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Im Rahmen dieser Arbeit wurden Anaylsenmethoden zur Quantifizierung von Ceramiden und Prostanoiden in verschiedenen biologischen Matrices unter Verwendung von Nano-LC gekoppelt mit Tandemmassenspektrometrie entwickelt und bei diversen biologischen Fragestellungen angewendet.
Die analytische Methode zu Quantifizierung der Ceramide ermöglichte deren Bestimmung in einem Probenvolumen von 2 μL CSF. Diese neu entwickelte Methode ist die erste publizierte Nano-LC-MS/MS-Methode zur Quantifizierung der Ceramide in biologischen Proben, gleichzeitig ist es auch diejenige analytische Methode mit der höchsten Empfindlichkeit [171]. Die beschriebene Methode umfasste die Substanzen C8:0, C16:0, C18:1, C18:0, C20:0, C24:1 und C24:0 Ceramid, als interner Standard wurde C17:0 Ce-ramid verwendet. Die Probenaufarbeitung bestand in einer einfachen Proteinfällung und Verdünnung mit Methanol, die chromatografische Trennung der Analyten erfolgte mit einer RP-C8 Säule unter Verwendung eines Gradientenprogramms. Die Methode wurde anhand von FDA-Richtlinien bezüglich Linearität, Bestimmungsgrenze, Präzision, Richtigkeit und Autosampler-Stabilität validiert. Die erreichten Bestimmungsgrenzen betrugen 0,225 pg auf der Säule (2,25 pg/μL CSF) für alle Ceramide außer C24:0 Ceramid, für das der Wert von 0,75 pg auf der Säule (7,5 pg/μL CSF) ermittelt wurde. Mit der durchgeführten Validierung wurde die Zuverlässigkeit der Methode für die Quantifizierung der Ceramide in CSF gezeigt. Mit einem Standardadditionsexperiment konnte belegt werden, dass PBS als Ersatzmatrix für CSF geeignet ist und somit die Ergebnisse der Validierung mit dotierten PBS-Proben auf CSF-Proben übertragbar sind. Das entwickelte Verfahren wurde für die Quantifizierung der Analyten in murinen CSF-Proben im Rahmen eines Projekts zur Erforschung der Rolle der Ceramide bei Multipler Sklerose angewendet. Anhand der Ergebnisse wurde die Hypothese bestätigt, dass die Konzentration von C16:0 Ceramid in CSF von EAE-Mäusen erhöht ist.
Die zweite entwickelte Nano-LC-MS/MS-Methode ermöglichte die Quantifizierung der Prostanoide PGE2, PGD2, 6-keto PGF1α, PGF2α und TXB2 in einer geringen Anzahl Immunzellen. Für eine erfolgreiche Bestimmung der Analyt-Konzentrationen waren nur 5.000 T-Zellen oder 40.000 Mastzellen erforderlich. Damit ist die beschriebene Methode geeignet für die Quantifizierung in Zellen, die durch Isolation aus tierischen Geweben oder Organen erhalten werden, ohne dass das Vereinigen mehrerer Proben erforderlich ist. Durch die Messung dieser bestimmten Zellpopulationen kann, im Unterschied zur Vermessung des gesamten Organs, eine differenziertere Analyse der Lokalisation der gemessenen Analyten erfolgen. Mittels der entwickelten Methode konnten die Prostanoide PGE2, PGD2, 6-keto PGF1α, PGF2α und TXB2 quantifiziert werden. Als interner Standard stand für jedes dieser Prostanoide ein vierfach deuteriertes Strukturanalogon zur Verfügung. Die Aufarbeitung der Immunzell-Proben erfolgte durch Flüssig-Flüssig-Extraktion mit Ethylacetat, die Chromatografie wurde mit einer RP-C8-Säule und einem Gradientenprogramm durchgeführt. Eine Validierung erfolgte für die Quantifizierung in T-Lymphozyten und Mastzellen für die Parameter Linearität, Bestimmungsgrenze, Präzision, Richtigkeit, Wiederfindung, Selektivität und Stabilität. Auch ein Standardadditionsexperiment mit beiden Matrices wurde durchgeführt. Die Bestimmungsgrenzen betrugen 75 fg auf der Säule für PGE2 und PGD2 sowie 112,5 fg für 6-keto PGF1α, PGF2α und TXB2, damit zeichnet sich die Methode durch höchste Empfindlichkeit aus. Die Me-thode wurde zur Messung der Prostanoid-Konzentration in T-Zellen, die im Rahmen eines Kontaktallergie-Modells aus dem Blut von unterschiedlich behandelten Mäusen isoliert worden waren, angewendet. Es konnte kein Unterschied in den Prostanoid-Konzentrationen in den T-Zellen sensibilisierter und nicht-sensibilisierter bzw. provozierter und nicht-provozierter Mäuse festgestellt werden. Bei einer zweiten Anwendung wurden die Prostanoide in murinen Mastzellen, die nach Zymosan-Injektion in die Hinterpfote zu verschiedenen Zeitpunkten nach dem Auslösen der Entzündung aus dem entstandenen Ödem isoliert worden waren, gemessen. Zusätzlich für diese Anwendung wurden einige Leukotriene in die Methode integriert. Es wurde festgestellt, dass die Konzentrationen von PGE2, PGD2 und PGF2α in Mastzellen nach der Injektion von Zymosan-Injektion ansteigen, wobei die gemessenen Konzentrationen für PGE2 48 Stunden nach der Injektion verglichen mit denen nach 24 Stunden, bezogen auf die anderen beiden Prostaglandine, am stärksten ansteigen. Außerdem wurde mittels der für die Immunzellen entwickelten Methode die Prostanoide in murinem Urin, humanem Plasma und humaner Tränenflüssigkeit quantifiziert.
