Speciation and Determination of Selenium and Mercury Accumulated

Chapter 22

Speciation and Determination of Selenium and Mercury Accumulated in a Dolphin Liver Kazuko Matsumoto

Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch022

Department of Chemistry, Waseda University, Okubo, Shinjuku-ku, Tokyo 169, Japan

Selenium and mercury species accumulated in a dolphin l i v e r were separated and p u r i f i e d using ultrafiltration and various chromatographic methods. One of the compounds identified is water-soluble ; has a molecular weight of < 1000; contains amino groups; and has a Se to Hg molar ratio of 1:1. Selenum and mercury in the various fractions were determined by carbon-furnance atomic absorption spectrometry with p a l l a d i u m matrix -modification. The addition of palladium to the sample solution eliminated the volatile l o s s of Hg and Se, and a l s o minimized the negative interference due to the biological matrix. Selenium i s an e s s e n t i a l element amd i s k n o w n t o be present i n some enzymes or proteins of vertebrates m a i n l y bound t o an amino acid, v i z . , selenocysteine (1 -3 ) · T h e t o x i c i t y o f m e r c u r y c o m p o u n d s consistently d e c r e a s e s i n t h e p r e s e n c e o f s e l e n i u m ( 4-- 7 ) « I t i s a l s o k n o w n t h a t many s e a a n i m a l s s u c h a s s e a l s a n d d o l p h i n s s o m e t i m e s a c c u m u l a t e Se a n d Hg a t a 1:1 molar r a t i o i n t h e i r l i v e r s ; t h e c o n c e n t r a t i o n s c a n be > 100 μ g/g (3-11 ) . Burk e t a l . (12) a n d Chen e t a l . (13) reported the formation of a high molecular weight ( m o l w t ) c o m p l e x c o n t a i n i n g S e a n d H g a t 1:1 molar r a t i o i n r a b b i t l i v e r s when t h e animals were s i m u l t a n e o u s l y a d m i n i s t e r e d w i t h mercuric c h l o r i d e and sodium s e l e n i t e . T h i s a c c u m u l a t i o n i s i m p o r t a n t from a bioinorganic viewpoint, because of the obvious e n v i r o n m e n t a l and t o x i c o l o g i c a l s i g n i f i c a n c e . However, the c h e m i c a l forms and t h e mechanism o f a c c u m u l a t i o n o f Se a n d Hg a r e n o t k n o w n . I n t h e p r e s e n t w o r k , we w i s h

0097-6156/91/0445-0278$06.00/0 © 1991 American Chemical Society

In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

22.

MATSUMOTO

Selenium and Mercury Accumulated in Dolphin Liver

to r e p o r t our s t u d i e s on the s e p a r a t i o n , p u r i f i c a t i o n and i d e n t i f i c a t i o n o f t h e S e a n d Hg s p e c i e s i n a dolphin liver. We a l s o r e p o r t a s e n s i t i v e c a r b o n f u r n a c e a t o m i c a b s o r p t i o n spectrοphotοmetric method w i t h P d m a t r i x - m o d i f i c a t i o n f o r t h e d e t e r m i n a t i o n o f Se a n d Hg.


