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Regular physical exercise has been investigated as a primary preventive measure of several chronic diseases and premature death. Moreover, it has been shown to synchronize responses across multiple organs. In particular, hepatic tissue has proven to be a descriptive matrix to monitor the effect of physical activity. In this study, we performed an untargeted metabolomics-based analysis of hepatic tissue extracts from rats that have undergone either lifelong or chronic exercise training. For this purpose, 56 hepatic samples were collected and were analyzed by UHPLC-TOF-MS in negative ionization mode. This approach involved untargeted metabolite detection on hepatic tissue extracts accompanied by an in-house retention time/accurate mass library enabling confident metabolite identification. Unsupervised (PCA) and supervised (OPLS-DA) multivariate analysis showed significant metabolic perturbation on a panel of 28 metabolites, including amino acids, vitamins, nucleotides, and sugars. The training regime employed in this study resulted in a probable acceleration of the bioenergetic processes (glycolysis, glycogen metabolism), promoted catabolism of purines, and supplied biosynthetic precursors via the pentose phosphate pathway and pentose and glucuronate interconversions. Overall, the applied methodology was able to discriminate the different training schedules based on the rat liver metabolome.

Carob (Ceratonia siliqua) is an exceptional source of significant bioactive compounds with great economic importance in the Mediterranean region, where it is widely cultivated. Carob fruit is used for the production of a variety of products and commodities such as powder, syrup, coffee, flour, cakes, and beverages. There is growing evidence of the beneficial effects of carob and the products made from it on a range of health problems. Therefore, metabolomics could be used to explore the nutrient-rich compounds of carob. Sample preparation is a crucial step in metabolomics-based analysis and has a great impact on the quality of the data obtained. Herein, sample preparation of carob syrup and powder was optimized, to enable highly efficient metabolomics-based HILIC-MS/MS analysis. Pooled powder and syrup samples were extracted under different conditions by adjusting pH, solvent type, and sample weight to solvent volume ratio (Wc/Vs). The metabolomics profiles obtained were evaluated using the established criteria of total area and number of maxima. It was observed that the Wc/Vs ratio of 1:2 resulted in the highest number of metabolites, regardless of solvent type or pH. Aqueous acetonitrile with a Wc/Vs ratio of 1:2 satisfied all established criteria for both carob syrup and powder samples. However, when the pH was adjusted, basic aqueous propanol 1:2 Wc/Vs and acidic aqueous acetonitrile 1:2 Wc/Vs provided the best results for syrup and powder, respectively. We strongly believe that the current study could support the standardization of the metabolomics sample preparation process to enable more efficient LC-MS/MS carob analysis.

Prediction of survival in very preterm infants is very important in terms of clinical medicine and parent counseling. In this prospective study involving 96 very preterm infants, we evaluated whether metabolomic analysis of gastric fluid and urine samples obtained soon after birth could predict survival on days of life (DOL) 3 and 15 as well as overall survival up to hospital discharge. Gas chromatography-mass spectrometry (GC-MS) profiling was used. Uni- and multivariate statistical analyses were carried out to evaluate significant metabolites and their prognostic value. Differences in several metabolites were identified between survivors and non-survivors at the time-points of the study. Binary logistic regression showed that certain metabolites in the gastric fluid including arabitol as well as succinic, erythronic and threonic acids was associated with DOL 15 and overall survival. Gastric glyceric acid was also associated with DOL 15 survival. Urine glyceric acid could predict survival on DOL 3 and overall survival. In conclusion, non-surviving preterm infants showed a different metabolic profile compared survivors showing a significant discrimination with the use of GC-MS-based gastric fluid and urine analysis. Results of this study support the usefulness of metabolomics in developing survival biomarkers in very preterm infants.

BACKGROUND/OBJECTIVES Religious fasting (RF) is practiced annually by millions of Christian and Muslim followers worldwide. Scarce data exist on the impact of RF on the metabolic and hematological profile of individuals with or without dyslipidemia. SUBJECTS/METHODS The present study included: (i) 60 Greek Orthodox participants, 30 with dyslipidemia and 30 without dyslipidemia, who abstained from meat, fish and dairy products for seven consecutive weeks, and (ii) 15 young, non-dyslipidemic Muslim participants abstaining totally from food and liquid from dawn till sunset during 30 days. Biochemical (iron, ferritin, vitamin B12, calcium, low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), triglyceride and fasting glucose) and hematological (hemoglobin, hematocrit) serum blood test results of study participants were measured pre- and post- RF (at weeks 0 and 7 for Orthodox participants and at weeks 0 and 4 for Muslim participants). RESULTS In dyslipidemic and non-dyslipidemic Orthodox participants, a significant reduction of fasting glucose, HDL, LDL and TC levels was found post-RF. Hemoglobin, hematocrit, iron and ferritin levels were significantly increased, while post-RF vitamin B12 and calcium levels were substantially decreased. Subanalysis between dyslipidemic and non-dyslipidemic Orthodox participants revealed a greater decrease of cholesterol levels in the former. In Muslim participants, triglyceride, LDL and total cholesterol levels were increased post-RF (all p values < 0.05). CONCLUSIONS Our study adds to the existing literature evidence about the significant impact of RF on metabolic and hematological profiles of Orthodox and Muslim followers. The prevention of calcium and B12 deficiency during Orthodox RF by supplement consumption as well as the protection from dehydration and dysregulation of lipid metabolism during Ramadan RF should concern both clinicians and dietician nutritionists. Nevertheless, studies with larger sample size and/or long-term follow-up are warranted before reaching definite conclusions about the effects of RF on human health.

