Arendt, Rainer M.

Gerbes, Alexander L.

Vollmar, Angelika M.

Xie, Yining

English

Universität München: Elektronischen Publikationen

LIFE SCIENCES 
L I F E S C I E N C E S 
TABLE ÜF CONTENTS 
VOLUME 43, NUMBER 19 
AIDS RESEARCH COMMUNICATIONS 
ILONA VATHY and ANNE M. 
ETGEN 
T.J. COLLINS, S. CHATTERJEE, 
L.S. LeGATE and T.K. BANERJI 
1493 Ovarian Steroids and Hypothalamic Nor-
epinephrine Release: Studies Using 
In Vivo Brain Microdialysis 
1501 Lithium: Evidence for Reduction in C i r -
culating Testosterone Levels i n Mice Fol-
lowing Chronic Administration 
E. GIRALDO, F. MARTOS, 
A. GOMEZ, A. GARCIA, M.A. 
VIGANO, H. LADINSKY and 
F. SANCHEZ DE LA CUESTA 
1507 Characterization of Muscarinic Receptor 
Subtypes in Human Tissues 
ALEXANDER L. GERBES, 
ANGELIKA Μ. VOLLMAR, 
YINING XIE and RAINER 
Μ. ARENDT 
1517 Presence of the A t r i a l N a t r i u r e t i c Fac­
tor (ANF) in Human A s c i t i c F l u i d 
G. CARO, M. BARRIOS and 
J.M. BAEYENS 
1523 Dose-Dependent and Stereoselective An­
tagonism by Diltiazem of Naloxone-Pre-
cipitated Morphine Abstinence After 
Acute Morphine-Dependence In Vivo and 
In Vitro 
JAN G.C. VAN AMSTERDAM, 
KAREL J.H. VAN BUUREN, 
MARCO J. KRIELAART, OBBE 
P. ZUIDERVELD and ROB 
P.J.H. TIJMS 
1529 Effect of Inhibitors of Enkephalin De­
gradation in the Isolated Guinea-Pig 
Ileum 
W. SUTANTO and E.R. 
DE KLOET 
L. ALESSIO, S. GOVONI, 
G. SALAR, F. BATTAINI, 
R. IAVICOLI and M. 
TRABUCCHI 
1537 ZK91587: A Novel Synthetic Antimineral-
ocorticoid Displays High A f f i n i t y for 
Corticosterone (Type I ) Receptors i n the 
Rat Hippocampus 
1545 Age-Related Changes of Methionine-Enke­
phalin and Beta-Endorphin / Beta-Lipo-
tropin Immunoreactivity in Human CSF 
SAT BIR S. KHALSA and 
GENE D. BLOCK 
HIROSHI SAITO, MITSUAKI 
NAKAMARU, TOSHIO OGIHARA, 
HIROMI RAKUGI, YUICHI 
KUMAHARA, TADASHI INAGAMI 
and KAZUAKI SHIMAMOTO 
1551 Phase-Shifts of the Bulla Ocular C i r c a -
dian Pacemaker in the Presence of C a l ­
modulin Antagonists 
1557 Effect of Vasodilator Prostaglandins on 
the Vascular Renin-Angiotensin System 
I N D E X E D IN: Current Contents, CABS, BIOSIS Database, Index Medicus, M E D L I N E , Psychol. Abstr., Nutr. 
Abstr., Energy Res. Abstr., Energy Data Base, Ophthal. Lit., Biological Abstr., Excerpta Medica, PASCAI^CNRS 
Database 
L I F S A K ISSN 0024-3205 
(280) 
L I F E S C I E N C E S 
PETER R. OELTGEN, SITA 1565 
P. NILEKANI, PAULA A. 
NUCHOLS, WILMA A. SPURRIER 
and TSUNG-PING SU 
I 
Further Studies on Opioids and Hiber-
nation: Delta Opioid Receptor Ligand 
S e l e c t i v i t y Induced Hibernation in 
Summer-Active Ground Squirrels 
Software Survey Section 
L i f e Sciences, Vol. 43, pp. 1517-1521 
Printed in the U.S.A. 
Pergaraon Press 
PRESENCE OF THE ATRIAL NATRIURETIC FACTOR (ANF) 
IN HUMAN ASCITIC FLUID 
* 
A l e x a n d e r L. Gerbes, A n g e l i k a Μ. Vollmar , Y i n i n g X i e , 
and R a i n e r Μ. Arendt 
M e d i z i n i s c h e K l i n i k I I and I , K l i n i k u m G r o s s h a d e r n and I n s t i t u t e 
of Pharmacology, V e t e r i n a r y S c h o o l , U n i v e r s i t y of Munich, Munich, 
F e d e r a l R e p u b l i c of Germany 
(Received in final, form September 12, 1988) 
Summary 
P r e s e n c e o f a t r i a l n a t r i u r e t i c f a c t o r ( A N F ) - l i k e mate­
r i a l was d e m o n s t r a t e d by r a d i o i m m u n o a s s a y i n a s c i t i c 
f l u i d o f 14 p a t i e n t s w i t h c i r r h o s i s o f t h e l i v e r . Im-
m u n o r e a c t i v e ANF c o n c e n t r a t i o n s (M+SEM) were 2.4 + 0.5 
f m o l / m l i n a s c i t e s , s i g n i f i c a n t l y l o w e r (p < 0.001) 
t h a n t h e c o r r e s p o n d i n g plasma c o n c e n t r a t i o n s o f 15.5 + 
2.6 f m o l / m l . High p e r f o r m a n c e g e l p e r m e a t i o n c h r o m a t o ­
g r a p h y and r e v e r s e phase h i g h p e r f o r m a n c e c h r o m a t o ­
g r a p h y o f t h e a s c i t i c ANF i m m u n o r e a c t i v i t y showed c o r ­
r e s p o n d e n c e t o t h e a l p h a human ANF( 99-1 26 ) . ANF l e v e l s 
i n a s c i t e s were s i g n i f i c a n t l y (ρ < 0.01) c o r r e l a t e d t o 
l e v e l s i n plasma ( r = 0 . 6 6 ) . 
