| Objective:Aluminum(A1) is ubiquitous in the environment,It is used in industry, medicine and normal life generally because of its best physico-chemistry nature.The chance for human to A1 exposure is frequently in normal life,Food additive,drugs and utensils comprising of aluminum are main sources of aluminum uptake in China. Aluminum has been speculated that its accumulation in the human brain might be linked to neurodegenerative disorders including Alzheimer's Diseases(AD),Dialysis encephalopathy,Amyotrophic lateral sclorosis and Parkinsonism dementia of Grum.It has been proved that A1 is the certain and unique causative agent of Dialysis encephalopathy.Amino acids in the central nervous system can be divided into excitatory neurotransmitter or inhibitory neurotransmitter depending on their function. Glu and Asp are the main excitatory neurotransmitter,Gly and GABA are the main inhibitory neurotransmitter,a growing body of evidence has been accumulating to indicate that changing of the concentration of amino acid neurotransmitters are closely related to neurodegeneration,brain impairment and learning and memory. Chelators have been useful to treat aluminium-overload diseases in clinical practice. In the following experiments,DFP(1,2-dimethyl-3-hydroxy-4-pyrid-one) is used as a new type of aluminum chelating agent in animals.We develop a simple and reliable reverse-phase high performance liquid chromatographic method to determinate the amino acids neurotransmitters and investigate the potential of DFP to improve the learning and memory ability and to recover the amino acids neurotransmitters in brain tissue,aimed to evaluate the protection of DFP against the central nervous system disorder in aluminum-exposed rats.At the same time,we would observe if DFP can reduce aluminum concentration in the animals and cause the removal or displacement of other essential metals to provide foundation for seeking a safe and efficacious chelator for aluminum overload.Methods1 The establishment of aluminum-intoxicated model in ratsThirty-five male wistar rats,adult and healthy,in the weight range of 180-220g were randomly divided into five groups by body weight:Negative control group, low-dose group,med-dose group,high-dose group,Al-loaded group.All the agents were administered by gavage.The other rats were treated with A1C13 except negative control group to establish the rats' model of aluminum intoxication.Negative control and Al-loaded group were treated with saline eight weeks later.At the same time,the low,medium and high-dose groups were intragastrically administered with DFP for two week.2 The effects of DFP on learning and memory impairment caused by aluminum in ratsTwo days before the administration ended,maze test was performed to investigate the effects of DFP on learning and memory impairment caused by aluminum in rats.3 Determination of the amino acids neurotransmitters in rats brain tissueThe amino acids were separated after precolumn derivatization with dansyl chloride,and then measured by HPLC with UV detection.The HPLC column was Hypersil-2 C18(4.6mm×250mm,5μm);The mobile phase A was methanol,the mobile phase B was a mixture of 1%tetrahydrofuran,15% methanol and 84%0.05mol/L sodium acetate(V/V/V= 1:15:84),Gradient elution was used,The wavelength used for measure the derivatives was 254 nm.The HPLC flow rate was 0.6 ml.min-1.All the measurements were performed at room temperature.4 The effects of DFP on amino acids neurotransmitters in rats brain tissue caused by aluminumThe rats were sacrificed by decapitation at the end of the treatment,Brains were rapidly removed and homogenized in ice bath,then mixed with methanol for protein precipitation,followed by centrifugation to remove the protein precipitate.2mol/L KHCO3(100μl) and 4g/L Dans.Cl-Acetone mixed solution(200μl) as derivatization reagent were orderly and successively added into the supernatant,The mixture was then sealed and allowed to stand for 40min at 80℃in a thermostatic water-bath in black condition.After precise 40min,the reaction was stopped by adding 0.1mol/L hydrochloric acid(200μl).The mixture was centrifuged and the supernatant 20μl of the reaction mixture was injected into the HPLC system.HPLC-UV test method was used to determine the amino acid neurotransmitters in rats brain.5 Effect of DFP removing aluminum from serum and brainThe rats were sacrificed by decapitation at the end of the treatment.Then the serum samples were obtained by collecting and centrafugating blood samples.The brain were removed,weighted,and frozen for later analysis of aluminum by graphite furnace atomic absorption spectrophotometry.6 Effects of