Accepted Manuscript

Title: Association between some endocrine-disruptingchemicals and childhood obesity in biological samples ofyoung girls: a cross-sectional study

Author: Haemin Choi Jinhee Eom Jiye Kim Sanghouck LeeYunje Kim

PII: S1382-6689(14)00088-XDOI: http://dx.doi.org/doi:10.1016/j.etap.2014.04.004Reference: ENVTOX 1980

To appear in: Environmental Toxicology and Pharmacology

Received date: 12-11-2013Revised date: 29-3-2014Accepted date: 1-4-2014

Please cite this article as: Choi, H., Eom, J., Kim, J., Lee, S., Kim, Y.,Associationbetween some endocrine-disrupting chemicals and childhood obesity in biologicalsamples of young girls: a cross-sectional study, Environmental Toxicology andPharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.04.004

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Highlights

- PA and MEP, DBP have a statistical association with the children obesity.

- DHEA has a statistical association with the children obesity.

- DHEA can affect the development of obesity.

*Highlights (for review)

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Association between some endocrine-

disrupting chemicals and childhood

obesity in biological samples of young

girls: a cross-sectional study

Haemin Choi1, Jinhee Eom

1, Jiye Kim

1, Sanghouck Lee

2, Yunje Kim

1*

1Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-

791, Republic of Korea

2Department of Health Environment, Hoseo Graduate School of Venture, Seoul, Republic of

Korea

Corresponding author : Yunje Kim

Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-

791, Republic of Korea.

Tel.: +82 2 958 5060

Fax: +82 2 958 5805

E-mail addresses : yjkim@kist.re.kr

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Abstract

Childhood obesity, a major public health concern, has increased worldwide. Endocrine-

disrupting chemicals (EDCs) have recently received attention as a cause of obesity. A cross-

sectional study using logistic regression was conducted to investigate the association between

some endocrine disrupting chemicals and obesity in young girls. Endogenous steroids

expected to be associated with EDCs were also investigated. The target compounds included

7 phthalates (MEP, DBP, MBP, DEHP, MEHP, PA and MBzP), 2 alkylphenols (4-NP and t-

OP), bisphenol A and 9 endogenous steroids (DHT, epi-T, T, DHEA, A, P, E1, E2 and E3). PA

in urine and MEP, DBP and PA in serum showed statistically significant differences between

the control and obese groups, those compounds were considered to be associated with obesity.

In addition, DHEA in serum showed a statistically significant difference between obese and

control groups. We concluded that these substances can affect the development of obesity.

Keywords: Childhood obesity, endocrine disrupting chemicals, phthalate, alkylphenol,

bisphenol A, endogenous steroids

1. Introduction

Overweight and obesity are defined by the WHO (World Health Organization) as abnormal

or excessive fat accumulation that presents a risk to health. Obesity has dramatically

increased worldwide in recent decades, becoming a major global health concern. Like adults,

the prevalence rate for child and adolescent obesity has increased all over the world.

Childhood obesity increased from 7 % in 1980 to nearly 20 % in 2008 in the United States,

and increased from 5.8 % in 1997 to 9.7 % in 2005 in Korea (Oh et al., 2008). Obesity is a

complex disease for which no single cause or cure exists. Obesity is most likely caused by a

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complicated interaction between genetic, behavioral, and environmental factors. However,

the most common causes are overeating, lack of physical activity and genetic susceptibility

(Ogden et al., 2012; Tang-Peronard et al., 2011). One environmental factor that has been

receiving attention recently is the contribution of endocrine-disrupting chemicals (EDCs)

such as phthalates, bisphenol A and alkylphenols to the high prevalence of obesity.

Endocrine-disrupting chemicals (EDCs) interfere by mimicking, blocking, or altering

hormones and endocrine systems. Children are more vulnerable than adults to the effects of

EDCs (Tang-Peronard et al., 2011).

Phthalates are industrial chemicals used in many consumer products, such as children's toys,

baby powder, food packaging, and building materials. Phthalates have anti-androgenic effects

in animal experiments, and some phthalate activities are dependent on PPARs, which are

receptors that regulate the development and maturation of adipogenesis (Hatch et al., 2010).

