臺灣博碩士論文加值系統

鄰苯二甲酸酯類(phthalic acid esters, PAEs)是一種合成的化學物質，存在於許多消費產品中，可經由飲食或直接接觸或使用含有塑化劑的產品而進入人體，最主要的暴露途徑是食入、吸入或皮膚接觸。在先前動物研究中指出，對男性生殖發育會產生不良健康效應。鄰苯二甲酸酯類是一種內分泌干擾物質(endocrine-disrupting chemicals, EDCs)，一旦進入到人體會干擾體內原本正常荷爾蒙的功能，包含可能改變青春期的發展。
本研究為一橫斷式研究，主要了解青春期學生體內鄰苯二甲酸酯代謝物濃度及鄰苯二甲酸酯的暴露與青春期學生第二性徵之關係。研究對象來自台灣北、中、南、東部，293名10~14歲青春期學生，並進行問卷及尿液檢體收集，其四種鄰苯二甲酸酯-Diethyl phthalate (DEP)、Dibutyl phthalate (DBP)、Di-(2-ethylhexyl) phthalate (DEHP)、Butyl benzyl phthalate (BBzP)，測量其鄰苯二甲酸單酯分別為Monoethyl phthalate (MEP)、Monobutyl phthalate (MBP)、Mono-2-ethylhexyl phthalate (MEHP)、 Monobenzyl phthalate (MBzP)， 定量方法是利用同位素稀釋法經固相萃取淨化及衍生反應後以GC-MS進行分析。
本研究中學生第二性徵發育情形，男學生出現比例最高為：青春痘(57.2%)、其次為體毛發育(50.0%)；女學生部分：乳房發育(87.9%)、青春痘(74.5%)、出現月經(68.8%)、平均初經年齡11.6±1.1歲。此外，在青春期學生體內MEP、MBP、MEHP、MBzP代謝物幾何平均濃度分別為 5.26、33.37、8.26、0.88 μg/g cr；尿中代謝物最高濃度除了MEHP外均以台灣北部的學童最高，其濃度分別為 12.46、44.16、9.57、1.44μg/g cr。
經迴歸模式校正年齡、BMI、睡眠習慣和運動習慣後，發現女生MEHP濃度在50到75百分位者相較於低暴露組，長青春痘的機率是0.26倍(p=0.04)；初經年齡在MEHP和MBzP濃度於高暴露組，其初經年齡會提早(β= -0.68年, p=0.01; β= -0.57年, p=0.00 )。另外，在高分子量代謝物(MEHP+MBzP)濃度較高的族群，其初經年齡會提早0.89歲(p<0.0001)。此外在男童中發現MEP高暴露組相較於低暴露組的人，發生聲音低沉的機率是3.06倍(p=0.01);另外在低分子量代謝物(MEP+MBP)濃度中也發現，高暴露組相較於低暴露組的人，發生聲音低沉的機率是3.8倍(p=0.02)；也發現在25到50百分位者相較於低暴露組發生夢遺情況的風險是0.15倍(P=0.01)。
本研究發現鄰苯二甲酸單酯與某些第二性徵是有關係的，而PAEs對於初經年齡的提早也扮演著類雌激素影響，在未來值得進一步探討可能影響初經年齡提早的相關機制及健康方面的問題。

Phthalates are synthetic chemicals that are used in numerous consumer products. People may be exposed to them when they use these products or via contaminated foodstuffs. Exposure routes include ingestion, inhalation and dermal contact. Several animal studies have demonstrated that phthalates can adversely affect male and female reproductive systems. They are endocrine-disrupting chemicals (EDCs), potentially interfering with the normal functions of human hormones, affecting the onset of pubertal development.
This cross-sectional study aims to evaluate levels of exposure of adolescent students to phthalates. Two hundreds and ninety-three adolescent students (aged from 10 to 14) were recruited from northern, central, southern, and eastern Taiwan. Their demographic data were collected using questionnaires; urine samples were also collected. In this study we measured four phthalate monoesters including monoethyl phthalate (MEP), monobutyl phthalate (MBP), mono-2-ethylhexyl phthalate (MEHP) and monobenzyl phthalate (MBzP) were analyzed by isotope dilution and gas chromatography-mass spectrometry (GC-MS). Urine samples were deconjugated and then solid-phase extraction and GC-MS.
