Nutritional Status Related to Selenium in Patients With Ataxia-Telangiectasia - A Case Control Study: an Association With Oxidative Stress and Risk of Atherosclerosis

Introduction: Ataxia-Telangiectasia (A-T) is a multi-system disorder that may be associated with endocrine changes, oxidative stress in addition to inammation. Studies suggest that selenium (Se) is a trace element related to protection against damage caused by oxidative stress; it is postulated that adequate consumption reduces the risk of some chronic diseases. Objective: To describe the concentrations of Se and glutathione peroxidase (GPx) in patients with A-T, to relate them to markers of the lipid prole. Methods: We evaluated, through a controlled cross-sectional study, 22 A-T patients matched by sex and age with healthy individuals, conjointly evaluating: nutritional status, food intake, serum selenium, glutathione peroxidase (activity), lipid metabolism biomarkers, inammation and lipid. Results: The median age in the A-T group was 12.2 years. A-T patients had greater impairment of lean body mass and GPx activity as well as lower abdominal circumference. A more atherogenic lipid prole was observed with higher concentrations of total cholesterol, non-HDL cholesterol, LDLox, Apo B, Apo B / Apo A-1 and LDL / HDL ratio; while a lower value was observed in the Apo A-1 / HDL ratio. It was also in the A-T group that statistical difference was detected in the three markers of liver function AST, ALT and GGT. In regard to food intake, A-T patients had lower values of carbohydrate, protein, monounsaturated fat, trans fat, and Se. Conclusion: The study showed cardiovascular risk in A-T patients. A-T patients appear to be at increased risk of reduced nutritional status, impaired liver function, dyslipidemia and inammation.


Introduction
Selenium (Se) is an essential micronutrient for antioxidant defense that integrates an important part of selenoproteins [1,2]. The most well-known selenoprotein is Glutathione peroxidase (GPx), which protects cells from damage caused by free radicals amongst reactive oxygen species (ROS) [3,4]. Selenium, in turn, blocks the activation of the nuclear transcription factor NFkB, a sensitive regulator that modulates the production of in ammatory mediators and adhesion molecules [5].
The trace element also stands out for its participation in immunological competence, particularly in cellmediated immunity, cognitive function, and protection against cardiovascular diseases (CVDs) [6,7].
Due to its antioxidant properties, Se plays an important role in lipid metabolism; however, the associations between serum Se concentrations and the risk of dyslipidemia are still controversial in the literature [3]. Some scienti c evidence has shown that Se de ciency occasionally increases the concentrations of total cholesterol (TC) [7] and triglycerides (TG) [8], elevating the risk of heart disease (HD), such as atherosclerosis, up to three times [9].
Patients with innate immunity errors (EII) have a higher susceptibility to infections, among other characteristics, hence the highest susceptibility to developing malignancies and autoimmunity. The release of in ammatory cytokines is remarkable along with many diseases belonging to this group. Thus, it is inferred that chronic in ammation might be associated with the pathogenesis of atherosclerosis [10][11][12][13]. Additional cardiovascular risk factors have been described in these diseases, such as increased oxidative stress and changes in the intestinal microbiota [14,15].
Ataxia-telangiectasia (A-T) is a rare disease (OMIM 208900) in which incidence ranges from 1: 40,000 to 1: 100,000, and it is caused by a mutation in the ATM gene (ataxia-telangiectasia mutated). The change induces di culty in repairing ruptures of double-stranded DNA, increases ROS production, mitochondrial dysfunction, with concomitant elevation of oxidative stress and consequent cell apoptosis [16,17]. DNA damage, combined with oxidative stress, can contribute to the pathogenesis of associated chronic diseases, including atherosclerosis [18,19].
Due to advances in the care of patients, different studies reveal increasing survival rates, with ensuing concern about the possible appearance of further associated diseases such as cardiovascular [20,21].
Dyslipidemia, a component of the metabolic syndrome, is one of the greatest risk factors with an impact on atherogenesis, which increases the risk of coronary failure two to three times [9]. To date, no studies are evaluating the risk of atherosclerosis in patients with A-T.
The present study aims to describe the concentrations of selenium and glutathione peroxidase in A-T patients and to relate them to markers of the lipid pro le.

