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+ + Co2

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*m = 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+ + Co2 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+ ≥ Ni2 ≈ 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 106

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

**10. Conclusion**

theoretically can produce up to 106

**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+ ≥ Zn2 . 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 106 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+ ≥ Ni2 ≈ Cu2+ ≈ Zn2+ [97].

32 Trace Elements - Human Health and Environment

the following order: Са2+ ≥ Mn2+ ≥ Мg2+ ≈ Сo2+ ≈ Ni2+ ≥ Cu2+ ≥ Zn2

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 106 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. Zaksas1 and Georgy A. Nevinsky2 \*

\*Address all correspondence to<EMAIL_ADDRESS>
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

[9] Becker JS, Matusch A, Wu B. Bioimaging mass spectrometry of trace elements – Recent advance and applications of LA-ICP-MS. Analytica Chimica Acta. 2014;**835**:1-18. DOI:

[10] Becker JS, Salber D. New mass spectrometric tools in brain research. Trends in Analytical

[11] Grolmusová Z, Horňáčková M, Plavčan J, Kopáni M, Babál P, Veis P. Laser induced breakdown spectroscopy of human liver samples with Wilson's disease. European Physical Journal Applied Physics. 2013;**63**:208-301. DOI: 10.1051/epjap/2013130030 [12] Motto-Ros V, Sancey L, Wang XC, Ma QL, Lux F, Bai XS, Panczer G, Tillement O, Yu J. Mapping nanoparticles injected into a biological tissue using laser-induced breakdown spectroscopy. Spectrochim Acta Part B. 2013;**87**:168-174. DOI: 10.1016/j.sab.2013.05.020 [13] Zheenbaev ZZ, Engelsht VS. Dvukhstruinyi Plazmatron (Two-Jet Plasmatron). Frunze:

[14] Schramel P. ICP and DCP emission spectrometry for trace element analysis in biomedical and environmental samples. Spectrochim Acta Part B. 1988;**43**:881-896. DOI:

[15] Meyer GA. Conical three-electrode d.c. plasma for spectrochemical analysis. Spectrochimica Acta, Part B: Atomic Spectroscopy. 1987;**42**:333-339. DOI: 10.1016/

[16] Zaksas NP. Comparison of excitation mechanisms in the analytical regions of a highpower two-jet plasma. Spectrochimica Acta, Part B: Atomic Spectroscopy. 2015;**109**:39-

[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:

[19] Yudelevich IG, Cherevko AS, Engelsht VS, Pikalov VV, Tagiltsev AP, Zheenbaev ZZ. A two-jet plasmatron for the spectrochemical analysis of geological samples. Spectrochim

[20] Shelpakova IR, Zaksas NP, Komissarova LN, Kovalevskij SV. Spectral methods for analysis of high-purity gallium with excitation of spectra in the two-jet arc plasmatron. Journal of Analytical Atomic Spectrometry. 2002;**17**:270-273. DOI: 10.1039/B109229B [21] Zaksas NP, Komissarova LN, Shelpakova IR.Analysis of indium and indium oxide using a two-jet arc plasmatron. Industrial Laboratory. Diagnostics of Materials (Russian).

Spectrometry. 2014;**29**:2204-2228. DOI: 10.1039/c4ja00250d

Chemistry. 2010;**29**:966-979. DOI: 10.1016/j.trac.2010.06.009

10.1016/j.aca.2014.04.048

Ilim (Russian); 1983. 200 p

10.1016/0584-8547(88)80194-0

43. DOI: 10.1016/j.sab.2015.04.012

2011;**66**:610-619. DOI: 10.1134/S1061934811050042

Acta, Part B. 1984;**39**:777-785. DOI: 1016/0584-8547(84)80086-5

0584-8547(87)80074-5

10.1016/j.sab.2013.06.013

2007:**73**:89-92. ISSN: 1028-6861

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

## **References**

[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 Spectrometry. 2014;**29**:2204-2228. DOI: 10.1039/c4ja00250d

