1984 sharma vima blood brain barrier brain research
TRANSCRIPT
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
1/14
Brain Re.~earch.
3 0 8 ( 1 9 8 4 ) 2 0 1 - 2 1 4 2{/1
E l s e v i e r
B R E 1 0 1 S 4
esearch eports
T h e o r e t i c a l In t e r p r e t a t io n o f E x t r a c ti o n i n B r a i n ) o f P e p t i d e s
I n c l u d i n g C o n c e n t r a t i o n V a r i a t io n s
R . R . S H A R M A a n d R . L . P . V I M A L
Department of Physics, University o[ lllinois at Chicago, Chicago, IL 60680 U.S.A.
( A c c e p t e d D e c e m b e r 2 0 t h , 1 98 3)
Key words:
e x t r a c t i o n - - p e p t i d e s - - b l o o d - b r a i n b a r r i e r b r a i n u p t a k e - - d i f f u s i o n , f a c i li t a te d
T h e t r a n s p o r t p r o p e r t i e s o f s e v e r a l p c p t i d e s a c r o s s b l o o d - b r a i n b a r r i e r ( B B B ) h a v e b e e n i n v e s t i g a t e d t h e o r e t ic a l l y i n t e r m s o f s im -
p l e d i ff u s i o n a n d f a c i li t a te d d i f f u s i o n p r o c e s s e s . C o m p a r i s o n o f t h e c a l c u l a t e d r e s u l ts f ro m t h e s i m p l e d i f f u s i o n a n d t h c e x p e r i m e n t a l
d a t a r e v e a l s t h e p r c s e n c e o f t h e f a c i li t a t ed d i f f u s i o n o f t h e s e s u b s t a n c e s w h i c h w e h a v e c o n c e i v e d o f a s a c a r r i e r - m e d i a t e d p r o c e s s . T h e
v a l u e s o f t h e p a r t i t i o n c o e f f i c i e n t s f f o r t h e s e p e p t i d e s w e r e i n t h e r a n g e 7 x l (I - a ~< f ~< 2 0 0 1 0 4 . T h e c a l c u l a t e d f v a l u e s g a v e p e r -
m e a b i l i t i e s . P , . in l ip i d s b e t w e e n l l) - r ~< p , ~ < 1 4 x 1 0 ~ c m / s . T h e s e v a l u e s w e r e t h e n u s e d t o e s t i m a t e t h e e x t r a c t i o n f o r p e p t i d e s f r o m
s i m p l e d i f f u s io n a lo n e w h i c h v a r y f r o m 0 . 3 t o 3 . 5 % c o m p a r e d w i t h t h e e x p e r i m e n t a l e x t r a c t i o n ( 0 . 4 - 1 2 % ) i n d i c a t i n g t h e i n a d e q u a c y
o f t h e s i m p l e d i f f u s io n a l o n e t o e x p l a i n t h e e x p e r i m e n t a l d a t a . A s f o r t h e c a r r i e r - m e d i a t e d f a c i li t a t ed d i f f u s i o n p r o c e s s w e h a v e u s e d
t h e a c t i v a t e d - c o m p l e x t h e o r y . T h e e x t r a c t i o n i n t h i s c a se d e p e n d s o n t h e m a x i m a l r a te o f t r a n s p o r t ( T m ax ) f a n d t h e r e c i p ro c a l o f th e a f -
f i n it y c o n s t a n t K , f o r t h e t r a n s p o r t o f p e p t i d e s t h r o u g h B B B . W e h a v e d e d u c e d t h a t ( T m ax ) f ~ 0 . 4 6 1 0 3 p m o l / g ' s a n d & ~ 0 . 3 5 n M
f or M e t - e n k e p h a l i n ( M e t - E N K ) , L e u - e n k e p h a l i n ( L e u - E N K ) , g l u t a t h i o n e , c a r n o s i n e , a - M S H a n d M I F a n d ( T ra de ) f ~ 1 0 1 0 3
p m o l / g ' s a n d K , - 7 n M f o r A V P , t i L T , f iE a n d a E t o e x p l a in t h e o b s e r v e d r e s u l t s . W e h a v e a l s o o b t a i n e d t h e q u a n t i t a t i v e v a r i a t i o n o f
e x t r a c t i o n w i th c o n c e n t r a t i o n o f p e p t i d e s i n t h e b r a i n - c a p il l a r y a n d h a v e e s t a b l i s h e d t h a t t h e e x t r a c t i o n d e c r e a s e s w i t h i n c r e a s i n g c o n -
c e n t r a t i o n o f p c p t i d e s , t e n d i n g t o a s m a l l c o n s t a n t v a l u e a t h i g h c o n c e n t r a t i o n s . I t h a s b e e n i n f e r r e d t h a t c a r r i e r - m e d i a t e d f a c i li t a te d
d i f t u s io n i s i m p o r t a n t f o r t h e t ra n s p o r t o f p e p t i d e s a c r o s s B B B .
I N T R O D U C F I O N
The knowledge of the transport of peptides across
blood-brain barrier (BBB) has been of considerable
interest~ :3 in recent years. Some pept ides effect cen-
tral nervous system (CNS) pharmacologically and
exert behavioral changes in animals and in menS,14,1~.
It has been reported ~,> that in bacteria and mamma-
lian intestines, the transport of peptides is via satu-
rable carrier systems.
in recent years, several measurements of the
transport of peptides across BBB have been report-
ed. The experimental data show large variations with
respect to concentration and nature of the peptides.
Some investigators~2,~3 have reported that the pep-
tides permeate readily across BBB with brain-uptake
index (BUI) values lying between 1 0-15 . On the
other hand, there have been observation s 5 which re-
port that the peptides are less permeable in BBB with
BUI values between 2. 4-3 .6 . In particular,
Greenbe rg et a1.12 have e mploy ed lower concent ra-
tions of peptides in their investigations and have re-
ported BUI values of peptides much higher than the
values deduced by Cor nford et al. 5 who have used
higher concentratio n of peptides.
A close analysis of the experimen tal resultsS, 12
shows that, in general, the extractions of peptides de-
crease with the increase of concentration of the pep-
tides approaching for large concentrations to a
(somewhat) small constant value characteristic of
saturation effect. These results suggest peptide-car-
tiers for the transport of peptides across the BBB.
Since the asymptotic value of the experimental ex-
traction (E) in various cases does not exactly ap-
Corres7)ondence:
R . R . S h a r m a , D e p a r t m e n t o f P h y s i c s, U n i v e r s i t y o f I ll i n o is at C h i c a g o , C h i c a g o , I L 6 0 6 8 0 , L I .S . A .
0 0 0 6 - 8 9 9 3, '8 4 / I ) 3 .0 0 1 9 8 4 E l s e v i e r S c i e n c e P u b l i s h e r s B . V .
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
2/14
2 2
p r o a c h t o z e r o , o n e is l e a d t o p r e d i c t t h e e x i s t e n c e o f
a n o t h e r p r o c e s s w h i c h c o u l d g i v e a c o n s t a n t , t h o u g h
s m a l l, c o n t r i b u t i o n t o E p a r t i c u l a r l y a t h i g h c o n c e n -
t r a ti o n s . I n f a c t , o u r p r e s e n t i n v e s t i g a ti o n s s h o w t h a t
t h e s i m p l e d i f f u s i o n p r o c e s s w h i c h d e p e n d s o n t h e
l ip i d s o l u b i l i t y o f p e p t i d e s ( p a r t i t i o n c o e f f i c i e n t ) c o n -
t r ib u t e s t o t h e s m a l l c o n s t a n t v a l u e o f t h e e x t r a c t i o n
o b s e r v e d a t h i g h c o n c e n t r a t i o n s .
T h e a i m o f t h e p r e s e n t p a p e r is t o e x a m i n e t h e t w o
i m p o r t a n t m e c h a n i s m s , n a m e l y , t h e c a rr i e r- m e -
d i a t e d f a c i l i t a t e d d i f f u s i o n a n d t h e s i m p l e d i f f u s i o n
w h i c h t o g e t h e r c o u l d b e r e s p o n s i b l e f o r t h e t r a n s p o r t
o f t h e p e p t i d e s a c r o s s t h e B B B . F o r s i m p l e d i f f u s io n
p r o c e s s e s o n e n e e d s t o t a k e i n t o a c c o u n t a p p r o p r i -
a t e l y t h e v a r i a t i o n o f t h e s o l u b i l it y o f v a r i o u s p e p -
t i d e s i n l i p i d s . I n t h e f o l l o w i n g s e c t i o n a t h e o r y r e l e -
v a n t t o th e t r a n s p o r t o f p e p t i d e s i n c o r p o r a t i n g e x -
p l ic i ty t h e p a r t i t io n c o e f f i c ie n t s a p p r o p r i a t e t o p e p -
t i d e s i n l i p id s h a s b e e n g i v e n . T h e e x p r e s s i o n f o r th e
p e r m e a b i l i t y f o r m a l l y c o m e s o u t t o b e t h e s a m e a s
t h e o n e w r it t en d o w n p h e n o m e n o l o g i c a l l y b y v a r io u s
a u t h o r s i n t h e l it e r a t u re , a n d d e p e n d s o n t h e c o n c e n -
t r a t io n o f t h e s u b s t a n c e i n t h e b r a i n - c a p i l la r y a n d o n
t w o k in e t ic c o n s ta n t s , ( T ~ ) f , t h e m a x i m a l r at e o f
t r a n s p o r t a n d
K
t h e M i c h a e l i s - M e n t e n c o n s t a n t
r e p r e s e n t i n g t h e r e c i p r o c a l o f th e a f f i n i ty o f b i n d i n g
o f th e s u b s t a n c e t o a c a r r ie r . W e h a v e u s e d t h i s e x -
p r e s s i o n t o e s t i m a t e t h e e x t r a c t i o n d u e t o t h e c a r r i e r -
m e d i a t e d f a c i l i t a t e d d i f f u s i o n a n d h a v e f o u n d t h a t i t
c a n e x p l a in i n c o n j u n c t i o n w i th t h e c o n t r i b u t i o n f r o m
s i m p l e d i f f u s i o n t h e o b s e r v e d e x p e r i m e n t a l d a t a a t
v a r i o u s c o n c e n t r a t i o n s .
T H E O R Y
A g e n e r a l e x p r e s s i o n f o r c a l c u l a t i n g th e e x t r a c t i o n
f o r c o m p l e x c a s e s i n v o l v i n g v a r i o u s d i f f u s i o n p r o c -
e s s es h a s n o t b e e n a v a i l a b l e in th e l i t e r a tu r e . H o w e v -
e r , a b a s i c f o r m u l a w h i c h c o n n e c t s t h e p e r m e a b i l i t y
( P ) a n d e x t r a c t i o n ( E ) is g i v e n b y :
P = - ~ In ( l - E )
w h e r e F is t h e r a t e o f b l o o d f l o w a n d S i s t h e s u r f a c e
a r e a o f th e b r a i n c a p i l l a ry . W e h a v e d e r i v e d e l s e -
w h e re 2 5 a n e x p r e s s i o n f o r E b y s o l v i n g m a t h e m a t i c a l
r e l a t i o n s d e s c r i b i n g t h e t e m p o r a l a n d s p a t i a l v a r i a -
t i o n s o f th e c o n c e n t r a t i o n o f a s u b s t a n c e i n t h e b r a i n
c a p i l la r y a n d e x t r a v a s c u l a r s p a c e o f th e b l a i n s t a r t m #
f r o m a g e n e r a l f o r m a l i s m T h e e x p r e s si o n ~ c o m c ~
o u t t o b e :
E = l - e x p - c , ; + K ; i I
w h e r e P , (c m / s ) is t h e p e r m e a b i l i t y o f t h e s u b s t a n c e
i n t h e b l o o d - b r a i n b a r r i e r d u e t o s im p l e d i f f u s i o n : S
i s i n c m Z/g o f b r a i n ; F i s i n c m L g s : c~ ( nM ) i s t he c on -
c e n t r a t i o n o f t h e l a b e l e d p e p t i d e i n t h e b o l u s o f s in g l e
i n j e c t io n t y p e ,
T m a x ) f
( in p m o l / g ' s : m M = u m o l / c m 3 )
is t h e m a x i m a l r a t e o f t r a n s p o r t d u e t o f a c i l i ta t e d d i l l
f u s i o n a n d K , ( in m M ) i s t h e M i c t i a e l i s - M e n t e n c o n -
s t a n t r e p r e s e n t i n g t h e r e c i p r o c a l o f th e a f f i n i t) o f a
p e p t i d e t o i ts c a r r i e r in th e b l o o d - b r a i n b a r r i e r
BBB).
