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Selenium status of industrial worker
NUTRITION RESEARCH,Vol. 3, pp. 805-817, 1983 0271-5317/83 $3.00 + .00 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
SELENIUM STATUSOF INDUSTRIAL WORKERI Helen W. Lane,2 Ph.D.,R.D., Doris C. Warren, Ph.D., Elaine Martin, and Jeanne McCowan, R.D. Program in Nutrition and Dietetics, The University of Texas Health Science Center, P.O. Box 20708, Houston, Texas, 77225 (HWL, JM) and Houston Baptist University (DCW, EM) 7502 Fondren Rd., Houston, Texas 77074 ABSTRACT The purpose of this study was to assess the selenium status of a well-defined industrially employed population: eighty-six oil refinery workers. Plasma selenium levels and erythrocyte GSH-Px activities were significantly lower in this industrial group (86 subjects) than in non-industrial group (174 subjects). In order to evaluate whether these lower values were responsive to supplementation, ten subjects from the industrial group were further studied for 12 weeks. Five of these subjects consumed a selenium supplement, 50 ~g Se as sodium selenite, once a day for eight weeks (weeks 2 to 10 of study). Diet histories revealed that these 10 workers consumed an average of 217+73 ~g Se/day indicating that they were consuming selenium levels aboTe the recommendations of the Food and Nutrition Board of the NRC. The supplemented group had significantly higher whole blood selenium levels and GSH-Px activity than the non-supplemented group and this difference was due to a drop in the levels found in the non-supplemented group at weeks 8 and 10. Thesedata suggest that selenium supplementation prevented the decreases in blood selenium levels and GSH-Px activity experienced by the non-supplemented subjects. Also, there was a positive correlation between blood selenium levels and GSH-Px (r = 0.45, p
1Supported by Science and Education Administration of the U.S. Department of Agriculture under Grant No. 5901-0401-8-00860 from the Competitive Research Office. 2Aaoress reprint requests.
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806
H.W. LANEet al. I NTRODUCTI ON
Selenium has been known to be an essential element for animals since the 1950's ( I ) . While recommended daily allowances are s t i l l under evaluation, the Food and Nutrition Board of the National Research Council recommended a safe and adequate intake of 50-200 ]~g/day (2). Since the difference between a therapeutic and a toxic level for selenium in humans is small (1), the selenium status in a healthy population must be cautiously determined prior to recommendation of selenium supplementation. Many symptoms, which occur with selenium deficiency in various species of animals, and the relationship of selenium with a vitamin E deficiency can be explained in part by the finding that selenium is essential for glutathione-peroxidase (GSH-Px) activity in animals (3) and humans (4). Today, the ,Dst accepted function of the selenoenzyme, GSH-Px, is to detoxify peroxides (5,6). An oxygen free radical may be metabolized through the action of superoxide dismutase to hydrogen peroxide (7). GSH-Pxcatalyzes a reaction in which the hydrogen peroxide is converted to water; therefore, damage from peroxides is prevented (6). I t has been demonstrated that animals raised on selenium and vitamin E deficient diets have low tolerance for various dietary oxidants (8). Using in vivo production of ethane as a measure of l i p i d peroxidation, rats consuming diets containing 0.2 ppm Se had less lipid peroxidation than rats consuming a selenium deficient diet (9). Blood selenium levels and GSH-Px activities have been evaluated in a variety of groups of healthy subjects from several different geographical regions: New Zealand children (10) and adults (10,11), urban and rural Ohioans (12), Texas elderly people and students and staff of a Texas university (13,14), pregnant women (15), and South Dakotans (16). New Zealanders consume less than 50 ug Se/day (lower value than the proposed RDA) and have blood selenium levels that were about half the level found in U.S. populations (10,11). Whenhea]thy New Zealanders consumed 100 ~g/day of supplemental selenium, their blood GSH-Px activity increased (17). Thomsonet. a l . (17) suggested that their intra-subject variation was due to variations in exercise level and adaptation to different selenium levels. WhenAmerican males were fed selenium depletion diets (about 30 )Jg Se/day), they had significant decreases in serum and urinary selenium concentrations while whole blood GSH-Px activity decreased slightly during the 45 day study (18). In terms of selenium status, one geographical area that has not been well studied is highly industrial regions. Glover (19) found that workers exposed to selenium from processing industries did not have different death rates than the genera] populations; however, Glover (18) did not report any measures of selenium status. Data from animal research suggests that chemical oxidant stress may affect selenium status (9). Thus, individuals working in an industrial setting may have different selenium status than individua]s living and working in a non-industrial setting. Therefore, the purpose of this study was to assess the selenium status of a well-defined industrial population and to study the effect of selenium supplementation in that population.
SELENIUM IN INDUSTRIAL WORKERS
807
MATERIALS AND METHODS Experimental Design. This study has two components: i. survey study and 2. treatment study. The purpose of the survey study was to determine i f oil refinery workers had different selenium status than a non-industrial population studied in the same region. The purpose of the treatment study was to determine i f the selenium status of oil refinery workers is influenced by selenium supplementation. In the survey study, eighty-six oil refinery employees (94% male, 6% female) were studied by measuring their plasma and erythrocyte selenium concentrations and erythrocyte GSH-Px a c t i v i t y . The nonindustrial population consisted of 174 subjects who were teachers, students, health workers, and retired e~loyees living in the same general geographic area but not working in the refine~. (Table 1) TABLE 1 Hematological and Nutritional Indices for Survey and Treatment Populations
Indices
Subjects Indus t r i a l
Industrial for treatment study
174
86
10
65
80
I0
109
6
0
38.5
34.7
Non-Indus t r i a l Number of subjects Sex
reale femaI e
Age
~ range
30.9 18-90
20-51
27-40
Hct %
~ + S.D.
