Akad~miai Kiad6, Budapest I N S T R U M E N T A L N E U T R O N A C T I V A T I O N A N A L Y S I S OF B L O O D S E R U M S. T a d j i k i , H.N. Erten, J. E r t e n * B i l k e n t U n i v e r s i t y , C h e m i s t r y D e p a r t m e n t , * H e a l t h Center, 06533 Ankara, T u r k e y R e c e i v e d 13 S e p t e m b e r 1994 A c c e p t e d 28 O c t o b e r 1994 I n s t r u m e n t a l n e u t r o n a c t i v a t i o n a n a l y s i s has been u s e d to d e t e r m i n e ]5 trace e l e - m e n t s in t w e l v e b l o o d serum samples t a k e n
from h e a l t h y s t u d e n t s at B i l k e n t U n i v e r s i t y in Ankara. The m e t h o d a l l o w e d the d e t e r m i - n a t i o n of Sc, Cr, Mn, Fe, Co, Zn, Se, Rb, Cs, Ce, Eu, Tb, Hf, Ta and Hg, w h i c h o c c u r at the ~g.ml-1 to n g . m l -I levels. T h e r e are no v a l u e s r e p o r t e d for Tb, Hf, Ce, Eu and Ta before. The o t h e r r e s u l t s are c o m p a r e d w i t h the v a l u e s r e p o r t e d in the litera- ture. M o s t are in the r a n g e of the r e p o r t e d v a l u e s e x c e p t for Fe, Zn, Se and Cs.
I N T R O D U C T I O N
The i m p o r t a n c e of trace e l e m e n t s in h u m a n h e a l t h has b e e n r e a l i z e d only in the last t w e n t y years. The b i o l o g - ical f u n c t i o n of t r a c e e l e m e n t s and the e f f e c t of t h e i r d e f i c i e n c y in h u m a n m e t a b o l i s m r e m a i n i m p o r t a n t q u e s - tions. A g r e a t m a j o r i t y of the t r a c e e l e m e n t s serve c h i e f l y as k e y c o m p o n e n t s in e n z y m e s y s t e m s c o n n e c t e d
0236-5731/95/US $ 9.S0
Copyright 9 1995 Akadgmiai Kiadd, Budapest
TADJIKI et al.: INAA OF BLOOD SERUM T A B L E I B i o l o g i c a l i m p o r t a n c e of some t r a c e e l e m e n t s E l e m e n t B i o l o g i c a l i m p o r t a n c e Se Cr Co Hg Zn M n Fe A n i n t e g r a l c o m p o n e n t of the e n z y m e g l u t a t h i o n e p e r o x i d a s e w h i c h n e u t r a l i z e s a c t i v e o x y g e n species such as H 2 0 2 and 05 .
A c o f a c t o r of sugar m e t a b o l i s m . E s s e n t i a l for the e f f e c t i v e m e t a b o l i s m of i n s u l i n in i n s u l i n s e n s i t i v e organs.
A c o m p o n e n t of v i t a m i n B12.
T o x i c element. C a u s e s severe h e a l t h problems.
A c o n s t i t u e n t of over 200 m e t a l l o e n z y m e s
(cabonic a n h y d r a s e , a l k a l i n e p h o s p h a t a s e , etc.). E s s e n t i a l for m a n y d i v e r s e f u n c t i o n s i n c l u d i n g g r o w t h a n d d e v e l o p m e n t , n o r m a l r e p r o d u c t i o n , immune and s e n s o r y functions, a n t i - o x i d a n t p r o t e c t i o n and s t a b i l i z a t i o n of m e m b r a n e s .
E s s e n t i a l for growth. A n a c t i v a t o r of some e n z y m e s such as R i b o n u c l e o t i d e r e d u c t a s e , P h o s p h o g l y c e r a t e and P h o s p h o m u t a s e and L - t h r e o n i n e d e h y d r o g e n a s e . E s s e n t i a l for growth. A n i n t e g r a l c o m p o n e n t of h e m o g l o b i n , h e m o p r o t e i n s and p r o t e i n B 2. to v i t a l p r o t e i n s y n t h e s i s . If the m e t a l is r e m o v e d the p r o t e i n u s u a l l y loses its f u n c t i o n a l capacity. T a b l e I s u m m a r i z e s the b i o l o g i c a l i m p o r t a n c e of some t r a c e ele- ments. B l o o d serum is o f t e n c o n s i d e r e d a c o n v e n i e n t
sample m a t e r i a l as it can be e a s i l y o b t a i n e d from large g r o u p s of subjects.
