Journal of Clinical Laboratory Analysis 6:375-378 (1992)

Enzyme lmmunoassay for Specific Analysis of Pancreatic Stone Proteins in Human Pancreatic Juice Tomoyuki Katsuzaki,’ Noriyuki Tatemichi,’ Chikako Takeichi,’ Shinobu Hayakawa,’ Tetsuo Hayakawa,2Tokimune Shibata,2 Hideo H a ~ a d aand , ~ Koji Ochi3 ‘Department of Clinical Chemistry, Maruko Pharmaceutical Co., Lfd., Kasugai, Japan; 2Second Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan; Department of Clinical Laboratory Medicine, Okayama University, Medical School, Okayama, Japan In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-SI converted from PSP-S2-5 on PSP determination and the assay method for PSP-SI precipitate after solubilizing PSP-Sl. When bovine trypsin was added to pancreatic juice, PSP-S, was converted from PSP-S2-5and precipitatedabout 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, Key words:

pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S, and PSP-S2-5.The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchangechromatographysuggested that the major component of PSP was PSP5 2 - 5 in pancreatic juice and PSP-S, in duodenal juice. o 1992 Wiley-Liss, Inc.

antibody, pancreatic thread protein, lithostathine, molecular form, duodenal juice, pancreatitis


from Pharmacia LKB Biotechnology, Ltd., Uppsala, Sweden. All other reagents were of special reagent grade.

Pancreatic stone protein (PSP) has been proposed to prevent crystal nucleation or growth of calcium salts in vitro (1). Patients and Collection of Pancreatic Abnormal values of PSP activity might play an important role and Duodenal Juices in the pathophysiology of chronic calcified pancreatitis ( 2 ) . PSP concentration in pancreatic juice and serum has been Pancreaticjuice samples were collected in ice-chilled tubes determined by radial immunodiffusion (3), fluorometric from the main pancreatic duct in 22 patients undergoing endoimmunoassay (4), radioimmunoassay (3,and enzyme im- scopic retrograde pancreatic cannulation (ERP) (7). Control munoassay (EIA) (5,6).Multigner et al. reported that signifi- subjects consisted of 1I patients complaining of vague abdomicantly lower levels of PSP were observed in patients with nal symptoms and subsequently found to be free from panchronic calcifying pancreatitis of different etiologies when creatic or hepatobiliary disease according to the results of serum compared with those in other pancreatic diseases and con- or urinary amylase, pancreozymin secretin test, or pancreatic trols (3). On the other hand, Schmiegel et al. showed no dif- imaging including ERP. Chronic pancreatitis patients were subference between the different pathologic groups (4). This classified into calcified (n = 4) and noncalcified pancreatitis discrepancy is due to the different specificities of antibodies (n = 7) by radiological evidence of pancreatic calcification. used in the assay systems (5). Therefore, we describe an assay Duodenal juice was aspirated from three control patients method solubilizing PSP-S and determined the concentra- during secretin test using Bartelheimer’s double balloon tube tion of PSP in pancreatic juice by the method having similar (8). All samples were stored at - 30°C until analyzed. reactivities toward PSP-S, and PSP-S*-s. In this investigation, molecular forms of PSP were also studied in pancreatic Purificationof PSP juice and duodenal juice. PSP-S, and PSP-S*-5 were purified from pancreatic juice as previously described (6). MATERIALS AND METHODS

Reagents Millex-GV filter (0.22 Fm) was purchased from Japan Millipore, Ltd., Tokyo, Japan. Mono S (HR5/5) was obtained 0 1992 Wiley-Liss, Inc.

Received April 7, 1992; accepted June 12, 1992. Address reprint requests to Dr. S. Hayakawa, Department of Clinical Chemistry, Maruko PharmaceuticalCo., Ltd., 1212 Teramae, Gejo-cho, Kasugai, 486, Japan.


Katsuzaki et al.

