BIOLOGICAL TRACE ELEMENT RESEARCH 5, 91-102 t l983)
The Selenium Status of Belgian Population Groups I. Healthy Adults M.
VERLINDEN,~'*"~
M.
VAN S P R U N D E L , 2 J . C . V A N DER
A U W E R A , - ' AND W . J. E Y L E N B O S C H 2
Departments of Pharmaceutical Sciences' and of Social Medicine and Epidemiology,: University of Antwerpen, Universiteitsplein I,
B-2610 Wilrijk, Belgium Received August 10, 1982; Accepted November 16, 1982
Abstract A moderate level of selenium (Se) was demonstrated in the blood (123 ng/mL), erythrocytes (RBC) (161 ng/mL), and plasma (97 ng/mL) of t 10 male and 54 healthy female Belgian adults by using hydride-generation atomic absorption spectrometry. These values agree well with those found in neighboring countries. The study is the first one to report on normal values for these parameters in Belgians. Glutathione peroxidase (GSH-Px) activity has also been assayed. The group is considered nondeficient. Neither the concentration of Se in blood, plasma, or RBC, nor the activity of GSH-Px in the latter, were related to age, sex, blood pressure, or smoking habits. Oral contraception was associated with elevated plasma Se levels as compared to controls (P < 0.01), A statistically significant positive relationship existed between the Se concentrations in whole blood or in red blood ceils and red blood celt GSH-Px activity (r = 0,31, P < 0.005). Index Entries: Selenium, in whole blood; selenium in red blood cells; erythrocyte selenium; plasma, selenium in; normal values for selenium; selenium and oral contraception; selenium and smoking; selenium and obesity; selenium and age; selenium and sex; selenium and blood pressure; glutathione peroxidase, in erythrocytes, glutathione peroxidase, and selenium concentration in blood fractions; Belgian adults, selenium in; selenium intake, in Belgium; selenium status; atomic absorption '*Present address: Janssen Pharmaceutica N.V.. Clinical R&D Department, B-2340 Beerse, Belgium.
91
9~
VERLINDEN ET AL.
spectrometry,, of selenium; contraceptives, and selenium; smoking, and selenium; obesity, and selenium; age, and selenium; sex, and selenium; blood pressure, and selenium.
Introduction It has been 25 yr since the discovery was made that selenium (Se) is an essential element in warm-blooded animals (1, 2). An increasing number of observations also indicate that there is an essential role for this ultramicro trace element in humans. Selenium, as selenocysteine, constitutes the active center of the enzyme glutathione peroxidase (GSH-Px) (3-5). The enzyme has been isolated from human e~throcytes (6). Selenium was shown to be required for growth of human fibroblasts (7). Furthermore, a selenium-responsive muscular syndrome has been reported in a patienton total parenteral nutrition (8). Several authors have published articles and comprehensive reviews on the role of Se and GSH-Px in human biology and pathology (9-15). Attention has also been focused on the possible involvement of selenium deficiency as a contributory factor in the causation of excessive cancer and cardiovascular deaths in low-selenium areas. There is well-established evidence of great variation in the geophysical occurrence of Se.(l 7). This variation is reflected in the concentration of blood selenium in the inhabitants (18). Animal experiments demonstrated the anticarcinogenic properties of Se in many tumor model systems, and epidemiologic studies present evidence that cancer mortalities, among other factors, are controlled by trace elements such as Se (19-25). Significantly lower blood or plasma Se levels were found in patients with carcinoma when compared to normals (26, 27). It has been proposed, accordingly, that an increase of the dietary Se intakes, either by change of diet or by supplementation, may decrease the cancer risk of some low-Se populations (25, 28). With regard to cardiovascular diseases it was noted that age-specific death rates for cardiovascular-renal, cerebrovascular, coronary, and hypertensive heart disease as well as for ischemic and hypertensive diseases were significantly lower in the very high-bioavailable Se areas than in low-Se areas (29, 30). Mean bloodbank Se concentrations were found to be inversely related to these sex- and agespecific death rates (31). Selenium has been shown to reverse Cd-induced hyptersension in rats (32). Keshan disease, a type of congestive heart disease endemic in some parts of China that are very low in Se (33), can be prevented or alleviated by the administration of sodium selenite (34). Selenium deficiency might be contributory to the myocardiopathy by influencing the natural history, of the infection or by predisposing individuals to clinical infections (35). It follows that research into the normal values of blood selenium concentrations in any particular country is essential. Lack of knowledge of the normal values of selenium parameters in healthy individuals may prevent the diagnosis of abnormal states or the identification of population groups that are at risk of developing serious diseases owing to Se deficiency. This paper deals with the determination of normal values for the
S E L E N I U M IN B E L G I A N A D U L T S
93
Se parameters: Se in blood, plasma, and red blood cells (RBC) and the activity of GSH-Px in a group of Belgian adults.