Zusammenfassend ermöglichen die entwickelten Methoden die Analyse geringer Ana-lytkonzentrationen in sehr kleinen Probenmengen und damit eine Reduktion von Versuchstierzahlen und Kosten.
Based on accumulating evidence of a role of lipid signaling in many physiological and pathophysiological processes including psychiatric diseases, the present data driven analysis was designed to gather information needed to develop a prospective biomarker, using a targeted lipidomics approach covering different lipid mediators. Using unsupervised methods of data structure detection, implemented as hierarchal clustering, emergent self-organizing maps of neuronal networks, and principal component analysis, a cluster structure was found in the input data space comprising plasma concentrations of d = 35 different lipid-markers of various classes acquired in n = 94 subjects with the clinical diagnoses depression, bipolar disorder, ADHD, dementia, or in healthy controls. The structure separated patients with dementia from the other clinical groups, indicating that dementia is associated with a distinct lipid mediator plasma concentrations pattern possibly providing a basis for a future biomarker. This hypothesis was subsequently assessed using supervised machine-learning methods, implemented as random forests or principal component analysis followed by computed ABC analysis used for feature selection, and as random forests, k-nearest neighbors, support vector machines, multilayer perceptron, and naïve Bayesian classifiers to estimate whether the selected lipid mediators provide sufficient information that the diagnosis of dementia can be established at a higher accuracy than by guessing. This succeeded using a set of d = 7 markers comprising GluCerC16:0, Cer24:0, Cer20:0, Cer16:0, Cer24:1, C16 sphinganine, and LacCerC16:0, at an accuracy of 77%. By contrast, using random lipid markers reduced the diagnostic accuracy to values of 65% or less, whereas training the algorithms with randomly permuted data was followed by complete failure to diagnose dementia, emphasizing that the selected lipid mediators were display a particular pattern in this disease possibly qualifying as biomarkers.
Hepatocellular carcinoma (HCC) shows a remarkable heterogeneity and is recognized as a chemoresistant tumor with dismal prognosis. In previous studies, we observed significant alterations in the serum sphingolipids of patients with HCC. This study aimed to investigate the in vitro effects of sorafenib, which is the most widely used systemic HCC medication, on the sphingolipid pathway as well as the effects of inhibiting the sphingolipid pathway in HCC. Huh7.5 and HepG2 cells were stimulated with sorafenib, and inhibitors of the sphingolipid pathway and cell proliferation, viability, and concentrations of bioactive metabolites were assessed. We observed a significant downregulation of cell proliferation and viability and a simultaneous upregulation of dihydroceramides upon sorafenib stimulation. Interestingly, fumonisin B1 (FB1) and the general sphingosine kinase inhibitor SKI II were able to inhibit cell proliferation more prominently in HepG2 and Huh7.5 cells, whereas there were no consistent effects on the formation of dihydroceramides, thus implying an involvement of distinct metabolic pathways. In conclusion, our study demonstrates a significant downregulation of HCC proliferation upon sorafenib, FB1, and SKI II treatment, whereas it seems they exert antiproliferative effects independently from sphingolipids. Certainly, further data would be required to elucidate the potential of FB1 and SKI II as putative novel therapeutic targets in HCC.
