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Antonelli, Giorgia (2009) Molecular endocrinology approach in patho-physiological conditions: new markers and alternative biological matrices. [Tesi di dottorato]

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Abstract (inglese)

The study of saliva for laboratory analyses is an increasing area of research with implications for basic and clinical purposes. Although this biological fluid is easy to manipulate and collect, attention must be directed to sample collection and storage, to method development and validation and to variability evaluation.
The analysis of saliva provides important information about the functioning of various organs within the body. In this respect, endocrine research certainly occupies a central role. In effect, some hormones commonly measured in plasma, such as steroids, non-steroid, peptide and protein hormones, can be detected in the oral fluid. The protein polypeptide hormones are maybe a new analytical approach of the medicine laboratory but, at present time, there are still too few investigations about protein and polypeptide hormone levels in saliva. Detection of steroid hormones is perhaps the most interesting application in salivary hormonal studies. Steroids have often been studied because salivary-free steroid hormones seem to reflect the serum-free levels. Among of these steroids, salivary cortisol measurement is today a widely accepted alternative to the determination in plasma.
In the section I of the present dissertation, a new ELISA method is explained. Research was carried out to study and validate an assay to measure salivary free IGF-I (sIGF-I) in human saliva. The detection range, the detection limit, the imprecision, the recovery and the specificity were evaluated. The pre-analytical variation was also studied. After the method validation, sIGF-I levels were measured in sedentary subjects and in athletes (protocol A and protocol C); moreover two different acute physical exercises in two groups of athletes were investigated to assess a possible effect on sIGF-I (protocol B and protocol C).
The section II describes an analysis method which used the chromatographic technique. A SPE-HPLC method with UV detection was developed and validated to simultaneously measure cortisol (sF) and cortisone (sE) in human saliva. The analytical performances, in terms of detection range, sensitivity, imprecision, recovery, were evaluated. The pre-analytical variation, with respect to collection strategy and storage conditions, was also examined. After validation, the sF and sE method was applied analyzing specimens collected from athletes, before and after a physical exercise (protocol C).
The results suggest further investigation from the laboratory point of view, taking into account the aspects related to the various forms and the specific and unspecific binding proteins (for sIGF-I assay) and to other steroid hormones and related metabolites identified and probably present in human saliva (for sF/sE assay).

Abstract (italiano)

Lo studio della saliva nelle analisi di laboratorio è un’area di ricerca in forte crescita, per le sue implicazioni nella ricerca di base ma anche a fini clinici. Sebbene questo fluido biologico sia facile da manipolare e da raccogliere, bisogna porre attenzione ai processi di raccolta e stoccaggio del campione, nonché allo sviluppo e alla validazione di metodi analitici, assieme alla valutazione delle variabilità.
L’analisi della saliva dà importanti informazioni sul funzionamento di vari organi del corpo. In relazione a questo, la ricerca endocrina occupa certamente un ruolo centrale. Infatti, alcuni ormoni normalmente misurati nel plasma, come ormoni steroidei, ma anche ormoni non steroidei, peptidici e proteici, possono essere identificati nel fluido orale. Un nuovo approccio analitico nella medicina di laboratorio è forse rappresentato dagli ormoni polipeptidici e proteici ma, tuttora, ci sono ancora troppi pochi studi su questi ormoni salivari. La misura degli ormoni steroidi, invece, rappresenta forse l’applicazione più interessante negli studi degli ormoni salivari. Spesso gli steroidi sono studiati perché la concentrazione salivare riflette i livelli sierici. Tra i vari steroidi, la misura del cortisolo salivare è oggi una alternativa alla sua determinazione plasmatici.
Nella I sezione viene spiegato un nuovo metodo ELISA. Sono stati sviluppati test sperimentali per studiare e validare un metodo per la misura dell’IGF-I libero salivare (sIGF-I). Sono stati studiati il range di misura, la sensibilità, l’imprecisione, il recupero e la specificità. Inoltre è stata studiata anche la variabilità pre-analitica. Dopo la validazione del metodo, sono stati misurati i livelli di sIGF-I in soggetti sedentari ed in atleti (protocollo A e protocollo C); inoltre è stato studiato il possibile effetto di due differenti esercizi fisici (in acuto) sulle concentrazioni di sIGF-I (protocollo B e protocollo C).
La II sezione prende in esame un metodo di analisi che usa la tecnica cromatografia. E’ stato sviluppato e valicato un metodo SPE-HPLC con rivelazione UV per la misura contemporanea del cortisolo (sF) e del cortisone (sE) nella saliva umana. Sono state calcolate le performance analitiche (range di misura, sensibilità, imprecisione, recupero). E’ stata considerata anche la variabilità pre-analitica con particolare attenzione alle condizioni di raccolta e conservazione del campione. Dopo la validazione, questo metodo è stato applicato a campioni raccolti da un gruppo di atleti, prima e dopo un esercizio fisico (protocollo C).
I risultati ottenuti suggeriscono ulteriori approfondimenti soprattutto da un punto di vista laboratoristico, tenendo presente la possibile presenza di varie forme e di specifiche ed aspecifiche proteine di legame (per sIGF-I) e altri ormoni steroidei e loro metaboliti identificati e probabilmente presenti nella saliva umana (per sF/sE).

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Tipo di EPrint:Tesi di dottorato
Relatore:Ragazzi, Eugenio
Correlatore:De Palo, Elio Franco
Dottorato (corsi e scuole):Ciclo 21 > Corsi per il 21simo ciclo > FARMACOLOGIA MOLECOLARE E CELLULARE
Data di deposito della tesi:20 Gennaio 2009
Anno di Pubblicazione:2009
Parole chiave (italiano / inglese):saliva, IGF-I, cortisol, cortisone, ELISA, HPLC
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/46 Scienze tecniche di medicina di laboratorio
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Medico Diagnostiche e Terapie Speciali
Codice ID:1347
Depositato il:20 Gen 2009
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