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Progastrin in serum from Zollinger-Ellison patients
GASTROENTEROLOGY
1990;93:142o-1426
Progastrin in Serum From Zollinger-Ellison Patients An Indicator of Malignancy? LINDA BARDRAM Department
of Surgical Gastroenterology
C, Rigshospitalet,
Progastrin and all of its processing products were measured in serum from 48 patients with ZollingerEllison syndrome, 42 patients with duodenal ulcers, and 34 normal subjects. A processing-independent gastrin analysis and a conventional radioimmunoassay for the biologically active a-amidated gastrins were used. In serum from normal subjects, 87% of all progastrin prod(median; range, 27%-160%) ucts were a-amidated gastrins, whereas they constituted only 39% (15%-130%) in serum from patients with duodenal ulcers (p < 0.01) and 46% (16%-100%) in serum from gastrinoma patients (p < 0.01). A significantly lower percentage of cu?amidated gastrin was found in patients with hepatic metastases (23 %) than in patients with apparently benign tumors (54%). Chromatography of serum showed that large progastrin molecules occurred mainly in patients with malignant tumors, whereas smaller glycine-extended precursors dominated in patients with benign tumors. The results indicate that the total progastrin product reflects tumor synthesis of gastrin better than conventional measurements of cy-amidated gastrin. Moreover, the results suggest that a low degree of processing of progastrin could serve as a predictor of a malignant clinical course at an early stage of the disease.
T
umor production of gastrin is responsible for the severe hypersecretion of acid that causes Zollinger-Ellison syndrome (1). Biologically active gastrins are products of several modifications of human progastrin (2,3) (Figure 1). These include cleavages at dibasic amino acids, partial sulfation of tyrosine 87 (4-6),phosphorylation of serine 99 (7,8),and the cr-carboxyamidation of phenylalanine 92, which is necessary for biological activity (9,lO). Thus, in addition to the bioactive a-amidated gastrins (gastrin 17, gastrin 34, and component I), inactive precursors with a COOH-terminal extension of glycine (ll,l~), as well
University of Copenhagen,
Denmark
as larger progastrins with further NH,- and COOHterminal extensions, exist [Figure 1). The degree of posttranslational processing of the gastrin precursor varies considerably in normal and of neoplastic cells (13-16). To study the synthesis progastrin and the processing products in different tissues, a radioimmunoanalysis by which all gastrin forms are measured independently of the degree of processing has been developed (17). Using this method, high concentrations of glycine-extended gastrins, as well as larger progastrins, were recently found in gastrinoma tissue (17a). The results of that study also indicated that inactive precursors were released to the blood. The occurrence of gastrin precursors as well as a-amidated gastrins in sera from a large group of Zollinger-Ellison patients, from patients with duodenal ulcers, and from normal subjects without gastrointestinal disease is examined in this study.
Materials and Methods Subjects Sera were obtained after an overnight fast from the following subjects. ZoJJinger-EJJison patients. Zollinger-Ellison patients included 12 women and 36 men with a median age of 49 yr (range, 25-73). Besides hypergastrinemia and severe hypersecretion of gastric acid [median basal acid output, 40 mmol H+/h; range, lo-106 mmol H+/h), the gastrinoma diagnosis was confirmed by immunohistochemistry in 26 patients, by radiological means in 3, and by persistent hypergastrinemia after total gastrectomi in 6. In 13 patients with typical clinical histories the gastrinoma has not yet been localized, but antral origin of hypergastrinemia was excluded. Further clinical details of the patients have been published elsewhere (l&19).
o 1990by the American Gastroenterological 0016.5035/90/$3.00
Association
June 1990
PROGASTRIN
Ab.3206 . a * Ab.2604
Ab.6017 AA
v//k
G-17
]YIY]
Preprogastrln 101
__r
m
G-17
lgty
y///h
G-17
I~IY/
Glycine ~
extended 2, __
Camp-I
G-34
G-17
___
w
G-17
F///h
G-17
1
vG_17
Carboxyamidated
Comp-I
__,8_
G-34
__
__
CLEAVAGE
1421
cu-amidated gastrins and nonamidated precursors (total progastrin product) irrespective of the degree of processing (17). All samples were assayed with ab 8017 both before and after tryptic cleavage. a-Amidated gastrins. Ab 2604 specifically binds the COOH-terminal bioactive site in the cr-amidated gastrins [Figure 1). It measures sulfated and nonsulfated gastrin 17, gastrin 34 and component I with almost equimolar potency (21.22). and there is no cross-reaction with nonamidated gastrins. All samples were assayed with ab 2604. Glycine-extended gastrins. Ab 3208 specifically binds the glycine-extended COOH terminus. It was used in selected cases to identify glycine-extended gastrins after fractionation by gel chromatography. In assays with ab 8017 and ab 2604, monoiodinated synthetic human gastrin 17 (24) was used as tracer and synthetic human gastrin 17 as standard. In assays with ab 3208, monoiodinated synthetic human gastrin 13-gly was used as tracer and synthetic human gastrin 13-gly as standard (23). Further details of the evaluation of the three antisera have been published (20,21,23).
G-17
Enzymatic
TRYPTIC
IN ZE PATIENTS
SITES
Figure 1. Schematic illustration of human progastrin and its main products. The sequence specificities of antisera 2664, 6017, and 3206 are shown. By tryptic cleavage, the sequence recognized by ab 6017 (hatched area) is made accessible to antibody binding in all progastrin products.
Treatment
Equal volumes of the sample and trypsin (TPCK, No. T-8642; Sigma, St. Louis, MO.), 100 rcg/ml 0.1 M sodium phosphate, pH 7.5, were incubated for 30 min at room temperature. The enzymatic reaction was terminated by boiling for 10 min. Recovery after enzymatic cleavage was at least 65oi.
Assay Procedure Duodenal ulcer patients. Each duodenal ulcer patient had an ulcer with a size of at least 5 mm, verified by endoscopy. There were 20 women and 22 men with a median #ageof 56 yr [range, 18-79). None of the patients had undergone gastric surgery except for simple closure of perforations. Blood was sampled at a time when antisecretory medication had been given for t5 days within the last 2 Wk.
Normal subjects. Twenty-seven women and 7 men from the medical staff with a median age of 35 yr (range, 22-60) who had no gastrointestinal disease constituted the reference group.
Eladioimmunoassays Three sequence-specific antisera (Figure 1) were used to measure the following. Total progastrin product. As illustrated in Figure 1, ab 8017 specifically binds the NH,-terminal sequence of gastrin 17. It measures all gastrin forms with a free gastrin 17 NH, terminus and binds sulfated and nonsulfated gastrin 17, gastrin l’i’-gly, and further COOH-terminal extended forms of gastrin 17 with equimolar potency (20). Because treatment with trypsin exposes the NH,-terminal sequence of gastrin 17 in a)) progastrin products [Figure l), ab 8017, used after tryptic cleavage, measures the total concentration of all
To 1.75 ml antiserum dilution was added 250 ~1 tracer (in a dilution corresponding to 1000 cpm) and 150 ~1 sample. After incubation for 72 h, the antibody-bound and free tracer were separated by addition of 500 ~1 charcoal solution and centrifugation. Standards were prepared in both assay buffer and hormone-free plasma. When undiluted serum or plasma was boiled to terminate tryptic cleavage, precipitation of proteins hampered the normal assay procedure. To overcome this difficulty, the samples [as well as standards and controls] were diluted l:3 and 450 ~1 was incubated with 1485 ~1 antiserum dilution and 215 ~1 tracer (=lOOOcpm).