E q n . 1 f o r g i s a p p r o p r i a t e l o th e t r a n s p o r t o / a
s u b s t a n c e f o r a s i n g l e t r a n s i t o f a b o l u s t h r o u g h b r a i n -
c a p i l la r y w h e n b o t h t h e s i m p l e a n d f a c i li t a te d d i ff u -
s i o n p r o c e s s e s a r e s i g n if i c an t . T h e b r a i n - u p t a k e i n-
de x i s r e l a t e d t o E by t h e e xp r e s s i o nZ ,~.
E
B U I
- EHOI_ t
( 2 )
w h e r e E H O H s t a n d s f o r t h e e x t r a c t i o n : o f w a t e r .
F o r p e n e t r a t i o n o f a p e p t i d e m o l e c u l e v i a s i m p l e
d i f fu s i o n t h r e e p o s s i b l e b a r r i e r s m a y b e c o n s i d e r e d
t o b e e n c o u n t e r e d b y th e m o l e c u l e a s h a v e b e e n d is -
c u s s e d b y D a n i e l l i 7 . T h e f i r s t b a r r i e r ( s e e F i g . 1 ) c o n -
s i d e r e d is a t t h e w a t e r - m e m b r a n e i n t e r fa c e w i t h a
b a r r i e r e n e r g y / ~ a f o r th e m o t i o n o f a m o l e c u l e f r o m
BL OOD-BRAIN BARRIER
A
EXTERNAL a e o . INTERNAL
/ I POTENTIAL BARRIER T :, i I a
AQUEOUS
MEDIUM MEDIUM
(BL OOD) . ,~ IA PEPT IDE MOL ECUL E ~ (BRAIN ECF :)
t
B D
F 4
Fig, 1. Schem atic diagram of a blood~b rain barrier w ith the
curve representing the potential barrier encou ntered by a pep-
t ide molecule. A B is the m embrane interface for diffusion from
water to the lipid medium With barrier-energy u~;/~c represents
the intermittent barrier height with:n inaxima, CD is the mem-
brane interface for the diffusion from l ipid to water medium
with barrier-energy #b' 2 is the wavelength (distance between
two maxima) and d is the thickness of the bloo d-b rain barrier .
EC F stands for extracellular f luid
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
3/14
t h e e x t e rn a l w a t e r - m e d i u m ( b l o o d s i d e ) to t h e i n te r -
n a l l i p id - m e d i u m in th e m e m b r a n e . T h e s e c o n d b a r -
r i e r e f f e c t i v e l y i n v o l v e s m a n y s u c c e s s i v e b a r r i e r s i n
t h e in t e r i o r o f t h e m e m b r a n e w i t h b a r r i e r e n e r g y / , ~ .
T h e t h i r d b a r r i e r i s l o c a t e d a t t h e o t h e r s i d e o f t h e
m e m b r a n e ( a g a i n , m e m b r a n e - w a t e r i n t e r f a c e ) w i t h
a b a r r i e r e n e r g y ,t4 ~. T h e e x p r e s s i o n f o r t h e p e r m e a -
b i li ty P , o f a m e m b r a n e t o a m o l e c u l e d u e t o s i m p l e d i f-
f u s i o n c a n b e w r i t t e n
as 7
e
P ~ - n b + 2 e (3 )
w h e r e
Z m V l R T
e=y~ ~exp -RT
b = , b V 2 ~ M e x p -
4 )
5 )
6 )
w h e r e 7 is a c ons t a n t , q : ', , , q :' c, e bb , a r e t he p r o ba b i l i -
t ie s fo r p e n e t r a t i o n o f a p e p t i d e m o l e c u l e a c r o s s t h e
ba r r i e r o f e ne r gy / ~ a , f~o a n d ,U b, r e s p e c t i v e l y ; n i s t he
n u m b e r o f b a rr i e r s in t h e i n te r i o r o f t h e m e m b r a n e .
R is t h e s t a n d a r d g a s c o n s t a n t ; M i s t h e m o l e c u l a r
w e i g h t o f th e d i f f u s i n g m o l e c u l e a n d T i s t h e a b s o l u t e
t e m p e r a t u r e . T h e e n e r g y ,u~, is e x p e c t e d t o b e n e a r l y
e q u a l t o t h e b a r r i e r e n e r g y / 4 , f o r a m o l e c u l e p e n -
e t r a ti n g th e l i p i d - w a t e r i n t e r f a c e . A c c o r d i n g l y , f r o m
E q n s . 5 a n d 6 w it h t h e a s s u m p t i o n : @ ~ = q < ,,
b = e ( 7 )
T h e n u m b e r o f m a x i m a n c an b e a p p r o x i m a t e l y w r it -
t e n a s ,
a
n ~ 8 1
w h e r e d i s t h e t h i c k n e s s o f t h e B B B a n d 2 i s t h e a v e r -
a g e w i d t h o f t h e i n t e r m i t t a n t b a r r i e r i n s id e t h e B B B .
M a k i n g us e o f d - ~ 78 A ( r e f . 26 ) a nd ~. - ~ 1
A ( r e f . 7 ) w e ge t 11 = 78 w h i c h i m p l i e s f o r t he B B B
t ha t ,
n b > > 2 e ( 9 )
C o n s e q u e n t l y , f r o m E q n . 3 o n e o b t a i n s .
203
P s _ f e ( 1 0 )
w h e r e
a (11)
f
b
T h e f a c t o r f i s t h e p a r t i t i o n c o e f f i c i e n t 7 r e p r e s e n t i n g
t h e r e l a t i v e s o l u b i l i t y o f t h e d i f f u s i n g m o l e c u l e i n t h e
l ip i d m e m b r a n e , t h a t i s , t h e f r a c t i o n o f m o l e c u l e s i n
t h e m e m b r a n e c o m p a r e d t o th o s e in w a t e r. F r o m
E q n s . 5 a n d 1 0 w e h a v e ,
f
P ~ = K / ~ ~ ( 12 )
w i t h K as t h e p r o p o r t i o n a l i t y c o n s t a n t d e f i n e d b y :
7_~ 'e 2~ ex p - (13)
I n t h e a b o v e K d e p e n d s o n t h e c h a r a c t e r is t i c s o f t h e
b a r r i e r s y s t e m , p a r t i c u l a r l y , o n t h e b a r r i e r h e i g h t ~ c
a n d th e m e m b r a n e t h i c k n e s s ( p r o p o r t i o n a l t o n) . A s
f~ e d o e s n o t v a r y s i g n i f i c a n t l y : ( f r o m o n e s u b s t a n c e t o
t h e o t h e r ) K is a s s u m e d t o b e t h e s a m e f o r t h e p e p -
t id e s c o n s i d e r e d . I n o r d e r t o e s t im a t e t h e v a l u e o f K
f o r p e p t i d e s w e h a v e a d o p t e d a n i n d i re c t m e t h o d
b a s e d o n s u c r o s e a s th e r e f e r e n c e m a t e r i a l , a s e x -
p l a i n e d i n C a l c u l a t i o n s a n d R e s u l t s .
I n E q n . 1 3 , D a n i e l l F h a s a r g u e d t h a t , b e s i d e s t h e
f a c t o r e x p ( - / ~ j R T ) , t h e f a c t o r 7 g '~ c o u ld b e t e m p e r -
a t u r e d e p e n d e n t . I n f a c t, t h e t e m p e r a t u r e d e p e n -
d e n c e o f t h e p r o d u c t 7 g k, e x p ( - u j R T ) is d e c i d e d b y
t h e sy s t e m u n d e r c o n s i d e r a t i o n . W e sh a ll a s s u m e
h e r e t h a t t h e p r o d u c t } , 0 e x p ( - , tk , / R T ) i s a s lo w l y
v a r y i n g f u n c t i o n o f t e m p e r a t u r e .
I n o u r c a s e t h e p a r t i t i o n c o e f f i c i e n t f r e p r e s e n t s t h e
s o l u b i l i ty o f a p e p t i d e m o l e c u l e i n t h e li p id m e d i u m
a s s o c i a t e d w i t h t h e B B B c o m p a r e d t o t h e m e d i u m
( a s s u m e d a q u e o u s ) o u t s i d e t h e B B B . I t h a s b e e n
f o u n d m t h a t t h e b e n z e n e r in g s a n d C H e - g r o u p s o f a
m o l e c u l e i n c r e a s e t h e v a l u e o f t h e p a r t i t i o n c o e f f i -
c i e n t f , w h e r e a s t h e g r o u p s s u c h as , - O H , a n d
- C H 2 C O N H d e c r e a s e its v a lu e .
A n e q u a t i o n u s e fu l f o r t h e e v a l u a t i o n o f f m a y b e
w r i t t e n a s 4 :
l gm fpcp = logm f rcf + n i b i (14)
wh r f pe p is th e p a r t i t i o n c o e f f i c i e n t o f a p e p t i d e , a n d
fre~ i s t he pa r t i t i on c oe f f i c i e n t o f a r e f e r e n c e s ub -
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
4/14
2 0 4
F A B L E I
b , c o n t r i b u t i o n s
List of i con t r ibu t ions o f va r ious s ing le g roups (componen ts~
cons t i tu t ing the pep t id e so lu te to log m f wh ere f is the pa r t i -
t i on c o e f f i c i en t o f t h e s o l u t e . T h e b , v a l u e o f t h e - O H g r o u p i s
n o t k n o w n a c c u r a t e l y a n d t h e r e f o r e t h e r e l e v a n t u n c e r t a m t ~
has been g iven .
G r o u p i n a s o l u t e ( i ) b i o f s o l u t e
- C H 2 ( w i t h t e r m i n a l m e t h y l g r o u p ) 0 .4 9
B e n z e n e r i ng
1 4 0
- O H - 0 . 6 0 + 0 .0 4
- C H 2 C O N H o r - C H R C O N H - 0 .8 4
s t a n c e ; n i r e p r e s e n t s t h e n u m b e r o f t h e i th g r o u p p r e s -
e n t i n a p e p t i d e ; b i i s t h e e f f e c t o f a s i n g l e i h g r o u p o n
l o g m f p~ p. T h e a p p r o x i m a t e v a l u e s 4 o f b i o f v a r i o u s
g r o u p s r e q u i r e d i n l o g m fp~ p a r e c o m p i l e d i n T a b l e 1
f o r e a s y r e f e r e n c e . F o r t h e r e f e r e n c e s o l u t e g l y ci n e
( H - C H C O O H N H 2 ) C o h n a n d E d s t a l l 4 c i te l o g m fC y
= - 3 . 3 9 1 . E q n . 1 4 t h e n g i v e s ,
l O g l 0 f pe p = - 3 . 3 9 1 + X n i b i ( 1 5 )
E g n s . 1 2 a n d 1 5 a r e u s e f u l f o r e s t i m a t i n g t o t h e e x -
t r a c t i o n f r o m s i m p l e d i f f u s i o n .
A s f o r o b t a i n i n g t h e c o n t r i b u t i o n t o E f r o m f a c i l i-
t a t e d d i f f u s i o n i t i s e v i d e n t f r o m E q n . 1 t h a t o n e
n e e d s t o k n o w t h e p a r a m e t e r s ( Tm a x) t a n d K , w h i c h
d e p e n d o n th e i n t e ra c t i o n b e t w e e n t h e s u b s t a n c e a n o
t h e c a r r i e r s i n t h e b l o o d - b r a i n b a r r i e r . A t p r e s e n t ~t
is n o t p o s s i b l e t o c a l c u l a t e t h e s e p a r a m e t e r s f r o m
f i r s t -p r i n c i p l e s , a n d t h e r e f o r e w e r e s o r t t o a n r e d i r e c t
m e t h o d o f e s t i m a t i n g ( Ti n, x) t a n d K , i n c o n l u n c n o n
w i t h t h e a v a i l a b l e d a t a f o r v a r i o u s p e p t i d e s . F o r t h i s
p u r p o s e o n e e x p r e s s e s E q n . 1 i n t h e f o l lo w i n g f o r m .
T h e r i g h t h a n d s i de o f t h e a b o v e e x p r e s s i o n d e p e n d s
o n l y o n t h e q u a n t i t i e s p l a y i n g p a r t m t h e f a c i l i t a t e d
d i f f u s i o n . T h i s e x p r e s s i o n w i lt b e u s e d b e l o w t o a n a -
l y z e t h e e x p e r i m e n t a l d a t a f o r E f o r v a r i o u s p e p t i d e s
a n d t o e x t r a c t i n f o r m a t i o n a s t o t h e c o n s t a n t s
( T ~ , , ) ~
a n d K t .
