Acute pancreatitis (AP) can be induced by various causes, but most frequently AP is induced by factors limiting out-flow of pancreatic juice and accumulation of fluids containing pancreatic secretory enzymes in the interstitium of pancreatic parenchyma. This effects reverse transport of some pancreatic enzymes to plasma. Therefore, assays of plasma (or serum) pancreatic amylase and lipase activities are commonly used as sensitive tests for detection of developing AP. An intraglanular activation of proteolytic and lipolytic enzymes of pancreatic juice induces injuries of pancreatic parenchyma. Pancreatic-type Poly C specific ribonuclease (P-RNase) is one of the few direct markers of pancreatic injury (1,2). P-RNase seems to indicate necrotic destruction of pancreatic parenchyma cells in contrary to other secretory enzymes such as amylase and lipase. Warshaw and Fournier (3) found that liberation of P-RNase from pancreatic slices occurs along with cell death and tissue hypoxia. Though, some amount of pancreatic P-RNase is always present in human plasma, an excessive release of this enzyme indicates pancreatic tissue destruction (1-3).
Deleterious effects of AP leading to injury of the pancreas, other organs and
tissues depends on several pathomechanisms; such as direct action of pancreatic
enzymes and enzyme digestion products on tissue, deviated kininogenesis, disturbances
in prostaglandin release, excessive complement activation and stimulation of
blood coagulation and fibrinolysis. Combined actions of these mechanisms may
contribute to rapid progression of the disease producing massive pancreatic
parenchymal cell destruction and extensive inflammatory reaction affecting other
organs of the abdominal cavity (4). Thus, the pancreatic inflammatory process
may develop to systemic inflammatory response (SIRS), multiple organ failure
(MOF) and subsequently death. The role of inflammatory cytokines in development
of AP have been extensively studied in recent years (5,6). In particular, use
of certain cytokines such as IL-6, IL-8, TNF
alpha
and TNF soluble receptors (sTNFR55/sTNFR75) as diagnostic markers and clinical
outcome prediction factors were examined (4-11). On the other hand, relationship
between P-RNase (which is a marker of pancreatic injury) (12) and levels of
inflammatory cytokines has not been studied so far. The objective of this study
was to follow changes of three major inflammatory cytokines:
IL-6, IL-8 and TNF-
alpha soluble receptors with
changes of P-RNase activity in the early period of acute pancreatitis.
MATERIAL AND METHODS
Studies were carried out on 56 patients (30 males, 26 females) with clinically verified diagnosis of acute pancreatitis, admitted to the I-st and the II-nd Departments of Surgery, Collegium Medicum, Jagiellonian University in Krakow. Mean age of the studied patients was 52,8±16,3 (range 22-92) years. Recruitment of patients to the studied group was based on direct examination of patients, ultrasound examination of pancreases and adjacent organs and laboratory examinations of: amylase in serum (assuming 3-fold increase as indicative for AP) (13). Beside, a set of other laboratory tests necessary for assessment of patient's clinical status were routinely performed as described elsewhere (14). Severity of disease was evaluated according to Atlanta Convention (15,16,17). Contrast-enhanced computed tomography is most important imaging tool for the diagnosis of severe acute pancreatitis due to its exellent capacity to demonstrate early inflammatory changes as well as complications, in particular pancreatic necrosis. Acute necrotizing pancreatitis was defined as a decreased enhancement of pancreatic parenchyma found on CT (14-17). The cause of AP were: gallstones in 35 patients, alcohol in 16 and unknown in 5 patients.
Blood samples for current laboratory examinations were collected on admission and then for 5 consecutive days from diagnose of disease. The collected blood was centrifuged (3000g for 10 min.) at approximately 4°C within 30 min from collection. Sera samples for P-RNase and cytokine assays were taken-up from surplus of material collected for routine laboratory examinations of patients and stored at -70°C for several weeks. All patients provided an informed consent on participation in the carried studies and all materials for the study were obtained from the surplus of patient's samples collected for routine monitoring of patients clinical status. The study was approved by the Committee for Ethics Research of the Jagiellonian University.
