Inorganic Chemistry in Biology and Medicine pdf

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.
ACS SYMPOSIUM SERIES
14 0
AMERICAN CHEMICAL SOCIETY
WASHINGTON, D.C. 1980
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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 therapy—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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Rights Reserved. The appearance of the code at the bottom of the first
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In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
ACS

Symposium
Series
M.
Joa
Advisory
Board
David
L.
Allara
Kenneth B. Bischoff
Donald G. Crosby
Donald D. Dollberg
Robert E.
Feeney
Jack
Halpern
Brian
M. Harney
Robert A. Hofstader
W.
Jeffrey Howe
James
D. Idol, Jr.
James
P. Lodge
Leon
Petrakis
F.
Sherwood Rowland
Alan

C. Sartorelli
Raymond B. Seymour
Gunter
Zweig
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
FOREWORD
The ACS SYMPOSIU M SERIE S
was founded in
197 4
to provide
a medium for publishin
format
of the Series parallels that of the continuing
ADVANCE S
IN CHEMISTR Y SERIE S
except
that in order to
save
time the
papers are not
typeset
but are reproduced as they are sub-
mitted by the authors in camera-ready
form.
Papers are re-
viewed under the supervision of the
Editors
with the
assistance

of
the Series Advisory
Board
and are
selected
to maintain the
integrity of the symposia; however, verbatim reproductions of
previously published papers are not accepted. Both reviews
and
reports of research are acceptable since symposia may
embrace both
types
of presentation.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
PREFACE
At its inception , the origina l plan for this symposiu m was to emphasiz e
the medica l aspect s of inorgani c chemistry , rather than to go over
once more new development s in bioinorgani c chemistry , importan t as the
subject is, since the latter topic has been treate d many times in recent
symposi a
reviews
and monographs . The objective s of this symposiu m
were
to
review
and interpre t the remarkabl e advance s that have occurre d recentl y
in
medica l inorgani c chemistr y and to stimulat e interes t on the part of
inorgani c chemist s to becom e involve d in the developin g researc h problem s

in
this area. The interaction
function s of metal ions in physiologica l systems are very complex , and the
precise nature of these interaction s and processe s are, for the most part,
unknown . In additio n to the application s of metal ions and complexe s for
medica l
purposes , extensiv e fundamenta l studie s are needed to understan d
the
basis
of these application s and thereb y make it possibl e to carry out
systemati c improvement s in curren t method s as well as to develo p new
approache s in this interestin g field.
Of
the approximatel y eighty metalli c elements , a considerabl e numbe r
have been identifie d as essentia l to life; many others have been indicate d
as possibl y essential , while a
large
numbe r of metals are of concer n
because
of
toxic effects that result
when
they are introduce d into the body
acci -
dentally or throug h environmenta l influences . Major meta l ions such as
Na
+
, K
+
, Mg

2+
, and
Ca
2+
are importan t in maintainin g electrolyt e concentra -
tion
in body fluids or as skeleta l constituents . Many of the transitio n metal
ions are essentia l in trace amount s for the activatio n of enzyme systems . In
many cases, these essentia l metal ions become toxic or even carcinogeni c
when
presen t at sufficien t levels to overwhel m the natura l ligands and
macromolecule s that functio n as carrier s for these ions, and thus more than
saturate the normal physiologica l processe s for their control . Under such
conditions ,
they may function , as do many unnatura l toxic metals , by
reacting with other biomolecules , distortin g or blockin g their essentia l
functions .
In many cases, the difference s
between
the essentia l and toxic
levels are surprisingl y
narrow.
This duality of behavio r
between
natura l
and toxic levels constitute s the
basis
of threshol d concentration s for several
carcinogeni c
metals—below

which these metals exist as essentia l and
noncarcinogeni c compounds . It also provide s a strong refutatio n of the
validit y
of the linear extrapolatio n metho d
stil l
in active use for the
interpretatio n of carcinogenicit y of compound s observe d at high concentra -
tion
levels in test animals .
vii
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
The topics covere d
in
this symposiu m
were
selecte d
so as to
provid e
example s
of
current and potentia l medica l application s
of
metal compounds .
The emphasi s
and
amount
of
attentio n given
were

in
many cases
not in
proportio n
to the
importanc e
or
activity levels
of
these applications ,
for a
number
of
reasons .
The use of
platinu m complexe s
for the
treatmen t
of
cancer
is
perhap s under-represente d becaus e severa l symposia , some
of
which have been published , have been held
on
this subject
in
recent years.
Similarly ,
iron nutrition , althoug h very important , has been omitted because

it
is
well covere d
by
periodi c
and
continuin g conference s
and
conferenc e
proceeding s devoted entirely
to
this field
of
research .
New
development s
of
ionophore s and on the use
of
chelatin g agents
for
the remova l
of
radioactiv e
metals from the body
were
not given
the
attentio n that they deserve
in

this
symposiu m becaus e these subjects
were
treated
in
separat e symposi a
at
the
same America n Chemica l
Because
of
the
large
numbe r and complexit y
of
the function s
of
metal
ions in physiologica l systems , the application s
of
complexe s
of
both essentia l
and unnatura l metal ions
for
medica l purpose s
are
expecte d
to
expand

dramaticall y
in the
next decade .
It is
hoped that this book will help
to
attract more inorgani c chemist s
to
this field,
to
provid e
the
expertis e
in
coordinatio n chemistr y neede d
for the
achievemen t
of
significan t
new
development s
in
this potentiall y importan t area
of
medicine .
The Editor wishe s
to
express
his
appreciatio n

for the
many helpful
suggestion s receive d from professiona l colleague s durin g
the
formativ e
stages
of
this symposium . Specia l thanks
are due to L. G.
Marzill i
for
assistanc e with subject matte r planning , and
to
J.
H.
Timmon s
for
valuabl e
editoria l
assistance .
Texas
A&M
University
College Station, Texas
August 7, 1980
A. E. MARTELL
viii
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1

