Some aspects of copper metabolism in Brindled Mice: Model for Menkes' disease

Some aspects of copper metabolism in Brindled mice

model for Menkes' disease

Some aspects of copper metabolism in Brindled Mice:

model for Menkes' disease

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Some aspects of copper metabolism in Brindled mice

model for Menkes' disease

P R O E F S C H R I F T ter verkrijging

van de graad van doctor in de

technische wetenschappen

aan de Technische Hogeschool Delft,

op gezag van de rector magnificus

prof. ir. B.P.Th. Veltman,

voor een commisie aangewezen

door het college van dekanen

te verdedigen op

donderdag 24 september 1981

te 16.00 uur door

H E N D R I K W I L L E M P R I N S

scheikundig ingenieur

geboren te Delft

Dit proefschrift is goedgekeurd door de promotor

PROF. IR. J . P . W . H O U T M A N

" D r . C . J . A . van den Hamer heeft in hoge mate bijgedragen

tot de begeleiding bij het tot stand komen van het proefschrift."

Aan mijn vader,

DANKWOORD

B i j h e t v e r s c h i j n e n van d i t p r o e f s c h r i f t w i l i k een i e d e r bedanken, d i e op

wat v o o r w i j z e dan ook, h e e f t b i j g e d r a g e n aan de t o t s t a n d k o m i n g e r v a n . I n h e t

b i j z o n d e r w i l i k m i j n promotor P r o f . i r . J.P.W. Houtman danken v o o r de

weten-s c h a p p e l i j k e v r i j h e i d d i e h i j m i j h e e f t g e l a t e n om h e t g e h e l e onderzoek n a a r

m i j n e i g e n i n z i c h t op t e z e t t e n en u i t t e v o e r e n .

Dr. D a v i d Hunt van h e t Queen Mary C o l l e g e i n Londen ben i k v e e l dank v e r

-s c h u l d i g d omdat h i j door h e t -schenken van de B r i n d l e d muizen h e t f e i t e l i j k e

on-d e r z o e k h e e f t m o g e l i j k gemaakt. De h e e r F.A.J. Angevaren en z i j n meon-dewerkers

van h e t C e n t r a a l P r o e f d i e r B e d r i j f TNO t e A u s t e r l i t z w i l i k dankzeggen v o o r h e t

o p z e t t e n v a n de i n t e e l t s t a m en h e t f o k k e n , onder " s p e c i f i c pathogen f r e e " c o n

-d i t i e s , van -de B r i n -d l e -d muizen ge-duren-de -de o n -d e r z o e k p e r i o -d e .

V o o r t s dank i k de Algemene D i e n s t van h e t I R I , met name N i t a H a l b e , Henk

van Dam, Bas S t i g t e r en Aad van d e r Kooy, en de l e d e n v a n de Vakgroep R a d i o

-chemie v o o r hun h u l p .

B i j z o n d e r v e e l dank ben i k v e r s c h u l d i g d aan a l l e medewerkers v a n de

werk-g r o e p N u c l e a i r e B i o t e c h n i e k . Ton, ook a l b r a c h t j e b e s c h e i d e n h e i d m i j vaak t o t

v e r t w i j f e l i n g , jouw h u l p i s v a n e s s e n t i e e l b e l a n g geweest. J e dogma o v e r de

t r a n s p o r t f u n k t i e v a n h e t " 3 0 , 0 0 0 - e i w i t j e " i s de k a t a l i s a t o r geweest van h e t

on-d e r z o e k n a a r h e t m e t a b o l i s m e van koper-metallothioneïne; jouw vakmanschap was

m e e s t e r l i j k . Ton van d e r Meer dank i k v o o r de v e l e k o p e r b e p a l i n g e n en v o o r z i j n

b e r e i d h e i d "nog even een kolommetje t e meten". L o u i s dank i k v o o r a l v o o r z i j n

" v a d e r l i j k e " b e z o r g d h e i d .

T e n s l o t t e bedank i k A n n e t t e v o o r h e t t y p e n van de v e l e m a n u s c r i p t e n en de

" c a m e r a - k l a r e " v e r s i e van h e t p r o e f s c h r i f t .

PREFACE

P u b l i s h i n g t h e r e s u l t s d u r i n g t h e r e s e a r c h p e r i o d , o f f e r s t h e o p p o r t u n i t y

t o e n t e r t h e d i s c u s s i o n on B r i n d l e d m i c e and on Menkes

d i s e a s e i n l i t e r a t u r e

and f a c i l i t a t e s t h e c o n t a c t s w i t h o t h e r r e s e a r c h w o r k e r s . F u r t h e r m o r e , t h e

d i s s e m i n a t i o n o f i n f o r m a t i o n by means o f p u b l i c a t i o n s i s much w i d e r t h a n by

means o f a " u s u a l " t h e s i s .

U n f o r t u n a t e l y , a c o m p i l a t i o n o f p a p e r s (see c h a p t e r V I I ) - t h e c o r e o f

t h i s t h e s i s - i s n o t e a s y t o r e a d . T h e r e f o r e , v a r i o u s c h a p t e r s a r e i n c l u d e d

w h i c h s h o u l d o f f e r t h e r e a d e r a s u r v e y . I n c h a p t e r I , GENERAL INTRODUCTION, t h e

r e l e v a n t l i t e r a t u r e i s r e v i e w e d . I n c h a p t e r I I , EVALUATION OF THE EXPERIMENTAL

WORK, i m p o r t a n t experimental r e s u l t s , d e s c r i b e d i n t h e p a p e r s , a r e e v a l u a t e d and

compared w i t h t h o s e d e s c r i b e d i n l i t e r a t u r e . F i n a l l y , i n c h a p t e r I I I , GENERAL

DISCUSSION, models a r e d i s c u s s e d w h i c h d e s c r i b e t h e c o p p e r m e t a b o l i s m i n n o r m a l

and m u t a t e d B r i n d l e d m i c e . S i n c e B r i n d l e d mice a r e t h e a n i m a l model f o r Menkes

d i s e a s e , s p e c i a l a t t e n t i o n i s p a i d t o d i f f e r e n c e s between t h e r e s u l t s o b t a i n e d

w i t h hemizygous B r i n d l e d m i c e and t h o s e o b t a i n e d w i t h Menkes p a t i e n t s .

LIST OF PAPERS

Paper I: Van den Hamer, C.J.A. & P r i n s , H.W. (1978). R e s u l t s o f a C u - l o a d i n g t e s t a p p l i e d t o p a t i e n t s w i t h an i n h e r i t e d d e f e c t i n t h e i r Cu-metabolism (Menkes' d i s e a s e ) . In: T r a c e Element Met a b o l i s m i n Man and Animal 3 ( K i r c h g e s s n e r , M., e d . ) , pp. 394396, A r b e i Met s k r e i s für T i e r e r n a h -rungsforschung, Weihenstephan, West Germany.

Paper I I : P r i n s , H.W. & Van den Hamer, C.J.A. (1978). Cucontent o f the l i v e r o f young mice i n r e -l a t i o n t o a d e f e c t i n the Cu-metabo-lism. In: T r a c e E-lement Metabo-lism i n Man and Anima-l - 3

( K i r c h g e s s n e r , M., e d . )( pp. 397-400, A r b e i t s k r e i s für T i e r e r n a h r u n g s f o r s c h u n g , Weihenstephan, West Germany.

Paper I I I : P r i n s , H.W. & Van den Hamer, C.J.A. (1979a). Primary b i o c h e m i c a l d e f e c t i n copper metab o l i s m i n mice with a r e c e s s i v e X - l i n k e d mutation analogous t o Menkes' d i s e a s e i n man. J . Inorg. Biochem. , 10^, 19-27.

Paper IV: Van den Hamer, C.J.A., P r i n s , H.W. & N o o i j e n , J . L . (1979). Menkes' d i s e a s e i n M o t t l e d mice and man. In: Models f o r the study o f i n b o r n e r r o r s o f metabolism (Hommes, F.A., e d . ) , pp.

95-102, E l s e v i e r , Amsterdam, The N e t h e r l a n d s .

Paper V: P r i n s , H.W. & Van den Hamer, C.J.A. (1979b). Impaired copper homeostasis i n B r i n d l e d mice L e t t e r t o the e d i t o r . J . Nutr., 109, 360-361.

Paper VI: P r i n s , H.W. & Van den Hamer, C.J.A. (1980). Abnormal c o p p e r - t h i o n e i n s y n t h e s i s and im-p a i r e d coim-pim-per u t i l i z a t i o n i n mutated B r i n d l e d mice: model f o r Menkes' d i s e a s e . J . N u t r . 110,

151-157.

Paper V I I : P r i n s , H.W. & Van den Hamer, C.J.A. Degradation o f 3 5S - l a b e l e d m e t a l l o t h i o n e i n i n the l i v e r and the kidney o f B r i n d l e d mice: model f o r Menkes' d i s e a s e . Accepted f o r p u b l i c a t i o n i n L i f e S c i .

Paper V I I I : P r i n s , H.W. & Van den Hamer, C.J.A. Comparative s t u d i e s o f copper metabolism i n l i v e r and k i d n e y o f normal and mutated B r i n d l e d mice - w i t h s p e c i a l emphasis on m e t a l l o t h i o n e i n . Accept-ed f o r p u b l i c a t i o n i n Comp. Biochem. P h y s i o l .

Paper IX: P r i n s , H.W. & Van den Hamer, C.J.A. C o p p e r - m e t a l l o t h i o n e i n metabolism i n the k i d n e y of B r i n d l e d mice. Paper accepted f o r p r e s e n t a t i o n a t the " I n t e r n a t i o n a l Symposium on T r a c e Element Metabolism i n Man and Animals (TEMA-4)", P e r t h , A u s t r a l i a .

Paper X: P r i n s , H.W. & Van den Hamer, C.J.A. Menkes' d i s e a s e i n the B r i n d l e d mice. I n v i t e d paper f o r p u b l i c a t i o n i n Am. J . P a t h o l , and i n Handbook: Animal Models o f Human Disease o f the R e g i s t r y o f Comparative Pathology, Washington, D.C., USA.