DeterminationMethod of PSP in PancreaticJuice In order to solubilize PSP-S1 precipitate in pancreatic juice, samples were first diluted 1: 100 with 0.1 M sodium acetate buffer, pH 4.0, and then further dilution was made to an appropriate concentration with 10 mM sodium phosphate, pH 7.0, containing 0.1 % bovine serum albumin, 0.125% gelatin, and 0.15 M NaCl. The diluted samples were provided to the EIA for PSP, by a procedure which has been described previously (6). The EIA system has similar reactivities toward PSP-SI and PSP-S2-5 (6). The total protein concentrations were determined by the Lowry method (9).

TABLE 1. Analytical Recovery of Standard PSP (150or 600 ng/ml) Added to Five Different Pancreatic Juice Samples, as Measured by the EIA

Sample no. 1

High Performance Liquid Chromatography (HPLC) Technique Human pancreatic juice from a patient with pancreatic cancer and duodenal juice from three control patients without definite pancreatic disease were diluted 1:lO with 20 mM sodium acetate buffer, pH 4.0, containing 20 mM CaC12. An aliquot of 2.0 ml was applied to a Mono S (HR5/5) column equilibrated with the buffer described above. The column was washed with the buffer for 5 min at a flow rate of 1.0 ml/min, and then eluted for 35 min with a linear gradient of 0- 1.O M NaC1.

Influence of Trypsin on PSP Measurement in PancreaticJuice To clarify the influence of transformation of the soluble form of PSP-S2-5 into the insoluble form of PSP-S1 by trypsin, 1 ml of pancreatic juice collected through a catheter kept in the pancreatic duct in a postoperative patient (underwent pancreaticoduodenectomy because of pancreatic head cancer) with pancreatic cancer was incubated with 320 p l of various concentrations (0.3 1- 10 mg/ml) of bovine trypsin (Boehringer Mannheim GmbH., Mannheim, Germany) at 37°C. Immediately after starting the incubation, an aliquot of the mixture was diluted 1:100 with 0.1 M sodium acetate buffer, pH 4.0. Another aliquot of the mixture was filtered with a Millex-GV filter (0.22 pm) to exclude precipitated PSP-Sl and then 50 p1 of the filtrate was diluted 1:lOO with the same buffer. The PSP concentration in the diluted solution with and without filtration was determined by the EIA. After 1 h incubation, the PSP determination was repeated in the same manner.

RESULTS Analytical Evaluationof EIA System for PSP in Pancreatic Juice Assay precision was studied at three concentrations of PSP in pancreatic juice. Within-run coefficients of variation (n = 12) were between 4.0% and 4.9% for pancreatic juice containing PSP at 0.304, 0.239, and 0.31 1 mg/ml. The recov-



(diluted 1: 1,000) (ngiml)

added (ngW



150 600

109.5 100. 1


150 600

110.3 101.4


150 600

89.7 99.0


150 600

106.7 98.5


100.8 aa -I




"Average recovery % ( fSD) = 101.6 2 6.0.

ery of PSP standard added to pancreatic juice samples was 101.6 6.0% (i ? SD) as shown in Table 1. Good linearity ( I = 0.998-0.999) was observed between PSP concentration (0.059-0.34 mg/ml) and sample volume of pancreatic juice (10-50 pl).


Elution Pattern of PSP in Human Pancreatic and Duodenal Juices on HPLC Human pancreatic juice was diluted and subjected to Mono S cation-exchange chromatography. PSP of each fraction was measured by the EIA. A single peak of immunoreactive PSP was eluted in a position corresponding to that of PSP-S2-5 (Fig. 1A). When human pancreatic juice (2.0 ml) was analyzed immediately after addition of trypsin (640 pl, 10 mg/ml), a peak of PSP-S2-5 disappeared and a single peak of immunoreactive PSP was observed at a position consistent with that of PSP-SI (Fig. 1B). After 1 h incubation, a single peak was observed again at the position of PSP-SI (Fig. 1C). In a duodenal juice sample, a single peak of PSP-SI was observed, as in trypsin-added pancreatic juice (Fig. 1D). Similar results were obtained with other two duodenal juice samples.

