Loss of Volatile Metabolites during Concentration of Metabolomic Extracts.

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.

Blood Plasma Circulating DNA-Protein Complexes: Involvement in Carcinogenesis and Prospects for Liquid Biopsy of Breast Cancer.

Circulating DNA (cirDNA) is a promising tool in translational medicine. However, studies of cirDNA have neglected its association with proteins, despite ample evidence that this interaction may affect the fate of DNA in the bloodstream and its molecular functions. The goal of the current study is to shed light on the differences between the proteomic cargos of histone-containing nucleoprotein complexes (NPCs) from healthy female (HFs) and breast cancer patients (BCPs), and to reveal the proteins involved in carcinogenesis. NPCs were isolated from the blood samples of HFs and BCPs using affinity chromatography. A total of 177 and 169 proteins were identified in NPCs from HFs and BCPs using MALDI-TOF mass spectrometry. A bioinformatics analysis revealed that catalytically active proteins, as well as proteins that bind nucleic acids and regulate the activity of receptors, are the most represented among the unique proteins of blood NPCs from HFs and BCPs. In addition, the proportion of proteins participating in ion channels and proteins binding proteins increases in the NPCs from BCP blood. However, the involvement in transport and signal transduction was greater in BCP NPCs compared to those from HFs. Gene ontology term (GO) analysis revealed that the NPC protein cargo from HF blood was enriched with proteins involved in the negative regulation of cell proliferation, and in BCP blood, proteins involved in EMT, invasion, and cell migration were observed. The combination of SPG7, ADRB1, SMCO4, PHF1, and PSMG1 NPC proteins differentiates BCPs from HFs with a sensitivity of 100% and a specificity of 80%. The obtained results indirectly indicate that, in tandem with proteins, blood cirDNA is an important part of intercellular communication, playing a regulatory and integrating role in the physiology of the body.

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.

Multifunctional fluorescent diarylethene: time-resolved study of photochemistry.

A study of luminescence and photochromic properties of (E)-2,3-bis(2,5-dimethylthiophen-3-yl)-5-(4-(pyrrolidin-1-yl)benzylidene)cyclopent-2-en-1-one, which is a diarylethene with a push-pull system between carbonyl and dimethylamino groups, was performed using time-resolved methods. The intramolecular charge transfer (ICT) process as well as 6π-electrocyclization and E-/Z-isomerization contribute to the complex light-induced properties of this molecule. Formation of unexpected short-lived intermediates was detected in the time range from 100 fs to 100 µs. A model based on two processes (additional photocyclization and interconversion between conformers) was proposed to rationalize this result. The key intermediates existing in the picosecond time domain are so-called precursors, which are proposed for both parallel (p) and anti-parallel (ap) isomers of the open form. In general, fast light-induced processes for the fluorescent diarylcyclopentenones are much more complicated than for the parent cyclopentenone-based DAE.

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.

Colitis-associated intestinal microbiota regulates brain glycine and host behavior in mice.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory disorders of the gastrointestinal tract with complex etiology and no strategies for complete cure. IBD are often complicated by mental disorders like anxiety and depression, indicating substantial shifts in the microbiota gut-brain axis. However, the mechanisms connecting IBD to mental diseases are still under debate. Here we use Muc2 knockout mouse model of chronic colitis to uncouple the effects of the intestinal microbiota on host behavior from chronic inflammation in the gut. Muc2 knockout male mice exhibit high exploratory activity, reduced anxiety-related behaviors, impaired sensorimotor gating, and altered social preference towards males and females. Microbial transfer to wild-type mice via littermate co-housing shows that colitis-associated microbiota rather than inflammation per se defines behavioral features in Muc2 colitis model. Metagenomic profiling and combination of antibiotic treatments revealed that bacterial species Akkermansia muciniphila is associated with the behavioral phenotype in mutants, and that its intestinal abundance correlates with social preference towards males. Metabolomic analysis together with pharmacological inhibition of Gly and NMDA receptors helped us to determine that brain glycine is responsible for the behavioral phenotype in Muc2 mice. Blood and brain metabolic profiles suggest that microbiota-dependent changes in choline metabolism might be involved in regulation of central glycine neurotransmission. Taken together, our data demonstrates that colitis-associated microbiota controls anxiety, sensorimotor gating and social behavior via metabolic regulation of the brain glycinergic system, providing new venues to combat neurological complications of IBD.