Determination of poly-C specific ribonuclease (P-RNase)
P-RNase activity was determined using Warshaw and Lee procedure, employing policytydylic acid (Poly-C) as the ribonuclease substrate (12). The assay protocol was as follows: in small centrifuge glass tubes, 20 µl of the studied serum sample and 180 µl of 100 mmol/L
Na-phosphate buffer pH 7.8 were applied. Then 200 µl of buffer Poly-C solution
was added to the whole series of prepared samples and the mixtures were immediately
transferred to a water bath with a temperature of 37°C (+ 0.1°C ). After 20
minutes of incubation the samples were placed in ice and 600 µL of ice cold
lanthanum nitrate (20 mmol/L) in perchloric acid (420 mmol/L) solution (precipitating
agent) were added. The samples were then left for 20 minutes to allow precipitated
Poly-C to coagulate, and latter centrifuged at 4°C for 15 minutes; next 100
µl of clear supernatant was taken and diluted in 1.4 ml of distilled water.
In the diluted supernatant light absorption at 278 nm was determined. To obtain
an increase in 278 nm light absorption underlying the determined P-RNase activity,
light absorption of blank assays, which were prepared exactly as the samples,
except for omission of the incubation step, were subtracted from the light absorption
values obtained for the samples. Each test was performed in duplicate, and final
P-RNase activity was calculated as a mean of two values. All values obtained
as an effect of the carried out procedure were expressed in arbitrary units.
One unit of P-RNase activity refers to such Poly-C hydrolyzing properties which
at standard assay conditions, liberate low molecular Poly C-degradation products
with 278 nm light absorption equal to 1.00 cm
-1.
Determination of plasma IL-8, IL-6 and soluble TNF-a receptors (sTNFR55 and sTNFR75) concentrations.
Determination of plasma IL-8 concentration was based on a solid phase Enzyme
Amplified Sensitivity Immunoassay (IL-8 EASIA
TM
, Biosource Europe S.A., Belgium). The assay is based on an oligoclonal antibodies
(MAB) against distinct epitopes of IL-8 are used. The use of number of distinct
MAbs avoids hyperspecificity and allows high sensitive assays with extended
standard range and short incubation time. Concentration of plasma IL-8 was expressed
as pg/mL, but minimum detectable concentration (MDC) is estimated to be 0,7
pg/mL and is defined as the IL-8 concentration corresponding to the average
optical density (OD) of 20 replicates of the zero standard + 2 standard deviation
(SD).
Plasma of IL-6 was measured in duplicate using the IL-6 EASIA
TM
test kit (Medgenics Diagnostics S.A.) concentration of plasma IL-6 was expressed
as pg/mL, but MDC of this assay was 2,0 pg/mL.
Serum sTNFR55 (sTNFRI) and sTNFR75 (sTNFRII) were measured using ELISA assay with monoclonal and polyclonal anti-sTNF55 and anti-sTNFR75 antibodies (MEDGENIX COMBO sTNFRI/sTNFRII kit, Biosource Europe S.A., Belgium). The COMBO sTNFRI/sTNFRII is an immunoenzymatic kit for the simultaneous quantitative measurement of sTNFRI and sTNFRII on the same sample, in the same well, during a single experiment. Purified sTNFR55 and sTNFR75 were used to construct standard curves. The lower limit of detection of the assay was 0,06 ng/mL for sTNFR55 and 0,02 ng/mL for sTNFR75 (MEDGENIX COMBO sTNFRI/sTNFRII kit, Biosource Europe S.A., Belgium). All cytokines assays are 96-well microtiter plate.
Statistical analysis
Relationship between the investigated variables and P-RNase were assessed by Pearson r correlation coefficients and linear regression functions. All variables with non-normal distribution are represented as medians and range. For comparison of independent samples we used Mann-Whitney U test but differences between all groups (healthy controls, MAP, SAP and patients with other acute abdominal diseases) were calculated using Kruskal-Wallis test. Values of p<0,05 we considered statistically significant.
Statistical calculations were performed with the Statistica 5,5 (Statsoft Inc., Tulsa, USA) package.
