MORFOLOGICZNA I KRYSTALOCHEMICZNA CHARAKTERYSTYKA KAMICY PRZEWODÓW TRZUSTKOWYCH

Oleksandr V. Kravets1, Ihor A. Danilenko2, Olga M. Smorodska3, Artem M. Piddubnyi3, Inna-Margaryta S. Zakorko3, Sergei N. Danilchenko4, Roman A. Moskalenko3, Mykola G. Kononenko2, Anatolii M. Romaniuk3

1 Department of General Surgery, Sumy State University, Sumy, Ukraine

2 Department of Surgery and Oncology, Sumy State University, Sumy, Ukraine

3 Department of Pathology, Sumy State University, Sumy, Ukraine

4 Institute of Applied Physics of National Academy of Science, Sumy, Ukraine

ABSTRACT

Introduction: Information on chemical and phase composition of pancreoliths is limited and discrepant. There are reports, that pancreoliths are composed by calcium, phosphate, calcium carbonate or combination of calcium with fatty acids

The aim of the work is studying of structural characteristics of pancreatoliths in 5 clinical cases.

Materials and methods: Morphological and crystal-chemical study of five cases of pathological biomineralization in the pancreas were conducted in the work.

Results: Two stones were located in the pancreatic duct, in other cases – in the ductal system of pancreas. Concretion sizes ranged from 0.5 to 1.5 cm in diameter. Pancreatic lithiasis’ form depended on the location: in the duct of Wirsung single concretions were found (they were relatively large, oval stones with smooth, regular edges); multiple, small concretions with irregular edges, coral-like stones dominated in the ductal system of pancreas. Histological study of pancreas showed the signs of chronic pancreatitis, tissue fibrosis, atrophy and edema of glandular component, system distension of ducts, nidal mix-cell inflammatory infiltrates, vessels’ plethora. Structural phase and chemical analysis of pathological biominerals responded calcite in all studied cases.

Conclusion: The presence of pancreatic lithiasis was found to be accompanied by significant morphological changes of the pancreas. The pancreatolith crystal phase was established to be calcium carbonate in the form of calcite.

Wiad Lek 2018, 71, 1 cz. II, -241

INTRODUCTION

Pancreatic lithiasis (PL), as a phenomenon of calculus formation in tissue and ductal system of the pancreas, was described by Graef in 1664 for the first time [1]. The majority of current research of PL and pancreolithes are dedicated to the management of this disease and to extracorporeal shockwave lithotripsy (and other methods of stones destruction) [2-9]. The main scientific works, which investigated causes and the mechanism of pancreolith formation, were written in the mid-twentieth century [10, 11]. But since that time the concept of development of pancreas pathology has changed and opportunities of scientific research have increased. This necessitates a complex research of mineral and tissue components of pathological biomineral formations in pancreas.

On average, the incidence of the disease is 0-4 in 1000 among the total world population. This index is known to be 4 per 1000 in the US and Brazil [12].

PL has geographical features related to nutritional factors. Thus, in developed countries (Western Europe, USA, Japan) PL occurs on the background of alcohol abuse (mostly men aged 30-50 years), on the other hand – in Asia and Africa, in some countries of Latin America PL is associated with chronic malnutrition (mostly women aged 20-30 years) [13].

Etiological factors of PL are not completely understood. They can be divided into several groups: nutritional (alcohol, malnutrition, smoking) [13], chronic inflammation (chronic pancreatitis, autoimmune pancreatitis, bacterial pancreatitis, surgery in pancreatobiliary zone) [5, 12], bile reflex (cholelithiasis and other diseases of biliary system) [12] and genetic factors, which are less common. [14]

The leading mechanism of stone formation is considered to be an increase of pancreas intraductal pressure, which is caused by increased mucin production [15]. In the case of genetically cased PL stone formation occurs with the participation of proteins, which have affinity to calcium [12, 14]. According to other studies data, the mechanism of stone formation in pancreas is closely concerned with two compounds: stone-forming protein (lithostatin) and fortoferin [16]. Studies data associate additionally stone formation with lactoferin and trypsinogen, but the leading role is still given to stone-forming protein [14, 15].

