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ARTICLE IN PRESS
JTEMB-25514; No. of Pages 7
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Epidemiology
Biomarkers of iron status and trace elements in welders夽 Dag G. Ellingsen a,∗ , Maxim Chashchin b,1 , Balazs Berlinger a , Tobias Konz c , Evgenij Zibarev b , Jan Aaseth d , Valery Chashchin b,e , Yngvar Thomassen a a
National Institute of Occupational Health, P.O. Box 8149 Dep, N-0033 Oslo, Norway Northwest Public Health Research Centre, 2-Sovetskaya 4, St. Petersburg 191036, Russia Department of Physical and Analytical Chemistry of the University of Oviedo, ES-33006, Spain d Department of Medicine, Innlandet Hospital Trust, N-2226 Kongsvinger, Norway e North-Western State Medical University, St. Petersburg 191015, Russia b c
a r t i c l e
i n f o
Article history: Received 20 December 2013 Accepted 3 March 2014 Keywords: Manganese Iron Carbohydrate deficient transferrin Hepcidin Ferritin
a b s t r a c t Iron status was studied in 137 welders exposed to a geometric mean (GM) air concentration of 214 g/m3 (range 1–3230) of manganese (Mn), in 137 referents and in 34 former welders. The GM concentrations of S-ferritin were 119 (3–1498), 112 (9–1277) and 98 (12–989) g/L (p = 0.24) in the three groups, respectively. Also the GM concentrations of S-hepcidin were not significantly different between the groups (8.4 g/L (2.8–117); 6.6 g/L (1.8–100); 6.5 g/L (1.2–22)) (p = 0.22). Multiple linear regression analysis including all welders and referents showed an increase in the concentration of S-ferritin associated with having serum carbohydrate deficient transferrin (S-CDT) above the upper reference limit of ≥1.7%, indicating high alcohol consumption. Serum C-reactive protein was not associated with exposure as welders, but an association with S-ferritin was shown. The GM S-ferritin concentrations among all welders and referents with S-CDT ≥ 1.7% were 157 g/L (95% CI 113–218) as compared to 104 g/L (95% CI 94–116) (p = 0.02) in those with S-CDT < 1.7%. The GM concentrations of Mn in biological fluids were higher in the welders as compared to the referents, while S-Fe, S-Co and B-Co were statistically significantly lower. This could suggest a competitive inhibition from Mn on the uptake of Fe and Co. Increasing concentrations of S-CDT was associated with higher S-Mn, S-Fe and B-Co in the multiple linear regression analysis. The association between S-CDT and S-Fe remained when all subjects with high S-CDT (≥1.7%) were excluded, suggesting increased uptake of Fe even at lower alcohol consumption. © 2014 Published by Elsevier GmbH.
Introduction Potentially high manganese (Mn) content in welding fumes may be a risk factor for neurological disturbances in welders [1]. Welding fume consists of primary particles that are agglomerated into chainlike structures, containing chemically complex compounds such as KMnF3 , Fe3 O4 , MnFe2 O4 or K2 MnO4 [2]. The predominant particle sizes of welding aerosols are below 1 m in aerodynamic
夽 The study was supported by Grant Number W81XWH-05-1-0239 from the U.S. Department of Defense, United States Army Medical Research and Material Command. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the United States Department of Defense, United States Army Medical Research and Material Command. ∗ Corresponding author. Tel.: +47 23195377; fax: +47 23195377. E-mail address: [email protected] (D.G. Ellingsen). 1 Current address: City Centre of Occupational Health, St. Petersburg 191014, Russia.
