DARWIN Digitale Dissertationen English Version Strich

FU Berlin
Digitale Dissertation

Graziano Pinna :
Untersuchungen über 3,5-Diiodothyronin- und 3,3'-Diiodothyronin-Konzentrationen im Gehirn und Serum von Menschen und Versuchstieren
3,5-Diiodothyronin- und 3,3'-Diiodothyronin-Konzentrationen im Serum und Gehirn
Investigation on 3,5-diiodothyronine and 3,3'-diiodothyronine concentrations in brain and serum of humans and experimental animals

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|Zusammenfassung| |Inhaltsverzeichnis| |Ergänzende Angaben|

Zusammenfassung

Die physiologischen Effekte von Schilddrüsenhormonen werden nach allgemeiner Ansicht über die Bindung des Hormons T3 an nukleäre Rezeptoren vermittelt. Eine wachsende Anzahl an Publikationen weist jedoch darauf hin, daß auch die Dijodthyronine 3,5-T2 und 3,3`-T2 physiologische Funktionen haben könnten. Das Ziel der vorliegenden Arbeit war es deshalb, sensitive Radioimmunoasay-Methoden zu entwickeln, mit denen die Messung dieser Hormone im Blut und in den Geweben von Menschen und Ratten unter physiologischen und pathologischen Verhältnissen möglich wird.

In dieser Arbeit werden die Serum- und Gewebekonzentrationen von 3,5-T2 und von 3,3`-T2 erstmals überhaupt verläßlich gemessen. Spezifische Veränderungen dieser Konzentrationen wurden im Serum von Patienten mit verschiedenen nicht-thyreoidalen Erkrankungen, nach pharmakologischen Interventionen (z.B. Antidepressiva) und unter physiologischen Bedingungen (z.B. zirkardiane Rhythmik) nachgewiesen.

Der Nachweis von meßbaren Konzentrationen dieser Hormone und ihrer Beeinflussung durch verschiedene Faktoren weist darauf hin, daß´die Dijodthyronine tatsächlich physiologische Funktionen haben könnten und daß eine weitere Erforschung der biochemischen Mechanismen solcher Effekte sinnvoll erscheint. Die Ergebnisse zeigen schließlich, daß Effekte von 3,5-T2 wesentlich wahrscheinlicher sind als solche von 3,3`-T2.

Inhaltsverzeichnis

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Titel
Inhaltsangabe

1.Introduction

1.1 Physiological effects of thyroid hormones 1

1.1.1 Effects of triiodothyronine (T3) 1

1.1.1.1 Effects of T3 at nuclear receptors 1

1.1.1.2 Non-nuclear effects of T3 3

1.1.2 Physiological effects of diiodothyronines 5

1.1.2.1 Physiological effects of 3,5-diiodothyronine (3,5-T2) 5

1.1.2.2 Physiological effects of 3,3'-diiodothyronine (3,3'-T2) 8

1.2 Rationale for the choice of experiments performed in this study 9

1.2.1 Diiodothyronine serum concentrations in humans with thyroidal and nonthyroidal illnesses 9

1.2.2 Diiodothyronine concentrations in normal brain tissue

and in brain tumors 11

1.2.3 Diiodothyronine concentrations in rat brain homogenates 11

1.2.4 Diiodothyronine concentrations in subcellular compartments 14

1.2.5 Effects of antidepressant treatment on diiodothyronine concentrations 14

1.2.6 Effects of circadian variations on diiodothyronine concentrations 15

1.3 Purpose of the study 15

 

2.Materials and Methods

2.1 Materials 17

2.1.1 Equipment for subcellular fractionation 17

2.1.2 Equipment for radiolabeling iodothyronines 17

2.1.3 Equipment for extracting, purifying, and separating iodothyronines 17

2.1.4 Equipment for RIA quantification 18

2.1.5 Chemicals and reagents for subcellular fractionation 19

2.1.6 Chemicals and reagents for radiolabeling iodothyronines 19

2.1.7 Chemicals and reagents for extracting, purifying, and separating iodothyronines 20

2.1.8 Chemicals and reagents for RIA quantification 20

2.1.9 Drugs 21

2.2 Studies in humans 21

2.2.1 Hormone determination in human serum 21

· 2.2.1.1 Healthy controls 21

· 2.2.1.2 Patients with thyroid disorders 22

· 2.2.1.3 Patients with different somatic, nonthyroidal diseases 22

· 2.2.1.3.1 Patients with sepsis........................................................22

