Rozprawy doktorskie na temat „Clenbuterol”

Afin de lutter efficacement contre les malversations du dopage, il apparaît essentiel de comprendre les mécanismes conduisant au remodelage musculaire. Dans ce but nous avons analysé les effets d’un β2-agoniste, le clenbutérol, sur le remodelage musculaire et les différentes voies de signalisation qui y sont associées. Nous nous sommes particulièrement intéressés au système des calpaïnes qui a souvent été associé à des phénomènes de remodelage musculaire, principalement dans des modèles d’atrophie. Nous avons montré une sollicitation précoce du système des calpaïnes lors d’un traitement chronique au clenbutérol chez le rat associé à une conversion phénotypique dans les muscles EDL et Soléaire et à une hypertrophie dans le muscle EDL uniquement. Puis, nous avons inhibé l’activité des calpaïnes en parallèle d’un traitement au clenbutérol. Les muscles ayant une activité des calpaïnes diminuée et soumis à un traitement au clenbutérol n’ont pas développé de remodelage musculaire. Ces premiers résultats renforcent l’idée d’une implication des calpaïnes dans le remodelage musculaire induit par un traitement chronique au clenbutérol
To fight doping in an effective manner, it is essential to understand the mechanisms leading to muscle remodeling. For this purpose we analyzed the effects of clenbuterol, on muscle remodeling and various associated signaling pathways. We were particularly interested with the calpain system which has often been associated with muscle remodeling phenomena, mainly in models of atrophy. We have shown that an early calpain system solicitation during chronic treatment with clenbuterol in rats was associated with a phenotypic conversion in the Soleus and EDL muscles and hypertrophy in the EDL muscle. We then inhibited the activity of calpains with a parallel clenbuterol treatment. The muscles with a reduced activity of calpain and treated with clenbuterol did not develop muscle remodeling. These initial results reinforce the idea of an involvement of calpain in the muscle remodeling induced by chronic treatment with clenbuterol

Clenbuterol Hydrochloride is a bronchodilator marketed as Spirospent® for Human Pharmaceuticals and as Ventipulmin® for Veterinary Pharmaceuticals by Boehringer Ingelheim.1 This research investigates formation of the 4-amino-α,α-dihydroxy-3,5-dichloroacetophenone impurity (Dihydroxy Impurity) in the synthesis of Clenbuterol Hydrochloride. The Dihydroxy Impurity has increased three-fold since the process was transferred to Boehringer Ingelheim Chemicals, Inc. in 1999.1 The Dihydroxy Impurity is proposed to be produced during an amination/reduction step from a reaction of water with the starting material.2 Additionally, the Dihydroxy Impurity can be formed by the reaction of water with an impurity, 4-amino-α,α-didibromo-3,5-dichloroacetophenone (Dibromo Impurity), that is generated in the previous step.2 The formation of the Dibromo Impurity was investigated through a series of Design of Experiments (DoE) analyses. The results from these analyses, presented within, determined the optimum bromination conditions to reduce the Dibromo Impurity. These conditions were able to reduce the Dibromo Impurity by 75%. A series of water spiking experiments with both the starting material of the amination/reduction step and the Dibromo Impurity were performed to investigate the formation of the Dihydroxy Impurity. Based on the results, a mechanism for the formation of the Dihydroxy Impurity is presented. The three-fold increase of the Dihydroxy Impurity was concluded to be due to ≥ 15% water in the amination reaction mixture reacting with the starting material. REFERENCES 1. Powner, T. H. Process Development Report: Preparation of Clenbuterol Hydrochloride USP, EP; Document No. DRCLEN Rev 0; Boehringer Ingelheim Chemicals, Inc.: Petersburg, VA, 2002; 1 – 194. 2. Brown, J. Clenbuterol Free Base C124A (Lot 1011974) Impurity; Boehringer Ingelheim Chemicals, Inc.: Petersburg, VA, 2007; 1 – 2.

Mitochondrial dysfunction is associated with numerous acute and chronic degenerative diseases. The beta-2 adrenergic receptor (beta(2)AR) agonist formoterol induces mitochondrial biogenesis (MB), but other beta(2)AR agonists, such as clenbuterol, do not. We sought to identify the MB signaling pathway of formoterol and the differences in signaling between these two ligands that result in the differential induction of MB. While formoterol and clenbuterol increased cAMP, only formoterol increased the phosphorylation of Akt and its downstream target eNOS. The increase in Akt phosphorylation was G beta gamma- and PI3K-dependent, and the increase in eNOS phosphorylation was G beta gamma- and Akt-dependent. Only formoterol increased cGMP. Formoterol induced MB as measured by increases in uncoupled cellular respiration and PGC-1 alpha and NDUFS1 mRNA expression and was blocked by inhibitors of G beta gamma, Akt, NOS, and soluble guanylate cyclase. To identify distinct receptor-ligand interactions leading to these differences in signaling, we docked formoterol and clenbuterol to six structures of the beta(2)AR. Compared to clenbuterol, the methoxyphenyl group of formoterol interacted more frequently with V114 and F193, while its formamide group interacted more frequently with C191. These data indicate that the unique structural features of formoterol allow it to interact with the beta(2)AR to activate the G beta gamma-Akt-eNOS-sGC pathway to induce MB.