RESULTS
Levels of serum amylase, P-RNase, IL-6, IL-8 and soluble TNF
alpha
receptors (sTNFR55/sTNFR75) in healthy individuals amounts to mean value 173,3
U/L (range 36,0-180,0); 18,7 U/L (3,5-37,0); <2,0 pg/mL; 2,25 pg/mL (0,07-4,11);
2,19 ng/mL (0,58-3,26) and 3,54 ng/mL (1,64-6,40), respectively.
As shown in
Table 1 level of P-RNase in patients with AP already in the
first day from diagnose of disease manifest about 2,5 fold increase in P-RNase
level, about 20 fold increase in IL-8 level and about 200 folds in IL-6 level,
respectively. In the further days inflammatory cytokines tend to decrease by
about 2,5 times for IL-8, 6 times for IL-6 and about 1,3 for soluble TNF
alpha
receptors. On the contrary to cytokines studied, P-RNase levels continue to
increase up to days 3-4 achieving levels by about 30 per cent higher than that
in the first day from diagnose (
Fig.2).
Table 1
P-RNase activities and cytokine concentrations in patients with AP during
5 days observations from diagnose of disease. |
|
P-RNase (poly
C specific ribonuclease), sTNFR55/sTNFR75 (soluble TNFa receptors), IL-8
(interleukin 8), IL-6 (interleukin 6) |
|
Fig. 1. Relationship between serum P-RNase (U/L)- x axis and IL-8 (pg/mL) - y axis; linear regression analysis: y = 5,835*x - 228,8; r = 0,75; n = 56; p<0,05. |
Studies of mutual relations between P-RNase levels and inflammatory cytokines
have shown a remarkably strong positive correlation between P-RNase and IL-6
and sTNFR75 and slightly less pronounced correlation for IL-8 (
Fig. 1)
and sTNFR55 (
Table 2). As shown in the
Table 2, IL-6 levels correlate
with P-RNase during 5 consecutive days from diagnose, whereas IL-8 correlate
on the 1
st and 2
nd
day and sTNFR55/sTNFR75 shown significant correlations with P-RNase only in
1
st day from admission to hospital.
|
Fig. 2. Time-dependent changes
in plasma P-RNase activity in patients with mild AP (
line) and in severe AP (
line); **p<0,001. |
Table 2
Pearson r correlation coefficient of P-RNase with cytokines studied in 5 consecutive days from admission to hospital in patients with AP. |
|
Significant
differences:* p<0,05; **p<0,001; NS- not significant; IL-8 (interleukin
8), IL-6 (interleukin 6), P-RNase ( poly-C specific ribonuclease), sTNFR55/sTNFR75
(soluble TNFalpha receptors). |
Selecting of studied AP patients on two groups varying in severity of disease
clinical course: group I of patients with mild form of AP and less severe clinical
course; and group of necrotic form of AP with severe clinical course of disease,
a set of significant differences in inflammatory cytokines, P-RNase and amylase
level were found (
Table 3).
Table 3
P-RNase, amylase activities and cytokine concentrations in patients with mild and severe forms of AP during 5 days from admission to hospital. |
|
*p<0,05;
**p<0,01; ***p<0,001 |
P-RNase and IL-8 levels are 2,5 and 3 folds higher and IL-6 is 70 folds higher
in patients with acute necrotizing pancreatitis than in patients with mild pancreatitis
(
Table 3).
Also sTNFR55 and sTNFR75 levels are about 2 times higher than that in patients
with mild pancreatitis. Comparing patients with acute necrotizing pancreatitis
with the reference group the most striking change in level of IL-6 in the 1
st
day from diagnose was observed (about 700 times) whereas IL-6 level increased
by 40-80 times in days 1 and 3 respectively. Soluble TNF
alpha
receptors: sTNFR55 and sTNFR75 levels increased by 5-7 times. The similar increase
rate was observed for level of P-RNase (about 3 times if compared to reference
group).
P-RNase correlated significantly with both sTNFR55, sTNFR75, IL-6 and IL-8,
with IL-6 having higher coefficients during 5 consecutive days of observation.
