**1. Introduction**

Ischemic stroke is a sudden disorder of cerebral blood flow that temporarily or permanently alters the function of a certain region of the brain. According to the World Stroke Organization (WSO), age-adjusted rate for ischemic stroke per 100,000 populations was 142.34 in 2016. Over 9.5 million new cases were diagnosed during that year [1]. Prevalence of stroke in US population younger than 60 years old is around 2%, but this proportion rises to 6% and 15% in people older than 60 and 80 years old, respectively [2]. In Spain, it has been estimated a prevalence of stroke of 6.4% in subjects older than 70 years [3].

Age-adjusted incidence ranges from 50 to 250 per 100,000 populations in France and Portugal, respectively [4]. In our country, for a similar period, incidence ranged from 99 to 206 per 100,000 populations, depending on the region in which the study was conducted [4]. The Canary Islands are the second region of Spain with the lowest adjusted rate of stroke (25.33 per 100,000 men and 19.66 per 100,000 women) [3].

Stroke is the second leading cause of death worldwide, and the third leading cause of disability [5]. In Spain, mortality has decreased considerably during the past decades, standing at around 50 per 100,000 inhabits for both sexes [6]. The Canary Islands are the second autonomous community in Spain with the lowest adjusted rate of cerebrovascular deaths (25.3 per 100,000 males and 19.7 per 100,000 women) [3].

The main risk factors for stroke are mostly modifiable factors such as hypertension, dyslipidemia, diabetes, smoking, low physical activity levels, unhealthy diet and abdominal obesity [2]. Age, gender, race/ethnicity or genetics also have an important role for the disease [7]. New risk factors for stroke have been proposed during the last decades. Some of them have emerged as protective factors (i.e., antiplatelet therapy) while others seem to increase the risk of stroke (i.e., sleep apnea or lipoprotein levels) [2]. However, their contribution to stroke risk is less well defined and understood. This is the case of environmental pollutants. In addition to gaseous and particulate air pollutants [8], persistent organic pollutants (POPs) seem to play an important role in development of stroke [9]. Although the association had been seen for years, it has recently been observed that elevated serum POPs levels were associated with an increased risk of stroke, specifically for organochlorine pesticides (p,p'-DDE) and polychlorinated biphenyls (PCB-118, -156 and -138) [10], possibly due to an association with hypertension [11,12] and obesity [13].

Among environmental pollutants, toxic heavy metals and metalloids are among the most dangerous because they are also not biodegradable and tend to accumulate in environmental compartments [14]. According to their high degree of toxicity, arsenic, cadmium, lead and mercury are usually highlighted among others [14]. However, the Agency for Toxic Substances and Disease Registry (ATSDR) publishes a list of priority chemicals that are determined to represent the most significant potential threat to human health because of their known or suspected toxicity, together with the potential human exposure. Additionally, there are a number of elements, the rare earth elements (REE) and other minor elements (ME), which are increasingly coveted due to the large number of technological applications for which they are already indispensable [15]. This set of elements is of growing concern because its enormous range of applications makes them mobilized from the few sites where they are abundant to be distributed all over the planet [16], especially once the useful life of the devices containing them ends. Although some of these elements are relatively abundant in the Earth's crust (i.e., cerium is as abundant as copper), REEs have been included among the new and emerging occupational and environmental health risks by several international organizations [17]. Di fferent studies have shown that some of these elements have an adverse e ffect on people's health [18–20], although the mechanisms of action are not clear [17,21].

Of all these inorganic elements, arsenic and lead have shown a relationship with stroke [22–25]. A significant dose-response relationship was observed between arsenic concentration in well water and prevalence of cerebrovascular disease [25] and a positive trend was reported between blood lead and stroke in a series of 88,000 workers from USA, Finland and UK [24]. However, the mechanisms of action are not clear and, for example, a potential role for arsenic methylation in the pathogenesis of stroke has been suggested [22].

