Metrology of Nail Clippings as Trace Element Biomarkers

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Metrology of Nail Clippings as Trace

Element Biomarkers

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Delft University of Technology

Faculty of Applied Sciences

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Metrology of Nail Clippings as Trace

Element Biomarkers

Proefschrift

ter verkrijging van de graad van doctor

aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus prof. ir. K C.A.M. Luyben,

voorzitter van het College voor Promoties,

in het openbaar te verdedigen op vrijdag 12 juli 2013 om 15:00 uur

door

Paulo César FAVARO

Master of Science,

University of São Paulo

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Samenstelling promotiecommissie:

Rector Magnificus voorzitter

Prof. Dr. H.T.Wolterbeek Technische Universiteit Delft, promotor Prof. Dr.Ir. M.de Bruin Technische Universiteit Delft

Prof. Dr. P.D.E.M.Verhaert Technische Universiteit Delft Prof. Dr.Ir.P.A.van den Brandt Universiteit Maastricht Prof. Dr.Ir.P.van ‘t Veer Wageningen Universiteit Prof. Dr. E.A. De Nadai Fernandes Universidade de São Paulo

Dr.Ir.P.Bode Gepensioneerd, voorheen Technische

ISBN 978-1-61499-287-5

Keywords: Human Nail, Neutron Activation Analysis, Micro Beam PIXE, Metrology, Biomarker, Nail Cleaning

Published and distributed by IOS Press under the imprint Delft University Press Publisher IOS Press Nieuwe Hemweg 6b 1013 BG Amsterdam The Netherlands tel: +31-20-688 3355 fax: +31-20-687 0019 email: info@iospress.nl www.iospress.nl www.dupress.nl LEGAL NOTICE

The publisher is not responsible for the use which might be made of the following information.

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Chapter 1 General introduction

Life expectancy has increased in all regions of the world during the last 2 decades (period 1990 – 2009), as was demonstrated by the World Health Organization [1]. This may be attributed to developments and availability of medicines and health care, reductions of, and anticipation on epidemic diseases and improvements in environmental conditions The related studies are partly based on assessments of the role of the chemical elements in the human body, e.g., relationships between the health status and nutrition or environmental exposure. The results have been obtained from measurements in large cohorts as the biological variation has to be taken into account. The analyses often imply the collection and measurement of the chemical elements in representative body parts, such as blood, urine, feces, sperm, sweat, earwax, exhaled breath condensate, saliva, body milk and tears, as well as in adipose tissue, meconium, hair and nail clippings. Often a preference exists for non-invasive collection of such biomarkers which explains the popularity of hair and nail clippings. [2-4].

Hair and nail clippings have the additional potential of providing a time-window: it is assumed that once the elements are immobilized in the nail keratin, so that the amount measured in a certain layer is a marker for that elements at the moment of its formation in the past. The materials may therefore act as a source of information on the long-term variations in the health status, on the impact of nutrition and on occupational exposure [5-10], as well as in forensic sciences. Nail clippings and hair can be easily obtained, even in post-mortem circumstances. The potential religious or cultural objection against providing hair samples can be overcome by collecting nail clippings.

The popularity of nail clippings started to grow towards the end of the 1970s when the interest for the element selenium was found to affect the effectiveness of antioxidant agents in the human body. Many studies have been undertaken to investigate the relation between selenium and cancer, and later on also between selenium, chromium and zinc and cardiovascular diseases [11-18].

It is remarkable that, in spite of the extensive usage, only a few studies have been carried to provide an answer to the fundamental question of what the trace elements in the clippings actually reflect. Also the analytical aspects such as the

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test portion preparation, particularly the separation of exogenous and endogenously present chemical elements have received limited attention. Sample preparation errors may easily result in misinterpretation of the results and consequently into a wrong conclusion e.g. on the health of man and/or animal involved.

1.1 Human nail as a biomarker

The term biomarker is used to describe the use of the human nail as a source of information on the human metabolism. The World Health Organization (WHO) defines biomarker as a chemical, its metabolite, or the product of an interaction between a chemical and some target molecule or cell that is measured in the human body [19]. The International Union of Pure and Applied Chemistry (IUPAC) has also used the term biomarker to describe the nail as metabolic indicator [20]. It is beyond the scope of this thesis to review these definitions, and the term ‘biomarker’ has been selected to describe the intended purpose

Keratin, as a protein, is the main constituent of the human nail. The nail is formed in a slow formation process (0.5 up to 1 mm per week) which implies that it takes 2-3 months after the formation in the root to reach the free edge of the fingertip where it can easily be sampled by clipping [11, 21].The (human) nail also contains many other chemical elements that all are incorporated already during its formation. Irregular nail formation, color, structure, shape, and strength of the nail may result from diseases as well as from disturbances in the body’s chemical element balance [22].

Both fingernails and toenails have been described as biomarkers, each with particular advantages and disadvantages. Fingernail grows faster than toenails, which allows for more frequent sampling. However, the fingernail is often more exposed to exogenous chemical elements, and such a contamination thus leads to an overestimation of the amounts of the endogenous chemical elements. On other hand, such an exposure can also turn into an opportunity for monitoring, e.g., the impact of handling toxic chemicals. Toenails may be more protected against exogenous exposure, and thus may be a better option for measurement of the relevant chemical elements.

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GENERAL INTRODUCTION CHAPTER 1 _________________________________________________________________________

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1.2 Chemical elements assessed by nail analysis

Selenium is the most measured chemical element in the human nail. The element selenium is part of the selenoprotein glutathion peroxidase, an essential enzyme of the antioxydative system that protects the body against the harmful effect of oxygen radicals. As such, selenium deficiencies are assumed to be related to cancer and cardiovascular disease. The amount of selenium in blood, nail and urine is considered as a marker for the selenium status in the human body. The selenium excreted by the urine represents about 60% of the selenium daily intake in the previous day [23, 24].

People taking food supplemented with selenium have a clear increase of the mass fraction of selenium in the nail if compared with a control group (0.904 mg.kg-1

with supplementation and 0.748 mg.kg-1 without supplementation). An experiment

reported by Longnecker et. al, which a group received selenium supplementation of 380 µg Se per day (using bread as a Se carrier) and the control group was at a dose of 32 µg Se per day. The Se concentrations in blood and serum of the group receiving the Se supplement started to reflect the administered dose after the second week and decreased again after the end of the supplementation period. The Se mass fraction in toenail clippings reflected the Se supplementation after 3 months and remained relatively elevated up until several months after completion of the supplementation [25]. In another study dealing with an extra intake of Zn, Cu and Mg, no increase on the respective trace element mass fractions in the nail was reported [26].

Other studies evaluated the relation between low selenium mass fractions in nail clippings and coronary heart disease and cancer. The results indicated that the selenium mass fraction in toenail does not support the thesis that selenium has a relation with coronary heart disease or cancer [11, 13, 14].