Materials and Methods
Sl~bjecIs Blood samples were taken from 164 Belgian residents, who were employed in a company manufacturing electronic equipment situated in Gent. They were not professionally exposed to selenium and were found in good general health by the occupational physician at the regular medical examination. One hundred and ten of these were men with an age range from 20 to 58 yr (mean 33 yr); 54 were women whose average age was 34 yr (ranging from 19 to 52 yr). People were questioned with regard to smoking habits, current use of medication, and oral contraceptives. Twenty-one out of 54 women were smokers; 4 of them smoked fewer than 5 cigarets a day, 3 were moderate smokers (between 5 and 14 cigarets a day) and 14 smoked heavily ( 15 cigarets a day or more). Of 75 questioned male participants, 21 did not smoke, 4 smoked lightly, 8 moderately, whereas 42 were heavy smokers. Regular consumers of drugs were excluded from the study. Out of 54 women, 23 used oral contraceptives. There was no difference in age between women practicing oral contraception and those who did not (P > 0.05). The length and weight of each participant was measured. The obesity index was calculated, as the ratio of weight and cubed length (W:L 3) (36). The average obesity indices for women and men did not differ significantly. A highly significant positive correlation existed between the obesity index and age [P < 0.0005, r = 0.395, n = 161]. This difference in obesity of participants in different age groups was more outspoken for males than for females. Participants were also examined with regard to some hematological parameters and blood pressure. Counts of red blood cells (RBC), white blood cells (WBC), and platelets were normal, as were the hematocrit (Hct) value, the hemoglobin concentration (Hb), and the mean corpuscular hemoglobin concentration (MCHC). Of 102 male and 54 female persons whose blood pressure was measured, 81 men and 50 women were considered normotensive (systolic pressure lower than 140 mm Hg and diastolic pressure lower than 90 mm Hg); 21 men and 4 women were hypertensive (systolic pressure 140 mm Hg or more and/or diastolic pressure 90 mm Hg or more). The average obesity index was slightly higher in hypertensives than in normotensives, but the difference was not statistically significant. Neither was there a significant difference in age between both blood pressure groups.
Estimation of Selenium Content and GSH-Px Activi~ Blood was taken by venipuncture in vacuum-containing tubes (BectonDickinson), on Li-heparin. In order to avoid differences in posture as possible sources of variations in plasma selenium concentrations (37), patients were placed in a supine position for at least 5 rain prior to puncture. Subjects were not required
94
~,ERI.INDEN E T A [ .
to be fasting. After centrifugation of a portion of the blood, Se was determined in blood and plasma by hydride-generation atomic-absorption spectrometry ~38). Sample pretreatment was by a wet HNO_~/HCIO~ digestion at 210~ as described in detail elsewhere (39). The selenium content of erythrocytes was calculated according to the hematocrit and the selenium contents of whole blood and plasma. In addition, the activity of euthrocyte GStt-Px was assayed at 37~ by a modification (40) of the method of Paglia and Valentine (41), with t-butylhydroperoxide as acceptor substrate. The activity was calculated in a coupled test procedure as the oxidation rate of NADPH in the presence of excess glutathione reductase. It is generally recognized that variations occur from time to time when the assay is carried out in different laboratories, or even within a given laboratory, and that it is essential for each laboratory to establish its own range of normal values (40). In this study' normal values were 45 +_ 20 U:v/g Hb (mean -+ 2 SD, n = 90), Within-day variability was less than 4% relative standard deviation; between-days variability less than 10% (42).
Statistical Evaluation The values of selenium concentrations in blood and plasma were distributed normally as was the activity of GSH-Px (~('-test for goodness of fit). The calculated erythrocyte Se concentrations slightly departed from normality. Differences between means were tested by the two-sample t-test, and by one-way analysis of variance. Data were occasionally subjected to correlation and linear regression analysis (43, 44).
