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

Analysis of Contaminants and Residues in Food

The food supply chain contains several steps that start with the farm and end with the forks of consumers [...

Analysis of Contaminants and Residues in Food

The food supply chain contains several steps that start with the farm and end with the forks of consumers [...

Development and validation of analytical methods for the determination of drugs and biomarkers in biological matrices

The objective of this thesis is the development and validation of analytical methods for the identification of biomarkers and drugs in biological samples through metabolomics-based approaches. Metabolomics constitutes an interdisciplinary field that combines modern instrumental analytical techniques (mass spectrometry, NMR spectroscopy) with advanced statistical modeling, aiming in the holistic profiling of metabolites that are present in a biological system. Metabolomics-based studies are applied in various scientific fields, such as life sciences, biology, chemistry, requiring the use of bioinformatics in order to statistically analyze large amounts of analytical data. In life sciences metabolomics is used as a useful tool for the investigation of the potential of metabolomic profiling in early disease diagnosis, personalized therapy or drugs efficacy and toxicity. Metabolomics may provide new biomarkers at molecular level that can be used as a more efficient, less invasive means for these purposes. However, despite the great applicability of such studies, significant limitations still occur, not allowing the implementation on large-scale analysis of large number of samples. For example, existing biomarkers used so far for some diseases for diagnosis and monitoring, such as e.g. disorders of neonatal thyroid gland (Congenital Hypothyroidism), renal impairment in infants and neonates with congenital narrowing of the pyelonephritis contribution (UPJO) and cardiovascular disease (CAD) are characterized by low specificity. Also, many of the metabolites and enzymes are widely distributed in neonates, especially in those born prematurely, giving false-negative results. Other limitations are focused on low analytical reproducibility of measurement or in the difficulty of analyzing specific categories of compounds, such as highly polar compounds or non-polar ones (e.g. lipids). Moreover, in some cases analysis of endogenous compounds is difficult due to the absence of analyte-free matrices, or of Certified Reference Material (CRM). Another problem is system contamination of biological matrices, especially in liquid chromatography that results in reduced efficiency of analysis. Therefore, the revocation of the above restrictions is of great importance in order to use metabolomics into final practices. Nowadays, scientific interest focuses in the establishment of validated metabolomics – based studies, as well as in the establishment of retention index libraries in LC, that will allow the rapid, effective and valid identification of unknown compounds in untargeted analyses. Retention index libraries will help overcoming the problems occurring from analytical standards, isotope-labeled standards or CRM absence. On the other hand, life sciences focus in the development of sensitive diagnostic tools for early diagnosis and treatment of various diseases, improving patients’ life. Thus, both targeted and untargeted approaches were conscripted for the determination of endogenous