N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
SLC30A3 and SEP15 gene polymorphisms influence the serum concentrations of zinc and selenium in mature adults Tatiane Jacobsen da Rocha a , Camila Korb b , Jaqueline Bohrer Schuch c , Daiani Pires Bamberg d , Fabiana Michelsen de Andrade e , Marilu Fiegenbaum f,⁎ a
Biomedical Health Sciences, University of Health Sciences of Porto Alegre–UFCSPA, Rio Grande do Sul, Brazil Biomedicine, Institute of Health Sciences, University Feevale, Rio Grande do Sul, Brazil c Biomedical Science, Feevale University, Rio Grande do Sul, Brazil d Psychology, Feevale University, Rio Grande do Sul, Brazil e Institute of Sciences, Letters and Arts and Institute of Health Sciences, University Feevale, Rio Grande do Sul, Brazil f Department of Basic Health Sciences, UFCSPA, Rio Grande do Sul, Brazil b
ARTI CLE I NFO
A BS TRACT
Article history:
Because of their numerous roles in several biological processes, zinc and selenium are the
Received 28 April 2014
most commonly studied micronutrients in the elderly. Therefore, we hypothesized that the
Revised 4 August 2014
polymorphisms in the genes that are responsible for the transport of zinc and selenium may
Accepted 22 August 2014
have a genotype-dependent effect on the serum concentration of these micronutrients. The objective of this study was to determine the effects of solute carrier family 30 member 3 (SLC30A3)
Keywords:
and 15-kd selenoprotein (SEP15) polymorphisms on zinc and selenium concentrations,
Zinc
respectively, in the serum. This cross-sectional study included 110 individuals who were
Selenium
aged 50 years or older. Serum micronutrient concentrations were determined by flame atomic
SLC30A3
absorption spectrophotometry (for zinc) and by atomic absorption spectrophotometry with a
SEP15
graphite furnace (for selenium). The single-nucleotide polymorphisms, rs73924411 and
Polymorphism
rs11126936 of the SLC30A3 gene and rs5859, rs5854, and rs561104 of the SEP15 gene, were
Aging
examined by real-time polymerase chain reaction. Regarding rs11126936, the serum zinc concentration was lower in CC homozygotes (0.75 ± 0.31 mg/L) than in A carriers (0.89 ± 0.28 mg/L, P = .016). Concerning rs561104, the serum selenium concentration was higher in CC homozygotes (5.65 ± 1.11 μg/dL) compared with T carriers (4.88 ± 1.25 μg/dL, P = .044). Our results demonstrate the influence of SLC30A3 and SEP15 gene polymorphisms on the serum concentrations of zinc and selenium, respectively. The effects of these associations should be further investigated to help elucidate the modes of action of trace elements and to identify biomarkers, which could ultimately define the optimal intake of these micronutrients at the molecular level. More research must be performed before the roles of these polymorphisms in the serum concentrations of zinc and selenium can be fully understood. © 2014 Elsevier Inc. All rights reserved.
Abbreviations: FFQ, food frequency questionnaire; IL-6, interleukin 6; SEP15, 15-kd selenoprotein; SLC30A3, solute carrier family 30 member 3; SNP, single-nucleotide polymorphisms; ZIP, ZRT/IRT-related proteins; ZnT, zinc transporter. ⁎ Corresponding author. Universidade Federal de Ciências da Saúde de Porto Alegre, Departamento de Ciências Básicas da Saúde–Genética Humana, Rua Sarmento Leite, 245 sala 403, CEP 90050-170–Porto Alegre–RS. Tel.: +55 51 33038773. E-mail addresses: [email protected] (T.J. da Rocha), [email protected] (C. Korb), [email protected] (J.B. Schuch), [email protected] (D.P. Bamberg), [email protected] (F.M. de Andrade), [email protected] (M. Fiegenbaum). http://dx.doi.org/10.1016/j.nutres.2014.08.009 0271-5317/© 2014 Elsevier Inc. All rights reserved.
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
1.