Stability of Metabolomic Content during Sample Preparation: Blood and Brain Tissues.

Thermal and enzymatic reactions can significantly change the tissue metabolomic content during the sample preparation. In this work, we evaluated the stability of metabolites in human whole blood, serum, and rat brain, as well as in metabolomic extracts from these tissues. We measured the concentrations of 63 metabolites in brain and 52 metabolites in blood. We have shown that metabolites in the extracts from biological tissues are stable within 24 h at 4 °C. Serum and whole blood metabolomes are also rather stable, changes in metabolomic content of the whole blood homogenate become apparent only after 1-2 h of incubation at 4 °C, and become strong after 24 h. The most significant changes correspond to energy metabolites: the concentrations of ATP and ADP decrease fivefold, and the concentrations of NAD, NADH, and NADPH decrease below the detectable level. A statistically significant increase was observed for AMP, IMP, hypoxanthine, and nicotinamide. The brain tissue is much more metabolically active than human blood, and significant metabolomic changes occur already within the first several minutes during the brain harvest and sample homogenization. At a longer timescale (hours), noticeable changes were observed for all classes of compounds, including amino acids, organic acids, alcohols, amines, sugars, nitrogenous bases, nucleotides, and nucleosides.

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.

A previously unknown way of heme detoxification in the digestive tract of cats.

Free heme is a highly toxic molecule for a living organism and its detoxification is a very important process, especially for carnivorous animals. Here we report the discovery of a previously unknown process for neutralizing free heme in the digestive tract of domestic cats. The cornerstone of this process is the encapsulation of heme into carbonated hydroxyapatite nanoparticles, followed by their excretion with faeces. This way of heme neutralization resembles the formation of insoluble heme-containing particles in the digestive tracts of other hematophagous species (for example, the formation of insoluble hemozoin crystals in malaria-causing Plasmodium parasites). Our findings suggest that the encapsulation of heme molecules into a hydroxyapatite matrix occurs during the transition from the acidic gastric juice to the small intestine with neutral conditions. The formation of these particles and their efficiency to include heme depends on the bone content in a cat's diet. In vitro experiments with heme-hydroxyapatite nanoparticles confirm the proposed scenario.

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.

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.

Application of EPR to porphyrin-protein agents for photodynamic therapy.

Recently, a new type of spin labels based on photoexcited triplet molecules was proposed for nanometer scale distance measurements by pulsed dipolar electron paramagnetic resonance (PD EPR). However, such molecules are also actively used within biological complexes as photosensitizers for photodynamic therapy (PDT) of cancer. Up to date, the idea of using the photoexcited triplets simultaneously as PDT agents and as spin labels for PD EPR has never been employed. In this work, we demonstrate that PD EPR in conjunction with other methods provides valuable information on the structure and function of PDT candidate complexes, exemplified here with porphyrins bound to human serum albumin (HSA). Two distinct porphyrins with different properties were used: amphiphilic meso-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) and water soluble cationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4); HSA was singly nitroxide-labeled to provide a second tag for PD EPR measurements. We found that TMPyP4 locates in a cavity at the center of the four-helix bundle of HSA subdomain IB, close to the interface with solvent, thus being readily accessible to oxygen. As a result, the photolysis of the complex leads to photooxidation of HSA by generated singlet oxygen and causes structural perturbation of the protein. Contrary, in case of mTHPP porphyrin, the binding occurs at the proton-rich pocket of HSA subdomain IIIA, where the access of oxygen to a photosensitizer is hindered. Structural data of PD EPR were supported by other EPR techniques, laser flash photolysis and protein photocleavage studies. Therefore, pulsed EPR on complexes of proteins with photoexcited triplets is a promising approach for gaining structural and functional insights into such PDT agents.

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.