Effects of Chemical Form of Selenium on Plasma Biomarkers in a High-Dose Human Supplementation Trial

804 Effects of Chemical Form of Selenium on Plasma Biomarkers in a High-Dose Human Supplementation Trial

Raymond F. Burk,1 Brooke K. Norsworthy,1 Kristina E. Hill,1 Amy K. Motley,1 and Daniel W. Byrne2 1Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, and 2Department of Biostatistics, Vanderbilt University School of Medicine, Nashville, Tennessee

Abstract

Intervention trials with different forms of selenium are under tation with selenomethionine and yeast raised the plasma way to assess the effects of selenium supplements on the selenium concentration in a dose-dependent manner. Selenite incidence of cancer and other diseases. Plasma selenium did not. The increased selenium concentration correlated with biomarkers respond to selenium administration and might be the amount of selenomethionine administered. Neither useful for assessing compliance and safety in these trials. The glutathione peroxidase activity nor selenoprotein P concen- present study characterized the effects of selenium supple- tration responded to selenium supplementation. Urinary mentation on plasma selenium biomarkers and urinary selenium excretion was greater after selenomethionine than selenium excretion in selenium-replete subjects. Moderate after selenite, with excretion after yeast being intermediate (f200 Mg/d) to large (f600 Mg/d) selenium supplements in the and not significantly different from either of the other two. forms sodium selenite, high-selenium yeast (yeast), and L- We conclude that plasma selenium concentration is useful in selenomethionine (selenomethionine) were administered. monitoring compliance and safety of selenium supplementa- Subjects were randomized into 10 groups (placebo and three tion as selenomethionine but not as selenite. Plasma selenium dose levels of each form of selenium). Plasma biomarkers seems to reflect the selenomethionine content of yeast but not (selenium concentration, selenoprotein P concentration, and the other yeast selenium forms. As judged by urinary glutathione peroxidase activity) were determined before selenium excretion, selenium in the form of selenomethionine supplementation and every 4 weeks for 16 weeks. Urinary is better absorbed than selenite. (Cancer Epidemiol Bio- selenium excretion was determined at 16 weeks. Supplemen- markers Prev 2006;15(4):804–10)

Introduction

In 1996, Clark et al. reported that a selenium supplement of Three biomarkers in plasma are used to assess selenium 200 Ag/d, administered to prevent a second nonmelanoma nutritional status and to predict toxicity. Two of them are skin cancer, reduced the risk of cancers of the prostate, lung, plasma selenoproteins (selenoprotein P and glutathione and colon (1). In response to that report, additional interven- peroxidase-3). Plasma levels of these selenoproteins are used tion trials have been initiated to assess the effectiveness of primarily as nutritional biomarkers of the element. Their selenium as a cancer chemoprevention agent (2). concentrations are depressed in selenium deficiency but Several forms of selenium are available for use in chemo- increase withincreasing selenium supplementation until prevention trials. L-Selenomethionine (selenomethionine), reaching plateaus at levels determined by genetic and which was chosen for use in the Selenium and Vitamin E environmental factors (6-8). The rationale for their status as Cancer Prevention Trial, is synthesized by plants and nutritional biomarkers is that they serve as representatives of consequently is the major form of selenium in the human all the selenoproteins in the body. diet. High-selenium yeast, a type of which was used in the The third biomarker is plasma selenium. It consists of Clark et al. study, is produced by growing yeast in a high- selenium in the forms of selenocysteine in the two selenopro- selenium medium. Analyses of high-selenium yeast indicate teins and selenomethionine present at methionine positions in that the major chemical form of selenium in it is selenome- all proteins plus small-molecule forms that contribute <3% of thionine, with several other compounds making up the the total (9, 10). Selenomethionine substitution for methionine remainder (3). Inorganic selenium in the form of selenite or seems to be a random process that is dependent on the selenate has been used successfully in animal chemopreven- availability of selenomethionine (9). Thus, these three bio- tion studies. markers all respond to selenium intake, but eachresponds The most frequently used dose of selenium in intervention differently and yields different information. A trials is 200 g/d. NorthAmericans taking sucha supplement At present, the recommended dietary allowance for North A will not exceed the safe upper limit of 400 g/d set by the American adults is 55 Ag selenium/d based on the intake Institute of Medicine (4). However, higher selenium doses that required to optimize plasma glutathione peroxidase activity might be toxic are sometimes employed in trials (5) and (4). Typical selenium intakes in NorthAmerica are 80 to 140 monitoring for selenium toxicity and compliance is needed in Ag/d. Intakes in many parts of the world, however, are lower all studies of selenium administration regardless of the largely due to low soil selenium levels. The lowest reported selenium dose. intakes of selenium are in some regions of China where people consume V10 Ag/d (8, 11). Intakes in Europe and New Zealand range from <30 to 60 Ag/d. Clearly, people in Received 12/16/05; accepted 2/14/06. Grant support: NIH grants R01 DK58763, M01 RR-00095, and P30 ES00267. some parts of the world do not meet their selenium The costs of publication of this article were defrayed in part by the payment of page charges. requirements. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. To use plasma biomarkers for assessing selenium status and Section 1734 solely to indicate this fact. to maintain safety during selenium supplementation trials, Requests for reprints: Raymond F. Burk, 1030C Medical ResearchBuilding IV, Vanderbilt Medical Center, Nashville, TN 37232-0252. E-mail: [email protected] knowledge of how the different supplements affect the Copyright D 2006 American Association for Cancer Research. biomarkers is needed. We conducted a randomized, placebo- doi:10.1158/1055-9965.EPI-05-0950 controlled trial in which healthy North Americans were

