Plasma Selenoprotein P Levels of Healthy Males in Different Selenium Status After Oral Supplementation with Different Forms of Selenium

Plasma selenoprotein P levels of healthy males in different selenium status after oral supplementation with different forms of selenium

M Persson-Moschos1, G Alfthan2 andBAÊ kesson1

1Department of Applied Nutrition and Food Chemistry, Center for Chemistry and Chemical Engineering, PO Box 124, University of Lund, S-221 00 Lund, Sweden and 2Department of Nutrition, National Public Health Institute, FIN-00300 Helsinki, Finland

Objective: To assess changes in selenoprotein P levels in plasma from subjects who had received oral supplements of different selenium forms. Design: The same study group participated in two similar selenium supplementation trials, Trial I in 1981 (Levander et al, 1983) and Trial II in 1987 (Alfthan et al, 1991). During Trial II the mean baseline intake of selenium in Finland was higher compared to that during Trial I (100 and 40 mg/d, respectively), due to a nation- wide supplementation of fertilisers which started in 1985. Subjects: Fifty healthy Finnish men, 36±60 y old. Intervention: The study group received daily placebo or oral supplements consisting of 200 mg selenium as selenium-enriched yeast, sodium selenate or selenium-enriched wheat (Trial I) or selenium-enriched yeast, sodium selenate or sodium selenite (Trial II). The duration of supplementation periods was 11 (Trial I) and 16 (Trial II) weeks. Results: In Trial I the mean plasma selenoprotein P values in all the supplemented groups increased signi®cantly, approaching a plateau at 2 weeks and reaching maxima at 4 weeks (mean increase 34%, P < 0.05). In Trial II the mean selenoprotein P levels of the supplemented groups were not signi®cantly different from each other or from the placebo group at the start or at any time point of the supplementation period. Conclusions: At a low selenium status the selenoprotein P levels increased in a similar fashion after supplementation with different forms of selenium, but at a high selenium status no signi®cant effects of supplementation with the same amount of selenium were observed. No differences in selenoprotein P levels were observed for inorganic and organic selenium supplements. Sponsorship: This study was supported by The Swedish Council for Forestry and Agricultural Research, the PaÊhlsson Foundation and the Swedish Nutrition Foundation. Descriptors: selenoprotein P; healthy men; selenium supplementation; selenium forms; selenium status

Introduction plasma re¯ects the selenium status in healthy subjects (Marchaluk et al, 1995; Persson-Moschos et al, 1995a; Hill Several chemical forms of selenium are present in the diet, et al, 1996) as well as in patients with low selenium status and the major forms are probably the selenium-containing (Persson-Moschos et al, 1995b; Rannem et al, 1996). amino acids selenocysteine and selenomethionine bound in Selenoprotein P is a selenium-rich protein, which contains proteins. Selenomethionine is likely the dominating form in up to ten selenocysteine residues per polypeptide chain, com- vegetables, while proteins of animal foods contain both pared to one selenocysteine residue in other characterised selenomethionine and selenocysteine in varying propor- selenoproteins. The function of selenoprotein P is unclear, tions (Burk, 1986). The inorganic forms selenite and but it is expressed by several tissues and it may serve as selenate are used in supplements but it is not certain if an extracellular oxidant defence (Burk & Hill, 1994). they occur in foods. Uncharacterised forms occur as well; Since the level of selenoprotein P may be related to for instance unknown low molecular-weight selenium susceptibility to oxidant-induced disease, it is important to compounds were demonstrated in ®sh (AÊ kesson & Sriku- know how it is in¯uenced by different forms of dietary mar, 1994). selenium. In this study, selenoprotein P was measured in In mammalian tissue the major functional form of plasma samples from subjects who had received oral selenium is selenocysteine in the primary structure of supplementation of different forms of selenium. Two simi- selenoproteins. At least ten different selenoproteins have lar supplementation trials have been carried out, and in the been identi®ed in mammals. Their concentrations in tissues second one the same subjects had acquired a higher are dependent on the dietary intake of selenium (Chen et al, selenium status through the nation-wide selenium supple- 1990; Behne et al, 1991; Marchaluk et al, 1995). mentation of fertilisers in Finland (Aro et al, 1997). The major selenoprotein in human plasma is selenoprotein P, which accounts for about 40% of the total plasma selenium Ê (Akesson et al, 1994; Hill et al, 1996). Its concentration in Methods

