SELENOPROTEIN P IN HEART FAILURE

Abstract

A method for assessing risk in a subject having heart failure that is (i) risk for getting a cardiovascular event and/or (ii) risk of worsening heart failure condition and/or (iii) assessing risk for mortality, and/or (iv) assessing risk of hospitalization or re-hospitalization due to heart failure, involves: a) determining the level and/or amount of Selenoprotein P and/or fragments thereof in a sample from the subject, b) correlating the determined level and/or the amount of Selenoprotein P and/or fragments thereof in a subject having heart failure with one or more of the risks (i) to (iv) mentioned above.

Subject matter of the present invention includes stratification of patients and treatment methods for heart failure patients at high risk (i) for getting a cardiovascular event and/or (ii) of worsening heart failure condition and/or (iii) for mortality, in particular cardiovascular mortality, and/or (iv) of hospitalisation or re-hospitalisation due to heart failure.

Claims

1. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure, comprising a) determining the level and/or the amount of Selenoprotein P and/or fragments thereof in a sample of said subject, b) correlating the determined level and/or the amount of Selenoprotein P and/or fragments thereof in a subject having heart failure with (i) the risk for getting a cardiovascular event and/or (ii) with the risk of worsening heart failure condition and/or (iii) with the risk for mortality, and/or (iv) with the risk of hospitalization or re-hospitalization due to heart failure.

2. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality, and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein in a subject having heart failure (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) the risk for mortality, and/or (iv) the risk of hospitalization or re-hospitalization due to heart failure is enhanced, when the determined level and/or the amount of Selenoprotein P and/or fragments thereof in a sample of said subject is below a threshold.

3. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality, and/or (iv) assessing the risk of hospitalization or re-hospitalization in due to heart failure according to claim 1, wherein in a subject having heart failure (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) the risk for mortality, and/or (iv) the risk of hospitalization or re-hospitalization due to heart failure is enhanced when said and/or the amount of Selenoprotein P and/or fragments thereof in said sample is below a threshold, wherein said threshold is between 2.0 and 4.4 mg/L, preferably between 2.3 and 3.8 mg/L, more preferably between 2.6 and 3.4 mg/L, more preferably between 3.0 and 3.3 mg/L, most preferred said threshold is 3.3 mg/L.

4. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality, and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein in a subject having heart failure (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) the risk for mortality, and/or (iv) the risk of hospitalization or re-hospitalization due to heart failure is enhanced when said level and/or the amount of Selenoprotein P and/or fragments thereof in said sample is below a threshold, wherein said threshold has been determined by the calculation of receiver operating characteristic curves (ROC curves), plotting the true positive rate (sensitivity, “disease” population e.g. subjects who did develop the condition) against the false positive rate (1-specificity, “normal” population e.g. subjects who did not develop the condition) at various threshold value settings.

5. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality, and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein in a subject having heart failure (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) the risk for mortality, and/or (iv) the risk of hospitalization or re-hospitalization due to heart failure is enhanced when said level and/or the amount of Selenoprotein P and/or fragments thereof in said sample is below a threshold, wherein said threshold is the lower range of a heart failure population e.g. below 4.4 mg/L, more preferred below 3.8 mg/L, even more preferred below 3.4 mg/L, most preferred equal to or below 3.3 mg/L.

6. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality, and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein said cardiovascular event is selected from a group comprising myocardial infarction, stroke, coronary re-vascularization, and heart failure and said mortality is cardiovascular mortality.

7. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein said mortality is cardiovascular mortality related to myocardial infarction, stroke or acute heart failure.

8. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein said level and/or amount of Selenoprotein P and/or fragments thereof has been determined by an immunoassay using at least one binder binding to SEQ ID No. 2.

9. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 8, wherein said at least one binder is an antibody or a fragment thereof.

10. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein said level and/or amount of Selenoprotein P and/or fragments thereof has been determined by mass spectroscopy.

11. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein said (i) risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) said risk for mortality is assessed for a period of time of up to one year.

12. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein said risk of hospitalization or re-hospitalization due to heart failure is assessed for a period of up to 30 days.

13. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein the sample is a bodily fluid.

14. A method for assessing a risk in a subject having heart failure that is (i) the risk for getting a cardiovascular event and/or (ii) the risk of worsening heart failure condition and/or (iii) assessing the risk for mortality and/or (iv) assessing the risk of hospitalization or re-hospitalization due to heart failure according to claim 1, wherein the sample is a bodily fluid selected from the group comprising whole blood, plasma, and serum.

15. A method for treating a subject having heart failure and having an enhanced risk for (i) getting a cardiovascular event and/or (ii) having an enhanced risk for worsening heart failure condition and/or (iii) having an enhanced risk for mortality and/or (iv) having an enhanced risk of hospitalization or re-hospitalization due to heart failure, comprising administering selenium to said subject.

16. A method of treating a subject having heart failure and having an enhanced risk for (i) getting a cardiovascular event and/or (ii) having an enhanced risk for worsening heart failure condition and/or (iii) having an enhanced risk for mortality and/or (iv) having an enhanced risk hospitalization or re-hospitalization due to heart failure as determined according to a method of claim 1, comprising administering selenium to said subject.

17. A method of treating a subject having heart failure and having an enhanced risk for (i) getting a cardiovascular event and/or (ii) having an enhanced risk for worsening heart failure condition and/or (iii) having an enhanced risk for mortality and/or (iv) having an enhanced risk of re-hospitalization due to heart failure as determined according to a method of claim 1, comprising: administering selenium to said subject, and wherein the determined level and/or the amount of Selenoprotein P and/or fragments thereof is below a threshold and wherein said threshold is between 2.0 and 4.4 mg/L.

Description

FIGURE DESCRIPTION

[0155] FIG. 1. Distribution of SePP (mg/L) within groups based on outcomes. A) no re-hospitalisation/survivor; B) re-hospitalised/survivor; C) no re-hospitalisation/deceased and D) re-hospitalised and deceased

[0156] FIG. 2. One-year survival within quartiles of SePP. Q1=quartile with lowest levels; Q4=quartile with highest levels. SePP levels within quartiles: Q1=1.8±0.4; Q2=2.6±0.2; Q3=3.4±0.2, Q4=4.7±0.8.

[0157] FIG. 3. 30-day re-hospitalisation within quartiles of SePP. Q1=quartile with lowest levels; Q4=quartile with highest levels. SePP levels within quartiles: Q1=1.8±0.4; Q2=2.6±0.2; Q3=3.4±0.2, Q4=4.7±0.8.

[0158] FIG. 4. Composite endpoint consisting of death or re-hospitalisation within 30 days, whichever came first, within quartiles of SePP. Q1=quartile with lowest levels; Q4=quartile with highest levels. SePP levels within quartiles: Q1=1.8±0.4; Q2=2.6±0.2; Q3=3.4±0.2, Q4=4.7±0.8.

[0159] FIG. 5. Comparison of the Selenoprotein P distribution in (A) a healthy reference population (MPP-study) and (B) heart failure patients (Harvest-study). Bold solid line shows the median of the respective population, broken line shows the first quartile of the healthy reference population.

EXAMPLES

Example 1: Assay Description

[0160] The Selenotest ELISA (Hybsier et al. 2017. Redox Biology 11: 403-414; Hybsier et al. 2015. Perspectives in Science 3: 23-24), a chromogenic enzyme-linked immunosorbent assay, for the quantitative determination of human Selenoproteinselenoprotein P in serum samples was used. The Selenotest ELISA is a sandwich enzyme immunoassay in 96 well plate format and uses two different Selenoproteinselenoprotein P specific monoclonal antibodies for the antigen capture and detection steps. The Selenoprotein P levels of the calibrators and controls were determined by measurements against serial dilutions of NIST SRM 1950 Standard Reference Material. Monoclonal antibodies (Ab) were generated by immunization of mice with an emulsion of purified recombinant Selenoprotein P. The specific monoclonal Ab5 was immobilized as capture-Ab, and the specific mAb2, was used as detection-Ab. The lower limit of quantification was determined at a Selenoprotein P level concentration of 11.6 μg/L, and the upper limit of quantification at 538.4 μg/L, thereby defining the working range at Selenoprotein P levels concentrations between 11.6 and 538.4 μg/L. The intersection at 20% CV defines the limit of detection, and was reached at a Selenoprotein P level concentration of 6.7 μg/L i.e., around 500-fold below average serum Selenoprotein P levels concentrations of well-supplied subjects. The signals were linear on dilution within the working range of the assay, and Selenoprotein P was stable in serum for 24 h at room temperature. For further details of the assay see Hybsier et al. 2017. Redox Biology 11: 403-414.

