NATURE OF IMMUNOREACTIVE GASTRIN EXTRACTED FROM TISSUES OF GASTROINTESTINAL TRACT

Transcription

1 G ASTROENTEROLOGY Copyright ~ 1971 by The Williams & Wilkins Co. Vol. 60. : Printed in U. S. A. NATURE OF IMMUNOREACTIVE GASTRIN EXTRACTED FROM TISSUES OF GASTROINTESTINAL TRACT SOLOMON A. BERSON, M.D., AND ROSALYN S. YALOW, PH.D. Radioisotope Service, Veterans Administration Hospital, Bronx, New York, and Departments 0/ Medicine, Mount Sinai School 0/ Medicine and The Mount Sinai Hospital, New York, New York Extracts of mucosa from human antrum, duodenum, and proximal jejunum obtained at surgery and at autopsy contain two immunoreactive components of gastrin, separable on the basis of size and charge. These components exhibit the same characteristics as the two plasma gastrin components identified earlier. The major plasma component (BG) has a less acidic charge and larger molecular size than heptadecapeptide gastrin; the minor plasma component (H-LG) resembles heptadecapeptide gastrin in both respects. H-LG predominates in most extracts of antral mucosa, but the relative abundance of BG increases distally, being the only detectable component (although at very low concentration) in proximal jejunal mucosal extracts. Both components are immunochemically indistinguishable from the two plasma components and from heptadecapeptide porcine gastrin. In previous studies, radioimmunoassay of gastrin in plasma fractions separated by Sephadex gel filtration and starch gel electrophoresis revealed two components of immunoreactive gastrin! On starch gel electrophoresis, the minor component migrates in the same zone as heptadecapeptide gastrin added to plasma, which is just in advance of free bromphenol blue and about Received July 16, Accepted September 25, Address requests for reprints to: Dr. Rosalyn S. Yalow, Nuclear Medicine Service, Veterans Administration Hospital, 130 West Kingsbridge Road, Bronx, New York The authors are indebted to Dr. R. A. Gregory and Dr. M. I. Grossman for providing the porcine gastrin I used in these studies. The technical assistance provided by Mrs. Barbara Smoliar, Mrs. Nancy Wu, and Miss Tokuko Saito is gratefully acknowledged. We also wish to thank Miss Elizabeth A. Walsh and Miss Gloria Spevacek for preparing the charts, Mr. Carl R. Bacot for the photography, and Miss Carol Montemurro, Mrs. Kathleen Mercado, Miss Barbara Jahss, and Mrs. Violet A. Mallory for their secretarial assistance. 215 twice as far from the origin as serum albumin; the major component migrates just in advance of serum albumin. On columns of Sephadex G-50 or a mixture of G-50 and G-25, the major component emerges between insulin (mol wt 5700) and proinsulin (mol wt 9000) and therefore corresponds to a molecular weight of about 7000 to 7500; the other component is eluted after insulin in the same region as heptadecapeptide gastrin added to plasma. It was concluded that the major component of immunoreactive human plasma gastrin is less acidic and of greater molecular size than heptadecapeptide gastrin. Subsequently, it was demonstrated, in a plasma containing almost equal amounts of the two components, that the less acidic component eluted from the starch gel behaves like the larger component (BG-big gastrin) on Sephadex gel filtration and that the heptadecapeptide-like gastrin (H-LG) eluted from starch gel emerges from the Sephadex columns in the same zone as authentic heptadecapeptide gastrin. 2 Other fractionation systems, including paper elec-