6. ZUSAMMENFASSUNG Charakterisierung b-adrenerger Rezeptoren auf Pferdelymphozyten und deren Regulation unter dem Einfluß von Clenbuterol und Dexamethason Getu Abraham Institut für Pharmakologie, Pharmazie und Toxikologie, Veterinärmedizinische Fakultät, Universität Leipzig, Deutschland Juni, 2001 86 S., 205 Lit., 7 Tab., 23 Abb. Über Vorkommen und Eigenschaften von b-Adrenozeptoren auf Pferdelymphozyten und ihre pharmakologische Regulation liegen bisher keine Untersuchungen vor. Mit der vorliegenden Arbeit sollten b-adrenerge Rezeptoren an intakten Pferdelymphozyten in Bindungsstudien mit dem Radioliganden (-)-(125I)-Iodocyanopindolol (ICYP), einem potenten b2-selektiven Adrenozeptorenantagonisten, charakterisiert und deren funktionelle Ansprechbarkeit durch Messung des Isoprenalin-induzierten Anstiegs des intrazellulären cAMP-Gehaltes mittels Radioimmunoassay bestimmt werden. Die Bindung von ICYP an intakten Pferdelymphozyten war schnell, sättigbar (maximale Anzahl der Bindungsstellen 320 ± 20 ICYP Bindungsstellen/Zelle, Mittelwert±SEM, n = 12) ) und hochaffin (KD-Wert für ICYP 14,37 ± 1,66 pmol/l, Mittelwert±SEM, n = 12). Verdrängungsexperimente mit Agonisten und Antagonisten zeigten eine Affinität und Stereoselektivität, wie sie bei b-Adrenozeptoren zu erwarten waren. Der nicht radioaktiv markierte b2-selektive Adrenozeptorantagonist ICI 118,551 verdrängte den Radioliganden ICYP aus seiner Bindung mit mehr als 1500fach höherer Affinität als der nicht radioaktiv markierte b1-selektive Adrenozeptorantagonist CGP 20712A. In Pferdelymphozyten können somit mehr als 90 % der b-Adrenozeptoren dem b2-Subtyp zugeordnet werden. Ein Anteil von weniger als 10 % an b1-Adrenozeptoren kann allerdings nicht ausgeschlossen werden. Die Bindungsstellen waren stereospezifisch, da das biologisch aktive (-)-Propranolol die Bindung von ICYP mit 40fach höherer Affinität verdrängte als (+)-Propranolol. b-adrenerge Agonisten verdrängten ICYP aus seiner spezifischen Bindung in der Rangfolge ihrer pharmakologischen Wirkungstärke, die typisch für den b2-Subtyp des Adrenozeptors ist: (-)-Isoprenalin > (-)-Adrenalin > (-)-Noradrenalin. Eine ähnliche Rangfolge wurde auch für die Agonist-induzierte cAMP-Bildung in Lymphozyten festgestellt. Aufgrund der dargestellten Befunde kann die Schlußfolgerung gezogen werden, daß ICYP ein geeigneter Radioligand ist, um b-adrenerge Rezeptoren an Pferdelymphozyten zu identifizieren und zu charakterisieren. Die Bindung von ICYP an intakte Lymphozyten stellt somit ein geeignetes Modell dar, um wichtige Informationen über die Funktion und Regulation des b-adrenergen Systems beim Pferd sowohl unter physiologischen (pathophysiologischen) Bedingungen als auch über Arzneimittel-induzierte Veränderungen in vivo zu gewinnen. Ausgehend von den beschriebenen Grundlagenuntersuchungen wurden in einer weiteren Versuchsreihe bei zwölf klinisch gesunden Pferden der Einfluß von Clenbuterol und Dexamethason lymphozytäre b2-Adrenozeptoren, hinsichtlich ihrer Dichte, ihrer Affinität zum Radioliganden und ihrer cAMP-Bildung (als Maß für die funktionelle Ansprechbarkeit der b2-Adrenozeptoren bestimmt über Stimulation durch 10 µmol/l Isoprenalin) untersucht. Während der 12tägigen Clenbuterolbehandlung (2 Ž 0,8 µg/kg/Tag, i. v.) fielen die Anzahl der b2-Adrenozeptoren an Lymphozyten und der durch (-)-Isoprenalin-induzierte Anstieg des intrazellulären cAMP-Gehaltes signifikant (p < 0,05, n = 8) unter die Ausgangswerte unbehandelter Tiere ab. Clenbuterol bewirkte in der therapeutischen Dosis bereits 48 Stunden nach der Behandlung einen Abfall der Anzahl der b2-Adrenozeptoren bzw. der cAMP-Bildung um etwa 30 - 40 %. Während der restlichen Behandlungsdauer unter Clenbuterol blieb die Rezeptorendichte auf diesem niedrigen Niveau. Bei einer weiteren Versuchsgruppe wurde der Effekt des Glukokortikoids Dexamethason auf die Clenbuterol-induzierte Desensibilisierung von b2-Adrenozeptoren untersucht. Dexamethasonverabreichung (1 Ž 0,1mg/kg/d, i. v.) unmittelbar nach der letzten Clenbuteroldosis über 5 Tage beschleunigte die Wiederherstellung der down-regulierten Anzahl der lymphozytären b2-Adrenozeptoren und des Isoprenalin-induzierten Anstiegs des intrazellulären cAMP-Gehaltes (p < 0,05, n = 4). Schon 24 Stunden nach der ersten Dexamethasongabe waren beide Parameter von den Durchschnittswerten am Tag vor Behandlungsbeginn statistisch nicht zu unterscheiden. Die Anzahl der lymphozytären b2-Adrenozeptoren stieg sogar ab dem dritten Tag der Dexamethasonbehandlung bis auf das Doppelte bei unbehandelten Tiere an. Nach dem Absetzen der Dexamethasonbehandlung kam es zu einer langsam Abnahme der Rezeptorendichte und ihrer Ansprechbarkeit, wobei die vor der Clenbuterolbehandlung gemessenen Werte erst nach 4 Tagen erreicht wurden. Bei gleichzeitiger Behandlung der Tiere mit Clenbuterol und Dexamethason konnte durch das Glukokortikoid die Clenbuterol-induzierte Abnahme der b2-Adrenozeptorendichte und deren Ansprechbarkeit vollständig verhindert werden. Weder durch die Clenbuterol-abhängige Down-Regulation noch durch die Dexamethason-bedingte Hochregulation kam es zu signifikanten Veränderungen der Dissoziationskonstante (KD) und somit der Affinität von ICYP zu b2-Adrenozeptoren. Die Ergebnisse zeigen, daß es unter Behandlung mit Clenbuterol zu einer schnell einsetzenden, umfangreichen Down-Regulation der b2-Adrenozeptorendichte an Lymphozyten des Pferdes kommt und daß diese Toleranzentwicklung durch Glukokortikoide vollständig wieder aufgehoben oder verhindert werden kann. Zur Therapie der COB von Pferden scheint somit die kombinierte Anwendung von b2-Mimetika und Glukokortikoiden von Vorteil zu sein
7. Summary Characterization of b-adrenergic Receptors on Equine Lymphocytes and their Regulation under the Influence of Clenbuterol and Dexamethasone Getu Abraham Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, Germany June, 2001 86 pp., 205 ref., 7 tab., 23 fig. b-Adrenoceptors and their regulation by pharmacological active substances have not yet been characterized in equine lymphocytes. In this study, b-adrenoceptors were identified and subclassified by (-)-[125I]-iodocyanopindolol (ICYP) binding, a potent b2-adrenergic antagonist, as well as by their isoprenaline-induced responsiveness using radioimmunoassay in intact viable equine lymphocytes. The specific ICYP binding was rapid, saturable (maximum number of binding sites 320 ± 20 ICYP binding sites/cell, means±SEM, n = 12) and of high affinity (KD-value for ICYP 14.37 ± 1.66 pmol/l, means±SEM, n = 12). The competition binding studies with agonists and antagonists showed the affinity and stereoselectivity to be expected for b-adrenergic receptors. The unlabelled selective b2-adrenoceptor antagonist ICI 118,551 was about 1500 times more potent in inhibiting ICYP binding than was the b1-selective adrenoceptor antagonist CGP 20712A. It is, therefore, concluded that in intact equine lymphocytes ICYP labels a class of functional b-adrenoceptors, that belong predominantly (> 90 %) to the b2-adrenoceptor subtype. A minor (< 10 %) b1-adrenoceptor component, however, cannot be completely ruled out. The binding sites were stereoselective, as the (-)-isomer of propranolol was about 40 times more potent to inhibit ICYP binding than was the (+)-isomer. Agonists competed for specific binding with a rank order of potency (-)-isoprenaline > (-)-adrenaline > (-)-noradrenaline, which is typical for a b2-subtype of adrenergic receptor; the same order of potency was obtained for agonist-induced stimulation of lymphocyte cyclic AMP content. Thus, it can be concluded that ICYP is a promising compound for the reliable determination of the binding characteristics of b-adrenoceptors of equine lymphocytes and besides that, this might give an insight in providing physiologically significant information about the regulation of the b-adrenergic receptor system. Based upon this study, we further investigated, in 12 healthy thoroughbred horses, the effects of the b2-agonist clenbuterol and the glucocorticoid dexamethasone on the lymphocyte b2-adrenenergic receptor density and affinity as well as on its responsiveness (assessed by lymphocyte cyclic AMP responses to 10 µmol/l (-)-isoprenaline). Clenbuterol (2Ž0.8µg/kg/d, i. v. for 12 days) decreased ICYP binding sites only 48 h after application by ~30-40 %; concomitantly, lymphocyte cAMP response to (-)-isoprenaline was significantly reduced (p < 0.05, n = 8). The values remained at these low levels throughout the following 10 days of treatment. After withdrawal of clenbuterol the density and responsiveness of b2-adrenoceptors gradually increased, reaching pre-drug levels after 4 days. Next the effects of dexamethasone on clenbuterol-induced desensitisation were studied. Administration of dexamethasone (1Ž0.1mg/kg/d, i. v. for five days) immediately after clenbuterol withdrawal accelerated b2-adrenergic receptor recovery (p < 0.05, n = 4): only 24 hours after administration dexamethasone restored the number of binding sites and cAMP response to (-)-isoprenaline to levels statistically indistinguishable from the values before starting clenbuterol treatment. Three days after dexamethasone administration the density of lymphocyte b2-adrenoceptors was further increased about two fold the pre-treatment values, and this increase declined gradually after dexamethasone withdrawal, reaching baseline values after 4 days. Furthermore, in groups simultaneously exposed to both drugs dexamethasone completely prevented clenbuterol-induced decrease in lymphocyte b2-adrenoceptor density and responsiveness. No significant change was observed in the dissociation constant and, thus, in the affinity of ICYP to b-adrenoceptors remained unchanged during or after treatment with either clenbuterol or dexamethasone. It is concluded that dexamethasone (glucocorticoids) can reverse and prevent clenbuterol-induced down-regulation of the lymphocyte b2-adrenoceptors and thus, a combined therapy with clenbuterol and dexamethasone may be potentially beneficial in COPD of horses

It was proposed that some O-alkyl analogues of the beta-adrenergic agonist clenbuterol would be effective structural and functional congeners of clenbuterol which may then be used for the production of clenbuterol-specific idiotypic antibodies. These antibodies could possibly then be used to generate anti-idiotypic antibodies that mimic the energy-repartitioning effects of clenbuterol. Therefore, the aim of this work was to synthesise and characterise these compounds, evaluate their physiological effects, characterise the specificity of antibodies produced in response to protein conjugates of two of the novel compounds, and then use this data to determine the utility of these compounds for the generation of anti-idiotype antibodies which mimic clenbuterol. The target compounds were synthesised in five steps from 3,5-dichloro-4-hydroxyacetophenone in overall yields of 5-28%. A synthetic scheme similar to that which has led to clenbuterol was used to form the phenylethanolamine backbone, with modifications to include the O-alkyl moiety via a modified Williamson ether synthesis, and elimination of a synthetic chlorination step. Overall, 15 new compounds were synthesised, which were characterised and their structure confirmed from proton and carbon-13 NMR, IR and mass spectral data. The two haptenic analogues were then conjugated to carrier proteins using carbodiimide-based chemistries. In conclusion, the results indicated that the O-alkyl analogues, although structurally similar, were ineffective functional mimics of clenbuterol. Therefore, the anti-clenbuterol antobodies produced from the novel O-alkyl analogues would appear to be unsuitable for production of anti-idiotypic antibodies that mimic the energy-repartitioning effects of clenbuterol since the antibodies were unable to distinguish between the compound which demonstrated energy-repartitioning effects (clenbuterol) and those that did not (O-alkyl clenbuterol analogues).