The most frequently occurring of secondary sexual characteristic was having pimples (57.2%); the second most frequently occurring was the development of pubic hair development (50.0%). For girls, the most frequently occurring secondary sexual characteristic was breast development (87.9%), which was followed by having pimples (74.5%), and menarche (68.8%).The average age for menarche was 11.6±1.1 years.
The geometric mean concentrations of MEP, MBP, MEHP, and MBzP in the urine samples of the students were 5.26, 33.37, 8.26, and 0.88 μg/g cr, respectively. The concentrations of the four metabolites, except that of MEHP, were highest in students from northern Taiwan, and were 12.4, 44.16, 9.57, 1.44 μg/g cr, respectively.
According to regression models, after adjusting for age, BMI, sleeping and exercise habits, the subjects with the third quartile of MEHP concentration exhibited a significantly lower occurrence of pimple than the reference group of girls (OR=0.26, p=0.04). The menarcheal age was significantly reduced in the group with the fourth quartile of MEHP concentration and for girls with MBzP above the median concentration (β= -0.68 years, p=0.01; β= -0.57 years, p=0.00). The menarcheal declined as the PAEs metabolite concentrations increased. The group with the highest HMW PAEs concentration, had a 0.89 year earlier menarcheal age (p<0.0001). For boys, high MEP concentration was associated with a significantly higher occurrence of voice change than in the reference group (OR=3.06, p=0.01). The onset of voice change was significantly higher for the group with the highest LMW concentration than for the reference group (OR=3.80, p=0.02). A significant inversed relationship existed between LMW concentration and nocturnal pollution (OR=0.15, p=0.01).
This study finds the relationship between exposure to PAEs and the sexual development of boys and girls. PAEs may reduce menarcheal age and have an estrogen-like effect on girls. This finding warrants future study to elucidate the causal relation and the associated mechanism. The effects of PAEs on the age of maturation and health require further investigation.

摘要 I
Abstract III
Contents V
List of Tables VII
List of Figures IX
Chapter 1. Introduction 1
1.1 Background 1
1.2 Study aims 4
Chapter 2. Literature Review 5
2.1 Phthalic acid esters (PAEs)5
2.1.1 Characteristics of PAEs 5
2.1.2 Exposure route and uses of phthalates 6
2.1.3 Metabolism of phthalates 7
2.2 Exposure sources 8
2.2.1 Environmental distribution 8
2.2.2 Human exposure 9
2.2.3 Toxicology of phthalates 11
2.3 Puberty 13
2.3.1 Definition of puberty 13
2.3.2 Secondary sexual characteristics 16
2.3.3 Relationship between urinary phthalate concentration and onset of puberty 17
Chapter 3. Materials and Methods 18
3.1 Instruments and reagents 18
3.1.1 Equipments 18
3.1.2 Chemicals 18
3.1.3 Instruments 19
3.2 Method 20
3.2.1 Study population 20
3.2.2 Experimental method 20
3.2.3 Determination of urinary creatinine 22
3.3 Method validation 24
3.3.1 Calibration curve 24
3.3.2 Recovery 24
3.3.3 QA/QC 24
3.3.4 Limit of detection (LOD)24
3.4 Statistical analyses 24
Chapter 4. Results and Discussion 26
4.1 Validity of methods and reproducibility of results 26
4.2 Demographic characteristics of subjects 28
4.3 Development of secondary sexual characteristics 35
4.4 Urinary PAEs metabolites in students 42
4.5 Factors correlated with urinary PAE concentrations 46
4.6 Association between physical development and urinary PAEs concentrations 50
4.7 Association between concentrations of urinary PAE metabolites and secondary sexual characteristics 52
4.7.1 Girls 52
4.7.2 Boys 57
4.8 Estimated daily intake 62
4.9 Limitations of study 66
Chapter 5. Conclusion 67
Reference 68
Appendix 87

Agarwal, D.K., Maronpot, R.R., Lamb, J.C.T., Kluwe, W.M., 1985. Adverse effects of butyl benzyl phthalate on the reproductive and hematopoietic systems of male rats. Toxicology, 35(3), 189-206.