Animal and human data have detected an association between phthalates-, such as MEP, and

increasing obesity and body size (Hatch et al., 2008). Bisphenol A (BPA) and alkylphenols

(APs) are the industrial chemicals that have received much attention due to their high

production and extensive usage. BPA is used in the production of polycarbonate plastic and

epoxy resins and is also found in baby bottles, microwave ovenware and the inner coating of

cans for food and beverages. BPA acts as an androgen receptor antagonist and, in

combination with insulin, accelerates adipocyte formation (Braun et al., 2009; Shankar et al.,

2012). In rodents, exposure to low doses of BPA leads to obesity and high lipid levels in utero

(Honma et al., 2002). Limited human data have suggested a positive association between

BPA exposure and obesity (Trasande et al., 2012; Wang et al., 2012). Alkylphenols are the

nonionic surfactants widely used in industry. The compound 4-nonlyphenol (4-NP) has been

investigated to determine whether there is an association with adipocytes in vitro, and

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perinatal exposure to 4-NP showed an increase in body weight and fat mass. The compound

4-NP also activated PPARγ and its target genes in adipose tissue. A few studies have

suggested that the increase in body weight was associated with developmental exposure to

some estrogenic chemicals (Lopez-Espinosa et al., 2009; Masuno et al., 2003). The

compound 4-tert-octylphenol (t-OP), an alkylphenols, is also a known estrogenic chemical

together with 4-nonylphenol and BPA.

In this study, we examined the association between obesiy classified by BMI and EDCs

which was suspected as one of the causes of obesity in young girls. We also examined the

change in the levels of endogenous steroids which were expected to be associated with EDCs.

Specially, we selected only the young girls in this experiment and examined the effects of

EDCs to estrogen. 7 Phthalates [mono-ethyl (MEP), di-n-butyl (DBP), mono-n-butyl (MBP),

di-2-ethylhexyl (DEHP), mono-2-ethylhexyl (MEHP), mono-benzyl (MBzP) phthalate,

phthalic acid (PA)], bisphenol A, and 2 alkylphenols [4-nonylphenol (4-NP) and 4-tert-

octylphenol (t-OP)], all of which are endocrine-disrupting chemicals, were assayed and

measured in terms of their levels in the urine and serum of a control group and an obese

group. And endogenous steroids were dehydroepiandrosterone (DHEA), androsterone (A),

estrone (E1), dihydrotestosterone (DHT), epitestosterone (epi-T), testosterone (T), estradiol

(E2), progesterone (P) and estriol (E3).

2. Materials and methods

2.1. Instruments and equipments

A GC/MS instrument consisting of a 6890 gas chromatograph (GC) and a 5975 mass

selective detector (MSD) from Agilent Technologies (Palo Alto, CA, USA) was used to detect

the target compounds. The column for the GC was an Ultra-1 (50 m x 0.2 mm i.d., 0.11 µm

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film thickness). Distilled water was purified through a Milli Q water system (Millipore;

Billerica, MA, USA). An HS501D shaker from IKA (Staufen, Germany) was used to mix the

samples and the organic solvents. A Varifuge-F system from Heraeus (Hanau, Germany) was

used for centrifugation. A vortex mixer from Scientific Industry (Bohemia, NY, USA) was

used for mixing, and a Turbovap® LV evaporator from Zymark (Hopkinton, MA, USA) and

a WB 2000 evaporator from Hiedolph (Schwabach, Germany) were used for drying. A DTC-

311 device from Daeil Biotech (Gyeonggi, Korea) was used to isolate the organic layer

during the extraction procedure.

.

2.2. Reagents

The following target compounds and internal standards (ISTDs) were purchased from

Sigma Aldrich (Saint Louise, MO, USA) : DEHP, DBP, MBP, PA, MEP, MBzP, 4-NP, t-OP,

BPA, DHEA, A, P, E1, E2, E3, terephthalic acid (t-PA, ISTD), benzyl butyl phthalate (BBP,

ISTD), n-octylphenol (n-OP, ISTD), bisphenol B (BPB, ISTD), d3-testosterone (d3-T, ISTD)

and ethinyl estradiol (EE2, ISTD). MEHP was purchased from AccuStandard (New Haven,

CT, USA). DHT, epi-T and T were purchased from Steraloid (Newport, RI, USA). All organic

solvents were of analytical and high - performance liquid chromatography grade. Each stock

solution of a reference standard was prepared at a concentration of 1000 μg / mL in methanol.