diameter, thus the particles can easily penetrate into the deeper parts of the lung [3,4]. The welding fume content of iron (Fe) is substantially higher than Mn in both manual and gas metal arc welding [5]. Mn and Fe share several transporters in animals and humans [6]. Non-heme Fe(II) and Mn(II) are absorbed in duodenal enterocytes through the divalent metal transporter 1 (DMT1). The cellular export of Fe(II) through ferroportin has also been proposed for Mn(II) [7,8]. Fe(III) and Mn(III) bind to transferrin and are taken up by cells through high affinity transferrin receptor (TfR) mediated endocytosis. After acidification, both metals are transported out of the endosome through DMT1 [9]. Hepcidin is important for the Fe metabolism by regulating ferroportin, perhaps through Fe sensors in the hepatocytes [10]. Thus, there is no surprise that high exposure to Mn may alter Fe metabolism. Some studies have addressed this issue. Oral administration of high amounts of Mn resulted in lower serum Fe and hemoglobin concentrations in lambs and lower gastrointestinal
http://dx.doi.org/10.1016/j.jtemb.2014.03.004 0946-672X/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Ellingsen DG, et al. Biomarkers of iron status and trace elements in welders. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.03.004
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2
absorption of Fe in humans [11,12]. Brain Mn concentrations were higher in Mn injected rats both at increased and at decreased dietary Fe as compared to the administration of normal dietary Fe [6]. Mn metabolism may also be altered by Fe deficiency, as evidenced by significantly higher concentrations of Mn in whole blood of Fe deficient women [13]. It also appears that Mn can interact with basic regulatory systems of Fe metabolism, e.g. inhibition of mitochondrial aconitase, probably by insertion of Mn instead of the labile Fe into its 4Fe-4S cluster [14], or by activation of the Fe responsive protein (IRP), perhaps by depleting Fe with concomitant reduction of intracellular ferritin [15]. A recent study suggested that Mn can competitively inhibit gastrointestinal Fe absorption through DMT1 [16]. Welders are exposed to Mn and Fe by inhalation. Pulmonary epithelial cells and alveolar macrophages have on their surfaces all key transporters of Fe such as TfR, DMT1 and ferroportin, as well as intracellular ferritin [17]. However, the pattern of pulmonary DMT1 expression is not altered in Fe-deficient rats in contrast to what is observed in enterocytes [18]. In contrast, Fe-loaded rats had reduced transport of Mn across the air–blood barrier after intratracheal instillation [19]. It has long been recognized that high alcohol consumption can result in liver Fe accumulation [20,21]. Studies of mice and rats have shown that high alcohol consumption results in down-regulation of hepcidin expression with a concomitant increase in the expression of DMT1 and ferroportin in duodenum [22,23]. Hepatocytic oxidative stress induced by alcohol has been proposed as a cause for reduced hepcidin expression [24]. Whether this may have an impact on the transport of other divalent metal ions, is to our knowledge not known. The potential for systemic Fe overload has been little studied in welders. Case reports have suggested that body Fe overload may occur in welders’ siderosis [25]. However, welders had similar serum ferritin concentrations when compared to referents, although the concentrations were associated with duration of exposure [26]. That study also reported reduced concentrations of soluble TfR in serum. A recent study did not show increased serum ferritin concentrations during a one week follow up of long term exposed welders [5]. This study is part of a larger study of nervous system impairment in welders. We have previously reported that these welders are exposed to high amounts of Fe, but mostly in a non-bioaccessible form, while the bioaccessibility was substantially higher for Mn [27]. The aim of this study is to assess whether Fe status biomarkers are altered in welders, to assess the impact of welding fume exposure on selected trace element concentrations and to study the impact of biomarkers of alcohol consumption and systemic inflammation on the above biomarkers. Some of the participants were also examined six years previously and results from that follow-up are also presented.
Materials and methods One hundred and forty-nine welders and 178 potential referents were invited to participate in this cross-sectional study, of whom 12 welders and 41 potential referents refused participation. In all, 137 welders and 137 referents were included (participation rates 91.9% and 77.0%, respectively). The study was restricted to men with at least one year of employment at one facility producing heavy machinery or at two shipyards in St. Petersburg (Russia). The referents were turners/fitters. The predominant welding methods in use were manual metal arc and gas metal arc welding. Criteria for exclusion have been published [27]. Sixty-three welders and 65 referents had been examined around six years earlier [28].