Introduction
Worldwide, the elderly populace is on a rise. In 2000, approximately 10% of the global population was elderly, and this number is predicted to increase above 20% by 2050. In Brazil, it is estimated that there will be 65 million elderly people by 2050. This change in the population profile requires the development of new scientific approaches to provide better living conditions for this growing group [1]. One of the consequences of this profound demographic shift is an increased awareness that nutritional influences on health should be optimized for older populations. This is especially important because the aging process is associated with physiologic changes, which increase predisposition to several diseases that could be prevented/delayed with proper nutritional guidance [2]. Human health can be greatly improved if nutritional requirements are correlated with the genetic profile of the individual and his stage of life [3,4]. The trace elements, selenium and zinc, are important micronutrients that have key roles in maintaining the health of the elderly via their numerous biological activities, including their antioxidant and neuroprotective effects [5-7]. More than 30 genes have been identified in the human genome that encode selenoproteins, which affect selenium uptake, metabolism, and excretion [8]. A 15-kd selenoprotein encoded by the SEP15 gene was recently identified. This protein is a member of the thioredoxin reductases, which are proteins that have antioxidant roles. The SEP15 protein contains an in-frame UGA codon and a sec insertion sequence element in the 3′ UTR. An examination of the available complementary DNA sequences for this protein revealed the polymorphisms rs5845 (C > T) and 5859 (G > A) within the 3′ UTR and rs561104 (T > C) in the first intron. These polymorphisms decrease the efficiency of the selenocysteine insertion sequence element, at higher concentrations of selenium. In addition, although the expression of SEP15 is influenced by the diet, its specific function is not known [9,10]. Fifteenkilodalton selenoproteins are shown to protect the cell against the action of reactive oxygen species, and increasing evidence indicates that oxidative stress is responsible for both biological aging and the emergence of age-associated diseases. Thus, studies involving the SEP15 gene could contribute to a better understanding of the role that gene-nutrient interactions play in healthy aging. Furthermore, the SEP15 gene is located on chromosome 1p31, at a locus that is often deleted or mutated in human cancers [11]. To date, most studies related to the SEP15 gene and the selenium concentration in serum have focused on an aggressive clinical outcome [12-14], and so the role of the SEP15 gene in determining the serum selenium concentration in mature adults and healthy elderly individuals has not been investigated. In this regard, the SEP15 gene was selected to determine its role on the serum selenium concentration in a healthy population. Zinc is an essential metal that is present in all biological tissues and the second most abundant metal. Furthermore, via its role as an antioxidant, zinc plays a fundamental physiologic role in the synthesis of proteins and nucleic acids, in intracellular signaling pathways, and in the prevention of
743
lipid peroxidation [15,16]. Factors that influence zinc metabolism include the diet as well as mutations and polymorphisms in genes encoding zinc transporter (ZnT) proteins; however, little is known about the effects that these polymorphisms have on the regulation of zinc homeostasis and the phenotypic consequences of this gene-nutrient interaction [17]. Zinc homeostasis in cells depends on zinc transport into or out of cellular compartments, and this transport is achieved by the action of 2 different solute-linked carrier families of ZnTs: ZnT or SLC30A and ZRT/IRT-related proteins (ZIP or SLC39A) [18]. The ZIP (SLC39A gene) family is responsible for increasing cytosolic zinc levels by importing zinc from either the extracellular space or from intracellular compartments. In contrast, the ZnT (SLC30A gene) family reduces the cytosolic zinc concentration by moving zinc out of the cell or into various intracellular compartments. Ten ZnT genes and 14 ZIP genes have been identified in the human genome, and zinc transport activity has been demonstrated for 7 ZnT and 9 ZIP. The solute carrier family 30 member 3 (SLC30A3) gene encodes the ZnT3 protein [19], which is localized to synaptic vesicles in glutamate synapses and known to be involved in cognitive function. In this study, we selected the 2 single-nucleotide polymorphisms (SNP), namely, in SLC30A3 because of their association to neurobiological implications and importance of zinc for the maintenance of synaptic activities and for the prevention of reactive oxygen species formation, which promotes healthy aging [15,20]. Thousands of genes encoding zinc transport proteins have been identified; however, information on the role of genes responsible for the concentration of zinc in serum is scarce. The purpose of this study was to test the hypothesis that the SLC30A3 and SEP15 gene polymorphisms affect the serum zinc and selenium concentrations, respectively, in mature and elderly adults. The specific research objectives were to (1) determine the effect of polymorphisms (rs11126936 and rs73924411) in the SLC30A3 gene, which encodes the ZnT3, on serum zinc concentration and (2) determine the effect of polymorphisms (rs5859, rs5845, and rs561104) in the SEP15 gene, which encodes the SEP15, on serum selenium concentration. Furthermore, we sought to determine whether age has a negative impact on the concentration of zinc and selenium in the serum.