The working solutions were made up in methanol at various concentrations as needed.

Acetonitrile (ACN) was used for deproteinization, and diethyl ether was used for the

extraction process. These chemicals were purchased from J. T. Baker (Phillipsburg, NJ, USA).

The trimethylsilylating reagents, N,O-bis(trimethylsilyl)trifluoroacetamide) (BSTFA) and

trimethylchlorosilane (TMCS) were purchased from Sigma Aldrich.

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2.3. Study population and data analysis

2.3.1. Study population

Urine and serum samples were collected from April 2011 to September 2011 from a medical

college located in Seoul. Urine and serum samples were obtained from 58 controls (age 6 ~12,

8.59 ± 1.23, BMI < 85 %), and 69 obese (age 6~14, 8.19 ± 1.53, 85% ≤ BMI) young Korean

girls. They were asked to provide a timed overnight urine sample. The diagnosis of obesity

was performed in a hospital. Overweight and obesity ranges were determined by using weight

and height to calculate - the body mass index (BMI in kg/m2). Height and weight were

respectively measured by nurses. They used stadiometers to measure height and electronic

digital scaled to measure weight. Children were weighted wearing only underpants.

Childhood obesity and overweight were defined as the 85th

and 95th

percentiles and above the

95th percentile of the body mass index based on 2007 child and adolescent physical growth

standards. Ethical approval has been granted by the ethical committee of the Inje University

Paik Hospital. Either participants or guardians provided a written informed consent for study

participation.

2.3.2. Statistics

A cross sectional study using logistic regression was performed to assess the association

between obesity and EDCs. The statistical calculations were performed on a personal

computer using the statistical software SPSS, version 18.0.0. Differences in proportions

between obesity groups and control groups were calculated using t-test. Two tailed values of

P < 0.05 were considered to indicate statistical significance.

2.4. Sample preparation

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Urine and serum were stored in glass containers and maintained at 20 until the analyzed

were performed. In total, 30 µL (10 ng/mL) of DPB, t-PA, BBP and n-OP as ISTDs were

spiked into 0.5 mL of serum and 2 mL of urine, respectively. ACN (5 mL) was added to the

serum sample for protein removal, and the mixture was then shaken and centrifuged at

2500rpm for 5 min. The upper part of the liquid was retrieved and evaporated completely

with nitrogen gas. Phosphate buffer (0.01 M, pH 3.0, 1.0 mL) and 25 μL of β-

glucuronidase/arylsulfatase from Helix pomatia (50 μL) were added. The resulting mixture

was incubated at 55°C for 90 min. After enzymatic hydrolysis, the solution was applied to a

C18 extraction column from Waters (Milford, MA, USA). The column cartridge had been

activated and conditioned previously with a methanol-distilled water (DW) solution (3 mL,

9:1 ratio). After application of the sample, the column was washed with DW (2 mL). The

analytes were then eluted twice with methanol (3 mL), and the eluent was evaporated to

dryness under a stream of nitrogen. The residue was reconstituted in a pH 3.0 phosphate

buffer (1 mL). This solution was extracted three times with diethyl ether (4 mL, each time for

10 min.). The organic solvent was evaporated in a N2 evaporator and further dried in a

vacuum desiccator over P2O5-KOH for more than 30 min. Finally, the dried residue was

derivatized with BSTFA/1% TMCS (50 µL) at 65°C for 30 min, and 2 µL of the resulting

solution was injected into the GC-MS.

2.5. GC/MS conditions

GC-MS analysis was carried out using an Agilent 6890 gas chromatograph interfaced with

an Agilent 5975 MSD. The ion source temperature was 230°C, and the resulting solution (2

µL) was injected in split mode (5:1) at an injector temperature of 280°C and separated using

an Ultra-1 capillary column (50 m x 0.2 mm i.d., 0.11 µm film thickness; Agilent

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Technologies). The initial oven temperature was 120°C; the temperature was, ramped up to

240°C at 15°C /min, ramp to 250°C at 1.5°C /min, ramp to 260°C at 0.5°C /min, and then to

300°C at 25°C /min, followed by, a final 4 min hold for a total run time of 40.26 min. Helium

(99.9999%) was used as the carrier gas (1.0 mL/min. constant flow at an oven temperature of

150°C). The mass spectrometer was operated at 70 eV in the electron ionization mode with

selected ion monitoring (SIM).

