To our knowledge, this is the first study to comprehensively analyze and compare plasma amino acid concentrations in multiple cetaceans. Comparisons of amino acid profiles will lead to a better understanding of the physiological role of their biochemical effects, and such effects may allow marine mammals to live entirely underwater. These data may also be useful indicators for assessing cetacean health conditions in the future. Since the aim of this study was to unveil the characteristic amino acid metabolism observed in cetaceans, compared with representative terrestrial animals, we chose mice as one of the most intensively investigated terrestrial model animals. Mice and rats are known to have similar amino acid metabolism, and both of them are frequently used as model animals to investigate amino acid metabolism 
Plasma amino acids can be viewed as a network that adapts to various physiological conditions and that may become perturbed in response to disease and/or physiological insults 
. In addition to the metabolic pathways within cells, there are various levels of networks within the body; one such level is represented by the transport of substrates such as amino acids between organ systems via the bloodstream. Our results () suggest that cluster analysis can be used to visualize a multitude of amino acid relationships among different species, which can help us understand the complex interrelations underlying metabolic adaptations to the environment. As shown in , aminograms of bottlenose dolphins, pacific white-sided dolphins, Risso's dolphins and false-killer whales formed a cetacean cluster that was markedly different from the cluster obtained from C57BL/6J and ICR mice, indicating that the physiological states of the cetaceans and the mice were markedly different. Among the 28 amino acids that we examined in this study, characteristic differences between the two clusters were found for ornithine, arginine, citrulline, threonine, lysine, serine, aspartate and glutamate (which were lower in cetaceans), and cystathionine, 3-MH, carnosine, urea and 1-MH (which were increased in cetaceans).
The most distinctive differences between cetaceans and mice were found in the concentrations of plasma 3-MH and carnosine (). Plasma levels of 3-MH in the cetaceans were 50 to 100 times greater than those in the terrestrial mammals. Most 3-MH is formed by the post-translational methylation of specific histidine residues in the myofibrillar proteins actin and myosin, and 3-MH cannot be reused for protein synthesis. In many terrestrial mammals, 3-MH does not undergo catabolism and is primarily excreted in the urine 
. However, since the urinary 3-MH levels of cetaceans were significantly lower than those of terrestrial mammals () 
, whereas plasma levels were significantly higher, it is likely that cetaceans reabsorb 3-MH. Some animals such as pigs, which have balenine in their skeletal muscle, reabsorb 3-MH for synthesizing balenine 
. Cetaceans might also reabsorb 3-MH for synthesis of balenine, and the further analyses whether balenine level in the cetacean skeletal muscle were upregulated or not will provide the physiological meaning of 3-MH reabsorption. At least, 3-MH is known to function as an endogenous antioxidant 
, and thus, these high concentrations of 3-MH in the plasma appear to play an important role in cetacean physiology. Although carnosine has not been detected in human 
or mouse plasma (), it was enriched in cetacean plasma. Also, carnosine is a naturally occurring antioxidant 
and transition metal ion-sequestering agent. It has been shown to act as an anti-glycating agent as well, inhibiting the formation of advanced glycation end products. Through its distinctive combination of antioxidant and anti-glycating properties, carnosine can attenuate cellular oxidative stress and inhibit the intracellular formation of ROS and reactive nitrogen species. As noted by Reddy et al. 
, “by controlling oxidative stress, suppressing glycation, and chelating metal ions, carnosine is able to reduce harmful sequelae such as DNA damage.” This has been supported by several studies 
. Carnosine and carnosine-related antioxidants have therefore recently attracted much attention as possible therapeutic agents for humans 
. Since anserine, which is carnosine-related metabolite, was not detected, it was likely that the metabolic system to uniquely upregulate carnosine level exist in cetaceans. Because diving mammals must cope with high rates of ROS generation due to alterations in apnea/reoxygenation and ischemia-reperfusion processes 
, high concentrations of these amino acids in the plasma could play an important antioxidant role and would be beneficial for aquatic life. Higher levels of urine carnosine in cetaceans than in mice () suggest the synthesis rate of carnosine might be enhanced in marine mammals.
Because characteristic differences were also found in urea and urea cycle-related amino acids, we performed a correlation analysis focusing on urea cycle-related amino acids ( and ). The urea cycle plays a major role in the generation of urea using amino acids derived from either dietary proteins or endogenous origins via conversion of deamination-derived ammonia in the liver. In the urea cycle, amino acids enter the pathway for urea synthesis via the transdeamination or transamination routes. In either route, α-ketoglutarate accepts an amino group from the donor amino acid to form glutamate. In the former pathway, glutamate is deaminated to form α-ketoglutarate and ammonium ions, and then the ammonium is incorporated into carbamoyl phosphate, which in turn reacts with ornithine to enter the urea cycle as citrulline. In the latter pathway, oxaloacetate accepts an amino group from glutamate to form aspartate. This aspartate now carries a second amino group into the urea cycle by condensing with citrulline to form argininosuccinate. Argininosuccinate is then cleaved to form fumarate and arginine. Finally, arginine is hydrolyzed to ornithine and urea 
. As shown in and , cetacean plasma levels of aspartate, glutamate, ornithine, arginine and citrulline, which are utilized to synthesize urea via the urea cycle, were much lower than those of mice, whereas the level of plasma urea itself was much higher in cetaceans. When all the proteinogenic amino acids are compared (), their plot distributions are seen to be less divergent between mice and cetaceans than those of the urea cycle-related amino acids (). This indicates that, in cetaceans, the urea and urea cycle-related amino acids are at unique homeostatic set points. Urea functions as a major blood osmolyte 
, and in some animals, blood osmotic pressure is primarily elevated through the retention of urea 
. For example, plasma urea levels are high in sharks; in a hyperosmotic environment, this adaptation prevents the loss of body water 
. It is possible that maintenance of a higher level of plasma urea, i.e., by converting urea cycle-related amino acids to urea by adjusting the set point of the urea cycle, would be advantageous for marine mammals. Such a metabolic adaptation might facilitate body water conservation in the hyperosmotic sea water environment.
Whereas our study provides the levels of amino acids in the plasma and urine of various cetaceans, variations in plasma amino acid levels due to species, sex, age, season, living situation and health have not yet been determined. Hematology, clinical blood chemistry profiles and behavioral indices are routinely used in marine mammals and in many terrestrial mammals to monitor health status. However, Waples and Gales 
reported a case of fatality of a bottlenose dolphin without associated changes in hematology or clinical blood chemistry profiles, suggesting that other physiological tests should be incorporated into the management techniques. Because plasma aminograms are used to distinguish abnormal subjects from healthy subjects in humans 
, it is likely that accumulated plasma and/or urine aminogram data will provide useful indicators for the assessment of cetacean health in artificial environments.
In conclusion, our results indicate that cetaceans and terrestrial mammals have different metabolic machinery for amino acids that facilitates adaptation to the ocean environment. To fully understand the metabolic adaptations of marine mammals required for a lifetime spent entirely underwater, further research is necessary regarding the distribution of amino acids in other marine mammals, such as Pinnipedia, which live both underwater and on land. The evolutionary closer terrestrial animals such as Artiodactyla will also be needed to be investigated in future. Network analyses of plasma aminograms in those animals will shed light on the origins of the unique homeostatic regulation of 3-MH, carnosine, urea and urea cycle-related amino acid levels observed in cetaceans.