Arendt, Rainer M.

Brenner, Barry M.

Gerbes, Alexander L.

Vollmar, Angelika M.

Xie, Yining

English

Universität München: Elektronischen Publikationen

7 
6 5 6 S E C T I O N V 
ATRIAL NATRIURETIC FACTOR IN ASCITES OF 
PATIENTS WITH CLRRHOSIS OF THE LLVER OR WITH 
MALIGNANT NEOPLASMS 
* Alexander L . Gerbes, **Angelika M. Vollmar, *Yining Xie, 
and t Rainer M . Arendt 
Departments o f M e d i c i n e *// and **/, K l i n i k u m Grosshadern, and tlnstitute o f 
Pharmacology, V e t e r i n a r y School, University o f M u n i c h , 
M u n i c h , F e d e r a l Republic o f Germany 
The presence of atrial natriuretic factor ( A N F ) - l i k e immunoreactivity was . I 
demonstrated in ascitic f luid ( A F ) of 14 patients with cirrhosis of the liver ( L C ) J 
and 10 patients with malignant neoplasms ( M N ) . A N F concentrations (mean ± J 
S E M , fmol/ml) in A F of L C / M N were 2.4 ± 0.5/3.3 ± 0.5, significantly (p < I 
0.001) lower than the corresponding concentrations in plasma (P) (15.5 ± 2.6/ | 
13.0 ± 2.9 fmol/ml). H igh Performance gel permeation and reverse phase 
chromatography of A N F immunoreactivity in A F showed correspondence to 
A N F 9 9 _ , 2 6 . A N F concentrations in A F were significantly (p < 0.01) correlated 
with concentrations in P in L C / M N (r = 0.66/0.90). Thus , elevations of A N F 
concentrations in P after infusion of A F would not seem to be due to high A N F 
content of A F . 
Increases of diuresis and natriuresis have been observed after intravenous 
reinfusion of ascitic fluid in patients with ascites due to cirrhosis of the l iver and 
in patients with malignant neoplasms (1,2). Recently, marked elevations of A N F 
plasma concentrations after reinfusion were reported and were considered to 
contribute to the ensuing diuresis (3-5). Al though the pathophysiological role of 
A N F in volume regulation has not been fully elucidated in cirrhosis (6,7) and 
has not received much attention in malignant ascites, the mechanism of A N F 
increase after ascites infusion deserves attention. The reinfused ascitic fluid it-
self might contribute to the observed rises of A N F plasma concentrations rather j 
than volume-stimulated A N F release alone. 
A N F has been demonstrated in various fluids of the human body, such as | 
plasma (8), urine (9), and cerebrospinal fluid (10). Asc i t i c f luid, however, has not 
been examined as yet for the occurrence of A N F . In this study, the presence of 
A N F in ascitic fluid was investigated. 
SUBJECTS AND M E T H O D S 
Twenty-four patients with ascites were investigated. Fourteen had biopsy-
proven liver cirrhosis (age 42 to 81, mean 61 ± 4 years) and 10 had primary or 
metastatic malignancy in the abdomen or pelvis (age 42 to 80, mean 64 ± 4 
years). 
A N P E F F E C T S O N THE K I D N E Y A N D L I V E R 6 5 7 
Determination of ANF by Radioimmunoassay (RIA) 
Simultaneously obtained samples of ascitic fluid and peripheral venous blood 
were drawn into precooled polystyrene tubes containing 500 K I U aprotinin and 
1 mg sodium ethylenediaminetetraacetic acid ( E D T A ) per m l . Samples were cen-
trifuged at 4°C, and the supernatant fluid was immediately frozen and stored at 
- 80°C. Ext rac t ion of ascites aliquots as wel l as R I A procedures were performed 
in the same way as for plasma samples; procedures have been detailed elsewhere 
