Vol. 273, Issue 4, R1451-R1456, October 1997
Use of feces to estimate isotopic abundance in
doubly labeled water studies in reindeer in summer and
winter
Geir
Gotaas1,
Eric
Milne2,
Paul
Haggarty2, and
Nicholas J. C.
Tyler1
1 Department of Arctic Biology
and Institute of Medical Biology, University of Tromsø, N-9037
Tromsø, Norway; and 2 Rowett
Research Institute, Bucksburn, Aberdeen AB21 9SB, United
Kingdom
 |
ABSTRACT |
The reliance on samples of blood or urine
to estimate isotopic abundance in studies of energy metabolism using
the doubly labeled water method has restricted application of the
technique to animals that are either tame or easy to catch. This is
generally not the case with large, free-ranging wild mammals. The use
of feces as a source of body water in which to measure the
concentration of isotopic markers was investigated in four female
reindeer in summer and in winter.
2H2O
and H218O
were injected to ~160 parts per million excess. Samples of plasma and
feces were then collected simultaneously for up to 456 h. Both isotopes
were equilibrated with body water at 8 h postdose. There were no
significant differences by animal between dilution spaces, rate
constants, rates of CO2
production, and total energy expenditure (TEE) calculated based on
samples of plasma or feces in any trial. Mean TEE was 3.557 W/kg (SD
0.907, n = 4) in summer and 1.865 W/kg
(SD 0.166, n = 4) in winter.
carbon dioxide production; cervid; seasonal physiology
| |
INTRODUCTION |
THE MAIN ADVANTAGE of the doubly labeled water (DLW)
technique (22, 23) over alternative methods of estimating energy expenditure is that it permits the study of subjects living
unrestricted in their natural environment. However, a reliance on
samples of blood or urine as the source of body water in which to
measure the concentrations of the isotopes used in the technique,
2H and
18O, has restricted application of
the method to animals that are either tame (1, 5) or easy to catch (14,
15), which is not the case with large, free-ranging wild mammals such
as reindeer. Feces contain a high proportion of water that is in equilibrium with the rest of the body water pool, and it has been shown
to be a reliable alternative when monitoring the concentration of
3H2O
in studies in reindeer (16). Although
3H has been widely used in studies
of water kinetics in large mammals (19),
2H, which is not radioactive, is
now the preferred hydrogen isotope in DLW studies. The present study
compared the concentration of two injected markers
(2H2O
and H218O) in
water extracted from parallel samples of feces and plasma. The aim of
the study was to assess the usefulness of feces as a source of body
water in DLW studies with reindeer. Because other studies have revealed
large seasonal differences in body water kinetics in reindeer (16, 21),
trials were conducted in both summer and winter.
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METHODS |
Experimental procedures.
In this study we used four adult (>4 yr old) nonpregnant,
nonlactating female Norwegian reindeer [Rangifer
tarandus tarandus; mean body mass 84.6 kg (SD 6.0, range 79.0-91.0 kg) in summer and 81.3 kg (SD 3.3, range
78.0-85.0 kg) in winter], which had been accustomed to
handling over several years. The study consisted of one summer
experiment (lasting 5 days between June 29 and July 13, 1993) and one
winter experiment (lasting 19 days between January 11 and February 8, 1994). Before and between experiments the animals were kept together in
an outdoor enclosure (~2,700
m2) at the Department of Arctic
Biology, University of Tromsø, where they had access to natural
vegetation including birch (Betula pubescens), willows (Salix
spp.), and sedges (Carex spp.) and were also
provided with a commercially available pelleted ration [RF-80
(3)] and snow or water ad libitum. One week before and throughout
each trial the animals were kept in individual semi-outdoors graveled
paddocks (~120 m2) with ad
libitum access to the pelleted ration and water or snow.