Molecular
and
Biological Properties of Ionophores
BERTON
C.
PRESSMAN,
GEORG E
PAINTER,
and
MOHAMMA D FAHIM
Department of Pharmacology, University of
Miami, Miami,
FL
33101
The
ionophores ar
which form lipid-solubl
complexe
transpor
cations across low polarity barriers such as organic
solvents
and
lipids (1).
From
a biological standpoint, the
most
important low
polarity
barrier
is the

lipid
bilayer which
lies
within biological
membranes; ionophores
possess
unique and
potent
biological proper-
ties
which derive from their ability to perturb transmembrane ion
gradients and electrical potentials.
Each
ionophore has its own
characteristic ion
selectivity
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 ability of ionophores to complex and transport
cations has an ever growing list of applications in experimental
biology and technology and may ultimately provide the
basis

for
novel cardiovascular drugs. Ionophores are
also
intriguing intel-
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 chirally
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
illustrated in Figure 1. All ionophores deploy an
array

of
liganding oxygen
atoms
about a cavity in
space
into which the com-
plexed cation fits.
X-ray
crystallography
reveals
that the
prin-
cipal
bonding energy is provided by induced dipolar interaction
between
the complexed cation and
those
specific
oxygens
which are
filled
in.
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
0-8412-0 5
8
8-4/ 80/47-140-003$05.00/ 0
© 1980 American Chemica l Society
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
4
INORGANI C CHEMISTR Y
IN
BIOLOG Y
AND
MEDICIN E
VANCOMYCIN
ENNIATIN
B MACROLIDE ACTINS
CYCLOHEXYL
ETHER
MONENSIN
NIGERICIN
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.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1.
PRESSMA N
ET AL.
Properties
of
Ionophores
5
dimensiona l
cage
which
accommodates
K
+
(r = 1.33 X) much more
snugl y
tha n
Na
+
(r = 0.95 ft)

resultin g
in a K
+
:Na
+
preferenc e
of
10,000:1
(4).
Enniati n
B is a
cycli c
hexadepsipeptide ;
the
smalle r rin g
result s
in a
relativel y
plane r arra y
of
ligandin g
oxygen
atoms;
th e
more
open
and more
flexibl e
cage
result s

in a K
+
:Na
+
discrimi -
natio n
of
onl y
3:1 (6).
A
new
featur e
appear s
in the
cycli c
tetraesters ,
the
macro-
lid e
nactins .
In
additio n
to the
este r carbonyls , fou r
hetero -
cycli c
ethe r
oxygens
participat e
in

complexation ;
the
oxygens
are
arrange d
at the
apice s
of a
cubi c
cage.
Fiv e varian t nactin s
are
known
dependin g
whether
0-4 of the R
groups
are
methyl s
(nonactin )
or ethyl s
(monactin ,
dinactin ,
trinactin ,
tetranactin)(7) .
While
the
aforementione d
ionophore s
are

Streptomyce s
metabo-
lites ,
the
crown
polyethers ,
the
depicte d
prototyp e
of
which
is
dicyclohexyl-18-crown-6
are
syntheti c (8)
Althoug h
the y
lac k
th e
intricat e
conformation
multipl e
asymmetric
carbo
ertie s
are
analogous .
Whil e
the y
are

les s
efficien t
ion
carriers ,
thei r
lac k
of
labil e
linkage s confer s increase d
chemica l
stability ;
they
fin d
extensiv e
use in
organi c synthesi s
for
solubilizin g
electrolytes ,
e.g.
enolates ,
in
nonpola r
solvent s
thereb y
pro -
vidin g
reactiv e
naked
anion s

(9 )
.
Th e
ionophore s
thu s
far
describe d lac k ionizabl e
groups
and
ar e
collectivel y
classifie d
as
neutra l
ionophores ;
thei r
complexes
acquir e
the net
charg e
of
whatever
ion is
complexed.
We
shal l
now
examine
two
representative s

of the
carboxyli c subclas s
of
iono -
phores .
Onl y
the
anioni c
form
of
thes e
ionophore s
complex
cations ,
hence
the y
form
electricall y
neutra l zwitterioni c
complexes.
Thi s
distinctio n
is
fundamenta l
for
explainin g
the
profoun d
difference s
i n

biologica l
behavio r
of the
ionophor e
subclasses ,
hence
we
pre -
fe r
carboxyli c
ionophor e
to the
term
polyethe r
antibioti c
used
by
Westle y
(5) .
The
latte r
term,
furthermore ,
lead s
to
functiona l
ambiguit y
wit h
the
etherea l

macrolid e
nactin s
and
crown
polyether s
which
are
neutra l
ionophores .
Th e naturall y occurrin g carboxyli c
ionophores ,
typifie d
by
monensin,
lac k
the
structura l
redundancy
of the
neutra l iono -
phores .
Monensin
consist s
of a
formall y linea r arra y
of
hetero -
cycli c
ether-containin g rings ,
however

the
molecula r
chiralit y
arisin g
from
the
ring s
and
asymmetric
carbon s
favor s
the
molecul e
assuming
a
quasi-cycli c configuration . Additiona l
stabilizatio n
of
the
rin g
is
conferre d
by
head-to-tai l
hydrogen
bonding .
In
additio n
to
it s ligandin g ethe r

oxygens,
monensin
has a
pai r
of
ligandin g
hydroxy l
oxygens
(10).
Th e
tai l
portio n
of
nigerici n closel y
resemble s
monensin,
however,
an
additiona l tetrahydropyrano l rin g thrust s
the
head
carboxy l
group
int o
the
complexatio n
sphere .
Thus,
in
additio n

to
th e
induce d
dipol e
ion
bonds
previousl y described , nigerici n
com-
plexe s
featur e
a
tru e ioni c
bond.
Despit e
major
similaritie s
in
structure , nigerici n prefer s
K
+
ove r
Na
+
by a
facto r
of 100
whil e
monensin
prefer s
Na

+
ove r
K"
1
" by a
facto r
of 10
(11 )
.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
6
INORGANIC
CHEMISTRY
IN
BIOLOGY
AND
MEDICINE
Dynamics
of
Ionophore-Mediate d
Transpor t
Neutra l
Ionophores .
The
relationshi p
between
equilibriu m
ionophor e
affinitie s

and
dynamic
biologica l
transmembrane
trans -
por t
is
detaile d
in
Figur e
2. The
transpor t cycl e catalyze d
by
neutra l
ionophore s
is
give n
on the
left .
Ionophore
added
to a
biologica l
membrane
partition s
predominatel y
int o
the membrane. A
portio n
of the

ionophor e
diffuse s
to the membrane
interfac e
where
i t
encounter s
a
hydrate d
cation .
A
loos e
encounte r
complex
is
formed
followe d
by
replacemen t
of the
cationi c hydratio n
spher e
by
engulfmen t
of the
catio n
by the
ionophore .
The
dehydrate d

com-
ple x
is
lipid-solubl e
and
hence
can
diffus e acros s
the membrane.
Th e catio n
is
the n
rehydrated , released ,
and the
uncomplexed
iono -
phore
free d
to
retur n
to
it s
initia l
stat e withi n
the membrane.
Th e
net
reactio n catalyze d
is the movement of an
io n

wit h
it s
charge
acros s
the membrane.
Two
independen t
factor
governin g
net
transpor t
tential ,
i.e . AE^B ,
and the
concentratio n gradient , [M+ ]
^/[M+ ]
B
•
At equilibrium ,
the
electrochemica l potentia l
(a
combined
functio n
of
electrica l
and
concentratio n
terms)
of M*" on

