Studies of Human Maximal and Minimal Safe Intake and Requirement of Selenium

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1 Studies of Human Maximal and Minimal Safe Intake and Requirement of Selenium G. YANO,,2, L. Gu', R. ZHOU', and S. YIN' 1 Introduction Although the geographical relationship between human cancer incidence and local environmental Se status has been repeatedly confirmed since 1969 (Shamberger and Frost 1969), the levels of Se used in animal studies, which support the role of Se as inhibiting cancer development, are potentially toxic. It was proposed that the justification for carrying out human intervention trials with Se to prevent cancer should be at best marginal or in the pharmacologic range. A promising approach to resolve the dose problem was the suggested use of levels similar to the intakes of populations residing in regions naturally high in Se (Clark and Combs 1986). Such an investigation has been launched since 1985 in a seleniferous area in Enshi (Yang et al a, b). A daily Se intake of 50 Ilg by extrapolation from animals was proposed to meet the human requirement. Since extremely low Se areas with Se-deficiency-related endemic diseases appear in China, the study of the human requirement has been conducted since 1983, and part of the work was recently confirmed in non-affected areas (Yang et al c). The results mentioned above are presented here. 2 Human Marginal and Maximal Safe Selenium Intakes 2.1 Study Design Three sites with low, medium and high Se levels in the seleniferous region in Enshi county were chosen for this study. Tho hundred to 300 subjects of different ages and sexes were included from each site. The chemical form of Se ingested was mainly in a natural food form. The subjects are life-long residents and cases of selenosis appeared in the high Se site. Field studies conducted in April and September 1986 followed the pilot study of November Foods consumed during the 3-day survey were collected for Se analysis. Blood, 24-h urine, breast milk ang. tissue samples were collected for Se, Hg, Cd, Zn, Cu or As determination. In order to determine the expected level of Se intake, individual Se intakes or tissue- 1 Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine, 29 Nan Wei Road, Beijing, Peoples' Republic of China 2 To whom correspondence should be addressed A. Wendel (ed.), Selenium in Biology and Medicine Springer-Verlag Berlin Heidelberg 1989

2 224 G. Yang et al. Table 1. Average daily Se intake (lj.g) of adults a Se status of site Male Female Average Ratio Low Medium High 70.5±4.8 (51) ± 22.9 (76) ± 76.3 (58) 62.0±3.6 (51) ±23.8 (82) ±64.6 (61) a Results are expressed as mean ± SE. Numbers in parentheses indicate the number of subjects examined. Se levels were correlated with either biochemical parameters or clinical signs. If the tissue-se level was known, it could be transferred to the respective Se intakes through regression equations. 2.2 Se-Intake and Tissue-Se Level The average daily Se intakes in medium and high Se sites were around 3- to 20-fold that of the low Se site (Thble 1). No significant differences were found between male' and female adults. Se intake and tissue or body fluid Se concentration were found to be significantly correlated, and similar relationships were also found between Se concentrations of tissue. Of the metals analyzed, only Cd and As were considered high either in the blood and hair or hair respectively of high Se site residents, but their effects on Se metabolism remain to be studied. 2.3 Clinical Diagnosis Diagnosis of selenosis was based mainly upon morphological alterations of the fingernails. Results indicated that the lowest blood-se level of selenosis diagnosed among 349 residents in the sites with different levels of blood-se, had a blood-se level of 1.02 Ilg ml- 1 (875 Ilg daily Se intake). More definite evidence obtained from five patients with long, persisting, distinct clinical signs showed a blood-se range of Ilg ml- 1 The minimal blood-se concentration of Ilg ml- 1 (909 Ilg daily Se intake) confirmed the previous result and would approximate the marginal level of Se toxicity which cannot be tolerated by the susceptible cases. 2.4 Percentage of Se Excretion from Se Intake The percentage varied within of a wide range of Se intakes until approximately 2000 Ilgday-l ( Ilg) was attained. However, such a homeostatic regulation weakened and the excretion percentage began to plateau as Se intake increased to near 800 Ilg day-l (blood-se level 0.95 Ilg ml- t ). As the Se intake further increased to over 1000 Ilg, the percentage excretion increases nearly to a