Gel Chromatography Serum samples (1 ml] were applied to Sephadex G-58 superfine columns (Pharmacia, Uppsala, Sweden] (10 x 1000 mm] and eluted with 0.125 M NH,HCO,, pH 8.2, at room temperature. The flow rate was 4 ml/h, and fractions of 1 ml were collected. Void volume and total volume were determined by addition of ‘251-albumen and ‘*NaCl. The columns were calibrated with sulfated and nonsulfated gastrin 17, gastrin 17-gly, the NH,-terminal 1-13 fragment of gastrin 17, and gastrin 34. All fractions were assayed with ab 2604. After tryptic cleavage, the fractions were assayed with ab 8017.
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LINDA BARDRAM
GASTROENTEROLOGY
Vol. 98, No. 6
Statistics The Mann-Whitney test was used to evaluate differences between groups and Spearman’s test to evaluate correlation. A probability value of
In normal subjects, serum concentrations of the total progastrin product were almost identical to the concentrations of a-amidated gastrins (26 and 25 pM, respectively) (Table 1). In a few subjects, the concentrations of a-amidated gastrins were significantly lower than the concentration of the total progastrin product, indicating that small amounts of inactive precursors might be present in the circulation. The a-amidated gastrins constituted 87% of the total progastrin product (median; range, 27%-160%) (Table 1). In patients with duodenal ulcer, the concentrations of a-amidated gastrins (18 PM) were of the same magnitude as in normal subjects, whereas the levels of total progastrin product were significantly higher (48 pM; p < 0.01) (Table 1). Measurements with ab 8017 without tryptic cleavage showed that the median concentration of gastrin forms with an intact gastrin 17 NH, terminus was 33.5 pM (range, 9-144 PM). In serum from gastrinoma patients, the concentrations of both a-amidated gastrins and total progastrin product were significtfntly higher than in normal subjects (p < 0.01) (Table 1). In 3 patients with histologically verified gastrinoma, the a-amidated gastrin concentrations were within the normal range, whereas measurements of the total progastrin product showed significantly increased concentrations. The ar-amidated gastrins constituted only 46% [range, 16%100%) of the total progastrin product (Table 1). The percentages of cy-amidated gastrins in all sera are shown in Figure 2. The percentages of Lu-amidated gastrins in duodenal ulcer patients (39%) and in gastrinoma patients (54% and 30%) were significantly Table 1. Concentrations (PM) of a-Amidated Gastrins tab 2604)and Total Progastrin Product [tib 8017 After Tryptic Cleavage] in Serum From Normal Subjects, Patients With DuodenalUlcer, and Patients
With Gastrinama cu-Aniidated gastrins
Normal [n = 34) Duodenal ulcer (n = 42) Gastrinomas (n = 48)
(5-6; (7-8; 390 (24-17.200)
Data expressed as median (range).
Total progastrin product
% a-Amidated of total
(10-9;
(2Z60,
(16-Z) 785 (76-84.000)
(153Y30) (16~;OO)
: .
8
8
¶b
3-
54%
i 39%
. . . a.
-t
Normal sutyects
?
A .
$oed.p
.
t3emgn gastrinomas
e
30%
r”
Malignant gastrinomas
Figure 2. The ratios (%) between concentrations of a-amidated gastrins (ah 2604)and total progastrin product [ah 6017 after tryptic cleavage) in serum from 34 normal subjects, 42 patients with duodenal ulcer, and 46 patients with gastrinomas (33 benign and 15 malignant). Median values are marked by solid lines. The ratios above 100% are explained by the small normal variations in the two assays and by the fact that recovery of peptide after tryptic cleavage was not always complete. Both factors will have increasing influence on the calculated ratios when the total concentrations of progastrin product are low and almost identical to the concentrations of u-amidated gastrins as in the normal subjects.
different from the percentage in normal subjects (87%; p < 0.01). Moreover, the percentage of a-amidated gastrins was significantly lower in patients with malignant gastrinomas (30%) than in patients with benign gastrinomas (54%; p < 0.01). In cases involving hepatic metastases, the percentage of a-amidated gastrins was 23% compared with 39% when only lymphnodes were involved [Table 2). There was no correlation between the percentage of cY-amidated gastrins and the concentration of the total progastrin product. One gastrinoma patient was followed up through different cIinica1 stages over a period of 6 yr. In the beginning, the gastrinoma was undetectable, but eventually a tumor became apparent in the tail of the pancreas which was subsequently resected. However, 18 mo after the operation there was recurrence with multiple hepatic metastases. Concentrations of the total progastrin product was significantly increased, and a low percentage of a-amidated gastrins (10%)
PROGASTRIN IN ZE PATIENTS
June 1990
Table :!. a-Amidated Gastrins tab 26041as a Percentage of Total Progastrin Product tab 8017 After Tryptic Cleavage) in Patients With Malignant and Benign Gastrinomas Median (%I All patknts (n = 48) Patients with hepatic metastases (n = 10) Patients with lymphnode metastases (n = 5) Patients without metastases [n = 33)
Range (%I
46
16-100
23
16-49
39
25-66
54
17-100
found already at an early stage of the disease when the diagnosis based on measurements of CXamidated gastrins was still uncertain (Table 3). Gel chromatography showed that serum from patients with malignant gastrinomas contained several gastrin precursors in addition to the cu-amidated gastrins, gastrin 17, gastrin 34, and component I. Two typical examples are shown in Figure 3A and B. The peak eluting at K,,) = 0.59 consisted mainly of gastrin 17-gly (confirmed by measurements with ab 3208; data not shown). The glycine-extended form of gastrin 34 was found in a peak eluting in front of the cu-amidated form. Finally, high concentrations of a large progastrin were seen at K, = 0.19. In sera from patients with apparently benign tumors (examples in Figure 4A and B), the majority of the nonamidated precursors were glycine-extended gastrins, whereas only small amounts of the large progastrin were found. In one patient with a gastrinoma located in a large cystic benign ovarian tumor, the only Lu-amidated gastrin found in circulation was gastrin 34, but the serum contained large amounts of glycine-extended gastrin 17, glycine-extended gastrin 34, and the large progastrin [Figure 5). was
Table 3. Concentrations (PM) of a-Amidated Gastrins (ab 2604) and Total Progastrin Product [ab 8037 After
Tryptic Cleavage) in Serum From a Gastrinoma Patient at Different Clinical Stages
Clinical stage Zollinger-Ellison syndr’ome suspected 3 yr later, no tumor found 4 yr later, after resection of pancreatic tumor 6 yr later, recurrence with hepatic metastases
a-Amidated gastrins
Total progastrin product
% a-Amidated of total
27
276
10
60
642
9
10
75
13
1500
7200
21
1423
