- Email: [email protected]
Toenail and Plasma Levels as Biomarkers of Selenium Exposure
Toenail and Plasma Levels as Biomarkers of Selenium Exposure JESSIE A. SATIA, PHD, MPH, IRENA B. KING, PHD, J. STEVEN MORRIS, PHD, KAYLA STRATTON, MS, AND EMILY WHITE, PHD
PURPOSE: Both blood and toenail selenium are used to assess selenium exposure in epidemiologic studies. Little is known about the relationship of these biomarkers with each other or about whether there are differences in the relationships of these biomarkers with diet, supplement use, or participant characteristics. METHODS: Data are from 220 participants in a large cohort study of supplement use and cancer risk. Measures of selenium exposure included supplement use (current and 10-year) from a self-administered questionnaire, an inventory of currently used supplements (multivitamins and single supplements), dietary intake from a food frequency questionnaire (FFQ), and selenium concentration in toenails and plasma. RESULTS: Plasma and toenail selenium concentrations were significantly correlated (rZ.56 [95% confidence interval: .46, .64]). Supplemental selenium was the strongest predictor of both selenium biomarkers, and these associations were slightly stronger when based on the supplement inventory and 10-year self-reported use compared to current self-reported use. Correlations of current and 10-year questionnaire dose and inventory dose with toenail selenium were .26, .36, and .33; for plasma selenium, these were .27, .36, and .36. Neither dietary selenium nor any participant characteristics, except smoking, was related to either biomarker. Current smokers had lower toenail, but not plasma, selenium levels compared to nonsmokers (.89 versus 1.03mg/g, p Z .03); however, the difference was not significant after control for supplement use (p Z .09). CONCLUSIONS: Both toenail and plasma selenium levels similarly reflect selenium intake exposure. There do not appear to be independent associations of toenail or plasma selenium with FFQ-derived selenium intakes, health-related behaviors, or demographic characteristics. Ann Epidemiol 2006;16:53–58. Ó 2006 Elsevier Inc. All rights reserved. KEY WORDS:
Selenium, Toenails, Plasma, Biomarkers, Supplements, Validity, Diet.
INTRODUCTION High intake of selenium, an essential trace nutrient, is associated with reduced risks of cancer and other chronic diseases (1–3). Assessing selenium exposure in epidemiologic studies is challenging. Dietary assessment instruments cannot accurately measure selenium intake from foods, because the selenium content of similar foods varies widely due to geographic variations in soil selenium concentrations (4, 5). Dietary supplements may also contribute substantially to selenium exposure; however, accurate From Department of Nutrition, University of North Carolina at Chapel Hill, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill (J.A.S.); Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle, WA (I.B.K., E.W.); University of Missouri–Columbia, Research Reactor Center, Columbia, MO (J.S.M.); and Department of Epidemiology, University of Washington, Seattle, WA (E.W.). Address correspondence and reprint requests to Jessie A. Satia, PhD, MPH, Amgen Inc., Department of Global Epidemiology, One Amgen Center Drive, 24-1-C, Thousand Oaks, CA 91320. Tel.: (805) 313-4097. fax: (805) 498-5593. E-mail: [email protected] This research was supported by National Cancer Institute Grant R01 CA74846 and K22 CA96556. Received October 1, 2004; accepted February 1, 2005. Ó 2006 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
assessment of supplement type, dose, and frequency of use requires detailed and lengthy questionnaires (4–6), and many of the dietary supplement assessment tools used in epidemiologic studies have not been adequately validated (4–7). For these reasons, selenium exposure is often assessed as its concentration in toenails or blood (4, 7–10). Toenail clippings are considered a superior marker of selenium status because they provide a time-integrated measure of exposure of up to a year, while blood levels are considered more appropriate as a short-term marker of selenium exposure (7, 8). However, there is limited knowledge about the relationships of selenium biomarkers with each other, and very few studies have examined whether relationships of demographic and behavioral characteristics between toenail and blood selenium differ. This information can be helpful when choosing and analyzing data from a selenium biomarker. Thus, the objectives of this report are to (1) describe associations between toenail and plasma selenium in the same set of participants, (2) assess associations of dietary and supplemental selenium intakes with toenail and plasma levels, and (3) examine demographic and behavioral correlates of toenail and plasma selenium concentrations. 1047-2797/06/$–see front matter doi:10.1016/j.annepidem.2005.02.011
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Satia et al. BIOMARKERS OF SELENIUM EXPOSURE
Selected Abbreviations and Acronyms FFQ Z food frequency questionnaire
