Endocrine hypertension, adrenal steroids and development of a saliva based aldosterone assay as a potential screening method [Elektronische Ressource] / vorgelegt von Jenny Manolopoulou

Jenny Manolopoulou

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 28
• Poids de l'ouvrage: 2 Mo

Extrait

Aus der Medizinischen Klinik, Innenstadt
der Ludwig-Maximillians-Universität München

Direktor: Prof. Dr. med. M. Reincke



Endocrine Hypertension, Adrenal Steroids and Development of
a Saliva Based Aldosterone Assay as a Potential Screening Method









Dissertation
Zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München




















vorgelegt von
Jenny Manolopoulou
aus
Hannover
2008






Mit Genehmigung der Medizinischen Fakultät
der Universität München




Berichterstatter: Prof. Dr. med. Martin Reincke
2. Berichterstatter: Priv. Doz. Dr. Christian Rust
Mitberichterstatter: Prof. Dr. Ulrich Pohl
Priv. Doz. Dr. Michael Vogeser
Mitbetreuung durch den
promovierten Mitarbeiter: Dr. med. Martin Bidlingmaier
Dekan: Prof. Dr. med. Dr.h.c. M. Reiser
Tag der mündlichen Prüfung: 09.10.2008 OUTLINE of CONTENTS
page

1. Introduction 10

1.1. The mineralocorticoid aldosterone 10
1.1.1. Adrenal cortex and production, mechanism of action 10
1.1.2. Control of release of aldosterone 13
1.1.3. Diurnal variation 16
1.1.4. The classical and local Renin-Angiotensin-Aldosterone system 17
1.1.5. Aldosterone and endocrine hypertension 20
1.2. Saliva and monitoring of hormones 24
1.2.1. Anatomy and physiology of salivary gland 25
1.2.2. Mechanisms of salivary secretion 26
1.2.3. Factors influencing flow rate and composition 27
1.2.4. Diagnostic application of saliva in measurement of steroid levels 29
1.2.5. Evidence for aldosterone in saliva until now 32
1.3. Immunoassay 34
1.3.1. Theory of competitive immunoassay using time-resolved fluorescence 34
1.3.2. Purification by reverse phase HPLC 37
1.4. Aims 40
1.4.1. Tracer production 40
1.4.2. Saliva assay 40
1.4.3. Validation 41
1.4.4. Application in human saliva, validation in human plasma 41
1.4.5. Application in small volume rodent serum/plasma 41

2. Materials and Methods 42

2.1. Experimental
2.1.1. Materials 42
2.1.1.1. Reagents
2.1.1.2. Antibodies 44
2.1.1.3. Buffers 45
32.1.2. Equipment 47
2.1.3. Miscellaneous 47
2.2. Clinical Studies 48
2.2.1. Materials 48
2.2.2. Equipment
2.3. Methods – Experimental 49
2.3.1. Synthesis of aldosterone 3-CMO biotin conjugate tracer 49
2.3.2. Reverse phase chromatographic purification of aldosterone tracer 49
2.3.2.1 Vertex-Säule Eurospher 100 C18 5µm –
Knauer Advanced Scientific Instruments 50
2.3.2.2. Synergi 4u Fusion-RP 80A, 250 x 4.6mm – Phenomenex Ltd. 50
2.3.3. Evaluation of fractions 51
2.3.3.1. Aldosterone content with RIA MAIA Adaltis 51
2.3.3.2. Fractions as assay tracer 51
2.3.3.3. Direct comparison of activity at equal concentration 52
2.3.3.4. Optimising concentration of chosen tracer fraction 53
2.3.4. Conditions of the assay 53
2.3.4.1. Optimisation of incubation time between tracer and standards 53
2.3.4.2. Optimising concentration of polyclonal capture antibody 53
2.3.4.3. Optim monoclonal capture antibody 54
2.3.5. Working dilution of antibodies 54
2.3.5.1. Primary coating – goat anti-rabbit immunoglobulins 54
2.3.5.2. Primary coating – rabbit anti-mouse immunoglobulins 55
2.3.5.3. Secondary coating - polyclonal rabbit anti-aldosterone 55
2.3.5.4. Secondary coating - monoclonal anti-aldosterone 55
2.3.6. Preparation of aldosterone calibrators 56
2.3.7. Final assay procedure 56
2.3.8. Extraction of aldosterone from saliva and plasma/serum 56
2.3.9. Saliva sampling 57
2.3.10. Assay validation 58
2.3.10.1. Cross-reactivity
2.3.10.2. Sensitivity - lower limit of detection 59
2.3.10.3. Linearity 59
2.3.10.4. Recovery 60
42.3.10.5. Precision (Reproducibility) 61
2.3.11. Salivary aldosterone determination with and without Sarstedt salivette 62
2.3.12. Pre-analytical storage stability of salivettes 62
2.3.13. Correlation with commercially available RIA 62
2.3.14. Extraction of saliva and plasma – ‘Acris assay’ 63
2.3.14.1. Salivary aldosterone 63
2.3.14.2. Plasma
2.3.15. Extraction of plasma – ‘A2E11 assay’ 64
2.4. Methods – Clinical 64
2.4.1. Clinical validation of salivary aldosterone 65
2.4.1.1. ‘Acris assay’ clinical validation - Day profile study 65
2.4.1.2. Mean aldosterone levels in PA and healthy participants 65
2.4.1.3. Posture test study 66
2.4.1.4. ACTH stimulation test 67
2.4.1.5. Aldosterone to cortisol ratio 68
2.4.1.6. ‘A2E11 assay’ clinical validation in saliva 68
2.4.2. Clinical validation in rodent plasma/serum using the ‘A2E11 assay’ 69
2.4.2.1. Basal aldosterone in male and female wild type mice 69
2.4.2.2. Suppression and stimulation of the HPA axis in mice 70
2.4.2.3. Effect of an increased potassium diet on the adrenal RAAS 70
2.5. Statistical analysis 71

