Study On Screening Sensitive Indexes To Evaluate The Copper Deficiency And Excess And Biology Effects Of Copper And Zinc

Copper is an essential trace element of human. Every organ of animal has a little copper, the copper content of human isl.4～2.1mg/kg body weight.From Wilson's and Menke's disease were found in 1912 and 1962 respectively, the conclusion of the copper deficiency, copper metabolism related to human disease was ascertained. The results of public investgation showed that the copper deficiency rate of infant and school age children was 3.48%～30.4% in our country, most of it was over 20%. The toxicity of copper was described at the end of 19 century. The copper toxicosis of human occurred when people ate excessive copper; in addition, we are anxious for the animal was fed high copper feed and remain copper in its musule and ogan. This indicated that the conditions of copper deficiency and copper excess exist in our country. So how to evaluate the conditions of copper deficiency or excess, especially marginal deficiency or excess are an important research intern.There were no sensitive indexes to evaluate the copper status of human up today. Screening indeal sensitive indexes to estimate copper marginal deficiency and excess is a very important and urgent work.In addition, researchers found that the ratio of copper to zinc of many tumour and cancer people rose during recently years, this indicated that copper cooperated with zinc to affect these diseases of human. But the mechanism of how it works remains unknown.Whereas the upper topics, the present thesis include the next 3 parts of works:Part I Screening sensitive biochemical indexes to evaluate the copper deficiency and excessExperimentation 1 : Pre-test of copper deficiencyObjective: Observation the various of biochemial indexes and the time of its begin to has significantly change of rats intake low copper diet. Methods: 12 male S.D rats aged 5 weeks randomly assigned to 2 groups (n=6), i.e.copper deficiency (CuD) and control group (control) respectively. The rats of copper deficiency group were fed semi-purified diet low in copper (1.0 mg/kg). The rats of control group were fed conventionality feed with copper level of 8.0mg/kg for 9 weeks. Rats had free access to both food and de-ionized water, took blood samples by cutting tail at the 3rd and 6th weeks, all of rats were sacrificed at the 9th weeks and collected blood samples. The plasma ceruloplasmin activity (PCP) and plasma ceruloplasmin content was determined. Result: The plasma ceruloplasmin activity and it's content of rats in copper deficiency group was significantly lower than that of rats in controul group(p<0.01). Plasma ceruloplasmin activity in blood samples collected at the 3rd weeks of rats in copper deficiency was significantly higher than that of samples collected at the 6th and 9th weeks (p<0.01) , the plasma ceruloplasmin activity of blood samples collected at the 6th and 9th weeks had no significantly defference (p>0.05) .The plasma ceruloplasmin content of rats in copper deficiency group was no significantly defferent among the blood samples collected at the 3rd, 6th and 9th weeks(p>0.05) .Conclusion: The plasma ceruloplasmin activity is a sensitive index of lower copper diet, and rat intake low copper diet for 6 week, plasma ceruloplasmin activity will decline to a low level.Experimentation 2: Copper deficiency and excess testObjective of this study was to establish copper deficiency model and screening sensitive biochemical indexes to evaluate the copper deficiency and excess in rats. Methods 45 male S.D rats aged 5 weeks randomly assigned to 5 groups (n=9), i.e.copper deficiency (CuD), copper marginal deficient (CuMD), copper marginal excess (CuME), copper excess (CuE) group and control group (control) respectively. All of the rats of the first 4 groups were fed semi-purified diet low in copper (0.73mg/kg), the rats of the control group were fed conventionality feed with copper level of 7.0mg/kg for 6 weeks. Every afternoon, all of rats were orally perfused copper sulphate solution, the copper concentration was 0, 0.033, 0.133, 0.800 and 0mg/ml respectively(the rats in which were perfused 0 level of copper were perfused with de-ionized water ). Rats had free access to both food and de-ionized water, and were sacrificed at the 42nd day. The blood and liver samples were taken for analysis of plasma ceruloplasmin activity (PCP) and its content (PPD value), content of liver metallothionein (LMT) and liver copper (LC), the activity of erythrocyte Cu-Zn superoxide dismutase (EC Cu-Zn SOD).Results Total variance analyzed showed the effects of copper to these 5 indices were significantly (p<0.05) . Except the