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High plasma pancreastatinlike immunoreactivity in a patient with malignant insulinoma
GASTROENTEROLOGY1989;97:1313-9
CASE
REPORT
High Plasma Pancreastatinlike Immunoreactivity in a Patient With Malignant Insulinoma KAYOKO TATEISHI, AKIHIRO FUNAKOSHI, ATSUO JIMI, SUSUMU FUNAKOSHI, HIROKAZU TAMAMURA, HARUAKI YAJIMA, and YUJI MATSUOKA The First Department of Biochemistry, School of Medicine, Fukuoka University, Fukuoka; National Kyushu Cancer Center, First Department of Pathology, Kurume University, Fukuoka; and Faculty of Pharmaceutical
Sciences, Kyoto University,
High levels of pancreastatinlike immunoreactivity were detected in the plasma (2.9 pmol/ml, >2OOfold the normal level), pancreas (2.9 nmol/g wet wt, >450-fold the normal level), and liver (1.6nmol/g wet wt) of a patient with pancreatic insulinoma with metastasis to the liver by a sensitive and specific radioimmunoassay for human pancreastatin. Antiserum was produced against the C-terminal fragment of human pancreastatin-(N-52), which was synthesized according to the sequence of human chromogranin A corresponding to that of pancreastatin. With the antiserum, intense immunocytochemical staining was detected in the tumors. Sephadex G-50 gel filtration showed that the tumors and plasma contained two molecular forms of pancreastatinlike immunoreactivity-a molecular form coeluted with synthetic human pancreastatin-52 and a larger molecular form (M, - 12,000-15,000). The smaller form eluted in the same position as synthetic human pancreastatin-52 on reverse-phase high-performance liquid chromatography.
Kyoto, Japan
fragment hPST-(24-52) and established a specific and sensitive radioimmunoassay (RIA) using antiserum produced against the synthetic peptide. This paper reports the presence of PST-L1 in both the plasma and tumors of a patient with malignant insulinoma, studies on its molecular forms, and its immunocytologic demonstration in the tumors. Materials
and Methods
Patient A 45-yr-old woman was admitted to the hospital because of a hypoglycemic attack and liver tumor in January 1985. The patient experienced relief after intravenous infusion of glucose. She was 165 cm in height and
weighed 57 kg. Her basal blood sugar level was 52 mg/dl and her basal concentration of immunoreactive insulin and C-peptide immunoreactivitywere 126.1 $J/ml (normal range, Cl0 pU/ml) and 6.1 ng/ml (normal range, <1.3
@ml), respectively. The level of antiinsulin antibody, expressed as specific binding of ‘251-porcine insulin to plasma, was 7.2% (normal range,
P
minal portion (positions 24-52) of hPST proposed by Konecki et al. (5). The sequence of hPST-52 shows 32% difference from that of porcine PST. To detect PST-like immunoreactivity (PST-LI) in human tissues and plasma, we synthesized the C-terminal
Abbreviations used in this paper: PST,pancreaeta~ PST&I, pencreastatinlike immunoreactivity; TFA, trihoroa~ acid. 0 1989 by the American Gastmenterological hsociation 0018~5085/89/$3.50
1314 TATEISHI ET AL.
with liver metastasis. Despite extensive therapy (prednisolone and streptozotocin), the patient died in March 1985. At autopsy, the tumors from the pancreas and liver were excised and stored at -80°C. Histologic examination revealed a nonencapsulated tumor composed of cells compatible with islet cells in both the pancreas and liver. The tumor cells in both the pancreas and liver reacted with antiinsulin antiserum. An atypical B granulelike appearance with and without a halo was seen by electron microscopy.
Peptides Human PST-52 and hPST-(24-52), which correspond to residues 250-301 and 273-301 of human chromogranin A, were synthesized by the Fmoc-based solidphase technique (Fmoc = fluoren-%ylmethoxycarbonyl) (7). N*-Tyrosyl-hPST-(24-52) was synthesized similarly. These synthetic peptides were purified to homogeneity by analytical high-performance liquid chromatography on a Cosmosil 5PhT-300 column as described (7), and their purities were determined by amino acid analysis. Synthetic porcine PST and its fragments, bovine PST (32-47), and other peptides were purchased from Peninsula Laboratories (Belmont, Calif.).
GASTROENTEROLOGYVol. 97. No. 5
Standard (hPST-52) or a sample was incubated with antiserum (final dilution, 1:330,000) for 48 h at 4°C in a total volume of 500 ~1 of 0.01 M phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin, 0.025 M ethylenediaminetetraacetic acid, 0.14 M NaCl, 0.05% (vol/vol) Tween 20, and 0.01% sodium azide. Then, 0.1 ml of tracer (-5000 cpm) was added and incubation was continued for 48 h at 4°C. The bound and unbound peptides were separated by adding goat-antirabbit immunoglobulin G. Standards of synthetic hPST-52 were prepared according to the net peptide content determined by amino acid analysis.
