Preface

Chapter 7 **A Simple and Highly Structured Procaine Hydrochloride as**

Chapter 8 **Biomonitoring of Trace Metals in the Coastal Waters Using**

**of Mercury Species in Water 133**

Shahawi

**VI** Contents

Sasikumar

**Bivalve Molluscs 153**

**Fluorescent Quenching Chemosensor for Trace Determination**

Dyab A. Al-Eryani, Waqas Ahmad, Zeinab M. Saigl, Hassan Alwael, Saleh O. Bahaffi, Yousry M. Moustafa and Mohammad S. El-

Periyadan K. Krishnakumar, Mohammad A. Qurban and Geetha

Trace elements, including metals, play an important role in many biological systems, both in normal or pathological processes. For the human metabolism, chromium, arsenic, cadmium, mercury, and lead are considered to be the notorious "top five" toxins among all trace ele‐ ments. Hexavalent chromium, for example, is highly toxic and carcinogenic; the same is val‐ id for mercury vapor and various bivalent mercury compounds. To understand the expedient toxicity of these five heavy metals mechanistically, one has to take into account that these metals exert outstanding affinity for strongly binding with sulfur; if present in the human body, they can easily bind to the thiol (–SH) groups of the amino acid cysteine in enzymes, thus inhibiting or even stopping important metabolic reactions, which deteriorates the health status of the affected person, and often ends in death.

Trace elements are resistant towards biodegradation; therefore, they undergo environmental accumulation, which enables their entrance into the food chain. Prior to mitigating trace ele‐ ment pollution, it is of importance to develop enhanced analytical systems to reliably and precisely determine the level of trace element contamination of soil, water, and air. Migra‐ tion of trace elements into areas originally not polluted by them typically occurs as dust, by leaching through soil, or by dispersing heavy metals containing sewage sludge.

We are tremendously optimistic that the exploratory and scientific attempts collected and summarized in this book will encourage researchers all over the globe to deepen their activi‐ ties in this field, and to attract the interest of undergraduates as well as of progressive repre‐ sentatives from relevant various sectors. Primarily, these activities will boost the impatiently desired breakthrough of advanced "trace element" identification and application processes.

Particular acknowledgment goes to the publishing process manager, Ms. Dajana Pemac, for her cooperation, exceptional efforts, and prompt response to my requests. Again, we would like to thank cordially all contributors to this issue for their supreme work.

**Hosam El-Din M. Saleh, Ph.D.**

Atomic Energy Authority Radioisotope Department Nuclear Research Center Giza, Egypt

**Section 1**

**Introduction to Trace Elements**

**Introduction to Trace Elements**

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: An Introduction to Trace**

**Introductory Chapter: An Introduction to Trace** 

DOI: 10.5772/intechopen.75010

"Trace elements" are such building blocks of our planet and all living organisms, which, although occurring in rather modest concentration levels, are indispensable for a plethora of metabolic processes. Especially since the last decades, we observe enormously increasing efforts devoted by the scientific community to investigate, characterize and quantify trace elements. So-called "essential trace elements" are important constituents of human food, animal fodder, plant fertilizers, or cultivation media to form biotechnologically relevant microbes. Trace elements travel from the soil through the food chain, starting from phytoplankton until reaching our dinner tables; apart from food, drinking water is another important source for trace element uptake by all organisms. This makes trace elements interesting for diverse scientists such as

analytical chemists, biochemists, geologists, physiologists, zoologists, and botanists [1].

In dependence on the scientific realm, different definitions for the terminus "trace elements" are found. An analytical chemist considers an element in a given sample with an average concentration of less than 100 ppm on an atomic counting basis or less than 100 μg/g on a mass basis, a "trace element". In contrast, biochemists define "trace elements" as those elements, which, although present only in tiny amounts, are needed to maintain the physiological balance of an organism, often acting as cofactors in enzymatic reactions; this biochemical definition encompasses various heavy metals (iron, copper, nickel, vanadium, cobalt, manganese, molybdenum, chromium, and zinc), some nonmetals (boron and iodine), and certain metalloids (selenium, silicon, and arsenic). This definition implies a daily requirement for "essential trace elements" by humans in amounts between 50 μg and 18 mg/day [2]. To become susceptible toward metabolizing by animals and humans, some trace elements need to undergo transformation by microbes into complex bioavailable forms, as observed in the case of cobalt, which is utilized mainly as cyanocobalamin (vitamin B12) [3, 4]. Moreover,

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Elements**

**1. Introduction**

**Elements**

Martin Koller and Hosam M. Saleh

Martin Koller and Hosam M. Saleh

http://dx.doi.org/10.5772/intechopen.75010

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Introductory Chapter: An Introduction to Trace Elements Introductory Chapter: An Introduction to Trace Elements**

DOI: 10.5772/intechopen.75010

Martin Koller and Hosam M. Saleh

Additional information is available at the end of the chapter Martin Koller and Hosam M. SalehAdditional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75010

## **1. Introduction**

"Trace elements" are such building blocks of our planet and all living organisms, which, although occurring in rather modest concentration levels, are indispensable for a plethora of metabolic processes. Especially since the last decades, we observe enormously increasing efforts devoted by the scientific community to investigate, characterize and quantify trace elements. So-called "essential trace elements" are important constituents of human food, animal fodder, plant fertilizers, or cultivation media to form biotechnologically relevant microbes. Trace elements travel from the soil through the food chain, starting from phytoplankton until reaching our dinner tables; apart from food, drinking water is another important source for trace element uptake by all organisms. This makes trace elements interesting for diverse scientists such as analytical chemists, biochemists, geologists, physiologists, zoologists, and botanists [1].

In dependence on the scientific realm, different definitions for the terminus "trace elements" are found. An analytical chemist considers an element in a given sample with an average concentration of less than 100 ppm on an atomic counting basis or less than 100 μg/g on a mass basis, a "trace element". In contrast, biochemists define "trace elements" as those elements, which, although present only in tiny amounts, are needed to maintain the physiological balance of an organism, often acting as cofactors in enzymatic reactions; this biochemical definition encompasses various heavy metals (iron, copper, nickel, vanadium, cobalt, manganese, molybdenum, chromium, and zinc), some nonmetals (boron and iodine), and certain metalloids (selenium, silicon, and arsenic). This definition implies a daily requirement for "essential trace elements" by humans in amounts between 50 μg and 18 mg/day [2]. To become susceptible toward metabolizing by animals and humans, some trace elements need to undergo transformation by microbes into complex bioavailable forms, as observed in the case of cobalt, which is utilized mainly as cyanocobalamin (vitamin B12) [3, 4]. Moreover,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

geologists define trace elements by concentrations not surmounting 1 pro mille of a rock or mineral. In addition, the term "trace element" is frequently used when analyzing the elemental composition of igneous rocks, hence those rocks formed by magma. In mineralogy, trace elements can substitute network-forming ions in mineral crystal structures; here, trace elements are not vital to a mineral's defined composition and do not appear in the mineral's chemical formula. However, as well known in the case of quartz, those metals occurring in trace quantities result in characteristic coloration of the minerals; for example, the substitution of silicon by iron in traces gives the quartz amethysts its famous purple coloration [5].

plants); to understand these uptake mechanism and how the plants develop the required resistance factors needed to withstand the high concentration of trace elements, which often are highly toxic heavy metals, needs highly precise detection devices [11, 12]. The determination of type and concentration of trace elements in nutrition, body tissue and liquids, coal, water, soil, and so on is regarded as the first and most important step to follow the mechanisms controlling the dispersal and accumulation of trace elements. Element speciation in different media (water, soil, food, plants tissue, coal, biological matter, food and fodder, minerals, etc.) is pivotal to assess an element's toxicity, bioavailability, environmental mobility, and biogeochemical performance. Classical methods for elemental analysis, such as gravimetric, titrimetric, calorimetric, and so on, do not sufficiently address the precision requirements when analyzing elements in trace concentrations. Therefore, new analytical techniques have been developed, which greatly simplified the quantitation of many trace elements and considerably extended their detection range. In this context, the development of reproducible and accurate techniques for trace element analysis in different media using spectroscopic instrumentation is continuously advanced. Here, inductively coupled plasma mass spectroscopy (ICP-MS) analysis of trace elements is of increasing importance; among the different ICP-MS techniques, modern developments like dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) was successfully used to simultaneously determine 17 trace elements in blood samples [13]. Similar techniques encompass sector field inductively coupled plasma mass spectrometry (SF-ICP-MS), which was successfully used for determination of aluminum, beryllium, cadmium, cobalt, chromium, mercury, manganese, nickel, lead, and vanadium in urine, serum, blood, and cerebrospinal fluid [14]. Moreover, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) constitutes a quantitative microbeam technique for rapid and accurate determinations of trace element concentrations in the sub-ppm range in various target materials [15]. A classical method, atom absorption spectroscopy (AAS), is still widely used to quantify various trace elements [16, 17], for example, the well-known graphite furnace atomic absorption spectrometry, which was, *inter alia*, successfully used for trace element determination in fish samples [18]. Prior to AAS measurement, biological samples to be investigated for trace elements need to be solubilized by different ashing techniques; here, a multitude of methods exists, with the use of nitric acid for "wet ashing" often being the method of choice [19]. Decades ago, X-ray fluorescence analysis was recognized as a viable tool to determine trace elements in geological samples [20–22]; in this context, a study reported by Leoni and Saitta in 1976 illustrated the viability of this technique to determine a total of 29 metals in rock and mineral samples in an astonishingly broad concentration range between 1 and 5000 ppm [20]. This AAS technique was later advanced to make it more custom-oriented and better applicable to different types of samples. Moreover, radiochemical trace element analysis is also widely used, mainly using radiochemical neutron activation analysis [23, 24]. By resorting to this method, trace elements were accurately determined in various samples, which are as diverse as human tissue [23], food products [16], and also in terrestrial, lunar,

Introductory Chapter: An Introduction to Trace Elements

http://dx.doi.org/10.5772/intechopen.75010

5

and meteoritic rock samples [24].

Keeping with trace elements playing a role in human metabolism, we find at least two of them as so-called "vital poisons" in the periodic system of elements, namely chromium and arsenic, well-known toxins, which, nevertheless, are essential for the functioning of our metabolism. Apart from the 14 undisputed essential trace elements mentioned in the above paragraphs, others, such as fluorine, strontium, or lithium, are suggested to also display biological functions in humans, which, although not clearly elucidated yet mechanistically, are evidenced by element deprivation effects in diverse metabolic studies [1, 6]. In addition, limited circumstantial evidence for certain benefits or biological function in mammals is reported in the case of the metals aluminum, rubidium, cadmium, germanium, tin, and lead, the so-called "ultra-trace elements" [7]. Only for prokaryotes, a physiological role of tungsten [8] and lanthanum [9] is generally accepted. What is not included in this list are all those elements which are present in significant amounts in our body, such as the four biological basic elements hydrogen, oxygen, carbon, and nitrogen and the macroelements sulfur, chlorine, phosphorus, magnesium, sodium, potassium, and calcium.

## **2. Determination of trace elements**

However, trace elements are not only blessing but also curse by generating environmental and health-related concerns when exceeding certain concentration levels. In the context of environmentally sensitive trace elements, it is often not easy to draw a clear line between the desired concentration range of a trace element and the range in which it already exerts toxicity [10]. Aquatic environments, soil, organisms, food, fodder, energy carriers like coal, and airborne particles are targets, which are potentially contaminated with trace elements; this, in turn, provokes the need for modern, reliable, and fast tools for determination of trace element in diverse matrices. Although many problems caused by trace elements are already well managed in the meanwhile, constant vigilance in environmental exposure, application, and nutritional supply of trace elements still displays the *conditio sine qua non*, and calls for intensified research in trace element determination. For example, during mining, beneficiation, and combustion of coal as well as during metal mining and follow-up treatments, a total of about 26 trace elements, among them toxic heavy metals like mercury, need to be considered and quantified [6]. In the biological field, many plants are described to heavily accumulate trace elements from soil (metal "hyperaccumulator" plants); to understand these uptake mechanism and how the plants develop the required resistance factors needed to withstand the high concentration of trace elements, which often are highly toxic heavy metals, needs highly precise detection devices [11, 12]. The determination of type and concentration of trace elements in nutrition, body tissue and liquids, coal, water, soil, and so on is regarded as the first and most important step to follow the mechanisms controlling the dispersal and accumulation of trace elements. Element speciation in different media (water, soil, food, plants tissue, coal, biological matter, food and fodder, minerals, etc.) is pivotal to assess an element's toxicity, bioavailability, environmental mobility, and biogeochemical performance. Classical methods for elemental analysis, such as gravimetric, titrimetric, calorimetric, and so on, do not sufficiently address the precision requirements when analyzing elements in trace concentrations. Therefore, new analytical techniques have been developed, which greatly simplified the quantitation of many trace elements and considerably extended their detection range. In this context, the development of reproducible and accurate techniques for trace element analysis in different media using spectroscopic instrumentation is continuously advanced. Here, inductively coupled plasma mass spectroscopy (ICP-MS) analysis of trace elements is of increasing importance; among the different ICP-MS techniques, modern developments like dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) was successfully used to simultaneously determine 17 trace elements in blood samples [13]. Similar techniques encompass sector field inductively coupled plasma mass spectrometry (SF-ICP-MS), which was successfully used for determination of aluminum, beryllium, cadmium, cobalt, chromium, mercury, manganese, nickel, lead, and vanadium in urine, serum, blood, and cerebrospinal fluid [14]. Moreover, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) constitutes a quantitative microbeam technique for rapid and accurate determinations of trace element concentrations in the sub-ppm range in various target materials [15]. A classical method, atom absorption spectroscopy (AAS), is still widely used to quantify various trace elements [16, 17], for example, the well-known graphite furnace atomic absorption spectrometry, which was, *inter alia*, successfully used for trace element determination in fish samples [18]. Prior to AAS measurement, biological samples to be investigated for trace elements need to be solubilized by different ashing techniques; here, a multitude of methods exists, with the use of nitric acid for "wet ashing" often being the method of choice [19]. Decades ago, X-ray fluorescence analysis was recognized as a viable tool to determine trace elements in geological samples [20–22]; in this context, a study reported by Leoni and Saitta in 1976 illustrated the viability of this technique to determine a total of 29 metals in rock and mineral samples in an astonishingly broad concentration range between 1 and 5000 ppm [20]. This AAS technique was later advanced to make it more custom-oriented and better applicable to different types of samples. Moreover, radiochemical trace element analysis is also widely used, mainly using radiochemical neutron activation analysis [23, 24]. By resorting to this method, trace elements were accurately determined in various samples, which are as diverse as human tissue [23], food products [16], and also in terrestrial, lunar, and meteoritic rock samples [24].

geologists define trace elements by concentrations not surmounting 1 pro mille of a rock or mineral. In addition, the term "trace element" is frequently used when analyzing the elemental composition of igneous rocks, hence those rocks formed by magma. In mineralogy, trace elements can substitute network-forming ions in mineral crystal structures; here, trace elements are not vital to a mineral's defined composition and do not appear in the mineral's chemical formula. However, as well known in the case of quartz, those metals occurring in trace quantities result in characteristic coloration of the minerals; for example, the substitution of silicon by iron in traces gives the quartz amethysts its famous purple coloration [5]. Keeping with trace elements playing a role in human metabolism, we find at least two of them as so-called "vital poisons" in the periodic system of elements, namely chromium and arsenic, well-known toxins, which, nevertheless, are essential for the functioning of our metabolism. Apart from the 14 undisputed essential trace elements mentioned in the above paragraphs, others, such as fluorine, strontium, or lithium, are suggested to also display biological functions in humans, which, although not clearly elucidated yet mechanistically, are evidenced by element deprivation effects in diverse metabolic studies [1, 6]. In addition, limited circumstantial evidence for certain benefits or biological function in mammals is reported in the case of the metals aluminum, rubidium, cadmium, germanium, tin, and lead, the so-called "ultra-trace elements" [7]. Only for prokaryotes, a physiological role of tungsten [8] and lanthanum [9] is generally accepted. What is not included in this list are all those elements which are present in significant amounts in our body, such as the four biological basic elements hydrogen, oxygen, carbon, and nitrogen and the macroelements sulfur, chlorine, phosphorus, magnesium, sodium, potassium,

However, trace elements are not only blessing but also curse by generating environmental and health-related concerns when exceeding certain concentration levels. In the context of environmentally sensitive trace elements, it is often not easy to draw a clear line between the desired concentration range of a trace element and the range in which it already exerts toxicity [10]. Aquatic environments, soil, organisms, food, fodder, energy carriers like coal, and airborne particles are targets, which are potentially contaminated with trace elements; this, in turn, provokes the need for modern, reliable, and fast tools for determination of trace element in diverse matrices. Although many problems caused by trace elements are already well managed in the meanwhile, constant vigilance in environmental exposure, application, and nutritional supply of trace elements still displays the *conditio sine qua non*, and calls for intensified research in trace element determination. For example, during mining, beneficiation, and combustion of coal as well as during metal mining and follow-up treatments, a total of about 26 trace elements, among them toxic heavy metals like mercury, need to be considered and quantified [6]. In the biological field, many plants are described to heavily accumulate trace elements from soil (metal "hyperaccumulator"

and calcium.

**2. Determination of trace elements**

4 Trace Elements - Human Health and Environment

## **Author details**

Martin Koller1 and Hosam M. Saleh2 \*

\*Address all correspondence to: hosamsaleh70@yahoo.com

1 Office of Research Management and Service, c/o Institute of Chemistry, University of Graz, Graz, Austria

[12] Chandra S, Gusain YS, Bhatt A. Metal hyperaccumulator plants and environmental pollution. In: Microbial Biotechnology in Environmental Monitoring and Cleanup. PA,

Introductory Chapter: An Introduction to Trace Elements

http://dx.doi.org/10.5772/intechopen.75010

7

[13] D'Ilio S, Violante N, Di Gregorio M, Senofonte O, Petrucci F. Simultaneous quantification of 17 trace elements in blood by dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) equipped with a high-efficiency sample introduction

[14] Bocca B, Alimonti A, Petrucci F, Violante N, Sancesario G, Forte G, Senofonte O. Quantification of trace elements by sector field inductively coupled plasma mass spectrometry in urine, serum, blood and cerebrospinal fluid of patients with Parkinson's

[15] Liu Y, Hu Z, Gao S, Günther D, Xu J, Gao C, Chen H. *In situ* analysis of major and trace elements of anhydrous minerals by LA-ICP-MS without applying an internal standard.

[16] Bermudez GM, Jasan R, Plá R, Pignata ML. Heavy metal and trace element concentrations in wheat grains: Assessment of potential non-carcinogenic health hazard through

[17] Singh M, Yadav P, Garg VK, Sharma A, Singh B, Sharma H. Quantification of minerals and trace elements in raw caprine milk using flame atomic absorption spectrophotometry and flame photometry. Journal of Food Science and Technology. 2015;**52**(8):5299-5304

[18] Tüzen M. Determination of heavy metals in fish samples of the middle Black Sea (Turkey) by graphite furnace atomic absorption spectrometry. Food Chemistry. 2003;**80**(1):119-123

[19] Clegg MS, Keen CL, Lönnerdal B, Hurley LS. Influence of ashing techniques on the analysis of trace elements in animal tissue. Biological Trace Element Research.

[20] Leoni L, Saitta M. X-ray fluorescence analysis of 29 trace elements in rock and mineral standards. Rendiconti della Societa Italiana di Mineralogia e Petrologia. 1976;**32**(2):

[21] Rowe H, Hughes N, Robinson K. The quantification and application of handheld energydispersive X-ray fluorescence (ED-XRF) in mudrock chemostratigraphy and geochemis-

[22] Margui E, Zawisza B, Sitko R. Trace and ultratrace analysis of liquid samples by X-ray fluorescence spectrometry. TrAC Trends in Analytical Chemistry. 2014;**53**:73-83 [23] Lievens P, Versieck J, Cornelis R, Hoste J. The distribution of trace elements in normal human liver determined by semi-automated radiochemical neutron activation analysis.

[24] Keays RR, Ganapathy R, Laul JC, Krähenbühl U, Morgan JW. The simultaneous determination of 20 trace elements in terrestrial, lunar and meteoritic material by radiochemi-

Journal of Radioanalytical and Nuclear Chemistry. 1977;**37**(1):483-496

cal neutron activation analysis. Analytica Chimica Acta. 1974;**72**(1):1-29

their consumption. Journal of Hazardous Materials. 2011;**193**:264-271

disease. Spectrochimica Acta Part B: Atomic Spectroscopy. 2004;**59**(4):559-566

USA: Hershey, IGI Global; 2018. pp. 305-317

Chemical Geology. 2008;**257**(1-2):34-43

try. Chemical Geology. 2012;**324**:122-131

1981;**3**(2):107-115

497-510

system. Analytica Chimica Acta. 2006;**579**(2):202-208

2 Radioisotope Department, Nuclear Research Center, Atomic Energy Authority, Cairo, Egypt

## **References**


[12] Chandra S, Gusain YS, Bhatt A. Metal hyperaccumulator plants and environmental pollution. In: Microbial Biotechnology in Environmental Monitoring and Cleanup. PA, USA: Hershey, IGI Global; 2018. pp. 305-317

**Author details**

6 Trace Elements - Human Health and Environment

and Hosam M. Saleh2

Science (Washington). 1948;**108**(2797):134

tive Ca2+ channels. Cell Calcium. 2007;**41**(2):97-106

China. Science of the Total Environment. 2017;**583**:421-431

Journal of Chemical Engineering. 2011;**2011**. Article ID 939161

\*Address all correspondence to: hosamsaleh70@yahoo.com

\*

1 Office of Research Management and Service, c/o Institute of Chemistry, University of Graz,

[1] Prashanth L, Kattapagari KK, Chitturi RT, Baddam VRR, Prasad LK. A review on role of essential trace elements in health and disease. Journal of Dr. NTR University of Health

[3] Fairweather-Tait SJ. Bioavailability of trace elements. Food Chemistry. 1992;**43**(3):213-217

[4] Rickes EL, Brink NG, Koniuszy PR, Wood TR, Folkers K. Vitamin B12, a cobalt complex.

[5] Lameiras FS, Nunes EHM, Vasconcelos WL. Infrared and chemical characterization of natural amethysts and prasiolites colored by irradiation. Materials Research.

[6] Swaine DJ. Why trace elements are important. Fuel Processing Technology. 2000;**65**:21-33

[7] Nielsen FH. Ultratrace minerals. In: Shils ME et al. USDA, ARS Source: Modern nutri-

[8] Andreesen JR, Makdessi K. Tungsten, the surprisingly positively acting heavy metal element for prokaryotes. Annals of the New York Academy of Sciences. 2008;**1125**(1):215-229

[9] Campbell AK, Naseem R, Wann K, Holland IB, Matthews SB. Fermentation product butane 2, 3-diol induces Ca2+ transients in *E. coli* through activation of lanthanum-sensi-

[10] Wang J, Liu G, Liu H, Lam PK. Multivariate statistical evaluation of dissolved trace elements and a water quality assessment in the middle reaches of Huaihe River, Anhui,

[11] Tangahu BV, Sheikh Abdullah SR, Basri H, Idris M, Anuar N, Mukhlisin M. A review on heavy metals (As, Pb, and Hg) uptake by plants through phytoremediation. International

tion in health and disease. Baltimore: Williams & Wilkins; 1999. pp. 283-303

[2] Mertz W. The essential trace elements. Science. 1981;**213**(4514):1332-1338

2 Radioisotope Department, Nuclear Research Center, Atomic Energy Authority, Cairo,

Martin Koller1

Graz, Austria

**References**

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2009;**12**(3):315-320

Egypt


**Section 2**

**Trace Elements in the Human Body**

**Trace Elements in the Human Body**

**Chapter 2**

**Provisional chapter**

**Minor and Trace Elements in Whole Blood, Tissues,**

**Minor and Trace Elements in Whole Blood, Tissues,** 

Microelements play different important roles in many physiological processes in all biological systems in both normal physiological and pathological conditions. They take part in the transport of nutrients and gases, support temperature, acid-base balance, homeostasis of the human organisms, maternal and child mental health, the functioning of enzymes, protein and DNA syntheses, cytoskeleton activation, etc. We have performed simultaneous determination of a number of minor and trace elements in whole blood and tissues of mammals by two-jet plasma atomic emission spectrometry (TJP-AES). TJP-AES allows direct analysis of powders without wet acid digestion and can be used for analysis of both large and small amount of the sample, which is important for biomedical investigations with humans and experimental animals. In addition, a content of different elements in preparations of human immunoglobulins was estimated by TJP-AES as well as using different physicochemical methods, the functional role of metal ions in antibodies functioning was analyzed. The analysis of the relative activity of antibodies with catalytic activity (abzymes) in the hydrolysis of DNA, RNA, proteins, peptides and oxidation-reduction reactions and the role of metal ions in the catalysis of these reactions

**Keywords:** two-jet plasma atomic emission spectrometry analysis, minor and trace

Minor and trace elements including metals play important roles in many systems, normal and pathologic processes. Nowadays, there are many methods for elemental analysis of different biological samples. Atomic absorption spectrometry (AAS), inductively coupled plasma atomic

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.75939

**Proteins and Immunoglobulins of Mammals**

**Proteins and Immunoglobulins of Mammals**

Natalia P. Zaksas and Georgy A. Nevinsky

Natalia P. Zaksas and Georgy A. Nevinsky

http://dx.doi.org/10.5772/intechopen.75939

by abzymes were carried out.

elements, functional role of metals

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals**

DOI: 10.5772/intechopen.75939

Natalia P. Zaksas and Georgy A. Nevinsky Natalia P. Zaksas and Georgy A. Nevinsky

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75939

#### **Abstract**

Microelements play different important roles in many physiological processes in all biological systems in both normal physiological and pathological conditions. They take part in the transport of nutrients and gases, support temperature, acid-base balance, homeostasis of the human organisms, maternal and child mental health, the functioning of enzymes, protein and DNA syntheses, cytoskeleton activation, etc. We have performed simultaneous determination of a number of minor and trace elements in whole blood and tissues of mammals by two-jet plasma atomic emission spectrometry (TJP-AES). TJP-AES allows direct analysis of powders without wet acid digestion and can be used for analysis of both large and small amount of the sample, which is important for biomedical investigations with humans and experimental animals. In addition, a content of different elements in preparations of human immunoglobulins was estimated by TJP-AES as well as using different physicochemical methods, the functional role of metal ions in antibodies functioning was analyzed. The analysis of the relative activity of antibodies with catalytic activity (abzymes) in the hydrolysis of DNA, RNA, proteins, peptides and oxidation-reduction reactions and the role of metal ions in the catalysis of these reactions by abzymes were carried out.

**Keywords:** two-jet plasma atomic emission spectrometry analysis, minor and trace elements, functional role of metals

## **1. Introduction**

Minor and trace elements including metals play important roles in many systems, normal and pathologic processes. Nowadays, there are many methods for elemental analysis of different biological samples. Atomic absorption spectrometry (AAS), inductively coupled plasma atomic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS) are usually used for analysis of blood and animal tissues [1, 2]. Generally, these methods require matrix destruction with concentrated acids. Using microwave-assisted wet acid digestion with temperature control and elevated pressure allows reducing the time of sample digestion and risk of element losses. For direct ICP-MS analysis of whole blood and serum, dilution with alkaline solutions containing EDTA, ammonia and Triton X-100 was also employed to lyse the blood cells and prevent blood clotting [3, 4]. To improve the analytical capabilities of the methods applied, the new analytical techniques and reagents are being developed. A collision/reaction cell technology allowed the removal of polyatomic interferences and extended the capabilities of ICP-MS for trace element determination [5]. To decrease limits of detection (LODs) of trace elements, different ways of their preconcentration were offered. For determining the low concentration of Pb in blood serum by flame AAS, Barbosa with coauthors [6] used oxidized carbon nanotubes covered with bovine serum albumin layers; preconcentration was performed in untreated blood serum and allowed getting LOD of Pb at the level of 2 μg/L. Mortada with coauthors [7] suggested hydroxyapatite nanorods prepared from recycled eggshell for solid phase extraction of Pb, Cu and Zn from solutions of different biological samples followed by AAS analysis. In spite of undeniable progress in developing the above-mentioned methods, the analysis of biological samples using solid sampling is very attractive as the analytical procedure is simple and risk of contamination and analyte losses is improbable. To adapt the above methods for direct analysis of solid biological samples, laser ablation (LA) and electrothermal vaporization (ETV) were applied. LA-ICP-MS has got significant attention over the last decade for the analysis of biological samples [8]. It has been mainly applied to produce images of element distributions in human and animal tissues, which is of great scientific interest [9]. The main challenge of facing the LA-ICP-MS application is fully quantitative analysis requiring complex strategies for producing reliable calibrating materials. Lack of certified reference materials (CRMs) with different biological matrices and complications of preparing matrix-matched calibration samples [10] often make it difficult to carry out quantitative multi-elemental analysis of different biological tissues. This problem is well known for ETV-ICP-AES, X-ray fluorescence and laser-induced breakdown spectrometry (LIBS).

and an ICP by a high power, which allows analysis of powdered samples without sample dissolution. Although the TJP appeared approximately at the same time as an ICP, TJP-AES was not generally recognized since only a few copies of the plasmatron were produced; it has not been modernized for decades. Nowadays, TJP-AES is experiencing a new stage in its development because a new modern plasmatron was designed at "VMK-Optoelektronika" (Russia). A photo-

**Figure 1.** (A) Plasma torch; (B) electrode unit and analytical regions of the plasma flow: 1, before the jet confluence; 2,

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Argon plasma jets are generated in non-consumable electrodes (copper anode and tungsten cathode); they join at the output to form plasma discharge. The argon consumption does not usually exceed 5 l/min. The power supply of the plasma generator, gas flow and automatic sample introduction systems are computer controlled. A TJP power can be fixed from 5 to 12 kW by varying the current strength in the range of 40–100 A. Current fluctuation does not exceed 1%. A new plasmatron is equipped with a concave diffraction grating (2400 lines/mm) and two multicrystal photodiode arrays allowing spectra to be measured in two spectral ranges: 190–350 and 390–450 nm. To transfer powders into the plasma, a powder introduction device was developed. The sample is placed in a Plexiglas beaker, inserted into the device and roiled with blast waves produced by a spark between zirconium electrodes over the surface of the powder. An aerosol obtained is delivered into the plasma with a carrier gas. The device allows the introduction of

There are two analytical regions in the TJP – before and after the jet confluence (**Figure 1**). The region of the confluence is not used for analysis due to high background emission. A study of behavior of a wide range of elements in the plasma has shown that the highest ratio of the

graph of the plasma torch and scheme of electrode unit are presented in **Figure 1**.

both small and large amount of the samples (5–500 mg).

**2.2. Analytical regions**

after the jet confluence.

In recent times, LIBS has been applied as a screening tool for trace element bio-imaging in human and animal organs. To detect Wilson's disease, the study of Cu distribution in human liver was carried out [11]. A low-cost approach allows the quick detection of pathological accumulation of Cu in the affected organs. The LIBS mapping of the mice kidney slices after injection of a solution containing Gd-based nanoparticles was performed [12]. Each of the above methods has both advantages and disadvantages, and the choice of the analytical method depends on the sample nature, analytical tasks and availability of appropriate device and calibration samples. In the present work, two-jet plasma atomic emission spectrometry (TJP-AES) was used for analysis of different biological samples.

## **2. Two-jet plasma atomic emission spectrometry**

#### **2.1. A two-jet plasmatron**

The TJP was developed by Zheenbaev and Engel'sht in the USSR (Kyrgyzstan Institute of Physics) in the mid-1970s [13]. It is a direct current (dc) plasma that differs from dc plasmas described [14, 15] Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals http://dx.doi.org/10.5772/intechopen.75939 13

**Figure 1.** (A) Plasma torch; (B) electrode unit and analytical regions of the plasma flow: 1, before the jet confluence; 2, after the jet confluence.

and an ICP by a high power, which allows analysis of powdered samples without sample dissolution. Although the TJP appeared approximately at the same time as an ICP, TJP-AES was not generally recognized since only a few copies of the plasmatron were produced; it has not been modernized for decades. Nowadays, TJP-AES is experiencing a new stage in its development because a new modern plasmatron was designed at "VMK-Optoelektronika" (Russia). A photograph of the plasma torch and scheme of electrode unit are presented in **Figure 1**.

Argon plasma jets are generated in non-consumable electrodes (copper anode and tungsten cathode); they join at the output to form plasma discharge. The argon consumption does not usually exceed 5 l/min. The power supply of the plasma generator, gas flow and automatic sample introduction systems are computer controlled. A TJP power can be fixed from 5 to 12 kW by varying the current strength in the range of 40–100 A. Current fluctuation does not exceed 1%. A new plasmatron is equipped with a concave diffraction grating (2400 lines/mm) and two multicrystal photodiode arrays allowing spectra to be measured in two spectral ranges: 190–350 and 390–450 nm.

To transfer powders into the plasma, a powder introduction device was developed. The sample is placed in a Plexiglas beaker, inserted into the device and roiled with blast waves produced by a spark between zirconium electrodes over the surface of the powder. An aerosol obtained is delivered into the plasma with a carrier gas. The device allows the introduction of both small and large amount of the samples (5–500 mg).

#### **2.2. Analytical regions**

emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS) are usually used for analysis of blood and animal tissues [1, 2]. Generally, these methods require matrix destruction with concentrated acids. Using microwave-assisted wet acid digestion with temperature control and elevated pressure allows reducing the time of sample digestion and risk of element losses. For direct ICP-MS analysis of whole blood and serum, dilution with alkaline solutions containing EDTA, ammonia and Triton X-100 was also employed to lyse the blood cells and prevent blood clotting [3, 4]. To improve the analytical capabilities of the methods applied, the new analytical techniques and reagents are being developed. A collision/reaction cell technology allowed the removal of polyatomic interferences and extended the capabilities of ICP-MS for trace element determination [5]. To decrease limits of detection (LODs) of trace elements, different ways of their preconcentration were offered. For determining the low concentration of Pb in blood serum by flame AAS, Barbosa with coauthors [6] used oxidized carbon nanotubes covered with bovine serum albumin layers; preconcentration was performed in untreated blood serum and allowed getting LOD of Pb at the level of 2 μg/L. Mortada with coauthors [7] suggested hydroxyapatite nanorods prepared from recycled eggshell for solid phase extraction of Pb, Cu and Zn from solutions of different biological samples followed by AAS analysis. In spite of undeniable progress in developing the above-mentioned methods, the analysis of biological samples using solid sampling is very attractive as the analytical procedure is simple and risk of contamination and analyte losses is improbable. To adapt the above methods for direct analysis of solid biological samples, laser ablation (LA) and electrothermal vaporization (ETV) were applied. LA-ICP-MS has got significant attention over the last decade for the analysis of biological samples [8]. It has been mainly applied to produce images of element distributions in human and animal tissues, which is of great scientific interest [9]. The main challenge of facing the LA-ICP-MS application is fully quantitative analysis requiring complex strategies for producing reliable calibrating materials. Lack of certified reference materials (CRMs) with different biological matrices and complications of preparing matrix-matched calibration samples [10] often make it difficult to carry out quantitative multi-elemental analysis of different biological tissues. This problem is well known for ETV-ICP-AES, X-ray fluorescence and laser-induced breakdown spectrometry (LIBS).

12 Trace Elements - Human Health and Environment

In recent times, LIBS has been applied as a screening tool for trace element bio-imaging in human and animal organs. To detect Wilson's disease, the study of Cu distribution in human liver was carried out [11]. A low-cost approach allows the quick detection of pathological accumulation of Cu in the affected organs. The LIBS mapping of the mice kidney slices after injection of a solution containing Gd-based nanoparticles was performed [12]. Each of the above methods has both advantages and disadvantages, and the choice of the analytical method depends on the sample nature, analytical tasks and availability of appropriate device and calibration samples. In the present work, two-jet plasma atomic emission spectrometry

The TJP was developed by Zheenbaev and Engel'sht in the USSR (Kyrgyzstan Institute of Physics) in the mid-1970s [13]. It is a direct current (dc) plasma that differs from dc plasmas described [14, 15]

(TJP-AES) was used for analysis of different biological samples.

**2. Two-jet plasma atomic emission spectrometry**

**2.1. A two-jet plasmatron**

There are two analytical regions in the TJP – before and after the jet confluence (**Figure 1**). The region of the confluence is not used for analysis due to high background emission. A study of behavior of a wide range of elements in the plasma has shown that the highest ratio of the

**Figure 2.** I<sup>l</sup> /Ib distribution along the plasma flow for Mn I 280.11, Al I 308.22; Cd I 228.80 and Fe I 302.06 lines: region (A) before the jet confluence, (B) after the jet confluence; 0 – point in the region of the confluence.

analytical line intensity to the background one (I<sup>l</sup> /Ib ) realizes in the region before the jet confluence both for atomic and ionic lines. As an example, the distribution of I<sup>l</sup> /Ib for analytical lines Mn I 280.11, Al I 308.22; Cd I 228.80 and Fe I 302.06 along the plasma flow is shown in **Figure 2**.

Analytical lines of Ag and Zn registered in the region before and after the jet confluence are shown in **Figure 3**.

As it is seen, the analytical signals are at the level of background fluctuations in the region after the jet confluence, but they are considerably higher than background in the region before one, which testifies to better LODs of elements in this region. One of the problems arising in the TJP-AES analysis of powdered samples is their incomplete evaporation which can lead to a systematic underestimation of the analysis results. The short residence time of the sample in the plasma is one of the reasons for the partial sample evaporation. In addition, evaporation efficiency depends on chemical composition, structure and particle size of powdered samples [17]. To demonstrate evaporation efficiency in the analytical zones of the TJP, silicon carbide (SiC) powders with an average particle size of 1, 3, 7.5, 17, 22 and 36 μm were used. SiC is a heat-resistant material having a high hardness close to the hardness of diamond. SiC evaporation was controlled by the intensity of weak Si I 212.30 line. To avoid signal self-absorption, the powders were diluted with graphite 100 times. The dependence of Si I 212.30 line intensity on the particle size is given in **Figure 4**.

The different behavior of silicon line is observed in the regions investigated. In the region before the jet confluence, there is an increase in the silicon line intensity along with a decrease in the particle size, the smaller particle size and the better evaporation efficiency. However, the complete sample evaporation does not occur even at a particle size of 3 μm. In the region after the jet confluence, the maximum intensity is achieved even at a particle size of 17 μm. The decrease in intensity at smaller particles seems to occur due to the introduction of light particles into the region after the confluence is complicated by a resistance of the consistent plasma jets. Nevertheless, the effect of particle size on evaporation efficiency is considerably weaker in the region after the jet confluence. Thus, the region before the jet confluence provides lower detection limits of elements than the region after one, but evaporation efficiency is better in the region after the jet confluence. Therefore, the choice of the analytical region depends on an analytical task and sample nature.

**Figure 3.** Analytical lines Ag I 328.07 (0.05 μg/g), Zn I 213.86 (0.5 μg/g) and Zn II 206.20 (1 μg/g) registered in the region

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(a) before the jet confluence and (b) after the jet confluence [16].

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals http://dx.doi.org/10.5772/intechopen.75939 15

analytical line intensity to the background one (I<sup>l</sup>

on the particle size is given in **Figure 4**.

**Figure 2**.

**Figure 2.** I<sup>l</sup>

/Ib

14 Trace Elements - Human Health and Environment

shown in **Figure 3**.

/Ib

distribution along the plasma flow for Mn I 280.11, Al I 308.22; Cd I 228.80 and Fe I 302.06 lines: region (A)

lines Mn I 280.11, Al I 308.22; Cd I 228.80 and Fe I 302.06 along the plasma flow is shown in

Analytical lines of Ag and Zn registered in the region before and after the jet confluence are

As it is seen, the analytical signals are at the level of background fluctuations in the region after the jet confluence, but they are considerably higher than background in the region before one, which testifies to better LODs of elements in this region. One of the problems arising in the TJP-AES analysis of powdered samples is their incomplete evaporation which can lead to a systematic underestimation of the analysis results. The short residence time of the sample in the plasma is one of the reasons for the partial sample evaporation. In addition, evaporation efficiency depends on chemical composition, structure and particle size of powdered samples [17]. To demonstrate evaporation efficiency in the analytical zones of the TJP, silicon carbide (SiC) powders with an average particle size of 1, 3, 7.5, 17, 22 and 36 μm were used. SiC is a heat-resistant material having a high hardness close to the hardness of diamond. SiC evaporation was controlled by the intensity of weak Si I 212.30 line. To avoid signal self-absorption, the powders were diluted with graphite 100 times. The dependence of Si I 212.30 line intensity

The different behavior of silicon line is observed in the regions investigated. In the region before the jet confluence, there is an increase in the silicon line intensity along with a decrease in the particle size, the smaller particle size and the better evaporation efficiency. However, the complete sample evaporation does not occur even at a particle size of 3 μm. In the region after the jet confluence, the maximum intensity is achieved even at a particle size of 17 μm. The decrease in intensity at smaller particles seems to occur due to the introduction of light particles into the region after the confluence is complicated by a resistance of the consistent plasma jets. Nevertheless, the effect of particle size on evaporation efficiency is considerably weaker in the region after the jet confluence. Thus, the region before the jet confluence provides lower detection limits of elements than the region after one, but evaporation efficiency is better in the region after the jet confluence. Therefore, the choice of the analytical region depends on an analytical task and sample nature.

fluence both for atomic and ionic lines. As an example, the distribution of I<sup>l</sup>

before the jet confluence, (B) after the jet confluence; 0 – point in the region of the confluence.

) realizes in the region before the jet con-

/Ib

for analytical

**Figure 3.** Analytical lines Ag I 328.07 (0.05 μg/g), Zn I 213.86 (0.5 μg/g) and Zn II 206.20 (1 μg/g) registered in the region (a) before the jet confluence and (b) after the jet confluence [16].

(**Figure 1**), and calibration samples similar to the analyzed ones were used. The region before

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The region before the jet confluence turned out to be suitable for analysis of high purity substances both by direct technique and after matrix separation. The TJP-AES techniques for analysis of gallium [20], indium and indium oxide [21] and tellurium dioxide [22] were developed. The direct techniques allow determination of about 30 elements using appropriate dilution of the sample with a spectroscopic buffer (graphite powder containing 15 wt.% NaCl) and unified calibration samples based on graphite powder with addition of 15 wt.% NaCl. As it was shown earlier, NaCl addition increases analytical line intensities and suppresses effects of a mineral matrix [23]. CRMs of graphite powder with different combinations of impurities are commercially available (Ural Federal University, Russia); in addition, preparing the reference sample with given element concentration in graphite is not a difficult task. Analysis of the above substances was carried out at the optimal conditions chosen for multi-elemental analysis of graphite powder (**Table 1**). Calibration curves (lgI-lgC) obtained for Cd and Hg in

A degree of sample dilution depends on the sample nature; a fourfold dilution is needed for analysis of indium oxide and gallium, and a twofold dilution is quite enough for analysis of tellurium dioxide. The preconcentration of impurities in gallium and indium was accomplished by matrix separation in the form of chlorides; tellurium dioxide was previously reduced by hydrogen to metal, and the preconcentration was performed by vacuum distillation of tellurium. The impurity concentrates contained a high concentration of matrix elements since incomplete matrix separation was applied to avoid the loss of a number of important impurities; they were analyzed by the same way as in the direct techniques. LODs of elements were at the level of 0.01–1 and 0.001–0.1 μg/g for direct analysis and after matrix separation, respectively. The possibility of analysis of such a different substances using unified calibration samples points at comparatively weak matrix affects this excitation source. For comparison, using such an approach for a dc arc with sample evaporation from a crater of graphite electrode requires a 100-, 50-, and 25-fold dilution of gallium, indium and tellurium oxides, respectively, which lead to worsening LODs of elements by more than an order of magnitude. Recently, the similar approach was used for analysis of different soils [24]. In spite of their complex and variable matrix composition, TJP-AES allowed direct determination of

the jet confluence was not practically used for analysis.

**3.1. Analysis of high purity substances**

graphite powder are presented in **Figure 5**.

**Parameter Value** Current strength 85 A Voltage 120 V Plasma gas flow 4 l/min Carrier gas flow 0.85 l/min Angle between the jets 60○

**Table 1.** Working conditions of the two-jet plasma.

Observation zone 4–5 mm lower than the point of the confluence

**Figure 4.** Dependence of Si I 212.30 line intensity on the particle size of SiC in the region (A) before the jet confluence (B) after the jet confluence.

