International Journal of Pancreatotogy, pp, 179-184 voL 11, no, 3, June 1992 9 Copyright 1992 by The Humana Pre~ Inc. All rights of any nature whatsoever reserved. 0169-4197/92/11:179-184/$2.00
Urinary Phospholipase A2 Excretion in Chronic Pancreatic Diseases C. Fabris, I D. Basso, 2 M. P. Panozzo, 3 G. D e l F a v e r o / To Meggiato, 3 M. Plebani, 2 C. F e r r a r a / P. F o g a r / M. Z a n i n o t t o / a n d R. Naccarato "gJ ~Cattedra di Medicina 1nterna, Universitfz degli Studi di Udine; 2lstituto di Medicina di Laboratorio and 3Cattedra di Malattie Apparato Digerente, Universit~ degIi Studi di Padova, Italy
Summary This study was performed to investigate the behavior of phospholipase A2 (PLA2) in serum and urine of patients with chronic pancreatic diseases and to ascertain whether any factors influenced the results. In 30 controls, 45 patients with pancreatic cancer, 54 with chronic pancreatitis, and 64 with extrapancreatic diseases, serum and urinary PLA2, pancreatic isoamylase and RNase, and urinary N-acetylglucosaminidase (NAG) were measured. Serum PLA2 levels were higher in patients with chronic pancreatitis than in all the other groups. In our patients, only occasionally was urinary PLA2 elevated, the increase occurring almost exclusively in the presence of an acute inflammatory disease, e.g., relapsed chronic pancreatitis or active inflammatory bowel disease. A correlation was found between serum PLA2 and serum RNase, an indicator of tissue damage, but not between serum PLA2 and pancreatic isoamylase. Urinary PLA2 output was correlated with its renal input and with RNase output. No correlation was found between PLA2 output and pancreatic isoamylase or NAG urinary excretion. In conclusion, (1) the determination of serum PLA2 activity may be an aspeeific test of pancreatic disease; (2) PLA2 urinary excretion occasionally increases, especially in the presence of severe phlogosis, which occurs in chronic pancreatitis, in particular during relapse; and (3) irrespective of the tissue origin of urinary PLA2, its increased excretion may be accounted for in part by its increased circulating levels. It is, however, more likely the consequence of a renal tubular dysfunction, which is sometimes found in patients with pancreatic diseases.
Key Words: Pancreatic cancer; chronic pancreatitis; renal damage.
Pancreatic PLA2 has been widely studied: It is a minor component of pancreatic secretion (less than 1%) and is synthesized as proenzyme, which is intraluminally activated by trypsin (3-5). Pancreatic PLA2 plays an important role in the digestion ofphospholipids and also promotes lipase action (1--6). Since PLA2 has a potentially damaging action on cell membranes, with the consequent production of toxic compounds, it has been suggested that this enzyme plays a key role in the pathogenesis of acute pancreatitis. High levels of this substance have been found in the
Introduction Phospholipases A2 form a group of enzymes distributed throughout the organism, where they play several physiological roles and are, in particular, involved in inflammatory reaction (1,2). Received November 14, 1990; Revised October 28, 1991; Accepted January 7, 1992 *Author to whom all correspondence and reprint requests should be addressed: Istituto di Medicina Interna, Cattedra di Malattie Apparato Digerente, Policlinico Universitario, Via Giustiniani, 2, 35100 Padova, Italy
i79
180 sera of patients with acute pancreatitis (7-11). Increased values have, however, also been found in chronic pancreatic diseases (12) and other inflammatory disorders and in renal failure (13,14). Like other pancreatic enzymes, PLA2 can be detected in urine, since it has a low mol wt and can be filtered by the kidney (15). In the literature, no exhaustive data are available either on the behavior of this enzyme in the urine of patients with chronic pancreatic diseases or on factors that could influence its urinary excretion. In this paper, we studied PLA2 activity in the urine of patients with pancreatic cancer and chronic pancreatitis in comparison with other diseases and investigated the relative role of pancreatic damage and renal alterations in influencing the results.
