Circulating levels of selenium species in Inuit adults from Nunavik
A. Achoubaa, P. Dumasb, N. Ouelleta, M. Lemirea b c, P. Ayottea b c*
a Axe santé publique et pratiques optimales en santé, Centre de recherche du CHU de Québec, Québec, Canada;
b Centre de toxicologie du Québec, Institut national de santé publique du Québec, Québec, Canada.
c Département de médecine sociale et préventive, Faculté de médecine, Université Laval, Québec, Québec, Canada
*Corresponding author: e-mail: pierre.ayotte@inspq.qc.ca, phone: 418-650-5115 ext.4654, fax: 418-654-2148
KEYWORDS: selenium, selenoproteins, isotope dilution analysis, Inuit, plasma, blood, mercury.
Abstract
Selenium (Se) is highly abundant in traditional marine foods consumed by Inuit and accordingly their Se intake is among the highest in the world. Some of these foods are also high in methylmercury (MeHg), which is bioaccumulated in fish and marine mammals. Se is an important element in the antioxidant defense system and therefore high Se intake may counteract some of the deleterious effects of MeHg exposure. However, elevated plasma Se concentrations have recently been associated to type 2 diabetes, hypercholesterolemia and/or hypertension. In this study, a method combining affinity chromatography and inductively coupled plasma-mass spectrometry with quantification by post-column isotope dilution was adapted in our laboratory and used to determine plasma levels of selenoproteins in archived plasma samples of Inuit adults who participated to the Nunavik Inuit Health Survey 2004. Mercury associated with selenoproteins was also quantified. Results show that glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and
selenoalbumin (SeAlb) represented respectively 25%, 52% and 23% of total plasma Se concentration (N = 852). In addition, small amount of Hg co-eluted with each Se- containing protein and 50% of plasma Hg was associated to SelP. A non-linear relationship was observed between plasma and blood Se levels in Inuit. Plasma Se concentration (median = 139 µg L-1; interquartile range (IQR) = 22.7 µg L-1) were markedly lower and less variable than these blood Se concentration (median = 261 µg L-1, IQR = 166 µg L-1). The non-linear relationship between plasma and blood Se suggests that a selenocompound, possibly selenoneine, accumulates in the blood cellular fraction of several Inuit adults.
1. Introduction
Selenium (Se) is an essential trace element for humans. It is mainly incorporated in selenoproteins in the form of selenocyteine (SeCys) which is encoded by the UGA codon in the human genome [20]. A total of 25 selenoproteins have been identified until now, which contain a SeCys residue in their active site and play several biological functions, notably protection from oxidative stress [20, 24]. In human plasma, three Se containing proteins, namely selenoprotein P (SelP), glutathione peroxidase 3 (GPx3) and selenoalbumin (SeAlb), are found so far [213]. Only SelP and GPx3 possess SeCys residues and belong to the family of selenoproteins while Se in SeAlb occurs unspecifically as selenomethionine (SeMet) by substitution of a methionine [214].
SelP is the most abundant plasma selenoprotein and contains more than 50% of the total plasma Se [24]. Although its expression is observed in many tissues, SelP is mainly secreted by the liver and can contain up to 10 SeCys residues [69]. It is mainly responsible for the transport of Se from the liver to peripheral tissues [64, 66] especially the brain and testis where specific receptors were recently identified [67, 68]. Further, SelP may also exhibit antioxidant functions, thereby protecting neuronal and astrocyte cells [215] and inhibiting the oxidation of low density lipoproteins [216]. GPx3 is secreted by kidneys [217] and plays a major role in the detoxification of extracellular hydrogen peroxides and fatty acid hydroperoxides [54]. It is also expressed in other tissues including the heart, where it is the third selenoprotein in terms of mRNA abundance [53]. SeAlb has no known
39 Se dependent biological function; it likely represents a form of Se storage which can be used by the liver to synthetize SelP [218].