2 216 BERSON AND YALOW Vol. 60, No.2 trophoresis, starch block electrophoresis, and aminoethyl cellulose chromatography, also readily distinguish BG from heptadecapeptide gastrin.2 Incubation or fractionation of plasmas in 8 M urea failed to alter BG but on incubation with trypsin BG rapidly disappeared, and a virtually quantitative increase in immunoreactivity appeared in zones corresponding largely to H-LG on starch gel electrophoresis and Sephadex gel filtration. 2 Whole plasma and each of the two plasma components separately,2 as well as highly purified native porcine gastrins I and II, and synthetic human gastrin 13 all show parallel inhibition of the binding of 125I-porcine gastrin I to guinea pig antiporcine gastrin antibodies. In this study, gastrin extracted from the mucosa of human gastrointestinal tissues obtained at surgery or at autopsy was subjected to the fractionation procedures used for plasma gastrin. Two major components of immunoreactive gastrin with characteristics similar to those of the plasma com- ponents were also found in the tissue extracts. The relative abundance of the two components differed considerably in different parts of the gastrointestinal tract. Materials and Methods Tissue samples and extraction procedures. Postmortem material was obtained 5 to 6 hr after death in 4 cases; mucosa was stripped from different portions of stomach, duodenum, and jejunum and extracted for gastrin in 10 volumes of boiling water for 30 min, and the residue was removed by centrifugation. Portions of stomach and duodenum were obtained during surgery in 6 subjects including 1 (Ro) with Zollinger-Ellison syndrome undergoing gastrectomy, 1 (C. S.) with carcinoma of the stomach, and 4 with duodenal ulcer. In 1 case (M. T.), as soon as the tissue was removed, a small piece of antral mucosa was stripped and frozen rapidly on Dry Ice. After quick weighings, two segments were extracted from the frozen state in boiling water, a third segment was extracted by maceration in ice water in a Teflon grinder, and a fourth segment was kept unfrozen at 4 C for 60 min before extraction in boiling water. In another case (J. W.), mucosa was stripped from one E " ḡ' 15 1 i,i ~ : Zw w'" ~ 0 8a: i5! Z (j) 0: ll ~ 0 ~ ANTRUM FREE BROMPHENOL r---- ~ i BLUE l ~ ~ I j! J8 MIDDLE N.S 1i.5 )JQ/gml DUODENUM ii... ~ ' PROXIMAL J,B DISTAL 0.1 ~ I! ~ J JEJUNUM.03L! ~ I \ i NS n g / ~ 1. _ \ I ' ~ ' ~. ' 4, m,...!.,ii..' ~ ~ / ~ MIDDLE RR 101!!I- ' I ~ R / m. ~ ' ~. II O~ l m " l II ::1 ::J PROXIMAL l.b MIDDLE 03 1 \/11 i r /.015 ' m ' GJ 05] I! ~! T l o B ~ : I ' m l ' 5 U d : Ii'! I l o ~ g m l '6 1 7 lsi IS IgllOI 1112 I l. - " ' ' ' 8 ~ 9 ~ ORIGIN ANOii ORIGIN ANODE ORIGIN ANODE ANODE FIG. 1. Distribution of immunoreactive gastrin, on starch gel electrophoresis, in extracts of antrum, duo denum, and proximal jejunum in postmortem material. The zones of migration of bromphenol blue stained albumin and of free bromphenol blue were noted prior to sectioning of the gels. Numbers at bottom refer to sections (each llz:inch wide) from gels which were eluted for radioimmunoassay as described in text. The to tal concentration of gastrin (boxed values) in the crude extract for each sample is expressed as micrograms or nanograms gastrin per g of mucosa. Since gel eluates from different gels were assayed at different dilutions the absolute concentrations in the individual segments are to be ignored, only the relative abundance of the com ponents in each gel being of importance.

