Introduction:

The content of metals in the hair is considered a good biomarker todetermine the chronic exposure to metals considered toxic and the concentration of essential metals. Within the indications of follow-upin countries of the first world it is recommended to have a biomarker that allows to evaluate the exposure to essential and non-essentialmetals during the uterine life. ^{(3, 4, 6, 15, 17, 18,19)} Exposure to toxic metals and deficiency of essential metals (which canbe potentially harmful) has been linked to various conditions, among which neurodegenerative diseases, cardiovascular diseases andcancer, among others ^{(2, 13, 8)}. The concentration of metals in the hair is a good biomarker for the evaluation of occupational, environmentaland nutritional exposure, due to the advantages it presents in comparison with other biological matrices that include: easy sampling,non-invasive and indicates a chronic exposure; additionally, the concentration of some metals present in the hair has been relatedto the aforementioned conditions in several case-control studies. Therefore, the importance of knowing the concentration of metals inthe hair as a biomarker of exposure and nutrition in early stages such as gestation is to be able to prevent alterations in the state of healthin later stages. ^{(1.9.14, 15, 18, 19.21)} Quantification of metals in hair by ICP-MSThe ICP-MS technique is a technique that has many advantages compared to other techniques such as optical emission spectrometrywith inductively coupled plasma (ICP-OES) and atomic absorption spectrometry (AAS), since multiple elements can be quantifiedsimultaneously. a short time, with little sample and low detection limits. Therefore, a good pre-treatment of the sample is necessary,this through the decomposition of the matrix by a digestion process to keep the ions in solution ^(10,19).The recommendations of the Environmental Protection Agency (EPA)for the microwave acid digestion of organic matrices is to have a minimum of 1 g of sample and bring it to a final volume of 10 ml ofoxidizing agent (USEPA, 1996) ; However, these conditions can bemodified according to the size and type of sample, and taking intoaccount the characteristics of each team. ^(1.21) Characteristics of the study population in relation to the concentrationof metals in the hair Hair color and shape are important characteristics because metalsbind to the functional groups of the two types of melanin, and hair shape determines the availability of SH groups of keratin-associatedproteins (7, 16,20). Goals
General purpose Determine the relationship of the concentration of the main metals(essential and non-essential) in hair samples of the mother and hernewborn child. Particular objectives
• Establish the pre-treatment conditions for acid digestion of hairsamples and the quantification of metals by ICP-MS. • Perform the quantification of metals in hair samples of the mothernewbor pair. Establish the relationship between the concentrations of the metals inthe hair samples of the mother and her son

Material and method

Collection of hair samples
The sample was taken for a period not longer than 48 hours afterdelivery, in a clean place using gloves and stainless steel scissors to cut approximately 100 to 150 mg of hair from newborns and mothersObtaining the maternal hair was as close to the scalp of the posterior area of the head, since in this region is where the hair grows more andmore uniformly, there being the largest number of active hair follicles,the same happened with the babies. Preparation of hair samples
Reagents
Triton X-100 was purchased from Sigma Aldrich (St. Louis, MO). Beginning
Prior to the quantification of the metals, a hair washing step wasperformed to eliminate the elements present on the surface of the hair that could interfere with the quantification of the elements of interest.
Process
The hair samples were rinsed with deionized water for 10 min, thenin a solution of triton X-100 0.1% for 20 min, then another rinse with deionized water (5 times), and finally dried at room temperature onpaper absorbent with little fluff (Kimwipes) (Foo et al., 1993).

The patient underwent the following diagnostic procedures:

The lesion started as a minute keratotic area related to the right alveolar ridge one year before the examination. During one year period, it expanded as a white patch to include the lateral surface of the tongue till the buccal vestibule. The lesion was not painful or ulcerated, but it was rough in texture and was annoying to the patient.