At each trial the animals were weighed to 0.1 kg on an electronic
balance (Alpha-100, Farmer Tronic, Denmark) and injected with
sufficient
2H218O
(~90 g) to enrich the body water
2H2O
and H218O by
~160 parts per million excess. A 50-cm silicone tube leading from a
50-ml syringe containing physiological saline was connected to a
catheter inserted into the left jugular vein. Pyrogen-free DLW, made
isotonic by adding 0.9% wt/vol NaCl, was injected from a syringe into
the lumen of the tube that was then flushed with saline before the
needle was withdrawn, thus ensuring complete administration of
isotopes. The exact amount of DLW injected was determined
gravimetrically by weighing the dose syringe with its needle to 0.001 g
before and after injection.
One sample of feces (~20 g wet wt) and one blood sample (10 ml) were
collected from each animal before injection of DLW for determination of
the background levels of
2H2O
and H218O.
Parallel samples of blood and feces were collected every hour for 16 h
after injection of the dose and then once a day for the remainder of
each trial (5 days postinjection in summer and 19 days postinjection in
winter). Feces were taken from the rectum and put immediately into
plastic vials that were then sealed. Blood was collected via an
indwelling catheter in the right jugular vein or by jugular
venipuncture (heparinized Vacutainer tubes, Beckton Dickinson
Vacutainer Systems Europe) and centrifuged (1,600 g, 15 min), and the plasma was
separated and stored in 5-ml cryotubes (Greiner Labortechnik). All
blood and feces samples were stored at 
20°C within 1 h of
collection. Water was extracted from two or three portions of each
sample of feces (2 g wet wt) and plasma (2 ml) by vacuum sublimation
(26), combined within tissue by portion and stored in 5-ml cryotubes at
20°C until analysis.
The fractionation of water in feces lying on the ground exposed to the
atmosphere (as might happen in a field study) was evaluated in summer
and winter. Samples of fresh feces obtained from animals after
administration of isotope (n = 2 per
season, each ~50 g) were divided into two similar portions. One was
stored immediately in a sealed vial at 20°C, whereas the
other was left outdoors on a petri dish for 5 min before being packed
and stored at 20°C.
Analyses.
The concentration of
2H2O
and H218O was
measured in a diluted solution of the dose, the diluting water, and in
water extracted from feces and plasma.
18O was determined after
equilibration with CO2 (28) on a
SIRA-12 isotope ratio mass spectrometer (VG Isogas, Middlewich,
UK).
2H2O
was converted to hydrogen gas by zinc reduction (30), and the
2H content was determined using a
SIRA-10 isotope ratio mass spectrometer. All analyses of
2H and
18O were performed in at least
three replicates, and the mean values were used in subsequent
calculations [within sample coefficient of variation (CV)
<0.415% for 2H and <0.099%
for 18O].
In all trials the background sample and the samples collected 6, 8, 10, and 12 h after injection of DLW and the sample collected the last day
of the trial were analyzed for both
2H and
18O. To facilitate the
investigation of the equilibration process, the samples collected 1, 2, 3, 4, 14, and 16 h after injection were also analyzed for
18O.
Equilibration of injected marker.
Rates of equilibration of
2H2O
and H218O
with body water were determined by visual inspection of the plots of
isotope concentration vs. time and assessed by regression analysis. For
each trial a series of linear models were developed in which the
natural logarithm of the concentration of isotope was regressed against
the time postinjection (hours). The first models included only data
from samples collected 12-16 h postinjection for
H218O and
8-12 h postinjection for
2H2O.
Thereafter each model was expanded by including data from samples
collected progressively closer to the time of injection. Sets of
regression coefficients from each series of models were plotted against
an arbitrary linear scale in which one equals the first model, two
equals the second model, and so forth. The point of inflection of the
resulting curve, determined by visual inspection, was taken as the
point of equilibration.
Comparison of
2H2O and
H218O concentration in water
from parallel samples of plasma and feces.
The patterns of wash-out of
2H2O
and H218O in
blood plasma and in feces were compared by the method of Bland and
Altman (2). The difference in the concentration of
2H2O
and H218O
within a pair of parallel samples of plasma and feces
(Dps) was
plotted against the mean concentration of that pair
(
ps). The
implication of each successive
Dps value was
then determined after calculating the bias, which is given by the mean
and the standard deviation of all the
Dps values.
Dps values that
fell outside two standard deviations of the mean were considered
significant, indicating that there was a difference in the
concentration of isotope in water extracted from that respective
parallel sample of blood plasma and feces.
