This article was downloaded by:[Kempson, Ivan M.]On: 13 December 2007Access Details: [subscription number 787587890]Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Toxicology andEnvironmental Health, Part BCritical ReviewsPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713667286

The Occurrence and Incorporation of Copper and Zincin Hair and their Potential Role as Bioindicators: AReviewIvan M. Kempson a; William M. Skinner a; K. Paul Kirkbride ba Ian Wark Research Institute, University of South Australia, Mawson Lakes,Australiab Forensic Science, S.A., Adelaide, South Australia, Australia

Online Publication Date: 01 December 2007To cite this Article: Kempson, Ivan M., Skinner, William M. and Kirkbride, K. Paul (2007) 'The Occurrence andIncorporation of Copper and Zinc in Hair and their Potential Role as Bioindicators: A Review', Journal of Toxicology andEnvironmental Health, Part B, 10:8, 611 - 622To link to this article: DOI: 10.1080/10937400701389917URL: http://dx.doi.org/10.1080/10937400701389917

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.

The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007

611

Journal of Toxicology and Environmental Health, Part B, 10:611–622, 2007Copyright © Taylor & Francis Group, LLCISSN: 1093-7404 print / 1521-6950 onlineDOI: 10.1080/10937400701389917

THE OCCURRENCE AND INCORPORATION OF COPPER AND ZINC IN HAIR AND THEIR POTENTIAL ROLE AS BIOINDICATORS: A REVIEW

Ivan M. Kempson1, William M. Skinner1, K. Paul Kirkbride2

1Ian Wark Research Institute, University of South Australia, Mawson Lakes, Australia, and 2Forensic Science, S.A., Adelaide, South Australia, Australia

This article reviews evidence that suggests Cu and Zn concentrations are not altered significantly by exogenous pro-cesses and may be useful in applications of hair analysis. The review attempts to identify what Cu and Zn concentra-tions may actually indicate biogenically and investigates the mechanisms by which they are incorporated into hair.Associations with specific hair components are proposed and avenues for development as a bioindicator are identi-fied. Areas of research that offer promise in application or confirming the use of Cu and Zn are also indicated. Cor-relations and relationships with other health disorders are reviewed. Endogenous blood concentrations may alsoexplain alterations in hair structure relating to breast cancer.

Hundreds of papers published over previous decades lie as testament to the interest and abilityto measure trace elemental concentrations in hair. Analytical techniques have progressed toprovide increasing sensitivity and selectivity, lower detection limits, and higher spatially resolvedanalyses. While these capabilities have improved immensely, any proven ability to apply elementalanalysis of hair has experienced minimal development.

The concept behind hair analysis offers benefits revolving around its growth behavior and easeof collection, transport, handling, and storage. Hair grows at a rate of approximately 1 cm permonth and incorporates xenobiotics from the body, primarily the blood supply. Analysis thereforerepresents a time period beyond that offered by urine and blood and can provide a temporal reflec-tion of endogenous products. The study of xenobiotics in hair has therefore promised to develop analternative means by which to monitor nutrition, health status, and exposure to toxins and pollut-ants (Revich, 1994; Samanta et al., 1999; Kales & Goldman, 2002) with respect to inorganic ana-lytes. This has many obvious important applications in occupational settings, environmentalmonitoring, medicine, biomonitoring, and forensic science. Owing to the amount of published dataon hair inorganic analysis, this review has been largely restricted to readily available, post 1990,peer-reviewed journal publications with significant discussion on Cu and Zn. Cu and Zn were cho-sen due to their importance for biological function and are discussed in terms of their incorpora-tion, potential for altering hair composition or structure, and relation to biogenic conditions. Thepapers referenced originate from a diverse range of studies and in some instances discuss very lim-ited data sets. These data have not been excluded from consideration in this review; however, theymay not accurately reflect all individuals. Research relating to hair analysis prior to 1990 has beenwell documented elsewhere (Valkovic, 1988).

CURRENT STATUS

Bulk analysis techniques are generally employed, such as atomic absorption spectroscopy(AAS), inductively coupled plasma mass spectrometry (ICP-MS), or inductively coupled plasma

This work was supported by the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australiaunder the Major National Research Facilities Program. Use of the Advanced Photon Source was supported by the U.S. Department ofEnergy, Office of Science, Basic Energy Sciences, under contract W-31-109-Eng-38. Support from the Canadian Light Source is alsogratefully acknowledged.

Current address for K. Paul Kirkbride is National Institute of Forensics, PO Box 13075, Law Courts Post Office Victoria, 8010, Australia.Address correspondence to Dr. Ivan M. Kempson, Ian Wark Research Institute, University of South Australia, Mawson Lakes, S.A.,

5095, Australia. E-mail: Ivan.Kempson@unisa.edu.au

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 612 I. M. KEMPSON ET AL.

atomic emission spectrometry (ICP-AES). More fundamental studies tend to use spatially resolvedtechniques, including particle-induced x-ray emission (PIXE), x-ray fluorescence (XRF), or time-of-flight secondary-ion mass spectrometry (ToF-SIMS). Each of these methods provides complementarydata in understanding hair elemental concentrations and population variables. However, servicesby commercial laboratories can be highly variable, misleading, and inaccurate (Seidel et al., 2001;Miekeley et al., 1998), even before any real issues regarding elemental incorporation have beenaddressed. Irrespective of instrumental and interpretative contradictions, before hair analysis can beapplied effectively, fundamental issues regarding the incorporation and loss mechanisms must bemore thoroughly scrutinized. It is clearly desirable to monitor exposure to pollutants or biogenicfunction using hair; however, there are underlying flaws in such analyses. These inadequacies stemfrom (1) lack of understanding regarding the incorporation mechanisms, (2) extent to which envi-ronmental contamination contributes, and (3) inability to discriminate contamination by eitherwashing procedures or the analytical technique. Contamination from exogenous sources is a majorissue (Kidwell & Blank, 1996), and washing (Borella et al., 1996) cannot be confirmed to remove alland only exogenous material. Incorporation mechanisms and the stability of elements, both bio-genic and anthropogenic, are poorly understood and inhibit the development of hair analysis as aroutine and reliable tool.

In areas of hair analysis dealing with organic compounds (such as drug analysis), “real” applica-tion is very promising and proven in many respects (Tagliaro et al., 1997). This basically stems fromthe fundamental issues regarding conclusively identifying hair content as being from endogenousuptake. In the case of drugs, and other organics, uncertainty arises in the analysis of parent com-pounds. A legal case, for example, will not succeed if it is argued that a drug is detected in hair thatcould have simply arisen due to exposure and contamination, rather than consumption. Instead ofpursuing the analysis of the parent compounds, metabolites are investigated that prove consump-tion. Mineral analysis has generally not benefited from such discrimination. Without some chemicaldifferentiation, such as organic metabolic products or potentially isotopic analysis, application isvery difficult. Organic forms of Hg (Ponce et al., 1998) and As (Mandal et al., 2003; Raab et al.,2002) are two elements that demonstrate promise in this respect.

Longitudinal analyses after significant dose intake have been proven to provide valuable data,such as for exposure to As (Nicolis et al., 2001; Lin et al., 1998) and Hg (Toribara, 1995, 2001). Inthese cases, concentrations exceed what is typical and exhibit trends that can be related to biogenicfunction rather than contamination. In addition, large population studies that might demonstratestatistical trends between groups of people are of interest. However, if these methods are applied asa diagnostic tool to classify individuals, there is generally little statistical confidence since the popu-lation groups overlap to such an extent that an individual cannot be placed into one group oranother.

