HEPTADECAPEPTIDE GASTRIN: MEASUREMENT IN BLOOD BY SPECIFIC RADIOIMMUNOASSAY

Transcription

1 GASTROENTEROLOGY 71: by The Williams & Wilkins Co. Vol. 71.!IIo. 6 Printed In U.S.A. HEPTADECAPEPTIDE GASTRIN: MEASUREMENT IN BLOOD BY SPECIFIC RADIOIMMUNOASSAY GRAHAM J. DOCKRAY, PH.D., AND IAN L. TAYLOR, M.R.C.P. Physiological Laboratory. University of Liverpool, Liverpool, England The characteristics are described of an antibody (designated L6) which.has virtally absolte specificity for heptadecapeptide gastrin. This antibody binds G17, bt does not bind peptide fragments or moleclar forms of gastrin comprising G17 with either amino acid deletions, or additions, at the carboxyl- and amino-terminals. In serm from patients with Zollinger-Ellison syndrome the only form of gastrin revealed by L6 was compatible with G17, and there was good agreement between estimated G17 concentrations in serm analyed by gel filtration and by direct radioimmnoassay sing L6. Using L6 in conjnction with antibodies specific for carboxyl- and amino-terminals of G 17 it has been possible to measre concentrations of different forms of gastrin in serm of normal sbjects after a meal in greater detail than previosly possible. After a light meal consisting of eggs, toast, and Oxo, serm concentrations of G 17 measred by L6 increased to a peak 2 min after feeding (A gastrin, 19 pmoles per liter; n = 17). In contrast, concentration of G34 peaked at 5 min (A gastrin, 27 pmoles per liter). Small amonts of amino-terminal fragments of G 17 were present throghot the digestive period. Applying the known ratio of biological potencies of G34 and G 17 for stimlation of acid secretion in man, it is estimated that G 17 acconts for abot 75% of the biological activity in blood after a meal, even thogh G34 is present in higher molar concentrations. Gregory and Tracy! first isolated the antral hormone, gastrin, in the form of a pair ofheptadecapeptide amides that were identical bt for the presence (G17-II) or absence (G 17 -I) of a slfate grop on their tyrosine reside. The sbseqent availability of pre natral and synthetic G17-I allowed the development of radioimmnoassays (RIA) sfficiently sensitive to measre conconcentrations of the hormone in normal blood. 2. Using a gastrin RIA, Yalow and Berson 5 discovered that in patients with elevated serm gastrin concentrations de to the Zollinger-Ellison syndrome (ZES), and in patients with pernicios anemia, the main circlating form of gastrin was larger and less acidic than G 17. This form of gastrin has been named big gastrin. Two tetratricontapeptides (G34) have now been isolated from ZES tmor tisse (gastrinomas) and hog antral mcosa which show the same chromatographic and immnochemical properties as big gastrin.' Like G17, the two larger Received Febrary 9, 1976, Accepted May 19, Address reqests for reprints to: G. J. Dockray, Department of PhYSiology, University of Liverpool, Brownlow Hill. P.O. Box 147, Liverpool L69 3BX, England. This work was spported by grants from the Medical Research COncil and the Nffield Fondation. The athors are indebted to Mrs. Elaine Clarke for skilled technical assistance. Samples of GI7-I and n, G34-I and 11 were generos gifts of Professor R. A. Gregory and Dr. H. J. Tracy. Le" G17 I was a gift from Professor E. Wiinsch. Gifts of 2 to 17 G17 I, 1 to 13 G17 I, and Pentagastrin were donated by Dr. J. Morley (lci, Alderly Park, Cheshire, England). Gifts of 14 to 17 G17 I and feline G17 I were from Professor G. Kenner and Dr. P. Richards (Department of Organic Chemistry, University of Liverpooil. peptides may be either slfated (G34-II) or nslfated (G34-1), and both yield G17 on treatment with trypsin. Several other moleclar forms of gastrin have also been