Pancreatic Stone Protein of Pancreatic Calculi in Chronic Calcified

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JOP. J. Pancreas (Online) 2002; 3(2):54-61.Pancreatic Stone Protein of

Pancreatic Calculi in Chronic CalcifiedPancreatitis in ManJOP. Journal of the

Pancreas ­ http://www.joplink.net ­ Vol. 3, No. 2 ­ March 200254Chun Xiang Jin1,

Satoru Naruse1, Motoji Kitagawa1, Hiroshi Ishiguro1, Takaharu Kondo1,Shinobu

Hayakawa2, Tetsuo Hayakawa11Second Department of Internal Medicine, Nagoya

University School of Medicine. Nagoya, Japan.2Department of Clinical Chemistry,

Maruko Pharmaceutical Co. Kasugai, JapanABSTRACTContext The role of protein

components of

pancreatic secretions has been controversial in

pancreatic stone formation.Objective To study the lithogenic role of

pancreatic stone protein and lactoferrin in

stone formation in chronicpancreatitis.Patients Pancreatic stones were collected

from 13 patients with alcoholic (n=6) and

nonalcoholic (n=7) chronic calcified

pancreatitis.Main outcome measures Pancreatic stone

extracts were analyzed for pancreatic stone

protein and lactoferrin using enzyme

immunoassay. The localization of pancreatic

stone protein immunoreactivity in the stone

was observed using immunogold staining and

scanning electron microscopy.Results Immunoreactivities for pancreatic

stone protein were detected in the stones from

all 13 patients with chronic calcified

pancreatitis and for lactoferrin in the stones

from five of the 13 patients. Pancreatic stone

protein immunoreactivity distributed diffusely

from the center to the periphery of the

pancreatic stones.Conclusions Involvement of pancreatic stone

protein seems to be constant from the initial

step of the stone formation to subsequent

steps of the stone growth. However,pancreatic stone protein is only one of the

precipitating proteins in pancreatic secretions

such as lactoferrin, trypsinogen, etc..INTRODUCTIONStone formation in the

pancreatic duct system

is common in chronic pancreatitis. However,

the mechanism of the stone formation has not

been fully elucidated. In a canine

experimental model of pancreatolithiasis,

persistent stasis of protein-rich pancreatic

juice secondary to partial obstruction in the

pancreatic duct leads to the calculus

formation [1, 2].

Plugs formed by the precipitation of the

protein within the interlobular and

intralobular ducts are one of the earliest

findings in chronic pancreatitis and the

protein plugs subsequently perpetuate

inflammation of the gland through repeated

obstruction of the pancreatic duct system. If

concentration-dependent precipitation is a

cause of the protein plug formation, some

proteins must be increased, at least, in their

concentration. Through the analysis of

pancreatic juice, the iron-binding protein,

lactoferrin, has been found to be secreted in

greater amounts by patients with chronic

pancreatitis [3, 4, 5, 6, 7]. Lactoferrin may

play a role in the formation of the protein

plugs frequently seen in chronic pancreatitis

because of its ability to produce an

aggregation of a large acidophilic protein,

such as albumin [8].JOP. J. Pancreas (Online) 2002; 3(2):54-61.JOP. Journal of

the Pancreas ­ http://www.joplink.net ­ Vol. 3, No. 2 ­ March 200255Pancreatic

stone protein (PSP) is a 16 kDa

acidic protein with an isoelectric point in the

range of pH 5.5-6. A truncated form of this

protein was originally isolated from calcium

carbonate stones surgically removed from the

main pancreatic duct of humans with chronic

pancreatitis [9]. PSP was believed to serve as

an inhibitor of calcium carbonate precipitation

in pancreatic juice and was called

" lithostathine " by some [10]. Increasing

numbers of studies suggest that PSP has no

more crystal inhibitory activity than

endogenous proteins such as serum albumin

and trypsinogen [11, 12]. PSP is highly

susceptible to trypsin cleavage at its ARG11-

ILE12 bond [13].

