BIOLOGICAL TRACE ELEMENT RESEARCH 5, t02~113 ~1983)

The Selenium Status of Belgian Population Groups II. Newborns, Children, and the Aged M.

V E R L I N D E N , " * " ~ M. VAN S P R U N D E L , 2, J. C. VAN DER A U W E R A , 2 AND W . J. E Y L E N B O S C H 2

Departments of Pharmaceutical Sciences" and of Social Medicine and Epidemiology,: Universi~ of Antwerpen (U.I.A.), Universiteitsplein 1, B-2610 Wilrijk, Belgium Received August I0, 1982; Accepted November 16, 1982

Abstract The selenium state of 40 elderly Belgian people, residing in geriatric homes, has been evaluated. Data are presented on the selenium (Se) contents of their blood, plasma, and erythrocytes. The activity of glutathione peroxidase (GSH-Px) has been assayed, All data were compared with those obtained for 164 young, working adults as presented in Part I of this study. Plasma selenium levels were significantly lower in the old (73 ng/mL) as compared to the young people (97 nNmL), but erythrocyte Se levels (200 ng/mL) and GSH-Px activity were significantly higher. The selenium concentration in plasma during infancy has also been estimated. The results reveal a very low Se level during the first months of life, with a gradual increase with age. The results are discussed in the light of literature data. Index Entries: Selenium, in whole blood; selenium, in red blood cells; erythrocyte selenium; plasma, selenium in; glutathione peroxidase, in erythrocytes; selenium status, of the elderly; selenium intake, in Belgium; geriatric residents, Se status in; plasma selenium, in newborns; plasma selenium, at delivery; plasma selenium, in children; age, and selenium; creatine kinase, and selenium; glutathione peroxidase, and selenium concentration in blood fractions.

Introduction As an integral part of the enzyme glutathione peroxidase (GSH-Px) (1, 2), selenium plays an important rote in preventing oxidative damage of tissues by lipid tPresent address: Janssen Pharmaceutica N.V., Clinical R&D Department. B-23,.10 Beerse, Belgium. 9 1983 by The Humana Press Inc. All rights of any nature whatsoever re~rved. 0163--4983i83/040(00103502.20

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peroxides. In animals, Se deficiency is well established and is reflected in low Se levels in biological fluids and tissues. In man, a low Se status has been associated with various pathological conditions, such as cardiovascular diseases (3) several types of cancer (3, 4), cirrhosis (5), neuronal ceroid Iipofuscinosis (6). As Se intake is merely by food proteins, low Se values have been repeatedly described in conditions where protein intake is suboptimal as, for example, in protein calorie malnutrition (7-9). Nutritive selenium deficiency may also occur in metabolic diseases under dietary treatment with synthetic amino acid mixtures, e.g., in maplesyrup-urine disease and in phenylketonuria (10, 11) or in patients on intravenous alimentation (12-14). It is proposed that on the basis of inadequate and/or imbalanced food consumption, groups of people may be identified that are likely to develop Se deficiency (14). Among them are children, people on restricted diets, elderly people, and surgical patients. Although the clinical relevance of low selenium values is still unclear, the current epidemiological knowledge cautions against long-term adverse health effects in these vulnerable population groups (15). In order to acquire some understanding of human Se state at old age, a group of elderly people was examined. Their daily dietary Se intake was estimated. In addition this paper reports the Se concentration in plasma samples of newborns and of children.

Materials and Methods

Subjects Elderly People.

Blood was taken from 40 well-nourished elderly Belgians, residing in geriatric homes in the Antwerp region and over 60-yr-old (mean age -+ SD, 82 - 7 yr, ranging from 60 to 99 yr). The group consisted of 9 male and 31 female subjects. On the average the time spent in the home since the date of entry was 3.5 yr. Hematological parameters were normal. Creatine phosphokinase (CPK) activities varied widely [mean activity -4- SD, 36.7 -+ 31.7 U, as determined by coupled reactions with ADP, phosphoenolpyruvate, and NADH (16)]. Almost every participant suffered to some extent from cardiovascular or cerebrovascular diseases. The same fish-free diets were served to all participants for seven consecutive days prior to blood sampling.