described; these inclde two forms larger than G34: big, big gastrin (BBG): and "component I," neither of which has yet been chemically or biologically character ied. Also a carboxyl-terminal fragment of G17, called "minigastrin," corresponding to the carboxyl-terminal 14 amino acids of G17 (G14), has been fond in low concentration in the tmors and blood of patients with ZES.8 In man and in dog, the clearance rate of exogenosly administered G34 is abot one sixth that of G17; in contrast, the potency of G34 (increment in molar con centration in blood reqired for a given gastric acid response) is abot 6 times less than G Becase different forms of gastrin have different biological activi ties, it is clear that any attempt to relate concentrations of endogenos hormone in the circlation to a particlar biological response mst take accont of the relative concentrations of different moleclar forms. Hitherto the most commonly sed way of estimating concentrations of the different forms of gastrin has been to fractionate samples by gel filtration, sally on Sephadex G-5, and measre gastrin in the coimn elates by RIA. Sch methods are laborios and fre qently involve loss of sensitivity de to diltion of the sample after passage throgh the colmn. Clearly, specific RIA for the principal moleclar forms in serm wold provide an attractive alternative to fractionation methods. 971

2 972 DOCKRA Y AND TA YLOR Vol. 71,No.6 Recently, it has been sggested that antibodies specific for the carboxyl-termins of gastrin (the most common type of gastrin antibody) cold be sed to measre G 17 and G34, whereas antibodies specific for the amino-termins of G17 wold measre this form alone. 12 However, the validity of this approach is dobtfl, for in the serm of patients with ZES and in normal antral mcosa there is one or more amino-terminal fragments of G 17 which cross-react with amino-terminal specific antibodies. IS This stdy reports now on the p r o p of r an t insal e s gastrin antibody that appears to be virtally absoltely specific for G17, that is to say it does not cross-react significantly with either carboxylterminal or amino-terminal fragments of G17, bt reqires the integrity of the entire molecle. This antibody offers for the first time the capacity to measre heptadecapeptide gastrin in serm withot prior fractionation, and withot problems arising from cross-reactions with fragments of G17 or larger molecles encompassing G17 within their strctre. Methods Sbjects. Stdies were made on the serm obtained from 6 patients w i ZES. h The diagnosis was established by elevated fasting serm gastrin concentrations (+5 pmoles per liter) together with the presence of pancreatic tmors containing immnoreactive gastrin and/or an anomolos response to secretin." Serm was also collected from 17 normal sbjects (4 female, 13 male, ages 18 to 31 years) who were either medical stdents or laboratory workers and were free from gastrointes tinal tract symptoms. After an overnight fast the normal sbjects ate, over a period of 1 min, a standard breakfast comprising two hard boiled eggs, one piece of toast and a cp of Oxo. Serm samples were obtained 15 min, and immediately before the meal period, then at lo min intervals for 6 min, and thereafter at 15-min intervals for a frther hor. All blood samples were allowed to clot at 4 C (3 min), and serm was separated by centrifging (2 x g, 1 min) and stored at -3 C before assay. RIA. Serm gastrin was measred by RIA according to pblished methods.",. Three antisera were sed. The charac. t e i sof t itwo c s of these, 1296 (specific for the carboxyl.ter. mms of G17) and 1295 (specific for the amino termins of G17) have already been described." The third antiserm, designated L6, was raised in