Inorganic Chemistry in Biology and Medicine pdf

Martell, EDITOR Texas A&M University Based on a symposium sponsored by the Division of Inorganic Chemistry at the 178th Meeting of the American Chemical Society, Washington, D.C., Se

Inorganic Chemistry

in Biology and Medicine

Arthur E Martell, EDITOR

Texas A&M University

Based on a symposium sponsored by the Division of Inorganic Chemistry

at the 178th Meeting of the American Chemical Society, Washington, D.C.,

September 10-11, 1979

AMERICAN CHEMICAL SOCIETY

Library of Congress CIP Data

Inorganic chemistry in biology and medicine

(ACS symposium series; 140 ISSN 0097-6156)

Includes bibliographies and index

1 Metals in the body—Congresses 2 Metals—

Therapeutic use—Congresses 3

Cancer—Chemother-apy—Congresses 4 Chelation therCancer—Chemother-apy—Congresses

5 Chemistry, Inorganic—Congresses

I Martell, Arthur Earl, 1916- II American

Chemical Society Division of Inorganic Chemistry

III Series IV Series: American Chemical Society

ACS symposium series; 140

QP532.I56 616 80-23248

ISBN 0-8412-0588-4 ACSMC8 140 1-436 1980

Copyright © 1980

American Chemical Society

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F Sherwood Rowland Alan C Sartorelli Raymond B Seymour Gunter Zweig

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PREFACE

A t its inception, the original plan for this symposium was to emphasize the medical aspects of inorganic chemistry, rather than to go over once more new developments in bioinorganic chemistry, important as the subject is, since the latter topic has been treated many times in recent symposia reviews and monographs The objectives of this symposium were

to review and interpret the remarkable advances that have occurred recently

in medical inorganic chemistry and to stimulate interest on the part of inorganic chemists to become involved in the developing research problems

in this area The interaction

functions of metal ions in physiological systems are very complex, and the precise nature of these interactions and processes are, for the most part, unknown In addition to the applications of metal ions and complexes for medical purposes, extensive fundamental studies are needed to understand the basis of these applications and thereby make it possible to carry out systematic improvements in current methods as well as to develop new approaches in this interesting field

Of the approximately eighty metallic elements, a considerable number have been identified as essential to life; many others have been indicated

as possibly essential, while a large number of metals are of concern because

of toxic effects that result when they are introduced into the body dentally or through environmental influences Major metal ions such as

acci-Na + , K + , M g 2 + , and C a 2 + are important in maintaining electrolyte tion in body fluids or as skeletal constituents Many of the transition metal ions are essential in trace amounts for the activation of enzyme systems In many cases, these essential metal ions become toxic or even carcinogenic when present at sufficient levels to overwhelm the natural ligands and macromolecules that function as carriers for these ions, and thus more than saturate the normal physiological processes for their control Under such conditions, they may function, as do many unnatural toxic metals, by reacting with other biomolecules, distorting or blocking their essential functions In many cases, the differences between the essential and toxic levels are surprisingly narrow This duality of behavior between natural and toxic levels constitutes the basis of threshold concentrations for several carcinogenic metals—below which these metals exist as essential and noncarcinogenic compounds It also provides a strong refutation of the validity of the linear extrapolation method still in active use for the interpretation of carcinogenicity of compounds observed at high concentra-

concentra-The topics covered in this symposium were selected so as to provide examples of current and potential medical applications of metal compounds The emphasis and amount of attention given were in many cases not in proportion to the importance or activity levels of these applications, for a number of reasons The use of platinum complexes for the treatment of cancer is perhaps under-represented because several symposia, some of which have been published, have been held on this subject in recent years Similarly, iron nutrition, although very important, has been omitted because

it is well covered by periodic and continuing conferences and conference proceedings devoted entirely to this field of research New developments of ionophores and on the use of chelating agents for the removal of radioactive metals from the body were not given the attention that they deserve in this symposium because these subjects were treated in separate symposia at the same American Chemical

Because of the large number and complexity of the functions of metal ions in physiological systems, the applications of complexes of both essential and unnatural metal ions for medical purposes are expected to expand dramatically in the next decade It is hoped that this book will help to attract more inorganic chemists to this field, to provide the expertise in coordination chemistry needed for the achievement of significant new developments in this potentially important area of medicine

The Editor wishes to express his appreciation for the many helpful suggestions received from professional colleagues during the formative stages of this symposium Special thanks are due to L G Marzilli for assistance with subject matter planning, and to J H Timmons for valuable editorial assistance

Texas A & M University

College Station, Texas

August 7, 1980

A E MARTELL

1 Molecular and Biological Properties of Ionophores

BERTON C PRESSMAN, G E O R G E PAINTER, and M O H A M M A D FAHIM

Department of Pharmacology, University of Miami, Miami, F L 33101

The ionophores ar

cations across low polarity barriers such as organic solvents and

l i p i d s (1) From a biological standpoint, the most important low polarity barrier is the l i p i d bilayer which l i e s within biological membranes; ionophores possess unique and potent biological proper- ties which derive from their a b i l i t y to perturb transmembrane ion gradients and e l e c t r i c a l potentials Each ionophore has i t s own characteristic ion s e l e c t i v i t y pattern arising from the interac- tion between the conformational options of the host ionophore and the effective atomic radius and charge density of the guest

cation The a b i l i t y of ionophores to complex and transport

cations has an ever growing l i s t of applications i n experimental biology and technology and may ultimately provide the basis for novel cardiovascular drugs Ionophores are also intriguing i n t e l - lectually as objects for study of chemical and physical complexa- tion processes at the molecular level and as challenges to the

state of the art of c h i r a l l y selective organic synthesis (2)

Several reviews are available for expanding the description of ionophores provided here (3,4,5)

General Structural Features of Ionophores

Several of the general structural features of ionophores are

i l l u s t r a t e d i n Figure 1 A l l ionophores deploy an array of

liganding oxygen atoms about a cavity i n space into which the plexed cation f i t s X-ray crystallography reveals that the p r i n - cipal bonding energy is provided by induced dipolar interaction between the complexed cation and those specific oxygens which are filled i n

com-Valinomycin consists of alternating residues of hydroxyacids and aminoacids constituting a cyclic dodecadepsipeptide In space the ring undulates defining a bracelet 4 Å high and 10 Å in diam- eter The liganding oxygens, the ester carbonyls, form a three

4 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

VANCOMYCIN ENNIATIN B MACROLIDE ACTINS

Figure 1 Structures of representative ionophores The oxygen atoms that x-ray crystallography indicates to be primarily involved in liganding to cations are filled in

1 PRESSMAN E T A L Properties of Ionophores 5

d i m e n s i o n a l cage which accommodates K + ( r = 1.33 X) much more snugly than N a + ( r = 0.95 ft) r e s u l t i n g i n a K + : N a + p r e f e r e n c e o f 10,000:1 ( 4 )

E n n i a t i n B i s a c y c l i c h e x a d e p s i p e p t i d e ; the s m a l l e r r i n g

r e s u l t s i n a r e l a t i v e l y p l a n e r a r r a y of l i g a n d i n g oxygen atoms; the more open and more f l e x i b l e cage r e s u l t s i n a K + : N a + d i s c r i m i -

o r e t h y l s (monactin, d i n a c t i n , t r i n a c t i n , t e t r a n a c t i n ) ( 7 )

While the aforementioned ionophores a r e Streptomyces

metabo-l i t e s , the crown p o metabo-l y e t h e r s , the d e p i c t e d p r o t o t y p e of which i s dicyclohexyl-18-crown-6 a r e s y n t h e t i c ( 8 ) Although they l a c k the i n t r i c a t e conformation

m u l t i p l e asymmetric carbo

e r t i e s a r e analogous While they a r e l e s s e f f i c i e n t i o n c a r r i e r s ,

t h e i r l a c k of l a b i l e l i n k a g e s confers i n c r e a s e d chemical s t a b i l i t y ; they f i n d e x t e n s i v e use i n o r g a n i c s y n t h e s i s f o r s o l u b i l i z i n g

e l e c t r o l y t e s , e.g e n o l a t e s , i n nonpolar s o l v e n t s thereby p r o

d i s t i n c t i o n i s fundamental f o r e x p l a i n i n g the profound d i f f e r e n c e s

i n b i o l o g i c a l b e h a v i o r of the ionophore s u b c l a s s e s , hence we p r e

-f e r c a r b o x y l i c ionophore to the term p o l y e t h e r a n t i b i o t i c used by Westley ( 5 ) The l a t t e r term, furthermore, l e a d s to f u n c t i o n a l ambiguity w i t h the e t h e r e a l m a c r o l i d e n a c t i n s and crown p o l y e t h e r s which a r e n e u t r a l ionophores

The n a t u r a l l y o c c u r r i n g c a r b o x y l i c ionophores, t y p i f i e d by monensin, l a c k the s t r u c t u r a l redundancy of the n e u t r a l i o n o -

phores Monensin c o n s i s t s of a f o r m a l l y l i n e a r a r r a y of h e t e r o

-c y -c l i -c e t h e r - -c o n t a i n i n g r i n g s , however the m o l e -c u l a r -c h i r a l i t y

a r i s i n g from the r i n g s and asymmetric carbons f a v o r s the molecule assuming a q u a s i - c y c l i c c o n f i g u r a t i o n A d d i t i o n a l s t a b i l i z a t i o n

of the r i n g i s c o n f e r r e d by h e a d - t o - t a i l hydrogen bonding I n

a d d i t i o n to i t s l i g a n d i n g ether oxygens, monensin has a p a i r o f

6 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

Dynamics o f Ionophore-Mediated T r a n s p o r t

N e u t r a l Ionophores The r e l a t i o n s h i p between e q u i l i b r i u m ionophore a f f i n i t i e s and dynamic b i o l o g i c a l transmembrane t r a n s -

p o r t i s d e t a i l e d i n F i g u r e 2 The t r a n s p o r t c y c l e c a t a l y z e d by

n e u t r a l ionophores i s g i v e n on the l e f t Ionophore added to a

b i o l o g i c a l membrane p a r t i t i o n s predominately i n t o the membrane A

p o r t i o n o f the ionophore d i f f u s e s to the membrane i n t e r f a c e where

i t encounters a hydrated c a t i o n A l o o s e encounter complex i s formed f o l l o w e d by replacement of the c a t i o n i c h y d r a t i o n sphere by engulfment o f the c a t i o n by the ionophore The dehydrated com-

p l e x i s l i p i d - s o l u b l e and hence can d i f f u s e a c r o s s the membrane The c a t i o n i s then rehydrated, r e l e a s e d , and the uncomplexed i o n o - phore f r e e d to r e t u r n to i t s i n i t i a l s t a t e w i t h i n the membrane The n e t r e a c t i o n c a t a l y z e d i s the movement o f an i o n w i t h i t s

charge a c r o s s the membrane

-systems but a l s o t h e i r transmembrane e l e c t r i c a l p o t e n t i a l s S i n c e the l a t t e r a r e so important i n b i o l o g i c a l c o n t r o l , i t i s not s u r -

p r i s i n g t h a t the n e u t r a l ionophores a r e e x c e e d i n g l y t o x i c towards

be h y d r a t e d and removed from the complex The r e s u l t a n t h i g h l y

p o l a r I " i s o b l i g e d to remain a t the i n t e r f a c e u n t i l a new charge

p a r t n e r , r e p r e s e n t e d by N+'R^O, a r r i v e s Once i n p o s i t i o n , N +

