Towards a more reliable identification of isomeric metabolites using pattern guided retention validation

Reliable analyte identification is critical in metabolomics experiments to ensure proper interpretation of data. Due to chemical similarity of metabolites (as isobars and isomers) identification by mass spectrometry or chromatography alone can be difficult. Here we show that isomeric compounds are quite common in the metabolic space as given in common metabolite databases. Further, we show that retention information can shift dramatically between different experiments decreasing the value of external or even in-house compound databases. As a consequence the retention information in compound databases should be updated regularly, to allow a reliable identification. To do so we present a feasible and budget conscious method to guarantee updates of retention information on a regular basis using well designed compound mixtures. For this we combine compounds in “Ident-Mixes”, showing a way to distinctly identify chemically similar compounds through combinatorics and principle of exclusion. We illustrate the feasibility of this approach by comparing Gas chromatography (GC)–columns with identical properties from three different vendors and by creating a compound database from measuring these mixtures by Liquid chromatography–mass spectrometry (LC–MS). The results show the high influence of used materials on retention behavior and the ability of our approach to generate high quality identifications in a short time

Altered emotion processing circuits during the anticipation of emotional stimuli in women with borderline personality disorder

Borderline personality disorder (BPD) is associated with disturbed emotion processing, typically encompassing intense and fast emotional reactions toward affective stimuli. In this study, we were interested in whether emotional dysregulation in BPD occurs not only during the perception of emotional stimuli, but also during the anticipation of upcoming emotional pictures in the absence of concrete stimuli. Eighteen female patients with a diagnosis of BPD and 18 healthy control subjects anticipated cued visual stimuli with prior known emotional valence or prior unknown emotional content during functional magnetic resonance imaging. Brain activity during the anticipation of emotional stimuli was compared between both groups. When anticipating negative pictures, BPD patients demonstrated less signal change in the left dorsal anterior cingulate cortex (dACC) and left middle cingulate cortex (MCC), and enhanced activations in the left pregenual ACC, left posterior cingulate cortex (PCC) as well as in left visual cortical areas including the lingual gyrus. During the anticipation of ambiguously announced stimuli, brain activity in BPD was also reduced in the left MCC extending into the medial and bilateral dorsolateral prefrontal cortex. Results point out that deficient recruitment of brain areas related to cognitive-emotional interaction already during the anticipation phase may add to emotional dysregulation in BPD. Stronger activation of the PCC could correspond to an increased autobiographical reference in BPD. Moreover, increased preparatory visual activity during negative anticipation may contribute to hypersensitivity toward emotional cues in this disorder

Optimized workflow for on-line derivatization for targeted metabolomics approach by gas chromatography-mass spectrometry

Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available

Exercise blood-drop metabolic profiling links metabolism with perceived exertion

BACKGROUND: Assessing detailed metabolism in exercising persons minute-to-minute has not been possible. We developed a “drop-of-blood” platform to fulfill that need. Our study aimed not only to demonstrate the utility of our methodology, but also to give insights into unknown mechanisms and new directions. METHODS: We developed a platform, based on gas chromatography and mass spectrometry, to assess metabolism from a blood-drop. We first observed a single volunteer who ran 13 km in 60 min. We particularly monitored relative perceived exertion (RPE). We observed that 2,3-bisphosphoglycerate peaked at RPE in this subject. We next expanded these findings to women and men volunteers who performed an RPE-based exercise protocol to RPE at Fi O 2 20.9% or Fi O 2 14.5% in random order. RESULTS: At 6 km, our subject reached his maximum relative perceived exertion (RPE); however, he continued running, felt better, and finished his run. Lactate levels had stably increased by 2 km, ketoacids increased gradually until the run’s end, while the hypoxia marker, 2,3 bisphosphoglycerate, peaked at maximum relative perceived exertion. In our normal volunteers, the changes in lactate, pyruvate, ß hydroxybutyrate and a hydroxybutyrate were not identical, but similar to our model proband runner. CONCLUSION: Glucose availability was not the limiting factor, as glucose availability increased towards exercise end in highly exerted subjects. Instead, the tricarboxylic acid?oxphos pathway, lactate clearance, and thus and the oxidative capacity appeared to be the defining elements in confronting maximal exertion. These ideas must be tested further in more definitive studies. Our preliminary work suggests that our single-drop methodology could be of great utility in studying exercise physiology

How does mindfulness modulate self-regulation in pre-adolescent children? : An integrative neurocognitive review