Discussion This study shows that small amounts of inactive gastrin precursors may be found only occasionally in the circulation in normal subjects. In almost half of the normal subjects, the concentration of a-amidated gastrins slightly exceeded the concentration of the total progastrin product (5-15 PM), resulting in ratios of a-amidated gastrins >lOO% (Figure 2). This is explained partly by the fact that peptid recovery after tryptic cleavage was not always complete and partly by the normal small variations in the two assays. Both of these factors play more important roles when the total concentrations of progastrin product are low and identical with concentrations of cu-amidated gastrins. In contrast, serum from patients with duodenal ulcers contained substantial amounts of nonamidated gastrins. A large part of these could also be measured with ab 8017 before tryptic cleavage. Thus they contain the free NH,-terminal sequence of gastrin 17. These results confirm earlier observations of increased NH,-terminal gastrin 17 immunoreactivity in ulcer patients (25.26). The majority of the nonamidated gastrins are presumably glycine-extended gastrin 17 or the NH,-terminal 1-13 fragment of gastrin 17. This fragment in the present assay is measured with 25% potency compared with gastrin 17 (20). Because neither gastrin 17-gly nor the NH,-terminal gastrin 1-13 affects the acid secretion in humans (271, a pathogenetic role in ulcer disease is not likely, but the increased amounts of nonamidated gastrins in the blood might reflect an abnormal posttranslational processing in the antral G cell. High concentrations of inactive gastrin precursors were found in serum from gastrinoma patients. Thus less than half of the total progastrin product were fully processed a-amidated gastrins. Gel chromatography showed the presence of gastrin 17, gastrin 34, and component I, as earlier described (28.29). In agreement with our earlier observations of increased concentrations of glycine-extended gastrins in serum from gastrinoma patients (30), large amounts of gastrin 17-gly, gastrin 34-gly, and component I-gly were also demonstrated. These peptides eluted in front of their corresponding a-amidated forms. The immunoreactivity in the peak at Kd = 0.59 could also be caused by the NH,-terminal l-13 fragment of gastrin 17, which in this system has the same elution constant as gastrin 17-gly. The presence of this fragment in serum of gastrinoma patients has been suggested previously (31,321. Measurements with ab 3208 in the present study showed that the peak contained predominantly gastrin 17-gly. Besides the glycine-extended gastrins, a large molecular progastrin (I& = 0.19) was demonstrated in serum from many of the patients. A similar progastrin has been shown in extracts of antral tissue, gastrinomas,
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GASTROENTEROLOGY Vol. 98. No. 6
LINDA BARDRAM
B
A
1
Progastrin
Progastrin 1
F
1.5
z c
G-34
z
I
G-34
G-17 s ns
G-17 s ns
I I
i
K
l.C I-
II I
5 0
Figure 3. Gel chromatography of serum from two patients with malignant gastrinomas. Serum samples (1 ml) were applied to Sephadex G-50 superfine columns (10 x 1000 mm) and eluted with 0.125 M NH,HCO,, pH 8.2, at room temperature. With a flow rate of 4 ml/h,
$ ii W
0.5
!I ii E
0.0
l-ml fractions were collected and assayed with ab 2604 [O--O) and, after tryptic cleavage, with ab 8017 (w).
CONSTANT (Kd)
and various other tumors (7,15-17a,33,34) and in sera from achlorhydric patients with pernicious anemia 1341. In gastrinoma tissue the increased synthesis of progastrin is often accompanied by incomplete posttranslational processing resulting in accumulation of unprocessed progastrin as well as glycine-extended
intermediates {8,17a,33,35]. The present study shows that large amounts of these precursors are released to the circulation. The ratio between the concentration of cr-amidated gastrins and the total progastrin product in serum will depend on differences in their release and metabolic clearance rates but will also reflect the degree of
A G-17gly G-17 I 3-
1 G-17gty
2-
Figure 4. Gel chromatography of serum from two patients with benign gastrinomas. Serum samples (1 ml) were applied to Sephadex G-50 superfine columns (10 x 1000 mm) and eluted with 0.125 M NH,HCO,, pH 8.2, at room temperature. With a flow rate of 4 ml/h, l-ml fractions were collected and assayed with ab 2604 (O--O) and, after tryptic cleavage, with ab 8017 (0-o)
0.1
l-
0.0
OI
0.0
I
I
0.5
I
I
1.0
I 0.0
ELUTION CONSTANT (Kd)
I
I 0.5
I
1 1.0
June 1990
PROGASTRIN IN ZE PATIENTS
G;34
I
I
0.0
ELMION
I
0.5
I
I
1.0
CONSTANT (Kd)
Figure 5. Gel chromatography of serum from a patient with a benign gastrinoma located in a large ovarian tumor. A serum sample (1 ml) was applied to a Sephadex G-60 superfhte column (10 x 1000 mm) and eluted with 0.125 M NH,HCO, pH 6.2 at room temperature. With a flow rate of 4 ml/h fractions of 1 ml were collected and assayed with ab 2604 (O---O) and after tryptic cleavage with ab 6017 (U).
progastrin processing in the tumor tissue. This ratio was found to be significantly lower in patients with malignant tumors than in patients with apparently benign disease (Table 2 and Figure 2). In contrast to earlier reports by Kothary et al. (32,36), measurements with the NH,-terminal antiserum (ab 8017) without tryptic cleavage did not distinguish between malignant and benign gastrinomas. Those measurements included only gastrin precursors with an intact gastrin 17 NH, terminus; as the present study shows, large amounts of longer gastrin precursors with extensions beyond the NH,-terminal sequence are present. Furthermore, the highest concentrations of the large progastrin (Kd = 0.19) were found in patients with hepatic metastases, whereas the smaller glycineextended gastrins predominanted in patients with benign disease. This indicates decreased processing with increasing dedifferentiation. In one patient with a malignant tumor, identical patterns of incomplete progastrin processing was found in the primary tumor and the metastases (data not shown). This suggests that poor processing may be a characteristic of the primary tumor and not only a consequence of metastatic growth. The finding of a very low degree of processing in a patient with an apparently benign tumor that eventually turned out to be malignant (Table 3) supports this hypothesis. Differentiation between malignant and benign gastrinomas depends on the demonstration of invasive growth and/or metastases. We cannot exclude the possibility that some of the benign tumors in the present study eventually will turn out to be malignant. In a few of the patients with tumors
1425
conceived as benign, the degree of precursor processing was as low as in the malignant patients. Only the continuing prospective study will show whether comparison of Lu-amidated gastrins with the total progastrin product has prognostic value. Posttranslational processing varies considerably between individual tumors. Therefore, measurements with a processing-independent radioimmunoassay that includes all precursors reflect peptide synthesis in tumors better than conventional methods. In the gastrinoma patients, only half of the circulating progastrin products are recognized by the COOH-terminalspecific antisera, which are most often used in conventional routine measurements. Moreover, the concentrations of the total progastrin product were significantly increased in four patients with a-amidated gastrins within the normal range. Prospective studies will show if measurements of the total progastrin product are superior in the differentiation of gastrinoma patients from patients with ulcers. The success rate in localizing gastrinomas by portal vein catheterization might also increase by the application of this method.
