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Association between several persistent organic pollutants in serum and adipokine levels in breast milk among lactating women of Korea Sunmi Kim, Jeongim Park, Hai-Joong Kim, Jeong Jae Lee, Gyuyeon Choi, Sooran Choi, Sung Joo Kim, Su Young Kim, Duk Hee Lee, Hyo-Bang Moon, Sungkyoon Kim, and Kyungho Choi Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 09 Jun 2015 Downloaded from http://pubs.acs.org on June 9, 2015
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Association between several persistent organic pollutants in serum and adipokine levels in
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breast milk among lactating women of Korea
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Sunmi Kima, Jeongim Parkb, Hai-Joong Kimc, Jeong Jae Leed, Gyuyeon Choid, Sooran Choie,
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Sungjoo Kimf, Su Young Kimg, Duk Hee Leeh, Hyo-Bang Mooni, Sungkyoon Kima, Kyungho Choia*
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a
School of Public Health, Seoul National University, Republic of Korea
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b
College of Natural Sciences, Soonchunhyang University, Republic of Korea
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c
College of Medicine, Korea University, Republic of Korea
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d
College of Medicine, Soonchunhyang University, Republic of Korea
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e
College of Medicine, Inha University, Republic of Korea
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f
College of Medicine, Hallym University, Republic of Korea
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g
College of Medicine, Jeju National University, Republic of Korea
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h
College of Medicine, Kyungpook National University, Republic of Korea
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i
College of Science and Technology, Hanyang University, Republic of Korea
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*Correspondence to: K. Choi
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Department of Environmental Health, School of Public Health, Seoul National University, 1,
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Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea. Tel.: +82 2 880 2738.
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E-mail address:
[email protected] 22 23
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Abstract
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Exposure to several persistent organic pollutants (POPs) has been linked to alteration of lipid
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metabolism. Adipokines, such as leptin and adiponectin, are hormones that play roles in lipid
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metabolism, and have been suggested as markers of health effects that may lead to obesity. To date,
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only serum adipokines have been associated with POPs exposure. In the present study, for the first
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time, the associations between leptin and adiponectin in breast milk, and several POPs in serum were
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investigated among 82 lactating Korean women between 25 and 46 years of age. Breast milk
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adipokine is important because adipokine intake of infant through breastfeeding may influence the
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growth of infants. The median concentrations of leptin and adiponectin in skimmed milk of the
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Korean lactating women were 17.9 ng/L and 16.5 µg/L, respectively. Leptin concentrations in breast
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milk were negatively associated with ∑hexachlorohexane (HCH), oxychlordane, ∑chlordane, or
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2,2',4,4',5,5'-hexachlorobiphenyl (CB 153) levels in maternal serum. Linear relationship between
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adiponectin and any POPs concentrations were not observed, however, non-monotonic relationship
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which showed generally positive associations was suggested for p,p’-
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dichlorodiphenyltrichloroethane (DDT) and ∑chlordane in quartile analysis. Our observations show
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that POPs at the current level of exposure may be associated with the alteration of lipid metabolism
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among lactating women. Implication of adipokine transfer to breastfed infants is of concern and
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deserves further investigation.
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Keyword: Leptin, adiponectin, POPs, obesity, lipid, metabolism
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1. Introduction
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Adipokines are hormones synthesized and secreted by adipocytes, and have been identified in
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both serum and breast milk. Adipokines are associated with regulation of food intake and energy
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balance.1 For this reason, adipokines, such as leptin and adiponectin, have been employed to
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understand the etiology of obesity among the human population.2-5 Leptin mainly acts to regulate
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energy balance, and decreased leptin may lead to increased adiposity. Adiponectin acts to increase
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uptake and catabolism of fatty acids, and to regulate energy expenditure. Among infants and children,
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blood adipokines have been associated with obesity. For example, perinatal leptin concentrations
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measured in maternal plasma at 26-28 weeks of gestation, and cord blood were negatively correlated
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with adiposity at 3 years of age, but leptin level at age 3 was positively correlated with adiposity at
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age 7.3 Adiponectin in cord blood was inversely associated with weight gain for the first 6 months
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after birth and adiposity at 3 years of age.5
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Recently, several persistent organic pollutants (POPs) have been suggested to cause obesity by
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alteration of lipid metabolism of humans.6, 7 For example, low-dose exposure to organochlorine
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pesticides (OCPs) and polychlorinated biphenyls (PCBs) has been associated with a greater risk of
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obesity8-11 even during childhood.12 Adipokines may play a role in the association between POPs
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exposure and obesity or type 2 diabetes.13
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POPs exposure has been associated with altered adipokine levels in many in vitro and in vivo
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experiments.13-16 Epidemiological observations are relatively limited but also increasing in number.