Pre-adolescence is a key developmental period in which complex intrinsic volitional methods of self-regulation are acquired as a result of rapid maturation within the brain networks underlying the self-regulatory processes of attention control and emotion regulation. Fostering adaptive self-regulation skills during this stage of development has strong implications for physical health, emotional and socio-economic outcomes during adulthood. There is a growing interest in mindfulness-based programmes for pre-adolescents with initial findings suggesting self-regulation improvements, however, neurodevelopmental studies on mindfulness with pre-adolescents are scarce. This analytical review outlines an integrative neuro-developmental approach, which combines self-report and behavioural assessments with event related brain potentials (ERPs) to provide a systemic multilevel understanding of the neurocognitive mechanisms of mindfulness in pre-adolescence. We specifically focus on the N2, error related negativity (ERN), error positivity (Pe), P3a, P3b and late positive potential (LPP) ERP components as indexes of mindfulness related modulations in non-volitional bottom-up self-regulatory processes (salience detection, stimulus driven orienting and mind wandering) and volitional top-down self-regulatory processes (endogenous orienting and executive attention)

Metabolom und Proteom basierte Analyse von Muskeldystrophien

Muscle represents the means to move. This movement needs energy, which is generated by the central carbon metabolism. Respective to function, different types of muscle have developed. Two main categories exist: Red (TI) oxidative slow twitch muscles optimized for long lasting contractions and white (TII) glycolytic fast twitch muscles, which are differently affected by aging and disease. Metabolic profiles are altered in muscle disease, by changed enzyme levels or regulation. I generated detailed metabolic and proteomic profiles from different muscle fiber types; Extensor digitorum longus, Tibialis anterior (both glycolytic), Quadriceps (glycolytic/oxidative) and Soleus (oxidative) of C57BL/6N mice. I found a distinct molecular make up for each muscle. While current classification holds expectedly true, my systems biology approach allows for a more detailed look at the interplay between metabolites and proteins. Dysferlinopathy, a hereditary muscular dystrophy, manifests in puberty, when glycolysis becomes prominent. In her thesis S. Keller subjected a mouse dysferlinopathy model (BLA/J) and human myotubes to MS-based metabolomics and proteomics and found a metabolic phenotype: Enzyme levels were mostly unchanged between diseased and control samples. Glycolysis intermediates including glucose-6-phosphate were reduced, polyol pathway members sorbitol and fructose were increased. Based on work by M. Pietzke and Ch. Zasada et al. I established label tracing methods including the glycogen pool in primary human myotubes. This allowed to demonstrate function loss of hexokinase II in dysferlinopathy. Label incorporation (LI) was slower in glycolytic intermediates and glycogen. LI into sorbitol was unchanged. Glycolysis impairment leads to more oxidative metabolism, causing oxidative stress, reflected in elevated pentose phosphate pathway poolsizes. In the cell the membrane repair protein Dysferlin is located in dysferlin vesicles. I subjected fractions enriched for dysferlin vesicles by S. Kunz [3] to proteomics and found well established interaction partners, but also glycolytic enzymes never discussed before in the literature. This further establishes a metabolic role of dysferlin. The techniques used in these studies require muscle tissue samples. Together with H. Kuich we performed a proof of principle study on a single human volunteer during exercise to assess the reflection of muscle metabolism in easier obtainable blood. 10 µL blood samples from the fingertip, similar to a diabetics blood glucose test, sufficed to recapitulate well established knowledge of sports medicine and find a possible molecular explanation for the phenomenon “Hitting the Wall”. The samples show relatedness according to “rate of perceived exhaustion” possibly reflecting energy availability in the muscle. We could attribute the relations to nine key metabolites. This workflow might lead to easier muscle disease diagnostics in health care.Muskel ist der Hauptverbraucher von Energie im Körper. Diese Energie wird mittels des zentralen Kohlenstoff Metabolismus gewonnen. Zwei Hauptkategorien von Muskeln existieren: oxidative, langsam kontrahierende (Typ 1) und glykolytische, schnell kontrahierende (Typ 2). Die verschiedenen Fasertypen zeigen unterschiedliche Anfalligkeiten für Krankheiten und den natürlichen Alterungsprozess. Dies macht die Unterschiede zwischen Muskeln auf molekularer Ebene interessant. Ich generierte Metabolom- und Proteomdaten von Muskeln mit spezifischen Eigenschaften; Extensor digitorum longus, Tibialis anterior (beide glykolytisch), Quadriceps (gemischt) und Soleus (oxidativ) von Mäusen. Es zeigte sich, das jeder Muskel eine spezifische molekulare Zusammensetzung besitzt. Die klassischen Kategorisierungsparameter bleiben bestehen, aber der systembiologische Ansatz erlaubt einen diffenrenzierteren Blick auf das Zusammenspiel von Metaboliten und Proteinen. Dysferlinopathie ist eine Erbkrankheit, die sich wahrend der Pubertät manifestiert, wenn sich der adulte glykolytische Stoffwechsel einstellt. Meine Vorgängerin S. Keller hatte Muskeln von BLA/J-Mäusen (ein Dysferlinopathie-Modell) und humane Myotuben analysiert und einen metabolischen Phanotyp der Krankheit gefunden: Glykolytische Enzyme waren unverandert, aber Glucose-6-phosphat und nachfolgende Metabolite waren vermindert, Sorbitol und Fructose erhöht. Basierend auf Arbeit von Pietzke und Zasada et al. etablierte ich eine Methode zur Messung der Labelinkorporation inklusive Glycogen in primären humanen Myotuben und konnte zeigen, dass tatsächlich die Funktion von Hexokinase II vermindert ist. Verminderte Glykolyseaktivitat führt zu einer Steigerung des oxidativen Metabolismus und erhöhtem oxidativen Stressniveau, was das beobachtete erhöhte Niveau an Pentosephosphatweg-Metaboliten zeigt. In der Zelle liegt das Membranreparaturprotein Dysferlin in Dysferlinvesikeln vor. Diese Vesikel wurden von S. Kunz isoliert. Im Vesikelproteom konnte ich etablierte Interaktionspartner und glykolytische Enzyme finden, die zwar z. T. in der Literatur bekannt sind, aber nie diskutiert wurden. Diese Techniken basieren alle auf Muskelbiopsien. Gemeinsam mit H. Kuich evaluierte ich im Rahmen einer Machbarkeitsstudie Blut als leichter erhältliches Probenmaterial, aus dem Muskelmetabolismus potentiell ablesbar ist. Aus 10 µL großen Blutproben, ähnlich einem Blutzuckertest, und gesammelt unter körperlicher Belastung, konnten wir etabliertes Wissen der Sportmedizin nachvollziehen und eine Erklärung für das Phänomen ”Hitting the Wall“ finden. Insbesondere zeigten die Proben größte Ähnlichkeit gemäß der selbst empfundenen Erschöpfung, was potentiell Rückschlüsse auf den Energiezustand des Muskels zulasst. Wir konnten 9 Schlüsselmetabolite bestimmen, auf denen diese Verwandtschaft beruht. Diese Analyse-Pipeline könnte eine Translation von Metabolomics in den klinischen Alltag der Muskeldiagnostik ebnen

