Identification of immunoreactive pancreatic stone protein in pancreatic stone, pancreatic tissue, and pancreatic juice

We first examined whether pancreatic stone protein (PSP) was present in pancreatic stone and normal pancreatic tissue. By using HPLC and Western blotting, a protein of Mr 13.5 kDa that reacted with monoclonal antibody against PSP was detected as a major component in EDTA-soluble fractions of pancreatic stone. In an in vitro experiment, this protein dose-dependently suppressed CaCO3 precipitation. PSP was immunohistochemically stained in the acinar cells of normal pancreatic tissue. Based on these findings, it seemed that PSP in pancreatic stone is probably a physiological secretory protein of the pancreas. We subsequently examined immunoreactive PSP in normal pancreatic juice by the Western blotting method. In all of the specimens, the band for immunoreactive PSP in pancreatic juice was found to correspond to 13.5 kDa, which thus agreed with that of purified PSP from a stone.     

Immunoreactive forms of pancreatic stone protein in six mammalian species

Secretory forms of the pancreatic stone protein (PSP S, Mr 17, 500-22,000) have been purified from human pancreatic juice. PSP S are inhibitors of CaCO3 crystal growth. The presence of similar proteins in bovine, canine, monkey, porcine, and rat pancreatic secretion was investigated in terms of biological role and immunological relationship. Pancreatic proteins were analyzed by electrophoretic separation and by subsequent immunoblotting with a rabbit polyclonal antibody against human PSP. A single immunoreactive form was detected in dog, pig, and rat (Mr 17,000), and two distinct immunoreactive forms were observed in cow and monkey (Mr 15,000 and 17,000). Inhibition of CaCO3 crystal growth was demonstrated in dog and rat. Further kinetic studies of the inhibition process in the rat showed that PSP S binds to the crystal surface according to a Langmuir adsorption isotherm with a dissociation constant (Kd) of 1.5 x 10(-6) M. These results suggest that proteins homologous to human PSP S are present in other mammalian species and may act as stabilizers of Ca(2+)-supersaturated pancreatic juice.     

Protein content of precipitates present in pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis

Chronic calcifying pancreatitis is characterized by the formation of intraductal protein plugs or precipitates and calcified stones in ducts. Similar precipitates may be collected by endoscopic retrograde catheterization of the main pancreatic duct. They are present in the pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis. Protein analysis of these precipitates was performed to try to elucidate the mechanism of stone formation. Two protein fraction were separated by extraction of precipitates. One fraction was easily soluble in saline and contained a small amount of most of the proteins of pancreatic juice. The other fraction was soluble in citrate or ethylenediaminetetraacetate and contained a few proteins with close isoelectric points and identical molecular weight (13,500). These proteins showed immunological identity with the "stone protein" isolated from human pancreatic calculi. Our data demonstrate that the major citrate-soluble protein of precipitates in pancreatic juice is identical with "stone protein". They are strongly support the concept that this protein is the organic matrix of pancreatic stones. Different mechanisms are proposed to explain the phenomenon of protein precipitation that frequently occurs in alcoholic subjects and patients with chronic calcifying pancreatitis.     

Fine structure of the organic matrix of human pancreatic stones

Pancreatic stones that were removed from the pancreatic ducts of patients with chronic calcifying pancreatitis were decalcified so the organic matrix could be studied by scanning and transmission electron microscopy. The observations made by scanning electron microscopy were compared with those made on undecalcified stones, and the findings were correlated with light microscopic observations. After the calcium carbonate was removed, the stones consisted of multiple partitions arranged like a sponge. They were embedded in a gel-like matrix. The organic partitions frequently were composed of dense surface layers and sparse central reticular accumulations, which had surrounded and bound calcium carbonate crystals. The organic matrix was heterogeneous in texture. Some areas had dense, regular, proteinaceous fibrous material. Deposits resembling fibrin were observed. Altered cellular constituents appeared to make up minor portions of the matrix. Calcium carbonate, which was precipitated in vitro in pancreatic juice, resembled the morphology of pancreatic stones more than that of pure calcium carbonate crystals. These results are consistent with the coformation of pancreatic stones from constituents in the pancreatic juice [including pancreatic stone protein (PSP), glycosaminoglycans, and occasional cells] and precipitated calcium carbonate.     