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Serum leptin showed negative associations with serum 2,2',3,4',5,5'-hexachlorobiphenyl (CB 138),
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2,2',3,4,4',5,5'-Heptachlorobiphenyl (CB 180), and 2,2nd ,4,4',5,-hexabromodiphenyl ether (BDE 153)
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levels in obese adults.17 Similarly, in boys of 8 to 9 years of age, serum leptin levels were inversely
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correlated with β-HCH or p,p’-dichlorodiphenyldichloroethylene (DDE) exposure.18 19, 20Among the
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obese population, serum adiponectin also showed a negative association with PCBs exposure,19, 20
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but among a population with normal BMI range, a positive association was reported between
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adiponectin and polybrominated diphenyl ethers (PBDEs) exposure.21
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Adipokines in breast milk can influence their circulating levels in breastfed infants.21, 22
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Furthermore, adiponectin in breast milk is reported to possess a greater biological activity and
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greater gastrointestinal absorption rate than the forms existing in blood.23 Therefore, breast milk
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adipokines have been recognized as important hormones that are associated with obesity.24-26
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Doneray et al.27 reported that breast milk leptin concentration is inversely associated with BMI
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increase during the first one month of age. Adiponectin levels in breast milk are positively associated
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with weight gain among 2 year old infants.28, 29 3 Environment ACS Paragon Plus
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Despite the importance of breast milk adipokines among infants, to our knowledge, its
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association with maternal POPs exposure has never been investigated. In the present study, we
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investigated the association between the breast milk adipokine levels and the serum POPs levels
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among lactating women of Korea. The utility of breast milk as an alternative medium to maternal
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serum for measuring adipokines could be assessed. In addition, since breast milk adipokines are
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important for infant growth and possibly for obesity in the later stages of the life, this study will also
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shed light on the implication of maternal POPs exposure on the potential adverse health outcome of
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her offspring through lactational transfer of adipokines.
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2. Materials and methods
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Study population and sample collection
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One-hundred and thirty-eight healthy pregnant women without chronic disease history were
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recruited before delivery from five university hospitals located in four cities of Korea, i.e., Seoul,
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Anyang, Ansan, and Jeju, from February to December 2011, as a part of the CHECK (Children’s
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Health and Environmental Chemicals in Korea) Panel. Details about the demographic characteristics
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of the participating women were shown in previous studies.30, 31 On arrival at the hospital for
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delivery (within 1 day before delivery), maternal blood samples were collected, separated for serum
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on site, and stored in polypropylene cryovials at −20 °C until analysis. Breast milk samples were
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collected from the same women at 7, 15 or 30 days after delivery, in polypropylene tubes following a
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detailed instruction with pictorial guide. The breast milk samples were then frozen and transported
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on ice to the laboratory. Samples were stored in the laboratory at −70 °C until further analysis.
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Among 138 recruited mothers collected for blood serum at delivery, 87 mothers provided breast milk
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samples but only 82 samples were analyzed for adipokines due to limited volume of breast milk
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sample. Seventy-four breast milk samples were collected at the 15th day after delivery; however, one
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and seven samples were collected at days 7 and 30 after delivery, respectively.
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One-on-one interviews with participating pregnant women was conducted at the time of
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enrollment. Demographic characteristics such as age, body weight, height, medical history, and
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pregnancy related records were obtained from this interview.
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The Institutional Review Boards of the School of Public Health, Seoul National University,
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and all participating university hospitals (Soonchunhyang University Seoul Hospital, Hallym
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University Sacred Heart Hospital, Korea University Ansan Hospital, and Jeju National University
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Hospital) approved the study. Written informed consents were obtained from the participating
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women. All samples and data were processed blind.