Calculations.
Body water pool size (N), rate constants
(k) for disappearance of
2H2O
and H218O,
water flux
(JH2O),
CO2 production
(rCO2), and total energy expenditure (TEE) were calculated using the two-point method. The difference in rates of disappearance of
2H2O
and H218O was
assessed from analysis of samples collected at 10 h postinjection and
on the last day of each trial according to the approach of Coward (8),
Cole et al. (6), Schoeller and Coward (29), and Haggarty et al. (17).
Calculations of all parameters were carried out using the isotopic
values for both plasma and feces.
The parameters
JH2O
and
rCO2
were calculated as follows
and
where
fractionation factors
f1
(2H2O
[vapor] to
2H2O
[liquid]),
f2
(H218O
[vapor] to
H218O
[liquid]), and
f3
(C18O2
[gas] to
H218O
[liquid]) were taken as 0.94, 0.99, and 1.04, respectively
(29). X denotes the proportion of the total water loss
assumed to undergo fractionation and was set at 0.2 (13, 26).
Corrected
JH2O
values were multiplied by 0.987 in summer and by 0.983 in winter to
compensate for the sequestration of
2H by incorporation into newly
synthesized fat, exchange with hydrogen in fecal solids, and by
incorporation into microbially produced methane (Gotaas et al.,
unpublished data). Energy expenditure was calculated from
rCO2
using a respiratory quotient of 0.8 and an energy equivalence of oxygen
of 20.1 kJ/l O2.
The subscripts F and P have been used in the text and in the legends to
indicate where parameters have been determined by analysis of samples
of feces or plasma, respectively. Thus dilution spaces for
2H2O
(ND) and
H218O
(NO) are denoted as
ND-F,
ND-P,
NO-F, and
NO-P and so forth.
Statistical analysis.
Dilution spaces, rate constants, rates of
CO2 production, and TEE calculated
from parallel samples of feces and plasma in each trial were compared
by paired t-tests. Dilution space
ratios were compared by analysis of variance. The potential bias
introduced into the calculations by fractionation of fecal water was
investigated by comparing the concentration of
2H2O
and H218O in
samples of feces stored immediately after collection and exposed
outdoors to the atmosphere for 5 min, respectively, using paired
t-tests. The potential variability in
rCO2
and TEE caused by fractionation of fecal water was investigated by a
simulation procedure. Sets of 1,500 normally distributed, random values
for the concentration of
2H2O
and H218O in
the background sample, the day 1 sample, and the last day sample in each trial were generated using the
respective three observed mean values and the standard deviations of
the concentration of both isotopes in feces exposed outdoors to the
atmosphere for 5 min in summer and winter, as appropriate. These values
were then used to calculate a set of 1,500 TEE values for each trial from which CVs of TEE were determined. The inherent variability of the
analytic procedure was assessed by comparing these CVs with the results
obtained from a second series of simulations that incorporated instead
the standard deviations of the concentration of isotopes in samples of
feces that had been stored immediately after collection.
H0 was rejected at
P 
0.05 in all tests.
| |
RESULTS |
Equilibration.
In all cases the concentration of
H218O was
higher in water extracted from plasma than in water extracted from
feces 1 h after injection of DLW. Thereafter, the concentration of
H218O in
plasma water declined and in fecal water increased until the two
converged on a common value. The concentration of
2H2O
in plasma and feces followed the same pattern. Both isotopes were
equilibrated with the body water pool ~8 h after injection in all
trials, both in summer and in winter (Fig.
1).
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Fig. 1.
Mean concentration of
H218O
[parts per million (ppm) excess] in water extracted from
parallel samples of plasma ( ) and feces ( ) from 1 to 456 h
postinjection in 1 reindeer in 1 winter trial. Arrow indicates point of
equilibration. DLW, doubly labeled water.
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|
Dilution spaces.