Several authors have reported that the mass fractions or concentrations of other potentially toxic elements such as cadmium, lead, mercury and arsenic in nail, hair, blood and urine, may be used as a marker for related diseases, environmental or occupational exposure. [6 - 8, 27-38].

Nail clippings (and hair) have been used in forensic research to evaluate cases of possible poisoning, drugs consumption, and ingestion of illegal substances. Nail clippings and hair can even be collect after death to solve the cause of death [9].

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Nickel in nail clippings and forearm skin was measured in volunteers exposed to a nickel solution daily during a 2 weeks period. The results showed an increase in the nickel mass fraction in the second week. [39].

The mass fractions of calcium and magnesium in female nail clippings were measured for correlation with bone mineral density. The results showed a low correlation (range from 0.03 to 0.18) for both elements [40].

There have been many other studies published in with the mass fraction of the chemical elements in nail clippings were used to assess if the elements can be used as a marker for specific disease patterns. There are indications that bromine is elevated in nail clippings from people suffering from Alzheimer’s disease though there is no correlation with the stage of the disease. Zinc and potassium mass fractions in nail clippings also increase due this disease, and here a correlation with the stage has been observed, whereas the mercury mass fractions decrease [41]. Low chromium mass fractions in toenail clippings are reported to be inversely correlated with myocardium infarction disease: patients have lower chromium mass fractions in nail when compared to health people controls (1.10 mg.kg-1 and

1.30 mg.kg-1 respectively) [17]. Trace elements mass fractions in fingernail, hair and

plasma of patients with chronic renal failure were evaluated and the results indicate that the values for zinc in samples from patients are lower than those from controls [42]. No correlation was found for the mass fractions of arsenic, copper, chromium, iron, and zinc in nail clippings and the breast cancer. [15].

The mass fraction of cerium level in toenail clipping is correlated to the diagnosis of first acute myocardial infarction, but the correlation is not yet fully understood [43].

1.3 Analytical aspects of trace element determination in nail clippings Many analytical techniques can be employed for measurement of chemical elements in nail clippings, each technique with specific advantages and also sample preparation mode. Instrumental Neutron Activation Analysis (INAA), techniques based in Inductively Coupled Plasma as Mass Spectrometry (MS) or Optical Emission (ES) and also Atomic Absorption Spectrometry (AAS) have been used [28, 31, 34, 40, 41].

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Neutron activation analysis is the preferred analytical technique in epidemiological studies utilizing nail clippings. Such studies are rather costly in design because often a large number of clippings have to be collected (projects involving several thousands of samples are not uncommon), as well as because of the time for obtaining additional information via questionnaires and other analyses, and the costs for analyses of such large quantities of samples. There have been several cases in which the results on the hypothesized key chemical element indicate that other chemical elements or constituents would be equally important to measure. This explains the preference for NAA. Moreover, the sensitivity for most elements of interest is adequate.

It can be derived from many such epidemiological studies that the differences in trace element levels between extreme cases, e.g. healthy people and people suffering from a disease, are sometimes rather small. Preferably, such differences between trace element mass fractions in nail clippings should not be due and/or influenced by anything else than by the uncertainty of measurement and by the biological variation (which may be the property of interest in epidemiological studies).

Differences between results of nail clipping analyses may also come from systematic errors in the sample preparation. In addition, analysis of large series of samples (ranging from hundreds to thousands) often require a long throughput time (which may vary from months to years) and differences may be introduced if during that period variations in the calibration status of the measurement system occur, which thus can be seen as a method error.

The method error in INAA can be assessed by regular analysis of certified reference materials and has been shown, over a period of about 20 years for the INAA facilities in Delft, to be (much) less than 5%, i.e., the degree of trueness in INAA ranges for most chemical elements from 0.95 to 1.05 [44].

In summary, the biological variation can only be properly assessed if, assuming the method is under statistical control and the uncertainty of measurement can be estimated, also the errors due to sample preparation are known and/or minimized.

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6 1.4 Objectives of the research

As has been outlined in the above, measurement results may be misinterpreted if the element mass fractions are biased by errors in the sample preparation, which may result in:

- Too high amounts of the elements by:

Addition of exogenous amounts of elements by contamination and insufficient cleaning.

Too rigorous drying procedures. - Too low amounts of the elements by:

Losses of endogenous amounts of elements by too rigorous cleaning; Losses of endogenous amounts of elements by evaporation and other effects during irradiation.

Insufficient drying of the sample.

It all may result in wrong conclusions and even wrong decisions.

A systematic evaluation of such effects towards the degree of trueness for the measurement of other elements does not exist. This marks the objectives of the research, described in this thesis.

1.5 Scope of the thesis

A research program has been conducted to assess the degree in which the endogenous amounts of elements in nail clippings may be overestimated or underestimated by errors in sample preparation of nail clippings for INAA.

An overview of the anatomy, growth, composition and structure of the human nail is firstly given in Chapter 2. In addition, the effect of nail diseases to changes in the structure and composition is discussed.

Nail polish and soil can be considered as the most common types of contamination to finger and toenail clippings, with an immediate impact on the assessment of the amounts of the endogenous chemical elements in the clipping. The ratio of the amounts of these exogenous elements and endogenous elements in the nail has

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been studied using INAA in view of the efficiency required of the cleaning procedures. This is described in Chapter 3.

There is no standard method for cleaning nail clippings prior analysis. Several cleaning procedures have been tested, not just chemical ones but also mechanical ones by which part of the nail surfaces is removed via scraping. One of the preconditions to the cleaning procedure is that the highest efficiency is required for removal of the exogenous chemical elements, without affecting, e.g. by leaching, the amounts of the endogenous elements. The research into the effectiveness of cleaning procedures for removal of the exogenous elements is described in Chapter 4.

In addition, such a possible effect of the cleaning procedures on the endogenous elements, e.g. by leaching but also by mechanical removal, has been studied by measurement of the distribution of the chemical elements along the thickness of the nail clipping. Microbeam PIXE has been used for this; the research is described in Chapter 5.

Whereas the first chapters in this thesis have been initiated by the potential errors due to unwanted addition of chemical elements, losses of chemical elements may also occur during the analysis of nail clippings. The well-known phenomenon of mercury losses by volatilization during neutron irradiation and subsequent migration through the walls of polyethylene vials receives attention in Chapter 6. Nail clippings are, like human hair, hygroscopic due to the keratinized structure. Cleaning procedures such a washing result in relatively high uptakes of moisture. This potential source of error is hardly explicitly mentioned in studies with nail clippings. Therefore, a study was undertaken to assess the typical mass changes due to this hygroscopic behavior, e.g. as a result of washing, drying and moisture uptake from the atmosphere during sample preparation. This has been described in Chapter 7.