compounds and a drug in biological matrices. The collected biological samples were analyzed with state-of-the-art analytical techniques, as gas and liquid chromatography coupled to mass spectrometry (GC-MS, UPLC-MS/MS, UHPLC-HRMS). Also, an attempt was made to calculate the retention index of a large number of endogenous compounds, in order to establish a universally applicable retention index library. Accurate determination of retention time is very important in confirming the detection of analytes, especially in complex matrices. Usually, the identification of a compound is based on the absolute match of the retention time and spectrum of the unknown compound to a standard reference. In liquid chromatography, variation in retention time is very common between different laboratories, instrumentation, chromatographic systems, etc., and the solution of using reference standards is not always feasible. Thus, an idea of overcoming this problem, is the establishment of universal retention index libraries. For this purpose, 95 endogenous metabolites, including amino acids, organic acids, carnitines, etc., and a reference material containing 20 homologous compounds (1-alkylpyrinium-3-sulfonate, APS) were analyzed using a RPLC-MS/MS method. Retention times for all analytes were recorded, and their respective retention indices were calculated based on the equation RI=RI0 + (RI1 – RI0) x [(RT-RT0)/ (RT1-RT0)]. A similar experimental procedure took place in 4 collaborating laboratories, using different instrumentation but exactly the same protocol of analysis regarding analytical column, mobile phases, column temperature and standards. Obtained data showed a linear correlation of R2> 0.8899 for all laboratories, proving results’ reproducibility and robustness despite different instrumentation but also demonstrating the applicability of a universal library among different laboratories. The next two studies were related to method development and validation of the analysis of drug in human urine and the analysis of four ceramides in human serum, respectively. Firstly, a NICI-GC-MS method was developed for the absolute quantification of acetazolamide and other sulfonamides in human urine. It was the first study, where sulfonamides derivatization with PFB-Br in mild conditions was reported. The method was based on an easy and quick sample preparation procedure including the evaporation of a small urine volume (50 μL), derivatization of sulfonamides and internal standard with 30% PFB-Br in ACN (60 min, 30 °C), evaporation and reconstitution with toluene. Quantification of acetazolamide and its respective internal standard was performed using the R-SO2-N (PFB)2 derivatives with m/z 581 and m/z 584, respectively. Method accuracy and precision were between 95.3% - 109% and 0.3% - 4.2%, respectively, while LOD and LOQ were 300 fmol and 1 μM, respectively. Regarding the other sulfonamides tested, only metazolamide and dorozolamide gave similar derivatives using PFB-Br, suggesting that the use of PFB-Br is limited to the size of the chain of this particular compound class. The developed and validated method was applied to real urine samples of an adult volunteer for the quantification of acetazolamide at different time points, after drug Acemit consumption. The second method was on the quantification of four specific ceramides, namely Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1), in human serum with a RPLC-MS/MS method. These specific ceramides were found to play an