Metabolomic Profiling Reveals Differences in Hypoxia Response between Far Eastern and Siberian Frogs.

Anoxia is a significant challenge for most animals, as it can lead to tissue damage and death. Among amphibians, the Siberian frog Rana amurensis is the only known species capable of surviving near-zero levels of oxygen in water for a prolonged period. In this study, we aimed to compare metabolomic profiles of the liver, brain, and heart of the Siberian frog exposed to long-term oxygen deprivation (approximately 0.2 mg/L water) with those of the susceptible Far Eastern frog (Rana dybowskii) subjected to short-term hypoxia to the limits of its tolerance. One of the most pronounced features was that the organs of the Far Eastern frog contained more lactate than those of the Siberian frog despite a much shorter exposure time. The amounts of succinate were similar between the two species. Interestingly, glycerol and 2,3-butanediol were found to be significantly accumulated under hypoxia in the Siberian frog, but not in the Far Eastern frog. The role and biosynthesis of these substances are still unclear, but they are most likely formed in certain side pathways of glycolysis. Based on the obtained data, we suggest a pathway for metabolic changes in the Siberian frog under anoxia.

Animal Metabolite Database: Metabolite Concentrations in Animal Tissues and Convenient Comparison of Quantitative Metabolomic Data.

The Animal Metabolite Database (AMDB, https://amdb.online) is a freely accessible database with built-in statistical analysis tools, allowing one to browse and compare quantitative metabolomics data and raw NMR and MS data, as well as sample metadata, with a focus on the metabolite concentrations rather than on the raw data itself. AMDB also functions as a platform for the metabolomics community, providing convenient deposition and exchange of quantitative metabolomic data. To date, the majority of the data in AMDB relate to the metabolite content of the eye lens and blood of vertebrates, primarily wild species from Siberia, Russia and laboratory rodents. However, data on other tissues (muscle, heart, liver, brain, and more) are also present, and the list of species and tissues is constantly growing. Typically, every sample in AMDB contains concentrations of 60-90 of the most abundant metabolites, provided in nanomoles per gram of wet tissue weight (nmol/g). We believe that AMDB will become a widely used tool in the community, as typical metabolite baseline concentrations in tissues of animal models will aid in a wide variety of fundamental and applied scientific fields, including, but not limited to, animal modeling of human diseases, assessment of medical formulations, and evolutionary and environmental studies.

Biochemical response of two earthworm taxa exposed to freezing.

Several earthworm species are known to be able to withstand freezing. At the biochemical level, this ability is based on cryoprotectant accumulation as well as several other mechanisms. In this study, we used 1H NMR to investigate metabolomic changes in two freeze-tolerant earthworm taxa, Dendrobaena octaedra and one of the genetic lineages of Eisenia sp. aff. nordenskioldi f. pallida. A total of 45 metabolites were quantified. High concentrations of glucose were present in frozen tissues of both taxa. No other putative cryoprotectants were found. We detected high levels of glycolysis end products and succinate in frozen animals, indicating the activation of glycolysis. Concentrations of many other substances also significantly increased. On the whole, metabolic change in response to freezing was much more pronounced in the specimens of Eisenia sp. aff. nordenskioldi f. pallida, including signs of nucleotide degradation.

Influence of Ecological Factors on the Metabolomic Composition of Fish Lenses.

Multiple stressors related to changes in environmental conditions (such as water temperature, salinity, and natural and anthropogenic pollution) may cause biological responses of aquatic organisms that lead to significant variations in the biochemical reactions in their tissues and thereby change the concentrations of metabolites. We used a quantitative NMR-based metabolomic analysis of the fish lens for the evaluation of the influence of environmental factors on metabolic processes in aquatic animals. For this purpose, three species of freshwater fish-Perca fluviatilis, Rutilus rutilus lacustris, and Gymnocephalus cernua-were caught at approximately the same time at three locations in Siberia (Russia) that differed in levels of dissolved oxygen (LDO) and water purity, and the concentrations of 57 major metabolites in the fish lenses were determined. We found that the metabolomic profiles of the fish lenses strongly depended on the location. The obtained data demonstrated that two typical stressors for aquatic animals-a reduced LDO and anthropogenic water pollution-caused a largely similar metabolic response in the fish lenses that led to an increase in the concentrations of several amino acids and a decrease in sarcosine and phosphoethanolamine. At the same time, the composition of the major lens osmolytes depended mostly on the oxygen level, while variations in AMP (decrease) and NAD (increase) corresponded to the water pollution. We suggest that the eye lens is a very convenient tissue for studying the impact of ecological factors on the metabolic state of aquatic animals, fish in particular.