Un traitement de 8 semaines au clenbuterol chez le rat, à des doses considérées comme dopantes, modifie profondément la composition corporelle des animaux avec une augmentation de la masse maigre et une diminution de la masse grasse. L'entraînement de force-résistance combiné au traitement accentue encore plus ces différences alors que l'exercice seul n'a qu'un effet modéré. Au niveau musculaire les principaux effets du traitement et/ou de l'entraînement sont une redistribution du type de fibres des muscles squelettiques lent (soleus), mixte (plantaris) et rapide (EDL) vers un phénotype encore plus rapide. Ceci s'accompagne d'une amélioration de la force maximale et d'une baisse de la résistance à la fatigue, avec des variations des taux plasmatiques d'IGF-1. Ces modifications sont retrouvées d'un point de vue métabolique, avec des changements variés des activités enzymatiques de la CK, de la LDH et de la CS en fonction du muscle squelettique considéré. Enfin, au niveau du métabolisme osseux, les résultats de cette étude indiquent qu'un traitement au clenbuterol diminue la BMC et la BMD fémorales chez les rats entraînés et sédentaires. Ces effets résultent probablement d'une augmentation de l'excrétion urinaire de DPD et une diminution des concentrations plasmatiques d'IGF-1 chez les animaux traités au clenbuterol. Il ressort que le clenbuterol utilisé à des doses dopantes peut effectivement générer un gain de force maximale, une augmentation de la masse maigre et une diminution du tissu adipeux, effets recherchés par les sportifs, mais aussi des effets néfastes, en particulier sur le métabolisme osseux où l'administration prolongée de ce béta2-agoniste risque de conduire à des fractures. Ces résultats, en particulier ceux obrenus sur le métabolisme osseux, nécessitent de nouvelles investigations pour confirmer ces effets délétères, notamment chez l'homme

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
A utilização ilícita de fármacos no desporto não é um fenómeno recente e é uma prática bem conhecida e difundida. São várias as substâncias que alteram a condição física do desportista e que podem melhorar o seu rendimento. A utilização de fármacos com finalidades não terapêuticas, além de ser eticamente reprovável, não é isenta de efeitos adversos na saúde dos atletas. A World Anti-Doping Agency (WADA) foi criada devido ao crescente número de casos de doping e de acontecimentos trágicos associados ao seu uso no desporto. A WADA é a entidade responsável pelo controlo anti-doping e pela publicação periódica de uma lista de substâncias e métodos proibidos. Esta lista tem vindo a aumentar e, neste momento, apresenta as seguintes classes de fármacos proibidas durante e fora das competições em competição: agentes anabólicos, hormonas e fatores de crescimento, agonistas, hormonas e modeladores metabólicos, diuréticos e agentes mascarantes. Estão ainda identificadas como classes de fármacos proibidas em competição os estimulantes, os narcóticos, os canabinóides e os glucocorticóides. Os -bloqueadores e o álcool estão identificados na lista, mas apenas são proibidas em desportos em particular. Segundo a literatura atual, os fármacos mais utilizados no doping são os agentes anabolizantes e os estimulantes. Este padrão de utilização do doping também tem sido observado no passado mais recente. Os dados estatísticos emitidos pela WADA desde 2003 mostram que os agentes anabolizantes são os dopantes mais frequentemente detetados e utilizados no desporto. Os atletas recorrem maioritariamente ao uso destas substâncias pelo facto destas proporcionarem um aumento do rendimento tanto nos treinos como em competição, conferindo-lhes uma evolução mais rápida e vantajosa perante os adversários. Também por estas razões, os desportos que apresentam maior taxa de utilização de agentes dopantes são o ciclismo, o atletismo e o futebol. Com este trabalho pretende-se analisar os dados mais recentes da utilização ilícita de fármacos no desporto e discutir os mecanismos de ação dos principais fármacos que podem conferir vantagem aos atletas e ainda as reações adversas decorrentes da sua utilização. The illicit use of drugs in sport is not a recent phenomenon and it is a well-known and widespread practice. There are several substances that alter the physical condition of the sportsman and can improve their performance. The use of drugs with no therapeutic purposes as well as being ethically reprehensible, isn't absent of adverse effects on the athletes' Health. The World Anti-Doping Agency (WADA) was created due to the increasing number of cases of doping and tragic events associated with its use in sport. The WADA is the entity responsible for anti-doping control and the periodic publication of an updated list of banned substances. This list has been increasing and at the moment, has the following classes of drugs: anabolic agents, hormones and growth factors, agonist’s -2, hormones and metabolic modulators, diuretics and masking agents. Still identified as classes of drugs prohibited in-competition are stimulants, narcotics, cannabinoids and glucocorticoids. Other classes such as -blockers and alcohol are identified in the list, but are only prohibited in particular sports. According to current literature, the pharmaceuticals which are more widely used in doping are anabolic and stimulant agents. This pattern of usage in doping has also been noticeable in the more recent past. The statistical data issued by WADA since 2003 shows that anabolic agents are the most frequently detected and used doping substances in sports. Athletes resort to the use of such substances mainly due to the fact that they provide a boost in performance, both in training and competition, providing them with a faster and more advantageous evolution than their opponents’. It is also because of the aforementioned reasons that the sports displaying a higher rate of usage of doping agents are cycling, athletics and football. With this project, one intends to analyse the most recent data concerning the illicit use of pharmaceuticals in sports and discuss the acting mechanisms of the main drugs which may provide athletes with an advantage, as well as the adverse effects arising from their use.

Stress activates a number of endocrine pathways that alter an animal's physiology in a manner which can result in undesirable meat quality. Animals frequently exhibit meat quality defects, including ecchymosis, at slaughter due to the stress of slaughter. This thesis explores how stress related hormones interact with adrenergic receptors to alter muscle and vascular physiology. Fallow deer were exposed to either a transciptional regulator (hydrocortisone), a beta adrenergic recptor agonist (clenbuterol) or a beta adrenergic receptor antagonist (propranolol). The administration of hydrocortisone resulted in a negative feed-back type reduction in circulating cortisol. Animals treated with propranolol and clenbuterol displayed less severe eccymosis. These results indicated that the beta 2 adrenergic receptor (B2AR) is important in controlling ecchymosis severity. B2AR was also found to be important in mediating vascular dynamics, growth and energy pathways. To investigate how adrenergic receptors alter skeletal muscle gene expression and meat quality, an in vivo wistar rat model was developed in conjunction with in vitro muscle cell (L6) experiments. Gene expression of B2AR, its associated kinase (BARK) and collagen type III, prolyl- 4-hydroxylase (P4Hy) was measured in rat muscle and L6 cells. Following exposure to clenbuterol and hydrocortisone, growth and meat quality were determined. The L6 experiments revealed that gene expression following exposure to hydrocortisone and B2AR ligands paralleled the in vivo rat changes in B2AR, BARK, collagen type III, and P4Hy gene expression. In both L6 and wistar rat models the B2AR and BARK genes are similarly expressed following clenbuterol exposure. Both rats and deer exposed to clenbuterol had significant increases in growth rate and a reduction of intramuscular fat. The B2AR therefore appears to be a major mediator of many interrelated events including energy distribution, growth and vascular response to stress. Habituating animals to stress stimuli may increase their coping ability and improve welfare and meat quality.
Doctor of Philosophy (PhD)

El análisis por Cromatografía Líquida de Alta Precisión (HPLC) de productos farmacéuticos es una necesidad y es de uso rutinario. Esta técnica evita minimiza los errores que conllevan a situaciones de riesgo al usuario, garantizando que el contenido en el producto sea el correcto. La validación es un proceso establecido que obtiene pruebas documentadas y demostrativas para que un método de análisis sea lo suficientemente fiable y reproducible para producir un resultado previsto dentro de los intervalos definidos. La validación de un método analítico es un requisito necesario para cumplir con las Buenas Prácticas de Manufactura (BPM) y así asegurar la calidad del medicamento. La Técnica Analítica desarrollada y propuesta en el presente trabajo plantea la cuantificación por el método de HPLC de un producto farmacéutico que contiene Clenbuterol Clorhidrato solución oral gotas. En el cual se procedió a la validación del método de análisis evaluando los parámetros que indican las obras oficiales, como son: selectividad, linealidad, precisión, exactitud y robustez. Posteriormente, se elaboró el Protocolo de validación del método de análisis, para lo cual se contó con el diseño experimental y los procedimientos estadísticos, concluyéndose así que el método analítico propuesto es selectivo, lineal, preciso, reproducible y exacto; comprobándose así su validez
The Analysis by High Precision Liquid Chromatography (HPLC) of pharmaceuticals is a necessity and is routinely used. This technique avoids minimizes errors that lead to situations of risk to the user, ensuring that the contents in the product is correct. Validation is a process established that evidence obtained to document and demonstrates an analytical method is sufficiently reliable and reproducible to produce an intended result within the defined intervals. The analytical method validation is a necessary requirement to comply with Good Manufacturing Practices (GMP) and thus ensure the quality of the product. The analytical technique developed and proposed in this paper discusses the quantification by HPLC of a pharmaceutical product containing Clenbuterol Hydrochloride oral solution drops. In which we proceeded to validate the analysis method evaluating the parameters that indicate the official works, such as: selectivity, linearity, precision, accuracy and robustness. Subsequently developed the Protocol for validation of analytical method, for which they received the experimental design and statistical procedures, concluding that the proposed analytical method is selective, linear, accurate, reproducible and accurate; see that they are valid

El análisis por Cromatografía Líquida de Alta Precisión (HPLC) de productos farmacéuticos es una necesidad y es de uso rutinario. Esta técnica evita minimiza los errores que conllevan a situaciones de riesgo al usuario, garantizando que el contenido en el producto sea el correcto. La validación es un proceso establecido que obtiene pruebas documentadas y demostrativas para que un método de análisis sea lo suficientemente fiable y reproducible para producir un resultado previsto dentro de los intervalos definidos. La validación de un método analítico es un requisito necesario para cumplir con las Buenas Prácticas de Manufactura (BPM) y así asegurar la calidad del medicamento. La Técnica Analítica desarrollada y propuesta en el presente trabajo plantea la cuantificación por el método de HPLC de un producto farmacéutico que contiene Clenbuterol Clorhidrato solución oral gotas. En el cual se procedió a la validación del método de análisis evaluando los parámetros que indican las obras oficiales, como son: selectividad, linealidad, precisión, exactitud y robustez. Posteriormente, se elaboró el Protocolo de validación del método de análisis, para lo cual se contó con el diseño experimental y los procedimientos estadísticos, concluyéndose así que el método analítico propuesto es selectivo, lineal, preciso, reproducible y exacto; comprobándose así su validez.
The Analysis by High Precision Liquid Chromatography (HPLC) of pharmaceuticals is a necessity and is routinely used. This technique avoids minimizes errors that lead to situations of risk to the user, ensuring that the contents in the product is correct. Validation is a process established that evidence obtained to document and demonstrates an analytical method is sufficiently reliable and reproducible to produce an intended result within the defined intervals. The analytical method validation is a necessary requirement to comply with Good Manufacturing Practices (GMP) and thus ensure the quality of the product. The analytical technique developed and proposed in this paper discusses the quantification by HPLC of a pharmaceutical product containing Clenbuterol Hydrochloride oral solution drops. In which we proceeded to validate the analysis method evaluating the parameters that indicate the official works, such as: selectivity, linearity, precision, accuracy and robustness. Subsequently developed the Protocol for validation of analytical method, for which they received the experimental design and statistical procedures, concluding that the proposed analytical method is selective, linear, accurate, reproducible and accurate; see that they are valid.
Tesis