**Author details**

Natalia P. Zaksas1

**References**

c5ja90001h

c7ja90005h

Sciences, Novosibirsk, Russia

34 Trace Elements - Human Health and Environment

Academy of Sciences, Novosibirsk, Russia

and Georgy A. Nevinsky2

\*Address all correspondence to<EMAIL_ADDRESS>
\*

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

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

[1] Taylor A, Day MP, Hill S, Marshall J, Patriarca M, White M. Atomic spectrometry update: Review of advances in the analysis of clinical and biological materials, foods and beverages. Journal of Analytical Atomic Spectrometry. 2015;**30**:542-579. DOI: 10.1039/

[2] Taylor A, Barlow N, Day MP, Hill S, Patriarca M, White M. Atomic spectrometry update: Review of advances in the analysis of clinical and biological materials, foods and beverages. Journal of Analytical Atomic Spectrometry. 2017;**32**:432-476. DOI: 10.1039/

[3] Barany E, Bergdahl IA, Schьtz A, Skerfving S, Oskarsson A. Inductively coupled mass spectrometry for direct multi-element analysis of diluted whole blood and serum. Journal of Analytical Atomic Spectrometry. 1997;**12**:1005-1009. DOI: 10.1039/A700904F

[4] Krachler M, Heisel C, Kretzer JP. Validation of ultratrace analysis of Co, Cr, Mo and Ni in whole blood, serum and urine using ICP-SMS. Journal of Analytical Atomic

[5] Kerger BD, Gerads R, Gurleyuk H, Urban A, Paustenbach DJ. Total cobalt determination in human blood and synovial fluid using inductively coupled plasma-mass spectrometry: Method validation and evaluation of performance variables affecting metal hip implant patient samples. Toxicological and Environmental Chemistry. 2015;**97**:1145-

[6] Barbosa VMP, Barbosa AF, Bettini J, Luccas PO, Figueiredo EC. Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry. Talanta. 2016;**147**:478-484. DOI:

[7] Mortada WI, Kenawy IMM, Abdelghany AM, Ismal AM, Donia AF, Nabieh KA. Determination of Cu2+, Zn2+ and Pb2+ in biological and food samples by FAAS after preconcentration with hydroxyapatite nanorods originated from eggshell. Materials

Science and Engineering: C. 2015;**52**:288-296. DOI: 10.1016/j.msec.2015.03.061

Spectrometry. 2009;**24**:605-610. DOI: 10.1039/b821913c

1163. DOI: 10.1080/02772248.2015.1092735

10.1016/j.talanta.2015.10.023

[22] Zaksas NP, Komissarova LN, Shelpakova IR. Atomic-emission spectral analysis of high purity tellurium dioxide with spectral excitation in a two jet arc plasmatron. Analytics and Control. (Russian) 2005;**9**:240-244. ISSN: 2073-1442 (Print). ISSN: 2073-1450 (Online)

[34] Ikhmyangan EN, Vasilenko NL, Sinitsina OI, Buneva VN, Nevinsky GA. Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities. Journal of

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

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

37

[35] Ikhmyangan EN, Vasilenko NL, Sinitsyna OI, Buneva VN, Nevinsky GA. The catalytic heterogeneity of G immunoglobulins with peroxidase activity from the blood of healthy Wistar rats. Immunopathology. Allergology and Infectology (Russian). 2006;**2**:32-48

[36] Tolmacheva AS, Blinova EA, Ermakov EA, Buneva VN, Vasilenko NL, Nevinsky GA. IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans. Journal of Molecular Recognition. 2015;**28**:565-580. DOI: 10.1002/jmr.2474

[37] Keinan E, editor. Catalytic Antibodies. Weinheim, Germany: Wiley-VCH Verlag GmbH

[38] Nevinsky GA, Kanyshkova TG, Buneva VN. Natural catalytic antibodies (abzymes) in

[39] Nevinsky GA, Buneva VN. Human catalytic RNA- and DNA-hydrolyzing antibodies.

[40] Nevinsky GA, Favorova OO, Buneva VN. Natural catalytic antibodies – New characters in the protein repertoire. In: Golemis E, editor. Protein-Protein Interactions; A Molecular Cloning Manual. New York, Cold Spring Harbor: Spring Harbor Lab. Press;

[41] Nevinsky GA, Buneva VN. Catalytic antibodies in healthy humans and patients with autoimmune and viral diseases. Journal of Cellular and Molecular Medicine. 2003;**7**:265-

[42] Nevinsky GA, Buneva VN. Natural catalytic antibodies-abzymes. In: Keinan E, editor. Catalytic Antibodies. Weinheim, Germany: Wiley-VCH Verlag GmbH and Co; 2005.

