AMINO TERMINAL GASTRIN FRAGMENT IN SERUM OF ZOLLINGER-ELLISON SYNDROME PATIENTS

Transcription

1 GASTROENTEROLOGY 68: , 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.2 Printed in U.S.A. AMNO TERMNAL GASTRN FRAGMENT N SERUM OF ZOLLNGER-ELLSON SYNDROME PATENTS G. J. DocK RAY, PH.D., AND J. H. WALSH, M.D. Veterans Administration Wadsworth Hospital Center, and University of California at Los Angeles School of Medicine, Los Angeles, California n addition to the previosly described moleclar forms of gastrin, a new component has been fond in high concentrations in the serm of6 patients with Zollinger-Ellison syndrome. Serm was fractionated by gel filtration, and ion exchange chromatography, and the new component was identified in elates by radioimmnoassay sing an antibody with specificity for the N -terminal portion of heptadecapeptide gastrin. The precise chemical natre of the new component is not known, bt its chromatographic behavior and its reactivity to varios antibodies is indistingishable from that of the natral or synthetic N -terminal 1 to 13 fragment of G-17. The new component is present in gastrinoma tmor tisse. ts concentration in serm of gastrinoma patients increases markedly when secretin is injected. The Zollinger-Ellison syndrome (ZES) is cased by the elaboration of gastrin from islet cell tmors. Elevated serm gastrin concentrations can be demonstrated rotinely in these patients by radioimmnoassay. Recently, it has been shown that total serm gastrin immnoreactivity is de to several related peptides (fig. 1), inclding " little" or heptadecapeptide gastrin (G- 17),1,2 "big" gastrin (G-34),1, 2 "minigas- Received May 13, Accepted Jly 29, Address reqests for reprints to: Dr. J. H. Walsh, Veterans Administration, Wadsworth Hospital Center, Bilding 115, Room 115, Los Angeles, California G. J. Dockray was in receipt of a United States Pblic Health Service nternational Postdoctoral Fellowship. The athors are indebted to Professor R. A. Gregory and Dr. H. J. Tracy for gifts of G-33-1, G-13-1, G-17 -, and G-17 - isolated from gastrinomas, and 1:13-G-17- isolated from hog antrm. Synthetic hman 1:13-G-l7-1 and 2:17-G-17- were donated by Dr. J. Morley (.C.., Alderly Park, Cheshire, England). R. Weld gave technical assistance. To avoid confsion between the two tridecapeptides sed in this stdy, the 1 to 13 N-terminal part of G-l7 will be designated l :13-G-17, and minigastrin, which in other pblications has been designated G-13, will be designated 5:17-G-17. trin" which corresponds with the C-terminal tridecapeptide G-17 (5:17-G-17),3 "bigbig" gastrin,4 and "Component." 3 n most patients with gastrinoma, the principal circlating form of gastrin is G-34. G-17, G-34, and 5:17 G-17 occr in the serm in both slfated () and nonslfated () forms, and all these forms have been isolated from gastrinomas and normal antral mcosa. 5-8 n addition, there have been isolated from hog antral mcosa a pair of peptides with amino acid composition corresponding to the N-terminal 1 to 13 seqences of porcine G-17- and G n this stdy, the moleclar species of gastrin in the serm of patients with ZES were separated by gel filtration and ion exchange chromatography, and concentrations were estimated by radioimmnoassay sing two antisera with different specificities. n addition to the previosly described forms of gastrin, a new component was identified in serm of ZES patients which was present in high concentrations, and which has properties like the 1 to 13 fragment of G-17. Methods Sera from 6 patients with gastrinoma were collected in fasting state and at intervals of time 222

