,'r 1985 by The Humana Press Inc. All n~hts of any nature whatsoever reserved. 0163-4984185i8008-0065502.00

Serum Selenium in Danish Blood Bank Donors E . B. T H O R L I N G , L* K. OVERVAD, ~ A. H E E R F O R D T , ~ AND A. F O L D S P A N G 2

The Institute of Cancer Research, The Danish Cancer Society, Radiumstationen, DK-8000 Aarhus C, Denmark and 2The University of Aarhus, Institute of Social Medicine, DK-8000 Aarhus C, Denmark Received November 24, 1984; Accepted March 18, 1985

ABSTRACT Serum selenium concentration was determined over a full year in healthy donors from the Blood Bank at the Municipal Hospital in Aarhus, Denmark. The age range of the donors, half of whom were women, was 20-60 yr. In months with even numbers, samples were obtained from women; in months with odd numbers, from men. For estimates of individual seasonal variation, a number of the laboratoD"s employees were observed with monthlv samples throughout a full year. The mean serum selenium concentration plus minus SD was 81 • 12 ~g/L for men (range, 42-145 ~g/L), and 79 • 9 ~.g/L for women (range, 51-110 ~g/L). This difference is statistically significant. We found no significant variation within the sexes due to age. The data on a possible seasonal variation are not conclusive, but suggest slightly lower values for late summer. Individuals appear to have fairly stable values over extended periods of time provided there are no major changes in lifestyle, selenium medication, or pregnancy. Index Entries: Serum selenium, in blood donors; age. and serum Se in blood donors; sex, and serum Se in blood donors; seasonal variation, of serum Se in blood donors; blood donors, seasonal variation of Se in; selenium, seasonal variation in blood. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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INTRODUCTION In 1957, selenium was s h o w n to be essential in animal nutrition (1), and recently the essentiality in h u m a n nutrition has been definitely established (2). The importance of selenium arises mainly from its being a constituent part of the enzyme, glutathione peroxidase, which plays a major role in reduction of peroxides (3). Attention was called to the importance of selenium as an anticarcinogen in animal experiments by Clayton and Bauman (4) as early as 1949. This interest was strongly amplified by elegant experiments in mice bearing the mouse m a m m a r y tumor virus by Schrauzer et al. (5) in 1980. The possibility that selenium might also be a protective agent against h u m a n cancers was suggested by epidemiological correlation studies by Shamberger (6) in 1976 and recently in more extensive studies in USA by Willett et al. (1983) (7) and in Finland by Salonen et al. (8) in 1984. Recent reports also associate relatively low selenium status (9-12) with cardiovascular degenerative diseases and cardiomyopathies. Human selenium status varies considerably from one part of the world to another because of differences in the selenium content of the diet. The Scandinavian countries have long been recognized as an area with relatively low selenium availability (13). From a Danish point of view, selen i u m is therefore of special interest. The present investigation was undertaken to establish a reference material for the central East Coast of Jutland.

MATERIAL AND M E T H O D S The material consists of healthy donors frequenting the Blood Bank at the Municipal Hospital in Aarhus, Denmark. The sampling of blood specimens began April, 1982, and was carried on t h r o u g h March, 1983. Every month, 20-30 serum samples w e r e obtained. In m o n t h s with even numbers, the samples were collected from w o m e n (a total of 134); and in months with odd numbers, from m e n (a total of 154). For all donors, age, sex, and date of sampling were registered. The blood sampling was d o n e between 9 and 12 AM. Ten mL of venous blood were d r a w n with no additives, and centrifuged after clot retraction. A serum aliquot of 3-4 mL was kept frozen until use. At the Blood Bank laboratories, a large pool of serum was collected, thoroughly mixed, and frozen in separate tubes of 3-4 mL for use in the day-to-day control of the assay. The samples were analyzed by a modification of the fluorometric m e t h o d described by Watkinson (14). In this, a strongly fluorescent corn-

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pound, a piazselenol, was formed by reaction between selenite and 2,3-diaminonaphthalene. Acid digestion was performed in a large reagent tube. One mL of the sample was treated with 10 mL of concentrated nitric acid. The tube was refluxed for 30 min, cooled, and 4 mL of concentrated perchloric acid were added. The nitric acid was removed by distillation, and the sample was refluxed with perchloric acid for 30 min to remove the nitric acid. The sample was then refluxed with 2 mL 1.25M HC1 for 5 rain. Five mL E D T A - N H 2 - O H solution was added and the pH was adjusted to 0.8-1.2 with 6M NH3 solution using cresol red as the pH indicator. Five mL of d i a m i n o n a p h t h a l e n e solution was added and the solution was left to react for 30 min in a water bath at 50~ The fluorescent c o m p o u n d was extracted into 5 mL of decalin. The decalin was washed with 50 mL of 0.1M HC1. The excitation wave-length was 365 n m and the m e a s u r e d wavelength was 525 nm. Standards used were 0.05, 0.100, and 0.200 ~g of sodium selenite. A Tecator heating block with 20 destructor tubes and a suction head for each tube was used.