Tissue
Normal human tissues were excised at autopsy within 5 h postmortem from patients with no diabetes mellitus or pancreatic diseases, and were promptly frozen and stored at -80°C. Frozen tissue specimens were weighed and boiled in 1 M acetic acid (5 ml/g tissue) for 5 min. They were then cooled, homogenized, and centrifuged for 30 min at 10,000 rpm, and the supernatants were collected.
Plasma Antiserum Synthetic hPST-(24-52) was conjugated with keyhole limpet hemocyanin using m-maleimidobenzoyl-Nhydroxysuccinimide ester according to the described procedure (8). The ratio of hPST-(24-52) to keyhole limpet hemocyanin in the conjugate was -6:l. Rabbits were immunized with the conjugate (100 pg as peptide) emulsified in complete Freund’s adjuvant. Booster injections of the same dose of conjugate in incomplete Freund’s adjuvant were administered at 2-wk intervals from 3 wk after the first immunization. The antiserum used for this study was obtained 10 days after the eighth immunization.
Immunocytochemistry Tissues were fixed in 10% formalin and embedded in paraffin. Sections of 5 pm were incubated with antiserum to PST at dilutions of 1:lOOO to 1:5000, and stained by the avidin-biotin peroxidase complex method. Control sections were treated with specific antiserum preabsorbed with hPST-52 (10 pg/rnl final dilution of antiserum). For electron microscopy, tissues were fixed in formalin, treated with a mixture of 4% formaldehyde and 1% glutaraldehyde, postfixed in 1% osmium tetroxide, embedded in Epon, and stained with 1% uranyl acetate.
Radioimmunoassay ‘251-labeled Tyr-hPST-(24-52) was prepared by the chloramine-T method, and purified by Sephadex G-10 chromatography and diethylaminoethyl-ion exchange chromatography (eluted with a gradient of O-l M NaCl in 0.01 M imidazole buffer, pH 7.5).
Extraction
Extraction
Plasma (1 ml) was mixed with an equal volume of acid solution [formic acid/trifluoroacetic acid (TFA)/water = 2:2:96] and passed through a Sep-Pak C,, cartridge at a flow rate of 0.5 mUmin. Adsorbed PST-L1 was eluted with 2.5 ml of a mixture of methanol, TFA, and water (80:1:19), dried under nitrogen gas, and reconstituted in 1.0 ml of assay buffer for RIA. Plasma for gel filtration was extracted similarly.
Gel Filtration Tumor extracts, extracts of plasma reconstituted with 0.5 ml of 1 M acetic acid, and plasma (0.5 ml) were applied to a Sephadex G-50 (fine) column (1.2 x 65.5 cm), equilibrated, and eluted with 1 M acetic acid containing 0.05% gelatin at a flow rate of 5 ml/h. Fractions of 2 ml were collected, lyophilized, and reconstituted in 0.5 ml assay buffer for RIA. The column was calibrated with blue dextran, cytochrome C (M, 12,384), synthetic hPST-52, hPST-(2P52), and lz51Na.
Reverse-Phase High-Performance Liquid Chromatography Fractions of plasma in the second peak from the Sephadex G-50 column were pooled, lyophilized, dissolved in 0.1% TFA/lS% acetonitrilejwater and applied to a reverse-phase high-performance liquid chromatography column (CLBondapak C18; Waters Co., Ltd., 3.9 mm x 30 cm) equilibrated with the same solvent. The column was eluted at 1.0 mllmin with a gradient of 15%-45% acetonitrile (in 0.1% TFA) over 40 min and 0.5-ml fractions were
PLASMA PANCREASTATINLIKE IMMUNOREACTIVITY 1315
November1989
Figure 1. Immunocytochemicallocalization of PST-LI in pancreatictumor. (Magnification,X100.)
collected. Each fraction was lyophilized and reconstituted in 0.5 ml of assay buffer for RIA.
Results Immunocytochemical
Staining
of Tumors
Immunocytochemical staining of PST was strongly positive in the cytoplasm of tumor ceils in the pancreas (Figure 1) and liver (Figure 2). Radioimmunoassay The antiserum (R711) used in this study reacted 100% with hPST-52 and 84% with hPST(24-52), and cross-reacted with porcine PST-49 (3.8%) and porcine PST-(33-49) (4.9%), but not with
Figure 2. Immunocytochemicallocalization of PST-LI in liver metastasis. (Magnification,X100.)
bovine PST-(32-47), [Arg’]-vasopressin, or human gastrin I (
of
As shown in Table 1, the PST-L1 levels in extracts of the pancreatic tumor and liver metastas’s were 250-460 times that in normal pancreas. d e PST-L1 level in peripheral plasma of the patient was 2.9 pmol/ml, which was 220 times the mean fasting level in normal subjects.