Palladium Addition Determinations

Method

f o r Selenium

and

Mercury

The m a i n o b s t a c l e t h a t d i s c o u r a g e s speciation studies o f S e a n d Hg c o m p o u n d s i n d o l p h i n l i v e r i s t h e l a c k o f s e n s i t i v e a n a l y t i c a l methods f o r these elements. Although carbon-furnace atomic absorption spectrometry u s u a l l y p r o v i d e s r a p i d and s e n s i t i v e methods f o r t h e s e elements, i t i s not a p p l i c a b l e t o sample s o l u t i o n s containing organic materials . Organic matrices suppress the a t o m i c a b s o r p t i o n s i g n a l o f Se and H g , and r e d u c e t h e i r s e n s i t i v i t i e s considerably. We f o u n d that these severe negative i n t e r f e r e n c e s c o u l d be removed b y a d d i t i o n o f 100 u g / m l P d t o t h e s a m p l e s o l u t i o n (14-16). T h e i n t e r f e r e n c e s d u e t o o r g a n i c m a t e r i a l s a n d t h e e f f e c t o f P d a d d i t i o n o n S e a n d Hg absorbance are e a s i l y observed i n F i g . 1. Although detection limits (aconcentration corresponding t o twice the r e l a t i v e standard d e v i a t i o n of the background) o f Se a n d Hg i n carbο η-furηace a t o m i c absorption spectrometry w e r e 10 n g / m l a n d 2 n g / m l , r e s p e c t i v e l y , for simple i n o r g a n i c s o l u t i o n s , no a b s o r p t i o n signal was o b s e r v e d when e x t r a c t s o l u t i o n spiked with Se o r Hg w e r e u s e d ( F i g . 1). These s i g n a l s were, however, r e s t o r e d to those o fthe o r i g i n a l i n o r g a n i c l e v e l s w i t h a 1 00 / / g / m l P d a d d i t i o n ( F i g . 1 ) . With thePd a d d i t i o n m e t h o d , t h e d e t e c t i o n l i m i t s f o r S e a n d Hg w e r e 17 n g / m l a n d 10 n g / m l , r e s p e c t i v e l y , e v e n i n the p r e s e n c e o f 1.5% o f a l b u m i n , a t y p i c a l o r g a n i c m a t r i x . F u r t h e r m o r e , we f o u n d t h a t t h e a d d i t i o n o f a l b u m i n o r any other organic m a t e r i a l t o the sample s o l u t i o n e n h a n c e d t h e Se s e n s i t i v i t y b y a p p r o x i m a t e l y a factor of two, p r o v i d e d t h e d r y i n g and a s h i n g c o n d i t i o n s were c a r e f u l l y s e l e c t e d (1_7) . This i s i n contrast t o the common o b s e r v a t i o n that organic materials accelerate the v o l a t i l i z a t i o n o f t h e a n a l y t e d u r i n g t h e a s h i n g stage and thereby reduce s e n s i t i v i t y ( 1_8 ) . T h e o p t i m i z e d t e m p e r a t u r e p r o g r a m we e m p l o y e d u s i n g o u r instrument, a H i t a c h i H-180-50 a t o m i c absorption spectrometer w i t h a H i t a c h i GA-3 g r a p h i t e furnace a t o m i z e r , w a s a s f o l l o w s : d r y , 30-150 °C , 40s; a s h , 150-400°C , 60s; 4-00-800°C , 60s; 8 0 0 - 1 200 V , 60s; a n d a t o m i z e , 24.00 X , 6s. The m e c h a n i s m o f t h e e n h a n c e m e n t e f f e c t w a s s t u d i e d w i t h e l e c t r o n m i c r o s c o p y (1_7) . The m i c r o s c o p y picture o fa v e r t i c a l section o f a carbon furnace, which h a d been applied with palladium and albumin solution and heated t o 1200 °Q , s h o w e d t h a t new


279

280

BIOLOGICAL TRACE ELEMENT RESEARCH

( I) 0.20h

0 . 15L

0 . lOf-


0.05^

0

11

25 50 75 100 Se Concentration (

ng/ml

0

(

)

extract solution of dolphin's l i v e r .

0.101

25 50 75 100 Se Concentration ng/ml

)

extract solution + Pd 100 u g / m l

(ID

0.08Γ

0.061

0.04f

0.02h

(A)

(B)

0

25

Hg

(C) 50

(D) 75

(Ε) 100

concentration (

ng/ml

)

il

(Λ·) 0

Hg

(Β') 25

(C) 50

(D«) (Ε') 5

100

concentration (

ng/ml

)

F i g . 1. Atomic Absorption Profiles of Selenium and Mercury i n Dolphin L i v e r E x t r a c t with and without Palladium Addition (I) selenium c a l i b r a t i o n curves (II) mercury c a l i b r a t i o n curves. ( A ) t ο (Ε) , r e s p e c t i v e l y a r e 0, 0 . 2 5 , 0 . 5 , 0 . 7 5 a n d 1, 3 g/ml m e r c u r y a d d i t i o n s w i t h o u t Pd m o d i f i c a t i o n . (A ) to ( Ε ) a r e t h e same a s ( A ) t o ( E ) , b u t w i t h 1 0 0 Ug/ml P d added. 1

1


22.

