Toenails as biomarker of exposure to essential trace metals: A review

Contents lists available at ScienceDirect Environmental Research journal homepage: www.elsevier.com/locate/envres Review article Toenails as biomarker of exposure to essential trace metals: A review. Enrique Gutiérrez-González a,b,∗∗ , Esther García-Esquinas b,c , Nerea Fernández de Larrea-Baz b,d , Inmaculada Salcedo-Bellido b,e , Ana Navas-Acien f , Virginia Lope b,d , José Luis Gómez-Ariza g , Roberto Pastor b,d , Marina Pollán b,d , Beatriz Pérez-Gómez a,b,d,∗ a Public Health & Preventive Medicine Teaching Unit, National School of Public Health, Carlos III Institute of Health, Monforte de Lemos 5, 28029, Madrid, Spain b Consortium for Biomedical Research in Epidemiology & Public Health (CIBER en Epidemiología y Salud Pública - CIBERESP), Monforte de Lemos 5, 28029, Madrid, Spain c Department of Preventive Medicine and Public Health, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo 4, 28029, Madrid, Spain d Department of Epidemiology of Chronic Diseases, National Centre for Epidemiology, Carlos III Institute of Health, Monforte de Lemos 5, 28029, Madrid, Spain e Department of Preventive Medicine and Public Health, University of Granada & Instituto de Investigación Biosanitaria de Granada, Av. de La Investigación, 11, 18016, Granada, Spain f Department of Environmental Health Sciences, Columbia University Mailman School of Public Health, 722 W 168th St, New York, NY, 10032, USA g Department of Chemistry, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Research Center on Health and Environment (RENSMA), C/ Menéndez Pelayo, 21002, Huelva, Spain ARTICLE INFO Keywords: Toenail Biomonitoring Biomarker Exposure Essential trace essential metals Systematic review ABSTRACT Health problems associated with essential trace metals can result from both inadequate (i.e., low intake) and excessive exposures (i.e., from environmental and/or occupational source). Thus, measuring the exposure level is a real challenge for epidemiologists. Among non-invasive biomarkers that intend to measure long-term exposure to essential trace metals, the toenail is probably the biological matrix with the greatest potential. This systematic review collects the current evidence regarding the validity of toenail clippings as exposure biomarker for trace metals such as boron, cobalt, copper, iron, manganese, molybdenum, selenium, silicon, vanadium and zinc. Special attention was paid to the time-window of exposure reflected by the toenail, the intraindividual variability in exposure levels over time in this matrix, and the relationship of toenail with other biomarkers, personal characteristics and environmental sources. Our search identified 139 papers, with selenium and zinc being the most studied elements. The variability among studies suggests that toenail levels may reflect different degrees of exposure and probably correspond to exposures occurred 3–12 months before sampling (i.e., for manganese/selenium). Few studies assessed the reproducibility of results over time and, for samples obtained 1–6 years apart, the correlation coefficient were between 0.26 and 0.66. Trace metal levels in toenails did not correlate well with those in the blood and urine and showed low-moderate correlation with those in the hair and fingernails. Available data suggests that for some elements (Se, Mn, Zn) toenail concentrations reflect long-term external exposures in fairly reproducible levels, while for other metals, this association has not yet been assessed. Among dietary factors, only toenail selenium showed clear associations with the intake of supplements or specific foods. The toenail levels could also represent occupational exposure, for instance, Mn exposure in welders. The scarcity of information on other essential trace elements, together with the great heterogeneity among studies makes the validation of the usage of toenails as biomarkers of exposure to these elements difficult. Standardization of sample collection, quality control, analytical techniques and reporting procedures might facilitate further research focused on the clear understanding of the significance of essential levels in this promising matrix and would enhance its utility in epidemiological research. https://doi.org/10.1016/j.envres.2019.108787 Received 8 July 2019; Received in revised form 26 September 2019; Accepted 30 September 2019 ∗ Corresponding author. Department of Epidemiology of Chronic Diseases, National Centre for Epidemiology, Carlos III Institute of Health, Monforte de Lemos 5, 28029, Madrid, Spain. ∗∗ Corresponding author. National School of Public Health, Carlos III Institute of Health, Avda. Monforte de Lemos, 5, 28029, Madrid, Spain. E-mail addresses: [email protected] (E. Gutiérrez-González), [email protected] (B. Pérez-Gómez). Environmental Research 179 (2019) 108787 Available online 07 October 2019 0013-9351/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T 1. Introduction Essential trace metals/metalloids either are needed to maintain physiologically important functions, or are part of organic structures with vital functions in humans (Mertz, 1981). However, in many cases, excessive levels of these elements also pose a serious health risk. The typical source of these elements is diet, but occupational (e.g., Mn in welders) and environmental sources (e.g., environmental release of Zn by metal production industries or from combustion of coal in power