1316
TATEISHI ET AL.
GASTROENTEROLOGY Vol. 97, No. 5
PST29 CYt Vo PST52 Vt I,!,’ I
0.4h a)
0.1, 10
, , , ““.,
0.30
. , ,,
loo fmol/ml PST-LX
,““1
1000
Figure 3. Standard curve of synthetic hPST-52 (o-o] and plots for serial dilutions of extracts of human pancreas (o----o), the insulinoma (A--A),liver metastasis (A---A), and plasma of the patient with insulinoma (O----O).
Molecular Characterization of Pancreastatinlike Immunoreactivity in Tumors The chromatographic profiles of extracts of the pancreatic tumor (Figure 4A) and liver metastasis (Figure 4B) on Sephadex G-50 were similar and showed two main peaks of PSI-LI. The peak eluted in the high molecular weight region (apparent molecular weight, -lZ,OOO-15,000) -40% of constituted the total immunoreactivity, and the other peak coeluted with synthetic PST-52 constituted -60% of the total immunoreactivity. Molecular Characterization of Pancreastatinlike Immunoreactivity in Plasma
L-_A!L
0.2
b)
0.1 0.30 U!h-
10
20 30 FRACTIONS
40
Figure 4. Elution profiles of PST-L1 in extracts of the insulinoma (a) and its liver metastasis (b) on a Sephadex G-50 fine column (1.2 X 85.5 cm) equilibrated with 1 M acetic acid containing 0.05% gelatin. Arrows indicate the elution volumes of markers: blue dextran (Vo); cytochrome C (Cyt; M, 12,384); synthetic hPST-52 (PST52); synthetic MST-(24-52) (PST29), and ‘*‘INa WI.
position as synthetic hPST-52 on reverse-phase high-performance liquid chromatography, as shown in Figure 6.
Discussion With the development of RIA and immunocytochemical techniques for various peptides, in-
Two forms of PST-L1 were also identified in plasma (Figure 5A) and an extract of plasma (Figure 5B), and corresponded to the same two peaks found in the tumors. Their relative amounts in total plasma (88% and 10% of the total PST-LI, respectively) and the plasma extract (79% and 20% of the total PST-LI, respectively) were, however, different from those in the tumors, the larger molecular weight form being more abundant in plasma. yaterial in the second peak (fractions 21-24) on gel chromatography (Figure 5A) eluted in the same
PST29 CYt Vo PST52 4141
vt I
LL- b)
Table
1. Pancreastatinlike Immunoreactivity in Tissue and Plasma of a Patient With Malignant lnsulinoma and Normal Subiects Malignant insulinoma
Tissue Pi3llcreaS Liver metastasis Plasma
2.9 nmol/g
wet wt 1.6 nmol/g wet wt 2.9 pmol/ml
Normal subjects (mean f SEM) 6.3 + 1.1 pmol/g wet wt (n = 7)
12.9 2 0.8 fmol/ml (n = 9)
0 10
FRA%ONS
30
40
Figure 5. Elution profiles of PST-L1 in plasma [(a) unextracted, (b] extracted with Sep-Pak C1s] of the patient with malignant insulinoma. Column conditions and markers are as described in Figure 4.
November
PLASMA PANCREASTATINLIKE IMMUNOREACTMTY
1989
hPST52 0.50.
1
-z 5 Y e
45
9
iow2,
35 i
3
25 ;
iii p.
15 * 0 20
Figure
60 40 FRACTIONS
80
6. Reverse-phase HPLC profile of the fraction coeluted with synthetic hPST-52 on Sephadex G-50 gel chromatography (Figure 5A). Trifluoroacetic acid-acetonitrile solvent system; column, flondapak C,, (3.9 mm x 30 cm]; flow rate 1 mbmin. The arrow indicates the elution position of synthetic hPST-52.
plasma and tumor extracts suggests that the two molecular forms identified in the tumor were secreted. The relative proportion of the molecular form corresponding to PST-52 in the plasma was, however, less than that in the tumor extracts. This difference may be due to faster degradation of PST52 than of the large molecular form in the plasma. Pancreastatin has been found to inhibit glucoseinduced insulin release both in vitro (1,14,15) and in vivo (16,17). The fact that the insulinoma contained a high level of PST-L1 is of interest because of the inhibitory effect of PST on insulin secretion. Further investigations are needed to clarify the biologic role of this new peptide.