Example 2: HARVEST-Malmö Study

[0161] The Swedish Heart and Brain Failure Investigation study (HARVEST-Malmo) is a prospective, on-going study undertaken in consecutive patients hospitalized for acute heart failure (either newly diagnosed or exacerbated chronic heart failure) in Malmö, Sweden. The only exclusion criterion was the inability to deliver consent. Baseline data including blood sample donations and clinical examination were collected between March 2014 and September 2018 in 324 subjects. Complete data was available in 295 patients. Data on one-year mortality (54 events), one-year cardiovascular-related mortality (44 events) and 30-day re-hospitalisation (61 events) was retrieved through national and regional registries. Selenoprotein P was measured upon admission, along with clinical examination.

Clinical Examination

[0162] Upon hospitalisation, fasting blood samples were drawn, blood pressure was measured and body mass index (BMI) was calculated as kilograms per square meter. Subjects' health status (symptoms, function, and quality of life) was evaluated using Kansas City Cardiomyopathy Questionnaire (KCCQ), a valid and reliable measure of health status in both heart failure with reduced ejection fraction and heart failure with preserved ejection fraction (Joseph, Novak et al. 2013) an instrument that has been validated in Swedish (Patel, Ekman et al. 2008). Prevalent diabetes was defined as prior physician diagnosis of type 1 or type 2 diabetes, or use of antidiabetic medication. Atrial fibrillation (AF) was defined as prior diagnosis of HF. Prior congestive heart failure was defined as prior hospitalisation for congestive heart failure, or a physician diagnosis of heart failure prior to inclusion in the study.

Laboratory Assays

[0163] Fasting N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed at the Department of Clinical Chemistry, Skåne University Hospital in Malmo, participating in a national standardisation and quality control system using a sandwich assay based on ElectroChemiLuminiscence Immunoassay (Cobas, Roche Diagnostic, Basel, Switzerland). As for analyses of Selenoprotein P, fasting blood samples were collected at admission in 4.5 ml EDTA-tubes and centrifuged at 1950 g for 10 minutes. Plasma was then aliquoted in 200 μl fractions into bar coded tubes (REMP, Brooks, Life Sciences, USA) and stored at −80° C. until analysis. Selenoprotein P was analysed with a validated ELISA immunoassay using monoclonal antibodies as described in Example 1.

Outcomes

[0164] KCCQ was used to quantify physical limitations, symptoms, self-efficacy, social interference and quality of life. An overall summary score <50 was considered as an indication of low health-related quality of life, whereas overall summary scores ≥50 are an indication of better health-related quality of life (Soto, Jones et al. 2004). Total one-year mortality was defined as all-cause mortality within one year from study inclusion, and obtained from Swedish total population register Statistics Sweden. Re-hospitalisation was defined as first of any unplanned readmissions for worsening heart failure within 30 days from study inclusion. A composite endpoint of death or re-hospitalisation, whichever came first, within 30 days from study inclusion was created.

Statistics

[0165] Prior to analyses, Selenoprotein P was normalized (z-standardised). NT-proBNP was the only variable with skewed distributions and therefore log transformed prior to analysis. Cross-sectional associations between Selenoprotein P and KCCQ were explored using logistic regression models, where the dependent variable KCCQ was dichotomized on <50 overall score points as a measure of low quality of life (higher points=better health-related quality of life) in a crude model, Model 1 (adjusted for age and sex) and Model 2 (further adjusted for BMI, systolic blood pressure (SBP), smoking, prevalent AF, prevalent diabetes, prior HF and log-transformed NT-proBNP). Cox regression models were carried out crude, in Model 1 (age and sex adjusted) and further adjusted for relevant risk factors in Model 2 (BMI, SBP, smoking, prevalent AF, prevalent diabetes, prior HF and log-transformed NT-proBNP) for one-year mortality, 30-day re-hospitalisation and a composite endpoint consisting of death or re-hospitalisation, whichever came first, within 30 days from study inclusion. Survival plots were computed using unadjusted Kaplan-Meier models. Length of hospital stay was analysed using crude linear regression models, adjusted for age and sex (Model 1) and further adjusted according to Model 2. All analyses were performed using IBM SPSS statistics version 25 (SPSS, Chicago, Ill.), except Harrell's C-statistics analyses that were performed using R 3.4.3.

[0166] A two-sided p-value <0.05 was considered statistically significant. Receiver operating curves (ROC) analysis was performed to determine threshold values with respective sensitivities and specificities.

Results

[0167] Baseline characteristics of the study population within quartiles of Selenoprotein P levels are presented in Table 1. Selenoprotein P was normally distributed in the population (median 3.4 mg/L).

Selenoprotein P and Quality of Life

[0168] Cross-sectional analyses of quality of life, based on a KCCQ overall score, revealed that each 1 SD increase of Selenoprotein P was associated with decreased risk of low health-related quality of life (n=47) defined as an overall summary score <50 in a crude model (OR 0.70; 95% CI 0.50-0.99, p=0.044), in Model 1 (OR 0.70; CI95% 0.50-0.99, p=0.043), and in fully adjusted Model 2 (OR 0.68; CI95% 0.47-0.97; p=0.035).

[0169] Due to disparities in sex between the lowest and the highest quartile of Selenoprotein P, an interaction analysis was performed. There was a significant interaction for sex, wherefore additional analyses were performed for each sex separately. Those revealed that the associations between Selenoprotein P levels and low health-related quality of life in the entire cohort were mainly driven by the male sex (n=205, 32 events, crude HR 0.67; CI 95% 0.45-0.99; p=0.048), whereas no significant associations were seen for females (n=89, 15 events, crude OR 0.82; CI 95% 0.40-1.67; p=0.581).

Selenoprotein P and One-Year Mortality

[0170] One-year mortality was higher in patients within the lowest quartile of Selenoprotein P (29.9%) as compared to patients with the highest levels of Selenoprotein P (8.8%). Selenoprotein P levels in relation to one-year mortality are illustrated in FIG. 1.

[0171] Cox regression analyses of one-year mortality are presented in Table 2, and reveal that each 1 SD increase in Selenoprotein P concentration was associated with lower risk of one-year mortality in crude analyses (HR 0.64; 95% CI 0.47-0.86; p=0.003), in Model 1 (HR 0.60; 95% CI 0.45-0.81, p=0.001), and further adjusted for BMI, SBP, smoking, prevalent AF, prevalent diabetes, log-transformed NT-proBNP and prior HF according to Model 2 (HR 0.65; 95% CI 0.48-0.88; p=0.005). C-index for Selenoprotein P was calculated to 0.628 (CI95% 0.553-0.703). Adding Selenoprotein P to the variables in Model 2 increases the (bootstrap-corrected) C-index from 0.736 to 0.751 (p for added value 0.004).

[0172] For better illustration, Selenoprotein P levels were divided into quartiles and related to one-year mortality (Table 3). Quartile analyses revealed that subjects in the quartile with lowest levels of Selenoprotein P (Q1) were at significantly higher risk of mortality within one year (HR 4.13; CI95% 1.64-10.4) as compared to subjects in Q4 (p for difference across quartiles 0.001) in fully adjusted Model 2. Kaplan Meier curves presenting survival within quartiles of Selenoprotein P are presented in FIG. 2.

[0173] Due to disparities in sex between the lowest and the highest quartile of Selenoprotein P, an interaction analysis was performed. There was a significant interaction for sex, wherefore additional analyses were performed for each sex separately. Those revealed that the associations between Selenoprotein P levels and mortality observed in the entire cohort were mainly driven by the male sex (n=208, 45 events, crude HR 0.60; CI 95% 0.44-0.82; p=0.001), whereas no significant associations were seen for females (n=92, 11 events, crude HR 0.72; CI 95% 0.35-1.52; p=0.391).