MATERIALS AND METHODS Data Collection Study procedures. Data are from a supplement questionnaire validation study within the VITamins And Lifestyle (VITAL) Study, a cohort investigation of dietary supplement use and cancer risk in western Washington State (11). Details on the validation study have been published (12). The mean age of the validation-study participants was 62 years; half were female, 95% were Caucasian, 5% were current smokers, and 17% were obese, with a body mass index (BMI) greater than 30kg/m2. Validation study participants completed a repeat VITAL baseline questionnaire and an in-person dietary supplement inventory with interview, and they also provided toenail clippings and blood samples. The study was approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center (FHCRC). Questionnaire and home interview data. All questionnaire data, including demographic and behavioral characteristics, food intake, and supplement use, were obtained from the repeat baseline questionnaire. Diet was assessed using a food frequency questionnaire (FFQ) developed at the FHCRC that asked about usual consumption of foods eaten during the past month (13). Supplement use was assessed by questionnaire and by inventory with interview. The questionnaire assessed both current use and use over the previous 10 years of 10 vitamins and 6 minerals from multivitamins, mixtures (e.g., antioxidant mixtures), and single supplements. Responses were closed-ended for frequency of use (1–2; 3–4; 5–6; 7 days per week), duration of use in the previous 10 years (1–3; 4–6; 7–9; 10 C years), daily dose (based on the most common formulations for each supplement, e.g., selenium at 25, 50, 100, and 200 mcg), and current use (yes or no) (12). For multivitamins, participants could report brands from two checklists: one for current and the other for past use. Dose of selenium in these brands was obtained from published sources (14) and inquiries to manufacturers. Participants whose current multivitamin brand was not on the checklist(s) were instructed to complete a section of the questionnaire on the amount of each nutrient, including selenium, in their multivitamin. For all supplements, participants were instructed to look at their supplement bottle labels when completing the questionnaire. During the home inventory, for all supplements used, interviewers used an open-ended format to query participants on frequency of use and number of pills, and they transcribed the vitamin or
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mineral dose per pill from each supplement bottle label. For these analyses, selenium supplement use was described by supplement type (i.e., none, multivitamin use only, or single supplement use only) and dose (from both multivitamins and single supplements). Compared to the home inventory (i.e., criterion measure), the questionnaire-derived measure of current supplemental selenium use had high validity (r Z .77) (12). Biological markers. Semi-fasting (R 6 hours) blood samples were processed within 2 hours of collection and stored at –80 C until analysis. Plasma selenium concentrations (mg/L) were measured using the electrothermal absorption spectrometry technique, based on previously described procedures (15). The intra-assay coefficient of variation (CV) was less than 3.7%, and the inter-assay CV was 3.8%. Toenail selenium concentrations (mg/g) were determined by instrumental neutron activation analysis at the University of Missouri Research Reactor Center (Columbia, MO). Details of these methods have been published (7–10). Statistical Analysis Pearson and Spearman correlations and their 95% confidence intervals (95% confidence interval [CI]), raw and adjusted for age, sex, BMI, race (white/nonwhite), and current smoking were computed to assess the association between toenail and plasma selenium concentrations. Linear regression and Pearson correlations were used to examine associations of toenail and plasma selenium with supplement use, diet, demographic characteristics, and health-related behaviors, controlling for age, sex, BMI, smoking, and supplement use. Dietary, supplemental, and biomarker selenium values were log-transformed and backtransformed into their original units. Analyses were conducted using SAS 8.2 (SAS Institute Inc., Cary, NC). Statistical significance was set at 0.05.
RESULTS Distributions of and associations between toenail and plasma selenium are given in Table 1. Mean plasma and toenail concentrations were 161 G 29 mg/L and 1.02 G .21 mg/g, respectively. Pearson and Spearman correlations (95% CI) between plasma and toenail selenium adjusted for covariates were .56 (.46, .64) and .46 (.32, .59), respectively. Table 2 gives associations of dietary, supplemental, and total (dietary plus supplemental) selenium intakes with toenail and plasma concentrations. For all measures of supplement use (current intake from the home inventory, selfreport of current use, and self-report of 10-year use), there were statistically significant and positive dose-response relationships between selenium intake from supplements
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and selenium biomarkers; however, the gradients in response were largely driven by the highest levels of intake. These associations were slightly stronger when selenium from supplements was based on current intake from the inventory and 10-year intake from questionnaire compared to current use from questionnaire. For example, the adjusted Pearson’s r for current supplemental intake from questionnaire, the supplement inventory, and 10-year intake from questionnaire with toenail selenium levels were .26, .33 and .36, respectively. Not unexpectedly, neither biomarker was associated with dietary selenium as assessed by a FFQ. Overall, the magnitudes of the correlations between each selenium intake measure and the biomarkers were similar for toenail and plasma, except for a stronger correlation of total (i.e., diet plus supplements) selenium intake with plasma (.38) compared to toenail (.31) selenium levels. Associations of various demographic and health-related behaviors with toenail and plasma selenium were also examined (data not shown). There were no differences in plasma or toenail selenium across participants categorized by sex, age, race, BMI, levels of physical activity, alcohol use, or consumption of foods high in selenium. Current cigarette smoking was associated with significantly lower toenail, but not plasma, selenium. After adjustment for age, sex, and BMI, toenail selenium was 13% lower among current smokers compared to non-smokers (.89 versus 1.03 mg/g, p Z .03); however, this relationship was no longer significant after control for selenium supplement use (p Z .09).
