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Vascular permeability factor in ascites from patients with spontaneous bacterial peritonitis
Hepatology Research 10 (1998) 185 – 192
Vascular permeability factor in ascites from patients with spontaneous bacterial peritonitis Nobuo Tomiyasu a,*, Keiichi Mitsuyama a, Atsushi Toyonaga a, Katsuhiko Matsuo b, Kyuichi Tanikawa a a
Second Department of Medicine, Kurume Uni6ersity School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830 -0011, Japan b Bioscience Research Department, Toagosei Co. Ltd., Tsukuba Research Laboratory, Ibaraki, Japan Received 18 September 1997; received in revised form 18 November 1997; accepted 21 November 1997
Abstract Ascites and serum levels of vascular permeability factor (VPF) were measured by enzymelinked immunosorbent assay in 30 patients with liver cirrhosis (19 had uninfected ascites and 11 had spontaneous bacterial peritonitis (SBP)) and in 15 patients with peritoneal carcinomatosis. Patients with SBP and peritoneal carcinomatosis had significantly higher ascites VPF levels than cirrhotic patients without SBP, whereas the serum levels did not differ between cirrhotic patients with and those without SBP. A paired analysis showed lower ascites VPF levels compared to the serum levels in cirrhotic patients without SBP and conversely, the levels were higher in patients with SBP. A longitudinal study revealed a marked increase in ascites VPF levels during the occurrence of SBP, which declined after successful treatment. Ascites VPF levels were found to correlate significantly with conventional markers of exudative effusions. These findings indicate that ascites VPF may be useful clinically as a marker of SBP. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascites; Liver cirrhosis; Spontaneous bacterial peritonitis; Vascular endothelial growth factor; Vascular permeability factor
* Corresponding author. Tel.: +81 942 317561; fax: + 81 942 342623. 1386-6346/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 1 1 4 - 9
186
N. Tomiyasu et al. / Hepatology Research 10 (1998) 185–192
1. Introduction Inflammation in serous cavities is often accompanied by the formation of an exudative effusion, which results mainly from the enhanced vascular permeability. Vascular permeability factor (VPF), also called vascular endothelial growth factor, is a glycoprotein with a molecular weight of 40 kDa. It possesses potent vascular permeability-enhancing activities including a 50 000-fold increase in strength compared to histamine on a molar basis and endothelial cell-specific mitogenic activities [1,2]. These powerful effects of VPF suggest that this molecule may be a candidate for the accumulation of effusions. Indeed in animal models, tumor- and oil-induced ascites contained detectable levels of VPF [3]. In human studies, high amounts of immunoreactive VPF were observed in pleural and peritoneal effusions of patients with malignancies [3]. Also, biologically active VPF was detected in ascites during ovarian hyperstimulation syndrome [4]. Spontaneous bacterial peritonitis (SBP) occurs as a complication in cirrhosis and ascites with a possible multifactorial pathogenesis [5]. We postulated that VPF may play an important role in the formation of ascites in SBP. Recent reports from our group and others have described a significant increase in serum VPF levels in patients with hepatocellular carcinoma compared to those with liver cirrhosis [6,7]. However, the appearance of VPF in ascites from patients with cirrhosis and the influence of SBP on VPF production has not yet been investigated. In the present study, we have used a sensitive enzyme-linked immunosorbent assay (ELISA) to measure VPF in ascites and sera from cirrhotic patients with and without SBP.
2. Material and methods
2.1. Patient population A total of 30 patients with liver cirrhosis who had evidence of ascites (19 patients had uninfected cirrhotic ascites and 11 had SBP) were included in this study. The diagnosis of cirrhosis was made on the basis of liver histology and clinical data. Three patients had B type cirrhosis, 25 with C type cirrhosis and two patients had alcoholic cirrhosis. None of the patients had hepatocellular carcinoma concurrently. SBP was defined by a polymorphonuclear cell count of less than 250/mm3 and a positive culture in ascites from patients with cirrhosis [8]. As disease controls, 15 patients with peritoneal carcinomatosis were examined. Peritoneal carcinomatosis was diagnosed when neoplastic cells were found in ascites by cytology or when a peritoneal malignancy was histologically proven at laparoscopy. This group included five patients with gastric cancer, two with colorectal cancer, four with pancreatic cancer, three with biliary tract cancer and one with a renal tumor. This project was performed according to the Helsinki Declaration and informed consent was obtained from every patient.