Data reported in the present thesis were obtained in two different projects. Accordingly, the thesis is divided into two chapters. The first chapter, which refers to papers I and II, reports a study aimed at unravelling the physiologic role of the cellular prion protein (PrPC) using an in vivo model of skeletal muscle regeneration. The second chapter refers to paper III in which the two dimensional electrophoresis (2DE) approach in combination with tandem mass spectrometry was used to identify potential biological markers of the illegal treatment of bulls with growth promoting agents (GPA). By focusing on the relationship between PrPC and skeletal muscle regeneration in a live model, in the first research line (Chapter I) we investigated if and how the protein influences the proliferation and the differentiation of muscle precursor cells. PrPC is a cell surface glycoprotein involved in the onset of rare and fatal neurodegenerative disorders, known as transmissible spongiphorm encephalopathies (TSE) or prion diseases. TSE occur when PrPC converts into a conformationally modified isoform that originates the prion, a novel infectious and neuro-pathogenic agent. Although much information is now available on the different routes of prion infection, both the mechanisms underlying prion neurotoxicity and the physiologic role of PrPC remain unclear. Nonetheless, use of different animal and cell models has suggested a number of putative functions for the protein, ranging from cell protection against oxidative and apoptotic challenge, to cell adhesion, proliferation and differentiation. Skeletal muscles express significant amounts of PrPC, and have been related to PrPC pathophysiology by several findings. Therefore, in order to clarify the physiologic role of PrPC, we employed a degeneration/regeneration protocol to the tibialis anterior muscle, which allowed us to compare the regeneration in mice expressing, or not, PrPC. The analyzed histological and biochemical parameters provided proof for the physiologic relevance of PrPC commitment in signalling events involved in muscle regeneration. Indeed, we observed that the absence of PrPC significantly delayed the regenerative process compared to WT muscles. In particular, we found that the lack of PrPC caused attenuation of the signalling pathway triggered by TNF-, which in turn decreased the activation of the p38 kinase pathway, and – consequently – later exit from the cell cycle, and differentiation, of myogenic precursor cells. Importantly, restoring PrPC expression completely rescued the PrP-KO muscle phenotype, highlighting that regulation of signalling pathways by PrPC has clear physiologic importance in an extraneural tissue. The second research line, described in Chapter II, was aimed at setting up a proteomic-based strategy to identify illicit drug treatments in bulls. Classical assays for detecting this kind of illegal practice are not suited to detect compounds either of unknown chemical structure, or present at levels below the quantification threshold of the presently used analytical techniques. The successful application of histological analyses of target organs, which are indirectly modified following these treatments, has suggested that approaches based on the biological effects of the molecules under consideration, rather than the direct detection of their residues, could be potentially valuable in the field. The most relevant advantage of this methodology is that cellular or tissue modifications by drugs remain evident long time after the end of illicit treatments, when chemical residues are no longer, or hardly detectable. On the other hand, this approach is significantly limited by subjective experience and evaluation skill of technicians. Thus new strategies are needed for detecting indirect biomarkers in animal fluids or tissues. These biomarkers can be naturally occurring molecules, such as proteins that are modified in structure, or in concentration, following variations of the normal condition of the animal. To identify possible biologic markers of illicit drug treatments of beef cattle, we adopted a proteomic approach, including 2D differential in gel electrophoresis (DIGE) and mass spectrometry analysis, to compare the protein expression pattern of muscle specimen from experimentally treated bulls and control animals. To this aim, bulls belonging to the treated cohort were subjected to three different pharmacological protocols, including use of growth promoting agents (GPA). Two of these treatments showed a remarkable anabolic effect compared to untreated animals, resulting in an altered skeletal muscle proteome. 2DE protein maps from treatment and control groups were compared using the DeCyder software for 2D-DIGE maps analysis. We then set out to identify, using a MALDI-tandem mass spectrometry (MS/MS) approach, all proteins showing a significant alteration in their expression levels following administration of GPA. Among differentially expressed 169 proteins, 29 were identified, most of which were found to be involved in muscle contraction and energy metabolism. These results corroborate previous findings on the mechanism of action of GPA, and may be useful to design new strategies for the discovery of illicit pharmacological treatments in bulls.
I dati riportati nella presente tesi sono stati ottenuti in due diversi progetti. Pertanto, la tesi è divisa in due distinti capitoli. Il primo capitolo, che si riferisce agli articoli I e II, riporta uno studio volto a chiarire il ruolo fisiologico della proteina prionica cellulare (PrPC) utilizzando un modello in vivo di rigenerazione del muscolo scheletrico. Il secondo capitolo si riferisce all'articolo III, in cui si è cercato di individuare possibili marcatori biologici di trattamento illecito di vitelloni con agenti promotori della crescita (GPA), utilizzando un approccio di elettroforesi bidimensionale (2DE), in combinazione con spettrometria di massa. Focalizzando l'attenzione sul rapporto tra PrPC e la rigenerazione del muscolo scheletrico in un modello in vivo, nella prima linea di ricerca (Capitolo I) abbiamo indagato se e come la proteina influenza la proliferazione e la differenziazione delle cellule precursori del muscolo. PrPC è una glicoproteina ancorata alla membrana esterna delle cellule coinvolta nella comparsa di malattie neurodegenerative rare e mortali, conosciute con il nome di encefalopatie spongiformi trasmissibili (EST) o malattie da prioni. L'evento alla base delle EST è la conversione della PrPC in una isoforma con una modificata conformazione che dà origine al prione, un agente infettivo neurotossico. Anche se ora sono disponibili molte informazioni sulle diverse vie di infezione da parte del prione, sia i meccanismi alla base della neurotossicità, sia il ruolo fisiologico della PrPC rimangono poco chiari. Tuttavia, l'uso di diversi modelli animali e cellulari ha suggerito molteplici funzioni putative per la PrPC, che vanno dalla protezione cellulare contro lo stress ossidativo e stimoli apoptotici, all'adesione, proliferazione e differenziazione cellulare. Il muscolo scheletrico esprime quantità significative di PrPC, e molti studi l'hanno correlato alla fisiopatologia della proteina. Pertanto, al fine di chiarire il ruolo fisiologico della PrPC in questo tessuto, abbiamo impiegato una protocollo di degenerazione/rigenerazione del muscolo tibiale anteriore, che ci ha permesso di confrontare il processo rigenerativo in topi che esprimono, o meno, PrPC. I parametri istologici e biochimici analizzati hanno fornito prove della rilevanza fisiologica della PrPC e del suo coinvolgimento negli eventi di segnalazione coinvolti nella rigenerazione muscolare. Infatti, è stato osservato che l'assenza della PrPC ritarda significativamente il processo di rigenerazione rispetto ai muscoli WT. In particolare, abbiamo trovato che la mancanza di PrPC causa un'attenuazione della via di segnalazione attivata dal TNF-alpha, che porta ad una ridotta attivazione della chinasi p38, e - conseguentemente - ritarda l'uscita dal ciclo cellulare e la differenziazione dei precursori miogenici. È importante sottolineare che il ripristino dell'espressione della PrPC abolisce completamente il fenotipo osservato nei muscoli di topi PrP-KO, sottolineando che la regolazione delle vie di segnalazione da parte PrPC ha una chiara importanza fisiologica anche in tessuti extraneuronali. La seconda linea di ricerca, descritta nel capitolo II, è stata volta a creare una strategia basata su tecniche di proteomica per l'identificazione di trattamenti farmacologici illeciti in vitelloni. L'approccio classico per la rilevazione di questa pratica illegale non è adatto ad individuare composti sia di struttura chimica sconosciuta, sia di farmaci presenti a livelli inferiori alla soglia di quantificazione delle tecniche analitiche attualmente impiegate. Il successo delle analisi istologiche di organi bersaglio, che vengono indirettamente modificati a seguito di questi trattamenti, ha suggerito che gli approcci basati sulla ricerca degli effetti biologici delle molecole in esame, piuttosto che sulla rilevazione diretta dei loro residui, potrebbero essere molto utili. Il vantaggio più rilevante di questa metodologia è che le modificazioni del tessuto indotte da un trattamento farmacologico rimangono evidenti molto tempo dopo la fine dei trattamenti illeciti, quando i residui chimici non sono più, o quasi, rilevabili. D'altra parte, questo approccio è notevolmente limitato dalla capacità di valutazione dei tecnici e l'analisi e influenzata dalla soggettività. Per questo, sono necessarie nuove strategie per il rilevamento dei biomarcatori indiretti presenti nei fluidi animali o nei tessuti. Questi biomarcatori possono essere molecole naturalmente presenti, come ad esempio proteine che abbiano subito modifiche nella struttura, o nella concentrazione, a seguito di variazioni della condizione fisiologica dell'animale. Per identificare tali marcatori biologici di trattamenti farmacologici illeciti nei bovini da carne, abbiamo adottato un approccio proteomico, mediante elettroforesi differenziale su gel in due dimensioni (2D-DIGE) e analisi in spettrometria di massa, al fine di confrontare i pattern di espressione proteica di muscolo scheletrico tra animali trattati farmacologicamente e di controllo. A questo scopo, i vitelloni appartenenti al gruppo di trattamento sono stati sottoposti a tre differenti protocolli farmacologici, mediante l'impiego di agenti promotori della crescita. Due di questi trattamenti hanno portato ad un notevole effetto anabolico rispetto agli animali non trattati, mostrando di conseguenza un'alterazione del proteoma del muscolo scheletrico. Le mappe proteiche dei campioni appartenenti ai gruppi di trattamento e di controllo sono state confrontate utilizzando il software DeCyder per analisi di dati derivanti da 2D-DIGE. Si è poi cercato di identificare, con un approccio di spettrometria di massa (MALDI) in tandem (MS/MS), tutte le proteine che mostrano una significativa alterazione nei loro livelli di espressione in seguito a somministrazione di agenti promotori della crescita. Tra le 169 proteine che cambiano in espressione in seguito al trattamento farmacologico, sono state identificate 29 proteine diverse, la maggior parte delle quali è coinvolta nella contrazione muscolare e nel metabolismo energetico. Questi risultati confermano i precedenti risultati sul meccanismo d'azione degli agenti promotori della crescita, e possono essere utili per sviluppare nuove strategie per l'identificazione di trattamenti farmacologici illeciti nei bovini da carne.