activity of EC Cu-Zn SOD of rats in copper excess group, the activity of plasma ceruloplasmin, the content of plasma ceruloplasmin, liver copper and liver metallothionein of rats in copper deficiency group was significantly lower than those of rats in control group and other 3 groups (p<0.05) .The analyze among groups showed the activity of plasma ceruloplasmin activity was significantly related to copper level of diet (P<0.01) , the activity of plasma ceruloplasmin of rats in copper deficiency group was significantly lower than that of other three group, and it rose with increasing copper level of diet, significant difference existed between any two groups (p<0.05) . The liver metallothionein concentration of rats was positive correlation to diet copper. The liver metallothionein concentration of rats in copper excess group was significant higher than that of rats in copper dificiency ,copper marginal deficiency and copper marginal excess groups (p<0.05); copper marginal excess group significantly higher than that of rats of copper deficiency group (p<0.05) .The content of plasma ceruloplasmin and liver copper of rats in copper deficiency significanlty lower than that of rats of other three groups (p<0.05) . The activity of erythrocyte Cu-Zn superoxide dismutase of rats in copper deficiency group and copper excess group was significantly lower than that of rats in copper marginal deficiency and copper marginal excess group (p<0.05) .Conclusion: Copper deficiency model in rats was successfully established after rat intaking low copper diet for 6 weeks. Plasma ceruloplasmin activity is the most sensitive index to evaluation copper nutrition status of rats.Part II Biology effects of rat intake copper deficiency and excess dietObjective: Biology effects were observated and analyzed of rats intake copper deficiency and excess diet. Method: 45 male S.D rats aged 5 weeks randomly assigned to 5 groups (n=9), i.e.copper deficiency (CuD), copper marginal deficient (CuMD), copper marginal excess (CuME), copper excess (CuE) group and control group (control) respectively. All of the rats in the first 4 groups were fed semi-purified diet low in copper (0.73mg/kg) for 6 weeks. The test designing was same as the first part. All of the rats were sacrificed at the 42nd day, the 6 kinds of organs were taken out and weighted, and a block of these organs of 5 rats in each group was taken out for histopathologic examination. The content of Cholesterin, Triglyceride, Very Low Density Lipoprotein, High Density Lipoprotein, IgM in plasm was determined. The composition of bone marrow cell and the density of thigh-bone were observed and analyzed.Results show either copper deficiency or excess will affect the growth of rats. A part of rats died in copper deficiency and excess group.The copper level significantly affected the heart weight coefficient (heart/body weight(HW/BW))(p<0.001); the HW/BWof rats in copper marginal excess group and copper excess group was significantly lower than that of rats in control and copper marginal deficiency group (p<0.01) , the brain weight coefficient (brain/body weight (KW/BW) )of rats in copper deficiency group was significantly lower than that of rats in control group.The cardiac muscle fibers of a part of rats in copper deficiency group are broke and eosinophilic. An arteriole in the lung shows thickening of the wall, obliteration of the lumen. The endothelium of small branch of coronary arteryof rats in copper marginal deficiency group had hyperplasia and swelling, the brain has bleeding, welling and form turbulence. The layer of middle smooth muscle of lung arteriole had manifold and the wall thicker of rats in copper marginal excess group. The heart fiber of rats in copper excess group had karyolysis, cell muscle acidphilia changed "acidphilia small body", cardiac plasm loosen, dissolve, heart ectoblast had a little incrassation.There was no significantly difference of the bone density among groups.The copper level of diet significantly affected the liver Fe and Cu (p<0.01) ,the content of liver iron of rats in copper deficiency and copper marginal excess group significantly higher than that of rat in control and copper excess group (p <0.05). Bone marrow cell hyperplasia were active and had no difference among groups.Red cell system of rats in copper excess group was higher than that of in copper deficiency and copper excess group (p<0.05).Conclusion: The biology effects of copper deficiency includ affecting the growth of rats, decrease the brain weight of rats, damnified the tissue structure of heart and lung. The biology effects of copper excess include affecting the growth of rats, decrease the heart weight of rats, damnified the