Parallel determinations of dilution spaces based on analysis of samples
of plasma and feces (ND-P and
ND-F) differed by no more than
2.01%, whereas NO-P and
NO-F differed by no more than 1.42% in any trial. The mean ND
values in summer were 3,251 mol (SD 302, n = 4) and 3,274 mol (SD 319, n = 4) based on samples of plasma and
feces, respectively. Corresponding values in winter were 2,932 mol (SD
196, n = 4) for
ND-P and 2,938 mol (SD 196, n = 4) for
ND-F (Table
1). There was no significant difference between either ND-P and
ND-F or
NO-P and
NO-F in either season (P > 0.05).
There were no significant differences between dilution space ratios
(ND to
NO) derived from analysis of
samples of plasma and feces within any trial in either summer
(F1,6 = 0.014, P > 0.05) or winter
(F1,6 = 2.075, P > 0.05). The mean
ratio ND to NO was 1.041 (SD 0.013, n = 8) in summer and 1.060 (SD 0.009, n = 8) in winter, respectively.
Disappearance of markers.
There were no significant differences in the concentration of isotopes
in water extracted from parallel samples of feces and blood plasma in
any of the trials after equilibration had been established. All
Dps values fell
within two standard deviations of
ps (Fig.
2). Consequently, neither
kD nor
kO differed significantly (P > 0.05) between
plasma and feces in either season. The mean
kD values in
summer were 0.159 day
1 (SD
0.016, n = 4) for
kD-P and 0.159 day1 (SD 0.019, n = 4) for
kD-F (Table
2). Mean
kO values in
summer were 0.194 day1 (SD
0.022, n = 4) and 0.195 day1 (SD 0.025, n = 4) for plasma and feces,
respectively. The corresponding mean rate constants in winter were
0.058 day1 (SD 0.011, n = 4) for
kD-P, 0.058 day1 (SD 0.010, n = 4) for
kD-F, 0.078 day1 (SD 0.013, n = 4) for
kO-P, and 0.079 day1 (SD 0.011, n = 4) for
kO-F (Table 2).
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Fig. 2.
Comparison and statistical evaluation of differences in the
concentration of
H218O in all
postequilibrium parallel samples of plasma and feces in 1 reindeer in 1 winter trial using the method of Bland and Altman (2).
Ordinate is mean concentration (ppm) of each pair of
samples, whereas abscissa is difference (ppm) within each
pair.
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Mean
JH2O
rates in summer were 516 mol/day (SD 94, n = 4) and 522 mol/day (SD 113, n = 4) calculated from samples of
plasma and feces, respectively (Table 2). Corresponding winter values
were 169 mol/day (SD 42, n = 4) for
JH2O-P
and 171 mol/day (SD 40, n = 4) for
JH2O-F
(Table 2). There was no significant difference between
JH2O-P
and
JH2O-F within either season (P > 0.05).
Exposure of feces samples to outdoor temperature and humidity.
There was no significant difference in the concentrations of either
isotope in water extracted from samples of feces that had been stored
immediately after collection and those that had been exposed to the
atmosphere for 5 min in either summer or winter, respectively
(P > 0.05, Table
3). Nevertheless, although not significant,
the concentrations of isotopes in the two sets of samples varied within
animals by up to 0.15% for 2H and
0.05% for 18O. The potential
effect of these differences was assessed using a simulation procedure
(see METHODS). The mean CV of the
TEE values generated in this way was 14.11% in summer and 8.29% in
winter, respectively. By comparison, the CV of TEE derived from the
simulation designed to test the inherent variability of the analytic
procedure were 10.55 and 4.81% in summer and winter, respectively.
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Table 3.
Enrichment of 2H2O and
H218O in replicate analyses of single samples
of feces frozen immediately after collection and after 5-min exposure
to atmosphere
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rCO2
and TEE.
Both
rCO2
and specific TEE differed substantially between animals both within and
between seasons (Table 4). The mean
TEEF was 3.557 W/kg (SD 0.907, n = 4) in summer and 1.865 W/kg (SD
0.166, n = 4) in winter. Corresponding
TEEP values were 3.457 W/kg (SD
1.059, n = 4) in summer and 1.912 W/kg
(SD 0.259, n = 4) in winter. There was
no significant difference between TEEP and
TEEF in either season
(P > 0.05).
| |
DISCUSSION |
Equilibration.