Children.

Paired samples of maternal and cord blood were obtained at 9 deliveries. Blood was also taken from 10 children less than 1-yr old, from 13 children between 1 and 6 yr of age, and from 15 children 6 to 15 yr. These children are considered healthy and were presented for routine blood analysis prior to minor operations.

Estimation of the Selenium Contents and of the GSH-Px Activity All participants were recumbent and fasting when venipuncture was done. Selenium concentrations were measured in whole blood and plasma of the elderly par-

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ticipants, in maternal and cord blood plasma, and in plasma of the children. The Se contents of food consumed daily by the elderly geriatric residents were assayed in duplicate portions on the 7d preceding blood sampling. Sample pretreatment was by a wet HNO3/HCIO4 digestion at 210~ and Se determination in the dissolved material was done by hydride-generation atomic absorption spectrometry, according to procedures described in detail in previous papers (17, 18). Erythrocyte Se concentrations were obtained by calculation. Erythrocyte GSH-Px activity was assayed at 37~ in a coupled test with t-butylhydroperoxide as the acceptor substrate (19, 20).

Statistical Evaluation Differences between means were tested by the two sample t-tests or by one-way analysis of variance. Maternal and cord plasma Se concentrations were evaluated by means of a t-test for paired observations. Occasionally simple linear regression and correlation analysis were performed.

Results The analysis of composite diets revealed an average daily Se intake by the elderly of 43 p,g (SD 10 ~zg) on days that fish was not included in the diet. Daily diets comprised approximately 70 g of meat, 400 g of bread and cereals, 150 g of potatoes, 100 g of dairy products, 100 g of fruits and vegetables, and 1 L of beverages. It is known that the frequency of fish, chicken, kidney, and liver consumption strongly affects blood Se levels. However, since none of these are frequently eaten in Belgium, they are not thought to have an especially marked influence on the average Se intake. The Se intake ranged from 33 to 64 ~g over the 7-d period. Assuming that healthy young workers consume approximately 150 g of meat a day, their average daily Se intake can be estimated 55 la,g. This agrees well with an average urinary Se excretion of 20.0 +- 8.75 i.zg/d as determined for4 young men and 2 young women (22-24). Blood, plasma, and red blood cell Se concentrations are represented graphically in Fig. 1 for a group of 164 healthy Belgian adults (20) and for the group of elderly people under investigation. Similarly, in Fig. 2, GSH-Px activities in several classes are compared. Since the categories 60--64 and 65-70 yr contain only two individuals, these data have been disregarded. Of 40 analyzed whole blood samples, nine were discarded for reasons of suspected contamination during sample digestion. Whole blood of 31 elderly people had a mean Se concentration of 130 ng/mL with a standard deviation and a relative standard deviation of 19 ng/mL and 14.6%. These values did not differ significantly from the 123 • 17 ng/mL blood Se values that were obtained in a study on 164 healthy Belgian subjects (20). Contrary to what could be anticipated, the elderly people did not exhibit lower hematocrit or hemoglobin concentration values as compared to the group of young people. The average plasma Se value in the group of elderly people amounted to 73 +_ 12 ng/mL and thus was significantly lower than the value of 97 +-- 12 ng/mL found in adults (P < 0.0005, one-sided t-test). Conversely, the calculated erythro-

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Fig. 1. Average erythrocyte, whole blood, and plasma Se concentrations in Belgian residents belonging to several age categories. Dotted lines indicate the mean of two individual results, both of residents over 90-yr-old.