References Tatemoto K, Efendic S, Mutt V, Makk G, Feistner GJ, Barchas JD. Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 1986;324:476-8. 2. Eiden LE. Is chromogranin a prohormone? Nature 1987; 325:301. 3. Huttner WB, Benedum UM. Chromogranin A and pancreastatin. Nature 1987;325:305. R, Koller KJ, Brownstein MJ, 4. Iacangelo AL, Fischer-Colbrie Eiden LE. The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin. Endocrinology 1988;122:2339-41. 5. Konecki DS, Benedum UM, Gerdes I-III, Huttner WB. The primary structure of human chromogranin A and pancreastatin. J Biol Chem 1987;282:17026-30. 6. Sekiya K, Ghatei MA, Minamino N, Bretherton-Watt D, Matsuo H, Bloom SR. Isolation of human pancreastatin fragment containing the active sequence from a glucagonoma. FEBS Lett 1988;228:153-6. 7. Funakoshi S, Tamamura H, Fujii N, et al. Combination of a new amide-precursor reagent and trimethylsilyl bromide deprotection for the Fmoc-based solid phase synthesis of human pancreastatin and one of its fragments. J Chem Sot Chem Commun 1988;1588-90. 8. Tateishi K, Hamaoka T, Sugiura N, Yanaihara C, Yanaihara N. A novel immunization procedure for production of anticholecystokinin-specific antiserum of low cross-reactivity, J Immunol Methods 1981;47:249-58. 9. Heitz PU, Kasper M, Polak JM, Kl6ppel G. Pancreatic endocrine tumors. Immunohistochemical analysis of 125 tumors. Hum Path01 1982;13:263-71. 10. Kloppel G, Heitz PU. Pancreatic endocrine tumors. Path01 Res Pratt 1988;183:155-68. 11. Tanaka Y, Nagamatsu Y, Morita Y, et al. A case of multihormone producing pancreatic islet-cell carcinoma (malignant insulinoma) with recurrent severe hypoglycemic attacks, J Biliary Tract Pancreas (Jpn) 1987;8:211-9. 12. O’Connor DT, Deftos LJ. Secretion of chromogranin A by peptide-producing endocrine neoplasms. N Engl J Med 1986; 314:1145-51. 13. Lloyd RV, Mervak T, Schmidt K, Warner TFCS, Wilson BS. Immunohistochemical detection of chromogranin and neuron-specific enolase in pancreatic endocrine neoplasms. Am J Surg Path01 1984;8:607-14. 14. Efendic S, Tatemoto K, Mutt V, @an C, Chang D, &tenson 1.
creasing numbers of endocrine tumors producing more than one peptide have been recognized ($10). The present case was recognized clinically to be insulinoma, which produces high levels of insulin and C-peptide and causes hypoglycemic attacks as we reported (11).Insulinoma has been considered to be a B-cell adenoma, but about half these tumors are mixed islet-cell tumors, as shown immunohistochemically (9). The present tumor produced PST-LI, as shown by a specific RIA using hPST-52 and hPST-(24-52), which were synthesized (7) according to the sequence deduced from the sequence of human chromogranin A (5). On immunocytochemical staining, intense immunoreactivity for PST was found in both the pancreatic tumor and the metastasis in the liver. This is the first report of detection of PST-L1 in the plasma. Significantly high levels of chromogranin A have been detected in plasma of patients with islet cell tumors and other neuroendocrine tumors (121, and chromogranin A has been proposed as an immunohistochemical marker for human tumors of endocrine origin (13). Therefore, the high PST-L1 in the plasma and the tumor tissues of the present patient is conceivable from the hypothesis that PST and its related peptides may be derived from chromogranin A (2-5). Pancreastatinlike immunoreactivity coeluted with synthetic hPST-52 on chromatography was detected in both the tumor extract and the plasma of the patient. This suggests the existence of a molecular form of PST-52 corresponding to porcine PST-49 in normal human plasma as well as the pancreas. The large molecular form of PST-L1 identified by gel chromatographic analysis may be an intermediate form derived from chromogranin A. Our finding of similar chromatographic profiles of
1317
1318 TATEISIB ET AL.
CG. Pancreastatin and islet hormone release. Proc Nat1 Acad Sci USA 1987;84:7257-60. 15. Funakoshi A, Jimi A, Yasunami Y, et al. Bioactivity of human pancreastatin and its localization in pancreas. Biochem Biophys Res Commun 1989;159:913-8. 16. Ahren B, Lindskog S, Tatemoto K, Efendic S. Pancreastatin inhibits insulin secretion and stimulates glucagon secretion in mice. Diabetes 1988;37:281-5. 17. Funakoshi A, Miyasaka K, Kitani K, Tatemoto K. Effect of
GASTROENTEROLOGY Vol. 97, No. 5
pancreastatin on pancreatic endocrine function scious rat. Regul Pept 1989;24:225-31.
in the con-
Received December 27,1988.Accepted April 14,1989. Address requests for reprints to: Dr. K. Tateishi, First Department of Biochemistry, School of Medicine, Fukuoka University 45-1, 7 chome, Nanakuma, Jonan-ku, Fukuoka 814-01, Japan. The authors thank Naoko Iwamoto for technical assistance and Keiko Fukushima for assistance in preparation of this manuscript.