Reagents Used Nitric acid, 65%, Merck Art. 452; 70% perchloric acid, Merck Art. 519; EDTA, Fluka Puriss; hydroxylamine as hydrochloride, EDTA-NHz--OH solution: 8 g EDTA and 25 g NONHBC1/L. NH3 was a d d e d to dissolve EDTA. Ammonia Cresol red, Merck Art. 5225, 2,3-diaminonaphthalene, Aldrich 17,613-3. Only the purest quality obtainable can be used without special purification. Responsible for most of the blank values. DAN solution: 1 g/L in 0.1M HC1. Decah y d r o n a p h t h a l e n e (decalin), Merck Art. 803101. Purified by passing through a 10 cm high column of A1203. Hydrochloric acid, Merck Art. 317. A1203, Merck Art. 1097. Sodiumselenit, Merck Art. 6607.

ReproducibiliO" The day-to-day variation of a serum pool was 2.5%. If the deviation exceeded 5%, the results were discharged, and the test was repeated.

Recovery A d d e d selenite could be recovered with 96% confidentiality.

Comparability to International Reference Standards Tests were run on "Seronorm" 102 and 103 samples obtained from Nyegaard and Co. AS, Oslo, Norway. We found 84 - 2 i~g/L (n -- 7) for batch 102 compared to 91 - 3 I~g/L, and 87 - 1 I~g/L (n = 8) for batch 103 compared to 93 • 3 p.g/L.

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Statistical Analysis In the statistical a~nalysis, analysis of variance a n d linear regression was e m p l o y e d (15). The analysis was carried out by use of the SPSS package (16), a n d the h a r d w a r e facilities at the Regional C o m p u t e r C e n t e r at the University of A a r h u s (RECAU). RESULTS Data on the s e r u m selenium level were obtained from a total of 288 blood d o n o r s , of w h i c h 134 were w o m e n and 154 men. The age distribution is s h o w n in Fig. 1. For the complete material, the s e r u m s e l e n i u m concentration, m e a n + s t a n d a r d deviation was 79.8 • 10.6 ~g/L (range for m e n , 42-145 ~g/L; range for w o m e n , 51-110 ~g/L). The data are close to normally distributed (Fig. 2), b u t after logarithmic t r a n s f o r m a t i o n a still better fit was found. Logarithmically transf o r m e d data were accordingly u s e d in the statistical analysis.

Influence of Gender The m e a n value for m e n was 80.8 ~g/L, a n d for w o m e n 78.6 ~g/L (Table 1). Based on a one-way analysis of variance of the raw data, a n d AGE

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TABLE 1 Serum Selenium Concentration (~.g/L) and Gender Serum selenium concentration, ~g/L Sex

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154 134 288

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Age Dependency The age d e p e n d e n c y was investigated using one-way a n d t w o - w a y analysis of variance as well as regression. No variation with age could be demonstrated. SERUM

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Seasonal Variation A o n e - w a y analysis of variance s h o w e d a signficant variation in the m o n t h l y m e a n values, especially for w o m e n . For w o m e n , the A u g u s t value was significantly lower t h a n the other m o n t h s ' (Fig. 3). The s a m e seasonal t e n d e n c y was a p p a r e n t for men, but to a lesser extent (Fig. 3). For July a n d A u g u s t combined, the variations were significantly lower than for the rest of the year, p = 0.0005. The statistical significance of this difference was confirmed in a regression analysis.

DISCUSSION S e r u m s e l e n i u m for a Danish reference p o p u l a t i o n was 81 +- 12 ~xg/L for m e n a n d 79 -+ 9 txg/L for w o m e n . This is at the same level as the other Scandinavian countries (17-19), and thereby further establishes Scandinavia as a relatively selenium-deficient area. The g e n d e r difference f o u n d has not been described in previous papers on reference populations, a n d the biological significance of this minor statistical difference might well be negligible. In the age range 20-60 yr, no significant variation from age was f o u n d . Previous works have d e m o n s t r a t e d low and variable values in early childhood, a n d decreasing values in old age (21). It s h o u l d therefore be stressed that this reference material concerns the age range 20-60 yr. The s e r u m s e l e n i u m concentration appears to be fairly stable over the year for both genders. There is, however, a statistically significant d r o p in w o m e n in the later s u m m e r , a n d a similar t e n d e n c y for m e n (Fig. 3). A n u m b e r of e m p l o y e e s at the Institute were followed for several m o n t h s in order to investigate the individual variations over p r o l o n g e d periods (Fig. 4). In these series we f o u n d remarkably stable values a n d no indication of any seasonal variation. Therefore, the slightly d e p r e s s e d ser u m s e l e n i u m concentration f o u n d in late s u m m e r may be taken as suggestive only of a possible seasonal variation, but the data collected so far do not warrant any definite statements.

ACKNOWLEDGMENTS We w a n t to express our thanks to the Head of the Blood Bank, Jan J6rgensen, MD, for his kind cooperation in providing us with the samples. The skilled expert laboratory assistance of Ms. Bente M a d s e n is highly appreciated.

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Serum selenium in danish Blood Bank donors.

Serum selenium concentration was determined over a full year in healthy donors from the Blood Bank at the municipal Hospital in Aarhus, Denmark. The a...
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