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

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 pollu- tion 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.


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][5][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][8][9][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][12][13][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][5][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.
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][5][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 store-operated Ca channels on the plasma membrane are understood to open when Ca 2+ stores are depleted at the endoplasmic reticulum (ER) [16][17][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 [ [4][5][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 (SO 4 2À ) 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][26][27].

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][5][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.

Concentration relations between serum and hair
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 [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.
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%  Figure 1). 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 ½Ca S ¼ ½Ca I þ ½Ca P ¼ 20 and ½Ca P ¼ ½Ca I ¼ 10 ðDried serumÞ 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.    . 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 (HPO 4 2À ), S (SO 4 2À ) 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. 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).

Hair elements with minor effects of air pollution before 2009
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 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( . 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].

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].  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)  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.

Store-operated Ca channel gating induced by the pollution
Ca contained in the polluted air, probably as calcium silicate, sulfate and carbonate, is breathed into the lung and must increase [Ca 2+ ] 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     channels results in activation of the store-operated Ca channels; all the female subjects in Figures 4 and 5 and almost all male subjects in Figure 6    this is by activation of the STIM proteins, inhibits voltage-gated Ca 2+ channels (Ca v 1.2) responsible for activating heart muscle cells [33]. This may be taken as a risk factor for cardiovascular mortality (especially in cardiac patients) and to a lesser degree, for all persons. in Table 1). However, the air pollution (apparently containing sulfur species) increases [SO 4 2À ] in serum in addition to the [Ca 2+ ] increase as have been seen in Figures 4-6 in serum.
[K] H is proportional to the intracellular [K], which strongly depends on K + transfer between cell and serum which is influenced by β-catecholamines, insulin, aldosterone, pH, and osmolality [36]. Each hair analysis gives the mean concentration for about 3 days (Section 2) and primarily shows the H + /K + exchange depending on pH. In acidosis, H + ions move into cells and, to maintain electrical balance, K + ions move out into the serum, resulting in a low can be reached by pumping K ions by the molecular pump, Na + /K + -ATPase, which expels three sodium ions from the cell and takes in two potassium ions. If the pumping power deteriorates, therefore, the decrease of the [K] H maximum level must occur with an increase of [Na] H . This can be seen in the typical example, Figure 2(b), for a hair sample (F150) having a high [Ti] H from PM pollution; the Na Kα peak at 1.04 keV appears clearly, in contrast to the nonpolluted samples in Figure 2(a). Despite the high internal absorbance of sodium's 1.04 keV X-ray emission, such Na peaks were observed for many subjects in Figures 4-6. It may be concluded that the function of Na + /K + -ATPase in cell membrane is deteriorated by the pollution.
Molecular ion pumps such as Na + /K + -ATPase work with the energy supply of fully phosphated ATP. Therefore, serum [P] is important in their deterioration. Both of P and Ca in serum are closely associated in an equilibrium relation with bone. The effect of the pollution on hair [P] H is seen in Figure 7; Figure 7 [20,22,32], and the deterioration of molecular pumps seems due to insufficient energy supply. Therefore, we can see the correlation between [P] H and [Na] H ; the low [P] H is accompanied by appearance of [Na] H peak as seen in Figure 7  [P] H for the male subjects in Figure 6; The serum [K] is regulated with free filtration at the glomerulus and by excretion-reabsorption in renal tubules. The reabsorption is produced by H + /K + -ATPase [36] which is also deteriorated by the insufficient ATP supply, resulting in the hypokalemia understood from the hair level. This pollution's effect is serious; even mild or moderate hypokalemia increases the risks of morbidity and mortality in patients with cardiovascular disease [36,37].
According to the meta-analysis for 57,158 patients with acute myocardial infarction (AMI) [37], primary ventricular fibrillation (PVF) is lethal and occurs without signs or symptoms of heart failure or cardiogenic shock. It was shown that patients with PVF had a lower serum K level before the event compared with patients without PVF. Though the weighted mean difference was small (À0.27 mmol/L), the finding of lower serum K before PVF was very consistent. The association between low [K] and PVF was confirmed in clinical settings. Patients with PVF have lower heart rates for interior AMI and higher rates for anterior AMI. It can be concluded that the hypokalemia due to PM causes a mortality increase in AMI [25,26] even 1 day after PM pollution increases. Also, it is well known that PVF results in blood stagnation in atrium to form clots having the risk for brain infarction.  Table 1. The decrease in serum Cu and Zn may be due to S inflow into serum from air pollution, which then forms compounds such as CuS and ZnS which may be excreted from the liver and pancreas, respectively. Both Cu and Zn are essential elements. For Zn, the normal serum concentration is in the range of 0.08-0.15 mg/dL, and its lower limit is 0.06-0.07 mg/dL. A decrease by half is therefore dangerous.
Zinc serves as a structural center for maintaining the higher-order structure of protein. Zinc participates in the activity of more than 70 enzymes, including carbonic anhydrase (CA) and dehydrogenase. Almost all proteins related to gene expression contain Zn as a DNA-binding motif "zinc finger." Zinc is essential for physiological functions such as growth, gestation, taste, and participates in immunity, brain development, insulin biosynthesis, and so on [38]. Therefore, the air-pollution's effect on serum Zn is serious to many aspects of health.
The reduction of serum [Cu] S due to the pollution can also give an answer to the question of the increase of myocardial infarction mortality. It is firmly established that the formation of elastin protein, which gives elasticity to blood vessels, is copper-dependent [39][40][41], and Cu deficiency degenerates smooth muscle cells in the aortic walls. It was observed that elastin increases at the damage sites (scar) on aortic walls [41]. This indicates that elastin is necessary to correct damages, which, if unrepaired, trigger both aortic rupture and infarction. Therefore, the Cu deficiency due to PM can cause a mortality increase of myocardial infarction [25].
Here, it should be noted that we encountered many subjects having the normal [S] H = 20 in