The addition of bovine trypsin to human

pancreatic juice enhances the conversion of

soluble isoforms of PSP S2-5into an insolubleisoform of PSP-S1[14]. The presence

of asmall amount of free proteolytic activity has

been reported in the pure pancreatic juice

obtained from patients with pancreatitis [15,

16, 17, 18]. Localization of the trypsin

immunostaining in the center of the

pancreatic stones was confirmed in our

previous report [19]. When the intraductal

activation of trypsinogen to trypsin occurs,

soluble PSP S2-5is converted into insolublePSP S1with the subsequent

precipitation ofPSP S1.In the present report, we studied the

lithogenic role of PSP and lactoferrin by

measuring the content of the proteins in

pancreatic stones using enzyme

immunoassay, immunostaining of PSP, and

scanning electron microscopy.METHODSCollection of Pancreatic StonesA total of

423 patients with chronic

pancreatitis (alcoholic calcified 124, alcoholic

non-calcified 135, non-alcoholic calcified 57,

non-alcoholic non-calcified 107) were

followed up in Nagoya University Hospital

and its affiliated hospitals during the period

1970-1991.

Pancreatic stones were collected from 13

patients (12 males, 1 female; mean age

51.0±15.4 years): 6 patients had alcoholic (A:

daily intake of more than 80 g of ethanol over

10 years) chronic calcified pancreatitis at

surgery or autopsy (one patient) and 7 patients

had non-alcoholic (NA) chronic calcified

pancreatitis at surgery as listed in Table 1.

Body mass index and serum albumin

exceeded 18.5 kg/m2and 3.0 g/dL,respectively, in all 13 patients without

clinical

evidence of malnutrition, although the

average diet compositions were unknown inTable 1. Total protein, pancreatic

stone protein and lactoferrin content in pancreatic stones.

Patient (Sex, age)Stone size*Weight(mg)Protein(µ

µ

µ

µg/mg stone)Pancreatic stoneprotein(ng/µ

µ

µ

µg protein)Lactoferrin(ng/µ

µ

µ

µg protein)Alcoholic pancreatitis

A1 (male, 71)Large244.23.610.40.1A2 (male, 52)Small16.028.8419.21.0A3 (male,

51)Small66.74.231.30A4 (male, 42)Large73.36.1105.00A5 (male,

41)Small8.587.00.13.0A6 (male, 34)Small61.76.9291.50Nonalcoholic pancreatitis

NA1 (male, 74)Large26.27.61.70NA2 (male, 70)Small16.413.71.30NA3 (female,

69)Large54.65.918.97.3NA4 (male, 50)Small46.47.80.10NA5 (male,

46)Large317.78.992.12.5NA6 (male, 35)Large.10NA7 (male,

29)Large92.76.719.10*Stone size: large stone indicates maximal diameter of stone

greater than 5 mm; small stone means all stones smallerthan or equal to 5 mm in

diameter.JOP. J. Pancreas (Online) 2002; 3(2):54-61.JOP. Journal of the Pancreas

­ http://www.joplink.net ­ Vol. 3, No. 2 ­ March 200256detail. The stones were

well-rinsed in saline

to remove contaminating superficial proteins,

then dried in air, and maintained in this

condition until they were prepared for this

study.Extraction of Proteins from StonesThe stones were ground in a mortar in 3

ml of

0.5M ethylenediaminetetracetic acid (EDTA),

pH 8.0, and kept overnight at 4 ºC after a 30-

min sonication. The next morning, the

suspension was ground again, then dialyzed

using two liters of distilled water three times

in semipermeable tubing with a cutoff of 3.5

kDa and centrifuged for 20 min at 18,000

rpm.Measurement of Protein, Pancreatic Stone

Protein and LactoferrinThe supernatant of the pancreatic stone

suspension was analyzed for protein by

Lowry's method [20], for human pancreatic

stone protein immunoreactivity [14, 21] and

lactoferrin [6, 7] by enzyme immunoassay

methods, as previously reported.Molecular Forms of Pancreatic Stone

Protein in Pancreatic StoneThe supernatant of the stone suspension was

applied to a Mono S (HR 5/5, Pharmacia.

