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Metabolic effects of bile lipids
Žížalová, Kateřina ; Leníček, Martin (advisor) ; Jirsa, Milan (referee) ; Dvořák, Karel (referee)
Bile acids act as important fat emulsifiers in the human body. However, it is increasingly becoming apparent that they also play a role as important signalling molecules. Bile acids are a relatively heterogeneous group differing also in their metabolic effects. Their individual proportion in the body is highly variable and strongly influenced by the composition of the microbiome. The importance of individual bile acids is not yet well enough described to be fully exploited in clinical practice. For this reason, the determination of bile acids by enzymatic methods seems to be considered sufficient in most departments. Another and more reliable option is the increasingly widespread use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). The aim of my work was to test the reliability of the enzymatic method and to introduce a method for the determination of bile acids, including the measurement of such atypical ones for example microbial conjugates and keto- and isoderivatives. Moreover, I aimed to test sample preparation for the measurement of 7α-hydroxy-4-cholesten-3-one, the bile acid biosynthesis marker. We used these methods to investigate the changes in bile acid composition changes in iron overload rats. In this rat model of genetically determined disorders of iron metabolism, bile...
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Study of redox and adsorption features of bile acids on hanging mercury drop electrode
Yershova, Polina ; Schwarzová, Karolina (advisor) ; Gajdár, Július (referee)
Bile acids are the end products of cholesterol metabolism and are important biological surfactants. The curved shape of their chains allows the cyclization of molecules, and the formation of a supramolecular structure. The goal of this thesis was to study the electrochemical and adsorption behavior of selected bile acids: lithocholic, deoxycholic and cholic acids. The measurements were carried out in the medium Brittonův-Robinson buffer:methanol in the ratio 9:1 using cyclic voltammetry and AC voltammetry methods and measuring the dependence of the differential capacitance Cd on the applied potential E. A hanging mercury drop electrode was used as a working electrode. The measurements showed that bile acids are adsorbed on the surface of the electrode and organizing themselves in self assembled monolayers (SAM). In our case we have observed formation of 2D condensed layers as specific form of SAM. Transfer techniques were used to demonstrate bile acid adsorption. A study of the behavior of lithocholic acid as a function of different pH values showed that only at pH 10.0 to 12.0 2D 2D condensation occurs, i. e. that at pH values in the range of 2.0 to 9.0 it is another type of adsorption. On AC voltammograms, there are a maximum of two areas in which peaks occur: the first is around -0.2 V and the...
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A new approach to the electroanalysis of primary bile acids and related steroids
Klouda, Jan ; Schwarzová, Karolina (advisor) ; Skopalová, Jana (referee) ; Vyskočil, Vlastimil (referee)
In this doctoral thesis, a novel method for the determination of primary bile acids cholic acid and chenodeoxycholic acid is presented. Bile acids play various vital roles in the mammalian body. Moreover, their determination is extremely helpful in liver and biliary disease diagnosis and management. These saturated organic compounds lack strong chromophores and fluorophores in their structure, and thus are usually hard to detect in spectroscopy. For this reason, either instrumentally advanced but expensive methods, such as mass spectrometry, or less reliable enzymatic methods are commonly employed in bile acids quantitation. Hence, the demand for simple and reliable methods for their determination is strong. Bile acids are also known to be virtually inert for direct electrochemical oxidation. Herein, a simple method for their chemical activation for electrochemical oxidation on bare electrode materials was developed, optimized and applied to cholic acid and chenodeoxycholic acid determination. The activation is based on a dehydration reaction of a primary bile acid with 0.1 mol L-1 HClO4 in acetonitrile (water content 0.55%) that introduces double bond(s) into the originally fully saturated steroid core. This naturally increases the electron density in the structure, and thus allows electrochemical...
