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Chromatographic and mass-spectrometric analysis of lipids of vernix caseosa
Vavrušová, Aneta ; Cvačka, Josef (advisor) ; Kuda, Ondřej (referee) ; Lísa, Miroslav (referee)
Lipidomics is a rapidly expanding research field that has captured extensive attention worldwide in the past few years due to the increasing awareness of the crucial roles of lipids in biological systems. The aim of lipidomics is to comprehensively analyze all lipids, to study their structure, biological function within the cell as well as interactions of lipids with other molecules. A combination of advanced analytical techniques, such as extraction, chromatography and mass spectrometry, is an effective tool for studying all aspects of lipidomics. This dissertation thesis is based on two journal publications and presents application of analytical strategies based on chromatography and mass spectrometry for investigation and characterization of new lipid classes of vernix caseosa. Firstly, the applicability of nonaqueous reversed-phase liquid chromatography atmospheric pressure ionization tandem mass spectrometry (LC- APCI-MS2 ) for structural characterization of cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids (Chl-ωOAHFAs) in vernix caseosa was investigated. For this purpose, a TLC chromatography method for the isolation of neutral Chl-ωOAHFAs from vernix caseosa was developed. Their general structure was established using multi-step mass spectrometric approach requiring transesterification...
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Metabolipidomic profiling of white adipose tissue by UPLC-MS/MS
Rombaldová, Martina ; Kuda, Ondřej (advisor) ; Šiklová, Michaela (referee) ; Čajka, Tomáš (referee)
Obesity is a serious problem in society today [1,2]. It might seems to have been caused simply by excess consumption of food compared to energy expenditure but obesity is actually a complex metabolic disorder centred on adipose lipid metabolism and cellular signalling systems linked to it [3]. Understanding the biology of adipose tissue (AT) is very important for the identification of novel and potential therapeutic targets in order to prevent and treat obesity-related disorders [4]. We utilized analytical approach liquid chromatography coupled to mass spectrometry (LC-MS) to study adipose tissue metabolism. Also, we were especially interested in the effect of omega-3 polyunsaturated fatty acids (PUFA) on that metabolism. Rodent and cell line experiments were performed and analyses were done of white adipose tissue (WAT), serum/plasma samples or cells as well as milk samples from mothers. At first, we established several ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for analysis of acylcarnitines (AC), amino acids (AmA) and other metabolites. Importantly, these methods were able to distinguish isobaric species of AC which is not usually possible. Using these approaches we uncovered several acylcarnitines, i.e. long chain AC, carnitine, acylcarnitine C4 and...
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Lipidomic analysis of vernix caseosa
Harazim, Eva ; Cvačka, Josef (advisor) ; Lísa, Miroslav (referee) ; Kuda, Ondřej (referee)
Methods of analytical chemistry are widely used in lipidomics. Separation techniques coupled to mass spectrometry or nuclear magnetic resonance are used very often. They make it possible to identify lipids present in the matrix in very small quantities. This work summarizes the application of modern analytical methods and instrumentation for identifying and characterizing lipids in vernix caseosa. It is shown how I contributed during the Ph.D. studies to the elucidation of the structure and characterization of unknown lipid classes followed by more detailed description of those lipid classes already identified in vernix caseosa. An integral part of my work was the application of the method enabling the localization of double bonds developed by our laboratory in triacylglycerols and 1,2-diol diesters in vernix caseosa. This analytical method is based on the formation of an acetonitrile adduct in an ionization source of a mass spectrometer enabling atmospheric pressure ionization. The complexity of the triacylglycerol class did not allow a complete characterization of the double bonds. However, the fragmentation mostly showed that double bonds up to n-12 position are present, but small peaks in some spectra also indicated double bonds at more distant positions from the chain termini. I have also...
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Regulation of lipogenesis in human adipose tissue: Effect of metabolic stress, dietary intervention and aging.
