INTEG.
AND
COMP. BIOL., 42:21–33 (2002)
The Analysis of 13C/12C Ratios in Exhaled CO2: Its Advantages and Potential Application to
Field Research to Infer Diet, Changes in Diet Over Time, and Substrate Metabolism in Birds1
KENT A. HATCH,2,* BERRY PINSHOW,*
AND
JOHN R. SPEAKMAN†
*Mitrani Department of Desert Ecology, Ben-Gurion University of the Negev, Midreshet Ben-Gurion 84990, Israel
†Department of Zoology, University of Aberdeen, Aberdeen Scotland, AB 242TZ UK
SYNOPSIS. Stable isotopes are becoming an increasingly powerful tool for studying the physiological ecology
of animals. The 13C/12C ratios of animal tissues are frequently used to reconstruct the diet of animals. This
usually requires killing the subjects. While there is an extensive medical literature on measuring the 13C/12C
ratio of exhaled CO2 to determine substrate digestion and oxidation, we found little evidence that animal
physiologists or physiological ecologists have applied 13C/12C breath analysis in their studies. The analysis
breath 13C/12C ratios has the advantage of being non-invasive and non-destructive and can be repeatedly
used on the same individual. Herein we briefly discuss the medical literature. We then discuss research
which shows that, not only can the breath13C/12C ratio indicate what an animal is currently eating, but also
the animal’s diet in the past, and any changes in diet have occurred over time. We show that naturally
occurring 13C/12C ratios in exhaled CO2 provides quantitative measure of the relative contribution of carbohydrates and lipids to flight metabolism. This technique is ripe for application to field research, and we
encourage physiological ecologists to add this technique to their toolbox.
13
INTRODUCTION
Stable isotopes have long been important tools for
plant physiologists and physiological ecologists. Currently, the use of stable isotopes in animal physiology
and physiological ecology is gaining more attention
and reviews of the topic appear with increasing frequency (Peterson and Fry, 1987; Rundel et al., 1988;
Griffiths, 1991; Macko, 1994; Gannes et al., 1997,
1998; Kelly and Finch, 1998; Hobson, 1999; Hobson
and Wassenaar, 1999; Kelly, 2000). The most common
uses of stable isotopes in ecology are the analyses of
15N/14N to establish trophic levels, food webs (e.g.,
Fry, 1988; Ambrose, 1991; Hobson and Welch, 1992,
1995; Jarman et al., 1996; Sydeman et al., 1997; Peterson, 1999), dietary analysis and reconstruction (e.g.,
Handley et al., 1991; Hobson and Sealy, 1991; Angerbjörn et al., 1994; Hobson, 1995; Alexander et al.,
1996; Hobson and Sease, 1998). Recently, stable isotopes have been used to discern animal movement patterns (Alisausakas and Ankney, 1992; Hobson and
Wassenaar, 1997; Hobson, 1999; Hobson et al., 1999).
While hair and feathers are occasionally used, usually
tissues such as liver, muscle, and collagen derived
from bones are analyzed for stable isotope ratios. This
is, of course, rather invasive and usually requires the
death of the study animals. Consequently, these techniques are not very useful with rare and endangered
species or for mark-recapture studies. The analysis of
13C/12C ratios in exhaled CO provides an alternative
2
method that has been little explored by animal physiological ecologists.
In contrast, there is an ample medical literature on
C breath tests. These tests are used both in basic
research and in clinical practice. Since little attention
has been given to 13C breath tests in animal physiological ecology, the purpose of this paper is to: 1) briefly review the existing medical literature, 2) discuss the
uses in field research to which 13C/12C analysis of
breath can currently be applied, and 3) discuss future
directions for research and applications of 13C/12C
breath analysis. As others have before us (Gannes et
al., 1997), we must extend a word of caution that
much work needs to be done in the field of stable
isotopes to validate the underlying assumptions.
13
C/12C ratios
Briefly, 13C/12C ratios are usually expressed as d13C
values (Craig, 1957). The more positive the d13C value,
the higher the 13C/12C ratio, the more negative the d13C
value, the lower the 13C/12C ratio. Changes in d13C values are expressed as D13C values (Craig, 1957). For a
more detailed discussion of the principles and terminology underlying research with stable isotopes, we
refer the reader to the paper by Gannes et al. (1998).
USES OF 13C/12C ANALYSIS OF
BREATH CO2 IN MEDICINE
The various analyses of the 13C/12C ratios in exhaled
CO2 are often called 13C breath tests in the medical
literature. There are several reviews of 13C breath tests
and related research (Braden et al., 1991; Harding and
Coward, 1998; Koletzko et al., 1998; Swart and Van
Den Berg, 1998). These tests are based on the assumption that the step of interest is the rate-limiting
step (Swart et al., 1998), thus a specific substrate is
carefully chosen with the given test in mind. The underlying principle is that if an individual is given a 13C
labeled substrate, and if that individual (or that individual’s bacteria) can metabolize it, then the exhaled
breath CO2 should be 13C enriched. Consequently, the
1 From the Symposium Taking Physiology to the Field: Advances
in Investigating Physiological Function in Free-Living Vertebrates
presented at the Annual Meeting of the Society for Integrative and
Comparative Biology, 3–7 January 2001, at Chicago, Illinois.
2 Present address of Kent Hatch is Department of Zoology, Brigham Young University, Provo, Utah 84602; E-mail: [email protected]
21
22
K. A. HATCH
results are usually expressed as a percentage of the 13C
recovered (PDR, Amarri and Weaver, 1995). We
should also add that when measuring PDR that it is
important to measure, or at least estimate CO2 production (Amarri et al., 1998).
Probably the most widely known 13C breath test is
for Helicobacter pylori, the bacterium responsible for
ulcers in humans. In this test 13C labeled urea is given
to the subject. H. pylori produces urease, and therefore
if it is present in the stomach, the breath of the subject
will become enriched in 13C. If the subject is not infected, then 13C enrichment of exhaled CO2 will not
occur. While this test is highly effective and there is
an extensive literature on the subject (Harding and
Coward, 1998; Kato et al., 1998; Koletzko et al., 1998;
Logan, 1998; Swart and Van Den Berg, 1998; Delvin
et al., 1999), it may not be of direct use to physiological ecologists. Likewise tests for small-bowel bacterial overgrowth (Solomons et al., 1977; Dellert et al.,
1997; Swart and Van Den Berg, 1998) may be of little
use. However, the principles involved may prove useful and there are a number of other breath tests that
should be of interest to physiological ecologists.