#### **2.3. Excitation mechanisms**

To solve the problems appearing in the analysis, it is important to have an idea about the processes occurring in the plasma. The probable mechanisms of an atom and ion excitation in the TJP were investigated [16, 18]. The Boltzmann distribution of excited energy levels for Fe atoms and singly ionized ions was found to take place in both analytical regions, which indicates the predominant excitation of atoms and ions by electron impact. Excitation temperature of Fe atoms and ions, Tatom and Tion, respectively, was measured along the plasma flow. In the optimal observation zone of the region before the jet confluence Tatom = 6000 K and Tion = 7900 K, and in the observation zone of the region after one Tatom = 7060 K and Tion = 8050 K. For atomic and ionic lines, the temperature deviation did not exceed 100 K in the optimal observation zones and was about 250 K near the jet confluence. The considerable difference in Tatom and Tion points at the departure from local thermodynamic equilibrium (LTE) in the plasma. The difference is 1900 K for the region before the jet confluence and 990 K for the region after one, which indicates that the region before the jet confluence is more non-equilibrium than the region after one. The disturbance of LTE in the plasma was shown to be due to metastable argon participation in atom ionization.

#### **3. Application of TJP-AES**

Originally, the TJP was intended for direct analysis of sparingly soluble geological samples [19], which considerably reduced the analysis time and element losses as compared with wet acid digestion. The spectra were registered in the analytical region after the jet confluence (**Figure 1**), and calibration samples similar to the analyzed ones were used. The region before the jet confluence was not practically used for analysis.

#### **3.1. Analysis of high purity substances**

**2.3. Excitation mechanisms**

16 Trace Elements - Human Health and Environment

after the jet confluence.

**3. Application of TJP-AES**

To solve the problems appearing in the analysis, it is important to have an idea about the processes occurring in the plasma. The probable mechanisms of an atom and ion excitation in the TJP were investigated [16, 18]. The Boltzmann distribution of excited energy levels for Fe atoms and singly ionized ions was found to take place in both analytical regions, which indicates the predominant excitation of atoms and ions by electron impact. Excitation temperature of Fe atoms and ions, Tatom and Tion, respectively, was measured along the plasma flow. In the optimal observation zone of the region before the jet confluence Tatom = 6000 K and Tion = 7900 K, and in the observation zone of the region after one Tatom = 7060 K and Tion = 8050 K. For atomic and ionic lines, the temperature deviation did not exceed 100 K in the optimal observation zones and was about 250 K near the jet confluence. The considerable difference in Tatom and Tion points at the departure from local thermodynamic equilibrium (LTE) in the plasma. The difference is 1900 K for the region before the jet confluence and 990 K for the region after one, which indicates that the region before the jet confluence is more non-equilibrium than the region after one. The disturbance of LTE in the plasma was shown to be due to metastable argon participation in atom ionization.

**Figure 4.** Dependence of Si I 212.30 line intensity on the particle size of SiC in the region (A) before the jet confluence (B)

Originally, the TJP was intended for direct analysis of sparingly soluble geological samples [19], which considerably reduced the analysis time and element losses as compared with wet acid digestion. The spectra were registered in the analytical region after the jet confluence The region before the jet confluence turned out to be suitable for analysis of high purity substances both by direct technique and after matrix separation. The TJP-AES techniques for analysis of gallium [20], indium and indium oxide [21] and tellurium dioxide [22] were developed. The direct techniques allow determination of about 30 elements using appropriate dilution of the sample with a spectroscopic buffer (graphite powder containing 15 wt.% NaCl) and unified calibration samples based on graphite powder with addition of 15 wt.% NaCl. As it was shown earlier, NaCl addition increases analytical line intensities and suppresses effects of a mineral matrix [23]. CRMs of graphite powder with different combinations of impurities are commercially available (Ural Federal University, Russia); in addition, preparing the reference sample with given element concentration in graphite is not a difficult task. Analysis of the above substances was carried out at the optimal conditions chosen for multi-elemental analysis of graphite powder (**Table 1**). Calibration curves (lgI-lgC) obtained for Cd and Hg in graphite powder are presented in **Figure 5**.

A degree of sample dilution depends on the sample nature; a fourfold dilution is needed for analysis of indium oxide and gallium, and a twofold dilution is quite enough for analysis of tellurium dioxide. The preconcentration of impurities in gallium and indium was accomplished by matrix separation in the form of chlorides; tellurium dioxide was previously reduced by hydrogen to metal, and the preconcentration was performed by vacuum distillation of tellurium. The impurity concentrates contained a high concentration of matrix elements since incomplete matrix separation was applied to avoid the loss of a number of important impurities; they were analyzed by the same way as in the direct techniques. LODs of elements were at the level of 0.01–1 and 0.001–0.1 μg/g for direct analysis and after matrix separation, respectively. The possibility of analysis of such a different substances using unified calibration samples points at comparatively weak matrix affects this excitation source. For comparison, using such an approach for a dc arc with sample evaporation from a crater of graphite electrode requires a 100-, 50-, and 25-fold dilution of gallium, indium and tellurium oxides, respectively, which lead to worsening LODs of elements by more than an order of magnitude. Recently, the similar approach was used for analysis of different soils [24]. In spite of their complex and variable matrix composition, TJP-AES allowed direct determination of


**Table 1.** Working conditions of the two-jet plasma.

**Figure 5.** Calibration curves for analytical lines Cd I 228.80 and Hg I 253.65 (C μg/g) [24].

As, B, Cd, Cu, Hg and P after a twofold and Be, Co, Cr, Ga, Nb, Pb and Zn after a 10-fold dilution with a spectroscopic buffer.

#### **3.2. Analysis of biological samples**

Solid sampling, with little or no chemical pretreatment, in the analysis of biological samples seems very attractive. The possibility of TJP-AES for direct analysis of biological samples using the same unified approach as for inorganic materials was investigated. First, starch was used to study the organic matrix influence on analytical signals of elements in the TJP [25]. It was found that the presence of 10 wt.% starch in graphite powder with introduced impurities did not affect analytical line intensities of elements while the decrease in intensities by a factor 2–5 was observed in a graphite dc arc. In the TJP, the effect was not observed even in the presence of 20 wt.% starch for many atomic lines of elements. The considerable decrease in intensities in a dc arc is due to a vigorous reaction of starch with air oxygen in an arc discharge with the release of gaseous products, which results in decreasing the residence time of the sample in the plasma. Although the TJP is an open system too, the oxidative reaction occurs less violently than in a dc arc since carrier argon partially displaces air from the excitation zone, and the sample is gradually introduced into the plasma. In addition, gaseous products seem to be retained in the excitation zone by argon flows. The experiments with starch gave hope to get positive results for more complex organic matrices.

#### *3.2.1. Analysis of animal organs*

The unified approach mentioned above was tested for animal organs, dried and finely powdered. Dry animal organs contain more than 50 wt.% proteins as well as fats, carbohydrates and others. The effect of such a complex matrix on the analytical signal of elements was studied by the analysis of a spiked sample based on graphite with the addition of 15 wt.% NaCl and 10 wt.% rat liver; concentration of elements introduced was 2.5 μg/g [26]. The analysis was carried out using calibration samples based on graphite powder with addition 15 wt.% NaCl; the spectra were observed in the region before the jet confluence. The analysis results are given in **Table 2**; the satisfactory recoveries were obtained for all investigated elements. Since the liver contained Co, Mn, Mo and Zn, the blank sample was prepared to estimate correctly the recovery of these elements. On the basis of the results obtained, a 10-fold dilution of powdered animal organs with buffer and calibration based on graphite powder were suggested for direct analysis of animal organs. To validate the technique, the results of direct analysis of bovine liver were compared with the results obtained after sample carbonization (500°C, 5 min) and autoclave digestion in a mixture of nitric acid and hydrogen peroxide [25]. The carbonized sample was analyzed at the conditions chosen for direct analysis. The solution obtained after acid digestion evaporated on graphite powder, diluted with buffer and analyzed by the same way. As it is seen from **Table 3**, the results of Al, Ca, Cu, Fe, Mg, Mn, Mo, P, Si and Zn satisfactory agree with each other. Only for Fe, Mn and Mo, the results obtained after autoclave digestion are lower than the results of the direct technique, which is likely to be due to their partial loss. The LODs of elements provided by the direct technique are at the level 0.1–10 μg/g, and they are lower by approximately one order of magnitude after carbonization. The use of the carbonization procedure allowed determining the low concentrations of Ag, Cd, Co, Cr, Pb and Ni in the liver.

**Element λ (nm) Concentrations of elements Recovery (%) Added (μg/g) Found (μg/g)<sup>a</sup>**

> 0.26 ± 0.8b 2.8 ± 0.6

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

0.80 ± 0.15b 3.5 ± 0.6

0.27 ± 0.10b 2.5 ± 0.3

6.8 ± 1.7b 9.3 ± 2.0

– 102

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19

– 108

– 89

100

Ag 338.29 2.5 2.4 ± 0.6 96 Ba 233.53 2.5 2.5 ± 1.0 100 Be 265.06 2.5 2.3 ± 0.8 92 Bi 306.77 2.5 2.6 ± 0.8 104 Cd 228.80 2.5 2.5 ± 0.3 100

Cr 283.56 2.5 2.8 ± 1.4 112 Ga 294.36 2.5 2.4 ± 0.4 96 Hg 253.65 2.5 2.6 ± 0.6 104

Ni 305.08 2.5 2.3 ± 0.7 92 Pb 283.31 2.5 2.5 ± 0.4 100 Sb 231.15 2.5 2.5 ± 0.7 100 Sn 284.00 2.5 2.3 ± 0.5 92

**Table 2.** Determination of elements in a spiked graphite powder containing 10% rat liver [26].

2.5

2.5

2.5

2.5

Co 345.35 –

Mn 280.11 –

Mo 317.03 –

Zn 213.85 –

Mean ± (95% confidence interval), n = 4.

Value obtained without spike addition.

a

b

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals http://dx.doi.org/10.5772/intechopen.75939 19


b Value obtained without spike addition.

**Figure 5.** Calibration curves for analytical lines Cd I 228.80 and Hg I 253.65 (C μg/g) [24].

hope to get positive results for more complex organic matrices.

tion with a spectroscopic buffer.

18 Trace Elements - Human Health and Environment

*3.2.1. Analysis of animal organs*

**3.2. Analysis of biological samples**

As, B, Cd, Cu, Hg and P after a twofold and Be, Co, Cr, Ga, Nb, Pb and Zn after a 10-fold dilu-

Solid sampling, with little or no chemical pretreatment, in the analysis of biological samples seems very attractive. The possibility of TJP-AES for direct analysis of biological samples using the same unified approach as for inorganic materials was investigated. First, starch was used to study the organic matrix influence on analytical signals of elements in the TJP [25]. It was found that the presence of 10 wt.% starch in graphite powder with introduced impurities did not affect analytical line intensities of elements while the decrease in intensities by a factor 2–5 was observed in a graphite dc arc. In the TJP, the effect was not observed even in the presence of 20 wt.% starch for many atomic lines of elements. The considerable decrease in intensities in a dc arc is due to a vigorous reaction of starch with air oxygen in an arc discharge with the release of gaseous products, which results in decreasing the residence time of the sample in the plasma. Although the TJP is an open system too, the oxidative reaction occurs less violently than in a dc arc since carrier argon partially displaces air from the excitation zone, and the sample is gradually introduced into the plasma. In addition, gaseous products seem to be retained in the excitation zone by argon flows. The experiments with starch gave

The unified approach mentioned above was tested for animal organs, dried and finely powdered. Dry animal organs contain more than 50 wt.% proteins as well as fats, carbohydrates and others. The effect of such a complex matrix on the analytical signal of elements was studied by the analysis of a spiked sample based on graphite with the addition of 15 wt.% NaCl and 10 wt.% rat liver; concentration of elements introduced was 2.5 μg/g [26]. The analysis was carried out using calibration samples based on graphite powder with addition 15 wt.% NaCl; the spectra were observed in the region before the jet confluence. The analysis results are given in **Table 2**; the satisfactory recoveries were obtained for all investigated elements. Since the liver contained Co, Mn, Mo and Zn, the blank sample was prepared to estimate correctly the recovery of these elements. On the basis of the results obtained, a 10-fold dilution **Table 2.** Determination of elements in a spiked graphite powder containing 10% rat liver [26].

of powdered animal organs with buffer and calibration based on graphite powder were suggested for direct analysis of animal organs. To validate the technique, the results of direct analysis of bovine liver were compared with the results obtained after sample carbonization (500°C, 5 min) and autoclave digestion in a mixture of nitric acid and hydrogen peroxide [25]. The carbonized sample was analyzed at the conditions chosen for direct analysis. The solution obtained after acid digestion evaporated on graphite powder, diluted with buffer and analyzed by the same way. As it is seen from **Table 3**, the results of Al, Ca, Cu, Fe, Mg, Mn, Mo, P, Si and Zn satisfactory agree with each other. Only for Fe, Mn and Mo, the results obtained after autoclave digestion are lower than the results of the direct technique, which is likely to be due to their partial loss. The LODs of elements provided by the direct technique are at the level 0.1–10 μg/g, and they are lower by approximately one order of magnitude after carbonization. The use of the carbonization procedure allowed determining the low concentrations of Ag, Cd, Co, Cr, Pb and Ni in the liver.


of investigation for living organisms. Whole blood quickly changes over time due to fast clotting, which troubles the sample cutting. The effect of anticoagulants also lasts a limited time. Therefore, for continuous biomedical experiments, freeze-dried whole blood which can be kept for a long time at normal conditions is very convenient. For determining the main essential elements (Fe, P, Ca, Mg, Zn and Cu) in freeze-dried whole blood, the direct technique developed for animal organs was applied [27]. To confirm the possibility of such an approach for blood analysis, the direct analysis of CRM of freeze-dried bovine blood (IAEA A-13) was carried out, and good agreement of the results with the certified values was obtained. In addition, the results of analysis of human and rat freeze-dried whole blood obtained using the different sample preparation procedures were compared (**Table 4**). The direct technique results satisfactory agreed with the results obtained after carbonization (400°C, 15 min) and autoclave digestion, which confirms the possibility of the unified approach for blood analysis. Simple sample preparation (dilution with buffer) and the possibility of analysis of small amount samples, 5–10 mg of blood powder (approximately 20–50 μL liquid blood), are of practical importance. For analysis of whole liquid blood, blood aliquots evaporated on graphite powder under an IR lamp and then carbonized at 400°C for 15 min. The remainder was ground in a mortar and analyzed as in the direct analysis. The techniques suggested were

**digestion**

Fe 296.67 2300 ± 200<sup>a</sup> 2200 ± 160 2200 ± 200 2600 ± 200 2400 ± 170 2500 ± 200 P 214.91 1700 ± 130 1600 ± 120 1500 ± 110 1400 ± 200 1400 ± 100 1300 ± 150 Ca 317.93 320 ± 20 340 ± 30 300 ± 20 240 ± 30 250 ± 20 280 ± 30 Mg 277.98 130 ± 15 150 ± 20 130 ± 10 120 ± 15 140 ± 15 150 ± 20 Zn 213.86 28 ± 1.6 26 ± 1.4 29 ± 1.7 29 ± 2.0 32 ± 1.5 32 ± 1.4 Cu 324.75 6.6 ± 0.5 6.2 ± 0.4 5.8 ± 0.4 3.6 ± 0.3 3.5 ± 0.3 3.3 ± 0.2

**Direct analysis**

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

**Carbonizing Autoclave** 

http://dx.doi.org/10.5772/intechopen.75939

**digestion**

21

**Element λ (nm) Human blood Rat blood Direct analysis Carbonizing Autoclave** 

**Table 4.** Concentration of elements in the freeze-dried blood samples (μg/g) [27].

validated for analysis of both freeze-dried and liquid blood serum and plasma.

respectively, and 10 wt.% water. Collagen and calcium hydroxyapatite Ca10(PO4)<sup>6</sup>

Bone is a highly mineralized mobile tissue which accumulates inorganic substances and diffuses them as the need arises. It contains 25 and 65 wt.% organic and inorganic substances,

main components of bone. Some elements are predominantly in the organic phase, and others are in the mineral phase of bone. It was found that a fourfold dilution of dry powdered bone was quite enough for element determination by the unified direct technique [28]. The results of "added-found" experiment and comparison of the direct technique results with the results of ICP-AES after wet acid digestion of the sample validated the technique. However, underestimating the Ba, Mg, and Sr concentration was obtained. These elements are strongly

(OH)<sup>2</sup>

are the

*3.2.3. Analysis of bone*

a

95% confidence interval, n = 4.

**Table 3.** Results of the TJP-AES analysis of bovine liver (μg/g) [25].

The technique suggested is very suitable for analysis of dry internals such as liver, kidney and spleen which are easily ground in a Plexiglas mortar to a powder with the particle size of 20–30 μm. However, the direct analysis of bovine and pork muscle CRMs and rat brain was found to provide the understated results [26]. These tissues are more thermostable than liver and have the particles of more than 100 μm. Flexible fibers of muscles and plastic consistence of brain make it difficult to obtain a powder with small particles. Incomplete evaporation of the samples is the most probable reason for the result underestimations. This problem was overcome by decreasing the consumption of a carrier gas, which increases the residence time of the sample in the excitation zone and concentration of air oxygen participating in organic matrix decomposition. It should be noted that the carbonization conditions are not all-purpose and depend on a kind of tissues. For brain and muscle tissues, the time of carbonization was increased up to 30 min. Thus, in spite of the unified approach to the analysis of organs, peculiarities of different tissues should be taken into account. For direct analysis, 5–10 mg of powdered sample is quite enough; for carbonization procedure, 50–100 mg of the sample is needed. Note that the ICP-AES and ICP-MS techniques with wet acid digestion of organs usually require 100–250 mg of the sample which is not always available. The relative standard deviation of the analysis results of animal organs usually is in the range of 3–12%.

#### *3.2.2. Analysis of whole blood*

The problem of availability of biological samples in ample quantity is particularly acute in experiments with living experimental animals (such as mice or rats). Blood is the main subject Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals http://dx.doi.org/10.5772/intechopen.75939 21


**Table 4.** Concentration of elements in the freeze-dried blood samples (μg/g) [27].

of investigation for living organisms. Whole blood quickly changes over time due to fast clotting, which troubles the sample cutting. The effect of anticoagulants also lasts a limited time. Therefore, for continuous biomedical experiments, freeze-dried whole blood which can be kept for a long time at normal conditions is very convenient. For determining the main essential elements (Fe, P, Ca, Mg, Zn and Cu) in freeze-dried whole blood, the direct technique developed for animal organs was applied [27]. To confirm the possibility of such an approach for blood analysis, the direct analysis of CRM of freeze-dried bovine blood (IAEA A-13) was carried out, and good agreement of the results with the certified values was obtained. In addition, the results of analysis of human and rat freeze-dried whole blood obtained using the different sample preparation procedures were compared (**Table 4**). The direct technique results satisfactory agreed with the results obtained after carbonization (400°C, 15 min) and autoclave digestion, which confirms the possibility of the unified approach for blood analysis. Simple sample preparation (dilution with buffer) and the possibility of analysis of small amount samples, 5–10 mg of blood powder (approximately 20–50 μL liquid blood), are of practical importance. For analysis of whole liquid blood, blood aliquots evaporated on graphite powder under an IR lamp and then carbonized at 400°C for 15 min. The remainder was ground in a mortar and analyzed as in the direct analysis. The techniques suggested were validated for analysis of both freeze-dried and liquid blood serum and plasma.

#### *3.2.3. Analysis of bone*

B 249.77 6.2 ± 2.0 2.7 ± 0.5 5.5 ± 1.0 Ca 317.93 130 ± 20 110 ± 30 130 ± 40 Cd 228.80 <0.4 0.17 ± 0.03 0.25 ± 0.10

Co 345.35 <1 0.31 ± 0.05 — Cu 324.75 12 ± 2 12 ± 4 10 ± 0.2 Cr 283.56 <1 0.2 ± 0.04 0.24 ± 0.06 Fe 302.06 250 ± 40 210 ± 70 150 ± 30 Mg 277.98 470 ± 80 460 ± 120 420 ± 90 Mn 279.83 16 ± 2 13 ± 3 9.5 ± 2.4 Mo 317.03 18 ± 3 20 ± 5 9.0 ± 2.3 Ni 305.08 <1 0.6 ± 0.1 0.77 ± 0.25 P, wt.% 214.91 0.99 ± 0.08 0.85 ± 0.25 0.76 ± 0.11 Pb 283.31 <1 0.40 ± 0.07 0.40 ±0.11 Si 288.16 22 ± 8 31 ± 14 22 ± 11 Zn 213.86 62 ± 15 55 ± 10 60 ± 8

The technique suggested is very suitable for analysis of dry internals such as liver, kidney and spleen which are easily ground in a Plexiglas mortar to a powder with the particle size of 20–30 μm. However, the direct analysis of bovine and pork muscle CRMs and rat brain was found to provide the understated results [26]. These tissues are more thermostable than liver and have the particles of more than 100 μm. Flexible fibers of muscles and plastic consistence of brain make it difficult to obtain a powder with small particles. Incomplete evaporation of the samples is the most probable reason for the result underestimations. This problem was overcome by decreasing the consumption of a carrier gas, which increases the residence time of the sample in the excitation zone and concentration of air oxygen participating in organic matrix decomposition. It should be noted that the carbonization conditions are not all-purpose and depend on a kind of tissues. For brain and muscle tissues, the time of carbonization was increased up to 30 min. Thus, in spite of the unified approach to the analysis of organs, peculiarities of different tissues should be taken into account. For direct analysis, 5–10 mg of powdered sample is quite enough; for carbonization procedure, 50–100 mg of the sample is needed. Note that the ICP-AES and ICP-MS techniques with wet acid digestion of organs usually require 100–250 mg of the sample which is not always available. The relative standard deviation of the analysis

The problem of availability of biological samples in ample quantity is particularly acute in experiments with living experimental animals (such as mice or rats). Blood is the main subject

a

95% confidence interval.

20 Trace Elements - Human Health and Environment

**Table 3.** Results of the TJP-AES analysis of bovine liver (μg/g) [25].

results of animal organs usually is in the range of 3–12%.

*3.2.2. Analysis of whole blood*

Bone is a highly mineralized mobile tissue which accumulates inorganic substances and diffuses them as the need arises. It contains 25 and 65 wt.% organic and inorganic substances, respectively, and 10 wt.% water. Collagen and calcium hydroxyapatite Ca10(PO4)<sup>6</sup> (OH)<sup>2</sup> are the main components of bone. Some elements are predominantly in the organic phase, and others are in the mineral phase of bone. It was found that a fourfold dilution of dry powdered bone was quite enough for element determination by the unified direct technique [28]. The results of "added-found" experiment and comparison of the direct technique results with the results of ICP-AES after wet acid digestion of the sample validated the technique. However, underestimating the Ba, Mg, and Sr concentration was obtained. These elements are strongly


chloride (CoCl<sup>2</sup>

CoCl<sup>2</sup>

u ≥ Al ≥ B. The 60 days treatment of mice with CoCl<sup>2</sup>

**Chemical element Control untreated mice (n = 19) Mice treated with CoCl2**

Protein 9.0–16.6 13.7 ± 1.9 2.0–7.7 6.2 ± 1.9

[29].

**Average value (μg/g) (value 1)**

gain, immunity, reproduction, etc. [29].

**Range of values (μg/g)**

Total protein, mg protein/ml of plasma

mice and animals treated with CoCl<sup>2</sup>

\*For each mouse, the mean of three repeats is used.

) on relative content of different metal ions in mouse plasma using TJP-AES

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

(daily dose 125 mg/kg) did not change

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23

**Ratio of (2) and (1)\***

**Average value (μg/g)** 

**(value 2)**

**Significance (***P***)**

and on the total protein content [29, 30]. Freeze-dried plasma (2–3 mg) was available for the TJP-AES analysis. On average the relative content of different elements in the plasma of 2-month-old mice balb/c (control group) decreased in the order: Ca > Mg > Si > Fe > Zn > C

appreciably the relative content (ReCo) Ca, Cu and Zn, while a 2.3-fold significant decrease in the ReCo of B and a significant increase in the content of Si (3.4-fold), Fe and Al (2.1-fold) and Mg (1.5-fold) was found (**Table 6**). The ReCo of Mo and Co for untreated mice was lower than test sensitivity. Mo in a detectable amount was determined only for two mice in the control group, but the plasmas of 9 out of 16 mice of analyzed group contained this metal. Cobalt treatment resulted in a 2.2-fold decrease in the concentration of total plasma protein and in 1.7-fold immunoglobulins. Clarification of the complex effects of Co2+ on its interactions *in vivo* with other trace elements is important for the explanation of cobalt toxicity and disturbances in homeostasis and physiological processes such as development, growth, weight

Homogeneous IgGs purified from sera of mice treated (t-IgGs) and non-treated (nt-IgGs) with

**(n = 16)**

**Table 6.** Relative content of different chemical elements and total protein in the freeze-dried blood plasma of control

Ca 540–1670 1099 ± 239 510–1670 1169 ± 288 1.17 0.53 Cu 6.0–20.5 10.1 ± 2.6 5.2–26.5 9.1 ± 2.7 0.99 0.46 Zn 14–53 24.2 ± 6.8 15–49 25.9 ± 5.9 1.18 0.56 B 1.2–6.7 5.8 ± 2.5 1.1–5.6 2.5 ± 0.79 0.47 3 x 10−4 Mg 190–590 423 ± 102 610–850 625 ± 75 1.76 1.2 х 10−7 Al 4–13 6.5 ± 2.9 8–25 13.8 ± 3.4 2.53 2.5 х 10−7 Fe 40–130 69 ± 17.0 72–270 147 ± 50 2.53 2.0 х 10−7 Si 50–190 101 ± 38 200–520 342 ± 92 4.07 1.0 х 10−9 Mo ~0 ~0 0–9 2.3 ± 2.2 — Co ~0 ~0 7.2–37 16.8 ± 6.5 —

**Range of values (μg/g)**

 containing intrinsically bound metal ions hydrolyze DNA with very low activity and lose this activity in the presence of EDTA [30]. The average relative DNase activity (RAs) of nt-IgGs increased after addition of external metal ions in the following order: Zn2+ < Ca2+ < Cu

**Table 5.** Limits of detection (LOD) of elements in bone [28].

bound with calcium hydroxyapatite, and its incomplete evaporation may lead to their understated concentrations. This effect took place even at a mean particle size of 30 μm. The strongest decrease in concentration was observed for Sr replacing Ca in hydroxyapatite. It is well known that strontium rachitis develops in the regions with a high content of radioactive Sr due to the formation of high concentration of strontium hydroxyapatite in bone, which results in the fragility of people and animal teeth and bones. Pretreatment of the samples with nitric acid followed by heating at 300°C or decrease of the consumption of carrier argon as in the case of brain and muscle tissues allowed getting valid results for Ba, Mg, and Sr. The satisfactory results obtained for other elements by direct technique point at their fractional volatilization from the particles in the plasma. These elements seem to be bound with the organic portion of bone or weakly bound with calcium hydroxyapatite. LODs of a number of elements in bone are given in **Table 5**.

Thus, on the example of different animal organs, whole blood and bone the possibility of TJP-AES for realizing the simple analytical techniques was shown. Solid sampling, unified calibration samples, the possibility of analysis of small amount samples are of great interest for experiments with different biological tissues.

## **4. Analysis of trace element changes in mice treated with CoCl2**

Transition-metal cobalt is an essential trace element required for vitamin B12 biosynthesis, enzyme activation, etc., but is toxic in high concentrations. We estimated the effect of cobalt chloride (CoCl<sup>2</sup> ) on relative content of different metal ions in mouse plasma using TJP-AES and on the total protein content [29, 30]. Freeze-dried plasma (2–3 mg) was available for the TJP-AES analysis. On average the relative content of different elements in the plasma of 2-month-old mice balb/c (control group) decreased in the order: Ca > Mg > Si > Fe > Zn > C u ≥ Al ≥ B. The 60 days treatment of mice with CoCl<sup>2</sup> (daily dose 125 mg/kg) did not change appreciably the relative content (ReCo) Ca, Cu and Zn, while a 2.3-fold significant decrease in the ReCo of B and a significant increase in the content of Si (3.4-fold), Fe and Al (2.1-fold) and Mg (1.5-fold) was found (**Table 6**). The ReCo of Mo and Co for untreated mice was lower than test sensitivity. Mo in a detectable amount was determined only for two mice in the control group, but the plasmas of 9 out of 16 mice of analyzed group contained this metal. Cobalt treatment resulted in a 2.2-fold decrease in the concentration of total plasma protein and in 1.7-fold immunoglobulins. Clarification of the complex effects of Co2+ on its interactions *in vivo* with other trace elements is important for the explanation of cobalt toxicity and disturbances in homeostasis and physiological processes such as development, growth, weight gain, immunity, reproduction, etc. [29].

**Element λ (nm) LOD (μg/g)**

Ag 328.07 0.1 Bi 306.77 1.2 Cd 228.80 0.5 Co 345.35 1.2 Cr 283.56 0.3 Cu 324.75 0.2 Fe 296.68 2.0 Ga 294.36 0.3 Mn 260.57 0.5 Mo 313.26 0.7 Ni 305.08 0.6 Pb 283.31 1.1 Sn 284.00 0.8 Zn 213.86 1.0

22 Trace Elements - Human Health and Environment

**Table 5.** Limits of detection (LOD) of elements in bone [28].

for experiments with different biological tissues.

bound with calcium hydroxyapatite, and its incomplete evaporation may lead to their understated concentrations. This effect took place even at a mean particle size of 30 μm. The strongest decrease in concentration was observed for Sr replacing Ca in hydroxyapatite. It is well known that strontium rachitis develops in the regions with a high content of radioactive Sr due to the formation of high concentration of strontium hydroxyapatite in bone, which results in the fragility of people and animal teeth and bones. Pretreatment of the samples with nitric acid followed by heating at 300°C or decrease of the consumption of carrier argon as in the case of brain and muscle tissues allowed getting valid results for Ba, Mg, and Sr. The satisfactory results obtained for other elements by direct technique point at their fractional volatilization from the particles in the plasma. These elements seem to be bound with the organic portion of bone or weakly bound with calcium hydroxyapatite. LODs of a number of elements in bone are given in **Table 5**.

Thus, on the example of different animal organs, whole blood and bone the possibility of TJP-AES for realizing the simple analytical techniques was shown. Solid sampling, unified calibration samples, the possibility of analysis of small amount samples are of great interest

Transition-metal cobalt is an essential trace element required for vitamin B12 biosynthesis, enzyme activation, etc., but is toxic in high concentrations. We estimated the effect of cobalt

**4. Analysis of trace element changes in mice treated with CoCl2**

Homogeneous IgGs purified from sera of mice treated (t-IgGs) and non-treated (nt-IgGs) with CoCl<sup>2</sup> containing intrinsically bound metal ions hydrolyze DNA with very low activity and lose this activity in the presence of EDTA [30]. The average relative DNase activity (RAs) of nt-IgGs increased after addition of external metal ions in the following order: Zn2+ < Ca2+ < Cu


**Table 6.** Relative content of different chemical elements and total protein in the freeze-dried blood plasma of control mice and animals treated with CoCl<sup>2</sup> [29].

2+ < Fe2+ < Mn2+ < Mg2+ < Co2+ < Ni2+. Interestingly, t-IgGs showed lower activity than nt-IgGs in the absence of external metal ions (2.7-fold) as well as in the presence of Cu2+ (9.5-fold), Co2+ (5.6-fold), Zn2+ (5.1-fold), Mg2+ (4.1-fold), Ca2+ (3.0-fold) and Fe2+ (1.3-fold). But t-IgGs were more active than nt-IgGs in the presence of Ni2+ (1.4-fold) and especially Mn2+ (2.2-fold), which are the best activators of t-IgGs. These data may be useful for an understanding of Co2+ toxicity, its effect on a change of metal-dependent specificity of mouse abzymes [30].

## **5. Abzymes with oxidation-reduction activities**

First, we have estimated the content of metals in the lyophilized plasmas of healthy Wistar rats (**Table 7**) by the TJP-AES method [31]. The relative amount of metals in the rat plasma decreased in the order: Ca > Mg > Fe > Cu ≥ Zn > Al ≥ Sr. > Ti ≥ Mo ≥ Mn (**Table 7**).


Nine plasmas of healthy Wistar rat's sera were used for purification of electrophoretically and immunologically homogeneous IgGs according to [31–35]. Homogeneous IgGs according to data of the TJP-AES method did not contain a detectable amount of Sr and Mo (**Table 8**) [31]. The relative amount of different metals bound to IgGs of SLE and MS patients in average decreased in the following order: Ca ≥ Zn ≥ Ti ≥ Mg ≥ Al ≥ Fe ≥ Cu ≥ Ni > Mn (**Table 8**). Thus, IgGs of individual rats can interact with metal ions showing a significant difference

**Table 8.** The relative content of metal ions in the lyophilized sle-IgGmix and MS-IgGmix samples from the sera of patients

Preparations sle-IgGmix and ms-IgGmix are mixtures of equal amounts of electrophoretically homogeneous IgGs from the

\*\*The content was determined by TJP-AES method; the errors of the values from two experiments were within 5–7%.

**sle-IgGmix ms-IgGmix**

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

http://dx.doi.org/10.5772/intechopen.75939

25

We have shown that rat IgGs lose most bound metal ions during the purification [31, 33]. From 26 to 39% of rat IgGs can interact with less or more efficiently with different metals ions. Interestingly, chromatography of IgGs from human plasmas on Chelex non-charged with metal ions led to the binding of a small amount of IgGs (~ 5%) bound with metal ions [36]. Chelex charged with Cu2+ ions additionally adsorbed ~ 38% of the total IgGs. In a number of publications, it was shown that all many catalytic activities are the intrinsic properties of mammalian antibodies and are not caused by impurities of any canonical enzymes [37–46]. For this purpose, in all cases, we have checked several previously developed strict criteria proving that all activities of Abs from blood sera and healthy donors

All higher organisms generate energy due to aerobic respiration, the process including a fourelectron stepwise reduction to water of molecular oxygen [47–51]. The partially reduced spe-

other components of different cells. Oxidative damage of cells components was regarded as

•−, are typical oxidants attacking proteins, lipids, DNA and

(**Table 8**) in spite of their comparable concentrations in the plasmas (**Table 7**).

and autoimmune patients belong to the Abs [37–46].

and O<sup>2</sup>

the significant factor of carcinogenesis and aging [47, 49, 51].

O2

**Metal Relative content (μg/g)**

sera of 12 SLE (sle-IgGmix) and 12 MS (ms-IgGmix) patients.

.

with SLE and MS, respectively\*

Al 7.0 8.0 Ca 10 120 Cu 8.0 4.0 Fe 4.0 9.0 Mg 4.0 17.0 Mn 0.2 ~0 Ni 6.5 0.7 Ti 2.0 27.0 Zn 37.0 11.0

cies include OH•, H<sup>2</sup>

\*

\* The content was determined by TJP-AES method; the relative standard deviation of the results from two experiments were within 5–7%.

\*\*The maximal and minimal values for each metal are marked in bold.

ῼSign ≤ in all cases means that the presence of metal in the samples is reliable, but its exact concentration cannot be determined; it can be in the range 0.1–1 μg/g.

**Table 7.** The relative content of different trace elements and metals in the lyophilized blood plasmas from nine rats.

<sup>\*\*\*</sup>Mean ± S.D.


2+ < Fe2+ < Mn2+ < Mg2+ < Co2+ < Ni2+. Interestingly, t-IgGs showed lower activity than nt-IgGs in the absence of external metal ions (2.7-fold) as well as in the presence of Cu2+ (9.5-fold), Co2+ (5.6-fold), Zn2+ (5.1-fold), Mg2+ (4.1-fold), Ca2+ (3.0-fold) and Fe2+ (1.3-fold). But t-IgGs were more active than nt-IgGs in the presence of Ni2+ (1.4-fold) and especially Mn2+ (2.2-fold), which are the best activators of t-IgGs. These data may be useful for an understanding of Co2+ toxic-

First, we have estimated the content of metals in the lyophilized plasmas of healthy Wistar rats (**Table 7**) by the TJP-AES method [31]. The relative amount of metals in the rat plasma decreased in the order: Ca > Mg > Fe > Cu ≥ Zn > Al ≥ Sr. > Ti ≥ Mo ≥ Mn (**Table 7**).

**Metal Relative content (μg/g)\* Average value (μg/g)**

The content was determined by TJP-AES method; the relative standard deviation of the results from two experiments

ῼSign ≤ in all cases means that the presence of metal in the samples is reliable, but its exact concentration cannot be

**Table 7.** The relative content of different trace elements and metals in the lyophilized blood plasmas from nine rats.

Ca 1700 1700 **1200** 1500 1700 1300 1200 1700 **2000** 1556 ± 274\*\*\* Mg 440 420 280 380 360 310 300 360 410 362 ± 56 Fe **14\*\*** 120 90 110 80 110 **80** 100 110 104 ± 19 Cu 29 25 **22** 24 34 22 25 **39** 33 28.1 ± 6.0 Zn 26 44 **22** 25 **28** 25 26 25 26 27.4 ± 6.4 Al 6.0 **15** 7.0 13 6.0 5.0 5.0 **4.0** 7.0 7.6 ± 3.8 Sr 5.5 6.2 5.3 6.2 8.4 **5.0** 6.2 6.5 **8.4** 6.4 ± 1.2 Ti **5.0** 3.0 3.0 3.0 3.0 4.0 **3.0** 4.0 4.0 3.6 ± 0.7 Mo 2.3 1.5 **1.1** 1.8 3.3 2.0 2.0 **3.4** 2.7 2.2 ± 0.8 Mn 1.2 **1.6** 1.1 **1.1** 1.3 1.4 1.2 1.4 1.3 1.3 ± 0.2 Pb <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 Cr 1.0 ≤1.0ῼ ≤1.0 ≤1.0 ≤1.0 **≤1.0** ≤1.0 ≤2.0 **≤6.0** ≤1.7 ± 1.6 Ni ≤1.0- ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 Co <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <0.1 Ag <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

**1 2 3 4 5 6 7 8 9**

ity, its effect on a change of metal-dependent specificity of mouse abzymes [30].

**5. Abzymes with oxidation-reduction activities**

Rat number

24 Trace Elements - Human Health and Environment

\*

were within 5–7%.

\*\*\*Mean ± S.D.

\*\*The maximal and minimal values for each metal are marked in bold.

determined; it can be in the range 0.1–1 μg/g.

\* Preparations sle-IgGmix and ms-IgGmix are mixtures of equal amounts of electrophoretically homogeneous IgGs from the sera of 12 SLE (sle-IgGmix) and 12 MS (ms-IgGmix) patients.

\*\*The content was determined by TJP-AES method; the errors of the values from two experiments were within 5–7%.

**Table 8.** The relative content of metal ions in the lyophilized sle-IgGmix and MS-IgGmix samples from the sera of patients with SLE and MS, respectively\* .

Nine plasmas of healthy Wistar rat's sera were used for purification of electrophoretically and immunologically homogeneous IgGs according to [31–35]. Homogeneous IgGs according to data of the TJP-AES method did not contain a detectable amount of Sr and Mo (**Table 8**) [31]. The relative amount of different metals bound to IgGs of SLE and MS patients in average decreased in the following order: Ca ≥ Zn ≥ Ti ≥ Mg ≥ Al ≥ Fe ≥ Cu ≥ Ni > Mn (**Table 8**). Thus, IgGs of individual rats can interact with metal ions showing a significant difference (**Table 8**) in spite of their comparable concentrations in the plasmas (**Table 7**).

We have shown that rat IgGs lose most bound metal ions during the purification [31, 33]. From 26 to 39% of rat IgGs can interact with less or more efficiently with different metals ions. Interestingly, chromatography of IgGs from human plasmas on Chelex non-charged with metal ions led to the binding of a small amount of IgGs (~ 5%) bound with metal ions [36]. Chelex charged with Cu2+ ions additionally adsorbed ~ 38% of the total IgGs. In a number of publications, it was shown that all many catalytic activities are the intrinsic properties of mammalian antibodies and are not caused by impurities of any canonical enzymes [37–46]. For this purpose, in all cases, we have checked several previously developed strict criteria proving that all activities of Abs from blood sera and healthy donors and autoimmune patients belong to the Abs [37–46].

All higher organisms generate energy due to aerobic respiration, the process including a fourelectron stepwise reduction to water of molecular oxygen [47–51]. The partially reduced species include OH•, H<sup>2</sup> O2 and O<sup>2</sup> •−, are typical oxidants attacking proteins, lipids, DNA and other components of different cells. Oxidative damage of cells components was regarded as the significant factor of carcinogenesis and aging [47, 49, 51].

Antioxidant enzymes (catalases, superoxide dismutases and glutathione peroxidases) are very important for preventing oxidative stress [52–56]. However, these enzymes are located inside of cells, and they undergo rapid inactivation in the blood [54]. Immunoglobulins (Igs) are significantly more stable molecules of blood. Therefore, it was interesting how metal ions can activate oxidation-reduction reactions catalyzed by antibodies. The catalysis of such reactions by the majority of canonical enzymes is dependent on metal ions with variable valence [50, 52–55]. First, we have shown that IgGs of healthy Wistar rats oxidize 3,3'-diaminobenzidine through a peroxidase activity in the presence of H<sup>2</sup> O2 and due to an oxidoreductase activity in the absence of H<sup>2</sup> O2 [31–35]. In the external metal ions absence, the specific peroxidase activity of IgGs of rats varied in the range 1.6–26% comparing with horseradish peroxidase (HRP, taken for 100%). The dialysis of IgGs against EDTA completely lost these activities. External metal ions activated significantly both activities of non-dialyzed (ND) and dialyzed (D) IgGs. The relative activities (RAs) in the presence of external Fe2+ or Cu2+ ions were increased up to 13–198% compared with that for HRP [31]. Cu2+ ions alone stimulated significantly both the oxidoreductase and peroxidase activities of dialyzed D-IgGs, but only at high concentration (≥2 mM) [31]. Mn2+ ions were weakly activated peroxidase activity but at >3 mM Mn2+ was good cofactor of the oxidoreductase activity at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of D-IgGs was revealed at 0.1–5 mM, but Fe2+ cannot activate their oxidoreductase activity. Al3+, Mg2+, Zn2+, Ca2+, and especially Ni2+ and Co2+ were not able to activate D-IgGs, but slightly activated ND-IgGs containing different intrinsic metal ions. Some metal ions activated IgGs especially ND-Abs in accordance with biphasic curves, which were specific for every individual Ab preparation [31]. The combinations of Fe2+ + Zn2+, Fe2+ + Mn2+, Cu2+ + Mn2+ and Cu2+ + Zn2+ and other metal ions led to the oxidation of substrates mainly with single-phase curves. In parallel to a significant increase of the activities comparing with Fe2+, Cu2+ or Mn2+ taken separately, the RAs of the oxidation reactions catalyzing by non-dialyzed and dialyzed IgGs, became to be comparable. Ni2+, Mg2+ and Co2+ sufficiently activated the Cu2+-dependent oxidation of substrates catalyzed by D-IgGs, while Ca2+ inhibited these reactions [31].

IgGs by two metal ions with variable oxidation state proceeds either using the second metal as

It was demonstrated that small fractions of IgGs from the sera of healthy humans as well

activities [36]. In contrast to rat antibodies, IgGs from human blood have both the dependent and independent on metal ions activities. After dialysis of human IgGs against EDTA and EGTA, the relative peroxidase and oxidoreductase activity dependently of IgG preparation decreased from 100 to ~10–85 and 14–83%, respectively. Addition of external metal ions to D-IgGs and ND-IgGs results in a significant increase in their activities. Separation of IgGs on Chelex results in Abs separation to many different subfractions with different affinities to the chelating resin. In the presence of Cu2+ external ions, the specific peroxidase RA of several human IgG subfractions after chromatography achieves 20–27% comparing with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of many IgG subfractions is ~

It was shown, that IgGs of rats and humans effectively oxidize not only DAB but also many other toxic, carcinogenic and mutagenic compounds such as phenol, *o-*phenylenediamine, α-naphthol, p-hydroquinone, etc. [34]. However, overall, the relative peroxidase and oxidoreductase activities of polyclonal rat IgGs in the presence of different metal ions is ~ 10–100-fold higher than those of polyclonal human IgGs. Interestingly, rats are known as the most resistant mammals to all harmful factors of an environment including carcinogens, mutagens and radiation. One cannot exclude that this is due to better protection of rats compared to peoples from harmful factors due to more active metal-dependent Abs with peroxidase and oxidoreductase activities.

