Analysis Of The Interaction Of β-agonists Residues In Foodstuff Of Animal Origin With Serum Albumin

Food safety has become a hot issue for the world and the effect of the veterinary medicine on food safety has become the focus of attention in recent years. When people eat foodstuff of animal origin, they begin to contact the hormones in animal.β-agonist steriod which is the growth acceleration hormone, also calledβ-agonist steriod or lean meat powder, is pure synthetic proteins assimilation hormone with fat distribution function. When this kind of hormone used for breeding, it can not only promote the growth rate and improve the feed conversion rate, reduce the carcass fat and increase lean meat, but also change the distribution of animal tissue. Due to the illegal use of animal feed after repeated prohibition, the serious residues ofβ-agonist which accumulate in animal tissues can pose a grave threat to human security, leading to serious outbreaks of human poisoning around the word. Spectroscopic (fluorescence, UV-vis and FT-IR) and voltammetric analysis combined with chemometrics were used to study the interaction mode, characteristic and mechanism of agonists steroids-serum albumin and understand and clarify the transmission, the metabolism and the toxicological action of agonists steroids in the body of living beings at a molecular level. The work was expected not only to provide guideline for further drug design and detoxication and emergency treatment but also to expound the source and cause of the residual veterinary drugs in the food from animals so as to supply useful information and data for food security and food quality and to authenticate and control the potential danger in food safety.This thesis is composed of six chapters.Chapter 1In the first section, the reason choosing the agonists steroids residues in foodstuff of animal origin as the research object was elaborated. Then, the meaning of the interaction between the small molecules and serum albumin was introduced. Furthermore, the research methods, research content and research object related with food such as food additives, pesticide and veterinary drug were emphatically reviewed. The superiority of the application of chemometrics in the field of the small molecules interact with serum albumin was discussed. At last, the importance and significance of the selected object of agonists steroids interaction with serum albumin was clarified.Chapter 2Under the imitated physiological conditions (pH=7.4), the interaction between clenbuteral (CLEN) and bovine serum albumin (BSA) was investigated by the fluorescence and UV-vis spectroscopy using the multivariate curve resolution-alternating least squares method (MCR-ALS). In this work, two different kinds of spectral methods with the same titration mode carried out. The concentration profiles and pure spectra for each species, the calculation of the [CLEN]:[BSA] molar ratio in the complex and the apparent equilibrium constant were extracted. The binding constants Ka and the number of binding sites n were also evaluated from the double-logarithm curve. The synchronous fluorescence spectra may represent that the conformation of BSA changed. From the result, the binding constant reach to 106 L mol-1 illustrate that the combination rate is high and it is difficult for CLEN in the body to eliminate. The effects of CLLN arc cumulative over extended period and is not easy to metabolize. The serious accumulative residues in internal organs and tissues is harm to food safety and human health.Chapter 3To investigate the interaction between Ractopamine (RAC). an animal growth promoter, and bovine serum albumin (BSA). three spectroscopic approaches (fluorescence. UV-vis and FT-IR) and three different experiments (two mole-ratio and a Job's methods) were used to monitor the biological kinetic interaction procedure. The spectra data matrix was also investigated with multivariate curve resolution—alternating least squares (MCR-ALS), and the concentration profiles and the pure spectra for three species (BSA, RAC and RAC-BSA) existed in the kinetic interaction procedure. The effect of RAC on the conformation of BSA was also studied by synchronous fluorescence and Fourier transform infrared (FT-IR). Although the low RAC residues in foodstuff of animal origin move into human body through food chain, the health of human beings is threatened seriously. The binding constant of CLEN and RAC reach to 106 L mol-1, too. Differing from both structure, the passivated halogeno in the benzene ring of CLEN so it is not easy to metabolize and is slow to eliminates in the body and the activate phenolic group in the benzene ring of RAC so it is easy to effect and metabolize and is relatively more short to maintain the effec time and it become the alternatives of CLEN.Chapter 4(3-Agonists such as ractopamine (RAC) and clenbuterol (CLEN), have similar effects as anabolic steroids i.e. they promote growth of muscular tissue and reduce body fat. They have been used successfully with animals and humans but have also been banned in many countries principally, because of their serious