2.
Methods and materials
2.1.
Study design and population
The study sample was recruited through advertisements and was composed of women and men (a total of 110 individuals but measurements limited to 56 women and 14 men due to sample size after recovery of serum) aged 50 years or older (Table 1). The individuals were interviewed between 2010 and 2012 at either Feevale University (Novo Hamburgo, Rio Grande do Sul, Brazil) or the Federal University of Health Sciences of Porto Alegre (Porto Alegre, Rio Grande do Sul, Brazil). This cross-sectional study involved the collection of blood and completion of a lifestyle questionnaire. The study excluded individuals who used vitamin supplements that contained micronutrients in their formulation. The Ethics Committees of Feevale University (no. 2.02.01.06.342)
744
N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
Table 1 – Characteristics of subjects by blood selenium and zinc concentrations n
Overall a Sex a Women Men Hormonal reposition, women only a Yes No Alcohol consumption a Yes No Smoking status b Never Past Current
Selenium P (micrograms per deciliter)
n
Zinc P (milligrams per liter)
70
5.02 ± 1.25
110 0.82 ± 0.30
56 14
5.13 ± 1.22 4.58 ± 1.31
.146
86 0.82 ± 0.30 24 0.83 ± 0.29
.860
39 17
5.01 ± 1.26 5.41 ± 1.12
.247
59 0.81 ± 0.32 27 0.86 ± 0.19
.420
43 27
5.37 ± 1.11 4.80 ± 1.29
.064
63 0.82 ± 0.33 47 0.82 ± 0.27
.980
48 21 1
5.07 ± 1.38 4.92 ± 0.93 5.02
.890
73 0.81 ± 0.31 32 0.85 ± 0.30 5 0.83 ± 0.12
.740
Selenium and zinc concentrations are shown as the means ± SD. Student t test. b Analysis of variance. a
2.4.
Statistical analyses
Continuous variables were expressed as the means ± SD. Allele frequencies were estimated by gene counting. The agreement of genotype frequencies with Hardy-Weinberg equilibrium expectations was tested using χ2 tests. The normality of serum zinc and selenium concentrations was tested using the Kolmogorov-Smirnov test, and both presented normal distributions with the investigated samples. The influence of sociodemographic variables on serum zinc and selenium concentrations was determined using Student t test or analysis of variance. The serum zinc and selenium concentrations were adjusted by age and sex through linear regression before comparisons were made between genotypes. Because of the lower frequency of homozygous genotypes, the genotypes of the investigated SNP gene were grouped (GA + AA at rs73924411 and AA + AC at rs11126936 for the SLC30A3 gene; and CT + TT at rs5845, GA + AA at rs5859, and CT + TT at rs561104 for the SEP15 gene). The adjusted serum zinc and selenium mean concentrations were then compared between genotypes using Student t test. The genotypic distributions between groups with low and normal/high zinc and selenium concentrations were compared by χ 2 or Fisher exact test. The correlation between age and serum zinc or selenium concentration was estimated by Pearson correlation coefficient. A P < .05 was considered significant. Statistical analysis was performed using SPSS19.0 (Armonk, NY, EUA) for Windows.
and Federal University of Health Sciences of Porto Alegre (no. 666.10) approved the study protocol. Each subject included in the sample population provided written informed consent.
3.
Results
2.2.
3.1.
Characteristics of the study population
Biochemical analyses
For the analyses of selenium and zinc in the serum, blood samples were stored in metal-free tubes (BD Vacutainer, Trace Elements Serum, São Paulo, SP, Brazil) and centrifuged within 2 hours after collection. After centrifugation, the serum was fractionated into specific transport tubes and frozen at −80°C until the completion of dosing. The serum zinc concentrations were measured by flame atomic absorption spectrophotometry as previously described [21], with a standard curve from 0.2 to 1.5 mg/L and linearity from 0.2 to 1.5 mg/L with a detection limit of 0.01 mg/L. The selenium concentrations were measured using atomic absorption spectrophotometry with a graphite furnace as previously described [22]; and the standard curve ranged from 4 to 40 μg/dL with linearity at 40 μg/dL and a detection limit of 0.1 μg/dL. The reference values for serum zinc and selenium concentrations ranged from 0.70 to 1.50 mg/L and from 4.6 to 14.3 μg/dL, respectively [23].
