Transthyretin ist ein multifunktionales Protein, welches hauptsächlich hinsichtlich seiner Rollen als Transportprotein, negatives Akutphasenprotein, biochemischer Marker, Enzym und als Komponente amyloidogener Prozesse bekannt ist. In der vorliegenden Dissertation soll der komplexe Zusammenhang von funktionellen und diagnostischen Aspekten in der TTR-Forschung dargestellt werden und dabei die Mikroheterogenität des Protein im Rahmen von drei Publikationen in den Vordergrund gestellt werden. Die erste Studie befasst sich mit der intrafollikuläre Eizellreifung während hormoninduzierter Superovulation (P1), eine weitere mit dem Ovarialkarzinom (P2) und die dritte mit polytraumatisierten Patienten (P3). Methodisch wurde nach einer Immunopräzipitation die massenspektrometrische Analytik genutzt, um die Qualität des Proteins abbilden zu können, für die quantitative Bestimmung von TTR wurde ein ELISA zur Anwendung gebracht. Die vier Hauptpeaks (natives TTR, S-cysteinyliertes TTR, S-cysteinylglyciniertes TTR und S-glutathionyliertes TTR) konnten in Serum, Follikelflüssigkeit und Aszites detektiert werden (P1, P2, P3). Eine vermehrte Dominanz des S-cysteinylglycinierten TTR konnte innerhalb der ersten 24 Stunden nach einem Trauma nachgewiesen werden (P3). Die Serumkonzentrationen von TTR sind assoziiert mit den CRP-Konzentrationen (P2, P3). Gesunde Kontrollen und Patienten unterscheiden sich hinsichtlich ihres Serumgehaltes an TTR (P2, P3). Posttranslationale Modifikationen des TTR treten am freien Cysteinrest in Position 10 des monomeren TTR (Cys10) auf12,13 und konnten sowohl in Serum und FF hormonstimulierter Frauen (P1), als auch in Serum und Aszites von Frauen mit einem Ovarialkarzinom (P2) und im Serum von Patienten nach einem Polytrauma (P3) nachgewiesen werden. Solche Modifikationen können Interaktionen mit anderen Plasmaproteinen beeinträchtigen und Einfluss auf Rezeptorbindung, Gewebeaufnahme, Abbau und Ausscheidung des Proteins nehmen. Bereits in früheren Studien wurde ein Zusammenhang zwischen Modifikationen der Mikroheterogenität von TTR und oxidativem Stress hergestellt. Die funktionellen Konsequenzen der passageren Änderungen des TTR-Moleküls nach einem Polytrauma sind derzeit noch unklar, es ist aber mit Spätfolgen zu rechnen. Kürzlich wurde eine Veränderung des Ratios von nativem TTR zu cysTTR als hilfreich für die Diagnose von Alzheimer postuliert. Weiterhin ist bewiesen, dass Modifikationen, wie sie bei den Polytraumapatienten beschrieben werden konnten, die Amyloidogenität von TTR beeinflussen. Zusammenfassend kann durch die drei Studien belegt werden, dass in jedem Fall posttranslationale Modifikationen am TTR Molekül auftreten, dass die Mikroheterogenität des Proteins in den verschiedenen untersuchten Körperflüssigkeiten identisch ist, und dass ein akuter oxidativer Stress Veränderungen im TTR-Molekül induziert, während diese beim Ovarialkarzinom nicht beobachtet werden konnten.