It was shown in many articles that electrophoretically and immunologically homogeneous polyclonal IgGs from sera of healthy volunteers and experimental mice are not active in the hydrolysis of DNA and RNA (for review see [37–46]). The occurrence of auto-Abs with catalytic activities is a distinctive feature of mammalian autoimmune diseases (reviewed in [37–46]). IgGs and/or IgMs abzymes hydrolyzing DNA and RNA were revealed in the sera of patients with several autoimmune and viral pathologies: SLE [57–61], multiple sclerosis [62–64], Hashimoto's thyroiditis and polyarthritis [65, 66], schizophrenia [67] and with three viral diseases viral hepatitis [68], acquired immunodeficiency syndrome [69] and tick-borne encephalitis [70]), as well as human milk [71–73], SLE mice [74, 75] and experimental autoimmune encephalomyelitis(EAE) mice [76, 77]. Antibodies with DNase activity from the blood of patients and mice with various diseases were dependent on different metal ions [57–70, 74–77], while human milk contains metal-dependent and independent DNase sIgAs and IgGs [71–73]. The RAs of IgGs from the sera of patients (and mice) with different AIDs vary significantly from patient to patient [57–70, 74–77]. **Figure 6** shows the cleavage of plasmid supercoiled (sc)DNA by 10 various IgGs bound with internal metal ions from the sera

**6. Dependence of DNA-hydrolyzing abzymes on metal ions**

fragments oxidize DAB through peroxidase and oxidoreductase

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Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

an electrophilic cofactor.

as their Fab and F(ab)<sup>2</sup>

4–6-fold higher than that for HRP [36].

of patients with various autoimmune diseases.

As mentioned above, the dependencies of the oxidoreductase and peroxidase activities of the ND-IgGs and D-IgGs on the concentrations of Fe2+, Cu2+ and Mn2+ were biphasic. This indicates that two different metal ions are likely to participate in the catalysis of these reactions. The canonical Cu, Zn superoxide dismutases usually use a Cu2+ ion together with a Zn2+ ion [53–55]. However, the only Cu2+ with variable valency participates in the oxidation of substrates, while Zn2+ serves as a second electrophilic metal cofactor of this enzyme [53–55]. The biphasic dependences can show for a similar function of the same second or another second metal ion. Since D-IgGs and ND-IgGs demonstrate a significantly higher activity in the presence of Cu2+ ions together with Mn2+ or Zn2+ ions, some fractions of IgGs can be Cu/Cu, Cu/Mn or Cu/Zn peroxidases or oxidoreductases. A remarkable increase in the IgGs activity by Cu2+ ions together with Co2+, Mg2+ or Ni2+ can speak in favor that these metal ions can also increase the oxidative function of Cu2+ to some extent as the second ions [31]. Only Fe2+ taken separately was activated the peroxidase activity of D-IgGs at low concentrations (<1 mM). However, FeCl<sup>2</sup> was completely unable to activate the oxidoreductase activity of D-IgGs. Most probably, Cu2+ + Mn2+ is an optimal pair for both the peroxidase and oxidoreductase reactions. It seems more likely that the activation of IgGs by two metal ions with variable oxidation state proceeds either using the second metal as an electrophilic cofactor.

Antioxidant enzymes (catalases, superoxide dismutases and glutathione peroxidases) are very important for preventing oxidative stress [52–56]. However, these enzymes are located inside of cells, and they undergo rapid inactivation in the blood [54]. Immunoglobulins (Igs) are significantly more stable molecules of blood. Therefore, it was interesting how metal ions can activate oxidation-reduction reactions catalyzed by antibodies. The catalysis of such reactions by the majority of canonical enzymes is dependent on metal ions with variable valence [50, 52–55]. First, we have shown that IgGs of healthy Wistar rats oxidize 3,3'-diaminoben-

oxidase activity of IgGs of rats varied in the range 1.6–26% comparing with horseradish peroxidase (HRP, taken for 100%). The dialysis of IgGs against EDTA completely lost these activities. External metal ions activated significantly both activities of non-dialyzed (ND) and dialyzed (D) IgGs. The relative activities (RAs) in the presence of external Fe2+ or Cu2+ ions were increased up to 13–198% compared with that for HRP [31]. Cu2+ ions alone stimulated significantly both the oxidoreductase and peroxidase activities of dialyzed D-IgGs, but only at high concentration (≥2 mM) [31]. Mn2+ ions were weakly activated peroxidase activity but at >3 mM Mn2+ was good cofactor of the oxidoreductase activity at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of D-IgGs was revealed at 0.1–5 mM, but Fe2+ cannot activate their oxidoreductase activity. Al3+, Mg2+, Zn2+, Ca2+, and especially Ni2+ and Co2+ were not able to activate D-IgGs, but slightly activated ND-IgGs containing different intrinsic metal ions. Some metal ions activated IgGs especially ND-Abs in accordance with biphasic curves, which were specific for every individual Ab preparation [31]. The combinations of Fe2+ + Zn2+, Fe2+ + Mn2+, Cu2+ + Mn2+ and Cu2+ + Zn2+ and other metal ions led to the oxidation of substrates mainly with single-phase curves. In parallel to a significant increase of the activities comparing with Fe2+, Cu2+ or Mn2+ taken separately, the RAs of the oxidation reactions catalyzing by non-dialyzed and dialyzed IgGs, became to be comparable. Ni2+, Mg2+ and Co2+ sufficiently activated the Cu2+-dependent oxidation of substrates catalyzed by D-IgGs, while Ca2+ inhib-

As mentioned above, the dependencies of the oxidoreductase and peroxidase activities of the ND-IgGs and D-IgGs on the concentrations of Fe2+, Cu2+ and Mn2+ were biphasic. This indicates that two different metal ions are likely to participate in the catalysis of these reactions. The canonical Cu, Zn superoxide dismutases usually use a Cu2+ ion together with a Zn2+ ion [53–55]. However, the only Cu2+ with variable valency participates in the oxidation of substrates, while Zn2+ serves as a second electrophilic metal cofactor of this enzyme [53–55]. The biphasic dependences can show for a similar function of the same second or another second metal ion. Since D-IgGs and ND-IgGs demonstrate a significantly higher activity in the presence of Cu2+ ions together with Mn2+ or Zn2+ ions, some fractions of IgGs can be Cu/Cu, Cu/Mn or Cu/Zn peroxidases or oxidoreductases. A remarkable increase in the IgGs activity by Cu2+ ions together with Co2+, Mg2+ or Ni2+ can speak in favor that these metal ions can also increase the oxidative function of Cu2+ to some extent as the second ions [31]. Only Fe2+ taken separately was activated the perox-

to activate the oxidoreductase activity of D-IgGs. Most probably, Cu2+ + Mn2+ is an optimal pair for both the peroxidase and oxidoreductase reactions. It seems more likely that the activation of

idase activity of D-IgGs at low concentrations (<1 mM). However, FeCl<sup>2</sup>

O2

[31–35]. In the external metal ions absence, the specific per-

and due to an oxidoreductase

was completely unable

zidine through a peroxidase activity in the presence of H<sup>2</sup>

O2

activity in the absence of H<sup>2</sup>

26 Trace Elements - Human Health and Environment

ited these reactions [31].

It was demonstrated that small fractions of IgGs from the sera of healthy humans as well as their Fab and F(ab)<sup>2</sup> fragments oxidize DAB through peroxidase and oxidoreductase activities [36]. In contrast to rat antibodies, IgGs from human blood have both the dependent and independent on metal ions activities. After dialysis of human IgGs against EDTA and EGTA, the relative peroxidase and oxidoreductase activity dependently of IgG preparation decreased from 100 to ~10–85 and 14–83%, respectively. Addition of external metal ions to D-IgGs and ND-IgGs results in a significant increase in their activities. Separation of IgGs on Chelex results in Abs separation to many different subfractions with different affinities to the chelating resin. In the presence of Cu2+ external ions, the specific peroxidase RA of several human IgG subfractions after chromatography achieves 20–27% comparing with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of many IgG subfractions is ~ 4–6-fold higher than that for HRP [36].

It was shown, that IgGs of rats and humans effectively oxidize not only DAB but also many other toxic, carcinogenic and mutagenic compounds such as phenol, *o-*phenylenediamine, α-naphthol, p-hydroquinone, etc. [34]. However, overall, the relative peroxidase and oxidoreductase activities of polyclonal rat IgGs in the presence of different metal ions is ~ 10–100-fold higher than those of polyclonal human IgGs. Interestingly, rats are known as the most resistant mammals to all harmful factors of an environment including carcinogens, mutagens and radiation. One cannot exclude that this is due to better protection of rats compared to peoples from harmful factors due to more active metal-dependent Abs with peroxidase and oxidoreductase activities.

## **6. Dependence of DNA-hydrolyzing abzymes on metal ions**

It was shown in many articles that electrophoretically and immunologically homogeneous polyclonal IgGs from sera of healthy volunteers and experimental mice are not active in the hydrolysis of DNA and RNA (for review see [37–46]). The occurrence of auto-Abs with catalytic activities is a distinctive feature of mammalian autoimmune diseases (reviewed in [37–46]). IgGs and/or IgMs abzymes hydrolyzing DNA and RNA were revealed in the sera of patients with several autoimmune and viral pathologies: SLE [57–61], multiple sclerosis [62–64], Hashimoto's thyroiditis and polyarthritis [65, 66], schizophrenia [67] and with three viral diseases viral hepatitis [68], acquired immunodeficiency syndrome [69] and tick-borne encephalitis [70]), as well as human milk [71–73], SLE mice [74, 75] and experimental autoimmune encephalomyelitis(EAE) mice [76, 77]. Antibodies with DNase activity from the blood of patients and mice with various diseases were dependent on different metal ions [57–70, 74–77], while human milk contains metal-dependent and independent DNase sIgAs and IgGs [71–73]. The RAs of IgGs from the sera of patients (and mice) with different AIDs vary significantly from patient to patient [57–70, 74–77]. **Figure 6** shows the cleavage of plasmid supercoiled (sc)DNA by 10 various IgGs bound with internal metal ions from the sera of patients with various autoimmune diseases.

**Figure 6.** Relative DNase activities of catalytic IgG-abzymes from sera of 10 different patients with various diseases in the hydrolysis of scDNA. Lanes 1–10 correspond to IgGs of 10 different patients; C<sup>1</sup> , DNA incubated alone; C<sup>2</sup> and C<sup>3</sup> , DNA incubated with Abs of two healthy donors.

During this time, some IgGs cause only single breaks in one strand of scDNA converting it to the relaxed form (lanes 1–3), when others make multiple breaks forming DNA linearization (lanes 4–6). The most active IgGs hydrolyze scDNA into medium- and short-length oligonucleotides (lanes 7–10). Polyclonal DNase IgGs from the sera of autoimmune-prone MRL mice were not after Abs dialysis against EDTA, but were activated by different externally added metal (Me2+) ions: Mn2+ ≥ Mg2+ > Ca2+ ≥ Cu2+ > Co2+ ≥ Ni2+ ≥ Zn2+ [74, 75]. Fe2+ ions could not stimulate the hydrolysis of scDNA by the Abs. The initial rate dependencies on the concentration of different Me2+ ions were mostly bell-shaped, having from one to four maxima at different concentrations of Me2+ ions. Mn2+, Ni2+ and Co2+ activated DNA hydrolysis. The Mn2+-dependent scDNA hydrolysis was activated by Ni2+, Ca2+, Co2+ and Mg2+, but was inhibited by Zn2+ and Cu2+. Only in the case of Mg2+and Mg2+ or Ca2+ as the second metal ions, an accumulation of linear DNA was observed. Affinity chromatography on DNA-cellulose separated DNase mouse IgGs to many subfractions having different affinities for DNA and varying levels of the relative activity (0–100%) in the presence of Mn2+, Ca2+ and Mg2+ ions. In contrast to all human DNases having 1 pH optimum, mouse IgGs hydrolyzing DNA showed several pronounced pH optima from 4.5 and 9.5; in the presence of Ca2+, Mn2+ and Mg2+ ions, these dependencies were different. These findings show the extreme diversity of the ability of metal-dependent mouse IgGs functioning at different pHs and to be activated by various optimal metal cofactors. At the same time, a similar situation on an extreme diversity of Me2+-dependent Abs was observed for DNase abzymes from sera of the patient with different autoimmune and viral diseases [70] including monoclonal light chains of human IgGs [78] (e.g., **Figure 7**).

Dependently on patient demonstrated different substrate specificity [63]. All the data obtained showed that polyclonal MS IgGs could contain different combinations of sequence-independent and sequence-dependent endo- and exonuclease activities [63]. The enzymatic properties of the DNA- and RNA-hydrolyzing IgGs of patients with various AIDs [37–46] distinguished them from all known canonical DNases and RNases [79–81].

Polyclonal DNase IgGs from sera of autoimmune patients, SLE mice, rabbits immunized with DNA and human milk are usually very heterogeneous in their affinity for DNA and can be separated into many subfractions by chromatography on DNA-cellulose [78, 82, 83]. An immunoglobulin light chain phagemid library was prepared using peripheral blood lymphocytes of patients with SLE [78, 82, 83]. Phage particles displaying light chains interacting with DNA were isolated by chromatography on DNA-cellulose; the fraction eluted by 0.5 M NaCl and acidic buffer (pH 2.6) were used for obtaining of individual monoclonal light chains (MLChs, 27–28 kDa) [78, 82, 83].

of all MLChs before their treatment with different specific inhibitors was taken for 100 %.

**Figure 7.** The RAs of MBP-hydrolyzing activity of twenty-two MLChs after their treatment with specific inhibitors of various type proteases. Different MLChs were preincubated in the absence of inhibitors (black bars, control-C), with 50 mM EDTA (gray bars) or 1.0 mM PMSF (white bars) before addition to the standard reaction mixture (A and B). Panel C demonstrates several examples of the RAs of MLChs with metal-dependent (1, 5, 12, 15, and 21) and serine-like activity (4 and 11), which no changing their activity after treatment with iodoacetamide; three MLChs (10, 14, and 18) showing negative response to EDTA and PMSF as well as MLCh-22 having positive answer to PMSF and EDTA after their preincubation with iodoacetamide resulting a significant decrease in the protease activity. Gray and white bars (panel C) correspond respectively to the activity after and before (control) these preparation treatment with iodoacetamide. The Ras

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**Figure 6.** Relative DNase activities of catalytic IgG-abzymes from sera of 10 different patients with various diseases in

During this time, some IgGs cause only single breaks in one strand of scDNA converting it to the relaxed form (lanes 1–3), when others make multiple breaks forming DNA linearization (lanes 4–6). The most active IgGs hydrolyze scDNA into medium- and short-length oligonucleotides (lanes 7–10). Polyclonal DNase IgGs from the sera of autoimmune-prone MRL mice were not after Abs dialysis against EDTA, but were activated by different externally added metal (Me2+) ions: Mn2+ ≥ Mg2+ > Ca2+ ≥ Cu2+ > Co2+ ≥ Ni2+ ≥ Zn2+ [74, 75]. Fe2+ ions could not stimulate the hydrolysis of scDNA by the Abs. The initial rate dependencies on the concentration of different Me2+ ions were mostly bell-shaped, having from one to four maxima at different concentrations of Me2+ ions. Mn2+, Ni2+ and Co2+ activated DNA hydrolysis. The Mn2+-dependent scDNA hydrolysis was activated by Ni2+, Ca2+, Co2+ and Mg2+, but was inhibited by Zn2+ and Cu2+. Only in the case of Mg2+and Mg2+ or Ca2+ as the second metal ions, an accumulation of linear DNA was observed. Affinity chromatography on DNA-cellulose separated DNase mouse IgGs to many subfractions having different affinities for DNA and varying levels of the relative activity (0–100%) in the presence of Mn2+, Ca2+ and Mg2+ ions. In contrast to all human DNases having 1 pH optimum, mouse IgGs hydrolyzing DNA showed several pronounced pH optima from 4.5 and 9.5; in the presence of Ca2+, Mn2+ and Mg2+ ions, these dependencies were different. These findings show the extreme diversity of the ability of metal-dependent mouse IgGs functioning at different pHs and to be activated by various optimal metal cofactors. At the same time, a similar situation on an extreme diversity of Me2+-dependent Abs was observed for DNase abzymes from sera of the patient with different autoimmune and viral diseases [70]

, DNA incubated alone; C<sup>2</sup>

 and C<sup>3</sup> ,

the hydrolysis of scDNA. Lanes 1–10 correspond to IgGs of 10 different patients; C<sup>1</sup>

including monoclonal light chains of human IgGs [78] (e.g., **Figure 7**).

them from all known canonical DNases and RNases [79–81].

Dependently on patient demonstrated different substrate specificity [63]. All the data obtained showed that polyclonal MS IgGs could contain different combinations of sequence-independent and sequence-dependent endo- and exonuclease activities [63]. The enzymatic properties of the DNA- and RNA-hydrolyzing IgGs of patients with various AIDs [37–46] distinguished

DNA incubated with Abs of two healthy donors.

28 Trace Elements - Human Health and Environment

**Figure 7.** The RAs of MBP-hydrolyzing activity of twenty-two MLChs after their treatment with specific inhibitors of various type proteases. Different MLChs were preincubated in the absence of inhibitors (black bars, control-C), with 50 mM EDTA (gray bars) or 1.0 mM PMSF (white bars) before addition to the standard reaction mixture (A and B). Panel C demonstrates several examples of the RAs of MLChs with metal-dependent (1, 5, 12, 15, and 21) and serine-like activity (4 and 11), which no changing their activity after treatment with iodoacetamide; three MLChs (10, 14, and 18) showing negative response to EDTA and PMSF as well as MLCh-22 having positive answer to PMSF and EDTA after their preincubation with iodoacetamide resulting a significant decrease in the protease activity. Gray and white bars (panel C) correspond respectively to the activity after and before (control) these preparation treatment with iodoacetamide. The Ras of all MLChs before their treatment with different specific inhibitors was taken for 100 %.

Polyclonal DNase IgGs from sera of autoimmune patients, SLE mice, rabbits immunized with DNA and human milk are usually very heterogeneous in their affinity for DNA and can be separated into many subfractions by chromatography on DNA-cellulose [78, 82, 83]. An immunoglobulin light chain phagemid library was prepared using peripheral blood lymphocytes of patients with SLE [78, 82, 83]. Phage particles displaying light chains interacting with DNA were isolated by chromatography on DNA-cellulose; the fraction eluted by 0.5 M NaCl and acidic buffer (pH 2.6) were used for obtaining of individual monoclonal light chains (MLChs, 27–28 kDa) [78, 82, 83]. About 45 of 451 and 33 of 687 individual colonies corresponding to peaks eluted with 0.5 M NaCl and acidic buffer, respectively, were randomly chosen for a study of MLChs with DNase activity. About 15 of 45 (*K*<sup>m</sup> = 260–320 nM) and 19 of 33 (*K*m = 3–9 nM) MLChs in the first and second case efficiently hydrolyzed DNA. All 34 MLChs demonstrated different optimal concentrations of KCl or NaCl and pH optima. All MLChs were metal-dependent DNases. The ratio of relative DNA-hydrolyzing activity in the presence of different metal ions was individual for each MLCh. For example, for monoclonal kappa light chain NGK-1 in optimal conditions the RAs decreased in the following order (%): Mn2+ (26.3) ≥ Ca2+ (23.0) ≥ Mg2+ (21.0) > Ni2+ (15.0) > Zn2+ (11.4) > Cu2+ (2.9) > Co2+ (0.0) [83]. But in average, the activity in DNA hydrolysis for all MLChs decreased in the following order: Mn2+ > Co2+ > Mg2+ > Ni2+ ≈ Ca2+ > Cu2+ > Zn2+ [78, 82, 83].

**8. Dependence of protein-hydrolyzing abzymes on metal ions**

[88–91].

For the first time, elevated levels of polyclonal antibodies to myelin basic protein (MBP) and abzymes hydrolyzing MBP were detected in the blood of MS [88–91] and then of SLE [92–95] patients. In the blood of healthy donors, no such abzymes have been detected [88–95]. It is believed that the mechanism of the pathogenesis of MS is associated with the destruction of myelin (including MBP), leading to inflammation processes associated with autoimmune reactions [96]. Some immunological and biochemical indicators of patients with MS and SLE are very similar [45]. First, we have shown that polyclonal IgGmix (a mixture of equimolar IgGs from 10 MS patients) can hydrolyze MBP in the presence better, than in the absence of different metal ions [88–91]. According to TJP-AES data, homogeneous IgG preparations of MS patients contained several intrinsic metal ions; Fe ≥ Ca > C u ≥ Zn ≥ Mg ≥ Mn ≥ Pb ≥ Co ≥ Ni [90]. Then, a minor Me2+-dependent fraction was obtained by chromatography of one IgG preparation on Chelex-100. This IgG fraction could not hydrolyze MBP in the absence of metal ions but was activated after addition of external Mg2+ > Mn2+ > Cu2+ > Ca2+ [90]. Proteolytic activities of individual IgGs from other MS patients were also activated by Fe2+, Ni2+, Zn2+, Co2+ and Pb2+, and especially Ni2+. Interestingly, specific proteolytic metal-dependent and independent activities of IgMs and IgAs from sera of MS patient were usually higher than those of IgGs [89]. A significant diversity of different fractions of polyclonal MS IgGs in their affinity for MBP and the hydrolysis of MBP at different optimal pHs (3–10.5) was demonstrating [91]. IgGs containing kappa- and lambda-light chains showed comparable RAs in the hydrolysis of MBP. IgGs of all four sub-classes were active, with their different average contribution to the total activity of abzymes in the hydrolysis of MBP: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%) [91]. The properties of MS abzymes demonstrating their significant catalytic diversity distinguish them from all known mammalian proteases including metal-dependent ones. These abzymes can attack MBP of the myelin-proteolipid shell of axons and play an important role in MS pathogenesis

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At the initial stage of SLE development antibodies against DNA, as well as DNA- and RNAhydrolyzing antibodies are mostly developed [37–46]. A little later, however, similar to MS pathology in the case of SLE patients the production of Abs against MBP and abzymes hydrolyzing this protein is happening [92–95]. The relative content of different metal ions in the preparations of lyophilized sle-IgGmix and ms-IgGmix from sera of patients with MS and SLE estimated by TJP-AES method to some extent comparable (**Table 8**). Ca2+ was the best activator of SLE-IgGmix and its activity increased in the order: Ca2+ > Mg2+ ≥ Co2+ ≥ Fe2+ ≥ Ni2+ ≥ Cu2+ ≥ Mn2+. Zn2+ inhibits the activity, while Fe2+ cannot activate sle-IgGmix. Ms-IgGmix before dialysis against EDTA showed another order of the activity: Mg2+ > Mn2+ ≥ Cu2+ ≥ Ni2+ ≥ Co2+ ≥ Ca2+, while Fe2+ and Zn2+ slightly inhibit its activity. Thus, on average, patients with MS and SLE develop abzymes hydrolyzing

MBP with different dependence on various metal ions. Combinations of Ca2+ + Co<sup>2</sup>

pathogenesis of MS and SLE patients [45, 46].

Mg2+ results in a significant increase in the MBP-hydrolyzing activity comparing to Ca2+, Mg2+ and Co2+ or ions taken separately [92]. Lambda-IgGs demonstrated higher RAs in the hydrolysis of MBP than kappa-IgGs [93]. The pH profiles of IgG4, IgG3, IgG2, IgG1 of SLE patients were unique; their RAs increased in the order: IgG4 < IgG2 < IgG3 < IgG1. Thus, the immune systems of SLE similarly to MS patients produce a variety of metal-dependent anti-MBP abzymes, which can hydrolyze MBP of the myelin-proteolipid shell of axons and can play important role in the

and Ca2+ +

It is known, that Co2+, Mn2+, Ca2+ and Ni2+ activate mammalian DNase I in much lesser degree than Mg2+ ions [80, 81]. Interestingly, human milk polyclonal sIgA DNase abzymes mainly Me2+-independent and they were only slightly activated by Mg2+, Mn2+ or Zn2+, and the cleavage of DNA substrates was inhibited by Ca2+ and Cu2+ [73]. The effect of metal ions on DNase activity of intact Abs from sera of MS patients decreased in the order: Mn2+ > Mg2+ > Zn2+ > Ca2+ [84]. The DNA-hydrolyzing activity of tick-borne encephalitis IgGs decreased in the following order: Mn2+ ≥ Co2+ ≥ Mg2+ > Ca2+, while Zn2+, Ni2+ and Cu2+ did not stimulate DNA hydrolysis [70]. Polyclonal intact IgGs from MRL mice following specific order of DNase activity activation by different metal ions: Mn2+ ≥ Mg2+ > Ca2+ ≥ Cu2+ > Co2+ ≥ Ni2+ ≥ Zn2+ [74]. Thus, the relative activity of metal-dependent abzymes hydrolyzing DNA depends on the type and timing of the disease as well as can be specific for every individual patient (or animal). On overall, relative metal-dependent DNase activity in blood of patients with different autoimmune and viral diseases increases in the following order: Diabetes < Viral hepatitis ≈ Tick-borne encephalitis < Polyarthritis ≤ Hashimoto's thyroiditis < Schizophrenia < AIDS ≤ Multiple sclerosis < SLE [85].

## **7. Dependence of RNA-hydrolyzing abzymes on metal ions**

First, it was shown that mouse SLE monoclonal IgGs directed against different DNA could effectively hydrolyze both DNA and RNA and cleavage of RNAs is 30–100-fold faster than DNA [86]. Later we have shown that immunization of rabbits with DNA, RNA, RNase A, DNase I or DNase II leads to the formation of abzymes that hydrolyze both DNA and RNA [85]. Interestingly, the substrate specificities of RNase IgGs from patients with autoimmune thyroiditis and polyarthritis [65], SLE [58], MS [62] and hepatitis [68] for different classic homopolynucleotides, cCMP and tRNAPhe with a stable compact structure [39, 59] were different and correlated with the type of disease and were well distinguishable from those of canonical RNases. The activity was strongly dependent on the patient and its disease, but in average increased in the order: hepatitis < polyarthritis < autoimmune thyroiditis < SLE≤MS.Abzymes of patients of SLE and MS patients demonstrate new RNase activity stimulated by Mg2+ ions [39, 59, 65, 87]. In the presence of Mg2 + ions, the abzymes produced products corresponding to new cleavage sites of mutant tRNALys, indicating its local structural or conformational changes compared to tRNALys from mitochondria. Thus, different metal ions play a very important role in the functioning of abzymes with DNase and RNase activities.

## **8. Dependence of protein-hydrolyzing abzymes on metal ions**

About 45 of 451 and 33 of 687 individual colonies corresponding to peaks eluted with 0.5 M NaCl and acidic buffer, respectively, were randomly chosen for a study of MLChs with DNase activity. About 15 of 45 (*K*<sup>m</sup> = 260–320 nM) and 19 of 33 (*K*m = 3–9 nM) MLChs in the first and second case efficiently hydrolyzed DNA. All 34 MLChs demonstrated different optimal concentrations of KCl or NaCl and pH optima. All MLChs were metal-dependent DNases. The ratio of relative DNA-hydrolyzing activity in the presence of different metal ions was individual for each MLCh. For example, for monoclonal kappa light chain NGK-1 in optimal conditions the RAs decreased in the following order (%): Mn2+ (26.3) ≥ Ca2+ (23.0) ≥ Mg2+ (21.0) > Ni2+ (15.0) > Zn2+ (11.4) > Cu2+ (2.9) > Co2+ (0.0) [83]. But in average, the activity in DNA hydrolysis for all MLChs decreased in the

It is known, that Co2+, Mn2+, Ca2+ and Ni2+ activate mammalian DNase I in much lesser degree than Mg2+ ions [80, 81]. Interestingly, human milk polyclonal sIgA DNase abzymes mainly Me2+-independent and they were only slightly activated by Mg2+, Mn2+ or Zn2+, and the cleavage of DNA substrates was inhibited by Ca2+ and Cu2+ [73]. The effect of metal ions on DNase activity of intact Abs from sera of MS patients decreased in the order: Mn2+ > Mg2+ > Zn2+ > Ca2+ [84]. The DNA-hydrolyzing activity of tick-borne encephalitis IgGs decreased in the following order: Mn2+ ≥ Co2+ ≥ Mg2+ > Ca2+, while Zn2+, Ni2+ and Cu2+ did not stimulate DNA hydrolysis [70]. Polyclonal intact IgGs from MRL mice following specific order of DNase activity activation by different metal ions: Mn2+ ≥ Mg2+ > Ca2+ ≥ Cu2+ > Co2+ ≥ Ni2+ ≥ Zn2+ [74]. Thus, the relative activity of metal-dependent abzymes hydrolyzing DNA depends on the type and timing of the disease as well as can be specific for every individual patient (or animal). On overall, relative metal-dependent DNase activity in blood of patients with different autoimmune and viral diseases increases in the following order: Diabetes < Viral hepatitis ≈ Tick-borne encephalitis < Polyarthritis ≤ Hashimoto's thyroiditis < Schizophrenia < AIDS ≤ Multiple sclerosis < SLE [85].

following order: Mn2+ > Co2+ > Mg2+ > Ni2+ ≈ Ca2+ > Cu2+ > Zn2+ [78, 82, 83].

30 Trace Elements - Human Health and Environment

**7. Dependence of RNA-hydrolyzing abzymes on metal ions**

activities.

First, it was shown that mouse SLE monoclonal IgGs directed against different DNA could effectively hydrolyze both DNA and RNA and cleavage of RNAs is 30–100-fold faster than DNA [86]. Later we have shown that immunization of rabbits with DNA, RNA, RNase A, DNase I or DNase II leads to the formation of abzymes that hydrolyze both DNA and RNA [85]. Interestingly, the substrate specificities of RNase IgGs from patients with autoimmune thyroiditis and polyarthritis [65], SLE [58], MS [62] and hepatitis [68] for different classic homopolynucleotides, cCMP and tRNAPhe with a stable compact structure [39, 59] were different and correlated with the type of disease and were well distinguishable from those of canonical RNases. The activity was strongly dependent on the patient and its disease, but in average increased in the order: hepatitis < polyarthritis < autoimmune thyroiditis < SLE≤MS.Abzymes of patients of SLE and MS patients demonstrate new RNase activity stimulated by Mg2+ ions [39, 59, 65, 87]. In the presence of Mg2 + ions, the abzymes produced products corresponding to new cleavage sites of mutant tRNALys, indicating its local structural or conformational changes compared to tRNALys from mitochondria. Thus, different metal ions play a very important role in the functioning of abzymes with DNase and RNase For the first time, elevated levels of polyclonal antibodies to myelin basic protein (MBP) and abzymes hydrolyzing MBP were detected in the blood of MS [88–91] and then of SLE [92–95] patients. In the blood of healthy donors, no such abzymes have been detected [88–95]. It is believed that the mechanism of the pathogenesis of MS is associated with the destruction of myelin (including MBP), leading to inflammation processes associated with autoimmune reactions [96]. Some immunological and biochemical indicators of patients with MS and SLE are very similar [45]. First, we have shown that polyclonal IgGmix (a mixture of equimolar IgGs from 10 MS patients) can hydrolyze MBP in the presence better, than in the absence of different metal ions [88–91]. According to TJP-AES data, homogeneous IgG preparations of MS patients contained several intrinsic metal ions; Fe ≥ Ca > C u ≥ Zn ≥ Mg ≥ Mn ≥ Pb ≥ Co ≥ Ni [90]. Then, a minor Me2+-dependent fraction was obtained by chromatography of one IgG preparation on Chelex-100. This IgG fraction could not hydrolyze MBP in the absence of metal ions but was activated after addition of external Mg2+ > Mn2+ > Cu2+ > Ca2+ [90]. Proteolytic activities of individual IgGs from other MS patients were also activated by Fe2+, Ni2+, Zn2+, Co2+ and Pb2+, and especially Ni2+. Interestingly, specific proteolytic metal-dependent and independent activities of IgMs and IgAs from sera of MS patient were usually higher than those of IgGs [89]. A significant diversity of different fractions of polyclonal MS IgGs in their affinity for MBP and the hydrolysis of MBP at different optimal pHs (3–10.5) was demonstrating [91]. IgGs containing kappa- and lambda-light chains showed comparable RAs in the hydrolysis of MBP. IgGs of all four sub-classes were active, with their different average contribution to the total activity of abzymes in the hydrolysis of MBP: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%) [91]. The properties of MS abzymes demonstrating their significant catalytic diversity distinguish them from all known mammalian proteases including metal-dependent ones. These abzymes can attack MBP of the myelin-proteolipid shell of axons and play an important role in MS pathogenesis [88–91].

At the initial stage of SLE development antibodies against DNA, as well as DNA- and RNAhydrolyzing antibodies are mostly developed [37–46]. A little later, however, similar to MS pathology in the case of SLE patients the production of Abs against MBP and abzymes hydrolyzing this protein is happening [92–95]. The relative content of different metal ions in the preparations of lyophilized sle-IgGmix and ms-IgGmix from sera of patients with MS and SLE estimated by TJP-AES method to some extent comparable (**Table 8**). Ca2+ was the best activator of SLE-IgGmix and its activity increased in the order: Ca2+ > Mg2+ ≥ Co2+ ≥ Fe2+ ≥ Ni2+ ≥ Cu2+ ≥ Mn2+. Zn2+ inhibits the activity, while Fe2+ cannot activate sle-IgGmix. Ms-IgGmix before dialysis against EDTA showed another order of the activity: Mg2+ > Mn2+ ≥ Cu2+ ≥ Ni2+ ≥ Co2+ ≥ Ca2+, while Fe2+ and Zn2+ slightly inhibit its activity. Thus, on average, patients with MS and SLE develop abzymes hydrolyzing MBP with different dependence on various metal ions. Combinations of Ca2+ + Co<sup>2</sup> and Ca2+ + Mg2+ results in a significant increase in the MBP-hydrolyzing activity comparing to Ca2+, Mg2+ and Co2+ or ions taken separately [92]. Lambda-IgGs demonstrated higher RAs in the hydrolysis of MBP than kappa-IgGs [93]. The pH profiles of IgG4, IgG3, IgG2, IgG1 of SLE patients were unique; their RAs increased in the order: IgG4 < IgG2 < IgG3 < IgG1. Thus, the immune systems of SLE similarly to MS patients produce a variety of metal-dependent anti-MBP abzymes, which can hydrolyze MBP of the myelin-proteolipid shell of axons and can play important role in the pathogenesis of MS and SLE patients [45, 46].

Phagemid library derived from lymphocytes of peripheral blood of patients with SLE was used for obtaining of MLChs hydrolyzing MBP [97–100]. About 22 of 72 MLChs hydrolyzing only MBP (not other control proteins) having various pH optima in a 5.7–9.0 range and different specificity in the hydrolysis of four various MBP oligopeptides [97]. Eleven MLChs were metalloproteases, while four and three MLChs showed serine-like and thiol-like proteolytic activities, respectively. The activity of three MLChs was suppressed by both PMSF and EDTA, while the other two by EDTA and iodoacetamide and one by EDTA, PMSF and iodoacetamide. The ratio of RAs in the presence of Mg2+, Ca2+, Mn2+, Zn2+, Cu2+, Ni2+ and Co2+ was very specific for all metal-dependent MLChs. For the total preparation of MLChs, the activity decreased in the order: Са2+ ≥ Сo2+ ≈ Мg2++ ≥ Mn2+ ≥ Ni<sup>2</sup> ≈ Cu2+ ≈ Zn2+ [97].

amino acid residues providing thiol, serine, acidic and metal-dependent proteases. But the

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

In addition, IgGs from sera of HIV-infected patients hydrolyze all human histones [106]. The RAs of IgGs in the hydrolysis of histones (H4, H3, H2a, H2b and H1) varied significantly for Abs of different patients. The effects of different external metal ions on the dialyzed polyclonal IgGs in the hydrolysis of five individual histones were very different. For example, maximal activation of one IgG preparation was observed in the hydrolysis of H4 by Zn2+ and Ni2+, H3 by Cu2+ and Ni2+, H2a by Cu2+, H2b by Co2+ and Ni2+, H1 by Cu2+ and Mn2+. Such an exceptional diversity in activation by different metals ions was observed for all 32 IgGs [106].

Using the TJP-AES method, we have estimated the relative contents of various trace elements, including metals in various organs, tissues and biological fluids of humans and animals, as well as in immunoglobulins from these sources., the maximal RAs of abzymes with different catalytic activities are most often achieved not in the presence of metal ions, which are contained in biological sources and antibodies in maximum quantities. Some specific abzymes show maximum activity in the presence of metal ions, which are minor elements of different organs and biological fluids. The question is why there are so many abzymes with very different properties including metal-dependent ones against the same protein. First, mammalian immune system

these Abs may be different. In addition, proteins and nucleic acids can adsorb ions of various metals including traces elements on their surfaces. Therefore, some specific antibodies (and abzymes) can be against fragments (antigenic determinants) of DNA and proteins containing no metal ions. Some other specific metal-dependent abzymes with nuclease and protease activities can be antibodies against sequences associated with one or more metal ions. In addition, not only antibodies against substrates imitating transition states of chemical reactions can possess catalytic activities, but also anti-idiotypic Abs against active centers of various enzymes. The activity of many various enzymes depends on the ions of different metals. Since secondary—anti-idiotypic antibodies against such active sites should contain all the structural components of an enzyme active center including amino acid residues for binding metal ions, they can be metal-dependent abzymes. In this chapter, we have analyzed not only the relative content of different metal ions in various biological substances but also analyzed a possible function of

This research was possible due to grant from the Russian Science Foundation (No. 16-15-

metal ions in the catalysis by autoantibodies of different chemical reactions.

variants of Abs against one antigenic determinant and all of

variants of Abs

33

http://dx.doi.org/10.5772/intechopen.75939

ratio of these abzymes activities may be individual for every HIV-infected patient.

Importantly, mammalian immune system theoretically can produce up to 10<sup>6</sup>

against one antigenic determinant and all of these Abs may be different.

**10. Conclusion**

theoretically can produce up to 10<sup>6</sup>

**Acknowledgements**

10,103) to G.A. Nevinsky).

In addition to these 22, were isolated other 3 MLChs, which were analyzed in more detailed. NGTA1-Me-pro (MLCh-23) was a typical metalloprotease inhibited only by EDTA [98]. The activity of MLCh-23 in the hydrolysis of MBP was reduced in the presence of ions of seven different metals in the following order: Са2+ > Мg2+ > Ni2+ ≥ Zn2+ ≥ Сo2+ ≥ Mn2+ > Cu2+. MLCh-23 has two active sites into the light chain with very distinct pH optima: pH 6.0 and 8.5 and different affinity for MBP [98]. Specific inhibitors of NGTA2-Me-pro-Tr (MLCh-24) were PMSF (42%) and EDTA (58%): it exhibits properties of a chimeric protease with serine and metal-dependent activities [99]. The addition of ions of different metals led to a decrease in the activity of MLCh-24 in the following order: Са2+ ≥ Mn2+ ≥ Мg2+ ≈ Сo2+ ≈ Ni2+ ≥ Cu2+ ≥ Zn<sup>2</sup> . NGTA2-Me-pro-Tr is the first example of an MLCh-23 having two combined centers with serine and metalloprotease activities.

It should be noted that all recombinant MLChs were obtained by affinity chromatography of phage particles on MBP-Sepharose. Taking this into account, a very unexpected result was obtained from analysis of NGTA3-pro-DNase (MLCh-25) [100]. Only 1 MLCh-25 of 25 recombinant MLChs effectively hydrolyzed not only MBP but also DNA. Preincubation of MLCh-25 with both PMSF (67%) and EDTA (36%) resulted in suppression of its protease activity. Ions of different metals activated MLCh-25 in the following order: Са2+ ≥ Ni2+ > Сo2+ ≈ Mn2+ ≥ Cu2+ ≈ Zn2+ ≥ Mg2+ [100]. The affinity of MLCh-25 metal-dependent and serine-like active centers for BMP was different. The DNase activity of MLCh-25 decreases in the following order: Mn2+ ≈ Сo2+ ≥ Мn2+ > Сu2+ ≈ Ni2+ ≥ Са2+ > Zn2+, which completely distinguishes MLCh-25 from canonical DNases [72]. Metal-dependent casein hydrolyzing sIgA antibodies from human milk were described [101]. The RA of sIgAs after removal of intrinsic metal ions increase their activity in the presence of external Fe2+ > Ca2+ > Co2+ ≥ Ni2+ and especially combinations of metals: Co2++Ca2+ < Mg2+ + Ca2+ < Ca2+ + Zn2+ < Fe2+ + Zn2+ < Fe2+ + Co2+ < Fe2+ + Ca2+ [101].

## **9. Catalytic activities of antibodies of HIV-infected patients**

Metal-dependent IgGs and/or IgMs from the blood of HIV-infected patients hydrolyzing DNA [69], viral reverse transcriptase [102] and integrase [103–105], and all histones [106] were described. Average activities of anti-IN IgGs in the hydrolysis of IN decreased in the order Mn2+ > Mg2+ ≈ Cu2+ > Co2+ while for IgMs in another order Cu2+ > Mn2+ > Co2+ ≫ Mg2+. Our findings show that active centers of anti-IN polyclonal abzymes of AIDS patients can contain amino acid residues providing thiol, serine, acidic and metal-dependent proteases. But the ratio of these abzymes activities may be individual for every HIV-infected patient.

In addition, IgGs from sera of HIV-infected patients hydrolyze all human histones [106]. The RAs of IgGs in the hydrolysis of histones (H4, H3, H2a, H2b and H1) varied significantly for Abs of different patients. The effects of different external metal ions on the dialyzed polyclonal IgGs in the hydrolysis of five individual histones were very different. For example, maximal activation of one IgG preparation was observed in the hydrolysis of H4 by Zn2+ and Ni2+, H3 by Cu2+ and Ni2+, H2a by Cu2+, H2b by Co2+ and Ni2+, H1 by Cu2+ and Mn2+. Such an exceptional diversity in activation by different metals ions was observed for all 32 IgGs [106]. Importantly, mammalian immune system theoretically can produce up to 10<sup>6</sup> variants of Abs against one antigenic determinant and all of these Abs may be different.

## **10. Conclusion**

Phagemid library derived from lymphocytes of peripheral blood of patients with SLE was used for obtaining of MLChs hydrolyzing MBP [97–100]. About 22 of 72 MLChs hydrolyzing only MBP (not other control proteins) having various pH optima in a 5.7–9.0 range and different specificity in the hydrolysis of four various MBP oligopeptides [97]. Eleven MLChs were metalloproteases, while four and three MLChs showed serine-like and thiol-like proteolytic activities, respectively. The activity of three MLChs was suppressed by both PMSF and EDTA, while the other two by EDTA and iodoacetamide and one by EDTA, PMSF and iodoacetamide. The ratio of RAs in the presence of Mg2+, Ca2+, Mn2+, Zn2+, Cu2+, Ni2+ and Co2+ was very specific for all metal-dependent MLChs. For the total preparation of MLChs, the activity

In addition to these 22, were isolated other 3 MLChs, which were analyzed in more detailed. NGTA1-Me-pro (MLCh-23) was a typical metalloprotease inhibited only by EDTA [98]. The activity of MLCh-23 in the hydrolysis of MBP was reduced in the presence of ions of seven different metals in the following order: Са2+ > Мg2+ > Ni2+ ≥ Zn2+ ≥ Сo2+ ≥ Mn2+ > Cu2+. MLCh-23 has two active sites into the light chain with very distinct pH optima: pH 6.0 and 8.5 and different affinity for MBP [98]. Specific inhibitors of NGTA2-Me-pro-Tr (MLCh-24) were PMSF (42%) and EDTA (58%): it exhibits properties of a chimeric protease with serine and metal-dependent activities [99]. The addition of ions of different metals led to a decrease in the activity of MLCh-24 in

example of an MLCh-23 having two combined centers with serine and metalloprotease activities. It should be noted that all recombinant MLChs were obtained by affinity chromatography of phage particles on MBP-Sepharose. Taking this into account, a very unexpected result was obtained from analysis of NGTA3-pro-DNase (MLCh-25) [100]. Only 1 MLCh-25 of 25 recombinant MLChs effectively hydrolyzed not only MBP but also DNA. Preincubation of MLCh-25 with both PMSF (67%) and EDTA (36%) resulted in suppression of its protease activity. Ions of different metals activated MLCh-25 in the following order: Са2+ ≥ Ni2+ > Сo2+ ≈ Mn2+ ≥ Cu2+ ≈ Zn2+ ≥ Mg2+ [100]. The affinity of MLCh-25 metal-dependent and serine-like active centers for BMP was different. The DNase activity of MLCh-25 decreases in the following order: Mn2+ ≈ Сo2+ ≥ Мn2+ > Сu2+ ≈ Ni2+ ≥ Са2+ > Zn2+, which completely distinguishes MLCh-25 from canonical DNases [72]. Metal-dependent casein hydrolyzing sIgA antibodies from human milk were described [101]. The RA of sIgAs after removal of intrinsic metal ions increase their activity in the presence of external Fe2+ > Ca2+ > Co2+ ≥ Ni2+ and especially combinations of metals:

Co2++Ca2+ < Mg2+ + Ca2+ < Ca2+ + Zn2+ < Fe2+ + Zn2+ < Fe2+ + Co2+ < Fe2+ + Ca2+ [101].