1 PRESSMAN E T A L Properties of Ionophores 1

exchanges i t s s o l v a t i o n H2O f o r the oxygen l i g a n d i n g system of I forming l i p i d compatible N+.I" which then d i f f u s e s a c r o s s the membrane There the process i s r e v e r s e d and N + i s exchanged f o r M+ The ionophore then r e e n t e r s the membrane as M+I"" thereby completing the c a t a l y t i c c y c l e The net r e a c t i o n i s the movement

of N+ a c r o s s the membrane i n exchange f o r M + without an ing net charge t r a n s l o c a t i o n T h i s i s presumably an e s s e n t i a l requirement f o r t o l e r a n c e o f a p p r e c i a b l e c o n c e n t r a t i o n s of i o n o - phores by animals, i e c a r b o x y l i c ionophores are r e l a t i v e l y non-

accompany-t o x i c compared accompany-to n e u accompany-t r a l ionophores In o accompany-t h e r words, accompany-the a b i l i accompany-t y

of c a r b o x y l i c ionophores to a l t e r p h y s i o l o g i c a l p r o c e s s e s i n a

p h a r m a c o l o g i c a l l y u s e f u l manner stems from t h e i r c a p a b i l i t y to

a l t e r transmembrane i o n g r a d i e n t s without d i r e c t l y s h o r t c i r c u i t ing the transmembrane p o t e n t i a l s o f e l e c t r i c a l l y a c t i v e c e l l s The f o r m a t i o n and d i s s o c i a t i o n of i o n o p h o r e - c a t i o n complexes

-i s e q u -i v a l e n t to the d-isplacement of the pr-imary c a t -i o n s o l v a t -i o n sphere by the ionophore

groups approach the s o l v a t e

They then i n t e r a c t v i a an a s s o c i a t i v e i n t e r c h a n g e mechanism a n a l o gous to an S N 2 mechanism (12) Formation of the t r a n s i t i o n s t a t e

-i n v o l v e s e x t e n s -i o n o f the c a t -i o n to both the e n t e r -i n g l -i g a n d and the d e p a r t i n g c a t i o n s o l v a t i o n sphere In the p r o c e s s , the l e s s

r i g o r o u s l y d e f i n e d s o l v a t i o n sphere o f the l i g a n d i s a l s o d i s charged The ionophore then e n g u l f s the c a t i o n , i t s l i g a n d i n g groups p r o g r e s s i v e l y d i s p l a c i n g the molecules o f the c a t i o n s o l v a -

-t i o n s h e l l i n a c o n c e r -t e d f a s h i o n In -the case o f -the c a r b o x y l i c ionophores, the i n i t i a l stage p r i o r to the f o r m a t i o n o f the t r a n s -

i t i o n complex i s a simple i o n p a i r

Although they v a r y w i d e l y i n s t r u c t u r e and conformation, the

c a r b o x y l i c ionophores f e a t u r e a v a r i e t y o f heteroatoms c o n s t i t u t ing a l i g a n d i n g system which o p e r a t e s by means of induced d i p o l e s The magnitude o f the d i p o l e s i n c r e a s e s p r o g r e s s i v e l y by i n d u c t i o n

-as approached by the c a t i o n and u l t i m a t e l y produces a s o l v a t i o n system s t r o n g e r than t h a t o f the b u l k phase s o l v e n t Whereas the

i n d i v i d u a l s o l v a t i o n m o l e c u l e s , w i t h i n the primary s o l v a t i o n sphere o f a c a t i o n , exchange independently w i t h the b u l k s o l v e n t , the l i g a n d s o f an ionophore, h e l d together by a common backbone, must behave i n a c o o p e r a t i v e manner I n t r a m o l e c u l a r hydrogen bonding and s u b s t i t u e n t s which f a v o r c y c l i c conformations ( e g

s p i r a n e systems) promote the s t a b i l i t y o f complexes Consequently, the v a r i o u s c a t i o n a f f i n i t y and s e l e c t i v i t y p a t t e r n s which c h a r a c -

t e r i z e each ionophore a r i s e from the p r e c i s e s p a c i a l depolyment o f

l i g a n d i n g heteroatoms as determined by m o l e c u l a r conformation (13,14)

Conformational S t u d i e s of a R e p r e s e n t a t i v e C a r b o x y l i c

Ionophore, S a l i n o m y c i n

S a l i n o m y c i n , a r e p r e s e n t a t i v e c a r b o x y l i c ionophore ( F i g u r e 3) (15), i s a p a r t i c u l a r l y s u i t a b l e model f o r s t u d y i n g the dynamic

8 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

Neu-the text

1 PRESSMAN E T AL Properties of Ionophores 9

c o n f o r m a t i o n a l aspects of complexation The c i r c u l a r d i c h r o i s m (CD) a r i s i n g from the n -> TT* t r a n s i t i o n of the C - l l carbonyl i s

s e n s i t i v e to m o l e c u l a r environment and serves as a probe to r e p o r t the c h i r a l i t y i n i t s v i c i n i t y CD enables us to e v a l u a t e the

c o n f o r m a t i o n a l p e r t u r b a t i o n s produced by a l t e r i n g the p o l a r and

p r o t i c p r o p e r t i e s of the s o l v e n t system Systematic p e r t u r b a t i o n

of the s o l u t i o n conformation of s a l i n o m y c i n by an a p p r o p r i a t e

c h o i c e of s o l v e n t s r e v e a l s that i o n a f f i n i t y and s e l e c t i v i t y are

v a r i a b l e , c o n f o r m a t i o n a l l y determined, p r o p e r t i e s

R e p r e s e n t a t i v e CD s p e c t r a of protonated s a l i n o m y c i n , i t s K +

complex and i t s uncomplexed a n i o n a r e presented i n F i g u r e 4 No

s i g n i f i c a n t s h i f t o f the n e g a t i v e 2 9 0 nm peak occurs with solvent change or l i g a n d i n g s t a t e ; Beer's law i s obeyed from 1 0 " ^ to

1 0 " " 6 M The f u n c t i o n most s u i t a b l e f o r r e l a t i n g CD spectra to the conformation of a molecule i s the r o t a t i o n a l s t r e n g t h ( R£) of the observed e l e c t r o n i c t r a n s i t i o ( 1 6 ) Sinc th Gaussia approxi mation appears to h o l d f o

F i g u r e 5 i l l u s t r a t e s the e f f e c t of solvent changes on the R£ of the ionophore f r e e a c i d and i t s anion Kosower's Z values proved e m p i r i c a l l y an e f f e c t i v e f u n c t i o n f o r r a n k i n g s o l v e n t s

a c c o r d i n g to t h e i r i n t e g r a t e d p o l a r and p r o t i c p r o p e r t i e s ( 1 8 )

The | R J | of the free a c i d decreases l i n e a r l y with a small p o s i t i v e

s l o p e as the Z values r i s e In contrast, the | R Q | of the complexed a n i o n , the s p e c i e s p a r t i c i p a t i n g i n complexation, drops

un-s h a r p l y between Z values of 8 0 and 8 3 , varying l i t t l e above and below these v a l u e s Thus, the conformation of the a n i o n tends toward one of two metastable s t a t e s depending upon s o l v e n t Z

v a l u e

The r o l e of the s o l v e n t i n determining e q u i l i b r i u m s o l u t i o n conformation can b e s t be understood i n terms o f f u n c t i o n a l group

s t a b i l i z a t i o n In p o l a r p r o t i c media the e q u i l i b r i u m conformation

of the uncomplexed a n i o n i c ionophore i s determined by the s o l v a

-t i o n of -the c a r b o x y l a -t e a n i o n and -the p o l a r l i g a n d i n g groups Thus, two d i s t i n c t s o l v e n t e f f e c t s are o p e r a t i v e , s o l v a t i o n o f the

p o l a r l i g a n d i n g groups r e s u l t i n g i n c o n f o r m a t i o n a l s t a b i l i z a t i o n due to decreased d i p o l e - d i p o l e r e p u l s i o n and maximization of the

s o l v a t i o n energy of the a n i o n The protonated ionophore responds

o n l y to the s o l v a t i o n of p o l a r l i g a n d i n g groups Thus, F i g u r e 5

p r o v i d e s i n s i g h t i n t o the r e l a t i v e importance of each o f these

f a c t o r s i n determining e q u i l i b r i u m s o l u t i o n conformation The

p e r t u r b a t i o n of conformation due to s o l v a t i o n of p o l a r l i g a n d i n g groups a l o n e , as i n the protonated ionophore, causes o n l y a s l i g h t change i n conformation, i e a s m a l l change i n |Rol> over a l a r g e range of Z values However, i o n i z a t i o n of the protonated form of the ionophore p r o f o u n d l y changes i t s response to s o l v e n t s A t Z

v a l u e s > 8 3 , the carboxylate i s s t a b i l i z e d by i t s p r o t i c , polar environment The r e s u l t i n g s o l v a t i o n sphere i n f l u e n c e s the con- formation s t r o n g l y as evidenced by the v e r y low | R Q | values

( F i g u r e 3 ) As the Z values f a l l , and the solvent becomes l e s s

10 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

Figure 4 CD spectra of the carboxylic

acid free anion and K + complex forms of

salinomycin The free anionic form was

generated by the addition of excess

tri-n-butylamine and the K + complex by the

addition of excess KSCN

M

10* -a

WmLiKT* (urn)

Figure 5 Rotational strengths of the

carboxylic acid and free anion forms of

salinomycin as a function of solvent Z

values

Figure 6 K + :Na* selectivity (l/K DNa +:

1/K Dk +) of salinomycin as a function of

solvent Z value

1 PRESSMAN E T A L Properties of Ionophores 11

a b l e to s t a b i l i z e the charge, s t a b i l i z a t i o n i s a c h i e v e d by a t i g h t

h e a d - t o - t a i l (C^-O-LyH) hydrogen bond The f o r m a t i o n of t h i s bond

r e s u l t s i n a compression of the l i g a n d i n g c a v i t y , the l i m i t of which i s determined by d i p o l e - d i p o l e r e p u l s i o n A p p l i c a t i o n o f the Octant Rule (16) to computer models o f the a n i o n c o r r o b o r a t e s

t h a t t i g h t e n i n g of the h e a d - t o - t a i l bond s h o u l d be accompanied by

a concomitant i n c r e a s e i n |R Q |

F i g u r e 4 i n d i c a t e s that CD can be employed to determine

com-p l e x a t i o n K D T s (see T a b l e I ) The r a t i o o f the Na + :K+ K D f s , i e

K + : N a + s e l e c t i v i t y , a l s o shows a sharp s h i f t between Z v a l u e s o f