Cancer Epidemiol Biomarkers Prev 2006;15(4). April 2006 Downloaded from cebp.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Epidemiology,Biomarkers & Prevention 805 supplemented withorganic and inorganic forms of selenium in collected in tubes containing EDTA as an anticoagulant. daily selenium doses up to f600 Ag. The effects of this Plasma was separated and stored at À70jC. An additional 5 supplementation on the plasma selenium biomarkers are mL blood was collected into a tube without EDTA. Serum was reported here. separated for albumin determination. Participants voided their first morning urine and then collected all urine for the following 24 hours. At the end of the Subjects and Methods collection period, the total volume of urine was recorded for eachindividual. Eachurine sample was mixed and aliquots Subjects. Volunteers ages z18 years were recruited at were stored at À70jC. Vanderbilt University Medical Center between February and Assays and Calculations. Albumin concentration was July 2003. Respondents to advertisements were eligible if they determined in serum at the Vanderbilt Clinic laboratory using were in good health, were neither pregnant nor planning a A the bromcresol green method (12). Glutathione peroxidase pregnancy, were not taking a selenium supplement of >25 g/ activity was determined in plasma using the assay we have d, and had not been diagnosed with liver disease. Eighty-eight employed in other human studies (13). The substrate was 0.25 subjects were enrolled after informed consent was obtained. mmol/L H2O2. The Vanderbilt Institutional Review Board approved the Plasma and urine selenium concentrations were measured protocol. Subjects were compensated for participation. using a fluorometric assay (14, 15). Our standard human Selenium Supplements. Selenite was purchased from plasma sample (16) was included in every assay to assess Kelatron Corp. (Ogden, UT) as sodium selenite. Dr. V. reproducibility. Based on 23 assays, this standard plasma had Badmaev (Sabinsa Corp., Piscataway, NJ) provided selenome- a selenium concentration of 121 F 5 Ag/L. The 24-hour urine thionine and Paul A. Willis (Cypress Systems, Inc., Fresno, CA) selenium excretion was calculated by adjusting the urine provided SelenoExcell High-Selenium Yeast. This yeast prep- selenium concentration to reflect the total urine volume. aration contained f1,200 Ag selenium/g and was estimated, Selenoprotein P was measured using a sandwichELISA based on proprietary analyses, to have f75% of its organic withtwo monoclonal antibodies, N22 and N11, whichwere selenium in the form of selenomethionine.3 provided by Dr. Takeshi Naruse (Kaketsuken, Kumamoto, Capsules ordered as placebos or to contain 200, 400, and Japan). Eachantibody recognizes a specific binding site in the 600 Ag selenium of eachform were made by Integrative NH2-terminal region of selenoprotein P. The capture anti- Therapeutics, Inc. (Wilsonville, OR). All capsules contained body, N22, was incubated on 96-well microtiter plates Fast-Flo Edible Lactose (Foremost Farms, Baraboo, WI) as the overnight at 4jC. The plates were washed with a solution filler. Capsules were assayed for selenium in our laboratory containing Block Ace (Dainippon Pharmaceuticals, Osaka, and the results are mean F SD (n = 3). Selenite capsules Japan) to block remaining binding sites. Coated plates were contained 202 F 30, 380 F 56, and 601 F 49 Ag selenium; stored at À20jC until needed. Plasma samples were prepared selenomethionine capsules contained 158 F 17, 338 F 34; and in two dilutions and eachdilution was measured in 507 F 25 Ag selenium; and yeast capsules contained 226 F 16, duplicate. Our human standard plasma was included on 439 F 27, and 703 F 48 Ag selenium. Placebos contained each plate. The diluted plasma was combined with the only the filler and their selenium content was not detectable biotinylated antibody, N11. Standard curves that ranged from (<0.05 Ag). 0.15 to 5 ng/mL were constructed using purified human Protocol. The study was a randomized, placebo-controlled selenoprotein P, which was also a gift of Dr. Takeshi Naruse intervention. After enrollment, subjects were admitted as (Kaketsuken). The sample or standard solution was then added to the N22-coated wells of the ELISA plate and inpatients to the General Clinical Research Center at Vander- j bilt University Medical Center. incubated at 37 C for 1 hour. Each well was rinsed and then Research nurses recorded height, weight, and medical incubated for an additional hour with streptavidin-horserad- ishperoxidase at 37j C. Again, the plate was rinsed, and histories of each participant. Subjects stayed overnight at the V V General Clinical ResearchCenter. After an overnightfast, 20 3,3 ,5,5 -tetramethylbenzidine base, a substrate of horseradish mL blood was taken by venipuncture and processed. Before peroxidase, was added to the plate. The plate was incubated discharge, subjects were given the study capsules and in the dark for 30 minutes. H2SO4 (0.5 mol/L) was added to instructed to take one capsule eachmorning withfood eachwell to stop thereaction. Absorbance at 450 nm was measured in eachwell and theselenoprotein P concentration beginning that morning. A Participants returned for outpatient visits after weeks 4, 8, of the plasma ( g/mL) was calculated for eachsample using 12, and 16 for fasting blood sampling and to exchange their pill the standard curve. Based on 56 readings, our human standard plasma was measured to have a mean selenoprotein bottle for a new one. Compliance was measured by counting F capsules remaining in the bottle and by questioning the P concentration of 5.5 0.8 mg/L. These assay results of the participants. At eachoutpatient visit, subjects completed a standard plasma show the reproducibility of the selenium brief questionnaire about how they were tolerating the study and selenoprotein P assays. capsules and, in particular, whether they had noticed any hair ‘‘Other’’ selenium is all the plasma selenium not in the loss or changes in nail condition, menstrual cycle, appetite, or selenoproteins. ‘‘Other’’ selenium was determined by calcula- gastrointestinal function. tion. We calculated the selenium content of each selenoprotein The study coordinator phoned participants midway be- by expressing plasma selenoprotein P and glutathione perox- tween appointments to encourage compliance and to remind idase concentrations as a fraction of their respective averages subjects to fast overnight before their appointments. Subjects in selenium-replete individuals: 5.5 mg/L for selenoprotein P completed a 24-hour urine collection just before their final (derived from analysis of the human standard plasma) and 159 blood sampling in the General Clinical Research Center. units/L for glutathione peroxidase (the initial value in this study of selenium-replete subjects). This fraction was then Sample Collection. At eachclinic visit, blood was sampled multiplied by the respective concentrations of selenium by venipuncture after an overnight fast. Fifteen milliliters were previously determined to be present within each selenoprotein in selenium-replete subjects (64 Ag/L for selenoprotein P and 17 Ag/L for plasma glutathione peroxidase; refs. 16, 17). We calculated ‘‘other’’ selenium by subtracting the selenium 3 P.A. Willis, personal communication. content of the two selenoproteins from the total selenium