stations) may also contribute to high exposure levels. Therefore, measurement of exposure at the individual level is a critical aspect in the research of the role of these elements in human health. According to the World Health Organization (WHO) (WHO, 1996), the list of essential trace metals/metalloids -which will be called as metals in this review paper, since most of the elements studied belong to this group-, includes chromium (III) (Cr) [now under discussion] (Nigra et al., 2016), copper (Cu), cobalt (Co), iron (Fe), molybdenum (Mo), selenium (Se), zinc (Zn), and [manganese (Mn), which was added in 2001 to this group (Institute of Medicine (US), 2001). Vanadium (V), silicon (Si) and boron (B), not strictly considered essential elements, are often studied with them due to their potential health benefits (WHO, 1996;US EPA O, 2014). A common approach to measure the exposure level to these essential trace metals is the use of biomarkers due to several reasons: they reflect both known and inadvertent exposures; they are not affected by recall bias, and they integrate all sources (i.e., diet, air and water) and routes of contact (Santonen et al., 2015). Among these, toenails are non-invasive matrices that have become quite popular in large epidemiological studies for their logistic advantages, namely very easy collection and storage and less influence of external contamination than fingernails and hair (Karagas et al., 2000). In addition, the slow rate of growth of toenails has made these suitable candidates for evaluating longer term exposures than other biomarkers such as blood or urine, a key issue in chronic diseases research (Hopps, 1977;Sukumar, 2006). However, the validity of these assumptions is unclear and the usefulness of measurement of essential trace metals in toenails as a biomarker of exposure is still uncertain. In this review, we condensed available data on essential trace metal levels in toenails and summarized the evidence on their validity as biological matrices of exposure. We paid special attention to a) stability over time and time-window of exposure, two aspects critical in evaluating long-term chronic exposures; b) the relationship with other commonly used biomarkers, such as blood and urine and; c) the association with personal characteristics and several sources, in order to better understand the factors that module or determine the levels of essential trace metals in this substrate. 2. Material and methods This review is reported in accordance with the PRISMA publication standards (Moher et al., 2009) and the protocol was registered in PROSPERO, the international prospective register of systematic reviews (registration number CRD42018085822) (University of York, 2018). 2.1. Identification of studies We conducted a systematic literature search for peer-reviewed papers providing original data on essential trace metal concentrations in toenails. The flow diagram of the study selection process is shown in Fig. A1 (Appendix A). Two reviewers (EG and BP) independently screened abstracts, reviewed full-text articles, extracted data and performed quality assessments. Initially, we searched the databases PubMed/MEDLINE, Web of Science and Scopus from inception to December 31, 2017, using the following exact searches: #1 Nail OR toenail; #2 Exposure OR biomonitoring OR biomarker; #3 Metals OR trace elements OR beryllium OR vanadium OR chromium OR cobalt OR nickel OR copper OR zinc OR arsenic OR selenium OR cadmium OR platinum OR lead OR uranium OR mercury OR thallium OR aluminium OR molybdenum OR manganese OR iron OR silicon OR boron; #4: #1 AND #2 AND #3. This search retrieved 2632 references. Then, we manually reviewed the reference lists of all included studies and identified 52 additional articles. Studies that reported original quantitative data on toenail metal concentrations in humans in English or Spanish were considered eligible. When the type of nail (fingernail/toenail) was not specified, the corresponding author was contacted for clarification. After exclusion of duplicates, 276 articles met the general inclusion criteria. From these, for the purpose of this review, we narrowed our scope to focus on the papers (139) with information on essential trace metals (Co, Cu, Fe, Mn, Mo, V and Zn) or metalloids (B, Se, Si); we did not include Cr due to its disputed essentiality (Nigra et al., 2016) and the carcinogenic role of Cr (VI) (IARC, 2012). 2.2. Data collection Data from included studies were extracted into a customized spreadsheet. According to a purpose-designed protocol, the following data were extracted from each study: a) basic information (first author, country, year of publication, research project, studied trace metals); b) design features (type of epidemiological study, main objective, sample size, population sample method, participant characteristics, informed consent request, ethical committee approval); c) sample and analytical information (toenail type (i.e., big toe, all toes), sample preparation, analytical method, quality control measures, limits of detection and availability of other biological specimens with trace metal data); d) metal concentrations, i.e., measures of central tendency (geometric or arithmetic mean, median (p50)) and dispersion (standard deviation (SD) or range), when available; and e) correlation with other biomarkers, with personal characteristics, with environmental data, or with previous toenail measures. Most studies reported metal levels in micrograms per gram (μg/g), but many authors chose to provide other units of concentration, namely nanograms per gram (ng/g); micromoles per kilogram (μmol/kg); nanomoles per gram (nmol/g); milligrams per kilogram (mg/kg); parts per million (ppm); and parts per billion (ppb). This variability in the units used to express metal concentrations was not related to any specific element or technique. For this review, we converted all units into μg/g to allow for an easier comparison among studies. 