纸尿裤尿液中激素分析方法的初步研究及应用
A preliminary methodological and application study on determining urine hormones from disposable diapers
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摘要:
背景 婴儿的环境内分泌干扰物暴露及健康效应已经引起了广泛的关注，但是受采样方法所限，婴儿体内激素水平的测定一直是大规模人群研究的难点。
目的 研究采用纸尿裤收集婴幼儿尿液是否可用于测定尿液中雌二醇（E2）、睾酮（T）、促卵泡生成素（FSH）、促黄体生成素（LH）的含量，为开展婴儿出生后早期环境内分泌干扰物暴露评估建立基础。
方法 将含不同浓度的激素加标尿液（E2：146、219、292、365、438、584、730 pmol/L；T：0.35、0.7、1.05、1.4、1.75、2.1、2.8 nmol/L；FSH和LH：0.2、0.4、0.6、0.8、1.0、1.2、1.6 IU/L）倾倒于某品牌纸尿裤，使其充分吸收，加氯化钙粉末释放纸尿裤吸附的尿液，使用BeckmanCoulter UniCelTM DxI 800全自动免疫分析系统测定尿液中4种激素的含量，计算测量结果的准确度和精密度。将含不同浓度的激素（E2：1 605.9、2049.9、2732.9pmol/L；T：1.79、2.50、3.20nmol/L；FSH：0.78、1.00、1.47IU/L；LH：0.47、0.61、0.83IU/L）的纸尿裤尿液样品于-20℃保存0、30、100d后测定4种激素的含量，计算相对标准偏差（RSD），考察其保存的稳定性。随机选取前期在浙江岱山采集的15例婴儿（男婴8例，女婴7例）出生后不同时期（出生时，出生后14 d、28 d、42 d、3个月和6个月）的纸尿裤，测定尿液中4种激素的含量，对方法进行初步验证。
结果 将激素加标尿液与纸尿裤释放尿液中E2、T、FSH和LH的浓度进行线性拟合，在146~730 pmol/L、0.35~2.8 nmol/L、0.2~1.6 IU/L和0.2~1.6 IU/L范围内，线性关系良好，决定系数（R2）分别为0.956、0.979、0.997和0.991；在不同激素水平下，4种激素的平均回收率（R）分别为95.4%、98.3%、69.7%和39.3%，RSD在5.5%~21.6%之间；-20℃条件下，不同浓度的4种激素在不同保存时间下的精密度在7.7%~31.0%之间。15例婴儿尿液中E2、T、FSH和LH含量范围分别为ND~44178pmol/L、ND~44.37nmol/L、ND~11.64IU/L和ND~8.88IU/L。
结论 该方法可较好地测定婴儿出生后早期尿液中E2、T、FSH和LH的含量，可考虑进一步应用于环境内分泌干扰物暴露对婴幼儿出生早期的内分泌干扰效应研究。
Abstract:Background Infants' exposure to environmental endocrine disruptors has raised great concern. But due to the limits of sampling techniques, it is difficult to measure the hormone levels of infants in large cohort studies.
Objective The study is designed to determine whether the urine collected by disposable diapers can be used to measure the concentrations of 17β-estradiol (E2), testosterone (T), folliclestimulating hormone (FSH), and luteinizing hormone (LH) in urine of infants, and to establish a basis for the assessment of environmental endocrine disruptor exposure in early postnatal period.
Methods The urine with different spike levels of hormones (E2:146, 219, 292, 365, 438, 584, and 730 pmol/L; T:0.35, 0.7, 1.05, 1.4, 1.75, 2.1, and 2.8 nmol/L; FSH and LH:0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.6IU/L) were poured into dry disposable diapers of a selected brand and absorbed completely. After the absorbed urine was collected with CaCl2 powder, the concentrations of four hormones in urine were measured with Beckman Coulter UniCelTM DxI 800 Access Immunoassay System, and the accuracy and precisions of the results were calculated. The stability of the four hormones at different levels (E2:1 605.9, 2 049.9, and 2732.9pmol/L; T:1.79, 2.50, and 3.20nmol/L; FSH:0.78, 1.00, and 1.47 IU/L; LH:0.47, 0.61, and 0.83IU/L) after different storage periods (0, 30, and 100 d) at -20℃ was also investigated by calculating the relative standard deviations (RSD). Wet disposable diaper samples were collected at different ages (at birth, 14d, 28d, 42d, 3 months, and 6 months) of 15 infants (eight male infants and seven female infants) who were selected randomly from a cohort study implemented earlier in Daishan, Zhejiang Province, and were analyzed for the four hormones by the established method for preliminary verification.
Results E2 (146-730pmol/L), T (0.35-2.8nmol/L), FSH (0.2-1.6IU/L), and LH (0.2-1.6IU/L) in standard urine and the urine collected from wet disposable diapers all showed good linearity with determination coefficients (R2) of 0.956, 0.979, 0.997, and 0.991, respectively. The average recoveries of the four kinds of hormones at different spike levels were 95.4%, 98.3%, 69.7%, and 39.3%, respectively, with RSDs between 5.5%-21.6%. The RSDs were between 7.7%-31.0% of the four hormones stored at -20℃ over different periods. The concentrations of E2, T, FSH, and LH of 15 infants were between ND-44178pmol/L, ND-44.37nmol/L, ND-11.64IU/L, and ND-8.88IU/L, respectively.
Conclusion This method can be used to measure the concentrations of E2, T, FSH, and LH in urine of infants in early postnatal period, and may be applied to studies on the endocrine disrupting effects in infants exposed to environmental endocrine disruptors.
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