CASE
REPORT
High Plasma Pancreastatinlike Immunoreactivity in a Patient With Malignant Insulinoma KAYOKO TATEISHI, AKIHIRO FUNAKOSHI, ATSUO JIMI, SUSUMU FUNAKOSHI, HIROKAZU TAMAMURA, HARUAKI YAJIMA, and YUJI MATSUOKA The First Department of Biochemistry, School of Medicine, Fukuoka University, Fukuoka; National Kyushu Cancer Center, First Department of Pathology, Kurume University, Fukuoka; and Faculty of Pharmaceutical
Sciences, Kyoto University,
High levels of pancreastatinlike immunoreactivity were detected in the plasma (2.9 pmol/ml, >2OOfold the normal level), pancreas (2.9 nmol/g wet wt, >450-fold the normal level), and liver (1.6nmol/g wet wt) of a patient with pancreatic insulinoma with metastasis to the liver by a sensitive and specific radioimmunoassay for human pancreastatin. Antiserum was produced against the C-terminal fragment of human pancreastatin-(N-52), which was synthesized according to the sequence of human chromogranin A corresponding to that of pancreastatin. With the antiserum, intense immunocytochemical staining was detected in the tumors. Sephadex G-50 gel filtration showed that the tumors and plasma contained two molecular forms of pancreastatinlike immunoreactivity-a molecular form coeluted with synthetic human pancreastatin-52 and a larger molecular form (M, - 12,000-15,000). The smaller form eluted in the same position as synthetic human pancreastatin-52 on reverse-phase high-performance liquid chromatography.
Kyoto, Japan
fragment hPST-(24-52) and established a specific and sensitive radioimmunoassay (RIA) using antiserum produced against the synthetic peptide. This paper reports the presence of PST-L1 in both the plasma and tumors of a patient with malignant insulinoma, studies on its molecular forms, and its immunocytologic demonstration in the tumors. Materials
and Methods
Patient A 45-yr-old woman was admitted to the hospital because of a hypoglycemic attack and liver tumor in January 1985. The patient experienced relief after intravenous infusion of glucose. She was 165 cm in height and
weighed 57 kg. Her basal blood sugar level was 52 mg/dl and her basal concentration of immunoreactive insulin and C-peptide immunoreactivitywere 126.1 $J/ml (normal range, Cl0 pU/ml) and 6.1 ng/ml (normal range, <1.3
@ml), respectively. The level of antiinsulin antibody, expressed as specific binding of ‘251-porcine insulin to plasma, was 7.2% (normal range,
P
minal portion (positions 24-52) of hPST proposed by Konecki et al. (5). The sequence of hPST-52 shows 32% difference from that of porcine PST. To detect PST-like immunoreactivity (PST-LI) in human tissues and plasma, we synthesized the C-terminal
Abbreviations used in this paper: PST,pancreaeta~ PST&I, pencreastatinlike immunoreactivity; TFA, trihoroa~ acid. 0 1989 by the American Gastmenterological hsociation 0018~5085/89/$3.50
1314 TATEISHI ET AL.
with liver metastasis. Despite extensive therapy (prednisolone and streptozotocin), the patient died in March 1985. At autopsy, the tumors from the pancreas and liver were excised and stored at -80°C. Histologic examination revealed a nonencapsulated tumor composed of cells compatible with islet cells in both the pancreas and liver. The tumor cells in both the pancreas and liver reacted with antiinsulin antiserum. An atypical B granulelike appearance with and without a halo was seen by electron microscopy.
Peptides Human PST-52 and hPST-(24-52), which correspond to residues 250-301 and 273-301 of human chromogranin A, were synthesized by the Fmoc-based solidphase technique (Fmoc = fluoren-%ylmethoxycarbonyl) (7). N*-Tyrosyl-hPST-(24-52) was synthesized similarly. These synthetic peptides were purified to homogeneity by analytical high-performance liquid chromatography on a Cosmosil 5PhT-300 column as described (7), and their purities were determined by amino acid analysis. Synthetic porcine PST and its fragments, bovine PST (32-47), and other peptides were purchased from Peninsula Laboratories (Belmont, Calif.).
GASTROENTEROLOGYVol. 97. No. 5
Standard (hPST-52) or a sample was incubated with antiserum (final dilution, 1:330,000) for 48 h at 4°C in a total volume of 500 ~1 of 0.01 M phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin, 0.025 M ethylenediaminetetraacetic acid, 0.14 M NaCl, 0.05% (vol/vol) Tween 20, and 0.01% sodium azide. Then, 0.1 ml of tracer (-5000 cpm) was added and incubation was continued for 48 h at 4°C. The bound and unbound peptides were separated by adding goat-antirabbit immunoglobulin G. Standards of synthetic hPST-52 were prepared according to the net peptide content determined by amino acid analysis.
