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Analysis of bilirubin's relevant photoproducts and their biological significance in the issue of neonatal jaundice
Křepelka, David ; Kozlík, Petr (advisor) ; Zelenka, Jaroslav (referee)
Neonatal jaundice occurs in almost 60% of full-term and 80% of premature babies, where a slightly increased concentration of bilirubin protects against oxidative stress just after birth. When a specific bilirubin level is exceeded in serum (usually above 340 µmol·l-1 ), bilirubin-could induce kernicterus. These negative states are prevented by blue-green light phototherapy (420-510 nm), which converts bilirubin into more polar photoproducts that are more easily excreted via bile and/or urine. Published data have shown that newborns with indicated phototherapy may develop clinical problems later in life (higher incidence of e.g. asthma, allergies, type 1 diabetes was observed at a later age). A possible reason for the occurrence of these diseases is the specific biological activity of photoproducts. This study aims to purify an unknown photoproduct (band P), formed after 8 hours of irradiation of a bilirubin solution with blue light, and to establish a quantitative analytical method for its measurement in relevant matrices. This product was isolated and separated by a thin layer and subsequent column flash chromatography. In the next part, an LC-MS/MS method was developed for quantification of band P. Finally, urine, plasma, and feces samples collected from 15 newborns before and after phototherapy...
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Study of bilirubin and its oxidation products
Capková, Nikola ; Dvořák, Aleš (advisor) ; Vejražka, Martin (referee) ; Holendová, Blanka (referee)
Phototherapy (PT) with blue-green light (420-490 nm) is the standard treatment for severe neonatal jaundice to prevent infants from toxic bilirubin (BR). Upon blue-green light exposure, BR is converted to more polar photoisomer (PI) lumirubin (LR) and the other oxidation products (mono-, di-, tripyrrols) which can be more easily disposed of the body via urine and/or bile. Although generally considered to be safe, PT is accompanied by an increased risk of various pathophysiological conditions (inflammatory processes, allergies, diabetes, and some types of cancer), in extremely low-birth-weight newborns. Thus, to account for these consequences, our study aimed to understand the mechanism of BR secretion in different tissues and cell lines and investigate the bioactive properties of BR and its main photooxidation product LR. At first, we focused on the detection of BR in the bile and feces of hyperbilirubinemic Gunn rats. Simultaneously, we tested the antioxidant and pro-oxidant effects of unconjugated BR in human hepatoblastoma (HepG2), proximal tubular (HK2), neuroblastoma (SH-SY5Y), and murine endothelial (H5V) cells by exposing them to progressively increasing concentrations of BR. To compare the BR and LR effects on metabolic and oxidative stress markers, the biological activities were...
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Surface-enhanced Raman spectral detection of bilirubin and temporally synchronized monitoring of its photochemical transformations in selected solvents by SERS and electronic absorption spectroscopy
Hrnčířová, Jana ; Vlčková, Blanka (advisor) ; Procházka, Marek (referee)
Surface-enhanced Raman scattering (SERS)-active systems based on macroscopic Ag nanosponge aggregates as well as the conditions of SERS spectral measurements were optimized for selective and sensitive detection of a biomedically important, amphiphilic bile pigment bilirubin (BR) in alkaline aqueous solutions and in its solutions in a selected water miscible solvent, namely dimethylsulfoxide (DMSO) and/or water-immiscible solvent, namely CH2Cl2. Ag nanosponges assembled by using HCl as a pre-aggregation agent were found to be the optimal SERS-active systems for a reliable detection of BR in all the above-mentioned solvents. In all cases, the protonated form of adsorbed BR has been detected upon BR incorporation into Ag nanosponges, and its marker bands have been established by SERS spectral probing at excitation wavelengths in the 445-780 nm range. The sensitivity of SERS spectral detection was evaluated in terms of the concentration values of SERS spectral detection limits (SERS LODs) of BR incorporated into Ag nanosponges. In particular, the SERS LOD for BR incorporated from its alkaline aqueous solution is 1 x 10-8 M (at 532 nm excitation), for BR incorporated from its solution in DMSO, its value is also 1 x 10-8 M (at 532 and/or 633 nm excitations), and for incorporation from the solution of BR...
