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Presence of the atrial natriuretic factor (ANF) in human ascitic fluid
Life Sciences, Vol. 43, pp. 1517-1521 Printed in the U.S.A.
Pergamon Press
PRESENCE OF THE ATRIAL NATRIURETIC FACTOR (ANF) IN HUMAN ASCITIC FLUID Alexander L. Gerbes, Angelika M. Vollmar and Rainer M. Arendt
, Yining Xie,
Medizinische Klinik I I and I , Klinikum Grosshadern and I n s t i t u t e of Pharmacology, Veterinary School, University of Munich, Munich, Federal Republic of Germany (Received in final form September 12, 1988)
Summary Presence of a t r i a l natriuretic f a c t o r ( A N F ) - l i k e material 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 in a s c i t i c fluid of 14 p a t i e n t s w i t h c i r r h o s i s of the l i v e r . Imm 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 fmol/ml in ascites, significantly lower (p < 0~001) than the 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 of 15.5 + 2.6 f m o l / m l . High p e r f o r m a n c e gel p e r m e a t i o n chromato T g r a p h y and r e v e r s e phase high 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 of the 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 respondence to the a l p h a human A N F ( 9 9 - 1 2 6 ) . ANF l e v e l s in a s c i t e s were s i g n i f i c a n t l y (p < 0 . 0 1 ) c o r r e l a t e d to l e v e l s in plasma ( r = 0 . 6 6 ) . In p a t i e n t s with cirrhosis of t h e l i v e r and a s c i t e s the p a t h o physiological r o l e of ANF has not been f u l l y elucidated (1,2). Following intravenous reinfusion of 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 to ANF r e lease stimulated by the hemodynamic changes following ascites infusion or due to e l e v a t e d ANF c o n c e n t r a t i o n s in ascitic fluid. Whereas the p r e s e n c e of a t r i a l natriuretic factor (ANF) has been d e m o n s t r a t e d in v a r i o u s f l u i d s of the 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 fluid (8), ascitic fluid has not been examined as y e t f o r the o c c u r e n c e of ANF. In t h i s s t u d y the p r e s e n c e of ANF in 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 Subjects Fourteen patients ( I I men, b i o p s y proven c i r r h o s i s of gated.
and Methods
3 women, the l i v e r
aged and
42 to 81 y e a r s ) w i t h a s c i t e s were i n v e s t i -
0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
1518
ANF is Present in Human Ascitic Fluid
Vol. 43, No. 19, 1988
D e t e r m i n a t i o n of ANF by r a d i o i m m u n o a s s a y . Simultaneously obtained samples of a s c i t i c fluid and p e r i p h e r a l 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 to p r e - c o o l e d polystyrene tubes containing 500 kallikrein inhibitor units aprotinin per ml and 1 mg sodium EDTA per m l . Samples were centrifuged at 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 at 80 ° C. E x t r a c t i o n of 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 in t h e same way as f o r plasma s a m p l e s ; p r o c e d u r e s were m o d i f i e d from (6) and have been d e t a i l e d e l s e w h e r e (9). Briefly, samples were e x t r a c t e d by a d s o r p t i o n to 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 titer of 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 : 1 2 0 0 0 0 , c r o s s r e a c t i v i t y to 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 fmol a l p h a human A N F / t u b e . The 50% b i n d i n g i n t e r c e p t of t h e s t a n d a r d c u r v e was I0 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 of ANF i m m u n o r e a c t i v i t y . Ascitic fluid 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 to h i g h p e r f o r m a n c e gel 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 high 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 gel 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 lized ascitic fluid ( I 0 ml) was d i s s o l v e d in 25NI column e l u e n s and a p p l i e d to a Spherogel TM TSK, 2000 SW column (lONm, 7.5mm x 300 mm, Beckman I n s t r u m e n t s , San Ramon, CA, USA), e l u t e d with 0.09% t r i f l 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 O.O05M Na2SO~, O.O02M NaH2PO. and 30% a c e t o n i t r i l e (flowrate: 0.3 m l / m i n ) . Calibration was c a r r i e d out w i t h b o v i n e serum a l b u m i n (BSA) (V~),9 - I v i t a m i n BI2 ( V ~ ) , r a t pro-ANF ( 2 - 1 2 6 } and a l p h a human ANF ( 2 6 ) . Immunoreactive ANF (IR-ANF) f r a c t i o n s ( O . 