[0174] Exemplary threshold values for determining the risk of one-year mortality with respective sensitivities and specificities are shown in Table 4.

Selenoprotein P and Risk of 30-Day Re-Hospitalisation

[0175] Thirty-day re-hospitalisation rate was higher in patients within the lowest quartile of Selenoprotein P (28.6%) as compared to patients with the highest levels of Selenoprotein P (7.4%). Selenoprotein P levels in relation to 30-day re-hospitalisation are illustrated in FIG. 1.

[0176] Cox regression analyses of 30-day re-hospitalisation (n=61) are presented in Table 2, and reveal that each 1 SD increment in Selenoprotein P concentration was associated with lower risk of re-hospitalisation within 30 days from study inclusion in crude analyses (HR 0.66; 95% CI 0.50-0.87; p=0.003), in Model 1 (HR 0.67; 95% CI 0.51-0.88, p=0.004), and further adjusted according to Model 2 (HR 0.67; 95% CI 0.51-0.89; p=0.005). C-index for Selenoprotein P was calculated to 0.617 (CI95% 0.552-0.682). Adding Selenoprotein P to the variables in Model 2 increases the C-ndex from 0.567 to 0.627 (p for added value 0.004). Bootstrap corrected C-index for model 2 is 0.48 (as none of the other variables contributes to prediction, the penalty is large and leaves the C-index below 0.5). Adding Selenoprotein P increases the boostrap corrected C-index to 0.547 (still less than Selenoprotein P alone, due to the penalty of adding 9 variables that have no predictive power).

[0177] Additionally, Selenoprotein P levels were divided into quartiles and related to 30-day re-hospitalisation (Table 3). Quartile analyses revealed that subjects in the quartile with lowest levels of Selenoprotein P (Q1) were at significantly higher risk of re-hospitalisation within 30 days from study inclusion (HR 4.29; CI95% 1.59-11.6) as compared to subjects in Q4 (p for difference across quartiles 0.004) in fully adjusted Model 2. Kaplan Meier curves presenting re-hospitalisation within quartiles of Selenoprotein P are presented in FIG. 3.

[0178] Due to disparities in sex between the lowest and the highest quartile of Selenoprotein P, an interaction analysis was performed. There was a significant interaction for sex, wherefore additional analyses were performed for each sex separately. Those revealed that the associations between Selenoprotein P levels and 30-day re-hospitalisation observed in the entire cohort were mainly driven by the male sex (n=205, 39 events, crude HR 0.68; CI 95% 0.49-0.93; p=0.017), whereas no significant associations were seen for females (n=90, 22 events, crude HR 0.65; CI 95% 0.38-1.11; p=0.116).

[0179] Exemplary threshold values for determining the risk of 30-day re-hospitalisation with respective sensitivities and specificities are shown in Table 5.

[0180] Selenoprotein P and Composite Endpoint (Re-Hospitalisation or Death Within 30 Days)

[0181] Death or re-hospitalisation within 30 days from study inclusion was more frequent in patients within the lowest quartile of Selenoprotein P (32.4%) as compared to patients with the highest levels of Selenoprotein P (7.4%). Selenoprotein P levels in relation to the composite endpoint of death or re-hospitalisation are illustrated in FIG. 1.

[0182] Cox regression analyses of associations of Selenoprotein P and the composite endpoint (68 events) are presented in Table 2, and reveal that each 1 SD increase in Selenoprotein P concentration was associated with lower risk of either death or re-hospitalisation within 30 days in crude analyses (HR 0.64 CI95% 0.49-0.83, p=0.001), in Model 1 (HR 0.65; CI95% 0.50-0.85; p=0.001), and further adjusted for BMI, SBP, smoking, prevalent AF, prevalent diabetes, log-transformed NT-proBNP and prior HF according to Model 2 (HR 0.66; 0.51-0.86; p=0.002). C-index for Selenoprotein P was calculated to 0.622 (CI95% 0.562-0.681). Adding Selenoprotein P to the variables in Model 2 increases the C-index from 0.584 to 0.632 (p for added value 0.002). Bootstrap corrected C-index for Model 2 is 0.507 (as none of the variables contributes to prediction). Adding Selenoprotein P increases the boostrap corrected C-index to 0.561 (still less than Selenoprotein P alone, due to the penalty of adding 9 variables that have no predictive power).

[0183] Further, Selenoprotein P levels were divided into quartiles and related to death or re-hospitalisation within 30 days (Table 3). Quartile analyses revealed that subjects in the quartile with lowest levels of Selenoprotein P (Q1) were at significantly higher risk of death or re-hospitalisation within 30 days (HR 4.80; CI95% 1.80-12.8) as compared to subjects in Q4 (p for difference across quartiles <0.002) in fully adjusted Model 2. Kaplan Meier curves presenting survival within quartiles of Selenoprotein P are presented in FIG. 4.

[0184] Distribution of Selenoprotein P within groups based on outcomes [A) no re-hospitalisation/survivor; B) re-hospitalised/survivor; C) no re-hospitalisation/deceased and D) re-hospitalised and deceased] is presented in FIG. 1.

Hospital Stay

[0185] Further analyses were carried out for Selenoprotein P and length of hospital stay, where each 1 SD increase in Selenoprotein P levels was associated with shorter hospital stay in crude analyses (β−0.95, p<0.001), in Model 1 (β−1.04, p<0.001), and Model 2 (β−0.96, p<0.001).

Discussion

[0186] This prospective study demonstrates that low plasma levels of Selenoprotein P are associated with lower health-related quality of life, higher one-year mortality risk, higher risk of 30-day readmission, and longer hospital stay upon admission for newly diagnosed or worsening acute heart failure. The prevalence of congestive heart failure, a common outcome to the majority of cardiac diseases, is steadily increasing worldwide, presumptively due to improved congestive heart failure survival and the ageing of the population (Savarese and Lund 2017).

[0187] The poor prognosis (Ponikowski, Voors et al. 2016), low quality of life (Hobbs, Kenkre et al. 2002) and the economic burden (Writing Group, Mozaffarian et al. 2016) posed to the society by the unplanned re-hospitalisations for worsening congestive heart failure make optimization of treatment of heart failure a top health priority. Up to date, no studies examining the associations of selenium deficiency (measured as low circulating levels of Selenoprotein P) and outcomes such as mortality and re-hospitalisation in a heart failure population have been presented.

[0188] Amongst 25 selenoproteins, Selenoprotein P has been suggested to act as a selenium transporter and to be essential in selenium metabolism and storage (Saito and Takahashi 2002, Labunskyy, Lee et al. 2011). In humans, levels of Selenoprotein P correlate with serum selenium levels (Andoh, Hirashima et al. 2005), and are usable as an index of the selenium nutritional status (Burk and Hill 2009). Selenium is an essential trace element that is involved in the control of the cell reduction-oxidation status and the immune system (McKenzie, Rafferty et al. 1998, Arthur, McKenzie et al. 2003, Huang, Rose et al. 2012), and is recognized as vital for the body's antioxidant defense mechanisms (Ahrens, Ellwanger et al. 2008). Increased oxidative stress has been proposed to contribute to pathogenesis of congestive heart failure (Givertz and Colucci 1998, Keith, Geranmayegan et al. 1998, Mallat, Philip et al. 1998, Singal, Khaper et al. 1998, Munzel and Harrison 1999, de Lorgeril and Salen 2006), and involvement of selenium in the protection from oxidative damage has been demonstrated in the cardiovascular system (Blankenberg, Rupprecht et al. 2003, Akbaraly, Arnaud et al. 2005, Ray, Semba et al. 2006, Joseph and Loscalzo 2013). As early as 1982, associations between low serum selenium levels and myocardial infarction and cardiovascular death were observed (Salonen, Alfthan et al. 1982). However, serum selenium is most likely a poor measure of the selenium status in the human body, whereas Selenoprotein P has been demonstrated to be valid biomarker of selenium status (Ashton, Hooper et al. 2009). In humans, Selenoprotein P has been demonstrated to be elevated in type 2 diabetes or prediabetes, as well as in overweight and obese subjects (Yang, Hwang et al. 2011) and Selenoprotein P expression levels have been shown to be severely up-regulated in subjects with type 2 diabetes (Misu, Takamura et al. 2010). No study has been able to conclude whether Selenoprotein P elevation in diabetes and prediabetes is a risk factor or a compensatory mechanism, given the fact that diabetes and insulin resistance are states of low-grade inflammation and oxidative stress. All of our analyses were adjusted for diabetes, implicating that associations of Selenoprotein P and low quality of life, one-year mortality and re-hospitalisation are independent of diabetes status.