(8). Br ief ly , samples were extracted by adsorption to Amber l i te X A D - 2 adsor-
bent resin. The final titer of the C-terminal-directed antibody Toni III was 
1:120,000, cross-reactivity to rat p r o - A N F was 48% and the assay sensitivity was 
0.5 fmol a-human A N F / t u b e . The 50% binding intercept of the Standard curve 
was 10 fmol. 
Chromatographie Analysis of ANF Immunoreactivity 
Asci t ic fluid extracts of six patients were subjected to high Performance gel 
permeation chromatography ( H P G P C ) and reverse phase high Performance 
chromatography ( R P - H P C ) . 
H P G P C 
Lyophi l ized ascitic fluid (10 ml) was dissolved in 25-|xl co lumn eluens and 
applied to a Spherogel T M T S K , 2000 S W column (10 p,m, 7.5 x 300 m m ; Beck-
mari Instruments), eluted with 0.09% trifluoroacetic acid ( T F A ) containing 0.005 
M N a 2 S 0 4 , 0.002 M N a H 2 P 0 4 , and 30% acetonitrile (flow rate, 0.3 ml/min). Ca l i -
bration was carried out with bovine serum albumin ( B S A ) ( V 0 ) , v i tamin B , 2 ( V t ) , 
rat p r o - A N F 2 _ l 2 6 , and a-human A N F 9 9 _ 1 2 6 . Immunoreactive A N F ( I R - A N F ) frac-
tions (0.6 ml), detected by R I A , were pooled and lyophi l ized . 
R P - H P C 
A n aliquot o f the pooled I R - A N F fractions of the H P G P C run was redissolved 
in 25 juul of 0 .1% T F A and loaded on a H P L C C-18 O D S Ultrasphere T M column 
(5 | L m , 2 x 150 m m ; Beckman Instruments), aecording to ref. 11. E lu t ion was 
c a r i e d out with a linear gradient of acetonitrile (10 to 80%, 45 min) in 0 .1% T F A 
(flosv rate, 0.2 ml/min). Cal ibrat ion of the column was performed with synthetic 
atmpeptin I and III, human A N F 9 9 _ l 2 6 , and rat p r o - A N F 2 _ , 2 6 . Fract ions (0.4 ml) 
weie assayed for A N F immunoreactivity. 
Statistical Evaluation 
Lifferences between ascitic and plasmatic A N F concentrations were com-
pand by paired r-test; the Pearson correlation coefficient was determined by the 
usml linear least Squares test. Data are presented as means ± S E M . 
6 5 8 S E C T I O N V 
RESULTS 
Serial dilutions of ascites were parallel to the Standard curve. Recover ies of 
7.8 and 15.6 fmol synthetic human a - A N F added to samples of ascites and 
plasma before extraction were 84% and 67% in ascites and 75% and 65% in 
plasma. Nonspecif ic binding, determined in the R I A without antiserum, was less 
than 5% in both ascites and plasma. In patients with cirrhosis , ascit ic A N F con-
centrations were 2.4 ± 0.5 fmol/ml , significantly (p < 0.001) lower than A N F 
concentrations in plasma (15.5 ± 2.6 fmol/ml). In patients wi th malignancies, 
A N F levels in ascites were 3.3 ± 0.5 fmol/ml , significantly (p < 0.001) lower 
than levels in plasma (13.0 ± 2.9 fmol/ml). A N F concentrations in ascites were 
significantly correlated to A N F plasma concentrations in cirrhosis (r = 0.66, p 
< 0.01) as well as in malignancies (r = 0.90, p < 0.01) (F ig . 1). 
When preextracted ascitic fluid was chromatographed on H P G P C and R P -
H P C , immunoreactive A N F was detected as a single peak coeluting with syn-
thetic human A N F 9 9 _ , 2 6 . N o significant amount o f higher-molecular-weight A N F -
like material such as precursor p r o - A N F was detected. 
DISCUSSION 
This study demonstrates the presence of A N F in ascitic fluid o f patients with 
cirrhosis of the l iver and with malignant neoplasms. Ext rac t ion procedures as 