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Table 1. Characteristics of subjects comparison procedures. Before-after comparisons were ana- t P lyzed withthepaired test or the Wilcoxon signed-rank test. Women Men All A general linear model repeated-measures ANOVA was No. subjects 59 22 81 employed to assess changes from baseline between groups. in study Correlations were tested withboththeSpearman rank- Age (y) 35.8 F 10.0 (59) 37.1 F 10.6 (22) 0.63 36.2 F 10.1 correlation test and the Pearson correlation. Linear regression Weight (kg) 74.7 F 18.8 (58) 82.6 F 11.7 (21) 0.01 76.8 F 17.5 F F F models were used to produce best-fit lines and equations for Body mass 27.6 6.5 (58) 26.2 3.5 (21) 0.76 27.2 5.9 relationship between the dose and selenium levels. Ps < 0.05 index Albumin 39 F 4 (48) 40 F 4 (19) 0.49 39 F 4 were considered statistically significant and all tests were (g/L plasma) two-tailed. Continuous variables are mean F SD unless otherwise indicated. NOTE: Data are mean F SD (n). Values for women and men were compared by Graphics and preliminary statistical analyses were done the Mann-Whitney test. on a Macintoshcomputer using Prism version 4 (GraphPad Software, Inc., San Diego, CA), Canvas version 8 (Deneba Systems, Inc., Miami, FL), and InStat version 3.0 (GraphPad present in the plasma (18): ‘‘Other’’ selenium (Ag/L) = plasma A À Software). Further statistical analyses were done on a personal selenium ( g/L) [glutathione peroxidase (units/L/159 computer withthestatistical package SPSS for Windows units/L)] Â 17 Ag/L À [selenoprotein P (mg/L/5.5 mg/L) Â A version 13.0 (SPSS, Chicago, IL) and the R statistical software 64 g/L]. (http://www.r-project.org). Creatinine was measured in the urine by an established method (19) adapted to a 96-well plate. The amount of creatinine excreted in 24 hours was determined by multiplying Results creatinine concentration by total urine volume. Participant characteristics are displayed in Table 1. Seventy- Compliance and Withdrawals. Eighty-two participants three percent of the subjects were female. Males and females completed the study. Two participants withdrew from the were not significantly different in age, body mass index, or study for reasons unrelated to the capsules and four due to the albumin concentration. odor of the capsules or minor gastrointestinal complaints. One subject’s data were excluded due to noncompliance, bringing Plasma Selenium Biomarkers before Supplementation. the final number of subjects analyzed to 81. Their compliance The initial plasma selenium biomarkers are presented in was estimated to be 98.2% witha range of 80.4% to 100%. Table 2. The mean plasma selenium concentration was similar Initial plasma glutathione peroxidase values were not to the mean U.S. serum selenium value of 125 Ag/L determined for 19 individuals due to a processing error. determined in the Third National Health and Nutrition Consequently, no initial glutathione peroxidase activities of Examination Survey (20). That value is considered to indicate these 19 subjects have been used in the analyses. Thus, fewer a selenium-replete status. Therefore, the values determined for subjects are included in the initial values for glutathione the two selenoproteins can be predicted to be optimal (or peroxidase activity and ‘‘other’’ selenium than in selenoprotein saturated) values. Of the selenium in plasma, 64% was P and selenium concentration. calculated to be in selenoproteins; therefore, 36% was ‘‘other’’ Four of the participants were considered to have incomplete selenium, presumably mostly selenomethionine in albumin or invalid urine collections based on 24-hour urine creatinine and other plasma proteins (9, 21). content; their urinary data were consequently excluded from Comparison of the biomarkers with one another (Table 3) the statistical analysis. This left 5 to 8 subjects in each revealed that neither selenoprotein correlated with plasma supplement group and 14 in the placebo group for the selenium nor did they correlate with each other. Therefore, statistical analysis of the urine data. although selenium biomarkers correlate with one another in selenium-deficient individuals (8, 13, 16), they did not in these Sample Size and Statistical Analysis. Based on our selenium-replete individuals. previous work,4 we estimated that to detect a difference in the mean plasma selenium levels among groups with different Plasma Selenium Biomarkers during Supplementation. forms of selenium supplementation a sample size of 10 in each Plasma selenium concentration increased markedly with group would have 87% power to detect a difference in means selenomethionine and yeast supplementation but much less of 41 Ag/L (the difference between the 200 Ag supplement withselenite supplementation (Fig. 1A). Thelargest increase group mean of 170 Ag/L and placebo group mean of 129 Ag/L) occurred in the first 4 weeks of supplementation. By week 16, assuming a common SD of 28 using a two-group t test witha the plasma selenium level seemed to have been approaching a 0.05 two-sided significance level. Subjects were randomized plateau. Supplementation with158, 338, and 507 Ag selenium within blocks of 22, with 4 placebo and 2 in each of the as selenomethionine caused plasma selenium concentration to 9 supplement groups. Therefore, the initial estimate was for increase significantly (P < 0.05) above the placebo value by 110 subjects total. Due to practical and financial constraints, a 71%, 108%, and 178%, respectively. Supplementation with226, decision was made to end the study after the 88th patient (the 439, and 703 Ag selenium as yeast caused plasma selenium fourth block of 22). This decision was made while the data concentration to increase significantly (P < 0.05) by 46%, 108%, were blinded and before any analyses. A Pearson m2 test or Fisher’s exact test was used to assess categorical comparisons between groups. Differences between Table 2. Initial plasma selenium biomarkers group means for continuous measurements were tested by the Student’s t test or the Mann-Whitney U test. Compar- Women Men P All isons among the four treatment groups were assessed with a Selenium (Ag/L) 121 F 14 (59) 123 F 10 (22) 0.44 122 F 13 one-way ANOVA or a Kruskal-Wallis test. The Student’s Selenoprotein P (mg/L) 5.2 F 1.0 (59) 5.7 F 0.7 (22) 0.02 5.3 F 0.9 Newman-Keuls and Tukey tests were used for multiple Glutathione peroxidase 154 F 28 (49) 176 F 41 (13) 0.13 159 F 32 (units/L) ‘‘Other’’ selenium 45 F 16 (49) 39 F 12 (13) 0.22 44 F 15