C A L C U L A T I O N S A N D R E S U L I S
T o c a l c u l a t e t h e e x t r a c t i o n E d u e t o s i m p l e d i f fu -
s i o n a l o n e o n e n e e d s t h e p e r m e a b i l i t y P ~ w h i c h c a n
b e e s t i m a t e d f r o m E q n s . 1 2 a n d 1 5. A s y e t t h e r e i s n o
f i r s t - p ri n c i p l e s m e t h o d a v a i l a b l e f o r e s t i m a t i n g K
a n d t h e r e f o r e w e a d o p t a n i n d i r e ct m e t h o d t o e s ti -
m a t e i t b y e m p l o y i n g t h e k n o w n p e r m e a b i l i t y P~ o f
s u c r o se . W e h a v e c h o s e n s u c r o s e a s a r e f e r e n c e s u b -
s t a n c e s i n c e i ts t r a n s p o r t p r o pe r ti e s6 , 22 a r e k n o w n t o
b e m a i n ly d u e t o s im p l e d i f f u s i o n s o t ha t t h e d e d u c e d
P , v a l u e is e x p e c t e d t o c o r r e s p o n d t o s i m p l e d i f f u-
T A B L E I I
L i s t o f th e v a l u e s o f v a r i o u s c o n s t a n t s u s e f u l f o r t h e c a l c u l a t i o n q f e x t r a c t i o n o p e p t i d e s i n th e t e x t a l o n g w i t h t h e r e l e v a n t r ~ je r e n c t' ~
C o n s t a n t s V a l u e . ~
Surface a rea S 240 cm2/g o f b ra in
Mo lecu la r we igh t o f g lyc ine , Mo t ~ 75
M o l e c u l a r w e i g h t o f a - M S H , M MSn 1681
M o l e c u l a r w e i g h t o f A V P , M A ; . v 1083
Mo lecu la r we igh t o f MI F , MMn. 301
Mo lecu la r we igh t o f suc rose , MsLr~ 342
M o l e c u l a r w e i g h t o f M e t - E N K , M M v:N 5 7 3
M o l e c u l a r w e i g h t o f c a r n o s i n e , M C AR 226
Mo lecu la r we igh t o f g lu ta th io ne , MGt ,T 307
M o l e c u l a r w e i g h t o f t i L T 1 00 0
Mo lecu la r we igh t o f f iE 3426
M o l e c u l a r w e i g h t o f a E 1 82 6
Par t i t ion coef f ic ien t o f suc rose , f sLc 1 .8 10 ~
Rat e of blo od flow , F 9.33 10- 3 cm,~/s.g of brain
Perm eab i l i ty o f suc rose 0 .26 10 7 cm/s
Ex trac t ion o f wa te r En o n 8 (I 5 ' :~
T h i c k n e ss o f B B B 7 8
A
Mo lecu la r we igh t o f L eu -E N K , Mt.~ :NK 591
Ref s
L u n d - A n d e r s e n ~
Me rck Index v~
M e r c k I n d e x w
Me rck In dex v'
M e r c k I n d e x >
M e r c k I n d e x >
M e r c k I n d e x ~
Me rck Index >~
Me rck In dex ~'
Rapopor t e t a l . :~
Ra pop or t e t a l . -q
R a p o p o r t e t a 1 2 ~
Ra pop or t e t a l . ~z
L u n d - A n d e r s e n ~
Rap opor t e t a l , :2
L u n d - A n d e r s c n ~
L u n d - A n d e r s e n t a n d S h a r m a a n d V i m a l 2~
M erck Inde x ~'~
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
5/14
2 0 5
T A B L E 1 I I
L i s t o f t he n u m b e r o f c o n s t i t u e n t g r o u p s i n a m i n o a c i d a n d n i
v alu es f o r M e t - E N K ( 7 ) , r - G l y - G l y - P h e - M e t )
G r o u p ( i) N u m b e r o f e f f e c ti v e g r o u p s i n n i
a m i n o a c i d p a rt s o f M e t - E N K
T y r G l v G l y P h e M e t
-CH ~ 1 - 1 3 5
Be nz e ne r ing 1 - - 1 2
OH 1 - - 1
- C H R ( O N t t 1 1 1 1 4
s i o n . T h i s al s o s u b j e c t s o u r p r o c e d u r e t o s el f - c o n s is -
t e n c y a s f a r a s t h e m e c h a n i s m ( s i m p l e d i f f u s i o n ) i s
c o n c e r n e d . A l t e r n a t e l y , o n e m a y d e c i d e to c h o o s e
g l y c i n e a s a r e f e r e n c e s u b s t a n c e b u t g l y c i n e , b e i n g a n
a m i n o a c id , i s i n a d e q u a t e a s i ts t r a n s p o r t m a y n o t
p u r e l y b e s i m p l e d i f f u s i o n . T h u s , t a k i n g t h e P ~ v a l u e
o f s u c r o s e x2 a s 0 . 2 6 1 0 - 7 c m / s w e o b t a i n K = 1 . 5 x
1 0 ~ c m / s X / ~ f r o m E q n . 1 2 u s i n g m o l e c u l a r w e i g h t
M o f s u c r o s e l~ = 3 4 2 , T = 3 1 0 K a n d e x p e r i m e n t a l f
o f s u c r o s e 22 = 1 . 8 1 0 a ( s e e T a b l e I I ) .
N e x t , t h e p a r t i t i o n c o e f f i c i e n t s f o f p e p t i d e s a r e
c a l c u l a t e d u s i n g E q n . 1 5 a n d t h e v a l u e s o f n i a n d b i a s
l i s te d in T a b l e s I I I a n d V - X f o l l o w i n g t h e d e t a i l s
T A B L E I V
g i v e n a b o v e . W i t h t h e a b o v e e s t i m a t e d K a n d T a b l e
I I f o r t h e m o l e c u l a r w e i g h t s r e q u i r e d i n E q n . 12 o n e
t h e n e s t i m a t e s t h e p e r m e a b i l i t i e s P~ f o r B B B t o p e p -
t i d e s d u e t o s i m p l e d i f f u s i o n ( a l s o l i s te d i n T a b l e I V ) .
I f o n e a s s u m e s t h a t f o r p e p t i d e s o n l y t h e s i m p l e d i f f u -
s i o n p r o c e s s is i m p o r t a n t , o n e m a y c a l c u l a t e t h e c o r -
r e s p o n d i n g E v a l u e s f r o m E q n . 1 b y t a k i n g ( Tm ~) t =
0 , t h e P ~ v a l u e s l i s t e d i n T a b l e I V , a n d S a n d F
v a l u e s 17 f r o m T a b l e I I . A s i t w i l l b e e v i d e n t i n s e c -
t i o n s A t o J t h e c a l c u l a t e d E v a l u e s f o r v a r i o u s p e p -
t i d e s d u e t o s i m p l e d i f f u s i o n a l o n e a r e t o o l o w t o e x -
p l a i n t h e e x p e r i m e n t a l r e s ul t s .
T h e t h e o r e t i c a l a n a l y s is o f E c o n s i d e r i n g b o t h t h e
s i m p l e d i f f u s i o n a n d f a c i l i t a t e d d i f f u s i o n i s a l s o p o s s i -
b l e ( s e c t io n K ) f r o m E q n . 1 6 b y e v a l u a t i n g t h e l e f t
h a n d s i d e o f E q n . 1 6 w i t h t h e c a l c u l a t e d v a l u e s o f P~
f r o m T a b l e I V a n d t h e a v a il a b l e e x p e r i m e n t a l d a t a
f o r E . T h i s p r o c e d u r e i s u s e f u l f o r e x t r a c t i n g i n f o r -
m a t i o n a s t o t h e r i g h t v a l u e s o f t h e k i n e t i c c o n s t a n t s
( T m a x ) f a n d K t t h a t c o u l d e x p l a i n c o n s i s t e n t l y t h e o b -
s e r v e d E v a l u e s . T h e d e t a i l s f o r v a r i o u s p e p t i d e s c o n -
c e r n i n g t h i s a r e g i v e n i n s e c t i o n s A t o K .
( ,4 ) M e t - E N K - - r n e t h i o n i n e - e n k e p h a l i n
M e t - E N K is a p e n t a p e p t i d e ( T y r - O l y - G l y -
P h e - M e t ) w h i c h a ct s a s a n i n h i b i t o r y n e u r o m m s m i t -
T a b u l a t i o n o f t h e c a l c u l a t e d p a r t i t i o n c o e f f i c i e n t ( f) , p e r m e a b i l i t y ( P , ) d u e t o s' im p l e d i f f u s i o n , t h e c o n t r i b u t i o n t o th e e . t tr a c t i o n d u e t o
s i m p l e d i f f u s i o n a l o n e ; a n d e x p e r i m e n t a l e x t r a c t io n ( E ) o f p e p t i d e s
The c a lc u la te d E due to s im ple d i f fus ion a lo ne i s signi f i c a nt ly lowe r tha n the e xpe r im e n ta l E indic a t ing the poss ib le e x i s te nc e of pe pt idc
c a r r i er in t h e b l o o d - b r a i n b a r r i e r .
P e p t i d e s P a r t i t i o n c o e f f i c i e n t P e r m e a b i l i t y d u e t o C a l c u l a t e d e x t r a c t i o n E x p e r i m e n t a l e x t r a c t i o n
f ( i n 1 0 4 ) s i m p l e d i f f u s i o n P , d u e t o s i m p l e E ( i n ( '( )
( i n 1 0 r c m / s ) d i f f u s i o n a l o n e ( in )
M e t - E N K 7 ) _ + 7 8 . 7 _ + 0 8 2 . 3 _ + 0 . 2 1 2 + 1 2 .5 _ +1 1 .Y ~ *
L e u - E N K 7 9 + 7 8 . 5 + 0 8 2 . 3 _ + 0 .2 2 . 7 _ + 0 .3 2 .7 _+ 1) .4 b,~
Glu ta th io nc 7 1 0 .3 0 .6 + {L5 0 .4 + () .1 h~,~'
Ca rno sine 17 3 0.8 1. I h
~t-MSH 212 ,+ 57 13.7 _+ 3 7 3.5 + 1 7.7 + 1.S,
M IF 63 9.6 2.5 11 = 1.2
AV P 7`) + 7 6.3 + 0 6 1.6 ,+ 0.2 1(I.5 * 2.9 ~
t iLT (f l l ipo trop in) 120 III 2.5 3.53 + 1.5 :
f ie ( f i -e nd or ph in ) 130 5.9 1.5 ) .5 _~. 2.9 ~
aE 0~- end orp hin ) 17 I . 1 (}.3 5.75 + 31
* High a nd low l im i t s a re du e to e f f e c t of c onc e nt ra t ion c ~ ( se e a l so Ta ble s X I a nd X I I ) .
~ Ca lc ula te d f rom BU I va lue f ro m Ka s t in e t a l . 14
b S e e C o r n f o r d e t a l / .
C a l c u l a t e d f r o m B U I v a l u e s f r o m G r e e n b e r g e t a l . 12
a C a lc ula te d f rom m e a su re d to ta l pe rm e a bi l i ty P - (14 _+ 6) x 10 7 c m /s (r e f . 21).
~ Ca lc ula te d f rom m e a su re d to ta l pe rm e a bi l i ty P = (39 _+ 12) 10 7 c m /s ( re f . 21 / .
f Ca lc ula te d f rom m e a sure d to ta l pe rm e a bi l i ty P = (23 + 13) x 10 : c m /s (r e f . 21 ) .
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
6/14
2 0 6
t e r o r n e u r o m o d u l a t o r i n t h e c e n t r a l n e r v o u s s y s t e m .
I t i n hi b i t s t h e a d r e n e r g i c , c h o l i n e r g i c a n d d o p a -
m i n e r g i c t r a n s m i s s i o n . T h e p a r t i t i o n c o e f f i ci e n t o f
M e t - E N K i s c a l c u l a t e d t o b e f = ( 7 9 + 7 ) x 1 0 '*
f r o m E q n . 15 u s in g T a b l e s I - I I l . T h o u g h i n M e t - -
E N K t h e s u l f ur g r o u p ( - S ) i s a l so p r e s e n t i n t h e
s t r u c t u r e , i t d o e s n o t c o n t r i b u t e t o b i s i n c e i t s e f f e c t i s
n u l l i f i e d b y t h e H o f t h e C H 3 - g r o u p a t t a c h e d t o i t , in
a n a l o g y w i t h t h e n u l l i f i c a t i o n o f t h e e f f e c t o f - S i n
c y s t e i n e ( C y s ) g r o u p a s d i s c u s s e d i n r e f . i 0 . T h e p e r -
m e a b i l i t y o f B B B t o M e t - E N K d u e t o s i m p l e d i ff u -
s i o n is t h e n ( f r o m E q n . 1 2 a n d T a b l e I I ) P~ = ( 8 . 7 _ +
0 . 8 ) x 1 0 -7 c m /s a t 3 7 C w h ic h w i th (T m ,~ 0 f = 0
y i e l d s t h e e x t r a c t i o n E M E N K = 2 . 3 --+ 0 . 2 % ( s e e a l s o
T a b l e I V ) d u e to s i m p l e d i f f u s i o n a l o n e f r o m E q n . 1.