sid e
A becomes
equa l
to the
electrochemica l potentia l
of on
sid e
B,
i.e .
PM A
=
PMB *
IN
TERMS
of
experimentall y
measurable
parameters ,
the
relationshi p
AE ^
= -59 mV log
[M
+
]
A
/[M
+
]
B

applies .
Thi s
signifie s
tha t
if the
electrica l
term,
AE^B ,
exceeds
the
concentratio n
term,
59 mV log
[M^/Mj],
the
io n
wil l
flo w
down the
potentia l gradien t
and
dissipat e
it
(electrophoreti c
transpor t
mode).
If the
concentratio n
term
exceeds

the
pre-exist -
in g
potentia l
term,
the movement of down
it s concentratio n
term
wil l
increas e
AE ^j g
(electrogeni c transport) .
The
relevan t
sig -
nificanc e
of
thi s
transpor t
mode
is
tha t neutra l
ionophore s
per -
tur b
not
onl y
the
transmembrane
ion

gradient s
of
biologica l
systems
but
als o
thei r
transmembrane
electrica l
potentials . Sinc e
th e
latte r
are so
importan t
in
biologica l
control ,
it is not
sur -
prisin g
tha t
the
neutra l
ionophore s
are
exceedingl y toxi c
towards
intac t
animals .
Carboxyli c

Ionophores .
Carboxyli c
ionophore-mediate d
trans -
por t
is
detaile d
on the
lef t
of
Figur e
2. The
form
assumed
withi n
th e
membrane at the
star t
of the
transpor t cycl e
is an
electri -
call y
neutra l zwitterion ,
M^-I";
anioni c fre e
I" is
presumably
too
pola r

to be
stabl e
at
tha t locus .
When
thi s
specie s diffuse s
to
th e
membrane
interface ,
it is
subjec t
to
solvation ;
the
catio n
can
be
hydrate d
and
removed
from
the
complex.
The
resultan t highl y
pola r
I" is
oblige d

to
remain
at the
interfac e
unti l
a new
charg e
partner , represente d
by
N+'R^O,
arrives .
Once in
position ,
N
+
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. PRESSMA N ET AL .
Properties
of
Ionophores
1
exchanges
it s
solvatio n
H2O fo r th e
oxygen
ligandin g
syste m of I
formin g

lipi d
compatibl e N+.I "
which
the n
diffuse s
acros s th e
membrane.
Ther e th e proces s i s reverse d and N
+
i s
exchanged
fo r
M+. Th e ionophor e the n reenter s th e
membrane
as M+I" " thereb y
completin g th e
catalyti c
cycle .
Th e ne t
reactio n
is th e
movement
o f
N+ acros s th e
membrane
in
exchange
fo r M
+
withou t an

accompany-
in g
ne t charg e
translocation .
Thi s is
presumably
an
essentia l
requiremen t fo r toleranc e of appreciabl e concentration s of iono -
phore s by animals , i.e .
carboxyli c
ionophore s ar e
relativel y
non-
toxi c
compared
to
neutra l
ionophores . I n othe r
words,
th e
abilit y
o f
carboxyli c
ionophore s to
alte r
physiologica l
processe s i n a
pharmacologicall y
usefu l

manner
stems
fro m
thei r
capabilit y
to
alte r
transmembrane
io n gradient s withou t
directl y
shor t
circuit -
in g
th e
transmembrane
potential s
of
electricall y
activ e
cells .
The formatio n an d
dissociatio n
of ionophore-catio n
complexes
i s
equivalen t to th e displacemen t o f th e primar y
catio n
solvatio n
spher e by th e ionophor e
groups

approac h th e solvate
They
the n
interac t
vi a an
associativ e
interchang e
mechanism
analo -
gous
to an S
N
2
mechanism
(12) . Formatio n of th e
transitio n
stat e
involve s
extensio n of th e
catio n
to bot h th e enterin g
ligan d
an d
the departin g
catio n
solvatio n
sphere . I n th e process , th e
les s
rigorousl y
define d

solvatio n
spher e of th e
ligan d
is
als o
dis -
charged . Th e ionophor e the n engulf s th e
cation ,
it s
ligandin g
groups
progressivel y
displacin g
th e molecule s of th e
catio n
solva -
tio n
shel l
in a concerte d fashion . I n th e cas e of th e
carboxyli c
ionophores , th e
initia l
stag e
prio r
to th e formatio n of th e
trans -
itio n
complex
is a simpl e io n
pair .

Althoug h the y var y widel y in
structur e
an d conformation , th e
carboxyli c
ionophore s featur e a
variet y
of
heteroatom s
constitut -
in g
a
ligandin g
syste m
which
operate s by
means
of induce d
dipoles .
The
magnitude
of th e dipole s increase s
progressivel y
by
inductio n
as approache d b y th e
catio n
an d
ultimatel y
produce s a
solvatio n

syste m stronge r tha n
tha t
of th e bul k
phase
solvent .
Whereas
th e
individua l
solvatio n
molecules , withi n th e primar y
solvatio n
spher e of a
cation ,
exchange
independentl y wit h th e bul k solvent ,
the ligand s of an ionophore , hel d togethe r by a
common
backbone,
must
behave
in a cooperativ e
manner.
Intramolecula r
hydrogen
bondin g an d substituent s
which
favo r
cycli c
conformation s (e.g .
spiran e

systems)
promote
th e
stabilit y
of
complexes.
Consequently ,
the variou s
catio n
affinit y
an d
selectivit y
pattern s
which
charac -
teriz e
eac h ionophor e
aris e
fro m th e
precis e
spacia l
depolyment
of
ligandin g
heteroatom s
as determine d by molecula r conformatio n
(13,14) .
Conformationa l Studie s of a Representativ e Carboxyli c
Ionophore , Salinomyci n
Salinomycin ,

a representativ e
carboxyli c
ionophor e (Figur e 3)
(15) ,
i s a
particularl y
suitabl e
model
fo r studyin g th e
dynamic
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
8
INORGANI C CHEMISTR Y
IN
BIOLOG Y
AND
MEDICIN E
NCITMl
\r
sMi
I
HI
TtMUKMIN •*
Sill
It
cmixYii d
V
I
r-i*M

