Relationship of Acylcarnitines to Myocardial Ischemic Remodeling and Clinical Manifestations in Chronic Heart Failure.

BACKGROUND: Progressive myocardial remodeling (MR) in chronic heart failure (CHF) leads to aggravation of systolic dysfunction (SD) and clinical manifestations. Identification of metabolomic markers of these processes may help in the search for new therapeutic approaches aimed at achieving reversibility of MR and improving prognosis in patients with CHF. METHODS: To determine the relationship between plasma acylcarnitine (ACs) levels, MR parameters and clinical characteristics, in patients with CHF of ischemic etiology (n = 79) and patients with coronary heart disease CHD (n = 19) targeted analysis of 30 ACs was performed by flow injection analysis mass spectrometry. RESULTS: Significant differences between cohorts were found for the levels of 11 ACs. Significant positive correlations (r > 0.3) between the medium- and long-chain ACs (MCACs and LCACs) and symptoms (CHF NYHA functional class (FC); r = 0.31-0.39; p < 0.05); negative correlation (r = -0.31-0.34; p < 0.05) between C5-OH and FC was revealed. Positive correlations of MCACs and LCACs (r = 0.31-0.48; p < 0.05) with the left atrium size and volume, the right atrium volume, right ventricle, and the inferior vena cava sizes, as well as the pulmonary artery systolic pressure level were shown. A negative correlation between C18:1 and left ventricular ejection fraction (r = -0.31; p < 0.05) was found. However, a decrease in levels compared to referent values of ACs with medium and long chain lengths was 50% of the CHF-CHD cohort. Carnitine deficiency was found in 6% and acylcarnitine deficiency in 3% of all patients with chronic heart disease. CONCLUSIONS: ACs may be used in assessing the severity of the clinical manifestations and MR. ACs are an important locus to study in terms of altered metabolic pathways in patients with CHF of ischemic etiology and SD. Further larger prospective trials are warranted and needed to determine the potential benefits to treat patients with CV diseases with aberrate AC levels.

Metabolomics of head and neck cancer in biofluids: an integrative systematic review.

INTRODUCTION: Head and neck cancer (HNC) is the fifth most common cancer globally. Diagnosis at early stages are critical to reduce mortality and improve functional and esthetic outcomes associated with HNC. Metabolomics is a promising approach for discovery of biomarkers and metabolic pathways for risk assessment and early detection of HNC. OBJECTIVES: To summarize and consolidate the available evidence on metabolomics and HNC in plasma/serum, saliva, and urine. METHODS: A systematic search of experimental research was executed using PubMed and Web of Science. Available data on areas under the curve was extracted. Metabolic pathway enrichment analysis were performed to identify metabolic pathways altered in HNC. Fifty-four studies were eligible for data extraction (33 performed in plasma/serum, 15 in saliva and 6 in urine). RESULTS: Metabolites with high discriminatory performance for detection of HNC included single metabolites and combination panels of several lysoPCs, pyroglutamate, glutamic acid, glucose, tartronic acid, arachidonic acid, norvaline, linoleic acid, propionate, acetone, acetate, choline, glutamate and others. The glucose-alanine cycle and the urea cycle were the most altered pathways in HNC, among other pathways (i.e. gluconeogenesis, glycine and serine metabolism, alanine metabolism, etc.). Specific metabolites that can potentially serve as complementary less- or non-invasive biomarkers, as well as metabolic pathways integrating the data from the available studies, are presented. CONCLUSION: The present work highlights utility of metabolite-based biomarkers for risk assessment, early detection, and prognostication of HNC, as well as facilitates incorporation of available metabolomics studies into multi-omics data integration and big data analytics for personalized health.

Anthropogenic Neighborhood Impact on Bacterial and Fungal Communities in Polar Bear Feces.

