Essential Trace Elements in Scalp Hair of Residents across the Caspian Oil and Gas Region of Kazakhstan

Most of the country’s oil and gas fields are situated in West Kazakhstan, mainly on the Caspian Sea coast, causing significant damage to the local environment and contributing to an imbalance in the trace element composition of the human body. The study is aimed to evaluate the relationship between the concentration of essential trace elements in scalp hair of the western Kazakhstan adult population and the remoteness of their residence from oil and gas fields. The concentration of essential trace elements (Co, Cu, Fe, I, Mn, Se, Zn) in the hair of 850 individuals aged 18–60 years was determined by inductively coupled plasma mass spectrometry. In residents of settlements located at a distance of >110 km from oil and gas fields, the concentration of Cu and I in hair was significantly higher than in those closer to 110 km (p < 0.001). The content of Cu and I were associated with the distance to oil and gas fields (0.072 (95% CI: 0.050; 0.094)) and (0.121 (95% CI: 0.058; 0.185)), respectively. We detected a significant imbalance in the distribution of some essential trace elements in residents’ scalp hair from the Caspian region of western Kazakhstan, living near oil and gas fields. The concentrations of Cu and I were significantly interrelated with the distance to oil and gas fields. The level of copper in the hair of both inhabitants of the area most remote from oil and gas facilities and the entire population of western Kazakhstan as a whole remains significantly low. The data obtained provide evidence of the possible impact of pollutants generated by the oil and gas facilities on a shortage of essential trace elements and associated subsequent health risks.


Introduction
The Republic of Kazakhstan (RK) ranks 8th in the world regarding proven oil reserves but 84th according to the Environmental Performance Index and 137th in the Life Expectancy Index [1]. Despite significant investments in public health and priority reforms to make health services more accessible and efficient, Kazakhstan lags behind high-income countries on health indicators [2]. and Zn in the hair of children living in a region free from oil production compared with a polluted area was described [25].
One should note that the right choice of biomarkers is crucial for assessing the impact of contaminants, including metals, on the human body. Hair samples are widely used to assess human exposure to various pollutants due to their many advantages, such as ease of sample collection and transportation, convenient storage, and higher concentrations of elements than in other bioindicators [26]. Hair deposits trace elements compared to urine, saliva, tear fluid, and blood. Also, hair provides a more accurate determination of trace elements and can reflect quantitative changes in many elements and related metabolic processes over a long period [27]. The correlation described by some authors between the concentration of elements in hair and their level in the body in a physiological or pathological state confirmed the usefulness of hair as a diagnostic tool [8,28]. Thus, there is a vast amount of literature on the adverse effects of heavy metals on human beings' health.
However, little is known about human exposure to heavy metals in western Kazakhstan's oil and gas production and processing areas. Only a few studies are available on the essential trace elements in residents living in those areas. This circumstance calls for the need to describe the trace element status of the population living around the fields.
The present study is the first on essential elements in scalp hair of residents from the Caspian region of western Kazakhstan, living near oil and gas fields. There have been no studies comparing the content of essential elements in residents' hair depending on the distance to the oil and gas fields in the Caspian region of western Kazakhstan.
Hence, the study's objective is to evaluate the relationship between the concentration of essential trace elements in scalp hair of the western Kazakhstan adult population and the remoteness of their residence from oil and gas fields.

Study Design and Sites Description
We carried out a cross-sectional study during research trips to Aktobe, Mangystau, and West Kazakhstan provinces (oblasts) to determine the trace element status of the adult population. The West Kazakhstan Medical University IRB approved the research (meeting No. 5 dated 13 May 2020), which was performed following the STROBE checklist and the principles of the Helsinki Declaration.
Description of the sites with and without oil and gas production. Western Kazakhstan is the country's largest oil and gas producing region, located between Eastern Europe and Central Asia. It consists of four provinces (oblasts): Atyrau, West Kazakhstan, Aktobe, and Mangystau. There is a coastline of considerable length, formed by the coast of the Caspian Sea, washing the Mangyshlak peninsula and the Caspian lowland.