Anderson, S.E., Dallal, G.E., &; Must, A. 2003. Relative weight and race influence average age at menarche: results from two nationally representative surveys of US girls studied 25 years apart. Pediatrics, 111(4), 844-850.
Api, A.M., 2001. Toxicological profile of diethyl phthalate: a vehicle for fragrance and cosmetic ingredients. Food and Chemical Toxicology, 39(2), 97-108.
Aschim, E.L., Haugen, T.B., Tretli, S.D., Anne K., Grotmol, T., 2004. Risk factors for hypospadias in Norwegian boys–association with testicular dysgenesis syndrome International journal of andrology, 27(4), 213-221.
Bartolomé, L., Cortazar, E., Raposo, J.C., Usobiaga, A., Zuloaga, O., Etxebarria, N., Fernández, L.A., 2005. Simultaneous microwave-assisted extraction of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters and nonylphenols in sediments. Journal of Chromatography A, 1068(2), 229-236.
Blanck, H.M., Marcus, M., Tolbert, P.E., Rubin, C., Henderson, A.K., Hertzberg, V.S., Cameron, L., 2000. Age at menarche and tanner stage in girls exposed in utero and postnatally to polybrominated biphenyl. Epidemiology, 11(6), 641-647.
Blount, B.C., Milgram, K.E., Silva, M.J., Malek, N.A., Reidy, J.A., Needham, L.L., Brock, J.W., 2000. Quantitative detection of eight phthalate metabolites in human urine using HPLC-APCI-MS/MS. Analytical chemistry, 72(17), 4127-4134.
Bornehag, C.G., Lundgren, B., Weschler, C. J., Sigsgaard, T., Hagerhed E.L., Sundell, J., 2005. Phthalates in Indoor Dust and Their Association with Building Characteristics. Environmental Health Perspectives, 113(10), 1399-1404. doi: 10.1289/ehp.7809
Bornehag, C.G., Sundell, J., Weschler, C.J., Sigsgaard, T., Lundgren, B., Hasselgren, M., Hägerhed E.L., 2004. The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case–control study. Environmental health perspectives, 112(14), 1393.
ChitSan, L., WenMing, M., Nadim, F., 2009. Identifying the potential sources of di-(2-ethylhexyl) phthalate contamination in the sediment of the Houjing River in southern Taiwan. Journal of hazardous materials, 161(1), 270-275.
Cho, S.C., Bhang, S.Y., Hong, Y.C., Shin, M.S., Kim, B.N., Kim, J.W., Kim, H.W., 2010. Relationship between environmental phthalate exposure and the intelligence of school-age children. Environmental health perspectives, 118(7), 1027.
Chou, Y.Y., Huang, P.C., Lee, C.C., Wu, M.H., Lin, S.J., 2009. Phthalate exposure in girls during early puberty. Journal of Pediatric Endocrinology and Metabolism, 22(1), 69-78.
Colón, I., Caro, D., Bourdony, C.J., Rosario, O., 2000. Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development. Environmental health perspectives, 108(9), 895-900.
David, R.M., 2000. Exposure to phthalate esters. Environmental Health Perspectives, 108(10), A440.
Den H.E., Schoeters, G., 2006. Endocrine disrupters and human puberty. International journal of andrology, 29(1), 264-271.
Durmaz, E., Ozmert, E.N., Erkekoglu, P., Giray, B., Derman, O., Hıncal, F., Yurdakok, K., 2010. Plasma phthalate levels in pubertal gynecomastia. Pediatrics, 125(1), E122-E129.
Duty, S.M., Singh, N.P., Silva, M.J., Barr, D.B., Brock, J.W., Ryan, L., Hauser, R., 2003. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay. Environmental Health Perspectives, 111(9), 1164.