In recent work (Kempson et al., 2006), a high-resolution synchrotron XRF microprobe was usedto examine elemental distributions in hair cross sections obtained from a smelter worker (primarilyprocessing Pb but also Ag, Au, Cu, and Zn). Analysis with highly advanced analytical techniquessuch as ToF-SIMS (Kempson & Skinner, 2005) and SXRF provided a combination of surface sensitivity,limits of detection, and spatial resolution that was not reported before, yielding new insight into theincorporation of metals in hair. Synchrotron sources offer significant advantages over conventionalinstrumentation as described elsewhere (Henderson & Baker, 2002; Bertsch & Hunter, 2001;Kempson et al., 2006). A spatial resolution of 130 nm was achieved at the Advanced Photon Source(Argonne National Laboratories, Argonne, IL) to examine hair sections from above and below thescalp level. High-resolution elemental maps provided an insight into possible incorporation mecha-nisms and associations of a variety of elements. Prior longitudinal analysis of the same individuals’hair revealed a strong dependence on exposure to the environment for Pb concentrations (Martinet al., 2005). Zn, however, remained constant along the hair. Ultimately, it was hoped that a thoroughanalysis of an individual’s hair and comparison with more general observations in the literature couldassist in the elucidation of some fundamental issues regarding the incorporation of metals into hair.The conclusions of these studies identified Cu and Zn as offering potential in application as a mon-itor reflecting endogenous uptake. What exactly the Cu and Zn concentrations may represent is not

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 CU AND ZN IN HAIR AND THEIR POTENTIAL ROLE AS BIOINDICATORS 613

clear. Endogenous trace elemental concentrations in hair have a dependence on blood concentra-tions; however, metabolic function within the hair follicle is also likely to play a role. Trace elemen-tal blood concentrations can be perturbed by other bodily function or dysfunction, leading tochanges in hair concentration. Thus if an element is proven to be endogenous, what does it repre-sent? Concentrations within the body, metabolism in the follicle, or metabolic function in a moregeneral sense depending on other organs and bodily processes are factors to consider. Forreference, some typical Cu and Zn concentration ranges in hair are provided in Table 1. Elementalconcentration ranges typically span orders of magnitude for most elements, but Cu and Zn exhibitlimited concentration ranges and comparatively smaller standard deviations (Batzevich, 1995;Kempson, 2004).

THE IMPORTANCE OF CU AND ZN AND THEIR INCORPORATION INTO HAIR

Cu and Zn play important roles in the formation and growth of hair and wool (Robbins, 2002),so the regulation of their concentration in hair is likely to be influenced by the biogenic activitywithin the follicle. Both influence metabolism of the growing structure. Cu acts as a catalyst in theoxidation of cysteine to cystine during fiber development, presumably at the tertiary and quaternarylevel of organization. Cell division and protein metabolism have a reliance on Zn.

The outer part of the hair is the cuticle, consisting of concentric layers of individual cuticle cells.This acts as a barrier to preserve the structural integrity of the fiber and is the first part of the hair tointeract with exogenous products. Two distinct elemental distributions exist on the surface of a hair,reflecting different affinities for mono- and divalent cations. This was highlighted by surface-sensitiveelemental mapping (Kempson et al., 2003; Kempson & Skinner, 2005). Chemical functionality ofthe cuticle is highly variable between the edges and flat surfaces of each cuticle cell (Swift, 1997).Monovalent cations have a strong preference for accumulation on the flat scale surfaces, while diva-lent cations exhibit a preference for the scale edges. Incorporation into hair from external sources isbelieved to be via migration at the scale edges rather than direct transverse diffusion. The outerlayer of each cuticle cell, the A-layer, is a highly cysteine-rich, chemically and mechanically toughprotein coated with a semipermeable proteinaceous material containing fatty acids, predominantly18-methyleicosanoic acid (Robbins, 2002). The scale edges reveal the transverse section of a cuticlecell comprising, moving radially out of the hair, the inner layer, endocuticle, and exocuticle. Theexocuticle is also cysteine rich, while the endocuticle is distinctly lacking in cysteine, but richer infree amino and carboxyl groups. The inner layer only has a minimal cross-sectional area and con-tributes minimally to the volume. Thus far, there does not appear to be any conclusive evidenceregarding the localized accumulation of Cu and Zn on cuticle scales. The cuticle, however, oftendoes not act as a barrier to prevent the incorporation of exogenous species. Metals can precipitateonto the surface and subsequently diffuse into the hair (Kempson et al., 2006; Bos et al., 1985). Instudies by the current authors, Cu and Zn have not appeared to do this.

Cu and Zn Localizations and Distributions in HairIn distal cross sections there are many localized concentrations of metals in the cuticle and in

the cortex (Merigoux et al., 2003; Kempson et al., 2006). Most of the localized concentrations are

TABLE 1. Typical concentrations of Cu and Zn in human head hair from population studies

n Cu, mean (SD)μg/g Zn, mean (SD)μg/g Notes Reference

58 13.170 (7.363) 207.919 (200.269) Male Khalique et al. (2005)30 24.506 (16.442) 251.369 (91.200) Female Khalique et al. (2005)83 12.352 (12.05) 156.48 (74.5) Urban industrialized population Chojnacka et al. (2005)266 7.96 (9.12) 129 (60.2) Nonindustrialized population Nowak (1998)114 25 (21) 142 (29) Urban population Rodushkin and Axelsson (2000)1091 44.1 (3.5) 156 (6) Miekeley et al. (1998)

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 614 I. M. KEMPSON ET AL.

colocalized by a variety of metals. The distal associations appear correlated with the general distri-bution of melanin in hair cross-sections but may also be associated with the nuclear remnants ofthe cortical cells. Different localized and colocalized distributions demonstrate that there aredifferent chemical affinities and associations related to these structural components. Melanin is apolyanionic polymer synthesized from tyrosine, possessing a large number of charged carboxylgroups and o-semiquinones. Subsequent binding between di- and trivalent metal ions and themelanin polymer are expected (Felix et al., 1978; Ito, 1986). Cortical cells are variable in their sizeand shape, but are quite long (~100 μm) and have a width of up to 6 μm (Robbins, 2002).Nuclear remnants are ~40-μm-long, spindle-shaped features 0.5 to 1 μm wide. As with melanin,the cystine-free nuclear remnants possess high concentrations of carboxyl functional groups as wellas amino groups (Swift, 1997) that may bind with cationic species. Another potential bindingmechanism may also exist with the cell membrane complex. The anionic free fatty acid content ofcell membrane complexes orientated with the fiber axis was attributed to the binding of Pb2+.Bertrand et al. (2003) investigated the lipid arrangement in hair fibers using x-ray diffraction (XRD).Results demonstrated an association of lead with the hair lipids and formation of lead soaps withthe free fatty acids.

In the hair root, S, Cu and Zn are the most notable elements (Kempson et al., 2006). S is repre-sentative of the hair matrix, originating from the cysteine and methionine content of the protein.The SXRF maps presented by Kempson et al. (2006) revealed a trend suggesting Cu and Zn arepotentially reliable bioindicators. Cu and Zn demonstrated less association with localized featuresand were more uniformly distributed through the hair, above and below the scalp.

