Radioimmunoassay of gastrointestinal hormones

991~5695/79/9974-9991$02.99/0 GMTEOENTE~LWY74~141-152, 1978 Copyright0 1978by the AmericanGa’stroenterological Amociation

PROGRESS RADIOIMMUNOASSAY EUGENE

Vol. 74, No. 1 Printedin USA.

IN GASTROENTEROLOGY

OF GASTROINTESTINAL

HORMONES

STRAUS

Veterans Administmtion New York, New York

Hospital, Bronx, New York, and The Mount Sinai School of Medicine,

The field of gastrointestinal peptide hormones is currently experiencing a great surge of productive research. Two fundamental and complementary scientific achievements have made this possible. The developing knowledge of peptide chemistry, accelerated over 50 years ago by the discovery of insulin, was exploited and expanded by Gregory and Tracy’, 2 and Jorpes and Mutt and their coworkers,5‘6 who succeeded in the isolation, purification, and sequencing of many gastrointestinal peptides. The availability of these peptides permitted the floodgates to burst when radioimmunoassay technology (RIA), originally developed by Yalow and Benson in the late 1950’s for the measurement of insulin,‘* * was ultimately applied first to the measurement of gastrin,+” and subsequently to other gastrointestinal peptides. RIA is an immensely powerful tool because it combines its exquisite sensitivity with the inherent specificity of the immune reaction. Shortly after its introduction for the measurement of insulin, RIA was broadly applied, making possible revolutionary developments in endocrinology, and furthering our current concept of the peptide hormones as a family sharing many common structural and physiological characteristics. It is not surprising then that RIA has rapidly opened the field of gastrointestinal hormones. At the recent First International Symposium on Gastrointestinal Hormones, over 60% of the abstracts offered data derived by the use of RIA. Five years ago Drs. Berson and Yalow prefaced their review of the impact of RIA in gastroenterology by saying that “the involvement of radioimmunoassay in gastroenterology is at present of such an embryonic nature that this brief review can aspire only to the status of an introduction to the subject rather than to that of a progress article, which, it is to be hoped, may legitimately occupy these pages a few years hence.“12 What progress has been made since this prior review? Gastrin, for both conceptual and technical reasons, remains the best studied of the three well-established gastrointestinal hormones. More precise criteria for the diagnosis of the Zollinger-Ellison (ZE) gastrinoma synReceived February 18, 1977. Accepted June 7, 1977. Address requests for reprints to: Eugene Straus, M.D., Veterans Administration Hospital, 130 West Kingsbridge Road, Bronx, New York 10468. This study was supported by the Medical Research Program of the Veterans Administration.

CUNY,

drome have emergedl”ls and gastrin assays are now readily available on a routine basis. The recent discovery that the ZE syndrome occurs in dogs should greatly facilitate the study of its protean manifestations as well as the mechanisms of its inheritance and a variety of therapeutic modalities. I9 Studies of secretagogues and inhibitors of gastrin release have improved our understanding of the regulation of circulating concentrations. 18,2o-22Data concerning the distribution of gastrin-like immunoreactivity in mammalian tissues,23-33 and the discovery of immunoreactive gas&in peptides in lower animal species,34-36 have provided new insight into the origin, evolution, and perhaps the physiological significance of gastrin. The subject of the molecular heterogeneity of circulating gastrin has received much attention.24-33, 37-47Although no physiologically significant new forms have been discovered since the previous review, a more complete understanding of the metabolism and biological potency of the two important biologically active circulating forms, heptadecapeptide (HG, G-17) and big gastrin (BG, G-24),2e2s*46*47has necessitated a more detailed analysis of the hormonal form of gastrin in physiological studies. Progress in the RIA of secretin and cholecystokinin (CCK) has been much slower than for gastrin for a variety of reasons. Among these has been the lack of clearly defined clinical problems. Although the ability to measure gastrin offered the enticing prospect of fresh insight into the world of acid-peptic disease, no such obvious rewards have underwritten the more arduous technical achievement necessary for sensitive, accurate measurement of secretin and CCK. Nevertheless, recently constructed RIA’s have been used to study the distribution of secretin and CCK in gastrointestinal mucosal tissues,4g50the fate of these hormones after intravenous administration,51-54and the effect of ingesting or artificially infusing the gastrointestinal tract with a variety of substances on the circulating hormonal concentrations.51-75 In addition to the three previously characterized hormones, an almost bewildering array of peptides has recently been extracted from gastrointestinal mucosal tissues. Many of these have been suggested as “candidate hormones”76 and some have been purified and synthesized. This profusion of peptides has aroused excitement and created the general impression that the gastrointestinal mucosa may be an extremely rich endocrine organ. The physiological significance of current

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per min per incubation tube but containing a chemical amount of tracer which is no more than the least amount of added hormone which produces a significant drop in the bound to free (B/F) ratio. It must also be appreciated that the immune reaction can be inhibited by a number of nonspecific factors such as the presence of high concentrations of salt or of other proteins, by enzyme inhibitors or anticoagulants, by changes in pH, etc. 82,83 When plasma is assayed virtually undiluted or in dilutions up to 15, not infrequently there are unknown and variable factors which mimic the presence of hormone by interfering in a nonspecific fashion with the immune reaction. It is therefore generally desirable to achieve sufficient sensitivity so that plasma can be measured at a dilution of 1:lO or greater. Body fluids other than plasma may present even more of a problem. For instance, because of its very acid pH, gastric juice, if not neutralized before assay, may dissociate antigen-antibody complexes. In addition the high proteolytic enzyme content of gastric or duodenal juices may destroy labeled antiTechnical Problems in the RIA of Gastrointestinal gen or specific antibody. In these cases, the resultant Hormones decrease in binding may be falsely interpreted as attribSince the prior review by Berson and Yalow in 1972, utable to the presence of hormone reacting in the new RIA’s have been developed for secretin, CCK, VIP, immune system. These effects must be considered when GIP, pancreatic polypeptide, and motilin. This section evaluating the validity of new assays per se or of the physiological findings obtained with these assays. reviews only the salient features of secretin, CCK, VIP, and GIP assays which have been used to derive Secretin. Secretin assays have recently been reported 63,86-8eImproved asdata relevant to physiological questions discussed in from many laboratories.51”3*55,58-80, succeeding sections of this review. These newer assays says have, for the most part, been attributable to the have been described as “sensitive and specific.” How- development of new and better antisera. The introducever, in terms of what is needed to measure steady tion of tyrosine into synthetic secretin to facilitate state hormonal concentrations they are, in most cases, labeling has proved not to be productive of a more not sensitive enough, and questions can be raised con- satisfactory labeled hormone.86 cerning their specificity. Chey and co-workers have described a secretin assay An assay, even if properly validated, may still be too using synthetic porcine secretin for iodination and as insensitive to detect the low concentrations of plasma standard.5g,64*8* 1251-secretinis purified by cation exhormone which may be present in the nonstimulated change on SP Sephadex C-25 co1umns.88Although these state. In RIA the minimal detectable hormone level is authors state that their assay can measure 2 to 3 pg per ml of plasma, 5g*64,88their published standard curves limited by the initial slope of the dose-response curve. This slope is dependent on the equilibrium constant manifest a significant drop in B/F only on the addition that characterizes the reaction of hormone with the of 5 pg of standard secretin per tube (3.3 pg per ml). predominant order of antibody-binding sites.@*85 Im- Because they assay plasma in a final dilution of 1:7.5, provement in sensitivity is achieved primarily by devel- it appears as if the minimal measurable secretin concenoping antisera of higher energy. However, with any tration is 25 pg per ml of plasma. Bloom and co-workers have published a number of given antiserum, the potential sensitivity of the RIA may not be achieved because of limitations imposed by papers concerned with measurements of circulating the quality or amount of the labeled hormone used as secretin.61,62Inasmuch as the methods sections of these tracer. papers refer to assay procedures published only in abstract forrng7 there is insufficient information for The quality of the tracer is relevant because overiodination or other chemical alterations in the labeled evaluation of their procedures. Rayford et al. have employed synthetic 6-tyrosyl peptide can result in diminished ability to bind to specific antibody and usually results in a less sensitive secretin for iodination and pure natural porcine secretin (GM, Gastrointestinal Hormone Research Laboratoassay. The specific activity of the labeled preparation may also be a limiting factor in very sensitive assays. ries, Karolinska Institutet, Stockholm, Sweden) as standard.51,63They assayed plasma in a final dilution In general the counting rate in the incubation tube must be sufficiently high to assure statistical accuracy, of 1:3. Although their standard curves are somewhat yet the chemical amount of the tracer must be such- variable, the optimal standard curve shown would perciently small so as not to occupy the major fraction of mit a minimal detectable secretin concentration of apthe high energy-binding sites. A useful rule of thumb proximately 150 pg per ml of plasma. would suggest a counting rate of at least 2000 counts Kolts and McGuigan have iodinated synthetic secreand future candidates remains to be evaluated by careful investigation in which RIA will play a central role. RIA’s have been developed for gastric inhibitory polypeptide (GIP),77 vasoactive intestinal polypeptide (VIP),78motilin,7g,8oand pancreatic polypeptide.81 RIA is now the central laboratory tool in any general investigational paradigm for the study of hormonal peptides. This approach includes the measurement of hormonal concentrations in tissue extracts, plasma, and other biological fluids. Whereas technical considerations involved in RIA have been well discussed,82-85 the rapid proliferation of this technology has resulted in significant stumbling over methodological barriers. This review attempts to focus attention on some areas of methodological distress, as well as to review new findings in the realm of hormonal physiology and heterogeneity. No attempt will be made here to discuss general considerations involved in the construction and validation of RIA systems, because this information is readily available elsewhere.82-85