Paper XI: P r i n s , H.W. (1979). Z i e k t e van Menkes: Koperdeficiëntie en K o p e r s t a p e l i n g . In: S t o f w i s s e l i n g van metalen - 2. Metaal-deficiëntie Z i e k t e n b i j de Mens (Van den Hamer, C.J.A. & Marx, J.J.M. e d s . ) , pp. 85-100, D e l f t s e U n i v e r s i t a i r e P e r s , D e l f t , Nederland.

CONTENTS

I GENERAL INTRODUCTION 1

1 Copper m e t a b o l i s m 1

1.1 Copper m e t a b o l i s m i n man and a n i m a l 1

1.2 A b n o r m a l i t i e s i n c o p p e r m e t a b o l i s m 3

2 Menkes

d i s e a s e 5

2.1 Copper m e t a b o l i s m i n Menkes

d i s e a s e 6

3 B r i n d l e d m i c e , a n i m a l model f o r Menkes

d i s e a s e 8

3.1 Copper m e t a b o l i s m i n B r i n d l e d mice 9

4 M e t a l l o t h i o n e i n 11

4.1 B i o c h e m i c a l f e a t u r e s o f m e t a l l o t h i o n e i n 12

4.1.1 I s o l a t i o n o f m e t a l l o t h i o n e i n 12

4.1.2 Amino a c i d c o m p o s i t i o n and sequence o f m e t a l l o t h i o n e i n 12

4.1.3 M e t a l - b i n d i n g i n m e t a l l o t h i o n e i n 13

4.1.4 O p t i c a l f e a t u r e s o f m e t a l l o t h i o n e i n 14

4.1.5 D e t e r m i n a t i o n o f m e t a l l o t h i o n e i n 14

4.1.6 O t h e r low m o l e c u l a r w e i g h t c o p p e r - b i n d i n g p r o t e i n s 15

4.2 M e t a b o l i s m o f m e t a l l o t h i o n e i n 16

4.2.1 O c c u r r e n c e o f m e t a l l o t h i o n e i n 16

4.2.2 I n d u c t i o n o f m e t a l l o t h i o n e i n 16

4.2.3 C a t a b o l i s m o f m e t a l l o t h i o n e i n 17

4.3 M e t a l l o t h i o n e i n and t r a c e e l e m e n t m e t a b o l i s m 18

I I EVALUATION OF THE EXPERIMENTAL WORK 20

1 Copper m e t a b o l i s m i n B r i n d l e d mice 20

1.1 L i v e r 21

1.2 K i d n e y 25

1.3 I n t e s t i n e 26

1.4 B r a i n 27

2 C o p p e r - m e t a l l o t h i o n e i n i n B r i n d l e d mice 28

2.1 C o p p e r - m e t a l l o t h i o n e i n m e t a b o l i s m i n t h e l i v e r 29

2.2 C o p p e r - m e t a l l o t h i o n e i n m e t a b o l i s m i n t h e k i d n e y 30

I I I GENERAL DISCUSSION 32

1 M o d e l s f o r c o p p e r m e t a b o l i s m i n l i v e r and k i d n e y o f n o r m a l

and mutated B r i n d l e d mice 32

1.2 K i d n e y 37 1.3 C o p p e r m e t a b o l i s m i n m u t a t e d B r i n d l e d m i c e 41 2 Menkes' d i s e a s e 45 2.1 D i f f e r e n c e s between t h e f e a t u r e s o f t h e m u t a t i o n i n Menkes p a t i e n t s and i n "Menkes" m i c e 46 IV SUMMARY 48 V SAMENVATTING 51 VI REFERENCES 55 V I I PAPERS 63 P a p e r I 63 P a p e r I I 67 P a p e r I I I 73 P a p e r IV 83 P a p e r V 93 P a p e r V I 97 P a p e r V I I 105 P a p e r V I I I 113 P a p e r IX 127 P a p e r X 131 P a p e r XI 137

CHAPTER I

GENERAL INTRODUCTION

1.1 Copper m e t a b o l i s m

D u r i n g t h e p o s t g l a c i a l p e r i o d (5000 t o 3000 B.C.) man d i s c o v e r e d t h e u s e

-f u l n e s s o -f copper and i t s a l l o y s -f o r g e n e r a l p u r p o s e s (Ren-frew, 1980). As e a r l y

as H i p p o c r a t e s (400 B.C.) c o p p e r compounds were u s e d i n t h e t r e a t m e n t o f a

v a r i e t y o f d i s e a s e s . Around 1800 A.D. c o p p e r was d e m o n s t r a t e d i n p l a n t s and

a n i m a l s b u t t h e o c c u r r e n c e was assumed t o be a c c i d e n t a l (Mason, 1979).

I n 1928 H a r t e t a l . p r o v e d t h a t c o p p e r was e s s e n t i a l f o r t h e b l o o d

forma-t i o n i n forma-t h e r a forma-t . Noforma-t o n l y f o r forma-t h e r a forma-t b u forma-t f o r many o forma-t h e r s p e c i e s i s c o p p e r an

e s s e n t i a l t r a c e e l e m e n t (Underwood, 1 9 7 7 ) . Many f a c t o r s have c o n t r i b u t e d t o t h e

e x t e n s i v e r e s e a r c h o f copper m e t a b o l i s m i n man and a n i m a l . The main f a c t o r s

a r e , f i r s t , t h e o c c u r r e n c e o f c o p p e r t o x i c i t y and copper d e f i c i e n c y i n farm

a n i m a l s and man, s e c o n d , t h e a v a i l a b i l i t y o f s e n s i t i v e and a c c u r a t e a n a l y t i c a l

methods ( s p e c t r o p h o t o m e t r y , a t o m i c a b s o r p t i o n s p e c t r o m e t r y , 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 ) f o r t h e d e t e r m i n a t i o n o f t h e c o p p e r c o n c e n t r a t i o n i n o r g a n s and body

f l u i d s , and t h i r d , t h e o c c u r r e n c e o f two g e n e t i c a l d e f e c t s i n c o p p e r m e t a b o l i s m

i n man, v i z . , W i l s o n ' s d i s e a s e and Menkes' d i s e a s e .

I.1.1 Copper m e t a b o l i s m i n man and a n i m a l

Copper m e t a b o l i s m i s e x t e n s i v e l y r e v i e w e d by S c h e i n b e r g and S t e r n l i e b

( 1 9 6 0 ) , by Underwood (1977) and, r e c e n t l y , by Mason (1979). Q u a l i t a t i v e l y ,

t h e r e a r e o n l y m i n o r d i f f e r e n c e s between c o p p e r m e t a b o l i s m i n man and a n i m a l .

T h e r e f o r e , t h e r e s u l t s o b t a i n e d w i t h a n i m a l s and humans a r e i n t e r m i n g l e d f o r

t h e d e s c r i p t i o n o f t h o s e f e a t u r e s o f c o p p e r m e t a b o l i s m , w h i c h a r e i n v o l v e d i n

t h e abnormal c o p p e r m e t a b o l i s m i n Menkes p a t i e n t s and mutated B r i n d l e d m i c e .

The m a j o r s i t e s o f a b s o r p t i o n o f a l i m e n t a r y copper a r e l o c a t e d i n t h e

stomach and t h e upper p a r t o f t h e s m a l l i n t e s t i n e (Van Campen, 1 9 7 1 ) . The p e r

c e n t a g e o f copper a b s o r b e d from t h e i n t e s t i n a l lumen and r e l e a s e d from t h e i n

-t e s -t i n a l mucosa i n -t o -t h e b l o o d c i r c u l a -t i o n v a r i e s be-tween 40 and 60 p e r c e n -t

( S t e r n l i e b , 1967; S t r i c k l a n d e t a l . , 1972; K i n g e t a l . , 1 9 7 8 ) . I n t h e a b s o r p

-t i v e c e l l s o f -t h e g a s -t r o i n -t e s -t i n a l -t r a c -t , -t h e s u r p l u s o f copper i s s -t o r e d as

c o p p e r m e t a l l o t h i o n e i n ( H a l l e t a l . , 1979) and i s e x c r e t e d when t h e c e l l d i s

-i n t e g r a t e s a f t e r d e s q u a m a t -i o n .

A f t e r r e l e a s e o f t h e a b s o r b e d c o p p e r i n t o t h e b l o o d c i r c u l a t i o n , c o p p e r i s l o o s e l y bound t o a l b u m i n and amino a c i d s . T h i s form o f c h e m i c a l l y bound c o p p e r i s c a l l e d f r e e - c o p p e r . F r e e - c o p p e r r e p r e s e n t s o n l y 7% o f the p l a s m a c o p p e r . The o t h e r f o r m o f c h e m i c a l l y bound p l a s m a c o p p e r i s c o p p e r , w h i c h i s i n c o r p o r a t e d i n c e r u l o p l a s m i n . T h i s p l a s m a p r o t e i n ( m o l e c u l a r w e i g h t 133,000 d a l t o n ) i s s y n t h e s i z e d by t h e l i v e r , and c o n t a i n s 0.3% c o p p e r . F r e e - c o p p e r i s t h e o n l y f o r m o f p l a s m a c o p p e r w h i c h i s i n t e r c h a n g e a b l e w i t h c o p p e r i n t i s s u e s and i n o t h e r body f l u i d s . I n t h e n e o n a t e t h e c o p p e r c o n c e n t r a t i o n i n p l a s m a i s low s i n c e a m i n o r amount o f c o p p e r i s p r e s e n t as c e r u l o p l a s m i n bound c o p p e r . However, t h e f r e e -c o p p e r -c o n -c e n t r a t i o n i s s l i g h t l y i n -c r e a s e d ( L i n d e r & Munro, 1 9 7 3 ) . S i n -c e apo-c e r u l o p l a s m i n i s i m m u n o l o g i apo-c a l l y d e t e apo-c t a b l e i n t h e p l a s m a o f t h e n e o n a t e (Matsuda e t a l . , 1974) t h e s e r e s u l t s i n d i c a t e t h a t t h e l i v e r i s , t o a low e x t e n t , a b l e t o i n c o r p o r a t e c o p p e r i n a p o c e r u l o p l a s m i n . W i t h i n a y e a r a f t e r b i r t h t h e c o p p e r and c e r u l o p l a s m i n c o n c e n t r a t i o n s o f t h e p l a s m a r e a c h t h e a d u l t l e v e l ( L i n d e r & Munro, 1973). C o p p e r , t a k e n up by t h e c e l l , i s among o t h e r t h i n g s i n c o r p o r a t e d i n v a r i o u s m e t a l l o p r o t e i n s and m e t a l l o e n z y m e s ( S c h e i n b e r g & S t e r n l i e b , 1960), v i z . , s u p e r o x i d e d i s m u t a s e , c y t o c h r o m e c o x i d a s e , t y r o s i n a s e , monoamine o x i -d a s e , an-d c e r u l o p l a s m i n ( o n l y i n t h e l i v e r ) . D e p e n d i n g on t h e c e l l t y p e , c o p p e r i s e x c r e t e d i n b i l e , u r i n e , s a l i v a , sweat and g a s t r o i n t e s t i n a l j u i c e . L i m i t e d r e s e a r c h i s done on t h e e x c r e t i o n o f c o p p e r t h r o u g h g a s t r o i n t e s t i n a l j u i c e , sweat and s a l i v a . The l a t t e r two a r e q u a n t i t a t i v e l y e q u a l t o t h e amount o f c o p p e r e x c r e t e d v i a t h e b i l e . However, t h e c o p p e r e x c r e t i o n t h r o u g h t h e b i l e i s t h e o n l y way o f e x c r e t i o n w h i c h i s u n d e r h o m e o s t a t i c c o n t r o l (Mason, 1979).