A l t h o u g h t h is v al u e a g r e e s w it h t h e e x p e r i m e n t a l E
( 2 - 3 % ) r e p o r t e d b y C o r n f o r d e t al .6 f o r t h e b o l u s
c o n c e n t r a t i o n c ; o f 2 1 - 2 7 0 n M , i t is s i g n i f i c a n t l y l o w -
e r c o m p a r e d t o t h e e x t r a c t i o n 1 2 -+ 1 % ( c a l c u l a t e d
f r o m E q n . 2 w i t h o b s e r v e d 13 B U I = 1 5 % a n d EH 6~H
= 8 0 + 5 % f o r c o n c e n t r a t i o n c ~ = 0 . 1 2 n M ) .
( B) L e u - E N K - - l e u c i n e - e n k e p h a l i n
L e u - E N K is a p e n t a p e p ti d e ( T y r - G l y - G l y -
P h e - L e u ) w h i c h a c ts a s a n i n h i b i t o r y n e u r o t r a n s m i l -
t e r in t h e c e n t r a l n e r v o u s s y s t e m . T h e p a r t i t i o n c o e f -
f i c i e n t o f L e u - E N K c o m e s o u t t o b e f = ( 7 9 _+ 7 )
10 - 4 f r o m E q n . 15 u s in g T a b l e V . T h e p e r m e a b i l i t y
o f B B B t o L e u - E N K d u e t o s i m p l e d if f u s io n is t h e n
( f r o m E q n . 1 2 a n d T a b l e I I ) p , = ( 8 . 5 + 0 . 8 ) x 1 0 -~7
c m / s a t 3 7 C w h i c h w i t h ( Tm a x) f = 0 g i v e s t h e e x t r a c -
t i o n EL EN K = 2 . 3 + 0 . 2 % d u e t o s i m p l e d i f f u s i o n
a l o n e f r o m E q n . 1 . T h e c a l c u l a t e d E L EN K i s c l o s e t o
t h e e x p e r i m e n t a P v a l u e 2 . 7 % ( s e e a l s o T a b l e I V ) a s -
s o c i a t e d w i t h t h e c o n c e n t r a t i o n c~ i n t h e s a t u r a t i o n
T A B L E V
L i s t o f t h e n u m b e r o f c o n s t i t u e n t g r o u p s i n a m i n o a c i d r e si d u es
a n d n i v alu es f o r L e u - E N K ( T y r - G l y - G l y - P h e - L e u )
G r o u p ( N u m b e r o f e f f ec t i ve g r o u p s i n n i
a m i n o a c id pa r ts o f L e u - E N K
T y r G l y G l y P h e L e u
1 1 3 5
C H ~
Benzene ring
1 - 1 - 2
- O H 1 . . . . I
- C H R C O N H 1 1 1 4
T A B L E V I
Lis t o f the n um be r o f c o ns t i tue nt group.~ m a tn ino ac id r es idues
a n d t h e c o r r e s p o n d i n g n , v a l u es f , r J u t a t h i o n e c G / u ~ ( ' v >
Gly )
In Cys-group we have not included the contribu tion from suilur
(S) since its effect is nullified bv the adlac cnl h vdro een, ~ di.~-
cussed in ref 5
Group i~
Nu mb er oj e t}e , -'m e group s i~t
a m i n e a c i d pa rt ~ o f g h a a t h i o n e
Glu ( w ( ;h'
- C H -
-C H R C O N H . . . .
r a n g e 2 2 - 5 5 0 n M . T h u s a t la r g e c o n c e n t r a t i o n s t h e
c a l c u l a t e d c o n t r i b u t i o n t o E d u e t o s i m p l e d i f f u s i o n
a g r e e s w i t h th e e x p e r i m e n t a l v a l u e a s i n M e t - E N K
( C t G l u t a t h i o n e
G l u t a t h i o n e ~s a t ri p e p t i d e ( G l u - C y s - G l y ) f o u n d
i n a n i m a l a n d p l a n t t i s s u e a s a c a r r i e r o f o x y g e n . I t
c o n t a i n s g l u t a m i c a c i d r e s i d u e j o i n e d i n a n u n u s u a l
p e p t i d e l i n k a g e i n v o l v i n g F - c a r b o x y l i n st e a d o f t h e
u s u a l a - c a r b o x y l g r o u p . T h e p a r t i t i o n c o e f f i c i e n t f o f
g l u t a t h i o n e i s c a l c u l a t e d t o b e f - 7 t 0 -`4 f r o m
E q n . 15 u s i n g T a b l e V I .
I t m u s t b e r e m a r k e d t h a t b e c a u s e o f t h e p e p t i d e
l i n k a g e i n v o l v i n g y - c a r b o x y l i n t h is c a s e t h e g r o u p
- C H R O C O N H b e t w e e n G l u a n d C v s c o n t r ib u t e s
d i f f e r e n t l y t o b i v a l u e t h a n t h e c o n t r i b u t i o n a r i s i n g
f r o m t h e n o r m a l a - c a r b o x y l g r o u p a s w e h a v e d i s c m -
e r e d b y e x p l a i n i n g t h e e x p e r i m e n t a l v a l u e 4 o f I og ,~
f ol u w h i c h i s - 2 . 9 9 2 . I t is b e c a u s e o f t h i s r e a s o n t h a t
w e h a v e a s s i g n e d n i - 0 . 5 f o r t h e l i n k a g e b e t w e e n
G l u a n d C y s i n T a b l e V I . T h e p e r m e a b i l i t y o f B B B
t o g l u t a t h i o n e ~ s e s t i m a t e d f r o m E q n . 1 2 a n d T a b l e
1 I to g iv e P , = 1 0 -7 c m /s a l 3 7 ~ C. E q n . 1 w i t h (T m . x ) t
- 0 t h e n y i e l d s t h e e x t r a c t i o n E o~ .~ = 0 . 3 % d u e t o
s i m p l e d i f f u si o n a l o n e . O n e n o t e s t h a t t h e c a l c u l a t e d
E G LT d u e t o s i m p l e d i f f u s i o n a l o n e ~s c l o s e t o t h e e x -
p e r i m e n t a l 5 v a l u e s w h i c h v a r i e s t r o m ( 1.6 t o 0 . 4 % [ o r
h i g h c o n c e n t r a t i o n s c ~ f r o m 1 00 0 t o 1 0 .0 0 0 n M ( s e e
a l s o T a b l e I V ) .
( D ) C a r n o s i n e
C a r n o s i n e t s a d i p e p t i d e o f a l a n i n e a n d h i s t id i n c
( A l a - H i s l o c c u r r i n g m m u s c l e s o f a n i m a l s a n d m e n .
O n e f i n d s t h e p a r t i t i o n c o e f f i c i e n t o f c a r n o s i n e f = 1
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
7/14
207
FABLE VII
Lis t o f the num be r o c ons t i tue nt groups in amin o ac id r e sidue .~
a n d t h e c o r r es p o n d i n g n i v a l u es f o r c a r n o s in e ( A l a - H i s )
Grou p ~ l) Nu mb e r c ) f e f f e c t i v e group s in n ,
a m i n o a c i d p ar t s o f c a r n o s i n e
A la His
-CH, 1 2 3
-CHRC ONtt 1 - 1
10 4 from Eq n. 15 (see also Tabl e Vl I). Th e per-
meability of BBB to carnosine due to simple diffusion
alone is estimated by using Eqn. 12 and Table II to
obt ain P~ = 3 10 7 cm/s at 37 C which fro m Eqn . 1
with the condition (Tm~,x) = 0 gives the extraction
Ec;,a = 1t.8 (due to simple diffusion alone )
agreeing with the experim ental value 1.1 for high
concentration ca = 38000 nM as in Met -E NK , Le u-
ENK and glutathione (see also Table IV).
( E ) a - M S H - - m e l a n o o , t e - s t i m u l a ti n g h o r m o n e
The struct ure I~ of a- MSH consists of first 13 (see
Table VIII) amino acids of adrenocorticotropic hor-
mone (A CTH) and an acetylated NH:-terminal on
one side and an amidated CO2H-terminal amino acid
on the other side. It is one of the most pot ent melano-
cyte-stimulating hormon es and has an importa nt
physiological pigment ary function. It has been estab-
lished that the injection of synthetic a-M SH im-
proves the performance in the Benton Visual Reten-
tion test and increases significantly the s omatosen so-
ry cortical evoked responses.
The partition coefficient of a-MSH comes out to
be f = (212 _+ 57) 10 ~ fro m Eqn. 15 us ing Tabl es I
and VIII. The permeab ility of a-MSH at 37 C is
then (fr om Eq n. 12 and Ta bl e I1) P~ = (13.7 _+ 3.7) x
10 -7 cm/s yiel ding , with (T .... )~ = (), t he ext rac tio n
EMSH = 3.5 + 1 due to simple diffusion alone which
is much l ower tha n the expe rime nta l ~: extra ction 7.7
_+ 1. 8~ ded uce d from BU112 = 9.6 _+ 2.3 usin g
Eqn. 2 and EHo H = 80 + 5 (see also Tabl e IV).
( F ) M I F - - M S H - r e l e a s e i n h i b i t i n g . fa c t or
MIF is a hypothalamic hormo ne which controls the
release of MSH from the pituitary and produces n eu-
rom odul ato ry effects~9. It is a tripe ptide with am ino
acid (AA) sequence Pro -Le u-G ly- NH> In this
case par tit ion coeff icien t of MIF is f = 63 x 10 ~ from
Eqn . 15 and Tab les l and IX and P~ = 9.6 10 -7 cm/s
at 37 C (from Eqn. 12 and Tabl e II) which with
(Tm,,~)f = 0 gives from Eqn . 1 the ex tr ac tion EMW =
2.5 due to simple diffusion, a value much lower
than the exp erim enta l value of extrac tion 11 _+ 1.2
deduce d from BU112 = 13.7 _+ 1.6~4 (see also Table
IV) corres pond ing to c~ = 0.14 nM and Euo u = 80 +
5 . We note that c~ correspo nding to the experime n-
tal E is very low and, in comparison, the calculated
extract ion EMW (due to simple diffusion alo ne) is sig-
nificantly lower than experim ental extraction.
( G ) A V P - - a r g i n i n e - v a so p r e s s in
AVP is antidiure tic hor mon e v~ which is extrac ted
from the posterior lobe of the pituitary of healthy do-
mestic animals. It produces neur otra nsmi ner- like ef-
fects when adminis tered at high concen tratio n in the
body and neurom odul ator- like effects at low concen-
tration. It has 9 amino acids (nanopeptide) and its
chemical formula is C4e Ho 5NIsO IeS 2 . The structural
formula of AVP indicating amino acid sequence is
TABLE VIII
Lis t o f the num be r ~ I ' c ons t i tue nt groups in amino ac id r e s idue s and the c or re spond ing n~ v alue s inv oh ' e d in Eqn . 15 . lor (*-MSH
( C H ~ ( ' O - S e r - f y r - S e r M e t - G l u - H i s - P h e - A r g T r p - G ly L y s '- P r o - V a l - N H 2)
G r o u p ( i) N u m b e r o f g r o u p s i n v a r i o u s a m i n o a c i d p a r t s o f ~ - M S H n ,
Se r Ty r Se r Me t G lu His Ph e Arg Trp Gh ' l. v~ Pro Val
-CH, 1 1 1 3 2 2 1 2 1 - 2 - 2 18
Benzene ring 1 - - 1 -- 4
OH 1 1 1 - - 3
CHRCONH 1 1 1 1 1 1 1 1 1 l 1 1 1 1 13
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
8/14
208
T A B L E I X
Lis t o f the numbe r o f c ons t i tue nt groups in amino ac id r e s iduc ~
a n d t h e c o r re s p o n d i n g n i v a l u e s f o r M I F ( P r o - L e u - G l v - - N t t ei
G r o u p ( i) N u m b e r o f e f f ec t i ve g r o u p s / i
i n a m i n o a c i d p a r t s in M I F
P r o L e u G l v
-C H~ 3 3.