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2.2. Sample collection Paracentesis was performed routinely via bedside inoculation using a 21-gauge needle under local anesthesia and ultrasound guidance. Aliquots of 50 ml ascites were separated for bacteriological culture, cytology and laboratory testing for VPF, lactate dehydrogenase (LDH), total protein, specific gravity and leukocyte and differential counts. For VPF estimation, the samples were centrifuged at 150× g for 20 min and supernatants were stored at − 20°C until analysis. Whenever possible, blood samples were obtained at the time of paracentesis to determine VPF levels and laboratory parameters.
2.3. VPF ELISA VPF levels were measured in duplicate using an ELISA by the method previously described [6]. This assay employed an anti-human VPF polyclonal antibody (Toagosei, Ibaraki, Japan) elicited in rabbits by immunizing recombinant VPF121 protein, which was prepared using a glutathione-S-transferase gene fusion system (Pharmacia Biotech., Tokyo, Japan). The sensitivity of this assay was assessed by measuring serially diluted recombinant human VPF121 (Toagosei) ranging from 4000 to 0 pg/ml. The intra- and inter-assay coefficients of variation were less than 9.0 and 5.0%, respectively. This ELISA detects all isoforms of VPF as well as the VPF121 isoform.
2.4. Statistics Due to the high rates of kurtosis and skewness, data were normalized by log transformation and parametric tests (Student’s t-test) were then applied. A one-way analysis of variance was performed by a three-group comparison. The Wilcoxonsigned rank test was used to compare paired data. Spearman’s rank correlation was used for correlation analysis. Differences were considered statistically significant for PB0.05. 3. Results Individual VPF levels are shown in Fig. 1. Detectable VPF levels were found in both ascites and sera from every subject. Ascites VPF levels were significantly higher in patients with SBP and those with peritoneal carcinomatosis than in cirrhotic patients without SBP. There was no significant difference in VPF levels between patients with SBP and peritoneal carcinomatosis. In serum, VPF levels were significantly higher in carcinomatosis patients than in cirrhotic patients without SBP, whereas the levels in cirrhotic patients with SBP were comparable to those in patients without SBP. Serial changes in five patients with cirrhosis showed that ascites VPF reached significantly higher levels during an episode of SBP and then declined after successful treatment (Fig. 2).
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Fig. 1. Levels of VPF in ascites (a) and sera (b) from cirrhotic patients with and without spontaneous bacterial peritonitis (SBP) and from patients with peritoneal carcinomatosis. Bars represent the mean and standard error of the mean.
A comparison of VPF levels between ascites and sera from the same cirrhotic patient showed that the VPF levels in ascites were significantly lower than those in serum in cirrhotic patients without SBP, whereas those in ascites were higher in patients with SBP (Fig. 3). No correlation was found between VPF levels in ascites and sera from 21 cirrhotic patients with both uninfected and SBP ascites.
Fig. 2. Serial changes in ascites VPF levels during the course of spontaneous bacterial peritonitis (SBP) in five patients with cirrhosis.
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189
Fig. 3. Analysis of VPF levels between ascites and sera from cirrhotic patients without (a) and with (b) spontaneous bacterial peritonitis and from patients with peritoneal carcinomatosis (c).
A positive correlation was noted in ascites from cirrhotic patients with both uninfected ascites and SBP between VPF levels and conventional markers for exudative effusion, such as leukocyte counts, total protein, LDH and the ratio of serum to ascites LDH [9] (Table 1).
4. Discussion Cirrhotic ascites contains a number of cytokines and growth factors, such as interleukin 6 [10,11], tumor necrosis factor-a [10], transforming growth factor-a [12] Table 1 Correlation between VPF levels and conventional parameters in ascites from cirrhotic patients both with and without spontaneous bacterial peritonitis
Specific gravity Total protein LDH Total protein ratio LDH ratio Leukocytes Polymorphonuclear cells
n
r
P
29 30 30 30 30 27 25
0.406 0.722 0.466 0.676 0.183 0.113 0.124
0.138 0.032 0.032 0.051 0.020 0.009 0.076
LDH, lactate dehydrogenase; LDH ratio, ascites-to-serum LDH level ratio [9]; total protein ratio, ascites-to-serum total protein level ratio [9].