well as characteristics of our highly sensitive and specific radioimmunoassay for 
determination of A N F in plasma have been previously described (8). App l i ca t ion 
of the extraction procedure to aliquots of ascitic fluid yielded high recovery 
rates, not different from those obtained in plasma. Validi ty of the R I A for A N F 
measurement in ascites was demonstrated by parallelity of serial d i lut ion curves 
with the Standard curve and by the absence of significant binding interference. 
In an attempt to characterize further A N F immunoreactivity in ascites, the 
ANF in plasma (fmol/ml) ANF in plasma (fmol/ml) 
FIG. 1. Significant correlation of ANF concentrations in ascites and in plasma of 14 pa-
tients with cirrhosis of the liver (r = 0.66, p < 0.01) (a) and 10 patients with malignant 
neoplasms (r = 0.90, p < 0.01) (b). 
A N P E F F E C T S O N THE K I D N E Y A N D L I V E R 6 5 9 
molecular weight pattern was investigated by H P G P C and reverse phase H P L C . 
Al though trace amounts of higher-molecular-weight A N F in plasma of patients 
with cirrhosis have been reported (8), A N F immunoreact ivi ty in ascites coeluted 
with synthetic A N F 9 9 _ l 2 6 , the main circulating form in human plasma. However , 
because of the low concentrations of A N F in ascites and a 48% cross-reactivity 
of our antibody to p r o - A N F (8), the presence o f small amounts of higher-molec-
ular-weight A N F in ascites cannot be excluded. 
A N F concentrations in ascites were significantly correlated with A N F plasma 
concentrations. This correlation as wel l as characterization of ascitic A N F as 
A N F 9 9 _ , 2 6 , the major circulating form of A N F , may indicate plasmatic origin o f 
ascitic A N F . A N F concentrations in ascites were significantly lower than in 
plasma. Thus, elevations of A N F levels in plasma after the infusion of ascitic 
fluid (3-5) seem not to be due to increased levels of A N F in ascitic fluid but 
rather due to increased release o f A N F after the hemodynamic changes of 
infusion. 
A C K N O W L E D G M E N T S 
A n i t a Fr iedr ich , Veit G ü l b e r g , and Fabian H ö p k e r are thanked for their Sup-
port. Parts of this study were supported by a grant from the funds of the M ü n -
chner Medizinische Wochenschrift (head: Prof. D r . Eyer ) . 
R E F E R E N C E S 
1. LeVeen, H . H . , Christoudias, G . , Ip, M . , et al. (1974): A n n . Surg., 180:580-591. 
2. Gough, I. R. (1984): Cancer, 54:2226-2230. 
3. Burghardt, W., Wernze, H . , and Diehl, K. L . (1986): K l i n . Wochenschr., 64(Suppl. 
VI): 103-107. 
4. Campbell, P., Blendis, L . M . , Skorecki, K . , et al. (1986): Hepatology, 6:1120. 
5. Witte, C. L . , Martinez, A . R, and Witte, M . H . (1987): N . E n g l . J. M e d . , 316:487. 
6. Gerbes, A. L . , Arendt, R. M . , Ritter, D. , et al. (1985): N . E n g l . J. M e d . , 313:1609-1610. 
7. Gerbes, A. L . , Arendt, R. M . , and Paumgartner, G . (1987): J. H e p a t o L , 5:123-132. 
8. Arendt, R. M . , Gerbes, A. L . , Ritter, D. , et al. (1986): K l i n . Wochenschr., 64(Suppl. 
6):97-102. 
9. Marumo, F., Sakamoto, H . , Ando, D. , et al. (1986): Biochem. Biophys. Res. C o m m u n . , 
137:231-236. 
10. Marumo, F., Masuda, T. , and Ando, K. (1987): Biochem. Biophys. Res. C o m m u n . , 
143:813-818. 
11. Vollmar, A. M . , Arendt, R. M . , and Schulz, R. (1987): E u r . J. P h a r m a c o l . , 143:315-
321. 