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tin (E. R. Squibb & Sons, Princeton, N. J.) at specific activities ranging from 75 to 100 &i per Fg5*,53 and use pure natural secretin (GIH) as standard. They claim to be able to measure 6.2 pg of secretin per ml. However their published data indicate that addition of this amount of secretin per milliliter results in reduction of binding of the 1251-secretinfrom 35 to 34%, a reduction which can hardly be considered significant. Four times as much hormone effects a reduction in binding to 31%. Because they assay plasma at a 15 dilution, it is unlikely that they can reliably measure concentrations less than 50 to 100 pg of secretin per ml of plasma. In our laboratory pure natural secretin (GIH) is used for iodination and as standard. Purification of ‘251-secretin is effected by adsorption to and elution from QUSO G-32.fiss 8fiBecause 1 to 2 pg of secretin per ml produce a significant drop in B/F (fig. l), and because plasma is assayed in a final dilution of 1:lO or 1:25, the minimal detectable concentration is 10 to 25 pg per ml of plasma. CCK. RIA for CCK has been hindered by many of the same problems encountered with the secretin assay. Although the recent availability of 99% pure CCK (GIH) for iodination and standards has been of considerable help, the need to iodinate a sulfated tyrosine moiety and the low specific activity of the 1251-CCK generally employed have presented problems in the development of highly sensitive assays. Harvey and co-workers have used highly purified CCK (GIH) for standards and for preparation of a tracer with a specific activity between 30 and 100 PCi per hg.7”’74Standard curves appear to show a significant reduction in B/F with the addition of 1 pg of CCK standard per tube (0.3 ml incubation volume), although the tracer employed ranges from 1 to 10 pg of lz51-CCK per tube. Because only 5 ~1 of plasma are assayed, the minimal detectable CCK cannot be less than 1 pg per 0.005 ml or 200 pg per ml of plasma. Nevertheless, the authors claim the ability to detect 5 pg per ml of plasma.73r74 Thompson et a1.71a 72 also employed highly purified CCK (GIH) in their assay. Their iodinated CCK had a specific activity ranging from 30 to 50 PCi per pg.71,72 With a typical counting system which has a sensitivity of 1.5 x 10”counts per min per pC!i of 1251,one obtains 40 x 1.5 x 10”counts per min per pg or 60 counts per min per pg of 1251-CCK.Because the tracer they use is as large as 12,000 counts per min per m1,7o* 71its chemical CCK concentration is 200 pg per ml. The minimal detectable concentration of the labeled hormone cannot be expected to be much less than the chemical concentration of the labeled hormone, thus this assay could hardly have a sensitivity better than 200 pg of CCK per ml, regardless of the quality of the antisera. Furthermore, as only 0.3 ml of plasma is assayed, the minimal detectable hormone appears to be greater than 670 pg of CCK per ml of plasma. VIP. Said and Faloona have iodinated pure porcine VIP, both natural and synthetic, achieving specific activities of 140 to 410 &i per pg.7s The standard curve demonstrates that the addition of 0.5 and 1.0 ng

SECRETIN STANDARD CURVE I :750.000 DILUTION OF ANTISERUM R I I .O

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FIG. 1. Ratio (B/F) of antibody (B) to free (F) *251-labeledsynthetic secretin as a function of the concentration of unlabeled pure natural secretin (GIH). Note that the secretin standard curve shown here is lo-fold less sensitive than the gastrin curve shown in figure 2.

of VIP per ml reduces the binding of ‘251-VIPto antibody from 55 to 51 and 50%, respectively. Because plasma is assayed at final dilutions of 1:4.5 and 1:9, the minimal detectable VIP concentration is about 2.2 to 4.5 ng per ml of plasma rather than the stated sensitivity of 100 to 200 pg per ml of plasma. GIP. Kuzio et al. have developed a RIA for GIP using purified GIP for iodination and as standard.77 The iodination mixture is purified by column chromatography on Sephadex G15. The 1251-GIPused as tracer has a specific activity estimated to be 270 $Zi per pg. The standard curve does show a detectable reduction in B/F with the addition of 20 pg of standard GIP per ml. However, the authors state that the region between 50 and 500 pg per ml is generally used for assay and that serum is assayed in 1:5 dilution. This suggests that 250 pg of GIP per ml of serum would be the lower limit of detectability. Yet they report a mean fasting concentration in 7 normal human subjects below this level, that is, a mean 237 + 14 pg per ml with a range from 74 to 500 pg per ml. Physiological studies. Grossman has categorized gastrointestinal peptides of hormonal interest into two group~.‘~ Perched comfortably, if not complacently, on top are the three well-established, “recognized,” hormones, secretin, gastrin, and CCK. Below these are the “candidate” hormones. Upward mobility has not yet been demonstrated. Molecules craving hormonal status have been frustrated by a lack of universal acceptance. Systems of taxonomy are, perforce, both good and bad. I believe that Grossman’s system has been highly influential because it recognizes several very central facts. First, that many of the candidate peptides were discovered without any knowledge of their possible physiological significance. Further, many established hormonal peptides have, in addition, distinct pharma-