Information on chemical and phase composition of pancreoliths is limited and discrepant. There are reports, that pancreoliths are composed by calcium, phosphate, calcium carbonate or combination of calcium with fatty acids [10, 11, 15].

THE AIM

The aim of the work is studying of structural characteristics of pancreatoliths in 5 clinical cases.

MATERIALS AND METHODS

Ethics Statement. A written informed consent was obtained from all patients. This research was approved by the Medical Ethics Committee of Sumy Regional Clinical Hospital and Medical Institute of Sumy State University (Protocol No.3/6, 07.06.16).

The surgery material Samples was fixed in 10% formaldehyde in PBS, dehydrated in ethanol, cleared by toluene and embedded in paraffin. Sections of 4 μm thickness were used for staining with hematoxylin-eosin.

The mineral component was isolated by heat treatment at 200 ° C for 1 hour. X-ray diffraction studies were performed on the diffractometer DRON4-07 (Burevestnik, Russia). We used CuKa radiation (wavelength 0.154 nm) under Bragg-Brentano focusing conditions (J-2J) (2J – Bragg angle). The current and voltage on the X-ray tube were respectively 20 mA and 30 kV. Samples were taken at the continuous recording mode (speed 2 °/min) in the range of 2J angles from 10 to 60°. All processing procedures of experimental data were performed using a licensed software package of experiment supporting and results processing (DIFWIN-1, LLP “Etalon PTTS”). The identification of crystalline phases was carried out by automatically comparing the obtained results with the cards of database Powder Diffraction File 2 without overlaying of restrictions on the elemental composition of the samples; the software package Crystallographica Search-Match (Oxford Cryosystems, www.crystallographica.co.uk) was used in the study.

RESULTS AND DISCUSSION

All cases of PL were represented by male patients. The 1st and the 2nd cases were accidental findings during autopsy studies. In the 3rd, 4th and 5th cases the diagnosis of PL was established at time, operations were conducted. In three cases pancreoliths were found in men, who abused alcohol and / or had cirrhotic liver damage.

Case 1

The man, aged 45, had a treatment in Surgical Department of Sumy Regional Clinical Hospital in October 2010. The patient complained about jaundice, itching, ascites, general weakness. He was found to suffer from cholelithiasis . choledochal stone led to obstructive jaundice, which was a reason of performed operations (cholecystectomy, Yurash’ choledochoduodenoanastomosis, drainage of abdominal cavity – 07.10.10). The general condition of the patient was complicated by surgery, comorbidities, mixed cirrhosis, diabetes, all that led to cardiac decompensation – acute cardiovascular failure, leading to death. At autopsy pancreas was found to be thick, edematous, to have such size 18,0х2,0х2,5 cm, with areas of fat deposits. In the pancreatic duct the stone was found, it was whitish with smooth rounded edges, in size 0,5х0,8 сm (Fig. 1A).

Case 2

The 43-year-old man had a treatment in Surgical Department of Sumy Regional Clinical Hospital in January 2013. The patient complained about coffee grounds vomiting, black stool, general weakness, an increase of the abdomen in the volume. Case history: excessive consumption of alcohol for more than 20 years, alcoholic cirrhosis for 8 years. After the examination the diagnosis was established: liver cirrhosis, varicose veins of the esophagus and gastrointestinal bleeding. A conservative therapy (infusion, blood-restoring, blood-substitutive) was performed, which did not give a positive result, the patient died on the first day after hospitalization.

Pathomorphological study of autopsy found, that changes in the liver correspond to mixed cirrhosis, changes in the upper gastrointestinal tract correspond to varicose veins of the esophagus and stomach and to erosive-ulcerative gastritis. At the same time, the pancreas was 12.5×1.5×2.8 cm in size, it was dense, atrophic, with sclerotic changes on a section, yellow, filled by coral white stones 0.4 to 1.5 cm in size, which were located in the ducts (Fig. 1 B).