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and hepatocyte growth factor [13]. In the present study, we examined the appearance of VPF in ascites from cirrhotic patients with and without SBP. We found that VPF is present in low amounts in uninfected ascites from patients with cirrhosis and that it is significantly increased in ascites from patients with SBP. The rise in ascites VPF levels is not specific for SBP since a similar result was observed in patients with peritoneal carcinomatosis, as shown in the present study as well as a previous report from Yeo et al. [3]. Further, a rise in VPF levels was not observed in serum but only in ascitic fluid of SBP patients, suggesting an importance of local VPF levels for the assessment of disease status. This is the first report of the measurement of VPF levels in ascites and sera from SBP patients. Recently, it has been reported that VPF is produced by megakaryocytes and platelets [14]. Thus, we must pay attention to the effect of these cells on VPF levels in serum. We also examined serial changes in ascites VPF levels during the course of SBP. We confirmed that an increase in VPF levels was indeed linked to the occurrence of peritonitis. The exact meaning of the increased VPF levels during SBP is unclear. However, VPF may contribute to the enhancement of vascular permeability and the subsequent development of exudative effusions. Clinically, serial measurements in the same individual may help determine disease prognosis and confirm treatment efficacy. An obvious next step is to examine whether high amounts of VPF detected in SBP ascites are biologically active. Using ascitic fluid, we further analyzed the link between VPF levels and conventional markers for inflammation and found that a significant relationship does exist. Although the design of this study did not permit further comparison, the measurement of VPF may help determine the degree of SBP. There are two arguments suggesting that an increase in ascites VPF during SBP is mainly due to the local production rather than the leakage from the vessel: (a) VPF levels in ascites were significantly higher than in serum; and (b) No correlation of VPF levels was found between ascites and serum. In addition, the possibility that decreased absorption of ascitic VPF through the diaphragm raises VPF levels cannot be excluded [15]. VPF that is produced locally from macrophages [16] or lymphocytes [17], which is potentially able to produce VPF, may be released into the peritoneal space. Indeed, peritoneal macrophages are activated in patients with SBP since their ascites contains large amounts of interleukin 6 [10,11] or tumor necrosis factor-a [10], a product from activated macrophages. What stimulates VPF production from these cells in SBP? It is well known in vitro that VPF production is markedly enhanced in hypoxic conditions [18]. A previous report shows that pO2 in ascitic fluid is lower in cirrhotic patients with SBP than in those without SBP [19], suggesting that this may, in part, induce the production of VPF. It has been also reported that SBP ascites contains large amounts of transforming growth factor-a [12], another stimulant of VPF release [20]. It is clear that the regulation of ascites VPF levels in SBP is complex. In addition, the possibility that an increase in the number of VPF-producing mononuclear cells may contribute to the rise of VPF levels cannot be excluded.
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In conclusion, ascites VPF may be useful clinically as a marker of SBP and this merits further investigation. Acknowledgements The authors wish to thank Drs Riko Tsuji, Tomoaki Minetoma, Yoshinobu Okabe and Takato Ueno, the Second Department of Medicine, Kurume University, for their invaluable co-operation. References [1] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvoark HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983 – 5. [2] Connolly DT. Vascular permeability factor: a unique regulator of blood vessel function. J Cell Biochem 1991;47:219–23. [3] Yeo KT, Wang HH, Nagy JA, et al. Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions. Cancer Res 1993;53:2912 – 8. [4] McClure N, Healy DL, Rogers PAW, et al. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994;344:235 – 6. [5] Conn HO, Fessel M. Spontaneous bacterial peritonitis in cirrhosis: variations on a theme. Medicine 1971;50:161–97. [6] Yoshiyama Y, Mitsuyama K, Ono N, Matsuo K, Tanikawa K. Vascular endothelial growth factor in patients with hepatocellular carcinoma: effects of transcatheter arterial embolization. Int J Oncol 1997;11:577–82. [7] Ohira Y, Miura H, Tojo J, Ohira H, Kuroda M, Obara K, Kasukawa R. High serum levels of vascular endothelial growth factor in patients with human hepatocellular carcinoma. Hepatol Res 1997;7:13–8. [8] Hoefs JC, Runyon BA. Spontaneous bacterial peritonitis. Dis Mon 1985;31:1 – 48. [9] Boyer TD, Kahn AM, Reynolds TB. Diagnostic value of ascitic fluid lactic dehydrogenase, protein and WBC levels. Arch Intern Med 1978;138:1103– 5. [10] Deviere J, Content J, Crusiaux A, Dupont E. IL-6 and TNFa in ascitic fluid during spontaneous bacterial peritonitis. Dig Dis Sci 1991;36:123 – 4. [11] Propst T, Propst A, Herold M, Schauer G, Judmaier G, Braunsteiner H, Stoffler G, Vogel W. Spontaneous bacterial peritonitis is associated with high levels of interleukin-6 and its secondary mediators in ascitic fluid. Eur J Clin Invest 1993;23:832 – 6. [12] Sairenji M, Suzuki K, Murakami K, Motohasi H, Okamoto T, Unmeda M. Transforming growth factor activity in pleural and peritoneal effusions from cancer and non-cancer patients. Jpn J Cancer Res 1987;78:814–8. [13] Shimizu I, Ichihara A, Nakamura T. Hepatocyte growth factor in ascites from patients with cirrhosis. J Biochem 1991;109:14–8. [14] Mohle R, Green D, Moore MA, Nachman RL, Rafii S. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA 1997;94:663–8. [15] Bona S, Gavelli A, Huguet C. The role of abdominal drainage after major hepatic resection. Am J Surg 1994;167:593–5. [16] Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994;55:410–22. [17] Freeman MR, Schneck FX, Gagnon ML, et al. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 1995;55:4140 – 5.
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[18] Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia-initiated angiogenesis. Nature 1992;359:843 – 5. [19] Garcia-Tsao G, Conn HO, Lerner E. The diagnosis of bacterial peritonitis: comparison of pH, lactate concentration and leukocyte count. Hepatology 1985;5:91 – 6. [20] Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW, Berse B, Dvorak HF. Overexpression of vascular permeability factor, vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 1994;180:1141 – 6.
.
Vascular permeability factor in ascites from patients with spontaneous bacterial peritonitis Nobuo Tomiyasu a,*, Keiichi Mitsuyama a, Atsushi Toyonaga a, Katsuhiko Matsuo b, Kyuichi Tanikawa a a
Second Department of Medicine, Kurume Uni6ersity School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830 -0011, Japan b Bioscience Research Department, Toagosei Co. Ltd., Tsukuba Research Laboratory, Ibaraki, Japan Received 18 September 1997; received in revised form 18 November 1997; accepted 21 November 1997
Abstract Ascites and serum levels of vascular permeability factor (VPF) were measured by enzymelinked immunosorbent assay in 30 patients with liver cirrhosis (19 had uninfected ascites and 11 had spontaneous bacterial peritonitis (SBP)) and in 15 patients with peritoneal carcinomatosis. Patients with SBP and peritoneal carcinomatosis had significantly higher ascites VPF levels than cirrhotic patients without SBP, whereas the serum levels did not differ between cirrhotic patients with and those without SBP. A paired analysis showed lower ascites VPF levels compared to the serum levels in cirrhotic patients without SBP and conversely, the levels were higher in patients with SBP. A longitudinal study revealed a marked increase in ascites VPF levels during the occurrence of SBP, which declined after successful treatment. Ascites VPF levels were found to correlate significantly with conventional markers of exudative effusions. These findings indicate that ascites VPF may be useful clinically as a marker of SBP. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascites; Liver cirrhosis; Spontaneous bacterial peritonitis; Vascular endothelial growth factor; Vascular permeability factor
* Corresponding author. Tel.: +81 942 317561; fax: + 81 942 342623. 1386-6346/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 1 1 4 - 9
186
N. Tomiyasu et al. / Hepatology Research 10 (1998) 185–192
1. Introduction Inflammation in serous cavities is often accompanied by the formation of an exudative effusion, which results mainly from the enhanced vascular permeability. Vascular permeability factor (VPF), also called vascular endothelial growth factor, is a glycoprotein with a molecular weight of 40 kDa. It possesses potent vascular permeability-enhancing activities including a 50 000-fold increase in strength compared to histamine on a molar basis and endothelial cell-specific mitogenic activities [1,2]. These powerful effects of VPF suggest that this molecule may be a candidate for the accumulation of effusions. Indeed in animal models, tumor- and oil-induced ascites contained detectable levels of VPF [3]. In human studies, high amounts of immunoreactive VPF were observed in pleural and peritoneal effusions of patients with malignancies [3]. Also, biologically active VPF was detected in ascites during ovarian hyperstimulation syndrome [4]. Spontaneous bacterial peritonitis (SBP) occurs as a complication in cirrhosis and ascites with a possible multifactorial pathogenesis [5]. We postulated that VPF may play an important role in the formation of ascites in SBP. Recent reports from our group and others have described a significant increase in serum VPF levels in patients with hepatocellular carcinoma compared to those with liver cirrhosis [6,7]. However, the appearance of VPF in ascites from patients with cirrhosis and the influence of SBP on VPF production has not yet been investigated. In the present study, we have used a sensitive enzyme-linked immunosorbent assay (ELISA) to measure VPF in ascites and sera from cirrhotic patients with and without SBP.