Pancreatic stone protein: quantification in pancreatic juice by enzyme-linked immunosorbent assay and comparison with other methods

To quantitate pancreatic stone protein (PSP), a competitive radioimmunoassay using monoclonal antibodies to PSP extracted from pancreatic stones and a sandwich enzyme-linked immunosorbent assay (ELISA) using monospecific polyclonal antibodies to the secretory forms of PSP (PSP S) were established. When PSP concentrations were measured in pancreatic juice by radioimmunoassay, no difference could be found between patients suffering from chronic calcifying pancreatitis and other diagnostic groups. Yet, with the ELISA technique involving polyclonal antibodies, decreased concentrations were found in chronic calcifying pancreatitis patients when compared to controls (p less than 0.001), chronic alcoholics without pancreatic symptoms, or obstructive pancreatitis patients. These discrepancies are discussed. The monoclonal antibodies recognizing the C-terminal part of PSS S (PSP S1), results from the radioimmunoassay indicate that the concentration of that polypeptide is identical in the juice of controls and patients. Results from the ELISA obtained with polyclonal antibodies raised against PSP S2-5 molecules, i.e., recognizing the PSP S1 part and the N-terminal portion of the molecule, indicate that the differences observed reflect differences in the juice concentration of that N-terminal peptide.     

Analysis of pancreatic stone protein gene of hereditary pancreatitis]

To investigate the pathogenesis of hereditary pancreatitis we determined whether pancreatic stone protein (PSP) gene was structurally altered in two independent families diagnosed as hereditary pancreatitis. Because, it has been shown that a decrease in the activity of PSP which inhibits CaCO3 crystal formation in pancreatic juice is closely related to the development of chronic calcifying pancreatitis. Southern blot analysis revealed neither a rearrangement nor a gross deletion of PSP gene in genomic DNA of affected members of both families. Furthermore, six exons of PSP gene amplified by polymerase chain reaction from genomic DNA was directly sequenced, while no apparent base mutation was observed. The immunohistochemical study utilizing monoclonal antibody to PSP showed the presence of immunoreactive PSP in the section of pancreatic tissue obtained from a patient affected with hereditary pancreatitis. However, the level of immunoreactive PSP in the remaining acinar cells of the patient pancreas was not reduced when compared with that of normal pancreas. Results, therefore, indicate that genetic alteration of PSP gene may not be responsible for the pathogenesis of hereditary pancreatitis.     

Immunocytochemical localization of pancreatic stone protein in the human digestive tract

Recently, in our laboratory, a protein extracted from human pancreatic stones was characterized and purified and a specific antibody was obtained. This pancreatic stone protein (PSP) was shown to have an inhibitory effect on the CaCO3 crystal growth in vitro. The cellular origin of such a protein and its repartition along the digestive tract were studied by immunolocalization (protein A-colloidal gold method) at the ultrastructural level. Surgical biopsies of pancreata from normal or chronic pancreatitis patients, needle liver biopsies, gastric mucosa, and jejunum and duodenum biopsies were minced and fixed in the Karnovsky medium or in buffered 4% paraformaldehyde. The specimens were washed in buffer, dehydrated through ethanol, and embedded in Epon 812. Ultrathin sections, collected on uncoated nickel grids, were submitted to the following reactives at room temperature: protein A 1 mg/ml, anti-PSP (1:2 to 1:100), and protein A-colloidal gold. The specificity of the localization was checked by substituting buffer or nonimmune rabbit serum to anti-PSP. The stone protein was markedly present in the zymogen granules and condensing vacuoles of the normal pancreatic acinar cells, the label was found in the acinar and ductal lumen. In chronic pancreatitis, the localization of PSP, when it occurred, was extremely weak in the acinar cells. No PSP was specifically characterized in hepatocytes, gastric mucosa, and enterocytes. However, a weak but specific reaction was found in the secretory granules of Paneth cells. These results in pancreas confirm the acinar secretory origin of the PSP and are in good agreement with its possible function in stabilizing pancreatic juice in vivo, which is normally supersaturated in calcium carbonate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pancreatic stone protein. II. Implication in stone formation during the course of chronic calcifying pancreatitis

Pancreatic stone protein, a novel protein isolated from pancreatic stones of patients suffering from chronic calcifying pancreatitis and secreted in normal human pancreatic juice, was measured by radial immunodiffusion in pure pancreatic juice. Patients with chronic calcifying pancreatitis of different etiologies had significantly lower levels of pancreatic stone protein when compared with other pancreatic diseases and controls. Pancreatic stone protein suppresses in vitro calcium carbonate precipitation and therefore stabilizes normally supersaturated pancreatic juice. The decreased pancreatic stone protein levels observed could be a key factor in the growth of calcium carbonate crystals and stone development during the course of chronic calcifying pancreatitis.     