43.8 + 4.6
44.5 + 4.0
46.5 + 3,6
Hgb g/dl
X + S.D.
13.7 + 1.4
14.9 _+ 1.6
14.5 +_ 0.9
Ten subjects from the industrial group were more extensively evaluated in the treatment study (Table l ) . For three months (84 days) bimonthly blood and urine selenium levels, blood GSH-Px a c t i v i t y , and dietary selenium levels from diet histories were determined for a total of six collections. The effect of daily supplementation of 50 ug seTenium was evaluated in five subjects. The selenium supplement was consumed daily between week 2 and week lO for a total period of eight weeks. The other five subjects did not consume a selenium supplement. The selenium supplement was in the form of selenite diluted with lactose given in capsule form.
808
H.W. L~E et alo TABLE 2 Description of the Ten ~lale Subjects in the Treatment Study
Parameter Age (yr) Weight(kg): Body fat (%r
X 34.7 84.1 23.5
S.D. 4.0 13.9 3.3
Range 27.0- 40.0 63.0-102.0 16.0- 28.7
acalculated from measures of four skin fold measures: triceps, biceps, suprailiac, and subscapula (27). All subjects completed medical histories and were free of any acute or chronic disease. All subjects signed an informed consent approved by the University of Texas Committee for the Protection of Human Subjects. Blood Preparation and Analysis. A vena puncture was performed with a syringe and needle. Immediately, hematocrits and hemoglobins were determined on a portion of the whole blood {20). The remaining blood was maintained at 4oc, treated with heparin, centrifuged, and the plasma removed and frozen. For the survey study, the cellular fraction was rinsed twice with saline to remove the bully coat (21). The cells were hemolyzed with cold deionized water. Hemoglobin values and selenium concentrations were measured on the hemolyzed blood. For the treatment study, the whole blood was diluted with deionized water and a fraction again diluted with phosphate buffer for GSH-Px analysis. Selenium and GSH-PxAnalysis. Selenium levels were determined on digestates of hemolyzed red bloods, plasma, whole blood, urine, or foods by the previously described fluorometric assay (13,14,22,23). All reported selenium levels are duplicated within a standard deviation of 0.0026. Results from NBS liver standard (1.1+0.05) were 1.0+0.02 ppm from five analyses completed at various- times throughout the study. Glutathione-peroxidase was measured by the coupled assay as previously described (13,14,24). For the survey study, hydrogen peroxide was the substrate. Due to controversy about appropriate substrates for the GSH-Px assay, both hydrogen peroxide and t-butyl peroxide was used for the treatment study. Enzymeactivity was expressed as iJmoIes of NADPHoxidized/min/g hemoglobin or ~moIes NADPH oxidized/rain/rag protein. Sta ti sti cal Methods. Analysis of variance (25,26) was used to evaluate overall differences between the industrial workers and the non-industrial subjects in the survey study. For the treatment study, analysis of variance with repeated measures was used. The i n i t i a l value for each the subject was used as a significant covariate.
SELENIUM IN INDUSTRIAL WORKERS
809
Diet Histories. For the treatment study, 24 hour dietary recalls were completed at each of the six collection times plus one i n i t i a l recall for a total of seven 24 hour dietary recalls every two weeks for three months. Each subject was interviewed by a dietitian to obtain each 24 hour diet history. Carbohydrate, protein, fat, calories, and selenium levels were calculated (23,28-30) from the histories. Because of variations in foods consumed and since geographical area can affect dietary selenium levels, 25 foods were analyzed for selenium (23) in our laboratory.
RESULTS Survey Study. .... The mean erythrocyte GSH-Px activity for the non-industrial Subjects was 35.0+15.3 units/g Hgb as compared to the significantly lower level of 28T9+13.7 units/g Hgb for the industrial group (Table 3) (p
TABLE 3 Selenium Indices for the Industrial and Non-Industrial Subjects
Subjects
Indices Non-Industrial Nude r
174
I ndust r i al 86
Erythrocyte GSH-Px Acti vi ty (Uni ts/g Hgb)c Se Concentration
35.0 + 15.3a'b
28.9 + 13.7
0.81 + 0.42 a'b
0.67 + O.IB
0.097 + 0.030 a'b
0.085 + 0.015
(pg/g of Hgb) Plasma Se Concentration (pg Se/g) a Values expressed as Man + standard deviation. b Values are significantly different (p
810
H.W. LANEet al.
127.1+54.1 g, and protein 90.2+23.8 g. There was a positive correlation (r = 0.64 p
27" 26" 2524" 23Whole 22. Blood
GBH-Px 21Activity 20. Unitslmg Protein
9
A
t
19" &
18-
9
9
17161514139 Supplemented Group
12"
9 Nonsupplemented Group
11-
r 045. p 0001
10-
~t
0.~}8
0.~)9
0.~0
0-~11
0.112
0.I13
O'14
0.115
0'16
!
0.17
Ug Se/ml of Whole Blood
Figure i A comparison of the whole blood selenium concentrated and GSH-Px activity in the ten subjects in the treatment study. The controls are the subjects not receiving the supplement.
SELENIUM IN INDUSTRIAL WORKERS
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~Nonsupplemented 28-
.....
