一、分析馬兜鈴酸藥材禁用後中藥從業人員及家屬血清中馬兜鈴酸之濃度;二、評估馬兜鈴酸藥材禁用後中藥從業人員血中鉛、汞及馬兜鈴酸與腎功能之相關性

Abstract

中文摘要<一> 研究背景與目的：馬兜鈴酸 (Aristolochic acids, AA) 為腎毒性物質且具突變性及致癌作用。職業醫學研究報告指出中藥從業人員腎臟癌之標準化死亡比（Standardized mortalityratio, SMR) 顯著高於一般民眾。中藥從業人員可能於藥材之收集、製程和配製過程中暴露馬兜鈴酸，且亦可能會服用較多含馬兜鈴酸之藥材。因此，本研究目的為偵測自2003年禁用馬兜鈴酸後，中藥從業人員及其家屬血清中馬兜鈴酸 (AA I /AA II) 之濃度，並評估中藥從業人員馬兜鈴酸之職業暴露情形。 材料與方法：本研究於2007 年5 - 6 月期間，收集共171 位中藥師公會成員(包含38 位家屬)之血液檢體。取250 μL 的血清樣本後，添加25 ng/mL 氟硝西泮-D7 內標準品，以850 μL 乙腈溶液，進行蛋白質沉澱。經由超音波震盪及高速離心，取上清液過濾後，以減壓離心濃縮至近乾，上機前以20 μL 甲醇回溶。定量AA I 與AA II 分析物，則採用Waters 極致液相層析(Acquity UPLC) 結合Waters Quattro Premier XE 三段式四極棒質譜儀 (triple-quadrupole mass spectrometer)。本研究使用BEH C18 管柱 (2.1 × 50 mm, 1.7μm)，流速為0.5 μL/min，層析移動相則為10 mM 甲酸銨緩衝溶液 (pH = 3.0) 與乙腈。分析物之游離化方式，則使用電灑游離法(electrospray ionization) 於正離子模式，進行選擇性反應偵測 (selected reaction monitoring, SRM)。 結果：本研究提供人體血清中微量濃度之馬兜鈴酸 (AA I/AA II) 定性兼定量檢測分析方法。分析方法之回收率達到95-112%。 AA I 與AA II 之偵測極限 (定義為 3 倍的訊號/雜訊比) 分別為 0.014 與 0.063 ng/mL。本研究進行人體血清檢體檢測結果顯示，AAI濃度高於偵測極限僅 28 位，AA II 則皆未檢出。於中藥從業人員及其家屬之血清檢體中AAI平均濃度分別為 0.064 ng/mL (n =24) 和0.044 ng/mL (n= 4)。24位偵測到AAI之中藥從業人員中，有20位處理過含馬兜鈴酸藥材，5位曾服用含馬兜鈴酸藥材。 結論：本研究是第一篇評估人體血清中馬兜鈴酸之暴露濃度，針對禁用含馬兜鈴酸藥材四年後所收集之血清檢體，仍有少數約16.4% 檢體偵測到極微量(濃度接近偵測極限)AA I，顯示國內中草藥之安全性仍需建立更嚴謹之把關機制。 中文摘要<二> 材料與方法：本研究於2007 年5-6 月期間，收集共138 位中藥師公會成員之血液檢與問卷。使用感應耦合電漿質譜分析儀分析血中重金屬濃度。採用極致液相層析-串聯質譜儀分析血清中馬兜鈴酸濃度。根據Modification of Diet in Renal Disease Study 的公式計算估計腎絲球過濾速率(eGFR)。若eGFR< 60 (ml/min/1.73m2)為慢性腎衰竭(Chronic kidney diseas)。 結果：共22位(15. 9%)中藥從業人員為慢性腎衰竭。血中重金屬平均濃度分別為砷(5.01 μg/L)、鎘(2.04 μg/L)、汞(19.92 μg/L)、鉛(51.06μg/L)。調整工作年資、性別、身體質量指數、抽菸、喝酒、收縮壓、糖尿病和尿酸後，汞(per log10 unit: β = -11.80,95% CI -22.5 to -1.0) 和鉛(per log10 unit: β= -9.79, 95% CI -16.7 to -2.9) 與eGFR呈負相關，且汞和鉛具顯著交互作用關係(p = 0.01)。中藥從業人員之血清檢體中AAI平均濃度為 0.06 ± 0.02 ng/mL。調整干擾因子及重金屬濃度後，處理馬兜鈴酸藥 材的工作年資(β = -0.01, 95% CI -0.2 to 0.1)與馬兜鈴酸之累積暴露量(β = -0.11,95% CI -2.5 to 2.3)和eGFR呈負相關但未達統計上顯著意義。 結論：相較於馬兜鈴酸禁用後之極低劑量暴露，中藥從業人員暴露至較高濃度之鉛、汞顯著降低其腎功能表現。顯示國內中草藥之安全性仍需建立更嚴謹之重金屬及馬兜鈴酸檢驗把關機制以保護中藥從業人員及一般民眾之健康。