Fisher, J.S., Macpherson, S., Marchetti, N., Sharpe, R.M., 2003. Human ‘testicular dysgenesis syndrome’: a possible model using in‐utero exposure of the rat to dibutyl phthalate. Human Reproduction, 18(7), 1383-1394.
Foster, P.M.D., Mylchreest, E., Gaido, K.W., Sar, M., 2001. Effects of phthalate esters on the developing reproductive tract of male rats. Apmis, 109(S103), S272-S277.
Frederiksen, H., Sorensen, K., Mouritsen, A., Aksglaede, L., Hagen, C.P., Petersen, J.H., Juul, A., 2012. High urinary phthalate concentration associated with delayed pubarche in girls. International journal of andrology, 35(3), 216-226.
Frederiksen, H., Skakkebaek, N.E., &; Andersson, A.M., 2007. Metabolism of phthalates in humans. Mol Nutr Food Res, 51(7), 899-911. doi: 10.1002/mnfr.200600243
Frederiksen, H., Sorensen, K., Mouritsen, A., Aksglaede, L., Hagen, C. P., Petersen, J. H., Juul, A., 2012. High urinary phthalate concentration associated with delayed
pubarche in girls. Int J Androl, 35(3), 216-226.
doi:10.1111/j.1365-2605.2012.01260.x Frederiksen, H., Aksglaede, L., Sorensen, K., Skakkebaek, N.E., Juul, A., Andersson,
A.M., 2011. Urinary excretion of phthalate metabolites in 129 healthy Danish
children and adolescents: estimation of daily phthalate intake. Environmental
research, 111(5), 656-663.
Frederiksen, H., Skakkebaek, N.E., Andersson, A.M., 2007. Metabolism of phthalates in humans. Molecular nutrition &; food research, 51(7), 899-911.
GerES, IV. Phthalate metabolites and bisphenol A in urine of German children Becker: Kerstin.
Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Prieto, P., 2003. ECVAM's response to the changing political environment for alternatives: consequences of the European Union chemicals and cosmetics policies. ATLA-NOTTINGHAM-, 31(5), 473-482.
Herman G., Marcia E., Slora, E.J., Wasserman, R.C., Bourdony, C.J., Bhapkar, M.V., Koch, G.G., Hasemeier, C.M., 1997. Secondary sexual characteristics and menses in young girls seen in office practice: a study from the Pediatric Research in Office Settings network. Pediatrics, 99(4), 505-512.
Houlihan, J., Brody, C., Schwan, B., 2002. Not too pretty. Phthalates, Beauty Products &; the FDA. Accessed May, 11, 2008.
Hsu, N.Y., Lee, C.C., Wang, J.Y., Li, Y.C., Chang, H.W., Chen, C.Y., Su, H.J., 2012. Predicted risk of childhood allergy, asthma, and reported symptoms using measured phthalate exposure in dust and urine. Indoor air, 22(3), 186-199.
Janjua, N.R., Frederiksen, H., Skakkebaek, N.E., Wulf, H.C., Andersson, AM., 2008. Urinary excretion of phthalates and paraben after repeated whole‐body topical application in humans. International journal of andrology, 31(2), 118-130.
Janjua, N.R., Mortensen, G.K., Andersson, A.M., Kongshoj, B., Skakkebæk, N.E., Wulf, H.C., 2007. Systemic uptake of diethyl phthalate, dibutyl phthalate, and butyl paraben following whole-body topical application and reproductive and thyroid hormone levels in humans. Environmental science &; technology, 41(15), 5564-5570.
Karpati, A.M., Rubin, C.H., Kieszak, S.M., Marcus, M., Troiano, R.P., 2002. Stature and pubertal stage assessment in American boys: the 1988–1994 Third National Health and Nutrition Examination Survey. Journal of Adolescent Health, 30(3), 205-212.
Kavlock, R.J., Daston, G.P., DeRosa, C., Fenner-Crisp, P., Gray, L.E., Kaattari, S., Maczka, C., 1996. Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the US EPA-sponsored workshop. Environmental health perspectives, 104(Suppl 4), 715.
Koch, H.M., Preuss, R., Angerer, J.D., 2006. Di (2‐ethylhexyl) phthalate (DEHP): human metabolism and internal exposure–an update and latest results1. International journal of andrology, 29(1), 155-165.
