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Synthesis and evaluation of human 6-hydroxyceramides
Majcher, Adam ; Opálka, Lukáš (advisor) ; Krátký, Martin (referee)
Title of the Master thesis: Synthesis and evaluation of human 6-hydroxyceramides Candidate: Adam Majcher Supervisor: PharmDr. Lukáš Opálka, Ph.D. Consultant: PharmDr. Andrej Kováčik, Ph.D. Charles University, Faculty of Pharmacy in Hradec Králové Skin Barrier Research Group, Department of Organic and Bioorganic Chemistry Ceramides (Cer), the members of sphingolipid family, occur in all human cells and play an important role in cell signaling. In high concentrations, Cer can also be found in the uppermost layer of epidermis called stratum corneum, along with free fatty acids and cholesterol (in equimolar ratio), where they form the intercellular multi-lamellar lipid matrix. The key function of stratum corneum is to ensure a permeability barrier, thus, to provide water and electrolyte homeostasis, and to prevent entry of harmful substances into the organism. Cer are composed of a sphingoid base and an acyl part derived from a long-chain fatty acid. Cer based on 6-hydroxysphingosine (H) are amongst the most unusual sphingolipids. In contrast to sphingosine-based Cer, 6-hydroxysphingosine-based Cer (H-Cer) are unique for the epidermis and, in addition, H-Cer are not typical for all mammals. Moreover, the function and biosynthesis of H-Cer in the skin is still not completely understood. Several...
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Evaluation of preparation of monolayer lipid skin barrier models
Růžičková, Karolína ; Švecová, Barbora (advisor) ; Vraníková, Barbora (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Karolína Růžičková Supervisor: PharmDr. Barbora Švecová, Ph.D. Consultant: Mgr. Anna Nováčková Title of thesis: Evaluation of preparation of monolayer lipid skin barrier models Skin, the protective barrier of human body, consists of several layers. The uppermost one is the stratum corneum, part of epidermis, whose extracellular matrix is composed mainly of ceramides, cholesterol and free fatty acids. The composition and arrangement of skin lipids are essential for the proper skin barrier function. Various multilayer and monolayer models are used to study skin lipids at the molecular level. Some of the evaluation methods are Langmuir monolayers at the air interface. In this work I dealt with the behavior of monolayer lipid models at four different pH values of the liquid subphase. Lipids isolated from human skin, lipid mixture prepared from the individual components, and a mixture of fatty acids were compared as well. Langmuir isotherms and the Brewster angle microscopy at different compression rates were used for this purpose. The results showed that pH of the subphase has no major effect on lipids arrangement. Lipids were most likely to form a tight monolayer at neutral pH 7,0, at a...
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Optimization of the skin barrier model with isolated ceramides of human stratum corneum
Dulanská, Lucia ; Pullmannová, Petra (advisor) ; Zbytovská, Jarmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Author: Lucia Dulanská Supervisor: Mgr. Petra Pullmannová, Ph.D Title of thesis: Optimization of the skin barrier model with isolated ceramides of human Stratum corneum Stratum corneum (SC), the uppermost layer of the skin, regulates transcutaneous water loss and protects against outer conditions and harmful substances. It consists of cornified cells - corneocytes and extracellular lipid matrix, which is responsible for the barrier functions. Corneocytes are covered with covalently bound lipids creating the corneocyte lipid envelope (CLE). CLE is considered to interconnect the extracellular lipids with corneocytes and to have a templating effect. We aimed to optimize a skin lipid model simulating also the presence of CLE. The lipidic part of the model was prepared from an equimolar mixture of isolated human skin ceramides (hCer), cholesterol and free fatty acids (FFA, either protonated or deuterated) with 5 weight % of cholesteryl sulfate. hCer were extracted from the isolated human SC and purified by the column chromatography. The composition of hCer was determined by the high- performance thin-layer chromatography. The reverse-phase and normal phase silica gel particles served as the CLE...