[0189] In cardiovascular disease, selenium deficiency led to a larger myocardial injury after myocardial ischemia-reperfusion in rats (Venardos, Harrison et al. 2004), data in line with findings in other studies (Pucheu, Coudray et al. 1995, Toufektsian, Boucher et al. 2000, Tanguy, Toufektsian et al. 2003). Moreover, rats that received high selenium intake showed reduced infarct size, improved functional recovery of the heart and decreased incidence of ventricular arrhythmias (Tanguy, Boucher et al. 1998, Tanguy, Morel et al. 2004, Rakotovao, Tanguy et al. 2005, Tanguy, Rakotovao et al. 2011). Those findings might serve as plausible explanations to the findings in our study, due to the fact that the majority of all heart failure cases (>50% in the United States of America) (Gheorghiade and Bonow 1998)—primarily in males—are caused by underlying coronary artery disease. In our cohort, we lacked complete data on the subjects' heart failure etiology, and could thus not analyze the associations of selenium deficiency with different predisposing etiological causes.

[0190] In analyses of Selenoprotein P association with risk of one-year mortality, as well as 30-day re-hospitalisation and quality of life measured by KCCQ, an interaction with sex was observed. Sensitivity analyses were then performed for each sex separately, revealing that the associations observed were mainly driven by the male subjects. Nevertheless, those data need to be interpreted with great caution, in view of the lower event rate among females.

[0191] Although research has identified extensive selenium dependent functions in the human body, the role of selenium supplementation in cardiovascular disease remains uncertain (Flores-Mateo, Navas-Acien et al. 2006, Rees, Hartley et al. 2013). Up to date, there are no studies on effects of selenium supplementation on outcome in a population with acute heart failure, with Keshans disease as the only exception (McKeag, McKinley et al. 2012). Our findings urge for studies exploring effects of selenium supplementation on outcomes in heart failure.

[0192] There are both strengths and limitations to this study. As we consecutively included patients admitted for new or worsening heart failure with inability to deliver consent to the study as only exclusion criteria, we most probably mimicked a representative heart failure population.

[0193] All analyses were adjusted for clinically relevant risk factors, so we believe that the data demonstrate that selenium deficiency is prognostic of poor outcome in a congestive heart failure setting. Our data was collected at a single regional center, which limits the applicability to other population. Moreover, out sample size was relatively small and the results need to be replicated in larger cohorts. Also, the subjects included in HARVEST-Malmo were at mainly Swedish descent, and the conclusions drawn might not be generalizable to all ancestries.

CONCLUSION

[0194] This study identifies Selenoprotein P as a novel marker of poor outcome in AHF and encourages future studies examining if supplementation of selenium might improve prognosis in CAHF-patients.

TABLE-US-00001 TABLE 1 Characteristics of the study population within quartiles of Selenoprotein P Total Q1 Q2 Q3 Q4 population 2.0 (0.8-2.3) 2.6 (2.3-3.0) 3.4 (3.0-3.8) 4.4 (3.8-6.9) n = 295 n = 77 n = 71 n = 79 n = 68 p Age (years) 74.4 ± 11.5 75.6 ± 10.5 73.8 ± 12.7 76.3 ± 10.9 73.0 ± 11.7 0.254 Sex (female; n (%))  90 (30.5)  29 (37.7)  27 (38.0)  22 (27.8)  12 (17.6) 0.025 BMI (kg/m.sup.2) 27.8 ± 6.0 27.6 ± 6.3 28.6 ± 7.2 27.0 ± 4.7 28.1 ± 5.5 0.425 SBP (mmHg)  136 ± 27  133 ± 23  138 ± 29  139 ± 25  136 ± 30 0.639 Smoking (n (%))  37 (12.5)   9 (11.7)  10 (14.1)  11 (13.9)   7 (10.3) 0.885 Prevalent AF (n (%))  143 (48.5)  37 (48.1)  37 (52.1)  41 (51.9)  28 (41.2) 0.535 Prevalent diabetes (n (%))  109 (36.9)  29 (37.7)  22 (31.0)  29 (36.7)  29 (42.6) 0.566 Prior CHF (n (%))  194 (65.8)  56 (72.7)  47 (66.2)  51 (64.6)  40 (58.8) 0.369 SePP (mg/L)  3.1 ± 1.1  1.8 ± 0.4  2.6 ± 0.2  3.4 ± 0.2  4.7 ± 0.7 <0.001 NT-proBNP pg/mL 4096 (2234- 4682 (2452- 3768 (2378- 4794 (2336- 3118 (1795- 0.181 8645) 11169) 8862) 7892) 6200) KCCQ score (<50 points  47 (15.9)  13 (15.6)  19 (26.8)   5 (6.3)  10 (14.7) 0.008 (%)) One-year mortality (n (%))  54 (18.3)  23 (29.9)  12 (16.9)  13 (16.5)   6 (8.8) 0.010 Re-hospitalisation (n (%))  61 (20.7)  22 (28.6)  18 (25.4)  16 (20.3)   5 (7.4) 0.010 Composite endpoint  68 (23.1)  25 (32.5)  20 (28.2)  18 (22.7)   5 (7.4) 0.001 (n (%)) Hospital stay (days)   7 (4-9)   8 (5-10)   7 (4-9)   6 (4-8)   6 (4-8) 0.002 Values are means ± standard deviations (SD) or medians (interquartile range (25-75)) in the whole population and within quartiles of Selenoprotein P. BMI = body mass index; KCCQ = Kansas City Cardiomyopathy Questionnaire; NT-proBNP = N-terminal prohormone of brain natriuretic peptide; SBP = systolic blood pressure; AF = atrial fibrillation; CHF = congestive heart failure; SePP = Selenoprotein P. Q1 = quartile with lowest SePP levels; Q4 = quartile with highest SePP levels.

TABLE-US-00002 TABLE 2 Associations of Selenoprotein P and risk of one-year mortality, 30-day re-hospitalisation and major adverse outcomes One-year mortality 30-day re-hospitalisation Composite endpoint (n = 54) (n = 61) (n = 98) HR (CI95%) p HR (CI95%) p HR (CI95%) p CRUDE SePP 0.64 (0.47-0.86) 0.003 0.66 (0.50-0.87) 0.003 0.65 (0.50-0.83) 0.001 MODEL 1 Age 1.06 (1.03-1.09) <0.001 1.01 (0.98-1.03) 0.540 1.01 (0.99-1.03) 0.336 Sex 0.38 (0.19-0.74) 0.004 1.13 (0.66-1.93) 0.655 1.23 (0.75-2.04) 0.523 SePP 0.60 (0.45-0.81) 0.001 0.67 (0.51-0.88) 0.004 0.65 (0.50-0.85) 0.001 MODEL 2 Age 1.07 (1.04-1.11) <0.001 1.01 (0.98-1.04) 0.524 1.02 (0.99-1.054 0.248 Sex 0.41 (0.21-0.82) 0.012 1.14 (0.65-1.99) 0.644 1.21 (0.72-2.05) 0.471 BMI 0.99 (0.93-1.06) 0.881 0.99 (0.94-1.05) 0.779 1.01 (0.96-1.05) 0.803 SBP 0.98 (0.97-0.99) 0.001 1.00 (0.99-1.01) 0.462 0.99 (0.98-1.00) 0.217 Smoking 1.33 (0.51-3.50) 0.562 1.03 (0.47-2.25) 0.943 1.09 (0.52-2.23) 0.820 Prevalent AF 0.59 (0.33-1.03) 0.063 1.05 (0.63-1.76) 0.852 0.99 (0.61-1.63) 0.985 Prevalent diabetes 1.76 (0.96-3.23) 0.068 0.97 (0.54-1.74) 0.925 0.99 (0.57-1.72) 0.204 Prior CHF 1.02 (0.51-2.06) 0.948 1.25 (0.69-2.25) 0.462 1.21 (0.69-2.11) 0.697 NT-proBNP 1.46 (1.07-1.98) 0.017 0.92 (0.70-1.19) 0.516 0.96 (0.75-1.23) 0.743 SePP 0.65 (0.48-0.88) 0.005 0.67 (0.51-0.89) 0.005 0.66 (0.51-0.86) 0.002 Values are hazard ratios (HR) and 95% confidence intervals. BMI = body mass index; SBP = systolic blood pressure; AF = atrial fibrillation; CHF = congestive heart failure, SePP = Selenoprotein P. The composite endpoint is defined as death or re-hospitalisation within 30 days from study inclusion, whichever came first. Model 1 is adjusted for age and sex. Model 2 is adjusted for age, sex, body mass index, systolic blood pressure, log-transformed NT-proBNP, smoking, prevalent atrial fibrillation, prevalent diabetes and prior CHF.