Nicotinamide adenine dinucleotide reduced (NADH) is a natural UV filter of certain bird lens.

In this work, we for the first time report the identification of UV filters in the bird eye lens. We found that lenses of some raptors (black kite, common buzzard) and waterfowl (birds from Podicipedidae family) contain unusually high levels of reduced nicotinamide adenine dinucleotide (NADH)-a compound with high absorption in the UV-A range with a maximum at 340 nm. The lens metabolome of these birds also features an extremely low [NAD +]/[NADH] ratio. Chemometric analysis demonstrates that the differences between the metabolomic compositions of lenses with low and high NADH abundances should be attributed to the taxonomic features of bird species rather to the influence of the low [NAD +]/[NADH] ratio. We attributed this observation to the low metabolic activity in lens fiber cells, which make up the bulk of the lens tissue. Photochemical measurements show that properties of NADH as a UV filter are as good as that of UV filters in the human lens, including strong absorption in the UV-A spectral region, high photostability under both aerobic and anaerobic conditions, low yields of triplet state, fluorescence, and radicals under irradiation. Lenticular UV filters protect the retina and the lens from photo-induced damages and improve the visual acuity by reducing chromatic aberrations; therefore, the results obtained contribute to our understanding of the extremely high acuity of the raptor vision.

The Application of Quantitative Metabolomics for the Taxonomic Differentiation of Birds.

In the current pilot study, we propose the use of quantitative metabolomics to reconstruct the phylogeny of vertebrates, namely birds. We determined the concentrations of the 67 most abundant metabolites in the eye lenses of the following 14 species from 6 orders of the class Aves (Birds): the Black kite (Milvus migrans), Eurasian magpie (Pica pica), Northern raven (Corvus corax), Eurasian coot (Fulica atra), Godlewski's bunting (Emberiza godlewskii), Great crested grebe (Podiceps cristatus), Great tit (Parus major), Hawfinch (Coccothraustes coccothraustes), Hooded crow (Corvus cornix), House sparrow (Passer domesticus), Rock dove (Columba livia), Rook (Corvus frugilegus), Short-eared owl (Asio flammeus) and Ural owl (Strix uralensis). Further analysis shows that the statistical approaches generally used in metabolomics can be applied for differentiation between species, and the most fruitful results were obtained with hierarchical clustering analysis (HCA). We observed the grouping of conspecific samples independently of the sampling place and date. The HCA tree structure supports the key role of genomics in the formation of the lens metabolome, but it also indicates the influence of the species lifestyle. A combination of genomics-based and metabolomics-based phylogeny could potentially resolve arising issues and yield a more reliable tree of life.

Metabolomic Analysis Reveals That the Moor Frog Rana arvalis Uses Both Glucose and Glycerol as Cryoprotectants.

The moor frog Rana arvalis is one of a few amphibians that can tolerate freezing to low temperatures, up to -16 °C. In this study, we performed metabolomic analysis of the liver and hindlimb muscles of frozen and control R. arvalis. We found that the moor frog synthesizes glucose and glycerol in similar concentrations as low molecular weight cryoprotectants. This is the first such case reported for the genus Rana, which was believed to use glucose only. We found that freezing upregulates glycolysis, with the accumulation of several end products: lactate, alanine, ethanol, and, possibly, 2,3-butanediol. To our knowledge, this is also the first report of ethanol as an end product of glycolysis in terrestrial vertebrates. We observed highly increased concentrations of nucleotide degradation products, implying high level of stress. The Krebs cycle arrest resulted in high concentrations of succinate, which is common for animals. However, we found almost no signs of adaptations to reoxygenation stress, with overall low levels of antioxidants. We also performed metabolomics analysis of subcutaneous ice that was found to contain glucose, glycerol, and several other substances.

Biochemical Response to Freezing in the Siberian Salamander Salamandrella keyserlingii.

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, ß-alanine, and ß-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.

Kinetic Studies of Antioxidant Properties of Ovothiol A.