2 Febrary 1975 AMNO TERMNAL GASTRN FRAGMENT 223 Pyro- (L, Z ' G1yZ' Pro J, Gln 1, His 1, Ser Z ' Vall' Ala 1, ASP1)-Lys -LyS 1 :lj G-17 -G1 n-gly-pro-trp-le-gl S -Ala-Tr-G1Y-Trp-Met-AsP-Phe-NH2\ 5:17 G-17 L- G_17 -' Pyro pyrogl tamyl R H or S03H 5:17 G-17 G-13 FG. 1. Partial amino acid seqence of G-34, showing strctral relations between this molecle and G-17, 5:17-G-17, 1:13-G-17. after rapid intravenos injection of pre natral porcine secretin (2.0 clinical nits per kg; Gastrointestinal Hormone Research Unit, KaroEnska nstittet, Stockholm, Sweden). The diagnosis was established in 5 patients by positive identification of typical islet cell tmors which contained immnoreactive gastrin. The remaining patient had increased basal gastrin after total gastrectomy (217 fmoles per mt) and a marked stimlation of serm gastrin by intravenos secretin. Extraction of gastrin in tmor samples was made by boiling the tisse in water (1:100 w/v) for 5 min, and then centrifging the extract (4 C, 3,300 g, 5 min) and discarding the pellet. The serm samples and tmor extracts were chromatographed on colmns of Sephadex G-50 sperfine (1 by 95 cm), eqilibrated, and elted with 0.02 M sodim barbital bffer, ph 8.4, containing 0.2 g per liter of sodim aide, and rn at 4 C. Colmns were calibrated with the following pre peptides: natral hman G-34-1, natral hman G-17-1 and G-17-, synthetic N -term inal tridecapeptide of G-17-1 (1: 13-G-17 -), natral N -terminal tridecapeptide of porcine G-17-1 (1:13-PG-17-), and natral hman 5:17-G All samples were applied to the colmns together with 1 pg of monoiodinated G-17-1 as a marker. The serm protein and salt peaks in the colmn elates were detected by optical density at 280 nmaod condctivity, respectively; the fraction nmber of the elates was described in terms of percentage of eltion volme from the protein peak (0%) to salt peak (100%). n the case of tmor extracts and standard peptides, 0.1 ml of hman serm was added to the samples before they were applied to the colmns in order to provide markers for the protein and salt peaks. on exchange chromatography of serm and tmor samples was carried ot on colmns of aminoethylcelllose (AE41, Whatman). Using methods recommended by the manfactrers, AE41 was freed from fines, eqilibrated with 0.05% ammonim bicarbonate, and packed into colmns (1 by 10 cm). The condctivity of samples to be chromatographed was adjsted to that of 0.05 M ammonim bicarbonate with distilled water, and 1 pg of G-17 was added to the samples to act as a marker in colmn elates. After the samples had been applied, the colmns were washed with 10 ml of 0.05 M ammonim bicarbonate, and then developed in a continos gradient to 0.5 M ammonim bicarbonate. Radioimmnoassay of gastrin in the colmn elates was carried ot with monoiodinated G-17-1," and with two antibodies (Ab) with different specificities. Both antibodies were raised in rabbits immnied with G-l7 - conjgated to bovine serm albmin by the carbodiimide techniqe. 10 Table 1 gives the relative inhibitory potencies for several gastrins and related peptides with the two antisera. Molecles with a C-terminal portion identical to that of G-17 (i.e., G-34, 2:17-G-17, 5:17-G-17) strongly inhibited binding of label to Ab 1296, bt the N -terminal 1 to 13 seqence of G-17 and desamido G-17 - only weakly inhibited the binding. With Ab 1295, however, 1:13-G-17 and desamido G-17 had relatively high immnoreactivity, bt G-34, 2:17-G-17, and 5:17-G-17 had low immnoreactivity. Ths, Ab 1295 had specificity for the intact N -termins of G-17, whereas Ab 1296 was specific for the C-termins of G-l7. However, with Ab 1296, increasing chain length did lead to an increase in immnoreactivity. The higher immnoreactivity