Effect of Trypsin on PSP in Pancreatic Juice Pancreatic juice was incubated with three different concentrations of trypsin at 37°C. Immediately after the start of the incubation, PSP concentration in the filtered samples was not substantially different from the initial concentration (pancreatic juice A: 0.243 mg/ml; B: 0.185 mg/ml). After 1 h incubation, the amount of immunoreactive PSP in the filtered samples was decreased significantly, especially with higher concentrations of trypsin, though the decrease was slight in the nonfiltered samples, as shown in Table 2. During 1 h incubation of pancreatic juice with trypsin (10 mg/ml), the PSP content decreased to 8 1% (A) and 85% (B) of the initial PSP

PSP in PancreaticJuice


TABLE 2. Effect of 'Ikypsin Addition Into Pancreatic Juices Pancreatic iuice

Trypsin concentration

PSP (mglrnl)




0.0 0.6 2.5 10.0

0.243 0.234 0.183 0.196

0.242 0.238 0.100 0.031

0.0 0.6 2.5 10.0

0.185 0.172 0.166 0. IS8

0.182 0.131 0.118

o 0x6

"Filtration was carried out with Millex-GV filter 1 h after trypsin addition.


protein. The normal range was 8.5 to 33.7 pg/mg protein. Although the PSP values (mean k 2 SD) in chronic calcified pancreatitis (17.2 & 7.2 pg/mg protein) and chronic noncalcified pancreatitis (27.5 k 2 1.O pg/mg protein) were not significantly different, abnormal values were observed in one patient (73.9 pg/mg protein) with chronic noncalcified pancreatitis.

DISCUSSION PSP levels in pancreatic juices from patients with chronic calcified pancreatitis were controversial (3-5). This discrepancy can be partly explained by different specificity of antibodies used in the assay system, or by the presence of PSP-S I in the form of precipitate in pancreatic juice. In a preliminary attempt to obtain precipitated PSP-S] we incubated pan= E creatic juice at 37°C for 5 h, but no precipitate was detected. Therefore, we added bovine trypsin to human pancreatic juice a (I) to enhance conversion of PSP-S2-5 into PSP-SI and could a obtain the precipitated PSP. In the present study, when pancreatic juice was incubated with high concentrations of trypsin for 1 h, the PSP level tended to be underestimated owing Fraction No. to precipitation of PSP-SI. Therefore, PSP-S1 was assayed after dilution with acetate buffer, pH 4.0, because its precipFig. 1. Cation exchange (Mono S) chromatography of human pancreatic itate was solubilized in acidic pH (2). Immediately after the juice and duodenal juice. A: Pancreatic juice (PSP-S2_, content 116 pg). B: start of the incubation of pancreatic juice with high concenPancreatic juice + trypsin (immediately after) (PSP-S1 content 114 pg). C: Pancreatic juice + trypsin (after 1 h) (PSP-S, content 101 pg). D: Duo- trations of trypsin, PSP-S2-s was completely converted to denal juice (PSP-s, content 7 pg). Closed circles and solid line indicate PSP-Sl (Fig. lB), but the immunoreactive PSP level remained amounts of immunoreactive PSP. Broken line indicates OD at 280 nm. Each unchanged from the initial value either before or after filtration fraction was collected at 0.5 ml/tube. of precipitate. These results suggested that the EIA had similar reactivity to PSP-SI and PSP-S2-5, and that PSP-SI content in pancreatic juice A and B without filtration, and remained soluble and was not precipitated in pancreatic juice the PSP content in filtered samples further decreased to 16% at least in a few minutes. (A) and 54% (B) of the PSP content in the corresponding Our proposed method for the determination of PSP in pannonfiltered samples. creatic juice after dissolving PSP precipitate with the acetate buffer excludes the influence of PSP-S precipitation caused PSP Values in Pancreatic Juices From Patients by endogenous trypsin activation, and both PSP-Sl and With and Without Pancreatic Disease PSP-S2-s can be measured to a similar extent. We can conThe PSP values in 11 control subjects were 21.1 6.3 sider that PSP levels in samples with and without the precippg/mgprotein(mean SD),rangingfrom 14.2t034.9 pg/mg itate filtration represent PSP-S2-5 and total PSP (PSP-SI +




Katsuzaki et al.

