Clinical Significance of Gastrin Radioimmunoassay John H. Walsh Serum gastrin radioimmunoassay (RIA) is a sensitive and specific method suitable for measurement of circulating concentrations of this peptide hormone, which is a major regulator of gastric acid secretion. When performed under optimal conditions this RIA permits measurement of low and normal serum gastrin levels and changes that occur after physiologic stimulation. Hypergastrinemia may be secondary to atrophy of the acid-secreting gastric mucosa. This form of hypergastrinemia is appropriate and leads to no serious consequences. Hypergastrinemia associated
with gastric acid hypersecretion is inappropriate. The major cause is a gastrinsecreting tumor (gastrinoma) that produces the clinical picture of the Zollinger-Ellison syndrome. The differential diagnosis of inappropriate hypergastrinemia includes antral G-cell hyperplasia and isolated retained antrum. Accurate diagnosis of these conditions may be aided by ancillary studies including feeding, secretin, and calcium stimulation tests. Distinction among these conditions is important in planning appropriate surgical treatment.
THE GASTRIN
R A D I O I M M U N O A S S A Y is similar to other RIA methJ. ods for peptide hormones. 13 - Antibodies are raised most successfully by conjugation of the gastrin molecule to bovine serum albumin. Labeled gastrin is prepared rather easily, is stable for 1-2 months, and retains biologic activity. Purification of labeled gastrin from free iodide can be achieved by simple molecular sieving on Sephadex G-10 or G-15, but a labeled product with the highest specific activity is obtained by ion-exchange chromatography on amino ethyl cellulose (AE-41, Whatman)) ,4 This procedure permits separation of unlabeled peptide from monoiodinated and diiodinated gastrin. Separation of antibody-bound from free labeled gastrin can be achieved by immunoprecipitation with a second antibody, by nonimmune precipitation, by charcoal absorption, or by absorption to an ion-exchange resin (IRP 58-M, Rohm and Haas). 5 The latter is efficient because gastrin is a highly acidic peptide and offers the advantage that early deterioration of label can be detected by an increase in nonspecific binding. The major pitfall of this method is interference in the separation introduced by heparin. Heavily heparinized plasma causes artifactually low gastrin values by increasing nonspecific binding. Antisera must have high association constants ( K = 1 x 10 -H) to permit accurate measurement of normal serum gastrin levels (10-100 x 10 -12 M). A number of such antisera have been produced. 6 The labeled hormone must have high specific activity; 2000 cpm of pure monoiodo-~25I-gastrin prepared by ionexchange chromatography contains approximately 1 pg gastrin. There are several forms of gastrin that are normally present in the plasma (Table 1). The longer half-lived G-34 form is the most abundant.
From the Department of Medicine, Universityof California, Los Angeles, Calif. John H. Walsh, M.D.: Department of Medicine. University of California, Los Angeles, Calif.
Supported by USPHS grant I-ROI-A M17294 and by grant A M17328 from the Center for Ulcer Research and Education. 9 1975 by Grune & Stratton, Inc. Seminars in Nuclear Medicine, Vol. 5, No. 3 (July), 1975
247
J O H N H. WALSH
248
Table 1.
Human big g a s t r i n 1
2
3
4
(G-34) 5
HW 3 8 3 9
6
7
8
9
10
11
12
13
14
15
16
17
32
33
34
Pyro-Leu-Gly-Pro-Gln-Gly-His-Pro-Ser-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys 18
19
20
21
22
23
24
25
26
27
28
29
30
31
-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH I R
Human h e p t a d e c a p e p t i d e
(little)
gastrin
(G-17,
18-34
of
2
G-34)
MW 2 0 9 8
i
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Pyro-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-A1y-Trp-Met-Asp-Phe-NH !
2
R
Human m i n i g a s t r i n
(G-13,
22-34
of
G-34,
5-17
of
G-17)
1
2
3
4
5
6
7
8
9
10
11
12
13
22
23
24
25
26
27
28
29
30
31
32
33
34
Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH
HW 1 6 4 7
2
I
R
C-terminal octapeptide of eholecystokinin 1
2
3
4
5
6
7
(CCK-8)
MW
1143
8
Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH
2
l SO3H
gastrin
I, R = H
gastrin
If,
Pyro
-
pyroglutamyl
R = S03H
ANTIBODY SPECIFICITIES
Antibodies may be raised with specificities to different portions of the gastrin molecule. 6,9 The most valuable antisera for clinical purposes have specificity for the biologically active, C-terminal portion ofgastrin; they recognize sulfated and nonsulfated forms of G-34, G-17, and G-13 on a roughly equimolar basis; and they discriminate between gastrin and cholecystokinin peptides. Specificity patterns of selected antisera are shown in Table 2. Squibb, 1296 (our laboratory), and 2604 (Dr. Jens Rehfeld, Copenhagen, Denmark) all are specific for the C-terminal region of gastrin and have varying cross-reactivity with cholecystokinin. An example is also given for an antibody with specificity for nonsulfated gastrins (NSS, Dr. Jack Hansky, Melbourne, Australia) and another antibody with specificity for the N-terminal region of G-17 (NTS, A b 1295, our laboratory). Antibodies with such unusual specificities are useful for research purposes, but C-terminal gastrin specificity is desirable for clinical purposes. It is apparent that values obtained for serum gastrin concentrations are dependent to a great extent on the degree to which the antibody used for RIA
249
GASTRIN RADIOIMMUNOASSAY
Table 2. Specifici~ of Different Antisera for Gastr|n and CCK Peptide$ Antibody Squibb
1296
2604
NS__SS
NT_~S
2.0
1.1
3.6
O.S
2.3
G-17-I
1.0
1.0
1.0
1.0
1.0
G-17-II
1.2
0.7
0.7
0.09
1 0
1.0
0.7
0.8
IDSO:
fmol/ml G-17-I
2-17,
G-17-I
1.0
l-IS,
G-17-1
0.09
with gastric acid hypersecretion is inappropriate. The major cause is a gastrinsecreting tumor (gastrinoma) that produces the clinical picture of the Zollinger-Ellison syndrome. The differential diagnosis of inappropriate hypergastrinemia includes antral G-cell hyperplasia and isolated retained antrum. Accurate diagnosis of these conditions may be aided by ancillary studies including feeding, secretin, and calcium stimulation tests. Distinction among these conditions is important in planning appropriate surgical treatment.