I n t h e c y t o p l a s m o f t h e l i v e r c o p p e r i s bound t o t h r e e p r o t e i n f r a c t i o n s w i t h a m o l e c u l a r w e i g h t on G-75 g e l f i l t r a t i o n o f 5 70,000 ( v o i d v o l u m e ) , 30,000 and 10,000 d a l t o n , r e s p e c t i v e l y ( P r i n s & Van den Hamer, 1979a: p a p e r I I I ; 1980: p a p e r V I ) . The p r o t e i n f r a c t i o n w i t h a m o l e c u l a r w e i g h t o f 30,000 d a l t o n c o n t a i n s s u p e r o x i d e d i s m u t a s e , a c o p p e r - , z i n c - e n z y m e . I t i s u n l i k e l y t h a t t h i s enzyme f u n c t i o n s a l s o a s a s p e c i f i c m e t a l s t o r a g e and t r a n s p o r t p r o t e i n (Bremner, 1980). The t h i r d c o p p e r - b i n d i n g p r o t e i n f r a c t i o n ( m o l e c u l a r w e i g h t 10,000 d a l t o n ) i s m e t a l l o t h i o n e i n (see s e c t i o n 1.4), a p r o t e i n w h i c h b i n d s t h e s u r p l u s o f c o p p e r w h i c h i s t a k e n up by t h e l i v e r . H a r d l y a n y t h i n g i s known a b o u t t h e c o p p e r - b i n d i n g p r o t e i n s i n t h e v o i d volume f r a c t i o n ( m o l e c u l a r w e i g h t > 70,000 d a l t o n ) . However, i t i s d o u b t f u l t h a t t h e c o p p e r i n t h i s f r a c -t i o n r e p r e s e n -t s newly s y n -t h e s i z e d c e r u l o p l a s m i n (Weiner & C o u s i n s , 1980) . A p a r t f r o m i t s p r e s e n c e i n t h e c y t o p l a s m , c o p p e r o c c u r s i n the s u b c e l l u l a r

f r a c t i o n s . The m i c r o s o m a l f r a c t i o n c o n t a i n s c o p p e r , w h i c h i s i n c o r p o r a t e d i n newly s y n t h e s i z e d p r o t e i n s . In t h e m i t o c h o n d r i a l f r a c t i o n c o p p e r i s bound t o , e.g., cytochrome c o x i d a s e . The l y s o s o m a l f r a c t i o n c o n t a i n s c o p p e r , w h i c h o r i g -i n a t e s from c a t a b o l -i z e d c o p p e r - p r o t e -i n s , and w h -i c h w -i l l be e x c r e t e d -i n t h e b i l e ( E v a n s , 1973). The n u c l e a r and l y s o s o m a l f r a c t i o n c o u l d s e r v e as a tempor a l s t o tempor a g e when t h e c a p a c i t y o f m e t a l l o t h i o n e i n i n t h e c y t o p l a s m i s i n s u f f i -c i e n t . I n t h e p e r i n a t a l p e r i o d c o p p e r a c c u m u l a t e s i n t h e l i v e r . I t depends on t h e i s o l a t i o n p r o c e d u r e w h e t h e r t h e c o p p e r a c c u m u l a t i o n i s f o u n d i n t h e c y t o p l a s m as m e t a l l o t h i o n e i n (Ryden & D e u t s c h , 1978) o r i n t h e m i t o c h o n d r i a l f r a c t i o n ( P o r t e r e t a l . , 1964) as m i t o c h o n d r o c u p r e i n ( p o l y - m e t a l l o t h i o n e i n ) . The accumu-l a t i o n i n f e t a accumu-l accumu-l i v e r c o u accumu-l d be e x p accumu-l a i n e d by the a b s e n c e o f b i accumu-l e e x c r e t i o n ( M e a r i c k & M i s t i l i s , 1969) o r t h e e x c e s s o f c o p p e r r e p r e s e n t s a r e s e r v e f o r r e q u i r e m e n t s i n t h e p o s t n a t a l p e r i o d (Mason, 1979), a p e r i o d , i n w h i c h t h e i n -t a k e o f c o p p e r from -t h e m i l k d i e -t (low i n copper) i s m a r g i n a l ( C a v e l l & Widdowson, 1964). In s e c t i o n I I . 1 . 1 t h e p h y s i o l o g i c a l i m p o r t a n c e o f the p e r i -n a t a l h e p a t i c c o p p e r a c c u m u l a t i o -n w i l l be f u r t h e r d i s c u s s e d .

I n t h e k i d n e y t h e c o p p e r d i s t r i b u t i o n o v e r t h e c y t o p l a s m i c c o p p e r - b i n d i n g p r o t e i n f r a c t i o n s i s q u a l i t a t i v e l y s i m i l a r t o t h a t i n t h e l i v e r ( P r i n s & Van den Hamer, 1979a: p a p e r I I I ) . F r e e c o p p e r bound t o amino a c i d s i s u l t r a f i l t r a t -ed i n t h e g l o m e r u l i and a p p e a r s i n t h e p r i m a r y u r i n e . V i a t h e t u b u l a r c e l l s t h i s c o p p e r i s r e a b s o r b e d i n t o t h e b l o o d c i r c u l a t i o n . I t i s n o t c l e a r t o w h i c h p r o t e i n f r a c t i o n t h e r e a b s o r b e d c o p p e r i s bound i n t h e t u b u l a r c e l l . The e x c r e -t i o n o f c o p p e r v i a -t h e u r i n e i s under n o r m a l c o n d i -t i o n s o f m i n o r i m p o r -t a n c e .

The c o p p e r m e t a b o l i s m i n t h e b r a i n i s p o o r l y i n v e s t i g a t e d ( P r o h a s k a & W e l l s , 1974). The u p t a k e o f r a d i o a c t i v e c o p p e r i n t h e b r a i n i s slow as compared t o t h a t i n t h e l i v e r . The c o p p e r - p r o t e i n s s u p e r o x i d e d i s m u t a s e , cytochrome c o x i d a s e , • d o p a m i n e - ^ - h y d r o x y l a s e (Hunt, 1977) and m e t a l l o t h i o n e i n (Chen & G a n t h e r , 1975) have been d e t e r m i n e d i n t h e b r a i n . However, h a r d l y a n y t h i n g i s known a b o u t t h e r e q u i r e m e n t f o r c o p p e r and t h e e f f e c t o f t h e c o p p e r s t a t u s on t h e morphology o f t h e b r a i n i n t h e p e r i n a t a l p e r i o d .

1.1.2 A b n o r m a l i t i e s i n c o p p e r m e t a b o l i s m

I n a n i m a l s c o p p e r d e f i c i e n c y o c c u r s as r e s u l t o f an i n s u f f i c i e n t amount o f c o p p e r i n t h e d i e t o r o f a l o w e r a v a i l a b i l i t y o f a l i m e n t a r y c o p p e r as r e s u l t o f d i e t a r y f a c t o r s . D i f f e r e n c e s i n t h e number and sequence o f t h e symptoms o f c o p p e r d e f i c i e n c y a r e due t o v a r i a t i o n s between s p e c i e s and g e n e t i c a l v a r i a t i o n w i t h i n s p e c i e s (Underwood, 1977) . I n man, n a t u r a l o c c u r r e n c e o f c o p p e r d e f i

