A simple enzymatic procedure for radioimmunochemical quantitation of the large molecular forms of gastrin and cholecystokinin

ANALYTICAL

BIOCHEMISTRY

10&126-

133 (1980)

A Simple Enzymatic Procedure for Radioimmunochemical Quantitation of the Large Molecular Forms of Gastrin and Cholecystokinin LAURA Institute

AND JENS F. REHFELD~

DE MAGISTRIS~

of Medical

Biochemistry,

University

of Aarhus.

Denmark

Received October 9, 1979 We have developed a simple enzymatic procedure for evaluation of antisera reactivity against the large molecular forms of gastrin and cholecystokinin (CCK). The procedure can be used for radioimmunochemical quantitation of the precursor molecules. The different molecular forms of gastrin or CCK in tissue extracts or plasma were separated by gel chromatography. The concentration of each form was then measured with 17 different antisera before and after tryptic cleavage. The ratio between the molar concentrations before and after tryptic cleavage varied from 0.32 to 1.00. Such variation can explain the variable hormone concentrations in serum and tissue measured with different radioimmunoassays. The present procedure can be performed with any biological fluid containing the precursor forms. It does not require the large molecular forms in pure state. In principle the procedure can be used for quantitation of all peptide precursors.

Like other peptide hormones, the two related hormones gastrin and cholecystokinin (CCK)3 are heterogenous; i.e., they each exist in tissue and blood in molecular forms of different size and biological activity (l-8). The must potent form is considered to be the principal hormonal form. Hence, with respect to gastric acid secretion the principal gastrin is the heptadecapeptide amide, gastrin- 17 (9); with respect to gallbladder contraction and pancreatic enzyme secretion the octapeptide amide, CCK-8, is the principal CCK (6,7,10). Recently, we have demonstrated that the larger forms of each hormone (Fig. 1) are biosynthetic precursors for the principal forms (11,12). Gastrin and CCK concentrations in biological fluids are measured by radioimmuno’ Present address: Cattedra di Gastroenterologia, II Clinica Medica Policiinico Umberto I, Universita’ de Roma, Roma, Italy. * To whom reprint requests should be addressed. o Abbreviations used: CCK, cholecystokinin; gastrin17, heptadecapeptide amide gastrin; gastrin-34, tetratriacontapeptide amide gastrin; CCK-33, triacontatriapeptide amide cholecystokinin; CCK-8, COOH-terminal octapeptide amide of CCK-33. OC!Q3-2697/80/030126-08$02.00/O Copyright 0 1980 by Academic Ress. Inc. All rights of reproduction in any form reserved.

126

assays, which most often have been developed for the principal molecular form only ( 13). Thus, the accuracy of the measurements is hampered by the lack of knowledge about the extent to which each assay measures the larger molecule forms. The problem is considerable. For instance, the predominant form of gastrin in human, porcine, and canine blood is the large precursor, gastrin34 (1,3,11,14). Moreover, antisera raised against gastrin-17 may vary from full (15) to no binding (16) of gastrin-34; such variation can explain the difference in measurement of basal gastrin concentrations in serum (for review, see Ref. (17)). Since most of the precursors are not yet, or in limited amounts only, available in pure form for calibration, most gastrin and CCK radioimmunoassays are still inaccurate. To overcome this problem, we previously proposed a procedure (13) based on two observations: (i) Gastrin-17 and CCK-8 constitute both monomeric parts of their respective precursors (Fig. 1). (ii) Gastrin-17 and CCK-8 are both at their NH, terminus linked to basic amino acid residues. Hence, the

QUANTITATION

OF HORMONE

CHOLECYSTOKININ

PRECURSORS

127

GASTRIN

FIG. 1. Amino acid sequences of porcine cholecystokinin (CCK, left side) and gastrin (right side). Shaded amino acid residues show the common COOH terminus, which constitutes the biological active site of both hormones. Arrows indicate the points of tryptic cleavage. As described in Refs. (l-7) gastrin and CCK are heterogenous. The largest molecular form, component I, has an unknown NH,-terminal extension (R camp-,) added to CCK-39 and gastrin-34, respectively. CCK component II corresponds to the COOH-terminal tritriacontapeptide amide, CCK-33, and gastrin component II to the COOH-terminal tetrattiacontapeptide amide, gastrin-34. Component III corresponds to the COOHterminal dodecapeptide amide, CCK-12, and the COOH-terminal heptadecapeptide amide, gastrin-17, respectively. Component IV corresponds to the COOH-terminal octapeptide amide, CCK-8. and the COOH-terminal tetradecapeptide amide, gastrin-14, respectively. The most active form with respect to stimulation of acid secretion is gastrin-17, and the most active form with respect to gallbladder emptying and secretion of pancreatic enzymes is CCK-8. Thus gastrin-17 is considered the principal gastrin and CCK-8 the principal CCK.