Benzene ring 1 -
- C H R C O N H 1 1 2
g i v e n b y
C y s - T y r - P h e - G l u - A s n - C y s - P r o - A r g - G l y - N H 2
W e h a v e e s t i m a t e d t h e p a r t i t i o n c o e f f i c i en t f o f
A V P f r o m E q n . 15 a n d T a b l e I I a n d f o u n d i t t o b e
( 7 9 + 7 ) x 1 0 4 a s l i s t e d i n T a b l e X . E q n . 1 2 a n d
T a b l e I I t h e n g i v e t h e p e r m e a b i l i t y o f A V P P~ = ( 6 .3
+ 0 . 6 ) x 1 0 -7 c m /s a t 3 7 C . T h i s v a lu e w i th (T m a x ) f =
0 c o r r e s p o n d s t o t h e e x t r a c t i o n E A V e = 1 .6 + 0 . 2 %
d u e t o s i m p l e d i f f u s i o n a l o n e a t 3 7 C ( u s i n g E q n . 1
a n d T a b l e I I ) . T h e c a l c u l a t e d E d u e t o s i m p l e d i f f u -
s i on is v e r y l o w c o m p a r e d w i t h t h e e x p e r i m e n t a l e x -
t r a c t io n 1 0. 5 + 2 . 9 % d e d u c e d f r o m t h e e x p e r i m e n t a l
B U I >" = 1 3 + 2 . 8 % ( f r o m E q n . 2 a n d u s i n g E H O H =
8 0 + 5 % ) c o r r e s p o n d i n g t o c ; = 6 . 7 n M , a l o w v a l u e
o f c o n c e n t r a t i o n ( s e e a ls o T a b l e I V ) .
( H ) f l L T - - f l - [ D - A l a 2 - 1 4 C - h o m o a r g O g ] l i p o t r o p i n 6 1 _ 6 9
t i L T i s a s y n t h e t i c p e p t i d e w h i c h i s a n a n a l o g o f t h e
n a t u r a l p e p t i d e f l -l ip o t ro p in 6 ~ _ 69 d e r i v e d f r o m t h e p i -
t u i t a r y h o r m o n e f l - l i p o t r o p i n . I t i s r e p o r t e d 2~ t o e x e r t
c e n t r a l e f f e c t s ( s u c h a s o p i a t i c e f f e c t ) w h e n i n j e c t e d
s y s t e m i c a l l y . B e c a u s e t h e s t r u c t u r e o f t i L T i s n o t
k n o w n d e f i n i t e l y w e a r c u n a b i c ~.~c a lc u la l e i t , ; p a r l J -
t i o n c o e f f i c i e n t u s in g o u r m e t h o d g i v e n i l l t h e t h e o r ~
s e c t i o n . F o r t u n a t e l y . t h e p a r t t t ~ o n c o e f f i c i e n I i n t h i s
c a s e i s k n o w n e x p e r i m e n t a l l y - ' , w h i c h i~ t -:. 1 2 f i x
1 0 4 T h e p e r m e a b i l i t y o f B B B t o f il _- l' d u e t o s i m p l e
d i f f u s io n a t 3 7 C i s t h e n P . -: J.l > l l ) " c m , s l t r o m
E q n 1 2 a n d T a b l e I1 ) . T h i s g ~ v c , ~ i lh ( l 'm , ,~ )t ~ ( i a n d
E q n . 1 E ~ l t = 2 . 5 % c l ue t o ~ m ~p te d i f f u s i o n a l o n e .
T h e e x p e r i m e n t a l v a l u e o f t h e e x t r a c t i o n f o r i l l / 1 is
n o t a v a i l a b l e f o r c o m p a r i s o n . H o w e v e r , t h e ex p e ri - -
m e n ta l 21 p e rm e a b i l i t y P i s I1 4 t~l x 1(1 " c m / s . T h i s
m e a s u r e m e n t c o r r e s p o n d s t o l h c a n l o u n l o l / ~L T
r a n g i n g f r o m 2 t o l ( I ,u C i i n j e c t e d i n t h e b r a i n c a p i l -
l a r y , t h e s p e c i f i c a c t i v i t y of / ;~ L I " 5 8 m C i / m M a n d
t h e f r a c t i o n r = 0 . 8 4 o f t i L T u n b o u n d t o b l o o d p l a s -
m a . T h e s e v a l u e s e n a b l e u s t o c s t i m a t e c~ t r o m
a m o u n t o f d r u g r e j e c t e d
c ~ = r x s p e c i f i c a c t i v i ty o f t h e d ru g / 1 7}
y i e l d i n g G = 8 7 n M f o r t h e a v e r a g e v a l u e o u C i o t
t i L T i n j e c t e d . I t m e a n s t h a t t h e m e a s u r e d P v a l u e ( 1 4
_+ 6 ) x 1 0 -7 c m /s c o r re s p o n d s to c i; - 8 7 r iM. T h i s P i s
t h e t o t a l p e r m e a b i l i t y a n d t h e r e f o r e i t y i e l d s t h e t o t a l
e x t r a c t i o n E = 3 . 5 3 ~_ 1 . 5c / r ( f o r f i L T a t c a = 8 7 n M ) .
w h i c h i s h i g h e r t h a n o u r c a l c u l a t e d v a l u e o f t h e e x -
t r a c t i o n 2 . 5 % a r i s i n g f r o m s i m p l e d i f f u s i o n a l o n e
see a l s o T a b l e I V ) .
(1) f i e f l - l D - A l a 2 - t 4 C - h o m o a r g V ] e n d o r p h i n
f i E i s a s y n t h e t i c 21 o p i o i d p e p t i d e , a n a n a l o g o f t h e
n a t u r a l p e p t i d e f l - e n d o r p h i n d e r i v e d f r o m t h e p i t u -
i t a r y h o r m o n e f l - l i p o tr o p i n . I t c o n t a i n s t h e s a m e a m i -
n o a ci d s e qu e n ce T y r - D - A l a - G l y - P h e - M e t a s
t i L T a n d a E . I t s p a r t i t i o n c o e f f ic i e n t f ha s b e e n m e a s -
u r e d 21 t o b e 1 30 x 1 0 - 4 . W e h a v e u s e d t h i s v a l u e o f f
T A B L E X
Lis t o f the nu mb e r o f c ons t i tue nt grou ps in am ino ac id r e s idue s an d the c or re spo nding n i v a lue s for A VP ( ):~'- , 71y r- P he -G tn - Ash ....
C y s - P r o - A r g - G l y - N H 2 )
G r o u p ( i) N u m b e r o f e f f e ct i v e g r o u p s i n v a r i o u s a c i d p a r t s o f A V P , .
- C H z
Benzene ring 1
- O H 1 . . . .
-C H R C O N H b e tw e en a m in o a cids 1 1 1 1
C y s T y r P h e G l u A s n C y s P r o A r g
1 1 1 2 1 1 2
1 I I
i I
GI)
~3
3
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
9/14
2119
T A B L E X I
List of the calculated values o f the total extraction (due to sim ple ffus ion and carrier-mediated facilitated diffusion) and experimental
ex tract ion for various concen tra tions c~ o f Me t-E NK . Le u- EN K , g luta th ione , carnos ine , a -M SH a nd MIF , us ing the max imal ra te o f
transport ( Tm,,Q - O. 46 x 10 --~ pm ol/ g.s and the reciprocal o f affinity K t (= 0.35 nM ) of peptide to its carrier which ha ve been e~'timated tLs-
ing exper imen tal ex trac tions for c ; equa l to 0 .12 nM and 50 nM o f M et- EN K, and ca lcu la ted ex tract ion E , = 2 .3~ '4 o f M et -E N K due to
fimple dif fusion (see Table IV)
Though the va lues o f the pa ram ete rs (Tmax) and K , exp la in a l l the expe r imen ta l re su l t s wi th in uncer ta in t ie s , the K~ va lues may ac tua l ly
be d i f fe ren t fo r d i f fe ren t subs tances .
Peptides Concentration c~ (in nM ) Extraction* Icalculated) (in ~ ) Extraction (experimental) (in C~ )
M et -E N K 0.12 12 _+ 1 12 +1 ~'
21 2.5 + 0.2 2.7 _+ 0.3 ~'
25 2.5_+ 0.2 3 _+1 h
50 2.4 + 0.2 2.4 ( .3~'
27 0 2.2 +_ 0. 2 " "S + I "~1,
L e u - E N K
22 2.4 + 1).2 2.7 + 0.3 t'
1311 2.2 _+ 0. 2 2.7 _+ 0.3 t'
550 2.2 + 0.2 2.7 + 0.4 ~'
Gl utat hion e 10()ll 0 .3 11.6 + 1 .5 h
10000 0.3 0.4 _+ 0.1 h
Ca rno sinc 38000 0.N 1.1 h
a- M SH 0.583 g.4 _+_1 7.7 + 1.gc
M IF 0 .14 11 .8 l l +_1 .2"
* T he uncer ta in t ie s in the ca lcu la ted E va lue s a re due to uncer ta in t ie s in ca lcu la ted P~ va lues .
Ca lcu la ted f rom BU I va lue f rom Kas t in e t a l . t3.
h Ca lcu la ted f rom BU I va lues f rom G reen berg e t a1.12.
c See Corn ford et al . 5 .
t o c a lc u l a te t h e p e r m e a b i l i t y o f B B B t o f i E d u e t o
s i m p l e d i f f u s i o n w h i c h c o m e s o u t t o b e P~ = 5 . 9 x
1 0 7 c m / s a t 3 7 C ( f r o m E q n . 1 2 a n d T a b l e I I ) . O n e
t h e n c a l c u l a t e s ( f r o m E q n . 1 w i t h ( T m a x) f = 0 ) t h e e x -
t r a c t i o n E f ~ E = 1 . 5 % ( s e e a l so T a b l e I V ) , a v a l u e
l o w e r t h an t h e e q u i v a l e n t e x p e r i m e n t a l e x t r a c t i o n
9 . 5 _+ 2 . 9 % w i t h t h e e x p e r i m e n t a l 2 1 t o t a l p e r m e a b i l i -
t y P = ( 3 9 _+ 1 2 ) 1 0 . 7 c m / s f o r E c o r r e s p o n d i n g t o c ;
= 6 . 8 n M e s t i m a t e d f r o m E q n . 1 7 w i t h t h e e x p e r i -
m e n t a l p a r a m e t e r s r = 0 . 2 , a m o u n t o f f i E i n j e c t e d =
2 / ~ C i a n d t h e s p e c i f ic a c t iv i t y 58 m C i / m M .
i ty 5 8 m C i / m M .
( J ) a E - - a - [ D - A l a 2 - 1 4 C - h o m o a r g J ] e n d o r p h i n
t e e i s a s y n t h e t i c 21 o p i o i d p e p t i d e r e p r e s e n t i n g t h e
n a t u r a l p e p t i d e a - e n d o r p h i n d e r i v e d f r o m t h e p i t u-
i t ar y h o r m o n e f l - l i p o t ro p i n . T h e m e a s u r e d p a r t i t io n
c o e f f i c i e n t o f E is f = 1 7 x 1 0 4 w h i c h y i e l d s t h e a s s o -
c i a t e d p e r m e a b i l i t y d u e t o s i m p l e d i f f u s i o n a l o n e a s
P ~ = 1 .1 1 0 - 7 c m / s a t 3 7 C ( u s i n g E q n . 1 2 a n d
T a b l e 11) a n d t h e c o r r e s p o n d i n g E = 0 . 3 % ( f r o m
E q n . 1 w i t h ( T ma ) f = 0 ) . T h e e x p e r i m e n t a l l y 2 1 m e a s -
u r e d t o t a l p e r m e a b i l i t y i n t h i s c a s e is P = ( 2 3 + 1 3 )
1 0 7 c m / s w h i c h g i v e s t h e e q u i v a l e n t e x p e r i m e n t a l
e x t r a c t i o n E = 5 . 7 5 _+ 3 % f o r c ; = 1 1 n M ( e s t i m a t e d
u s i n g E q n . 1 7 w i t h r = 0 . 3 4 , a m o u n t i n j e c t e d = 2 # C i
a n d t h e s p e c i f i c a c t i v i ty = 5 8 m C i / m M ) . W e n o t e t h a t
t h e c a l c u l a t e d E ( 0 . 3 % ) d u e t o s i m p l e d i f f u s io n a l o n e
i s m u c h l e s s t h a n t h e e x p e r i m e n t a l E ( 5 , 7 5 +_ 3 % )
( s e e al s o T a b l e I V ) .
