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Selenium in human hair in relation to age, diet, pathological condition and serum levels
The Science of the Total Environment, 24 (1982) 41--49
41
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SELENIUM IN HUMAN HAIR IN RELATION TO AGE, DIET, PATHOLOGICAL CONDITION AND SERUM LEVELS*
S A R O J A THIMAYA and SEETHA N. GANAPATHY
Nutrition Research Program, North Carolina Agricultural and Technical State University, Greensboro, NC 27411 (U.S.A) (Received December 9th, 1981; accepted January 8th, 1982)
ABSTRACT Approximately 0.5 g of hair samples free from medicated shampoos, bleaches and dyes were collected from 115 subjects from the occipitonuchal region. Prior to the analyses, the samples were thoroughly washed and dried in a gravity convection oven until consistent weight was obtained. A 100-mg portion of the cut up and thoroughly mixed sample in duplicate was used for the analysis of selenium (Se) by a fluorometric technique. The results were treated statistically. The level varied from 0.55 in the elderly to 0.76 #g/g of hair in adolescents. A decline of Se was noted in the groups of ages 16--40 yrs, the levels being 0.64 to 0.59pg/g of hair. From groups 41---60 yrs no increase was seen in the Se content, the lowest level noted was in group 61--70 yrs. These differences between groups were found to be statistically significant (P ~ 0.05). No significant correlation was found between the Se content of hair and 'diet. The levels in hair and serum of patients with cardiovascular disease were significantly low (P ~ 0.05), the levels being 0.49 pg/g of hair and 0.10 pg]ml of serum. It was also noted that there was no correlation between Se in hair and serum of the same individuals. Further work using larger samples is necessary to shed more light on these relationships.
INTRODUCTION
Although selenium (Se) has been found to be essential to many animal species, its role in human nutrition has been difficult to define. Its interrelationship with Vitamin E suggests that it might function as an anti-oxidant. The role of Se in the mammalian systems, in the enzyme glutathione peroxidase, has been fully d o c u m e n t e d [1]. A recent study shows that it might function in electron transport mechanisms [2]. The deficiency of Se in animals results in many Se responsive diseases such as muscular dystrophy, exudative diathesis and hepatic necrosis [3--5]. It has been noted to prevent several chemically induced cancers in animals. Studies show that the incidence of human cancer and heart disease is lower in high Se areas [6, 7]. * Supported in part by USDA-SEA/CR grant. 0048-9697/82/0000--0000/$02.75 ©1982
Elsevier Scientific Publishing Company
42 It is believed that Keshan's disease, a type of congestive heart failure could be due to Se deficiency, the most susceptible to the disease being women of child-bearing age and children below 15 years [ 8]. While considering suitable indicators to determine the Se status, hair seems to be a potential biopsy material. Hair has been noted to provide a valuable index of copper and chromium status in man [9, 10]. Many serious ailments have been diagnosed by preliminary hair analysis. In phenylketonuria, cystic fibrosis and protein-calorie malnutrition, the mineral content of hair was observed to change [11]. Hair concentration of elements such as copper, cadmium and lead have already been observed to be low in the elderly [12]. The following work is an a t t e m p t to study the level of Se in human hair in relation to age, diet, pathological condition and serum levels. The correlation between the concentration of Se in hair and serum of the same individual has also been studied. Cancer and cardiovascular disease were the two selected pathological conditions.
METHODS The study, conducted in Greensboro, NC, was divided into two parts. The first part deals with the relationship of hair Se content to age and dietary intake of Se, while the second involves the Se levels in the selected pathological conditions such as cancer and cardiovascular disease. The first part involved 115 local subjects, free from health problems. The information such as age, sex, health condition and dietary data was obtained with the help of a questionnaire. The hair of each participating subject was c u t and placed in numbered plastic bags which were sealed and attached to the appropriate questionnaire. The second part involved 59 hospital patients, for twenty-three of w h o m matching hair and serum samples were available for correlation study. The normals in this part consisted of five subjects free from any health problems.
The questionnaire Besides collecting the above mentioned personal data such as age, sex, diet and health conditions, the questionnaire was also designed to collect information on whether dyes, bleaches and medicated shampoos were used for hair. This helped to avoid the inclusion of chemically treated hair. The results of a pilot study helped in modifying certain items on the questionnaire. As it was realised that subjects were inclined to give inaccurate information regarding their age, it was decided to have age groups with intervals of ten years. For the first t w e n t y years however, the subjects were grouped in five-year intervals. The f o o d data was obtained by means of a seven-day dietary recall.
Preparation of the hair sample for analysis The samples measured 3--5 cm and weighed approximately 0.5 g and were
43 cut from the occipitonuchal region of the head, close to the hair root. They were then stored in sealed plastic bags until analyses. Prior to the analyses, the hair samples were washed with detergent, rinsed several times with deionised, distilled water and dried in a gravity convection oven at 45°C till consistent weight was obtained. The dried hair samples were then cut into small parts, thoroughly mixed in order to obtain a homogenous sample and a 100-mg portion in duplicate was used for the analysis of Se. The Se assay was c o n d u c t e d according to a slightly modified method of Whetter and Ullrey [ 1 3 ] . The instrument used for the measurement of fluorescence was the Perkin-Elmer fluorescence s p e c t r o p h o t o m e t e r Model 203. All chemicals and glassware were obtained from Fisher Scientific Company except 2,3-diaminonaphthalene which was purchased from Aldrich Chemicals. The glassware was washed with detergent, soaked in concentrated nitric acid, rinsed in deionised distilled water and dried before use.