Material and Methods The study was performed on 193 subjects, 45 of which had pancreatic cancer of duct cell origin (24 males, 21 females, age range 28-78 yr) that was histologically confirmed by means of surgical or autoptic specimens (16). Fifty-four had chronic pancreatitis (44 males, 10 females, age range 18-77 yr), which was diagnosed on the basis of the clinical picture and positive findings from at least two of the following: plain abdomen X-ray for pancreatic calcifications, pancreatic ultrasonography, computed axial tomography, and endoscopic retrograde pancreatography. Sixty-four had extrapancreatic diseases, of gastrointestinal origin (35 males, 29 females, age range 17-83 yr). The diagnoses were based on the clinical picture and on the results of specific radiological and histological procedures. The numbers of gastrointestinal benign diseases were: irritable colon (5 cases), Crohn's disease (5), ulcerative colitis (2), duodenitis (1), celiac disease (1), hiatus hernia (1), and diverticulosis of the colon (2). The numbers of benign liver bitiary tract diseases were: choledocholithiasis (8 cases), liver cirrhosis (7), sclerosing cholangitis (5), chronic hepatitis (3), gallstones (3), benign stenosis of the papilla of Vater (3), acute hepatitis (2), liver steatosis (2), chronic cholangitis (1), benign adenoma of the papilla of Vater (1), and intrahepatic lithiasis (1). Extrapancreatic gastrointestinal malignancies included: carcinoma of the ampulla of Vater (5 cases), carcinoma of the main bite duct International Journal of Pancreatology
Fabris et al. (2), carcinoma of the colon (1), carcinoma of the esophagus (1), carcinoma of the gallbladder (1), and hepatocellul ar carcinoma (1). The control group consisted of 30 healthy members of the medical staffor blood donors (12 males, 18 females, age range 23-54 yr) without previous or present gastrointestinal diseases. None of the subjects presented evidence of an overt renal tubular disease, and none lind serum creatinine above the upper normal limit or a frank glomerular proteinuria (less than 1.0 g/die). Phospholipase A2 activity was determined in serum and urine by means of a fluorimetric assay (Thuren's method) (17). This method evaluates the activity of PLA2 on a fluorescent phospholipid analog [ 1-octacosanyl-2-(pyren- 1-yl)hexanoyl-sn-glycero- 3-phosphatidyl monomethyl ester]. The reaction product [(pyren- 1-yl)hexanoic acid] is measured fluorimetrically after liquid-liquid phase partition. Pancreatic ribonuclease (RNase) was measured in serum and urine using Reddi and Holland's method (18) in the conditions described elsewhere (19). Serum and urinary pancreatic isoamylase was measured according to a double monoclonal antibody (MAb) assay (20)~N-acetylglucosaminidase in urine was measured by means of Linko-Lopponen' s method (21). The statistical analysis was made using one-way analysis of variance (ANOVA), Bonferroni's test for pair,vise comparisons (22), and Student's t-test. Before statistical tests were performed, a logarithmic transformation of urinary data was always done to reduce their scattering and to enhance their symmetry in the distribution of the results.
Results Figure 1 shows the individual values and the statistical analysis of serum PLA2 in the material studied. A correlation was tbund between the serum levels of PLA2 and creatinine values (r = 0.170, p < 0.05); the former did not correlate with serum pancreatic isoamylase (r = 0.120, p = ns). Figure 2 reports the urinary data for PLA2. No significant differences were found between the PLA2 serum and urinary values of chronic pancreatitis patients with relapse and of those under remission (t = t .566, p = ns, and t = 1.043, p = ns, respectively). Volume 11, 1992
Phospholipase A2 Excretion
181 350.
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} O
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4o II]
,223,[-
3:307
..