[43] Nevinsky GA. Natural catalytic antibodies in norm and in autoimmune diseases. In: Brenner KJ, editor. Autoimmune Diseases: Symptoms, Diagnosis and Treatment.

[44] Nevinsky GA. Natural catalytic antibodies in norm and in HIV-infected patients. In: Kasenga FH, editor. Understanding HIV/AIDS Management and Care—Pandemic

[45] Nevinsky GA. Autoimmune processes in multiple sclerosis: Production of harmful catalytic antibodies associated with significant changes in the hematopoietic stem cell differentiation and proliferation. In: Conzalez-Quevedo A, editor. Multiple Sclerosis. Rijeka,

[46] Nevinsky GA. Catalytic antibodies in norm and systemic lupus erythematosus. In: Khan WA, editor. Lupus. Rijeka, Croatia: InTech; 2017. pp. 41-101. DOI: 10.5772/67790

Approaches the 21st Century. Rijeka, Croatia: InTech; 2011. pp. 151-192

normalcy and pathology. Biochemistry (Moscow). 2000;**65**:1245-1255

Molecular Recognition. 2006;**19**:432-440. PMID: 16835846

and Co; 2005. 586 p

2002. pp. 532-534

Journal of Immunology. 2002;**269**:235-249

276. DOI: 10.1111/j.1582-4934.2003.tb00227.x

pp. 505-569. DOI: 10.1002/3527603662.ch19

Croatia: InTech; 2016. pp. 100-147

New York, USA: Nova Science Publishers; 2010. pp. 1-107

[34] Ikhmyangan EN, Vasilenko NL, Sinitsina OI, Buneva VN, Nevinsky GA. Substrate specificity of rat sera IgG antibodies with peroxidase and oxidoreductase activities. Journal of Molecular Recognition. 2006;**19**:432-440. PMID: 16835846

[22] Zaksas NP, Komissarova LN, Shelpakova IR. Atomic-emission spectral analysis of high purity tellurium dioxide with spectral excitation in a two jet arc plasmatron. Analytics and Control. (Russian) 2005;**9**:240-244. ISSN: 2073-1442 (Print). ISSN: 2073-1450 (Online)

[23] Zaksas NP, Shelpakova IR, Gerasimov VA. Determination of trace elements in different powdered samples by atomic emission spectrometrywith spectral excitation in a two-jet arc plasmatron. Journal of Analytical Chemistry. 2004;**59**:222-228. DOI:

[24] Zaksas NP, Veryaskin AF. Solid sampling in analysis of by soils two-jet plasma atomic emission spectrometry. Analytical Sciences. 2017;**33**:605-609. DOI: 10.2116/analsci.33.605

[25] Zaksas NP, Sultangazieva TT, Korda TM. Using a two-jet arc plasmatron for determining the trace element composition of powdered biological samples. Journal of Analytical

[26] Zaksas NP, Nevinsky GA. Solid sampling in analysis of animal organs by two-jet plasma atomic emission spectrometry. Spectrochimica Acta, Part B: Atomic Spectroscopy. 2011;**66**:

[27] Zaksas NP, Gerasimov VA, Nevinsky GA. Simultaneous determination of Fe, P, Ca, Mg, Zn and Cu in whole blood by two-jet plasma atomic emission spectrometry. Talanta.

[28] Zaksas NP, Sultangazieva TT, Gerasimov VA. Determination of trace elements in bone by two-jet plasma atomic emission spectrometry. Analytical and Bioanalytical Chemistry.

[29] Zaksas N, Gluhcheva Y, Sedykh S, Madzharova M, Atanassova N, Nevinsky G. Effect of

[30] Legostaeva GA, Zaksas NP, Gluhcheva YG, Sedykh SE, Madzharova ME, Atanassova NN, Buneva VN, Nevinsky GA. Effect of CoCl₂ on the content of different metals and a relative activity of DNA-hydrolyzing abzymes in the blood plasma of mice. Journal of

[31] Tolmacheva AS, Zaksas NP, Buneva VN, Vasilenko NL, Nevinsky GA. Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions. Journal of Molecular Recognition. 2009;**22**:26-37. DOI:

[32] Ikhmyangan EN, Vasilenko NL, Buneva VN, Nevinsky GA. IgG antibodies with peroxidase-like activity from the sera of healthy Wistar rats. FEBS Letters. 2005;**579**:3960-3964.