Contamination, cleaning, hygroscopic effects and element losses all have an impact on the assessment of the ‘true’ amounts of the endogenous chemical elements. Wrong corrections for these effects have an impact on the classification of samples – and thus of their donors – in epidemiological studies, e.g. as ‘healthy’ or ‘with disease’. The potential effect of the cleaning procedures tested in this research (and described in Chapter 4) to such a classification has been evaluated. This is described in Chapter 8.

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All results are finally projected against the objectives of this research project and discussed in Chapter 9 of this dissertation.

As a complement to the evaluation of possible losses of mercury during irradiation presented in Chapter 6, an investigation into the mercury chemical species present in the nail was conduct by the measurement of methyl mercury in the nail samples from donors that consume fish frequently and donors that never consume fish. The experiment was performed using gas chromatography ICP-MS technique. The results are given in Appendix 1.

1.6 References

[1] World Health Organization. Life Expectancy. WHO. 2012. Ref Type: Internet Communication

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assessing persistent organic pollutants. Environ Int 33, 685-693 (2007).

[3] B. Wu and T. Chen, Changes in hair arsenic concentration in a population exposed

to heavy pollution: Follow-up investigation in Chenzhou City, Hunan Province, Southern China. J Environ Sci (China) 22, 283-289 (2010).

[4] M. Esteban and A. Castaño, Non-invasive matrices in human biomonitoring: A

review. Environ Int 35, 438-449 (2009).

[5] M. Garland, J. S. Morris, B. A. Rosner, M. J. Stampfer, V. L. Spate, C. J. Baskett, W. C. Willett and D. J. Hunter, Toenail trace element levels as

biomarkers: reproducibility over a 6-year period. Cancer Epidemiol Biomarkers

Prev 2, 493-497 (1993).

[6] B. L. Gulson, Nails: concern over their use in lead exposure assessment. Sci Total Environ 177, 323-327 (1996).

[7] T. H. Lin, Y. L. Huang and M. Y. Wang, Arsenic species in drinking water, hair,

fingernails, and urine of patients with blackfoot disease. J Toxicol Environ Health

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[8] B. Nowak and J. Chmielnicka, Relationship of Lead and Cadmium to Essential

Elements in Hair, Teeth, and Nails of Environmentally Exposed People. Ecotoxicol

Environ Safety 46, 265-274 (2000).

[9] C. Ralph Daniel III, B. M. Piraccini and A. Tosti, The nail and hair in forensic

science. J Am Acad Dermatol 50, 258-261 (2004).

[10] R. Mehra and M. Juneja, Fingernails as biological indices of metal exposure. J Biosci 30, 253-257 (2005).

[11] P. S. H. van Noord, Selenium and Human Cancer Risk nail keratin as a tool in metabolic epidemiology, pp. 1-167, State University Utrecht, 1993.

[12] J. S. Morris, T. Rohan, C. L. Soskolne, M. Jain, T. L. Horsman, V. L. Spate, C. K. Baskett, M. M. Mason and T. A. Nichols, Selenium status and cancer

mortality in subjects residing in four Canadian provinces. J Radioanal Nucl Chem 249, 421-427 (2001).

[13] S. Rajpathak, E. Rimm, J. S. Morris and F. Hu, Toenail Selenium and

Cardiovascular Disease in Men with Diabetes. J Am Coll Nutr 24, 250-256 (2005).

[14] K. Yoshizawa, A. Ascherio, J. S. Morris, M. J. Stampfer, E. Giovannucci, C. K. Baskett, W. C. Willett and E. B. Rimm, Prospective Study of Selenium Levels in

Toenails and Risk of Coronary Heart Disease in Men. Am J Epidemiol 158,

852-860 (2003).

[15] M. Garland, J. S. Morris, G. A. Colditz, M. J. Stampfer, V. L. Spate, C. K. Baskett, B. A. Rosner, F. E. Speizer, W. C. Willett and D. J. Hunter, Toenail

trace element levels and breast cancer: a prospective study. Am J Epidemiol 144,

653-660 (1996).

[16] P. Xun, K. Liu, J. S. Morris, M. L. Daviglus and K. He, Longitudinal association

between toenail selenium levels and measures of subclinical atherosclerosis: The CARDIA trace element study. Atherosclerosis 210, 662-667 (2010).

[17] E. Guallar, F. J. Jiménez, P. van 't Veer, P. Bode, R. A. Riemersma, J. Gómez-Aracena, J. D. Kark, L. Arab, F. J. Kok, J. M. Martín-Moreno and for the EURAMIC-Heavy Metals and Myocardial Infarction Study Group, Low

Toenail Chromium Concentration and Increased Risk of Nonfatal Myocardial Infarction. Am J Epidemiol 162, 157-164 (2005).

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[18] J. M. Martín-Moreno, L. Gorgojo, R. Riemersma, J. Gómez-Aracena, J. Kark, J. Guillen, J. Jimenez, J. Ringstad, J. Fernández-Crehuet, P. Bode, F. Kok and Heavy Metals and Myocardial Infarction Study

[19] WHO. Biomarkers and human biomonitoring. World Health Organization . 2013.

Ref Type: Internet Communication

[20] H. Ole, A. A. M. D. L. Frank, R.-N. Ole, S. J. Steem, G. David, H. Olf, P. Sara, P. Finn and O. Erik, Human exposure to outdoor air pollution. Pure Appl Chem

73, 933-958 (2001).

[21] T. Kitahara and H. Ogawa, The extraction and characterization of human nail

keratin. J Dermatol Sci 2, 402-406 (1991).

[22] U. Runne and C. E. Orfanos, The human nail: structure, growth and pathological

changes . Curr Probl Dermatol 9, 102-149 (1981).

[23] M. P. Longnecker, D. O. Stram, P. R. Taylor, O. A. Levander, M. Howe, C. Veillon, P. A. McAdam, K. Y. Patterson, J. M. Holden, J. S. Morris, C. A. Swanson and W. C. Willett, Use of selenium concentration in whole blood, serum,

toenails, or urine as a surrogate measure of selenium intake. Epidemiology 7,

384-390 (1996).

[24] M. M. Manson, J. S. Morris, V. L. Spate, C. J. Baskett, T. A. Nichols, T. L. Horsman, L. Le Marchand, L. N. Kolonel and S. Yukimoto, Comparison of

whole blood, plasma and nails as monitors for the dietary intake of selenium. J

Radioanal Nucl Chem 236, 29-34 (1998).

[25] M. P. Longnecker, M. J. Stampfer, J. S. Morris, V. Spate, C. Baskett, M. Mason and W. C. Willett, A 1-y trial of the effect of high-selenium bread on selenium

concentrations in blood and toenails. Am J Clin Nutr 57, 408-413 (1993).

[26] J. Brockman, J. Guthrie, J. Morris, J. Davis, R. Madsen and J. Robertson,

Analysis of the toenail as a biomonitor of supranutritional intake of Zn, Cu, and Mg. J Radioanal Nucl Chem 279, 405-410 (2009).