80 and 83 ( c f F i g u r e 6 ) Thus, the a b i l i t y of the complexing form o f the ionophore to d i s c r i m i n a t e between i o n s depends

s t r o n g l y upon environmental i n f l u e n c e s on c o n f o r m a t i o n Changes

i n i n t e r - l i g a n d d i s t a n c e s and l i g a n d o r i e n t a t i o n s e f f e c t e d by changes i n ionophore conformation m a n i f e s t themselves by a d e t e r -

m i n a t i v e a l t e r a t i o n o f the f r e e energy o f complexation

CD was u t i l i z e d t

f o r m a t i o n of the c a t i o n - i o n o p h o r

t i o n isotherms were p l o t t e d from l i n e a r computer f i t s o f

1 / [ c a t i o n ] v e r s u s 1 / A R £ ; the slopes y i e l d e d Kpj's while e x t r a p o l a

-t i o n of R J to i n f i n i t e c a t i o n c o n c e n t r a t i o n provided the R^'s of

the c a t i o n - s a t u r a t e d ionophore I t i s important to note t h a t the

c a t i o n i t s e l f i s a s i g n i f i c a n t v i n c i n a l moiety, which by v i r t u e o f

i t s charge, p o l a r i z a b i l i t y and l o c a t i o n w i t h r e s p e c t to the

chromophore o f concern, can modify the r o t a t i o n a l s t r e n g t h of the chromophore

Comparison of the | R £ | values f o r the N a + and K + complexes o f

s a l i n o m y c i n i n T a b l e I w i t h the | R J | values f o r salinomycin anion

i n F i g u r e 5 shows an i n c r e a s e i n the magnitude of | R £ | upon

com-p l e x a t i o n i n a l l s o l v e n t s T h i s correscom-ponds to a change i n

con-f o r m a t i o n upon complexation, i e r e o r i e n t a t i o n ocon-f the ionophore about the c a t i o n A p p l i c a t i o n of the Octant Rule to computer generated models o f s a l i n o m y c i n i n d i c a t e s t h a t t h i s r e o r i e n t a t i o n

i s a c o n s t r i c t i o n of the l i g a n d i n g oxygens which surround the

l e a s t to the o t h e r n a t u r a l l y o c c u r r i n g c a r b o x y l i c ionophores The

i n f l u e n c e of ionophore environment, e.g s o l v e n t , on ionophore conformation i s p a r t i c u l a r l y s i g n i f i c a n t when c o n s i d e r i n g the

environmental continuum encountered by an ionophore when t r a n s

-v e r s i n g a b i o l o g i c a l membrane

1 PRESSMAN E T A L Properties of Ionophores 13

The e x t e n s i o n to ionophore s e l e c t i v i t y o f a h y p o t h e s i s based

on analogy w i t h the r i g i d m a t r i c e s of i o n s e l e c t i v e g l a s s e s (19)

i s i n c o n s i s t e n t w i t h the dynamic c o n f o r m a t i o n a l aspect of i o n

s e l e c t i v i t y developed i n the p r e s e n t paper Furthermore, the

c o n f o r m a t i o n a l o p t i o n s of ionophores are not n e c e s s a r i l y a graded

f u n c t i o n of environmental p o l a r i t y but may d i s p l a y sudden s h i f t s between metastable s t a t e s over narrow p o l a r i t y ranges E l e c t r o -

s t a t i c i n t e r a c t i o n s between i o n s and induced d i p o l e s undoubtedly

p l a y a d e t e r m i n a t i v e r o l e i n c a t i o n complexation by ionophores, but the a b i l i t y of the ionophore to a l t e r i t s conformation cannot

be i g n o r e d as i t i s i n the assumption o f i s o s t e r i s m ( 1 9 )

P h a r m a c o l o g i c a l P r o p e r t i e s of C a r b o x y l i c Ionophores

P h a r m a c o l o g i c a l E f f e c t s Although both n e u t r a l and

carboxy-l i c ionophores have bee e x t e n s i v e carboxy-l empcarboxy-loyed t o o l f o i

v i t r o s t u d i e s of b i o l o g i c a

v i o u s l y , o n l y the c a r b o x y l i ionophore y

by i n t a c t animals to produce w e l l d e f i n e d p h a r m a c o l o g i c a l

responses We i n i t i a l l y examined the c a r d i o v a s c u l a r e f f e c t s of

l a s a l o c i d because o f i t s a b i l i t y to t r a n s p o r t the key b i o l o g i c a l

c o n t r o l agents, C a ^ + and catecholamines (20,21) However, we

l a t e r d i s c o v e r e d t h a t c a r b o x y l i c ionophores s e l e c t i v e f o r a l k a l i ions were even more potent i n evoking the same responses (22)

F i g u r e 7 i l l u s t r a t e s the two d i s t i n c t primary c a r d i o v a s c u l a r

e f f e c t s produced by monensin A t low c o n c e n t r a t i o n s , 50 yg/kg,

i t produces a d i r e c t d i l i t a t i o n , i e r e l a x a t i o n of the smooth muscle of the coronary a r t e r i e s , m a n i f e s t e d by a m u l t i f o l d i n - crease i n coronary b l o o d flow A t t h i s l e v e l or below, no o t h e r

e f f e c t s o c c u r I f the dose i s i n c r e a s e d to 0.2 mg/kg, an i n o

-t r o p i c response f o l l o w s -the i n i -t i a l coronary d i l i -t a -t i o n T h i s response, an i n c r e a s e i n c a r d i a c c o n t r a c t i l i t y , can be monitored

as the maximum r a t e of r i s e of p r e s s u r e i n the l e f t v e n t r i c l e ,

LV dP/dt max Other parameters p a r a l l e l the i n o t r o p i c e f f e c t

F o l l o w i n g an i n i t i a l drop caused by d i l i t a t i o n of the systemic

a r t e r i e s , mean b l o o d p r e s s u r e r i s e s as does p u l s e p r e s s u r e , the

i n t e r v a l between lowest ( d i a s t o l i c ) and h i g h e s t ( s y s t o l i c ) t r a n

-s i e n t p r e -s -s u r e -s ; the r a t e of b l o o d pumped by the h e a r t ( c a r d i a c output) a l s o r i s e s

The two d i s t i n c t e f f e c t s are thus an i n c r e a s e i n coronary flow, which r a p i d l y f o l l o w s i n j e c t i o n o f the ionophore, f o l l o w e d

by an i n o t r o p i c response, which o n l y appears a t h i g h e r doses The r e s o l u t i o n by dosage of the two ionophore responses i s

c l e a r l y apparent i n the dose-response p l o t of F i g u r e 8 Coronary flow r i s e s p r o g r e s s i v e l y u n t i l i t p l a t e a u s a t 10-50 yg/kg

monensin Higher doses cause a secondary i n c r e a s e i n flow r e

-f l e c t i n g the r i s e i n a t r i a l p r e s s u r e which d r i v e s b l o o d through the c o r o n a r i e s Only 2.5 yg/kg ( i e 2.5 ppb) a r e s u f f u c i e n t to double the b a s a l flow r a t e I t i s p o s s i b l e to d e t e c t the i n - creased flow of 1 yg/kg (1 ppb) w i t h s t a t i s t i c a l c o n f i d e n c e

14 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

1.15 Ml/Kg M Bt/Kl

Figure 7 Cardiovascular response of a typical anesthetized dog to monensin A low dose (0.05 mg/kg) was

interval of an hour to permit

(0.2 mg/kg) was administered The lowest tracing (mean LAD CF.) is the averaged flow measured by a magnetic flow probe encircling the left anterior descending coronary artery The AP trace gives the diastolic-systolic pressure range recorded from a catheter in the aorta LV dP/dt max, the index of cardiac con- tractility, was obtained from a manometer-tipped catheter inserted in the left ventricle The measured pressure was converted to its derivative to record dP/dt

1 PRESSMAN E T A L Properties of Ionophores 15

Mechanism of the P h a r m a c o l o g i c a l E f f e c t s T a b l e I I compares the jln v i t r o i o n c a r r y i n g c a p a c i t y of a s e r i e s of ionophores w i t h

t h e i r i n o t r o p i c potency A p p r e c i a b l e r a t e s o f C a 2 + o r c a t e c h o l amine (norepinephrine) t r a n s p o r t a r e observed o n l y f o r l a s a l o c i d , the ionophore of the group w i t h the poorest i n o t r o p i c potency Extremely wide ranges of C a 2 + and n o r e p i n e p h r i n e t r a n s p o r t

-c a p a -c i t y are seen w i t h no -c o r r e l a t i o n w i t h i n o t r o p i -c poten-cy The

C a 2 + - s e l e c t i v e A-23187 g i v e s o n l y a s p o r a t i c i n o t r o p i c response

w i t h the i n t a c t dog The c o r r e l a t i o n between i n o t r o p i c potency and N a + t r a n s p o r t c a p a c i t y i s l e s s n e g a t i v e and i s w i t h i n the realm of l i k e l y d i f f e r e n c e s between the p r o p e r t i e s of the e x p e r i - mental s o l v e n t b a r r i e r system and those of a c t u a l b i o l o g i c a l mem- branes When the a c t i v i t i e s of ionophores are compared on the

b a s i s o f the q u a n t i t y r e q u i r e d to r e l e a s e a standard amount o f K +

from e r y t h r o c y t e s , c h i e f l y i n exchange f o r N a + , the c o r r e l a t i o n with i n o t r o p i c potency i s even b e t t e r

i e n t l y measured r e l e a s e of K+, i s more s i g n i f i c a n t than the l o s s

of K + per se An a d d i t i o n a l f a c t o r i s t h a t d i f f e r e n t b i o l o g i c a l membranes, e.g e r y t h r o c y t e s and m i t o c h o n d r i a , respond d i f f e r e n t l y

to ionophores (23) A l l t h i n g s taken i n t o c o n s i d e r a t i o n , the data

of Table I I are reasonably s u p p o r t i v e o f a mechanism o f a c t i o n of ionophores i n v o l v i n g i n i t i a t i o n of an i n c r e a s e i n i n t r a c e l l u l a r

-Increased i n t r a c e l l u l a r C a 2 + a c t i v i t y a l s o a c t i v a t e s s e c r e tory c e l l s (25) I n h i b i t i o n s t u d i e s i n d i c a t e that the i n o t r o p i c

e f f e c t of monensin i s mediated i n p a r t by the r e l e a s e of c a t e c h o l amines from the a d r e n a l s and/or the h e a r t i t s e l f (22) Monensin

-a l s o d i s c h -a r g e s c-atechol-amines from d i s -a g g r e g -a t e d b o v i n e chrom-af-

chromaff i n c e l l s i n c u l t u r e (26,27), and induces the r e l e a s e ochromaff a c e t y l

-c h o l i n e a t the neuromus-cular j u n -c t i o n (28) Thus, the s e -c r e t i o n

s t i m u l a t o r y a c t i v i t y of monensin a l s o supports the concept t h a t

1 PRESSMAN E T A L Properties of Ionophores 17

l a r C a 2 + are p l a u s i b l e One would be an e x c h a n g e - d i f f u s i o n

c a r r i e r i n the plasma membrane p e r m i t t i n g the l a r g e C a 2 + a c t i v i t y

g r a d i e n t (a 10"3 M e x t r a c e l l u l a r , a 10" 7 M i n t e r i o r ) to permit

e n t r y of C a 2 + i n t o the c e l l i n exchange f o r N a + (On thermodynamic grounds one would expect the exchange r a t i o to be 3-4 N a + e x p e l l e d

f o r each C a 2 + taken up) Thus, making more i n t r a c e l l u l a r N a +

a v a i l a b l e f o r exchange, or i n thermodynamic terms reducing the

g r a d i e n t a g a i n s t which N a + must move (a 1 0 ~ 2 M i n t r a c e l l u l a r , a 10-1 M e x t r a c e l l u l a r ) , would favor the entry of C a 2 + A c r i t i c a l

e v a l u a t i o n of t h i s h y p o t h e s i s has appeared i n a r e c e n t review (29) An a l t e r n a t e mechanism would be the r e l e a s e of i n t r a c e l l u -

l a r ^ bound C a 2 + by displacement by Na+ T h i s i s f e a s i b l e s i n c e the gross chemical C a 2 + i n t r a c e l l u l a r c o n c e n t r a t i o n i s c a 10"" 3 M

