Biomonitoring of 33 Elements in Blood and Urine Samples from Coastal Populations in Sanmen County of Zhejiang Province

ZHANG Su－jing，LUO Ru－xin，MA Dong，ZHUO Xian－yi（Shanghai Key Laboratory of Forensic Medicine，Institute of Forensic Science，Ministry of Justice，P.R.China，Shanghai 200063，China）

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Biomonitoring of 33 Elements in Blood and Urine Samples from Coastal Populations in Sanmen County of Zhejiang Province

ZHANG Su－jing，LUO Ru－xin，MA Dong，ZHUO Xian－yi
（Shanghai Key Laboratory of Forensic Medicine，Institute of Forensic Science，Ministry of Justice，P.R.China，Shanghai 200063，China）

Abstract:Objective To determine the normal reference values of 33 elements，Ag，Al，As，Au，B，Ba，Be，Ca，Cd，Co，Cr，Cs，Cu，F(xiàn)e，Ga，Hg，Li，Mg，Mn，Mo，Ni，Pb，Rb，Sb，Se，Sr，Th，Ti，Tl，U，V，Zn and Zr，in the blood and urine samples from the general population in Sanmen County of Zhejiang province，a typical coastal area of eastern China.M ethods The 33 elements in 272 blood and 300 urine samples were determ ined by inductively coupled plasma-mass spectrometry（ICP-MS）.The normality test of data was conducted using SPSS 17.0 Statistics.The data was compared w ith other reports.Results The normal reference values of the 33 elements in the blood and urine samples from the general population in Sanmen County were obtained，which of some elements were found to be sim ilar w ith other reports，such as Co，Cu，Mn and Sr，while As，Cd，Hg and Pb were generally found to be higher than those previously reported.There was a w ide variation between the reports from different countries in blood Ba. Conclusion The normal reference values of the 33 elements in the blood and urine samples from the general population in Sanmen County are established，and successfully applied to two poisoning cases.

Key words:forensic toxicology；elements；inductively coupled plasma-mass spectrometry；blood；urine；coastal area

Introduction

Element biomonitoring in the human body is important to environmental health，and clinical and forensic toxicology et al.［1-4］For human biomonitoring，the conventional samples are blood and urine［5-6］；blood Pb is the commonly used indicator of exposure［6-7］，and urine is often used in the case of essential elements intoxication［8］.In order to assess whether individuals are more exposed，reference values are often established in researches［4］.While environment and life style can influence the levels of elements in the human body［9-12］.Therefore，reference values should be established by region.

The present work was to investigate the reference values of 33 elements，Ag，Al，As，Au，B，Ba，Be，Ca，Cd，Co，Cr，Cs，Cu，F(xiàn)e，Ga，Hg，Li，Mg，Mn，Mo，Ni，Pb，Rb，Sb，Se，Sr，Th，Ti，Tl，U，V，Zn and Zr，in blood and urine samples from the coastal general population in Sanmen County of Zhejiang Province，China.Of the 33 elements，severalwere focused in terms of environmental and occupational health，some were of clinical interest，and some were applied to forensic toxicology.

M aterials and M ethods

Instrument

For determination was used an Agilent 7500Ce inductively coupled plasma-mass spectrometry（ICPMS）（Agilent Technologies，Japan）w ith an AXL500 autosampler（Agilent Technologies）.

Chem icals and reagents

An environmental-element calibration standard solution（1 000mg/L:Fe，K，Ca，Na，Ma；10mg/L: Ag，Al，As，Ba，Be，Cd，Co，Cr，Cu，Mn，Mo，Ni，Pb，Sb，Se，Tl，V，Zn，Th，U）was purchased from Agilent（USA）.ICP multi-element standard solutionsⅥ（1 000mg/L:Ca；100mg/L:As，B，Be，Ca，F(xiàn)e，Se，Zn；10mg/L:Ag，Al，Ba，Bi，Cd，Co，Cr，Cu，Ga，K，Li，Mg，Mn，Mo，Na，Na，Ni，Pb，Rb，Sr，Te，Tl，U，V），ⅩⅦ（100mg/L:Hf，Ir，Sb，Sn，Ta，Ti，Zr）and cesium ICP standard solution（1 000mg/L）were from Merck（Germany）.Mercury ICP standard solution（1 000mg/L）and gold ICP standard solution （1 000mg/L）were obtained from NSI（USA）.Furthermore，65%HNO3（Merck，Darmstadt，Germany）was diluted w ith deionized water purified using a M illi-Q system（M illipore，Massachusetts，USA）to obtain 5%HNO3.29%-32%H2O2was obtained from Alfa Aesar（USA）.15m L polypropylene tube was obtained from Corning（New York，USA）.