The Caspian region includes three oil and gas basins: Pre-Caspian, Ustyurt-Bozashi, and Mangyshlak. Oil production volumes reach up to 100-120 million tons per year. At the same time, oil production is expanding due to the giant and large Paleozoic and Mesozoic fields of the Caspian region (Tengiz, Karachaganak, Kashagan and Zhanazhol groups, Uzen, Kalamkas, Zhetybay, Imashevskoye), geotectonically related to the Caspian basin and the west of the Turan plate. Overall, two hundred thirty-three fields have been discovered in the Caspian region-162 sites in the Caspian basin, 55 in Mangyshlak, and 18 in Ustyurt-Bozashi including 8 in the Northern and Middle Caspian waters [29].
Aktobe province (oblast) is located in the northwestern part of Kazakhstan. The annual precipitation ranges from 125 to 300 mm. Aktobe is a large industrial region of Kazakhstan. The main industrial sectors are mining, chemical industry, and ferrous metallurgy. The tense environmental situation in the region is associated with the activities of chromium mining and processing companies and oil and gas facilities. A stable natural-technogenic boron-chromium geochemical province has formed in the area [30]. The West Kazakhstan province is located northwest and occupies 151.34 thousand sq. km. The climate is sharply continental. The annual rainfall ranges from 250 up to 400 mm. The oil and gas industry (Karachaganak and Chinarevskoye fields) and engineering enterprises negatively impact the environment [31]. Mangistau province is located in southwest Kazakhstan, toward the east of the Caspian Sea on the Mangyshlak plateau. The region's territory constitutes the wormwood-saline desert, extremely arid, with rainfall of about 100-150 mm per year. The Mangistau oblast is a developed industrial region where 25% of Kazakhstan's oil is produced. The main adverse factors are the lack of freshwater, infertile soils, and constant fluctuations in the level of the Caspian Sea [32].

Sampling
We used a cluster sample for this cross-sectional study. In each cluster, we formed a group with a random sample of recruited volunteers aged 18-60, permanently residing in the study area. A total of 850 individuals, men 350 (41.2%), and women 500 (58.8%), were included in the study. The surveyed population involved in the study came from 32 settlements in western Kazakhstan. These settlements, their distance from the oil fields, and the number of participants in each are enlisted in Table S1 (Supplementary Materials).
Exclusion criteria were the following: acute infectious, surgical, or traumatic diseases, decompensated chronic somatic diseases, having metal implants (including amalgam fillings), following a vegetarian diet, intake of vitamin and mineral supplements, pregnancy, childbirth less than one year ago, and breastfeeding.
The spatial distribution of the locations is reflected in Figure 1. We compared the content of trace elements in the hair of residents living near the oil and gas fields fewer than 16 km, from 16 to 110, and more than 110 km, respectively.
A distance of up to 16 km is considered the distance at which the consequences of the operation of the oil and gas facilities can affect the human body [33]. When comparing the content of trace elements, for the results of those living out of 110 km, we assumed them to be relatively uncontaminated [34]. Distances from the settlements to fields were calculated using Google Maps. For each location, we applied the coordinates taken from Wikipedia.
Each participant was enrolled after clearly explaining the research goals, the method used to analyze the collected samples, and how her/his identity and privacy would have been protected. The informed consent forms were provided in the Kazakh and Russian languages, of the participant's choice. These forms are available on reasonable request.

Data Collection
We collected samples from November 2020 to February 2021. Hair samples of at least 0.1 g were obtained by cutting with clean stainless steel scissors from three to five areas at the back of the head [35,36]. In the case of long hair, only the proximal terminus was collected. In these hair samples, we estimated the content of essential trace elements: Co, Cu, Fe, I, Mn, Se, and Zn.
The elemental composition of hair we studied using inductively coupled plasma mass spectrometry (ICP MS) on a NexION 300D mass spectrometer (PerkinElmer Inc., Shelton, CT, USA) equipped with an ESISC-2 DX4 automatic dispenser (Elemental Scientific Inc., Omaha, NE, USA). The ICP-MS operating parameters are shown in Table S2, while the limits of detection (LoD), limits of quantification (LoQ), and background equivalent concentration (BEC) are presented in Table S3.
Hair samples were subjected to preparation by washing and microwave decomposition. The hair strands were washed with acetone, rinsed three times with deionized water, and dried in air at a temperature of 60 • C. After preliminary preparation and taking of weighted amounts (no less than 10 mg) of each hair sample, bio substrates were transferred into chemically stable Teflon tubes with high purity nitric acid.