Supplemented Group p< 0.05
26Whole
Blood
Group
2.4-
-
GSH-Px Activity
22
Unitslmg Protein
20 /
181B141210i
i
i
4
6
B
10
Weeks Se Supplementation ~
1'2
/ J
Figure 2 The mean and standard deviations for whole blood selenium concentrations over the 12 weeks.
p
0.17 t
o16~ 015ug Se/mt of Whole Blood 014"
iI
\\
f~.,.. . . /Ii/ ,.. Z~
0.13-
~T ~'. ~
IT II
0,12 0.11" 0. I 0 " ~Nonsupplemented Group
0.09" 0,08"
-- -- Supplemented Group I
2 I
I
4
I
6 Weeks
1
8
110
I
t2
" Se Supplementation
Figure 3 The mean and standard deviation of whole blood GSH-Px activity over the 12 weeks.
812
H.W. LANEet al. TABLE 4 Measures of Selenium Status for the Ten Male Subjects During The Treatment Study
Subjects a
Indices Non-Supplemented
Suppl ementedb
0.10 + 0.08 c
0.14 + 0.05
43.7 + 2.0 c 16.3 + 2.2 c
59.6 +_ 2.1 18.5 + 2.4
0~08 + 0.01
0.08 + 0.01
89.4 + 20.2
96.4 + 23.2
228 + 121
203 + 106
Whole Blood Se Concentration
(pglml) GSH-Px Activi ~ d (units Img protein) H202 t-butyl P1asma Se Concentration
(pglml) Urine Se Concentration (pglday) Dietary Se (pg/day) a Values expressed as means + standard deviations. b For the supplemented subjects, the values are the means for the eight weeks during supplementation (4 collections), weeks 4 through 10. c SignifiCantly different from values found for supplemented subjects (p
DISCUSSION The influence of environment on selenium indices was further documented by this research. Employees of a large oil refinery had lower indices for selenium status than the non-industria| group living in the same geographical region. The selenium and GSH-Px levels of both the industrial and non-industrial subjects in our survey group were similar to levels found in Ohio (12), were above levels found in New Zealand (10,11), and below levels found in South Dakota (15).
SELENIUM IN INDUSTRIAL WORKERS
One possible explanation for the lower selenium and GSH-Px values of the oil refinery workers, as compared to the non-induslrial subjects, may be that their diets were low in selenium. This hypothesis was investigated in the treatment study. I t has been postulated that adequate selenium intake is dependent on the subject's a b i l i t y to ingest adequate calories as carbohydrate and protein along with nutrient density, ~g Se/lOOO kcal. Also, several studies suggest that selenium from food is well absorbed (31, 32) so dietary selenium may be a reasonable predictor of selenium absorption. Thus, as well as calculating the total daily selenium intake and selenium density, the diets were evaluated in terms of energy, carbohydrate and protein. The results from the dietary histories indicate that the ten subjects from the industrial population consumed about 200 ug Se/day which is near the upper range suggested by the Food and Nutrition Board (2) and generally above those levels reported for other populations (i0-17). The selenium density was approximately g4 ~g Se/ 1,000 kcal and this density was above that of 54 and 71 ~g Se/lO00 kcal found for free-living and institutional elderly, respectively (14). These industrial workers consumed caloric levels within the recommended energy range suggested by the Food and Nutrition Board (2). The weight and percent body fat results support the fact that these subjects generally were consuming adequate calories (Table 2). Strong positive correlations were found between selenium intake and energy from carbohydrate and protein. These positive correlations have been reported previously by us (14) and others (33) and further support the concept that a U.S. diet containing adequate calories and protein and a variety of foods w i l l provide selenium within the recommended levels (2). The industrial subjects from the treatment study (10 male oil refinery workers) were consuming adequate calories, protein, and selenium as well as a high selenium density diet, so their lower selenium indices can not be explained by diet. Results from the treatment study indicate that there was a positive correlation between whole blood selenium concentration and GSH-Px activity for both the selenium supplemented and nonsupplemented subjects (Fig, I ) , This correlation has been observed by others (10) but studies from New Zealand (10) suggest that GSH-Px a c t i v i t y plateaus when blood selenium levels are 0.10 ~g Se/ml. This plateau did not occur in this industrial population suggesting that the level of GSH-Px activity is not optional at the same level as found in the New Zealand subjects, In order to determine i f selenium supplementation would increase the levels of the selenium indices, five industrial subjects consumed 50 ~g Se/day as selenite for eight weeks. I t appears from the mean values reported in Table 4 that supplementation resulted in higher values for the selenium indices. However, when the results are reported bimonthly (Fig. 3-4), the supplemented group maintained their levels of selenium and GSH-Px activities with a significant decrease in whole blood selenium and GSH-Px levels in the non-supplemented group especially at weeks 8 and 10. These data suggest that selenium supplementation may have prevented a decrease in selenium status.