C a r b o x y l i c Ionophores and E f f i c i e n c y of Feed C o n v e r s i o n by

L i v e s t o c k A s t r o n g note of r e l e v a n c e to s t u d i e s of the chemical and p h a r m a c o l o g i c a l p r o p e r t i e s of c a r b o x y l i c ionophores d e r i v e s from the l a r g e s c a l e use of monensin as a l i v e s t o c k f e e d a d d i t i v e The r a t i o n a l e i s t h a t c a r b o x y l i c ionophores c o n t r o l endemic

c o c c i d i o s i s i n the p o u l t r y gut (30) and promote a more f a v o r a b l e

f e r m e n t a t i o n of c e l l u l o s e i n the b o v i n e rumen (31) In e i t h e r case, the net r e s u l t i s the e c o n o m i c a l l y important i n c r e a s e d

e f f i c i e n c y of c o n v e r s i o n o f feed i n t o meat

P h a r m a c o k i n e t i c s of Ionophore A b s o r p t i o n We have developed

a s e n s i t i v e chemical assay f o r c a r b o x y l i c ionophores (which w i l l

be p u b l i s h e d elsewhere) based on t h e i r a b i l i t y to form l i p i d s o l u

-b l e complexes w i t h c a t i o n s We can d e t e c t as l i t t l e as 1 p a r t per

b i l l i o n (ppb) monensin i n 2 ml of blood plasma o r t i s s u e For a comparison y a r d s t i c k , c u r r e n t f e e d i n g regimens c a l l f o r ca 30

p a r t s per m i l l i o n (ppm) i n c a t t l e feed (32) and as much as 100 ppm

i n p o u l t r y feed (33)

T y p i c a l l y , a cow i n g e s t s about 0.3 g (^ 1 ppm) monensin/day

As p r e v i o u s l y observed i n F i g u r e 7, as l i t t l e as 1 ppl> (based on body weight) produces a d e t e c t a b l e p h y s i o l o g i c a l e f f e c t on the dog

In o r d e r to e s t a b l i s h the pharmacokinetic r e l a t i o n s h i p s

between o r a l l y i n g e s t e d and i n t r a v e n o u s l y i n j e c t e d monensin, we

c a r r i e d out p r e l i m i n a r y s t u d i e s of monensin b l o o d l e v e l s i n the dog In F i g u r e 9 we see t h a t i n j e c t e d monensin c l e a r s from the plasma w i t h a t ^ of ^ 2.5 minutes which we presume i s too r a p i d

f o r the o p e r a t i o n of normal e l i m i n a t i o n mechanisms Hence, i t i s

18 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

MIREHSIR f 1IMI/K(

I.I IKE

2SII 20N it!i\

ISM sec

1000

2**1/11 HAL USE

60 90 120 ISO MINUTES AFTER 00SE

Figure 9 Pharmacokinetics of monensin in the dog In the upper trace, 100 fig/kg monensin was injected into a barbiturate-anesthetized dog with a manometer-tipped catheter in the left ventricle to measure dP/dt Blood samples were taken at various periods and 2 mL samples of plasma obtained by centrifugation for ionophore assay Note that the monensin cleared the blood rapidly and that the cardiac responses persisted Subsequent assays revealed the monensin entered the dog tissues, par- ticularly the lungs The lower trace compares the pharmacokinetics of the injected dose with those obtained from a nonanesthetized dog that received the monensin orally (2 mg/kg) as a concentrate applied to a small quantity of feed The plasma levels obtained by administration of an oral dose approached those obtained by injection, indicating that the major portion of the oral dose passed through the

plasma and into the tissues before being eliminated

1 PRESSMAN E T A L Properties of Ionophores 19

reasonable to assume that the ionophore l e a v i n g the plasma i s

taken up by the t i s s u e s T h i s would not a t a l l be unexpected

con-s i d e r i n g the h i g h l i p i d : w a t e r p a r t i t i o n c o e f f i c i e n t of ionophorecon-s

I t i s supported by the delayed and p e r s i s t e n t e l e v a t i o n of the

i o n o p h o r e s e n s i t i v e c a r d i a c f u n c t i o n parameter, LV dP/dt P r e

-l i m i n a r y t r i a -l s of a v a r i a t i o n of our assay adapted f o r who-le

t i s s u e s i n d i c a t e that i n the r a b b i t the major p o r t i o n of monensin appears i n the t i s s u e s w i t h i n 10 minutes f o l l o w i n g i v i n j e c t i o n ,

a t c o n c e n t r a t i o n s roughly p a r a l l e l i n g the degree o f b l o o d p e r f u

-s i o n : lung > h e a r t > kidney > l i v e r , mu-scle, f a t

The lower graph of F i g u r e 8 compares the time course of pearance i n the plasma of i n j e c t e d and o r a l l y a d m i n i s t e r e d

ap-monensin doses i n the dog The o r a l dose appears i n the blood more s l o w l y but produces more s u s t a i n e d ionophore b l o o d l e v e l s The time c o n c e n t r a t i o n i n t e g r a l g i v e s an index of the q u a n t i t y of the drug which passes through the plasma; r a t e of e n t r y and c l e a r ance from the b l o o d a f f e c

net i n t e g r a l The i n t e g r a

the i n t e g r a l o f a known dose a d m i n i s t e r e d d i r e c t l y i n t o the b l o o d Although d i f f e r e n t animals and d i f f e r e n t dose l e v e l s were used, the r a t i o of the i v i o r a l dose i n t e g r a l s are approximately p r o -

p o r t i o n a l to the 1:20 r a t i o s of the net doses a d m i n i s t e r e d T h i s

s i g n i f i e s t h a t a major p o r t i o n , i f not a l l of the o r a l l y i n g e s t e d monensin dose, passes through the b l o o d stream of the dog b e f o r e being e l i m i n a t e d In the r a b b i t , a h e r b i v o r e , one might p r e d i c t

a b s o r p t i o n o f o r a l doses would be slower We can d e t e c t o r a l l y

a d m i n i s t e r e d monensin doses i n r a b b i t plasma, but o n l y a f t e r a couple of hours f o l l o w i n g i n g e s t i o n We have not y e t completed the more prolonged plasma l e v e l - t i m e p r o f i l e s i n t h i s s p e c i e s

The Need f o r Increased S u r v e i l l a n c e of the Exposure of Man

to Ionophores From the l i p i d s o l u b i l i t y of monensin and o t h e r ionophores, we would p r e d i c t they should have no t r o u b l e e q u i l i -

b r a t i n g a c r o s s b i o l o g i c a l membrane systems i n c l u d i n g the gut T h i s

i s c e r t a i n l y the case f o r the two d i v e r s e s p e c i e s observed, the dog, a c a r n i v o r e , and the r a b b i t , a h e r b i v o r e A c c o r d i n g l y , we

i n f e r that there i s ample o p p o r t u n i t y f o r monensin and o t h e r

c a r b o x y l i c ionophores administered o r a l l y to l i v e s t o c k to d i s t r i b ute s y s t e m i c a l l y and e x e r t a p h a r m a c o l o g i c a l e f f e c t on the r e c i p i - ent a n i m a l Furthermore, the r e s u l t a n t p h y s i o l o g i c a l e f f e c t s may

-be p a r t of the mechanism by which ionophores produce t h e i r proved feed c o n v e r s i o n e f f i c i e n c y

im-There are f u r t h e r i n f e r e n c e s which d i r e c t l y a f f e c t man I f the ionophores do pervade the t i s s u e s , i t i s p o s s i b l e t h a t man may become exposed to p h a r m a c o l o g i c a l l y competent and p o t e n t i a l l y

d e t r i m e n t a l l e v e l s of ionophores through h i s meat supply

Based on l i m i t e d pharmacokinetic and t o x i c o l o g i c a l data, the F.D.A has s e t upper p e r m i s s i b l e l e v e l s of 0.05 ppm i n meat f o r human consumption (34) The i s o t o p e r e s i d u e s t u d i e s of Herberg

e t a l r e p o r t t h a t under c u r r e n t f e e d i n g procedures c a t t l e l i v e r

20 INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE may accumulate over ten times t h i s l e v e l o f monensin as a combina-

t i o n o f parent compounds and m e t a b o l i t e s o f unknown

pharmacologi-c a l e f f e pharmacologi-c t s (35) T h i s d a t a was o b t a i n e d 12 hours a f t e r

adminis-t r a adminis-t i o n of adminis-tagged monensin One mighadminis-t surmise adminis-t h a adminis-t r e s i d u e s would be a p p r e c i a b l y h i g h e r f o r an animal butchered a s h o r t e r

p e r i o d of time f o l l o w i n g i t s l a s t exposure to monensin T h i s i s

p a r t i c u l a r l y s i g n i f i c a n t i n t h a t l i t e r a t u r e s u p p l i e d to farmers

a d v i s e s t h a t no withdrawal p e r i o d i s n e c e s s a r y

C u r r e n t l y a v a i l a b l e methods f o r a s s a y i n g monensin i n v o l v e cumbersome e x t r a c t i o n procedures, t h i n l a y e r chromatography and

d e t e c t i o n by means of bioautographs w i t h microorganisms whose

s e n s i t i v i t y to ionophores and t h e i r m e t a b o l i t e s (36) may o r may not p a r a l l e l mammalian s e n s i t i v i t y The simple c h e m i c a l assay method we have developed can p r o v i d e a more r a t i o n a l b a s i s f o r

a s s i g n i n g p e r m i s s i b l e r e s i d u e l e v e l s , f o r r o u t i n e l y m o n i t o r i n g products a r r i v i n g a t the market and a s c e r t a i n i n g whether s t i p u -

l a t e d ionophore withdrawa

A d d i t i o n a l c o m p l i c a t i o n

n o t a b l y poor b i o d e g r a d a b i l i t y of monensin Reports i n d i c a t e that

c a t t l e f e c a l l y e l i m i n a t e 75% o f i n g e s t e d monensin without

degrad a t i o n Furthermore, 6070% o f the monensin s u r v i v e s 10 weeks i n

c u b a t i o n a t 37° ( 3 4 ) C u r r e n t manuring p r a c t i c e s render i t p r u dent to determine whether crops o r garden produce take up s i g n i f i - cant q u a n t i t i e s of c a r b o x y l i c ionophores o r whether the o b v i o u s l y