M
Figure
2. Different
modes
of
ionophore-mediated transmembrane transport.
Neu-
tral
ionophore-mediated transport
is
depicted
on the
left
and carboxylic ionophore-
mediated transport,
on the
right.
The
individual
transport
steps
are
detailed
in
the
text.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. PRESSMA N E T AL .
Properties

of
Ionophores
9
conformationa l aspect s o f complexation . Th e
circula r
dichrois m
(CD )
arisin g
from
th e n ->
TT *
transitio n
o f th e C-l l carbony l i s
sensitiv e
t o molecula r
environmen t
an d serve s a s a
probe
t o repor t
the
chiralit y
i n it s
vicinity .
C D enable s u s t o evaluat e th e
conformationa l perturbation s
produce d
by
alterin g
th e pola r an d
proti c

propertie s o f th e solven t
system.
Systemati c perturbatio n
o f
th e
solutio n
conformatio n o f salinomyci n by a n appropriat e
choic e o f solvent s reveal s tha t io n
affinit y
an d
selectivit y
ar e
variable ,
conformationall y determined ,
properties .
Representativ e C D spectr a of protonate d salinomycin , it s K
+
complex
an d it s
uncomplexed
anio n ar e presente d i n Figur e 4 . N o
significan t
shif t
o f th e negativ e
29 0
nm
peak
occur s wit h solven t
change
or ligandin g

state ;
Beer' s
la w i s
obeyed
from
10" ^
t o
10"" 6
M .
Th e functio n
most
suitabl e
fo r
relatin g
C D spectr a t o th e
conformatio n o f a molecul e i s th e
rotationa l
strengt h ( R£) of th e
observe d
electroni c
transitio
(16)
Sinc th Gaussia approxi
mation
appear s
t o hol d fo
calculate d
from
[8 ] an d (17) .
Figur e

5
illustrate s
th e
effec t
o f solven t
changes
o n th e
R £
o f th e ionophor e
fre e
aci d
an d it s anion .
Kosower's
Z value s
prove d
empiricall y
a n
effectiv e
functio n fo r rankin g solvent s
accordin g t o
thei r
integrate d pola r an d
proti c
propertie s (18) .
The
| RJ |
of th e
fre e
aci d
decrease s

linearl y
wit h a smal l
positiv e
slop e
a s th e Z value s
rise .
I n contrast , th e
| RQ |
o f th e un -
complexed
anion , th e specie s
participatin g
i n complexation ,
drop s
sharpl y
between
Z value s o f 80 an d 83 , varyin g
littl e
above
an d
below
thes e values .
Thus,
th e conformatio n o f th e anio n tend s
toward
on e o f tw o metastabl e state s
dependin g
upon
solven t Z
value .

The
rol e
o f th e solven t i n determinin g equilibriu m
solutio n
conformatio n ca n bes t b e understoo d i n
terms
o f
functiona l
group
stabilization .
I n pola r
proti c
media
th e equilibriu m conformatio n
o f
th e
uncomplexed
anioni c ionophor e i s determine d b y th e solva -
tio n
o f th e carboxylat e anio n an d th e pola r ligandin g
groups .
Thus,
tw o
distinc t
solven t
effect s
ar e operative ,
solvatio n
o f th e
pola r

ligandin g
groups
resultin g
i n conformationa l
stabilizatio n
due t o decrease d dipole-dipol e repulsio n an d maximizatio n o f th e
solvatio n
energ y
o f th e anion . Th e protonate d ionophor e
respond s
onl y
t o th e
solvatio n
o f pola r ligandin g
groups .
Thus,
Figur e 5
provide s
insigh t
int o
th e
relativ e
importanc e
o f
each
o f thes e
factor s
i n determinin g equilibriu m
solutio n
conformation . Th e

perturbatio n
o f conformatio n du e t o
solvatio n
o f pola r ligandin g
groups
alone , a s i n th e protonate d ionophore ,
cause s
onl y a
sligh t
change
i n conformation , i.e . a smal l
change
i n
|Rol>
ove r a larg e
range
o f Z values .
However,
ionizatio n
o f th e protonate d
form
o f
the ionophor e profoundl y
changes
it s respons e t o solvents . A t Z
value s > 83 , th e carboxylat e i s
stabilize d
b y it s
protic ,
pola r

environment .
Th e
resultin g
solvatio n
spher e influence s th e
con-
formatio n strongl y a s evidence d b y th e ver y lo w
| RQ |
value s
(Figur e
3) . A s th e Z value s
fall ,
an d th e solven t
becomes
les s
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
10
INORGANI C
CHEMISTR Y
IN
BIOLOG Y
AND
MEDICIN E
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
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1.
PRESSMA N
ET AL. Properties of
Ionophores
11
abl e
to
stabiliz e
the charge ,
stabilizatio n
is achieve d by a
tigh t
head-to-tai l
(C^-O-LyH)
hydrogen
bond.
The formatio n of
thi s

bond
result s
in a compressio n of the
ligandin g
cavity ,
the
limi t
of
which
is determine d by
dipole-dipol e
repulsion .
Applicatio n
of
the Octan t Rule (16) to
computer
models
of the anio n corroborate s
tha t
tightenin g
of the
head-to-tai l
bond
shoul d be
accompanied
by
a concomitan t increas e in
| RQ| .
Figur e
4

indicate s
tha t
CD can be
employed
to determin e
com-
plexatio n
K
D
T
s
(see Table I). The
rati o
of the
Na
+
:K+
K
D
f
s,
i.e.
K
+
:Na
+
selectivity ,
als o
shows
a sharp

shif t
between
Z value s of
80 and 83 (cf. Figure 6).
Thus,
the
abilit y
of the complexin g
form of the ionophor e to
discriminat e
between
ions
depends
strongl y
upon
environmenta l
influence s
on conformation .
Changes
i n
inter-ligan d
distance s and
ligan d
orientation s
effecte d
by
changes
in ionophor e conformatio n manifes t themselve s by a deter -
minativ e
alteratio n

of the
fre e
energ y of complexation
CD was
utilize d
t
formatio n
of the cation-ionophor
tio n
isotherm s
were
plotte d
from
linea r
computer
fit s
of
1/[cation ]
versu s
1/ AR £;
the slope s
yielde d
Kpj's while extrapola -
tio n
of RJ to
infinit e
catio n
concentratio n provide d the R^'s of
the cation-saturate d ionophore . It is importan t to note
tha t

the
catio n
itsel f
is a
significan t
vincina l
moiety ,
which
by
virtu e
of
it s
charge ,
polarizabilit y
and
locatio n
with respec t to the
chromophore
of concern , can modif y the
rotationa l
strengt h of the
chromophore.
Comparison
of the
| R £ |
value s for the Na
+
and K
+
complexes

of
salinomyci n
in Table I with the
| RJ |
value s for salinomyci n anion
i n
Figur e 5
shows
an increas e in the
magnitude
of |R£|
upon
com-
plexatio n
in all
solvents .
Thi s correspond s to a
change
in con-
formatio n
upon
complexation , i.e .
reorientatio n
of the ionophor e
about the
cation .
Applicatio n
of the Octant Rule to
computer
generate d

models
of salinomyci n
indicate s
tha t
thi s
reorientatio n
i s
a
constrictio n
of the
ligandin g
oxygens
which
surroun d the
cation .
The exten t of
thi s
constrictio n
correlate s
with the
stabilit y
of the
complex
indicate d
by its (cf . Tabl e I).
X-ray
crystallographi c
studie s
confir m
tha t