Uppsala, Sweden) column equilibrated with

0.02 M sodium acetate buffer (pH 4.0)

containing 0.02 M CaCl2. The column waswashed with the buffer for 5 min at a

flow

rate of 1.0 mL/min, then eluted for 35 min

with a linear gradient of 0-1.0 M NaCl as

previously reported [14, 21, 22]. Enzyme

immunoassay of PSP S2-5and PSP S1in eachfraction was carried out using the

method

described previously [14, 21, 22].Localization of Human Pancreatic Stone

Protein Immunoreactivity in Pancreatic

StonesLocalization of pancreatic stone protein

immunoreactivity in pancreatic stones wasstudied using immunogold staining and

scanning electron microscopy as previously

reported [19]. Pancreatic stones were

embedded in Epon. The epoxy blocks were

polished using a grinding machine (EMP-2,

Eiko Engineering. Nakaminato, Japan) at 280

rpm with a polish powder of aluminum oxide

(diameter 0.05 µm) in order to expose theinner part of the stones. The exposed

surface

of the pancreatic stone was immunostained

using the first antibody (mouse anti-human

pancreatic stone protein, the same as used in

the enzyme immunoassay for human

pancreatic stone protein) and colloidal gold

(20 nm gold protein) conjugated goat anti-

mouse 1gG (EY Laboratories Inc. San Mateo,

CA, USA). After ion-sputter-coating with

gold-palladium, the immunostained surface

was observed with a scanning electron

microscope (S-800, Hitachi. Tokyo, Japan) at

5 kV as previously reported [19].ETHICSInformed consent was obtained from each

patient and the study protocol was approved

by the Human Research Ethics Committee of

the Nagoya University School of Medicine.STATISTICSData are presented as mean

and standard

deviation. ANOVA was applied for statistical

comparison. The correlation coefficient was

calculated by the least-squares method. P

values less than 0.05 were considered

statistically significant.RESULTSProtein, Pancreatic Stone Protein and

Lactoferrin Content in Pancreatic StonesProtein, human pancreatic stone protein

immunoreactivity and lactoferrin

immunoreactivity in pancreatic stones were

determined and presented as µg/mg of stonefor protein and ng/µg of protein for

pancreaticstone protein and lactoferrin

immunoreactivities (Table 1). The proteinJOP. J. Pancreas (Online) 2002;

3(2):54-61.JOP. Journal of the Pancreas ­ http://www.joplink.net ­ Vol. 3, No. 2

­ March 200257content was not significantly (P=0.285)

different between alcoholic (22.8±32.9 µg/mgstone) and non-alcoholic (8.9±2.8

µg/mgstone) pancreatitis.

The pancreatic stone protein

immunoreactivity was measured in pancreatic

stones in all 13 patients, ranging from 0.1 to

419.2 ng/µg protein, but did not differsignificantly (P=0.238) between alcoholic

(142.9±173.4 ng/µg protein) and non-alcoholic (51.2±83.2 ng/µg

protein)pancreatitis.

There was a significant correlation (P=0.010)

between protein and pancreatic stone protein

in pancreatic stones when the alcoholic

pancreatitis patient (A5) was excluded

because the pancreatic stone of the patient

was predominantly composed of fatty acidcalcium [19] (Figure 1). No further

significant

correlation was detectable between pancreatic

stone protein and lactoferrin (P=0.894).