TABLE-US-00003 TABLE 3 Quartile analyses of Selenoprotein P in relation to one-year mortality and 30-day re-hospitalisation One-year 30-day re- Composite mortality hospitalisation endpoint HR (CI95%) HR (CI95%) HR (CI95%) Crude Q1 3.94 (1.60-9.67) 4.35 (1.65-11.5) 5.06 (1.94-13.2) Q2 2.04 (0.76-5.42) 3.90 (1.44-10.5) 4.45 (1.67-11.9) Q3 1.95 (0.74-5.12) 2.97 (1.09-8.10) 3.43 (1.27-9.24) Q4 Referent Referent Referent p for trend 0.001 0.002 0.001 Model 1 Q1 4.66 (1.88-11.5) 4.22 (1.59-11.2) 4.80 (1.83-12.6) Q2 2.26 (0.84-6.03) 3.80 (1.40-10.3) 4.26 (1.59-11.4) Q3 1.77 (0.67-4.67) 2.89 (1.05-7.89) 3.29 (1.22-8.89) Q4 Referent Referent Referent p for trend <0.001  0.003 0.001 Model 2 Q1 4.13 (1.64-10.43) 4.29 (1.59-11.6) 4.80 (1.80-12.8) Q2 2.07 (0.76-5.63) 3.87 (1.42-10.6) 4.33 (1.60-11.7) Q3 1.79 (0.67-4.81) 2.97 (1.07-8.22) 3.37 (1.24-9.20) Q4 Referent Referent Referent p for trend 0.001 0.004 0.002 Values are hazard ratios (HR) and 95% confidence intervals (95% CI) for quartiles of Selenoprotein P in relation to mortality within one year. Q1 = quartile with lowest levels of Selenoprotein P; Q4 = quartile with highest levels of Selenoprotein P. Model 1 is adjusted for age and sex. Model 2 is adjusted for age, sex, body mass index, systolic blood pressure, log-transformed NT-proBNP, smoking, prevalent atrial fibrillation, prevalent diabetes and prior CHF. Selenoprotein P levels within quartiles: Q1 (1.8 ± 0.4); Q2 (2.6 ± 0.2); Q3 (3.4 ± 0.2) Q4 (4.7 ± 0.8).

TABLE-US-00004 TABLE 4 Receiver operating curve (ROC) characteristics of Selenoprotein P thresholds for one-year mortality with respective sensitivities and specificities SePP (mg/L) Specificity (in %) Sensitivity (in %) 2.0 87.6 27.8 2.3 77.6 42.6 2.6 62.7 50.0 3.0 53.1 64.8 3.3 43.2 79.6 3.8 25.7 88.9 4.4 14.5 90.7

TABLE-US-00005 TABLE 5 Receiver operating curve (ROC) characteristics of Selenoprotein P thresholds for 30-days re-hospitalisation with respective sensitivities and specificities SePP (mg/L) Specificity (in %) Sensitivity (in %) 2.0 87.2 24.6 2.3 76.5 36.1 2.6 63.2 50.8 3.0 53.8 65.6 3.3 43.2 77.0 3.8 26.9 91.8 4.4 12.4 95.1

Example 3: MPP-Study

Study Description

[0195] The population-based Malmo Preventive Project (MPP) is a Swedish single-center prospective population-based study. Between 1974 and 1992, a total of 33,346 men and women of the homogenous ethnic background from the Malmo city area were recruited and screened for traditional risk factors of all-cause mortality and cardiovascular disease (CVD). A detailed description of baseline procedures may be found elsewhere (Fedorowski et al. 2010. Eur Heart J 31: 85-91; Berglund et al. 1996. J Intern Med 239: 489-97). In the years 2002-2006, all survivors from the original MPP cohort were invited for a reexamination. Of these, 18,240 participants (n=6,682 women) responded to the invitation and were reexamined including blood sampling and immediate −80° C. storage of EDTA plasma aliquots. The reexamination in 2002-2006 represents the baseline time point in the current study.

[0196] The 5060 of 18240 subjects tested for Selenoprotein P is a random sample (mean age 69 years). 4366 subjects were free from prior CVD (myocardial infarction, stroke and coronary re-vascularizations). Mean follow-up time of patients was 9.3 years, with deaths (n=1111), CVD deaths (n=351) and first CVD event (n=745). Selenoprotein P was measured with a validated ELISA immunoassay using monoclonal antibodies as described in Example 1. Baseline characteristics of the cohort are shown in table 6.

TABLE-US-00006 TABLE 6 Baseline characteristics of the MPP study population Variable n = 4366 Age 69.4 (6.2) gender male 3008 (68.9%) Current Smoking 835 (19.1%) AHT 1476 (33.8%) HDL 1.4 (0.4) LDL 3.7 (1.0) BMI 27.1 (6.2) SBP 146.6 (20.3) prevalent Diabetes 466 (10.7%) Deaths 1111 (25.4%) CVD Deaths 351 (8%) first CVD event 745 (17.1%) SePP (mg/L) 5.5 (range 0.4-20.0)

[0197] During a median (interquartile range) follow-up time of 9.3 (8.3-11) years, a total of 1111 deaths occurred. The largest number of deaths was observed in Selenoprotein P quintile 1 (n=314; 3.7 mg/L with a range between 0.4 and 4.3 mg/L). Similar patterns were observed for the endpoint analyses of cardiovascular mortality (351 events) and risk of a first cardiovascular event (745 events), respectively, with significantly higher risk in Selenoprotein P quintile 1.

[0198] The frequency distribution of Selenoprotein P in this healthy population ranges from 0.4 to 20.0 mg/L with a median concentration of 5.5 mg/L (FIG. 5A). Threshold ranges of Selenoprotein P to assess the risk of healthy subjects for getting a first cardiovascular event or cardiovascular mortality are between 4.0 and 5.5 mg/L. When compared to the healthy population from MPP, the Selenoprotein P concentration of the heart failure population (HARVEST study), is a much lower concentration ranging between 0.8 and 6.9 mg/L and a median of 3.0 mg/L, where the majority of values are well below a threshold for healthy subjects (e.g. 97.3% of heart failure patients are below 5.5 mg/L and 79.7% of heart failure patients are below 4.0 mg/L) (FIG. 5B). Heart failure patients have Selenoprotein P concentrations that are compareable to healthy patients having a risk of getting a cardiovascular event, as those patients have already suffered a cardiovascular event (namely heart failure). Surprisingly, and according to the present invention the low Selenoprotein P concentrations in heart failure patients can further be divided into subgroups, whereas Selenoprotein P concentrations at the lower end of the distribution in heart failure patients have a higher risk of e.g. worsening heart failure or rehospitalization due to heart failure or mortality according to the present invention (see Example 2).