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Chemical analysis in blood serum Maternal serum samples were processed and measured for 19 PCB congeners, 19 PBDEs
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congeners, and 19 OCPs, as shown in a previous study.31 For POPs quantification, 13C-labeled 8
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PCBs (EC9605-SS; Wellington Laboratories, Guelph, ON, Canada), 13C-labelled 6 PBDEs (MBDE-
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MXE; Wellington) and 13C-labeled 19 OCPs (ES-5349-L; Cambridge Isotope Laboratories, Andover,
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MA, USA) were used as surrogate internal standards. Details about the POPs measurement among
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the present population are reported elsewhere.31 The experimental procedures for serum POPs
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analysis were optimized following previous studies with some modifications.33, 34 Details for sample
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extraction and instrumental analysis are provided in Supporting Information. A high-resolution gas
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chromatography interfaced with a high-resolution mass spectrometer (HRGC/HRMS; JMS 800D,
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JEOL, Tokyo, Japan) was used for the identification and quantification of OCPs, PCBs and PBDEs.
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Details of instrumental parameters have been reported elsewhere.32, 33
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POPs concentrations were normalized by lipid weight of serum. Total lipid (mg/dL) was
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calculated from concentrations of total cholesterol and triglyceride that were analyzed by enzymatic
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methods in a commercial clinical laboratory (Samkwang Laboratory, Seoul, Korea), by the following
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equation34:
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Total lipid = 2.27 * total cholesterol + triglyceride + 62.3
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The recoveries of spiked 13C-labeled POPs compounds were 91 ± 10% (average± SD) for
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OCPs, 62 ± 5.5% for PCBs and 87 ± 13% for PBDEs. Cross contamination between the samples was
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negligible. Procedural blanks (n = 10) were processed with every set of 15 serum samples to check
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laboratory contamination. Blanks did not contain quantifiable amounts of target contaminants. Limit
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of quantification (LOQ) was calculated as 10 times the signal to noise ratio. The respective LOQs for
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OCPs, PCBs and PBDEs were from 0.7 to 1.7 ng/g lipid weight (lw), from 0.8 to 8.3 ng/g lw and
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from 0.2 to 0.8 ng/g lw.
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Adipokines detection in breast milk The breast milk samples were thawed at room temperature. After being shaken, 2 ml of breast
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milk was taken from each sample tube. The breast milks were then centrifuged at 10,000 rpm for 1
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minute to separate fat layers. Only the water phase of the samples (skimmed milk) were collected 5 Environment ACS Paragon Plus
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and diluted to within the assay concentration range before being measured for adipokines.
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Commercially available enzyme-linked immunosorbent assay (ELISA) kit (Human Leptin DuoSet®,
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and Human Adiponectin/Acrp30 DuoSet®, R&D systems, Minneapolis, MN, USA) was applied.
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The analytical method was validated following the manufacturer's instructions. Recoveries were
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calculated by comparing to the spiked internal standard, and were 86 % and 95 % for leptin and
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adiponectin, respectively. Recoveries for linearity within spiked matrix, 2-, 4-, and 8-fold diluted
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matrix ranged between 88 and 92 % for leptin assay, and between 130 and 140 % for adiponectin
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assay. Standard curves were generated in 96-well plates with R2 > 0.99 and R2 > 0.96 for leptin and
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adiponectin measurements, respectively. All assays were performed in duplicate. Detection limits for
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leptin and adiponectin were 15.63 and 62.5 ng/L, respectively.
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Statistical analysis
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Hexachlorobenzene (HCB), β-HCH, p,p’-DDE, p,p’- dichlorodiphenyltrichloroethane (DDT),
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oxychlordane, trans-Nonachlordane, 2.2’-4,4’-tetrabromodiphenyl ether (BDE 47), CB 138, and CB
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153, and the sum of each group (ΣPCB, ΣPBDE, ΣDDT, Σchlordane, and ΣHCH), were used in
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statistical analysis. However, we did not report the results regarding β-HCH, because β-HCH
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occupies a predominant portion of ΣHCH, and the statistical results were almost the same. For POPs
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that were detected ≥ 75% of the population, a proxy value, i.e., LOQ divided by square root 2, was
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used to replace the non-detects.35 For HCB that was detected in =60%, statistical analysis
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was conducted with detected values only.31, 36
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Because of a possibility of a decline of adipokine concentration over a duration of breast
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feeding, the dataset with breast milk at 15 days postpartum only (n=74) was chosen to minimize
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potential variation of adipokine levels by duration of lactation. Then, four extreme outliers with the
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values > 1.5 fold interquartile range were identified and excluded from each dataset. Thus, final
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dataset for statistical analysis was limited to 70 subjects. Multiple linear regression analysis was
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conducted following natural log transformation to minimize the effect of a highly right-skewed data.