I n p a t i e n t s w i t h c i r r h o s i s o f t h e l i v e r and a s c i t e s t h e p a t h o ­
p h y s i o l o g i c a l r o l e o f ANF has n o t been f u l l y e l u c i d a t e d ( 1 , 2 ) . 
F o l l o w i n g i n t r a v e n o u s r e i n f u s i o n o f a s c i t e s e l e v a t e d ANF plasma 
l e v e l s have been r e p o r t e d ( 3 , 4 , 5 ) . T h i s m i g h t be due t o ANF r e ­
l e a s e s t i m u l a t e d by t h e hemodynamic changes f o l l o w i n g a s c i t e s 
i n f u s i o n or due t o e l e v a t e d ANF c o n c e n t r a t i o n s i n a s c i t i c f l u i d . 
Whereas t h e p r e s e n c e o f a t r i a l n a t r i u r e t i c f a c t o r (ANF) has been 
d e m o n s t r a t e d i n v a r i o u s f l u i d s o f t h e human body, such as plasma 
( 6 ) , u r i n e ( 7 ) and c e r e b r o s p i n a l f l u i d ( 8 ) , a s c i t i c f l u i d has n o t 
been examined as y e t f o r t h e o c c u r e n c e o f ANF. I n t h i s s t u d y t h e 
p r e s e n c e o f ANF i n a s c i t i c f l u i d was i n v e s t i g a t e d . 
S u b j e c t s and Methods 
F o u r t e e n p a t i e n t s (11 men, 3 women, aged 42 t o 81 y e a r s ) w i t h 
b i o p s y p r o v e n c i r r h o s i s o f t h e l i v e r and a s c i t e s were i n v e s t i ­
g a t e d . 
0024-3205/88 $3.00 + .00 
Copyright (c) 1988 Pergamon Press pic 
1518 ANF i s Present in Human A s c i t i c Fluid Vol. 43, No. 19, 1988 
D e t e r m i n a t i o n o f ANF by r a d i o i m m u n o a s s a y . S i m u l t a n e o u s l y o b t a i n e d 
samp 1 es 5T a s c i t i c f l u i d and p e r i p h e r a 1 venous b l o o d were drawn 
i n t o p r e - c o o l e d s y r i n g e s and i m m e d i a t e l y t r a n s f e r r e d t o p r e - c o o l e d 
p o l y s t y r e n e t u b e s c o n t a i n i n g 500 k a l l i k r e i n i n h i b i t o r u n i t s 
a p r o t i n i n per ml and 1 mg sodium EDTA p e r m l . Samples were 
c e n t r i f u g e d a t 4° C, t h e s u p e r n a t a n t was i m m e d i a t e l y f r o z e n and 
s t o r e d a t - 80° C. E x t r a c t i o n o f a s c i t e s a l i q u o t s as w e l l as RIA 
p r o c e d u r e s were p e r f o r m e d i n t h e same way as f o r plasma samples; 
p r o c e d u r e s were m o d i f i e d f r o m ( 6 ) and have been d e t a i l e d e l s e w h e r e 
( 9 ) . B r i e f l y , samples were e x t r a c t e d by a d s o r p t i o n t o A m b e r l i t e 
XAD-2 a d s o r b e n t r e s i n . The f i n a l t i t e r o f t h e C - t e r m i n a l d i r e c t e d 
a n t i b o d y Toni I I I was 1:120000, crossreactiνity t o r a t - p r o ANF was 
48% and t h e assay s e n s i t i v i t y was 0.5 f m o l a l p h a human ANF/tube. 
The 50% b i n d i n g i n t e r c e p t o f t h e s t a n d a r d c u r v e was 10 f m o l . 
C h r o m a t o g r a p h i c a n a l y s i s o f ANF i m m u n o r e a c t i v i t y . A s c i t i c f l u i d 
e x t r a c t s of 4 p a t i e n t s were s u b j e c t e d t o h i g h p e r f o r m a n c e g e l p e r ­
m e a t i o n c h r o m a t o g r a p h y (HPGPC) and r e v e r s e phase h i g h p e r f o r m a n c e 
c h r o m a t o g r a p h y (RP-HPC). 
High p e r f o r m a n c e g e l p e r m e a t i o n c h r o m a t o g r a p h y (HPGPC): L y o p h i -
l i z e d a s c i t i c f l u i d (10 m l ) was d i s s o l v e d i n 25μ 1 column e l u e n s 
and a p p l i e d t o a S p h e r o g e l TM TSK, 2000 SW column (ΙΟμπι, 7.5mm χ 
300 mm, Beckman I n s t r u m e n t s , San Rarnon, CA, USA), e l u t e d w i t h 
0.09% t r i f 1 u o r o a c e t i c a c i d (TFA) c o n t a i n i n g 0.005M Na 2S0., 0.002M 
NaHpPO, and 30% a c e t o n i t r i l e ( f l o w r a t e : 0.3 m l / m i n ) . C a l i b r a t i o n 
was c a r r i e d o u t w i t h b o v i n e serum a l b u m i n (BSA) ( V ) , v i t a m i n B12 
( V . ) , r a t pro-ANF ( 2 - 1 2 6 ) and a l p h a human ANF ( 9 9 - 1 2 6 ) . Immuno-
r e a c t i v e ANF (IR-ANF) f r a c t i o n s ( 0 . 6 m l ) , d e t e c t e d by RIA, were 
p o o l e d and l y o p h i 1 i zed. 