Late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) are major causes of neonatal morbidity and mortality. In this prospective, case-control study, we evaluated the metabolic profile of neonates with LOS and NEC. Blood samples were collected from 15 septic neonates and 17 neonates with NEC at the clinical suspicion of the specific diseases. Sixteen gestational and postnatal age-matched neonates without sepsis/NEC served as controls. Serum metabolic profiles were assessed using liquid chromatography–quadrupole time-of-flight mass spectrometry. Metabolomic analysis revealed significant differences in the metabolic profile of neonates with LOS or NEC compared to controls. More specifically, a number of molecules possibly identified as phosphatidylcholines or lysophosphatidylcholines were found to be significantly reduced both in neonates with LOS and those with NEC compared to controls. Additionally, L-carnitine could efficiently discriminate NEC cases from controls. The results of the current study suggest that certain phospholipids and their derivatives could possibly be used as biomarkers for the early detection of LOS and NEC.

Background Coronary artery disease (CAD) remains one of the leading causes of mortality and morbidity worldwide. As oxygen and nutrient supply to the myocardium significantly decrease during ischemic periods, important changes occur regarding myocardial intermediary energy metabolism. Metabolomics is an emerging field in systems biology, which quantifies metabolic changes in response to disease progression. This study aims to evaluate the diagnostic utility of plasma metabolomics-based biomarkers for determining the complexity and severity of CAD, as it is assessed via the SYNTAX score. Methods Corlipid is a prospective, non-interventional cohort trial empowered to enroll 1065 patients with no previous coronary intervention history, who undergo coronary angiography in University Hospital AHEPA, Thessaloniki. Venous blood samples are collected before coronary angiography. State-of the-art analytical methods are performed to calculate the serum levels of novel biomarkers: ceramides, acyl-carnitines, fatty acids, and proteins such as galectin-3, adiponectin, and the ratio of apolipoprotein B/apolipoprotein A1. Furthermore, all patients will be categorized based on the indication for coronary angiography (acute coronary syndrome, chronic coronary syndrome, preoperative coronary angiography) and on the severity of CAD using the SYNTAX score. Follow-up of 12 months after enrollment will be performed to record the occurrence of major adverse cardiovascular events. A risk prediction algorithm will be developed by combining clinical characteristics with established and novel biomarkers to identify patients at high risk for complex CAD based on their metabolite signatures. The first patient was enrolled in July 2019 and completion of enrollment is expected until May 2021. Discussion CorLipid is an ongoing trial aiming to investigate the correlation between metabolic profile and complexity of coronary artery disease in a cohort of patients undergoing coronary angiography with the potential to suggest a decision-making tool with high discriminative power for patients with CAD. To our knowledge, Corlipid is the first study aspiring to create an integrative metabolomic biomarkers-based algorithm by combining metabolites from multiple classes, involved in a wide range of pathways with well-established biochemical markers. Trial registration CorLipid trial registration: ClinicalTrials.gov number: NCT04580173. Registered 8 October 2020—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04580173.

The chronic and acute effect of ethanol administration on the metabolic phenotype of mouse brain was studied in a C57BL/6 mouse model of ethanol abuse using both untargeted and targeted ultra performance liquid chromatography-tandem mass spectrometry. Two experiments based on either chronic (8 week) exposure to ethanol of both male and female mice or acute exposure of male mice for 11 days, plus 2 oral gavage doses of 25% ethanol, were undertaken. Marked differences were found in amino acids, nucleotides, nucleosides, and related metabolites as well as a number of different lipids. Using untargeted metabolite profiling, acute ethanol exposure found significant decreases in several metabolites including nucleosides, fatty acids, glycerophosphocholine, and a number of phospholipids, while chronic exposure resulted in increases in several amino acids with notable decreases in adenosine, acetylcarnitine, and galactosylceramides. Similarly, targeted metabolite analysis, focusing on the hydrophilic fraction of the brain tissue extract, identified significant decreases in the metabolism of amino acids and derivatives, as well as purine degradation especially after chronic exposure to ethanol.

Recent studies support that acylcarnitines exert a significant role in cardiovascular disease development and progression. The aim of this metabolomics-based study was to investigate the association of serum acylcarnitine levels with coronary artery disease (CAD) severity, as assessed via SYNTAX Score. Within the context of the prospective CorLipid trial (NCT04580173), the levels of 13 circulating acylcarnitines were accurately determined through a newly developed HILIC-MS/MS method in 958 patients undergoing coronary angiography in the AHEPA University Hospital of Thessaloniki, Greece. Patients presenting with acute coronary syndrome had significantly lower median acylcarnitine C8, C10, C16, C18:1 and C18:2 values, compared to patients with chronic coronary syndrome (p = 0.012, 0.007, 0.018, 0.011 and 22 (p = 0.002, 0.024, 0.044 and 0.012, respectively), indicating a potential prognostic capability of those metabolites and of the ratio C4/C18:2 for the prediction of CAD severity. In conclusion, serum acylcarnitines could serve as clinically useful biomarkers leading to a more individualized management of patients with CAD, once further clinically oriented metabolomics-based studies provide similar evidence.