[33] Ikhmyangan EN, Vasilenko NL, Buneva VN, Nevinsky GA. Metal ions-dependent peroxidase and oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats.

Journal of Molecular Recognition. 2006;**19**:91-105. PMID: 16416456

Molecular Recognition 2013;**26**:10-22. DOI: 10.1002/jmr.2217. PMID: 23280613

 treatment on major and trace elements metabolism and protein concentration in mice. Journal of Trace Elements in Medicine and Biology. 2013;**27**:27-30. DOI: 10.1016/j.

Chemistry. 2006;**61**:632-637. DOI: 10.1134/S1061934806060128

2010;**80**:2187-2190. DOI: 10.1016/j.talanta.2009.10.046

2008;**391**:687-693. DOI: 10.1007/s00216-008-2050-8

10.1023/B:JANC.0000018963.03529.e0

36 Trace Elements - Human Health and Environment

861-865. DOI: 10.1016/j.sab.2011.11.001

CoCl2

jtemb.2012.07.005

10.1002/jmr.923

PMID: 15993881

[47] Ames BN. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science. 1983;**221**:1256-1264. PMID: 6351251

blood and cerebrospinal fluid of patients with multiple sclerosis effectively hydrolyze

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

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

39

DNA and RNA. Biochemistry (Moscow). 1998;**63**:1239-1248. PMID: 9864461/

Immunology Letters. 2001;**76**:163-167. PMID: 11306143

DOI: 10.1371/journal.pone.0093001

2015;**5**:150064. DOI: 10.1098/rsob.150064

9369225

PMID: 10214697

[63] Baranovskii AG, Ershova NA, Buneva VN, Kanyshkova TG, Mogelnitskii AS, Doronin BM, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies from the sera of patients with multiple sclerosis.

[64] Parkhomenko TA, Doronin VB, Castellazzi M, Padroni M, Pastore M, Buneva VN, Granieri E, Nevinsky GA. Comparison of DNA-hydrolyzing antibodies from the cerebrospinal fluid and serum of patients with multiple sclerosis. PLoS One. 2014;**9**:e93001.

[65] Vlasov AV, Baranovskiĭ AG, Kanyshkova TG, Prints AV, Zabara VG, Naumov VA, Breusov AA, Giege R, Buneva VN, Nevinskiĭ GA. Substrate specificity of DNA- and RNA-hydrolyzing antibodies from blood of patients with polyarthritis and Hashimoto's thyroiditis. Molekulyarnaya Biologiya (Moscow). 1998;**32**:559-569. PMID: 9720080 [66] Nevinsky GA, Breusov AA, Baranovskii AG, Prints AV, Kanyshkova TG, Galvita AV, Naumov VA, Buneva VN. Effect of different drugs on the level of DNA-hydrolyzing polyclonal IgG antibodies in sera of patients with Hashimoto's thyroiditis and nontoxic

[67] Ermakov EA, Smirnova LP, Parkhomenko TA, Dmitrenok PS, Krotenko NM, Fattakhov NS, Bokhan NA, Semke AV, Ivanova SA, Buneva VN, Nevinsky GA. DNA-hydrolysing activity of IgG antibodies from the sera of patients with schizophrenia. Open Biology.

[68] Baranovsky AG, Matushin VG, Vlassov AV, Zabara VG, Naumov VA, Giege R, Buneva VN, Nevinsky GA. DNA- and RNA-hydrolyzing antibodies from the blood of patients with various forms of viral hepatitis. Biochemistry (Moscow). 1997;**62**:1358-1366. PMID:

[69] Odintsova ES, Kharitonova MA, Baranovskiĭ AG, Siziakina LP, Buneva VN, Nevinskiĭ GA. DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immunodeficiency syndrome. Molecular Biology (Moscow). 2006;**40**:857-864. PMID: 17086987

[70] Parkhomenko TA, Buneva VN, Tyshkevich OB, Generalov II, Doronin BM, Nevinsky GA. DNA-hydrolyzing activity of IgG antibodies from the sera of patients with tick-

[72] Buneva VN, Kanyshkova TG, Vlassov AV, Semenov DV, Khlimankov DYu, Breusova LR, Nevinsky GA. Catalytic DNA- and RNA-hydrolyzing antibodies from milk of healthy human mothers. Applied Biochemistry and Biotechnology. 1998;**75**:63-76.