NOTE: Data are mean F SD (n). Values for men and women were compared by 4 R.F. Burk et al., unpublished results. the Mann-Whitney test.

Table 3. Correlations among initial plasma selenium biomarkers

Plasma biomarkers All subjects Subjects in lowest tertile of plasma selenium*

n R2 PnR2 P

Selenium vs selenoprotein P 81 <0.01 0.63 27 <0.01 0.58 Selenium vs glutathione peroxidase 62 0.03 0.15 22 0.07 0.22 Selenoprotein P vs glutathione peroxidase 62 <0.01 0.61 22 0.12 0.12

NOTE: Correlations and Ps are based on the Pearson correlation. *Lowest tertile of baseline plasma selenium was defined as V116.24 Ag/L. and 182%, respectively. In contrast, the two lower doses of Urinary Selenium Excretion after 16 Weeks of Supple- selenium as selenite (202 and 380 Ag) did not raise plasma mentation. Excretion of selenium in the urine by the placebo selenium significantly. The highest dose (601 Ag) raised group was 55 F 22 Ag/24 hours (n = 13). Selenium is also lost plasma selenium after 16 weeks only by 26% (P < 0.05). The from the body in the stool and in sloughed skin cells. Thus, the plasma selenium concentration of the placebo group did not average daily selenium loss, and therefore intake (because this change over the 16 weeks of supplementation (P = 0.63, group was under steady-state conditions), was above the repeated-measures ANOVA). recommended dietary allowance of 55 Ag. This is another The other two plasma selenium biomarkers, glutathione confirmation of the selenium-replete status of the subjects. peroxidase and selenoprotein P, showed no tendency to Urinary selenium excretion after 16 weeks of supplemen- increase during the study in any of the treatment groups tation is shown in Fig. 3. When corrected for placebo (Fig. 1B and C). This lack of response to the selenium excretion level and expressed as percentage of administered supplements confirms that the study subjects were selenium selenium, the percentage of the dose excreted did not vary replete before supplementation was begun. Because seleno- significantly between the doses of a given form of the element protein concentration was not changed by the supplements, (data not shown). However, it differed (P < 0.05) between the the increase observed in total plasma selenium concentration forms selenomethionine (60 F 26%; n = 26) and selenite (41 F was caused by enlargement of the nonspecific selenium pool 15%; n = 20). Percent urinary excretion of selenium designated as ‘‘other’’ selenium. administered as yeast was 52 F 23% (n = 18), which was To allow comparison of the forms of selenium administered, not significantly different from percent excretion of either of the plasma selenium concentration achieved after 16 weeks of the other forms of selenium. Thus, a greater fraction of the supplementation was compared withselenium dose adjusted dose of selenium administered was excreted in the urine for body weight (Fig. 2). Each form of selenium raised the when the form was selenomethionine than when the form plasma selenium witha different efficiency. Slopes of thebest- was selenite. fit lines were selenomethionine > yeast > selenite. Setting the The slope of each best-fit line in Fig. 3 is another indicator of selenomethionine slope at 100%, the yeast slope was 73% and the fraction of administered selenium that was excreted in the selenite slope was 11%. This gives an indication of the urine. Setting the line-of-best-fit slope of the selenomethionine relative increases in plasma selenium concentration that were group at 100%, the slope of the yeast group was 77% and that achieved with each form of the element. of the selenite group was 60%. The urinary excretion results

Figure 1. Plasma selenium biomarkers in subjects supplemented daily with selenium in three forms. , placebo; , , and , selenium doses as selenite (202, 380, and 601 Ag/d, respectively); , , and , selenium doses as selenomethionine (158, 338, and 507 Ag/d, respectively); , , and , selenium doses as yeast (226, 439, and 703 Ag/d, respectively). A. Selenium concentrations (n = 6-8 with placebo n =15).B. Plasma glutathione peroxidase activities (n = 4-7 with placebo n = 12). C. Plasma selenoprotein P concentrations (n = 6-8 with placebo n = 15). Points, mean; bars, 1SD.