There are effective breath tests for the gastric emptying rates of both liquids and solids in humans. The
former entails solubilizing 13C labeled octanoic acid in
egg yolk, making it into an omelet and serving the
omelet to the subject. The omelet passes through the
stomach and is rapidly digested in the duodenum. Thus
the signal appears in the breath after the solid food has
cleared the stomach (Maes et al., 1994). Likewise,
both 13C-acetate (Braden et al., 1995) and 13C-glycine
(Maes et al., 1994) have been used to label the liquid
phase for measuring the gastric emptying rate of liquids. It is also possible to measure intestinal transit
time (Heine et al., 1995; Swart and Van Den Berg,
1998). Since lactose-13C ureides are poorly absorbed
by the small intestine, they pass to the colon where
they are hydrolyzed by bacteria. Shortly thereafter the
13C signal shows up in the breath of the subject.
13C breath tests can effectively show the digestion
of lipids (Hoshi et al., 1992; Amarri and Weaver, 1995;
Jeukendrip et al., 1995; Amarri et al., 1998; Harding
and Coward, 1998; Koletzko et al., 1998; Swart and
Van Den Berg, 1998; Schmidt et al., 1999), proteins
(Evenepoel et al., 1998; Swart and Van Den Berg,
1998) and carbohydrates (Duchesne et al., 1982; Lacroix et al., 1982; Hiele et al., 1989, 1990; Dewit et
al., 1992; Swart and Van Den Berg, 1998). In addition,
by giving subjects 13C labeled glucose, fructose, lactose or starch, for example, the digestion of those specific carbohydrates can be studied (Swart and Van Den
Berg, 1998). One might also look at the digestibility
of cellulose in different animals by feeding them a diet
in which the cellulose is 13C labeled. While we are not
aware of any such test or experiment, it should not be
difficult to do. Finally, Tanis et al. (1996) published a
breath test for measuring the depletion of liver glycogen using a naturally 13C-enriched carbohydrate diet.
These studies have primarily been done in humans
ET AL.
and rats. Their broader applicability has not been demonstrated, nor have they been adapted for use in the
field. However, these techniques may be of interest to
physiologists and physiological ecologists interested in
questions concerning animal nutrition.
PRESENT AND POTENTIAL APPLICATION OF 13C/12C
ANALYSIS OF BREATH CO2 IN THE FIELD
Field metabolic rate
Doubly labeled water has long been virtually the
sole technique for measuring field metabolic rates
(FMR). However, enough time must pass for a measurable amount of deuterium (or tritium) and 18O to be
eliminated through expired CO2 and the loss of body
water. This makes measurements of FMR possible
only over relatively long periods of days to weeks.
Recently, it has become possible to measure FMR over
periods of minutes to hours. This is done by administering a bolus dose of 13C labeled bicarbonate and
taking breath samples to determine the washout kinetics (Junghans et al., 1997; Speakman and Thomson,
1997).
Infer diet
To use 13C/12C ratios in the field to infer the composition of the animal’s diet, there must be two possible food resources, and these two resources must differ isotopically. For example, C3 and C4 plants differ
dramatically in their carbon isotope ratios (see
O’Leary, 1981; Gannes et al., 1998). Marine and Terrestrial food sources also differ quite substantially in
13C/12C ratios (Hobson, 1986, 1999; Mizutani et al.,
1990; Hobson and Sealy, 1991; Angerbjörn et al.,
1994). A two end-point mixing model is generally assumed to calculate the percent contribution to the diet
of the two dietary sources (Schroeder, 1983; Kline et
al., 1990; Gannes et al., 1997). If the sources are spatially disparate, inferences can be made about the animal’s movement and the time spent foraging in each
location. Such inferences of diet and movement are
usually based on the 13C/12C ratios of such tissues as
skeletal muscle, liver, or bone collagen.
There are two major disadvantages in using the carbon isotope ratios of such tissues as the basis of dietary inferences. First, they require the destruction of
the animal. This is problematic if one would like to
make repeated measures on the same animal or if the
animal is rare or endangered. The second problem is
that the carbon isotope ratio of all tissues does not
represent that of the diet equally well (DeNiro and
Epstein, 1978; Tieszen and Fagre, 1993; Ambrose and
Norr, 1993). Typically, there is some fractionation that
occurs between the diet and the tissue. By fractionation
we mean a change in the 13C/12C ratio caused by physical or chemical processes, in this case processes involved in the synthesis and construction of the tissue.
To an extent, this can be compensated for by experimentally determining the fractionation factors for the
desired tissues. However, few of these data exist.
Even when these data do exist, the experimentally
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C/12C BREATH ANALYSIS
23
FIG. 2. Variation in breath d13C of captive pigeons and wild-caught
blackcaps. Captive pigeons ate a mostly C4 diet (with some C3
wheat). The blackcaps ate almost exclusively C3 plants (with one
possible exception).
FIG. 1. The d13C values of the tissues of rats relative to the d13C
values of the diets the rats were raised on. The d13C value of all the
diets was set to zero (heavy horizontal line). The symbols represent
the fractionation of the tissue from the diet. The effects of nutrient
routing can be seen. Adapted from Tieszen and Fagre, 1993.
determined fractionation factor may not be valid for
all diets that the animal might eat. In one of the few
studies of its kind, Tieszen and Fagre (1993) demonstrated that the tissues of mice fed diets consisting of
different components derived from either C3 or C4
plants had vastly different fractionation factors. For
example, when the mice were fed a completely C3 or
a completely C4 diet, the 13C/12C ratio of the liver and
muscle were almost identical to that of the diet. The
fractionation factors were very small. However, when
mice were fed a diet containing C4 protein, with the
remainder of the diet coming from C3 plants, the carbon isotope ratio of bone collagen, liver and muscle
did not reflect that of the bulk diet, rather it reflected
the carbon isotope ratio of the protein portion of the
diet (Fig. 1). Thus the fractionation factors for these
tissues were several times larger for mice raised on the
C3 diet with C4 protein than it was for mice raised on
purely C3 or (nearly pure) C4 diets. Tieszen and Fagre
(1993) showed that one cannot assume that a given
tissue has a constant fractionation factor for all possible diets. These results were confirmed in a similar
study by Ambrose and Norr (1993).