TABLE-US-00007 SEQUENCE LISTING SEQ ID NO. 1: Selenoprotein P including signal sequence (amino acid 1 to 381) MWRSLGLALA LCLLPSGGTE SQDQSSLCKQ PPAWSIRDQD PMLNSNGSVT VVALLQASUY LCILQASKLE DLRVKLKKEG YSNISYIVVN HQGISSRLKY THLKNKVSEH IPVYQQEENQ TDVWTLLNGS KDDFLIYDRC GRLVYHLGLP FSFLTFPYVE EAIKIAYCEK KCGNCSLTTL KDEDFCKRVS LATVDKTVET PSPHYHHEHH HNHGHQHLGS SELSENQQPG APNAPTHPAP PGLHHHHKHK GQHRQGHPEN RDMPASEDLQ DLQKKLCRKR CINQLLCKLP TDSELAPRSU CCHCRHLIFE KTGSAITUQC KENLPSLCSU QGLRAEENIT ESCQURLPPA AUQISQQLIP TEASASURUK NQAKKUEUPS N SEQ ID NO. 2: secreted Selenoprotein P (amino acid 20 to 381) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCCHCRHLIF EKTGSAITUQ CKENLPSLCS UQGLRAEENI TESCQURLPP AAUQISQQLI PTEASASURU KNQAKKUEUP SN SEQ ID NO. 3: Selenoprotein P (amino acid 20 to 346) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCCHCRHLIF EKTGSAITUQ CKENLPSLCS UQGLRAEENI TESCQUR SEQ ID NO. 4: Selenoprotein P (amino acid 20 to 298) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPR SEQ ID NO. 5: Selenoprotein P (amino acid 20 to 299) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS SEQ ID NO. 6: Selenoprotein P (amino acid 20 to 300) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS U SEQ ID NO. 7: Selenoprotein P (amino acid 20 to 301) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UC SEQ ID NO. 8: Selenoprotein P (amino acid 20 to 302) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCC SEQ ID NO. 9: Selenoprotein P (amino acid 20 to 303) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCCH SEQ ID NO. 10: Selenoprotein P (amino acid 20 to 304) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCCHC SEQ ID NO. 11: Selenoprotein P (amino acid 20 to 305) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCCHCR SEQ ID NO. 12: Selenoprotein P (amino acid 20 to 306) ESQDQSSLCK QPPAWSIRDQ DPMLNSNGSV TVVALLQASU YLCILQASKL EDLRVKLKKE GYSNISYIVV NHQGISSRLK YTHLKNKVSE HIPVYQQEEN QTDVWTLLNG SKDDFLIYDR CGRLVYHLGL PFSFLTFPYV EEAIKIAYCE KKCGNCSLTT LKDEDFCKRV SLATVDKTVE TPSPHYHHEH HHNHGHQHLG SSELSENQQP GAPNAPTHPA PPGLHHHHKH KGQHRQGHPE NRDMPASEDL QDLQKKLCRK RCINQLLCKL PTDSELAPRS UCCHCRH SEQ ID NO. 13: Selenoprotein P (amino acid 1 to 235) MWRSLGLALA LCLLPSGGTE SQDQSSLCKQ PPAWSIRDQD PMLNSNGSVT VVALLQASUY LCILQASKLE DLRVKLKKEG YSNISYIVVN HQGISSRLKY THLKNKVSEH IPVYQQEENQ TDVWTLLNGS KDDFLIYDRC GRLVYHLGLP FSFLTFPYVE EAIKIAYCEK KCGNCSLTTL KDEDFCKRVS LATVDKTVET PSPHYHHEHH HNHGHQHLGS SELSENQQPG APNAP SEQ ID NO. 14: Selenoprotein P (amino acid 279 to 381) KRCINQLLCK LPTDSELAPR SUCCHCRHLI FEKTGSAITU QCKENLPSLC SUQGLRAEEN ITESCQURLP PAAUQISQQL IPTEASASUR UKNQAKKUEU PSN SEQ ID NO. 15: Selenoprotein P (amino acid 312 to 381) TGSAITUQCK ENLPSLCSUQ GLRAEENITE SCQURLPPAA UQISQQLIPT EASASURUKN QAKKUEUPSN

