| Manganese(Mn) is one of the essential trace elements in biologic metabolism. With Proper quantity, Mn acts as some enzymes' active groups and reactivators, at the same time participates in the physiological functions of central nervous system. Manganese and its chemical compounds are also important material in industry with extensive application, frequently used in electric welding to improve the hardness and strength. Overdose exposure to Mn in industry may result in occurrence of occupational manganism, which can cause damage to many aspects of the body such as nerve, immunity and reproduction. The consumption of manganese in our country exceeded ten million ton per year, occupational chronic manganism has high incidence rate, which results in enormous economic loss in our country. There is no effective treating method to chronic manganism, besides no effective standard in diagnosis. Most researches before all detected the avearge concentration of manganese in the working place, and neglected the actual exposure dosage of individual. The research is to explore the correlation of individual 's actual exposure concentration and index in biomaterial for the suitable biomarkers of manganese exposure evaluation.ObjectiveTo establish a HPLC-FLD method for simultaneous determination of homovanillic acid (HVA) and vanilmandelic acid (VMA) in human urine. To explore the relationship between the individual actual exposure dosage of manganese-exposed workers and biological index in order to looking for suitable biomarkers. MethodsThis study selected 270 electric welding workers who exposed to manganese in certain locomotive and coach wagon factory of JINAN, among which 238 male and 32 female, all eliminated various kinds of liver or kidney disease, without other poison contacting and long-term medicine usage. Collecting these 270 persons' blood and urine to analyze.To grope and verify the methods of determination of HVA and VMA in urine. Accroding to the documents, high performance liquid chromatogram with fluorescence detector was chosen. The samples of urine were carried with the mobile phase comprised of methanol-0.1 mol/L phosphate buffered solution (20:80,V/V). The flow rate was 1ml/min, injection volumn was 10μl, the detection was taken at 277 nm of excitation wave and 320 nm of emission wave.The manganese in blood and urine was determined with flame atomic absorption spectrometry,HVA and VMA in urine with HPLC-FLD method, and DNA was extracted from blood to analyze the gene polymorphism of glutamic acid decarboxylase 67 (GAD67) with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and heme oxygenase-1 (HO-1) with examing the genotypic frequencies of (GT)n repeats. The correlation, one-way ANOVA and t-test was used for the data analysis.ResultsThe determination of HVA and VMA in urine was finished in fifteen minutes, and the retention time was 3.18 min for VMA and 6.54 min for HVA separately. The detection limit of HVA was 0.15μg/ml, the linear range was 0-25μg/ml,recovery rates were between 84.5%-102.1%, relative standard deviation (RSD) was less than 3.44%. The detection limit of VMA was 0.13μg/ml, the linear range was 0-20μg/ml, recovery rates were between 94.7%~101.3%, relative standard deviation (RSD) was less than 2.16%.There was no correlation between mangane concentration in blood and urine and exposure dosage, which hint the blood and urine manganese concentration neither could be the exposure biomarkers of manganese exposed.There was significant negative correlation between the exposure dosage of workers and their HVA concentration in urine (Pearson correlation coefficient=-0.168,P<0.01), as well as the exposure dosage of workers and the mean concentration of HVA and VMA in urine(Pearson correlation coefficient=-0.172, P<0.01), which prompt the HVA concentration and mean of these two acids in urine descended accompany the raise of exposure dosage, so they could action some effective biomarker of occupational manganism and evaluate the exposure condition of manganese for workers.After amplified and cut with Mnl I enzyme, one hundred and thirty two DNA samples was classified into three genotypes by CC,TC and TT. These samples was divided into two groups according to dividual exposure concentration, the difference of HVA and mean of these two acids in urine among every genotype was analyzed in each group, and there is no statistics difference, which hint the site of GAD67 Mnl I could not be the susceptible biomarker of manganese exposure.After amplified and silver stained, these samples were classified into six genotypes by SS,SM,SL,MM,ML and LL. These samples were divided into two groups according to dividual exposure concentration, the difference of HVA and mean of these two acids in urine among every genotype was analyzed in each group, and there is no statistics difference. Then the samples in these two groups was divided into two forms according to whether including L allele or not, then contrast the difference between these two forms in each group with t-test, and the differences were both statistical significance, which hint that the L allele of HO-1 could be the susceptibility biomarker of manganese exposed.ConclusionsThe method of determining HVA and VMA in urine was simple processing, stable sample, long saving time, high sensitivity, and great reproduciblity. It demanded fifteen minutes to finish one analysis, the standard curve has good linear. All the index of this method are consistent with the requirements of "Manufacture Criterion of Biomaterial Analytical Method" and can determine the contents of these two acids in human urine.Manganese in blood and urine neither could not act as the exposed biomarker of manganese exposed; HVA and mean of HVA and VMA in urine both could act as the effective biomarkers of manganese exposed. Age and gender are important influencing factors of manganese exposure. The GAD67 Mnl I site could not be the susceptible biomarker of manganese exposure. The L allele of HO-1 could be the susceptibility biomarker of manganese exposed.
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