Tieszen and Fagre (1993) concluded from their results that the 13C/12C ratio of breath CO2 is generally
a more reliable estimator of bulk diet than are tissues.
Despite the fact that 13C/12C breath analysis is noninvasive and repeatable, few studies can be found in
the animal physiology or ecology literature where the
carbon isotope ratio of breath CO2 is used to make
inferences about diet. Nor are there many studies in
which breath tests like those used on humans are used
on animal subjects.
Hatch et al. (unpublished data) found a clear difference between the 13C/12C ratio of the exhaled CO2 of
captive pigeons fed a mixed C3–C4 diet when compared to wild-caught blackcaps known to eat an almost
entirely C3 diet (Fig. 2). The authors were also able to
calculate the percentage of C3 and C4 seeds eaten by
individual pigeons from the 13C/12C ratio of the breath.
Klein et al. (1988) used a breath test combined with
an oral dose of 13C labeled glucose to determine glucose metabolism in fed and fasted horses. There is at
least one field study in which the authors suggest that
breath CO2 carbon isotope ratios might be better than
carbon isotope ratios tissues for indicating diet (Hobson and Stirling, 1997).
Nutrient routing
The reason that the 13C/12C ratio of the liver and
muscle of the rats in Tieszen and Fagre’s (1993) study
reflected that of the C4 protein rather than the 13C/12C
ratio of the bulk diet is that the dietary protein was
used directly for the synthesis of the proteins in the
liver, muscle, and other organs. This phenomenon of
routing specific dietary components to specific tissues
is termed ‘‘nutrient routing’’ (Ambrose and Norr,
1993) or ‘‘isotopic routing’’ (Schwarcz and Schoeninger, 1991).
Tieszen and Fagre (1993) found that, generally
speaking, the carbon isotope ratio of breath CO2 was
a more reliable indicator of the carbon isotope ratio of
bulk diet. The one diet where Tieszen and Fagre
(1993) did not find the carbon isotope ratio of breath
CO2 to be representative of bulk diet when the diet
was C3 based but contained C4 cellulose (Fig. 1). The
reason for this is obvious. While cellulose contributed
to the 13C/12C ratio of the diet as a whole, the rats were
unable to digest it, and therefore cellulose did not contribute to the 13C/12C ratio of the exhaled CO2.
While it is very unlikely that one would find a natural situation in which there would be substantial differences in 13C/12C ratios between the bulk diet and
cellulose in the diet, this result in their study makes a
very important point. The 13C/12C ratios found in ex-
24
K. A. HATCH
haled CO2 represent the 13C/12C ratio of that portion of
the diet metabolized by the animal for energy. Thus,
Wolf et al. (2000) suggest that by combining the isotopic analysis of breath CO2 with the analysis of various tissues, one may be able to determine which portion of the diet is being routed to the synthesis of proteins, the deposition of fat or metabolism for energy.
One may also be able to determine if an animal in the
field relies on different foods to supply different macronutrients and the extent to which it does so. To our
knowledge, no such studies have been done.
The phenomenon of nutrient routing also suggests
one other situation in which the 13C/12C ratio of the
breath may not be representative of that of the diet.
Gannes et al. (1998) suggest that the 13C/12C ratio of
exhaled breath CO2 may underestimate the contribution of protein in the diet of animals eating low protein
diets, because in this situation, almost the entire protein component of the diet would be routed to protein
synthesis. However, when Tieszen and Fagre (1993)
fed rats a diet with the normal amount of protein or a
diet with twice the normal protein (again the protein
was C4 based, while the remainder of the diet was C3),
they found little difference in the 13C/12C ratio of the
rats’ breath. Thus, Tieszen and Fagre’s (1993) data
suggest that exhaled CO2 is a reasonable estimator of
the 13C/12C ratio of the diet in most situations.
Changes in diet over time
Several studies have suggested that the comparison
of carbon or nitrogen isotope ratios in tissues with different turnover rates might provide an indication of
changes in diet over time (Tieszen et al., 1983; Hobson
and Clark, 1992a; Gannes et al., 1998). The idea is
that there is an expected relationship between the stable isotope ratios (either of C or N) of the different
tissues of an animal raised on a constant diet and a
change in the relative isotopic ratios of the tissues suggests a change in diet. Tissues that turn over slowly
can be used as a baseline with which to compare the
stable isotope ratios of tissues that turn over more
quickly. For instance, Hobson and Clark (1992a)
showed that bone collagen turns over very slowly.
Whole blood also turns over relatively slowly, whereas
muscle and liver turn over more quickly. Thus, if one
finds that the stable isotope ratios of muscle and liver
are much different than one would expect given the
stable isotope ratios in bone collagen and whole blood,
one could infer that a change in diet has occurred within the time that muscle and liver tissues turn over. In
the case of Japanese quail, that would be between 3
and 12 days (Hobson and Clark, 1992a).
While the 13C/12C analysis of different tissues to infer dietary history has been suggested in the literature
(Tieszen et al., 1983; Kelly and Finch, 1998), we are
unable to find any examples of its actual use in the
field. Tissue analyses have the disadvantages of requiring the killing of the subject and of time consuming tissue preparation. Hatch et al. (2002) showed that
recent changes in diet over time can also be deter-
ET AL.
mined through the analysis of the 13C/12C ratios of a
subject’s exhaled CO2.
The authors used 12 pigeons that had been raised
on a predominantly C4 diet. This diet consisted of 60%
sorghum (C4), 15% corn (C4), 15% wheat (C3), and
20% pellets. The pellets were 90% corn and 10%
wheat gluten. Studies by Tieszen and Fagre (1993) and
others (DeNiro and Epstein, 1978; Tieszen et al., 1983;
Hobson and Clark, 1992a, b; Hobson, 1995) show that
the carbon isotope ratio of the tissues generally reflect
that of the diet. Consequently, the tissues of these 12
pigeons had a strong C4 signature. To further enhance
that signature, the authors fasted these pigeons for
three days and then fed the pigeons corn for 30 days.