Reverse phase h i g h p e r f o r m a n c e c h r o m a t o g r a p h y (RP-HPC): An a l i q u o t 
of t h e p o o l e d IR-ANF f r a c t i o n s o f t h e HPGPC r u n was r e d i s s o l v e d i n 
25 μΐ 0 . 1 % TFA and l o a d e d on a HPLC C 18 ODS U l t r a s p h e r e TM column 
(5 μ m 9 2mmx15 0mm, Beckman I n s t r u m e n t s , San Ramon, USA) a c c o r d i n g t o 
( 1 0 ) . E l u t i o n was c a r r i e d o u t w i t h a l i n e a r g r a d i e n t o f a c e t o n i ­
t r i l e ( 1 0 - 8 0 % , 45 m i n ) i n 0.1%TFA ( f l o w r a t e : 0 . 2 m l / m i n ) . C a l i ­
b r a t i o n o f t h e column was p e r f o r m e d w i t h s y n t h e t i c a t r i o p e p t i n I 
and I I I , a l p h a human ANF ( 9 9 - 1 2 6 ) and r a t pro-ANF ( 2 - 1 2 6 ) . 
F r a c t i o n s ( 0 . 4 m l ) were assayed f o r ANF i m m u n o r e a c t i v i t y . 
S t a t i s t i c a l e v a l u a t i o n . D i f f e r e n c e s between a s c i t i c and p l a s m a t i c 
ANF c o n c e n t r a t i o n s were compared by p a i r e d t - t e s t ; t h e Pearson 
c o r r e l a t i o n c o e f f i c i e n t was d e t e r m i n e d by t h e u s u a l l i n e a r l e a s t 
s q u a r e s t e s t . Data a r e p r e s e n t e d as mean and s t a n d a r d e r r o r 
(M + SEM) . 
R e s u l t s 
Radi o i mmunoas s ay 
S e r i a l d i l u t i o n s o f a s c i t e s were p a r a l l e l t o t h e s t a n d a r d c u r v e , 
as shown i n f i g u r e 1 . R e c o v e r i e s o f 7.8 and 15.6 f m o l s y n t h e t i c 
human a l p h a ANF added t o samples o f a s c i t e s and plasma p r i o r t o 
e x t r a c t i o n were 84% and 67% i n a s c i t e s and 75% and 65% i n ρ l a s in a. 
N o n s p e c i f i c b i n d i n g , d e t e r m i n e d i n t h e RIA w i t h o u t a n t i s e r u m was 
l e s s t h a n 5% i n b o t h a s c i t e s and plasma. A s c i t i c ANF c o n c e n ­
t r a t i o n s were 2.4 + 0.5 f m o l / m l , s i g n i f i c a n t l y ( p < 0.001) l o w e r 
t h a n ANF c o n c e n t r a t i o n s i n plasma (15.5 + 2.6 f m o l / m l ) . ANF l e v e l s 
Vol. 43, No. 19, 1988 ANF i s Present in Human A s c i t i c Fluid 1519 
i n a s c i t e s were s i g n i f i c a n t l y c o r r e l a t e d t o ANF plasma l e v e l s ( r = 
0.56, ρ < 0 . 0 1 ; f i g u r e 2 ) . 
factor of dilution 
10 10C 1000 10000 
A N F ( f m o l / t u b e ) 
FIG. 1 
A s t a n d a r d c u r v e f o r r a ­
dio i m m u n o a s s a y o f a l p h a 
human ANF ( c i r c l e s ) i n 
c o m p a r i s o n t o s e r i a l d i ­
l u t i o n s o f a s c i t i c f l u i d 
( t r i a n g l e s ) . Each p o i n t 
i s t h e mean o f two expe­
r i m e n t s . D i l u t i o n c u r v e s 
p a r a l l e l t h e s t a n d a r d 
c u r v e s . 
HPLC c h a r a c t e r i z a t i o n o f i m m u n o r e a c t i v e ANF 
When p r e - e x t r a c t e d a s c i t i c f l u i d was c h r o m a t o g r a p h e d on HPGPC and 
r e v e r s e phase HPLC, i m m u n o r e a c t i v e ANF was d e t e c t e d as a s i n g l e 
peak cοeιut i ηg w i t h s y n t h e t i c a l p h a human ANF. No s i g n i f i c a n t 
amounts o f h i g h e r m o l e c u l a r w e i g h t A N F - l i k e m a t e r i a l such as p r e ­
c u r s o r pro-ANF was d e t e c t e d , as d e m o n s t r a t e d i n f i g u r e 3. 
1520 ANF i s Present i n Human A s c i t i c Fluid Vol. 43, No. 19, 1988 
Ζ 
< 
ά 
Τ 
20 ~30~ 
-80 
-40 
c ο 
φ ο <0 
f r a c t i o n n u m b e r 
FIG. 3 
HPLC a n a l y s i s o f i m m u n o r e a c t i v e ANF i n a s c i t i c f l u i d 
A: HPGPC: The v o i d (V ) and t o t a l ( V . ) volumes o f t h e column were 
d e t e r m i n e d by BSA and v i t a m i n Β T2, r e s p e c t i v e l y . The e l u t i o n 
p o s i t i o n s o f s y n t h e t i c r a t - p r o - A N F ( 2 - 1 2 6 ) (p-ANF) and a l p h a 
human ANF (ANF) are i n d i c a t e d by a r r o w s . F r a c t i o n s were t e s t e d 
f o r i m m u n o r e a c t i v e ANF by RIA. 
Β: Reverse phase HPLC: Ar r o w s i n d i c a t e t h e e l u t i o n p o s i t i o n s o f 
a t r i o p e p t i n I (AP I ) , a t r i o p e p t i n I I I (AP I I I ) , a l p h a human ANF 
(ANF) and r a t p r o - ANF ( 2 - 1 2 6 ) (p-ANF). I m m u n o r e a c t i v e ANF was 
e v a l u a t e d by RIA. 
