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INAUGURAL-DISSERTATION Zur Erlangung Des Doktorgrades Der Medizin

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INAUGURAL-DISSERTATION Zur Erlangung Des Doktorgrades Der Medizin Aus der Universitätsklinik für Kinderheilkunde und Jugendmedizin Abteilung Kinderheilkunde III mit Poliklinik Ärztliche Direktorin: Frau Professor Dr. I. Krägeloh-Mann Bestimmung von sphingolipidabbauenden Enzymen in Granulozyten, Monozyten und Lymphozyten zur Optimierung der Labordiagnostik bei Sphingolipid-Speichererkrankungen INAUGURAL-DISSERTATION zur Erlangung des Doktorgrades der Medizin der MEDIZINISCHEN FAKULTÄT der Eberhard-Karls-Universität zu Tübingen vorgelegt von SEBASTIAN GEORG CHRISTOPHER STROBEL aus Burlington/USA 2005 Dekan: Professor Dr. med. C. D. Claussen 1. Berichterstatter: Professor Dr. rer. nat. G. Bruchelt 2. Berichterstatter: Privatdozentin Dr. rer. nat. H. Schmid Für meine Eltern und Geschwister 4 INHALTSVERZEICHNIS Inhalt Seite 1. Einleitung 8 1.1. Einführung 8 1.2. Lysosomale Speicherkrankheiten 10 1.2.1. Stoffwechsel der Sphingolipide 10 1.2.1.1. Funktion der Sphingolipide 10 1.2.1.2. Biosynthese der Sphingolipide 11 1.2.1.3. Abbau der Sphingolipide 14 1.2.1.4. Störungen im lysosomalen Sphingolipidabbau: Lipidspeicherkrankheiten 15 1.2.1.5. Synthese und Reifung lysosomaler Enzyme 17 1.2.1.6. Speicherung und Freisetzung der lysosomalen Enzyme 19 1.2.2. Erkrankungen durch Störungen des Sphingolipidabbaus 20 1.2.2.1. Metachromatische Leukodystrophie (MLD) 23 1.2.2.2. ß-Galaktosidase-Mangel (GM1 Gangliosidose, Morquio B) 27 1.2.2.3. GM2 Gangliosidosen 35 1.2.3. Diagnostik der Sphingolipidosen 40 1.2.3.1. Biochemische Diagnostik 40 1.2.3.1.1. Analyse der gespeicherten Materialien aus Biopsien 40 1.2.3.1.2. Enzymatische Nachweisverfahren 41 1.2.3.1.3. Metabolische Untersuchungen 43 1.2.3.2. Molekulare Nachweisverfahren und genetische Diagnostik 44 1.2.3.3. Pränatale Diagnostik 44 1.2.4. Probleme der enzymatischen Nachweisverfahren 45 1.3. Aufgabenstellung 47 INHALTSVERZEICHNIS 5 1.3.1. Trennung der Leukozyten in Granulozyten, Monozyten und Lymphozyten mit Messung des jeweiligen Anteils an der Gesamtenzymaktivität 47 1.3.2. Versuche zur Degranulation der Enzyme aus den Granulozyten und Bestimmung der Enzyme im Überstand und Zellpellet 47 1.3.3. Vergleich der Ausschüttung von Myeloperoxidase und den sphingolipidabbauenden Enzymen nach Stimulation der Granulozyten 48 2. Material und Methoden 49 2.1. Material 49 2.1.1. Chemikalien und Substanzen 49 2.1.2. Lösungen, Puffer und Medien 50 2.1.3. Verbrauchsmaterialien 54 2.1.4. Geräte 54 2.2. Methoden 55 2.2.1. Bestimmung des Differentialblutbildes und des MPXI- Wertes mit Hilfe des Bayer ADVIA 120 55 2.2.2. Leukozytenisolierung aus Vollblut mittels Dextransedimentation 60 2.2.3. Isolation der Granulozyten und mononukleären Blutzellen aus Vollblut 61 2.2.4. Auftrennung der mononukleären Blutzellen mittels MACS in Monozyten und Lymphozyten 62 2.2.5. Anfertigung, Färbung und Auszählung von Zytospinpräparaten 66 2.2.6. Übersicht über die Funktionsteste (Degranulation, Enzymaktivitätsbestimmung) 67 2.2.7. Degranulationsuntersuchungen an Granulozyten 68 2.2.8. MPO-ELISA 72 2.2.9. Degranulationsuntersuchungen an Monozyten 75 6 INHALTSVERZEICHNIS 