3. Results 3.1. Study characteristics We identified a total of 139 manuscripts published between 1975 and 2017, which provided levels of essential metals in toenails, from 89 different research projects (number of papers per research project ranged from 1 to 9; 25 projects had 2 or more manuscripts). Almost 40% of the articles evaluated exposure either in the USA (49 papers, with data from 25 research projects) or in Canada (6 manuscripts, with data from 4 research projects) and another 39% of the papers included information from studies conducted in Europe (the Netherlands −13 manuscripts from 4 research projects-; Italy −7 papers from 4 projects-; and Poland −5 manuscripts from 5 research projects-were the most common locations). The remaining studies (21%) presented toenail essential metal levels from other countries, with a higher proportion of Asian countries in the recent years. One research project combined E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 2 populations from more than one continent (EURAMIC study). The number of manuscripts has increased in the last decade (See Fig. A2, Appendix A), evidencing a growing interest in this biomarker. The characteristics of each paper, sorted by research study and year of publication, are detailed in Appendix A, Table A1. Search results are summarized in Fig. 1. The majority of the research projects (n = 48) were focused on a single element, usually selenium. Overall, Se was the most studied essential trace element (n = 94 reports), followed by Zn (n = 44), Cu (n = 32), Mn (n = 31), Fe (n = 24), Co (n = 20), V (n = 9) and Mo (n = 8), while no data were found about toenail concentrations of B or Si. Regarding the aims of the articles, 71 used toenails as biomarkers of exposure to one or more essential metals or tried to identify their determinants, while 78 studied the association between toenail essential metal levels, mainly Se and Zn, and different health problems or biological characteristics (i.e., cholesterol levels). The most common outcome studied was cancer (Garland et al., 1995;Ghadirian et al., 2000), although other articles evaluated cardiovascular (Kardinaal et al., 1997), metabolic (Park and Seo, 2016), or hematologic effects (Lee et al., 2016), as well as neurologic (Meramat et al., 2017) or mentalhealth related problems (Colangelo et al., 2014) (Appendix A, Table A1). The number of participants in each study was highly variable: 33 research projects included less than 100 individuals, 46 studies, between 100 and 1000 and 9 recruited more than 1000 participants (range: 12–9267). In one project (only 1 paper published), the sample size was not specified (Abdulrahman et al., 2012). Baseline characteristics of these subjects were heterogeneous across studies and depended on the aim of each investigation. Thus, each paper provided information describing some features of the specific population under study, such as age, sex, race, socioeconomic status, occupation, or dwelling place (See Appendix A, Table A1). However, in some cases, this description was limited to geographical area of recruitment, omitting basic data such as age or sex of the participants. Regarding study design, most papers presented cross-sectional analyses (n = 59, which also included baseline levels in longitudinal projects), followed by case-control (30 reports) studies; prospective designs (case-cohort: 9 reports; nested case-controls: 13 reports; cohort: 25 reports) and randomized trials (3 reports). Convenience sampling was the typical strategy for recruitment and only 17 research projects reported participation rates, ranging from 41.8% (O’Rorke et al., 2012) to 98% 22 ; usually, in case-control studies participation was lower among controls. Concerning ethical considerations, 47 articles declared having approval of an ethics committee and informed consent explicitly appeared as an inclusion criterion in 49 papers. 3.2. Analytical methodology: sample collection, nail preparation, analysis and quality control (Table A1) Toenails clippings -which are the last end of the nail and constitute only a fraction of the total nail-were obtained using stainless-steel clippers or scissors. In regard to sample storage, studies that included information about this issue indicated that were kept in paper envelopes or in plastic bags/vials (polyethylene or non-specified), usually at room temperatures. The effects of long-term storage (6 years) were evaluated only for Se by St-Pierre et al., who concluded that the loss of humidity occurred with long-term storage led to increased Se concentrations (St-Pierre et al., 2006). The influence of sampling date has not been commonly studied. Isolated reports have suggested that Zn levels might be higher in winter (Campos et al., 2008) and that there might be significant differences in metal toenail levels depending on the season of sampling collection, for Cu, but not for Zn (Wilhelm et al., 