9
I0o
c's CS
PC
cP
GIBD LBBD
EI~M
Fig 1. Individual values of P L A 2 in the material studied. The continuous line represents the upper normal limit (mean + 2 SD o f our controls). CS = control s u b j e c t s ; PC = p a n c r e a t i c c a n c e r ; C P = c h r o n i c pancreatitis; G I B D = gastrointestinal benign diseases; L B B D = liver b i l i a r y b e n i g n diseases; E P M = extrapancreatic malignancies; and O = relapsed chronic pancreatitis. Analysis of variance: F = 15.05; p < 0.001 Bonferroni's test for pairwise comparisons: * = p < 0.05 as compared to CP. ** = p < 0.001 as compared to all the other groups.
Table 1 reports mean values, standard errors, and the percentages of pathological findings for serum and minary isoamylase and urinary NAG. Figure 3 illustrates individual values and reports results of the statistical analysis of urinary pancreatic ribonuclease. The serum levels of this enzyme were found to be correlated with serum PLA2 (r = 0.160, p < 0.05). Table 2 shows the correlations between urinary PLA2 and the indices reported, considering the subjects as a whole. International Journal of Pancreatology
r
c'P
Fig. 2. Individual values o f urinary PLA2 outputs in the material studied. The continuous line represents the upper normal limit (mean + 2 SD o f our controls). CS = control subjects; PC = pancreatic cancer; CP = chronic pancreatitis; G I B D = gastrointestinal benign diseases; L B B D = liver b i l i a r y b e n i g n d i s e a s e s , E P M = extrapancreatic malignancies; Units = pmol rain -~ mL-~; and O = relapsed c h r o n i c pancreatitis. Analysis o f variance: F = 1.29, p = ns.
Discussion Immunological assays are available for the measurement and quantification of PLA2 in biological fluids (5,23). These methods, however, measure both proenzyme and active form, which is the true mediator of the damage from PLA2 to plasma membranes. We therefore utilized a fluorimetric assay to measure the enzymatic activity of PLA2. Nevertheless, with this method, we determined all PLA2 activity without a distinction between enzymes of different sources, including the pancreas and inflammatory cells (1,2). Increased serum PLA2 values were found in the vast majority of patients with chronic pancreatic disVolume 11, 1992
182
Fabris et al. 3"000-
Table 1 Pathological Results of Serum and Urinary Pancreatic Isoamylase and Urinary NAG Mean Serum pancreatic isoamylase CS PC CP EPD
52.67 105.27 145.82 75.96
SE
%
4.55 36.73 38.92 11.25
0 27 46 33
2"000
Urinary pancreatic isoamylase output CS PC CP EPD
0.68 1.77 0.53 0.31
1.09 0.63 0.51 0.50
16 38 38 25
0.53 0.34 0.24 0.27
12 45 27 25
0
1'000
9
Urinary NAG output CS PC CP EPD
-0.11 1.10 0.50 -0.08
Urinary data were logarithmically transformed before making the analyses, because o f the scattered distribution o f the results. CS = control subjects; PC = pancreatic cancer; C P = chronic pancreatitis; EPD = extrapancreatic diseases,
eases, whether of a neoplastic or inflammatory nature. However, elevated values were also found in patients with benign or malignant extrapancreatic diseases. This suggests that although the increase of this enzyme in serum is particularly pronounced in pancreatitis, this finding is aspecific (13). Two considerations emerge: First, the high PLA2 values found in diseases other than those of the pancreas may be related to the wide distribution of this enzyme in the organism, although we cannot rule out a consensual pancreatic involvement in some of these patients; and second, although in pancreatic diseases PLA2 may be derived mainly from the damaged pancreas, the elevated values may also be caused by release of the enzyme from other cells. This hypothesis is supported by the observation that no difference was found between patients with chronic pancreatitis studied in a relapsed phase and those studied during a quiescent phase. Theretbre, the activity measured in our patients' sera may depend upon enzymes released not only from the pancreas. Moreover, International Journal of Pancreatology