-l a r g e s o i -l burdens o f such compounds f i n d t h e i r way i n t o water

s u p p l i e s

We have l o n g been i n t e r e s t e d i n the p o s s i b i l i t y t h a t the

c a r d i o v a s c u l a r e f f e c t s of c a r b o x y l i c ionophores c o u l d be harnessed

to p r o v i d e new drugs f o r the treatment of d i s e a s e s t a t e s such as

h e a r t f a i l u r e and shock There may, however, be s u b p o p u l a t i o n s o f man f o r whom ionophores may be p a r t i c u l a r l y t o x i c For example,

a t o x i c i n t e r a c t i o n between monensin and d i g i t a l i s on the dog

h e a r t has been r e p o r t e d ( 3 7 ) Our o r a l a b s o r p t i o n d a t a do i n d i

u t i l i z e d to d e s i g n a simple assay procedure which g i v e s promise

f o r p r o v i d i n g more r a t i o n a l safeguards f o r man i n the widespread use of ionophores i n food p r o d u c t i o n L a s t l y , i n view of the burgeoning i n c r e a s e s i n the s c a l e of commercial ionophore usage,

1 PRESSMAN E T A L Properties of Ionophores 21

i t appears urgent t h a t we i n c r e a s e our understanding i n depth o f the p h y s i o l o g i c a l and m e t a b o l i c e f f e c t s o f ionophores and t h e i r

p h a r m a c o l o g i c a l and t o x i c o l o g i c a l r a m i f i c a t i o n s

Acknowledgements

We wish to acknowledge the a s s i s t a n c e o f Ms Georgina

Del V a l l e and Mr Frank L a t t a n z i o i n the development o f the i o n o phore assay and D r s L A l l e n and M K o l b e r i n h e l p i n g program the computer s t u d i e s We are i n d e b t e d to E l i L i l l y f o r samples o f monensin and A.H Robbins and Kaken Chemical Co (Japan)

-f o r s a l i n o m y c i n These s t u d i e s were supported i n p a r t by NIH

grant HL-23932 and a grant from the F l o r i d a A f f i l i a t e o f the American H e a r t A s s o c i a t i o n

Literature Cited

1 Pressman, B.C.; Harris,

Proc Natl Acad Sci U.S.A., 1969, 58, 1949-1956

2 Fukuyama, T.; Akasaka, K.; Karanewsky, D.S.; Wang, C.-L.J.; Schmid, G.; Kishi, Y J Am Chem Soc., 1979, 101, 262-263

3 Pressman, B.C Ann Rev Biochem., 1976, 45, 501-530

4 Ovchinnikov, Yu.A.; Ivanov, V.T.; Shkrob, A.M Active Complexones"; Elsevier:New York, 1975; Vol 12

"Membrane-5 Westley, J.W "Kirk-Othmer Encyclopedia of Chemistry and Technology"; Wiley:New York, 1978; pp 47-64

6 Shemyakin, M.M., Ovchinnikov, V.T., Ivanov, V.K., Antanov, A.M., Shkrob, A.M., Mikholeva, I.I., Enstratov, A.V.;

Malenkov, G.G Biochem Biophys Res Commun., 1967, 29, 834-841

7 Hanada, M.; Nanata, Y.; Hayashi, T.; Ando, K.J Antibiotics,

1974, 27, 555-557

8 Pedersen, C.J J Am Chem Soc., 1967, 89, 7017

9 Liotta, C.L.; Harris, H.P J Am Chem Soc., 1973, 95, 225

10 Pinkerton, M.; Steinrauf, L.K J Mol Biol., 1970, 49,

533-546

11 Pressman, B.C Fed Proc., 1968, 27, 1283-1289

12 Burgess, J "Metal Ions in Solution"; Wiley:New York, 1978;

pp 318-326

13 Urry, D.W "Enzymes of Biological Membranes"; Plenum Pub Corp.:New York, 1976; Vol I; ed Martinosi, A., pp 31-69

14 Urry, D.W J Am Chem Soc., 1974, 94, 77-81

15 Kinashi, H., Ōtake, N., Yonehara, H Acta Chrystallographica,

22 INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE

19 Eisenman, G.; Ciani, S.; Szabo, G J Membrane Biol., 1969,

1, 294-345

20 Pressman, B.C Fed Proc., 1973, 32, 1698-1705

21 deGuzman, N.T.; Pressman, B.C Circulation, 1974, 69,

24 Shlafer, M.; Somani, P.; Pressman, B.C.; Palmer, R.F

J Mol Cell Cardiol., 1978, 10, 333-346

25 Douglas, W.W "Secretory Mechanisms of Exocrine Glands"; Munksgaard:Copenhagen, 1974; p 116

26 Hochman, J.; Perlman, R.L Biochem Biophys Acta, 1976, 421, 168-175

27 Rubin, R.W.; Corcoran, J.; Pressman, B.C J Cell Biol., 1979,

83, 434a

28 Kita, H., Van der

29 van Breemen, C.; Aaronson, P.; Loutzenhiser, R Pharmacol Rev., 1979, 30, 167-208

30 Shunnard, R.F.; Callender, M.E "Antimicrobial Agents in

33 Chappel, L.R.; Babcock, W.E Poultry Sci., 1979, 58, 304-307

34 Feinman, S.E.; Matheson, J.C "Draft Environmental Impact Statement: Subtherapeutic Antibacterial Agents in Animal

Feeds"; available from Hearing Clerk, Food and Drug

Administration, Room 4-65, 5600 Fishers Lane, Rockville, Maryland 20857, 1978; pp A100-A108

35 Herberg, R.; Manthey, J.; Richardson, L.; Cooley, C.;

Donoho, A J Agric Food Chem., 1978, 26, 1087-1089

36 Donoho, A.; Manthey, J.; Occolowitz, J.; Zornes, L J Agric Food Chem., 1978, 26, 1090-1095

37 Saini, R.K.; Hester, R.K.; Somani, P.; Pressman, B.C

2

Possible Functions and Medical Significance of the Abstruse Trace Metals

FORREST H NIELSEN

United States Department of Agriculture, Science and Education Administration,

Human Nutrition Laboratory, Grand Forks, ND 58202

S i n c e 1970, a numbe

metals present i n minut

e s s e n t i a l n u t r i e n t s The t r a c e metals i n c l u d e cadmium, l e a d ,

n i c k e l , t i n and vanadium F i n d i n g s suggesting t h a t cadmium,

l e a d and t i n a r e e s s e n t i a l have come from one l a b o r a t o r y (1,2,3) and have not been confirmed i n another l a b o r a t o r y Minor growth

d e p r e s s i o n i n s u b o p t i m a l l y growing r a t s was the main c r i t e r i o n

f o r demonstrating the e s s e n t i a l i t y of cadmium, l e a d and t i n

That c r i t e r i o n i s of q u e s t i o n a b l e p h y s i o l o g i c a l s i g n i f i c a n c e The evidence i s more s u b s t a n t i a l f o r the e s s e n t i a l i t y of n i c k e l and vanadium A l s o , apparent p r o g r e s s has been made i n

determining e s s e n t i a l f u n c t i o n s f o r those elements Thus, i n

t h i s chapter the p o s s i b l e m e d i c a l s i g n i f i c a n c e and e s s e n t i a l

f u n c t i o n s of n i c k e l and vanadium are emphasized

N i c k e l

E s s e n t i a l i t y N i c k e l i s an e s s e n t i a l n u t r i e n t f o r animals and p r o b a b l y f o r humans Signs of n i c k e l d e p r i v a t i o n have been

d e s c r i b e d f o r f i v e animal s p e c i e s - c h i c k , r a t , m i n i p i g , goat and sheep B r i e f l y , the s i g n s of d e f i c i e n c y i n c l u d e the

f o l l o w i n g :

I (4) r e p o r t e d t h a t the s i g n s of n i c k e l d e p r i v a t i o n i n

c h i c k s i n c l u d e d depressed l e v e l s of l i v e r p h o s p h o l i p i d s ,

o x i d a t i v e a b i l i t y o f the l i v e r i n the presence of

α-glycerophosphate, y e l l o w lipochrome pigments i n the shank s k i n ,

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

24 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

summarized r e c e n t l y , Schnegg and K i r c h g e s s n e r ( 6 ) developed a

s e t of n i c k e l d e p r i v a t i o n s i g n s f o r the r a t that appear

d i v e r g e n t t o those of N i e l s e n et a l ( 5 ) Schnegg and

K i r c h g e s s n e r found t h a t , a t age 3 0 days, r a t s exhibited

s i g n i f i c a n t l y depressed growth, h e m a t o c r i t s , hemoglobin l e v e l s ,

e r y t h r o c y t e counts, l e v e l s of u r e a , ATP and g l u c o s e i n serum,

l e v e l s of t r i g l y c e r i d e s , glucose and glycogen i n l i v e r , l e v e l s

of i r o n , copper and z i n c i n l i v e r , kidney and s p l e e n , and

a c t i v i t i e s of s e v e r a l l i v e r and kidney enzymes They a l s o

found t h a t the s i g n s of n i c k e l d e p r i v a t i o n were l e s s severe i n

o l d e r r a t s and i n r a t s f e d 1 0 0 yg instead of 5 0 yg of iron/g of

d i e t Schnegg and K i r c h g e s s n e r suggested that some of the s i g n s

r e s u l t e d from impaired i r o n a b s o r p t i o n induced by n i c k e l

d e p r i v a t i o n

Anke et a l (_7 ,J3) found that n i c k e l - d e p r i v e d minipigs and

goats e x h i b i t e d depressed growth delayed e s t r u s e l e v a t e d

p e r i n a t a l m o r t a l i t y , u n t h r i f t i n e s

and s c a l y and c r u s t y s k i n

s k e l e t o n and of z i n c i n l i v e r , h a i r , r i b and b r a i n Spears e t

a l ( 9 , 1 0 ) found t h a t n i c k e l - d e p r i v e d lambs showed depressed growth, t o t a l serum p r o t e i n s , e r y t h r o c y t e counts, and t o t a l

l i p i d s and c h o l e s t e r o l i n l i v e r , and copper i n l i v e r I r o n contents were e l e v a t e d i n l i v e r , s p l e e n , lung and b r a i n

The d i s c u s s e d f i n d i n g s show t h a t n i c k e l meets the

requirements f o r e s s e n t i a l i t y as d e f i n e d by Mertz ( 1 1 ) That

a m e t a l l o p r o t e i n They f r a c t i o n a t e d human serum by column

chromatography and found a m e t a l l o p r o t e i n that contained n i c k e l , but n o n d e t e c t a b l e l e v e l s of Ca, Mg, S r , Ba, F e , Zn and Mn

Nomoto jet a l ( 1 3 ) used a technique b a s i c a l l y the same as that

of Himmelhoch et: a l t o demonstrate the presence of a n i c k e l

-c o n t a i n i n g m a -c r o g l o b u l i n , whi-ch they named " n i -c k e l o p l a s m i n " , i n

r a b b i t serum Subsequently, Sunderman ejt a l ( 1 4 ) i s o l a t e d

n i c k e l o p l a s m i n from human serum O r i g i n a l l y , Sunderman e t a l

( 1 4 ) stated that the nickeloplasmin of humans and rabbits was

an a2-macroglobulin L a t e r , however, immunologic studies by

Nomoto et_ a l ( 1 5 ) i n d i c a t e d that r a b b i t serum nickeloplasmin

r e a c t s as an a\-macroglobulin that i s a p p a r e n t l y homologous to

human a 2 - m a c r o g l o b u l i n They c a u t i o n e d , however, that the

2 NIELSON Abstruse Trace Metals 25

apparent r e l a t i o n s h i p between r a b b i t a i - m a c r o g l o b u l i n and human