2. Material and methods
2.1. Patient population A total of 30 patients with liver cirrhosis who had evidence of ascites (19 patients had uninfected cirrhotic ascites and 11 had SBP) were included in this study. The diagnosis of cirrhosis was made on the basis of liver histology and clinical data. Three patients had B type cirrhosis, 25 with C type cirrhosis and two patients had alcoholic cirrhosis. None of the patients had hepatocellular carcinoma concurrently. SBP was defined by a polymorphonuclear cell count of less than 250/mm3 and a positive culture in ascites from patients with cirrhosis [8]. As disease controls, 15 patients with peritoneal carcinomatosis were examined. Peritoneal carcinomatosis was diagnosed when neoplastic cells were found in ascites by cytology or when a peritoneal malignancy was histologically proven at laparoscopy. This group included five patients with gastric cancer, two with colorectal cancer, four with pancreatic cancer, three with biliary tract cancer and one with a renal tumor. This project was performed according to the Helsinki Declaration and informed consent was obtained from every patient.
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2.2. Sample collection Paracentesis was performed routinely via bedside inoculation using a 21-gauge needle under local anesthesia and ultrasound guidance. Aliquots of 50 ml ascites were separated for bacteriological culture, cytology and laboratory testing for VPF, lactate dehydrogenase (LDH), total protein, specific gravity and leukocyte and differential counts. For VPF estimation, the samples were centrifuged at 150× g for 20 min and supernatants were stored at − 20°C until analysis. Whenever possible, blood samples were obtained at the time of paracentesis to determine VPF levels and laboratory parameters.
2.3. VPF ELISA VPF levels were measured in duplicate using an ELISA by the method previously described [6]. This assay employed an anti-human VPF polyclonal antibody (Toagosei, Ibaraki, Japan) elicited in rabbits by immunizing recombinant VPF121 protein, which was prepared using a glutathione-S-transferase gene fusion system (Pharmacia Biotech., Tokyo, Japan). The sensitivity of this assay was assessed by measuring serially diluted recombinant human VPF121 (Toagosei) ranging from 4000 to 0 pg/ml. The intra- and inter-assay coefficients of variation were less than 9.0 and 5.0%, respectively. This ELISA detects all isoforms of VPF as well as the VPF121 isoform.
2.4. Statistics Due to the high rates of kurtosis and skewness, data were normalized by log transformation and parametric tests (Student’s t-test) were then applied. A one-way analysis of variance was performed by a three-group comparison. The Wilcoxonsigned rank test was used to compare paired data. Spearman’s rank correlation was used for correlation analysis. Differences were considered statistically significant for PB0.05. 3. Results Individual VPF levels are shown in Fig. 1. Detectable VPF levels were found in both ascites and sera from every subject. Ascites VPF levels were significantly higher in patients with SBP and those with peritoneal carcinomatosis than in cirrhotic patients without SBP. There was no significant difference in VPF levels between patients with SBP and peritoneal carcinomatosis. In serum, VPF levels were significantly higher in carcinomatosis patients than in cirrhotic patients without SBP, whereas the levels in cirrhotic patients with SBP were comparable to those in patients without SBP. Serial changes in five patients with cirrhosis showed that ascites VPF reached significantly higher levels during an episode of SBP and then declined after successful treatment (Fig. 2).