813
814
~H.W. LANEet al. U.S. males consuming selenium depletion diets (less than 50 ~g/day for 45 days) had significant drops in serum and urinary selenium levels with significant increases with repletion (18). There was nochange in GSH-Px activity during depletion or repletion. In contrast, the non-supplemented industrial subjects had;a drop in whole blood GSH-Px and Se levels but no change in plasma or urinary selenium levels. The comparison of the two studies further indicates that the lower selenium indices found i n the industrial subjects was no__tt due to diet. However, there may be several explanations for these results. Skin is one of the tissues containing reasonable selenium levels1 (35) and these industrial workers may be losing more selenium through loss in perspiration. This study was completed during Texas summer months (May through August). Thus, sweat losses may contribute to total selenium losses in outside workers. This needs to be studied further. Another possible explanation is that exposure to environmental oxidant may affect selenium status. There is some evidence from animal studies that environmental hazards such as polychlorinated biphenyls (PCB) and herbicides such as paraquat may be involved in tissue peroxidation (8, 34). Basically, these chemicals exacerbate the symptoms of selenium-vitamin E deficiency, I t may be that individuals residing around and working with potential peroxidants may have a higher requirement for selenium and thus the addition of 50~g Se/day may aid in maintaining blood levels. Further research on both selenium losses in sweat and the influence of potential chemical oxidants needs to be completed. Finally, selenium supplementation of New Zealanders resulted in wide variation in the data (17). These investigators suggest that the variations may be due to environmental changes as well as variation in exercise levels. Furthermore, the selenomethionine supplementation resulted in higher i n i t i a l increases in selenium indicies than selenite. There may have been a more dramatic difference between the supplemented and non-supplemented groups i f selenomethionine had been used. Three conclusions can be made from this study, i. The industrial workers' indices for selenium status were lower than that found in a non-industrial group living in the same region. 2. Lower selenium indices of the industrial group cannot be explained by diet. In fact these data reconfirm the hypothesis that U.S. diets adequate in calories and protein" provided recommended dietary selenium levels. 3. The reason for the statistical effect of selenium supplementation was due to a decrease in the measures of selenium status in the non-supplemented industrial group. Probably for complex reasons industrial workers
1Skin selenium levels from 6 autopsies~(violent injury deaths) were 0.112+_0.041 ppm, (mean ~ standard deviation), unpublished.
SELENIUM IN INDUSTRIAL WORKERS
815
may have a higher requirement for selenium than suggested by research with non-industrial subjects. ACKNOWLEDGEMENTS The author wishes to thank the following organization for f a c i l itating the study: Houston Baptist University, Shell Oil Company, Oil, Chemical and Atomic Workers International Union, Local No. 4-367, Delta Epsilon Sorority Alumni, Retired Workers Association, and T i t l e IIIC Feeding Site. In addition we want to express our appreciation to Donna Covell, Diane Siaz, Barbara Taylor, Judith Ragdale, and John Barber for their technical assistance. Statistical support was provided by Dennis Johnston and Pat O'Sullivan.
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NATIONAL RESEARCHCOUNCIL. Selenium in Nutrition. Academy of Sciences. Washington, D.C., 1977.
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FOODAND NUTRITIONBOARD, Recommended Dietary Allowances. 7th ed.
3.
ROTRUCK, J.P., POPE, A.L., GANTHER, H.E., HAFEMAN, D.G., and HOEKSTRA, W.G. Selenium: biochemical role as a component of glutathione-peroxidase. Science, 179:588-592, 1973.
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AWASTHI, V.C., BUETHER, E. and SRIVASTAVA, S.K. Purification and protective properties of human erythrocyte glutathioneperoxidase. J. Biol. Chem.; 240:5144-5149, 1975.
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GANTHER, H.E., HAFEMAN, D.G., LAWRENCE, R.A., SERFASS, R.E. and HOEKSTRA, W.G. Selenium and glutathione peroxidase in health and disease-A-Review In: Trace Elements In HumanHealth and Disease Vol. I I . A.S. Prassad ted), Academic Press, New York, 165-218, 1976.
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HAFEMAN, D.G. and HOEKSTRA, W.G. Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. ~. Nutr. 107:666-672, 1977.
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HALLIWELL, B. Superoxidedismutase, catalase and glutathione peroxidase: Solutions to the problems of l i v i n g with oxygen. New Phytol. 73:1075-1086, 1974.
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COMBS, G.F. and SCOTT, M.L. Polychlorinated biphenyl-stimulated selenium deficiency in the chick. Poultry Sci.. 54:1152-1158, 1975.
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HAFEMAN, D.G. and HOEKSTRA, W.G. Protection against carbon tetrachloride-induced l i p i d peroxidation in the rat by dietary vitamin E, selenium and methionine as measured by ethane evolution. J. Nutr. 107:656-665, 1977.
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THOMSON, C.D., REA, H.M., DOESBURG, V.M., ROBINSON, M.F. Selenium concentrations and glutathione-peroxidase activities in whole blood of New Zealand residents. Br. J. Nutr. 37:457460, 1977.
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KAY, R.G. Blood selenium values in an adult Auckland population group. New Zealand Med J. 90:11-13, 1979.
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SNOOK, J .T. Selenium status of rural and urban Ohio populations. Fed. Proc. 39:338, 1980.
13. LANE, H.W., DUDRICK, S. and WARREN, D.C. Blood selenium levels and glutathione-peroxidase a c t i v i t i e s in university and chronic intravenous hyperalimentation subjects. Proc. Soc. Exp. Biol. and Med. 167:338~390, 1981. 14.
LANE, H.W., WARREN, D.C., TAYLOR, B.J. and STOOL, E. Blood selenium and glutathione-peroxidase levels and dietary selenium of free-living and institutionalized elderly subjects. Proc. Soc. Ex_xj~.Biol. and Med. 173:87-95, 1983.
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BUTLER, J.A., WHANGER, P.D. ant TRIPP, M.J. Blood selenium and glutathione peroxidase a c t i v i t y in pregnant women: Comparative assays in primates and other animals. Am. J. Clin. Nutr. 36: 15-23, 1982.