DISCUSSION We found a moderate correlation between plasma and toenail selenium concentrations. We also found little difference between associations of supplemental selenium intake with plasma selenium compared to supplemental intake with toenail selenium. Unexpectedly, associations with both biomarkers were slightly stronger for questionnaire-based 10-year compared to current supplement use. Finally, neither FFQ-derived selenium intake, frequency of eating selected foods with high selenium content (cereals, fish, and red meat), nor any demographic or behavioral characteristics was significantly associated with either selenium biomarker after control for age, sex, BMI, smoking, and selenium supplement use. As in previous studies, selenium intake, particularly from supplements, was strongly associated with biomarker levels; in other reports, correlations have ranged from .32 to .74 (7, 8, 16). The fact that toenail and plasma selenium levels were higher for single compared to multivitamin users is not surprising, as most commonly used multivitamins typically
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TABLE 1. Distributions and associations of toenail and plasma selenium (n Z 220), 2001
Mean G SD 25th %tile 50th %tile 75th %tile Raw Pearson’s r (95% CI) Partial Pearson’s r (95% CI)2 Raw Spearman’s r (95% CI) Partial Spearman’s r (95% CI)2
Toenail selenium (mg/g)1
Plasma selenium (mg/L)1
1.02 G.21 0.86 0.99 1.13
161 G 29 144 156 169 .55 (.45, .64) .56 (.46, .64) .43 (.30, .56) .46 (.32, .59)
NOTE: All correlations significant at p ! 0.0001. 1 Plasma selenium values were log transformed, toenail selenium was not transformed. 2 Partial Pearson’s and Spearman’s correlations are adjusted for age, sex, race, body mass index, and current smoking. All variables were treated as continuous except for sex, race, and smoking.
contain lower doses of selenium than do single supplements (14). Also not surprisingly, correlations with the selenium biomarkers were somewhat stronger for supplemental selenium intakes from the inventory plus interview than from self-report (i.e., the questionnaire), suggesting that the inventory is a superior measure of selenium intake. However, compared to the self-administered questionnaire, the inventory is a more detailed approach and requires an in-person interview, which may not be practical for large epidemiologic studies. The stronger association for questionnaire-derived 10-year compared to current supplement use with both toenail and plasma selenium challenges the hypothesis or notion that blood levels of selenium are reflective of shortterm, but not long-term, intake (7, 17, 18). Furthermore, correlations of toenail and plasma concentrations with 10-year supplement use were identical (Pearson’s r Z .36), suggesting that both biomarkers are valid measures of long-term selenium exposure in this study sample. Nonetheless, we note that plasma selenium may only reflect long-term intake among long-standing and consistent selenium supplement users (such as the participants in our study) because although plasma is more sensitive than toenails to day-to-day differences in intake, regular use of a supplement over an extended period may attenuate such variability. Therefore, plasma levels may not be reflective of long-term selenium intake in all populations. As expected, FFQ-derived selenium intakes were not associated with toenail or plasma selenium levels, consistent with other studies (7, 8, 19) showing that self-reported dietary selenium is not a useful measure of selenium exposure.
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TABLE 2. Associations of selenium intakes from diet and supplements with toenail and plasma concentrations (n = 220), 2001 Adjusted means1 Selenium intake Dietary intake (self-report) 0 ! – 80 mcg 81 – 104 mcg 105 – 141 mcg 142 – 286 mcg P for trend Pearson’s r1,3 (p-value) Current supplement use (supplement inventory) Nonusers Multivitamin users only4 Single supplement users5 P value Current supplement dose (supplement inventory) Nonusers 0 ! – 20 mcg 21 – 45 mcg 46 – 186 mcg 187 – 3300 mcg P for trend Pearson’s r1,3 (95% CI) Current supplement use (self-report) Nonusers Multivitamin users only4 Single supplement users5 P value Current supplement dose (self-report) Non-users 0 ! – 20 mcg 21 – 39 mcg 40 – 100 mcg 101 – 250 mcg P for trend Pearson’s r1,3 (95% CI) Dietary intake plus current supplement dose (self-report) 0 ! – 100 mcg 101 – 140 mcg 141 – 200 mcg 201 – 536 mcg P for trend Pearson’s r1,3 (p-value) 10-year supplement use (self-report) Nonusers Multivitamin users only4 Single supplement users5 P value 10-year supplement dose (self-report) Nonusers 0 ! – 10 mcg 11 – 20 mcg 21 – 44 mcg 45 – 244 mcg P for trend Pearson’s r1,3 (95% CI)
%
Mean intakes (mcg)
Toenail Selenium (mg/g)
Plasma selenium2 (mg/L)
25.4 24.4 25.4 24.9
63.9 92.5 121.0 185.5
1.00a 1.03a 1.03a 1.02a .45 .09 (ÿ.05, .22)
160a 155a 160a 162a .58 .08 (ÿ.06, .21)
33.8 46.1 20.1
0.0 116.0 224.9
33.8 24.7 12.8 12.3 16.4
0 17.9 37.2 97.6 471.4
.97a .97a .92a 1.09b 1.20c .0001 .33 (.20, .44)
153a 152a 152a 159a 189b .0001 .36 (.24, .47)
34.3 48.9 16.9
0.0 45.7 141.3
.99a .98a 1.18b .0001
156a 154a 181b .0001
34.3 23.7 9.1 19.6 13.2
0.0 17.7 26.1 67.8 198.4
.99a .95a .99a 1.04a 1.20b .0001 .26 (.13, .38)
156a 151a 151a 160a 188b .0001 .27 (.15, .39)
25.0 25.0 25.0 25.0
79.3 11.8 164.9 283.1
.99a .98a .97a 1.13b .0003 .31 (.18, .43)
154a 151a 156a 175b !.0001 .38 (.26, .49)
25.6 51.6 22.8
0.0 22.3 69.5
.97a .97a 1.19b .0001
152a 155a 175b .0001
25.6 20.6 19.2 16.4 18.3
0.0 5.1 17.1 30.9 98.4
.97a .93a .99a 1.02a 1.21b .0001 .36 (.24, .47)
152a 151a 157a 157a 182b .0001 .36 (.24, .47)
.97a 1.03a 1.08a .03
153a 160a 166a .01
NOTE: Plasma selenium was log-transformed, toenail selenium was not transformed. a-c For each selenium intake variable, means with different superscripts are statistically significantly different from each other (p ! 0.05) 1 Means and Pearson’s correlation coefficients adjusted for age, sex, body mass index, and current smoking. For partial Pearson’s correlations, all variables were treated as continuous except for sex, race, and smoking. 2 Least square means of plasma selenium values were log transformed and then back-transformed to the original units. 3 The dietary, supplement use, or total (dietary and supplemental) intake variables were log-transformed and back-transformed into their original units. 4 Multivitamin users only: current self-report, n Z 107; current interview, n Z 101, 10-year self-report, n Z 113. 5 Single supplement users: current self-report, n Z 37; current interview, n Z 44, 10-year self-report, n Z 50.