[73] Nevinsky GA, Kanyshkova TG, Semenov DV, Vlassov AV, Gal'vita AV, Buneva VN. Secretory immunoglobulin a from healthy human mothers' milk catalyzes nucleic

borne encephalitis. Biochimie. 2010;**92**:545-554. DOI: 10.1016/j.biochi.2010.01.022 [71] Kanyshkova TG, Semenov DV, Khlimankov DY, Buneva VN, Nevinsky GA. DNAhydrolyzing activity of the light chain of IgG antibodies from milk of healthy human

mothers. FEBS Letters. 1997;**416**:23-26. PMID: 9369225

nodal goiter. Medical Science Monitor. 2001;**7**:201-211. PMID: 11257722

blood and cerebrospinal fluid of patients with multiple sclerosis effectively hydrolyze DNA and RNA. Biochemistry (Moscow). 1998;**63**:1239-1248. PMID: 9864461/

[63] Baranovskii AG, Ershova NA, Buneva VN, Kanyshkova TG, Mogelnitskii AS, Doronin BM, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies from the sera of patients with multiple sclerosis. Immunology Letters. 2001;**76**:163-167. PMID: 11306143

[47] Ames BN. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative

[48] Cutler RG. Antioxidants and aging. The American Journal of Clinical Nutrition.

[49] Beckman KB, Ames BN. The free radical theory of aging matures. Physiological Reviews.

[50] Feuers RJ, Weindruch R, Hart RW. Caloric restriction, aging, and antioxidant enzymes.

[52] Mates JM, Perez-Gomez C, Nunez de Castro I. Antioxidant enzymes and human dis-

[53] Mates JM, Sanchez-Jimenez F. Antioxidant enzymes and their implications in pathophysiologic processes. Frontiers in Bioscience. 1999;**4**:D339-D345. PMID: 10077544 [54] Zenkov NK, Lankin VZ, Men'shikova EB. Oxidative Stress. Biochemical and Pathophysiological Aspects. Moscow: MAIK, Nauka/Interperiodica; 2001. pp. 3-343

[55] Dhaunsi GS, Gulati S, Singh AK, Orak JK, Asayama K, Singh I. Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evi-

[56] Kirkman HN, Gaetani GF. Catalase: A tetrameric enzyme with four tightly bound molecules of NADPH. In: Proceedings of the National Academy of Sciences of the United

[57] Shuster AM, Gololobov GV, Kvashuk OA, Bogomolova AE, Smirnov IV, Gabibov AG. DNA hydrolyzing autoantibodies. Science. 1992;**256**:665-667. PMID: 1585181

[58] Buneva VN, Andrievskaia OA, Romannikova IV, Gololobov GV, Iadav RP, Iamkovoĭ VI, Nevinskii GA. Interaction of catalytically active antibodies with oligoribonucleotides.

[59] Vlassov A, Florentz C, Helm M, Naumov V, Buneva V, Nevinsky G, Giegé R. Characterization and selectivity of catalytic antibodies from human serum with RNase activity.

[60] Andrievskaya OA, Buneva VN, Naumov VA, Nevinsky GA. Catalytic heterogeneity of polyclonal RNA-hydrolyzing IgM from sera of patients with lupus erythematosus.

[61] Andrievskaya OA, Buneva VN, Baranovskii AG, Gal'vita AV, Benzo ES, Naumov VA, Nevinsky GA. Catalytic diversity of polyclonal RNA-hydrolyzing IgG antibodies from the sera of patients with lupus erythematosus. Immunology Letters. 2002;**81**:191-198.