all
cationi c
complexes
of
carboxyli c
ionophore s
have
thei r
ligandin g
atoms
ori -
ented towar d a
centra l
cavity .
The exten t to
which
thi s
conforma-
tio n
would
be
altere d
in the
absence
of a
bound
catio n
due to the
mutual
electrostati c

repulsio n
of the
dipola r
oxygen
atoms
would,
i n
turn ,
be
modulated
by the
mobilit y
of the
backbone
supportin g
the
ligands .
We conclud e
tha t
the
dynamics
of molecula r conformatio n asso-
ciate d
with salinomyci n complexatio n in all
likelihoo d
exten d at
leas t
to the othe r
naturall y
occurrin g

carboxyli c
ionophores . The
influenc e
of ionophor e environment , e.g . solvent , on ionophor e
conformatio n is
particularl y
significan t
when
considerin g the
environmenta l
continuum
encountere d by an ionophor e
when
trans -
versin g
a
biologica l
membrane.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
T + +
Tabl e
I
Effec t
of
Solven t
Z
Valu e
on and R
Q

of Na and K
Complexes
of Salinomyci n
SOLVENT
Z
K
D
Na
+
K
D
K
+
|R
T
|Na
+
xl0
38
1
o
1
|R
T
|K
+
xl0
38
1
o

1
50%
DI0XANE/H
2
0
87.6
1.84x10"
•3
3.
87x10"
•4
1.17
1.21
MeOH
83.6
4.89x10"
•4
1.03x10"
4
1.47
1.75
80%
DI0XANE/H
2
0
80.2
3.12x10"
•5
1.17x10"
•5

1.77
1.80
EtOH*
79.6 5.69x10"
•5
5.52x10"
•5
1.69
1.73
90%
DI0XANE/H
2
0*
76.7
5.45x10"
•5
5.48x10"
•5
1.71
1.70
*
A
prior i
we
would
expec t
a
progressiv e
drop
in K^'s as the

solven t
Z
value s
decreas e
sinc e
the
energie s require d
to
desolvat e
the
cation s
(12) and
ionophor e
(38 )
prio r
to
complexatio n
decreas e
progressively .
The
ris e
in
apparen t
value s
in
solvent s
of low Z
value s
can be
accounte d

for
by
progressiv e increase s
i n
ion
pairin g
which
reduc e
the
actua l catio n concentration , i.e . catio n
ac-
tivity ,
availabl e
for
complexation .
Preliminar y correction s
for ion
pairin g
by
means of
Bjerrum' s
equation ,
however,
do not
significantl y
alte r
the
catio n
selectivit y
pattern s reporte d here .

2
o
*>
o
>
o
o
X
m
§
GO
H
2
a
5
r
1
i
>
a
w
2
o
2
w
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. PRESSMA N ET AL.
Properties
of

Ionophores
13
The extensio n to ionophor e
selectivit y
of a hypothesi s base d
on analog y wit h th e
rigi d
matrice s of io n
selectiv e
glasse s (19 )
i s
inconsisten t
wit h th e
dynamic
conformationa l aspec t of io n
selectivit y
develope d in th e presen t paper . Furthermore , th e
conformationa l
option s of ionophore s ar e no t
necessaril y
a grade d
functio n
of environmenta l
polarit y
bu t ma y
displa y
sudden
shift s
between
metastabl e

state s
ove r
narrow
polarit y
ranges .
Electro -
stati c
interaction s
between
ion s and induce d
dipole s
undoubtedl y
pla y
a determinativ e
rol e
in
catio n
complexatio n by ionophores ,
but the
abilit y
of th e ionophor e to
alte r
it s conformatio n canno t
be ignore d as it is in the assumptio n o f
isosteris m
(19) .
Pharmacologica l
Propertie s
of Carboxyli c Ionophore s
Pharmacologica l

Effects .
Althoug h bot h
neutra l
an d carboxy -
li c
ionophore s
have
bee
extensivel
employed
tool
fo i
vitr o
studie s
of
biologica
viously ,
onl y th e
carboxyli
ionophore
y
by
intac t
animal s to produc e
wel l
define d pharmacologica l
responses . We
initiall y
examined
th e

cardiovascula r
effect s
of
lasaloci d
becaus e o f it s
abilit y
to
transpor t
th e ke y
biologica l
contro l
agents , Ca^
+
an d catecholamine s (20,21) .
However,
we
late r
discovere d
tha t
carboxyli c
ionophore s
selectiv e
fo r
alkal i
ion s
were
even
more
poten t in evokin g th e
same

response s (22) .
Figur e
7
illustrate s
th e tw o
distinc t
primar y
cardiovascula r
effect s
produce d by
monensin.
At lo w concentrations , 50
yg/kg,
i t
produce s a
direc t
dilitation ,
i.e .
relaxatio n
of th e
smooth
muscle
of the coronar y
arteries ,
manifeste d by a
multifol d
in -
creas e
in coronar y bloo d flow . A t
thi s

leve l
or
below,
no othe r
effect s
occur . I f th e
dose
is increase d to 0. 2
mg/kg,
an
ino -
tropi c
respons e
follow s
th e
initia l
coronar y
dilitation .
Thi s
response , an increas e in
cardia c
contractility ,
ca n be monitore d
as th e
maximum
rat e
of
ris e
of pressur e in the
lef t

ventricle ,
LV dP/d t max . Othe r parameter s
paralle l
th e
inotropi c
effect .
Followin g
an
initia l
dro p cause d by
dilitatio n
of th e systemi c
arteries ,
mean
bloo d pressur e
rise s
as
does
puls e pressure , the
interva l
between
lowes t
(diastolic )
an d highes t
(systolic )
tran -
sien t
pressures ; th e
rat e
of bloo d

pumped
by the hear t
(cardia c
output )
als o
rises .
The tw o
distinc t
effect s
ar e thu s an increas e in coronar y
flow ,
which
rapidl y
follow s
injectio n
of th e ionophore , followe d
by an
inotropi c
response ,
which
onl y appear s a t highe r doses .
The
resolutio n
by
dosage
of the two ionophor e response s is
clearl y
apparen t in th e dose-respons e
plo t
of Figur e 8. Coronar y

flo w
rise s
progressivel y
unti l
it plateau s at 10-5 0
yg/kg
monensin.
Highe r dose s caus e a secondar y increas e in flo w re -
flectin g
th e
ris e
in
atria l
pressur e
which
drive s
bloo d throug h
the coronaries . Onl y 2. 5
yg/kg
(i.e .
2. 5 ppb ) ar e
suffucien t
to
doubl e th e basa l flo w
rate .
I t is
possibl e
to detec t the in -
crease d
flo w of 1