Ovothiol A (OSH) is one of the strongest natural antioxidants. So far, its presence was found in tissues of marine invertebrates, algae and fish. Due to very low pKa value of the SH group, under physiological conditions, this compound is almost entirely present in chemically active thiolate form and reacts with ROS and radicals significantly faster than other natural thiols. In biological systems, OSH acts in tandem with glutathione GSH, with OSH neutralizing oxidants and GSH maintaining ovothiol in the reduced state. In the present work, we report the rate constants of OSH oxidation by H2O2 and of reduction of oxidized ovothiol OSSO by GSH and we estimate the Arrhenius parameters for these rate constants. The absorption spectra of reaction intermediates, adduct OSSG and sulfenic acid OSOH, were obtained. We also found that OSH effectively quenches the triplet state of kynurenic acid with an almost diffusion-controlled rate constant. This finding indicates that OSH may serve as a good photoprotector to inhibit the deleterious effect of solar UV irradiation; this assumption explains the high concentrations of OSH in the fish lens. The unique antioxidant and photoprotecting properties of OSH open promising perspectives for its use in the treatment of human diseases.

Most abundant metabolites in tissues of freshwater fish pike-perch (Sander lucioperca).

Quantitative metabolomic analysis was performed for eleven tissues of freshwater fish pike-perch (Sander lucioperca), including gill, heart, liver, kidney, spleen, muscle, brain, milt, lens, aqueous (AH) and vitreous (VH) humors with the use of NMR spectroscopy. The absolute values of concentrations were determined for more than 65 most abundant metabolites in every tissue. It was found that from the metabolomic viewpoint, kidney and gill are the most similar tissues, while the metabolomic compositions of ocular tissues-lens, AH, and VH significantly differ from that of other tissues. The combinations of intracellular osmolytes and antioxidants are specific for every tissue. In particular, the concentration of antioxidant ovothiol A in the lens is much higher than in any other tissue, while the brain enjoys the elevated level of ascorbate. The most abundant osmolyte in the fish spleen, muscle, and heart is taurine, and in the brain, gill, and lens-myo-inositol. Other important osmolytes specific for particular tissues are N-acetyl-histidine, N-acetyl-aspartate, betaine, threonine-phosphoethanolamine, and serine-phosphoethanolamine. The quantitative data obtained in the present work can be used as the baseline metabolite concentrations in the fish tissues to evaluate the influence of seasonal, ecological and other factors on the fish metabolism.

Comparative Metabolomic Profiling of Rat Embryonic and Induced Pluripotent Stem Cells.

Metabolomic profiles of somatic cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) reflect their metabolic phenotypes. The comparative study of metabolomes of these cells is important for understanding the differences in metabolism between somatic and pluripotent cells, and also the possible differences between ESCs and iPSCs. Here, we performed for the first time the metabolomic analysis of rat ESCs, iPSCs, and embryonic fibroblasts (EFs) at both quantitative and semi-quantitative levels using NMR spectroscopy and liquid chromatography with mass spectrometric detection, respectively. The total of 106 metabolites has been identified, and the concentrations of 51 compounds have been measured. It is found that the reprogramming of rat EFs into iPSCs affects virtually all metabolic pathways and causes drastic changes in the cell metabolomic profile. The difference between ESCs and iPSCs is much less pronounced: the concentrations of the majority of metabolites in ESCs and iPSCs are similar, and significant differences were observed for only several compounds, including adenosine, cysteic acid, glycerophosphoglycerol, inositol phosphate, glucose, myo-inositol, phosphoserine, xanthosine, guanosine. The observed differences between the metabolomic compositions of ESCs and iPSCs do not influence the pluripotent ability of iPSCs. Graphical Abstract.

Metabolic response of the Siberian wood frog Rana amurensis to extreme hypoxia.

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.

Post-mortem changes in metabolomic profiles of human serum, aqueous humor and vitreous humor.