Microwave digestion was carried out for 20 min at a temperature of 170-180 • C in a "Berghof Speedwave 4 system" (Berghof Products + Instruments, GmbH, 72800 Eningen, Germany). After cooling and equalizing the pressure in the system, the solutions obtained during decomposition were transferred into tubes, and the volume was adjusted to 15 mL with distilled deionized water. The final solution was used for chemical analysis. The system was calibrated using the Universal Data Acquisition Standards Kit (Perkin Elmer Inc., Shelton, CT, USA). Internal online standardization was performed using a Yttrium-89 isotope solution obtained from Yttrium (Y) "Pure Single Element Standard" (Perkin Elmer Inc., Shelton, CT, USA). A certified human hair standard GBW09101 "Humanhair" issued by the Shanghai Institute of Nuclear Research (China) was used for quality control.

Statistical Processing
The distribution of the data concentrations of essential trace elements (Co, Cu, Fe, I, Mn, Se, Zn) in the scalp hair was non-Gaussian. The content of essential trace elements in hair is presented with the geometric means (GM), arithmetic means (AM), median (Me), percentiles (P2.5%; P97.5%), maximum (Max), and minimum (Min) values. Qualitative variables are presented as absolute values and percentages.
We analyzed the differences in the content of essential trace elements in three groups depending on the place of residence (<16 km, 16-110 km, and >110 km) using the Kruskal-Wallis H-test. For a posteriori comparisons for these three groups, a new critical level of p < 0.017 was used [37].
Multiple linear regression analysis was applied to assess the relationship between concentrations of essential trace elements in the hair (dependent variable) and the settlement's remoteness from the oil and gas production site (independent variable). We transformed the data using the natural logarithm Ln(X) to perform the multiple linear regression model. Verification of the distribution of variables after the logarithm was carried out using de-scriptive statistics and graphical methods. The following covariates self-reported by the participants in the questionnaire were included in the models as covariates to adjust for potential confounding: age (continuous), gender (two categories), body mass index (BMI, continuous), and tobacco smoking (two categories).
For statistical testing hypotheses, the critical significance level p was taken equal to 0.05 with a 95% confidence interval. SPPS.v.25 Modeler (IBM) and Statistica.v.10 (StatSoft) software were used for statistical analysis.

Results
The main features of the study participants are presented in Table S1. The study included n = 850 adults from western Kazakhstan. There were 350 men (41.2%) and 500 (58.8%) women. The average age of men was 42 (31.0; 53.0) years, and women were older by an average of 10 years. The body mass index for men and women was 25.7 (22.5; 28.7) and 25.5 (22.7; 29.1). Among the total sample, smokers were 12.7%, and the city inhabitants were 239 (28.1%). The remoteness of settlements (n = 32) from oil and gas production/processing areas ranged from 2.3 to 475 km (Table S1).
Data on the concentration of essential trace elements in residents' scalp hair are summarized in Table 1. Data are presented as the arithmetic mean (AM), geometric mean (GM), median (Me), percentiles (P2.5%; 97.5%), and maximum (Max) and minimum (Min) values. We analyzed comparatively the three groups depending on the distance from oil and gas fields: group 1-less than 16 km; group 2-from 16 to 110 km and group 3-more than 110 km. Findings are presented in Table 2. We observed a significant change in Cu, I, Mn, and Se concentrations. The highest hair concentrations of Cu = 10.96 µg/g, I = 0.391 µg/g, and Mn = 0.559 µg/g have been recorded in residents from the settlements at a distance more than 110 km away from the oil and gas production sites. A posteriori comparisons showed that the content of Cu and I in their hair was significantly higher than that of residents whose settlements belonged to groups 1 and 2 (p < 0.001). The content of Mn = 0.361 µg/g in the hair of residents living less than 16 km from the oil and gas production sites was significantly less than that of groups 2 and 3. Concerning Se, we observed the opposite effect. Its concentration decreased with the distance from the fields to 0.540 µg/g; 0.509 µg/g; 0.471 µg/g, respectively, and reliably differed in all three studied groups.
