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Se-Metabolism inside the mammalian organism fed Se-supplemented Brassica napus forage
Žíla, Ondřej ; Čadková, Zuzana (advisor) ; Václav, Václav (referee)
The aim of the thesis was to determine whether the individual Se-speciation in the mammalian organism are affected by the form of received selenium. Selenium is an essential micronutrient important for humans and animals. It plays an important role in the antioxidant protection of the organism and in the conversion of thyroid hormones. In our experiment the laboratory Wistar rats were divided into three groups. Each group had a different diet. The rats were fed with selenium in the form of soy, sodium selenite and extracted rapeseed meal. Urine samples were regularly collected during the four-week experiment and in the end of the feeding study, the blood serum was also collected. The total selenium content was measured by ICP-MS, while the individual Se-speciation in urine and serum by HPLC coupled with ICP-MS. In the urine the identified speciation were methylselenocystein (MeSeCys), trimethylselenium (TMSe) and selenosugar 1 and 3. In the blood serum the measured speciation were TMSe, selenite, selenate and selenosugar 1. For the group fed with sodium selenite the measured values in the urine were generally higher, this might be due to a higher overall intake and also an inorganic form of selenium with a lower absorbency. Groups that received selenium from plant sources took in several Se-compounds and the total measured content of Se-speciation and secretion dynamics were not significantly different. Additionally speciation of selenosugar 2 was measured for the group fed with rapeseed meals, which in the other groups did not appear. When receiving selenium from plant sources the biotransformation in the mammalian organism differs in comparison to receiving selenium from mineral salts. The initial hypothesis that Se-speciation is influenced by the form of selenium administered in the diet was confirm by our results. Since the group fed rapeseed showed similar results as the group fed a standard feed with soy, the extracted rapeseed meal could serve as a good source in livestock nutrition.
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Versatile use of liquid chromatography and mass spectrometry in drug metabolism studies
Suchanová, Bohumila ; Wsól, Vladimír (advisor) ; Solich, Petr (referee) ; Lemr, Karel (referee)
Human organism has always been exposed to a vast array of chemicals encountered in the environment. Chemical revolution has significantly influenced biological evolution of humans leading to serious unpredictable toxicities. In response to continual chemical stress they have developed a variety of enzymes to transform these xenobiotics. Xenobiotics are mostly highly lipophilic and cannot readily be excreted from the body without metabolism to more hydrophilic, water-soluble metabolites. Not only environmental chemicals represent xenobiotics but also drugs, dietary components etc. Biotransformation studies play an important role in the drug discovery and development process. Usually data from drug metabolism is required before a new substance can advance towards the development stages of a new therapeutic agent. Data on metabolism is frequently used to optimize drug candidates, suggest more active compounds or support toxicology studies. The increased flux of new chemical entities into drug discovery has placed an increased need for fast and reliable information on the metabolism of these substances. Liquid chromatography coupled with mass spectrometry can meet demands for rapid drugs and metabolites analysis imposed by modern drug discovery strategies. This dissertation thesis presents an evidence...
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Explant cultures of Higher plants 27
Kostřiba, Jan ; Dušková, Jiřina (advisor) ; Tůmová, Lenka (referee)
Kostřiba J.: In vitro cultures of higher plants XXVII. Diploma Paper, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, 2007, p. 61 The work aimed at biotransformation of exogenous precursors of arbutin with in vitro cultures of Centella asiatica (L.). The precursors of arbutin were added into the medium (hydroquinone, tyrosine, p-coumaric acid, hydroxybenzoic acid). The precursors were used in a concentration of 200 mg/l and the period of their action was 6, 12, 24, 48 and 168 hours (1 week). The cultivation of calluses was created by two different ways - by suspension culture and by culture cultivated on the filter-paper bridge. The cultivation was prosecuted on the light. Positive results (both TLC and HPLC analysis) in arbutin production were obtained in all suspension cultures after an addition of hydroquinone. The largest amount of arbutin in callus cultures was measured after a 48 hours cultivation (0,043 %). After a 168 hours cultivation was amount of arbutin only 0,038 %. In this experimental variant arbutin was also released into the medium (0,096 %). Biotransformation experiments with the others precursors of arbutin were not successful. The way of cultivation by the filter-paper bridges was also unsuccessful.