**9. Catalytic activities of antibodies of HIV-infected patients**

Metal-dependent IgGs and/or IgMs from the blood of HIV-infected patients hydrolyzing DNA [69], viral reverse transcriptase [102] and integrase [103–105], and all histones [106] were described. Average activities of anti-IN IgGs in the hydrolysis of IN decreased in the order Mn2+ > Mg2+ ≈ Cu2+ > Co2+ while for IgMs in another order Cu2+ > Mn2+ > Co2+ ≫ Mg2+. Our findings show that active centers of anti-IN polyclonal abzymes of AIDS patients can contain

. NGTA2-Me-pro-Tr is the first

decreased in the order: Са2+ ≥ Сo2+ ≈ Мg2++ ≥ Mn2+ ≥ Ni<sup>2</sup> ≈ Cu2+ ≈ Zn2+ [97].

32 Trace Elements - Human Health and Environment

the following order: Са2+ ≥ Mn2+ ≥ Мg2+ ≈ Сo2+ ≈ Ni2+ ≥ Cu2+ ≥ Zn<sup>2</sup>

Using the TJP-AES method, we have estimated the relative contents of various trace elements, including metals in various organs, tissues and biological fluids of humans and animals, as well as in immunoglobulins from these sources., the maximal RAs of abzymes with different catalytic activities are most often achieved not in the presence of metal ions, which are contained in biological sources and antibodies in maximum quantities. Some specific abzymes show maximum activity in the presence of metal ions, which are minor elements of different organs and biological fluids. The question is why there are so many abzymes with very different properties including metal-dependent ones against the same protein. First, mammalian immune system theoretically can produce up to 10<sup>6</sup> variants of Abs against one antigenic determinant and all of these Abs may be different. In addition, proteins and nucleic acids can adsorb ions of various metals including traces elements on their surfaces. Therefore, some specific antibodies (and abzymes) can be against fragments (antigenic determinants) of DNA and proteins containing no metal ions. Some other specific metal-dependent abzymes with nuclease and protease activities can be antibodies against sequences associated with one or more metal ions. In addition, not only antibodies against substrates imitating transition states of chemical reactions can possess catalytic activities, but also anti-idiotypic Abs against active centers of various enzymes. The activity of many various enzymes depends on the ions of different metals. Since secondary—anti-idiotypic antibodies against such active sites should contain all the structural components of an enzyme active center including amino acid residues for binding metal ions, they can be metal-dependent abzymes. In this chapter, we have analyzed not only the relative content of different metal ions in various biological substances but also analyzed a possible function of metal ions in the catalysis by autoantibodies of different chemical reactions.

## **Acknowledgements**

This research was possible due to grant from the Russian Science Foundation (No. 16-15- 10,103) to G.A. Nevinsky).

## **Author details**

Natalia P. Zaksas<sup>1</sup> and Georgy A. Nevinsky<sup>2</sup> \*

\*Address all correspondence to: nevinky@niboch.nsc.ru

1 Nikolaev Institute of Inorganic Chemistry, Siberian Division, Russian Academy of Sciences, Novosibirsk, Russia

[8] Pozebon D, Scheffler GL, Dressler VL, Nunes MAG. Review of the applications of laser ablation inductively coupled plasma mass spectrometry. Journal of Analytical Atomic

Minor and Trace Elements in Whole Blood, Tissues, Proteins and Immunoglobulins of Mammals

http://dx.doi.org/10.5772/intechopen.75939

35

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[17] Cherevko AS. Mechanism of the evaporation of particles of powder test materials in the discharge of a two-jet argon arc plasmatron. Journal of Analytical Chemistry.

[18] Zaksas NP, Gerasimov VA. Consideration on excitation mechanisms in a high-power two-jet plasma. Spectrochimica Acta, Part B: Atomic Spectroscopy. 2013;**88**:174-179. DOI:

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2 Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, Novosibirsk, Russia

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**Author details**

Natalia P. Zaksas<sup>1</sup>

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c5ja90001h

c7ja90005h

Sciences, Novosibirsk, Russia

34 Trace Elements - Human Health and Environment

Academy of Sciences, Novosibirsk, Russia

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\*Address all correspondence to: nevinky@niboch.nsc.ru

\*

1 Nikolaev Institute of Inorganic Chemistry, Siberian Division, Russian Academy of

2 Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian

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**Chapter 3**

Provisional chapter

**Trace Elements in Hair: Relevance to Air Pollution**

DOI: 10.5772/intechopen.74373

Elemental concentrations of single hair samples taken from 2003 to 2012 had been evaluated by X-ray fluorescence for the assessment of the relation between calcium and cancer. Early results implied a mechanism linking hair and serum element concentrations with a shift in element levels over time. After 2009, pollution-attributable differences were seen in the levels of Ca, Sr, P, Cl, Br, K, S, elements under renal control by parathyroid hormone (PTH), as well as Cu, Zn, Ti. Especially, hair taken from February to March 2011 showed low [Cu] and [Zn] indicating about half of the normal serum level and often three orders of magnitude higher [Ti] than typical. These specimens also showed higher serum [S] than usual, and except for one patient with PTH-related disease, all the subjects had the normal or lower hair calcium than typical for earlier years. Almost all the subjects showed store-operated Ca channel gating. The pollution era is associated with an increase in hair Na, a decrease in K, and

can be attributed to increases in serum Ca and S coincident with breathing the polluted air; the incorporated Ca closes the ion channels of hair matrix cells but may be moved with P to bone, resulting in the abnormal P deficiency, likely producing an ATP shortage in serum. This insufficient ATP supply may result in inactivated molecular pumps and hypokalemia contributing to fatal ventricular fibrillation in patients with myocardial infarction. The pollution increase [S] in serum may be excreted by forming sulfide compounds with Cu and Zn, resulting in Cu deficiency necessary for making elastin to repair damage in blood vessels. The K and Cu deficiencies observed appear to account for the reported increase in infarction

/K+

/K<sup>+</sup>

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.



abnormally low P, suggesting a functional deterioration of Na+

Keywords: calcium, sulfur, ion channels, parathyroid hormone, Na<sup>+</sup>

Trace Elements in Hair: Relevance to Air Pollution

Jun-ichi Chikawa, Jeremy Salter, Hiroki Shima,

Jun-ichi Chikawa, Jeremy Salter, Hiroki Shima,

Kousaku Yamada and Shingo Yamamoto

Kousaku Yamada and Shingo Yamamoto

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Takaaki Tsuchida, Takashi Ueda,

Takaaki Tsuchida, Takashi Ueda,

http://dx.doi.org/10.5772/intechopen.74373

mortality after high-pollution days.

hypokalemia, yellow haze, copper, zinc

Abstract

#### **Trace Elements in Hair: Relevance to Air Pollution** Trace Elements in Hair: Relevance to Air Pollution

DOI: 10.5772/intechopen.74373

Jun-ichi Chikawa, Jeremy Salter, Hiroki Shima, Takaaki Tsuchida, Takashi Ueda, Kousaku Yamada and Shingo Yamamoto Jun-ichi Chikawa, Jeremy Salter, Hiroki Shima, Takaaki Tsuchida, Takashi Ueda, Kousaku Yamada and Shingo Yamamoto

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74373

#### Abstract

Elemental concentrations of single hair samples taken from 2003 to 2012 had been evaluated by X-ray fluorescence for the assessment of the relation between calcium and cancer. Early results implied a mechanism linking hair and serum element concentrations with a shift in element levels over time. After 2009, pollution-attributable differences were seen in the levels of Ca, Sr, P, Cl, Br, K, S, elements under renal control by parathyroid hormone (PTH), as well as Cu, Zn, Ti. Especially, hair taken from February to March 2011 showed low [Cu] and [Zn] indicating about half of the normal serum level and often three orders of magnitude higher [Ti] than typical. These specimens also showed higher serum [S] than usual, and except for one patient with PTH-related disease, all the subjects had the normal or lower hair calcium than typical for earlier years. Almost all the subjects showed store-operated Ca channel gating. The pollution era is associated with an increase in hair Na, a decrease in K, and abnormally low P, suggesting a functional deterioration of Na+ /K+ -ATPase. These results can be attributed to increases in serum Ca and S coincident with breathing the polluted air; the incorporated Ca closes the ion channels of hair matrix cells but may be moved with P to bone, resulting in the abnormal P deficiency, likely producing an ATP shortage in serum. This insufficient ATP supply may result in inactivated molecular pumps and hypokalemia contributing to fatal ventricular fibrillation in patients with myocardial infarction. The pollution increase [S] in serum may be excreted by forming sulfide compounds with Cu and Zn, resulting in Cu deficiency necessary for making elastin to repair damage in blood vessels. The K and Cu deficiencies observed appear to account for the reported increase in infarction mortality after high-pollution days.

Keywords: calcium, sulfur, ion channels, parathyroid hormone, Na<sup>+</sup> /K<sup>+</sup> -ATPase, hypokalemia, yellow haze, copper, zinc

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## 1. Introduction

Synchrotron X-ray fluorescence analysis of single hair segments was achieved by Iida and Noma [1]. Today, elemental distribution in hair microstructures has been observed especially in cosmetic studies [2] and occasionally in environmental studies [3]. In our previous work [4–6], the study of the relations between elemental concentrations in serum and hair (total concentration instead of structural distribution) was employed for our inquiries into links between cancer and calcium metabolism. X-ray fluorescence (XRF) detects many elements, and in time it became clear that the spectra acquired for cancer assessment also showed pollution-related trends.

Pollution particles smaller than 10 and 2.5 μm suspended in the atmosphere are referred to as "PM 10" and "PM 2.5" (fine Particulate Matter). An association between daily mean PM concentration and daily mortality was shown by a statistical model [7–10]. Also, correlations between PM and cancer were investigated [9]. The PM 2.5 is mainly attributed to soot particles contained in gas emissions from diesel vehicles and manufacturing facilities and contains various substances; combustion-associated gases include sulfur and nitrogen oxides. The PM includes wind-blown yellow sand that often comes to Japan [10] by desertification in the continent. Such particulates have reached California, British Columbia, and the French Alps [11–14]. Today, air pollution PM 2.5 is a universal problem, routinely affecting most of the world, although most obvious in and downwind of industrial areas. PM 2.5 is too small to be filtered by the conventional gauze mask and can deposit materials in capillary vessels in the lung [15].

In this study, to explore the possible origins of the mortality increase observed among cardiac patients, elemental inflow from polluted air into blood has been investigated by review of Xray fluorescence analyses (XFA) of hair using the ratio of characteristic X-ray peaks to background scatter [4–6]. Since the scatter is a function of the mass of the sample within the beam, the peak-to-scatter ratio (P/S) can be used as a unitless relative concentration of each detected element, independent of hair thickness and shape. With the relationships of serum and hair elements given by the equations in Table 1 previously established [5], we obtained useful approximations of element levels in serum.

the normal intracellular [Ca]. Thus, hair elements constitute a record of the combination of

Numerical values of peak/scatter as apparent concentrations of element X defined by Eq. (2). Eq. (1): [X]S = [X]I + [X]P. Subscripts: H = hair, S = serum, I = serum Ion, P = serum Protein, OH = Hair produced during Open ion channels,

Dried serum was measured to be [Na]S = 10 by the definition [X] = P/S Eq. (2), and hair usually shows no Na peak, that is, [Na]H = 1. This resulting ratio corresponds to established values: [Na] = 142 mmol/L in serum and [Na] = 14 mmol/L in cell [23]. The standard concentration [X]H given by Eq. (2), of course, can be converted into the conventional expression such

[Fe]S = [Fe]P [Fe]H = [Fe]P = 15 (4) [Cu]S = [Cu]P [Cu]H = [Cu]P = 20 (5)

[S]P = [S]I [S]HC = [S]P = 20 (7) [S]HO = (1/2)[S]I

[Ca]P = [Ca]I [Ca]HC = [Ca]P = 10 (9) [Ca]HO = (1/2)[Ca]I

[Sr]P = [Sr]I [Sr]HC = [Sr]P = 10 (11)

[Cl]P = 0.04[Cl]I [Cl]HC = [Cl]P = 10 (13) [Cl]HO = {2[Cl]I}

[Br]P = 0.04[Br]I [Br]HC = [Br]P = 10 (15) [Br]HO = {2[Br]I}

[K]S = [K]I [K]HC = 7.1[K]S = 300 (17) [P]P = [P]I = (1/4)[P]S [P]HC ≧ 10 (18) [P]HO = 5 [P]I

[Sr]HO = (1/2){[Sr]P+[Sr]I}

<sup>2</sup> = 400 (6)

<sup>2</sup> = 200 (8)

<sup>2</sup> = 50 (10)

1/2 = 22 (14)

1/2 = 22 (16)

1/2 = 5 (19)

<sup>2</sup> = 200 (12)

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 47

The store-operated Ca channels on the plasma membrane are understood to open when Ca2+ stores are depleted at the endoplasmic reticulum (ER) [16–18]. "Store-operated ion channels" adjust the intracellular Ca ion level toward the normal, and an XFA scan along hair shows that the normal [Ca]HC = 10 was maintained despite the ongoing channel gating revealed by the noted fluctuation in [Sr]H [6]. Therefore, the store-operated channels gate due to shortage of Ca stored in serum protein [5, 6], corresponding to hair Ca less than the normal ([Ca]HC < 10 by

However, when the serum Ca deficiency further proceeds beyond the range covered by the store-operated Ca entry (SOCE), the deficiency increases the secretion of parathyroid hormone (PTH), which causes PTH-activated Ca channel gating. Such channel activity is due to membrane voltage and propagates from channel to channel, resulting in the upper range [Ca]H > 10

serum elements and ion channel function.

Table 1. Standards of hair [X]H and dried serum [X]S.

HC = Hair produced during Closed ion channels.

as [Ca]HC = 0.49 mg/g and [Ca]HO = 2.5 mg/g [5].

[Zn]S = [Zn]P [Zn]H = [Zn]P

Eq. (9) in Table 1).

XFA for scalp hair has revealed ion channel gating of hair matrix (HM) cells [4]. By the principle of "inflow-outflow equality," the content of an element in growing hair must be equal to the inflow into the HM cells from blood [4, 5], and the elements admitted through ion channels occur in hair at two distinct levels produced by the closing or opening (gating) of the relevant channels (in vivo). By such channel gating, homeostasis of cellular essential elements can be maintained. Especially, Ca is an overall regulator of ion gating. We had given ten subjects 2-week oral calcium supplementation; the (dried) serum samples showed a single value for Ca [5]. Similarly, no significant variation was seen among the subjects for Sr, Cl, Br, K, S, and P, elements under renal homeostatic control, although in nonsupplemented subjects, considerable variation is usual [4–6]. Calcium is the central player for the homeostatic control of the elements, and usually, therefore, store-operated Ca channels are activated to maintain


Numerical values of peak/scatter as apparent concentrations of element X defined by Eq. (2). Eq. (1): [X]S = [X]I + [X]P. Subscripts: H = hair, S = serum, I = serum Ion, P = serum Protein, OH = Hair produced during Open ion channels, HC = Hair produced during Closed ion channels.

Dried serum was measured to be [Na]S = 10 by the definition [X] = P/S Eq. (2), and hair usually shows no Na peak, that is, [Na]H = 1. This resulting ratio corresponds to established values: [Na] = 142 mmol/L in serum and [Na] = 14 mmol/L in cell [23]. The standard concentration [X]H given by Eq. (2), of course, can be converted into the conventional expression such as [Ca]HC = 0.49 mg/g and [Ca]HO = 2.5 mg/g [5].

Table 1. Standards of hair [X]H and dried serum [X]S.

1. Introduction

46 Trace Elements - Human Health and Environment

pollution-related trends.

in capillary vessels in the lung [15].

approximations of element levels in serum.

Synchrotron X-ray fluorescence analysis of single hair segments was achieved by Iida and Noma [1]. Today, elemental distribution in hair microstructures has been observed especially in cosmetic studies [2] and occasionally in environmental studies [3]. In our previous work [4–6], the study of the relations between elemental concentrations in serum and hair (total concentration instead of structural distribution) was employed for our inquiries into links between cancer and calcium metabolism. X-ray fluorescence (XRF) detects many elements, and in time it became clear that the spectra acquired for cancer assessment also showed

Pollution particles smaller than 10 and 2.5 μm suspended in the atmosphere are referred to as "PM 10" and "PM 2.5" (fine Particulate Matter). An association between daily mean PM concentration and daily mortality was shown by a statistical model [7–10]. Also, correlations between PM and cancer were investigated [9]. The PM 2.5 is mainly attributed to soot particles contained in gas emissions from diesel vehicles and manufacturing facilities and contains various substances; combustion-associated gases include sulfur and nitrogen oxides. The PM includes wind-blown yellow sand that often comes to Japan [10] by desertification in the continent. Such particulates have reached California, British Columbia, and the French Alps [11–14]. Today, air pollution PM 2.5 is a universal problem, routinely affecting most of the world, although most obvious in and downwind of industrial areas. PM 2.5 is too small to be filtered by the conventional gauze mask and can deposit materials

In this study, to explore the possible origins of the mortality increase observed among cardiac patients, elemental inflow from polluted air into blood has been investigated by review of Xray fluorescence analyses (XFA) of hair using the ratio of characteristic X-ray peaks to background scatter [4–6]. Since the scatter is a function of the mass of the sample within the beam, the peak-to-scatter ratio (P/S) can be used as a unitless relative concentration of each detected element, independent of hair thickness and shape. With the relationships of serum and hair elements given by the equations in Table 1 previously established [5], we obtained useful

XFA for scalp hair has revealed ion channel gating of hair matrix (HM) cells [4]. By the principle of "inflow-outflow equality," the content of an element in growing hair must be equal to the inflow into the HM cells from blood [4, 5], and the elements admitted through ion channels occur in hair at two distinct levels produced by the closing or opening (gating) of the relevant channels (in vivo). By such channel gating, homeostasis of cellular essential elements can be maintained. Especially, Ca is an overall regulator of ion gating. We had given ten subjects 2-week oral calcium supplementation; the (dried) serum samples showed a single value for Ca [5]. Similarly, no significant variation was seen among the subjects for Sr, Cl, Br, K, S, and P, elements under renal homeostatic control, although in nonsupplemented subjects, considerable variation is usual [4–6]. Calcium is the central player for the homeostatic control of the elements, and usually, therefore, store-operated Ca channels are activated to maintain the normal intracellular [Ca]. Thus, hair elements constitute a record of the combination of serum elements and ion channel function.

The store-operated Ca channels on the plasma membrane are understood to open when Ca2+ stores are depleted at the endoplasmic reticulum (ER) [16–18]. "Store-operated ion channels" adjust the intracellular Ca ion level toward the normal, and an XFA scan along hair shows that the normal [Ca]HC = 10 was maintained despite the ongoing channel gating revealed by the noted fluctuation in [Sr]H [6]. Therefore, the store-operated channels gate due to shortage of Ca stored in serum protein [5, 6], corresponding to hair Ca less than the normal ([Ca]HC < 10 by Eq. (9) in Table 1).

However, when the serum Ca deficiency further proceeds beyond the range covered by the store-operated Ca entry (SOCE), the deficiency increases the secretion of parathyroid hormone (PTH), which causes PTH-activated Ca channel gating. Such channel activity is due to membrane voltage and propagates from channel to channel, resulting in the upper range [Ca]H > 10 and reaching the maximum [Ca]HO = 50 and [Sr]HO = 200 [Eqs. (10) and (12) in Table 1]. Ca oral supplementation immediately transits this highest level into the normal or less than normal [Ca]HC≦10 and [Sr]HC≦10; PTH regulates the Ca channel(s) of HM cells [4–6]. As a typical example, de Groot et al. [19] elucidated the epithelial Ca channel TRPV-5 activated by PTH, which is responsible for Ca reabsorption in renal tubule [20]. Today, for utilizing the channel on-off action for biosensing applications, artificial cell membrane systems have been widely investigated [21].

Thus, calcium has a very high essentiality and homeostasis. The serum standard values for subjects with Ca supplementation are given in Table 1. The hair standards were calculated from the serum standards and confirmed by the P/S FXA [5, 6]. For this work, Ca may be treated as the major regulator of ion channels and may react to a change in serum Ca level in just 15–20 min [22].

Because sulfur-containing amino acids, methionine and cysteine are components of almost all protein molecules, sulfur has also high essentiality and homeostasis and is contained as the ionic sulfur species (SO4 <sup>2</sup>) in serum [23, 24]. Its relation between serum and hair is similar to that of Ca as seen from Eqs. (7)–(10) in Table 1, although the sulfate ion channels have not been reported to the knowledge of the authors [5].

We have observed elemental levels in the hair of thousands of people from 2003 to the present day. Samples after 2009 show significant changes in hair elements, and today the intrinsic hair element levels cannot be seen any more. However, the homeostasis of Ca and S is maintained even with their great inflow into serum from the polluted air at the sacrifice of other important elements responsible for heart activity, resulting in the increased mortality in myocardial infarction cases up to 7 days following short-term pollution [25–27].

3. Concentration relations between serum and hair

whether the ion channels for element X are gating or closing;

into the hair.

standard levels. Female subject age: 62.

Serum contains water at 90–92% and Ca at 8.5–10.2 mg/dL; these Ca values correspond to 92 and 90% water contents, respectively, with negligible [Ca] variations in dried serum. Dried serum is calculated to have Ca at 1 mg/g. Hair can be regarded as serum protein containing mineral elements. For example, Cu exists mainly not as free ions, only bound to serum protein, and the reported serum [Cu]S of 17 μmol/L [28] becomes dried serum concentration of 207 μmol/kg, which is in good agreement with the reported hair Cu concentration of 190–200 μmol/kg. Therefore, any element in serum protein has about the same concentration in hair, if no ions flow

Figure 1. Typical Ca deficiency observed along a single hair strand by scanning 0.05-mm-width XFA. Transitions of DE type (PTH-regulated Ca entry), DA-EE type (Ca channel closing with a low [K]H), and DA type (store-operated Ca entry, SOCE) can be seen with the [Ca]H variation due to the Ca channel gating, resulting in synchronized variations for [Cu]H, [Ti]H, [K]H, [Cl]H, and [Br]H. In the SOCE region (DA type), [Ti]H and [Sr]H are still high, and other elements have the

In most cases, the total concentration of element X in dried serum, [X]S, is the sum of

[X]I and [X]P, the concentrations of ionic and protein-bound X, respectively. By the principle of the inflow-outflow equality, X's hair concentrations [X]H can occur in two levels depending on

½ � X <sup>S</sup> ¼ ½ � X <sup>I</sup> þ ½ � X <sup>P</sup> (1)

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 49

## 2. Samples and method

All the hair and blood samples were collected after informed consent was obtained in accordance with the ethical standards of Hyogo College of Medicine.

X-ray fluorescence analysis (XFA) of the hair samples was carried out using synchrotron radiation at the Photon Factory's (PF) BL4A beamline in the same way as previously [4–6]: excitation energy was 17.4 keV (0.71 Å), the hair was suspended into the X-ray beam in vacuum, and fluorescence detected using a lithium-drifted silicon detector. Because almost all our subjects dye their hair, to avoid hair contamination, only dye-free fresh hair root (length about 1 mm) was used for single-point evaluation. Datum was typically collected for a minute in the range of 0.5–17.5 keV, and the fluorescence peaks evaluated on the log-scale plot by height, all k-alphas except the Ca K-beta peak at 4.0 keV used to avoid interferences. Since hair grows at about 1 cm per month, each data point corresponds to about 3 days of hair growth. The variation of concentrations with time in Figure 1 was observed by scanning along hair strands using a beam width of 0.05 mm.

Figure 1. Typical Ca deficiency observed along a single hair strand by scanning 0.05-mm-width XFA. Transitions of DE type (PTH-regulated Ca entry), DA-EE type (Ca channel closing with a low [K]H), and DA type (store-operated Ca entry, SOCE) can be seen with the [Ca]H variation due to the Ca channel gating, resulting in synchronized variations for [Cu]H, [Ti]H, [K]H, [Cl]H, and [Br]H. In the SOCE region (DA type), [Ti]H and [Sr]H are still high, and other elements have the standard levels. Female subject age: 62.

## 3. Concentration relations between serum and hair

and reaching the maximum [Ca]HO = 50 and [Sr]HO = 200 [Eqs. (10) and (12) in Table 1]. Ca oral supplementation immediately transits this highest level into the normal or less than normal [Ca]HC≦10 and [Sr]HC≦10; PTH regulates the Ca channel(s) of HM cells [4–6]. As a typical example, de Groot et al. [19] elucidated the epithelial Ca channel TRPV-5 activated by PTH, which is responsible for Ca reabsorption in renal tubule [20]. Today, for utilizing the channel on-off action for biosensing applications, artificial cell membrane systems have been widely

Thus, calcium has a very high essentiality and homeostasis. The serum standard values for subjects with Ca supplementation are given in Table 1. The hair standards were calculated from the serum standards and confirmed by the P/S FXA [5, 6]. For this work, Ca may be treated as the major regulator of ion channels and may react to a change in serum Ca level in

Because sulfur-containing amino acids, methionine and cysteine are components of almost all protein molecules, sulfur has also high essentiality and homeostasis and is contained as the

that of Ca as seen from Eqs. (7)–(10) in Table 1, although the sulfate ion channels have not been

We have observed elemental levels in the hair of thousands of people from 2003 to the present day. Samples after 2009 show significant changes in hair elements, and today the intrinsic hair element levels cannot be seen any more. However, the homeostasis of Ca and S is maintained even with their great inflow into serum from the polluted air at the sacrifice of other important elements responsible for heart activity, resulting in the increased mortality in myocardial

All the hair and blood samples were collected after informed consent was obtained in accor-

X-ray fluorescence analysis (XFA) of the hair samples was carried out using synchrotron radiation at the Photon Factory's (PF) BL4A beamline in the same way as previously [4–6]: excitation energy was 17.4 keV (0.71 Å), the hair was suspended into the X-ray beam in vacuum, and fluorescence detected using a lithium-drifted silicon detector. Because almost all our subjects dye their hair, to avoid hair contamination, only dye-free fresh hair root (length about 1 mm) was used for single-point evaluation. Datum was typically collected for a minute in the range of 0.5–17.5 keV, and the fluorescence peaks evaluated on the log-scale plot by height, all k-alphas except the Ca K-beta peak at 4.0 keV used to avoid interferences. Since hair grows at about 1 cm per month, each data point corresponds to about 3 days of hair growth. The variation of concentrations with time in Figure 1 was observed by scanning along hair

<sup>2</sup>) in serum [23, 24]. Its relation between serum and hair is similar to

investigated [21].

48 Trace Elements - Human Health and Environment

just 15–20 min [22].

ionic sulfur species (SO4

2. Samples and method

strands using a beam width of 0.05 mm.

reported to the knowledge of the authors [5].

infarction cases up to 7 days following short-term pollution [25–27].

dance with the ethical standards of Hyogo College of Medicine.

Serum contains water at 90–92% and Ca at 8.5–10.2 mg/dL; these Ca values correspond to 92 and 90% water contents, respectively, with negligible [Ca] variations in dried serum. Dried serum is calculated to have Ca at 1 mg/g. Hair can be regarded as serum protein containing mineral elements. For example, Cu exists mainly not as free ions, only bound to serum protein, and the reported serum [Cu]S of 17 μmol/L [28] becomes dried serum concentration of 207 μmol/kg, which is in good agreement with the reported hair Cu concentration of 190–200 μmol/kg. Therefore, any element in serum protein has about the same concentration in hair, if no ions flow into the hair.

In most cases, the total concentration of element X in dried serum, [X]S, is the sum of

$$[\mathbf{X}]\_{\rm S} = [\mathbf{X}]\_{\rm I} + [\mathbf{X}]\_{\rm P} \tag{1}$$

[X]I and [X]P, the concentrations of ionic and protein-bound X, respectively. By the principle of the inflow-outflow equality, X's hair concentrations [X]H can occur in two levels depending on whether the ion channels for element X are gating or closing;

[X]HO = hair concentration in the case of the gating (open) ion channels.

[X]HC = hair concentration in the case of the closing ion channels.

For specimen mass M within the excitation X-ray beam in XFA, the peak height P for element X must be P = kM[X], with k, a proportional constant, so log[X] = logP – logkM. Logarithmic spectra for thick and thin specimens having the same concentration can be superimposed by moving one to the other vertically, that is, logkM = logS, and.

$$
\log\left[\mathbf{X}\right] = \log P - \log \mathbf{S} = \log\left(P/\mathbf{S}\right),
\tag{2}
$$

ion collision probability, {[Ca]CI}

mental results (see Figure 1).

½Ca�

[Ca]P in steady-state hair growth).

<sup>S</sup> ¼ ½Ca�

<sup>I</sup> þ ½Ca�

<sup>H</sup> ¼ ½Ca�

½Ca�

the ranges of [Ca]H≦10 and [Ca]H > 10, respectively.

can express as.

gating.

[Ca]C; the rates are equal, 2r[Ca]H = q{[Ca]CI}

[Ca]HO = 50. Therefore, q = r and [Ca]HO = (1/2)[Ca]I

previously, that is, [Sr]CI = [Sr]CP. Similarly, we have

2

½Ca�

½Sr�

atoms in the protein is proportional to twice the [Ca]H. [Ca]H is in equilibrium with cell

When the channel gating frequency is high enough to result in [Ca]CI = [Ca]I (=10), we have

<sup>H</sup> ¼ ð1=2Þ½Ca�

Sr is assumed to be distributed equally as ion and protein-bound atom in HM cells, as established

According to the definition [X]H = P/S by Eq. (2), the congener proportionality is expressed as [Ca]CI = [Sr]CI = [Sr]CP, and the Sr/Ca ratio becomes [Sr]H/[Ca]H = 4. This agrees with experi-

As seen above, when Ca channel gating takes place, the [Sr]H/[Ca]H = 4. For Ca channel closing, only the Ca and Sr on serum protein are incorporated into HM cells, resulting in [Sr]H = [Ca]H = 10 by Eqs. (9) and (11), [Sr]H/[Ca]H = 1. Thus, in this work, [Sr]H is a useful marker for channel

Here, we first consider the hair elements without pollution. For dried serum from Ca-supplemented five male and five female subjects, a single concentration value for each element in Table 1 was found. Ca appears to be the central and overriding player in the regulation of various elements; by ensuring normal serum Ca with a Ca supplement, all other elements

<sup>P</sup> ¼ ð1=2Þ ½Ca�

with closed Ca channels (only serum protein can enter the HM cells, so [Ca]H must be equal to

Unfortunately, almost all humans have "ordinary deficiencies" in Ca in varying degrees [30, 31]. Calcium balance is maintained in kidney; Ca is freely filtered by the glomerulus, and the quantity of Ca filtered each day of over 10 g is far greater than the content of the entire ECF compartment [20]. Therefore, ~98% of the filtered Ca must be reabsorbed along the renal tubule. Approximately 70% of filtered Ca is reabsorbed passively in the proximal tubule. The remaining reabsorption is controlled by the Ca-sensor, CaSR, on the basolateral membrane of the tubular cells. Consequently, we have a tendency to fall into Ca deficiency. The ranges of [Ca]H < 10 and [Ca]H > 10 are due to Ca deficiency with deviations toward acidosis [Cl]H > 10 and alkalosis [Cl]H < 10, respectively. Two kinds of known Ca channels account for the results seen in hair analysis; the store-operated channel and PTH-operated channel are activated in

under renal control (Sr, Cl, Br, K, S, P) become normal [5]. We have the Ca standard

<sup>P</sup> ¼ 20 and ½Ca�

<sup>H</sup> ¼ ð1=2Þf½Sr�

2

CI 2 :

CIþ½Sr�

<sup>P</sup> ¼ ½Ca�

<sup>S</sup> ¼ 10 ðHairÞ

<sup>I</sup> ¼ 10 ðDried serumÞ

CPg<sup>2</sup> :

2

, in the HM cell. The dissociation rate of two Ca pair

, where r and q are the proportional constants.

. For a channel gating frequency, we

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 51

independently of the specimen thickness, where S is the background due to X-rays scattered by the specimen. Eq. (2) is valid even for thick specimens because the X-ray absorption occurs for P and S in the same way. (The hair was suspended in the X-ray beam in vacuum, so there is no other mass to contribute background scatter.) Using this definition for [X], the relations between [X]H and [X]S obtained in the previous paper [5] are listed in Table 1 (also, see Figure 3).

As seen from the foregoing argument, the background S is due to serum (hair) protein (nonprotein solids: 3–4%). According to the Gamble [23] gram, the ionic element species are controlled to maintain electric neutrality with [Protein�] in serum. Then, the ratio of the number of X atoms to the number of the protein molecules within the excitation X-ray beam in XFA, which is proportional to [X] = P/S, is under homeostatic control. If congener elements, X and Z, have a proportionality, we have [X] = [Z] by the definition Eq. (2), as [Cl]H = [Br]H in Table 1. The concentration by Eq. (2) can be considered to use the standard value of each element as a unit and results in a new concentration system to show the relationship of elements in dried serum, cell, and hair.

For an element Z such as Cu, Fe, and Zn being protein-bound and having no ion in serum, the value log[Z]H = [logP – logS]H is normalized for comparing their concentrations by.

$$\log \left[ Z \right]\_{\rm H} = \left[ \log P - \log S \right]\_{\rm H} / \left( \left[ \log P - \log S \right]\_{\rm H} \right)\_{\rm st} \tag{3}$$

using the standard ([logP – logS]H)st for hair. The standard values for the Ca-supplemented healthy subjects are [Cu]H = 20, [Fe]H = 15 and [Zn]H = 400 (Table 1).

By the principle of "inflow-outflow equality," hair composition reveals ion channel closing and opening. There may be many kinds of ion channels and receptors, some yet unknown. Though sulfate ion channels have not been reported, we observed upper [S]HO and lower [S]HC levels in hair, suggesting the existence of S-related ion channels [5] (designated as S or SO4 <sup>2</sup>� channels).

Although channel gating produces a short pulsed inflow into the cell each time, the messenger protein binds with one of the associated receptors; such gating can occur so frequently as to bring the intracellular ion concentration [X]CI to serum ion concentration [X]I. Since the [X]CI is kept at the serum [X]I by the channels' flow, [X]HO is determined by [X]I (see Table 1).

In HM cells, Ca can enter primarily as ionic ([Ca]CI) with few protein-bound atoms ([Ca]CP = 0). The cellular Ca concentration [Ca]C is equal to cell ion concentration [Ca]CI, that is, [Ca]C = [Ca]CI. Ca is incorporated as a pair of atoms into the hair protein molecules formed in the HM cells [29]. The reaction rate of the pair formation is proportional to the ion collision probability, {[Ca]CI} 2 , in the HM cell. The dissociation rate of two Ca pair atoms in the protein is proportional to twice the [Ca]H. [Ca]H is in equilibrium with cell [Ca]C; the rates are equal, 2r[Ca]H = q{[Ca]CI} 2 , where r and q are the proportional constants. When the channel gating frequency is high enough to result in [Ca]CI = [Ca]I (=10), we have [Ca]HO = 50. Therefore, q = r and [Ca]HO = (1/2)[Ca]I 2 . For a channel gating frequency, we can express as.

[X]HO = hair concentration in the case of the gating (open) ion channels.

For specimen mass M within the excitation X-ray beam in XFA, the peak height P for element X must be P = kM[X], with k, a proportional constant, so log[X] = logP – logkM. Logarithmic spectra for thick and thin specimens having the same concentration can be superimposed by

independently of the specimen thickness, where S is the background due to X-rays scattered by the specimen. Eq. (2) is valid even for thick specimens because the X-ray absorption occurs for P and S in the same way. (The hair was suspended in the X-ray beam in vacuum, so there is no other mass to contribute background scatter.) Using this definition for [X], the relations between

As seen from the foregoing argument, the background S is due to serum (hair) protein (nonprotein solids: 3–4%). According to the Gamble [23] gram, the ionic element species are controlled to maintain electric neutrality with [Protein�] in serum. Then, the ratio of the number of X atoms to the number of the protein molecules within the excitation X-ray beam in XFA, which is proportional to [X] = P/S, is under homeostatic control. If congener elements, X and Z, have a proportionality, we have [X] = [Z] by the definition Eq. (2), as [Cl]H = [Br]H in Table 1. The concentration by Eq. (2) can be considered to use the standard value of each element as a unit and results in a new concentration system to show the relationship of elements in dried serum, cell, and hair.

For an element Z such as Cu, Fe, and Zn being protein-bound and having no ion in serum, the

log Z½ �<sup>H</sup> ¼ ½ � log P – log S <sup>H</sup>= ½ � log P – log S <sup>H</sup>

using the standard ([logP – logS]H)st for hair. The standard values for the Ca-supplemented

By the principle of "inflow-outflow equality," hair composition reveals ion channel closing and opening. There may be many kinds of ion channels and receptors, some yet unknown. Though sulfate ion channels have not been reported, we observed upper [S]HO and lower [S]HC levels in

Although channel gating produces a short pulsed inflow into the cell each time, the messenger protein binds with one of the associated receptors; such gating can occur so frequently as to bring the intracellular ion concentration [X]CI to serum ion concentration [X]I. Since the [X]CI is

In HM cells, Ca can enter primarily as ionic ([Ca]CI) with few protein-bound atoms ([Ca]CP = 0). The cellular Ca concentration [Ca]C is equal to cell ion concentration [Ca]CI, that is, [Ca]C = [Ca]CI. Ca is incorporated as a pair of atoms into the hair protein molecules formed in the HM cells [29]. The reaction rate of the pair formation is proportional to the

value log[Z]H = [logP – logS]H is normalized for comparing their concentrations by.

hair, suggesting the existence of S-related ion channels [5] (designated as S or SO4

kept at the serum [X]I by the channels' flow, [X]HO is determined by [X]I (see Table 1).

healthy subjects are [Cu]H = 20, [Fe]H = 15 and [Zn]H = 400 (Table 1).

[X]H and [X]S obtained in the previous paper [5] are listed in Table 1 (also, see Figure 3).

log X½ �¼ log P – log S ¼ log ð Þ P=S , (2)

st (3)

<sup>2</sup>� channels).

[X]HC = hair concentration in the case of the closing ion channels.

50 Trace Elements - Human Health and Environment

moving one to the other vertically, that is, logkM = logS, and.

$$[\mathbf{C}\mathbf{a}]\_{\mathbf{H}} = (1/2)[\mathbf{C}\mathbf{a}]\_{\mathbf{C}\mathbf{l}}{}^2.$$

Sr is assumed to be distributed equally as ion and protein-bound atom in HM cells, as established previously, that is, [Sr]CI = [Sr]CP. Similarly, we have

$$\left[\mathbf{S}\mathbf{r}\right]\_{\mathrm{H}} = \left(1/2\right)\left\{\left[\mathbf{S}\mathbf{r}\right]\_{\mathrm{CI}} + \left[\mathbf{S}\mathbf{r}\right]\_{\mathrm{CP}}\right\}^2.$$

According to the definition [X]H = P/S by Eq. (2), the congener proportionality is expressed as [Ca]CI = [Sr]CI = [Sr]CP, and the Sr/Ca ratio becomes [Sr]H/[Ca]H = 4. This agrees with experimental results (see Figure 1).

As seen above, when Ca channel gating takes place, the [Sr]H/[Ca]H = 4. For Ca channel closing, only the Ca and Sr on serum protein are incorporated into HM cells, resulting in [Sr]H = [Ca]H = 10 by Eqs. (9) and (11), [Sr]H/[Ca]H = 1. Thus, in this work, [Sr]H is a useful marker for channel gating.

Here, we first consider the hair elements without pollution. For dried serum from Ca-supplemented five male and five female subjects, a single concentration value for each element in Table 1 was found. Ca appears to be the central and overriding player in the regulation of various elements; by ensuring normal serum Ca with a Ca supplement, all other elements under renal control (Sr, Cl, Br, K, S, P) become normal [5]. We have the Ca standard

$$[\mathbf{[Ca]}\_{\mathrm{S}} = [\mathbf{Ca}]\_{\mathrm{I}} + [\mathbf{Ca}]\_{\mathrm{P}} = 20 \text{ and } [\mathbf{Ca}]\_{\mathrm{P}} = [\mathbf{Ca}]\_{\mathrm{I}} = 10 \text{ (Dried serum)}]$$

$$[\mathbf{[Ca]}\_{\mathrm{H}} = [\mathbf{Ca}]\_{\mathrm{P}} = (1/2) \ [\mathbf{Ca}]\_{\mathrm{S}} = 10 \text{ (Hair)}$$

with closed Ca channels (only serum protein can enter the HM cells, so [Ca]H must be equal to [Ca]P in steady-state hair growth).

Unfortunately, almost all humans have "ordinary deficiencies" in Ca in varying degrees [30, 31]. Calcium balance is maintained in kidney; Ca is freely filtered by the glomerulus, and the quantity of Ca filtered each day of over 10 g is far greater than the content of the entire ECF compartment [20]. Therefore, ~98% of the filtered Ca must be reabsorbed along the renal tubule. Approximately 70% of filtered Ca is reabsorbed passively in the proximal tubule. The remaining reabsorption is controlled by the Ca-sensor, CaSR, on the basolateral membrane of the tubular cells. Consequently, we have a tendency to fall into Ca deficiency. The ranges of [Ca]H < 10 and [Ca]H > 10 are due to Ca deficiency with deviations toward acidosis [Cl]H > 10 and alkalosis [Cl]H < 10, respectively. Two kinds of known Ca channels account for the results seen in hair analysis; the store-operated channel and PTH-operated channel are activated in the ranges of [Ca]H≦10 and [Ca]H > 10, respectively.

decreases to the low-level [Ca]H = 10 standard, with [Sr]H/[Ca]H = 1 due to Ca channel closing, for about 2 months. Furthermore, the standard [Ca]H = 10 is maintained for about 1.5 months by gating the store-operated Ca channels (SOCE) with [Sr]H/[Ca]H~2; [Ti]H is as high as [Ti]H~10, with open Cl channels [Cl]H = [Br]H = 22 (acidosis: DA type). At the follicle (root), the concentra-

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 53

The observed process can be divided into three regions by the abrupt Sr change; the regions labeled "SOCE," DA-EE type with Ca channel closing, and "PTH regulated Ca entry." The store-operated Ca entry (SOCE) channel system is for maintaining the intracellular Ca at the normal by channel gating when the depletion of Ca storage in the cell occurs. For hair, therefore, the store-operated Ca channels must work for [Ca]H≦10 so as to keep the normal [Ca]H = 10, as reported earlier [6]. In contrast, the "PTH-regulated Ca entry" region indicates greater inflow of Ca and Sr through the gating Ca channels; the electrical signal initiated by the activation of the receptor PTH1R propagates rapidly over the surface of the cell due to the opening of other ion channels that are sensitive to the voltage change caused by the initial channel opening, as observed for renal epithelial Ca channel (TRPV5) as a typical example by de Groot et al [19]. For the hair Ca level, the channel gating frequency depends on serum [PTH] and causes the downward deviations from [Ca]H = 50 and [Sr]H = 200. Based on such results, the deduced type of Ca channel is referred to as "PTH-regulated Ca channels." Typical XFA spectra in Figure 2(a) show Ca channel closing and opening (gating) accompanied by levels

tions become singular values, which will be reported elsewhere.

<sup>2</sup>), S (SO4

high-level [Ca]H > 10 due to the Ca2+ inflow into HM cells.

result in a high [K]H and a low [S]H ([5]. Also, see Section 5.2.)

<sup>2</sup>) and K<sup>+</sup>

the Ca patterns typical in the region before PM pollution was notable.

.

By taking into account, both the PTH-regulated and store-operated Ca channels, Ca deficiency can be classified into five distinct types, as summarized in Table 2. These five types describe

DE type has Ca deficiency for excitation of the PTH-regulated Ca channel gating resulting in a

The Ca2+ and Sr2+ inflow through the gating channels may become toxic [30, 31], resulting in abnormally high [Cu]H and/or [Ti]H and the inequality [Cl]H < [Br]H (disordered chloride shift), which can be taken as the deterioration of hepatocytes and erythrocytes, respectively. In Figure 1, when the PTH-regulated Ca channels are closed, [Cu]H and [Cl]H become normal for the EE-type ([Cu]H = 10, [Cl]H = 10). In the store-operated SOCE region, the [Ti]H is high, that is, [Ti]H~10 (for normal [Ti]H ~1), suggesting deterioration of Ti excretion as a consequence

When the gating accumulates [Ca2+] and [Sr2+] to ionic serum levels in HM cells, that is, [Ca]CI = [Ca]I = 10 and [Sr]CI = [Sr]I = 10, we have the maximum [Ca]HO = 50 and [Sr]HO = 200 in Table 1. An 8-month lasting [Ca]HO = 50 was observed for chronic Ca deficiency [6], as referred to as LD type (long-term Ca deficiency). "LD type" often causes fatty liver [31].