From the internal standard solution 10mg/L（Agilent，USA），20μg/L Y，In and Tb were obtained by dilution w ith 5%HNO3.Calibration curves for different concentrations of elements were prepared w ith standard solutions diluted w ith 5%HNO3.

Sample collection and preparation

A total number of 272 blood samples for 163 females from 18 to 69 years old and 109 males from 18 to 68 years old，and 300 urine samples for 154 females from 18 to 67 years old and 146 males from 16 to 61 years old were obtained from the general population in Sanmen County of Zhejiang Province.The county is a typical hilly area in south-eastern coastal China，the m iddle and western area of which are geographically characterized by low mountains and hilly areas，and the eastern area of which is of coastal plain.This region is rich in aquatic resources.The blood samples were collected in 2m L vacuum blood collection tubes w ith EDTAK2as anticoagulation，and the urine samples，in 15m L polyethylene vials.All the samples were stored in a refrigerator at-20℃before analysis.All subjects agreed to participate in the current study，w ith ethical approval acquired from the Ethics Comm ittee of the Institute of Forensic Science，M inistry of Justice，P.R.China.W ith 0.8m L 65%HNO3and 0.2m L 29%-32%H2O2，0.25m L blood was added into a 15m L polypropylene tube to be digested at 90℃for 3 h. The solution was diluted to 10m L w ith deionized water.0.25m L urine was diluted to 10m L w ith 5% HNO3in a 15m L polypropylene tube.These procedures were time-efficient and highly applicable to large numbers of samples for biological monitoring.

For real samples analyses，the control samples，the pooled blood and urine spiked w ith elements standard solutions，were assayed after calibration every the 20thsample.

Data processing

The statistical analysis was performed using SPSS 17.0.Distribution normality was checked by Kolmogorov-Sm irnov test.For the calculation，the measured concentrations below the lim its of quantitation（LOQ）were assumed to be LOQ/2［13］.

Results and Discussion

M ethod Validation

The lim its of detection ranged from 0.000 2μg/L （U）to 21.8μg/L（Ca）in blood and 0.000 1μg/L （U）to 4.64μg/L（Ca）in urine，respectively.The LOQ were the lowest points of the element calibrations，ranging from 0.001μg/L（U）to 50.0μg/L（Ca）for both blood and urine.All the linearities had a correlation coefficient higher than 0.999.The additional recoveries both for blood and urine ranged from 90%to 112%；intra-day precisions（6 samples at each concentration level on the same day），from 0.7%to 8.9%；and inter-day precisions（6 replicates on 3 days），from 1.8%to 9.1%.

Sam ple Determ ination

According to normality test，most of the datasets gathered for the 33 elements were observed to be non-normally distributed（P<0.05）.Median concentrations and selected percentiles of the 33 elements in 272 blood and 300 urine samples were measured and the proportions of the measured values below the LOQ of each element were listed（Table 1-2）.

The comparisons were made of the blood and urine elements levels obtained in the current study and the values reported by the previous studies，respectively（Table 1-2）.According to themedian and P5-P95（or P95）（Table 1-2），the sim ilarities and differences were observed as follows:

（1）The current study contained Mg，Ca，Ti，F(xiàn)e，Zr and Th，on which few reports were made in the literature［1，8，12，14］.

（2）Some elements levels obtained in the current study were sim ilar to those reported previously［1，8，12，14］，such as Co，Cu，Mn and Sr.

（3）Some elements levels were quite different w ith others；such significant differences could be mainly ascribed to different regions and living styles，as indicated by the P95concentrations of blood Bawhich was 156μg/L in the current study，77.6μg/L reported by a French study［1］，and 1.2μg/L reported by a German study［14］.Moreover，the current levels of As，Cd and Pb were higher than the previously reported ones，and the current level of Hg was higher than that by some other reports［4，11，14］.The reason behind such significant differences can be more consumption of aquatic products by the population in the current study.A Spain study［15］reported that the fish and shellfish contained the highest As，Cd，Hg and Pb in 11 groups of foods.Similarly，one Korea's survey［12］showed that the high concentrations of Hg and As in its population w ith great seafood consumption.As indicated by the comparisons，the current study provides valuable related data on some certain area.

