{"paper_id":"57b1d375-3113-4f40-b9eb-c06a5d012637","body_text":"S H O R T R E P O R T Open Access\nElevated levels of whole blood nickel in a group\nof Sri Lankan women with endometriosis: a case\ncontrol study\nNalinda Silva 1*, Hemantha Senanayake 2 and Vajira Waduge 3\nAbstract\nBackground: Endometriosis is characterized by the persistence of endometrial tissue in ectopic sites outside the\nuterine cavity. Presence of nickel, cadmium and lead in ectopic endometrial tissue has been reported previously.\nWhile any association between blood levels of nickel and endometriosis is yet to be described in literature,\nconflicting reports are available with regards to cadmium and lead levels in blood and urine.\nFindings: In fifty patients with endometriosis and fifty age-matched controls confirmed by laparoscopy or\nlaparotomy, whole blood samples were collected and digested using supra pure 65% HNO 3. Whole blood levels of\nnickel and lead were measured using Total Reflection X-ray Fluorescence (TXRF) while cadmium levels were\nevaluated using graphite furnace atomic absorption spectroscopy (GFASS). Women with endometriosis had\nsignificantly higher ( P=0.016) geometric mean (95% CI) whole blood nickel levels [2.6(1.9-3.3) μg/L] as compared to\nwomen without endometriosis [0.8 (0.7-0.9) μg/L]. Whole blood levels of cadmium and lead were similar between\nthe two groups.\nConclusions: Although women with endometriosis in this study population had higher levels of nickel in whole\nblood compared to controls, whether nickel could be considered as an aetiological factor in endometriosis remains\ninconclusive in view of the smaller sample that was evaluated.\nKeywords: Endometriosis, Metalloestrogens, Nickel\nFindings\nBackground\nEndometriosis is an estrogen dependent chronic inflam-\nmatory disease characterized by the survival of endomet-\nrial tissue outside the uterine cavity [1]. Endometriosis\nwhich affects an estimated 6-10% of women of the repro-\nductive age group, is a considerable burden to affected\nwomen, their families and healthcare systems [2].\nAmong the etiological factors that promote the survival\nof ectopic tissue, estrogenic compounds that promote pro-\ntracted continuation these tissues are gaining prominence\nsupported by persuasive evidence from in vitro , in vivo\nand human studies [3].\nCompelling molecular [4], in vitro[5] and in vivo[6] evi-\ndence for the ability of metal ions to activate the estrogen\nreceptor [7] has defined a group of xenoestrogens termed\nmetalloestrogens [8]. While evidence for the purported\nestrogenicity of cadmium are in plenty, estrogenic proper-\nties of other metals such as nickel have also been demon-\nstrated by some research groups [9].\nIn vivo experiments by Krugner-Higby et al. [10] have\ndemonstrated that the occurrence of endometriosis was\nhigher in six female rhesus monkeys exposed to a daily\nintake of 5 to 10 mg/Kg of lead as compared to a control\ngroup (n= 4) given lead-free water over a period of 19\nyears, but the difference was not statistically significant,\nprobably due to the small sample size [10].\nWe have previously demonstrated the presence of cad-\nmium, lead and nickel in ectopic endometrial tissue in a\ngroup of Sri Lankan women [11]. Heavy metals enter the\nhuman body either by ingestion or inhalation and are sub-\nsequently transferred to the vascular compartment (blood)\n[12]. Metals derived from the circulation get deposited in\nthe tissues by various mechanisms [13]. We hypothesized\n* Correspondence: yasanga@sjp.ac.lk\n1Lecturer in Physiology, Faculty of Medical Sciences University of Sri\nJayewardenepura, Nugegoda, Sri Lanka\nFull list of author information is available at the end of the article\n© 2013 Silva et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative\nCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and\nreproduction in any medium, provided the original work is properly cited.\nSilva et al. BMC Research Notes 2013, 6:13\nhttp://www.biomedcentral.com/1756-0500/6/13\n\nthe hematogenous route (blood) as a possible source of\nmetals in the ectopic endometrial tissue.