Correspondence: Dr M Persson-Moschos. Study group Received 2 November 1997; revised 26 January 1998; accepted 31 January As described in previous reports (Levander et al, 1983; 1998 Alfthan et al, 1991) the study group consisted of healthy Plasma selenoprotein P levels of healthy males M Persson±Moschos et al 364 Finnish men, aged 36±60 y. Fifty men were recruited for Bonferroni's test and Duncan's multiple range test. P- the ®rst supplementation study during 1981±1982 (Trial I) values < 0.05 were considered statistically signi®cant. Cor- and the same subjects except ®ve also participated in the relations were assessed by Pearson's two-tailed signi®cance second study (Trial II) six years later, when they were test. The statistical analysis were performed by using SPSS divided into the same supplementation groups as before. 6.1 for Windows (SPSS Inc, 1994). The mean daily dietary intake of selenium of adults increased from approximately 40 mg in 1981 to 100 mgin 1987 as a result of the country-wide supplementation of Results fertilisers with selenium starting in Finland in 1985 (Aro In Trial I the mean levels of selenoprotein P in the et al, 1997). supplemented groups (yeast, selenate and wheat) increased The subjects were divided into one placebo group (n 15 signi®cantly in a similar way and approached a plateau and 20, respectively) and three treatment groups (n 10 for already after two weeks of supplementation (Figure 1; each) that received 200 mg selenium per day as selenium- Table 1). The supplemented groups reached their maximal enriched yeast, sodium selenate or selenium-enriched wheat selenoprotein P value at 4 weeks (1.4 (0.1) au) (Trial I) or selenium-enriched yeast, sodium selenate or (mean (s.d.)). The selenoprotein P levels among the sup- sodium selenite (Trial II). For analysis of selenoprotein P, plemented groups were not signi®cantly different from plasma samples from Trial I were available at the time points each other at any time-point, except at 21 weeks when 0 (before intervention), 2, 4, 7 and 11 weeks (supplementation the group supplemented with selenate had signi®cantly period), and 21 weeks (10 weeks after the end of supplemen- lower levels than the group that received yeast (P < 0.05 tation period). From Trial II plasma samples at the time points in Duncan's test) (Figure 1). The more rapid post-supple- 0 (before intervention), 4, 7 and 16 weeks (supplementation mentational decline in selenoprotein P level in the selenate period) were available. group is most probably explained by the higher tissue Data on plasma and erythrocyte selenium, and plasma and amounts of unspeci®cally incorporated selenium which platelet glutathione peroxidase activity were available from had accumulated in the subjects given organic selenium. previous studies (Levander et al, 1983; Alfthan et al, 1991). During the post-supplementation period more selenium was thus liberated during protein turnover in the subjects given Methods organic selenium than in those given selenate and to some Selenoprotein P levels in the plasma samples were mea- extent utilized for selenoprotein synthesis. The mean seleno- sured with a radioimmunoassay (Persson-Moschos et al, protein P level of the placebo group stayed constant 1995a), and the concentration of selenoprotein P was throughout the supplementation period, but in the post- expressed in arbitrary units (au) relative to a standard of supplementation period it increased by 21% from 11±21 pooled plasma. The intra-assay coef®cient of variation (cv) weeks (P < 0.05). The most plausible explanation of this was 3.8% and the inter-assay cv was 6.6%. The standard phenomenon is that selenium-rich grain was imported deviations of mean selenoprotein P values of all groups in during this period and the selenium intake of the subjects Trial I varied from 0.09±0.24 au, and in Trial II the s.d. was increased accordingly (Levander et al, 1983; Mutanen varied from 0.16±0.35 au. & Koivistoinen, 1983), although unknown factors may also be involved. Statistics In Trial II the study group had acquired a higher Differences between selenoprotein P mean levels in differ- selenium status and the mean concentration of selenopro- ent groups and their changes after selenium supplementa- tein P at baseline (0 weeks) was 74% higher than in Trial I tion were evaluated by one-way ANOVA followed by (1.8 (0.3) and 1.0 (0.2)) au, respectively (Figure 1). The