Table 1　Comparisons of 33 elements in blood samp les w ith other studies　?。é蘥/L）

Table 2　Com parisons of 33 elements in urine sam p les w ith other studies?。é蘥/L）

Cases

Case 1:A woman had her facial skin turn into blue gray，because for a long time she had taken a kind of drug for her psoriasis.The histopathological and ophthalmological exam ination revealed suspected metal substance deposition in the skin and eyes，respectively.While the medication had been discontinued at that time，the patient's skin and eyes had already been seriously damaged，w ith metal deposition found in the liver，kidney，lung，heart，brain，bone and other organs，at different degrees.We assayed her blood and urine using the established methods，the results show ing whole blood Ag at 100μg/L and urine Ag at 1.35μg/L.According to the values in Table 1（P95of blood Ag:0.37μg/L， 4.51μg/L［1］and 0.236μg/L［14］）and in Table 2（P95of urine Ag:0.26μg/L and 0.03μg/L［8］），Ag content in the woman's blood and urine went beyond our reference values and ones reported previously in some other countries.Thus，a diagnosis was made of argyrosis，heavy metal poisoning，in the case of the woman.

Case 2:A man，who was adm itted to hospital for severe corrosion in the esophagus and acute renal damage，had his blood and urine multi-elements screened by ICP-MS.The results showed the whole blood Hg was 19.30μg/L and urine Hg was 22.45μg/L.When compared w ith the values of ours and ones previously reported in some other countries in Table 1（P95of blood Hg:7.49μg/L，8.13μg/L［1］，3.3μg/L［14］and 11.00μg/L［12］）and in Table 2（P95of urine Hg:2.80μg/L and 2.21μg/L［1］），both the blood and urine Hg were higher，the urine Hg about 10 times higher than that of the reports.In the case of man，a diagnosis was made of mercury poisoning.After 11 days of treatment，the levels dropped to 10.35μg/L in blood and 10.69μg/L in urine，respectively.A month later，such levels closed to the reference values w ith 6.82μg/L in blood and 3.90μg/L in urine，respectively.Every physiological index turned to normal.

Conclusion

The reference values of the 33 elements of the general population living in Sanmen County，coastal area of eastern China，were established.Establishing reference values of some certain area were valuable. The methods and results were successfully applied to two metal poisoning cases.

Acknow ledgments

The current study was supported by the Major Projects of Shanghai（15DZ1207500-2），the National Natural Science Foundation of China（81302613），Shanghai Technical Standards Special Foundation （13DZ0503001），and the Council of Shanghai Key Laboratory of Forensic Medicine（14DZ2270800），the Grants from the National Key Technology R&D Program of the M inistry of Science and Technology of the People's Republic of China（2012BAK16B02）.

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（Editor:YAN Hui）

·案例分析·

Author:ZHANG Su-jing（1986—），forensic toxicologist；E-mail:zhangsj@ssfjd.cn

文章編號：1004－5619（2016）02－0114－05

浙江省三門縣沿海地區(qū)人群血液和尿液中33種元素的生物監(jiān)測（英文）

張素靜，駱如欣，馬棟，卓先義
（司法部司法鑒定科學(xué)技術(shù)研究所上海市法醫(yī)學(xué)重點(diǎn)實(shí)驗(yàn)室，上海200063）

摘要：目的確立華東典型沿海地區(qū)浙江省三門縣一般人群血液和尿液中33種元素（Ag、Al、As、Au、B、Ba、Be、Ca、Cd、Co、Cr、Cs、Cu、Fe、Ga、Hg、Li、Mg、Mn、Mo、Ni、Pb、Rb、Sb、Se、Sr、Th、Ti、Tl、U、V、Zn和Zr）的正常值參考范圍。方法采用電感耦合等離子體質(zhì)譜法檢測272例血液樣本和300例尿液樣本中33種元素。采用SPSS 17.0軟件對所得數(shù)據(jù)進(jìn)行正態(tài)性檢驗(yàn)，將所得數(shù)據(jù)與已有文獻(xiàn)報道進(jìn)行對比。結(jié)果建立了三門縣一般人群血液和尿液中33種元素的正常值參考范圍，其中Co、Cu、Mn和Sr等元素的正常值參考范圍與其他報道相似，而As、Cd、Hg和Pb與其他研究報道存在差異，一般為高于其他報道，而血液中的Ba元素在各國文獻(xiàn)報道之間差異較大。結(jié)論建立了三門縣人群血液和尿液中33種元素的正常值參考范圍，并成功應(yīng)用于兩例中毒案例的檢測。

關(guān)鍵詞：法醫(yī)毒理學(xué)；元素；電感耦合等離子體質(zhì)譜法；血液；尿；沿海地區(qū)

Received date:（2015-04-14）

中圖分類號：DF795.1文獻(xiàn)標(biāo)志碼：A

doi：10.3969/j.issn.1004－5619.2016.02.009

Corresponding author:ZHUO Xian-yi，research fellow，postgraduatementor in forensic toxicology；E-mail:zhuoxy@ssfjd.cn MA Dong，associate research fellow in forensic toxicology；E-mail:madong@ssfjd.cn