Daily intake o f selenium The values of Se in various c o m m o n foods, reported in a previous study c o n d u c t e d in this laboratory were used in the calculation of the daily intake of Se [ 1 4 ] . The items such as fruits, fruit juices and vegetables whose Se levels are negligible were n o t included for the calculation. This level of Se in the diet was used to find the correlation with the Se c o n t e n t in hair. For the second part of the study only the patients on regular diets were included. The obtained data were collected, coded and transferred to opscan sheets and IBM-key punched. The c o m p u t e r program tool e m p l o y e d was the SPSS [15]. The Student's T-test was used to find the difference in the Se level in the various age groups and the Pearson's correlation was used to find the relationship of Se in hair to that in diet and serum.
RESULTS AND DISCUSSION The findings regarding the influence of age on hair Se c o n t e n t and the correlation between diet and hair Se levels are presented and discussed in Part I, while levels of Se in hair and serum under pathological conditions are given and discussed in Part II.
Part I All age groups were represented with the majority in the group 21--30 yrs. The details are presented in Table 1. The variation observed in the Se levels in the groups can be n o t e d in Fig. 1. Significantly high levels (P ( 0 . 0 5 ) were noted in the 1 1 - - 1 5 y r group comprising the adolescents, when compared to the other groups. The Se c o n t e n t varied from 0.55 to 0.76 #g/g of hair. A gradual decline of Se was f o u n d in 16--20 through 31--40 yr groups, b e y o n d which up to the 51--60 yr group no increase was noted. The level was lowest in the 61--70 yr group. These group differences were statistically significant (P ( 0 . 0 5 ) . These observations indicate that hair Se c o n t e n t is influenced by
44 TABLE 1 HAIR SELENIUM LEVELS OF THE D I F F E R E N T AGE GROUPS
Age g r o u p
Age (years)
No. o f cases
1 2 3 4 5 6 7 8 9 10
5 and b e l o w 6--10 11--15 16--20 21--30 31--40 41--50 51--60 61--70 a b o v e 70
6 12 13 21 26 9 7 9 8 4
S e l e n i u m level a (pg/g) 0.66 0.64 0.76 0.64 0.62 0.59 0.62 0.64 0.55 0.62
-+ 0.07 -+ 0.04 b -+ 0.04 c + 0.04 + 0.03 + 0.04 -+ 0.04 + 0.06 -+ 0.03 + 0.03
a Mean s e l e n i u m levels -+SEM. Statistically significant (P < 0.05) d i f f e r e n c e s in the levels were f o u n d in t h e following groups. b B e t w e e n groups 2 and 3. c B e t w e e n groups 3 and 4. T h e t-test p e r f o r m e d b e t w e e n s u b s e q u e n t g r o u p s gave significant d i f f e r e n c e s (P < 0.05) b e t w e e n t h e groups i n d i c a t e d above. Significant d i f f e r e n c e s (P < 0.05) were also n o t e d w h e n g r o u p 3 was c o m p a r e d w i t h g r o u p s 5, 6, 7, 9 a n d 10.
7~ g *~
0.9
0.8
0.7
o
.E 0,6 o
~- 0.5
0,0
. . o . .~. . o
~
o
i
i
~o ~
i
~ Age
o
6
~
o
'
o
'
.
i
i
i
i
i
0
~
m
,¢
~
to
'
group ~n yrs
Fig. 1. Mean Se c o n c e n t r a t i o n -+ SEM for t h e age groups. The h i g h e s t c o n c e n t r a t i o n was seen in t h e 1 1 - - 1 5 y r g r o u p and t h e l o w e s t in t h e 6 1 - - 7 0 y r group.