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 l i z e d . Reverse phase high 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 of t h e HPGPC run was r e d i s s o l v e d in 25 N1 0.1% TFA and loaded on a HPLC C 18 ODS U l t r a s p h e r e TM column (5~m, 2mmxl5Omm, Beckman I n s t r u m e n t s , San Ramon, USA) a c c o r d i n g to (I0). E l u t i o n was c a r r i e d out w i t h a l i n e a r g r a d i e n t of a c e t o n i trile (10-80%, 45 min) in O.I%TFA ( f l o w r a t e : O.2ml/min). Calib r a t i o n of the column was p e r f o r m e d w i t h s y n t h e t i c atriopeptin I and III, alpha h u m a n ANF ( 9 9 - 1 2 6 ) and rat pro-ANF (2-126). F r a c t i o n s ( O . 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 . Statistical evaluation. 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 ~ the Pearson correlation coefficient was d e t e r m i n e d by the usual l i n e a r least squares t e s t . Data are presented as m e a n and s t a n d a r d error (M+SEM). Results Radioimmunoassay Serial dilutions of a s c i t e s were p a r a l l e l to the s t a n d a r d c u r v e , as shown in f i g u r e I . R e c o v e r i e s of 7 . 8 and 15.6 fmol s y n t h e t i c human a l p h a ANF added to samples of a s c i t e s and plasma p r i o r to extraction were 84% and 67% in a s c i t e s and 75% and 65% in plasma. 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 in the RIA w i t h o u t a n t i s e r u m was l e s s than 5% in both ascites and plasma. A s c i t i c ANF concentrations were 2.4 + 0.5 f m o l / m l , significantly (p < 0 . 0 0 1 ) l o w e r than ANF c o n c e n t r a t i o n s in plasma ( 1 5 . 5 + 2.6 f m o l / m l ) . ANF l e v e l s
Vol. 43, No. 19, 1988
ANF is Present in Human Ascitic Fluid
1519
in 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 to ANF plasma l e v e l s ( r = 0.66, p < O.Ol; f i g u r e 2). FIG.
1
factor of dilution 100
A standard curve f o r radioimmunoassay of alpha human ANF ( c i r c l e s ) in comparison to s e r i a l d i l u t i o n s of a s c i t i c f l u i d (triangles). E a c h point i s the mean of two experiments. D i l u t i o n curves parallel the standard curves.
75
~
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1
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1000
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FIG. E
6-
Significant correlation o f ANF c o n c e n t r a t i o n s in ascites and i n p l a s m a o f 14 p a t i e n t s with cirrhosis of the liver (r = 0.66, p < 0.01).
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of
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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 chromatographed on HPGPC and reverse phase HPLC, immunoreactive ANF was detected as a s i n g l e peak c o e l u t i n g with s y n t h e t i c alpha human ANF. No s i g n i f i c a n t amounts of higher molecular weight ANF-like m a t e r i a l such as precursor pro-ANF was detected, as demonstrated in f i g u r e 3.
1520
ANF is Present in Human Ascitic Fluid
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Vol. 43, No. 19, 1988
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i m m u n o r e a c t i v e ANF in
ascitic
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HPGPC: The v o i d (V o) and t o t a l (V~) volumes of the column were d e t e r m i n e d by BSA and v i t a m i n B ~2, r e s p e c t i v e l y . The e l u t i o n positions of s y n t h e t i c rat-pro-ANF (2-126) (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.