Acylcarnitines are essential diagnostic markers for complex diseases and fatty acid metabolism disorders, and play an important role in cardiovascular diseases. Herein, a HILIC-MS/MS method was developed and validated for the rapid quantitation of the acylcarnitines C2, C3, C4, C5, C6, C8, C10, C12, C14, C16, C18, C18:1 and C18:2 in human serum. RPLC and HILIC modes were tested, and HILIC was selected since it provided optimum analyte separation. Intra- and interday accuracy ranged from 90.4% to 114% and from 96% to 112%, respectively, while intra- and interday precision ranged from 0.37% to 13.7% and from 1.3% to 9.5%, respectively. A limit of quantitation (LOQ) of 78.1 ng/mL was found for C2, 2.4 ng/mL for C3, C18:1 and C18:2, and 1.2 ng/mL for C4, C5, C6, C8, C10, C12, C14, C16, and C18. Method validation was performed in accordance with bioanalytical method guidelines. Subsequently the method was applied in the analysis of approximately 1040 samples from patients with coronary artery disease.

H γήρανση είναι μία αναπόφευκτη διαδικασία φθίνουσας εξέλιξης, όλου του οργανισμού. Η μη εξαντλητική, αερόβια, μακροχρόνια άσκηση, έχει βρεθεί ότι πιθανόν μειώνει το φαινότυπο της γήρανσης. Στα πλαίσια της παρούσας διατριβής αναλύθηκαν για πρώτη φορά, διαφορετικά βιολογικά δειγμάτα επιμύων που ασκούνταν ή ήταν καθιστικά, με 3 αναλυτικές τεχνικές, για διάστημα 21 μηνών. Η χρήση δειγμάτων κοπράνων για την εξέταση της επίδρασης της άσκησης στο μεταβόλομα επιμύων παράλληλα με τη γήρανση έγινε για πρώτη φορά. Επιπλεόν, στα πλαίσια της μελέτης, αναλύθηκαν δείγματα ιστού εντέρου, με σκοπό να συμπληρώσουν το πάζλ της επίδρασης της άσκησης στο μεταβολικό αποτύπωμα των επιμύων. Για την υλοποίηση της μελέτης, 60 θηλυκοί επίμυες της φυλής Wistar χωρίστηκαν τυχαία σε 4 ομάδες, ανάλογα με τη διάρκεια της άσκησης. Την πρώτη ομάδα αποτελούσαν οι επίμυες που δεν πραγματοποίησαν καθόλου άσκηση σε όλη τη διάρκεια της ζωής τους, τη δεύτερη ομάδα οι επίμυες που ασκήθηκαν δια βίου (δηλαδή από τον 5 μήνα της ζωής τους μέχρι τον 21), την τρίτη ομάδα οι επίμυες που ασκήθηκαν από τον 5 μέχρι τον 15 μήνα και τέλος, την τέταρτη ομάδα οι επίμυες που ασκήθηκαν από τον 15 μήνα μέχρι τον 21. Η άσκηση που εκτέλεσαν οι επίμυες ήταν κολύμβηση με ανάρτηση βάρους στην ουρά, από 4- 0 % του σωματικού τους βάρους (μειούμενο με την ηλικία). Η άσκηση των επιμύων πραγματοποιούνταν 2-5 φορές την εβδομάδα, για 15-18 λεπτά την ημέρα, ανάλογα με την ηλικία. Δείγματα ούρων, αίματος και κοπράνων συλλέγονταν σε προγραμματισμένα, τακτά χρονικά διαστήματα και ιστοί συλλέχθηκαν μετά την ευθανασία των πειραματόζωων. Στα πλαίσια της διατριβής, πραγματοποιήθηκε για πρώτη φορά μελέτη προκατεργασίας δειγμάτων κοπράνων επιμύων, τόσο για μη στοχευμένη ανάλυση με φασματοσκοπία NMR και GC–MS, όσο και για στοχευμένη ανάλυση με HILIC-MS/MS. Οι κρίσιμες παράμετροι που βελτιστοποιήθηκαν, ήταν o λόγος εκχύλισης, η φύση και η τιμή pΗ του διαλύτη εκχύλισης. Ως βέλτιστες συνθήκες εκχύλισης των δειγμάτων κοπράνων κρίθηκαν ο λόγος 1/2 υπό ουδέτερες συνθήκες, για τη μη στοχευμένη και στοχευμένη μεταβολομική ανάλυση, με διαλύτη εκχύλισης το ακετονιτρίλιο. Ακόμη αναλύθηκαν δείγματα κοπράνων των ασκούμενων και των καθιστικών επιμύων της μελέτης από διάφορα χρονικά σημεία δειγματοληψίας με φασματοσκοπία NMR και με τη στοχευμένη μέθοδο HILIC-MS/MS. Υπό την επίδραση της άσκησης και της γήρανσης, στο μεταβολικό αποτύπωμα των κοπράνων, παρατηρήθηκαν αλλαγές στο μεταβολισμό των αμινοξέων και στα σχετιζόμενα με τα ενεργειακά «καύσιμα» μονοπάτια. Κυρίως αμινοξέα και λιπαρά οξέα βρέθηκαν να ευθύνονται για το διαχωρισμό των δειγμάτων των ασκούμενων από τα δείγματα των καθιστικών επιμύων με βάση τα μεταβολικά προφίλ που λήφθηκαν από την ανάλυση των δειγμάτων εντέρου. Σε όλα τα εξεταζόμενα σημεία δειγματοληψίας δειγμάτων ούρων, η επίδραση της άσκησης έγινε εμφανής και μάλιστα ήταν ανάλογη του χρόνου άσκησης. Το ολικό αίμα αποδείχθηκε κατάλληλο υπόστρωμα για τη μελέτη της επίδρασης της άσκησης. Οι αλλαγές στο μεταβολισμό των αμινοξέων, στους μεταβολίτες του κύκλου του Krebs και στα παράγωγα των πουρινών και των πυριμιδινών, δείχνουν ότι και οι δύο εξεταζόμενοι παράγοντες επηρέασαν σημαντικά αλλά και παρόμοια βιοχημικά μονοπάτια.

Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging.

Necrotizing enterocolitis (NEC) is a leading cause of gastrointestinal morbidity and mortality in preterm neonates. The aim of this pilot study was to explore using metabolomics alternations in the urine metabolites related to NEC that could possibly serve as diagnostic biomarkers of the disease. Urine samples were prospectively collected at the day of initial evaluation for NEC from 15 diseased preterm neonates (five Bell's stage I and ten stage II/III) and an equal number of matched controls. Urine metabolic profiles were assessed using non-targeted nuclear magnetic resonance spectroscopy and targeted liquid chromatography-tandem mass spectrometry monitoring 108 metabolites. Multivariate statistical models with data from either analytical approach showed clear separation between the metabolic profiles of neonates with NEC and controls. Twenty-five discriminant metabolites were identified belonging to amino and organic acids, sugars and vitamins. A number of metabolite combinations were found to have an excellent diagnostic performance in detecting neonates developing NEC. Our results show that the metabolic profile of neonates with NEC differs significantly from that of controls, making possible their separation using urine metabolomic analysis. Nevertheless, whether the small set of significant metabolites detected in this investigation could be used as early diagnostic biomarkers of NEC should be validated in larger studies.

Intraventricular hemorrhage (IVH) is a major cause of morbidity and mortality in preterm neonates. Elucidation of the mechanisms underlying IVH and/or development of disease biomarkers is essential. The aim of the study was to investigate the urine metabolic profile of preterm neonates (gestational age 32 weeks) IVH and explore the role of metabolomics in understanding pathophysiological mechanisms of the disease from which novel biomarkers could be derived. In this single-center, prospective, case-control study, urine samples were collected from seven preterm infants with early IVH (IVH group) and from 11 preterm ones without IVH (control group) on days 1, 3 and 9 of life. Urine metabolites were evaluated using targeted liquid chromatography-tandem mass spectrometry. Demographic and perinatal-clinical characteristics were recorded. Univariate and multivariate statistical analyses were performed. Orthogonal Partial Least Squares-Discriminant Analysis showed that the study groups differed significantly due to alternation in 20 out of the 40 metabolites detected in the urine. Elevated differentiated metabolites included energy intermediates and other important compounds, whereas reduced ones various amino acids, hypoxanthine and nicotinamide. A set of metabolites showed high performance as indicators of IVH, especially during day 1. As evidenced by metabolomics, preterm neonates with IVH demonstrate significant metabolism perturbations. Potentially, a selected panel of metabolites could be used as urine biomarkers of IVH development and/or progression in high-risk preterm infants.

Developing risk assessment tools for CAD prediction remains challenging nowadays. We developed an ML predictive algorithm based on metabolic and clinical data for determining the severity of CAD, as assessed via the SYNTAX score. Analytical methods were developed to determine serum blood levels of specific ceramides, acyl-carnitines, fatty acids, and proteins such as galectin-3, adiponectin, and APOB/APOA1 ratio. Patients were grouped into: obstructive CAD (SS > 0) and non-obstructive CAD (SS = 0). A risk prediction algorithm (boosted ensemble algorithm XGBoost) was developed by combining clinical characteristics with established and novel biomarkers to identify patients at high risk for complex CAD. The study population comprised 958 patients (CorLipid trial (NCT04580173)), with no prior CAD, who underwent coronary angiography. Of them, 533 (55.6%) suffered ACS, 170 (17.7%) presented with NSTEMI, 222 (23.2%) with STEMI, and 141 (14.7%) with unstable angina. Of the total sample, 681 (71%) had obstructive CAD. The algorithm dataset was 73 biochemical parameters and metabolic biomarkers as well as anthropometric and medical history variables. The performance of the XGBoost algorithm had an AUC value of 0.725 (95% CI: 0.691–0.759). Thus, a ML model incorporating clinical features in addition to certain metabolic features can estimate the pre-test likelihood of obstructive CAD.

