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RADIOIMMUNOASSAY OF CHOLECYSTOKININ
IN TISSUE AND PLASMA
Studies in normal subjects and in patients with coeliac
disease and pancreatic insufficiency
A a n Ria
R e m o o en A l e x a n d e r
A a n m i j n oud er s
RADIOIMMUNOASSAY OF CHOLECYSTOKININ
IN TISSUE AND PLASMA
Studies in normal subjects and in patients with coeliac
disease and pancreatic insuffïciency
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN
DOCTOR IN DE GENEESKUNDE
AAN DE KATHOLIEKE UNIVERSITEIT TE NIJMEGEN
OP GEZAG VAN DE RECTOR MAGNIFICUS
PROF. DR. J. H. G. I. GIESBERS
VOLGENS BESLUIT
VAN HET COLLEGE VAN DEKANEN
IN HET OPENBAAR TE VERDEDIGEN
OP VRIJDAG 20 JANUARI 1984
DES NAMIDDAGS TE 2.00 UUR PRECIES
DOOR
JOHANNES BERNARDUS
MARIA JOSEPHUS
JANSEN
GEBOREN
TE
AARLE-RIXTEL
krips repro meppel
1984
Promotor : Dr. J. H. M. van Tongeren
Co-referent : Dr. C. B. H. W. Lamers
The studies presented in this thesis were supported by a grant (13-37-32) from the Foundation for Medical
Research FUNGO and were carried out under the direction of Dr CBHW Lamers in the Laboratory of
Gastroinlestinal Hormones;
Division of Gastroenterology (hcad Dr JH M van Tongeren), Department of Medicine (head Prof Dr A van
’t Laar), St Radboud Hospital, Umversity of Nijmegen, Nijmegen, The Netherlands.
CONTENTS
Chapter
I
Chapter
II
Introduction
Radioimmunoassay
duction
Chapter
III
a nd o u t l i n e
investigations
of a n t i b o d i e s
of Bol t o n - H u n t e r
27
labelled
l e c y s t o k i n i n -33 to c a r b o x y l - t e r m i n a l
cho­
CCK-
antibodies
Chapter
IV
tokinin
Chapter
Chapter
V
VI
VII
employing
V II I
IX
39
brai n
in p o r c i n e
Radioimmunoassay
X
of c h o l e c y s t o k i n i n 59
of c h o l e c y s t o k i n i n
in hu-
tissue and plasma
63
Ef f e c t of i n s u l i n - h y p o g l y c a e m i a
a nd
pancreatic
on p l a s ma
polypeptide
77
E f f e c t of b o m b e s i n on p l a s m a c h o l e c y s t o ­
in n o r m a l
subjects
zed
patients
fic
radioimmunoassays
measured
and g a s t r e c t o m i -
by s e q u e n c e
plasma
during
infusion
Plasma cholecystokinin
patients with
speci83
M o l e c u l a r f o r m s of c h o l e c y s t o k i n i n
man
Chapter
cholecystoki­
47
evidence
kinin
Chapter
different
to c h o l e c y s ­
1 abel s
cholecystokinin
Chapter
of a n t i s e r a
Immunological
man
Chapter
37
Characterization
nin
7
of c h o l e c y s t o k i n i n : P r o -
and e v a l u a t i o n
Low binding
of
in h u-
of bombe s i n
concentrations
pancreatic
insufficiency
95
in
105
5
Chapter
XI
Plasma
cholecystokinin
responses
and ga ll bladder
to i n t r a d u o d e n a l
tients with
coeliac
fat
in pa-
disease
121
Summary
135
Samenvatting
139
Woorden
143
van W a a r d e r i n g
C u r r i c u l u m Vitae
Publications
on G a s t r o i n t e s t i n a l
145
Hormones
by t h e A u t h o r
146
Chapter I
Introductlon
and
Outline of Investigations
INTRODUCTION
History
F or a v e r y l o n g p e r i o d it w as g e n e r a l l y a c c e p t e d t h a t act i v i t i e s in t he b o d y w e r e e n t i r e l y c o - o r d i n a t e d b y the n e r v o u s
system. T h e d i s c o v e r y of s e c r e t m in 1902 by B a y l i s s & Sta r l ing (1) t h e r e f o r e , c a n b e c o n s i d e r e d as a t u r m n g - o o i n t in
h i s t o r y o f c o - o r d i n a t i n g functions.
messenger"
S e c r e t i n is a "ch e m i c a l
s h a n n g w i t h the n e r v o u s s y s t e m the q u a l i t y of co-
o r d i n a t i n g f u n c t i o n s in o t h e r o r g a n s t h a n the p l a c e w e r e the
s t i m u l u s is r e c e i v e d . B a y l i s s & S t a r l i n g r e a l i z e d , t h a t ït
w a s u n l i k e l y t h a t s e c r e t i n was the o n l y e n d o g e n o u s s u b s t a n c e
t h a t a c t e d as a " c h e m i c a l m e s s e n g e r " b e t w e e n d i f f e r e n t organs,
a n d t h a t o t h e r s w o u l d s o o n be found. T h e y c r e a t e d a n e w w o r d
to d e s e n b e s u c h m e s s e n g e r s : hormone, d e r i v e d f r o m the G r eek
a n d m e a n i n g "to e x c i t e " (2). B a y l i s s & S t a r l i n g w e r e c o r ­
r e c t in a s s u m i n g t h a t m a n y h o r m o n e s w o u l d s o o n be d i s c o v e r e d .
In 1904, F l e i g d i s c o v e r e d t h a t a c i d a n d s o a p w e r e able to r e ­
lease a h o r m o n e f r o m t h e d u o d e n u m s t i m u l a t i n g b i l e s e c r e t i o n
(3). In 1905, E k i n s d i s c o v e r e d g a s t n n ,
f r o m the a n t r a l m u c o s a ,
a hormone o n g i n a t i n g
g i v m g g a s t n c a c i d s e c r e t i o n w h e n ïn-
]ected intravenously into anaesthetized cats
(4). F u r t h e r evi-
d e n c e t h a t g a l l b l a d d e r c o n t r a c t i o n w a s r e g u l a t e d bv a h o r m o n e
w a s f o u n d b y Ivy & O l d b e r g in 1928
(5). T h i s h o r m o n e wa s n a m e d
c h o l e c y s t o k i n i n . In 1943, H a r p e r & R a p e r
(9) d i s c o v e r e d a h o r ­
m o n e g i v i n g p a n c r e a t i c e n z y m e secret ion, w h i c h t h e y n a m e d o a n creozymin. Although other gastrointestinal hormones,
tilin
too,
like m o -
(6) a n d s u b s t a n c e P (7) w e r e d i s c o v e r e d in that p e r i o d
s e c r e t i n , g a s t n n a n d c h o l e c y s t o k i n i n c o n s t i t u t e the c l a s -
sic a l t r i a d of g u t h o r m o n e s , b e c a u s e for a l o n g period, t h e s e
3 h o r m o n e s w e r e c o n s i d e r e d s u f f i c i ë n t to e x p l a i n th e e n t i r e
e n d o e n n e r e g u l a t i o n of d i g e s t i v e f u n c t i o n s
(8).
H a l f a c e n t u r y e l a p s e d b e f o r e the ï m o l i c a t i o n s of the gast r o i n t e s t i n a l h o r m o n e s w e r e r e alized. Th e r e a s o n s for s u c h s l o w
p r o g r e s s i n c l u d e d c o n t r o v e r s y o v e r the v e r y e x i s t a n c e of several h o r m o n e s o n o ne hand,
8
a n d d i f f i c u l t i e s in ï s o l a t m g t h e m
on the o t her. O n l y in the 6 0 ' s, b i o c h e m i c a l t e c h n i q u e s b e c a m e
s o l i d e n o u g h to p u r i f y t i s s u e e x t r a c t s for s t r u c t u r a l w o r k
(12,
13,14). D u r i n g p u r i f i c a t i o n of the c h o l e c y s t o k i n e t i c s u b s t a n c e
f r o m p o r c i n e s m a l l i n t e s t i n e , J o r p e s & M u t t d i s c o v e r e d that
t his c h o l e c y s t o k i n e t i c s u b s t a n c e w a s a l s o r e s p o n s i b l e for the
pancreozymin activity, and that cholecystokinin and pancreoz y mi n w e r e in f a c t t he same h o r m o n e
d en c e in d i s c o v e r y ,
kinin
(10). In v i e w of a n t e c e -
t h i s p o l y p e p t i d e is n o w n a m e d c h o l e c y s t o ­
(11). A f t e r 1960 t he d e v e l o p m e n t in b i o c h e m i c a l t e c h n i ­
ques a d v a n c e d in a w a y t h a t n o w a d a y s m a n y p o l y p e p t i d e s are
purified and structurally characterized, before their biological f u n c t i o n s a re e l u c i d a t e d
Recently,
( 1 5 , 1 6 , 1 7,18).
a d r a m a t i c n e w p e r i o d in the h i s t o r y of c h o l e ­
c y s t o k i n i n s t a r t e d , d u e t o the d e v e l o p m e n t of r a d i o i m m u n o a s s a y
and immunohistochemical techniques.
In 1975, V a n d e r h a e g h e n et
al d i s c o v e r e d a g a s t r i n - l i k e s u b s t a n c e in the c e n t r a l n e r v o u s
s y s t e m (19), t h a t t u r n e d o u t to be m a i n l y c h o l e c y s t o k i n i n - l i k e .
(20,21,22).
It b e c a m e c l e a r that c h o l e c y s t o k i n i n wa s n o t o n l y
c o n f i n e d to the g a s t r o i n t e s t i n a l tract, bu t was a l s o w i d e l y
d i s t r i b u t e d t h r o u g h o u t the c e n t r a l a n d p e r i f e r a l n e r v o u s s y s ­
t e m (21,23) .
Heterogeneity
L i k e m a n y o t h e r p o l y p e p t i d e h o r m o n e s , c h o l e c y s t o k i n i n is
h e t e r o g e n e o u s . T h i s m e a n s that p e p t i d e h o r m o n e s e x i s t in a numb e r o f d i f f e r e n t m o l e c u l a r forms a l l c o n t a i n i n g the biologic-ally
active region
(size-heterogeneity)(24,25,26,27), while between
species amino acid variations
may occur
ty) (28, 29, 30) . In m a m m a l i a n species,
(s p e c i e s - h e t e r o g e n e i -
at l e a s t 5 m o l e c u l a r v a r -
ian t s of c h o l e c y s t o k i n i n h a v e b e e n d e m o n s t r a t e d in s m all intestinal mucosa extracts
(31). So far 3 of t h e s e m o l e c u l a r forms
have been isolated and structurally characterized from m a m m a ­
lian i n t e s t i n a l m u c o s a : T he o r i g i n a l l y i s o l a t e d p o r c i n e t r i a contatriapeptide
(CCK 33) (14), a m o l e c u l a r v a r i a n t e x t e n d e d by
a f u r t h e r 6 a m i n o a c i d r e s i d u e s at the a m i n o - t e r m i n u s , p o r c i n e
triacontanonapeptide
(CCK 39) (32), a n d a m o l e c u l a r f o r m c o n t a i -
9
n i n g 58 a n n o a c i d r e s i d u e s
(CCK 58), p r o b a b l y c o m p a r a b l e w i t h
porcine component I cholecystokinin
(31), w h i c h h a s r e c e n t l y
b e e n i s o l a t e d f r o m c a n i n e small m t e s t i n a l m u c o s a
(33). Al-
t h o u g h at p r e s e n t o n l y l a r g e r m o l e c u l a r f o r m s of c h o l e c y s t o k i ­
n i n h a v e b e e n i s o l a t e d f r o m u p p e r small m t e s t i n a l mucosa,
ït
is b e l i e v e d f r o m r a d i o i m m u n o a s s a y data, t h a t s m a l l e r m o l e c u l a r
forms predominate
(31,34,35). H o w e v e r , u n t i l n o w none of the s e
small molecular v a n a n t s have been structurally c h a r a c t e n z e d
f r o m sma l l i n t e s t i n e . A l t h o u g h ït is g e n e r a l l y bel i e v e d ,
s m a l l e r m o l e c u l a r f o r m s of c h o l e c y s t o k i n i n ,
tide, p r e d o m i n a t e in s m a l l m t e s t i n a l m u c o s a
that
like the o c t a p e p (31, 34,35), the
c o r r e c t p r o p o r t i o n b e t w e e n the d i f f e r e n t m o l e c u l a r v a n a n t s
is
s t i l l q u e s t i o n a b l e , a n d some r e c e n t s t u d i e s e v e n s h o w r a t i o s
in f a v o u r of l a r g e r m o l e c u l a r f o r m s
(71,72). A m a j o r re a s o n
for t h i s c o n t r o v e r s y is the d i f f e r e n c e in a n t i b o d y a f f i n i t y
fo r the v a n o u s m o l e c u l a r for m s of c h o l e c y s t o k i n i n . M a n y a n t i s e r a r a i s e d a g a i n s t s m a l l forms of c h o l e c y s t o k i n i n m a y s h o w
diminished
b i n d i n g to l a r g e r m o l e c u l a r v a n a n t s
(36), w h i l e
some antisera raised against larger molecular forms may show
d i m i n i s h e d b i n d i n g to s m a l l e r m o l e c u l a r v a r i a n t s
p r o b l e m c a n be r e l a t e d to d i f f e r e n c e s m
(78). This
amino acid chain
lenght and charge be t w e e n v a n o u s molec u l a r cholecystokinin
variants, promoting conformational changes between smaller and
l a r g e r m o l e c u l a r forms. A l t h o u g h the c o m p l e t e a n t i b o d y b i n d i n g
sit e is p r e s e n t in d i f f e r e n t m o l e c u l a r for m s of a peptide, c o n ­
f o r m a t i o n a l c h a n g e s m a y r e s u l t in d i f f e r e n c e s in a n t i b o d y a f ­
finity
(91).
T h e h e t e r o g e n e i t y is the m o r e i m p o r t a n t , b e c a u s e d i f f e r e n t
m o l e c u l a r for m s m a y p o s s e s s d i f f e r e n t b i o l o g i c a l a c t i v i t i e s .
It has b e e n r e p o r t e d t h a t s u l p h a t e d c h o l e c y s t o k i n i n o c t a p e p t i d e
is
2- 10
t i m e s m o r e p o t e n t on a m o l a r b a s e t h a n c h o l e c y s t o ­
kinin t n a c o n t a t n a p e p t i d e
in s e v e r a l systems,
b l a d d e r c o n t r a c t i o n in v i v o a n d v i t r o
to s m a l l m o l e c u l a r v a r i a n t s ,
i n c l u d i n g gal l -
(85,89,90).
In c o n t r a s t
large m o l e c u l a r f o r m s of c h o l e c y s ­
t o k i n i n like t he t n a c o n t a t n a a n d t r i a c o n t a n o n a p e p t i d e h a v e
p o s i t i v e l y c h a r g e d a m i n o a c i d r e s i d u e s in e x c e s s . T h i s is not
10
o n l y i m p o r t a n t f or e x t r a c t i o n p r o c e d u r e s
(83), b u t it a l s o m a -
kes l a r g e r m o l e c u l a r forms o f c h o l e c y s t o k i n i n v e r y s u s c e p t i b l e
to a d h e r e to p l a s t i c s u r f a c e s l i k e r a d i o i m m u n o a s s a y t u b e s and
infusion materials
(84). H o w e v e r , w h e n s u f f i c i ë n t a m o u n t s of
al b u m i n w e r e a d d e d to i n f u s a t e s to p r e v e n t a d h e r e n c e , c h o l e c y s ­
to k i n i n o c t a p e p t i d e a n d t r i a c o n t a t r i a p e p t i d e t u r n e d o u t to be
e q u i p o t e n t in c o n t r a c t i n g g u i n e a p i g g a l l b l a d d e r in v i t r o
(87)
a n d in s t i m u l a t i n g p a n c r e a t i c e n z y m e s e c r e t i o n in v i v o a n d in
v i tr o
In n e r v o u s tissue, c h o l e c y s t o k i n i n is h e t e r o g e n e o u s too.
S m a l l e r m o l e c u l a r f o r m s p r e d o m i n a t e e v e n m o r e (22,31,36). A
small f o r m o f c h o l e c y s t o k i n i n c o n t a i n i n g the c a r b o x y l - t e r m i n a l
o c t a p e p t i d e is t he o n l y m o l e c u l a r f o r m s t r u c t u r a l l y c h a r a c t e ­
r i z e d in b r a i n , h i t h e r t o
nological evidence,
(37). H o w e v e r , t h e r e is s t r o n g immu-
t h a t l a r g e r m o l e c u l a r v a r i a n t s of c h o l e c y s ­
t o k i n i n a re p r e s e n t in b r a i n t o o
(31,38). The e x a c t b i o l o g i c a l
f u n c t i o n s o f t h e s e m o l e c u l a r v a r i a n t s in the n e r v o u s s y s t e m
stil l h a v e to be e l u c i d a t e d , b u t it is g e n e r a l l y a c c e p t e d , t h a t
larg e r m o l e c u l a r for m s of c h o l e c y s t o k i n i n in b r a i n are p r e c u r ­
sors of s m a l l e r ones,
tetrapeptide
like the o c t a p e p t i d e a n d p o s s i b l y the
(31).
T h e m e s s e n g e r - R N A of c h o l e c y s t o k i n i n has n o t y e t b e e n
isolated, b u t it c a n be d e r i v e d f r o m s t u d i e s on the r e l a t e d
p e p t i d e g a s t r i n , w h e r e m e s s e n g e r - R N A c o d e s for a p e p t i d e of
some 110 - 140 a m i n o a c i d r e s i d u e s
(39,40)
that t h e r e is r o o m
for a d d i t i o n a l h e t e r o g e n e i t y a n d e x t e n s i o n s of the k n o w n m o ­
le c u l a r forms, n o t o n l y at the a m i n o - t e r m i n u s , b u t a l s o at the
carboxyl-terminus
(32).
C h o l e c y s t o k i n i n in the n e r v o u s s y s t e m
C h o l e c y s t o k i n i n is w i d e l y d i s t r i b u t e d t h r o u g h o u t the n e r ­
v o u s s y s t e m (40) . B o t h in c e n t r a l a n d p e r i p h e r a l n e r v o u s tissue,
c h o l e c y s t o k i n i n o c c u r s n ot o n l y in c e l l b o dies, b u t a l s o in
n e rv e e n d i n g s . T h e r e is i m m u n o c y t o c h e m i c a l e v i d e n c e , t h a t c h o ­
l e c y s t o k i n i n is m a i n l y c o n c e n t r a t e d in n e r v e t e r m i n a l s e s p e c iall y in s y n a p t o s o m e s and s y n a p t i c v e s i c u l e s
(42). It has a l s o
11
b e e n s h o w n t h a t d e p o l a r i z a t i o n of n e r v e s in r a t s induces a c a l ­
cium dependent cholecystokinin release
(42). F u r t h e r m o r e , c h o ­
l e c y s t o k i n i n o c t a p e p t i d e a n d t e t r a p e p t i d e a p n l i c a t e d to the
p o s t s y n a p t i c m e m b r a n e in t h e h i p p o c a m p e s w e r e a b l e to e x c i t a t e
neurones
(43). T a k e n t o g e t h e r , t h e s e e x p e r i m e n t s full f i l l the
m a j o r c r i t e r i a for c h o l e c y s t o k i n i n to be a c c e p t e d as a n e u r o ­
t r a n s m i t t e r in the n e r v o u s s y s t e m (44). Th e e x a c t f u n c t i o n s of
c h o l e c y s t o k i n i n in c e n t r a l a n d p e n f e r a l n e r v o u s t i s s u e have
s t i l l t o be e l u c i d a t e d a n d a re c h a l l e n g e s for r e s e a r c h in the
future.
C h o l e c y s t o k i n i n in t h e s m a l l m t e s t i n a l m u c o s a
It is g e n e r a l l y a c c e p t e d ,
hormone.
However,
t h a t c h o l e c y s t o k i n i n acts as a
t h i s c o n c e p t has so far b e e n b a s e d o n s t u d ­
ies in w h i c h the e v i d e n c e h a s b e e n i n d i r e c t
(45,46,47,48).
In
sum m a r y , n u t n e n t s p e r f u s e d i n t r a d u o d e n a l l y a r e able to c a u s e
gallbladder contraction
cretion
(45,46,48)
a n d p a n c r e a t i c e n z y m e se-
( 4 5 , 4 6, 4 7 , 4 8 ) . F r o m t h e s e e x p e r i m e n t s it wa s c o n c l u d e d
t h a t i n t r a d u o d e n a l a d m i n i s t r a t i o n of n u t n e n t s act by r e l e a sin g c h o l e c y s t o k i n i n f r o m the d u o d e n a l m u c o s a . However,
it h a s
a l s o b e e n shown, t h a t c h o l i n e r g i c r e f l e x e s w e r e able to c a u s e
pancreatic enzyme secretion d u n n g
i n t r a d u o d e n a l p e r f u s i o n of
s o d i u m o l e a t e a n d a m i n o a c i d s and t h at c h o l e c y s t o k i n i n was n o t
of n e c e s s i t y for t h i s a c t i o n
(51,49,50). W h e t h e r n e u r a l or
h o r m o n a l m e c h a n i s m s a r e t he m a j o r p h y s i o l o g i c a l s t i m u l u s for
p a n c r e a t i c e n z y m e s e c r e t i o n a f t e r a m e a l ha s s t i l l to be e l u ­
cidated. Furthermore, conflictang results have been published
a b o u t the a c t i o n of c h o l e c y s t o k i n i n on t a r g e t organs. For ïnstance,
it h as b e e n s h o w n t h a t l ow d o s e s of c h o l e c y s t o k i n i n
l e a d t o m u s c u l u s s p h i n c t e r O d d i r e l a x a t i o n , b u t h i g h d o s e s to
contraction
(52). T h e s e p r o b l e m s c an o n l y be u n n d d l e d by de -
m o n s t r a t i o n t h a t t a r g e t c e l l r e s p o n s e s c a n be m i m i c k e d be e x o g e n o u s i n f u s i o n of c h o l e c y s t o k i n i n in a m o u n t s t h a t p r o d u c e
p l a s m a c o n c e n t r a t i o n s s i m i l a r to t h o s e o b s e r v e d d u n n g the end o g e n o u s r e l e a s e of c h o l e c y s t o k i n i n b y m e a l s t i m u l a t i o n
(53,
54). T h e o n l y s u i t a b l e t e c h n i q u e a v a i l a b l e for this p u r p o s e at
12
TABLE I
peripheral biological actions of cholecystokinin
-
Increases
pancreatic
- Gallbladder
- Relaxation
secretion
contraction
of m u s c u l u s
- Augmentation
- Inhibition
enzyme
of
of
the
gastric
- Increases
intestinal
- Decreases
lower
sphincter
action
of
Oddi
secretin
emptying
peristalsis
oesophageal
- Inhibition
of
gastrin
- Inhibition
of
intestinal
pressure
stimulated
water
- Trophic
effect
on
pancreas
- Trophic
effect
on
intestinal
- Effects
on
gastric
and
acid
electrolyte
secretion
absorption
mucosa
satiety
TABLE II
amino acid sequence of the gastrin-cholecystokinin family
Pyro-Leu-Gly-Pro-Gln-GlyhGastrin 1-34 =
pCCK
1-39: H j N - T y r - I l e - G l n - G l n - A l a - A r g - L y s - A l a - P r o - S e r - G l y -
hGastrin 1-34:
pCCK
1-39: A r g - V a l - S e r - M e t - I l e - L y s - A s n - L e u - G l n - S e r - L e u - A s p (s o 3h )
hGastrin 1-34: G l y - P r o - T r p - L e u - G l u - G l u - G l u - G l u - G l u - A l a - T y r - G l y pCCK
1-39:
SO^H
hGastrin 1-34:
PCCK
1-39:
13
the m o m e n t is r a d i o i m m u n o a s s a y . A ll r e p o r t e d p h a r m a c o l o g i c a l
a c t i o n s of c h o l e c y s t o k i n i n
(table I ) , h a v e to b e v a l i d a t e d for
t h i s c r i t e r i u m to be c o n s i d e r e d of p h y s i o l o g i c a l s i g n i f i c a n c e .
H o w e v e r , r a d i o i m m u n o a s s a y of c h o l e c y s t o k i n i n ha s p r o v e n to be
extremely difficult and conflicting data have been published
(55,56,57,58).
P R O B L E M S IN C H O L E C Y S T O K I N I N R A D I O I M M U N O A S S A Y
Antibodies
M a n y p r o b l e m s in t he r a d i o i m m u n o a s s a y of c h o l e c y s t o k i n i n
c a n b e r e l a t e d to t he s t r u c t u r e of t he p e p t i d e , d i s p l a y i n g
some r e m a r k a b l e f e a t u r e s
(table I I ) . M o s t s t r i k i n g is the si-
m i l a r i t y of t h e b i o l o g i c a l l y a c t i v e p a r t of c h o l e c y s t o k i n i n and
g a s t r i n . C h o l e c y s t o k i n i n a n d g a s t r i n sha r e an i d e n t i c a l c a r b o x y l - t e r m i n a l p e n t a p e p t i d e amide. T h i s s i m i l a r i t y s u g g e s t s a common ancestor
(63) . T h e a m i n o - t e r m i n a l e x t e n s i o n of this p e n t a ­
p e p t i d e t h e r e f o r e g o v e r n s the b i o l o g i e a c t i v i t y of these p e p t i d e s t o w a r d s d i f f e r e n t t a r g e t organs.
In t h i s way, g a s t r i n is
r e s p o n s i b l e f or g a s t r i c a c i d s e c r e t i o n , w h i l e c h o l e c y s t o k i n i n
c a u s e s g a l l b l a d d e r c o n t r a c t i o n a n d p a n c r e a t i c e n z y m e secret i o n .
B e c a u s e t h i s b i o l o g i c a l l y a c t i v e p e n t a p e p t i d e is h i g h l y i m m u n o ge n i c ,
it t u r n e d o u t t h a t t h i s s i m i l a r i t y d i s p l a y e d c o n s i d e r a -
b l e p r o b l e m s in r a i s i n g s p e c i f i c a n t i s e r a , e s p e c i a l l y for c h o ­
l e c y s t o k i n i n . A n t i s e r a f or c h o l e c y s t o k i n i n d i r e c t e d t o w a r d s
t h i s b i o l o g i c a l l y a c t i v e r e g i o n of the m o l e c u l e are u n s p e c i f i c ,
b e c a u s e t h e y s h o w full c r o s s - r e a c t i v i t y w i t h g a s t r i n
(58,64,65).
S i n c e g a s t r i n p r o b a b l y c i r c u l a t e s in h i g h e r c o n c e n t r a t i o n s
t h a n c h o l e c y s t o k i n i n , c r o s s - r e a c t i v i t y b e t w e e n t h e s e two
p e p t i d e s is h i g h l y u n d e s i r a b l e . T h i s p r o b l e m w a s t r i e d to
o v e r c o m e by s e p a r a t i n g p l a s m a c h o l e c y s t o k i n i n f r o m g a s t r i n
u s i n g c h r o m a t o g r a p h i c t e c h n i q u e s in c o m b i n a t i o n w i t h t h ese u n ­
specific antibodies
(58,64,65). H o w e v e r , t h e s e t e c h n i q u e s t u r ­
n e d o u t to b e i n s u f f i c i ë n t in s e p a r a t i n g l a r g e r forms of c h o ­
lecystokinin from gastrin and therefore only allowed estimafeions o n s m a l l e r m o l e c u l a r forms o f c h o l e c y s t o k i n i n . U s i n g
14
these methods,
it a p p e a r e d t h a t i n f u s i o n of c h o l e c y s t o k i n i n
o c t a p e p t i d e r e s u l t i n g in p l a s m a c o n c e n t r a t i o n s c o m p a r a b l e to
th os e f o u n d a f t e r a m i x e d m e a l w e r e i n s u f f i c i ë n t to a c c o u n t
for full p h y s i o l o g i c a l r e g u l a t i o n of p a n c r e a t i c e n z y m e s e c r e ­
tion
(65). A m a j o r h o r m o n a l r o l e f o r c h o l e c y s t o k i n i n o c t a p e p ­
tide is a l s o d i s c u t a b l e b e c a u s e t h e r e is e v i d e n c e t h a t this
p e p t i d e is c l e a r e d to a large e x t e n t f r o m the c i r c u l a t i o n w h e n
it p a s s e s t h e l i v e r
(66,67,68). D a t a on c h o l e c y s t o k i n i n in
plasma should therefore be based on radioimmunoassays using
a n t i b o d i e s b i n d i n g to all b i o l o g i c a l l y a c t i v e c h o l e c y s t o k i n i n
v a r i a n t s p r e s e n t in c i r c u l a t i o n , w i t h o u t c r o s s - r e a c t i v i t y w i t h
gastnn.
T h e s e d a t a c an o n l y b e o b t a i n e d u s i n g a n t i s e r a n o t
d i r e c t e d t o w a r d s the r e g i o n t h a t c h o l e c y s t o k i n i n an d g a s t n n
h a v e in c o m m o n . H o w e v e r , b e c a u s e t h e s e a n t i b o d i e s are n o t d i ­
r e c t e d t o w a r d s the b i o l o g i c a l l y a c t i v e p a r t of the m o l e c u l e , the
p o s s i b i l i t y is m t r o d u c e d that b i o l o g i c a l l y i n a c t i v e f r a g m e n t s
may be bound.
In o r d e r to e x c l u d e t h i s p o s s i b i l i t y , t h e s e a n ­
t i s e r a h a v e t o be c h a r a c t e n z e d w i t h g r e a t care, u s i n g t i s s u e
e x t r a c t s a d e q u a t e l y s e p a r a t e d on m o l e c u l a r w e i g h t a n d c h a rge,
in c o m b i n a t i o n w i t h a n t i b o d i e s d i r e c t e d t o w a r d s the b i o l o g i c a l l y
a c t i v e p e n t a p e p t i d e . F u r t h e r m o r e , a n t i s e r a d i r e c t e d t o w a r d s reg i o n s o u t s i d e the b i o l o g i c a l l y a c t i v e p e n t a p e p t i d e do n o t b i n d
to s m all b i o l o g i c a l l y a c t i v e v a r i a n t s m i s s i n g the a m i n o a c i d
s e q u e n c e a g a i n s t w h i c h the a n t i s e r u m is r a i s e d
(69). Th e m o r e
this b i n d i n g r e g i o n in c h o l e c y s t o k i n i n is l o c a t e d in d i s t a n t
f r o m the b i o l o g i c a l l y a c t i v e p e n t a p e p t i d e , the less b i o l o g i c a l l y
a c t i v e f r a g m e n t s of c h o l e c y s t o k i n i n a r e bound. F o r t u n a t e l y ,
it
t u r n e d o u t t h a t t h e s u l p h a t e d t y r o s y l r e g i o n in v i c i n i t y to
the c a r b o x y l - t e r m m a l p e n t a p e p t i d e ,
is o n e of t h e r e g i o n s o u t ­
side the b i o a c t i v e p e n t a p e p t i d e w i t h th e h i g h e s t i m m u n o r e a c t i vity
(70,71,72). T h i s s u l p h a t e d t y r o s y l r e s i d u e is no t o n l y
h i g h l y i m m u n o r e a c t i v e , b u t a l s o g o v e r n s b i o l o g i c a l a c t i v i t y of
cholecystokinin
(73,74). A n t i s e r a d i r e c t e d t o w a r d s this r e g i o n
h a v e the a d v a n t a g e of b i n d i n g to s u l p h a t e d c h o l e c y s t o k i n i n o c ­
t a p e p t i d e . H o w e v e r , b e c a u s e t h i s s u l p h a t e d t y r o s y l r e g i o n in
c h o l e c y s t o k i n i n a l s o sho w s s t n k i n g s i m i l a n t i e s w i t h s u l p h a -
15
t e d m o l e c u l a r for m s o f g a s t r i n , t h e s e a n t i s e r a h a v e to be c h a r a c t e r i z e d w i t h s u l p h a t e d a n d n ot o n l y w i t h u n s u l p h a t e d m o l e ­
c u l a r v a r i a n t s of g a s t r i n ,
cificity
(70,71,72,88).
in o r d e r to d e t e r m i n e a n t i b o d y spe-
It has a l s o b e e n s h o w n that some a n t i ­
s e r a d i r e c t e d t o w a r d s as y e t u n c h a r a c t e r i z e d b i o l o g i c a l l y inact i v e r e g i o n s in p o r c i n e c h o l e c y s t o k i n i n m a y s h o w d i m i n i s h e d
b i n d i n g to c h o l e c y s t o k i n i n in o t h e r s p e c i e s
(75) , s u g g e s t i n g
s p e c i e s d i f f e r e n c e s in t h i s r e g i o n of c h o l e c y s t o k i n i n . C o n s i d e r i n g t h e s e data, m o s t r e l i a b l e r e s u l t s o n c h o l e c v s t o k i n i n can
be o b t a i n e d b y u s i n g a p a n e l of w e l l c h a r a c t e r i z e d antisera.
R a d i o i o d i n a t i o n of c h o l e c y s t o k i n i n
T h e r e s p o n s e p a r a m e t e r in r a d i o i m m u n o a s s a y s is p e r c e n t a g e
a n t i b o d y b i n d i n g a n d d i s p l a c e m e n t of l a b e l l e d pept i d e . Intro125
d u c t i o n of
I i n t o t h e p e p t i d e w i l l c h a n g e the a n t i g e n e t i c
p r o p e r t i e s o f the p e p t i d e in one w a y or a n o t h e r . T h i s m a y res u l t in d i m i n i s h e d i m m u n o r e a c t i v i t y of the a n t i g e n by the lab e l l i n g t e c h n i q u e , m a s k i n g the t r u e q u a l i t y of a n t i s e r a . C o n v e n t i o n a l l a b e l l i n g t e c h n i q u e s are b a s e d o n o x i d a t i o n u s i n g
125
c h l o r a m i n e T or l a c t o p e r o x i d a s e to i n t r o d u c é
I into the
p e p t i d e , p r e f e r a b l y i n t o an u n s u l p h a t e d t y r o s y l r e s i d u e of a
polypeptide. However, the originally isolated triacontatriapeptide,
in c o n t r a s t t o t h e t r i a c o n t a n o n a p e p t i d e , does not
p o s s e s s s u c h an u n s u l p h a t e d t y r o s y l r e s i d u e , h i n d e r i n g the
125
introduction of
I i n t o t he m o l e c u l e by t h i s m e thod. F u r t h e r m o re , b o t h g a s t r i n a n d c h o l e c y s t o k i n i n c o n t a i n 1 a n d 2 m e t h i o n y l r e s i d u e s in t h e b i o l o g i c a l l y a c t i v e p a r t of the m o l e c u l e .
T h e s e r e s i d u e s are h i g h l y s e n s i t i v e to o x i d a t i o n , r e s u l t i n g in
d i m i n i s h e d b i o a c t i v i t y f or c h o l e c y s t o k i n i n
for g a s t r i n
(70,76)
as w e l l as
(77) . It h a s b e e n r e p o r t e d t h a t o x i d a t i v e r a d i o ­
iodination techniques also abolish cholecystokinin immunore­
activity
(70). T h i s m a y b e an e x p l a n a t i o n f o r t h e lack of sen-
s i t i v i t y in a f e w p r e l i m i n a r y r e p o r t e d r a d i o i m m u n o a s s a y s
56,5 7 ) . H o w e v e r ,
in o t h e r studies,
(55,
l a b e l s p r e p a r e d by o x i d a t i v e
m e t h o d s w e r e s u c c e s s f u l l y e m p l o y e d in r a d i o i m m u n o a s s a y s of
both cholecystokinin
16
( 6 9 , 6 4 , 65,78,79)
and gastrin
(80). In o r ­
der to p r e v e n t o x i d a t i v e l a b e l l i n g d a mage, R e h f e l d (70) in t r o 125
I-hydroxyphenylpropion-
duced a conjugation technique using
ic a c i d - s u c c i n i m i d e e s t e r
( B o l t o n - H u n t e r reagent,
81)
for io-
d i n a t i o n o f c h o l e c y s t o k i n i n . T h i s e s t e r b i n d s to o n e of the
free a m i n o g r o u p s in c h o l e c y s t o k i n i n , p r e f e r a b l y to l y s y l side
chains. H o w e v e r ,
it h a s b e e n s h o w n in albumin,
that this c o n ­
jugation technique interferes with protein conformation,
re-
s u l t i n g in d i m i n i s h e d i m m u n o r e a c t i v i t y for s o m e a n t i s e r a
(82).
In a n a logy,
t h i s t e c h n i q u e m a y a l s o r e s u l t in d i m i n i s h e d i m ­
m u n o r e a c t i v i t y for s o m e c h o l e c y s t o k i n i n a n tisera. T h e r e f o r e ,
a n t i s e r a r a i s e d a g a i n s t c h o l e c y s t o k i n i n s h o u l d be c h a r a c t e r i ­
zed u s i n g b o t h l a b e l s p r e p a r e d b y o x i d a t i v e a n d c o n j u g a t i o n
techniques,
in o r d e r to d e t e c t the m o s t s u i t a b l e c h o l e c y s t o ­
k i n i n a n t i b o d i e s . A s p e c i a l p r o b l e m in the d e v e l o p m e n t of rad i o i m m u n o a s s a y for c h o l e c y s t o k i n i n ,
c o n c e r n s th e q u a l i t y of
p e p t i d e s used. T h e p e p t i d e r e q u i r e d as S t a n d a r d a n d t r a c e r has
to b e p u r e a n d t h e r e f o r e , p r e f e r a b l y s y n t h e t i c f r a g m e n t s h a v e
to b e used. H o w e v e r ,
l a r g e r m o l e c u l a r f o rms of c h o l e c y s t o k i n i n
are o n l y a v a i l a b l e in l i m i t e d a m o u n t s as h i g h l y p u r i f i e d p o r ­
cin e e x t r a c t s
(14,32). T h i s p r o b l e m m a y a l s o a c c o u n t for the
d i f f i c u l t i e s e x p e r i e n c e d in s o m e p r e l i m i n a r y r e p o r t e d c h o l e ­
cystokinin radioimmunoassays.
C o n s i d e r i n g t h e s e data,
it h a s to be e m p h a s i z e d t h a t con-
f o r m a t i o n a l c h a n g e s b e t w e e n m o l e c u l a r v a r i a n t s of c h o l e c y s t o ­
k inin and conformational changes introduced by labelling t e c h ­
ni q u e s m a y b e i n v o l v e d in d i f f e r e n c e s in c h o l e c y s t o k i n i n
immunoreactivity.
OUTLINE OF INVESTIGATIONS
T h e a i m o f t h i s s t u d y w a s t o d e v e l o p a s p e c i f i c a n d sens i t i v e r a d i o i m m u n o a s s a y for c h o l e c y s t o k i n i n .
D a t a a b o u t the p r o d u c t i o n a n d e v a l u a t i o n of a n t i s e r a are
p r e s e n t e d in c h a p t e r II.
125
Introduction of
I i n t o a p o l y p e p t i d e m a y r e s u l t in
c h a n g e s in i m m u n o r e a c t i v i t y . T h e e v a l u a t i o n of a n t i s e r a t e s t e d
w i t h cholecy s t o k i n i n labels prepared by d ifferent techniques
is p r e s e n t e d in c h a p t e r s III a n d IV.
It a p p e a r e d t h a t o n e o f the a n t i s e r a w a s s p e c i f i c for the
t r i a c o n t a n o n a p e p t i d e of c h o l e c y s t o k i n i n
(CCK 39). This u r g e d
us to i n v e s t i g a t e w h e t h e r o r n o t c h o l e c y s t o k i n i n - 3 9 i m m u n o r e a c ­
t i v i t y c o u l d be d e m o n s t r a t e d in p o r c i n e brain. T h e s e d a t a are
p r e s e n t e d in c h a p t e r V.
T h e r e l i a b i l i t y of a h i g h l y s e n s i t i v e a n d s p e c i f i c c h o l e ­
c y s t o k i n i n r a d i o i m m u n o a s s a y for m e a s u r e m e n t s in h u m a n tissue
a n d b l o o d is e x t e n s i v e l y e v a l u a t e d in c h a p t e r VI.
In c h a p t e r s II a n d I V it w a s s h o w n t h a t o r a l and i n t r a ­
d u o d e n a l a d m i n i s t r a t i o n of fat s t i m u l a t e d p l a s m a c h o l e c y s t o k i ­
nin secretion.
In c h a p t e r s VII a n d V I I I the e f f e c t of o t h e r
s t i m u l i for p a n c r e a t i c p r o t e i n s e c r e t i o n o n p l a s m a c h o l e c v s t o k i n i n c o n c e n t r a t i o n s are p r e s e n t e d .
I n c h a p t e r V I I the e f f e c t of i n s u l i n - h y p o g l y c a e m i a and in
c h a p t e r V I I I the e f f e c t o f i n f u s i o n of b o m b e s i n o n p l a s m a c h o ­
l e c y s t o k i n i n w e r e s t u died.
T o d e t e r m i n e t he r o l e s of g a s t r i n a n d g a s t r i c acid in the
e f f e c t o f b o m b e s i n o n p l a s m a CCK, b o t h n o r m a l s u b j e c t s and p a ­
t i e n t s w i t h p r e v i o u s p a r t i a l g a s t r e c t o m y w e r e investi g a t e d .
T h e m o l e c u l a r for m s of c h o l e c y s t o k i n i n in p l a s m a d u r i n g
i n f u s i o n of b o m b e s i n in m a n are r e p o r t e d in c h a p t e r IX.
In c h a p t e r s X a n d XI p l a s m a c h o l e c y s t o k i n i n is m e a s u r e d
in p a t i e n t s w i t h c l i n i c a l c o n d i t i o n s ,
in w h i c h p r e v i o u s l y ab-
normal CCK values have been suggested.
In c h a p t e r X p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y , du e
to a l c o h o l o r c y s t i c f i b r o s i s are studied, w h i l e in c h a p t e r
XI t he e f f e c t of i n t r a d u o d e n a l fat o n p l a s m a C C K an d on g a l l b l a d d e r c o n t r a c t i o n in p a t i e n t s w i t h c o e l i a c d i s e a s e is r e p o r ­
ted.
18
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M.
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related pep­
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23
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75;
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M U T T , V., 1964.
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79.
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R. & W A L D U M , H . L . , 1 9 8 2 . I o d i n a t i o n w i t h I o d o - g e n a n d r a ­
d i o i m m u n o a s s a y o f c h o l e c y s t o k i n i n in a c i d i f i e d p l a s m a , C C K
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R U B I N , B. & E N G E L , S . L . , 1 9 7 3 . S o m e b i o c h e m i c a l c h a r a c t e r i s t i c s of c h o l e c y s t o k i n i n a n d a n a l o g u e s . In: F r o n t i e r s in
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L A M E R S , C . B . H . W . , P O I T R A S , P., J A N S E N , J . B . M . J . & W A L S H ,
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DEBAS,
24
B e h a v i o r of s e c r e t i n , c h o l e c y s t o k i n i n a n d
oxidation with hydrogen peroxide. Acta
2185-2186.
& C H E Y , W. Y. , 1983. R a d i o i m m u n o a s s a y
D i g . D i s . S c i . 28: 4 5 6 - 4 6 8 .
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F. & R E H F E L D , J . F . , 1 9 7 2 . P r e p a r a t i o n a n d e v a l u a 1 2 5 I- s y n t h e t i c human g a s t r i n I for r a d i o i m m u n o a n S c a n d . J. C l i n . L a b . I n v e s t . 3 0 : 3 6 1 - 3 6 8 .
H.T.
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1973.
Pure
1981.
Regul.
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R U B I N , B. E N G E L , S . L . , D R U G I S , A . M . , D Z E L A L U S , M . , G R I G A S ,
E. O . , W A N G H , M . H . & Y I A C A S , E., 1969 . C h o 1 e c y s t o k i n i n - 1 ike
a c t i v i t i e s in g u i n e a p i g s a n d d o g s of the C 0 0 H - t e r m i n a l
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C R U M P T O N , M . J . & W I L K I N S O N , J . M . , 1965. T h e i m m u n o 1o g i c a 1
a c t i v i t y of the c h y m o t r y p t i c p e p t i d e s of s p e r m - w h a l e m y o g l o b i n . B i o c h e m . J. 94: 5 4 5 - 5 5 6 .
