The Siberian salamander Salamandrella keyserlingii Dybowski, 1870 is a unique amphibian that is capable to survive long-term freezing at -55 °C. Nothing is known on the biochemical basis of this remarkable freezing tolerance, except for the fact that it uses
glycerol as a low molecular weight cryoprotectant. We used 1H-NMR analysis to study quantitative changes of multiple metabolites in liver and hindlimb muscle of S. keyserlingii in response to freezing. For the majority of molecules we observed significant changes in concentrations.
Glycerol content in frozen organs was as high
as 2% w/w, which confirms its role as a cryoprotectant. No other putative cryoprotectants were detected. Freezing resulted in
ischemia manifested as increased concentrations of glycolysis products:
lactate and
alanine. Unexpectedly, we detected no increase in concentrations of
succinate, which accumulates under
ischemia in various tetrapods. Freezing proved to be a dramatic stress with reduced
adenosine phosphate pool and high levels of
nucleotide degradation products (
hypoxanthine, β-
alanine, and β-aminoisobutyrate). There was also significant increase in the concentrations of
choline and glycerophosphocholine, which may be interpreted as the degradation of biomembranes. Thus, we found that freezing results not only in macroscopical damage due to
ice formation, but also to degradation of
DNA and biomembranes.