3 February 1971 NATURE OF EXTRACTED IMMUNOREACTIVE GASTRIN 217 Ro Z E SYNDROME J. L. DUODENAL ULCER ANTRUM lj... o C/) 0.2 ~ E «... ::::>0>...JC: W, 0.1 c/)z ZW QtE I-:W «C/) 0:: ~ uj 0.4 W<.9 U I Z U ::: U<[ ~ t : i 0:: I-: C/) <3 ANTRUM 0.11 ANTRUM n, C. S. Ca. STOMACH D. B. DUODENAL ULCER PYLORUS ANTRUM I I I I I I I I I I ~ tl' 2 ' 3 ' 4 ' 5 ' 6 ' 7 ' 8 ' ~ ORIGIN ANODE J. W DUODENAL ULCER FIG. 2. Same as figure 1 for specimens obtained during surgery. All extracts shown were obtained by boil. ing pieces of mucosa that had been stripped from the refrigerated organs. segment of antrum, frozen rapidly on Dry Ice, and extracted from the frozen state in boiling water. The remainder of the antrum and the proximal duodenum from J. W. and the en tire tissues from 4 other cases were placed on ice (not Dry Ice) and the mucosa was stripped 30 to 60 min later and stored at - 15 C until extracted. A pancreatic tumor removed from a patient with a Zollinger-Ellison syndrome had been at ambient temperature for an unknown period of time (at least several hours) before extraction by boiling in water for 20 min. The boiled tissue was then re-extracted by boiling for another 20 min. Fractionation of tissue gastrin extracts. Since starch gel electrophoresis' is the most convenient method for separating the two components of plasma gastrin, all tissue extracts were subjected to this procedure as previously described for plasma fractionation. I Thirty microliters of extract were pipetted into each of one to four slits of a starch gel. In some experiments bromphenol blue-stained plasma containing added heptadecapeptide gastrin was pipetted into another slit. Following electrophoresis, gel segments were cut from the slab and frozen for at least 4 hr. After thawing, 0.3 to 2 ml of Veronal buffer, 0.02 M, ph 8.4, containing 2.5 mg per ml of human serum albumin, were added to each segment and the protein contained therein was eluted by maceration. Gel particles were removed by centrifugation and the supernatant solution was immunoassayed for gastrin concentration. By comparison with the concentration in the unfractionated extracts it was found that recovery from the starch gel averaged about 75%. Eluates of segments from a starch gel electrophoresis of an antral mucosal extract were refractionated on a Sephadex G-50 column. I"I-Porcine gastrin and 131I-labeled insulin as well as 131 I-albumin and 131 I were added to the components fractionated on Sephadex. A purified extract of porcine antra supplied by Dr. S. Tauber was dissolved at 5 #g per ml in distilled water, assayed for gastrin concentration, and fractionated on starch gel electrophoresis. Radioimmunoassay. Radioimmunoassay was

4 218 BERSON AND YALOW Vol. 60, No.2 carried out as previously described 3 using a new, more sensitive antiserum. l All concentrations obtained by radioimmunoassay are expressed in units equivalent to the same immunoreactivity of heptadecapeptide porcine gastrin I. Two recent preparations of synthetic human gastrin I prepared by Dr. K. L. Agarwal (Dr. M. I. Grossman provided us with a sample of this preparation) and Dr. J. S. Morley reacted in the immunoassay system with a potency about 80% that of porcine gastrin I. All incubation mixtures were made up to a total of 2.5 ml; bound and free labeled gastrin were separated using Rohm and Haas Amberlite resin IRP 58M. Crude tissue extracts were assayed at dilutions of 1: 500 to 1: 2500, and starch gel and Sephadex fractions at 1: 2.5 to 1: 50, diluent in all cases being 0.02 M Veronalalbumin buffer, ph 8.4, which is the same buffer used for eluting gastrin from the gels. Results The distribution of immunoreactive gastrin components in eluates from starch gel segments is shown in figure 1 (postmortem tissues) and