important key-role to various disease states, such as diabetes, cardiovascular diseases, cancer, etc. Different sample preparation methodologies were tested for accurate and efficient extraction of ceramides from human plasma, with liquid-liquid extraction using dichloromethane: methanol, 2:1 v/v being selected. Supported liquid extraction also presented satisfactory results, regarding accuracy, precision, extraction recovery and matrix effect. Final sample preparation protocol included extraction of the compounds of interest into organic phase, evaporation to dryness and reconstitution in the mobile phase. For the quantification of the endogenous compounds the use of surrogate matrix was also tested. As such five % of bovine serum albumin was compared to both an external and a standard addition curves, providing satisfactory results. The proposed method showed good linearity for all compounds (R2>0.99), as well as satisfactory accuracy and precision. Extraction recovery ranged between 82.2% to 120.2% for all ceramides. The developed method was then applied to real serum samples of CAD patients, in order to confirm method’s suitability into clinical practice. The followed three chapters were related to the application of metabolomics-based studies for biomarkers discovery into two clinical studies in human cohorts and one nutritional intervention in animals. The first study focused in the metabolic profiling of dried blood spots (DBS) of infants with congenital hypothyroidism. In total, 36 sampled of infants with CH and 24 samples of healthy matched controls, were analysed with both an untargeted gas chromatography-mass spectrometry method and a targeted liquid chromatography tandem mass spectrometry method. From the target analysis only 14 metabolites were detected in DBS, including creatinine, benzoic acid, nicotinamide and others, probably due to low sensitivity. Statistical analysis failed to highlight any statistically significant difference between the tested cohorts. Thus, untargeted GC-MS analysis was performed. Three hundred and forty-seven features were detected and identified using AMDIS and GaVIn software, with 96 of them passing all analytical criteria set. Univariate statistical analysis performed highlighted four amino acids, namely glutamate, glutamine, lysine and methionine to significant up-regulated or down-regulated in hypothyroidism group. The results are in agreement with the literature, confirming the fact that amino acids are key-compounds directly related to the thyroid gland state. The next study targeted in the analysis of a specific class of compounds, in order to distinguish the metabolic profile of serum and urine of infants with severe or mild ureteropelvic junction obstruction (UPJO) compared to healthy matched controls. Two targeted methodologies were applied using HILIC-MS/MS for the determination of 100 polar endogenous compounds and GC-MS for the analysis of amino acids and derivatives, as well as for nitrite, nitrate and malondialdehyde. Both univariate and multivariate statistical analyses highlighted a panel of metabolites significantly altered between the groups tested. From the differentiated metabolites great importance is given in homocysteine, choline, isoleucine, homoarginine, malondialdehyde and asymmetric dimethylarginine in serum and in glutamic acid, xanthine and hypoxanthine in urine. The results indicate that a panel of validated metabolites can be used in clinical practice for early and accurate