Koniecki, D., Wang, R., Moody, R. P., Zhu, J., 2011. Phthalates in cosmetic and personal care products: concentrations and possible dermal exposure. Environ Res, 111(3), 329-336. doi: 10.1016/j.envres.2011.01.013
Latini, G., 2005). Monitoring phthalate exposure in humans. Clin Chim Acta, 361(1-2), 20-29. doi: 10.1016/j.cccn.2005.05.003
Lin, S., Ku, H.Y., Su, P.H., Chen, J.W., Huang, P.C., Angerer, J., Wang, S.L., 2011. Phthalate exposure in pregnant women and their children in central Taiwan. Chemosphere, 82(7), 947-955.
Lorz, P.M., Towae, F.K., Enke, W., Jackh, R., Bhargava, N., 2002. Phthalic acid and derivates: Ullmann’s encyclopedia of industrial chemistry, Weinheim: wiley-VCH.
Louis, G.M.B., Gray, L.E., Marcus, M., Ojeda, S.R., Pescovitz, O.H., Witchel, S. F., Tyl, R.W., 2008. Environmental factors and puberty timing: expert panel research needs. Pediatrics, 121(Supplement 3), S192-S207.
Ma, H.M., Chen, S.K., Chen, R.M., Zhu, C, Xiong, F., Li, T., Liu, L., 2011. Pubertal development timing in urban Chinese boys. International journal of andrology, 34(5pt2), e435-e445.
Ma, H.M., Du, M.L., Luo, X.P., Chen, S.K., Liu, L., Chen, R.M., 2009. Genetic Disease, Chinese Medical Association. Onset of breast and pubic hair development and menses in urban chinese girls. Pediatrics, 124(2), e269-277. doi: 10.1542/peds.2008-2638
Ma, H.M., Du, M.L., Luo, X.P., Chen, S.K., Liu, L., Chen, R.M., Wang, W., 2009. Onset of breast and pubic hair development and menses in urban Chinese girls. Pediatrics, 124(2), e269-e277.
Mackintosh, C.E., Maldonado, J.A., Ikonomou, M.G., Gobas, F.A.P.C., 2006. Sorption of phthalate esters and PCBs in a marine ecosystem. Environmental science &; technology, 40(11), 3481-3488.
Marshall, W.A., Tanner, J.M., 1969. Variations in pattern of pubertal changes in girls. Archives of disease in childhood, 44(235), 291.
Marshall, W.A., &; Tanner, J.M., 1970. Variations in the pattern of pubertal changes in boys. Archives of disease in childhood, 45(239), 13-23.
Mieritz, M.G., Frederiksen, H., Sorensen, K., Aksglaede, L., Mouritsen, A., Hagen, C.P., Juul, A., 2012. Urinary phthalate excretion in 555 healthy Danish boys with and without pubertal gynaecomastia. International journal of andrology, 35(3), 227-235.
Niino, T., Ishibashi, T., Itho, T., Sakai, S., Ishiwata, H., Yamada, T., Onodera, S., 2002. Simultaneous determination of phthalate di-and monoesters in poly (vinylchloride) products and human saliva by gas chromatography–mass spectrometry. Journal of Chromatography B, 780(1), 35-44.
Parks, L.G., Ostby, J.S., Lambright, C.R., Abbott, B.D., Klinefelter, G.R., Barlow, N.J., Gray, L.l., 2000. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicological Sciences, 58(2), 339-349.
Pinyerd, B., Zipf, W.B., 2005. Puberty-timing is everything! J Pediatr Nurs, 20(2), 75-82. doi: 10.1016/j.pedn.2004.12.011
Schettler, T., 2006. Human exposure to phthalates via consumer products. International journal of andrology, 29(1), 134-139.
Schoeters, G., Den H., Elly, D., Willem, V., Larebeke, N., Leijs, M., 2008. Endocrine disruptors and abnormalities of pubertal development. Basic &; clinical pharmacology &; toxicology, 102(2), 168-175.