1991) or Se (Baskett et al., 1995). Most of the projects (n = 40) did not identify the specific type of toenails analyzed. Among those that did, the majority (n = 36 studies) collected clippings from all toes; 8 projects used nails from big toes and, in another two studies, specimens from big toes and from the other toes Fig. 1. Graphical summary of the systematic review results. Sankey diagram. The first column represents the total number of articles included in the review. The second column shows the geographical distribution according to the place where the studies were conducted. The third column illustrates the number of articles that assessed each element in toenails (many articles deal with more than one element). The last column shows the main objective of the articles included in the review (some studies may share both objectives). E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 3 were evaluated separately. Two projects provided partial information, reporting sampling from both feet (Ghadirian et al., 2000;Vinceti et al., 2005) or from right foot (Vinceti et al., 2012), or using nails from different feet for analysis of each metal, without specifying which toes were considered (Vinceti et al., 2005). Possible differences in metal levels among toes have only been explored for Se, comparing the big toe to the rest of toes (Baskett et al., 2001;Kok et al., 1989;Longnecker et al., 1993) or with a pool of all toes (van ’t Veer et al., 1990), without finding significant variations. Also for this element, a high correlation between Se concentrations in the right and left toenails was observed in one study (r = 0.74) (Steven Morris et al., 1983). In regard to the amount of bio-specimen, most papers (n = 105) did not provide any summary or descriptive data (i.e., mean or range) about the toenail mass used for the analyses. However, several authors (Kardinaal et al., 1997;Steven Morris et al., 1983;Mordukhovich et al., 2012;Platz et al., 2002;Sanders et al., 2014) remarked that the limit of detection (LOD) for trace metal determinations was dependent on toenail mass and at least 25 reports indicated that a minimum weight was required for the analyses, which was only specified in 15 papers (i.e., 10 mg in 12 of them). In some studies (Lee et al., 2016; Abdulrahman et al., 2012;Al-Saleh and Billedo, 2006;Goullé et al., 2009;Hartman et al., 2002;Michaud et al., 2002;Morris and Crane, 2013;Saat et al., 2013;Wongwit et al., 2004), researchers stated they analyzed aliquots of toenail within a mass range (mostly 10–50 mg). It should be noted that, according to some investigators, the weight of the clippings not only limits the possibility of measurement of trace metals, but also bias the levels obtained. Thus, Baskett et al. described an apparent systematic bias in metal concentrations (Se, in this case) that were inversely proportional to the mass of the sample (Baskett et al., 1995), which occurred in clipping samples weighing 23 mg or less. Additionally, Saint Pierre et al. (St-Pierre et al., 2006) found an inverse correlation between sample weight and Se concentration, while Colangelo et al. reported that toenail mass was positively associated with toenail Se levels (Colangelo et al., 2014). In some of the studies, this possible influence of nail weight in trace metal concentrations was considered and was corrected for. Thus, some authors adapted analytical procedures to fit small samples (Baskett et al., 1995); in other studies, toenail weight was considered as a potential confounding factor (Colangelo et al., 2014), or measured levels were normalized based on sample weight (Brockman et al., 2009). Also, regression models were fitted with log-transformed toenail element levels (the dependent variable) against the nail weight (the independent variable) (St-Pierre et al., 2006), or the residual of each observation was added to the predicted mean toenail value and the result was exponentiated (Garland et al., 1993,1995,1996). In most studies, toenails were subjected to pretreatment after clipping and storage, before analysis, and the methods used were highly variable. Table A1 (Appendix A) presents a summary of this information, derived from each report included in the review. Nail polish in samples was generally removed with acetone and visible dirt was manually cleaned. Direct effects of nail polish (Ghadirian et al., 2000; van ’t Veer et al., 1990;Krogh et al., 2003) or debris (van ’t Veer et al., 1990) was assessed only for Se, and these factors did not seem to influence toenail Se concentrations. In order to reduce external contamination, toenails were cleaned by washing with detergent, deionized water, methanol, Triton solution, acetone, or sodium dodecylsulfate, assisted by a sonicator or an ultrasound bath. Subsequently, samples were dried in an oven, air-dried or freeze-dried. Neutron activation analysis (NAA or instrumental NAA) was the most commonly used technique for element measurement (46 studies), followed by inductively coupled plasma spectrometry (ICP-MS, ICPOES, ICP-AES) (29 studies) and atomic absorption spectrometry (AAS) (14 studies) (Appendix A, Table A1). In these two last cases, digestion of samples was performed in nitric acid, with or without chlorhydric acid, in perchloric acid, or in hydrogen peroxide, when needed; some reports specified a microwave digestion system or a sonicator. Certain research projects, like CARDIA, CLUE II, or ORDET, employed different techniques depending on the metals studied. Less common techniques used were thermal neutron flux (Graham et al., 1991) and acid digestion fluorometry (Hartman et al., 2002;Michaud et al., 2002;Alfthan et al., 1992;Männistö et al., 2000;Ovaskainen et al., 1993). Sixty reports mentioned quality control procedures, which included the use of certified reference material (urine or hair) or homemade nail reference material, recovery analysis, procedural blanks, duplicate samples, or spike samples. 