A balanced sphingolipid rheostat is indispensable for dendritic cell function and survival and thus initiation of an immune response. Sphingolipid levels are dynamically maintained by the action of sphingolipid enzymes of which sphingosine kinases, S1P phosphatases (SGPP-1/2) and S1P lyase (SGPL-1), are pivotal in the balance of S1P and sphingosine levels. In this study, we present that SGPP-1 and SGPL-1 are regulated in inflammatory dendritic cells and contribute to S1P fate. TLR-dependent activation caused SGPL-1 protein downregulation with subsequent decrease of enzymatic activity by two-thirds. In parallel, confocal fluorescence microscopy revealed that endogenous SGPP-1 was expressed in nuclei of naive dendritic cells and was translocated into the cytoplasmatic compartment upon inflammatory stimulation resulting in dephosphorylation of S1P. Mass spectrometric determination showed that a part of the resulting sphingosine was released from the cell, increasing extracellular levels. Another route of diminishing intracellular S1P was possibly taken by its export via ATP-binding cassette transporter C1 which was upregulated in array analysis, while the S1P transporter, spinster homolog 2, was not relevant in dendritic cells. These investigations newly describe the sequential expression and localization of the endogenous S1P regulators SGPP-1 and SGPL-1 and highlight their contribution to the sphingolipid rheostat in inflammation.
Pathophysiological role of prostanoids in coagulation of the portal venous system in liver cirrhosis
(2019)
Background: Prostanoids are important regulators of platelet aggregation and thrombotic arterial diseases. Their involvement in the development of portal vein thrombosis, frequent in decompensated liver cirrhosis, is still not investigated.
Methods: Therefore, we used pro-thrombotic venous milieu generation by bare metal stent transjugular intrahepatic portosystemic shunt insertion, to study the role of prostanoids in decompensated liver cirrhosis. Here, 89 patients receiving transjugular intrahepatic portosystemic shunt insertion were included in the study, and baseline levels of thromboxane B2, prostaglandin D2 and prostaglandin E2 were measured in the portal and the hepatic vein.
Results: While the hepatic vein contained higher levels of thromboxane B2 than the portal vein, levels of prostaglandin E2 and D2 were higher in the portal vein (all P<0.0001). Baseline concentrations of thromboxane B2 in the portal vein were independently associated with an increase of portal hepatic venous pressure gradient during short term follow-up, as an indirect sign of thrombogenic potential (multivariable P = 0.004). Moreover, severity of liver disease was inversely correlated with portal as well as hepatic vein levels of prostaglandin D2 and E2 (all P<0.0001).
Conclusions: Elevated portal venous thromboxane B2 concentrations are possibly associated with the extent of thrombogenic potential in patients with decompensated liver cirrhosis.
Trial registration: ClinicalTrials.gov identifier: NCT03584204.
Inflammatory activation of astroglia adds to the pathology of various neurological diseases. Astrocytes respond to microglia-derived cytokines such as interleukin-1α (IL-1α) with enhanced inflammatory signaling. This provokes pro-inflammatory gene expression of, among others, the eicosanoid-generating enzyme prostaglandin endoperoxide synthase 2 (Ptgs2). Whereas metabolic regulation of innate immune cell inflammatory responses is intensely studied, pathways related to how metabolism modulates inflammatory signaling in astrocytes are underexplored. Here, we examined how mitochondrial oxidative phosphorylation affects inflammatory responses towards IL-1α and tumor necrosis factor α in neonatal rat astrocytes. Blocking respiratory complex I and III or adenosine triphosphate (ATP) synthase did not affect activation of inflammatory signaling by IL-1α, but did elicit differential effects on inflammatory gene mRNA expression. Remarkably, mRNA and protein expression of Ptgs2 by IL-1α was consistently up-regulated when oxidative phosphorylation was inhibited. The increase of Ptgs2 resulted from mRNA stabilization. Mitochondrial inhibitors also increased IL-1α-triggered secretion of eicosanoids, such as prostaglandin E2, prostaglandin F2α, and 6-keto-prostaglandin F1α, as assessed by liquid chromatography/mass spectrometry. Mechanistically, attenuating oxidative phosphorylation elevated adenosine monophosphate (AMP) and activated AMP-activated protein kinase (AMPK). AMPK silencing prevented Ptgs2 up-regulation by mitochondrial inhibitors, while AMPK activators recapitulated Ptgs2 mRNA stability regulation. Our data indicate modulation of astrocyte inflammatory responses by oxidative metabolism, with relevance towards eicosanoid production.