MATSUMOTO

Selenium and

Mercury Accumulated in Dolphin Liver


g r a p h i t e l a y e r s w e r e f o r m e s f r o m a l b u m i n on t h e surface of the tube. A combined study using e l e c t r o n m i c r o s c o p y and e n e r g y - d i s p e r s i v e X - r a y s p e c t r o m e t r y of the tube s u r f a c e c l e a r l y i n d i c a t e d the e x i s t e n c e of b o t h Se a n d Pd a s f i n e p a r t i c l e s d i s p e r s e d on t h e tube s u r f a c e ; a l s o , the Se to Pd molar r a t i o was apporximately 1 : 1 . T h i s f a c t p r o v e d t h a t Se was r e t a i n e d on t h e t u b e s u r f a c e a t h i g h e r t e m p e r a t u r e s a s palladium selenide. In a d d i t i o n , thermal stability of the p a l l a d i u m selenide eliminated v o l a t i l i z a t i o n loss d u r i n g t h e a s h i n g s t a g e and i n c r e a s e d the sensitivity o f Se d e t e r m i n a t i o n . The formation of g r a p h i t e l a y e r possibly contributed to the r e t e n t i o n of palladium s e l e n i d e i n or between the l a y e r s t r u c t u r e . E x t r a c t i o n and Compounds f r o m

U l t r a f i l t r a t i o n of a Dolphin L i v e r

Se-

and

Hg-Containing

A d o l p h i n l i v e r was homogenized w i t h the a d d i t i o n of a c e t o n e ( 1 0 ml/g l i v e r ) a n d was dried. The Se a n d Hg c o n c e n t r a t i o n s were 5 6 . 9 and 138 yMg/g, r e s p e c t i v e l y , and t h e Hg t o Se m o l a r r a t i o w a s 0 . 9 5 · The liver p o w d e r was tested for e x t r a c t i o n e f f i c i e n c y with various solvents ( s e e b e l o w ) and the e x t r a c t - s o l u t i o n was subjected to u l t r a f i l t r a t i o n w i t h an A m i c o n YM-2 membrane f o r o b t a i n i n g a l o w - m o l - w t ( 1 0 0 0 ) fraction (12).

Distribution of H i g h c o n t a i n i n g Compounds

and

Low-mol-wt

Hg-

and

Se-

S i x s o l v e n t s were tested for their suitability to extract the Hgand Secontaining compounds from dolphin liver as s h o w n i n T a b l e I . When inorganic solutions without s u r f a c e - a c t i v e a g e n t s were used f o r e x t r a c t i o n , t h e e x t r a c t i o n e f f i c i e n c y was l e s s than 15%; among t h e inorganic solutions, 0.2M ammonium a c e t a t e g a v e t h e h i g h e s t y i e l d o f 14·% f o r b o t h Hg and Se. The y i e l d s o f Hg a n d Se w e r e a l w a y s n e a r l y equal i n any e x t r a c t i o n s o l u t i o n and i n any retentate or filtrate after ultrafiltration. This implied that Hg a n d Se w e r e p r e s e n t i n m o r e t h a n one c o m p o u n d , a l l of w h i c h c o n t a i n e d Hg a n d Se a t a 1 : 1 m o l a r r a t i o . On the other hand, sodium dodecyl s u l p h a t e (SDS) o r T r i t o n X1 0 0 enhanced the e x t r a c t i o n e f f i c i e n c y , but mainly of the high-mol-wt(>1000) fractions. Also these results s h o w e d t h a t t h e c o m p o u n d s c o n t a i n i n g Hg a n d Se a t a 1 : 1 molar r a t i o e x i s t i n the l o w - m o l - w t ( < 1 0 0 0 ) fractions. I t i s thus c o n c e i v a b l e that the low-mol-wt molecule i s the b a s i c s t r u c t u r a l u n i t e x i s t i n g a l s o i n the highmol-wt fractions. Therefore, further purification p r o c e d u r e s were c a r r i e d out o n l y f o r the low-mol-wt compounds.