B: Reverse phase HPLC: Arrows i n d i c a t e the e l u t i o n positions of atriopeptin I (AP I ) ~ a t r i o p e p t i n III (AP I I I ) , a l p h a human ANF (ANF) and r a t p r o - ANF ( 2 - 1 2 6 ) ( ) - A N F ) . 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. Discussion T h i s s t u d y d e m o n s t r a t e s the p r e s e n c e of ANF i n a s c i t i c fluid of patients with cirrhosis of the l i v e r . Extraction p r o c e d u r e s as well as c h a r a c t e r i s t i c s of our highly sensitive and s p e c i f i c radioimmunoassay for determination of ANF in plasma have been previously described (6,9). Application of the e x t r a c t i o n procedure to a l i q u o t s of a s c i t i c f l u i d y i e l d e d high r e c o v e r y r a t e s , not different from t h o s e o b t a i n e d in plasma. V a l i d i t y of the RIA f o r ANF measurement in 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 of serial dilution c u r v e s w i t h the s t a n d a r d c u r v e and by the absence of s i g n i f i c a n t binding interference. In an a t t e m p t to f u r t h e r characterize ANF i m m u n o r e a c t i v i t y in ascites, the 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 high p e r f o r m a n c e gel 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 of h i g h e r m o l e c u l a r w e i g h t ANF in plasma of patients with cirrhosis had been r e p o r t e d (9), ANF immunoreactivity in 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, the main c i r c u l a t i n g form in human plasma. However, due to the low c o n c e n t r a t i o n s of ANF in a s c i t e s and to a 48% o n l y c r o s s r e a c t i v i t y of our a n t i b o d y to pro-ANF ( 9 ) , the presence of s m a l l amounts of h i g h e r m o l e c u l a r w e i g h t ANF in a s c i t e s cannot be e x c l u d e d .
Vol. 43, No. 19, 1988
ANF is Present in Human Ascitic Fluid
1521
ANF concentrations in ascites were found to be s i g n i f i c a n t l y corr e l a t e d to ANF plasma concentrations. This c o r r e l a t i o n as well as c h a r a c t e r i z a t i o n of a s c i t i c ANF as alpha ANF, the major c i r c u l a t i n g form of ANF, may allow the speculation that a s c i t i c ANF is of plasma o r i g i n . ANF concentrations in ascites were found to be s i g n i f i c a n t l y lower than in plasma. Thus, elevations of ANF levels in plasma observed f o l l o w i n g infusion of a s c i t i c f l u i d (3,4,5) seem not to be due to increased levels of ANF in a s c i t i c f l u i d , but rather due to increased release of ANF f o l l o w i n g the hemodynamic changes of i n f u s i o n .
Acknowledgments Anita F r i e d r i c h , Tobias Eisenhut, Veit GUlberg and Fabian H~pker are thanked f o r t h e i r support. Parts of t h i s study were provided for by a grant from the F r i e d r i c h - B a u r - S t i f t u n g of the Faculty of Medicine, U n i v e r s i t y of Munich (head: Prof. Dr. Buchborn). References I.
A.L. GERBES, R.M. ARENDT, D. RITTER, D. JUNGST, J. ZAHRINGER and G. PAUMGARTNER, N. Engl. J. Med. 313 1609-1610 (1985). 2. A.L. GERBES, R.M. ARENDT and G. PAUMG~-ITTNER, J. Hepatology 5 123-132 (1987). 3. W. BURGHARDT, H. WERNZE and K.L. DIEHL, K l i n . Wochenschr. 64 (Suppl Vl) I03-I07 (1986). 4. P. CAMPBELL, L . M . BLENDIS, K. SKORECKI, A. LOGAN, P.Y. WONG and P. GREIG, Hepatology 6 l l 2 0 (1986). 5. C.L. WITTE, A.P. MARTINEZ-and M.H. WITTE, N. Engl. J. Med. 316 487 (1987). 6. R . M . ARENDT, E. STANGL, J. ZAHRINGER, D . C . LIEBISCH and A. HERZ, FEBS L e t t . 189 57-61 (1985). 7. F. MARUMO, H. SAK~TO, K. ANDO, T. ISHIGAMI and M. KAWAKAMI, Biochem. Biophys. Res. Comm. 137 231- 236 (1986). 8. F. MARUMO, T. MASUDA and K. A ~ , Biochem. Biophys. Res. Comm. 143 813-818 (1987). 9. R-TI~. ARENDT, A.L. GERBES, D. RITTER and E. STANGL, K l i n . Wochenschr. 64 (suppl Vl) 97-I02 (1986). lO. A.M. VOLLMAR~R.M. ARENDT and R. SCHULZ, Eur. J. Pharmacol. 143 315-321 (1987).