Tissue
Normal human tissues were excised at autopsy within 5 h postmortem from patients with no diabetes mellitus or pancreatic diseases, and were promptly frozen and stored at -80°C. Frozen tissue specimens were weighed and boiled in 1 M acetic acid (5 ml/g tissue) for 5 min. They were then cooled, homogenized, and centrifuged for 30 min at 10,000 rpm, and the supernatants were collected.
Plasma Antiserum Synthetic hPST-(24-52) was conjugated with keyhole limpet hemocyanin using m-maleimidobenzoyl-Nhydroxysuccinimide ester according to the described procedure (8). The ratio of hPST-(24-52) to keyhole limpet hemocyanin in the conjugate was -6:l. Rabbits were immunized with the conjugate (100 pg as peptide) emulsified in complete Freund’s adjuvant. Booster injections of the same dose of conjugate in incomplete Freund’s adjuvant were administered at 2-wk intervals from 3 wk after the first immunization. The antiserum used for this study was obtained 10 days after the eighth immunization.
Immunocytochemistry Tissues were fixed in 10% formalin and embedded in paraffin. Sections of 5 pm were incubated with antiserum to PST at dilutions of 1:lOOO to 1:5000, and stained by the avidin-biotin peroxidase complex method. Control sections were treated with specific antiserum preabsorbed with hPST-52 (10 pg/rnl final dilution of antiserum). For electron microscopy, tissues were fixed in formalin, treated with a mixture of 4% formaldehyde and 1% glutaraldehyde, postfixed in 1% osmium tetroxide, embedded in Epon, and stained with 1% uranyl acetate.
Radioimmunoassay ‘251-labeled Tyr-hPST-(24-52) was prepared by the chloramine-T method, and purified by Sephadex G-10 chromatography and diethylaminoethyl-ion exchange chromatography (eluted with a gradient of O-l M NaCl in 0.01 M imidazole buffer, pH 7.5).
Extraction
Extraction
Plasma (1 ml) was mixed with an equal volume of acid solution [formic acid/trifluoroacetic acid (TFA)/water = 2:2:96] and passed through a Sep-Pak C,, cartridge at a flow rate of 0.5 mUmin. Adsorbed PST-L1 was eluted with 2.5 ml of a mixture of methanol, TFA, and water (80:1:19), dried under nitrogen gas, and reconstituted in 1.0 ml of assay buffer for RIA. Plasma for gel filtration was extracted similarly.
Gel Filtration Tumor extracts, extracts of plasma reconstituted with 0.5 ml of 1 M acetic acid, and plasma (0.5 ml) were applied to a Sephadex G-50 (fine) column (1.2 x 65.5 cm), equilibrated, and eluted with 1 M acetic acid containing 0.05% gelatin at a flow rate of 5 ml/h. Fractions of 2 ml were collected, lyophilized, and reconstituted in 0.5 ml assay buffer for RIA. The column was calibrated with blue dextran, cytochrome C (M, 12,384), synthetic hPST-52, hPST-(2P52), and lz51Na.
Reverse-Phase High-Performance Liquid Chromatography Fractions of plasma in the second peak from the Sephadex G-50 column were pooled, lyophilized, dissolved in 0.1% TFA/lS% acetonitrilejwater and applied to a reverse-phase high-performance liquid chromatography column (CLBondapak C18; Waters Co., Ltd., 3.9 mm x 30 cm) equilibrated with the same solvent. The column was eluted at 1.0 mllmin with a gradient of 15%-45% acetonitrile (in 0.1% TFA) over 40 min and 0.5-ml fractions were
PLASMA PANCREASTATINLIKE IMMUNOREACTIVITY 1315
November1989
Figure 1. Immunocytochemicallocalization of PST-LI in pancreatictumor. (Magnification,X100.)
collected. Each fraction was lyophilized and reconstituted in 0.5 ml of assay buffer for RIA.
Results Immunocytochemical
Staining
of Tumors
Immunocytochemical staining of PST was strongly positive in the cytoplasm of tumor ceils in the pancreas (Figure 1) and liver (Figure 2). Radioimmunoassay The antiserum (R711) used in this study reacted 100% with hPST-52 and 84% with hPST(24-52), and cross-reacted with porcine PST-49 (3.8%) and porcine PST-(33-49) (4.9%), but not with
Figure 2. Immunocytochemicallocalization of PST-LI in liver metastasis. (Magnification,X100.)
bovine PST-(32-47), [Arg’]-vasopressin, or human gastrin I (
of
As shown in Table 1, the PST-L1 levels in extracts of the pancreatic tumor and liver metastas’s were 250-460 times that in normal pancreas. d e PST-L1 level in peripheral plasma of the patient was 2.9 pmol/ml, which was 220 times the mean fasting level in normal subjects.