Results The mean (-+ SD) blood, plasma, and e~throcyte Se concentrations (Cb~,• Cpt~_~,,~, and CRuc) and the enzyme activity are shown in "Fable 1. These parameters are represented as a function of age in Table 2, whereas the histogram in Fig. 1 (a-d) visualizes the distribution for the entire group under investigation. The slight deviation from normality of the distribution of erythrocyte Se may occur because these values do not represent measurements, but are rather rough estimates obtained by calculation. There was no significant variation in blood, plasma, or RBC-Se or in GSH-Px activity with age, in either males or females. Neither was there a variation with sex, obesity, smoking habits, or blood pressure. Differences in hematocrit, WBC or RBC count, or hemoglobin concentration did not produce differences in the values of the Se indicators. Neither were they affected by smoking. Oral contraception had no influence on the Se content of whole blood and erythrocytes. However, those women taking oral contraceptives exhibited plasma Se levels that were slightly, but significantly, higher than plasma levels of women who did not, as demonstrated in Table 3 (t = 2.62. n = 23, P < 0.0l, one-sided t-test). In almost every instance, most of the blood Se was concentrated in the cell compartment. On a cell basis, leukocytes contain larger quantities of selenium than ewthrocytes, but account for only less than 5% of the
95
SELENIUM IN BELGIAN ADULTS
TABLE 1 Mean Concentration ( • Standard Deviation) o f Se in Blood, Plasma, and Erythrocytes" Parameter
Sex
n
,~ • x
st, %
Range
Median
Se in blood, ng/mL
M F M + F
109 53 162
122 • 17 125 • 17 123 • 17
13.9 13.6 13,8
8 2 - 176 99 - 178 82 - 178
120 121 121
Se in plasma, ng/mL
M F M + F
110 53 163
97 • 12 97 • 12 97 • 12
12.4 12.4 12.4
67 - 123 7 2 - 119 67 - 123
98 97 97
Se in RBC, ng/rnL
M F M + F
108 52 160
159 • 31 165 +-- 32 161 --+ 32
19.5 19.3 19.9
94 - 263 104 - 259 94 - 263
154 163 157
GSh-Px, U37/g Hb
M F M • F
107 52 159
48.14 • 10.85 46.17 • 1t.39 47.50 • 11,07
22.5 24.7 23.3
23.8 - 78.0 1 9 . 6 - 70.6 19.6 - 78.0
47.90 45.10 47.80
Se in cell traction % o f total
M F M • F
108 52 160
55.3 • 6.2 54.2 +__ 5.5 54.9 • 6.0
11.2 10,2 10.9
47 - 26 43 - 68 41 - 71
55.0 54,5 55.0
activity of GSH-Px. M, male; F. female; n, number of subjects: Sr, relative standard deviation.
~
total S e p r e s e n t in w h o l e b l o o d (45). H e n c e , for p r a c t i c a l p u r p o s e s , the c a l c u l a t e d cell S e w a s c o n s i d e r e d to be l o c a t e d in r e d b l o o d c e i l s o n l y . O n the a v e r a g e , R B C c o n t a i n e d 1.26 t i m e s m o r e S e than the p l a s m a f r a c t i o n , i . e . , a p p r o x i m a t e l y 5 5 % o f b l o o d S e w a s a s s o c i a t e d with the e r y t h r o c y t e s . E x c e p t for b l o o d h e m o g l o b i n c o n c e n t r a t i o n , n o n e o f the factors m e n t i o n e d a b o v e ( s e x , a g e , e t c . ) s h o w e d a r e l a t i o n s h i p to the ratio o f cell Se o v e r p l a s m a Se. H o w e v e r , an i n c r e a s e o f the h e m o g l o b i n TABLE 2 Mean Concentration ( • Standard Deviation) of Se in Blood, Plasma, and Erythrocytes and of Erythrocyte GSH-Px Activity as a Function of Age ~ Selenium concentration, ng/mL Blood Age, yr <~ 19 20-24 25-29 30--34 35-39 40---44 9 45-49 50--54 55-59
n 1 27 30 41 22 23 11 4 3
~f • s 112 122 119 126 122 126 128 126 102
--- 19 --+ 15 • 18 • 17 • 16 • 13 • 17 • 6
~Symbols as in Table 1.
Red blood cells n
~•
1 27 29 41 22 23 11 3 3
130 160 147 166 162 172 173 164 106
s • • -+ • +_ • -+-
30 22 33 32 31 35 31 14
Activity glutathione-
Plasma n 1 29 30 42 22 23 11 3 3
.4• s 99 96 97 98 93 95 96 107 99
• 12 __+ 13 • 14 • 11 --+ 12 --- 9 __+ 6 • 9
peroxidase,U37/g Hb n 1 28 29 40 22 22 10 4 3
.~• s 27.5 45.0 43.9 50,3 46.0 53.8 46.9 44.2 45.9
-+ 10.4 -+ 9.3 • 12.8 • 9.8 • 9.8 • 7.4 -+- 18.0 • 5.0
96
VERLINDEN
%
ET AL.