2.3.
DNA extraction and genotyping
Genomic DNA was isolated from peripheral blood leukocytes by a standard salting-out procedure, which was developed by Lahiri and Nurberger [24]. The rs5859, rs5854, and rs561104 SNP of the SEP15 gene and the rs73924411 and rs11126936 SNP of the SLC30A3 gene were identified by allelic discrimination with TaqMan 5′-nuclease assays (Real Time PCR; Applied Biosystems, Foster, CA, USA).
For this cross-sectional study, 110 individuals (78% women) were selected who were aged 50 years or older (64.86 ± 8.75 years). Data for the comparison of selenium and zinc levels, according to the descriptive characteristics of the subjects, are presented in Table 1, which included 56 women and 14 men (Table 1). No significant difference was observed between sex, alcohol consumption, or smoking status and the serum concentrations of selenium and zinc (P > .05). However, age was negatively correlated with the serum zinc concentration (r = −0.25, P = .008) but not the serum selenium concentration (r = 0.177, P = .143). In addition, 29 subjects in the sample (26.4%) had a serum zinc level that was below the reference value, whereas 11 subjects (15.7%) had selenium concentrations that were less than the recommended amount (data not shown).
3.2. The association of SLC30A3 and SEP15 gene polymorphisms with zinc and selenium concentrations In the genetic and biochemical analyses of zinc, 110 samples were analyzed to determine zinc levels, and the SLC30A3 gene was genotyped. Serum assays for selenium were performed on 70 samples (56 women and 14 men), all of which had SEP15 genotype information available. This difference in the number of samples from subjects (110 vs 70) occurred due to the amount of serum obtained
745
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
Table 2 – Solute carrier family 30 member 3 gene polymorphisms and serum zinc concentrations of subjects SLC30A3 polymorphisms
n
rs73924411 GG GA + AA P rs11126936 AA + AC CC P a b c d
Zinc (milligrams per liter) a
Low (
Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
SLC30A3 and SEP15 gene polymorphisms influence the serum concentrations of zinc and selenium in mature adults Tatiane Jacobsen da Rocha a , Camila Korb b , Jaqueline Bohrer Schuch c , Daiani Pires Bamberg d , Fabiana Michelsen de Andrade e , Marilu Fiegenbaum f,⁎ a
Biomedical Health Sciences, University of Health Sciences of Porto Alegre–UFCSPA, Rio Grande do Sul, Brazil Biomedicine, Institute of Health Sciences, University Feevale, Rio Grande do Sul, Brazil c Biomedical Science, Feevale University, Rio Grande do Sul, Brazil d Psychology, Feevale University, Rio Grande do Sul, Brazil e Institute of Sciences, Letters and Arts and Institute of Health Sciences, University Feevale, Rio Grande do Sul, Brazil f Department of Basic Health Sciences, UFCSPA, Rio Grande do Sul, Brazil b
ARTI CLE I NFO
A BS TRACT
Article history:
Because of their numerous roles in several biological processes, zinc and selenium are the
Received 28 April 2014
most commonly studied micronutrients in the elderly. Therefore, we hypothesized that the
Revised 4 August 2014
polymorphisms in the genes that are responsible for the transport of zinc and selenium may
Accepted 22 August 2014
have a genotype-dependent effect on the serum concentration of these micronutrients. The objective of this study was to determine the effects of solute carrier family 30 member 3 (SLC30A3)
Keywords:
and 15-kd selenoprotein (SEP15) polymorphisms on zinc and selenium concentrations,
Zinc
respectively, in the serum. This cross-sectional study included 110 individuals who were
Selenium
aged 50 years or older. Serum micronutrient concentrations were determined by flame atomic
SLC30A3
absorption spectrophotometry (for zinc) and by atomic absorption spectrophotometry with a
SEP15
graphite furnace (for selenium). The single-nucleotide polymorphisms, rs73924411 and
Polymorphism
rs11126936 of the SLC30A3 gene and rs5859, rs5854, and rs561104 of the SEP15 gene, were
Aging
examined by real-time polymerase chain reaction. Regarding rs11126936, the serum zinc concentration was lower in CC homozygotes (0.75 ± 0.31 mg/L) than in A carriers (0.89 ± 0.28 mg/L, P = .016). Concerning rs561104, the serum selenium concentration was higher in CC homozygotes (5.65 ± 1.11 μg/dL) compared with T carriers (4.88 ± 1.25 μg/dL, P = .044). Our results demonstrate the influence of SLC30A3 and SEP15 gene polymorphisms on the serum concentrations of zinc and selenium, respectively. The effects of these associations should be further investigated to help elucidate the modes of action of trace elements and to identify biomarkers, which could ultimately define the optimal intake of these micronutrients at the molecular level. More research must be performed before the roles of these polymorphisms in the serum concentrations of zinc and selenium can be fully understood. © 2014 Elsevier Inc. All rights reserved.