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cological activity. And finally, that physiological significance can be established only by careful work which is reproducible in many laboratories. The system has served us well. Nevertheless, because secretin and CCK assays are, like those for VIP, GIP, motilin, and pancreatic polypeptide, in relatively early stages of development, we are only beginning to satisfy for these “well-established” hormones the criteria for hormonal status required of the candidates. The hormonal status of secretin and CCK is “accepted” primarily because they are highly potent mediators of their major biological activities as determined by target organ response to exogenous doses. When presumably appropriate secretagogues cause target organ responses it is then assumed that these hormones are mediators. Direct RIA measurements are needed to establish that these assumptions are correct. With the exception of gastrin then, much of the measurement of gastrointestinal peptides in plasma has been directed toward determination of steady state concentrations and evaluation of their primary physiological effects. Gastrin determinations are important clinically for diagnosing well-established syndromes of gastrin excess. The gastrointestinal hormones are generally involved in modulating secretory, motor, absorptive, or other processes directed toward the digestion of a meal. Most of these digestive activities, such as gastric acid secretion, biliary and pancreatic exocrine flow, and a variety of motor adaptations, are relatively or absolutely quiescent during the interdigestive, or steady state period. Furthermore, because exogenous administration of relatively small amounts of these peptides, usually of the order of 5 to 10 kg or so, produce a definite physiological effect, it can be expected that the unstimulated plasma concentrations of the biologically active forms of these hormones must be quite low and that their measurement requires assays of high sensitivity. It is therefore not surprising that the newer assays for most of the gastrointestinal hormones require extensive refinement before reliable determinations of plasma levels in the nonstimulated state can be made. With increase in assay sensitivity there is usually a tendency for the reported unstimulated hormone concentrations to fall until the true value, agreed on in many laboratories, is finally achieved. The higher levels reported initially can often be attributed to the interpretation of random variations in binding of the labeled hormone as being caused by hormone with a concentration corresponding to the minimal detectable in the assay. Nonetheless, even in the absence of good measurements of basal levels, it is often possible to evaluate the relative effectiveness of potential secretagogues, because in the stimulated state plasma hormone levels may increase as much as 3-fold or greater over basal levels. Plasma levels obtained by RIA should be reasonably consistent with what is known about the physiological or pharmacological reaction of the particular hormone. For instance, if the unstimulated hormonal level obtained by RIA is reported to be greater than the value one might anticipate after exogenous administration of an amount of the hormone which is known to produce a

physiological effect, and yet the physiological events mediated by the hormone are quiescent in the unstimulated state, then the measured plasma concentration is in error or the immunoreactive form must be of low bioactivity. This caveat must be kept in mind in evaluating the physiological findings associated with the newly described RIA’s for the gastrointestinal hormanes . Gash-in. Many laboratories have developed sensitive gastrin RIA’s and the data accumulated regarding the regulation of circulating gastrin concentrations in health and disease, its distribution in tissues, its heterogeneous molecular forms, and the clinical usefulness of gastrin measurements serve as a guide for the study of other gastrointestinal peptides. Several outstanding reviews of the ZE syndrome16~ l7 and the gastrin field18 have appeared in the recent medical literature and the following section represents a selective discussion of the recent advances. With improvements in gastrin RIA procedures most laboratories now report the ability to measure immunoreactive gastrin at concentrations of about 1 pg per ml. Fasting gastrin concentrations in normal man, although tending to “bottom out,” as has been previously discussed, l2 are generally reported to be in the range of 30 to 70 pg per ml.‘*, s0-s2Recent improvements in the sensitivity of our gastrin assay permit the measurement of 0.1 pg of gastrin per ml (fig. 2). In this system we can detect as little as 2.5 pg per ml of plasma. With the improved sensitivity we are finding concentrations somewhat lower than those generally reported, that is, levels above 20 pg per ml in only about one-quarter of subjects with no known gastrointestinal disease. The most important developments in the gastrin field relate to improved applications in the diagnosis of gastrin-secreting tumors. This has come from a more detailed appreciation of the hypergastrinemic hyperchlorhydric state and the description of provocative GASTRIN

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FIG. 2. Ratio (B/F) of antibody-bound (B) to free (F) 1251-labeled human gastrin I as a function of the concentration of unlabeled human gastrin I.

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FIG. 3. Basal plasma gastrin concentrations in patients with proved Zollinger-Ellison (ZE) syndrome (left) and in nontumorous hypergastrinemic hyperchlorhydria (NT-HH) (right). Note the breaks in scale. (From Straus and Yalow.9

which exploit the finding that tumor gastrin and mucosal gastrin have different release properties. Figure 3, left, illustrates the broad range of fasting plasma gastrin concentrations found in a group of 48 patients with ZE.15Proof of the diagnoses was obtained either by histological examination of primary and/or metastatic tumor tissue, or by persistent hypergastrinemia after total gastrectomy. Most importantly, although the majority of duodenal ulcer patients have fasting gastrin levels that are within the normal range, some hyperchlorhydric subjects with no evidence of gastrinoma have fasting levels which overlap the ZE range (fig. 3, right). The occurrence of hypergastrinemia and hyperchlorhydria in the absence of a gastrinoma has been referred to as nontumerous hypergastrinemic hyperchlorhydria (NT-HH)15 and probably reflects a d&function in feedback-inhibition of gastrin release similar to, but more severe than that which occurs in most duodenal ulcer patients.12Other investigators have subsequently found similar patients and used the terms ZE type Is3or antral G-cell hyperplasia.@ To avoid confusion, the term ZE should clearly be restricted to gastrin-secreting tumors, and use of the term antral G-cell hyperplasia would, in most cases rely upon anatomical assumptions regarding the source of the circulating gastrin. We therefore prefer a more physiologically descriptive nomenclature (NT-HH). The diagnostic differentiation of NT-HH from ZE is what necessitates provocative testing. Accurate diagnosis is crucial because surgical intervention in ZE requires a total gastrectomy. The most commonly used provocative tests are a standard test meal (STM),12,I5a calcium infusiorQ4or bolus challenge,15 and a secretin

challenge. I3 In normal subjects mean plasma gastrin generally more than doubles after a STM and there is no significant response to calcium or secretin injection (fig. 4). The same pattern of response to these stimuli is seen in the usual duodenal ulcer patient with fasting gastrin in or near the normal range, but the integrated postprandial gastrin release is generally greater (fig. 5).12,15* s1 Patients with NT-HH and marked elevation of fasting gastrin, respond similarly to provocation, suggesting that in this group, too, the gastrin is of mucosal origin. l5 In contrast, plasma gastrin in ZE patients shows a marked increase after intravenous secretin or calcium but not after a STM.12 Although Creutzfeld et al. have reported postprandial gastrin concentrations in excess of 100% above fasting levels in 3 of 9 ZE patients,“j most reports are in agreement with the finding that postprandial gastrin release in ZE patients, when it occurs, results in considerably less than a 100% increase above fasting plasma levels.Poor response to a STM in patients with ZE, even those with relatively low fasting gastrin levels, may be attributed to the inhibition of mucosal gastrin release by the greatly increased load of gastric acid. The different responses of ZE and NT-HH patients are shown in figure 6. Careful diagnostic evaluation of acid-peptic disease must always consider basal gastrin within the context

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FIG. 4. Plasma gastrin concentrations in the fasting state and in response to three provocative stimuli in normal subjects. (From Straus and Yalow.9. 1601

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FIG. 5. Plasma gastrin concentrations in the fasting state and in response to three provocative stimuli in patients with duodenal ulcer who had basal gastrin concentrations less than 0.1 mg per ml. (From Straus and Yalow.9

