. 2), which was in air, that transference from air to nitrogen had very little effect on the rate of carbon dioxide production. Both these samples were preclimacteric. At the point marked A the sample in nitrogen was transferred to an atmosphere of 100% carbon dioxide in nitrogen from which all traces of oxygen had been removed. The carbon dioxide caused a slight, though definite, depression in respiratory activity. At B when the sample was returned to nitrogen without carbon dioxide, oxygen still being absent, the respiratory activity rose again slightly. The second sample (no. 2), which was in air at the outset, began to show its climacteric rise after about 7 days. On the ioth day, before the climacteric rise had fully developed, it was transferred to nitrogen at the point C. The respiratory activity in nitrogen remained on the pre-climacteric level. At the point D when it was transferred to i o % carbon

Marine turtles often have extremely high water turnover accompanied by a low field metabolic rate (FMR), a combination that can contraindicate the use of doubly labelled water (DLW). Therefore, we conducted a validation study to assess the suitability of the DLW technique for determining FMR of marine turtles. Six green turtles (22.42+/-3.13 kg) were injected with DLW and placed in a tank of seawater with a respirometer for continuous monitoring of oxygen consumption (MR) over a 5-day period. Trials were conducted for turtles in both fed and fasted states. Respiratory exchange ratio (RER) was determined in a dry respirometer and used to calculate energy expenditure. For fed and fasted turtles, total body water (TBW) was 66.67+/-3.37% and 58.70+/-7.63% of body mass, and water flux rates were 9.57+/-1.33% and 6.14+/-0.65% TBW day(-1), respectively. Water turnover in fasted turtles was 36% lower than that of fed turtles but MR (from oxygen consumption) of fasted turtles (13.77+/-1.49 kJ kg(-1) day(-1)) was 52% lower than in fed turtles (28.66+/-5.31 kJ kg(-1) day(-1)). Deuterium to oxygen-18 turnover rate (k(d):k(o)) ratios averaged 0.91+/-0.02 for fed turtles and 1.07+/-0.16 for fasted turtles. Fed turtles had a mean group difference of 8% and a mean individual difference of 53% between DLW and respirometry. The DLW method gave negative MR values in fasted turtles and could not be compared with respirometry data. Researchers should use caution when applying the DLW method in marine reptiles, especially when high water flux causes >90% of the labeled oxygen turnover to be due to water exchange.

The method of choice for measuring total energy expenditure in free-living individuals is the doubly labeled water (DLW) method. This experiment examined the behavior of natural background isotope abundance fluctuations within and between individuals over time to assess possible methods of accounting for variations in the background isotope abundances to potentially improve the precision of the DLW measurement. In this work, we measured natural background variations in (2)H, (18)O and (17)O in water from urine samples collected from 40 human subjects who resided in the same geographical area. Each subject provided a urine sample for 30 consecutive days. Isotopic abundances in the samples were measured using Off-Axis Integrated Cavity Output Spectroscopy. Autocorrelation analyses demonstrated that the background isotopes in a given individual were not temporally correlated over the time scales of typical DLW studies. Using samples obtained from different individuals on the same calendar day, cross-correlation analyses demonstrated that the background variations of different individuals were not correlated in time. However, the measured ratios of the three isotopes (2)H, (18)O and (17)O were highly correlated (R(2)=0.89-0.96). Although neither specific timing of DLW water studies nor intraindividual comparisons were found to be avenues for reducing the impact of background isotope abundance fluctuations on DLW studies, strong inter-isotope correlations within an individual confirm that use of a dosing ratio of 8‰:1‰ (0.6 p.p.m.: p.p.m.) optimizes DLW precision. Theoretical implications for the possible use of (17)O measurements within a DLW study require further study.

Abstract. Although the ecology of diapause has been widely studied in the field, the underlying physiological responses occurring in tropical diapausing insects remain virtually unexplored. This is especially the case with rates of respiration in diapausing tropical insect species. The present study compares rates of metabolism, as assessed by measurement of carbon dioxide production, between two species of diapausing and reproductively active tropical butterflies, Euploea core (W.S Macleay) and Euploea sylvester (Fabricius) , independent of temperature. Measurement of metabolism over a day‐time/night‐time regime confirms that these tropical butterflies display a diurnal rhythm in accordance with many other tropical and temperate insect species, regardless of developmental state. In addition, diapausing Euploea butterflies display rates of carbon dioxide production only 28% lower than those of reproductively active butterflies, and can terminate diapause within days of receiving the correct cues. Maintaining a similar metabolic rate throughout diapause, as well as a rapid termination of diapause, would enable these tropical butterflies to respond immediately to larval host plant resources, without the disadvantage of missing optimum conditions, allowing the species to maximize their reproductive potential.