Histopathologic and immunohistochemical studies on alcoholic pancreatitis and chronic obstructive pancreatitis: special emphasis on ductal obstruction and genesis of pancreatitis

We compared alcoholic pancreatitis (AP), which is thought to be caused by protein plugs, and chronic obstructive pancreatitis (COP), distal to carcinoma, both histopathologically and immunohistochemically. Eighteen cases of AP showed marked and irregularly distributed interlobular fibrosis. The exocrine parenchyma and its immunoreactivity against anti-amylase were rather well preserved, except in the advanced stages of the disease. Protein plugs and pancreatic stones were found. Fifteen cases of COP showed a uniform distribution of inter- and intralobular fibrosis, marked destruction of the exocrine parenchyma, and loss of amylase concentration. Neither protein plugs nor pancreatic stones were found. Anti-collagen immunoreactivity was found in both types of pancreatitis. Although obstruction of the main or small pancreatic ducts is considered to be the principal factor in the genesis of both AP and COP, the histological features of the two diseases are quite distinct from one another. Therefore, duct obstruction caused by protein plugs appears not to be the main factor in the genesis of alcoholic pancreatitis.     

Idiopathic chronic calcifying pancreatitis with diabetes mellitus

Summary We present a case of a 27-year-old female suffering from chronic calcifying pancreatitis with diabetes mellitus. Radiographic examinations and exocrine pancreatic function tests revealed considerable dilatation of pancreatic ducts with large intraductal calculi and exocrine pancreatic insufficiency, respectively. Recent literature indicates that a decrease in the activity of pancreatic stone protein (PSP), which inhibits CaCO₃ crystal formation in pancreatic juice, is closely related to the development of chronic calcifying pancreatitis. The patient had no apparent cause or family history of pancreatitis. We therefore investigated the possibility that alterations in the PSP gene might explain the chronic pancreatitis seen in this patient. Six exons of the PSP gene amplified by polymerase chain reaction were directly sequenced, but there was no apparent base mutation observed. Furthermore, Southern blot analysis revealed neither rearrangement nor deletion of the PSP gene in the genomic DNA of this case. However, this genetic approach will be useful for future study of the etiology of hereditary pancreatitis.     

Conformational changes of pancreatitis-associated protein (PAP) activated by trypsin lead to insoluble protein aggregates

Pancreatitis-associated protein (PAP), a secretory acute-phase protein of the pancreatic acinar cell, is highly up-regulated early in acute pancreatitis. PAP expression returns to undetectable levels when the pancreas recovers. In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis. Their functions remain obscure. Pancreatic stone protein (PSP/reg), which shows strong sequence homology to PAP, is secreted into pancreatic juice under physiologic and pathologic conditions. PSP/reg is highly susceptible to trypsin cleavage at its ARG11-ILE12 bond. Cleavage results in an N-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP). PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro. PTP can be found in protein plugs or stones from patients with chronic pancreatitis. Rat PAP contains a trypsin cleavage site at the same position as PSP/reg. We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH. To test our hypothesis, we generated and purified recombinant PAP. Here we report the production of rat PAP I, II, and III in a yeast expression system using Pichia pastoris. We demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis-associated thread protein (PATP). PATP displays pH-dependent solubility characteristics very similar to those of PTP.     

Quantification of human lithostathine by high performance liquid chromatography.

Pancreatic stones of patients with chronic calcifying pancreatitis (CCP) are mostly made up of CaCO3 crystals. Formation and growth of such crystals is inhibited in vitro by lithostathine, a protein present in normal pancreatic juice. Decreased lithostathine activity was therefore suspected in patients with CCP, but comparison by immunoassay of lithostathine concentrations in the pancreatic juices of patients and controls led to conflicting results. This study shows that these discrepancies might have been caused in part by a remarkably high susceptibility of the protein to trypsin like cleavage, resulting in important structural changes and concomitant modifications of the epitopes. A novel lithostathine assay in juice was developed, based on separation of secretory proteins by high performance liquid chromatography. The chromatographic separation of lithostathine was based on hydrophobic interactions at pH 5.0 using a Phenyl-TSK column. This study showed with this assay that lithostathine concentrations (microgram/mg of total protein) were similar in CCP patients with alcoholic aetiology (mean (SD) 6.3 (2.7)) and other aetiologies (7.2 (3.7)), but one third of those estimated in patients without pancreatic disease (16.7 (4.3)). Similar concentrations were found, however, in chronic alcoholic patients without CCP (6.6 (3.3)) and in patients with CCP. It was concluded that decreased lithostathine concentration is associated with CCP, although such a decrease is not sufficient by itself for the disease to occur.     