LITERATURE

[0199] Ahrens, I., C. Ellwanger, B. K. Smith, N. Bassler, Y. C. Chen, I. Neudorfer, A. Ludwig, C. Bode and K. Peter (2008). “Selenium supplementation induces metalloproteinase-dependent L-selectin shedding from monocytes.” J Leukoc Biol 83(6): 1388-1395. [0200] Akbaraly, N. T., J. Arnaud, I. Hininger-Favier, V. Gourlet, A. M. Roussel and C. Berr (2005). “Selenium and mortality in the elderly: results from the EVA study.” Clin Chem 51(11): 2117-2123. [0201] Akesson, B., T. Bellew and R. F. Burk (1994). “Purification of selenoprotein P from human plasma.” Biochim Biophys Acta 1204(2): 243-249. [0202] Alehagen, U., J. Aaseth and P. Johansson (2015). “Reduced Cardiovascular Mortality 10 Years after Supplementation with Selenium and Coenzyme Q10 for Four Years: Follow-Up Results of a Prospective Randomized Double-Blind Placebo-Controlled Trial in Elderly Citizens.” PLoS One 10(12): e0141641. [0203] Alehagen, U., P. Johansson, M. Bjornstedt, A. Rosen and U. Dahlstrom (2013). “Cardiovascular mortality and N-terminal-proBNP reduced after combined selenium and coenzyme Q10 supplementation: a 5-year prospective randomized double-blind placebo-controlled trial among elderly Swedish citizens.” Int J Cardiol 167(5): 1860-1866. [0204] Andoh, A., M. Hirashima, H. Maeda, K. Hata, O. Inatomi, T. Tsujikawa, M. Sasaki, K. Takahashi and Y. Fujiyama (2005). “Serum selenoprotein-P levels in patients with inflammatory bowel disease.” Nutrition 21(5): 574-579. [0205] Arroyo, M., S. P. Laguardia, S. K. Bhattacharya, M. D. Nelson, P. L. Johnson, L. D. Carbone, K. P. Newman and K. T. Weber (2006). “Micronutrients in African-Americans with decompensated and compensated heart failure.” Transl Res 148(6): 301-308. [0206] Arthur, J. R., R. C. McKenzie and G. J. Beckett (2003). “Selenium in the immune system.” J Nutr 133(5 Suppl 1): 1457S-1459S. [0207] Ashton, K., L. Hooper, L. J. Harvey, R. Hurst, A. Casgrain and S. J. Fairweather-Tait (2009). “Methods of assessment of selenium status in humans: a systematic review.” Am J Clin Nutr 89(6): 2025S-2039S. [0208] Ballihaut, G., L. E. Kilpatrick, E. L. Kilpatrick and W. C. Davis (2012). “Multiple forms of selenoprotein P in a candidate human plasma standard reference material.” Metallomics 4(6): 533-538. [0209] Blankenberg, S., H. J. Rupprecht, C. Bickel, M. Torzewski, G. Hafner, L. Tiret, M. Smieja, F. Cambien, J. Meyer, K. J. Lackner and I. AtheroGene (2003). “Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease.” N Engl J Med 349(17): 1605-1613. [0210] Bleys, J., A. Navas-Acien and E. Guallar (2008). “Serum selenium levels and all-cause, cancer, and cardiovascular mortality among US adults.” Arch Intern Med 168(4): 404-410. [0211] Burk, R. F. and K. E. Hill (2009). “Selenoprotein P-expression, functions, and roles in mammals.” Biochim Biophys Acta 1790(11): 1441-1447. [0212] Burk, R. F., B. K. Norsworthy, K. E. Hill, A. K. Motley and D. W. Byrne (2006). “Effects of chemical form of selenium on plasma biomarkers in a high-dose human supplementation trial.” Cancer Epidemiol Biomarkers Prev 15(4): 804-810. [0213] Chen, M., B. Liu, D. Wilkinson, A. T. Hutchison, C. H. Thompson, G. A. Wittert and L. K. Heilbronn (2017). “Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance.” Obes Res Clin Pract 11(2): 227-232. [0214] Clark, A. L., M. Cherif, T. A. McDonagh and I. B. Squire (2018). “In-hospital worsening heart failure: a clinically relevant endpoint?” ESC Heart Fail 5(1): 9-18. [0215] de Lorgeril, M. and P. Salen (2006). “Selenium and antioxidant defenses as major mediators in the development of chronic heart failure.” Heart Fail Rev 11(1): 13-17. [0216] Flores-Mateo, G., A. Navas-Acien, R. Pastor-Barriuso and E. Guallar (2006). “Selenium and coronary heart disease: a meta-analysis.” Am J Clin Nutr 84(4): 762-773. [0217] Ghaemian, A., E. Salehifar, H. Shiraj and Z. Babaee (2012). “A Comparison of Selenium Concentrations between Congestive Heart Failure Patients and Healthy Volunteers.” J Tehran Heart Cent 7(2): 53-57. [0218] Gharipour, M., M. Sadeghi, M. Salehi, M. Behmanesh, E. Khosravi, M. Dianatkhah, S. Haghjoo Javanmard, R. Razavi and A. Gharipour (2017). “Association of expression of selenoprotein P in mRNA and protein levels with metabolic syndrome in subjects with cardiovascular disease: Results of the Selenegene study.” J Gene Med 19(3). [0219] Gheorghiade, M. and R. O. Bonow (1998). “Chronic heart failure in the United States: a manifestation of coronary artery disease.” Circulation 97(3): 282-289. [0220] Givertz, M. M. and W. S. Colucci (1998). “New targets for heart-failure therapy: endothelin, inflammatory cytokines, and oxidative stress.” Lancet 352 Suppl 1: SI34-38. [0221] Hill, K. E., R. S. Lloyd and R. F. Burk (1993). “Conserved nucleotide sequences in the open reading frame and 3′ untranslated region of selenoprotein P mRNA.” Proc Natl Acad Sci USA 90(2): 537-541. [0222] Hill, K. E., Y. Xia, B. Akesson, M. E. Boeglin and R. F. Burk (1996). “Selenoprotein P concentration in plasma is an index of selenium status in selenium-deficient and selenium-supplemented Chinese subjects.” J Nutr 126(1): 138-145. [0223] Hill, K. E., J. Zhou, L. M. Austin, A. K. Motley, A. J. Ham, G. E. Olson, J. F. Atkins, R. F. Gesteland and R. F. Burk (2007). “The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium.” J Biol Chem 282(15): 10972-10980. [0224] Himeno, S., H. S. Chittum and R. F. Burk (1996). “Isoforms of selenoprotein P in rat plasma. Evidence for a full-length form and another form that terminates at the second UGA in the open reading frame.” J Biol Chem 271(26): 15769-15775. [0225] Hirashima, M., T. Naruse, H. Maeda, C. Nozaki, Y. Saito and K. Takahashi (2003). “Identification of selenoprotein P fragments as a cell-death inhibitory factor.” Biol Pharm Bull 26(6): 794-798. [0226] Hobbs, F. D., J. E. Kenkre, A. K. Roalfe, R. C. Davis, R. Hare and M. K. Davies (2002). “Impact of heart failure and left ventricular systolic dysfunction on quality of life: a cross-sectional study comparing common chronic cardiac and medical disorders and a representative adult population.” Eur Heart J 23(23): 1867-1876. [0227] Hollenbach, B., N. G. Morgenthaler, J. Struck, C. Alonso, A. Bergmann, J. Kohrle and L. Schomburg (2008). “New assay for the measurement of selenoprotein P as a sepsis biomarker from serum.” J Trace Elem Med Biol 22(1): 24-32. [0228] Huang, Z., A. H. Rose and P. R. Hoffmann (2012). “The role of selenium in inflammation and immunity: from molecular mechanisms to therapeutic opportunities.” Antioxidants & redox signaling 16(7): 705-743. [0229] Hybsier, S., T. Schulz, Z. Wu, I. Demuth, W. B. Minich, K. Renko, E. Rijntjes, J. Kohrle, C. J. Strasburger, E. Steinhagen-Thiessen and L. Schomburg (2017). “Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P.” Redox Biol 11: 403-414. [0230] Joseph, J. and J. Loscalzo (2013). “Selenistasis: epistatic effects of selenium on cardiovascular phenotype.” Nutrients 5(2): 340-358. [0231] Joseph, S. M., E. Novak, S. V. Arnold, P. G. Jones, H. Khattak, A. E. Platts, V. G. Davila-Roman, D. L. Mann and J. A. Spertus (2013). “Comparable performance of the Kansas City Cardiomyopathy Questionnaire in patients with heart failure with preserved and reduced ejection fraction.” Circ Heart Fail 6(6): 1139-1146. [0232] Keith, M., A. Geranmayegan, M. J. Sole, R. Kurian, A. Robinson, A. S. Omran and K. N. Jeejeebhoy (1998). “Increased oxidative stress in patients with congestive heart failure.” J Am Coll Cardiol 31(6): 1352-1356. [0233] Kosar, F., I. Sahin, C. Taskapan, Z. Kucukbay, H. Gullu, H. Taskapan and S. Cehreli (2006). “Trace element status (Se, Zn, Cu) in heart failure.” Anadolu Kardiyol Derg 6(3): 216-220. [0234] Labunskyy, V. M., B. C. Lee, D. E. Handy, J. Loscalzo, D. L. Hatfield and V. N. Gladyshev (2011). “Both maximal expression of selenoproteins and selenoprotein deficiency can promote development of type 2 diabetes-like phenotype in mice.” Antioxid Redox Signal 14(12): 2327-2336. [0235] Liu, H., H. Xu and K. Huang (2017). “Selenium in the prevention of atherosclerosis and its underlying mechanisms.” Metallomics 9(1): 21-37. [0236] Lubos, E., C. R. Sinning, R. B. Schnabel, P. S. Wild, T. Zeller, H. J. Rupprecht, C. Bickel, K. J. Lackner, D. Peetz, J. Loscalzo, T. Munzel and S. Blankenberg (2010). “Serum selenium and prognosis in cardiovascular disease: results from the AtheroGene study.” Atherosclerosis 209(1): 271-277. [0237] Ma, S., K. E. Hill, R. M. Caprioli and R. F. Burk (2002). “Mass spectrometric characterization of full-length rat selenoprotein P and three isoforms shortened at the C terminus. Evidence that three UGA codons in the mRNA open reading frame have alternative functions of specifying selenocysteine insertion or translation termination.” J Biol Chem 277(15): 12749-12754. [0238] Mallat, Z., I. Philip, M. Lebret, D. Chatel, J. Maclouf