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To determine the associations between POPs and adipokine measurements, age (continuous), infant
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sex and pre-pregnancy BMI (categorical) were included as explanatory variables in the models. We
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also evaluated group difference according to the quartiles of the serum POPs levels with ANCOVA
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to consider the possible non-monotonic dose-responses.9, 37, 38 Percent changes in adipokine levels
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expected for an interquartile range (IQR) increase of a given chemical concentration were back-
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calculated from the regression coefficients for each target chemical.39 Statistical significance was
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defined at p < 0.05. SAS 9.3 (SAS Institute, Cary, NC, USA) was used for statistical analyses.
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3. Results
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Characteristics and POPs exposure level of study population
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The participating pregnant women were generally in their early 30s (median 33 years of age)
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and approximately a half of the participating women were primiparae (Table 1). About 70% of the
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participating women gave birth to babies by normal spontaneous vaginal delivery. Before pregnancy,
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most women (61%) were within normal BMI range with a median of 20.5 kg/m2.
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Of the 57 POPs analyzed, nine chemicals were detected in > 60% of samples (Table 2). p,p’-
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DDE and CB 153 was detected in > 90% of the serum samples. ΣHCH, ΣDDT, and Σchlordane were
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detected at a median of 9.20 (IQR of 5.82-13.3), 67.3 (47.8-92.7), and 3.8 ng/g lw (2.77-5.06),
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respectively. Median serum concentrations of ΣPCB and ΣPBDE were determined at 27.3 ng/g lw
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(16.2-34.7) and 2.20 ng/g lw (1.49-4.93), respectively.
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Leptin and adiponectin concentrations in breast milk
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Leptin was detected with a median of 17.9 ng/L (range ND-3356 ng/L) in the breast milk
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samples analyzed in the present study (n=82). For adiponectin, the median breast milk level was 16.5
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µg/L (range 2.6-91.0 µg/L, Table 3). A significant negative correlation was observed between pre-
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pregnancy BMI and breast milk leptin levels (Spearman’s rho=-0.24, n=77, p 60%
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were shown here. ΣPCB is the sum of all target PCB congeners (CB 18, 28, 33, 44, 52, 70, 101, 105,
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118, 128, 138, 153, 170, 180, 187, 194, 195, 199 and 206), and ΣPBDE is the sum of all target PBDE
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congeners (BDE 17, 28, 47, 49, 66, 71, 77, 85, 99, 100, 119, 126, 138, 153, 154, 156, 183, 184 and
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191). ΣHCH included α-, β-, γ- and δ-HCH, ΣDDTs included p,p′-DDE, o,p′-DDE, p,p′-
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Dichlorodiphenyldichloroethane (DDD), o,p′-DDD, p,p′-DDT and o,p′-DDT, and Σchlordanes
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included oxychlordane, trans-chlordane, cis-chlordane, trans-Nonachlordane and cis-nonachlordane.
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Table 3. Concentrations of leptin and adiponectin in skimmed breast milk samples in Korea and other countries
Country
Study year
n
Sampling time (months)
GM
Median
AM±STD
Range
GM
Median
AM±STD
Range
Leptin (ng/L)
Adiponectin (µg/L)
Reference
Germany
20002001
674
1-2
164
-
281±381
ND-4119
10.55
-
12.8±10.1
-
Weyermann et al. 2007
Spain
2003
28
1
-
-
156±39
ND-853
-
-
-
-
Miralles et al., 2006
19982005
30
1-13
-
400
-
ND-2200
-
16.6
-
6.9-80.4
USA
199
1-8
-
-
-
-
-
17.7
-
4.2-87.9
37
1
-
-
-
-
-
11.7
-
6.2-39.8
Martin et al. 2006
45
1-7
-
-
-
-
-
-
25.6±8.4
-
Woo et al. 2009
277
1-7
-
-
-
-
-
-
23.7±8.6
-
Woo et al. 2009
72
1
-
-
200±30
-
-
-
22.0±0.6
-
72
3
-
-
100±10
-
-
-
20.5±0.6
-
72
6
-
-
100±20
-
-
-
21.4±0.8
-
Mexico USA Mexico
Czech republic
19982005 19982005 19982005 20062008
Martin et al. 2006
Bronsky et al., 2011
Italy
-
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