Se is exceptionally abundant in certain traditional marine foods of Inuit and accordingly, Se intake in this population is among the highest in the world [198]. However, some marine foods also contain high concentrations of methylmercury (MeHg) which is bioaccumulated by marine mammals and predatory fish species in the Arctic [198, 200]. MeHg with its capacity to cross the blood-brain and placental barriers and its gastrointestinal absorption near to 100% is a neurotoxic threat for Arctic residents [219]. This toxicity can be influenced by certain elements present in foods which may offset some of the deleterious effects of MeHg [220]. Data from several epidemiological studies suggest that an adequate Se intake could play a protective role against the toxic effects of MeHg [208, 221], to which the Inuit population is highly exposed. Several researchers have hypothesized that Se is directly linked to the demethylation of MeHg and that granules of HgSe would be the final product of this degradation [177]. These granules have already been identified in marine mammals and are located in the cytoplasm of macrophages in the liver [190, 191]. However in other populations, an elevated Se status has been recently associated to type 2 diabetes, hypercholesterolemia and/or hypertension [8, 155]. Additionally, in some seleniferous areas in China (County of Enshi, Hubei Province) where selenosis symptoms were observed in the population, SeMet was found to be the main Se species involved [222].
Typically, the Se status is assessed by measuring the Se content of the plasma/serum or blood. To prevent human toxicity, the U.S. Environmental Protection Agency set Se blood levels corresponding to the no observable adverse effect level (NOAEL) and the lowest observable adverse effect level (LOAEL) at 1000 µg L-1 and 1350 μg L-1 respectively. At the opposite, deficiency occurs when plasma Se status is below 70-90 μg L-1[223].
All these findings suggest that, to better understand the impacts of high Se status on human health of Inuit, new biomarkers other than the commonly measured total plasma Se must be studied. The circulating levels of plasma selenoproteins may help to better characterize the
Se status and largely improve our capacity to identify the risks and benefit linked to the exceptionally high Se status of the Inuit population.
Here, we adapted in our laboratory the method of Li et al (2011) combining affinity column (AF) chromatography and inductively coupled plasma mass spectrometry (ICP-MS) with post-column isotope dilution (ID-AF-ICP-MS) to determine plasma levels of GPx3, SelP and SelAlb in a representative sample of the Inuit population of Nunavik, in order to better assess their Se status.
2.
Experimental
2.1. Study population
In the fall of 2004, a health survey was conducted among the 14 communities of Nunavik living along the coasts of the East Hudson Bay, Hudson Straight and Ungava Bay. The Nunavik territory covers a third of the total surface area of Quebec (Canada) and is home to approximately 11000 Inuit [196]. The aim of this study was to gather social and health information for this population and verify the evolution of its health status since the last survey conducted in 1992 [224]. The targeted individuals were the permanent Inuit residents of Nunavik aged between 18 and 74 years. The study design and the sampling procedure were published elsewhere [210]. Briefly, a two-stage stratified random sampling method was used. In the first stage, a proportional random sample selection of private Inuit households was carried out, taking into account the size of each village. In the second stage, eligible members of each household were asked to participate. Participants were invited to fill out several questionnaires and attend a clinical session during which blood samples and different clinical and anthropometric measurements were collected. The overall participation rate was 50% and a total of 889 participants completed the clinical session.
Our study sample consisted of 852 participants from whom plasma samples were available. After blood collection and centrifugation, plasma was isolated then aliquoted in Sarstedt
41 (Nümbrecht, Germany) 1.5 mL tubes and stored at -80 °C at the Centre de Toxicologie du Québec (CTQ) of the Institut National de Santé Publique du Québec, Canada (INSPQ).
2.2. Reagents and chemicals
Ammonium acetate (ACS grade) was purchased from Anachemia (Montreal, QC, Canada), while methanol and ethanol (Omnisolv grade) were obtained from EMD (Omaha, NB) and Commercial Alcohols (Brampton, ON, Canada) respectively. The enriched solution of 77Se (98%) was obtained from Cambridge Isotope Laboratories (Andover, MA) and 204 Hg (98%) from Isoflex (San Francisco, CA). Milli-Q water was purified by the advantage A10 ultrapure water system (Merck Millipore, Billerica, MA).
Two Se certified human serum reference materials (CRM) (BCR-637 and SRM-1950) were used throughout. The SRM-1950 was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) and the BCR-637 from the Institute for Reference Materials and Measurements (Geel, Belgium). Proficiency testing material (PTM) (QMEQAS 11-S-04) with a consensus value for Hg and Se were also used; they were obtained from the quality assessment scheme for trace elements operated by the INSPQ.