DO type (Ca deficiency with Overplus in serum) has Ca channels closed in [Ca]H > 10 by an increase of serum [Ca2+] with bone resorption [22, 30, 31], which results in serum alkalosis accompanied by a high [Ca]P (=[Ca]H > 10 for closed Ca channels). Consequently, we have [Ca]H = [Sr]H > 10 with [Cl]H = [Br]H < 10. In this case, the serum [PTH] becomes so low as to

changes for P (HPO4

of Sr inflow into hepatocytes.

Figure 2. X-ray fluorescence spectra of single hair samples. (a) With ion channel closing and gating of Ca, K and S (SO4 <sup>2</sup>) in the hair matrix cells without pollution. Compare the peak heights with the levels listed in Table 1. (b) Typical example of hair with the pollution (F150 in Figure 5) showing a very high Ti peak and clearly recognized Na peak [Compare with (a)]. The [Ca] is lower than the normal, with store-operated Ca channel gating. The k-alpha peaks are labeled (see Section 2) (16.5–17.5 keV omitted to preserve useful scale).

Figure 1 gives a typical example showing the effects of both the store-operated and PTHregulated Ca channels. The single strand from a 62-year-old female was analyzed from root to tip with the 0.05-mm-width excitation beam. As time progresses from right to left, the Ca deficiency is improving; the high-level [Ca]H = 50 ([Sr]H = 200) continues for a long term (12 months) from the tip, with deviations downward, keeping [Sr]H/[Ca]H = 4 and then abruptly decreases to the low-level [Ca]H = 10 standard, with [Sr]H/[Ca]H = 1 due to Ca channel closing, for about 2 months. Furthermore, the standard [Ca]H = 10 is maintained for about 1.5 months by gating the store-operated Ca channels (SOCE) with [Sr]H/[Ca]H~2; [Ti]H is as high as [Ti]H~10, with open Cl channels [Cl]H = [Br]H = 22 (acidosis: DA type). At the follicle (root), the concentrations become singular values, which will be reported elsewhere.

The observed process can be divided into three regions by the abrupt Sr change; the regions labeled "SOCE," DA-EE type with Ca channel closing, and "PTH regulated Ca entry." The store-operated Ca entry (SOCE) channel system is for maintaining the intracellular Ca at the normal by channel gating when the depletion of Ca storage in the cell occurs. For hair, therefore, the store-operated Ca channels must work for [Ca]H≦10 so as to keep the normal [Ca]H = 10, as reported earlier [6]. In contrast, the "PTH-regulated Ca entry" region indicates greater inflow of Ca and Sr through the gating Ca channels; the electrical signal initiated by the activation of the receptor PTH1R propagates rapidly over the surface of the cell due to the opening of other ion channels that are sensitive to the voltage change caused by the initial channel opening, as observed for renal epithelial Ca channel (TRPV5) as a typical example by de Groot et al [19]. For the hair Ca level, the channel gating frequency depends on serum [PTH] and causes the downward deviations from [Ca]H = 50 and [Sr]H = 200. Based on such results, the deduced type of Ca channel is referred to as "PTH-regulated Ca channels." Typical XFA spectra in Figure 2(a) show Ca channel closing and opening (gating) accompanied by levels changes for P (HPO4 <sup>2</sup>), S (SO4 <sup>2</sup>) and K<sup>+</sup> .

By taking into account, both the PTH-regulated and store-operated Ca channels, Ca deficiency can be classified into five distinct types, as summarized in Table 2. These five types describe the Ca patterns typical in the region before PM pollution was notable.

DE type has Ca deficiency for excitation of the PTH-regulated Ca channel gating resulting in a high-level [Ca]H > 10 due to the Ca2+ inflow into HM cells.

The Ca2+ and Sr2+ inflow through the gating channels may become toxic [30, 31], resulting in abnormally high [Cu]H and/or [Ti]H and the inequality [Cl]H < [Br]H (disordered chloride shift), which can be taken as the deterioration of hepatocytes and erythrocytes, respectively. In Figure 1, when the PTH-regulated Ca channels are closed, [Cu]H and [Cl]H become normal for the EE-type ([Cu]H = 10, [Cl]H = 10). In the store-operated SOCE region, the [Ti]H is high, that is, [Ti]H~10 (for normal [Ti]H ~1), suggesting deterioration of Ti excretion as a consequence of Sr inflow into hepatocytes.

When the gating accumulates [Ca2+] and [Sr2+] to ionic serum levels in HM cells, that is, [Ca]CI = [Ca]I = 10 and [Sr]CI = [Sr]I = 10, we have the maximum [Ca]HO = 50 and [Sr]HO = 200 in Table 1. An 8-month lasting [Ca]HO = 50 was observed for chronic Ca deficiency [6], as referred to as LD type (long-term Ca deficiency). "LD type" often causes fatty liver [31].

DO type (Ca deficiency with Overplus in serum) has Ca channels closed in [Ca]H > 10 by an increase of serum [Ca2+] with bone resorption [22, 30, 31], which results in serum alkalosis accompanied by a high [Ca]P (=[Ca]H > 10 for closed Ca channels). Consequently, we have [Ca]H = [Sr]H > 10 with [Cl]H = [Br]H < 10. In this case, the serum [PTH] becomes so low as to result in a high [K]H and a low [S]H ([5]. Also, see Section 5.2.)

Figure 1 gives a typical example showing the effects of both the store-operated and PTHregulated Ca channels. The single strand from a 62-year-old female was analyzed from root to tip with the 0.05-mm-width excitation beam. As time progresses from right to left, the Ca deficiency is improving; the high-level [Ca]H = 50 ([Sr]H = 200) continues for a long term (12 months) from the tip, with deviations downward, keeping [Sr]H/[Ca]H = 4 and then abruptly

Figure 2. X-ray fluorescence spectra of single hair samples. (a) With ion channel closing and gating of Ca, K and S (SO4

2) (16.5–17.5 keV omitted to preserve useful scale).

52 Trace Elements - Human Health and Environment

in the hair matrix cells without pollution. Compare the peak heights with the levels listed in Table 1. (b) Typical example of hair with the pollution (F150 in Figure 5) showing a very high Ti peak and clearly recognized Na peak [Compare with (a)]. The [Ca] is lower than the normal, with store-operated Ca channel gating. The k-alpha peaks are labeled (see Section

<sup>2</sup>)


Table 2. Classification of Ca deficiency observed in hair with Eqs. (2) and (3).

In summary, for [Ca]H > 10, hair Ca is by PTH-regulated Ca channels, DE for gating and DO type for closing.

In [Ca]H < 10, the subjects have store-operated Ca channel gating with [Sr]H/[Ca]H = 4 (DA type). In this case, PTH-regulated channels are closed, and the hair Ca is due to Ca bound on serum protein, that is, [Ca]H = [Ca]P < 10. If the serum [PTH] is still high enough to inhibit the H+

exchangers from excreting H+ in renal proximal convoluted tubular cells [32], serum [H+

increases; this deviation of serum pH toward acidosis ([Cl]H = [Br]H> > 10 "DA type) ionizes the protein-bound Ca to increase serum [Ca2+]. Therefore, Ca in serum protein is a stockpile that the body can tap in cases of Ca deficiency. Oral calcium supplementation shows no immediate

Normally, molecular pumps on cell membrane expel Ca2+ ions (closing the Ca channels) so that the intracellular [Ca2+] is nearly zero. This creates a state for signal Ca2+ inflow through the associated channels. However, when Ca2+ stores are depleted at the endoplasmic reticulum (ER), the store-operated Ca channels open [17, 18, 33], as Cahalan [16] illustrated clearly; STIM proteins in the ER open the store-operated "Orai" Ca2+ channels and inhibit voltage-gated CaV1.2 channels in plasma membrane. Because of the ubiquitous Ca-sensing STIM proteins, this store-operated Ca channel gating may be assumed to be similar to that we observed in the hair [5, 6]. Then, Ca depletion in the ER likely occurs with the depletion of the Ca stored on

By 2-month supplementation of Ca 900 mg/day (3ACa) [34], LD type recovers to DO to DA type; the level of Ca deficiency is in the decreasing order of LD, DE, DO, and DA (see Table 2).

In Japan, notable air pollution had not been frequent until 2009. Figure 3 shows results from hair samples obtained before 2009, and elemental concentrations evaluated for the hair roots of 50 randomly selected subjects of each sex between their 30s and 70s; the labels' Roman

By the criteria listed in Table 2, we can determine the type of each subject in Figure 3 as labeled in Figure 3(b). Examining Figure 3(a) and (b), the high [Cu]H and [Ti]H are parallel with Srindicated gating (10 < [Ca]H ≤ 50 with [Sr]H/[Ca]H = 4), and thus related to opening of PTHregulated Ca channels (DE type) causing deterioration of hepatic Cu and Ti excretion. Similar results can be seen for some DA-type subjects indicated by "LD-DA," which are the DA type with [Ca]H < 10 with [Cl]H> > 10 at present and were LD type in past. This means that recovery from Ca entry takes a long time. There are also many intrinsic DA-type subjects ([Ca]H < 10 with [Cl]H> > 10) having the normal Cu level. Many have [Sr]H/[Ca]H = 4 by the opening of store-

In this way, we obtained the results showing all the subjects with Ca deficiency in varying degrees; 24 out of 50 are DA type, 20 are DO type, and 4 are DE type, 2 are LD-DA type for the male, and 31 out of 50 are DA type, 9 are DO type, and 6 are DE type, 4 are LD-DA type for the female. This means that DA type, the least severe type of Ca deficiency, occurs in half of the subjects, regardless of gender. The second level in severity (and the second step in cases of

effects on [Ca]H < 10 and [Sr]H/[Ca]H = 4 (2-month Ca supplementation is needed).

serum proteins ([Ca]P = [Ca]H < 10 with closed PTH-regulated Ca channels).

4. Hair elements with minor effects of air pollution before 2009

numerals stand for each age period, and the bar graph is in order of age.

operated Ca channels.

/Na+

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373

]

55

At [Ca]H = 10, if the normal state of [Ca]H = [Sr]H = 10 continues steadily, we can call this EE type (Ca ever enough to close Ca channels). Some healthy subjects have been found to keep EE type more than several months.

Usually, however, transitions between open and closed Ca channels occur at [Ca]H = 10, resulting in 1 < [Sr]H/[Ca]H < 4. Scanning the 50-μm width X-ray beam along the hair, in earlier work [6], we observed the [Ca]H maintained at [Ca]H = 10 (normal) during the variation of the [Sr]H by Ca channel gating. Therefore, the observed Ca channel gating is due to some kind of store-operated type to be activated at [Ca]H = [Ca]P≦10. It was concluded that transition between [Ca]I-sensitive (PTH-regulated) and [Ca]P-sensitive (store-operated) channel gating occurs at [Ca]H = 10.

In [Ca]H < 10, the subjects have store-operated Ca channel gating with [Sr]H/[Ca]H = 4 (DA type). In this case, PTH-regulated channels are closed, and the hair Ca is due to Ca bound on serum protein, that is, [Ca]H = [Ca]P < 10. If the serum [PTH] is still high enough to inhibit the H+ /Na+ exchangers from excreting H+ in renal proximal convoluted tubular cells [32], serum [H+ ] increases; this deviation of serum pH toward acidosis ([Cl]H = [Br]H> > 10 "DA type) ionizes the protein-bound Ca to increase serum [Ca2+]. Therefore, Ca in serum protein is a stockpile that the body can tap in cases of Ca deficiency. Oral calcium supplementation shows no immediate effects on [Ca]H < 10 and [Sr]H/[Ca]H = 4 (2-month Ca supplementation is needed).

Normally, molecular pumps on cell membrane expel Ca2+ ions (closing the Ca channels) so that the intracellular [Ca2+] is nearly zero. This creates a state for signal Ca2+ inflow through the associated channels. However, when Ca2+ stores are depleted at the endoplasmic reticulum (ER), the store-operated Ca channels open [17, 18, 33], as Cahalan [16] illustrated clearly; STIM proteins in the ER open the store-operated "Orai" Ca2+ channels and inhibit voltage-gated CaV1.2 channels in plasma membrane. Because of the ubiquitous Ca-sensing STIM proteins, this store-operated Ca channel gating may be assumed to be similar to that we observed in the hair [5, 6]. Then, Ca depletion in the ER likely occurs with the depletion of the Ca stored on serum proteins ([Ca]P = [Ca]H < 10 with closed PTH-regulated Ca channels).

By 2-month supplementation of Ca 900 mg/day (3ACa) [34], LD type recovers to DO to DA type; the level of Ca deficiency is in the decreasing order of LD, DE, DO, and DA (see Table 2).

## 4. Hair elements with minor effects of air pollution before 2009

In summary, for [Ca]H > 10, hair Ca is by PTH-regulated Ca channels, DE for gating and DO


At [Ca]H = 10, if the normal state of [Ca]H = [Sr]H = 10 continues steadily, we can call this EE type (Ca ever enough to close Ca channels). Some healthy subjects have been found to keep EE

Usually, however, transitions between open and closed Ca channels occur at [Ca]H = 10, resulting in 1 < [Sr]H/[Ca]H < 4. Scanning the 50-μm width X-ray beam along the hair, in earlier work [6], we observed the [Ca]H maintained at [Ca]H = 10 (normal) during the variation of the [Sr]H by Ca channel gating. Therefore, the observed Ca channel gating is due to some kind of store-operated type to be activated at [Ca]H = [Ca]P≦10. It was concluded that transition between [Ca]I-sensitive (PTH-regulated) and [Ca]P-sensitive (store-operated) channel gating

type for closing.

PTH inhibits H+

high [H<sup>+</sup>

low [SO4

occurs at [Ca]H = 10.

type more than several months.

LD type: Long-term continuation of DE type due to chronic Ca deficiency.

[Cu]H > 10 and/or high [Ti]H by deteriorated metal excretion in hepatocytes. [Cl]H > [Br]H or [Cl]H < [Br]H by deteriorated chloride shift in erythrocytes

/H<sup>+</sup> exchange in cells, and [K]H≲10.

<sup>2</sup>] in serum, [S]H = 200 by gating sulfate ion channels Eq. (8).

DO type: PTH-regulated Ca channel closing with bone resorption by Ca deficiency. [Ca]H = [Sr]H≳10 with [Cl]H = ([Br]H) < 10 (Alkalosis), [K]H = 200, [S]H = 20. Ca in serum protein increases with the alkalosis ([Ca]H = [Ca]P).

EE type: Ever closing of Ca channels by Ca sufficient "Ever Enough".(No deficiency).

DA type: PTH-regulated Ca channel closing and Store-operated channel gating. [Ca]H = 10 with 1 < [Sr]H/[Ca]H≦4 and [Ca]H < 10 with [Sr]H/[Ca]H = 4.

Table 2. Classification of Ca deficiency observed in hair with Eqs. (2) and (3).

/H+ exchange in cells, and [K]H≲10.

<sup>2</sup>] in serum, [S]H = 200 by gating sulfate ion channels Eq. (8).


DE type: PTH-regulated Ca channel gating. 10 < [Ca]H ≤ 50 with [Sr]H/[Ca]H = 4.

54 Trace Elements - Human Health and Environment

/Na<sup>+</sup>

] in serum, K+

PTH inhibits H+

high [H<sup>+</sup>

low [SO4

Ca (Sr) inflow into cells deteriorates their functions:

Serum: [Ca]I≲10 and [Ca]P≲10. High [PTH]. Acidosis.

Serum: [Ca]I > 10 and [Ca]P > 10. [PTH]~0. Highly alkalosis.

Serum: [Ca]I = 10 and [Ca]P = 10. [PTH]~0. Slightly alkalosis.


([Cu]H = 10, [Cl]H > 10, [Ti]H: high)

Serum: [Ca]I≲10 and [Ca]P≲10. Mean [PTH]. Acidosis.

/Na<sup>+</sup>

] in serum, K+

[Ca]H = [Sr]H = 10, [Cl]H = ([Br]H)≲10, [K]H = 200, [S]H = 20, [P]H≳10.


In Japan, notable air pollution had not been frequent until 2009. Figure 3 shows results from hair samples obtained before 2009, and elemental concentrations evaluated for the hair roots of 50 randomly selected subjects of each sex between their 30s and 70s; the labels' Roman numerals stand for each age period, and the bar graph is in order of age.

By the criteria listed in Table 2, we can determine the type of each subject in Figure 3 as labeled in Figure 3(b). Examining Figure 3(a) and (b), the high [Cu]H and [Ti]H are parallel with Srindicated gating (10 < [Ca]H ≤ 50 with [Sr]H/[Ca]H = 4), and thus related to opening of PTHregulated Ca channels (DE type) causing deterioration of hepatic Cu and Ti excretion. Similar results can be seen for some DA-type subjects indicated by "LD-DA," which are the DA type with [Ca]H < 10 with [Cl]H> > 10 at present and were LD type in past. This means that recovery from Ca entry takes a long time. There are also many intrinsic DA-type subjects ([Ca]H < 10 with [Cl]H> > 10) having the normal Cu level. Many have [Sr]H/[Ca]H = 4 by the opening of storeoperated Ca channels.

In this way, we obtained the results showing all the subjects with Ca deficiency in varying degrees; 24 out of 50 are DA type, 20 are DO type, and 4 are DE type, 2 are LD-DA type for the male, and 31 out of 50 are DA type, 9 are DO type, and 6 are DE type, 4 are LD-DA type for the female. This means that DA type, the least severe type of Ca deficiency, occurs in half of the subjects, regardless of gender. The second level in severity (and the second step in cases of

In Figure 3(d), almost all the females in our studies have the maximum [S]H = 200 (SO4

can be achieved by pumping K+ into the HM cells with Na<sup>+</sup>

around the standard; [Zn]H is consistent with [Zn]H = [Zn]S

5. Air pollution observed by hair analysis in February 2011

Cl, Br, P, K, S, Cu, and Zn changed their levels in hair from those in Figure 3.

Ca contained in the polluted air, probably as calcium silicate, sulfate and carbonate, is breathed into the lung and must increase [Ca2+] in serum. As seen in Figures 4–6(b), all the subjects have [Ca]H levels normal or less than the normal, that is, [Ca]H≲10, except the subject labeled "F073" having parathyroid gland dysfunction. This means that PTH-regulated Ca channels are closed with the air pollution. Comparison with Figure 3(b) indicates that breathing the

5.1. Store-operated Ca channel gating induced by the pollution

hair protein also exists as a pair of atoms) [5].

desert [35].

channel opening) due to Ca deficiency, and the normal [S]H = 20 cannot be seen. For the males, [S]H values are slightly lower than the maximum, the two subjects MV-8 and MVII-2 (DO type) have the standard [S]H = 20, and 10 subjects have the normal [K]H = 200 (in DO type), which

Figure 3(e) shows hair [Zn]H and [Fe]H for the same subjects. Zinc values are well regulated

serum protein are incorporated into the hair protein in pairs, in the same manner as Ca (Ca in

The effect of air pollution on hair elements in Japan is discernible from 2009 onward. Especially, from February to March 2011, very high contents of Ti in hair were observed, as seen in Figures 4–6, corresponding to a period of yellow haze, and indicating PM from the mainland

Figures 4 and 5 are from female subjects under 60 years old and above, respectively. Figure 6 is from male subjects between 23 and 83. The bar graphs are in order of age. Almost all hair samples were taken in February 2011. Three-orders of magnitude higher [Ti]H can be seen. Such high levels of [Ti]H were also observed for hair samples obtained in Tokyo area in this period. All the people in the area must have breathed the polluted air containing Ti. However, about 80% of the female subjects younger than 60 (Figure 4) have [Ti]H > 10, and less than 30% of the male subjects showed this effect. Figures 4–6(c) show the [Cl]H and [Br]H for the same subjects. The correspondence between the high [Ti]H and high [Cl]H (and [Br]H) is clear. The high [Ti]H should be attributed to the deterioration of the liver's function to excrete Ti into bile caused by Sr2+ inflow into hepatocytes. The [Cl]H abnormality can be attributed to Ca2+ inflow (in past) into erythrocytes through open Ca2+ channels (a PTH effect) ultimately caused by Ca deficiency. Recovery of Ca levels requires months of oral supplementation. Therefore, the observed [Ti]H and [Cl]H abnormalities mean that the affected subjects were LD type in past even if Ca is sufficient when the hair was taken. The observed difference between the sexes is due to most of the male subjects being DO type. Although all the subjects breathed the polluted air, the pollution effect depends on the Ca-deficiency history of the individuals. However, all elements Ca, Sr,

/K<sup>+</sup>

2


. This implies that Zn atoms on the

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373

2

57

Figure 3. Hair concentrations observed for 50 randomly selected male and female subjects aged between 30 and 70. The hair samples were obtained before 2009 and show little or minor effects of air pollution. The bars graphed are in order of age and the Roman numerals in the subject labels stand for age period. (a) [Cu]H and [Ti]H. The essential element Cu shows most values close to the homeostatic standard at [Cu]H = 10 by Eq. (3), while Ti has a variety in [Ti]H by Eq. (2). (b) [Ca]H and [Sr]H having two separate standard levels with close and open Ca channels, [Ca]H = 10 vs. [Ca]H = 50 and [Sr]H = 10 vs. [Sr]H = 200, respectively. (c) [Cl]H and [Br]H having two separate standard levels at [Cl]H = [Br]H = 10 vs. [Cl]H = [Br]H = 22. (d) [K]H with the normal [K]H = 200 and [S]H having two separate standard levels, [S]H = 20 vs. [S]H = 200. (e) [Zn]H and [Fe]H showing values close to the standard at [Zn]H = 10 and [Fe]H = 10 normalized by Eq. (3).

increasing deficiency), DO type, is more common for the male, and LD type is more common for the female, that is, the male appears to tolerate Ca deficiency by bone resorption, and the female by Ca channel gating.

In Figure 3(d), almost all the females in our studies have the maximum [S]H = 200 (SO4 2 channel opening) due to Ca deficiency, and the normal [S]H = 20 cannot be seen. For the males, [S]H values are slightly lower than the maximum, the two subjects MV-8 and MVII-2 (DO type) have the standard [S]H = 20, and 10 subjects have the normal [K]H = 200 (in DO type), which can be achieved by pumping K+ into the HM cells with Na<sup>+</sup> /K<sup>+</sup> -ATPase.

Figure 3(e) shows hair [Zn]H and [Fe]H for the same subjects. Zinc values are well regulated around the standard; [Zn]H is consistent with [Zn]H = [Zn]S 2 . This implies that Zn atoms on the serum protein are incorporated into the hair protein in pairs, in the same manner as Ca (Ca in hair protein also exists as a pair of atoms) [5].

## 5. Air pollution observed by hair analysis in February 2011

The effect of air pollution on hair elements in Japan is discernible from 2009 onward. Especially, from February to March 2011, very high contents of Ti in hair were observed, as seen in Figures 4–6, corresponding to a period of yellow haze, and indicating PM from the mainland desert [35].

Figures 4 and 5 are from female subjects under 60 years old and above, respectively. Figure 6 is from male subjects between 23 and 83. The bar graphs are in order of age. Almost all hair samples were taken in February 2011. Three-orders of magnitude higher [Ti]H can be seen. Such high levels of [Ti]H were also observed for hair samples obtained in Tokyo area in this period.

All the people in the area must have breathed the polluted air containing Ti. However, about 80% of the female subjects younger than 60 (Figure 4) have [Ti]H > 10, and less than 30% of the male subjects showed this effect. Figures 4–6(c) show the [Cl]H and [Br]H for the same subjects. The correspondence between the high [Ti]H and high [Cl]H (and [Br]H) is clear. The high [Ti]H should be attributed to the deterioration of the liver's function to excrete Ti into bile caused by Sr2+ inflow into hepatocytes. The [Cl]H abnormality can be attributed to Ca2+ inflow (in past) into erythrocytes through open Ca2+ channels (a PTH effect) ultimately caused by Ca deficiency. Recovery of Ca levels requires months of oral supplementation. Therefore, the observed [Ti]H and [Cl]H abnormalities mean that the affected subjects were LD type in past even if Ca is sufficient when the hair was taken. The observed difference between the sexes is due to most of the male subjects being DO type. Although all the subjects breathed the polluted air, the pollution effect depends on the Ca-deficiency history of the individuals. However, all elements Ca, Sr, Cl, Br, P, K, S, Cu, and Zn changed their levels in hair from those in Figure 3.

#### 5.1. Store-operated Ca channel gating induced by the pollution

increasing deficiency), DO type, is more common for the male, and LD type is more common for the female, that is, the male appears to tolerate Ca deficiency by bone resorption, and the

Figure 3. Hair concentrations observed for 50 randomly selected male and female subjects aged between 30 and 70. The hair samples were obtained before 2009 and show little or minor effects of air pollution. The bars graphed are in order of age and the Roman numerals in the subject labels stand for age period. (a) [Cu]H and [Ti]H. The essential element Cu shows most values close to the homeostatic standard at [Cu]H = 10 by Eq. (3), while Ti has a variety in [Ti]H by Eq. (2). (b) [Ca]H and [Sr]H having two separate standard levels with close and open Ca channels, [Ca]H = 10 vs. [Ca]H = 50 and [Sr]H = 10 vs. [Sr]H = 200, respectively. (c) [Cl]H and [Br]H having two separate standard levels at [Cl]H = [Br]H = 10 vs. [Cl]H = [Br]H = 22. (d) [K]H with the normal [K]H = 200 and [S]H having two separate standard levels, [S]H = 20 vs. [S]H = 200. (e) [Zn]H and [Fe]H showing values close to the standard at [Zn]H = 10 and [Fe]H = 10 normalized by Eq. (3).

female by Ca channel gating.

56 Trace Elements - Human Health and Environment

Ca contained in the polluted air, probably as calcium silicate, sulfate and carbonate, is breathed into the lung and must increase [Ca2+] in serum. As seen in Figures 4–6(b), all the subjects have [Ca]H levels normal or less than the normal, that is, [Ca]H≲10, except the subject labeled "F073" having parathyroid gland dysfunction. This means that PTH-regulated Ca channels are closed with the air pollution. Comparison with Figure 3(b) indicates that breathing the

Figure 4. Effect of the air pollution on elements in hair root obtained in February 2011 from female subjects younger than 60. The bar graphs are in order of age from 23. Compare with Figure 3. (a) [Cu]H and [Ti]H. Although the subjects younger than 42 (F019) have the normal [Cu]H = 10 by Eq. (3), the [Cu]H level for the older is lower than the normal. The [Ti]H level by Eq. (2) is high for all the subjects; [Ti]H > 1000 for the several subjects with the maximum of 2000 for F036 (age: 48), reflecting the atmosphere polluted with Ti. The high levels for [Cu]H and/or [Ti]H are consistent with deterioration of liver function. (b) [Ca]H and [Sr]H. All the subjects have [Ca]H≦10 due to PTH-regulated Ca channel closing except F073 (parathyroid gland dysfunction, [Sr]H/[Ca]H = 4 by PTH). (c) [Cl]H and [Br]H. As a whole, [Cl]H (and [Br]H) is lower than that for Figure 3 by a deviation to alkalosis with a high [Ca2+] in serum. The very high [Ti]H in (a) is associated with the high [Cl]H ([Br]H) (acidosis). (d) [K]H and [S]H. Many subjects have the lower standard [S]H = 20 with closing the ion channels attributable to breathing S in the polluted air. (e) [Zn]H and [Fe]H normalized by Eq. (3). All the subjects have low [Zn]H values (indicating 1/4 of the normal hair level), while the standard [Fe]H is seen for many subjects. The pollution effects shown here are for hair samples collected in Hyogo Prefecture. The similar effects are confirmed for those in Yokohama.

polluted air (yellow sands) has an effect as if Ca supplements were taken. However, to increase Ca stored in serum protein to the normal [Ca]H = [Ca]P = 10, long-term Ca supplementation over 2 months with 900 mg/day is required. Therefore, the closing of PTH-regulated Ca

have low [Zn]H values (1/4 of the normal), with the standard [Fe]H for many subjects.

Figure 5. Effect of the air pollution on elements in hair root obtained in February 2011 from female subjects aged from 60 to 83. The bar graphs are in order of age. Compare with Figure 3. (a) [Cu]H and [Ti]H. Almost all the subjects have [Cu]H < 10 by Eq. (3). The [Ti]H level by Eq. (2) is high for all the subjects; [Ti]H > 1000 for the several subjects with the maximum of 3000 for F122 (age: 69). (b) [Ca]H and [Sr]H. All the subjects have [Ca]H≦10 due to PTH-regulated Ca channel closing. (c) [Cl]H and [Br]H. As a whole, [Cl]H ([Br]H) is lower than that for Figure 3 by a deviation to alkalosis with a high [Ca2+] in serum. Also, see the correspondence between very high [Ti]H in (a) and high [Cl]H ([Br]H). (d) [K]H and [S]H. The lower standard [S]H = 20 is due to breathing S in the polluted air. (e) [Zn]H and [Fe]H normalized by Eq. (3). All the subjects

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 59

Figure 5. Effect of the air pollution on elements in hair root obtained in February 2011 from female subjects aged from 60 to 83. The bar graphs are in order of age. Compare with Figure 3. (a) [Cu]H and [Ti]H. Almost all the subjects have [Cu]H < 10 by Eq. (3). The [Ti]H level by Eq. (2) is high for all the subjects; [Ti]H > 1000 for the several subjects with the maximum of 3000 for F122 (age: 69). (b) [Ca]H and [Sr]H. All the subjects have [Ca]H≦10 due to PTH-regulated Ca channel closing. (c) [Cl]H and [Br]H. As a whole, [Cl]H ([Br]H) is lower than that for Figure 3 by a deviation to alkalosis with a high [Ca2+] in serum. Also, see the correspondence between very high [Ti]H in (a) and high [Cl]H ([Br]H). (d) [K]H and [S]H. The lower standard [S]H = 20 is due to breathing S in the polluted air. (e) [Zn]H and [Fe]H normalized by Eq. (3). All the subjects have low [Zn]H values (1/4 of the normal), with the standard [Fe]H for many subjects.

Figure 4. Effect of the air pollution on elements in hair root obtained in February 2011 from female subjects younger than 60. The bar graphs are in order of age from 23. Compare with Figure 3. (a) [Cu]H and [Ti]H. Although the subjects younger than 42 (F019) have the normal [Cu]H = 10 by Eq. (3), the [Cu]H level for the older is lower than the normal. The [Ti]H level by Eq. (2) is high for all the subjects; [Ti]H > 1000 for the several subjects with the maximum of 2000 for F036 (age: 48), reflecting the atmosphere polluted with Ti. The high levels for [Cu]H and/or [Ti]H are consistent with deterioration of liver function. (b) [Ca]H and [Sr]H. All the subjects have [Ca]H≦10 due to PTH-regulated Ca channel closing except F073 (parathyroid gland dysfunction, [Sr]H/[Ca]H = 4 by PTH). (c) [Cl]H and [Br]H. As a whole, [Cl]H (and [Br]H) is lower than that for Figure 3 by a deviation to alkalosis with a high [Ca2+] in serum. The very high [Ti]H in (a) is associated with the high [Cl]H ([Br]H) (acidosis). (d) [K]H and [S]H. Many subjects have the lower standard [S]H = 20 with closing the ion channels attributable to breathing S in the polluted air. (e) [Zn]H and [Fe]H normalized by Eq. (3). All the subjects have low [Zn]H values (indicating 1/4 of the normal hair level), while the standard [Fe]H is seen for many subjects. The pollution effects shown here are for hair samples collected in Hyogo Prefecture. The similar effects are confirmed for those in

Yokohama.

58 Trace Elements - Human Health and Environment

polluted air (yellow sands) has an effect as if Ca supplements were taken. However, to increase Ca stored in serum protein to the normal [Ca]H = [Ca]P = 10, long-term Ca supplementation over 2 months with 900 mg/day is required. Therefore, the closing of PTH-regulated Ca

this is by activation of the STIM proteins, inhibits voltage-gated Ca2+ channels (Cav1.2) responsible for activating heart muscle cells [33]. This may be taken as a risk factor for cardiovascular

Both the [K]H and [S]H for the same subjects are shown in Figures 4–6(d). As seen in Figure 3(d), almost all subjects without air pollution have low [K]H < 10 and high [S]H = 200 due to the PTH

in Table 1). However, the air pollution (apparently containing sulfur species) increases [SO4

in serum in addition to the [Ca2+] increase as have been seen in Figures 4–6(b), resulting in the lower level [S]H = 20 due to ion channel closing. In Figures 4–6(d), many subjects have the normal [S]H = 20, which had been seldom seen without air pollution (before 2009) due to common dietary Ca deficiencies. We can conclude that the air pollution overfills [Ca2+] and [SO4

[K]H is proportional to the intracellular [K], which strongly depends on K<sup>+</sup> transfer between cell and serum which is influenced by β-catecholamines, insulin, aldosterone, pH, and osmolality [36]. Each hair analysis gives the mean concentration for about 3 days (Section 2) and

and, to maintain electrical balance, K<sup>+</sup> ions move out into the serum, resulting in a low [K]H, and vice-versa in alkalosis. The pollution increases the serum [Ca2+] and shifts the subject from acidosis to alkalosis. Therefore, there seen many high values of [K]H in Figures 4–6(d). However, the [K]H values never reach the maximum level of [K]H = 200 seen in Figure 3(d). Since it

maximum indicates the serum [K]S at about a half of the normal, that is, hypokalemia defined as a [K]S < 3.5 mmol/L, as opposed to the normal 5 mmol/L [23]. The maximum [K]H = 200 can

sodium ions from the cell and takes in two potassium ions. If the pumping power deteriorates, therefore, the decrease of the [K]H maximum level must occur with an increase of [Na]H. This can be seen in the typical example, Figure 2(b), for a hair sample (F150) having a high [Ti]H from PM pollution; the Na Kα peak at 1.04 keV appears clearly, in contrast to the nonpolluted samples in Figure 2(a). Despite the high internal absorbance of sodium's 1.04 keV X-ray emission, such Na peaks were observed for many subjects in Figures 4–6. It may be concluded

phosphated ATP. Therefore, serum [P] is important in their deterioration. Both of P and Ca in serum are closely associated in an equilibrium relation with bone. The effect of the pollution on hair [P]H is seen in Figure 7; Figure 7(a)–(d) shows [P]H values observed for the hair samples in Figures 3–6. As has been reported [5], hair has the upper level of [P]H≳10 and lower level of [P]H = 5 by Eq. (2). In Figure 7(a) without pollution, 12 out of 50 males and 7 out of 50 female subjects have the upper level [P]H≳10. Usually, [P]H < 5 lower than the standard cannot be observed without air pollution [Figure 7(a)]. With the pollution, however, many hair samples

/K<sup>+</sup>

/H+ exchange in the cells) and the reabsorption of SO4

<sup>2</sup> inflows into HM cells to give [S]H = 200 (maximum by Eq. (8)

/K<sup>+</sup> exchange depending on pH. In acidosis, H+ ions move into cells

/K<sup>+</sup>



<sup>2</sup> in serum causes ion channel gating

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373

] [5], the [K]H~100 observed as the


2

61

<sup>2</sup>]

2]

mortality (especially in cardiac patients) and to a lesser degree, for all persons.

5.2. Deterioration of molecular pumps in membrane by the pollution

inhibition of both the excretion of H+ (K+

into cells, through which SO4

primarily shows the H+

that the function of Na<sup>+</sup>

in serum.

in renal tubules, respectively [32]. The deficiency of SO4

is the maximum that is proportional to the serum [K+

/K<sup>+</sup>

Molecular ion pumps such as Na<sup>+</sup>

be reached by pumping K ions by the molecular pump, Na+

Figure 6. Effect of the air pollution on elements in hair root obtained in February 2011 from male subjects aged from 26 to 87. The bar graphs are in order of age. Compare with Figure 3. (a) [Cu]H and [Ti]H. Almost all the subjects have [Cu]H < 10 by Eq. (3). The high [Ti]H > 1000 is seen for several subjects with the maximum of 3000 for M385 (age: 75). (b) [Ca]H and [Sr]H. All the subjects have [Ca]H≦10 due to PTH-regulated Ca channel closing. (c) [Cl]H and [Br]H. As a whole, [Cl]H ([Br]H) is lower than that for Figure 3 by a deviation to alkalosis with a high [Ca2+] in serum. Also, see the correspondence between very high [Ti]H in (a) and high [Cl]H ([Br]H). (d) [K]H and [S]H. The lower standard [S]H = 20 is due to breathing S in the polluted air. (e) [Zn]H and [Fe]H normalized by Eq. (3). All the subjects have low [Zn]H values (1/4 of the normal), with the standard [Fe]H for many subjects.

channels results in activation of the store-operated Ca channels; all the female subjects in Figures 4 and 5 and almost all male subjects in Figure 6 have [Ca]H < 10 with [Sr]H/[Ca]H = 4 or [Ca]H = 10 with 1 < [Sr]H/[Ca]H≦4 produced by store-operated Ca channel opening. In other words, the pollution mainly changes the Ca channel gating into the store-operated type, which, if this is by activation of the STIM proteins, inhibits voltage-gated Ca2+ channels (Cav1.2) responsible for activating heart muscle cells [33]. This may be taken as a risk factor for cardiovascular mortality (especially in cardiac patients) and to a lesser degree, for all persons.

#### 5.2. Deterioration of molecular pumps in membrane by the pollution

Both the [K]H and [S]H for the same subjects are shown in Figures 4–6(d). As seen in Figure 3(d), almost all subjects without air pollution have low [K]H < 10 and high [S]H = 200 due to the PTH inhibition of both the excretion of H+ (K+ /H+ exchange in the cells) and the reabsorption of SO4 2 in renal tubules, respectively [32]. The deficiency of SO4 <sup>2</sup> in serum causes ion channel gating into cells, through which SO4 <sup>2</sup> inflows into HM cells to give [S]H = 200 (maximum by Eq. (8) in Table 1). However, the air pollution (apparently containing sulfur species) increases [SO4 <sup>2</sup>] in serum in addition to the [Ca2+] increase as have been seen in Figures 4–6(b), resulting in the lower level [S]H = 20 due to ion channel closing. In Figures 4–6(d), many subjects have the normal [S]H = 20, which had been seldom seen without air pollution (before 2009) due to common dietary Ca deficiencies. We can conclude that the air pollution overfills [Ca2+] and [SO4 2] in serum.

[K]H is proportional to the intracellular [K], which strongly depends on K<sup>+</sup> transfer between cell and serum which is influenced by β-catecholamines, insulin, aldosterone, pH, and osmolality [36]. Each hair analysis gives the mean concentration for about 3 days (Section 2) and primarily shows the H+ /K<sup>+</sup> exchange depending on pH. In acidosis, H+ ions move into cells and, to maintain electrical balance, K<sup>+</sup> ions move out into the serum, resulting in a low [K]H, and vice-versa in alkalosis. The pollution increases the serum [Ca2+] and shifts the subject from acidosis to alkalosis. Therefore, there seen many high values of [K]H in Figures 4–6(d). However, the [K]H values never reach the maximum level of [K]H = 200 seen in Figure 3(d). Since it is the maximum that is proportional to the serum [K+ ] [5], the [K]H~100 observed as the maximum indicates the serum [K]S at about a half of the normal, that is, hypokalemia defined as a [K]S < 3.5 mmol/L, as opposed to the normal 5 mmol/L [23]. The maximum [K]H = 200 can be reached by pumping K ions by the molecular pump, Na+ /K<sup>+</sup> -ATPase, which expels three sodium ions from the cell and takes in two potassium ions. If the pumping power deteriorates, therefore, the decrease of the [K]H maximum level must occur with an increase of [Na]H. This can be seen in the typical example, Figure 2(b), for a hair sample (F150) having a high [Ti]H from PM pollution; the Na Kα peak at 1.04 keV appears clearly, in contrast to the nonpolluted samples in Figure 2(a). Despite the high internal absorbance of sodium's 1.04 keV X-ray emission, such Na peaks were observed for many subjects in Figures 4–6. It may be concluded that the function of Na<sup>+</sup> /K<sup>+</sup> -ATPase in cell membrane is deteriorated by the pollution.

Molecular ion pumps such as Na<sup>+</sup> /K<sup>+</sup> -ATPase work with the energy supply of fully phosphated ATP. Therefore, serum [P] is important in their deterioration. Both of P and Ca in serum are closely associated in an equilibrium relation with bone. The effect of the pollution on hair [P]H is seen in Figure 7; Figure 7(a)–(d) shows [P]H values observed for the hair samples in Figures 3–6. As has been reported [5], hair has the upper level of [P]H≳10 and lower level of [P]H = 5 by Eq. (2). In Figure 7(a) without pollution, 12 out of 50 males and 7 out of 50 female subjects have the upper level [P]H≳10. Usually, [P]H < 5 lower than the standard cannot be observed without air pollution [Figure 7(a)]. With the pollution, however, many hair samples

channels results in activation of the store-operated Ca channels; all the female subjects in Figures 4 and 5 and almost all male subjects in Figure 6 have [Ca]H < 10 with [Sr]H/[Ca]H = 4 or [Ca]H = 10 with 1 < [Sr]H/[Ca]H≦4 produced by store-operated Ca channel opening. In other words, the pollution mainly changes the Ca channel gating into the store-operated type, which, if

with the standard [Fe]H for many subjects.

60 Trace Elements - Human Health and Environment

Figure 6. Effect of the air pollution on elements in hair root obtained in February 2011 from male subjects aged from 26 to 87. The bar graphs are in order of age. Compare with Figure 3. (a) [Cu]H and [Ti]H. Almost all the subjects have [Cu]H < 10 by Eq. (3). The high [Ti]H > 1000 is seen for several subjects with the maximum of 3000 for M385 (age: 75). (b) [Ca]H and [Sr]H. All the subjects have [Ca]H≦10 due to PTH-regulated Ca channel closing. (c) [Cl]H and [Br]H. As a whole, [Cl]H ([Br]H) is lower than that for Figure 3 by a deviation to alkalosis with a high [Ca2+] in serum. Also, see the correspondence between very high [Ti]H in (a) and high [Cl]H ([Br]H). (d) [K]H and [S]H. The lower standard [S]H = 20 is due to breathing S in the polluted air. (e) [Zn]H and [Fe]H normalized by Eq. (3). All the subjects have low [Zn]H values (1/4 of the normal),

The serum [K] is regulated with free filtration at the glomerulus and by excretion-reabsorption

by the insufficient ATP supply, resulting in the hypokalemia understood from the hair level. This pollution's effect is serious; even mild or moderate hypokalemia increases the risks of

According to the meta-analysis for 57,158 patients with acute myocardial infarction (AMI) [37], primary ventricular fibrillation (PVF) is lethal and occurs without signs or symptoms of heart failure or cardiogenic shock. It was shown that patients with PVF had a lower serum K level before the event compared with patients without PVF. Though the weighted mean difference was small (0.27 mmol/L), the finding of lower serum K before PVF was very consistent. The association between low [K] and PVF was confirmed in clinical settings. Patients with PVF have lower heart rates for interior AMI and higher rates for anterior AMI. It can be concluded that the hypokalemia due to PM causes a mortality increase in AMI [25, 26] even 1 day after PM pollution increases. Also, it is well known that PVF results in blood stagnation in atrium to

The [P]H decrease as observed in Figure 7 never takes place with oral Ca supplementation, which leads to the upper level [P]H≧10 with [Cl]H = [Br]H = 10 [5]. The direct Ca intake by breathing the polluted air into the lung is essentially different from absorption through the GI

In observation of [Na] and [P] by FXA, absorption of the Na Kα and P Kα by the specimen must greatly reduce the number of 1.04 and 2.01 keV photons reaching the detector. However, although low keV X-rays are received from only the hair surface, the calculation of concentration of P/S by Eq. (2) is valid because the absorption for the peak P and background S is produced by the same matrix. The observation of [Na]H increase and [P]H decrease is consistent with the emerging pattern and should not be dismissed because of the low sampling volume inherent.

Another effect of the pollution is the decrease of [Cu]H = [Cu]S and [Zn]H = [Zn]S

definition by Eq. (2)]. As seen from the comparison of Figures 4–6(a) and (e) with Figure 3(a) and (e) showing their normalized values by Eq. (3), most subjects have the normal [Fe]H = 10 independent of pollution, but decreases of Cu and Zn, [Cu]H < 10 and [Zn]H < 10, are seen with the pollution. The [Cu]H decrease depends upon age, and many subjects older than 60 have [Cu]H = [Cu]S < 5 less than a half of normal. The [Zn]H decrease takes place about equally for the male and female populations studied. We estimate the average hair zinc level in Figures 4– 6(e) to be 1/4 of normal, that is, the air pollution therefore decreases serum [Zn]S to half of normal by Eq. (6) in Table 1. The decrease in serum Cu and Zn may be due to S inflow into serum from air pollution, which then forms compounds such as CuS and ZnS which may be excreted from the liver and pancreas, respectively. Both Cu and Zn are essential elements. For Zn, the normal serum concentration is in the range of 0.08–0.15 mg/dL, and its lower limit is

/K<sup>+</sup>


Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 63

<sup>2</sup> and K<sup>+</sup>

.