3.3. Essential trace metal concentrations in toenails across populations Tables B1–B8 (Appendix B) present the levels of each essential metal in each study and the subgroup of participants (mean (SD), median, range) extracted from the 139 studies, included in this review. Their main data are shown graphically in Fig. 2. Only 18 (9.5%) research projects, mostly those which used IPC-MS, provided information on LOD, and even fewer (n = 4), specified limits of quantification (LOQ). The LODs were calculated from procedural blank analyses, usually as three standard deviations from the mean blank reading (Al-Saleh and Billedo, 2006;Krogh et al., 2003;Herman et al., 2013;Przybylowicz et al., 2012;Ntihabose et al., 2017;Bouchard et al., 2017). Among the studies that reported LODs for specific metals, for Co, LODs ranged from 0.0003 (Goullé et al., 2009) to 0.01 μg/g (Chanpiwat et al., 2015); for Cu, from 0.009 (Przybylowicz et al., 2012) to 0.12 μg/g (Chanpiwat et al., 2015); for Fe, from 1.55 (Przybylowicz et al., 2012) to 2.93 μg/g (Chanpiwat et al., 2015); for Mn, from 0.001 (Goullé et al., 2009) to 0.33 μg/g (Chanpiwat et al., 2015); for Mo, from 0.0004 (Goullé et al., 2009) to 0.02 μg/g (Chanpiwat et al., 2015); for Se, from 0.0022 (Emmanuelle et al., 2012) to 0.02 μg/g (Goullé et al., 2009); and for Zn, from 0.01 (Goullé et al., 2009) to 0.279 μg/g (Przybylowicz et al., 2012). Only one study specified the LOD for V, which was 0.001 μg/g (Goullé et al., 2009). The percentage of samples with quantities under LOD, when available, depended on the element studied. It was low or inexistent for Zn and Se; in contrast, some studies reported a significant proportion of samples with quantities under the LOD for other metals, i.e., Cu, Fe, Mn and V (See Appendix B, Tables B1–B8). Among the elements studied, Co, Mo, Se and V had the lowest levels, and most studies found mean values < 1 μg/g (Fig. 2a,e,f and g; Tables B1, B5, B.6, B.7). However, a couple of studies reported exposed populations with higher mean Co concentrations: metal workers in two towns of northern Italy (AMs: 18.90 and 53.79 μg/g (Sabbioni et al., 1994),); and young adults living near a mining area in Zambia (GM: 1.39 μg/g) (Ndilila et al., 2014). Morris et al. also reported exceptionally high Se levels (AM 8.27 μg/g) among people in USA who consumed misformulated supplements with excessive Se concentrations (Morris and Crane, 2013). In general, mean Se levels were higher in studies from USA-Canada, when compared with other countries. Fe toenail concentrations (Table B3 and Fig. 2c) showed the highest variability across studies, with median levels ranging from 8.8 μg/g in a group of 60–80 year-old women from Spain (Sureda et al., 2017) to exceptionally high levels (median: 1434 μg/g) in subjects living in contaminated areas of Cambodia (Chanpiwat et al., 2015). Mean Cu and Mn levels were usually below 10 μg/g (Tables B.2 and B.4,Fig. 2b–d, respectively), while mean Zn levels (Table B.8,Fig. 2h) ranged mostly from 50 to 200 μg/g. The highest Cu, Mn and Zn concentrations were found among subjects living in rural areas near a highly industrialized city in Pakistan (AM: 26.2 μg/g for Cu, 52.1 for Mn and 298 for Zn) (Mohmand et al., 2015). High concentrations of Mn E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 4 were observed in individuals living in highly polluted areas of Cambodia (AM: 43.9 μg/g) (Chanpiwat et al., 2015) and among welders from a local boilermaker union in the USA (AM: 26.9–55.5 μg/g) (Laohaudomchok et al., 2011). 3.4. Toenail as a biomarker of integrated long-term exposure: time-window of exposure and intraindividual stability over time (Tables 1 and 2) The time-frame of exposure covered by toenails, investigated only for Se and Mn, was explored in studies that identified a specific moment in which the exposure occurred, which allowed to calculate the time elapsed until detection in toenail samples. Selenium was the most studied element and was evaluated in three research projects. In the USA, 12 young men participated in a research conducted to evaluate the effect of one-year intake of Se-supplemented whole wheat bread on Se toenail levels. The participants were classified into three groups: those with high-dose (4.91 μmol Se/d), medium-dose (2.61 μmol Se/d) and low-dose (0.41 μmol Se/d), and the concentration of Se in toenail clippings was measured every 1–2 weeks for 2 years (Longnecker et al., 1993). Se peak levels were detected in the big toenail after 24–37 weeks vs. 12–24 weeks in other toes; this difference was attributed to the shorter nail bed in smaller nails. Selenium concentration in the big toenail was still high, even after one year of intake termination and remained over baseline for two years; this suggests that toenail clippings could provide a time-integrated measure of Se intake for over 26–52 weeks, serving as a good biomarker of Se intake for retrospective studies. Other studies