\nThe present study was conducted with the objective of\nexploring further the association between endometriosis\nand whole blood levels of cadmium, lead and nickel in the\nsame group of women with endometriosis who were pre-\nviously studied. We compared the whole blood levels of\nthe cadmium, lead and nickel in women having endomet-\nriosis (in whom these metals were detected in ectopic\nendometrial tissue -cases) with the blood levels of same\nmetals in women who had no evidence of endometriosis\n(controls).\nMethods\nThis case control study was conducted among women of\nthe reproductive age group, at the Professorial Gynecology\nUnit of the National Hospital, Colombo, Sri Lanka.\nPatients awaiting elective laparotomy or laparoscopy for\ndiagnostic and/or treatment purposes were included.\nThose who were diagnosed visually as having endome-\ntriosis subsequent to laparotomy or laparoscopy were\nselected as cases (n=50). Apart from the ectopic endomet-\nrial tissue sample that was collected as described in the\nprevious study [11], simultaneous venous blood sample\nwas collected from each case. Women matched for age in\nwhom endometriosis had been excluded by laparoscopy\nor laparotomy were recruited as controls (n=50). Indica-\ntions for laparoscopy or laparotomy in controls were sub-\nfertility, dysmenorrhea, chronic pelvic pain or detection of\nan ovarian mass in ultrasound scan.\nA sample of venous blood was collected from all partici-\npants during intravenous cannulation at the time of induc-\ntion of anesthesia into polypropelene tubes containing\nethylene diamine tetraacetic acid (EDTA). Pre-operatively,\ninformed written consent was obtained from all the parti-\ncipants. All the blood samples were stored in a −20°C\nfreezer until analysis.\nWhole blood samples were digested using a protocol\ndescribed previously [14] with some modifications. Briefly,\nmatched blood of cases and controls were allowed to\nreach the room temperature. Then whole blood samples\nwere transferred to pre-treated, acid washed glass beakers.\nThe wet weight of each sample was determined using a\nchemical balance. Each sample was prepared in duplicate.\nAll the blood samples were digested using 65% supra pure\nNitric acid (HNO\n3, Merck, USA) while maintaining a uni-\nform temperature. The final solution was made in 2mL of\n5% HNO 3 that was prepared by diluting the 65% supra\npure HNO3 with double distilled deionized water.\nThe total-reflection X-ray fluorescence (TXRF) machine\navailable at the Atomic Energy Authority (AEA) of Sri\nLanka was utilized to measure metals apart from cad-\nmium. TXRF is a multi element analysis technique [15]\ncapable of detecting an array of elements at detection\nlevels of picograms per liter (pg/L) [15]. An internal stand-\nard, Gallium, allows quantification of metals using the\nAxil software. However, the TXRF that was used had an\ninherent weakness in measuring cadmium since it had a\nMolybdenum x-ray tube. Therefore, we used atomic ab-\nsorption spectroscopy (model GBC 933AA) together with\na graphite furnace (model GBC GF 3000) available at the\nInstitute of Fundamental Studies (IFS), Kandy, Sri Lanka\nto estimate cadmium levels as described elsewhere[16].\nBoth AEA and IFS [16] are national level apex institutions\nin Sri Lanka that have international certifications for trace\nelement analysis where regular quality assurance pro-\ngrams are conducted.\nQuality control and validation were performed using\nreference material supplied by the International Atomic\nEnergy Authority (IAEA-A-13), Seronorm\n™ trace ele-\nments in whole blood levels 1 (MR 4210) and National In-\nstitute of Standards & Technology (NIST) Gaithersburg,\nUSA water sample with trace elements (SRM 1643e). For\n96% of the determinations, repeatability error did not ex-\nceed 10%. The detection limits for nickel, lead and cad-\nmium in whole blood were as follows 0.05, 1.0, 0.01 μg/\nL. The precision for nickel, lead and cadmium in the range of\nthe samples analyzed in this study was + 2, 3 and 6%,\nrespectively.\nSPSS version 13 for Windows was used for statistical\nanalysis. Log transformation of metal levels was done and\nmeans were compared using t-test.\nEthical clearance was obtained from the Ethical Review\nCommittees of the Faculty of Medical Sciences, University\nof Sri Jayewardenepura and the National Hospital of\nSri Lanka.