figure 2 (surgical material). The same components are observed in the tissue extracts as were previously described in the plasma. In two of the four antra obtained postmortem and in one of the six antra obtained during surgery, almost all of the extracted gastrin was comprised of H-LG. However, substantial ICE WATER EXTRACTION BOILING WATER EXTRACTION ORIGIN FIG. 3. Distribution of immunoreactive gastrin, on starch gel electrophoresis, in an ice water extract of antral mucosa (surgical specimen; M. T.) compared with extract of adjacent piece of mucosa obtained by boiling (J) ~ E «' 3g' w' ~ 20 ~ (J)W z ~ Q ~ ~ (J) 10 a::...j ~ w ~ (! ) U z::i: OU ua:: z 0.1 ~ ii:(j) ~ u. (J)O «(!) EXTRACT OF TUMOR RESIDUAL TUMOR REEXTRACTED FIG. 4. Distribution of immunoreactive gastrin on starch gel electrophoresis in extracts of a Zollinger Ellison pancreatic tumor and in plasma obtained from same patient (Do). The concentration in the first crude extract was 44 p.g per g; concentration of gas trin in re-extract of residual piece of tumor was 9 Jl.g per g. amounts of BG ranging up to more than 60% of the total were found in the remaining seven antra. In 1 surgical case, an ice water extract of antral mucosa yielded only about one-fourth as much gastrin as that extracted by boiling, but BG was about 15% in both extracts (figure 3). The pyloric segment of antral mucosa obtained during surgery from 1 patient with duodenal ulcer (J. L. fig. 2) contained about 20% of the gastrin in the form of BG. Proximal duodenum in 2 postmortem (fig. 1) and 2 surgical cases (fig. 2) contained about equal concentrations of BG and H-LG and middle duodenum in postmortem cases (fig. 1) contained principally BG. Distal duodenal and proximal jejunal gastrin from postmortem material was composed almost entirely of BG (fig. 1). Antral and proximal duodenal extracts from a patient with Zollinger-Ellison syndrome contained substantial amounts of

5 February 1971 NATURE OF EXTRACTED IMMUNOREACTIVE GASTRIN 219 total gastrin but less than that obtained in any of the other surgical specimens from the same regions (fig. 2). The extract from the pancreatic tumor, removed from another patient with Zollinger-Ellison syndrome, contained about equal amounts of BG and H-LG; re-extraction yielded about 20% as much as the first extraction and the distribution of BG and H-LG was about the same. A plasma sample obtained from this patient prior to surgery contained less than 10% H-LG (fig. 4). The two gastrin components obtained from starch gel electrophoresis of the extracts of a surgical specimen of antral mucosa were subjected to Sephadex G-50 filtration. The less anodal component corresponded almost exclusively to BG on gel filtration and almost all of the H-LG component from the gel emerged in the zone heptadecapeptide gastrin on gel filtration (fig. 5). In the 2 cases (M. T. and J. W.) in which antral mucosa was quickly stripped from surgical specimens and frozen on Dry Ice, the yield of gastrin was considerably greater than from whole tissues kept on ice...j w :r: '" 1.5 ~ E ;:! u>g' STARCH GEL ELECTROPHORESIS (j) z o ~ 250 0:: I- 200 Z w_ U E 150 z' og u ~ 100 z ~ n:: (j)w <!.-I <.9 w (.!) C;S 200 Cl <{ 5: 150 W (fj 6; 100 SEPHADEX GEL FILTRATION GSO COLUMN I ~ ANODE ~ :::J c E, <J) C :::J u PERCENT OF ELUTION VOLUME BETWEEN ALBUMIN-I I 31 AND SALT PEAK (lodide I31 ) FIG. 5. Distribution of immunoreactive gastrin on starch gel electrophoresis in extract of antral mucosa obtained during surgery (top) and in eluates from starch gel segment no 6 (middle) and starch gel segment no 4 (bottom) on Sephadex gel filtration. Gastrin concentrations are shown in stippled bars. Distribution of labeled substances added to eluates prior to Sephadex filtration are also shown.