25
Chapter II
Radioimmunoassay of Cholecystokinin
Production and Evaluation of Antibodies
J.B.M.J. Jansen
and
C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory
Division of Gastroenterology
St. R a d b o u d H o s p i t a l
University of Nijmegen
Nijmegen
The N e t h e r l a n d s
J. Clin.
Chem.
B io ahem.
(1983)
21: 387-
Jansen and Lamers Radioimmunoassay of cholecystokinin
J C lin Chem Clin Biochem
Vol 21 1983, pp 387-394
Radioimmunoassay of Cholecystokinin:
Production and Evaluation of Antibodies
By J B M J Jansen and C B H W Lamers')
Gastrointestinal Hormone Laboratory, Division o f Gastroenterology, St Radboud Hospital,
University o f Nijmegen, Nijmegen, The Netherlands
(Received January '1, 1982/January 10 1983)
Summary: In order to produce antibodies for the radioimmunoassay of cholecystokinin the following animals
were immunized 10 rabbits and 3 guinea pigs with cholecystokinin 1-33 coupled to bovine serum albumin, 9
rabbits and 2 guinea pigs with uncoupled cholecystokinin 1-33, 5 rabbits with cholecystokinin 10-20
coupled to bovine serum albumin and 4 rabbits with sulphated cholecystokinin 26—33 coupled to bovine
serum albumin The titer, the binding energy and the specificity of the antibodies were evaluated Cholecysto­
kinin 1-33, coupled to [l25I]hydroxyphenylpropiomc acid-succinimide ester (Bolton-Hunter reagent) to a
specific activity of 8 1-14 7 MBq/(ig (219-398 nCi/^g) (n = 8), was used as label After the fourth ïmmunization 5 rabbits immunized with albumin coupled cholecystokinin 1-13 had antibody titers between 1 22500
and 1 80000, while one rabbit immunized with uncoupled cholecystokinin 1-33 had a titer of 1 52000 The
affinity constant of these 6 antibodies ranged from K = 2 0 x 10'° to 72 3 x 10"’ 1/mol One antibody was
specific for all sulphated forms of cholecystokinin, while the other 5 antibodies were specific for the large
molecular forms of cholecystokinin In the guinea pigs immunized with cholecystokinin 1-33 and in the
rabbits immunized with cholecystokinin fragments antibody titers were less than 1 500 Concentrations of
cholecystokinin in an aqueous-acid extract of human and hog upper small intestme were 48 7 ± 6 7 pmol/g
and 46 2 ± 3 4 pmol/g, when measured with antibody T204 specific for sulphated forms of cholecystokinin,
and 49 7 ± 1 9 pmol/g and 39 2 ± 1 6 pmol/g, when measured with antibody 1703 specific for largc forms of
cholecystokinin After ingestion of 60 ml corn oil in 6 normal subjects, plasma cholecystokinin increased from
2 7 ± 0 5 pmol/1 to 6 8 ± 1 7 pmol/1 (p < 0 05) when measured with antibody T204, and from 1 0 ± 0 3
pmol/1 to 5 7 ± 1 0 pmol/1 (p < 0 05) when measured with antibody 1703
In conclusion, immunization of rabbits with cholecystokinin 1-33 resulted in high titer antibodies which
could be used in sensitive and specific radioimmunoassays for cholecystokinin
Radioimmunassay fur Cholecystokinin
Cewinnung und Bewertung von Antikorpern
Zusanunenfassung: Z ur Gewinnung von Antikorpern fur einen Radioimmunoassay fur Cholecystokinin
wurden immunisiert 10 Kamnchen und 3 Meerschweinchen mit an Rinderserumalbumin gekoppcltcm
Cholecystokinin 1-33, 9 Kamnchen und 2 Meerschweinchen mit ungekoppeltem Cholecystokinin 1-33,
5 Kamnchen mit an Rinderserumalbumin gekoppeltem Cholecystokinin 10-20 und 4 Kamnchen mit an R in ­
derserumalbumin gekoppeltem, sulfatiertem Cholecystokinin 26-33 Titer, Bindungsenergie und Spezifitat
der Antikorper wurden bestimmt Cholecystokinin 1-33, gekoppelt an [l25I]Hydroxyphenylpropionsauresuccimmid ester (Bolton-Hunter-Reagenz) mit einer spezifischen Aktivital von 8,1-14,7 MBq/|xg (219-398
nCi/(ig) (n = 8), wurde als Label benutzt Nach der vierten Immumsierung hatten 5 der mit an Rinderserum­
albumin gekoppeltem Cholecystokinin 1-33 immumsierten Kamnchen Antikorpertiter zwischen 1 22500
und 1 80000, wahrend ein mit ungekoppéltem Cholecystokinin 1-33 ïmmumsiertes Kamnchen einen Titer
') Supported by the Foundation for Medical Research FUNGO (grant No 13-37 32)
28
J Clin Chem Clin Biochem / Vol 21, 1983 /N o 6
Jansen and Lamers Radioimmunoassay of cholecystokinin
von 1 *52000 aufwies Die Affinitatskonstante dieser 6 Antikorper lag zwischen K = 2,0 x 10'° und 72,3 x
10"' l/mol Ein Antikorper war spezifisch fur alle sulfatierten Cholecystokinine, wahrend die anderen 5 A nti­
korper spezifisch fur die groBmolckularen Cholecystokinine waren Bei den mit Cholecystokinin 1-33 ïmmumsicrten Meerschweinchcn und bei den mit Cholecystokinm-Fragmenten immunisierten Kamnchen waren
die Antikorpertiter kleiner als 1 500
Die Cholecvstokimn-Konzcntrationen in einem waBng-sauren Extrakt aus Schlcimhaut des oberen Dunndarms vom Menschen und vom Schwcin betrugen 48,7 ± 6,7 pmol/g bzw 46,2 ± 3,4 pmol/g, wenn sic mit
Antikorper T204 bestimmt wurden, der fur die sulfatierten Cholecystokinine spezifisch ïst Wenn sie mit dem
fur die groOmolekularen Cholecystokinine spezifischen Antikorper 1703 gemessen wurden, lagen sie bei 49,7
+ 1,9 pmol/g bzw 39,2 ± 1,6 pmol/g Nach Emnahme von 60 ml Maisol stieg bei 6 gesunden Probanden die
Konzentration von Cholecystokinin ïm Plasma von 2,7 ± 0,5 pmol/1 auf 6,8 ± 1,7 pmol/1 (p < 0 05, Messung
mit Antikorper 1204), und von 1,0 ± 0,3 auf 5,7 ± 1,0 pmol/1 (p < 0 05, Messung mit Antikorper 1703)
W ir folgern, daB die Immunisierung von Kamnchen mit Cholecystokinin 1-33 zur Bildung von hohen Antikorpertitern fuhrt, die fur empfindliche und spezifische Radioimmunoassays fur Cholecystokinin benutzt
werden konnen
Introduction
Cholecystokinin is one of the classical gut hormones
(1) This peptide hormone stimulatcs gall bladder
contrattion and secretion of cnzymes from the pan­
creas (2, 3) In extracts of gut and brain cholecysto­
kinin was found to be present in several molecular
forms (4) Onginally cholecystokinin was isolated
from acid extracts of hog small intestine as a basic
molecule containing 33 amino acid residues (chole­
cystokinin 1-33) with the chemical structure LysAla-Pro-Ser-Gly-Arg-Val-Ser-Met-lle-Lys-AsnLeu-Gln-Ser-Leu-Asp-Pro-Ser-His-Arg-Ile-SerAsp-Arg-Asp-Tyr(SOi)-Met-Gly-Trp-Met-AspPhe (4, 5) A molecular form extended at the N H 2 terminus by 6 amino acid residues, Tyr-Ile-Gln-GInAla-Arg (cholecystokinin 1-39), was desenbed
some years later (6) Recently, it was shown that in
neutral extracts of sheep brain a smaller molecular
form containing 8 amino acid residues (cholecysto­
kinin 26-33) predominates (4, 7)
Even though the first radioimmunoassay of chole­
cystokinin was reported as early as 1969 (8), only
few radioimmunoassays have so far been developed,
despite much effort Several factors contnbute to the
difficulties in developing a rcliable radioimmunoas­
say for cholecystokinin
1 difficulties in producing antibodies duc to the low
immunogemcity (9),
2 an unacceptably high degree of cross-reactivity of
COOH-terminal antibodies with gastnn, because
both peptides share the same COOH-terminal
pentapeptide sequence,
3 suspected species diffcrcnccs in the NHi-terminal
sequence of cholecystokinin 1—33 resulting in
antibodies reacting with porcine cholecystokinin
but not with cholecystokinin from other species
( 10, 11),
4
availability of only limited amounts of pure
cholecystokinin, and
5 difficulties in preparing ïmmuno-reactive chole­
cystokinin labels It has been shown that the conventional oxidizing labelling methods, such as the
chloramine T method and the lactoperoxidase
method, result in cholecystokinin labels of poor
immunoreactivity, probably due to the deleterious effects of oxidation of the methiomne re­
sidues in cholecystokinin (12) Recently, the la­
belling problem has been overcome by using a
non-oxidizing
labelling
method
Using
[12<I]hydroxyphenylpropionic
acid-succinimide
ester (Bolton-Hunter reagent) Rehfeld was able
to produce highly ïmmunoreactive cholecysto­
kinin 1-33 labels, and he suggested that chole­
cystokinin 1-33 was a good immunogen (12,
13)
In this report we present our experience with the
production of antibodies to cholecystokinin in rabbits and guinea pigs Furthermore, the titer, the
binding energy and the specificity of the antibodies
were evaluated in radioimmunoassays using BoltonHunter labelled cholecystokinin 1-33 In addition,
we have measured cholecystokinin concentrations in
extracts of human and hog upper small intestine and
in human plasma after ingestion of fat, using ra­
dioimmunoassays employing an antibody specific for
sulphated forms of cholecystokinin and an antibody
specific for large forms of cholecystokinin
29
J Clin Chem Clin Biochem / Vol 21, 1983 / No 6
Jansen and Lamere Radioimmunoassay of cholecystokinin
Materials and Methods
peptides
99% pure porcine cholecystokinin 1-19 and 30% and 99% pure
poreme cholecystokinin 1-33 were obtained from Professor
Muti, Karolinska Institutc. Stockholm, Sweden, synthetic sulphat­
ed and unsulphated cholecystokinin 26-33 from the Squibb Institute Pnncetown N J , USA, synthetic unsulphated human gastnn
1-17 from lmpenal Chemical Industries, ('heshire Fngland, vasoactive intestinal polypcptide from Penmsula Laboratories, San
Carlos, Ca, USA, gastric inhibitory polypcptide from Professor
Brown, Vancouver Canada, sulphated human gastnn 1-17 and
sulphated and unsulphated gastnn 1-34 from Professor Grossman Los Angeles, Ca, USA, secretin synthesized by the Squibb
Institutc, Princetown, N J USA. through the courtesy of Profes­
sor Groisman caerulein from Farmitalia, Milan, Italy. synthetic
cholecystokinin 10—20 from UCB Brussels. Bclgium, porcine
pancreatic polypeptide porcine glucagon and monocomponent
insulin from Novo Industry ('openhagen. Denmark, synthetic
cholecystokinin 30-33 (tetragastnn) from Sigma, St Louis, Mo,
USA
O th e r reagents
(l2SI)hydroxvphenylpTopionic acid succinimide ester (Bolton
Hunter reagent) was purchased from New bngland Nuclcar Bos­
ton Ma. USA. Sephadex G 50 SF and SP-Sephadex C 25 from
Pharmacia, Uppsala Sweden, 1-ethyl 3 (3 dimethylaminoprop
yl) carbodumide hydrothJonde from JCN, Plainview. NY, USA,
hreund's complete and incomplete adjuvant from Difco l aboratones, Detroit, Mi, USA, Bordetella pertussis vaccine from RIV,
Bilthovcn. Fhe Netherlands, human and bovinc scrum albumin
from Behnngwerke AG, Marburg/I ahn West Germany, dextran
T 70 and bovine serum albumin (RIA-grade) from Sigma St
Louis, Mo, USA, trypsin IPCK from Worthington Biochcmical
Corporation, Freehold, NJ. USA. sodium ethylmercunthiosalicylate from Aldnch-Turope. Beerse, Belgium, double antibody solid phase from Organon, Oss. The Netherlands, all other reagents
from E Merck. Darmstadt, FRG
A n im a ls
28 randomly bred rabbits and 5 randomly bred guinea pigs were
immumzcd
A p p ara tus
Automatic y-scintillation counter Philips PW 4800, Philips, Eind­
hoven, The Netherlands, digital dilutcr dispenser, Hamilton, Bonaduz, Switzerland
tiple sites into the back of the animals, while booster tnjections
were given subcutaneously with four weeks intervals on 4 sites
The doses of antigen injected are givcn in table 1 Seven days after
each booster injection blood was drawn from an ear vein in rabbits
and by cardiac puncture in guinea pigs
Tab 1 Type (BSA = bovine serum albumin coupled) and
amount of antigen used for production of antibodies to
cholecystokinin (CCK) in rabbits and guinea pigs
Animal
species
Rabbit
Rabbit
Rabbit
Rabbit
(fuinea pig
Guinea pig
Num-Antigen
ber
of
ani~
mals
10
9
5
4
3
2
Doseof
antigen
(nmol)
initial boost­
er
30% CCK 1-33-BSA
30% CCK 1-33
CCK 10-20-BSA
sulphated CCK 26-33-BSA
30% CCK 1-33-BSA
30% CCK 1-33
20
20
120
120
10
10
10
10
60
60
5
5
R a d io io d in a iio n
37 MBq (1 mCi) (12,iI]hydroxyphenyIpropiomc acid-succimmide
ester (Bolton-Hunter reagent) was dned under a gentle stream of
mtrogen according to the instructions of the manufacturer Five
Hg 99% pure cholecystokinin 1-33 dissolved in 5 \
i\0 05 mol/l
acetic acid was added to the bot lom of the vial After addition of
15 ul 0 05 mol/1 sodium borate pH 10. the vial was gently agitated
for 30 minutes in an ice bath To remove excess unreacted
[l2Sï]hydroxyphenylpropiomc acid-succimmtde ester, 0 2 mol/l
glycine in 500 ^1 0 05 mol/l sodium borate pH 8 5 was added to
the mixture and the vial was agitated for 5 minutes The reaction
mixture was chromatographed at 4°C on a Sephadex G 50 SF
column, 100 x 0 9 cm. using 0 5 mol/1 acetic acid containmg 2 g/1
gelatine as eluant Every hour 5 fractions of 1 ml were collected
The elution profile of the radioactivity is shown in figure 1 Only
the second radioactive peak showed immunoreactivity The fraction with the highest radioactivity in peak 11 was diluted in the
elutmg buffer and stored at -20°C This label retained its immu­
noreactivity for at least 3 months Label damage was between 2
P ro du c tion o f antibod ies
Uncouplcd cholecystokinin 1-33 30% pure porcine cholecysto­
kinin I -33 dissolved in 0 05 mol/1 sodium phosphate buffer pH
7 5 was uscd without modification
Albumin coupled peptides
30% pure porcine cholecystokinin 1-33. synthetic cholecystokinin 10-20, and synthetic sulphated cholecystokinin 26-33 were
dissolved in 0 05 mol/1 sodium phosphate buffer pH 7 5 and conjugated to bovine serum albumin by reacting with 1-ethyl- 3-(3-dimcthylammopropyl) carbodumide hydrochloride for 2 hours at
room temperature and a further 20 hours at 4 °C The ratio between peptide, bovine serum albumin and l-ethyl-3-(3-dimethyl
aminopropyl) carbodumide hydrochtonde on a molar base was
1 0 2 462 The antigen solution was emulsified with equal vo­
lumes of Freund's adjuvant for the initial immumzations and with
equal volumes of Freund's incomplete adjuvant for booster injections At the initial immunization 0 5 ml Bordetella pertussis vac­
cine was admimstered intramuscularly Fach rabbit was immuntzcd with 2 ml and each guinea pig with 1 ml of the emulsified
antigen The initial injections were given intracutaneously on mul-
30
J Clin Chem Clin Biochem / Vol 21, 1981 / No 6
F ra c tio n n u m b e r
Fig 1 Elution profile of l25l radioactivity after application of
reaction mixture of cholecystokinin 1-33 with
[l2Sl|hydroxyphenylpropiomc
acid-succimmide
ester
(Bolton-Hunter reagent) to a Sephadex G 50 SF column
Peak II represents labelled cholecystokinin 1-33 For de­
tails see lexl
Jansen and Lamers Radioimmunoassay of cholecystokinin
and 7% The specific activity of the label, determmed according to
Stadil & Rehfeld (14), ranged from 8 1 to 14 7 MBq/jig (219 to
198 nCi/|ig) (n = 8) When the label was rcchromatographed on
SP-Sephadex C 25 using a 0 OS to 1 0 mol/l acetic acid gradiënt,
the radioactivuy elutcd as a single peak
In c u b a tio n co n d ilio n s
Antibody titers were determmed as follows The assays were set
up in plastic tubes in an ite bath using 0 02 mol/l sodium barbital
buffer pH 8 4 containing 0 01 mmol/1 human serum albumin and
0 1)6 mmol/1 sodium ethylmercunthiosdhcylatc The 2 2 ml ïncubation volume consisted of 1 0 ml of approximately 2000 counts/
min label 0 2 ml Standard solution, and I 0 ml diluted antiserum
The tubes were incubated for 96 hours at 4 °C To determine the
binding cnergy and cross-reactivity of the antibodies a pre-equihbrium method was used 0 2 ml Standard solution was incubated
with 0 l ml diluted antiserum for 72 hours at 4 °C After addition
of approximately 2000 counts/min Holton-Hurtter labelled cholecystokinin 1-33 in 0 2 ml buffer, the mixture was incubated for
another 48 hours at 4°C All dilutions in this pre-equilibnum assay were made in 0 0S mol/l sodium phosphate buffer pH 7 4 con­
taining 0 08 m m oll human serum albumin and 0 06 mmol/l sodi­
um ethylmercurithiosahc>late
The dned supernatant was reconstituted in assay buffer a n d 200
|xl were incubated with 100 fil diluted antiserum for 72 hours at
4 °C After addition of 200 ^1 labelled cholecystokinin 1-33 con>
taining approximately 2000 counts/min and incubation for another 20 hours at 4°C, free and antibody-bound peptide were separated and counted tn a y-sciniiJJation counter Cholecysfokmin
1-33 was used as S ta n d ard , and antibodies 1703 and T204 as
antisera 0 05 mol/l sodium phosphate buffer pH 7 4 containing
0 08 mmol/1 human serum albumin and 0 06 mmol/1 sodium
ethylmercunthiosalicylate was used as assay buffer Recovery of
cholecystokinin added to hormone-free plasma was 85 4 ± 2 0%
(mean ± SD, n = 11) Separation using plasma-coated charcoal
or the double-antibody solid phase technique gave identical results The delection limit of the assays was between 0 5 and 1 0
pmol/1 plasma Intra-assay vanation ranged from 4 6-8 4% and
intcr-assay vanation from 11 3-15 4%
H um an studies
After an ovcmight fasl 6 normal subjects (4 males and 2 fcmales,
mean age 30 years, range 23 —38 vears) ingested 60 ml corn oil
Blood samples were obtained at -5, 0, 5, 10, 15, 20, 30, 40, 50,
60, 75 and 90 minutes
Statistics
Separation procedure
Antibody-bound radioactivuy was separated from free label by
addition of a 1 S ml suspension of 0 05 mol/l sodium phosphate
buffer pH 7 4 containing 25 mg activatcd charcoal, 2 5 mg dextran T 70 and 16 mg bovine serum albumin The tubes were vortexed and immediately ccntrifuged (3000 g for 10 minutcs) In the
double-antibod> separation techmque 1 ml of 1 20 diluted sheep
and rabbil immunoglobulm was added to the tube and incubated
for 4 to 8 hours at 4 "C before ccntnfugation Both the supernatant and the pellet were counted in an automatic y-scintillation
counter
Tryptic cleavage
230 (imol/1 99% pure cholecystokinin 1-33 was incubated with 5
g/l trypsin for 60 minutcs at 37 °C The action of trypsm was terminated by boiling for 10 minutcs The completeness of cleavage
of cholecystokinin 1-33 to the COOH-terminal octapeptide (sul­
phated cholecystokinin 26-33) was assessed by gel filtration of
cholecystokinin 1 33 before and after trypsmization on a Sephadex G 50 SF column. 200 x I 5 cm, using 0 02 molA sodium
barbital buffer pH 8 4 as eluant at 4 eC The eluate was measured
by radioimmunoassay using a C O O II terminal cholecystokinin
antibody and l2SI labelled non-sulphatcd cholecystokinin 26-33
(15)
Tissue extraction
Pieces of freshly obtained human (n = 3) and hog (n = 3) upper
small intcstinc were extracted in boiling water (1 g/10 ml) for 10
minutes After homogenization and ccntnfugation the supernatant was decanted and the pellet was re-extracted in 0 5 mol/l acctic acid (1 g/10 ml) for 10 minutes After centrifugation the supernatant was added to the supernatant of the aqueous extraction,
rapidly frozen. lyophilized, dissolved in 0 05 mol/l sodium phos­
phate buffer and measured by radioimmunoassay
M easurem ent of ch o le cy sto k in in in plasm a
Blood samples were collected in ice-chilled glass tubes containing
2 g/i cthylenediammetetraaceiale (EDTA) After centrifugation
the plasma was frozen at —20 °C Cholecystokinin was extracted
from plasma by addition of 2 volumes of 960 ml/1 ethanol to 1
volume of plasma After mixing and centrifugation the supernatant was evaporatcd to dryness under a stream of nitrogen at 37 °C
Results were expressed as mean ± SEM, unless otherwise stated
Student's t-test for paircd data was used for analysis of the plasma
cholecystokinin results
Results
The titers of the antibodies after the second, third
and fourth immunization are presented in table 2.
Antibodies with titers greatcr than 1:10000 were not
detected after the second immunization, while after
the third immunization 3 ammals and after the
fourth immunization 6 ammals had produced antibo­
dies with titers of more than 1:10000. O f the 10 rab­
bits immunized with albumin-coupled cholecysto­
kinin 1-33, 5 had antibody titers of more than
1:10000 ranging from 1:22500 to 1:80000. O f the 9
rabbits immunized with uncoupled cholecystokinin
1-33, one produced a high titer antibody, 1:100000
after the third and 1:52000 after the fourth immuni­
zation. Immunization with synthetic cholecystokinin
fragments (cholecystokinin 10—20 and sulphated
cholecystokinin 26-33) did not result in antibodies
detectable in radioimmunoassays employing BoltonHunter labelled cholecystokinin 1-33. None of the
guinea pigs produced cholecystokinin antibodies
with titers of more than 1:500.
In radioimmunoassays using Bolton-Hunter labelled
cholecystokinin 1-33 the binding energy of 6 high
titer antibodies after the fourth immunization and of
one antibody after the third immunization (1703)
were determmed. The affinity constants indicating
the binding cnergy between the antibodies and
cholecystokinin 1-33 (16) are presented in table 3.
31
J Clin Chem Clin Biochem / Vol 21, 1983 / No. 6
Jansen and Lamers. Radioimmunoassay of cholecystokinin
Four antibodies had very high affimty constants of
more than 5 x 10" 1/mol. These high binding energies were reflected in low IDjo’s (inhibition doseso)
between 2.8 and 5.0 pmol/1 in the radioimmunoas­
says. With these sensitive radioimmunoassays for
cholecystokinin 1-33, very low detection limits
ranging from 0.5 to 3 pmol/1 sample were obtained.
Tab 2 The dilution of antibodies to cholecystokinin (CCK) in
the incubation mixture binding 50% of 1 fmol BoltonHunter labelled cholecystokinin 1-33
All antibodies cross-reacted with cholecystokinin
1-39 (table 3). Five antibodies (1204, 1304, 1404,
T104 and 1703/1704) did not cross-react with either
synthetic cholecystokinin fragments or cholecysto­
kinin fragments produced by trypsinization (table 3).
Furthermore, these antibodies did not show any
binding to sulphated or non-sulphated gastrms, to
COOH-terminal
tetragastrin
(cholecystokinin
30-33), to caerulein or to structurally unrelatcd
peptides such as insulin, glucagon, pancreatic poly­
peptide, gastnc inhibitory polypeptide, vasoactive
intestinal polypeptide or secretin. Antibody T 204
bound to cholecystokinin 33, cholecystokinin 39,
sulphated cholecystokinin 26-33, caerulein and to
trypsinized cholecystokinin 33 (table 3). Binding to
sulphated gastrin 1-17 and sulphated gastrin 1-34
was low, while binding to non-sulphated forms of
cholecystokinin or gastrin and to unrelated peptides
was absent. A ll 6 antibodies did not only bind to por­
cine cholecystokinin, bul also to cholecystokinin in
human tissue extracts.
Reciprocal of antibody dilution
aftcr
Animal number
immunizationll
immum7ation III
ïmmumzation IV
Rabbit
30% CCK 1—33-BSA
11
12
13
14
Tl
12
T3 through T6
1000
5000
2000
2000
<500
n t
<500
1800
6000
10000
60000
<500
3700
<500
2750
27000
75000
50000
22500
80000
<500
30% CCK 1-33
15
16
17
18
T7
T8 through Tl 1
<500
<500
2000
<500
<500
<500
1700
2400
100000
1800
<500
<500
<500
1500
52000
2500
2600
<500
CCK 10-20-BSA
1 through 5
<500
<500
<500
Sulphated CCK 26-33-BSA
7 through 10
<500
<500
<500
Cutnea ptg
30% CCK 1-33-BSA
G1 through G3
<500
<500
<500
30% CCK 1-33
G4 and G5
<500
<500
<500
Standard curves for both types of antibodies are
shown in figures 2 and 3.
Concentrations of cholecystokinin in an aqueous
acid extract of human (n = 3) and hog (n = 3) upper
small intestine were 48.7 ± 6.7 pmol/g and 46.2 ±
3.4 pmol/g, when measured with antibody T204, and
49.7 ± 1 .9 pmol/g and 39.2 ± 1 .6 pmol/g, when
measured with antibody 1703.
n t = not tesled
BSA = bovine serum albumin coupled
Tab 3. Binding energy, inhibition dose 50 and cross-reactivity of high titer cholecystokinin (CCK) antibodies***
Antibody
number
K*
ID W"
CCK
1-33
CCK
1-39
CCK
10-20
Caerulein Sul­
phated
CCK
26-33
Non-sulphated
CCK
26-33
Trypsinïzcd
CCK
1-33
CCK
30-33
Sul­
phated
gastrin
1-17
Sul­
phated
gastrin
1-34
1204
1304
1404
Tl 04
T204
1703
1704
20
72 3
28
55 4
52 2
59 4
31 6
26.0
45
24 0
50
33
2.8
51
10
I 0
I0
10
10
10
10
0.00
0.49
0 80
0 50
1 12
0 89
0 88
<0
<0
<0
<0
<0
<0
<0
<0 001
<0.001
<0 001
<0 001
0 20
<0 001
<0 001
<0
<0
<0
<0
<0
<0
<0
<0 001
<0 001
<0 001
< 0 001
0 88
<0 001
< 0 001
< 0 001
< 0 001
<0 001
<0 001
<0 001
<0 001
<0.001
<0.001
<0 001
<0 001
<0.001
0016
<0 001
<0 001
< 0 001
<0 001
<0 001
<0 001
0018
<0 001
<0 001
”
001
001
001
001
001
001
001
<0.001
< 0 001
<0 001
<0 001
0 93
<0 001
<0 001
001
001
001
001
001
001
001
* Affinity constant K (x 1010 l/mol)
Inhibition dose 50- dose of cholecystoktnm 1-33 which reduces binding between label and antibody by 50% (pmol/1)
None of the antibodies showed cross-reactivity with non-sulphated gastrin 17. non-sulphated gastrin 34 or unrelated regulatory pep­
tides
32
J Clin. Chem Clin. Biochem. / Vol 21, 1983 / No 6
Jansen and Immers* Radioimmunoassay of cholecystokinin
060
-
050
-
8
-
c [pmol/ll
Fig 2 Standard curves of antibody 1703 using Bollon-Hunter la­
belled cholecystokinin 1-33 The antibody binds to
cholecystokinin 1-33 ( • ) and cholecystokinin 1-39 (A),
but not to sulphated gastnn (▲), unsulphated gastnn (V),
sulphatcd cholecystokinin 26-33 (x), unsulphated chole­
cystokinin 26—33 (O), trypsinized cholecystokinin 1-33
(■) or caerulein (n)
Fig 4 The effect of ingestion of 60 ml corn oil on plasma chole­
cystokinin concentrations in 6 normal subjects Open circles indicate antibody T204 and filled circles antibody
1703 Asterisks indicate significant differences from basal
value (p < 0 05)
Discussion
-i------- ------ 1_____ i_____________i_____ I____________ i_____ 1___
1
5
10
50
100
500 1000
c [pmol/l]
Fig 3 Standard curvcs of antibody T204 using Bolton-Hunter la­
belled cholecystokinin 1-33 The antibody binds to
cholecystokinin-gastnn peptides containing the sulphated
tyrosinc region cholccystokinm 1-39 (A), cholecystokin­
in 1-33 ( • ) , trypsinized cholecystokinin 1-33 (■), sul­
phated cholecystokinin 26-33 (x), caerulein (□) and sul­
phatcd gastnn (▲) Binding to cholccystokinm-gastrin
peptides lacking the sulphated tyrosine region is absent
unsulphated cholecystokinin 26—33 (O), and unsulphated
gastnn (V)
Basal plasma cholecystokinin concentrations were
very low, 2.7 ± 0.5 pmol/l as measured with anti­
body T204, and 1.0 ± 0.3 pmol/l as measured with
antibody 1703. Ingestion of corn oil induced signifi­
cant increases in plasma cholecystokinin (p < 0.05).
The peak plasma cholecystokinin concentrations
were found 20 minutes after ingestion of the corn oil.
The increases in plasma cholecystokinin were similar
for both antibodies (fig. 4).
This study shows that cholecystokinin 1-33 is an ex­
cellent immunogen when Bolton-Hunter labelled
cholecystokinin 1-33 is employed as the radioactive
peptide in radioimmunoassays Immunization of
rabbits with albumin-coupled cholecystokinin 1-33
produced high titer antibodies in 5 of 10 animals,
while immunization of 9 rabbits with uncoupled
cholecystokinin 1 —33 produced high titer antibodies
in one animal Four of these antibodies had high
binding energies with affinity constants of more than
5 x 10 '1 1/mol. No antibodies were obtained in guinea pigs immunized with uncoupled or albumincoupled cholecystokinin 1-33 or in rabbits immun­
ized with albumin-coupled cholecystokinin fragments. Therefore, immunization of rabbits with al­
bumin-coupled cholecystokinin 1—33 seems to be
the most suitable method to produce antibodies for
the radioimmunoassay of cholecystokinin. In con­
trast to a previous report (9), our results confirm
Rehfeld's finding that cholecystokinin 1—33 has
good immunogenic properties (12). It has been
claimed by Rehfeld that cholecystokinin 1-33 is a
good immunogen provided cholecystokinin is not exposed to oxidation during the labelhng procedure
(12). However, high titer cholecystokinin antibodies
have been desenbed in radioimmunoassays using
cholecystokinin 1-39 or nonsulphated cholecysto­
kinin 26-33 labelled with 125I by the chloramine T
method (15, 17, 18). On the other hand, it seems to
be extremely difficult to raise high titer antibodies
against cholecystokinin 1—33 when an oxidizing labelling method is used. Thompson's group has pro­
duced an antibody against cholecystokinin 1-33
33
J Clin Chem Clin Biochcm / Vol 21, 1983 / No 6
Jansen and Lamers Radioimmunoassay of cholecystoktnin
which binds 25 to 35% of cholecystokmin 1-39, la­
belled with 12,I by the chloramine T method, at a
final dilution of 1 100000(17) In most radioimmunoassays using cholecystokmin 1-33 or cholecystokinin 1-39, labelled by the chloramine T method,
the titers of the antibodies are extremely low (8, 10,
19, 20, 21) From the present study ït cannot be concluded whether the excellent immunogenicity of
cholecystokmin 1-33 in our hands is attnbutable to
the immunization schedule employed or to the use of
a non-oxidizing labelling method Using BoltonHunler labelled cholecystokmin 1-33, Rehfeld reported cholecystokmin antibodies with a titer between 1 10000 and 1 30000 m 3 of 10 rabbits ïmmunized with albumin-coupled cholecystokmin
1 —33 (12) Howevcr, none of these antibodies had
affimty constants above 5 x 10“ 1/mol Byrnes et al
(22) immunized 16 rabbits with albumin-coupled
cholecystokmin 1-33 and only one of the ammals
produced an antibody with a titer of 1 10000 When
Bolton-Hunter labelled cholecystokmin 1-33 was
uscd in the assay the affimty constant was 5 5 x 10"
l/mol (22) Rehfeld suggested that cholecystokmin
1-33 is particularly ïmmunogenic in guinea pigs
(12) Twelve of 30 ammals produced antibodies with
titers between 1 10000 and 1 50000 Ih re e of these
antibodies had affimty constants of more than 5 x
10" 1/mol While Rehfeld (12) and Byrnes et al (22)
did not fmd antibody titers higher than 50000, we
found this level of titers in 4 rabbits Some authors
advocate an immunization schedule in which crude
cholecystokmin 1-33 is used for the mitial ïmmumzations followed by 99% pure cholecystokmin 1-33
for the final immunization (17) In our study excel­
lent antibodies wcre obtained after immunization
with 30% pure cholccystokinin 1-33 99% pure
cholecystokmin 1—33 is difficult to obtain and our
data do not support the need for highly punfied
cholecystokmin 1—33 as immunogen
The high binding energy of 4 antibodies (K > 5 x
10" 1/mol) in the present study was reflccted in the
high sensitivity of the radioimmunoassays The dose
of cholecystokmin 1-33 at which the binding be­
tween label and antibody was inhibited by 50%
(IDso) ranged from 2 8 to 5 0 pmol/1 The detection
limit of these assays was between 0 5 and 3 pmol/1
sample These assays were extremely sensitive and
they were able to measure the very low concentrations of cholecystokmin present in the circulation
(fig 4)
34
J Clin Chem Chn Biothem / Vol 21 1983 / No 6
Five antibodies bound to cholecystokmin 1-33 and
cholecystokmin 1—39, but not to synthetic cholccystokinin fragments (sulphated and non-sulphated
cholecystokmin 26 —33, cholecystokmin 30-33.
cholecystokmin 10-20) or cholecystokmin frag­
ments prepared by trypsmization of cholecystokmin
1-33 (cholecystokmin 1-6, cholecystokmin 7-11,
cholecystokmin 12-21, cholecystokmin 22-25. sul­
phated cholecystokmin 26-33) Co et al (10) and
Straus & Yalow (11) produced antibodies to chole­
cystokmin 1-33 which reacted only with porcine
cholecystokmin. but not with cholecystokmin from
othcr species They suggested that there might be
species differences in the structure of cholecystokinm 1—33 at the NH;-terminus Our antibodies not
only bound to porcine cholecystokmin but also to
human cholecystokmin Since the antibodies do not
cross-react with gastrin or with structurallv unrclatcd
peptides, these antibodies are extremely suitable for
quantifying large forms of cholecystokmin in blood
and tissue One antibody (T204) seems to be directed to the sulphated tyrosine region of the molecule
It binds to large cholecystokmin and cholecystokmin
fragments contaming the sulphated tyrosine region
(cholecystokmin 1-39, cholecystokmin 1-33, caerulein, sulphated cholecystokmin 26-33, trypsinized
cholecystokmin 33) but not to unsulphated cholccystokinin fragments (non-sulphated cholecvstokinin
26-33, cholecystokmin 10-20, cholecystokmin
30-33) Since the binding to sulphated gastrin 1-17
and sulphated gastnn 1-34 is low and the binding to
non-sulphated gastrin 1-17 and gastrin 1-34 and
structurally unrelated peptides is absent, this anti­
body is suitable for quantifying the sulphated forms
of cholecystokmin in blood and tissue Since only
sulphated molecular forms of cholecystokmin possess biological activity, this assay will quantify only
the biological activc forms of cholecystokmin
In conclusion, immunization of rabbits with chole­
cystokmin 1 -33 resulted in antibodies which can be
used in sensitive and specific radioimmunoassays for
cholecystokmin
Acknowledgements
The authors are endebted to Mr J Koedam and Dr W van der
Gulden Central Animal Laboratory and to Mr H J Jansen for
thcir asvistance in immumzing the ammals
Jansen and Lamers Radioimmunoassay of cholecystokimn
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H E , Hershman, J M & Walsh J H (1980) Am J Physiol
239, E232 —L235
16 Scatchard, G (1949) Ann N Y Acad Sci 51, 660-672
17 Miyaia, M , Rayford, P L Sl Thompson, J C (1980)
Surgery 87, 209-215
18 Dockray, G J (1980) Brain Res 188, 155-165
19 Reeder, D D , Becker, H D , Smith, N J , Rayford, P L &
Thompson, J C (1973) Ann Surg 178, 304-310
20 Harvey, R F , Dowsett, L , Hartog, M & Read, A E (1974)
Gut 15, 690-699
21 Schlegel W , Raptis, S , Grube, D 8l Pfeiffer, E F (1977)
Chn Chim Acta 80, 305 316
22 Byrnes, D J , Henderson, L , Borody, T & Rehfeld, J F
(1981) Chn Chim Acta 111, 81-89
J Clin Chem Cltn Biochem / Vol. 21, 1983 /N o 6
Chapter III
Low binding of Bolton-Hunter labelled
cholecystokinin-33 to carboxyl-terminal
CCK-antibodies
J.B.M.J, Jansen
and
C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory
Division of gastroenterology
St. R a d b o u d H o s p i t a l
University of Nijmegen
Nijmegen
The N e t h e r l a n d s
Journal of Immunologiaal Methods
(1982)
51: 223-230.
37
J o u rn a l o f Im m u n olo gic a l M ethods, 51 (1982) 223—230
Elsevier Biom edical Press
LOW BINDING OF BOLTON-HUNTER-LABELLED
CHOLECYSTOKININ-33 TO CARBOXYL-TERMINAL
CCK-ANTIBODIES
J.B .M .J. JA N S E N 1 and C.B H.W . L A M E R S 2
Gastrointestinal H orm one Laboratory, Division o f Gastroenterology, St. R ad bo ud H o spi­
tal, University o f Nijmegen, Nijmegen, The Netherlands
(Received 27 Ju ly 1981, accepted 3 December 1981)
Labelling o f cholecystokinin-33 (C C K 33) w ith [125I]hydrox yphenylpropionic acidsuccinim ide ester (Bolton-Hunter reagent) results in labels w ith excellent immunoreactivity when tested w ith non-carboxyl-terminal CCK-antibodies b u t extremely low binding to
carboxyl-terminal CCK-antibodies This finding m ight explain some o f the difficulties
experienced w ith pro ductio n o f im m unoreactive C CK 33 labels prepared by the BoltonHunter m ethod.
Key words: cholecystokinin assay — Bolton-Hunter-labellmg reagent — horm one assay
IN T R O D U C T IO N
Cholecystokinin (CCK) is one of the classical gut hormones (Rayford et
al., 1976). In intestinal extracts of man CCK is present in different molecular
forms including CCK33 and CCK8 (Rehfeld, 1978b). This peptide hormone
is a potent stimulant of pancreatic protein secretion and gall bladder contraction. Despite its important physiological role few reliable radioimmunoassays
for CCK have been published. One of the major problems in developing a
radioimmunoassay for CCK is the production of immunoreactive CCK-labels.
Preparation of [lJ5I]CCK33 by conventional oxidizing methods presents dif­
ficulties, because the single tyrosine residue in CCK33 is sulphated (Mutt and
Jorpes, 1971). In addition, it has been shown that both the chloramine T
and the lactoperoxidase methods result in great loss in immunoreactivity of
CCK (Rehfeld, 1978a). This has been overcome by labelling CCK33 by a
non-oxidizing method (Rehfeld, 1978a). With [155I]hydroxyphenylpro-
1 Supported by the F o u n d a tio n for Medical Research F U N G O (G ran t No. 13-37-32).
2 Correspondence to: C. Lamers, M .D ., Ph.D ., Division o f G astroenterology, St. R ad bo ud
Hospital, 6500 H B Nijmegen, The Netherlands.
0022-1759/82/0000—0 0 0 0 /$ 0 2.75 © 1982 Elsevier Biom edical Press
33
pionic acid-succinimide ester (Bolton-Hunter reagent) CCK33 labels of good
quality could be obtained (Rehfeld, 1978a; Sankaran et al., 1979; Beinfeld
et al., 1981; Byrnes et al., 1981).
In the present study we report that such [12SI]Bolton-Hunter-CCK33
labels may show extremely low binding to carboxyl-terminal CCK-antibodies.
M A T E R IA L S A N D M E T H O D S
Radioiodination procedures
[nsI]Bolton-Hunter reagent ([125I]hydroxyphenylpropionic acid-succini­
mide ester, New England Nuclear, Boston, MA, U.S.A.) was conjugated to
99% pure porcine CCK33 (V. Mutt, Karolinska Institutet, Stockholm,
Sweden) as described by Sankaran et al. (1979). The label was subsequently
purified on Sephadex G-50 SF columns, 100.0—1.0 cm (Pharmacia, Uppsala,
Sweden) using 0.5 M acetic acid and 0.2% gelatin as eluant. Peak II in Fig. 1
represents labelled CCK33. This Bolton-Hunter-labelled CCK33 eluted as a
single peak after refiltration on SP-Sephadex G-25 (Pharmacia, Uppsala,
Sweden) with a 0.5 M—1.5 M sodium acetate pH 4.0 gradiënt as eluting buf­
fer. The label retained its immunoreactivity when tested with non-carboxylterminal CCK-antibodies for at least 3 months. Label damage in the absence
Fig. 1. E lu tio n profile o f 125I-radioactivity after application o f reaction m ix ture o f
CCK33 w ith [125I]hy d rox y p he n y l propionic acid-succinimide ester (Bolton-Hunter
reagent) to a Sephadex G-50 superfine colum n. Peak I I represents labelled C C K 33. For
details see text.
39
cpno(»'06 ) —
2 0 - i
!\
t r a c t io n n u m b e r
Fig. 2. E lu tio n profile o f 125I-radioactivity after a p p lic atio n o f reaction m ixture o f
C C K 8 I w ith [125I]N a to D E 52 cellulose. The first peak represents unreacted 1JSI and the
second peak [12SI]C C K 8 I. For details see text.
of antibody was 2—7%. The specific activity of the label ranged from 219 to
398 iiC i/jug (n = 3).
Non-sulphated CCK8 (CCK8I, Squibb Institute, Princeton, NJ, U.S.A.)
was labelled with 12SI (Behringwerke A.G., Marburg, F.R.G.) by the chloramine T method (Dockray, 1980). The labelled peptide was purified on
DE52 cellulose columns, 1 cm X 10 cm (Whatman, Maidstone, England)
with a 0.05 M to 0.5 M ammonium carbonate gradiënt as eluant (Fig. 2).
This [125I]CCK8I label retained its immunoreactivity when tested with
carboxyl-terminal antibodies for at least 2 months. Label damage in the
absence of antibody was less than 2%.
Antisera
The following non-carboxyl-terminal CCK-antibodies were tested: anti­
body T104 was raised against CCK33 coupled to bovine serum albumin
(Behringwerke A.G., Marburg, F.R.G.) using l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (I.C.N., Plainview, NJ, U.S.A.), and antibody 1703 was
raised against uncoupled CCK33 (Fig. 3). When Bolton-Hunter-labelled
CCK33 was used, these antibodies reacted with CCK33 and CCK39, but not
with CCK8II, CCK8I, caerulein (Farmitalia, Milan, Italy), gastrin 17, gastrin
14-17, CCK 10-20 (U.C.B., Brussels, Belgium) or CCK33 fragments prepared
by trypsinization.