Sphingosine‐1‐phosphate (S1P) regulates pathophysiological processes, including liver regeneration, vascular tone control, and immune response. In patients with liver cirrhosis, acute deterioration of liver function is associated with high mortality rates. The present study investigated whether serum S1P concentrations are associated with disease severity in patients with chronic liver disease from compensated cirrhosis (CC), acute decompensation (AD), or acute‐on‐chronic liver failure (ACLF). From August 2013 to October 2017, patients who were admitted to the University Hospital Frankfurt with CC, AD, or ACLF were enrolled in our cirrhosis cohort study. Tandem mass spectrometry was performed on serum samples of 127 patients to assess S1P concentration. Our study comprised 19 patients with CC, 55 with AD, and 51 with ACLF, aged 29 to 76 years. We observed a significant decrease of S1P according to advanced liver injury from CC and AD up to ACLF (P < 0.001). S1P levels further decreased with progression to ACLF grade 3 (P < 0.05), and S1P highly inversely correlated with the Model for End‐Stage Liver Disease score (r = −0.508; P < 0.001). In multivariate analysis, S1P remained an independent predictor of 7‐day mortality with high diagnostic accuracy (area under the curve, 0.874; P < 0.001). Conclusion: In patients with chronic liver disease, serum S1P levels dramatically decreased with advanced stages of liver disease and were predictive of early mortality. Because S1P is a potent regulator of endothelial integrity and immune response, low S1P levels may significantly influence progressive multiorgan failure. Our data justify further elucidation of the diagnostic and therapeutic role of S1P in ACLF.
Patients after orthopic liver transplantation (OLT) are at risk of developing graft dysfunction. Sphingolipids (SL’s) have been identified to play a pivotal role in the regulation of hepatocellular apoptosis, inflammation and immunity. We aimed to investigate the serum SL profile in a prospective real-world cohort of post-OLT patients. From October 2015 until July 2016, 149 well-characterized post-OLT patients were analyzed. SL’s were assessed in serum probes via Liquid Chromatography/Tandem Mass Spectrometry. Twenty-nine (20%) patients had a biopsy proven graft rejection with decreased C20-ceramide (Cer) (p = 0.042), C18-dihydroceramide (DHC) (p = 0.022) and C24DHC (p = 0.060) levels. Furthermore, C18DHC (p = 0.044) and C24DHC (p = 0.011) were significantly down-regulated in patients with ischemic type biliary lesions (ITBL; n = 15; 10%). One-hundred and thirty-three patients (89%) have so far received tacrolimus as the main immunosuppressive agent with observed elevations of C14Cer (p = 0.052), C18Cer (p = 0.049) and C18:1Cer (p = 0.024). Hepatocellular carcinoma (HCC) pre-OLT was associated with increases in C24:1Cer (p = 0.024) and C24:1DHC (p = 0.024). In this large prospective cross-sectional study of patients, post-OLT serum levels of (very-)long chain (dihydro-)ceramides associate with graft rejection, ITBL, tacrolimus intake and HCC pre-OLT. Hence, serum SL’s may be indicative of graft complications. Further research is necessary to identify their diverse mechanistic role in regulating immunity and inflammation in patients post-OLT.
Objectives: Sphingolipids (SLs) have been implicated as potent regulators of the hepatitis B virus (HBV) life cycle. We investigated the SL biomarker potential regarding virologic endpoints in a prospective subgroup of patients with HBeAg-negative chronic HBV infection.
Methods: From 2009–2016 98 patients with HBeAg-negative HBV infection were prospectively followed over four years. Clinical, laboratory and imaging data were evaluated annually. SLs were assessed in available serum probes via liquid chromatography coupled to tandem mass spectrometry.
Results: Of those 98 patients, 10 (10.2%) showed HBV reactivation, 13 (13.2%) lost HBsAg and 9 (9.1%) gained status of HBsAg-/HBsAb-coexistence, whereas 66 (67.3%) had no events. Within the four-year analysis sphingosine (p = 0.020), sphinganine (p<0.001), dhS1P (p<0.001), C16DHC (p<0.01) and C20Cer (p<0.001) showed a significant upregulation in patients without virologic events, C18Cer significantly decreased (p<0.001). At baseline decreased S1P-, dhS1P- and C16Cer-levels were observed in patients with upcoming status of HBsAg-/HBsAb-coexistence. S1P and dhS1P levels were elevated HBV genotype D infected patients.
Conclusions: In a prospective cohort of patients with a HBeAg-negative HBV infection, serum SLs associated with the virologic course and HBV genotype D. Further studies are required to elucidate SLs as potential novel predictors of the course of HBeAg-negative HBV infection.
Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the ‘endocannabinoidome’ in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn’s disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.