diagnosis and prognosis of the disease. In the final chapter two metabolomics-based methodologies were applied for the evaluation of the nutritional effects of carob in everyday diet. Carob is considered to be of highly nutritional value, as it is rich in vitamins, tannins and nutrients. The beneficial effects of carob against cancer, metabolic syndrome, diabetes, diarrhea, hyperlipidemia and gastro esophageal reflux disease are only a few of its therapeutic actions. For this reason, 8 male Wistar rats were treated with carob powder for a 15-day period and compared to 8 non-treated rats. Fecal and urine samples were collected at 5 time points (0, 1, 5, 10 and 15 days) and were analysed with both HILIC-MS/MS method and RP LC-HRMS method. Based on the targeted approach a clear group separation was observed after day one and day fifteen on fecal metabolome, while a mild one was observed on day one on urine metabolome. Twenty-one fecal metabolites were differentiated, including amino acids and their derivatives, vitamins and organic acids, and 7 metabolites were found to be altered in rat urine samples. On the other hand, untargeted approach was able to separate the two groups based on fecal metabolome at day fifteen. Twenty-one features were altered in positive ionization mode, among which phenylalanine and dodecanedioic acid were identified. Metabolic alterations in fecal samples could be attributed to physiological and biochemical adaptations derived from the nutritional intervention. Fecal targeted metabolomics were proven to be suitable for highlighting such alterations.Αντικείμενο της παρούσας διδακτορικής διατριβής αποτέλεσε η ανάπτυξη και επικύρωση αναλυτικών μεθόδων για την εύρεση φαρμάκων και βιοδεικτών σε βιολογικά δείγματα, μέσω της μεταβολομικής ανάλυσης. Η μεταβολομική αποτελεί ένα διεπιστημονικό πεδίο έρευνας που συνδυάζει σύγχρονες αναλυτικές τεχνικές, όπως φασματομετρία μάζας, φασματοσκοπία NMR, με προηγμένα στατιστικά μοντέλα, με στόχο την ολιστική περιγραφή των ενδογενών ενώσεων που εμπεριέχονται στα διάφορα βιολογικά υποστρώματα (βιολογικά υγρά, ιστοί, τρόφιμα, κ.α.). Βρίσκει εφαρμογή σε πληθώρα επιστημών, όπως οι επιστήμες ζωής, η βιολογία, η χημεία, κ.α., ενώ παράλληλα απαιτεί τη χρήση της βιοπληροφορικής προκειμένου να αναλυθεί ο μεγάλος όγκος δεδομένων που προκύπτει. Στις επιστήμες ζωής, η μεταβολομική χρησιμοποιείται ως πολυδύναμο εργαλείο για τη διερεύνηση του μεταβολικού προφίλ στην έγκαιρη διάγνωση νόσων, στην εξατομικευμένη θεραπεία, καθώς και στην αποτελεσματικότητα φαρμάκων ή στην τοξικότητα αυτών. Οι νέοι, εξαγόμενοι βιοδείκτες μπορούν να χρησιμοποιηθούν ως περισσότερο αποτελεσματικό και λιγότερο επεμβατικό μέσο για τον σκοπό αυτό. Ωστόσο, υπάρχουν σημαντικοί περιορισμοί, που δυσχεραίνουν την εφαρμογή της μεταβολομικής σε μεγάλης κλίμακας αναλύσεις σημαντικού αριθμού δειγμάτων. Για παράδειγμα, υπάρχοντες βιοδείκτες που χρησιμοποιούνται μέχρι στιγμής για ορισμένες ασθένειες διάγνωσης και παρακολούθησης, όπως π.χ. διαταραχές του θυρεοειδούς αδένα (συγγενής υποθυρεοειδισμός), νεφρική δυσλειτουργία σε βρέφη και νεογνά με συγγενή στένωση της πυελοουρητηρικής συμβολής (ΣΠΟΥΣ) και των καρδιαγγειακών παθήσεων (CAD) χαρακτηρίζονται από χαμηλή ειδικότητα. Επίσης, πολλοί από τους μεταβολίτες και τα ένζυμα κατανέμονται ευρέως στα νεογνά, ειδικά σε εκείνα που γεννιούνται πρόωρα, δίνοντας ψευδώς αρνητικά αποτελέσματα. Άλλοι περιορισμοί έγκεινται στη χαμηλή αναπαραγωγιμότητα της μέτρησης ή στη δυσκολία ανάλυσης συγκεκριμένων κατηγοριών ενώσεων, όπως πολύ πολικών ενώσεων ή μη πολικών (π.