The Experimental Study Of Several Important Problems About Magnesium In Rat Induced By Purified Water

With the improvement of people's living standard,consuming high-energy and refined food is common in the world.Then the mineral matter in the food kept on decreasing. However,drinking soft water is prevailed in the world.And the hard effect due to low calcium or magnesium of soft water is still debated.Some one supports the hypothesis that the purpose of drinking water is supplement water,because the mineral mainly came from diet.The other supports hypothesis that the mineral matter in water is more bioavailability to a higher content,soft water can accelerate the mineral water elimination from body and increase dietary intake of toxic metals,people may be sickness due to drinking purified water long period.Then,study the biological effect of soft water on rat is useful to guide drinking waterThe research in our department reported that purified water can not affect on the growth and development of rat when the mineral mater sufficient in food,but it can induce blood rat,liver weight,liver to body weight,MDA,ET-1 increased significantly.Another study indicated a significant trend toward a decreased risk of having a child of very low birth weight(VLBW) with increasing magnesium levels in drinking water.It suggested that soft water might exert on the reproduce function and induce oxidative stress.Furthermore, our department reported that the serum magnesium of female rat was decreased significantly but other serum elements were not,such as Ca,Mg,K,Na,Fe,Mn,P,Zn,Cu,Sr,Cr,Mo,F,Li,Ni.In order to gain a better understanding of the effect of the soft drinking water(especially the magnesium in water),we study the effect of the purified water on rat with dietary magnesium deficient,magnesium sufficient and magnesium plentiful.Methods:1.The model of the magnesium content controlled strict in animal feeds(according to AIN-93G):Control diet:all the micronutrients are sufficient for good health,according to AIN-93M-GX Mineral Mix(DYETS #210050) except Mg2+content is 0.4g/kg,according to Estimated Average Requirement(EAR,from Nutrition Rehabilitation Centre,NRC) for rats.Magnesium deficient-diet:Mg2+ content is approx.0.2g/kg,a half of the Estimated Average Requirement(EAR,from NRC) for rats.Control drinking water:total dissolved solids(TDS) 175mg/L,conductivity 350μS/cm,chemical oxygen demand(COD) under detection.Purified drinking water.TDS 1.16mg/L,conductivity 2.28μS/cm,COD under detection.Weaning SD rat(68 female,36male) were assigned into 4 group by the randomization method,group 1 fed with control diet and control water,group 2 fed with control diet and purified water,group 3 fed with magnesium deficient diet and control water,group 4 fed with magnesium deficient diet and purified water throughout the entire study respectively.The animals were weighed individually on experimental day 0,7,14,21,28, and 35 during the preconception period.At 9 weeks of age(the rats were sexual mature), two females were paired with one male(2:1)from the same treatment group for a period of 14 days until mating was confirmed by observation of a copulatory plug or the presence of sperm in a vaginal rinse.The day that mating was confirmed was recorded as gestation day (GD) 0.The mated females were weighed on days 0,6,12,18 of gestation and 0,4,7,14 and 21 of lactation.All offspring were examined for behavior development during the lactation period.The body weight,reproduction(fertility rate of male,conception rate, delivery time,number of litters,number of pups,number of live pups,umber of dead pups, number of external abnormalities,average litter size,total sex ratio),offspring's physical development parameters(pinna detachment,incisor eruption,development of fur,eye opening,auditive channel opening),behavioral parameters(surface righting ability,negative geotaxis,air righting reflex,walking test and forepaw grip strength),spatial learning were observed in the prenatal toxicity test.The serum protein,serum magnesium concentration was assayed with biochemical method.The magnesium content in organ was detected with atomic absorption spectrophotometer.The spleen cell proliferation was determined with MTT.The function of peritoneal macrophage was determined by Indian ink.And the host's resistance was determined by bacterium attack.Statistical analyses were performed using SPSS 12.0 Statistical Software.The data for quantitative,continuous variables(e.g.,body weights) collected from all rats were analyzed by a factorial model to examine the main effects of diet(control or Mg deficient diet) and water(control or purified water) and their interactions.For the hidden platform test,a