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Of the demographic and behavioral characteristics examined, only smoking was significantly associated with toenail selenium, similar to the finding reported by others (7, 16, 17, 19). In this study, however, the lower toenail selenium concentration among current smokers is largely due to lower selenium intakes (diet plus supplements) among current smokers compared to non-smokers: 115 and 164 mcg, respectively, p Z .04 (data not shown). Consequently, after control for supplement use, the association between smoking and toenail selenium was no longer significant (p Z .09). Nonetheless, we are unable to explain why these differences in selenium intake across categories of smokers were reflected in toenails but not in plasma. Swanson and colleagues similarly observed that smoking was an independent determinant of toenail, but not serum or whole blood selenium concentrations (16). There have been reports of positive significant associations of other demographic and behavioral characteristics, including younger age (7), female sex (19–21), and nonuse of alcohol (22) with higher selenium biomarker levels. However, because there is no indication that selenium supplement use was controlled for in these analyses, it is not clear that these variables were truly independently associated with the selenium biomarkers in these studies. A major strength of this study is that it is, to our knowledge, the first to describe, directly, the relationship between toenail and plasma selenium concentrations and to compare their associations with both current and longterm selenium intake. Specifically, both biomarker measures were available from the same set of study participants, thereby allowing the estimation of their correlation and a direct, within-study comparison of which biomarker may more accurately reflect selenium status. In addition, we examined a wide array of potential demographic, lifestyle, and behavioral correlates. Potential limitations of the study include the possibility of a limited range of selenium intake among participants, the limited number of current smokers in the study population, and the challenges of assessing selenium exposure from diet and self-reported supplement use. Studies with more accurate assessments of selenium from diet have found dietary selenium to be the primary determinant of tissue selenium concentrations (8, 16). In conclusion, our findings suggest that both toenail and plasma selenium concentrations are equally valid measures of current, as well as long-term selenium intakedin particular, high supplement use. The data also show that selenium from supplements is the strongest predictor of biomarker levels, whereas selenium intakes from an FFQ and most demographic characteristics and health-related behaviors do not appear to be independently associated with toenail or plasma selenium levels.
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REFERENCES 1. Thomson CD. Assessment of requirements for selenium and adequacy of selenium status: A review. Eur J Clin Nutr. 2004;58:391–402. 2. Li H, Stampfer MJ, Giovannucci EL, Morris JS, Willett WC, Gaziano JM, et al. A prospective study of plasma selenium levels and prostate cancer risk. J Natl Cancer Inst. 2004;96:696–703. 3. Daniels LA. Selenium: Does selenium status have health outcomes beyond overt deficiency? Med J Aust. 2004;180:373–374. 4. Mayne ST. Antioxidant nutrients and chronic disease: Use of biomarkers of exposure and oxidative stress status in epidemiologic research. J Nutr. 2003;(suppl 3):933S–940S. 5. Neuhouser ML. Dietary supplement use by American women: Challenges in assessing patterns of use, motives and costs. J Nutr. 2003Jun;133: 1992S–1996S. 6. Patterson RE, Kristal AR, Levy L, McLerran D, White E. Validity of methods used to assess vitamin and mineral supplement use. Am J Epidemiol. 1998;148:643–649. 7. Hunter DJ, Morris JS, Chute CG, Kushner E, Colditz GA, Stampfer MJ, et al. Predictors of selenium concentration in human toenails. Am J Epidemiol. 1990;132:114–122. 8. Longnecker MP, Stram DO, Taylor PR, Levander OA, Howe M, Veillon C, et al. Use of selenium concentration in whole blood, serum, toenails, or urine as a surrogate measure of selenium intake. Epidemiology. 1996; 7:384–390. 9. Morris JS, Stampfer MJ, Willett W. Dietary selenium in humans: Toenails as an indicator. Biol Trace Elem Res. 1983;5 539–537. 