The equilibration of isotopes in different body water compartments
observed in this study closely resembles the pattern found in other
studies in ruminants where different compartments have been sampled at
short intervals for up to 20 h after injection of isotopes of hydrogen
and oxygen (11, 16, 26). In all of these studies there was an initial
peak in the concentration of isotopes in the body water compartment
into which the dose was injected (plasma in the present study),
followed by a rapid decline and a concomitant increase in the
concentration of isotope in the nonlabeled compartment (feces in the
present study) until the concentrations in each compartment converged
at a common level.
As in the study of sheep by Midwood (26), we detected no difference in
the rates of equilibration of
2H2O
and H218O.
Furthermore, the equilibration time of ~8 h for each isotope is
similar to that reported for
3H2O
in reindeer (16). These observations confirm that there is a large and
rapid exchange of water between different body water compartments in
ruminants as in other vertebrates (7, 10, 18). Fancy et al. (13)
reported that
3H2O
equilibrated more slowly than
H218O in
reindeer and attributed this to an extra mixing of
H218O due to
rapid recycling of bicarbonate between plasma and rumen water via
saliva. However, neither we nor Midwood (26) have found evidence of any
difference in the rates of equilibration of hydrogen- and
oxygen-labeled water.
Dilution spaces.
The mean ratios ND-F to
NO-F reported here of 1.041 in
summer and 1.064 in winter correspond well with the theoretical range. Midwood et al. (27) reported ratios
ND to
NO in sheep ranging from 1.033 to
1.082 (n = 4) (using
2H2O
and H218O),
indicating a very small difference between reindeer and sheep in this
respect. Based on studies in humans, Culebras and Moore (9) calculated
the theoretical maximum upward distortion of the total body water
measurements by isotope dilution due to exchange of
2H2O
with
1H2O
in free fatty acids, protein, and carbohydrate to be 5.22%. With the
assumption that NO correctly
predicts body water volume, the theoretical ratio
ND to
NO will then be 1.055 in humans
(8). On the basis of this value, the International Atomic Energy Agency recommended that ratios ND to
NO within the range
1.015-1.060 should be considered acceptable in humans (8). After
the procedure of calculation outlined by Culebras and Moore (9) and
assuming body composition in reindeer of 60-75% water,
5-15% fat, 15-20% protein, and 5% carbohydrate, the
theoretical range of the ratio ND
to NO is 1.038-1.062.
Protein, fat, and carbohydrate do not demonstrate their theoretical
maximum exchange rates in vivo (9), and the hydrogen pool size will in
practice therefore usually be less than 3% greater than the total body
water volume (25).
Fancy et al. (13) reported ratios
ND to
NO in reindeer and caribou ranging
from 1.061 to 1.112 (n = 3) in summer
and from 0.973 to 1.128 (n = 5) in
winter (using
3H2O
and H218O).
The seasonal extreme values of 1.128 and 0.973 suggest that the
experimental animals in which these values were calculated may have
been unusually fat or lean, respectively.
Disappearance of markers.
There was no significant difference in the rate constants for
2H2O
or H218O,
respectively, measured in the plasma and feces within any trial once
isotope equilibrium had been established. Likewise, we found no
difference in the rate constants for
3H2O
dilution in water extracted from plasma, rumen fluid, or feces (16),
and Martin and Ehle (24) found no difference in the rate constants for
2H2O
dilution in water extracted from samples of blood, milk, urine, and
feces in cattle. It is evident that a dynamic equilibrium between
different body water compartments exists and maintains the uniform
concentration of isotopes throughout the body water pool.
The large seasonal differences in both
kD and
kO reported here
confirm results from other studies with reindeer (13, 16, 21) and
reflect seasonal changes in
JH2O
and energy expenditure found in these and other cervids (13, 16, 21).
Exposure of feces samples to outdoor temperature and humidity.