z
Urinary Phospholipase A2 Excretion in Chronic Pancreatic Diseases C. Fabris, I D. Basso, 2 M. P. Panozzo, 3 G. D e l F a v e r o / To Meggiato, 3 M. Plebani, 2 C. F e r r a r a / P. F o g a r / M. Z a n i n o t t o / a n d R. Naccarato "gJ ~Cattedra di Medicina 1nterna, Universitfz degli Studi di Udine; 2lstituto di Medicina di Laboratorio and 3Cattedra di Malattie Apparato Digerente, Universit~ degIi Studi di Padova, Italy
Summary This study was performed to investigate the behavior of phospholipase A2 (PLA2) in serum and urine of patients with chronic pancreatic diseases and to ascertain whether any factors influenced the results. In 30 controls, 45 patients with pancreatic cancer, 54 with chronic pancreatitis, and 64 with extrapancreatic diseases, serum and urinary PLA2, pancreatic isoamylase and RNase, and urinary N-acetylglucosaminidase (NAG) were measured. Serum PLA2 levels were higher in patients with chronic pancreatitis than in all the other groups. In our patients, only occasionally was urinary PLA2 elevated, the increase occurring almost exclusively in the presence of an acute inflammatory disease, e.g., relapsed chronic pancreatitis or active inflammatory bowel disease. A correlation was found between serum PLA2 and serum RNase, an indicator of tissue damage, but not between serum PLA2 and pancreatic isoamylase. Urinary PLA2 output was correlated with its renal input and with RNase output. No correlation was found between PLA2 output and pancreatic isoamylase or NAG urinary excretion. In conclusion, (1) the determination of serum PLA2 activity may be an aspeeific test of pancreatic disease; (2) PLA2 urinary excretion occasionally increases, especially in the presence of severe phlogosis, which occurs in chronic pancreatitis, in particular during relapse; and (3) irrespective of the tissue origin of urinary PLA2, its increased excretion may be accounted for in part by its increased circulating levels. It is, however, more likely the consequence of a renal tubular dysfunction, which is sometimes found in patients with pancreatic diseases.
Key Words: Pancreatic cancer; chronic pancreatitis; renal damage.
Pancreatic PLA2 has been widely studied: It is a minor component of pancreatic secretion (less than 1%) and is synthesized as proenzyme, which is intraluminally activated by trypsin (3-5). Pancreatic PLA2 plays an important role in the digestion ofphospholipids and also promotes lipase action (1--6). Since PLA2 has a potentially damaging action on cell membranes, with the consequent production of toxic compounds, it has been suggested that this enzyme plays a key role in the pathogenesis of acute pancreatitis. High levels of this substance have been found in the
Introduction Phospholipases A2 form a group of enzymes distributed throughout the organism, where they play several physiological roles and are, in particular, involved in inflammatory reaction (1,2). Received November 14, 1990; Revised October 28, 1991; Accepted January 7, 1992 *Author to whom all correspondence and reprint requests should be addressed: Istituto di Medicina Interna, Cattedra di Malattie Apparato Digerente, Policlinico Universitario, Via Giustiniani, 2, 35100 Padova, Italy
i79
180 sera of patients with acute pancreatitis (7-11). Increased values have, however, also been found in chronic pancreatic diseases (12) and other inflammatory disorders and in renal failure (13,14). Like other pancreatic enzymes, PLA2 can be detected in urine, since it has a low mol wt and can be filtered by the kidney (15). In the literature, no exhaustive data are available either on the behavior of this enzyme in the urine of patients with chronic pancreatic diseases or on factors that could influence its urinary excretion. In this paper, we studied PLA2 activity in the urine of patients with pancreatic cancer and chronic pancreatitis in comparison with other diseases and investigated the relative role of pancreatic damage and renal alterations in influencing the results.