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TIME INMINUTES FIG.6.Plasma gastrin concentrations in the fasting state and in response to three provocative stimuli in gastrin hypersecretors; patient Ha (left) has Zollinger-Ellison (ZE) syndrome; subject Iv (Fight) is in the nontumorous hypergastrinemic hyperchlorhydria (NT-HH) group. (From Straus and Yalow.15)

of acid secretory capacity, and provocative testing should be employed, wherever possible, when the diagnosis of ZE is considered. In recent years a more complete understanding of the nature of circulating immunoreactive gastrin has emerged through an appreciation that gastrin appears to be quite unique among the peptide hormones in that there are two forms,24-2B differing about 2-fold in molecular weight,97,98which have virtually the same biological potency as determined by dose-acid response studies.47Because of the relevance of these findings to the interpretation of the significance of plasma immunoreactive gastrin there has been a great deal of work in the field of gastrin heterogeneity (see Reference 29 for review). In spite of much effort, since the discovery of BG no new forms have been found which contribute significantly to bioactive circulating or tissue gastrin, although several additional gastrin components have been described.27,3745 Component I of Rehfeld30+ has been studied extensively. This component has an elution volume on Sephadex gel filtration less than BG and corresponding to that of proinsulin. It accounts for less than 1%of antral gastrin and is not found in duodenal or jejunal extracts31 Component I has been reported to contribute only an average of 6% to the total gastrin immunoreactivity in fasting plasma from normal subjects and 3% of duodenal ulcer patients,3l and is not released by feeding. 41There are no data relating to its biological activity. The suggestion39that component I is a precursor of BG remains speculative. However it has been pointed out that consideration of the apparent net charge of BG and component I make it unlikely that the highly basic BG could be contained within a molecule (component I) that appears to be as acidic as HG.SB Yalow and Berson have reported on a still larger form of gastrin, (big big gastrin). It elutes in the void volume on Sephadex G50 gel filtration, is a minor component, generally less than 1 to 2% in extracts of

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ZE tumors or in the plasma of gastrin hypersecretors with ZE or pernicious anemia, and is virtually undetectable in antral or duodenal extracts.*‘*43 A component with similar Sephadex gel filtration properties often is the predominant form in the plasma of fasting man, dog, or pig. 27There has been some controversy as to whether some or all of the void volume immunoreactivity found in the plasma of normal or antrectomized man and animals is identical with the major component of void volume immunoreactivity partially purified from ZE plasma or tumor. 29,33,99The nature of the plasma void volume immunoreactivity has certainly not been fully elucidated but there is evidence that at least some fraction of it is distinguishable from the tumor BBG extracted and partially purified by Gregory and Tracy. Thus on boiling there is no striking alteration of either total immunoreactive gastrin or the Sephadex gel filtration pattern of plasma fortified with tumor BBG, although the total and the void volume immunoreactivity of fasting dog plasma is markedly decreased2B(fig. 7). Because plasma HG and BG are unaffected by boiling,24-2s it might be worthwhile to assay boiled rather than unboiled plasma to determine the immunoreactive content attributable to the biologically significant forms. Fragments of gastrin smaller than HG have been described. Minigastrin, the carboxyl-terminal gastrin tetradecapeptide, has been reported by Rehfeld to account for only a small percentage of plasma gastrin immunoreactivity.30*31 Dockray and Walsh3’ have obtained a very interesting antiserum with which they claim to have identified in plasma the presence of an amino-terminal gastrin fragment resembling the 1-13 peptide. This peptide would have no biological activity and is not detected in radioimmunoassay except with this very special antiserum. As yet there have been no reports concerning the presence in plasma of the residual carboxyl-terminal tetrapeptide amide. Knowledge of the hormonal form(s) of circulating gastrin is essential to the interpretation of gastrin

FIG. 7. Sephadex G50 gel filtration. Dog plasma was diluted 12 in saline and applied to column before boiling (Upper lef?) and after boiling for 10min (lower left) which precipitated the serum proteins. Big big gastrin (BBGa) (void volume immunoreactivity from a gastrinoma) was added to the dog plasma, which was then diluted 1:2 in saline and applied to column before (upper right) and after boiling for 10min (lower right). (From Yalow and Straus.?

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radioimmunoassay in clinical situations. Because of the difference in turnover times of BG and HG,46,41 their relative biological activities, as defined by the traditional dose-response method, are certainly different from that defined by plasma concentration-response data. It is therefore important to consider the hormonal forms in attempting to relate plasma immunoreactive gastrin concentration to its biological effectiveness. Secretin. Recently secretin assays have been reported from a number of laboratories. 51-53* 55,5wo,63,8*89 In spite of considerable activity in this field there remains a paucity of reliable data on fasting secretin concentrations and the response of this peptide to what might be considered physiological stimuli. The problems in this regard are undoubtedly generated by the need, as yet usually unmet, for assays proved to be of sufficient sensitivity. To estimate the sensitivity required one must appreciate that bolus administration of 20 pg of GIH secretin to a 70-kg man results in an hourly total bicarbonate response of approximately 25 mEq.‘O”This dose produces a plasma level at 3 minutes averaging about 500 pg/ml which subsequently falls with a halftime of less than 3 minutes. This dose is comparable to that of gastrin II, about 50 pg, which given as a single intravenous injection produces a maximal acid response.‘O’Thus it is likely that levels of secretin in the fasting state and after stimulation by endogenous secretagogues would not exceed gastrin levels. Nonetheless, steady state circulating concentrations in normal human subjects have been reported over an extremely wide range from 0.6 pg per mls8 to greater than 600 pg per ml. 52For instance Chey and co-workers have reported in several papers that fasting concentrations in normal subjects average about 55 to 70 pg per m1.59,~4,*9 More recently they have reported values below 8 pg per ml@ with no indication as to what can account for the striking reduction in mean fasting levels in the absence of any change in the standards or antiserum used. Release of secretin into the circulation in response to intraduodenal infusion of 10 ml per min of 0.1 N hydrochloric acid has been demonstrated by several investigators.51-53, 5~7 Th is pharmacological maneuver tells us little about the role of acid-induced secretin reponse in normal physiology. However, subjects with marked basal hyperchlorhydria manifest distinct hypersecretinemia (fig. S).“s This observation is consistent with the earlier reports that pancreatic bicarbonate secretion in patients with ordinary duodenal ulcer is related to their gastric acid secretion,1o2and that basal pancreatic volume and total bicarbonate secretion are markedly increased in ZE syndrome. lo39 lo4 The role of secretin, a potential inhibitor of gastrinstimulated gastric acid secretion, in duodenal ulcer disease is of considerable interest. Bloom and WardG1 have reported that, in response to duodenal infusions of hydrochloric acid, patients with duodenal ulcer release less secretin than do normal subjects. However Isenberg et aL6* have been unable to confirm this finding. In Bloom’s first report61basal secretin levels were found to be significantly lower in duodenal ulcer subjects

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FIG. 8. Concentration of immunoreactive secretin in the fasting state in man and dog. (From Straus and Yalow.@‘)

than in normal persons, whereas in the latter study, using the same author’s immunoassay, basal levels were significantly higher in duodenal ulcer subjects6* Proof of the role of secretin as a digestive hormone requires evidence that it is released in response to feeding stimuli which call forth those digestive processes which it is presumed to mediate. Several laboratories have reported their failure to observe postprandial secretin release, even though they claim to have reliable assays with sticient sensitivity to determine fasting levels.59*64,67*89,lo5 With the sensitivity of our assay, -25 pg of secretin per ml of plasma, we are currently unable to measure fasting secretin concentrations in most normal and duodenal ulcer subjects.69 Nonetheless in a few subjects without marked basal hyperchlorhydria, but in whom fasting secretin is measurable, we have reported that there is a modest increase in circulating secretin after our standard test meal (fig. 9).69The limited sensitivity of our assay may have resulted in a failure to detect small postprandial increases in the majority of these subjects. However, if the secretin response to feeding is not a direct effect of ingested secretagogues but is simply secondary to changes in duodenal acidification, then it may well be that in many subjects ingestion of the standard test meal does not cause sufficient change in duodenal pH to provoke secretin release. Patients with ZE or other hyperchlorhydric subjects with markedly elevated fasting secretin levels respond to feeding with a decrease in circulating secretin levels (fig. 9),6g presumably caused by a reduction in the rate of acid delivery to the duodenum. CCK. The ~~~ continuous infusion dose for the pancreozyminic effect of pure CCK in the dog has been given as about 245 pmoles per kg per h.‘06 This is to be compared with ~~~ continuous infusion gastrin dose of 200 pmoles per kg per hr. lo7. Thus one might expect that the fasting and stimulated plasma levels of gastrin and CCK should also be comparable. However the reported levels of CCK in the basal state have been widely divergent. Harvey and co-workers reported a mean fasting concentration of 26 pg per ml with a