[62] Baranovskii AG, Kanyshkova TG, Mogelnitskii AS, Naumov VA, Buneva VN, Gusev EI, Boiko AN, Zargarova TA, Favorova OO, Nevinsky GA. Polyclonal antibodies from

dence. The Journal of Biological Chemistry. 1992;**267**:6870-6873. PMID: 1551895

[51] Allen RG. Free Radicals in Aging. Boca Raton, FL: CPC Press; 1993. pp. 12-23

eases. Clinical Biochemistry. 1999;**32**:595-603. PMID: 10638941

diseases. Science. 1983;**221**:1256-1264. PMID: 6351251

Mutation Research. 1993;**295**:191-200. PMID: 7507557

States of America. 1984;**81**:4343-4347. PMID: 6589599

Molecular Biology (Moscow). 1994;**28**:738-743. PMID: 7990801

Nucleic Acids Research. 1998;**26**:5243-5250. PMID: 9826744

Medical Science Monitor. 2000;**6**:460-470. PMID: 11208354

PMID: 11947924

1991;**53**:373S-3379. PMID: 1985414

38 Trace Elements - Human Health and Environment

1998;**78**:547-581. PMID: 9562038

acid hydrolysis. Applied Biochemistry and Biotechnology. 2000;**83**:115-129. PMID: 10826954

[85] Buneva VN, Nevinsky GA. Exceptional diversity of catalytic antibodies with varying activity in the blood of autoimmune and viral disease patients. Molekuliarnaia Biologiia

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

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

41

[86] Andrievskaia OA, Kanyshkova TG, Iamkovoi VI, Buneva VN, Nevinskii GA. Monoclonal antibodies to DNA hydrolyze RNA better than DNA. Doklady Akademii Nauk

[87] Vlassov AV, Helm M, Florentz C, Naumov VA, Breusov AA, Buneva VN, Giege R, Nevinsky GA. Variability of substrate specificity of serum antibodies obtained from patients with different autoimmune and viral diseases in reaction of tRNA hydrolysis.

[88] Polosukhina DI, Kanyshkova TG, Doronin BM, Tyshkevich OB, Buneva VN, Boiko AN, Gusev E., Favorova OO, Nevinsky GA. Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis. Journal of Cellular and

[89] Polosukhina DI, Buneva VN, Doronin BM, Tyshkevich OB, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Hydrolysis of myelin basic protein by IgM and IgA antibodies from the sera of patients with multiple sclerosis. Medical Science Monitor.

[90] Polosukhina DI, Kanyshkova TG, Doronin BM, Tyshkevich OB, Buneva VN, Boiko AN, Gusev EI, Favorova OO, Nevinsky GA. Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis. Immunology Letters.

[91] Legostaeva GA, Polosukhina DI, Bezuglova AM, Doronin BM, Buneva VN, Nevinsky GA. Affinity and catalytic heterogeneity of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with multiple sclerosis. Journal of Cellular and Molecular

[92] Bezuglova AM, Konenkova LP, Doronin BM, Buneva VN, Nevinsky GA. Affinity and catalytic heterogeneity and metal-dependence of polyclonal myelin basic proteinhydrolyzing IgGs from sera of patients with systemic lupus erythematosus. Journal of

[93] Bezuglova AM, Konenkova LP, Buneva VN, Nevinsky GA. IgGs containing light chains of the λ-and κ-type and of all subclasses (IgG1-IgG4) from the sera of patients with systemic lupus erythematosus hydrolyze myelin basic protein. International Immunology.

[94] Bezuglova AM, Dmitrenok PS, Konenkova LP, Buneva VN, Nevinsky GA. Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus. Peptides. 2012;**37**:69-78. DOI: 10.1016/j.

Medicine. 2010;**14**:699-709. DOI: 10.1111/j.1582-4934.2009.00738.x

Molecular Recognition. 2011;**24**:960-974. DOI: 10.1002/jmr.1143

(Moscow). 2017;**51**:969-984. DOI: 10.7868/S002689841706009X

Russian Journal of Immunology. 1999;**4**:25-32. PMID: 12687113

Molecular Medicine. 2004;**8**:359-368. PMID: 15491511

2005;**11**:BR266-BR272. PMID: 16049372

2006;**103**:75-81. DOI: 10.1016/j.imlet.2005.10.018

2012;**24**:759-770. DOI: 10.1093/intimm/dxs071

peptides.2012.07.003

(Moscow). 1997;**355**:401-403. PMID: 9376783

[85] Buneva VN, Nevinsky GA. Exceptional diversity of catalytic antibodies with varying activity in the blood of autoimmune and viral disease patients. Molekuliarnaia Biologiia (Moscow). 2017;**51**:969-984. DOI: 10.7868/S002689841706009X

acid hydrolysis. Applied Biochemistry and Biotechnology. 2000;**83**:115-129. PMID:

[74] Kuznetsova IA, Orlovskaya IA, Buneva VN, Nevinsky GA. Activation of DNAhydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions. Biochimica et Biophysica Acta 2007;**1774**:884-896. PMID: 17561457 [75] Kostrikina IA, Kolesova ME, Orlovskaya IA, Buneva VN, Nevinsky GA. Diversity of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL/MpJ-lpr mice.

[76] Doronin VB, Parkhomenko TA, Korablev A, Toporkova LB, Lopatnikova JA, Alshevskaja AA, Sennikov SV, Buneva VN, Budde T, Meuth SG, Orlovskaya IA, Popova NA, Nevinsky GA. Changes in different parameters, lymphocyte proliferation and hematopoietic progenitor colony formation in EAE mice treated with myelin oligodendrocyte glycoprotein. Journal of Cellular and Molecular Medicine. 2016;**20**:81-94. DOI: 10.1111/

[77] Aulova KS, Toporkova LB, Lopatnikova JA, Alshevskaya AA, Sennikov SV, Buneva VN, Budde T, Meuth SG, Popova NA, Orlovskaya IA, Nevinsky GA. Changes in haematopoietic progenitor colony differentiation and proliferation and the production of different abzymes in EAE mice treated with DNA. Journal of Cellular and Molecular Medicine.

[78] Kostrikina IA, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: Molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different catalytic properties. Biochimica et Biophysica Acta. 2014;**1840**:1725-1737. DOI: 10.1016/j.

[80] Suck D. DNA recognition by DNase I. Journal of Molecular Recognition. 1994;**7**:65-70.

[81] Bernardi G. Spleen aciddeoxyribonuclease. In: Boyer PD, editor. The enzymes, 3rd ed.,

[82] Botvinovskaya AV, Kostrikina IA, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: Molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties. Journal of Molecular Recognition. 2013;**26**:450-460.

[83] Kostrikina IA, Odintsova ES, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: Molecular cloning and analysis of recombinant DNase monoclonal κ light chain NGK-1.

[84] Baranovskii AG, Buneva VN, Doronin BM, Nevinsky GA. Immuniglobulins from blood of patients with multiple sclerosis like catalytic heterogeneous nucleases. Russian

International Immunology. 2014;**26**:439-450. DOI: 10.1093/intimm/dxu047

Journal of Molecular Recognition. 2011;**24**:557-569. DOI: 10.1002/jmr.1067

10826954

40 Trace Elements - Human Health and Environment

jcmm.12704

bbagen.2014.01.027

PMID: 7826675

DOI: 10.1002/jmr.2286

2017;**21**:3795-3809. DOI: 10.1111/jcmm.13289

New York: Academic Press; 1971;**4**:271-287

Journal of Immunology. 2008;**2**:405-419

[79] Shapot VS. Nucleases. Moscow: Medicine Press; 1968. pp. 1-162

[95] Timofeeva AM, Dmitrenok PS, Konenkova LP, Buneva VN, Nevinsky GA. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus. PLoS One. 2013;**8**:e51600. DOI: 10.1371/journal. pone.0051600

[105] Baranova SV, Buneva VN, Kharitonova MA, Sizyakina LP, Zakharova OD, Nevinsky GA. Diversity of integrase-hydrolyzing IgGs and IgMs from sera of HIV-infected patients. Biochemistry (Moscow). 2011;**76**:1300-1311. DOI: 10.1134/S0006297911120030

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

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

43

[106] Baranova SV, Buneva VN, Nevinsky GA. Antibodies from the sera of HIV-infected patients efficiently hydrolyze all human histones. Journal of Molecular Recognition.