Correlations of Cu and Zn ConcentrationsCaroli et al. (1998) examined hair from individuals associated with goldsmithing. Cu and Zn

produced concentrations in the test group (13.27 and 172.3 μg/g respectively, n=73) greaterthan any other trace metal examined. The control group similarly produced high results (11.08and 168.9 μg/g, n=22). No significance was found between the two populations for Zn. The Cuvalues however, produced a significant difference. On inspection of the data, it was found thatthe mean value of the Cu concentrations for the test group was substantially reduced by theremoval of 2 individuals from the test group of 73. In this case, these two data points might possi-bly be removed on the grounds of being outliers, however for the development of hair analysis itwould be worthy to discover the reason for their extraneous values. A small number of individu-als producing very high Cu concentrations, much greater than average concentrations, were alsofound elsewhere (Altaf et al., 2004). These individuals were restricted to a group suffering frommanic depression. The reason for their deviation may or may not give cause to be legitimatelyremoved (if contamination could be confirmed) but does give cause for speculation. Again, itcannot be shown that these differences are due to either contamination or increased biogenicconcentrations. Cu and Zn were selected within their study because they are present in the alloysto which the individuals may be exposed, hence it would be expected that the test group mayhave elevated levels in hair, which was not the case for Zn. In another study of a much largerpopulation (n=1802) (Batzevich, 1995), Zn and Cu were classed together as showing the lowestintra- and intergroup variability.

In a study by Trunova et al. (2003) it was concluded that only S, Ti, Co, Cu, Zn, Ga, and Se outof 20 elements studied (S, Cl, K, Ca, Sc, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Se, Br, Rb, Sr, Hg, andPb) were potentially reliable indicators of endogenous consumption. In another study, Rodushkinand Axelsson (2000) examined the correlations between elements. Cu and Zn exhibited no signifi-cant correlations. Such correlation studies are very useful for developing conclusions regarding theincorporation of elemental species. It is interesting to note that in their work, each of these metalswere positively correlated, suggesting that there are no competitive mechanisms by which one ionis replaced in preference for another (at least for the concentrations that occurred in their study).This is surprising since it is common in natural environments that certain species are displaced byother species with greater affinities for a particular binding site. The implications of this for hair anal-ysis are twofold: (1) Incorporated elements from endogenous sources are securely bound within the

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 CU AND ZN IN HAIR AND THEIR POTENTIAL ROLE AS BIOINDICATORS 615

hair and are not lost upon exposure to the environment (except in the case of Al when consideringother results from Kempson and Skinner [2005]); and (2) contamination is sufficiently bound withinthe hair as to not be removed easily (to the detriment of hair analysis). True endogenous competi-tion may exist but is masked by exogenous components and hence real inverse correlations may bedisguised and not detected.

The Impact of Exogenous MechanismsA major concern relates to the stability of Cu and Zn concentrations once the hair is exposed to

the environment. Cu and Zn were found to be stable in longitudinal analyses along hairs (Martinet al., 2005; Dombovari et al., 1999; Sky-Peck, 1990; Rodushkin & Axelsson, 2003), suggesting thatthere is no detectable influence from the external environment. It was also shown to be constant fordifferent areas of the head (Trunova et al., 2003). Why Cu and Zn appear to be stable along thelength of the hair and unaffected by environmental contamination is uncertain. It might beexpected that other metals, such as Mn, Fe, etc., that are chemically similar should exhibit the sametrend. Cu and Zn were not found to be correlated with any other elements considered by Rodushkinand Axelsson (2000) or Bermejo-Barrera et al. (1998). In contrast, Khalique et al. (2005) discoveredthat Zn was correlated with Fe in male subjects. In females, Zn and Ca, and Cu and Cr were corre-lated. Rodushkin and Axelsson (2000) considered significance at p < .01, however, while Khaliqueet al. considered significance at p < .05. This could be a reflection of either what is consideredsignificant or gender differences. What is surprising here is that Zn is correlated with Ca, since thetrends exhibited by Ca are not similar to Zn, e.g., longitudinal exogenous accumulation and highaffinity for the cuticle. Similar to the results of Rodushkin and Axelsson (2000), concentration varia-tions were small. Out of the elements studied, Cu exhibited the least variation in values in maledonors, while Zn was least variable in the female subjects.

Nicolis et al. (2000) measured Zn variations along individual hairs to monitor Zn status duringnutritional supplementation. They concluded thatg synchrotron XRF analysis of hair was a reliablemethod for temporal monitoring of Zn. Cu and Zn concentrations in the hair of South Americanmummies dated pre ~500 BC were effectively identical to modern data ranges (Du et al., 1996). Inanother case of a 3200-yr-old sample, low Zn may be related to a dietary deficiency (Baptista et al.,1981). These results provide evidence that Cu and Zn are resistant to exogenous accumulation andin some capacity reflect biogenic activity.

Confirmation of the discrimination between endogenous and exogenous Cu and Zn may beassisted if there are organic compounds that reflect metabolism. The authors are not aware of anyrelevant research in this area. Alternatively, isotopic analysis may assist in corroborating theevidence. Ohno et al. (2005) measured Zn isotope ratios in red blood cells and hair. In red bloodcells δ66Zn was 0.39 to 0.47‰, while in hair it was –0.16‰. In another pooled hair measurement,a δ66Zn of –0.46 ± 0.040‰ was measured (Stenberg et al., 2004). Zn was not noted to accumulatelongitudinally. If the bulk of Zn was endogenous and anthropogenic Zn had to accumulate suffi-ciently to reduce the Zn isotope ratio sufficiently, the contribution of the bulk Zn measurementmight be observed to increase along the hair. It was proposed that transport of Zn across biologicalmembranes favours light isotopes (Weiss et al., 2005). This would then be consistent with the iso-tope shift observed between blood and hair. As an indication of isotope ratios in minerals and ores,δ66Zn in sphalerite samples varies from −0.17 to 1.33‰ (Wilkinson et al., 2005), and, with theexception of one sample (which measured −0.19‰), δ66Zn varied between 0.02 and 0.44‰ in aselection of ores and sedimentary material (Marechal et al., 1999). These values demonstrate that itwould be unlikely for contamination to sufficiently influence hair isotopic ratios to those that weremeasured. However, industrial processing of Zn appears to fractionate isotopes such that anthropo-genic sources may exhibit alternative ratios (Tanimizu et al., 2002; Mason et al., 2004). These havenot been sufficiently identified as yet so other exogenous sources, such as pollution from smelting,cannot be commented on. There is even less information regarding Cu. In an early publication byDever and Bresse (1989) rats were fed different forms of Cu, the isotope ratios were subsequentlymeasured in the hair. Their most precise measurements indicated the lighter isotope to remainroughly constant or enhanced.

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 616 I. M. KEMPSON ET AL.

CU AND ZN BINDING IN HAIR

Understanding the incorporation mechanisms of Cu and Zn may be assisted by results pub-lished regarding Ca (Merigoux et al., 2003; Kempson et al., 2003). Merigoux et al. (2003) observedthat Ca associated with granules in the hair cortex was easily removed by HCl. A second type ofbinding for Ca consisted of deposits located in the medulla wall, probably also in the cuticle, andrather uniformly in the cortex, none of which were easily removed using HCl. It was speculated thatthese latter calcium atoms may be bound to proteins. Perhaps Cu and Zn have a higher affinity forS or other groups rather than O functional groups, whereas Ca may bind with both S and O siteswith only one of these sites being easily removed by acid. Merigoux et al. (2003) indicated that theeasily removed Ca is associated with granular features likely to be comprised primarily of lipids. Thebound Ca that is not easily removed exhibits a more delocalized distribution and is spread moreuniformly through the cortex and has greater association with the medulla wall. It was proposedthat this form of Ca is associated with the hair proteins. Perhaps the difference between Ca, and Cuand Zn is that the latter do not form soaps with lipids. The importance of proteins for bindingmetals, in particular in the medulla, was previously demonstrated (Kempson & Skinner, 2005;Kempson et al., 2003).