[27] J. S. Morris, M. J. Stampfer and W. C. Willett, Dietary selenium in humans

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[28] W. I. Mortada, M. A. Sobh, M. M. El-Defrawy and S. E. Farahat, Reference

Intervals of Cadmium, Lead, and Mercury in Blood, Urine, Hair, and Nails among Residents in Mansoura City, Nile Delta, Egypt. Environ Res 90, 104-110 (2001).

[29] M. R. Karagas, T. D. Tosteson, J. Blum, B. Klaue, J. E. Weiss, V. Stannard, V. Spate and J. S. Morris. Measurement of low levels of arsenic exposure: a comparison of water and toenail concentrations. American Journal of Epidemiology 152[1], 84-90. 2000.

[30] A.H. Bu-Olayan, S. N. Al-Yakoob and S. Alhazeem, Lead in drinking water

from water coolers and in fingernails from subjects in Kuwait City, Kuwait. Sci

Total Environ 181, 209-214 (1996).

[31] M. Wilhelm, B. Pesch, J. Wittsiepe, P. Jakubis, P. Miskovic, T. Keegan, M. J. Nieuwenhuijsen and U. Ranft, Comparison of arsenic levels in fingernails with

urinary As species as biomarkers of arsenic exposure in residents living close to a coal-burning power plant in Prievidza District, Slovakia. J Expo Anal Environ

Epidemiol 15, 89-98 (2005).

[32] M. Wilhelm, D. Hafner, I. Lombeck and F. K. Ohnesorge, Monitoring of

cadmium, copper, lead and zinc status in young children using toenails: comparison with scalp hair. Sci Total Environ 103, 199-207 (1991).

[33] C. A. Helsby, Determination of mercury in finger nails and body hair. Anal Chim Acta 82, 427-430 (1976).

[34] K. L. Chen, C. J. Amarasiriwardena and D. C. Christiani. Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry. Biol Trace Elem Res 67[2], 109-125. 1999.

[35] G. V. Alfthan, Toenail mercury concentration as a biomarker of methylmercury

exposure. Biomarkers 2, 233-238 (1997).

[36] T. Suzuki, S. Watanabe and N. Matsuo, Comparison of hair with nail as index

media for biological monitoring of mercury. Sangyo Igaku 31, 235-238 (1989).

[37] E. Platz, K. Helzlsouer, S. Hoffman, J. Morris, C. Baskett and G. Comstock,

Prediagnostic toenail cadmium and zinc and subsequent prostate cancer risk.

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[38] B. K. Mandal, Y. Ogra and K. T. Suzuki, 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 (2003).

[39] J. Kristiansen, J. M. Christensen, T. Henriksen, N. H. Nielsen and T. Menné,

Determination of nickel in fingernails and forearm skin (stratum corneum). Anal

Chim Acta 403, 265-272 (2000).

[40] C. M. VechtHart, P. Bode, W. T. Trouerbach and H. J. A. Collette, Calcium and

magnesium in humans’ toenails do not reflect bone mineral density. Anal Chim

Acta 236, 1-6 (1995).

[41] D. E. Vance, W. D. Ehmann and W. R. Markesbery, A search for a longitudinal

variations in trace element levels in nail of Alzheimer's disease patients. Biol Trace

Elem Res 26-7, 461-470 (1990).

[42] F. Marumo, Y. Tsukamoto, S. Iwanami, T. Kishimoto and S. Yamagami, Trace

element concentrations in hair, fingernails and plasma of patients with chronic renal failure on hemodialysis and hemofiltration . Nephron 38, 267-272 (1984).

[43] J. Gómez-Aracena, R. A. Riemersma, M. Gutiérrez-Bedmar, P. Bode, J. D. Kark, A. Garcia-Rodríguez, L. Gorgojo, P. v. Veer, J. Fernández-Crehuet, F. J. Kok and J. M. Martin-Moreno, Toenail cerium levels and risk of a first acute

myocardial infarction: The EURAMIC and heavy metals study. Chemosphere 64,

112-120 (2006).

[44] P. Bode and M. Blaauw, Performance and robustness of a user,

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Chapter 2 Overview of the human nail

2.1 Introduction

An overview of the anatomy of the human nail is provided in this Chapter, with an outline of the composition, structure and other relevant information such as nail diseases that can contribute to changes in the structure and composition of the human nail.

2.2 Anatomy

The basic anatomy of the fingertip and the nail formation and structure is shown in the Figure 2.1, providing a general overview of all parts of the fingertip that are involved in the formation of the human nail, pushing it to until the free edge appears from which the nail clipping is collected.

2.2.1 The fingertip

The fingertip is the region supporting the nail root and the base for the nail plate. The blood circulation in the root, nail bed and nail fold region comes from two branches derived from the main two arteries that supply the extreme part of the finger. The network of vessels in the nail region is distributed in papillar, pseudopapillar, reticular and subdermical ones. The morphology and density of these networks of vessels can vary depending on their location. The blood provides the nutrients and chemical elements that the nail root uses to produce the nail keratin. The nervous system in this region follows the same course of the arteries over the distal finger tip [1-2].

2.2.2 Production of the keratin in the matrix

The matrix of the nail, which is located under the eponychium and in the proximal nail fold region, produces the keratin protein, which is the main component of the human nail. Still in the nail matrix, keratin and dead cells are grouped, forming thin layers of “biological plastic”. Several of these layers are superposed forming the hard nail structure called nail plate. The keratin produced in the root represents about 79% of the keratin present in the nail plate [3-4].

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Keratin is composed from mainly the amino acid cysteine and 17 other amino acids. The formation of the nail structure begins by the connection of two cysteine amino acids by disulfide bonding (S-S). The structure of the keratin protein is folded in α-helix shape and the disulfide bonds provide high stability toenail, making it a hard structure with high chemical and mechanical resistance. The hydrophobic properties of the disulfide bonds facilitate low interaction between the proteins and an aqueous environment, which contributes to the resistance of the nail. Hydrogen bonding is also found in the keratin structure.

The keratin present in the matrix and nail bed was found encoded by two multigene families, the acidic proteins (type I) and basic or neutral proteins (type II). The hard keratin structure is a result of the combination of one gene from each family. Skin diseases and altered skin morphology can cause a change in the genetic coding. Berker et al report that the keratins K14, K1 and K10 can be found in the matrix and part of the nail bed region, and the keratins K16, K17, K6 and K14 in the nail bed up to the hyponychium region. The hard keratin, mainly composed by the nail plate, correspond to 90% of the keratin in the nail plate and the other 10% represent soft-keratin, composed by the connective tissue of the nail bed and nail plate [5-8].

The final composition of the nail is determined during the nail production. The matrix uses the chemical elements present in the blood adding them to the keratin layers. There is also a contribution by interaction from the nail bed to composition of the nail but this has not yet well studied and quantified.