and A. Tedgui (1998). “Elevated levels of 8-iso-prostaglandin F2alpha in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure.” Circulation 97(16): 1536-1539. [0239] McKeag, N. A., M. C. McKinley, J. V. Woodside, M. T. Harbinson and P. P. McKeown (2012). “The role of micronutrients in heart failure.” J Acad Nutr Diet 112(6): 870-886. [0240] McKenzie, R. C., T. S. Rafferty and G. J. Beckett (1998). “Selenium: an essential element for immune function.” Immunol Today 19(8): 342-345. [0241] Meplan, C., L. K. Crosley, F. Nicol, G. J. Beckett, A. F. Howie, K. E. Hill, G. Horgan, J. C. Mathers, J. R. Arthur and J. E. Hesketh (2007). “Genetic polymorphisms in the human selenoprotein P gene determine the response of selenoprotein markers to selenium supplementation in a gender-specific manner (the SELGEN study).” Faseb j 21(12): 3063-3074. [0242] Misu, H., T. Takamura, H. Takayama, H. Hayashi, N. Matsuzawa-Nagata, S. Kurita, K. Ishikura, H. Ando, Y. Takeshita, T. Ota, M. Sakurai, T. Yamashita, E. Mizukoshi, T. Yamashita, M. Honda, K. Miyamoto, T. Kubota, N. Kubota, T. Kadowaki, H. J. Kim, I. K. Lee, Y. Minokoshi, Y. Saito, K. Takahashi, Y. Yamada, N. Takakura and S. Kaneko (2010). “A liver-derived secretory protein, selenoprotein P, causes insulin resistance.” Cell Metab 12(5): 483-495. [0243] Munzel, T. and D. G. Harrison (1999). “Increased superoxide in heart failure: a biochemical baroreflex gone awry.” Circulation 100(3): 216-218. [0244] Patel, H., I. Ekman, J. A. Spertus, S. M. Wasserman and L. O. Persson (2008). “Psychometric properties of a Swedish version of the Kansas City Cardiomyopathy Questionnaire in a Chronic Heart Failure population.” Eur J Cardiovasc Nurs 7(3): 214-221. [0245] Ponikowski, P., A. A. Voors, S. D. Anker, H. Bueno, J. G. F. Cleland, A. J. S. Coats, V. Falk, J. R. Gonzalez-Juanatey, V. P. Harjola, E. A. Jankowska, M. Jessup, C. Linde, P. Nihoyannopoulos, J. T. Parissis, B. Pieske, J. P. Riley, G. M. C. Rosano, L. M. Ruilope, F. Ruschitzka, F. H. Rutten, P. van der Meer and E. S. C. S. D. Group (2016). “2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) Developed with the special contribution of the Heart Failure Association (HFA) of the ESC.” Eur Heart J 37(27): 2129-2200. [0246] Pucheu, S., C. Coudray, N. Tresallet, A. Favier and J. de Leiris (1995). “Effect of dietary antioxidant trace element supply on cardiac tolerance to ischemia-reperfusion in the rat.” J Mol Cell Cardiol 27(10): 2303-2314. [0247] Rakotovao, A., S. Tanguy, M. C. Toufektsian, C. Berthonneche, V. Ducros, A. Tosaki, J. de Leiris and F. Boucher (2005). “Selenium status as determinant of connexin-43 dephosphorylation in ex vivo ischemic/reperfused rat myocardium.” J Trace Elem Med Biol 19(1): 43-47. [0248] Ray, A. L., R. D. Semba, J. Walston, L. Ferrucci, A. R. Cappola, M. O. Ricks, Q. L. Xue and L. P. Fried (2006). “Low serum selenium and total carotenoids predict mortality among older women living in the community: the women's health and aging studies.” J Nutr 136(1): 172-176. [0249] Rayman, M. P. and S. Stranges (2013). “Epidemiology of selenium and type 2 diabetes: can we make sense of it?” Free Radic Biol Med 65: 1557-1564. [0250] Read, R., T. Bellew, J. G. Yang, K. E. Hill, I. S. Palmer and R. F. Burk (1990). “Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum.” J Biol Chem 265(29): 17899-17905. [0251] Rees, K., L. Hartley, C. Day, N. Flowers, A. Clarke and S. Stranges (2013). “Selenium supplementation for the primary prevention of cardiovascular disease.” Cochrane Database Syst Rev(1): Cd009671. [0252] Reeves, M. A. and P. R. Hoffmann (2009). “The human selenoproteome: recent insights into functions and regulation.” Cell Mol Life Sci 66(15): 2457-2478. [0253] Renko, K., P. J. Hofmann, M. Stoedter, B. Hollenbach, T. Behrends, J. Kohrle, U. Schweizer and L. Schomburg (2009). “Down-regulation of the hepatic selenoprotein biosynthesis machinery impairs selenium metabolism during the acute phase response in mice.” Faseb j 23(6): 1758-1765. [0254] Renko, K., M. Werner, I. Renner-Muller, T. G. Cooper, C. H. Yeung, B. Hollenbach, M. Scharpf, J. Kohrle, L. Schomburg and U. Schweizer (2008). “Hepatic selenoprotein P (SePP) expression restores selenium transport and prevents infertility and motor-incoordination in Sepp-knockout mice.” Biochem J 409(3): 741-749. [0255] Saito, Y. and K. Takahashi (2002). “Characterization of selenoprotein P as a selenium supply protein.” Eur J Biochem 269(22): 5746-5751. [0256] Saliba, W., R. El Fakih and W. Shaheen (2010). “Heart failure secondary to selenium deficiency, reversible after supplementation.” Int J Cardiol 141(2): e26-27. [0257] Salonen, J. T., G. Alfthan, J. K. Huttunen, J. Pikkarainen and P. Puska (1982). “Association between cardiovascular death and myocardial infarction and serum selenium in a matched-pair longitudinal study.” Lancet 2(8291): 175-179. [0258] Savarese, G. and L. H. Lund (2017). “Global Public Health Burden of Heart Failure.” Cardiac failure review 3(1): 7-11. [0259] Singal, P. K., N. Khaper, V. Palace and D. Kumar (1998). “The role of oxidative stress in the genesis of heart disease.” Cardiovasc Res 40(3): 426-432. [0260] Soto, G. E., P. Jones, W. S. Weintraub, H. M. Krumholz and J. A. Spertus (2004). “Prognostic value of health status in patients with heart failure after acute myocardial infarction.” Circulation 110(5): 546-551. [0261] Strauss, E., J. Tomczak, R. Staniszewski and G. Oszkinis (2018). “Associations and interactions between variants in selenoprotein genes, selenoprotein levels and the development of abdominal aortic aneurysm, peripheral arterial disease, and heart failure.” PLoS One 13(9): e0203350. [0262] Tanguy, S., F. Boucher, S. Besse, V. Ducros, A. Favier and J. de Leiris (1998). “Trace elements and cardioprotection: increasing endogenous glutathione peroxidase activity by oral selenium supplementation in rats limits reperfusion-induced arrhythmias.” J Trace Elem Med Biol 12(1): 28-38. [0263] Tanguy, S., S. Morel, C. Berthonneche, M. C. Toufektsian, M. de Lorgeril, V. Ducros, A. Tosaki, J. de Leiris and F. Boucher (2004). “Preischemic selenium status as a major determinant of myocardial infarct size in vivo in rats.” Antioxid Redox Signal 6(4): 792-796. [0264] Tanguy, S., A. Rakotovao, M. G. Jouan, C. Ghezzi, J. de Leiris and F. Boucher (2011). “Dietary selenium intake influences Cx43 dephosphorylation, TNF-alpha expression and cardiac remodeling after reperfused infarction.” Mol Nutr Food Res 55(4): 522-529. [0265] Tanguy, S., M. C. Toufektsian, S. Besse, V. Ducros, J. De Leiris and F. Boucher (2003). “Dietary selenium intake affects cardiac susceptibility to ischaemia/reperfusion in male senescent rats.” Age Ageing 32(3): 273-278. [0266] Toufektsian, M. C., F. Boucher, S. Pucheu, S. Tanguy, C. Ribuot, D. Sanou, N. Tresallet and J. de Leiris (2000). “Effects of selenium deficiency on the response of cardiac tissue to ischemia and reperfusion.” Toxicology 148(2-3): 125-132. [0267] Venardos, K., G. Harrison, J. Headrick and A. Perkins (2004). “Effects of dietary selenium on glutathione peroxidase and thioredoxin reductase activity and recovery from cardiac ischemia-reperfusion.” J Trace Elem Med Biol 18(1): 81-88. [0268] Writing Group, M., D. Mozaffarian, E. J. Benjamin, A. S. Go, D. K. Arnett, M. J. Blaha, M. Cushman, S. R. Das, S. de Ferranti, J. P. Despres, H. J. Fullerton, V. J. Howard, M. D. Huffman, C. R. Isasi, M. C. Jimenez, S. E. Judd, B. M. Kissela, J. H. Lichtman, L. D. Lisabeth, S. Liu, R. H. Mackey, D. J. Magid, D. K. McGuire, E. R. Mohler, 3rd, C. S. Moy, P. Muntner, M. E. Mussolino, K. Nasir, R. W. Neumar, G. Nichol, L. Palaniappan, D. K. Pandey, M. J. Reeves, C. J. Rodriguez, W. Rosamond, P. D. Sorlie, J. Stein, A. Towfighi, T. N. Turan, S. S. Virani, D. Woo, R. W. Yeh, M. B. Turner, C. American Heart Association Statistics and S. Stroke Statistics (2016). “Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.” Circulation 133(4): e38-360. [0269] Xia, Y., K. E. Hill, D. W. Byrne, J. Xu and R. F. Burk (2005). “Effectiveness of selenium supplements in a low-selenium area of China.” Am J Clin Nutr 81(4): 829-834. [0270] Yang, J. G., K. E. Hill and R. F. Burk (1989). “Dietary selenium intake controls rat plasma selenoprotein P concentration.” J Nutr 119(7): 1010-1012. [0271] Yang, S. J., S. Y. Hwang, H. Y. Choi, H. J. Yoo, J. A. Seo, S. G. Kim, N. H. Kim, S. H. Baik, D. S. Choi and K. M. Choi (2011). “Serum selenoprotein P levels in patients with type 2 diabetes and prediabetes: implications for insulin resistance, inflammation, and atherosclerosis.” J Clin Endocrinol Metab 96(8): E1325-1329. [0272] Yang, X., K. E. Hill, M. J. Maguire and R. F. Burk (2000). “Synthesis and secretion of selenoprotein P by cultured rat astrocytes.” Biochim Biophys Acta 1474(3): 390-396.