Journal of Perinatology (2008) 28, 258–263 r 2008 Nature Publishing Group All rights reserved. 0743-8346/08 $30 www.nature.com/jp ORIGINAL ARTICLE Maternal bisphenol-A levels at delivery: a looming problem? V Padmanabhan1,2, K Siefert3, S Ransom4, T Johnson2, J Pinkerton2, L Anderson2, L Tao5 and K Kannan5 1Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA; 2Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; 3School of Social Work, University of Michigan, Ann Arbor, MI, USA; 4University of North Texas Health Science Center, Fort Worth, TX, USA and 5Wadsworth Center, New York State Department of Health and Department of Environmental Health Sciences, School of Public Health, State University of New York, Empire State Plaza, Albany, NY, USA estrogenic activity in landfill leachates.4 BPA is used in the Objective: The objective was to determine whether bisphenol-A (BPA) is manufacture of epoxy resins and polycarbonate plastics that are found in maternal circulation of pregnant women in the US population and used in dental fillings, plastic food and commercially available is related to gestational length and birth weight. water containers, baby bottles and food wrap, as well as in the Method: Circulating levels of BPA were quantified by high performance lining of beverage and food cans, thus presenting a large number 5–8 liquid chromatography-tandem mass spectrometry at delivery in 40 of possibilities for human exposure. Small quantities of BPA 9–11 southeastern Michigan mothers and correlated with gestational length have been detected in river water and sediments, and more 12 and birth weight of offspring. recently, in indoor air and dust. There is long-standing evidence that BPA can bind to the Result: Maternal levels of unconjugated BPA ranged between 0.5 and estrogen receptor and induce estrogen receptor-mediated gene 22.3 ng mlÀ1 in southeastern Michigan mothers. There was no expression13–15 although recent evidence suggests that BPA can correlation between BPA concentrations and gestational length or birth also bind to the thyroid receptor and affect thyroid hormone weight of offspring. signaling.16 BPA does not bind to plasma estrogen-binding proteins Conclusion: This is the first study to document measurable levels of BPA that normally limit the bioavailability of estradiol.17 Accumulation in maternal blood of the US population. Long-term follow-up studies of of BPA appears to occur in pregnant adult female subjects,3 more offspring are needed to validate or refute concerns over human fetal likely in fat and other tissues.7,18,19 BPA is particularly potent exposure to synthetic exogenous steroids. during fetal and neonatal development as the liver has limited Journal of Perinatology (2008) 28, 258–263; doi:10.1038/sj.jp.7211913; capacity to deactivate BPA in fetuses and newborns, especially in published online 14 February 2008 humans.17 It is becoming increasingly evident that inappropriate exposure Keywords: endocrine disrupting chemicals; pregnancy to sex steroids/steroid mimics has an impact on fetal growth and organ differentiation.20,21 For instance, fetal exposure to excess Introduction prenatal testosterone, an estrogen precursor, from days 30 to 90 of gestation (term 147 days), resulted in intrauterine growth Environmental endocrine disrupting chemicals are hormonally restriction (IUGR) and low birth weight offspring in sheep.22–24 On active compounds that interfere with the normal functioning of the 1 the contrary, prenatal treatment with dihydrotestosterone, a endocrine system. Exposure to environmental endocrine nonaromatizable androgen, did not reduce birth weight disrupting chemicals appears to be a significant and contentious 2 (Padmanabhan V, unpublished). Combined with the fact that public health issue. In recent years, attention has focused on in utero exposure to diethylstilbestrol (DES), an estrogenic agent, human exposure to bisphenol-A (BPA), a widely used industrial is associated with IUGR25 and prenatal BPA treatment leads to low plasticizer. Every year, over six billion pounds of BPA are used in birth weight offspring in sheep,26 these findings support the the manufacture of epoxy resins and polycarbonate plastics used in 3 hypothesis that increased estrogen signaling during inopportune a wide variety of domestic products. BPA accounts for most of times of fetal development can lead to IUGR. As BPA can bind 13,15 Correspondence: Professor V Padmanabhan, Pediatrics and Communicable Diseases, estrogen receptors, continued exposure to this compound University of Michigan, Room 1109, 300 North Ingalls Building, Ann Arbor, MI 48109-0404, during gestation is likely to have an impact on the developmental USA. trajectory of the fetus. E-mail: [email protected] Received 9 July 2007; revised 9 November 2007; accepted 3 December 2007; published online The widespread presence of BPA and increased susceptibility of 14 February 2008 the developing fetus to estrogen mimics necessitates comprehensive Maternal BPA levels in the US population V Padmanabhan et al 259 evaluation of exposure