After 30 days breath samples were taken. The d13C of
the pigeons’ breath was similar to the d13C value of
their corn diet. The authors then fed 6 of the pigeons
wheat while the remaining six pigeons continued to
eat corn. After 48 hr the exhaled d13C from corn-fed
pigeons was unchanged, while that from wheat-fed pigeons had become more negative, approaching the
d13C of wheat. The breath d13C clearly indicated a
change from the corn diet to the wheat diet. Three of
the wheat-fed pigeons were then fasted for three days,
after which their exhaled d13C increased, approaching
the d13C the breath of pigeons that continued to eat
corn. This showed that the wheat-fed pigeons had been
eating corn when their lipid reserves were formed.
This study showed that one can determine changes in
diet over time between two isotopically different foods
by repeatedly taking breath samples and analyzing
their 13C/12C ratios (Fig. 3).
More importantly, the study also showed that one
can determine a change in diet over time from a single
capture event. If the animal has recently eaten and a
breath sample is taken immediately after capture, this
breath sample will reflect the 13C/12C ratio of the diet.
One then fasts the animal long enough that one is confident it has metabolized its food and is now metabolizing endogenous energy reserves (24 hr in pigeons,
Rashotte et al., 1995) and takes a second breath sample. This second sample reflects the 13C/12C ratio of the
endogenous stores it is burning for energy while fasting. In most cases this is primarily endogenous lipid
stores (Cherel et al., 1988; Castellini and Rea, 1992).
If the animal is eating a diet that is isotopically similar
to the diet it was eating when its lipid reserves were
formed, the 13C/12C ratios of the two breath samples
should differ by approximately a d13C value of 3 ‰.
This is because endogenous lipids are approximately
3 ‰ lighter (i.e., have a lower 13C/12C ratio) than the
substrate from which they were formed (DeNiro and
Epstein, 1977). In contrast, if there is a larger difference in the 13C/12C ratios of the two samples one can
infer that the diet that the animal was eating when it
formed its endogenous energy reserves was isotopically different from its present diet, and a change in
diet has occurred. Thus, from the 13C/12C ratios of exhaled CO2 one could determine that some pigeons had
recently eaten a C3 diet (wheat) while others had re-
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C/12C BREATH ANALYSIS
25
FIG. 4. Comparison of d13C of the breath of pigeons fasted for 72
hours with the d13C of the breath of the same pigeons after they had
been fed ad libitum for 48 hours. The high degree of correlation
shows that, not only are there differences in diet selection between
pigeons, but the individual pigeons are highly consistent in their diet
selection over time.
FIG. 3. Experimental design (A) and results of 13C/12C breath analysis showing change in diet (B). Twelve pigeons had been raised on
a predominantly C4 diet. Thirty-three days previous to the experiment all 12 pigeons were fasted for three days and then fed only
corn for 30 days (A). At day zero, the first breath samples were
taken and then six pigeons were fed a wheat diet. At day two, three
of the corn-fed pigeons and three of the wheat-fed pigeons were
fasted. The results (B) clearly show that the switch from the corn to
the wheat diet was apparent in the 13C/12C ratio in the pigeons’
breath. More importantly, when three wheat-fed pigeons were fasted,
the 13C/12C ratio of their breath became identical to that of pigeons
fed corn and fasted. Thus, from 13C/12C breath of a fasted animal it
is possible to determine the isotopic nature of their diet in the past,
when they laid down their lipid stores. Error bars are not given for
clarity’s sake. Adapted from Hatch et al. (2002).
cently eaten a C4 diet (corn). When these pigeons were
then fasted and breath samples taken, the breath samples showed that all the fasted pigeons had eaten corn
in the past, when their lipid stores were formed.
Hatch et al. (unpublished data) also found differences in food selection among pigeons housed together
in a single loft and fed a common diet. These differences were consistent over time as well.
That individual pigeons differ in food choice is well
established (Moon and Zeigler, 1979; Inman et al.,
1987). When housed in groups, competition and dominance hierarchies enhance individual differences in
food choice. Lower ranking pigeons tend to consume
more of the less desirable seeds, while higher-ranking
pigeons will consume more of the seeds they prefer
(Inman et al., 1987; Robichaud et al., 1996). Breath
samples were taken from a set of 12 pigeons after
these pigeons had fasted for 72 hr. These pigeons had
been raised and fed on the mixed corn, wheat, sorghum, pellet diet described above. They then allowed
these pigeons to feed ad libitum for 48 hr, after which
a second breath sample was taken from each pigeon.
The high correlation between the fasted and fed breath
d13C values showed that, while there were substantial
differences between individual birds in diet choice, the
mixture of C3 and C4 grains chosen by individual pigeons over time was quite consistent (Fig. 4).
Again, there are several advantages to using breath
samples over using tissue samples. Breath samples are
easier to collect and prepare for analysis. Collection is
non-invasive and can be repeated on the same subject.
In addition, if the possible locations of the isotopically
different diets differ spatially, one can also make inferences about the movement of the animal through
space and time.
Determining exogenous vs. endogenous substrate use
Since the 13C/12C ratio of exhaled CO2 can be used
to determine both past and present diet, we decided to
explore whether 13C breath analysis would be useful
in determining exogenous vs. endogenous substrate
metabolism. Specifically, we decided to look at the
effect of different periods of food deprivation on substrate metabolism in flying and resting pigeons. Researchers usually use respiratory quotient (RQ) to determine metabolic substrate use. This, however, requires the collection of exhaled CO2 to determine the
ratio of CO2 produced to O2 consumed. In flying birds
this necessitates that a trained bird be flown in a wind
tunnel with a mask over its face (e.g., Butler et al.,
1977; Rothe et al., 1987; Nachtigall, 1990). This is not
a natural situation and it is often difficult to train birds
to fly for long periods under these conditions. Consequently, blood metabolite levels are frequently used
to indicate substrate metabolism in wild-caught birds.
However, the analysis of blood metabolite concentrations gives qualitative, not quantitative results. Since
26
K. A. HATCH
endogenous lipids have a lower 13C/12C ratio than the
substrate from which they were formed (DeNiro and
Epstein, 1977), we thought that the 13C/12C ratio in
exhaled CO2 might give a more quantitative picture of
substrate use in flying birds. We now present this study
and its results.