χ. λιπίδια). Επιπροσθέτως, σε ορισμένες περιπτώσεις η ανάλυση ενδογενών ενώσεων είναι δύσκολη λόγω της απουσίας “λευκών” υποστρωμάτων ή κάποιου Πιστοποιημένου Υλικού Αναφοράς (Certified Reference Material, CRM), ενώ πολλές φορές σημαντικό πρόβλημα αποτελεί και η μόλυνση του αναλυτικού συστήματος από τα διάφορα βιολογικά υποστρώματα, ιδίως στην περίπτωση της υγρής χρωματογραφίας, οδηγώντας έτσι σε μειωμένη αποτελεσματικότητα της μέτρησης. Επομένως, η αποτελεσματική αντιμετώπιση των παραπάνω περιορισμών, έχει μεγάλη σημασία για την εφαρμογή της μεταβολομικής στην πράξη. Τα τελευταία χρόνια, το ενδιαφέρον της επιστημονικής κοινότητας, επικεντρώνεται στην ανάπτυξη επικυρωμένων μεθόδων μεταβολομικής, καθώς και στην καθιέρωση βιβλιοθηκών δεικτών συγκράτησης στην υγρή χρωματογραφία, με στόχο την γρήγορη, αποτελεσματική και έγκυρη ταυτοποίηση άγνωστων ενώσεων σε μη στοχευμένες αναλύσεις μεταβολομικής. Η δημιουργία τέτοιων βιβλιοθηκών θα βοηθήσει στην αντιμετώπιση των προβλημάτων που προκύπτουν από την δυσκολία εύρεσης ή ακόμα και απουσίας αναλυτικών προτύπων, επισημασμένων προτύπων, καθώς και Πιστοποιημένων Υλικών Αναφοράς. Από την άλλη πλευρά, οι επιστήμες ζωής επικεντρώνονται στην ανάπτυξη ευαίσθητων διαγνωστικών εργαλείων για την έγκαιρη διάγνωση και θεραπεία διαφόρων ασθενειών με στόχο τη βελτίωση της ποιότητας ζωής των ασθενών. Για τους παραπάνω λόγους, επιστρατεύτηκαν τόσο στοχευμένες όσο και μη στοχευμένες μελέτες μεταβολομικής για τον προσδιορισμό ενδογενών ενώσεων και ενός φαρμάκου σε διάφορα βιολογικά υποστρώματα. Όλα τα συλλεχθέντα βιολογικά δείγματα αναλύθηκαν με προηγμένες, “state-of-the-art” αναλυτικές πλατφόρμες, όπως είναι η αέρια και υγρή χρωματογραφία συζευγμένη με φασματομετρία μάζας (GC-MS, UPLC-MS/MS, UHPLC-HRMS). Ακόμα, έγινε προσπάθεια εύρεσης του δείκτη συγκράτησης μιας πληθώρας ενδογενών ενώσεων, με σκοπό τη δημιουργία βιβλιοθήκης δεικτών συγκράτησης, η οποία θα μπορούσε να εφαρμοστεί καθολικά. Ο ακριβής προσδιορισμός του χρόνου συγκράτησης είναι πολύ σημαντικός στην επιβεβαίωση μιας άγνωστης ένωσης, ιδίως σε πολύπλοκα υποστρώματα. Συνήθως, η ταυτοποίηση μιας ένωσης βασίζεται στην απόλυτη αντιστοίχιση του χρόνου συγκράτησης και του φάσματος μαζών της άγνωστης ένωσης με μια αντίστοιχη πρότυπη ένωση. Ωστόσο, στην υγρή χρωματογραφία η διακύμανση του χρόνου συγκράτησης είναι συχνό φαινόμενο μεταξύ διαφορετικών εργαστηρίων, οργάνων, αναλυτικών συστημάτων, κτλ., ενώ η χρήση πρότυπων ενώσεων αναφοράς δεν είναι πάντα εφικτή. Για αυτούς τους λόγους, μια πρόταση για την αντιμετώπιση του συγκεκριμένου προβλήματος, αποτελεί η δημιουργία – καθιέρωση βιβλιοθηκών δεικτών συγκράτησης καθολικού χαρακτήρα. Για το σκοπό αυτό, 95 ενδογενείς μεταβολές, συμπεριλαμβανομένων αμινοξέων, οργανικών οξέων, καρνιτινών, κ.α., καθώς και ένα υλικό αναφοράς αποτελούμενο από 20 ενώσεις μιας ομόλογης σειράς, αναλύθηκαν με μέθοδο υγρής χρωματογραφίας αντίστροφης φάσης συζευγμένη με φασματομετρία μάζας (RPLC-MS/MS). Ακολούθησε καταγραφή των χρόνων συγκράτησης για όλες τις ενώσεις, ενώ οι αντίστοιχοι δείκτες συγκράτησης υπολογίστηκαν με βάση την εξίσωση RI = RI0 + (RI1 - RI0) x [(RT-RT0)/(RT1-RT0)]. Παρόμοια πειραματική διαδικασία επιστρατεύτηκε και από άλλα 4 συνεργαζόμενα εργαστήρια, χρησιμοποιώντας διαφορετικές αναλυτικές πλατφόρμες, αλλά ακριβώς ίδιο αναλυτικό πρωτόκολλο, αναφορικά με την αναλυτική στήλη, την κινητή φάση, τη θερμοκρασία στήλης, τη ροή και τις πρότυπες ενώσεις. Τα δεδομένα που προέκυψαν, υπέδειξαν μια