2.3. Instrumentation
The complete chromatography separation of the three selenoproteins was carried out using an Acquity ultra performance liquid chromatography system (UPLC) (Waters, Milford, MA) equipped with two 1-mL affinity columns heparin-sepharose and blue-sepharose, both purchased from GE Healthcare (Uppsala, Sweden). The columns were connected to a six- way Rheodyne automated switching valve (Model MXP9900-000; Oak Harbor, WA). A NexION 300s ICP-MS (Perkin-Elmer, Waltham, MA) was coupled to the UPLC system for Se and Hg detection and quantification by post-column isotope dilution. A daily performance solution containing 1 µg L-1 of Be, Ce, Co, Fe, In, Li, Mg, Mn, Pb and U in 1% (v/v) HNO3 was used for instrument optimization. The kinetic energy discrimination
mode (KED) in the collision cell using H2 gaswas employed to eliminate the polyatomic interferences upon detection of both Se and Hg. The isotope solution containing 77Se and 204Hg was continuously infused using the main ICP-MS peristaltic pump and mixed to the UPLC column outflow through a T connector. UPLC and ICP-MS operating conditions are presented in Table 3.
2.4. Analytical procedures
2.4.1. Speciation analysis
The method was adaptated from the previous published work of Li et al [211].Plasma samples were injected through the UPLC system following the conditions described in table 3 and schematic representation of the procedure is given by Figure 10. While the switching valve was in position 1, the flow passed through both heparin and blue sepharose columns using the mobile phase A. The Heparin-sepharose column has the ability to retain selectively the SelP while the blue-sepharose can retain both SelP and SeAlb. At this stage, the SelP and SeAlb were retained by heparin and blue sepharose columns respectively while the GPx and small Se compounds were the first eluted. Switching the valve to position 2 excluded the blue-sepharose column and permitted the elution of the SelP using mobile phase B. Finally the system was switched back to position 1 to elute SeAlb using the mobile phase B.
Post-column isotope dilution was performed using two stock solutions of 77Se and 204Hg prepared as follows: 4.92 mg of 77Se was weighed and dissolved in 10 mL of 5% (v/v) HNO3; and 1 mg of 204Hg was dissolved in 5 mL of 10% (v/v) HNO3 then completed to 100 mL with 10% (v/v) HNO3 containing 1 mL of Au at 1000 mg L-1. Using the previous stock solutions, a daily isotope solution was prepared for speciation analysis in 0.5 % (v/v) HNO3 containing 2.95 µg L-1 of 77Se, 5 µg L-1 of 204Hg and 8% (v/v) MeOH.
ICP-MS data were treated with the IDSoft post column ID software [225]. After the input of all ID parameters including the isotopic abundance, detector dead time and mass bias,
43 (78Se/77Se) and (200Hg/204Hg) chromatographic ratios were transformed to mass flow charts and the integration of peaks yielded concentrations.
2.4.2. Blood Se and Hg concentrations analysis
The measurement of whole blood Se and Hg concentrations was performed at the toxicology laboratory of INSPQ and the complete analytic method is detailed elsewhere [226].
2.5. Statistical analysis
Descriptive statistics were used to present the distributions of blood Hg, Se and plasma Hg, Se biomarkers. Normality was tested for both Se and Hg: Se in plasma follows a normal distribution and mean concentrations were used for comparison. In contrast, blood Hg and blood Se follow a log-normal distribution and accordingly medians were used. The relationship between plasma and blood Se was tested using the Pearson correlation coefficient.
3. Results
Figure 11 shows a mass flow chromatogram obtained after the complete separation of selenoproteins by ID-AF-ICP-MS. The speciation time analysis was 20 min and the three plasma Se-containing proteins (GPx3, SelP and SeAlb) were well separated. As shown in Figure 11, there was a Hg signal associated with each Se-containing protein. To quantify both Se and Hg, (78Se/77Se) and (200Hg/204Hg) ratios were used for isotope dilution analysis respectively. Data on the analytic performance of the method are presented in table 4. For Se-containing protein quantification, the limits of detection (LODs) were 0.70, 1.10 and 2.20 µg Se L-1 for GPx3, SelP and SeAlb respectively. The repeatability in terms of relative standard deviation (RSD %) was 5.84% for GPX3, 3.36% for SelP and 2.65% for SeAlb. Both SRM-1950 and BCR-637 had a certified value for total plasma Se and the method
shows a LOD of 3.71 µg L-1 and between-day precisions of 2.30% and 3.22% for SRM- 1950 and BCR-637 respectively. The quantification of Hg concentration associated with Se-containing proteins was validated through a PTM with a consensus value for total plasma Hg. The LODs were 0.02 µg L-1 for all Hg associated to selenoproteins (Hg-GPx3, Hg-SelP and Hg-SeAlb) and 0.04 µg L-1 for the total plasma Hg concentration. The repeatability was 3.49% for total plasma Hg and 19.14%, 7.15% and 19.37% for Hg associated to GPx3, SelP and SeAlb respectively.