Parkinsons sjukdom (PD) är ett tillstånd som ger en försvårad och försämrad livskvalité. I dagsläget finns det endast symtomatiska läkemedel men ingen bot med vilken sjukdomen upphör eller som bromsar förloppet. Pågående forskningsarbete utgår bland annat från att ta fram nya läkemedel men även också undersöka om redan befintliga läkemedel går att använda som behandling av PD. Många av de redan befintliga läkemedlen som testas är de som har förmågan att påverka proteinet α-synuklein (α-syn) och dess aggregering, som visats vara en central orsak till uppkomsten av PD. I föreliggande litteraturstudie undersöktes på vilket sätt en del läkemedel vars indikation är PD och även andra sjukdomstillstånd såsom astma, påverkar (ex. påskyndar eller inhiberar) in-vitro aggregering av α-syn. Därutöver genomfördes en detaljerad analys av de utvalda läkemedlen och deras effekt på α-syn aggregering utifrån deras kemiska egenskaper med avseende på löslighet (hydrofila, lipofila, amfifila) och inbindning till α-syn. Här kunde det visas att aggregering av α-syn inhiberades av alla utvalda läkemedel förutom dexametason, som istället påskyndade aggregeringskinetiken för proteinet. Dessutom uppvisade fasudil, ceftriaxon, dopamin, entakapon och tolkapon inbindning till delar av (hydrofila, hydrofoba eller amfifila) vilka delade samma fysikalkemiska egenskaper som α-syn. Därtill uppvisade utvalda läkemedel med till viss del plana strukturer (ex. aromatiska ringar) direkt inbindning till α-syn, vilka också rapporterats ha en något högre grad av transport över blod-hjärnbarriären, dock måste dessa fynd mer noggrant undersökas. Sammanfattningsvis visade alla utvalda läkemedel förutom dexametason anti-aggregeringsegenskaper (hämmande) mot α-syn genom att antingen indirekt eller direkt binda till proteinet och därmed hindra proteinet från att börja binda till sig själv. Mer studier måste genomföras för att studera effekten av läkemedelsexponering på α-syn för att identifiera viktiga segment av proteinet som kan utgöra läkemedelsmål för inhibering av α-syn aggregering.
Parkinson's disease (PD) is a condition that leads to an aggravated and worsened quality of life. At present, there are only symptomatic drugs for PD but no cure that eradicate the disease nor halter the disease progression have been found. Current research is being carried out to develop new drugs, but efforts also investigate whether existing drugs can be used as treatment for PD. Many of the already existing drugs being tested are those that have the ability to interact with a protein called α-synuclein (α-syn), that has been implicated to be a major player for onset of PD. In the present literature study, it was investigated in what way some drugs, whose indication is PD but also other diseases such as asthma, affect (i.e. propagate or inhibit) the in-vitro aggregation kinetics of αsyn. Additionally, a detailed analysis of the investigated drugs and their effect on the aggregation pathway was made to characterize common chemical features of the selected drugs based upon choice of solvents and binding to α-syn. Here, it could be shown that aggregation of α-syn is inhibited upon exposure to all selected drugs except dexametason which instead propagated aggregation of α-syn. In addition, fasudil, ceftriaxone, dopamine, entacapone and tolcapone was found to bind to parts (hydrophilic, hydrophobic or amphiphilic) of α-syn similar to their solubility features. Moreover, the selected drugs that were found to bind to α-syn seemed to exhibit planar in structure (i.e. aromatic rings) and also be associated to pass the blood-brain barrier to a greater extent, however these findings need to be more thoroughly investigated. In summary, all drugs but dexametason were shown to inhibit aggregation of α-syn invitro by either indirectly or directly affecting the aggregation of the protein. Further investigations need to be carried out to study the effect of drug exposure on α-syn aggregation in order to propose key segments of α-syn that can act as drug targets for inhibition of protein aggregation.

Stress activates a number of endocrine pathways that alter an animal's physiology in a manner which can result in undesirable meat quality. Animals frequently exhibit meat quality defects, including ecchymosis, at slaughter due to the stress of slaughter. This thesis explores how stress related hormones interact with adrenergic receptors to alter muscle and vascular physiology. Fallow deer were exposed to either a transciptional regulator (hydrocortisone), a beta adrenergic recptor agonist (clenbuterol) or a beta adrenergic receptor antagonist (propranolol). The administration of hydrocortisone resulted in a negative feed-back type reduction in circulating cortisol. Animals treated with propranolol and clenbuterol displayed less severe eccymosis. These results indicated that the beta 2 adrenergic receptor (B2AR) is important in controlling ecchymosis severity. B2AR was also found to be important in mediating vascular dynamics, growth and energy pathways. To investigate how adrenergic receptors alter skeletal muscle gene expression and meat quality, an in vivo wistar rat model was developed in conjunction with in vitro muscle cell (L6) experiments. Gene expression of B2AR, its associated kinase (BARK) and collagen type III, prolyl- 4-hydroxylase (P4Hy) was measured in rat muscle and L6 cells. Following exposure to clenbuterol and hydrocortisone, growth and meat quality were determined. The L6 experiments revealed that gene expression following exposure to hydrocortisone and B2AR ligands paralleled the in vivo rat changes in B2AR, BARK, collagen type III, and P4Hy gene expression. In both L6 and wistar rat models the B2AR and BARK genes are similarly expressed following clenbuterol exposure. Both rats and deer exposed to clenbuterol had significant increases in growth rate and a reduction of intramuscular fat. The B2AR therefore appears to be a major mediator of many interrelated events including energy distribution, growth and vascular response to stress. Habituating animals to stress stimuli may increase their coping ability and improve welfare and meat quality.

碩士
國立中興大學
獸醫學系
87
The objective of this study was to determine the concentrations of clenbuterol in equine urine and serum after a continuously oral dose of clenbuterol?HCl. Horses were fed with 0.8mg of clenbuterol/kg BW twice a day (therapeutic dose). Urine and serum were collected separately after the latest treatment. Blood samples were given at 10min, 15min, 20min, 30min, 1h, 2h, 4h, 6h, 12h, 1d, 2d after dosing; urine were collected at 20min, 30min, 1h, 2h, 4h, 6h, 12h, 1d, 2d after dosing. Samples from treated animal were analysed and the clenbuterol concentrations were comparative to those obtained by ELISA and capillary electrophoresis (CE), using solid phase extraction for sample clean-up. The highest concentration of clenbuterol found in urine was 72.5 ng/ml (at therapeutic dose) and 2.9 ng/kg in serum (5X therapeutic dose). The initial half-life of serum and urine clenbuterol residues in horses were estimated to be 3.5 and 8 h; the slower elimination for serum and urine approximately 24 and 48 h, respectively. A biphasic pattern of clenbuterol elimination is also characteristic for plasma and urine. Recoveries of clenbuterol from fortified 0.1, 1, 10 ng/ml in serum and urine samples carried through the entire procedure were about 80% relative to pure standards. ELISA was identified in our reserch to detect clenbuterol in equine serum and urine after oral administration. The SPE method developed in our experiment were appropriate to others in higher recovery rate.

碩士
國立中興大學
獸醫學系
90
Clenbuterol is one kind of β2 selective agonists. It binds withβ2 receptors then via series of signal transductions activating protein kinase to make pharmalogical effect. Clenbuterol clinically uses as bronchodilators by enhancing the clearance of mucosal cilliary, but it improves the carcass and lowers the feed efficacy. Illegal abusement using as growth promoter is common now. An ELISA was developed for the detection of clenbuterol in diversity. The clenbuterol derived diazotized clenbuterol and was conjugated with HSA to produce antigen. Anticlenbuterol serum was obtained after several times. Enzyme conjugate was produced with horseradish peroxidase (HRP). Checkerboard method was used to determine optimal concentrations of antibodies and enzyme conjugate. A standard curve was established suggesting that it is a competitive immunoassay. Working antiserum dilution was 8000x, and 2000x for enzyme conjugate. The detectable range was 0.5-50ng/ml in PBS and horse serum, and 0.1-50ng/ml in urine. The cross reactivity with salbutamol and terbutaline was 5.469% and 4.534%, respectively. The clenbuterol antiserum cross-reacted with other endogenous catecholamines and their metabolites less than 0.1%. Intra- and interassay variations were less than 10% in PBS, horse urine and horse serum. Horse serum concentration 9-15 hours following clenbuterol administration was maximum (0.84ng/ml), and horse urine 3 hours after administration concentration was maximum (97ng/ml). The both values declined rapidly, and they remained detectable for 216 hours. Results show that a sensitive and simple ELISA was developed for detection in horse urine and serum without extraction.