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20 30 40 50 60 70 80 90 100 Erythrocyte glutathione peroxidase activity in healthy adults and in elderly

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cyte Se concentration of 200 + 42 ngtmL in the elderly was considerably higher than the average red blood cell Se concentration in the young (161 m 32 ng/mL). By conversion of whole blood Se values to cell and plasma Se it was found that red blood cells of the old people contained 2.13 times more Se than the plasma fraction, i.e., 67% of blood Se was concentrated in the erythrocytes. This is a significant shift of blood Se to the cell compartment in comparison to the younger subjects. The elevated cell Se concentration was associated with a high enzymatic activity of 52.86 U37/g Hb (m 11.40 U37ig Hb) which was significantly higher than the activity of 47.50 -+ 11.07 U37/g Hb, found in young adults (20). No differences in blood, erythrocyte, or plasma Se concentrations or in GSH-Px activity were found that could be attributed to differences in sex or age. The activity of creatine phosphokinase, which is often considered a diagnostic tool in patients with myocardial infarction (16), was not related to the plasma Se level. Contrary to the observations made in young healthy adults (20), no correlation was detected between erythrocyte or blood Se content and erythrocyte GSH-Px activity, but a significant positive relationship was apparent between the plasma Se content of the elderly and their red blood cell GSH-Px activity (n = 40, r = 0.33, P < 0.05). These combined observations reveal a common trend: a positive correlation between erythrocyte GSH-Px activity and Se content was only present when the latter was low. With respect to observations made on plasma Se levels in infants, newborns exhibited significantly lower plasma Se concentrations than did their mothers, as is illustrated by the data in Table I (P < 0.005, one-sided paired t-test). Table 2 shows values obtained for children of various ages and are visualized in Fig. 3. Eight babies, one-half year old or less, had even lower plasma Se levels than newborns, the lowest value found being 21 ng/mL. Sucklings, toddlers, school chiI-

TABLE 1 Plasma Selenium Concentration in ng/mL of Nine Paired Maternal and Cord Blood Samples at the Time of Delivery Plasma selenium concentration, ng/mL No. I

2 3 4 5 6 7 8

9 Mean • SD RSD

Mother

Newborn

70 86 78 88 93 83 76 97 121 88 -+ 15 17%

45 75 57 74 56 76 56 90 66 66 • 14 21%

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TABLE 2 Selenium Concentration in the Plasma of Children of Various Ages" Age Birth 2 Months 3 Months 4 Months 5 Months 6 Months 7-12 Months 1-1.5 Years 1.5--2 Years 2-3 Years 3-4 Years 4-5 Years 5--6 Years 7-15 Years Adult women" at time of delivery

Number of children 9 1 1 3 1 2 2 f 2 3 3 3 1 15 9

Mean plasma Se + SD in ng/mL 66 21 27 33 32 23 48 43 40 61 59 52 62 65

-+_+14

.+- 9

--+ 14 --- 12 -+ 2 +-- I3

88 --+ 15

*Mothers of the newborns mentioned at the top of the table.

Fig. 3. Plasma Se concentrations in children of various ages. M indicates mean plasma Se concentration of nine mothers at the time of delivery.

dren, and teenagers had ~ a d u a l l y higher plasma Se levels. Although the method used is a cross-sectional one and the data are too limited to be conclusive, they seem to indicate an age-dependency o f Se concentrations in plasma. At the age of 15, mature Se values seem not to be reached as yet.