D i s c u s s i o n 
T h i s s t u d y d e m o n s t r a t e s t h e p r e s e n c e o f ANF i n a s c i t i c f l u i d o f 
p a t i e n t s w i t h c i r r h o s i s o f t h e l i v e r . E x t r a c t i o n p r o c e d u r e s as 
w e l l as c h a r a c t e r i s t i c s o f our h i g h l y 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 f o r d e t e r m i n a t i o n o f ANF i n plasma have been 
p r e v i o u s l y d e s c r i b e d ( 6 , 9 ) . A p p l i c a t i o n o f t h e e x t r a c t i o n p r o c e ­
d u r e t o a l i q u o t s o f a s c i t i c f l u i d y i e l d e d h i g h r e c o v e r y r a t e s , n o t 
d i f f e r e n t f r o m t h o s e o b t a i n e d i n plasma. V a l i d i t y o f t h e RIA f o r 
ANF measurement i n a s c i t e s was d e m o n s t r a t e d by p a r a l l e l i t y o f 
s e r i a l d i l u t i o n c u r v e s w i t h t h e s t a n d a r d c u r v e and by t h e absence 
o f s i g n i f i c a n t b i n d i n g i n t e r f e r e n c e . 
I n an a t t e m p t t o f u r t h e r c h a r a c t e r i z e ANF i m m u n o r e a c t i v i t y i n 
a s c i t e s , che m o l e c u l a r w e i g h t p a t t e r n was i n v e s t i g a t e d by h i g h 
p e r f o r m a n c e g e l p e r m e a t i o n c h r o m a t o g r a p h y and r e v e r s e phase HPLC. 
Whereas t r a c e amounts o f h i g h e r m o l e c u l a r w e i g h t ANF i n plasma o f 
p a t i e n t s w i t h c i r r h o s i s had been r e p o r t e d ( 9 ) , ANF immunoreac-
t i ν i t y i n a s c i t e s c o e l u t e d w i t h s y n t h e t i c a l p h a human ANF, t h e 
main c i r c u l a t i n g f o r m i n human plasma. However, due t o t h e low 
c o n c e n t r a t i o n s o f ANF i n a s c i t e s and t o a 48% o n l y c r o s s r e a c t i v i t y 
o f our a n t i b o d y t o pro-ANF ( 9 ) , t h e p r e s e n c e o f s m a l l amounts o f 
h i g h e r m o l e c u l a r w e i g h t ANF i n a s c i t e s c a n n o t be e x c l u d e d . 
Vol. 43, No. 19, 1988 ANF i s Present i n Human A s c i t i c Fluid 1521 
ANF c o n c e n t r a t i o n s i n a s c i t e s were f o u n d t o be s i g n i f i c a n t l y c o r -
r e l a t e d t o ANF plasma c o n c e n t r a t i o n s . T h i s c o r r e l a t i o n as w e l l as 
c h a r a c t e r i z a t i o n o f a s c i t i c ANF as a l p h a ANF, t h e m a j o r c i r c u -
l a t i n g f o r m o f ANF, may a l l o w t h e s p e c u l a t i o n t h a t a s c i t i c ANF i s 
of plasma o r i g i n . ANF c o n c e n t r a t i o n s i n a s c i t e s were f o u n d t o be 
s i g n i f i c a n t l y l o w e r t h a n i n plasma. Thus, e l e v a t i o n s o f ANF l e v e l s 
i n p lasma o b s e r v e d f o l l o w i n g i n f u s i o n o f a s c i t i c f l u i d ( 3 , 4 , 5 ) 
seem n o t t o be due t o i n c r e a s e d l e v e l s o f ANF i n a s c i t i c f l u i d , 
b u t r a t h e r due t o i n c r e a s e d r e l e a s e o f ANF f o l l o w i n g t h e hemo-
dynamic changes o f i n f u s i o n . 
Ac know!edgment s 
A n i t a F r i e d r i c h , T o b i a s E i s e n h u t , V e i t Gülberg and F a b i a n Höpker 
are t h a n k e d f o r t h e i r s u p p o r t . P a r t s o f t h i s s t u d y were p r o v i d e d 
f o r by a g r a n t f r o m t h e F r i e d r i c h - B a u r - S t i f t u n g o f t h e F a c u l t y o f 
M e d i c i n e , U n i v e r s i t y o f Munich ( h e a d : P r o f . Dr. B u c h b o r n ) . 
R e f e r e n c e s 
1. A.L. GERBES, R.M. ARENDT, D. RITTER, D. JÜNGST, J. ZÄHRINGER 
and G. PAUMGARTNER, Ν. E n g l . J. Med. 31 3 1 6 0 9 - 1 61 0 ( 1 9 8 5 ) . 
2 . A.L. GERBES, R.M. ARENDT and G. PAUMSMTNER, J. H e p a t o l o g y 5 
1 2 3 - 1 3 2 ( 1 9 8 7 ) . 
3 . W. BURGHARDT, Η. WERNZE and K.L. DIEHL, K l i n . Wochenschr. 64 
( S u p p l V I ) 1 0 3 - 1 0 7 ( 1 9 8 6 ) . ~~~ 
4 . P. CAMPBELL, L.M. BLENDIS, K. SKORECKI, A. LOGAN, P.Y. WONG 
and P. GREIG, H e p a t o l o g y 6 1 1 2 0 ( 1 9 8 6 ) . 
5 . C.L. WITTE, A.P. MARTINEZ and M.H. WITTE, Ν. E n g l . J. Med. 316 
4 8 7 ( 1 9 8 7 ) . 
6 . R.M. ARENDT, Ε. STANGL, J. ZÄHRINGER, D.C. LIEBISCH and Α. 
HERZ, FEBS L e t t . 1 8 9 5 7 - 6 1 ( 1 9 8 5 ) . 
7 . F. MARUMO, H. SAKMTJTO, K. ANDO, T. ISHIGAMI and M. KAWAKAMI , 
Biochem. B i o p h y s . Res. Comm. 137 2 3 1 - 236 ( 1 9 8 6 ) . 
