Zollinger Ellison syndrome (ZES) is caused by release. The Zollinger Ellison Syndrome and Mismeasurement of Gastrin. Patients and Methods Patients

Transcription

1 GASTROENTEROLOGY 2011;140: The Zollinger Ellison Syndrome and Mismeasurement of Gastrin JENS F. REHFELD,* MARIE HÉLÈNE GINGRAS, LINDA BARDRAM, LINDA HILSTED,* JENS P. GOETZE,* and PIERRE POITRAS Departments of *Clinical Biochemistry and Surgical Gastroenterology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; and Department of Gastroenterology, CHUM - Hospital Saint-Luc, Université de Montréal, Montréal, Quebec, Canada See editorial on page BACKGROUND & AIMS: Zollinger Ellison syndrome (ZES) is characterized by hypersecretion of gastric acid, severe peptic ulcerations in the upper small intestine, and diarrhea. It is usually diagnosed by measuring increased levels of gastrin in plasma. METHODS: We examined the accuracy of commercial kits to measure gastrin (7 radioimmunoassays and 5 enzyme-linked immunosorbent assays), using plasma from 40 patients suspected or known to have ZES. Each sample was analyzed using the 12 kits and a reference assay that measures bioactive gastrin in plasma, irrespective of size and amino acid derivatization. Known concentrations of peptides with identical sequences to circulating gastrins were also assessed by all assays. Molecular patterns in plasma from patients with ZES were examined by chromatography and monitored by kits that measure false-low or falsehigh concentrations of gastrin. RESULTS: Failure to diagnose gastrinomas has serious consequences. Four kits found false-low concentrations of gastrin in 20% to 80% of the patients. Specificity assessment showed that the antibodies used in these kits bound only gastrin-17. Three kits found false-high concentrations of gastrin, because the reagents had increased reactions to sulfated gastrins or to unspecific factors in plasma. Thus, only 5 of 12 kits tested accurately measure plasma concentrations of gastrin. CONCLUSIONS: Seven of 12 tested commercial kits inaccurately measure plasma concentrations of gastrin; these assays used antibodies with inappropriate specificity that were insufficiently validated. Misdiagnosis of gastrinoma based on lack of specificity of assays for gastrin results in ineffective or inappropriate therapy for patients with ZES. Keywords: Diagnostic Assays; Gastrinoma; Antibody; Gastrin Quantitation. View this article s video abstract at org. Zollinger Ellison syndrome (ZES) is caused by release of gastrin from endocrine tumors. 1,2 Most gastrinomas originate in the pancreas or the duodenal mucosa. 3 6 The syndrome is characterized by hypersecretion of gastric acid, severe peptic ulceration in the upper small intestine, and often diarrhea. The diagnosis is usually established by measuring elevated gastrin concentrations in plasma. 4,5,7 13 Classically, 0.3% to 3% of patients with ZES have been reported to have normal or low gastrin concentrations A case of ZES with normal gastrin concentrations might be explained by unidentified acid stimulatory factors that are different from gastrin 17,18 or simply by assays unable to measure gastrin levels correctly. The latter possibility is worth examining because gastrin is not a single molecule but circulates as several peptides of various lengths and amino acid modifications (Figure 1A). 2,19 21 Moreover, the profile of circulating gastrins in hypergastrinemia deviates from that of healthy subjects (Figure 1B and C) The gastrin assays used for diagnosis thus have to be able to measure the pattern present in patients with ZES. Today, diagnostic measurements of gastrin are performed mainly using commercial immunoassay kits. 13,25 We describe here 2 patients suspected to have ZES and the consequences experienced by these patients because gastrin levels in plasma were reported to be normal or only slightly elevated. Motivated by these cases, we compared the diagnostic reliability of available gastrin kits with that of an immunoassay that has been fully characterized with respect to the pattern of gastrins in patients with gastrinoma. Finally, we characterized the specificity of the kits and are now able to explain false low and high concentrations in terms of reactivity with the different forms of gastrin. Patients and Methods Patients Two patients from Montreal motivated this study. Their stories are summarized in Results. Abbreviations used in this paper: CT, computed tomography; ELISA, enzyme-linked immunosorbent assay;, radioimmunoassay; ZES, Zollinger-Ellison syndrome by the AGA Institute /$36.00 doi: /j.gastro