40
a /F
°
100-i
Fig. 3. Standard curve o f a non-carboxyl-terminal CCK *antibody (17 03), showing no
cross-reactivity w ith C CK 8 or gastrin. [ 12SI]Bolton-Huntcr-CCK33 used as label.
The following carboxyl-terminal CCK-antibodies were tested: antibody
L48, raised against sulphated CCK8 (CCK8II) coupled to bovine serum albumin using the carbodiimide method (Dockray, 1980), was purchased from
Merseyside Laboratories, Woolston, U.K.; antibody 5135, raised against
CCK8I coupled to bovine serum albumin by the carbodiimide method
(Rosenquist and Walsh, 1980), was a gift from Drs. Rosenquist and Walsh
(Fig. 4); antibody LAL was raised against non-sulphated gastrin (gastrin 171)
coupled to bovine serum albumin using the carbodiimide method. When
[125I]CCK8I was used as label, all these antibodies reacted with CCK39,
CCK33, CCK8II, CCK8I, gastrin 17 and gastrin 14-17. All antibodies were
raised in rabbits.
Radioimmunoassay procedure
Radioimmunoassays were performed in plastic tubes in an ice-bath using
0.02 M sodium barbital buffer pH 8.4 containing 30 ^mol/1 human serum
albumin (Behringwerke A.G., Marburg, F.R.G.) and 0.6 mmol/1 thiomersal
41
B / F •/. -
Fig. 4. Standard curve o f a carboxyl-terminal CCK-antibody (5135), showing crossreactivity w ith CCK 8 and gastrin. [125I]C C K 8 I used as label.
(Aldrich, Beerse, Belgium). The 2.2 ml incubation mixture contained 1.0 ml
of approximately 2000 cpm label, 0.2 ml Standard solution, and 1.0 ml
diluted antiserum. The tubes were incubated for 96 h at 4°C. Antibodybound radioactivity was separated from unbound label using a suspension
containing 25 mg activated charcoal (Merck, Darmstad, F.R.G.), 2.5 mg dextran T70 (Sigma, St. Louis, MO, U.S.A.), and 16 mg bovine serum albumin
(Sigma, St. Louis, MO, U.S.A.). The tubes were vortexed and immediately
centrifuged (3000 X#, 10 min). Both the supematant and the pellet were
counted in an automatic gamma scintillation counter (Philips PW4800;
Philips, The Netherlands).
RESULTS
[12SI]Bolton-Hunter-CCK33 showed excellent binding to non-carboxyl-terminal CCK-antibodies. The antisera could be used in final dilutions
of 1 : 22,500 and 1 : 100,000 (Table 1). The antibodies did not show sig­
nificant binding to [12SI]CCK8I. Using [lïsI]Bolton-Hunter-CCK33, the
concentration of Standard CCK33 that produced 50% inhibition of binding
42
TABLE1
Binding o f Bolton-Hunter-labelled CCK 33 ([ 12SI]BH-CCK33) and [n s I]C C K 8 I to noncarboxyl-terminal and to carboxyl-terminal specific CCK-antibodies.
Titer a
[ 125I ]BH-CCK33
Non-carboxyl term inal
CCK-antibodies
T l 04
1703
Carboxyl-terminal C C K
antibodies
L48
5135
LAL
1 : 22 500
1 : 100 000
< 500
< 500
<500
ID ,0 b
(pmol/1)
[1JSI)C C K 8 I
15.5
5.9
<500
<500
1 : 100 000
1 ■ 2 000 000
1
•
C C K 33
1 000 000
9.1
7.7
16 0
C C K 8 II
>10 000
>10 000
3.1
3.1
3.6
a Final d ilutio n o f antiserum giving 50% binding between antibody and label
b In h ib itio n doseso: concentration o f peptide giving 50 % in h ib itio n o f the binding
between label and an tibo dy (pmol/1 incubation m ixture).
between label and antibody (ID 50) ranged from 5.9 to 15.5 pmol/1, while
there was no binding to CCK8I or CCK8II (Table 1).
[12SI]CCK8I showed excellent binding to carboxyl-terminal CCK-anti­
bodies (Table 1). These carboxyl-terminal CCK-antibodies could be used in
final dilutions of 1 : 100,000—1 : 2,000,000. The IDS0 for CCK8II ranged
from 3.1 to 3.6 pmol/1, and for CCK33 from 7.7 to 16.0 pmol/1. Although
unlabelled CCK33 displaced [125I]CCK8I from these antibodies, there was
negligible binding of [12SI]Bolton-Hunter-CCK33 to the carboxyl-terminal
CCK-antibodies. Even when the carboxyl-terminal CCK-antibodies were used
in excess (1 : 500) binding to [12SI]Bolton-Hunter-CCK33 was less than 50%.
These findings were consistently obtained using 3 different [12SI]BoltonHunter-CCK33 preparations. The binding of [12SI]Bolton-Hunter-CCK33 to
excess antibody (1 : 500) ranged for antibody L48 from 17.2 to 17.8%,
for antibody 5135 from 1.3 to 10.5%, and for antibody LAL from 13.6
to 16.2%.
D IS C U SS IO N
Comparable values for ID S0 for CCK33 could be obtained with 2 differ­
ent types of radioimmunoassays: (1) with [12,T]Bolton-Hunter-CCK33 and
non-carboxyl-terminal CCK-antibodies, and (2) with [125I]CCK8I and car­
boxyl-terminal CCK-antibodies. Although CCK33 displaced [125I]CCK8I
from carboxyl-terminal CCK-antibodies, [125I]Bolton-Hunter-CCK33 did not
43
show significant binding to carboxyl-terminal antibodies, even when these
antibodies were used in excess. Therefore, labelling of CCK33 with BoltonHunter reagent as described in this study may interfere with binding to carboxyl-terminal antibodies. Bolton-Hunter reagent is known to bind to basic
amino acid residues (Bolton and Hunter, 1973). However, the carboxyl-ter­
minal portion of CCK33 is deprived of basic amino acid residues (Mutt and
Jorpes, 1971). Therefore, the decrease in immunoreactivity of CCK33 is not
simply due to binding of the Bolton-Hunter reagent to amino acid residues
in the carboxyl-terminal portion of the molecule. Further we found that
another CCK-antibody (T204), specific for the region containing the sul­
phated tyrosine residue but not for the carboxyl-terminus (binding to
CCK39, CCK33, CCK8II and caerulein, but not to CCK8I, gastrin 17 or
gastrin 14-17), showed excellent binding to [125I]Bolton-Hunter-CCK33
(data not shown). In contrast to our findings, Rehfeld (1978a) and Beinfeld
et al. (1981) reported good binding of [125I]Bolton-Hunter-CCK33 to anti­
bodies against gastrin 17 and against CCK8II. In our study, carboxyl-ter­
minal CCK-antibodies raised against gastrin 17, CCK8I and CCK8II did not
show significant binding to [12SI]Bolton-Hunter-CCK33. The reason for this
discrepancy is not apparent. It is possible that carboxyl-terminal CCK-antibodies differ in their sensitivity to alterations in the conformation of the
CCK33 molecule. Jacobson et al. (1972) have shown that blocking lysine
residues by succinylation alters the tertiary structure and immunochemical
reactivity of albumin. By analogy, conformational changes of CCK33
induced by Bolton-Hunter labelling might interfere with binding to some
carboxyl-terminal CCK-antibodies.
Previously, we and other workers have experienced considerable diffi­
culties in producing immunoreactive [125I]Bolton-Hunter-CCK33 when
tested with carboxyl-terminal CCK-antibodies (Rosenquist, Walsh, personal
Communications). This might be related to the present finding that [12SI]Bolton-Hunter-CCK33 showed excellent binding to non-carboxyl-terminal
CCK-antibodies and negligible binding to some carboxyl-terminal antibodies.
Another interesting finding in the present study is that [125I]CCK8I labels
with excellent immunoreactivity can be obtained with the chloramine-T
method. This is in agreement with other studies (Dockray, 1980; Lamers
et al., 1980), but contrasts with Rerifeld’s finding of large losses in immuno­
reactivity by this oxidizing method (Rehfeld, 1978a). Since immunoreactive
[125I]CCK39 labels have also been produced by the chloramine T or lactoperoxidase methods (Schlegel et al., 1977; Bernard et al., 1981; Lonovics
et al., 1981), the effect of the use of oxidizing methods on the immuno­
reactivity of CCK labels needs further investigation.
REFERENCES
Beinfeld, M .C., D .K . Meyer, R .L . Eskay, R.T . Jensen and M .J. Brownstcin, 1981, Brain
Res. 212, 51.
44
Bernard, C., R . R am blière, N. Stremdoerfer and J.A . Chayvialle, 1981, Gastroenterology
80, 1110.
B olton, A .E . and W .M . H unter, 1973, Biochem . J. 133, 529.
Byrnes, D .J., L. Henderson, T. Borody and J.F . R ehfeld, 1981, Clin. C h im . A cta 111, 81.
Dockray, G .J., 1980, Brain Res. 188, 155.
Jacobson, C., L. F u nd ing , N.P. M aller and J. Steensgaard, 1972, Eur. J. Biochem . 30,
392.
Lamers, C.B., J.E . M orley, P. Poitras, B. Sharp, H.E. Carlson, J.M . Hershman and J.H .
Walsh, 1980, A m . J. Physiol. 239, E232.
Lonovics, J., S. G u zm a n , P.G. D evitt, K .E . H ejtm ancik, R .L . S u d d ith , P.L. R ayford and
J.C . T hom pson, 1981, Endocrinology 108, 1925.
M utt, V. and E. Jorpes, 1971, Biochem . J. 125, 57P.
Rayford, P.L., T h.A . M iller and J.C . T hom pson, 1976, New Engl. J. Med. 294, 1093.
Rehfeld, J .F ., 1978a, J. Biol. Chem . 253, 4016.
Rehfeld, J .F ., 1978b, J. Biol. Chem . 253, 4022.
Rosenquist, G .L . and J.H . Walsh, 1980, in: Gastrointestinal Horm ones, ed. G .B . J. Glass
(Raven Press, New Y o rk ) p. 769.
Sankaran, H ., C.W. Deveney, I.D . G o ld fin e and J.A . W illiams, 1979, J. Biol. Chem . 254,
9349.
Schlegel, W ., S. Raptis, D. G rube and E.F. Pfeiffer, 1977, Clin. C him . A cta 80, 305.
45
Chapter IV
Characterization of Antisera to Cholecystokinin
Employing Different Cholecystokinin Labels
J.B.M.J. Jansen
and
C.B.H.W, Lamers
Gastrointestinal Hormone Laboratory
D i v i s i o n of G a s t r o e n t e r o l o g y
St. R a d b o u d H o s p i t a l
University of Nijmegen
Nijmegen
The N e t h e r l a n d s
Journal of Immunologieal Methods:
in press.
47
IV CHARACTERIZATION OF ANTISERA TO CHOLECYSTOKININ EMPLOYING
DIFFERENT CHOLECYSTOKININ LABELS
Abstract
A n t i s e r a r a i s e d a g a i n s t s y n t h e t i c s u l p h a t e d C C K 26-33
a n d a g a i n s t 30% p u r e p o r c i n e C C K
(n=4)
(n=ll) w e r e c h a r a c t e r i z e d e m p l o -
y i n g d i f f e r e n t C C K - l a b e l s . C C K 39 a n d C C K 26-33 w e r e c o u p l e d to
125 I u s i n g t h e c h l o r a m i n e - T m e t h o d , w h i l e C C K 33 w a s c o n j u g a t e d
to 125 I-hydroxyphenylpropionic acid-succinimide ester (BoltonH u n t e r r e a g e n t ) • A n t i s e r a r a i s e d a g a i n s t C C K 26-33 b o u n d to
(
12 5
I )C C K 26- 3 3 only. O f t he a n t i s e r a r a i s e d a g a i n s t 30% p u r e
CCK, 2 b o u n d to a ll 3 l a b e l s , 4 to (1 2 5 I ) B H - C C K 33 an d (1 2 5 I ) C C K
26-33,
3 to (1 2 5 I ) C C K 39 a n d
26-33 o n l y , a n d o n e to
acting with
(1 2 5 I ) B H - C C K 33, o n e to (1 2 5 I ) C C K
(^2 ^ I ) B H - C C K 33 only. T h e a n t i b o d i e s re-
(1 2 ^ I ) C C K 2 6 - 3 3 a l s o b o u n d to
(1 2 ^ I ) G a s t r i n 1-17.
D i f f e r e n t C C K - l a b e l s b o u n d to d i f f e r e n t b i n d i n g sites in the
same a n t i s e r u m
( a n t i b o d y h e t e r o g e n e i t y ). T h e p a t t e r n of r e a c t i -
v i t y o f t h e a n t i s e r u m to C C K - p e p t i d e s w a s d e p e n d e n t o f th e t y p e
of l a b e l u s ed. U s i n g t h e s e d i f f e r e n t l a bels, a n t i b o d i e s s p e c i f i c
for C C K 39, for C C K 33 a n d C C K 39, f or s u l p h a t e d forms of CCK,
a n d for all C C K - p e p t i d e s a n d g a s t r i n c o u l d be detected.
It is c o n c l u d e d t h a t a n t i s e r a r a i s e d a g a i n s t C C K s h o u l d b e
characterized employing different CCK-labels.
Introduction
Cholecystokinin
(CCK) t o g e t h e r w i t h s e c r e t i n and g a s t r i n
c o n s t i t u t e t he c l a s s i c a l t r i a d of g u t - h o r m o n e s
1976). C C K s t i m u l a t e s g a l l b l a d d e r c o n t r a c t i o n
1928) a n d p a n c r e a t i c e n z y m e s e c r e t i o n
(Rayford et al,
(Ivy & O l d b e r g ,
(Harper & Raper, 1943).
O r i g i n a l l y , C C K w a s i s o l a t e d f r o m a c i d e x t r a c t s of h o g small
i n t e s t i n e as a m o l e c u l e c o n t a i n i n g 33 a m i n o a c i d r e s i d u e s
(Mutt & J o r p e s ,
1967). A m o l e c u l a r f o r m e x t e n d e d at the a m i n o -
terminus by a further 6 amino acid residues
be d some years later
(Mutt,
(Rehfeld,
(CCK 39) w a s d e s c r i -
1976). In i n t e s t i n a l e x t r a c t s of man,
C C K is p r e s e n t in d i f f e r e n t m o l e c u l a r forms,
CCK 26-33
(CCK 33)
i n c l u d i n g C C K 33 a n d
1978b), W h i l e the r o l e s of s e c r e t i n an d g a s ­
trin, in t he m e a n t i m e , h a v e b e e n w e l l e s t a b l i s h e d , the p h y s i o l o 48
g i c a l a n d p a t h o p h y s i o l o g i c a l s i g n i f i c a n c e of C C K h a v e r e m a i n e d
sp e c u l a t i v e , m a i n l y d u e to t h e l a c k o f s e n s i t i v e a n d s p e c i f i c
r a d i o i m m u n o a s s a y s . O n e of the m a j o r p r o b l e m s in d e v e l o p i n g s p e ­
c i f i c r a d i o i m m u n o a s s a y s f or C C K is the p r o d u c t i o n of i m m u n o r Ê a c 125
tive C C K - l a b e l s . P r e p a r a t i o n of (
I ) C C K 33 b y c o n v e n t i o n a l oxid i z i n g m e t h o d s p r e s e n t s d i f f i c u l t i e s b e c a u s e th e s i n g l e t y r o s y l
r e s i d u e in C C K 33 is s u l p h a t e d
(Mutt,
1976).
In a d d i t i o n , it has
b e e n s h o w n t h a t t r e a t m e n t of C C K w i t h c h l o r a m i n e - T or l a c t o p e r o x i d a s e s t r o n g l y d i m i n i s h e s t h e b i o a c t i v i t y of the p e p t i d e , p r o b a b l y d u e to t h e d e l e t e r i o u s e f f e c t s o f o x i d a t i o n on the m e t h i o nyl r e s i d u e s in C C K
feld
(1978a)
(Mutt, 1964; R e h f e l d ,
1978a). R e c e n t l y , R e h ­
reported that oxidative radioiodination techniques
a l s o a b o l i s h e d C C K - i m m u n o r e a c t i v i t y , w h i l e l a b e l l i n g b y a nonoxidizing conjugation method
Hunter ,
1973)
(Bolton-Hunter reagent)(Bolton &
r e s u l t e d in C C K - l a b e l s w i t h e x c e l l e n t i m m u n o r e a c -
tivity. H o w e v e r , in o t h e r s t u d i e s C C K - l a b e l s p r e p a r e d b y o x i d a ­
t ive r a d i o i o d i n a t i o n t e c h n i q u e s w e r e s u c c e s s f u l l y e m p l o y e d in the
r a d i o i m m u n o a s s a y of C C K (Dockray,
1980; t«alsh et al, 1982; L i l j a
et al, 1982; C h a n g & C h ey, 1983). F u r t h e r m o r e , it has r e c e n t l y
b e e n r e p o r t e d t h a t B o l t o n - H u n t e r l a b e l l e d C C K 33 o f t e n s h o w s low
b i n d i n g to c a r b o x y l - t e r m i n a l C C K - a n t i b o d i e s
(Jansen & L a m e r s ,
1982).
In t he p r e s e n t s t u d y w e h a v e t e s t e d a n t i s e r a r a i s e d a g a i n s t
30% p u r i f i e d p o r c i n e C C K a n d s y n t h e t i c C C K 26-33 e m p l o y i n g la b e l s
prepared by both oxidative and conjugative labelling techniques
using different CCK-peptides.
Materials
99% p u r e p o r c i n e C C K 33, 99% p u r e p o r c i n e C C K 39 a n d 30%
p u r e p o r c i n e C C K w e r e o b t a i n e d f r o m prof. V. M u t t , K a r o l i n s k a
I nstit u t e ,
S t o c k h o l m , Sweden;
s y n t h e t i c s u l p h a t e d an d u n s u l p h a -
t ed C C K 2 6 - 3 3 f r o m t he S q u i b b I n s t i t u t e , P r i n c e t o w n , N.J., USA;
s y n t h e t i c u n s u l p h a t e d h u m a n g a s t r i n 1-17 f r o m I m p e r i a l C h e m i c a l
125
In d u s t r i e s , C h e s h i r e , E n g l a n d ; (
I)hydroxyphenylpropionic
acid-succinimide ester
( B o l t o n - H u n t e r reagent) f r o m N e w E n g l a n d
125
N u c l e a r , B o s t o n , Ma, USA; (
I ) N a f r o m B e h r i n g w e r k e AG, M a r b u r g /
L a hn, FRG; s o d i u m e t h y l m e r c u r i t h i o s a l y c i l a t e f r o m A l d r i c h - E u r o p e ,
49
Be e r s e , B e l g i u m ;
S e p h a d e x G 50 SF a n d C M - S e p h a d e x C 25 f r o m
Pharmacia, Uppsala,
Sweden; A E 41 and DE 52 c e l l u l o s e f r o m W h a t -
man, M a i d s t o n e , E n g l a n d ; d e x t r a n T 70 an d b o v i n e s e r u m a l b u m i n
( R I A - g r a d e ) , f r o m S i g m a St. L o u i s , Mo, USA; h u m a n and b o v i n e s e ­
r u m a l b u m i n f r o m B e h r i n g w e r k e AG, M a r b u r g / L a h n , FRG; all o t h e r
r e a g e n t s f r o m E. M e r c k , D a r m s t a d t , FRG.
Antisera
A m o n g 29 r a b b i t s a n d 5 g u i n e a p i g s i m m u n i z e d w i t h p a r t l y
p urified porcine CCK or synthetic CCK fragments
1983),
(Jansen & L a m e r s ,
15 r a b b i t s p r o d u c e d a n t i s e r a b i n d i n g to at l e ast on e of
the C C K - l a b e l s s t u d i e d in a d i l u t i o n of m o r e t h a n 1:500
TABLE
Titers
Anti-
Imm-
'no .
gen
7
8
9
10
BSAECDCCK
26-33°
of
CCK-antisera
I
employing
different
Antiserum
12 5 I C C K3 9
(table I).
12 5 I B H C C K 3 3 b
1 25
labels
titer3
12 5
ICCK26-33
I g a s t r i n l - 17
<500
<500
<500
<500
<500
<500
<500
<500
660
20,000
9 ,900
17,500
<500
12 , 0 0 0
10,000
20,000
11
12
13
14
Tl
T2
BSAECDCCK
(3 0 % ) °
<500
<500
65,000
<500
45,000
15,000
2,750
27,000
75,000
50,000
22,500
80 ,000
19,000
3,500
38,000
70,000
<500
<500
25,000
5 ,000
39 ,000
40,000
<500
<500
15
16
17
18
T3
CCK
(30%)
<500
600
<500
32 , 0 0 0
<500
<500
1 ,500
52,000
2 ,500
2 ,600
28,000
<500
5,000
8 ,500
<500
30,000
<500
2 ,500
2 ,500
<500
a.
b.
c.
50
F i n a l d i l u t i o n of
b o d y and label.
antiserum
giving
50%
binding
12 5
.
.
C C K - 3 3 c o u p l e d to
I-hydroxyphenylprop ionic
ester (Bolton-Hunter reagent).
C C K - p e p t i d e s c o u p l e d to b o v i n e s e r u m a l b u m i n
(3-dimetylaminopropyl)-carbodiimide.
between
anti­
acid- succinimideusing
l-ethyl-3-
F o u r a n i m a l s w e r e i m m u n i z e d w i t h s y n t h e t i c C C K 2 6 - 3 3 c o u p l e d to
bo v i n e s e r u m a l b u m i n u s i n g t he c a r b o d i i m i d e m e t h o d
(120 n m o l at
the i n i t i a l - a n d 60 n m o l a t b o o s t e r i n j e c t i o n s ) , 6 w i t h 30% pure
porcine CCK
(20 n m o l a t t he i n i t i a l - a n d 10 n m o l a t b o o s t e r in­
jections) . T h e a n t i s e r a t e s t e d in t he p r e s e n t s t u d y w e r e o b t a i ned a f t e r the f o u r t h i m m u n i z a t i o n . B i n d i n g of the a n t i s e r a to
(1 2 s I)C C K 39,
(1 2 5 I ) B H - C C K 33,
(1 2 5 I ) C C K 26-33 a n d
(1 2 5 I )g a s t r i n
1-17 w a s d e t e r m i n e d . W h e n the t iter, d e f i n e d as the d i l u t i o n of
the a n t i s e r u m g i v i n g 50% b i n d i n g of the l a b e l l e d p e p t i d e , w a s
h i g h e r t h a n 1 : 1 5 , 0 0 0 , d i s p l a c e m e n t s t u d i e s w i t h C C K - p e p t i d e s and
gastrin were performed.
Radioiodination
N o n - s u l p h a t e d C C K 26- 3 3 w a s l a b e l l e d w i t h
c h l o r a m i n e - T m e t h o d as d e s c r i b e d p r e v i o u s l y
(^2 ^I) b y the
(Jansen & L a m e r s ,
1982). T h e l a b e l l e d p e p t i d e w a s p u r i f i e d on DE 52 c e l l u l o s e
c o l um n s
(1 c m x 10 cm) u s i n g a 0.05 M to 0.5 M a m m o n i u m c a r b o -
n a t e pH 5.0 g r a d i ë n t as elua n t . T h e l a b e l l e d p e p t i d e r e t a i n e d
its i m m u n o r e a c t i v i t y for at l e a s t 2 m o n ths. L a b e l d a m a g e in the
a b s e n c e o f a n t i b o d y w a s less t h a n 2%. T h e s p e c i f i c r a d i o a c t i v i ­
ty of the l a b e l r a n g e d f r o m 2 6 0 - 5 0 0 y C i / y g
(n=5).
N o n - s u l p h a t e d g a s t r i n 1-17 w a s l a b e l l e d w i t h
chloramine-T method
(
125
I) b y the
(Stadil & R e h f e l d , 1972) . T h e l a b e l l e d p e p ­
tide w a s p u r i f i e d o n A E 41 c e l l u l o s e c o l u m n s
(1 c m x 10 cm) w i t h
a 0.05 M to 0.2 M a m m o n i u m b i c a r b o n a t e pH 8.4 g r a d i ë n t as eluant.
Th e l a b e l l e d p e p t i d e r e t a i n e d its i m m u n o r e a c t i v i t y f o r a t least
2 months. L a b e l d a m a g e in the a b s e n c e of a n t i b o d y r a n g e d b e t w e e n
1-5% a n d the s p e c i f i c r a d i o a c t i v i t y f r o m 2 7 5 - 5 2 5 y C i / y g
(n=10).
C o n j u g a t i o n o f C C K 33 w i t h B o l t o n - H u n t e r r e a g e n t w a s per125
f o r m e d a s d e s c r i b e d b e f o r e (Jansen & L a m e r s , 1982).
I-hydroxyp h e n y l p r o p i o n i c a c i d - s u c c i n i m i d e e s t e r wa s d r i e d u n d e r a g e n t l e
s t r e a m of n i t r o g e n a c c o r d i n g to the i n s t r u c t i o n s of the m a n u f a c turer. 5 y g 99% p u r e C C K 33, d i s s o l v e d in 5 yl 0.05 M a c e t i c a c i d
w a s a d d e d to t h e b o t t o m of t h e vial. A f t e r a d d i t i o n o f 15 yl 0.05
M s o d i u m b o r a t e p H 10, the v i a l w a s g e n t l y a g i t a t e d in an ice bath.
A f t e r o ne h o u r 0.2 M g l y c i n e in 500 yl 0.05 M s o d i u m b o r a t e pH 8.5
51
w a s a d d e d to the m i x t u r e a n d the v i a l w a s a g i t a t e d for a n o t h e r
5 m i n u t e s . T h e r e a c t i o n m i x t u r e w a s t h e n c h r o m a t o g r a p h e d at 4
°C on a S e p h a d e x G 50 SF c o l u m n
(100 c m x 1 cm) u s ing 0.5 M
a c e t i c a c i d c o n t a i n i n g 1 g /1 g e l a t i n as e l uant. E v ery hour 5
f r a c t i o n s of 1 m l w e r e c o l l e c t e d . T h e f r a c t i o n w i t h the h i g h e s t
r a d i o a c t i v i t y of the i m m u n o r e a c t i v e p e a k w a s d i l u t e d in 0.05 M
a c e t i c a c i d a n d s t o r e d a t -20 °C. T h i s
( ^ ^ I ) B H - C C K 33 label
r e t a i n e d its i m m u n o r e a c t i v i t y f or at l e a s t 2 m o n t h s . L a b e l dam a ge in t he a b s e n c e of a n t i b o d y r a n g e d b e t w e e n 2-7%. Th e s p e c i f i c
r a d i o a c t i v i t y of the l a b e l r a n g e d f r o m 2 6 0 - 4 8 5 y C i / y g ( n = 8 ) .
125
9 9% p u r e p o r c i n e C C K 39 w a s l a b e l l e d w i t h
I by the ch l o r a m i n e - T m e t h o d . 0.25 m C i 125 I d i s s o l v e d in 5 yl 0.25 M s o d i u m
p h o s p h a t e b u f f e r p H 7.5 w a s m i x e d v i g o r o u s l y w i t h 5 yg 99% p u r e
C C K 39, d i s s o l v e d in 5 yl 0.05 M s o d i u m p h o s p h a t e b u f f e r p H 7.5
a n d 10 y g c h l o r a m i n e - T d i s s o l v e d in 10 yl s o d i u m p h o s p h a t e b u f f e r
p H 7.5 in a g l a s s tube. A f t e r 20 s e c o n d s the r e a c t i o n w a s termin a t e d b y a d d i t i o n of 20 y g s o d i u m m e t a b i s u l p h i t e d i s s o l v e d in 10
yl s o d i u m p h o s p h a t e b u f f e r p H 7.5. T h e l a b e l l e d p e p t i d e w a s p u r i f i e d o n C M - S e p h a d e x C 25 c o l u m n s
(1 c m x 10 cm) w i t h a 0.05 M to
125
0.5 M a m m o n i u m a c e t a t e g r a d i ë n t as e l u a n t (fig 1). T h i s (
I)CCK
39 l a b e l r e t a i n e d its i m m u n o r e a c t i v i t y for a t l e a s t 2 months.
L a b e l d a m a g e in the a b s e n c e of a n t i b o d y w a s less than 5%. The
s p e c i f i c a c t i v i t y of the l a b e l r a n g e d f r o m 2 5 0 - 4 1 0 y C i / y g
(n=3).
Incubation conditions
R a d i o i m m u n o a s s a y s w e r e p e r f o r m e d in p l a s t i c t u bes in an ice
bath.
0.05 M s o d i u m p h o s p h a t e b u f f e r pH 7.4 c o n t a i n i n g 0.08 m m o l / 1
h u m a n s e r u m a l b u m i n a n d 0.06 m m o l / 1 s o d i u m e t h y l m e r c u r i t h i o s a l i c y l a t e w a s u s e d as a s s a y b u f f e r . T h e i n c u b a t i o n m i x t u r e c o n t a i n e d
0.2 m l of a p p r o x i m a t e l y 33 d ps l a b e l l e d p e p t i d e , 0.2 ml S t a n d a r d
s o l u t i o n a n d 0.1 ml d i l u t e d a n t i s e r u m . T h e t u b e s w e r e i n c u b a t e d
for 9 6 h o u r s a t 4 °C t o d e t e r m i n e a n t i b o d y t i t e r s , w h i l e a pree q u i l i b r i u m a s s a y w a s u s e d to set u p S t a n d a r d c u r v e s w i t h CCKpeptides or gastrin.
In t h i s p r e - e q u i l i b r i u m assay, a d d i t i o n of
l a b e l l e d p e p t i d e w a s d e l a y e d u n t i l 20 h o u r s b e f o r e s e p a r a t i o n of
free from antibody b ound peptide was performed.
52
S e p a r a t i o n of
free and a n t i b o d y b o u n d p e p t i d e w a s p e r f o r m e d b y a d d i t i o n of a
1.5 ml c h a r c o a l s u s p e n s i o n , c o n t a i n i n g 25 m g a c t i v a t e d c h a r c o a l ,
2.5 mg d e x t r a n T 70 a n d 16 m g b o v i n e s e r u m a l b u m i n (RIA-grade)
in the a s s a y b u f f e r . T h e t u b e s w e r e v o r t e x e d a n d c e n t r i f u g e d
(3000 g, 10 m i n u t e s ) . B o t h s u p e r n a t a n t a n d p e l l e t w e r e c o u n t e d
in an a u t o m a t i c y - s c i n t i l l a t i o n c o u n t er.
Fraction n um ber
Figure 1
p r o f i l e o f 1 2 5 I - r a d i o a c t i v i ty a f t e r a p p l i c a t i o n o f t h e
r e a c t i o n m i x t u r e o f C C K 39 w i t h 1 2 5 I N a t o a C M - S e p h a d e x C 25
c o l u m n (1 c m x 10 c m ) u s i n g a 0 . 0 5 M to 0 . 5 M a m m o n i u m a c e t a t e
b u f f e r pH 5.0 g r a d i ë n t . The fir st p e a k r e p r e s e n t s u n r e a c t e d
1251 and t h e s e c o n d p e a k ( 1 2 5 I ) C C K 39. F o r d e t a i l s s e e text .
Elution
Results
A n t i s e r a r a i s e d a g a i n s t C C K 2 6 - 3 3 b o u n d to (
b u t not to
when
(
3I ) C C K 39 or (
D I ) B H - C C K 33
IOC
I ) C C K 26-33,
(table X). Howe v e r ,
(1 ^ I ) C C K 2 6 - 3 3 w a s u s e d as l abel, b o t h C C K 39 a n d C C K 33
w e r e a b l e to d i s p l a c e the l a b e l f r o m the a n t i s e r a
(table I I ) .
53
TABLE
DISPLACEMENT
OF D I F F E R E N T
A n t i-
L a b e l led
serum
peptide
CCK39
CCK33
1 . 00
0 ,.81
9.,38
8. 3 I
8 . 69
4 ., 1 6
5 ., 1 0
5 .,56
1.00
1,,25
< 0 .,01
N ., T . C
N .,T.°
8
1 . 00
1., 14
1 .,20
1 , 14
1 .,08
39
1 .00
1., 1 9
<0.,01
<0,,01
< 0 , 01
11
125i - c c k
12
12 5 i - b h - CC K
13
125i - c c k
12 5 i - c c k
T 1
-CCK
12 5 i - b
h
T2
12 5 i - b
h
-CCK
33
33
33
39
- CCK
12 5 i - c c k
39
15
12 5 i - c c k
8
17
12 5 i - b
18
12 5 i - c c k
h
33
8
- CCK
125i -c c k
CCK26-33
Gastrinl-17
unsulpha­
ted.
1.,08
1 .00
1 .00
h
binding3
1.,00
8
8
125i -b
CCK-PEPTIDES
5 .,56
12 5 i - c c k
h
CCK26-33
sulphated
BY
4 .,51
10
125i -b
CCK-ANTISERA
3 .,53
8b
125I-CCK
125i-c c k
LABELS FROM
AND GASTRIN
Relative
8
14
II
-CCK
33
33
39
1 .0 0
2 ,. 1 4
<0,,01
<0,.01
<0,,01
1 .00
1,. 1 I
2 .,9 0
2 , 60
2 , 10
1 .00
1,,25
<0.,0 1
N ,T .
N ,T .
N .T .
N..T.
1.00
2 ,.00
< 0 , 01
N .,T .
1 . 00
1,.43
<0,,01
N,.T .
1 .0 0
0,.89
0 ,. 83
< 0 ,. 01
<0,.01
1 .00
0 . 90
0 ,. 79
<0,.01
<0,.01
1 . 00
1 . 12
0,,84
0,.75
0,.70
1 .00
1 . 14
< 0 .01
<0,.01
<0,.01
1 .00
<0,.01
<0,.01
< 0 ,.01
<0..01
a. The r e l a t i v e b i n d i n g of C C K - p e p t i d e s and g a s t r i n to a n t i s e r a
was c a l c u l a t e d f r o m the ID-50 for each p e p t i d e (Inhibition
d o s e 50 (ID-50) is the dose of the p e p t i d e that reduces b i n ­
ding b e t w e e n label and a n t i b o d y by 50%).
12 5
b. (
I ) C C K 26-33.
c. N.T.: not tested.
O f t h e a n t i s e r a r a i s e d a g a i n s t 30% p u r e p o r c i n e CCK, 2 b o u n d to
a ll 3 C C K - l a b e l s ,
4 to
(1 2 5 I )B H - C C K 33 a n d
(1 2 5 I)C C K 26-33, 3 to
(1 2 5 I ) C C K 39 a n d (1 2 5 I ) B H - C C K 33, o n e to (1 2 5 I ) C C K 26-33 only,
125
a n d o n e to (
I ) B H - C C K 33 o n l y (table I). T h e 2 a n t i s e r a b i n d i n g
to al l 3 C C K - l a b e l s s h o w e d d i f f e r e n t d i s p l a c e m e n t s cu r v e s for
54
C C K - p e p t i d e s w h e n t e s t e d w i t h d i f f e r e n t C C K - l a b e l s (table I I ) .
12 5
Vvhen t e s t e d w i t h (
I ) C C K 2 6-33, a n t i s e r u m 13 b o u n d to a l l CCKp e p t i d e s a n d g a s t r i n 1-17, w h e r e a s t h is a n t i s e r u m b o u n d to CC K 33
an d C C K 39 o n l y w h e n t e s t e d w i t h
(1 2 5 i )CCK 39 o r
(1 2 5 I ) B H - C C K 33
(table II; f i g 2). W h e n a n t i s e r u m 18 w a s t e s t e d w i t h
it w a s f o u n d to b e s p e c i f i c f o r C C K 39
bodies reacting with both
(
1? S
(1 2 5 I ) C C K 39,
(fig 3). O f the a anti-
I ) B H - C C K 33 a n d
1 ? ■ï
(
I ) C C K 26- 3 3
one b o u n d to a l l C C K - p e p t i d e s a n d g a s t r i n , 2 b o u n d to C C K 39 and
C C K 33 only, w h i l e a n t i s e r u m 14 b o u n d to al l C C K - p e p t i d e s an d
1 TC
gastrin when tested with (
I ) C C K 2 6 -33, an d w i t h C C K 39 and
C C K 33 only, w h e n t e s t e d w i t h
(1 2 5 I ) B H - C C K 33
(table II).
Figure 2
D i f f e r e n t p a t t e r n s of
d i s p l a c e m e n t of d i f f e r e n t
C C K - l a b e l s f r o m the same
a n t i s e r u m ( n o . 13) b y
CCK-peptides.
pM
O f the a n t i s e r a r e a c t i n g w i t h b o t h
(1 2 5 I ) C C K 39 a n d
(1 2 5 I ) B H - C C K
33, a n t i s e r u m Tl b o u n d to C C K 39 a n d C C K 33 only, w h i l e a n t i s e r u m
T2 b o u n d to C C K 39, C C K 33 a n d s u l p h a t e d C C K 26-33, b u t n o t to
n o n - s u l p h a t e d C C K 2 6 -33 a n d g a s t r i n
.
r e a c t i n g to
125
(
(table II). A n t i s e r u m 15,
I ) C C K 26-33 b o u n d to a l l C C K p e p t i d e s a n d g a s t r i n .
A l l a n t i s e r a r e a c t i n g to (1 2 5 I ) C C K 26- 33 b o u n d a l s o to
(1 2 5 I)
g a s t r i n 1-17. E x c e p t for a n t i s e r u m T2, al l a n t i s e r a r e a c t i n g to
55
s u l p h a t e d C C K 2 6 - 3 3 b o u n d a l s o ,u n s u l p h a t e d C C K 26-33 an d gast r i n .
B/F (% )
T----- 1----------- 1----- 1-5
10
50
100
pM
Figure
Standard
curve
of
3
I2 5
a n t i b o d y 18 u s i n g (
a s s a y is s p e c i f i c f o r
I ) C C K 39
C C K 39.
as
label.
The
Discussion
This study shows that antisera raised against CCK may bind
to C C K - l a b e l s p r e p a r e d b y b o t h o x i d a t i v e a n d n o n - o x i d a t i v e r a d i o iodination methods.
It c a n t h e r e f o r e b e c o n c l u d e d that, in c o n ­
t r a s t to a p r e v i o u s r e p o r t
(Rehfeld, 1 9 7 8 a ) , C C K - l a b e l s p r e p a r e d
b y o x i d a t i v e t e c h n i q u e s d o n o t n e c e s s a r i l y lose t h eir i m m u n o r e a c t i v e p r o p e r t i e s . T h i s is in a g r e e m e n t w i t h p r e v i o u s r e p o r t s on
r a d i o i m m u n o a s s a y s f or C C K e m p l o y i n g C C K - l a b e l s p r e p a r e d b y the
chloramine-T method
al,
(Dockray,
1980; W a l s h et al,
1982; L i l j a et
1982; C h a n g & C h e y , 1983); T h e d a t a p r e s e n t e d in the p r e s e n t
study confirms our previous observation that antibodies binding
125
to (
I ) C C K 26- 3 3 a n d t h e r e f o r e d i r e c t e d t o w a r d s the c a r b o x y l t e r m i n a l r e g i o n of C C K o f t e n p o s s e s s l o w a f f i n i t y to B o l t o n H u n t e r l a b e l l e d C C K 33
(Jansen & L a m e r s , 1982). T w o a n t i s e r a
125
13 a n d no. 14), h o w e v e r , b o u n d in h i g h t i t e r s to b o t h (
I)
1 2R
CCK 26-33 and (
I ) B H - C C K 33 (table I). T h e s e a n t i b o d i e s s h o w e d
(no.
d i f f e r e n t d i s p l a c e m e n t p r o f i l e s for C C K - p e p t i d e s and g a s t r i n w h e n
studied with
tested with
(1 2 5 I ) B H - C C K 33 o r (1 2 5 I ) C C K 2 6 -33 (table II). W h e n
125
(
I ) C C K 2 6-33, the a n t i s e r a b o u n d to all C C K - p e p t i -
d e s a n d g a s t r i n , w h i l e t h e y b o u n d to C C K 39 a n d C C K 33 o n l y w h e n
tested with
56
(^2 ^ I ) B H - C C K 33. It is t h e r e f o r e l i k e l y t h a t
(*2 ^I)
CC K 26-33 a n d
(
1? R
3 I ) B H - C C K 33 b i n d to d i f f e r e n t a n t i b o d y p o p u l a -
tions in t he 2 a n t i s e r a . O n e a n t i b o d y p o p u l a t i o n c a n b e d e t e c t e d
by both
( ^ ^ I ) C C K 26- 3 3 a n d
(*2 ^I) g a s t r i n 1-17, w h e r e a s the o t h e r
125
(
I)
a n t i b o d y p o p u l a t i o n in t h e s e a n t i s e r a c a n b e d e t e c t e d b y
B H - C C K 33. T h e f i r s t a n t i b o d y p o p u l a t i o n is n o t s p e c i f i c for C C K
b e c a u s e o f c r o s s - r e a c t i v i t y w i t h g a s t r i n , w h i l e th e l a t t e r anti»b o d y is s p e c i f i c f o r t he l a r g e r f o r m s of C C K
F i v e a n t i s e r a b o u n d to ( * ^ I ) C C K 39
(table II, f i g 2).
(table I). A n t i s e r a Tl
an d T2 s h o w e d s i m i l a r d i s p l a c e m e n t p r o f i l e s fo r C C K - p e p t i d e s
when tested w i t h
(1 2 5 I ) B H - C C K 33 a n d
(1 2 5 I ) C C K 39
(table II),
howev e r , t he b i n d i n g s i tes f or C C K o f b o t h a n t i b o d i e s d i f f e r ,
s i n c e a n t i s e r u m Tl d o e s n o t b i n d to C C K 26-33, w h i l e a n t i s e r u m
T2 bi n d s to s u l p h a t e d C C K 26-33 b u t n o t to u n s u l p h a t e d C C K 26-33
and g a s trin.
It is t h e r e f o r e l i k e l y t h a t a n t i s e r u m T2 is
d i r e c t e d t o w a r d s t he s u l p h a t e d t y r o s y l r e g i o n of CCK.
F r o m the d i s p l a c e m e n t p r o f i l e s it c a n b e d e r i v e d t h a t a n t i s e r u m
Tl is d i r e c t e d t o w a r d s e i t h e r the a m i n o - t e r m i n u s o r th e m i d r e g i o n
of CCK.
It is, h o w e v e r , u n l i k e l y t h a t a n t i s e r u m Tl is d i r e c t e d
to war d s t he a m i n o - t e r m i n u s of CCK, s i nce the a n t i s e r u m sho w s si­
m i l a r b i n d i n g p r o p e r t i e s to C C K 39 and C C K 33 w h i c h d i f f e r f r o m
each o t h e r in t he a m i n o - t e r m i n a l r e g i o n
s e r u m 18 w a s t e s t e d w i t h
(Mutt,
1976). W h e n a n t i ­
(1 2 ^ I ) C C K 39, C C K 39 w a s t h e o n l y p e p t i ­
de th a t d i s p l a c e s t h e lab e l f r o m t he a n t i s e r u m .
It is t h e r e f o r e
lik e l y t h a t t h i s a n t i s e r u m is d i r e c t e d to a r e g i o n in C C K 39 that
IOC
C C K 39 a n d C C K 33 h a v e n o t in common. E m p l o y i n g (
I ) C C K 39 as
label a n d 99% p u r e p o r c i n e C C K 39 as Sta n d a r d , a n t i s e r u m 18 can
b e us e d in a r a d i o i m m u n o a s s a y s p e c i f i c for C C K 39
( J a nsen & La-
mers, 1983b). A n t i s e r a b i n d i n g to (1 2 ^ I ) C C K 39 d o n o t n e c e s s a r i l y
125
b i n d to (
I ) B H - C C K 33. A n e x p l a n a t i o n f o r this p h e n o m e n o n is
n ot a p p a r e n t .