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HOWE, M, Selenium in the blood of South Dakotan. Arch Environ. Health; 34:444-448, 1979.
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THOMSON, C.D., ROBINSON, M.F., CAMPBELL, D.R. and REA, H.M. Effect of prolonged supplementation with d a i l y supplements of selenomethionine and sodium selenite on glutathione peroxidase a c t i v i t y in blood of New Zealand residents. Am. J. Clin. Nutr. 32:24-31, 1982.
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LEVANDER, D.A., SUTHERLAND, B., MORRIS, V.C. and KING, J.C. Selenium balance in young men during selenium depletion and repletion. Am. J. Clin. Nutr. 34:2662-2669, 1981.
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SAUBERLICH, H.E., DOWDY, R.P. and SKAL, J.H. Laboratory Tests for the Assessment of Nutritional Status. CRC Press, Cleveland, OH, 1977.
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OH, S.H., GANTHER, H.L., HOEKSTRA, W.G. Selenium as a component of glutathione-peroxidase isolated from ovine erythrocyte. Biochem. 13:1825-1828, 1974.
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LANE, H.W., TAYLOR, B,J., STOOL, E., SERVANCE, D. and WARREN, D. Selenium content of selected foods. JADA. 82:24-28, 1983.
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PAGLIA, D.E. and VALENTINE, W.N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione-peroxidase. J. Lab. and Clin. Med. 70:158-169, 1967.
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SNEDECOR, G.W. and COCHPd~N,W.G. St at i st i cal methods. Iowa State University Press, Ames, Iowa, 273-275, 1972.
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DURNIN, J.V.G.A. and WOMERSLEY, J. Body fat assessed from total body density and it s estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br. J. Nutr. 32:77-97, 1974.
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HIGGS, D.J., MORRIS, V.C. and Levander, O.A. Effect of cooking on selenium content of foods. ~. Agri. Food Chem. 20:678-680, 1972.
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BURK, R.F. Selenium in Man. In: Trace Elements in Human Health and Disease, Vol. I I . A.S. Prasad (ed), Aca-d-em-i"EPress, New York, New York. p. 105-133, 1976.
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GREGOR, J.L., SMITH, S.A. and SNEDEKER, S.M. Effect of Dietary Calcium and Phosphorus Levels on the U t i l i z a t i o n of Calcium, Phosphorus, Magnesium, Managanese, and Selenium by Adult Males. Nutr. Res. 1:315-325, 1981.
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Selenium content of foods, J.
817
SELENIUM STATUSOF INDUSTRIAL WORKERI Helen W. Lane,2 Ph.D.,R.D., Doris C. Warren, Ph.D., Elaine Martin, and Jeanne McCowan, R.D. Program in Nutrition and Dietetics, The University of Texas Health Science Center, P.O. Box 20708, Houston, Texas, 77225 (HWL, JM) and Houston Baptist University (DCW, EM) 7502 Fondren Rd., Houston, Texas 77074 ABSTRACT The purpose of this study was to assess the selenium status of a well-defined industrially employed population: eighty-six oil refinery workers. Plasma selenium levels and erythrocyte GSH-Px activities were significantly lower in this industrial group (86 subjects) than in non-industrial group (174 subjects). In order to evaluate whether these lower values were responsive to supplementation, ten subjects from the industrial group were further studied for 12 weeks. Five of these subjects consumed a selenium supplement, 50 ~g Se as sodium selenite, once a day for eight weeks (weeks 2 to 10 of study). Diet histories revealed that these 10 workers consumed an average of 217+73 ~g Se/day indicating that they were consuming selenium levels aboTe the recommendations of the Food and Nutrition Board of the NRC. The supplemented group had significantly higher whole blood selenium levels and GSH-Px activity than the non-supplemented group and this difference was due to a drop in the levels found in the non-supplemented group at weeks 8 and 10. Thesedata suggest that selenium supplementation prevented the decreases in blood selenium levels and GSH-Px activity experienced by the non-supplemented subjects. Also, there was a positive correlation between blood selenium levels and GSH-Px (r = 0.45, p
1Supported by Science and Education Administration of the U.S. Department of Agriculture under Grant No. 5901-0401-8-00860 from the Competitive Research Office. 2Aaoress reprint requests.