<sup>2</sup> [with the

in renal tubules. The reabsorption is produced by H<sup>+</sup>

form clots having the risk for brain infarction.

morbidity and mortality in patients with cardiovascular disease [36, 37].

tract and produces deficiency in the intracellular main ions [23], HPO4

5.3. Reduction of serum Cu and Zn with the pollution

0.06–0.07 mg/dL. A decrease by half is therefore dangerous.

Figure 7. Association between [P]H and [Na]H with inactive Na<sup>+</sup> /K<sup>+</sup> -ATPase due to the air pollution. (a) [P]H without air pollution for the male and female subjects in Figure 3. The upper and lower levels are seen at [P]H≳10 and [P]H = 5, respectively. [Na]H is not seen by the normal Na<sup>+</sup> /K + -ATPase. (b) and (c) show [P]H and [Na]H for the female subjects in Figures 4 and 5, respectively. [P]H lower than the usual lower level is seen with the appearance of [Na]H peak. (d) [P]H for the male subjects in Figure 6; the low [P]H is accompanied by the [Na]H peak. The low [P]H values are often seen for the male over 60 (M351: 63). The abnormally low [P]H indicates insufficient energy supply to the Na+ /K<sup>+</sup> - ATPase. Breathing the polluted air increases serum [Ca2+] which moves to bone, accompanied by P to cause P deficiency.

in Figure 7(b)–(d) showed [P]H = 1.5–3.5, much lower than the usual lower level. Also, the upper level [P]H≳10 is not seen, except for the subject labeled "M306," in Figure 7(b)-(d). As a whole, [P]H is decreased by the air pollution; by Eq. (19) in Table 1, serum [P]I ([HPO4 <sup>2</sup>]) is calculated to be a half of the normal for [P]H = 3.5 and 1/4 for [P]H = 2.5, taking [P]H = 5 for the lower standard. This serum [P]I decrease may be explained as the pollution-induced excess Ca moving with P to bone by forming insoluble Ca PO4; serum HPO4 <sup>2</sup> moving to bone must lower serum [P]S [20, 22, 32], and the deterioration of molecular pumps seems due to insufficient energy supply. Therefore, we can see the correlation between [P]H and [Na]H; the low [P]H is accompanied by appearance of [Na]H peak as seen in Figure 7(b)-(d). ([Na]H peaks were not seen in Figure 7(a) without pollution).

The serum [K] is regulated with free filtration at the glomerulus and by excretion-reabsorption in renal tubules. The reabsorption is produced by H+ /K<sup>+</sup> -ATPase [36] which is also deteriorated by the insufficient ATP supply, resulting in the hypokalemia understood from the hair level. This pollution's effect is serious; even mild or moderate hypokalemia increases the risks of morbidity and mortality in patients with cardiovascular disease [36, 37].

According to the meta-analysis for 57,158 patients with acute myocardial infarction (AMI) [37], primary ventricular fibrillation (PVF) is lethal and occurs without signs or symptoms of heart failure or cardiogenic shock. It was shown that patients with PVF had a lower serum K level before the event compared with patients without PVF. Though the weighted mean difference was small (0.27 mmol/L), the finding of lower serum K before PVF was very consistent. The association between low [K] and PVF was confirmed in clinical settings. Patients with PVF have lower heart rates for interior AMI and higher rates for anterior AMI. It can be concluded that the hypokalemia due to PM causes a mortality increase in AMI [25, 26] even 1 day after PM pollution increases. Also, it is well known that PVF results in blood stagnation in atrium to form clots having the risk for brain infarction.

The [P]H decrease as observed in Figure 7 never takes place with oral Ca supplementation, which leads to the upper level [P]H≧10 with [Cl]H = [Br]H = 10 [5]. The direct Ca intake by breathing the polluted air into the lung is essentially different from absorption through the GI tract and produces deficiency in the intracellular main ions [23], HPO4 <sup>2</sup> and K<sup>+</sup> .

In observation of [Na] and [P] by FXA, absorption of the Na Kα and P Kα by the specimen must greatly reduce the number of 1.04 and 2.01 keV photons reaching the detector. However, although low keV X-rays are received from only the hair surface, the calculation of concentration of P/S by Eq. (2) is valid because the absorption for the peak P and background S is produced by the same matrix. The observation of [Na]H increase and [P]H decrease is consistent with the emerging pattern and should not be dismissed because of the low sampling volume inherent.

#### 5.3. Reduction of serum Cu and Zn with the pollution

in Figure 7(b)–(d) showed [P]H = 1.5–3.5, much lower than the usual lower level. Also, the upper level [P]H≳10 is not seen, except for the subject labeled "M306," in Figure 7(b)-(d). As a whole, [P]H is decreased by the air pollution; by Eq. (19) in Table 1, serum [P]I ([HPO4

air pollution for the male and female subjects in Figure 3. The upper and lower levels are seen at [P]H≳10 and [P]H = 5,

in Figures 4 and 5, respectively. [P]H lower than the usual lower level is seen with the appearance of [Na]H peak. (d) [P]H for the male subjects in Figure 6; the low [P]H is accompanied by the [Na]H peak. The low [P]H values are often seen for the male over 60 (M351: 63). The abnormally low [P]H indicates insufficient energy supply to the Na+

ATPase. Breathing the polluted air increases serum [Ca2+] which moves to bone, accompanied by P to cause P

/K<sup>+</sup>

calculated to be a half of the normal for [P]H = 3.5 and 1/4 for [P]H = 2.5, taking [P]H = 5 for the lower standard. This serum [P]I decrease may be explained as the pollution-induced excess Ca

lower serum [P]S [20, 22, 32], and the deterioration of molecular pumps seems due to insufficient energy supply. Therefore, we can see the correlation between [P]H and [Na]H; the low [P]H is accompanied by appearance of [Na]H peak as seen in Figure 7(b)-(d). ([Na]H peaks

moving with P to bone by forming insoluble Ca PO4; serum HPO4

were not seen in Figure 7(a) without pollution).

Figure 7. Association between [P]H and [Na]H with inactive Na<sup>+</sup>

respectively. [Na]H is not seen by the normal Na<sup>+</sup>

62 Trace Elements - Human Health and Environment

deficiency.

<sup>2</sup>]) is

/K<sup>+</sup> -

<sup>2</sup> moving to bone must


/K + -ATPase. (b) and (c) show [P]H and [Na]H for the female subjects

Another effect of the pollution is the decrease of [Cu]H = [Cu]S and [Zn]H = [Zn]S <sup>2</sup> [with the definition by Eq. (2)]. As seen from the comparison of Figures 4–6(a) and (e) with Figure 3(a) and (e) showing their normalized values by Eq. (3), most subjects have the normal [Fe]H = 10 independent of pollution, but decreases of Cu and Zn, [Cu]H < 10 and [Zn]H < 10, are seen with the pollution. The [Cu]H decrease depends upon age, and many subjects older than 60 have [Cu]H = [Cu]S < 5 less than a half of normal. The [Zn]H decrease takes place about equally for the male and female populations studied. We estimate the average hair zinc level in Figures 4– 6(e) to be 1/4 of normal, that is, the air pollution therefore decreases serum [Zn]S to half of normal by Eq. (6) in Table 1. The decrease in serum Cu and Zn may be due to S inflow into serum from air pollution, which then forms compounds such as CuS and ZnS which may be excreted from the liver and pancreas, respectively. Both Cu and Zn are essential elements. For Zn, the normal serum concentration is in the range of 0.08–0.15 mg/dL, and its lower limit is 0.06–0.07 mg/dL. A decrease by half is therefore dangerous.

Zinc serves as a structural center for maintaining the higher-order structure of protein. Zinc participates in the activity of more than 70 enzymes, including carbonic anhydrase (CA) and dehydrogenase. Almost all proteins related to gene expression contain Zn as a DNA-binding motif "zinc finger." Zinc is essential for physiological functions such as growth, gestation, taste, and participates in immunity, brain development, insulin biosynthesis, and so on [38]. Therefore, the air-pollution's effect on serum Zn is serious to many aspects of health.

The observed relation between the sexes leads to the conclusion that the serum element levels

Hair calcium depends on both PTH-regulated and store-operated Ca2+ channels; oral Ca supplementation transits hair Ca level from the Ca upper level ([Ca]H = 50) to the lower level (normal [Ca]H = 10) by closing PTH-regulated Ca channels of hair matrix cells (HM cells), and gives no effects on the store-operated channels which are activated by the decrease of stock Ca bound on serum protein. In-vitro studies ([16–18] show store-operated "Orai" Ca channels activate when Ca2+ stores are depleted at the endoplasmic reticulum (ER). Together with hair studies (in vivo), our analysis concludes that the store-operated and PTH-regulated Ca channels activate with depletion of [Ca]P and [Ca]I in Eq. (1), respectively, both of which are

Without air pollution before 2009, a half of the 100 subjects were classified as mildly calciumdeficient DA type ([Ca]H = [Ca]P < 10 with [Cl]H> > 10), and the other half are of more severe Ca

Consequently, except one patient with parathyroid gland dysfunction, all the subjects had the normal or lower [Ca]H≲10 (with [Cl]H < 10) produced by closed PTH-regulated Ca channels, and almost all the subjects had store-operated Ca channel gating, which, if done by the STIM

The overfilled Ca and S in serum has consequences: we expect that Ca is removed as the phosphate to bone and S is removed to bile and pancreatic fluid as excretable metal sulfides, resulting in loss of needed Zn and Cu. Serum [K] as well as [Cu] and [Zn] decrease to half of the normal level with air pollution. The excessive Ca2+ results in a notable deficiency of serum [P]. It must create a shortage of ATP, which deteriorates cell membrane molecular pumps such

hypokalemia to produce fatal ventricular fibrillation in patients with myocardial infarction accompanied with heart rate variability [26]. It is well known that even mild or moderate hypokalemia increases the risks of mortality in patients with cardiovascular diseases. Abnormal intracellular [K] and [Na] due to the inactive molecular pumps may be also responsible for heart rate variability. (K+ and Na+ regulate and form the electric pulses commanding myocar-

The excessive [S] in serum may decrease toward the normal by forming sulfide compounds with Cu and Zn, which are excreted from the liver and pancreas, resulting in deficiency of Cu necessary for the formation of elastin protein to repair damage in blood vessels. Thus, the sulfur species in the pollution may provide a second increase the mortality in myocardial


<sup>2</sup>]) in serum.

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 65

The air pollution from February to March 2011 overfilled Ca ([Ca2+]) and S ([SO4

proteins, inhibits voltage-gated Ca channels and worsens cardiac risks.

deficiency related to PTH-regulated Ca channels ([Ca]I < 10).

/K<sup>+</sup>

change so as to eliminate the excess Ca and S inhaled from the air pollution.

7. Conclusion

regulated by PTH.

as H<sup>+</sup> /K<sup>+</sup>

dial movement.)

infarction [25].


The reduction of serum [Cu]S due to the pollution can also give an answer to the question of the increase of myocardial infarction mortality. It is firmly established that the formation of elastin protein, which gives elasticity to blood vessels, is copper-dependent [39–41], and Cu deficiency degenerates smooth muscle cells in the aortic walls. It was observed that elastin increases at the damage sites (scar) on aortic walls [41]. This indicates that elastin is necessary to correct damages, which, if unrepaired, trigger both aortic rupture and infarction. Therefore, the Cu deficiency due to PM can cause a mortality increase of myocardial infarction [25].

Here, it should be noted that we encountered many subjects having the normal [S]H = 20 in Figures 4–6(d), in contrast to Figure 3(d) showing [S]H = 200. This observation indicates that the serum [S] remains normal by excreting the excess [S] incorporated from the pollution together with Cu, and Zn [implying the essentiality for some sulfur species, referred to as sulfate, having its own or accessible ion channels. See Figure 2(a)].

## 6. Differences in pollution effect between the sexes

The normal levels of hair elements are the same for male and female. However, the pollution effects show differences as seen clearly in Figures 4–7. As described in Section 4, male and female deal with Ca deficiency differently; DO type is more common for the mail, and LD type is more common for the female, that is, males tolerate Ca deficiency with Ca channels closed by bone resorption, and females have a tendency to open the PTH-regulated Ca channels without bone resorption. The resulting Ca and/or Sr inflow through the channels causes deterioration of the hepatocytes' function to excrete pollutants into bile; a dysfunction that persists for months. Consequently, the pollution effect appears greater for the female; more female subjects have the high [Ti]H as seen in Figures 4–6(a).

In Figure 7, the appearance of [Na]H due to the excess serum Ca by breathing the polluted air shows difference between the sexes clearly; male can accommodate the excess Ca on serum protein as expected from the fact that DO type is more common for male.

The pollution's effect to decrease [Cu]H and [Zn]H can be recognized in Figures 4–6(a) and (e). However, no difference between the sexes is clear; this implies these elements have no relation to the Ca metabolism, suggesting an association with the excess [S] in serum due to the pollution. By forming sulfide species, zinc and copper are excreted mainly with pancreatic fluid and bile into the gut, respectively.

The observed relation between the sexes leads to the conclusion that the serum element levels change so as to eliminate the excess Ca and S inhaled from the air pollution.

## 7. Conclusion

Zinc serves as a structural center for maintaining the higher-order structure of protein. Zinc participates in the activity of more than 70 enzymes, including carbonic anhydrase (CA) and dehydrogenase. Almost all proteins related to gene expression contain Zn as a DNA-binding motif "zinc finger." Zinc is essential for physiological functions such as growth, gestation, taste, and participates in immunity, brain development, insulin biosynthesis, and so on [38].

The reduction of serum [Cu]S due to the pollution can also give an answer to the question of the increase of myocardial infarction mortality. It is firmly established that the formation of elastin protein, which gives elasticity to blood vessels, is copper-dependent [39–41], and Cu deficiency degenerates smooth muscle cells in the aortic walls. It was observed that elastin increases at the damage sites (scar) on aortic walls [41]. This indicates that elastin is necessary to correct damages, which, if unrepaired, trigger both aortic rupture and infarction. Therefore, the Cu deficiency due to PM can cause a mortality increase of myocardial infarc-

Here, it should be noted that we encountered many subjects having the normal [S]H = 20 in Figures 4–6(d), in contrast to Figure 3(d) showing [S]H = 200. This observation indicates that the serum [S] remains normal by excreting the excess [S] incorporated from the pollution together with Cu, and Zn [implying the essentiality for some sulfur species, referred to as

The normal levels of hair elements are the same for male and female. However, the pollution effects show differences as seen clearly in Figures 4–7. As described in Section 4, male and female deal with Ca deficiency differently; DO type is more common for the mail, and LD type is more common for the female, that is, males tolerate Ca deficiency with Ca channels closed by bone resorption, and females have a tendency to open the PTH-regulated Ca channels without bone resorption. The resulting Ca and/or Sr inflow through the channels causes deterioration of the hepatocytes' function to excrete pollutants into bile; a dysfunction that persists for months. Consequently, the pollution effect appears greater for the female; more

In Figure 7, the appearance of [Na]H due to the excess serum Ca by breathing the polluted air shows difference between the sexes clearly; male can accommodate the excess Ca on serum

The pollution's effect to decrease [Cu]H and [Zn]H can be recognized in Figures 4–6(a) and (e). However, no difference between the sexes is clear; this implies these elements have no relation to the Ca metabolism, suggesting an association with the excess [S] in serum due to the pollution. By forming sulfide species, zinc and copper are excreted mainly with pancreatic

sulfate, having its own or accessible ion channels. See Figure 2(a)].

6. Differences in pollution effect between the sexes

female subjects have the high [Ti]H as seen in Figures 4–6(a).

fluid and bile into the gut, respectively.

protein as expected from the fact that DO type is more common for male.

Therefore, the air-pollution's effect on serum Zn is serious to many aspects of health.

tion [25].

64 Trace Elements - Human Health and Environment

Hair calcium depends on both PTH-regulated and store-operated Ca2+ channels; oral Ca supplementation transits hair Ca level from the Ca upper level ([Ca]H = 50) to the lower level (normal [Ca]H = 10) by closing PTH-regulated Ca channels of hair matrix cells (HM cells), and gives no effects on the store-operated channels which are activated by the decrease of stock Ca bound on serum protein. In-vitro studies ([16–18] show store-operated "Orai" Ca channels activate when Ca2+ stores are depleted at the endoplasmic reticulum (ER). Together with hair studies (in vivo), our analysis concludes that the store-operated and PTH-regulated Ca channels activate with depletion of [Ca]P and [Ca]I in Eq. (1), respectively, both of which are regulated by PTH.

Without air pollution before 2009, a half of the 100 subjects were classified as mildly calciumdeficient DA type ([Ca]H = [Ca]P < 10 with [Cl]H> > 10), and the other half are of more severe Ca deficiency related to PTH-regulated Ca channels ([Ca]I < 10).

The air pollution from February to March 2011 overfilled Ca ([Ca2+]) and S ([SO4 <sup>2</sup>]) in serum. Consequently, except one patient with parathyroid gland dysfunction, all the subjects had the normal or lower [Ca]H≲10 (with [Cl]H < 10) produced by closed PTH-regulated Ca channels, and almost all the subjects had store-operated Ca channel gating, which, if done by the STIM proteins, inhibits voltage-gated Ca channels and worsens cardiac risks.

The overfilled Ca and S in serum has consequences: we expect that Ca is removed as the phosphate to bone and S is removed to bile and pancreatic fluid as excretable metal sulfides, resulting in loss of needed Zn and Cu. Serum [K] as well as [Cu] and [Zn] decrease to half of the normal level with air pollution. The excessive Ca2+ results in a notable deficiency of serum [P]. It must create a shortage of ATP, which deteriorates cell membrane molecular pumps such as H<sup>+</sup> /K<sup>+</sup> -ATPase and Na+ /K<sup>+</sup> -ATPase, responsible for the renal K reabsorption, resulting in hypokalemia to produce fatal ventricular fibrillation in patients with myocardial infarction accompanied with heart rate variability [26]. It is well known that even mild or moderate hypokalemia increases the risks of mortality in patients with cardiovascular diseases. Abnormal intracellular [K] and [Na] due to the inactive molecular pumps may be also responsible for heart rate variability. (K+ and Na+ regulate and form the electric pulses commanding myocardial movement.)

The excessive [S] in serum may decrease toward the normal by forming sulfide compounds with Cu and Zn, which are excreted from the liver and pancreas, resulting in deficiency of Cu necessary for the formation of elastin protein to repair damage in blood vessels. Thus, the sulfur species in the pollution may provide a second increase the mortality in myocardial infarction [25].

In summation, hair analysis shows that post-2009 air pollution causes serious deficiencies in serum K, P, Cu, and Zn, and is likely responsible for various diseases.

[4] Chikawa J, Yamada K, Akimoto T, Sakurai H, Yasui H, Yamamoto H, Okabe S, Ebara M.

Trace Elements in Hair: Relevance to Air Pollution http://dx.doi.org/10.5772/intechopen.74373 67

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Finally, it is emphasized that the deterioration of Na<sup>+</sup> /K<sup>+</sup> -ATPase (Figure 7) is a serious pollution effect because Na+ /K<sup>+</sup> -ATPase exists in all cells, for example, more than half of brain energy dissipation is due to Na+ /K<sup>+</sup> -ATPase; the pollution effect on neurodegenerative disease [42] is conceivable.

## Acknowledgements

This work has been performed under the approval of the Photon Factory (Proposal No. 2005R15, 2006G408, 2009Y011, 2009Y022, 2010Y023) in collaboration with industry (Health Analysis Laboratory, Ltd.). The authors would like to express their sincere thanks to Professor A. Iida, Photon Factory, for his great help with the experiments at BL-4A. His sophisticated instrumentation made it possible to analyze the hair roots from thousands of people.

## Author details

Jun-ichi Chikawa1 \*, Jeremy Salter<sup>1</sup> , Hiroki Shima<sup>2</sup> , Takaaki Tsuchida<sup>3</sup> , Takashi Ueda<sup>4</sup> , Kousaku Yamada1 and Shingo Yamamoto<sup>5</sup>

\*Address all correspondence to: chikawa@hyogosta.jp


## References


In summation, hair analysis shows that post-2009 air pollution causes serious deficiencies in

This work has been performed under the approval of the Photon Factory (Proposal No. 2005R15, 2006G408, 2009Y011, 2009Y022, 2010Y023) in collaboration with industry (Health Analysis Laboratory, Ltd.). The authors would like to express their sincere thanks to Professor A. Iida, Photon Factory, for his great help with the experiments at BL-4A. His sophisticated

instrumentation made it possible to analyze the hair roots from thousands of people.

, Hiroki Shima<sup>2</sup>

/K<sup>+</sup>


, Takaaki Tsuchida<sup>3</sup>



, Takashi Ueda<sup>4</sup>

,

serum K, P, Cu, and Zn, and is likely responsible for various diseases.

/K<sup>+</sup>

Finally, it is emphasized that the deterioration of Na<sup>+</sup>

/K<sup>+</sup>

\*, Jeremy Salter<sup>1</sup>

\*Address all correspondence to: chikawa@hyogosta.jp

2 Shima Institute in Quantum Medicine, Osaka, Japan

1 Hyogo Science and Technology Association, Himeji, Japan

5 Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

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[2] Ito A, Inoue T, Kawai T, Taki Y, Inoue S, Shimizu T, Shinohara K. AIP Conference Pro-

[3] Noguchi T, Itai T, Kawaguchi M, Takahasshi S, Tanabe S. Applicability of human hair as a bioindicator for trace elements exposure. In: Kawaguchi M, Misaki K, Sato H, Yokokawa T, Itai T, Nguyen TM, Ono J, Tanabe S, editors. Environmental Chemistry. Vol. 6. Tokyo:

Kousaku Yamada1 and Shingo Yamamoto<sup>5</sup>

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4 Ueda Heart Clinic, Tatsuno-shi, Hyogo, Japan

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[42] is conceivable.

Author details

Jun-ichi Chikawa1

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**Chapter 4**

**Provisional chapter**

**Relation of Trace Elements on Dental Health**

As a result, TEs have significant effects on healthy tooth formation.

**Relation of Trace Elements on Dental Health**

DOI: 10.5772/intechopen.75899

Trace elements (TEs) play an important role in human health. Toxic effects are caused by deficiency or excess of TEs. TEs have significant effects on both dental health and human health. It participates in important biological polyphosphate compound functions such as ATP, DNA, and RNA. TEs are present at different concentrations in the tooth structure. Changes in the density of some TEs affect tooth. The alteration of the density of some TEs makes the teeth more susceptible to caries. Others are protective against caries formation. Important TEs zinc (Zn), phosphorus (P), and magnesium (Mg) have important effects on dental health. Measuring the TE values through tissue sampling to identify and correct these effects has an important effect. In general, tissue samples such as blood, urine, teeth, nails, and hair are used in TE studies. Teeth are accepted as appropriate indication of TEs.

**Keywords:** trace elements, teeth health, human health, dental caries, dental structure

About 96% of life materials consist of carbon, hydrogen, and nitrogen elements. Almost 50% of the known elements are at measurable concentrations in life system. In humans and other mammals, physiological activities of 23 elements are known, 11 of which are classified as trace elements (TEs). TEs consist of transition elements [vanadium, chromium, manganese (Mn), iron (Fe), cobalt, copper (Cu), zinc (Zn), and molybdenum] and non-metal elements [selenium (Se), fluorine, and iodine]. TEs are, unlike sodium, calcium, magnesium, potassium, and chlorine, which are considered as macronutrients and required at larger amounts, fall into the micro-nutrient category, which is required at negligible levels (usually lower than 100 mg/ day). Major and TEs play an essential role in human health. Lack or abundance of these elements due to natural or man-made reasons can lead to critical clinic consequences [1–4].

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75899

Mehmet Sinan Doğan

Mehmet Sinan Doğan

**Abstract**

**1. Introduction**

#### **Relation of Trace Elements on Dental Health Relation of Trace Elements on Dental Health**

DOI: 10.5772/intechopen.75899

#### Mehmet Sinan Doğan Mehmet Sinan Doğan

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75899

**Abstract**

Trace elements (TEs) play an important role in human health. Toxic effects are caused by deficiency or excess of TEs. TEs have significant effects on both dental health and human health. It participates in important biological polyphosphate compound functions such as ATP, DNA, and RNA. TEs are present at different concentrations in the tooth structure. Changes in the density of some TEs affect tooth. The alteration of the density of some TEs makes the teeth more susceptible to caries. Others are protective against caries formation. Important TEs zinc (Zn), phosphorus (P), and magnesium (Mg) have important effects on dental health. Measuring the TE values through tissue sampling to identify and correct these effects has an important effect. In general, tissue samples such as blood, urine, teeth, nails, and hair are used in TE studies. Teeth are accepted as appropriate indication of TEs. As a result, TEs have significant effects on healthy tooth formation.

**Keywords:** trace elements, teeth health, human health, dental caries, dental structure

## **1. Introduction**

About 96% of life materials consist of carbon, hydrogen, and nitrogen elements. Almost 50% of the known elements are at measurable concentrations in life system. In humans and other mammals, physiological activities of 23 elements are known, 11 of which are classified as trace elements (TEs). TEs consist of transition elements [vanadium, chromium, manganese (Mn), iron (Fe), cobalt, copper (Cu), zinc (Zn), and molybdenum] and non-metal elements [selenium (Se), fluorine, and iodine]. TEs are, unlike sodium, calcium, magnesium, potassium, and chlorine, which are considered as macronutrients and required at larger amounts, fall into the micro-nutrient category, which is required at negligible levels (usually lower than 100 mg/ day). Major and TEs play an essential role in human health. Lack or abundance of these elements due to natural or man-made reasons can lead to critical clinic consequences [1–4].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A tooth consists of hard tissue (enamel, dentine, and cement) and soft tissue (pulp and periodontal ligaments), and has TE in its structure. A tooth has a multicellular structure which can cooperate functionally with maxillofacial area [5].

The inorganic component of cement is similar to bone, dentin, and enamel. The basic mineral component of the cement is hydroxyapatite with amorphous calcium phosphate (Ca10 (PO4) 6 (OH) 2). The crystallinity of the cement inorganic component is lower than other calcifying tissues [16]. As a result, cement is decalcified more easily, while it has a tendency to coalesce for the adsorption of surrounding ions (i.e., fluoride). In general, the cement of adult mature teeth has higher fluoride content compared to other calcifying tissues. Mg content of the cement is about half of that in the dentin. There is a gradual increase in Mg in the deep layers of the cement [14].

Relation of Trace Elements on Dental Health http://dx.doi.org/10.5772/intechopen.75899 73

Tooth pulp developing from dental papilla consists of odontoblast, fibroblast, blood, and neural veins [17]. Odontoblast cells are the most important cells of the pulp. As the cells responsible for the construction of dentin and pre-dentin, they are also responsible for reparative dentin make-up in pathological cases [5, 18]. Pulp cells, especially fibroblasts, produce several inflammatory mediators such as IL-8, IL-6, and vascular endothelial growth factor in cases which threaten the health of the pulp. Tooth pulp performs a series of biological activities such as nutrition, sensitiveness, construction, and protection. The change in blood pressure and flow in the veins coming from apical region essentially affects the health of the pulp. When tooth pulp is damaged due to mechanical, chemical, thermal and microbial irritants, local tissue reactions and lymphatic, vascular inflammatory responses occur. The existence of dentin affected by oral bacteria which lead to the formation of caries is most important

Dental caries is a microbiological infectious disease of the teeth that results in the destruction of dental calcified tissues. There must be three factors for the formation of dental caries; bacteria (from mutans Streptococci and Lactobacillus species), susceptible tooth surface (host), and nutrient (diet) to provide bacterial growth [20, 21]. Changes in the density of TEs due to some environmental and genetic impacts have some effects on human and dental health. The change in density of some elements can lead to dental caries. Based on the previous studies on humans and animals, some classifications have been made on the impact of TE on dental caries [4, 9].

In addition, there is another classification with regard to the impact of TE on dental health:

**Cariostatic elements**: Molybdenum, Vanadium, Fluoride, Strontium, Lithium.

The effect on dental and oral tissues of elements and their features.

**Caries-promoting elements**: Selenium, Cadmium, Lead, Manganese, Copper, Zinc.

**5. Dental pulp**

reasons for pulp inflammation [19].

**Cariostatic elements:** Fluoride (F), Phosphorus (P).

**Mildly cariostatic:** Mo, V, Cu, Sr., B, Li, Au.

**Caries promoting:** Se, Mg, Cd, Pt, Pb, Si.

**Doubtful:** Be, Co, Mn, Sn, Zn, Br, I. **Caries inert:** Ba, Al, Ni, Fe, Pd, Ti.

## **2. Enamel**

Enamel is the hard tissue that covers the surface of the tooth. The function of this layer is to protect dentine-pulp complex. Enamel is the hardest and most resistant tissue in the body. It consists of 95% inorganic material (calcium hydroxyapatite crystals), 2% organic material (proteins such as amylogenic, enameline, ameloblastin, and tuftelin, among others), and 3% water [6–8]. A negligible part of the 95% inorganic material is represented by TEs. As a result of the analyses conducted using different methods in tooth enameling several chemical components are observed. These components include phosphorus (P), calcium (Ca), magnesium (Mg), zinc (Zn), lead (Pb), cobalt (Co), fluorine (F), iron (Fe), aluminum (Al), and selenium (Se). Inorganic structure of enamel consists of 36.1 Ca, 17.3 P, 3.0 carbon oxide, 0.5 Mg, 0.2 Na, 0.3 potassium (K), 0.016 F, 0.1 sulfur (S), 0.01 copper (Cu), 0.016 Zn, 0.003 silicon (Si); and low levels of silver (Ag), strontium (Sr), barium (Ba), chromium (Cr), manganese (Mn), vanadium (V), aluminum (Al), lithium (Li), and selenium (Se). TE is placed in the human tooth enamel from the environment during and after the mineralization and maturation period of the tooth [9].

## **3. Dentin**

The dentin consists of 70% inorganic material (hydroxyapatite crystals and TEs), 18% organic material (type I collagen fiber and proteins such as osteonectin, osteopontin, osteoclastin-like dentin Gla protein, dentin phosphorene, dentin matrix protein, and dentin sialoprotein) and 12% water [6–8].

The inorganic material of dentin contains about 40 elements, ranging from 1000 ppm (i.e., Zn, Sr., Fe, Al, B, Ba, Pb, etc.) to 100 ppb (i.e., Ni, Li, Ag, As, Se, Nb, Hg, etc.) [10–12].

The ratio of TE in human dentin varies according to age and sex. Cobalt can be higher in women, while lead can be higher in men [13].

## **4. Cementum**

Cementum is a special connective tissue that connects the periodontal ligament to the root surface, covering the outermost layer of the calcite matrix on the root surface [14].

Cement is a vascular and unlimited mineralized tissue. It is the interface between dentin and periodontal ligament and contributes to the repair and renewal of periodontal tissue after injury [15].

The inorganic component of cement is similar to bone, dentin, and enamel. The basic mineral component of the cement is hydroxyapatite with amorphous calcium phosphate (Ca10 (PO4) 6 (OH) 2). The crystallinity of the cement inorganic component is lower than other calcifying tissues [16]. As a result, cement is decalcified more easily, while it has a tendency to coalesce for the adsorption of surrounding ions (i.e., fluoride). In general, the cement of adult mature teeth has higher fluoride content compared to other calcifying tissues. Mg content of the cement is about half of that in the dentin. There is a gradual increase in Mg in the deep layers of the cement [14].

## **5. Dental pulp**

A tooth consists of hard tissue (enamel, dentine, and cement) and soft tissue (pulp and periodontal ligaments), and has TE in its structure. A tooth has a multicellular structure which can

Enamel is the hard tissue that covers the surface of the tooth. The function of this layer is to protect dentine-pulp complex. Enamel is the hardest and most resistant tissue in the body. It consists of 95% inorganic material (calcium hydroxyapatite crystals), 2% organic material (proteins such as amylogenic, enameline, ameloblastin, and tuftelin, among others), and 3% water [6–8]. A negligible part of the 95% inorganic material is represented by TEs. As a result of the analyses conducted using different methods in tooth enameling several chemical components are observed. These components include phosphorus (P), calcium (Ca), magnesium (Mg), zinc (Zn), lead (Pb), cobalt (Co), fluorine (F), iron (Fe), aluminum (Al), and selenium (Se). Inorganic structure of enamel consists of 36.1 Ca, 17.3 P, 3.0 carbon oxide, 0.5 Mg, 0.2 Na, 0.3 potassium (K), 0.016 F, 0.1 sulfur (S), 0.01 copper (Cu), 0.016 Zn, 0.003 silicon (Si); and low levels of silver (Ag), strontium (Sr), barium (Ba), chromium (Cr), manganese (Mn), vanadium (V), aluminum (Al), lithium (Li), and selenium (Se). TE is placed in the human tooth enamel from the environment during and after the mineralization and maturation period of the tooth [9].

The dentin consists of 70% inorganic material (hydroxyapatite crystals and TEs), 18% organic material (type I collagen fiber and proteins such as osteonectin, osteopontin, osteoclastin-like dentin Gla protein, dentin phosphorene, dentin matrix protein, and dentin sialoprotein) and

The inorganic material of dentin contains about 40 elements, ranging from 1000 ppm (i.e., Zn,

The ratio of TE in human dentin varies according to age and sex. Cobalt can be higher in

Cementum is a special connective tissue that connects the periodontal ligament to the root

Cement is a vascular and unlimited mineralized tissue. It is the interface between dentin and periodontal ligament and contributes to the repair and renewal of periodontal tissue after

Sr., Fe, Al, B, Ba, Pb, etc.) to 100 ppb (i.e., Ni, Li, Ag, As, Se, Nb, Hg, etc.) [10–12].

surface, covering the outermost layer of the calcite matrix on the root surface [14].

cooperate functionally with maxillofacial area [5].

72 Trace Elements - Human Health and Environment

**2. Enamel**

**3. Dentin**

12% water [6–8].

**4. Cementum**

injury [15].

women, while lead can be higher in men [13].

Tooth pulp developing from dental papilla consists of odontoblast, fibroblast, blood, and neural veins [17]. Odontoblast cells are the most important cells of the pulp. As the cells responsible for the construction of dentin and pre-dentin, they are also responsible for reparative dentin make-up in pathological cases [5, 18]. Pulp cells, especially fibroblasts, produce several inflammatory mediators such as IL-8, IL-6, and vascular endothelial growth factor in cases which threaten the health of the pulp. Tooth pulp performs a series of biological activities such as nutrition, sensitiveness, construction, and protection. The change in blood pressure and flow in the veins coming from apical region essentially affects the health of the pulp. When tooth pulp is damaged due to mechanical, chemical, thermal and microbial irritants, local tissue reactions and lymphatic, vascular inflammatory responses occur. The existence of dentin affected by oral bacteria which lead to the formation of caries is most important reasons for pulp inflammation [19].

Dental caries is a microbiological infectious disease of the teeth that results in the destruction of dental calcified tissues. There must be three factors for the formation of dental caries; bacteria (from mutans Streptococci and Lactobacillus species), susceptible tooth surface (host), and nutrient (diet) to provide bacterial growth [20, 21]. Changes in the density of TEs due to some environmental and genetic impacts have some effects on human and dental health. The change in density of some elements can lead to dental caries. Based on the previous studies on humans and animals, some classifications have been made on the impact of TE on dental caries [4, 9].

**Cariostatic elements:** Fluoride (F), Phosphorus (P).

**Mildly cariostatic:** Mo, V, Cu, Sr., B, Li, Au.

**Doubtful:** Be, Co, Mn, Sn, Zn, Br, I.

**Caries inert:** Ba, Al, Ni, Fe, Pd, Ti.

**Caries promoting:** Se, Mg, Cd, Pt, Pb, Si.

In addition, there is another classification with regard to the impact of TE on dental health:

**Cariostatic elements**: Molybdenum, Vanadium, Fluoride, Strontium, Lithium.

**Caries-promoting elements**: Selenium, Cadmium, Lead, Manganese, Copper, Zinc.

The effect on dental and oral tissues of elements and their features.

## **6. Fluoride (F)**

Fluoride is an essential part of the organized matrix in hard tissues such as teeth and bones and is found in the form of fluorapatite. In addition, it can combine with calcium and stimulate osteoblastic activity. The daily recommended amount of fluoride is 0.7 mg for 1–3 ages, 1 mg for 4–8 ages, 2 mg for 9–13 ages, 3 mg for 14–18 ages, 4 mg for males above 18 years of age and 3 mg for females above 18 years of age.

calcium element in its properties; like calcium, it is taken up and is preferably implanted into the bone. Strontium may have both beneficial and deleterious effects on humans, depending

Relation of Trace Elements on Dental Health http://dx.doi.org/10.5772/intechopen.75899 75

High strontium content is related to low caries incidence. Epidemiological studies conducted on males determined that dental caries cases involve high strontium content. One study compared good and decayed enamels and found out that strontium was higher in the good enamel. In addition, it is reported that strontium ratio in tooth decreased with aging; stron-

Apatites with strontium settlement are more difficult to remove from enamel compared to pure calcium component and it is stated that solution of enamel remineralized without strontium is more difficult than enamel remineralized with strontium. For this reason, it is believed that strontium adds resistance to hydroxyapatites against the solution. Strontium settlement in tooth enamel makes apatite crystal more resistant to caries due to the hetero-ionic change of calcium. The resistance of apatite crystals against demineralization which occurs as a result

More than 90% of lithium is eliminated from the human body through the kidney. The human

Lithium exposure through drinking water and other environmental sources can also affect thyroid function. Lithium is used in the treatment of bipolar disorders. Lithium is found to have an indirect relationship with tooth caries. It is reported that dental caries incidence reduces in the existence of lithium. One study conducted to control the relation between lithium and dental caries determined that it reduced the dental caries incidence in humans [4]. It has also been reported the histopathologic changes in the structure of salivary glands [30]. Lithium exposure through drinking water and other environmental sources can also affect

Copper plays an essential role in our metabolism as it is involved in the functions of several

Copper is defined as a material which increases caries. In decayed teeth, a higher level of copper is found compared to healthy teeth. Higher caries prevalence is found to be related to the

Conducted studies showed that serum copper level is significantly higher in patients with oral leukoplakia and oral submucous fibrosis and also malignant tumors such as squamous

tium ratio is found to be higher in young people compared to the elderly [4].

serum has been reported to have a lithium half-life of less than 24 hours [29].

on the amount received [27].

of acid attacks is increased [28].

**9. Lithium (li)**

thyroid function [31**]**.

**10. Copper (cu)**

cell carcinoma [22].

critical enzymes [28, 32].

existence of copper in water, food, soil or vegetables [4].

Fluoride need of the body is met by drinking water, food, and tea [22].

The most well-known function of fluorine is a prevention of tooth caries. As a result of the changes created by this function dental structure, the resistance of enamel increases. In addition, it prevents the proliferation of bacteria in dental plaque. It also accelerates remineralization [4, 23].

However, during calcification of the teeth, it can lead to dental fluorosis due to excessive fluorine concentration. Dental fluorosis is a kind of enamel hypoplasia. Dental fluorosis has varying strengths from lesions in the form of white small spots on the enamel to loss of material in dental structure. The impact of excessive fluoride intake on the dental structure is a function of such factors as the fluoride concentration in drinking water, exposure amount, exposure period, and development period of the tooth [24, 25].

## **7. Vanadium (V)**

Vanadium is found naturally in the soil, water, and air. Natural sources of vanadium include continental dust, sea aerosol, and volcanic emissions. The release of vanadium is associated with industrial sources such as oil refineries and power plants, especially using vanadiumrich petroleum and coal. Most foods contain naturally low vanadium concentrations. Sea products generally contain vanadium at a higher concentration than the meat of land animals. Daily vanadium uptake was reported in the range of 0.01–0.02 mg [26].

Although the role of vanadium in the development of dental caries is not clear, animal studies showed that it reduces dental caries. It is seen that when hamsters on Cariogenic diet are given vanadium, the formation of dental caries decreased. In addition, a study conducted on rats reported that when they are given intraperitoneal vanadium, the formation of dental caries reduced. On the contrary, some studies on monkeys showed that monkeys fed with water including vanadium content suffered from higher dental caries incidents [4].

## **8. Strontium (Sr)**

Strontium is an element found everywhere in the environment. Stable and radioactive strontium compounds are used in many industrial processes and find applications in research and medical fields. Although strontium is not considered an important element and does not have a known biological role, it is present in all living organisms. Strontium resembles calcium element in its properties; like calcium, it is taken up and is preferably implanted into the bone. Strontium may have both beneficial and deleterious effects on humans, depending on the amount received [27].

High strontium content is related to low caries incidence. Epidemiological studies conducted on males determined that dental caries cases involve high strontium content. One study compared good and decayed enamels and found out that strontium was higher in the good enamel. In addition, it is reported that strontium ratio in tooth decreased with aging; strontium ratio is found to be higher in young people compared to the elderly [4].

Apatites with strontium settlement are more difficult to remove from enamel compared to pure calcium component and it is stated that solution of enamel remineralized without strontium is more difficult than enamel remineralized with strontium. For this reason, it is believed that strontium adds resistance to hydroxyapatites against the solution. Strontium settlement in tooth enamel makes apatite crystal more resistant to caries due to the hetero-ionic change of calcium. The resistance of apatite crystals against demineralization which occurs as a result of acid attacks is increased [28].

## **9. Lithium (li)**

**6. Fluoride (F)**

74 Trace Elements - Human Health and Environment

tion [4, 23].

**7. Vanadium (V)**

**8. Strontium (Sr)**

age and 3 mg for females above 18 years of age.

period, and development period of the tooth [24, 25].

Fluoride need of the body is met by drinking water, food, and tea [22].

Daily vanadium uptake was reported in the range of 0.01–0.02 mg [26].

including vanadium content suffered from higher dental caries incidents [4].

Fluoride is an essential part of the organized matrix in hard tissues such as teeth and bones and is found in the form of fluorapatite. In addition, it can combine with calcium and stimulate osteoblastic activity. The daily recommended amount of fluoride is 0.7 mg for 1–3 ages, 1 mg for 4–8 ages, 2 mg for 9–13 ages, 3 mg for 14–18 ages, 4 mg for males above 18 years of

The most well-known function of fluorine is a prevention of tooth caries. As a result of the changes created by this function dental structure, the resistance of enamel increases. In addition, it prevents the proliferation of bacteria in dental plaque. It also accelerates remineraliza-

However, during calcification of the teeth, it can lead to dental fluorosis due to excessive fluorine concentration. Dental fluorosis is a kind of enamel hypoplasia. Dental fluorosis has varying strengths from lesions in the form of white small spots on the enamel to loss of material in dental structure. The impact of excessive fluoride intake on the dental structure is a function of such factors as the fluoride concentration in drinking water, exposure amount, exposure

Vanadium is found naturally in the soil, water, and air. Natural sources of vanadium include continental dust, sea aerosol, and volcanic emissions. The release of vanadium is associated with industrial sources such as oil refineries and power plants, especially using vanadiumrich petroleum and coal. Most foods contain naturally low vanadium concentrations. Sea products generally contain vanadium at a higher concentration than the meat of land animals.

Although the role of vanadium in the development of dental caries is not clear, animal studies showed that it reduces dental caries. It is seen that when hamsters on Cariogenic diet are given vanadium, the formation of dental caries decreased. In addition, a study conducted on rats reported that when they are given intraperitoneal vanadium, the formation of dental caries reduced. On the contrary, some studies on monkeys showed that monkeys fed with water

Strontium is an element found everywhere in the environment. Stable and radioactive strontium compounds are used in many industrial processes and find applications in research and medical fields. Although strontium is not considered an important element and does not have a known biological role, it is present in all living organisms. Strontium resembles More than 90% of lithium is eliminated from the human body through the kidney. The human serum has been reported to have a lithium half-life of less than 24 hours [29].

Lithium exposure through drinking water and other environmental sources can also affect thyroid function. Lithium is used in the treatment of bipolar disorders. Lithium is found to have an indirect relationship with tooth caries. It is reported that dental caries incidence reduces in the existence of lithium. One study conducted to control the relation between lithium and dental caries determined that it reduced the dental caries incidence in humans [4]. It has also been reported the histopathologic changes in the structure of salivary glands [30].

Lithium exposure through drinking water and other environmental sources can also affect thyroid function [31**]**.

## **10. Copper (cu)**

Copper plays an essential role in our metabolism as it is involved in the functions of several critical enzymes [28, 32].

Copper is defined as a material which increases caries. In decayed teeth, a higher level of copper is found compared to healthy teeth. Higher caries prevalence is found to be related to the existence of copper in water, food, soil or vegetables [4].

Conducted studies showed that serum copper level is significantly higher in patients with oral leukoplakia and oral submucous fibrosis and also malignant tumors such as squamous cell carcinoma [22].