\nResults and discussion\nMean (±SD) age in cases and controls were 33.0 (±5 .4)\nand 32.7(±5.4) years respectively. Cases and controls were\nsimilar in body mass index while none of the women who\nparticipated in the study were current smokers. Other\ndemographic, biological and dietary characteristics of this\ngroup of women with endometriosis have been described\npreviously [17].\nCadmium, lead and nickel were detected in whole blood\nof all the participants. The whole blood nickel levels in\ncases were significantly higher compared to controls.\nCases had lower whole blood cadmium levels and higher\nTable 1 Levels of metalloestrogens in whole blood ( μg/L)\nof cases and controls\nMetalloestrogen Cases (n=50) Controls (n=50) P value†\nNickel 2.6 (1.9-3.3) 0.8 (0.7-0.9) 0.016\nLead 11.0 (8.6-13.3) 6.9 (5.7-8.0) 0.389\nCadmium 0.7 (0.7-0.9) 0.8 (0.6-1.0) 0.423\nData expressed as geometric mean (95% CI).\n† t-test between blood levels of cases and controls.\nSilva et al. BMC Research Notes 2013, 6:13 Page 2 of 4\nhttp://www.biomedcentral.com/1756-0500/6/13\n\nlead levels as compared to controls; however these diffe-\nrences were not statistically significant (T able 1).\nEnvironmental pollution has been identified as a poten-\ntial cause for the increase in the prevalence of endome-\ntriosis following industrialization [18]. Heavy metals are\ngaining prominence as pollutants that could affect human\nhealth profoundly, especially in the face of escalating levels\nof environmental contamination [19]. Women are suscep-\ntible to heavy metal toxicity due to differences in kinetics,\nmode of action, susceptibility and exposure [20,21].\nThe demonstration of higher levels of nickel in whole\nblood in women with endometriosis as compared to con-\ntrols in our study is a novel finding. Hitherto, there is no\npublished literature that has investigated the nexus bet-\nween nickel and endometriosis. The whole blood nickel\nlevels we are reporting are comparable to those reported\nfrom women in the general population in different coun-\ntries [22,23]. Thus the levels we have detected are essen-\ntially non toxic levels although cases had higher nickel\nlevels which may have contributed towards the presence\nof nickel in ectopic endometrial tissue.\nNickel similar to other heavy metals, primarily enters\nthe human body either by ingestion or inhalation [24]. In\naddition, nickel is a common metal found in accessories\nfrequently used by women [20] and its absorption via in-\ntact or damaged skin has been demonstrated [25]. Martin\net al. [26] assessed the estrogenic potency of metals using\nthe 50% effective concentration (EC\n50)o fd i f f e r e n tm e t a l s\nas determined from dose –response curves performed in\nMCF-7 cells transiently transfected with the luciferase re-\nporter construct. They found that nickel had a relative\npotency of 1.0 compared to estradiol [26] implying that\nnickel is a potent metalloestrogen.\nHumans studies that attempted to elicit the association\nbetween cadmium and lead and endometriosis have yiel-\nded conflicting results. In a population based study in the\nUnited States, high blood concentrations of cadmium\nwere associated with endometriosis [27]; while a study\ndone in Belgium reported similar levels of cadmium and\nlead in blood in both cases and controls [28]. Urinary\ncadmium concentrations were similar in cases with endo-\nmetriosis and controls [28,29]. The results of this study\nare in agreement with the previously published research\napart from that of Jackson et al. In the study conducted by\nJackson and co workers that probed the association bet-\nween cadmium and endometriosis, a considerable number\nof women with endometriosis were smokers [27]. In con-\ntrast, in our cohort of women none were current smokers.\nIn conclusion in this group of women with endometriosis\nhigher blood levels of nickel were observed. Whether nickel\nc o u l db ec o n s i d e r e da sa ne t i o logical factor in endometri-\nosis remains inconclusive considering two factors, namely\nthe small sample size evaluated in this study and the ab-\nsence of a previous report of such a link. However based on\nthe available scientific evidence for occupational and envir-\nonmental exposure of women to metals including nickel,\nfuture research would prove to be invaluable in further ex-\nploring the association between nickel and endometriosis.