805
806
H.W. LANEet al. I NTRODUCTI ON
Selenium has been known to be an essential element for animals since the 1950's ( I ) . While recommended daily allowances are s t i l l under evaluation, the Food and Nutrition Board of the National Research Council recommended a safe and adequate intake of 50-200 ]~g/day (2). Since the difference between a therapeutic and a toxic level for selenium in humans is small (1), the selenium status in a healthy population must be cautiously determined prior to recommendation of selenium supplementation. Many symptoms, which occur with selenium deficiency in various species of animals, and the relationship of selenium with a vitamin E deficiency can be explained in part by the finding that selenium is essential for glutathione-peroxidase (GSH-Px) activity in animals (3) and humans (4). Today, the ,Dst accepted function of the selenoenzyme, GSH-Px, is to detoxify peroxides (5,6). An oxygen free radical may be metabolized through the action of superoxide dismutase to hydrogen peroxide (7). GSH-Pxcatalyzes a reaction in which the hydrogen peroxide is converted to water; therefore, damage from peroxides is prevented (6). I t has been demonstrated that animals raised on selenium and vitamin E deficient diets have low tolerance for various dietary oxidants (8). Using in vivo production of ethane as a measure of l i p i d peroxidation, rats consuming diets containing 0.2 ppm Se had less lipid peroxidation than rats consuming a selenium deficient diet (9). Blood selenium levels and GSH-Px activities have been evaluated in a variety of groups of healthy subjects from several different geographical regions: New Zealand children (10) and adults (10,11), urban and rural Ohioans (12), Texas elderly people and students and staff of a Texas university (13,14), pregnant women (15), and South Dakotans (16). New Zealanders consume less than 50 ug Se/day (lower value than the proposed RDA) and have blood selenium levels that were about half the level found in U.S. populations (10,11). Whenhea]thy New Zealanders consumed 100 ~g/day of supplemental selenium, their blood GSH-Px activity increased (17). Thomsonet. a l . (17) suggested that their intra-subject variation was due to variations in exercise level and adaptation to different selenium levels. WhenAmerican males were fed selenium depletion diets (about 30 )Jg Se/day), they had significant decreases in serum and urinary selenium concentrations while whole blood GSH-Px activity decreased slightly during the 45 day study (18). In terms of selenium status, one geographical area that has not been well studied is highly industrial regions. Glover (19) found that workers exposed to selenium from processing industries did not have different death rates than the genera] populations; however, Glover (18) did not report any measures of selenium status. Data from animal research suggests that chemical oxidant stress may affect selenium status (9). Thus, individuals working in an industrial setting may have different selenium status than individua]s living and working in a non-industrial setting. Therefore, the purpose of this study was to assess the selenium status of a well-defined industrial population and to study the effect of selenium supplementation in that population.
SELENIUM IN INDUSTRIAL WORKERS
807
MATERIALS AND METHODS Experimental Design. This study has two components: i. survey study and 2. treatment study. The purpose of the survey study was to determine i f oil refinery workers had different selenium status than a non-industrial population studied in the same region. The purpose of the treatment study was to determine i f the selenium status of oil refinery workers is influenced by selenium supplementation. In the survey study, eighty-six oil refinery employees (94% male, 6% female) were studied by measuring their plasma and erythrocyte selenium concentrations and erythrocyte GSH-Px a c t i v i t y . The nonindustrial population consisted of 174 subjects who were teachers, students, health workers, and retired e~loyees living in the same general geographic area but not working in the refine~. (Table 1) TABLE 1 Hematological and Nutritional Indices for Survey and Treatment Populations
Indices
Subjects Indus t r i a l
Industrial for treatment study
174
86
10
65
80
I0
109
6
0
38.5
34.7
Non-Indus t r i a l Number of subjects Sex
reale femaI e
Age
~ range
30.9 18-90
20-51
27-40
Hct %
~ + S.D.
43.8 + 4.6
44.5 + 4.0
46.5 + 3,6
Hgb g/dl
X + S.D.
13.7 + 1.4
14.9 _+ 1.6
14.5 +_ 0.9
Ten subjects from the industrial group were more extensively evaluated in the treatment study (Table l ) . For three months (84 days) bimonthly blood and urine selenium levels, blood GSH-Px a c t i v i t y , and dietary selenium levels from diet histories were determined for a total of six collections. The effect of daily supplementation of 50 ug seTenium was evaluated in five subjects. The selenium supplement was consumed daily between week 2 and week lO for a total period of eight weeks. The other five subjects did not consume a selenium supplement. The selenium supplement was in the form of selenite diluted with lactose given in capsule form.
808
H.W. L~E et alo TABLE 2 Description of the Ten ~lale Subjects in the Treatment Study
Parameter Age (yr) Weight(kg): Body fat (%r
X 34.7 84.1 23.5
S.D. 4.0 13.9 3.3
Range 27.0- 40.0 63.0-102.0 16.0- 28.7
acalculated from measures of four skin fold measures: triceps, biceps, suprailiac, and subscapula (27). All subjects completed medical histories and were free of any acute or chronic disease. All subjects signed an informed consent approved by the University of Texas Committee for the Protection of Human Subjects. Blood Preparation and Analysis. A vena puncture was performed with a syringe and needle. Immediately, hematocrits and hemoglobins were determined on a portion of the whole blood {20). The remaining blood was maintained at 4oc, treated with heparin, centrifuged, and the plasma removed and frozen. For the survey study, the cellular fraction was rinsed twice with saline to remove the bully coat (21). The cells were hemolyzed with cold deionized water. Hemoglobin values and selenium concentrations were measured on the hemolyzed blood. For the treatment study, the whole blood was diluted with deionized water and a fraction again diluted with phosphate buffer for GSH-Px analysis. Selenium and GSH-PxAnalysis. Selenium levels were determined on digestates of hemolyzed red bloods, plasma, whole blood, urine, or foods by the previously described fluorometric assay (13,14,22,23). All reported selenium levels are duplicated within a standard deviation of 0.0026. Results from NBS liver standard (1.1+0.05) were 1.0+0.02 ppm from five analyses completed at various- times throughout the study. Glutathione-peroxidase was measured by the coupled assay as previously described (13,14,24). For the survey study, hydrogen peroxide was the substrate. Due to controversy about appropriate substrates for the GSH-Px assay, both hydrogen peroxide and t-butyl peroxide was used for the treatment study. Enzymeactivity was expressed as iJmoIes of NADPHoxidized/min/g hemoglobin or ~moIes NADPH oxidized/rain/rag protein. Sta ti sti cal Methods. Analysis of variance (25,26) was used to evaluate overall differences between the industrial workers and the non-industrial subjects in the survey study. For the treatment study, analysis of variance with repeated measures was used. The i n i t i a l value for each the subject was used as a significant covariate.