Pergamon Press
PRESENCE OF THE ATRIAL NATRIURETIC FACTOR (ANF) IN HUMAN ASCITIC FLUID Alexander L. Gerbes, Angelika M. Vollmar and Rainer M. Arendt
, Yining Xie,
Medizinische Klinik I I and I , Klinikum Grosshadern and I n s t i t u t e of Pharmacology, Veterinary School, University of Munich, Munich, Federal Republic of Germany (Received in final form September 12, 1988)
Summary Presence of a t r i a l natriuretic f a c t o r ( A N F ) - l i k e material 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 in a s c i t i c fluid of 14 p a t i e n t s w i t h c i r r h o s i s of the l i v e r . Imm 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 fmol/ml in ascites, significantly lower (p < 0~001) than the 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 of 15.5 + 2.6 f m o l / m l . High p e r f o r m a n c e gel p e r m e a t i o n chromato T g r a p h y and r e v e r s e phase high 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 of the 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 respondence to the a l p h a human A N F ( 9 9 - 1 2 6 ) . ANF l e v e l s in a s c i t e s were s i g n i f i c a n t l y (p < 0 . 0 1 ) c o r r e l a t e d to l e v e l s in plasma ( r = 0 . 6 6 ) . In p a t i e n t s with cirrhosis of t h e l i v e r and a s c i t e s the p a t h o physiological r o l e of ANF has not been f u l l y elucidated (1,2). Following intravenous reinfusion of 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 to ANF r e lease stimulated by the hemodynamic changes following ascites infusion or due to e l e v a t e d ANF c o n c e n t r a t i o n s in ascitic fluid. Whereas the p r e s e n c e of a t r i a l natriuretic factor (ANF) has been d e m o n s t r a t e d in v a r i o u s f l u i d s of the 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 fluid (8), ascitic fluid has not been examined as y e t f o r the o c c u r e n c e of ANF. In t h i s s t u d y the p r e s e n c e of ANF in 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 Subjects Fourteen patients ( I I men, b i o p s y proven c i r r h o s i s of gated.
and Methods
3 women, the l i v e r
aged and
42 to 81 y e a r s ) w i t h a s c i t e s were i n v e s t i -
0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
1518
ANF is Present in Human Ascitic Fluid
Vol. 43, No. 19, 1988
D e t e r m i n a t i o n of ANF by r a d i o i m m u n o a s s a y . Simultaneously obtained samples of a s c i t i c fluid and p e r i p h e r a l 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 to p r e - c o o l e d polystyrene tubes containing 500 kallikrein inhibitor units aprotinin per ml and 1 mg sodium EDTA per m l . Samples were centrifuged at 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 at 80 ° C. E x t r a c t i o n of 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 in t h e same way as f o r plasma s a m p l e s ; p r o c e d u r e s were m o d i f i e d from (6) and have been d e t a i l e d e l s e w h e r e (9). Briefly, samples were e x t r a c t e d by a d s o r p t i o n to 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 titer of 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 : 1 2 0 0 0 0 , c r o s s r e a c t i v i t y to 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 fmol a l p h a human A N F / t u b e . The 50% b i n d i n g i n t e r c e p t of t h e s t a n d a r d c u r v e was I0 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 of ANF i m m u n o r e a c t i v i t y . Ascitic fluid 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 to h i g h p e r f o r m a n c e gel 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 high 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 gel 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 lized ascitic fluid ( I 0 ml) was d i s s o l v e d in 25NI column e l u e n s and a p p l i e d to a Spherogel TM TSK, 2000 SW column (lONm, 7.5mm x 300 mm, Beckman I n s t r u m e n t s , San Ramon, CA, USA), e l u t e d with 0.09% t r i f l 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 O.O05M Na2SO~, O.O02M NaH2PO. and 30% a c e t o n i t r i l e (flowrate: 0.3 m l / m i n ) . Calibration was c a r r i e d out w i t h b o v i n e serum a l b u m i n (BSA) (V~),9 - I v i t a m i n BI2 ( V ~ ) , r a t pro-ANF ( 2 - 1 2 6 } and a l p h a human ANF ( 2 6 ) . Immunoreactive ANF (IR-ANF) f r a c t i o n s ( O . 