The GRACE score constitutes a useful tool for risk stratification in patients with acute coronary syndrome (ACS), while the SYNTAX score determines the complexity of coronary artery disease (CAD). This study sought to correlate these scores and assess the accuracy of the GRACE score in predicting the extent of CAD. A total of 539 patients with ACS undergoing coronary angiography were included in this analysis. The patients were classified into those with a SYNTAX score &lt, 33 and a SYNTAX score ≥ 33. Spearman’s correlation and receiver operator characteristic analysis were conducted to investigate the role of the GRACE score as a predictor of the SYNTAX score. There was a significantly positive correlation between the SYNTAX and the GRACE scores (r = 0.32, p &lt, 0.001). The GRACE score predicted severe CAD (SYNTAX ≥ 33) moderately well (the area under the curve was 0.595 (0.522–0.667)). A GRACE score of 126 was documented as the optimal cut-off for the prediction of a SYNTAX score ≥ 33 (sensitivity = 53.5% and specificity = 66%). Therefore, our study reports a significantly positive correlation between the GRACE and the SYNTAX score in patients with ACS. Notably, NSTEMI patients with a high-risk coronary anatomy have higher calculated GRACE scores. A multidisciplinary approach by a heart team could possibly alter the therapeutic approach and management in patients presenting with ACS and a high calculated GRACE score.

Congenital anomalies of the urinary tract, and particularly of obstructive nephropathy such as ureteropelvic junction obstruction (UPJO) in infants, can later lead to chronic kidney disease and hypertension. Fundamental questions regarding underlying mechanisms remain unanswered. The aim of the present study was to quantitate the systemic amino acids metabolome in 21 UPJO infants requiring surgery (Group A) and 21 UPJO infants under conservative treatment (Group B). Nineteen healthy age-matched infants served as controls (Group C). Serum amino acids involved in several pathways and representative metabolites, including the L-arginine-derived nitric oxide (NO) metabolites nitrite and nitrate and the lipid peroxidation biomarker malondialdehyde (MDA) were measured by gas chromatography–mass spectrometry (GC–MS) methods using their stable-isotope labeled analogs as internal standards after derivatization to their methyl esters N-pentafluoropropionic amides (amino acids) and to their pentafluorobenzyl derivatives (nitrite, nitrate, MDA). The concentrations of the majority of the biomarkers were found to be lower in Group A compared to Group B. Statistical analysis revealed clear differentiation between the examined study groups. Univariate statistical analysis highlighted serum homoarginine (q = 0.006), asymmetric dimethylarginine (q = 0.05) and malondialdehyde (q = 0.022) as potential biomarkers for UPJO infants requiring surgery. Group A also differed from Group B with respect to the diameter of the preoperative anterior–posterior renal pelvis (AP) as well as regarding the number and extent of inverse correlations between AP and the serum concentrations of the biomarkers. In Group A, but not in Group B, the AP diameter strongly correlated with hydroxy-proline (r = −0.746, p = 0.0002) and MDA (r = −0.754, p = 0.002). Our results indicate a diminished amino acids metabolome in the serum of UPJO infants requiring surgery comparing to a conservative group.

ST-elevation myocardial infarction (STEMI) remains one of the leading causes of mortality worldwide. The identification of novel metabolic and imaging biomarkers could unveil key pathophysiological mechanisms at the molecular level and promote personalized care in patients with acute coronary syndromes. We studied 38 patients with STEMI who underwent primary percutaneous coronary intervention and thrombus aspiration. We sought to correlate serum ceramide levels with micro-CT quantified aspirated thrombus volume and relevant angiographic outcomes, including modified TIMI thrombus grade and pre- or post-procedural TIMI flow. Higher ceramide C16:0 levels were significantly but weakly correlated with larger aspirated thrombus volume (Spearman r = 0.326, p = 0.046), larger intracoronary thrombus burden (TB; p = 0.030) and worse pre- and post-procedural TIMI flow (p = 0.049 and p = 0.039, respectively). Ceramides C24:0 and C24:1 were also significantly associated with larger intracoronary TB (p = 0.008 and p = 0.001, respectively). Receiver operating characteristic analysis demonstrated that ceramides C24:0 and C24:1 could significantly predict higher intracoronary TB (area under the curve: 0.788, 95% CI: 0.629–0.946 and 0.846, 95% CI: 0.706–0.985, respectively). In conclusion, serum ceramide levels were higher among patients with larger intracoronary and aspirated TB. This suggests that quantification of serum ceramides might improve risk-stratification of patients with STEMI and facilitate an individualized approach in clinical practice.

Life expectancy has risen in the past decades, resulting in an increase in the number of aged individuals. Exercise remains one of the most cost-effective treatments against disease and the physical consequences of aging. The purpose of this research was to investigate the effects of aging, long-term and lifelong exercise on the rat urinary metabolome. Thirty-six male Wistar rats were divided into four equal groups: exercise from 3 to 12 months of age (A), lifelong exercise from 3 to 21 months of age (B), no exercise (C), and exercise from 12 to 21 months of age (D). Exercise consisted in swimming for 20 min/day, 5 days/week. Urine samples collection was performed at 3, 12 and 21 months of life and their analysis was conducted by liquid chromatography-mass spectrometry. Multivariate analysis of the metabolite data did not show any discrimination between groups at any of the three aforementioned ages. However, multivariate analysis discriminated the three ages clearly when the groups were treated as one. Univariate analysis showed that training increased the levels of urinary amino acids and possibly protected against sarcopenia, as evidenced by the higher levels of creatine in the exercising groups. Aging was accompanied by decreased levels of urinary amino acids and signs of increased glycolysis. Concluding, both aging and, to a lesser degree, exercise affected the rat urinary metabolome, including metabolites related to energy metabolism, with exercise showing a potential to mitigate the consequences of aging.