· 2.2.1.3.2 Patients with head and/or brain injury............................22

· 2.2.1.3.3 Patients with brain tumors and metastases....................23

· 2.2.1.3.4 Patients with liver diseases.............................................23

· 2.2.1.4 Acute stress 24

· 2.2.1.5 Sleep deprivation 24

2.2.2 Hormone determination in human brain tissue 25

· 2.2.2.1 Healthy donors 25

· 2.2.2.2 Samples of human brain tumors and metastases 26

2.3 Studies in experimental animals 26

2.3.1 Animals 26

2.3.2 Group 1: Control animals 26

· 2.3.2.1 Homogenates of different brain areas, pituitary glands,

and liver of the rat 26

· 2.3.2.2 Subcellular fractions of rat brain regions 27

2.3.3 Group 2: Antidepressant treatment with desipramine 27

2.3.4 Group 3: Circadian variations of iodothyronines 27

2.4 Hormone determination 28

2.4.1 RIA buffers 28

2.4.2 Synthesis of 3,5-T2 tracer 28

2.4.3 Synthesis of 3,3'-T2 tracer 28

2.4.4 Preparation of 3,5-T2-binding antibody 30

2.4.5 Preparation of 3,3'-T2 -binding antibody 31

2.4.6 RIA procedure for 3,5-T2 in serum and tissue samples 31

2.4.7 RIA procedure for 3,3'-T2 in serum and tissue samples 32

2.4.8 Serum determination of other iodothyronines and thyrotropin (TSH) 32

2.4.9 Tissue determination of other iodothyronines 33

2.5 Preparation of animal and human tissue samples 34

2.5.1 Brain dissection in the rat 34

2.5.2 Subcellular fractionation 37

· 2.5.2.1 Homogenization of the single areas of the rat brain 38

· 2.5.2.2 Centrifugation of the homogenates 38

· 2.5.2.3 Isolation of the nuclear fraction 38

· 2.5.2.4 Isolation of the mitochondrial fraction 39

· 2.5.2.5 Isolation of the synaptosomal fraction 39

· 2.5.2.6 Isolation of the myelin 39

· 2.5.2.7 Isolation of the microsomal fraction 39

· 2.5.2.8 Electron microscopic characterization of the subcellular

fractions 40

· 2.5.2.9 Characterization of the subcellular fractions by biochemical

markers 40

2.5.3 Extraction of iodothyronines from the subcellular fractions 43

· 2.5.3.1 Suspension of the subcellular fractions 43

· 2.5.3.2 Extraction of the subcellular fractions 43

· 2.5.4.3 Preparation of the subcellular fractions and homogenate

extracts for HPLC 45

2.5.5 Extraction of diiodothyronines from serum 45

2.5.6 Protein quantification 45

2.6 HPLC 46

2.6.1 Preparation of the tissue sample extracts for the autosampler 46

2.6.2 Purification and separation of the extracted iodothyronines by HPLC 47

2.6.3 Iodothyronine collection for RIA quantification 47

2.7 Data analysis 47

 