Citrate and calcium secretion in the pure human pancreatic juice of alcoholic and nonalcoholic men and of chronic pancreatitis patients

Citrate, calcium and protein have been estimated in pure pancreatic juice after a secretin and a CCK injection in 4 patients presenting with alcoholic calcified pancreatitis (ACP), 10 controls without evidence of pancreatic disease, drinking more than 130 g alcohol/day, and 10 controls without evidence of pancreatic disease, drinking less than 20 g alcohol/day. Citrate is normally secreted in the pancreatic juice and this secretion increases in parallel with protein after CCK injection. Citrate secretion is significantly decreased in the two alcoholic groups. Calcium secretion is increased in the ACP, and reasons are presented to suggest that this may be due to lesions of the ducts. These modifications could play a role in the formation of pancreatic stones which are mostly built up of calcium carbonate.     

Molecular forms of serum pancreatic stone protein in acute pancreatitis.

CONCLUSION: Elevation of serum pancreatic stone protein-(PSP) S1 suggests activation of trypsinogen in the pancreas. This information would prompt the start of intensive treatment and may improve prognosis of acute pancreatitis (AP). BACKGROUND: PSP exists in two molecular forms, PSP-S2-5 and PSP-S1. PSP-S1 is produced by enzyme cleavage of PSP-S2-5 by trypsin. Total serum PSP rose in AP, but little is known about its molecular forms. In this study, we characterized the molecular forms of serum PSP in AP. METHODS: Sera were taken from 8 patients with severe acute pancreatitis (sAP) and from 11 patients with mild acute pancreatitis (mAP). Serum PSP was characterized by high-performance liquid chromatography (HPLC) followed by the specific enzyme immunoassay (EIA). RESULTS: The total serum PSP in sAP was higher than in mAP, but the difference was not significant. The PSP-S1 was detected in serum in all (7/7) patients in sAP and in 72% (8/11) of patients in mAP. Serum level of PSP-S1 was significantly higher in sAP than that in mAP (p < 0.05), and the cutoff value to distinguish the two groups was 30 ng/mL. Serum PSP-S1 did not show significant correlation with total PSP, immunoreactive trypsin, or C-reactive protein.     

Regulation of PSP/reg in rat pancreas: immediate and steady-state adaptation to different diets.

Pancreatic stone protein/reg protein (PSP/reg) is a secretory pancreatic protein of hitherto unknown function. It is precursor to a spontaneously precipitating peptide called pancreatic thread protein, which is found in protein plugs within the pancreatic ductal system. Increasing PSP/reg concentrations in pancreatic juice might augment the risk of intraductal plug formation and therefore be a condition predisposing to chronic pancreatitis. Malnutrition is associated with a high incidence of chronic pancreatitis in tropical countries. In a diet study with rats, we tested the hypothesis that protein malnutrition leads to increased PSP/reg concentrations in pancreatic juice. A highly sensitive and reliable enzyme-linked immunosorbent assay (ELISA) for rat PSP/reg was newly established. Male Sprague-Dawley rats were allocated to three nearly isocaloric experimental diets, which contained 0, 45, or 82% casein, respectively, or to a control diet (22% casein). We evaluated PSP/reg expression under these four dietary conditions on the RNA and on the protein level, performing a time-course study over a period of 28 days. Our results demonstrate that PSP/reg expression is not increased because of a protein-deficient diet if investigated under steady-state conditions. After a temporary increase in PSP/reg levels due to a carbohydrate-deficient high-protein diet, we could not find signs of a diet-dependent regulation of this protein. The regulation of PSP/reg thus differs from that of most other pancreatic secretory proteins. Our findings contradict earlier reports that had drawn conclusions based solely on messenger RNA levels.     

Duodenal total and ionised calcium secretion in normal subjects, chronic alcoholics, and patients with various stages of chronic alcoholic pancreatitis.

Previous studies have shown increased secretion of total calcium in the duodenal juice of patients with chronic alcoholic pancreatitis compared with healthy subjects. In order to get more detailed information on calcium secretion and pancreatic stone formation in chronic alcoholic pancreatitis, ionised and total calcium concentrations were determined in the duodenal juice of normal subjects, chronic alcoholics, and patients with various stages of chronic alcoholic pancreatitis. Total calcium secretion was in agreement with previously published data. Chronic alcoholics presented a significant increase of ionised calcium. In the course of pancreatitis all calcium fractions increased progressively revealing highest concentrations in patients with severe exocrine insufficiency. In non-calcified and calcified pancreatitis all calcium fractions were identical. It is suggested that the increase of ionised calcium originates from serum ionised calcium passing by diffusion into the damaged pancreatic duct system.     