Results from the blood Se analysis and application of the adapted method for Se speciation on 852 samples from Inuit adults are presented in table 5. High concentrations of blood Se are observed in this population (mean = 350 µg L-1) and 50% of participants had a blood Se level ≥ 261 µg L-1 (interquartile range (IQR) = 166). The maximum and minimum concentrations registered for blood Se were 3355 µg L-1 and 119 µg L-1 respectively. Total plasma Se concentrations were lower than the blood Se levels; concentrations ranged between 84.5 to 229 µg L-1 (mean = 140 µg L-1) with a median of 139 µg L-1 (IQR =22.7). Speciation analysis shows that 52% of total plasma Se was in the form of SelP (mean = 72.4 µg Se L-1) while GPx3 (mean = 35.6 µg Se L-1) and SeAlb (mean = 32.6 µg Se L-1) represented 25% and 23% of total plasma Se, respectively.
Results of Hg analysis in both blood and plasma are shown in table 6. As expected, blood Hg levels were higher than corresponding plasma levels in our participants (median in blood = 11.8 µg L-1, range = 0.08 to 241 µg L-1 vs median in plasma = 1.78µg L-1, range = 0.02 to 24.5 µg L-1). Similarly to Se, more than half of total plasma Hg was associated to SelP (mean = 1.46 µg L-1) and the rest was shared equally between GPx3 (mean = 0.70 µg L-1) and SeAlb (mean =0.55 µg L-1).
The relationship between total blood and plasma Se concentrations in this population was found to be non-linear (r = 0.33, p < 0.001) (Figure12). Plasma Se levels reach a plateau at approximately 175 µg L-1 while blood Se continues to increase.
45
4. Discussion
4.1. Method development and validation
In this study, we developed a method for the simultaneous quantification of Se proteins in human plasma and associated Hg content by ID-AF-ICP-MS. Although several methods were published using the same approach for Se proteins [211, 227, 228], none of them allowed to simultaneously quantify Se proteins and Hg. The use of H2 gas with KED mode in the collision cell, permitted to eliminate argon (Ar) induced polyatomic interferences (Ar38Ar40) on isotope 78Se. We selected post-column isotope dilution for quantification because standards for selenoproteins were unavailable. Moreover, this method provides the most accurate results for Se quantification when performed correctly [229]. Indeed, the accuracy and repeatability of our method for both CRMs and PTM were excellent (less than 5% in overall for total Se and Hg). We also investigated the possibility of using standards which were provided in some ELISA kits to elaborate the standard curves, but no Se was detected when analyzed by ICP-MS in those standards as they were fragments of recombinant selenoproteins. Therefore, the validation process was also affected by the lack of selenoprotein standards so that the evaluation of matrix effects and recovery could not be performed.
4.2. Biomarkers of Se and Hg status among the Inuit of Nunavik
Our study is the first to conduct plasma Se speciation in a population with a very high selenium status and also the largest one in terms of participant number (n = 852). To our knowledge, this is the second population study focusing on the Se speciation in an apparently healthy population, following that conducted in 2010 by Letsiou et al. in the Greek population [230].