8 . F. MARUMO, T. MASUDA and Κ. Α N M , Biochem. B i o p h y s . Res. Comm. 
1 43 81 3 - 8 1 8 ( 1 9 8 7 ) . 
9 . ΤΓΓΜ. ARENDT, A.L. GERBES, D. RITTER and E. STANGL, K l i n . 
Wochenschr. 64_ ( s u p p l V I ) 9 7 - 1 02 ( 1 9 8 6 ) . 
1 0 . A.M. VOLLMAR, R.M. ARENDT and R. SCHULZ, Eur. J. Ph a r m a c o l . 
1 43 31 5 - 3 2 1 ( 1 9 8 7 ) . 


Presence of the atrial natriuretic factor (ANF) in human ascitic fluid

Open Access LMU

LIFE SCIENCES L I F E S C I E N C E S TABLE ÜF CONTENTS VOLUME 43, NUMBER 19 AIDS RESEARCH COMMUNICATIONS ILONA VATHY and ANNE M. ETGEN T.J. COLLINS, S. CHATTERJEE, L.S. LeGATE and T.K. BANERJI 1493 Ovarian Steroids and Hypothalamic Nor-epinephrine Release: Studies Using In Vivo Brain Microdialysis 1501 Lithium: Evidence for Reduction in C i r -culating Testosterone Levels i n Mice Fol-lowing Chronic Administration E. GIRALDO, F. MARTOS, A. GOMEZ, A. GARCIA, M.A. VIGANO, H. LADINSKY and F. SANCHEZ DE LA CUESTA 1507 Characterization of Muscarinic Receptor Subtypes in Human Tissues ALEXANDER L. GERBES, ANGELIKA Μ. VOLLMAR, YINING XIE and RAINER Μ. ARENDT 1517 Presence of the A t r i a l N a t r i u r e t i c Fac­tor (ANF) in Human A s c i t i c F l u i d G. CARO, M. BARRIOS and J.M. BAEYENS 1523 Dose-Dependent and Stereoselective An­tagonism by Diltiazem of Naloxone-Pre-cipitated Morphine Abstinence After Acute Morphine-Dependence In Vivo and In Vitro JAN G.C. VAN AMSTERDAM, KAREL J.H. VAN BUUREN, MARCO J. KRIELAART, OBBE P. ZUIDERVELD and ROB P.J.H. TIJMS 1529 Effect of Inhibitors of Enkephalin De­gradation in the Isolated Guinea-Pig Ileum W. SUTANTO and E.R. DE KLOET L. ALESSIO, S. GOVONI, G. SALAR, F. BATTAINI, R. IAVICOLI and M. TRABUCCHI 1537 ZK91587: A Novel Synthetic Antimineral-ocorticoid Displays High A f f i n i t y for Corticosterone (Type I ) Receptors i n the Rat Hippocampus 1545 Age-Related Changes of Methionine-Enke­phalin and Beta-Endorphin / Beta-Lipo-tropin Immunoreactivity in Human CSF SAT BIR S. KHALSA and GENE D. BLOCK HIROSHI SAITO, MITSUAKI NAKAMARU, TOSHIO OGIHARA, HIROMI RAKUGI, YUICHI KUMAHARA, TADASHI INAGAMI and KAZUAKI SHIMAMOTO 1551 Phase-Shifts of the Bulla Ocular C i r c a -dian Pacemaker in the Presence of C a l ­modulin Antagonists 1557 Effect of Vasodilator Prostaglandins on the Vascular Renin-Angiotensin System I N D E X E D IN: Current Contents, CABS, BIOSIS Database, Index Medicus, M E D L I N E , Psychol. Abstr., Nutr. Abstr., Energy Res. Abstr., Energy Data Base, Ophthal. Lit., Biological Abstr., Excerpta Medica, PASCAI^CNRS Database L I F S A K ISSN 0024-3205 (280) L I F E S C I E N C E S PETER R. OELTGEN, SITA 1565 P. NILEKANI, PAULA A. NUCHOLS, WILMA A. SPURRIER and TSUNG-PING SU I Further Studies on Opioids and Hiber-nation: Delta Opioid Receptor Ligand S e l e c t i v i t y Induced Hibernation in Summer-Active Ground Squirrels Software Survey Section L i f e Sciences, Vol. 43, pp. 1517-1521 Printed in the U.S.A. Pergaraon Press PRESENCE OF THE ATRIAL NATRIURETIC FACTOR (ANF) IN HUMAN ASCITIC FLUID * A l e x a n d e r L. Gerbes, A n g e l i k a Μ. Vollmar , Y i n i n g X i e , and R a i n e r Μ. Arendt M e d i z i n i s c h e K l i n i k I I and I , K l i n i k u m G r o s s h a d e r n and I n s t i t u t e of Pharmacology, V e t e r i n a r y S c h o o l , U n i v e r s i t y of Munich, Munich, F e d e r a l R e p u b l i c of Germany (Received in final, form September 12, 1988) Summary P r e s e n c e o f a t r i a l n a t r i u r e t i c f a c t o r ( A N F ) - l i k e mate­r i a l was d e m o n s t r a t e d by r a d i o i m m u n o a s s a y i n a s c i t i c f l u i d o f 14 p a t i e n t s w i t h c i r r h o s i s o f t h e l i v e r . Im-m u n o r e a c t i v e ANF c o n c e n t r a t i o n s (M+SEM) were 2.4 + 0.5 f m o l / m l i n a s c i t e s , s i g n i f i c a n t l y l o w e r (p < 0.001) t h a n t h e c o r r e s p o n d i n g plasma c o n c e n t r a t i o n s o f 15.5 + 2.6 f m o l / m l . High p e r f o r m a n c e g e l p e r m e a t i o n c h r o m a t o ­g r a p h y and r e v e r s e phase h i g h p e r f o r m a n c e c h r o m a t o ­g r a p h y o f t h e a s c i t i c ANF i m m u n o r e a c t i v i t y showed c o r ­r e s p o n d e n c e t o t h e a l p h a human ANF( 99-1 26 ) . ANF l e v e l s i n a s c i t e s were s i g n i f i c a n t l y (ρ < 0.01) c o r r e l a t e d t o l e v e l s i n plasma ( r = 0 . 