To understand the fundamental mechanisms of metal binding within hair, it is desirable toidentify which chemical and structural features are relevant. Thus, the tendency of elemental andmolecular species to reflect endogenous activity may be deduced. Hair is comprised of manydifferent components such as proteins, lipids, and melanin, exhibiting variable forms with hairstructure. Hair is well known to contain high-S proteins, in addition to glycine-tyrosine-rich proteinsand acidic and neutral to basic side-chained proteins comprising the intermediate filaments. Theinter-microfibrillar matrix is also a cystine-rich protein. The lipid content comprises multiple com-ponents but in particular 18-methyleicosanoic acid with minor quantities of cholesterol sulfate,cholesterol, and fatty alcohol. All of the cells within hair are coated by lipid layers covalently boundto the protein. Various melanin monomers exist in the aggregation of eumelanin and pheomelaningranules, which each have variations in chemical reactivity.

It is not known how Cu or Zn are specifically bound within hair. Cu+ will preferentially bindwith sulfhydryl groups. Cu2+ and Zn2+ show a preference for the generation of ligands with amines(an N bound to C and possibly H atoms), thiolates, and ionized peptides and carboxylates, respec-tively. Each form of Cu can exist in biological systems, but Cu+ can be a better for a generalizedmodel of complexation with S, although some Cu(II)–S organometallics, particularly in mixed coor-dination with N, do exist (Pal et al., 1968; Palaniandavar, 2004; Besic et al., 2005). The absoluteoxidation state of Cu bound to S is difficult to measure, as in this environment it can exhibit mixedd-character (Goh et al., 2006). Hair contains roughly 16% of its N as −NH2 amines; therefore, hair islikely to provide preferable sites for the binding and retention of Cu and Zn. As an indication ofreactivity, stability constants of Cu2+- and Zn2+-complexed as amines are 13.00 and 9.65, respec-tively (Williams & Frausto da Silva, 1996), indicating a high affinity for such chemical features. Inaddition, the Zn2+ ion has a high affinity for cysteine (log K=9.17) and Cu2+ for histadine (logK=10.20). Solubility products for CuS (Ksp=6×10-36) and ZnS (4.5×10-24) (Williams & Frausto daSilva, 1996) indicate that if S groups are available for undergoing complexation with Cu and Zn,their resulting incorporation is likely to be of a reasonably secure nature. However, Ca (which isreadily influenced by external contamination) has a preference for carboxyl, phosphate, and someneutral O-donors. Melanin comprises o-semiquinone functional groups, which aid in the binding ofmetal ions such as Ca. Future analysis by the current authors will investigate how Cu and Zn arebound within hair, which will most likely indicate a variety of complexes dependant on local chem-istry and will be compared with other elements such as Ca.

Trichohyalin proteins may also influence the incorporation of metals. These granular featuresoccur in the medulla and can also occur in the cortex (Robbins, 2002). It is believed that it is thesefeatures reflected in an SEM image provided elsewhere (Kempson et al., 2006). This type of protein,with an excess of acidic residues (approximately 75% of its composition comprises glutamic acid,glutamine, arginine and leucine residues), is hydrophilic. Approximately 57% of the amino acids

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 CU AND ZN IN HAIR AND THEIR POTENTIAL ROLE AS BIOINDICATORS 617

possess ionizable side chains (recall that Cu and Zn have an affinity for ionized peptides). However,the NH2 terminus has an overall hydrophobic nature and actually resembles a family of otherCa-binding proteins. It was proposed that trichohyalin comprises the bulk of protein in a maturemedulla (Stark et al., 1990), which may explain the localizations of metals within the medullaobserved (Kempson & Skinner, 2005). In sheep wool, trichohyalin is comprised of 26.1 mol% and17.2 mol% of glutamine and glutamate respectively. These have functional groups terminating witha −CONH2 (amide) and −COOH, respectively (recall that Ca has an affinity for carboxyl groupswhile Cu and Zn have affinity for amines). The structure of trichohyalin was identified to possess Cabinding domains, and it was postulated that the association was related to Ca-dependent enzymeactivity within the follicle (Fietz et al., 1993). The binding of Ca may be primarily driven by theglutamate functional group. However, the Ca concentrations once exposed to the environment areno longer sufficiently reliable to be representative of such action. It is conceivable then to expectthat Zn and Cu may bind with the free amine groups associated with the trichohyalin protein,presumably largely contributed to by the high glutamine content. The authors are not aware of anyevidence in the literature to demonstrate whether Cu and Zn are concentrated in the medulla or not.

Unfortunately, the sample studied in the work of Kempson et al. (2006) did not possess amedulla. It would be interesting to see whether the medullar proteins contribute to higher retentionof Zn and Cu than cortical features. Medullar proteins appear to be quite variable in their chemicalassociations (Kempson et al., 2003). There is a lack of literature describing sufficiently sensitive anal-yses of transverse distributions of Cu and Zn. Radial analyses of Zn distributions were previouslypresented (Dombovari et al., 1999; Martin et al., 2005); however, in each case, insufficient sensitivityor an inability to confirm the presence of a medulla did not reveal the influence of the medulla. Inany case, the cortical proteins still appear to contribute to a higher retention of Cu and Zn than theydo for other elements. If Cu and Zn are not associated with the medulla, this may be reflective ofactivity within the follicle. For example, Cu and Zn are likely to be incorporated into the hair via theblood supply within the dermal papilla. If Cu and Zn are securely incorporated only at the begin-ning of hair formation, they may not be exposed to features present in the denatured medullarycanal at a subsequent stage of hair development.

Pb, Cd, Zn, Se, and Cu were specifically studied in hair from people exposed to pollution in thework place (Wasiak et al., 1996; Srivastava et al., 1997). Pb and Cd were able to accumulate in thehair in high concentrations. This supports the observations of Pb accumulating in the hair fromexposure to contamination. An interesting observation, however, was reduced Zn concentrationswith higher Pb or Cd levels (Pb and Cd are other elements that were not identified to have any cor-relations by Rodushkin and Axelsson [2000]). If Pb is the cause of the reduced Zn concentrations,there are two possible explanations for this: (1) Pb contamination displaces Zn in a competitivebinding role; or (2) endogenous Pb interferes in the metabolic mechanisms by which Zn is incorpo-rated. These results represent some of the few negative correlations published in the literature. Thissecond possibility is consistent with Pb and Cd playing a disruptive role in the biogenic activityinvolving Zn. Pb is well known to impair mental and physical development, decrease hemoglobinsynthesis, and, among other things, alter DNA in exposed children (Yanez et al., 2003). The sideeffects from exposure to Pb may play a role in altering Zn and Cu concentrations in hair due to itsimpact on metabolic activity. Blood Pb may also replace Zn in Zn-containing heme enzymes; theyexhibit an inverse relationship in blood concentrations (Goyer, 1997).