Abbreviations: FFQ, food frequency questionnaire; IL-6, interleukin 6; SEP15, 15-kd selenoprotein; SLC30A3, solute carrier family 30 member 3; SNP, single-nucleotide polymorphisms; ZIP, ZRT/IRT-related proteins; ZnT, zinc transporter. ⁎ Corresponding author. Universidade Federal de Ciências da Saúde de Porto Alegre, Departamento de Ciências Básicas da Saúde–Genética Humana, Rua Sarmento Leite, 245 sala 403, CEP 90050-170–Porto Alegre–RS. Tel.: +55 51 33038773. E-mail addresses: [email protected] (T.J. da Rocha), [email protected] (C. Korb), [email protected] (J.B. Schuch), [email protected] (D.P. Bamberg), [email protected] (F.M. de Andrade), [email protected] (M. Fiegenbaum). http://dx.doi.org/10.1016/j.nutres.2014.08.009 0271-5317/© 2014 Elsevier Inc. All rights reserved.
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
1.
Introduction
Worldwide, the elderly populace is on a rise. In 2000, approximately 10% of the global population was elderly, and this number is predicted to increase above 20% by 2050. In Brazil, it is estimated that there will be 65 million elderly people by 2050. This change in the population profile requires the development of new scientific approaches to provide better living conditions for this growing group [1]. One of the consequences of this profound demographic shift is an increased awareness that nutritional influences on health should be optimized for older populations. This is especially important because the aging process is associated with physiologic changes, which increase predisposition to several diseases that could be prevented/delayed with proper nutritional guidance [2]. Human health can be greatly improved if nutritional requirements are correlated with the genetic profile of the individual and his stage of life [3,4]. The trace elements, selenium and zinc, are important micronutrients that have key roles in maintaining the health of the elderly via their numerous biological activities, including their antioxidant and neuroprotective effects [5-7]. More than 30 genes have been identified in the human genome that encode selenoproteins, which affect selenium uptake, metabolism, and excretion [8]. A 15-kd selenoprotein encoded by the SEP15 gene was recently identified. This protein is a member of the thioredoxin reductases, which are proteins that have antioxidant roles. The SEP15 protein contains an in-frame UGA codon and a sec insertion sequence element in the 3′ UTR. An examination of the available complementary DNA sequences for this protein revealed the polymorphisms rs5845 (C > T) and 5859 (G > A) within the 3′ UTR and rs561104 (T > C) in the first intron. These polymorphisms decrease the efficiency of the selenocysteine insertion sequence element, at higher concentrations of selenium. In addition, although the expression of SEP15 is influenced by the diet, its specific function is not known [9,10]. Fifteenkilodalton selenoproteins are shown to protect the cell against the action of reactive oxygen species, and increasing evidence indicates that oxidative stress is responsible for both biological aging and the emergence of age-associated diseases. Thus, studies involving the SEP15 gene could contribute to a better understanding of the role that gene-nutrient interactions play in healthy aging. Furthermore, the SEP15 gene is located on chromosome 1p31, at a locus that is often deleted or mutated in human cancers [11]. To date, most studies related to the SEP15 gene and the selenium concentration in serum have focused on an aggressive clinical outcome [12-14], and so the role of the SEP15 gene in determining the serum selenium concentration in mature adults and healthy elderly individuals has not been investigated. In this regard, the SEP15 gene was selected to determine its role on the serum selenium concentration in a healthy population. Zinc is an essential metal that is present in all biological tissues and the second most abundant metal. Furthermore, via its role as an antioxidant, zinc plays a fundamental physiologic role in the synthesis of proteins and nucleic acids, in intracellular signaling pathways, and in the prevention of