3. Results and discussions

3.1. Validation of analytical assays of the target compounds

The experimental method for analyzing the urine and serum samples was validated in terms

of precision, recovery and reproducibility using GC/MS. For this investigation, 3 different

concentrations were used. (n=5) In the case of the EDCs, DEHP and MBzP were used at a

concentration of 5, 50 and 100 ng/mL., and the others were used at concentrations of 2, 20,

and 100 ng/mL. Endogenous steroids were used at a concentration of 1, 10 and 50 ng/mL.

The recovery rates of the EDCs were 83.18 % (20 ng/mL of MBP) - 123.34 % (20 ng/mL of

DBP) from the urine samples and 90.48 % (100 ng/mL of PA) - 112.07 % (2 ng/mL of MEP)

from the serum samples. The recovery rates of the endogenous steroids were 89.60 % (1

ng/mL of A) - 116.95 % (10 ng/mL of T) from the urine samples and 87.08 % (1 ng/mL of T)

- 119.68 % (10 ng/mL of A) from the serum samples. The precision and accuracy rates of the

measurement of the EDCs were 92.95 % (2 ng/mL of MBzP) – 114.03 % (2 ng/mL of 4-NP)

and 0.40% (100 ng/mL of PA) – 10.56% (2 ng/mL of MBzP), respectively, from the urine

samples and 77.76% (2 ng/mL of DBP) – 119.55 % (2 ng/mL of MBP) and 0.57 % (100

ng/mL of DEHP) – 15.32% (2 ng/mL of DBP), respectively, from the serum samples. The

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precision and accuracy rates of the measurements of the endogenous steroids were 82.53 %

(1 ng/mL of E1) – 125.19 % (1 ng/mL of DHT) and 0.65% (1 ng/mL of DHT) – 9.63% (1

ng/mL of T) from the urine samples and 78.98 % (1 ng/mL of T) – 133.70 % (1 ng/mL of epi-

T) and 0.52 % (50 ng/mL of DHEA) – 9.14 % (10 ng/mL of P) in the serum samples. The

calibration curves for each target compound showed usable correlation coefficients (For the

EDCs : r2=0.9958 - 0.9997 for the urine samples and 0.9977 - 0.9998 for the serum samples,

and, for the endogenous steroids, r2=0.9978 - 0.9997 for the urine samples and 0.9987 -

0.9998 for the serum samples). The limit of detection (LOD) and the limit of quantitation

(LOQ) were established at signal to noise ratios of 3 and 10, respectively. The LOD and LOQ

of the EDCs were 0.19 – 1.17 ng/mL and 0.64 – 3.87 ng/mL in urine samples, and 0.11 – 1.46

ng/mL and 0.37 – 4.82 ng/mL in serum samples, respectively. The LOD and LOQ of the

endogenous steroids were 0.08 – 0.27 ng/mL and 0.27 – 0.90 ng/mL in urine samples, and

0.05 – 0.23 ng/mL and 0.18 – 0.75 ng/mL in serum samples, respectively.

3.2. Association between the EDCs and the occurrence of childhood obesity in young girl

We measured the level of endocrine-disrupting chemicals (EDCs) in urine and serum

samples of the control group and the obese group by established analytical methods. The

target EDCs were 7 phthalates (MEP, DBP, MBP, DEHP, MEHP, PA and MBzP), BPA and 2

alkylphenols (4-NP and t-OP). Table 1 (a) shows the average, maximum and minimum levels

for EDCs in urine and serum samples of the control and obese groups.

(Table 1)

3.2.1. Phthalates

Previous studies to detect phthalates in urine samples were performed by Teitelbaum et al.