tissue structure of heart and lung, affected the obsorption of iron, may promoted the iron deficiency anemia of rats.Part III Study on biology effect of rat intaking difference level of copper and zinc.Objective: Biology effects were observated of rat intaking diet with different level of copper and zinc. Method: 36 male S.D rats aged 5 weeks randomly assigned to 4 groups (n=9), i.e. Control Group(Control), Low Copper Group(LCG), Low Zinc Group(LZG) and Low Copper Low Zinc Group(LCLZG), respectively. The rats of the control group were fed conventionality feed with copper level of 7.0mg/kg, the rats of others three groups were fed semi-purified diet low in copper (0.73mg/kg) for 6 weeks. Every afternoon, all of rats were orally perfused copper sulphate solution, the copper concentration was 0, 0, 0.0625 and 0 mg/ml respectively(the rats in which were perfused 0 level of copper were perfused with de-ionized water ). The copper and zinc of diet of 1-4 groups was 7.0,15.0; 0.73,15.17; 5.73,3.17 and 0.73,3.17mg/kg, respectively. Rats had free access to both food and de-ionized water, and were sacrificed at the 42nd day. The blood and liver samples were taken for analysis of plasma ceruloplasmin activity and it's content, liver metallothionein, the activity of erythrocyte Cu-Zn superoxide dismutase and liver copper content. The 6 kinds of organs were taken out and weighted, and a block of these organs of 5 rats in each group was taken out for histopathologic examination. The content of cholesterin, triglyceride, very low density lipoprotein, high density lipoprotein, IgM in serum was determined. The composition of bone marrow cell and the density of thigh-bone were observed and analyzed. Result: The plasma ceruloplasmin activity of rats in controul group was significantly higher than that of rats in other three groups, and the plasma ceruloplasmin activity of rats in low copper group was significantly lower than that of rats in others three group (p<0.01); the plasma ceruloplasmin activity of rat in low zinc group was significantly higher than that of rats in low copper low zinc group.The liver metallothionein of rats in low copper group was significantly lower than that of rats in others three groups (p<0.01or p<0.05), that of rat in low zinc group was significantly higher than that of rats in low copper and low zinc group (p<0.05) .The activity of erythrocyte Cu-Zn superoxide dismutase of rats in control group was significantly higher than that of rats in low copper group and lower than that of rats in low zinc group and low copper low zinc group, but rats in low copper group was significantly lower than that of rats in others three group (p<0.001) . The liver copper rose with the ratio of copper to zinc of diet.The contents of cholesterin, triglyceride, high density lipoprotein, IgM in serum of rats in low zinc and low copper low zinc group were significantly declined (p< 0.05) . The bone marrow granulocyte system and the ratio of granulocyte system to erythrocyte system was significantly higher than those of rats in control and low copper group,lymphocyte cell was significantly lower than that of rats in control and low copper group (p<0.05) .The kidney, brain and liver weight of rats in low copper and low zinc group was significantly lower than that of rats in control group (p<0.05 or p<0.01) . There was no obvious damnify of the tissue structure of rats in low copper and low zinc group and control group, the rat in low zinc group had mild myocarditis. The damnification of tissue structure of rats in low copper group see the part II.Conclusion: The liver metallothionein and liver copper content correlated positively with the ratio of Cu/Zn, the plasma ceruloplasmin activity and plasma ceruloplasmin content also rose with the ratio of Cu/Zn, but its fall at excess level. The content triglyceride, cholesterin, high density lipoprotein and IgM of rat intook low level of zinc significantly decline. The heart muscle was destroyed when rat intook diet low in zinc or copper, but there was no distinctly destroyed when rats intaken diet low in copper and zinc.All of the upper results showesIn this thesis, after building up the copper deficiency model via the rat intook low copper diet, the feeding tests of rat intaking different level of copper and different copper and zinc were conduted for 6 weeks, the sensitive index to evaluate the copper deficiency and copper excess was found, the the biology effects of the weight of organ and it's pathology changes, the composition of bone marrow cell and the density of thigh-bone were observed and analyzed. These results provide important base for early diagnosis and prevention for copper deficiency and copper excess.