age. T h e v a r i a t i o n s seen f o r d i f f e r e n t ages m a y be a s s o c i a t e d w i t h p h y s i o l o g i c a l c h a n g e s t h a t t a k e p l a c e d u r i n g life. S o m e t r a c e e l e m e n t s such as lead a n d zinc h a v e b e e n n o t e d t o d e c r e a s e in h u m a n hair w i t h age [ 1 2 ] . A p i l o t s t u d y
45 conducted by the investigators also revealed that hair Se tended to decrease with age; however only twenty samples were taken for the study. The lipid peroxidation theory of aging suggests that oxidation of the lipids in the cell membranes destroys its integrity leading to the destruction of the cells and the accumulation of oxidised lipoproteins [16]. Selenium, an integral part of the enzyme glutathione peroxidase, plays a role in the removal of the metabolically generated peroxides from the cells [1]. The high Se content in the hair of adolescents seems to imply that Se could be involved in growth and development. According to a study, Se has been found to be required for the growth and development of human fetal tissue [17]. There was no difference found in the hair concentration of Se between whites and non-whites, the level observed for both was 0.64+0.02#g/g. A significant difference ( P < 0.05) was found between males and females, the levels being 0.67+ 0.02 and 0.54+ 0.02#g/g of hair respectively. A weak correlation of r = +0.02 was found between the Se content in diet and hair. A study of the scatter plot in Fig. 2 revealed that the points are scattered somewhat randomly. At this time as not much information 200
S c a t t e r plot f o r Se in d i e t a n d h a i r
180
160
L 140
•
o
•%
It
120
.=>"l o o
g
a•
• •
80
:.:'.y. : 60
Q
4O •l
0:4
o'.6 Hair
o'.8
1;o
Se ( / J g / g )
Fig. 2. Scatter plot for Se in the diet and hair. The daily intake of Se is plotted on the ordinate and the hair Se level on the abscissa. It can be noted that the points are scattered s o m e w h a t r a n d o m l y s h o w i n g no relationship b e t w e e n the t w o levels.
46
TABLE 2 DIET AND HAIR LEVELS OF SELENIUM Age group
Hair selenium levela
Diet selenium b
5 and below 6--10 11--15 16--20 21--30 31--40 41--50 51--60 61--70 above 70
0.66 + 0.07 0.64 + 0.04 0.76 + 0.04 0.64 + 0.04 0.62 + 0.03 0.59 + 0.04 0.62 + 0.04 0.64 + 0.06 0.55 -+0.03 0.62+ 0.03
61 + 22 c 87 + 25 93 + 28 109 + 40 87 + 38 78 +-22 d 73 -+ 20 d 75 + 18 63 + 21 65 + 13
a Mean levels + SEM (pg/g). b Mean levels + SD (pg intake/day). d Correlations found between the levels were almost significant. c Two-third the serving was taken for calculating the Se level for this group. The above table shows the calculated amount of selenium in the diets of different age groups. The highest consumption is seen in the 16--20 yr group. Lower amounts were consumed by children and the elderly.
exists o n the bioavailability o f Se f r o m f o o d s , w h i c h m i g h t be an i m p o r t a n t f a c t o r t o c o n s i d e r in similar studies in t h e f u t u r e . Table 2 gives the details o f hair and diet c o n t e n t o f Se o b s e r v e d in the groups. T h e m e a n value o f the d i e t a r y Se was f o u n d t o be 87 + 3 p g / d a y , ranging f r o m a low 25 to a high o f 2 0 4 p g / d a y . T h e l o w value n o t e d was d u e t o p o o r d i e t a r y habits o f o n e o f the subjects. T h e m e a n value seems to be close t o t h a t r e p o r t e d in the findings o f a previous s t u d y c o n d u c t e d in this l a b o r a t o r y [ 1 8 , 1 9 ] . A c c o r d i n g to these r e p o r t s the m e a n Se c o n t e n t o f t h e d i f f e r e n t diets ranged f r o m 82 to 93 p g / d a y . These a b o v e findings are c o m p a r a b l e to t h e results o f o t h e r studies [ 2 0 , 2 1 ] w h e r e levels varied a c c o r d i n g t o the geographical l o c a t i o n and f o o d habits.
Part H The s e c o n d p a r t o f the s t u d y involved hospital patients. Figures 3 and 4 s h o w t h e Se levels in hair a n d s e r u m u n d e r n o r m a l a n d p a t h o l o g i c a l conditions. T h e hair Se c o n t e n t ( p g / g + SEM) observed u n d e r the selected p a t h o l o g i c a l c o n d i t i o n s was as follows, 0.51 + 0 . 0 2 in cancer, 0.49 + 0 . 0 3 in cardiovascular disease a n d 0.55 + 0.05 in o t h e r diseases. In the n o r m a l s it was f o u n d t o be 0.63 -+0.06 pg/g o f hair. T h e Se levels in t h e s e r u m ( p g / m l + S E M ) were 0 . 1 4 + 0 . 0 0 6 in t h e n o r m a l s , 0.11 + 0 . 0 0 9 in t h e c a n c e r patients, 0.10 + 0 . 0 0 8 in t h e cardiac patients and 0.12 + 0 . 0 0 6 in patients with o t h e r p a t h o l o g i c a l c o n d i t i o n s . T h e hair Se levels in t h e n o r m a l s f o u n d
47
0.7 O.E
..m 0.5 v)
0.4 0.3 0.2 i
o.oi
Normal
Cancer Cardiovascular Other Disease
Fig. 3. Mean hair levels of Se(pg/g +-SEM) for the selected pathological conditions. Significant low levels (P < 0.05) were observed in the hair of patients with cardiovascular disease.
°'14I
I °'~2I
"~'~0'm10I :kO.gOI 0"06I O.04J',
,, o.oi
Normal
Cancer Cardiovascular Other Disease
Fig. 4. Mean serum levels of Se(pg/ml-+ SEM) for the pathological conditions. The level of Se in the serum of the cardiac patients was found to be significantly low (P < 0.05).
in the second part of the study seem close to those noted in the first part of the study, the levels being 0.63 and 0.64/~g/g of hair respectively.