Ureteropelvic junction obstruction (UPJO) constitutes the predominant cause of obstructive nephropathy in both neonates and infants. Fundamental questions regarding UPJO's mechanism, assessment, and treatment still remain unanswered. The aim of the present study was to elucidate potential differences through serum metabolic profiling of surgical cases of infants with UPJO compared to both nonsurgical cases and healthy age-matched controls. Early diagnosis of renal dysfunction in this cohort based on highlighted biomarkers was the ultimate goal. Thus, serum samples were collected from 20 patients preoperatively, 19 patients with mild stenosis treated conservatively, and 17 healthy controls. All samples were subjected to targeted metabolomics analysis by hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC LC-MS/MS). Both univariate and multivariate statistical analyses were performed. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) score plots showed that the studied groups differed significantly, with a panel of metabolites, including creatinine, tryptophan, choline, and aspartate, distinguishing patients who required surgery from those followed by systematical monitoring as well as from healthy controls, showing high performance as indicators of UPJO disease.

Aim: Physical exercise can reduce adverse conditions during aging, while both exercise and aging act as metabolism modifiers. The present study investigates rat fecal and cecal metabolome alterations derived from exercise during rats’ lifespan. Methods & results: Groups of rats trained life-long or for a specific period of time were under study. The training protocol consisted of swimming, 15–18 min per day, 3–5 days per week, with load of 4–0% of rat's weight. Fecal samples and cecal extracts were analyzed by targeted and untargeted metabolic profiling methods (GC–MS and LC–MS/MS). Effects of exercise and aging on the rats’ fecal and cecal metabolome were observed. Conclusion: Fecal and cecal metabolomics are a promising field to investigate exercise biochemistry and age-related alterations.

Alcoholic liver disease (ALD) as a consequence of ethanol chronic consumption could lead to hepatic cirrhosis that is linked to high morbidity and mortality. Disease diagnosis is still very challenging and usually clear findings are obtained in the later stage of ALD. The profound effect of ethanol on metabolism can be depicted using metabolomics; thus, the discovery of novel biomarkers could shed light on the initiation and the progression of the ALD, serving diagnostic purposes. In the present study, Hydrophilic Interaction Liquid Chromatography tandem Mass Spectrometry HILIC-MS/MS based metabolomics analyisis of urine and fecal samples of C57BL/6 mice of both sexes at two sampling time points was performed, monitoring the effect of eight-week ethanol consumption. The altered hepatic metabolism caused by ethanol consumption induces extensive biochemical perturbations and changes in gut microbiota population on a great scale. Fecal samples were proven to be a suitable specimen for studying ALD since it was more vulnerable to the metabolic changes in comparison to urine samples. The metabolome of male mice was affected on a greater scale than the female metabolome due to ethanol exposure. Precursor small molecules of essential pathways of energy production responded to ethanol exposure. A meaningful correlation between the two studied specimens demonstrated the impact of ethanol in endogenous and symbiome metabolism.

The term metabolomics or metabonomics, “Biochemistry's new side of view,” refers to the study of small molecules derived from biochemical reactions in cells, organs, and organisms and has more recently been generalized to metabolic phenotyping. Quantitative and qualitative metabolic phenotyping (providing either absolute or relative quantitation) delivers a map sometimes with detailed cartographic information of the small molecule content in biological samples. GC-MS metabolic phenotyping is considered a “gold standard” for the analysis of small (semi)volatile molecules. The high separation capacity, the reliable identification based on spectral libraries, and the precise quantification of metabolites provide advantages that overcome the hurdle of derivatization reactions needed for the enhancement of the chromatographic performance of polar metabolites. The existence of validated workflows of a plethora of specimens bolsters the use of GC-MS. For the successful application of such workflows, derivatization and chromatographic parameters should be fully controllable, otherwise errors and biases may be introduced, affecting data quality and the validity of the obtained results. In data treatment, peak annotation and metabolite identification remains a challenging task; however, it is by far at the most advanced stage compared to the other metabolic phenotyping platforms. This is due to the high reproducibility of the ionization mode, which renders spectra directly comparable among various instruments or laboratories. As a consequence, validated spectral libraries have been generated (both commercial and web-based) and are widely used, enhancing the identification of unknown peaks in complex data. Data handling remains a demanding process but sophisticated platforms facilitate the process from peak picking to peak alignment and deconvolution of the signals. Validated multivariate statistics is usually performed to efficiently express the extracted information. Biochemical interpretation is assisted by online metabolome and pathway databases. Finally, GC × GC-MS technology provides an interesting alternative, which offers the highest separation capabilities among the various analytical separation technologies.