a New Zealand white rabbit after immniation with a mixtre of approximately eqal amonts of natral porcine G17-1 and -II conjgated to bovine serm albmin (molar ratio of G17 to albmin in the conjgate was 8:1)." The conjgate was dissolved in saline, emlsified in Frend's c o m p adjvant, l t e and a dose eqivalent to 5 pg of G17 was admmlstered by mltiple intradermal injection over the dorsal srface; simltaneosly,.15 ml of pertssis vaccine was administered intramsclarly.' At 6 to 8-week intervals the.ra.bbit received booster shots of the same conjgate administered as before, bt withot the pertssis vaccine. Bleedings were taken 7 to 1 days after each boost and stdies described here were made sing antiserm taken after the forth boost. In view of the special characteristics of this antiserm it is worth pointing ot that rabbit L6 was 1 of 6 animals receiving this conjgate nder the same conditions. Of the remaining 5 rabbits, 2 failed to prodce antisera, 1 prodced an amino-terminal specific antibody, 1 prodced a carboxyl terminal specific antibody and the last rabbit pro dced a mixed poplation of antibodies. lui-labeled G17 or related peptides were prepared by the chloramine-t method, prified by ion exchange chromatogra phy,,. and peaks corresponding to monoiodinated peptide were sed. Rotinely, 1U1 labeled G17-1 was sed in assays and a standard of G17-I was employed. Where concentrations of G34 were estimated a correction was applied to allow for slight differences in immnoreactivity of G17 and G34.13 Unless otherwise stated, all peptides and standards were of hman origin. Assays made with 1296 and 1295 were able to detect.25 p oper l liter e of standard G 17 -I, whereas sensitivity of assays smg L6 was.5 to.6 pmole per liter. Antibody specificity. The specificity of L6 was established in two ways: (1) binding of labeled peptides to antibody, and (2) inhibition of binding of "51-labeled hman G17 I by nlabeled peptides. In table 1 are recorded the diltions of antisera reqired to bind 5% of varios labeled gastrins. In confirma tion of previos reslts it is clear that 1296 bond 5% of labeled G17 (.5 fmole) and other peptides with a similar a r b o x y - t(g34, e r 2 m to i n17 G17) s at high titers, bt was mcapable of binding 5% of labeled amino-terminal fragment (1 to 13 G 17) even at 25- to 4-fold lower diltions. In contrast, 1 bond 9 5 5% oflabeled hman G17 and 1 to 13 G17 at high hters, bt at 2-fold lower diltions failed to bind 5% of labeled carboxyl terminal fragment (2 to 17 G17) or G34. The binding spectrm of L6 was distinct from that of either 1296 or 1295; L6 bond 5% of labeled hman G17 and molecles closely related to it at high titers, bt at 8-fold lower diltions was nable to bind 5% of labeled carboxyl- and amino-termi nal fragments ofgl7. A similar pattern of immnoreactivity emerged from the second series of experiments in which 15 peptides of the gastrin series were stdied for their ability to inhibit binding of IUI-GI7 to antibody. Typical binding crves for L6 are shown in figre 1; it is clear that the binding of label was inhibited by nlabeled hman G 17 and serm from a patient with ZES, and crves were parallel. In contrast, G34, 2 to 17 G17 and 1 to 13 G17 showed no appreciable inhibition of binding. In other experiments, hman and porcine G17's in either slfated or nslfated forms, cat GI7.1, and Le" hman G17-1 showed high immnoreactivity with all three antisera and inhibition c with e s each antiserm were parallel (not shown); comparison of doses reqired for 5% inhibition of binding are given in table 2. Natral porcine and hman G34-1 or -II, 5 to 17 GI7-I, and other peptides with carboxyl termini closely resembling G17 showed high immnoreactivity with 1296 bt not with 1295 or L6. However, amino-terminal fragments of G17 showed immnoreactivity with 1295 bt not with 1296 or L6. Taken together these reslts illstrate the carboxyl termi nal specificity of 1296 and the amino-terminal specificity of 1295, and demonstrate the differences between these two antibodies and 1.6. In particlar, it is clear that the specificity of L6 is directed toward the intact G17 molecle and that TABLE 1. Diltion of antibody binding 5% of trace labeled monoiociogostrin Label Antibody L6 G17-1 1:37. 