2 May 2011 GASTRIN MISMEASUREMENT IN PATIENTS WITH GASTRINOMA 1445 Figure 1. (A) The overall structure of human progastrin and its bioactive products in plasma, that is, carboxyamidated gastrin-71, -52, -34, -17, and -14. The major processing sites in progastrin at basic amino acid residues (R, arginine; K, lysine) are indicated. Note that part of the bioactive gastrins (ie, carboxyamidated) exist in a tyrosyl-sulfated (Y, tyrosine) form, as indicated by SO 3 in parentheses. 37 (B) Pattern of circulating gastrins secreted from G cells in healthy subjects (data from Stadil et al 38 ). Note that the gel chromatography used does not distinguish gastrin-71 from gastrin-52. (C) Pattern of circulating gastrins secreted from gastrinoma cells in patients with ZES (data from Rehfeld and Stadil 23 ). Plasma Samples Samples from 40 Danish patients with proven or suspected ZES were examined. They included 21 men and 19 women with a median age of 53 years (range, years). Seven had localized gastrinoma in the pancreas or duodenum, 10 had metastatic gastrinoma, 10 fulfilled the clinical criteria of ZES with no tumor found yet, 7 were measured in the follow-up period after removal of one or more gastrinomas, and 6 were under suspicion but not finally proven to have a gastrinoma. The suspicion of a ZES diagnosis was corroborated by measurements of gastrin in plasma, using a fully characterized gastrin radioimmunoassay (), 26,27 and subsequently confirmed by the usual diagnostic procedures (computed tomography [CT] scan, Octreoscan (Octreoscan, Covidien PLC, Dublin, Ireland), surgery, histologic analysis, basal and pentagastrin-stimulated gastric acid secretion). In 5 of the patients, the gastrinoma was part of a MEN-1 syndrome, but none had been subjected to parathyroidectomy at the time of plasma sampling. Active disease was defined by plasma concentrations of gastrin and histologic analysis. The study was approved by the local ethics committee. Synthetic Peptides Human gastrin-17 (sulfated and nonsulfated), gastrin-34 (nonsulfated), gastrin-14 (sulfated and nonsulfated), gastrin-52 (nonsulfated), and cholecystokinin (CCK-8 sulfated) were synthesized by CRB (Cambridge Research Biochemicals, Billingham, Cleveland, UK) or Sigma (Sigma- Aldrich Chemie GmbH, St Louis, MO). The largest molecular form in human plasma, gastrin-71, 21 was not available for calibration. The quantity and structure of each synthetic peptide was controlled by high-performance liquid chromatography, mass spectrometry, and amino acid analysis.

3 1446 REHFELD ET AL GASTROENTEROLOGY Vol. 140, No. 5 Table 1. Assay Information From Manufacturers of Gastrin Immunoassay Kits Manufacturer Human gastrins used for specificity test Immunoassay technology Upper reference limit Diagnostic use Described limitations Biohit ELISA Hypergastrinemia Human plasma recommended Correlate Gastrin-13 (ns) ELISA For research use only; not for use in diagnostic procedures DiaSorin DRG Diagnostics Euro-Diagnostica Siemens Immulite 2000 MP Biomed Peninsula Phoenix EIA Phoenix Siemens a Gastrin 17 (s) Gastrin 34 (s) 108 pg/ml or 52 pmol/l (guideline) Hypergastrinemia Lipemia 111 pg/ml or Hypergastrinemia None 53 pmol/l (guideline) 60 pmol/l Hypergastrinemia None ELISA 115 pg/ml or 55 pmol/l (guideline) Hypergastrinemia None 111 pg/ml or 53 pmol/l (guideline) Hypergastrinemia None For research use only; not for use in diagnostic procedures ELISA Hypergastrinemia None Hypergastrinemia Extraction recommended; intended for in vitro use 90 pg/ml or 41 Hypergastrinemia None pmol/l (guideline) US Biological ELISA Not for use in diagnosis of gastrinoma ns, nonsulfated; s, sulfated. a Mini gastrin also tested without indication of length or sulfation. Gastrin Kits Seven and 5 enzyme-linked immunosorbent assays (ELISA) kits were purchased. They constituted all the gastrin kits available according to a catalog and Internet search. The kits were used according to the manufacturers instructions by a technician with more than 20 years of experience in measurement of gastrin. Each sample was assayed twice in duplicate. As indicated in Table 1, the instructions of 3 kits state For research only. Not for use in diagnostic procedures. We have, nevertheless, included them because correct measurement is always crucial, both in diagnosis and research. Reference Assay and Additional Assays Used for Characterization The results of plasma measurements obtained with the kits were compared with those of an in-house based on antiserum no. 2604, which binds the bioactive sequence of all amidated gastrins. 26 The antiserum binds human gastrin-14, -17, -34, -52, and -71 with equimolar potency, irrespective of degree of sulfation. Moreover, the cross-reactivity with CCK is negligible. 26 Under the conditions used, there is no interference from plasma proteins, salt, or other unrelated substances. 27 Monoiodinated human nonsulfated gastrin-17 was used as tracer, 28 and the same batch of synthetic gastrin-17 in its nonsulfated form was used as standard. The assay setup and conditions of incubation have been detailed elsewhere. 27 The assay has been used for 4 decades for diagnostic measurement in Scandinavian countries. 9 To corroborate the results obtained with kits that measured only gastrin-17, we repeated the measurements with another in-house using antiserum no , which is specific for gastrin-17 without reactivity with longer or shorter forms of gastrin (but 4 times higher affinity for sulfated gastrin-17). Moreover, for the chromatographic examination, we used a gastrin based antiserum no that is specific for N-terminus of