PSP-S2-5), respectively. The decrease of PSP content was roughly estimated to be from 50% to 80% in the samples after filtration of the precipitate, but the total content of PSP, dissolving the precipitate with 0.1 M acetate buffer, was up to 20% after 1 h incubation with trypsin. Therefore, differential measurement of PSP can be achieved to some extent by comparison between the two PSP values with and without filtration. A single peak of PSP-S, in duodenal juice reflects complete conversion from PSP-S2_5 into PSP-SI in the duodenum by activated trypsin. PSP content in duodenal juice was about one-tenth to one-hundredth of that in pancreatic juice. The low PSP content can be explained by digestion and dilution in the duodenal lumen. The proposed method is recommended for pancreatic juice, not for duodenal juice. PSP values in patients with chronic pancreatitis were not significantly different from normal values in the present study, as reported by Schmiegel et al. (4). More elaborate assays such as differential determination of PSP-SI and PSP-S2-5 using appropriate antibodies to PSP or filtering PSP-SI precipitate as in the present study will be required to elucidate roles of PSP in pancreatitis.

ACKNOWLEDGMENTS This study was supported by a grant of the Intractable Pancreatic Disease Research Committee from the Health and Welfare Ministry of Japan.

REFERENCES 1. Multigner L, De Car0 A, Lombardo D, Campese D, Sarles H: Pancreatic stone protein, a phosphoprotein which inhibits calcium carbonate precipitation from human pancreatic juice. Biochem Biophys Res Commun 110~69-74, 1983. 2. De Car0 A, Multigner L, Dagom JC, Sarles H: The human pancreatic stone protein. Biochirnie 70:1209-1214, 1988. 3. Multigner L, Sarles H, Lombardo D, De Car0 A: Pancreatic stone protein. 11. Implication in stone formation during the course of chronic calcifying pancreatitis. Gastroenterology 89:387-391, 1985. 4. Schmiegel W, Burchert M, Kalthoff H, Roeder C, Biitzow G, Grimm H, Kremer B, Soehendra N, Schreiber HW, Thiele HG, Greten H: Immunochemical characterization and quantitative distribution of pancreatic stone protein in sera and pancreatic secretions in pancreatic disorders. Gastroenterology 99: 1421- 1430, 1990. 5. Provansal-Cheylan M, Mariani A, Bernard JP,Sarles H, Dupuy P Pancreatic stone protein: Quantification in pancreatic juice by enzyme-linked immunosorbent assay and comparison with other methods. Pancreas 4:680-689, 1989. 6 . Katsuzaki T, Tatemichi T, Takeichi C, Hayakawa S , Hawakawa T, Shibata T, Nakae Y, Naruse S: Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum. J Clin Lob Anal 6:143-147, 1992. 7. Hayakawa T, Harada H, Noda A, Kondo T: Lactofenin in pure pancreaticjuice in chronic pancreatitis. Am JGastroenterol78:222-224, 1983. 8. Hayakawa T, Kondo T, Shibata T,Kitagawa M, Sakai Y, Sobajima H, lshiguro H, Nakae Y, Kato K : Exocrine pancreatic function in chronic liver diseases. AmJ Gastroenterol86:201-204, 1991. 9. Lowry OH, Rosebrough NJ, Fan AL, Randall RJ: Protein measurement with Fohn phenol reagent. JBiol Chem 193:265-275, 1951.

Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice.

In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form o...
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