DFP removing other essential elementsTo investigate copper,zinc,calcium,iron and magnesium contents,The brains were digested to analyze the above elements concentration by flame atomic absorption spectrophotometry.7 Pathomorphological observation of the brain tissueThe brain of the rats were fixed with 4%paraformaldehyde,and then did hematoxylin-eosin staining.Pathomorphological observations were carried out to investigate the morphological changes.Results1 The effects of DFP on learning and memory impairment caused by aluminum in ratsThe maze test showed that the total reaction time(TRT) of Al-loaded group was significantly more than that of negative control group(P＜0.01).The TRT of low-dose group was more than negative control group(P＜0.05),The TRT of the three different dose groups(Al+DFP) was less than Al-loaded group(P＜0.01),and there was no statistical significance among mid-dose group,high-dose group and the negative control group.The error numbers of Al-loaded group and low-dose group were significantly more than that of negative control group(P＜0.01).The three different dose groups(Al+DFP) were less than Al-loaded group(P＜0.01),and there was no statistical significance among mid-dose group,high-dose group and the negative control group.2 Technology test of the HPLC method of the amino acids neurotransmittersIn the condition of this experiment,four kinds of amino acids neurotransmitters were separated perfectly in 35min.the retention time were:Asp 10.48min,Glu 11.92min,Gly 30.76min,GABA 33.45min.A good linear response was obtained for the four amino acids neurotransmitters over the range of 20～200μg/ml with correlation coefficients not less than 0.999.The detection limits were:Asp 1.5×10-3mg/L,Glu 1.8×10-3mg/L,Gly 0.3×10-3mg/L,GABA 0.5×10-3mg/L;the recoveries were:Asp 84.1%～108.8%,Glu 82.4%～109.2%,Gly 87.1%～109%,GABA 83.1%～104.9%;The inter-day RSD were:Asp 0.52%～2.9%,Glu 1.23%～2.37%,Gly 1.01%～3.15%,GABA 0.72%～3.84%;the intra-day RSD were:Asp 1.6%～4.66%,Glu 3.04%～5.43%,Gly 1.23%～4.87%,GABA 1.60%～5.76%.3 The effects of DFP on amino acids neurotransmitters in rats brain tissue caused by aluminumThe content of Asp and Glu of the Al-loaded group was significantly higher than that of negative control group(P＜0.01),the three different dose groups(Al+DFP) were significantly higher than the Al-loaded group(P＜0.01) and lower than the negative control group(P＜0.01).The content of Gly and GABA of the Al-loaded group was significantly lower than that of negative control group(P＜0.01),the three different dose groups(Al+DFP) were significantly lower than the Al-loaded group (P＜0.01) and higher than the negative control group(P＜0.01).4 Effect of DFP removing aluminum from serum and brainAluminum loading produced significant elevation of A1 level compared to control rats in serum and brains(P＜0.01).Aluminum contents of high-dose group in serum were lower than that of negative control whereas the same condition was not found in the low-,medium-dose group.Aluminum contents of the three doses groups in brain were significantly lower Al-loaded group(P＜0.01).There was no statistical significance between high-dose group and the negative control group.5 Effects of DFP removing other essential elementsZinc content of Al-loaded group were significantly higher negative control (P＜0.05).There was no significant difference among the three dose groups and negative control group,and,No significant differences existed between all the DFP groups and Al-loaded group.There were no significant differences among various groups in the contents of copper iron,calcium and magnesium contents in brains.6 Pathomorphological observation of the brainThe brain damage had been found among Al-loaded rats in the pathological examination.Neurons of the DFP-treated groups recovered to normal as compared with Al-loaded rats.The high dose group recovered better.Conclusions1 DFP could improve the learning and memory ability induced by aluminum.2 The estabished method in our research is proved to be simple,sensitive,rapid,and accurate,and it was a more reliable and sensitive method to measure the amino acid neurotransmitters in rat brain.3 DFP could increase the excitatory amino acids neurotransmitters and decrease the inhibitory amino acids neurotransmitters of rats induced by aluminum,and effectively protect the central nervous system.4 DFP could chelated aluminum and reduces aluminum contents in rats and has no obvious effect on other essential elements such as zinc,magnesium,copper et al.5 DFP could significantly improve the pathological damages of brain tissue induced by aluminum.
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