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Figure 2. Plasma selenium concentrations shown in relation to the daily dose of selenium/kg body weight in subjects supplemented for 16 weeks with selenium in three forms. 0 week, subjects receiving placebos. The best-fit lines were calculated using linear regression and were as follows: selenite, y = 3.12x + 122; selenomethionine, y = 28.5x + 132; yeast, y = 20.9x + 125. The R2s are 0.40 for selenite, 0.91for selenomethionine, and 0.89 for yeast. Ps for all three (significantly different from 0) are <0.001. The 95% confidence interval for the slopes are as follows: selenite, 1.83-4.45; selenomethionine, 25.13-31.89; yeast, 18.6-23.8. are compatible withsupplemental selenium being absorbed to differ in selenium nutritional status at least as indicated a greater extent in the form of selenomethionine than in the by plasma selenoprotein P or glutathione peroxidase. Thus, forms of yeast and selenite. factors that lead to an increase in ‘‘other’’ selenium in plasma, suchas consumption of plant foods richin selenomethionine, need to be considered as the cause of differences in Discussion plasma selenium and as potential influences on any clinical events found to correlate withit. Plasma Biomarkers before Selenium Supplementation. Plasma Biomarkers and Forms of Selenium Administered. Like other healthy North Americans, the subjects were Selenium concentration was the only plasma selenium selenium replete when they entered this study. This conclusion biomarker that was affected by supplementation (Fig. 1). is based on the initial plasma selenium concentration being Because supplementation did not cause an increase in above the level at which plasma selenoproteins are optimized selenoprotein P or glutathione peroxidase, the category of (7, 8), the mean daily urinary selenium excretion by the ‘‘other’’ selenium accounted for the increase. Figure 4 depicts placebo group indicating an intake greater than the recom- some established relationships of ingested selenium forms mended dietary allowance, and, most importantly, the lack of with plasma forms of the element. ‘‘Other’’ plasma selenium effect of selenium supplementation on the plasma selenopro- comprises selenomethionine in albumin and other proteins teins (Fig. 1B and C). This last observation implies that the and small-molecule forms, such as the selenosugar synthesized selenium administered as supplements was not needed to in the liver and bound for the urine. Small-molecule forms support the synthesis of these selenoproteins. Thus, the initial account for <3% of plasma selenium (22), indicating that the selenium biomarker values can be considered to indicate a ‘‘other’’ selenium is mostly selenomethionine. selenium-replete state. Forms, suchas selenite and selenocysteine (in selenopro- Epidemiology studies often rank subjects according to their teins), enter directly into the selenium pool (Fig. 4). They plasma selenium concentrations (e.g., in tertiles). The incidence cannot be converted to selenomethionine and therefore cannot of pathologic conditions or responses to chemoprevention in enter the methionine pool and contribute to the ‘‘other’’ each tertile is then determined. Table 3 shows that plasma selenium. This accounts for the relative inability of selenite selenium did not correlate with either selenoprotein in these supplements to raise plasma selenium in this study (Fig. 1A). selenium-replete subjects. Even when biomarkers of the lowest tertile of plasma selenium concentrations were compared with Bioavailability of Selenium in the Supplements. An earlier one another, there were still no correlations. This indicates that study carried out by our group in selenium-deficient Chinese forming groups of selenium-replete subjects according to subjects indicated that selenium given in the form of plasma selenium concentration will not constitute groups that selenomethionine had almost twice the bioavailability of