6 6 ) . I n p a t i e n t s w i t h c i r r h o s i s o f t h e l i v e r and a s c i t e s t h e p a t h o ­p h y s i o l o g i c a l r o l e o f ANF has n o t been f u l l y e l u c i d a t e d ( 1 , 2 ) . F o l l o w i n g i n t r a v e n o u s r e i n f u s i o n o f a s c i t e s e l e v a t e d ANF plasma l e v e l s have been r e p o r t e d ( 3 , 4 , 5 ) . T h i s m i g h t be due t o ANF r e ­l e a s e s t i m u l a t e d by t h e hemodynamic changes f o l l o w i n g a s c i t e s i n f u s i o n or due t o e l e v a t e d ANF c o n c e n t r a t i o n s i n a s c i t i c f l u i d . Whereas t h e p r e s e n c e o f a t r i a l n a t r i u r e t i c f a c t o r (ANF) has been d e m o n s t r a t e d i n v a r i o u s f l u i d s o f t h e human body, such as plasma ( 6 ) , u r i n e ( 7 ) and c e r e b r o s p i n a l f l u i d ( 8 ) , a s c i t i c f l u i d has n o t been examined as y e t f o r t h e o c c u r e n c e o f ANF. I n t h i s s t u d y t h e p r e s e n c e o f ANF i n a s c i t i c f l u i d was i n v e s t i g a t e d . S u b j e c t s and Methods F o u r t e e n p a t i e n t s (11 men, 3 women, aged 42 t o 81 y e a r s ) w i t h b i o p s y p r o v e n c i r r h o s i s o f t h e l i v e r and a s c i t e s were i n v e s t i ­g a t e d . 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press pic 1518 ANF i s Present in Human A s c i t i c Fluid Vol. 43, No. 19, 1988 D e t e r m i n a t i o n o f ANF by r a d i o i m m u n o a s s a y . S i m u l t a n e o u s l y o b t a i n e d samp 1 es 5T a s c i t i c f l u i d and p e r i p h e r a 1 venous b l o o d were drawn i n t o p r e - c o o l e d s y r i n g e s and i m m e d i a t e l y t r a n s f e r r e d t o p r e - c o o l e d p o l y s t y r e n e t u b e s c o n t a i n i n g 500 k a l l i k r e i n i n h i b i t o r u n i t s a p r o t i n i n per ml and 1 mg sodium EDTA p e r m l . Samples were c e n t r i f u g e d a t 4° C, t h e s u p e r n a t a n t was i m m e d i a t e l y f r o z e n and s t o r e d a t - 80° C. E x t r a c t i o n o f a s c i t e s a l i q u o t s as w e l l as RIA p r o c e d u r e s were p e r f o r m e d i n t h e same way as f o r plasma samples; p r o c e d u r e s were m o d i f i e d f r o m ( 6 ) and have been d e t a i l e d e l s e w h e r e ( 9 ) . B r i e f l y , samples were e x t r a c t e d by a d s o r p t i o n t o A m b e r l i t e XAD-2 a d s o r b e n t r e s i n . The f i n a l t i t e r o f t h e C - t e r m i n a l d i r e c t e d a n t i b o d y Toni I I I was 1:120000, crossreactiνity t o r a t - p r o ANF was 48% and t h e assay s e n s i t i v i t y was 0.5 f m o l a l p h a human ANF/tube. The 50% b i n d i n g i n t e r c e p t o f t h e s t a n d a r d c u r v e was 10 f m o l . C h r o m a t o g r a p h i c a n a l y s i s o f ANF i m m u n o r e a c t i v i t y . A s c i t i c f l u i d e x t r a c t s of 4 p a t i e n t s were s u b j e c t e d t o h i g h p e r f o r m a n c e g e l p e r ­m e a t i o n c h r o m a t o g r a p h y (HPGPC) and r e v e r s e phase h i g h p e r f o r m a n c e c h r o m a t o g r a p h y (RP-HPC). High p e r f o r m a n c e g e l p e r m e a t i o n c h r o m a t o g r a p h y (HPGPC): L y o p h i -l i z e d a s c i t i c f l u i d (10 m l ) was d i s s o l v e d i n 25μ 1 column e l u e n s and a p p l i e d t o a S p h e r o g e l TM TSK, 2000 SW column (ΙΟμπι, 7.5mm χ 300 mm, Beckman I n s t r u m e n t s , San Rarnon, CA, USA), e l u t e d w i t h 0.09% t r i f 1 u o r o a c e t i c a c i d (TFA) c o n t a i n i n g 0.005M Na 2S0., 0.002M NaHpPO, and 30% a c e t o n i t r i l e ( f l o w r a t e : 0.3 m l / m i n ) . C a l i b r a t i o n was c a r r i e d o u t w i t h b o v i n e serum a l b u m i n (BSA) ( V ) , v i t a m i n B12 ( V . ) , r a t pro-ANF ( 2 - 1 2 6 ) and a l p h a human ANF ( 9 9 - 1 2 6 ) . Immuno-r e a c t i v e ANF (IR-ANF) f r a c t i o n s ( 0 . 6 m l ) , d e t e c t e d by RIA, were p o o l e d and l y o p h i 1 i zed. Reverse phase h i g h p e r f o r m a n c e c h r o m a t o g r a p h y (RP-HPC): An a l i q u o t of t h e p o o l e d IR-ANF f r a c t i o n s o f t h e HPGPC r u n was r e d i s s o l v e d i n 25 μΐ 0 . 