-c i e n -c y i s o n l y d e s -c r i b e d i n p r e m a t u r e i n f a n t s (Graham & Cordano, 1976). T h e s e i n f a n t s l a c k t h e c o p p e r s t o r e s w h i c h a r e b u i l t up i n t h e l a s t p e r i o d o f g e s t a -t i o n . S h o r -t l y a f -t e r b i r -t h , -t h e l i m i -t e d c o p p e r s -t o r e s a r e e x h a u s -t e d , w h i c h i s a c c e l e r a t e d by m i l k d i e t s , w h i c h a r e low i n c o p p e r , and t h e f i r s t symptoms o f c o p p e r d e f i c i e n c y become m a n i f e s t . See a l s o s e c t i o n I I . 1 . 1 . I n d i s e a s e s i n w h i c h p a t h o l o g i c a l c o n d i t i o n s , e.g., i n t e s t i n a l m a l a b s o r p -t i o n , c h r o n i c d i a r r h e a , o r s h o r -t bowel r e s e c -t i o n , i n -t e r f e r e w i -t h -t h e c a p a c i -t y o f t h e a l i m e n t a r y c o p p e r a b s o r p t i o n , c o p p e r d e f i c i e n c y can o c c u r (Mason, 1979). C o p p e r d e f i c i e n c y i s c h a r a c t e r i z e d by n e u t r o p e n i a , o s t e o p o r o s i s , depigmen-t a depigmen-t i o n o f h a i r , n e u r o l o g i c a l a b n o r m a l i depigmen-t i e s , h y p o c u p r e m i a and depigmen-t h e r e s p o n s e depigmen-t o t h e r a p e u t i c a l l y g i v e n c o p p e r . A l l symptoms o f c o p p e r d e f i c i e n c y d i s a p p e a r w i t h t h e r e s t o r a t i o n o f t h e p r o p e r c o p p e r s t a t u s (Underwood, 1977). C o p p e r t o x i c i t y o c c u r s a f t e r e x c e s s i v e i n t a k e o f c o p p e r , o r a f t e r b l o c k i n g o f t h e b i l i a r y e x c r e t i o n o f c o p p e r . E x c e s s i v e i n t a k e c o u l d r e s u l t f r o m a c c i d e n -t a l l y d r i n k i n g o f c o p p e r s o l u -t i o n s , from d r i n k i n g c o p p e r s o l u -t i o n s as a mechan-ism f o r s u i c i d e , f r o m d i a l y s i s w i t h c o p p e r - c o n t a i n i n g membranes, o r f r o m t r e a t m e n t o f t h e denuded s k i n w i t h c o p p e r s u l f a t e c r y s t a l s ( S c h e i n b e r g & S t e r n l i e b , 1976; Mason, 1979). I n sheep, t o x i c amounts o f c o p p e r a r e a c c u m u l a t e d i n t h e l i v e r as r e s u l t o f l i v e r damage due t o f e e d i n g HeZiotropium enropaewn, a p l a n t w h i c h c o n t a i n s h e p a t o t o x i c a l k a l o i d s (Underwood, 1977). I n man, a h e p a t i c a c c u m u l a t i o n o f c o p p e r can o c c u r u n d e r c h r o n i c c h o l e s t a t i c c o n d i t i o n s ( J a n s s e n s & Van den Hamer, 1980). I n l i t e r a t u r e , a b n o r m a l i t i e s i n c o p p e r m e t a b o l i s m a r e a l s o d e s c r i b e d f o r g e n e t i c d e f e c t s . W i l s o n ' s d i s e a s e ( S c h e i n b e r g & S t e r n l i e b , 1965) i s an a u t o s o -mal r e c e s s i v e d i s e a s e c h a r a c t e r i z e d by a c c u m u l a t i o n o f c o p p e r i n t h e l i v e r ( h e m o l y t i c c r i s e s ) , i n t h e b r a i n ( n e u r o l o g i c a l d i s o r d e r s ) , and i n t h e c o r n e a ( K a y s e r - F l e i s c h e r r i n g ) and by h y p o c u p r e m i a due t o an i m p a i r e d c e r u l o p l a s m i n s y n t h e s i s . The a c c u m u l a t i o n o f c o p p e r i s p r o b a b l y c a u s e d by an i m p a i r e d b i l i a r y e x c r e t i o n o f c o p p e r ( S t e r n l i e b e t a l . , 1973). The d e c r e a s e d c e r u l o p l a s m i n s y n -t h e s i s c o u l d be r e l a -t e d -t o -t h e d y s f u n c -t i o n o f -t h e l i v e r . T h e r e e x s i s -t -two ways o f t h e r a p y , i . e . , by i n c r e a s i n g t h e u r i n a r y e x c r e t i o n o f c o p p e r o r by d e c r e a s i n g t h e c o p p e r a b s o r p t i o n f r o m t h e i n t e s t i n e . The u r i n a r y e x c r e t i o n i s i n -c r e a s e d by a d m i n i s t r a t i o n o f D - p e n i -c i l l a m i n e (Walshe, 1956). F r e e - -c o p p e r i s s e q u e s t e r e d by D - p e n i c i l l a m i n e and t h e c o p p e r complex i s p r e f e r e n t i a l l y ex-c r e t e d i n t h e u r i n e . The D - p e n i ex-c i l l a m i n e t h e r a p y a l s o r e s u l t s i n a d e ex-c r e a s e d u p t a k e o f c o p p e r by t h e t i s s u e s and t h u s p r e v e n t s c o p p e r i n t o x i c a t i o n . The r e s o r p t i o n o f a l i m e n t a r y c o p p e r can be d e p r e s s e d by e n l a r g e m e n t o f t h e t r a p p i n g c a p a c i t y o f i n t e s t i n a l m e t a l l o t h i o n e i n f o r c o p p e r . T h e r e f o r e , the amount o f i n

t e s t i n a l m e t a l l o t h i o n e i n i s i n c r e a s e d by i n d u c t i o n o f the s y n t h e s i s o f m e t a l l o -t h i o n e i n w i -t h o r a l l y g i v e n z i n c (Hoogenraad e -t a l . , 1978).

The a n i m a l model f o r W i l s o n ' s d i s e a s e i s p r o b a b l y t h e B e d l i n g t o n t e r r i e r . These dogs have a d e f e c t w h i c h r e s u l t s i n a t o x i c a c c u m u l a t i o n o f c o p p e r i n t h e l i v e r (Twedt e t a l . , 1979).

Menkes1 d i s e a s e (Menkes e t a l . , 1962; Danks e t a l . , 1972a) i s a n o t h e r h e r e d i t a r y d i s e a s e w i t h an i m p a i r e d c o p p e r m e t a b o l i s m (see s e c t i o n 1.2). The i n b o r n e r r o r o f Menkes' d i s e a s e i s s u b j e c t o f t h i s t h e s i s . An a n i m a l model f o r Menkes' d i s e a s e i s t h e m u t a t e d B r i n d l e d mouse (Hunt, 1974). The m u t a t i o n i n B r i n d l e d m i c e , one o f t h e f i v e m o t t l e d m u t a n t s o f t h e house mouse, i s homolo-gous t o t h e m u t a t i o n i n Menkes' d i s e a s e . The B r i n d l e d m i c e a r e u s e d as an ex-p e r i m e n t a l model f o r r e s e a r c h o f t h e c o ex-p ex-p e r m e t a b o l i s m i n Menkes' d i s e a s e (see s e c t i o n 1.3).

1.2 Menkes' d i s e a s e

I n 1962, Menkes e t a l . d e s c r i b e d a new d e g e n e r a t i v e d i s e a s e o f the c e n t r a l n e r v o u s system as a " s e x - l i n k e d r e c e s s i v e d i s o r d e r w i t h r e t a r d a t i o n o f g r o w t h , p e c u l i a r h a i r and f o c a l c e r e b r a l and c e r e b e l l a r d e g e n e r a t i o n " . T h i s d i s o r d e r w i l l be r e f e r r e d t o as Menkes' d i s e a s e . Synonyms f o r Menkes' d i s e a s e a r e " k i n k y - h a i r d i s e a s e " , "Menkes* k i n k y - h a i r syndrome", " s t e e l y h a i r d i s e a s e " , " t r i c h o p o l i o d y s t r o p h y " , and "X-chromosome l i n k e d c o p p e r m a l a b s o r p t i o n (X-cLCM)". Up t o now a t l e a s t 60 Menkes p a t i e n t s a r e d e s c r i b e d i n l i t e r a t u r e ( D o b r e s c u e t a l . , 1980). A s s u m i n g 80% a s c e r t a i n m e n t t h e e s t i m a t e d f r e q u e n c y i s 1 i n 35,000 l i v e b i r t h s (Danks e t a l . , 1974). F o r a r e v i e w see F r e n c h ( 1 9 7 7 ) .

The f i r s t symptoms a p p e a r w i t h i n t h e f i r s t month a f t e r b i r t h . A l t h o u g h t h e sequence o f a p p e a r a n c e and t h e number o f symptoms a r e n o t t h e same f o r a l l Menkes p a t i e n t s , most symptoms have been o b s e r v e d i n e a c h Menkes p a t i e n t .

Symptoms f o r Menkes' d i s e a s e a r e : H a i r : p i l i t o r t i ( t i g h t t w i s t i n g o f t h e h a i r a r o u n d t h e l o n g i t u d i n a l a x i s ) , m o n i l e t h r i x ( p e r i o d i c i n c r e a s e s i n c r o s s d i a m e t e r a l o n g i t s l e n g t h ) , t r i c h o r r h e x i s nodosa ( f r a c t u r e s o f t h e h a i r s h a f t w h i c h a r e n o d u l a r and f r a y e d ) , d e p i g m e n t a t i o n ; B r a i n : c o n v u l s i v e s e i z u r e s and f a i l u r e t o t h r i v e ; m i l e s t o n e s as head c o n t r o l and s m i l i n g a r e u s u a l l y a c h i e v e d b u t a r e e v e n t u a l l y l o s t and t h e p a t i e n t g e t s i n t o a v e g e t a t i v e s t a t e ; d e g e n e r a t i v e changes i n c e r e b r u m and c e r e b e l l u m , g l i o s i s and a b e r r a t i o n o f P u r k i n j e c e l l s ;

S k e l e t o n : Wormian bones i n t h e s k u l l and b i l a t e r a l m e t a p h y s e a l s p u r r i n g

o f t h e l o n g bones;

B l o o d v e s s e l s : t o r t u o s i t y o f t h e v e s s e l s i n b r a i n and abdomen i n c o m b i n a t i o n

w i t h d e f e c t s i n t h e i n t e r n a l e l a s t i c l a m i n a and a h y p e r p l a s i a

o f t h e o v e r l y i n g i n t i m a .

O t h e r symptoms a r e poor w e i g h t g a i n , h y p o t h e r m i a and m e t a c h r o m a s i a o f i n v i t r o

c u l t u r e d f i b r o b l a s t s .