ofnatra5:17-g-17, compared with synthetic 2:17-G-17, with Ab 1295, may be attribted to a trace contamination of the former with G-17. Similarly, with Ab 1296, the higher immnoreactivity of natral 1:13-PG-17-1 relative to synthetic l:13-g-17-1 can be acconted for by contamination of the former with G-17. Confirmation of the specificities of the two antibodies was obtained in stdies sing synthetic 1:13-G-17-1 and 2:17-G-17-1 labeled with odination was performed by the chloramine-t method, and reaction mixtres were separated on AE celllose." Peaks corresponding to monoiodinated gastrins were sed. Table 2 shows that G-17 and :17-G-17 were strongly bond by Ab 1296, bt 12'1 1: 13-G-17 was only weakly bond. With Ab 1295, however, :13-G-l7 and G-17 were strongly bond, bt :17-G-17 was not. Figre 2 shows that with Ab 1296, the inhibition crves of G-l7-, G-34-, and ZE serm were parallel; likewise, with Ab 1295, inhibition crves of G-17-, 1:13-G-17-, and ZE serm were parallel. The concentration of the different compo-

3 224 DOCKRA Y AND WALSH Vol. 68, No.2 TABLE 1. mmnoreactive potencies of gastrins and related peptides (relative to G-17-l) with antibodies (Ab) 1295 and 1296 a Peptide Sorce Ab 1296 Ab 1295 G-17-1 Gastrinoma G-17- Gastrinoma G-34-1 Gastrinoma :17-G-17-1 Gastrinoma :17-G-17-1 Synthetic :13-G-17-1 Synthetic :13-PG-17- Hog antrm Desamido G-17-1 Synthetic % CCKb Hog dodenm < Pentagastrin Synthetic < Tetrapeptide Synthetic < < (14:17-G-17) a For many of the peptides, more than one preparation was stdied, and the vales given represent the range of immnoreactive potency which was encontered. b CCK, cholecystokinin. TABLE 2. Ratio of bond-free "', for monoiodinated G-17, 1:13-G-17, and 2:17-G-17 with antibodies (Ab) 1296 and 1295 a "'1-G :13-G-17 2:17-G-17 Control (no antibody) Ab :20, : D Ab :20, :150, a Antibody sed at two concentrations: one was a working diltion, the other a high concentration to indicate maximal binding of label. nents in serm and tisse has been expressed in two ways. n the first, concentration was expressed with respect to a standard of pre natral hman G-17 -, in order to determine the relative contribtion made by the different components to total immnoreactivity. However, when expressed like this, estimates of the concentration of G-34 and N-terminal fragments "of G-17 are probably nderestimates, becase the immnoreactivity of G-34 and fragments, sch as 1:13-G-17 and desamido G-17, were less than that of G-17. n addition, therefore, we have sed standards of pre natral hman G-34- in estimating concentration of G-34, and synthetic hman 1:13-G-17 for determining concentrations of the N-terminal fragment. Reslts Figre 3 shows the separation of gastrin components in the serm of a patient with gastrinoma that was obtained by gel filtration on colmns of Sephadex G-50. Also shown are reslts from several rns in which standard pre gastrins were chromatographed on the same colmn nder identical conditions, and elates assayed with both antibodies. Ab 1296 revealed the presence of two main peaks of immnoreactivity in the elates of ZE serm. The first peak corresponded in position to G-34, while the second peak emerged from the colmn in the same position as nlabeled G-17 - and slightly in front of the G-17 marker. The small peak which elted jst ahead of G-34 corresponds to the component of Rehfeld and Stadil. 3 n this patient, there was no immnoreactivity in the void volme of the colmn, so that the big, big gastrin of Yalow and Berson 4 was absent, althogh in other ZE patients we have observed this component. With Ab 1295, no peak of immnoreactivity was fond in the G-34 region. The G-17 peak was detected eqally well by Ab 1295 and by Ab However, the main peak of immnoreactivity detected by Ab 1295 was between the G-34 and G-17 regions. ts eltion volme (50%) was the same as that of synthetic 1:13-G-17 (49%, mean of three experiments) and less than that of desamido G-17 (55%, mean of two experiments), and G-17-1 (62%, mean of for experiments). Detection of this peak only by the antibody with N-terminal specificity sggests that this material has an