% 20-
20M:109
=~15-
I
M:110 s F:53 I
!
F:53
cr
]
g ~> 10-
~r.
:
}5ng Se/ml blood 100
125
ng O-
L","50 175
l .
,
75
, ,
, ,
t00
%
:i-'i
Se/rnl plasma
125
b
%
20.
20" M:108 ~ ~ ] F:52 I l
15-
~?'715-
10-
'- 1 0 -
-~ 5ng Se/ml RBC O-
''1''~'
100
'
1
150
E
200
'1
250
'~
l
O-
300
25
c
50
75
d
Fig. 1. Histogram of the distribution of Se contents in blood (a), plasma (b), red blood cells (c), and of GSH-Px activity (d) for the entire population analyzed. concentration was associated with an increase of this ratio. The influence of hematocrit values on cell Se concentration was not investigated. Regression analysis of the data on Se concentrations yielded the following equation: Cblood
= 64.37 + 0.61 cp,a~,,,~
in n g / m L
TABLE 3 Influence of Oral Contraception on Values of Se Parameters in a Group of Healthy Belgian Women Oral contraception Parameter Se in whole blood, ng/mL Se in plasma, ng/mL Se in red blood cells, ng/mL GSH-Px, U37/g Hb
-
1 2 2 - 14 (30) 93 +-- 12 (29) 168 • 29 (28) 44.45 • 12.45 (28)
+ 126 101 157 48.85
• 17 (23) --+ 10 (23) -+ 30 (23) _+_ 11.09 (23)
97
SELENIUM IN BELGIAN ADULTS
Fig. 2. residents.
Erythrocyte Se concentrations and GSH-Px activities in 155 healthy Belgian
Erythrocyte and plasma Se concentrations (ng/mL) were not correlated. A significant positive relationship was seen between the blood or erythrocyte Se levels and erythrocyte GSH-Px activity. The scatter diagram of the latter is shown in Fig. 2 (n = 155, r = 0.31, P < 0.0005). The enzyme activity could be predicted from erythrocyte Se levels by the equation: GSH-Px = 30.34 + 0.11 CRBC No relation was found between erythorcyte GSH-Px activity and plasma Se concent.ration.
Discussion On the basis of the selenium content of soil and crops, several geographic areas have been delineated. The selenium content of plant and animal products generally corresponds to the soil selenium levels (12). The Se content in soils is usually reflected in the Se levels of blood and blood fractions of the inhabitants and the activity of erythrocyte glutathione peroxidase (18, 46). Thus, Guatemala (47), Venezuela (48), Canada (49), and some parts of the United States (17, 18) have been considered rich in Se. In these areas Se concentrations in whole blood average 0.18-0.22 ixg/mL (17, 18, 24, 47, 49). However, values as high as 0.813 Ixg/mL were found in the blood of I 11 children living in a seteniferous zone of Venezuela (48). In Japanese adults, plasma and RBC Se values on the order of 0.25--0.30 ~g/mL, respectively have been observed (50). Other countries, e.g., New Zealand (15, 46, 51-53), parts of Finland (54), and Italy (55), have been identified as poor in Se. In New Zealand, selenium deficiency causes pathological conditions in cattle and sheep (56). Human blood and plasma Se levels are typically 0.06-0.08 Ixg/mL and 0.04-0.06 Ixg/mL, respectively
(51-55).
98
V E R L I N D E N ET AL.