Also 34 former welders (four females) that had been diagnosed with manganism caused by welding were examined, of whom 17 had been examined around six years earlier [28]. The latter subjects belonged to a group of 27 patients examined at that time, of whom two had died, seven had moved out of the region and were thus not invited to participate and one declined the request to participate. Additional 20 patients were identified from the patient records kept at the clinic of the Northwest Public Health Research Centre (NWPHRC). They were invited to participate, but three subjects declined. Thus, the participation rate among the eligible patients was 89.5%. Background data of all participants are presented (Table 1). The Norwegian Regional Ethical Committee for Medical Research (REK2) approved the study that was also approved by the Ethics Committee of the NWPHRC (St. Petersburg, Russia) and the Office of Research Protection, US Army Medical Research and Material Command (Fort Detrick, MD, USA). Participation was strictly voluntary and informed written consent was obtained from all participants. Collection of biological samples Welders and referents went through a structured interview and collection of biological samples at the occupational health clinics of the respective plants, while the patients were examined at the NWPHRC. First voided morning urine samples were collected in 10 mL Sarstedt® polypropylene (PP) tubes (Sarstedt AG, Nümbrecht, Germany). Blood samples were collected from the cubital vein between 8.30 and 9.30 the same morning after cleaning of the skin. Whole blood for the determination of trace elements was collected in 4 mL Lithium-Heparine Vacuette® vacutainers. Nine mL Vacuette® vacutainers without additives (Greiner Labortechnik GmbH, Austria) were used for the harvest of serum. After a wait of 30 min the tubes were centrifuged for 10 min at 1500 × g. Serum was pipetted into 1.5 mL Sarstedt® cryotubes (Sarstedt AG, Nümbrecht, Germany) for storage. The samples were kept frozen at −20 ◦ C until analysis. Ten referents refused to give a urine sample, while 14 referents declined blood sampling. Three former and one current welder refused to give urine samples. Due to lack of serum, trace elements in serum was determined in 124 welders, 33 former welders and 83 referents. For the same reason, serum hepcidin was measured in 45 welders, 19 former welders and 35 referents. Determination of biomarkers Serum ferritin (S-ferritin) was measured by an immunoturbimetric method using Advia 2400 CapillarysTM (Siemens Healthcare Diagnostics Inc. NY, US) at Fürst Medical Laboratory (Oslo, Norway). The method’s reproducibility was 2.3%. Serum hepcidin (S-hepcidin) was determined by a quantitative ELISA sandwich immunoassay (BlueGene Biotech, Shanghai) at the University of Oviedo (Spain). The assay used a microtiter plate pre-coated with a monoclonal antibody specific for hepcidin. Standards and samples were added to the plate wells and finally, a second polyclonal antibody conjugated to horseradish peroxidase was added. The color change in the plate was measured spectrophotometrically at 450 nm. The immunoassay was previously cross-validated. The method’s DL was 0.1 ng mL−1 and the intraassay CV was
ARTICLE IN PRESS
JTEMB-25514; No. of Pages 7
Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Epidemiology
Biomarkers of iron status and trace elements in welders夽 Dag G. Ellingsen a,∗ , Maxim Chashchin b,1 , Balazs Berlinger a , Tobias Konz c , Evgenij Zibarev b , Jan Aaseth d , Valery Chashchin b,e , Yngvar Thomassen a a
National Institute of Occupational Health, P.O. Box 8149 Dep, N-0033 Oslo, Norway Northwest Public Health Research Centre, 2-Sovetskaya 4, St. Petersburg 191036, Russia Department of Physical and Analytical Chemistry of the University of Oviedo, ES-33006, Spain d Department of Medicine, Innlandet Hospital Trust, N-2226 Kongsvinger, Norway e North-Western State Medical University, St. Petersburg 191015, Russia b c
a r t i c l e
i n f o
Article history: Received 20 December 2013 Accepted 3 March 2014 Keywords: Manganese Iron Carbohydrate deficient transferrin Hepcidin Ferritin
a b s t r a c t Iron status was studied in 137 welders exposed to a geometric mean (GM) air concentration of 214 g/m3 (range 1–3230) of manganese (Mn), in 137 referents and in 34 former welders. The GM concentrations of S-ferritin were 119 (3–1498), 112 (9–1277) and 98 (12–989) g/L (p = 0.24) in the three groups, respectively. Also the GM concentrations of S-hepcidin were not significantly different between the groups (8.4 g/L (2.8–117); 6.6 g/L (1.8–100); 6.5 g/L (1.2–22)) (p = 0.22). Multiple linear regression analysis including all welders and referents showed an increase in the concentration of S-ferritin associated with having serum carbohydrate deficient transferrin (S-CDT) above the upper reference limit of ≥1.7%, indicating high alcohol consumption. Serum C-reactive protein was not associated with exposure as welders, but an association with S-ferritin was shown. The GM S-ferritin concentrations among all welders and referents with S-CDT ≥ 1.7% were 157 g/L (95% CI 113–218) as compared to 104 g/L (95% CI 94–116) (p = 0.02) in those with S-CDT < 1.7%. The GM concentrations of Mn in biological fluids were higher in the welders as compared to the referents, while S-Fe, S-Co and B-Co were statistically significantly lower. This could suggest a competitive inhibition from Mn on the uptake of Fe and Co. Increasing concentrations of S-CDT was associated with higher S-Mn, S-Fe and B-Co in the multiple linear regression analysis. The association between S-CDT and S-Fe remained when all subjects with high S-CDT (≥1.7%) were excluded, suggesting increased uptake of Fe even at lower alcohol consumption. © 2014 Published by Elsevier GmbH.