included 10 middle-aged men and women provided with Se-76 supplementation, and the peak Se concentrations in toenails were observed at 16–32 (Baskett et al., 1995, 2001) and 16–50 (Baskett et al., 1998) weeks. Peak concentrations were also reported by Morris et al. at 34 weeks after ingestion of high Se misformulated products, while, in this study, baseline levels were recovered after 59 weeks (Morris and Crane, 2013). For Mn, the time-frame of exposure was evaluated in 3 studies conducted among occupationally exposed workers (welders). Exposure time reflected by toenail Mn concentrations was estimated between 7 and 9 (Grashow et al., 2014) and 7–12 months (Laohaudomchok et al., 2011;Ward et al., 2017) (Table 2). Since one year is an insufficient period for the study of exposure for most chronic diseases, it is crucial to identify the extent to which individual point measurements of trace metals in toenails represent toenail concentration levels in the same person in the previous years. This stability of essential trace metals over time was evaluated in 7 reports by estimating the within-person correlation among samples obtained at different moments (Table 1) (Baskett et al., 1995;Garland et al., 1993;Krogh et al., 2003;Hunter et al., 1990a;Rodrigues et al., 2015;Xun et al., 2010a). The usage of correlation coefficients to assess reproducibility is not ideal, but no studies used the intra-class correlation coefficient. Fig. 2. Essential metal levels [mean or median (μg/g dry weight] in human toenails (1975–2017). E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 5 Table 1 Reproducibility over time of essential metals in toenails and correlation with levels in other biological specimen. Toenail metal Author Year N Toenails reproducibility over time * Hair Urine Whole blood Serum Plasma Finger nail Other toenails Feces Cord blood Placenta Saliva Co Garland e 1993 127 r = 0.35 (6-year) Sabbioni 1994 115 r = 0.38 (ns) r = 0.25 (ns) Chanpiwat e 2015 180 r = 0.20 r = −0.08 (ns) Cu Wilhelm 1991 461 r = 0.18 Garland e 1993 127 r = 0.26 (6-year) Herman 2013 30 r = 0.26 Kuiper 2014 239 (ns) Chanpiwat e 2015 180 r = 0.10 (ns) r = −0.08 (ns) Fe Garland 1993 127 r = 0.43 (6-year) Chanpiwat e 2015 180 r = 0.36 r = −0.03 (ns) Mn Wongwit f 2004 135 r = 0.05 (ns) (ns) Kuiper 2014 239 (ns) Coelho e 2014 122 r = 0.23 Chanpiwat e 2015 180 r = 0.32 r = −0.10 (ns) Rodrigues e 2015 711 r = 0.37–0.41 (6/7 months) b r = 0.14 Mother-infant r = 0.39 r = 0.14 Hassani g 2016 83 r = 0.65 r = 0.65 Ntihabose 2017 268 r = 0.40 r = 0.14 Mo Kuiper 2014 239 (ns) Chanpiwat e 2015 180 r = 0.08 (ns) r = 0.00 (ns) Se Hunter 1990b 868 r = 0.60 (5-year) Morris 1983 62 r = −0.3 (ns) r = 0.30 (ns) Left-Right r = 0.74 Kok 1989 168 Big-Small (nd) Van't Veer 1990 372 Big-All (nd) Longnecker 1991 142 r = 0.60 r = 0.91 r = 0.89 Alfthan 1992 132 r = 0.57 Garland 1993 127 r = 0.48 (6-year) Longnecker 1993 12 Big-Small (nd) Baskett 2001 11 Left-Right ** Krogh 2003 80 r = 0.57 (1-year) c Satia e 2006 220 r = 0.55 Al Saleh d 2006 691 r = −0.16 Xun 2010b 69 r = 0.56 (20-year) Vinceti e 2012 105 r = 0.27 Coelho e 2014 122 r = 0.33 Kuiper f 2014 239 (ns) Rayman e 2015 230 r = 0.45 Punshon ae 2016 554 h V Rainska 2007 33 r = 0.61 Zn Mckenzie 1979 110 (ns) (ns) (ns) Wilhelm a 1991 461 r = 0.03 (ns) Garland 1993 127 r = 0.58 (6-year) Chanpiwat e 2015 180 r = −0.02 (ns) r = 0.08 (ns) Punshon a 2016 554 (ns) N = number of participants (samples) in the study; r = correlation coefficient; (ns) = p > 0.05 except in Champiwat (2015) (p > 0.01) or author only stated that correlation was not statistically significant; (nd) = author stated that there were no differences. * = between parentheses is expressed the intervale of time between both measurements. a Log transformed. b Pregnant women: first trimester vs one month post-partum. c Not modified by age, menopausal status, smoking or acetone treatment. d No significant differences between mean levels in both specimens using t-test. e Spearman correlation. f Pearson correlation. g Partial correlation test. h = strong positive significant correlation with maternal toenails. Not significant with infant toenails. E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 6 For most elements, this information was obtained from a single analysis carried out in 127 female nurses participating in the Nurse Health Study I (NHSI), which measured 5 trace metals (Co, Cu, Fe, Se and Zn) in 2 samples of toenails collected 6 years apart. Correlation values were around 0.5 for Zn, Se and Fe and less than 0.4 for Co and Cu (Garland et al., 1993). In the case of Se and Mn, our search identified additional studies that evaluated the reproducibility of their measurements in different moments of time. For Se, the studies we identified reported a Pearson correlation coefficient of 0.57 among samples collected around 1 year apart from 80 women in the ORDET prospective study of whom 40 were pre-menopausal and 40 were postmenopausal (Krogh et al., 2003); a correlation coefficient of 0.60 using samples collected approximately 5 years apart, from the NHS I cohort (Hunter et al., 1990a); and a correlation coefficient of 0.56 using toenail Se samples collected 20 years apart from 64 participants in the CARDIA project (Xun et al., 2010a). Another exploratory research stated that Se levels were very stable in toenail samples from 2 men and 2 women, followed up for 15 months prior to Se supplementation (Baskett et al., 1995). For Mn, two studies have been conducted in this context: a study which reported correlation coefficients between 0.37 and 0.41 in pregnant women, with samples taken during the first trimester of pregnancy and one month after delivery (Rodrigues et al., 2015) and another exploratory analysis that evaluated clippings of the five toenails from the same individual, collected four times over a 9-month period, with a large variability in the Mn levels (Guthrie et al., 2008). 