Positive correlation were found between IL-6 level and P-RNase activities at
all days studied. The Pearson r correlation coefficient for this relationship
was 0,86 at 1
st day from admission to hospital,
and r 0,79; r 0,70; r 0,60 and r 0,57 respectively (all p<0,001) (
Table 2).
In contrast, correlation coefficients between increase in P-RNase activity and
sTNFR55 as well as sTNFR75 concentrations were observed, but only at 1
st
day from diagnose of disease (r 0,55 and r 0,73; p<0,05) for sTNFR55 and sTNFR75,
respectively. The Pearson correlation coefficient r for the relation between
increase in P-RNase and IL-8 release was significant only during first two days
from admission to hospital ( r 0,62 at 1
st and
r 0,75 on 2
nd day; p<0,001) (
Table 2).
Results obtained in the carried study shows that P-RNase level parallels changes
of major inflammatory cytokines and support view that P-RNase is specific early
marker of pancreatic tissue injury.
DISCUSSION
The carried out studies have shown that in the initial phase of acute pancreatitis,
pronounced increase in concentration levels of P-RNase and the inflammatory
cytokines occurs; also there is a set of significant correlations between the
P-RNase and levels of cytokines studied. The most remarkable interrelation occurring
during the initial 5 days from the onset of the disease was found between IL-6
and P-RNase. However, less pronounced interrelations between P-RNase and IL-8
and sTNFR55/sTNFR75 were observed. A possible explanation of this observed phenomenon
may point to early events occurring in AP development. Assuming that the acute
inflammatory process in AP is triggered by primary activation of pancreatic
proteolytic enzymes effecting pancreatic tissue degradation and release of peptides
and proteins to interstitial fluid (11,18), P-RNase is also released as a specific
marker of pancreatic tissue destruction (3,12,19). Thus, it is conceivable that
increase in P-RNase indicates an initial output of pro-inflammatory peptides
from the pancreas subsequently inducing activation of the immune-cell release
of inflammatory cytokines: interleukin-6, interleukin-8, tumour necrosis factor
alpha and other inflammatory cytokines. Therefore
interrelations between P-RNase and inflammatory cytokines are noticeable in
the first day from diagnose of disease when inflammatory reaction occurs in
place of primary proteolytic injury. However, further extensive destruction
of pancreatic parenchyma due to infiltration of phagocytes, eventually may induce
a "hyperinflammatory" reaction affecting other organs of the abdominal cavity
(20,21). P-RNase which is a marker of pancreatic parenchyma cell destruction,
continues to rise, independently of inflammatory cytokine release. Interrelations
between the P-RNase and cytokines studied disappears in further days of disease.
Increase of P-RNase level in patients with AP remains related to the severity
of disease (12,19). The inflammatory cytokines on the other hand, play an important
part both in progression of acute pancreatitis from the mild form to severe
life-threatening disease or in attenuation of pancreatic damage and recovery
from acute pancreatitis (22, 23). It is of interest how much disequilibrium
between proinflammatory and anti-inflammatory cytokines contributes to pancreatic
tissue necrosis, systemic inflammatory syndrome and multiple organ failure (4-9).
This is in accord with our present finding that both elevation of P-RNase (12,19)
and increase in inflammatory cytokines is most remarkably increased in patients
who developed pancreatic necrosis with a severe disease clinical course (4,7,10,20,24).
These results are in accord with formerly published information that IL-6 and
IL-8 increase in the early period of AP development (4-9,25-26); however, our
studies have shown a specific day to day-change pattern for the studied inflammatory
cytokines. Moreover, a particularly strong correlation between the P-RNase activity
and IL-6 in consecutive days of the disease and in the entire studied period
was found. They increase upon contact with bacterial liposaccharides, and necrotic
tissue (27); thus pancreatic cell death and destruction seems to be a primary
cause of P-RNase release and the studied cytokine output.
As the studies presented in this paper refer to an early period of AP development, findings of this paper may be of some practical use for early selection of patients who have developing pancreatic necrotic injuries in order to foresee future poor clinical outcome due to severe manifestation of AP, haemorrhage and pancreatic necrosis.
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