1 % TFA and l o a d e d on a HPLC C 18 ODS U l t r a s p h e r e TM column (5 μ m 9 2mmx15 0mm, Beckman I n s t r u m e n t s , San Ramon, USA) a c c o r d i n g t o ( 1 0 ) . E l u t i o n was c a r r i e d o u t w i t h a l i n e a r g r a d i e n t o f a c e t o n i ­t r i l e ( 1 0 - 8 0 % , 45 m i n ) i n 0.1%TFA ( f l o w r a t e : 0 . 2 m l / m i n ) . C a l i ­b r a t i o n o f t h e column was p e r f o r m e d w i t h s y n t h e t i c a t r i o p e p t i n I and I I I , a l p h a human ANF ( 9 9 - 1 2 6 ) and r a t pro-ANF ( 2 - 1 2 6 ) . F r a c t i o n s ( 0 . 4 m l ) were assayed f o r ANF i m m u n o r e a c t i v i t y . S t a t i s t i c a l e v a l u a t i o n . D i f f e r e n c e s between a s c i t i c and p l a s m a t i c ANF c o n c e n t r a t i o n s were compared by p a i r e d t - t e s t ; t h e Pearson c o r r e l a t i o n c o e f f i c i e n t was d e t e r m i n e d by t h e u s u a l l i n e a r l e a s t s q u a r e s t e s t . Data a r e p r e s e n t e d as mean and s t a n d a r d e r r o r (M + SEM) . R e s u l t s Radi o i mmunoas s ay S e r i a l d i l u t i o n s o f a s c i t e s were p a r a l l e l t o t h e s t a n d a r d c u r v e , as shown i n f i g u r e 1 . R e c o v e r i e s o f 7.8 and 15.6 f m o l s y n t h e t i c human a l p h a ANF added t o samples o f a s c i t e s and plasma p r i o r t o e x t r a c t i o n were 84% and 67% i n a s c i t e s and 75% and 65% i n ρ l a s in a. N o n s p e c i f i c b i n d i n g , d e t e r m i n e d i n t h e RIA w i t h o u t a n t i s e r u m was l e s s t h a n 5% i n b o t h a s c i t e s and plasma. A s c i t i c ANF c o n c e n ­t r a t i o n s were 2.4 + 0.5 f m o l / m l , s i g n i f i c a n t l y ( p < 0.001) l o w e r t h a n ANF c o n c e n t r a t i o n s i n plasma (15.5 + 2.6 f m o l / m l ) . ANF l e v e l s Vol. 43, No. 19, 1988 ANF i s Present in Human A s c i t i c Fluid 1519 i n a s c i t e s were s i g n i f i c a n t l y c o r r e l a t e d t o ANF plasma l e v e l s ( r = 0.56, ρ < 0 . 0 1 ; f i g u r e 2 ) . factor of dilution 10 10C 1000 10000 A N F ( f m o l / t u b e ) FIG. 1 A s t a n d a r d c u r v e f o r r a ­dio i m m u n o a s s a y o f a l p h a human ANF ( c i r c l e s ) i n c o m p a r i s o n t o s e r i a l d i ­l u t i o n s o f a s c i t i c f l u i d ( t r i a n g l e s ) . Each p o i n t i s t h e mean o f two expe­r i m e n t s . D i l u t i o n c u r v e s p a r a l l e l t h e s t a n d a r d c u r v e s . HPLC c h a r a c t e r i z a t i o n o f i m m u n o r e a c t i v e ANF When p r e - e x t r a c t e d a s c i t i c f l u i d was c h r o m a t o g r a p h e d on HPGPC and r e v e r s e phase HPLC, i m m u n o r e a c t i v e ANF was d e t e c t e d as a s i n g l e peak cοeιut i ηg w i t h s y n t h e t i c a l p h a human ANF. No s i g n i f i c a n t amounts o f h i g h e r m o l e c u l a r w e i g h t A N F - l i k e m a t e r i a l such as p r e ­c u r s o r pro-ANF was d e t e c t e d , as d e m o n s t r a t e d i n f i g u r e 3. 1520 ANF i s Present i n Human A s c i t i c Fluid Vol. 43, No. 19, 1988 Ζ < ά Τ 20 ~30~ -80 -40 c ο φ ο <0 f r a c t i o n n u m b e r FIG. 3 HPLC a n a l y s i s o f i m m u n o r e a c t i v e ANF i n a s c i t i c f l u i d A: HPGPC: The v o i d (V ) and t o t a l ( V . ) volumes o f t h e column were d e t e r m i n e d by BSA and v i t a m i n Β T2, r e s p e c t i v e l y . The e l u t i o n p o s i t i o n s o f s y n t h e t i c r a t - p r o - A N F ( 2 - 1 2 6 ) (p-ANF) and a l p h a human ANF (ANF) are i n d i c a t e d by a r r o w s . F r a c t i o n s were t e s t e d f o r i m m u n o r e a c t i v e ANF by RIA. Β: Reverse phase HPLC: Ar r o w s i n d i c a t e t h e e l u t i o n p o s i t i o n s o f a t r i o p e p t i n I (AP I ) , a t r i o p e p t i n I I I (AP I I I ) , a l p h a human ANF (ANF) and r a t p r o - ANF ( 2 - 1 2 6 ) (p-ANF). I m m u n o r e a c t i v e ANF was e v a l u a t e d by RIA. D i s c u s s i o n T h i s s t u d y d e m o n s t r a t e s t h e p r e s e n c e o f ANF i n a s c i t i c f l u i d o f p a t i e n t s w i t h c i r r h o s i s o f t h e l i v e r . E x t r a c t i o n p r o c e d u r e s as w e l l as c h a r a c t e r i s t i c s o f our h i g h l y 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 f o r d e t e r m i n a t i o n o f ANF i n plasma have been p r e v i o