2.2.3 Nail fold

The skin around the nail plate is denoted as posterior and lateral nail fold (Figure 2.1), and the keratinized skin called cuticle serves as protection of the matrix against irritants, allergens and bacterial and fungal pathogens. It also protects the border of the nail and keeps the nail connected with the skin. The posterior and lateral nail folds are composed by skin that covers the nail matrix. The cuticle is composed by a modified stratum corneum, and the connection with upper surface of the nail plate avoids the passage of contamination to the matrix [9].

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OVERVIEW OF THE HUMAN NAIL CHAPTER 2 _________________________________________________________________________

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16 2.2.4 Lunula

The lunula is the white half-moon portion of the distal nail matrix and extends beyond the proximal nail fold. The origin and the function of this part of the nail plate are not clearly defined. Some studies have controversial information about the formation of the lunula. Some show that the region of the lunula is poor vascularised and consequently the lunula has a pale color; others describe the lunula as not completely connected to tissues whereas the nail bed over the lunula has rich and regular vascular network. Alterations in the shape and color of the lunula can be an indication of cutaneous disorders [1, 10-11].

2.2.5 Nail plate and nail bed

The nail plate starts after the formation in the matrix, in the region of the proximal nail fold and connected on the nail bed. The nail plate is pushed towards the hyponychium and at this point, it gets separated from the skin in the free edge part. The speed of growth of the nail plate varies depending on the finger (fingernails and toenail), sex, season and diseases [3].

The nail plate is composed of three different horizontal layers: a thin dorsal, a thicker intermediate layer and a ventral part that is connected to the nail bed, as shown in Figure 2.2 [9]. The three layers can be distinguished because of the orientation of the nail fibers. . On the dorsal side, the orientation appears to be parallel to the growth axis, and on the intermediate layer the position shifts. The orientations of the keratin cells are partially responsible for the hardness of the nail structure [2]. The three different parts of the nail plate can be observed using the scanning electron microscopy (SEM), as shown in Figure 2.3. The dorsal and intermediate layers appear as a hard and compact structure, while the ventral part has a soft and open structure. The intermediate and the ventral layers have different orientation.

The phospholipids, present mainly in the dorsal and intermediate layers, contribute to flexibility of the nail plate [9]. A SEM picture of the dorsal nail surface of a newborn (see Figure 2.4a and b at the magnification) shows the keratin layers of the nail plate, and the compactness of the nail structure. SEM pictures of dorsal nail surface from adults, shown in Fig. 2.4c and d, give a similar picture as those from the newborn baby, viz., the keratin in the layers and closed structure. The ventral surface of the nail shows an open and soft structure as can be seen from Figure 2.3.

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The nail bed is an epidermal part of the fingertip, where the nail plate lays and is held by connective tissue produced in the nail bed. The bed has a network of elastic fibers, scattered fat cells, a well-vascularised area, but there is no fat in the subcutaneous nail bed. The nail bed epidermal layer is usually not more than two or three cells thick, and serves as connective tissue to the nail plate [12].

The nail bed produces 21% of the nail plate. The nail bed cells do not have independent movements, but continuously produce keratin. The longitudinal orientation of the bed epithelium is the biological solution for the continuous attachment and movement of the nail plate [2, 4, 9, 13].

The nail plates from toenail and fingernail have different growth rates. Publications report that, in average, the fingernail grows 0.5 to 1.2 mm per week, and that the toenail has one quarter to half of this growth rate. Nutritional deprivation, environmental temperature, age, diseases and traumas can alter the growth rate [14]. The thickness of the nail plate varies typically from 0.4 to 0.75 mm in fingernail and up to 1.0 mm in toenails. Diseases and dysfunctions from the nail bed or in the matrix can also contribute to thickening of the nail plate [2-3, 5].

Figure. 2.2 Schematic representation of the nail formation and movement of the nail plate

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Figure 2.3 Scanning Electron Microscopy (SEM) picture of the transversal length of the nail plate.

Ventral

Intermediate

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Figure 2.4 A and B Scanning Electron Microscopy (SEM) picture of the newborn nail plate keratin layers showing the compacted structure of the nail plate. C and D are the pictures of dorsal nail surface from adults.

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20 2.3 Composition

Most chemical elements have been measured in human nail clippings. Some elements can thus be seen as major constituents, but most of them are present at a trace level.

Rodushkin and Axelsson published an overview of the elements mass fraction observed in fingernail samples (at least n=96), given in Table 2.1. Some extremely high values should be considered with skepticism, mainly for the most abundant chemical elements in the environment as Fe, Cl, Ca, Na and S [15].

2.3.1 Variation in the trace element mass fraction due to age

The amounts of trace elements vary with the age. Bromine, cobalt, iron, sodium, chromium, and antimony have a negative correlation with age. Zinc increases during childhood period and maintains a positive correlation after it. Mercury levels are practically constant up to 20-25 years of age, and increase between 25 and 60 years. Amalgam fillings present in the mouth and the fish consumption may explain the increase of the mercury in the nails. Thorium appears higher in childhood period, decreasing up to 50 years old and increase again over 60 years old. Probably, thorium comes from the contact with the soil. During the childhood, it is not uncommon that there are several activities with soil, by the hand and the mouth, and the hygienic habits are not always fully followed [15-16]. Bu-Olayan et al studied the influence of lead in drinking water on fingernails in Kuwait city, and an influence of age on the retention of lead in the nail was found. The results show that the highest mass fraction of lead in nail is found between 16 and 35 years of age and decreasing after this period. The reason for the decrease is unknown [17]. 2.3.2 Variation in the trace element mass fraction due to gender

Gender has a significant influence on the nail composition. Silicon, gold, bismuth, and zinc are higher in females than males, and sodium and potassium are higher in males. The following median mass fractions in μg g-1_{were found for male and}

female, respectively: gold, 0.035 and 0.065; bismuth, 0.016 and 0.037; sodium, 215 and 112; magnesium, 100 and 85; silicon, 41 and 54; potassium, 145 and 101 [15-16]. 2.3.3 Influence of smoking habit on the nail composition

Rodushkin and Axelsson report that the mass fraction of cadmium in nails from smokers from Swedish was 10 times higher than in non-smokers (0.760±0.870 μgg-1

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vs. 0.073±0.088 μg g-1). The chemical elements La, Ce, Pr and Nd also showed some

increase in nails from smokers [15].

Contradictory to Rodushkin and Axelsson, Mortada et al reported no significant changes for Cd (1.44±0.81 μg g-1 and 1.23±0.83 μg g-1), Pb (4.75±2.69 μg g-1 and

4.9±2.73 μg g-1) and Hg (1.06±0.12 μg g-1 and 0.98±0.17 μg g-1) in the nails,

respectively from smokers and non-smokers [18].

