Microbore affinity-HPLC (AF-HPLC) hyphenated to inductively coupled plasma-sector field mass spec... more Microbore affinity-HPLC (AF-HPLC) hyphenated to inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) was applied to the simultaneous speciation analysis of glutathione peroxidase (GPx), selenoprotein P (SelP) and seleno-albumin (SeAlb) in 5 μL of (human) serum. ICP-SFMS (high resolution mode, HR, M/ΔM = 10,000) was used for the quantification of selenium in GPx, SelP and SeAlb after their separation by AF-HPLC, without prior sample preparation or mathematical correction of the spectral interferences. In order to compensate for the loss in sensitivity due to the detection in HR mode and the very low amount of sample taken for analysis, a high-efficiency sample introduction system was used as an interface between AF-HPLC and ICP-SFMS. This led to a signal-to-noise ratio enhancement of about one order of magnitude compared to the conventional experimental ICP-SFMS set-up. Apart from the very low sample consumption, another major advantage of the method described here is the significant reduction of the analysis time (≤7 min). Quantification of GPx, SelP and SeAlb was carried out using on-line (post column) isotope dilution; comparison with on-line external calibration by using Se-L-cystine standard is also addressed. The method accuracy for the determination of total protein bound Se was assessed by the analysis of a human serum reference material (BCR-637) certified for total Se content.
The association between the concentration/activity of selenium/selenoproteins in plasma and type ... more The association between the concentration/activity of selenium/selenoproteins in plasma and type 2 diabetes mellitus is still a matter of debate. This cross-sectional pilot study evaluates whether patients with diabetes present a different plasma selenoproteins status than a healthy control group and examines whether the introduction of clinical parameters allows the detection of correlations and further grouping criteria. For this purpose, the levels of plasma glutathione peroxidase (GPx), selenoprotein P (SelP), and seleno-albumin (SeAlb) present in 40 patients affected by type 2 diabetes mellitus were determined simultaneously and accurately by a newly developed analytical method. The results show that patients with diabetes demonstrate significantly lower levels of GPx and SeAlb with respect to healthy subjects (11 6 3 ng/mL and 9 6 2 ng/mL vs 18 6 8 ng/mL and 11 6 2 ng/mL, respectively). Significant negative correlations were revealed among GPx, SeAlb, and clinical parameters including fasting plasma glucose, hemoglobin A1c, and the albuminto-creatinine ratio. Our findings suggest an association between the individual selenoproteins concentration and the presence of diabetes, including associated clinical parameters. It currently cannot be ascertained whether the altered selenoproteins status is a consequence or a causative factor for diabetes. This study demonstrates the potential of a method for individual selenoproteins determination for investigating the biochemical relationship between selenium and diabetes. (Translational Research 2010;156:242-250) Abbreviations: ACR ¼ Albumin-to-creatinine ratio; ANCOVA ¼ analysis of covariance; BMI ¼ body mass index; CL ¼ confidence limits; FA ¼ factorial analysis; FPG ¼ fasting plasma glucose; GPx ¼ glutathione peroxidase; HbA1c ¼ hemoglobin A1c; HDL ¼ high-density lipoprotein; HPLC ¼ high-performance liquid chromatography; ICP-MS ¼ inductively coupled plasma-mass spectrometer; K-S ¼ Kolmogorov-Smirnov; LDL ¼ low-density lipoprotein; LRA ¼ logistic regression analysis; OR ¼ odds ratio; SD ¼ standard deviation; Se ¼ selenium; SeAlb ¼ seleno-albumin; SelP ¼ selenoprotein P; S-W ¼ Shapiro-Wilk A series of studies have shown recently that selenium (Se) has insulin-mimetic properties both in vitro and in vivo, including the stimulation of glucose uptake 1,2 as well as the regulation of glycolysis, gluconeogenesis, fatty acid synthesis, and the pentose phosphate pathway. 3,4 Se also prevents or alleviates the adverse effects that diabetes has on cardiac, 5-7 renal, and platelet functions 8,9 as well as atherosclerosis progression. 10
In this case-control pilot study a recent method based on HPLC hyphenated to ICP-MS was employed ... more In this case-control pilot study a recent method based on HPLC hyphenated to ICP-MS was employed for the quantification of serum glutathione peroxidase type 3 (GPx3), seleno-protein P (SelP) and seleno-albumin (SeAlb) in 42 patients with colorectal cancer (CRC) and 20 controls. Patients with early cancer stage (TNM I) showed a significantly higher level of SeAlb (19 ± 3 ng/mL) in respect to both metastatic CRC patients (TNM IV, 16± 4 ng/mL) and healthy controls (16 ± 3 ng/mL). Classification models based on logistic regression analysis, classification trees and artificial neural networks were constructed using seleno-proteins concentrations as predictors. Neural networks lead to the best performances, up to 95% of corrected predictions in TNM I vs. controls discrimination. These results suggest a potential association between individual selenoproteins and CRC progression. Age and radiochemoteraphy were assessed as confounding factors, showing no significant effects. Still, SeAlb level tended to reduce with the age in healthy persons, but did not in CRC patients. Seleno-proteins concentration was also compared with a number of clinical parameters considered as prognostic factors in CRC. Significant Spearman's correlations were revealed between SelP and SeAlb, and presence of peritumoural lymphocytic infiltration (ρ = −0.57 and ρ = −0.37, respectively); and SeAlb and degree of cellular differentiation (grading, ρ = −0.37). This study marks the importance to systematically introduce speciation analysis and multidisciplinary approaches in the investigation of the role of seleno-proteins as a potential combined biomarker for CRC.