48
It was n o t e d t hat hair and serum Se levels were low in cardiovascular disease as c o m p a r e d to the levels of normals; the values were 0.49 and 0.63 pg/g respectively. The difference was statistically significant (P < 0.05). The Se c o n t e n t in blood has been f o u n d to be low in several states in the U.S.A., where cardiac mortality is high [ 2 2 ] . Other studies show that the c o n t e n t o f Se in blood of cancer patients is significantly lower when compared to the normals [ 2 3 ] . A weak correlation (r = ÷ 0 . 0 1 ) was f o u n d between the Se levels in hair and serum of the same individuals. This result might be due to hair representing a long-term dietary state whereas the level of serum indicates a recent dietary intake. In conclusion it can be stated t h a t age seems to influence the level of Se in hair o f humans. There was no correlation f o u n d between the Se c o n t e n t o f diet and hair. Similar results were obtained between hair and serum levels o f the same individuals. The hair and serum levels o f Se seem to be lower in patients with cardiovascular disease. While comparing the Se in hair and serum, it can be n o t e d that hair is a m ore stable indicator than serum.
REFERENCES 1 L. FIohe, W. A. Gunzler and H. H. Schock, Glutathione Peroxidase: a Selenoenzyme, Fd. Eur. Soc. Lett., 32 (1973) 132--134. 2 O. A. Levander, E. C. Morris and D. J. Higgs, Selenium as a catalyst for the reduction of Cytochrome c by glutathione. Biochemistry, 12 (1973) 4591--4595. 3 O. H. Muth, J. E. Oldfield, L. F. Remnert and J. R. Schubert, Effect of selenium and Vitamin E on white muscle disease. Science, 128 (1958). 4 E. L. Patterson, R. Milstrey and E. L. R. Stokstad, Effect of selenium in preventing exudative diathesis in chicks. Proc. Soc. Exptl. Biol. Med., 95 (1957) 617--620. 5 K. Schwarz and C. M. Foltz, Selenium as an integral part of Factor 3 against dietary liver necrotic degeneration. J. Am. Chem. Soc., 79 (1957) 3292. 6 R. J. Shamberger and D. V. Frost, Possible protective effect of Selenium against human cancer. Can. Med. Assoc. J., 100 (1969) 682. 7 H. Marjanen and S. Soini, Possible causal relationships between nutritional imbalances especially Manganese deficiency and susceptibility to cancer in Finland. Ann. Agric. Fenn., 11 (1972) 394--406. 8 R.J. Shamberger, Selenium in the environment. Sci. Total Environm., 17 (1981) 70. 9 L. M. Klevay, Hair as biopsy material. II. Assessment of copper nutriture. Am. J. Clin. Nutr., 23 (1970) 1194--1202. 10 K. M. Hambidge, M. L. Franklin and H. A. Jacobs, Hair and Chromium concentration. Effects of sample washing and external environment. Am. J. Clin Nutr., 25 (1972) 384--389. 11 A. Brown (Ed.), The First HumanHair Symposium.Medcom Press, New York, 1974. 12 V. Vlado, Trace Elements in HumanHair. Garland PublishingInc., New York, 1977, p. 61. 13 P. A. Whetter and D. E. Ullrey, Improved fluorometrie method for determining Selenium. J. Assoc. Offic. Anal. Chem., 61 (1978) 927. 14 S.N. Ganapathy, B. T. Joyner and D. R. Sawyerr, Seleniumcontent of representative foods of the major food groups. In press. 15 N. H. Nie, C. H. Hull, J. G. Jenkins, K. Steinhrenner and D. H. Bent, Statistical Package for the Social Sciences. McGraw-Hill, New York, 1975, 2nd edn.
49 16 17
18 19
20 21 22 23
H.A. Guthrie, Introductory Nutrition. C. V. Mosby, St. Louis, 1975, 3rd edn. p. 419. W. L. McKeehan, W. G. Hamilton and R. G. Ham, Selenium as an essential trace element for the growth of WI-38 human diploid fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2023. S. N. Ganapathy and R. Dhanda, Selenium content of omnivorous and cafetaria diets. Ind. J. Nutr. Diet., 17 (1980) 53--59. S. N. Ganapathy, L. K. Booker, R. Craven and C. H. Edwards, Trace minerals, amino acids and plasma proteins in adult men fed wheat diets. J. Am. Diet. Assoc., 78 (1981) 490--497. J. H. Watkinson, The Selenium status of New Zealanders. N.Z. Med. J. 80 (1974} 202. M. C. Mudragon and W. G. Jaffe, The ingestion of selenium in Caracass compared with other cities. Arch. Latinoam. Nutr., 26 (1976) 341. R. J. Shamberger, C. E. Willis and L. J. McCormack, Blood selenium and cardiac mortality in 19 states. Federation Proceedings, 1979. K. P. McConnell, W. L. Broghamer, Jr., A. J. Blotcky and O. J. Hurt, Selenium levels in h u m a n blood and tissues in health and disease. J. Nutr., 105 (1975) 1026.