The lactoferrin immunoreactivity was

detectable in 5 of the 13 patients, ranging

from 0.1 to 7.3 ng/µg protein. There was nodefinite common etiology or clinical

feature

to explain the high lactoferrin content in

patients A5, NA3, and NA5. The stone in the

A5 patient was predominantly composed of

fatty acid calcium. The stones in the NA3 and

NA5 patients were large and were obtained

from the markedly dilated main pancreatic

duct.Molecular Forms of Pancreatic Stone

Protein in Pancreatic StonesTo identify the molecular forms of pancreatic

stone protein immunoreactivity in pancreatic

stones, the pancreatic stone extracts from two

patients (A1 and NA6) were applied to the

Mono S column, The elution peak of the

pancreatic stone protein immunoreactivity

was not identical to that of pancreatic stone

protein S2-5or S1previously reported [14, 23](Figure 2).Localization of

Pancreatic Stone Protein in

Pancreatic StonesLocalization of the pancreatic stone protein

immunoreactivity in pancreatic stones was

observed by immunogold staining and

scanning electron microscopy (Figures 3 and

4). The exposed inner surface of the

pancreatic stone from patient NA6 looked

amorphous at the center of the stone with

concentric laminar layers partly in the

periphery (Figure 3). Gold particles were

distributed diffusely from the center to the

periphery of the pancreatic stone.DISCUSSIONAnalysis of the protein components

in the

pancreatic study is important in clarifying the

mechanism of stone formation in the

pancreatic duct. In the present study we have

shown that pancreatic stone protein is a majorFigure 1. Correlation between

pancreatic stone protein

(PSP) and protein in pancreatic stones from 12 patients

with chronic pancreatitis excluding one patient (A5)

having a pancreatic stone predominantly composed of

fatty acid calcium.Figure 2. Fractionation pattern of pancreatic stone

protein in stone extracts from two patients (A1: open

circles with dotted line; NA6: closed circles with solid

line; see Table 1).JOP. J. Pancreas (Online) 2002; 3(2):54-61.JOP. Journal of

the Pancreas ­ http://www.joplink.net ­ Vol. 3, No. 2 ­ March 200258protein

component of the pancreatic stone.

Pancreatic stone protein was detectable in

pancreatic stones from all the 13 patients

studied, ranging widely from only a trace

amount to 1.21%, when expressed as a

percentage of the stone weight. ni et al.

[24] reported that the smallest amount was

0.013% and the largest was 0.296% of the

stone weight. The percentages obtained in our

study showed wider variation than that of

ni et al. [24]. In the present study, a

significant correlation was obtained between

total protein and pancreatic stone protein

when patient A5 was excluded because the

pancreatic stone was composed

predominantly of fatty acid calcium and not

of calcium carbonate. A wide range of

percentages of pancreatic stone protein in the

total protein (ranging from 0.01 to 41.9 %)

suggests that the mechanisms and protein

components involved in the stone formation

are multifactorial, and that pancreatic stone

protein is not the sole protein involved in

stone formation [3, 4, 5, 6, 7, 13, 15, 19, 25].

In our previous study, trypsin

immunoreactivity was detectable in

pancreatic stones in 11 of the 13 patients with

chronic pancreatitis [18]. The stones without

detectable trypsin immunoreactivity were

from two of the three patients having a highcontent of lactoferrin in the

stones. Of the two

patients, one (A5) had a pancreatic stone

predominantly composed of fatty acid

calcium and the other (NA5) had large stones

migrating in the markedly dilated pancreatic

duct. In these patients, recurrent infection

with or without persistent stasis in the

pancreatic secretion might be involved in the

stone formation. Reports of bacterial growth

in stone debris [26] and impaired antibacterial

activity in pure pancreatic juice from patients

with chronic pancreatitis [27] support the

involvement of the infection in the stone

formation. However, pancreatic stone protein

contents were very low in A5 but moderately

high in NA5. The inconsistency in protein

constituents might reflect the complexity of

mechanisms and precipitable proteins

involved in the stone formation.