It m i g h t , h o w e v e r , b e p o s s i b l e t h a t s t r u c t u r a l or
c o n f o r m a t i o n a l c h a n g e s in t he C C K - m o l e c u l e , i n t r o d u c e d b y d i f f e ­
r e n t l a b e l l i n g t e c h n i q u e s , a r e r e s p o n s i b l e for d i f f e r e n t b i n d i n g
of a n t i s e r a t o the 2 labels.
All antisera reacting with
(
125
I ) CCK 2 6 -33 b o u n d a l s o to
gastrin. T h e c r o s s - r e a c t i v i t y of c a r b o x y l - t e r m i n a l C C K - a n t i s e r a
w i t h g a s t r i n is p r o b a b l y r e l a t e d to s t r u c t u r a l s i m i l a r i t i e s in
the c a r b o x y l - t e r m i n a l r e g i o n s of C C K an d g a s t r i n
(Mutt & J o rpes,
57
1967). T h e r e f o r e ,
c a r b o x y l - t e r m i n a l C C K - a n t i s e r a are n o t s u i t a b l e
for s p e c i f i c m e a s u r e m e n t s of C C K - c o n c e n t r a t i o n s .
In c o n c l u s i o n , this s t u d y d e m o n s t r a t e s the i m p o r t a n c e of test i n g a n t i s e r a r a i s e d a g a i n s t C C K w i t h l a b e l s p r e p a r e d by d i f f e r e n t
labelling techniques using different CCK-pe p t i d e s .
REFERENCES
BOLTON
CHANG
AE
TM
DOCKRAY
& H U N T E R W M , 1973,
& CHEY
GJ,
HARPER
AA
IVY
& OLDBERG
AC
W Y , 1983,
1980,
Brain
& R A P E R MS,
E,
Dig Dis
Res
188,
1943,
1928,
Biochem
J
Sci
133,
28,
J Physiol
102,
115.
86,
599.
Am J P hysiol
JBMJ
& LAMERS
CBHW,
1982,
JANSEN
387 .
JBMJ
& LAMERS
CBHW,
1983a,
J Clin
JANSEN
JBMJ
& LAMERS
CBHW,
1983b,
Life
MUTT
V,
F A G A N CJ,
JC, 1982,
1964,
Acta
MUTT
V & JORPES
MUTT
V,
1976,
JE,
Clin
J
Immunol
Chem
Scand
1967,
18,
Biochem
Endocrinol
5,
Biophys
J Biol
Chem
253 , 4016.
REHFELD
JF,
J Biol
Chem
253,
58
F & REHFELD
LAMERS
CB
TA & THOMPSON
JF,
1972,
JC,
Scand
& VALENZUELA
223.
Clin Biochem
32,
21 ,
911.
LC,
RAYFORD
PHL
&
Res
Commun
26,
392.
175s.
J F , 1978a,
JH,
51,
2185.
PHL,
STADIL
Chem
Sci
REHFELD
1978b,
Methods
W I E N E R I, I N O U E K, W A T S O N
G a s t r o e n t e r o l o g y 83, 256.
RAYFORD
WALSH
438 .
MILLER
456.
155.
JANSEN
L I L J A P,
THOMPSON
529.
1976,
N Eng
J Med
294,
1093.
4022.
J Clin
JE,
Lab
Invest
30,
361.
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82,
Chapter V
Immunological Evidence of Cholecystokinin-39
in Porcine Brain
J.B.M.J. Jansen
and
C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory
D i v i s i o n of G a s t r o e n t e r o l o g y
St. R a d b o u d H o s p i t a l
U n i v e r s i t y of N i j m e g e n
Nijmegen
The N e t h e r l a n d s
L if e S a i e n o e
(1982) 32: 911-913.
59
Life Sciences, Vol. 32, pp. 911-913
Printed in the U.S.A.
Pergamon Press
IMMUNOLOGICAL EVIDENCE OF CH0LECYST0KININ-39
IN PORCINE BRAIN
J.B.M.J. Jansen and C.B.H.W. Lamers
Laboratory for Gastrointestinal Hormones,
Division of Gastroenterology, St.Radboud
Hospital, University of Nijmegen,
Nijmegen, The Netherlands
(Received in final form November 15, 1982)
Summary
Using a sensitive and specific radioimmunoassay for cholecystokinin-39 (CCK-39), CCK-39 was demonstrated in aqueous-acid
extracts of procine brain. The highest concentration of CCK-39
was found in the cortex (6.1±1.5 pmol/g). In the cortex CCK-39
comprised 21% of total CCK-inmunoreactivity and 51% of large
CCK-imnunoreactivity
Cholecystokinin (CCK) has been isolated from porcine intestines by Mutt and
Jorpes (1). These workers found that intestinal CCK consisted of 33 amino
acid residues (CCK-33). In addition, they characterized a CCK-peptide of 39
amino acid residues corresponding to the form of 33 residues extended at
the NH2-terminus by 6 residues (CCK-39) (2). Furthermore, a small mole­
cular form CCK-peptide, suggested to contain 8 amino acid residues, was extracted from small intestine of several marmialians (3,4). Recently, CCKlike peptides were discovered in brain extracts (5). It is suggested that
these CCK-like peptides in the brain play a role as neuromodulator or neuro­
transmitter. A small molecular form of CCK was demonstrated in cerebral ex­
tracts from different species (3,4,6,7,8,9). This small molecular form CCK
from sheep brain was chemically characterized and it was found to be identical to the 8 C-terminal amino acid residues of porcine CCK (10). Using inrnuno-chemical methods some workers have demonstrated larger forms of CCK in
cerebral extracts (4,7,8), whereas other workers using similar techniques
were unable to demonstrate these large molecular forms of CCK (3,9).
Although CCK-39 was isolated and chemically characterized already in 1976
(2) almost no attention has been paid to this molecular form of CCK. The
lack of data on CCK-39 is due to the absence of specific radioimnunoassays
for CCK-39 and to the fact that CCK-39 cannot be separated from CCK-33 by
conventional separation techniques, such as Sephadex G50 column chromatography.
We have developed a specific and sensitive radioimmunoassay for CCK-39. This
assay enabled us to demonstrate the presence of CCK-39 in porcine brain. Furthermore, the concentrations of CCK-39 in different regions of the brain were
compared with CCK-concentrations obtained with 3 other radioimmunoassays
using antibodies with different specificities.
Methods
Fresh porcine brains were obtained from the local abattoir. Specimens from
different regions of the brain were extracted in boiling water
(1 g/1Oml)
0024-3205/83/080911-03$03.00/0
Copyright (c) 1983 Pergamon Press Ltd.
60
Vol
C h o l ecystokinin-39 in Porcine B r a m
32, No. 8, 1983
for 10 minutes. After homogenïzation and centrifugation the supernatant was
decanted and the pellet was re-extracted in 0.5 M boiling acetic acid (1 g/
10 ml) for another 10 minutes. After centrifugation the supernatant was a d ­
ded to the supernatant of the aqueous extraction, rapidly frozen, lyophilized and redissolved in assay buffer prior to the assay.
The concentrations of CCK in tissue extracts were measured by 4 radioimmunoassays with different specificities.
TABLE 1
CCK39
CCK33
CCK8 sulfated
CCK8 non-sulfated
Ab.1804
Ab.1703
Ab.T204
1.00
<0.001
<0.001
<0.001
1.00
1.12
<0.001
<0.001
1.00
0.89
0.83
<0.001
Ab.5135
1.00
1.09
1.20
1.20
Binding of antibodies 1804, 1703, T204 and 5135
to different molecular forms of cholecystokinin
Antibody 1804 is specific for CCK 39, antibody
1703 for large molecular forms of CCK, antibody
T204 for sulphated forms of CCK, while antibody
5135 binds to all molecular forms of CCK
All antibodies were raised in rabbits; Ab 1703 against 30% pure porcine CCK,
Ab 1804 and Ab T204 against 30% pure porcine CCK coupled to bovine serum alburnin, and Ab 5135 against synthetic non-sulfated CCK-8 coupled to bovine
serum albumin. In the assay using Ab 1804 the label was prepared by coupling
99% pure CCK-39 to 125i by the chloramine T method (11), for Ab 1703 and
Ab T204 by coupling 99% pure CCK-33 to 125i-hydroxyphenylpropiomc acidsuccinimide ester (12), and for Ab 5135 by coupling synthetic non-sulfated
CCK-8 to 125i by the chloramine T method (8,11).99% pure CCK-39 was used as
Standard in all assays. 0.05 M sodium phosphate buffer pH 7.4 containing 80
ymol/1 human serum albumin and 60 umol/1 sodiumethylmercurithiosalicylate was
used as assay buffer. Separation of free and bound label was performed by ad­
dition of an activated charcoal suspension. The 50% inhibition dose for CCK39 (ID50) was 4.5 fmol/ml for Ab 1804, 2.8 fmol/ml for Ab 1703, 3.3 fmol/ml
for Ab T204, and 4.0 fmol/ml for Ab 5135. Intra-assay variations were less
than 10%
Results
TABLE 2
Ab.1804
B rain(n=5)
Cortex
Thalamus
Hypothalamus
Pons
Cerebellum
6.1±1.5
0.2±0.1
0.4±0.1
0.1±0.1
<0.1
Ab.1703
Ab.T204
Ab.5135
11.8±0.5
2.0±0.6
2.6±0.6
1.8+0.6
<0.1
24.1+3.0
2.4±0.8
3.0±0.7
2.1±0.7
<0.1
28.4+5.1
3.3±0.5
3.5±0.5
2.U0.5
<0.1
Concentrations of Cholecystokimn-like ïmnunoreactivity (pmol/g) in aqueous-acid extracts of
porcine brain as measured by radioimmunoassays
using antibodies with different specificities.
Dilution curves of all tissue extracts were paral­
lel to the Standard curves
61
Vol. 32, No. 8, 1983
C holecystokinin-39 in P o r cine Brain
Discussion
By using a sensitive and specific radioimmunoassay for CCK-39 we have shown
for the first time that CCK-39 is present in porcine brain. It was further
shown that the highest concentrations of CCK-39 are found in the cortex. Thus,
the distribution of CCK-39 is not different from other molecular forms of
CCK. In the cortex CCK-39 comprised 20-25% of total CCK-irminoreactivity as
measured by assays specific for the C-terminus (Ab 5135) and for the sulfated
tyrosine region (Ab T204) and 507» of large CCK-iimunoreactivity as measured
by radioimmunoassay using Ab 1703. From this study it cannot be derived
whether CCK-39 plays a physiological role in the brain or that it merely re­
presents a precursor of smaller biologically active forms of CCK. Since the
concentration of CCK in the cortex as measured by Ab 1703 is about twice
that measured by the CCK-39 specific antibody (Ab 1804), it is likely that
in addition to CCK-39 other large molecular forms of CCK are present in the
brain.
Acknowledgments:
This study was supported by the Foundation for Medical Research FUNG0
(grant nr. 13-37-32). Antibody 5135 was a gift of Drs. G. Rosenquist
and J. Walsh.
References
1. V. MUTT
and E. JORPES, Biochem.J.
125 75P-76P (1971).
2. V. MUTT, Clin. Endocrinol. 5 175S-183S (1976).
3. G.J. DOCKRAY, Nature 270 359-361 (1977).
4. J.F. REHFELD, J.Biol.Chem. 253 4022-4030 (1978).
5. J.J. VANDERHAGEN, J.C. SIGNEAU, and W. GEPTS, Nature 257 604-605 (1975).
6. G.J. DOCKRAY, Nature 264 568-570 (1976).
7. E. STRAUS, and R.S. YALOW, Proc.Natl.Acad.Sci. USA 75 486-489 (1978).
8. C.B. LAMERS, J.E. MORLEY, P. POITRAS, B. SHARP, H.E. CARLSON, J.M. HERSFMAN,
and J.H. WALSH, Am.J.Physiol. 239 E232-E235 (1980).
9. F.L. GE0LA, J.M. HERSHMAN, R. WARWICK, J.R. REEVE, J.H. WALSH and
W.W. T0URTELL0TTE. J.Cl in.Endocrinol.Metab. 53 270-275 (1981).
10. G.J. DOCKRAY, R.A. GREG0RY, J.I. HARRIS, J. HUTCHINSON, and M.J. RUNSWICK,
J.Physiol. 280 16P (1978).
11. W.M. HUNTER, F.C. GREENWOOD. Nature J94 495-496 (1972)
12. J.F. REHFELD, J.Biol.Chem. 253 4016-4021 (1978)
62
Chapter VI
Radioirnmunoassay of Cholecystokinin in Human
Tissue and Plasma
J.B.M.J. Jansen
and
C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory
Division of Gastroenterology
St. R a d b o u d H o s p i t a l
University of Nijmegen
Nijmegen
The N e t h e r l a n d s
Clin.
Chim. A c t a
(1983)
131: 306-316 .
63
Chntca Chimica Acta, 131 (1983) 305-316
Elsevier
C C A 2570
Radioimmunoassay of cholecystokinin in human
tissue and plasma
J.B.M.J. Jansen and C.B.H.W. Lamers *
Gaslromleslinal Hormone Laboratory, Dwision of Gastroenterology, St Radboud Hospual, UnwersUy of
Nijmegen, Nijmegen (The Nelherlands)
(Received Septem ber 3rd, 1982, revision M arch 17th, 1983)
Sumniary
A highly sensitive radioimmunoassay for cholecystokinin (CCK) without any
cross-reactivity with gastrin is described. The antibody was raised in a rabbit by
immunisation with 30% CCK and bound to all COOH-terminal CCK-peptides
containing at least 14 amino acid residues. The affinity constant of the antibody was
59.4 X 10'° 1/m ol.
CCK 33 conjugated to [l25I]hydroxyphenylpropionic acid-succinimide ester was
used as label. The binding between label and antibody was inhibited by 50% (ID50)
at a concentration of 2.8 pmol/1 cholecystokinin 33. The detection limit of the assay
was between 0.5 and 1.0 pmol/1 plasma. Concentrations of CCK in aqueous acid
extracts of human upper small intestine were 36.5 ± 9.8 pm ol/g and of human
cerebral cortex 28.2 + 2.5 pm ol/g tissue. Plasma samples were extracted in 96%
ethanol prior to assay. No advantage was obtained by adding aprotinin to the tubes.
When frozen at —20°C plasma CCK was stable for at least 6 months. Basal plasma
CCK concentrations in 30 normal subjects were very low, 0.9 ± 0.1 pmol/1, range
0.5 to 3.1 pmol/1. Intraduodenal administration of fat induced significant increases
in plasma CCK from 1.1 ± 0.1 to 8.2 ± 1.3 pmol/1 ( p = 0.01). Infusion of exogenous
CCK, resulting in plasma CCK levels slightly lower than those measured during
administration of fat, induced pancreatic enzyme secretion and gallbladder contraction. The reliability of this radioimmunoassay for measurements of CCK in human
plasma was extensively evaluated.
Introduction
Even though the first radioimmunoassay of cholecystokinin (CCK) was reported
as long ago as 1969 [1], no specific radioimmunoassays sufficiently sensitive to
* C orrespondence lo Dr. C. Lamers, Division of G astroenterology, St. Radboud Hospital, 6500 HB
Nijm egen, The N etherlands.
0009-8981/83/S 03.00 © 1983 Elsevier Science Publishers B.V.
64
measure CCK concentrations in human plasma have been published. All published
assays either show some cross-reactivity with gastrin or the cross-reactivity especially
with sulphated gastrins was not adequately tested [1 —10]. Furthermore, some radio­
immunoassays of CCK are extremely sensitive to non-specific interference by
plasma factors [5,8]. In fact, basal plasma CCK concentrations varied from less than
0.8 pmol/1 to 1 200 pmol/1 and postprandial plasma CCK levels from 2.0 to 2 275
pmol/1, as measured by the different radioimmunoassays.
We report on a highly sensitive radioimmunoassay for CCK without any cross-reactivity with gastrin. The reliability of this assay for measurements of CCK in
human tissue and plasma has been extensively evaluated.
Materials and methods
Peptides
99% pure porcine CCK 39 and CCK 33 and 30% pure porcine CCK were
purchased from Prof. V Mutt, Karolinska Institute, Stockholm, Sweden; synthetic
CCK-peptides were obtained from Prof. N. Yanaihara, Shizuoka College of
Pharmacy, Oshika, Japan; the Squibb Institute, Princetown, NJ, USA; UCB, Brus­
sels, Belgium; Bachem, Torrance, CA, USA; and Sigma, St. Louis, MO, USA.
Gastrin-peptides were obtained from Prof. M Grossman, Los Angeles, CA, USA
and Imperial Chemical Industries, Cheshire, UK.
Reagents and other materials
[125I]Hydroxyphenylpropionic acid-succinimide ester (Bolton Hunter reagent)
was purchased from New England Nuclear, Boston, MA, USA; Sephadex G50
Superfine from Pharmacia, Uppsala, Sweden; human serum albumin from Behringwerke AG, Marburg am Lahn, FRG; sodium ethylmercurithiosalicylate from
Aldrich-Europe, Beerse, Belgium; dextran T70 and bovine serum albumin (RIAgrade) from Sigma, St. Louis, MO, USA; double antibody solid phase (DASP) from
Organon, Oss, The Netherlands; aprotinin (Trasylol®) from Bayer, Leverkusen,
FRG; Intralipid from Vitrum AB, Stockholm, Sweden; all other reagents from E.
Merck, Darmstadt, FRG.
Apparatus
Automatic gamma scintillation counter Philips PW 4800, Philips, Eindhoven, The
Netherlands.
Standard
99% pure natural pojcine CCK 33 dissolved in assay buffer was used as Standard.
Labelled peptide
CCK 33 was coupled to [12SI]hydroxyphenylpropionic acid-succinimide ester
(Bolton-Hunter reagent) as described before [11].
Antibodies
Ten rabbits and three guinea pigs were immunised with 30% pure porcine CCK
65
coupled to bovine serum albumin, nine rabbits and two guinea pigs with uncoupled
30% CCK, five rabbits with synthetic CCK 10-20 coupled to bovine serum albumin
and four rabbits with sulphated CCK 26-33 coupled to bovine serum albumin. In the
initial immunisations the dose of antigen was 10-20 nmol for CCK and 120 nmol
for CCK-fragments, while at booster injections half of the initial dose was administered. After the fourth immunisation five rabbits immunised with albumin-coupled
CCK had antibody titres between 1 :22 500 and 1 :80000, while one rabbit im­
munised with uncoupled CCK had a titre of 1 :52000. The affinity constants of
these six antibodies ranged from 2.0 X 10'° to 72.3 X 1010 1/mol. In the guinea pigs
immunised with CCK and in the rabbits immunised with CCK-fragments antibody
titres were less than 1 :500. In the present study antibody 1703, obtained from a
rabbit after the third immunisation with uncoupled CCK, was used. This antibody
was used in a dilution of 1: 100000. The affinity constant of the antibody was
59.4 X l O 10 1/mol and the ID50 was 2.8 pmol/1. Antibody 1703 bound to all
COOH-terminal CCK-peptides containing at least 14 amino acid residues and did
not show any binding to gastrins (Table I). The lack of cross-reactivity with gastrin
was validated by measurements of samples with very high concentrations of endogenous human gastrin (extracts of antral and gastrinoma tissue and plasma from
gastrectomised Zollinger-Ellison patients). Results obtained with antibody 1703 were
compared with those obtained with antibody T204. Antibody T204 was harvested
from a rabbit after the fourth immunisation with albumin-coupled CCK and was
used in a dilution of 1 : 80000. The affinity constant of this antibody was 52.2 X 1010
1/mol and the ID50 was 3.3 pmol/1. The antibody was specific for the sulphated
TABLEI
Relative potencies of CCK -peptides and gastnns to antibodies 1703 and T204
C C K 39
C C K 33
C C K 20-33
C C K 22-33
C C K 24-33
C C K 26-33 sulphated
C C K 26-33 unsulphated
C C K 30-33
C C K 1-21
C C K 1-15
C C K 10-20
C C K 16-27
C C K 20-27
gastrin 34 sulphated
gasln n 34 unsulphated
gastrin 17 sulphated
g astn n 17 unsulphated
66
A ntibody
1703
Antibody
T204
0.91
1.00
0 62
<0.01
<0.01
<0.01
<0.01
<001
<001
<0.01
<0.01
0.01
0.01
<0.01
<0.01
<0.01
< 0.01
1.09
1.00
1 69
1.71
0.78
0.65
<0.01
< 0.01
<0.01
<0.01
<0.01
0.01
0.02
0.02
<0.01
0.02
<0.01
tyrosine region in the CCK molecule (Table I). Antibody T204 showed low binding
to sulphated gastrins but did not react with unsulphated gastrins (Table I). The
antibodies were further characterised by measurement of the eluates from a Sephadex G-50 column to which an aqueous-acid extract of human small intestine was
applied. No binding with structurally unrelated polypeptide hormones was found.
Conditions for incubation
The optimum incubation time was determined. Equilibrium and non-equilibrium
systems with delayed addition of labelled peptide were lested. Furthermore, the
optimum pH was determined and interference by different concentrations of sodium
chloride, albumin and urea was assessed.
Separation procedure
Two separation techniques were compared: (1) adsorption of free peptide to
charcoal, and (2) binding of antibody-bound peptide to a second antibody. Different
amounts of charcoal (1 to 128 mg per tube) in a solution containing fixed amounts
of bovine serum albumin and dextran T70 were added to the incubation tubes.
Furthermore, the effect of delaying separation after the addition of the charcoal
suspension for periods of 5 to 60 min were tested. In the double-anlibody separation
technique 1 ml of 1 :20 diluted sheep anti-rabbit immunoglobulin (DASP) was
added to the tube and incubated for 4 to 8 h at 4°C before centrifugation.
Tissue extraction
Gastrinoma and antral tissues were extracted in boiling water, whereas brain and
small intestinal tissues were first extracted in water followed by extraction in acid.
The aqueous-acid extracts were prepared as follows. The tissue was extracted in
boiling water (1 g/10 ml) for 10 min. After homogenisation and centrifugation the
supernatant was decanted and the pellet was re-extracted in 0.5 mol/1 acetic acid (1
g /10 ml) for 10 min. After centrifugation the supernatant was added to the
supernatant of the aqueous extraction, rapidly frezen, lyophilised, dissolved in 0.05
mol/1 sodium phosphate buffer, pH 7.4, and applied to a 100 X 1 cm Sephadex
G-50 Superfine column eluted with 0.05 mol/1 sodium phosphate buffer, pH 7.4,
containing 0.06 mmol/1 sodium ethylmercurithiosalicylate.
Treatment o f blood samples
In order to evaluate the stability of CCK, blood samples were collected in both
glass tubes at room temperature and in ice-chilled glass tubes containing 2 g/1
ethylenediaminetetraacetate (EDTA) and 1000 K U /m l aprotinin (Trasylol®). The
blood samples were either allowed to clot at room temperature or were kept on ice
until centrifugation within 2 h after collecting the samples. Both serum and plasma
samples were frozen at —20°C. Plasma samples without added aprotinin containing
endogenous CCK in concentrations from 1.0 to 19.0 pm ol/l were re-assayed after
storage for 6 months at —20° C.
67
Extractwn o f samples
Since initial experiments showed that hormone-free plasma induced non-specific
interference in the assay, CCK was extracted from plasma by addition of ethanol,
methanol or acetone. The extracts were centrifuged at room temperature at 3000 X g
for 15 min. The supernatant was decanted and dried under nitrogen at 37°C. The
dried product was stored at - 20°C and reconstituted to the original volume in assay
buffer. In order to determine whether ethanol-extracted plasma interfered with the
antigen-antibody binding, Standard curves of CCK 33 were run in assay buffer or in
ethanol-extracted hormone-free plasma. Hormone-free plasma was prepared by the
addition of charcoal to the plasma (20 g/1). Subsequently, the plasma was vigorously
stirred, centrifuged and filtered until no charcoal was visible in the filtrate.
Reliabihty o f the assay
Accuracy. The recovery of known amounts of CCK 33 and sulphated CCK 8
added to hormone-free plasma was determined. In addition, recovery of CCK 33
coupled to [l25I]hydroxyphenylpropionic acid-succinimide ester added to hormonefree plasma was studied. Standard CCK 33 solutions added to hormone-free plasma
bef ore and after ethanol extraction were chromatographed on a 100 X 1 cm Sep­
hadex G-50 Superfine column to exclude conversion of CCK 33 to smaller molecular
forms. Serial dilutions of aqueous-acid extracts of human brain and gut, and of
plasm a containing a high concentration of endogenous CCK were measured and
com pared with the dilution curve of CCK 33 Standard.
Precision. Intra-assay and inter-assay variations were determined by 12-fold
measurements of samples containing between 0.4 to 25 pm ol/1 CCK.
Detection limit. The detection limit was defined as the smallest amount of CCK
which could be differentiated from zero hormone concentration with 95% confidence.
Human studies
Endogenous CCK. Fasting concentrations of CCK were measured in 30 normal
subjects (20 male, 10 female; age 42 + 3 yr, range 21-69 yr). In four normal subjects
(all male, 23 ± 1 yr) the effect of intraduodenal adm inistration of fat (250 ml 20%
Intralipid® over 1 h) on plasma CCK concentrations was studied.
Exogenous CCK. To evaluate whether plasma CCK concentrations comparable
to those measured during administration of fat were able to stimulate pancreatic
enzyme secretion and gallbladder contraction, plasma CCK concentrations and
bilirubin and amylase output into the duodenum were measured during infusion of
12.5 pmol • k g - 1 • h ~ 1 CCK.
68
Statistical analysis
Results were expressed as the mean + 1 SEM, unless otherwise stated. Statistical
analysis was done by Student’s /-test.
Results
Antibody characterisation with endogenous human peptides
Radioimmunoassay using antibody 1703 showed CCK-like immunoreactivity in
brain (28.2 ± 2.5 p m o l/g , n = 3) and upper small intestine (36.5 ± 9.8 p m ol/g,
n = 3). In five of six gastrectomised patients with gastrin-producing tumours (Zollinger-Ellison syndrome) and very high plasma gastrin concentrations (262-50000
pmol/1) plasma CCK concentrations were less than 1 pmol/1, while one such patiënt
had a basal plasma CCK concentration of 1.4 pmol/1. No CCK was demonstrable
in aqueous extracts of two gastrin-producing tumours and five antral extracts with
gastrin concentrations ranging from 10310 to 130475 pmol/1.
Fig. 1 shows the elution profile of an aqueous-acid extract of human small
intestine applied to a Sephadex G-50 column as measured by radioimmunoassay
using antibody 1703 and antibody T204. Radioimmunoassay with antibody 1703
revealed four and with antibody T204 five distinct peaks of CCK-like immunoreac­
tivity. The peak co-eluting with the CCK 33 S tandard and the peak intermediate
between Standard CCK 33 and Standard CCK 8 com prised the majority of the
Fig. 1. Elution profile of an aqueous-acid extract of hum an small intestine applied to a Sephadex G-50
colum n as m easured by radioim m unoassay using antibody 1703 (upper panel) and antibody T204 (lower
panel).
69
CCK-like im m unoreactivity The peak co-eluting with the CCK 8 Standard was only
detectable by radioim m unoassay usm g antibody T204
Incubation conditions
Equilibrium between antibody-bound and free labelled peptide was achieved after
incubation for 96 h However, the most sensitive radioimmunoassay was obtained
when a non-equilibnum system with 72 h pre-incubation followed by 20 h incuba­
tion with labelled peptide was used The optimum pH of the assay buffer was
between 6 and 8 Sodium chloride interfered with the binding between peptide and
antibody when present in concentrations above 220 mmol/1, while interference by
albumin was found when the concentration exceeded 0 33 mmol/1 However, when
album in was present in concentrations of less than 0 04 mmol/1, a marked decrease
in binding between peptide and antibody was observed Urea concentrations exceeding 800 mmol/1 also produced non-specific interference in the assay For routine
measurements of CCK 200 ftl of sample or Standard were incubated with 100 jul
diluted antiserum for 72 h at 4°C After addition of 200 fi\ labelled CCK 33
containing approximately 2000 dpm and incubation for another 20 h at 4°C, free
and antibody-bound peptide were separated 0 05 m o l/l sodium phosphate buffer,
pH 7 4, containing 0 08 m mol/1 human serum albumin and 0 06 mmol/1 sodium
ethylmercunthiosalicylate was used as assay buffer
Separation procedure
Optimal separation between free and antibody-bound peptide was obtained when
between 10 and 32 mg charcoal was added to each tube Delay of centrifugation
after imxing for penods of up to 60 min did not change the equilibrium between free
and bound peptide However, when the mixture was centrifuged within 5 min after
addition of the charcoal suspension, marked differences in the ratio between free
and bound peptide were found Delay of decantation of the supernatant after
centrifugation for penods of up to 60 min did not mterfere with the equilibrium In
Ihe absence of antibody between 2 and 1% of the radioactive label was not absorbed
to the charcoal pellet and thus measured as aspecifically bound radioactivity
Separation by the double antibody solid phase (DASP) techmque was as effective as
the charcoal separation method
In routine assays separation was performed by the addition of 16 mg charcoal, 2 5
mg dextran T70 and 15 mg bovine serum alburrun, dissolved in 1 5 ml 0 05 m o l/l
sodium phosphate buffer, pH 7 4, to each test tube in an ïce-bath The mixture was
centnfuged for aboul 10 mm (3000 X g for 15 min), the supernatant was subsequently decanted, and both the supernatant and the pellet were counted in a gamma
spectrometer
Treatment o f blood samples
Concentrations of CCK were not influenced by the treatment of the blood
samples Sinular results were obtained when blood was collected in ïce-chilled tubes
containing ethylenediaminetetraacetate and aprotimn, or in tubes without additions
at room temperature ( y = 1 06x —0 12, r = 0 983, n = 19) Storage of plasma sam70
pies containing 1.0 to 19.0 pm ol/1 CCK for 6 months at - 2 0 ° C did not affect the
CCK concentration measured (_y = 1.1 lx - 0.14; r = 0.977; n = 13).
For routine purpocp? blood samples were collecled in ice-chilled tubes containing
2 g/1 ethylenediaminetetraacetate. All samples were measured in duplicate.
Extraction procedure
N o advantage was obtained by using methanol or acetone over ethanol. For
routine extractions 2 vols. of 96.% ethanol were added to 1 vol. of plasma or serum.
A fter mixing and centrifugation (3000 X g for 15 min) the supernatant was evaporated
to dryness under nitrogen al 37°C. The dried supernatant was reconstituted in assay
buffer to the original sample volume immediately before the assay. Standard curves
in assay buffer and hormone-free plasma were identical, indicating that extracted
hormone-free plasma did not interfere in the binding between peptide and antibody.
Reliabihty o f the assay
Accuracy. Recovery of CCK 33 added to hormone-free plasma in concentrations
from 1.2 to 20.0 pm ol/1 was 85.4 ± 2.0% (mean + S D ; n = 11). When 0.8 to 43.0
pm ol/1 sulphated CCK 8 was added to hormone-free plasma, recovery was 89.8 ±
1.7% as measured with antibody T204 (n = 7). Recovery of different am ounts of
labelled CCK 33 (1000 to20 0 0 0 d p m ) added to hormone-free plasma was 83.0 ± 1.3%
( n = 12). CCK 33 was not converted to smaller molecular forms as shown by
Fig. 2. Senal dilulions of Standard CC K 33 ( • --------- • ) , of an aqueous-acid extract of hum an small
intestine (□ --------- □), of an aqueous-acid extract of hum an brain ( O ----------O ), and of plasm a containing a high concentration of endogenous CC K ( a --------- a ).
Fig. 3. Intra-assay ( O --------- O ) and inter-assay ( • ---------- • ) vanations of CCK m easurem ents.
71
measurement of immunoreactivity in the eluates of a Sephadex G-50 column to
which CCK 33 in hormone-free plasma before and after ethanol extraction was
applied (data not shown). Senal dilutions of an aqueous-acid extract of human bram
and upper small ïntestme and of plasma containing high concentrations of endogenous CCK were superimposable on the Standard curve (Fig 2)
Precision
Intra-assay and inter-assay precision are presented in Fig 3
Detection limit The delection limit of the assay was approximately 0 5 pmol
CCK per 1 plasma when a freshly prepared CCK label was used and about 1.0
p m o l/l when a CCK label, prepared more than 8 weeks before the assay, was used.
This reduction in sensitivity was due to a change in the slope of the dose-response
curvc.
Human studies
Fasting plasma CCK concentrations in 30 normal subjects were 0 9 ± 0 1 p m o l/l
with a range of 0.5 to 3 1 p m o l/l (Fig 4). In the majonty of subjects basal plasma
CCK concentrations were less than 1 p m o l/l. Sirrular results were obtained when the
samples were measured with antibody T204 after correction for the slight cross-reactivity of thjs antibody with sulphated gastnn. There was no significant difference in
plasma CCK concentrations between men and women Furthermore, no relation
between basal plasma CCK concentration and age was observed Intraduodenal
instillation of fat induced significant increases in plasma CCK from 1 1 ± 0 1 to
8.2 ± 1.3 p m o l/l ( p = 0 01, Fig 5) The increase in plasma CCK as measured with
antibody T204 was similar to that measured using antibody 1703 (Fig. 5). Infusion
of exogenous CCK in a dose of 12 5 pmol kg - 1 • h " 1 resulted in increases in plasma
CCK to levels of about 6 p m o l/l (Fig. 6). This increase in plasma CCK was
accompanied by an increase in pancreatic amylase secretion from 9 to 32 K U /1 5
min and an increase in duodenal bilirubin to 18 m m ol/15 min indicating gallbladder
contraction (Fig. 6).
N
A b 1703
25
20
norm al
subjects
(n * 3 0 )
<■
15
lO
5
ry,
-----------
"...
0
1
2
3
4
( pmol /l )
Fig 4 Fasting plasma CCK concentrations in 30 norm al subjects
72
CCK LI (pmol/ 1 )
---------------
C C K - LI
(p m o l/ l)
bihnjbm mmol/15 rmn
15
arnykise kU/15mm
30 t
60
90
T im e (m m )
15
30
45 60
mmutes
Fig. 5 The effect of intraduodenal adm irustration of fat on plasma CC K concentrations m 4 norm al
subjects as m easured by radioimm unoassay using antibody 1703 (upper panel) and antibody T204 (lower
panel). A stensks indicate significant ( p < 0 05) differenceS from basal value
Fig 6 The effect of infusion of exogenous C C K on plasm a C C K concentrations, and on am ylase and
bihrubin outputs into the duodenum .
Discussion
The present study describes a highly sensitive radioimmunoassay for CCK in
human plasma without any cross-reactivity with gastrin. The absence of cross-reactivity with gastrin was substantiated by measurements of extracts of hum an antral
and gastrinoma tissues and of plasma from gastrectomised patients with
Zollinger-Ellison syndrome, all containing very large amounts of gastrin. By column
chromatography of an aqueous-acid extract of human small intestine it was shown
that the antibody bound to all molecular forms of CCK larger than CCK 8. Since all
four molecular forms of CCK measured by the assay also reacted with an antibody
specific for the sulphated tyrosine region in CCK (T204), it can be concluded that all
these components contain the sulphated tyrosine region and consequently possess
biological activity. Using this assay it was found that basal plasma CCK concentra-
73
tions are very low. It is interesting to note that secretin, another hormone rcleased
from the upper small intestine with stimulatory actions on the pancreas, circulales in
similarly low concentrations [12]. Basal plasma concentrations of other gastrointestinal hormones, e.g. gastrin, pancreatic polypeptide, glucagon, insulin, and gastric
inhibitory polypeptide, are much higher than those of CCK and secretin. The
finding ihat plasma CCK concentrations are very low compared to those in most
previous reports is probably attributable to the absence of cross-reactivity with
gastrin and to the lack of non-specific interference in the present assay. The low
concentrations of CCK are not due to the absence of cross-reactivity with CCK 8,
since similar results were obtained by radioimmunoassay using an antibody (T204)
binding to all molecular forms of CCK containing the sulphated tyrosine region. In
two recent studies in which CCK was partly separated from gastrin by column
chromatography prior to measurement by a CCK-assay highly cross-reacting with
gastrin, it was found that both CCK 8 and larger molecular forms of CCK were
undetectable in basal plasma [9,10]. In another recent study it was found that the
mean basal plasma concentration of CCK in man was 12 pm ol/1 [8]. However,
chromatography and charcoal treatment showed that this apparent CCK-like immunoreactivity was almost exclusively due to non-specific interference in that assay
[8]. The finding that plasma CCK concentrations in man are so low compared to
those of gastrin makes the application of radioimmunoassays for CCK cross-reacting
with gastrin undesirable. Several methods have been employed in an attem pt to
separate CCK from gastrin in order to overcome the problem of interference of
gastrin in the CCK assay, such as column chromatography [9,10], saturation of the
antibodies with gastrin [6] or extraction of the sample in ethanol [6]. However, none
o f these methods have been proven to be successful in separating larger forms of
C C K from gastrin. Furtherm ore, the suggestion that gastrin is poorly extracted in
96% ethanol could not be confirmed in our laboratory. The cross-reactivity of
antibodies with sulphated gastrin 17 and non-sulphated and sulphated gastrin 34 has
been reported in only a few studies [9,10]. This is the more troublesome because
antibodies specific for the sulphated tyrosine region in CCK usually do not cross-react with unsulphated gastrins but often do with sulphated gastrins and because
gastrin 34 is the predom inant molecular form of gastrin in fasting plasma [13].
It was further shown that intraduodenal adm inistration of fat, a potent stimulus
of pancreatic enzyme secretion and gallbladder contraction, resulted in significant
increases in plasma CCK concentrations. Since the assay measures all molecular
forms of CCK larger than CCK 8, it is evident that the increase in plasma CCK has
to be atlributed to larger or intermediate forms of CCK. Increases in CCK measured
by an antibody specific for the sulphated tyrosin region in CCK and therefore
cross-reacting with CCK 8 (T204) were identical, indicating that the majority of
CCK in the circulation is larger than CCK 8. This finding, although contrasting with
some previous studies [8-10], is not unexpected since smaller gastrin-CCK like
peptides are degradated by passage through the liver [14]. It has previously been
suggested that CCK 4 is a major molecular form of CCK in tissue and possibly in
blood [15]. Since our assays do not cross-react with CCK 4, we are unable to confirm
o r to reject this suggestion. However, CCK 4 is very unlikely to play a hormonal role
74
in the stimulation of pancreatic enzyme secretion and gallbladder contraction, since
CCK 4 is effectively inactivated by the liver [14].
The finding that infusion of exogenous CCK, resulting in slightly lower plasma
CCK concentrations as during administration of fat, induces pancreatic enzyme
secretion and gallbladder contraction indicates that CCK may play a physiological
role in the regulation of pancreatic secretion and gallbladder contraction in man.
In conclusion, using a highly sensitive and specific radioimmunoassay for CCK it
was shown that basal plasma CCK concentrations are very low, that ïntraduodenal
administration of fat induces increases in plasma CCK and that CCK may play a
physiological role in the regulation of pancreatic enzyme secretion and gallbladder
contraction in man.
Acknowledgement
This study was supported by the Foundation for Medical Research FU N G O
(G rant No. 13-37-32).
References
1 Y oung JD . Lazarus L, Chisholm DJ Radioim m unoassay of pancreozym in cholecyslokmin in hum an
serum. J Nucl M ed 1969, 10 743-745.
2 Reeder D D , Becker H D , Smith NJ, Rayford PL, T hom pson JC M easurem ent of endogenous release
o f cholecystokinin by radioim m unoassay Ann Surg 1973. 178 304-310.
3 Harvey R F, D owsett L, H arlog M, Read AE. Radioim m unoassay of cholecystokimn-pancreozym in
G ut 1974; 15: 690-699.
4 Schlegel W, R aptis S, G rube D, Pfeiffer EF. E stim adon of cholecystokm in-pancreozym in (C C K ) in
hum an plasm a and tissue by a specific radioimm unoassay and the immunohistochem ical Id e n tific a tio n
of pancreozym in-producing cells m the duodenum Clin Chim Acta 1977, 80. 305-316
5 Burhol PG, Rayford PL, Jorde R, W aldum HL, Schulz TB. T hom pson JC. R adioim m unoassay of
plasm a cholecystokinin, duodenal release of CCK, dium al vanation of plasm a CCK. and im­
m unoreactive plasm a C C K com ponenls in man. H epato-G astroenterology 1980, 27. 300-309
6 Byrnes DJ, H enderson L, Borody T, Rehfeld JF. Radioim m unoassay of cholecystokinin in hum an
plasma. Clin Chim Acta 1981; 111: 81-89
7 W iener I, Inoue K, Fagan C, Lilja P, W atson L, Thom pson J. Release of cholecystokinin in m an Ann
Surg 1981; 194: 321-327.
8 Burhol PG, Jensen T G , Lygren I, Schullz TB, Jorde R, W aldum HL. Iodination with lodo-gen and
radioim m unoassay of cholecystokinin in acidified plasm a, C C K release, and molecular CC K components in man. Digestion 1982, 23: 156-168.
9 Calam J, Ellis A, D ockray G J. Identification and m easurem ent of molecular variants of cholecys­
tokinin in duodenal mucosa and plasma. J Clin Invest 1982, 69. 218-225
10 W alsh JH , Lam ers CB, Valenzuela JE. Cholecystokinin-octapeptide im m unoreactivity in hum an
plasm a. G astroenterology 1982; 82. 438-444
11 Jansen JBMJ, Lam ers CBHW Low binding of Bolton-Hunter-labelled cholecystokjnin-33 to
carboxyl-term inal CCK-antibodies. J Im munol M ethods 1982, 51: 223-230
12 Schaffalitzky de M uckadell OB, Fahrenkrug J. Secretion pattern of secretin in man- regulation by
g astnc acid. G ut 1978, 19. 812-818
13 Lamers CB, W alsh JH , Jansen JB, H am son AR, Ippoliti A F, Van Tongeren JH . Evidence that gastrin
34 is preferentially released from the hum an duodenum . G astroenterology 1982, 83. 233-239.
14 Strunz U T, Thom pson M R, Elashoff J, G rossm an M l. H epatic inactivation o f gastnns of vanous
chain lengths in dogs. G astroenterology 1978, 74 550-554.
15 Rehfeld JF T etn n . In: Bloom SR, Polak JM , eds. G u t horm ones. London' Churchill Livingstone
1981. 240-247.
75
Chapter VII
Effect of Insulin-Hypoglycaemia on Plasma
Cholecystokinin and Pancreatic Polypeptide
J.B.M.J. Jansen
and
C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory
Division of Gastroenterology
St. R a d b o u d H o s p i t a l
JJniversity o f N i j m e g e n
Nijmegen
The N e t h e r l a n d s
Soand.
J. G a s t r o e n t e r o l . (1983)
82: 21 9-221.
77
VII EFFECT OF INSULIN-HYPOGLYCAEMIA ON PLASMA CHOLECYSTOKININ
AND PANCREATIC POLYPEPTIDE
Introduction
P a n c r e a t i c e n z y m e s e c r e t i o n d u r i n g a m e a l is at least p a r t l y
m e d i a t e d by v a g a l - c h o l i n e r g i c m e c h a n i s m s
(1). T h i s v a g a l - c h o l i n e r -
gic s t i m u l a t i o n of p a n c r e a t i c e n z y m e s e c r e t i o n m a y be e i t h e r d i ­
rect o r i n d i r e c t t h r o u g h the r e l e a s e of c h o l e c y s t o k i n i n
(CCK)
f r o m the u p p e r s m a l l i n t e s t i n a l m u c o s a . Th e l a t t e r m e c h a n i s m is
s u p p o r t e d by the r e c e n t f i n d i n g t h a t c h o l e c y s t o k i n i n r e l e a s e d u ­
r i n g f e e d i n g is i n h i b i t e d by atropine (2). T h e p r e s e n t s t udy was
u n d e r t a k e n to d e t e r m i n e w h e t h e r a c t i v a t i o n of v a g a l - c h o l i n e r g i c
m e c h a n i s m s by i n s u l i n - h y p o g l y c a e m i a i n d u c e s the r e l e a s e of CC K
into t he c i r c u l a t i o n of man.