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FIG. 9. Mean (~SEM)plasma secretin before and after completion of test meal in 3 patients with Zollinger-Ellison (ZE) syndrome, and 2 duodenal ulcer (DU) patients with basal acid output (BAO) ~15 mEq per hr, and 8 subjects with normal gastric acid secretion. (From Straus, E.: In Med Clin North Am, 62, in press).

range from 5 to 80 pg per ml in 25 normal subjects in one 8tudy,73and in another a mean of 60 pg per ml, with a range from 5 to 800 pg per ml in 50 other 8ubjects.74Thompson et al. have reported levels more than lo-fold higher, i.e., a mean fasting concentration of 730 pg per ml in 16 normal 8ubjects.71 The reports by these groups of postprandial increases in circulating CCK concentrations are even more divergent. Harvey et al. 73have reported that the ingestion of 1 pint of milk initiated a dramatic rise in circulating CCK levels, peaking in about 30 min at from 10 to 16 ng per ml, about 100 times the food-stimulated gastrin response to a test meal. Plasma CCK then fell precipitously, approaching basal levels within 15 min, suggesting an acute suppression of its release.73 In marked contrast, Thompson’s group described a very modest response of about 50% above basal concentrations after ingestion of a high protein, high carbohydrate liquid meal.” The kinetics of response was also very different because, rather than a precipitous decline following peak levels, Thompson et aI.‘l found that CCK levels were still maximal 240 min after ingestion of the meal. Reports from these two groups are also in disagreement concerning CCK levels in duodenal ulcer patients. Thompson et al.‘l find lower levels (-600 pg per ml) in 12 duodenal ulcer patient8 than in 16 normal subjects (700 pg per ml). Harvey et a1.75find that CCK is markedly elevated to 300 pg per ml in 10 ulcer patients compared to a mean value of 26 pg per ml in one group of 25 normal persons73 or a mean of 60 pg per ml in another group of 50 normal subjects.74They attributed the elevated CCK in ulcer patient8 to stimulation of their small intestinal mucosa because of hyperacidity, and reported that metiamide therapy affected a dramatic decrease in basal CCK.75

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Thompson et al.‘l reported CCK excess in 11 patients with ZE, all of whom were studied after total gastrectomy when gastric hyperacidity could not have been a factor. They postulated therefore that the tumor may have been the source of the circulating CCK although they found no significant CCK in gastrinoma extracts. These inconsistent reports are unlikely to stand the test of time so that the CCK assay8 already reported have added little to our knowledge of the physiology or pathophysiology of this hormone. VIP. Recent studies have provided data linking VIP with the Verner-Morrison, or “pancreatic cholera,” syndrome.7s*lo8,losThis disorder, with a broad spectrum of variably expressed clinical manifestations, has generally been thought to be caused by excessive concentrations of some hormonal substance in association with pancreatic neoplasia or hyperplasia. Bloom and Polak have repeatedly stated that “plasma VIP levels are elevated in all cases of the classical Verner-Morrison syndrome.“lo9,110These workers have recently pointed out that earlier diagnosis could prevent metastatic spread from the primary pancreatic tumor and have claimed that, “Estimations of a single fasting plasma sample for VIP now enables the diagnosis to be made very rapidly, and no errors have been noted where adequately taken samples for VIP estimation have been available.“110Inasmuch as therapeutic intervention as radical as total pancreatectomy may be recommended on the basis of apparent elevation of a single fasting VIP level, it is imperative to have a well-established range for normal subjects and other control groups in order to be able to determine with confidence when concentrations are clearly excessive. This is often not the case with the peptide hormones. For instance, as just described, the range of plasma gastrin concentrations associated with gastrinomas is very wide and overlaps levels found in nontumorous states. Thus positive diagnosis not infrequently requires u8e of appropriate stimulation or suppression tests rather than reliance on assay of a single fasting sample. Analysis of current literature does not support conclusions linking all cases of “classical” Verner-Morrison syndrome with VIP excess. Bloom and co-workers have found the normal range for plasma VIP to be between 0 and 100 pg per ml, but give no detailed information concerning their assay nor their procedure for establishing the normal range. los,logSaid and Faloona’* report a mean steady state VIP concentration for 25 normal persons as 79 +: 64 (SD) pg per ml. This level was established by averaging 22 samples with undetectable levels which were taken as 0 ng per ml; one sample presumed to have 0.2 ng per ml, and two with detectable levels of 2.5 and 3.6 ng per ml. As indicated above, the minimal detectable plasma VIP in their assay system appears to be about 2.5 ng per ml. One can conclude from their data that normal plasma VIP levels are generally below the level of detectability but may range up to 3.6 ng per ml. Yet they report “elevated” plasma VIP levels, between 0.6 and 9.0 ng per ml, in 26 of 28 subjects with chronic watery diarrhea. In 4 of these

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cases no pathological lesion could be found and in 7 others the malignancy was not of pancreatic origin. Furthermore, as was discussed earlier, 51 and 50% binding of 1251-VIPto antibody corresponds to plasma concentrations of 2.5 or 5.0 ng per ml, respectively. From this point of view, one can see that virtually all of the reportedly elevated VIP levels differed from what might be considered the upper limit of normal range (3.6 ng per ml) by considerably less than 5% reduction in bound 1251-VIP. Because of the variability in the clinical expression of watery diarrhea1 syn$omes it is quite possible that we are dealing here with a “mixed bag” containing cases caused by the excessive secretion of one or more humoral substances which stimulate intestinal and/or exocrine glandular secretions. In fact, cases have now been reported in which VIP levels were normal, whereas elevated levels of prostaglandins of the E series were found.“‘, 11*Although some, or even all, cases of Verner-Morrison syndrome may be associated with VIP excess, it seems clear that, until more conclusive data are obtained, therapeutic decisions in cases of watery diarrhea syndromes should be made on clinical grounds alone until better assays are available that give confidence in laboratory reports of VIP concentrations. GIP. The enterogastrone-like action of exogenous porcine GIP has been demonstrated in dogs.l13,‘14Maximum acid inhibition was obtained with GIP at an infusion rate of 1.0 pg per kg per hr against a background of stimulant doses sufficient to produce 60 to 70% of maximum acid secretion. Recently attention has focused on the insulin-releasing effects of GIP. Pederson et a1.‘15 have presented data indicating release of immunoreactive GIP after oral ingestion of glucose or fat in a dose-related fashion in dogs. The immunoreactive insulin response to oral glucose (1.0 g per kg) was shown to peak 30 to 45 min post-ingestion and plasma GIP rose simultaneously from undetectable (~0.1 ng per ml) to a mean peak of approximately 1.0 ng per ml. The insulin response to a glucose infusion (0.6 g per kg per hr) was significantly augmented by a continuous GIP infusion (2.0 pg per kg per hr) started 1 hr earlier. By the beginning of the glucose infusion, the GIP infusion had produced serum GIP levels of approximately 2.5 ng per ml, a value of about 2- to 3fold higher than the maximal serum GIP level achieved after oral glucose. Whether a similar augmentation of insulin response would have occurred with a smaller GIP infusion begun simultaneously with the glucose infusion was not tested. It appears from the data of Pederson et al.“” that GIP is not an insulin secretagogue per se, inasmuch as infusion of porcine GIP in the dog is not accompanied by insulin release. Furthermore, ingestion of fat is not associated with insulin release, although serum GIP is greater than that after glucose ingestion.l15 However, fat ingestion does augment the insulin response to intravenous glucose. 115 These studies115certainly present some support for the role of GIP as an incretin. Nonetheless there remain