1:17. 1:13. PorcineGl7 J 1:29, 1:18, 1:81, Le"GI7 1 1:4. 1:18, 1:18, 2-17 G17-1 1:4, <1:1, 1:1, G34-1 1:26, <1:1, <1:1, 1-13 G17 1 <1:1, 1:23, <1:1,

3 December 1976 HEPTADECAPEPTlDE GASTRIN IN BLOOD 973 FINAL DILUTION ZE SERUM (",./mll O I W " INITIAL IIF 4 HO-17-1 HO HO HO-17-1 o ZE SERUM 2 CONCENTRATION OF PEPTIDE (p mol/ii FIG. 1. Competitive inhibition crves for hman (H) G17-I, G34-I, 2 to 17 G17-I. and 1 to 13 G17-1 with L6 (1:1,) and a label of '''I-GI7-I. Also shown is an inhibition crve for the addition of graded amonts of serm from a patient with Zollinger-Ellison syndrome (ZES). Ordinate: ratiq of bond (B) to free (F) "'1-GI7-1 as a percentage of ratio in the absence of nlabeled peptide or serm. Abscissa: concentration of nlabeled peptide or serm. 1 1 TABLE 2. Potencies of pep tides relative to hman G 17-1 ( 1.) in inhibiting finding of '25/-G17-1 to antibody (n = natral peptide. s synthetic peptide) Peptides Anhod\' ;; L6 Heptadecapeptide gastrins n G n GI7-II n Porcine GI7-I (Met' hman) s Feline G17-1 (Ala' hman) s Le"GI7-I Carboxyl-terminal gastrin peptides n G34-I.44 <.1 <.3 n G34-II.44.4 <.3 n Porcine G34-II.64.2 <.3 s 5-17 G <.1 <.3 s 2-17 G s Pentagastrin.2 <.1 <.1 s Cholecystokinin carboxyl-.27 <.1 <.1 terminal octapeptide Amino-terminal gastrin peptides s Desamido G17-I s 1-13 GI7-I <.2.88 <.3 Mixtres s 1-13GI7-I} s 14-17GI7-I Eqimolar amonts. immunoreactivity is almost abolished by alterations at either termins. whether in the form of amino' acid deletions. or extensions of the chain. Minor amino acid sbstittions within the body of the peptide chain. sch as distingish feline, porcine. and hman G17's, are tolerated withot significant change in immnoreactivity. bt the importance of the peptide backbone is emphasied by the fact that a combination eqimolar amonts of 1 to 13 G17 and 14 to 17 G17 did not case significant inhibition. Validation. Data presented in detail in the Reslts section indicate that in serm of ZES patients and normal sbjects after feeding. G17 is the only form of immnoreactive gastrin measred by L6; these observations provide the basis for sing L6 as a specific antibody for G17 estimations by RIA. The validity of this proposition was established in three ways. First, in a series of serm samples with gastrin concentrations in the normal range, serm G 17 was estimated both by specific RIA sing L6, and by fractionation of serm on Sephadex G-5 and application of 1296 in RIA of the colmn elates. Colmns of Sephadex G-5 sperfine (1 by 1 em) were rn at 4 C in.2 M sodim barbital bffer. ph 8.4, containing.2% sodim aide. All samples applied to the colmn were fortified with markers of '''I-GI7, l2s1-iabeled bovine serm albmin (to indicate void volme), and Na l26 1 (salt peak); it shold be noted that emerges from the colmn with an eltion volme of abot 112% compared to 1% when condctivity is sed to estimate salt peak, so that the eltion volmes of standards in this stdy are slightly less than those previosly reported. U On separate occasions the colmns were calibrated with standards of G34-I, G 17 -I. and -II; in these experiments 9 to 11% of immnoreactive gastrin applied to the colmn was recovered in the elates. and there were no differences in this respect between G34 and G 17. The recovery of immnoreactive gastrin in serm samples rn on Sephadex G-5 estimated by RIA with sing standard of G17. was 5 to 15/, of that applied (mean. 11%); estimations of the amont of G 17 in the serm samples were adjsted to take accont of the recovery of total immnoreactivity. Second. the three antisera were sed to estimate gastrin concentration in a series of serm samples to which had been