Pancreatic stone protein – sepsis and the riddles of the exocrine pancreas

Pancreatic stone protein (PSP), discovered in the 1970ies, was first associated with stone formation during chronic pancreatitis. Later, the same protein was independently detected in islet preparations and named regenerating protein 1 (REG1). Additional isoforms of PSP, including pancreatitis-associated protein (PAP), belong to the same protein family. Although the names indicate a potential function in stone formation or islet regeneration, involvements in cellular processes were only suggestive and never unequivocally proven. We established an association between PSP levels in patient blood samples and the development of sepsis. In this review, written in connection with receiving the Lifetime Achievement Award of the European Pancreatic Club, the evolution of the sepsis aspect of PSP is described. We conclude that the true functional properties of this fascinating pancreatic protein still remain an enigma.     

The immunohistochemical evaluation of PSP/ reg -protein in normal and diseased human pancreatic tissues

Summary In order to elucidate the characteristics ofreg-protein, which is identical to pancreatic stone protein (PSP/reg-protein), and the relationship between the generation and evolution of chronic pancreatitis and the expression of PSP/reg-protein in the pancreas, we investigated the expression of PSP/reg-protein in normal and diseased human pancreatic tissues by immunohistochemistry. The PSP/reg-protein was expressed in all cases with normal pancreas or chronic pancreatitis, and in 70.6% of cases with pancreatic cancer. This protein was present in the cytoplasm of acinar cells and, in some cases, in the intraluminal contents of ductules in nonmalignant tissues. From the view of distribution and cellular localization, PSP/reg-protein was expressed more broadly and densely in chronic pancreatitis with mild to moderate injury than in the normal pancreas. However, the protein was less expressed in severely damaged chronic pancreatitis tissue, such as calcifying pancreatitis, than in the normal pancreas. These findings suggest that mild to moderate injury to pancreatic tissue may stimulate the synthesis of PSP/reg-protein, whereas more severe injury tends to depress it.     

An insight into the role of human pancreatic lithostathine

Human lithostathine was initially isolated from pancreatic stones in patients with alcoholic calcifying chronic pancreatitis. It is secreted into the pancreatic juice where it was believed to inhibit stone formation. The N-terminal undecapeptide was assumed to play an important role in the mechanism, by adsorption to the crystal surface. Later, the role of lithostathine in calcite formation and growth was questioned, together with the associated mechanism of action. In particular, although lithostathine adsorbs on calcite crystal, this property does not now seem to be specific. Moreover, the N-terminal undecapeptide is not likely to have, by itself, the function of the entire protein. The different aspects of this controversy are reviewed and discussed, particularly in the light of recent structural biology. Comparative biological data now available allow us to draw an interesting parallel between lithostathine and other related proteins. Finally, lithostathine might affect stone formation and may also have another function which could be investigated in the other proteins belonging to the same structural family.     

Purification and assay of secretory lithostathine in human pancreatic juice by fast protein liquid chromatography.

Impaired secretion of lithostathine, a pancreatic glycoprotein capable of inhibiting the growth of CaCO3 crystals, has been reported in chronic calcifying pancreatitis. Controversial results were obtained, however, using immunoassays with different antibodies. The aim of this study was to purify and to measure juice lithostathine by a non-immunological method. Fast protein liquid chromatography (FPLC) on a cation exchange column eluted by a sodium chloride gradient, was used. The conditions appropriate to separate secretory (S) from hydrolysed (H) isoforms of immunopurified lithostathine were also used for juice analysis. Pancreatic juice was collected by endoscopic cannulation of the major pancreatic duct, after secretin stimulation, from eight patients with chronic pancreatitis (CP) and from eight controls. In all samples, S-isoforms of lithostathine (ranging from 16 to 19 Mr at SDS-PAGE) were the only constituent of two of the 15 peaks in which FPLC resolved the pancreatic proteins. The nature of these two peaks was confirmed by their coelution with immunopurified S-lithostathine and by immunoblot analysis with polyclonal anti-lithostathine antibodies. The ratio between the area of S-lithostathine peaks and the total area of proteic eluates, was always lower in CP patients (5.3 micrograms/mg of protein, median value; 0.2-15.4, range) than in controls (35.2 micrograms/mg; 16.6-55.9). It is concluded that lithostathine can be purified and measured in pancreatic juice by FPLC. Our results with a nonimmunological assay confirm a reduced secretion of lithostathine in patients with CP.