The next step constituted a multiple linear regression analysis (Table 3). We introduced the essential trace elements' concentrations natural algorithm into the analysis as a dependent variable. The distance from the place of residence to oil and gas production facilities, age, gender, BMI, and smoking were introduced as independent variables. We found that regardless of age, gender, BMI, and smoking, with the distance from the oil and gas production locations (every 100 km), the concentration of Cu and I in residents' hair increased. An inverse relationship was observed for Co, Fe, Se, and Zn, but for Mn, a direct one. Nonetheless, statistical significance for these trace elements was not found.

Discussion
Overall, we established a significant difference between the three groups of examined residents of the Caspian region in the concentrations of Cu, I, Se (p < 0.001), and Mn (p = 0.002). The level of Cu, I, and Mn in the hair of residents living in the closest proximity to active oil and gas fields (<16 km) appeared to be significantly lower. On the contrary, the distribution of Se in residents' hair demonstrates the opposite trend, reaching a maximum near the working oil and gas facilities (Table 1).
When reporting the elemental status of residents in oil-producing regions, attention is usually focused on the groups of toxic and potentially toxic elements associated with these elements' pollution of the oil-producing area [38][39][40]. The existing data on the content of essential elements in the hair of residents from oil-producing regions are not enough to perform a comparative analysis. The change in the range of essential elements in the body accompanied by changes in their level in hair can be affected by various factors. The story of essential elements in the body can be influenced by the natural geochemical features of terrains where the population lives and by pollution of the territory of various origins (natural, industrial). In addition, the trace element status reflects multiple biochemical and physiological processes, including those associated with antagonistic or synergistic interactions between elements belonging to both the same and different groups. There are conflicting data on the hair content of manganese, which belongs to the group of essential elements due to its participation in critical physiological processes. At the same time, it is one of the chemical elements associated with the pollution of oil and gas production areas. Some studies confirm the contamination of soils in oil and gas production areas with manganese [18,41], and some report no change in manganese concentration in soils of such regions. [17,42] Studies report an increase in the concentration of Mn in the hair of children living under conditions of industrial pollution [43] and the hair and urine of pregnant women from the regions of gas production by fracturing method [23]. The study by Moon et al. is of great interest as being one of the oldest research for determining the concentration of chemical elements in the hair of residents from oil-producing regions [25]. The authors found a rise in the concentration of manganese, along with other elements from the group of essential ones-iron and zinc, as well as the concentration of calcium and magnesium in the hair of children living in an area classified as unpolluted. In our study, manganese concentration is significantly lower in residents' hair from the area of oil fields ( Table 2).
On the one hand, this can indirectly confirm the absence of significant contamination of this area with manganese compounds. On the other hand, a higher concentration of manganese in the hair of residents of zones remote from the oil and gas facilities ( Table 2) could likely be associated with large industrial cities in western Kazakhstan. The economic potential of these cities is formed precisely by ferrous and non-ferrous metallurgy, resulting in the active entry of ecologically dangerous substances into the environment. These substances include recognized pollutants As, Hd, Cd, Be, and heavy metals, classified as essential trace elements-Cu, Fe, Zn, Co, and Mn according to the physiological classification. Albeit the study by Baubekova et al. does not classify western Kazakhstan as a region with a high level of industrial pollution of Fe, Zn, Co, and Mn [44].
When evaluating the content of essential trace elements in the hair of residents of western Kazakhstan, we should realize that the entire territory of settlement belongs to the zone of dry steppes, semi-deserts, and deserts, forming a biogeochemical zone characterized by an excess of zinc and boron and a deficiency of copper, iodine, and cobalt [45].
The decrease in the level of copper is most pronounced in the group of residents from zones located near oil and gas fields (<16 km) (Table 2, Figure 1). Researchers from Poland obtained similar findings in a study conducted in industrially polluted areas. Nowak et al. found a reduced concentration of copper in biological substrates, including hair, among residents of contaminated terrains. The authors attribute the decrease in copper to an increase in the concentration of lead and cadmium in the environment and in the examined individuals' bodies. As known, these heavy metals belong to the toxic group and are antagonists of copper [46].