Results

The mothers who agreed to participate in the study were 96, andthe general characteristics of the 96 mother-newborn pairs are presented in tables 3 and 4. The newborns had an average age of 39weeks, a weight of 3073.13 g, a size of 50 cm, a head circumference of 34.98 cm and the APGAR (Appearance, Pulse, Gesture, Activityand Breathing) was 9 for 99% and 8 for 1%. Weight, height, cephalic perimeter, gestational age and APGAR were used to include newbornsin the study, because an inclusion criterion was that the newborns had a good state of health. The hair shape of the newborns was lanky in81.25% and wavy in 18.75% of the children. 87.5% of newborns had dark colored hair and 12.5% light colored hair. (Table 1)

Table 1 : Characteristics of newborns..

The average age of the mothers was 23 years. For 33% of the mothers, it was their first delivery and 67% of the mothers had had more than one birth. No mother presented her hair dyed. The hair shape of the mothers was lax in 53%, wavy in 41% and curly in 6% of the participants. The hair color of the mothers was 1% blond, 54% brown and 45% black. (table2)

Table 2 : Internal and external quality control

The digestion technique was adjusted to 25 and 100 mg of hair sample for the newborn and the mother, respectively, as described in sections 2.6.2 and 2.6.3 of Methods. From the reference sample that was used as internal quality control, the following elements were quantified: Be, B, Ca, Zn, Cu, As, Se, Cd, Sb, La, Ce, Pr, Pb, Tb, Ho, Lu, Hg, Tl, Bi, Th and U (Table 5). The coefficient of variation of the concentration of these elements was from 0.5 to 15.7%, and the recoveries were from 74 to 116%, depending on the element. For the report of the concentrations of elements in the hair samples of the mother-child binomial, rare earths were omitted. (table3)

Table 3: Concentration of the metals present in the reference sample of trace elements in human hair (NCS DC73347A).

Table 4 :Concentration of metals in samples of donated hair, added with 10 ppb of the multi-element standa

Table 5 :Concentration of the metals present in one of the samples of the inter-calibration exercise for trace elements in human hair from the INSPQ Laboratory, Canada

The recovery of the multi-element standard metals added to the hair samples was 78-120% for Be, As, Cd, Sb, La, Ce, Pr, Pb, Lu, Hg, Tl, Bi and U, and the coefficient of variation was less than 15% (Table 6). From the inter-laboratory exercise in which they participated (as external quality control) with two hair samples provided by the INSPQ Laboratory, Canada, the following elements were validated: Be, Zn, As, Se, Cd, Sb, Hg , Ba and Pt, the results of one of the samples are presented in Table 7. Recoveries were obtained from 78 to 114% and the coefficient of variation was from 0.5 to 2.5%, depending on the element.

figure1: Concentratión of metals esentiales in hair. The results represent the Geométric mean And the IC to 9 .

figure 2: Concentratión of metals no esentiales in Hair. The results represent the Geométric mean and IC al 95 %.a) Concentratión en ppb y b) concentratión en ppm (n=96). * p< 0.05, ** p< 0.001.

figure 3: Correlatión enter la concentratión de Ca, Zn y Cu in the mother´s hair and neewborn. We aply test of Spearman, the líne represent tendens of relatión of date, n=96, *p<0.05, **p<0.001.

Tabla 6 :Correlatión between metals presents in Mother´s hair.

n=96, * p ≤0.05, ** p ≤ 0.001

Tabla 7 :Correlatión between metals presents in newborn´s hair.

n=96, * p ≤0.05, ** p ≤ 0.001

Table 8:Correlatión between metals presents in the hair of mother and newborn binomious.

R< 0.3=low, r=0.3 - 0.69=moderathe, r> 0.69=hight. In bold we present the correlations significants (p< 0.05) in binomious.