unanswered questions as to its role under physiological rather than pharmacological dose levels. The relative unavailability of GIP has precluded confhmation of these studies by other laboratories. Hormone concentrations in gastrointestinal fluids. Until recently RIA of peptide hormones was primarily carried out by endocrinologists and physiologists concerned with plasma and tissue concentrations of hormones within the traditional province of endocrinology. Because gastroenterologists are interested in many hollow, as well as solid organs, clinical and investigational applications of gastrointestinal hormone assays involve the determination of concentrations in widely disparate tissue extracts and biological fluids. Included among these are gastric and intestinal secretions, bile, and stool water. In comparison with the more commonly assayed tissue extracts and blood constituents, these fluids introduce unique concentrations of salts, bile constituents, acids, enzymes, and other materials into the assay system which may result in artifact, owing to (1) interference of chemicals in the fluid with the antigen-antibody reaction, and/or (2) degradation by fluid contents of the antigen and/or antibody. As a general rule then, the more concentrated the fluid being assayed, the more likely to occur are a&factual distortions in the assay. Reports of extremely high gastrin concentrations in gastric juiceli6, 11’have prompted the suggestion that the clearance of gastrin from the circulation may be accomplished by its secretion into the lumen of the gut,‘16 and further speculation concerning the physiological significance of secretion via this route.117* 118 Assay of gastric juice is complicated by the possibility of introducing artifact attributable to interference with the antigen-antibody reaction, as well as by destruction of both antigen and antibody.11sIf labeled antigen or antibody is destroyed during incubation, immune precipitation is reduced, and this reduction is likely to be interpretable erroneously as caused by a high hormonal content, Recent studies have demonstrated that the presence of enzymes in unboiled duodenal and acid gastric secretions damages both labeled hormone and antibody even when these secretions are diluted 1:250 in the incubation mixture.11s When artifact related topH and enzyme activity are eliminated, gastric and duodenal secretions collected under the usual conditions of sampling do not contain significant amounts of gastrin. lls The recent preliminary report of high levels of VIP in cholera stool water,‘*O although provocative, should be followed by further studies which include data relative to the possibility of artifact in these determinations. Similar circumstances may obtain when working with bile, succus entericus, and unboiled tissue extracts. Conclusion The functions of the gastrointestinal tract are modulated by the coordinated actions of nerves and hormones. “Nervism” continues to contribute to our under-

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standing of physiology, and “hormonism” is on the rise again. Those who are interested in hormonal physiology now have a very powerful tool, but one which must be finely honed. The rapid expansion of the gastrointestinal hormone field has been accompanied by inevitable methodological problems and this critical evaluation has focused on these in an attempt to facilitate further productivity. REFERENCES 1. Gregory RA, Tracy HJ: The preparation and properties of gastrin. J Physiol (Land) 156:523-543, 1961 2. Gregory RA, Tracy HJ: The constitution and properties of two gastrins extracted from hog antral mucosa. I. The isolation of two gastritis from hog antral mucosa. Gut 5103-117, 1964 Jorpes JE, Mutt V: The preparation of secretin. Biochem J 52:328-330, 1952 Jorpes JE, Mutt V: On the biological activity and amino acid composition of secretin. Acta Chem Stand 15:513-519, 1970 Brown JC, Pederson RA, Jorpes JE, et al: Preparation of highly active enterogastrone. Can J Physiol Pharmacol47:113114, 1969 Said SI, Mutt V: Polypeptide with broad biological activity: isolation from small intestine. Science 1691217-1218, 1970 Berson SA, Yalow RS: Recent studies on insulin-binding antibodies. Ann NY Acad Sci 82:338-344, 1959 Yalow RS, Berson SA: Assay of plasma insulin in human subjects by immunological methods. Nature 184:1648-X49, 1959 9. McGuigan JE: Studies of the immunochemical specificity of some antibodies to human gastrin. Gastroenterology 56:429438, 1969 10. Hansky J, Cain MD: Radioimmunoassay of gastrin in human serum. Lancet 2:1388-1390,1969 11. Yalow RS, Berson SA: Radioimmunoassay of gastrin. Gastroenterology 5&l-14,1970 12. Berson SA, Yalow RS: Radioimmunoassay in gastroenterology. Gastroenterology 62:1061-1084, 1972 13. Isenberg JI, Walsh JH, Passaro E Jr, et al: Unusual effect of secretin on serum gastrin, serum calcium, and gastric acid secretion in a patient with suspected Zollinger-Ellison syndrome. Gastroenterology 62:626-631, 1972 14. Passaro E Jr, Basso N, Walsh JH: Calcium challenge in the Zollinger-Ellison syndrome. Surgery 72:60-67, 1972 15. Straus E, Yalow RS: Differential diagnosis of hypergastrinemia. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 99-113 16. Creutzfeldt W, Arnold R, Creutzfeldt C, et al: Pathomorphologic, biochemical, and diagnostic aspects of gastrinomas (Zollinger-Ellison syndrome). Hum Path01 647-76, 1975 17. Isenberg JI, Walsh JH, Grossman MI: Zollinger-Ellison syndrome. Gastroenterology 65140-165, 1973 18. Walsh JH, Grossman MI: Gastrin. N Engl J Med 292:13241332, 1975 19. Straus E, Johnson GF, Yalow RS: Canine Zollinger-Ellison syndrome. Gastroenterology 72:380-381,1977 20. Hansky J, Soveny C, Korman MG: Effect of secretin on serum gastrin as measured by immunoassay. Gastroenterology 61:6268, 1971 21. Thompson JC, Reeder DD, Bunchman HH, et al: Effect of secretin on circulating gastrin. Ann Surg 176:384-392, 1972 22. Straus E, Greenstein AJ, Yalow RS Effect of secretin on release of heterogeneous forms of gastrin. Gut 16999-1005, 1975 23. Nilsson G, Yalow RS, Berson SA: Distribution of gastrin in the gastrointestinal tract of human, dog, cat and hog. In