The mean whole blood and plasma Se concentrations of 0. 123 ~g/mL (SD 0.017 txg/mL) and 0.097 Ixg/mL (SD 0.012 I.tg/mL) found in this study compare well with the moderate Se values that have been noted in Germany (573, Holland (58). Norway (59), and Sweden (60). Nineteen healthy German adults exhibited an average plasma Se level of 0. 102 p,g/mL (57), whereas blood Se concentrations averaged 0.14 I~g/mL in a group of more than 1000 Dutch, 19-21 yr old men (58), 0. I 1-0.13 I,zg/mL in Norway (59) and Sweden (60). Versieck et al. (69) have found 0.13 ~g/mL to be the mean Se concentration in serum from l 9 Belgian men and 17 Belgian women, with values ranging from 0.09 to 0.18 p.g/mL. Instrumental neutron activation was the analytical technique for Se determinations. These investigators found an average erythrocyte Se concentration of 0.16 ~g/g (ranging from 0.09 to 0.21 Ixg/g). After conversion to volume yields, this corresponds to about 0.17 p,g/mL RBC. The values calculated in this study are somewhat lower. Since nutritional myopathy caused by selenium deficiency in pasture occurs in Belgian calves (62), it may be suspected that the Belgian human population also receives too little selenium, but until now. actual intakes have not been determined. Schrauzer and White showed that a linear correlation exists between dietary Se intakes and blood Se levels (63). On the basis of their work one can caculate that a mean whole bIood Se concentration of 0.123 I.Lg/mL corresponds to an average daily Se intake of about 70 p,g/d (range 40-120 ~g). This is in good agreement with our chemical analysis of 24 h diets. An average Se intake of 55 ~g/d for healthy Belgian adults could thus be estimated (64). Welsh et al. (65) determined Se intakes by analyzing 132 diet composites of 22 Maryland residents. They found a mean daily Se intake of 81.0 p,g (SD 41 Ixg). Of these diets, 80% had Se levels within the 50-200 p.g/d range that is set by the Food and Nutrition Board of the American National Academy of Sciences as "estimated safe and adequate daily dietary intakes" for selenium (66). A survey of reported daily intakes for different parts of the United States and for some other countries was presented by these authors (65). Chemical analysis of diet duplicates yielded an average dietary Se intake of 93 Ixg/d in North-Carolina, approximately 220 I.zg/d in South Dakota and in some parts of Canada, 6-33 Ixffd in New Zealand, excluding fish and organ meat, or 34--70 p,g/d when the latter are included (65). Levander et al. have carried out depletion/repletion studies for estimating human Se requirements (67). They found that adult North American males need about 54 I.Lg/d of Se to replace fecal and urinary losses and to maintain plasma levels and body stores, ff 80% of the food Se is absorbed this corresponds to a dietary intake of about 70 Ixg/d. In the light of these findings, many diets may not provide adequate amounts of Se. However, as pointed out by the authors, dietary Se recommendations for North Americans may not be appropriate for other groups, as demonstrated by the low amount of Se (24 ~g/d) needed to achieve balance in New Zealand residents (67). From the limited data on dietary Se intakes and on blood Se concentrations, it seems reasonable to classify the group of people that was studied among those population groups that have neither a high nor a very. low Se status. This classification does not preclude the occurrence of Se deficiency syndromes in livestock. As pointed out by other investigators, geographic differences in soil Se concentrations are more pronounced than those in blood Se because of, e.g.,
SELENIUM IN BELGIAN ADULTS
99
interregional movements of human food (18). This argument holds especially for small countries with a high degree of industrialization, such as Belgium, where locally produced foods do not constitute the bulk of the diet. Plasma Se concentrations are generally lower than the concentrations in blood and in RBC (15, 37, 46, 68). Our data resemble those of Dickson and Tomlinson (49) and of Behne and Wolters (69). The first authors tbund RBC to contain 1.26 times more of the total blood Se content than plasma (49), where Behne and Wolters reported 55% of whole blood Se to be present as RBC Se (69). Differences in age are usually not reflected in differences of Se blood or plasma values (18, 51, 53, 70), at least not in the age groups 20-60 yr. No differences were found between males and females regarding plasma Se, blood Se, RBC Se, or GSH-Px activity (46, 51, 54). Blood Se concentrations were also the same in patients with and without hypertension (70, 71). Kay and Knight (53) previously showed that smoking does not produce alterations in blood Se. These authors also did not see a significant effect on blood Se levels in women taking oral contraceptives (53), in accord with our observations. However, we found plasma levels to be approximately 