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Study of thiol addition to biliverdine, the synthesis of labelled bilirubine
Felklová, Veronika ; Lešetický, Ladislav (advisor) ; Smrček, Stanislav (referee)
Biliverdin and bilirubin are bile pigments which are degradation products of heme. Biliverdin (BV) is greenish-blue pigment and is reduction product of tetrapyrrolic core of heme by influence of the hemoxygenase (HO). The final product of this degradation is yellowish-brown pigment bilirubin (BR) which forms from BV thanks to the biliverdinreduktase (BVR). Normal and slightly raised level of bilirubin in plasma has cytoprotective effects whereas high levels are cytotoxic often. In severe unconjugated hyperbilirubinemia cases (newborn children) unconjugated bilirubin (UCB) accumulates in central nervous system (CNS) and causes bilirubin-induced neurologic dysfunction (BIND). Unfortunately there is a limitation for finding UCB pathophysiology caused by difficult determination of UCB content and distribution in tissues and biological fluids. So the main purpose of this thesis is to find and integrate isolated methods which will serve as the basis of finding bilirubin distribution. This progress would have a significant effect on studies of bilirubin neurotoxicity on newborn children. This method is based on radioactive labeling of UCB. Preferentially atom C 10 used for binding suitable functional group (thiols) because conformation of indicated bilirubin shouldn't change in this position. And then the...
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Bilirubin influence on the progression of inflammatory bowel disease.
Patková, Anna ; Nachtigal, Petr (advisor) ; Hronek, Miloslav (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Bilirubin influence on the progression of inflammatory bowel disease Diploma thesis Anna Patková Supervisor: doc. PharmDr. Petr Nachtigal, Ph.D. Background: Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gut caused by an interaction of genetic and environmental factors. It is thought that tissue damage is also partly caused by an oxidative stress. Heme oxygenase-1 and bilirubin are strong antioxidants and both of them provide an anti-inflammatory effect in various tissues. The aim of this diploma thesis was to detect changes of expression of HO-I in the large intestine of normobilirubinemic and hyperbilirubinemic rats after the induction of acute or chronic experimental colitis. Methods: We used Gunn rats with hereditary defect of UDP-glucuronyltransferase, which causes hyperbilirubinemia. The control group of animals was made up of heterozygous littermates of the Gunn rats, which have normal serum bilirubin levels. All animals were treated by dextran sulfate sodium in order to induce an experimental colitis. Rats were divided into two groups. Each of them contained hyperbilirubinemic and normobilirubinemic...
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Inherited Disorders of Bilirubin Metabolism
Šlachtová, Lenka ; Martásek, Pavel (advisor) ; Baxová, Alice (referee)
Inherited disorders of bilirubin metabolism - hereditary hyperbilirubinemias - are metabolic disorders manifested in early childhood. Unconjugated hyperbilirubinemias result from the defect of the enzyme uridine diphosphoglucuronosyltransferase (UGT1A1). UGT1A1 mediates the conjugation of bilirubin with glucuronid acid in hepatocytes and its elimination to water soluble compound. In the next step of bilirubin degradation the transport of conjugated bilirubin from hepatocyte into the bile occure. It is caused by the ATP dependent transporters ABCC2, ATP1B1 and OATP1B3. Mutations in the genes coding the bilirubin transporters results in conjugated hyperbilirubinemia Dubin-Johnson or Rotor syndrome. This study is focused on unconjugated hyperbilirubinemia in adolescents including the non-typical manifestations and the defects of ABCC2 transporter and their phenotype in humans.
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Application of Mass Spectrometry for Analysis of Biologically Active and Clinically Significant Compounds.
Štícha, Martin ; Jelínek, Ivan (advisor) ; Smrček, Stanislav (referee) ; Tůma, Petr (referee)
- 8 - ABSTRACT (EN) The thesis is submitted as a commented set of reviewed publications documenting and depicting the possibilities of mass spectrometry in the field of chemical, biological and pharmaceutical research; namely for the purposes of structure elucidation of selected organometallic complexes, analyses of drugs and their metabolites, monitoring of important biological markers. In course of experimental work, the following objectives were studied and solved: Proposal and realization of micro-scale preparation of selected rhenium complexes with aromatic ligands, utilizing tetrabutyammonium tetrachlorooxorhenate as a starting material; preparation and structure characterization of oxorhenium(V) complexes with 1,2-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2,3- dihydroxynaphtalene as ligands by means of ESI/MS, APPI/MS and LDI-MS; ESI/MS and UV/Vis study of kinetic behavior of complexes arising from the reaction of tetrabutylamonnium tetrachlooxorhenate with pyrogallol and catechol as ligands. Special aim was devoted to the study of subsequent chemical transformation of primarily formed Re(V) complexes; structure characterization of selected ferrocene complexes with copper, gold and silver by means of ESI/MS. Proposal of methodology of structure characterization and quantification of the...