ικανοποιητική γραμμική συσχέτιση (R2> 0,8899) μεταξύ όλων των εργαστηρίων, αποδεικνύοντας με αυτόν τον τρόπο την αναπαραγωγιμότητα, την ευρωστία των αποτελεσμάτων, αλλά και την εφαρμοσιμότητα μιας καθολικής βιβλιοθήκης μεταξύ διαφορετικών εργαστηρίων με διαφορετική οργανολογία. Οι επόμενες δυο μελέτες αφορούσαν την ανάπτυξη μεθόδων και επικύρωση αυτών για την ανάλυση ενός φαρμάκου στα ανθρώπινα ούρα και την ανάλυση τεσσάρων κηραμιδίων στον ανθρώπινο ορό, αντίστοιχα. Αρχικά, αναπτύχθηκε μέθοδος αέριας χρωματογραφίας – φασματομετρία μάζας (NICI-GC-MS) για τον ποσοτικό προσδιορισμό της ακεταζολαμίδης και άλλων σουλφοναμιδίων στα ανθρώπινα ούρα. Για πρώτη φορά γίνεται χρήση του αντιδραστηρίου παραγωγοποίησης PFB-Br σε ήπιες συνθήκες για τον προσδιορισμό σουλφοναμιδίων. Η μέθοδος περιελάβανε μια έυκολη και γρήγορη προκατεργασία μικρού όγκου δείγματος ανθρώπινων ούρων (50 μL), παραγωγοποίηση των ενώσεων ενδιαφέροντος με 30% PFB-Br σε ACN (60 min, 30 °C), εξάτμιση μέχρι ξηρού και ανασύσταση με τολουόλιο. Ο προσδιορισμός της ακεταζολαμίδης και του αντίστοιχου εσωτερικού της προτύπου πραγματοποιήθηκε βάσει των παραγώγων R-SO2-N(PFB)2 με m/z 581 και m/z 584, αντίστοιχα. Η ακρίβεια και επαναληψιμότητα της μεθόδου κυμάνθηκε μεταξύ 95,3% - 109% και 0,3% - 4,2%, αντίστοιχα, ενώ το LOD και το LOQ ήταν 300 fmol και 1 μM, αντίστοιχα. Με την προτεινόμενη μέθοδο συν προσδιορίστηκαν τα σουλφοναμίδια μεθαζολαμίδη και δορζολαμίδη, υποδηλώνοντας ότι η χρήση του PFB-Br περιορίζεται στο μέγεθος της αλυσίδας της συγκεκριμένης κατηγορίας ενώσεων. Η μέθοδος εφαρμόστηκε σε πραγματικά δείγματα ούρων ενήλικα εθελοντή, όπου πραγματοποιήθηκε προσδιορισμός της ακεταζολαμίδης σε διάφορα χρονικά σημεία πριν και μετά την κατανάλωση του φαρμακευτικού σκευάσματος Acemit. Η δεύτερη μέθοδος αφορούσε τον ποσοτικό προσδιορισμό τεσσάρων κηραμιδίων και συγκεκριμένα των Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0 και Cer d18:1/24:1, σε ανθρώπινο ορό με την τεχνική RPLC-MS/MS. Τα συγκεκριμένα κηραμίδια βρέθηκε ότι διαδραματίζουν σημαντικό ρόλο σε διάφορες παθολογικές καταστάσεις, όπως ο διαβήτης, οι καρδιαγγειακές παθήσεις, ο καρκίνος κ.α.. Διάφορα πρωτόκολλα προκατεργασίας δείγματος δοκιμάστηκαν για την ικανοποιητική εκχύλιση των κηραμιδίων από τον ανθρώπινο ορό, με την εκχύλιση υγρού-υγρού με διχλωρομεθάνιο:μεθανόλη, 2:1 v/v να παρουσιάζει τα βέλτιστα αποτελέσματα. Ωσττόσο, και η SLE παρουσίασε ικανοποιητικά αποτελέσματα, αναφορικά με την ακρίβεια, την επαναληψιμότητα, την ανάκτηση εκχύλισης και την επίδραση του υποστρώματος. Το τελικό πρωτόκολλο προκατεργασίας δείγματος περιελάβανε εκχύλιση των ενώσεων ενδιαφέροντος από το υπόστρωμα, εξάτμιση της οργανικής φάσης μέχρι ξηρού και την ανασύσταση στην κινητή φάση. Για τον ποσοτικό προσδιορισμό των κηραμιδιων εξετάστηκε επίσης η χρήση υποκατάστατου υποστρώματος (surrogate matrix). Για το λόγο αυτό, 5% αλβουμίνη βόειου ορού συγκρίθηκε τόσο με μία εξωτερική καμπύλη, όσο και με αντίστοιχη καμπύλη σταθερής προσθήκης, δίνοντας ικανοποιητικά αποτελέσματα. Η μέθοδος παρουσίασε καλή γραμμικότητα για όλες τις ενώσεις (R2>0,99), καθώς και ικανοποιητική ακρίβεια και επαναληψιμότητα. Η ανάκτηση της εκχύλισης κυμάνθηκε από 82,2% έως 120,2% για όλα τα κηραμίδια. Και σε αυτή τη περίπτωση, η μέθοδος εφαρμόστηκε σε πραγματικά δείγματα ασθενών με κάποια καρδιαγγειακή πάθηση προκειμένου να επιβεβαιωθεί η καταλληλότητα της μεθόδου σε αναλύσεις ρουτίνας. Τα επόμενα κεφάλαια παρουσιάζουν εφαρμογές της μεταβολομικής για την εύρεση βιοδεικτών σε δύο κλινικές μελέτες