A m o n g the v a r i o u s a n a l y t i c a l techniques, I n s t r u m e n t a l N e u t r o n A c t i v a t i o n A n a l y s i s (INAA) is one of the m o s t p o w e r f u l for the s i m u l t a n e o u s d e t e r m i n a t i o n of s e v e r a l e l e m e n t s in h u m a n b l o o d serum and o t h e r b i o l o g i c a l ma- t e r i a l s I-7.
In t h i s w o r k c o n c e n t r a t i o n s of f i f t e e n t r a c e e l e m e n t s h a v e b e e n d e t e r m i n e d in h u m a n b l o o d s e r u m u s i n g I N N A m e t h o d . F i v e of t h e s e e l e m e n t s are r e p o r t e d for t h e f i r s t time. E X P E R I M E N T A L The b l o o d s a m p l e s w e r e t a k e n from a g r o u p of h e a l t h y s t u d e n t s at B i l k e n t U n i v e r s i t y in A n k a r a u s i n g s t a i n - less s t e e l d i s p o s a b l e n e e d l e s . No a n t i c o a g u l a n t w a s used. T h e s e r u m w a s s e p a r a t e d f r o m the e r y t h r o c y t e s by c e n t r i f u g i n g at 1000 g for 10 m i n and t h e s e r u m p a r t w a s d e c a n t e d i n t o p r e c l e a n e d p o l y c a r b o n a t e tubes. The s e r u m s a m p l e s w e r e t h e n f r e e z e - d r i e d . T h e y w e r e i n t r o d u c e d i n t o q u a r t z t u b e s w h i c h w e r e c l e a n e d a n d s t e r i l i z e d by i m m e r s i n g i n t o a b o i l i n g s o l u t i o n of c o n c e n t r a t e d H N O 3 and H 2 S O 4 a c i d s a n d r i n s i n g s e v e r a l t i m e s w i t h d o u b l y d i s t i l l e d , d e i o n i z e d w a t e r . T h e s e r u m s a m p l e s w e r e s e a l e d and i r r a d i a t e d in the R e a c t o r at the N u c l e a r R e s e a r c h C e n t e r in K. ~ e k m e c e (Istanbul) t o g e t h e r w i t h s t a n d a r d a n d b l a n k s a m p l e s , for 8 h at a flux of 4 x 1 0 1 3 n . c m - 2 . s -I. A f t e r 17 d of c o o l - ing, a c t i v i t y m e a s u r e m e n t s w e r e d o n e u s i n g a h y p e r p u r e c o a x i a l Ge d e t e c t o r and a m u l t i c h a n n e l a n a l y z e r . T h e r e s o l u t i o n (FWHM) of the d e t e c t o r w a s 0.790 k e V f o r the 122 k e V y - l i n e of 5 8 C o a n d 1.80 k e V for the 1330 k e V T - l i n e of 60Co. E f f i c i e n c y c a l i b r a t i o n w a s d o n e u s i n g s t a n d a r d s o u r c e s . T h e s a m p l e s w e r e s y s t e m a t i c a l l y ana- l y z e d a c c o r d i n g to a c o u n t i n g p r o g r a m for a b o u t 9 m o n t h s . T h e b a c k g r o u n d c o n t r i b u t i o n of the b l a n k s a m p l e w a s sub- t r a c t e d f r o m e a c h m e a s u r e m e n t . A l l a c t i v i t i e s w e r e cor- r e c t e d to the e n d of i r r a d i a t i o n u s i n g s t a n d a r d g r o w t h a n d d e c a y e q u a t i o n s .
