POZIOM KALLISTATYNY U PACJENTÓW Z PRZEWLEKŁYM ZAPALENIEM TRZUSTKI I WSPÓŁWYSTĘPUJĄCĄ MIAŻDŻYCĄ

Elizaveta S. Sirchak, Svetlana M. Opalenyk, Natalia Yu. Kurchak

Uzhhorod National University, Uzhhorod, Ukraine

Abstract

Introduction: The article is devoted to the optimization of complex diagnosis in patients with combination of chronic pancreatitis and atherosclerosis.

The aim: To study the level of serum kallistatin in patients with combination of chronic pancreatitis and atherosclerosis.

Materials and methods: 89 patients with chronic pancreatitis were examined. The serum kallistatin level was determined by immunoassay using the Human Serpin A4 ELISA Kit from RayBiotech according to the application method. To determine the excretory pancreatic insufficiency all patients underwent 13C-amylase and 13C-mixed triglyceride breathing tests.

Results: Analysis of the study results showed that patients with less severe excretory pancreatic insufficiency and lipid profile disorders had higher kallistatin level than the control group (33.66±4.55 ng/ml). Instead, in patients with severe excretory pancreatic insufficiency and dyslipidemia, it was significantly lower – 14.69±9.21 ng/ml.

Conclusions: The interrelation between the severity of the chronic pancreatitis course with atherosclerosis and the serum kallistatin level was observed. It has been established that studies of serum kallistatin level can serve as a marker of the chronic pancreatitis` severity in combination with atherosclerosis.

 

Wiad Lek 2018, 71, 2 cz. I, -318

 

Introduction

Kallistatin (KS) is an endogenous protein that has pleiotropic effect, including vasodilation and inhibition of inflammation, oxidative stress, apoptosis and fibrosis. Kallistatin was studied in human plasma as tissue kallikrein–binding protein. Through its 2 functional domains — an active site and a heparin-binding site — kallistatin regulates different signaling pathways and a wide spectrum of biological functions. Kallistatin’s active site is a key for tissue kallikrein activity inhibition and also it stimulates the release of endothelial`s nitric oxide (NO). It is proved that kallistatin protects the vascular wall from damage and has therapeutic potential in vascular and inflammatory diseases [1].

Activation of the kallikrein-kinin system probably leads to the development of inflammatory disorders in pancreas. The pancreas has high amount of kallikrein, which may be prematurely activated during acute pancreatitis. So, kinins consequently released by kallikrein can mediate vascular changes such as vasodilation and increased permeability [2].

Study by Griesbacher et al. 1993; Weidenbach et al. 1995; Ericson and Sjoback 1996; Hoffmann et al. 1996 on rats showed that the kallikrein-kinin system is involved in development of the pain syndrome, hypotension, edema, and shock associated with acute pancreatitis. Another recent study on rat model demonstrated that tissue`s kallikrein increases in alcoholic`s and ischemic`s acute pancreatitis by 2.8-fold and 3-fold, respectively. Kallistatin has a protective inhibitory activity by regulating excessive kallikrein activity in tissues [3].

Chronic inflammatory process leads to the vascular wall injury, which in sequence cause decrease in the synthesis or secretion of vasoactive substances by the endothelium. Prolonged inflammation can lead to endothelial dysfunction and become a factor that contributing to hypertension and heart disease development [2].

Recently, it has been found that KS inhibits endothelial inflammation by increasing endothelial NO synthetase expression and NO formation in endothelial cells. KS thus inhibits inflammation by two unique and independent mechanisms: increases endothelial NO synthase expression and decreases TNFα – induced signaling pathways. As a result KS has an unique position in the development of new strategies for treating a wide spectrum of states and diseases caused by vascular inflammation and endothelial dysfunction, including chronic pancreatitis [2,5].

The aim

The aim of the research – to investigate the level of serum kallistatin in patients with combination of chronic pancreatitis and atherosclerosis.

Materials and methods

89 patients with chronic pancreatitis were examined in the gastroenterological department of the Transcarpathia Regional Clinical Hospital named after A.Novak and in family ambulatory by gastroenterologist. The control group consisted of 30 practically healthy persons without signs of pancreatic-duodenal zone injury.

The diagnosis of chronic pancreatitis was based on complaints, anamnestic data, laboratory-instrumental methods of examination in accordance with the Marseilles-Roman`s criteria (1989), supplemented by Ya.S. Zimmermann (1995), as well as on the basis of the points system M-ANNHEIM.

The diagnosis of atherosclerosis was based on measuring the thickness of the intima media layer of the carotid artery and presence of the lipidogram violations (increased blood plasma total cholesterol, triglycerides, low density lipoprotein, atherogenicity and decrease in high density lipoprotein). Biochemical parameters were determined with automatic biochemical analyzer ChemWell, Awareness Technology INC (USA). Also, the coefficient of atherogenisity (CA) was calculated according to the equation: CA = (total cholesterol – high density lipoprotein)/high density lipoprotein.

All patients underwent ultrasound diagnostics of the abdominal cavity with the determination of the size, contours, structure and echogenicity of the hepatic-biliary system. The ultrasound was performed with Philips HDI-1500 with a 3.5 mHz scan sensor.