που αφορούν την πληθυσμιακή ομάδα των παιδιών και νεογνών και μία διατροφική παρέμβαση σε επίμυες. Η πρώτη μελέτη επικεντρώθηκε στο μεταβολικό προφίλ αποξηραμένων κηλίδων αίματος (dried blood spots, DBS) παιδιών με συγγενή υποθυρεοειδισμό (ΣΥ). Συνολικά, 36 δείγματα παιδιών με ΣΥ και 24 δείγματα υγιών μαρτύρων, αναλύθηκαν τόσο με στοχευμένη μέθοδο υγρής χρωματογραφίας – φασματομετρίας μάζας όσο και με μη στοχευμένη μεταβολομική ανάλυση αέριας χρωματογραφίας – φασματομετρίας μάζας. Από την στοχευμένη μεταβολομική ανάλυση, μόνο 14 μεταβολίτες ανιχνεύτηκαν στο συγκεκριμένο υπόστρωμα συμπεριλαμβανομένων της κρεατινίνης, του βενζοϊκού οξέος, του νικοτιναμιδίου και άλλων, πιθανώς λόγω χαμηλής ευαισθησίας. Η στατιστική ανάλυση απέτυχε να αναδείξει οποιαδήποτε στατιστικά σημαντική διαφορά μεταξύ των δύο ομάδων. Από την μη στοχευμένη ανάλυση που ακολούθησε, 347 μεταβλητές εξήχθησαν με τη χρήση των λογισμικών AMDIS και GaVIn, με τις 96 από αυτές να πληρούν όλα τα αναλυτικά κριτήρια. Από τη στατιστική ανάλυση που πραγματοποιήθηκε, τέσσερα αμινοξέα ξεχώρισαν και συγκεκριμένα το γλουταμινικό οξύ, η γλουταμίνη, η λυσίνη και η μεθειονίνη, με τα επίπεδά τους να είναι σημαντικά χαμηλότερα στην ομάδα του ΣΥ. Τα εξαγόμενα αποτελέσματ

The Contribution of Lipidomics in Ovarian Cancer Management: A Systematic Review

Lipidomics is a comprehensive study of all lipid components in living cells, serum, plasma, or tissues, with the aim of discovering diagnostic, prognostic, and predictive biomarkers for diseases such as malignant tumors. This systematic review evaluates studies, applying lipidomics to the diagnosis, prognosis, prediction, and differentiation of malignant and benign ovarian tumors. A literature search was performed in PubMed, Science Direct, and SciFinder. Only publications written in English after 2012 were included. Relevant citations were identified from the reference lists of primary included studies and were also included in our list. All studies included referred to the application of lipidomics in serum/plasma samples from human cases of OC, some of which also included tumor tissue samples. In some of the included studies, metabolome analysis was also performed, in which other metabolites were identified in addition to lipids. Qualitative data were assessed, and the risk of bias was determined using the ROBINS-I tool. A total of twenty-nine studies were included, fifteen of which applied non-targeted lipidomics, seven applied targeted lipidomics, and seven were reviews relevant to our objectives. Most studies focused on the potential application of lipidomics in the diagnosis of OC and showed that phospholipids and sphingolipids change most significantly during disease development. In conclusion, this systematic review highlights the potential contribution of lipids as biomarkers in OC management

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

Advanced Glycation End-Products (AGEs) of Lysine and Effects of Anti-TCR/Anti-TNF-α Antibody-Based Therapy in the LEW.1AR1-iddm Rat, an Animal Model of Human Type 1 Diabetes