Climate changes cause a dramatical increase in the ice-free season in the Arctic, forcing polar bears ashore, closer to human settlements associated with new and non-natural food objects. Such a diet may crucially transform the intestinal microbiome and metabolism of polar bears. The aim of this study was to characterize changes in the gut bacterial and fungal communities resulting from the transition to anthropogenic food objects by the means of 16S and ITS metabarcoding. Thus, rectal samples from 16 wild polar bears from the Kara-Barents subpopulation were studied. Human waste consuming resulted in a significant increase in the relative abundance of fermentative bacteria (Lactobacillaceae, Leuconostocaceae, and Streptococcaceae) and a decrease in proteolytic Enterobacteriaceae. However, the alpha-diversity parameters remained similar. Also, for the first time, the composition of the fungal community of the polar bear intestine was determined. Diet change is associated with the displacement of eurybiontic fungi (Thelebolus, Dipodascus, Candida (sake), and Geotrichum) by opportunistic Candida (tropicalis), Kazachstania, and Trichosporon. Feeding on human waste does not cause any signs of dysbiosis and probably leads to adaptive changes in the bacterial microbiome. However, the emergence of fungal facultative pathogens increases the risk of infections.

Targeted metabolomic profiling as a tool for diagnostics of patients with non-small-cell lung cancer.

Lung cancer is referred to as the second most common cancer worldwide and is mainly associated with complex diagnostics and the absence of personalized therapy. Metabolomics may provide significant insights into the improvement of lung cancer diagnostics through identification of the specific biomarkers or biomarker panels that characterize the pathological state of the patient. We performed targeted metabolomic profiling of plasma samples from individuals with non-small cell lung cancer (NSLC, n = 100) and individuals without any cancer or chronic pathologies (n = 100) to identify the relationship between plasma endogenous metabolites and NSLC by means of modern comprehensive bioinformatics tools, including univariate analysis, multivariate analysis, partial correlation network analysis and machine learning. Through the comparison of metabolomic profiles of patients with NSCLC and noncancer individuals, we identified significant alterations in the concentration levels of metabolites mainly related to tryptophan metabolism, the TCA cycle, the urea cycle and lipid metabolism. Additionally, partial correlation network analysis revealed new ratios of the metabolites that significantly distinguished the considered groups of participants. Using the identified significantly altered metabolites and their ratios, we developed a machine learning classification model with an ROC AUC value equal to 0.96. The developed machine learning lung cancer model may serve as a prototype of the approach for the in-time diagnostics of lung cancer that in the future may be introduced in routine clinical use. Overall, we have demonstrated that the combination of metabolomics and up-to-date bioinformatics can be used as a potential tool for proper diagnostics of patients with NSCLC.

Target Metabolome Profiling-Based Machine Learning as a Diagnostic Approach for Cardiovascular Diseases in Adults.

Metabolomics is a promising technology for the application of translational medicine to cardiovascular risk. Here, we applied a liquid chromatography/tandem mass spectrometry approach to explore the associations between plasma concentrations of amino acids, methylarginines, acylcarnitines, and tryptophan catabolism metabolites and cardiometabolic risk factors in patients diagnosed with arterial hypertension (HTA) (n = 61), coronary artery disease (CAD) (n = 48), and non-cardiovascular disease (CVD) individuals (n = 27). In total, almost all significantly different acylcarnitines, amino acids, methylarginines, and intermediates of the kynurenic and indolic tryptophan conversion pathways presented increased (p < 0.05) in concentration levels during the progression of CVD, indicating an association of inflammation, mitochondrial imbalance, and oxidative stress with early stages of CVD. Additionally, the random forest algorithm was found to have the highest prediction power in multiclass and binary classification patients with CAD, HTA, and non-CVD individuals and globally between CVD and non-CVD individuals (accuracy equal to 0.80 and 0.91, respectively). Thus, the present study provided a complex approach for the risk stratification of patients with CAD, patients with HTA, and non-CVD individuals using targeted metabolomics profiling.

Pharmacokinetic Properties of the Novel Synthetic Cannabinoid 5F-APINAC and Its Influence on Metabolites Associated with Neurotransmission in Rabbit Plasma.