There are few papers on iodine content in the biological substrates of the inhabitants of petrochemical provinces. Kudabayeva et al. reported a decrease in iodine concentration in the children's hair in western Kazakhstan. However, the authors have not focused on the terrain zoning according to the distance of settlements from oil and gas fields [47][48][49]. In the Aral Sea region adjacent to western Kazakhstan, a decline in the iodine in the biological substrates of residents has also been established [50]. The frequency of thyroid diseases associated with decreased function is relatively high in Kazakhstan west, primarily because the region is classified as an iodine-deficient biogeochemical province. Considering that oil and gas fields are located in rural areas, the diet of inhabitants of the settlements around the fields may differ from the diet of urban residents, and a lessening of iodine in their hair may be a consequence of a reduction of iodine intake from food. It has been found that iodine deficiency is common for terrains where the population consumes local agricultural products grown on soils poor in iodine. [51,52]. Formal studies to determine the dietary intake of iodine in western Kazakhstan have not been performed yet. The decrease in hair iodine concentration, probably reflecting the decline in iodine content in the bodies of the population living near oil and gas fields, may result from iodine interactions with antagonists, particularly with Pb, which is a recognized environmental pollutant in oil production [17,34]. Our study also found a decrease in the concentration of Cu in the scalp hair of the oil production zone inhabitants. It is known that copper deficiency can reduce the absorption of iodine by the body and its utilization by the thyroid gland [53,54].
Notably, the regression analysis confirmed the increase in selenium concentration in residents' hair from the area most affected by the oil and gas industry (Tables 1 and 2). However, the studied region (western Kazakhstan) is not among the biogeochemical provinces characterized by increased selenium content. Researchers have identified and described selenium-bearing areas in many parts of the world: the eastern part of Russia [55], China [56], Taiwan [57], Punjab [58], and Amazonia [59]. Their studies have assessed the relationship between chronic exposure to environmental selenium and human health. Human health can be affected by abnormally low and excessively high levels of selenium in the environment-in soil, food of local origin, and drinking water [60].
Some authors suppose that selenium levels in the blood and hair are the best biomarkers of exposure because the content of selenium in the blood serum and the hair is strongly correlated. [58]. However, Kousa et al. could not establish such a correlation [61]. According to some researchers, Se in urine is the best tool for determining Se status in the body [62]. Although, it is commonly accepted that selenium, a chemical analog of sulfur, accumulates in nails and hair, as the sulfur-containing amino acids cysteine and methionine comprise hair and nail. This property of selenium is believed to entail a negative effect on the human body, associated with a violation of the functions of proteins due to the replacement of sulfur in amino acids [63,64]. An increase in selenium content in hair can be noted in many pathological conditions. For example, Tinkov et al. found that the Se content in the hair of obese individuals significantly exceeded the control values [65].
However, in the present study, the increased level of selenium in the hair of residents from the oil and gas production zone within 0-16 km, compared with those living in zones at a distance of 16 to 110 km and more than 110 km, did not go beyond the reference values established by the laboratory (Tables 1 and 4). It may be associated with increased dietary intake of selenium-containing foods such as beef [56], fish [57], or white bread [55]. Some sources evidence a slight increase in the content of selenium in food (meat, dairy products, wheat) in the neighboring region of the Russian Federation [55]. The results of comparing the range of essential trace elements Co, Cu, Fe, I, Mn, Se, and Zn in the hair of residents living in areas with varying degrees of remoteness from pollution sources associated with the operation of oil-and-gas-producing/processing facilities, we confirmed through the multiple linear regression analysis. According to our findings, the increased concentration of Cu and I in hair is associated with increased distance from oil and gas fields. But for Se, the unadjusted regression analysis model describes an inverse relationship (Table 3). When conducting a regression analysis, the food intake and environment are mostly believed to be leading factors influencing the concentration of essential elements in hair. However, additional endogenous and exogenous reasons may play a significant role. Linear regression models created considering confounding factors such as age, gender, BMI, and smoking, confirmed the above patterns described for Cu and I ( Table 2).  Table 4 presents comparative results on the hair concentration of essential trace elements Co, Cu, Fe, I, Mn, Se, and Zn, obtained in different populations. These data indicate a significant difference in the distribution of essential elements due to the residence of people in territories belonging to various biogeochemical provinces, characterized by an excess or deficiency of certain elements.