4 974 DOCKRAYAND TAYLOR Vol. 71, No.6 added varying amonts of G34-I, G 17 -I, and 1 to 13 G 17 -J. The concentrations of G 17 were estimated directly by RIA sing L6, allowing for immnoreactive gastrin in the original serm sample (blank assay). Concentrations ofg34 and 1 to 13 G17 were determined by sbtraction of estimates made by L6 from those made with 1296 and 1295, respectively. Concentration of G17 estimated by L6 varied from 72 to 15% (mean, 89%) of that added. The range of recoveries of G34 were 72 to 11% (mean, 97%), and for 1 to 13 G17 were 86 to 125% (mean, 94%) (table 3). Third, the inflence of nonspecific factors in serm on RIA's with L6 and 1296 were assessed by measrements of immnoreactive gastrin in serm samples before and after selective removal of gastrin by an immnosorption method. The immnosorbent was prepared by copling carboxyl-terminal specific gastrin antisera to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), sing methods recommended by the manfactrers. The Sepharose beads conjgated with antibody were packed into small colmns (1 by 2 cm) and serm samples were passed throgh the colmn. Immnoreactive gastrin in serm was specifically bond by the immobilied antibody and the gastrin-stripped serm was collected from the colmn otflow. Extraction of gastrin was checked by spiking each sample with 126I-GI7; in 1 serm samples from 5 sbjects (5 fasting samples and 5 taken 3 min after the standard meal) the efficiency of extraction of "'I-G17 was 93. ± 5.9%. Reslts Zollinger-Ellison Syndrome Figre 2 shows the separation of immnoreactive gastrins in the serm of 2 patients after fractionation on Sephadex G-5. There were striking differences in the patterns of immnoreactive gastrin revealed by the three antisera. In both patients the only peak of immnoreactive gastrin revealed by L6 had an eltion volme typical of G17, and 1296 and 1295 also indicated immnoreactive gastrin in this region of the colmn elates. In addition, however, the two latter antibodies also revealed other forms of gastrin. There was a peak of immnoreactivity compatible with G34 that was revealed by 1296 bt not by 1295 or L6; this peak was a major component in the serm from one patient (lower panel, fig. 2) bt a minor component in the other (pper panel, fig. 2). Frthermore, in the serm of one patient (pper panel, fig. 2), althogh not in the other, there was a large peak of immnoreactive material (eltion volme 5.- coo S- o e a. 3 '-" tjl 2 ::J 1 II: I II: I- IJ) c( I:) 1 5 O:r i G-3C-I G-17-n t + f G L' io 4' 6' Jo 1& % ELUTION VOLUME FROM "'I BSA TO "'I FIG. 2. Eltion profiles obtained when serm from 2 patients with Zollinger-Ellison syndrome (ZES) was fractionated on Sephadex G-5 (1 by 1 em), and colmn elates assayed with three antisera. Rate of flow, 6 ml per hr; fraction volme, 1. ml. Arrows at top indicate the eltion volmes of standard peptides determined in separate colmn rns. TABLE 3. Recovery of standard GI7, G34, and 1 to 13 G17 added to serm" Blank Serm "am pies A B C Peptide added G17 J G34 J GI7.{ lmmnoreactive gastrin, by assay ± ± ± ± ± ± ± ± 19 L6 7 ± ± ± ± 14 Estimated peptide added G G34 J H3GI7 { "All vales (inclding the original standards) based on standard G17 1. Blank refers to serm withot added standard. Units. picomoles per liter; means ± SEM, n 7.