Concentration of metals in the hair of mothers and newborns

The concentrations of essential and non-essential metals in hair samples of mothers and newborns are presented in Figures 4 and 5, respectively. Significant differences were observed in the concentrations of all metals in the mothers with respect to the concentrations of the metals in the newborns, with the exception of Sb. Relationship between the concentrations of non-essential metals in the hair of mothers and newborns
To determine the relationship between the concentrations of the same non-essential element between mothers and newborns, the Spearman rank correlation was performed. Significant positive correlations were obtained between hair concentrations of mothers and newborns, which for the case of Hg was high (r = 0.7061, p <0.001), moderate for As (r = 4825, p <0.001) and B ( r = 0.6324, p <0.001), and low for U (r = 0.2198, p <0.05) (Figure 7). Correlation of quantified metals in the hair of mothers and newborns
The bi-varied analyzes between the elements were carried out, both in the hair metal concentrations of the mothers (Table 6) and of the newborns (Table 7). On the other hand, Table 10 presents a comparative summary of statistically significant positive correlations between the hair elements of mothers and newborns. In the case of potentially toxic metals, significant associations among the following elements stand out: As-B, Pb-As, Pb-Cd, Pb-Sb and Pb-U, both in the hair of the mother and in the hair of her newborn son, being more robust the association in the hair of the mother. For the case of the essential elements, associations were observed between Ca-Cu and Cu-Zn, in both types of hair being Cu-Zn more robust in the hair of the newborn. Finally, when evaluating the possible correlation between non-essential and essential elements, a correlation was observed for U-Ca in both types of hair (Table 8).

figure 4:Correlatión between concentratión of Hg, As, U y B in the mothers and newborn. We aply test of correlation to Spearman, the líne represent the tendens of relation of the date, n=96, *p<0.05, **p<0.001.

Discussion

Exposure to toxic metals and deficiency of essential metals (which canbe potentially harmful) has been linked to various ailments, including neurodegenerative, cardiovascular and cancer diseases, among others(24). The concentration of metals in the hair is a good biomarker for the evaluation of occupational, environmental and nutritional exposure,due to the advantages it presents in comparison with other biologicalmatrices that include: easy sampling, non-invasive and indicates a chronic exposure; In addition, the concentration of some metalspresent in the hair has been related to the aforementioned conditions in several case-control studies ^(13,24). Therefore, the importance ofknowing the concentration of metals in the hair as a biomarker of exposure and nutrition in early stages such as gestation is to be ableto prevent alterations in the state of health in later stages. Quantification of metals in hair by ICP-MSThe ICP-MS technique is a technique that has many advantagescompared to other techniques such as optical emission spectrometrywith inductively coupled plasma (ICP-OES) and atomic absorption spectrometry (AAS), since multiple elements can be quantifiedsimultaneously. A short time, with little sample and low detection limits. Therefore, a good pre-treatment of the sample is necessary,this through the decomposition of the matrix by a digestion process to keep the ions in solution ^(18,19) in this work the pre-treatment ofthe sample of hair with some modifications that included different amounts of the sample, addition of different volumes of HNO3 andH2O2 and variation of the conditions of temperature and time of digestion, being finally the following conditions: 3 ml of HNO3, 300 μlof H2O2, 150 ° C and 25 min. Very similar digestion conditions have been described (final volume of 3 ml and digestion temperatures) and withof the Environmental Protection Agency (EPA) for the microwave acid digestion of organic matrices is to have a minimum of 1 g of sampleand bring it to a final volume of 10 ml of oxidizing agent (USEPA, 1996); However, these conditions can be modified according to thesize and type of sample, and taking into account the characteristics of each team. The amount of newborn hair sample was the main reasonto adjust the aforementioned conditions and be able to quantify the elements of interest present in trace levels. For quality control,we used the reference material DC73347A Human hair, which has previously been used as a reference material for the determinationof Hg and As (Gault et al., 2008, Martínez et al., 2012), highlighting that it was achieved Validate the quantification of 21 elements, fromwhich good recoveries were obtained, ranging from 74 to 116% and coefficients of variation less than 15.7%. With this, we were able toensure the quantification of 11 elements in 96 pairs of hair samples from mothers and children, using the concentrations of metals in hairas an indicator of exposure in the gestational stage