repeated measure ANOVA was used to determine the significance of the difference between the groups.The frequency data were analyzed by nonparametric x2-test.Unless otherwise noted,data presented were for the mean values±standard error of the mean.In all the experiments,P≤0.05 was the criterion for statistical significance.2.The model of the common animal feeds(according to GB14924-2001):Weaning SD rat(20 male) were assigned into 2 groups by the randomization method,group 1 fed with control water,group 2 fed with purified water 28 weeks.The Rat liver proteins were detected with 2D electrophoresis and MALDI-TOF-MS.The main results and conclusions are summarized as follows:Part one:The model of the magnesium content controlled strict in animal feeds (according to AIN-93G):The effect of the purified water was decrypted as follows:1.The model of magnesium deficiency was established successfully.The magnesium concentration of male's liver,female's serum and bone and offspring's liver decreased significantly in the rat fed with magnesium deficient diet but not in the control diet.2.The effect of purified water on the magnesium content(1) Male rat:Magnesium deficiency in animal feeds decreased the liver magnesium content significantly.There was a decreased trend in the serum magnesium content, increased trend in the liver and spleen magnesium content in the rat drinking purified water, although this did not achieve statistical significance.It suggested that the magnesium redistribute,and it is hard to observe the effect ion of magnesium in water.(2) Female rat:Magnesium deficiency in animal feeds decreased the serum and bone magnesium content significantly.There was a decreased trend in the serum and bone magnesium content in the rat drinking purified water,although this did not achieve statistical significance.It suggested that the magnesium redistribute,and study the biological effect on bone of female is interesting.(3) Offspring:Magnesium deficiency in maternal animal feeds decreased the pups' liver magnesium content significantly.There were no significantly differences in the magnesium content of the pups which maternal drinking purified water whatever fed with control diet or magnesium deficient diet.It suggested that the maternal can not afford the quantity of the magnesium for pups' development when maternal magnesium deficiency.3.The effect of purified water on the reproduction functionNo differences were noted for number of litters,pups,live pups,dead pups,average litter size,average litter weight,total sex ratio,and average sex ratio in the groups.It suggested that the maternal can afford the quantity of the magnesium to maintain the reproduction function.4.The effect of purified water on the F1-pupsMagnesium deficiency in maternal animal feeds decreased the offspring's body weight. The behavior deviations in F1-pups,defined by decreases in the performance of cliff avoidance reflex(day),air righting(degree),walking tests(score),grip strength(s),learning and memory show a decreased trend in the groups,although there were no significantly differences.It seems that maternal fed with magnesium deficiency diet can retard the offspring's behavior development;drinking purified might aggravate the retardation.5.The effect of purified water on the immune functionMagnesium deficiency in animal feeds decreased serum protein significantly in F0-female,but it had no effect on the serum protein in F0-male and pups.Magnesium deficiency and drinking purified water in maternal animal feeds had no effect on the spleen cell proliferation,the function of peritoneal macrophage and the host's resistance.But we observed maternal drinking purified water can decrease the function of peritoneal macrophage although this did not achieve statistical significance.It suggested studying the immune function of purified water is useful.6.The effect of purified water on the albuminMagnesium deficiency in animal feeds decreased albumin significantly in F0-female, but it had no effect on the albumin in F0-male alone.Magnesium deficiency diet with purified water decreased albumin significantly in F0-male(there were interaction between the magnesium deficiency diets with purified water).Part two:The model of the magnesium content plentiful in animal feeds:The effect of the purified water was decrypted as follows: 1.It had no effect on the body weight,organ-to-body weight.2.We identify 304 proteins with different expressing level between group fed with control water and group fed with purified water by 2D electrophoresis.We chosen 15 proteins to identify by MALDI-TOF-MS,and got 14 differential proteins,including MMSDH,Arginase -1,ATP synthase beta subunit,CAT,bFGF,regucalcin.