10. Yoshizawa K, Ascherio A, Morris JS, Stampfer MJ, Giovannucci E, Baskett CK, et al. Prospective study of selenium levels in toenails and risk of coronary heart disease in men. Am J Epidemiol. 2003;158: 852–860. 11. White E, Patterson RE, Kristal AR, Thornquist M, King I, Shattuck AL, et al. VITamins And Lifestyle cohort study: Study design and characteristics of supplement users. Am J Epidemiol. 2004;1;159:83–93. 12. Satia-Abouta J, Patterson RE, King IB, Stratton KL, Shattuck AL, Kristal AR, et al. Reliability and validity of self-report of vitamin and mineral supplement use in the vitamins and lifestyle study. Am J Epidemiol. 2003;15;157:944–954. 13. Patterson RE, Kristal AR, Tinker LF, et al. Measurement characteristics of the Women’s Health Initiative food frequency questionnaire. Ann Epidemiol. 1999;9:178–187. 14. Physicians’ desk reference for nonprescription drugs and dietary supplements. 23rd ed. Montvale, NJ: Medical Economics Data Publication Co; 2002. 15. Goodman GE, Schaffer S, Bankson DD, Hughes MP, Omenn GS, Carotene and Retinol Efficacy Trial Co-Investigators. Predictors of serum selenium in cigarette smokers and the lack of association with lung and prostate cancer risk. Cancer Epidemiol Biomarkers Prev. 2001;10: 1069–1076. 16. Swanson CA, Longnecker MP, Veillon C, Howe M, Levander OA, Taylor PR, et al. Selenium intake, age, gender, and smoking in relation to indices of selenium status of adults residing in a seleniferous area. Am J Clin Nutr. 1990;52:858–862. 17. Krogh V, Pala V, Vinceti M, Berrino F, Ganzi A, Micheli A, et al. Toenail selenium as biomarker: reproducibility over a one-year period and factors influencing reproducibility. J Trace Elem Med Biol. 2003;17: 31–36. 18. Willett W. Nutritional epidemiology: issues and challenges. Int J Epidemiol. 1987 Jun;16:312–317. 19. van den Brandt PA, Goldbohm RA, van’t Veer P, Bode P, Hermus RJ, Sturmans F. Predictors of toenail selenium levels in men and women. Cancer Epidemiol Biomarkers Prev. 1993 Mar-Apr;2: 107–112.
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20. Zeegers MP, Goldbohm RA, Bode P, van den Brandt PA. Prediagnostic toenail selenium and risk of bladder cancer. Cancer Epidemiol Biomarkers Prev. 2002;11:1292–1297. 21. Morris JS, Tohan T, Soskolne CL, Jain M, Horsman TL, Spate VL, et al. Selenium status and cancer mortality in subjects residing in four Canadian
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PURPOSE: Both blood and toenail selenium are used to assess selenium exposure in epidemiologic studies. Little is known about the relationship of these biomarkers with each other or about whether there are differences in the relationships of these biomarkers with diet, supplement use, or participant characteristics. METHODS: Data are from 220 participants in a large cohort study of supplement use and cancer risk. Measures of selenium exposure included supplement use (current and 10-year) from a self-administered questionnaire, an inventory of currently used supplements (multivitamins and single supplements), dietary intake from a food frequency questionnaire (FFQ), and selenium concentration in toenails and plasma. RESULTS: Plasma and toenail selenium concentrations were significantly correlated (rZ.56 [95% confidence interval: .46, .64]). Supplemental selenium was the strongest predictor of both selenium biomarkers, and these associations were slightly stronger when based on the supplement inventory and 10-year self-reported use compared to current self-reported use. Correlations of current and 10-year questionnaire dose and inventory dose with toenail selenium were .26, .36, and .33; for plasma selenium, these were .27, .36, and .36. Neither dietary selenium nor any participant characteristics, except smoking, was related to either biomarker. Current smokers had lower toenail, but not plasma, selenium levels compared to nonsmokers (.89 versus 1.03mg/g, p Z .03); however, the difference was not significant after control for supplement use (p Z .09). CONCLUSIONS: Both toenail and plasma selenium levels similarly reflect selenium intake exposure. There do not appear to be independent associations of toenail or plasma selenium with FFQ-derived selenium intakes, health-related behaviors, or demographic characteristics. Ann Epidemiol 2006;16:53–58. Ó 2006 Elsevier Inc. All rights reserved. KEY WORDS:
Selenium, Toenails, Plasma, Biomarkers, Supplements, Validity, Diet.