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 l i z e d . Reverse phase high 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 of t h e HPGPC run was r e d i s s o l v e d in 25 N1 0.1% TFA and loaded on a HPLC C 18 ODS U l t r a s p h e r e TM column (5~m, 2mmxl5Omm, Beckman I n s t r u m e n t s , San Ramon, USA) a c c o r d i n g to (I0). E l u t i o n was c a r r i e d out w i t h a l i n e a r g r a d i e n t of a c e t o n i trile (10-80%, 45 min) in O.I%TFA ( f l o w r a t e : O.2ml/min). Calib r a t i o n of the column was p e r f o r m e d w i t h s y n t h e t i c atriopeptin I and III, alpha h u m a n ANF ( 9 9 - 1 2 6 ) and rat pro-ANF (2-126). F r a c t i o n s ( O . 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 . Statistical evaluation. 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 ~ the Pearson correlation coefficient was d e t e r m i n e d by the usual l i n e a r least squares t e s t . Data are presented as m e a n and s t a n d a r d error (M+SEM). Results Radioimmunoassay Serial dilutions of a s c i t e s were p a r a l l e l to the s t a n d a r d c u r v e , as shown in f i g u r e I . R e c o v e r i e s of 7 . 8 and 15.6 fmol s y n t h e t i c human a l p h a ANF added to samples of a s c i t e s and plasma p r i o r to extraction were 84% and 67% in a s c i t e s and 75% and 65% in plasma. 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 in the RIA w i t h o u t a n t i s e r u m was l e s s than 5% in both ascites and plasma. A s c i t i c ANF concentrations were 2.4 + 0.5 f m o l / m l , significantly (p < 0 . 0 0 1 ) l o w e r than ANF c o n c e n t r a t i o n s in plasma ( 1 5 . 5 + 2.6 f m o l / m l ) . ANF l e v e l s
Vol. 43, No. 19, 1988
ANF is Present in Human Ascitic Fluid
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in 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 to ANF plasma l e v e l s ( r = 0.66, p < O.Ol; f i g u r e 2). FIG.
1
factor of dilution 100
A standard curve f o r radioimmunoassay of alpha human ANF ( c i r c l e s ) in comparison to s e r i a l d i l u t i o n s of a s c i t i c f l u i d (triangles). E a c h point i s the mean of two experiments. D i l u t i o n curves parallel the standard curves.
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Significant correlation o f ANF c o n c e n t r a t i o n s in ascites and i n p l a s m a o f 14 p a t i e n t s with cirrhosis of the liver (r = 0.66, p < 0.01).
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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 chromatographed on HPGPC and reverse phase HPLC, immunoreactive ANF was detected as a s i n g l e peak c o e l u t i n g with s y n t h e t i c alpha human ANF. No s i g n i f i c a n t amounts of higher molecular weight ANF-like m a t e r i a l such as precursor pro-ANF was detected, as demonstrated in f i g u r e 3.
1520
ANF is Present in Human Ascitic Fluid
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Vol. 43, No. 19, 1988
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HPGPC: The v o i d (V o) and t o t a l (V~) volumes of the column were d e t e r m i n e d by BSA and v i t a m i n B ~2, r e s p e c t i v e l y . The e l u t i o n positions of s y n t h e t i c rat-pro-ANF (2-126) (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.
