Diploma thesis is devoted to the analysis of triacylglycerol estolides (TG-est) by HPLC/MS. These lipids contain four ester bonds. TG-est are part of some vegetable oils and in mammals function as a storage form of metabolically important fatty acid estolides. The aim of this thesis was to prove the existence of TG-est in vernix caseosa and explore the possibilities of analysis in this material. Standards of TG-est were studied with using high-resolution and tandem mass spectrometry. Was suggested and optimized HPLC method for TG-est in a reverse nonaqueous system with the use of column Kinetex C18 and gradient elution with acetonitrile and 2-propanol. This method was used for analysis vernix caseosa fraction, and it was shown that TG-est are present in the sample. Attempts to simplify the lipid sample before analysis HPLC/MS by TLC fractionation were not very successful.

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...

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...

Abnormalities of lipid metabolism are considered risk factors for cardiovascular, metabolic, nephrologic diseases amd some malignancies, as well. Nowadays, a lot of effort is devoted to study new risk factors and surrogate markers of conditions mentioned above to improve their prognosis and decrease mortality. The aim of this thesis was to provide a comprehensive survey of lipid metabolism, characteristics of different lipid compounds in health and diseases and of possibilities of utilization of selected lipid parameters in the diagnostics of pathological conditions listed above. Selected lioid parameters were observed in several studies, focused on specific pathological conditions. Besides conventional lipid analytes, the composition of fatty acids in plasma lipid pools was studied in healthy controls, and in the patients suffering from metabolic syndrome, chronic pancreatitis, and pancreatic cancer, as well. Markers of an oxidative stress (oxidatively modified LDL particles and conjugated dienes in precipitated LDL) were assessed in healthy controls, patients with metabolic syndrome, chronic pancreatitis, pancreatic cancer, and in the patients with different concentrations of plasma apoB-48, too. LDL particles subfraction were investigated in healthy controls, in the patients with different...

In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...