Figure 1 Changes in selenoprotein P levels in plasma from healthy Finnish men that had received different forms of selenium supplements (200 mg selenium/d) in two previous studies, Trial l in 1981±1982 (Levander et al, 1983) and Trial II in 1987 (Alfthan et al, 1991). In Trial II the subjects had acquired a higher selenium status through a nation-wide selenium supplementation of fertilisers starting in 1985. Supplementation was started at week 0 and stopped at week 11 (Trial I) or at week 16 (Trial II). Each symbol represents the mean selenoprotein P value of nine to twenty subjects (Trial I) or of nine to ®fteen subjects (Trial II). The standard deviations of mean selenoprotein P values of all groups in Trial I varied from 0.09±0.24 au (arbitrary units), and in Trial II the SD varied from 0.16±0.35 au. Plasma selenoprotein P levels of healthy males M Persson±Moschos et al 365 Table 1 Percentage change in selenium status variables of healthy men after supplementation with different selenium forms

Selenium forms

Selenium status variable Wheat Yeast Selenate Selenite Placebo

Trial IÐ% change from 0±11 weeks P-Sea . 139*** . 144*** . 66*** Ð . 10 RBC-Sea . 111*** . 113*** . 19* Ð . 6 P-GSHPxa . 15* . 10** . 16 Ð . 5 Plat-GSHPxa . 79*** . 61*** . 106*** Ð . 10 P-SeP . 36*** . 27** . 30*** Ð . 2 Trial IIÐ% change from 0±16 weeks P-Seb Ð . 49*** . 2* . 12 72 RBC-Seb Ð . 72*** . 4* . 6 73 Plat-Seb Ð . 10 . 47** . 29*** . 3 P-SeP Ð . 10 . 5 . 7 . 1