Atrial natriuretic factor (ANF) in ascites of patients with cirrhosis of the liver or with malignant neoplasms

Open Access LMU

7 6 5 6 S E C T I O N V ATRIAL NATRIURETIC FACTOR IN ASCITES OF PATIENTS WITH CLRRHOSIS OF THE LLVER OR WITH MALIGNANT NEOPLASMS * Alexander L . Gerbes, **Angelika M. Vollmar, *Yining Xie, and t Rainer M . Arendt Departments o f M e d i c i n e *// and **/, K l i n i k u m Grosshadern, and tlnstitute o f Pharmacology, V e t e r i n a r y School, University o f M u n i c h , M u n i c h , F e d e r a l Republic o f Germany The presence of atrial natriuretic factor ( A N F ) - l i k e immunoreactivity was . I demonstrated in ascitic f luid ( A F ) of 14 patients with cirrhosis of the liver ( L C ) J and 10 patients with malignant neoplasms ( M N ) . A N F concentrations (mean ± J S E M , fmol/ml) in A F of L C / M N were 2.4 ± 0.5/3.3 ± 0.5, significantly (p < I 0.001) lower than the corresponding concentrations in plasma (P) (15.5 ± 2.6/ | 13.0 ± 2.9 fmol/ml). H igh Performance gel permeation and reverse phase chromatography of A N F immunoreactivity in A F showed correspondence to A N F 9 9 _ , 2 6 . A N F concentrations in A F were significantly (p < 0.01) correlated with concentrations in P in L C / M N (r = 0.66/0.90). Thus , elevations of A N F concentrations in P after infusion of A F would not seem to be due to high A N F content of A F . Increases of diuresis and natriuresis have been observed after intravenous reinfusion of ascitic fluid in patients with ascites due to cirrhosis of the l iver and in patients with malignant neoplasms (1,2). Recently, marked elevations of A N F plasma concentrations after reinfusion were reported and were considered to contribute to the ensuing diuresis (3-5). Al though the pathophysiological role of A N F in volume regulation has not been fully elucidated in cirrhosis (6,7) and has not received much attention in malignant ascites, the mechanism of A N F increase after ascites infusion deserves attention. The reinfused ascitic fluid it-self might contribute to the observed rises of A N F plasma concentrations rather j than volume-stimulated A N F release alone. A N F has been demonstrated in various fluids of the human body, such as | plasma (8), urine (9), and cerebrospinal fluid (10). Asc i t i c f luid, however, has not been examined as yet for the occurrence of A N F . In this study, the presence of A N F in ascitic fluid was investigated. SUBJECTS AND M E T H O D S Twenty-four patients with ascites were investigated. Fourteen had biopsy-proven liver cirrhosis (age 42 to 81, mean 61 ± 4 years) and 10 had primary or metastatic malignancy in the abdomen or pelvis (age 42 to 80, mean 64 ± 4 years). A N P E F F E C T S O N THE K I D N E Y A N D L I V E R 6 5 7 Determination of ANF by Radioimmunoassay (RIA) Simultaneously obtained samples of ascitic fluid and peripheral venous blood were drawn into precooled polystyrene tubes containing 500 K I U aprotinin and 1 mg sodium ethylenediaminetetraacetic acid ( E D T A ) per m l . Samples were cen-trifuged at 4°C, and the supernatant fluid was immediately frozen and stored at - 80°C. Ext rac t ion of ascites aliquots as wel l as R I A procedures were performed in the same way as for plasma samples; procedures have been detailed elsewhere (8). Br ief ly , samples were extracted by adsorption to Amber l i te X A D - 2 adsor-bent resin. The final titer of the C-terminal-directed antibody Toni III was 1:120,000, cross-reactivity to rat p r o - A N F was 48% and the assay sensitivity was 0.5 fmol a-human A N F / t u b e . The 50% binding intercept of the Standard curve was 10 fmol. Chromatographie Analysis of ANF Immunoreactivity Asci t ic fluid extracts of six patients were subjected to high Performance gel permeation chromatography ( H P G P C ) and reverse phase high Performance chromatography ( R P - H P C ) . H P G P C Lyophi l ized ascitic fluid (10 ml) was dissolved in 25-|xl co lumn eluens and applied to a Spherogel T M T S K , 2000 S W column (10 p,m, 7.5 x 300 m m ; Beck-mari Instruments), eluted with 0.09% trifluoroacetic acid ( T F A ) containing 0.005 M N a 2 S 0 4 , 0.002 M N a H 2 P 0 4 , and 30% acetonitrile (flow rate, 0.3 ml/min). Ca l i -bration was carried out with bovine serum albumin ( B S A ) ( V 0 ) , v i tamin B , 2 ( V t ) , rat p r o - A N F 2 _ l 2 6 , and a-human A N F 9 9 _ 1 2 6 . Immunoreactive A N F ( I R - A N F ) frac-tions (0.6 ml), detected by R I A , were pooled and lyophi l ized . R P - H P C A n aliquot o f the pooled I R - A N F fractions of the H P G P C run was redissolved in 25 juul of 0 .1% T F A and loaded on a H P L C C-18 O D S Ultrasphere T M column (5 | L m , 2 x 150 m m ; Beckman Instruments), aecording to ref. 11. E lu t ion was c a r i e d out with a linear gradient of acetonitrile (10 to 80%, 45 min) in 0 .1% T F A (flosv rate, 0.2 ml/min). Cal ibrat ion of the column was performed with synthetic atmpeptin I and III, human A N F 9 9 _ l 2 6 , and rat p r o - A N F 2 _ , 2 6 . Fract ions (0.4 ml) weie assayed for A N F immunoreactivity. Statistical Evaluation Lifferences between ascitic and plasmatic A N F concentrations were com-pand by paired r-test; the Pearson correlation coefficient was determined by the usml linear least Squares test. Data are presented as means ± S E M . 