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Development of electroanalytical methods for detection of bile acids possessing 7α hydroxyl group
Jelšíková, Kristýna ; Schwarzová, Karolina (advisor) ; Dejmková, Hana (referee)
This master's thesis contains a study of electrochemical processes of selected bile acids possessing 7 hydroxyl group (cholic, chenodeoxycholic and −muricholic). The measurements were performed on boron−doped diamond electrode in the non-aqueous medium of acetonitrile and perchloric acid (water content 0.55 %) by cyclic voltammetry. It is known that the electrochemical activity of 7 bile acids is increased by a dehydration reaction between perchloric acid and the 7 bile acid. The subject of the study was the stability of the voltammetric response of chemically activated bile acids in the region of negative potentials. It was found that the presence of oxygen in the measured solution is an important factor for obtaining the cathodic signal of 7 bile acids. It probably performs a regenerative function; the product of the electrochemical reduction is re-oxidized in its presence, which leads to an increase in the voltammetric response. At the same time, it is important that the direction of the scan in cyclic voltammetry first proceeds to positive values. A potential of +2.0 V (vs. Ag/AgNO3 in acetonitrile) must be reached for the HO● radicals to be formed. It is these radicals that presumably lead to the formation of the product(s) of bile acids electrochemical oxidation that can be subsequently...
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Development of voltammetric methods for detection of bile acids and their conjugates
Petráňová, Karolína ; Schwarzová, Karolina (advisor) ; Nesměrák, Karel (referee)
The aim of this thesis is to study the electrochemical properties of bile acids and their conjugates. Specifically, cholic acid, chenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, glycocholic acid, and glycochenodeoxycholic acid were studied. The measurements were carried out on a boron-doped diamond electrode in an environment of acetonitrile and perchloric acid, with a water content of 0.55% in the solution by method of cyclic voltammetry. In order for bile acids to be electrochemically active, they must first be dehydrated, which takes place in a reaction with perchloric acid. This reaction requires a relatively long time, so it was accelerated by heating the compounds. The goal of this work was to determine the conditions under which the dehydration products of bile acids can be detected. The water content that can be added to the solution after dehydration to keep the voltammetric signal stable was determined. Furthermore, it was found that the voltammetric determination can be carried out even at a pH that is not extremely acidic in which is the the dehydration carried out. Finally, calibration dependencies were compiled.
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The use of mercury-based electrode materials for the study of electrochemical reduction of selected bile acids
Hulová, Dagmar ; Schwarzová, Karolina (advisor) ; Navrátil, Tomáš (referee)
The electrochemic's behaviour of the bile acids (cholic, glycocholic, deoxycholic, ursodeoxycholic and lithocholic acid) was studied on the meniscus-modified silver solid amalgam electrode (m-AgSAE) by differential pulse voltammery. Bile acids provide in the solution of the Britton - Robinson buffer and methanol (9:1) in the pH range 3.0 to 12.0 a cathodal signal in the high negative potentials: cholic acid, deoxycholic acid, ursodeoxycholic acid and lithocholic acid about −1400 mV and glycocholic acid, which alone is the conjugate with glycine, about −1500 mV. Cholic acid, glycocholic acid, deoxycholic acid and ursodeoxycholic acid provide the highest peaks to pH 5.0, approximately in their pKa values. Lithocholic acid provides peaks from pH 7.0. It was demonstrated by the cyclic voltammetry that the electrochemical behavior is influenced by the adsorption of the bile acids to the electrode; presumed reaction at the working electrode - a reduction of a proton of a carboxylic group, is controlled by the diffusion and the process is quasireversible. Utilization of the electrochemical reduction of bile acids for the voltammetric determination does not seem very suitable. It has been proven that the presence of the methanol deteriorates the measuring results for glycocholic acid. In the presence of...
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Interaction between adipocytes and immune cells in pathogenesis of obesity related pro-inflammatory state of adipose tissue
Mališová, Lucia ; Rossmeislová, Lenka (advisor) ; Flachs, Pavel (referee) ; Kazdová, Ludmila (referee)
Obesity is considered to be a worldwide epidemic disease characterized by an accumulation of AT. Increased adiposity can perturb normal metabolic functions and lead to the development of diseases like insulin resistance and other metabolic disorders. A large amount of clinical studies have been shown that changes in inflammatory signaling in adipose tissue cells, increased infiltration of immune cells into AT as well as stress of endoplasmic reticulum belong to the key molecular steps leading to the development of metabolic disturbances associated with this disease. Adverse metabolic effects of AT accumulation can be diminished by calorie restriction resulting in weight loss. In addition, stress of endoplasmic reticulum could be alleviated by chemical chaperones including bile acids. These two approaches for the treatment of obesity or the obesity-associated disturbances were basis for this PhD thesis. In the first part of this work, we studied inflammation status of gluteal in comparison with abdominal AT and differentiation and secretory capacity of adipocytes after weight loss in obese patients. We revealed that inflammatory profile of gluteal AT, estimated by mRNA level of macrophages and cytokines as markers of inflammatory status of the body, did not explain the different clinical impact of...
