Factors influencing biomarkers of selenium status determined with a novel established and calibrated Selenoprotein P ELISA

Selen (Se) ist ein essentielles Spurenelement, das in Form der modifizierten Aminosäure Selenocystein Bestandteil der Selenoproteine ist. Dort stellt es bei Enzymen der antioxidativen Abwehr und des Schilddrüsenmetabolismus einen Teil des aktiven Zentrums dar. Das über die Nahrung aufgenommene Se wird über Selenoprotein P (SELENOP) von der Leber zu den extrahepatischen Organen transportiert. Eine Unterversorgung mit Se, so wie es bei vielen Europäern der Fall ist, erhöht das Krankheitsrisiko für Inflammation, Krebs, Fertilitätsstörungen und Autoimmunität. Daher ist die Erfassung von Biomarkern des Se-Status von großer Bedeutung. Die SELENOP-Konzentration im Serum bzw. Plasma spiegelt die Versorgungslage der Organe mit dem spezifisch regulierten Se-Pool wider. Jedoch zeigen Publikationen zu SELENOP-Konzentrationen von gesunden Menschen Diskrepanzen um annähernd das Zehntausendfache, was in der Verwendung kommerzieller nicht validierter und unkalibrierter Immunassays begründet liegt. Daher stand die Etablierung eines ELISAs, dessen Charakterisierung und Kalibrierung an einem international zugänglichen NIST Standard Referenz Material im Vordergrund dieser Arbeit. Diese Aufgabe wurde bewältigt und der neue Assay zeigt innerhalb seines großen Arbeitsbereiches (11,6 - 538,4 µg/L) eine sehr gute Richtigkeit (2,9 %) und gute Präzision (9,3 %). Der Vergleich jüngerer und älterer Probanden offenbarte einen altersspezifischen Geschlechtsdimorphismus. Bei jungen Frauen war der lineare Zusammenhang von Gesamt-Se und SELENOP im Serum auffällig gering, und der Quotient aus Se / SELENOP war höher als bei jüngeren oder älteren Männern oder älteren Frauen. Durch diesen Befund bereichert diese Arbeit die Biomarker des Se-Status um den Se / SELENOP-Quotienten. Überdies wird zum ersten Mal gezeigt, dass dieser Quotient durch die Verwendung von Antibiotika negativ beeinflusst werden kann. Bei den hier untersuchten gesunden Teilnehmern liegt er beim isolierten SELENOP mit 5,4 ± 0,5 (MW ± SD) deutlich unter dem theoretisch möglichen Wert von 10 Atomen pro Molekül. Zukünftige Studien mit hochqualitativen Proben in validierten und kalibrierten Methoden sind notwendig, um pathophysiologische Resultate in Zusammenhang mit Se-Biomarkern vor dem Hintergrund einer altersabhängigen geschlechtsspezifischen Regulation zu interpretieren. Weiterhin muss geklärt werden, inwieweit der Se / SELENOP-Quotient zusätzlich klinisch relevante Informationen bei der Diagnose von Erkrankungen oder Beurteilung von Mortalitätsraten wie z.B. bei konnatalen Infektionen in der Pädiatrie oder schwerer Sepsis liefert.Selenium (Se) is an essential trace element inserted in selenoproteins in the form of the modified amino acid selenocysteine. In the active site of enzymes, Se is involved in processes of antioxidant defense and thyroid metabolism. The dietary Se is transported via selenoprotein P (SELENOP) from the liver to the extrahepatic tissues for the synthesis of other selenoproteins. A low Se intake like in Europe increases the risk of inflammation, cancer, fertility problems, and autoimmunity, which makes the detection of biomarkers of the Se status very important. In contrast to total Se, SELENOP in serum or plasma represents the supply of the tissues with the regulated Se-pool. However, publications on SELENOP concentrations of healthy humans differ by almost factor ten thousand, which are outside reasonable limits. These contradictions complicate the comparability of clinical studies. Reasons are the usage of commercial non-validated and uncalibrated immunoassays. Consequently, the establishment of an ELISA, its characterization, and calibration against the internationally available NIST Standard Reference Material SRM1950 is the central part of this work. The new assay shows excellent accuracy (2.9 %) and good precision (9.3 %) within its large working range (11.6 - 538.4 μg / L). The comparison of younger and older subjects showed an age-specific sexual dimorphism. In serum, the correlation between total Se and SELENOP was remarkably low in young women, and the Se / SELENOP ratio was higher than that of younger or older men or older women. Because of this finding, this work enriches the biomarkers of Se status by the Se / SELENOP ratio. Moreover, it is shown for the first time that this quotient can be adversely affected using antibiotics. In healthy participants studied here on average 5.4 +/- 0.5 (mean +/- SD) Se atoms per molecule were found in the isolated SELENOP which is below the theoretically possible value of 10. Future studies with high-quality samples in validated and calibrated methods are necessary to interpret pathophysiological results associated with Se biomarkers considering an age-dependent gender-specific regulation. Furthermore, it must be clarified to what extent the Se / SELENOP ratio provides additional clinically relevant information in disease diagnostics or prediction of mortality risks, e.g., in conatal infections in pediatrics or severe sepsis

Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine

Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7–15 Sec residues depending on species. Assessing an individual’s selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6–538.4 µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status

Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P

Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6–538.4 µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status

Association of selenoprotein and selenium pathway gnotypes with risk of colorectal cancer and interaction with selenium status