In another study, Nowak and Chmielnicka (2000) focused specifically the impact of Cd and Pbon the concentrations of essential elements (Fe, Zn, Cu, and Ca) in hair, teeth, and fingernails. Dataindicated that Pb concentrations in hair were inversely correlated to Ca and Fe concentrations.In nails, Pb was believed to be related to a decrease in Cu and Zn. While a correlation between Pband Cu or Zn was not observed in hair samples, it is interesting that in the case of the fingernails,Pb was inversely related to Cu and Zn concentrations. This evidence suggests that Pb may interferein biological functions that involve Cu and Zn, providing this correlation can be assumed to beunrelated to exogenous mechanisms. Not all studies observed the same trends in results. Somedependence on the population groups studied may account for differences in the correlationsobserved by Nowak and Chmielnicka (2000) as compared to the study by Wasiak et al. (1996).

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 618 I. M. KEMPSON ET AL.

Rodushkin and Axelsson (2000) did not observe any correlations between S, P, Zn, Cu, Se, Cd, Pb,As, and Tl. Of these elements, some appear to occur primarily due to contamination (e.g., Pb andpossibly P and Se) and others primarily due to endogenous uptake (e.g., Zn and Cu).

The observations presented earlier are interesting in that it is well known that elemental speciesmay easily accumulate on and in hair once exposed to the environment. The suggestion thatCu and Zn do not accumulate via exogenous sources, while so many other elements do, raisesinteresting questions. For instance, the loci for the Cu and Zn must be either saturated or no longeraccessible once hair has formed. How this relates to the composition and formation of hair fibersand the role of Cu and Zn within the follicle will likely prompt further dedicated research.

CU AND ZN AS BIOINDICATORS

While Zn may not be indicative of exposure in the environment, in some instances it may per-haps be used as a monitor for metabolic differences; i.e., the variability of Zn measured in hair is adirect result of the body’s use of Zn rather than the quantity available. However, this might not beapplicable to individuals with dietary deficiencies, which may also be reflected by hair concentra-tions. Similarly, while Cu concentrations are largely regulated by metabolism during formation ofthe hair, it appears that this metal is prone to deviate significantly from mean values (Caroli et al.,1998). Other data suggest that there are influences on Zn hair concentrations attributed to breastcancer and health conditions that result in dietary deficiency (Altaf et al., 2004). Cu concentrations(along with Mg, Mn, and in particular Ni) were statistically altered in hair of esophageal cancerpatients, while Zn (along with Fe, Se, and Pb) were not altered (Azin et al., 1998). Kilic et al. (2004)reported a reduced Zn concentration in a group suffering from breast cancer, along with anincrease in Cu. Kolmogorov et al. (2000) also reported a decrease in Zn concentrations (along withSe, and an increase in Cr) in a group of breast cancer patients.

Total reflection XRF was used to report on the analysis of Cu, Fe, Se, and Zn in blood serum(Hernandez-Caraballo et al., 2005). In that study, blood serum samples were analyzed from a groupof 27 individuals who were diagnosed with cancer and a group of 32 individuals who received anegative diagnosis. Based on statistical analysis of the elemental concentrations, the healthy(specificity=90−100%) and unhealthy individuals (sensitivity=100%) were distinguished. TheZn:Cu ratio was particularly important in the discrimination between the two groups. These resultsprovide evidence that cancers may alter blood elemental concentrations. It appears that the alter-ation in Cu and Zn concentrations may impact on the concentrations incorporated into hair, whichobtains its nutrition from the blood supply.

These observations also relate to the diagnostic potential of hair proposed by James et al.(2005), where hair diffraction patterns were proposed to indicate individuals afflicted with breastcancer. In that study, a blind test reported on the analysis of hair from 503 individuals (210 ofwhom were diagnosed with breast cancer). No false negatives were reported, while 47 false posi-tives occurred (specificity=84%). The negative results might be misleading, however, as there ispotential for the individuals given a negative diagnosis to still develop breast cancer (James, 2003b),indicating that this method may provide an earlier diagnosis than conventionally possible. After thisdiagnostic analysis was first proposed by James et al. (1999) there has been considerable debateregarding the accuracy in its application (Amenitsch et al., 1999; Chu et al., 1999; Schroer et al.,1999; Howell et al., 2000; Meyer et al., 2000; James, 2003a). These latest results, however, remainconvincing, and such structural alterations are supported by the discussion presented here. Itappears that modified metabolic activity in the follicle as a result of altered blood trace elementalconcentrations may affect the structure of the hair.

Other metabolic processes may be indicated by hair Cu and Zn concentrations that relate totheir binding and transport within the body. For instance, metallothioneins are small, cysteinyl-rich proteins that bind divalent ions and are important in the transportation of metals, especiallyZn. Pb and Cd compete for such binding opportunities and inhibit the transportation of othermetals including Cu and Zn. It was proposed that metallothionein dysfunction may be a funda-mental cause of autism (Walsh et al., 2001). Zn concentrations were found to be low in blood

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 CU AND ZN IN HAIR AND THEIR POTENTIAL ROLE AS BIOINDICATORS 619

plasma and erythrocytes as well as in the hair of autistic children (Yorbik et al., 2004). In addition,serum albumin was shown to increase in cases of autism (Croonenberghs et al., 2002). Albumin isa Cu- and Zn-binding protein and was conversely shown to be reduced in blood serum inpatients suffering from dementia (Molaschi et al., 1996). Molaschi et al. (1996) observed a trendof lower concentrations of Cu and Zn in blood of individuals suffering from dementia, althoughthey did not place significance on their measurements. Albumin levels were also found to bealtered in the serum of retarded children (Sanchez-Lastres et al., 2003). Cu concentrations in hairwere associated with retardation in children (Man & Zheng, 2002), and Cu and Zn concentra-tions also appeared to be altered in hypertensive patients (Afridi et al., 2006). These examplesdemonstrate that impaired metabolic function within the body may impact on the Cu and Znaccessible by the hair’s dermal papilla and hence alter hair concentrations. Malabsorption,Menkes and Wilson syndromes, inflammation, and infection may also be reflected by altered Cuand/or Zn levels.

Statistical differences in Cu and Zn concentrations from control levels tend to be restricted topopulations that suffer from ailments (Azin et al., 1998; Altaf et al., 2004), rather than those whoare occupationally or environmentally exposed (Caroli et al., 1998). Presuming that Cu and Znprovide accurate indications of metabolic or biogenic activity, it may represent either activity withinthe follicle only, or within the greater system of the body or parts thereof.

SUMMARY

The first step in developing hair analysis as an applied tool is to identify what is of endogenousorigin and discriminate it from anthropogenic sources. If a point is reached whereby elementalconcentrations can be confirmed to have originated endogenously, other factors still need to beconsidered, such as age, gender, hair color, and hair treatments (Sky-Peck, 1990; Altaf et al., 2004;Malter et al., 2005; Chojnacka et al., 2006). Meng (1996) measured higher Zn concentrations inthe hair of females compared to males and concentrations increased until the onset of pubertybefore decreasing with age. However, Bertazzo et al. (1996) did not detect any gender differencesfor Cu or Zn, and any age differences were predominantly in females (Cu decreased in the over 60 yrold demographic and Zn increased between the 2–5 and 20–40 yr old groups). Cu concentrationswere lowest in white hair and no significant difference was detected for Zn between different haircolors. Various processes lead to altered chemical structures in hair that influence elemental con-centrations and need to be considered in the development of any application. Oxidative processeslead to the formation of cysteic acid salts (from peroxide bleaching) or Bunte salts (from sulfiteperms and sunlight oxidation). Some effects on elemental concentrations due to bleaching weredemonstrated elsewhere (Kempson & Skinner, 2005) but did not include Cu and Zn. Underalkaline conditions, other metal complexes can form (−CO2M). These factors also need to beconsidered in any serious attempt to apply hair mineral analysis for biomonitoring. It is likely thatsome, if not most, chemical alterations of hair will diminish the usefulness of Cu and Zn data.