1316
TATEISHI ET AL.
GASTROENTEROLOGY Vol. 97, No. 5
PST29 CYt Vo PST52 Vt I,!,’ I
0.4h a)
0.1, 10
, , , ““.,
0.30
. , ,,
loo fmol/ml PST-LX
,““1
1000
Figure 3. Standard curve of synthetic hPST-52 (o-o] and plots for serial dilutions of extracts of human pancreas (o----o), the insulinoma (A--A),liver metastasis (A---A), and plasma of the patient with insulinoma (O----O).
Molecular Characterization of Pancreastatinlike Immunoreactivity in Tumors The chromatographic profiles of extracts of the pancreatic tumor (Figure 4A) and liver metastasis (Figure 4B) on Sephadex G-50 were similar and showed two main peaks of PSI-LI. The peak eluted in the high molecular weight region (apparent molecular weight, -lZ,OOO-15,000) -40% of constituted the total immunoreactivity, and the other peak coeluted with synthetic PST-52 constituted -60% of the total immunoreactivity. Molecular Characterization of Pancreastatinlike Immunoreactivity in Plasma
L-_A!L
0.2
b)
0.1 0.30 U!h-
10
20 30 FRACTIONS
40
Figure 4. Elution profiles of PST-L1 in extracts of the insulinoma (a) and its liver metastasis (b) on a Sephadex G-50 fine column (1.2 X 85.5 cm) equilibrated with 1 M acetic acid containing 0.05% gelatin. Arrows indicate the elution volumes of markers: blue dextran (Vo); cytochrome C (Cyt; M, 12,384); synthetic hPST-52 (PST52); synthetic MST-(24-52) (PST29), and ‘*‘INa WI.
position as synthetic hPST-52 on reverse-phase high-performance liquid chromatography, as shown in Figure 6.
Discussion With the development of RIA and immunocytochemical techniques for various peptides, in-
Two forms of PST-L1 were also identified in plasma (Figure 5A) and an extract of plasma (Figure 5B), and corresponded to the same two peaks found in the tumors. Their relative amounts in total plasma (88% and 10% of the total PST-LI, respectively) and the plasma extract (79% and 20% of the total PST-LI, respectively) were, however, different from those in the tumors, the larger molecular weight form being more abundant in plasma. yaterial in the second peak (fractions 21-24) on gel chromatography (Figure 5A) eluted in the same
PST29 CYt Vo PST52 4141
vt I
LL- b)
Table
1. Pancreastatinlike Immunoreactivity in Tissue and Plasma of a Patient With Malignant lnsulinoma and Normal Subiects Malignant insulinoma
Tissue Pi3llcreaS Liver metastasis Plasma
2.9 nmol/g
wet wt 1.6 nmol/g wet wt 2.9 pmol/ml
Normal subjects (mean f SEM) 6.3 + 1.1 pmol/g wet wt (n = 7)
12.9 2 0.8 fmol/ml (n = 9)
0 10
FRA%ONS
30
40
Figure 5. Elution profiles of PST-L1 in plasma [(a) unextracted, (b] extracted with Sep-Pak C1s] of the patient with malignant insulinoma. Column conditions and markers are as described in Figure 4.
November
PLASMA PANCREASTATINLIKE IMMUNOREACTMTY
1989
hPST52 0.50.
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Figure
60 40 FRACTIONS
80
6. Reverse-phase HPLC profile of the fraction coeluted with synthetic hPST-52 on Sephadex G-50 gel chromatography (Figure 5A). Trifluoroacetic acid-acetonitrile solvent system; column, flondapak C,, (3.9 mm x 30 cm]; flow rate 1 mbmin. The arrow indicates the elution position of synthetic hPST-52.
plasma and tumor extracts suggests that the two molecular forms identified in the tumor were secreted. The relative proportion of the molecular form corresponding to PST-52 in the plasma was, however, less than that in the tumor extracts. This difference may be due to faster degradation of PST52 than of the large molecular form in the plasma. Pancreastatin has been found to inhibit glucoseinduced insulin release both in vitro (1,14,15) and in vivo (16,17). The fact that the insulinoma contained a high level of PST-L1 is of interest because of the inhibitory effect of PST on insulin secretion. Further investigations are needed to clarify the biologic role of this new peptide.