THE GASTRIN
R A D I O I M M U N O A S S A Y is similar to other RIA methJ. ods for peptide hormones. 13 - Antibodies are raised most successfully by conjugation of the gastrin molecule to bovine serum albumin. Labeled gastrin is prepared rather easily, is stable for 1-2 months, and retains biologic activity. Purification of labeled gastrin from free iodide can be achieved by simple molecular sieving on Sephadex G-10 or G-15, but a labeled product with the highest specific activity is obtained by ion-exchange chromatography on amino ethyl cellulose (AE-41, Whatman)) ,4 This procedure permits separation of unlabeled peptide from monoiodinated and diiodinated gastrin. Separation of antibody-bound from free labeled gastrin can be achieved by immunoprecipitation with a second antibody, by nonimmune precipitation, by charcoal absorption, or by absorption to an ion-exchange resin (IRP 58-M, Rohm and Haas). 5 The latter is efficient because gastrin is a highly acidic peptide and offers the advantage that early deterioration of label can be detected by an increase in nonspecific binding. The major pitfall of this method is interference in the separation introduced by heparin. Heavily heparinized plasma causes artifactually low gastrin values by increasing nonspecific binding. Antisera must have high association constants ( K = 1 x 10 -H) to permit accurate measurement of normal serum gastrin levels (10-100 x 10 -12 M). A number of such antisera have been produced. 6 The labeled hormone must have high specific activity; 2000 cpm of pure monoiodo-~25I-gastrin prepared by ionexchange chromatography contains approximately 1 pg gastrin. There are several forms of gastrin that are normally present in the plasma (Table 1). The longer half-lived G-34 form is the most abundant.
From the Department of Medicine, Universityof California, Los Angeles, Calif. John H. Walsh, M.D.: Department of Medicine. University of California, Los Angeles, Calif.
Supported by USPHS grant I-ROI-A M17294 and by grant A M17328 from the Center for Ulcer Research and Education. 9 1975 by Grune & Stratton, Inc. Seminars in Nuclear Medicine, Vol. 5, No. 3 (July), 1975
247
J O H N H. WALSH
248
Table 1.
Human big g a s t r i n 1
2
3
4
(G-34) 5
HW 3 8 3 9
6
7
8
9
10
11
12
13
14
15
16
17
32
33
34
Pyro-Leu-Gly-Pro-Gln-Gly-His-Pro-Ser-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys 18
19
20
21
22
23
24
25
26
27
28
29
30
31
-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH I R
Human h e p t a d e c a p e p t i d e
(little)
gastrin
(G-17,
18-34
of
2
G-34)
MW 2 0 9 8
i
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Pyro-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-A1y-Trp-Met-Asp-Phe-NH !
2
R
Human m i n i g a s t r i n
(G-13,
22-34
of
G-34,
5-17
of
G-17)
1
2
3
4
5
6
7
8
9
10
11
12
13
22
23
24
25
26
27
28
29
30
31
32
33
34
Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH
HW 1 6 4 7
2
I
R
C-terminal octapeptide of eholecystokinin 1
2
3
4
5
6
7
(CCK-8)
MW
1143
8
Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH
2
l SO3H
gastrin
I, R = H
gastrin
If,
Pyro
-
pyroglutamyl
R = S03H
ANTIBODY SPECIFICITIES
Antibodies may be raised with specificities to different portions of the gastrin molecule. 6,9 The most valuable antisera for clinical purposes have specificity for the biologically active, C-terminal portion ofgastrin; they recognize sulfated and nonsulfated forms of G-34, G-17, and G-13 on a roughly equimolar basis; and they discriminate between gastrin and cholecystokinin peptides. Specificity patterns of selected antisera are shown in Table 2. Squibb, 1296 (our laboratory), and 2604 (Dr. Jens Rehfeld, Copenhagen, Denmark) all are specific for the C-terminal region of gastrin and have varying cross-reactivity with cholecystokinin. An example is also given for an antibody with specificity for nonsulfated gastrins (NSS, Dr. Jack Hansky, Melbourne, Australia) and another antibody with specificity for the N-terminal region of G-17 (NTS, A b 1295, our laboratory). Antibodies with such unusual specificities are useful for research purposes, but C-terminal gastrin specificity is desirable for clinical purposes. It is apparent that values obtained for serum gastrin concentrations are dependent to a great extent on the degree to which the antibody used for RIA
249
GASTRIN RADIOIMMUNOASSAY
Table 2. Specifici~ of Different Antisera for Gastr|n and CCK Peptide$ Antibody Squibb
1296
2604
NS__SS
NT_~S
2.0
1.1
3.6
O.S
2.3
G-17-I
1.0
1.0
1.0
1.0
1.0
G-17-II
1.2
0.7
0.7
0.09
1 0
1.0
0.7
0.8
IDSO:
fmol/ml G-17-I
2-17,
G-17-I
1.0
l-IS,
G-17-1
0.09