4 Febrary 1975 AMNO TERMNAL GASTRN FRAGMENT rl (; f 0.8 " 0.6 o ::> :i! 0.4 Q!i a: 0.2 o FNAL DLUTON ZE SERUM ()111m!) Qp5 0;5 10 :;g.o 0;1 Lp ' b Ab :136'17-1 & ZE SERUM 0.'1.b do 000 PEPTDE CONCENTRATON (f.monn!) FG. 2. Competitive inhibition crves for G-17 -, G-34-1, 1:13-G-17-, and serm from patient with gastrinoma, with antibody (Ab) 1296 (final diltion 1:300,0(0) and Ab 1295 (final diltion 1:180,000). Trace was monoiodinated G-17-. ZE, Zollinger Ellison. N -terminal partian clasely resembling that.of G-17, and the same "N-terminal peptide.of G-17" (NT-G-17) is adapted ta describe it. After gradient eltian chramatagraphy (AE 41).of serm fram a patient with ZES, Ab 1296 indicated the presence.of twa majar peaks ( and ) and twa minar peaks ( and V) in the elates (fig. 4). Ab 1295 revealed peaks.of immnareactivity carrespanding ta peaks and V.only G-17, which had been applied with the sample, appeared in the elates between peaks and V. The fractians carrespanding ta each.of the far peaks in the AE elates were separately paaled, lyaphilied, and rechramatagraphed an Sephadex G-50. mmnareactive material in AE peak had an eltion valme an Sephadex G-50 similar ta that.of G-34 (fig. 5). n ather experiments, it was fand that AE peak behaved identically ta AE peak an Sephadex G-50. The chromatagraphic and immnoreactive praperties.of AE peaks and are therefare characteristic.of G-34-1 and G-34-, respectively. When AE peak was applied ta Sephadex G-50 twa main campanents appeared in the elates,.one with an eltian valme and pattern.of immnareactivity characteristic.of G-17 -, and the ather characteristic.of the NT -G-17 previasly identified (fig. 5). Rechramatagraphy.of AE peak V an Sephadex G-50 failed ta resalve twa distinct immnareactive peaks; hawever, the immnareactivity detected by Ab 1295 emerged fram the calmn distinctly earlier than that revealed by Ab Fractians taken from the ascending slape.of the peak (x) and fram the descending slape (y) were separately rern an Sephadex G-50 and were fand ta yield single peaks.of esr-;entially hamageneas reactivity typical.of NT-G-17 and G-17-, respectively (fig. 6). The imperfect separatian.obtained an Sephadex G-50 reflects the clase similarity in the eltian valmes.of NT -G-17 and G-17- which is apparent fram a stdy.of figre 2. The relative cancentratians.of different farms.of gastrin in the fasting serm.of 6 patients with gastrinamas was.obtained by integratian.of the peaks in Sephadex G lao!; 60 ::: Q!i 30 a: a: 10 en 5 "'" D A R D --D % EWTON \QLUME FROM PROTEN TO SALT PEAKS FG. 3. Upper trace, record of five separate colmn rns in which standard samples of G-34-, 1:13-G-17-1, G-17-, G-17-, and 5:17-G-17-1 were applied to the same Sephadex colmn and elted nder conditions identical to those for gastrinoma serm shown in lower panel. Lower trace, eltion pattern obtained when 0.5 ml of serm of patient with gastrinoma was chromatographed on Sephadex G-50, sperfine (1 by 95 em). Serm obtained at srgery. Flow: 6 ml per hr, fraction sie l.0 m!. All the tbes in the elates were assayed with both Ab 1295 (- - -) and AB 1296 (--), and immnoreactivity is expressed in terms of a standard of G-17-. ZE, Zollinger-Ellison.

