This work represents the first comprehensive report on quantitative metabolomic composition of tissues of pike-perch (Sander lucioperca) and Siberian roach (Rutilus rutilus lacustris). The total of 68 most abundant metabolites are identified and quantified in the fish lenses and gills by the combination of LC-MS and NMR. It is shown that the concentrations of some compounds in the lens are much higher than that in the gills; that indicates the importance of these metabolites for the adaptation to the specific living conditions and maintaining the homeostasis of the fish lens. The lens metabolome undergoes significant seasonal changes due to the variations of dissolved oxygen level and fish feeding activity. The most season-affected metabolites are osmolytes and antioxidants, and the most affected metabolic pathway is the histidine pathway. In late autumn, the major lens osmolytes are N-acetyl-histidine and threonine phosphoethanolamine (Thr-PETA), while in winter the highest concentrations were observed for serine phosphoethanolamine (Ser-PETA) and myo-inositol. The presence of Thr-PETA and Ser-PETA in fish tissues and their role in cell osmotic protection are reported for the first time. The obtained concentrations can be used as baseline levels for studying the influence of environmental factors on fish health.
Tissue protection from oxidative stress by antioxidants is of vital importance for cellular metabolism. The lens mostly consists of fiber cells lacking nuclei and organelles, having minimal metabolic activity; therefore, the defense of the lens tissue from the oxidative stress strongly relies on metabolites. Protein-free extracts from lenses and gills of freshwater fish, Sander lucioperca and Rutilus rutilus lacustris, were subjected to analysis using high-field 1H NMR spectroscopy and HPLC with optical and high-resolution mass spectrometric detection. It was found that the eye lenses of freshwater fish contain high concentrations of ovothiol A (OSH), i.e., one of the most powerful antioxidants exciting in nature. OSH was identified and quantified in millimolar concentrations. The concentration of OSH in the lens and gills depends on the fish genus and on the season. A possible mechanism of the reactive oxygen species deactivation in fish lenses is discussed. This work is the first to report on the presence of OSH in vertebrates. The presence of ovothiol in the fish tissue implies that it may be a significantly more common antioxidant in freshwater and marine animals than was previously thought.
the Siberian wood frog Rana amurensis is a recently discovered example of extreme hypoxia tolerance that is able to survive several months without oxygen. We studied metabolomic profiles of heart and liver of R. amurensis exposed to 17 days of extreme hypoxia. Without oxygen, the studied tissues experience considerable stress with a drastic decrease of ATP, phosphocreatine, and NAD+ concentrations, and concomitant increase of AMP, creatine, and NADH. Heart and liver switch to different pathways of glycolysis with differential accumulation of lactate, alanine, succinate, as well as 2,3-butanediol (previously not reported for vertebrates as an end product of glycolysis) and depletion of aspartate. We also observed statistically significant changes in concentrations of certain osmolytes and choline-related compounds. Low succinate/fumarate ratio and high glutathione levels indicate adaptations to reoxygenation stress. Our data suggest that maintenance of the ATP/ADP pool is not required for survival of R. amurensis, in contrast to anoxia-tolerant turtles. Anoxia is a huge stress for the majority of vertebrates. Amphibians are considered to be relatively anoxia-intolerant, in contrast to a few turtle species and certain fish species 1-3 : the most resistant species are known to survive anoxia for a few days at most. However, a recent study 4 proved that the Siberian wood frog Rana amurensis can tolerate almost complete anoxia at 2-3 °C (below 0.2 mg/L oxygen, i.e., less than 1.5% of the normal concentration at this temperature) for several months. This is on par with the best vertebrate models from other classes. Moreover, while red-eared slider turtles, the most studied anoxia-tolerant tetrapods, are dormant under anoxia, the Siberian wood frog is able to react to stimuli, e.g., to flee when disturbed. R. amurensis is thus a unique model of extreme hypoxia tolerance among terrestrial vertebrates. The natural range of R. amurensis includes Northeastern China and Siberia from the Urals to the coast of the Okhotsk sea; its northern distribution is limited by about 71° N. It overwinters under ice in water bodies. Many of these lakes are shallow (< 3 m) due to thermokarst origin, i.e. resulting from local melting of permafrost. Experimental studies 4 demonstrated that during the winter oxygen is depleted in overwintering sites till almost complete anoxia. Siberian wood frogs overwinter for 6 to 7 months in different regions under hypoxia until they can leave the lakes in April-early May. The mechanisms of adaptation to the lack of oxygen are totally unknown for R. amurensis. Here we made an attempt to study this phenomenon using 1 H-NMR-based quantitative metabolomics. This method simultaneously yields concentrations of multiple metabolites 5. At the present, the detailed quantitative metabolomic composition is known for many human tissues, but for very few animals 6-8. To the best of our knowledge, this is the first report on the quantitative metabolomic analysis of amphibian tissues. We determined concentrations of ove...
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