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Asymmetric Dimethylarginine and Its Relation to Atherogenesis
Široká, Romana ; Racek, Jaroslav (advisor) ; Teplan, Vladimír (referee) ; Hyánek, Josef (referee)
ASYMMETRIC DIMETHYLARGININE - COMPARSION OF CHROMATOGRAPHY AND IMMUNOMETRIC METHODS Objective:Asymmetric dimethylarginine (ADMA) is often discussed in connection with hyperhomocysteinemia and its toxic effect on vessel wall. ADMA concentration is usually measured by HPLC (High Performance Liquid Chromatography) after previous derivatisation. Recently, ELISA (Enzym Linked Immuno Assay) methods for ADMA determination were introduced and ELISA kits are commercially available. Method and Result:The aim of the study was to compare HPLC and ELISA methods for ADMA determination. For HPLC determination we used equipments from Thermo separation product (Florida, USA). After solid-phase extraction on polymer cation-exchange column and the following derivatisation with o-phthaldialdehyde the samples were separated using C18 column (mobile phase 8.7% acetonitril, 50 mmol/l phosphate buffer, pH 6.5) and a fluorescence detector. NG-monomethyl-L-arginine was used as an internal standard. ADMA® ELISA kit, based on a competitive principle, was obtained from DLD Diagnostika, Hamburg, Germany. ADMA was measured in EDTA plasma of 40 healthy blood donors and 40 hemodialysis patients with hyperhomocysteinemia. Conclusion: In spite of different principles both methods showed a very good correlation (r = 0.944, p<0.0001). ELISA...
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Monitoring compensation of patients with cystinuria by spectrophotometric method
Leitner, Martin ; Hyánek, Josef (referee) ; Chrastina, Petr (advisor)
The presented bachelor's thesis deals with the issue of cystinuria, hereditary metabolic disorder of transport of dibasic amino acids. The objective of the thesis was to select a spectrophotometric method of determining cystine in urine, to find the most suitable conditions for determining cystine in a clinical laboratory and to apply the method on samples of urine of patients with cystinuria. For determining cystine in urine, the method using coloured red and violet product of cystine reaction was selected after reduction to cystine with ninhydrin agent in acid environment. Absorption spectra of standard of cystine and urine in a patient with cystinuria were measured with the maximum at 560 nm. Interference of ornithine and other amino acids was excluded. Kinetics of reduction of cystine to cysteine was measured. The most suitable time of reduction is 5 minutes, in longer time cysteine is oxidized to cystine. Kinetics of creation of a coloured product was measured. The maximum absorption in heating the reaction mix with ninhydrin to 100řC was reached after 10 minutes, later only degradation processes and absorbance reduction takes place. Stability of a coloured product of reaction in light was tested. The product is stable for ten minutes, then absorbance drops by 8% after 45 minutes. Linearity of...
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The study of mitochondrial biogenesis during fetal development
Pejznochová, Martina ; Zeman, Jiří (advisor) ; Hyánek, Josef (referee) ; Brdička, Radim (referee)
Postnatal adaptation of neonate to extrauterine life is among others dependent on effective mitochondrial biogenesis during fetal development. Therefore the study of mitochondrial biogenesis on molecular and biochemical level may improve the care of very premature neonates, especially critically ill premature neonates. This thesis has been worked out in The laboratory for study of mitochondrial disorders (Department of Pediatrics, 1st faculty of Medicine, Charles University in Prague). The thesis is based on molecular genetic analyses, which are focus on characterization of expression of gene involved in mitochondrial biogenesis including gene of oxidative phosphorylation system (OXPHOS) and on changes in mtDNA content during human fetal development. The results than enable : Efective analysis of the mRNA expression level by quantitative real-time PCR method in fetal tissues. Analysis of the changes in the mtDNA content in different fetal tissues To understand and to explain the tissue-specific differences in expression of the OXPHOS genes and of the genes involved in mtDNA transcription and in regulation of mtDNA content during second trimester of gestation.
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Study of mitochondrial ultrastructure and functions in selected mitochondrial and lysosomal storage disorders
Kostková, Olga ; Hansíková, Hana (advisor) ; Šmíd, František (referee) ; Hyánek, Josef (referee)
This thesis has been worked out in The laboratory for study of mitochondrial disorders (Departement of Pediatrics, 1st Faculty of Medcine, Chales university in Prague) and in cooperation with The Institute of Inherited Metabolic Disorders. Mitochondrial disorders represent a heterogeneous group of diseases with the onset at any age from neonatal period till adulthood, mostly presented with very severe clinical courses of disease. The mammalian organism is fully dependent on mitochondrial oxidative phosphorylation system as on the major energy producer of the cell. Therefore the mitochondrial disorders affect mainly high energy demanded tissues such as brain, heart or muscle. Simillar phenotype is observed in many lysosomal storage disorders. Despite of expanding knowledge of molecular basis of mitochondrial and lysosomal disorders, it may be still difficult to explain the exact pathogenesis of disease as well as the prognosis for patients and their families. Mitochondrial functions affect more than just energy production; they contribute in initiation of apoptosis, in cellular calcium homeostasis, and in production of reactive oxygene species. Disturbed mitochondria become a goal of autophagy mediated by the lysosomal compartement. The results of our study enable: 1. better understanding of the tissue...
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Examining the significance of peptides regulating the intake of food and the nutritional state of children and adolescents
Bronský, Jiří ; Průša, Richard (advisor) ; Pelouch, Václav (referee) ; Hyánek, Josef (referee)
Introduction: Regulation of food intake and nutritional status is mediated by complex interactions of regulatory peptides of the central nervous system, gastrointestinal tract and adipose tissue. These systems are connected by feedback loops which inform the centre about amount of ingested food and energy reserves in the organism. Dysfunction of any of these regulatory areas may lead to changes in nutritional status of the organism. Methods: We used radioimmunoassay to measure plasma levels of orexin A, total ghrelin and serum levels of leptin and enzyme immunoassay to measure serum levels of adiponectin in healthy subjects and in children with obesity, anorexia nervosa, Crohn's disease and celiac disease and we evaluated the influence of nutritional therapy on these levels. Moreover, we evaluated relationship of these regulatory peptides to other biochemical and anthropometrical factors of nutritional status. We also measured plasma levels of total and unreduced amylin by enzyme immunoassay with immunofluorescence detection in adult patients with osteoporosis, type II diabetes mellitus and in the control group. Results: During reduction of body weight in obese children and adolescents, there were statistically significant changes of plasma orexin A levels and total ghrelin levels, but we haven't seen any...
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