5 226 DOCKRA Y AND WALSH Vol. 68, No. 2 1! " 6()() ii :;; ;; o ZE SERUM ON AE CELWLOSE " 1\ FRACTON NU M BER FG. 4. Eltion profile of 0.1 ml of serm from gastrinoma patient on aminoethylcelllose (AE41). Colmn (1 by 10 cm) eqilibrated with 0.05 M ammonim bicarbonate and from fraction 10 developed with a continos gradient to 0.5 M ammonim bicarbonate. Volme of mixing vessel 125 m!. Rate of flow, 6 ml per hr; fraction sie, 1.0 m!. Tbes were assayed as in figre 3. Peaks identified by Roman nmerals. Fractions corresponding to each of the peaks were pooled and lyophilied. ZE, Zollinger-Ellison. elates (table 3). The concentration of the different moleclar forms have been expressed as percentage of total immnoreactivity with each antibody sing G-17 - as a standard, so that an assessment can be made of the contribtion of each of the moleclar forms to total immnoreactivity measred by the two antibodies. n all patients, G-34 was the main circlating component estimated by Ab n 2 patients, NT-G-17 was the major component estimated by Ab Total serm immnoreactivity estimated by Ab 1295 was abot 20 to 50% that estimated with Ab 1296 (5 of6 patients). The component of Rehfeld and Stadil,3 and big, big gastrins were only minor components in each of the patients stdied here. None of the serm samples examined appeared to contain significant amonts of 5:17-G-17-; this may be de either to low concentrations present in the serm, or to the fact that the colmns sed (1 by 95 cm) were not long enogh to yield the clear ct separation of 5: 17 -G-17 from G-17 that can be obtained with longer colmns (1 by 200 cm).s After rapid intravenos injection of secretin (in 4 patients), there was a 2- to 8-fold increase in serm gastrin concentration estimated by the two antibodies. The gastrin concentration estimated by both antibodies reached a peak at 5 min after injection and declined to basal levels 30 to 45 min after injection. n all 4 patients, the increases in serm gastrin concentration : l 20 = '" j <!) 20 r.\ \ -G-17 ' 1 " c: E t 2OO ;)\ 100! o " ELUTON FROM PROTEN TO SALT PEAKS FG. 5. Peaks in elates of aminoethylcelllose (A E) colmn re-rn on Sephadex G-50. Samples reconstitted in 1.0 ml of saline and aliqots applied to colmns together with 0.1 ml of hman serm and 1 pg of G-17. Fractions identified as "x" and "y" rechromatographed on Sephadex G-50. Conditions as in figre 3., 100 FRACTON Y g ffl 60!;t G 17!\ t5 A _ - w 8 60 i; 20 «<!) 100 FRACTON 'X".. : \.,.! \ : \ / =.E t o Z :> o % ELUTON OLUME FROM PROTEN TO SAlT PfAKS FG. 6. Rechromatography of fractions "x" and "y" (fig. 4) on Sephadex G-50. Conditions as in figre 3. 8

6 Febrary 1975 AMNO TERMNAL GASTRN FRAGMENT 227 TABLE 3. Relative concentration of gastrin components in the basal serm of 6 patients with gastrinoma estimated from immnoassays with antibodies (Ab) 1296 and 1295 a Patient Total gastrin Ab 1296 Ab 1295 % of total BBG' Comp' G-34 G-17 Total gastrin %oftotal BBG G-N'" G-17 {malelml {malelml W. E S , , H.A... 26, , S. T.... ' P. A M. C.. 8, , a Concentrations were calclated from eltion patterns on Sephadex colmns, sing G-17 - as the standard. BBG, "big-big" gastrin. C Comp, component 1. d NT, N-terminal. cold be attribted to increases in G-34, G-17, and NT-G-17 (fig. 7, table 4). n order to determine whether NT-G-17 was generated from either G-34 or G-17 de to action of serm enymes, pre samples of G-34- and G-17- were incbated with freshly collected hman serm or with saline for 30 min at 37 C. Figre 8 shows that no changes were apparent in the immnoreactivity and gel filtration properties of G-34 and G-17 after incbations in serm or saline. mmnoreactive material with properties corresponding to the NT -G-17 of serm was fond in extracts of the tmors of 5 patients. When an extract of