Methods
Through the medium of a cross-sectional controlled study, we evaluated 22 A-T patients of both genders, between 3 and 27 years of age, who were diagnosed with A-T according to the criteria of the European Society for Immunode ciencies (ESID) [22].
Recruited from Primary Health Care Service, the control group was composed of 18 healthy volunteers matched in age and gender, comparing biochemical markers related to cardiovascular risk and food intake.
The study was approved by The Research Ethics Committee from the Federal University of São Paulo, nanced by The São Paulo Research Foundation -FAPESP nº 2015/13308-9, and signed informed consents were obtained from all participants (or a responsible guardian in the case of children).
The demographic, clinical, and treatment data were obtained from the patients' charts. The family history risk of atherosclerosis was assessed for patients and controls.
At the time of sample collection, none of the subjects had an acute infectious disease, nor had they been using corticosteroids for at least 3 months. Howbeit, one patient was using antifungal and ve were using antibiotics.

Anthropometric evaluation and food intake
The anthropometric evaluation involved measurements of weight, height, mid-upper arm circumference (MUAC), and skinfold thickness (tricipital, subscapular, bicipital, and sacroiliac) as proposed by the World Health Organization (WHO) and Frisancho (1990) [23,24]. The patients who were unable to stand upright had their weight measured in the wheelchair, on a speci c scale for wheelchair-users (Micheletti -capacity 1100 lbs -serial: 2161058). Recumbent height was measured with the patient lying on a at and rm surface, using an inextensible tape graduated in millimeters.
To assess the body mass index (BMI) and height for age (H/A) of children and adolescents, expressed as Z-scores, the WHO [25] criteria and the classi cation proposed by De Onis et al. were adopted [26]. For adults, the cut-off point of the WHO for BMI was chosen instead [23]. The sum of skinfold thickness and MUAC was used to estimate children's body composition [27][28][29]. While for adults, the estimation of body composition was based on the sum of the four different skinfolds [30]. The body fat percentage was classi ed according to the method previously described [28,29].
The pubertal stage was evaluated according to Marshall and Tanner [31].
The assessment of food intake was performed using a 24 h dietary recall (R24hs), applied at 3 times, with an interval of 15 days between them. The calculation of nutrients in the diet was performed by the use of the Software Dietwin ®, comparing the cases to the controls [32,33].
Considering that food composition tables available in some software do not have complete data on Se content in food, these data were included manually based on the article by Ferreira et al. (2002) [34].
Thereby, only one of A-T patients had feeding tubes.
Selenium was determined by graphite furnace atomic absorption spectrometry. For classi cation, the cutoff point ≤ 45 µg/L was adopted for inadequacy. Glutathione peroxidase activity was measured by the enzymatic method.
The lipid pro le, including the triglyceride, total cholesterol and high-density lipoprotein cholesterol (HDLc) was measured with enzymatic-colorimetric tests. Low-density lipoprotein cholesterol (LDL-c) and verylow-density lipoprotein cholesterol (VLDL-c) were calculated using the formula by Friedewald et al. (1972) [35].
For classi cation, the cut-off points suggested by the American Academy of Pediatrics [36] and the National Cholesterol Education Program (NCEP) [37] were adopted. The presence of dyslipidemia was considered when the TC> 170 mg / dL for children / adolescents and> 200 mg / dL for adults and / or LDL-c> 110 mg / dL for children / adolescents and> 129 mg / dL for adults and / or triglycerides> 100 mg / dL for children / adolescents and> 150 mg / dL for adults and / or HDL-c <35 mg / dL for children / adolescents, <40 mg / dL for women and <50 mg / dL for men.
The non-HDLc (NHDL-c) values were obtained by subtracting the HDL-c rates from the TC levels and thereafter classi ed according to the work of Bogalusa [38] and NCEP. The following ratios were also calculated: total cholesterol/ HDL-c, Apo B/Apo A-1, LDL-c/Apo B, LDL-c/HDL-c [39], HDL-c/Apo A-1 [40]. Complete blood counts, MDA, and us-CRP were measured by the methods of Colorimetric and Turbidimetric, respectively. The cutoff point used to indicate elevation was us-CRP ≥ 8.
Glycemia was measured by enzymatic reference method with hexokinase, while insulin was quanti ed by electrochemiluminescence. Using the fasting glucose and insulin values, the HOMA-IR (Homeostasis Model Assessment of Insulin Resistance) rate was calculated utilizing the following formula: HOMA-IR = fasting glucose (mmol / L) x fasting insulin (µU / mL) / 22.5. HOMA-IR was considerably changed> 3.16 [41].