References 1. Zollinger RM, Ellison EH. Primary peptic ulcerations of the jejunum associated with islet cell tumors of the pancreas. Ann Surg 1955;142:709-723. 2. Boel E, Vuust J, Norris F, Norris K, Wind A, Rehfeld JF. Marcker KA. Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication. Proc Nat1 Acad Sci USA 1983;80:2866-2869. 3. Kato K, Himeno S, Takahashi Y. Wakabayashi T, Tarui S, Matsubara K. Molecular cloning of human gastrin precursor cDNA. Gene 1983;26:53-57. 4. Noe BD. Converting enzymes. In: Polak JM, Bloom SR, eds. Current trends in tumor pathology: Endocrine tumors. Volume I. Edinbourgh: Churchill Livingstone, 1985:57-81. 5. Gregory RA. Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;5:103-117. 6. Rehfeld JF, Stadil F. Vikels#e J. Immunoreactive gastrin components in human serum. Gut 1974;15:102-111. 7. Dockray GJ, Varro A, Desmond H, Young J, Gregory H, Gregory RA. Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment. J Biol Chem 1987;262:8643-8647. 8. Desmond H, Pauwels S, Varro A, Gregory H, Young J, Dockray GJ. Isolation and characterization of the intact gastrin precursor from a gastrinoma. FEBS Lett 1987;210:185-188. 9. Morley JS, Tracy HJ, Gregory RA. Structure-function relationships in the active C-terminal tetrapeptide amide sequence of gastrin. Nature [London) 1965;207:1356-1360. 10. Bradbury AF, Finnie MDA, Smyth DG. Mechanism of Cterminal amide formation by pituitary enzymes. Nature (London] 1982;298:686-688. 11. Sugano K, Aponte GW, Yamada T. Identification and characterization of glycine-extended posttranslational processing intermediates of progastrin in porcine stomach. J Biol Chem 1985;260: 11724-11729. 12. Hilsted L, Rehfeld JF. a-Amidation of antral progastrin: relation to other posttranslational modifications. J Biol Chem 1987;262: 16953-16957.
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LINDA BARDRAM
13. Rehfeld /F. Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs. Evidence of posttranslational control of cell differentiation. J Biol Chem 1986;261:5841-5847. 14. Rehfeld JF, Bardram L, Cantor P, Hilsted L, Schwartz TW. Cell specific processing of pro-cholecystokinin and progastrin. Biochimie 1988;70:25-31. 15. Bardram L, Hilsted L. Rehfeld JF. Cholecystokinin, gastrin and their precursors in pheochromocytomas. Acta Endocrinol (Copenh) 1989;120:479-484. 16. Rehfeld JF, Bardram L, Hilsted L. Gastrin in human bronchogenic carcinomas: Constant expression but variable processing of progastrin. Cancer Res 1989;49:2840-2843. 17. Bardram L. Rehfeld JF. Processing-independent radioimmunoanalysis: a general analytical principle applied to progastrin and its products. Anal Biochem 1988;175:537-543. 17a. Bardram L. Gastrin in non-neoplastic pancreatic tissue from patients with and without gastrinomas. Stand J Gastroenterol 1990 (in press). 18. Bardram L, Stadil F. Effects of Omeprazole on acid secretion and acid-related symptoms in patients with Zollinger-Ellison syndrome. Stand J Gastroenterol1989;26(suppl):98-103. 19. Bardram L, Stadil F. The place of surgery in the treatment of gastrointestinal APUD-omas. In: Buchanan KD, ed. Gastrointestinal APUDomas. International Congress and Symposium series no. 138. London: Royal Society of Medicine Services Limited, 1988:37-45. 20. Bardram L, Rehfeld JF. Production and evaluation of monospecific antibodies for a processing-independent sequence of human progastrin. Stand J Clin Lab Invest 1989;49:173-182. 21. Rehfeld JF, Stadil F, Rubin B. Production and evaluation of antibodies for the radioimmunoassay of gastrin. Stand J Clin Lab Invest 1972;30:221-232. 22. Rehfeld JF, Magistris LD, Andersen BN. Sulfation of gastrin: effect on immunoreactivity. Regul Pept 1981;2:333-342. 23. H&ted L, Rehfeld JF. Measurement of precursors for aamidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage. Anal Biothem 1986;152:119-126. 24. Stadil F, Rehfeld JF. Preparation of ‘2”I-labelled synthetic human gastrin-I for radioimmunoanalysis. Stand J Clin Lab Invest 1972;30:361-368. 25. Petersen B, Rehfeld JF. The NH,-terminal tridecapeptide fragment of gastrin-17 in serum from duodenal ulcer patients. Stand J Gastroenterol1980;15:29-31. 26. Kothary PC, Vinik AI. NH,-terminal of gastrin-17 in duodenal
27.
28.
29.
30.
31.
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33.
34.
35.
36.
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ulcer disease: identification of progastrin-17. Biochem Biophys Res Comm 1987;146:884-888. Hilsted L, Hint K, Christiansen J, Rehfeld JF. Neither glycineextended gastrin nor the l-13 fragment of gastrin-17 influences gastric acid secretion in humans. Gastroenterology 1988;94:96102. Rehfeld JF, Stadil F. Gelfiltration studies on immunoreactive gastrin in sermn from Zollinger-Ellison patients. Gut 1973:14:369* 373. Dockray GJ, Walsh JH, Passaro E Jr. Relative abundance of big and little gastrins in tumours and blood of patients with Zollinger-Ellison syndrome. Gut 1975;16:353-358, H&ted L, Bardram L. C-terminally glycine-extended gastrins in serum and tumors from patients with the Zollinger-Ellison syndrome (abstr). Can J Physiol Pharmacol1986;136-137. Dockray GJ, Walsh JH. Aminoterminal gastrin fragment in serum of Zollinger-Ellisons syndrome patients. Gastroenterology 1975;68:222-232. Kothary PC, Fabri PJ. Gower W, O’Dorisio TM, Ellis J, Vinik AI. Evaluation of NH,-terminal gastrins in gastrinoma syndrome. J Clin Endocrinol Metab 1986;62:970-974. Pauwels S, Desmond H, Dimaline R, Dockray GJ. Identification of progastrin in gastrinomas, antrum and duodenum by a novel radioimmunoassay. J Clin Invest 1986;77:376-381. Jensen S. Borch K, Hilsted L, Rehfeld JF. Progastrin processing in hypersecretory G-cells in humans. Gastroenterology 1989:96: 1063-1070. DelValle J, Sugano K, Yamada T. Progastrin and its glycineextended posttranslational processing intermediates in human gastrointestinal tissues. Gastroenterology 1987;92:1908-1912. Kothary PC, Mahoney WC, Vinik AI. Identification of gastrin molecular variants in gastrinoma syndrome. Regul Pept 1987;17: 71-84.
Received June 26,1989. Accepted November 15.1989. Address requests for reprints to: Linda Bardram, M.D., Department of Surgical Gastroenterology C, Rigshospitalet, Blegdamsvej 9 DK-2100, Copenhagen, Denmark. The study was supported by grants from the Danish Cancer Union. The encouragement from Professor Flemming Stadil and the skillful technical assistance of Inge Mortensen, Winna Staunstrup, and Hanne Nordvig are gratefully acknowledged. The author thanks Drs. K. Lauridsen (Odense]. and J. Rask Madsen (Copenhagen] for sera from duodenal ulcer patients.