(1) Arginase -1 participates in the urea cycle.It catalyzes arginine to ornithin and urea. NOS can catalyze arginine to NO.And NO decreased in the rat which drinking purified water.So,Arginase -1 express lower in the group 2 might inhibit the urea cycle,increase the NO.(2) ATP synthase beta subunit is an ingredient of ATP synthase.ATP synthase beta subunit express in group 2 but not in group 1.It might be the mitochondrial membrane damaged by oxidative stress in rat liver induced by purified water,and then the ATP synthase activity decreased feedback to increase the ATP synthase beta subunit expression.(3) MMSDH is a Coenzyme A dependent mitochondrial enzyme,a member of aldehyde dehydrogenase.It is important in the MDA metabolism.It expressed in the group 1but not in group 2,indicated that MDA would aggregation in liver.It is similar to our previous study.(4) BFGF is a peptide to regulate the cell growth.It increased in the inflammatory disease.It express in group 2 but not in group 1,indicated there were inflammatory reaction in the rat liver which drinking purified water.Our previous study also reported hepatic cell was focal necrosis and leukocyte infiltration.(5) Regucalcin is an calcium binding protein.In liver,it maintains calcium homeostasis in hepatocyte intracytoplasm.It express higher in group 2 indicated the absorption of calcium decreased or the calcium homeostasis disturbance due to the membrane damaged by oxidative stress.(6) CAT participate in the ROS metabolic process。It expressed in the group 1 but not in group 2,indicated that relation to the oxidative stress in liver induced by purified water.Conclusion:1.Magnesium deficiency in diets and drinking purified water might induce the magnesium redistribute in rat body,and the magnesium level of the organ changed in different degree.The maternal serum magnesium and bone magnesium decreased significantly should be pay more attention,it suggested the bone constituent of females especially which experience pregnancy and breast feed are easy to change due to drinking purified water.2.F0-female mobilize the organism magnesium to maintain the reproduction and nurse offspring,so there were no significant differences in the rat whatever fed with magnesium deficient diet or control diet and purified water or control water.But at later period of the lactation day,maternal fed with magnesium deficient diet cannot afford magnesium to the need of pups,so the pups weight gain and development delayed.Maternal drinking purified water whatever fed with control diet or magnesium deficient diet might lag the pups weight gain and development.3.Magneium exerts an important role in the immune function.Magnesium deficient diet can decreased the F0-rat(especially the female rat) immune function significantly. Although maternal fed with magnesium deficient diet,it can mobilize the magnesium to afford the pups,the pups immune function did not decrease.But maternal drinking purified water might decrease the pups' immune function,and the mechanism was not unknown.4.The albumin decreased significantly in F0-male but not in F0-female which fed with magnesium deficient diet,the reason might be the need of the magnesium in female more than the male during gestation and lactation period.Because the magnesium level in water was lower,so we did not observed the albumin decreased in the female rat which drinking purified water whatever having control diet or magnesium deficiency diet.But the albumin decreased significantly in F0-male rat drinking purified water and consuming magnesium deficient diet.5.It might be oxidative stress,calcium homeostasis disturbance and inflammatory reaction in liver of the rat drinking purified water under common diet.Because we observed the MMSDH,CAT expression disappeared in the rat liver which drinking purified water, which might induce MDA accumulate,lipid peroxidation(oxidative stress),then damage the cell membrane.The mitochondrial membrane damaged might affect the ATP synthase activity decreased,then feedback to increase the ATP synthase beta subunit expression.And the membrane damaged might induce calcium homeostasis disturbance,so the regucalcin express higher in the rat liver which drinking purified water.NO decreased in the rat which drinking purified water,then Arginase -1 express lowers in the rat liver which drinking purified water inorder to inhibit the urea cycle,increase the NO.It is reported that oxidative stress are relate to inflammatory reaction,we also observed the bFGF express in the rat liver which drinking purified water but not in the rat which drinking control water.In a word,drinking purified water should be carefully considered,especially for susceptible population.