dc.contributor.author
Pinna, Graziano
dc.date.accessioned
2018-06-08T00:59:44Z
dc.date.available
2001-05-28T00:00:00.649Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/12796
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-16994
dc.description
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
dc.description.abstract
It is widely accepted that the principle physiological actions of thyroid
hormones are exerted by the binding of T3 to its nuclear receptors. However,
an increasing number of reports suggest that the diiodothyronine 3,5-T2 and
3,3'-T2 may also have physiological effects. The purpose of this study was
therefore to develop sensitive radioimmunoassays for the measurement of these
hormones in blood and tissues of humans and rats during physiological and
pathological conditions.
This study reported, for the first time, reliable serum and tissue
concentrations of 3,5-T2 and 3,3'-T2 in humans and rats. Specific changes in
their concentrations could be demonstrated during pathological conditions such
as nonthyroidal illnesses, after pharmacological treatment (such as
antidepressants), or even under physiological conditions (such as circadian
variations). The detection of clearly measurable tissue levels of these
hormones which are affected by different physiological and pathological
circumstances makes it plausible that these hormones may indeed have
physiological functions and provides a reasonable basis for the further
investigation of their biochemical mechanisms underlying these effects. The
data indicate that biochemical and physiological effects of 3,5-T2 are much
more likely than those of 3,3'-T2.
de
dc.description.abstract
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.
de
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
thyroid hormones
dc.subject
nonthyroidal illness
dc.subject
brain and serum concentrations
dc.subject
circadian variations
dc.subject
antidepressant treatment
dc.subject.ddc
600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit::610 Medizin und Gesundheit
dc.title
Investigation on 3,5-diiodothyronine and 3,3'-diiodothyronine concentrations
in brain and serum of humans and experimental animals
dc.contributor.firstReferee
Prof. Dr. Andreas Baumgartner
dc.contributor.furtherReferee
Prof. Dr. Jörg-Wilhelm Oestmann
dc.date.accepted
2001-04-24
dc.date.embargoEnd
2001-05-28
dc.identifier.urn
urn:nbn:de:kobv:188-2001000803
dc.title.subtitle
3,5-diiodothyronine and 3,3'-diiodothyronine concentrations in serum and brain
tissue
dc.title.translated
Untersuchungen über 3,5-Diiodothyronin- und 3,3'-Diiodothyronin-
Konzentrationen im Gehirn und Serum von Menschen und Versuchstieren
de
dc.title.translatedsubtitle
3,5-Diiodothyronin- und 3,3'-Diiodothyronin-Konzentrationen im Serum und
Gehirn
de
refubium.affiliation
Charité - Universitätsmedizin Berlin
de
refubium.mycore.fudocsId
FUDISS_thesis_000000000402
refubium.mycore.transfer
http://www.diss.fu-berlin.de/2001/80/
refubium.mycore.derivateId
FUDISS_derivate_000000000402
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