Statistical Analysis
The statistical package SPSS 25.0 was used for the analysis. Continuous variables were tested for normality. For comparisons between nonparametric variables, the Mann-Whitney or Kruskal-Wallis test was applied, and, for the parametric variables, the t-Student or ANOVA was the chosen test. To analyze the association between qualitative variables, either the Chi-square test or Fisher's exact test was used. A signi cance level of 5% (p < 0.05) was adopted.

Results
The classi cation of nutritional status by BMI of the patients and the controls is summarized in Table 1. Both groups had similar socioeconomic conditions, per capita income, and pubertal stage (data not shown). Table 2. Therein is identi ed the median time since diagnosis of the disease as 7 years and 2 months (0.7-20.2 years). Twelve out of 22 patients (54.5%) received regular intravenous immunoglobulin (IVIG) treatment and vitamin supplement was used by 17/22 (77.3%) convalescents. Over 60.0% patients had dyslipidemia, while 3/22 (13.6%) had pneumopathy, and 1/22 (5.4%) had constipation. Selenium alterations were found in 9/22 (40.9%) of cases. All patients had an increase in alpha-fetoprotein (AFP) to a normal value of up to 5.8 IU / mL, a pathognomonic characteristic of the disease. Whereas there was no correlation between AFP concentrations and biomarkers associated with the atherosclerosis risk (data not shown in the table).

The characterization of patients with A-T is in
The biochemical variables that assess nutritional status relative to selenium, in ammation biomarkers, glycemic metabolism, and liver function are presented in Table 3.
A lower total intake of energy, protein, and carbohydrate was observed in A-T group, compared to the control group (Table 4). Although total fat intake did not show any statistical difference between groups, con ictingly results were found the A-T group. The intake of polyunsaturated, monounsaturated and trans fat was lower, as well as that of selenium.
The markers of the lipid pro le are shown in Table 5. A more atherogenic lipid pro le with higher concentrations of total cholesterol, non-HDL cholesterol, oxidized LDL, Apo B, Apo B / Apo A-1, LDL / HDL and less than Apo A-1 / HDL was observed in the A-T group.