5 December 1976 HEPTADECAPEPTIDE GASTRIN IN BLOOD % from void to lui peaks) which emerged before standard G17-II (eltion volme 52%) and which was revealed by 1295, bt not by 1296 or L6. The gel filtration properties and pattern of immnoreactivity of this material correspond with those of the biologically inactive 1 to 13 fragment of G 17.'8 Table 4 smmaries estimates of serm gastrin concentration in 6 patients with ZES made with the three antibodies, and also gives the concentration of G 17 estimated after gel filtration. The concentration of G 17 in serm measred directly by L6 varied from 72 to 13% (mean, 92%) of the concentration determined by the fractionation method. Normal Sbjects Comparison of G17 estimated by gel filtration and by L6. The concentration of G 17 in 15 serm samples, with total immnoreactive gastrin concentrations in the range encontered after feeding (1296: 3 to 25 pmoles per liter) was measred directly by RIA with L6, and after fractionation on Sephadex G-5 (fig. 3). For 12 of the 15 samples there was good agreement between the two methods of estimation (±5%). Fasting concentrations of gastrin. In 17 normal sbjects, fasting serm gastrin measred by L6 was 11.8 ± 2.4 pmoles per liter (mean ± SE) and was not significantly different from that measred by 1296 (12.4 ± 2.2 TABLE 4. Total immnoreactive gastrin in serm of 6 patients with Z.ES compared with G 17 concentrations estimated by gel filtration" Patient Immnoreactive serm gastrm L6 G17 concentration Ha 29, 251, 76, 93, Se 4,3 19,5 3,8 2,9 Hi 1,65 5, 1,1S 1,24 McG 1, S 53 B 57,7 4,8 6,66 7,S2O Kn 136,4 26, 12, 13.3 " All vales are expressed as picomoles per liter, G 17 -I standard., 1 1 \OJ 1/1 1/1 C( 75 Z \OJ '" ' = ' :E ::. -;- o a. 5 III: - C( Z III: \OJ U Z U ;: Z5 U \:J \OJ 1/ G-17 CONCENTRATION ESTIMATED BY GEL FILTRATION (p moi/l) FIG. 3. Comparison of G 17 in 15 samples of serm estimated by L6 (ordinate) and by fractionation on Sephadex G-5 (abscissa). The line indicates eqal vales in the two assay systems. pmoles per liter), and 1295 (11.4 ± 2.7 pmoles per liter). Yalow and W 7 have reported that the major form of circlating gastrin in the fasting state emerges in the void volme of Sephadex colmns, and have called this form BBG. Becase of the relatively low sensitivity of L6 it was not possible to se this antibody to assay colmn elates obtained after rnning normal fasting serm on Sephadex G-5, and so to identify the immnoreactive components in fasting blood. Ths the possibility that L6 measres BBG is not yet resolved. However, the otcome of this qestion shold not affect the validity of measrements of the increment in serm gastrin measred by L6 with feeding, as BBG does not increase after a meal. 7 Insight into the natre of immnoreactivity in fasting blood is provided by stdies on serm stripped of immnoreactive gastrin by immnosorption. In 5 sbjects concentrations of immnoreactive gastrin in fasting serm measred by 1296 were redced by abot 4% after stripping gastrin by immnosorption (table 5), bt the difference was not significant; there was also a redction of abot 3% in immnoreactivity measred by L6, which again failed to reach statistical significance. The apparent immnoreactivity remaining in serm after immnosorption can be attribted to nonspecific factors, e.g., serm proteins, which inhibit binding of antibody and label bt do not directly bind to gastrin antibodies. The reslts sggest that after allowing for nonspecific inhibition of binding, the "tre" concentration of gastrin in serm measred by L6 is abot 3 to 4 pmoles per liter. Moreover, the apparent immnoreactivity measred by L6 after stripping gastrin did not increase with feeding (table 5), so that the nonspecific factors do not affect the validity of measrements of the increment in gastrin concentrations with feeding. There was a slight increase after feeding in apparent immnoreactivity of stripped serm measred by 1296, bt this was not significant and acconted for only abot 15 to 2% of the total increment in gastrin concentrations. Responses to feeding. The postprandial increments in serm gastrin measred by RIA sing the three antisera are shown in figre 4. Antiserm 1296 indicated a peak increment in serm gastrin immnoreactivity at 2 min which maintained a platea ntil 5 min before declin- TABLE 5. Concentration of immnoreactive gastrin in serm estimated by 1296 and L6 before and after extraction with an immnosorbent" Fasting serm Preimmnosorption Postimmnosorption Serm, 3-min postprandial Preimmnosorption Postimmnosorption 1:! ± ± ± ± 2.1 Ant.hody L ILl ± ± 2.S IS.6 ± ± 2.3 "Vales are expressed as picomoles per liter G 17 { st andard; mean, ± SE. n = 5.