( K ) C a l c u l a t i o n o f ( T , ,~ x ) f a n d K ,
T a b l e I V c o n t a i n s t h e e x t r a c t i o n s f o r v a r i o u s p e p -
t i d e s c a l c u l a t e d a b o v e f r o m s i m p l e d i f f u s i o n a l o n e ,
w h i c h a r e si g n i f i c a n tl y l o w e r t h a n t h e e x p e r i m e n t a l
v a l u e s in d i c a t i n g t h a t t h e m e r e s i m p l e d i f f u s io n p r o c -
e s s c a n n o t e x p l a i n t h e t r a n s p o r t o f p e p t i d e s t h r o u g h
B B B . F u r t h e r , t h e e x p e r i m e n t a l e x t r a c t i o n s a r e
f o u n d t o d e p e n d o n t h e c o n c e n t r a t i o n s o f p e p t i d e s i n
t h e b o l u s (s e e T a b l e s I V , X I a n d X I I ) . W i t h t h e i n-
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
10/14
21(1
T A B L E X I I
Lis t of the calculated values o f total extraction using ( T,na~) = l O :
pmo l /g . s and K t = 7 nM an d exper imen ta l ex tra c t ions /br vari -
ous concentrat ion c~ o f A VP , t iL T , f iE and a E
(Tmax) and K have bee n estimated from extractions due to sim-
ple diffusion and experimental extraction of t iLT and aE .
Peptides Concentration Extrac tion Extraction
c~ (in nM ) (calculated) (experimental)
(in ) (in )
AV P (11.7) 6.7 9 10.5 _+ 2.9 ~
tiL T 87 3.53 3.53 _+ 1.5 b
fiE 6.8 (20.6 ) 8.9 9.5 _+ 2.9 ~'
aE ll (35) 6 5.75 _+ 3u
Calculated from BU I values of Gre enb erg et al . J2.
Calcula ted f rom perm eabi l i ty values f rom R apo por t e t a l .:~ .
t e n t o f d e t e r m i n i n g t h e b a s i c m e c h a n i s m i n h e r e n t i n
t h e t r a n s p o r t o f p e p t i d e s w e n o w s h o w i n F ig . 2 th e
p o i n t s r e p r e s e n t i n g t h e l e f t h a n d s i d e o f E q n . 1 6 f o r
r e l e v a n t l o g m c ;, t a k i n g f o r E t h e k n o w n e x p e r i m e n -
t a l e x t r a c t i o n s f o r d i f f e r e n t p e p t i d e s . F i g . 2 s h o w s
t h a t t h e l e f t h a n d s i d e o f E q n . 1 6 w h i c h r e p r e s e n t s
t h e e x t r a c t i o n w i t h o u t s i m p l e d i f f u s i o n ( s e e t h e o r y
s e c ti o n ) d e c r e a s e s w i t h c o n c e n t r a t i o n c~ a p p r o a c h i n g
a s y m p t o t i c a l l y t o z e r o i n d i c a t i n g a s a t u r a t i o n e f f e c t
2O
o~
5
1
',i 4
5 q
o I ' ~- '~-.-~,+ $ # - - 4 - ~ '.
1 3 4 5 6 7 8 9
I o g ~o C O C o i n p M )
F ig . 2 . T he sem i loga r it hm ic p lo t o f I - [ (1 -E ) / e xp( -P sS /F ) ] vs
logm c~ fo r M e t -E N K ( liD ), L eu -E N K (T ) , g lu t a th ione
(O ) , ca rnos ine (X ) , a -M S H (O ) , M IF (@ ) , A V P (zS) ,
t iL T (& ) , f iE (V ) and aE (11 ) . D o t t ed cu rve is the t heo re t -
ical p lot of
] v s
(Kr+ c~)F )I l gl c~l
with (Tmax)~ = 0.4 6x i0 -3 pmo l /g . s and K = 0.35 nM, which
sa ti sfy approx im a te ly he expe r im en ta l po in t s co r r e spond ing t o
M e t - E N K , L e u - E N K , g l ut at h io n e , c a m o si n e, a - M S H a n d
M IF .
a n d h e n c e t h e p r e s e n c e o f p e p t l d e c a r r i e r s f o r t ra ns .-
p o r t .
A s p o i n t e d o u t i n t h e t h e o r y s e c t i o n i t i s n o t p o s s i -
b l e t o c a l c u l a t e d i r e c t l y (T m ax ) ~ a n d K t a t p r e s e n t .
F o r t u n a t e l y , i t i s p o s s i b l e t o d e t e r m i n e ( T . . . . )f a n d K ,
i n d i r e c t l y b y u s in g a n y t w o e x p e r i m e n t a l E v a l u e s f o r
d i f f e r e n t c o n c e n t r a t i o n s i n E q n . 1. A c c o r d i n g l y , w e
u t i li z e t h e E v a lu e s o f M e t - E N K i n E q n . i f o r t w o
c o n c e n t r a t i o n s c ; e q u a l to 0 .1 2 n M a n d 5 0 n M a n d o b -
t a in t w o s i m u l t a n e o u s e q u a t i o n s w h i c h y i e l d ( T .. .. . )t
= 0 . 4 6 I O 3 p m o l / g ' s a n d h , = 0 . 3 5 n M ( u s i n g
T a b l e s I I , IV a n d X I f o r r a t e o f b l o o d f l o w ( F ) = 9 .3 3
x 10 -3 cm 3/g . s , S = 2 4 0 c m : / g , P , = 8 . 7 1 0 7 c m / s ,
E = 1 2 % a t c a = 0 . 1 2 n M a n d E = 2 . 4 % f o r c ; - - 5 0
n M ) . K n o w i n g ( T m ax ) f a n d K , w c n o w a t t e m p t t o c x -
p l a i n t h e o b s e r v e d E v a l u e s o f p e p t i d e s f o r d i f f e r e n t
c o n c e n t r a t i o n s . F o r t h is p u r p o s e w e h a v e a l s o p l o t t e d
i n F i g . 2 t h e r i g h t h a n d s i d e o f E q n . 1 6 v e r s u s l o g > e l;
( s h o w n a s a d o t t e d c u r v e ) . W e f i n d t h a t t h e d o t t e d
c u r v e f a l ls c lo s e t o a l l th e ~ x p e r i m e n t a l p o i n t s
( F i g . 2 ) e x c e p t t o t h o s e f o r A V P , t i L T , f i E a n d < z E
w h i c h r e q u i r e a d i f f e r e n t s e t o f K : a n d ( T . .. . )~ v a l u e s .
I n f a c t , t h e m e t h o d a n a l o g o u s t o t h e o n e d e s c r i b e d
ab ov e l e ad s t o K t = 7 nM an d (T m ax) t = 1(1 ] (} a
p m o l / g ' s a p p r o p r i a t e t o A V P , t i L T , f i E a n d a E ( s e e
a l s o T a b l e X l l ) .
W i t h t h e d e d u c e d v a l u e s o f ( Tm ax ) f a n d
K ,
i t i s in-
t e r e s t i n g t o c a l c u l a t e E f o r v a r i o u s p e p t i d e s f r o m
E q n . 1 u s i n g t h e r e q u i r e d p a r a m e t e r s f r o m T a b l e s 11
a n d I V . T a b l e X I l i s ts t h e c a l c u l a t e d E f o r M e t -
E N K , L e u - E N K . g l u t a t h i o n c, c a r n o si n e , a - M S H
a n d M I F f o r d i f fe r e n t c o n c e n t r a n o n s a n d c o m p a r e s
w i t h t h e e x p e r i m e n t a l d a t a . A s f o r t h e t o t a l E f o r
A V P , t i L T , f i E a n d a E w e h a v e l is t e d t h e m i n T a b l e
X I I a s o b t a i n e d f r o m E q n . l u s i n g P, t r o m F a b l e
I V , ( T ma x) I = 1 0 x 1 0 - 3 p m o l / g s a n d K . = - n M .
T a b l e X I I a l s o l is t s t h e e x p e r i m e n t a l E f o r c o m p a r i -
s o n .
I f o n e i n c l u d e s t h e e x p e r i m e n t a l e r r o r s i n th e
m e a s u r e m e n t o f t h e e x t r a c t i o n s , o n e f i n d s l a r g e e r-
r o r s i n t h e a b o v e v a l u e s o f i T ...... ~ a n d K , . E x p l i c i t l y K ,
= 0 . 3 5 + 0 . 3 1 n M a n d ( T m ax ) f = ( 0 . 4 6 - 0 . 2 9 ) x 1 0 - 3
p m o l / g ' s f or t h e f ir st g r o u p o f p e p t i d e s ( M e t - E N K ,
L e u - E N K . g l u t a t h i on e , c a r o si n e , a - M S H a n d M I F )
an d K t = l l _ 11 nM . (T m ax) I = (15 - 15 ) x 10 -~
p m o l / g ' s f o r t h e s e c o n d g r o u p ( A V P , t i L T . ~ E a n d
a E ) o f p e p t i d e s . T h e l a r g e e r r o r s i n (T m~ xJf a n d K , d e -
m a n d m o r e p r e c i s e m e a s u r e m e n t s o f E v a l u e s .
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
11/14
T h e v a r i a t i o n s o f t h e t o t a l c a l c u l a t e d e x t r a c t i o n E
( f r o m b o t h s i m p l e a n d f a c i l i t a t e d d i f f u s i o n ) w i t h c o n -
c e n t r a t i o n c~ a r e d e p i c t e d i n F i g. 3 f o r M e t - E N K
a n d L e u - E N K ( c u r v e I ) , g l u t a t h i o n e ( c u r v e 1 I ) , c a r -
n o s i n e ( c u r v e 1 1 I) , (z - M S H ( c u r v e I V ) , a n d M I F
( c u r v e V ) b y u s in g a p p r o p r i a t e l y P~ f r o m T a b l e I V , S
a n d F f r o m T a b l e I I , K ~ = 0 . 3 5 n M a n d Tma x 0 .46 x
1 0 - 3 p m o l / g s i n E q n . 1 . I n F i g . 3 t h e e x p e r i m e n t a l
e x t r a c t i o n s f o r a ll t h e p e p t i d e s c o n s i d e r e d a r e a l s o
s h o w n f o r c o m p a r i s o n . T h e c u r v e s i n F i g . 3 gi v e t h e
r ig h t b e h a v i o r o f E v s l o g > c ; a n d a r e i m p o r t a n t f o r
p r e d i c t in g t h e E v a l u e s f o r a n y c o n c e n t r a t i o n o f p e p -
t i de s .
I ) I S ( U S S I O N A N D P R E D I C T I O N O F N O R M A L CON
CENTRAT 1ON
O F P E P T I D E S
W e h a v e e s t i m a t e d a b o v e t h e p a r t i t i o n c o e f f i c i e n t s
o f w m o u s p e p t i d e s i n e t h a n o l w i t h g l y c in e a s a r e f e r-
e n c e . A s s u m i n g t h a t t h e c a l c u l a t e d v a l u e s o f t h e p a r -
t i ti o n c o e f f i c i e n t s a p p r o x i m a t e c l o s e l y t h e v a l u e s a p -
p r o p r i a t e f o r p e p t i d e s in b l o o d - b r a i n b a r r i e r, w e
h a v e c a l c u l a t e d f i rs t th e e x t r a c t i o n v a l u e s d u e t o s i m -
p le d if fu si on a lo n e fo r M e t - E N K , L e u - E N K , g lu ta -
t h i o n e , c a r n o s i n e , a - M S H , M I F , A V P , t i L T (/:~-
[ D - A l a - ' - I a C - h o m o a r g ] l i p o t r o p i n ~ , l
~,~),
t i e
( / :
7 2 C - -
uJ
Z 15
: - ~ - T _ ~ - - . .
d 1 0
~ 1 2 3 4 5 6 7 8 9
, o g C o , C o i n p M
F i g . 3 . ] 'h e s e m i l o g a r i t h m i c p l o t o f th e v a r i a t i o n o f t h e e x t r a c -
t io n ( in ) o f M e t - E N K , L e u - E N K , g l u ta t h io n e a n d c a rn o -
s i n c w i t h c a p i l l a r y - c o n c e n t r a t i o n ( l o g [ . c ~. c ~ i n p M ) . C u r v e I , is
a theore t ica l ext rac t ion E for Met -ENK and Leu-ENK as a
function of c~ (see parts A and B in the calculations an d results
section): curves If, Ill, IV and V are glutathione, carnosine
(z-MSH and MIF . respectively. { T m a x f = 0.46 x 10 3 pmo l/g's
and /~, 0. 35 nM have been use d. The exper imental points arc
indicated by ) , x, O. '~. , , , /~, ~ and ~\ for M et-
ENK, L cu -E NK , glutathione, carm)sine, c~-MSH, MIF, AV P,
dE , ~zE and/4ELL respectively.