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Frontiers in Gastrointestinal Hormone Research. Stockholm, Sweden, Almqvist & Wiksell, 1973, p 95-101 24. Yalow RS, Berson SA: Size and charge distinctions between endogenous human plasma gastrin in peripheral blood and heptadecapeptide gastrins. Gastroenterology 58:609-615, 1970 25. Berson SA, Yalow RS Nature of immunoreactive gastrin extracted from tissues of gastrointestinal tract. Gastroenterology 69215222,197l 26. Yalow RS, Berson SA: State of endogenous gastrin in blood and tissues. In Frontiers in Gastrointestinal Hormone Research. Stockholm, Sweden, Almqvist & Wiksell, 1973, p 83-91 27. Yalow RS, Wu N: Additional studies on the nature of big big gastrin. Gastroenterology 65:19-27, 1973 28. Creutzfeldt W, Arnold R, Creutzfeldt C, et al: Mucosal gastrin concentration, molecular forms of gastrin, number and ultrastructure of G-cells in patients with duodenal ulcer. Gut 17:745754,1976 29. Yalow RS, Straus E: Heterogeneity of gastrointestinal hormones. In Hormonal Receptors in Digestive Tract Physiology. Edited by G Rosselin. Amsterdam, The Netherlands, Elsevierl North-Holland Publishing Co, 1977, p 79-93 30. Malmstrom J. Stadil F, Rehfeld JF: Gastrins in tissue: concentration and component pattern in gastric, duodenal, and jejunal mucosa of normal human subjects and patients with duodenal ulcer. Gastroenterology 70:697-703,1976 31. Rehfeld JF: What is gastrin? A progress report on the heterogeneity of gastrin in serum and tissue. Digestion 11:397-405, 1974 32. Rehfeld JF, Stadil F, Malmstrom J, et al: Gastrin heterogeneity in serum and tissue: a progress report. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 43-58 33. Track NS, Arnold R. Creutzfeldt C, et al: The different forms of immunoreactive gastrin in blood and tissue. Verh Dtsch Ges Inn Med 80:361-368,1974 34. Straus E, Gainer H, Yalow RS: Molluscan gastrin: concentration and molecular forms. Science 190:687-689, 1975 35. Hansen D: Evidence of a gastrin-like substance in Rhino&us productus. Comp Biochem Physiol [A] 52:61-63,1975 36. Dockray GJ: Molecular evolution of gut hormones: application of comparative studies on the regulation of digestion. Gastroenterology 72:344-358, 1977 37. Dockray GJ, Walsh JH: Amino terminal gastrin fragment in serum of Zollinger-Ellison syndrome patients. Gastroenterology 68:222-230, 1975 38. Gregory RA, Tracy HJ: Isolation of two minigastrins from Zollinger-Ellison tumour tissue. Gut 15683~685,1974 39. Rehfeld JF: Three components of gastrin in human serum. Gel filtration studies on the molecular size of immunoreactive serum gastrin. Biochim Biophys Acta 285:364-372,1972 40. Rehfeld JF, Stadil F, Vikelsoe J: Immunoreactive gastrin components in human serum. Gut 15:102-l&l974 41. Stadil F, Rehfeld JF, Christiansen LA, et al: Patterns of gastrin components in serum during feeding in normal subjects and duodenal ulcer patients. Stand J Gastroenterol l&863868, 1975 42. Yalow RS: Gas&ins: small, big, and big big. In Endocrinology of the Gut. Edited by WY Chey, FP Brooks. Thorofare NJ, CB Slack Inc, 1974, p 261-276 43. Yalow RS, Berson SA: And now, “big, big” gastrin. Biochem Biophys Res Commun 48:391-395,1972 44. Gregory RA: The gastrointestinal hormones: a review of recent advances. J Physiol (Land) 241:1-32, 1974 45. Walsh JH, Trout HH III, Debas HT, et al: Immunochemical and biological properties of gastrins obtained from different species and of different molecular species of gastrins. In Endocrinology of the Gut. Edited by WY Chey, FP Brooks. Thorofare

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NJ, CB Slack Inc, 1974, p 277-289 46. Straus E, Yalow RS: Studies on the distribution and degradation of heptadecapeptide, big, and big big gastrins. Gastroenterology 66:936-943, 1974 47. Walsh JH, Debas HT, Grossman MI: Pure human big gastrin: immunochemical properties, disappearance half time, and acidstimulating action in dogs. J Clin Invest 541477-485,1974 48. Straus E, Urbach H, Yalow RS: Secretin RL4: methodology and application to studies of distribution and molecular forms of secretin in tissues (abstr). Endocrinology 96 (suppl):A-269, 1975 49. Bloom SR: Hormones of the gastrointestinal tract. Br Med Bull 30:62-67, 1974 50. Bloom SR, Bryant MG: Distribution of radioimmunoassayable gastrin, secretin, pancreozymin, and enteroglucagon in rat, dog, and baboon gut. J Endocrinology 59XIIV, 1973 51. Rayford PL, Curtis PJ, Fender HR, et al: Radioimmunoassay measurement of disappearance half-time of secretin in dogs. Surg Forum 26:385-386, 1975 52. Kolts BE, McGuigan JE: Radioimmunoassay of secretin: serum concentrations and half-life in man (abstr). Gastroenterology 66:849, 1974 53. Kolts BE, McGuigan JE: Radioimmunoassay measurement of secretin half-life in man. Gastroenterology 7255-60, 1977 54. Rayford PL, Fender HR, Ramus NH, et al: Disappearance half-time of exogenous cholecystokinin from circulation in man and dogs (abst). Gastroenterology 68:971, 1975 55. Boden G, Chey WY; Preparation and specificity of antiserum to synthetic secretin and its uses in a radioimmunoassay. Endocrinology 92:1617-1624, 1973 56. Boden G, Essa N. Owen 0: Effects of intraduodenal amino acids, fatty acids, and sugars on secretin concentrations. Gastroenterology 68:722-727, 1975 57. Boden G, Essa N, Owen OE, et al: Effects of intraduodenal administration of HCl and glucose on circulating immunoreactive secretin and insulin concentrations. J Clin Invest 53:11851193, 1974 58. Bloom SR: The development of a radioimmunoassay for secretin. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 257-268 59. Chey Wy, Tai HH, Rhodes R, et al: Radioimmunoassay of secretin: further studies. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 269-281 60. Byrnes DJ, Marjason JP: Radioimmunoassay of secretin in plasma. Horm Metab Res 8:361-365,1976 61. Bloom SR, Ward AS: Failure of secretin release in patients with duodenal ulcer. Br Med J 1:126-127, 1975 62. Isenberg JI, Cano R, Bloom SR: Effect of graded amounts of acid instilled into the duodenum on pancreatic bicarbonate secretion and plasma secretin in duodenal ulcer patients and normal subjects. Gastroenterology 72:6-8, 1977 63. Raytord PL, Curtis PJ, Fender HR, et al: Plasma levels of secretin in man and dogs: validation of a secretin radioimmunoassay. Surgery 79658-665, 1976 64. Rhodes RA, Tai HH, Chey WY: Observations on plasma secretin levels by radioimmunoassay in response to duodenal acidification and to a meat meal in humans. Am J Dig Dis 21:873879, 1976 65. Chey WY, Lee KY, Tai HH, et al: Plasma secretin response to duodenal pH in patients with Zollinger-Ellison syndrome and in dogs with prolonged duodenal acidification. Symposium on Hormones and Ulcer and 1st International Symposium on Gastrointestinal Hormones, Abstract 025, 1976 66. Chey WY, Tai HH, Rhodes R, et al: Release of secretin in normal and abnormal state. Symposium on Hormones and Ulcer and 1st International Symposium on Gastrointestinal