Abstract <1> Background and Objectives: Aristolochic acids (AAs) are known to be strongly nephrotoxic and carcinogenic. Occupational studies showed that the SMR for kidney cancer were significantly higher for the Chinese herbalists. Herbalists are potentially exposed to aristolochic acids while gathering plants, preparing or applying herbal remedies and may also consume more AAs contained herbs. This study was to determine the AAs levels in serum of herbalists and family members to access the exposure of aristolochic acid after the ban of herbs containing AAs in Taiwan. Methods: A total of 171 subjects were members of the Occupational Union of Chinese Herbalists, recruited between May and June 2007 in Taiwan. Serum samples were processed with protein precipitation using acentonitrile mixed with an internal standard,flunitrazepam-D7 and were analyzed by ultra-high-pressure liquid chromatography tandem mass spectrometry. The instrument is operated in selected reaction monitoring (SRM) with an electrospray ionization source in positive ionization mode. The analytes were separated within4 min by a Acquity BEH C18 column (2.1mm×50mm,1.7μm) with 10 mM ammonium formate (pH = 3) / acentonitrile gradient elution at a flow rate of 0.5 mL/min. Results: The method exhibited good linearity over the concentration range of 0.05–100 ng/mL and 0.4–100 ng/mL for AA I and AA II. Intra- and inter-day precisions of calibration were less than 9.3% relative standard deviation. Accuracy was tested by spiking three 4 different concentrations (0.4, 1.0 and 10 ng/mL) in bovine serum and the recoveries ranged from 95 to 112%. The limits of detction (LODs) were 0.014 and 0.063 ng/mL for AAI and AAII respectively, which were sensitive enough for determining AAs levels in human serum. Under optimum UHPLC–MS/MS conditions, a total of 28 subjects’ AAI levels were detected above the LOD (0.014 ng/mL). AAII wasn’t detected in any serum samples. The mean concentration of AAI in herbalists and family members were 0.064 ng/mL(n=24) and 0.044 ng/mL(n=4), respectively. Moreover, 20 herbalists who was detected AAI in serum have ever processed herbs containing aristolochic acids. Conclusions: The present study is, to our knowledge, the first study determining the AAs levels in human serum. AAI was still detected in 28 (16.4%) serum which was collected in 2007, four years after the ban of herbs containing aristolochic acids in Taiwan.

Abstract <2> Objective: We examined the association between multiple heavy metals and aristolochic acid levels and the estimated glomerular filtration rate (eGFR) in herbalists. Methods: A total of 138 subjects were members of the Occupational Union of Chinese Herbalists, recruited between May and June 2007 in Taiwan. Heavy metals in blood samples were analyzed by Agilent 7500C inductively coupled plasma-mass spectrometry. Aristolochic acids (AAs) in serum were measured by UHPLC-MS/MS. Renal function was assessed by using a simplified Modification of Diet in Renal Disease Study equation to estimate glomerular filtration rate (eGFR). Subjects with eGFR levels below 60 (ml/min/1.73m2) were regarded as having chronic kidney disease. Results: There were 22 (15.9%) subjects with chronic kidney disease. Heavy metal 44 concentrations of arsenic, cadmium, mercury and lead in blood were 5.01 ± 4.8, 2.04 ± 2.3, 19.92 ± 14.8, and 51.06 ± 54.0 μg/L, respectively. After adjusting for work years gender, body mass index, systolic pressure, diabetes, cigarette smoking, alcohol drinking and uric acid, in multi-metal analysis, there were significant negative effects for eGFR levels in mercury (per log10 unit: β = -11.80, 95% CI -22.5 to -1.0) and lead (per log10 unit: β= -9.79, 95% CI -16.7 to -2.9) with a significant interaction (p = 0.01) between mercury and lead. Aristolochic acid I was detected in 24 (17.4%) herbalists, the mean concentration was 0.06 ± 0.02 ng/mL, and there was negative correlation between eGFR and AAI concentration but without significant (r = -0.40, p=0.07, n=24). Duration of processing AA-herbs (β = -0.01, 95% CI -0.2 to 0.1 and cumulative AA exposure (β = -0.11, 95% CI -2.5 to 2.3) were negatively associated with eGFR after adjusting other confounders and heavy metal levels but without significant effect. Conclusions: We found the independent renal effects of mercury and lead in herbalists after the ban of herbs containing aristolochic acids in Taiwan. Although the AAs exposure did not show a significantly negative effect on eGFR in this study, the rigorous monitoring of toxic heavy metals and aristolochic acids in herbs is still needed to protect not only herbalists but also the general population who take herbs.