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A new approach to the electroanalysis of primary bile acids and related steroids
Klouda, Jan
In this doctoral thesis, a novel method for the determination of primary bile acids cholic acid and chenodeoxycholic acid is presented. Bile acids play various vital roles in the mammalian body. Moreover, their determination is extremely helpful in liver and biliary disease diagnosis and management. These saturated organic compounds lack strong chromophores and fluorophores in their structure, and thus are usually hard to detect in spectroscopy. For this reason, either instrumentally advanced but expensive methods, such as mass spectrometry, or less reliable enzymatic methods are commonly employed in bile acids quantitation. Hence, the demand for simple and reliable methods for their determination is strong. Bile acids are also known to be virtually inert for direct electrochemical oxidation. Herein, a simple method for their chemical activation for electrochemical oxidation on bare electrode materials was developed, optimized and applied to cholic acid and chenodeoxycholic acid determination. The activation is based on a dehydration reaction of a primary bile acid with 0.1 mol L-1 HClO4 in acetonitrile (water content 0.55%) that introduces double bond(s) into the originally fully saturated steroid core. This naturally increases the electron density in the structure, and thus allows electrochemical...
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A new approach to the electroanalysis of primary bile acids and related steroids
Klouda, Jan
In this doctoral thesis, a novel method for the determination of primary bile acids cholic acid and chenodeoxycholic acid is presented. Bile acids play various vital roles in the mammalian body. Moreover, their determination is extremely helpful in liver and biliary disease diagnosis and management. These saturated organic compounds lack strong chromophores and fluorophores in their structure, and thus are usually hard to detect in spectroscopy. For this reason, either instrumentally advanced but expensive methods, such as mass spectrometry, or less reliable enzymatic methods are commonly employed in bile acids quantitation. Hence, the demand for simple and reliable methods for their determination is strong. Bile acids are also known to be virtually inert for direct electrochemical oxidation. Herein, a simple method for their chemical activation for electrochemical oxidation on bare electrode materials was developed, optimized and applied to cholic acid and chenodeoxycholic acid determination. The activation is based on a dehydration reaction of a primary bile acid with 0.1 mol L-1 HClO4 in acetonitrile (water content 0.55%) that introduces double bond(s) into the originally fully saturated steroid core. This naturally increases the electron density in the structure, and thus allows electrochemical...
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Development of electroanalytical methods for detection of bile acids possessing 7α hydroxyl group
Jelšíková, Kristýna ; Schwarzová, Karolina (advisor) ; Dejmková, Hana (referee)
This master's thesis contains a study of electrochemical processes of selected bile acids possessing 7 hydroxyl group (cholic, chenodeoxycholic and −muricholic). The measurements were performed on boron−doped diamond electrode in the non-aqueous medium of acetonitrile and perchloric acid (water content 0.55 %) by cyclic voltammetry. It is known that the electrochemical activity of 7 bile acids is increased by a dehydration reaction between perchloric acid and the 7 bile acid. The subject of the study was the stability of the voltammetric response of chemically activated bile acids in the region of negative potentials. It was found that the presence of oxygen in the measured solution is an important factor for obtaining the cathodic signal of 7 bile acids. It probably performs a regenerative function; the product of the electrochemical reduction is re-oxidized in its presence, which leads to an increase in the voltammetric response. At the same time, it is important that the direction of the scan in cyclic voltammetry first proceeds to positive values. A potential of +2.0 V (vs. Ag/AgNO3 in acetonitrile) must be reached for the HO● radicals to be formed. It is these radicals that presumably lead to the formation of the product(s) of bile acids electrochemical oxidation that can be subsequently...
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