Contamination from the environment is the major contributor to several, if not most, elementsmeasured in hair (e.g., Ca and Pb). As such, these elements do not currently offer to accuratelyreflect the endogenous function of individuals for routine analysis, largely due to the fact that con-tamination is not distinguishable. Recent results and the pooling of information from the literatureoffer valuable information to assist in understanding the incorporation of metals into hair. Cu andZn have been targeted as demonstrating promise as reflecting biogenic activity. While it is likely thatexternal processes will still impact on concentrations of Cu and Zn, this appears to be much less sig-nificant than what occurs for other elements.

It appears, however, that in some cases hair concentrations have a dependence on biogenicactivity rather than being purely a function of biogenic concentrations (such as blood concentra-tions). For example, Pb may interfere with the metabolism in the hair follicle and alter Cu and Znhair concentrations from the values expected based on blood concentrations. Potential still existsfor Cu and Zn deficiencies to be represented in these cases. Large, anomalous Cu concentrations(such as those measured by Caroli et al. [1998] and Altaf et al. [2004]) that deviate significantly from

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 620 I. M. KEMPSON ET AL.

typical concentrations may also reflect a medical disposition, or simply a particular cosmetic treat-ment or exogenous source, which remains unclear.

Evidence in the literature indicates that metabolic activity involving Cu and Zn may be altereddue to the health of an individual. Pb and Cd appear to impact on metabolism and alter Cu and Znconcentrations. Other evidence exists to demonstrate that Cu and Zn concentrations in componentsof blood or their transport deviate from normal values in instances of autism, dementia, mental retar-dation and cancer. This in turn appears to result in two things: (1) Hair Cu and Zn concentrationsare likewise altered; and (2) in the case of breast cancer sufferers, these altered concentrationsimpact on the growth of hair and lead to the altered structures observed in x-ray diffraction analysis.Potential in the analysis of Zn and Cu does exist and deserves further specific study.

REFERENCESAfridi, H. I., Kazi, T. G., Jamali, M. K., Kazi, G. H., Arain, M. B., Jalbani, N., and Shar, G. Q. 2006. Analysis of heavy metals in scalp hair

samples of hypertensive patients by conventional and microwave digestion methods. Spectroscopy Lett. 39:203–214.Altaf, W. J., Akanle, O. A., Admans, L. L., Beasley, D., Butler, C., and Spyrou, N. M. 2004. The University of Surrey database of elemental

composition of human hair. J. Radioanal. Nucl. Chem. 259:493–498.Amenitsch, H., Rappolt, M., Laggner, P., Bernstorff, S., Moslinge, R., Fleischmann, E., Wagner, T., Lax, S., and Petru, S. 1999. Synchrotron

x-ray study at Trieste: No correlation between breast cancer and hair structure. Synchrotron Rad. News 12:32–34.Azin, F., Raie, R. M., and Mahmoudi, M. M. 1998. Correlation between the levels of certain carcinogenic and anticarcinogenic trace

elements and esophageal cancer in northern Iran. Ecotoxicol. Environ. Safety 39:179–184.Baptista, G. B., Montenegro, E. C., Paschoa, A. S., and Barros, L. C. 1981. Analysis of trace elements in human hair by PIXE. Nucl.

Instrum. Methods 181:263–267.Batzevich, V. A. 1995. Hair trace element analysis in human ecology studies. Sci. Total Environ. 164:89–98.Bermejo-Barrera, P., Moreda-Pineiro, A., Moreda-Pineiro, J., and Bermejo-Barrera, A. 1998. Acid predigestion as a slurry pretreatment

for the determination of Ca, Cu, K, Mg, Na and Zn in human scalp hair by flame atomic absorption/emission spectrometry with ahigh-performance nebulizer. Fresen. J. Anal. Chem. 360:707–711.

Bertazzo, A., Costa, C., Biasiolo, M., Allegri, G., Cirrincione, G., and Presti, G. 1996. Determination of copper and zinc levels in humanhair: Influence of sex, age, and hair pigmentation. Biol. Trace Elem. Res. 52:37–53.

Bertrand, L., Doucet, J., Simionovici, A., Tsoucaris, G., and Walter, P. 2003. Lead-revealed lipid organization in human hair. Biochim.Biophys. Acta 1620:218–224.

Bertsch, P. M., and Hunter, D. B. 2001. Applications of synchrotron-based x-ray microprobes. Chem. Rev. 101:1809–1842.Besic, E., Gomzi, V., Sankovic, K., Herak, J. N., and Krilov, D. 2005. EPR study of a copper impurity center in a single crystal of 2-

thiothymine. Spectrochim. Acta A 61:2803–2808.Borella, P., Rovesti, S., Caselgrandi, E., and Bargellini, A. 1996. Quality control in hair analysis: a systematic study on washing procedures

for trace element determinations. Mikrochim. Acta 123:271–280.Bos, A. J. J., van der Stap, C. C. A. H., Valkovic, V., Vis, R. D., and Verheul, H. 1985. Incorporation routes of elements into human hair;

Implications for hair analysis used for monitoring. Sci. Total Environ. 42:157–169.Caroli, S., Senofonte, O., Violante, N., D'llio, S., Camai, S., Chiodo, F., and Menditto, A. 1998. Diagnostic potential of hair analysis as

applied to the goldsmith sector. Microchem. J. 59:32–44.Chojnacka, K., Gorecka, H., Chojnacki, A., and Gorecki, H. 2005. Inter-element interactions in human hair. Environ. Toxicol. Pharmacol.

20:368–374.Chojnacka, K., Gorecka, H., and Gorecki, H. 2006. The effect of age, sex, smoking habit and hair color on the composition of hair. Environ.

Toxicol. Pharmacol. 22:52–57.Chu, B., Fang, D., and Hsiao, B. S. 1999. Hair test results at the advanced polymer beamline (X27C) at the NSLS. Synchrotron Rad. News

12:36.Croonenberghs, J., Wauters, A., Devreese, K., Verkerk, R., Scharpe, S., Bosmans, E., Egyed, B., Deboutte, D., and Maes, M. 2002.

Increased serum albumin, y globulin, immunoglobulin IgG, and IgG2 and IgG4 in autism. Psychol. Med. 32:1457–1463.Dever, M., and Bresse, R. R. 1989. Enriched copper isotope tracer for dietary copper incorporation into hair using neutron activation

analysis in conjunction with direct-current argon plasma spectrometry. Anal. Chim. Acta 225:185–192.Dombovari, J., Papp, L., Uzonyi, I., Borbely-Kiss, I., Elekes, Z., Varga, Z., Matyus, J., and Kakuk, G. 1999. Study of cross-sectional and

longitudinal distribution of some major and minor elements in the hair samples of haemodialysed patients with micro-PIXE. J. Anal.Atom. Spectrom. 14:553–557.

Du, A. Y., Mangelson, N. F., Rees, L. B., and Matheny, R. T. 1996. Pixe elemental analysis of South American mummy hair. Nucl.Instrum. Methods B 109/110:673–676.

Felix, C. C., Hyde, J. S., and Sarna, T. 1978. Interactions of melanin with metal ions: Electron spin resonance evidence for chelatecomplexes of metal ions with free radicals. J. Am. Chem. Soc. 100:3922–3926.