(2012) analyzed the association between nine phthalate metabolites (MEP, MBP, MCPP,

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MBzP, MiBP, MEHP, MEOHP, MECPP and MEHHP) and BMI and WC in New York City

children, and Hatch et al. (2008) analyzed the association between six phthalate metabolites

(MEP, MEHP, MBP, MBzP, MEHHP and MEOHP) and BMI and WC in eight age/gender

subgroups. However, MBP showed no clear trends among children and adolescents, and

MBzP showed a no major trends in females. Teitelbaum et al. (2012) found an association

between MEP and low MWP (low molecular weight phthalate metabolites, MEP, MBP and

MiBP) and BMI and WC among overweight children, and Hatch et al. (2008) found a

positive association of MEP (p=0.03) in female adolescents and MBzP, MEOHP, MEHHP,

MEP, and MBP (p=0.0002, for MBzP) in adult males (age 20-59) with BMI and WC.

In the present study, MEP in the urine of the obese group was 1.37 times higher than MEP in

the urine of the control group. Unlike the results of Teitelbaum et al. (2012) and Hatch et al.

(2008), MEP showed no association with childhood obesity. DBP and MBP in the urine of the

obese group were also 1.40 and 1.59 times higher than DBP and MBP in the urine of the

control group; however DBP and MBP showed no association with childhood obesity.

Furthermore, PA in the urine of the obese group was 2.35 times high respectively, than PA in

the urine of the control group. The compounds showed statistically significant relationships

with childhood obesity (PA (p=0.008)).

MEP in the serum of the obese group was 3.70 times higher than MEP in the serum of the

control group. Unlike MEP in the urine, MEP in the serum (p=0.000) showed a statistically

significant association with childhood obesity. DBP in the serum of the obese group was 1.18

times higher than DBP in the serum of the control group and showed a statistically significant

association with childhood obesity (DBP (p = 0.009)). The level of PA in serum of the obese

group was also 1.39 times higher than level of PA in the serum of the control group and

showed a significant association with childhood obesity (p = 0.024).

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3.2.2. BPA

We found no associations between urinary BPA concentrations and childhood obesity in

young girls (p = 0.599), which is consistent with the results of Yi et al. (2009) and Galloway

et al. (2010). We examined the previous studies regarding the relationship between urinary

BPA concentrations and childhood obesity, but the previous studies had conflicting results. Yi

et al. (2009) analyzed the BPA levels of obese and normal children in Seoul, Wang et al.

(2012) analyzed the BPA concentrations of 2,921 students in primary and middle schools in

the Changning District of Shanghai City in China, and Trasande et al. (2012) analyzed the

BPA concentrations of a nationally representative subsample of 2,838 participants (age 6~19)

in a cross-sectional analysis. Yi et al. (2009) found no significant association between BPA

levels and childhood obesity, despite showing higher BPA levels in obese children than in

normal children (p = 0.22). In contrast, Wang et al. (2012) found that BPA concentrations and

daily intake estimates of BPA were positively associated with BMI in females and in the 8-11

year age group and Trasande et al. (2012) found that high BPA concentration was associated

with obesity in their cross-sectional study of children and adolescents.

We also examined the previous studies regarding a relationship between urinary BPA

concentrations and adult obesity. Galloway et al. (2010) analyzed daily excretion of BPA

among 715 Italian adults (age 20~74), and Carwile et al. (2011) analyzed the BPA

concentration in 2,747 adults (age 18-74) in the United States. Similar to many studies

dealing with childhood obesity, conflicting results have been obtained. Galloway et al. (2010)

found no association between BPA concentrations and obesity in an overweight group

(p=0.296) and two obese groups (p=0.369, BMI 30.1-34.9 and p=0.738, BMI ≥ 35). In

contrast, Carwile et al. (2011) found association between BPA exposure and obesity.

In this study, we found no association between serum BPA concentrations and childhood

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obesity (p = 0.357), which is consistent with the results of Newbold et al. (2007). Takeuchi et

al. (2004) reported a higher serum BPA concentration in obese women than in non-obese

women and found a significant positive association between serum BPA concentrations and

BMI. Masuno et al. (2002) examined the effects on adipose cells treated with BPA in vitro,

showing that BPA in combination with insulin can accelerate the conversion of a adipose

cells. Newbold et al. (2007) observed the weight changes of controls and female mice treated

with BPA. In contrast to the two in vitro studies, these authors found that body weights were

not significantly different between controls and female mice treated with BPA. Many groups

have studied the relationship between BPA and obesity, but the relationship remains unclear.