gastrinoma was analyed by gradient eltion chromatography on AE 41, for peaks were identified in the elates which corresponded closely with the for peaks fond in serm (fig. 9). Rechromatography of gastrinoma peak on Sephadex G-50 gave two peaks with the properties identical to G-17 -, and the NT-G-17 of serm. An analysis has been rr'ide of the concentrations of NT -G-17, G-34, and G-17 in gastrinomas of 5 patients (table 5). Discssion This stdy has shown that in addition to the previosly identified gastrin components in the serm of ZES patients, there is another component with some immnochemical properties of gastrin. Becase of its pattern of immnoreactivity, it is assmed that the new component has an N-terminal portion closely resembling that ofg-17. An antibody with specificity for the amino terminal portion of gastrin similar to that of Ab 1295 has previosly been described by Agarwal et at. 11 The specificity of Ab 1296 (carboxyl termins of gastrin) is essentially similar to that of antibodies sed in other gastrin immnoassay At the start of this stdy, it had been hoped that, by sing Ab 1296 and Ab 1295 separately to measre total serm gastrin immnoreactivity, it wold be possible to make estimates of the relative concentrations of G-34 and G-17 withot prior fractionation. However, the discovery of NT -G-17 in serm means that immnoreactivity with an antibody having N-terminal specificity cannot be sed as an index of G-17 concentration. NT-G-17 was not separated from G-17 by ion exchange chromatography on AEcelllose, indicating that the two components have a similar charge. However, NT-G-17 was fond to emerge from the Sephadex colmns between G-34 and G-17. Theoretically, large molecles shold travel faster on Sephadex colmns than smaller ones, so that at first sight NT -G-17 wold appear to be intermediate in sie between G-34 and G-17. However, the 1 to 13 fragment of hman G-17 - and of porcine G-17- were both fond to migrate on Sephadex colmns between G-34 and G-17, indicating that gastrin-related molecles do not behave entirely as predicated.

7 228 DOCKRA Y AND WALSH Vol. 68, No.2 E. '0 E Min POST SECRETN /\-1 G 17-1 / ; i!\, '\ : /..:.... : Ul /-,,'. j, :3 c Q./ /. /: \. C( :: 10 / i \\\_'\., L.. /' -,'. ' ' ' -. - w 0 0!!!!!:.,'" :: 1\ \ Ul /\. J \c., c C :l '....,. '-.::::,.., % ELUTON VOLUME FROM SERUM PROTEN TO SAlT PrAKS FG. 7. Lower trace, eltion pattern obtained with 1.0 ml of basal serm from patient with gastrinoma. Conditions as in figre 1. Upper trace, serm (1.0 m\) from same patient, 5 min after the injection of pre natral porcine secretin (2.0 clinical nits per kg). Conditions as in figre 3. TABLE 4. Concentration of moleclar forms of gastrin in serm of gastrinoma patimts before and 5 min after rapid intravenos injection of secretin" Patient Basal gastrin concentrations 5 min postsecretin gastrin concentrations Comp G 34 G-l7 NT-G- l7 Comp G-34 G-l7 NT-G-l7 fmoles/ml fmoles/m W. E , , ,728 2,115 4,925 S. T , ,830 P. A ,949 1,341 1,000 M. C ,255 1,656 7,800 14,530 4,770 14,550 a Concentrations were estimated from eltion patterns on Sephadex G 50 sing antibody (Ab) 1296, (component, G-34, G-17) and Ab 1295 (NT-G-l7). G-17- was sed as the standard for estimating G-17 and component concentration. Standards of G-34 and synthetic 1:13-G-17 were sed for estimating concentrations of G-34 and NT -G-17, respectively. " Big-big" gastrin was identified in only 2 patients, and its concentration did not change after secretin. NT, N-terminal. One possible explanation for the anomalos behavior of G-17 relat ive to 1: 13-G-17 is that G-17 contains two tryptophan and one phenylalanine reside, whereas 1:13-G-17 has only one tryptophan. Peptides with a high proportion of aromatic amino acids are known to be retarded on Sephadex.