Case 3

56-year-old man was hospitalized in Surgery Department of Sumy City Clinical Hospital №5 in December 2016. The patient complained about itching, discoloration of stool, the yellow color of the mucous membranes and skin, general weakness and malaise. From case history: the patient had worsening of chronic pancreatitis several times during past 10 years. Significant worsening of health was observed for last two months. After examination such diseases were diagnosed: chronic recurrent pseudotumorous calculous pancreatitis; Susp Ca of pancreatic head; jaundice, wirsungolithiasis.

Surgical treatment component of that patient was carrying out the surgery: laparotomy, a longitudinal pancreatojejunostomy and cholecystojejunostomy on the “excluded by Roux“ small bowel loops, drainage of the abdominal cavity. Obtained concretions were 1 cm in a diameter, they had a rough surface, dense texture and shape of duct of Wirsung copy.

The postoperative period passed normal. The patient was discharged from the hospital with recovery.

Case 4

37-year-old man was hospitalized in Sumy City Clinical Hospital №5 in November 2016 with complains about upper abdominal pain radiating to back, general weakness, yellowish color of sclera. Similar symptoms occurred periodically during last 7 years. During examination: the slightly edematous abdomen, yellow color of sclera, slight muscle tension of anterior abdominal wall in the epigastrium and the presence of dense formation (13 cm in a diameter) in the left hypochondrium. After examination such diagnosis was established: chronic calculous pancreatitis, wirsungolithiasis; cyst of the pancreas tail. After stabilizing the patient state surgery was performed: laparotomy, cystojejunostomy and cholecystojejunostomy on the “excluded by Roux“ small bowel loops, drainage of the abdominal cavity. Obtained panreoliths filled densely the duct of Wirsung, almost completely blocking bile and pancreatic juice flow, they had gray-yellow color, dense texture, irregular shape with a rough surface and, their size was 0.3 cm in a diameter.

The postoperative period passed normal. The patient was discharged from the hospital with recovery.

Case 5

47-year-old patient was hospitalized in Surgery Department of Sumy City Clinical Hospital №5. He complained about upper abdominal pain, nausea, vomiting, general weakness. From case history: patient was treated for acute and worsening of chronic pancreatitis during last 5 years, occasionally drank hard alcohol, recommended diet was not followed. After examination such diagnosis was established: chronic calculous pancreatitis, wirsungolithiasis. As a result of absence of positive dynamics and the growing lack of exocrine pancreatic function, surgery was performed: laparotomy, longitudinal pancreatojejunostomy, drainage of the abdominal cavity. A significant number of biominerals were found intraoperatively, which were located in the pancreatic ducts. They had a rounded shape, yellow-pink color, dense texture, smooth surface.

Histological examination of pancreas tissues of all clinical cases showed the similar results. Thus, pancreas was characterized by strong development of fibrous tissue, chronic inflammatory infiltration of the stroma and glands (Fig. 2A, B). Significant edematous tissue, histoarchitectonics abnormality, atrophy of glandular system were observed in pancreas with PL in both clinical cases (Fig. 2 C, D). In addition, the extension of pancreatic ductal system was revealed, remains of PAS-positive substance were observed in some duct lumen (Fig. 2 C). Pancreas tissue with pancreatolithes contained focal fat deposits and point hemorrhages.

Research of biomineralith using the methods of applied materials science

The XRD recorded for one of the samples of pancreatic calculi is shown in Fig. 3. By comparing the location of the peaks in the XRD with the JCPDS data on inorganic compounds, the peaks were identified as due to calcium carbonate (CaCO3) in calcite form. No other phases are observed other than the reported one.