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The Bachelor Thesis deals with an activation of heterocyclic aromatic amines (HAA) which have numerous carcinogenic effects on studied animals and human cells. These carcinogens are formed during the heat processing of meat and during the smoking. However, further transformation of the compounds is required to gather their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP), especially its forms 1A1 and 1A2. The products of this activation - N-hydroxylamines - are further activated in conjugation reactions. In this work, we were focused on the transformation of N-hydroxylamines to more reactive acetate esters and sulphate esters, which is catalyzed by sulphotranspherase (SULT) and N-acetyltranspherase (NAT), respectively. The affection of these enzymes can control the formation of carcinoma. For example, some dietary compounds, such as caffeic acid and quercetin, are the most common inhibitors of these enzymes: caffeic acid is considered as a strong inhibitor of phenolic sulphotranspherase (P-PST), whereas quercetin is a good inhibitor of NAT. On the other hand, some dietary compounds can also induce an opposite effect: for instance, phenol acids induce the P-PST. (in Czech) Key words: Heterocyclic aromatic amines, biotransformation, cytochrome P450,...
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Ovine hemonchosis-results of its laboratory control in course of experimental infection
Lochmanová, Eva ; Lamka, Jiří (advisor) ; Ducháček, Lubomír (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lochmanová Eva Supervisor: prof. RNDr. Jiří Lamka, CSc. Title of diploma thesis: Ovine hemonchosis - results of its laboratory control in course of experimental infection The anthelmintic resistance is a matter of interest in many professional workplaces, as it creates high losses for practical farm animals breeding and it represents a potentially limiting factor for the existence of their own holdings. The mechanisms leading to the anthelmintic resistance emergence are studied, and among them there is also the possibility of induction of biotransformation enzymes, that the parasites (round worms) use to overcome the contact with what is xenobiotics for themselves. Parasitic models and in the long term also Haemonchus contortus are used. Three strains of the parasite are used in the Department of Biochemical Sciences Faculty of Pharmacy, Charles University in Hradec Kralove: fully susceptible, fully resistant, resistant only to benzimidazole anthelmintics. Parasites are experimentally reared in sheep. The aim of this thesis was to experimentally document parasitoses experimental course all of these strains by parasitological methods. L3 larvae Haemonchus contortus were given to...
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Endoparasites of cattle under various breeding management
HOLUBOVÁ, Nikola
Samples of excrements for parasitic investigation was taken away in 16 breeding, when it was about 3 breeding - when animals were been regularly milking, 11 breeding of fatcattle, one breeding of wisents and one breeding of aurochs. It was been observing the influence of technology of breeding, when it was about ecology breeding. Utility type of fatcattle was been permanently grazing, milk animal was 6 months grazing and 6 months stabled. Hobby breeding of wisents and aurochs was been breeded whole year on the pasture. According the lokality, where the breeding is, was noticed the altitude. Statistic analysis proved that the decrease of prevalence kryptosporids infection was in the straight relationship with the increase of altitude. The most infected was herds breeded to the 500 metres altitude. Ossurrence of endoparasites was observed on the dependence of seasonal occurrence. The most low prevalence of parasites infections was found out at miking animals, so that means stabled animals and grazing. Statistic analysis proved animals breeded in system of whole year grazing are statistical to much more infected by parasites than animals breed like technology combined grazing and stabling. Pursuance of occurence and risk of infection of individual species of parasites were consensus statistical comparison found out that animal whole year grazing are 7,25× frequently infected by fluke of Paramphistomum genus (?2=16,4; d.f.=1; P<0,001). In the comparison was no found out diference beetwen occurrence of cocsids genus Eimeria, infusorian of Buxtonella genus, nematode Trichostrongylidea family and fluke Fasciola hepatica species in the dependence of cattle breeding technology. In the breeding was used anti-parasites medicamets IVOMEC SUPER and HELMIGAL. Pursuance of detailed analysis I grew up the end that application of anti-parasites medicamets had no influence on occurrence and prevalence of Fasciola hepatica fluke s pecies and pulmonary nemathods Dictyocaulus genus. On the contrary animals - which weren´t cured anti-parasites medicamets ? were 4,85× frequently inficated by gastrointestinal nemathods (GIN).