3 Studies of Human Maximal and Minimal Safe Intake and Requirement of Selenium 225 Table 2. Prothrombin time of residents in relation to concentration of blood-se Whole blood Se level Number of Average time a Samples with prolonged prothrombin (/lg rni- 1) subjects (s) time (2:14s) Cases (0,70 ) < ±0.2a ±0.2a ±0.3b ±0.3 b a Results are expressed as mean ± SE. Means not sharing a common superscript letter are significantly different (p<0.01). constant. This seems to indicate that as Se intake increases to around 800 Jlg day -1, the kidney would begin failing to regulate the excretion of Se in the urine. No notable change of the (CH 3 hse + content in urine was observed. 2.5 Plasma Prothrombin Time Excessive ingestion of Se may damage the parenchymal cells of the liver and impair the hepatic synthesis of clotting factor(s). Blood samples of inhabitants from different sites were collected for measurement of plasma prothrombin time. It was found that as blood-se increased to a level above 1 Jlg ml- 1 (850 Jlg Se intake), cases with a prolonged prothrorribin time increased abruptly (Table 2). 2.6 Other Biochemical Parameters As Se intake increased to a high level, changes of other biochemical parameters were observed: (1) decrease of the plasma-se to erythrocyte-se ratio may indicate an increased burden of Se to the body. (2) Decrease of whole blood OSH concentration may indicate the shifting of blood OSH metabolism towards a higher oxidation state. (3) No indication of anemia was observed from hemoglobin, hematocrit or erythrocyte-free proporphyrin content determination. (4) Severe liver damage was again not detected either from analyses of biochemical indicators such as serum OPT, ZnTT, TTT, icteric index and alkaline phosphatase activity or through supersonic B morphologic examination. (5) White cell count increased stepwise from the low to high Se site; its significance is not clear. 2.7 Summary of the Results Results are summarized in Table 3. It shows that clinical signs may occur at a marginal blood-se level of approximately ;::: 1 Jlg ml- 1 or a corresponding marginal daily Se-intake of 850 Jlg day-i, while biochemical alterations suggested a mar-

4 226 a. Yang et al. Table 3. Evidence obtained for the safe level of Se intake a Evidence Se intake (Jlg day- ') Blood Se level (Jlg ml-') Manifestation of clinical signs Long persistence of clinical signs Delay of prothrombin time Reduction of whole blood ash content Plateau of percentage of Se excretion to Se intake Remarkable reduction of plasma-se to erythrocyte-se ratio ~ ( ) ~ ( ) ~ ( ) - (853) ~ ~ 1.02 ~ 1.05 ~ ~ ( ) -(0.9) a Numbers in parentheses are calculated from regression equations. ginal blood-se level of around :5 1.0 IJ,g ml-i or an approximate, marginal, daily safe Se intake of 750 IJ,g day-i. As a safety factor of 1.3 is used, a maximal daily safe Se intake of 550 IJ,g day-l is suggested for inhabitants in seleniferous area. 3 The Selenium Requirement of Humans (Yang et al. 1987, 1988c) 3.1 Minimal Se Requirement It is estimated as the approximate, minimal Se intake required to protect the inhabitants in low Se areas against KD (Keshan disease). Such a Se intake was estimated to average 17 IJ,g day - 1 for adults (Yang et 'al. 1987). This result is in agreement with the suggestion from a previous balance study on New Zealand women indicating that the minimal Se requirement is probably not more than 20 IJ,g day-l (Stewart et al. 1975). This is also in agreement with the statement that Keshan disease appeared only under extremely poor Se status in the presence of environmental or host stress (Levander 1987). 3.2 Physiological Se Requirement It is estimated as the level of Se intake sufficient to maintain plasma GSH-Px activity at a maximal, constant level. A previous study indicated that as graded levels of Se as selenomethionine was supplemented to male adults living in a low Se area, the plasma GSH-Px activity plateaued at a daily Se intake of : IJ,g (Yang et al. 1987). Since more than of the Se intake is quantitatively supplemented, individual variations in Se intakes cannot be obtained in this study. Levander (1987) used a safety factor of 1.3 as the coefficient of variation. Thus, a daily Se intake of 53 IJ,g was obtained for male adults in this study. It was accidentally found recently that the blood-se level of the institute workers in Beijing were marginal. In view of the variation of values in food availability and the complicated factors shown to influence the GSH-Px activities in tissues, observation was thus made on such a population. The 12 subjects were