211
[ D - A l a 2 - 1 4 C - h o m o a r g g ] e n d o r p h i n ) a n d a E ( a -
[ D - A l a 2 - 1 4 C - h o m o a r g ] e n d o r p h i n ) i n t h e c a l c u l a -
t i o n s a n d r e s u l t s s e c t i o n . O u r t h e o r e t i c a l e s t i m a t e s
o f e x t r a c t i o n s a r e 2 . 3 , 2 . 3 , 0 . 3 , 0 . 8 , 3 . 5 , 2 . 5 , 1 . 6 ,
2 . 5 , 1 .5 a n d 0 . 3 % ( se e T a b l e I V ) f o r M e t - E N K , L e u -
E N K , g l u ta t h i o n e , c a r n o s in e , ( z -M S H , M I F , A V P ,
t i L T , t i E a n d a E , r e s p e c t i v e l y , d u e t o s i m p l e d i f f u -
s i o n a l o n e .
E x p l i c i t l y , t h e c a l c u l a t e d E d u e t o s i m p l e d i f f u s i o n
a l on e f o r M e t - E N K is 2 . 3 % w h i c h is m u c h l o w e r
t h a n t h e v a l u e o f 12 + 1 % ( c a l c u l a t e d f r o m t h e e x -
p e r i m e n t a l B U I = 1 5 % o b s e r v e d b y K a s t in e t a l .H ) .
T h e v al ue f or E f o r M e t - E N K , n a m e l y 3 % q u o t e d
b y C o r n f o r d e t a l . S a p p e a r s t o b e f o r h i g h e r c o n c e n -
t r a ti o n c ; ( 2 1 - 2 7 0 n M ) t h a n t h a t ( 0 .1 2 n M ) u s e d b y
K a s t i n e t a l . ~ 4 s o t h a t t h e e x t r a c t i o n v a l u e f o r M e t -
E N K i s h i g h f o r l o w c o n c e n t r a t i o n a n d l o w f o r h i g h
c o n c e n t r a t i o n . T h i s is t h e p h e n o m e n a a n a l o g o u s t o
t h e s a t u r a t i o n e f f e c t l e a d i n g t o t h e s p e c u l a t i o n o f t h e
d o m i n a n c e o f c a r r i e r - m e d i a t e d p r o c e s s f o r t h e t r a n s-
p o r t o f M e t - E N K . S i m i la r a ss e r t io n h a s a ls o b e e n
m a d e f o r o t h e r p e p t i d e s b y p l o t ti n g t h e l e f t a n d r i g h t
h a n d s i d e s o f E q n . 1 6 a s a f u n c t i o n o f l o g > c~ i n
F i g . 2 a s d i s c u s s e d i n p a r t K a b o v e .
S i n c e t h e E v a l u e s f r o m s i m p l e d i f f u s i o n a r e t o o
l o w t o e x p la i n t h e e x p e r i m e n t a l d a t a , w e h a v e c a l c u -
l a t e d E n o t o n l y f r o m s i m p l e d i f f u s i o n b u t a l s o f r o m
t h e c o m b i n e d s i m p l e d i f f u s i o n a n d c a r r i e r - m e d i a t e d
f a c i l i t a t e d d i f f u s i o n . T h e k i n e t i c c o n s t a n t s , t h e r e c i p -
r o c a l o f th e a f f i n i ty o f p e p t i d e t o i t s c a r r i e r K , a n d
m a x i m a l r a t e o f t r a n s p o r t ( T . . . . )~, w h i c h a r e n e e d e d
t o c a lc u l a te E , h a v e b e e n e s t i m a t e d b y a n i n d ir e c t
m e t h o d a s e x p l a i n e d a b o v e . T h e v a l u e s w e h a v e o b -
t a i ne d a r e ( Tm , ,x )f = 0 .46 x 10 ~ pm ol / g s a n d K r =
0 . 3 5 n M a p p r o p r i a t e t o e x p l a in a l l t h e a v a i l a b l e e x -
p e r im e n t al d at a f o r M e t - E N K , L e u - E N K , g lu ta -
t h i o n e , c a rn o s i n e , a - M S H a n d M I F . F o r e x p l a i n i n g
t h e d a ta f o r A V P , t i L T , # E a n d ( hE w e h a v e d e d u c e d
( Tm ~x)f = 10 x 10 s pm ol / g - s a n d K , = 7 nM. T h i s d i f -
f e r e n c e i n ( Tm ~, )f a nd K ~ va l u e s f o r t h e t w o g r o up s o f
p e p t i d e s a p p e a r s t o b e p a r t l y d u e t o t h e d i f f e r e n c e i n
t h e n a t u r e o f p e p t i d e s r e q u i r i n g d i f f e r e n t c a r r ie r s a n d
p a r t l y d u e t o t h e u n c e r t a i n t i e s i n t h e e x p e r i m e n t a l
d a t a . T a b l e s X I a n d X I I c o m p i l e t h e c a l c u la t e d E
v a l u es f o r v a r io u s c o n c e n t r a t i o n s o f th e p e p t i d e s
a l o n g w it h t h e o b s e r v e d v a l u e s .
I n c l u d i n g th e e x p e r i m e n t a l u n c e r t a i n t i e s i n t h e
m e a s u r e d e x t r a c t i o n s a n d u n c e r t a i n t i e s i n P~ v a l u e s ,
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
12/14
212
w e h a v e f o u n d l a rg e u n c e r t a i n t i e s i n Tmax) f a n d K t s o
t h a t
Tmax) f =
( 0 .46 _+ 0 .29 ) x 10 - 3 pm ol / g . s a nd K t =
0 .3 5 + 0 .3 1 n M fo r M e t - E N K , L e u - E N K , g lu t a-
t h i o n e , c a r n o s in e , a - M S H a n d M I F a n d (T m~ xh =
( 15 + 15 ) X 10 - 3 pm ol / g ' s a nd K t = ( 11 + 11 ) nM f o r
t h e A V P ,
t i L T t i e
a n d c t E .
R e m a r k a b l y , i t i s p o s s i b l e t o e x p l a i n a l l a v a i l a b l e
e x p e r i m e n t a l d a t a f o r d i f f e r e n t p e p t i d e s b y a s s u m i n g
o n l y t w o s e t s o f v a l u e s o f Tmax) f a n d K t. I n f a c t , o n e
m a y e x p e c t d i f f e r e n t ( Tm :,x )f a n d
K t
f o r d i f fe r e n t p e p -
t id e s , p a r t i c u l a rl y , b e c a u s e d i f f e r e n t c a r r ie r s m a y b e
i n v o l v e d w i th d i f fe r e n t p e p t id e s . H o w e v e r , o w i n g t o
t h e l a rg e e x p e r i m e n t a l u n c e r t a i n t i e s i n t h e o b s e r v e d
E v a l u e s , a t p r e s e n t i t i s n o t p o s s i b l e t o m a k e o u t d i f -
f e r e n c e s i n t h e ( T ma x) a n d
Kr
v a l u e s f o r v a r i o u s p e p -
t id e s . I t c o u l d h o w e v e r b e r e m a r k e d t h a t , i f t h e n a -
t u r e o f a ll p e p t i d e s i n a g r o u p ( a s w e l l as t h e n a t u r e o f
c a r r i e r s i n v o l v e d i n t h e g r o u p ) i s t h e s a m e , a s i n g l e
s e t o f Tmax) f a n d K , v a l u e s i s p e r t i n e n t t o t h a t g r o u p .
K n o w i n g t h e v a lu e s o f th e p a r a m e t e r s i n v o l v e d in
t h e e x p r e s s i o n f o r E ( E q n . 1 ) w e h a v e p l o t t e d t h e
c a l c u l a t e d E a s a f u n c t i o n o f t h e c o n c e n t r a t i o n c ; in
t h e b r a i n - c a p i l l a r y a n d c o m p a r e d w i t h t h e o b s e r v e d
E v a l u e s ( F ig . 3 ) f o r v ar i o u s p e p t id e s , M e t - E N K
e t c . ( u s i n g t h e m o s t a p p r o p r i a t e v a l u e s ( T m ax )f = 0 . 4 6
t 0 -3 p m o l / g ' s , K r = 0 . 3 5 n M a n d t h e P~ v a l u e s f r o m
T a b l e I V ) . O n e n o t e s t h a t t h e e x p e r i m e n t a l E v a l u e s
c a n b e e x p l a i n e d b y o u r p r e s e n t t h e o r e t i c a l t r e a t -
m e n t . A l s o , f o r c~
< >
Kr
t h e e x t r a c t i o n b e c o m e s a l m o s t i n -
d e p e n d e n t o f t h e c o n c e n t r a t i o n o f p e p ti d e s w i t h th e
a s y m p t o t i c v a lu e a p p r o a c h i n g t h e o n e f r o m s im p l e
d i f f u s i o n . T h i s s i g n i f i e s t h a t t h e c a r r i e r s i n v o l v e d a r e
s a t u r a t e d a t h i g h c o n c e n t r a t i o n s t r a n s p o r t i n g t h e
p e p t id e s w i t h m a x i m u m c a p a c it y .
F i g . 3 s h o w s t h e s h a r p v a r i a t i o n o f E v s lo g l 0 c ; in
t h e r a n g e 1 02 < c ; < 1 04 ( i n p M ) . T h i s i s i n t e r e s t i n g a s
i t c a n b e u s e d t o e x t r a c t i n f o r m a t i o n a s t o t h e p e p t i d e
c o n c e n t r a t i o n t h a t s h o u l d b e p r e s e n t n o r m a l l y i n th e
b r a i n - c a p i l la r y f o r t h e e f f i ci e n t w o r k i n g o f t h e B B B
i n n a t u r e . S i n c e t h e v a r i a ti o n o f t h e e x t r a c t i o n w i t h
c o n c e n t r t io n is l a r g e n e a r t h e p o i n t o f i n f lc c u o r~
( F ig . 3 ) w e d e d u c e t h e n o r m a l c o n c e m r a t i o n ~: o [ a
p e p t i d e f r o m t h e e x p r e s s i o n
[ J l
. . . .
y = ) 18)
[ d ( l o g , o co) ] c~ = C:
w h i c h w i t h E q n . 1 g i v e s ,
- 2 F + K r 1 + 2 F K ( 19 )
(Tmax)l
- K t fo r 2~FK~- < < 1 12t/ )
U s i n g ( Tm a x) = 0 . 4 6 x l () - ~ p m o l / g ' s a n d K ; = 0 , 3 5
n M f o r M e t - E N K e t c .. a n d F = 9 . 3 3 x t 0 -3 c m 3/ ~'~
w e o b t a i n
(Tm x)' - 0.07 < < 1
2 F K t
w h i c h m e a n s t h a t e ~ K r = 0 . 3 5 n M a n d . h e n c e , t h e
n o r m a l c o n c e n t r a t i o n o f t h e p e p t i d e s i n t h e b r a i n is c ,,
~ - E e ~ E K , ~ ( 8 % ) ( 0 .3 5 n M ) = 0 . 0 3 n M . T h e im -
p o r t a n t i n f o rm a t i o n w h i c h w e h a v e d e d u c e d , n a m e -
ly, e ~- K t is u su a l l y ta k e n f o r g r a n t e d a n d c o n s t i t u t e s
a g o o d a p p r o x i m a t i o n , a s p o i n t e d o u t b y G r e e n -
b e r g ~ l, t h o u g h n o v a l i d j u s t i f i c a t i o n f o r i t a p p e a r s t o
h a v e b e e n g i v e n p r e v i o u s ly .