012-macroglobulin was complicated when Saunders ejt a l (16) found

t h a t f i v e components of human a2-macroglobulin can be

d i s t i n g u i s h e d on the b a s i s of e l e c t r o p h o r e t i c and enzyme-binding

p r o p e r t i e s Other c h a r a c t e r i s t i c s of n i c k e l o p l a s m i n were an estimated m o l e c u l a r weight of 7.0 x 1 0 5 , n i c k e l content of 0.90

as an o r g a n i c complex that i s not s y n t h e s i z e d r e a d i l y by the

r a b b i t jLn v i v o The f i n d i n g s of Decsy and Sunderman (17)

suggested t h a t n i c k e l o p l a s m i n was a t e r n a r y complex of serum

a i - m a c r o g l o b u l i n w i t h a N i - c o n s t i t u e n t of serum Sunderman (18) noted t h a t Haupt et: a l (19) i s o l a t e d from human serum a 9.55-oti-glycoprotein t h a t s t r o n g l y bound N i ( I I ) and thus

suggested t h a t n i c k e l o p l a s m i n might r e p r e s e n t a complex of the

urease from s e v e r a l p l a n t s and microorganisms i s a n i c k e l

metalloenzyme (20-25) Dixon e t a l (20) found t h a t h i g h l y

p u r i f i e d urease (E.C.3.5.1.5) from j a c k beans ( C a n a v a l i a

e n s i f o r m i s ) c o n t a i n e d s t o i c h i o m e t r i c amounts of n i c k e l ,

2 0 + 0 3 g atom of n i c k e l per 105,000 g of enzyme The a c t i v e

-s i t e n i c k e l i o n wa-s t i g h t l y bound, b e i n g -s i m i l a r to the z i n c i o n

i n y e a s t a l c o h o l dehydrogenase (E.C.1.1.1.1) and manganous i o n

i n c h i c k e n l i v e r pyruvate c a r b o x y l a s e (E.C.6.4.1.1) Jack bean urease was s t a b l e and f u l l y a c t i v e i n the presence of

0.5 mM EDTA a t n e u t r a l pH The n i c k e l i o n was removed only upon e x h a u s t i v e d i a l y s i s i n the presence of c h e l a t i n g agents (21), and then i t was not p o s s i b l e to r e s t o r e n i c k e l w i t h

r e c o n s t i t u t i o n of enzymatic a c t i v i t y Jack bean urease has

r e l a t i v e l y low r e a c t i v i t y of the a c t i v e - s i t e s u l f h y d r y l group (26) A c c o r d i n g to Dixon ^ t a l (21), t h i s c o u l d be e x p l a i n e d

by c o o r d i n a t i o n of the a c t i v e - s i t e n i c k e l w i t h the u n r e a c t i v e

c y s t e i n e

The b i o l o g i c a l r o l e of urease a p p a r e n t l y i s the c o n v e r s i o n

26 INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE

of u r e a to i n o r g a n i c ammonia t h a t can be used by p l a n t s ( 2 4 , 2 5 )

Dixon et a l ( 2 1 ) suggested the following mechanism for that

c o n v e r s i o n : The amide n i t r o g e n of urea c o o r d i n a t e s w i t h the enzyme-bound n i c k e l N u c l e o p h i l i c a t t a c k or g e n e r a l base

c a t a l y s i s by a s u i t a b l e a c t i v e - s i t e group would then l e a d to an

a c t i v e - s i t e , nickel-ammonia complex

Thus, a s p e c i f i c b i o l o g i c a l r o l e i s known f o r n i c k e l i n

p l a n t s No such s p e c i f i c r o l e has been d e f i n e d f o r animals

N i c k e l can a c t i v a t e many enzymes i n v i t r o (Table I ) , but i t s

r o l e as a s p e c i f i c c o f a c t o r f o r any enzyme has not been shown

i n animals

The s p e c i f i c manner i n which n i c k e l a c t s i n animals i s

unknown, but r e c e n t f i n d i n g s suggest t h a t i t has a r o l e i n the

p a s s i v e a b s o r p t i o n of the F e ( I I I ) i o n I found i n r a t s t h a t the form of d i e t a r y i r o n might e x p l a i n the apparent d i f f e r e n c e s i n data f o r growth and h e m a t o c r i t s between my e a r l y s t u d i e s ( 5 ) and

the s t u d i e s of Schnegg an

s t u d i e s ( 5 ) , I supplied

d i s s o l v e d i n H C 1 (determined to be f e r r i c c h l o r i d e ) , whereas

Schnegg and K i r c h g e s s n e r (6) supplied 50 yg of iron/g of d i e t as

the s u l f a t e Schnegg and K i r c h g e s s n e r i n d i c a t e d , by p e r s o n a l communication, t h a t they had used f e r r o u s s u l f a t e , but I ( 2 7 )

c o u l d not o b t a i n growth and h e m a t o c r i t f i n d i n g s s i m i l a r to

t h e i r s u n l e s s i r o n was s u p p l i e d as f e r r i c s u l f a t e When I

s t u d i e d the r e l a t i o n s h i p between n i c k e l and i r o n f u r t h e r i n

f a c t o r i a l l y designed experiments, n i c k e l and i r o n i n t e r a c t e d to

a f f e c t h e m a t o c r i t and hemoglobin, but a p p a r e n t l y only when

d i e t a r y i r o n was mostly i n a r e l a t i v e l y u n a v a i l a b l e form, such

as f e r r i c s u l f a t e

In t h r e e experiments, female weanling r a t s were fed a

b a s a l d i e t c o n t a i n i n g about 10 ng of n i c k e l and 2 3 yg of

i r o n / g and supplemented w i t h graded l e v e l s of n i c k e l and i r o n

I r o n was supplemented to the d i e t at 0 , 2 5 , 50 and 1 0 0 yg/g i n

a l l experiments I r o n was s u p p l i e d as F e 2 (S0t+) 3-n^O i n

Experiments 1 and 3 , and as a mixture of 40% F e S 0 i +#n H 2 0 and

60% F e2( S 0l +) 3 - n H20 i n Experiment 2 An extra l e v e l , 1 2 5 yg/g,

was added i n Experiment 3 In a l l experiments, n i c k e l was

supplemented to the d i e t a t 0 , 5 and 50 yg/g A f t e r 9 - 1 0 weeks,

e s p e c i a l l y when the d i e t a r y i r o n supplement was only f e r r i c

s u l f a t e , the i n t e r a c t i o n between i r o n and n i c k e l a f f e c t e d

s e v e r a l parameters examined Data f o r h e m a t o c r i t and hemoglobin appear i n T a b l e s I I and I I I In Experiments 1 and 3 , when

d i e t a r y f e r r i c s u l f a t e was low, h e m a t o c r i t and hemoglobin were lower i n n i c k e l - d e p r i v e d than -supplemented r a t s , e s p e c i a l l y when i r o n / g of d i e t was 25 yg Experiment 1 n i c k e l - d e p r i v e d

r a t s had an average h e m a t o c r i t of 3 6 3 % and hemoglobin l e v e l

of 1 0 0 9 g/100 ml, whereas r a t s fed n i c k e l at 5 and 50 yg/g of

d i e t had h e m a t o c r i t s of 4 0 8 % and 4 2 0 % and hemoglobin l e v e l s

of 1 1 7 7 and 1 2 0 9 g/100 ml, r e s p e c t i v e l y In Experiment 3 ,

n i c k e l - d e p r i v e d r a t s had an average h e m a t o c r i t of 2 6 8 % and

NIELSON Abstruse Trace Metals

T a b l e I Enzymes " a c t i v a t e d " by n i c k e l

R a b b i t muscle

P r o t e u s v u l g a r i s Bovine pancreas

C o m p i l e d by N i e l s e n ( 3 3 )

INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

Nickel e f f e c t means - 30 s-test 30 - 1.2

-l e v e -l s of supp-lements i n d i e t : Ni (nicke-l ch-loride) and Fe

( f e r r i c sulfate) i n Experiments 1 and 3; Fe was a mixture of

40% ferrous and 60% f e r r i c s u l f a t e i n Experiment 2

^The Scheffe test (28) i s a method for performing multiple

comparisons between group means Means d i f f e r i n g by more

than the value given are s i g n i f i c a n t l y d i f f e r e n t (P < 0.05)

As i t assumes a l l possible comparisons are performed, i t i s

regarded as a conservative test

NIELSON Abstruse Trace Metals

Table I I I

E f f e c t s on rats of n i c k e l , i r o n , and t h e i r i n t e r a c t i o n on

hemoglobin levels

Treatment Hemoglobin Level

Ni Fe Experiment 1 Experiment 2 Experiment 3

The Scheffe test (28) i s a method for performing multiple comparisons between group means Means d i f f e r i n g by more than the value given are s i g n i f i c a n t l y d i f f e r e n t (P < 0.05)

As i t assumes a l l possible comparisons are performed, i t i s regarded as a conservative test

INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

hemoglobin l e v e l of 6.19 g/100 ml; whereas r a t s f e d n i c k e l at

5 and 50 yg/g of d i e t had h e m a t o c r i t s of 32.1% and 33.8% and hemoglobin l e v e l s of 8.31 and 8.92 g/100 ml, r e s p e c t i v e l y The

d i f f e r e n c e between n i c k e l - d e p r i v e d and supplemented r a t s i n

Experiments 1 and 3 were s i g n i f i c a n t by the S c h e f f e t e s t ( 2 8 )

D i e t a r y n i c k e l a p p a r e n t l y d i d not a f f e c t h e m a t o c r i t or

hemoglobin when the d i e t c o n t a i n e d 100 yg of i r o n / g N i c k e l and

i r o n d i d not i n t e r a c t to a f f e c t h e m a t o c r i t and hemoglobin when

i r o n was s u p p l i e d as f e r r i c - f e r r o u s s u l f a t e

The form of d i e t a r y i r o n a l s o i n f l u e n c e d the e f f e c t of

n i c k e l on h e m a t o c r i t and hemoglobin When f e r r i c s u l f a t e was

f e d (Experiments 1 and 3 ) , both parameters were s i g n i f i c a n t l y lower i n n i c k e l - d e p r i v e d than -supplemented r a t s In

Experiment 2 the e f f e c t of n i c k e l was much l e s s marked than i n Experiments 1 and 3 In Experiment 2, the g r e a t e s t d i f f e r e n c e was i n r a t s f e d no supplemental i r o n

There were some d i f f e r e n c e

e s p e c i a l l y when the d i e

Experiment 3, h e m a t o c r i t and hemoglobin l e v e l s were

s i g n i f i c a n t l y depressed i n a l l groups f e d 25 yg i r o n / g of d i e t , although the d e p r e s s i o n was l e s s severe i n nickel-supplemented than - d e p r i v e d r a t s In Experiment 1, w i t h 25 yg of i r o n / g of

d i e t , h e m a t o c r i t and hemoglobin were depressed only i n n i c k e l

-d e p r i v e -d r a t s ; v a l u e s were near normal i n r a t s f e -d 5 or 50 yg of

n i c k e l / g o f d i e t I n Experiment 3, the h e m a t o c r i t and

hemoglobin data i n d i c a t e d that r a t s f e d 50 yg of i r o n / g of d i e t

as f e r r i c s u l f a t e were s t i l l s l i g h t l y i r o n - d e f i c i e n t In