41
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SELENIUM IN HUMAN HAIR IN RELATION TO AGE, DIET, PATHOLOGICAL CONDITION AND SERUM LEVELS*
S A R O J A THIMAYA and SEETHA N. GANAPATHY
Nutrition Research Program, North Carolina Agricultural and Technical State University, Greensboro, NC 27411 (U.S.A) (Received December 9th, 1981; accepted January 8th, 1982)
ABSTRACT Approximately 0.5 g of hair samples free from medicated shampoos, bleaches and dyes were collected from 115 subjects from the occipitonuchal region. Prior to the analyses, the samples were thoroughly washed and dried in a gravity convection oven until consistent weight was obtained. A 100-mg portion of the cut up and thoroughly mixed sample in duplicate was used for the analysis of selenium (Se) by a fluorometric technique. The results were treated statistically. The level varied from 0.55 in the elderly to 0.76 #g/g of hair in adolescents. A decline of Se was noted in the groups of ages 16--40 yrs, the levels being 0.64 to 0.59pg/g of hair. From groups 41---60 yrs no increase was seen in the Se content, the lowest level noted was in group 61--70 yrs. These differences between groups were found to be statistically significant (P ~ 0.05). No significant correlation was found between the Se content of hair and 'diet. The levels in hair and serum of patients with cardiovascular disease were significantly low (P ~ 0.05), the levels being 0.49 pg/g of hair and 0.10 pg]ml of serum. It was also noted that there was no correlation between Se in hair and serum of the same individuals. Further work using larger samples is necessary to shed more light on these relationships.
INTRODUCTION
Although selenium (Se) has been found to be essential to many animal species, its role in human nutrition has been difficult to define. Its interrelationship with Vitamin E suggests that it might function as an anti-oxidant. The role of Se in the mammalian systems, in the enzyme glutathione peroxidase, has been fully d o c u m e n t e d [1]. A recent study shows that it might function in electron transport mechanisms [2]. The deficiency of Se in animals results in many Se responsive diseases such as muscular dystrophy, exudative diathesis and hepatic necrosis [3--5]. It has been noted to prevent several chemically induced cancers in animals. Studies show that the incidence of human cancer and heart disease is lower in high Se areas [6, 7]. * Supported in part by USDA-SEA/CR grant. 0048-9697/82/0000--0000/$02.75 ©1982
Elsevier Scientific Publishing Company
42 It is believed that Keshan's disease, a type of congestive heart failure could be due to Se deficiency, the most susceptible to the disease being women of child-bearing age and children below 15 years [ 8]. While considering suitable indicators to determine the Se status, hair seems to be a potential biopsy material. Hair has been noted to provide a valuable index of copper and chromium status in man [9, 10]. Many serious ailments have been diagnosed by preliminary hair analysis. In phenylketonuria, cystic fibrosis and protein-calorie malnutrition, the mineral content of hair was observed to change [11]. Hair concentration of elements such as copper, cadmium and lead have already been observed to be low in the elderly [12]. The following work is an a t t e m p t to study the level of Se in human hair in relation to age, diet, pathological condition and serum levels. The correlation between the concentration of Se in hair and serum of the same individual has also been studied. Cancer and cardiovascular disease were the two selected pathological conditions.
METHODS The study, conducted in Greensboro, NC, was divided into two parts. The first part deals with the relationship of hair Se content to age and dietary intake of Se, while the second involves the Se levels in the selected pathological conditions such as cancer and cardiovascular disease. The first part involved 115 local subjects, free from health problems. The information such as age, sex, health condition and dietary data was obtained with the help of a questionnaire. The hair of each participating subject was c u t and placed in numbered plastic bags which were sealed and attached to the appropriate questionnaire. The second part involved 59 hospital patients, for twenty-three of w h o m matching hair and serum samples were available for correlation study. The normals in this part consisted of five subjects free from any health problems.
The questionnaire Besides collecting the above mentioned personal data such as age, sex, diet and health conditions, the questionnaire was also designed to collect information on whether dyes, bleaches and medicated shampoos were used for hair. This helped to avoid the inclusion of chemically treated hair. The results of a pilot study helped in modifying certain items on the questionnaire. As it was realised that subjects were inclined to give inaccurate information regarding their age, it was decided to have age groups with intervals of ten years. For the first t w e n t y years however, the subjects were grouped in five-year intervals. The f o o d data was obtained by means of a seven-day dietary recall.
Preparation of the hair sample for analysis The samples measured 3--5 cm and weighed approximately 0.5 g and were
43 cut from the occipitonuchal region of the head, close to the hair root. They were then stored in sealed plastic bags until analyses. Prior to the analyses, the hair samples were washed with detergent, rinsed several times with deionised, distilled water and dried in a gravity convection oven at 45°C till consistent weight was obtained. The dried hair samples were then cut into small parts, thoroughly mixed in order to obtain a homogenous sample and a 100-mg portion in duplicate was used for the analysis of Se. The Se assay was c o n d u c t e d according to a slightly modified method of Whetter and Ullrey [ 1 3 ] . The instrument used for the measurement of fluorescence was the Perkin-Elmer fluorescence s p e c t r o p h o t o m e t e r Model 203. All chemicals and glassware were obtained from Fisher Scientific Company except 2,3-diaminonaphthalene which was purchased from Aldrich Chemicals. The glassware was washed with detergent, soaked in concentrated nitric acid, rinsed in deionised distilled water and dried before use.