3.5. Correlation of trace elements in toenails with other biological matrices (Table 1) To better understand toenail metal levels as biomarkers of exposure to essential trace elements, it is important to explore their relationship with other substrates commonly used in this field, especially blood and urine. A total of 20 studies with a variable number of participants (range: 30–868) evaluated the correlation of trace elements in toenails with other biological matrices collected concurrently (i.e., whole blood, serum, plasma, urine, hair, fingernails, feces, cord blood, placenta and saliva) (Table 1). Hair: Hair was the biological matrix more often compared with toenails (n = 10), probably due to its shared non-invasiveness and its similar keratin-based composition. For Mo, Se or Zn, no significant associations have been observed between levels in this substrate and in toenails (Wilhelm et al., 1991;Steven Morris et al., 1983;Chanpiwat et al., 2015;Kuiper et al., 2014;McKenzie, 1979). Other metals, such as Co and Cu, showed weak positive (< 0.30) or null correlation between both biomarkers (Wilhelm et al., 1991;Herman et al., 2013;Chanpiwat et al., 2015;Sabbioni et al., 1994). In contrast, the association was moderate (between 0.32 and 0.40) for Mn (Ntihabose et al., 2017; Chanpiwat et al., 2015) and Fe (Chanpiwat et al., 2015) and strong (r = 0.61) for V, (Rainska et al., 2007), although the interpretation of the latter result was limited by the small sample size of the study (n = 33). Urine: Our search identified two studies which revealed strong positive correlations between metal levels in toenails and urine for Mn (r = 0.65 (Hassani et al., 2016)) and Se (r = 0.60 (Longnecker et al., 1991)). In contrast, Co, Cu, Fe, Mo and Zn in toenails and in urine were not found to be correlated in other studies (Chanpiwat et al., 2015; Kuiper et al., 2014;McKenzie, 1979). Blood: Surprisingly, there were only data comparing these two matrices for Se, Mn, Co and Zn. In the case of Se, four studies showed positive and significant correlations with whole blood (r = 0.91 (Longnecker et al., 1991) and r = 0.45 (Rayman et al., 2015)); serum (r = 0.89 (Longnecker et al., 1991)); and plasma (r = 0.55 (Satia et al., 2006)). However, in the study by Al Saleh et al. a negative correlation between log-transformed Se concentrations in toenails and serum was reported (r = −0.16 (Al-Saleh et al., 2006)). Regarding Mn, Hassani et al. described a strong correlation between toenails and whole blood levels (r = 0.65 (Hassani et al., 2016)), but both biomarkers were not found to be related in a sample of welders studied by Wongwit et al. (2004); moreover, Rodrigues et al. reported a low correlation between infant toenails and cord blood concentrations (r = 0.14 (Rodrigues et al., 2015)). Finally, whole blood Co (Sabbioni et al., 1994) and serum Zn (McKenzie, 1979) levels were not significantly correlated with the corresponding toenail concentrations. Fingernails: Only three studies evaluated the correlation between toenails and fingernails, showing a low correlation for Mn (r = 0.23 (Coelho et al., 2014)) and a medium correlation for Se (r = 0.33 (Coelho et al., 2014) and r = 0.57 (Alfthan et al., 1992)). In these studies, as well as in the studies by Baskett et al. for Se (Baskett et al., 1995,1998,2001), fingernail concentrations were almost twice as high as those from toenails. Other matrices: For Mn levels in toenails, a weak correlation has been reported with the concentration in saliva (Ntihabose et al., 2017) and no correlation with levels in feces (Wongwit et al., 2004). A moderate correlation has been reported between infant and maternal toenail Mn levels (r = 0.39 (Rodrigues et al., 2015)). In the case of Se, a positive correlation was observed between maternal toenail and placenta Se concentrations (Punshon et al., 2016). 3.6. Correlation of essential metals with other trace metals in toenails (Table A2) The correlation among essential trace elements in toenails or with other toxic metals was explored in 14 studies (Table A2, Appendix A). Most of the reported correlations were low-moderate (below 0.40) or non-significant, both for essential and non-essential elements. Among the essential metals group, the strongest correlations were observed between Fe and Co (r = 0.81) (Rainska et al., 2007) and between Mn and V (r = 0.40) (Masironi et al., 1976). Regarding the association between essential and toxic metals, Mn had the highest proportion of strong correlations (with aluminium (Al) (Guthrie et al., 2008), arsenic (As) (Mordukhovich et al., 2012;Sanders et al., 2014), barium (Ba) (Kuiper et al., 2014), cadmium (Cd) (Mordukhovich et al., 2012; Sanders et al., 2014), chromium (Cr) (Sanders et al., 2014), lead (Pb) (Mordukhovich et al., 2012) and uranium (U) (Kuiper et al., 2014)). Other strong correlations were reported for Co and tungsten (W) (Sabbioni et al., 1994), Cu and Cd (Wilhelm et al., 1991), Cu and Pb (Wilhelm et al., 1991;Kuiper et al., 2014), Se and As (negative correlation) (Burgess et al., 2014), V and Al (Masironi et al., 1976) and V and scandium (Sc) (Rainska et al., 2007). 