yg/kg
(1 ppb ) wit h
statistica l
confidence .
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
14
INORGANI C
CHEMISTR Y
IN
BIOLOG Y
AND
MEDICIN E
1.1 5
Ml/K g
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 time-
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
directly.
0 10 20 30 40 SO 60 70 80 90. lAo
MONENSIN INJECTED
Figure
8. Dose-response curve of coronary flow vs. monensin in the dog. Data
replotted from Ref. 37, as a function of dose at a fixed time interval of 5 min after
injection.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. PRESSMA N E T AL .
Properties
of
Ionophores
15
Mechanism
o f th e Pharmacologica l
Effects .
Tabl e I I
compares
th e jl n
vitr o

io n carryin g capacit y o f a serie s o f
ionophore s
wit h
thei r
inotropi c
potency .
Appreciabl e rate s o f
Ca
2+
o r catechol -
amine
(norepinephrine ) transpor t ar e
observe d
onl y fo r
lasalocid ,
th e
ionophor e
o f th e
group
wit h th e poores t inotropi c
potency .
Extremel y
wide
range s
o f
Ca
2
+
an d norepinephrin e transpor t
capacit y ar e

seen
wit h n o correlatio n wit h inotropi c
potency .
Th e
Ca2+-selectiv e
A-23187
give s onl y a sporati c inotropi c
respons e
wit h th e
intac t
dog . Th e correlatio n
between
inotropi c
potenc y
an d Na
+
transpor t capacit y i s les s negativ e an d i s withi n th e
real m
o f
likel y
difference s
between
th e propertie s o f th e experi -
mental
solven t barrie r
system
an d
thos e
o f actua l
biologica l

mem-
branes .
When
th e
activitie s
o f
ionophore s
ar e
compared
o n th e
basi s o f th e quantit y require d t o releas e a standar d
amount
o f K
+
from
erythrocytes ,
chiefl y
i n
exchange
fo r Na
+
, th e correlatio n
wit h inotropi c
potenc y
i s
even
better .
Cell s
i n genera l contai
electrolyte s

containin g
mediate d
exchange-diffusio n transpor t
thermodynamicall y
favor s
los s
o f
intracellula r
K
+
fo r a roughl y equivalen t
amount
o f Na
+
.
Sinc e th e
relativ e
increas e i n
cellula r
Na
+
induce d b y
ionophore s
i s
considerabl y greate r
tha n
th e
relativ e
los s o f K
+

, w e
infe r
tha t
th e gai n i n
intracellula r
Na
+
,
reflecte d
b y th e
more
conven-
ientl y
measured
releas e o f K+ , i s
more
significan t
tha n
th e los s
of K
+
pe r se . A n additiona l facto r i s tha t
differen t
biologica l
membranes,
e.g . erythrocyte s an d mitochondria ,
respon d
differentl y
to
ionophore s

(23) . Al l thing s
take n
int o consideration , th e dat a
of Tabl e I I ar e reasonabl y supportiv e o f a
mechanism
o f actio n o f
ionophore s
involvin g
initiatio n
o f a n increas e i n
intracellula r
Na
+
.
Many
o f th e effect s o f
ionophore s
appear
t o involv e a n in -
creas e i n
intracellula r
Ca
2
+.
Increase d
contractilit y
implie s a n
increase d
availabilit y
o f

intracellula r
Ca^ + t o trigge r th e
inter -
actio n
o f
acti n
an d
myosin.
A t highe r concentrations ,
monensin
progressivel y
induce s
contractio n o f th e restin g hear t (contrac -
ture ) indicatin g tha t
Ca
2+
activit y
becomes
to o elevate d t o allo w
normal
relaxatio n (24) .
Increase d
intracellula r
Ca
2+
activit y
als o activate s secre -
tor y
cell s
(25) . Inhibitio n studie s indicat e tha t th e inotropi c

effec t
o f
monensin
i s
mediate d
i n par t b y th e releas e o f catechol -
amines
from
th e adrenal s
and/or
th e hear t
itsel f
(22) .
Monensin
als o
discharge s
catecholamine s
from
disaggregate d
bovin e
chromaf-
fi n
cell s
i n cultur e
(26,27) ,
an d
induce s
th e releas e o f acetyl -
cholin e a t th e
neuromuscula r

junctio n (28) .
Thus,
th e secretio n
stimulator y
activit y
o f
monensin
als o
support s
th e
concep t
tha t
increase d
intracellula r
Na
+
activit y
produce s
a
ris e
i n
intra -
cellula r
Ca
2+
activit y
sufficien t
t o stimulat e Ca
2+
-activabl e

cells .
Two
hypothese s
fo r th e conversio n o f a
primar y
increas e i n
intracellula r
Na
+
activit y
t o a
subsequen t
increas e i n
intracellu -
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
Tabl e
II
Compariso n
of
Inotropi c
Potenc y
of
Ionophore s
wit h jl n
vitr o
Transpor t Propertie s
ON
Ionophor e
Inotropi c

Potenc y
Ca
2+
Transpor t
Norepinephrin e
Transpor t
Na
Transpor t
Erythrocyt e
K
+
Releas e
Lasaloci d
(1.0) (1.0)
(1.0)
(1.0)
(1.0)
Lysocelli n
1.5
-
-
-
4.1
Septamyci n
2.0
-
-
- -
Nigerici n
2.8

.000009
0.001
1.4
16.4
Dianemyci n
4.3
.00015 .1
27
18.0
Monensi n
6.1
.000009 .003
31
7.2
X-206
7.7
.000025
.002
2
10.9
Salinomyci n
12.1
- -
-
10.0
A-204
13.1
.000025
.01
20

41
A-23187
+
.37
low
.002
-
Inotropi c
potencie s
were compared
as the
invers e
of the
ionophor e dose
require d
to
doubl e
LV
max
dP/dt . Ca
2+
,
norepinephrin e
and Na
+
transpor t rate s
were
obtaine d
in the
verti -

call y
stacke d thre e
phase syste m
describe d
in
ref.
(39).
Erythrocyt e
K
+
releas e
potenc y
was
measured
as the
invers e
of the
concentratio n require d
to
releas e
10 mM K
+
from
washed
human
erythrocyte s
suspende d
in mock
plasm a
containin g

5 mM KC1, 145 mM
NaCl
and 10 mM
TRIS
chloride ,
pH 7.4.
2
o
>
=
g
H
3
w
3
r
8
>
a
w
a
o
3
w
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. PRESSMA N E T AL .
Properties
of
Ionophores

17
la r
Ca
2+
ar e plausible . On e
would
b e a n exchange-diffusio n
carrie r
i n th e
plasma
membrane
permittin g th e larg e
Ca
2+
activit y
gradien t ( a 10" 3 M
extracellular ,
a 10"
7
M
interior )
t o permi t
entr y o f
Ca
2
+
int o th e
cel l
i n
exchange

fo r Na
+
. (O n
thermodynamic
grounds
on e
would
expec t
th e
exchange
rati o
t o b e 3- 4 Na
+
expelle d
fo r
each
Ca
2+
take n
up) .
Thus,
making
more
intracellula r
Na
+
availabl e
fo r
exchange,
o r i n

thermodynamic
terms
reducin g th e
gradien t agains t
which
Na
+
must
move
( a 10~
2
M
intracellular ,
a
10- 1 M
extracellular) ,
would
favo r th e entr y o f
Ca
2+
.
A
critica l
evaluatio n
o f
thi s
hypothesi s ha s
appeare d
i n a recen t
revie w