Daily intake o f selenium The values of Se in various c o m m o n foods, reported in a previous study c o n d u c t e d in this laboratory were used in the calculation of the daily intake of Se [ 1 4 ] . The items such as fruits, fruit juices and vegetables whose Se levels are negligible were n o t included for the calculation. This level of Se in the diet was used to find the correlation with the Se c o n t e n t in hair. For the second part of the study only the patients on regular diets were included. The obtained data were collected, coded and transferred to opscan sheets and IBM-key punched. The c o m p u t e r program tool e m p l o y e d was the SPSS [15]. The Student's T-test was used to find the difference in the Se level in the various age groups and the Pearson's correlation was used to find the relationship of Se in hair to that in diet and serum.
RESULTS AND DISCUSSION The findings regarding the influence of age on hair Se c o n t e n t and the correlation between diet and hair Se levels are presented and discussed in Part I, while levels of Se in hair and serum under pathological conditions are given and discussed in Part II.
Part I All age groups were represented with the majority in the group 21--30 yrs. The details are presented in Table 1. The variation observed in the Se levels in the groups can be n o t e d in Fig. 1. Significantly high levels (P ( 0 . 0 5 ) were noted in the 1 1 - - 1 5 y r group comprising the adolescents, when compared to the other groups. The Se c o n t e n t varied from 0.55 to 0.76 #g/g of hair. A gradual decline of Se was f o u n d in 16--20 through 31--40 yr groups, b e y o n d which up to the 51--60 yr group no increase was noted. The level was lowest in the 61--70 yr group. These group differences were statistically significant (P ( 0 . 0 5 ) . These observations indicate that hair Se c o n t e n t is influenced by
44 TABLE 1 HAIR SELENIUM LEVELS OF THE D I F F E R E N T AGE GROUPS
Age g r o u p
Age (years)
No. o f cases
1 2 3 4 5 6 7 8 9 10
5 and b e l o w 6--10 11--15 16--20 21--30 31--40 41--50 51--60 61--70 a b o v e 70
6 12 13 21 26 9 7 9 8 4
S e l e n i u m level a (pg/g) 0.66 0.64 0.76 0.64 0.62 0.59 0.62 0.64 0.55 0.62
-+ 0.07 -+ 0.04 b -+ 0.04 c + 0.04 + 0.03 + 0.04 -+ 0.04 + 0.06 -+ 0.03 + 0.03
a Mean s e l e n i u m levels -+SEM. Statistically significant (P < 0.05) d i f f e r e n c e s in the levels were f o u n d in t h e following groups. b B e t w e e n groups 2 and 3. c B e t w e e n groups 3 and 4. T h e t-test p e r f o r m e d b e t w e e n s u b s e q u e n t g r o u p s gave significant d i f f e r e n c e s (P < 0.05) b e t w e e n t h e groups i n d i c a t e d above. Significant d i f f e r e n c e s (P < 0.05) were also n o t e d w h e n g r o u p 3 was c o m p a r e d w i t h g r o u p s 5, 6, 7, 9 a n d 10.
7~ g *~
0.9
0.8
0.7
o
.E 0,6 o
~- 0.5
0,0
. . o . .~. . o
~
o
i
i
~o ~
i
~ Age
o
6
~
o
'
o
'
.
i
i
i
i
i
0
~
m
,¢
~
to
'
group ~n yrs
Fig. 1. Mean Se c o n c e n t r a t i o n -+ SEM for t h e age groups. The h i g h e s t c o n c e n t r a t i o n was seen in t h e 1 1 - - 1 5 y r g r o u p and t h e l o w e s t in t h e 6 1 - - 7 0 y r group.
age. T h e v a r i a t i o n s seen f o r d i f f e r e n t ages m a y be a s s o c i a t e d w i t h p h y s i o l o g i c a l c h a n g e s t h a t t a k e p l a c e d u r i n g life. S o m e t r a c e e l e m e n t s such as lead a n d zinc h a v e b e e n n o t e d t o d e c r e a s e in h u m a n hair w i t h age [ 1 2 ] . A p i l o t s t u d y
45 conducted by the investigators also revealed that hair Se tended to decrease with age; however only twenty samples were taken for the study. The lipid peroxidation theory of aging suggests that oxidation of the lipids in the cell membranes destroys its integrity leading to the destruction of the cells and the accumulation of oxidised lipoproteins [16]. Selenium, an integral part of the enzyme glutathione peroxidase, plays a role in the removal of the metabolically generated peroxides from the cells [1]. The high Se content in the hair of adolescents seems to imply that Se could be involved in growth and development. According to a study, Se has been found to be required for the growth and development of human fetal tissue [17]. There was no difference found in the hair concentration of Se between whites and non-whites, the level observed for both was 0.64+0.02#g/g. A significant difference ( P < 0.05) was found between males and females, the levels being 0.67+ 0.02 and 0.54+ 0.02#g/g of hair respectively. A weak correlation of r = +0.02 was found between the Se content in diet and hair. A study of the scatter plot in Fig. 2 revealed that the points are scattered somewhat randomly. At this time as not much information 200
S c a t t e r plot f o r Se in d i e t a n d h a i r
180
160
L 140
•
o
•%
It
120
.=>"l o o
g
a•
• •
80
:.:'.y. : 60
Q
4O •l
0:4
o'.6 Hair
o'.8
1;o
Se ( / J g / g )
Fig. 2. Scatter plot for Se in the diet and hair. The daily intake of Se is plotted on the ordinate and the hair Se level on the abscissa. It can be noted that the points are scattered s o m e w h a t r a n d o m l y s h o w i n g no relationship b e t w e e n the t w o levels.
