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Determination of selected components in human urine with electrophoresis in short capillary.
Makrlíková, Anna
Capillary zone electrophoresis is frequently used in various analyses. In this diploma thesis a hydrodynamic sample introduction method controlled by pressure pulse has been proposed for short-capillary electrophoresis. The base electrolyte flushes sample from the loop of a six-way sampling valve and is carried to the injection end of the capillary. At the time when the sample zone reached the capillary, a short pressure impulse is generated in the electrolyte stream, which provides injection of the sample into the capillary. Then the electrolyte flow is stopped and the separation voltage is turned on. The amount of sample introduced to the capillary is controlled by the duration of the pressure pulse. This new sample introduction method was tested in the determination of ammonia, histidine, creatinine, uric acid and hippuric acid in human urine and for rapid screening of the contents of the inorganic ions in cerebrospinal fluid and blood plasma. The determination was performed in a capillary with an overall length of 10,5 cm and two base electrolytes was tested - 50 mM MES + 5 mM NaOH (pH 5,10) and 1 M acetic acid + 1,5 mM crown ether 18-crown-6 (pH 2,40). Using dual detection techniques contactless conductivity and UV spectrometric detection, anorganic and organic substances in the sample could...
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Analysis of cell-free nucleic acids in urine of urological patients.
Šantorová, Šárka ; Korabečná, Marie (advisor) ; Brdička, Radim (referee) ; Drábek, Jiří (referee)
The two studies follow free nucleic acids in urine in search for biomarkers to distinguish urinary bladder cancer patients from controls. Bladder cancer forms 4 % of newly diagnosed oncological diseases in the Czech Republic. Nowadays, there is no accredited non-invasive method for its diagnosis, which is sufficiently accurate. Urine supernatant, which is washing the bladder mucosa and which does not contain cell debris, seems to be an appropriate source of biomarkers for non-invasive diagnosis. miRNAs, as a non-invasive biomarker of urinary bladder cancer, were studied in one of the studies. miRNAs are short noncoding RNA, which block the process of translation. miRNAs occur in all body fluids and are relatively stable. A study with three phases was assessed to find a suitable miRNA marker. 109 individuals were examined in total (36 controls and 73 bladder cancer patients). The analysis of miRNAs was based on RT-PCR (Reverse Transcription Polymerase Chain Reaction). In the first phase, the urine of 59 individuals was analyzed on TaqMan array card with 381 miRNAs. In the second phase, the results of the first phase were confirmed on the same cohort by a single miRNA assay. In the third phase, a new cohort was used (23 controls and 27 bladder cancer patients), analyzed by a single miRNA assay again....
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Saturace koní jodem v Jižních Čechách
SLAVÍKOVÁ, Sofia
The main aim of this thesis was to evaluate the current state of iodine levels in horses from selected farms in South Bohemia. Out of the sample group, no animal with iodine deficiency was detected. Additional objective of this thesis was to assess whether selected horse owners supplement their horses with iodine and what form of supplementation they choose.
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Determination of selected components in human urine with electrophoresis in short capillary.
Makrlíková, Anna
Capillary zone electrophoresis is frequently used in various analyses. In this diploma thesis a hydrodynamic sample introduction method controlled by pressure pulse has been proposed for short-capillary electrophoresis. The base electrolyte flushes sample from the loop of a six-way sampling valve and is carried to the injection end of the capillary. At the time when the sample zone reached the capillary, a short pressure impulse is generated in the electrolyte stream, which provides injection of the sample into the capillary. Then the electrolyte flow is stopped and the separation voltage is turned on. The amount of sample introduced to the capillary is controlled by the duration of the pressure pulse. This new sample introduction method was tested in the determination of ammonia, histidine, creatinine, uric acid and hippuric acid in human urine and for rapid screening of the contents of the inorganic ions in cerebrospinal fluid and blood plasma. The determination was performed in a capillary with an overall length of 10,5 cm and two base electrolytes was tested - 50 mM MES + 5 mM NaOH (pH 5,10) and 1 M acetic acid + 1,5 mM crown ether 18-crown-6 (pH 2,40). Using dual detection techniques contactless conductivity and UV spectrometric detection, anorganic and organic substances in the sample could...
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Voltammetric Determination of Tumor Biomarkers using Flow Injection Analysis with Amperometric Detection
Němečková-Makrlíková, Anna ; Matysik, F.-M. ; Navrátil, Tomáš ; Barek, J. ; Vyskočil, V.
Three tumor biomarkers (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindole-3-acetic acid) have been determined by flow injection analysis with amperometric detection at screen-printed carbon electrodes in optimum medium of Britton-Robinson buffer (0.04 mol l(-1), pH = 2.0). Dependences of the peaks current on the concentration of biomarkers were linear in the tested concentration region from 0.05 to 100 mu mol l(-1), with the limits of detection of 0.065 mu mol l(-1) for homovanillic acid, 0.053 mu mol l(-1) for vanillylmandelic acid, and 0.033 mu mol l(-1) for 5-hydroxyindole-3-acetic acid (calculated from heights), and 0.024 mu mol l(-1) for homovanillic acid, 0.020 mu mol l(-1) for vanillylmandelic acid, and 0.012 mu mol l(-1) for 5-hydroxyindole-3-acetic acid (calculated fromareas), respectively.