In this study, we deprived pigeons of food for 2, 12,
24, 48, and 72 hr. The pigeons were normally fed once
every 24 hr, so only the longer periods of food deprivation constituted serious fasts. However, for ease of
discussion we will refer to all the periods of food deprivation as ‘‘fasts.’’ Following each fast some pigeons
flew for 4 hr while others rested for 4 hr. We made
the following hypotheses: 1) As pigeons shifted from
metabolizing food to metabolizing endogenous energy
stores, lipid metabolism would increase with increased
fast duration. 2) Protein would decrease with increased
fast duration as pigeons shifted to stage two fasting
and began sparing proteins (Cherel et al., 1988; Castellini and Rea, 1992). 3) Carbohydrate metabolism
would decrease with increased fast duration, again as
pigeons shifted from burning exogenous carbohydrates
to endogenous lipids. 4) Both lipid and protein metabolism would be greater in flying pigeons than in resting pigeons. This is because their greater need for energy would require more lipids to fuel flight and their
higher metabolic rate would require more protein to
supply the citric acid cycle (Jenni and Jenni-Eiermann,
1998).
We decided to measure free fatty acid (FFA) and
uric acid (UA) levels in the blood plasma in addition
to taking breath samples. Since FFA is used as an indicator of lipid metabolism (Hurley et al., 1986; JenniEiermann and Jenni, 1992) and UA is used as an indicator of protein metabolism (Mori and George, 1978;
Robin et al., 1988), the following predictions stemmed
from our hypotheses. 1) FFA levels would be greater
in flying pigeons than in resting pigeons. 2) FFA levels
would increase with increased fast duration. 3) UA
levels would be greater in flying pigeons than in resting pigeons. 4) Initially there would be a decrease in
UA levels with increased fast duration. However, we
also thought that this might be followed by an increase
in UA levels after the 72 hr fast if a fast of this length
exhausts their lipid reserves, forcing the pigeons to use
endogenous proteins for energy. 5) Breath 13C/12C ratios would decrease with increased fast duration, again
as pigeons shifted from using exogenous carbohydrates to endogenous lipids for fuel. 6) 13C/12C ratios
would be lower in flying pigeons than in resting pigeons, because flying pigeons would need more lipids
to fuel flight.
MATERIAL
AND
METHODS
Experimental animals
We used 13 adult tippler pigeons, a breed of Columba livia bred to fly overhead in large circles for long
periods of time (mean mass 5 258 g). We normally
fed the pigeons once a day, at eight o’clock each morn-
ET AL.
ing. We rang a bell before giving the food to the birds
so that they would associate feeding with the ringing
of the bell. We allowed the pigeons to feed for 15 min
and then removed the food. We provided the pigeons
with water ad libidum. Tippler pigeons were maintained at the Jacob Blaustein Institute for Desert Research, Midreshet Ben-Gurion, Israel (308529N,
348469E: 476 m asl). The pigeons were housed together in an out door loft (3 m 3 2 m 3 2 m).
Pigeon flights
We trained the pigeons to fly for 4–5 hours without
stopping and then to land at the ringing of a bell. In
order to assess the effect of feeding and fasting on
metabolic substrate use in flight, we withheld food
from pigeons for 2, 12, 24, 48, or 72 hours preceding
the test flight. Each fasting period and the associated
flight were conducted separately. We allowed the pigeons to feed ad libitum for the 48 hours following
each fast and its associated flight. Then we returned
the pigeons to their normal feeding schedule until the
beginning of the next fast.
For each flight we randomly assigned pigeons to
either the flying group, or the resting group. Later, we
repeated the fast and the associated flight with the
groups reversed, i.e., those that had rested the first time
flew the second time, and those that flew the second
time rested the first time. This allowed the pigeons to
serve as their won controls. The first 12, 24, 48 and
72 hour fasts with their associated flights were conducted in July of 1998. We randomly assigned the order of the fast. The second 12, 24, 48 and 72 hour
fasts with their associated flights were conducted in
September and October of 1998. Because it was very
difficult too keep pigeons from landing for four hours
when they had been fed only two hours before the
flight, we only conducted one such flight. This was
done in October 1998. All the flights were between
8:00 a.m. and 1:00 p.m. During the flight, resting pigeons were held in their loft without access to food or
water.
Breath and blood sampling
We took both pre- and post-flight breath and blood
samples. We selected each pigeon at random before
the flight, irrespective of whether the pigeon was assigned to fly or to rest. First a 1 ml blood sample was
taken from the pigeon and then a breath sample. Blood
samples were taken from the leg by venipuncture. The
blood sample was placed in a microcentrifuge tube and
immediately placed in a refrigerator. Following the
flight, the sampling procedure was repeated, with the
exception that we sampled the flying pigeons first and
then the resting pigeons. We did this so that the blood
and breath samples of the flying pigeons would reflect
their condition in flight as nearly as possible. All the
flying pigeons were sampled within 30 minutes of
landing.
After post-flight samples were taken, we separated
the blood plasma in a microcentrifuge (5 min, 12,000
APPLYING
13
C/12C BREATH ANALYSIS
27
FIG. 5. Balloon, mask and syringe system used to collect breath sample from birds. The balloon is first flushed with pure oxygen. Then the
mask is placed over the mouth of the bird and the bird is allowed to rebreathe the oxygen for 30 sec.
g). We then removed the blood plasma from the microcentrifuge tube, placed it in a second microcentrifuge tube and stored the blood plasma samples at
2808C until we analyzed the samples. We analyzed
blood plasma samples for uric acid and free fatty acids
using spectrophotometric assays from Sigma Diagnostics (St. Louis, Mo) and Wako Chemicals (Dallas) respectively.
Breath sampling method and analysis
The sampling device consisted of a latex party balloon (unstretched volume 30 ml) a syringe needle (18
gauge) and a mask (unpublished data, K.A.H.). Each
was connected to a different stem of a small three-way
stopcock (e.g., Cole-Parmer 1999–2000 catalog, p.
1189, cat. no. 30600-03; Fig. 5).
Before taking a sample, we flushed the balloon 5–
6 times with pure oxygen, then filled the balloon with
oxygen until it was slightly expanded (40–45 ml for
our balloons). We then lined the edge of the mask with
a gel (Ultra Gel—Aquarius 101, Mediate Ltd., Israel;
a water-soluble gel for ultrasound use) to prevent leakage around the edge of the mask. For accurate 13C/12C
analysis of exhaled CO2 with an isotope ratio mass
spectrometer, a 10 ml sample containing 1–6% CO2
was required. Because of the small size and lung volume of our birds, we allowed each pigeon to rebreathe
the oxygen in the balloon for 30–40 sec. We used a
single balloon for each experiment. We then aspirated
the exhaled CO2 sample from the balloon into an evacuated Exetainert tube (Labco Ltd., Buckinghamshire,
UK).
