| OBJECTIVE: The study of the Speciation in trace elements in biological systems is of great importance in nutriological,toxicological,and pharmacological studies. A procedure based on isoelectric focusing(IEF)separation and synchrotron radiation X-ray fluorescence ( SRXRF) analysis for the speciation in trace elements in biological samples Was preliminarily established.As well as the analysis in quantity is also discussed. The use of IEF and SRXRF for the speciation of protein-bound trace elements in cytosol of human hepatocellular carcinoma and the surrounding normal tissue and those of fish liver collected from mercury miner area will make a great sense in human health and environment protection .The development of the study offers the technical support in the Speciation in trace elements in biological systems .It also opens up new opportunities for the application of modern Nuclear Analysis Technique in the science of biology .METHODS:1. Making a change of the content in gel matrix component by addition of the same amount of metal sample ,then the fluorescence intensity is detected by SRXRF .We can investigate the effect on the relative content of metal sample.2. proteins in cytosol of human hepatocellular carcinoma and the surrounding normal tissue are separated with thin layer isoelectric focusing(IEF).Drying of gel and making analysis of the content of metalions in protein bands by SRXRF.3. proteins in cytosol of fish liver collected from mercury miner area are separated with thin layer isoelectric focusing(IEF).Drying of gel and making analysis of the content of metalions in protein bands by SRXRF.RESULTS:1. Our results indicate that the relative content of Fe ,Cu, Zn detected by SRXRF is reduced with the increasing of the content of Tris-Hcl , acrylamide, glycerin, respectively.2. The contents of iron,zine,and copper in bands from the surrounding normal tissue were generally higher than that from hepatoma tissue,especially in Fe-containing proteins with pl of 6.06,5.81,5.32,3.45, 5.07,Cu-containing proteins with pl of 6.06,5.81,In addition,several zinc-containing proteins were present in the pI range of 4.32—6.44, However, Fe contents in Fe—containing proteins of 5.94 from the hepatoma tissue were slight higher than that from the surrounding normal tissue.3. The results indicate that there are four main zinc-containing bands with isoelectric points (pl) of 4.94,5.05, 4.82, 5.17 and two main Hg– containing bands with isoelectric points (pl)of6.2 3.67 present in the sample. In the two main Hg -containing bands , high level of As,Se are determined at the same time .CONCLUSIONS:1. The relative contents of the same amount of the metal sample in different gel matrix detected by SRXRF have some distinction .Moreover There are some rules in it .The results offer significant information for the establishment of mathematic model and matrix correction program between the intensity of fluorescence and contents .2. The result showed that the metal—containing proteins detected in the two samples were very much alike , but their distribution patterns were easily distinguishable.A number of research groups have been active in developing the interaction between MT and cancer. The content of zinc- containing proteins with isoelectric points (p I) of 4.32—6.44 from the surrounding normal tissue are slight higher than that from the hepatoma tissue. It was reported that the expression of MT was lower in the hepatoma tissue which is similar to our results. The main zine- containing proteins are MT and its polymer.The study demonstrated that the enzyme activity of Cu,Zn-superoxide Dismutase(Cu,Zn-SOD).was drastically suppressed In hepatoma tissue. It was reflected a high degree of damage in the function of antioxidant .The zinc-containing proteins with isoelectric points (p I )of 6.06,5.81,5.56,5.44 In the surrounding normal tissue also have a high level of copper .these should be Cu,Zn-superoxide dismutase (Cu,Zn-SOD). It was reported that the isoelectric points (pI )of Cu,Zn-SOD in human liver was 5.37,5.56,5.66.3.A certain interaction exists between selenium and many heavy metals such as mercury, arsenic,cadmium and plumbum . our results confirm it is evident as the mercury-exposed level is higher in fish liver .Meanwhile ,the result probably depends on the time of accumulation. The distinction of specific and nourishing condition also have influence on the results .The powerful evidence can be obtained by increasing the number of sample .In addition ,there is correlation between Hg and As which exist in the same isoelectric points (p I ) proteins .This relationship is not always consequence but concomitance. MT participate in all kinds of biological functions ,such as deposition ,transportation ,metabolism in trace elements(Cu ,Zn) and detoxification in heavy metal (Cd ,Hg)in biological systems .It was reported that the isoelectric points (pI) is in the range of 3.36-5.57 when the subtype of MT combine with different number metalions . The main zinc-containing proteins in cytosol of fish liver are MT and its polymer .The study is useful for the investigation of the interaction between Hg and MT.SRXRF is an advanced multielement technique which offers the analysis of highest sensitivity and good mass resolution, Especially in keeping track of trace elements in biological samples after an electrophoretic separation。Further studies are necessary to validate the universality and the biological meaning of the pattern yet.
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