TADJIKI et al.: INAA OF BLOOD SERUM R E S U L T S A N D D I S C U S S I O N F i g u r e I s h o w s two y - r a y s p e c t r a of b l o o d s e r u m i r r a - d i a t e d for 8 h a n d c o u n t e d for 2 6 0 0 s. T h e t o p s p e c t r u m w a s t a k e n 27 d a f t e r i r r a d i a t i o n a n d t h e b o t t o m s p e c t r u m 118 d later. A l l t h e p e a k s w e r e w e l l r e s o l v e d e x c e p t for t h e 2 7 9 . 2 k e V y - p e a k of 2 0 3 H g w h i c h c o u l d n o t be s e p a - r a t e d f r o m the 2 7 9 . 5 k e V y - p e a k of 75Se. T h e n e t a c t i v - i t y of 2 0 3 H g w a s c a l c u l a t e d by s u b t r a c t i n g the c o n t r i - 75 b u t i o n o f Se f r o m its 2 6 4 . 7 k e V y - p e a k . F i g u r e 2 i l - l u s t r a t e s t h e h a l f - l i f e d e t e r m i n a t i o n of the r a d i o - n u c l i d e s u s e d in t h e a n a l y s i s . T h e d e c a y o f the y - p e a k s w a s f o l l o w e d by c o u n t i n g the s a m p l e s s e v e r a l t i m e s . T h e h a l f - l i v e s o b t a i n e d a g r e e w e l l w i t h t h e l i t e r a t u r e v a l u e s in all c a s e s . T a b l e 2 g i v e s t h e l i s t of r a d i o e l e m e n t s , t h e i r y - p e a k s , a b s o l u t e i n t e n s i t i e s a n d h a l f - l i v e s u s e d in t h e a n a l y s i s t o g e t h e r w i t h the c o n c e n t r a t i o n s of f i f t e e n t r a c e e l e - m e n t s d e t e r m i n e d in e a c h of the 12 b l o o d s e r u m s ~ n p l e s . E x p e r i m e n t a l m e a n v a l u e s o f e a c h e l e m e n t , t h e i r r a n g e s 8 a n d a v a i l a b l e l i t e r a t u r e r a n g e s a r e g i v e n . F o r t h e t r a c e e l e m e n t s , Hf, Tb, Ta, E u a n d Ce no l i t e r a t u r e v a l u e s are a v a i l a b l e . T h e w i d e r a n g e s r e p o r t e d r e f l e c t a n a l y t i c a l d i f f i c u l t i e s in d e t e r m i n i n g s u c h low l e v e l s of e l e m e n t s . O u r r e s u l t s f o r Cr, Hg, Co, Rb, M n a n d Sc a r e w i t h i n t h e r a n g e s r e p o r t e d in t h e l i t e r a t u r e . T h o s e of Zn a n d Se a r e s o m e w h a t lower. T h e low v a l u e s of Zn a n d Se m a y be i m p o r t a n t as b o t h o f t h e s e a r e e s s e n t i a l t r a c e e l e - m e n t s . T h e d i s t r i b u t i o n of Zn a n d Se t e n d to s h o w re- g i o n a l d i f f e r e n c e s d e p e n d i n g on e n v i r o n m e n t a l a n d a g r i - c u l t u r a l c o n d i t i o n s 9 ' 1 0 It m a y be w o r t h w h i l e to p u r s u e t h e r e a s o n s for l o w l e v e l s of Zn and Se b y e x a m i n i n g t h e s u b j e c t s f u r t h e r . T h e l e v e l s of F e a n d Cs a r e h i g h e r 312
x 103 4 3 2 1 0 x 103 lHf 755e § 203Hg
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T A D J I K I e t al.: I N A A O F B L O O D S E R U M ('q -,-4 ~) fi~-~4 - m ~ m ~4 ,-4 ~ ~ . ~ O -.4 ~ ,--4 43 N r3 ~ q4 (D4J ~ ~ m O ,.~ . - 4 0 . ~ 4 ~ ~ o ~ . ~ ~. 4~ ~ ' ~ r ~ O 0 0 ~ ~ - ~ I ~ ~ ~ I r'~ ~ 0 ~ tn,--4 o c6 0 ~ -rmr3 ,,-4 ~ , . Q q 3 , - q O ~ n3 ~ r t0 0) g 4 ~ q q3 r -m 0 -~4 0 ~ ~ ~ -,~ -,4 0,--4 r~.C~ I 44 f~ o~ ~ . . . ~ " 0 0 0 0 0 0 0 0 0 0 CO 0 0 0 0 ~ U'~ 0 I ,I I t I I I | / Lr5 I I I I I I m co S ~ d N ~ ~ m N ~ 4 ~ ~ ~ m ~ d O~ 0 O~ '..0 0 TM, 0"~ C~, 'DO O~ 0 ~ ~ Oq 9 9 9 9 9 ~ 9 9 9 , ~ 9 0 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