Subjects and methods
A f t e r an o v e r n i g h t f a s t an i n t r a v e n o u s b o l u s of 0.1 U / k g
Insulin Actrapid was administered to 5 h ealthy volunteers
2 F ; a g e 29-61 y r ) . P l a s m a CCK, p a n c r e a t i c p o l y p e p t i d e
(3M,
(PP) an d
g l u c o s e w e r e m e a s u r e d a t -10, 0, 10, 20, 30, 40, 50, 60, 70, 80
a n d 90 min. P l a s m a C C K w a s m e a s u r e d b y r a d i o i m m u n o a s s a y u s i n g
two antibodies with different s p e c i f i c i t i e s . Cross-reactivity
s t u d i e s w i t h p u r i f i e d p o r c i n e C C K p r e p a r a t i o n s , C C K fragments,
a n d i n t e s t i n a l a n d c e r e b r a l e x t r a c t s s h o w e d t h a t a n t i b o d y 1703
b i n d s to c o m p o n e n t I-CCK, CCK-39, C C K - 3 3 a n d i n t e r m e d i a t e CCK,
w h i l e a n t i b o d y T 204 , d i r e c t e d to the s u l p h a t e d t y r o s v l region,
b i n d s a l s o to s u l p h a t e d C C K - 8
p o r c i n e C C K - 3 3 c o n j u g a t e d to
(fig 1). In the a s s a y 99% pure
1 25
1-hydroxyphenylpropionic acid
s u c c i n i m i d e e s t e r w a s u s e d as label a n d 99% p u r e p o r c i n e CC K - 3 3
as S t a n d a r d .
I n t r a - a s s a y p r e c i s i o n w a s less t h a n 8% a n d inter-
a s s a y p r e c i s i o n less t h a n 15%. T he d e t e c t i o n lim i t of the a s s a y
wa s a b o u t 0.5 p m o l / 1 p l a s m a . The a s s a y s for C C K w e r e f u r t h e r v a l i d a t e d b y the d e m o n s t r a t i o n of i n c r e a s e s in p l a s m a C C K a f t e r
o r a l i n g e s t i o n of f at o r a m i x e d meal, d u r i n g i n t r a d u o d e n a l adm i n i s t r a t i o n of fat, a n d d u r i n g i n f u s i o n of b o m b e s i n
(3). P l a s ­
m a PP w a s m e a s u r e d b y r a d i o i m m u n o a s s a y s as d e s c r i b e d p r e v i o u s l y
78
(4).
pm o l /1
Fig 1 C ro ss-re a ctiv ity o f a n tib o d y T 204 a n d 1703 w ith p u n fic d p o rcin e C C K p re p a ra tio n s , C C K -fra g m e n ts a n d
g a s tn n .
79
glucose (mmol/l)
Figure 2
E f f e c t of ï n s u l i n h y p o g l y c a e m i a on
plasma glucose, p l a s ­
ma C C K a n d p l a s m a PP
in 5 n o r m a l s u b j e c t s .
p la sm a C C K -L I (p m o l/l) ■
5 -i
3 -
2
-
A b 1703
1
-
I
I
0
80
"T
1I
I
30
I
I
I
60
I
I
1
"
9 0 m in u te s
Re s u l t s
As s h o w n in f ig 2 i n j e c t i o n o f 0.1 U / k g i n s u l i n i n d u c e d a
s i g n i f i c a n t d e c r e a s e in p l a s m a g l u c o s e an d a s i g n i f i c a n t r i s e in
p l a s m a PP. H o w e v e r , p l a s m a C C K c o n c e n t r a t i o n s did no t c h a n g e during insulin-hyp o g l y c a e m i a .
Discussion
The p r e s e n t s t u d y s h o w s that p l a s m a C C K c o n c e n t r a t i o n s in
m a n are v e r y low. T h i s is in a g r e e m e n t w i t h a r e c e n t s t u d y show i n g that the a p p a r e n t C C K - l i k e i m m u n o r e a c t i v i t y in b a s a l h u m a n
p l a s m a of 12 p m o l / l w a s a l m o s t e x c l u s i v e l y due to n o n - s p e c i f i c
i n t e r f e r e n c e in t h a t a s s a y
(5). The s l i g h t l y h i g h e r c o n c e n t r a ­
t i on s m e a s u r e d w i t h antibocjy
coinP a r e d w i t h the l e v e l s ob-
^204
t a i n e d w i t h a n t i b o d y 1 703 are p r o b a b l y a t t n b u t a b l e to a h i g h e r
s e n s i t i v i t y to n o n - s p e c i f i c f a c t o r s of a n t i b o d y
anc^ no t to
^204
the p r e s e n c e of s u l p h a t e d C C K - 8 in p l asma. R e c e n t s t u d i e s h a v e
s h ow n t h a t the a m o u n t of C C K - 8 m
b a s a l p l a s m a is n e g l i g i b l e
(6,7). I n s u l i n - h y p o g l y c a e m i a d i d n o t ind u c e i n c r e a s e s in p l a s m a
CCK. On t he o t h e r hand, p l a s m a PP c o n c e n t r a t i o n s w e r e s i g m f i c a n t l y i n c r e a s e d d u r i n g i n s u l i n - h y p o g l y c a e m i a . T h i s i n c r e a s e in
p l a s m a PP in the a b s e n c e of a r i s e in p l a s m a C C K is n o t e w o r t h y ,
since PP is s u g g e s t e d to c o u n t e r a c t the e f f e c t s of C C K on p a n ­
c r e a s a n d g a l l b l a d d e r , w h i l e C C K is p r o b a b l y i n v o l v e d m
the
p o s t p r a n d i a l PP r e l e a s e .
REFERENCES
1.
H A R P E R A A , S C R A T C H E R D T. I n : T h e e c o c r i n e
H S A R L E S , Eds) W B S A U N D E R S , L o n d o n , 1979,
2.
MATON
1125.
3.
JANSEN
JBMJ,
LAMERS
CBHW.
4.
LAMERS
CBHW,
DIEMEL
J M , JANSEN
5.
B U R H O L PG, J E N S E N TG, L Y G R E N
D i g e s t i o n 198 2 , 23, 156.
6.
WALSH
438 .
JH,
7.
CALAM
J,
PN,
SELDEN
LAMERS
ELLIS
A,
AC,
CHADWICK
VS.
Gastroentero 1ogy
Gastroenterology
I,
C B , VALENZUELA
DOCKRAY
pancreas.
p 80.
GJ. J
JBMJ.
Surgery
SCHULZ
JE.
Clin
1982,
TB,
82,
1093.
91,
R,
1982,
288.
WALDUM
HL.
1982,
82,
Gastr o e n t e r o 1ogy
Invest
HOWAT,
1982 , 82 ,
1982,
GORDE
(HT
69,
218.
81
Chapter VIII
Effect of Bombesin on Plasma Cholecystokinin in
Normal Subjects and Gastrectomized Patients
Measured by Sequence Specific Radioimmunoassays
Jan B.M.J. Jansen
and
Cornelis B.H.W, Lamers
Gastrointestinal Hormone Laboratory
D i v i s i o n of G a s t r o e n t e r o l o g y
St. R a d b o u d H o s p i t a l
U n i v e r s i t y of N i j m e g e n
Nijmegen
The N e t h e r l a n d s
Surgery:
aaaepted for publiaation
83
VIII
E F F E C T O F B O M B E S I N O N P L A S M A C H O L E C Y S T O K I N I N IN
NORMAL SUBJECTS AND GASTRECTOMIZED PATIENTS
M E A SURED BY SEQUENCE SPECIFIC RADIOIMMUNOASSAYS
Abstract
S i n c e b o m b e s i n is k n o w n to s t i m u l a t e p a n c r e a t i c e n z y m e s e ­
cretion and gallbladder contraction, we have measured plasma
cholecystokinin
(CCK) c o n c e n t r a t i o n s d u r i n g b o m b e s i n i n f u s i o n
u s i n g s e q u e n c e - s p e c i f i c r a d i o i m m u n o a s s a y s . Antibody 1703 b i n d s
to C O O H - t e r m i n a l C C K - p e p t i d e s c o n t a i n i n g at l e a s t 14 a m i n o a c i d
residues, while antibody
^204
•'■
s s p e c i f i c for the s u l p h a t e d t y ­
r o s y l r e g i o n of CCK. In 9 n o r m a l s u b j e c t s i n f u s i o n of i n c r e a s i n g
doses of bombesin
(2.4 p m o l / k g . 2 0 min;
k g . 2 0 m i n a n d 60 p m o l / k g . 2 0 min)
plasma CCK-responses
(58.5 + 9.7; 70.5 + 12.2;
101 . 4 + 15.4 p m o l / 1 . 20 m i n
+ 13.4;
6 p m o l / k g . 2 0 min;
18 p m o l /
induced dose-related integrated
(an t i b o d y 1703)
79.5 + 11.1 and
a n d 50.4 + 11.9; 62.0
74.7 + 9.2 a n d 116.1 + 11.3 p m o l / 1 . 20 m i n
(antibody T 2 0 4 ^ -
I n f u s i o n o f 60 p m o l / k g . 2 0 m i n b o m b e s i n in 8 p a t i e n t s w i t h p a r t i a l
g a s t r e c t o m y r e s u l t e d in s i m i l a r i n c r e a s e s in p l a s m a C C K (8.1 +
1.8 p m o l / 1 ,
a n t i b o d y 1703;
8 normal control subjects
9.5 + 2.0 p m o l / 1 , a n t i b o d y T 2 0 4 ) as in
(6.5 + 0.9 p m o l / 1 , a n t i b o d y 1703, 8.8
+ 1.0 D m o l / 1 , a n t i b o d y T 2 0 4 ^ • D u r in 9 i n f u s i o n of b o m b e s i n p l a s m a
g a s t r i n i n c r e a s e d f r o m 16.7 + 1.4 to 4 9.6 + 8.1 p m o l / 1
(p<0.005)
in n o r m a l s u b j e c t s , w h i l e t h e r e w a s no s i g n i f i c a n t cha n g e in p l a s ­
ma
g a s t r i n in g a s t r e c t o m i z e d p a t i e n t s .
In the 3 p a t i e n t s w i t h
p a r t i a l g a s t r e c t o m y s t u d i e d , b o m b e s i n d i d n o t s i g n i f i c a n t l y inf luence gastric acid secretion.
It is c o n c l u d e d t h a t i n f u s i o n o f b o m b e s i n r e l e a s e s C C K in
ma n by a gastrin-independent mechanism.
Introduction
Bombesin, a tetradecapeptide originally isolated from amphib i a n skin, p o s s e s s e s p o w e r f u l l s t i m u l a t o r y a c t i o n s on s e veral
gastrointestinal functions
(1). R e c e n t l y , a m a m m a l i a n 27 a m i n o
acid polypeptide sharing structural and functional similarities
with bombesin
(gastrin r e l e a s i n g D e p t i de)
th e g a s t r o i n t e s t i n a l t r a c t
84
ha s b e e n i s o l a t e d f r o m
(18). B o m b e s i n - l i k e i m m u n o r e a c t i v i t y
ha s
b e e n d e m o n s t r a t e d in the g a s t r o i n t e s t i n a l t r a c t o f m a n a n d it
h a s b e e n s u g g e s t e d t h a t b o m b e s i n - l i k e p e p t i d e s m a y be i m p l i c a t e d
in the p h y s i o l o g i c a l r e g u l a t i o n of g a s t r o i n t e s t i n a l f u n c t i o n s
(4,
21). S i n c e b o m b e s i n - l i k e p e p t i d e s a p p e a r to be c o n f i n e d to n e r v e s
(7) , it is l i k e l y t h a t t h e s e s u b s t a n c e s m a y h a v e a n e u r o t r a n s m i t ­
t e r r a t h e r t h a n an e n d o c r i n e f u n c t i o n .
Several studies suggest
that b o m b e s i n - l i k e p e p t i d e s are i m p l i c a t e d in the p h y s i o l o g i c a l
r e g u l a t i o n o f g a s t r i n s e c r e t i o n f r o m the a n t r u m
(10,23,25). O n e of
the m o s t s t r i k i n g p r o p e r t i e s o f b o m b e s i n is its a b i l i t y to s t i m u late p a n c r e a t i c e n z y m e s e c r e t i o n a n d g a l l b l a d d e r c o n t r a c t i o n in ma n
(2). B a s e d o n e x p e r i m e n t s u s i n g a n t r e c t o m y an d e n t e r e c t o m y in dogs
it w a s s u g g e s t e d t h a t b o m b e s i n s t i m u l a t e s p a n c r e a t i c e m z y m e s e c r e ­
t i o n b y r e l e a s i n g C C K f r o m the u p p e r s m a l l i n t e s t i n e
(14). Howe v e r ,
the p r e s e n c e of s p e c i f i c r e c e p t o r s for b o m b e s i n on the a c i n a r c e lls
s u g g e s t s a d i r e c t a c t i o n of b o m b e s i n o n the p a n c r e a s
more,
(6). F u r t h e r -
the l o c a l i z a t i o n of b o m b e s i n i n n e r v e s m a y be s u g g e s t i v e of
a rol e for b o m b e s i n in a c t i v a t i n g a n e n t e r o p a n c r e a t i c r e f l e x
(16).
U s i n g s e n s i t i v e s e q u e n c e - s p e c i f i c radioiirununoassays for CCK, w e
w e r e a b l e to d e m o n s t r a t e t h a t b o m b e s i n r e l e a s e s C C K in man. T o exc l u d e the p o s s i b i l i t y t h a t t he i n c r e a s e in p l a s m a C C K w a s s e c o n d a r y to g a s t r i n - s t i m u l a t e d g a s t r i c a c i d s e c r e t i o n in r e s p o n s e to
b o mbes i n , b o t h p l a s m a C C K a n d p l a s m a g a s t r i n w e r e m e a s u r e d d u r i n g
i n f u s i o n o f b o m b e s i n in p a t i e n t s w i t h p a r t i a l g a s t r e c t o m y .
Subjects and methods
A f t e r a n o v e r n i g h t f a s t i n c r e a s i n g d o s e s of b o m b e s i n
6; 18; 60 p m o l/kg) w e r e a d m i n i s t e r e d to 9 n o r m a l s u b j e c t s
(2.4;
(8M,
1F, ag e 22-54 y e a r s ) . T h e d o s e s of b o m b e s i n w e r e i n f u s e d d u r i n g
20 m i n u t e
p e r i o d s s e p a r a t e d f r o m e a c h o t h e r b y i n f u s i o n of s a l i n e
for 4 5 m i n u t e s . B l o o d s a m p l e s f or m e a s u r e m e n t of C C K w e r e o b t a i n e d
at -5, 0, 5, 10, 15 a n d 20 m i n u t e s d u r i n g e a c h d o s e of b o m b e s i n .
F u r t h e r m o r e , t h e p l a s m a C C K a n d g a s t r i n r e s p o n s e s to 60 pmol / k g .
20 m i n b o m b e s i n i n 8 p a t i e n t s w i t h p a r t i a l g a s t r e c t o m y
(6M, 2F,
age 35-68 years) w e r e c o m p a r e d to t h o s e o b t a i n e d in 8 n o r m a l s u b ­
jects
(5M, 2F, age 36-69 y e a r s ) . F i v e p a t i e n t s h a d B i l l r o t h - I I
a n a s t o m o s i s a n d 3 B i l l r o t h - I a n a s t o m o s i s . B l o o d s a m p l e s for m e a s u ­
r e m e n t of C C K a n d g a s t r i n w e r e o b t a i n e d at -5, 0, 5, 10, 15 a n d 20
85
minutes.
In 3 p a t i e n t s w i t h p a r t i a l g a s t r e c t o m y an d R o u x - Y ana-
stomosis
(3M, age 47- 5 4 years)
g a s t r i c a c i d s e c r e t i o n wa s m e a s u ­
r e d d u r i n g i n f u s i o n of s a l i n e f or 1 h o u r f o l l o w e d by i n f u s i o n of
180 p m o l / k g b o m b e s i n f o r a n o t h e r hour.
Plasma CCK concentrations were measured by 2 sequence-spec i f i c r a d i o i m m u n o a s s a y s e m p l o y i n g a n t i b o d y 1703 and a n t i b o d y
^204
• A n t i b o d y 1703 b i n d s to all C O O H - t e r m i n a l C C K - p e p t i d e s
c o n t a i n i n g at lea s t 14 a m i n o a c i d r e s i d u e s and d o e s not s h o w a n y
cross-reactivity with gastrin
(table 1). A n t i b o d y ^204 b i n|3s to
all C C K - p e p t i d e s c o n t a i n i n g the s u l p h a t e d t y r o s y l region. This
a n t i b o d y s h o w s l o w b i n d i n g to s u l p h a t e d g a s t r i n s b u t it d o e s n o t
b i n d to u n s u l p h a t e d g a s t r i n s
(table 1). T h e a n t i b o d i e s do not
b i n d to s t r u c t u r a l l y u n r e l a t e d r e g u l a t o r y p e p t i d e s , i n c l u d i n g
insulin, glucagon, pancreatic polypeptide,
somatostatin,
secretin,
gastric inhibitory polypeptide, vasoactive intestinal polypeptide,
n e u r o t e n s i n or b o m b e s i n . M e a s u r e m e n t of the e l u a t e of a S e p h a d e x
G50 SF c o l u m n to w h i c h an a q u e o u s - a c i d e x t r a c t of h u m a n u p p e r small
i n t e s t i n e w a s a p p l i e d s h o w e d t h a t b o t h a n t i b o d i e s b i n d to c o m p o ­
n e n t I - CCK, C C K 33, C C K 39, a n d i n t e r m e d i a t e f o r m s of CCK, w h i l e
a n t i b o d y T 2 q^ b i n d s a l s o to C C K 8 (12) . C C K 33 c o u p l e d to 1 2 5 Ihydroxyphenylpropionic acid-succinimide ester
gent) w a s u s e d as l a b e l
*
Standard.
(Bolton-Hunter r e a ­
(11). 99% p u r e p o r c i n e C C K 33 w a s u s e d as
0.05 m o l / 1 s o d i u m p h o s p h a t e b u f f e r p H 7.4 c o n t a i n i n g
0.08 m m o l / 1 h u m a n s e r u m a l b u m i n a n d 0.06 m m o l / 1 s o d i u m e t h y l m e r c u r i t h i o s a l i c y l a t e w a s u s e d as a s s a y buffer. A n o n - e q u i l i b r i u m syst e m w a s u s e d w i t h 72 h o u r p r e i n c u b a t i o n f o l l o w e d b y 24 h o u r s i n c u ­
b a t i o n after addition of the labelled peptide. Separation b etween
free a n d a n t i b o d y b o u n d h o r m o n e w a s p e r f o r m e d b y a d s o r p t i o n of the
free p e p t i d e to p l a s m a - c o a t e d c h a r c o a l . T h e 50% i n h i b i t i o n d o s e
(ID^q) w a s 2.8 p m o l / 1 f o r a n t i b o d y 1703 an d 3.3 p m o l / 1 for a n t i ­
b o d y ^204' p la s m a s a m p l e s w e r e e x t r a c t e d in 96% ethanol, d r i e d und e r n i t r o g e n at 37 °C a n d r e c o n s t i t u t e d in a s s a y b u f f e r to t h e ori g i n a l v o l u m e b e f o r e t he assay. R e c o v e r y of C C K 33 an d s u l p h a t e d
C C K 8 a d d e d t o h o r m o n e f r e e p l a s m a w a s 85.4 + 2.0%
+ 1.7%
(n=ll)
an d 89.8
( n = 7 ) , r e s p e c t i v e l y . T h e d e t e c t i o n l i m i t of b o t h a s s a y s w a s
a b o u t 0.5 p m o l / 1 p l a s m a . T h e i n t r a - a s s a y v a r i a t i o n w a s b e t w e e n 4.6
86
and 11.5%, a n d the i n t e r - a s s a y v a r i a t i o n r a n g e d f r o m 11.3 to 26.1%.
D i l u t i o n c u r v e s of p l a s m a s a m p l e s c o n t a i n i n g a h i g h c o n c e n t r a t i o n
of e n d o g e n o u s C C K w e r e p a r a l l e l t o t he S t a n d a r d curve. P l a s m a
g a s t r i n c o n c e n t r a t i o n s w e r e m e a s u r e d b y r a d i o i m m u n o a s s a y as p r e viously described
(15). S y n t h e t i c b o m b e s i n - 1 4 w a s p u r c h a s e d f r o m
B o e h r i n g e r M a n n h e i m B i o c h e m i c a l s , I n d i a n a p o l i s , Indiana.
R e s u l t s w e r e e x p r e s s e d as the m e a n + SEM. Th e i n t e g r a t e d p l a s ­
m a C C K s e c r e t i o n d u r i n g i n f u s i o n o f b o m b e s i n wa s d e t e r m i n e d b y
c a l c u l a t i n g the a r e a u n d e r the c u r v e a f t e r s u b t r a c t i o n of b a s a l
value. S t a t i s t i c a l a n a l y s i s w a s d o n e b y S t u d e n t ' s t - t e s t for paired and u n p a i r e d r e s u l t s .
I n f o r m e d .consent w a s o b t a i n e d f r o m all
s u b j e c t s studied.
Table 1
R e l a t i v e b i n d i n g of C C K - p e p t i d e s a n d g a s t n n s
to a n t i b o d y 1703 a n d a n t i b o d y T 2 04
antibody
C C K 39
C C K 33
CCK 20-33
CCK 22-33
CCK 24-33
CCK 26-33
sulphated
CCK 26-33 unsul p h a t e d
CCK 30-33
CCK
1-2 1
CCK
1-15
CCK 10-20
CCK 16-27
CCK 20-27
Gast rin34
sulphated
G a s t n n 3 4 unsulphated
G a s t r i n 17
sulphated
G a s t r i n 17 un s u l p h a t e d
1703
0.
.9 1
1,
.00
0,
. 62
< 0 .0 1
<0 ,
.0 1
<0 ,
.0 1
<0..01
< 0 . 01
<0 .
. 01
< 0 .0 1
< 0 .0 1
0 ,
.0 1
0 .0 1
<0 .0 1
<0 .0 1
<0 .
,0 1
<0 .
,0 1
antibody
T 2Q4
1 .09
1 .00
1 .6 9
1 .7 1
0 .7 8
0,
.65
<0 .0 1
<0 ,
.0 1
<0 ,
.0 1
. 01
<0 .
< 0 .0 1
0 .0 1
. 02
0.
0.
,0 2
<0 .
.01
0.
,02
,01
<0 .
R e sul t s
I n f u s i o n of i n c r e a s i n g d o s e s o f b o m b e s i n into 9 n o r m a l s u b ­
jects i n d u c e d d o s e - r e l a t e d i n c r e a s e s of p l a s m a C C K
(fig 1). Th e
i n t e g r a t e d p l a s m a C C K r e s p o n s e t o i n f u s i o n of 18 p m o l / k g . 2 0 m i n
a n d 60 p m o l / k g . 2 0 m i n b o m b e s i n w a s s i g n i f i c a n t l y g r e a t e r t h a n
the r e s p o n s e t o 2.4 p m o l / k g . 2 0 m i n b o m b e s i n
(p<0.05 a n d p < 0.01,
87
r e s p e c t i v e l y ), w h e r e a s the C C K s e c r e t i o n i n d u c e d by 6 pmol/kg.
20 m i n b o m b e s i n w a s not s i g n i f i c a n t l y d i f f e r e n t f r o m the C C K - r e l e a s e d u r i n g i n f u s i o n of 2.4 p m o l / k g . 20 m i n b o m b e s i n
(fig 1).
B a s a l p l a s m a C C K c o n c e n t r a t i o n s in 8 p a t i e n t s w i t h p a r t i a l
gastrectomy
(1.4 + 0.4 p m o l / 1 , a n t i b o d y 1703;
1.8 + 0.4 p m o l/1,
a n t i b o d y T 2 q 4 ^ w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h ose m e a ­
s u r e d in 8 n o r m a l c o n t r o l s u b j e c t s (1.2 + 0.3 pmol/1, a n t i b o d y
1703;
1.6 + 0.3 p m o l / 1 ,
a n t i b o d y T 2 Q 4 ). I n f u s i o n of 60 pmol/kg.
20 m i n b o m b e s i n i n d u c e d s i g n i f i c a n t i n c r e a s e s in p l a s m a C C K b o t h
in p a t i e n t s w i t h p a r t i a l g a s t r e c t o m y and in n o r m a l subj e c t s
(fig 2 a n d 3).
24
60
180
60 0
B B S ( p m o l/k g - 2 0 m i n )
Figure l
I n t e g r a t e d p l a s m a C C K s e c r e t i o n d u r i n g i n f u s i o n of i n c r e a s i n g
d o s e s of b o m b e s i n i n t o 9 n o r m a l s u b j e c t s . T h e u p p e r p a n e l r e p r e ­
s e n t s the r e s u l t s o b t a i n e d w i t h a n t i b o d y 1703 a nd the l o w e r p a ­
nel thos e o b t a i n e d w i t h a n t i b o d y
88
C C K -L I .
(p m o l/ l)
C C K -LI (pmol/1 )
10 -
10 -
Bombesin 100ng/kg
Bombesin 1 0 0 n g /k g
8-
6-
A-
4-
2-
Bombesm 100ng/kg
Bombesm 1 0 0 n g /k g
10 n
10- |
8-
8-
6-
6-
4-
A-
2
2-
-
-I-5
0
— I--------------1--------------1
10
15
20
Tim e (min)
Figure 2
T he e f f e c t of i n f u s i o n o f 60
p m o l / k g . 2 0 m i n b o m b e s i n on
p l a s m a C C K c o n c e n t r a t i o n s in
8 normal subjects. The upper
panel r e p r esents the results
o b t a i n e d w i t h a n t i b o d y 1703
and the l o w e r p a n e l tho se
o b t a in ed with a n t i b o d y T204.
* p < 0 . 0 5 , ** p < 0 . 0 1 , ***
p < 0 .005.
0J
r~
-5
~r~
5
" " l------------ 1------------ 1
10
15
20
Tim e (min)
Figure 3
T h e e f f e c t o f i n f u s i o n o f 60
p m o l / k g . 2 0 m in b o m b e s i n on
p l a s m a C C K c o n c e n t r a t i o n s in
8 patients with partial gastrectomy. The upper panel r e ­
presents the results obta i n e d
w i t h a n t i b o d y 1703 and the l o ­
wer panel those obtained with
a n t i b o d y T 2 0 4 . * p < 0 . 0 5 , **
p < 0 . 0 1 , *** p < 0 . 0 0 5 .
89
T h e i n c r e m e n t s in p l a s m a C C K d u r i n g i n f u s i o n of b o m b e s i n in the
gastrectomized patients
(8.1 + 1.8 p m o l / l ,
a n t i b o d y 1703;
9.5 +
2.0 p m o l / l , a n t i b o d y Tjg^) w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m
t h o s e in n o r m a l s u b j e c t s
(6.5 + 0.9 p m o l / l ,
a n t i b o d y 1703; 8.8 +
1.0 p m o l / l , a n t i b o d y T 2 Q 4 ). S i m i l a r l y , th e i n t e g r a t e d p l a s m a CCK
s e c r e t i o n d u r i n g b o m b e s i n in the g a s t r e c t o m i z e d p a t i e n t s
18.5 p m o l / l . 20 min, a n t i b o d y 1703;
(81.5 +
1 2 1.5 + 25.5 p m o l / l . 20 min,
a n t i b o d y T 2 Q 4 ) d i d n ot s i g n i f i c a n t l y d i f f e r f r o m that in the n o r ­
mal subjects
(89.0 + 27.0 p m o l / l . 20 min,
a n t i b o d y 1703; 122.8 +
28.0 p m o l / l . 20 min, a n t i b o d y T j q ^). I n f u s i o n of b o m b e s i n into n o r ­
m a l s u b j e c t s i n d u c e d s i g n i f i c a n t i n c r e a s e s in p l a s m a g a s t r i n from
16.7 + 1.4 to 49.6 + 8.1 p m o l / l
(fig 4). O n th e o t h e r hand,
in the
g a s t r e c t o m i z e d p a t i e n t s p l a s m a g a s t r i n c o n c e n t r a t i o n s w e r e not
significantly affected by bombesin
(fig 4).
Plasma gastnn
( p m o l/ I )
5
10
15
20
T im e (m in )
Figure 4
P l a s m a g a s t r i n c o n c e n t r a t i o n s d u r i n g i n f u s i o n of 60 p m o l / k g . 2 0
m i n b o m b e s i n in 8 n o r m a l s u b j e c t s ( u p p e r p a n e l ) a n d i n 8 p a t i e n t s
w i t h p a r t i a l g a s t r e c t o m y ( l o w e r p a n e l ) . * p < 0 . 0 5 ; **
p<
0.01; *** p <0.0 05 .
90
I n f u s i o n of 180 p m o l / k g b o m b e s i n o v e r 1 h o u r in 3 p a t i e n t s w i t h
p a r t i a l g a s t r e c t o m y a n d R o u x - Y a n a s t o m o s i s d i d not s i g n i f i c a n t l y
influence gastric acid secretion.
In 2 of the p a t i e n t s g a s t r i c
a c i d w a s u n c h a n g e d , w h i l e in the t h i r d p a t i ë n t g a s t r i c a c i d s e ­
c r e t i o n i n c r e a s e d s l i g h t l y f r o m 0.1 to 0.4 mmol/h. T h i s p a t i ë n t ,
howev e r , s h o w e d an i n c r e a s e in s e r u m g a s t r i n f r o m 10 to 24 pg/ml.
I n f u s i o n of b o m b e s i n w a s w e l l - t o l e r a t e d .
Discussion
T he p r e s e n t s t u d y s h o w e d t h a t i n f u s i o n of b o m b e s i n i n t o hum a n s i n d u c e d s i g n i f i c a n t i n c r e a s e s in p l a s m a CC K as m e a s u r e d by
s e q u e n c e - s p e c i f i c r a d i o i m m u n o a s s a y s . S i n c e C C K is a p o t e n t s t i ­
m u l u s of p a n c r e a t i c e n z y m e s e c r e t i o n an d g a l l b l a d d e r c o n t r a c t i o n
(2), it is p o s s i b l e t h a t the e f f e c t s of b o m b e s i n on p a n c r e a s and
g a l l b l a d d e r are m e d i a t e d b y CCK. In fact, b o m b e s i n is t h e first
d e s c r i b e d a n d o n l y k n o w n p e p t i d e c a p a b l e to r e l e a s e C C K in man.
S i n c e it h a s b e e n r e p o r t e d t h a t i n s t i l l a t i o n of h y d r o c h l o r i c
a c i d into t he d u o d e n u m r e l e a s e s C C K
(5,19), it m i g h t be p o s s i b l e
that the b o m b e s i n - i n d u c e d i n c r e a s e in p l a s m a C C K is m e d i a t e d by
s t i m u l a t i o n of g a s t r i n a nd s u b s e q u e n t l y of g a s t r i c a c i d d u r i n g
i n f u s i o n of b o m b e s i n . H o w e v e r ,
antrectomy abolishing the plasma
g a s t r i n r e s p o n s e to b o m b e s i n d i d n o t a f f e c t the b o m b e s i n - s t i m u l a ted i n c r e a s e in p l a s m a CCK. It has b e e n r e p o r t e d t h a t in p a t i e n t s
w i t h p a r t i a l g a s t r e c t o m y i n f u s i o n o f b o m b e s i n d o e s not r e s u l t in
i n c r e a s e s in p l a s m a g a s t r i n a nd g a s t r i c a c i d s e c r e t i o n
(3). S i n c e
i n f u s i o n of b o m b e s i n p r o v o k e s g a l l b l a d d e r c o n t r a c t i o n ,
it m i g h t
b e p o s s i b l e t h a t t h e a b s e n c e of s t i m u l a t i o n of g a s t r i c a c i d b y
b o m b e s i n is d u e t o n e u t r a l i z a t i o n b e c a u s e of an i n c r e a s e d r e f l u x
of b i l e to t he s t o m a c h . T h e r e f o r e , w e h a v e s t u d i e d the e f f e c t of
b o m b e s i n on g a s t r i c a c i d s e c r e t i o n in p a t i e n t s w i t h p a r t i a l g a s ­
t r e c t o m y a n d R o u x - Y a n a s t o m o s i s , p r e v e n t i n g the r e f l u x of bile.
A l s o in t h e s e p a t i e n t s i n f u s i o n of b o m b e s i n d i d n o t i n f l u e n c e
g a s t r i c a c i d s e c r e t i o n . Thus, the i n c r e a s e in p l a s m a C C K d u r i n g
i n f u s i o n of b o m b e s i n is n o t s e c o n d a r y to a g a s t r i n - i n d u c e d i n c r e a ­
se in g a s t r i c a c i d s e c r e t i o n .
It h a s b e e n s h o w n t h a t C C K in t i s s u e e x t r a c t s is p r e s e n t in
se v e r a l m o l e c u l a r f o r m s
(12,22). A t p r e s e n t it is no t k n o w n w h i c h
91
m o l e c u l a r forms o f C C K a re r e l e a s e d i nto the c i r c u l a t i o n d u r i n g
i n f u s i o n of b o m b e s i n .
In this s t u d y w e f o u n d t h a t b o m b e s i n indu-
c e d i n c r e a s e s in p l a s m a CCK, as m e a s u r e d b y r a d i o i m m u n o a s s a y s
using 2 antibodies with different specificities. Antibody
^204
is s p e c i f i c for the s u l p h a t e d t y r o s y l r e g i o n of CCK. S i n c e sulp h a t i o n of the t y r o s y l r e s i d u e in p o s i t i o n 27 of the C C K m o l e c u l e
is r e q u i r e d for b i o l o g i c a l a c t i v i t y ,
it is l i k e l y t h a t the m o l e ­
c u l a r f o rms of C C K r e l e a s e d d u r i n g i n f u s i o n of b o m b e s i n p o s s e s s
b i o l o g i c a l a c t i v i t y . A n t i b o d y 1703 b i n d s to a l l C O O H - t e r m i n a l
C C K - p e p t i d e s c o n t a i n i n g at l e ast 14 a m i n o a c i d r e sidues,
sugges-
t i n g t h a t l a r g e o r i n t e r m e d i a t e f o r m s of C C K ar e p r e s e n t in the
c i r c u l a t i o n d u r i n g i n f u s i o n o f b o m b e s i n . T h i s f i n d i n g is in ag r e e m e n t w i t h a p r e v i o u s s t u d y in d o g s s h o w i n g that i n f u s i o n of
b o m b e s i n i n c r e a s e s p l a s m a C C K l e v e l s as m e a s u r e d w i t h an a n t i b o d y
s p e c i f i c for l a r g e m o l e c u l a r f o r m s of C C K
(20). G h a t e i a n d co-
w o r k e r s r e p o r t e d i n c r e a s e s in p l a s m a C C K d u r i n g i n f u s i o n of b o m ­
b e s i n i n t o h u m a n s b y a r a d i o i m m u n o a s s a y s y s t e m u s i n g an a n t i b o d y
fully cross-reacting w ith gastrin
(8). H o w e v e r , t h e r e l e a s e of
l a r g e a m o u n t s of g a s t r i n d u r i n g b o m b e s i n i n f u s i o n m a k e s t h e int e r p r e t a t i o n of the r e s u l t s o f t h a t s t u d y v e r y d i f f i c u l t
(8).
T h e f i n d i n g t h a t b o m b e s i n r e l e a s e s C C K in m a n m a y h a v e p h y s i o l o g i c a l s i g n i f i c a n c e . T h e l o c a l i z a t i o n of b o m b e s i n in n e r v e s
o f t h e g u t s u g g e s t s t h a t the p e p t i d e is i n v o l v e d in th e n e r v o u s
c o n t r o l of the g a s t r o i n t e s t i n a l t r a c t
(7,23). R e c e n t s t u d i e s have
p o i n t e d to an i m p o r t a n t r o l e for b o m b e s i n - l i k e p e p t i d e s in the
r e l e a s e o f g a s t r i n f r o m t he a n t r u m
(10,23,25). S i m i l a r l y ,
it is
p o s s i b l e t h a t b o m b e s i n - l i k e p e p t i d e s in s m a l l i n t e s t i n a l n e r v e s
a r e i n v o l v e d in t h e r e l e a s e o f C C K f r o m t h e gut.
F u r t h e r m o r e , t h e f i n d i n g t h a t b o m b e s i n r e l e a s e s C C K in m a n
s u g g e s t s t h a t t he C C K r e s p o n s e to b o m b e s i n c a n be u s e d as a simp l e a n d s a f e t e s t t o s t u d y the f u n c t i o n o f C C K - c e l l s in d i s o r d e r s
w i t h s u g g e s t e d o r s u s p e c t e d a b n o r m a l i t i e s of C C K relea s e , s u c h as
c o e l i a c d i s e a s e , e x t e n s i v e s m all b o w e l r e s e c t i o n , g a l l b l a d d e r and
pancreatic diseases
92
( 9 , 1 3 , 17,24).
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17 .
LOW-BEER
of s e r u m
disease.
18 .
M C D O N A L D T J , J Ó R V A L L N, N I L S S O N G, V A G N E M, G H A T E I M A , B L O O M
S R , M U T T V. C h a r a c t e n z a t i o n o f a g a s t r i n r e l e a s i n g p e p t i d e
from p o r cine n o n - a n t r a l g a s t n c tissue. B i o c h e m Biophys Res
C o m m u n 1979; 90: 2 2 7 - 2 3 3 .
19 .
M E Y E R JH. R e l e a s e o f s e c r e t i n a n d c h o l e c y s t o k i n i n .
S O N JC, E d i t o r : G a s t r o ï n t e s t i n a 1 H o r m o n e s . A u s t i n ,
v e r s i t y of Tex a s Press, pp 475-489.
20.
M I Y A T A M, R A Y F O R D P L , T H O M P S O N J C . H o r m o n a l ( g a s t r i n , s e c r e ­
tin, c h o l e c y s t o k i n i n ) and s e c r e t o r y e f f e c t s of b o m b e s i n and
d u o d e n a l a c ï d i f i e a t i o n in d o g s . S u r g e r y 1 9 8 0 ; 87: 2 0 9 - 2 1 5 .
21.
P O L A K J M , B L O O M S R , H O B B S S, S O L C I A E, P E A R S E A G E . D i s t n b u t i o n of a b o m b e s i n - 1 i k e p e p t i d e in h u m a n g a s t r o i n t e s t i n a 1
t r a c t . L a n c e t 1 9 7 6 ; I: 1 1 0 9 - 1 1 1 0 .
22.
R E H F E L D JF. I m m u n o 1 o g i c a 1 s t u d i e s
C h e m 1978; 253: 4 0 2 2 - 4 0 3 0 .
23.
S C H U B E R T ML, B I T A R K N , M A K H L O U F G M . R e g u l a t i o n of g a s t r i n
s o m a t o s t a t i n s e c r e t i o n by c h o l i n e r g i c a n d n o n - c h o l i n e r g i c
i n t r a m u r a l n e u r o n s . A m J P h y s i o l 1982; 243: G 4 4 2 - G 4 4 7 .
24.
S E A L A M , D E B A S H T , R E Y N O L D S C, S A I D S I , T A I L O R I L . G a s t n c a n d
pancreatic h y p o s e c r e t i o n following massive small bowel resect i o n . D i g D i s S c i 1 9 8 2 ; 27: 1 1 7 - 1 2 3 .
25.
V A R N E R AA, M O D L I N IM, W A L S H JH. H i g h p o t e n c y of b o m b e s i n f o r
s t i m u l a t i o n of h u m a n g a s t r i n release and g a s t n c acid s e c r e ­
t i o n . R e g u l P e p t i d e s 1 9 8 1 ; 1: 2 8 9 - 2 9 6 .
94
T S , H A R V E Y R F , R H Y S D A V I E S E, R E A D A E . A b n o r m a 1 1 1 1 e s
c h o l e c y s t o k i n i n a n d g a l l b l a d d e r e m p t y i n g in c e l i a c
N Engl J M e d 1975; 292: 9 6 1 - 9 6 3 .
on
In: T H O M P ­
1975, Uni-
cholecystokinin.
J
Biol
and
Chapter IX
Molecular Forms of Cholecystokinin in Human
Plasma During Infusion of Bombesin
J.B.M.J. Jansen
and
C.B.H.W. Lamers
GastrointesLinal Hormone Laboratory
D i v i s i o n of G a s t r o e n t e r o l o g y
St. R a d b o u d H o s p i t a l
U n i v e r s i t y of N i j m e g e n
Nijmegen
The N e t h e r l a n d s
Life Science
(1983) 33: 2197-2205.
95
MOLECULAR FORMS OF CHOLECYSTOKININ IN HUMAN
PLASMA DURING INFUSION OF BOMBESIN
J.B.M.J. Jansen and C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory,
Division of Gastroenterology, St. Radboud
Hospital, University of Nijmegen,
Nijmegen, The Netherlands
Summary
Bombesin is a tetradecapeptide with stimulatory actions on several
gastrointestinal functions. Infusion of bombesin (60 pmol/kg. 20 min)
into 7 normal subjects induced significant increases in plasma chole­
cystokinin (CCK) as measured with 2 sequence-specific radioinmunoassays.
Employing antibody 1703, specific for carboxyl-terminal CCK-peptides
containing at least 14 amino acid residues, plasma CCK concentrations
rose from D.8±0.2 pmol/1 to 9.9 ± 1.7 pmol/1 (p<0.005), while using anti­
body T204, specific for the sulfated tyrosine region of CCK, plasma CCK
levels increased from 2.9±0.5 pmol/1 to 12.4±1.3 pmol/1 (p<0.005). Plasma
samples obtained from 3 subjects during bombesin infusion were
fractionated by Sephadex column chromatography. Fractionation revealed
4 molecular forms of CCK: peak I eluted in the void volume and comprised
0-7% of CCK-like inmunoreacti vity, peak II eluted at 35% and comprised
8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised
44-61% of CCK-like iinnunoreactivity, and peak IV eluted at 75% and com­
prised 15-27% of CCK-like immunoreactivity. Radioinmunoassay with a
carboxyl-terminal CCK-antibody fully cross-reacting with gastrin did not
reveal additional molecular forms of CCK. Since both the carboxyl-terminus
and the sulfated tyrosine region are required for biological activity of
CCK, it is likely that al 1 these molecular forms of CCK possessbiological
activity.
Cholecystokinin (CCK) is a 33 amino acid polypeptide hormone isolated
from hog small intestine (1). The hormone is suggested to play an important
role in postprandial gal 1 bladder contraction and enzyme secretion from the
pancreas (1,2). In addition, a polypeptide of 39 amino acid residues corresponding to the form of 33 residues extended at the amino-terminus by 6 resi­
dues (CCK39) has been characterized (3). Column chromatography of human small
intestinal extracts has revealed additional CCK-peptides, both larger and
smaller than those chemically characterized (4).
Previous studies have suggested that bombesin, a tetradecapeptide iso­
lated from amphibian skin with powerful actions on the gastrointestinal tract,
releases CCK from the small intestine (5). This suggestion was based upon the
finding that infusion of bombesin induced gallbladder contraction and pan­
creatic enzyme secretion (5,6). In the absence of reliable radioinmunoassays
for CCK other mechanisms for the action of bombesin on gallbladder contraction
and pancreatic enzyme secretion have been proposed. A direct action of bombesin
on the pancreas was supported by the finding of receptors for bombesin on the
36
pancreatic acinar cells and on the gallbladder (7,8), while the localization
of bombesin in small intestinal nerves suggested a role in the activation of
a duodenopancreatic reflex-mechanism (9).
We have developed sequence-specific radioimmunoassays sufficiently sensitive to measure the low CCK concentrations in human plasma. Using these assays
we showed that infusion of bombesin into healthy humans releases large amounts
of CCK into the circulation. Furthermore, we characterized the molecular forms
of CCK in human plasma during infusion of bombesin by column chromatography
and sequence-specific radioinmunoassays.