Discussion
The present study showed that selenium concentrations did not differ signi cantly between patients and controls. However, it must be pointed out that 41% of A-T patients had selenium concentrations below the reference value. and that MDA concentrations and plus activity of GPx were higher and lower in the A-T group compared to controls, respectively. Also, A-T patients had an atherogenic pro le (> NHDL-c,> LDLox,> Apo B and <Apo A-1 / HDL-c) and there was a relevant and positive correlation of selenium concentrations with TC/ HDL-c, LDL-c/ HDL-c and TG / HDL-c ratios.
To our knowledge, there are no current publications evaluating the association between selenium concentrations and GPx activity with those of lipid metabolism biomarkers in A-T patients. Additionally, in vitro A-T cells are under a constant state of oxidative stress and have an abnormal response to agents inducing this state [42]. Oxidative stress is a central mechanism in the pathogenesis of the disease, especially neurodegeneration [43] and other morbidities associated with the disease, such as liver disorders and dyslipidemias [44,45] [46] demonstrated that ATM protein de ciency accelerates the atherosclerotic process via systemic effects (regulation of NF-κB expression) and on the vascular endothelium. The authors concluded that damage to mitochondrial DNA, increased production of free radicals and reduced oxidative phosphorylation, in which effects are directly related to changes in lipid and glucose metabolism as well as presented in A-T patients.
Nonetheless, Squadrone et al (2015) [47] evaluated the concentrations of trace elements, including selenium and the antioxidant enzymes, associated with them in 16 individuals with A-T and controls. The essayists also found no differences in selenium concentrations, similarly to what we have observed before, between patients and controls. They described a break in trace element homeostasis with an increase in copper concentrations and a reduction in zinc volume with a consequent reduction in the expression of SOD1 and SOD2 and an increase in the risk of cell apoptosis in A-T patients. GPx activity did not differ between groups. During oxidative stress, selenoprotein P binds to the endothelium of in amed tissues and it is mobilized to other compartments to exercise its antioxidant function. There is a reduction in its hepatic synthesis, induced by in ammatory cytokines. The synthesis and activity of glutathione, in turn, can be reduced when there is malnutrition, hyperglycemia, corticotherapy, skeletal muscle inactivity; situations frequently observed in A-T patients [48]. Some hypotheses may explain the higher MDA values and the lower GPx activity, pointing to an exacerbation of oxidative stress in our patients: a) older age (median 12.2 years) b) a signi cant change in liver enzymes, malnutrition level, and impaired lean mass (not mentioned in the study by Squadrone et al.).
Individuals with A-T demonstrated changes in lipid metabolism biomarkers compatible with an atherogenic pro le, with an emphasis on elevating NHDL-c and LDLox. LDL-c is easily susceptible to oxidation under conditions of oxidative stress, reveled in our study by the elevation of MDA (lipid peroxidation marker) and reduction of GPx in A-T convalescents, which results in LDLox, and has some atherogenic characteristics. A recent meta-analysis and systematic review described, based on observational studies, the association between LDLox concentrations and the development of atherosclerotic cardiovascular diseases [49].
The ndings of lower selenium consumption in the A-T group combined with about 40% of serum concentrations below the reference value may re ect on neurodegeneration. Se is vital for the central nervous system and it is involved in various functions, such as motor performance, coordination, memory and cognition. The role of Se and selenoproteins in neurotransmission goes beyond their antioxidant properties and involves the regulation of in ammation, in uencing the phosphorylation of proteins and ion channels, modifying calcium homeostasis, and cholesterol metabolism in the brain. Furthermore, it plays a direct role in signaling by means of selenoprotein P and its interaction with 2 synaptic post receptors of Apolipoprotein (ApoER2) [50]. Future studies evaluating the role of selenium in neurodegeneration-related outcomes in A-T patients appear promising.
Regarding the association of selenium with total cholesterol and LDL-c, some studies show a direct association in the general population [51, 52], while others display a negative correlation [53,54] or no association at all [55]. This inconsistency may be due to the sample size or even the lack of adjustment for confounding variables. In our study, there was a signi cant and positive connection between selenium and the TC/ LDL-c, LDL-c/ HDL-c, and TG/ HDL-c ratios. Therefore, caution should be exercised when proposing selenium supplementation in A-T patients with an open view to distinct outcomes.
In conclusion, the presence of selenium below the reference value by nearly 40% and lower GPx activity in individuals with A-T reinforces the importance of assessing the nutritional status of selenium in those patients. Particularly due to the positive correlation between the lipid pro le ratios related to cardiovascular risk and selenium concentrations. Consequently, it is thus possible to deduce that A-T patients are most prone to present increased risk of other morbidities by reason of impaired nutritional status, impaired liver function, dyslipidemia and oxidative stress. The study was approved by The Research Ethics Committee from the Federal University of São Paulo (CEP/UNIFESP -972.812 11/03/2015) and signed informed consents were obtained from all participants (or a responsible guardian in the case of children).

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The study was nanced by The São Paulo Research Foundation -FAPESP nº 2015/13308-9. The Foundation FAPESP did not in uence in the design of the study and collection, analysis, and interpretation of data or the writing of the manuscript.
Authors' contributions IGAA à collected, analyzed and interpreted data from patients A-T and the control group and was a major contributor in writing the manuscript.
FISS à analyzed and interpreted data from patients A-T and the control group and contributed to the correction of the manuscript writing.
FLAF à performed the analysis of laboratory tests and contributed to the correction of the manuscript writing.
CSAL à analyzed and interpreted data from patients A-T and the control group and contributed to the correction of the manuscript writing. ROSS à analyzed and interpreted data from patients A-T and the control group and was a major contributor in writing the manuscript.  Signi cance level of the Chi-square test (1) , Fisher's exact test (2) ; p<0.05      Correlation matrix between selenium concentrations and the lipid pro le of A-T patients. Signi cance level of Spearman correlation (p < 0,05)