P-Se plasma selenium; RBC-Se red blood cell selenium; P-GHSPx plasma glutathione peroxidase; Plat-GSHPx platelet glutathione peroxidase; P-SeP plasma selenoprotein P. a Data were obtained from Levander et al, 1983. b Data were obtained from Alfthan et al, 1991. Signi®cance of changes in selenium status variables: *P < 0.05; **P < 0.01; ***P < 0.001 (Paired t-test). mean selenoprotein P levels among the supplemented and that it increased more when the selenium supplement was groups (yeast, selenate and selenite) were not at any time yeast (144%) or selenium-rich wheat (139%) compared with point signi®cantly different from each other or from that in selenate (66%) (Levander et al, 1983) (Table 1). In Trial II the the placebo group. study group had acquired a higher basal plasma selenium Plasma selenium and selenoprotein P concentrations in concentration (1.4 mmol/l) through the nation-wide supple- the whole study group were correlated at baseline in both mentation offertilizers (Alfthanet al, 1991).Supplementation trial I and II (r 0.34, P 0.015, and r 0.30, P 0.048, with yeast selenium resulted in considerably elevatedlevels of respectively). As expected, the correlations between plasma plasma selenium (49%), and only a small but signi®cant selenium and selenoprotein P increased after supplementa- increase was observed after supplementation with selenite tion in Trial I, being usually >0.70 (P < 0.001) at different but not with selenate. In both trials the maximal level of time points. In Trial II the correlations decreased with time plasma selenium was the same (2.1 mmol/l) in the groups from 0.30 (P 0.048) to 0.16 (not signi®cant). In Trial I the supplemented with yeast selenium. Signi®cant increases in correlations between plasma glutathione peroxidase and plasma selenium following ingestion of selenomethionine- selenium or selenoprotein P were not signi®cant in most containing supplements have also been observed in several cases, r always being < 0.35. human (Tarp et al, 1985; NeÁve et al, 1988; Thomson et al, We also calculated the ratio of selenoprotein P/plasma 1993) and animal experiments (Whanger & Butler, 1988; selenium. In Trial I the ratio decreased rapidly during Butler et al, 1990). Since approximately half of the total supplementation from approx. 15 to approx. 8 in the amount of selenium in the selenium-rich yeast used in the wheat and yeast selenium groups but was unchanged in Finnish study was in the form of selenomethionine (Korhola et the selenate group the ®rst four weeks after supplementa- al, 1986), a likely cause of the large increase in plasma tion, and thereafter it decreased somewhat. In the placebo selenium in the groups given these supplements is that sele- group selenoprotein P/plasma selenium ratio was constant nomethionine is nonspeci®cally incorporated into all tissue with time. In Trial II this ratio also decreased in the yeast proteins, in plasma mainly into albumin. Organic selenium selenium group from 15 to 11, but was unchanged in the forms therefore increase plasma selenium much more than the groups supplemented with inorganic selenium. inorganic forms selenate and selenite which enter the selenide pool and are thereby available only to synthesis of speci®c selenoproteins.Itisnoteworthythatthemaximalmeanplasma Discussion selenium concentration in the selenate-supplemented group There are indications that supplementation with selenium was the same in the two trials (1.4 mmol/l). or nutrient combinations containing selenium will decrease Concerning selenoprotein P, the results in Trial I indi- the incidence of cancer (Blot et al, 1993; Clark et al, 1996). cated that the three selenium forms were equally ef®cient in Moreover, selenium supplementation has been shown to increasing its levels in plasma (Figure 1). The maximum have a prophylactic effect on Keshan disease (Ge & Yang, levels after supplementation were reached at four weeks at 1993). The number of cases has been radically reduced by plasma selenium levels in the range 1.2±1.7 mmol/l. How- selenium enrichment of table salt. Selenium supplementa- ever, in Trial II no increase in selenoprotein P levels was tion has been tried in several other disease conditions but observed in any supplemented group. The baseline seleno- usually without preventive or therapeutic effect (Johnsson protein P levels had probably already reached a maximum et al, 1997). It is unknown whether the possible anti- level due to the higher initial selenium status of the carcinogenic effects are mediated by changes in the con- subjects. A tendency towards plateauing of selenoprotein centrations or actions of different selenoproteins. Recently P levels at plasma selenium concentrations in the range several new selenoproteins have been identi®ed, and it is 1.2±1.5 mmol/l was also observed previously in a cross- important to study the effect of different forms of dietary sectional investigation of 414 European subjects (Mar- selenium on the levels of each selenoprotein. chaluk et al, 1995). On the other hand, when Chinese Previous results in Trial I showed that the baseline plasma men having a low initial plasma selenium level selenium in the study group was moderately low (0.9 mmol/l), (0.5 mmol/l) were supplemented with 200 mg selenium per Plasma selenoprotein P levels of healthy males M Persson±Moschos et al 366 day (as selenate) for two weeks, selenoprotein P levels selenite increased the platelet glutathione peroxidase activ- tended to approach a plateau after two weeks at a plasma ity which reached a plateau at a plasma selenium concen- selenium concentration of only 0.9 mmol/l (Hill et al, 1996). tration of 1.4 mmol/l (Alfthan et al, 1991). NeÁve (1994) Several factors can in¯uence the plasma selenium level at summarized the responses in platelet glutathione peroxi- which selenoprotein P reaches a plateau. One is that the dase activities in various studies after selenium supplemen- dietary intake of selenomethionine and its unspeci®c incor- tation. If selenite or selenate was used for supplementation, poration into plasma proteins would be expected to vary in platelet GSHPx plateaued when the plasma