NH,-terminal peptide bonds can be cleaved by trypsin (Fig. 1). We have now developed the procedure. METHODS

Materials Gastrins. Large molecular forms of gastrin (components I and II) were extracted from a human pancreatic gastrinoma. Tumor tissue, 7.0 g, was in the frozen state cut into pieces of a few milligrams, which were immersed into boiling water, 10 ml/g tissue, pH 6.6, for 20 min. After homogenization

for 5 min and centrifugation at 10,000 rpm, the supernatant was decanted and stored at -20°C. Highly purified human tetratriacontapeptide gastrin (gastrin-34 = component II) and heptadecapeptide gastrin (gastrin- 17 = component III) for calibrations were generously donated by R. A. Gregory, University of Liverpool, United Kingdom. Synthetic human gastrin- 17 for immunization, isotope labeling, and standard was purchased from Imperial Chemical Industries, Alderly Park, Cheshire, United Kingdom. Cholecystokinins. Large molecular forms of CCK (components I, II, and III) were

128

DE MAGISTRIS

extracted from porcine jejunal mucosa. The tissue was obtained from a local abbatoir and immediately frozen. Mucosal tissue, 7.8 g, was in the frozen state cut into pieces of a few milligrams which were immersed into boiling water, pH 6.6, 10 ml/g tissue, for 20 min. After homogenization, centrifugation, and decantation of the supernatant (which contains mainly the small molecular forms of CCK, the octapeptide (CCK-8) and the tetrapeptide amide (CCK4) (7)), the pellet was extracted with 0.5 M acetic acid, 10 ml/g tissue, for 5 min. After another homogenization and centrifugation (10,000 rpm), the supernatant was decanted and stored at -20°C. Highly purified porcine tritriacontapeptide CCK (CCK-33 = component II) for calibrations, isotope labeling, and standard was generously donated by V. Mutt, Karolinska Institute, Stockholm, Sweden. Synthetic porcine CCK-8 for calibration and standards was generously donated by M. Ondetti, Squibb Institute of Medical Research, Princeton, New Jersey. Other reagents. Trypsin TPCK (Lot 3742, 37M 788) was purchased from Worthington Biochemical Corporation Freehold, New Jersey; Sephadex G-50 superfine from Pharmacia, Uppsala, Sweden; and 1251-labeled human albumin and 22NaC1 from Radiochemical Centre, Amersham, England. Experimental

AND REHFELD

sequence of gastrin and CCK using antiserum 2609 (18), 1251-gastrin- 17 (19), and synthetic human gastrin-17 or synthetic CCK-8 as standards. This assay consequently measures all molecular forms of gastrin and CCK that contain the common COOH terminus (20). Fractions corresponding to the peak of gastrin and CCK components I and II were pooled (Figs. 2 and 3). For control of unspecific effects of the trypsin preparation, fractions of the gastrin17-like and CCK-&like peaks were also pooled. Tryptic cleavage. Half of each gastrin pool was incubated at 37°C for 30 min with 1 mg trypsin/ml. The action of trypsin was terminated by boiling for 10 min. The CCK components were incubated under similar circumstances except that the large molecular form of CCK required a higher concen-

Procedures

Chromatography. Samples of 6-8 ml tissue extract (1% of bed volume) were applied to Sephadex G-50 superfine columns (25 x 2000 mm), eluted with 0.25 M ammonium bicarbonate, pH 8.2, at 4°C with a flow rate of 20 ml/h. Fractions of 3.5 ml were collected. The columns were calibrated with 1251albumin for indication of void volume (V,), with **NaCl for indication of total volume ( VJ, and with gastrin-34 ( V,-,,), gastrin-17 (VG--17), CCK-33 ( VCCK-&, and CCK-8 (V,,,-,). The elutions were monitored by a radioimmunoassay, which is specific for the common COOH-terminal pentapeptide amide

L 06

02 EL”TIOf&OL”ME III

FIG. 2. Gel chromatography of a boiling water extract of a human gastrin-producing tumor on a Sephadex G-SO superfine column (25 x 2000 mm) eluted with 0.25 M ammonium bicarbonate, pH 8.2, at 4°C with a flow rate of 20 ml/h. Fractions of 3.5 ml were collected. The columns were calibrated with 1Z51-albumin (V,), human gastrin-34, gas&in-17, and 22NaCI ( Vt). Roman numbers (I-III) indicate the three largest components from which the encircled fractions were pooled.