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Fig. 1. Levels of VPF in ascites (a) and sera (b) from cirrhotic patients with and without spontaneous bacterial peritonitis (SBP) and from patients with peritoneal carcinomatosis. Bars represent the mean and standard error of the mean.
A comparison of VPF levels between ascites and sera from the same cirrhotic patient showed that the VPF levels in ascites were significantly lower than those in serum in cirrhotic patients without SBP, whereas those in ascites were higher in patients with SBP (Fig. 3). No correlation was found between VPF levels in ascites and sera from 21 cirrhotic patients with both uninfected and SBP ascites.
Fig. 2. Serial changes in ascites VPF levels during the course of spontaneous bacterial peritonitis (SBP) in five patients with cirrhosis.
N. Tomiyasu et al. / Hepatology Research 10 (1998) 185–192
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Fig. 3. Analysis of VPF levels between ascites and sera from cirrhotic patients without (a) and with (b) spontaneous bacterial peritonitis and from patients with peritoneal carcinomatosis (c).
A positive correlation was noted in ascites from cirrhotic patients with both uninfected ascites and SBP between VPF levels and conventional markers for exudative effusion, such as leukocyte counts, total protein, LDH and the ratio of serum to ascites LDH [9] (Table 1).
4. Discussion Cirrhotic ascites contains a number of cytokines and growth factors, such as interleukin 6 [10,11], tumor necrosis factor-a [10], transforming growth factor-a [12] Table 1 Correlation between VPF levels and conventional parameters in ascites from cirrhotic patients both with and without spontaneous bacterial peritonitis
Specific gravity Total protein LDH Total protein ratio LDH ratio Leukocytes Polymorphonuclear cells
n
r
P
29 30 30 30 30 27 25
0.406 0.722 0.466 0.676 0.183 0.113 0.124
0.138 0.032 0.032 0.051 0.020 0.009 0.076
LDH, lactate dehydrogenase; LDH ratio, ascites-to-serum LDH level ratio [9]; total protein ratio, ascites-to-serum total protein level ratio [9].
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N. Tomiyasu et al. / Hepatology Research 10 (1998) 185–192
and hepatocyte growth factor [13]. In the present study, we examined the appearance of VPF in ascites from cirrhotic patients with and without SBP. We found that VPF is present in low amounts in uninfected ascites from patients with cirrhosis and that it is significantly increased in ascites from patients with SBP. The rise in ascites VPF levels is not specific for SBP since a similar result was observed in patients with peritoneal carcinomatosis, as shown in the present study as well as a previous report from Yeo et al. [3]. Further, a rise in VPF levels was not observed in serum but only in ascitic fluid of SBP patients, suggesting an importance of local VPF levels for the assessment of disease status. This is the first report of the measurement of VPF levels in ascites and sera from SBP patients. Recently, it has been reported that VPF is produced by megakaryocytes and platelets [14]. Thus, we must pay attention to the effect of these cells on VPF levels in serum. We also examined serial changes in ascites VPF levels during the course of SBP. We confirmed that an increase in VPF levels was indeed linked to the occurrence of peritonitis. The exact meaning of the increased VPF levels during SBP is unclear. However, VPF may contribute to the enhancement of vascular permeability and the subsequent development of exudative effusions. Clinically, serial measurements in the same individual may help determine disease prognosis and confirm treatment efficacy. An obvious next step is to examine whether high amounts of VPF detected in SBP ascites are biologically active. Using ascitic fluid, we further analyzed the link between VPF levels and conventional markers for inflammation and found that a significant relationship does exist. Although the design of this study did not permit further comparison, the measurement of VPF may help determine the degree of SBP. There are two arguments suggesting that an increase in ascites VPF during SBP is mainly due to the local production rather than the leakage from the vessel: (a) VPF levels in ascites were significantly higher than in serum; and (b) No correlation of VPF levels was found between ascites and serum. In addition, the possibility that decreased absorption of ascitic VPF through the diaphragm raises VPF levels cannot be excluded [15]. VPF that is produced locally from macrophages [16] or lymphocytes [17], which is potentially able to produce VPF, may be released into the peritoneal space. Indeed, peritoneal macrophages are activated in patients with SBP since their ascites contains large amounts of interleukin 6 [10,11] or tumor necrosis factor-a [10], a product from activated macrophages. What stimulates VPF production from these cells in SBP? It is well known in vitro that VPF production is markedly enhanced in hypoxic conditions [18]. A previous report shows that pO2 in ascitic fluid is lower in cirrhotic patients with SBP than in those without SBP [19], suggesting that this may, in part, induce the production of VPF. It has been also reported that SBP ascites contains large amounts of transforming growth factor-a [12], another stimulant of VPF release [20]. It is clear that the regulation of ascites VPF levels in SBP is complex. In addition, the possibility that an increase in the number of VPF-producing mononuclear cells may contribute to the rise of VPF levels cannot be excluded.
