研究背景與目的：雙酚A(BPA)、壬基酚(NP)與辛基酚(OP)為工業上常用之化學物
質，也時常存在日常生活的用品中。塑膠奶瓶、玩具、水瓶、罐頭食品、清潔劑…
等，皆會釋放出微量的酚類化合物。一般來說，這些酚類物質大多經由飲食而進 入人體，飲食攝入也是日常生活中最主要的暴露途徑。這些酚類物質也被歸類為 環境賀爾蒙之汙染物，研究指出此種物質可能會影響生殖系統之發育或胎兒之生 長與發育。然而，目前對於產前暴露酚類物質與其胎兒之出生結果與往後生長發
育之相關性仍不清楚。因此，本研究目的為分析臍帶血中雙酚A、壬基酚與辛基
酚濃度，並探討其與胎兒出生結果與往後生長發育之相關性。
材料與方法：本研究之401 位產後婦女與其單胞胎之胎兒，皆選自於台北出生世
代追蹤研究(Taiwan Birth Panel Study)。產後利用結構式問卷收集產婦產前之生活 習慣調查，並於生產時收集胎兒臍帶血。以極致液相層析─串聯質譜儀
(UPLC-MS/MS)分析臍帶血中酚類化合物之濃度。並進一步使用迴歸模式分析臍 帶血中酚類化合物與其胎兒之出生結果與往後生長發育之相關性。
結果：臍帶血中雙酚A、壬基酚與辛基酚之中位數濃度分別為 1.50、60.97 以及
2.30 ng/mL。在迴歸模式中，調整相關之潛在干擾因子後，發現壬基酚和出生時 的頭圍(per ln unit: β = -0.13, 95% CI: -0.23, -0.03)呈顯著之負相關；關於往後生長 發育的部分，發現壬基酚和頭圍(per ln unit: β = -0.10, 95% CI: -0.19, -0.01 for 0-18 months)與身高(per ln uint: β = -0.05, 95% CI: -0.10, -0.002 for 0-6 years)呈負相關。
結論：本研究結果顯示，臍帶血中壬基酚濃度和胎兒之出生頭圍與其往後的頭圍 和身高之發展呈負相關。但仍需要更多完整且長期的研究來進一步探討產前酚類
Abstract
Background and objective: Bisphenol A, nonylphenol, and octylphenol are widely
used in our life, and they all classified as endocrine-disrupting compounds (EDCs) which could lead the adverse effect on children’s growth. Previous studies investigated the association between prenatal phenols exposure and birth outcomes, and the findings were inconsistent. In addition, most of these studies measured the levels of phenols in maternal blood or urine during pregnancy to regard as the prenatal exposure.
Few studies measured the phenols levels in cord blood to represent the prenatal exposure of fetus. The objective of this study was to measure the phenols levels in cord blood and explore the effects of prenatal phenols exposure on birth size and growth.
Methods: The study was a part of the Taiwan Birth Panel Study. A total of 401 pairs
of parents and their infants were recruited in this study. We used the ultra-performance liquid chromatography – tandem mass spectrometry (UPLC/MS/MS) to measure the free form BPA, NP and OP in cord blood. Birth outcomes were obtained after delivery, and we followed up these children to obtain the growth data up to 6 years old. The association between phenols levels in cord blood and child growth was assessed using linear regression and mixed models.
Results: The median of BPA, NP, and OP concentration in cord blood were 3.10, 74.4,
and 2.30 ng/mL, respectively. For birth outcomes, after adjusting the confounders, head circumference decreased by 0.13 cm (95% CI: -0.23, -0.03) in association with a 1-unit increase in ln-transformed NP concentration. Regarding the child growth, a 1-unit increase of ln-NP was significantly associated with a decrease in head circumference (β = -0.11, 95% CI: -0.21, -0.02 for 0-18 months), and height z-score (β
= -0.06, 95% CI: -0.11, -0.01 for 0-6 years).