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Study of free sphingoid bases in skin barrier
Jarešová, Zuzana ; Kováčik, Andrej (advisor) ; Vraníková, Barbora (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Zuzana Jarešová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: STUDY OF FREE SPHINGOID BASES IN SKIN BARRIER The skin barrier, localized in the stratum corneum (SC), consists of corneocytes and an intercellular matrix formed from three types of lipids - ceramides, free fatty acids, and cholesterol, represented in an equimolar ratio. The overall arrangement of lipids is organized and highly specialized. Ceramides are structurally formed from the fatty acid acyl attached to a sphingoid base. In minor but not insignificant amounts, free sphingoid bases can also be found in the skin barrier. Several studies show that there is an increased concentration of free sphingoid bases in skin barrier disorders, such as atopic dermatitis. Although it is assumed that the presence of free sphingoid bases affects the skin barrier, it is not elucidated the way of their participation till today. The lack of studies or their diverse results leads us to the main goal of this thesis - to clarify how free sphingoid bases influence the skin barrier. In this work, the model membranes were prepared by the isolation of human SC ex vivo. Sphingosine (S),...
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Synthesis and evaluation of human 6-hydroxyceramides
Majcher, Adam ; Opálka, Lukáš (advisor) ; Krátký, Martin (referee)
Title of the Master thesis: Synthesis and evaluation of human 6-hydroxyceramides Candidate: Adam Majcher Supervisor: PharmDr. Lukáš Opálka, Ph.D. Consultant: PharmDr. Andrej Kováčik, Ph.D. Charles University, Faculty of Pharmacy in Hradec Králové Skin Barrier Research Group, Department of Organic and Bioorganic Chemistry Ceramides (Cer), the members of sphingolipid family, occur in all human cells and play an important role in cell signaling. In high concentrations, Cer can also be found in the uppermost layer of epidermis called stratum corneum, along with free fatty acids and cholesterol (in equimolar ratio), where they form the intercellular multi-lamellar lipid matrix. The key function of stratum corneum is to ensure a permeability barrier, thus, to provide water and electrolyte homeostasis, and to prevent entry of harmful substances into the organism. Cer are composed of a sphingoid base and an acyl part derived from a long-chain fatty acid. Cer based on 6-hydroxysphingosine (H) are amongst the most unusual sphingolipids. In contrast to sphingosine-based Cer, 6-hydroxysphingosine-based Cer (H-Cer) are unique for the epidermis and, in addition, H-Cer are not typical for all mammals. Moreover, the function and biosynthesis of H-Cer in the skin is still not completely understood. Several...
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Study of sphingosine, dihydrosphingosine and phytosphingosine in skin barrier models
Kubátová, Denisa ; Kováčik, Andrej (advisor) ; Svačinová, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Denisa Kubátová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: Study of sphingosine, dihydrosphingosine and phytosphingosine in skin barrier models The stratum corneum (SC), the uppermost layer of the skin, localized in the uppermost part of the epidermis, represents the skin barrier of the organism. SC is composed of corneocytes and an intercellular lipid matrix, which is formed by ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol) in an equimolar ratio. Substances from the group of sphingolipids - Cer, are sphingoid bases (for example, sphingosine (S), dihydrosphingosine (dS), phytosphingosine (P)) acylated with a fatty acid (for example, lignoceric acid (LIG)). In the lipid matrix, the metabolic products of Cer (free sphingoid bases) are also present, but their role in SC barrier functions is not clear. Some studies show that Cer with different sphingoid bases, and increased presence of free sphingoid bases, can lead to a change in the permeability of the skin barrier. This work aimed to study the effect of permeability of sphingoid bases on the model membrane permeability. Nine types of membranes were prepared; they...
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Study of substances affecting permeability of the skin barrier
Nováčková, Anna ; Zbytovská, Jarmila (advisor) ; Cvačka, Josef (referee) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Anna Nováčková Supervisor doc. Mgr. Jarmila Zbytovská, Dr. rer. nat. Title of Doctoral Thesis Study of substances affecting permeability of the skin barrier The skin barrier plays a vital role in protecting the human body and enables mammals' life on dry land. The epidermis has the primary barrier function due to several cells' layers, which gradually differentiate to their final stage, the stratum corneum (SC). SC is formed by stratified keratinocytes (known as corneocytes) surrounded by a lipid matrix. This intercellular matrix consists of an approximately equimolar ratio of ceramides, free fatty acids and cholesterol. These are particular substances formed in the epidermis from their precursors during the keratinocyte's differentiation, and their arrangement into the multilamellar structure is essential for the impermeability of the skin barrier. However, some substances or factors can disrupt the skin barrier. It is usually an undesirable process of lipid disbalance resulting in disorders or diseases of the skin barrier. On the other hand, specific substances have been developed for a reversible disruption of the skin barrier (so-called enhancers) to allow drug...