INTRODUCTION High intake of selenium, an essential trace nutrient, is associated with reduced risks of cancer and other chronic diseases (1–3). Assessing selenium exposure in epidemiologic studies is challenging. Dietary assessment instruments cannot accurately measure selenium intake from foods, because the selenium content of similar foods varies widely due to geographic variations in soil selenium concentrations (4, 5). Dietary supplements may also contribute substantially to selenium exposure; however, accurate From Department of Nutrition, University of North Carolina at Chapel Hill, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill (J.A.S.); Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle, WA (I.B.K., E.W.); University of Missouri–Columbia, Research Reactor Center, Columbia, MO (J.S.M.); and Department of Epidemiology, University of Washington, Seattle, WA (E.W.). Address correspondence and reprint requests to Jessie A. Satia, PhD, MPH, Amgen Inc., Department of Global Epidemiology, One Amgen Center Drive, 24-1-C, Thousand Oaks, CA 91320. Tel.: (805) 313-4097. fax: (805) 498-5593. E-mail: [email protected] This research was supported by National Cancer Institute Grant R01 CA74846 and K22 CA96556. Received October 1, 2004; accepted February 1, 2005. Ó 2006 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
assessment of supplement type, dose, and frequency of use requires detailed and lengthy questionnaires (4–6), and many of the dietary supplement assessment tools used in epidemiologic studies have not been adequately validated (4–7). For these reasons, selenium exposure is often assessed as its concentration in toenails or blood (4, 7–10). Toenail clippings are considered a superior marker of selenium status because they provide a time-integrated measure of exposure of up to a year, while blood levels are considered more appropriate as a short-term marker of selenium exposure (7, 8). However, there is limited knowledge about the relationships of selenium biomarkers with each other, and very few studies have examined whether relationships of demographic and behavioral characteristics between toenail and blood selenium differ. This information can be helpful when choosing and analyzing data from a selenium biomarker. Thus, the objectives of this report are to (1) describe associations between toenail and plasma selenium in the same set of participants, (2) assess associations of dietary and supplemental selenium intakes with toenail and plasma levels, and (3) examine demographic and behavioral correlates of toenail and plasma selenium concentrations. 1047-2797/06/$–see front matter doi:10.1016/j.annepidem.2005.02.011
54
Satia et al. BIOMARKERS OF SELENIUM EXPOSURE
Selected Abbreviations and Acronyms FFQ Z food frequency questionnaire
MATERIALS AND METHODS Data Collection Study procedures. Data are from a supplement questionnaire validation study within the VITamins And Lifestyle (VITAL) Study, a cohort investigation of dietary supplement use and cancer risk in western Washington State (11). Details on the validation study have been published (12). The mean age of the validation-study participants was 62 years; half were female, 95% were Caucasian, 5% were current smokers, and 17% were obese, with a body mass index (BMI) greater than 30kg/m2. Validation study participants completed a repeat VITAL baseline questionnaire and an in-person dietary supplement inventory with interview, and they also provided toenail clippings and blood samples. The study was approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center (FHCRC). Questionnaire and home interview data. All questionnaire data, including demographic and behavioral characteristics, food intake, and supplement use, were obtained from the repeat baseline questionnaire. Diet was assessed using a food frequency questionnaire (FFQ) developed at the FHCRC that asked about usual consumption of foods eaten during the past month (13). Supplement use was assessed by questionnaire and by inventory with interview. The questionnaire assessed both current use and use over the previous 10 years of 10 vitamins and 6 minerals from multivitamins, mixtures (e.g., antioxidant mixtures), and single supplements. Responses were closed-ended for frequency of use (1–2; 3–4; 5–6; 7 days per week), duration of use in the previous 10 years (1–3; 4–6; 7–9; 10 C years), daily dose (based on the most common formulations for each supplement, e.g., selenium at 25, 50, 100, and 200 mcg), and current use (yes or no) (12). For multivitamins, participants could report brands from two checklists: one for current and the other for past use. Dose of selenium in these brands was obtained from published sources (14) and inquiries to manufacturers. Participants whose current multivitamin brand was not on the checklist(s) were instructed to complete a section of the questionnaire on the amount of each nutrient, including selenium, in their multivitamin. For all supplements, participants were instructed to look at their supplement bottle labels when completing the questionnaire. During the home inventory, for all supplements used, interviewers used an open-ended format to query participants on frequency of use and number of pills, and they transcribed the vitamin or
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mineral dose per pill from each supplement bottle label. For these analyses, selenium supplement use was described by supplement type (i.e., none, multivitamin use only, or single supplement use only) and dose (from both multivitamins and single supplements). Compared to the home inventory (i.e., criterion measure), the questionnaire-derived measure of current supplemental selenium use had high validity (r Z .77) (12). Biological markers. Semi-fasting (R 6 hours) blood samples were processed within 2 hours of collection and stored at –80 C until analysis. Plasma selenium concentrations (mg/L) were measured using the electrothermal absorption spectrometry technique, based on previously described procedures (15). The intra-assay coefficient of variation (CV) was less than 3.7%, and the inter-assay CV was 3.8%. Toenail selenium concentrations (mg/g) were determined by instrumental neutron activation analysis at the University of Missouri Research Reactor Center (Columbia, MO). Details of these methods have been published (7–10). Statistical Analysis Pearson and Spearman correlations and their 95% confidence intervals (95% confidence interval [CI]), raw and adjusted for age, sex, BMI, race (white/nonwhite), and current smoking were computed to assess the association between toenail and plasma selenium concentrations. Linear regression and Pearson correlations were used to examine associations of toenail and plasma selenium with supplement use, diet, demographic characteristics, and health-related behaviors, controlling for age, sex, BMI, smoking, and supplement use. Dietary, supplemental, and biomarker selenium values were log-transformed and backtransformed into their original units. Analyses were conducted using SAS 8.2 (SAS Institute Inc., Cary, NC). Statistical significance was set at 0.05.