46
TABLE 2 DIET AND HAIR LEVELS OF SELENIUM Age group
Hair selenium levela
Diet selenium b
5 and below 6--10 11--15 16--20 21--30 31--40 41--50 51--60 61--70 above 70
0.66 + 0.07 0.64 + 0.04 0.76 + 0.04 0.64 + 0.04 0.62 + 0.03 0.59 + 0.04 0.62 + 0.04 0.64 + 0.06 0.55 -+0.03 0.62+ 0.03
61 + 22 c 87 + 25 93 + 28 109 + 40 87 + 38 78 +-22 d 73 -+ 20 d 75 + 18 63 + 21 65 + 13
a Mean levels + SEM (pg/g). b Mean levels + SD (pg intake/day). d Correlations found between the levels were almost significant. c Two-third the serving was taken for calculating the Se level for this group. The above table shows the calculated amount of selenium in the diets of different age groups. The highest consumption is seen in the 16--20 yr group. Lower amounts were consumed by children and the elderly.
exists o n the bioavailability o f Se f r o m f o o d s , w h i c h m i g h t be an i m p o r t a n t f a c t o r t o c o n s i d e r in similar studies in t h e f u t u r e . Table 2 gives the details o f hair and diet c o n t e n t o f Se o b s e r v e d in the groups. T h e m e a n value o f the d i e t a r y Se was f o u n d t o be 87 + 3 p g / d a y , ranging f r o m a low 25 to a high o f 2 0 4 p g / d a y . T h e l o w value n o t e d was d u e t o p o o r d i e t a r y habits o f o n e o f the subjects. T h e m e a n value seems to be close t o t h a t r e p o r t e d in the findings o f a previous s t u d y c o n d u c t e d in this l a b o r a t o r y [ 1 8 , 1 9 ] . A c c o r d i n g to these r e p o r t s the m e a n Se c o n t e n t o f t h e d i f f e r e n t diets ranged f r o m 82 to 93 p g / d a y . These a b o v e findings are c o m p a r a b l e to t h e results o f o t h e r studies [ 2 0 , 2 1 ] w h e r e levels varied a c c o r d i n g t o the geographical l o c a t i o n and f o o d habits.
Part H The s e c o n d p a r t o f the s t u d y involved hospital patients. Figures 3 and 4 s h o w t h e Se levels in hair a n d s e r u m u n d e r n o r m a l a n d p a t h o l o g i c a l conditions. T h e hair Se c o n t e n t ( p g / g + SEM) observed u n d e r the selected p a t h o l o g i c a l c o n d i t i o n s was as follows, 0.51 + 0 . 0 2 in cancer, 0.49 + 0 . 0 3 in cardiovascular disease a n d 0.55 + 0.05 in o t h e r diseases. In the n o r m a l s it was f o u n d t o be 0.63 -+0.06 pg/g o f hair. T h e Se levels in t h e s e r u m ( p g / m l + S E M ) were 0 . 1 4 + 0 . 0 0 6 in t h e n o r m a l s , 0.11 + 0 . 0 0 9 in t h e c a n c e r patients, 0.10 + 0 . 0 0 8 in t h e cardiac patients and 0.12 + 0 . 0 0 6 in patients with o t h e r p a t h o l o g i c a l c o n d i t i o n s . T h e hair Se levels in t h e n o r m a l s f o u n d
47
0.7 O.E
..m 0.5 v)
0.4 0.3 0.2 i
o.oi
Normal
Cancer Cardiovascular Other Disease
Fig. 3. Mean hair levels of Se(pg/g +-SEM) for the selected pathological conditions. Significant low levels (P < 0.05) were observed in the hair of patients with cardiovascular disease.
°'14I
I °'~2I
"~'~0'm10I :kO.gOI 0"06I O.04J',
,, o.oi
Normal
Cancer Cardiovascular Other Disease
Fig. 4. Mean serum levels of Se(pg/ml-+ SEM) for the pathological conditions. The level of Se in the serum of the cardiac patients was found to be significantly low (P < 0.05).
in the second part of the study seem close to those noted in the first part of the study, the levels being 0.63 and 0.64/~g/g of hair respectively.