6. ZUSAMMENFASSUNG Charakterisierung b-adrenerger Rezeptoren auf Pferdelymphozyten und deren Regulation unter dem Einfluß von Clenbuterol und Dexamethason Getu Abraham Institut für Pharmakologie, Pharmazie und Toxikologie, Veterinärmedizinische Fakultät, Universität Leipzig, Deutschland Juni, 2001 86 S., 205 Lit., 7 Tab., 23 Abb. Über Vorkommen und Eigenschaften von b-Adrenozeptoren auf Pferdelymphozyten und ihre pharmakologische Regulation liegen bisher keine Untersuchungen vor. Mit der vorliegenden Arbeit sollten b-adrenerge Rezeptoren an intakten Pferdelymphozyten in Bindungsstudien mit dem Radioliganden (-)-(125I)-Iodocyanopindolol (ICYP), einem potenten b2-selektiven Adrenozeptorenantagonisten, charakterisiert und deren funktionelle Ansprechbarkeit durch Messung des Isoprenalin-induzierten Anstiegs des intrazellulären cAMP-Gehaltes mittels Radioimmunoassay bestimmt werden. Die Bindung von ICYP an intakten Pferdelymphozyten war schnell, sättigbar (maximale Anzahl der Bindungsstellen 320 ± 20 ICYP Bindungsstellen/Zelle, Mittelwert±SEM, n = 12) ) und hochaffin (KD-Wert für ICYP 14,37 ± 1,66 pmol/l, Mittelwert±SEM, n = 12). Verdrängungsexperimente mit Agonisten und Antagonisten zeigten eine Affinität und Stereoselektivität, wie sie bei b-Adrenozeptoren zu erwarten waren. Der nicht radioaktiv markierte b2-selektive Adrenozeptorantagonist ICI 118,551 verdrängte den Radioliganden ICYP aus seiner Bindung mit mehr als 1500fach höherer Affinität als der nicht radioaktiv markierte b1-selektive Adrenozeptorantagonist CGP 20712A. In Pferdelymphozyten können somit mehr als 90 % der b-Adrenozeptoren dem b2-Subtyp zugeordnet werden. Ein Anteil von weniger als 10 % an b1-Adrenozeptoren kann allerdings nicht ausgeschlossen werden. Die Bindungsstellen waren stereospezifisch, da das biologisch aktive (-)-Propranolol die Bindung von ICYP mit 40fach höherer Affinität verdrängte als (+)-Propranolol. b-adrenerge Agonisten verdrängten ICYP aus seiner spezifischen Bindung in der Rangfolge ihrer pharmakologischen Wirkungstärke, die typisch für den b2-Subtyp des Adrenozeptors ist: (-)-Isoprenalin > (-)-Adrenalin > (-)-Noradrenalin. Eine ähnliche Rangfolge wurde auch für die Agonist-induzierte cAMP-Bildung in Lymphozyten festgestellt. Aufgrund der dargestellten Befunde kann die Schlußfolgerung gezogen werden, daß ICYP ein geeigneter Radioligand ist, um b-adrenerge Rezeptoren an Pferdelymphozyten zu identifizieren und zu charakterisieren. Die Bindung von ICYP an intakte Lymphozyten stellt somit ein geeignetes Modell dar, um wichtige Informationen über die Funktion und Regulation des b-adrenergen Systems beim Pferd sowohl unter physiologischen (pathophysiologischen) Bedingungen als auch über Arzneimittel-induzierte Veränderungen in vivo zu gewinnen. Ausgehend von den beschriebenen Grundlagenuntersuchungen wurden in einer weiteren Versuchsreihe bei zwölf klinisch gesunden Pferden der Einfluß von Clenbuterol und Dexamethason lymphozytäre b2-Adrenozeptoren, hinsichtlich ihrer Dichte, ihrer Affinität zum Radioliganden und ihrer cAMP-Bildung (als Maß für die funktionelle Ansprechbarkeit der b2-Adrenozeptoren bestimmt über Stimulation durch 10 µmol/l Isoprenalin) untersucht. Während der 12tägigen Clenbuterolbehandlung (2 Ž 0,8 µg/kg/Tag, i. v.) fielen die Anzahl der b2-Adrenozeptoren an Lymphozyten und der durch (-)-Isoprenalin-induzierte Anstieg des intrazellulären cAMP-Gehaltes signifikant (p < 0,05, n = 8) unter die Ausgangswerte unbehandelter Tiere ab. Clenbuterol bewirkte in der therapeutischen Dosis bereits 48 Stunden nach der Behandlung einen Abfall der Anzahl der b2-Adrenozeptoren bzw. der cAMP-Bildung um etwa 30 - 40 %. Während der restlichen Behandlungsdauer unter Clenbuterol blieb die Rezeptorendichte auf diesem niedrigen Niveau. Bei einer weiteren Versuchsgruppe wurde der Effekt des Glukokortikoids Dexamethason auf die Clenbuterol-induzierte Desensibilisierung von b2-Adrenozeptoren untersucht. Dexamethasonverabreichung (1 Ž 0,1mg/kg/d, i. v.) unmittelbar nach der letzten Clenbuteroldosis über 5 Tage beschleunigte die Wiederherstellung der down-regulierten Anzahl der lymphozytären b2-Adrenozeptoren und des Isoprenalin-induzierten Anstiegs des intrazellulären cAMP-Gehaltes (p < 0,05, n = 4). Schon 24 Stunden nach der ersten Dexamethasongabe waren beide Parameter von den Durchschnittswerten am Tag vor Behandlungsbeginn statistisch nicht zu unterscheiden. Die Anzahl der lymphozytären b2-Adrenozeptoren stieg sogar ab dem dritten Tag der Dexamethasonbehandlung bis auf das Doppelte bei unbehandelten Tiere an. Nach dem Absetzen der Dexamethasonbehandlung kam es zu einer langsam Abnahme der Rezeptorendichte und ihrer Ansprechbarkeit, wobei die vor der Clenbuterolbehandlung gemessenen Werte erst nach 4 Tagen erreicht wurden. Bei gleichzeitiger Behandlung der Tiere mit Clenbuterol und Dexamethason konnte durch das Glukokortikoid die Clenbuterol-induzierte Abnahme der b2-Adrenozeptorendichte und deren Ansprechbarkeit vollständig verhindert werden. Weder durch die Clenbuterol-abhängige Down-Regulation noch durch die Dexamethason-bedingte Hochregulation kam es zu signifikanten Veränderungen der Dissoziationskonstante (KD) und somit der Affinität von ICYP zu b2-Adrenozeptoren. Die Ergebnisse zeigen, daß es unter Behandlung mit Clenbuterol zu einer schnell einsetzenden, umfangreichen Down-Regulation der b2-Adrenozeptorendichte an Lymphozyten des Pferdes kommt und daß diese Toleranzentwicklung durch Glukokortikoide vollständig wieder aufgehoben oder verhindert werden kann. Zur Therapie der COB von Pferden scheint somit die kombinierte Anwendung von b2-Mimetika und Glukokortikoiden von Vorteil zu sein.
7. Summary Characterization of b-adrenergic Receptors on Equine Lymphocytes and their Regulation under the Influence of Clenbuterol and Dexamethasone Getu Abraham Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, Germany June, 2001 86 pp., 205 ref., 7 tab., 23 fig. b-Adrenoceptors and their regulation by pharmacological active substances have not yet been characterized in equine lymphocytes. In this study, b-adrenoceptors were identified and subclassified by (-)-[125I]-iodocyanopindolol (ICYP) binding, a potent b2-adrenergic antagonist, as well as by their isoprenaline-induced responsiveness using radioimmunoassay in intact viable equine lymphocytes. The specific ICYP binding was rapid, saturable (maximum number of binding sites 320 ± 20 ICYP binding sites/cell, means±SEM, n = 12) and of high affinity (KD-value for ICYP 14.37 ± 1.66 pmol/l, means±SEM, n = 12). The competition binding studies with agonists and antagonists showed the affinity and stereoselectivity to be expected for b-adrenergic receptors. The unlabelled selective b2-adrenoceptor antagonist ICI 118,551 was about 1500 times more potent in inhibiting ICYP binding than was the b1-selective adrenoceptor antagonist CGP 20712A. It is, therefore, concluded that in intact equine lymphocytes ICYP labels a class of functional b-adrenoceptors, that belong predominantly (> 90 %) to the b2-adrenoceptor subtype. A minor (< 10 %) b1-adrenoceptor component, however, cannot be completely ruled out. The binding sites were stereoselective, as the (-)-isomer of propranolol was about 40 times more potent to inhibit ICYP binding than was the (+)-isomer. Agonists competed for specific binding with a rank order of potency (-)-isoprenaline > (-)-adrenaline > (-)-noradrenaline, which is typical for a b2-subtype of adrenergic receptor; the same order of potency was obtained for agonist-induced stimulation of lymphocyte cyclic AMP content. Thus, it can be concluded that ICYP is a promising compound for the reliable determination of the binding characteristics of b-adrenoceptors of equine lymphocytes and besides that, this might give an insight in providing physiologically significant information about the regulation of the b-adrenergic receptor system. Based upon this study, we further investigated, in 12 healthy thoroughbred horses, the effects of the b2-agonist clenbuterol and the glucocorticoid dexamethasone on the lymphocyte b2-adrenenergic receptor density and affinity as well as on its responsiveness (assessed by lymphocyte cyclic AMP responses to 10 µmol/l (-)-isoprenaline). Clenbuterol (2Ž0.8µg/kg/d, i. v. for 12 days) decreased ICYP binding sites only 48 h after application by ~30-40 %; concomitantly, lymphocyte cAMP response to (-)-isoprenaline was significantly reduced (p < 0.05, n = 8). The values remained at these low levels throughout the following 10 days of treatment. After withdrawal of clenbuterol the density and responsiveness of b2-adrenoceptors gradually increased, reaching pre-drug levels after 4 days. Next the effects of dexamethasone on clenbuterol-induced desensitisation were studied. Administration of dexamethasone (1Ž0.1mg/kg/d, i. v. for five days) immediately after clenbuterol withdrawal accelerated b2-adrenergic receptor recovery (p < 0.05, n = 4): only 24 hours after administration dexamethasone restored the number of binding sites and cAMP response to (-)-isoprenaline to levels statistically indistinguishable from the values before starting clenbuterol treatment. Three days after dexamethasone administration the density of lymphocyte b2-adrenoceptors was further increased about two fold the pre-treatment values, and this increase declined gradually after dexamethasone withdrawal, reaching baseline values after 4 days. Furthermore, in groups simultaneously exposed to both drugs dexamethasone completely prevented clenbuterol-induced decrease in lymphocyte b2-adrenoceptor density and responsiveness. No significant change was observed in the dissociation constant and, thus, in the affinity of ICYP to b-adrenoceptors remained unchanged during or after treatment with either clenbuterol or dexamethasone. It is concluded that dexamethasone (glucocorticoids) can reverse and prevent clenbuterol-induced down-regulation of the lymphocyte b2-adrenoceptors and thus, a combined therapy with clenbuterol and dexamethasone may be potentially beneficial in COPD of horses.