selenium con- different populations. A subject with a low selenium intake centration was 1.2±1.5 mmol/l. When organic selenium would be expected to have less selenomethionine incorpo- forms were administered the plateau was reached at a rated in plasma proteins that a subject with a higher plasma selenium of 1.4±1.7 mmol/l. selenium intake. If these two subjects are supplemented In Trial I there were no signi®cant changes in plasma with saturating amounts of selenate, both would be glutathione peroxidase activity during supplementation expected to obtain similar plateaus of selenoprotein P and (Levander et al, 1983). Similar ®ndings were observed in other selenoproteins since selenate would not be incorpo- Dutch subjects with plasma levels in the same range as the rated into selenomethionine. The subject with initially subjects in Trial I, after supplementation with selenium-rich higher selenium intake would likely reach a higher meat or bread (van der Torre et al, 1991). On the other hand, plasma selenium level after supplementation than the sub- supplementation of New Zealanders with a plasma selenium ject with initially low selenium intake. Dietary methionine level of 0.7 mmol/l with 200 mg/d selenium as selenate or may also in¯uence the amount of selenium available for selenium-rich yeast, resulted in increased plasma glutathione synthesis of selenoproteins since it competes with seleno- peroxidase activities plateauing at selenium concentrations of methionine for incorporation into proteins (Luo et al, 1985; 1.2 mmol/l and 2.5 mmol/l, respectively (Thomson et al, Waschulewski & Sunde, 1988; Behne et al, 1991). Since 1993). In Chinese men with a plasma selenium concentration there were no signi®cant differences in selenoprotein P of 0.3 mmol/l, supplementation with 200 mg/d as sodium levels between subjects given organic and inorganic forms selenate or selenium-rich yeast resulted in similar increases of selenium in Trial I, the methionine supply of the subjects in plasma glutathionine peroxidase in both groups, reaching was probably suf®cient making possible a high utilisation plateaus at plasma selenium concentrations of 0.9 mmol/l and of selenium from the selenomethionine supplement for 1.4 mmol/l, respectively (Xia et al, 1992). Moreover, when synthesis of selenoprotein P. plasma glutathione peroxidase and selenium data from dif- The difference between organic and inorganic selenium ferent cross-sectional studies were combined, a tendency supplements can also be interpreted from the selenoprotein/ towards a plateau of plasma glutathione peroxidase was selenium ratios. Although plasma selenium and selenoprotein reached at a plasma selenium concentration of approximately P were correlated in Trial I, the selenoprotein P/selenium ratio 1 mmol/l (Huang, 1996). varied in different supplementation groups with time. Wheat It is noteworthy that supplementation with yeast sele- and yeast selenium supplementation increased plasma selenium and selenate gives similar responses in erythrocyte nium much more than did selenate supplementation and in and plasma glutathione peroxidase (Thomson et al, 1993) these groups the ratios decreased rapidly. The fact that in the and selenoprotein P (this study), whereas platelet glu- selenate group the selenoprotein P/selenium ratio was tathione peroxidase increases much more after selenate unchanged in the four ®rst weeks of the supplementation than after yeast selenium (Levander et al, 1983; Alfthan period is compatible with the interpretation that most of the et al, 1991; Thomson et al, 1993). This difference remains increase in plasma selenium was due to increases in seleno- to be explained. Several mechanisms are involved in the protein P and maybe other selenoproteins. homeostatic regulation of selenoprotein levels both with An interesting point is that the mean baseline level of respect to the rate of change for individual proteins and selenoprotein P in Trial II (1.8 au) was higher than its mean their maximum concentrations in tissues. Although regula- maximum level after supplementation in Trial I (1.4 au). tion at the transcriptional level has been observed for When Trial I was started, the subjects' basal intake of selenoproteins (O'Prey et al, 1993), there is little evidence selenium was 62 mg/d (plasma selenium 0.9 mmol/l), and that selenium supply exerts its in¯uence via such mechan- together with the daily selenium supplements of 200 mg isms (Burk, 1997). The biosynthesis of individual seleno- selenium, the total intake was approximately 262 mg/d. proteins in different selenium states is tissue- and When Trial II was started, the basal dietary intake of selenium selenoprotein-speci®c (Behne et al, 1991), which may be was approximately 100 mg/d (plasma selenium 1.4 mmol/l), mediated by tissue-speci®c changes in selenocysteine and the subjects had probably had this higher level of tRNA:s (Diamond et al, 1993) and differences in the selenium intake more than one year before the supplementa- selenocysteine insertion sequences (SECIS) for different tion study. The ®nding that selenoprotein P starting levels in selenoproteins (Kollmus et al, 1996). Moreover, the Trial II were higher than the plateaus of Trial I is dif®cult to response in mRNA levels of individual selenoproteins to interpret, but it may be due to long-term redistribution and changes in selenium supply is both tissue-speci®c and accumulation of selenium in different organs which may differs in magnitude and direction (Bermano et al, 1995). increase the synthesis of selenoprotein P. Selenium supplementation can also affect non-selenopro- The response in selenoprotein P levels after supplemen- tein genes and gene products (Christensen & Pusey, 1994; tation can be compared to the corresponding responses in Nelson et al, 1996). The importance of all these mechan- other selenoprotein levels. In Trial I platelet glutathione isms in the human body is not yet known. peroxidase activity increased rapidly and reached a plateau at four weeks at a plasma selenium concentration of 1.3± 1.5 mmol/l (Levander et al, 1983). 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