We analyzed the samples using an ISOCHROM-VG
isotope ratio mass spectrometer (laboratory of John
Speakman, University of Aberdeen, Aberdeen, Scotland). We adjusted each batch of 10 samples using a
working standard enrichment and expressed all 13C/12C
ratios as d13C values, relative to the international standard, Pee Dee Belemnite (PDB, Craig, 1957). Initially,
we ran the samples from each Exetainert in triplicate
to establish the precision of replicate analyses (established precision was be 0.01 ‰). Thereafter we ran
each sample in duplicate. All samples were analyzed
in a blind experimental protocol.
Statistical analysis
We analyzed plasma UA, plasma FFA and 13C of
exhaled CO2 separately using repeated measures ANOVAs (type III) for each. We present the multivariate
repeated measures analysis of FFAs for the 12, 24, and
72 hour fast because there were results from only one
2 hr fasted flight and we were missing FFA results for
one 48 hour flight. However, the univariate analysis
using all the data provided similar results. We present
28
K. A. HATCH
ET AL.
TABLE 1. Results of a multivariate repeated measures analysis comparing blood plasma free fatty acid levels of pigeons that flew for 4 hours
following a 12, 24 and 72 hour fast with the same pigeons when resting for 4 hours following a 12, 24 and 72 hour fast.
Source
Treatment
Pigeon ID
Subject (Group)
Time
Time∗Treatment
Time∗Pigeon ID
Time∗Subject (Group)
df
Sum of
squares
Mean
square
1
12
12
2
2
24
24
16.038
2.881
1.448
3.562
0.772
3.950
5.635
16.038
0.240
0.121
1.781
0.386
0.165
0.235
F-value
P-value
132.887
1.989
0.001
0.1239
7.585
1.644
0.701
0.0028
0.2142
0.8049
G-G
0.0045
0.2182
0.7885
Dependent: FFA.
the multivariate results for the UA analysis using data
from 12, 24, 48 and 72 hour fasts. Here, the univariate
results differed slightly. These differences are presented in the discussion section. Finally, since we needed
to use all the data for the 13C/12C analysis of breath
CO2, we provide the univariate repeated measures results. We included ‘‘treatment’’ and ‘‘pigeon ID’’ as
factors in all analyses, since the pigeons were their
own controls. While blood metabolite concentrations
can change quickly after a flight (e.g., Jenni-Eiermann
and Jenni, 1991; Schwilch et al., 1996) ANCOVA did
not find the time between landing and sampling to be
a significant covariate (P . 0.1). Thus it was not included in any of the models. Likewise, pre-flight UA,
FFA and 13CO2 were not found to be significant covariates (P . 0.1) and were eliminated from the models.
The reduced models are presented in Tables 1, 2 and
3. We used paired contrasts within the univariate repeated measures ANOVA to compare flying and resting pigeons within each fast period. However, this
could not be calculated for the two-hour fast results
because only one flight was completed. For this fast
we compared flying and resting pigeons using a separate t-test. In this case the pigeons did not act as their
own controls. For the 2 hour fasted flight, the flying
and resting pigeons were different birds.
RESULTS
Blood metabolites
FFA levels were higher in the pigeons when they
flew than when they rested. There was also a significant increase in FFA levels with increased fast duration. Since the time by treatment interaction was not
significant, FFA levels of both resting and flying pigeons increased at the same rate (Fig. 6.; Table 1).
Likewise, UA levels were higher in flying pigeons
than in resting pigeons. However, we found no evidence of an increase in UA levels with increased fast
duration (Fig. 6.; Table 2). Paired contrasts of flying
and resting pigeons for each fast period within a univariate repeated measures ANOVA showed that both
FFA and UA levels were higher after each individual
fast period in the pigeons when they flew than when
they rested (P , 0.5). For the two-hour flight, t-tests
showed FFA (t11 5 5.9, P . 0.0001) and UA (t11 5
5.0, P . 0.001) levels to be greater in flying pigeons
than in resting pigeons.
13
C/12C breath analysis
We found the effect of treatment on the breath 13C/
12C ratios to be significant. Paired contrasts showed the
breath of resting pigeons to have a higher 13C/12C after
a 12 hour fast than that of flying pigeons (P , 0.05).
Resting pigeons had a higher 13C/12C than flying pigeons in their breath after the 2 hour fast as well (t11
5 3.9, P . 0.01). However, paired contrasts showed
no difference in the breath 13C/12C ratios of resting and
flying pigeons after the 24, 48, and 72 hour fasts. Thus
both time and the time by treatment interactions were
significant (Fig. 6.; Table 3)
DISCUSSION
Blood metabolites
Blood plasma FFA levels indicated higher levels of
lipid metabolism in flying pigeons than in resting pigeons. The increase in FFA levels with increased fast
TABLE 2. Results of a multivariate repeated measures analysis comparing blood plasma Uric acid levels of pigeons that flew for 4 hours
following a 12, 24, 48 and 72 hour fast with the same pigeons when resting for 4 hours following a 12, 24, 48 and 72 hour fast.
Source
Treatment
Pigeon ID
Subject (Group)
Time
Time∗Treatment
Time∗Pigeon ID
Time∗Subject (Group)
Dependent: UA.
df
Sum of
squares
Mean
square
1
12
11
3
3
36
33
3,845,624
2,002,703
1,736,926
202,790
111,132
1,496,145
1,004,583
3,845,624
166,892
157,902
67,597
37,044
41,560
30,442
F-value
P-value
24.354
1.057
0.0004
0.4669
2.221
1.217
1.365
0.1042
0.3190
0.1846
G-G
0.1242
0.3172
0.2185
APPLYING
13
C/12C BREATH ANALYSIS
29
TABLE 3. Results of a univariate repeated measures analysis comparing breath d13C values of pigeons that flew for 4 hours following a 2,
12, 24, 48 and 72 hour fast with the same pigeons when resting for 4 hours following a 2, 12, 24, 48 and 72 hour fast.