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Hormones. Abstract 026, 1976 67. Lee KY, Tai HH, Chey WY: Plasma secretin and gastrin responses to a meat meal and duodenal acidification in dogs. Am J Physiol230:784-789,1976 68. Straus E, Urbach HJ, Yalow RS: Alcohol-stimulated secretin secretion. N Engl J Med 293:1031-1032,1975 69. Straus E, Yalow RS: Hypersecretinemia associated with marked basal hyperchlorhydria in man and dog. Gastroenterology 72:992-994, 1977 70. Reeder DD, Becker HD, Smith NJ, et al: Measurement of endogenous release of cholecystokinin by radioimmunoassay. Ann Surg 178:304-310, 1973 71. Thompson JC, Fender HR, Ramus NI, et al: Cholecystokinin metabolism in man and dogs. Ann Surg 182:496-504, 1975 72. Rayford PL, Fender HR, Ramus NI, et al: Release and half-life of CCK in man. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 301-318 73. Harvey RF, Dorosett L, Hartog M, et al: A radioimmunoassay for cholecystokinin-pancreozymin. Lancet 2:826-828, 1973 74. Harvey RF, Dorosett L, Hartog M, et al: Radioimmunoassay of cholecystokinin-pancreozymin. Gut 15:690-699,1974 75. Spence RW, Celestin LR, Harvey RF: Effect of metiamide on basal and stimulated serum cholecystokinin levels in duodenal ulcer patients. Gut 17:920-923, 1976 76. Grossman MI and others: Candidate hormones of the gut. Gastroenterology 67:730-755, 1974 77. Kuzio M, Dryburgh JR, Malloy KM, et al: Radioimmunoassay for gastric inhibitory polypeptide. Gastroenterology 66:357-364, 1974 78. Said S, Faloona GR: Elevated plasma and tissue levels of vasoactive intestinal polypeptide in the water-diarrhea syndrome due to pancreatic, bronchogenic and other tumors. N Engl J Med 293:155-160, 1975 79. Dryburg JR, Brown JC: Radioimmunoassay of motilin. Gastroenterology 681169-1176, 1976 80. Brown JC, Dryburgh JR Current status of motilin. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 549-554 81. Polak JM, Bloom SR, Adrian TE, et al: Pancreatic polypeptide in insulinomas, gastrinomas, vipomas, and glucagonomas. Lancet 1:328-330,1976 82. Berson SA, Yalow RS General radioimmunoassay. In Methods in Investigative and Diagnostic Endocrinology. Edited by SA Berson, RS Yalow. Amsterdam, The Netherlands, North-Holland Publishing Co, 1973, p 84-120 83. Yalow RS: Radioimmunoassay: practices and pitfalls. Circ Res 32:1116-1128,1973 84. Berson SA, Yalow RS Quantitative aspects of reaction between insulin and insulin-binding antibody. J Clin Invest 381996 2016, 1959 85. Yalow RS, Berson SA: Radioimmunoassay. In Statistics in Endocrinology. Edited by JW McArthur, T Colton. Cambridge, Mass, The MIT Press, 1970, p 327-344 86. Straus E, Urbach HJ, Yalow RS: Comparative reactivities of ‘*‘Isecretin and L251-6-tyrosylsecretin with guinea pig and rabbit antisecretin sera. Biochem Biophys Res Commun 64:1036-1040, 1975 87. Bloom SR, Ogawa 0: Radioimmunoassay of human peripheral plasma secretin. J Endocrinol58:XXIV-XXV, 1973 88. Tai HH, Korsch B, Chey WY: Preparation of ‘ZSI-labeledsecretin of high specific radioactivity. Anal Biochem 6934-42, 1975 89. Tai H-H, Chey WY: Simultaneous radioimmunoassay of secretin and gastrin. Anal Biochem 74:12-24, 1976 90. Farooq 0, Walsh JH: Atropine enhances serum gastrin response to insulin in man. Gastroenterology 68:662-666,1975 91. McGuigan JE, Trudeau WL: Differences in rates of gastrin

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release in normal persons and patients with duodenal ulcer disease. N Engl J Med 288:64-66,1973 92. Straus E, Gerson CD, Yalow Rs: Hypersecretion of gastrin associated with the short-bowel syndrome. Gastroenterology 661%180,1974 93. Hansky J, Soveny C, Korman MG: Effect of secretin on serum gastrin as measured by immunoassay. Gastroenterology 61:6268, 1971 94. Walsh JH, Grossman MI: Circulating gastrin in peptic ulcer disease. Mt Sinai Med J NY 50:374-381,1973 95. Lamers CBH: The Zollinger-Ellison syndrome. Observations on 18 patients. In Some Aspects of the Zollinger-Ellison Syndrome and Serum Gastrin. Edited by CBH Lamers. Amsterdam, The Netherlands, Krips Repro Meppel, 1976, p 17-48 96. Hansky J: Hypergastrinaemia, hyperacidity and peptic ulceration (abstr). Rendic Gastroenterol9:11, 1977 97. Gregory RA, Tracy HJ: Isolation of two “big gastrins” from Zollinger-Ellison tumour tissue. Lancet 2:797-799,1972 98. Gregory RA, Tracy HJ: The chemistry of the gastrins: some recent advances. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 13-24 99. Yalow RS, Straus E: Heterogeneity of gastrin. In Progress in Gastroenterology. Edited by GBJ Glass. New York, Grune & Stratton, Inc (in press) 100. Hartley RC, Gambill EE, Summerskill WHJ: Pancreatic volume and bicarbonate output with augmented doses of secretin. Gastroenterology 48312-317, 1965 101. Makhlouf GM, McManus. JAP, Card WI: Action of gastrin II on gastric secretion in man. In Gastrin. Edited by MI Grossman. Berkeley, Los Angeles, Calif, University of California Press, 1966, p 139-169 102. Petersen H: The relationship between gastric and pancreatic secretion in man. Stand J Gastrcenterol4:345-351,1969 103. Dyke WI’: Pancreatic hypersecretion in the Zollinger-Ellison syndrome. Gastroenterology 60:90-95,197l 104. Dreiling DA, Greenstein A: Pancreatic function in patients with the Zollinger-Ellison syndrome. Observations concerning acid-bicarbonate secretion. Med Cir Dig l:l-4, 1972 105. Rayford FL, Miller TA, Thompson JC: Secretin, cholecystokinin and newer gastrointestinal hormones. N Engl J Med 294:1093-1101,1976 106. Debas HT, Grossman MI: Pure cholecystokinin: pancreatic protein and bicarbonate response. Digestion $469-481, 1973

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107. Walsh JH: Biologic activity and disappearance rates of big, little, and mini-gastrins in dog and man. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 75-83 108. Bloom Sr, Polak JM, Pearse AGE: Vasoactive intestinal peptide and water-diarrhea syndrome. Lancet 2:14-16,1973 109. Bloom SR, Polak JM: The role of VIP in pancreatic cholera. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 635-642 110. Pearse AGE, Polak JM, Bloom SR: The newer gut hormones. Cellular sources, physiology, pathology, and clinical aspects. Gastroenterology 72:746-761, 1977 111. Jaffe BM, Condon S: Prostaglandins E and F in endocrine diarrheagenic syndromes. Ann Surg 184:516-524,1976 112. JaiTeeBM, Condon S: Plasma concentrations of PGE in patients with the WDHA syndrome. Symposium on Hormones and Ulcer and 1st International Symposium on Gastrointestinal Hormones, Abstract 072,1976 113. Pederson RA, Brown JC: Inhibition of histamine-, pentagastrin-, and insulin-stimulated canine gastric secretion by pure “gastric inhibitory polypeptide.” Gastroenterology 62:393-400, 1972 114. Brown JC, Dryburgh JR, Moccia P, et al: The current status of GIP. In Gastrointestinal Hormones. Edited by JC Thompson. Austin, Texas, University of Texas Press, 1975, p 537-547 115. Pederson RA, Schubert HE, Brown JC: Gastric inhibitory polypeptide. Its physiologic release and insulinotrophic action in the dog. Diabetes 24:1050-1056,1975 116. Jordan PH, Bianca SS, Yip MD: The presence of gastrin in fasting and stimulated gastric juice of man. Surgery 72:352356, 1972 117 Uvnas-Wallensten K, Rehfeld J: Molecular forms of gastrin in antral mucosa, plasma and gastric juice during vagal stimulation in anesthetized cats. Acta Physiol Stand 98:217-226, 1976 118. Creutzfeldt W: Hormone-producing cells of the gastrointestinal tract. In Endocrinology of the Gut. Edited by NS Track. Ontario, Canada, Wm S Merrell Co, 1976, p 22-33 119. Straus E, Yalow RS Artifacts in the radioimmunoassay of peptide hormones in gastric and duodenal secretions. J Lab Clin Med 87:292-298,1976 120. Bloom SR, Nalin DR, Mitchell SJ, et al: High levels of VIP in cholera stool water, Symposium on Hormones and Ulcer and 1st International Symposium on Gastrointestinal Hormones, Abstract 018, 1976