1.2.1 Copper m e t a b o l i s m i n Menkes

d i s e a s e

Danks e t a l . , (1972a) a s s o c i a t e d t h e changes i n t h e a r t e r i a l w a l l o f

c o p p e r d e f i c i e n t c h i c k s and t h e d e p i g m e n t a t i o n o f w o o l p r o d u c e d by c o p p e r

de-f i c i e n t sheep w i t h t h e s i m i l a r symptoms o de-f Menkes' d i s e a s e and i n v e s t i g a t e d

t h e c o p p e r m e t a b o l i s m o f Menkes p a t i e n t s . The serum c o n c e n t r a t i o n o f c o p p e r

and c e r u l o p l a s m i n was low. T o g e t h e r w i t h t h e d e c r e a s e d c o p p e r c o n c e n t r a t i o n o f

t h e b r a i n and t h e l i v e r i t was c o n c l u d e d t h a t Menkes p a t i e n t s s u f f e r e d f r o m

c o p p e r d e f i c i e n c y .

O r a l l y a d m i n i s t e r e d c o p p e r t e m p o r a r i l y i m p r o v e d t h e c l i n i c a l s t a t u s .

64

S t u d i e s w i t h r a d i o a c t i v e c o p p e r ( Cu) r e v e a l e d t h a t t h e p l a s m a c l e a r a n c e o f

64 64

i n t r a v e n o u s l y g i v e n Cu was n o r m a l b u t o r a l l y g i v e n Cu h a r d l y appeared i n

t h e p l a s m a o f Menkes p a t i e n t s (Danks e t a l , 1972b). I n a d d i t i o n w i t h t h e i n

-c r e a s e d -c o p p e r -c o n -c e n t r a t i o n i n b i o p s i e s o f d u o d e n a l mu-cosa (Danks e t a l . ,

1973), Danks h y p o t e s i z e d t h a t " . . . e v i d e n c e f o r a d e f e c t i n t h e i n t e s t i n a l

a b s o r p t i o n o f c o p p e r i s g i v e n . T r e a t m e n t o f Menkes' syndrome may become p o s s i

-b l e as r e s u l t o f t h e s e f i n d i n g s . " (Danks e t a l . , 1972-b).

S i n c e i n t r a v e n o u s l y g i v e n copper i s n o r m a l l y t a k e n up by t h e t i s s u e s a

v a r i e t y o f c o p p e r complexes were p a r e n t e r a l l y g i v e n as t h e r a p y f o r Menkes'

d i s e a s e (Mason, 1979). Copper s u l f a t e , c o p p e r a c e t a t e , copper a l b u m i n , copper

e d t a , C u C l ^ + h i s t i d i n e and c e r u l o p l a s m i n were u s e d d u r i n g 7 t o 427 d a y s ,

s t a r t i n g even t h r e e days a f t e r b i r t h . However, even a f t e r a t e m p o r a l i m p r o v e

-ment, a l l t r e a t m e n t s f a i l e d as a t h e r a p y f o r Menkes' d i s e a s e .

I n 1975, Heydorn e t a l . measured t h e c o p p e r c o n c e n t r a t i o n i n o r g a n s o f a

male f e t u s o f a woman who had p r e v i o u s l y g i v e n b i r t h t o two Menkes p a t i e n t s .

The c o p p e r c o n c e n t r a t i o n s i n k i d n e y , s p l e e n , p a n c r e a s and p l a c e n t a were s i g n i f

-i c a n t l y -i n c r e a s e d as compared t o t h o s e o f age matched c o n t r o l s , whereas t h e

h e p a t i c c o p p e r c o n c e n t r a t i o n was s i g n i f i c a n t l y d e c r e a s e d . However, t h e t o t a l

amount o f c o p p e r i n t h e f e t u s was a t l e a s t e q u a l t o t h a t o f age matched

con-t r o l s . T h i s i n d i c a con-t e s con-t h a con-t even i n u con-t e r o con-t h e d i s con-t r i b u con-t i o n o f c o p p e r o v e r con-t h e

t i s s u e s i s changed. T a b l e 1 g i v e s t h e c o p p e r c o n c e n t r a t i o n i n t i s s u e s o f

T a b l e 1. Copper concentration in tissues of Menkes and control fetuses

The r e s u l t s a r e e x p r e s s e d as UgCu/g wet t i s s u e .

(Casey & R o b i n s o n , 1978; Heydorn e t a l . , 1975; N o o i j e n e t a l . , 1981; Van den Hamer e t a l . , 1979). T i s s u e Menkes C o n t r o l s L i v e r 2.8 - 11.8 19 - 119 B r a i n ( c e r e b r a l c o r t e x ) 0.17 0.27 - 1.7 K i d n e y ( c o r t e x ) 5.9; 17.3 0.5 - 1.14 P a n c r e a s 15.3 1.27 - 1.74 Lung 1.84 0.3 - 0.57 M u s c l e and s k i n 1.7 0.25 - 1.7 P l a c e n t a 12; 14.5 0.76 - 0.86

Menkes f e t u s e s and age matched c o n t r o l s . N o t o n l y i n t h e f e t u s b u t a l s o i n Menkes p a t i e n t s t h e c o p p e r c o n c e n t r a t i o n o f v a r i o u s o r g a n s i s i n c r e a s e d ( T a b l e 2 ) . As r e s u l t o f c o p p e r t h e r a p y c o p p e r a c c u m u l a t e s f u r t h e r i n t h e s e o r g a n s , t h e h e p a t i c c o p p e r c o n c e n t r a t i o n i s n o r m a l i z e d b u t t h e c o p p e r c o n c e n t r a t i o n o f t h e b r a i n r e m a i n s a t a subnormal l e v e l . The c o p p e r c o n c e n t r a t i o n o f t h e b r a i n w i l l be d i s c u s s e d i n more d e t a i l i n s e c t i o n I I I . 2 . 1 . S t u d i e s o f t h e c o p p e r m e t a b o l i s m o f c u l t u r e d f i b r o b l a s t s and o f a m n i o t i c f l u i d c e l l s o f Menkes p a t i e n t s r e v e a l e d t h a t t h e c o p p e r c o n c e n t r a t i o n i n t h e s e c e l l s i s s i g n i f i c a n t l y i n c r e a s e d (Goka e t a l . , 1976; C a m a k a r i s e t a l . , 1980). 64 A f t e r i n c u b a t i o n i n a medium w h i c h c o n t a i n s r a d i o a c t i v e c o p p e r , Cu i s accumu-64

l a t e d (Horn, 1976). The enhanced i n c o r p o r a t i o n o f Cu i n a m n i o t i c f l u i d c e l l s o f Menkes p a t i e n t s i s now u s e d as a g e n e t i c marker o f Menkes1 d i s e a s e f o r p r e -n a t a l d i a g -n o s i s o f Me-nkes p a t i e -n t s ( G a l j a a r d , 1980).

Due t o t h e p r e n a t a l d i a g n o s i s t h e number o f Menkes p a t i e n t s d e c r e a s e s . However, c u l t u r e d f i b r o b l a s t s o f Menkes p a t i e n t s o f f e r t h e o p p o r t u n i t y t o p e r -f o r m -f u r t h e r r e s e a r c h on Menkes' d i s e a s e . I n Menkes -f i b r o b l a s t s t h e a b n o r m a l amount o f c o p p e r i s s t o r e d i n a c y t o p l a s m i c p r o t e i n f r a c t i o n w i t h a m o l e c u l a r w e i g h t o f 10,000 d a l t o n , w h i c h i s p r o b a b l y m e t a l l o t h i o n e i n ( B e r a t i s e t a l . , 1978; G a r n i c a e t a l . , 1978). In c o n c l u s i o n , t h e m u t a t i o n f o r Menkes' d i s e a s e r e s u l t s i n an abnormal d i s t r i b u t i o n o f c o p p e r and i s a l r e a d y a c t i v e i n t h e p r e n a t a l p e r i o d o f l i f e . C o p p e r a c c u m u l a t e s i n v a r i o u s t i s s u e s , e . g . , k i d n e y , p a n c r e a s , m u s c l e , i n t e s -t i n e , l u n g , s k i n and p l a c e n -t a . L i v e r and b r a i n a r e -t i s s u e s w i -t h a d e c r e a s e d c o p p e r c o n c e n t r a t i o n .

The symptoms o f Menkes' d i s e a s e c a n be e x p l a i n e d by t h e i n a c t i v i t y o f 7

T a b l e 2. Copper concentration in body fluids and tissues of Menkes patients

- with and without copper therapy - and age matched controls

( A o k i & N a k a h a s h i , 1977; D a i s h e t a l . , 1978; Danks e t a l . , 1974; Dekaban &

S t e u s i n g , 1974; G a r n i c a & F l e t c h e r , 1975; G a r n i c a e t a l . , 1977; G r o v e r &

S c r u t t o n , 1975; Horn e t a l . , 1978; L o t t e t a l . , 1975; Van den Hamer e t a l . ,

1979; W a l k e r - S m i t h e t a l . , 1973; Wehinger e t a l . , 1975; W i l l i a m s e t a l . , 1977;

W i l l i a m s e t a l . , 1979).

Body f l u i d , t i s s u e Menkes p a t i e n t s C o n t r o l s

w i t h o u t w i t h

c o p p e r c o p p e r

t h e r a p y t h e r a p y

Plasma (ugCu/ml) 0.1 - 0.5 0 . 8 - 2.0 0.9 - 1.1

C e r u l o p l a s m i n (mg/100 ml)

0

-

7 1 4 - 3 8 1 8 - 3 9

U r i n e (UgCu/24h) 5 - 1 5 3 9 - 9 5 3 - 2 8

L i q u o r (ugCu/ml) 0.02 - 0.04 - 0.03 - 0.05

A m n i o t i c f l u i d (UgCu/ml) 0.26 - 0.03 - 0.09

L i v e r (pgCu/g wet t i s s u e ) 2.0 - 3.7 5 - 1 0 3.4 - 58

B r a i n ( c e r e b r a l c o r t e x )

(UgCu/g wet t i s s u e ) 0.2 - 0.6 0.8 - 1.4 2.3 - 2.9

K i d n e y ( c o r t e x )

(UgCu/g wet t i s s u e ) 8 - 100 48 - 150 2.0 - 2.5

c o p p e r - c o n t a i n i n g enzymes. The f a i l u r e o f copper t h e r a p y i n d i c a t e s t h a t , i n

c o n t r a s t t o n a t u r a l o c c u r r i n g c o p p e r d e f i c i e n c y , p a r e n t e r a l l y g i v e n c o p p e r i s

n o t a b l e t o r e s t o r e a l l c o p p e r d e p l e t e d compartments o r t h a t t h e c o p p e r d e f i

c i e n c y c a u s e d i r r e v e r s i b l e damage w h i c h c a n n o t be r e c o v e r e d by c o p p e r s u p p l e

-t i o n .