It has to be highlighted that some inorganic elements are neurological disruptors with the capacity to cross the blood brain barrier [18]. While small concentrations of some elements are needed for life, most are considered non-essential and some are very toxic even at very low concentrations. Some elements follow a hormetic dose–response curve and may cause, at a very low dose, the opposite effect to a high dose [26]. Thus, the presence of these elements into neurons, even at low concentrations, seems to be able to modify brain homeostasis. This is the case of gadolinium and tantalum whose tissue concentrations were higher among patients with brain cancer compared to a control group [18]. The aim of the present study was to evaluate the contribution of 45 inorganic elements—including trace elements, elements included in the priority list of substances of the Agency for Toxic Substances and Disease Registry (ATSDR), and REE and other elements used in electronic devices—as factors associated with stroke.

#### **2. Patients and Methods**

#### *2.1. Study Design and Participants*

We designed a case-control study aimed to disclose the role of inorganic elements in the stroke. The study was approved by the Research Ethics Committee of the CHUIMI on 23 February 2017 (ID number: CEIm-CHUIMI-2017/907). For that, a total of 92 patients diagnosed with ischemic stroke and admitted into the Complejo Hospitalario Insular-Materno Infantil CHUIMI for rehabilitation were included in the study. The control group consisted of patients admitted to the rehabilitation service for other causes. The final number of control subjects was 83. The recruitment was made between April 2017 and April 2018. The inclusion criteria for cases were: (i) having been diagnosed of stroke in the 12 months prior to being referred to the Rehabilitation Service, (ii) ability to agree to participate in the study (signed informed consent) and (iii) being over 18 years old. The inclusion criteria for controls were: (i) admission diagnosis unrelated with stroke (i.e., traumatic diseases), (ii) never having been diagnosed with stroke, (iii) ability to agree to participate in the study (signed informed consent) and (iv) being over 18 years. Cases and controls were matched in terms of age and gender making a selection of cases in relation to the demographic characteristics of the cases.

Patients—cases and controls—were contacted and asked to participate in the study. All patients signed the informed consent before entering the study. The study was approved by the Research Ethics Committee of the CHUIMI (study number CEIm-CHUIMI-2017/907).

Barthel index, an instrument widely used to evaluate independency and measures the capacity of the person for the execution of ten basic activities in daily life [27], was recorded three months after the admission in the rehabilitation service. The demographic and clinical data of the patients included in the study were collected from the corresponding medical records and are shown in Table 1.


**Table 1.** Demographic characteristics of study participants.

Abbreviations: SD, standard deviation. a Chi-squared test (two tail). b Mann–Whitney's U test (two tail). c 1 missed data.

Blood samples were obtained from all of the participants. Samples of blood were collected in 4 mL heparinized tubes (BD Vacutainer, LH 68 I.U. Lithium Heparin, BD-Plymouth, PL6 7BP, UK) and maintained at 4 ◦C. An aliquot of blood was stored at −80 ◦C until the chemical analysis, performed in the Toxicology Unit of the ULPGC.

#### *2.2. Selection of Elements and Sample Preparation*

Blood concentration levels of 45 inorganic elements were analyzed. We determined trace elements, heavy metals, rare earth elements (REEs) and other elements used in electronic devices, as we have previously reported [15,28].

Then, 100 mg of whole blood was weighed into quartz digestion tubes and then digested into 1 mL of acid solution (65% HNO3) using a Milestone Ethos Up equipment (Milestone, Bologna, Italy). The digestion conditions were programmed as follows (power (W)–temperature (◦C)–time (min): step 1: 1800–100–5; step 2: 1800–150–5; step 3: 1800–200–8; and step 4: 1800–200–7. After cooling, the digested samples were transferred and diluted. An aliquot of each sample was taken and the internal standard (ISTD) was added for the analysis.

The ISTD solution included scandium, germanium, rhodium and iridium (20 mg/mL each). Elements of standard purity (5% HNO3, 100 mg/L) were purchased from CPA Chem (Stara Zagora, Bulgaria). Two standard curves (range = 0.005–20 ng/mL) were made: (a) one used a commercial multi-element mixture (CPA Chem Catalog number E5B8·K1.5N.L1, 21 elements) containing all the trace elements and the main heavy metals and (b) the other multi-element mixture included individual elements (CPA Chem) that contained the REEs and other elements used in electronic devices [18,19].