RESULTS Distributions of and associations between toenail and plasma selenium are given in Table 1. Mean plasma and toenail concentrations were 161 G 29 mg/L and 1.02 G .21 mg/g, respectively. Pearson and Spearman correlations (95% CI) between plasma and toenail selenium adjusted for covariates were .56 (.46, .64) and .46 (.32, .59), respectively. Table 2 gives associations of dietary, supplemental, and total (dietary plus supplemental) selenium intakes with toenail and plasma concentrations. For all measures of supplement use (current intake from the home inventory, selfreport of current use, and self-report of 10-year use), there were statistically significant and positive dose-response relationships between selenium intake from supplements
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and selenium biomarkers; however, the gradients in response were largely driven by the highest levels of intake. These associations were slightly stronger when selenium from supplements was based on current intake from the inventory and 10-year intake from questionnaire compared to current use from questionnaire. For example, the adjusted Pearson’s r for current supplemental intake from questionnaire, the supplement inventory, and 10-year intake from questionnaire with toenail selenium levels were .26, .33 and .36, respectively. Not unexpectedly, neither biomarker was associated with dietary selenium as assessed by a FFQ. Overall, the magnitudes of the correlations between each selenium intake measure and the biomarkers were similar for toenail and plasma, except for a stronger correlation of total (i.e., diet plus supplements) selenium intake with plasma (.38) compared to toenail (.31) selenium levels. Associations of various demographic and health-related behaviors with toenail and plasma selenium were also examined (data not shown). There were no differences in plasma or toenail selenium across participants categorized by sex, age, race, BMI, levels of physical activity, alcohol use, or consumption of foods high in selenium. Current cigarette smoking was associated with significantly lower toenail, but not plasma, selenium. After adjustment for age, sex, and BMI, toenail selenium was 13% lower among current smokers compared to non-smokers (.89 versus 1.03 mg/g, p Z .03); however, this relationship was no longer significant after control for selenium supplement use (p Z .09).
DISCUSSION We found a moderate correlation between plasma and toenail selenium concentrations. We also found little difference between associations of supplemental selenium intake with plasma selenium compared to supplemental intake with toenail selenium. Unexpectedly, associations with both biomarkers were slightly stronger for questionnaire-based 10-year compared to current supplement use. Finally, neither FFQ-derived selenium intake, frequency of eating selected foods with high selenium content (cereals, fish, and red meat), nor any demographic or behavioral characteristics was significantly associated with either selenium biomarker after control for age, sex, BMI, smoking, and selenium supplement use. As in previous studies, selenium intake, particularly from supplements, was strongly associated with biomarker levels; in other reports, correlations have ranged from .32 to .74 (7, 8, 16). The fact that toenail and plasma selenium levels were higher for single compared to multivitamin users is not surprising, as most commonly used multivitamins typically
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TABLE 1. Distributions and associations of toenail and plasma selenium (n Z 220), 2001
Mean G SD 25th %tile 50th %tile 75th %tile Raw Pearson’s r (95% CI) Partial Pearson’s r (95% CI)2 Raw Spearman’s r (95% CI) Partial Spearman’s r (95% CI)2
Toenail selenium (mg/g)1
Plasma selenium (mg/L)1
1.02 G.21 0.86 0.99 1.13
161 G 29 144 156 169 .55 (.45, .64) .56 (.46, .64) .43 (.30, .56) .46 (.32, .59)
NOTE: All correlations significant at p ! 0.0001. 1 Plasma selenium values were log transformed, toenail selenium was not transformed. 2 Partial Pearson’s and Spearman’s correlations are adjusted for age, sex, race, body mass index, and current smoking. All variables were treated as continuous except for sex, race, and smoking.
contain lower doses of selenium than do single supplements (14). Also not surprisingly, correlations with the selenium biomarkers were somewhat stronger for supplemental selenium intakes from the inventory plus interview than from self-report (i.e., the questionnaire), suggesting that the inventory is a superior measure of selenium intake. However, compared to the self-administered questionnaire, the inventory is a more detailed approach and requires an in-person interview, which may not be practical for large epidemiologic studies. The stronger association for questionnaire-derived 10-year compared to current supplement use with both toenail and plasma selenium challenges the hypothesis or notion that blood levels of selenium are reflective of shortterm, but not long-term, intake (7, 17, 18). Furthermore, correlations of toenail and plasma concentrations with 10-year supplement use were identical (Pearson’s r Z .36), suggesting that both biomarkers are valid measures of long-term selenium exposure in this study sample. Nonetheless, we note that plasma selenium may only reflect long-term intake among long-standing and consistent selenium supplement users (such as the participants in our study) because although plasma is more sensitive than toenails to day-to-day differences in intake, regular use of a supplement over an extended period may attenuate such variability. Therefore, plasma levels may not be reflective of long-term selenium intake in all populations. As expected, FFQ-derived selenium intakes were not associated with toenail or plasma selenium levels, consistent with other studies (7, 8, 19) showing that self-reported dietary selenium is not a useful measure of selenium exposure.