Characteristics of the study population in relation to the concentrationof metals in the hair Hair color and shape are important characteristics because metalsbind to the functional groups of the two types of melanin, and hair shape determines the availability of SH groups of keratin-associatedproteins ⁽²²⁾. However, these characteristics did not present significant differences in our population, except for the color that had a significantdifference with respect to the concentration of Ca in the hair of the newborn, finding higher concentrations of Ca in dark hair (1902.16 vs1565.43 ppm ), similar results were obtained in melanosomes of dark hair finding higher concentration of Ca, due to the binding of Ca to thecarboxyl groups present in DHICA (5,6-dihydroxyindole-2-carboxylic acid) of eumelanin, a compound lacking in the pheomelanin (Hong& Simon, 2007). Of the characteristics of the mothers, the number of births presented a significant difference with respect to the content ofZn, observing higher concentrations of Zn in the hair of mothers with a delivery (203.08 ppm), than in mothers with more than one birthin 2002, where he mentions that women with multiple pregnancies are more at risk of developing Zn deficiency during pregnancy, perhapsdue to the low availability of accumulated Zn in mothers with multiple births (25).Concentration of the elements in the hair of the mother-child binomialIn this study the following number of times was obtained plus the concentration of toxic elements in the hair of the mother with respectto that of the newborn: 0.81 of Cu, 0.43 of Hg, 1.24 of As, 6.39 of Pb, 7.51 of U and 6.63 of V. The rest of the metals were presented ingreater proportion in the newborn than in the mother. Similar results were observed in a population of mothers and children in the UnitedKingdom (n = 82), where Cu, Cd and Pb were found mostly in the hair of the mother and Zn in the newborn (Razagui & Ghribi, 2005). Inanother population of Tehran, Iran (n = 6) environmentally exposed to high concentrations of metals reported higher concentrations of Cu,Zn, B, Cd and Hg in the newborn compared to the mother, only the Zn and B behaved similarly to the present study ⁽²¹⁾.

The concentrations of the essential elements observed in the hair of mothers and newborns living in Ecatepec were in the following order:Ca> Zn> Cu (Figure 4). As regards the potentially toxic elements, the concentration in the mothers was V> Pb> Hg> U> Sb> As> Cd, and inthe hair of the newborns they were Hg> Sb> V> Pb> Cd> As> U (Figure 5). In a study conducted in populations of eleven cities in China notexposed to a specific source of metals, Pb> As> Cd was found in hair (26), following this same order of concentration in the mothers. Accordingto data obtained in our laboratory, the levels of metals adsorbed in the PM of the Ecatepec region showed the presence of several metalsin the ambient air: up to 0.5 μg / m3 of Mn, Pb and Cu, and up to 0.15 μg / m3 of V, Ni, Cr and Cd (Alvarado-Cruz et al., 2017). This order ofabundance in the environment for Cu, V and Cd coincides with thelevels of metals in the hair present in both the mother and her son, suggesting that hair is a good indicator of environmental exposure.Comparing the concentrations of metals in the hair of newborns with the values established for children of three years, we found that thegeometric means of the concentrations of As, Cd, V and Cu in the hair of newborns were found below the minimum reference value,however in all these elements were atypical values, except for the As that is within the reference range, the concentrations of Pb and Hgwere lower than established as a maximum (3 and 1 ppm, respectively) , while Ca and Zn were above the maximum concentration. For themothers, the geometric means of the concentrations of As, Cd, Hg, Cu and Zn were below the maximum value, but atypical values werefound in As, Cd, Cu and Zn. For the case of V the geometric mean is almost four times above the maximum reference value for adults).

Similarly there are concentrations in the hair for some elementsconsidered at risk in exposed populations (ATSDR, 2010). The toxic concentration in the hair for As, Hg and Pb is of 1, 5 and 8 ppm,respectively, comparing these concentrations with those quantified in the hair of the mother-child binomial of this study, these were foundfar below the values considered toxic. For the normal values reported by the INSPQ, Canada, the geometric means of all the elements arewithin the reference limit both mothers and newborns, but individually comparing the values of mothers and children with the normal valuesfor the INSPQ are atypical data for 22.9% of newborns in Sb. Regarding the atypical concentrations in mothers, 57.3, 15.6, 6.2 and 1% weretaken for V, U, Zn and Cu, respectively. Because specific hair reference values are lacking for newborns and pregnant women, it is difficult to make a comparison for all the elements, since the mineral composition of the hair is dependent on age and gender. Analysis The concentrations of some elements were higher in newborns thanin mothers, as is the case of Ca (56% higher), Zn (7%), B (66%) and Sb (26%). , the other elements were found in greater proportion in themother than in the newborn. On the other hand, a positive correlation was observed between the concentrations of Ca, Cu, Zn, As, B, Hg andU in the hair of the mother and that of the newborns. They presented higher concentrations in 1.5 times.