Element Range µg g-1 _Element _{Range µg g}-1

Ag 0.003 - 6.5 Na 3 - 960 Al 12 - 137 Nb 0.003 - 0.067 As 0.009 - 2.57 Ni 0.14 - 6.95 Au 0.006 - 2.6 P 82 - 625 B 0.12 - 60 Pb 0.04 - 240 Ba 0.28 - 3.99 Pd <.00006 - 0.0098 Be <.0002 - 0.0066 Pt 0.00002 - 0.0011 Bi 0.004 - 0.543 Rb 0.042 - 750 Br 2 - 49.9 Re <.000005 - 0.00014 Ca 345 - 5900 Rh <.00001 - 0.000089 Cd 0.013 - 1.9 Ru <.00002 - 0.000054 Ce 0.024 - 0.771 S 23400 - 43500 Cl 2020 - 22600 Sb 0.001 - 0.128 Co 0.006 - 3 Sc 0.0013 - 0.04 Cr 0.224 - 6.7 Se 0.62 - 63 Cs 0.0008 - 0.347 Si 13 - 5400 Cu 4.2 - 81 Sn 0.11 - 2.56 Fe 12 - 7300 Sr 0.16 - 3.3 Ga 0.003 - 0.053 Ta <.002 - 0.068 Ge <.0022 - 0.024 Te <.00007 - 0.0019 Hf 0.002 - 0.192 Th 0.002 - 0.063 Hg 0.028 - 2.8 Ti 0.16 - 16.1 I 0.077 - 0.81 Tl 0.0003 - 0.0058 Ir <.00001 - 0.00026 U 0.002 - 0.047 K 17 - 3010 W 0.003 - 0.053 La 0.015 - 0.425 V 0.018 - 0.476 Li 0.013 - 0.255 Y 0.007 - 0.094 Mg 23 - 191 Zn 73 - 3080 Mn 0.19 - 3.3 Zr 0.054 - 7.89 Mo 0.015 - 0.16

Table 2.1 Published mass fraction of the chemical elements found in the fingernail (Rodushkin and Axelsson [15]).

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2.3.4 Changes in the nail composition due to diseases

The influence of diseases on the nail is well studied in dermatology, like on color and structure changes of the nail plate. However, not many studies have been done to evaluate the variation in the nail’s chemical composition due to diseases. Vance et al studied the chemical composition of the nail in Alzheimer’s disease patients. The results show that bromine, potassium and zinc are higher in patients compared to the controls, whereas mercury is lower than in the controls. Potassium and zinc have the tendency to increase with the age or the time after the patient acquires the disease [19]. Garland et al studied arsenic, copper, chromium, iron and zinc in toenail of women with breast cancer and controls, but none of these trace elements can support that the breast cancer changes the composition of the nail [20]. Sukumar and Subramanian studied the relation between trace elements in nail and coronary heart disease (CHD), hypertension and diabetes of residents of New Delhi. The chemical elements Cd, Cr, Cu, Mn, Ni, Pb and Zn showed insignificant correlation between patients and controls [21].

2.3.5 Correlation among trace elements

Statistics indicate that some elements present in the nail can result in increases or decreases of the other trace elements. The reasons of such effects are not clearly understood but it has been observed during changes of the trace elements amount in the environment or changes in the metabolism. Garland et al observed that the increase of calcium and selenium in toenail results in a decrease of copper, iron, sulfur and zinc levels [22]. Nowak and Chmielnicka reported that an increase of lead in nail also leads to a decrease of copper and zinc [23].

2.4 Nail moisture and transport of water through the nail plate

The nail keratin is a hygroscopic material and the nail plate becomes soft after contact with water. The water content in the nail plate is supplied by the nail bed, and the amount of the natural water in the nail can vary from 7 up to 25% [3, 6, 24] (see also Chapter 8 of this dissertation). The moisture in the nail maintains the nail structure healthy and avoids the nail to become brittle. As the nail is very sensible in relation to the humidity in the air, some studies report that during winter the water in the nail is reduced, mainly because of the cold air that carries less water, and the heat systems that dry out much more the air, and the free edge part of the nail is more susceptible to become brittle [6].

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Near-Infrared Spectrometry was used by Egawa et al to evaluate the moisture in the human nail in different conditions of relative humidity in the air. The results confirm the close correlation between the relative humidity in the air with the moisture in the nail, mainly to relative humidity in the air over80%, but no correlation was observed between the thickness of the nail and the amount of water present in the nail sample. In the same experiment by Egawa et al, the amount of water in the nail was evaluated after the nail had been soaked in water for some time and then kept at room humidity conditions. The results indicate a strong reduction of the water content in the nail, almost achieving the original moisture amount, as was present before immersion in the water [24].

Wessel et al and Gniadecka et al, using Near-Infrared-FT-Raman spectroscopy, found that the water molecules in the nail structure interact with the keratin structure by forming bonds. The same study suggests that, after the nails have been soaked in water, the water bonds with the structure of the nail thereby changing the geometry of the proteins bonds. This causes the nail to become soft [8, 25].

Walters et al studied the permeation of water and alcohols through the nail plate, and observed that the coefficient of permeation to water is 16.5 X 10-3_{cm h}-1_and

the coefficient of permeation to alcohols varies conform the molecule structure, e.g., 5.6 X 10-3 cm h-1 for methanol, 5.8 X 10-3 cm h-1 for ethanol until 0.27 X 10-3

cm h-1 for n-octanol. The study suggests that the hydrated human nail plate

behaves like a hydrogel of high ionic strength to the polar and semi-polar alcohols. [27] The flux of water through the nail plate is higher than the skin, but because of the low level of lipids in the nail plate the structure does not retain the water, then the human nail has low water content [27].

2.5 Nail disorders

Several studies have claimed that the nail can be used as a chemical monitor to nutritional aspects for the human body, and may help to avoid future diseases. But the nail is submitted to some drastic changes due to disorders of the body. Frequently the disorders in the nail plate are the result of some cutaneous problems, and different diseases can result in the same change on the pattern of the nail plate. As such, the diagnosis can be difficult since some diseases or infection can share the same aspect in the nail. The most common changes in the nail are thickening or thinning of the plate, ridging or pitting, discoloration of the plate,

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separation of the nail plate from the nail bed (Lysis), complete shedding of the nail, subungual hyperkeratosis, change in the consistency or configuration of the nail, proximal or lateral nail fold inflammation and separation [2].

The items below explain briefly the problems found in human nail and their possible origin.

2.5.1 Genetic disorders of the nail

Nail deformity, as variation on the nail shape, color and nail plate structure can be a result of differences in the gene expression. The Table 2.2 shows some genetic disorders that can have an influence on the nail formation. The genetic alteration can be congenital or hereditary, and more specific tests must be applied to evaluate such genetic alteration. Besides the nail be a possible indicator other variation on the metabolism can result in a similar nail deformity.

Teeth, nail and hair are formed during the embryonic stage and some disorders observed in the nail related to genetic problems can be also observed in the teeth and hair.

A genetic disorder observed in the nail (such as change in the nail coloration and/or nail thickening) can also be linked to other disorders such as mental retardation, deafness and onycho-osteodystrophy, the DOOR syndrome, juvenile cataracts, defective hair and teeth and light sensitivity, as well as Rothmund-Thomson syndrome [2, 28].