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The effects of glucosyl sphingosine on barrier function of skin and complex skin model
Yanok, Oksana ; Kováčik, Andrej (advisor) ; Svačinová, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of pharmeceutical technology Candidate: Oksana Yanok Supervisor: Pharm.Dr. Andrej Kováčik, Ph.D. Title of Diploma Thesis: The effects of glucosyl sphingosine on barrier function of skin and complex skin model. The skin barrier, which provides protection from water loss and harmful environmental influences is located in the stratum corneum. The dominant group of lipids within the stratum corneum are ceramides (Cer), which also have the most important role in ensuring the barrier properties of the skin. The enzymes sphingomyelin deacylase and glucosylceramide deacylase hydrolyze the amide bond of Cer precursors, which leads to highly polar metabolites, called lysolipids. The increased activity of these enzymes is considered to be one of the major factors leading to the development of a number of skin diseases characterized by a skin barrier disorder (for example atopic dermatitis). In this study we prepared model membranes mimicking a healthy skin barrier as an equimolar mixtures of human Cer, cholesterol, free fatty acids with the addition of 5 % cholesterol sulfate. We also prepared models in which the amount of Cer was gradually reduced and replaced by the hydrophilic lysolipid glucosylsphingosine. The permeability was measured...
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Study of substances affecting permeability of the skin barrier
Nováčková, Anna ; Zbytovská, Jarmila (advisor) ; Cvačka, Josef (referee) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Anna Nováčková Supervisor doc. Mgr. Jarmila Zbytovská, Dr. rer. nat. Title of Doctoral Thesis Study of substances affecting permeability of the skin barrier The skin barrier plays a vital role in protecting the human body and enables mammals' life on dry land. The epidermis has the primary barrier function due to several cells' layers, which gradually differentiate to their final stage, the stratum corneum (SC). SC is formed by stratified keratinocytes (known as corneocytes) surrounded by a lipid matrix. This intercellular matrix consists of an approximately equimolar ratio of ceramides, free fatty acids and cholesterol. These are particular substances formed in the epidermis from their precursors during the keratinocyte's differentiation, and their arrangement into the multilamellar structure is essential for the impermeability of the skin barrier. However, some substances or factors can disrupt the skin barrier. It is usually an undesirable process of lipid disbalance resulting in disorders or diseases of the skin barrier. On the other hand, specific substances have been developed for a reversible disruption of the skin barrier (so-called enhancers) to allow drug...
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Optimization of the skin barrier model with isolated ceramides of human stratum corneum
Dulanská, Lucia ; Pullmannová, Petra (advisor) ; Zbytovská, Jarmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Author: Lucia Dulanská Supervisor: Mgr. Petra Pullmannová, Ph.D Title of thesis: Optimization of the skin barrier model with isolated ceramides of human Stratum corneum Stratum corneum (SC), the uppermost layer of the skin, regulates transcutaneous water loss and protects against outer conditions and harmful substances. It consists of cornified cells - corneocytes and extracellular lipid matrix, which is responsible for the barrier functions. Corneocytes are covered with covalently bound lipids creating the corneocyte lipid envelope (CLE). CLE is considered to interconnect the extracellular lipids with corneocytes and to have a templating effect. We aimed to optimize a skin lipid model simulating also the presence of CLE. The lipidic part of the model was prepared from an equimolar mixture of isolated human skin ceramides (hCer), cholesterol and free fatty acids (FFA, either protonated or deuterated) with 5 weight % of cholesteryl sulfate. hCer were extracted from the isolated human SC and purified by the column chromatography. The composition of hCer was determined by the high- performance thin-layer chromatography. The reverse-phase and normal phase silica gel particles served as the CLE...
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