Ceramides are key-role lipids involved in numerous central cellular processes. A plethora of studies have demonstrated that the levels of ceramides in blood circulation are related to different disease states, such as type 2 diabetes, cardiovascular diseases, ovarian cancer, multiple sclerosis and others. Herein, a RPLC-MS/MS method for the rapid quantification of ceramides Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1) in human blood serum was developed and validated. Different sample preparation strategies including SLE, LLE and QuECheRS were tested with the aim to attain effective, accurate and reproducible determination of ceramides in serum samples. Intra and inter-day accuracy were found to be between 80.0–111% and 87.8–106%, respectively, for all ceramides, while intra and inter-day precision were found to vary from 0.05% to 10.2% %RSD and 2.2% to 14.0% %RSD, respectively. The lower limits of quantification were 2.3 ng/mL for Cer(d18:1/16:0) and Cer(d18:1/18:0) and 1.4 ng/mL for Cer(d18:1/24:0) and Cer(d18:1/24:1). The method was evaluated in accordance to bioanalytical method guidelines and was used for the determination of serum ceramides of patients with coronary artery disease to evaluate its utility in clinical analyses.

Fecal metabolomics-based analysis indisputably constitutes a very useful tool for elucidating the biochemistry of digestion and absorption of the gastrointestinal system. Fecal samples represent the most suitable, non-invasive, specimen for the study of the symbiotic relationship between the host and the intestinal microbiota.It is well established that the balance of the intestinal microbiota changes in response to some stimuli, physiological such as gender, age, diet, exercise and pathological such as gastrointestinal and hepatic disease. Fecal samples have been analyzed using the most widespread analytical techniques, namely, NMR spectroscopy, GC-MS, and LC-MS/MS. Rat fecal sample is a frequently used and particularly useful substrate for metabolomics-based studies in related fields. The complexity and diversity of the nature of fecal samples require careful and skillful handling for the effective quantitative extraction of the metabolites while avoiding their deterioration. Parameters such as the fecal sample weight to extraction solvent volume, the nature and the pH value of the extraction solvent, and the homogenization process are some important factors for the optimal extraction of samples, in order to obtain high-quality metabolic fingerprints, using either untargeted or targeted metabolomics.

Fecal metabolomics-based analysis indisputably constitutes a very useful tool for elucidating the biochemistry of digestion and absorption of the gastrointestinal system. Fecal samples represent the most suitable, non-invasive, specimen for the study of the symbiotic relationship between the host and the intestinal microbiota.It is well established that the balance of the intestinal microbiota changes in response to some stimuli, physiological such as gender, age, diet, exercise and pathological such as gastrointestinal and hepatic disease. Fecal samples have been analyzed using the most widespread analytical techniques, namely, NMR spectroscopy, GC-MS, and LC-MS/MS. Rat fecal sample is a frequently used and particularly useful substrate for metabolomics-based studies in related fields. The complexity and diversity of the nature of fecal samples require careful and skillful handling for the effective quantitative extraction of the metabolites while avoiding their deterioration. Parameters such as the fecal sample weight to extraction solvent volume, the nature and the pH value of the extraction solvent, and the homogenization process are some important factors for the optimal extraction of samples, in order to obtain high-quality metabolic fingerprints, using either untargeted or targeted metabolomics.

The global metabolic profiling of feces represents a challenge for both analytical chemistry and biochemistry standpoints. As a specimen, feces is complex, not homogenous and rich in macromolecules and particulate, non-digested, matter that can present problems for analytical systems. Further to this, the composition of feces is highly dependent on short-term dietary factors whilst also representing the primary specimen where co-metabolism of the host organism and the gut-microbiota is expressed. Thus the presence and the content of metabolites can be a result of host metabolism, gut microbiota metabolism or co-metabolism. Successful sample preparation and metabolite analysis require that the methodology applied for sample preparation is adequate to compensate for the highly variable nature of the sample in order to generate useful data and provide insight to ongoing biochemical processes, thereby generating hypotheses. The current practices for processing fecal samples for global metabolic profiling are described with emphasis on critical aspects in sample preparation: e.g., homogenization, filtration, centrifugation, solvent extraction and so forth and also conditions/parameter selection are discussed. The different methods applied for feces processing prior to metabolite analysis are summarized and illustrated using selected examples to highlight the effect of sample preparation on the metabolic profile obtained.

Ceratonia siliqua L. Fabaceae, commonly known as the carob tree, is native to the eastern Mediterranean countries and its products are widely used in the diet of people living in Mediterranean Europe, Middle East and North Africa. Carobs are considered to be of high nutritional value, as they are virtually fat-free, rich in proteins, antioxidants, vitamins and contain several important minerals. Different types of carob products are available in the local market, such as carob syrup, powder, flour, snack, cream, etc. However, the potential positive health effects of carob-containing products are largely unknown and have not been extensively studied. The aim of this study was to determine significant urine and fecal metabolome alterations in 8 rats treated with carob powder for 15 days as compared to 8 non-treated ones (controls) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to underly specific metabolites that changed according to the treatment. Urine and fecal samples were collected in five time points during a 15 day period of treatment with carob powder throughout water consumption (10 g powder / L). A targeted HILIC-UPLC-MS/MS method was applied for the determination of 101 polar metabolites (sugars, amino acids, organic acids, amines, etc) in a single run of 40 min in both rat urine and feces. Chromatographic separation was performed on an Aquity BEH amide column (2.1 x 100 mm, i.d. 1.7 μm); the mobile phase was consisted of A: Acetonitrile:H2O 95:5 v/v (+ 10 mM ammonium formate) and B: H2O:Acetonitrile 70:30 v/v (+10 mM ammonium formate). The solvents flow rate was set at 0.5 mL/min. Mass spectrometry parameters were optimized for each of the 101 pre-selected analytes. Approximately 55 urinary and fecal metabolites were identified in both specimens. Data were further processed with multivariate (SIMCA 13) and univariate statistics (ANOVA). The differentiation of treated rats and controls was highlighted using discriminant multivariate models. Acknowledgements: The authors would like to thank the “Black Gold” project financially supported by the University of Cyprus.