Subjects and methods
Seven healthy subjects (6 male, 1 female, mean age 46 years, range 33-56
years) were studied. After an overnight fast 60 pmol/kg bombesin (Boehringer
Mannheim Biochemicals, Indianapolis, Indiana, USA) was infused over 20 minutes
and plasma samples for CCK were obtained a t -5,0,5,10,15 and 20 minutes. Plas­
ma CCK concentrations were measured by radioimmunoassay using 2 sequencespecific antibodies 1703 and T204 (10). In addition, plasma samples (4-5 ml)
from 3 subjects during infusion of bombesin were fractionated by Sephadex G50
column chromatography. These plasma samples were extracted in 96% ethanol,
evaporated to dryness under a stream of nitrogen at 37°C, dissolved in 1 ml of
the elution buffer and applied to the column. Furthermore, 1 ml of a 1:10
diluted aqueous-acid extract of human upper small intestine was fractionated
by Sephadex G50 column chromatography. A freshly obtained biopsy of upper small
intestine from a normal subject was extracted in boiling water ( 1g / 10 ml) for
10 minutes. After centrifugation the supernatant was decanted and the pellet
was re-extracted in 0.5 M boiling acetic acid (Ig/IOml) for another 10 minutes.
After centrifugation the supernatant was added to the supernatant of the aqueous
extraction, rapidly frozen, lyophilized, and redissolved in the elution buffer.
The columns (100 x 1 cm) were eluted with 0.05 mol/1 sodium phosphate buffer pH
7.4 containing 0.06 mmol/1 sodiumethylmercurithiosalicylate. To prevent adherence
of peptides to the glass wall of the column, the column was previously coated
with dichlordimethylsilane. The fractions eluting from the column were measured
by radioimmunoassay using antibodies 1703, T204 and 5135. Antibody 1703 binds
to carboxyl-terminal CCK-peptides containing at least 14 amino acid residues
and the antibody does not show any cross-reactivity with gastrins. Antibody
T204 binds to all CCK-peptides containing the sulfated tyrosine region of CCK
and the antibody shows low binding to sulfated gastrins but it does not crossreact with unsulfated gastrins. Antibody 5135 binds to carboxyl-terminal CCKpeptides and shows full cross-reactivity with both sulfated and unsulfated
gastrins. The binding of the 3 antibodies to highly purified porcine CCK preparations, to synthetic CCK fragments and to gastrin is reported in table 1.
None of the antibodies showed cross-reactivity with structurally unrelated
regulatory peptides. Bombesin, studied in concentrations of up to 3000 pmol/1,
did not bind to the antibodies. In the assays using antibodies 1703 and T204
99% pure CCK33 coupled to l25i_hy droxyphenylpropionic acid-succinimide ester
(Bolton-Hunter reagent) was used as label, whereas in the assay using anti­
body 5135 unsulfated CCK 8 coupled to 125i was employed (11). 0.05 mol/1 sodium
phosphate buffer pH 7.4 containing 0.08 mmol/1 human serum albumin and 0.06
mmol/1 sodiumethylmercurithiosalicylate was used as assay buffer. A nonequilibrium system was used with 72 hours preincubation followed by 24 hours
incubation after addition of the labeled peptide. Separation between free
and antibody bound hormone was performed by adsorption of the free peptide
to plasma-coated charcoal for antibodies 1703 and T204 and to amberlite IRP-67
for antibody 5135. The 50% inhibition dose (ID 5 0 ) was 2.8 pmol/1 for antibody
1703, 3.3 pmol/1 for antibody T204 and 4.0 pmol/1 for antibody 5135. Plasma
samples were extracted in 96% ethanol, dried under nitrogen at 37°C and
97
Table 1
CCK 39
CCK 33
CCK 20-33
CCK 22-33
CCK 24-33
CCK 26-33 sulfated
CCK 26-33 unsulfated
CCK 30-33
CCK 1-21
CCK 1-15
CCK 10-20
CCK 16-27
CCK 20-27
gastrin 34 sulfated
gastrin 34 unsulfated
gastrin 17 sulfated
gastrin 17 unsulfated
antibody
1703
antibody
T204
antibody
5135
0.91
1.09
1.06
1.00
1. 00
1.00
0.62
1.69
1.71
0.78
0.65
2.89
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.01
0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.01
0.02
0.02
< 0.01
0.02
< 0.01
2.01
1.92
2.49
2.59
<0.01
< 0.01
< 0.01
< 0.01
0.09
0.06
2.21
2.29
2.38
2.32
Relative binding of cholecystokininpeptides and gastrins to antibodies 1703,
T 204 and 5135
reconstituted in assay buffer to the original volume before the assay. Recovery of
CCK33 and sulfated CCK 8 added to hormone-free plasma was 85.4 ± 2.0% (n=11)
and 89.8 ± 1.7% (n=7), respectively. The detection limit of the assay employing
antibody 1703 and antibody T204 was about 0.5 pmol/1 sample and for antibody
5135 about 1 pmol/1 sample. The intra-assay variation was between 4.6 and 11.5%
and the inter-assay variation ranged from 6 . 8 to 26.1%. Dilution curves of plasma
samples containing a high concentration of endogenous CCK were parallel to the
Standard curve.
Results were expressed as the mean ± 1 SEM. The integrated CCK secretion
during infusion of bombesin was determined by calculating the area under the
curve after subtraction of the basal values. Statistical analysis was done by
Student 's t-test. Informed consent was obtained from all subjects studied.
Resul ts
Basal plasma CCK concentrations in 7 normal subjects were very low (0.8
± 0.2 pmol/1, antibody 1703; 2.9 ± 0.5 pmol/1, antibody T204). Infusion of 60
pmol/kg bombesin over 20 minutes induced significant (p<0.005) increases in
plasma CCK to peak levels of 9.9 ± 1.7 (antibody 1703) and 12.4 ± 1.3 pmol/1
(antibody T204) (fig.1). The peak increments in plasma CCK were not significantly different when measured with antibody 1703 (8.7 ± 1.6 pmol/1) or with
antibody T204 (9.5 ± 1.2 pmol/1). Similarly, the integrated plasma CCK secre­
tion measured with antibody 1703 (117.5 ± 19.0 pmol/1.20 min) did not significantly differ from that determined with antibody T204 (114.3 ± 10.7 pmol/1.
20 min.). Infusion of saline did not significantly change plasma CCK concen­
trations. Basal plasma CCK concentrations were 0.9 ± 0.2 pmol/1 (antibody 1703)
and 2.2 ± 0.3 pmol/1 (antibody T204), while peak concentrations were 1.0 ± 0.2
pmol/1 (antibody 1703) and 2.3 ± 0.3 pmol/1 (antibody T204).
98
FIG. 1
The effect of infusion of 60 pmol/kg.20 min
bombesin into 7 normal subjects on plasma
cholecystokinin as measured with antibody
1703 (left panel) and antibody T204 (right
panel). Asterisks denote significant
(p<0.005) differences from basal value
TABLE II
peak
peak
peak
peak
I
II
III
IV
antibody
1703
antibody
T204
5( 0- 7)
23( 8-41)
51(44-61)
21(15-25)
3( 0- 5)
17( 8-21)
59(58-61)
21(16-27)
Percentage distribution (mean and range)
of molecular forms of cholecystokinin in
plasma form 3 normal subjects during in­
fusion of bombesin
99
F ra c tio n n u m b e r
FIG. 2
Elution profile of cholecystokinin in human plasma during bombesin
stimulation fractionated by Sephadex G50 column chromatography. The
upper panel represents the results obtained with antibody 1703, the
middle panel with antibody T204, and the lower panel with antibody
5135. The column was previously calibrated with albumin (V ), ’
“I
(Vt ), pure porcine cholecystokinin 33 (50%) and synthetic Sulfated
cholecystokinin 8 (112%). Recovery of cholecystokinin-1ike inmunoreactivity was between 73 and 94%.
100
Fraction n u m b er
FIG. 3
Elution profile of an aqueous-acid extract of human upper small intestine
fractionated by Sephadex G50 column chromatography. The upper panel represents
the results obtained with antibody 1703, the middle panel with antibody T204,
and the lower panel with antibody 5135. The column was previously calibrated
with albumin (V0 ).
(Vt), pure porcine cholecystokinin 33 (50%) and synthetic sulfated cholecystokinin 8 (112%). Recovery of cholecystokinin-1ike
iimunoreactivity was between 76 and 81%.
101
Fractionation of basal plasma by Sephadex column chromatography did not
reveal any peaks when measured with antibodies 1703 and T204. Fractionation of
plasma during bombesin infusion revealed 4 seperate peaks demonstrable with
antibodies 1703 and T204 (fig. 2, table 2). Peak I eluted in the void volume
and comprised 0-7% of CCK-like immunoreactivity, peak II eluted at 35% and comprised 8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised
44-61% of CCK-like immunoreactivity, and peak IV eluted at 75% and comprised
15-27% of CCK-like immunoreactivity. Interpretation of the results obtained
with antibody 5135 was hampered by the cross-reactivity of the antibody to
gastrins (table 1). The elution positions of gastrin 34 and gastrin 17 overlapped with those of the CCK-peptides. However, no additional peaks were observed employing antibody 5135. Comparison of the molecular forms of CCK in
plasma with those demonstrated in an aqueous-acid extract of human upper small
intestine revealed an additional peak of CCK-like immunoreactivity in the
intestinal extract eluting at 112% of the elution volume (fig. 2 and 3). This
peak was observed with antibodies T204 and 5135, but not with antibody 1703.
Discussion
The present study showed that infusion of bombesin into humans stimulates
plasma CCK secretion as measured by sequence-specific radioimmunoassays. One
previous study on the effects of bombesin on plasma CCK in man has been reported
(12). However, the antibody used in that study showed full cross-reactivity
with gastrin (12). Since plasma gastrin concentrations are several times higher
than those of CCK and large amounts of gastrin are released during infusion of
bombesin (5,13), cross-reactivity of the antibody with gastrin makes interpre­
tation of the results of that study very difficult. Bombesin has been demon­
strated in the human gastrointestinal tract (14,15). Since bombesin in the
gastrointestinal tract is localized in nerves (16), it is suggested that bom­
besin acts as a neurotransmitter (2,16). Recent studies have pointed to an
important role for bombesin in the stimulation of gastrin release from the
antrum (5,13,17). Similarly, it might be possible that bombesin plays a role in
the physiological regulation of CCK secretion from the upper small intestine.
Infusion of bombesin is known to stimulate pancreatic enzyme secretion and
gaïlbladder contraction in man (6). Since CCK is a potent stimulus for pan­
creatic enzyme secretion and gaTlbladder contraction (1,2), it is possible
that the effects of bombesin on pancreas and gaTlbladder are mediated by CCK.
Furthermore, it might be possible that other effects of bombesin, such as inhibition of gastric emptying (18) and induction of satiety (19), are similarly
mediated by the release of CCK. However, a direct action of bombesin on the
pancreas and gaTlbladder cannot be excluded.
The present study showed that CCK in human plasma during infusion of bom­
besin is heterogenous. Four sepera'te peaks of CCK-like immunoreactivity were
found. All 4 peaks were demonstrated by antibodies 1703 and T204. Because anti­
body 1703 binds to carboxyl-terminal CCK-peptides containing at least 14 amino
acid residues and antibody T204 is specific for the sulfated tyrosine region
of CCK, it can be concluded that these molecular forms consist of at least the
14 carboxyl-terminal amino acid residues and contain the sulfated tyrosine
region of CCK. Since the presence of both the carboxyl-terminus and the sulfated
tyrosine region are required for biological activity (1,2), it is likely that
all these 4 molecular forms of CCK demonstrated in plasma possess biological
activity. The majority of CCK eluted in peaks II, III and IV. Peak II corresponds to so-called component I CCK found in the present and other studies in
small intestinal extracts (4,10,20). In a recent study the partial structure
of this large form of CCK, extracted from canine small intestine, was
elucidated and was found to consist of 58 amino acid residues (20). Based on
the elution position it is likely that peak III corresponds to CCK 33 and
102
CCK 39, which CCK-peptides are not separated from each other by Sephadex G50
column chromatography. The CCK-peptide eluting at 75% of the elution volume
(peak IV) corresponds to intermediate CCK in intestinal extracts (4,10). The
chemical structure of intermediate CCK has not been elucidated, but the pre­
sent study suggests that it consists of at least 14 amino acid residues. Comparison of the molecular forms of CCK in plasma during bombesin infusion and
in an extract of human small intestine showed that all 4 molecular forms found
in plasma were also present in the small intestine. In addition, a small mole­
cular form eluting after the salt peak was demonstrated in the intestinal ex­
tract. Since this peptide bound to antibodies T204 and 5135 and not to anti­
body 1703, it can be derived that it must contain the carboxyl-terminus and
the sulfated tyrosine region of CCK and that it consists of less than 14 amino
acid residues. Although the chemical structure of this CCK-peptide has not been
characterized, it is generally accepted that it represents the 8 carboxylterminal amino acid residues of CCK (CCK 8 ) (2,4). Several reports suggest that
CCK 8 is the predominant molecular form of CCK in postprandial plasma (21,22,
23), while these studies were unable to demonstrate large forms of CCK in
plasma after feeding. These findings suggest that the molecular forms of CCK
in plasma during infusion of bombesin differ from those found in postprandial
plasma. However, in most reports the small form of CCK in postprandial plasma
eluted earlier farm the column than synthetic CCK 8 or the small form of CCK
present in intestinal extracts (21,23). This finding suggests that either
small CCK in postprandial plasma is larger than CCK 8 or that the elution
position of CCK 8 is altered by plasma factors. The failure to demonstrate
large forms of CCK in postprandial plasma can be attributed to several
factors, such as low binding of many carboxyl-terminal antibodies to large
forms of CCK (24,25), overlap in elution position between large forms of CCK
and gastrins when samples are fractionated by Sephadex chromatography, and
low recovery of large forms of CCK from columns due to adherence of large CCKpeptides to plastic or glass. In a recent study, in which large CCK was sepa­
rated from gastrins by high pressure liquid chromatography and the concentration of large CCK was corrected for the lower binding of CCK 33 to the carboxyl-terminal antibody used, it was found that concentrations of large and
small forms of CCK in postprandial plasma were about equal (26). However,
from that study no conclusions could be drawn about the presence of other
molecular forms of CCK in postprandial plasma (26).
It is concluded that infusion of bombesin into humans stimulates secretion of CCK into the circulation and that CCK in plasma during infusion of
bombesin is heterogenous.
Acknowledgments
This study was supported by the Foundation for Medical Research FUN60
(grant nr. 13-37-32). Antibody 5135 was a gift of Drs. G. Rosenquist and
J. Walsh, and some of the synthetic cholecystokinin-peptides were obtained
from prof. N. Yanaihara.
Referencens
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59-144, Raven Press, New York (1981).
103
3. V. MUTT, Clin.Endocrinol. 5 175S-183S (1976).
4. J.F. REHFELD, J.Biol.Chem. 253 4022-4030 (1978).
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6 . N. BASSO, S. GIRI, G. IMPROTA, E. LEZOCHE, P. MELCHIORRI, M. PERCOCO
and V. SPERANZA, Gut J 6 994-998 (1975).
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Y.C. LEE, N.C. CHRISTOFIDES, D.L. SARSON, K. MASHITER, I. MACINTYRE
and S.R. BLOOM, J.Clin.Endocrinol Metab 54 980-985 (1982).
13. A.A. VARNER, I.M. MODLIN and J.H. WALSH, Regul.Peptides ^ 289-296 (1981).
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1109-1110 (1976).
15. S.R. BLOOM, M.A. GHATEI, J.W. WHARTON, J.M. POLAK and M.R. BROWN, Gastro­
enterology 76 1103 (1979).
16. G.J. DOCKRAY, C. VAILLANT and J.H. WALSH, Neuroscience 4 1561-1568 (1979).
17. M.L. SCHUBERT, K.N. BITAR and G.M. MAKHLOUF, Am.J.Physiol. 243 G442-G447
(1982).
18. J.H. WALSH, V. MAXWELL, J. FERRARI and A.A. VARNER, Peptides 2 (Suppl.2)
193-198 (1981).
“
19. D.B. WEST, R.H. WILLIAMS, D.J. BRAGET and S.C. WOODS, Peptides 3 61-67
(1982).
~
20. V.E. EYSSELEIN, J.R. REEVE, J.E. SHIVELEY, D. HAWKE and J.H. WALSH,
Peptides 3 687-691 (1982).
21. J.H. WALSH, C.B. LAMERS and J.E. VALENZUELA, Gastroenterology 82 438-444
(1982).
—
22. J. CALAM, A. ELLIS and G.J. DOCKRAY, J.Clin.Invest. 69 218-225 (1982).
23. D.J. BYRNES, T. BORODY, G. DASKALOPULOS, M. BOYLE and I. BENN, Peptides
2 (Suppl. 2) 259-262 (1981).
24. G.J. DOCKRAY, Brain Res. 188 155-165 (1980).
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26. P.N. MATON, A.C. SELDEN and V.S. CHADWICK, Regul. Peptides 4 251-260 (1982).
104
Chapter X
Plasma Cholecystokinin Concentrations in Patients
with Pancreatic Insufficiency
J.B.M.J. Jansen
W.P.M. Hopman and C.B.H.W. Lamers
Gastrointestinal Hormone Laboratory
D i v i s i o n of G a s t r o e n t e r o l o g y
St. R a d b o u d H o s p i t a l
JJniversity o f N i j m e g e n
Nijmegen
The N e t h e r l a n d s
S u b m i t t e d for P u b l i e a t i o n
10 5
X
P L A S M A C H O L E C Y S T O K I N I N CONC EN TR AT IO NS
IN PATIENTS WITH
PANC RE ATI C IN SU FF ICIENCY
Abstract
It h a s b e e n c l a i m e d that p l a s m a c h o l e c y s t o k i n i n
(CCK) c o n ­
c e n t r a t i o n s are r a i s e d in p a t i e n t s w i t h n a n c r e a t i c ï n s u f f i c i e n cy. Wc h a v e m e a s u r e d p l a s m a C C K c o n c e n t r a t i o n s m
with pancreatic insuffïciency
c y s t i c f i brosis)
usmg
(22
32 p a t i e n t s
a l c o h o l i c p a t i e n t s an d 1 0
a n d in 30 n ormal s u b j e c t s b y r a d i o i m m u n o a s s a y
a n t i b o d i e s w i t h d i f f e r e n t s p e c i f ï c i t i e s . A n t i b o d y 1703
b i n d s to C O O H - t e r m i n a l f o r m s of C C K c o n t a i n i n g at least 14 amino a c i d
r e s i d u e s a n d d o e s not c r o s s - r e a c t w i t h g a s trins. A n t i ­
b o d y T 2 q 4 b i n d s to a ll C C K - p e p t i d e s c o n t a i n i n g the s u l p h a t e d
t y r o s y l r e g i o n a n d s h o w s l ow c r o s s - r e a c t i v i t y w i t h s u l p h a t e d
g a s t r i n s b u t n o b i n d i n g to n o n - s u l p h a t e d g a s t r i n s . A n t i b o d y
5135 b i n d s to all C O O H - t e r m i n a l C C K - p e p t i d e s a n d shows full
c r o s s - r e a c t i v i t y w i t h gas t r i n s .
In o a t i e n t s w i t h p a n c r e a t i c ïn-
sufficiency plasma CCK concentrations
b o d y 1703;
(1.2 ± 0.1 pmol/1, a n t i ­
2.0 ± 0.2 p m o l / 1 , a n t i b o d y T 2 0 4 ' 12.5 ± 1.4 pmol/1,
a n t i b o d y 5135) w e r e n o t s i g n i f ï c a n t l y d i f f e r e n t f r o m tho s e in
normal subjects
p m o l/ 1 ,
(1.1 + 0.1 pmol/1,
a n t i b o d y 1703;
a n t i b o d y T 2 0 4 ' 10.5 ± 0*9 p m o l /1,
2.2 ± 0.3
a n t i b o d y 5135). Fur-
t h e r m o r e , p l a s m a C C K c o n c e n t r a t i o n s in p a t i e n t s w i t h p a n c r e a t i c
i n s u f f ï c i e n c y due t o a l c o h o l i c p a n c r e a t i t i s ( 1 . 2 ± 0 . 1 o m o l / 1 ,
a n t i b o d y 1703; 1.9 + 0.2 pmol/1, a n t i b o d y T 2 Q 4 ; 14.0 ± 0.1 pmol/1,
a n t i b o d y 5135) w e r e n o t s i g n i f ï c a n t l y d i f f e r e n t f r o m tho s e in
patients with cystic fibrosis
2.4 ± 0.4 pmol / 1 ,
antibody
(1.2 ± 0.2 p m o l / 1 , a n t i b o d y 1703;
~ ^ ‘^ P m °l/lf a n t i b o d y 5135).
C r o s s - r e a c t i v i t y w i t h g a s t r i n a c c o u n t e d for a l m o s t all C C K - l i k e
i m m u n o r e a c t i v i t y m e a s u r e d w i t h a n t i b o d y 5135. O r a l i n g e s t i o n of
250 m l 20% i n t r a l i p i d i n d u c e d s i m i l a r i n c r e a s e s in p l a s m a CC K in
6 patients with pancreatic
insuffïciency
(4.3 ± 1 . 5 pmol/1, a n ­
t i b o d y 1703; 2.9 ± 0.6 pmol/1, a n t i b o d y T 2 Q 4 ) as in 7 nor m a l
subjects
(5.0 ± 0.9 p m o l / 1 , a n t i b o d y 1703;
tibody t 2 Q 4 ) .
106
4.3 ± 0.9 pmol/1, a n ­
It is c o n c l u d e d t h a t C C K c o n c e n t r a t i o n s m
fasting plasma
are v e r y low, th a t o r a l fat i n d u c e s s i g n i f i c a n t i n c r e a s e s in
p l a s m a CCK, a nd t h a t m
patients with pancreatic insufficiency
plasma CCK levels are not increased.
Introduction
It has b e e n c l a i m e d t h a t p l a s m a c h o l e c y s t o k i n i n
(CCK) c o n ­
c e n t r a t i o n s are e l e v a t e d in p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i ­
ency
(1-5). It w a s s u g g e s t e d t h a t t h e s e h i g h CC K lev e l s r e s u l t -
e d f r o m an i m p a i r e d i n h i b i t i o n of C C K s e c r e t i o n by p a n c r e a t i c
enzymes. H o w e v e r , c n t i c i s m on the r a d i o i m m u n o a s s a y m e t h o d emp l o y e d has b e e n r a i s e d
(6,7). C o n f i r m a t i o n of the i n c r e a s e d
p l a s m a C C K c o n c e n t r a t i o n s in p a t i e n t s w i t h p a n c r e a t i c i n s u f f i ­
c i e n c y b y r e l i a b l e r a d i o i m m u n o a s s a y s for CC K w o u l d s u p p o r t the
p o s s i b i l i t y to d i a g n o s e p a n c r e a t i c i n s u f f i c i e n c y by m e a s u r e m e n t
of C C K m
a s i n g l e b l o o d sample.
We h a v e d e v e l o p e d r a d i o i m m u n o a s s a y s for C C K u s i n g a n t i b o ­
dies w i t h d i f f e r e n t s p e c i f ï c i t i e s . T he as s a y s w e r e s u f f i c i c n t l y
s e n s i t i v e to e n a b l e m e a s u r e m e n t of l o w C C K c o n c e n t r a t i o n s in
plasma.
In t he p r e s e n t s t u d y w e h a v e m e a s u r e d b a s a l p l a s m a C C K
c o n c e n t r a t i o n s in p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y du e to
a l c o h o l i c p a n c r e a t i t i s or c y s t i c f i b r o s i s and in n o r m a l subjects.
Furthermore,
t h e p l a s m a C C K r e s p o n s e s to oral i n g e s t i o n of fat
w e r e d e t e r m i n e d in p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y an d
no r m a l sub j e c t s .
Subjects and methods
Basal p l a s m a C C K c o n c e n t r a t i o n s w e r e m e a s u r e d m
32 p a ­
ti e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y an d 30 n o r m a l s u b j e c t s .
patients
(16M, 6 F; m e a n age 45 y e a r s , r a n g e 26-75 years)
p a n c r e a t i c i n s u f f i c i e n c y d ue to a l c o h o l i c p a n c r e a t i t i s .
22
ha d
8 of
t h ese p a t i e n t s h a d d i a b e t e s m e l l i t u s an d all w e r e t r e a t e d w i t h
pancreatic enzyme supplements.
years, r a n g e 17-37 years)
10 p a t i e n t s
(7M, 3F; m e a n ag e 22
h a d c y s t i c fibrosis. Al l t h e s e p a ­
tient s w e r e t r e a t e d w i t h p a n c r e a t i c e n z y m e s u p p l e m e n t s , bu t
n o n e of t h e m h a d d i a b e t e s m e l l i t u s .
In add i t i o n ,
30 n o r m a l
107
subjects
(25M, 5F; m e a n age 41 years, r a n g e 19-69 years) w i t h ­
o u t k n o w n g a s t r o i n t e s t i n a l or m e t a b o l i c d i s e a s e s were studied.
In 6 p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y
43 y e a r s , r a n g e 2 5 -52 years)
m e a n a ge 41 y ears,
(5M,
1F; m e a n age
and in 7 n o r m a l s u b j e c t s
r a n g e 24-60 years)
(6 M,
1F;
plasma CCK concentrations
w e r e m e a s u r e d by r a d i o i m m u n o a s s a y u s i n g a n t i b o d i e s 1703 and
T 2 Q 4 a f t e r o r a l i n g e s t i o n of 250 m l 20% i n t r a l i p i d
Stockholm,
(Vitrum AB,
S w e d e n ) . A l l s u b j e c t s w e r e s t u d i e d a f t e r an o v e r n i g h t
fast. P a n c r e a t i c e n z y m e t h e r a p y w a s d i s c o n t i n u e d for at l e ast
18 h ours.
P l a s m a C C K c o n c e n t r a t i o n s w e r e m e a s u r e d by r a d i o i m m u n o a s s a y
using 3 antibodies with different specificities
(8,9). A n t i b o d y
1703 b i n d s to all C O O H - t e r m i n a l C C K - p e p t i d e s c o n t a i n i n g at lea s t
14 a m i n o a c i d r e s i d u e s a n d the a n t i b o d y d o e s no t show any c r o s s
reactivity with g a s t n n s .
A n t i b o d y ^204 t,ln<3s to all CC K p e p t i ­
d e s c o n t a i n i n g t he s u l p h a t e d t y r o s y l r e g i o n of C C K and the a n ­
t i b o d y s h o w s l ow b i n d i n g to s u l p h a t e d f o rms of g a s t r i n bu t it
does not react with unsulphated g a s t n n s .
A n t i b o d y 5135 b i n d s
to C O O H - t e r m i n a l C C K - p e p t i d e s a n d s h ows full c r o s s - r e a c t i v i t y
with both sulphated and unsulphated g a s t n n s .
N o n e of the a n t i ­
b o d i e s s h o w e d s i g n i f i c a n t b i n d i n g to C C K - 4 . T h e b i n d i n g of the
3 a n t i b o d i e s to p o r c i n e C C K p r e p a r a t i o n s , to s y n t h e t i c C C K
f r a g m e n t s a n d to g a s t n n s is p r e s e n t e d m fig 1 a n d tab l e 1 .
N o n e o f t he a n t i b o d i e s s h o w e d c r o s s - r e a c t i v i t y w i t h s t r u c t u r a l ly u n r e l a t e d p e p t i d e s . M e a s u r e m e n t o f the e l u a t e on a S e p h a d e x
G 50 SF c o l u m n to w h i c h an a q u e o u s - a c i d e x t r a c t of h u man u p p e r
small intestine was applied showed that all 3 antibodies bound
to c o m p o n e n t - I CCK, C C K - 3 3 / C C K - 3 9 a n d i n t e r m e d i a t e CCK(s),
w h i l e a n t i b o d i e s T_„. a n d 5135 b o u n d a l s o to C C K - 8 . In the as204
says u s i n g a n t i b o d i e s 1703 a nd T „ n . 99% p u r e p o r c i n e CCK - 3 3
125
c o u p l e d to
I-hydroxyphenylpropionic acid-succinimide ester
( B o l t o n - H u n t e r reagent)
w a s u s e d as label, w h e r e a s m
u s i n g a n t i b o d y 5135 u n s u l p h a t e d C C K - 8 c o u p l e d to
ployed
1 25
the a s s a y
I wa s em -
(9). 0.05 m o l / l s o d i u m p h o s p h a t e b u f f e r pH 7.4 c o n t a i n ­
ing 0.08 m m o l / 1 h u m a n s e r u m a l b u m i n a n d 0.06 m m o l / 1 s o d i u m e t h y l m e r c u r i t h i o s a l y c i l a t e was u s e d as a s s a y b u f fer. A n o n - e q u i -
108
109
1000
Pla sma ( pmol/1)
Standard
curves
of
Figure 1
a n t i b o d y 1703 (left p a n e l ) , a n t i b o d y
and anti b o d y 5135 ( n g h t panel) .
10 0 0 0
Pl as m a ( p m o l / l )
T204
(middle
panel)
RELATIVE
RING THE
P O T E N C I E S OF C C K - P E P T I D E S T O A N T I B O D I E S 1703, T2 0 4 A N D 5135 D E T E R M I N E D BY C O M P A I N H I B I T I O N D O S E - 5 0 (ID-50) F O R T H E D I F F E R E N T P E P T I D E S AS P R E S E N T E D B E T W E E N
B R A C K E T S
ANTIBODY
( P M O L / L ) .
1703
T204
5 1 35
CCK
39
0 .91
(
3 . 1)
1 .09
(
3 .0)
1 . 06
(
12
0)
CCK
33
1 . 00
(
2 .8)
1 .00
(
3 .3)
1 . 00
(
12
0)
CCK
2 0 - 33
0 .62
(
4 .7 )
1 .69
(
2 .0)
2 .89
(
3 5)
CCK
2 2 - 33
<0 . 0 1
(
933
)
1 .7 1
(
2 .0)
2 .0 1
(
5 0)
CCK
2 4 - 33
<0 . 0 1
(> 1 0 0 0
)
0 .7 8
(
5 .5)
1 .92
(
5
(
6 .6)
CCK
2 6 - 33
sulphated
<0 . 0 1
(> 1 0 0 0
)
0 .65
CCK
2 6 - 33
nonsulphated
<0 . 0 1
(>1000
)
<0 .01
CCK
3 0 - 33
CCK
1- 21
<0 . 0 1
(>1000
)
<0 . 0 1
(>1000
)
CCK
1- 15
CCK
1 0 - 20
<0 .01
(>1000
)
<0 .01
(>1000
CCK
1 6 - 27
0 .01
(
280
1)
2 .49
(
4 0)
(>1000
)
2 . 59
(
3 8)
<0 .01
(>1000
)
<0 . 0 1
(>1000
)
<0 . 0 1
(>1000
)
<0 . 0 1
(> 1 0 0 0
)
<0 .01
(>1000
)
<0 . 0 1
(> 1 0 0 0
)
)
<0 .01
(>1000
)
<0 . 0 1
(>1000
)
)
0 .0 1
(
330
)
0 .09
(
)
(
165
)
2 .2 1
(
4 5)
(>1000
)
2 .29
(
4
3)
(
165
)
2 .38
(
4
2)
(>1000
)
2 . 32
(
4 3)
Gas trin
34
sulphated
<0 . 0 1
(>1000
)
0 .02
Gastrin
34
nonsulphated
<0 .01
(>1000
)
<0 .01
Gastrin
17
s u l p h a te d
<0 . 0 1
(>1000
)
0 .02
Ga s t rin
17 n o n s u l p h a t e d
<0 .01
(>1000
)
<0 . 0 1
T A B L E
1
111
l i b r i u m s y s t e m w a s u s e d w i t h 72 h o u r s p r e i n c u b a t i o n f o l l o w e d by 24
h o u r s i n c u b a t i o n a f t e r a d d i t i o n of t h e l a b e l l e d p e p t i d e .
Separa­
t i o n b e t w e e n f r e e a n d a n t i b o d y b o u n d h o r m o n e was p e r f o r m e d by ads o r p t i o n of the f r e e p e p t i d e to c h a r c o a l for a n t i b o d i e s
1703 and
^204 anc^ to a m b e r l i t e I R P - 6 7 for a n t i b o d y 5135. Th e 50% i n h i b i t i o n
d o s e for C C K - 3 3 w a s 2.8 p m o l / l for a n t i b o d y 1703,
a n t i b o d y ^204 ant^ 12 •0 p m o l / l for a n t i b o d y 5135
s a m p l e s w e r e e x t r a c t e d in 96% e t h a n o l ,
3.3 p m o l / l for
(table 1). P l a s m a
d n e d u n d e r n i t r o g e n at 37
°C a n d r e c o n s t i t u t e d in a s s a y b u f f e r to the o n g i n a l v o l u m e b e f o ­
re the assay.
R e c o v e r y of C C K - 3 3 a n d s u l p h a t e d C C K - 8
h o r m o n e - f r e e p l a s m a w as 85.4 ± 2.0%
a d d e d to
{n =11) a n d 89.8 ± 1.7%
(n = 7)
r e s p c c t i v e l y . T h e d e t e c t i o n limit of the a s s a y s e m p l o y i n g a n t i b o d y
1703 and a n t i b o d y T 2 Q 4 w a s a *3°ut 0.5 p m o l / l p l a s m a a n d for a n t i ­
b o d y 5135 a b o u t 1 p m o l / l plasma. T h e i n t r a - a s s a y v a n a t i o n w a s
11.5% at 1.2 p m o l / l ,
8.4% at 2.5 p m o l / l an d 4.6% at 8.0 p m o l / l
(n=5) . The i n t e r - a s s a y v a n a t i o n w a s 2 6. 1 % at 1.3 p m o l / l ,
at 2.6 p m o l / l a n d 15.4% at 8 . 8 p m o l / l
11.3%
(n=5). D i l u t i o n c u r v e s of
p l a s m a s a m p l e s c o n t a i n i n g a h i g h c o n c c n t r a t i o n of e n d o g e n o u s C C K
w e r e p a r a l l e l t o t he S t a n d a r d curve.
R e s u l t s w e r e e x p r e s s e d as the m e a n ± 1 SEM. T h e i n t e g r a t e d
C C K s e c r e t i o n a f t e r i n g e s t i o n of f a t w a s d e t e r m i n e d b y c a l c u l a t i n g
the a r e a u n d e r t he c u r v e a f t e r s u b t r a c t i o n of b a s a l v a l u e s .
S t a t i s t i c a l a n a l y s i s w as done by S t u d e n t ' s t - t e s t for p a i r e d a n d
unpaired results.
I n f o r m e d c o n s e n t w a s o b t a i n e d f r o m all s u b j e c t s
studied.
Results
F a s t i n g p l a s m a C C K c o n c e n t r a t i o n s in 30 n o r m a l s u b j e c t s were
1.1
± 0.1 p m o l / l w h e n m e a s u r e d w i t h a n t i b o d y 1703 a n d 2.2 ± 0.3
p m o l / l w h e n m e a s u r e d w i t h a n t i b o d y T 2 0 4 ’ p la s m a C C K l e v e l s m
patients with pancreatic insuffïcicncy
(1 . 2
±0.1
32
pmol/l, a n t i ­
b o d y 1703 a n d 2.0 ± 0.2 pmol/l, a n t i b o d y ^ 2 0 4 ^ w e r e no t s i g m f i c a n t l y d i f f e r e n t f r o m t h o s e in n o r m a l s u b jects. F u r t h e r m o r e ,
there were no significant differences between plasma CC K c o n c e n ­
t r a t i o n s in 2 2 p a t i e n t s w i t h p a n c r e a t i c i n s u f f ï c i e n c y d u e to alcoholic pancreatitis
(1.2 ± 0.1 p m o l / l ,
a n t i b o d y 1703;
1.9 ± 0.2
111
pmol/1, antibody T 2 Q 4 ) and those in 10 patients with pancreatic
i n s u f ficiency due to cystic f i b r o s i s '(1.2 ± 0.2 pmol/1, antibody
1703; 2.4 ± 0.4 pmol/1, antibody
Plasma CCK-like immu n o ­
rea c t i v i t y m e a sured with antibody 5135 was markedly higher in
all subjects, but was not significantly different between the
groups of subjects studied {10.5 ± 2.9 pmol/1 in normal subjects,
14.0 ± 1.9 pmol/1 in patients with alcoholic pancreatitis and
9.1 ± 1.0 pmol/1 in patients with cystic f i b r o s i s ) . However, measurement of the samples with antibody 2604, specific for gastrin
wi t h o u t c r o s s - reactivity with CCK (10), showed that almost all
i m m u n o reactivity meas u r e d with antibody 5135 could be attributed
to gastrin. The distribution of plasma CCK concentrations as
m e a s u r e d with the 3 antibodies is depicted in figure 2.
C y s tic
fib ro s is
(n =10)
20
25
30
p m o l/ l
D i s t r i b u t i o n o f b a s a l p l a s m a C C K c o n c e n t r a t i o n s i n 30 n o r m a 1 s u b —
j e c t s , i n 22 p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y d u e t o a l c o ­
h o l i c p a n c r e a t i t i s , a n d i n 10 p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y due to c y s t i c f i b r o s i s , as m e a s u r e d w i t h a n t i b o d y 1703 (left
p a n e 1) , a n t i b o d y T
204 (middle panel) a n d a n t i b o d y 5135 (right
panel) .
112
I n g e s t i o n of 250 ml 20% i n t r a l i p i d i n d u c e d s i g n i f i c a n t i n ­
c r e a s e s in p l a s m a C C K m b o t h n o r m a l s u b j e c t s a n d p a t i e n t s w i t h
p a n c r e a t i c i n s u f f ï c i e n c y (figure 3).
P la s m a CCK
( p m o l/1 )
T im e (m in)
T im e (m in)
Figure 3
T h e e f f e c t of i n g e s t i o n of 250 ml 2 0 % I n t r a l i p i d on p l a s m a C C K
in 7 n o r m a l s u b j e c t s (•---- •) a n d 6 p a t i e n t s w i t h p a n c r e a t i c ï n s u f f ï c i e n c y (o---- o) a s m e a s u r e d w i t h a n t i b o d y 1 7 0 3
(left p a n e l )
a n d a n t i b o d y T 2Q4 ( r i g h t p a ne l)
P o s t p r a n d i a l p l a s m a C C K c o n c e n t r a t i o n s in n o r m a l s u b j e c t s w e r e
s i g n i f ï c a n t l y h i g h e r t h a n b a s a l v a l u e s at 1 0 , 2 0 , 30, 40, 50, 60,
75 a n d 105 m i n u t e s
(p<0.05 - p < 0 .005) w h e n m e a s u r e d w i t h a n t i ­
b o d y 1703 a n d at 10, 20, 30, 40, 50, 60 and 90 m i n u t e s
(p<0.05 -
p < 0 .005) w h e n m e a s u r e d w i t h a n t i b o d y ^ 2 0 4 ’ In P a t l e n t s w i t h p a n ­
c r e a t i c i n s u f f ï c i e n c y p l a s m a C C K l e v e l s at 10, 20, 30
minutes
a n d 40
(p<0.05; a n t i b o d i e s 1703 a n d l^g^) w e r e s i g n i f ï c a n t l y
higher than basal concentrations.
f e r e n c e s b e t w e e n the two g r o u p s m
T h e r e w e r e no s i g n i f i c a n t diffat-stimulated plasma CCK
c o n c e n t r a t i o n s . T h e p e a k i n c r e m e n t s in p l a s m a C C K in n o r m a l s u b ­
ject s
(5.0 ± 0.9 p m o l / 1 , a n t i b o d y 1703 and 4.3 ± 0.9 o m o l / 1 ,
an-
113
tibody T 2 o 4 > were not sic?n;i-f icantly different from those in patients with pancreatic insufficiency (4.3 ± 1.5 pmol/1, antibody
1703 and 2.9 + 0.6 pmol/1 antibody T 2 Q 4 ). Similarly, there were
no significant differences in the integrated plasma CCK secretions after ingestion of fat between the normal subjects
(264 ±
58 p m o l / 1 . 120 min., antibody 1703 and 175 ± 25 p m o l / 1 . 120 min.,
an t i b o d y T 2 Q 4 ) and the patients with pancreatic insufficiency
(217 ± 99 p m o l / 1 . 120 min, antibody 1703 and 125 ± 38 p m o l / 1 . 120
min., antibody T 2 Q 4 ) (figure 4).
Integrated
plasma CC K
p m o l / 1 . 120
minutes.
200 _
100
-
300 -
200
-
100
-
normal
subjects
pancreatic
insufficiency
Figure 4
I n t e g r a t e d p l a s m a C C K s e c r e t i o n a f t e r oral i n g e s t i o n of 250 ml
2 0% I n t r a l i p i d in 7 n o r m a l s u b j e c t s a n d 6 p a t i e n t s w i t h p a n c r e ­
a t i c i n s u f f i c i e n c y as m e a s u r e d w i t h a n t i b o d y T 2 0 4 (up per p a n e l )
a n d a n t i b o d y 1703 ( l o w e r p a n e l )
D i s cussion
In the present study plasma CCK concentrations were measured
in normal subjects and in patients with pancreatic insufficiency
by radioimmunoassay using antibodies with different s p e c i f i c i t i e s .
The development of a radioimmunoassay for CCK in plasma has proven
to be very difficult. Several factors contribute to the d i f f i c u l ­
ties in developing reliable radioimmunoassays for CCK, such as
114
d i f f ï c u l t i e s in p r o d u c i n g a n t i b o d i e s
(1 1 ), c r o s s - r e a c t i v i t y of
the a n t i b o d i e s w i t h g a s t r i n d ue to s i m i l a n t i e s in s t r u c t u r e bet w e e n g a s t r i n a n d CCK,
man and porcine CCK
suspected species differences between h u ­
(12,13),
lack of p u n f i e d or s y n t h e t i c h u m a n
C C K a n d a v a i l a b i l i t y of o n l y l i m i t e d a m o u n t s of p u r e p o r c i n e C C K
and d i f f i c u l t i e s in p r e p a r i n g i m m u n o r e a c t i v e C C K - l a b e l s
(9,14).
In the p r e s e n t s t u d y it w a s f u r t h e r s h own that C C K c o n c e n t r a t i o n s
in p l a s m a m e a s u r e d w i t h s p e c i f i c a n t i b o d i e s w e r e so l o w t h a t they
ca n o n l y be d e t e c t e d by v e r y s e n s i t i v e r a d i o i m m u n o a s s a y s . Since
p l a s m a g a s t r i n c o n c e n t r a t i o n s are m a n y t i m e s h i g h e r t h a n tho s e
of C C K (15), r a d i o i m m u n o a s s a y s s i g n i f ï c a n t l y c r o s s - r e a c t i n g w i t h
g a s t r i n a re n o t s u i t a b l e for m e a s u r e m e n t of C C K in p l a s m a . For
example, c r o s s - r e a c t i v i t y w i t h g a s t r i n a c c o u n t e d for a l m o s t all
i m m u n o r e a c t i v i t y m e a s u r e d in p l a s m a w i t h a n t i b o d y 5135. U n f o r tunat e l y , in s o m e p r e v i o u s r e p o r t s a n t i b o d i e s s h o w e d full cr o s s r e a c t i v i t y w i t h g a s t r i n , w h i l e in o t h e r s t u d i e s the c r o s s - r e a c ti v i t y of the a n t i b o d i e s w a s not t e s t e d for s u l p h a t e d g a s t r i n s
or g a s t r m - 3 4
(16-20). F a i l u r e to d e t e r m i n e the c r o s s - r e a c t i v i t y
of C C K a n t i b o d i e s w i t h s u l p h a t e d g a s t r i n s or g a s t r i n - 3 4 is the
more d i s t u r b i n g ,
first, b e c a u s e a n t i b o d i e s s p e c i f i c for the s u l ­
p h a t e d t y r o s y l r e g i o n of C C K u s u a l l y do not c r o s s - r e a c t w i t h u n ­
sulphated gastrins but often do with sulphated gastrins
(19,20),
second, b e c a u s e g a s t r i n - 3 4 is the p r e d o m i n a n t m o l e c u l a r f o r m of
g a s t r i n in p l a s m a o f f a s t i n g h u m a n s
(15).