Introduction Potentially high manganese (Mn) content in welding fumes may be a risk factor for neurological disturbances in welders [1]. Welding fume consists of primary particles that are agglomerated into chainlike structures, containing chemically complex compounds such as KMnF3 , Fe3 O4 , MnFe2 O4 or K2 MnO4 [2]. The predominant particle sizes of welding aerosols are below 1 m in aerodynamic
夽 The study was supported by Grant Number W81XWH-05-1-0239 from the U.S. Department of Defense, United States Army Medical Research and Material Command. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the United States Department of Defense, United States Army Medical Research and Material Command. ∗ Corresponding author. Tel.: +47 23195377; fax: +47 23195377. E-mail address: [email protected] (D.G. Ellingsen). 1 Current address: City Centre of Occupational Health, St. Petersburg 191014, Russia.
diameter, thus the particles can easily penetrate into the deeper parts of the lung [3,4]. The welding fume content of iron (Fe) is substantially higher than Mn in both manual and gas metal arc welding [5]. Mn and Fe share several transporters in animals and humans [6]. Non-heme Fe(II) and Mn(II) are absorbed in duodenal enterocytes through the divalent metal transporter 1 (DMT1). The cellular export of Fe(II) through ferroportin has also been proposed for Mn(II) [7,8]. Fe(III) and Mn(III) bind to transferrin and are taken up by cells through high affinity transferrin receptor (TfR) mediated endocytosis. After acidification, both metals are transported out of the endosome through DMT1 [9]. Hepcidin is important for the Fe metabolism by regulating ferroportin, perhaps through Fe sensors in the hepatocytes [10]. Thus, there is no surprise that high exposure to Mn may alter Fe metabolism. Some studies have addressed this issue. Oral administration of high amounts of Mn resulted in lower serum Fe and hemoglobin concentrations in lambs and lower gastrointestinal
http://dx.doi.org/10.1016/j.jtemb.2014.03.004 0946-672X/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: Ellingsen DG, et al. Biomarkers of iron status and trace elements in welders. J Trace Elem Med Biol (2014), http://dx.doi.org/10.1016/j.jtemb.2014.03.004
G Model JTEMB-25514; No. of Pages 7
ARTICLE IN PRESS D.G. Ellingsen et al. / Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx
2
absorption of Fe in humans [11,12]. Brain Mn concentrations were higher in Mn injected rats both at increased and at decreased dietary Fe as compared to the administration of normal dietary Fe [6]. Mn metabolism may also be altered by Fe deficiency, as evidenced by significantly higher concentrations of Mn in whole blood of Fe deficient women [13]. It also appears that Mn can interact with basic regulatory systems of Fe metabolism, e.g. inhibition of mitochondrial aconitase, probably by insertion of Mn instead of the labile Fe into its 4Fe-4S cluster [14], or by activation of the Fe responsive protein (IRP), perhaps by depleting Fe with concomitant reduction of intracellular ferritin [15]. A recent study suggested that Mn can competitively inhibit gastrointestinal Fe absorption through DMT1 [16]. Welders are exposed to Mn and Fe by inhalation. Pulmonary epithelial cells and alveolar macrophages have on their surfaces all key transporters of Fe such as TfR, DMT1 and ferroportin, as well as intracellular ferritin [17]. However, the pattern of pulmonary DMT1 expression is not altered in Fe-deficient rats in contrast to what is observed in enterocytes [18]. In contrast, Fe-loaded rats had reduced transport of Mn across the air–blood barrier after intratracheal instillation [19]. It has long been recognized that high alcohol consumption can result in liver Fe accumulation [20,21]. Studies of mice and rats have shown that high alcohol consumption results in down-regulation of hepcidin expression with a concomitant increase in the expression of DMT1 and ferroportin in duodenum [22,23]. Hepatocytic oxidative stress induced by alcohol has been proposed as a cause for reduced hepcidin expression [24]. Whether this may have an impact on the transport of other divalent metal ions, is to our knowledge not known. The potential for systemic Fe overload has been little studied in welders. Case reports have suggested that body Fe overload may occur in welders’ siderosis [25]. However, welders had similar serum ferritin concentrations when compared to referents, although the concentrations were associated with duration of exposure [26]. That study also reported reduced concentrations of soluble TfR in serum. A recent study did not show increased serum ferritin concentrations during a one week follow up of long term exposed welders [5]. This study is part of a larger study of nervous system impairment in welders. We have previously reported that these welders are exposed to high amounts of Fe, but mostly in a non-bioaccessible form, while the bioaccessibility was substantially higher for Mn [27]. The aim of this study is to assess whether Fe status biomarkers are altered in welders, to assess the impact of welding fume exposure on selected trace element concentrations and to study the impact of biomarkers of alcohol consumption and systemic inflammation on the above biomarkers. Some of the participants were also examined six years previously and results from that follow-up are also presented.