2.2.10 Messung der enzymkatalysierten Hydrolasereaktionen 77 3. Ergebnisse 81 3.1. Erstellung eines Differentialblutbildes mittels Bayer ADVIA 120 81 3.1.1. Vergleich der Bayer ADVIA 120 Ergebnisse im Vollblut, nach Dextransedimentation und nach Histopaque- Isolierung 81 3.2. Bestimmung der sphingolipidabbauenden Enzymaktivität bei Gesamtleukozyten (Dextransedimentation), isolierten Granulozyten (Histopaque-Technik) und mononukleären Blutzellen (Histopaque-Technik) 87 3.3. Bestimmung der Aktivität sphingolipidabbauender Enzyme bei Monozyten und Lymphozyten im Vergleich zu Granulozyten 90 3.3.1. Reinheit der isolierten Zellfraktionen nach MACS- Isolierung 91 3.3.2. Messung der sphingolipidabbauenden Enzymaktivität bei den einzelnen Zellfraktionen 93 3.4. Degranulationsversuche an Granulozyten 100 3.4.1. Einfluss des Isolationsverfahrens auf das Degranulationsverhalten der Granulozyten 101 3.4.2. Degranulation von Granulozyten vor und nach Behandlung mit GM-CSF, Cytochalasin B und N-fMLP 103 3.4.2.1. Ergebnisse der ADVIA 120 Messungen 105 3.4.2.2. Bestimmung der aus Granulozyten freigesetzten MPO mit Hilfe des MPO-ELISA 110 3.4.3. Messung der sphingolipidabbauenden Enzymaktivitäten im Überstand und Zellpellet der degranulierten Granulozyten 114 3.4.4. Korrelation MPO-ELISA – sphingolipidabbauende Enzyme in Granulozyten 121 INHALTSVERZEICHNIS 7 3.5. Einfluss von Zytokinen und Chemotaktika auf das Degranulationsverhalten von Monozyten 125 3.5.1. Isolierung der Monozyten aus Vollblut 127 3.5.2. Degranulation der Monozyten unter dem Einfluß von Cyt-B bzw. N-fMLP 128 3.5.3. Messung der Gesamthexosaminidase im Überstand und Zellpellet der degranulierten Monozyten 129 4. Diskussion 131 4.1. Einleitung 131 4.2. Interpretation der Ergebnisse 132 4.2.1. Verteilung der Enzymaktivität in den einzelnen Leukozytenfraktionen 132 4.2.1.1. Beurteilung des Differentialblutbildes der Leukozytenfraktion am Bayer ADVIA 120 132 4.2.1.2. Verteilung der sphingolipidabbauenden Enzyme innerhalb der Leukozytenfraktionen 135 4.2.2. Degranulationsversuche an Granulozyten und Monozyten 140 4.2.2.1. Degranulation von sphingolipidabbauenden Enzymen bei Granulozyten/Monozyten nach Inkubation mit bekannten Stimuli 141 5. Zusammenfassung 145 6. Abkürzungen 147 7. Literaturverzeichnis 149 8 EINLEITUNG 1 EINLEITUNG 1.1 Einführung In dieser Arbeit sollten Bestimmungsverfahren für Sphingolipidosen bezüglich der Bezugssysteme optimiert werden. Spingolipidosen sind Lipidspeicherkrankheiten, die in den meisten Fällen mit dem Tod enden. Ursachen sind vielfältige genetische Defekte, die dazu führen, dass verschiedene, sonst am Abbau der Sphingolipide beteiligte Enzyme, in einer nicht funktionsfähigen Form vorliegen. Über die Bestimmung dieser Enzymaktivitäten aus Extrakten von Leukozyten oder nach Erhalt von Hautbiopsien, aus denen Fibroblastenkulturen angelegt werden können, werden die verschiedenen Sphingolipidosen charakterisiert. Für die Enzymmessungen werden Leukozyten mittels einer Dextransedimentation aus Vollblut gewonnen. Die EDTA-Blutproben werden teilweise auf dem Postweg versandt, sodass diese längere Zeit unterwegs sind. Aus den Zellextrakten - und nicht wie sonst in der Labormedizin oft üblich aus Serum oder Plasma - werden die Enzymaktivitäten meist über photometrische