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Method Development for the Analysis of Steroids, Steroid Acids and Sterol Sulfates in Biological Samples

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Method Development for the Analysis of Steroids, Steroid Acids and Sterol Sulfates in Biological Samples Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Method development for the analysis of steroids, steroid acids and sterol sulfates in biological samples Julia Christina Junker aus Erlangen 2021 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Franz Bracher betreut. Eidesstattliche Versicherung Diese Dissertation wurde selbstständig und ohne unerlaubte Hilfe erarbeitet. München, den 19.02.2021 _________________ Julia Christina Junker Dissertation eingereicht am: 22.02.2021 1. Gutachter: Prof. Dr. Franz Bracher 2. Gutachter: Prof. Dr. Michael Vogeser Mündliche Prüfung am: 23.03.2021 Danksagung Mein Dank gilt zuallererst Herrn Prof. Dr. Franz Bracher dafür, dass er mich als “Analytikerin” in seinen Arbeitskreis aufgenommen hat und meine Arbeit von Anfang an unterstützt und gefördet hat. Desweiteren gilt mein Dank Prof Dr. Vogeser für die freundliche Übernahme des Koreferats. Ihm und allen anderen Mitgliedern der Prüfungskommission danke ich für Ihre Zeit und Hilfe. Ganz besonders bedanken möchte ich mich bei Dr. Christoph Müller, der mir immer mit Rat und Tat zur Seite stand, für seine engagierte fachliche Unterstützung und die vielen konstruktiven Gespräche. Meinen Kooperationspartnern Prof. Dr. Harald Steiner und Dr. Frits Kamp möchte ich für die Unterstützung und Förderung des Projekts danken. Ebenso möchte ich mich bei allen Mitgliedern des Arbeitskreises ganz herzlich für die schöne gemeinsame Zeit bedanken. Besonders bedanken möchte ich mich bei Kathi, Alex, Bini, Desi und Susi für die hervorragende Zusammenarbeit im 8. Semster. Außerdem danke ich Sonja und Anna für die tolle Arbeitsatmosphäre im Analytiklabor. Bei meinen Freunden und Simon möchte ich mich für vor allem für die moralische Unterstützung und den starken Rückhalt ganz herzlich bedanken. Mein größter Dank gebührt meiner Familie und besonders meine Eltern, die mich immer uneingeschränkt und in allem unterstützt haben. Table of contents 1. Introduction .................................................................................................................... 1 1.1. Significance of natural sterols .................................................................................. 1 1.2. Steroid biosynthestic pathways ............................................................................... 2 1.2.1. Cholesterol ..................................................................................................... 2 1.2.2. Oxysterols ...................................................................................................... 5 1.2.3. Steroid acids ................................................................................................... 6 1.2.4. Neurosteroids ................................................................................................. 9 1.2.5. Sterol sulfates ................................................................................................10 1.3. Steroids and Alzheimer’s disease ...........................................................................11 1.4. Steroid analysis ......................................................................................................11 2. Topic .............................................................................................................................13 3. Sterol sulfate analysis ...................................................................................................14 3.1. Summary ................................................................................................................14 3.2. Personal contribution ..............................................................................................15 3.3. Article .....................................................................................................................17 3.4. Supplementary material .........................................................................................37 4. Analysis of neutral steroids, steroid acids and sterol sulfates ........................................39 4.1. Summary ................................................................................................................39 4.2. Personal contribution ..............................................................................................40 4.3. Article .....................................................................................................................41 5. Characterization of inhibitors of cholesterol biosynthesis ...............................................80 5.1. Summary ................................................................................................................80 5.2. Personal contribution ..............................................................................................81 5.3. Article .....................................................................................................................82 5.4. Supplementary material ....................................................................................... 111 6. Traceless isoprenylation .............................................................................................. 152 6.1. Summary .............................................................................................................. 152 6.2. Personal contribution ............................................................................................ 153 I 6.3. Article ................................................................................................................... 154 6.4. Supplementary material ....................................................................................... 164 7. Summary ..................................................................................................................... 208 8. Abbreviations .............................................................................................................. 210 9. References .................................................................................................................. 211 II 1. Introduction 1. Introduction 1.1. Significance of natural sterols Sterols are omnipresent in living nature, whereby the most abundant sterols are cholesterol in mammals, β-sitosterol in plants and ergosterol in fungi (Figure 1a) [1]. Figure 1 a: Structures of cholesterol, β-sitosterol and ergosterol, b: Numbering and ring letters of steroids according to IUPAC [2] These sterols are synthesized de novo in most organisms starting from acetyl-CoA (coenzyme A) and isoprenoids, but they can also be acquired from diet [3]. The latter was shown for invertebrates like insects that lack an own de novo biosynthesis, but are able to synthesize the required sterols from dietary precursors [4]. The fact that sterols are vital for so many different species and that the enzyme squalene monooxygenase, which performs the initial step for sterol biosynthesis, has several conserved motifs throughout the different species [5, 6] gives a hint to the importance of sterols for early eukaryotic evolution. It has been suggested, see e.g. [6, 7], that sterols played an important role in the evolution from prokaryotes to eukaryotes. This evolutionary step is characterized by the development of cell compartments and the differentiation of cell organelles as well as the endocytosis and exocytosis [5]. All these processes involve rapid deformation of cell membranes, and sterols play a crucial role for membrane fluidity and function [5]. Due to their amphiphilic properties they are stored in between the phospholipids in the cell membranes. Cholesterol, for example, is oriented perpendicular to the membrane plane and causes an increase of the membrane’s stability [8]. Hence, sterols are essential building materials and a prerequisite for the function of every cell. Despite of their important physical properties, sterols like cholesterol act as precursors for further essential steroids or bile acids [3, 9]. The focus of this work is the mammalian sterol cholesterol and its biosynthesis and metabolism. 1 1. Introduction 1.2. Steroid biosynthetic pathways 1.2.1. Cholesterol Cholesterol de novo biosynthesis in mammals generally can take place in every tissue. However, the majority is synthesized in the liver [9, 10]. It is then distributed throughout the body via the blood circulation, when bound to specific transport proteins as very low density lipoprotein (VLDL) and low density lipoprotein (LDL) [9-11]. In the form of LDL, cholesterol is taken up into cells by LDL receptor mediated endocytosis, and excess cholesterol can be redistributed to the liver in the form of high density lipoprotein (HDL) [9, 11]. One exception to this cholesterol dissemination is the central nervous system (CNS). Almost all CNS cholesterol is synthesized de novo within the brain and it is remarkable that the CNS, which accounts for about 2% of the body mass, accounts for about one quarter of the whole unesterified cholesterol [11]. Cholesterol biosynthesis can be divided into pre- and post-squalene pathway. In Figure 2 the pre-squalene section and the involved enzymes and intermediates are shown. First, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) (C6) is synthesized from three acetyl CoA (C2) building blocks. Following reduction of HMG-CoA leads to mevalonate (C6). After activation of mevalonate by conjugation with pyrophosphate and subsequent decarboxylation, isopentenyl
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