(29) . A n alternat e
mechanism
would
b e th e releas e o f
intracellu -
lar ^
bound
Ca
2+
b y displacemen t b y Na+ . Thi s i s
feasibl e
sinc e
th e gros s chemica l
Ca
2+
intracellula r
concentratio n i s ca . 10" " 3 M
whil e i t require s onl y 10"~ 6 - 10~
5
M
Ca
2+
activit y
t o activat e
contractio n
o r secretion .
There
might
wel l
exis t

purposefu l
Ca
2+
-
Na
+
ion-exchang e
site s
withi n
cell s
s o designe d tha t onl y a smal l
relativ e
Na
+
activit y
chang
relativ e
Ca
2+
activit y
chang
sufficien t
t o activat e
Ca
2+
-dependent
intracellula r
processes .
Impact
o f

Ionophore s
o n Ma n an d
Animal s
Carboxyli c
Ionophore s
an d
Efficienc y
o f
Feed
Conversio n b y
Livestock . A stron g
note
o f relevanc e t o studie s o f th e chemica l
an d pharmacologica l propertie s o f carboxyli c
ionophore s
derive s
from
th e larg e scal e us e o f
monensin
a s a livestoc k fee d additive .
Th e rational e i s tha t carboxyli c
ionophore s
contro l
endemic
coccidiosi s
i n th e poultr y gu t (30 ) an d
promote
a
more
favorabl e

fermentatio n o f
cellulos e
i n th e
bovin e
rumen
(31) . I n eithe r
case , th e ne t
resul t
i s th e economicall y importan t increase d
efficienc y
o f conversio n o f fee d int o
meat.
Pharmacokinetic s
o f
Ionophore
Absorption . W e
have
develope d
a sensitiv e chemica l
assa y
fo r carboxyli c
ionophore s
(whic h
wil l
be publishe d
elsewhere )
based
o n
thei r
abilit y

t o
form
lipi d
solu -
bl e
complexes
wit h cations . W e ca n detec t a s
littl e
a s 1 par t pe r
billio n
(ppb )
monensin
i n 2 m l o f bloo d
plasma
o r
tissue .
Fo r a
compariso n
yardstick , curren t feedin g
regimens
cal l
fo r ca . 3 0
part s pe r
millio n
(ppm ) i n
cattl e
fee d (32 ) an d a s
much
a s 10 0 pp m
i n

poultr y fee d (33) .
Typically ,
a co w ingest s
about
0. 3 g ( ^ 1 ppm )
monensin/day.
As previousl y
observe d
i n Figur e 7 , a s
littl e
a s 1
ppl>
(base d o n
body
weight)
produce s
a detectabl e physiologica l
effec t
o n th e
dog .
In orde r t o establis h th e pharmacokineti c relationship s
between
orall y
ingeste d an d intravenousl y injecte d
monensin,
w e
carrie d
ou t preliminar y studie s o f
monensin
bloo d

level s
i n th e
dog . I n Figur e 9 w e se e tha t injecte d
monensin
clear s
from
th e
plasma
wit h a t ^ o f ^ 2. 5
minutes
which
w e
presume
i s to o rapi d
fo r
th e operatio n o f
normal
eliminatio n
mechanisms.
Hence,
i t is
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
18
INORGANI C
CHEMISTR Y
IN
BIOLOG Y
AND
MEDICIN E

MIREHSIR
f
1IMI/K(
I.I. IKE
2SII
20N
it!i\
ISM
sec
100 0
2**1/11
HAL USE
60
90 120 ISO
MINUTE S
AFTER
00S E
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.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. PRESSMA N E T AL .
Properties
of
Ionophores
19
reasonabl e t o
assume
tha t th e
ionophor e
leavin g th e
plasma
i s
take n
u p b y th e tissues . Thi s
would
no t a t al l be
unexpecte d
con-
siderin g
th e hig h lipid:wate r
partitio n
coefficien t
o f ionophores .
I t
i s supporte d b y th e delaye d an d persisten t elevatio n o f th e
ionophore-sensitiv e cardia c functio n
parameter ,
L V
dP/dt.

Pre -
liminar y
trial s
o f a
variatio n
o f ou r
assa y
adapte d
fo r
whole
tissue s
indicat e tha t i n th e rabbi t th e
major
portio n o f
monensin
appears
i n th e tissue s withi n 1 0
minutes
followin g i.v .
injection ,
at
concentration s roughl y
parallelin g
th e
degree
o f bloo d perfu -
sion :
lun g > hear t > kidne y >
liver ,
muscle,

fat .
Th e
lowe r
graph
o f Figur e 8
compares
th e
time
cours e o f ap -
pearanc e
i n th e
plasma
o f injecte d an d
orall y
administere d
monensin
doses
i n th e dog . Th e
ora l
dose
appear s
i n th e bloo d
more
slowl y bu t
produce s
more
sustaine d
ionophor e
bloo d
levels .

The
time
concentratio n
integra l
give s a n inde x o f th e quantit y o f
th e
dru g
which
passe s
throug h
th e
plasma;
rat e o f entr y an d
clear
ance
from
th e bloo d
affec
ne t
integral .
Th e
integra
th e
integra l
o f a
known
dose
administere d
directl y
int o th e bloo d

Althoug h
differen t
animal s
an d
differen t
dose
level s
were
used,
th e
rati o
o f th e
i.v.iora l
dose
integral s ar e approximatel y pro -
portiona l
t o th e 1:2 0
ratio s
o f th e ne t
doses
administered . Thi s
signifie s
tha t a
major
portion , i f no t al l of th e
orall y
ingeste d
monensin
dose,
passe s

throug h
th e bloo d
strea m
o f th e do g befor e
bein g eliminated . I n th e rabbit , a herbivore , on e
might
predic t
absorptio n o f
ora l
doses
would
b e slower . W e ca n detec t
orall y
administere d
monensin
doses
i n rabbi t
plasma,
bu t onl y
afte r
a
coupl e o f
hours
followin g ingestion . W e
have
no t ye t
complete d
th e
more
prolonge d

plasma
level-tim e
profile s
i n
thi s
species .
Th e
Need
fo r Increase d Surveillanc e o f th e
Exposure
o f Ma n
to
Ionophores .
From
th e
lipi d
solubilit y
o f
monensin
an d othe r
ionophores , w e
would
predic t the y shoul d
have
n o troubl e
equili -
bratin g
acros s
biologica l
membrane