Tierartidentifizierung in Tiermehl mit MALDI/TOF-MS

According to Commission Regulation 999/2001 [1] supplemented by 1234/2003 [2]it is prohibited to feed Meat and Bone Meal (MBM) or Processed Animal Protein(PAP), respectively to any kind of farm animals in the European Union. Today theonly method approved by the European Union relies on microscopic evaluation. Asalternatives immunological, techniques based on polymerase chain reaction (PCR)and spectroscopic (NIR) methods are currently investigated.Microscopic methods are quite subjective and require a great deal of experienceby the personnel. Due to rather harsh conditions for treatment of MBM, speciedby Commission Regulation 1774/2002 (e. g. 20 min at 3 bar, 133 C). Resulting instrong denaturation, immunological methods are not sensitive enough. PCR basedmethods on the other hand are not able to distinguish between allowed components(e. g. egg, milk) and MBM. As a result alternative methods are still underinvestigation.Osteocalcin is a rather durable protein which occurs only in the tissue of vertebrates,almost only in the bone matrix. It has a highly conserved region whichprovides the possibility for reliable identication, but has some species specic variationsas well.In this work a procedure was developed to isolate and characterize osteocalcinfrom MBM. The clean up involves mainly HPLC, but also a protocol includingthe usage of solid phase extraction was developed. It is possible to specify thending of 10% of bovine MBM in porcine MBM, which may not be the limit; butsince the aim of this work was not to push the detection limit but to prove principalapplicability for the rst time, obviously there is room for improvement. As a resultbefore considering statistical validation, the method will have to be improved anddeveloped much further

Towards a More Reliable Identification of Isomeric Metabolites Using Pattern Guided Retention Validation

Reliable analyte identification is critical in metabolomics experiments to ensure proper interpretation of data. Due to chemical similarity of metabolites (as isobars and isomers) identification by mass spectrometry or chromatography alone can be difficult. Here we show that isomeric compounds are quite common in the metabolic space as given in common metabolite databases. Further, we show that retention information can shift dramatically between different experiments decreasing the value of external or even in-house compound databases. As a consequence the retention information in compound databases should be updated regularly, to allow a reliable identification. To do so we present a feasible and budget conscious method to guarantee updates of retention information on a regular basis using well designed compound mixtures. For this we combine compounds in “Ident-Mixes”, showing a way to distinctly identify chemically similar compounds through combinatorics and principle of exclusion. We illustrate the feasibility of this approach by comparing Gas chromatography (GC)–columns with identical properties from three different vendors and by creating a compound database from measuring these mixtures by Liquid chromatography–mass spectrometry (LC–MS). The results show the high influence of used materials on retention behavior and the ability of our approach to generate high quality identifications in a short time.</jats:p