3.Results

3.1 Method validation 49

3.1.1 HPLC 49

· 3.1.1.1 Separation of iodothyronines by HPLC 49

· 3.1.1.2 Recovery of iodothyronines after extraction and HPLC 50

3.1.2 RIA for 3,5-T2 50

· 3.1.2.1 RIA sensitivity for 3,5-T2 50

· 3.1.2.2 Cross-reactivity of 3,5-T2 antibody with iodothyronines 50

· 3.1.2.3 Cross-reactivity of 3,5-T2 antibody with drugs 52

· 3.1.2.4 Recovery of "cold" 3,5-T2 53

· 3.1.2.5 Inter- and intra-assay coefficients of variation 53

3.1.3 RIA for 3,3'-T2 54

· 3.1.3.1 RIA sensitivity for 3,3'-T2 54

· 3.1.3.2 Cross-reactivity of 3,3'-T2 antibody with iodothyronines 55

· 3.1.3.3 Cross-reactivity of 3,3'-T2 antibody with drugs 56

· 3.1.3.4 Recovery of "cold" 3,3'-T2 57

· 3.1.3.5 Inter- and intra-assay coefficients of variation 57

3.2 Clinical Studies 58

3.2.1 Serum concentrations of 3,5-T2 58

· 3.2.1.1 Healthy controls 58

· 3.2.1.2 Patients with thyroid disorders 59

· 3.2.1.3 Patients with different somatic, nonthyroidal diseases 60

· 3.2.1.4 Acute stress 60

· 3.2.1.5 Sleep deprivation 61

3.2.2 Serum concentrations of 3,3'-T2: 61

· 3.2.2.1 Healthy controls 61

· 3.2.2.2 Patients with thyroid disorders 62

· 3.2.2.3 Patients with different somatic, nonthyroidal diseases 62

· 3.2.2.4 Acute stress 63

· 3.2.2.5 Sleep deprivation 63

3.2.3 Serum concentrations of other iodothyronines and thyrotropin (TSH) 64

3.2.4 Tissue levels of 3,5-T2 65

· 3.2.4.1 Human brain areas of healthy donors 65

· 3.2.4.2 Human brain tumors and metastases 67

3.2.5 Tissue levels of 3,3'-T2 67

· 3.2.5.1 Human brain areas of healthy donors 67

· 3.2.5.2 Human brain tumors and metastases..............................................68

3.2.6 Tissue levels of other iodothyronines in human brain areas

of healthy donors 68

3.2.7 Tissue levels of other iodothyronines in human brain tumors

and metastases 69

3.3 Animal studies 71

3.3.1 Serum concentrations of 3,5-T2 and 3,3'-T2 in control animals 71

3.3.2 Tissue levels of 3,5-T2 71

· 3.3.2.1 Homogenates of various brain areas of the rat 71

· 3.3.2.2 Subcellular fractions of brain areas of the rat 73

3.3.3 Tissue levels of 3,3'-T2 74

· 3.3.3.1 Homogenates of various brain areas of the rat 74

· 3.3.3.2 Subcellular fractions of brain areas of the rat 74

3.3.4 Tissue levels of other iodothyronines 76

· 3.3.4.1 Homogenates of various brain areas of the rat 76

· 3.3.4.2 Subcellular fractions of brain areas of the rat 78

3.3.5 Effects of antidepressant drugs on brain subcellular concentrations

of 3,5-T2 and 3,3'-T2 79

3.3.6 Effects of circadian variation on concentrations of 3,5-T2 and other iodothyronines 82

 

4.Discussion

4.1 3,5-T2 serum and tissue concentrations in humans 87

4.2 3,3'-T2 serum and tissue concentrations in humans 91

4.3 3,5-T2 serum and tissue concentrations in rats 94

4.4 3,3'-T2 serum and tissue concentrations in rats 100

5.Summary 103

6.Reference List 106

 

Curriculum Vitae 117

List of Publications 119

Poster and Slide Presentation...............................................................................121

Acknowledgment 124

Ergänzende Angaben:

Online-Adresse: http://www.diss.fu-berlin.de/2001/80/index.html
Sprache: Englisch
Keywords: thyroid hormones, nonthyroidal illness, brain and serum concentrations, circadian variations, antidepressant treatment
DNB-Sachgruppe: 33 Medizin
Datum der Disputation: 24-Apr-2001
Entstanden am: Fachbereich Humanmedizin, Freie Universität Berlin
Erster Gutachter: Prof. Dr. Andreas Baumgartner
Zweiter Gutachter: Prof. Dr. Jörg-Wilhelm Oestmann
Kontakt (Verfasser): gpinna@gmx.net
Kontakt (Betreuer): abaum@ukbf.fu-berlin.de
Abgabedatum:28-May-2001
Freigabedatum:28-May-2001

 

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