Accepted_test

The Siberian wood frog Rana amurensis Boulenger, 1886 is a unique amphibian capable of surviving several months in water with very low oxygen content (up to 0.2 mg/L). Our aim was to study metabolomic changes of this species at the onset of hypoxia (1 day), as well as in 1 hour of reoxygenation following long-term hypoxia exposure using ¹H nuclear magnetic resonance (NMR). Organs (liver, heart, and brain) were extracted as quickly as possible, and quantitative metabolomic analysis was performed using ¹H NMR. The results were compared to the samples kept under normal oxygen content, as well as those exposed to 30 days of hypoxia. While succinate in most studied vertebrates accumulates under hypoxia and is believed to undergo rapid conversion upon oxygen restoration, our study revealed a decrease in succinate in the brain in reoxygenation, while it remained unchanged in the liver. This observation suggests the existence of a mechanism that inhibits succinate conversion. Furthermore, we observed intriguing disparities concerning two substances with unclear functions: glycerol and 2,3-butanediol. Glycerol exhibited rapid accumulation during hypoxia and equally swift processing during reoxygenation. In contrast, 2,3-butanediol required an extended period to accumulate, yet persisted after reoxygenation. Of the identified metabolites pool, only a few molecules exhibited continuous dynamics with rapid accumulation or decrease in response to hypoxia and subsequent reversal upon reoxygenation. Certain substances grouped together into unit with similar patterns. To gain a comprehensive understanding of specific substances, additional time points are necessary.