743
lipid peroxidation [15,16]. Factors that influence zinc metabolism include the diet as well as mutations and polymorphisms in genes encoding zinc transporter (ZnT) proteins; however, little is known about the effects that these polymorphisms have on the regulation of zinc homeostasis and the phenotypic consequences of this gene-nutrient interaction [17]. Zinc homeostasis in cells depends on zinc transport into or out of cellular compartments, and this transport is achieved by the action of 2 different solute-linked carrier families of ZnTs: ZnT or SLC30A and ZRT/IRT-related proteins (ZIP or SLC39A) [18]. The ZIP (SLC39A gene) family is responsible for increasing cytosolic zinc levels by importing zinc from either the extracellular space or from intracellular compartments. In contrast, the ZnT (SLC30A gene) family reduces the cytosolic zinc concentration by moving zinc out of the cell or into various intracellular compartments. Ten ZnT genes and 14 ZIP genes have been identified in the human genome, and zinc transport activity has been demonstrated for 7 ZnT and 9 ZIP. The solute carrier family 30 member 3 (SLC30A3) gene encodes the ZnT3 protein [19], which is localized to synaptic vesicles in glutamate synapses and known to be involved in cognitive function. In this study, we selected the 2 single-nucleotide polymorphisms (SNP), namely, in SLC30A3 because of their association to neurobiological implications and importance of zinc for the maintenance of synaptic activities and for the prevention of reactive oxygen species formation, which promotes healthy aging [15,20]. Thousands of genes encoding zinc transport proteins have been identified; however, information on the role of genes responsible for the concentration of zinc in serum is scarce. The purpose of this study was to test the hypothesis that the SLC30A3 and SEP15 gene polymorphisms affect the serum zinc and selenium concentrations, respectively, in mature and elderly adults. The specific research objectives were to (1) determine the effect of polymorphisms (rs11126936 and rs73924411) in the SLC30A3 gene, which encodes the ZnT3, on serum zinc concentration and (2) determine the effect of polymorphisms (rs5859, rs5845, and rs561104) in the SEP15 gene, which encodes the SEP15, on serum selenium concentration. Furthermore, we sought to determine whether age has a negative impact on the concentration of zinc and selenium in the serum.
2.
Methods and materials
2.1.
Study design and population
The study sample was recruited through advertisements and was composed of women and men (a total of 110 individuals but measurements limited to 56 women and 14 men due to sample size after recovery of serum) aged 50 years or older (Table 1). The individuals were interviewed between 2010 and 2012 at either Feevale University (Novo Hamburgo, Rio Grande do Sul, Brazil) or the Federal University of Health Sciences of Porto Alegre (Porto Alegre, Rio Grande do Sul, Brazil). This cross-sectional study involved the collection of blood and completion of a lifestyle questionnaire. The study excluded individuals who used vitamin supplements that contained micronutrients in their formulation. The Ethics Committees of Feevale University (no. 2.02.01.06.342)
744
N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
Table 1 – Characteristics of subjects by blood selenium and zinc concentrations n
Overall a Sex a Women Men Hormonal reposition, women only a Yes No Alcohol consumption a Yes No Smoking status b Never Past Current
Selenium P (micrograms per deciliter)
n
Zinc P (milligrams per liter)
70
5.02 ± 1.25
110 0.82 ± 0.30
56 14
5.13 ± 1.22 4.58 ± 1.31
.146
86 0.82 ± 0.30 24 0.83 ± 0.29
.860
39 17
5.01 ± 1.26 5.41 ± 1.12
.247
59 0.81 ± 0.32 27 0.86 ± 0.19
.420
43 27
5.37 ± 1.11 4.80 ± 1.29
.064
63 0.82 ± 0.33 47 0.82 ± 0.27
.980
48 21 1
5.07 ± 1.38 4.92 ± 0.93 5.02
.890
73 0.81 ± 0.31 32 0.85 ± 0.30 5 0.83 ± 0.12
.740
Selenium and zinc concentrations are shown as the means ± SD. Student t test. b Analysis of variance. a
2.4.