r r) r - - CO o'~ 0 4 Lf3 F ~ F'- t " ~ P CO " ~ P.I ~:P O-1
9 0 9 9 9 * 9 F " - - O 9 OO OO O,1 0 3 ~'~ I ' ~ v"- k O x'-- O ~14 x--- F ~ C'I t "~ OO ( ' q CO C ) w - v ' - O,I k O x--- CO C.I 0 4 w - ~-- x--- 0 3 o 3 O O CO ['-- 0 3 Lr3 ~ 0 3 t.O F ~ Oq tX~ o ' ) ~ ~ '.D ~ u Y ~ 8 ~ ~ v-- w-- v-- v ~ ~ ~ r 3 1 4
g . ~ - / 4 0 100 200 Time, d Fig. 2. I l l u s t r a t i o n of t h e h a l f - l i f e d e t e r m i n a t i o n of s o m e r a d i o n u c l i d e s u s e d in the a n a l y s i s . Exp. h a l f - l i f e , d ~: 4 6 S c 71 .8+10 9 : 5 1 C r 26.5+_0.4 o : 5 9 F e 43.4+-0.03 O : ~ 6 0 T b 72.5+_0.9 8 1 H f D: 43.3_+I 4 A" 5 4 M n 274.5+_35 ~: 6 0 C o I .2x103+_512 +: 6 5 Z n 255.6+_13 t h a n t h e r e p o r t e d r a n g e s . In t h e c a s e of Fe, e r y t h r o c y t e c o n t a m i n a t i o n o f s o m e s a m p l e s m a y c a u s e a h i g h e r v a l u e . T h e d e t e c t i o n l i m i t of a r a d i o n u c l i d e is an i m p o r t a n t p a r a m e t e r in n e u t r o n a c t i v a t i o n a n a l y s i s . It is u s u a l l y d e f i n e d as the s m a l l e s t p h o t o - p e a k t h a t can be d e t e c t e d w i t h c e r t a i n c o n f i d e n c e a b o v e t h e b a c k g r o u n d c o n t i n u u m . 11 C u r r i e s h o w e d t h a t the l i m i t of d e t e c t i o n , L D (counts), for a c e r t a i n r a d i o a c t i v i t y is e q u a l to 3 . 2 9 OB' w h e r e OB is the s t a n d a r d d e v i a t i o n of the p e a k of i n t e r e s t in the b l a n k sample. T h e m i n i m u m d e t e c t a b l e m a s s , M D is g i v e n b y M D = L D / K , w h e r e K is the s e n s i t i v i t y f a c t o r
TADJIKI et al.: INAA OF BLOOD SERUM ( c o u n t s . g - l ) . In t h i s w o r k , K w a s f o u n d f r o m the s l o p e of the r a d i o n u c l i d e a c t i v i t y vs. w e i g h t (g) of t r a c e e l e - m e n t p l o t u s i n g 12 s a m p l e s . T h e ~ v a l u e s a n d the s e n s i - t i v i t y f a c t o r s K for e a c h e l e m e n t a n a l y z e d a r e a l s o l i s t e d in T a b l e 2. We t h a n k the T u r k i s h A t o m i c E n e r g y C o m m i s s i o n for R e a c t o r I r r a d i a t i o n s a n d Z. G ~ k m e n o ~ l u f o r h e r c o n t r i - b u t i o n s in t h e i n i t i a l p h a s e s of t h i s w o r k . R E F E R E N C E S I. R.A. N a d k a r n i , G.H. M o r r i s o n , R a d i o c h e m . R a d i o a n a l . L e t t . , 24 (1976) 103. 2. D. B e h n e , H. J u r g e n s e n , J. R a d i o a n a l . C h e m . , 42 (1978) 447. 3. V. B l e k a s t a d , J. J o n s e n , E. S t e i n n e s , K. H e l g e l a n d , A c t a Med. S c a n d . , 216 (1984) 25. 4. L. X i l e i , D. V a n R e n t e r g h e m , R. C o r n e l i s , L. M e e s , A n a l y t i c a Chem. A c t a, 211 (1988) 231. 5. N. Lavi, Z.B. A l f a s s i , A n a l y s t, 115 (1990) 817. 6. R.O. A l l e n , E. S t e i n n e s , M.D. B a k e r , N u c l e a r A c t i v a - t i o n T e c h n i q u e , Proc. L i f e S c i e n c e S y m p o s i u m , 1978, I A E A V i e n n a , 447 (1979). 7. B.L. De La Cruz, L.M. L a n s a n g a n , G.A. A s p e r , R.R. P a r a d e r o , J. P h i l i p p . Med. A s s o c . , 51 (1975) 173. 8. H.J.M. Bowen, J. R a d i o a n a l . C h e m . , 19 (1974) 215.
9. R.S. G i b s o n , F o o d and Nutr. Sci., 13 (1989) 67.
10. G. L o c k i t c h , Crit. Rev. in Clin. Lab. S c i . , 27 (1989) 483.
11. L.A. C u r r i e , Anal. C h e m . , 40 (1968) 580.