*)

1.3 B r i n d l e d mice , a n i m a l model f o r Menkes' d i s e a s e

The B r i n d l e d a l l e l e (Mo'

) a r o s e s p o n t a n e o u s l y i n t h e C57BL i n b r e d s t r a i n

( F r a s e r e t a l . , 1953). The B r i n d l e d a l l e l e i s one o f t h e s i x a l l e l e s o f t h e

m o t t l e d l o c u s on t h e X-chromosome ( F a l c o n e r , 1953; Hunt, 1974). F o u r g e n o t y p e s

A l l r e s e a r c h has been c a r r i e d o u t w i t h B r i n d l e d m i c e . U n f o r t u n a t e l y , i n t h e

f i r s t two p a p e r s t h e mice have been m e n t i o n e d e r r o n e o u s l y as B l o t c h y . See

a l s o P r i n s & Van den Hamer, 1979a: p a p e r I I I .

are d i s t i n g u i s h e d , v i z . , normal male and female, heterozygous female (Mo^

/

)

and hemizygous male (Mo*

^) .

The hemizygous males are almost devoid of pigment. The v i b r i s s a e are

strongly curled and the coat i s wavy. The mice have a s l i g h t tremor, an

uncoor-dinated manner of walking and clasp the hind feet when held up by the t a i l . The

b r / y

hemizygous B r i n d l e d male (Mo ) w i l l be r e f e r r e d to as "Menkes" mice. They

die u s u a l l y between 14-21 days post partum but a few are v i a b l e and f e r t i l e

(Yajima & Suzuki, 1979a).

The heterozygous females (Mo^

) have a variegated coat c o l o r , the

v i b r i s s a e are c u r l y but the coat i s not waved. They are v i a b l e and f e r t i l e .

The "Menkes" mice have an i n h e r i t e d d e f i c i e n c y of the neurotransmitter

noradrenaline (Hunt & Johnson, 1972). The d e f i c i e n c y was explained by a

defec-t i v e hydroxyladefec-tion of dopamine defec-to noradrenaline a r i s i n g as r e s u l defec-t of a defecdefec-t

i n the enzyme dopamineghydroxylase or as r e s u l t of the presence of an i n h i b i

-t o r of -t h i s enzyme.

Research on the connective t i s s u e abnormalities of the "Menkes" mice

revealed that the low t e n s i l e strength was due to an impaired formation of

c r o s s - l i n k s i n collagen and e l a s t i n (Rowe et a l . , 1974), i n d i c a t i n g that the

"Menkes" mice are not able to generate a l l y s i n e required f o r c r o s s - l i n k

forma-t i o n . A l i n k was made wiforma-th forma-the connecforma-tive forma-t i s s u e abnormaliforma-ties i n Menkes

patients and i n copper d e f i c i e n t p i g s . Both s u f f e r from an i n a c t i v e

copper-containing enzyme, v i z . , l y s y l oxidase.

The neuronal degeneration of the b r a i n of "Menkes" mice was extensively

i n v e s t i g a t e d . Neuronal degeneration was found i n the c e r e b r a l cortex and i n the

thalamic n u c l e i (Yajima & Suzuki, 1979d). The degenerating c o r t i c a l neurons

contained enlarged mitochondria. These abnormal mitochondria were also apparent

i n c o r t i c a l neurons which appeared normal (Yajima & Suzuki, 1979c). T h i s could

i n d i c a t e that an i n a c t i v i t y of cytochrome c oxidase i n the mitochondria

preceded the neuronal degeneration.

In the long-surviving hemizygous Brindled mice the neuronal degeneration

gradually disappeared i n the c e r e b r a l cortex and proceeded i n the underlying

white matter (Yajima & Suzuki, 1979a; 1979b). The l a t t e r s i t u a t i o n i s

compara-ble t o that described f o r Menkes' disease (Ghatak et a l . , 1972; Williams et

a l . , 1978). The cause of the change i n s i t e of the neuronal degeneration i n

r e l a t i o n to age i n "Menkes" mice remains obscure.

1.3.1 Copper metabolism i n Brindled mice

i n i t i a l u p t a k e o f Cu i n t h e i n t e s t i n e o f normal and "Menkes" m i c e was f o u n d t o be e q u a l . B u t , i n t h e l a t t e r , the c o p p e r c o n c e n t r a t i o n was i n c r e a s e d i n t h e i n t e s t i n a l w a l l and i n t h e k i d n e y and was d e c r e a s e d i n l i v e r and b r a i n (Hunt, 1976). In v i t r o , i t was f o u n d t h a t t h e c o p p e r m e t a b o l i s m o f c u l t u r e d f i b r o -b l a s t s o f hemizygous B r i n d l e d m i c e and Menkes p a t i e n t s were a l t e r e d s i m i l a r l y

(Camakaris e t a l . , 1980). A l l t h e s e r e s u l t s i n d i c a t e t h a t t h e m u t a t i o n s i n "Menkes" mice and i n Menkes p a t i e n t s a r e p r o b a b l y homologous (Danks, 1977).

B i o c h e m i c a l r e s e a r c h o f c o p p e r enzymes r e v e a l e d t h a t t h e enzyme a c t i v i t y o f c y t o c h r o m e c o x i d a s e , s u p e r o x i d e d i s m u t a s e and 6 a m i n o l e v u l i n a t e d e h y d r a -t a s e i n -t h e l i v e r o f "Menkes" m i c e i s n o r m a l (Hun-t, 1977). A l -t h o u g h -t h e c o p p e r c o n c e n t r a t i o n i s d e c r e a s e d i n t h e l i v e r o f t h e s e m i c e a s compared t o age matched normal B r i n d l e d m i c e , s u f f i c i e n t c o p p e r i s a v a i l a b l e f o r t h e i n t r a -c e l l u l a r -c o p p e r enzymes. I n t h e b r a i n t h e a c t i v i t i e s o f t h e c o p p e r - c o n t a i n i n g enzymes cytochrome c o x i d a s e and s u p e r o x i d e d i s m u t a s e a r e s i g n i f i c a n t l y d e c r e a s e d b u t t h e mono-amine o x i d a s e a c t i v i t y i s n o r m a l . The e l e v a t i o n o f t h e a c t i v i t y o f dopmono-amine- dopamine-(3-hydroxylase i n v i t r o i s e x p l a i n e d by t h e a c t i v a t i o n o f p r e v i o u s l y i n a c t i v e enzyme by a d d i t i o n o f c o p p e r t o t h e a s s a y s y s t e m . I n v i v o , c o p p e r i o n s i n a c t i -v a t e t h e i n h i b i t o r s o f d o p a m i n e - 8 - h y d r o x y l a s e . I n t h e "Menkes" mice t h e enzyme c o n c e n t r a t i o n c o u l d be i n c r e a s e d i n o r d e r t o c o u n t e r a c t t h e d i m i n i s h e d e f f e c t o f t h e i n a c t i v a t i o n o f t h e i n h i b i t o r s by c o p p e r i o n s a s r e s u l t o f t h e c o p p e r d e f i c i e n c y . However, t h e s u b n o r m a l n o r a d r e n a l i n e c o n c e n t r a t i o n i n d i c a t e s t h a t t h e i n c r e a s e o f t h e amount o f enzyme i s i n s u f f i c i e n t (Hunt, 1977).

The d e p i g m e n t a t i o n o f t h e c o a t o f "Menkes" m i c e i s c a u s e d by an i n a c t i -v a t i o n o f t h e c o p p e r - c o n t a i n i n g enzyme t y r o s i n a s e ( H o l a t e i n e t a l . , 1979). R e s t o r a t i o n o f t h e a c t i v i t y i s a c h i e v e d a f t e r i n v i t r o a d d i t i o n o f c o p p e r t o h a i r - b u l b s u p e r n a t a n t s o f "Menkes" and h e t e r o z y g o u s m i c e . T h i s i n d i c a t e s among o t h e r t h i n g s , t h a t i n t h e h e t e r o z y g o t e s t h e c o p p e r m e t a b o l i s m i s a l t e r e d as r e s u l t o f t h e m u t a t i o n .

The e f f e c t o f c o p p e r t h e r a p y has been s t u d i e d i n "Menkes" m i c e . Copper t h e r a p y overcomes t e m p o r a r i l y t h e l e t h a l i t y o f t h e m u t a t i o n and c a u s e s a p i g m e n t a t i o n o f t h e f u r and a s t r a i g h t e n i n g o f t h e v i b r i s s a e . The c o p p e r c o n c e n -t r a -t i o n i n c r e a s e s f u r -t h e r i n -t h e k i d n e y and i s s l i g h -t l y e l e v a -t e d -t o a subnormal l e v e l i n l i v e r and b r a i n . The n o r a d r e n a l i n e c o n c e n t r a t i o n n o r m a l i z e s b u t t h e c y t o c h r o m e c o x i d a s e a c t i v i t y r e m a i n s a t a subnormal l e v e l i n t h e b r a i n (Hunt,

1976).

t h e l a t t e r t h e l y s y l o x i d a s e a c t i v i t y i n c r e a s e d t o 70% o f t h e n o r m a l v a l u e b u t was u n d e t e c t a b l e when t h e m i c e were 60 d a y s o l d .