In r a d i o i m m u n o a s s a y s for C C K in p l a s m a e i t h e r g a s t r i n - a n t i b o d i e s w i t h d i f f e r e n t d e g r e e s of c r o s s - r e a c t i v i t y w i t h C C K
(21—
24) o r m o r e o r less s p e c i f i c C C K - a n t i s e r a h a v e b e e n e m p l o y e d
(19.25.26). T h r e e s t u d i e s u s i n g r a d i o i m m u n o a s s a y s w i t h g a s t r i n C C K - a n t i b o d i e s in c o m b i n a t i o n w i t h c h r o m a t o g r a p h i c s e p a r a t i o n
t e c h n i q u e s s h o w e d t h a t b a s a l p l a s m a C C K c o n c e n t r a t i o n s w e r e belo w the d e t e c t i o n l i m i t of the a s s a y
(21-23), w h i l e o n e s t udy
r e p o r t e d v e r y h i g h b a s a l p l a s m a C C K l e vels of m o r e t h a n 100 p m o l / 1
(24). In a s s a y s u s i n g m o r e or less s p e c i f i c C C K a n t i b o d i e s b a s a l
CC K c o n c e n t r a t i o n s in h u m a n p l a s m a r a n g e d f r o m 5 to 12 p m o l / 1
(19.25.26). H o w e v e r ,
m
n o n e of t h e s e s t u d i e s the p r e s e n c e of
s i g n i f i c a n t a m o u n t s of C C K in b a s a l p l a s m a c o u l d be c o n f i r m e d by
115
c h r o m a t o g r a p h y . In the p r e s e n t study, u s i n g s p e c i f i c r a d i o i m m u n o a s s a y s f or C C K b a s a l p l a s m a C C K l e v e l s w e r e m u c h lower.
generally accepted,
It is
t h a t h o r m o n e c o n c e n t r a t i o n s are lower when
measured by radioimmunoassays employing highly immunoreactive
labels and high affinity antibodies
(8 ). The h i g h q u a l i t y of l a ­
b e l a n d a n t i b o d i e s u s e d in the p r e s e n t s t u d y is r e f l e c t e d in the
v e r y l o w i n h i b i t i o n d o s e 50 ( I D ^ )
assay
a n d d e t e c t i o n limit of the
(fig 1, t a b l e 1). T h e h i g h e r c o n c e n t r a t i o n s of C C K in bas a l
p l a s m a m e a s u r e d b y t he o t h e r a s s a y s
(19,25,26) m a y be due to
c r o s s - r e a c t i v i t y with gastrins, but may also result from a higher
s e n s i t i v i t y of the a s s a y s for n o n - s p e c i f i c factors.
In fact,
in
o n e o f t h e s t u d i e s it w a s s h o w n b y c o l u m n c h r o m a t o g r a p h y a n d c h a r ­
c o a l t r e a t m e n t t h a t t he a p p a r e n t C C K - l i k e i m m u n o r e a c t i v i t y of 12
pmol/ 1
in b a s a l p l a s m a w a s a l m o s t e x c l u s i v e l y d u e to n o n - s p e c i f i c
i n t e r f e r e n c e in t h a t a s s a y
(25). It is i n t e r e s t i n g to note that
s e c r e t i n , a n o t h e r p o l y p e p t i d e h o r m o n e r e l e a s e d f r o m the u p p e r
s m a l l i n t e s t i n e w i t h s t i m u l a t o r y a c t i o n on the p a n c r e a s c i r c u l a tes in s i m i l a r l o w c o n c e n t r a t i o n s as C C K
(27). B a s a l p l a s m a c o n ­
c e n t r a t i o n s of m o s t o t h e r g a s t r o i n t e s t i n a l h o r m o n e s are m a n y tim e s h i g h e r t h a n t h o s e of C C K a n d s e c r e tin.
T h e r e is c o n s i d e r a b l e c o n t r o v e r s y r e l a t e d to the m o l e c u l a r
f o r m s o f C C K in p o s t p r a n d i a l p l a s m a .
In the p r e s e n t study oral
i n g e s t i o n of i n t r a l i p i d i n d u c e d s i g n i f i c a n t i n c r e a s e s in p l a s m a
C C K as m e a s u r e d w i t h two s p e c i f i c C C K - a n t i b o d i e s .
S i nce a n t i b o d y
1703 b i n d s to C O O H - t e r m i n a l C C K - p e p t i d e s c o n t a i n i n g at least 14
a mino acid residues,
it c a n be c o n c l u d e d t h a t l a rge or i n t e r m e -
d i a t e f o r m s o f C C K are p r e s e n t in p l a s m a a f t e r i n g e s t i o n of fat.
M e a s u r e m e n t w i t h a n t i b o d y T^g^ i n d i c a t e s t h a t s u l p h a t e d forms of
C C K a re r e l e a s e d d u r i n g s t i m u l a t i o n . W e h a v e r e c e n t l y shown by
c o l u m n c h r o m a t o g r a p h y t h a t s e v e r a l m o l e c u l a r for m s of CCK, b o t h
l a r g e r a n d s m a l l e r t h a n CCK - 3 3 , a re r e l e a s e d d u r i n g i n f u s i o n of
b o m b e s i n in m a n
(9). S t u d i e s u s i n g r a d i o i m m u n o a s s a y t e c h n i q u e s
e m p l o y i n g g a s t r i n - a n t i b o d i e s w i t h d i f f e r e n t d e g r e e s of cr o s s reactivity with CCK have suggested that a small molecular form
of CCK, p o s s i b l y C C K - 8 , p r e d o m i n a t e s in p o s t p r a n d i a l p l a s m a
22,24 ) .
11 6
(21,
However, radioimmunoassays using more specific CCK-anti-
bo d i e s or o t h e r s e p a r a t i o n t e c h n i q u e s i n d i c a t e t h a t c o n s i d e r a b l e
a m o u n t s of l a r g e r f o r m s o f C C K a re r e l e a s e d a f t e r f e e d i n g
(19,
23,26). The f a i l u r e to d e m o n s t r a t e large m o l e c u l a r f o r m s of CCK
in the c i r c u l a t i o n b y r a d i o i m m u n o a s s a y s e m p l o y i n g g a s t r i n / C C K a n t i b o d i e s a n d c o l u m n c h r o m a t o g r a p h y c a n be a t t n b u t e d to s e v e r a l
factors,
s u c h as a r e l a t i v e l o w b i n d i n g of C O O H - t e r m i n a l a n t i ­
bo d i e s to l a r g e f o r m s of C C K
(18), o v e r l a p b e t w e e n l a r g e forms
of CC K a nd g a s t r i n s in e l u t i o n p o s i t i o n f r o m S e p h a d e x c o l u m n s
(22) and l o w r e c o v e r y of lar g e f o rms of C C K f r o m c o l u m n s du e to
a d h e r e n c e of the p e p t i d e to p l a s t i c o r glass. It h a s r e c e n t l y
b e e n shown b y h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y a n d r a d i o i m m u ­
no a s s a y u s i n g a g a s t n n / C C K - a n t i b o d y that a f t e r c o r r e c t i o n for
the lower b i n d i n g of t he a n t i b o d y t o C C K - 3 3 the m o l a r c o n c e n t r a ­
t i o n s of C C K - 3 3 - a n d C C K - 8 - l i k e ï m m u n o r e a c t i v i t y in p o s t p r a n d i a l
plasma were similar
(23). In t h i s r e s p e c t it is i n t e r e s t i n g to
n o t e that a f t e r f e e d i n g in d o g s s m a l l forms of C C K p r e d o m i n a t e
in po r t a l b l o o d , w h i l e l a r g e f o r m s o f C C K p r e d o m i n a t e in p e r i pheral plasma
(28). In a n o t h e r s t u d y by the same g r o u p it was
s h own t h a t t he d i f f e r e n t r a t i o s b e t w e e n large a n d sma l l f o r m s of
C C K in p o r t a l a n d p e r i p h e r a l p l a s m a r e s u l t e d f r o m p r e f e r e n t i a l
e l i m i n a t i o n o f s m a l l f o r m s of C C K b y p a s s a g e t h r o u g h the l i ver
(29) . In a n a l o g y ,
it is v e r y u n l i k e l y that C C K-4 c o n t n b u t e s to
C C K in p o s t p r a n d i a l p l a s m a , as s u g g e s t e d in some s t u d i e s
(24,30),
since C C K-4 is e f f e c t i v e l y i n a c t i v a t e d by p a s s a g e t h r o u g h the
live r
(31). In a n y case, C C K - 4 d o e s n o t c o n t r i b u t e to C C K ï m m u n o ­
r e a c t i v i t y m e a s u r e d in t he p r e s e n t s t u d y s i nce n o n e of t h e a n t i ­
bodies showed significant cross-reactivity with this peptide.
Th e p r e s e n c e of l a r g e f o r m s of C C K in the c i r c u l a t i o n is of phys i o l o g i c a l s i g n i f i c a n c e , b e c a u s e r e c e n t s t u d i e s h a v e s h o w n that
s m all and l a r g e f o r m s of C C K h a v e s i m i l a r p o t e n c i e s in c o n t r a c t ïng the g a l l b l a d d e r a n d s t i m u l a t m g p a n c r e a t i c e n z y m e s e c r e t i o n
(32,33). In t he a b s e n c e of a g r e e m e n t on the m o l e c u l a r for m s of
C C K in p o s t p r a n d i a l p l a s m a ,
it is e s s e n t i a l to m e a s u r e p l a s m a
CCK-concentrations by several assays using sequence-specifie an ­
t i b o d i e s as d o n e in t he p r e s e n t study.
In c o n t r a s t t o o r e v i o u s r e p o r t s
(1-5), this s t u d y s h o w e d that
117
p l a s m a C C K - c o n c e n t r a t i o n s w e r e n ot i n c r e a s e d in p a t i e n t s w i t h
pancreatic insuffï ci en cy. Both patients with alcoholic pancreatitis and those with cystic fibrosis had similar plasma CCKc o n c e n t r a t i o n s as n o r m a l s u b j e c t s . S i n c e the 3 a n t i b o d i e s u s e d
in t he a s s a y s s h o w c d b i n d i n g to d i f f e r e n t r e g i o n s of the CC K
molecule,
it is u n l i k e l y t h a t the f a i l u r e to f i n d e l e v a t e d p l a s ­
m a C C K l e v e l s in p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y is rel a t e d to the s p e c i f i c i t y of the a n t i b o d i e s . The i n c r e a s e d p l a s m a
C C K c o n c e n t r a t i o n s r e p o r t e d p r e v i o u s l y w e r e s u g g e s t e d to r e s u l t
f r o m i m p a i r e d i n h i b i t i o n o f C C K r e l e a s e by the lack of s u f f i c i ë n t
a m o u n t s of p a n c r e a t i c e n z y m e s in the u p p e r s m all int e s t i n e in p a ­
tients with pancreatic i n s u f f i c i e n c y . However,
a feed b a c k c o n t r o l
m e c h a n i s m b e t w e e n p a n c r e a t i c e n z y m e s in the u p p e r small i n t e s t i n e
a n d p a n c r e a t i c s e c r e t i o n s e e m s to e x i s t in the rat, b u t no t in
the d o g or in m a n
(34). F u r t h e r m o r e ,
it w a s s h o w n in the p r e s e n t
s t u d y t h a t i n g e s t i o n o f f a t i n d u c e d s i g n i f i c a n t i n c reases in p l a s ­
m a C C K in b o t h n o r m a l s u b j e c t s a n d in p a t i e n t s w i t h p a n c r e a t i c
i n s u f f i c i e n c y . The n s e s
in p l a s m a C C K in the p a t i e n t s w i t h p a n ­
c r e a t i c i n s u f f i c i e n c y w e r e s i m i l a r to t h o s e in no r m a l s u b jects.
It is t h e r e f o r e c o n c l u d e d t h a t in p a t i e n t s w i t h p a n c r e a t i c ïnsufficiency neither basal nor fat-stimulated plasma C C K - c o n c e n ­
t r a t i o n s are i n c r e a s e d m
c o m p a n s o n w i t h n o r m a l subjects.
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ïn-
Chapter XI
Plasma Cholecystokinin and Gallbladder Responses
to Intraduodenal Fat In Patients with
Coeliac Disease
Jan B.M.J, Jansen
Wim P.M, Hopman and Cornelis B.H.W, Lamers
Gastrointestinal Hormone Laboratory
Division of Gastroenterology
St. R a d b o u d H o s p i t a l
üniversity of Nijmegen
Nijmegen
The N e t h e r l a n d s
Submitted for Publioation
121
C h a p t e r XI P LA SM A C H O L E C Y S T O K I N I N A ND G A L L B L A D D E R RESPONSES TO
IN TR AD UODENAL FAT IN PATIE NT S WI T H COELIAC DISEASE.
Abstract
Plasma cholecystokinin
(CCK) r e s p o n s e s to i n t r a d u o d e n a l m -
s t i l l a t i o n of 60 ml c o r n o il w e r e s t u d i e d in 9 p a t i e n t s w i t h a
flat ] e ] u n a l m u c o s a due to c o e l i a c d i sease,
in 5 c o e l i a c p a t i e n t s
w i t h a n o r m a l m u c o s a on a g l u t e n - f r e e d i e t a n d in 7 normal s u b ­
jects. B a s a l p l a s m a C C K c o n c e n t r a t i o n s w e r e s i m i l a r in the 3
groups studies
pmol/1 m
jects ) .
(2.4 ± 0.5 p m o l / 1 in u n t r e a t e d pa t i e n t s , 2.2 ± 0.4
t r e a t e d p a t i e n t s a nd 2.1 ± 0.3 p m o l / 1 in normal s u b ­
I n t r a d u o d e n a l a d m i n i s t r a t i o n of fat f a i l e d to incr e a s e
p l a s m a C C K in u n t r e a t e d p a t i e n t s , w h e r e a s s i g n i f i c a n t
i n c r e a s e s w e r e o b s e r v e d in t r e a t e d p a t i e n t s
a n d in n o r m a l s u b j e c t s
(3.3 ± 1.1 p m o l/1).
(p<0.31)
(3.4 + 0.9 pmol/1)
In the 3 u n t r e a t e d
p a t i e n t s s t u d i e d by u l t r a s o n o g r a p h y g a l l b l a d d e r v o l u m e was una f f e c t e d b y a d m i n i s t r a t i o n of fat, w h e r e a s g a l l b l a d d e r v o l u m e
d e c r e a s e d b y 70 ± 7% in 4 t r e a t e d p a t i e n t s a n d by 83 + 5% in 7
n o r m a l sub j e c t s .
It is c o n c l u d e d t h a t m
patients with untreated coeliac d i s ­
e a s e i n t r a d u o d e n a l i n s t i l l a t i o n of fat does no t incr e a s e p l a s m a
C C K c o n c e n t r a t i o n s a n d d o e s n ot i n d u c e g a l l b l a d d e r con t r a c t i o n ,
w h e r e a s c o e l i a c p a t i e n t s on a g l u t e n - f r e e d i e t s h o w no r m a l in­
c r e a s e s in p l a s m a C C K a c c o m p a n i e d b y c o n t r a c t i o n of the g a l l b l a d ­
der. T h e s e s t u d i e s p o i n t to an i m p o r t a n t role for C C K in the reg u l a t i o n of g a l l b l a d d e r c o n t r a c t i o n in r e s p o n s e to i n t r a d u o d e n a l
fat.
Introduction
C o e l i a c d i s e a s e is c h a r a c t e r i z e d b y a g l u t e n - i n d u c e d h y p e r r e g e n e r a t i v e v i l l o u s a t r o p h y of t he s ma l l i n t e s t i n a l m u c o s a res u l t i n g in m a l a b s o m t i o n a n d n u t r i t i o n a l d e f i c i e n c y
(1). M o s t
coeliac patients show a favourable clinical and histological r e s ­
p o n s e t o w i t h d r a w a l of g l u t e n f r o m t h e i r d i e t
(1). It has p r e v i -
ously been reported that coeliac patients often show a reduced
p a n c r e a t i c e n z y m e s e c r e t i o n a n d an i m p a i r e d g a l l b l a d d e r c o n t r a c -
1 22
tion in r e s p o n s e to i n t e s t i n a l s t i m u l i
(2,3). Th e m e c h a n i s m for
this i m p a i r e d p a n c r e a t i c s e c r e t i o n a n d g a l l b l a d d e r c o n t r a c t i o n
has n o t b e e n e l u c i d a t e d . B a s e d on h i g h c i r c u l a t i n g c h o l e c y s t o k i ­
nin
(CCK) c o n c e n t r a t i o n s a nd a r e d u c e d p a n c r e a t i c r e s p o n s e to
stimulation with exogenous hormones,
it wa s s u g g e s t e d t h a t the
g a l l b l a d d e r a n d p a n c r e a s in c o e l i a c p a t i e n t s w e r e less s e n s i t i v e
to h o r m o n a l s t i m u l a t i o n
(3). A n o t h e r study, howe v e r ,
s h o w e d that
in c o e l i a c patieijts g a l l b l a d d e r a n d p a n c r e a t i c r e s p o n s e s to e x o ­
genous hormones were normal
(2). It w a s t h e r e f o r e c o n c l u d e d that
the i m p a i r e d g a l l b l a d d e r a n d p a n c r e a t i c r e s p o n s e s to i n t e s t i n a l
n u t r i e n t s w e r e s e c o n d a r y to a r e d u c e d s e c r e t i o n of C C K f r o m the
u p p e r small i n t e s t i n e
(2). In the a b s e n c e of r e l i a b l e r a d i o i m m u ­
no a s s a y s for C C K t h i s c o n t r o v e r s y c o u l d not be sett l e d . We have
r e c e n t l y d e v e l o p e d a s e n s i t i v e a n d s p e c i f i c r a d i o i m m u n o a s s a y for
CCK
(4). U s i n g t h i s a s s a y w e h a v e s t u d i e d p l a s m a C C K r e s p o n s e s to
i n t r a d u o d e n a l fat in p a t i e n t s w i t h a flat j e j u n a l m u c o s a du e to
coeliac disease,
in c o e l i a c p a t i e n t s w i t h a n o r m a l m u c o s a on a
g l u t e n - f r e e d i e t a n d in n o r m a l s u b j e c t s.
In ad d i t i o n ,
m
some of
the s u b j e c t s a a l l b l a d d e r v o l u m e b e f o r e and at r e g u l a r i n t e r v a l s
a f t e r i n t r a d u o d e n a l i n s t i l l a t i o n o f f at w a s m e a s u r e d b y u l t r a sonography.
Patients and methods
Pl a s m a C C K c o n c e n t r a t i o n s w e r e m e a s u r e d in 9 c o e l i a c p a t i e n t s
w i t h a flat j e j u n a l m u c o s a b e f o r e g l u t e n - w i t h d r a w a l
(3M, 6 F, age
27 - 70 years) , in 5 c o e l i a c p a t i e n t s w i t h a n o r m a l m u c o s a d u n n g
gluten-free diet
jects
(1M, 4F, age 21 - 51 years)
an d in 7 n o r m a l s u b ­
(5M, 2F, a ge 21 - 28 y e a r s ) . T w o of the p a t i e n t s w e r e s t u ­
d ied b e f o r e g l u t e n - w i t h d r a w a l a n d d u n n g c o m p l e t e n o r m a l i s a t i o n
of small b o w e l m u c o s a a n d l a b o r a t o r y p a r a m e t e r s on a g l u t e n - f r e e
diet. In 3 of t h e u n t r e a t e d p a t i e n t s ,
in 4 of the t r e a t e d p a t i e n t s
an d in 7 n o r m a l s u b j e c t s g a l l b l a d d e r v o l u m e s w e r e s t u d i e d b y ultrasonography.
A f t e r an o v e r n i g h t f a s t the t i p o f a d u o d e n a l t u b e w a s p o s i t i o n e d in the t h i r d p a r t of t he d u o d e n u m u n d e r f l u o r o s c o p i c c o n t r o l . 60 m l c o r n oil w a s s u b s e q u e n t l y a d m i n i s t e r e d i n t r a d u o d e n a l l y
123
w i t h i n 5 m i n u t e s . B l o o d s a m p l e s for C C K w e r e d r a w n from an ïnd w e l l i n g v e n o u s c a t h e t e r at -10, 0, 10, 20, 30, 40, 50, 60, 75
a n d 90 m i n u t e s . B l o o d w a s c o l l e c t e d in ï c e - c h i l l e d g l ass t u b e s
c o n t a i n i n g 2 g /1 e t h y l e n e d i a m m e t e t r a a c e t a t e . S a m p l e s were c e n t n f u g e d a n d t he p l a s m a w a s s t o r e d at -20 °C w i t h i n 2 h o u r s a f ­
t e r c o l l e c t i o n . P l a s m a C C K c o n c e n t r a t i o n s w e r e m e a s u r e d by a s e n ­
sitive and specific radioimmunoassay
(4). A n t i b o d y ^ 204' r a l s e d
m a r a b b i t a f t e r the f o u r t h i m m u n i z a t i o n w i t h a l b u m i n - c o u n l e d
C C K (5), w a s u s e d in a d i l u t i o n of 1:80,000. T h e a n t i b o d y b i n d s
to a ll c a r b o x y l - t e r m i n a l C C K - p e p t i d e s c o n t a i n i n g the s u l p h a t e d
tyrosyl region
(fig 1).
T h e a n t i b o d y s h o w s v e r y small c r o s s -
reactivity with sulphated gastrins
(about 2 %), b u t does no t s h o w
a n y b i n d i n g to u n s u l p h a t e d g a s t r i n s or s t r u c t u r a l l y u n r e l a t e d
regulatory peptides
(4).
Figure 1
B i n d i n g of h i g h l y p u r i f i e d
porcine and synthetic CCK
p e p t i d e s to a n t i b o d y T204.
99% pu r e p o r c i n e C C K - 3 3
c o u p l e d to 1 2 5 I - h y d r o x y p h e n y l p r o p i o m c acidsuccinimide ester (BoltonHunter reagent) was used
as l a b e l .
10
124
100
1000
(p m o l / 1)
99% p u r e p o r c i n e C C K 33 w a s u s e d as S t a n d a r d . T h e d e t e c t i o n l i ­
m i t o f t he a s s a y w a s b e t w e e n 0.5 a n d 1.0 p m o l / l p l a sma. T h e int r a - a s s a y p r e c i s i o n w a s 11.5% at 1.2 p m ol/l, 8.4% at 2.5 p m o l / l
an d 4.6% a t 8.0 p m o l / l (n=5), w h i l e t he i n t e r - a s s a y o r e c i s i o n was
26.1% a t 1.3 p m o l / l , 11.3% a t 2.6 p m o l / l a n d 15.4% at 8 . 8 p m ol/l.
P l a s m a s a m p l e s w e r e m e a s u r e d in d u p l i c a t e .
G a l l b l a d d e r v o l u m e s w e r e m e a s u r e d b e f o r e a n d d u r i n g the
f i r s t h o u r o f the s t u d y b y u l t r a s o n o g r a p h y u s i n g the su m of cyl i n d e r s m e t h o d as d e s c r i b e d b y E v e r s o n
(6 ). T h e m e a n of 3 m e a -
s u r e m e n t s w a s u s e d for c a l c u l a t i o n . T h e v a r i a t i o n of this m e t h o d
for t he v o l u m e s m e a s u r e d r a n g e d f r o m 10.0 to 17.5%.
R e s u l t s w e r e e x p r e s s e d as m e a n ± 1 SEM. T h e i n t e g r a t e d p l a s ­
m a C C K c o n c e n t r a t i o n s w e r e d e t e r m i n e d b y c a l c u l a t i n g the a r e a u n ­
d e r the c u r v e a f t e r s u b t r a c t i o n o f t he b a s a l va l u e . S t a t i s t i c a l
a n a l y s i s w a s d o n e b y S t u d e n t ' s t - t e s t for p a i r e d a n d u n p a i r e d
resul t s .
I n f o r m e d c o n s e n t w a s o b t a i n e d f r o m all s u b j e c t s studied.
Results
B a s a l p l a s m a C C K c o n c e n t r a t i o n s w e r e s i m i l a r in all 3 g r o u p s
studied
(2.4 ± 0.5 p m o l / l in u n t r e a t e d p a t i e n t s , 2.2 ± 0.4 p m o l / l
in t r e a t e d p a t i e n t s a n d 2.1 ± 0.3 p m o l / l in n o r m a l s u b j e c t s ) .
I n t r a d u o d e n a l i n s t i l l a t i o n of c o r n o i l in n o r m a l s u b j e c t s i n d u c e d
s i g n i f i c a n t i n c r e a s e s in p l a s m a C C K at 20, 30, 40, 50, 60 an d 75
minutes
( p<0.05 - p<0.001).
In u n t r e a t e d c o e l i a c p a t i e n t s i n t r a ­
d u o d e n a l i n s t i l l a t i o n of c o r n oil f a i l e d to i n c r e a s e p l a s m a C C K
concentrations
(fig 2). In c o n t r a s t , c o e l i a c p a t i e n t s on a g l u t e n
f r e e d i e t s h o w e d p l a s m a C C K r e s p o n s e s to c o r n oi l s i m i l a r to those f o u n d in n o r m a l s u b j e c t s
(fig 3). In t h e s e p a t i e n t s p l a s m a C C K
l e v e l s w e r e s i g n i f i c a n t l y e l e v a t e d o v e r b a s a l v a l u e at 30, 40, 50,
60 a n d 75 m i n u t e s
(p<0.05 - p < 0 . 0 0 5 ) . T h e p e a k i n c r e m e n t s in p l a s ­
m a C C K in u n t r e a t e d p a t i e n t s
ly l o w e r
pmol/l)
( p < 0 .01)
(0.1 ± 0.5 pmol/l) w e r e s i g n i f i c a n t ­
t h a n t h o s e f o u n d in n o r m a l s u b j e c t s
a n d in t r e a t e d p a t i e n t s
(3.3 ± 1.1
(3.4 ± 0.9 p m o l / l ) . S i m i l a r l y ,
t h e i n t e g r a t e d p l a s m a C C K c o n c e n t r a t i o n s in r e s p o n s e to c o r n oil
in th e u n t r e a t e d p a t i e n t s
(p<0.001)
(11.8 ± 8.3 p m o l / l . h r ) w a s s i g n i f i c a n t l y
l o w e r t h a n in n o r m a l s u b j e c t s
(110.2 ± 29.1 p m o l / l . h r )
125
a n d in the t r e a t e d c o e l i a c p a t i e n t s
(137.2 ± 39.8 p m o l / 1 . h r ) .
T h e i n t e g r a t e d C C K s e c r e t i o n in the t r e a t e d p a t i e n t s w a s n o t
s i g n i f i c a n t l y d i f f e r e n t f r o m the n o r m a l s u b j e c t s
(fig 4). In the
no r m a l s u b j e c t s t he i n c r e a s e in p l a s m a C C K a f t e r i n t r a d u o d e n a l
c o r n oil w a s a c c o m p a n i e d b y s i g n i f i c a n t d e c r e a s e s in g a l l b l a d d e r
volume
(fig 5). T h e g a l l b l a d d e r v o l u m e s at 30, 40, 50 an d 60
minutes were significantly lower
(p<0.05 - p<0.001)
than basal
vo l u m e . T h e r e w a s a h i g h l y s i g n i f i c a n t i n v e r s e c o r r e l a t i o n b e ­
tween plasma CCK concentrations and gallbladder volumes
(r=
0.931, p < 0 . 0 1 ) .
Time (min)
Figure
2
P l a s m a C C K r e s p o n s e s to i n t r a d u o d e n a l i n s t i l l a t i o n of 60 m l c o r n
o i l in 9 c o e l i a c p a t i e n t s w i t h a f l a t m u c o s a ( o p e n ci r c l e s ) a n d
7 normal subjects (closed circles). Asterisks denote significant
d i f f e r e n c e s b e t w e e n the two g r ou p s.
126
Th e 3 u n t r e a t e d c o e l i a c p a t i e n t s t e s t e d showed, in a d d i t i o n to
the a b s e n c e in i n c r e a s e s in p l a s m a CCK, a f a i l u r e of g a l l b l a d d e r
c o n t r a c t i o n in r e s p o n s e to i n t r a d u o d e n a l c o r n oil
(fig 5).
In
con t r a s t , in t he 4 t r e a t e d c o e l i a c p a t i e n t s s t u d i e d , the i n c r e a ­
ses in p l a s m a C C K w e r e a c c o m p a n i e d b y d e c r e a s e s in g a l l b l a d d e r
v o l u m e s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e f o u n d in n o r m a l
subjects
(fig 6 ) .
Time (min)
Figure 3
P l a s m a C C K r e s p o n s e s to i n t r a d u o d e n a l i n s t i l l a t i o n o f 6 0 m l c o r n
o i l in 5 c o e l i a c p a t i e n t s w i t h a n o r m a l m u c o s a o n a g l u t e n - f r e e
diet (open circles) and 7 normal subjects (closed circles).
127
I n t e g r a t e d g a l l b l a d d e r v o l u m e s i n t he u n t r e a t e d c o e l i a c p a t i e n t s
3
(-1.1 ± 0.5 c m .hr) w e r e s i g n i f i c a n t l y (p<0.001) low e r t h a n t h ose
3
in n o r m a l s u b j e c t s (161.0 ± 29.5 c m .hr) a n d in the t r e a t e d patients
3
(132.9 ± 16.9 c m . h r ) . N o n e o f th e s u b j e c t s e x p e r i e n c e d
a d v e r s e r e a c t i o n s d u r i n g the study.
Integrated CCK re sp o n se
( p m o l / l . hr)
norm al
subjects
(n = 7 )
celiacs
flat
m u co sa
(n = 9 )
Figure
celiacs
norm al
m u co sa
(n = 5 )
4
I n t e g r a t e d p l a s m a C CK s e c r e t i o n in r e s p o n s e to i n t raduodenal
i n s t i l l a t i o n of 60 ml c o r n oil in 7 n o r m a l subjects, 9 coeliac
p a t i e n t s w i t h a flat m u c o s a and 5 c o e l i a c p a t i e n t s with a n o r ­
mal m u c o s a on a g l u t e n - f r e e diet.
128
0
10
20
30
*0
50
eo
T m * (m in)
Figure 5
G a l l b l a d d e r v o l u m e r e s p o n s e s to i n t r a d u o d e n a l i n s t i l l a t i o n of
c o r n o i l in 3 c o e l i a c p a t i e n t s w i t h a f l a t m u c o s a a n d in 7 n o r m a 1 sub)ec t s .
G a l l b l a d d e r v o l u m e r e s p o n s e s to i n t r a d u o d e n a l i n s t i l l a t i o n of
c o r n o i l in 3 u n t r e a t e d c o e l i a c p a t i e n t s a n d 4 t r e a t e d c o e l i a c
patients. A s t e . n s k s denote significant differences between
the 2 groups.
129
Discussion
T h e p r e s e n t s t u d y s h o w e d t h a t in p a t i e n t s w i t h a flat m u c o s a
du e to c o e l i a c d i s e a s e i n t r a d u o d e n a l i n s t i l l a t i o n of fat f a i l e d
to i n d u c e i n c r e a s e s in p l a s m a CCK.
2 p r e v i o u s s t udies on the e f ­
fect of n u t r i e n t s o n p l a s m a C C K in c o e l i a c p a t i e n t s have been
reported
(3,7). L o w - B e e r et al
(3) f o u n d t h a t b a s a l p l a s m a CCK
c o n c e n t r a t i o n s in p a t i e n t s w i t h c o e l i a c d i s e a s e w e r e a b o u t 15
t i m e s h i g h e r t h a n t h o s e in n o r m a l s u b jects, w h i l e the peak p l a s ­
m a C C K c o n c e n t r a t i o n s a f t e r an o r a l fat m e a l w e r e not s i g n i f i c a n t l y d i f f e r e n t . T he r e l i a b i l i t y of the r a d i o i m m u n o a s s a y for
C C K u s e d in t h a t s t u d y h a s r e p e a t e d l y b e e n q u e s t i o n e d
the o t h e r hand, C a l a m et al
(8,9). On
(7) f o u n d t h a t C C K - 8 was u n d e t e c t a -
bl e in b a s a l p l a s m a f r o m b o t h c o e l i a c p a t i e n t s a n d normal s u b ­
jects. A f t e r i n g e s t i o n of a m i x e d m e a l C C K - 8 wa s d e t e c t a b l e in
p l a s m a f r o m all 5 n o r m a l s u bjects, b u t in p l a s m a from o n l y one
of 6 c o e l i a c p a t i e n t s s t u d i e d
(7). In the l a t t e r study CC K was
m e a s u r e d b y r a d i o i m m u n o a s s a y u s i n g a C C K - a n t i b o d y fully c r o s s r e a c t i n g w i t h g a s t r i n in c o m b i n a t i o n w i t h c o l u m n c h r o m a t o g r a p h y .
By t h i s a s s a y o n l y C C K - 8
in p l a s m a
like i m m u n o r e a c t i v i t y c o u l d be d e t e c t e d
(7). S e v e r a l r e c e n t s t u dies, h o w e v e r , h a v e i n d i c a t e d
t h a t m o l e c u l a r f o r m s l a r g e r than C C K - 8 are p r e s e n t in h u m a n and
canine plasma
(10-13). T he a n t i b o d y u s e d in the p r e s e n t s t udy
b i n d s to b o t h C C K - 8 a nd to l a r g e r m o l e c u l a r f o r m s of CC K
(6,13) .
Th e f i n d i n g of i m p a i r e d C C K s e c r e t i o n in r e s p o n s e to intest i n a l n u t r i e n t s in c o e l i a c p a t i e n t s is n o t u n e x p e c t e d ,
since a
r e d u c e d s e c r e t i o n of 2 o t h e r p o l y p e p t i d e h o r m o n e s p r o d u c e d in
the u p p e r s m a l l i n t e s t i n a l m u c o s a h as p r e v i o u s l y b e e n r e p o r t e d
(14). I n t r a d u o d e n a l i n s t i l l a t i o n of a c i d r e s u l t e d in a r e d u c e d
s e c r e t i n p r o d u c t i o n , w h i l e i n g e s t i o n of a m i x e d m e a l i n d u c e d a
s u b n o r m a l r e l e a s e of g a s t r i c i n h i b i t o r y p o l y p e p t i d e
(GIP).
S i n c e C C K is k n o w n to s t i m u l a t e g a l l b l a d d e r c o n t r a c t i o n
(15),
the f a i l u r e of c o e l i a c p a t i e n t s to c o n t r a c t t h e i r g a l l b l a d d e r a f ­
t e r i n t r a d u o d e n a l fat is n o t u n e x p e c t e d . U s i n g i s o t o p e c h o l e c y s t o g r a p h y , L o w - B e e r et al
(3) s h o w e d t h a t i n g e s t i o n of a fatty
meal induced subnormal but significant gallbladder contraction
in c o e l i a c p a t i e n t s , w h i l e D i M a g n o et al
130
(2) f o u n d that b i l i r u b i n
o u t p u t into t h e d u o d e n u m d i d n o t r e s p o n d to d u o d e n a l p e r f u s i o n
w i t h e s s e n t i a l a m i n o acids.
In the p r e s e n t s t u d y t h e a b s e n c e of
g a l l b l a d d e r c o n t r a c t i o n w a s d e m o n s t r a t e d by u l t r a s o n o g r a p h y , a
s e n s i t i v e t e c h m q u e o b v i a t i n g the n e e d for i n t u b a t i o n or the use
of i s o t o p e s
(6 ). I m p a i r e d g a l l b l a d d e r c o n t r a c t i o n h a s b e e n s u g ­
g e s t e d to p l a y a p o s s i b l e r o l e in the p a t h o g e n e s i s of g a l l s t o n e s
(16). H o w e v e r ,
to o u r k n o w l e d g e n o i n c r e a s e d p r e v a l e n c e of c h o l e -
li t h i a s i s in c o e l i a c p a t i e n t s has b e e n r e ported.
It h a s b e e n spe-
cu l a t e d t h a t u n d e r s a t u r a t i o n of g a l l b l a d d e r b i l e as a r e s u l t of
the i n t e s t i n a l l e s i o n p r e v e n t s the f o r m a t i o n of g a l l s t o n e s m
such p a t i e n t s
(17).
C o e l i a c p a t i e n t s w i t h n o r m a l ^ e j u n a l m u c o s a on a g l u t e n - f r e e
d i e t s h o w e d n o r m a l p l a s m a C C K r e s p o n s e s to m t r a d u o d e n a l
mstil-
lation of fat. In t h e s e p a t i e n t s f a t - i n d u c e d i n c r e a s e s in p l a s m a
CCK were accompanied by effective gallbladder contraction. These
findi n g s s u g g e s t t h a t the i m p a i r m e n t of p l a s m a C C K a n d g a l l b l a d ­
de r r e s p o n s e s to fat in c o e l i a c p a t i e n t s is r e v e r s i b l e . T h i s sugg e s t i o n w a s s u b s t a n t i a t e d in t h e 2 p a t i e n t s w h o c o u l d be s t u d i e d
before and after gluten withdrawal.
R e c e n t l y , t h e e x i s t e n c e of an e n t e r o c h o l e c y s t i c r e f l e x m e c h a n i s m for the s t i m u l a t i o n of g a l l b l a d d e r c o n t r a c t i o n in r e s p o n s e
to i n t e s t i n a l s t i m u l i h a s b e e n s u g g e s t e d
(18). T h e p r e s e n t study,
h owever, p o i n t s to an i m p o r t a n t r o l e for CC K in th e s t i m u l a t i o n
of g a l l b l a d d e r c o n t r a c t i o n a f t e r m t r a d u o d e n a l fat. T h i s f i n d i n g
is in a g r e e m e n t w i t h a p r e v i o u s s t u d y s h o w i n g t h a t i n f u s i o n of
C C K to p l a s m a c o n c e n t r a t i o n s s i m i l a r to tho s e f o u n d a f t e r a m e a l
in duce s g a l l b l a d d e r c o n t r a c t i o n as m e a s u r e d b y u l t r a s o n o g r a p h y
In c o n c l u s i o n ,
(19).
the p r e s e n t s t u d y s hows that in c o e l i a c p a ­
ti e n t s w i t h a f l a t je;junal m u c o s a , p l a s m a C C K and g a l l b l a d d e r r e s ­
p o n s e s to m t r a d u o d e n a l fat a r e g r e a t l y i m p aired, w h e r e a s such
f u n c t i o n s a re n o r m a l in c o e l i a c p a t i e n t s on a g l u t e n - f r e e diet.
T h e s e f i n d i n g s p o i n t to a n i m p o r t a n t r o le for C C K m
the r e g u l a -
tion of g a l l b l a d d e r c o n t r a c t i o n a f t e r m t r a d u o d e n a l fat.
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cholecystokinin.
Dig
profile
contrac­
W I L L I A M S L F . T h e r o l e of
of c h o l e s t e r o l g a l l s t o n e s .
THOMPSON
J C . Correlation
of
re-
lease and
vagotomy.
19.
actions
Surgery
of c h o l e c y s t o k i n i n
1 9 8 3 ; 93: 7 8 6 - 7 9 1 .
in d o g s
before
B Y R N E S D J , B O R O D Y T, D A S K A L O P O U L O S G, B O Y L E M,
c y st o ki n i n and ga l lb l ad d e r contraction: effect
P e p t i d e s 1 9 8 1 ; 2 ( s u p p l 2): 2 5 9 - 2 6 2 .
and
after
B E N N I. C h o l e ­
of C C K infusi on .
1 33
SUMMARY
135
SUMMARY
C h o l e c y s t o k i n i n t o g e t h e r w i t h s e c r e t i n and g a s t r i n c o n s t i t u t e t he c l a s s i c a l t r i a d of g u t h o r m o n e s . W h i l e the rol e s of
s e c r e t i n a n d g a s t r i n hav e , in t he m e a n t i m e , b e e n w e l l e s t a b l i s h ed, t h e p h y s i o l o g i c a l a n d p a t h o p h y s i o l o g i c a l r o l e s of CCK h a v e
r e m a i n e d s p e c u l a t i v e , m a i n l y d u e to the l a c k of s p e c i f i c a n d rel i a b l e r a d i o i m m u n o a s s a y s f or CCK. S e v e r a l r e a s o n s d i s c u s s e d in
c h a p t e r I m a y a c c o u n t f or this s i t u a t i o n , like size and s p e c i e s
h e t e r o g e n e i t y , s i m i l a r i t y b e t w e e n the b i o l o g i c a l l y ac t i v e p a r t of
CCK and gastrin,
l a b e l l i n g p r o b l e m s a nd s u s p e c t e d c o n f o r m a t i o n a l
c h a n g e s b e t w e e n d i f f e r e n t m o l e c u l a r f o rms of CCK. F u r t h e r m o r e ,
C C K is n o t o n l y p r e s e n t in t h e sma l l b o w e l m u c o s a , but a l s o in
n e r v e s i n t he g a s t r o i n t e s t i n a l tract, i n t r o d u c i n g the p o s s i b i l i ty t h a t C C K n o t o n l y a c t s as a h o r m o n e , b u t also as a n e u r o t r a n s ­
mitter,
at l e a s t in s o m e r e g i o n s of the gut.
T h e a i m of the s t u d i e s p r e s e n t e d in this thesis was to dev e l o p a s p e c i f i c a n d s e n s i t i v e r a d i o i m m u n o a s s a y for CCK. D a t a
a b o u t t h e p r o d u c t i o n a n d e v a l u a t i o n of a n t i s e r a w e r e p r e s e n t e d
in c h a p t e r II. I m m u n i z a t i o n of r a b b i t s w i t h C C K - 3 3 r e s u l t e d in
h i g h t i t e r a n t i b o d i e s w h i c h c o u l d be u s e d in s e n s i t i v e and s p e ­
c i f i c r a d i o i m m u n o a s s a y s f or CCK.
125
I n t r o d u c t i o n of
I i n t o a p o l y p e p t i d e m a y re s u l t in c h a n ­
g e s in i m m u n o r e a c t i v i t y . T h e e v a l u a t i o n of a n t i s e r a t e s t e d w i t h
C C K - l a b e l s p r e p a r e d b y d i f f e r e n t t e c h n i q u e s w a s p r e s e n t e d in
c h a p t e r s III a n d IV. In c h a p t e r III it w a s s h o w n that l a b e l l i n g
125
with
I-hydroxyphenylpropionic acid-hydroxysuccinimide ester
( B o l t o n - H u n t e r reagent)
r e s u l t e d in l a b e l s w i t h e x c e l l e n t immu-
noreactivity when tested with non-carboxyl-terminal CCK-antibo­
d i es , b u t e x t r e m e l y l o w b i n d i n g to c a r b o x y l - t e r m i n a l a n t i b o d i e s ,
w h i l e in c h a p t e r I V it w a s s h o w n t h a t d i f f e r e n t C C K - l a b e l s b o u n d
to d i f f e r e n t b i n d i n g s i t e s in the s a m e a n t i s e r u m
(antibody h e t e ­
r o g e n e i t y ) . T h e p a t t e r n o f i m m u n o r e a c t i v i t y of the a n t i s e r u m to
C C K - p e p t i d e s w a s d e p e n d e n t o n the t y p e of lab e l used. T h e r e f o r e ,
antis e r a raised against CCK should be characterized employing
d ifferent CCK-labels.
1 36
It a p p e a r e d t h a t o n e of the a n t i s e r a w a s s p e c i f i c for the
t r i a c o n t a n o n a p e p t i d e of C C K
( C C K - 3 9 ) . U s i n g this a n t i s e r u m in a
s e n s i t i v e a n d s p e c i f i c r a d i o i m m u n o a s s a y for C C K - 3 9 , C C K - 3 9 - l i k e
i m m u n o r e a c t i v i t y w a s d e m o n s t r a t e d in e x t r a c t s of p o r c i n e brain.
T h e s e d a t a w e r e p r e s e n t e d in c h a p t e r V.