碩士
國立清華大學
化學系
92
In this thesis, we develop a method of using headspace solid-phase microextraction (HS-SPME) combined with gas chromatograph-ion trap mass spectrometer (GC-ITMS) to detect a β-agonist : clenbuterol. We discuss and optimize eight parameters of extraction and desertion procedure, including types of fibers, extraction temperature, extraction time, desorption temperature, desorption time, stir rate, ionic strength, pH of sample and cooling system. The method show good linearity over the concentration range 1–1000 ng/mL and gives detection limits below ng/mL level. Precision is also good (3.89%). Finally, the method is successfully applied to analyze clenbuterol in urine.

碩士
中山醫學大學
生物醫學科學學系碩士班
103
Clenbuterol (CLE) and Salbutamol (Sal), β2 agonist similar to Ractopamine (Rac), have been widely used in livestock due to theirs remarkable effectiveness of muscle growth and protein-to-fat ratio improvement. CLE and Sal may cause acute toxic responses, such as cardiac palpitation, tachycardia, nervousness, muscle tremors and confusion. In addition, the toxicity of Sal was less than the CLE. For detecting the level of CLE and Sal in pork or beef, rabbit polyclonal antibodies for CLE and Sal were produced in our studies. Using the antibodies from the rabbit immunized with CLE-γ-globulin or Sal-γ-globulin, both sensitive indirect and direct competitive enzyme-linked immunosorbent assays (ci and cdELISA) were developed. In the ciELISA of CLE, the concentration causing 50% inhibition (IC50) of binding of antibodies to the solid-phase Sal-OVA by free CLE was 0.77 ng/mL. In the ciELISA of Sal, the concentration causing 50% inhibition (IC50) of binding of antibodies to the solid-phase Sal-OVA by free Sal was 35 ng/mL. In the cdELISA of CLE, the concentration causing 50% inhibition (IC50) of binding of Salbutamol-horseradish peroxidase (Sal-HRP) to the antibody by CLE was calculated to be 0.3 ng/mL. In the cdELISA of Sal, the concentration causing 50% inhibition (IC50) of binding of Salbutamol-horseradish peroxidase (Sal-HRP) to the antibody by Sal was calculated to be 35 ng/mL. The antibody-gold nanoparticle immunochromatographic strip (immunostrip) for CLE was established for detecting the level of CLE and Sal in pork or beef. The detection limit of strip were 10 ng/mL and 20 ng/mL for CLE and Sal , respectively. In addition, Immunostrip of Sal was established for detecting the level of Sal and CLE in pork or beef. The detection limit of strip were both 100 ng/mL for CLE and Sal. Therefore, we have been successfully generated polyclonal antibodies, developed sensitive ELISAs for detection of CLE or Sal and established an immunostrip for rapid on-site detection of CLE and Sal in beef or pork samples.

碩士
中國文化大學
生活應用科學研究所
90
A simple and rapid method with reverse phase high performance liquid chromatographic (HPLC) to determine clenbuterol in muscle and liver of swine and cattle. After homogenizing the tissue with 0.4N perchloric acid, the supernatant was extracted with diethyl ether, followed by back-extraction in 0.2M sulphuric acid, and finally determined by HPLC. The separation was performed with a Waters 5C18-MS (2.0×150 mm)(Cosmosil) column on 0.05M NaH2PO4 (pH3.0):acetonitrile (80:20, v/v) as a mobile phase, and clenbuterol was detected by absorbance at 212nm. The recovery of this method was studied spiking 0.0005, 0.001, 0.002, 0.005 and 0.01 ppm levels in swine muscle, swine liver and bovine muscle and the results showed recoveries at the ranges of 80.9~88.8﹪, 80.3~86.9﹪ and 80.6~90.6﹪，respectively. The coefficients variation of average recoveries was between 0.7and 6.3﹪. The limitation of detection by this method was 0.0001 ppm in these three commodities，which meets the requirement for detecting clenbuterol residues in these commodities at the officials allowed levels. Complying with this method, 60 samples were purchased at markets. The result showed two samples were detected but non of them were violated the regulation.

It was proposed that some O-alkyl analogues of the beta-adrenergic agonist clenbuterol would be effective structural and functional congeners of clenbuterol which may then be used for the production of clenbuterol-specific idiotypic antibodies. These antibodies could possibly then be used to generate anti-idiotypic antibodies that mimic the energy-repartitioning effects of clenbuterol. Therefore, the aim of this work was to synthesise and characterise these compounds, evaluate their physiological effects, characterise the specificity of antibodies produced in response to protein conjugates of two of the novel compounds, and then use this data to determine the utility of these compounds for the generation of anti-idiotype antibodies which mimic clenbuterol. The target compounds were synthesised in five steps from 3,5-dichloro-4-hydroxyacetophenone in overall yields of 5-28%. A synthetic scheme similar to that which has led to clenbuterol was used to form the phenylethanolamine backbone, with modifications to include the O-alkyl moiety via a modified Williamson ether synthesis, and elimination of a synthetic chlorination step. Overall, 15 new compounds were synthesised, which were characterised and their structure confirmed from proton and carbon-13 NMR, IR and mass spectral data. The two haptenic analogues were then conjugated to carrier proteins using carbodiimide-based chemistries. In conclusion, the results indicated that the O-alkyl analogues, although structurally similar, were ineffective functional mimics of clenbuterol. Therefore, the anti-clenbuterol antobodies produced from the novel O-alkyl analogues would appear to be unsuitable for production of anti-idiotypic antibodies that mimic the energy-repartitioning effects of clenbuterol since the antibodies were unable to distinguish between the compound which demonstrated energy-repartitioning effects (clenbuterol) and those that did not (O-alkyl clenbuterol analogues).
Doctor of Philosophy (PhD)

There were two main objectives for this study. The first of these was to develop a research methodology with which both the affinity and efficacy of B-adrenoceptor agonists on protein degradation in skeletal muscle could be studied.

B2-Adrenoceptors were isolated from toad skeletal muscle and were characterised in radioligand binding experiments. The resultant affinity constants for 14 drugs were compared with values obtained from bovine Bradrenoceptors, and paired t-test analysis indicated that no significant difference existed between these two populations of B2-adrenoceptors. Thus, toad skeletal muscle B2-adrenoceptors provide a suitable model for screening B-ligand affinities, and values may be extrapolated to the corresponding receptors in cattle (target species).

Recombinant Escherichia coli expressing human B1,-and human B2-adrenoceptors were also characterised in radioligand binding experiments. The data obtained verified that each clone provided a source of homogeneous receptors, which displayed the ligand binding affinities appropriate for the respective receptor subtype. Thus, recombinant bacteria are a useful tool for screening the subtype-selectivity of ligands.

The bacterial clone expressing human B2-adrenoceptors displayed ligand binding affinities that were not significantly different to drug affinity determinations performed with bovine B2-adrenoceptors. Thus, the B2-strain is a useful model of the Bradrenoceptor expressed in the skeletal muscle of cattle. Additionally, recombinant B2-adrenoceptors exhibited differential binding behaviour for agonists and antagonists: all antagonists tested displayed monophasic displacement curves, whereas agonist molecules having molecular weight of less than 250 g/mol and an octanol/water partition coefficient (log P) less than zero, displayed biphasic binding behaviour. From these experiments, the cloned Escherichia coli cell lines were selected as the preferred source of B2-adrenoceptors for radioligand binding screens.

The B-adrenoceptor agonist efficacy assay selected for this study was the isolated perfused rat hemicorpus. In the first experiment, the rate of 3-methylhistidine (3-MH) release from perfused hemicorpora was examined: the 3 MH efflux rate of 0.227 ± 0.009 Amo1/30 g/3 hours corresponded to a protein degradation rate of 8.7 ± 0.3 %/day. While this value was comparable to other estimates reported in the literature, the HPLC analytical technique used to quantify 3-MH was at the lower limit of detection sensitivity. This author was not confident that the small changes in 3-MH release that may result from B-agonist treatment would be detected using this method. Hence, an alternative approach of measuring proteolysis as a function of other essential amino acids was examined. With this second method, reincorporation of amino acid markers into protein was prevented by the protein synthesis inhibitor, cycloheximide. In the presence of radiolabelled phenylalanine and leucine, the addition of 40 Amo1/1 cycloheximide to the perfusion medium was found to inhibit protein synthesis by 78 to 90%, without influencing the composition of the free amino acid pools within muscle. Furthermore, this radiolabel incorporation data (for rat hindlimbs perfused in the absence of cycloheximide) enabled the protein synthetic rate of the muscle to be estimated at between 2.0 and 3.06 %/day.