6 976 DOCKRAYAND TAYLOR Vol. 71. No.6 35 $ r. 3.! ii c as " 2 :I :> II: 15! 1 OJ :I OJ a:! 5 o o eo 1' 12 TIME (min.' FIG. 4. Increment in serm gastrin measred by three antibodies (Ab) after a light meal. Each point is the mean of 17 sbjects. and verticallinel indicate 8. ing toward basal levels. In contrast, L6 showed a peak of immnoreactive gastrin at 2 min which was immediately followed by a decline toward basal vales, and at 4 and 5 min the increase serm gastrin was significantly less than at 2 min (paired t-test, P <.5). The peak increment seen with L6 was approximately 6% of that recorded with The time corse and magnitde of the response measred by 1295 was intermediate between the L6 and 1296 responses. The difference between L6 and 1296 can be acconted for by gastrin components similar at their carboxyl-termins to G17, principally G34, bt also inclding trace amonts of component I and G14 (for convenience this fraction is designated "G34"). In the same way the difference between L6 and 1295 can be attribted to molecles with an amino-terminal portion resembling G17. The G34 fraction reached a peak abot 5 min after feeding when it acconted for the major portion of immnoreactive gastrin in the serm (fig. 5). The concentrations of the amino-terminal gastrin component (NT G17) were relatively low and appeared to be present throghot the digestive period (fig. 5). Althogh the data in figre 5 represent estimates of the molar concentration of different forms of gastrin in blood after a meal they give no indication of the relative biological activities of these different forms. An estimate of the relative importance of the gastrin components for acid secretion has been obtained by recalclating the reslts and expressing them with respect to a standard of G 17 derived from biological activity" (fig. 6). In this way the reslts take into accont the 6-fold difference in serm concentrations of G17 and G34 reqired to prodce half-maximal rates of acid secretion, II and are therefore analogos to the reslts one might have anticipated had a bioassay been sed instead of an immnoassay. By comparing figres 5 and 6 it is clear that G 17 acconts for the greater part of the biological activity in blood even thogh G34 is present in higher concentrations. Discssion Many peptide hormones have been shown to exist in several different moleclar forms, and in this regard gastrin is no exception. Becase the different forms of gastrin have different biological activities and clearance rates, it is clearly desirable to measre concentrations separately; bt in the past only relatively insensitive and time-consming chromatographic methods have been available for the estimation of different forms of gastrin in the circlation. However, dring a rotine examination of gastrin antibodies an nsal antiserm was discovered with virtally absolte specificity for G17, and the se of this antiserm in measrements of G 17 in blood is reported here. The specificity of antibody L6 was established by stdying the direct binding to it of labeled peptides, and by stdies on the inhibition of binding of l2&i-g 17 to antibody by nlabeled peptides. The reslts of the two 25 2 o 5 a: 15 o :; 1 a: OJ til 5 ",.. ", ",. - -.,. ' " " '...!"'--'" : to eo tol '2! 8 TIM' (lftlft.) FIG. Increment in serm concentrations of different forms of gastrin after a light meal. Data calclated from figre 4. G 17 is the same as 1.6; "G34" is given in qotation marks to indicate that it is estimated indirectly (1.6 sbtracted from 1296). NT G17 is amino-ter minal gastrin immnoreactivity estimated indirectly (1.6 sbtracted from 1295). G-17 o so to 75 to E!!!J TIME (min) FIG. 6. Same data as figre 5. recalclated to take into accont the 6-fold difference in concentrations of G34 and G17 needed to give half maximal stimlation of acid secretion. Ordinate: increase in serm gastrin concentration based on a standard of the biological activity of G17. The reslts are analogos to those npected from a bioassay.