The r e l i a b i l i t y of h i g h l y s e n s i t i v e an d s p e c i f i c r a d i o i m ­
m u n o a s s a y s f o r m e a s u r e m e n t of C C K in h u m a n t i s s u e a n d b l o o d u s ­
ing two a n t i b o d i e s
(1703 and T 2 0 4 ) , w a s e x t e n s i v e l y e v a l u a t e d
in c h a p t e r VI. T h e b i n d i n g b e t w e e n label an d a n t i b o d y w a s inhib i t e d b y 50%
(ID j-q ) at a c o n c e n t r a t i o n of 2.8 p m o l / 1 C C K - 3 3 for
a n t i b o d y 1703, a n d a t a c o n c e n t r a t i o n of 3.3 p m o l / 1 C C K - 3 3 for
a n t i b o d y T204. T h e d e t e c t i o n l i m i t of the a s s a y w a s b e t w e e n 0.5
and 1.0 p m o l / 1 p l a s m a . P l a s m a s a m p l e s w e r e e x t r a c t e d in 96% e ­
t hano l p r i o r to a ssay.
Infusion of exogenous CCK-33,
resulting
in p l a s m a C C K - l e v e l s s l i g h t l y l o w e r t han t h o s e m e a s u r e d d u r i n g
a d m i n i s t r a t i o n of fat,
i n d u c e d p a n c r e a t i c e n z y m e s e c r e t i o n and
g a l l b l a d d e r c o n t r a c t i o n . T h e r e l i a b i l i t y of t h e s e r a d i o i m m u n o as s a y s f o r m e a s u r e m e n t s in h u m a n p l a s m a wa s e x t e n s i v e l y e v a l u ­
ated.
In c h a p t e r s II a n d I V it w a s s h o wn that oral a n d i n t r a d u ­
o d e n a l a d m i n i s t r a t i o n of fat s t i m u l a t e d p l a s m a C C K s e c r e t i o n .
In c h a p t e r s V I I a n d V I I I the e f f e c t of o t h e r s t i m u l i for p a n ­
c r e a t i c e n z y m e s e c r e t i o n on p l a s m a C C K c o n c e n t r a t i o n s w e r e studied. In c h a p t e r V I I the e f f e c t o f i n s u l i n - h y p o g l y c a e m i a w a s
studied.
It w a s d e m o n s t r a t e d t h a t a c t i v a t i o n of v a g a l c h o l i n e r -
gic m e c h a n i s m s b y i n s u l i n - h y p o g l y c a e m i a i n d u c e d a s i g n i f i c a n t
rise in p l a s m a p a n c r e a t i c p o l y p e p t i d e . Howev e r , p l a s m a C C K c o n ­
centrations did not significantly change during insulin-hypoglycae m i a .
In c h a p t e r V I I I t he e f f e c t of b o m b e s i n on p l a s m a CCK
wa s studied. B o m b e s i n is a t e t r a d e c a p e p t i d e w i t h s t i m u l a t o r y actions on a n t r a l g a s t r i n r e l e a s e .
I n f u s i o n of b o m b e s i n in no r m a l
s ubj e c t s a n d g a s t r e c t o m i z e d p a t i e n t s s h o w e d t h a t b o m b e s i n r e l e a s ed C C K in m a n b y a g a s t r i n a n d g a s t r i c a c i d i n d e p e n d e n t m e c h a nism.
The m o l e c u l a r f o r m s o f C C K in p l a s m a d u r i n g i n f u s i o n of
b o m b e s i n in m a n w e r e r e p o r t e d in c h a p t e r IX. F r a c t i o n a t i o n by
137
S e p h a d e x G 50 S F c o l u m n c h r o m a t o g r a p h y r e v e a l e d 4 m o l e c u l a r
f o r m s of C C K in B B S - s t i m u l a t e d p l a s m a. P e a k I e l u t e d in the
v o i d v o l u m e a n d c o m p r i s e d 0-7% of t o t a l C C K - l i k e i m m u n o r e a c t i vity.
P e a k II e l u t e d a t 35% a n d c o m p r i s e d 8-41% of total CCK-
l i k e i m m u n o r e a c t i v i t y . P e a k III e l u t e d at 50% a n d c o m p r i s e d
4 4 - 6 1 % o f t o t a l C C K - l i k e i m m u n o r e a c t i v i t y . P e a k IV eluted at
7 5% a n d c o m p r i s e d 1 5 - 2 7 % o f t o t a l C C K - l i k e i m m u n o r e a c t i v i t y .
In c h a p t e r s X a nd XI p l a s m a C C K w a s m e a s u r e d in p a t i e n t s
w i t h c l i n i c a l c o n d i t i o n s , in w h o m p r e v i o u s l y a b n o r m a l CCK value s h a v e b e e n s u g g e s t e d .
In c h a p t e r X p a t i e n t s w i t h p a n c r e a ­
tic i n s u f f i c i e n c y d u e to a l c o h o l o r c y s t i c f i b r o s i s w e r e s t u ­
die d .
It w a s s h o w n t h a t C C K c o n c e n t r a t i o n s in f a s t i n g pl a s m a
w e r e v e r y low, t h a t o r a l f a t i n d u c e d s i g n i f i c a n t i n c reases in
p l a s m a CCK, a n d t h a t in p a t i e n t s w i t h p a n c r e a t i c i n s u f f i c i e n c y
pla s m a CCK levels were not increased.
In c h a p t e r XI the e f f e c t
of i n t r a d u o d e n a l fat o n p l a s m a C C K a n d o n g a l l b l a d d e r cont r a c t i o n in p a t i e n t s w i t h c o e l i a c d i s e a s e w a s r e p o r t e d . In p a t i e n t s
w i t h u n t r e a t e d c o e l i a c d i s e a s e i n t r a d u o d e n a l i n s t i l l a t i o n of
fat d i d n o t i n c r e a s e p l a s m a C C K c o n c e n t r a t i o n s an d d i d not ind u c e g a l l b l a d d e r c o n t r a c t i o n , w h e r e a s c o e l i a c p a t i e n t s on a
g l u t e n - f r e e d i e t s h o w e d n o r m a l i n c r e a s e s in p l a s m a C C K a c c o m p a n i e d b y c o n t r a c t i o n of the g a l l b l a d d e r . T h e s e s t u d i e s p o i n t e d
to a n i m p o r t a n t r o l e for C C K in t he r e g u l a t i o n of g a l l b l a d d e r
contraction.
1 38
SAMENVATTING
Cholecystokinine
( C C K ) , s e c r e t i n e en g a s t r i n e z i j n d e d r i e
r.ieest b e k e n d e m a a g - d a r m h o r m o n e n . D e f u n c t i e s v a n g a s t r i n e en s e ­
c r e t i n e z i j n i n m i d d e l s g r o t e n d e e l s a c h t e r h a a l d , m a a r de f y s i o l o ­
g i s c h e en p a t h o f y s i o l o g i s c h e b e t e k e n i s v a n C C K is n o g v r i j w e l o n ­
be k e n d . D i t k o m t h o o f d z a k e l i j k o m d a t er t o t v o o r k o r t g e e n b e ­
trouwbare en specifieke r a d ioimmunologische b e p a l i n g e n voor CCK
be s t o n d e n . H i e r v o o r z i j n v e r s c h i l l e n d e o o r z a k e n a a n te w i j z e n
w a a r v a n d e b e l a n g r i j k s t e in h o o f d s t u k I w e r d e n b e s p r o k e n . V o o r a l
h e t v o o r k o m e n v a n C C K in m o l e c u l a i r e v o r m e n v a n v e r s c h i l l e n d e
grootte
( h e t e r o g e n i t e i t in m o l e c u u l g r o o t t e ) ; v e r s c h i l l e n in am i n o -
zuursamenstelling van CCK tussen diverse species
(s p e c i e s h e t e r o g e -
n i t e i t ) ; o v e r e e n k o m s t in s t r u c t u u r v a n he t b i o l o g i s c h a c t i e v e g e ­
d e e l t e v a n C C K e n g a s t r i n e ; p r o b l e m e n bij h e t m a k e n v a n r a d i o a c ­
tief g e m e r k t C C K e n v e r m e e n d e v o r m v e r a n d e r i n g e n in t e r t i a i r e s t r u c ­
tuur tussen v erschillende moleculaire vormen van CCK zouden h i e r ­
v o o r v e r a n t w o o r d e l i j k k u n n e n zijn. B o v e n d i e n is C C K n i e t a l l e e n
a a n w e z i g in h e t s l i j m v l i e s , m a a r o o k in h e t z e n u w w e e f s e l v a n het
m a a g - d a rm ka na al , waardoor de mogelijkheid bestaat dat CCK niet al­
l e e n als h o r m o o n , m a a r o o k als n e u r o t r a n s m i t t e r in s o m m i g e d e l e n
v a n h e t m a a g - d a r m k a n a a l a c t i e f is.
H e t d o e l v a n d i t o n d e r z o e k w a s e e n s p e c i f i e k e en g e v o e l i g e
r a d i o i m m u n o l o g i s c h e b e p a l i n g v o o r C C K te o n t w i k k e l e n . G e g e v e n s
o v e r d e p r o d u c t i e e n e v a l u a t i e v a n a n t i s e r a w e r d e n in h o o f d s t u k II
beschreven.
I m m u n i s a t i e v a n k o n i j n e n m e t C C K - 3 3 r e s u l t e e r d e in de
p r o d u c t i e v a n a n t i l i c h a m e n m e t e e n h o g e titer, d i e g e b r u i k t k o n d e n
w o r d e n in g e v o e l i g e e n s p e c i f i e k e r a d i o i m m u n o l o g i s c h e b e p a l i n g e n
125
v o o r CCK. K o p p e l i n g v a n
I aan een polypeptide kan veranderingen
in i m m u n o r e a c t i v i t e i t v a n d a t p o l y p e p t i d e t e w e e g b r e n g e n . E v a l u a ­
ti e v a n d e b i n d i n g v a n a n t i s e r a aan C C K d a t m e t b e h u l p v a n v e r s c h i l 125
l e n d e t e c h n i e k e n r a d i o a c t i e f is g e m e r k t m e t
I w e r d in d e h o o f d ­
s t u k k e n III e n IV b e s c h r e v e n .
In h o o f d s t u k III w e r d g e v o n d e n da t
125
er na c o n j u g a t i e v a n C C K - 3 3 m e t d e s u c c i n i m i d e e s t e r v a n
I-hydroxyphenylpropionzuur
( B o l t o n - H u n t e r reagens)
radioactief gemerkt
CCK-33 o n t stond dat u i t stekend bond aan a n tilichamen die niet g e ­
richt waren tegen het biologisch actieve COOH-terminale gedeelte
139
v a n CC K , m a a r d a t zeer s l e c h t b o n d a a n a n t i l i c h a m e n g e r i c h t t e g e n
h e t C O O H - t e r m i n a l e g e d e e l t e d a t C C K g e m e e n h e e f t m e t gas t r i n e .
In
h o o f d s t u k IV w e r d b e s c h r e v e n d a t CCK, w a a r a a n o p v e r s c h i l l e n d e ma12 5
nieren
I w a s g e b o n d e n , zich b o n d a a n v e r s c h i l l e n d e a n t i l i c h a m e n
binnen hetzelfde antiserum
(antilichaamheterogeniteit). De binding
v a n e e n a n t i s e r u m a a n v e r s c h i l l e n d e m o l e c u l a i r e v o r m e n va n C C K w a s
a f h a n k e l i j k v a n h e t g e b r u i k t e t y p e r a d i o a c t i e f g e m e r k t CCK. D a a r o m
b e h o r e n a n t i s e r a o p g e w e k t t e g e n C C K g e t e s t te w o r d e n m e t op v e r ­
s c h i l l e n d e m a n i e r e n r a d i o a c t i e f g e m e r k t CCK, t e n e i n d e de m e e s t g e ­
s c h i k t e a n t i g e n - a n t i l i c h a a m c o m b i n a t i e o p te sporen.
Het b l eek dat een van de antisera specifiek gericht was tegen
CCK-39. Gebruik makend van dit a ntiserum kon een gevoelige bepaling
worden ontwikkeld specifiek voor CCK-39, waarmee voor het eerst
C C K - 3 9 in d e h e r s e n e n k o n w o r d e n a a n g e t o o n d . D e z e g e g e v e n s w e r d e n
in h o o f d s t u k V b e s c h r e v e n . D e b e t r o u w b a a r h e i d v a n g e v o e l i g e en s p e ­
c i f i e k e r a d i o i m m u n o l o g i s c h e b e p a l i n g e n g e b r u i k m a k e n d va n t w e e a n t i ­
lichamen
(1703 en T 2 0 4 ) , v o o r h e t m e t e n v a n C C K in he t m e n s e l i j k
w e e f s e l e n b l o e d w e r d u i t v o e r i g b e s c h r e v e n in h o o f d s t u k VI. 2.8
p m o l / 1 C C K - 3 3 w a s in s t a a t de b i n d i n g t u s s e n a n t i s e r u m 1703 en de
125
met
I - B o l t o n - H u n t e r r e a g e n s g e m e r k t e C C K - 3 3 label m e t 50% te
v e r l a g e n . V o o r a n t i s e r u m T 2 0 4 w a s h i e r v o o r 3.3 p m o l / 1 C C K - 3 3 nodig.
De k l e inste hoe v e e l h e i d CCK-33 die nog b e t r o u w b a a r gemeten kon w o r ­
d e n l a g t u s s e n 0.5 en 1.0 p m o l / l p l a s m a . P l a s m a m o n s t e r s w e r d e n a l ­
v o r e n s g e m e t e n t e w o r d e n e e r s t b e h a n d e l d m e t e e n d u b b e l v o l u m e 96%
e t h a n o l , t e n e i n d e e i w i t t e n te p r e c i p i t e r e n .
I n f u s i e v a n C C K - 3 3 tot
p l a s m a w a a r d e n d i e iets l a g e r l a g e n d a n d i e g e m e t e n na t o e d i e n i n g
v a n vet, s t i m u l e e r d e p a n c r e a s e n z y m s e c r e t i e en g a l b l a a s c o n t r a c t i e .
De betrouwbaarheid van deze radioimmunologische bepalingen voor
h e t m e t e n v a n C C K in m e n s e l i j k w e e f s e l en p l a s m a w e r d u i t v o e r i g
onderzocht.
In d e h o o f d s t u k k e n II en IV w e r d b e s c h r e v e n d a t o r a l e en intraduodenale toediening van maisolie het plasma CCK gehalte deed
s tij g e n .
In de h o o f d s t u k k e n V I I e n V I I I w e r d h e t e f f e c t v a n a n d e r e
s t i m u l i v a n d e p a n c r e a s e n z y m s e c r e t i e o p h e t p l a s m a CC K g e h a l t e b e ­
studeerd.
In h o o f d s t u k V I I w e r d h e t e f f e c t v a n i n s u l i n e h y p o g l y c a e -
mie bestudeerd. Aangetoond werd dat stimulering van het cholinerg-
ische s y s t e e m d o o r i n s u l i n e h y p o g l y c a e m i e e e n d u i d e l i j k e s t i j g i n g
va n h e t p l a s m a p a n c r e a s p o l y p e p t i d e g e h a l t e gaf. Het p l a s m a CC K
gehalte veranderde tijdens insulinehypoglycaemie echter niet sig­
nific a n t .
In h o o f d s t u k V I I I w e r d h e t e f f e c t v a n b o m b e s i n e
(BBS)
op he t p l a s m a C C K g e h a l t e b e s t u d e e r d . BB S is e e n u i t 14 a m i n o z u ­
ren b e s t a a n d p o l y p e p t i d e , d a t in s t a a t is g a s t r i n e u i t h e t ant r u m v a n d e m a a g vrij te m a k e n .
I n f u s i e v a n BBS bij g e z o n d e v r i j ­
w i l l i g e r s en b ij p a t i e n t e n m e t e e n m a a g r e s e c t i e t o o n d e aan da t
BBS in s t a a t w a s C C K vrij te m a k e n o n a f h a n k e l i j k v a n g a s t r i n e - en
maagzuursecretie.
De m o l e c u l a i r e v o r m e n v a n C C K d i e in he t p l a s m a v r i j k o m e n
ti j d e n s i n f u s i e v a n b o m b e s i n e w e r d e n b e s c h r e v e n in h o o f d s t u k IX.
N a s c h e i d i n g v a n B B S - g e s t i m u l e e r d p l a s m a o p e e n S e p h a d e x G 50 SF
k o l o m k o n d e n bij 3 p r o e f p e r s o n e n t e n m i n s t e 4 v e r s c h i l l e n d e m o l e ­
cu l a i r e v o r m e n v a n C C K w o r d e n a a n g e t o o n d . De e e r s t e v o r m e l u e e r d e
in h e t z o g e n a a m d e " v o i d - v o l u m e " e n b e v a t t e 0-7% v a n de t o t a a l g e ­
m e t e n h o e v e e l h e i d CCK. D e t w e e d e v o r m e l u e e r d e o p 35% t u s s e n h e t
"v o i d - v o l u m e " en h e t " t o t a l e - v o l u m e " v a n de k o l o m e n b e v a t t e 8-41%
v a n de t o t a a l g e m e t e n h o e v e e l h e i d CCK. D e d e r d e v o r m e l u e e r d e op
50% t u s s e n h e t " v o i d - v o l u m e " en h e t " t o t a l e v o l u m e "
p l a a t s w a a r C C K - 3 3 e n C C K - 3 9 elueren)
(dit is o o k de
en b e v a t t e 4 4 - 6 1 % v a n de t o ­
taal g e m e t e n h o e v e e l e i d CCK. D e v i e r d e v o r m e l u e e r d e op 75% t u s ­
sen h e t " v o i d - v o l u m e " e n h e t " t o t a l e - v o l u m e " en b e v a t t e 15-27%
v a n de t o t a a l g e m e t e n h o e v e e l h e i d CCK.
In de h o o f d s t u k k e n X en XI w e r d e n de r e s u l t a t e n b e s c h r e v e n
va n s t u d i e s v e r r i c h t bij p a t i e n t e n w a a r b i j d o o r a n d e r e o n d e r z o e ­
kers e en g e s t o o r d e C C K s e c r e t i e w e r d v e r o n d e r s t e l d .
In h o o f d s t u k
X w e r d e n p a t i e n t e n m e t p a n c r e a s i n s u f f i c i e n t i e te n g e v o l g e va n
a l k o h o l m i s b r u i k of m u c o v i s c o ï d o s e b e s t u d e e r d . Bij d e z e p a t i e n t e n
w e r d e n l a g e n u c h t e r e C C K - w a a r d e n g e m e t en.
Na orale toediening
v a n v e t t r a d er e en d u i d e l i j k e s t i j g i n g v a n h e t p l a s m a - C C K - g e h a l t e
op. Z o w e l b a s a l e a ls g e s t i m u l e e r d e p l a s m a C C K - w a a r d e n bij p a t i e n t ­
en m e t p a n c r e a s i n s u f f i c i e n t i e w a r e n n i e t s i g n i f i c a n t v e r s c h i l l e n d
va n de w a a r d e n g e v o n d e n b ij g e z o n d e v r i j w i l l i g e r s .
In h o o f d s t u k
XI w e r d h e t e f f e c t v a n i n t r a d u o d e n a a l t o e g e d i e n d v e t op h e t p l a s ­
m a C C K - g e h a l t e e n d e g a l b l a a s s a m e n t r e k k i n g bij p a t i e n t e n m e t
141
d a r m s p r u w b e s t u d e e r d . Bij p a t i e n t e n m e t o n b e h a n d e l d e d a r m s p r u w
ontstond na intraduodenale toediening van vet geen stijging van
h e t p l a s m a C C K - g e h a l t e en t r a d er o o k g e e n g a l b l a a s s a m e n t r e k k i n g
op, t e r w i j l bij p a t i e n t e n m e t d a r m s p r u w d i e g o e d r e a g e e r d e n op
b e h andeling met een glutenvrij dieët normale stijgingen van het
p l a s m a C C K - g e h a l t e w e r d e n g e v o n d e n in c o m b i n a t i e m e t g a l b l a a s c o n t r a c t i e . D e z e o n d e r z o e k e n w e z e n o p e e n b e l a n g r i j k e rol v o o r
C C K bij h e t r e g u l e r e n v a n g a l b l a a s c o n t r a c t i e .
1 42
WOORDEN VAN W A A RDERING
A l l e n , d i e h e b b e n b i j g e d r a g e n a a n h e t to t s t a n d k o m e n va n
di t p r o e f s c h r i f t w i l ik v a n h a r t e danken.
D e p r a k t i s c h e v a a r d i g h e d e n v o o r he t v e r w e r k e n v a n d e m e e r
dan 200.000 reageerbuizen, die nodig waren voor het verr i c h t e n
va n d e s t u d i e s b e s c h r e v e n in d i t p r o e f s c h r i f t ,
l e e r d e ik v a n de
heer W R o e f f e n .
De h e e r J K o e d a m v a n h e t C e n t r a a l D i e r e n l a b o r a t o r i u m
(hoofd:
Dr WJI v d Gul d e n ) w a s b e h u l p z a a m bij het i m m u n i s e r e n v a n k o n i j n e n
en cavia's.
Mevr M H u y b r egts-vd Manaker bestelde het radioactieve jodium
w a a r m e e o p h e t Ir R e i c h e r t l a b o r a t o n u m
(hoofd: Dr F M H Corstens)
c h o l e c y s t o k i n i n e w e r d g e l a b e l l e d . De d e s k u n d i g e a d v i e z e n v a n de
heer W v d B r o e k w a r e n n o d i g o m s t e e d s m i n d e r s p o r e n v a n d e z e lab e l l i n g a c h t e r te laten. E v e n m i n m a g de b e r e i d w i l l i g e h u l p die
ik k r e e g v a n d e h e e r G G r u t t e r s
Centraal Dierenlaboratorium)
(I s o t o p e n l a b o r a t o n u m v a n het
bij d e p r a k t i s c h e u i t v o e r i n g v a n
een a a n t a l e x p e r i m e n t e n o n v e r m e l d b l i jven.
Mevr M Hessels van het Klinisch Chemisch L a b o r a t o r i u m van
de Kliniek voor Inwendige Ziekten
(hoofd: P r o f Dr A J a n s e n ) , b e ­
paalde m e t v e e l zorg p a n c r e a s e n z y m a c t i v i t e i t e n .
In h e t b i j z o n d e r g a a t m i j n d a n k u i t n a a r p a t i e n t e n en g e z o n ­
de v r i j w i l l i g e r s , d i e b e r e i d w a r e n a a n h e t o n d e r z o e k m e e te w e r ­
ken. C o l l e g a W H o p m a n en de m e d e w e r k s t e r s v a n de a f d e l i n g R a d i o ­
diagnostiek
(hoofd: P r o f D r G Rose n b u s ch)
b e n ik z e e r e r k e n t e l i j k
v o o r de n a u w k e u r i g e e c h o g r a f i s c h e m e t i n g e n e n b e r e k e n i n g e n v a n
galblaasvolumina.
De m e d e w e r k e r s v a n d e a f d e l i n g K l i n i s c h e F a r m a c i e w a r e n b e ­
h u l p z a a m bij h e t b e r e i d e n v a n b o m b e s i n e - i n f u s a t e n .
De g e r i n g e h o e v e e l h e d e n v a n v e r s c h i l l e n d e p o l y p e p t i d e n d i e
voor vele experimenten nodig waren werden op uiterst nauwkeurige
wijze afgewogen door de heer J Diersmann
(Faculteit W & N ) .
K o l o m c h r o m a t o g r a f i e bij d e j u i s t e t e m p e r a t u u r w a s m e d e m o g e ­
lijk d o o r h e t b e r e i d w i l l i g b e s c h i k b a a r s t e l l e n v a n d e d a a r v o o r
geschikte
r u i m t e in d e k o u d e k a m e r v a n he t O o g h e e l k u n d i g L a b o r a ­
143
torium
(hoofd: D r R M B r o e k h u y s e ) .
De h e ren H Berris en C Nicolasen va n de afdeling Medische
I l l u s t r a t i e v e r v a a r d i g d e n m e t v e e l z o r g de t e k e n i n g e n v o o r d i t
proefschrift, die vervolgens door de afdeling Medische F otogra­
f ie
(hoofd: A T A Reynen) d r u k k l a a r w e r d e n g e m a a k t . De m e d e w e r k ­
ers v a n d e M e d i s c h e B i b l i o t h e e k
(hoofd E d e G r a a f f } , w a r e n b e ­
h u l p z a a m b ij h e t v e r z a m e l e n v a n d e l i t e r a t u u r .
H et snelle en accurate typewerk v an m e v r Pat M e y - F u l l r o t h ,
s e c r e t a r e s s e v a n d e a f d e l i n g M a a g - , D a r m - en L e v e r z i e k t e n , is
r e e d s in v e l e p r o e f s c h r i f t e n g e r o e m d . O o k h a a r d e l e g e r e n d e
k w a l i t e i t e n , w a a r d o o r ik d e i n d r u k h e b g e k r e g e n d a t noch T i n e
R e i n t j e s n o c h E l s T e u n e s s e n zich a a n t y p e w e r k v a n een v a n de
versies van enig artikel hebben kunnen onttrekken, dienen niet
o n v e r m e l d te b l i j v e n .
Alle
( o u d - ) m e d e w e r k e r s v a n h e t L a b o r a t o r i u m v o o r Maag-,
D a r m - e n L e v e r z i e k t e n b e d a n k ik v o o r d e p r e t t i g e sa m e n w e r k i n g .
H e t b e l a n g v a n s t e l l i n g X u i t h e t p r o e f s c h r i f t v a n Dr H T M C u y p e rs, d a t h e t i n s t e l l e n v a n e e n " v e r p l i c h t " u u r t j e ri k k e n in de
m i d d a g p a u z e in p l a a t s v a n e en " d i s c u s s i e " u u r t j e b i j d r a a g t tot
e e n p r e t t i g e w e r k s f e e r , d i e n t h i e r b i j n o g eens b e n a d r u k t te w o r ­
den .
De uitgave van dit proefschrift werd m o g e l i j k gemaakt door
f i n a n c i ë l e s t e u n v a n d e f i r m a SK&F.
H e l a a s l a a t h e t p r o m o t i e r e g e l e m e n t v a n d e z e u n i v e r s i t e i t mij
n i e t toe, hem, d i e d o o r z i j n v e l e b i j z o n d e r e k w a l i t e i t e n h e t m e e s t
a a n h e t t o t s t a n d k o m e n v a n d i t p r o e f s c h r i f t h e e f t b i j g e d r a g e n , op
d e z e p l a a t s te b e d a n k e n .
1 44
CURRICULUM VITAE
De s c h r i j v e r v a n d i t p r o e f s c h r i f t w e r d op 23 a u g u s t u s 1948
te A a r l e - R i x t e l g e b o r e n .
In 1967 b e h a a l d e hij a a n h e t Dr. K n i p p e n b e r g c o l l e g e te
H e l m o n d h e t e i n d d i p l o m a HBS-B.
V a n a f 1967 s t u d e e r d e hij g e n e e s k u n d e aa n de K a t h o l i e k e
U n i v e r s i t e i t te N i j m e g e n .
In 1973 w e r d he t d o c t o r a a l e x a m e n en
in 1975 h e t a r t s e x a m e n b e h a a l d . D a a r n a b e g o n zijn o p l e i d i n g tot
i n t e r n i s t bij Dr. M. v a n Zoe r e n , in h e t M i l i t a i r H o s p i t a a l
"Dr. A. M a t h i j s e n " te U t r e c h t
(hoofd o p l e i d i n g d e s t i j d s : D r . C.
va n B e l l e ^ ) . In 1977 w e r d d e z e o p l e i d i n g v o o r t g e z e t a a n de U n i ­
v e r s i t e i t s k l i n i e k v o o r I n w e n d i g e Z i e k t e n v a n het R a d b o u d z i e k e n huis te N i j m e g e n
(hoofd d e s t i j d s : Prof. Dr. C.L.H. M a j o o r ^ , h u i ­
di g hoofd: Prof. Dr. A. v a n
't L a a r ) . In n o v e m b e r 1980 w e r d hij
i n g e s c h r e v e n in h e t s p e c i a l i s t e n r e g i s t e r .
V a n 1 m a a r t 1980 tot 1 s e p t e m b e r 1983 w a s hij a l s w e t e n ­
s c h a p p e l i j k m e d e w e r k e r in d i e n s t v a n de S t i c h t i n g Z u i v e r W e t e n ­
schappelijk Onderzoek
D a r m - en L e v e r z i e k t e n ,
(FUNGO) v e r b o n d e n a a n de a f d e l i n g Maag-,
t i j d e n s w e l k e p e r i o d e he t o n d e r z o e k
p l a a t s v o n d d a t r e s u l t e e r d e in d i t p r o e f s c h r i f t . V a n a f 1 s e p t e m ­
b er 1983 is hij a ls w e t e n s c h a p p e l i j k m e d e w e r k e r in d i e n s t v a n
het K o n i n g i n W i l h e l m i n a F o n d s v e r b o n d e n aan de a f d e l i n g Maag-,
D a r m - en L e v e r z i e k t e n v a n h e t R a d b o u d z i e k e n h u i s te N i j m e g e n .
145
PUBLICATIONS
1.
JANSEN
ten
JBMJ
LAMERS
na
& CBHW
CBHW
LAMERS
C,
& JBMJ
JI
4.
ROTTER,
JANSEN
of
M SAMLOFF
JBMJ
LAMERS
tion
21:
the
23:
Common
van
algemene
aspec­
23:
antrum-
13-19.
29-33.
& J JANSEN,
1980.
syndromes.
Academic
Francisco:
1981.
serum
AUTHOR.
achtergebleven
Gastrointestinal
San
LAMERS,
THE
C h o 1e c y st o k i n i n e : Huidige
& DL R I M O I N .
Sidney,
& CBHW
CBHW,
in
1980.
P FROELING
on borabesin-stiraulated
5.
BY
Bombesine:
hyperchlorhydria
Toronto,
Digestion
1980.
aantonen
JANSEN,
C DIEMEL,
Heterogeneïty
London,
het
T Gastroenterol
hypergastrmemic
and
LAMERS,
voor
HORMONES
maagresectie. T Gastroenterol
inzichten.
3.
GASTROINTESTINAL
en b e t e k e n i s
weefsel
2.
ON
The
gastrin
Heredity
In:
of
Genetics
Disorders.
Press.
New
(Eds)
York,
81-87.
effect
and
of
somatostatin
gastric
acid
in m a n .
193-197.
JBMJ
JANSEN
diagnosis
of
& W ROEFFEN,
gastrinoma.
1981.
Secretin
ïnjec-
Gastroenterology
80:
1615-1616.
6.
BOLLEN
ECM,
T E N , 1981.
7.
ovanan
JANSEN
JBMJ
JANSEN
JBMJ
Sci
JANSEN
calcium
14 6
LAMERS,
27:
JBMJ
on
Regul
& CBHW
and
JANSEN,
syndrome
c y s t a d e n o c a r c i n o m a . Br
& CBHW
in m a n .
bombesin
9.
JBMJ
bombesïn-stimulated
cretion
Dis
LAMERS,
Z o l 1 i n g e r - E l 1 1 son
ducing
hibit
8.
CBHW
food
1981.
due
to
Calcitonm
1:
1982.
in p a t i e n t s
LARSSON
J Surg
serum gastrin
Peptides
LAMERS,
LI
a gastrin
pro-
68 : 7 7 6 - 7 7 7 .
and
and
& HJM JOOS-
secretin
gastric
acid
inse­
415-421.
Serum gastrin
with
responses
to
h y p e r g a s t r m a e m i a . Dig
303-307.
& CBHW
LAMERS,
1982.
secretin-stimulated
Effect
serum
of
changes
gastrin
in
serum
in p a t i e n t s
with
the
10.
Z o l 1 i n g e r - E l 1 ison
LAMERS
JHM
CB,
van
tially
JH WALSH,
TONGEREN,
released
syndrome.
JB
JANSEN,
1982.
from
Gastroenterology
AR
Evidence
the
human
HARRISON,
that
AF
83:
IPPOLITI
gastnn-34
duodenum.
173-178.
&
is p r e f e r e n -
Gastroenterology
83:
233-239 .
11.
12.
JANSEN
JBMJ
J Immun
Meth
LAMERS
roth
CB,
SPETH
51:
CM
DIEMEL
PAJ,
dumping
15.
JBMJ
and
JBMJ
Proc
First
Exerpta
& CBHW
LAMERS,
& CBHW
Production
Biochem
JBMJ
21:
& CBHW
in h u m a n
Intern
Bolton-Hunter
CCK-antibodies.
pancreatic
in p a t i e n t s
in n o r m a l
and
1982.
Symp
on
Amsterdam:
1983.
LAMERS,
JBMJ
cystokinin
Serum
and
LAMERS,
Medica,
JANSEN
JANSEN
secretin
& CBHW
in p o r c i n e
Clin
1982.
gastrectomies
JANSEN
syndrome.
cystokinin:
16.
food
II
c h o 1 e c y s t o k in i n - 3 9
Chem
of
with
polyBill-
subjects.
288-292.
W CREUTZFELDT.
JANSEN
Low binding
& JB JANSEN,
to
Billroth
91:
1982.
to c a r b o x y l - t e r m i n a l
223-230.
responses
I and
Surgery
14.
LAMERS,
c h o l e c y s t o k i n i n - 33
peptide
13.
& CBHW
labeled
Life
Acarbose.
of
the
(Ed)
Sci
evidence
32:
Radioimmunoassay
evaluation
in
524-526.
Immunological
brain.
1983.
Acarbose
of
911-913.
of
chole­
antibodies.
J Clin
387-394.
LAMERS,
tissue
1983.
and
Radioimmunoassay
plasma.
Clin
Chim
of
chole­
Acta
131:
3 0 5 - 3 16.
17.
JANSEN
JBMJ
caemia
on
peptjde.
18.
LAMERS
& CBHW
plasma
Scand
CBHW,
JM
LAMERS,
1983.
cholecystokinin
J Gastroenterol
DIEMEL,
JBMJ
Effect
and
82:
of
plasma
insulïn-hypoglypancreatic
poly-
219-221.
JANSEN,
E van
LEER,
R van
LEUSEN
147
& JJ
PEETOOM,
derateLy
Scand
19.
elevated
CBHW,
potencies
s u re d by
82:
2 0.
22.
PAJ,
LAMERS
m
CBHW
JANSEN
by
JANSEN
and
& JH
with
mo-
concentrations.
WALSH,
1983.
Relatl-
c h o l e c y s t o k i n i n -8 m e a ­
bioassay.
on
JANSEN,
1983.
acid
Scand
J Gastroenterol
W P M , JBMJ
to
JANSEN
oral
JBMJ
fat
Ann
& CBHW
in h u m a n
The
effect
of
& CBHW
with
and
and
a gastrïn-recep-
serum gastrin
Plasma
Billroth
in
5:
21-24.
on p l a s m a
chole­
gastrectomized
LAMERS.
study
pectin
Gastroenterol
of b o m b e s i n
and
with
798-802.
radioimmunoassays.
patients
Surgery,
mea­
in p r e s s .
cholecystokinin
I and
Billroth
in p r e s s .
LAMERS,
plasma
24:
J Clin
in p a t i e n t s
Surg,
acarbose
Gut
Effect
subjects
1983 . C o m p a r a t i v e
of
secretion
syndrome.
& CBHW LAMERS.
gastrectomy.
JANSEN
gastnc
in n o r m a l
LAMERS,
syndrome.
sequence-specific
1983.
Molecular
during
infusion
& CBHW
LAMERS.
of
forms
of
cholecys­
bombesin.
Life
Sci
2197-2205.
HOPMAN
WPM,
plasma
cholecystokinin
Digestion,
JBMJ
JANSEN
response
to
Effect
of a t r o p i n e
intraduodenal
fat
on
in m an .
in p r e s s .
LAMERS
CBHW,
plasma
pancreatic
bombesin
148
JBMJ
8, C B H W
dumping
& JBMJ
JBMJ
HOPMAN
33:
subjects
203-204.
a combination
Z o l 1 i n g e r - E l 1 1 son
tokinin
2 6.
JANSEN
pectin,
antagonist
response
25.
JBMJ
the
sured
2 4.
in
polypeptide
of c h o l e c y s t o k i n m - 3 3 a n d
tor
II
82:
P POITRAS,
the
cystokinin
2 3.
serum pancreatic
radioimmunoassay
acarbose,
placebo
2 1.
suppression
191-192.
SPETH
of
Atropine
J Gastroenterol
LAMERS
ve
1983.
CM D I E M E L
in n o r m a l
& JBMJ
polypeptide
subjects
JANSEN.
Comparative
responses
and patients
to
food,
with
study
of
secretin
chromc
and
pancrea-
27.
titis.
Dig
HOPMAN
W P M , JBMJ
of
equimolar
Chain
der
28.
Dis
Sci,
JANSEN,
amounts
trïglycerides
contraction.
LAMERS
CBHW,
sinogen
in p r e s s .
of
JBMJ
chain
on p l a s m a
Am J Clin
JANSEN,
I in h e r i d i t a r y
s y n d r o m e . In:
G ROSENBUSCH
long
Genetic
LAMERS.
cholecystokinin
Nutr,
JI
& CBHW
triglycerides
and
Effect
medium
gallblad­
in p r e s s .
ROTTER
& IM
S A M L O F F . Serum
hyperpeps1 nogenaemic
and
and
Clinical
Aspects
peptic
of
pep-
ulcer
Pepsinogen,
in
press.
29.
LAMERS
CBHW
& JBMJ
Histopathology
and T h e r a p e u t i c
30.
HAFKENSCHEID
trypsin,
normal
Clin
31.
and
and patients
Recent
atrophy:
Advances
Endoscopy,
in D i a g n o s t i c
JBMJ
JANSEN
during
with
& CBHW
bombesin
pancreatic
LAMERS.
Serum
stimulation
m
insufficiency.
in p r e s s
& CBHW
LAMERS.
employing
J
m
Meth,
mucosa
in p r e s s .
lipase
cholecystokinin
Immunol
In:
J C M , M HESSELS,
subjects
JBMJ
Antral
Gastrin.
Endoscopy,
a-amylase
Chim Acta,
JANSEN
and
JANSEN.
C h a r a c t e r 1 z a 1 1 on
different
of
antisera
cholecystokinin
to
labels.
press.
149
STELLINGEN
I
H et
in h e t s l i j m v l i e s
tokinine
v an h e t m a a g - d a r m k a n a a l
aanwezige cholecys-
is ee n h o r m o o n .
II
De b e t e k e n i s
van
t o k i n i n e a ls
perifeer circulerend
h et b i o l o g i s c h
actieve
octapeptide
va n c h o l e c y s -
hormoon wordt overschat.
- C a l a m J, E l l is A & D o a k r a y G J . J Clin I n v e s t 1982; 69: 2 1 8 - 2 2 5
- Wal s h <TH, L a m e r s CB & V a l e n z u e l a JE. G a s t r o e n t e r o l o g y 1982; 82
438-444.
- Dit p r o e f s o h r i f t .
III
Immunologische eigenschappen
belangrijk
gedeelte bepaald
v an c h o l e c y s t o k i n i n e w o r d e n
d o o r de c o n f o r m a t i e
v o o r e en
van v e r s c h i l l e n d e
fragmenten•
- Dit p r o e f s c h r i f t .
IV
V o o r h e t v e r k l a r e n van
de
in testinale fase
pancreatische
de p a n c r e a s - e n z y m s e c r e t i e
van e e n m a a l t i j d
d ie o p t r e e d t t i j d e n s
is h e t b e s t a a n
van e en e n t e r o -
reflex overbodig.
- S o l o m o n T E & G r o s s m a n MI. Am J P h y s i o l 1979; 226: E 1 8 6 - E 1 9 0 .
- S i n g e r MV, S o l o m o n TE
<3
G r o s s m a n MI. A m J P h y s i o l 1980; 238:
G 1 8 - G 2 2 en G23-G29.
- Dit p r o e f s c h r i f t .
Pancreasenzymen
derdrukken
spelen
bij
V
de m e n s g e e n b e l a n g r i j k e
rol
bij
he t o n ­
van de c h o l e c y s t o k i n i n e - s e c r e t i e .
VI
Indien
tijdens
s t e a t o r r h o e bij
patienten met da rms pruw o n v old oend e
b e h a n d e l i n g m e t e en g l u t e n v r i j
pancreasenzymen
overwogen
te w o r d e n .
dieët,
dient
verbetert
toediening
van
VII
Pancreas-enzymsecretie,
va n b o m b e s i n e ,
tokinine
d ie o p t r e e d t
kan v e r k l a a r d w o r d e n
tijdens
int ra v e n e u z e
d o o r h et v r i j k o m e n
toediening
v an c h o l e c y s -
in de p e r i f e r e c i r c u l a t i e .
V I II
Bij
h et b e o o r d e l e n
v an e en
ferentiaal -diagnose
diteit,
di ent men
"secretine-test"
v an h y p e r g a s t r i n e m i e
geinformeerd
te z i j n o v e r
- J a n s e n J B M J & L a m e r s CBHW.
in h et k a d e r van
in c o m b i n a t i e m e t
de d i f ­
hyperaci-
het s e r u m ca 1c i u m g e h a 1 te .
G a s t r o e n t e r o l o g y 1982; 83: 438-444.
IX
Elke
p a t i ë n t m e t ee n e r n s t i g
gische
van
behandeling wordt
zijn
peptisch ulcuslijden,
voorgesteld,
heeft
die voor c h i r u r ­
r e c h t op h e t b e p a l e n
nuchter serum g a s t r i n eg e h a l te .
X
Aangezien
reeds
geringe
bij
gezonde
bij
de b e h a n d e l i n g
personen
hoeveelheden
diarrhee
medium-chain-triglycerids
veroorzaken,
dient
van p a t i ë n t e n m é t d i a r r h e e
de w a a r d e
in t w i j f e l
(MCT)
van M C T
te w o r d e n
getrokken.
- H o p m a n WPM, J a n s e n J B M J & L a m e r s CBHW. A m J C li n Nutr,
in press.
XI
H et
selectief vrijkomen
van b o m b e s i n e ,
maakt
toire an t r u m f u n c t i e
v an g a s t r i n e
deze
uit het a n t r u m t i j d e n s
stof g e s c h i k t om s t o o r n i s s e n
infusie
in de s e c r e -
op te s p o r e n .
- Eigen w a a r n e m i n g e n .
XII
De h o g e
impact-factor toegekend
de w a a r d e
pelijke
van ee n c i t a t i e - i n d e x
kwaliteit
van
aan
"review"tijdschriften
bij
de b e o o r d e l i n g
publicaties.
- Sai J o u r n a l C i t a t i o n Reports.
vermindert
v an de w e t e n s c h a p ­
XIII
H et p u b l i c e r e n
m en
levert
medische
van c o n g r e s s e n en s y m p o s i a
geen
in v e e l a l
z i n n i g e b i j d r a g e aan de t o c h
l i t e r a t u u r en d i e n t
slechts
al
f ra ai e b o e k v o r ­
e x p l o s i e v e groei
ter v e r fra aiin g
van
van een c u r r i ­
c u l u m vi t a e .
- M i t a h e l l JRA.
Br M e d J 1983; 287: 418.
XIV
Nu ee n
aantal
"maag-darmhormonen"
zijn aange too nd,
d o o r de m a a g " ,
zijn maag
z ij n g e z e g d e s
"ergens
zitten"
zijn
ook
zo al s
b u i k vol
in h e t c e n t r a l e z e n uw s t e l se l
"de l i e f d e
van h e b b e n "
o ok n a t u u r w e t e n s c h a p p e l i j k
van
en
de man gaat
" met iets in
i n t e r e s s a n t g e w o r d en .
XV
Kaarten
tijdens
spannend,
door
omdat
"nakaarten"
Nijmegen,
de m i d d a g p a u z e
g e e n enk el
is v o o r l a b o r a t o r i u m m e d e w e r k e r s o n t ­
spelletje
reproduceerbaar
is en a l l ee n
g e a n a l y s e e r d kan w o r d e n .
20 j a n u a r i
1984.
J.B.M.J.
J ansen.