Study On Effects Of High Fluoride Exposure On OPG, RANKL And OPG/RANKL In Human Serums

PrefaceFluorine is an important trace element in the human body. It plays an irreplaceable role in physiological metabolism. Fluoridation is conducive to the deposition of calcium and phosphorus in the bone, accelerate bone formation, promote bone growth, increase the hardness of bone to maintain bone health. However, when fluoride obtained from outside exceed the normal physiological need, it will lead to acute or chronic fluorosis. In china, fluorosis mainly results from drinking water and the number of patients is large. Endemic fluorosis has become a threat to working people in physical and mental health, especially people in the poor region.One of the most serious clinical manifestations of chronic fluorosis is skeletal fluorosis. The pathological manifestation of skeletal fluorosis has diversity, including osteosclerosis, osteomalacia, osteoporosis and bone degenerative cartilage. Active osteoblast play a primary role in early skeletal fluorosis. The continuous dynamic balance of the active osteoblasts and osteoclasts maintain bone health. Any factors that damage the balance will result in changes in markers of bone metabolism. In blood, urine and other body fluids, biochemical markers of bone metabolism, can indirectly reflect the physiological state of bone.RANKL is a soluble KB-activating factor receptor ligand and belongs to tumor necrosis factor receptor family. RANKL plays an important role in the process of osteoclast differentiation, activation, and apoptosis. RANKL is a main promoting factor in mature osteoclast formation and survival. OPG is a osteoclasts negative regulatory factor in the form of soluble protein. By combinating with RANKL, OPG may inhibit RANKL/RANK signal transduction in osteoclasts, inhibit bone remodeling and bone loss, and play a role of anti-osteoporosis.So far, the studies on the OPG and RANKL were animal experiments with fluoride exposure. Human serum OPG and RANKL levels in high fluoride exposure in drinking water has not been reported. The purpose of this study is to explore whether high fluoride exposure has some relationship with human serum OPG and RANKL.Materials and Methods1. Study subjectsWe randomly selected Wang shanbao country of Kang ping, Jianye country of Linghai, Fengtian country of Zhangwu, and Huang huangdi country of Faku in Liaoning Province as our survey field spots. We investigated 461 local residents aged over 17 years old. Collected demographic information including age, gender, degree of education, occupation, smoking, drinking alcohol and health condition. We gathered demographic information and collected their urine and fasting blood samples. Accroding to the WS of People's Republic of China on the normal level of urinary fluoride of population, the subjects were divided into two groups, high fluoride exposure group (fluoride group for short) with urinary fluoride concentration higher than 1.6 mg/L and control group with urinary fluoride concentration equal to or lower than 1.6 mg/L.2. Urine fluoride testWe collected the urine samples and recorded the name, number and volume, stored the samples in-20℃refrigerator, measuring urine fluoride level with a CSB-F-I fluoride ion electrode.3. Serum OPG and RANKL level testsBlood samples of 5 ml was collected, standed two hours in room temperature, The serum was separated with 3000 rpm centrifugation for 20 minutes, and then was stored in-70℃refrigerator. We used double-antibody sandwich ABC-ELISA method to test the levels of human serum OPG and RANKL. Two test kits were bought from Biomedica Medizinpordukte GmbH & Co KG. Operations were strictly carried out following up kit instructions. All the test values should minus the blank value. Serum OPG and RANKL concentration were obtained by fitting the standard curve. Serum OPG and RANKL concentration units used pg/ml.4. Statistical analysisWe use non-parametric test, t test or correlation analysis to do the statistical analysis with the software of SPSS 13.0.Results1. Urine fluoride concentration in fluoride and control groupsThere were 47 persons in fluoride exposure group. The median of urine fluoride was 3.40mg/L and the interquartile range of ne fluoride was 1.22 mg/L in fluoride exposure group. There were 39 persons in control group. The median of urine fluoride was 0.60mg/L and the interquartile range of ne fluoride was 0.22 mg/L in fluoride exposure group.2. Comparison of demographic informations between two groupsThere was no significant sex difference between fluoride group and control. There was no significant age difference between fluoride group and control.3.Serum OPG,RANKL levels and OPG/RANKL ratio compared by urinary fluoride levelThere were no significant differences in serum OPG and RANKL levels and OPG/RANKL ratio divided by urinary fluoride levels.4. Serum OPG and RANKL levels and OPG/RANKL compared by genderThere were no significant differences in the levels of serum OPG and RANKL levels and OPG/RANKL ratio in different gender group.5. Serum OPG and RANKL levels and OPG/RANKL ratio compared by ageThere was significant difference in serum OPG level and OPG/RANKL ratio in male and female. Serum OPG level in person above 50 years old was higher than that in person below 50 years old.6.Correlation analysis on serum OPG with ageThe serum OPG level were positively correlated with age in different fluoride exposure human.Conclutions1,There were no significant difference in serum OPG and RANKL levels and OPG/RANKL ratio divided by urinary fluoride levels. 2,Male and female serum OPG level were ranged with age. The serum OPG level was positively correlated with the age.