Conclusions

1. The methodology for the digestion of the hair with acid digestion in closed system was standardized for 25 and 100 mg of sample of thenewborn and the mother, respectively.
2. The quantification of 21 elements of the reference material by ICPMS (including some rare earths) was validated, with good percentages of recovery (74 to 119%) and low coefficients of variation (less than 15.7%).
3. Significant differences were observed between the concentrationsof Cu, Ca, B, As, Hg, Cd, Pb, V, U and Zn in the hair samples of the mother-newborn pair, with the exception of Sb.
4. A positive correlation was observed between the concentrations ofAs, B, Hg, U, Ca, Cu and Zn in the hair of the mother with those of the hair of the newborn, indicating that the mother’s exposure is reflectedin her son.
5. The concentrations of metals in the hair of the mother and thenewborn suggest that the metals are transferred during pregnancy and stored in the hair of the newborn, which represents a risk to thehealth of the children.
6. Of the toxic elements in the hair of the mothers, 15.6 and 57.3% ofthe samples are above the normal value of U and V, respectively. For the hair of the newborns, 22.9% of the samples were above the normalvalue of Sb. However, information on concentrations considered toxic of these elements in hair is lacking.
7. The concentrations of metals in the hair of the mother can be a goodindicator of the exposure of the fetus during pregnancy for at least seven elements: As, B, Ca, Cu, Hg, Zn and U.
8. The positive correlations observed in the mother-newborn pairoffers the advantage that by knowing the concentration of elements in the pregnant mother’s hair, the exposure of the developing childcan be estimated.

References:

1. Agency for Toxic substances and Disease Registry (ATSDR). (2012). No Title. Retrieved July 25, 2015, from www.atsdr.cdc.gov/toxprofiles
2. Al-Ayadhi, L. (2005). Heavy metals and trace elements in hair samples of autistic children in central Saudi Arabia. Neurosciences, 10(3), 213–218.
3. Byrne, R., & Benedik, L. (1991). Uranium content of blood , urine and hair of exposed and non-exposed persons determined by radiochemical neutron activation analysis , with emphasis on quality control. Science of the Total Environment, 107(1), 143–157.
4. Bridges, C., & Zalups, K. (2005). Molecular and ionic mimicry and the transport of toxic metals. Toxicology and Applied Pharmacology, 204(3), 274–308.
5. Calderón, L., Franco, M., D’Angiulli, A., Rodríguez, J., Blaurock, E., Busch, Y., Chao, C., Thompson, C., Mukherjee, P., Torres, R. & Perry, G. (2015). Mexico City normal weight children exposed to high concentrations of ambient PM2.5 show high blood leptin and endothelin-1, vitamin D deficiency, and food reward hormone dysregulation versus low pollution controls. Relevance for obesity and Alzheimer disease. Environmental Research, 140(1), 579-592.
6. Chen, G., Chen, X., Yan, C., Wu, X., & Zeng, G. (2014). Surveying Mercury Levels in Hair, Blood and Urine of under 7-Year Old Children from a Coastal City in China. International Journal of Environmental Research and Public Health, 11(11), 12029–12041.
7. Dawber, R. (1996). Hair: Its structure and response to cosmetic preparations. Clinics in Dermatology, 14(1), 105–112.
8. Foo, S., Khoo, N., Heng, A., Chua, L., Chia, S., Ong, C., Ngim, C. & Jeyaratnam, J. (1993). Metals in hair as biological indices for exposure. International Archives of Occupational and Environmental Health, 65(1), S83–S86.
9. Gellein, K., & Lierhagen, S. (2008). Trace element profiles in single strands of human hair determined by HR-ICP-MS. Biological Trace Element Research, 123(1-3), 250-260.
10. González, M., Peña, A., & Meseguer, I. (2008). Monitoring heavy metal contents in food and hair in a sample of young Spanish subjects. Food and Chemical Toxicology, 46(9),
11. H. Ayuntamiento de Ecatepec de Morelos. (2013). No Title. Retrieved July 23, 2015, from www.ecatepec.gob.mx/Cuaderno de Inf. Est. y Geog. No. 3-ok.pdf
12. Instituto Nacional de Estadística y Geografía (INEGI). (2013). No Title. Retrieved July 23, 2015, from www3.inegi.org.mx/sistemas/mexicocifras/?e=15&mun=033
13. Manduca, P., Naim, A., & Signoriello, S. (2014). Specific association of teratogen and toxicant metals in hair of newborns with congenital birth defects or developmentally premature birth in a cohort of couples with documented parental exposure to military attacks: observational study at Al Shifa Hospit. International Journal of Environmental Research and Public Health, 11(5), 5208–23.
,li>Mckenzie, J. (1979). Content of zinc in serum, urine, hair, and toenails of New Zealand adults. The American journal of clinical nutrition, 32(3), 570-579.
14. Nowak, B. (1998). Contents and relationship of elements in human hair for a non-industrialised population in Poland. Science of the Total Environment, 209(1), 59–68.
15. Ortonne, J., & Prota, G. (1993). Hair melanins and hair color: ultrastructural and biochemical aspects. The Journal of Investigative Dermatology. 101(1 Suppl), 82S-89S.
16. Razagui, I., & Ghribi, I. (2005). Maternal and neonatal scalp hair concentrations of zinc, copper, cadmium, and lead: relationship to some lifestyle factors. Biological Trace Element Research, 106(1), 1–28.
17. Rodrigues, J., Batista, B., Nunes, J., Passos, C. & Barbosa, F. (2008a). Evaluation of the use of human hair for biomonitoring the deficiency of essential and exposure to toxic elements. Science of the Total Environment, 405(1-3), 370–376.
18. Rodrigues, J., Nunes, J., Batista, B., Simiao de Souza, S. & Barbosa Jr, F. (2008b). A fast method for the determination of 16 elements in hair samples by inductively coupled plasma mass spectrometry (ICP-MS) with tetramethylammonium hydroxide solubilization at room temperature. Journal of Analytical Atomic Spectrometry, 23(7), 992–996.
19. Sakamoto, M., Man Chan, H., Domingo, J. L., Kubota, M., & Murata, K. (2012). Changes in body burden of mercury, lead, arsenic, cadmium and selenium in infants during early lactation in comparison with placental transfer. Ecotoxicology and Environmental Safety, 84(1), 179–184.
20. Savabieasfahani, M., Hoseiny, M., & Goodarzi, S. (2012). Toxic and essential trace metals in first baby haircuts and mother hair from imam hossein hospital Tehran, Iran. Bulletin of Environmental Contamination and Toxicology, 88(2), 140–144 Secretaria del Medio Ambiente. (2013). Zona Metropolitana del Valle de México. Inventario de emisiones de contaminantes y de efecto invernadero 2012, Primera ed.
21. Shimomura, Y., & Ito, M. (2005). Human Hair Keratin-Associated Proteins. Journal of Investigative Dermatology Symposium Proceedings, 10(3), 230–233.
22. Suliburska, J. (2011). A comparison of levels of select minerals in scalp hair samples with estimated dietary intakes of these minerals in women of reproductive age. Biological Trace Element Research, 144(1-3), 77–85.
23. Wołowiec, P., Michalak, I., Chojnacka, K., & Mikulewicz, M. (2013). Hair analysis in health assessment. Clinica Chimica Acta, 419, 139–171.
24. Salgueiro, M. J., Zubillaga, M. B., Lysionek, A. E., Caro, R. A., Weill, R., & Boccio, J. R. (2002). The role of zinc in the growth and development of children. Nutrition, 18(6), 510-519.
25. Zhou, T., Li, Z., Zhang, F., Jiang, X., Shi, W., Wu, L., & Christie, P. (206). Concentrations of arsenic, cadmium and lead in human hair and typical foods in eleven Chinese cities. Environmental Toxicology and Pharmacology, 48, 150-156.