1990;93:142o-1426
Progastrin in Serum From Zollinger-Ellison Patients An Indicator of Malignancy? LINDA BARDRAM Department
of Surgical Gastroenterology
C, Rigshospitalet,
Progastrin and all of its processing products were measured in serum from 48 patients with ZollingerEllison syndrome, 42 patients with duodenal ulcers, and 34 normal subjects. A processing-independent gastrin analysis and a conventional radioimmunoassay for the biologically active a-amidated gastrins were used. In serum from normal subjects, 87% of all progastrin prod(median; range, 27%-160%) ucts were a-amidated gastrins, whereas they constituted only 39% (15%-130%) in serum from patients with duodenal ulcers (p < 0.01) and 46% (16%-100%) in serum from gastrinoma patients (p < 0.01). A significantly lower percentage of cu?amidated gastrin was found in patients with hepatic metastases (23 %) than in patients with apparently benign tumors (54%). Chromatography of serum showed that large progastrin molecules occurred mainly in patients with malignant tumors, whereas smaller glycine-extended precursors dominated in patients with benign tumors. The results indicate that the total progastrin product reflects tumor synthesis of gastrin better than conventional measurements of cy-amidated gastrin. Moreover, the results suggest that a low degree of processing of progastrin could serve as a predictor of a malignant clinical course at an early stage of the disease.
T
umor production of gastrin is responsible for the severe hypersecretion of acid that causes Zollinger-Ellison syndrome (1). Biologically active gastrins are products of several modifications of human progastrin (2,3) (Figure 1). These include cleavages at dibasic amino acids, partial sulfation of tyrosine 87 (4-6),phosphorylation of serine 99 (7,8),and the cr-carboxyamidation of phenylalanine 92, which is necessary for biological activity (9,lO). Thus, in addition to the bioactive a-amidated gastrins (gastrin 17, gastrin 34, and component I), inactive precursors with a COOH-terminal extension of glycine (ll,l~), as well
University of Copenhagen,
Denmark
as larger progastrins with further NH,- and COOHterminal extensions, exist [Figure 1). The degree of posttranslational processing of the gastrin precursor varies considerably in normal and of neoplastic cells (13-16). To study the synthesis progastrin and the processing products in different tissues, a radioimmunoanalysis by which all gastrin forms are measured independently of the degree of processing has been developed (17). Using this method, high concentrations of glycine-extended gastrins, as well as larger progastrins, were recently found in gastrinoma tissue (17a). The results of that study also indicated that inactive precursors were released to the blood. The occurrence of gastrin precursors as well as a-amidated gastrins in sera from a large group of Zollinger-Ellison patients, from patients with duodenal ulcers, and from normal subjects without gastrointestinal disease is examined in this study.
Materials and Methods Subjects Sera were obtained after an overnight fast from the following subjects. ZoJJinger-EJJison patients. Zollinger-Ellison patients included 12 women and 36 men with a median age of 49 yr (range, 25-73). Besides hypergastrinemia and severe hypersecretion of gastric acid [median basal acid output, 40 mmol H+/h; range, lo-106 mmol H+/h), the gastrinoma diagnosis was confirmed by immunohistochemistry in 26 patients, by radiological means in 3, and by persistent hypergastrinemia after total gastrectomi in 6. In 13 patients with typical clinical histories the gastrinoma has not yet been localized, but antral origin of hypergastrinemia was excluded. Further clinical details of the patients have been published elsewhere (l&19).
o 1990by the American Gastroenterological 0016.5035/90/$3.00
Association
June 1990
PROGASTRIN
Ab.3206 . a * Ab.2604
Ab.6017 AA
v//k
G-17
]YIY]
Preprogastrln 101
__r
m
G-17
lgty
y///h
G-17
I~IY/
Glycine ~
extended 2, __
Camp-I
G-34
G-17
___
w
G-17
F///h
G-17
1
vG_17
Carboxyamidated
Comp-I
__,8_
G-34
__
__
CLEAVAGE
1421
cu-amidated gastrins and nonamidated precursors (total progastrin product) irrespective of the degree of processing (17). All samples were assayed with ab 8017 both before and after tryptic cleavage. a-Amidated gastrins. Ab 2604 specifically binds the COOH-terminal bioactive site in the cr-amidated gastrins [Figure 1). It measures sulfated and nonsulfated gastrin 17, gastrin 34 and component I with almost equimolar potency (21.22). and there is no cross-reaction with nonamidated gastrins. All samples were assayed with ab 2604. Glycine-extended gastrins. Ab 3208 specifically binds the glycine-extended COOH terminus. It was used in selected cases to identify glycine-extended gastrins after fractionation by gel chromatography. In assays with ab 8017 and ab 2604, monoiodinated synthetic human gastrin 17 (24) was used as tracer and synthetic human gastrin 17 as standard. In assays with ab 3208, monoiodinated synthetic human gastrin 13-gly was used as tracer and synthetic human gastrin 13-gly as standard (23). Further details of the evaluation of the three antisera have been published (20,21,23).
G-17
Enzymatic
TRYPTIC
IN ZE PATIENTS
SITES
Figure 1. Schematic illustration of human progastrin and its main products. The sequence specificities of antisera 2664, 6017, and 3206 are shown. By tryptic cleavage, the sequence recognized by ab 6017 (hatched area) is made accessible to antibody binding in all progastrin products.
Treatment
Equal volumes of the sample and trypsin (TPCK, No. T-8642; Sigma, St. Louis, MO.), 100 rcg/ml 0.1 M sodium phosphate, pH 7.5, were incubated for 30 min at room temperature. The enzymatic reaction was terminated by boiling for 10 min. Recovery after enzymatic cleavage was at least 65oi.
Assay Procedure Duodenal ulcer patients. Each duodenal ulcer patient had an ulcer with a size of at least 5 mm, verified by endoscopy. There were 20 women and 22 men with a median #ageof 56 yr [range, 18-79). None of the patients had undergone gastric surgery except for simple closure of perforations. Blood was sampled at a time when antisecretory medication had been given for t5 days within the last 2 Wk.
Normal subjects. Twenty-seven women and 7 men from the medical staff with a median age of 35 yr (range, 22-60) who had no gastrointestinal disease constituted the reference group.
Eladioimmunoassays Three sequence-specific antisera (Figure 1) were used to measure the following. Total progastrin product. As illustrated in Figure 1, ab 8017 specifically binds the NH,-terminal sequence of gastrin 17. It measures all gastrin forms with a free gastrin 17 NH, terminus and binds sulfated and nonsulfated gastrin 17, gastrin l’i’-gly, and further COOH-terminal extended forms of gastrin 17 with equimolar potency (20). Because treatment with trypsin exposes the NH,-terminal sequence of gastrin 17 in a)) progastrin products [Figure l), ab 8017, used after tryptic cleavage, measures the total concentration of all
To 1.75 ml antiserum dilution was added 250 ~1 tracer (in a dilution corresponding to 1000 cpm) and 150 ~1 sample. After incubation for 72 h, the antibody-bound and free tracer were separated by addition of 500 ~1 charcoal solution and centrifugation. Standards were prepared in both assay buffer and hormone-free plasma. When undiluted serum or plasma was boiled to terminate tryptic cleavage, precipitation of proteins hampered the normal assay procedure. To overcome this difficulty, the samples [as well as standards and controls] were diluted l:3 and 450 ~1 was incubated with 1485 ~1 antiserum dilution and 215 ~1 tracer (=lOOOcpm).