The hereditary genetic disorder Subtotal Leuconychia, can stimulate the change in the coloration of the nail plate, which then becomes white, and can also change the structure and hardness of the nail plate [29].

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Disease observed only in the nail

Diseases of multiple organ systems of ectodermal and /or mesodermal origin

CONGENITAL (present at birth)

Anonychia Hidrotic congenital ectodermal dysplasia (Clouston) Racket nails Anhidrotic congenital ectodermal dysplasia

Koilonychia Pachyonychia congenital

Congenital pitting Chondroectodermal dysplasia

Ridging Focal dermal hypoplasia (Goltz)

Micronychia Oculo-dento-digital syndrome

Polynychia DOOR syndrome (deafness, onycho-osteodystrophy. Mental retardation)

Onychoheterotropia Coddins-Siris syndrome

HEREDITARY (nail changes usually occurring after birth) Hereditary clubbing Darier’s disease

Periodic shedding Tuberous sclerosis Hereditary leuconychia Epidermolysis bullosa

Nail-patella-elbow syndrome Progeria

Dyskeratosis congenita Pachydermoperiostosis

Poikiloderma congenital (Rothmund-Thomson) Incontinentia pigmenti

Peutz-Jeghers syndrome Acrodermatitis enteropathica Wilson’s disease

Table 2.2 Genetic disorders that can influence the nail formation.

2.5.2 Infectious diseases

The onychomycosis or fungal infection is a common disorder of the nail. A combination of the right humidity and temperature, as e.g., is present in closed shoes, can be ideal for fungal colonization. The dermatophytes, molds and yeasts are the onychomycosis found in the infected nail, seen in Table 2.3. The onychomycosis can infect the nail through the surface, distal subungual and proximal subungual. The infection on the surface of the nail is characterized by white nail or nail with discoloration. The nail fold and nail bed are not contaminated in this case. This kind of fungus can be easily removed by scraping-off the dorsal part of the nail. The distal subungual onychomycosis is the

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contamination of the hyponychium and lateral nail fold, with extension to the nail bed. The symptoms are the hyperkeratosis and change of the color to yellow-brown. The nail plate can thicken, crumble and separation of the nail plate from nail bed can occur. Antibiotics are used to treat this kind of onychomycosis. The proximal subungual onychomycosis is the contamination of the nail bed through the proximal nail fold, followed by contamination of the nail bed. The characteristic is a white-yellow nail.

Localized process Systemic process

Fungal

Dermatophyte infection

T. rubrum T. mentagrophytes E. floccosum

Superficial infection of nail plate surface by both dermatophytes and nondermatophyte molds

Yeast infection Candidiasis

Candida albicans most common Intermediate and deep fungal infections including mycetoma, chromoblatomycosis

Systemic mycoses

Bacterial

Abscess, paronuchial inflammation or nail plate discoloration, or both

Septicemia and septic emboli (splinter hemorrhages)

Staphylococcus Streptococcus Pseudomonas

Plus mixed bacterial and fungal infections

Spirochetal Syphilis, yaws, pinta

Viral

Warts, molluscum contagiosum, herpes simplex (whitlow)

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Paronychia is another class of infection, characterized by contamination mainly of the fingernails by a mixture of fungus and bacteria, mainly the Candida albicans. The nail folds are the main entrance to the fungus and bacteria, and the yellow-brown coloration, swelling and purulent materials are characteristics of this infection [2].

2.5.3 Neoplasm

The tumors’ in the nail region are another cause for changes of the nail plate. Some benign and malign neoplasms in the nail region are given in Table 2.4. The diagnosis of neoplasm can be difficult and too late since the nail plate sometimes overlay the tumor. Also inflammation and nail change can be attributed to nail disorder [2].

Benign Malignant

Local Systemic

Mucous cyst Bowen’s disease Metastatic carcinoma

Fibroma Squamous cell

carcinoma

Lymphoma Acquired digital fibrokeratoma Basal cell

epithelioma Recurring digital fibrous tumor of

childhood

Melanoma

Glomus tumor Sarcoma

Angioma

Pyogenic granuloma Neuroma

Giant cell tumor of tendon sheath Eccrine poroma Actinic keratosis Keratoacanthoma Clavus Enchondroma Osteochondroma Exostosis Epidermid Supernumerary

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2.5.4 Systemic disease and hormone variation

Several systemic diseases can affect the nail formation, structure, coloration and hardness. The diabetes mellitus can affect several organs of the body, inclusive the skin and nail. The diabetes can develop yellowish, thickened, fragile, ridges and brittle nails. Some diabetics develop vesicles and bulla on the toes, sometimes associated with onychomycosis, that promote change structure of the nail [30-32]. The thyroid hormone deficiency or surplus can affect the nail, hair and skin. The hyperthyroidism can be associated to the yellow nail syndrome, slow growing, and absence of lunula and cuticles can be observed. The hypothyroidism can promote dry, brittle and lusterless nails; sometimes longitudinally ridge is associated to hypothyroidism [30, 33-34].

An increase of the nail growth is observed during the pregnancy, and a decrease during lactation, the cause is not well known, but hormonal variation could be the reason. Haenggi et al discuss the variation in the hormone rates during the period of premenopausal and postmenopausal, and the differences in the capillary diameter and blood flow in the nail fold region. Some women observed that the nail changes in the postmenopausal period: the nails become thinner, softer, brittle and peeling [30, 35-36].

2.5.5 Changes in the structure of the nail

Koilonychia is the change in the shape of the nail plate, and a concavity of the nail is similar to a spoon shape. This disorder is related to trauma, exposure of the hands to petroleum solvents, iron deficiency and Raynaud’s disease [37].

Pitting is a disorder that changes the structure of the nail plate, which appear as a small depression over the nail plate. This disorder is related to the Reiter’s syndrome, sarcoidosis, pemphigus, alopecia areata, and incontinentia pigmenti [37].

Onycholisis is the separation of the nail plate from nail bed. The main reason is some mechanical trauma suffered by the nail. Because of the damage in the nail bed, this disorder can be followed by some onychomycosis and the nail color can change to white [37].

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Beau’s lines are transversal linear depression in the nail plate, and can be due to traumas in the nail bed and plate, and can be associated with arsenic poisoning [37].

Some other disorders due to chemical, mechanical trauma and other systemic diseases that can have influence on the disorders of the nails are given in Table 2.5, and the relation between infection, drugs and diseases with the change in the color of the nail region is shown in Table 2.6 [2].

