9423920 Raymond Freezing resistance in polar fishes is usually attributed to noncolligative peptide antifreezes. In 1991, it was found that some arctic fishes increase the freezing point depression of their body fluids by producing high concentrations of glycerol in winter. Subsequent studies of this phenomenon have led to a realization that other inorganic ions and organic osmolytes have important roles in freezing resistance in many polar fishes, and possibly other roles as well. Prominent among these solutes are trimethylamine oxide (TMAO) and urea, which are more widely distributed in polar fishes than glycerol. These studies have lead to several unusual findings concerning the synthesis, roles, conservation and distribution of these osmolytes. Therefope, the overall objective of this study is to better understand the biochemistry and physiology of glycerol, TMAO and urea in arctic fishes. Previous studies have shown that significant glycerol losses occur in smelt and that these losses are compensated for by a synthetic pathway (via pyruvate) that appears to differ from that in other cold-hardy, glycerol-producing animals. New studies will focus on various amino acid substrates that may be involved in this pathway, and the seasonal variation in activity of three key enzymes that are also thought to be involved. The unexpected occurrence of high serum TMAO levels in many arctic fishes also raises several questions. TMAO is usually acquired by teleosts through the diet, but in the smelt family, it appears to be synthesized in response to cold. TMAO synthesis will be tested by an assay for TMAO oxidase in warm and cold-acclimated fishes. Inorganic ion concentrations in polar fishes are high enough to interfere with intracellular processes, such as enzyme activities. The possibility that TMAO may partially counteract this salt inhibition--for which there is some preliminary evidence--will be investigated. A possible role of TMAO in reducing epithelial membrane permeabilities to low molecular weight osmolytes will also be investigated. Preliminary evidence that TMAO itself is conserved, rather than excreted as it is in most fishes, will be further tested in smelt and other arctic species. Conservation of TMAO would provide further evidence that it has a physiological role. Urea synthesis in teleosts is normally accomplished through the purine pathway, but in fishes with high urea levels (including a few teleosts), it can occur by the ornithine cycle. Both pathways will be assayed in the smelt to determine if their high urea levels are also the result of an ornithine cycle.
