| ObjectiveThe thesis evaluates the changement of Glutathione(GSH) and Cysteine(Cys) in plasma and excitatory amino acid(EAA) and inhibitory amino acids(IAA) in brain tissue of acute thallium poisoning dogs treated with different methods by the testing method of high-performance liquid chromatography (HPLC). The damage mechanism of lipid peroxidation and neurotoxic caused by the combination of thallium in vivo with a mercapto group are explosed. The results provide a theoretical basis for the treatment of acute thallium poisoning.Methods1. Animal grouping:24 beagle dogs are divided randomly into four groups, male and female each half(n=6), which includes the control group (A), Prussian blue treatment group (B), hemoperfusion group (C) and Prussian blue combined hemoperfusion group (D). Each group is given thallium nitrate (TINO3) median lethal dose (LD50=45 mg/kg) gavage. Group A receives no treatment after exposure; Group B receives orally Prussian blue after the first four hours exposure at a dose of 467 mg/kg/d, and three times a day for five consecutive days; Group C begins hemoperfusion after the first four hours exposure, one time a day, two hours each time for five consecutive days; Group D is given the treatment of Group B plus Group C after the first four hours exposure. Body weight, mental state and diet are observed for each animal during the ten days. Then, each brain region is taken after them are killed.2. Measure the content of Glutathione and Cysteine in plasma by HPLC:four groups of animals are blood 1 ml at pre-exposed and exposed 1 h,2h,4h,6h,8h,12h,24 h, 48 h,72 h,96 h,120 h,144 h,168 h,192 h,216 h,240 h. Then, all the bloods are centrifuged immediately after ice bath. In the end, the supernatant are took away. The testing standards of HPLC in this part are:penicillamine as the internal standard, derivatization with DTNB, UV detector is used to evaluate GSH and Cys in plasma. After the rest, the plasma is placed at the temperature of-79℃ for future determination.3. Measure the content of the excitatory and inhibitory amino acids (EAA and IAA) of brain tissue by HPLC:the animals are observed for ten days and then are killed. Then, at each part of cortex, striatum, hippocampus, hypothalamus, cerebellum and brainstem.0.1 g brain tissue are taken. The samples are weighed and then homogenized by sonication in a solution of 0.1 M HC1O, all in proportion of 1:10. After that, samples are centrifuged at (16000 r,4℃) for 15 mins. Then, take 500 μl supernatant. The supernatant is mixed with 0.1 M NaOH to bring the pH to 9.0, and volume to 1 ml. The testing standards of HPLC in this part are:Column ZORBAX SB-C.8(4.6×250 nm,5μm), O-phthalaldehyde (OPA) is the derivatine reagent, a moblie phase consisting of B.0.1 mol/L NaH2PO3-0.5 mmol/L Ethylenediamine-t etraacetic acid (EDTA) (adjusted pH with phosphoric acid to 4.5) and methanol(85:15) and A(Acetonitrile), the flow rate of A gradiented is 1ml/min, colume temperature is 26℃, a detection wave with a length of 230/450 nm by fluorescence detector is used to evaluate EAA and IAA in brain tissue.ResultsTo establish a model for the quick determination of glutathione and Cysteine in plasma. The GSH, Cys and penicillamine are isolated at 2.9 min,4.0 min and 9.0 min. The peak is good and the separation degree is high.GSH at the range of 0.714-14.286 μg/ml, the linearity is good:The standard curve equation is Y= 15.153X+8.043 with the correlation coefficient 0.9993. RSD<7.61% for the stability test; day precision is smaller than 8.422; days of recovery is 100.38%～103.47%; day precision <8.518%; daytime recovery is 98.16%～104.11%.Cys at the rangeof 0.714～14.286 μg/ml, the linearity is good. The standard curve equation is Y=17.813X+653.89, and the correlation coefficient is 0.9994; Stability test RSD<8.01%; day precision<5.965%; days of recovery is 97.41%～101.48%; Day precision is<7.611%; daytime recovery is 99.79%～108.68%.To establish an HPLC method for the determination of Asp, Glu, Gly and GABA in brain tissue, Asp, Glu, Gly and GABA are isolated at 3.5 min,5.0 min,5.6 min and 18.4 min. The peak is good and the separation degree is high.Asp at the range of 25～500 μg/ml,the linearity is good. The standard curve equation is Y=373.95X+3654.60, and the correlation coefficient is 0.9998. Stability test RSD is<8.965%; day precision is<7.526%; days of recovery is 99.68%～105.69%; Day precision is<7.806%; daytime recovery is 94.78%～97.33%.Glu at the range of 25～500μg/ml,the linearity is good.The standard curve equation is Y=343.83X+1575.80, and the correlation coefficient is 0.9994; Stability test RSD is<6.512%; day precision is<7.308%; days of recovery is 97.80%～101.50%; Day precision is<7.599%; daytime recovery is 95.50%～107.50%.Gly at the range of 25～500μg/ml,the linearity is good. The standard curve equation is Y=989.64X+4972.90, and the correlation coefficient is 0.9995,Stability test RSD is<5.793%; day precision is<5.653%; days of recovery is 98.57%～107.67%; Day precision is<6.632%; daytime recovery is 98.23%～104.64%.GABA at the range of 25～500 μg/ml,the linearity is good. The standard curve equation is Y=697.23X+4612.40, and the correlation coefficient is 0.9997. Stability test RSD is<7.925%; day precision is<8.018%; days of recovery is 98.86%～109.46%; Day precision is<8.890%; daytime recovery is 92.66%～104.34%.Between control group, PB group, HP group and PB+HP, GSH and Cys in plasma are different. For the main effect test P<0.05, it shows that the concentration of GSH and Cys are different from each other at different times. For the main effect test P<0.05, it shows that there are significant differences between GSH and Cys of each treatment group and control group, in which the prussian blue which combined hemoperfusion(PB+HP) decreases significantly.Between control group, PB group, HP group and PB+HP,the Content of Asp and Glu in the cortex, hypothalamus, and hippocampus are different P<0.05 and other brain areas remain no significant changes. Between control group, PB group, HP group and PB+HP, the content of IAA in the brain regions do not change significantly.ConclusionAn HPLC method for the determination of GSH and Cys in plasma and Asp, Glu, Gly and GABA in brain tissue is established. The method is accurate, sensitive with high stability, which can be used for the detection of GSH and Cys of plasma and four amino acids of brain tissues in thallium poisoning dogs rapidly. In acute thallium poisoning dogs, the GSH and Cys in plasma decrease obviously. After PB, HP and their combination treatment, the GSH and Cys decline slower than control group and PB+HP group shows the most obviously. The content of EAA and IAA are different between each brain area in acute thallium poisoning dogs, and after PB, HP and their combination treatment, the content of EAA in cortex, hypothalamus and hippocampus decreases comparing with control group, in which, PB+HP declines more obviously. IAA changes no obvious changes. In summary, GSH, Cys and EAA play a significant role in the pathogenesis of acute thallium poisoning. The treatment of PB, HP and PB+HP are all effective, and PB+HP performance more obviously. The method proposed in this thesis may be the best means to treat acute thallium poisoning.
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