Gel Chromatography Serum samples (1 ml] were applied to Sephadex G-58 superfine columns (Pharmacia, Uppsala, Sweden] (10 x 1000 mm] and eluted with 0.125 M NH,HCO,, pH 8.2, at room temperature. The flow rate was 4 ml/h, and fractions of 1 ml were collected. Void volume and total volume were determined by addition of ‘251-albumen and ‘*NaCl. The columns were calibrated with sulfated and nonsulfated gastrin 17, gastrin 17-gly, the NH,-terminal 1-13 fragment of gastrin 17, and gastrin 34. All fractions were assayed with ab 2604. After tryptic cleavage, the fractions were assayed with ab 8017.
1422
LINDA BARDRAM
GASTROENTEROLOGY
Vol. 98, No. 6
Statistics The Mann-Whitney test was used to evaluate differences between groups and Spearman’s test to evaluate correlation. A probability value of
In normal subjects, serum concentrations of the total progastrin product were almost identical to the concentrations of a-amidated gastrins (26 and 25 pM, respectively) (Table 1). In a few subjects, the concentrations of a-amidated gastrins were significantly lower than the concentration of the total progastrin product, indicating that small amounts of inactive precursors might be present in the circulation. The a-amidated gastrins constituted 87% of the total progastrin product (median; range, 27%-160%) (Table 1). In patients with duodenal ulcer, the concentrations of a-amidated gastrins (18 PM) were of the same magnitude as in normal subjects, whereas the levels of total progastrin product were significantly higher (48 pM; p < 0.01) (Table 1). Measurements with ab 8017 without tryptic cleavage showed that the median concentration of gastrin forms with an intact gastrin 17 NH, terminus was 33.5 pM (range, 9-144 PM). In serum from gastrinoma patients, the concentrations of both a-amidated gastrins and total progastrin product were significtfntly higher than in normal subjects (p < 0.01) (Table 1). In 3 patients with histologically verified gastrinoma, the a-amidated gastrin concentrations were within the normal range, whereas measurements of the total progastrin product showed significantly increased concentrations. The ar-amidated gastrins constituted only 46% [range, 16%100%) of the total progastrin product (Table 1). The percentages of cy-amidated gastrins in all sera are shown in Figure 2. The percentages of Lu-amidated gastrins in duodenal ulcer patients (39%) and in gastrinoma patients (54% and 30%) were significantly Table 1. Concentrations (PM) of a-Amidated Gastrins tab 2604)and Total Progastrin Product [tib 8017 After Tryptic Cleavage] in Serum From Normal Subjects, Patients With DuodenalUlcer, and Patients
With Gastrinama cu-Aniidated gastrins
Normal [n = 34) Duodenal ulcer (n = 42) Gastrinomas (n = 48)
(5-6; (7-8; 390 (24-17.200)
Data expressed as median (range).
Total progastrin product
% a-Amidated of total
(10-9;
(2Z60,
(16-Z) 785 (76-84.000)
(153Y30) (16~;OO)
: .
8
8
¶b
3-
54%
i 39%
. . . a.
-t
Normal sutyects
?
A .
$oed.p
.
t3emgn gastrinomas
e
30%
r”
Malignant gastrinomas
Figure 2. The ratios (%) between concentrations of a-amidated gastrins (ah 2604)and total progastrin product [ah 6017 after tryptic cleavage) in serum from 34 normal subjects, 42 patients with duodenal ulcer, and 46 patients with gastrinomas (33 benign and 15 malignant). Median values are marked by solid lines. The ratios above 100% are explained by the small normal variations in the two assays and by the fact that recovery of peptide after tryptic cleavage was not always complete. Both factors will have increasing influence on the calculated ratios when the total concentrations of progastrin product are low and almost identical to the concentrations of u-amidated gastrins as in the normal subjects.
different from the percentage in normal subjects (87%; p < 0.01). Moreover, the percentage of a-amidated gastrins was significantly lower in patients with malignant gastrinomas (30%) than in patients with benign gastrinomas (54%; p < 0.01). In cases involving hepatic metastases, the percentage of a-amidated gastrins was 23% compared with 39% when only lymphnodes were involved [Table 2). There was no correlation between the percentage of cY-amidated gastrins and the concentration of the total progastrin product. One gastrinoma patient was followed up through different cIinica1 stages over a period of 6 yr. In the beginning, the gastrinoma was undetectable, but eventually a tumor became apparent in the tail of the pancreas which was subsequently resected. However, 18 mo after the operation there was recurrence with multiple hepatic metastases. Concentrations of the total progastrin product was significantly increased, and a low percentage of a-amidated gastrins (10%)
PROGASTRIN IN ZE PATIENTS
June 1990
Table :!. a-Amidated Gastrins tab 26041as a Percentage of Total Progastrin Product tab 8017 After Tryptic Cleavage) in Patients With Malignant and Benign Gastrinomas Median (%I All patknts (n = 48) Patients with hepatic metastases (n = 10) Patients with lymphnode metastases (n = 5) Patients without metastases [n = 33)
Range (%I
46
16-100
23
16-49
39
25-66
54
17-100
found already at an early stage of the disease when the diagnosis based on measurements of CXamidated gastrins was still uncertain (Table 3). Gel chromatography showed that serum from patients with malignant gastrinomas contained several gastrin precursors in addition to the cu-amidated gastrins, gastrin 17, gastrin 34, and component I. Two typical examples are shown in Figure 3A and B. The peak eluting at K,,) = 0.59 consisted mainly of gastrin 17-gly (confirmed by measurements with ab 3208; data not shown). The glycine-extended form of gastrin 34 was found in a peak eluting in front of the cu-amidated form. Finally, high concentrations of a large progastrin were seen at K, = 0.19. In sera from patients with apparently benign tumors (examples in Figure 4A and B), the majority of the nonamidated precursors were glycine-extended gastrins, whereas only small amounts of the large progastrin were found. In one patient with a gastrinoma located in a large cystic benign ovarian tumor, the only Lu-amidated gastrin found in circulation was gastrin 34, but the serum contained large amounts of glycine-extended gastrin 17, glycine-extended gastrin 34, and the large progastrin [Figure 5). was
Table 3. Concentrations (PM) of a-Amidated Gastrins (ab 2604) and Total Progastrin Product [ab 8037 After
Tryptic Cleavage) in Serum From a Gastrinoma Patient at Different Clinical Stages
Clinical stage Zollinger-Ellison syndr’ome suspected 3 yr later, no tumor found 4 yr later, after resection of pancreatic tumor 6 yr later, recurrence with hepatic metastases
a-Amidated gastrins
Total progastrin product
% a-Amidated of total
27
276
10
60
642
9
10
75
13
1500
7200
21
1423