Differences in pollution effect between the sexes
The normal levels of hair elements are the same for male and female. However, the pollution effects show differences as seen clearly in Figures 4-7. As described in Section 4, male and female deal with Ca deficiency differently; DO type is more common for the mail, and LD type is more common for the female, that is, males tolerate Ca deficiency with Ca channels closed by bone resorption, and females have a tendency to open the PTH-regulated Ca channels without bone resorption. The resulting Ca and/or Sr inflow through the channels causes deterioration of the hepatocytes' function to excrete pollutants into bile; a dysfunction that persists for months. Consequently, the pollution effect appears greater for the female; more female subjects have the high [Ti] H as seen in Figures 4-6(a).
In Figure 7, the appearance of [Na] H due to the excess serum Ca by breathing the polluted air shows difference between the sexes clearly; male can accommodate the excess Ca on serum protein as expected from the fact that DO type is more common for male. Figures 4-6(a) and (e). However, no difference between the sexes is clear; this implies these elements have no relation to the Ca metabolism, suggesting an association with the excess [S] in serum due to the pollution. By forming sulfide species, zinc and copper are excreted mainly with pancreatic fluid and bile into the gut, respectively.

The pollution's effect to decrease [Cu] H and [Zn] H can be recognized in
The observed relation between the sexes leads to the conclusion that the serum element levels change so as to eliminate the excess Ca and S inhaled from the air pollution.

Conclusion
Hair calcium depends on both PTH-regulated and store-operated Ca 2+ channels; . It must create a shortage of ATP, which deteriorates cell membrane molecular pumps such as H + /K + -ATPase and Na + /K + -ATPase, responsible for the renal K reabsorption, resulting in hypokalemia to produce fatal ventricular fibrillation in patients with myocardial infarction accompanied with heart rate variability [26]. It is well known that even mild or moderate hypokalemia increases the risks of mortality in patients with cardiovascular diseases. Abnormal intracellular [K] and [Na] due to the inactive molecular pumps may be also responsible for heart rate variability. (K + and Na + regulate and form the electric pulses commanding myocardial movement.) The excessive [S] in serum may decrease toward the normal by forming sulfide compounds with Cu and Zn, which are excreted from the liver and pancreas, resulting in deficiency of Cu necessary for the formation of elastin protein to repair damage in blood vessels. Thus, the sulfur species in the pollution may provide a second increase the mortality in myocardial infarction [25].
In summation, hair analysis shows that post-2009 air pollution causes serious deficiencies in serum K, P, Cu, and Zn, and is likely responsible for various diseases.
Finally, it is emphasized that the deterioration of Na + /K + -ATPase ( Figure 7) is a serious pollution effect because Na + /K + -ATPase exists in all cells, for example, more than half of brain energy dissipation is due to Na + /K + -ATPase; the pollution effect on neurodegenerative disease [42] is conceivable.