Figure 3. Urinary selenium excretion after 16 weeks of selenium supplementation in three forms. Each value has had the placebo mean of 55 Ag subtracted and is shown in relation to the daily selenium supplement. The best-fit lines were calculated by linear regression analysis and their equations were as follows: selenite, y = 0.45x + 45; selenomethionine, y = 0.74x + 14; yeast, y = 0.57x + 33. The R2s are 0.52 for selenite, 0.59 for selenomethionine, and 0.54 for yeast. Ps for all three (significantly different from 0) are <0.001. The 95% confidence interval for the slopes are as follows: selenite, 0.24-0.65; selenomethionine, 0.43-1.05; yeast, 0.31-0.83.

beings than selenium in the form of selenite. Selenite selenium raised plasma selenoproteins in selenium-deficient Chinese with f60% of the effectiveness of selenomethionine selenium (8). In the present study, a greater proportion of administered selenium was excreted in the urine by subjects given selenomethionine (60%) than by subjects given selenite (41%), and similar results on urinary selenium excretion were obtained in the study carried out in China.5 When averaged, these findings suggest that absorption of selenite selenium is approximately two-thirds of the absorption of selenomethionine selenium. Animal studies have generally shown similar bioavailabilities of these two forms of selenium (23), but stable isotope studies in human beings have indicated that only f Figure 4. Relationship of ingested forms of selenium with plasma 50% of selenite selenium is absorbed (24) and >90% of forms of the element. Selenomethionine in the methionine pool is selenomethionine selenium is absorbed (25). Thus, it seems metabolized via the trans-sulfuration pathway and selenocysteine reasonable to assign relative selenium bioavailabilities of 1.0 h-lyase (28) to yield metabolically active selenium in the selenium for selenomethionine and 0.64 (average of estimated relative absorption in this study and bioavailability in the China study) pool. The selenosugar (1h-methylseleno-N-acetyl-D-galactosamine) does not accumulate in plasma because it is filtered into the urine by for selenite. This difference in bioavailabilities has implications the kidney (29). for formulation of selenium supplements. Selenite selenium is muchless effective thanselenome- thionine selenium in raising plasma selenium concentration selenium given as selenite based on the biomarkers seleno- in selenium-replete individuals (Fig. 2). Part of this difference protein P and glutathione peroxidase (8). Those biomarkers can be attributed to less efficient absorption, but the slope of cannot be used to estimate bioavailability in the present study plasma selenium concentration versus dose of supplementa- because they were optimized when the study started and tion for selenite is only 11% that of selenomethionine (Fig. 2). therefore did not respond to selenium supplementation. Thus, selenite acts as predicted by Fig. 4 in not being Neither can plasma selenium concentration be used here to available to the methionine pool. This limits its plasma estimate bioavailability because the forms administered had incorporation to the selenoproteins and the tiny small- different characteristics of incorporation into plasma. This molecule fraction. leaves only urinary excretion as an estimate of bioavailability The yeast preparation used contained an estimated 75% of (or absorption) of selenium in this study. its selenium as selenomethionine. Urinary excretion of If the assumption is made that a steady state in the selenium by this group was intermediate between the metabolism of selenium and selenomethionine existed after selenomethionine and the selenite groups (Fig. 3), suggesting the 16 weeks of supplementation, urinary excretion of the that absorption of selenium in the form of yeast is greater than element should have been proportional to absorbed selenium. selenite selenium but less than selenomethionine selenium. Urinary excretion will actually be less than the amount of Yeast was also intermediate between the other two forms in selenium absorbed because some endogenous selenium is lost raising plasma selenium concentration (Fig. 2). The slope of in sloughed skin cells and in feces. More selenium was selenium concentration versus dose of selenium as yeast was excreted in the urine when it was administered as selenome- 73% that of selenomethionine. This value closely approximates thionine than when it was administered as selenite. Therefore, the fraction of selenium estimated by the manufacturer to be we conclude that selenium was better absorbed, and conse- selenomethionine in yeast. Both these findings are compatible quently more bioavailable, when administered as selenome- with the minor forms of selenium in the yeast, having little thionine than when given as selenite. This leads to the effect on plasma selenium and perhaps not even being conclusion that selenium in the form of selenomethionine has absorbed. Thus, additional work on yeast is needed to greater bioavailability than selenium as selenite in selenium- determine the absorption and metabolism of its different replete NorthAmericans as well as in selenium-deficient chemical forms of selenium. Chinese (8). Yeast seems to be intermediate between the other Implications for Safe Upper Levels of Selenium Intake. two forms possibly because most of the selenium present in Some of the subjects in this study ingested >800 Ag selenium/d yeast is selenomethionine. for 16 weeks. This is considerably more than the Institute of Use of Plasma Selenium to Estimate Compliance in Medicine’s tolerable upper level of 400 Ag/d (4). No signs of Selenium Supplementation Trials. The results of this study selenium toxicity (hair loss and nail changes) were observed, show that plasma selenium can be used to estimate compliance in agreement withChinese observations in subjects with of selenomethionine intake but not of selenite intake. Figure 2 intakes of f800 Ag (26). Moreover, plasma selenium concen- shows that administration of selenomethionine for 16 weeks trations (Fig. 1A) were lower than levels determined in healthy raised plasma selenium concentration according to the farmers in Enshi County,5 an area in China with high selenium equation: DPlasma selenium concentration = 28.5 kg/L Â in the soil where selenium toxicity is not recognized now but daily selenium dose (Ag/kg). was reported in the past (27). The use of this equation requires that the presupplementa- tion plasma selenium concentration be known. Thus, the expected increase in plasma selenium is 285 Ag/L for a daily Conclusions dose of 10 Ag selenium as selenomethionine/kg. Such a This study of selenium-replete subjects compared the effects of measurement could be used to assess compliance and guard supplementing three forms of selenium in moderate to high against toxicity. doses on selenium biomarkers. Plasma selenium concentration Biological Properties of the Forms of Selenium Supple- was responsive to the supplements in proportion to their mented. This study and our recent study carried out in China (8) allow several inferences about the forms of selenium that we used for supplementation. The first is that selenium in the form of selenomethionine has greater bioavailability in human 5 Y. Xia et al., unpublished observations.