\nAbbreviations\nAEA: Atomic Energy Authority; EDTA: Ethylene diamine tetraacetic acid;\nIFS: Institute of Fundamental Studies; IAEA: International Atomic Energy\nAuthority; NIST: National Institute of Standards & Technology; TXRF: Total-\nreflection X-ray fluorescence.\nCompeting interests\nAuthors declare that they have no competing interests.\nAuthors’ contributions\nNS carried out the sample collection, analysis and drafted the manuscript. HS\nidentified the study participants and carried out the surgical procedures. VW\nassisted in the TXRF analysis of the samples and interpreted the data. All\nauthors read and approved the final manuscript.\nAcknowledgements\nWe wish to acknowledge the staff at the Operating Theater, National\nHospital, Sri Lanka for the support in sample collection. Dr. M.S.M Iqbal and\nMs. D Aluthpatabendi at the Institute of Fundamental Studies, Kandy, Sri\nLanka are kindly acknowledged for assistance in cadmium analysis. This\nstudy was supported by the University of Sri Jayewardenepura (grant ASP/6/\nRe/2008/06), National Coordinating Committee on Reproductive Health\nResearch of Sri Lanka and by the University of Alabama at Birmingham\nInternational Training and Research in Environmental and Occupational\nHealth program, Grant Number 5 D43 TW05750, from the National Institute\nof Health-Fogarty International Center (NIH-FIC). The content is solely the\nresponsibility of the authors and do not necessarily represent the official\nviews of the NIH-FIC.\nAuthor details\n1Lecturer in Physiology, Faculty of Medical Sciences University of Sri\nJayewardenepura, Nugegoda, Sri Lanka. 2Professor in Obstetrics and\nGynecology, Department of Obstetrics and Gynecology, Faculty of Medicine,\nUniversity of Colombo, Colombo, Sri Lanka. 3Head of Life Sciences Division,\nAtomic Energy Authority, Colombo, Sri Lanka.\nReceived: 21 November 2012 Accepted: 11 January 2013\nPublished: 14 January 2013\nReferences\n1. Donnez J: Endometriosis: enigmatic in the pathogenesis and\ncontroversial in its therapy. Fertil Steril 2012, 98(3):509–510.\n2. Markovic M, Manderson L, Warren N: Endurance and contest: women's\nnarratives of endometriosis. Health (London) 2008, 12:349–367.\n3. Garai J, Molnar V, Varga T, Koppan M, Torok A, Bodis J: Endometriosis:\nharmful survival of an ectopic tissue. Front Biosci 2006, 11:595–619.\n4. Stoica A, Katzenellenbogen BS, Martin MB: Activation of estrogen receptor-\nalpha by the heavy metal cadmium. Mol Endocrinol 2000, 14(4):545–553.\n5. Garcia-Morales P, Aceda M, Kenney N, Kims N, Salomon DS, Gottardisn MM,\nSolomonn HB, Shollern PF, Jordan C, Martin MB: Effect of cadmium on\nestrogen receptor levels and estrogen-induced responses in human\nbreast cancer cells. J Biol Chem 1994, 269(24):16896–16901.\n6. Johnson MD, Kenny N, Stoica A, Hilakivi-Clarke L, Singh B, Chepko G, Clarke\nR, Sholler PF, Lirio AA, Foss C, : Cadmium mimics the in vivo effects of\nestrogen in the uterus and mammary glands. Nat Med 2003,\n9(8):1081–1084.\n7. Safe S: Cadmium disguise dupes the estrogen receptor. Nat Med 2003,\n9(8):1000–1001.\n8. Darbre PD: Metalloestrogens: an emerging class of inorganic\nxenoestrogens with potential to add to the oestrogenic burden of the\nhuman breast. J Appl Toxicol 2006, 26(191):197.\n9. Auino NB, Sevigny MB, Sabangan J, Louie MC: The role of cadmium and\nnickel in estrogen receptor signaling and breast cancer:metalloestrogens\nor not? J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 2012,\n30(3):189–224.\nSilva et al. BMC Research Notes 2013, 6:13 Page 3 of 4\nhttp://www.biomedcentral.com/1756-0500/6/13\n\n10. Krugner-Higby L, Rosenstein A, Handschke L, Luck M, Laughlin NK, Mahvi D,\nGendron A: Inguinal hernias, endometriosis, and other adverse outcomes\nin rhesus monkeys following lead exposure. Neurotoxicol Teratol 2003,\n25:561–570.\n11. Silva N, Senanayake H, Peiris-John R, Wickremasinghe R, Sathiakumar N,\nWaduge V: Presence of metalloestrogens in ectopic endometrial tissue.\nJ Pharmaceut Biomedsci 2012, 24(24):1–5.