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Study of chiral aspects of drug metabolism using instrumental analytic methods
Holmanová, Veronika ; Nobilis, Milan (advisor) ; Kovaříková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Candidate: Bc. Veronika Holmanová Supervisor: Doc. PharmDr. Milan Nobilis, CSc. Title of diploma thesis: Study of chiral aspects of drug metabolism using instrumental analytic methods For the evaluation of stereospecificity of rat and human cytosolic carbonyl reductases and activity of microsomal oxidases involved in nabumetone biotransformation, a new chiral high-performance liquid chromatographic method was developed. The prepared LLE-HPLC-PDA method enabled extraction, separation and ultraviolet detection of prochiral nabumetone and its five phase I metabolites including enantiomers of two chiral biotransformation products of carbonyl reduction. Methyl ester of naproxen served as an internal standard. Diethyl ether was used for liquid-liquid extraction (LLE) of biomatrices. Chiralcel OD-R 250 mm×4.6 mm column with a mobile phase methanol-1M NaClO4/HClO4 aqueous solution pH=3 (75:25, v/v) were employed in isocratic sufficient separation of nine analytes. The whole analysis lasted 60 minutes at the flow rate of 0.5 ml/min. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at λ=265 nm). The results of in vitro nabumetone...
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Metabolism of anthelmintics in plants
Pavlík, František ; Szotáková, Barbora (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: František Pavlík Supervisor: Prof. Ing. Barbora Szotáková, Ph.D. Consultant: RNDr. Radka Podlipná, Ph.D. Title of diploma thesis: Metabolism of anthelmintics in plants Anthelmintics enter into environment mainly through excretion of treated animals. They may cause a lot of adverse effects in ecosystem as they can affect non- target animals or they can accumulate in plants. Further, after consumption of these plants by livestock on pastures the resistence of helminths can be induced. Therefore, the present study was designed to find out biotransformation of frequently used anthelmintics flubendazole (FLU), albendazole (ABZ), fenbendazole (FBZ), ivermectine (IVM) and monepantel (MOP) by the common meadow plants harebell (Campanula rotundifolia) and plantain (Plantago lanceolata), which may often come into contact with anthelmintics through the excrements of treated animals. As model systems, suspensions of harebell and plantain cells and in vitro regenerants of plantain were used. There was no toxic effect of anthelmintics for these plants; they were able to metabolize anthelmintics into a wide scale of various compounds. These metabolites were identified by ultrahigh-performance liquid...
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Identification of metabolites of anthelminthics in helminth parasites using mass spectrometry
Komrska, Jan ; Vokřál, Ivan (referee) ; Skálová, Lenka (advisor)
If we want be successful in the treatment of parasitic disease, it is important to get the utmost information on the fate of given drug. The discovery of the metabolism of anthelmintics and in cosequence of participating enzymes can help us fight the rising resistance to given drugs. The aim of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole (ABZ), flubendazole (FLU) and mebendazole (MEB) formed in ex vivo incubations by parasitic helminth Dicrocoelium dendriticum, using liquid chromatography-mass spectrometric techniques. In the ex vivo study, D. dendriticum adults were incubated in cultivating medium in presence of benzimidazole drug for 24 hours. After incubation of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid phase extraction. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS). We detected in phase I albendazole sulphoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methylderivatives of flubendazole, reduced flubendazole and reduced mebendazole were observed.
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The assay of peroxidases activities and their comparison in various helminths
Sulanová, Lenka ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lenka Sulanová Supervisor: Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The assay of peroxidases activities and their comparison in various helminths The aim of diploma thesis was to search for simple methods for determination of peroxidases activities, to choose a suitable method, optimize it and compare it with the method used in the workplace. The 3,3′,5,5′-tetramethylbenzidine (TMB) has been used as a substrate in selected technique. I have optimized this method and compared it with the formely applied method using substrate O-phenylenediamine dihydrochloride (OPD). Method working with TMB is more precise, more comfortable but more expensive than method using OPD. I have applied both of methods for assay of peroxidases activities in the subcellular fractions of the two species of helminths: Dicrocoelicum dendriticum and Haemonchus contortus. I found out that activities of peroxidases are higher at Haemonchus contortus than at Dicrocoelicum dendriticum. Different activities of peroxidases were observed in the subcellular fractions of the Haemonchus contortus strains sensitive and resistant to anthelmintics. The ex vivo contact of Dicrocoelicum dendriticum with...
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