Volatile metabolites can be lost during the preanalytical stage of metabolomic analysis. This work is aimed at the experimental and theoretical study of mechanisms of volatile substance evaporation and retention in the residues during the drying of extract solutions. We demonstrate that solvent evaporation leads to the unavoidable loss of nondissociating volatile metabolites with low boiling points and high vapor pressures (such as acetone and ethanol). The retention of dissociating volatile compounds (primarily organic acids RH) during the evaporation depends on the presence of buffer salts in solution, which are responsible for maintaining the neutral pH. An acid remains in the solution as long as it is present predominantly in the dissociated R– state. At the very last stage of solvent evaporation, buffer salts precipitate, forming a solid matrix for metabolite trapping in the residue. At the same time, buffer precipitation leads to a decrease of the solution pH, increase of the portion of RH in associated state, and acceleration of RH volatilization. The RH recovery is thus determined by the competition between the solute volatilization in the associated RH form and metabolite trapping in the solid matrix. The retention of volatile acids in the residue after extract drying can be improved either by adding buffer salts to maintain high pH or by incomplete sample drying.
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.
Modern evolutionary biology offers a wide variety of methods to explore the evolution of species and to describe their relationships. The methods of DNA/RNA sequence analysis have been developing for decades and have become increasingly popular and reasonably reliable. Nevertheless, final phylogenetic trees for many taxa are still under debate because both classical and genomics-based approaches have their own limitations for phylogenetic tree reconstruction. Here, we propose the use of younger ‘omics’ methods, namely quantitative metabolomics, to aid the phylogeny reconstruction of vertebrates. We show that metabolomics-based hierarchical clustering analysis trees match, although not perfectly, to the genomics-based trees.
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.
In metabolomics, high-performance liquid chromatography with mass spectrometry detection (HPLC-MS) analyzes a greater variety of metabolites in biological tissues or fluids: the number of metabolites is in the hundreds or even thousands. Manual analysis of such data is very difficult, so usually automation using specialized algorithms is resorted to for HPLC-MS data analysis. Unfortunately, the reliability and accuracy of modern algorithms are not high enough; according to some estimates, the share of false signals can reach 20-50%. For the first time in our laboratory we have implemented an algorithm for HPLC-MS data processing based on the principles of BigData analysis using modern machine learning methods - neural networks. The use of neural networks (CNN) allowed us to significantly increase the reliability and accuracy of peak detection and integration, and to achieve a quality at which the proportion of false signals does not exceed 5%.Peakonly is freely available on GitHub (https://github.com/arseha/peakonly) under an MIT license.
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. 
Detailed investigation of photophysical processes, occurring in kynurenine molecule under UV irradiation, revealed that the ultrafast internal conversion is the main channel of the excited S1 state decay. Our results speak in favor of intermolecular hydrogen bonding interactions to be responsible for very fast radiationless transition S1->S0. In aprotic solvents, in absence of intermolecular hydrogen bonds, the lifetime of S1 state tremendously increases, which results in the augmentation of the yield of reactive triplet state. Our experiments revealed the detailed mechanism of spontaneous decomposition of kynurenine and its derivatives in aqueous solutions under physiological conditions. It has been shown that decomposition products, carboxyketoalkenes, can covalently attach to the amino acid residues of lens proteins; the rate constants of the covalent attachment reactions were measured. Our results demonstrate that glutathione can bind reactive molecules of carboxyalkenes and, thus, this antioxidant could be regarded as the main protecting agent, preventing undesired modifications of lens proteins.
The mechanisms of photochemical reactions of kynurenine and its derivatives were studied within last several years. For the first time, it has been found that the triplet state of kynurenine could be populated via the direct photolysis. This triplet state is photochemically active and it can react with amino acid residues (tryptophan and tyrosine) via electron transfer mechanism. It has been shown that ascorbate is the most effective quencher of kynurenine triplet state among all antioxidants present in the human eye lens. The covalent attachment of molecular UV filters to proteins markedly augments their photochemical activity. The content of kynurenine and other molecular UV filters was determined in the human lenses with various degrees of the cataract. For the first time, the products of kynurenine decomposition were found in the human cataractous lenses. It has been shown that the low level of antioxidant glutathione is responsible for the presence of these molecules. The obtained results support a key role of chemical reactions with participation of kynurenine and its derivatives in the lens protein modification and the cataract development.