Copper concentration is found to be higher compared to the enamel of healthy and primary teeth than the enamel of decayed teeth [25].

concentrations varying from 0.3 to 2.9 ug manganese/g. Tissues rich in mitochondria and pigments (for example, retina, dark skin) tend to have high manganese concentrations. Bones, livers, pancreas, and kidneys typically have higher manganese concentrations than other tissues. The most important manganese store is in the bones. There are 49 elements in enamel hydroxyapatite crystals; one of them is manganese, which is usually in very slight percentages. The manganese concentrations in enamel are between 0.08 and 20 ppm, equivalent to 0.08–20 mg/kg and dentine between 0.6 and 1000 ppm. Mn concentration is at enamel-dentin limit at the external surface of

Relation of Trace Elements on Dental Health http://dx.doi.org/10.5772/intechopen.75899 77

Manganese is a TE which can be included in enamel through food, air, and water. In addition, Mn has the potential of changing Ca place at HAP. Several studies reported that Mn has the

Manganese is increasingly related to decay prevalence. One study found out that in areas where manganese content is higher, dental caries incidence in males increased. Therefore, it

There are 2–4 grams of zinc scattered throughout the human body. Zinc is stored in the prostate, eye parts, brain, muscles, bones, kidney, and liver. It is the second most abundant transition metal in organisms after iron and is the only metal seen in all enzyme classes. In blood plasma, zinc is carried and bound to albumin (60%) and transferrin (10%). Zinc concentration

The daily average requirement of zinc is 15–20 mg/day. Approximately 2–5 mg/day is expelled through pancreas and intestines. Plasma zinc level decreases in such cases as pregnancy, loss of liquid, oral contraceptive usage, blood loss, acute myocardial infarction, infections, and

Zinc plays an essential role in cell reproduction, differentiation, and metabolic activities. Zinc also supports normal growth in pregnancy, childhood, and adolescence periods [28, 42, 43]. Zinc is mostly found in animal products like meat, milk, and in fishes. Zinc bio adjustment is

The role of zinc in the development of dental caries is controversial. One study which analyzed the existence of TEs in children found zinc levels of children with more dental caries to be higher. In addition, it has been found out that zinc concentration in caries enamel of milk teeth was higher. However, another study showed that existence of zinc in saliva decreased

Contrary to the foregoing, zinc is added to oral health products in order to control plaques, reduce halitosis and delay tartar development. The zinc released from mouthwash solutions and toothpastes can continue to exist in plaque and saliva for a long time. Low zinc concentrations can reduce enamel demineralization. However, their anticariogenic impact is still controversial. Zinc deficiency is reported to be a potential risk factor for oral and

in blood plasma always remains unchanged regardless of the zinc intake [22, 39, 40].

enamel and higher at permanent dentition compared to primary dentition [29, 30].

ability to include in synthetic HAP without degrading the crystal area size [34, 38].

is emphasized that manganese encourages caries [4].

**13. Zinc (Zn)**

malignity [41].

low in phytonutrients [22].

the development of dental caries [4, 44, 45].

Recommended daily copper level is 340 mcg/day for 1–3 ages, 440 mcg/day for 4–8 ages, 700 mcg/day for 9–13 ages, 890 mcg/day for 14–18 ages, 900 mcg/day for males and females above 18 years of age, and 1000 mcg/day for pregnant and 1300 mcg/day for nursing women.

Copper is mostly found in oysters, sea animals with shell, whole grains, hazelnuts, potatoes, greeneries with dark leaves, dried fruits and animal products such as kidneys and livers [22].

## **11. Selenium (se)**

Selenium is a vital trace element which is an essential component of antioxidant enzymes. Selenium salts are required for several cellular functions in the human body but their excessive amount is toxic [33].

Selenium is found in liver, kidneys, sea products, meat, grains, grain products, milk products, fruits, and vegetables. The recommended daily intake is 70 micrograms [22].

Selenium is a non-metallic element which is found epidemically in nature and absorbed by the body through food or inhalation. Selenium is reported to be involved in synthetic hydroxyapatite as anionic Se+4 through phosphate change with selenite. The ionic radius of Se+4 (0.50 Å) is higher than P+5 (Phosphate) (0.35 Å) value. For this reason, the fabric parameter increases after it is settled in synthetic hydroxyapatites [34–36].

It is found out that selenium leads to structural changes in dental dentin and mandibular condyles. Increase in dental caries emerged in the case of selenium intake. Some studies show that there is a direct relationship between caries sensitiveness and selenium ejected with urine [4].

It is reported that selenium is settled in the micro-crystal structure of the enamel at the beginning of the decay and made it more sensitive toward dissolution [25].

In addition, it is reported that decrease in selenium level in the body leads to oxidative stresses. A recent study found out that patients with oral mucositis due to a high level of chemotherapy effectively reduced the term and seriousness of oral mucositis and, in addition, sufficient selenium reinforcement can produce cytoprotective impact and antiulcer activity [22, 37].

## **12. Manganese (Mn)**

Manganese content in food products varies considerably. It is highest in peanuts and grains; it is found in lowest concentration in milk products, meat, poultry, fish, and sea products. In addition, manganese can be found in coffee and tea which constitute 10% of daily intake. The body of an adult has 15 mg manganese on average which is typically seen in the nucleic acid. The daily requirement is approximately 2–5 mg/day. Manganese functions are considered as an enzyme activator and a part of metalloenzymes. Manganese is found in all mammal tissues at concentrations varying from 0.3 to 2.9 ug manganese/g. Tissues rich in mitochondria and pigments (for example, retina, dark skin) tend to have high manganese concentrations. Bones, livers, pancreas, and kidneys typically have higher manganese concentrations than other tissues. The most important manganese store is in the bones. There are 49 elements in enamel hydroxyapatite crystals; one of them is manganese, which is usually in very slight percentages. The manganese concentrations in enamel are between 0.08 and 20 ppm, equivalent to 0.08–20 mg/kg and dentine between 0.6 and 1000 ppm. Mn concentration is at enamel-dentin limit at the external surface of enamel and higher at permanent dentition compared to primary dentition [29, 30].

Manganese is a TE which can be included in enamel through food, air, and water. In addition, Mn has the potential of changing Ca place at HAP. Several studies reported that Mn has the ability to include in synthetic HAP without degrading the crystal area size [34, 38].

Manganese is increasingly related to decay prevalence. One study found out that in areas where manganese content is higher, dental caries incidence in males increased. Therefore, it is emphasized that manganese encourages caries [4].

## **13. Zinc (Zn)**

Copper concentration is found to be higher compared to the enamel of healthy and primary

Recommended daily copper level is 340 mcg/day for 1–3 ages, 440 mcg/day for 4–8 ages, 700 mcg/day for 9–13 ages, 890 mcg/day for 14–18 ages, 900 mcg/day for males and females above 18 years of age, and 1000 mcg/day for pregnant and 1300 mcg/day for nursing women. Copper is mostly found in oysters, sea animals with shell, whole grains, hazelnuts, potatoes, greeneries with dark leaves, dried fruits and animal products such as kidneys and livers [22].

Selenium is a vital trace element which is an essential component of antioxidant enzymes. Selenium salts are required for several cellular functions in the human body but their exces-

Selenium is found in liver, kidneys, sea products, meat, grains, grain products, milk products,

Selenium is a non-metallic element which is found epidemically in nature and absorbed by the body through food or inhalation. Selenium is reported to be involved in synthetic hydroxyapatite as anionic Se+4 through phosphate change with selenite. The ionic radius of Se+4 (0.50 Å) is higher than P+5 (Phosphate) (0.35 Å) value. For this reason, the fabric parameter

It is found out that selenium leads to structural changes in dental dentin and mandibular condyles. Increase in dental caries emerged in the case of selenium intake. Some studies show that there is a direct relationship between caries sensitiveness and selenium ejected with urine [4]. It is reported that selenium is settled in the micro-crystal structure of the enamel at the begin-

In addition, it is reported that decrease in selenium level in the body leads to oxidative stresses. A recent study found out that patients with oral mucositis due to a high level of chemotherapy effectively reduced the term and seriousness of oral mucositis and, in addition, sufficient selenium reinforcement can produce cytoprotective impact and antiulcer activity [22, 37].

Manganese content in food products varies considerably. It is highest in peanuts and grains; it is found in lowest concentration in milk products, meat, poultry, fish, and sea products. In addition, manganese can be found in coffee and tea which constitute 10% of daily intake. The body of an adult has 15 mg manganese on average which is typically seen in the nucleic acid. The daily requirement is approximately 2–5 mg/day. Manganese functions are considered as an enzyme activator and a part of metalloenzymes. Manganese is found in all mammal tissues at

fruits, and vegetables. The recommended daily intake is 70 micrograms [22].

increases after it is settled in synthetic hydroxyapatites [34–36].

ning of the decay and made it more sensitive toward dissolution [25].

teeth than the enamel of decayed teeth [25].

76 Trace Elements - Human Health and Environment

**11. Selenium (se)**

sive amount is toxic [33].

**12. Manganese (Mn)**

There are 2–4 grams of zinc scattered throughout the human body. Zinc is stored in the prostate, eye parts, brain, muscles, bones, kidney, and liver. It is the second most abundant transition metal in organisms after iron and is the only metal seen in all enzyme classes. In blood plasma, zinc is carried and bound to albumin (60%) and transferrin (10%). Zinc concentration in blood plasma always remains unchanged regardless of the zinc intake [22, 39, 40].

The daily average requirement of zinc is 15–20 mg/day. Approximately 2–5 mg/day is expelled through pancreas and intestines. Plasma zinc level decreases in such cases as pregnancy, loss of liquid, oral contraceptive usage, blood loss, acute myocardial infarction, infections, and malignity [41].

Zinc plays an essential role in cell reproduction, differentiation, and metabolic activities. Zinc also supports normal growth in pregnancy, childhood, and adolescence periods [28, 42, 43]. Zinc is mostly found in animal products like meat, milk, and in fishes. Zinc bio adjustment is low in phytonutrients [22].

The role of zinc in the development of dental caries is controversial. One study which analyzed the existence of TEs in children found zinc levels of children with more dental caries to be higher. In addition, it has been found out that zinc concentration in caries enamel of milk teeth was higher. However, another study showed that existence of zinc in saliva decreased the development of dental caries [4, 44, 45].

Contrary to the foregoing, zinc is added to oral health products in order to control plaques, reduce halitosis and delay tartar development. The zinc released from mouthwash solutions and toothpastes can continue to exist in plaque and saliva for a long time. Low zinc concentrations can reduce enamel demineralization. However, their anticariogenic impact is still controversial. Zinc deficiency is reported to be a potential risk factor for oral and periodontal patients. Parakeratotic changes in cheeks, tongue, and esophagus are indicators of zinc deficiency. Serum zinc level has been found to be at lower levels in patients with potentially premalignant disorders such as oral leukoplakia [22, 43, 46].

**16. Iron (Fe)**

Unlike other TE, iron (Fe) is abundant in nature and a biologically essential component of every living organism. Nevertheless, despite geological abundance, when oxygen contacts iron, hardly soluble oxides are created. For this reason, it is not easily received by organisms [55].

These are most common dietary resources for iron: liver, meat, poultry products and fish;

As an essential element, iron mostly enters the body with green vegetables. It is reported that

The amount of Fe is 4–5 gm in healthy individuals. Iron (Fe) is a trace metal that is necessary to ensure that almost all organisms survive. Participation in heme and iron–sulfur cluster containing proteins allows Fe to participate in a variety of vital functions such as oxygen

Some studies reported that iron can act as preventive for dental caries. The study reported

In addition, some iron addition products used for iron deficiency anemia are reported to have a cariostatic effect and delayed the emergence of dental caries in human teeth. As a result of iron deficiency, angular cheilitis, atrophic glossitis, diffused oral mucosal atrophy, candidal infections, oral premalignant lesions, and stomatitis can be seen in the oral region [22].

Besides all these explanations, some factors change the density of the TEs on the teeth such as exposure to electromagnetic fields, Wi-Fi, radio frequencies emitted from mobile phones, environmental pollution, excessive fertilization of soil, natural disasters, and dental anoma-

[1] Fraga CG. Relevance, essentiality and toxicity of trace elements in human health. Molecular

O to acidic drinks reduced mineral loss and human enamel

Relation of Trace Elements on Dental Health http://dx.doi.org/10.5772/intechopen.75899 79

cereals, green leafy vegetables, pulses, nuts, oilseeds, and dried fruits [22].

transport, DNA synthesis, metabolic energy, and cellular respiration [56].

.7H2

iron concentrations are low in enamel [34].

preserved the surface microhardness [57, 58].

Address all correspondence to: dtlider@hotmail.com

Aspects of Medicine. 2005;**26**(4-5):235-244

Faculty of Dentistry, Harran University, Pediatric Dentistry, Şanlıurfa, Turkey

that adding 2 mmol/L FeSO<sup>4</sup>

lies [3, 27, 49, 50].

**Author details**

**References**

Mehmet Sinan Doğan

## **14. Cadmium (Cd)**

Cadmium is found in some vegetables (leaved vegetables, potatoes, grains, and seeds) and animal food (liver and kidney). The moment cadmium enters the body, it accumulates in the liver and bones and is expelled very slowly (cadmium reference). Cadmium is an active element in soil and can be received by plants easily. As a result of being received by plants and entering the food chain or possibility of reaching water environment by being washed from the soil, it creates a significant environmental problem. In addition, the downward carriage of cadmium from the soil with chelating agents accelerates and it can lead to pollution in drinking and irrigation waters as it enters underground water sources [47, 48]. Exposure to cadmium is related to some various systematic health impacts such as kidney failure, skeleton disorders, and cardiovascular diseases [49]. Cadmium can be released from intraoral alloys in dentistry patients and accumulate in teeth and mouth tissues, which are strictly bound to metallothioneins [50]. The relation has been found between cadmium and the increase in decay prevalence. However, it is stated that settlement of cadmium in teeth after growth is not effective on caries. Some studies conducted on test animals indicate that there is a strong relationship between the formation of dental caries and cadmium intake in dental development period [4]. The increase of exposure to and dispersion of this toxic material is becoming increasingly important on the systematic and oral health of sensitive populations such as children [49].

#### **15. Lead (Pb)**

People can be exposed to lead through contaminated food and beverage, resulting from industrial activity [51]. Lead is added to the food chain especially through vegetables growing on contaminated soil. Lead can be transferred with plants and grass from contaminated soil, which potentially leads to the accumulation of toxic metals in vegetating ruminants and especially in cattle. The accumulation of lead creates toxic effects in cattle; it also leads to toxic effects in people who consume meat and milk contaminated with toxic metals [52].

It is a harmful and toxic metal for the human body. Lead has the ability to translocation with Ca+2 at the HAP of teeth. For this reason, it downsizes the HAP crystals [34, 53].

Lead is transferred to the tissues of the body such as teeth through environment or nutrition. It is determined to have an encouraging effect on dental caries. In addition, it is found out that lead increases the formation of enamel hypoplasia. A positive correlation has been found between saliva lead levels and decay development of children with early childhood decays. Thus, lead plays an essential role in the development of new caries lesions [4, 53, 54].

## **16. Iron (Fe)**

periodontal patients. Parakeratotic changes in cheeks, tongue, and esophagus are indicators of zinc deficiency. Serum zinc level has been found to be at lower levels in patients with

Cadmium is found in some vegetables (leaved vegetables, potatoes, grains, and seeds) and animal food (liver and kidney). The moment cadmium enters the body, it accumulates in the liver and bones and is expelled very slowly (cadmium reference). Cadmium is an active element in soil and can be received by plants easily. As a result of being received by plants and entering the food chain or possibility of reaching water environment by being washed from the soil, it creates a significant environmental problem. In addition, the downward carriage of cadmium from the soil with chelating agents accelerates and it can lead to pollution in drinking and irrigation waters as it enters underground water sources [47, 48]. Exposure to cadmium is related to some various systematic health impacts such as kidney failure, skeleton disorders, and cardiovascular diseases [49]. Cadmium can be released from intraoral alloys in dentistry patients and accumulate in teeth and mouth tissues, which are strictly bound to metallothioneins [50]. The relation has been found between cadmium and the increase in decay prevalence. However, it is stated that settlement of cadmium in teeth after growth is not effective on caries. Some studies conducted on test animals indicate that there is a strong relationship between the formation of dental caries and cadmium intake in dental development period [4]. The increase of exposure to and dispersion of this toxic material is becoming increasingly important on the systematic and oral health of sensitive

People can be exposed to lead through contaminated food and beverage, resulting from industrial activity [51]. Lead is added to the food chain especially through vegetables growing on contaminated soil. Lead can be transferred with plants and grass from contaminated soil, which potentially leads to the accumulation of toxic metals in vegetating ruminants and especially in cattle. The accumulation of lead creates toxic effects in cattle; it also leads to toxic

It is a harmful and toxic metal for the human body. Lead has the ability to translocation with

Lead is transferred to the tissues of the body such as teeth through environment or nutrition. It is determined to have an encouraging effect on dental caries. In addition, it is found out that lead increases the formation of enamel hypoplasia. A positive correlation has been found between saliva lead levels and decay development of children with early childhood decays.

effects in people who consume meat and milk contaminated with toxic metals [52].

Thus, lead plays an essential role in the development of new caries lesions [4, 53, 54].

Ca+2 at the HAP of teeth. For this reason, it downsizes the HAP crystals [34, 53].

potentially premalignant disorders such as oral leukoplakia [22, 43, 46].

**14. Cadmium (Cd)**

78 Trace Elements - Human Health and Environment

populations such as children [49].

**15. Lead (Pb)**

Unlike other TE, iron (Fe) is abundant in nature and a biologically essential component of every living organism. Nevertheless, despite geological abundance, when oxygen contacts iron, hardly soluble oxides are created. For this reason, it is not easily received by organisms [55].

These are most common dietary resources for iron: liver, meat, poultry products and fish; cereals, green leafy vegetables, pulses, nuts, oilseeds, and dried fruits [22].

As an essential element, iron mostly enters the body with green vegetables. It is reported that iron concentrations are low in enamel [34].

The amount of Fe is 4–5 gm in healthy individuals. Iron (Fe) is a trace metal that is necessary to ensure that almost all organisms survive. Participation in heme and iron–sulfur cluster containing proteins allows Fe to participate in a variety of vital functions such as oxygen transport, DNA synthesis, metabolic energy, and cellular respiration [56].

Some studies reported that iron can act as preventive for dental caries. The study reported that adding 2 mmol/L FeSO<sup>4</sup> .7H2 O to acidic drinks reduced mineral loss and human enamel preserved the surface microhardness [57, 58].

In addition, some iron addition products used for iron deficiency anemia are reported to have a cariostatic effect and delayed the emergence of dental caries in human teeth. As a result of iron deficiency, angular cheilitis, atrophic glossitis, diffused oral mucosal atrophy, candidal infections, oral premalignant lesions, and stomatitis can be seen in the oral region [22].

Besides all these explanations, some factors change the density of the TEs on the teeth such as exposure to electromagnetic fields, Wi-Fi, radio frequencies emitted from mobile phones, environmental pollution, excessive fertilization of soil, natural disasters, and dental anomalies [3, 27, 49, 50].

## **Author details**

Mehmet Sinan Doğan

Address all correspondence to: dtlider@hotmail.com

Faculty of Dentistry, Harran University, Pediatric Dentistry, Şanlıurfa, Turkey

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(SeO3


**Chapter 5**

Provisional chapter

**Trace Elements in the Human Milk**

Trace Elements in the Human Milk

Additional information is available at the end of the chapter

Human breast milk is considered to be the perfect food for infants, specifically adapted to their needs. Before birth, the mother transfers all the nutrients and bioactive components to the fetus through the placenta. After birth, these substances have to be transferred through colostrum and milk. In particular, human breast milk is supposed to provide all the essential trace elements that are required by the normal term newborn infant. Therefore, the composition of human breast milk and its changes during lactation is a topic of major importance and has been the subject for intensive research. Conversely, human milk can also be a transfer medium of undesirable (toxic) elements from the mother to the infant. An extensive review of the most recent literature was carried out focusing on the current trace elements levels and their changes during lactation. For several elements, there is a consistent knowledge of their characteristic concentrations throughout the various stages of lactation, their dependence on maternal nutritional status, inter-individual and geographical variability, metabolic pathways, inter-elemental relationships, and effects on child development. For

DOI: 10.5772/intechopen.76436

Keywords: breast milk, breastfeeding, micronutrients, trace elements, temporal changes

The nutritional requirements during breastfeeding are among the most important in human development. The production of 750–1000 ml of human milk per day represents the transfer from the mother to the infant of approximately 2100–2520 kJ, as energy-producing macronu-

Likewise, all the vitamins and minerals (both macrominerals and trace elements) needed to

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

support the child's growth and development are transferred in the same way.

many other elements, this knowledge does not exist or is quite limited.

Additional information is available at the end of the chapter

Manuel de Rezende Pinto and

Manuel de Rezende Pinto and

http://dx.doi.org/10.5772/intechopen.76436

Agostinho A. Almeida

Agostinho A. Almeida

Abstract

1. Introduction

trients (carbohydrates, proteins, and lipids) [1].

**Chapter 5** Provisional chapter

#### **Trace Elements in the Human Milk** Trace Elements in the Human Milk

Manuel de Rezende Pinto and Agostinho A. Almeida Manuel de Rezende Pinto and Agostinho A. Almeida

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76436

#### Abstract

Human breast milk is considered to be the perfect food for infants, specifically adapted to their needs. Before birth, the mother transfers all the nutrients and bioactive components to the fetus through the placenta. After birth, these substances have to be transferred through colostrum and milk. In particular, human breast milk is supposed to provide all the essential trace elements that are required by the normal term newborn infant. Therefore, the composition of human breast milk and its changes during lactation is a topic of major importance and has been the subject for intensive research. Conversely, human milk can also be a transfer medium of undesirable (toxic) elements from the mother to the infant. An extensive review of the most recent literature was carried out focusing on the current trace elements levels and their changes during lactation. For several elements, there is a consistent knowledge of their characteristic concentrations throughout the various stages of lactation, their dependence on maternal nutritional status, inter-individual and geographical variability, metabolic pathways, inter-elemental relationships, and effects on child development. For many other elements, this knowledge does not exist or is quite limited.

DOI: 10.5772/intechopen.76436

Keywords: breast milk, breastfeeding, micronutrients, trace elements, temporal changes

#### 1. Introduction

The nutritional requirements during breastfeeding are among the most important in human development. The production of 750–1000 ml of human milk per day represents the transfer from the mother to the infant of approximately 2100–2520 kJ, as energy-producing macronutrients (carbohydrates, proteins, and lipids) [1].

Likewise, all the vitamins and minerals (both macrominerals and trace elements) needed to support the child's growth and development are transferred in the same way.

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

According to current recommendations, the newborn should whenever possible be exclusively breastfed for the first 6 months of life [2]. Breast milk is then the only nutritional source of the child at this stage. Among these nutrients, macrominerals and essential trace elements are particularly noteworthy.

the baby and the child. Many of these micronutrients are also involved in the body's defense functions [1]. Minerals are closely related to the action of the remaining nutrients, affecting

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Contrarily to the generality of the macronutrients, it is documented that when maternal nutrition is inadequate, significant changes in milk composition may occur for some of its micronutrients (e.g., some fatty acids and vitamins, sodium, potassium, chloride, phosphorus, copper, zinc, manganese, and iron). The correlation between the levels of minerals in breast milk and the mother's diet is variable from element to element, being in some cases strongly

These changes have important implications for the growth and development of the breastfed infants [5]. It is also documented that there are differences regarding the milk composition

Little is known about the biochemical function of boron in human tissues. Signs of boron deficiency depend on the nutritional levels of aluminum, calcium, cholecalciferol, magnesium, methionine, and potassium. It affects calcium and magnesium concentrations in plasma and tissues, plasma alkaline phosphatase, and bone calcification [7]. The toxicity of boron is very low orally. There is no clear definition of symptoms of chronic boron intoxication in humans [7]. The levels of boron in breast milk seem to increase during lactation [8]. Average concen-

Chromium, as trivalent chromium, is an essential nutrient that enhances the action of insulin and, consequently, the metabolism of carbohydrates, lipids, and proteins. It has been suggested that the active form of chromium, the so-called "glucose tolerance factor," is a complex of chromium, nicotinic acid, and the amino acids glycine, cysteine, and glutamic acid. Biochemically, it affects the ability of the transmembrane insulin receptor to interact with insulin [7]. Chromium deficiency causes glucose intolerance similar to diabetes mellitus, elevated plasma free fatty acids levels, changes in nitrogen metabolism, weight loss, neuropathy, and respiratory depression [7]. The toxicity of trivalent chromium is so low that no effects of administering excessive amounts of this species could be observed. Hexavalent chromium, by contrast, is extremely toxic [7]. The concentrations of chromium in breast milk are very low and present a great variability [8, 9]. They may be increased during 21–89 and 90–180 days of

their absorption, metabolism, and excretion [1].

linked to the mother's intake and body stores [4].

between the different races [6].

3.1. Essential trace elements

3.1.1. Boron

3.1.2. Chromium

lactation [8, 9].

3. Trace elements in human milk

tration of 0.14 μg/L was observed in mature milk [8].

Maternal nutritional deficiencies in these elements may occur during lactation, affecting the mother's health. If this is reflected in the volume and quality of the milk, it will lead to nutritional deficiencies in the child, consequently affecting its development and health status.

The characterization of the composition of the human milk, in relation to these elements, is therefore of the utmost importance and has attracted much attention for many years.

On the other hand, breast milk can be a source of exposure of the child to various xenobiotics, including toxic trace elements.

A systematization of the current knowledge about the typical ("normal") levels of trace elements in human milk and the major factors responsible for their variability was the main objective of the present review. It is expected to be a useful tool for future studies and in the formulation of breast milk substitutes.

## 2. Human breast milk

Human milk is a complex secretion produced by the mammary glands of postpartum women [3]. It is generally assumed that the average volume of breast milk ingested by a nursing infant is about 600 ml/day.

#### 2.1. Macronutrients

The composition of breast milk in terms of macronutrients varies during lactation. According to a recent review [4], the mean concentration of main macronutrients in mature milk from full-term women is estimated to be approximately 0.9–1.2 g/dL for protein, 3.2–3.6 g/dL for lipids, and 6.7–7.8 g/dL for lactose. The total energy is estimated to be approximately 65–70 kcal/dL, and is highly correlated with the lipids content. These macronutrients have three different sources: synthesis in the lactocyte, dietary origin, and maternal stores [4].

The nutritional quality of breast milk tends to be highly conserved for most of the macronutrients, independently of the maternal diet [4].

#### 2.2. Micronutrients

Micronutrients, usually considered as the vitamins and the minerals (both the macrominerals and the trace elements), are fundamental for the proper development of the child. They are essential in the formation and regeneration of tissues, as well as in regulating most of the functions of the body's systems, leading its deficiency to disease, and serious malformations in the baby and the child. Many of these micronutrients are also involved in the body's defense functions [1]. Minerals are closely related to the action of the remaining nutrients, affecting their absorption, metabolism, and excretion [1].

Contrarily to the generality of the macronutrients, it is documented that when maternal nutrition is inadequate, significant changes in milk composition may occur for some of its micronutrients (e.g., some fatty acids and vitamins, sodium, potassium, chloride, phosphorus, copper, zinc, manganese, and iron). The correlation between the levels of minerals in breast milk and the mother's diet is variable from element to element, being in some cases strongly linked to the mother's intake and body stores [4].

These changes have important implications for the growth and development of the breastfed infants [5]. It is also documented that there are differences regarding the milk composition between the different races [6].

## 3. Trace elements in human milk

#### 3.1. Essential trace elements

#### 3.1.1. Boron

According to current recommendations, the newborn should whenever possible be exclusively breastfed for the first 6 months of life [2]. Breast milk is then the only nutritional source of the child at this stage. Among these nutrients, macrominerals and essential trace elements are

Maternal nutritional deficiencies in these elements may occur during lactation, affecting the mother's health. If this is reflected in the volume and quality of the milk, it will lead to nutritional deficiencies in the child, consequently affecting its development and health status. The characterization of the composition of the human milk, in relation to these elements, is

On the other hand, breast milk can be a source of exposure of the child to various xenobiotics,

A systematization of the current knowledge about the typical ("normal") levels of trace elements in human milk and the major factors responsible for their variability was the main objective of the present review. It is expected to be a useful tool for future studies and in the

Human milk is a complex secretion produced by the mammary glands of postpartum women [3]. It is generally assumed that the average volume of breast milk ingested by a nursing infant

The composition of breast milk in terms of macronutrients varies during lactation. According to a recent review [4], the mean concentration of main macronutrients in mature milk from full-term women is estimated to be approximately 0.9–1.2 g/dL for protein, 3.2–3.6 g/dL for lipids, and 6.7–7.8 g/dL for lactose. The total energy is estimated to be approximately 65–70 kcal/dL, and is highly correlated with the lipids content. These macronutrients have three different sources: synthesis in the lactocyte, dietary origin, and maternal stores [4].

The nutritional quality of breast milk tends to be highly conserved for most of the macronutri-

Micronutrients, usually considered as the vitamins and the minerals (both the macrominerals and the trace elements), are fundamental for the proper development of the child. They are essential in the formation and regeneration of tissues, as well as in regulating most of the functions of the body's systems, leading its deficiency to disease, and serious malformations in

therefore of the utmost importance and has attracted much attention for many years.

particularly noteworthy.

86 Trace Elements - Human Health and Environment

including toxic trace elements.

2. Human breast milk

is about 600 ml/day.

2.1. Macronutrients

2.2. Micronutrients

formulation of breast milk substitutes.

ents, independently of the maternal diet [4].

Little is known about the biochemical function of boron in human tissues. Signs of boron deficiency depend on the nutritional levels of aluminum, calcium, cholecalciferol, magnesium, methionine, and potassium. It affects calcium and magnesium concentrations in plasma and tissues, plasma alkaline phosphatase, and bone calcification [7]. The toxicity of boron is very low orally. There is no clear definition of symptoms of chronic boron intoxication in humans [7]. The levels of boron in breast milk seem to increase during lactation [8]. Average concentration of 0.14 μg/L was observed in mature milk [8].

#### 3.1.2. Chromium

Chromium, as trivalent chromium, is an essential nutrient that enhances the action of insulin and, consequently, the metabolism of carbohydrates, lipids, and proteins. It has been suggested that the active form of chromium, the so-called "glucose tolerance factor," is a complex of chromium, nicotinic acid, and the amino acids glycine, cysteine, and glutamic acid. Biochemically, it affects the ability of the transmembrane insulin receptor to interact with insulin [7]. Chromium deficiency causes glucose intolerance similar to diabetes mellitus, elevated plasma free fatty acids levels, changes in nitrogen metabolism, weight loss, neuropathy, and respiratory depression [7]. The toxicity of trivalent chromium is so low that no effects of administering excessive amounts of this species could be observed. Hexavalent chromium, by contrast, is extremely toxic [7]. The concentrations of chromium in breast milk are very low and present a great variability [8, 9]. They may be increased during 21–89 and 90–180 days of lactation [8, 9].


Time after delivery<sup>1</sup>

0.4 75–90 d 17

0.581 18–46 d M 73 0.320 4–6 mo M 100

0.0423 75–90 d 17

0.0049 1 mo M 19

0.127 2 w T 32 0.125 4 w M 22 0.123 6 w M 26 0.127 8 w M 22 0.108 12 w M 9

0.13094 1–7 w M 6 NAM 0.18687 2–6 w M 23 POL 0.21104 3–7 w M 21 ARG

Milk type2

0.16952 2–6 w M 20 USA ICP-MS [48] 2017

0.3–0.6 — ——— — [20] 2001 1.72 1–7 d C 50 BRA TXRF [76] 2002 1.19 — M — JPN ICP-AES [9] 2005 0.5 30–45 d M 31 USA (Texas) AAS [19] 2008

0.36 — — 27 IRN FAAS [77] 2015 0.558 5–17 d T 55 GTM ICP-MS [72] 2016

0.047 — — 12 AUS ICP-MS [51] 2016 1.27 2–6 w M 20 USA ICP-MS [48] 2017

0.0478 30–45 d M 31 USA (Texas) NAA [19] 2009

0.113 — — 12 AUS ICP-MS [51] 2016

0.000929 1–20 mo M 205 ARE ICP-MS [8] 2008 0.011 — M — JPN ICP-AES [9] 2005 0.0077 2 d C 34 PRT ICP-MS [12] 2008

0.012 5–17 d T 55 GTM ICP-MS [72] 2016

1.53 1–7 w M 6 NAM ICP-MS 1 2–6 w M 23 POL ICP-MS 0.99 3–7 w M 21 ARG ICP-MS

I 0.098–0.247 14 d–3.5 y — 14–24 — — [20] 2001

Mn 0.003–0.01 — ——— — [20] 2001

0.133 1 w C 44 KOR GFAAS

Fe 0.38<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

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Time after delivery<sup>1</sup>

0.00072 1 mo M 19

0.056 3 d C 41

0.498 1 mo M 19

0.489 2 w T 32 0.384 4 w M 22 0.356 6 w M 26 0.303 8 w M 22 0.301 12 w M 9

0.460 18–46 d M 73 0.262 4–6 mo M 100

Milk type2

B 0.000145 1–20 mo M 205 ARE ICP-MS [8] 2008 Co 0.00019<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

Cr 0.0243<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

Cu 0.4<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

0.0002–0.0007 — ——— — [20] 2001 0.00085 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 0.000009 1–20 mo M 205 ARE ICP-MS [8] 2008 0.00069 2 d C 34 PRT ICP-MS [12] 2008

0.0009–0.0012 0–7 mo — 6–45 — — [20] 2001 0.0108 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 0.017–0.076 1–365 d — — JPN ICP-AES [9] 2005 0.000689 1–20 mo M 205 ARE ICP-MS [8] 2008 0.000173 1–191 d M 79 JPN ICP-MS [75] 2008

0.11–0.62 1–293 d — 6–50 — — [20] 2001 0.54 1–7 d C 50 BRA TXRF [76] 2002 0.162 2 mo M 32 TUR FAAS [53] 2005 0.35 — M — JPN ICP-AES [9] 2005 0.066 1 mo M 41 IND ICP-AES [37] 2006

0.41519 1–20 mo M 205 ARE ICP-MS [8] 2008 0.403 — — 120 ARE ICP-MS [54] 2008 0.760 2 d C 34 PRT ICP-MS [12] 2008

0.3 — — 27 IRN FAAS [77] 2015 0.587 5–17 d T 55 GTM ICP-MS [72] 2016

0.220 — — 12 AUS ICP-MS [51] 2016

0.506 1 w C 44 KOR GFAAS

n Country<sup>3</sup> Analytical

technique4

(Zeeman)

[14] 2012

[Ref] year


Time after delivery<sup>1</sup>

0.276 1 mo M 41

2 75–90 d 17

2.785 1 mo M 19

9.1 2 w T 32 7.2 4 w M 22 8 6 w M 26 7.4 8 w M 22 6.6 12 w M 9

3.47 18–46 d M 73 1.44 4–6 mo M 100

0.03657 5–10 d T 45 0.01811 30–35 d M 45 0.01344 60–65 d M 45

0.0058 1 mo M 19

Milk type2

7.8 1 w C 44 KOR GFAAS

0.255 3 d C 41 IND ICP-AES [37] 2006

2.1 30–45 d M 31 USA (Texas) FAAS [19] 2008

1.468 1–20 mo M 205 ARE ICP-MS [8] 2008 2.730 — — 120 ARE ICP-MS [54] 2008 12.137 2 d C 34 PRT ICP-MS [12] 2008

2.34 — — 27 IRN FAAS [77] 2015 4.36 5–17 d T 55 GTM ICP-MS [72] 2016

1.390 — — 12 AUS ICP-MS [51] 2016

0.00078 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 0.000005 1–20 mo M 205 ARE ICP-MS [8] 2008

0.007056 1–20 mo M 205 ARE ICP-MS [8] 2008 0.05645 1–4 d C 45 TWN GFAAS [43] 2014

0.00025–0.003 — ——— — [20] 2001 0.000089 1–20 mo M 205 ARE ICP-MS [8] 2008 0.000196 — — 120 ARE ICP-MS [54] 2008 0.0078 2 d C 34 PRT ICP-MS [12] 2008

0.00150 1–4 d C 45 TWN GFAAS [43] 2014

Ag 0.00041<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

Al 0.067 — — 27 AUT ICP-SFMS [45] 2000

As 0.0067<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

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Time after delivery<sup>1</sup>

0.011 18–46 d M 73 0.0077 4–6 mo M 100

0.0157 75–90 d 17

0.0321 1 mo M 19

0.0114 2 w T 32 0.0127 4 w M 22 0.0114 6 w M 26 0.0108 8 w M 22 0.0105 12 w M 9

0.017 18–46 d M 73 0.013 4–6 mo M 100

Milk type2

0.0116 1–7 w M 6 NAM ICP-MS 0.00161 2–6 w M 23 POL ICP-MS 0.00762 3–7 w M 21 ARG ICP-MS

Mo 0.0002–0.017 1–293 d — 6–46 — — [29] 2000

Se 0.0056–0.08 1–869 d — 5–241 — — [29] 2000

0.00137 — — 12 AUS ICP-MS [51] 2016 0.00271 2–6 w M 20 USA ICP-MS [48] 2017

0.00072 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 0.000348 1–20 mo M 205 ARE ICP-MS [8] 2008 0.000542 1–191 d M 79 JPN ICP-MS [75] 2008 0.00037 — — 12 AUS ICP-MS [51] 2016

0.017<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000 0.0163 2–3 mo M 31 ESP ICP-AES [28] 2003 0.017 — M — JPN ICP-AES [9] 2005 0.010623 1–20 mo M 205 ARE ICP-MS [8] 2008 0.0159 30–45 d M 31 USA (Texas) NAA [19] 2008

0.0722 2 d C 34 PRT ICP-MS [12] 2008

0.017 5–17 d T 55 GTM ICP-MS [72] 2016

0.0143 — — 12 AUS ICP-MS [51] 2016

6.97 1–7 d C 50 BRA TXRF [76] 2002 1.2 2 mo M 32 TUR FAAS [53] 2005 1.45 — M — JPN ICP-AES [9] 2005

Zn 0.8–4.7 2 d–2 y — 6–71 — — [20] 2001

0.0118 1 w C 44 KOR GFAAS

n Country<sup>3</sup> Analytical

technique4

(Zeeman)

[14] 2012

[Ref] year


Time after delivery<sup>1</sup>

0.0058 1 mo M 19

0.00094 1 mo M 19

0.00892 5–10 d T 45 0.01172 30–35 d M 45 0.00293 60–65 d M 45

1.05 18–46 d M 73 0.810 4–6 mo M 100

Milk type2

0.00289–0.01333 — — 10–15 IRN DC/Au-amal [80] 2012 0.000008 1–20 mo M 205 ARE ICP-MS [8] 2008

0.000002 1–20 mo M 205 ARE ICP-MS [8] 2008

0.010–0.020 — ——— — [20] 2001 0.48 2 mo M 32 TUR FAAS [53] 2005 0.0076 2 d C 34 PRT ICP-MS [12] 2008

0.002581 1–20 mo M 205 ARE ICP-MS [8] 2008

0.000019 1–20 mo M 205 ARE ICP-MS [8] 2008 0.00151 — — 120 ARE ICP-MS [54] 2008 0.00155 2 d C 34 PRT ICP-MS [12] 2008

0.01322 1–4 d C 45 TWN GFAAS [43] 2014

0.00077 2–6 w M 20 USA ICP-MS [48] 2017

0.01817 — — 74 LBN GFAAS [47] 2018

32.176<sup>a</sup> 2–15 d T 40 IRN NAA [71] 2014 1.12 5–17 d T 55 GTM ICP-MS [72] 2016

Pt < 0.00001<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000 Rb 0.3–1.2 — ——— — [20] 2001

Ru 0.00015 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002

0.00215 1–7 w M 6 NAM ICP-MS 0.00102 2–6 w M 23 POL ICP-MS 0.00059 3–7 w M 21 ARG ICP-MS

Pb 0.001–0.005 — ——— — [20] 2001

La 0.00007 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002

Li 0.000005 1–20 mo M 205 ARE ICP-MS [8] 2008 Nb 0.00004 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 Ni 0.00079<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

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Time after delivery<sup>1</sup>

0.00068 5–10 d T 45 0.00027 30–35 d M 45 0.00016 60–65 d M 45

0.00065 5–10 d T 45 0.00049 30–35 d M 45 0.00034 60–65 d M 45

Milk type2

0.00668 1–7 w M 6 NAM ICP-MS 0.00386 2–6 w M 23 POL ICP-MS 0.00451 3–7 w M 21 ARG ICP-MS

0.00347 2–6 w M 20 USA ICP-MS [48] 2017

0.00236 — — 74 LBN GFAAS [47] 2018

0.0001–0.0021 — —— AUT ICP-SFMS [59] 2000

0.000097 2 mo M 32 TUR FAAS [53] 2005 0.000003 1–20 mo M 205 ARE ICP-MS [8] 2008 0.00027 — — 120 ARE ICP-MS [54] 2008 0.00137 1–4 d C 45 TWN GFAAS [43] 2014

0.00087 — — 74 LBN GFAAS [47] 2018

0.0000154 — — 11 DEU ICP-MS [56] 2010

0.000146–0.000237 — — 33 CAN — [79] 1997 0.001–0.003 — ——— — [20] 2001 0.000008 1–20 mo M 205 ARE ICP-MS [8] 2008 0.000115 — — 120 ARE ICP-MS [54] 2008

Ce 0.00012 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002

Cs 0.001–0.005 — ——— — [20] 2001 Ga 0.00052 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 Hg 0.000030–0.00062 — — 17 CAN CV-AFS [78] 1994

0.0000157 — — 51 DEU ICP-MS 0.0000139 — — 26 ESP ICP-MS

Au 0.00029<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

Ba 0.000017 1–20 mo M 205 ARE ICP-MS [8] 2008 Be 0.000008 1–20 mo M 205 ARE ICP-MS [8] 2008 Bi 0.000002 1–20 mo M 205 ARE ICP-MS [8] 2008 Br 0.812 — — 12 AUS ICP-MS [51] 2016 Cd <0.001 — ——— — [20] 2001

n Country<sup>3</sup> Analytical

technique4

[Ref] year


involved include the use of oxygen in cellular respiration and the synthesis of essential biomolecules such as the complex proteins of the skeleton and blood vessels connective tissues and a variety of neuroactive compounds of the central nervous system. It is estimated that an adult individual contains between 50 and 120 mg of copper in the whole body [7]. Copper in the blood is distributed by plasma and erythrocytes, of which 60% is associated with metalloenzyme Cu,Zn-superoxide dismutase (SOD), the rest being linked to other proteins and amino acids [7]. Plasma copper levels in the adult range between 0.8 and 1.2 mg/L and are not significantly influenced by feeding. In women, they are about 10% higher, and may be three times higher during pregnancy [7]. In plasma, about 93% of copper is bound to ceruloplasmin, a protein with multiple functions, particularly involved in the metabolism of iron [7]. In adulthood, copper deficiency is associated with hypochromic anemia, neutropenia, hypopigmentation, deficient bone formation with osteoporosis, and vascular deficiencies [7]. The deficiency of this element in the infant is related to identical symptoms [7, 14]. Copper is mainly accumulated in the liver, especially during the third trimester of pregnancy. Premature infants tend to have lower copper stores compared to full-term children [14]. There is a decrease in copper concentration in milk during lactation, reaching a minimum of 0.08– 0.10 mg/L in mature milk between 6 months and 1 year [15]. This trend is consistent with the majority of published results by other authors during this same period of lactation [8, 9, 12, 15]. One author reported a slight increase in copper concentration in breast milk in the first month, from 0.25–0.29 mg/L in colostrum to 0.37–0.41 mg/L in mature milk [15]. The effect of iron dietary supplementation of the mother in serum and milk cooper levels is not clear. Some authors reported a decrease in cooper milk associated with iron supplementation [16, 17], but a

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Iodine is an essential constituent of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). These hormones are involved in the regulation of various enzymes and metabolic processes. The organs most subject to its regulation are the developing brain, muscles, heart, pituitary gland, and kidneys [19, 20]. Iodine is thus absolutely essential for life in mammals [20]. The increase in perinatal mortality associated with iodine deficiency and a reduction in birth weight are described [7]. Since the newborn's brain has only reached about one-third of its size, continuing its accelerated growth until 2 years of age, thyroid hormones and iodine keep playing a key role throughout this period. Populations with severe iodine deficiencies present a high risk of mental retardation and cretinism [7, 19, 21]. Iodine adequate intake is 110 and 130 μg/day for first and second semester of life, respectively [22]. The concentration of iodine in breast milk is strongly correlated with the dietary intake of the mother [19]. The nutritional intake of iodine in the nursing woman (recommended dietary allowance) should be of 220 μg/day [22]. Iodine content in breast milk as shown to be strongly

Iron is an essential component of several proteins, including enzymes, cytochromes, myoglobin, and hemoglobin. Nearly two-thirds of body iron is found in the hemoglobin of circulating

noncorrelation have also been reported [18].

reduced by mother's smoking habits [23].