10% higher in women on oral contraceptives than in controls, whereas Behne et al. noted lowering effects of sex hormones and pregnancy on hair and serum Se concentrations, respectively (69, 73). Neither erythrocyte Se nor GSH-Px activity were changed in pregnant compared to nonpregnant women, but a decrease was seen for both parameters in plasma (69)..These changes were not caused by alterations in the protein/water ratio during pregnancy. Rudolph and Wong found lower plasma Se levels and GSH-Px activities in pregnancy. Although erythrocyte Se concentrations were the same, erythrocyte GSH-Px activities were significantly higher (74). In New Zealand a very significant positive relationship was found between the activity of erythrocyte GSH-Px and the blood Se concentration (15, 7l, 75) when the latter were below 0.1 and 0.14 Ixg/mL, respectively. In spite of Se levels much higher than these New Zealand limits, Rudolph and Wong noted a very close positive correlation between Se levels and GSH-Px activity, both in red blood cells and in plasma, of 24 paired maternal-cord samples together with samples from healthy men and women (74). Workers in the United States (63. 76) and Germany (69, 77), however, found no correlation between Se in blood or blood fractions and GSH-Px. This was considered a logical consequence of the fact that only 10-11% of the total Se is bound to GSH-Px (69, 77). Our results seem to support the New Zealand findings. The mean erythrocyte Se concentration was 0.16 txg/mL, i.e., approximately the limit set by New Zealand investigators for a correlation with GSH-Px activity. This may explain why the correlation, although highly significant, was weak (r = 0.31, P < 0.0005). In view of the results obtained in elderly people, this relationship is further discussed elsewhere (64). At present, Se concentrations in biological fluids as well as erythrocyte GSH-Px activity are considered indicative of Se status. On this basis, the subjects investigated in the present study cannot be considered Se-deficient. Hewever, it remains possible that the current criteria of Se adequacy will have to be revised in the future and that more specific indicators of selenium status will be discovered. The existence of a non-Se-dependent form of GSH-Px in erythrocytes (78) also cautions
1.00
VERLINDEN ET AL,
against extending conclusions with regard to ewthrocyte (Se-dependent) GSH-Px to the overall Se status.
Acknowledgments One of us (MV) wishes to express her gratitude to the Belgian National Fund for Scientific Research whose research assistant she was at the time of the investigation. Furthermore the authors are greatly indebted to Dr. G. S. Fell (Glasgow Royal Infirmary) for having made assays of the GSH-Px activity possible and to Prof. M. Peetermans and Dr. M. Van Der Plancken, hematologists (Academic Hospital Antwerpen). The department of pharmaceutical sciences (UIA), especially Prof, H. Deelstra, is acknowledged for having provided technical facilities for atomic absorption spectrometric measurements.
References 1. K. Schwarz and C. M. Foltz, J. Am Chem. Soc. 79, 3292 (1957). 2. E. L. Patterson, R. Milstrey, and E. L. R. Stokstad, Proc. Soc. Exp. Biol. Med. 95, 617 (1957). 3. J.T. Rotruck, A. L. Pope, H. E. Ganther, D. G. Hafeman, and W. G. Hoekstra. Science 179, 588 (1973). 4. L. Floh6, W. A. GiJnzler, and H. H. Schock, FEBS Lett. 32, 132 (1973). 5. R. Ladenstein, O. Epp, R. Huber, and A. Wendel, Proceedings of the 3rd International Symposium on Organic Selenium and Tellurium Compounds, D. Cagniant and G. Kirsch, eds., Universit6 de Metz, 1979, pp. 147-153. 6. Y. C. Awasthi, E. Beutler, and S. L. Srivastava, J. Biol. Chem. 250, 5144 (1975). 7. W. L. McKeehan, W. G. Hamiltcn, and R. G. Ham, Proc. Natl. Acad. Sci. 73, 2023 (1976). 8. A. M. van Rij, C. D. Thomson, J. M. McKenzie, and M. F Robinson. Am. J. Clin. Nutr. 32, 2076 (1979). 9. W. G. Hoekstra, In Trace Element Metabolism in Animals-2, J. W. Suttie and H. E. Ganther, eds., University Park Press, Baltimore, 1974, pp. 61-77. 10. D. V. Frost and P. M. Lish, Annu. Rev. Pharmacol. Toxicol. 15, 259 (1975). 11. W. G. Hoekstra, Fed. Proc. 34(11), 2083 (1975). 12. R. F. Burk. in Trace Elements in Human Health and Disease. vol. II, A. S. Prasad and D. Oberleas, eds., Academic Press, New York, 1976, pp. 1(34-133. 13. H. E. Ganther, D. G. Hafeman, R. A. Lawrence, R. E. Serfass, and W. G. Hoekstra, in Trace Elements in Human Health and Disease, vol. II, A. S. Prasad and D. Oberleas, eds., Academic Press, New York, NY, 1976, pp. 165-234. 14. K. Schwarz, Med. Clin. North America 60(4), 745 (1976). 15. C. D. Thomson and M. F. Robinson, Am. J. Clin. Nutr. 33, 303 (1980). 16. R. J. Shamberger, Sci. Total Environ. 17, 59 (1981). 17. J. Kubota, W. H. Allaway, D. L. Carter, E. E. Cary, and V. A. Lazar, J. Agric. Food Chem. 15, 448 (1967). 18. W. H. Allaway, J. Kubota, F. Losee, and M. Roth, Arch. Environ. Health 16, 342 (1968).