H a r d l y any c o n c l u s i o n on t h e e f f e c t i v e n e s s o f c o p p e r t h e r a p y can be drawn f r o m t h e s e s t u d i e s s i n c e t h e c o p p e r t h e r a p y c o n s i s t e d o f one s i n g l e i n j e c t i o n o f c o p p e r . U n f o r t u n a t e l y , no r e s e a r c h has been p e r f o r m e d o f t h e e f f e c t o f c o p p e r t h e r a p y on t h e n e u r o n a l d e g e n e r a t i o n c a u s e d by t h e c o p p e r d e f i c i e n c y . I n s e c t i o n I I . 1 . 4 t h e e f f e c t i v e n e s s o f c o p p e r t h e r a p y w i l l be f u r t h e r d i s c u s s e d .

1.4 M e t a l l o t h i o n e i n

I n 1957 m e t a l l o t h i o n e i n was d i s c o v e r e d as a cadmium- and z i n c - b i n d i n g p r o t e i n i n t h e e q u i n e k i d n e y (Margoshes & V a l l e e ) . R e f e r r i n g t o t h e c h a r a c t e r -i s t -i c f e a t u r e s o f m e t a l - b -i n d -i n g and h -i g h c y s t e -i n e c o n c e n t r a t -i o n t h -i s p r o t e -i n was named m e t a l l o t h i o n e i n ( K a g i & V a l l e e , 1960).

The t e r m i n o l o g y employed f o r d i f f e r e n t m e t a l l o t h i o n e i n s i s c o n f u s i n g . In t h i s t h e s i s t h e recommendations f o r t h e n o m e n c l a t u r e o f m e t a l l o t h i o n e i n , as d e s c r i b e d by K a g i & N o r d b e r g (1979), a r e u s e d : - t h e t e r m m e t a l l o t h i o n e i n r e f e r s t o a l l p r o t e i n s w h i c h a r e c h a r a c t e r i z e d by: - m o l e c u l a r w e i g h t o f 6,000 - 7,000 d a l t o n (on g e l f i l t r a t i o n an a p p a r e n t m o l e c u l a r w e i g h t o f 10,000 d a l t o n ) ; - h i g h m e t a l c o n t e n t ; - o p t i c a l f e a t u r e s o f m e t a l t h i o l a t e s ; - u n i q u e amino a c i d sequence; t h e t e r m a p o m e t a l l o t h i o n e i n i s u s e d t o s p e c i f y the a p o p r o t e i n (the p o l y p e p -t i d e c h a i n denuded o f a l l m e -t a l s ) ; - more s p e c i f i c t e r m s s u c h as " c a d m i u m - m e t a l l o t h i o n e i n " and " c o p p e r - t h i o n e i n " a r e u s e d when m e t a l l o t h i o n e i n c o n t a i n s p r e d o m i n a n t l y cadmium and c o p p e r , r e s p e c t i v e l y ;

- i n c o n t e x t s where s p e c i f i c a t i o n s o f t h e m e t a l c o m p o s i t i o n i s n o t a v a i l a b l e o r i s o f no s p e c i a l i n t e r e s t , t h e t e r m " m e t a l l o t h i o n e i n " i s u s e d .

F o r unambigous i d e n t i f i c a t i o n i t i s recommended t h a t t h e r e s e m b l a n c e o f a s p e c i f i c p r o t e i n t o e q u i n e r e n a l m e t a l l o t h i o n e i n i s documented by an a n a l y s i s o f i t s amino a c i d c o m p o s i t i o n o r o f i t s amino a c i d s e q u e n c e . The term " i s o -m e t a l l o t h i o n e i n " i s a p p l i e d t o t h o s e for-ms o f -m e t a l l o t h i o n e i n w h i c h o c c u r i n a

s i n g l e o r g a n i s m b u t a r e c o d e d by d i f f e r e n t g e n e s : t h e y d i f f e r i n amino a c i d c o m p o s i t i o n .

R e s e a r c h was f o c u s s e d on m e t a l l o t h i o n e i n when P i s c a t o r (1964) p o s t u l a t e d t h a t t h e s y n t h e s i s o f t h i s p r o t e i n was i n d u c e d by cadmium and t h a t i t s e r v e d as an i n t r a c e l l u l a r c a d m i u m - d e t o x i f y i n g p r o t e i n .

R e c e n t l y , a g r o u p o f p a r t i c i p a n t s o f t h e " F i r s t I n t e r n a t i o n a l M e e t i n g on

M e t a l l o t h i o n e i n and o t h e r Low M o l e c u l a r Weight M e t a l b i n d i n g P r o t e i n s " has r e

-v i e w e d t h e knowledge o f t h e s e p r o t e i n s ( K a g i & N o r d b e r g , 1979). M e t a l l o t h i o n e i n

i s now t h o u g h t t o b i n d cadmium, z i n c , c o p p e r , m e r c u r y , s i l v e r , b i s m u t h and

p r o b a b l y g o l d and l e a d d e p e n d i n g on t h e m e t a l t o w h i c h t h e o r g a n i s m has been

e x p o s e d . A number o f b i o l o g i c a l f u n c t i o n s has been p r o p o s e d f o r m e t a l l o t h i o

n e i n , v i z . , s t o r a g e , d e t o x i f i c a t i o n and t r a n s p o r t o f m e t a l s , immune r e s p o n s e

and t h e m e t a b o l i s m o f e s s e n t i a l t r a c e e l e m e n t s .

1.4.1 B i o c h e m i c a l f e a t u r e s o f m e t a l l o t h i o n e i n

1.4.1.1 I s o l a t i o n o f m e t a l l o t h i o n e i n

M e t a l l o t h i o n e i n has been i s o l a t e d from v a r i o u s t i s s u e s o f a l a r g e v a r i e t y

o f s p e c i e s r a n g i n g f r o m m i c r o o r g a n i s m s and C r u s t a c e a t o m i c e , r a t and man

( K a g i & N o r d b e r g , 1979).

A f t e r h o m o g e n i z a t i o n and c e n t r i f u g a t i o n , t h e s t a n d a r d i s o l a t i o n p r o c e d u r e

(Webb, 1979) c o n t i n u e s w i t h t h e s e p a r a t i o n o f t h e c y t o p l a s m i c p r o t e i n s a c c o r d

-i n g t o t h e -i r m o l e c u l a r w e -i g h t on Sephadex G-75. The p r o t e -i n f r a c t -i o n w -i t h a

m o l e c u l a r w e i g h t o f 10,000 d a l t o n , w h i c h c o n t a i n s m e t a l l o t h i o n e i n , i s f u r t h e r

p u r i f i e d by i o n exchange c h r o m a t o g r a p h y on DEAE Sephadex A-25, a p p l y i n g a

g r a d i e n t o f T r i s - b u f f e r o r o f N a C l i n T r i s - b u f f e r . N o r m a l l y , cadmium- and

z i n c - m e t a l l o t h i o n e i n a r e s e p a r a t e d i n two i s o m e t a l l o t h i o n e i n s ( C a i n & H o l t ,

1979) and c o p p e r m e t a l l o t h i o n e i n i n t h r e e d i f f e r e n t i s o m e t a l l o t h i o n e i n s

(Bremner & Young, 1976). However, d i f f e r e n t i o n exchange e l u t i o n p a t t e r n s have

been d e s c r i b e d i n l i t e r a t u r e ( S u z u k i & Yamamura, 1979; Webb, 1 9 7 9 ) . The d i f f e r

-e n c -e s i n t h -e -e l u t i o n p a t t -e r n s can o r i g i n a t -e from d i f f -e r -e n c -e s i n t h -e c o p p -e r t o

z i n c r a t i o o f t h e i s o m e t a l l o t h i o n e i n s ( C a i n & H o l t , 1 9 7 9 ) .

R e c e n t l y , a new i s o l a t i o n p r o c e d u r e has been d e s c r i b e d u s i n g h i g h p r e s s u r e

l i q u i d c h r o m a t o g r a p h y ( S u z u k i , 1980; S u z u k i & Yamamura, 1980a). I n one s t e p t h e

m e t a l l o t h i o n e i n f r a c t i o n o f a c r u d e s u p e r n a t a n t i s s e p a r a t e d i n t o t h e v a r i o u s

i s o m e t a l l o t h i o n e i n s u s i n g a g e l p e r m e a t i o n column (TSK GEL SW 3 0 0 0 ) . The t i m e

f o r i s o l a t i o n i s r e d u c e d from 2 days t o l e s s t h a n 1 h o u r .

1.4.1.2 Amino a c i d c o m p o s i t i o n and sequence o f m e t a l l o t h i o n e i n

M e t a l l o t h i o n e i n has a t y p i c a l amino a c i d c o m p o s i t i o n ( K o j i m a & K a g i , 1978).