QUANTITATION

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OF HORMONE

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129

PRECURSORS

Radioimmunoanalysis. The antisera were raised in rabbits against synthetic human gastrin (1-17) (antisera Nos. 2716-2720), synthetic human gastrin (2- 17) (antisera Nos. 2601-2606, 2609, and 4556-4563), or synthetic human gastrin (6- 13) (antisera Nos. 4710 and 4713) covalently coupled by carbodiimide to bovine serum albumin as described in detail previously (18). Antisera Nos. 2609 and 2717, which were highly specific for the COOH-terminal pentapeptide amide common for gastrin and CCK, were used also to measure CCK (20). Monoiodinated synthetic human gastrin-17 was used as tracer (19) and synthetic human gastrin-17 300

200

ELUTION

VOLUME (11

3. Gel chromatography of an acetic acid extract (upper panel) and a neutral boiling water extract (lower panel) of porcine jejunal mucosa. The gel chromatography was performed as described in the legend to Fig. 2. In addition the columns were calibrated with porcine CCK-33, CCK-8, and the COOH-terminal tetrapeptide amide (CCK-4). The encircled fractions were pooled. FIG.

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tration of trypsin (5 mg/ml) for cleavage to the COOH-terminal octapeptide (Fig. 4). After trypsin incubation the concentration of immunoreactivity in seven different dilutions of each half-pool was measured radiochemically using 17 different antisera. The measurements were related to the concentrations in the corresponding nontrypsinated half-pools that were not incubated with trypsin. The concentrations of immunoreactivity were expressed in equivalents of synthetic human gastrin-17 or synthetic porcine CCK-8. The incubation period of 30 min was not critical using trypsin concentrations of 1 mg/ml (for gastrin) and 5 mg/mI (for CCK). Incubation for 15 min was equally efficient.

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ELUTION

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FIG. 4. Gel chromatography of 100 ng porcine CCK33 Incubated with 1 mg trypsin (upper panel) or 5 mg trypsin (lower panel). After inactivation of trypsin by boiling the samples were applied to Sephadex G-50 superfine columns (10 x 1000 mm) eluted with 0.25 M ammonium bicarbonate, pH 8.2, at 4°C with a flow rate of 5 ml/h. Fractions of 1 ml were collected and assayed. The columns were calibrated as described in Figs. 2 and 3

130

DE MAGISTRIS

AND

or synthetic porcine CCK-8 was used as standard. Antisera, tracer, and standards or samples were incubated in volumes of 2000, 250, and 150 ~1, respectively, for 7 days at 4°C to ensure equilibrium. The antisera used were characterized by their titer (defined as the dilution at which the antisera can bind 50% of 2 fmol tracer at equilibrium), avidity (expressed by the “effective” equilibrium constant, ZQf (21)), and heterogeneity (expressed by the index of heterogeneity (22)). Validation of the procedure. The reproducibility of the procedure for three of the antisera (Nos. 2604, 2609, and 2720) was evaluated by determination of the ratio for gastrin component II concentrations before and after tryptic cleavage in 10 different pools assayed on the same days (intraassay precision) or in one pool assayed 10 times over a period of 2 months (interassay preComponent

I

REHFELD

cision). The precision was expressed by the coefficient of variation. The accuracy of the procedure was evaluated by gel chromatography of the trypsin-digested fragments (Fig. 4), and by the ability of fragment to displace the tracer from the antisera. The specificity of the antisera has been evaluated in detail previously (18,20). It is important to realize that the antisera were all raised against the principal hormone form of gastrin or characteristic fragments of this form, and moreover that only the principal form (gastrin- 17) was used as tracer. Hence, the assays could not measure NH,-terminal tryptic fragments of the large molecular forms of gastrin or CCK. Unspecijc interference from tissue or plasma proteins and salt was avoided by using a small sample volume (maximal 6.25% of the incubation volume) in the assays as reported in detail previously (17,23).

Component (Gastrln-3L-I\ke)

I

Component (Gastnn-17-IIke)

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100

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30 IMMUNOREACTIVE (pmoi

10 GASTRIN equlv

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30 BEFORE gastrln-17

TRYPTIC

10 CLEAVAGE

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FIG. 5. Comparison of the reactivity of two antisera (Nos. 2720 (A-C) and 2602 (D-F)) with the three larger gastrin components (I-III) before and after tryptic cleavage. The concentration of each component was measured in seven different dilutions. The solid lines show the regression line for the different dilutions, indicated also by the slope x/y. The broken lines correspond to x = y.