SELENIUM IN BELGIAN ADULTS
lO[
19. G. N. Schrauzer, J. E. McGinness, and K. Kuehn, Proceedings of the 3rd International Symposium on Organic" Selenium and Tellurium Compounds. D. Cagniant and 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
30.
31.
32.
33. 34. 35. 36.
37. 38. 39. 40. 41. 42. 43. 44. 45.
46.
G. Kirsch. eds., Universit6 de Metz, 1979, pp. 171-183. G. N. Schrauzer, D. A. White, and C. J. Schneider, Bioinorg. Chem. 7, 35 (1977). C. C. Clayton and C. A. Baumann, Cancer Res. 9, 575 (1949). J. F. Riley, Experientia 24, 1237 (1969). R. J. Shamberger, J. Natl. Cancer Inst. 44, 931 (1971). R. J. Shamberger and C. E. Willis, CRC Crit. Rev. Clin. Lab. Sci. 2, 211 (1971). G. N. Schrauzer, D. A. White, and C. J. Schneider, Bioinorg. Chem. 7, 23 (11977). R. J. Shamberger, E. Rukovena, A. K. Longfield, S. A. Yytko, S. Deohar, and C_ E. Willis, J. Natl. Cancer Inst. 50(4), 863 (1973). W. L. Broghamer, K. P. McConnell, and A. L. Blotcky, Cancer 37, 1384 (1976). M. G. Lafond and E. J. Calabrese, Med. Hypotheses 5(8), 877 (1979). R.J. Shamberger. S. A. Tytko, and C. E. Willis, in Trace Substances in Environmental Health, 1X, D. D. Hemphill, ed., University of Missouri, Columbia, Mo., 1975, pp. 15-22. R. J. Shamberger,/Vl. S. Gunsch. C. E. Willis, and L. J. McCormack, in Trace Substances in Environmental Health, XII, D. D. Hemphill, ed.. University of Missouri, Columbia, Mo., 1978, pp. 48-52. R. J. Shamberger, C. E. Willis, and L. J. McCormack, in Trace Substances in Environmental Health, XIII, D. D. Hemphill, ed., University of Missouri, Columbia, Mo., 19"79, pp. 59-63. H.M. Perry, E. F. Perry, and M. W. Erlangen, in Trace Substances in Environmental Health, VIII, D. D. Hemphill, ed., University 0f Missouri, Columbia, Mo., 1974, pp. 51-57. Keshan Disease Research Group of the Chinese Academy of Medical Science Beijing, Chin. Med. J. 92(7), 477 (1979). Keshan Disease Research Group of the Chinese Academy of Medical Science Beijing, Chin. Med. J. 92(7), 471 (1979). H. Guanqing, Chin. Med. J. 92(6), 416 (1979). A. Norden and B. Schersten. in Nutrition and Old Age, B. Borgstr6m, A. Nord6n, B. ,~kesson, M. Abdulla, and M. J/igerstad, eds., Scand. J. Gastroenterol. 14, suppl. 52, 42 (1979). H. JiJrgensen and D. Behne, J. Radioanal. Chem. 37, 375 (1977). M. Verlinden, J. Baart, and H. Deelstra, Talanta 27, 633 (1980). M. Verlinden, Talanta 29, 875 (1982). E. Beutler, K. G. Blum, J. C. Kaplan, G. W. L6hr, B. Ramot, and W. N. Valentine, Br. J. Haematol. 35, 331 (1977). D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). G. S. Fell, personal communication. W. J. Dixon and F. J. Masey, eds., Introduction to Statistical Analysis, 3rd ed., McGraw-Hill Book Company, New York, 1969, p. 638. K. A. Yeomans, ed., Statistics tbr the Social Scientist. Applied Statistics. Penguin Educati.on, Harmondsworth, 1976, p. 397. M. Verlinden, W. Cooreman, and H. Deeelstra, in Trace Element Analytical Chemist~' in Medicine and Biology, P. Br/itter and P. Schramel, eds., Walter de Gruyter & Co., Berlin, 1980, pp. 513-522. M. F. Robinson, P. J. Godfrey, C. D. Thomson. H. M. Rea, and A. M. van Rij, Am. J. Clin. Nutr. 32, 1477 (1979).