u s l y d e s c r i b e d ( 6 , 9 ) . A p p l i c a t i o n o f t h e e x t r a c t i o n p r o c e ­d u r e t o a l i q u o t s o f a s c i t i c f l u i d y i e l d e d h i g h r e c o v e r y r a t e s , n o t d i f f e r e n t f r o m t h o s e o b t a i n e d i n plasma. V a l i d i t y o f t h e RIA f o r ANF measurement i n a s c i t e s was d e m o n s t r a t e d by p a r a l l e l i t y o f s e r i a l d i l u t i o n c u r v e s w i t h t h e s t a n d a r d c u r v e and by t h e absence o f s i g n i f i c a n t b i n d i n g i n t e r f e r e n c e . I n an a t t e m p t t o f u r t h e r c h a r a c t e r i z e ANF i m m u n o r e a c t i v i t y i n a s c i t e s , che m o l e c u l a r w e i g h t p a t t e r n was i n v e s t i g a t e d by h i g h p e r f o r m a n c e g e l p e r m e a t i o n c h r o m a t o g r a p h y and r e v e r s e phase HPLC. Whereas t r a c e amounts o f h i g h e r m o l e c u l a r w e i g h t ANF i n plasma o f p a t i e n t s w i t h c i r r h o s i s had been r e p o r t e d ( 9 ) , ANF immunoreac-t i ν i t y i n a s c i t e s c o e l u t e d w i t h s y n t h e t i c a l p h a human ANF, t h e main c i r c u l a t i n g f o r m i n human plasma. However, due t o t h e low c o n c e n t r a t i o n s o f ANF i n a s c i t e s and t o a 48% o n l y c r o s s r e a c t i v i t y o f our a n t i b o d y t o pro-ANF ( 9 ) , t h e p r e s e n c e o f s m a l l amounts o f h i g h e r m o l e c u l a r w e i g h t ANF i n a s c i t e s c a n n o t be e x c l u d e d . Vol. 43, No. 19, 1988 ANF i s Present i n Human A s c i t i c Fluid 1521 ANF c o n c e n t r a t i o n s i n a s c i t e s were f o u n d t o be s i g n i f i c a n t l y c o r -r e l a t e d t o ANF plasma c o n c e n t r a t i o n s . T h i s c o r r e l a t i o n as w e l l as c h a r a c t e r i z a t i o n o f a s c i t i c ANF as a l p h a ANF, t h e m a j o r c i r c u -l a t i n g f o r m o f ANF, may a l l o w t h e s p e c u l a t i o n t h a t a s c i t i c ANF i s of plasma o r i g i n . ANF c o n c e n t r a t i o n s i n a s c i t e s were f o u n d t o be s i g n i f i c a n t l y l o w e r t h a n i n plasma. Thus, e l e v a t i o n s o f ANF l e v e l s i n p lasma o b s e r v e d f o l l o w i n g i n f u s i o n o f a s c i t i c f l u i d ( 3 , 4 , 5 ) seem n o t t o be due t o i n c r e a s e d l e v e l s o f ANF i n a s c i t i c f l u i d , b u t r a t h e r due t o i n c r e a s e d r e l e a s e o f ANF f o l l o w i n g t h e hemo-dynamic changes o f i n f u s i o n . Ac know!edgment s A n i t a F r i e d r i c h , T o b i a s E i s e n h u t , V e i t Gülberg and F a b i a n Höpker are t h a n k e d f o r t h e i r s u p p o r t . P a r t s o f t h i s s t u d y were p r o v i d e d f o r by a g r a n t f r o m t h e F r i e d r i c h - B a u r - S t i f t u n g o f t h e F a c u l t y o f M e d i c i n e , U n i v e r s i t y o f Munich ( h e a d : P r o f . Dr. B u c h b o r n ) . R e f e r e n c e s 1. A.L. GERBES, R.M. ARENDT, D. RITTER, D. JÜNGST, J. ZÄHRINGER and G. PAUMGARTNER, Ν. E n g l . J. Med. 31 3 1 6 0 9 - 1 61 0 ( 1 9 8 5 ) . 2 . A.L. GERBES, R.M. ARENDT and G. PAUMSMTNER, J. H e p a t o l o g y 5 1 2 3 - 1 3 2 ( 1 9 8 7 ) . 3 . W. BURGHARDT, Η. WERNZE and K.L. DIEHL, K l i n . Wochenschr. 64 ( S u p p l V I ) 1 0 3 - 1 0 7 ( 1 9 8 6 ) . ~~~ 4 . P. CAMPBELL, L.M. BLENDIS, K. SKORECKI, A. LOGAN, P.Y. WONG and P. GREIG, H e p a t o l o g y 6 1 1 2 0 ( 1 9 8 6 ) . 5 . C.L. WITTE, A.P. MARTINEZ and M.H. WITTE, Ν. E n g l . J. Med. 316 4 8 7 ( 1 9 8 7 ) . 6 . R.M. ARENDT, Ε. STANGL, J. ZÄHRINGER, D.C. LIEBISCH and Α. HERZ, FEBS L e t t . 1 8 9 5 7 - 6 1 ( 1 9 8 5 ) . 7 . F. MARUMO, H. SAKMTJTO, K. ANDO, T. ISHIGAMI and M. KAWAKAMI , Biochem. B i o p h y s . Res. Comm. 137 2 3 1 - 236 ( 1 9 8 6 ) . 8 . F. MARUMO, T. MASUDA and Κ. Α N M , Biochem. B i o p h y s . Res. Comm. 1 43 81 3 - 8 1 8 ( 1 9 8 7 ) . 9 . ΤΓΓΜ. ARENDT, A.L. GERBES, D. RITTER and E. STANGL, K l i n . Wochenschr. 64_ ( s u p p l V I ) 9 7 - 1 02 ( 1 9 8 6 ) . 1 0 . A.M. VOLLMAR, R.M. ARENDT and R. SCHULZ, Eur. J. Ph a r m a c o l . 1 43 31 5 - 3 2 1 ( 1 9 8 7 ) . 

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Presence of the atrial natriuretic factor (ANF) in human ascitic fluid

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