Source
Treatment
Pigeon ID
Subject (Group)
Time
Time∗Treatment
Time∗Pigeon ID
Time∗Subject (Group)
df
Sum of
squares
Mean
square
1
12
12
4
3
46
34
23.111
164.969
9.214
299.872
22.736
86.054
38.297
23.111
13.747
0.768
74.968
7.579
1.871
1.126
F-value
P-value
30.099
17.904
0.0001
0.0001
66.556
6.728
1.661
0.0001
0.0011
0.0627
Dependent: d13C.
duration indicated a corresponding increase in lipid
metabolism.
Blood plasma UA levels indicated higher levels of
protein metabolism in flying pigeons than in resting
pigeons. In contrast to Gannes et al. (2001), we did
not find evidence of increasing protein metabolism
with increased fast duration. This may be due to a lack
of sensitivity in the multivariate repeated measures
analysis. The univariate repeated measures analysis,
taking advantage of all the data, found the effect of
time to be significant, but found no time by treatment
interactions. However, the trend appears to be a decrease in UA, driven by the relatively high UA levels
found in pigeons after only a 2 hour fast (Fig. 6). This
suggests that between a two and a twelve hour fast the
pigeons entered stage II fasting, decreasing their protein catabolism. There is little evidence to suggest that
a 72-hr fast had caused either flying or resting pigeons
to reach stage III fasting (Cherel et al., 1988; Castellini
and Rea, 1992).
There are two problems with using blood metabolites to measure changes in substrate metabolism in
birds. First, blood metabolites are qualitative, not
quantitative. Second, birds strictly regulate their blood
glucose levels, and these don’t seem to vary with either fast duration or flight duration (Swain, 1992;
Schwilch et al., 1996). Here measuring the 13C/12C ratios of exhaled CO2 provides the needed extra information.
13
C/12C breath analysis
FIG. 6. Blood plasma free fatty acid (FFA) levels (A) in pigeons
following a 4 hour flight (black squares) and FFA levels of the same
pigeons following a 4 hour rest (open circles). Blood plasma uric
acid (UA) levels are indicated in ‘‘B’’ and d13C values of exhaled
CO2 are indicated in ‘‘C.’’ Each 4 hour flight or rest period followed
a period of food deprivation (2, 12, 24, 48, 72 hours). The open
circles are slightly offset for clarity. The solid black line indicates
the least squares means of the fliers and the dashed lines indicate
the least squares means of the pigeons when they rested.
Resting pigeons after a 2 and a 12 hour fast had
higher 13C/12C ratios than did flying pigeons. This suggests that the resting pigeons were using more carbohydrates while the flying pigeons were using more lipids to fuel metabolism. However, in both flying and
resting pigeons the 13C/12C ratios decreased, converging on the same value after a 24 hour fast. This value
then did not change with increased fast duration, remaining the same for both flying and resting pigeons.
This strongly suggests that the pigeons were relying
primarily on lipid reserves to fuel metabolism. This is
supported by the observation of Rashotte et al. (1995)
that pigeons shift to phase II fasting after a 24 hour
fast. And while the absolute amount of lipids metabolized may have increased with increasing fast dura-
30
K. A. HATCH
FIG. 7. Percentage of carbohydrate vs. lipid metabolism in flying
and resting pigeons after different fast durations calculated from d13C
values using a two-endpoint model. After a 24 hour fast both flying
and resting pigeons are assumed to be burning ‘‘100%’’ lipids. The
pigeons resting after a 2 hour fast are assumed to be burning
‘‘100%’’ carbohydrates (we ignore protein metabolism for these calculations). This is probably a reasonable assumption because the
mean breath d13C for these pigeons lies between the calculated mean
d13C for the diet and the maximum possible d13C (for pigeons eating
only the C4 grains corn and sorghum).
tion, as indicated by the FFA results, the fact that the
13C/12C ratios remained constant show that ratio of lipid to carbohydrate metabolism did not change.
It is important to note that the carbon isotope ratios
in the exhaled CO2 do not give a measure of absolute
carbohydrate or lipid metabolism. Rather, they are an
integration of the 13C/12C ratios of the substrates being
metabolized, and as such indicate the ratio of lipid to
carbohydrate or lipid to protein metabolism. However,
if we assume that the mean d13C value of both flying
and resting pigeons after the 24, 48, and 72 hour fasts
indicates 100% lipid metabolism (ignoring protein for
the time being), and if we assume that the mean d13C
value of pigeons that rested after a two hour fast represents 100% carbohydrate metabolism, we can calculate the percent lipid vs. carbohydrate metabolized
for pigeons that flew after the 2 hour and 12 hour fasts
and for pigeons that rested after the 12 hour fast. We
used a two-endpoint mixing model for these calculations (Schroeder, 1983; Kline et al., 1990; Gannes et
al., 1997). Since our two endpoints are derived directly
from the exhaled CO2, we didn’t need to worry about
a fractionation factor (Wolf and Martinez del Rio,
2000). The estimates are presented in Figure 7. Thus
the analysis of 13C/12C ratios in the breath of pigeons
provides us with a quantitative measurement of lipid
vs. carbohydrate metabolism that was impossible to get
otherwise.
To this point we’ve ignored the effect of protein
metabolism on the 13C/12C ratios in the breath. This
seems fairly reasonable, since protein accounts for a
small percentage of the fuel used until the animal
reaches stage III fasting (Cherel et al., 1988; Castellini
and Rea, 1992). However, the 13C/12C ratios of exhaled
ET AL.
CO2 do suggest something about the relative amount
of protein metabolized in resting and flying pigeons.
Endogenous proteins usually have a higher 13C/12C ratio than endogenous lipids (Tieszen and Fagre, 1993).
The fact that the 13C/12C ratio of the exhaled CO2 remained the same for both flying and resting pigeons
after a 24, 48, and 72 hour fast suggests that, while
levels of both lipid and protein metabolism were higher for flying than resting pigeons and levels of lipid
and protein metabolism may have increased with increased fast duration, the ratio of lipid to protein metabolism did not change. After a 24 hour fast, neither
flight nor fast duration had any effect on the ratio of
lipid to proteins metabolized by the pigeons. This conclusion supports the hypothesis of Jenni and JenniEiermann (1998) that protein and lipid metabolism are
closely tied through the citric acid cycle. However, we
did not measure the 13C/12C ratio of the pigeons’ endogenous proteins, therefore we cannot exclude the
possibility that pigeons’ endogenous proteins had the
same 13C/12C ratio as their lipid reserves. This seems
unlikely given the evidence demonstrating differences
between the d13C values of lipids and proteinaceous
tissues (Tieszen and Fagre, 1993). Nevertheless, if the
13C/12C ratios of endogenous lipids and proteins were
similar in these pigeons, isotopic analysis of breath
CO2 would be unable to reveal any changes in the ratio
of lipids to proteins metabolized.