6 220 BERSON AND YALOW Vol. 60, No.2 for 1/2 to 1 hr prior to extraction. The quick-frozen antral mucosa from J. W. yielded 11 Ilg per g with 13% BG (fig. 6), whereas the iced antrum from which mucosa was removed 1 hr later yielded 2.6 Ilg per g with 30% BG (fig. 2). Also, two pieces of quick-frozen mucosa from M. T., extracted directly from the frozen state, yielded significantly greater amounts of gastrin (30 J,Lg per g and 25 J,Lg per g) than an adjacent piece subsequently kept at 4 C for 1 hr, which gave almost the same yield (7 Ilg per g) as the ice water extract of the quick-frozen material. The porcine antral extract supplied by Dr. Tauber contained 1.06% gastrin by radioimmunoassay using porcine gastrin I as standard. On starch gel electrophoresis there was negligible immunoreactivity except in the zone of heptadecapeptide gastrin (fig. 7). BG and H-LG components of the Zollinger-Ellison tumor extract fractionated on starch gel electrophoresis inhibited the COt«:ENTRATION OF EXTRACT 5P9/ml GASTRIN IMMUNOREACTIVITY 3'9/011 FREE P H E : N O L FIG. 6. Same as figure 1 and 2 for extract of quickfrozen antral mucosa (J. W.) Compare with figure 2 for distribution of gastrin components in adjacent piece of antral mucosa that had been stripped from antrum refrigerated for 1 hr. FIG. 7. Distribution of immunoreactive gastrin components of Tauber-Madison gastrin extract on starch gel electrophoresis. If) 0.6 C\J GP :170X10 3 DILUTION OF ANTISERUM HEPTADECAPEPTIDE PORCINE GASTRIN Do TUMOR EXTRACT ELUATES FROM STARCH GEL SEGMENTS.. 7, z 0.5 0: I- (f) <{ W z 0.3 U a:: 0 Q 0.2 mill 0.1 i f iii PORCINE GASTRIN CONCENTRATION-pg/ml FIG. 8. Ratio of antibody-bound 125I-porcine gastrin I to free 125I-porcine gastrin I as a function of the concentration of unlabeled porcine gastrin I ( e). The gastrin concentrations in eluates from starch gel segments 4 and 7 shown in figure 4 (top) were determined by radioimmunoassay at a dilution of 1 :600 by reference to the standard curve of porcine gastrin I; concentrations in all other dilutions of gel eluates were then calculated from the dilution factors.

7 February 1971 NATURE OF EXTRACTED IMMUNOREACTIVE GASTRIN 221 binding of 12'I-porcine gastrin I to guinea pig anti porcine gastrin antibodies in a parallel fashion not distinguishable from the inhibition produced by unlabeled porcine gastrin I (fig. 8). Discussion Biologically active gastrin extracted from porcine' and human 6 antra and from a Zollinger-Ellison tumor 7 8 has been shown by Gregory and his co-workers to be a heptadecapeptide of molecular weight 2,100. Tauber and Madison have obtained a peptide from hog antra 9 and from a Zollinger-Ellison tumor 10 estimated to have a molecular weight about 12,500 with an amino acid composition inconsistent with the presence of heptadecapeptide gastrin because of the absence of tryptophan. 10 The BG component of the present study could not be identified with the Tauber-Madison product. Evidence that BG appears to be a biologically active as well as an immunologically reactive gastrin component is presented elsewhere. 2 We earlier suggested that H-LG is the heptadecapeptide itself and that BG is a larger molecule that may contain within it the heptadecapeptide linked firmly to another substance of about 5000 molecular weight. 1 This suggestion was further supported by the liberation of H-LG from BG during incubation of plasma with trypsin. 2 Final proof of the nature of BG can be established only when it has been purified and its composition determined, but it can be concluded from the results of the present study that BG is present within the mucosae of stomach and proximal small bowel as well as in the plasma. Elsewhere,l1 we have shown that a preparation of Jorpes-Mutt cholecystokinin-pancreozymin (CCK-PZ) containing 10% CCK-PZ by weight, which cross-reacts weakly in t}1e gastrin radioimmunoassay system, exhibits a different electrophoretic mobility from BG on starch gel electrophoresis. Over 75% of the total immunoreactivity of CCK-PZ was obtained from starch gel eluates at or immediately anodal to the origin, which is consistent with the basic nature of CCK-PZ. None of our jejunal extracts showed immunoreactivity in this region. It is therefore unlikely that jejunal BG immunoreactivity can be attributed to CCK-PZ contamination. A small fraction of the total immunoreactivity of CCK-PZ was found in the eluates immediately in front of the serum albumin region. At a CCK-PZ concentration of 5 J,l.g per ml the total immunoreactivity corresponded to 10 ng of