levels in pregnant women. Studies from and (2) recovery of BPA from blood, plasma and serum. These industrialized nations such as Germany and Japan, suggest that validations were carried out using sheep blood. measurable levels of BPA are found in maternal circulation.27–29 Similar studies have not been undertaken with pregnant women in Transfer with glass or Pasteur pipettes the United States. The only available information in the US To determine whether transferring plasma with plastic transfer population relates to urinary levels of BPA metabolites in the pipettes resulted in leaching of BPA into the sample, 10 ml blood general population.30,31 A recent CDC survey found higher BPA samples were collected by venipuncture from three female sheep metabolites (inactive) in the urine of 6 to 9-year-old girls with and transferred to EDTA tubes containing 112 ml of a stock solution body mass index (BMI) <85th percentile compared to those with of BPA (stock concentration: 0.45 mg mlÀ1; Aldrich #239658, BMI >85th percentile.32 Urinary measures provide an index as to 99 þ % pure; Sigma-Aldrich, St Louis, MO, USA) in absolute whether humans are exposed to BPA but do not indicate ethanol (Aaper Alcohol and Chemical Co, Shelbyville, KY, USA) to circulating levels. Assuming environmental exposure levels are yield a final concentration of 5 ng mlÀ1. Syringes and needles used similar, high levels of urinary metabolites would imply lower for procurement of blood samples were from Becton Dickinson circulating levels of parent compound and vice versa. Considering (Franklin Lakes, NJ, USA). The blood was then split in half using the susceptibility of fetuses to endocrine-disrupting chemicals, it is glass pipettes and samples were spun down. The resulting plasma important to gain an understanding of maternal circulating levels was aspirated using either a glass (Fisher #13-678-7C flint glass of BPA in the United States. Therefore, the objective of this study pipettes; Fisher Scientific, Pittsburgh, PA, USA) or polyethylene was to determine if BPA is found in the circulation of pregnant plastic (Fisher #12-711-7 polyethylene) pipette into glass tubes and women in the US population. stored until BPA measurement. Recovery of BPA from whole blood, plasma or serum Methods Blood samples were collected from female sheep by venipuncture Maternal sample collection into glass tubes to which 5, 10 and 20 ng mlÀ1 BPA was added. The study involved the use of maternal blood samples collected at Five milliliters from each BPA-spiked sample were then transferred the time of delivery from 40 pregnant mothers as part of standard to; (1) a heparinized glass tube and spun down for plasma; (2) a clinical hospital procedures. Blood samples were collected glass tube with no additives, allowed to clot for 2 h at room beginning 4 August 2006 and continued until 2 November 2006 temperature and then spun down to recover serum and (3) a glass from pregnant women delivering at the University of Michigan tube with no additives added, left as whole blood. Blood, plasma Hospital in Ann Arbor, MI, USA. Maternal blood samples were and serum samples were frozen and stored at À20 1C. The initial drawn by venipuncture directly into a vacutainer tube, which BPA spiking process took less than a minute to avoid complications contained EDTA. After procuring blood samples from the hospital, from clotting. Recovery of BPA from whole blood, serum and samples were placed in a cooler and transported to the research plasma was then compared. laboratory. Maternal blood samples were centrifuged at 3000 r.p.m. and plasma was transferred to a glass tube and placed in À80 1C BPA measurements freezer until time for BPA measurement. Human blood samples BPA levels in samples were quantified using a high-performance were handled according to the guidelines for safe laboratory liquid chromatography (HPLC) coupled with API 2000 electrospray practices. Identical collection and processing protocol were followed triple-quadruple mass spectrometer (ESI-MS/MS). Briefly, a 0.8 to for all subjects. Information on first visitFweight, height, weight 1 ml aliquot of blood was transferred into a 15 ml polypropylene at time of delivery, gestational length and birth weightFwere tube and 10 ml of 1-ppm butylphenol was added as an internal obtained for each subject. Confidentiality was maintained by standard. Samples were extracted twice with 5 ml of ethyl ether by substituting a subject identification number in place of name and shaking in an orbital shaker for 30 min. The ethyl ether patient identification and thereby eliminating any link to the (2 Â 5 ml) was pooled and evaporated to dryness under a gentle patient. All materials collected from a given subject and computer stream of nitrogen. The sample extract was then reconstituted with data were identified with this number. The Institutional Review 0.5 ml of methanol. Standards of BPA were prepared in methanol Board of the University of Michigan reviewed and approved the at concentrations ranging from 0.2 to 100 ng mlÀ1 for calibration.