5 Studies of Human Maximal and Minimal Safe Intake and Requirement of Selenium 227 Table 4. Influence of Se supplementation as Na 2 Se0 3 on Se levels and GSH-Px activities of blood, erythrocytes and plasma of male and female adults Week Se supplemented" Blood Se Erythrocyte Se (Ilg day-i) (Ilg ml- I) (Ilg ml- I ) o ± ± ± ± ± ± ± ±0.054 Plasma Se (Ilg ml- I ) Plasma GSH-Px activityb ± ± S± ± S± ± ± ±0.03 " Results are expressed as mean ± SE. Supplemented to a basal diet providing an average dietary Se intake of 45.5 Ilg day-l for both sexes. b nmol NADPH min- l ml- l. Table 5. Influence of Se supplementation as Se-methionine on Se levels and GSH-Px activities of blood, erythrocytes and plasma of male and female adults Week Se supplemented" Blood Se Erythrocyte Se (Ilg day-i) (Ilg ml- I) (Ilg ml- l ) o ± S± ± ±0.01O 0.21S± ± ± S5 ± Plasma Se (Ilgml- l) Plasma GSH-Px activityb 0.102± ± ± ± O.OS ± ±O.OS ± ± 0.07 " Results are expressed as mean ± SE. Supplemented to a basal diet providing an average dietary Se intake of 45.4 Ilg day-l for both sexes. b nmole NADPH min -I ml- l. middle-aged and young laboratory workers with equal numbers of both sexes. They were divided into two groups of eight and four each. The average body weight was 60 kg for males and 56 kg for females. Short-term supplementation of higher doses of Se either as sodium selenite or as selenomethionine was made. Blood samples were collected for Se and enzyme activity estimation. The results showetl that plasma GSH-Px activities of both groups remained unchanged during the period of Se supplementation (Thbles 4 and 5). The amount of average Se intake provided from the natural diet was 48.3 Jlg, which is within the variation range of previous studies on male adults in a low Se area in which a safe daily Se intake of 53 Jlg was able to maintain the plasma GSH-Px activity at plateau. This study confirmed the previous result. However, as a safety factor of 1.3 was used in this study, the safety intake was 62 Jlg for males and 55 Jlg for females consuming a natural diet. Another approach is to determine the amount of supplemented Se as sodium selenite necessary to maintain the plasma Se level at plateau (Yang et al. 1988c). A preliminary experiment indicated that a 20 Jlg Se supplementation daily (or a daily Se intake of 68.3 Jlg) could raise the plasma Se to a constant level. As the safety factor 1.3 was used, the daily Se intake should approximate 90 Jlg day - 1 for 60 kg male adults. The results are summarized in Table 6.

6 228 G. Yang et al.: Studies 'of Human Maximal and Minimal Safe Intake Table 6. Daily selenium requirement and suggested dietary allowance for humans Se intake Method of estimation Experimental value (l1g day-i) Safe intake" (l1g day-i) Minimal requirement Physiological requirement Suggested optimum dietary allowance range 1. Dietary survey in KD area 2. Calculated from marginal Se level of cereal 1. Suppl. Se-Met. to max. plasma GSH-Px activity 2. Natural dietary Se intake to max. plasma GSH-Px activity 3. Suppl. selenite to max. plasma Se level Summation of the data obtained 19.1 ± 5.5 (M) 13.3 ±4.1 (F) 19.2 (M) 16.0 (F) :s40.9±0.6 (M) :s 48.3 ± 20.2 (M) :s 42.8 ± 20.4 (F) ± 20.2 (M) ± 20.4 (F) 50 (M) 60 (M) 55 (F) 90(M) 80 (F) "Results are expressed as mean±sd. A safety factor of 1.3 is used after Levander, OA [21]. M, male; F, female. Acknowledgements. The Enshi work was funded by the US National Cancer Institute and supported by the Research Agreement from the US Department of Agriculture. References Clark LC, Combs CF Jr (1986) Selenium compounds and the prevention of cancer: research needs and public health implications. J Nutr 116: Levander OA (1987) A global view of human selenium nutrition. Annu Rev Nutr 7: Shamberger RJ, Frost DV (1969) Possible protective effect of selenium against human cancer. Can Med J 100:682 Stewart RDH, Griffiths NM, Thomson CD, Robinson MF (1978) Quantitative selenium metabolism in normal New Zealand women. Br J Nutr 40:45-54 Yang GQ, Zhu LZ, Uu SJ, Gu LZ, Qian PC, Huang JH, Lu MD (1987) Human selenium requirements in China. In: Combs GF Jr, Spallholz JE, Levander OA, Oldfield JE (eds) 3rd. Int Symp Selenium BioI Med, part B. Van Nostrand Reinhold, New York, pp Yang GQ, Zhou RH, Yin SA, Gu LZ, Yan BW, Uu YQ (1988a) Studies of maximal daily selenium intake of safety in seleniferous area in China. 1. Selenium intake and tissue selenium levels of the inhabitants. J 'll"ace Element Electrolyt Health Dis (submitted) Yang GQ, Zhou RH, Yin SA, Gu LZ, Yan BW, Uu YQ (1988b) Studies of maximal daily selenium intake of seleniferous area in China. 2. Relation between selenium intake and the manifestation of clinical signs and some biochemical alterations. J Trace Element Electrolyt Health Dis (submitted) Yang GQ, Zhou RH, Yin SA, Gu LZ (1988c) Further studies on the physiological selenium requirement of humans in China, and a brief discussion of the results. J Hyg Res 1988 (ih press) (in Chinese)