Fietz, M. J., McLaughlan, C. J., Campbell, M. T., and Rogers, G. E. 1993. Analysis of the sheep trichohyalin gene: Potential structural andcalcium-binding roles of trichohyalin in the hair follicle. J. Cell Biol. 121:855–865.

Goh, S. W., Buckley, A. N., Lamb, R. N., Rosenberg, R. A., and Moran, D. 2006. The oxidation states of copper and iron in mineralsulfides, and the oxides formed on initial exposure of chalcopyrite and bornite to air. Geochim. Cosmochim. Acta 70:2210–2228.

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 CU AND ZN IN HAIR AND THEIR POTENTIAL ROLE AS BIOINDICATORS 621

Goyer, R. A. 1997. Toxic and essential metal interactions. Annu. Rev. Nutr. 17:37–50.Henderson, G. S., and Baker, D. R., eds. 2002. Synchrotron radiation: Earth, environmental and material sciences applications. Short

Course Series 30. St. Johns, Newfoundland, Canada: Mineralogical Association of Canada.Hernandez-Caraballo, E. A., Rivas, F., Perez, A. G., and Marco-Parra, L. M. 2005. Evaluation of chemometric techniques and artificial

networks for cancer screening using Cu, Fe, Se and Zn concentrations in blood serum. Anal. Chim. Acta 533:161–168.Howell, A., Grossman, J. G., Cheung, K. C., Kanbi, L., Evans, D. G., and Hasnain, S. S. 2000. Can hair be used to screen for breast

cancer? J. Med. Genet. 37:297–298.Ito, S. 1986. Reexamination of the structure of eumelanin. Biochem. Biophys. Acta 883:155–161.James, V., Corino, G., Robertson, T., Dutton, N., Halas, D., Boyd, A., Bentel, J., and Papadimitriou, J. 2005. Early diagnosis of breast

cancer by hair diffraction. Int. J. Cancer 114:969–972.James, V., Kearsley, J., Irving, T., Amemiya, Y., and Cookson, D. 1999. Using hair to screen for breast cancer. Nature 398:33–34.James, V. J. 2003a. The traps and pitfalls inherent in the correlation of changes in the fibre diffraction pattern of hair with breast cancer.

Phys. Med. Biol. 48:L5–L9.James, V. J. 2003b. False-positives in studies of changes in fibre diffraction of hair from patients with breast cancer may not be false. J.

Nat. Cancer. Inst. 95:170–171.Kales, S. N., and Goldman, R. H. 2002. Mercury exposure: Current concepts, controversies, and a clinic's experience. J. Occup. Environ.

Med. 44:143–154.Kempson, I. M. 2004. ToF-SIMS measurements of elemental and molecular distributions in human hair. Ian Wark Research Institute,

University of South Australia, Adelaide.Kempson, I. M., and Skinner, W. M. 2005. ToF-SIMS analysis of elemental distributions in human hair. Sci. Total Environ. 338:213–227.Kempson, I. M., Skinner, W. M., and Kirkbride, K. P. 2006. Advanced analysis of metal distributions in human hair. Environ. Sci. Technol.

40:3423–3428.Kempson, I. M., Skinner, W. M., and Kirkbride, P. K. 2003. Calcium distributions in human hair by ToF-SIMS. Biochim. Biophys. Acta 1624:1–5.Khalique, A., Ahmad, S., Anjum, T., Jaffar, M., Shah, M. H., Shaheen, N., Tariq, S. R., and Manzoor, S. 2005. A comparative study based

on gender and age dependence of selected metals in scalp hair. Environ. Monit. Assess. 104:45–57.Kidwell, D. A., and Blank, D. L. 1996. Environmental exposure—The stumbling block of hair testing. In Drug testing in hair, ed. P. Kintz,

pp. 17–68. Boca Raton, FL: CRC Press.Kilic, E., Demiroglu, A., Saraymen, R., and Ok, E. 2004. Comparative quantative analysis of zinc, magnesium, and copper content in the

scalp hair of healthy people and breast cancer patients. J. Trace Elem. Exp. Med. 17:175–180.Kolmogorov, Y., Kovaleva, V., and Gonchar, A. 2000. Analysis of trace elements in scalp hair of healthy people, hyperplasia and breast

cancer patients with XRF method. Nucl. Instrum. Methods A 448:457–460.Lin, T.-H., Huang, Y.-L., and Wang, M.-Y. 1998. Arsenic species in drinking water, hair, fingernails, and urine of patients with blackfoot

disease. J Toxicol. Environ. Health A 53:85–93.Malter, R., Rendon, C., and Aalund, R. 2005. A developmental study of sex difference in hair tissue mineral analysis patterns at ages six,

twelve and eighteen. J. Orthomol. Med. 20:245–254.Man, C. K., and Zheng, Y. H. 2002. Analysis of trace elements in scalp hair of mentally retarded children. J. Radioanal. Nucl. Chem.

253:375–377.Mandal, B. K., Ogra, Y., and Suzuki, K. T. 2003. Speciation of arsenic in human nail and hair from arsenic-affected area by HPLC-inductively

coupled argon plasma mass spectrometry. Toxicol. Appl. Pharmacol. 189:73–83.Marechal, C. N., Telouk, P., and Albarede, F. 1999. Precise analysis of copper and zinc isotopic compositions by plasma-source mass

spectrometry. Chem. Geol. 156:251–273.Martin, R. R., Kempson, I. M., Naftel, S. J., and Skinner, W. M. 2005. Preliminary synchrotron analysis of lead in hair from a lead smelter

worker. Chemosphere 58:1385–1390.Mason, T. F. D., Weiss, D. J., Horstwood, M., Parrish, R. R., Russell, S. S., Mullane, E., and Coles, B. J. 2004. High-precision Cu and Zn

isotope analysis by plasma source mass spectrometry Part 2. Correcting for mass discrimination effects. J. Anal. Atom. Spectrom.19:218–226.

Meng, Z. 1996. Age- and sex-related difference in zinc and lead levels in human hair. Biol. Trace Elem. Res. 61:79–87.Merigoux, C., Briki, F., Sarrot-Reynauld, F., Salome, M., Fayard, B., Susini, J., and Doucet, J. 2003. Evidence for various calcium sites in

human hair shaft revealed by sub-micrometer x-ray fluorescence. Biochim. Biophys. Acta 1619:53–58.Meyer, P., Goergl, R., Botz, J. W., and Fratzl, P. 2000. Breast cancer screening using small-angle x-ray scattering analysis of human hair.

JNCI 92:1092–1093.Miekeley, N., Carneiro, M. T. W. D., and Dasilveira, C. L. P. 1998. How reliable are human hair reference intervals for trace elements?

Sci. Total Environ. 218:9–17.Molaschi, M., Ponzetto, M., Bertagna, B., Berrino, E., and Ferrario, E. 1996. Determination of selected trace elements in patients

affected by dementia. Arch. Gerontol. Geriatr. Suppl. 5:39–42.Nicolis, I., Deschamps, P., Curis, E., Chevallier, P., Corriol, O., Colomb, V., and Benazeth, S. 2000. Using hair as a diagnostic tool: X-ray

fluorescence monitoring of the Zn content of hairs of patients under parenteral nutrition. J. Trace Microprobe Tech. 18:511–516.Nicolis, I., Deschamps, P., Curis, E., Corriol, O., Acar, V., Zerrouk, N., Chaumeil, J. C., Guyon, F., and Benazeth, S. 2001. XAS applied to

pharmaceuticals: drug administration and bioavailability. J. Synchrotron Radiat. 8:984–986.Nowak, B. 1998. Contents and relationship of elements in human hair for a non- industrialised population in Poland. Sci. Total Environ.