Sharman, M., Read, W.A., Castle, L., Gilbert, J., 1994. Levels of di‐(2‐ethylhexyl) phthalate and total phthalate esters in milk, cream, butter and cheese. Food Additives &; Contaminants, 11(3), 375-385.
Shea, K.M., 2003. Pediatric exposure and potential toxicity of phthalate plasticizers. Pediatrics, 111(6), 1467-1474.
Shibko, S.I., Blumenthal, H., 1973. Toxicology of phthalic acid esters used in food-packaging material. Environmental health perspectives, 3, 131.
Silva, M.J., Barr, D.B., Reidy, J.A., Malek, N.A., Hodge, C.C., Caudill, S.P., Calafat, A.M., 2004. Urinary levels of seven phthalate metabolites in the US population from the National Health and Nutrition Examination Survey (NHANES) 1999-2000. Environmental Health Perspectives, 112(3), 331.
Skakkebaek, N.E., Rajpert-De M.E., Main, K.M., 2001. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects: Opinion. Human reproduction, 16(5), 972-978.
Swan, S.H., Main, K.M., Liu, F., Stewart, S.L., Kruse, R.L., Calafat, A.M., Sullivan, S., 2005. Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environmental health perspectives, 113(8), 1056.
Tan, G.H., 1995. Residue levels of phthalate esters in water and sediment samples from the Klang River basin. Bulletin of Environmental Contamination and Toxicology, 54(2), 171-176.
Teitelbaum, S.L., Britton, J.A., Calafat, A.M., Ye, X., Silva, M.J., Reidy, J.A., Wolff, M.S., 2008. Temporal variability in urinary concentrations of phthalate metabolites, phytoestrogens and phenols among minority children in the United States. Environmental research, 106(2), 257-269.
Toppari, J., Larsen, J.C., Christiansen, P., Giwercman, A., Grandjean, P., Guillette J., Louis J., Keiding, N., (1996). Male reproductive health and environmental xenoestrogens. Environmental health perspectives, 104(Suppl 4), 741.
Trasande, L., Attina, T.M., Sathyanarayana, S., Spanier, A.J., Blustein, J. 2013. Race/Ethnicity-Specific Associations of Urinary Phthalates with Childhood Body Mass in a Nationally Representative Sample. Environmental health perspectives.
Uche-Nwachi, E.O., Odekunle, A., Gray, J., Bethel, T., Burrows, Y., Carter, J., Stubbs, L., 2007. Mean age of menarche in trinidad and its relationship to body mass index, ethinicity and mothers age of menarche. OnLine Journal of Biological Sciences, 7(2), 66.
Vitali, M., Guidotti, M., Macilenti, G., Cremisini, C., 1997. Phthalate esters in freshwaters as markers of contamination sources—a site study in Italy. Environment International, 23(3), 337-347.
Wang, H., Zhou, Y., Tang, C., He, Y., Wu, J., Chen, Y., Jiang, Q., 2013. Urinary Phthalate Metabolites Are Associated with Body Mass Index and Waist Circumference in Chinese School Children. PloS one, 8(2), e56800.
Whincup, P.H., Gilg, J.A., Odoki, K., Taylor, S.J.C., Cook, D.G., 2001. Age of menarche in contemporary British teenagers: survey of girls born between 1982 and 1986. Bmj, 322(7294), 1095-1096.
Wolff, M.S., Teitelbaum, S.L., Pinney, S.M., Windham, G., Liao, L., Biro, F., Rybak, M.E., 2010. Investigation of relationships between urinary biomarkers of phytoestrogens, phthalates, and phenols and pubertal stages in girls. Environmental health perspectives, 118(7), 1039.
Yuan, S.Y., Liu, C., Liao, C.S., Chang, B.V., 2002. Occurrence and microbial degradation of phthalate esters in Taiwan river sediments. Chemosphere, 49(10), 1295-1299.
Zuckerman, D., 2001. When little girls become women: Early onset of puberty in girls. The ribbon, 6.
吳文豪: 台北學齡女童初經年齡與身高、體重及身體質量指數之關聯. 北市醫學雜誌 2005, 2: 1098-1106.