Thiele, Herbert
Lade...
Preferred name
Thiele, Herbert
Official Name
Thiele, Herbert
4 Ergebnisse
Gerade angezeigt 1 - 4 von 4
- Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Lipidanalyse von Bioflüssigkeiten durch kombinierte SPE/HPLC-, MS- und NMR-Techniken(2013-05-07); ; ; Body fluids like blood plasma are easy to gain and reflect the metabolic state of an organism. One of the challenging tasks is the analysis of intact and underivatized lipids, which have various important functions in organism (energy storage, membrane building block, cellular signalling, precursor of relevant biomolecules etc.). The lipid composition consists of different, structural diverse lipid classes with several subclasses including numerous lipid species. These species vary in chain length, degree of saturation and/or kind of bonding of the attached fatty acid(s). Anomalous lipid compositions were correlated to different diseases like diabetes and many others. In the present work a hyphenated approach consisting of different separation (SPE, HPLC) and analytical techniques (MS, NMR) was applied for the investigation of lipids in body fluids. Such an approach combines the advantages of the different techniques resulting in increased selectivity, sensitivity and structure elucidation power. The thesis shows the way from finding a suitable lipid extraction method to the identification of individual lipid species in blood plasma and serum samples with a main focus on method development and optimization. Beside several SPE and LC-MS methods for the separation of various lipid groups, classes or species, a new isocratic HILIC-ESI-MS method for the class separation of phospholipids is mentionable. Furthermore, the analytic procedure was used for lipid analysis of diabetic mouse samples (diabetes mellitus type 2) to demonstrate the application for samples with pathological background. Detailed experimental features and results obtained on different lipids are part of this work.Dissertation448 295 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Metabolism in Hepatocytes and Adipocytes under inhibition of carbonic anhydrases studied with multinuclear NMR-spectroscopy(2009-06-17); ; ; Obesity is defined as an intense overweight condition and is an increasingly serious problem for the health of population, particularly in the modern Western countries. The excessive body weight is associated with various diseases, especially cardiovascular diseases, diabetes mellitus type 2, certain types of cancer, sleep apnea, and osteoarthritis.There are therefore already a number of therapeutic procedures aimed at the treatment or prophylaxis of obesity. A novel therapeutic procedure for the treatment and/or prophylaxis of obesity, which can complement the previously known form of therapy, intends the inhibition of de novo lipogenesis (DNL). DNL means the synthesis of endogenous fatty acids from carbohydrate in the mammalian organism. There are various conceivable possibilities to inhibite DNL in mammalian cells, all of which aim to reduce the turnover of the TCA cycle. The inhibition of carbonic anhydrases (CAs) isozymes involving in several steps of the de novo lipogenesis can inhibit the DNL. The CAs catalyze a very simple physiological reaction, the interconversion of carbon dioxide and bicarbonate. It is comprehensible that inhibition of mitochondrial CA (i.e. CA V) and/or cytosolic CA (i.e. CA II) subsequently affect pyruvate carboxylation. The carbohydrates are not only used to synthesis lipids, but also TCA cycle intermediates and TCA cycle related amino acids such as glutamate. Sulphonamide/ Sulphamate are carbonic anhydrase inhibitors (CAIs) and good candidates to inhibit DNL on the step of pyruvate carboxylation in mitochondria and/or in cytosol. Inhibition of CA will reduce the influx into the anaplerotic activity of pyruvate carboxylase (pc) and subsequently into the citric acid (TCA) cycle, which will modify the intermediates of the TCA cycle and its effluxes (in particular glutamate) and lipid synthesis. The inhibition of carboanhydrase affects also the synthesis of alanine and lactate. To study the effect of CAIs upon the synthesis of lipids and other metabolites in HEP-G2 and 3T3-L1 cells multinuclear NMR spectroscopy was used. To investigate the effect of CAIs on the cellular metabolism, the HEP-G2 and 3T3-L1 cells were incubated for 6, 12, or 24h in the presence or absence of CAIs, and metabolic alterations of labeled substrates were followed up using NMR spectroscopy. The data of the present study clearly show that topiramate (TPM) and acetazolamide (ACT) are nonspecific inhibitors of CA isozymes (IC50 ≥1000 à �à µM), but DNL in the presence of lower concentrations of ethoxyzolamide (ETZ) is more effectively inhibited. The results also show that compound 1 (a newly synthesised sulfonamide) is a very efficient candidate to inhibit CA II and CA V and possibly also other CA isozymes. The data of this study show also that compound 1 has no effect on DNL, if labelled acetate is used as tracer. Therefore, the effect of compound 1 on DNL operates at the level of CA V and/or CA II and not at the level of acetyl-CoA carboxylase (ACC).HEP-G2 cells were also incubated for 6h with compound 5 (another newly synthesised sulfonamide) and three follower compounds of compound 1 (i.e. compound 2, compound 3, compound 4) or drug vehicle (0.1% DMSO) to study the CA inhibitory effect on bicarbonate fixation in cultured HEP-G2 cells. The results of this study clearly show that compound 5 is the most efficient CAI whereas the followers of compound 1 are mediocer CAIs for DNL. DNL is inhibited to 100% in the presence of 50 à �à µM compound 5 or 100 à �à µM compound 1, thus the effects of 50 à �à µM compound 5 on bicarbonate fixation in cultured HEP-G2 cells is comparable with the inhibitory effect of 100 à �à µM compound 1. The HEP-G2 cells were incubated in the presence of CAIs or drug vehicle (0.1% DMSO) for control with [U-13C]glucose for 6 or 24h. The results of this study show that after 6h the effect of 50 à �à µM compound 5 and 100 à �à µM compound 1 on the glucose influx into the TCA cycle via pc and on pdh are similar. However after 24h in the presence of 50 à �à µM compound 5, the influx of