The strong psychoactive effects of synthetic cannabinoids raise the need for the deeper studying of their neurometabolic effects. The pharmacokinetic properties of 5F-APINAC and its influence on metabolomics profiles associated with neurotransmission were investigated in rabbit plasma. Twelve rabbits divided into three groups received 1-mL 5F-APINAC at 0.1, 1 and 2 mg/kg. The intervention groups were compared with the controls. Sampling was performed at nine time points (0-24 h). Ultra-high-performance liquid chromatography-tandem mass spectrometry was used. The pharmacokinetics were dose-dependent (higher curve at a higher dose) with a rapid biotransformation, followed by gradual elimination within 24 h. The tryptophan concentrations abruptly decreased (p < 0.05) in all tested groups, returning to the basal levels after 6 h. 5-hydroxylindole acetic acid increased (p < 0.05) in the controls, but this trend was absent in the treated groups. The aspartic acid concentrations were elevated (p < 0.001) in the treated groups. L-kynurenine was elevated (p < 0.01) in the intervention groups receiving 1 mg/kg to 2 mg/kg. Dose-dependent elevations (p < 0.01) were found for kynurenic acid, xanthurenic acid and quinolinic acid (p < 0.01), whereas the anthranilic acid trends were decreased (p < 0.01). The indole-3-propionic acid and indole-3-carboxaldehyde trends were elevated (p < 0.05), whereas the indole-3-lactic acid trajectories were decreased (p < 0.01) in the intervention groups. 5F-APINAC administration had a rapid biotransformation and gradual elimination. The metabolites related to the kynurenine and serotonergic system/serotonin pathways, aspartic acid innervation system and microbial tryptophan catabolism were altered.

In Vivo Targeted Metabolomic Profiling of Prostanit, a Novel Anti-PAD NO-Donating Alprostadil-Based Drug.

Prostanit is a novel drug developed for the treatment of peripheral arterial diseases. It consists of a prostaglandin E1 (PGE1) moiety with two nitric oxide (NO) donor fragments, which provide a combined vasodilation effect on smooth muscles and vascular spastic reaction. Prostanit pharmacokinetics, however, remains poorly investigated. Thus, the object of this study was to investigate the pharmacokinetics of Prostanit-related and -affected metabolites in rabbit plasma using the liquid chromatography-mass spectrometry (LC-MS) approach. Besides, NO generation from Prostanit in isolated rat aorta and human smooth muscle cells was studied using the Griess method. In plasma, Prostanit was rapidly metabolized to 1,3-dinitroglycerol (1,3-DNG), PGE1, and 13,14-dihydro-15-keto-PGE1. Simultaneously, the constant growth of amino acid (proline, 4-hydroxyproline, alanine, phenylalanine, etc.), steroid (androsterone and corticosterone), and purine (adenosine, adenosine-5 monophosphate, and guanosine) levels was observed. Glycine, aspartate, cortisol, and testosterone levels were decreased. Ex vivo Prostanit induced both NO synthase-dependent and -independent NO generation. The observed pharmacokinetic properties suggested some novel beneficial activities (i.e., effect prolongation and anti-inflammation). These properties may provide a basis for future research of the effectiveness and safety of Prostanit, as well as for its characterization from a clinical perspective.

A simple and robust method for broad range screening of hair samples for drugs of abuse using a high-throughput UHPLC-Ion Trap MS instrument.