281


Extraction

a

Sodium

4

3

dodecyl

4

3

1% N a C l 0.20M p h o s p h a t e (pH 7) 0.20M p h o s p h a t e containing 1% 0.20M p h o s p h a t e c o n t a i n i n g 2% Χ-Ί 00 0.20M CH COONH 0.20M CH COONH c o n t a i n i n g 4% a

sulfate.

SDS

a

buffer SDS buffer Triton

buffer

solution

I.

Extraction

Table

1 6

8.2

1 6

6.3

5.6

Hg

—

1 7

43

5.4

—

1 .0

0.59

Hg

1 5

8.1

ratio,%

Extraction

41

5.5

—

1 8

1 .0

0.60

Se

R e t e n t a t e of ultrafiltration (B)

1 7

6.0

4.9

Se

F i l t r a t e of ultrafiltration (A)

Extraction

R a t i o s o f Hg and Se i n V a r i o u s


59

1 4

31

7.3

6.2

Hg

Total (A)+(B)

—

35

7.0

5.5

Se

58

1 4

Solutions

22.

MATSUMOTO


Chromatographic

Behavior

of

the

Hg-

and

Se-Containing


Species B o t h Hg a n d Se w e r e d e t e c t e d m a i n l y i n u n a d s o r b e d f r a c t i o n s and numbers 85-95 i n Dowex 5 0 W - X 4 c h r o m a t o g r a p h y , as shown i n F i g . 2. The m e r c u r y - t o s e l e n i u m m o l a r r a t i o s w e r e n o t 1:1 i n the unadsorbed fractions. S e l e n i u m i n t h e s e f r a c t i o n s c o u l d be s i m p l e compounds such as i n o r g a n i c s e l e n i t e or selenocysteinic acid. On t h e o t h e r h a n d , Hg a n d Se e x i s t a t a 1:1 molar r a t i o i n f r a c t i o n s 85-95· A s e p a r a t e e x p e r i m e n t u s i n g s t a n d a r d amino a c i d s showed t h a t t h i s e l u t i o n p o s i t i o n was j u s t a f t e r t h e p o s i t i o n at w h i c h l e u c i n e was e l u t e d . F r a c t i o n s 90-95 w e r e p o o l e d a n d a p p l i e d t o S e p h a d e x G-15 gel filtration. M e r c u r y and s e l e n i u m a p p e a r e d i n f r a c t i o n s 50-59 a t a 1:1 m o l a r r a t i o ( F i g . 3). From t h e elution pattern, the f r a c t i o n s w e r e d i v i d e d i n t o t h r e e g r o u p s , 48-53> 54-56, a n d 5 7 - 5 9 , a n d e a c h p o o l e d f r a c t i o n was further p u r i f i e d by p a p e r chromatography. In t h e p a p e r chromatograms s h o w n i n F i g . 4, Hg a n d Se w e r e f o u n d a t R 0.3-0.6. M e r c u r y and s e l e n i u m i n t h e s e c a s e s w e r e p r e s e n t a t a 1:1 molar r a t i o . The materials from R 0.3-0.6 and 0.3-0.5 i n F i g . 4 were combined and f u r t h e r p u r i f i e d u s i n g a n i o n exchange chromatography. B o t h Hg a n d Se a p p e a r e d i n f r a c t i o n s 4-7 a t a 1:1 m o l a r r a t i o i n Dowex 1-X2 a n i o n e x c h a n g e chromatography (Fig. 5). Most of the histidine p r e s e n t as the main impurity c o u l d be r e m o v e d b y t h i s chromatographic procedure. F r a c t i o n s 4-7 were p o o l e d and further p u r i f i e d by C h e l e x - 1 0 0 i o n - e x c h a n g e chromatography. f

f

B e h a v i o r o f t h e Hg- a n d S e - C o n t a i n i n g C o m p o u n d s R e v e r s e d - P h a s e HPLC a n d C h e l e x - 1 0 0 Chromatography