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The study of new risk factors of the cardiovascular diseases
Eremiášová, Lenka ; Danzig, Vilém (advisor) ; Vrablík, Michal (referee) ; Rosolová, Hana (referee)
Bilirubin is a major product of the heme catabolism in the vascular bed with substantial antioxidant properties. These importantly contribute to pathogenesis of diseases associated with increased oxidative stress, including cardiovascular or cancer diseases. In the first part of this PhD project serum bilirubin concentrations were examined in the 1 % representative sample of the general Czech population, together with determination of the prevalence of Gilbert's syndrome. Bilirubin concentrations were determined also within individual polymorphisms of the UGT1A1 gene (OMIM*191740) responsible for bilirubin biotransformation in the liver, including their association with the basic risk factors for atherosclerosis. We also assessed the activity of the standard liver enzymes (representing another significant risk factor for the development of cardiovascular diseases) with surprisingly high proportion of subjects with elevated values. Simultaneously, we determined the concentrations of serum bilirubin in a group of patients with an acute coronary syndrome, who manifested with significantly lower concentrations as compared to general population. In the second part of this research project, the relationship between plasma concentrations of bilirubin and individual variants of UGT1A1 gene polymorphisms...
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Bilirubin secretory pathway and its disorders.
Sticová, Eva ; Jirsa, Milan (advisor) ; Bronský, Jiří (referee) ; Jirásek, Tomáš (referee)
Identification and functional characterization of numerous transport systems at the sinusoidal and canalicular membrane of hepatocytes have significantly expanded our understanding of bilirubin metabolism and contributed to elucidation of molecular basis of hereditary jaundice. Moreover, dysregulation of hepatobiliary transport systems could explain jaundice in many acquired liver disorders. This thesis is focused on the new aspects of bilirubin handling in hepatocytes based on elucidation of the molecular basis of Rotor syndrome. The first study is focused on the antioxidative properties of bilirubin in liver tissue in a model of obstructive cholestasis. In the second part of the thesis we present several novel mutations in ABCC2, the gene associated with Dubin-Johnson syndrome, identified in patients selected for the Rotor locus mapping study. In the key third study concerned with Rotor syndrome we demonstrated that biallelic inactivating mutations causing complete absence of transport proteins OATP1B1 and OATP1B3 result in disruption of hepatic reuptake of bilirubin, which is the molecular basis of Rotor-type jaundice. These results indicate that apart from secretion of conjugated bilirubin into bile, a significant fraction of bilirubin glucuronide is secreted via MRP3 into sinusoidal blood and...
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Antiproliferative effects of heme catabolic pathway's products
Koníčková, Renata ; Vítek, Libor (advisor) ; Haluzík, Martin (referee) ; Farghali, Hassan (referee)
Presented work is focused on heme metabolism with the main interest in bile pigments. Recent data indicate that bilirubin is not only a waste product of the heme catabolic pathway, but also emphasize its important biological impacts, including possible antiproliferative effects. Until today metabolism of bilirubin has not been completely elucidated, which has prevented detailed evaluation of its potential anticancer action. The aim of this study was to clarify some aspects of heme catabolism with respect for antiproliferative properties of its products. Based on the fact that bilirubin potently affects carcinogenesis of the intestine, we initially investigated not properly known bilirubin metabolism by intestinal bacteria. We studied bilirubin neurotoxic effects in hyperbilirubinemic Gunn rats - its distribution in the brain tissue and its degradation during pathological conditions, such as severe newborn jaundice or Crigler-Najjar syndrome. Possible approaches to improve the treatment of severe unconjugated hyperbilirubinemias, combination of the phototherapy and human albumin administration were also investigated. The main reason of these studies was the fact that mechanisms of neurotoxic effects of bilirubin are predominantly identical with those, by which bilirubin inhibits cancer cells growth....
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