A Cohort Study of Gastric Fluid and Urine Metabolomics for the Prediction of Survival in Severe Prematurity.

Predicting survival in very preterm infants is critical in clinical medicine and parent counseling. In this prospective cohort study involving 96 very preterm infants, we evaluated whether the metabolomic analysis of gastric fluid and urine samples obtained shortly after birth could predict survival in the first 3 and 15 days of life (DOL), as well as overall survival up to hospital discharge. Gas chromatography-mass spectrometry (GC-MS) profiling was used. Uni- and multivariate statistical analyses were conducted 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 gastric fluid, including arabitol, and succinic, erythronic and threonic acids, were associated with 15 DOL and overall survival. Gastric glyceric acid was also associated with 15 DOL survival. Urine glyceric acid could predict survival in the first 3 DOL and overall survival. In conclusion, non-surviving preterm infants exhibited a different metabolic profile compared with survivors, demonstrating significant discrimination with the use of GC-MS-based gastric fluid and urine analyses. The results of this study support the usefulness of metabolomics in developing survival biomarkers in very preterm infants

Correlation of Serum Acylcarnitines with Clinical Presentation and Severity of Coronary Artery Disease

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 <0.001, respectively). Among CAD subgroups, median C5 levels were significantly decreased in unstable angina compared to STEMI (p = 0.026), while median C10, C16, C18:1 and C18:2 levels were higher in stable angina compared to STEMI (p = 0.019 p = 0.012, p = 0.013 and p < 0.001, respectively). Moreover, median C2, C3, C4 and C8 levels were significantly elevated in patients with diabetes mellitus (p < 0.001, <0.001, 0.029 and 0.011, respectively). Moreover, short-chain acylcarnitine C2, C4, C5 and C6 levels were elevated in patients with heavier calcification and lower left ventricular ejection fraction (LVEF) % (all p-values less than 0.05). With regard to CAD severity, median C4 and C5 levels were elevated and C16 and C18:2 levels were reduced in the high CAD complexity group with SYNTAX Score > 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

Metabolomics-based methods for the discovery of exercise and aging biomarkers in mammals

Aging is an inevitable descending process of the whole organism. Training and especially mild life-long exercise has been found to reduce the severity of aging. Aging and exercise are therefore two factors that strongly affect the whole metabolome. In this dissertation, samples from exercise and sedentary rats were studied for the first time, in a period of 21 months. Metabolomics-based analysis using GC-MS and LC-MS/MS of rat fecal and cecal samples in order to investigate the alterations according to exercise and along with aging, is also a novel aspect of this study. Serving the aims of this dissertation, 60 female rats were separated randomly in four groups: one sedentary (group A) and 3 exercise groups with different exercise time periods. The exercise groups were submitted to a training protocol when they reached adulthood. The exercise groups were consisted of animals (group B) that were exercised for the whole period of their life (15-16 months), animals (group C) that were exercised for 10 months and then rested for the next period of their life and animals (group D) that were exercised for 6 months in the last period of their life. The training protocol was consisted of 15–18 min of swimming, 2–5 days per week, with a load of weight (4– 0 % of their body weight) attached to rats’ tails depending on their age. Urine, blood and fecal samples were collected in strictly scheduled time points and tissues were collected post mortem. Optimization of fecal sample preparation for both untargeted NMR and GC-MS and for targeted HILIC-MS / MS analysis was also performed for the first time. The critical parameters that were optimized, were the extraction ratio, the nature and pH value of the extraction solvent. The ratio 1/2 (Wf/Vs) provided richer results under neutral conditions, for both non-targeted and targeted approaches. Extraction with acetonitrile showed greater metabolite coverage, by LC-MS/MS analysis. Fecal samples of sedentary and exercise rats were analyzed by NMR spectroscopy and by targeted HILIC-MS / MS method. Exercise affected amino acids related pathways as long as other hallmarks of energy fuels, along with the intensity and duration in the rat fecal metabolic profiles. Aging also affected fecal metabolome in almost the same manner. Changes in the gut microbiota were reflected also on rat cecal metabolic profiles, in which amino acids and lipids alterations were found. Exercise resulted in the differentiation of sedentary versus exercise rats in every examined time point of urine collection. Perturbed lipids', amino acids', carbohydrates' metabolism summarized the findings of the effect of exercise and aging on rat urine metabolic profiles. Whole blood samples were also analyzed in order to extract information on the effect of exercise along with aging. Changes in the metabolism of amino acids, in Kreb's cycle metabolites and in purines' and pyrimidines' biochemical pathways concluded the alterations on rat whole blood samples.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

Impact of Exercise and Aging on Rat Urine and Blood Metabolome. An LC-MS Based Metabolomics Longitudinal Study

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

Effects of Aging, Long-Term and Lifelong Exercise on the Urinary Metabolic Footprint of Rats

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

A Prospective, Case-Control Study of Serum Metabolomics in Neonates with Late-Onset Sepsis and Necrotizing Enterocolitis

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

Correlation of Serum Acylcarnitines with Clinical Presentation and Severity of Coronary Artery Disease

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 p = 0.026), while median C10, C16, C18:1 and C18:2 levels were higher in stable angina compared to STEMI (p = 0.019 p = 0.012, p = 0.013 and p p p-values less than 0.05). With regard to CAD severity, median C4 and C5 levels were elevated and C16 and C18:2 levels were reduced in the high CAD complexity group with SYNTAX Score > 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

Evaluation of Cocaine Effect on Endogenous Metabolites of HepG2 Cells Using Targeted Metabolomics

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

Machine Learning Algorithm to Predict Obstructive Coronary Artery Disease: Insights from the CorLipid Trial

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