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Discussion The data obtained on Se in blood and blood fractions in healthy adults (20) and in elderly people seem to deny that age could bear a relationship on Se parameters. In few of the studies published until now with regard to the effect of age on the Se state (15, 21, 25, 26), the influence of age was investigated in age groups above 60 yr. Dickson and Tomlinson suggested that the average selenium content of the blood of persons in different age groups might decrease with increasing age, but they admitted that there were insufficient individuals in each of their groups to give statistical significance to such a conclusion (27). Investigators in New Zealand found that blood Se concentrations for adults decreased after 60 yr of age (27, 28). They also found significantly lower plasma and erythrocyte Se levels (27, 28) and GSH-Px activities (28) in elderly people compared to controls. However, it may be difficult to distinguish between effects resulting from the biological process of aging and those from differences in eating habits that are associated with aging. It has been well-established that plasma Se values are to a large extent influenced by recent changes in dietary Se intake and thus are a sensitive index of the short-term Se status of a person (24). Erythrocytes and whole blood show a slower response to changes in dietary intake (24), as incorporation of in: gested Se into red blood cell proteins only occurs during erythropoiesis (29). Thus, erythrocyte Se is considered to give a long-term index of Se status because of the long-life span of red blood cells (24). Our findings on plasma in elderly people lend support to the theory that their reduced dietary Se intake as compared to young people is reflected in lower plasma Se levels. We cannot offer an explanation for the markedly increased red cell Se concentration and GSH-Px activity in our group of aged persons. It could for instance be hypothesized that aging causes plasma proteins to partially loose their Se binding properties, thus allowing accumulation of whole blood Se in the cell fraction. The high activity of GSH-Px may then reflect a larger store of red blood cell Se available for incorporation during the synthesis of GSH-Px molecules, which is known to be enhanced by the availability of Se (30). It appears that a significant correlation between erythrocyte Se content and its GSH-Px activity only exists when the latter is below a threshold value. Above this concentration, GSH-Px activity is not noticeably increased, suggesting that this Se concentration is optimal and that Se intakes that maintain this concentration are adequate, or that above this concentration other factors might play a greater role in influencing GSH-Px levels (31). This may explain why no correlation was found between erythrocyte Se and GSH-Px levels in our group of elderly people, whereas such a relationship was present in a group of younger adults (20). It may also explain why in the latter case this correlation was much weaker in Belgian adults than in New Zealand residents. Nine women presented an average plasma Se concentration of 88 ng/mL (SD 15 ng/mL) at delivery, which is lower than the mean concentration of 97 ng/mL (SD 12 ng/mL) found in 53 healthy nonpregnant women (20). When the value of subject nine is omitted, the difference becomes highly significant (P < 0.02, twosided t-test). Thus pregnant full-term women had plasma levels 9.5% lower than

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those of nonpregnant controls. Rudolph and Wong have reported exactly the same concentration ratio (32). Our findings alike, these authors observed that plasma Se levels of babies at birth are always markedly lower than those of their mothers. Both in their study and in ours, fetal plasma contained 75% of the amount of Se present in plasma of the mothers. They did not only note low plasma and red blood cell Se values in cord blood, but also the activities of both plasma and erythrocyte GSH-Px were lower (32). It was suggested that placental transport of Se is limited, resulting in low fetal Se concentrations and hence low GSH-Px activities. Alternatively, it was not excluded that low enzyme activity represents a phase of development adapted to the low intrauterine oxygen environment with a decreased need for peroxide detoxification by GSH-Px and hence a decreased uptake of Se from maternal plasma (32). It is well known that the GSH-Px plays an important role in the resistance of erythrocytes to oxidative stress. The erythrocytes of newborn infants, especially of premature ones, are susceptible to oxidative damage, with oxidation of fetal hemoglobin as the main threat (33). It has repeatedly been shown that the concentration of whole blood Se is markedly lower in deceased premature and fullterm infants than in living full-term infants (33, 34). in some cases neonatal jaundice and hemolytic anemia have been associated with decreased erythrocyte GSH-Px activity (33). Money was the first to suggest that vitamin E and/or Se deficiency could be causative of the sudden infant death syndrome (SIDS) (34). Rhead et al., however, found no difference in the whole blood Se concentration of 12 SID cases (0.1.00 +-- 0.036 t~g/mL) and of three hospitalized and one accidentally deceased infants (0.090 • 0.02l I.zg/mL) (35). SIDS plasma specimen contained 0.69 p,g/mL of Se (SD 0.022 txg/mL) and this was similar to levels reported for Guatemalan children with kwashiorkor (8). In our study 13 healthy infants of comparable age (2-24 months) had a mean plasma Se concentration of 34 ng/mL (median 33 ng/mL), which is 50% lower than the SIDS levels mentioned above, but identical to the median plasma Se concentration of 12 healthy German infants, 1--4 months old, as reported by Lombeck et al. (36). Although Rhead et al. concluded that Se deficiency probably has not a primary role in the etiology of SIDS, they thought it capable of contributing to it by leading to diminished capillary resistance and hence to increased susceptibility to respiratory infections (35). Although the data in this study are too scarce to establish a clear correlation between plasma Se and age during infancy, they seem to support the age-dependence of plasma Se as described by Lombeck et al. (36). These authors reported a median Se concentration of 34 ng/mL for 12 babies 1--4 months old with an increase to 58 ng/mL (n = 13) in the second half-year of life. Our results are slightly lower, but they exhibit a very similar trend. These authors noted a further rise in toddlers and in youngsters up to the age of maturity. Our values are a little higher than those reported by Westermarck et al. (37) in Finland, where soil Se content is very low. Several reasons may underly a low Se status during early infancy. Although it is thought by some that selenium stores increase with maturity, possibly in proportion to plasma proteins and hemoglobin (7), low dietary Se intakes are likely to play a major role. German (36) and American (38) investigators have shown that