References Tatemoto K, Efendic S, Mutt V, Makk G, Feistner GJ, Barchas JD. Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 1986;324:476-8. 2. Eiden LE. Is chromogranin a prohormone? Nature 1987; 325:301. 3. Huttner WB, Benedum UM. Chromogranin A and pancreastatin. Nature 1987;325:305. R, Koller KJ, Brownstein MJ, 4. Iacangelo AL, Fischer-Colbrie Eiden LE. The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin. Endocrinology 1988;122:2339-41. 5. Konecki DS, Benedum UM, Gerdes I-III, Huttner WB. The primary structure of human chromogranin A and pancreastatin. J Biol Chem 1987;282:17026-30. 6. Sekiya K, Ghatei MA, Minamino N, Bretherton-Watt D, Matsuo H, Bloom SR. Isolation of human pancreastatin fragment containing the active sequence from a glucagonoma. FEBS Lett 1988;228:153-6. 7. Funakoshi S, Tamamura H, Fujii N, et al. Combination of a new amide-precursor reagent and trimethylsilyl bromide deprotection for the Fmoc-based solid phase synthesis of human pancreastatin and one of its fragments. J Chem Sot Chem Commun 1988;1588-90. 8. Tateishi K, Hamaoka T, Sugiura N, Yanaihara C, Yanaihara N. A novel immunization procedure for production of anticholecystokinin-specific antiserum of low cross-reactivity, J Immunol Methods 1981;47:249-58. 9. Heitz PU, Kasper M, Polak JM, Kl6ppel G. Pancreatic endocrine tumors. Immunohistochemical analysis of 125 tumors. Hum Path01 1982;13:263-71. 10. Kloppel G, Heitz PU. Pancreatic endocrine tumors. Path01 Res Pratt 1988;183:155-68. 11. Tanaka Y, Nagamatsu Y, Morita Y, et al. A case of multihormone producing pancreatic islet-cell carcinoma (malignant insulinoma) with recurrent severe hypoglycemic attacks, J Biliary Tract Pancreas (Jpn) 1987;8:211-9. 12. O’Connor DT, Deftos LJ. Secretion of chromogranin A by peptide-producing endocrine neoplasms. N Engl J Med 1986; 314:1145-51. 13. Lloyd RV, Mervak T, Schmidt K, Warner TFCS, Wilson BS. Immunohistochemical detection of chromogranin and neuron-specific enolase in pancreatic endocrine neoplasms. Am J Surg Path01 1984;8:607-14. 14. Efendic S, Tatemoto K, Mutt V, @an C, Chang D, &tenson 1.
creasing numbers of endocrine tumors producing more than one peptide have been recognized ($10). The present case was recognized clinically to be insulinoma, which produces high levels of insulin and C-peptide and causes hypoglycemic attacks as we reported (11).Insulinoma has been considered to be a B-cell adenoma, but about half these tumors are mixed islet-cell tumors, as shown immunohistochemically (9). The present tumor produced PST-LI, as shown by a specific RIA using hPST-52 and hPST-(24-52), which were synthesized (7) according to the sequence deduced from the sequence of human chromogranin A (5). On immunocytochemical staining, intense immunoreactivity for PST was found in both the pancreatic tumor and the metastasis in the liver. This is the first report of detection of PST-L1 in the plasma. Significantly high levels of chromogranin A have been detected in plasma of patients with islet cell tumors and other neuroendocrine tumors (121, and chromogranin A has been proposed as an immunohistochemical marker for human tumors of endocrine origin (13). Therefore, the high PST-L1 in the plasma and the tumor tissues of the present patient is conceivable from the hypothesis that PST and its related peptides may be derived from chromogranin A (2-5). Pancreastatinlike immunoreactivity coeluted with synthetic hPST-52 on chromatography was detected in both the tumor extract and the plasma of the patient. This suggests the existence of a molecular form of PST-52 corresponding to porcine PST-49 in normal human plasma as well as the pancreas. The large molecular form of PST-L1 identified by gel chromatographic analysis may be an intermediate form derived from chromogranin A. Our finding of similar chromatographic profiles of
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1318 TATEISIB ET AL.
CG. Pancreastatin and islet hormone release. Proc Nat1 Acad Sci USA 1987;84:7257-60. 15. Funakoshi A, Jimi A, Yasunami Y, et al. Bioactivity of human pancreastatin and its localization in pancreas. Biochem Biophys Res Commun 1989;159:913-8. 16. Ahren B, Lindskog S, Tatemoto K, Efendic S. Pancreastatin inhibits insulin secretion and stimulates glucagon secretion in mice. Diabetes 1988;37:281-5. 17. Funakoshi A, Miyasaka K, Kitani K, Tatemoto K. Effect of
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pancreastatin on pancreatic endocrine function scious rat. Regul Pept 1989;24:225-31.
in the con-
Received December 27,1988.Accepted April 14,1989. Address requests for reprints to: Dr. K. Tateishi, First Department of Biochemistry, School of Medicine, Fukuoka University 45-1, 7 chome, Nanakuma, Jonan-ku, Fukuoka 814-01, Japan. The authors thank Naoko Iwamoto for technical assistance and Keiko Fukushima for assistance in preparation of this manuscript.