Disorders of the nail due to chemical or mechanical trauma Occupational marks Self-induced

Weavers Nail biting

Musicians Habit tic

Photographers Excessive manicuring

Bartenders Furriers Athletes

Nail changes accompanying systemic disease but relationship uncertain Finding Systemic disease

Koilonychia (Spoon nails) Microcytic anemia, Raynaud’s disease, nail-patella syndrome

White nail Cirrhosis

Clubbing Cardiopulmonary disorders, cirrhosis,

congenital heart disease, inflammatory bowel disease

Shell nails Bronchiectasis

Onycholysis Hyperthyroidism

Yellow nails Lymphedema, pleural effusions, pulmonary

diseases

Hal and half nails Renal and liver disease

Double white bands Hypoalbuminemia

Acral psoriasiform changes Gastrointestinal and upper respiratory tract cancer

Table 2.5 Disorders of the nail due to chemical or mechanical trauma and nail changes accompanying systemic disease but relationship uncertain. Table adapted from Ref. 2, 37.

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Nail color Color location

Local conditions

Drugs and other agents

Mercury products Gray-black Nail plate

Resorcinol Brown Nail plate

Vioform Yellow Nail plate

Picric acid Yellow-brown Nail plate

Nicotine Yellow-brown Nail plate

Hair dyes Black Nail plate

Photographic developer Yellow-brown Nail plate

Hydroquinone Orange-brown Nail plate

Nail lacquer Red-brown Nail plate

Infections Green Nail plate

Pseudomonas Yellow-white Nail bed, plate

Dermatophytes Yellow-brown Nail plate

Candida Green, Yellow, black Nail bed, plate

Molds Miscellaneous

Melanocytic hyperplastia Black-brown Matrix, plate

Melanoma Black Nail bed, matrix,

plate

Post-radiation Black Matrix, nail plate

Systemic conditions

Drugs

Tetracyclines Yellow-brown Nail plate

Antimalarials (quinacrine, chloroquine) Blue Nail plate, bed

Arsenic White bands Nail plate

Chlorpromazine Blue-black Nail bed

Phenolphthalein Gray-black Lunula

Gold salts Black-brown Nail plate

Cytoxic drugs (blemycin, doxorubicin, Cyclophosphamide, melohalan, 5-fluorouracil)

Horizontal or vertical brown-black bands

Nail bed and or plate

Diseases

Argyria Slate gray Lunula

Wilson’s disease Blue Lunula

Yellow-nail syndrome Yellow Nail bed

Addison’s disease Brown Nail bed, matrix

Peutz-Jeghers syndrome Brown-black bands Nail bed, matrix Laugier-Hunziker syndrome Brown-black bands Nail bed

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Table 2.6 Disorders of the nail due infection, drugs and diseases related with the change in the color of the nail. Table adapted from Ref. 2, 37.

2.6 Brief explanation about chemical elements in the metabolic system

Chemical elements are important for the human metabolism and are responsible for chemical reactions, production of cellular structures, metabolism adjustments, and to keep the organism alive. The human body is a complex structure, and a good nutrition is the key to have a health body and avoid diseases. The maintenance of the human body depends on the vitamins, minerals and trace elements supplied to the body through the right alimentation. The roles of the vitamins and minerals to the biological systems are well defined in the literature and will not be discussed here. The trace elements defined by the World Health Organization (WHO) as essential to the humans are iron, zinc, copper, chromium, iodine, cobalt, molybdenum and selenium. The criteria that define which are the essential trace elements (ETE) to the humans is described as a chemical that causes dysfunction or abnormalities in the humans metabolism due to the abstinence or deficiency of the trace element, but that can be reversed after its administration. The chemical elements silicon, manganese, nickel, boron and vanadium have been studied to evaluate the beneficial aspects to the humans, but until now they are not recognized as essential elements.

The assimilation of the ETE by the body depends on the bioavailability of the trace element. There are many factors such as chemical form or species of the ETE, nutritional state, age, gender, food source, pathological conditions, and interactions with other substance, that contribute to the bioavailability of the ETE to the body. The risk of death due deficiency or excess of ETE have been positively reported,

Vitamin B12 deficiency Black Nail bed

Pinta Black Nail bed

Vitiligo Brown Nail bed

Cushing’s syndrome Black Nail bed, matrix

Cirrhosis White Nail bed

Hypoalbuminemia Double white bands (horizontal)

Nail bed Chronic renal disease Half white, half pink

(horizontal)

Nail bed Hereditary subtotal leuconychia White Nail plate

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but the lethal dose is not well defined. The combination of the deficiency or high exposure of ETE with diseases and variation in the metabolism, contribute to the variation on the determination of the lethal dose [38].

2.6.1 Effects of the ETE deficiency and excess

Anemia is a common case of iron deficiency, and low iron intake can reduce the maximum oxygenation of the body reducing the physical performance and causing fatigue. Iron deficiency can increase adverse pregnancy outcomes. The iron excess can induce gastrointestinal problems, liver cirrhosis, risk of cardiomyopathy and hepatic cancer. Iron can help athletes with low ferritin, and increase the weight of the infants with iron deficiency [38-41].

Zinc is involved in the structural maintenance of proteins and regulation of gene expression and the zinc deficiency is related to changes in the metabolism linked with diseases in the heart, diabetes, and high blood pressure. Homeostatic mechanism regulates the zinc concentration in plasma and tissues, and reduce lost of zinc when the intake is reduced. Zinc excess can decrease the erythrocyte superoxide dismutase. A reduction of ferritin concentrations is caused by the effects of zinc on copper and iron. In general, zinc has been positively correlated in the improvement of the symptoms of dysfunctions or diseases like anorexia, depression, bladder cancer, rheumatoid arthritis [38-39, 42-49].

Copper has a relation with metalloenzymes that reduce the molecular oxygen. The copper deficiency results in a decrease in ceruloplasmin levels and superoxide dismutase. This may cause defective tissue synthesis and oesteogenisis, nutropena and iron-resistant anemia. Copper excess can result in gastrointestinal problems eventually leading to multiple organs failure and death [38, 42].

Low selenium intake may cause changes in thyroid hormone function, also can result in incomplete saturation of glutathione present in plasma, erythrocytes and platelets. Low selenium is also related with Keshan disease, which sometimes in combination with infection may produce myocardial abnormalities. Excess of selenium may cause nausea, vomiting and subsequent neurological disease, and hair, nail and skin change. Selenocysteine is the active component of oxidase. Selenium has been reported to prevent asthma and increase the performance in athletes. Selenium is pointed as chemo-preventive substance against free-radicals and in the prevention of the cancer [3, 38-39, 50-52].

Metrology of Nail Clippings as Trace Element Biomarkers

Metrology of Nail Clippings as Trace

Element Biomarkers

Delft University of Technology

Faculty of Applied Sciences

Metrology of Nail Clippings as Trace

Element Biomarkers

Proefschrift

ter verkrijging van de graad van doctor

aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus prof. ir. K C.A.M. Luyben,

voorzitter van het College voor Promoties,

in het openbaar te verdedigen op vrijdag 12 juli 2013 om 15:00 uur

door

Master of Science,

Contents

Chapter 1

General introduction

Chapter 2

Overview of the human nail

Ventral

Intermediate