Shikonin and its enantiomer alkannin, which are natural products, have been extensively studied in vitro and in vivo for, among others, their antitumor activity. The investigation of the molecular pathways involved in their action is of interest, since they are not yet clearly defined. Metabolic profiling in cells can provide a picture of a cell's phenotype upon intervention, assisting in the elucidation of the mechanism of action. In this study, the cytotoxic effect of shikonin on a human hepatocarcinoma cell line was studied. Huh7 cells were treated with shikonin at 5 μM, and it was found that shikonin markedly inhibited cell growth. Metabolic profiling indicated alterations in the metabolic content of the cells and the culture media upon treatment, detecting the metabolic response of the cells. This study demonstrates the potential of metabolomics to improve knowledge on the mechanisms involved in shikonin's antitumor action.

Aging is an inevitable condition leading to health deterioration and death. Regular physical exercise can moderate the metabolic phenotype changes of aging. However, only a small number of metabolomics-based studies provide data on the effect of exercise along with aging. Here, urine and whole blood samples from Wistar rats were analyzed in a longitudinal study to explore metabolic alterations due to exercise and aging. The study comprised three different programs of exercises, including a life-long protocol which started at the age of 5 months and ended at the age of 21 months. An acute exercise session was also evaluated. Urine and whole blood samples were collected at different time points and were analyzed by LC-MS/MS (Liquid Chromatography–tandem Mass Spectrometry). Based on their metabolic profiles, samples from trained and sedentary rats were differentiated. The impact on the metabolome was found to depend on the length of exercise period with acute exercise also showing significant changes. Metabolic alterations due to aging were equally pronounced in sedentary and trained rats in both urine and blood analyzed samples.

Exercise is important in the prevention and treatment of the metabolic syndrome (MetS), a cluster of risk factors that raises morbidity. Metabolomics can facilitate the optimization of exercise prescription. This study aimed to investigate whether the response of the human urinary metabolic fingerprint to exercise depends on the presence of MetS or exercise mode. Twenty-three sedentary men (MetS, n = 9, and Healthy, n = 14) completed four trials: resting, high-intensity interval exercise (HIIE), continuous moderate-intensity exercise (CME), and resistance exercise (RE). Urine samples were collected pre-exercise and at 2, 4, and 24 h for targeted analysis by liquid chromatography-mass spectrometry. Time exerted the strongest differentiating effect, followed by exercise mode and health status. The greatest changes were observed in the first post-exercise samples, with a gradual return to baseline at 24 h. RE caused the greatest responses overall, followed by HIIE, while CME had minimal effect. The metabolic fingerprints of the two groups were separated at 2 h, after HIIE and RE, and at 4 h, after HIIE, with evidence of blunted response to exercise in MetS. Our findings show diverse responses of the urinary metabolic fingerprint to different exercise modes in men with and without metabolic syndrome.

Exercise plays a beneficial role in the treatment of metabolic syndrome (MetS). Metabolomics can provide new insights and facilitate the optimization of exercise prescription. This study aimed to investigate whether the response of the human serum metabolic fingerprint to exercise depends on exercise mode or the presence of MetS. Twenty-three sedentary men (nine with MetS and fourteen healthy) completed four trials: Resting, high-intensity interval exercise (HIIE), continuous moderate-intensity exercise (CME), and resistance exercise (RE). Blood samples were collected pre-exercise, immediately after exercise, and 1 h post-exercise for targeted metabolomic analysis in serum by liquid chromatography&ndash, mass spectrometry. Time exerted the strongest differentiating effect, followed by exercise mode. The largest changes from baseline were found in the immediate post-exercise samples. RE caused the strongest responses overall, followed by HIIE, while CME had minimal effect. Unlike previous results in urine, no valid model could separate the two groups in serum. Exercise exerted a beneficial effect on prominent serum biomarkers of metabolic risks, such as branched-chain amino acids, alanine, acetylcarnitine, choline, and betaine. These findings contribute to the ongoing research efforts to map the molecular responses to exercise and to optimize exercise guidelines for individuals at cardiometabolic risk.

In the present article we report our results on the development of a selective automated method for the determination of histamine in seafood using the concept of zone fluidics. The method is based on the sequential on-line reaction of the analyte with o-phthalaldehyde in the absence of a nucleophilic reagent, followed by acidification. The careful selection of the chemical and instrumental variables enabled the determination of the analyte with adequate sensitivity at the low micromolar level and with specificity against other biogenic amines and amino acids such as histidine. The LOD was 0.05 μmol L(-1) (0.6 mg kg(-1)) and linearity was obeyed in the range of 0.5-15 μmol L(-1) (5.5-170 mg kg(-1)). The proposed method offers a satisfactory sampling rate of 15 h(-1) and adequate accuracy and precision for the analysis of seafood products after minimum sample preparation and without employing a separation technique.