side effects. However, their illegal use persists. Thus, their interaction with biomolecules such as bovine serum albumin (BSA) is of significance, especially the co-operative reaction of mixed ligands with the protein. Fluorescence and UV-vis spectra of complex mixtures of individual ligands, binary and ternary complexes with BSA resulted in significantly overlapping spectral profiles. Qualitative and quantitative information about the various complex ligand-protein species formed, was obtained with the resolution of the excitation-emission fluorescence three-way data matrices by chemometrics methods-MCR-ALS and PARAFAC. Individual spectra of the ligands, their binary complexes with BSA and their ternary complexes were extracted, and quantitative concentration profiles for each species in a particular interaction were constructed. Such analyses made it possible to interpret the role and behaviour of each reaction component. It was found that both ligands, RAC and CLEN, bound co-operatively in site I of the BSA. This was confirmed with the use of site markers such as warfarin (siteⅠ) and ibuprofen (siteⅡ). However, CLEN formed a 1:1 CLEN-BSA complex, while RAC formed a 2:1 RAC2-BSA binary species. Interestingly, when CLEN or RAC was added to RAC2-BSA or CLEN-BSA, respectively, ternary complexes were produced such as RAC2-BSA-CLEN. Significantly, the presence of the second ligand in such an interaction in excess appeared to increase of the affinity of the added ligand for BSA. This may have consequences on the amount of steroid required to achieve a desired tissue growth effect.Chapter 5As new agonist steroid, levodopa and dopamine added to animal feed can promote the growth of liestock, improve the conversion of feed. It has the high concealment performance and usually will not be included in conventional detection. However, levodopa and dopamine can produce serious side effects to people and livestock. An analytical method was researched for the simultaneous determination of reactants and products during the binding of important small molecules such as levodopa (LD) with biopolymers such as bovine serum albumin (BSA). Voltammetry and fluorescence spectroscopy were used to obtain the analytical profiles from different reactant mixtures as a function of concentration. This enabled the extraction of the equilibrium constants (KSV) which are reported for the first time. Voltammetric results supported the formation of the LD-BSA complex but not that with dopamine. Further information of the LD-BSA system was unattainable because the measured composite profiles could not be extracted. New information was obtained when the extended data matrix was resolved by the MCR-ALS method. The previously unavailable extracted voltammogram profile of LD-BSA complex indicated that the complex was electroactive; this was unexpected if the LD-BSA system was in its folded state, and hence, it was suggested that the protein must be unfolded. The observation that the drug: BSA stoichiometry was 3:1 i.e. (levodopa)3-BSA, which was obtained from the MCR-ALS extracted concentration profiles for all three reaction components, supported this suggestion. The experiment indicated that when human and livestock contact the two substances, the side effect of levodopa is stronger.Chapter 6The surfactant is widely used in the medium of human body contact such as food, medicine, cosmetic and personal health supplies. Effect of ionic surfactant, sodium dodecyl sulfate (SDS)/N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB) addition on the interaction betweenβ-agonist - salbutamol (SAL) and BSA has been studied by intrinsic fluorescence and chemometrical method - multivariate curve resolution - alternating least squares (MCR-ALS) and parallel factor analysis algorithm (PARAFAC). The mechanism and binding site for the binding of SAL with bovine serum albumin (BSA) was explored by fluorescence spectroscopy. The thermodynamic parameters like enthalpy (AH) and entropy (AS) corresponding to the ligand binding process implied the role of van der Waals force and hydrogen bonds interaction in stabilizing the SAL - BSA complex. The ligand replacement with warfarin and ibuprofen confirms that SAL binds to the sub-domain IIA of the protein. The observation that the ligand:BSA stoichiometry were 2:1 i.e. (SAL)2-BSA and 4:1 i.e. (CTAB)4-BSA, which were obtained from the MCR-ALS extracted concentration profiles for all three reaction components, supported this suggestion. Quantitative information about the complex CTAB - protein - SAL species formed was obtained with the resolution of the excitation-emission fluorescence three-way data matrices by PARAFAC. It is appeared to increase the affinity of the added SAL in a given CTAB4 - BSA mixture interaction so that it is difficult for SAL to metabolite and to the extent that the urine testing of SAL is harder to be detected. It is difficult for a SAL ligand to bind with BSA in a giving SDS3-BSA mixture and the combination state of SAL with BSA is little and the Free State is more so the SAL is easier to metabolite and to be detected in the urine testing.