A s i n f e r r e d b y G r e e n b e r g e~ a l. ~2 f r o m s y n a p t o -
s o m e e x p e r i m e n t s t h e p e p t i d e s c t -M S H . M I F a n d
A V P d o n o t h a v e s p e c i f i c b i n d i n g i n t h e m a j o r r e -
g i o n s , f o r e b r a i n , c e r e b r a l c o r t e x a n d h y p o t h a t a m u s ,
o f t h e b r a i n . H o w e v e r . i t is k n o w n ~:,2~ t h a t p e p t i d e s
e x e r t e f f ec t (s u c h as p r o d u c e d b v n e u r o m o d u l a t o r s
o r o p i a t i c e f f e c t , e t c . ) in t h e c e n t r a l n e r v o u s s y s t e m
i n d i c a ti n g t h e e x i s t e n c e o f s o m e r e g i o n s o f c e n t r a l
n e r v o u s s y st e m ( C N S ) w h e r e p e p t i d e s m a y h a v e s p e -
c i fi c b in d i n g s ~ L T h e p o s s i b l e a r e a s I I f o r s p e c i f i c b i n d -
i n gs o f p e p t i d e s a r e l o c u s c o e r u l e u s , r a p h e n u c l e u s ,
b r a i n s t e m r e t i c u l a r f o r m a t i o n , t h a l a m u s a n d l i m b i c
s y s te m . I n t h e s e p a r t ic u l a r a r e a s ~ c o n c e n t r a t i o n o f
p e p t id e s m a y b e h i g h ev e n t h o u g h t h e t ot a l a m o u n t m
t h e w h o le b r a i n - v o l u m e m a y b e m u c h l o w e r.
I t c o u l d b e p o s s i b le t h a t t h e p e p t i d e s n d o n o t g e t
m e t a b o l i z e d i n t h e n o n - s p e c i f i c r e g i o n s o f t h e b r a i n
a f t e r i n j e c t i o n s o t h a t t h e y m a y s t a y i n t h e n e u r o n a l
c e l l - m e m b r a n e s ( in b r a i n ) f o r 2 - - 3 r a i n ( u n l i k e w a t e r
f o r w h i c h t h i s t i m e i s i n s e c o n d s ) a n d s u b s e q u e n t l y
g e t r e l e a s e d f r o m t h e c e l l - m e m b r a n e a n d r e t u r n t o
b l o o d . T h i s s p e c u l a t i o n s e e m s v a l i d a s a c u t e i n j e c t io n
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
13/14
of some peptides cures l: Parkinsonism only for a
short duration after which it (Parkinsonism) comes
back to its original symptom 11. This implies that for
gaining continuous pharmacological effect one
should infuse the peptides continuously rather than
administer acutely. This can explain why the acute
peptide administration in some experiments (intra-
peritoneally or intraventricularly) does not produce
significant pharmacological effects.
Rapoport et al.:l have concluded that the trans-
port of peptides across blood-brain barrier (BBB) is
a function of the solubility of peptides in BBB com-
pared to aqueous medium. According to our present
investigation, the transport of peptides is mainly due
to the carrier-mediated facilitated diffusion: the sim-
ple diffusion, though dependent on the solubility of
the peptides in BBB, contributes only about 1()-2()/(
(Table IV) of the total extraction.
As for the actual carrier-mediated process the pep-
tide-carrier complex (activated complex) may be
conceived of diffusing (or rot ating in certain cases of
transport -- not of present concern) in BBB from
blood-side to brain-side, relieving the peptide on the
brain-side and subsequently returning to blood-side
to repeat the process. Gree nbe rg et a1.12 and Kastin
et al.l~ have observed that peptides cross BBB read-
ily, as opposed to conclusion of Cornford et al. 5 who
obtained low extractions of peptides. This apparent
contradiction can be resolved easily in view of our
present theoretical calculations (Fig. 3) which pre-
dict that at low concentrations the extraction is high
and at high concentration the extraction is low. In
fact, if c~ ~< K r one expects to observe high E as
against low E if c~ ~> K,. Furthermore, the order of
magnitude of K, (~< 22 nM) conjec tured by Cornford et
al.5 is consistent with our estimates of K . The varia-
tions of E with log m ca for various peptides given in
Fig. 3 are important for predicting the E values for
any concentration of peptides.
CONCLUSION
In the present paper we have studies comparative-
ly the simple diffusion (of peptides) which depends
on the solubility of peptides in lipids, and the carrier-
mediated facilitated diffusion to estimate the extrac-
tion (Eqn. I ) of peptides across blo od- bra in barrier
(BBB).
213
The extraction due to simple diffusion depends on
the permeability P~ of BBB to peptides which has
been expressed in terms of the partition coefficient f
(Eqns. 12 and 15) representing the solubility of a
peptide in BBB compared to water. The partition co-
efficient f and permeability P~ have been calculated
above for various peptides and fall in thc range 7
10-a~
-
8/11/2019 1984 Sharma Vima Blood Brain Barrier Brain Research
14/14
2 1 4
p l ai n s t h e o b s e r v e d t r a n s p o r t o f p e p t i d e s a c r o ss
B B B .
I t h a s b e e n i n d i c a t e d t h a t t h e p r e s e n t t r e a t m e n t i s
i m p o r t a n t f o r u n d e r s t a n d i n g t h e b i o lo g i c a l a n d n e u -
r o p h a r m a c o l o g i c a l e f f e c t s o f p e p t i d e s. W e h a v e b e e n
a b l e t o d e r i v e a g e n e r a l e x p r e s s i o n f o r th e e x p e c t e d
n o r m a l c a p i l l a r y c o n c e n t r a t i o n ~: o f a p e p t i d e
( E q n . 1 9 ) i n t e r m s o f K t b y i n v o k i n g a m a t h e m a t i c a l
c o n d i t i o n a p p r o p r i a t e t o o p t i m u m e f f i ci e n c y o f a n a t-
u r a l s y s t e m .
F o r t h e p e p t i d e s i n v e s t i g a t e d w e h a v e d e d u c e d a n
i m p o r t a n t r e s u l t t h a t e = K t ( E q n . 2 0 ) . I n a d d i t i o n ,
w e h a v e d i s c u s s e d b r i e f l y th e n o n - s p e c i f i c a n d s p e c i f -
i c b i n d i n g s o f p e p t i d e s i n t h e c e n t r a l n e r v o u s s y s t e m
i n t h e d i s c u s s i o n s e c t i o n w h i c h l e a d u s t o c o n c l u d e
t h a t , i n o r d e r t o h a v e m o r e s i g n i f i c a n t a n d l o n g e r
p h a r m a c o l o g i c a l e f f e ct s c o m p a r e d t o tr a ns ie n ~ c [ -
f e c t s o f a c u t e a d m i n i s t r a t i o n , o n e n t u st i n f u s e ~ he
p e p t i d e s c o n t i n u o u s l y .
T h e p r e s e n t t h e o r e t i c a l m o d e l c o u l d v e r y w e ll b e
e x t e n d e d w i t h o u t f u r t h e r m o d i f i c a t i o n s t o o b t a i n e x -
t r a c t i o n s o r b r a i n u p t a k e s f o r s u b s t a n c e s s u c h a s a m ~ -
n o a c i d s , h e x o s e s , u r e a , a m i n e s a n d o t h e r m o l e c u l e s
h a v i n g s i m i l a r t r a n s p o r t p r o p e r t i e s .
A C K N O W L E D G E M E N T S
T h i s w o r k h a s b e e n s u p p o r t e d i n p a r t b y th e R e -
s e a r c h B o a r d o f t h e U n i v e r s i t y o f I l l i no i s at C h i c a g o
u n d e r B i o m e d i c a l R e s e a r c h P r o j e c t s . T h e a u t h o r s
a r e t h a n k f u l t o P r o f e s s o r R . G r e e n b e r g f o r v a l u a b l e
d i s c u s s i o n s .
R E F E R E N C E S
1 Barak , Z . and Gi lva ry , C . . Pep t ide t ranspo r t , B i o m e m -
branes 7 (1975) 167.
2 Barke r , J . L . , Pep t ides : ro les in neuron a l exc i tab i l i ty , Phys-
iol. Rev. 56 (1976) 435-452.
3 B a s s i n g t h w a i g h t e , J . B . ; A c o n c u r r e n t f l o w m o d e l f o r e x-
t rac t ion dur ing t ranscap i l la ry passage , Circulat. Res. 35
(1974) 483-503.
4 Cohn , E . J . and Eds ta l l , J . T . , Pro te ins Am ino Ac ids and
Peptides R e i n h o l d P u N . C o . , N e w Y o r k , 1 9 5 9 , p p .
196-216.
5 C o r n f o r d , E . M . , B r a u n , L . D . , C r a n e , P . D . a n d O l d e n -
d o f f , W . H . , B l o o d - b r a i n b a r r i e r r e s tr i c t io n o f p e p t i d e s
a n d t h e l o w u p t a k e o f e n k e p h a l i n s , Endocr ino logy 103, 4 .
1978, pp. 1297-1303.
6 C r o n e , C . , T h e p e r m e a b i l i t y o f c a p il l a ri e s in v a r i o u s o rg a n s
a s d e t e r m i n e d b y u s e o f t h e i n d i c a t o r d if f u s i o n m e t h o d ,
Acta . phys io l scand . 58 (1963) 292-305.
7 D a v s o n , H . a n d D a n i e l l i , J . F . , The Permeabili ty of Natura l
M e m b r a n e s Ha fner PUN . , 197(I , pp . 324-33 5 .
8 D e W i e d , D . , The Neurosc iences Th ird S tudy Program
Cam br idge , M A, M IT Press , (1974) p . 653 .
9 G o l d s t e i n , G . W . , C s e j t e y , J . a n d D i a m o n d , I . , C a r r i e r m e -
d ia ted g lucose t ranspor t in cap i l la r ie s i so la ted f rom ra t
b r a i m
J . Neurochem.
28 (1977) 725-728.
1 0 G o r e s k y , C . A . , Z i e g l e r , W . H . a n d B a c h , G . G . , C a p i l l a ry
exchan ge mode l l ing , ba r r ie r - l im i ted and f low- l im i ted d is t r i-
bu t ion , Circulat. Res. 27 (1970) 739-764.
11 G r e e n b e r g , R . ( p e rs o n a l c o m m u n i c a t i o n ) .
1 2 G r e e n b e r g , R . , W h a l l y , C . E . , J o u r d i k i a n , F . , M e n d e l s o n ,
I . S . , Wal te r , R . , Nico l ic s , K. , Coy , D. H. , Scha l ly , A. V.
a n d K a s t i n , A . , P e p t i d e s r e a d i l y p e n e t r a t e t h e b l o o d - b r a i n
bar r ie r : up ta ke o f pep t ides by synap toso mes in pass ive , J .
Pharmaco l . B iochem. Behav . 5 (1976) 151 -158.
13 Kas t in , A. J . , Nissen , J . C . , Scha l ly , A. V. and Coy , D. H. ,
B l o o d - b r a i n b a r r i e r h a l f - t i m e d i s a p p e a r a n c e a n d b r a i n d i s-
t r i b u ti o n o f l a b e l e d e n k e p h a l i n a n d p o t e n t a n a l o g , Bra in
Res. Bull . 1 (1976) 583.
14 K a st in . A . J . , S e n d m a n C . A . . S t r a t t o n L . O . , S c h a t l v , A .
V . a n d M i l l er . L . H . . B e h a v i o r a l e l e c t r o g r a p h i c c h a n g e s i n
ra ts and man a f te r MSH. Progress in Brain Research Vol.
42. Elsev ie r Sc ien t i f ic , Am ste rd am , 1975 . pp. 143-150
15 Laidler. K. J . Theories of Chemical Rates. M c G r a w - H i l t .
New York . 1969 , pp . 41 -55 .
16 Lehn ing er , F . L . .
Biochemis try .
2nd edn . Wor th Pub t . .
New York , 1975. pp . 1 -24 8 .
1 7 L u n d - A n d e r s o n , H . . T r a n s p o r t o f g l u c o s e f r o m b l o o d t o
bra in . Physiol. Rev. . 59 (19791 305-352.
18 Marx . J . L . . Learn ing behav iour f I~ e f fec ts o f p l tmta ry
h o r m o n e s . Science 19011975) 367-37lL
19 Merck Index . Merck , NJ . 1968 .
2 0 O l d e n d o r f . W . H . . B r a i n u p t a k e o t r a d i o l a b e l e d a m i n o
ac ids , amines and hexoses a f te r a r te r ia l in lecuon . A m e r J .
Phvsiol. 2 21 ( 1 9 7 1 ) 1 6 2 9 - 1 6 3 9
2 1 R a p o p o r t , S . I . . K l e e . W A . . P e t u g r c w . K . D . a n d O h n o ,
K . . E n t r y o f o p i o i d p e p t i d e s i n t o t h e C N S . Science. 207
(1980) 84-8 6 .
2 2 R a p o p o r t . S . I .. O h n o , J . a n d P e t t t g r c w , K . D . . D r