Proteins and peptides in human follicular fluid (FF) are from plasma or produced by follicular structures. Composition changes reflect oocyte maturation and can be used as diagnostic markers. Aim of the study was to determine protein and peptide pattern of paired serum and FF samples of women undergoing IVF. METHODS: Surface enhanced laser desorption and ionization-time of flight-mass spectrometry (SELDI-TOF-MS) was used to obtain characteristic protein pattern. RESULTS: 186 individual mass signals were obtained from a combination of enrichment on strong anion exchanger (110), weak cation exchanger (52) and normal phase surfaces (24). Based on molecular masses, isoelectric points and immunoreactivety, four signals were identified as haptoglobin, haptoglobin 1 and transthyretin (TTR). Immunological and MS characteristics of the TTR:retinol-binding protein transport complex revealed no microheterogeneity differences between serum and FF. Discriminatory pattern based on decision-tree-based classification and regression analysis distinguished between serum and FF with a sensitivity and specifity of 100%. CONCLUSION: Quantitative and qualitative differences indicate selective transport processes rather than mere filtration across the blood-follicle- barrier. Identified proteins as well as characteristic peptide and/or protein signatures might emerge as potential candidates for diagnostic markers of follicle and/or oocyte maturation and thus oocyte quality. Background: Transthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR. Methods: Subjects were 48 women with primary invasive epithelial ovarian cancer or recurrent ovarian carcinoma. The control group consisted of 20 postmenopausal women. TTR and retinol-binding protein (RBP) levels were measured by enzyme-linked immunoassay (ELISA) and C-reactive protein (CRP) levels by a high-sensitivity latex particle turbidimetric assay. The molecular heterogeneity of TTR was analysed using immunoprecipitation and matrix-associated laser desorption ionization time-of- flight mass spectrometry (MALDI-TOF-MS). Presence of TTR in tumor tissue was determined with indirect peroxidase immunostaining. Results: TTR and RBP (µg/ml) levels in serum were 148.5 ± 96.7 and 22.5 ± 14.8 in affected women compared to 363.3 ± 105.5 and 55.8 ± 9.3 in healthy postmenopausal women (p < 0.01). In ascitic fluid, levels were 1.02 ± 0.24 and 4.63 ± 1.57 µg/ml, respectively. The mean levels of TTR and RBP in serum showed a tendency to decrease with the severity of the disease and were lower in affected women whose CRP levels were > 40 mg/ml (p = 0.08 for TTR; p < 0.05 for RBP). No differences in TTR microheterogeneity were observed between TTR isolated from serum of affected and healthy women or from ascitic fluid. TTR occurred rather consistently in four variants. Mass signals were at 13758 ± 7, 13876 ± 13 (greatest intensity), 13924 ± 21 and 14062 ± 24 Da, representing native, S-cysteinylated, S-cysteinglycinylated and glutathionylated TTR, respectively. Serum of healthy and affected women as well as ascitic fluid contained the truncated fragment of TTR (12828 ± 11 Da). No immunoreactive TTR was observed in the tumor sites. Conclusion: The severity of the cancer associated catabolism as well as the inflammation status affect serum TTR and RBP levels. Neither TTR nor its truncated form originates from tumor tissue and its occurrence in ascites may well reflect the filtration from blood into ascitic fluid. Transthyretin (TTR) which exists in different isoforms, is a valid marker for acute phase response and subclinical malnutrition. The aim of the study was to investigate the relationship between inflammation, oxidative stress and the occurrence of changes in microheterogeneity of TTR. A prospective, observational study at a level-I trauma center of a large urban medical university was performed. Patients were severely injured (n=18; injury severity score (ISS): 34-66), and were observed within the first 24 hours as and over the following days until day 20 after injury. 20 healthy subjects, matched by age and sex, were used as controls. TTR was enriched by immunoprecipitation. Microheterogeneity of TTR was determined by linear matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI- TOF-MS). Four major mass signals were observed for TTR representing native, S-cysteinylated, S-cysteinglycinylated and S-glutathionylated TTR, respectively. In the course of their ICU stay, 14 out of 18 patients showed a transient change in microheterogeneity in favour of the S-cysteinglycinylated form of TTR (p < 0.05 vs. controls). The occurrence of this variant was not associated with the severity of trauma or the intensity of the acute-phase response, but was associated with oxidative stress as evidenced by Trolox. Our results demonstrate that changes in microheterogeneity of TTR occur in a substantial number of ICU trauma patients. The diagnostic values of this changes remains to be elucidated. It is speculated that TTR modification may well be the mechanism underlying the morphological manifestation of amyloidose or Alzheimer’s diseases in patients surviving multiple trauma.