Despite its very low level in humans, selenium plays an important and unique role among the (semi... more Despite its very low level in humans, selenium plays an important and unique role among the (semi)metal trace essential elements because it is the only one for which incorporation into proteins is genetically encoded, as the constitutive part of the 21st amino acid, selenocysteine. Twenty-five selenoproteins have been identified so far in the human proteome. The biological functions of some of them are still unknown, whereas for others there is evidence for a role in antioxidant defence, redox state regulation and a wide variety of specific metabolic pathways. In relation to these functions, the selenoproteins emerged in recent years as possible biomarkers of several diseases such as diabetes and several forms of cancer. Comprehension of the selenium biochemical pathways under normal physiological conditions is therefore an important requisite to elucidate its preventing/therapeutic effect for human diseases. This review summarizes the most recent findings on the biochemistry of active selenium species in humans, and addresses the latest evidence on the link between selenium intake, selenoproteins functionality and beneficial health effects. Primary emphasis is given to the interpretation of biochemical mechanisms rather than epidemiological/observational data. In this context, the review includes the following sections: (1) brief introduction; (2) general nutritional aspects of selenium; (3) global view of selenium metabolic routes; (4) detailed characterization of all human selenoproteins; (5) detailed discussion of the relation between selenoproteins and a variety of human diseases.
A study of selenium (Se) speciation in rat colon tissues is presented. Four different procedures ... more A study of selenium (Se) speciation in rat colon tissues is presented. Four different procedures for the extraction of Se compounds were evaluated in terms of recovery and species preservation. Total Se in tissue and extracts was determined by ICP-MS and isotope dilution analysis. The selected and optimized protocol allowed an extraction of 43% of Se, while continuously bubbling nitrogen in the solution during the procedure was mandatory to prevent the oxidative degradation of selenoproteins. Speciation analysis was then performed on the extracts using size exclusion-and anion exchange-HPLC for species separation. A number of Se compounds were detected in rat colon extracts, and individually quantified by coupling HPLC-ICP-MS and species-unspecific on-line (post-column) isotope dilution analysis. Among the isolated selenospecies, the two major proteins glutathione peroxidase type 2 and thioredoxin reductase type 1 have been potentially identified by their molecular weight using
Se is one of the most investigated essential trace elements in the past years, mostly due to its ... more Se is one of the most investigated essential trace elements in the past years, mostly due to its cancer prevention properties. Nevertheless, the accurate determination of its biologically active species, such as the selenoproteins (SeProt) in human serum, is currently a challenging task. This is because of the lack of appropriate quantification standards, certified reference materials (CRMs), and/or reference measurement methods. Additionally, most of the methods developed so far for the determination of SeProt were applied to the analysis of control (volunteers) serums, which are not available to other laboratories, therefore making methods intercomparison virtually impossible. We present here for the first time indicative levels of SeProt in a commercially available human serum, namely the BCR-637 CRM with certified level of total Se. The concentrations of selenium associated with glutathione peroxidase (GPx), selenoprotein P (SelP) and selenoalbumin (SeAlb) in this serum were calculated using the results obtained by 13 different analytical methods (literature and non-published data) on the basis of (affinity) high-performance liquid chromatography (AF-HPLC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). The indicative levels of SeProt in the BCR-637 serum can be used for validation of methods dealing with the determination of these proteins in human serum.