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Optimization of urinary exosome isolation for proteomic analysis in kidney disease diagnosis
Ulrychová, Lucie ; Přikryl, Petr (advisor) ; Kučerová, Zdenka (referee)
Extracellular vesicles (exosomes) are the subject of current nephrology proteomics research as they are considered as a promising source of potential biomarkers of kidney disease. This work is focused on discovery of the most appropriate procedure for the urinary exosomes isolation. We have compared already described methods, based on different physicochemical principles of isolation: hydrostatic filtration dialysis (HFD), differential ultracentrifugation, ultrafiltration through a 100 kDa filter, or sample precipitation with Total Exosome Isolation (from urine) kit. Characterization of individual isolated exosomal fractions was performed using SDS-PAGE method (presence of contaminating proteins), western blot analysis (detection of exosomal markers TSG101, alix), nanoparticle tracking analysis (NTA, vesicle size and concentration) or transmission electron microscopy (TEM, vesicles morphology). Due to the presence of contaminating proteins in urine samples, which could distort the results of subsequent proteomic assays, the conditions for the cleavage of undesirable proteins by proteinase K prior to their own isolation were optimized. It has been found that the best yield and purity of the isolated exosomal fractions were provided by a process combining HFD with differential ultracentrifugation...
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Gene expression of selected genes in heart of long-term cold acclimated rat under mild conditions
Marvanová, Aneta ; Žurmanová, Jitka (advisor) ; Nováková, Olga (referee)
Beneficial effect of hardening or cold adaptation for human health has been known for many decades. Beneficial or detrimental effect depends on the regimen and intensity of the adaptation and thus it is very important to pay attention to them. It has been shown in previous studies that cold adaptation of animals at a low temperature 0-4 žC is accompanied by dehydration and the damage of kidneys. As our best knowledge, the effect of mild cold adaptation on the molecular mechanisms in the heart has not been studied until now. The first aim of this study was to set up a new model of cold adaptation without any negative effects on the organism. The second aim was to assess mRNA levels of selected genes related to the important mechanisms associated with energy metabolism and cardio-protective signaling pathways in the left ventricle. Results obtained using the new model for cold adaptation at 102 řC did not show any negative changes in the blood or urine parameters and body fluid balance was maintained. Screening of mRNAs showed that chronic adaptation might affect the phenotype of the heart, transcriptional activity of the antioxidant system and of the pro-apoptotic genes, lowers adrenergic signaling and moderate glucose metabolism in cardiomyocytes. Key words: cold adaptation, heart, gene...
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In-matrix esterification of polar carboxylic acids in urine
Anýžová, Petra ; Mráz, Jaroslav (advisor) ; Feltl, Ladislav (referee)
A set of 15 carboxylic acids of various structural types was used to test a new procedure of esterification of these substances in the urine without previous extraction. The acids were first measured by a reference method without the matrix, and then by a procedure, where lyophilized urine was treated with derivatizing reagent alcohol·HCl, which was then evaporated, the residue dissolved in water, esters extracted into an organic solvent and subsequently determined by GC/FID and GC/MS. The work is mainly focused on 2-hydroxyethyl- and carboxymethyl-mercapturic acids, but the procedure was also tested on di-, tri- and hydroxy-carboxylic acids, known as physiological urinary components or markers of metabolic disorders. Key words: carboxylic acids mecapturic acids freeze-dried urine esterification gas chromatography
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Determination of selected components in human urine with electrophoresis in short capillary.
Makrlíková, Anna ; Opekar, František (advisor) ; Jelínek, Ivan (referee)
Capillary zone electrophoresis is frequently used in various analyses. In this diploma thesis a hydrodynamic sample introduction method controlled by pressure pulse has been proposed for short-capillary electrophoresis. The base electrolyte flushes sample from the loop of a six-way sampling valve and is carried to the injection end of the capillary. At the time when the sample zone reached the capillary, a short pressure impulse is generated in the electrolyte stream, which provides injection of the sample into the capillary. Then the electrolyte flow is stopped and the separation voltage is turned on. The amount of sample introduced to the capillary is controlled by the duration of the pressure pulse. This new sample introduction method was tested in the determination of ammonia, histidine, creatinine, uric acid and hippuric acid in human urine and for rapid screening of the contents of the inorganic ions in cerebrospinal fluid and blood plasma. The determination was performed in a capillary with an overall length of 10,5 cm and two base electrolytes was tested - 50 mM MES + 5 mM NaOH (pH 5,10) and 1 M acetic acid + 1,5 mM crown ether 18-crown-6 (pH 2,40). Using dual detection techniques contactless conductivity and UV spectrometric detection, anorganic and organic substances in the sample could...
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Capillary electrophoresis with contactless conductivity detection for rapid determination of meldonium in urine
Šlampová, Andrea ; Kubáň, Pavel
A simple, rapid and cheap capillary electrophoresis-capacitively coupled contactless\nconductivity detection (CE-C4D) method for determination of meldonium (MEL) in\nurine samples was developed. Sample pretreatment was minimized to dilution of urine\nsamples with DI water and MEL was determined by CE-C4D in background electrolyte\nconsisting of 2 M acetic acid (pH 2.3) in ng mL−1 to mg mL−1 concentration range.\nSample-to-sample analysis time was less than 4 min, limit of detection and\nquantification was 0.015 and 0.05 μg mL−1, respectively, and the method showed\nexcellent linearity (r2 ≥ 0.9998), recovery (97.6–99.9%) and intraday as well as interday\nrepeatability (RSD ≤ 4.7%).
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