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). Previously, we have shown that combination with anti-TCR/anti-TNF-α antibody-based therapy re-established normoglycemia and increased proteinic arginine-dimethylation in the spleen, yet not in the pancreas. High blood glucose is often associated with elevated formation of advanced glycation end-products (AGEs) which act via their receptor (RAGE). Both anti-TCR and anti-TNF-α are inhibitors of RAGE. The aim of the present work was to investigate potential biochemical changes of anti-TCR/anti-TNF-α therapy in the LEW.1AR1-iddm rat. We determined by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) the content of free and proteinic AGEs and the Nε-monomethylation of lysine (Lys) residues in proteins of pancreas, kidney, liver, spleen and lymph nodes of normoglycemic control (ngCo, n = 6), acute diabetic (acT1D, n = 6), chronic diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Analyzed biomarkers included Lys and its metabolites Nε-carboxymethyl lysine (CML), furosine and Nε-monomethyl lysine (MML). Other amino acids were also determined. Statistical methods including ANOVA, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to evaluate the effects. Most statistical differences between the study groups were observed for spleen, pancreas and kidney, with liver and lymph nodes showing no such differences. In the pancreas, the groups differed with respect to proteinic furosine (p = 0.0289) and free CML (p = 0.0023). In the kidneys, the groups differed with respect to proteinic furosine (p = 0.0076) and CML (p = 0.0270). In the spleen, group differences were found for proteinic furosine (p = 0.0114) and free furosine (p = 0.0368), as well as for proteinic CML (p = 0.0502) and proteinic MML (p = 0.0191). The acT1D rats had lower furosine, CML and MML levels in the spleen than the rats in all other groups. This observation corresponds to the lower citrullination levels previously measured in these rats. PCA revealed diametric associations between PC1 and PC2 for spleen (r = −0.8271, p < 0.0001) compared to pancreas (r = 0.5805, p = 0.0073) and kidney (r = 0.8692, p < 0.0001). These findings underscore the importance of the spleen in this animal model of human T1D. OPLS-DA showed that in total sixteen amino acids differed in the experimental groups

Development, Validation and Application of an Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC-MS/MS) Method after QuEChERS Cleanup for Selected Dichloroanilines and Phthalates in Rice Samples

Dichloroanilines and phthalic acid esters (phthalates) are food contaminants, stable in solution even at high temperatures, which exhibit considerable toxic effects, while acting as endocrine disruptors. In the present study, a quick and easy UHPLC-MS/MS method for simultaneously analyzing two dichloroanilines (3,4-DCA and 3,5-DCA) and six phthalates (DMP, DnBP, BBP, DnOP, DEHP, and mBP) in commercial rice samples was developed, validated, and applied. For the cleanup process, the methodology of quick, easy, cheap, effective, rugged, and safe (QuEChERS) was applied, whereas different dispersants (GCB, C18, and PSA) were tested. What was developed and presented had limits of detection ranging from 0.017 up to 0.12 mg/kg, recoveries (trueness) below 120%, and relative standard deviations (RSD; precision) <15% for all target analytes, whilst no significant matrix effects occurred for all analytes. It was determined that the rice samples analyzed using this developed technique did not contain any of the two dichloroaniline compounds (3,4-DCA and 3,5-DCA) nor two of the six phthalate (DMP and mBP) compounds analyzed, while the levels of other phthalates (DEHP, BBP, DnBP and DnOP) were within the legal limits. The current method ensures a fast and easy approach for the high-throughput quantification of the selected food contaminants in rice

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