3.7. Determinants of essential metals in toenails Identification of the exposure sources or factors related to them is crucial in understanding the information provided by the measurement of essential metal levels in toenails. Most of the studies reported on this issue, although the huge variability in the variables studied, populations, designs and indicators made the calculation of quantitative summary estimators practically impossible. Notwithstanding, we have tried to summarize this information in order to give a general view of the published results. For this purpose, we have designed two tables that present, for each combination of metal and factor studied, the identification (i.e., author and date) of each report and the information about their possible relationship. Table 2 includes data for non-dietary factors and Table 3 includes the corresponding associations with intake of supplements, dietary estimates, foods and nutrients. 3.7.1. Personal and anthropometric characteristics (Table 2) Age: For toenail Se, most of the studies showed either no changes with age (Kardinaal et al., 1997;Colangelo et al., 2014;van ’t Veer et al., 1990;Alfthan et al., 1992;Ovaskainen et al., 1993;Satia et al., 2006;Everson et al., 2017;Hashemian et al., 2017;Kristal et al., 2014; E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 7 Table 2 Studies (Author year) reporting association of toenail essential trace metals to personal characteristics and non-dietary exposures. (continued on next page) E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 8 Lipsky et al., 2004;Morris, 2001;Morris et al., 2006;Swanson et al., 1990;van den Brandt et al., 1993;Xun et al., 2010b), or lower levels in elder individuals (Garland et al., 1995;Park and Seo, 2016;Hunter et al., 1990b;Jang et al., 2017;Park et al., 2011;Żukowska et al., 2009). Toenail concentrations of V were also inversely associated with age (Masironi et al., 1976;Rakovic et al., 1997;Slotnick et al., 2005) and a similar result was reported in the only study evaluating Co and Fe (Rakovic et al., 1997). The effect of age was inconsistent for Cu, Mo, Mn and Zn. Sex: Women generally had higher toenail Se concentrations than men (Lee et al., 2016;Baskett et al., 2001;Xun et al., 2010a;Rainska et al., 2007;Morris, 2001;Morris et al., 2006;Swanson et al., 1990;van den Brandt et al., 1993;Xun et al., 2010b;Żukowska et al., 2009). For Cu, Mo, Mn and V, the studies showed either higher levels in women (Coelho et al., 2014;Rakovic et al., 1997;Slotnick et al., 2005) or no variation by sex (Masironi et al., 1976;McKenzie et al., 1978;Reis et al., 2015). Fe levels were similar in both sexes (Sureda et al., 2017; Rakovic et al., 1997). For Zn, the available data showed very inconsistent results, with some reports finding no differences by sex (Park and Seo, 2016;Meramat et al., 2017;Przybylowicz et al., 2012;Sureda et al., 2017;Hashemian et al., 2017;Rakovic et al., 1997;McKenzie et al., 1978), while others showed higher concentrations either in women (Lee et al., 2016;Rainska et al., 2007;Bergomi et al., 2002) or in men (Campos et al., 2008;Gonzalez et al., 2008). Cobalt was the only metal for which levels were consistently higher in men (Sureda et al., 2017;Rakovic et al., 1997;Slotnick et al., 2005). Differences by race/ethnic group have been evaluated for Mn and Se in a few papers. White people usually had higher toenail levels of Mn than other groups (Mordukhovich et al., 2012;Laohaudomchok et al., 2011); for Se, in some studies, levels were found to be higher in whites (Everson et al., 2017;Kristal et al., 2014), while other studies found lower concentrations in African-Americans (Colangelo et al., 2014; Kristal et al., 2014;Xun et al., 2010b,2011). One study did not find any differences with respect to ethnicity (Satia et al., 2006). The influence of body mass index (BMI) was studied only for Se, Zn and Fe. Neither Se (Kardinaal et al., 1997;Colangelo et al., 2014; Ovaskainen et al., 1993;Satia et al., 2006;Hashemian et al., 2017; Kristal et al., 2014;Lipsky et al., 2004;Morris et al., 2006;van den Brandt et al., 1993;Hunter et al., 1990b), nor Zn (Hashemian et al., 2017;Park et al., 2016;Vinceti et al., 2015) varied with this factor, while one study reported a positive link between Fe in female toenails and BMI (Garland et al., 1996). Only a few studies evaluated the effect of reproductive factors, showing that parity may be positively associated with toenail Mn (Rodrigues et al., 2015) and Se concentrations (van Noord et al., 1993), while a later age at first birth may be associated to lower Fe levels (Garland et al., 1996). An older age at menarche was found to be associated with higher Se concentrations (Hunter et al., 1990a), but menopause did not modify levels of this element in toenails (Krogh et al., 2003). 3.7.2. Non-dietary lifestyle & social factors (Table 2) Educational level was positively correlated with higher Se concentrations (Colangelo et al., 2014;Xun et al., 2010a,2011;Rayman Table 2 (continued) E. Gutiérrez-González, et al. Environmental Research 179 (2019) 108787 9 van den Brandt, P.A., Goldbohm, R.A., van’t Veer, P., Bode, P., Hermus, R.J., Sturmans, F., 1993. Predictors of toenail selenium levels in men and women. 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