Cancer Epidemiol Biomarkers Prev 2006;15(4). April 2006 Downloaded from cebp.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. 810 Selenium Supplements and Plasma Biomarkers selenomethionine content. We propose that the increment in 10. Waschulewski IH, Sunde RA. Effect of dietary methionine on tissue selenium and glutathione peroxidase (EC 1.11.1.9) activity in rats fed plasma selenium concentration during supplementation can be selenomethionine. Br J Nutr 1998;60:57 – 68. used to estimate compliance and ensure safety of selenome- 11. Moreno-Reyes R, Suetens C, Mathieu F, et al. Kashin-Beck osteoarthropathy thionine supplements given in chemoprevention studies. in rural Tibet in relation to selenium and iodine status. N Engl J Med 1998; Bioavailability of selenium, as estimated by urinary excre- 339:1112 – 20. 12. Dumas BT, Watson WA, Biggs HG. Albumin standards and the measure- tion after 16 weeks of supplementation, was greatest for ment of serum albumin withbromcresol green. Clin ChimActa 1997;258: selenomethionine, less for yeast, and least for selenite. Yeast 21 – 30. contains selenium mostly as selenomethionine but has a 13. Xia Y, Hill KE, Burk RF. Biochemical studies of a selenium-deficient significant amount of selenium in other forms. The results of population in China: measurement of selenium, glutathione peroxidase and oxidant defenses in blood. J Nutr 1989;119:1318 – 26. this study are compatible with sharply differing fates of 14. KohTS, Benson TH. Critical re-appraisal of fluorometric methodfor different yeast selenium forms. determination of selenium in biological materials. J Assoc Off Anal Chem Some of the subjects in this study had total daily intakes of 1983;66:918 – 26. over 800 Ag selenium for 16 weeks. No signs of selenium 15. Sheehan TMT, Gao M. Simplified fluorometric assay of total selenium in plasma and urine. Clin Chem 1990;36:2124 – 6. toxicity were detected in them. Thus, we conclude that total ˚ A 16. Hill KE, Xia Y, Akesson B, Boeglin ME, Burk RF. Selenoprotein P intakes of selenium in the range of 800 g/d can be used safely concentration in plasma is an index of selenium status in selenium-deficient in studies of limited duration if the subjects are monitored and selenium-supplemented Chinese subjects. J Nutr 1996;126:138 – 45. closely for signs of selenium toxicity. 17. Avissar N, Whitin JC, Allen PZ, Palmer IS, Cohen HJ. Antihuman plasma glutathione peroxidase antibodies: immunologic investigations to determine plasma glutathione peroxidase protein and selenium content in plasma. Acknowledgments Blood 1989;73:318 – 23. 18. Burk RF, Early DS, Hill KE, Palmer IS, Boeglin ME. Plasma selenium in We thank the staff at the General Clinical Research Center at the patients withcirrhosis. Hepatology 1998;27:794 – 8. Vanderbilt University Medical Center for their work on this study, Sue 19. Heinegard D, Tiderstrom G. Determination of serum creatinine by a direct Taylor and Rebecca Klinger for having randomized the subjects and colorimetric method. Clin Chim Acta 1973;43:305 – 10. prepared the bottles of supplements, and the study participants. 20. Niskar AS, Paschal DC, Kieszak SM, et al. Serum selenium levels in the US population: Third National Health and Nutrition Examination Survey, 1988- 1994. Biol Trace Elem Res 2003;91:1 – 10. 