6 5 8 S E C T I O N V RESULTS Serial dilutions of ascites were parallel to the Standard curve. Recover ies of 7.8 and 15.6 fmol synthetic human a - A N F added to samples of ascites and plasma before extraction were 84% and 67% in ascites and 75% and 65% in plasma. Nonspecif ic binding, determined in the R I A without antiserum, was less than 5% in both ascites and plasma. In patients with cirrhosis , ascit ic A N F con-centrations were 2.4 ± 0.5 fmol/ml , significantly (p < 0.001) lower than A N F concentrations in plasma (15.5 ± 2.6 fmol/ml). In patients wi th malignancies, A N F levels in ascites were 3.3 ± 0.5 fmol/ml , significantly (p < 0.001) lower than levels in plasma (13.0 ± 2.9 fmol/ml). A N F concentrations in ascites were significantly correlated to A N F plasma concentrations in cirrhosis (r = 0.66, p < 0.01) as well as in malignancies (r = 0.90, p < 0.01) (F ig . 1). When preextracted ascitic fluid was chromatographed on H P G P C and R P -H P C , immunoreactive A N F was detected as a single peak coeluting with syn-thetic human A N F 9 9 _ , 2 6 . N o significant amount o f higher-molecular-weight A N F -like material such as precursor p r o - A N F was detected. DISCUSSION This study demonstrates the presence of A N F in ascitic fluid o f patients with cirrhosis of the l iver and with malignant neoplasms. Ext rac t ion procedures as well as characteristics of our highly sensitive and specific radioimmunoassay for determination of A N F in plasma have been previously described (8). App l i ca t ion of the extraction procedure to aliquots of ascitic fluid yielded high recovery rates, not different from those obtained in plasma. Validi ty of the R I A for A N F measurement in ascites was demonstrated by parallelity of serial d i lut ion curves with the Standard curve and by the absence of significant binding interference. In an attempt to characterize further A N F immunoreactivity in ascites, the ANF in plasma (fmol/ml) ANF in plasma (fmol/ml) FIG. 1. Significant correlation of ANF concentrations in ascites and in plasma of 14 pa-tients with cirrhosis of the liver (r = 0.66, p < 0.01) (a) and 10 patients with malignant neoplasms (r = 0.90, p < 0.01) (b). A N P E F F E C T S O N THE K I D N E Y A N D L I V E R 6 5 9 molecular weight pattern was investigated by H P G P C and reverse phase H P L C . Al though trace amounts of higher-molecular-weight A N F in plasma of patients with cirrhosis have been reported (8), A N F immunoreact ivi ty in ascites coeluted with synthetic A N F 9 9 _ l 2 6 , the main circulating form in human plasma. However , because of the low concentrations of A N F in ascites and a 48% cross-reactivity of our antibody to p r o - A N F (8), the presence o f small amounts of higher-molec-ular-weight A N F in ascites cannot be excluded. A N F concentrations in ascites were significantly correlated with A N F plasma concentrations. This correlation as wel l as characterization of ascitic A N F as A N F 9 9 _ , 2 6 , the major circulating form of A N F , may indicate plasmatic origin o f ascitic A N F . A N F concentrations in ascites were significantly lower than in plasma. Thus, elevations of A N F levels in plasma after the infusion of ascitic fluid (3-5) seem not to be due to increased levels of A N F in ascitic fluid but rather due to increased release o f A N F after the hemodynamic changes of infusion. A C K N O W L E D G M E N T S A n i t a Fr iedr ich , Veit G ü l b e r g , and Fabian H ö p k e r are thanked for their Sup-port. Parts of this study were supported by a grant from the funds of the M ü n -chner Medizinische Wochenschrift (head: Prof. D r . Eyer ) . R E F E R E N C E S 1. LeVeen, H . H . , Christoudias, G . , Ip, M . , et al. (1974): A n n . Surg., 180:580-591. 2. Gough, I. R. (1984): Cancer, 54:2226-2230. 3. Burghardt, W., Wernze, H . , and Diehl, K. L . (1986): K l i n . Wochenschr., 64(Suppl. VI): 103-107. 4. Campbell, P., Blendis, L . M . , Skorecki, K . , et al. (1986): Hepatology, 6:1120. 5. Witte, C. L . , Martinez, A . R, and Witte, M . H . (1987): N . E n g l . J. M e d . , 316:487. 6. Gerbes, A. L . , Arendt, R. M . , Ritter, D. , et al. (1985): N . E n g l . J. M e d . , 313:1609-1610. 7. Gerbes, A. L . , Arendt, R. M . , and Paumgartner, G . (1987): J. H e p a t o L , 5:123-132. 8. Arendt, R. M . , Gerbes, A. L . , Ritter, D. , et al. (1986): K l i n . Wochenschr., 64(Suppl. 6):97-102. 9. Marumo, F., Sakamoto, H . , Ando, D. , et al. (1986): Biochem. Biophys. Res. C o m m u n . , 137:231-236. 10. Marumo, F., Masuda, T. , and Ando, K. (1987): Biochem. Biophys. Res. C o m m u n . , 143:813-818. 11. Vollmar, A. M . , Arendt, R. M . , and Schulz, R. (1987): E u r . J. P h a r m a c o l . , 143:315-321. 

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Atrial natriuretic factor (ANF) in ascites of patients with cirrhosis of the liver or with malignant neoplasms

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