Statistical analyses
Continuous variables were expressed as the means ± SD. Allele frequencies were estimated by gene counting. The agreement of genotype frequencies with Hardy-Weinberg equilibrium expectations was tested using χ2 tests. The normality of serum zinc and selenium concentrations was tested using the Kolmogorov-Smirnov test, and both presented normal distributions with the investigated samples. The influence of sociodemographic variables on serum zinc and selenium concentrations was determined using Student t test or analysis of variance. The serum zinc and selenium concentrations were adjusted by age and sex through linear regression before comparisons were made between genotypes. Because of the lower frequency of homozygous genotypes, the genotypes of the investigated SNP gene were grouped (GA + AA at rs73924411 and AA + AC at rs11126936 for the SLC30A3 gene; and CT + TT at rs5845, GA + AA at rs5859, and CT + TT at rs561104 for the SEP15 gene). The adjusted serum zinc and selenium mean concentrations were then compared between genotypes using Student t test. The genotypic distributions between groups with low and normal/high zinc and selenium concentrations were compared by χ 2 or Fisher exact test. The correlation between age and serum zinc or selenium concentration was estimated by Pearson correlation coefficient. A P < .05 was considered significant. Statistical analysis was performed using SPSS19.0 (Armonk, NY, EUA) for Windows.
and Federal University of Health Sciences of Porto Alegre (no. 666.10) approved the study protocol. Each subject included in the sample population provided written informed consent.
3.
Results
2.2.
3.1.
Characteristics of the study population
Biochemical analyses
For the analyses of selenium and zinc in the serum, blood samples were stored in metal-free tubes (BD Vacutainer, Trace Elements Serum, São Paulo, SP, Brazil) and centrifuged within 2 hours after collection. After centrifugation, the serum was fractionated into specific transport tubes and frozen at −80°C until the completion of dosing. The serum zinc concentrations were measured by flame atomic absorption spectrophotometry as previously described [21], with a standard curve from 0.2 to 1.5 mg/L and linearity from 0.2 to 1.5 mg/L with a detection limit of 0.01 mg/L. The selenium concentrations were measured using atomic absorption spectrophotometry with a graphite furnace as previously described [22]; and the standard curve ranged from 4 to 40 μg/dL with linearity at 40 μg/dL and a detection limit of 0.1 μg/dL. The reference values for serum zinc and selenium concentrations ranged from 0.70 to 1.50 mg/L and from 4.6 to 14.3 μg/dL, respectively [23].
2.3.
DNA extraction and genotyping
Genomic DNA was isolated from peripheral blood leukocytes by a standard salting-out procedure, which was developed by Lahiri and Nurberger [24]. The rs5859, rs5854, and rs561104 SNP of the SEP15 gene and the rs73924411 and rs11126936 SNP of the SLC30A3 gene were identified by allelic discrimination with TaqMan 5′-nuclease assays (Real Time PCR; Applied Biosystems, Foster, CA, USA).
For this cross-sectional study, 110 individuals (78% women) were selected who were aged 50 years or older (64.86 ± 8.75 years). Data for the comparison of selenium and zinc levels, according to the descriptive characteristics of the subjects, are presented in Table 1, which included 56 women and 14 men (Table 1). No significant difference was observed between sex, alcohol consumption, or smoking status and the serum concentrations of selenium and zinc (P > .05). However, age was negatively correlated with the serum zinc concentration (r = −0.25, P = .008) but not the serum selenium concentration (r = 0.177, P = .143). In addition, 29 subjects in the sample (26.4%) had a serum zinc level that was below the reference value, whereas 11 subjects (15.7%) had selenium concentrations that were less than the recommended amount (data not shown).
3.2. The association of SLC30A3 and SEP15 gene polymorphisms with zinc and selenium concentrations In the genetic and biochemical analyses of zinc, 110 samples were analyzed to determine zinc levels, and the SLC30A3 gene was genotyped. Serum assays for selenium were performed on 70 samples (56 women and 14 men), all of which had SEP15 genotype information available. This difference in the number of samples from subjects (110 vs 70) occurred due to the amount of serum obtained
745
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 4 2–7 48
Table 2 – Solute carrier family 30 member 3 gene polymorphisms and serum zinc concentrations of subjects SLC30A3 polymorphisms
n
rs73924411 GG GA + AA P rs11126936 AA + AC CC P a b c d
Zinc (milligrams per liter) a
Low (