gastrin Eq per m!. The quantity of immunoreactivity detected in the BG region would correspond to a contamination with jejunal BG in the CCK-PZ preparations not exceeding 1 part in 600 by weight, assuming BG to have a molecular weight of about The absolute concentrations of gastrin in the mucosal tissues of the gastrointestinal tract cannot be considered established from the results of the present study. The considerably higher yields obtained from quick-frozen antral mucosa than from iced or refrigerated antrum raise the suspicion that there may be an initially rapid loss of gastrin in excised unfrozen tissue. Emfis and Fyro12 observed a marked reduction in antral gastrin (measured by bioassay) when cat cadavers were stored, initially at room temperature for 4 hr, prior to removal of antrum. We have observed reductions of 33 and 90% in the quantity of gastrin extractable from porcine antral mucosa after storage for 16 hr at 4 C and 27 C respectively (unpublished observations). However, since material obtained 5 to 6 hr postmortem did not contain significantly smaller amounts of extractable gastrin than surgical speciments refrigerated for 30 to 60 min, we cannot exclude the possibility that random variation among a small number of samples may account for the differences between the frozen and refrigerated surgical material studied here. The apparently higher ratio of BG to H-LG generally observed in plasma than in antral extracts could be a consequence of the preferential secretion of BG, more rapid removal of H-LG from plasma, or

8 222 BERSON AND YALOW Vol. 60, No.2 significant duodenal-jejunal contribution to plasma gastrin. In other studies 2 an increase in both components in plasma was observed following feeding in 2 patients with pernicious anemia. Whereas the increment was principally BG in 1 case, H-LG first appeared only after feeding in the 2nd case and then disappeared more rapidly than BG. Clearly, further studies are necessary to clarify the patterns of response to feeding and the rates of clearance of the two components from the plasma. Uvnas has suggested that the duodenum may be a more important source of gastrin than is usually assumed. 13 Although the possibility that duodenum and jejunum contribute significantly to plasma gastrin cannot be supported by the very low basal levels of plasma gastrin and the absence of response to feeding in patients who have undergone subtotal gastrectomy,14 it can hardly be supposed that duodenum and jejunum respond normally in this circumstance. REFERENCES 1. Yalow RS, Berson SA: Size and charge distinctions between endogenous human plasma gastrin in peripheral blood and heptadecapeptide gastrins. Gastroenterology 58: , Yalow RS, Berson SA: Further studies on the nature of immunoreactive gastrin in human plasma. Gastroenterology 60: , Yalow RS, Berson SA: Radioimmunoassay of gastrin. Gastroenterology 58:1-14, Smithies 0: Zone electrophoresis in starch gels and its application to studies of serum proteins. Advances Protein Chern 114:65-113, Gregory RA, Tracy HJ: The constitution and properties of two gastrins extracted from hog antral mucosa. I. The isolation of two gastrins from hog antral mucosa. Gut 5: , Gregory RA, Tracy HJ, Grossman MI: Isolation of two gastrins from human antral mucosa. Nature (London) 209:583, Gregory RA, Grossman MI, Tracy HJ, et al: Nature of the gastric secretagogue in Zollinger Ellison tumors. Lancet 2: , Gregory RA, Tracy HJ, Agarwal KL, et al: Amino acid constitution of two gastrins isolated from Zollinger-Ellison tumor tissue. Gut 10: , Tauber S, Madison LL: The isolation and characterization of porcine gastrin. J BioI Chern 240: , Tauber SD: Characterization of a pure gastrin. Edited by M. l. Grossman. Berkeley, University of California Press, 1966, p Nilsson G, Yalow RS, Berson SA: Distribution of gastrin in the gastrointestinal tract of human, dog, cat and hog, Symposium on Frontiers in Gastrointestinal Hormone Research, Special Nobel Symposia Series. Uppsala, Almqvist and Wiksell (in press) 12. Emas S, Fyro B: Antral gastrin activity in duodenal and gastric ulcers. Gastroenterology 46:1-7, Uvnas B: In discussion of Elwin CE, Uvnas B. Distribution and local release of gastrin. Gastrin. Edited by M. l. Grossman. Berkeley, University of California Press, 1966, p Berson SA, Walsh JH, Yalow RS: Radioimmunoassay of gastrin in human plasma and regulation of gastrin secretion, Symposium on Frontiers in Gastrointestinal Hormone Research, Special Nobel Symposia Series. Uppsala, Almqvist and Wiksell, (in press)