Experiment 1, h e m a t o c r i t and hemoglobin a p p a r e n t l y were normal

i n r a t s f e d 50 yg of i r o n / g of d i e t P o s s i b l y , the i r o n

supplement was most h i g h l y contaminated w i t h the f e r r o u s form

i n Experiment 1 The i r o n supplement was a s c e r t a i n e d to be 92%

i n the f e r r i c form i n Experiment 3, but was not t e s t e d i n

Experiment 1

The o b s e r v a t i o n s t h a t the form of d i e t a r y i r o n a p p a r e n t l y

a f f e c t e d the response of r a t s t o n i c k e l d e p r i v a t i o n and n i c k e l and i r o n i n t e r a c t e d suggest that n i c k e l a f f e c t s i r o n a b s o r p t i o n The apparent dependence of that i n t e r a c t i o n upon the r e l a t i v e l y

i n s o l u b l e f e r r i c s a l t suggests t h a t n i c k e l has a r o l e i n the

t h a t form h i g h - s p i n complexes and thereby i n c r e a s e the e l e c t r o d e

p o t e n t i a l s t a b i l i z e F e ( I I ) over F e ( I I I ) Most other b i o l i g a n d s lower the e l e c t r o d e p o t e n t i a l and thus enhance the s t a b i l i t y of the F e ( I I I ) s t a t e T h e r e f o r e , the p r e f e r r e d c h e l a t e d s t a t e of

i r o n jLn v i v o i s probably F e ( I I I ) and the r e d u c t i o n to F e ( I I ) occurs spontaneously o n l y i n the presence of h i g h l o c a l

c o n c e n t r a t i o n s o f a r e d u c i n g m e t a b o l i t e , or under the i n f l u e n c e

2 NIELSON Abstruse Trace Metals 31

of s p e c i a l enzyme mechanisms N i c k e l might i n t e r a c t w i t h i r o n through one of those mechanisms but probably does not The

f i n d i n g t h a t 50 yg of n i c k e l / g of d i e t was not much b e t t e r than

5 yg i n improving h e m a t o c r i t s and hemoglobin l e v e l s i n n i c k e l

-d e p r i v e -d r a t s fe-d low l e v e l s of i r o n as f e r r i c s u l f a t e i s

a p p a r e n t l y i n c o n s i s t e n t w i t h the p o s s i b i l i t y t h a t n i c k e l acts

as, or p a r t o f , a r e d u c i n g agent c o n v e r t i n g F e ( I I I ) t o F e ( I I ) The i d e a t h a t n i c k e l might act i n a s p e c i a l enzyme mechanism

t h a t converts F e ( I I I ) t o F e ( I I ) i s a t t r a c t i v e , but no such

mechanism i s known

The most a t t r a c t i v e p o s s i b i l i t y i s t h a t n i c k e l promotes the

a b s o r p t i o n of F e ( I I I ) per se by enhancing i t s complexation to a

l i p o p h i l i c molecule Evidence shows t h a t both a c t i v e and

of the l i p o p h i l i c F e ( I I I ) complexes i n at l e a s t two ways

N i c k e l might e i t h e r act i n an enzymatic r e a c t i o n that forms a

l i p o p h i l i c i r o n t r a n s p o r t molecule or simply p r e s e r v e a

t r a n s p o r t l i g a n d , such as c i t r a t e , by complexing w i t h i t u n t i l

r e p l a c e d by the F e ( I I I ) i o n

The h y p o t h e s i s t h a t n i c k e l has a r o l e i n the p a s s i v e

d i f f u s i o n of F e ( I I I ) i s supported by my data f o r h e m a t o c r i t and

hemoglobin d i s c u s s e d p r e v i o u s l y Dowdle et a l (31) suggested

t h a t the a c t i v e t r a n s p o r t mechanism f o r i r o n would become

important i f p a s s i v e d i f f u s i o n were r e s t r i c t e d Thus, at the lower l e v e l s of i r o n supplementation as a f e r r i c - f e r r o u s

m i x t u r e , there was some f e r r o u s i o n s a v a i l a b l e f o r a c t i v e

t r a n s p o r t , and n i c k e l d e p r i v a t i o n d i d not s i g n i f i c a n t l y a f f e c t

l e v e l s of h e m a t o c r i t or hemoglobin On the other hand, when

o n l y f e r r i c i r o n was f e d , the a c t i v e t r a n s p o r t mechanism c o u l d not operate, and i n n i c k e l d e p r i v a t i o n , the p a s s i v e d i f f u s i o n

of l i p o p h i l i c F e ( I I I ) complexes a p p a r e n t l y was i n h i b i t e d As a

r e s u l t , l e v e l s of h e m a t o c r i t and hemoglobin d i f f e r e d between

n i c k e l - d e p r i v e d and -supplemented r a t s at low l e v e l s of i r o n supplementation At h i g h l e v e l s of supplementation, perhaps

t h e r e was enough F e ( I I ) p r e s e n t i n the d i e t to prevent any

d i f f e r e n c e s as the i r o n supplement was approximately 92% F e ( I I I )

M e d i c a l S i g n i f i c a n c e An i n i t i a l impression i s t h a t n i c k e l

n u t r i t u r e would not be of p r a c t i c a l s i g n i f i c a n c e I (4)

r e p o r t e d t h a t 50 yg of n i c k e l / k g of d i e t s a t i s f i e d the d i e t a r y

n i c k e l requirement of c h i c k s , and Schnegg and K i r c h g e s s n e r (6)

r e p o r t e d a s i m i l a r requirement f o r r a t s I f animal data were

e x t r a p o l a t e d to man, the d i e t a r y n i c k e l requirement of humans

32 INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE would p r o b a b l y be i n the range of 16-25 yg/1000 C a l (32)

L i m i t e d s t u d i e s i n d i c a t e t h a t the o r a l i n t a k e of n i c k e l by

humans ranges between 170 and 700 yg per day (33) which would

be ample to meet the h y p o t h e s i z e d n i c k e l requirement

However, the f i n d i n g t h a t n i c k e l may be important i n the

a b s o r p t i o n and metabolism of i r o n might help d e f i n e s i t u a t i o n s

i n which n i c k e l would have m e d i c a l s i g n i f i c a n c e I am d e f i n i n g

m e d i c a l s i g n i f i c a n c e as the u n i n t e n t i o n a l p r o d u c t i o n of a

n u t r i t i o n a l d i s o r d e r i n humans P o s s i b l y f o r i n d i v i d u a l s who consume u n a v a i l a b l e , or d e f i c i e n t amounts o f , i r o n , or have an

e l e v a t e d need f o r i r o n , n i c k e l n u t r i t u r e might be of concern For example, many women consume inadequate i r o n N i c k e l a l l e r g y

e x e r c i s e d w i t h such treatment to assure that proper n i c k e l and

i r o n n u t r i t u r e i s maintained to avoid adverse consequences

Vanadium

E s s e n t i a l i t y Evidence f o r the n u t r i t i o n a l e s s e n t i a l i t y

of vanadium i s not c o n c l u s i v e S t r a s i a (37) found that r a t s

f e d l e s s than 100 ng of vanadium/g of d i e t e x h i b i t e d slower

growth, h i g h e r plasma and bone i r o n , and h i g h e r h e m a t o c r i t s

than c o n t r o l s f e d 0.5 yg of vanadium/g of d i e t However,

W i l l i a m s (38) was unable to d u p l i c a t e the f i n d i n g s of S t r a s i a (37), even i n the same l a b o r a t o r y under s i m i l a r c o n d i t i o n s

Schwarz and M i l n e (39) r e p o r t e d t h a t a vanadium supplement of

S t u d i e s w i t h c h i c k s a l s o gave i n c o n s i s t e n t s i g n s of

d e f i c i e n c y Hopkins and Mohr (41,42) found that

vanadium-d e p r i v e vanadium-d c h i c k s e x h i b i t e vanadium-d s i g n i f i c a n t l y vanadium-depressevanadium-d wing anvanadium-d t a i l

f e a t h e r development, depressed plasma c h o l e s t e r o l at age 28 days, e l e v a t e d plasma c h o l e s t e r o l at age 49 days, and, i n a

subsequent study (40), e l e v a t e d plasma t r i g l y c e r i d e s at age 28 days I r e p o r t e d t h a t vanadium-deprivation depressed growth,

e l e v a t e d h e m a t o c r i t s and plasma c h o l e s t e r o l , and a d v e r s e l y

a f f e c t e d bone development (43)

I became concerned about the i n c o n s i s t e n c y of the e f f e c t

of vanadium d e p r i v a t i o n on c h i c k s and r a t s , and attempted to

e s t a b l i s h a d e f i n i t e s e t of s i g n s of vanadium d e p r i v a t i o n f o r these s p e c i e s In 16 experiments, i n which c h i c k s were fed

2 NIELSON Abstruse Trace Metals

composition a f f e c t s the form of d i e t a r y vanadium Vanadium has

a r i c h and v a r i e d chemistry, e s p e c i a l l y i n the (IV) and (V)

s t a t e The form of vanadium, u s u a l l y an oxyanion ( i e VO3"",

V 0 2 + ) , depends upon i t s c o n c e n t r a t i o n i n and pH o f the medium (45) Perhaps, one for

a b s o r p t i o n , or a c t i v e i

d i e t t h a t i s r e l a t i v e l y low i n vanadium might be n u t r i t i o n a l l y

e i t h e r d e f i c i e n t or adequate depending on the form of the

vanadium

Nonetheless, because the evidence i s i n c o n s i s t e n t , f u r t h e r

s t u d i e s are necessary to d e f i n i t e l y e s t a b l i s h vanadium as an

f i r s t t o r e p o r t t h a t vanadium p o t e n t l y i n h i b i t s (Na, K)-ATPase,