Conclusion: In this study, we observed an evidence of prenatal NP exposure may have
an adverse effect on head circumference at birth and child growth in the early childhood. However, among these young children, we didn’t find an effect of prenatal BPA and OP exposure on child growth. Therefore, further studies are needed to explore the association between prenatal phenols exposure and child growth
Key words: prenatal exposure, phenol, fetal and child growth, young children
Materials and Methods ... 30 Study population ... 30 Measurement of BPA, NP, OP in cord blood ... 31 Chemicals and reagents ... 31 Sample preparation and calibration experiments ... 31 Instrumental analysis ... 33 Evaluation of matrix effect and extraction efficiency of sample pretreatment 34 Method validation and quantification ... 35 Infant’s birth outcomes and child growth ... 37 Statistical analysis ... 37 Results ... 39 Method performance, matrix effect, recovery and method validation ... 39 Phenols levels in cord blood ... 40 Prenatal phenols exposure and child growth ... 41 Discussion ... 43
Figure of contents
Figure 1. The flow chart of study population ... 51 Figure 2. The flow chart of sample preparation ... 52 Figure 3. UPLC/MS/MS chromatogram of solvent blank and matrix blank ... 53 Figure 4. UPLC/MS/MS chromatogram of bovine plasma and human cord blood ... 54
Table of contents
Table 1. Selected reaction monitoring (SRM) transitions, individual collision energy and cone voltage of the analytes ... 55 Table 2. Instrumental parameters of the mass spectrometer ... 56 Table 3. Matrix effect factor (%), extraction efficiency and recovery percentages of analytes with different concentration in bovine plasma ... 57 Table 4. Intra- and Inter-day and precision for BPA, NP, and OP in bovine plasma .... 58 Table 5. Quantification of the spiked samples in human cord blood plasma ... 59 Table 6. The detection rate and concentration of BPA, NP, OP in cord blood plasma . 60 Table 7. Characteristics of 401 mother-infants pairs included in this study ... 61 Table 8. Phenols levels distribution in maternal and infant’s characteristics ... 62 Table 9. Results from univariable linear/mixed model analysis ... 63 Table 10. Linear regression models of phenols levels in cord blood and birth outcomes ... 64 Table 11. Mixed models of phenols levels in cord blood and child growth from birth to 12 months ... 65 Table 12. Mixed models of phenols levels in cord blood and child growth from birth to 2 years ... 66 Table 13. Mixed models of phenols levels in cord blood and child growth from birth to 5 years ... 67 Table 14. Mixed models of phenols levels in cord blood and child growth from birth to 6 years ... 68 Table 15. Dose-response of prenatal NP exposure and child growth ... 69
Introduction
Bisphenol A (BPA) used in the manufacture of polycarbonate plastics (PC) and epoxy resins. BPA had been found to release from the plastic products, including toys, baby bottles, dental sealant, food and water containers, also the lining of beverages and food cans (Cao et al., 2009; Maragou et al., 2008). Nonylphenol (NP) and octylphenol (OP) are widely used and the most common surfactants in domestic detergents, pesticide formulations, industrial products, and release from the wax used for coating fruits and vegetables, plastic food packaging (Ying et al., 2002). BPA, NP, and OP are widely spread in the environment, they all classified as endocrine-disrupting compounds (EDCs). Endocrine disruptors are the chemicals that can interfere with hormone system in animals and humans, and then affect the natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes (Kavlock, 1999).
Exposure sources and routes are ubiquitous make human exposure nearly universal, especially in dietary ingestion. Specific products or activities had been examined for potential exposure of human, including canned food, microwave containers
dietary ingestion for general population, especially for preschool children. Dietary ingestion of BPA accounted for > 95% of the children excreted amounts of urinary BPA (Morgan et al., 2011). The average daily NP intake in Taiwan is at least 4-fold higher than daily intake in western countries due to the dietary habit of Taiwanese (Lu et al., 2007). However, phenols exposure of pregnant mothers is an important issue and should be concerned about.
Phenols levels had been measured in maternal blood, placental tissue and cord blood of pregnant mothers in previous studies (Chen et al., 2008; Schönfelder et al., 2002). These findings supported that phenols can accumulate in the maternal-fetal-placental unit, and have the maternal-fetal transfer when the barrier of placenta does not exist. Placental transfer can lead to the prenatal exposure to fetus, and the potential effect of prenatal phenols exposure on fetal and children growth is a concern.
Previous studies which investigated the association between prenatal phenols exposure and birth outcomes were limited and inconsistent. In the general population, few studies found the adverse effect of prenatal BPA exposure on birth outcomes such as gestational age and birth weight (Chou et al., 2011; Cantonwine et al., 2010).
Increased BPA exposure was associated with slightly increase in head circumference (Philippat et al., 2012). Another study didn’t find the association between prenatal BPA
exposure and birth outcomes (Wolff et al., 2008). In addition, the effect of prenatal NP exposure on the birth size of offspring was only shown in the animal studies (Jie et al., 2010). Regarding the body growth at postnatal age, the effect of prenatal BPA exposure on body weight was only shown in animal studies (Honma et al., 2002; Rubin et al., 2001). Overall, the association between prenatal phenols exposure and child growth is not explicit. Additionally, most of these previous studies measured the levels of phenols in maternal blood or urine during pregnancy to regard as the prenatal exposure of fetus. Few studies used the phenols levels in cord blood to represent the prenatal exposure.
According to the metabolism of bisphenol A in human, BPA were rapidly absorbed from the gastrointestinal tract in human. Conjugation with glucuronic acid in the liver, and rapidly cleared from blood by elimination with urine (Völkel et al., 2002).
The half-life of BPA and NP from the blood was 5.3 and 2-3 hours, respectively (Völkel et al., 2002; Müller et al., 1998). Although the half-life of phenols is short, the exposure sources and routes are ubiquitous in our life. BPA glucuronide does not exert hormonal activity (Matthews, 2001; Snyder et al., 2000), therefore only unconjugated BPA is possibly expected the hormonal effects and cause the health effect. Also, there
potential estrogenic effects. Furthermore, the deconjugation of BPA glucuronide in
utero by β-glucuronidase, an enzyme that is present in high concentrations in placenta
and various other tissues (Ginsberg and Rice, 2009). This creates the potential for local activation of the conjugated from back to free BPA in numerous tissues and may be important to resultant fetal exposure. The risk of phenols exposure does not be negligible despite the rapid metabolism in human.
In this study, we measured the free form BPA, NP and OP levels in cord blood.
Moreover, we explored the effect of prenatal phenols exposure on birth size and child growth among the young children.