Šrámková, Veronika ; Rossmeislová, Lenka (advisor) ; Cahová, Monika (referee) ; Kuda, Ondřej (referee) ; Tanti, Jean-Francois (referee)
Adipose tissue (AT) is a complex organ specialised in safe storage and release of energy as lipids. The adipose organ is therefore essential for the maintenance of energy homeostasis. The prototypical cells of AT are adipocytes, emerging from the precursors in a process called adipogenesis. Adipogenesis itself is tightly connected with lipogenesis, i.e. with the synthesis of fatty acids and triglycerides. Various stimuli can disturb adipocyte differentiation and lipogenesis and thus contribute to AT dysfunction and development of associated metabolic diseases. This thesis was endoplasmicfocused on the investigation of lipogenesis in the context of reticulum stress (ERS), calorie restriction and aging. In Project A, we showed that exposition of adipocytes to high acute ERS inhibits expression of lipogenic genes and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to ERS impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in adipocytes. These effects of ERS could therefore contribute to the worsening of AT function seen in obesity. The capacity of AT to store lipids decreases in aging, possibly due to the accumulation of senescence cells or higher ERS. In Project B, we investigated lipogenic capacity of...
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Role of phospholipases A2 in the mechanism of cardioprotection induced by adaptation to chronic hypoxia
Míčová, Petra ; Novotný, Jiří (advisor) ; Kuda, Ondřej (referee) ; Kazdová, Ludmila (referee)
Cardiovascular diseases, particularly acute myocardial infarction, are the leading causes of death in developed countries including the Czech Republic. One of the ways to increase cardiac resistance against acute ischemia/reperfusion (I/R) injury is adaptation to chronic hypoxia. However, changes at the molecular level associated with this adaptation have still not been fully explored. It is obvious that the myocardial function depends on maintaining membrane integrity and cellular homeostasis of cardiomyocytes. From this perspective, phospholipases A2 (PLA2) are the key enzymes that take part in the remodeling and repairing of the cell membranes. Moreover, PLA2 are also involved in generation of lipid signaling molecules - free long chain fatty acids (FA) and 2-lysophopholipids. In myocardium, members of three major PLA2 classes are present: cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2) and secretoric PLA2 (sPLA2). This thesis aimed to determine the following in the left ventricular myocardium of adult male Wistar rats: 1) The effect of intermittent hypobaric hypoxia (IHH; 8 hours/day, 5 days/week, 5 weeks, ~ 7000 m) on the expression of total cPLA2α and its phosphorylated form (p-cPLA2α, Ser505 ), and further iPLA2 and sPLA2IIA, as well as signaling proteins activating cPLA2α enzyme...
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Targeted analysis of branched-chain faty acid esters by LC/MS
Březinová, Marie ; Kuda, Ondřej (advisor) ; Vrkoslav, Vladimír (referee)
Lipidomics as a part of metabolomics is a fast-growing area of research due to the improvement in analytical techniques. This master thesis is focused on lipid extraction techniques optimization, using liquid liquid extraction and solid phase extraction as pre-separation methods and ultra performance liquid chromatography coupled with mass spectrometry for extraction and subsequent identification of branched-chain fatty acid esters (FAHFA - branched-chain Fatty Acid esters of Hydroxy Fatty Acids). This newly discovered class of lipid molecules is associated with insulin secretion, which could improve whole body and local glucose metabolism, providing potential for diabetes 2 type treatment. Solid phase extraction of biological samples was optimized on columns regarding to sorbent composition using reversed phase columns with modified styrene divinylbenzene polymer or octadecyl-bonded polymer and normal phase columns packed with silica gel. Column Strata SI-1 Silica was the most effective for FAHFA separation from biological samples. Chromatographic separation of FAHFA was performed on UPLC Ultimate 3000 RSLC equipped with Kinetex C18 1,7 µm, 2,1 x 150 mm column using gradient program. UPLC was coupled to QTRAP 5500/SelexION, a hybrid, triple quadrupole, linear ion trap mass spectrometer equipped...