3.2.3. Alkylphenols

Research involving the relationship between alkylphenols and childhood obesity has been

insufficient until the present studies.

We examined the previous studies regarding the relationships between 4-NP, t-OP and obesity

using in vitro studies and animal studies. Masuno et al. (2002) observed the effect of 4-NP on

cell proliferation and adipocyte formation in cultures of fully differentiated 3T3-L1 cells.

Their results indicated that 4-NP had the ability to stimulate the proliferation of 3T3-L1 cells

but, inhibited the adipocyte formation of 3T3-L1 cells. Hao et al. (2012) tested the dose-

dependent effects of 4-NP on adipocyte differentiation using the 3T3-L1 cell model. In

contrast to the results of Masuno et al. (2002) , Hao et al. (2012) showed that 4-NP induced

adipogenesis and activated PPARγ in adipose tissue. They suggested that perinatal exposure

to 4-NP could increase the incidence of obesity in offspring.

In this study, we found no association between 4-NP and t-OP in urine and serum and

childhood obesity, which is consistent with the results of Masuno. et al. (2002).

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3.3. Association between the EDCs and the levels of endogenous steroids.

We supposed that EDCs is expected to cause a change of the levels of endogenous steroids.

Therefore, we examined the change of the levels of endogenous steroids expected to be

affected by EDCs. The 9 endogenous steroids that were targeted were DHEA, A, E1, DHT,

epi-T, T, E2, P and E3. Table 1 (b) shows the average, maximum and minimum levels for

endogenous steroids in the urine and serum samples of the control and obese groups. In this

study, even though the levels of the endogenous steroids (except P) in the urine of the obese

group ranged from 1.00 (DHEA) to 3.65 (E1) times higher than the levels of the endogenous

steroids (except P) of the control group, we found no significant difference between the

control and the obese groups. Estrogen levels, especially in the urine of the obese group, were,

on average, 3.11 times higher than estrogen levels of the control group. Estrogen is known to

regulate adipose development and deposition in females (Cooke and Naaz, 2004). Previous

studies have examined the association between estrogen and obesity. Dieudonne et al. (2000)

studied the role of sex hormones including estrogens in adipogenesis. Dieudonne et al. (2000)

found that estrogens elicited, in vitro, a mitogenic response in superficial preadipocytes from

female rats and found a more than 2-fold increase in proliferation of superficial preadipocytes

in female rate. Karim et al. (2009) studied the association between two endogenous steroids

(estrone and estradiol) and obesity in postmenopausal women. Serum estrone showed no

association with BMI, but serum total estradiol (p = 0.01) and free estradiol (p = 0.002)

showed an association with BMI. We estimate that EDCs may affect the level of estrogen and

that high levels of estrogen can affect obesity.

DHEA and P levels in the serum of the obese group were 1.12 and 1.22 times higher than

DHEA and P levels of the control group. DHEA showed a statistically significant difference

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between the control and the obese group (p = 0.012) in serum. We suggest that EDCs may

have an effect on the elevation of DHEA levels.

4. Conclusion

We studied the association between childhood obesity and endocrine-disrupting chemicals

that were detected in urine and serum. PA in urine showed a statistically significant

relationship to childhood obesity. MEP, DBP and PA in serum showed a statistically

significant relationship to childhood obesity. We concluded that these substances can affect

the development of obesity. In the case of endogenous steroids, DHEA (p=0.012), in

particular, showed a statistically significant difference in serum between the control and the

obese group. We conclude that EDCs may affect the level of estrogens and DHEA that high

levels of estrogens and DHEA may affect the development of obesity.