In Figure 2, we presented the distribution of copper concentrations in hair of the adult population of western Kazakhstan living in areas of different distances from oil and gas production zones compared to similar data on the child population of the same area [67], as well as the populations of Italy (Sicily) [74], Brazil [8], Russia [66], Sweden [72], Poland [73], and Canada [70]. This graph also includes data from the research by Iyengar et al. and Caroli et al., with accumulated findings from several studies [68,69]. Of the presented populations, the exposed ones included the population of children from Sicily living in the region of industrial pollution. The rest of the populations were not exposed or had both the exposed and the unexposed individuals (Iyengar et al., Caroli et al.). Noteworthy is the decrease in the median (10.35 µg/g) compared to the rest of the populations (Figure 2), except for a study also performed in western Kazakhstan, where the concentration of copper in children's hair was found to be 9.5 µg/g. The decrease in the level of copper in hair is most pronounced in the zone of oil and gas production (<16 km), and as moving away from the sources of pollution, the median concentrations of copper at the distances of 16-110 km and >110 km increase significantly. Despite this, copper concentrations in the inhabitants' hair from terrains most remote from oil and gas facilities, such as the entire population of western Kazakhstan, remain significantly low.
Supposing changes in copper concentration in the hair are associated with the oil industry is convincing, given the distance difference between the three zones. Yet, the decrease in the copper level in the population of western Kazakhstan as a whole, especially compared to other populations, needs additional rationale. Likely, this phenomenon also suggests reducing the copper concentration related to the biogeochemical province of a zone of dry steppes, deserts, and semi-deserts, to which this region belongs. Figure 3 shows that the median distribution of iodine concentration in adults' hair in western Kazakhstan, described in this work, is significantly lower than the same values in the child population from the same region [67], the study by Iyengar et al. [69] and Sweden research [72]. There is also a significant difference in the level of hair iodine depending on the remoteness of oil and gas production enterprises. The hair concentration of iodine is minimal in the zone <16 km and gradually increases, taking the values typical for most inhabitants living at a distance of >110 km from the oil production area.
The present study has some limitations. Heterogeneity of age and gender distribution within and between settlements was the main limitation of the study. Most men were more likely to have short or seldom hair, which was inconvenient for collecting samples. Besides this, many were reluctant to participate in the study for unspoken reasons. As for women, they were more willing to be enrolled in the study. The study format did not imply receiving complete information about the content of trace elements in the environment of western Kazakhstan (soil, water, air). Furthermore, dietary habits also influence trace element status, but we could not scope these habits within the research presented. Although some studies show that while the idea of measuring trace elements in hair is attractive, hair is not a suitable biomarker for assessing I, Cu, and Mn deficiency. In addition, residents of the Atyrau region of the western part of Kazakhstan are not included. Studies solely focused on recording changes in the level of essential elements in hair toward a decrease or an increase cannot explain the nature of these metamorphoses. Therefore, significant fluctuations in the concentration of essential elements in residents' hair from the studied region should become the basis for more comprehensive research involving influencing factors as far as possible. The protocol of further examination should include blood tests and determining the amount of trace elements ingested with food. It was also necessary to analyze blood and urine samples to compare and confirm f toxic effects.
Notwithstanding, our data at least confirmed a change in the content of essential elements depending on the distance to/from oil and gas producing facilities. Significant changes in the concentration level of essential elements in the hair of residents of the studied region should be the basis for additional research, including analysis of the content of toxic, potentially toxic trace elements, antagonistic, and synergistic interactions between them. In future research, it is important to study the content of essential elements in the air, water, and soil of the considered region of Kazakhstan and evaluate their relationship with adverse health effects on people living in the region of oil and gas production in western Kazakhstan.

Conclusions
In the inhabitants' hair from the oil and gas production region in western Kazakhstan, the concentrations of Cu and I are significantly interrelated with the distance to oil and gas fields (approaching them). In zones with varying degrees of remoteness from oil and gas fields, we revealed a reduction in Cu, I, and Mn content at a distance of 0-16 km near such sites. The Se concentration decreased with distance from the fields. The data obtained indicate the possible impact of pollutants associated with the activities of oil and gas producing/processing enterprises on a shortage of essential trace elements and consequent health risks.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxics10070364/s1, Table S1: Brief description of the study sites and population; Table S2: System characteristics and settings for trace element analysis; Table S3

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of the West Kazakhstan Medical University (meeting No. 5 dated 13 May 2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data are available from the corresponding author upon request.