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TABLE 2. Associations of selenium intakes from diet and supplements with toenail and plasma concentrations (n = 220), 2001 Adjusted means1 Selenium intake Dietary intake (self-report) 0 ! – 80 mcg 81 – 104 mcg 105 – 141 mcg 142 – 286 mcg P for trend Pearson’s r1,3 (p-value) Current supplement use (supplement inventory) Nonusers Multivitamin users only4 Single supplement users5 P value Current supplement dose (supplement inventory) Nonusers 0 ! – 20 mcg 21 – 45 mcg 46 – 186 mcg 187 – 3300 mcg P for trend Pearson’s r1,3 (95% CI) Current supplement use (self-report) Nonusers Multivitamin users only4 Single supplement users5 P value Current supplement dose (self-report) Non-users 0 ! – 20 mcg 21 – 39 mcg 40 – 100 mcg 101 – 250 mcg P for trend Pearson’s r1,3 (95% CI) Dietary intake plus current supplement dose (self-report) 0 ! – 100 mcg 101 – 140 mcg 141 – 200 mcg 201 – 536 mcg P for trend Pearson’s r1,3 (p-value) 10-year supplement use (self-report) Nonusers Multivitamin users only4 Single supplement users5 P value 10-year supplement dose (self-report) Nonusers 0 ! – 10 mcg 11 – 20 mcg 21 – 44 mcg 45 – 244 mcg P for trend Pearson’s r1,3 (95% CI)
%
Mean intakes (mcg)
Toenail Selenium (mg/g)
Plasma selenium2 (mg/L)
25.4 24.4 25.4 24.9
63.9 92.5 121.0 185.5
1.00a 1.03a 1.03a 1.02a .45 .09 (ÿ.05, .22)
160a 155a 160a 162a .58 .08 (ÿ.06, .21)
33.8 46.1 20.1
0.0 116.0 224.9
33.8 24.7 12.8 12.3 16.4
0 17.9 37.2 97.6 471.4
.97a .97a .92a 1.09b 1.20c .0001 .33 (.20, .44)
153a 152a 152a 159a 189b .0001 .36 (.24, .47)
34.3 48.9 16.9
0.0 45.7 141.3
.99a .98a 1.18b .0001
156a 154a 181b .0001
34.3 23.7 9.1 19.6 13.2
0.0 17.7 26.1 67.8 198.4
.99a .95a .99a 1.04a 1.20b .0001 .26 (.13, .38)
156a 151a 151a 160a 188b .0001 .27 (.15, .39)
25.0 25.0 25.0 25.0
79.3 11.8 164.9 283.1
.99a .98a .97a 1.13b .0003 .31 (.18, .43)
154a 151a 156a 175b !.0001 .38 (.26, .49)
25.6 51.6 22.8
0.0 22.3 69.5
.97a .97a 1.19b .0001
152a 155a 175b .0001
25.6 20.6 19.2 16.4 18.3
0.0 5.1 17.1 30.9 98.4
.97a .93a .99a 1.02a 1.21b .0001 .36 (.24, .47)
152a 151a 157a 157a 182b .0001 .36 (.24, .47)
.97a 1.03a 1.08a .03
153a 160a 166a .01
NOTE: Plasma selenium was log-transformed, toenail selenium was not transformed. a-c For each selenium intake variable, means with different superscripts are statistically significantly different from each other (p ! 0.05) 1 Means and Pearson’s correlation coefficients adjusted for age, sex, body mass index, and current smoking. For partial Pearson’s correlations, all variables were treated as continuous except for sex, race, and smoking. 2 Least square means of plasma selenium values were log transformed and then back-transformed to the original units. 3 The dietary, supplement use, or total (dietary and supplemental) intake variables were log-transformed and back-transformed into their original units. 4 Multivitamin users only: current self-report, n Z 107; current interview, n Z 101, 10-year self-report, n Z 113. 5 Single supplement users: current self-report, n Z 37; current interview, n Z 44, 10-year self-report, n Z 50.
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Of the demographic and behavioral characteristics examined, only smoking was significantly associated with toenail selenium, similar to the finding reported by others (7, 16, 17, 19). In this study, however, the lower toenail selenium concentration among current smokers is largely due to lower selenium intakes (diet plus supplements) among current smokers compared to non-smokers: 115 and 164 mcg, respectively, p Z .04 (data not shown). Consequently, after control for supplement use, the association between smoking and toenail selenium was no longer significant (p Z .09). Nonetheless, we are unable to explain why these differences in selenium intake across categories of smokers were reflected in toenails but not in plasma. Swanson and colleagues similarly observed that smoking was an independent determinant of toenail, but not serum or whole blood selenium concentrations (16). There have been reports of positive significant associations of other demographic and behavioral characteristics, including younger age (7), female sex (19–21), and nonuse of alcohol (22) with higher selenium biomarker levels. However, because there is no indication that selenium supplement use was controlled for in these analyses, it is not clear that these variables were truly independently associated with the selenium biomarkers in these studies. A major strength of this study is that it is, to our knowledge, the first to describe, directly, the relationship between toenail and plasma selenium concentrations and to compare their associations with both current and longterm selenium intake. Specifically, both biomarker measures were available from the same set of study participants, thereby allowing the estimation of their correlation and a direct, within-study comparison of which biomarker may more accurately reflect selenium status. In addition, we examined a wide array of potential demographic, lifestyle, and behavioral correlates. Potential limitations of the study include the possibility of a limited range of selenium intake among participants, the limited number of current smokers in the study population, and the challenges of assessing selenium exposure from diet and self-reported supplement use. Studies with more accurate assessments of selenium from diet have found dietary selenium to be the primary determinant of tissue selenium concentrations (8, 16). In conclusion, our findings suggest that both toenail and plasma selenium concentrations are equally valid measures of current, as well as long-term selenium intakedin particular, high supplement use. The data also show that selenium from supplements is the strongest predictor of biomarker levels, whereas selenium intakes from an FFQ and most demographic characteristics and health-related behaviors do not appear to be independently associated with toenail or plasma selenium levels.
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