systems
includin g th e gut . Thi s
i s
certainl y
th e
cas e
fo r th e tw o divers e specie s
observed ,
th e
dog , a carnivore , an d th e rabbit , a herbivore . Accordingly , w e
infe r
tha t ther e i s
ample
opportunit y fo r
monensin
an d othe r
carboxyli c
ionophore s
administere d
orall y
t o livestoc k t o
distrib -
ut e systemicall y an d exer t a pharmacologica l
effec t
o n th e
recipi -
en t animal .
Furthermore ,
th e resultan t physiologica l
effect s

ma y
be par t o f th e
mechanism
b y
which
ionophore s
produce
thei r
im -
prove d
fee d conversio n
efficiency .
There
ar e furthe r inference s
which
directl y
affec t
man . I f
th e
ionophore s
d o
pervade
th e tissues , i t i s possibl e tha t ma n ma y
become
exposed
t o pharmacologicall y
competent
an d
potentiall y
detrimenta l

level s
o f
ionophore s
throug h
hi s
meat
supply .
Based
o n
limite d
pharmacokineti c an d
toxicologica l
data , th e
F.D.A.
ha s se t
upper
permissibl e
level s
o f 0.0 5 pp m i n
meat
fo r
human
consumptio n
(34) . Th e isotop e residu e studie s o f
Herber g
et
al . repor t tha t
under
curren t feedin g
procedure s

cattl e
live r
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
20
INORGANI C CHEMISTR Y IN BIOLOG Y AN D MEDICIN E
may accumulat e ove r ten time s
thi s
leve l
of
monensin
as a
combina-
tio n
of paren t
compounds
and metabolite s of
unknown
pharmacologi -
ca l
effect s
(35) .
Thi s
dat a was obtaine d 12 hour s
afte r
adminis -
tratio n
of tagge d
monensin.
On e migh t surmis e

tha t
residue s
would
be
appreciabl y
highe r for an anima l butchere d a
shorte r
perio d
of tim e
followin g
it s
las t
exposur e to
monensin.
Thi s
is
particularl y
significan t
in
tha t
literatur e
supplie d
to farmer s
advise s
tha t
no withdrawa l
perio d
is necessary .
Currentl y
availabl e

methods
for assayin g
monensin
involv e
cumbersome
extractio n
procedures ,
thi n
laye r
chromatograph y and
detectio n
by
means
of bioautograph s wit h microorganism s
whose
sensitivit y
to ionophore s and
thei r
metabolite s (36 ) may or may
not
paralle l
mammalian
sensitivity .
Th e simpl e chemica l assa y
method
we
have
develope d can provid e a
more
rationa l

basi s
for
assignin g
permissibl e
residu e
levels ,
fo r
routinel y
monitorin g
product s
arrivin g
at the
market
and
ascertainin g
whether
stipu -
late d
ionophor e withdrawa
Additiona l
complication
notabl y
poo r
biodegradabilit y
of
monensin.
Report s
indicat e
tha t
cattl e

fecall y
eliminat e
75% of
ingeste d
monensin
withou t degra -
dation .
Furthermore ,
60-70%
of the
monensin
survive s
10
weeks
in-
cubatio n
at 37 ° (34) . Curren t
manuring
practice s
rende r it pru -
dent to determin e
whether
crop s or garde n produc e tak e up
signifi -
can t
quantitie s
of
carboxyli c
ionophore s or
whether

the obviousl y
larg e
soi l
burden s of suc h
compounds
fin d
thei r
way
int o
wate r
supplies .
We
have
lon g
been
intereste d
in the
possibilit y
tha t
the
cardiovascula r
effect s
of
carboxyli c
ionophore s
coul d
be harnesse d
to
provid e new drug s fo r th e treatmen t of diseas e
state s

suc h as
hear t
failur e
and shock . Ther e may ,
however,
be subpopulation s of
man fo r
whom
ionophore s may be
particularl y
toxic .
Fo r
example,
a
toxi c
interactio n
between
monensin
and
digitali s
on th e dog
hear t
has
been
reporte d (37) . Our
ora l
absorptio n data do
indi -
cat e
tha t

if a
usefu l
human
therapeuti c
applicatio n
can be es -
tablished ,
ionophore s
coul d
be administere d as drug s
orally .
Summary
We
have
describe d
how th e uniqu e
physica l
propertie s
of iono -
phor e molecule s
lea d
to
bette r
understandin g of
thei r
uniqu e
bio -
logica l
effects .
Ionophore s

have
been
applie d
as
tool s
for
biologica l
research , as commerciall y importan t
livestoc k
fee d
additive s
for
increasin g
the
efficienc y
of
meat
production , and in -
vestigate d
as
potentiall y
usefu l
drug s in man.
Expertis e
derive d
from
studie s
of th e molecula r
propertie s
of ionophore s has

been
utilize d
to desig n a simpl e assa y procedur e whic h give s promis e
fo r
providin g
more
rationa l
safeguard s fo r man in the widesprea d
use of ionophore s in foo d production .
Lastly ,
in view of the
burgeonin g
increase s
in the
scal e
of commercia l ionophor e usage ,
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1.
PRESSMA N
ET
AL.
Properties
of
Ionophores
21
i t
appear s urgen t
tha t
we

increas e
our
understandin g
in
depth
of
the
physiologica l
and
metaboli c
effect s
of
ionophore s
and
thei r
pharmacologica l
and
toxicologica l
ramifications .
Acknowledgements
We
wish
to
acknowledge
the
assistanc e
of Ms.
Georgin a
Del
Vall e

and Mr.
Fran k
Lattanzi o
in
the developmen t
of the
iono -
phore assa y
and
Drs.
L.
Alle n
and M.
Kolbe r in
helpin g
progra m
the
computer
studies .
We
are indebte d
to
Eli
Lill y
for sample s
of
monensin
and A.H.
Robbin s
and

Kaken
Chemica l
Co.
(Japan )
fo r
salinomycin . Thes e
studie s
were
supporte d
in
par t
by NIH
gran t
HL-23932
and a
gran t fro m the
Florid a
Affiliat e
of the
America n Hear t
Association .
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.
"Membrane-
Active
Complexones";
Elsevier:New York, 1975; Vol. 12.
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,
1977, B31,
part
10,
2411-2415.
16. Djerassi, C. "Optical
Rotatory
Dispersion: Applications to
Organic
Chemistry";
McGraw-Hill:New
York, 1960; pp.
41-48.
17. Moffitt, W.;
Woodward,
R.B.; Moscowitz, A.; Klyne, W.;
Djerassi, C. J. Am.
Chem.

Soc., 1961, 83,
4013-4018.
18.
Kossower,
E.M. J. Am.
Chem.
Soc., 1958, 80,
3253-3260.
In Inorganic Chemistry in Biology and Medicine; Martell, A.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.