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Structural and Functional Interactions of Mitochondrial ADP-Phosphorylating Apparatus
Nůsková, Hana ; Houštěk, Josef (advisor) ; Kolarov, Jordan (referee) ; Kuda, Ondřej (referee) ; Panicucci Zíková, Alena (referee)
The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...
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Targeted analysis of eicosanoids by LC/MS
Moravcová, Marie ; Kuda, Ondřej (advisor) ; Kozlík, Petr (referee)
Eicosanoids represent large group of biologically active lipid metabolites syntesized from polyunsaturated fatty acids that play an important role in many physiological processes. To describe complex metabolism of these compounds, analytical methods including extraction from a biological sample using solid phase extraction and liquid chromatography coupled with mass spectrometer detection were used. Solid phase extraction of biological samples was optimized on four types of reverse phase columns of which column Strata X 60 mg/3 ml, 33 um (Phenomenex , USA) was the most effective. Also, alternative solid phase extraction of eicosanoids using columns with ion exchange sorbents and a column with normal phase were tested, but proved to be unsuitable for targeted analysis of eicosanoids. The extraction method yielding the best results - Strata X 60 mg/3 ml, 33 µm (Phenomenex, USA) was used for the separation of eicosanoids from mouse gonadal fat samples. Eicosanoids were analyzed by liquid chromatography and the separation mechanisms were tested on three UPLC core-shell columns of different lengths (50 mm, 100 mm, 150 mm). The most effective separation of prostaglandin E2 and prostaglandin D2 was achieved using the longest column Kinetex 150 mm × 2,1 mm, 2,6 µm. Furthermore, ionization parameters, such...
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Obesity its causes and cosequences
Ondráčková, Anna ; Vybíral, Stanislav (advisor) ; Kuda, Ondřej (referee)
Obesity is one of the most common health problems in the Word. Over 500 million adults and over 40 million children are obese. The aim of this work is to summarize findings of causes and consequences of this health problem. Even though in some cases only genetic mutations are responsible for obesity for example mutation in ob gene or Prader-Willi syndrome most cases are caused by lifestyle of people -excessive food intake and insufficient physical activity. There are many health problems accompanying obesity. The most serious problems are metabolic syndrome, diabetes mellitus, cardiovascular diseases and cancer. The consequences of obesity are, however, not confined only to health. We can also observe the economic consequences. Financial cost of treatment of obesity and related diseases are estimated in billions of dollars. Pharmacological treatment has so far proved to be ineffective. If drugs are effective, the side effects are so severe that their use is not possible. So far, the adipose tissue hormones that regulate food intake provide the best hope.
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Effects of n-3 polyunsaturated fatty acids, rosiglitazone, low caloric diet and environmental pollutants: obesity and related disorders
Kuda, Ondřej ; Kopecký, Jan (advisor) ; Bendlová, Běla (referee) ; Pravenec, Michal (referee)
4. CONCLUSIONS Conceming the specific aims of the thesis, the following conclusions may be formulated: l.Additionalbeneficia|effectsofthecombinedfieatmentbyn_3PUFAof marine origin and anti-diabetic drug rosiglitazone on obesity and related disorders were proven in mice fed high-fed diet. In comparison with either n- 3 PUFA or TZD effects alone, the combined treatment was more potent in amelioration of the following adverse pheÍrotypes: a. body weight, body weight gain and body lipid content; b. dYsliPidaemia; c. muscle insulin resistance demonstrated as enhanced muscle glycogen synthesis; obesity-associated inflammation of adipose tissue; and inaddition,additiveinductionofadiponectinwasfound,especiallyits multimeric form, which induces insulin sansitivity' These results on mouse modet suggest possible application for human medicine, where fish oil concentrates may enhance efficacy ofthe treatment byTZD,and reduce the risk of the adverse effect of the TzD-therapy' 2. Relatively specific induction oflipid oxidation by n-3 PUFA in the intestine was found, which may be important for e'g' anti-obesity and hypolipidemic effects ofthese liPids. 3. Changes in adiponectin concentration as well as distribution of its multimeric forms as a result oflow caloric diet were found in human patients' 4. Association between...
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