7 December 1976 HEPTADECAPEPTIDE GASTRIN IN BLOOD 977 approaches complemented each other, for in both instances G 17 or molecles differing from it in only trivial amino acid sbstittions within the body of the peptide chain, showed cross-reactivity. In contrast, peptides with amino acid deletions at either the amino-termins of G17 (2 to 17 G17, 5 to 17 G17) or at the carboxyl-termins (1 to 13 G17), and peptides in which the amino-termins was extended into a longer chain (G34) exhibited minimal binding properties. It is generally agreed that antigenic determinants in peptides are 6 to 7 amino acid resides in length, and a determinant of 17 amino acids wold appear nlikely. The precise immnochemical basis for the specificity of L6 is not yet known and stdies in this direction are contining. However, one possibility is that throgh torsion of the heptadecapeptide chain L6 binds both amino- and carboxyl-terminal regions simltaneosly. It is interesting in this context to note that in other proteins it has been observed that two antigenically reactive "regions" (analogos in this case to the carboxyl- and amino-terminal parts ofgl7), which are separated along a peptide chain can nevertheless be broght into apposition and incorporated into a single antigenic reactive "site" throgh a three-dimensional rearrangement of the molecle.1o Althogh the immnochemical fondation for the specificity of L6 is not yet clear we have shown that in practice this antibody can be sed with high specificity for estimations of Gl? in serm. Ths, in the serm of patients with ZES and in normal sbjects after feeding, the concentrations of G17 estimated by L6 agree closely with those determined by 1296 after fractionation on Sephadex. Moreover, the only peak of immnoreactive gastrin revealed by L6 when serm from ZES patients was rn on Sephadex had the properties of G 17. Using L6 it has been possible to stdy the time corse of the Gt7 response to a meal in normal sbjects in greater detail than was previosly practical. The reslts indicate that G 17 concentrations peak abot 2 min, after feeding and then decline toward basal levels; at its peak G 17 acconts for abot half of the carboxyl-terminal immnoreactivity in the circlation. By sbtracting estimates made with L6 from those with 1296 (carboxylterminal specific) it has been possible to estimate the concentration of other components with carboxyl-terminal immnoreactivity, of which the most important is G34. The peak concentration of this component is achieved at abot 5 min after feeding and is abot 5% higher than the peak G 17 concentration. The longer time to peak of G34 can be attribted to its longer half-life in blood, and conseqently the greater time needed to reach a maximm dring continos secretion from the antrm. In dodenal lcer patients Walsh et al. have shown that the concentration of G34 in serm reqired for half-maximal rate of acid secretion (145 pmoles per liter) is abot 6 times greater than G17 (25 pmoles per liter), II and a similar ratio has been observed in dogs. 1 Applying this information to the present reslts (fig. 6), it is calclated that at all times after a meal G 17 is the principal biologically active form of gastrin in the blood, and that G34 probably contribtes only 2 to 3% of the acid-stimlating capacity, even thogh its molar concentration in blood may be higher than that of G 17. Frther stdies are now needed to relate concentrations of endogenos G 17 and G34 after a meal to biological activity of exogenosly administered gastrins in the same sbjects. Moreover, application of antibody L6 to the measrement of gastrin in patients with varios digestive diseases shold clarify the possible role of an imbalance in the ratio of different forms of gastrin in blood in the pathogenesis of gastrointestinal disease. REFERENCES 1. Gregory RA. Tracy HJ: The constittion and properties of two gastrins extracted from hog antral mcosa. Gt 5: McGigan JE: Immnochemical stdies with synthetic hman gastrin. Gastroenterology 54: Yalow RS. Berson SA: Radioimmnoassay of gastrin. Gastroenter. ology 58: Gangli PC. Hnter WM: Radioimmnoassay of gastrin in hman plasma. J Physiol (Lond) 22: Yalow RS. Berson SA: Sie and charge distinctions between endogenos hman plasma gastrin in peripheral blood and hep. tadecapeptide gastrin. Gastroenterology 58:6!HH5 6. Gregory RA. Tracy HJ: Isolation of two "big gastrins" from Zollinger-Ellison tmor tisse. Lancet 2: Yalow RS. W M: Additional stdies on the natre of big big gastrin. Gastroenterology 65: Rehfeld JF. 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