Proving of Bread Dough II: Measurement of Gas Production and Retention

Doubly-labeled water (H/sup 3/H/sup 18/O) has been used to determine water flux and energy metabolism in a variety of vertebrates. This study examines the applicability of this technique to arthropods. The theory of the technique depends upon the assumption that doubly-labeled water introduced into the animal's body water equilibrates with water and carbon dioxide by the action of carbonic anhydrase. Tritium (/sup 3/H) is lost from the animal only with water while oxygen-18 is lost with both water and carbon dioxide. The difference bwtween the rates of loss of the two isotopes is proportional to CO/sub 2/ loss rate. Validation of the use of tritiated water for measuring water flux was accomplished by comparing gravimetric measurements of water gain with flux rates determined by loss of tritiated water. At room humidity, an overestimate for influx calculated from labeled water calculations was found, averaging 12 mg H/sub 2/O (g.d)/sup -1/. Comparison of CO/sub 2/ loss rate determined isotopically with rates of CO/sub 2/ loss determined by standard metabolic rates also yielded overestimates for the isotopic technique, overestimates ranging between 20 and 30%. The relevance of this for studies using labeled water for studying water fluxes and free metabolism of free-ranging arthropods is discussed.

Variation of the dilution space ratio (Nd/No) between deuterium ((2)H) and oxygen-18 ((18)O) impacts the calculation of total energy expenditure (TEE) by doubly labeled water (DLW). Our aim was to examine the physiological and methodological sources of variation of Nd/No in humans. We analyzed data from 2,297 humans (0.25-89 yr old). This included the variables Nd/No, total body water, TEE, body mass index (BMI), and percent body fat (%fat). To differentiate between physiologic and methodologic sources of variation, the urine samples from 54 subjects were divided and blinded and analyzed separately, and repeated DLW dosing was performed in an additional 55 participants after 6 mo. Sex, BMI, and %fat did not significantly affect Nd/No, for which the interindividual SD was 0.017. The measurement error from the duplicate urine sample sets was 0.010, and intraindividual SD of Nd/No in repeats experiments was 0.013. An additional SD of 0.008 was contributed by calibration of the DLW dose water. The variation of measured Nd/No in humans was distributed within a small range and measurement error accounted for 68% of this variation. There was no evidence that Nd/No differed with respect to sex, BMI, and age between 1 and 80 yr, and thus use of a constant value is suggested to minimize the effect of stable isotope analysis error on calculation of TEE in the DLW studies in humans. Based on a review of 103 publications, the average dilution space ratio is 1.036 for individuals between 1 and 80 yr of age.

Doubly Labeled Water Is a Validated and Verified Reference Standard in Nutrition Research

Energy Expenditure and Water Flux in Three Sympatric Desert Rodents

BackgroundThe doubly-labeled water (DLW) method is the gold standard methodology for determination of free-living, total daily energy expenditure (TEE). However, there is no single accepted approach for either the sampling protocols (daily vs. two-point, in which samples are collected after dosing and at the end of the measurement period) or the calculations used in the determination of the rate of carbon dioxide production (rCO2) and TEE. Moreover, fluctuations in natural background abundances introduce error in the calculation of rCO2 and TEE. The advent of new technologies makes feasible the possibility of including additional isotope measures (17O) to account for background variation, which may improve accuracy.MethodsSixteen subjects were studied for 7 consecutive days in a whole-room indirect calorimeter (IC) with concurrent measurement of TEE by DLW. Daily urine samples were obtained and isotope ratios determined using off-axis integrated cavity output spectroscopy (OA-ICOS).ResultsWe determined the best combination of approaches for estimating dilution spaces, elimination rates, and calculated average daily carbon dioxide production (VCO2) using six different published equations. Using the best combination, multi-point fitting of isotope elimination rates using the daily urine samples substantially improved the average precision (4.5% vs. 6.0%) and accuracy (−0.5% vs. −3.0%) compared with the two-point method. This improvement may partly reflect the less variable day-to-day chamber measurements of energy expenditure. Utilizing 17O measurements to correct for errors due to background isotope fluctuations provided additional but minor improvements in precision (4.2% vs. 4.5%) and accuracy (0.2% vs. 0.5%).ConclusionsThis work shows that optimizing sampling and calculation protocols can improve the accuracy and precision of DLW measurements.

Ecosystem carbon dioxide, energy, and water fluxes were measured using eddy covariance in a fresh clear-cut surrounded by a mixed spruce-birch-aspen forest in the boreal zone of European Russia. Measurements were initiated in spring 2016 following timber harvest and continued for five months. The influence of surrounding forest on air flow and turbulent fluxes within the clear-cut were examined using a process-based two-dimensional (2D) hydrodynamic turbulent exchange model. The clear-cut was a source of CO2 to the atmosphere prior to onset of vegetation growth during early spring. During this period the mean daily latent (LE) and sensible (H) heat fluxes were very similar and the Bowen ratio (β = H/LE) averaged about 1.0. Daily net ecosystem exchange of CO2 (NEE) was around 0 gC m−2 d−1 following onset of vegetation growth from mid-spring through summer, while β declined to 0.6–0.7. There was strong diurnal variability in NEE, LE and H over the measurement period that was governed by solar radiation and temperature as well as the leaf area index (LAI) of regrown vegetation. Modeled vertical CO2 and H2O fluxes along a transect that crossed the clear-cut and coincided with the dominate wind direction showed that the clear-cut strongly influenced turbulent fluxes within the atmospheric surface layer. Furthermore, modeled atmospheric dynamics suggested that the clear-cut had a large influence on turbulent fluxes in the downwind forest, but little impact on the upwind side. An aggregated approach including field measurements and process-based models can be a useful approach to estimate energy, water and carbon dioxide fluxes in non-uniform forest landscapes.