3. Result 71

3.1. Reverse phase chromatographic purification of the aldosterone
3-CMO-biotin conjugate 71
3.1.1. Vertex-Säule Eurospher - Knauer Advanced Scientific Instruments 71
3.1.2. Synergi 4u Fusion-RP - Phenomenex Ltd. 73
3.2. Evaluation of fractions 74
3.2.1. Aldosterone content with RIA MAIA Adaltis 74
3.2.2. Fractions as assay tracer 76
3.2.3. Direct comparison of activity at equal concentration 77
3.2.4. Conditions of the assay 78
3.2.4.1. Optimisation of tracer concentration and incubation time
5 between tracer and standards 78
3.2.4.2. Optimising final concentration of polyclonal antibody and tracer 80
3.2.4.3. Optimization final concentration of monoclonal capture antibody 81
3.3. Assay validation 83
3.3.1. Cross-reactivities 83
3.3.1.1. Polyclonal rabbit anti-aldosterone antibody (Acris) 83
3.3.1.2. Monoclonal anti-aldosterone antibody (A2E11) 83
3.3.2. Sensitivity 84
3.3.2.1. Polyclonal rabbit anti-aldosterone antibody (Acris) 84
3.3.2.2. Monoclonal anti-aldosterone antibody (A2E11) 85
3.3.3. Linearity 86
3.3.3.1. Polyclonal rabbit anti-aldosterone antibody (Acris) 86
3.3.3.2. Monoclonal anti-aldosterone antibody (A2E11) 86
3.3.4. Recovery 88
3.3.4.1. Polyclonal rabbit anti-aldosterone antibody (Acris) 88
3.3.4.2. Monoclonal anti-aldosterone antibody (A2E11) 88
3.3.5. Precision 89
3.3.5.1. Polyclonal rabbit anti-aldosterone antibody (Acris) 89
3.3.5.2. Monoclonal anti-aldosterone antibody (A2E11) 91
3.4. Salivary aldosterone determination with and without Sarstedt salivette 93
3.5. Pre-analytical storage stability of saliva samples 94
3.6. Correlation of calibrators with commercially available RIA 95
3.7. Extraction of saliva and plasma – Acris assay 98
3.7.1. Salivary aldosterone 98
3.7.2. Plasma aldosterone 98
3.8. Extraction of plasma – A2E11 assay 102
3.9. Salivary aldosterone – Acris assay clinical validation 103
3.9.1. Day Profile Study results – Saliva and plasma 103
3.9.2. Mean levels in PA and healthy participants 104
3.10. Salivary aldosterone TRFIA measurements compared
to plasma measured with a commercial RIA 106
3.11. Posture test study 106
3.12. ACTH stimulation test 108
3.13. Salivary aldosterone to cortisol ratio 110
63.14. A2E11 assay salivary aldosterone clinical validation 113
3.15. A2E11 assay – validation in rodent serum/plasma 113
3.15.1. Basal aldosterone in wild type mice 113
3.15.2. Suppression and stimulation of the HPA axis in wild type mice 116
3.15.3. Effect of an increased potassium diet on the adrenal RAAS 116

4. Discussion 118

4.1. Aldosterone in hypertension - Primary aldosteronism 118
4.2. Aldosterone in saliva – application of the assay 119
4.3. Establishment of the assay 121
4.4. Clinical validation 123
4.4.1. Salivary aldosterone monitors changes in plasma 123
4.4.2. Posture test findings and future application 125
4.4.3. ACTH stimulation test findings and future application 127
4.4.4. Aldosterone to cortisol ratio – extra confirmation for diagnosis 128
4.4.5. Monitoring fluctuatio