When proteolysis is measured as a function of the release of essential amino acids, the rate of myofibrillar protein breakdown is indistinguishable from non-myofibrillar breakdown. Thus, specific increases in myofibrillar degradation may potentially be undetected. However, the probability of detecting significant changes in degradation may be improved by co-analysing a total of eight essential and semi-essential amino acids. This approach was endorsed when the amino acid efflux rate from rat hemicorpora perfused in the presence and absence of cycloheximide were examined: logically, the blockade of protein synthesis would be expected to result in elevated rates of amino acid efflux. Indeed, the amino acid efflux rates from cycloheximide treated tissues were consistently, though not necessarily significantly, higher than the rates determined in cycloheximide-free tissues. However, when a paired t-test analysis was performed with all eight amino acids, the level of statistical significance improved dramatically.

The results from perfusion experiments indicated that all subsequent studies of protein degradation should be performed in the presence of 40/Imola cycloheximide; and the essential amino acids, valine, methionine, tryptophan, phenylalanine, isoleucine, lysine, leucine, and the semi-essential amino acid tyrosine should be used as co-indices of the rate of protein degradation.

When rat hemicorpora were perfused in the presence of 40 Amo1/1 cycloheximide, the mean ± SEM rate of potassium released from 30 grams of skeletal muscle was 740 ± 60 Amo1/3 hours. Likewise, the mean ± SEM rate of glucose consumed by 30 grams of skeletal muscle was 330 ± 20 Amo1/3 hours. HPLC analysis of muscle amino acids derived from both intact rats, and from hemicorpora which had been perfused for three hours, revealed that the intracellular free amino acid content of muscle was not significantly altered by the perfusion process. Thus, the rate of protein degradation in perfused muscle tissue was estimated directly from the rate of amino acid efflux for eight essential/semi-essential amino acids.

The second major objective of this study was to use the rat hemicorpus preparation to determine whether the B2-adrenoceptor agonist, clenbuterol, mediates its growth promoting effects on skeletal muscle through a reduction in the rate of myofibrillar protein degradation.

Clenbuterol treatment (4 mg/kg diet, 265 μg/kg BWT/day) resulted in net protein accretion, and significantly enhanced the growth rate of treated animals. In mature male rats, clenbuterol treatment in the diet increased the rate of weight gain to 260% of the untreated level. However, the magnitude of the growth response declined after six days of treatment, which is consistent with B-adrenoceptor desensitisation. Clenbuterol-induced changes in blood flow to different tissues was evident from the change in the colour of adipose tissue in abdominal fat depots.

Clenbuterol treatment had no significant effect on either the perfusate pressure range or the rate of glucose uptake in perfused rat hemicorpora. In contrast, potassium efflux rates in chronically treated hindlimb preparations were elevated by 95%. The increase in potassium efflux may have been an artefact of the cycloheximide-induced inhibition of protein synthesis. However, more recent evidence of elevated plasma potassium levels in cows chronically treated with clenbuterol indicate that this may be a real effect of long-term B-agonist treatment.

When the rate of protein degradation was evaluated in terms of the sum of essential amino acids effluxed from perfused rat hemicorpora, acute (three hours) clenbuterol treatment caused a significant, but transient increase in proteolysis (11%). In contrast, longer-term clenbuterol treatment (6 and 12 days) effected a non-significant decrease in the rate of protein degradation (11.8 and 7.6%, respectively), and this effect was diminished over time, in a manner consistent with B-adrenoceptor desensitisation. Furthermore, the withdrawal of clenbuterol from chronically-treated tissues resulted in an immediate and significant increase (25.1%) in the rate of protein degradation, which indicates that the continued presence of clenbuterol is required to maintain the effect of reduced proteolysis.

Clearly, the reduction in the rate of protein degradation observed with chronic clenbuterol treatment is insufficient to account for the 160% increase seen in the growth rate of treated rats. Consequently, it can be concluded that protein synthetic rates must have also been elevated. Indeed, it has been estimated that the 160% increase in the growth rate of clenbuterol-treated rats could be accounted for by a net 16.4% change in the component processes of protein turnover (ie an 11.8% reduction in protein breakdown, and a 4.6% increase in protein synthesis). This is equivalent to a 10.25% change in protein turnover for every 100% increase in growth rate, and compares exceptionally well with the 10 to 13% change estimated by Kim and Sainz (1992).

From these studies, it was concluded that the mechanism of clenbuterol-induced muscle accretion includes components of both increased protein synthesis and decreased protein degradation. In tropical cattle, this saving in metabolic energy resulting from decreased proteolysis may offer an advantage to the animal during the dry winter months where seasonal weight losses are often observed.

The aim of this project was to investigate physiological methods for increasing growth rate in beef cattle. The effect of glucocorticoids with or without clenbuterol on B₂- adrenoceptors (B₂-AR) in skeletal muscle and the effect of fasting on serum leptin were studied.

Muscle growth can be promoted in meat producing animals, humans, and experimental animals by the use of B-agonists. However, these anabolic effects rapidly attenuate due to a decrease in B₂-AR density in skeletal muscle following treatment. Glucocorticoids were demonstrated to increase B₂-AR density in rat lung. Thus, the first section of the project was designed to determine whether glucocorticoids could prevent clenbuterol-induced downregulation of B₂-ARs.

Corticosterone, dexamethasone and clenbuterol were tested for effects on muscle growth and B₂-AR density in male rats. Corticosterone caused a dose-related decrease in total weight gain and gain in gastrocnemius/plantaris bundle (GP muscle), but failed to increase B-AR density in the muscle at any of doses tested (6.25-50mg/kg, sc 5d). GP muscle and the ratio of gain/food intake were increased in the group treated with clenbuterol and decreased in the group treated with dexamethasone when administrated daily for 10d. B-AR density in GP muscle was decreased by both clenbuterol and dexamethasone. In contrast, B-AR density in rat lung was increased by dexamethasone and decreased by clenbuterol. hi concurrent treatment group, dexamethasone prevented clenbuterol-induced downregulation of B₂-ARs in rat lung, but not in GP muscle. Thus, dexamethasone does not enhance the anabolic effect of clenbuterol, but causes a marked catabolic effect instead. The results demonstrate that a B-agonist/glucocorticoid combination would not be useful in animal production, and the discovery that B₂-drenoceptors are regulated differently in different tissues warrants further investigation.

The leptin system is postulated to be a key regulatory hormonal pathway associated with the regulation of body weight through effects on metabolism and appetite both in humans and in rodents. Thus, in the second section of this study, an immunological method for detecting bovine leptin and the effects of fasting on circulating leptin, cortisol, glucose and FFA in beef cattle were investigated.

Polyclonal antibodies against bovine recombinant leptin were produced by immunizations of mice, rats, sheep and cattle. Immunization resulted in a positive response with maximum titres assayed by ELISA of 800 in 6/16 of the C57 BL/6 mice and 4/8 of cattle, 3000 in 7/16 rat, and 102400 in 3/3 sheep. No immune response was observed in BALB/c mice. Spleen lymphocytes derived from immunized rats were fused with the murine myeloma cells. Eight hundred hybridomas were cultured. Unfortunately, no leptin specific monoclonal antibody was found on screening the supernatants from these clones.

Cattle and sheep antibodies were affinity purified and conjugated with horseradish peroxidase or biotin. Two immunoassays, avidin-biotin ELISA and competitive ELISA, were developed for determining bovine leptin. The detection limit for recombinant bovine leptin was 500ng/m1 in the avidin-biotin ELISA and 5ng/m1 in the competitive ELISA. The application of the competitive ELISA developed in this study was tested by measuring leptin concentrations of bovine serum and serum plus standards. The results showed that the leptin concentration of different diluted serum was 45.4 ± 3.31 ng/ml.

However, the concentrations of leptin of 1:2 diluted serum plus each standard were all higher than expected, possibly due to the presence of reserve leptin binding proteins in the serum. Thus, after the problem of putative interference of binding protein in serum is resolved, determination of large numbers of sample from beef cattle, rapidly and economically using the competitive assay, will be possible.

The effect of 24 h fasting on serum leptin levels was studied in beef cattle. Serum leptin 4.17 ± 0.11 ng human equivalent /ml was detected in these cattle using a human leptin RIA kit. The leptin levels of fasting animals decreased as compared to the animals before fasting and re -feeding. Fasting caused a 72.33 ± 28.18 % increase in serum cortisol and 203.01 ± 30.94 % increase in plasma FFA. Plasma glucose did not change. A positive correlation between serum leptin concentrations and body weight were observed in seven cattle (before fasting: r2 = 0.663, P = 0.026; after re-feeding: r2 = 0.902, P = 0.0011), but one other animal was outlying. These results indicated that depriving cattle of food caused a decrease of serum leptin and an increase of stress steroids and plasma FFA, while glucose level maintained normal, which demonstrated that leptin levels are readily influenced by physiological changes and are consistent with a homeostatic signaling function for this hormone.

According to the findings of the fasting study, further work would be needed: (1) to investigate whether there are some relationships between leptin and FFA, glucose, insulin and other hormones in circulation in beef cattle as in rats and mice; and (2) to establish whether immunization of cattle with leptin will decrease serum leptin and whether lower leptin level will stimulate food intake, increase growth and enhance the productivity of beef cattle.