158
Marine Mammal Physiology: Requisites for Ocean Living
Worthy et al. 1992; Crocker et al. 1998; Houser et al. 2001) that helped establish the foundation for its use in other labs studying similar interests in marine mammals (i.e., Ortiz et al.
1999; Champagne et al. 2005; Houser et al. 2007).
In theory, the application of the technique is relatively easy now that it has been
well established. The biggest challenges are the cost of the isotopes, especially deuterium and 18 O (if studying energetics by doubly labeled water) and, if the animals are
very large, handling the animals to obtain blood or tissue for subsequent analyses. In
practicum, the dilution, or equilibration, of the isotope with the body’s total water pool,
or compartment, provides a relatively accurate estimate of the actual TBW pool. In general, an initial blood sample is taken prior to administration of the isotope to determine
background levels of the isotope being used since many of the isotopes are naturally
present in the animal’s environment. If background levels are detected, they should be
subtracted from the subsequent levels measured post-dosing. Animals then receive a
mass-specific dose of the isotope, thus weighing the animals prior to dosing is necessary
and can provide an additional challenge depending on size and temperament of the
animal. For these reasons, sedation of the animal, especially with most marine mammals is necessary, and thus, can induce additional expenses. To ensure accuracy of the
method, the vehicle used for dosing should be pre-weighed to the greatest degree of
accuracy of the scale and again after dosing to accurately calculate the actual amount of
isotope infused. Furthermore, route of dosing (i.e., gavage or intravenous) should also
be flushed with sterile saline to further ensure complete administration of the isotope.
After dosing of the isotope, subsequent blood (or urine) samples are obtained at specific
and well-defined intervals to develop a dilution (or disappearance) curve. If using an
isotope of hydrogen in the form of water, the labeled water must then be extracted from
the blood samples (i.e., as demonstrated in Ortiz et al. 1978) for later quantification of
the isotope. The labeled water is lyophilized from the whole blood samples and a freeze
trap method is common (Ortiz et al. 1978; Byers 1979). The labeled water is now ready
for measurement (i.e., D 2 O can be measured by infrared analyzer or 3 H by scintillation).
Once the values from each sample at each time point have been obtained, the instantaneous dilution space (IDS) of the isotope is calculated as the y-intercept (T 0 ) from a multipoint (depending on the number of post-dosing samples) regression of natural log of the
isotope concentration versus time (Ortiz et al. 1978; Byers and Schelling 1986; Ortiz et al.
1999). Isotopic dilution space (usually in liters) is calculated as: IDS = D/CF∗[T 0 ], where
D is the administered dose of isotope, CF is the correction factor for the difference in the
mass of unlabeled water versus that of the labeled water (i.e., 1.105 is the correction for
the density of D 2 O versus unlabeled H 2 O), and [T 0 ] is the concentration of the isotope at
time zero, or the IDS (Byers and Schelling 1986; Ortiz et al. 1999). The IDS provides an
estimate of TBW. Water turnover rate (r H O
2 ) can then be estimated from the product of
TBW and the slope (K) of the multi-point regression constructed from the dilution curve.
In cases where animals lose body mass during the study period, r H O
2
can be estimated
using the equations presented by Nagy and Costa (1980) that account for changes in the
size of the water pool since TBW scales with body mass. The isotopic half time (t 1/2 ) of the
isotope in the body pool is calculated as 0.693/K, where 0.693 is a constant.
While a vast majority of tracer studies in marine mammals assess the dilution of a
single- or dual-label isotope technique, isotopic analysis by nuclear magnetic resonance
(NMR) provides a very important and useful alternative approach. The cost and availability of an NMR may be problematic and challenging its use has been successfully deployed
in the study of glucose metabolism in elephant seal pups (Champagne et al. 2012). Spectral
isotopic analysis by NMR allows for the simultaneous determination of the enrichment of
Marine Mammal Physiology: Requisites for Ocean Living
Worthy et al. 1992; Crocker et al. 1998; Houser et al. 2001) that helped establish the foundation for its use in other labs studying similar interests in marine mammals (i.e., Ortiz et al.
1999; Champagne et al. 2005; Houser et al. 2007).
In theory, the application of the technique is relatively easy now that it has been
well established. The biggest challenges are the cost of the isotopes, especially deuterium and 18 O (if studying energetics by doubly labeled water) and, if the animals are
very large, handling the animals to obtain blood or tissue for subsequent analyses. In
practicum, the dilution, or equilibration, of the isotope with the body’s total water pool,
or compartment, provides a relatively accurate estimate of the actual TBW pool. In general, an initial blood sample is taken prior to administration of the isotope to determine
background levels of the isotope being used since many of the isotopes are naturally
present in the animal’s environment. If background levels are detected, they should be
subtracted from the subsequent levels measured post-dosing. Animals then receive a
mass-specific dose of the isotope, thus weighing the animals prior to dosing is necessary
and can provide an additional challenge depending on size and temperament of the
animal. For these reasons, sedation of the animal, especially with most marine mammals is necessary, and thus, can induce additional expenses. To ensure accuracy of the
method, the vehicle used for dosing should be pre-weighed to the greatest degree of
accuracy of the scale and again after dosing to accurately calculate the actual amount of
isotope infused. Furthermore, route of dosing (i.e., gavage or intravenous) should also
be flushed with sterile saline to further ensure complete administration of the isotope.
After dosing of the isotope, subsequent blood (or urine) samples are obtained at specific
and well-defined intervals to develop a dilution (or disappearance) curve. If using an
isotope of hydrogen in the form of water, the labeled water must then be extracted from
the blood samples (i.e., as demonstrated in Ortiz et al. 1978) for later quantification of
the isotope. The labeled water is lyophilized from the whole blood samples and a freeze
trap method is common (Ortiz et al. 1978; Byers 1979). The labeled water is now ready
for measurement (i.e., D 2 O can be measured by infrared analyzer or 3 H by scintillation).
Once the values from each sample at each time point have been obtained, the instantaneous dilution space (IDS) of the isotope is calculated as the y-intercept (T 0 ) from a multipoint (depending on the number of post-dosing samples) regression of natural log of the
isotope concentration versus time (Ortiz et al. 1978; Byers and Schelling 1986; Ortiz et al.
1999). Isotopic dilution space (usually in liters) is calculated as: IDS = D/CF∗[T 0 ], where
D is the administered dose of isotope, CF is the correction factor for the difference in the
mass of unlabeled water versus that of the labeled water (i.e., 1.105 is the correction for
the density of D 2 O versus unlabeled H 2 O), and [T 0 ] is the concentration of the isotope at
time zero, or the IDS (Byers and Schelling 1986; Ortiz et al. 1999). The IDS provides an
estimate of TBW. Water turnover rate (r H O
2 ) can then be estimated from the product of
TBW and the slope (K) of the multi-point regression constructed from the dilution curve.
In cases where animals lose body mass during the study period, r H O
2
can be estimated
using the equations presented by Nagy and Costa (1980) that account for changes in the
size of the water pool since TBW scales with body mass. The isotopic half time (t 1/2 ) of the
isotope in the body pool is calculated as 0.693/K, where 0.693 is a constant.
While a vast majority of tracer studies in marine mammals assess the dilution of a
single- or dual-label isotope technique, isotopic analysis by nuclear magnetic resonance
(NMR) provides a very important and useful alternative approach. The cost and availability of an NMR may be problematic and challenging its use has been successfully deployed
in the study of glucose metabolism in elephant seal pups (Champagne et al. 2012). Spectral
isotopic analysis by NMR allows for the simultaneous determination of the enrichment of