Tympner demonstrated increases of viscosity,

trypsin activity, lactoferrin and total protein in

pure pancreatic juice obtained from patients

with chronic pancreatitis [28]. Renner et al.

also observed high concentrations of protein

through the period of secretin stimulation and

the sporadic appearance of free proteolytic

activity in many 1-min specimens through the

collection period of pure pancreatic secretions

in patients with acute pancreatitis. A small

amount of precipitate in several 1-min

collections of pancreatic secretions appeared

to precede and coincide with the presence ofFigure 3. Scanning electron

microscopic view of the

exposed inner surface of the pancreatic stone obtained

from a patient (NA6) with chronic calcified

pancreatitis. The center of the stone looks amorphous

and is partially surrounded by a concentric laminar

layer in the periphery of the stone.Figure 4. Scanning electron microscopic

picture of the

inner surface of the stone from a patient (NA6).

Distribution of gold particles is diffuse from the center

to the periphery of the stones.JOP. J. Pancreas (Online) 2002; 3(2):54-61.JOP.

Journal of the Pancreas ­ http://www.joplink.net ­ Vol. 3, No. 2 ­ March

200259free proteolytic activity [16, 17]. In our

previous studies, both activation of the

pancreatic juice with enteropeptidase and the

addition of trypsin to the pancreatic juice

converted the soluble forms of pancreatic

stone protein S2-5into the insoluble form ofpancreatic stone protein S1and about

45-85%precipitated out after 1 h [14, 22]. In a study

by Provansal-Cheylan et al., analysis of the

clogging material in occluded pancreatic

endoprosthesis revealed the presence of

trypsinogen, amylase and pancreatic stone

protein S1[29].The localization of the pancreatic stone

protein immunoreactivity in the pancreatic

stone was demonstrated by immunostaining

using an immunogold technique and scanning

electron microscopy. Gold particles,

indicating the presence of pancreatic stone

protein immunoreactivity, distributed

diffusely from the center to the periphery of

the pancreatic stones. In our previous study,

trypsinogen immunoreactivity was distributed

more densely in the center of the pancreatic

stone than the periphery of the stone [19].

From the density of the distribution of gold

particles, the involvement of trypsinogen in

the initial step of the stone formation seems to

be more intense than in the subsequent steps

of stone growth. On the other hand,

involvement of pancreatic stone protein seems

to be constant from the initial step to the

subsequent steps of stone growth. In

physiological conditions, pancreatic stone

protein may play an inhibitory role in calcium

carbonate precipitation as one of several

components in pancreatic juice which

normally prevents calcium carbonate

precipitation. In pathological conditions

where local activation of pancreatic zymogen

occurs [16, 17, 18, 25], pancreatic stone

protein plays a lithogenic role by

transforming the soluble forms of pancreatic

stone protein S2-5into the insoluble form of S1rather than playing an inhibitory

role in stone

formation.

We cannot conclude which protein is more

important for the precipitate and stone

formation. The most likely candidate for

promoting precipitation would be the localactivation of pancreatic zymogens.

Active

trypsin would further enhance precipitation by

cleavage of soluble pancreatic stone protein

S2-5to insoluble protein S1. However,pancreatic stones containing only trace

amounts of pancreatic stone protein and

trypsinogen suggest that additional candidate

proteins and mechanisms will be required in

stone formation.Received December 31st, 2001 - AcceptedJanuary 17th, 2002Key

words Calculi; Lactoferrin; Pancreatic

Ducts; Pancreatitis; ProteinsAbbreviations PSP: pancreatic stone

proteinAcknowledgement Supported in part by a

research grant for intractable pancreatic

disease from the Ministry of Health and

Welfare of JapanCorrespondence

Tetsuo Hayakawa

Second Department of Internal Medicine

Nagoya University School of Medicine

Tsuruma-cho 65

Showa-ku

Nagoya

Japan 466-8550

Phone: +81-52-744.2164

Fax: +81-52-744.2173

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