on

The Hg a n d Se s p e c i e s w e r e n o t a d s o r b e d on r e v e r s e d phase columns ( S e n s h u Pak SC-8-1251 o r Sedox-DE-613). The h y d r o p h i l i c i t y o f t h e c o m p o u n d s was m o r e t h a n t h a t of t h e amino a c i d s u s u a l l y f o u n d i n p r o t e i n s , w h i c h w e r e a d s o r b e d on t h e c o l u m n u n d e r t h e same c o n s i t i o n s . M e r c u r y and s e l e n i u m were d e t e c t e d i n f r a c t i o n s 18-29 a t a 1:1 m o l a r r a t i o i n C h e l e x - 1 0 0 c h r o m a t o g r a p h y . The e l u t i o n p o s i t i o n s f o r Hg a n d Se w e r e a l m o s t i d e n t i c a l with those of C a a n d Mg . T h e r e f o r e , t h e Hgand S e - c o n t a i n i n g c o m p o u n d was w e a k l y a d s o r b e d on the chelate resin. The a d s o r p t i o n b e h a v i o r was distinctly d i f f e r e n t f r o m , and weaker t h a n t h a t o f s i m p l e Hg ion, s h o w i n g t h a t Hg i n t h e H g - , and Se-containing c o m p o u n d was t i g h t l y b o u n d t o t h e m o l e c u l e a n d could not be e a s i l y r e m o v e d . In a l l c h r o m a t o g r a p h i c s t u d i e s , e x c e p t f o r t h e u n a d s o r b e d f r a c t i o n s i n Dowex 50W-X4 c h r o m a o t g r a p h y , Hg a n d Se a p p e a r e d a t t h e same p o s i t i o n s , w i t h a 1:1 m o l a r ratio. The m o s t p u r i f i e d m a t e r i a l c o n t a i n e d 0.22 mol o f b o t h Hg a n d S e . I n d u c t i v e l y coupled plasma atomic 2 +


283


F i g . 2. I o n Exchange C h r o m â t o g r a m o n a Dowex 50W-X4 C o l u m n ( 2 χ 1 5 0 cm) o f a L o w - m o l - w t F r a c t i o n ( < 1 0 0 0 ) o f Dolphin Liver Extract. B u f f e r s o l u t i o n s used were: (1) 0.2M p y r i d i n e f o r m a t e , p H 2 . 7 4 ; ( 2 ) 0.2M p y r i d i n e , pH 3 . 9 8 ; ( 3 ) 1.0M p y r i d i n e a c e t a t e , pH 4 . 7 6 ; (U) 2 . 0M pyridine acetate, pH 4 . 7 3 ; ( 5 ) 2.0M p y r i d i n e acetate, pH 5 . 1 0 ; a n d ( 6 ) 2.0M p y r i d i n e a c e t a t e , pH 7 . 0 0 . Flow r a t e : 20 m l / h , 20 m l / f r a c t i o n . ( )Hg; ( )Se ; ( )pH ( R e p r o d u c e d w i t h p e r m i s s i o n from R e f . 19. C o p y r i g h t 1 9 8 6 Humana P r e s s ) .


22.


MATSUMOTO

1.5 Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch022

NaCl methionine

Ε 60 Ά 1.0

c ο

-0.5

«β U •U c

«I

ο c ο ο

Blue Dextran

25

30

35

40

45

50 Fraction

55

60

65

70

Number

F i g . 3. Elution P a t t e r n s o f Hg a n d Se o n S e p h a d e x G15. Sample f r a c t i o n s 90-95 on i o n e x c h a n g e c h r o m o t o g r a p h y (Dowex 50W-X4.) o f d o l p h i n l i v e r l o w - m o l wt f r a c t i o n ( < 1 0 0 0 ) ; c o l u m n : S e p h a d e x G-15 ( 2 χ 80 c m ) ; buffer: 0.1M C H ^ C O O N H , ( p H 7 ) ; f l o w r a t e : 6 0 m l / h , )Se (Reproduced with 3ml/fraction. ( )Hg; ( C o p y r i g h t 1 9 8 6 Humana P r e s s ) . p e r m i s s i o n f r o m R e f . 19


285

286

BIOLOGICAL TRACE ELEMENT RESEARCH

1201(No.

48-53)


80 K -

40

0.2

c

•

1 2 0

0.4

0.6

0.8

(No.

54-56)

0.6

0.8

_

CO

^ a:

80 40 ι ι

Ο

2

0.2

Ο

K / /

0.4

M

Speciation and Determination of Selenium and Mercury Accumulated

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