To determine the excretory pancreatic insufficiency, all patients were given 13C-amylase and 13C-mixed triglyceride breathing tests. Results were analyzed according to the 13 breathing samples. In all breath samples, the concentration of 13CO2 (IZINTA, Hungary) was determined by infrared spectrometry. The maximal concentration of 13CO2 between 150 and 210 minutes at a level of more than 8 %, and a total concentration of 360 minutes – 30-35%, was considered normal for the 13C-mixed triglyceride breath test. The saved excretory function of the pancreas was considered at total concentration of 13CO2 to the end of the test (360 min.), more than 10% for the 13C-amylase breath test.

The serum`s kallistatin level (Serpin A4) was determined by immunoassay using the Human Serpin A4 ELISA Kit from RayBiotech, according to the application method. An analysis of the results was carried out at a wavelength of 450 nm.

The methodology of studies corresponds to the Helsinki Declaration of 1975 and its revision of 1983. The statistical processing of the patients` results was carried out using program STATISTICA 10.0 (firm StatSoft Inc., USA).

Results and discussion

Depending on the presence of dyslipidemia all patients with chronic pancreatitis were divided into 2 groups. The first group included 38 patients with chronic pancreatitis without atherosclerotic changes (CA < 3), and the second group – 51 patients with combination of chronic pancreatitis and atherosclerotic changes (CA > 3). Table I shows lipid metabolism indices in patients with chronic pancreatitis, in comparison with control group (p <0.05).

To evaluate the degree of excretory pancreatic insufficiency, all patients were conducted 13C-amylase and 13C-mixed triglyceride breathing tests. Table ІІ shows the results of respiratory tests in patients with chronic pancreatitis and in the control group.

The level of serum kallistatin in practically healthy subjects group (n = 30) was within the range of 25.38±4.07 ng/ml. These indices were accepted as normal kallistatin`s values. Picture 1 shows the results of our research.

According to the results shown in picture 1, all patients have violations of the excretory insufficiency of the pancreas based on the results of 13C-mixed triglycerides` breathing test. In first group patients, the maximal concentration of 13СО2 between 150 and 210 minutes was 6.9±0.6%. The total concentration of 13СО2 at the end of the study (360 minutes) was at the level of 18.2±0.7% (p<0,05). In second group patients, these indicators were at the level of 5.8±1.2% and 14.0 ± 0.8% respectively (p<0.05).

Instead, according to the results of the 13C-amylase`s breathing test, the pronounced excretory insufficiency was observed in patients with combination of CP with atherosclerotic changes (total concentration 13СО2 at the end of the study (360 minutes) was 7.1±1.1%). In patients with CP without atherosclerotic changes, this indicator was at the lower limit of norm and amounted to 11.2±1.3 %, respectively (p<0,05).

The analysis of study results showed that in patients with less pronounced disorders of the excretory pancreatic insufficiency (decrease in the total concentration of 13СО2 at the end of the study (360 min) on the results of the 13C-amylase`s breathing test to 11.2±1.3% and 13С-triglycerides` mixed breathing test to 18.2±0.7%) and without violations of the lipid profile, the level of kallistatin was 33.66±4.55 ng/ml.

Instead, the examined patients had pronounced violation of the excretory pancreatic insufficiency (decrease in the total concentration of 13CO2 at the end of the study (360 min) based on the results of the 13С-triglycerides` mixed breathing test to 14.0±0.8% and –amylase`s breathing test to 7.1±1.1%) and dyslipidemia, this indicator was at 14.69±9.21 ng/ml.

In this study in patients with combination of chronic pancreatitis and atherosclerosis were measured the level of the anti-inflammatory and vasodilator marker kallistatin, for the first time. An increase in the level of kallistatin in patients without severe excretory pancreatic insufficiency is likely to be explained by the activation of the protective mechanisms cascade of the body in response to the inflammatory process in the pancreatic tissue. In patients with more pronounced excretory pancreatic insufficiency and atherosclerotic changes, there is a decrease in serum`s kallistatin level compared to the control group, which allows us to assume the depletion of kallistatin reserves in response to the long course of CP.

Research of one group of authors showed that kallistatin has unique protective function in endothelial injury, independent of its interaction with tissue`s kallikrein. Kallistatin exerts multiple protective effects against cardiovascular system dysfunction by inhibiting oxidative stress, inflammation, apoptosis and elevating NO levels in experimental animal models and cultured cells [6].

Kallistatin is a significant maintainer of vascular homeostasis through its antioxidant`s actions. Kallistatin gene transfer reduced aortic oxidative stress, whereas kallistatin`s antibody injection increased it [7,]. Oxidative stress decreases kallistatin expression in cultured endothelial cells, leading to insufficient kallistatin levels to maintain healthy vascular homeostasis [8]. Our study confirms the authors’ data as we have found a significant decrease in the kallistatin level in patients with combination of chronic pancreatitis and atherosclerosis.