21. Butler JA, Thomson C, Whanger PD, Robinson MF. Selenium distribution in References blood fractions of New Zealand women taking organic or inorganic 1. Clark LC, Combs GF, Jr., Turnbull BW, et al. Effects of selenium selenium. Am J Clin Nutr 1991;53:748 – 54. supplementation for cancer prevention in patients withcarcinoma of the 22. Burk RF. Binding of 75Se to human plasma proteins. Biochim Biophys Acta skin. A randomized controlled trial. Nutritional Prevention of Cancer Study 1974;372:255 – 65. Group. JAMA 1996;276:1957 – 63. 23. Sunde RA, Gutzke GE, Hoekstra WG. Effect of dietary methionine on the 2. Lippman SM, Goodman PJ, Klein EA, et al. Designing the Selenium and biopotency of selenite and selenomethionine in the rat. J Nutr 1981;111: Vitamin E Cancer Prevention Trial (SELECT). J Natl Cancer Inst 2005;97: 76 – 88. 94 – 102. 24. Van Dael P, Davidsson L, Munoz-Box R, Fay LB, Barclay D. Selenium 3. Ip C, Birringer M, Block E, et al. Chemical speciation influences comparative absorption and retention from a selenite- or selenate-fortified milk-based activity of selenium-enriched garlic and yeast in mammary cancer formula in men measured by a stable-isotope technique. Br J Nutr 2001;85: prevention. J Agric Food Chem 2000;48:2062 – 70. 157 – 63. 4. Institute of Medicine. Selenium. In: Dietary reference intakes for vitamin C, 25. Mangels AR, Moser-Veillon PB, Patterson KY, Veillon C. Selenium vitamin E, selenium, and carotenoids. Washington (DC): National Academy utilization during human lactation by use of stable-isotope tracers. Am J Press; 2000. p. 284 – 324. Clin Nutr 1990;52:621 – 7. 5. Reid ME, Stratton MS, Lillico AJ, et al. A report of high-dose selenium 26. Yang G-Q, Zhou R-H. Further observations on the human maximum safe supplementation: response and toxicities. J Trace Elem Med Biol 2004;18: dietary selenium intake in a seleniferous area of China. J Trace Elem 69 – 74. Electrolytes HealthDis 1994;8:159 – 65. 6. Duffield AJ, Thomson CD, Hill KE, et al. An estimation of selenium 27. Yang GQ, Wang S, Zhou R, Sun S. Endemic selenium intoxication of humans requirements for New Zealanders. Am J Clin Nutr 1999;70:896 – 903. in China. Am J Clin Nutr 1983;37:872 – 81. 7. Persson-Moschos M, Alfthan G, Akesson B. Plasma selenoprotein P levels of 28. Esaki N, Nakamura T, Tanaka H, Soda K. Selenocysteine lyase, a novel healthy males in different selenium status after oral supplementation with enzyme that specifically acts on selenocysteine. Mammalian distribution different forms of selenium. Eur J Clin Nutr 1998;52:363 – 7. and purification and properties of pig liver enzyme. J Biol Chem 1982;257: 8. Xia Y, Hill KE, Byrne DW, Xu J, Burk RF. Effectiveness of selenium 4386 – 91. supplements in a low-selenium area of China. Am J Clin Nutr 2005;81:829–34. 29. Kobayashi Y, Ogra Y, Ishiwata K, Takayama H, Aimi N, Suzuki KT. 9. Burk RF, Hill KE, Motley AK. Plasma selenium in specific and non-specific Selenosugars are key and urinary metabolites for selenium excretion within forms. Biofactors 2001;14:107 – 14. the required to low-toxic range. Proc Natl Acad Sci U S A 2002;99:15932 – 6.

Cancer Epidemiol Biomarkers Prev 2006;15(4). April 2006 Downloaded from cebp.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Effects of Chemical Form of Selenium on Plasma Biomarkers in a High-Dose Human Supplementation Trial

Raymond F. Burk, Brooke K. Norsworthy, Kristina E. Hill, et al.

Cancer Epidemiol Biomarkers Prev 2006;15:804-810.

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