Material and Methods The study was performed on 193 subjects, 45 of which had pancreatic cancer of duct cell origin (24 males, 21 females, age range 28-78 yr) that was histologically confirmed by means of surgical or autoptic specimens (16). Fifty-four had chronic pancreatitis (44 males, 10 females, age range 18-77 yr), which was diagnosed on the basis of the clinical picture and positive findings from at least two of the following: plain abdomen X-ray for pancreatic calcifications, pancreatic ultrasonography, computed axial tomography, and endoscopic retrograde pancreatography. Sixty-four had extrapancreatic diseases, of gastrointestinal origin (35 males, 29 females, age range 17-83 yr). The diagnoses were based on the clinical picture and on the results of specific radiological and histological procedures. The numbers of gastrointestinal benign diseases were: irritable colon (5 cases), Crohn's disease (5), ulcerative colitis (2), duodenitis (1), celiac disease (1), hiatus hernia (1), and diverticulosis of the colon (2). The numbers of benign liver bitiary tract diseases were: choledocholithiasis (8 cases), liver cirrhosis (7), sclerosing cholangitis (5), chronic hepatitis (3), gallstones (3), benign stenosis of the papilla of Vater (3), acute hepatitis (2), liver steatosis (2), chronic cholangitis (1), benign adenoma of the papilla of Vater (1), and intrahepatic lithiasis (1). Extrapancreatic gastrointestinal malignancies included: carcinoma of the ampulla of Vater (5 cases), carcinoma of the main bite duct International Journal of Pancreatology
Fabris et al. (2), carcinoma of the colon (1), carcinoma of the esophagus (1), carcinoma of the gallbladder (1), and hepatocellul ar carcinoma (1). The control group consisted of 30 healthy members of the medical staffor blood donors (12 males, 18 females, age range 23-54 yr) without previous or present gastrointestinal diseases. None of the subjects presented evidence of an overt renal tubular disease, and none lind serum creatinine above the upper normal limit or a frank glomerular proteinuria (less than 1.0 g/die). Phospholipase A2 activity was determined in serum and urine by means of a fluorimetric assay (Thuren's method) (17). This method evaluates the activity of PLA2 on a fluorescent phospholipid analog [ 1-octacosanyl-2-(pyren- 1-yl)hexanoyl-sn-glycero- 3-phosphatidyl monomethyl ester]. The reaction product [(pyren- 1-yl)hexanoic acid] is measured fluorimetrically after liquid-liquid phase partition. Pancreatic ribonuclease (RNase) was measured in serum and urine using Reddi and Holland's method (18) in the conditions described elsewhere (19). Serum and urinary pancreatic isoamylase was measured according to a double monoclonal antibody (MAb) assay (20)~N-acetylglucosaminidase in urine was measured by means of Linko-Lopponen' s method (21). The statistical analysis was made using one-way analysis of variance (ANOVA), Bonferroni's test for pair,vise comparisons (22), and Student's t-test. Before statistical tests were performed, a logarithmic transformation of urinary data was always done to reduce their scattering and to enhance their symmetry in the distribution of the results.
Results Figure 1 shows the individual values and the statistical analysis of serum PLA2 in the material studied. A correlation was tbund between the serum levels of PLA2 and creatinine values (r = 0.170, p < 0.05); the former did not correlate with serum pancreatic isoamylase (r = 0.120, p = ns). Figure 2 reports the urinary data for PLA2. No significant differences were found between the PLA2 serum and urinary values of chronic pancreatitis patients with relapse and of those under remission (t = t .566, p = ns, and t = 1.043, p = ns, respectively). Volume 11, 1992
Phospholipase A2 Excretion
181 350.
8(,~
J.
O
3041
12C O
O
~o
3 c
"g_
~
O
9
.
.
II
9
G,'~
L.i~
2so~
i 80
} O
o.
4o II]
,223,[-
3:307
..