I t has a h i g h c o n c e n t r a t i o n o f c y s t e i n e (2530%) and a r e l a t i v e l y h i g h c o n c e n

-t r a -t i o n o f l y s i n e (10-15%) and s e r i n e ( 7 - 1 7 . 5 % ) . A r o m a -t i c amino a c i d s and

h i s t i d i n e a r e a b s e n t . The d i f f e r e n c e s i n t h e amino a c i d c o m p o s i t i o n o f m e t a l l o

-t h i o n e i n d e s c r i b e d i n l i -t e r a -t u r e a r e p r o b a b l y c a u s e d by d i f f e r e n c e s i n -t i s s u e s

from w h i c h m e t a l l o t h i o n e i n h a s been i s o l a t e d , and i n p u r e n e s s o f t h e p r e p a r a -t i o n u s e d f o r -t h e d e -t e r m i n a -t i o n o f -t h e amino a c i d c o m p o s i -t i o n (Webb, 1979). The m e t a l l o t h i o n e i n p o l y p e p t i d e c h a i n c o n t a i n s 61 amino a c i d s ( m o l e c u l a r w e i g h t 6,000 - 7,000 d a l t o n ) among w h i c h 20 c y s t e i n y l r e s i d u e s , one m e t h i o n i n e r e s i d u e as t h e N - a c e t y l d e r i v a t i v e a t t h e N - t e r m i n a l s i d e and an a l a n i n e r e s i d u e a t t h e C t e r m i n a l s i d e o f t h e p o l y p e p t i d e c h a i n . The c y s t e i n y l r e s i -dues o c c u r i n t h r e e a r r a n g e m e n t s , v i z . , c y s - x - c y s ( 7 ) , c y s - c y s ( 3 ) , and c y s - x - x - c y s ( 3 ) ; x r e p r e s e n t s an amino a c i d o t h e r t h a n c y s t e i n e and t h e f i g u r e i n p a r e n t h e s e s r e p r e s e n t s t h e number o f t h e a r r a n g e m e n t s . B a s i c amino a c i d s and s e r i n e a r e p r e d o m i n a n t l y l o c a t e d a t t h e j u x t a p o s i t i o n o f t h e c y s t e i n y l r e s i -d u e s . M e t a l l o t h i o n e i n -does n o t c o n t a i n f r e e s u l f h y -d r y l o r -d i s u l f i -d e g r o u p s . The d i s c r e p a n c y i n t h e m o l e c u l a r w e i g h t o f m e t a l l o t h i o n e i n b a s e d on g e l f i l t r a t i o n e x p e r i m e n t s (10,000 d a l t o n ) a n d t h e m o l e c u l a r w e i g h t o f 6,000 - 7,000 d a l t o n c a l c u l a t e d from t h e amino a c i d c o m p o s i t i o n i s c a u s e d b y t h e n o n - g l o b u l a r shape o f m e t a l l o t h i o n e i n , w h i c h r e s u l t s i n a h i g h e r a p p a r e n t m o l e c u l a r w e i g h t i n g e l f i l t r a t i o n c h r o m a t o g r a p h y (Ryden & D e u t s c h , 1978).

R e c e n t l y , S u z u k i and Yamamura (1980b) d e s c r i b e d a d i m e r o f cadmium-m e t a l l o t h i o n e i n . The d i cadmium-m e r s a r e forcadmium-med by an i n t e r cadmium-m o l e c u l a r bond between two monomers and c o n s i s t o f t h e t h r e e c o m b i n a t i o n s o f t h e two i s o m e t a l l o t h i o n e i n s . Whether t h e d i m e r s a r e i s o l a t i o n a r t i f a c t s o r a r e p r e s e n t under p h y s i o l o g i c a l c o n d i t i o n s r e m a i n s o b s c u r e .

1.4.1.3 M e t a l - b i n d i n g i n m e t a l l o t h i o n e i n

Z i n c and cadmium a r e p r o b a b l y bound t o m e t a l l o t h i o n e i n t h r o u g h m e r c a p t i d e bonds w i t h a c y s x c y s a r r a n g e m e n t . The m e t a l s a r e c h e l a t e d t o a t h i r d c y s t e i n y l r e s i d u e as r e s u l t o f t e r t i a r y s t r u c t u r e f o l d i n g . The r a t i o o f t h e c y s t e i n y l r e s i d u e s t o t h e sum o f m e t a l i o n s i s 3, i n d i c a t i n g t h a t 67 m e t a l -b i n d i n g ' s i t e s a r e a v a i l a -b l e f o r t h e s e m e t a l s (We-b-b, 1979). A r a t i o o f 2 f o u n d f o r c o p p e r s u g g e s t s t h a t t h e r e a r e 10 c o p p e r - b i n d i n g s i t e s .

From t h e f u n g u s , Neurospora orassa, a c o p p e r b i n d i n g p r o t e i n has been i s o -l a t e d w i t h a m o -l e c u -l a r w e i g h t o f 2,200 d a -l t o n (25 amino a c i d s ) . The p o -l y p e p t i d e c h a i n i s s i m i l a r t o t h e N t e r m i n a l r e g i o n o f t h e p o l y p e p t i d e c h a i n o f m e t a l l o -t h i o n e i n . The r a -t i o o f -t h e c y s -t e i n y l r e s i d u e s -t o c o p p e r i s c l o s e -t o 1 f o r -t h i s p r o t e i n ( L e r c h , 1980). M e t a l s d i f f e r i n t h e i r a f f i n i t y f o r m e t a l l o t h i o n e i n . The a f f i n i t y d e -c r e a s e s i n t h e o r d e r : Hg > Ag > Cu > Cd > Zn. I n v i v o and i n v i t r o , t h e m e t a l s bound t o m e t a l l o t h i o n e i n a r e e x c h a n g e a b l e f o r m e t a l s w i t h a h i g h e r a f f i n i t y . The a p o p r o t e i n i s o b t a i n e d b y a c i d i f i c a t i o n o f z i n c - o r c a d m i u m - m e t a l l o t h i o n e i n t o pH o f 4.53.5 and 3.52.5, r e s p e c t i v e l y . Copper i s removed a t pH 1 by t r e a t

-ment w i t h m e r c a p t o e t h a n e o r e d t a (Webb, 1979). No p r o c e d u r e s a r e d e s c r i b e d t o o b t a i n a p o m e t a l l o t h i o n e i n f r o m m e r c u r y - m e t a l l o t h i o n e i n .

1.4.1.4 O p t i c a l f e a t u r e s o f m e t a l l o t h i o n e i n

The t y p i c a l amino a c i d c o m p o s t i o n and t h e m e r c a p t i d e m e t a l bonds o f m e t a l l o t h i o n e i n a r e r e f l e c t e d i n t h e e l e c t r o n i c s p e c t r a . S i n c e m e t a l l o t h i o n e i n c o n -t a i n s no a r o m a -t i c amino a c i d s -t h e a b s o r p -t i o n a -t 280 nm i s low. The m e -t a l m e r c a p t i d e complexes show a b s o r p t i o n bands a t 220 nm, 250 nm, and 270 nm f o r z i n c , cadmium and c o p p e r , r e s p e c t i v e l y (Rupp & Weser, 1978). I n v e s t i g a t i o n s o f t h e i s o m e t a l l o t h i o n e i n s o f h o r s e , human and sheep r e v e a l e d t h a t t h e p r o t o n n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a o f t h e i s o m e t a l l o t h i o n e i n s a r e s i m i l a r b u t t h e z i n c and c a d m i u m m e t a l l o t h i o n e i n m o l e c u l e s have d i f f e r e n t t e r t i a r y s t r u c -t u r e s (Vasak e -t a l . , 1980). Seven n o n - e q u i v a l e n -t c a d m i u m - b i n d i n g s i -t e s a r e d e t e c t a b l e w i t h * ^ C d n u c l e a r m a g n e t i c r e s o n a n c e ( S a d l e r e t a l . , 1978; S u z u k i & M a i t a n i , 1978). F o r c o p p e r - m e t a l l o t h i o n e i n no e l e c t r o n p a r a m a g n e t i c r e s o n a n c e a b s o r p t i o n i s o b s e r v e d , i n d i c a t i n g t h a t c o p p e r i s p r o b a b l y bound t o m e t a l l o -t h i o n e i n as c u p r o u s c o p p e r (Rupp & Weser, 1979).

1.4.1.5 D e t e r m i n a t i o n o f m e t a l l o t h i o n e i n T h e r e i s no a n a l y t i c a l p r o c e d u r e i n g e n e r a l use f o r t h e d e t e r m i n a t i o n o f m e t a l l o t h i o n e i n i n a n i m a l t i s s u e s and i n b i o c h e m i c a l s a m p l e s . The q u a n t i t a t i v e d e t e r m i n a t i o n o f m e t a l l o t h i o n e i n i s s u b j e c t o f c o n s i d e r a b l e c o n t r o v e r s y . Due t o t h e u n i q u e amino a c i d c o m p o s i t i o n (see s e c t i o n 1.4.1.2) v a r i o u s a s s a y s f o r t h e d e t e r m i n a t i o n o f p r o t e i n s a r e n o t a p p l i c a b l e f o r m e t a l l o t h i o n e i n . The F o l i n r e a g e n t i s n o t s u i t a b l e s i n c e a r o m a t i c amino a c i d s and i n p a r t i c u l a r t y r o s i n e , w h i c h f o r m a c o l o r e d complex w i t h t h e r e a g e n t , a r e a b s e n t i n m e t a l l o t h i o n e i n . The d a t a o b t a i n e d a r e m e r e l y t h e r e s u l t o f t h e p r e s e n c e o f i m p u r i t i e s w i t h h i g h e r r e s p o n s e t o t h e r e a g e n t (Van d e r M a l l i e & G a r v e y , 1979; Webb, 1979). The f l u o f o m e t r i c method o f B o h l e n e t a l . , (1973) i s b a s e d on t h e r e a c t i o n o f f l u -o r e s c a m i n e w i t h f r e e amin-o g r -o u p s . H-owever, many b i -o c h e m i c a l samples c -o n t a i n T r i s - b u f f e r w h i c h r e a c t s w i t h f l u o r e s c a m i n e . O t h e r a s s a y s measure t h e amount o f s u l f h y d r y l g r o u p s o f p r o t e i n s . P i o t r o w s k i e t a l . (1973) d e v e l o p e d a method i n 203 w h i c h t h e s u l f h y d r y l g r o u p s a r e l a b e l e d w i t h Hg ( K o t s o n i s & K l a a s s e n , 1 9 7 7 ) . 203 203 A l l H g - l a b e l e d p r o t e i n s , e x c e p t H g - m e t a l l o t h i o n e i n , a r e p r e c i p i t a t e d w i t h t r i c h l o r o a c e t i c a c i d (TCA) and t h e amount o f m e t a l l o t h i o n e i n i s c a l c u l a t e d f r o m

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t h e r a d i o a c t i v i t y o f Hg i n t h e T C A - s o l u b l e f r a c t i o n . The method depends on 203

t h e a s s u m p t i o n s t h a t Hg b i n d s q u a n t i t a t i v e l y t o a l l s u l f h y d r y l g r o u p s o f m e t a l l o t h i o n e i n , t h a t low m o l e c u l a r w e i g h t l i g a n d s , p r e s e n t i n t h e T C A - s o l u b l e

203