102
v~Rt, I.~DE~ EV .~,L.
47. R. F. Burk, W. N. Pearson, R. P. Wood, and F. Viteri, Am. J. Clin. Nutr. 20, 723 (1967). 48. W. G. Jaffa, M. Ruphael, M. C. Modragon. and M. A. Cuevas, Arch. Lat. Amer. Nutr. 22, 595 (1972). 49. R. C. Dickson and R. H. Tomlinson, Clin. Chim. Ac'ta 16, 311 (1967). 50. K. Kurahashi, S. Inoue, S. Yonekura, Y. Shimoishi. and K. T6ei, Analyst 105,690 (1980). 51. N. M. Griffiths and C. D. Thomson. NZ Med. J. 80, 199 (1974). 52. C. D. Thomson, H. M. Rea, V. M. Doesburg, and M. F. Robinson, ,r~r. J. Nutr. 37, 457 (1977). 53. R. G. Kay and G S. Knight. N Z M e d . J. 90, 11 (1979). 54. T. Westermarck, P. Raunu. M. Kit~jarinta, and 1.. Lappalainen. ,4cm Pharmacol. Toxicol. 40, 465 (1977). 55. I... C. Rossi, G F. Clemente. and G. Santaroni, Arch. Environ. Health 31, 160 (19761). 56. W. J. Hartley and A. B. Grant. Fed. Proc. 20, 679 (1961). 57. I. Lombeck, K. Kasperek, H. D. Harbisch, L. E. Feinendegen, and H. J. Bremer, Eur. J. Pediatr. 125, 81 (1977). 58. J. J. M. de Goeij, personal communication. 59. K. Saeed, Y. Thomassen, and F. J. kangmyhr, Anal. Chim. Acta 110, 285 (1979). 60. D. Brune. K. Samsahl, and P. O. Wester, Clin. Chim. Acta 13, 285 (1966). 61. J. Versieck, J. Hoste, F. Barbier. H Michels, and J. De Rudder, Clin. Chem. 23, 1301 (t077). 62. F. Lomba, G. Chauvaux, A. Massip, and V. Bienfet, Ann. Med. Vet. 117, 481 (1973). 63. G. N. Schrauzer and D. A. White, Bioinorg. Chem. 8,. 303 (1978). 64. M. Verlinden~ M. Van Sprunde[, J. C. Van der Auwera, and W. J. Eylenbosch, Biol. Trace Elem. Res., 5, 103 (1983). 65. S. O. Welsh, J. M. Holden, W. R. Wolf, and O. A. Levander, J. Am. Dietet. Assoc. 79, 277 (1981). 66. Food and Nutrition Board: Recommended Dietary Allowances, 9th rev. ed.. National Academy of Sciences, Washington, DC, 1980. 67. O. A. Levander, B. Sutherland. V. C. Morris, and J. C. Kind, Am. J. Clin. Nutr. 34, 2662 (1981). 68. R. L. McKenzie, H. M. Rea, C. D. Thomson, and M. F. Robinson, Am. J. Ctin. Nutr. 31, 1413 (1978). 69. D. Behne and W. Wolters, J. Clin. Chem. Clin. Biochem. 17, 133 (1979). 70. J. G. Sullivan, A. J. Blotcky. N. M. Jetton, H. K. J. Hahn, and R. E. Burch, J. Nutr. 109, 1432 (1979). 71. C. D. Thomson, H. M. Rea, M. F. Robinson, and F. O. Simpson. Proc. Univ. Ota#o Med. Sch. 56, 31 (1978). 72. S. Sundberg, P. Luukka, K. L. Andersen, A. W. Eriksson, and P. Siltanen, Ann. Clin. Res. 7, 17 (1975). 73. D. Behne, W. Elger, W. Schmelzer, and M. Witte, Bioinorg. Chem. 5, 199 (1976). 74. N. Rudolph and S. L. Wong, Pediatr. Res. 12, 789 (1978). 75. C. D. Thomson, H. M. Rea, M. F. Robinson, and O. W. Chapman, Proc. Univ. Otago Med. Sch. 55, 18 (1977). 76. J. L. Valentine, H. K. Kang, P. M. Dang, and M. Schluchter, J. Toxicol. Environ. Health 6, 731 (1980). 77. K. Schmidt and W. Heller. Blut 33, 247 (1976). 78. R. A. Lawrence and R. F. Burk, J. Nutr. 108, 211 (19781.