Discussion This study shows that small amounts of inactive gastrin precursors may be found only occasionally in the circulation in normal subjects. In almost half of the normal subjects, the concentration of a-amidated gastrins slightly exceeded the concentration of the total progastrin product (5-15 PM), resulting in ratios of a-amidated gastrins >lOO% (Figure 2). This is explained partly by the fact that peptid recovery after tryptic cleavage was not always complete and partly by the normal small variations in the two assays. Both of these factors play more important roles when the total concentrations of progastrin product are low and identical with concentrations of cu-amidated gastrins. In contrast, serum from patients with duodenal ulcers contained substantial amounts of nonamidated gastrins. A large part of these could also be measured with ab 8017 before tryptic cleavage. Thus they contain the free NH,-terminal sequence of gastrin 17. These results confirm earlier observations of increased NH,-terminal gastrin 17 immunoreactivity in ulcer patients (25.26). The majority of the nonamidated gastrins are presumably glycine-extended gastrin 17 or the NH,-terminal 1-13 fragment of gastrin 17. This fragment in the present assay is measured with 25% potency compared with gastrin 17 (20). Because neither gastrin 17-gly nor the NH,-terminal gastrin 1-13 affects the acid secretion in humans (271, a pathogenetic role in ulcer disease is not likely, but the increased amounts of nonamidated gastrins in the blood might reflect an abnormal posttranslational processing in the antral G cell. High concentrations of inactive gastrin precursors were found in serum from gastrinoma patients. Thus less than half of the total progastrin product were fully processed a-amidated gastrins. Gel chromatography showed the presence of gastrin 17, gastrin 34, and component I, as earlier described (28.29). In agreement with our earlier observations of increased concentrations of glycine-extended gastrins in serum from gastrinoma patients (30), large amounts of gastrin 17-gly, gastrin 34-gly, and component I-gly were also demonstrated. These peptides eluted in front of their corresponding a-amidated forms. The immunoreactivity in the peak at Kd = 0.59 could also be caused by the NH,-terminal l-13 fragment of gastrin 17, which in this system has the same elution constant as gastrin 17-gly. The presence of this fragment in serum of gastrinoma patients has been suggested previously (31,321. Measurements with ab 3208 in the present study showed that the peak contained predominantly gastrin 17-gly. Besides the glycine-extended gastrins, a large molecular progastrin (I& = 0.19) was demonstrated in serum from many of the patients. A similar progastrin has been shown in extracts of antral tissue, gastrinomas,
1424
GASTROENTEROLOGY Vol. 98. No. 6
LINDA BARDRAM
B
A
1
Progastrin
Progastrin 1
F
1.5
z c
G-34
z
I
G-34
G-17 s ns
G-17 s ns
I I
i
K
l.C I-
II I
5 0
Figure 3. Gel chromatography of serum from two patients with malignant gastrinomas. Serum samples (1 ml) were applied to Sephadex G-50 superfine columns (10 x 1000 mm) and eluted with 0.125 M NH,HCO,, pH 8.2, at room temperature. With a flow rate of 4 ml/h,
$ ii W
0.5
!I ii E
0.0
l-ml fractions were collected and assayed with ab 2604 [O--O) and, after tryptic cleavage, with ab 8017 (w).
CONSTANT (Kd)
and various other tumors (7,15-17a,33,34) and in sera from achlorhydric patients with pernicious anemia 1341. In gastrinoma tissue the increased synthesis of progastrin is often accompanied by incomplete posttranslational processing resulting in accumulation of unprocessed progastrin as well as glycine-extended
intermediates {8,17a,33,35]. The present study shows that large amounts of these precursors are released to the circulation. The ratio between the concentration of cr-amidated gastrins and the total progastrin product in serum will depend on differences in their release and metabolic clearance rates but will also reflect the degree of
A G-17gly G-17 I 3-
1 G-17gty
2-
Figure 4. Gel chromatography of serum from two patients with benign gastrinomas. Serum samples (1 ml) were applied to Sephadex G-50 superfine columns (10 x 1000 mm) and eluted with 0.125 M NH,HCO,, pH 8.2, at room temperature. With a flow rate of 4 ml/h, l-ml fractions were collected and assayed with ab 2604 (O--O) and, after tryptic cleavage, with ab 8017 (0-o)
0.1
l-
0.0
OI
0.0
I
I
0.5
I
I
1.0
I 0.0
ELUTION CONSTANT (Kd)
I
I 0.5
I
1 1.0
June 1990
PROGASTRIN IN ZE PATIENTS
G;34
I
I
0.0
ELMION
I
0.5
I
I
1.0
CONSTANT (Kd)
Figure 5. Gel chromatography of serum from a patient with a benign gastrinoma located in a large ovarian tumor. A serum sample (1 ml) was applied to a Sephadex G-60 superfhte column (10 x 1000 mm) and eluted with 0.125 M NH,HCO, pH 6.2 at room temperature. With a flow rate of 4 ml/h fractions of 1 ml were collected and assayed with ab 2604 (O---O) and after tryptic cleavage with ab 6017 (U).
progastrin processing in the tumor tissue. This ratio was found to be significantly lower in patients with malignant tumors than in patients with apparently benign disease (Table 2 and Figure 2). In contrast to earlier reports by Kothary et al. (32,36), measurements with the NH,-terminal antiserum (ab 8017) without tryptic cleavage did not distinguish between malignant and benign gastrinomas. Those measurements included only gastrin precursors with an intact gastrin 17 NH, terminus; as the present study shows, large amounts of longer gastrin precursors with extensions beyond the NH,-terminal sequence are present. Furthermore, the highest concentrations of the large progastrin (Kd = 0.19) were found in patients with hepatic metastases, whereas the smaller glycineextended gastrins predominanted in patients with benign disease. This indicates decreased processing with increasing dedifferentiation. In one patient with a malignant tumor, identical patterns of incomplete progastrin processing was found in the primary tumor and the metastases (data not shown). This suggests that poor processing may be a characteristic of the primary tumor and not only a consequence of metastatic growth. The finding of a very low degree of processing in a patient with an apparently benign tumor that eventually turned out to be malignant (Table 3) supports this hypothesis. Differentiation between malignant and benign gastrinomas depends on the demonstration of invasive growth and/or metastases. We cannot exclude the possibility that some of the benign tumors in the present study eventually will turn out to be malignant. In a few of the patients with tumors
1425
conceived as benign, the degree of precursor processing was as low as in the malignant patients. Only the continuing prospective study will show whether comparison of Lu-amidated gastrins with the total progastrin product has prognostic value. Posttranslational processing varies considerably between individual tumors. Therefore, measurements with a processing-independent radioimmunoassay that includes all precursors reflect peptide synthesis in tumors better than conventional methods. In the gastrinoma patients, only half of the circulating progastrin products are recognized by the COOH-terminalspecific antisera, which are most often used in conventional routine measurements. Moreover, the concentrations of the total progastrin product were significantly increased in four patients with a-amidated gastrins within the normal range. Prospective studies will show if measurements of the total progastrin product are superior in the differentiation of gastrinoma patients from patients with ulcers. The success rate in localizing gastrinomas by portal vein catheterization might also increase by the application of this method.