SELENIUM IN INDUSTRIAL WORKERS
809
Diet Histories. For the treatment study, 24 hour dietary recalls were completed at each of the six collection times plus one i n i t i a l recall for a total of seven 24 hour dietary recalls every two weeks for three months. Each subject was interviewed by a dietitian to obtain each 24 hour diet history. Carbohydrate, protein, fat, calories, and selenium levels were calculated (23,28-30) from the histories. Because of variations in foods consumed and since geographical area can affect dietary selenium levels, 25 foods were analyzed for selenium (23) in our laboratory.
RESULTS Survey Study. .... The mean erythrocyte GSH-Px activity for the non-industrial Subjects was 35.0+15.3 units/g Hgb as compared to the significantly lower level of 28T9+13.7 units/g Hgb for the industrial group (Table 3) (p
TABLE 3 Selenium Indices for the Industrial and Non-Industrial Subjects
Subjects
Indices Non-Industrial Nude r
174
I ndust r i al 86
Erythrocyte GSH-Px Acti vi ty (Uni ts/g Hgb)c Se Concentration
35.0 + 15.3a'b
28.9 + 13.7
0.81 + 0.42 a'b
0.67 + O.IB
0.097 + 0.030 a'b
0.085 + 0.015
(pg/g of Hgb) Plasma Se Concentration (pg Se/g) a Values expressed as Man + standard deviation. b Values are significantly different (p
810
H.W. LANEet al.
127.1+54.1 g, and protein 90.2+23.8 g. There was a positive correlation (r = 0.64 p
27" 26" 2524" 23Whole 22. Blood
GBH-Px 21Activity 20. Unitslmg Protein
9
A
t
19" &
18-
9
9
17161514139 Supplemented Group
12"
9 Nonsupplemented Group
11-
r 045. p 0001
10-
~t
0.~}8
0.~)9
0.~0
0-~11
0.112
0.I13
O'14
0.115
0'16
!
0.17
Ug Se/ml of Whole Blood
Figure i A comparison of the whole blood selenium concentrated and GSH-Px activity in the ten subjects in the treatment study. The controls are the subjects not receiving the supplement.
SELENIUM IN INDUSTRIAL WORKERS
811
~Nonsupplemented 28-
.....
Supplemented Group p< 0.05
26Whole
Blood
Group
2.4-
-
GSH-Px Activity
22
Unitslmg Protein
20 /
181B141210i
i
i
4
6
B
10
Weeks Se Supplementation ~
1'2
/ J
Figure 2 The mean and standard deviations for whole blood selenium concentrations over the 12 weeks.
p
0.17 t
o16~ 015ug Se/mt of Whole Blood 014"
iI
\\
f~.,.. . . /Ii/ ,.. Z~
0.13-
~T ~'. ~
IT II
0,12 0.11" 0. I 0 " ~Nonsupplemented Group
0.09" 0,08"
-- -- Supplemented Group I
2 I
I
4
I
6 Weeks
1
8
110
I
t2
" Se Supplementation
Figure 3 The mean and standard deviation of whole blood GSH-Px activity over the 12 weeks.
812
H.W. LANEet al. TABLE 4 Measures of Selenium Status for the Ten Male Subjects During The Treatment Study
Subjects a
Indices Non-Supplemented
Suppl ementedb
0.10 + 0.08 c
0.14 + 0.05
43.7 + 2.0 c 16.3 + 2.2 c
59.6 +_ 2.1 18.5 + 2.4
0~08 + 0.01
0.08 + 0.01
89.4 + 20.2
96.4 + 23.2
228 + 121
203 + 106
Whole Blood Se Concentration
(pglml) GSH-Px Activi ~ d (units Img protein) H202 t-butyl P1asma Se Concentration
(pglml) Urine Se Concentration (pglday) Dietary Se (pg/day) a Values expressed as means + standard deviations. b For the supplemented subjects, the values are the means for the eight weeks during supplementation (4 collections), weeks 4 through 10. c SignifiCantly different from values found for supplemented subjects (p
DISCUSSION The influence of environment on selenium indices was further documented by this research. Employees of a large oil refinery had lower indices for selenium status than the non-industria| group living in the same geographical region. The selenium and GSH-Px levels of both the industrial and non-industrial subjects in our survey group were similar to levels found in Ohio (12), were above levels found in New Zealand (10,11), and below levels found in South Dakota (15).