labelled glucose into the TCA cycle via pc is decreased by only 20% and pdh activity is not affected, whereas after 24h the pc and pdh activities are reduced in the presence of 100 μM compound 1 to 100% and 80% of control respectively. The results of experiments with [2-13C]acetate in the presence of compound 5 show that compound 5 has possibly an indirect inhibitory effect on pdh. The results of this study also show that, similar to compound 1, the effect of compound 5 on DNL is at the level of CA and not at the level of acetyl-CoA carboxylase.Dissertation266 123 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Strukturelle und quantitative Identifizierung der Einzelkomponenten in Lipidgemischen(2014-10-15); ; ; Lipidomics aims to qualitatively and quantitatively define lipid classes, including their molecular species, in biological systems. Continuous technical advances in instrumentation enable a high level of sensitivity and precision. Due to the broad diversity of chemical structures of lipids, different methods and strategies are being applied for the investigation of lipids. In this study lipophilic extracts of pig brain (liver, kidney, heart and human blood plasma) were examined with Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS) after separation with solid phase extraction (SPE) in different lipid fractions. The aim of this work was the identification and quantification of the individual components in lipid mixtures. After the class separation the individual components of phosphatidylethanolamine and galactosylcerebroside were further separated with an isocratic High Performance Liquid Chromatography(HPLC)method. At first an optimized dual phase extraction procedure for tissues was developed in this thesis which enables a quantitative isolation of analytes under mild conditions. The focus of this work was the SPE method development. The SPE technique is easy, rapid, and reliable. The growing popularity of SPE is in part due to the operational simplicity and cost reduction in solvents and because it is easy to automate. The developed SPE method for phospholipid class separation with the use of silica gel cartridge allowed the separation of neutral lipids, galactocerebroside, sulfatide, cardiolipin, phosphatidylethanolamine and plasmalogen, as well as the approximately separation of phosphatidylcholine and sphingomyeline. The method proved to be reproducible and showed recoveries higher 73%. The individual components of the phosphatidylethanolamine fraction from each tissue were analyzed with the separation by RP-HPLC-MS. A total of 25 phosphatidylethanolamine components were baseline separated in a period of 20 min (eluting 6.8 min - 18.6 min). Here also the differences of the lipid components of the various organs were analyzed and discussed in relation to their functions. With the same method 19 galactocerebroside were separated. This confirms the potential of the method developed in this work for the detailed characterization of heterogeneous lipid compositions.Dissertation290 139 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, MR-spektroskopische Metabolitenanalyse von Biopsatproben für die Tumordiagnose(2011-09-21); ; ; Gliomas are the most common primary brain tumours among adults and reveal an altered metabolism which can be analyzed by NMR spectroscopy. The tumour delineation is still a challenging task because gliomas are invasively growing tumours which tend to relapse after neurosurgical resection due to (unrecognized) remaining tumour cells. Therefore, the aim of this study was to characterize the regional metabolic profiles of different tumour sections of malignant gliomas. For this purpose specimens of active, viable tumour core, tumour margin and more distant tumour margin were extracted with an optimized dual phase extraction procedure and hydrophilic as well as lipophilic tissue extracts were recorded by NMR spectroscopy. Different pattern recognition methods (partial least squares regression, linear discriminant analysis and self organizing maps) were used to recognize relevant metabolites for an intratumoral discrimination and to visualize the high dimensional data. Furthermore, the metabolic profiles of the different glioma types were compared. The tumours revealed characteristic metabolic profiles which reflected typical neoplastic alterations, i.e. glycine and phosphocholine were markers of malignancy and elevated in tumour core extracts of glioblastomas. Statistical analysis showed that NAA, N-Acetylgalactosamine and creatine were relevant metabolites for an intratumoral discrimination and decreased from more distant tumour margin towards the tumour core because of a displacement of neuronal structures as well as an altered glycoconjugate and energy metabolism in the tumour cells. Furthermore, the latter caused elevated levels of alanine and branched chain amino acids in the tumour core extracts. Among lipids dolichol(phosphate), triacylglycerides and cholesteryl esters were relevant components which were preferentially detected in the tumour core. Also polyunsaturated fatty acids (i.e. arachidonic and linoleic acid) were elevated in the tumor core. In addition, the ratio of galactosyl cerebrosides to sphingomyelins represented a good metabolic discriminator. In order to get further insights in tumour lipid metabolism a solid phase extraction for the separation of neural lipids was developed. Lipid extracts of the three different tumour regions were separated in free fatty acids, neutral and acidic phospholipids, cholesteryl esters, triacylglycerides, cholesterol, di- and monoacylglycerides. Results showed a correlation of polyunsaturated fatty acids with polar lipids in the tumour core confirming a higher demand of membrane fluidity and disordered signaling pathways. In conclusion, hydrophilic as well as lipophilic extracts of the different tumour regions showed distinct alterations in their metabolic profiles which enable an intratumoral discrimination. Metabolic differences are caused by a variable fraction of neoplastic cells in the biopsies, which decreases from tumour core over tumour margin to the more distant tumour margin, and further towards healthy cells with a tumour affected metabolism.Dissertation229 174