48
It was n o t e d t hat hair and serum Se levels were low in cardiovascular disease as c o m p a r e d to the levels of normals; the values were 0.49 and 0.63 pg/g respectively. The difference was statistically significant (P < 0.05). The Se c o n t e n t in blood has been f o u n d to be low in several states in the U.S.A., where cardiac mortality is high [ 2 2 ] . Other studies show that the c o n t e n t o f Se in blood of cancer patients is significantly lower when compared to the normals [ 2 3 ] . A weak correlation (r = ÷ 0 . 0 1 ) was f o u n d between the Se levels in hair and serum of the same individuals. This result might be due to hair representing a long-term dietary state whereas the level of serum indicates a recent dietary intake. In conclusion it can be stated t h a t age seems to influence the level of Se in hair o f humans. There was no correlation f o u n d between the Se c o n t e n t o f diet and hair. Similar results were obtained between hair and serum levels o f the same individuals. The hair and serum levels o f Se seem to be lower in patients with cardiovascular disease. While comparing the Se in hair and serum, it can be n o t e d that hair is a m ore stable indicator than serum.
REFERENCES 1 L. FIohe, W. A. Gunzler and H. H. Schock, Glutathione Peroxidase: a Selenoenzyme, Fd. Eur. Soc. Lett., 32 (1973) 132--134. 2 O. A. Levander, E. C. Morris and D. J. Higgs, Selenium as a catalyst for the reduction of Cytochrome c by glutathione. Biochemistry, 12 (1973) 4591--4595. 3 O. H. Muth, J. E. Oldfield, L. F. Remnert and J. R. Schubert, Effect of selenium and Vitamin E on white muscle disease. Science, 128 (1958). 4 E. L. Patterson, R. Milstrey and E. L. R. Stokstad, Effect of selenium in preventing exudative diathesis in chicks. Proc. Soc. Exptl. Biol. Med., 95 (1957) 617--620. 5 K. Schwarz and C. M. Foltz, Selenium as an integral part of Factor 3 against dietary liver necrotic degeneration. J. Am. Chem. Soc., 79 (1957) 3292. 6 R. J. Shamberger and D. V. Frost, Possible protective effect of Selenium against human cancer. Can. Med. Assoc. J., 100 (1969) 682. 7 H. Marjanen and S. Soini, Possible causal relationships between nutritional imbalances especially Manganese deficiency and susceptibility to cancer in Finland. Ann. Agric. Fenn., 11 (1972) 394--406. 8 R.J. Shamberger, Selenium in the environment. Sci. Total Environm., 17 (1981) 70. 9 L. M. Klevay, Hair as biopsy material. II. Assessment of copper nutriture. Am. J. Clin. Nutr., 23 (1970) 1194--1202. 10 K. M. Hambidge, M. L. Franklin and H. A. Jacobs, Hair and Chromium concentration. Effects of sample washing and external environment. Am. J. Clin Nutr., 25 (1972) 384--389. 11 A. Brown (Ed.), The First HumanHair Symposium.Medcom Press, New York, 1974. 12 V. Vlado, Trace Elements in HumanHair. Garland PublishingInc., New York, 1977, p. 61. 13 P. A. Whetter and D. E. Ullrey, Improved fluorometrie method for determining Selenium. J. Assoc. Offic. Anal. Chem., 61 (1978) 927. 14 S.N. Ganapathy, B. T. Joyner and D. R. Sawyerr, Seleniumcontent of representative foods of the major food groups. In press. 15 N. H. Nie, C. H. Hull, J. G. Jenkins, K. Steinhrenner and D. H. Bent, Statistical Package for the Social Sciences. McGraw-Hill, New York, 1975, 2nd edn.
49 16 17
18 19
20 21 22 23
H.A. Guthrie, Introductory Nutrition. C. V. Mosby, St. Louis, 1975, 3rd edn. p. 419. W. L. McKeehan, W. G. Hamilton and R. G. Ham, Selenium as an essential trace element for the growth of WI-38 human diploid fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2023. S. N. Ganapathy and R. Dhanda, Selenium content of omnivorous and cafetaria diets. Ind. J. Nutr. Diet., 17 (1980) 53--59. S. N. Ganapathy, L. K. Booker, R. Craven and C. H. Edwards, Trace minerals, amino acids and plasma proteins in adult men fed wheat diets. J. Am. Diet. Assoc., 78 (1981) 490--497. J. H. Watkinson, The Selenium status of New Zealanders. N.Z. Med. J. 80 (1974} 202. M. C. Mudragon and W. G. Jaffe, The ingestion of selenium in Caracass compared with other cities. Arch. Latinoam. Nutr., 26 (1976) 341. R. J. Shamberger, C. E. Willis and L. J. McCormack, Blood selenium and cardiac mortality in 19 states. Federation Proceedings, 1979. K. P. McConnell, W. L. Broghamer, Jr., A. J. Blotcky and O. J. Hurt, Selenium levels in h u m a n blood and tissues in health and disease. J. Nutr., 105 (1975) 1026.