The results of our studies are original and suggest that fluctuations in serum kallistatin levels in relation to the norm can serve as a criterion for determining the severity of chronic pancreatitis and predicting the course of this disease. Further studies of the dynamics of the kallistatin level are necessary for the improvement of the pathogenic mechanisms of the pancreatic insufficiency formation and progression in patients with vascular pathology.

Conclusions

1. In patients with chronic pancreatitis without pronounced excretory insufficiency of the pancreas, an increase in the serum kallistatin level was observed.

2. In patients with chronic pancreatitis with pronounced excretory insufficiency of the pancreas and atherosclerosis – decrease in serum kallistatin level was observed.

REFERENCES

1. Julie Chao, Grant Bledsoe, Lee Chao. Protective Role of Kallistatin in Vascular and Organ Injury. Hypertension 2016; 68:533-541.

2. Hang Yin, Lin Gao, Bo Shen et al. Kallistatin Inhibits Vascular Inflammation by Antagonizing Tumor Necrosis Factor –Induced Nuclear Factor кB Activation. Hypertension 2010; 56:260-267.

3. William C. Wolf, Russell A. Harley, Dan Sluce Lee Chao et al. Cellular localization of kallistatin and tissue kallikrein in human pancreas and salivary glands. Histochem Cell Biol 1998; 110:477–484.

4. Allison Ross Eckard, Soohee Chо, Mary Ann O’Riordan et al. Kallistatin Levels in HIV-infected Patients and Effects of Statin Therapy. Biomarkers 2016; 1354-750X (Print) 1366-5804. http://dx.doi.org/10.1080/1354750X.2016.1204002.

5. K.F. Huang, H.Y. Yang,Y.M. Xing et al. Recombinant Human Kallistatin Inhibits Angiogenesis by Blocking VEGF Signaling Pathway. Journal of Cellular Biochemistry 2014; 115:575–584.

6. Shen B, Gao L, Hsu YT, Bledsoe G, Hagiwara M, Chao L, Chao J. Kallistatin attenuates endothelial apoptosis through inhibition of oxidative stress and activation of akt-enos signaling. Am J Physiol Heart Circ Physiol. 2010; 299: H1419–H1427.

7. Liu Y, Bledsoe G, Hagiwara M, Shen B, Chao L, Chao J. Depletion of endogenous kallistatin exacerbates renal and cardiovascular oxidative stress, inflammation, and organ remodeling. Am J Physiol Renal Physiol. 2012; 303: F1230–F1238.

8. Shen B, Chao L, Chao J. Pivotal role of jnk-dependent foxo1 activation in downregulation of kallistatin expression by oxidative stress. Am J Physiol Heart Circ Physiol. 2010; 298:H1048–H1054.

 

Scientific research is a fragment state budget topic № 851 “Mechanisms of the formation of complications in hepatic and pancreas`s diseases, methods of their treatment and prevention”, state registration number: 0115U001103, performed at the departments of surgical diseases and propaedeutic of internal diseases of the Medical Faculty in Uzhhorod National University.

Authors are responsible for the content and it does not necessarily represent the official views of the National Institutes.

ADDRESS FOR CORRESPONDENCE

Opalenyk Svetlana

v. Kamyanytsia, st. Kiltseva, 5, 89411, Uzhhorod, Ukraine

tel.: +380990409917

e-mail: kyzma16011993@ukr.net

Received: 20.02.2018

Accepted: 10.04.2018

Table I. Changes in lipid metabolism indices in patients with chronic pancreatitis

Indicator

Control group (n=30)

Patients with CP without atherosclerotic changes (n=38)

Patients with combination of CP and atherosclerotic changes (n=51)

Triglycerides (mmol/l)

1,1±0,12

1,65±0,41

3,1±0,45

Total cholesterol (mmol/l)

4,38±0,41

4,77±0,43

7,19±0,33

Low density lipoprotein (mmol/l)

3,0±0,24

3,3±0,17

4,1±0,65

Very low density lipoprotein (mmol/l)

0,17±0,62

0,61±0,12

2,3±0,76

High density lipoprotein (mmol/l)

2,3±0,35

1,85±0,22

0,83±0,38

CА

1,65±0,22

1,88±0,31

4,19±0,56

Table II. Results of 13C-amylase and 13C-mixed triglyceride breath tests in the examined patients with chronic pancreatitis and control group

Indicator

Control group (n=30)

Patients with CP without atherosclerotic changes (n=38)

Patients with combination of CP and atherosclerotic changes (n=51)

13C- mixed triglyceride breath test:

– maximal concentration of 13СО2 between 150 and 210 minutes

18,0±1,2 %

6,9±0,6 %*

5,8±1,2 %*

– total concentration 13СО2 at the end of the study (360 min.)

30,9±2,1 %

18,2±0,7 %*

14,0±0,8 %*

13C-amylase breath test:

– total concentration 13СО2 at the end of the study (360 min.)

22,7±0,9 %

11,2±1,3 %**

7,1±1,1 %*

Note: * – p <0,05 – the difference between the indicators in patients and the control group is reliable;
the rates in patients with the CP of the first group and the second group are significantly different: ** – p <0,05.

Fig. 1. Concentration of serum kallistatin in patients with chronic pancreatitis and control group.