Mean plasma Se concentration, which represents the biomarker of Se status most often used, was 140 µg L-1, with values ranging from 84.5 to 229 µg L-1. The mean concentration in Inuit was similar to those reported in general populations of North America (USA mean
= 136.4 µg L-1)[149], and higher than those found in UK (mean = 85.3 µg L-1) [231], Russia (mean = 93.6 µg L-1)[232], Korea (mean = 112 µg L-1) [233], Australia (mean = 103 µg L-1) [234], Japan (mean = 119.8 µg L-1) [235], France (mean = 88.0 µg L-1) [236] and even in Finland (mean = 110.5 µg L-1) where soils are fertilized with selenium since 1984 [237]. The 25th and 75th percentiles of plasma Se concentrations were 128 and 151 µg L-1 respectively, indicating a very narrow distribution of Se plasma levels (IQR =22.7). With regard to plasma Se distribution in selenoproteins, SelP contained 52% of total plasma Se while GPx3 and SeAlb represented 25 and 23% respectively. These results are in agreement with those noted in the Greek population, except for SeAlb which represented only 11% of total plasma Se (25% in GPx3, 53% in SelP, mean plasma Se = 91 µg L-1, n = 399). The variation of results for SeAlb can be explained by the difference in SeMet status between the populations, since Se in SeAlb is mainly incorporated unspecifically in the form of SeMet substituting for Met [238]. Moreover, our results were also in complete agreement with those reported in previous published studies involving a limited number of healthy participants, using the same analytical method, in which Se status was above 100 µg L-1. Jitaru et al (2008) found Se incorporated at 23%, 56% and 20% in GPX3, SelP and SeAlb respectively (plasma Se mean =110 µg L-1, n=3) [239] while Reyes et Al (2003) found 19%, 55% and 15% (plasma Se mean =100 µg L-1, n=5) [240]. However, when comparing our results to those in which Se status was relatively low (control groups in Roman et al studies (2010) mean Se = 85 µg L-1, n = 15) results were inconsistent and the percentage of Se-GPx3 tends to decrease with respect to SelP which remains the dominant Se-containing protein [241]. Furthermore, more than 75% of our participants exhibited plasma Se levels above 125 µg L-1, a Se status high enough to maximize activities and concentrations of selenoproteins [242]. These findings taken together support the existence of a regulation mechanism for plasma Se status. At high Se levels where selenoproteins activity and concentration are maximized, little variation of Se distribution among Se- containing proteins can be observed between populations. In contrast, variation may occur at low Se status as GPx3 is known to be more sensitive to Se status, in contrast to SelP which ensure the delivery of Se to organs and therefore it is less sensitive to the Se status [243].
47 Blood Hg levels in Nunavik (median = 11.2 µg L-1 IQR=17.9) is the highest when compared to other populations in Canada (median = 0.81 µg L-1, IQR =1.28) [244], USA (median = 0.64 µg L-1, Q1< LOD Q3 = 2.87 µg L-1) [245], Korea (median = 3.37 µg L-1, IQR = 3) [246], Hong Kong (median = 4.93 µg L-1, IQR = 3.23)[247] and Norway (median = 4.0 µg L-1) [248]. However, it remains lower than levels founds in population of both Brazilian Amazon (median= 57.2 µg L-1, IQR = 55.9) [249] and Inuit of Greenland (median=18 µg L-1, IQR = 25.3) [250].This elevated blood Hg comes mostly from the consumption of marine mammals which bioaccumulate MeHg in their tissues [200]. Lemire et al (2012) previously reported that beluga meat constitutes the most important source of MeHg, contributing up to two thirds of MeHg intake in this population [198].
Blood Se concentrations are exceptionally high in this population (median =261 µg L-1, IQR=166 µg L-1), with values comparable to those of the Inuit of Greenland (GM = 285.11 range = 68 to 5600 µg L-1) [250] and populations in the Brazilian Amazon (median = 284.3 μg L-1, IQR=161.9 µg L-1) [249]. However, when compared to general populations including Canadians (median = 199 µg L-1, IQR=36.7 µg L-1) [251], Americans (median = 189 μg L-1, IQR= 31.3 µg L-1) [252] and other populations in Europe [253, 254], blood Se levels in Nunavik are much higher. These high blood Se levels are also related to the consumption of traditional marine foods in Nunavik. Beluga mattaaq, seal liver and walrus meat were found to be the most important sources of Se in this population [198].
In this study, Hg was monitored and quantified simultaneously along with Se speciation. Indeed, our results show that plasma Hg accounted for 15% of blood Hg (mean = 2.59 µg L-1). In addition, this plasma Hg was found to be associated with the three peaks of Se proteins (Figure 12). Having said that, the first peak includes all unretained molecules and therefore Hg may not be associated to GPx3. However, Hg appears to bind to both SelP and SeAlb since it is retained and eluted with these Se-containing proteins selectively.