2016;**29**:346-362. DOI: 10.1002/jmr.2534

[105] Baranova SV, Buneva VN, Kharitonova MA, Sizyakina LP, Zakharova OD, Nevinsky GA. Diversity of integrase-hydrolyzing IgGs and IgMs from sera of HIV-infected patients. Biochemistry (Moscow). 2011;**76**:1300-1311. DOI: 10.1134/S0006297911120030

[95] Timofeeva AM, Dmitrenok PS, Konenkova LP, Buneva VN, Nevinsky GA. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus. PLoS One. 2013;**8**:e51600. DOI: 10.1371/journal.

[96] O'Connor KC, Bar-Or A, Hafler DA. The neuroimmunology of multiple sclerosis: Possible roles of T and B lymphocytes in immunopathogenesis. Journal of Clinical

[97] Timofeeva AM, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein. Journal of Molecular Recognition.

[98] Timofeeva AM, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: Molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA1-Me-pro with two metalloprotease active centers. Molecular BioSystems. 2016;**12**:3556-3566.

[99] Timofeeva AM, Ivanisenko NV, Buneva VN, Nevinsky GA. Systemic lupus erythematosus: Molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA2-Me-pro-Tr possessing two different activities-trypsin-like and metalloprote-

ase. International Immunology. 2015;**27**:633-645. DOI: 10.1093/intimm/dxv042

protease and DNase. Lupus: Open Access. 2017;**2**(2):1-12

[100] Timofeeva AM, Buneva VN, Nevinsky GA. SLE: Unusual recombinant monoclonal light chain ngta3-pro-dnase possessing three different activities trypsin-like, metallo-

[101] Odintsova ES, Zaksas NP, Buneva VN, Nevinsky GA. Metal dependent hydrolysis of β-casein by sIgA antibodies from human milk. Journal of Molecular Recognition.

[102] Odintsova ES, Kharitonova MA, Baranovskii AG, Sizyakina LP, Buneva VN, Nevinsky GA. Proteolytic activity of IgG antibodies from blood of acquired immunodeficiency

[103] Odintsova ES, Baranova SV, Dmitrenok PS, Rasskazov VA, Calmels C, Parissi V, Andreola ML, Buneva VN, Zakharova OD, Nevinsky GA. Antibodies to HIV integrase catalyze site-specific degradation of their antigen. International Immunology.

[104] Baranova SV, Buneva VN, Kharitonova MA, Sizyakina LP, Calmels C, Andreola ML, Parissi V, Zakharova OD, Nevinsky GA. HIV-1 integrase-hydrolyzing IgM antibodies from sera of HIV-infected patients. International Immunology. 2010;**22**:671-680. DOI:

syndrome patients. Biochemistry (Moscow). 2006;**71**:251-261. PMID: 16545061

pone.0051600

42 Trace Elements - Human Health and Environment

Immunology. 2001;**21**:81-92. PMID: 11332657

2015;**28**:614-627. DOI: 10.1002/jmr

2011;**24**:45-59. DOI: 10.1002/jmr.1022

2011;**23**:601-612. DOI: 10.1093/intimm/dxr065

10.1093/intimm/dxq051

DOI: 10.1039/c6mb00573j

[106] Baranova SV, Buneva VN, Nevinsky GA. Antibodies from the sera of HIV-infected patients efficiently hydrolyze all human histones. Journal of Molecular Recognition. 2016;**29**:346-362. DOI: 10.1002/jmr.2534

**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+

© 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+

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+ /K+ -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}

2 = 400 (6)

2 = 200 (8)

2 = 50 (10)

1/2 = 22 (14)

1/2 = 22 (16)

1/2 = 5 (19)

2 = 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 2) 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 S ¼ ½ � X I þ ½ � X P (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

2) 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).

S ¼ ½Ca�

I þ ½Ca�

H 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

H 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

P 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

P ¼ 20 and ½Ca�

H ¼ ð1=2Þf½Sr�

2

CI 2 :

CIþ½Sr�

P ¼ ½Ca�

S ¼ 10 ðHairÞ

I ¼ 10 ðDried serumÞ

CPg2 :

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 2� 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 �H ¼ ½ � log P – log S H= ½ � log P – log S H

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)

2� 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.

2), 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.)

2) and K+

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 2) 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 2), S (SO4 2) and K+ .

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

2)

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+

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+ exchange in cells, and [K]H≲10.

2] 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.

2] 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+

] in serum, K+

PTH inhibits H+

high [H+

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+

] 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+

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+

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+ /K+ -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.