Exposure of feces to the atmosphere for 5 min had no significant effect
on the concentration of
2H2O
and H218O in
water extracted from them. Although delays in collecting feces may
result in an increase in the variability of the results, the absolute
increase in the CV of our estimates of TEE based on samples exposed to
the atmosphere was small, and the values (CV = 8.29 and 14.11%) are
similar to those reported in other DLW studies [e.g., 8% in red
deer (Cervus elaphus); P. Haggarty, J. J. Robinson, J. Ashton, E. Milne, C. L. Adam, C. E. Kyle, S. L. Christie, and A. J. Midwood, unpublished data]. In
our experience with reindeer the risk of fractionation is small because
feces can usually be collected from the ground within a few seconds of
dropping. The risk of contamination by ground water or snow is minimal
because reindeer feces are usually dropped in a clump, and it is a
simple matter to discard all pellets at the periphery and to retrieve
only clean pellets from the core.
rCO2
and TEE.
The large individual variation in
rCO2
and specific TEE in both seasons observed in the present study might
reflect different levels of activity between animals. Individual
differences of similar magnitude have been reported in both ruminants
and monogastric animals (17, 20, 27). Likewise, the large seasonal difference in TEE measured in the present study, where the ratio of
average specific TEEF-summer to
TEEF-winter was 1.91, is similar to values reported by Fancy (12) and Boertje (4) based on estimates of
TEE made using factorial models. These authors reported ratios of 1.59 and 1.88, respectively.
Conclusions.
Feces represent a reliable source of body water for DLW studies with
free-ranging reindeer. In field application of the method, feces should
be collected as soon as possible after dropping to minimize
fractionation and the risk of contamination by ambient water. In
circumstances where changes in the body water volume of the animal
during the measurement period cannot be assumed to be negligible, it
may be necessary to capture and weigh it at the end of the trial. This
does not obviate the potential advantages of our method; feces are very
easy to collect and may still be preferred as the source of body water.
In addition, in studies where body water parameters are calculated
using the multipoint method, several samples can be collected
throughout the trial without any disturbance to the animal before it is
finally caught at the end.
The standard error of the mean of the specific TEE was within 15% of
the mean in both summer and winter. Thus, given the observed variance
in specific TEE, it would be necessary to use not less than 10 animals
to ensure that the standard error fell within 10% of the mean.
| |
ACKNOWLEDGEMENTS |
We thank two anonymous referees for their constructive comments on
an earlier version of the manuscript.
| |
FOOTNOTES |
This study was supported by the Norwegian Reindeer Husbandry Research
Fund, the University of Tromsø, and the Scottish Office Agriculture, Environment and Fisheries Department. G. Gotaas is grateful for a Joseph-Bech-Europa-Studienreisestipendium awarded by the
F.V.S. Foundation, Hamburg, Germany.
Present address of N. J. C. Tyler: Dept. of Ecology/Zoology, Inst. of
Biology, Univ. of Tromsø, N-9037 Tromsø, Norway.
Address for reprint requests: G. Gotaas, Dept. of Arctic Biology, Univ.
of Tromsø, N-9037 Tromsø, Norway.
Received 11 February 1997; accepted in final form 16 June 1997.
| |
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AJP Regul Integr Compar Physiol 273(4):R1451-R1456
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Abstract
Introduction
![<IT>J</IT><SUB>H<SUB>2</SUB>O</SUB> = <FR><NU><IT>k</IT><SUB>D</SUB> ⋅ N<SUB>D</SUB></NU><DE>[( <IT>f</IT><SUB>1</SUB> ⋅ X) + (1 − X)]</DE></FR>](/content/vol273/issue4/fulltext/R1451/img002.gif)
![<IT>r</IT><SUB>CO<SUB>2</SUB></SUB> = <FR><NU><IT>k</IT><SUB>O</SUB> ⋅ N<SUB>O</SUB> − [( <IT>f</IT><SUB>2</SUB> ⋅ <IT>X</IT> ⋅ <IT>J</IT><SUB>H<SUB>2</SUB>O</SUB>) + (1 − <IT>X</IT>) ⋅ <IT>J</IT><SUB>H<SUB>2</SUB>O</SUB>]</NU><DE><IT>2 ⋅ f</IT><SUB>3</SUB></DE></FR>](/content/vol273/issue4/fulltext/R1451/img003.gif)