Microbore affinity-HPLC (AF-HPLC) hyphenated to inductively coupled plasma-sector field mass spec... more Microbore affinity-HPLC (AF-HPLC) hyphenated to inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) was applied to the simultaneous speciation analysis of glutathione peroxidase (GPx), selenoprotein P (SelP) and seleno-albumin (SeAlb) in 5 μL of (human) serum. ICP-SFMS (high resolution mode, HR, M/ΔM = 10,000) was used for the quantification of selenium in GPx, SelP and SeAlb after their separation by AF-HPLC, without prior sample preparation or mathematical correction of the spectral interferences. In order to compensate for the loss in sensitivity due to the detection in HR mode and the very low amount of sample taken for analysis, a high-efficiency sample introduction system was used as an interface between AF-HPLC and ICP-SFMS. This led to a signal-to-noise ratio enhancement of about one order of magnitude compared to the conventional experimental ICP-SFMS set-up. Apart from the very low sample consumption, another major advantage of the method described here is the significant reduction of the analysis time (≤7 min). Quantification of GPx, SelP and SeAlb was carried out using on-line (post column) isotope dilution; comparison with on-line external calibration by using Se-L-cystine standard is also addressed. The method accuracy for the determination of total protein bound Se was assessed by the analysis of a human serum reference material (BCR-637) certified for total Se content.
The association between the concentration/activity of selenium/selenoproteins in plasma and type ... more The association between the concentration/activity of selenium/selenoproteins in plasma and type 2 diabetes mellitus is still a matter of debate. This cross-sectional pilot study evaluates whether patients with diabetes present a different plasma selenoproteins status than a healthy control group and examines whether the introduction of clinical parameters allows the detection of correlations and further grouping criteria. For this purpose, the levels of plasma glutathione peroxidase (GPx), selenoprotein P (SelP), and seleno-albumin (SeAlb) present in 40 patients affected by type 2 diabetes mellitus were determined simultaneously and accurately by a newly developed analytical method. The results show that patients with diabetes demonstrate significantly lower levels of GPx and SeAlb with respect to healthy subjects (11 6 3 ng/mL and 9 6 2 ng/mL vs 18 6 8 ng/mL and 11 6 2 ng/mL, respectively). Significant negative correlations were revealed among GPx, SeAlb, and clinical parameters including fasting plasma glucose, hemoglobin A1c, and the albuminto-creatinine ratio. Our findings suggest an association between the individual selenoproteins concentration and the presence of diabetes, including associated clinical parameters. It currently cannot be ascertained whether the altered selenoproteins status is a consequence or a causative factor for diabetes. This study demonstrates the potential of a method for individual selenoproteins determination for investigating the biochemical relationship between selenium and diabetes. (Translational Research 2010;156:242-250) Abbreviations: ACR ¼ Albumin-to-creatinine ratio; ANCOVA ¼ analysis of covariance; BMI ¼ body mass index; CL ¼ confidence limits; FA ¼ factorial analysis; FPG ¼ fasting plasma glucose; GPx ¼ glutathione peroxidase; HbA1c ¼ hemoglobin A1c; HDL ¼ high-density lipoprotein; HPLC ¼ high-performance liquid chromatography; ICP-MS ¼ inductively coupled plasma-mass spectrometer; K-S ¼ Kolmogorov-Smirnov; LDL ¼ low-density lipoprotein; LRA ¼ logistic regression analysis; OR ¼ odds ratio; SD ¼ standard deviation; Se ¼ selenium; SeAlb ¼ seleno-albumin; SelP ¼ selenoprotein P; S-W ¼ Shapiro-Wilk A series of studies have shown recently that selenium (Se) has insulin-mimetic properties both in vitro and in vivo, including the stimulation of glucose uptake 1,2 as well as the regulation of glycolysis, gluconeogenesis, fatty acid synthesis, and the pentose phosphate pathway. 3,4 Se also prevents or alleviates the adverse effects that diabetes has on cardiac, 5-7 renal, and platelet functions 8,9 as well as atherosclerosis progression. 10
In this case-control pilot study a recent method based on HPLC hyphenated to ICP-MS was employed ... more In this case-control pilot study a recent method based on HPLC hyphenated to ICP-MS was employed for the quantification of serum glutathione peroxidase type 3 (GPx3), seleno-protein P (SelP) and seleno-albumin (SeAlb) in 42 patients with colorectal cancer (CRC) and 20 controls. Patients with early cancer stage (TNM I) showed a significantly higher level of SeAlb (19 ± 3 ng/mL) in respect to both metastatic CRC patients (TNM IV, 16± 4 ng/mL) and healthy controls (16 ± 3 ng/mL). Classification models based on logistic regression analysis, classification trees and artificial neural networks were constructed using seleno-proteins concentrations as predictors. Neural networks lead to the best performances, up to 95% of corrected predictions in TNM I vs. controls discrimination. These results suggest a potential association between individual selenoproteins and CRC progression. Age and radiochemoteraphy were assessed as confounding factors, showing no significant effects. Still, SeAlb level tended to reduce with the age in healthy persons, but did not in CRC patients. Seleno-proteins concentration was also compared with a number of clinical parameters considered as prognostic factors in CRC. Significant Spearman's correlations were revealed between SelP and SeAlb, and presence of peritumoural lymphocytic infiltration (ρ = −0.57 and ρ = −0.37, respectively); and SeAlb and degree of cellular differentiation (grading, ρ = −0.37). This study marks the importance to systematically introduce speciation analysis and multidisciplinary approaches in the investigation of the role of seleno-proteins as a potential combined biomarker for CRC.