Selenoprotein genetic variations and suboptimal selenium (Se) levels may contribute to the risk of colorectal cancer (CRC) development. We examined the association between CRC risk and genotype for single nucleotide polymorphisms (SNPs) in selenoprotein and Se metabolic pathway genes. Illumina Goldengate assays were designed and resulted in the genotyping of 1040 variants in 154 genes from 1420 cases and 1421 controls within the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Multivariable logistic regression revealed an association of 144 individual SNPs from 63 Se pathway genes with CRC risk. However, regarding the selenoprotein genes, only TXNRD1 rs11111979 retained borderline statistical significance after adjustment for correlated tests (PACT = 0.10; PACT significance threshold was P < 0.1). SNPs in Wingless/Integrated (Wnt) and Transforming growth factor (TGF) beta-signaling genes (FRZB, SMAD3, SMAD7) from pathways affected by Se intake were also associated with CRC risk after multiple testing adjustments. Interactions with Se status (using existing serum Se and Selenoprotein P data) were tested at the SNP, gene, and pathway levels. Pathway analyses using the modified Adaptive Rank Truncated Product method suggested that genes and gene x Se status interactions in antioxidant, apoptosis, and TGF-beta signaling pathways may be associated with CRC risk. This study suggests that SNPs in the Se pathway alone or in combination with suboptimal Se status may contribute to CRC development

Association of Selenoprotein and Selenium Pathway Genotypes with Risk of Colorectal Cancer and Interaction with Selenium Status

Selenoprotein genetic variations and suboptimal selenium (Se) levels may contribute to the risk of colorectal cancer (CRC) development. We examined the association between CRC risk and genotype for single nucleotide polymorphisms (SNPs) in selenoprotein and Se metabolic pathway genes. Illumina Goldengate assays were designed and resulted in the genotyping of 1040 variants in 154 genes from 1420 cases and 1421 controls within the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Multivariable logistic regression revealed an association of 144 individual SNPs from 63 Se pathway genes with CRC risk. However, regarding the selenoprotein genes, only TXNRD1 rs11111979 retained borderline statistical significance after adjustment for correlated tests (P-ACT = 0.10; P-ACT significance threshold was P <0.1). SNPs in Wingless/Integrated (Wnt) and Transforming growth factor (TGF) beta-signaling genes (FRZB, SMAD3, SMAD7) from pathways affected by Se intake were also associated with CRC risk after multiple testing adjustments. Interactions with Se status (using existing serum Se and Selenoprotein P data) were tested at the SNP, gene, and pathway levels. Pathway analyses using the modified Adaptive Rank Truncated Product method suggested that genes and gene x Se status interactions in antioxidant, apoptosis, and TGF-beta signaling pathways may be associated with CRC risk. This study suggests that SNPs in the Se pathway alone or in combination with suboptimal Se status may contribute to CRC development.Peer reviewe

Hypoxia reduces and redirects selenoprotein biosynthesis

Selenium deficiency constitutes a risk factor for the incidence and negative course of severe diseases including sepsis, stroke, autoimmune diseases or cancer. In this study, hypoxia is identified as a powerful stimulus to redirect selenoprotein biosynthesis causing reduced selenoprotein P expression and diminished selenium export from hepatocytes in favour of increased biosynthesis of the essential protective intracellular phospholipid hydroperoxide glutathione peroxidase GPX4. Specifically, hypoxia decreases transcript concentrations of central factors controlling selenium and selenocysteine metabolism including selenophosphate synthetase-2, phosphoseryl-tRNASerSec kinase and selenocysteine lyase, which are all proven to be rate-limiting enzymes in selenoprotein biosynthesis. These effects are paralleled by a general decline of selenoprotein expression; however, not all selenoproteins are affected to the same extent by hypoxia, and GPX4 constitutes an exception as its expression becomes slightly increased. Supplemental selenium is able to overcome the hypoxia-dependent down regulation of selenoprotein expression in our cell culture model system, supporting the concept of using selenium as an adjuvant treatment option in severe diseases. Although it remains to be tested whether these effects constitute a hepatocyte-specific response, the selenium-dependent decline of selenoprotein P biosynthesis under hypoxic conditions may explain the progressive selenium deficit developing in severe diseases.Peer Reviewe

Hypoxia reduces and redirects selenoprotein biosynthesis

Selenium deficiency constitutes a risk factor for the incidence and negative course of severe diseases including sepsis, stroke, autoimmune diseases or cancer. In this study, hypoxia is identified as a powerful stimulus to redirect selenoprotein biosynthesis causing reduced selenoprotein P expression and diminished selenium export from hepatocytes in favour of increased biosynthesis of the essential protective intracellular phospholipid hydroperoxide glutathione peroxidase GPX4. Specifically, hypoxia decreases transcript concentrations of central factors controlling selenium and selenocysteine metabolism including selenophosphate synthetase-2, phosphoseryl-tRNASerSec kinase and selenocysteine lyase, which are all proven to be rate-limiting enzymes in selenoprotein biosynthesis. These effects are paralleled by a general decline of selenoprotein expression; however, not all selenoproteins are affected to the same extent by hypoxia, and GPX4 constitutes an exception as its expression becomes slightly increased. Supplemental selenium is able to overcome the hypoxia-dependent down regulation of selenoprotein expression in our cell culture model system, supporting the concept of using selenium as an adjuvant treatment option in severe diseases. Although it remains to be tested whether these effects constitute a hepatocyte-specific response, the selenium-dependent decline of selenoprotein P biosynthesis under hypoxic conditions may explain the progressive selenium deficit developing in severe diseases.Peer Reviewe