209:59–68.Nowak, B., and Chmielnicka, J. 2000. Relationship of lead and cadmium to essential elements in hair, teeth, and nails of environmentally

exposed people. Ecotoxicol. Environ. Safety 46:265–274.

Dow

nloa

ded

By:

[Kem

pson

, Iva

n M

.] A

t: 22

:55

13 D

ecem

ber 2

007 622 I. M. KEMPSON ET AL.

Ohno, T., Shinohara, A., Chiba, M., and Hirata, T. 2005. Precise Zn isotopic ratio measurements of human red blood cell and hairsamples by multiple collector-ICP-mass spectrometry. Anal. Sci. 21:425–428.

Pal, A. K., Mitra, S. N., and Sengupta, P. 1968. Studies on the paramagnatism of sulfur-coordinated copper diethyldithiocarbamate. J.Chem. Phys. 48:5219–5222.

Palaniandavar, M. 2004. Synthetic models for electron transfer blue copper proteins: Structure, spectra and redox properties of certaincopper(II) complexes of bis (benzimidazolyl)-thioether ligands. Proc. Indian Nat. Sci. Acad. A 70:293–310.

Ponce, R. A., Bartell, S. M., Kavanach, T. J., Woods, J. S., Griffith, W. C., Lee, R. C., Takaro, T. K., and Faustman, E. M. 1998. Uncer-tainty analysis methods for comparing predictive models and biomarkers: A case study of dietary methyl mercury exposure. Reg.Toxicol. Pharmacol. 28:96–105.

Raab, A., Hansen, H. R., Zhuang, L. Y., and Feldmann, J. 2002. Arsenic accumulation and speciation analysis in wool from sheepexposed to arsenosugars. Talanta 58:67–76.

Revich, B. A. 1994. Lead in hair and urine of children and adults from industrialized areas. Arch. Environ. Health 49:59–62.Robbins, C. R. 2002. Chemical and physical behavior of human hair, 4th ed. New York: Springer.Rodushkin, I., and Axelsson, M. D. 2000. Application of double focusing sector field ICP-MS for multielemental characterization of

human hair and nails. Part II. A study of the inhabitants of northern Sweden. Sci. Total Environ. 262:21–36.Rodushkin, I., and Axelsson, M. D. 2003. Application of double focusing sector field ICP-MS for multielemental characterisation of

human hair and nails. Part III. Direct analysis by laser ablation. Sci. Total Environ. 305:23–39.Samanta, G., Chowdhury, T. R., Mandal, B. K., Biswas, B. K., Chowdhury, U. K., Basu, G. K., Chanda, C. R., Lodh, D., and Chakraborti,

D. 1999. Flow injection hydride generation atomic absorption spectrometry for determination of arsenic in water and biologicalsamples from arsenic-affected districts of West Bengal, India, and Bangladesh. Microchem. J. 62:174–191.

Sanchez-Lastres, J., Eiris-Punal, J., Otero-Cepeda, J. L., Pavon-Belinchon, P., and Castro-Gago, M. 2003. Nutritional status of mentallyretarded children in northwest Spain: II. Biochemical indicators. Acta Paediatr. 92:928–934.

Schroer, K., DeRisis, D., Kastrow, K., Busch, E., Volkow, N., and Capel, M. 1999. Hair test results at the NSLS. Synchrotron Rad. News12:34–35.

Seidel, S., Kreutzer, R., Smith, D., McNeel, S., and Gilliss, D. 2001. Assessment of commercial laboratories performing hair mineral anal-ysis. J. Am. Med. Assoc. 285:67–72.

Sky-Peck, H. H. 1990. Distribution of trace elements in human hair. Clin. Physiol. Biochem. 8:70–80.Srivastava, A. K., Gupta, B. N., Bihari, V., Gaur, J. S., and Mathur, N. 1997. Hair selenium as a monitoring tool for occupational expo-

sures in relation to clinical profile. J. Toxicol. Environ. Health 51:437–445.Stark, H.-J., Breitkreutz, D., Limat, A., Ryle, C. M., Roop, D., Leigh, I., and Fusenig, N. E. 1990. Keratins 1 and 10 or homologues as reg-

ular constituents of inner root sheath and cuticle cells in the human hair follicle. Eur. J. Cell Biol. 52:359–372.Stenberg, A., Andren, H., Malinovsky, D., Engstrom, E., Rodushkin, I., and Baxter, D. C. 2004. Isotopic variations of Zn in biological

materials. Anal. Chem. 76:3971–3978.Swift, J. A. 1997. Morphology and histochemistry of human hair. In Formation and structure of human hair, ed. P. Jolles, H. Zahn, and H.

Hocker, pp. 149–176. Basel: Birkhauser Verlag.Tagliaro, F., Smith, F. P., Debattisti, Z., Manetto, G., and Marigo, M. 1997. Hair analysis, a novel tool in forensic and biomedical

sciences—Chromatographic and electrophoretic/electrokinetic analytical strategies. J. Chromatogr. Biomed. 689:261–271.Tanimizu, M., Asada, Y., and Hirata, T. 2002. Absolute isotopic composition and atomic weight of commercial zinc using inductively

coupled plasma mass spectrometry. Anal. Chem. 74:5814–5819.Toribara, T. Y. 1995. Information from lateral scans of single hairs by x-ray fluorescence. Instrum. Sci. Technol. 23:217–226.Toribara, T. Y. 2001. Analysis of single hair by XRF discloses mercury intake. Hum. Exp. Toxicol. 20:185–188.Trunova, V., Parshina, N., and Kondratyev, V. 2003. Determination of the distribution of trace elements in human hair as a function of

the position on the head by SRXRF and TXRF. J. Synchrotron Radiat. 10:371–375.Valkovic, V. 1988. Human hair, vol I/II. Boca Raton, FL: CRC Press.Walsh, W. J., Usman, A., and Tarpey, J. 2001. Disordered metal metabolism in a large autism population. Paper read at the American

Psychiatric Association Annual Meeting, New Orleans.Wasiak, W., Ciszewska, W., and Ciszewski, A. 1996. Hair analysis. Part 1. Differential pulse anodic stripping voltammetric determination

of lead, cadmium, zinc and copper in human hair samples of persons in permanent contact with a polluted workplace environ-ment. Anal. Chim. Acta 335:201–207.

Weiss, D. J., Mason, T. F. D., Zhao, F. J., Kirk, G. J. D., Coles, B. J., and Horstwood, M. S. A. 2005. Isotopic discrimination of zinc inhigher plants. New Phytol. 165:703–710.

Wilkinson, J. J., Weiss, D. J., Mason, T. F. D., and Coles, B. J. 2005. Zinc isotope variation in hydrothermal systems: Preliminary evidencefrom the Irish midlands ore field. Econ. Geol. 100:583–590.

Williams, R. J. P., and Frausto da Silva, J. J. R. 1996. The natural selection of the chemical elements. Oxford: Clarendon Press.Yanez, L., Garcia-Nieto, E., Rojas, E., Carrizales, L., Mejia, J., Calderon, J., Razo, I., and Diaz-Barriga, F. 2003. DNA damage in blood

cells from children exposed to arsenic and lead in a mining area. Environ. Res. 93:231–240.Yorbik, K., Akay, C., Sayal, A., Cansever, A., Sohmen, T., and Cavdar, A. O. 2004. Zinc status in autistic children. J. Trace Elem. Exp. Med.

17:101–107.