157
Chapter seven: Water balance
electrolyte responses and changes in water flux rates to exogenous manipulations.
Descriptions of the osmoregulatory mechanisms of marine mammals were based on
inferences from these comparative studies and our existing knowledge of these mechanisms gleaned from detailed studies in humans and common laboratory models. These
studies that measured primarily plasma and urine concentrations of variables used
commonly employed techniques and methodologies such as antibody-based (radioimmunoassays followed by enzyme-linked immunosolvent assay) and photometric
methods. Furthermore, early histological analyses of the kidneys of marine mammals
employed traditional methods commonly used in other vertebrates. The advent of technological advancements has provided the use of time-resolved fluorescent immunoassay (TR-FIA), molecular techniques such as qRT-PCR and Western blotting, kinetic
enzyme activity assays, and immunohistochemistry. While none of these techniques
and methods is considered especially advanced in today’s laboratory, obtaining fresh
or in vivo/in situ renal tissue or gaining access to the kidney to make real-time renal
functional studies is extremely challenging. Therefore, assessing the renal responses
as measured by changes in urinary variables to exogenous perturbations of water and
electrolytes in addition to the quantification of expressions of renal genes and proteins
(most likely from postmortem samples) remain principal methodologies to further
e lucidate the renal mechanisms. However, inferences from basic renal studies in more
traditional mammalian models will continue to be important to help interpret discoveries made in marine mammals.
7.3.2 Isotopic dilution
The use of isotopic dilution to estimate total body water (TBW) pool size, water turnover rates
(r H O
2
), and energetics has been applied across a broad range of animals and has provided
a significant tool for the study of water and energy metabolism (kinetics) (Hevesy and
Hofer 1934; Lifson and McClintock 1966; Lifson et al. 1997; Nagy and Costa 1980; Schoeller
et al. 1986; Speakman 1997; Ortiz et al. 1999). While this technique requires the acceptance
of a number of assumptions and the validation for its specific application, and has some
degree of error of estimation (Lifson and McClintock 1966; Culebras and Moore 1977; Nagy
and Costa 1980; Wong et al. 1987; Speakman et al. 1993; Schoeller and Hnilicka 1996), it
continues to be well received and widely used technique. The measurement of TBW is
based on the fact that both of the hydrogen atoms of H 2 O are completely exchangeable
with isotopic H 2 O labeled with either deuterium (D or 2 H) or tritium ( 3 H) (Culebras and
Moore 1977). While deuterium is a stable, non-radioactive isotope of hydrogen ( 1 H), 3 H is
radioactive; regardless, both behave exactly as the hydrogens of water, and thus, serve as
ideal tracers for studying the kinetics of water within the body. The excellent work of the
earlier investigators (H. Hevesy, E. Hofer, N. Lifson, R. McClintock, F.D. Moore, K.A. Nagy,
D.A. Schoeller, J.R. Speakman, W.W. Wong, and others) helped establish the framework
for applying this technique in marine mammals. The first application of isotopic dilution
to measure TBW and r H O
2
was performed in fasting northern elephant seal pups by Ortiz
et al. (1978). While the use of 24 Na in delphinids was reported in 1970, this technique was
not performed in the animals directly but rather added to their tank water to track the
appearance of Na + in the animals after 24 h (Telfer et al. 1970). Nonetheless, this work
provided a useful and alternative application of isotopic dilution for studying water flux
in a group of marine mammals. Soon after the work of Ortiz et al. (1978), a number of
papers employing the use of isotopic dilution to measure water kinetics and metabolism
emanated from the team at the University of California, Santa Cruz (Costa et al. 1986, 1989;
Chapter seven: Water balance
electrolyte responses and changes in water flux rates to exogenous manipulations.
Descriptions of the osmoregulatory mechanisms of marine mammals were based on
inferences from these comparative studies and our existing knowledge of these mechanisms gleaned from detailed studies in humans and common laboratory models. These
studies that measured primarily plasma and urine concentrations of variables used
commonly employed techniques and methodologies such as antibody-based (radioimmunoassays followed by enzyme-linked immunosolvent assay) and photometric
methods. Furthermore, early histological analyses of the kidneys of marine mammals
employed traditional methods commonly used in other vertebrates. The advent of technological advancements has provided the use of time-resolved fluorescent immunoassay (TR-FIA), molecular techniques such as qRT-PCR and Western blotting, kinetic
enzyme activity assays, and immunohistochemistry. While none of these techniques
and methods is considered especially advanced in today’s laboratory, obtaining fresh
or in vivo/in situ renal tissue or gaining access to the kidney to make real-time renal
functional studies is extremely challenging. Therefore, assessing the renal responses
as measured by changes in urinary variables to exogenous perturbations of water and
electrolytes in addition to the quantification of expressions of renal genes and proteins
(most likely from postmortem samples) remain principal methodologies to further
e lucidate the renal mechanisms. However, inferences from basic renal studies in more
traditional mammalian models will continue to be important to help interpret discoveries made in marine mammals.
7.3.2 Isotopic dilution
The use of isotopic dilution to estimate total body water (TBW) pool size, water turnover rates
(r H O
2
), and energetics has been applied across a broad range of animals and has provided
a significant tool for the study of water and energy metabolism (kinetics) (Hevesy and
Hofer 1934; Lifson and McClintock 1966; Lifson et al. 1997; Nagy and Costa 1980; Schoeller
et al. 1986; Speakman 1997; Ortiz et al. 1999). While this technique requires the acceptance
of a number of assumptions and the validation for its specific application, and has some
degree of error of estimation (Lifson and McClintock 1966; Culebras and Moore 1977; Nagy
and Costa 1980; Wong et al. 1987; Speakman et al. 1993; Schoeller and Hnilicka 1996), it
continues to be well received and widely used technique. The measurement of TBW is
based on the fact that both of the hydrogen atoms of H 2 O are completely exchangeable
with isotopic H 2 O labeled with either deuterium (D or 2 H) or tritium ( 3 H) (Culebras and
Moore 1977). While deuterium is a stable, non-radioactive isotope of hydrogen ( 1 H), 3 H is
radioactive; regardless, both behave exactly as the hydrogens of water, and thus, serve as
ideal tracers for studying the kinetics of water within the body. The excellent work of the
earlier investigators (H. Hevesy, E. Hofer, N. Lifson, R. McClintock, F.D. Moore, K.A. Nagy,
D.A. Schoeller, J.R. Speakman, W.W. Wong, and others) helped establish the framework
for applying this technique in marine mammals. The first application of isotopic dilution
to measure TBW and r H O
2
was performed in fasting northern elephant seal pups by Ortiz
et al. (1978). While the use of 24 Na in delphinids was reported in 1970, this technique was
not performed in the animals directly but rather added to their tank water to track the
appearance of Na + in the animals after 24 h (Telfer et al. 1970). Nonetheless, this work
provided a useful and alternative application of isotopic dilution for studying water flux
in a group of marine mammals. Soon after the work of Ortiz et al. (1978), a number of
papers employing the use of isotopic dilution to measure water kinetics and metabolism
emanated from the team at the University of California, Santa Cruz (Costa et al. 1986, 1989;