B: Reverse phase HPLC: Arrows i n d i c a t e the e l u t i o n positions of atriopeptin I (AP I ) ~ a t r i o p e p t i n III (AP I I I ) , a l p h a human ANF (ANF) and r a t p r o - ANF ( 2 - 1 2 6 ) ( ) - A N F ) . 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. Discussion T h i s s t u d y d e m o n s t r a t e s the p r e s e n c e of ANF i n a s c i t i c fluid of patients with cirrhosis of the l i v e r . Extraction p r o c e d u r e s as well as c h a r a c t e r i s t i c s of our highly sensitive and s p e c i f i c radioimmunoassay for determination of ANF in plasma have been previously described (6,9). Application of the e x t r a c t i o n procedure to a l i q u o t s of a s c i t i c f l u i d y i e l d e d high r e c o v e r y r a t e s , not different from t h o s e o b t a i n e d in plasma. V a l i d i t y of the RIA f o r ANF measurement in 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 of serial dilution c u r v e s w i t h the s t a n d a r d c u r v e and by the absence of s i g n i f i c a n t binding interference. In an a t t e m p t to f u r t h e r characterize ANF i m m u n o r e a c t i v i t y in ascites, the 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 high p e r f o r m a n c e gel 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 of h i g h e r m o l e c u l a r w e i g h t ANF in plasma of patients with cirrhosis had been r e p o r t e d (9), ANF immunoreactivity in 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, the main c i r c u l a t i n g form in human plasma. However, due to the low c o n c e n t r a t i o n s of ANF in a s c i t e s and to a 48% o n l y c r o s s r e a c t i v i t y of our a n t i b o d y to pro-ANF ( 9 ) , the presence of s m a l l amounts of h i g h e r m o l e c u l a r w e i g h t ANF in a s c i t e s cannot be e x c l u d e d .
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ANF is Present in Human Ascitic Fluid
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ANF concentrations in ascites were found to be s i g n i f i c a n t l y corr e l a t e d to ANF plasma concentrations. This c o r r e l a t i o n as well as c h a r a c t e r i z a t i o n of a s c i t i c ANF as alpha ANF, the major c i r c u l a t i n g form of ANF, may allow the speculation that a s c i t i c ANF is of plasma o r i g i n . ANF concentrations in ascites were found to be s i g n i f i c a n t l y lower than in plasma. Thus, elevations of ANF levels in plasma observed f o l l o w i n g infusion of a s c i t i c f l u i d (3,4,5) seem not to be due to increased levels of ANF in a s c i t i c f l u i d , but rather due to increased release of ANF f o l l o w i n g the hemodynamic changes of i n f u s i o n .
Acknowledgments Anita F r i e d r i c h , Tobias Eisenhut, Veit GUlberg and Fabian H~pker are thanked f o r t h e i r support. Parts of t h i s study were provided for by a grant from the F r i e d r i c h - B a u r - S t i f t u n g of the Faculty of Medicine, U n i v e r s i t y of Munich (head: Prof. Dr. Buchborn). References I.
A.L. GERBES, R.M. ARENDT, D. RITTER, D. JUNGST, J. ZAHRINGER and G. PAUMGARTNER, N. Engl. J. Med. 313 1609-1610 (1985). 2. A.L. GERBES, R.M. ARENDT and G. PAUMG~-ITTNER, J. Hepatology 5 123-132 (1987). 3. W. BURGHARDT, H. WERNZE and K.L. DIEHL, K l i n . Wochenschr. 64 (Suppl Vl) I03-I07 (1986). 4. P. CAMPBELL, L . M . BLENDIS, K. SKORECKI, A. LOGAN, P.Y. WONG and P. GREIG, Hepatology 6 l l 2 0 (1986). 5. C.L. WITTE, A.P. MARTINEZ-and M.H. WITTE, N. Engl. J. Med. 316 487 (1987). 6. R . M . ARENDT, E. STANGL, J. ZAHRINGER, D . C . LIEBISCH and A. HERZ, FEBS L e t t . 189 57-61 (1985). 7. F. MARUMO, H. SAK~TO, K. ANDO, T. ISHIGAMI and M. KAWAKAMI, Biochem. Biophys. Res. Comm. 137 231- 236 (1986). 8. F. MARUMO, T. MASUDA and K. A ~ , Biochem. Biophys. Res. Comm. 143 813-818 (1987). 9. R-TI~. ARENDT, A.L. GERBES, D. RITTER and E. STANGL, K l i n . Wochenschr. 64 (suppl Vl) 97-I02 (1986). lO. A.M. VOLLMAR~R.M. ARENDT and R. SCHULZ, Eur. J. Pharmacol. 143 315-321 (1987).