Materials and methods One hundred and forty-nine welders and 178 potential referents were invited to participate in this cross-sectional study, of whom 12 welders and 41 potential referents refused participation. In all, 137 welders and 137 referents were included (participation rates 91.9% and 77.0%, respectively). The study was restricted to men with at least one year of employment at one facility producing heavy machinery or at two shipyards in St. Petersburg (Russia). The referents were turners/fitters. The predominant welding methods in use were manual metal arc and gas metal arc welding. Criteria for exclusion have been published [27]. Sixty-three welders and 65 referents had been examined around six years earlier [28].
Also 34 former welders (four females) that had been diagnosed with manganism caused by welding were examined, of whom 17 had been examined around six years earlier [28]. The latter subjects belonged to a group of 27 patients examined at that time, of whom two had died, seven had moved out of the region and were thus not invited to participate and one declined the request to participate. Additional 20 patients were identified from the patient records kept at the clinic of the Northwest Public Health Research Centre (NWPHRC). They were invited to participate, but three subjects declined. Thus, the participation rate among the eligible patients was 89.5%. Background data of all participants are presented (Table 1). The Norwegian Regional Ethical Committee for Medical Research (REK2) approved the study that was also approved by the Ethics Committee of the NWPHRC (St. Petersburg, Russia) and the Office of Research Protection, US Army Medical Research and Material Command (Fort Detrick, MD, USA). Participation was strictly voluntary and informed written consent was obtained from all participants. Collection of biological samples Welders and referents went through a structured interview and collection of biological samples at the occupational health clinics of the respective plants, while the patients were examined at the NWPHRC. First voided morning urine samples were collected in 10 mL Sarstedt® polypropylene (PP) tubes (Sarstedt AG, Nümbrecht, Germany). Blood samples were collected from the cubital vein between 8.30 and 9.30 the same morning after cleaning of the skin. Whole blood for the determination of trace elements was collected in 4 mL Lithium-Heparine Vacuette® vacutainers. Nine mL Vacuette® vacutainers without additives (Greiner Labortechnik GmbH, Austria) were used for the harvest of serum. After a wait of 30 min the tubes were centrifuged for 10 min at 1500 × g. Serum was pipetted into 1.5 mL Sarstedt® cryotubes (Sarstedt AG, Nümbrecht, Germany) for storage. The samples were kept frozen at −20 ◦ C until analysis. Ten referents refused to give a urine sample, while 14 referents declined blood sampling. Three former and one current welder refused to give urine samples. Due to lack of serum, trace elements in serum was determined in 124 welders, 33 former welders and 83 referents. For the same reason, serum hepcidin was measured in 45 welders, 19 former welders and 35 referents. Determination of biomarkers Serum ferritin (S-ferritin) was measured by an immunoturbimetric method using Advia 2400 CapillarysTM (Siemens Healthcare Diagnostics Inc. NY, US) at Fürst Medical Laboratory (Oslo, Norway). The method’s reproducibility was 2.3%. Serum hepcidin (S-hepcidin) was determined by a quantitative ELISA sandwich immunoassay (BlueGene Biotech, Shanghai) at the University of Oviedo (Spain). The assay used a microtiter plate pre-coated with a monoclonal antibody specific for hepcidin. Standards and samples were added to the plate wells and finally, a second polyclonal antibody conjugated to horseradish peroxidase was added. The color change in the plate was measured spectrophotometrically at 450 nm. The immunoassay was previously cross-validated. The method’s DL was 0.1 ng mL−1 and the intraassay CV was