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the lower Se concentrations found in serum of infants between 1 and 6 months old, compared to newborns and to older children, may reflect their low Se intake. Infants fed commercially available milk formula and baby foods have markedly lower Se intakes than their breast-fed counterparts. Processing of cow milk and of cereals, fruits, and vegetables may account for large losses of Se (36). According to Lombeck et al. (36) a West-European formula-fed infant receives about 3.5 ~g/d of Se in the first months of life, while a breast-fed infant gets about 13.3 p,g/d. Similarly, Smith et al. (38) have reported intakes of 7.22 Ixg and 10.08 ~tg/d respectively, for North American babies. They attributed 18% of the variation in serum Se to Se intake (38). Inadequate Se intake has also been found causative of the low Se status characteristic of children with inborn errors of amino acid metabolism, who are under dietary therapy with semisynthetic amino acid mixtures or protein hydrolyzates (11). The general protein depletion that occurs in protein-energy malnutrition probably explains the reduced Se stores associated with these conditions (39). At present, the clinical relevance of the reduced Se stores in these diseases is not fully understood. Nevertheless, several studies indicate a correlation between inadquate Se intakes and certain diseases, and a beneficial effect of Se supplementation. Especially children may be at risk of developing diseases that might be prevented by supplementation of their diet with Se. Of paramount importance is a form of cardiomyopathy that affects children with a very low Se status. The disease is known as Keshan disease and is endemic in some parts of the People's Republic of China. It is a selenium-responsive condition that affects most frequently children under 15 yr of age, who live in areas where soil and crops are extremely poor in Se. Blood Se values in affected areas are generally below 0.01 p,g/mL. Oral administration of 0.5-1.0 mg sodium selenite weekly dramatically lowered the incidence of Keshan disease and improved its prognosis. Nutritive Se deficiency is thought to contribute to the development of this biogeochemical disease, by failing to protect the child against myocardio-necrosis brought about by lipid peroxides (40). The Keshan disease research group estimates that minimum adequate blood Se values for prevention of the disease are within the range of 0.03-0.04 ~g/mL and that the minimum daily requirement of Se is around 30 p,g (40). Recently, similar cases of cardiomyopathy and Se deficiency in children have been reported in the West (41, 42). Se deficiency may also lead to muscular syndromes as has been reported in New Zealand (13). In some children with kwashiokor an increased weight gain was noted after Se supplementation (43, 44). Both patients with the infantile- and the juvenile-type neuronal ceroid lipofuscinosis have been shown to have low blood and plasma Se concentrations. Selenium supplementation of such children reportedly lead to a transitory physical and mental improvement in some of them

(6). Current knowledge thus indicates that there may be several serious diseases that are aggravated by Se deficiency. Our results confirm that the Se plasma concentration in young children is very low and that children may therefore be at risk of developing Se deficiency-associated diseases.

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Acknowledgments The authors are indebted to the following persons for their valuable cooperation during some stages of the investigation: Dr. G, Van Vlasselaer, Mr. Lucas, and the nursing staff of the geriatric homes; Dr. J. Van der Heyden, obstetrician (U. I.A.), Prof. M. Peetermans and Dr. M. Van der Plancken, hematologists (Academic Hospital Antwerpen). They are very grateful to Dr. G. S. Fell (Glasgow Royal Infirmary) who enabled GSH-Px assays. Marleen Verlinden wishes to thank the National Fund for Scientific Research (Belgium) whose fellow she was at the time of the investigation. Finally, the department of pharmaceutical sciences (U .I.A. ), especially Prof. H. Deelstra, is acknowledged for having provided facilities for atomic absorption spectrometric measurements.

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