INTRODUCTION: Application of metabolomic methods to forensic studies may expand the limits of the post-mortem interval (PMI) estimation, and improve the accuracy of the estimation. To this end, it is important to determine which tissue is the most suitable for analysis, and which compounds are the most promising candidates for PMI estimation. OBJECTIVES: This work is aimed at the comparison of human serum, aqueous humor (AH), and vitreous humor (VH) as perspective tissues for metabolomic-based PMI estimation, at the determination of most promising PMI biomarkers, and at the development of method of PMI estimation based on the measurement of concentrations of PMI biomarkers. METHODS: Quantitative metabolomic profiling of samples of the human serum, AH, and VH taken at different PMIs has been performed with the use of NMR spectroscopy. RESULTS: It is found that the metabolomic changes in anatomically isolated ocular fluids are slower and smoother than that in blood. A good positive time correlation (Pearson coefficient r > 0.5) was observed for several metabolites, including hypoxanthine, choline, creatine, betaine, glutamate, and glycine. A model for PMI estimation based on concentrations of several metabolites in AH and VH is proposed. CONCLUSIONS: The obtained results demonstrate that the metabolomic analysis of AH and VH is more suitable for the PMI estimation than that of serum. The compounds with good positive time correlation can be considered as potential PMI biomarkers.

Seasonal Variations and Interspecific Differences in Metabolomes of Freshwater Fish Tissues: Quantitative Metabolomic Profiles of Lenses and Gills.

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.

Ovothiol A is the Main Antioxidant in Fish Lens.

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.

A novel method of sample homogenization with the use of a microtome-cryostat apparatus.

Quantitative metabolomics places high demands on sample preparation, including a high degree of metabolite extraction and controlled sample weight. In respect to elastic collagen-rich tissues, the existing methods of sample homogenization poorly fit these demands due to incomplete homogenization, sample material loss, or metabolite degradation. Herein, a novel method based on the use of a microtome-cryostat apparatus is proposed. The performance of the cryotome method is compared with the results obtained with the use of a vortex bead beating. NMR-based metabolomic analysis shows that the extraction efficiency and the data scattering for both methods of sample preparation are similar. However, the heat generation during the bead beating causes the destruction of thermally-unstable compounds; besides, it may cause protein hydrolysis, leading to an artificial increase in the amino acid level. The cryotome method of sample homogenization does not cause sample heating, and it seems to be ideal for elastic tissues.

Optical properties of the human lens constituents.

The absorption and fluorescence properties of the metabolomic (MET), water-soluble and urea-soluble protein fractions from the middle-age, aged, and cataractous human lenses have been measured. At 280nm and 300nm the major lens absorbers are crystallins, which absorb more than 90% of light in the UV-B region (280-315nm). In middle-aged lenses, the absorption at 360nm is mostly provided by UV filters contained in the MET fraction. With aging, and especially with the cataract development, the absorption of MET fraction in UV-A region (315-400nm) decreases due to the drop of the UV filter concentration, while the absorption of protein fractions increases due to the accumulation of post-translational modifications. Consequently, the contribution of the MET fraction into the total lens absorption at 360nm decays from 63% in middle-aged lenses to 25% in aged lenses to 3% in cataractous lenses. The fluorescence yield of the MET fraction from cataractous lenses also significantly increases. Therefore, the protection of the lens tissue against UV radiation in aged and cataractous lenses weakens: the absorption of UV-A light is mostly provided by modified crystallins and non-UV-filter metabolites, which are photochemically more active than the UV filters. The obtained data indicate that the aged and cataractous human lenses are more vulnerable to UV-A light than the middle-aged lenses.

Photochemistry of aqueous solutions of kynurenic acid and kynurenine yellow.

The photophysics and photochemistry of kynurenic acid (KNA) and kynurenine yellow (KNY) in neutral aqueous solutions were investigated using time-resolved optical spectroscopy. Both molecules have similar quinoline-like structures, the only difference being the absence of conjugation in the nitrogen containing cycle in KNY. The main channel of S(1) excited state decay in the case of partially-unconjugated KNY is the solvent assisted S(1) → S(0) radiationless transition via intermolecular hydrogen bonds (Φ(IC) = 0.96), whereas, in the case of fully-conjugated KNA, it is intersystem crossing to the triplet state (Φ(T) = 0.82). The major intermediate products of the singlet excited KNY deactivation are the triplet state (Φ(T) = 0.022) and, most probably, the enol form (Φ(enol) = 0.012), which decay with the formation of 2,3-dihydro-4-hydroxyquinoline and 4-hydroxyquinoline, respectively. The results obtained show that KNA and KNY, which are products of the decomposition of the UV filter kynurenine, are significantly more photoactive and less photostable than the parent molecule.