Verfahren bestimmt, wozu vielfach nicht die normalen Sphingolipide verwendet werden, sondern „künstliche“, analoge Verbindungen. Als Ergebnis wird der Extinktionswert bezogen auf 105 Zellen angegeben. Ist kein Defekt vorhanden, erhält man einen Messwert, der sich deutlich vom Leerwert unterscheidet. Folgende Probleme zeigen sich bei diesem Testverfahren in der täglichen Anwendung: 1. Der lange Transportweg wirkt als „Stress“ auf die Blutzellen, v.a. auf die Granulozyten, die mit einer Abgabe von Teilen ihres Enzymgehaltes in das Plasma reagieren. Im nachfolgenden Test können somit eventuell falsch-niedrige Enzymaktivitäten in den Zellen gemessen werden. EINLEITUNG 9 2. Die verschiedenen Fraktionen der Leukozyten (Granulozyten, Monozyten und Lymphozyten) besitzen unterschiedlich starke Enzymaktivitäten. Daher kann z.B. bei gleicher Qualität der Blutzellen bei von einander differierender Leukozytenzusammensetzung eine unterschiedliche Enzymaktivität (bezogen auf Gesamtleukozyten) gemessen werden. Ziel der Arbeit war es daher auf diese Probleme einzugehen, die v.a. bei Grenzwerten („Restaktivitäten“) an Bedeutung gewinnen. Es sollte untersucht werden, welchen Anteil die einzelnen Leukozytenfraktionen an der Gesamtenzymaktivität besitzen und wie dieselben auf „Stress“ reagieren. Die Hauptfraktion der isolierten Leukozyten stellen neutrophile Granulozyten dar, die als Markerenzym Myeloperoxidase (MPO) besitzen. Daher sollte ein Zusammenhang zwischen der Degranulation der MPO während der Aufarbeitung und der für den Sphingolipidabbau verantwortlichen Enzyme hergestellt werden, da sich diese wahrscheinlich in den gleichen Granula befinden. Dank dieses Zusammenhangs ließe sich dann ein Rückschluss auf den Enzymverlust ziehen, da mittels des Blutanalysegerätes Bayer ADVIA 120 die Myeloperoxidaseaktivität in den neutrophilen Granulozyten und Monozyten bestimmt werden kann. Zunächst soll auf die Krankheiten, die mittels dieser Testverfahren untersucht werden, näher eingegangen werden. Im Anschluss daran folgt eine genauere Beschreibung der Testverfahren. 10 EINLEITUNG 1.2 Lysosomale Speicherkrankheiten 1.2.1 Stoffwechsel der Sphingolipide 1.2.1.1 Funktion der Sphingolipide Sphingolipide sind Bestandteile der Glykokalix tierischer Zellen, d.h. zusammen mit Glykoproteinen und Glykosaminglykanen bilden sie die Oberfläche der Plasmamembran einer Zelle. Sphingolipide besitzen einen bipolaren Aufbau: einerseits einen hydrophoben Ceramid(N-Acylsphingosin)-Rest, mit dem sie in der Zellmembran verankert sind, und andererseits einen hydrophilen Anteil, der nach außen in den Extrazellulärraum ragt. Die an die Sphingolipide gekoppelten Oligosaccharide, die zusammen die sogenannten Glykosphingolipide bilden, decken die Zelloberfläche teilweise ab [Sandhoff et Quintern, 1988]. Das Oligosaccharidmuster der Glykokalix einer Zelle ist sehr vielfältig und für diese spezifisch. Aufgrund der unterschiedlichen Verknüpfung der Monosaccharide zu Oligosacchariden entsteht die enorme Vielfalt. Die Glykokalix einer Zelle besitzt viele Aufgaben, darunter die gegenseitige Erkennung gleicher Zellen, die dadurch in die Lage versetzt werden, Zellverbände zu bilden. Sie bildet auch Bindungsstellen für Toxine [Walton et al.,
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