SELENIUM IN INDUSTRIAL WORKERS
One possible explanation for the lower selenium and GSH-Px values of the oil refinery workers, as compared to the non-induslrial subjects, may be that their diets were low in selenium. This hypothesis was investigated in the treatment study. I t has been postulated that adequate selenium intake is dependent on the subject's a b i l i t y to ingest adequate calories as carbohydrate and protein along with nutrient density, ~g Se/lOOO kcal. Also, several studies suggest that selenium from food is well absorbed (31, 32) so dietary selenium may be a reasonable predictor of selenium absorption. Thus, as well as calculating the total daily selenium intake and selenium density, the diets were evaluated in terms of energy, carbohydrate and protein. The results from the dietary histories indicate that the ten subjects from the industrial population consumed about 200 ug Se/day which is near the upper range suggested by the Food and Nutrition Board (2) and generally above those levels reported for other populations (i0-17). The selenium density was approximately g4 ~g Se/ 1,000 kcal and this density was above that of 54 and 71 ~g Se/lO00 kcal found for free-living and institutional elderly, respectively (14). These industrial workers consumed caloric levels within the recommended energy range suggested by the Food and Nutrition Board (2). The weight and percent body fat results support the fact that these subjects generally were consuming adequate calories (Table 2). Strong positive correlations were found between selenium intake and energy from carbohydrate and protein. These positive correlations have been reported previously by us (14) and others (33) and further support the concept that a U.S. diet containing adequate calories and protein and a variety of foods w i l l provide selenium within the recommended levels (2). The industrial subjects from the treatment study (10 male oil refinery workers) were consuming adequate calories, protein, and selenium as well as a high selenium density diet, so their lower selenium indices can not be explained by diet. Results from the treatment study indicate that there was a positive correlation between whole blood selenium concentration and GSH-Px activity for both the selenium supplemented and nonsupplemented subjects (Fig, I ) , This correlation has been observed by others (10) but studies from New Zealand (10) suggest that GSH-Px a c t i v i t y plateaus when blood selenium levels are 0.10 ~g Se/ml. This plateau did not occur in this industrial population suggesting that the level of GSH-Px activity is not optional at the same level as found in the New Zealand subjects, In order to determine i f selenium supplementation would increase the levels of the selenium indices, five industrial subjects consumed 50 ~g Se/day as selenite for eight weeks. I t appears from the mean values reported in Table 4 that supplementation resulted in higher values for the selenium indices. However, when the results are reported bimonthly (Fig. 3-4), the supplemented group maintained their levels of selenium and GSH-Px activities with a significant decrease in whole blood selenium and GSH-Px levels in the non-supplemented group especially at weeks 8 and 10. These data suggest that selenium supplementation may have prevented a decrease in selenium status.
813
814
~H.W. LANEet al. U.S. males consuming selenium depletion diets (less than 50 ~g/day for 45 days) had significant drops in serum and urinary selenium levels with significant increases with repletion (18). There was nochange in GSH-Px activity during depletion or repletion. In contrast, the non-supplemented industrial subjects had;a drop in whole blood GSH-Px and Se levels but no change in plasma or urinary selenium levels. The comparison of the two studies further indicates that the lower selenium indices found i n the industrial subjects was no__tt due to diet. However, there may be several explanations for these results. Skin is one of the tissues containing reasonable selenium levels1 (35) and these industrial workers may be losing more selenium through loss in perspiration. This study was completed during Texas summer months (May through August). Thus, sweat losses may contribute to total selenium losses in outside workers. This needs to be studied further. Another possible explanation is that exposure to environmental oxidant may affect selenium status. There is some evidence from animal studies that environmental hazards such as polychlorinated biphenyls (PCB) and herbicides such as paraquat may be involved in tissue peroxidation (8, 34). Basically, these chemicals exacerbate the symptoms of selenium-vitamin E deficiency, I t may be that individuals residing around and working with potential peroxidants may have a higher requirement for selenium and thus the addition of 50~g Se/day may aid in maintaining blood levels. Further research on both selenium losses in sweat and the influence of potential chemical oxidants needs to be completed. Finally, selenium supplementation of New Zealanders resulted in wide variation in the data (17). These investigators suggest that the variations may be due to environmental changes as well as variation in exercise levels. Furthermore, the selenomethionine supplementation resulted in higher i n i t i a l increases in selenium indicies than selenite. There may have been a more dramatic difference between the supplemented and non-supplemented groups i f selenomethionine had been used. Three conclusions can be made from this study, i. The industrial workers' indices for selenium status were lower than that found in a non-industrial group living in the same region. 2. Lower selenium indices of the industrial group cannot be explained by diet. In fact these data reconfirm the hypothesis that U.S. diets adequate in calories and protein" provided recommended dietary selenium levels. 3. The reason for the statistical effect of selenium supplementation was due to a decrease in the measures of selenium status in the non-supplemented industrial group. Probably for complex reasons industrial workers
1Skin selenium levels from 6 autopsies~(violent injury deaths) were 0.112+_0.041 ppm, (mean ~ standard deviation), unpublished.
SELENIUM IN INDUSTRIAL WORKERS
815
may have a higher requirement for selenium than suggested by research with non-industrial subjects. ACKNOWLEDGEMENTS The author wishes to thank the following organization for f a c i l itating the study: Houston Baptist University, Shell Oil Company, Oil, Chemical and Atomic Workers International Union, Local No. 4-367, Delta Epsilon Sorority Alumni, Retired Workers Association, and T i t l e IIIC Feeding Site. In addition we want to express our appreciation to Donna Covell, Diane Siaz, Barbara Taylor, Judith Ragdale, and John Barber for their technical assistance. Statistical support was provided by Dennis Johnston and Pat O'Sullivan.
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FOODAND NUTRITIONBOARD, Recommended Dietary Allowances. 7th ed.
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ROTRUCK, J.P., POPE, A.L., GANTHER, H.E., HAFEMAN, D.G., and HOEKSTRA, W.G. Selenium: biochemical role as a component of glutathione-peroxidase. Science, 179:588-592, 1973.
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HAFEMAN, D.G. and HOEKSTRA, W.G. Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. ~. Nutr. 107:666-672, 1977.
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HALLIWELL, B. Superoxidedismutase, catalase and glutathione peroxidase: Solutions to the problems of l i v i n g with oxygen. New Phytol. 73:1075-1086, 1974.
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COMBS, G.F. and SCOTT, M.L. Polychlorinated biphenyl-stimulated selenium deficiency in the chick. Poultry Sci.. 54:1152-1158, 1975.
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817