FUTURE DIRECTIONS
Tissue sampling is currently the predominant method of inferring diet or changes in diet over time and
generally requires killing the animal. The next obvious
step is begin using 13C/12C analysis of breath CO2 in
the field, either as a non-invasive, non-destructive replacement for tissue sampling, or in concert with tissue
sampling to make inferences about nutrient routing
within the animal. The 13C/12C ratio of exhaled CO2
provides a good indication of the 13C/12C in an animal’s
diet, can indicate changes in diet over time, either
through repeatedly capturing and taking breath samples from an individual over time and, if the possible
diets differ spatially, changes in diet can also suggest
animal movement and behavior.
We have also demonstrated that it is possible to determine the ratio of lipid to carbohydrate metabolism
in both flying and resting pigeons. However, because
of differences in diet selection, it was necessary to use
the pigeons as their own controls. There may be situations in the field where it is possible to use animals
as their own controls too, such as marking and recapturing resident birds or birds at a stopover site. One
may not need to repeatedly sample the same animal to
make inferences of substrate metabolism (endogenous
vs. exogenous, lipids vs. carbohydrates) if one is sure
the animal’s diet is very consistent. However, this
method should be further developed so that it is useful
in a variety of field conditions.
It should also be possible to capture an animal and
feed it a diet that will label its tissues differently from
APPLYING
13
C/12C BREATH ANALYSIS
TABLE 4. 13C/12C ratios in blood macronutrients and breath CO2 in
fasted rats (SD, ‰).
Sample
Blood protein
Blood glucose
Blood lipid
Breath CO2
d13C after 4
hr fast
220.1
217.6
224.9
219.7
(0.2)
(0.9)
(0.1)
(0.7)
d13C after 48
hr fast
220.1
218.5
223.9
223.3
(0.2)
(1.4)
(0.3)
(0.4)
* From Schoeller et al., 1984.
its natural diet. Then one could release the animal,
recapture it, and from the 13C/12C ratio of the breath
determine the ratio of natural food to endogenous
stores that the animal is burning. One could also isotopically label a single component of the animal’s endogenous energy stores, the protein component for example. The animal can then be released and later recaptured. Breath samples can then be taken to determine the contribution of the labeled component to the
animal’s metabolism. Of course, the animal must be
held and fed a labeled diet long enough that the label
shows up in its tissues. The animal must also be recaptured in a relatively short period of time, before the
label is lost. This probably means that a radio transmitter needs to be attached to the animal as well.
More promising is the possibility of combining the
13C/12C analysis of exhaled CO with the 13C/12C anal2
ysis of macronutrients in the blood plasma. Schoeller
et al. (1984) fasted rats for four hours, at which point
they took breath and blood samples. They found 13C/
12C ratio of the breath to be intermediate between that
of blood lipids and blood glucose. They then took
breath and blood samples from the rats after the rats
had fasted for 48 hr. They found that the 13C/12C of
the breath was nearly identical to that of the blood
lipids, indicating that the rats had shifted to metabolizing lipids for energy (Table 4).
Similarly, one may be able to capture an animal take
a breath and blood sample and analyze these for their
13C/12C ratios. From these one should be able to determine the ratio of lipids to carbohydrates that the
animal is burning for energy. One can then fast the
animal, take a second breath and blood sample and
analyze the 13C/12C ratios. By comparing these with
the first breath and blood samples, one should be able
to determine whether the lipids and carbohydrates metabolized to CO2 in the first sample came from diet or
endogenous stores, since the breath CO2 and blood metabolites in the second sample must come from endogenous stores.
We listed several 13C breath tests developed as noninvasive clinical tests for the metabolism of a specific
substrate. Some of these may be adaptable for use in
the field and some may not. They all, however, should
be of interest to physiological ecologists interested in
the ability of animals to metabolize a specific substrate. We hope that physiological ecologists will become more aware of the applications of stable isotope
analysis for physiological and nutritional investiga-
31
tions that are found in the medical literature. We stress
that these need to be validated in the laboratory for
use with the particular study animal.
This brings us to probably the most important future
direction for physiological ecologists with regards to
stable isotope technology. Many of the current uses by
animal ecologists of stable isotopes have not been experimentally validated in the laboratory. Their assumptions are based on correlations observed in the
field. We repeat the call of Gannes et al. (1997) for
experimental validation of present and future techniques using stable isotopes.
CONCLUSIONS
The analysis of the 13C/12C ratios in exhaled CO2
has many advantages over the traditional tissue analysis. It is non-invasive and non-destructive. Samples
can be taken from the same individual repeatedly.
Sampling is simple and quick. It does not require that
the animal be killed, nor does it require the dissection
and preparation that tissue sampling does. Carbon isotope ratio of the breath is, in a fed animal, representative of the diet as a whole (Tieszen and Fagre, 1993).
More specifically, it is representative of that portion of
the diet being metabolized for energy. It should therefore be possible to combine breath analysis with tissue
analysis to determine which dietary components are
used for energy and which are used for tissue synthesis. We have shown that breath samples taken before
and after a fast can reveal dietary history over time.
We have also shown that it is possible to estimate the
relative energetic contribution of exogenous carbohydrate to endogenous lipids in resting and flying birds.
There is great potential for the use of 13C/12C breath
analysis in the field as well as in the laboratory. We
hope that this technique will be used more extensively
and developed for further applications in the future.
ACKNOWLEDGMENTS
We extend special thanks to Peter Thomson and
Jane McLaren for technical assistance with the mass
spectrometry and thanks to C. Richard Tracy, Christopher Gienger, Joan Wright, Ken Nussear, Eric Simandle, Micheal Sears, Denise Jones and Szabolcs
Lengyel for their comments and suggestions. During
the study KH was supported by a Fulbright Post-doctoral Fellowship through the United States–Israel Educational Foundation. This study was supported by
United States–Israel Binational Science Foundation
grant number 93-00232. This is paper number 341 of
the Mitrani Department for Desert Ecology.
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