QUANTITATION

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HORMONE

131

PRECURSORS TABLE

RESULTS

The effect of tryptic cleavage on the measurement of the large molecular forms of gastrin is shown in Fig. 5 and Table 1. The figure shows measurement with antiserum 2720 having a low reactivity against the large components and with antiserum 2602 having a higher reactivity. The variation for all antisera ranged from 32% (Ab. 2720) to 100% (Ab. 2604, Tables 1 and 2). Trypsin per se did not influence the measurements in that the gastrin-17- and CCKHike components were measured with the same potency irrespective of the presence of trypsin (Fig. 5, Tables 1 and 2). The “trypsin ratio” between the component concentrations before and after cleavage was not related to antibody avidity (expressed by the effective equilibrium constant, K&(range: 0.2-5.4 x lo’* M-l), antibody heterogeneity (expressed by Sip’s index of heterogeneity,

THE RATIO BETWEEN CONCENTRATIONS WITH

Antiserum No. 2609 2717

CHOLECYSTOKININ BEFORE AND AFTER

TRYPS~N Two

Component I 0.37 0.60

2

AS MEASURED ANTISERA

Component II (CCK33-like) 0.56 1.00

COMPONENT INCUBATION WITH

Component IV (CCK8-like) 1.0 1.0

ff (range: 0.46- 1.OO)), or titer (range: 12,0004,000,000). The intra- and interassay precision for antisera 2604,2609, and 2720 were 9, 10, and 8% and 19, 18, and 20%, respectively. The trypsin-digested fragments of the large gastrins and CCKs were diluted in parallel with the standards, synthetic gastrin-17 and CCK-8, respectively (Fig. 6). DISCUSSION

TABLE

THE

RATIO

CENTRATIONS TRYPSIN ANTISERA

Antiserum No. 2601 2602 2604 2605 2606 2609 2716 2717 2718 2720 4556 4559 4560 4562 4563 4710 4713

BETWEEN BEFORE

AS MEASURED

Component I 0.70 0.65 1.00 0.87 0.51 0.52 0.50 0.66 0.76 0.40 0.66 0.62 0.70 0.70 0.76 0.58 0.82

1

GASTRIN AND

AFTER

WITH

COMPONENT

CON-

INCUBATION

WITH

17 DIFFERENT

Component II (gastrin34-like) 0.78 0.95 0.94 0.78 0.68 0.63 0.62 0.74 0.80 0.32 0.65 0.66 0.53 0.64 0.88 0.46 0.62

GASTRIN

Component III (gastrin17-like) 1.0 1.0 1.0 1.0 I.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

The present study shows that a simple and reliable procedure can be used to determine the extent to which antisera raised against a small hormonal form can bind the larger precursor forms. Consequently, the procedure allows accurate quantitation of the different molecular forms of gastrin and CCK in biological fluids. Assessment of antiserum reactivity against the large molecular forms has so far not been possible, because the largest forms of gastrin and CCK have not yet been purified; gastrin-34, CCK-39, and CCK-33 are not available in quantities sufficient to test all antisera in the various laboratories. Apart from overcoming these problems, the present procedure has other advantages. First, since crude material, tissue extracts or plasma, is used, precursor molecules from any species can be quantitated. In contrast the purified larger forms are of either porcine or human origin. Second, the large molecular forms in blood, urine, or cerebrospinal fluid may be degraded and hence may not necessarily be identical with molecular forms

132

DE MAGISTRIS Ab

no

AND REHFELD

2716

Ab

I

I

0

IMMUNOREACTIVE (“molequl”

h”rncl”

GASTRIN gastr,n-17

II1

no

2717

I

I

02

IMMUNOREACTIVE (nmol

equlv

I

04

CHOLECYSTOKININ porctns

CCK-811)

FIG. 6. Displacement of the tracer from two antisera by trypsin-digested fragments of gastrin component I (A) and II (0) from gastrinoma tissue in four dilutions ((l:l, 1:2, 1:4, and 1:8) left-hand diagram), and of CCK components I (A) and II (0) from porcine jejunal mucosa in four corresponding dilutions (right-hand diagram). The fragments displaced the tracer in parallel with the standards, synthetic human gastrin-17 and synthetic porcine CCK-8, respectively (0).