9
I0o
c's CS
PC
cP
GIBD LBBD
EI~M
Fig 1. Individual values of P L A 2 in the material studied. The continuous line represents the upper normal limit (mean + 2 SD o f our controls). CS = control s u b j e c t s ; PC = p a n c r e a t i c c a n c e r ; C P = c h r o n i c pancreatitis; G I B D = gastrointestinal benign diseases; L B B D = liver b i l i a r y b e n i g n diseases; E P M = extrapancreatic malignancies; and O = relapsed chronic pancreatitis. Analysis of variance: F = 15.05; p < 0.001 Bonferroni's test for pairwise comparisons: * = p < 0.05 as compared to CP. ** = p < 0.001 as compared to all the other groups.
Table 1 reports mean values, standard errors, and the percentages of pathological findings for serum and minary isoamylase and urinary NAG. Figure 3 illustrates individual values and reports results of the statistical analysis of urinary pancreatic ribonuclease. The serum levels of this enzyme were found to be correlated with serum PLA2 (r = 0.160, p < 0.05). Table 2 shows the correlations between urinary PLA2 and the indices reported, considering the subjects as a whole. International Journal of Pancreatology
r
c'P
Fig. 2. Individual values o f urinary PLA2 outputs in the material studied. The continuous line represents the upper normal limit (mean + 2 SD o f our controls). CS = control subjects; PC = pancreatic cancer; CP = chronic pancreatitis; G I B D = gastrointestinal benign diseases; L B B D = liver b i l i a r y b e n i g n d i s e a s e s , E P M = extrapancreatic malignancies; Units = pmol rain -~ mL-~; and O = relapsed c h r o n i c pancreatitis. Analysis o f variance: F = 1.29, p = ns.
Discussion Immunological assays are available for the measurement and quantification of PLA2 in biological fluids (5,23). These methods, however, measure both proenzyme and active form, which is the true mediator of the damage from PLA2 to plasma membranes. We therefore utilized a fluorimetric assay to measure the enzymatic activity of PLA2. Nevertheless, with this method, we determined all PLA2 activity without a distinction between enzymes of different sources, including the pancreas and inflammatory cells (1,2). Increased serum PLA2 values were found in the vast majority of patients with chronic pancreatic disVolume 11, 1992
182
Fabris et al. 3"000-
Table 1 Pathological Results of Serum and Urinary Pancreatic Isoamylase and Urinary NAG Mean Serum pancreatic isoamylase CS PC CP EPD
52.67 105.27 145.82 75.96
SE
%
4.55 36.73 38.92 11.25
0 27 46 33
2"000
Urinary pancreatic isoamylase output CS PC CP EPD
0.68 1.77 0.53 0.31
1.09 0.63 0.51 0.50
16 38 38 25
0.53 0.34 0.24 0.27
12 45 27 25
0
1'000
9
Urinary NAG output CS PC CP EPD
-0.11 1.10 0.50 -0.08
Urinary data were logarithmically transformed before making the analyses, because o f the scattered distribution o f the results. CS = control subjects; PC = pancreatic cancer; C P = chronic pancreatitis; EPD = extrapancreatic diseases,
eases, whether of a neoplastic or inflammatory nature. However, elevated values were also found in patients with benign or malignant extrapancreatic diseases. This suggests that although the increase of this enzyme in serum is particularly pronounced in pancreatitis, this finding is aspecific (13). Two considerations emerge: First, the high PLA2 values found in diseases other than those of the pancreas may be related to the wide distribution of this enzyme in the organism, although we cannot rule out a consensual pancreatic involvement in some of these patients; and second, although in pancreatic diseases PLA2 may be derived mainly from the damaged pancreas, the elevated values may also be caused by release of the enzyme from other cells. This hypothesis is supported by the observation that no difference was found between patients with chronic pancreatitis studied in a relapsed phase and those studied during a quiescent phase. Theretbre, the activity measured in our patients' sera may depend upon enzymes released not only from the pancreas. Moreover, International Journal of Pancreatology
z