8 Febrary 1975 AMNO TERMNAL GASTRN FRAGMENT i 100 "- ] 80!:.60 l:l 40!;; :3 20 '" fi 100 0::! 80 r o NCUBATED SERUM /1"1.'17 " ' " " il j""'" i i r G \-G-17 \ (OO'! [100 Ul o o ro % ELUTON VOLUME FROM PROTEN TO SALT PEAKS FG. 8. Upper trace, eltion pattern obtained when 10.0 pmoles of G-34- and 10.0 pmoles of G-17- incbated in 1 ml of hman serm at 34 C for 30 min, and then chromatographed on Sephadex G-50. Conditions as in figre 3. Lower trace, as pper trace, bt G-34- and G-17- were incbated in 1 ml of normal saline. All the moleclar forms of gastrin which occr in the tmor and circlation of gastrinoma patients also occr in normal antral mcosa. Since the 1 to 13 fragment of G-17 has been isolated from hog antrm and NT-G-17 has immnochemical and gel filtration properties indistingishable from 1: 13-G-17, it seems reasonable to sggest that NT -G-17 may be the 1 to 13 fragment of G-17. t is nlikely that NT-G-17 is a mch smaller fragment than the tridecapeptide, for it wold then emerge later in the colmn elates; on the other hand, a larger fragment of gastrin wold be expected to contain the, tryptophan at position 14, and oght therefore to behave like G-17. n this stdy, we have sed G-17 - as a standard for estimating concentrations of NT-G-17. This was done in order to obtain an estimate of the contribtion made by NT-G-17 to total immnoreactivity measred by Ab 1295 in immnoassays of serm. However, there was abot a 5-fold difference in immnoreactivity of G-17-1 and synthetic N -terminal fragments, sch as 1:13-G-17-1 and desamido G-17-1, so that concentrations of the N -terminal fragment in serm, which were estimated from a standard of G-17-, may well be nderestimates. We have also sed 1:13-G-17- as a standard to determine NT-G-17 concentrations in serm and gastrinoma, and the vales so obtained are probably close to the actal concentration of the N -terminal peptide. However, ntil NT-G-17 is flly characteried, estimates of its concentra- 400 (/) w!c[ 100 ::J...J W 0 Q!c[ a: w (/) <l C> 20 o GASTRNOMA EXTRACT ON AE CELLULOSE eo 100 FRACTON NO. AE PEAK. ON G \ 'Y. ELUTON VOLUME FROM PROTE'" TO SAl' P[A<S r 100 [0.i e 200' f ' o o o 6 FG. 9. Upper trace, eltion profile of gastrinoma extract on aminoethylcelllose (AE41). Conditions as in figre 4. Lower trace, fractions corresponding to peak -AE elates were pooled, lyophilied, and reside was dissolved in 1.0 ml of normal saline, 0.1 ml chromatographed in Sephadex G-50. Conditions as in figre 3. TABLE 5. Concentration of G-34, G-17, and NT-G-17 in tmor extracts of 5 patients a Patient G-34 G-l7 NT-G-17 nmole/g nmole/g nmole/g W.E S H.A P. A M. C a Concentrations were estimated from eltion patterns on Sephadex G-50, and from radioimmnoassay sing antibody (Ab) 1296 (G-34 and G-17), and Ab 1295 (NT-G-l7). Standard for assays as in table 4. Component and "big-big" gastrin were not detected in any of the extracts. NT, N-terminal.