\n12. Schweinberg F, Von-Karsa L: Heavy metal concentration in humans.\nComp Biochem Physiol 1990, 95c(2):117–123.\n13. Bridges CC, Zalups RK: Molecular and ionic mimicry and the transport of\ntoxic metals. Toxicol Appl Pharmacol 2005, 204:274–308.\n14. Nasiadek M, Krawczyk T, Sapotal A: Tissue levels of cadmium and trace\nelements in patients with myoma and uterine cancer. Hum Exp Toxicol\n2005, 24:623–630.\n15. Wobrauschek P: Total reflection x-ray fluorescence analysis —a review.\nX Ray Spectrom 2007, 36:289–300.\n16. Bandara JMRS, Senevirathna DMAN, Dasanayake DMRSB, Herath V, Bandara\nJMRP, Abeysekara T, Rajapaksha KH: Chronic renal failure among farm\nfamilies in cascade irrigation systems in Sri Lanka associated with\nelevated dietary cadmium levels in rice and freshwater fish (Tilapia).\nEnviron Geochem Health 2008, 30:465–478.\n17. Silva N, Senanayake H, Peiris-John R, Wickremasinghe R: Demographic,\nbiological and dietary characteristics associated with endometriosis in a\ngroup of Sri Lankan women. Sri Lanka J Obstet Gynecol 2011, 33:91–97.\n18. Heilier JF, Donnez J, Nackersd F, Rousseaud R, Verougstraetea V,\nRosenkranza K, Donnezc O, Grandjeanc F, Lisona D, Tongletd R:\nEnvironmental and host-associated risk factors in endometriosis and\ndeep endometriotic nodules: a matched case –control study. Environ Res\n2007, 103:121–129.\n19. World Health Organization: Cadmium.I n Environmental Health Criteria.\nVolume 134 . Geneva: IPCS -International Program on Chemical Safety; 1992.\n20. Butter ME: Are women more vulnerable to environmental pollution?\nJ Hum Ecol 2006, 20(3):221–226.\n21. Vahter M, A·kessona A, Lide ’n C, Ceccatellia S, Berglunda M: Gender\ndifferences in the disposition and toxicity of metals. Environ Res 2007,\n104:85–95.\n22. Ikeda M, Ohashi F, Fukui Y, Sakuragi S, Moriguchi J: Cadmium, chromium,\nlead, manganese and nickel concentrations in blood of women in non-\npolluted areas in Japan, as determined by inductively coupled plasma-\nsector field-mass spectrometry. Int Arch Occup Environ Health 2011,\n84(2):139–150.\n23. Nunes JA, Batistaa BL, Rodriguesa JL, Caldasb NM, Netob JAG, Barbosa F:\nSimple method based on ICP-MS for estimation of background levels of\narsenic, cadmium, copper, manganese, nickel, lead, and selenium in\nblood of the Brazilian population. J Toxicol Environ Health A 2010,\n73:878–887.\n24. Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological\nprofile for Nickel .: ; 2005.\n25. Filon FL, D ’Agostin F, Crosera M, Adami G, Bovenzi M, Maina G: In vitro\nabsorption of metal powders through intact and damaged human skin.\nToxicol Vitr 2009, 23:574–579.\n26. Martin MB, Reiter R, Pham T, Avellet YR, Camara J, Lahm M, Pentecost E,\nPrathap K, Gilmore BA, Diverkar S, : Estrogen-like activity of metals in\nMcf-7 breast cancer cells. Endocrinology 2003, 144(6):2425–2436.\n27. Jackson LW, Zullo MD, Goldberg JM: The association between heavy\nmetals, endometriosis and uterine myomas among premenopausal\nwomen: National Health and Nutrition Examination Survey 1999 –2002.\nHum Reprod 2008, 23(3):679–687.\n28. Heilier JF, Donnez J, Verougstraete V, Donnez O, Grandjean F, Haufroid V,\nNackers F, Lison D: Cadmium, lead and endometriosis. Int Arch Occup\nEnviron Health 2006, 80:149–153.\n29. Itoh H, Iwasaki M, Nakajima Y, Endo Y, Hanaoka T, Sasaki H, Tanaka T, Yang\nB, Tsugane S: A case –control study of the association between urinary\ncadmium concentration and endometriosis in infertile Japanese women.\nSci Total Environ 2008, 402:171–175.\ndoi:10.1186/1756-0500-6-13\nCite this article as: Silva et al. : Elevated levels of whole blood nickel in a\ngroup of Sri Lankan women with endometriosis: a case control study.\nBMC Research Notes 2013 6:13.\nSubmit your next manuscript to BioMed Central\nand take full advantage of: \n• Convenient online submission\n• Thorough peer review\n• No space constraints or color figure charges\n• Immediate publication on acceptance\n• Inclusion in PubMed, CAS, Scopus and Google Scholar\n• Research which is freely available for redistribution\nSubmit your manuscript at \nwww.biomedcentral.com/submit\nSilva et al. BMC Research Notes 2013, 6:13 Page 4 of 4\nhttp://www.biomedcentral.com/1756-0500/6/13","source_license":"CC0","license_restricted":false}