The use of chromatography hyphenated with mass spectrometry is a well-established approach in clinical and forensic toxicology, particularly for the analysis of the so-called alternative matrices (hair, nails, oral fluid, sweat). This procedure for the quantitative determination of targeted analytes has been reported since the early 1980s and today is the golden standard in analytical toxicology. However this technology has not found wide application the broad spectrum preliminarily screening of samples which is still mostly based on immunoassays. The aim of the present work was to test a recent instrumental approach based on UHPLC-Ion Trap-MS (Toxtyper®, Bruker Daltonics) intended to be used in routine contexts for the analysis of drug of abuse applied to hair toxicological analysis. The reported analytical method is based on a simple hair pre-treatment consisting of an overnight acid incubation in 0.1 mol/L HCl, followed by direct injection, after neutralization with equimolar amount of NaOH. The separation was then performed using a reverse phase column with a rapid gradient elution of 11 min (from 1% acetonitrile in 0.1% ammonium formate to 95% acetonitrile in 0.1% ammonium formate). Detection was by a fast ion trap analyzer (32,500 m/z sec-1) operating in the mass range 70-800 m/z. The chromatographic retention time and MS2/MS3 data were used for compound identification using a proprietary database which allowed to screen for up to 987 compounds. The tested analytical method showed limits of detection in the range between 0.01 and 0.09 ng/mg of hair matrix for a panel of 16 drugs of abuse (except for MDA, morphine, 6-MAM and norketamine, which showed limits of detection of 0.25, 0.15, 0.15 and 0.25 ng/mg, respectively). The method was validated according to international guidelines on a selected panel of drugs of abuse. The analytical performance of the instrument was assessed by analyzing 968 hair samples from forensic cases. A good concordance with a reference confirmatory method based on GC-MS was found in terms of classification of both negative and positive samples. Finally, the method was also successfully tested by analyzing 12 proficiency test samples containing not only common drugs of abuse but also new psychoactive substances, including fentanyls and cathinones.

Thanatochemistry at the crime scene: a microfluidic paper-based device for ammonium analysis in the vitreous humor.

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (µPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of "RGB distance" by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4 mmol L-1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n = 25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2 > 0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene.

A novel low-cost approach for the semi-quantitative analysis of carbohydrate-deficient transferrin (CDT) based on fluorescence resonance energy transfer (FRET).

BACKGROUND AND AIM: The increase of the carbohydrate-deficient transferrin (CDT) as results of an heavy intake of alcohol for at least two weeks, is a well-known biochemical modification since the middle '70s. Notwithstanding the first commercial kit for the diagnosis of chronic alcohol abuse based on this biomarker was commercially accessible already thirty years ago, only expensive analytical methods are currently available for its determination. The present paper shows a new approach intrinsically sensitive and specific, based on a specific derivatization of transferrin, and not requiring sophisticated instrumentation. METHODS: The proposed procedure is based on a selective chelation of terbium (III) by transferrin followed by detection using an characteristic Fluorescence Resonance Transfer Energy (FRET) phenomenon (ex 298 nm - em 550 nm). RESULTS: The proposed procedure showed a limit of detection of 2.5 pmol/mL and a reproducibility intra-day and inter-days <15% and 20%, respectively. The results obtained analyzing 40 serum samples using the developed method, were compared with those obtained with HPLC-Vis and an R2 = 0.8854 was found. CONCLUSIONS: Considering its main features (low-cost, ease of operation, minimum need of instrumentation) the present method is suitable for application in screening contexts and in non-strictly regulated environments (e.g. clinical diagnosis) as well as in developing countries or remote areas.

Development of a low cost gas diffusion device for ammonia detection in the vitreous humor and its preliminary application for estimation of the time since death.

A simple and low cost analytical device is described for the determination of ammonium in the vitreous humor suitable for inferring the post mortem interval in forensic cases. The device is based on ammonia formation from ammonium ion by means of NaOH addition to the vitreous humor sample and its detection with a pH chemical indicator in the gas phase above the vitreous humor sample. From the gas phase, ammonia diffuses through a polymeric membrane and it is trapped and detected with a droplet of pH indicator thymol blue. The color change of the droplet is measured using a smartphone camera. Under optimal conditions, the device showed a limit of detection of 0.2mM, with between days precision of ≤ 15% expressed as relative standard deviation, and an accuracy between days from 88.3% to 114.5%. This homemade gas diffusion analytical device was successfully used for the determination of ammonia in vitreous humor samples from forensic autopsies. The results obtained with the proposed method, although for a limited number, showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover a significant correlation was also found between the results of the present method and the time elapsed since death by a simple evaluation of the color intensity. In conclusion, this preliminary study showed that the proposed device, after adequate validation, could be a promising tool for a presumptive estimation of the time since death directly at the crime scene.

LC-MS/MS Identification and Structural Characterization of Main Biodegradation Products of Nitroproston - A Novel Prostaglandin-based Pharmaceutical Compound.