References 1. Zollinger RM, Ellison EH. Primary peptic ulcerations of the jejunum associated with islet cell tumors of the pancreas. Ann Surg 1955;142:709-723. 2. Boel E, Vuust J, Norris F, Norris K, Wind A, Rehfeld JF. Marcker KA. Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication. Proc Nat1 Acad Sci USA 1983;80:2866-2869. 3. Kato K, Himeno S, Takahashi Y. Wakabayashi T, Tarui S, Matsubara K. Molecular cloning of human gastrin precursor cDNA. Gene 1983;26:53-57. 4. Noe BD. Converting enzymes. In: Polak JM, Bloom SR, eds. Current trends in tumor pathology: Endocrine tumors. Volume I. Edinbourgh: Churchill Livingstone, 1985:57-81. 5. Gregory RA. Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;5:103-117. 6. Rehfeld JF, Stadil F. Vikels#e J. Immunoreactive gastrin components in human serum. Gut 1974;15:102-111. 7. Dockray GJ, Varro A, Desmond H, Young J, Gregory H, Gregory RA. Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment. J Biol Chem 1987;262:8643-8647. 8. Desmond H, Pauwels S, Varro A, Gregory H, Young J, Dockray GJ. Isolation and characterization of the intact gastrin precursor from a gastrinoma. FEBS Lett 1987;210:185-188. 9. Morley JS, Tracy HJ, Gregory RA. Structure-function relationships in the active C-terminal tetrapeptide amide sequence of gastrin. Nature [London) 1965;207:1356-1360. 10. Bradbury AF, Finnie MDA, Smyth DG. Mechanism of Cterminal amide formation by pituitary enzymes. Nature (London] 1982;298:686-688. 11. Sugano K, Aponte GW, Yamada T. Identification and characterization of glycine-extended posttranslational processing intermediates of progastrin in porcine stomach. J Biol Chem 1985;260: 11724-11729. 12. Hilsted L, Rehfeld JF. a-Amidation of antral progastrin: relation to other posttranslational modifications. J Biol Chem 1987;262: 16953-16957.
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LINDA BARDRAM
13. Rehfeld /F. Accumulation of nonamidated preprogastrin and preprocholecystokinin products in porcine pituitary corticotrophs. Evidence of posttranslational control of cell differentiation. J Biol Chem 1986;261:5841-5847. 14. Rehfeld JF, Bardram L, Cantor P, Hilsted L, Schwartz TW. Cell specific processing of pro-cholecystokinin and progastrin. Biochimie 1988;70:25-31. 15. Bardram L, Hilsted L. Rehfeld JF. Cholecystokinin, gastrin and their precursors in pheochromocytomas. Acta Endocrinol (Copenh) 1989;120:479-484. 16. Rehfeld JF, Bardram L, Hilsted L. Gastrin in human bronchogenic carcinomas: Constant expression but variable processing of progastrin. Cancer Res 1989;49:2840-2843. 17. Bardram L. Rehfeld JF. Processing-independent radioimmunoanalysis: a general analytical principle applied to progastrin and its products. Anal Biochem 1988;175:537-543. 17a. Bardram L. Gastrin in non-neoplastic pancreatic tissue from patients with and without gastrinomas. Stand J Gastroenterol 1990 (in press). 18. Bardram L, Stadil F. Effects of Omeprazole on acid secretion and acid-related symptoms in patients with Zollinger-Ellison syndrome. Stand J Gastroenterol1989;26(suppl):98-103. 19. Bardram L, Stadil F. The place of surgery in the treatment of gastrointestinal APUD-omas. In: Buchanan KD, ed. Gastrointestinal APUDomas. International Congress and Symposium series no. 138. London: Royal Society of Medicine Services Limited, 1988:37-45. 20. Bardram L, Rehfeld JF. Production and evaluation of monospecific antibodies for a processing-independent sequence of human progastrin. Stand J Clin Lab Invest 1989;49:173-182. 21. Rehfeld JF, Stadil F, Rubin B. Production and evaluation of antibodies for the radioimmunoassay of gastrin. Stand J Clin Lab Invest 1972;30:221-232. 22. Rehfeld JF, Magistris LD, Andersen BN. Sulfation of gastrin: effect on immunoreactivity. Regul Pept 1981;2:333-342. 23. H&ted L, Rehfeld JF. Measurement of precursors for aamidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage. Anal Biothem 1986;152:119-126. 24. Stadil F, Rehfeld JF. Preparation of ‘2”I-labelled synthetic human gastrin-I for radioimmunoanalysis. Stand J Clin Lab Invest 1972;30:361-368. 25. Petersen B, Rehfeld JF. The NH,-terminal tridecapeptide fragment of gastrin-17 in serum from duodenal ulcer patients. Stand J Gastroenterol1980;15:29-31. 26. Kothary PC, Vinik AI. NH,-terminal of gastrin-17 in duodenal
27.
28.
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31.
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33.
34.
35.
36.
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ulcer disease: identification of progastrin-17. Biochem Biophys Res Comm 1987;146:884-888. Hilsted L, Hint K, Christiansen J, Rehfeld JF. Neither glycineextended gastrin nor the l-13 fragment of gastrin-17 influences gastric acid secretion in humans. Gastroenterology 1988;94:96102. Rehfeld JF, Stadil F. Gelfiltration studies on immunoreactive gastrin in sermn from Zollinger-Ellison patients. Gut 1973:14:369* 373. Dockray GJ, Walsh JH, Passaro E Jr. Relative abundance of big and little gastrins in tumours and blood of patients with Zollinger-Ellison syndrome. Gut 1975;16:353-358, H&ted L, Bardram L. C-terminally glycine-extended gastrins in serum and tumors from patients with the Zollinger-Ellison syndrome (abstr). Can J Physiol Pharmacol1986;136-137. Dockray GJ, Walsh JH. Aminoterminal gastrin fragment in serum of Zollinger-Ellisons syndrome patients. Gastroenterology 1975;68:222-232. Kothary PC, Fabri PJ. Gower W, O’Dorisio TM, Ellis J, Vinik AI. Evaluation of NH,-terminal gastrins in gastrinoma syndrome. J Clin Endocrinol Metab 1986;62:970-974. Pauwels S, Desmond H, Dimaline R, Dockray GJ. Identification of progastrin in gastrinomas, antrum and duodenum by a novel radioimmunoassay. J Clin Invest 1986;77:376-381. Jensen S. Borch K, Hilsted L, Rehfeld JF. Progastrin processing in hypersecretory G-cells in humans. Gastroenterology 1989:96: 1063-1070. DelValle J, Sugano K, Yamada T. Progastrin and its glycineextended posttranslational processing intermediates in human gastrointestinal tissues. Gastroenterology 1987;92:1908-1912. Kothary PC, Mahoney WC, Vinik AI. Identification of gastrin molecular variants in gastrinoma syndrome. Regul Pept 1987;17: 71-84.
Received June 26,1989. Accepted November 15.1989. Address requests for reprints to: Linda Bardram, M.D., Department of Surgical Gastroenterology C, Rigshospitalet, Blegdamsvej 9 DK-2100, Copenhagen, Denmark. The study was supported by grants from the Danish Cancer Union. The encouragement from Professor Flemming Stadil and the skillful technical assistance of Inge Mortensen, Winna Staunstrup, and Hanne Nordvig are gratefully acknowledged. The author thanks Drs. K. Lauridsen (Odense]. and J. Rask Madsen (Copenhagen] for sera from duodenal ulcer patients.