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Table Legend

Table 1. Average, maximum, and minimum concentrations, standard deviations of the target compounds in the urine and plasma sample and

p-value between the control and obese group

Table

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Table 1. Average, maximum, and minimum concentrations, standard deviations of the target compounds in the urine and plasma sample and

p-value between the control and obese group

(a) EDCs

Urine Serum

Average

(ng/mL)

Max

(ng/mL)

Min

(ng/mL)

SD (%) P-value Average

(ng/mL)

Max

(ng/mL)

Min

(ng/mL)

SD (%) P-value

MEP Control 4.56 23.61 0.55 4.56 0.725 5.38 16.87 N.Q 4.47 0.000*

Obesity 6.23 56.95 0.90 8.82 19.93 57.08 2.88 14.43

DBP Control 28.64 116.32 4.48 22.98 0.119 207.70 320.60 113.99 54.79 0.009*

Obesity 38.36 248.24 5.88 37.37 244.99 470.65 132.13 83.76

MBP Control 55.68 315.79 13.00 50.24 0.294 95.29 140.06 56.58 27.77 0.000

Obesity 88.79 467.43 29.16 75.20 78.77 168.37 46.18 22.68

DEHP Control 121.34 494.77 34.44 77.21 0.579 61.80 244.97 18.57 44.55 0.742

Obesity 104.96 397.98 18.56 87.07 154.84 477.11 32.20 116.91

MEHP Control 10.72 45.55 N.Q 9.24 0.006 13.68 31.90 5.31 6.74 0.811

Obesity 9.10 21.62 N.Q 5.17 25.14 227.15 4.82 34.99

PA Control 90.76 298.51 24.45 67.77 0.008* 93.48 192.27 51.33 33.94 0.024*

Obesity 213.37 845.63 33.70 198.30 129.72 258.92 80.94 34.04

MBzP Control 263.50 724.38 45.64 196.15 0.004 357.42 1059.76 153.28 222.42 0.433

Obesity 170.00 676.36 19.60 159.23 528.78 1974.60 45.16 506.51

n-NP Control 10.87 110.31 N.D 17.01 0.624 3.34 6.39 1.59 1.27 0.554

Obesity 10.77 73.54 N.D 14.52 3.95 8.29 1.56 1.60

t-OP Control 9.04 48.89 N.D 10.10 0.692 40.33 67.00 18.36 13.94 0.059

Obesity 7.48 36.81 N.D 7.44 44.15 74.39 24.51 13.82

BPA Control 3.66 19.73 N.D 3.23 0.599 14.44 29.45 6.25 6.88 0.357

Obesity 2.72 8.83 N.D 1.82 13.27 38.05 5.44 7.50

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(b) Endogenous steroids

Urine Serum

Average

(ng/mL)

Max

(ng/mL)

Min

(ng/mL)

SD (%) P-value Average

(ng/mL)

Max

(ng/mL)

Min

(ng/mL)

SD (%) P-value

DHEA Control 4.47 27.21 N.D 6.27 0.317 13.26 37.51 N.D 10.51 0.012*

Obesity 4.48 40.55 N.D 6.38 14.81 50.71 N.Q 10.69

A Control 5.46 11.17 N.D 3.92 0.115 - N.Q N.D - -

Obesity 8.81 31.47 N.D 10.91 - 97.06 N.D -

E1 Control 0.98 2.48 N.Q 0.60 0.747 - 20.72 N.D - -

Obesity 3.58 25.21 N.D 6.72 - 5.09 N.D -

DHT Control 1.56 5.17 N.D 1.25 0.691 - 5.99 N.D - -

Obesity 1.84 5.87 N.D 1.48 - 1.02 N.D -

epi-E Control 4.28 31.00 N.D 5.07 0.214 3.22 7.40 N.D 1.57 0.355

Obesity 5.00 36.48 0.68 5.79 3.17 9.15 N.D 1.76

T Control 6.74 23.34 1.92 4.94 0.872 - 5.63 N.D - -

Obesity 9.38 82.07 1.67 13.32 - 5.42 N.D -

E2 Control 1.59 10.01 N.D 1.90 0.330 - 2.33 N.D - -

Obesity 5.58 121.74 N.D 19.82 - N.D N.D -

P Control 3.69 14.88 N.D 4.22 0.557 19.48 37.80 6.74 8.86 0.171

Obesity 3.68 13.55 N.D 3.24 23.72 47.89 4.92 10.12

E3 Control 1.35 3.58 N.D 0.90 0.969 - 0.59 N.D - -

Obesity 2.95 33.70 N.D 6.15 - N.Q N.D -

SD : Standard Deviation,

N.D : Not detected

N.Q : Not quantified

* Correlation is significant at the 0.05 p value