BACKGROUND: Nitroproston is a novel prostaglandin-based compound modified by NOdonating groups with potential application in obstructive respiratory diseases such as asthma and obstructive bronchitis. Nitroproston has been extensively studied using various pharmacological models. Its biological stability is still uncertain. OBJECTIVE: The aim of the present study was to evaluate Nitroproston stability in vitro, as well as to identify and characterize its major biodegradation products. METHODS: The principal biodegradation products of Nitroproston were identified in vitro using liquid chromatography/ion trap - time-of-flight mass-spectrometry. The postulated structure of metabolites was confirmed using authentic reference standards. Rat, rabbit and human plasma and human whole blood samples were used for comparative in vitro degradation study. Nitroproston and its biodegradation products in biological samples were measured by liquid chromatography/triple -stage quadrupole mass spectrometry. RESULTS: Nitroproston is rapidly hydrolyzed in rat plasma to generate glycerol-1,3-dinitrate and prostaglandin E2. The latter can undergo conversion to cyclopentenone prostaglandins A2 and B2. Thereby less than 5% of the parent compound was observed in rat plasma at the first moment of incubation. A similar pattern was observed for rabbit plasma where half-life (T1/2) of Nitroproston was about 2.0 minutes. Nitroproston biodegradation rate for human plasma was the slowest (T1/2 = 2.1 h) among tested species, occurred more rapidly in whole blood (T1/2 = 14.8 min). CONCLUSION: It was found that Nitroproston is rapidly hydrolyzed in rodent compared to human plasma incubations. Whereas Nitroproston is relatively stable in human plasma an enhanced hydrolytic activity was observed in whole human blood incubations. Extensive metabolism of Nitroproston in human whole blood was mainly associated with red blood cells. The observed interspecies variability highlights the need of suitable animal model selection for Nitroproston follow-up PK/PD studies.

Rabbit plasma metabolomic analysis of Nitroproston®: a multi target natural prostaglandin based-drug.

INTRODUCTION: Nitroproston® is a novel multi-target drug bearing natural prostaglandin E2 (PGE2) and nitric oxide (NO)-donating fragments for treatment of inflammatory and obstructive diseases (i.e., asthma and obstructive bronchitis). OBJECTIVES: To investigate the effects of Nitroproston® administration on plasma metabolomics in vivo. METHODS: Experimental in vivo study randomly assigning the target drug (treatment group) or a saline solution without the drug (vehicle control group) to 12 rabbits (n = 6 in each group). Untargeted (5880 initial features; 1869 negative-4011 positive ion peaks; UPLC-IT-TOF/MS) and 84 targeted moieties (Nitroproston® related metabolites, prostaglandins, steroids, purines, pyrimidines and amino acids; HPLC-QQQ-MS/MS) were measured from plasma at 0, 2, 4, 6, 8, 12, 18, 24, 32 and 60 min after administration. RESULTS: PGE2, 13,14-dihydro-15-keto-PGE2, PGB2, 1,3-GDN and 15-keto-PGE2 increased in the treatment group. Steroids (i.e., cortisone, progesterone), organic acids, 3-oxododecanoic acid, nicotinate D-ribonucleoside, thymidine, the amino acids serine and aspartate, and derivatives pyridinoline, aminoadipic acid and uric acid increased (p < 0.05 AUCROC curve > 0.75) after treatment. Purines (i.e., xanthine, guanine, guanosine), bile acids, acylcarnitines and the amino acids L-tryptophan and L-phenylalanine were decreased. Nitroproston® impacted steroidogenesis, purine metabolism and ammonia recycling pathways, among others. CONCLUSION: Nitroproston®, a multi action novel drug based on natural prostaglandins, altered metabolites (i.e., guanine, adenine, cortisol, cortisone and aspartate) involved in purine metabolism, urea and ammonia biological cycles, steroidogenesis, among other pathways. Suggested mechanisms of action, metabolic pathway interconnections and useful information to further understand the metabolic effects of prostaglandin administration are presented.