9 230 DOCKRA Y AND WALSH Vol. 68, No. 2 tion in the serm mst necessarily be relative. senberg et al. 16 first demonstrated the stimlatory action of secretin infsions on serm gastrin concentration in gastrinoma patients. The rapid increase in the concentration of serm gastrin components, which occrred after intravenos bols secretin injection, sggests that secretin acts by releasing the stores of gastrin in the tmor cells, rather than by increasing the rate of gastrin synthesis. Since NT-G-17 was identified in tmor extracts, and since its concentration in serm increased rapidly after secretin, it wold seem that this molecle is stored by the tmor together with G-17 and G-34. Conceivably, the fragment may be synthesied in its own right, or, it may be prodced from the breakdown of G-17 or G-34 within the synthesiing cell. Additional stdies of NT -G-17 may be expected to yield information on the synthesis and metabolism of gastrin. N-terminal fragments of G-17 have no known biological activity. However, the possibility that NT -G-17 may be the 1 to 13 fragment of G-17 is of particlar biological interest, since the fragment of G-17 which with 1 to 13 makes p the whole G-17 molecle is the C-terminal tetrapeptide. t is well known that the C-terminal tetrapeptide of gastrin is the minimal fragment of the molecle which has appreciable biological activity. f the 1 to 13 fragment of G-17 occrs natrally then it is reasonable to sggest that tetrapeptide also occrs. Althogh antibodies with specificity to the C-termins of gastrin are widely sed in immnoassays, these do not bind well to tetrapeptide alone, and there have, so far, been no reports of natrally occrring C-terminal tetrapeptide. This stdy i-ndicates the desirability of a search for gastrin tetrapeptide. REFERENCES 1. Yalow RS, Berson SA: Sie and charge distinctions between endogenos hman plasma gastrin in peripheral blood and heptadecapeptide gastrin_ Gastroenterology 58: , Berson SA, Yalow RS : Natre of immnoreactive gastrin extracted from tisses of gastrointestinal tract. Gastroenterology 60: , Rehfeld JF, Stadil F: Gel filtration stdies on immnoreactive gastrin in serm from Zollinger Ellison patients. Gt 14: , Yalow RS, Berson SA: And now "big, big" gastrin. Biochem Biophys Res Commn 48: , Gregory RA, Tracy HJ, Agarwal KL, et al: Aminoacid constittion of two gastrins isolated from Zol1inger-El1ison tmor tisse. Gt 10: , Gregory RA, Tracy HJ: solation of two " big gastrins" from Zollinger-Ellison tmor tisse. Lancet 2: , Gregory RA, Tracy HJ: The constittion and properties of two gastrins extracted from hog antral mcosa. Gt 5: , Gregory RA: The gastrointestinal hormones: a review of recent advances. J Physiol 241:1-32, Stadil F, Rehfeld JF: Preparation of "'-labelled synthetic hman gastrin for radioimmnoanalysis. Scand J Clin Lab nvest 30: , McGigan JE: mmnochemical stdies with synthetic hman gastrin. Gastroenterology 54: , Agarwal KL, Grdinski S, Kenno GW, et ll: mmnochemical differentiation between gastrin and related peptide hormones throgh a novel conjgation of peptide to proteins. Experientia 27: , McGigan JE: mmnochemical stdies with synthetic hman gastrin. Gastroenterology 54: , Yalow RS, Berson SA: Radioimmnoassay of gastrin. Gastroenterology 58:1-14, Rehfeld JF, Stadil F, Rbin B: Prodction and evalation of antibodies for the radioimmnoassay of gastrin. Scand J Clin Lab nvest 30: , Walsh JH, Debas HT, Grossman M: Pre hman big gastrin; immnochemical properties, half-life and acid-stimlating action in dogs. J Clin nvest 54: , senberg J, Walsh JH, Passaro E Jr, et al: Unsal effect of secretin on serm gastrin, serm calcim, and gastric acid secretion in a patient with sspected Zollinger-Ellison syndrome. Gastroenterology 62: , 1972