3.1.5. Iodine

3.1.6. Iron

1 d: day(s); w: week(s); mo: month(s); y: year(s); t: term; pt: pre-term.

2 C: colostrum; T: transition milk; M: mature milk.

3 Countries according to ISO 3166-1 A3.

4 ICP-MS: inductively coupled plasma mass spectrometry; ICP-SFMS: inductively coupled plasma sector field mass spectrometry; TXRF: total reflection X-ray fluorescence; FAAS: flame atomic absorption spectroscopy; ICP-AES: inductively coupled plasma atomic emission spectroscopy; GFAAS: graphite furnace atomic absorption spectroscopy; NAA: neutron activation analysis; CV-AFS: cold vapor atomic fluorescence spectroscopy; DC/Au-amal: direct combustion/Au amalgam.

a mg/kg, as dry matter.

b median.

Table 1. Summary of available data on trace element levels in human milk.

#### 3.1.3. Cobalt

Cobalt is a constituent of vitamin B12 and has been linked to the synthesis of antibodies and phagocytic activity in neutrophils and macrophages [10, 11]. Increased cobalt levels were observed throughout lactation [10, 12]. It has been speculated that this increase would be related to the increased needs arising from the infants' production of humoral antibodies, which starts during the third and fourth month of life, when the passage of the passive acquired immunity to active acquired immunity occurs [13]. Another study reported no significant changes in cobalt levels in breast milk throughout the various stages of lactation [8]. Plasma cobalt was higher in blood plasma of 12–14-week-old infants fed breast milk compared to healthy adults [8]. Cobalt levels in maternal plasma have a negative correlation with their concentration of breast milk [8].

#### 3.1.4. Copper

Copper is present in biological tissues mainly in the form of organic complexes, most of them being enzymatic systems. The metabolic processes in which copper dependent enzymes are involved include the use of oxygen in cellular respiration and the synthesis of essential biomolecules such as the complex proteins of the skeleton and blood vessels connective tissues and a variety of neuroactive compounds of the central nervous system. It is estimated that an adult individual contains between 50 and 120 mg of copper in the whole body [7]. Copper in the blood is distributed by plasma and erythrocytes, of which 60% is associated with metalloenzyme Cu,Zn-superoxide dismutase (SOD), the rest being linked to other proteins and amino acids [7]. Plasma copper levels in the adult range between 0.8 and 1.2 mg/L and are not significantly influenced by feeding. In women, they are about 10% higher, and may be three times higher during pregnancy [7]. In plasma, about 93% of copper is bound to ceruloplasmin, a protein with multiple functions, particularly involved in the metabolism of iron [7]. In adulthood, copper deficiency is associated with hypochromic anemia, neutropenia, hypopigmentation, deficient bone formation with osteoporosis, and vascular deficiencies [7]. The deficiency of this element in the infant is related to identical symptoms [7, 14]. Copper is mainly accumulated in the liver, especially during the third trimester of pregnancy. Premature infants tend to have lower copper stores compared to full-term children [14]. There is a decrease in copper concentration in milk during lactation, reaching a minimum of 0.08– 0.10 mg/L in mature milk between 6 months and 1 year [15]. This trend is consistent with the majority of published results by other authors during this same period of lactation [8, 9, 12, 15]. One author reported a slight increase in copper concentration in breast milk in the first month, from 0.25–0.29 mg/L in colostrum to 0.37–0.41 mg/L in mature milk [15]. The effect of iron dietary supplementation of the mother in serum and milk cooper levels is not clear. Some authors reported a decrease in cooper milk associated with iron supplementation [16, 17], but a noncorrelation have also been reported [18].

#### 3.1.5. Iodine

3.1.3. Cobalt

1

2

3

4

b median.

amalgam. a

mg/kg, as dry matter.

Element Average or interval (mg/L)

94 Trace Elements - Human Health and Environment

Time after delivery<sup>1</sup>

0.046 18–46 d M 73 0.046 4–6 mo M 100

d: day(s); w: week(s); mo: month(s); y: year(s); t: term; pt: pre-term.

Table 1. Summary of available data on trace element levels in human milk.

C: colostrum; T: transition milk; M: mature milk.

Countries according to ISO 3166-1 A3.

Milk type2

Sb 0.00014 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002

Sc 0.00019<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000 Sn <0.001- < 0.002 — ——— — [20] 2001 Sr 0.044 5–17 d T 55 GTM ICP-MS [72] 2016

Ti 0.0063<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

Th 0.00002 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 U 0.00003 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002 V 0.00018<sup>b</sup> — — 27 AUT ICP-SFMS [45] 2000

0.270 2–8 w M 19 CZE DEU POL ICP-MS [46] 2002

ICP-MS: inductively coupled plasma mass spectrometry; ICP-SFMS: inductively coupled plasma sector field mass spectrometry; TXRF: total reflection X-ray fluorescence; FAAS: flame atomic absorption spectroscopy; ICP-AES: inductively coupled plasma atomic emission spectroscopy; GFAAS: graphite furnace atomic absorption spectroscopy; NAA: neutron activation analysis; CV-AFS: cold vapor atomic fluorescence spectroscopy; DC/Au-amal: direct combustion/Au

0.000352 1–20 mo M 205 ARE ICP-MS [8] 2008

n Country<sup>3</sup> Analytical

technique4

[Ref] year

3.1.4. Copper

concentration of breast milk [8].

Cobalt is a constituent of vitamin B12 and has been linked to the synthesis of antibodies and phagocytic activity in neutrophils and macrophages [10, 11]. Increased cobalt levels were observed throughout lactation [10, 12]. It has been speculated that this increase would be related to the increased needs arising from the infants' production of humoral antibodies, which starts during the third and fourth month of life, when the passage of the passive acquired immunity to active acquired immunity occurs [13]. Another study reported no significant changes in cobalt levels in breast milk throughout the various stages of lactation [8]. Plasma cobalt was higher in blood plasma of 12–14-week-old infants fed breast milk compared to healthy adults [8]. Cobalt levels in maternal plasma have a negative correlation with their

Copper is present in biological tissues mainly in the form of organic complexes, most of them being enzymatic systems. The metabolic processes in which copper dependent enzymes are Iodine is an essential constituent of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). These hormones are involved in the regulation of various enzymes and metabolic processes. The organs most subject to its regulation are the developing brain, muscles, heart, pituitary gland, and kidneys [19, 20]. Iodine is thus absolutely essential for life in mammals [20]. The increase in perinatal mortality associated with iodine deficiency and a reduction in birth weight are described [7]. Since the newborn's brain has only reached about one-third of its size, continuing its accelerated growth until 2 years of age, thyroid hormones and iodine keep playing a key role throughout this period. Populations with severe iodine deficiencies present a high risk of mental retardation and cretinism [7, 19, 21]. Iodine adequate intake is 110 and 130 μg/day for first and second semester of life, respectively [22]. The concentration of iodine in breast milk is strongly correlated with the dietary intake of the mother [19]. The nutritional intake of iodine in the nursing woman (recommended dietary allowance) should be of 220 μg/day [22]. Iodine content in breast milk as shown to be strongly reduced by mother's smoking habits [23].

#### 3.1.6. Iron

Iron is an essential component of several proteins, including enzymes, cytochromes, myoglobin, and hemoglobin. Nearly two-thirds of body iron is found in the hemoglobin of circulating red blood cells, involved in oxygen transport. About 25% is stored as rapidly mobilizable reserves and the remaining 15% is in muscle myoglobin [22]. The individual iron reserves have a great influence on its absorption. Elevated reserves inhibit the gastrointestinal absorption of iron. Absorption occurs in the small intestine [22]. Iron deficiency results in anemia, which represents the most frequent nutritional deficiency of essential elements. The main symptoms are reduced work capacity and delayed psychomotor development in the child, with cognitive deficit [22]. Anemia is also the most prevalent disease in children [24]. During the first 6 months after delivery, iron concentration in breast milk is relatively stable, ranging from 0.21 to 0.27 mg/L. Some authors have reported that, after this period, iron levels in breast milk tend to fall very significantly to values between 0.08 and 0.10 mg/L [15]. Other authors observed quite constant levels throughout lactation [9]. No differences were found associated with the mother's age, number of children, or number of previously breastfed infants [15]. Likewise, the iron content in the maternal diet did not showed a significant correlation with iron concentration in breast milk [19, 25].

3.1.9. Selenium

of the mother and the lactation phase [12, 28, 35].

3.1.10. Zinc

The best known metabolic role of selenium in mammals was as a component of the enzyme glutathione peroxidase, which, together with vitamin E, catalase, and superoxide dismutase, is a key player of the body's antioxidant defense system. More recently, its role in Pselenoprotein has been discovered, and there is also increasing evidence of the involvement of a selenoprotein in the synthesis of the hormone triiodothyronine from thyroxine [20, 27]. Keshan's disease is a cardiomyopathy associated with selenium deficiency, which mainly affects children and women of childbearing age. Also, Kashin-Beck disease, an osteoarthropathy, is associated with selenium deficiency in soils (and consequently in food), affecting children between 5 and 13 years [7, 28, 29]. Chronic selenium toxicity is characterized primarily by hair loss and changes in nail morphology. In some cases, there is the development of skin lesions and central nervous system disturbances, being the biochemical mechanism unknown [20, 28]. There is evidence of a negative correlation between the supply of selenium and the prevalence of breast, prostate, colon, pancreatic, lung, and bladder cancer [30]. The concentration of selenium in breast milk is directly dependent on the mother's selenium dietary intake [9, 31– 33]. In one study, the selenium breast milk concentrations were studied according to the region of residence of the lactating mother. Two selenium-rich regions (Portuguesa) and one control region (Yaracuy) were compared. A significant increase of selenium was observed, from 42.9 μg/L for the control region to 56.6 and 112.2 μg/L for the two seleniferous regions [31]. Other authors reported selenium levels in the first month between 12.7 and 32.1 μg/L [12, 14, 19]. There appears to be a significant inverse correlation between selenium and zinc concentrations in breast milk. Also, mothers with higher dietary selenium intakes present lower concentrations of zinc in breast milk [31]. Studies of zinc binding compounds in breastmilk allowed the identification of six compounds with affinity for selenium too. It was possible to identify one of those compounds as the citrate, which is the main low molecular weight zinc binder. The decrease in zinc concentration in breast milk is then related to the concentration of citrate, which in turn depends on the concentration of selenium in plasma. High levels of selenium observed in seleniferous regions induced reduction of citrate in milk, probably by the suppression of the mechanism of citrate production in the Golgi apparatus described by Linzell [31, 34]. In the case of premature infants, since their reserves of selenium at birth are low, it is of the utmost importance the supply of this element in adequate concentrations through breast milk, which is clearly dependent, as mentioned above, on geographical aspects, the nutritional intake

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In the cell, zinc is involved in catalytic, structural, and regulatory functions [20]. The biochemical role of zinc results from its presence in hundreds of enzymatic systems and as a stabilizer of the molecular structure of cellular substructures. Examples of zinc metalloenzymes are RNA polymerase, alcohol dehydrogenase, carbonic anhydrase, and alkaline phosphatase [20]. Zinc is involved in the synthesis and degradation of carbohydrates, lipids, proteins, and nucleic acids. It plays an essential role in gene expression [7]. The skeletal muscle contains about 60% of the total zinc of the body, being the bone mass

#### 3.1.7. Manganese

Manganese is an essential nutrient, involved in the formation of bone tissue and in specific reactions related to the metabolism of amino acids, cholesterol, and carbohydrates. Manganese metalloenzymes include arginase, glutamine synthetase, phosphoenolpyruvate decarboxylase, and Mn-superoxide dismutase (Mn-SOD), the mitochondrial correspondent to Cu,Zn-SOD (cytoplasmic) [7]. Only a small part of the manganese ingested is absorbed. Absorption occurs through an active transport process [7]. Although there are symptoms associated with manganese deficiency, it was not possible to clearly establish a relationship between low dietary intakes and health problems. Manganese toxicity causes central nervous system effects similar to those of Parkinson's disease [14, 22]. In one study in Japanese women, the concentrations of manganese in breast milk showed a very low variability throughout the lactation period [9]. Other studies however reported that breast milk concentrations decrease throughout lactation [8]. Also, a direct correlation between maternal plasma levels and breast milk concentration has been observed [8]. A significant reduction in milk manganese content during lactation was also observed in a small study of 29 wellnourished mothers in the United Arab Emirates [8, 12].

#### 3.1.8. Molybdenum

Molybdenum is a cofactor of a small number of enzymes, such as sulfite oxidase, xanthine oxidase, and aldehyde oxidase, involved in the catabolism of sulfur amino acids and heterocyclic compounds such as purines and pyrimidines [20]. In all Mo-enzymes, functional molybdenum is present in the form of an organic component, molybdopterin [20]. Dietary molybdenum deficiencies are associated with growth problems, neurological disorders, and premature death [26]. On the other hand, its excess is associated with an increase in susceptibility to gout, hyperuricemia, and xanthuria [26]. Concentrations of molybdenum in breast milk increase significantly during lactation [8].

#### 3.1.9. Selenium

red blood cells, involved in oxygen transport. About 25% is stored as rapidly mobilizable reserves and the remaining 15% is in muscle myoglobin [22]. The individual iron reserves have a great influence on its absorption. Elevated reserves inhibit the gastrointestinal absorption of iron. Absorption occurs in the small intestine [22]. Iron deficiency results in anemia, which represents the most frequent nutritional deficiency of essential elements. The main symptoms are reduced work capacity and delayed psychomotor development in the child, with cognitive deficit [22]. Anemia is also the most prevalent disease in children [24]. During the first 6 months after delivery, iron concentration in breast milk is relatively stable, ranging from 0.21 to 0.27 mg/L. Some authors have reported that, after this period, iron levels in breast milk tend to fall very significantly to values between 0.08 and 0.10 mg/L [15]. Other authors observed quite constant levels throughout lactation [9]. No differences were found associated with the mother's age, number of children, or number of previously breastfed infants [15]. Likewise, the iron content in the maternal diet did not showed a significant correlation with iron concentra-

Manganese is an essential nutrient, involved in the formation of bone tissue and in specific reactions related to the metabolism of amino acids, cholesterol, and carbohydrates. Manganese metalloenzymes include arginase, glutamine synthetase, phosphoenolpyruvate decarboxylase, and Mn-superoxide dismutase (Mn-SOD), the mitochondrial correspondent to Cu,Zn-SOD (cytoplasmic) [7]. Only a small part of the manganese ingested is absorbed. Absorption occurs through an active transport process [7]. Although there are symptoms associated with manganese deficiency, it was not possible to clearly establish a relationship between low dietary intakes and health problems. Manganese toxicity causes central nervous system effects similar to those of Parkinson's disease [14, 22]. In one study in Japanese women, the concentrations of manganese in breast milk showed a very low variability throughout the lactation period [9]. Other studies however reported that breast milk concentrations decrease throughout lactation [8]. Also, a direct correlation between maternal plasma levels and breast milk concentration has been observed [8]. A significant reduction in milk manganese content during lactation was also observed in a small study of 29 well-

Molybdenum is a cofactor of a small number of enzymes, such as sulfite oxidase, xanthine oxidase, and aldehyde oxidase, involved in the catabolism of sulfur amino acids and heterocyclic compounds such as purines and pyrimidines [20]. In all Mo-enzymes, functional molybdenum is present in the form of an organic component, molybdopterin [20]. Dietary molybdenum deficiencies are associated with growth problems, neurological disorders, and premature death [26]. On the other hand, its excess is associated with an increase in susceptibility to gout, hyperuricemia, and xanthuria [26]. Concentrations of molybdenum in breast milk increase

tion in breast milk [19, 25].

96 Trace Elements - Human Health and Environment

nourished mothers in the United Arab Emirates [8, 12].

3.1.7. Manganese

3.1.8. Molybdenum

significantly during lactation [8].

The best known metabolic role of selenium in mammals was as a component of the enzyme glutathione peroxidase, which, together with vitamin E, catalase, and superoxide dismutase, is a key player of the body's antioxidant defense system. More recently, its role in Pselenoprotein has been discovered, and there is also increasing evidence of the involvement of a selenoprotein in the synthesis of the hormone triiodothyronine from thyroxine [20, 27]. Keshan's disease is a cardiomyopathy associated with selenium deficiency, which mainly affects children and women of childbearing age. Also, Kashin-Beck disease, an osteoarthropathy, is associated with selenium deficiency in soils (and consequently in food), affecting children between 5 and 13 years [7, 28, 29]. Chronic selenium toxicity is characterized primarily by hair loss and changes in nail morphology. In some cases, there is the development of skin lesions and central nervous system disturbances, being the biochemical mechanism unknown [20, 28]. There is evidence of a negative correlation between the supply of selenium and the prevalence of breast, prostate, colon, pancreatic, lung, and bladder cancer [30]. The concentration of selenium in breast milk is directly dependent on the mother's selenium dietary intake [9, 31– 33]. In one study, the selenium breast milk concentrations were studied according to the region of residence of the lactating mother. Two selenium-rich regions (Portuguesa) and one control region (Yaracuy) were compared. A significant increase of selenium was observed, from 42.9 μg/L for the control region to 56.6 and 112.2 μg/L for the two seleniferous regions [31]. Other authors reported selenium levels in the first month between 12.7 and 32.1 μg/L [12, 14, 19]. There appears to be a significant inverse correlation between selenium and zinc concentrations in breast milk. Also, mothers with higher dietary selenium intakes present lower concentrations of zinc in breast milk [31]. Studies of zinc binding compounds in breastmilk allowed the identification of six compounds with affinity for selenium too. It was possible to identify one of those compounds as the citrate, which is the main low molecular weight zinc binder. The decrease in zinc concentration in breast milk is then related to the concentration of citrate, which in turn depends on the concentration of selenium in plasma. High levels of selenium observed in seleniferous regions induced reduction of citrate in milk, probably by the suppression of the mechanism of citrate production in the Golgi apparatus described by Linzell [31, 34]. In the case of premature infants, since their reserves of selenium at birth are low, it is of the utmost importance the supply of this element in adequate concentrations through breast milk, which is clearly dependent, as mentioned above, on geographical aspects, the nutritional intake of the mother and the lactation phase [12, 28, 35].

#### 3.1.10. Zinc

In the cell, zinc is involved in catalytic, structural, and regulatory functions [20]. The biochemical role of zinc results from its presence in hundreds of enzymatic systems and as a stabilizer of the molecular structure of cellular substructures. Examples of zinc metalloenzymes are RNA polymerase, alcohol dehydrogenase, carbonic anhydrase, and alkaline phosphatase [20]. Zinc is involved in the synthesis and degradation of carbohydrates, lipids, proteins, and nucleic acids. It plays an essential role in gene expression [7]. The skeletal muscle contains about 60% of the total zinc of the body, being the bone mass responsible for about 30% [36]. Severe zinc deficiency is associated with clinical symptoms such as delayed growth, delayed sexual and skeletal maturation, dermatitis, diarrhea, alopecia, poor appetite, behavioral changes, and increased susceptibility to disease, as result of immune system malfunction [20, 37]. Generally, mild zinc deficiency occurs without a convenient diagnosis. When detected, it is usually related to a reduced growth rate and poor resistance to infections. Acute zinc poisoning is rare. The manifestations are nausea, vomiting, diarrhea, fever, and lethargy. In the case of chronic exposure to zinc, interference with other elements, particularly with copper, occurs. The zinc/copper interaction causes an excess of zinc to result in a reduction of copper levels (by decreasing its absorption at the gastrointestinal tract) [7]. Zinc metabolism is still subject to interference with other elements. For example, if iron is present at high levels in diet, it may decrease the absorption of zinc, and this aspect should be considered when, during pregnancy and lactation, the control of iron levels is attempted through the use of dietary supplements. Other authors observed that dietary supplementation of the mother with iron did not significantly interfere with their plasma zinc levels nor with zinc levels in breast milk [18, 19, 38]. Also, calcium and phosphorus may interfere with the absorption of zinc, and there are also contradictory studies of the effect of these two elements [20]. Due to the abovementioned zinc/copper antagonism, high concentrations of zinc in breast milk may result in copper deficiency, since zinc can competitively inhibit copper absorption in the gastrointestinal tract of the child. Metallothionein appears to play a relevant role in this process [39]. As for selenium, zinc is accumulated by the fetus during the third trimester of pregnancy [14]. Zinc may be about 15 times more concentrated in breast milk than in the mother's plasma, evidencing an active transport mechanism, and its essential role for the child's development [12, 31]. There is a rapid and significant decrease in the concentration of zinc in breast milk throughout the lactation period, significantly during the first month [12]. This evidence is supported by several published studies [8, 14, 15, 40]. There were no differences associated with the mother's number of children or lactation history. Regarding the age of the mother, it was found that the concentration of zinc tends to increase (higher values for ages greater than 30 years) [15]. Significantly higher zinc values were observed in breast milk between the third and seventh days in mothers with preterm infants, compared to those with full-term infants [37].

3.2.2. Antimony

3.2.3. Arsenic

3.2.4. Barium

3.2.5. Beryllium

3.2.6. Bismuth

3.2.7. Bromide

3.2.8. Cadmium

tion of 0.812 mg/L [51].

been reported in breast milk [8].

associated with cereal and fish intake [47].

The calculated transfer factor from food into human milk, calculated as the element concentration in food (g/kg) divided by the element concentration in milk (g/L), has been determined as 13.2 [46]. The reported concentration in human breast milk is between 0.14 and 0.35 μg/L.

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Arsenic occurs in trivalent and pentavalent forms in food, water, and the environment. The biological effects of arsenic strongly depend on the actual chemical specie it is present, with inorganic forms being more toxic than most organic forms. Inorganic forms are methylated in the human body, giving rise to less toxic compounds, which are then excreted through urine [7]. Acute intoxication with arsenic is rare and is characterized by nausea, vomiting, diarrhea, and acute abdominal pain. Chronic intoxication is caused by exposure to natural sources, contaminated food, or water, or other accidental source [7]. In a recent study, presence of arsenic was observed in 63.51% of samples, with an average concentration of 2.36 μg/L [47], consistent with results from other authors [12, 20, 45, 48]. Arsenic presence in human milk is

No nutritional requirements are set for barium in mammals, and its origin is probably associated with plant sources, following metabolic pathways similar to the elements of the same group in the periodic table, such as calcium and strontium [11]. Barium levels in milk may be

Beryllium compounds are very toxic [49], and chronic beryllium disease has been observed in children living near a beryllium factory [50]. There is a high probability that this element can be transferred from mother to child through breast milk [50]. Concentration of 0.008 μg/L has

Described as having an important function in tissue development, cellular structure and membrane integrity, its presence in breast milk was reported in a recent study at a concentra-

The main source of exposure to cadmium is tobacco smoke. It is estimated that about 10% of mothers smoke during pregnancy, and the percentage of those who return to smoking during

dependent of mother diet [11]. Concentrations of 0.017 μg/L have been reported [8].

This element has been reported in breast milk at a concentration of 0.002 μg/L [8].

#### 3.2. Non-essential/toxic trace elements

#### 3.2.1. Aluminum

Brain function of children exposed to this metal can be seriously affected [41]. Relevant sources of exposure are not only breast milk of mothers exposed to relevant levels of aluminum, but also infant Al-adjuvanted vaccination, like hepatitis B [41, 42]. Aluminum milk levels decrease significantly over the nursing, ranging from 0.056 mg/L in colostrum to 0.013 mg/L in mature milk [43]. Older mothers' (>25 years) milk present a higher aluminum concentration than the younger ones [44]. There is no relevant data available related to aluminum exposure during early life, and its correlation with brain function. Concentrations in human milk varying between 7.056 and 67 μg/L are described [8, 43, 45].

#### 3.2.2. Antimony

responsible for about 30% [36]. Severe zinc deficiency is associated with clinical symptoms such as delayed growth, delayed sexual and skeletal maturation, dermatitis, diarrhea, alopecia, poor appetite, behavioral changes, and increased susceptibility to disease, as result of immune system malfunction [20, 37]. Generally, mild zinc deficiency occurs without a convenient diagnosis. When detected, it is usually related to a reduced growth rate and poor resistance to infections. Acute zinc poisoning is rare. The manifestations are nausea, vomiting, diarrhea, fever, and lethargy. In the case of chronic exposure to zinc, interference with other elements, particularly with copper, occurs. The zinc/copper interaction causes an excess of zinc to result in a reduction of copper levels (by decreasing its absorption at the gastrointestinal tract) [7]. Zinc metabolism is still subject to interference with other elements. For example, if iron is present at high levels in diet, it may decrease the absorption of zinc, and this aspect should be considered when, during pregnancy and lactation, the control of iron levels is attempted through the use of dietary supplements. Other authors observed that dietary supplementation of the mother with iron did not significantly interfere with their plasma zinc levels nor with zinc levels in breast milk [18, 19, 38]. Also, calcium and phosphorus may interfere with the absorption of zinc, and there are also contradictory studies of the effect of these two elements [20]. Due to the abovementioned zinc/copper antagonism, high concentrations of zinc in breast milk may result in copper deficiency, since zinc can competitively inhibit copper absorption in the gastrointestinal tract of the child. Metallothionein appears to play a relevant role in this process [39]. As for selenium, zinc is accumulated by the fetus during the third trimester of pregnancy [14]. Zinc may be about 15 times more concentrated in breast milk than in the mother's plasma, evidencing an active transport mechanism, and its essential role for the child's development [12, 31]. There is a rapid and significant decrease in the concentration of zinc in breast milk throughout the lactation period, significantly during the first month [12]. This evidence is supported by several published studies [8, 14, 15, 40]. There were no differences associated with the mother's number of children or lactation history. Regarding the age of the mother, it was found that the concentration of zinc tends to increase (higher values for ages greater than 30 years) [15]. Significantly higher zinc values were observed in breast milk between the third and seventh days in mothers with pre-

term infants, compared to those with full-term infants [37].

Brain function of children exposed to this metal can be seriously affected [41]. Relevant sources of exposure are not only breast milk of mothers exposed to relevant levels of aluminum, but also infant Al-adjuvanted vaccination, like hepatitis B [41, 42]. Aluminum milk levels decrease significantly over the nursing, ranging from 0.056 mg/L in colostrum to 0.013 mg/L in mature milk [43]. Older mothers' (>25 years) milk present a higher aluminum concentration than the younger ones [44]. There is no relevant data available related to aluminum exposure during early life, and its correlation with brain function. Concentrations in human milk varying

3.2. Non-essential/toxic trace elements

98 Trace Elements - Human Health and Environment

between 7.056 and 67 μg/L are described [8, 43, 45].

3.2.1. Aluminum

The calculated transfer factor from food into human milk, calculated as the element concentration in food (g/kg) divided by the element concentration in milk (g/L), has been determined as 13.2 [46]. The reported concentration in human breast milk is between 0.14 and 0.35 μg/L.

## 3.2.3. Arsenic

Arsenic occurs in trivalent and pentavalent forms in food, water, and the environment. The biological effects of arsenic strongly depend on the actual chemical specie it is present, with inorganic forms being more toxic than most organic forms. Inorganic forms are methylated in the human body, giving rise to less toxic compounds, which are then excreted through urine [7]. Acute intoxication with arsenic is rare and is characterized by nausea, vomiting, diarrhea, and acute abdominal pain. Chronic intoxication is caused by exposure to natural sources, contaminated food, or water, or other accidental source [7]. In a recent study, presence of arsenic was observed in 63.51% of samples, with an average concentration of 2.36 μg/L [47], consistent with results from other authors [12, 20, 45, 48]. Arsenic presence in human milk is associated with cereal and fish intake [47].

#### 3.2.4. Barium

No nutritional requirements are set for barium in mammals, and its origin is probably associated with plant sources, following metabolic pathways similar to the elements of the same group in the periodic table, such as calcium and strontium [11]. Barium levels in milk may be dependent of mother diet [11]. Concentrations of 0.017 μg/L have been reported [8].

#### 3.2.5. Beryllium

Beryllium compounds are very toxic [49], and chronic beryllium disease has been observed in children living near a beryllium factory [50]. There is a high probability that this element can be transferred from mother to child through breast milk [50]. Concentration of 0.008 μg/L has been reported in breast milk [8].

#### 3.2.6. Bismuth

This element has been reported in breast milk at a concentration of 0.002 μg/L [8].

## 3.2.7. Bromide

Described as having an important function in tissue development, cellular structure and membrane integrity, its presence in breast milk was reported in a recent study at a concentration of 0.812 mg/L [51].

#### 3.2.8. Cadmium

The main source of exposure to cadmium is tobacco smoke. It is estimated that about 10% of mothers smoke during pregnancy, and the percentage of those who return to smoking during lactation is even higher [23]. Smoking contributes to increased levels of some metals in breast milk. Cadmium is the one of highest concern because it is an IARC type 1 carcinogen, altering the metabolism of other micronutrients, such as copper, iron, magnesium, selenium, and zinc. Cadmium levels are about four times higher in the breast milk of smoking mothers compared to non-smoking ones [23]. It has been reported that metallothionein levels are about half in smoking mothers. This seems to indicate a protective mechanism to the child, since the toxicity of the metallothionein-Cd complex is higher than that of inorganic cadmium [52]. Concentrations of cadmium in human milk varying between 0.003 and 1.37 μg/L are described [8, 20, 43, 47, 53, 54].

occurrence of intellectual development deficits (decreased IQ) is well known [60, 61]. In the human body, most of the lead is deposited in the bones (bones and teeth contain more than 90% of the body's total lead load) [62]. Lead mobilization of the human skeleton during pregnancy and lactation has been described [63, 64], resulting in a transgenerational transfer from the mother to the child. The mother consumption of calcium-rich foods reduces the risk of increased concentrations of lead in breast milk (>100 μg/L) [65, 66]. Iron also interferes with the absorption and toxicity of lead in children, causing a reduction of lead absorption. The hematological effects and intellectual deficit caused by lead are antagonized by an iron-rich diet [60]. A great inter-individual variability was observed in the concentration of lead in breast milk, which may be three orders of magnitude [67]. Concentrations varying between

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Although clinical recommendations discourage the treatment of lactating mothers with lithium for bipolar disorder, studies demonstrate a concentration reduction in half from mother serum to milk, and also from milk to infant plasma, in the same proportions, with no serious adverse effects [68]. Concentrations of 0.005 μg/L in human milk are

Mercury is a highly toxic metal. Like lead, it causes depletion of glutathione as well as the protein sulfhydryl binding groups, resulting in the production of reactive oxygen species, such as superoxide anion and hydroxyl radicals [69]. Mercury levels in breast milk vary considerably depending on the mother's place of residence, lactation stage, age, and diet. An increase in mercury levels in mother's plasma and breast milk during lactation is described [8]. Con-

Nickel role in some human physiological function is unknown [11]. However, it is reported that in case of severe nickel depletion, growth and hematopoiesis are depressed [7]. Due to its high homeostatic regulation, the symptoms of nickel poisoning are simply related to gastrointestinal irritation [7]. A decrease in nickel concentration throughout lactation is described [12].

The calculated transfer factor from food into human milk was determined as 20.7 [46]. A mean

Concentrations varying between 0.79 and 480 μg/L are reported [8, 12, 20, 45, 53].

0.019 and 18.17 μg/L are reported [8, 12, 20, 43, 47, 48, 54].

centrations varying between 0.008 and 3 μg/L are reported [70].

concentration of 0.04 μg/L in human milk was reported [46].

Undetectable levels were reported for platinum in human milk [45].

3.2.15. Lithium

reported [8].

3.2.16. Mercury

3.2.17. Nickel

3.2.18. Niobium

3.2.19. Platinum

## 3.2.9. Cerium

There is no evidence that it is an essential element [11], neither transport from mother to infant through milk [55, 56]. Concentrations in human milk varying between 0.0139 and 0.12 μg/L are described [46, 56].

## 3.2.10. Cesium

There is no evidence that it is an essential element [11]. It has been reported 137Cs in breast milk after the reactor accident at Chernobyl [57]. Concentrations in human milk varying between 1 and 5 μg/L are described [20].

#### 3.2.11. Gallium

The presence of 67Ga in human breast milk has been described [58]. A concentration of 0.52 μg/L was reported [46].

#### 3.2.12. Gold

Traces of gold have been found in breast milk, and it is assumed an association with the use of jewelry or dental amalgams [59]. Concentrations varying between 0.1 and 2.1 μg/L are described [45, 59].

#### 3.2.13. Lanthanum

There is no evidence that it is an essential element [11]. The calculated transfer factor from food into the human milk has been determined as 13.8 [46]. The reported concentration in human breast milk is between 0.002 and 0.07 μg/L [8, 46].

#### 3.2.14. Lead

Lead blood levels in the worldwide population have been decreasing significantly since the 1970s, largely due to the reduction of sources of environmental contamination, mainly the virtual elimination of this element from automobile fuels, but also from other sources of exposure such as paints, plumbing, ceramics, cosmetics, welding of food cans, etc. [60]. In young American children, it is reported that the levels of lead above 100 μg/L will mainly be related to exposure to particulate matter resulting from the degradation of paints used in homes [60]. The relationship between elevated levels of lead in children's blood and the occurrence of intellectual development deficits (decreased IQ) is well known [60, 61]. In the human body, most of the lead is deposited in the bones (bones and teeth contain more than 90% of the body's total lead load) [62]. Lead mobilization of the human skeleton during pregnancy and lactation has been described [63, 64], resulting in a transgenerational transfer from the mother to the child. The mother consumption of calcium-rich foods reduces the risk of increased concentrations of lead in breast milk (>100 μg/L) [65, 66]. Iron also interferes with the absorption and toxicity of lead in children, causing a reduction of lead absorption. The hematological effects and intellectual deficit caused by lead are antagonized by an iron-rich diet [60]. A great inter-individual variability was observed in the concentration of lead in breast milk, which may be three orders of magnitude [67]. Concentrations varying between 0.019 and 18.17 μg/L are reported [8, 12, 20, 43, 47, 48, 54].

#### 3.2.15. Lithium

lactation is even higher [23]. Smoking contributes to increased levels of some metals in breast milk. Cadmium is the one of highest concern because it is an IARC type 1 carcinogen, altering the metabolism of other micronutrients, such as copper, iron, magnesium, selenium, and zinc. Cadmium levels are about four times higher in the breast milk of smoking mothers compared to non-smoking ones [23]. It has been reported that metallothionein levels are about half in smoking mothers. This seems to indicate a protective mechanism to the child, since the toxicity of the metallothionein-Cd complex is higher than that of inorganic cadmium [52]. Concentrations of cadmium in human milk varying between 0.003 and 1.37 μg/L are described [8, 20, 43,

There is no evidence that it is an essential element [11], neither transport from mother to infant through milk [55, 56]. Concentrations in human milk varying between 0.0139 and 0.12 μg/L are

There is no evidence that it is an essential element [11]. It has been reported 137Cs in breast milk after the reactor accident at Chernobyl [57]. Concentrations in human milk varying between 1

The presence of 67Ga in human breast milk has been described [58]. A concentration of 0.52 μg/L

Traces of gold have been found in breast milk, and it is assumed an association with the use of jewelry or dental amalgams [59]. Concentrations varying between 0.1 and 2.1 μg/L are

There is no evidence that it is an essential element [11]. The calculated transfer factor from food into the human milk has been determined as 13.8 [46]. The reported concentration in human

Lead blood levels in the worldwide population have been decreasing significantly since the 1970s, largely due to the reduction of sources of environmental contamination, mainly the virtual elimination of this element from automobile fuels, but also from other sources of exposure such as paints, plumbing, ceramics, cosmetics, welding of food cans, etc. [60]. In young American children, it is reported that the levels of lead above 100 μg/L will mainly be related to exposure to particulate matter resulting from the degradation of paints used in homes [60]. The relationship between elevated levels of lead in children's blood and the

47, 53, 54].

3.2.9. Cerium

described [46, 56].

and 5 μg/L are described [20].

100 Trace Elements - Human Health and Environment

3.2.10. Cesium

3.2.11. Gallium

3.2.12. Gold

was reported [46].

described [45, 59].

3.2.13. Lanthanum

3.2.14. Lead

breast milk is between 0.002 and 0.07 μg/L [8, 46].

Although clinical recommendations discourage the treatment of lactating mothers with lithium for bipolar disorder, studies demonstrate a concentration reduction in half from mother serum to milk, and also from milk to infant plasma, in the same proportions, with no serious adverse effects [68]. Concentrations of 0.005 μg/L in human milk are reported [8].

#### 3.2.16. Mercury

Mercury is a highly toxic metal. Like lead, it causes depletion of glutathione as well as the protein sulfhydryl binding groups, resulting in the production of reactive oxygen species, such as superoxide anion and hydroxyl radicals [69]. Mercury levels in breast milk vary considerably depending on the mother's place of residence, lactation stage, age, and diet. An increase in mercury levels in mother's plasma and breast milk during lactation is described [8]. Concentrations varying between 0.008 and 3 μg/L are reported [70].

#### 3.2.17. Nickel

Nickel role in some human physiological function is unknown [11]. However, it is reported that in case of severe nickel depletion, growth and hematopoiesis are depressed [7]. Due to its high homeostatic regulation, the symptoms of nickel poisoning are simply related to gastrointestinal irritation [7]. A decrease in nickel concentration throughout lactation is described [12]. Concentrations varying between 0.79 and 480 μg/L are reported [8, 12, 20, 45, 53].

#### 3.2.18. Niobium

The calculated transfer factor from food into human milk was determined as 20.7 [46]. A mean concentration of 0.04 μg/L in human milk was reported [46].

#### 3.2.19. Platinum

Undetectable levels were reported for platinum in human milk [45].

#### 3.2.20. Rubidium

A relatively abundant element in body fluids and tissues, rubidium is also present in breast milk, having a behavior similar to that of potassium, although no rubidium-dependent biochemical functions are known [71]. Concentrations of rubidium in human milk ranging from 0.3 to 1.2 μg/L have been reported [20, 71, 72].

3.2.28. Uranium

3.2.29. Vanadium

4. Concluding remarks

decrease during lactation.

edge about these aspects.

results much more reliable.

Acknowledgements

50006/2013.

studies on this topic are very scarce.

The calculated transfer factor from food into human milk for uranium was determined as 21.3

Trace Elements in the Human Milk

103

http://dx.doi.org/10.5772/intechopen.76436

No defined biochemical function has been identified for vanadium in the higher animals. Vanadium is a relatively toxic element, with the most frequent symptoms being intestinal disturbances and greenish tongue [7]. A concentration of 0.18 μg/L in human milk was reported [45].

Breast milk is for many children, in the first months of life, their unique source of nutrients,

Although by definition trace elements are present in breast milk at relatively low concentra-

All of them show a great inter-individual variability in the breast milk, generally tending to

The variability of trace element levels in breast milk related to geographic, environmental, and dietary factors is well studied for several of them. For others, there is still insufficient knowl-

The advances in sensitivity and specificity of the analytical instrumentation occurred in recent years have made it possible to determine several elements which had not been studied until then. On the other hand, the higher reliability of current analytical techniques and the higher awareness about the importance of contamination control makes the more recently published

It should be noted that the nutritional value of breast milk regarding a particular trace element does not depend on its total analytical concentration but on its bioavailable fraction; however,

Knowing to what extent the mother's supplementation can adequately correct any deficiencies in milk quality in terms of trace element levels is another important issue that should be studied.

This work received financial support from the European Union (FEDER funds POCI/01/ 0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/

including trace elements essential for their healthy growth and development.

tions, they play a crucial role in multiple physiological processes.

[46]. A concentration of 0.03 μg/L in the human milk was reported [46].

#### 3.2.21. Ruthenium

The calculated transfer factor from food into human milk was determined as 4.1 [46]. Concentrations of 0.15 μg/L in human milk have been reported [46].

## 3.2.22. Scandium

Median concentrations of scandium in human milk of 0.19 μg/L are reported [45].

#### 3.2.23. Silver

Silver has been found in breast milk, and it is speculated that it also originates from the use of jewelry or dental amalgams [59]. Concentrations varying between 0.005 and 0.78 μg/L are reported [8, 45, 46].

#### 3.2.24. Strontium

There is no evidence of the nutritional importance of this element, although it is known that it is concentrated in the bone mass [11]. Biokinetic model for strontium in the lactating woman from bone to milk have been described [73]. Concentrations in human milk varying between 44 and 46 μg/L are reported [72].

#### 3.2.25. Thorium

The calculated transfer factor from food into human milk was determined as 20.2 [46]. A concentration of 0.2 μg/L in human milk was reported [46].

#### 3.2.26. Tin

There is no evidence that tin is an essential element [11]. Signs of chronic exposure to inorganic tin include decreased growth and anemia [7]. Also, organic tin compounds were studied and no significant transport could be observed from mother diet to milk [74]. Undetectable levels were reported [20].

#### 3.2.27. Titanium

The calculated transfer factor from food into human milk for titanium was determined as 5.6 [46]. Concentrations in human milk varying between 6.3 and 270 μg/L are reported [45, 46].

#### 3.2.28. Uranium

3.2.20. Rubidium

102 Trace Elements - Human Health and Environment

3.2.21. Ruthenium

3.2.22. Scandium

3.2.23. Silver

reported [8, 45, 46].

3.2.24. Strontium

3.2.25. Thorium

3.2.26. Tin

were reported [20].

3.2.27. Titanium

44 and 46 μg/L are reported [72].

0.3 to 1.2 μg/L have been reported [20, 71, 72].

trations of 0.15 μg/L in human milk have been reported [46].

concentration of 0.2 μg/L in human milk was reported [46].

A relatively abundant element in body fluids and tissues, rubidium is also present in breast milk, having a behavior similar to that of potassium, although no rubidium-dependent biochemical functions are known [71]. Concentrations of rubidium in human milk ranging from

The calculated transfer factor from food into human milk was determined as 4.1 [46]. Concen-

Silver has been found in breast milk, and it is speculated that it also originates from the use of jewelry or dental amalgams [59]. Concentrations varying between 0.005 and 0.78 μg/L are

There is no evidence of the nutritional importance of this element, although it is known that it is concentrated in the bone mass [11]. Biokinetic model for strontium in the lactating woman from bone to milk have been described [73]. Concentrations in human milk varying between

The calculated transfer factor from food into human milk was determined as 20.2 [46]. A

There is no evidence that tin is an essential element [11]. Signs of chronic exposure to inorganic tin include decreased growth and anemia [7]. Also, organic tin compounds were studied and no significant transport could be observed from mother diet to milk [74]. Undetectable levels

The calculated transfer factor from food into human milk for titanium was determined as 5.6 [46]. Concentrations in human milk varying between 6.3 and 270 μg/L are reported [45, 46].

Median concentrations of scandium in human milk of 0.19 μg/L are reported [45].

The calculated transfer factor from food into human milk for uranium was determined as 21.3 [46]. A concentration of 0.03 μg/L in the human milk was reported [46].

## 3.2.29. Vanadium

No defined biochemical function has been identified for vanadium in the higher animals. Vanadium is a relatively toxic element, with the most frequent symptoms being intestinal disturbances and greenish tongue [7]. A concentration of 0.18 μg/L in human milk was reported [45].

## 4. Concluding remarks

Breast milk is for many children, in the first months of life, their unique source of nutrients, including trace elements essential for their healthy growth and development.

Although by definition trace elements are present in breast milk at relatively low concentrations, they play a crucial role in multiple physiological processes.

All of them show a great inter-individual variability in the breast milk, generally tending to decrease during lactation.

The variability of trace element levels in breast milk related to geographic, environmental, and dietary factors is well studied for several of them. For others, there is still insufficient knowledge about these aspects.

The advances in sensitivity and specificity of the analytical instrumentation occurred in recent years have made it possible to determine several elements which had not been studied until then. On the other hand, the higher reliability of current analytical techniques and the higher awareness about the importance of contamination control makes the more recently published results much more reliable.

It should be noted that the nutritional value of breast milk regarding a particular trace element does not depend on its total analytical concentration but on its bioavailable fraction; however, studies on this topic are very scarce.

Knowing to what extent the mother's supplementation can adequately correct any deficiencies in milk quality in terms of trace element levels is another important issue that should be studied.

## Acknowledgements

This work received financial support from the European Union (FEDER funds POCI/01/ 0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/ 50006/2013.

## Author details

Manuel de Rezende Pinto and Agostinho A. Almeida\*

\*Address all correspondence to: aalmeida@ff.up.pt

LAQV, REQUIMTE, Departamento de Ciências Químicas, Laboratório de Química Aplicada, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

[12] Almeida AA et al. Trace elements in human milk: Correlation with blood levels, interelement correlations and changes in concentration during the first month of lactation.

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**Section 3**

**Environmental Trace Elements**