Despite its very low level in humans, selenium plays an important and unique role among the (semi... more Despite its very low level in humans, selenium plays an important and unique role among the (semi)metal trace essential elements because it is the only one for which incorporation into proteins is genetically encoded, as the constitutive part of the 21st amino acid, selenocysteine. Twenty-five selenoproteins have been identified so far in the human proteome. The biological functions of some of them are still unknown, whereas for others there is evidence for a role in antioxidant defence, redox state regulation and a wide variety of specific metabolic pathways. In relation to these functions, the selenoproteins emerged in recent years as possible biomarkers of several diseases such as diabetes and several forms of cancer. Comprehension of the selenium biochemical pathways under normal physiological conditions is therefore an important requisite to elucidate its preventing/therapeutic effect for human diseases. This review summarizes the most recent findings on the biochemistry of active selenium species in humans, and addresses the latest evidence on the link between selenium intake, selenoproteins functionality and beneficial health effects. Primary emphasis is given to the interpretation of biochemical mechanisms rather than epidemiological/observational data. In this context, the review includes the following sections: (1) brief introduction; (2) general nutritional aspects of selenium; (3) global view of selenium metabolic routes; (4) detailed characterization of all human selenoproteins; (5) detailed discussion of the relation between selenoproteins and a variety of human diseases.
A study of selenium (Se) speciation in rat colon tissues is presented. Four different procedures ... more A study of selenium (Se) speciation in rat colon tissues is presented. Four different procedures for the extraction of Se compounds were evaluated in terms of recovery and species preservation. Total Se in tissue and extracts was determined by ICP-MS and isotope dilution analysis. The selected and optimized protocol allowed an extraction of 43% of Se, while continuously bubbling nitrogen in the solution during the procedure was mandatory to prevent the oxidative degradation of selenoproteins. Speciation analysis was then performed on the extracts using size exclusion-and anion exchange-HPLC for species separation. A number of Se compounds were detected in rat colon extracts, and individually quantified by coupling HPLC-ICP-MS and species-unspecific on-line (post-column) isotope dilution analysis. Among the isolated selenospecies, the two major proteins glutathione peroxidase type 2 and thioredoxin reductase type 1 have been potentially identified by their molecular weight using
Se is one of the most investigated essential trace elements in the past years, mostly due to its ... more Se is one of the most investigated essential trace elements in the past years, mostly due to its cancer prevention properties. Nevertheless, the accurate determination of its biologically active species, such as the selenoproteins (SeProt) in human serum, is currently a challenging task. This is because of the lack of appropriate quantification standards, certified reference materials (CRMs), and/or reference measurement methods. Additionally, most of the methods developed so far for the determination of SeProt were applied to the analysis of control (volunteers) serums, which are not available to other laboratories, therefore making methods intercomparison virtually impossible. We present here for the first time indicative levels of SeProt in a commercially available human serum, namely the BCR-637 CRM with certified level of total Se. The concentrations of selenium associated with glutathione peroxidase (GPx), selenoprotein P (SelP) and selenoalbumin (SeAlb) in this serum were calculated using the results obtained by 13 different analytical methods (literature and non-published data) on the basis of (affinity) high-performance liquid chromatography (AF-HPLC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). The indicative levels of SeProt in the BCR-637 serum can be used for validation of methods dealing with the determination of these proteins in human serum.
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