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191
In conclusion, ascites VPF may be useful clinically as a marker of SBP and this merits further investigation. Acknowledgements The authors wish to thank Drs Riko Tsuji, Tomoaki Minetoma, Yoshinobu Okabe and Takato Ueno, the Second Department of Medicine, Kurume University, for their invaluable co-operation. References [1] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvoark HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983 – 5. [2] Connolly DT. Vascular permeability factor: a unique regulator of blood vessel function. J Cell Biochem 1991;47:219–23. [3] Yeo KT, Wang HH, Nagy JA, et al. Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions. Cancer Res 1993;53:2912 – 8. [4] McClure N, Healy DL, Rogers PAW, et al. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994;344:235 – 6. [5] Conn HO, Fessel M. Spontaneous bacterial peritonitis in cirrhosis: variations on a theme. Medicine 1971;50:161–97. [6] Yoshiyama Y, Mitsuyama K, Ono N, Matsuo K, Tanikawa K. Vascular endothelial growth factor in patients with hepatocellular carcinoma: effects of transcatheter arterial embolization. Int J Oncol 1997;11:577–82. [7] Ohira Y, Miura H, Tojo J, Ohira H, Kuroda M, Obara K, Kasukawa R. High serum levels of vascular endothelial growth factor in patients with human hepatocellular carcinoma. Hepatol Res 1997;7:13–8. [8] Hoefs JC, Runyon BA. Spontaneous bacterial peritonitis. Dis Mon 1985;31:1 – 48. [9] Boyer TD, Kahn AM, Reynolds TB. Diagnostic value of ascitic fluid lactic dehydrogenase, protein and WBC levels. Arch Intern Med 1978;138:1103– 5. [10] Deviere J, Content J, Crusiaux A, Dupont E. IL-6 and TNFa in ascitic fluid during spontaneous bacterial peritonitis. Dig Dis Sci 1991;36:123 – 4. [11] Propst T, Propst A, Herold M, Schauer G, Judmaier G, Braunsteiner H, Stoffler G, Vogel W. Spontaneous bacterial peritonitis is associated with high levels of interleukin-6 and its secondary mediators in ascitic fluid. Eur J Clin Invest 1993;23:832 – 6. [12] Sairenji M, Suzuki K, Murakami K, Motohasi H, Okamoto T, Unmeda M. Transforming growth factor activity in pleural and peritoneal effusions from cancer and non-cancer patients. Jpn J Cancer Res 1987;78:814–8. [13] Shimizu I, Ichihara A, Nakamura T. Hepatocyte growth factor in ascites from patients with cirrhosis. J Biochem 1991;109:14–8. [14] Mohle R, Green D, Moore MA, Nachman RL, Rafii S. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA 1997;94:663–8. [15] Bona S, Gavelli A, Huguet C. The role of abdominal drainage after major hepatic resection. Am J Surg 1994;167:593–5. [16] Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994;55:410–22. [17] Freeman MR, Schneck FX, Gagnon ML, et al. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 1995;55:4140 – 5.
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[18] Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia-initiated angiogenesis. Nature 1992;359:843 – 5. [19] Garcia-Tsao G, Conn HO, Lerner E. The diagnosis of bacterial peritonitis: comparison of pH, lactate concentration and leukocyte count. Hepatology 1985;5:91 – 6. [20] Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW, Berse B, Dvorak HF. Overexpression of vascular permeability factor, vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 1994;180:1141 – 6.
.