All patients with PL were men aged 43 to 56 years. Three of five patients had a history of alcohol abuse, partly confirming the data literature about negative effects of alcohol on the PL development [13]. Pancreoliths morphology depended on their localization in pancreas: single were detected in the pancreatic duct, they were relatively large, oval stone with a smooth, regular edges; multiple, small concretions with irregular edges, coral-like stones dominated in small ducts and glandular parenchyma of pancreas. Two stones were located in the pancreatic duct, in other cases – in the ductal system of pancreas. Concretions sizes ranged from 0.5 to 1.5 cm in diameter. Obviously, the shape of biomineral deposits was influenced by their location and local conditions.

Histological examination of pancreas showed the signs of chronic pancreatitis, tissue fibrosis, atrophy and edema of glandular component, system extension of pancreas ducts, focal mix-cell inflammatory infiltrates, vessels’ plethora. Of course, chronic inflammation provides the main conditions for pathological biomineralization in pancreas, but the possibility of secondary processes in the surrounding tissues must be remembered, as a response to irritation and injury by stones. Overall, diffuse fibrosis of pancreas, atrophy of glandular parenchyma and chronic inflammation of the gland are the results of PL.

Structural phase of pathological biominerals was represented by calcite in all studied cases. It should be noted, that calcite forms are in a human body only in the inner ear (otolithes, norm) and in gallbladder (some stones, pathology); hydroxyapatite mainly forms in other tissues [17-19]. The main reason of the calcite formation in pancreas tissues is a presence of a great number of bicarbonate ions, which are a defining building material for pancreoliths [20].

Due to the limited number of samples in this study, it was not possible to find a possible connection between the chronic pancreatitis etiology and biomineral depositions [20].

Analysing the clinical results, patients, in whom PL was established in time and surgery was conducted, have recovered. Deaths of patients, in whom PL were accidentally discovered during autopsy, were characterized by severe primary pathology (cirrhosis).

CONCLUSION

Thus, the presence of pancreoliths was found to be accompanied by significant morphological changes in pancreas. Histological examination of pancreas revealed the signs of chronic pancreatitis, fibrosis, atrophy, edema of glandular tissue, system extension of pancreatic ducts, focal mix-cell inflammatory infiltrates, vessels’ plethora.

The crystalline phase of pancreoliths is calcium carbonate in the form of calcite.

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14. Chun J CX, Naruse1 S. et al. Pancreatic Stone Protein of Pancreatic Calculi in Chronic Calcified Pancreatitis in Man. JOP. J. Pancreas (Online) 2002; 3(2):54-61.

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20. Cros J, Bazin D, Kellum A et al. Investigation at the micrometer scale of pancreatic calcifications in chronic pancreatitis by µFTIR spectroscopy and field emission scanning electron microscopy. Comptes Rendus Chimie. 2015; 1-14. Doi:10.1016/j.crci.2015.06.015.

Address for correspondence

Moskalenko Roman

tel.+380979802731

e-mail. r.moskalenko@med.sumdu.edu.ua

Received: 15.10.2017

Accepted: 18.02.2018

Fig. 1. A. Concretion from the duct of Wirsung of the pancreas (case 1).

B. Coral-like concretion of pancreas ductal system (case 2, after heat treatment).

A. Fibrosis, extension of gland ducts of pancreas, hematoxylin-eosin staining, magnifying x100.

B. Chronic inflammatory infiltration of pancreas tissue, hematoxylin-eosin staining, magnifying. x400.

C. Fibrosis, histoarchitectonics abnormality of pancreas, ducts extension with secret remains. PAS – reaction, magnifying x100.

D. Edema and desquamation of glandular epithelium. PAS – reaction, magnifying x400.

Fig.2. Histological examination of the pancreas.

Fig. 3. Original diffraction pattern of mineral material of pancreatic lithiasis

Table. I. Some clinical and pathological characteristics of patients.

Case

Age

Alcohol abuse

Pancreolith localization

Mineral phase

Treatment result

1

43

+

Pancreatic duct

calcite

death

2

45

+

Small ducts

calcite

death

3

56

Pancreatic duct

calcite

recovery

4

37

Pancreatic duct

calcite

recovery

5

47

+

Small ducts

calcite

recovery