Cant l e y et_ a l (47) were f i r s t t o f i n d that pentavalent

orthovanadate was a n a t u r a l l y o c c u r r i n g i n h i b i t o r of t h a t

enzyme Vanadate was shown to i n h i b i t (Na, K) ATPase from

kidney (46,_47,4 L 8), b r a i n (48) , h e a r t (_48,_49) , red blood c e l l s (50,51), shark r e c t a l gland and e e l e l e c t r o p l a x (49) ATP-

phosphohydrolase from v a r i o u s dynein f r a c t i o n s , commonly known

as dynein ATPase, a l s o was p o t e n t l y i n h i b i t e d by vanadate

(52,53,54) Josephson and C a n t l e y (55) found that vanadate

d i d not p o t e n t l y i n h i b i t other ATPase systems, such as

Ca-ATPase, m i t o c h o n d r i a l c o u p l i n g f a c t o r F, and actomyosin Cande and Wolniak (54) found t h a t vanadate d i d not p o t e n t l y

i n h i b i t g l y c e r i n a t e d m y o f i b r i l c o n t r a c t i o n or myosin ATPase

a c t i v i t y Those f i n d i n g s suggest that vanadate would be an

i d e a l s p e c i f i c i n h i b i t o r of (Na, K)-ATPase or dynein ATPase Magnesium and potassium f a c i l i t a t e vanadate i n h i b i t i o n of (Na, K)-ATPase a c t i v i t y and they both appear to b i n d

s y n e r g i s t i c a l l y w i t h vanadate (56) ATP depressed vanadate

i n h i b i t i o n of enzyme a c t i v i t y (48) On the other hand, Gibbons

34 INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

et a l (53) found t h a t dynein ATPase i n h i b i t i o n by vanadate d i d not depend upon the magnesium c o n c e n t r a t i o n or on the presence

or absence of potassium Furthermore, ATP had no obvious

a f f e c t on vanadate i n h i b i t i o n of dynein ATPase

C a n t l e y e t a l (50) found t h a t vanadate binds to one h i g h

-a f f i n i t y -and one low -a f f i n i t y s i t e per (N-a, K)-ATP-ase enzyme molecule The l o w - a f f i n i t y s i t e was a p p a r e n t l y r e s p o n s i b l e f o r

i n h i b i t i o n of (Na, K)-ATPase a c t i v i t y and was the h i g h - a f f i n i t y ATP s i t e where sodium-dependent p r o t e i n p h o s p h o r y l a t i o n o c c u r s

C a n t l e y e_t a l (56) proposed t h a t the u n u s u a l l y h i g h a f f i n i t y

of vanadate f o r (Na, K)-ATPase was due t o i t s a b i l i t y to form

a t r i g o n a l b i p y r a m i d a l s t r u c t u r e analogous to the t r a n s i t i o n

s t a t e f o r phosphate h y d r o l y s i s

C a n t l e y et_ a l (50) found t h a t vanadate was t r a n s p o r t e d

to the r e d b l o o d c e l l where i t i n h i b i t e d the sodium pump by

b i n d i n g to (Na, K)-ATPase from the c y t o p l a s m i c s i d e (the s i t e

mechanism of i n h i b i t i o n i s not the same i n each enzyme The

i n h i b i t i o n of RNase and a l k a l i n e phosphatase i s g r e a t e r by

oxyvanadium (IV) than by vanadium (V)

Thus, the f i n d i n g s to date suggest that vanadium has a

b i o l o g i c a l f u n c t i o n i n c o n t r o l l i n g one or more enzymatic

r e a c t i o n s concerned w i t h phosphate metabolism However, f u r t h e r

i n v i v o s t u d i e s are n e c e s s a r y b e f o r e a c o n c l u s i v e statement can

be made

M e d i c a l S i g n i f i c a n c e The m e d i c a l s i g n i f i c a n c e of vanadium

i s u n c l e a r because knowledge i s incomplete of the c o n d i t i o n s

n e c e s s a r y to produce vanadium d e f i c i e n c y , d i e t a r y components

t h a t a f f e c t vanadium metabolism, and i t s b i o l o g i c a l f u n c t i o n

I t i s d i f f i c u l t to suggest a vanadium requirement f o r animal

s p e c i e s , i n c l u d i n g humans However, at l e a s t f o u r independent

l a b o r a t o r i e s have found t h a t d i e t s w i t h l e s s than 25 ng of

vanadium/g a d v e r s e l y a f f e c t r a t s and c h i c k s under c e r t a i n

c o n d i t i o n s I f animal d a t a could be e x t r a p o l a t e d to humans, then a 70 kg man consuming 1 kg of d i e t per day (dry b a s i s )

would have a d a i l y requirement of about 25 yg of vanadium

under c e r t a i n d i e t a r y c o n d i t i o n s

Recent s t u d i e s have shown t h a t the vanadium content of

most foods i s v e r y low (60,61,62,63,64), g e n e r a l l y not more

than a nanogram/g Myron et^ a l (63) r e p o r t e d that nine

i n s t i t u t i o n a l d i e t s s u p p l i e d 12.4-30.1 yg of vanadium d a i l y ,

2 NIELSON Abstruse Trace Metals 35

and i n t a k e averaged 20 yg Byrne and K o s t a (64) s t a t e d that the d i e t a r y i n t a k e of vanadium i s i n the order of a few tens of micrograms and may vary w i d e l y T h i s suggests t h a t vanadium

i n t a k e i s not always o p t i m a l i n humans

In a d d i t i o n to n u t r i t i o n a l d e f i c i e n c y , n u t r i t i o n a l vanadium

t o x i c i t y may have m e d i c a l s i g n i f i c a n c e The f i n d i n g s d i s c u s s e d

p r e v i o u s l y suggest t h a t because vanadium i s a potent i n h i b i t o r

of s e v e r a l enzymes, any undue e l e v a t i o n i n t i s s u e vanadium

content might a d v e r s e l y a f f e c t b i o c h e m i c a l systems t h a t depend upon normal phosphate metabolism Even r e l a t i v e l y s m a l l

amounts of d i e t a r y vanadium c o u l d be t o x i c i n some s i t u a t i o n s For example, Hunt (65) found t h a t the a d d i t i o n of 500 yg of chromium as the a c e t a t e / g of d i e t made 5 yg of vanadium/g of

d i e t t o x i c to c h i c k s Those c h i c k s e x h i b i t e d depressed growth and h e m a t o c r i t s , e l e v a t e d plasma c h o l e s t e r o l , kidney (Na, K) ATPase, and l i v e r / b o d y weight r a t i o Morphology of t h e i r

p r o x i m a l t i b i a e was d r a s t i c a l l

abnormally t h i c k and th

t h i n Metaphyseal bone was n o n e x i s t e n t T r a n s m i s s i o n e l e c t r o n

m i c r o s c o p i c examinations r e v e a l e d a d i s o r g a n i z e d growth p l a t e and the presence of an abnormal, e l e c t r o n - d e n s e m a t r i x

component around the chondrocytes i n the p r o l i f e r a t i v e zone

F i v e yg of vanadium/g of d i e t without chromium supplementation had no obvious e f f e c t on c h i c k s

Cadmium, Lead, and T i n

E s s e n t i a l i t y At p r e s e n t , the evidence suggesting

t h a t cadmium, l e a d and t i n are e s s e n t i a l does not f u l f i l l the requirements f o r e s s e n t i a l i t y as d e f i n e d by Mertz (11)

Although d i e t a r y supplements of cadmium, l e a d , or t i n s l i g h t l y improved the growth of s u b o p t i m a l l y growing r a t s , these

supplements d i d not r e s u l t i n o p t i m a l growth (1,_2 >3) Thus, i t cannot be s t a t e d u n e q u i v o c a l l y t h a t cadmium, l e a d , or t i n

d e f i c i e n c y r e p r o d u c i b l y r e s u l t s i n an impairment of a f u n c t i o n from o p t i m a l to suboptimal

A p p a r e n t l y , the suboptimal growth i n a l l r a t s i n the

cadmium, l e a d and t i n s t u d i e s was due to r i b o f l a v i n d e f i c i e n c y (66) U n f o r t u n a t e l y , the death of the p r i n c i p a l i n v e s t i g a t o r

of cadmium, l e a d and t i n e s s e n t i a l i t y (Klaus Schwarz) prevented

f u r t h e r s t u d i e s which would have answered the q u e s t i o n whether

d e f i c i e n c i e s of those elements would depress growth i n r a t s which were not r i b o f l a v i n - d e f i c i e n t T h i s q u e s t i o n may remain unanswered f o r some time because, to my knowledge, s t u d i e s

concerned w i t h the e s s e n t i a l i t y of cadmium, l e a d , and t i n are not c u r r e n t l y pursued i n another l a b o r a t o r y

The r e p o r t s which suggest the e s s e n t i a l i t y of cadmium,

l e a d and t i n can a l s o be c r i t i c i z e d i n the f o l l o w i n g manner:

1 The b a s a l d i e t s were not adequately d e s c r i b e d , thus

p r e v e n t i n g the c o n f i r m a t i o n of the growth f i n d i n g s i n another

36 INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE

s i g n i f i c a n t f i n d i n g s a p p a r e n t l y were obtained through the

method of combining experiments, thus i n c r e a s i n g the s t a t i s t i c a l term n (no of animals) (2,3) Combining experiments b e f o r e

s t a t i s t i c a l treatment of the data i s i n a p p r o p r i a t e

3 The s m a l l growth d i f f e r e n c e between " d e f i c i e n t - c o n t r o l s " and supplemented r a t s (about 5 to 7 grams a f t e r 25 to 30 days

on experiment) may be of q u e s t i o n a b l e p h y s i o l o g i c a l meaning Perhaps t h i s growth response was due to the supplemental metals

p a r t i a l l y p r e v e n t i n g the breakdown of some e s s e n t i a l n u t r i e n t such as r i b o f l a v i n , or s u b s t i t u t i n f o t r a c element

l a c k i n g i n the d i e t

4 The a d d i t i o n o suggeste

was of no apparent b e n e f i t to d e f i c i e n t - c o n t r o l animals i n

subsequent s t u d i e s For example, i n the t i n s t u d i e s , the

d e f i c i e n t c o n t r o l s gained about 1.3 to 1.9 g/day; t i n

-supplemented r a t s , 1.7 to 2.2 g/day However, even w i t h the

a d d i t i o n of t i n , and some other elements subsequently found

p o s s i b l y e s s e n t i a l , such as f l u o r i n e and s i l i c o n , the d e f i c i e n t

-c o n t r o l s i n the l e a d study s t i l l gained only 1.5 to 2.1 g/day; lead-suppiemented r a t s , 1.6 to 2.2 g/day D e f i c i e n t - c o n t r o l and cadmium-supplemented r a t s a l s o e x h i b i t e d s i m i l a r d a i l y

weight g a i n s No e x p l a n a t i o n was g i v e n f o r the f i n d i n g t h a t

d e f i c i e n t - c o n t r o l s weighed the same i n each of the t i n , l e a d and cadmium s t u d i e s , even though one would expect the d e f i c i e n t -

c o n t r o l s would show b e t t e r growth r a t e s i n l a t t e r s t u d i e s

because t h e i r d i e t s c o n t a i n e d more e s s e n t i a l elements

Because of the p r e v i o u s l y d i s c u s s e d q u e s t i o n s and

c r i t i c i s m s , I conclude t h a t cadmium, l e a d and t i n should not be

i n c l u d e d i n the l i s t of e s s e n t i a l t r a c e metals at the present time

B i o l o g i c a l F u n c t i o n and M e d i c a l S i g n i f i c a n c e U n t i l more

c o n c l u s i v e evidence i s found s u g g e s t i n g cadmium, l e a d and t i n are e s s e n t i a l , the d e s c r i p t i o n of any p o s s i b l e b i o l o g i c a l

f u n c t i o n seems i n a p p r o p r i a t e The t o x i c o l o g i c aspects of

cadmium, l e a d and t i n are of m e d i c a l s i g n i f i c a n c e However,

a proper d i s c u s s i o n of the t o x i c o l o g y of those elements i s

beyond the scope of t h i s p r e s e n t a t i o n and i s adequately done elsewhere (67,68,69)

Summary

The evidence to date has e s t a b l i s h e d n i c k e l as an e s s e n t i a l

n u t r i e n t f o r s e v e r a l animal s p e c i e s The e s s e n t i a l i t y of