purified from tissue. Hence, the present procedure allows a more accurate determination of the large molecular forms in the fluid under study. The procedure is simple. It can be used in all radioimmunoassay laboratories, since it requires only one simple fractionation like gel filtration. The material required, tissue extracts or plasma, can be obtained elsewhere. Moreover, when an antiserum is characterized once, the procedure need not be repeated when material of the same kind is studied. Also the fractions containing the precursor forms can be stored for future evaluations of new antisera. A modification of the procedure can be used to quantitate large versus small forms of gastrin or CCK without fractionation or use of antisera specific for different molecular forms. Thus, the increase in hormone concentration after tryptic cleavage measured by an antiserum, which binds the larger forms with reduced potency, corresponds to

the reduction in binding potency for the large hormonal forms. For instance, Ab. 2720 measured a serum concentration of 28 pmol eq gastrin-17/liter before and 52 pmol/liter after tryptic cleavage. Since Ab. 2720 measures the larger gastrins with a potency of 33% compared to that of gastrin-17 (Table 1) the increase of 24 pmoVliter will correspond to 67% of the large gas&ins in the sample. Hence, the sample contained 36 pmol large gastrins/liter (component I and gastrin-34) and 16 pmol small gastrin/liter (gastrin-17 and -14). An even simpler version without tryptic cleavage can be applied using two antisera of which the first measures the large forms with full potency, and the second measures with reduced potency. Applications of such methods are now in progress in our laboratory. The principle of the procedure can be applied to all polypeptides, for which quantitation of precursor concentrations is desirable. Trypsin may not always be the en-

QUANTITATION

OF HORMONE

zyme of choice, although many precursors contain trypsin-sensitive peptide bonds at the linkage of the principal form to the remaining part of the precursor (24). The optimal enzyme(s) are those that cleave the ribosomal precursor product during the posttranslational modification in vivo. ACKNOWLEDGMENTS The present study was supported by grants from the Danish MRC.

REFERENCES 1. Yalow, R. S., and Berson, S. A. (1970) Castroenterology 58, 609-615. 2. Gregory, R. A., and Tracy, H. J. (1972) Lancer 2, 797-799. 3. Rehfeld, J. F. (1972) Biochim. Biophvs. Actu 285, 364-372. 4. Rehfeld, J. F., and Stadil, F. (1973) Girt 14, 369373. 5. Mutt, V. (1976) Clin. Endocrinol. 5, 175s-183s. 6. Dockray, G. J. (1977) Nature (London) 270, 358-361. 7. Rehfeld, J. F. (1978) J. Biol. Chem. 253, 40224030. 8. Walsh, J. H. (1975) in Gastrointestinal Hormones (Thompson, J. C., ed.), pp. 75-84, Univ. of Texas Press, Austin/London.

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PRECURSORS

9. Gregory, R. A., and Tracy, H. J. (1964) Cur 5, 101-117. 10. Rubin, B., and Engel, S. L. (1973) in Frontiers in Gastrointestinal Hormone Research (Andersson, S., ed.), pp. 41-55, Almquist and Wiksell, Stockholm. II. Rehfeld, J. F., and Uvnls-Wallensten, K. (1978) J. Physiol.

283, 379-396.

12. Goltermann, N. R., Rehfeld, J. F., and RoigaardPetersen, H. (1980) J. Biol. Chem., in press. 13. Rehfeld, J. F. (1978) in Gut Hormones (Bloom, S. R., ed.), pp. 145- 148, Churchill Livingstone, Edinburgh/London/New York. 14. Rehfeld, J. F., Stadil, F., and Vikelsoe, J. (1974) Gut 15, 102-111. 15. Rehfeld, J. F. (1976) J. C/in. Invest. 58, 41-49. 16. Dockray. G. J.. and Taylor, I. L. (1976) Gastroenteratogy 71, 971-977. 17. Stadil, F., and Rehfeld, J. F. (1973) Stand. J. Gusfroenferol. 8, IOl- 112. 18. Rehfeld, J. F., Stadil, F., and Rubin, B. (1972) Scund.

J. Clin.

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Invesi.

30, 221-232.

19. Stadil, F.. and Rehfeld, J. F. (1972) Scund. J. C/in. Lab. Invest. 30, 361-368. 20. Rehfeld, J. F. (1978) J. Biol. Chem. 253, 40164021. 21. Ekins, R., and Newman, B. (1970) Acta Endocrinol. Suppl. 147, 11-36. 22. Sips, R. (1948) J. Chem. Phys. 16, 490-501. 23. Rehfeld, J. F., Schwartz, T. W., and Stadil, F. ( 1977) Gustroenterology 72, 469-477. 24. Tager, H. S., and Steiner, D. F. (1974) Annu. Rev.

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43, 509-538.