4 May 2011 GASTRIN MISMEASUREMENT IN PATIENTS WITH GASTRINOMA 1447 human gastrin Finally, for the chromatographic examination of the reactivity to sulfation, we used antiserum no. 2605, which binds only nonsulfated gastrins. 26,30 Chromatography For characterization of the gastrins in gastrinoma plasma, samples were applied to Sephadex (Sephadex, GE Healthcare, Little Chalfont, Buckinghamshire, UK) G50 superfine colums. 23 The elutions were monitored using selected immunoassay kits and in-house sequence specific with antibodies as described previously. Statistics For assay comparison, we used Deming regression analysis, which determines the line of best fit for a 2-dimensional data set. Bland Altman bias plots were also calculated; all comparisons were performed using Graph- Pad Prism software (La Jolla, CA). Results Case Report 1 A 49-year-old man with diarrhea and profuse vomiting was hospitalized in the intensive care unit for hypovolemic shock and acute renal failure. A nasogastric tube drained 3Lofliquids daily, and he was treated with intravenous pantoprazole 40 mg twice daily. Computed axial tomography (CT scan) showed 2 minor hepatic lesions and a large 12-cm lesion with septae and cysts as well as severe fluid accumulation in intestinal loops. Table 2. Gastrin Levels in Plasma From Patients Suspected or in Control for Gastrinomas Commercial gastrin kits Reference assay Biohit Correlate DiaSorin DRG Euro- Diagnostica Immulite MP Biomed Peninsula Phoenix EIA Phoenix Siemens US Biological GroupA GroupB GroupC , , ,400 11, ,600 11, ,000 18,900 44,500 11,300 22, ,500 10,500 20,500 16, ,500 62, ,000 1,260, , ,000 87,400 3,140, , ,000 1,129, , , ,000 Upper concentration limit for healthy subjects (see also Table 1) NOTE. Values are expressed as pmol/l. The patients in group A had concentrations 100 pmol/l (reference assay). a Several in this group had their gastrinomas removed. The patients in group B had concentrations between 100 and 400 pmol/l (reference assay). These patients were in treatment with proton pump inhibitors. The patients in group C had concentrations 400 pmol/l (reference assay). The upper concentration limits (pmol/l) for normal fasting subjects are indicated in the last line of the table for kits providing this information. a The upper reference concentration for healthy fasting subjects is approximately 50 pmol/l, but because patients with dyspeptic symptoms are often treated with proton pump inhibitors with ensuing moderate hypergastrinemia up to 100 pmol/l, concentrations above this level are in the gastrinoma range. Consequently, concentrations 100 pmol/l are false low when the reference assay results are 100 pmol/l.

5 1448 REHFELD ET AL GASTROENTEROLOGY Vol. 140, No. 5 Gastrointestinal endoscopy revealed mild esophagitis and prominent gastric folds but no ulcers. While still being treated with pantoprazole, the patient s serum gastrin level was 67 pmol/l (normal, 0 50 pmol/l; GammaDab Gastrin Kit, DiaSorin, Stillwater, MN). During a secretin test, the gastrin concentrations were again almost normal (53 64 pmol/l). Based on the normal gastrin levels, the diagnosis of ZES was discarded. Attempts to stop pantoprazole treatment led to profuse acid vomiting and multiple esophageal strictures, prohibiting oral feeding and gastric secretory studies. Somatostatin receptor scintigraphy (Octreoscan) suggested a neuroendocrine tumor in liver segments V, VI, and VII. Considering a diagnosis of ZES, the dosage of intravenous pantoprazole was increased to 80 mg every 8 hours before it could be substituted for an oral suspension of esomeprazole 120 mg twice daily. Remeasurement with the DiaSorin kit still revealed low gastrin concentration (64 pmol/l). The same serum sample measured with an Immulite 1000 kit (Diagnostic Products Corp, Llanberis, Caernafon, Gwynedd, UK) showed an elevated gastrin level (271 pmol/l) suspicious of gastrinoma. An exploratory laparotomy revealed bilobar hepatic neuroendocrine tumor metastases, but the primary tumor was not identified. Chemoembolization and treatment with octreotide stabilized tumor progression. Four years later, esomeprazole 80 mg twice daily is still required to maintain basal acid secretion less than 2 mmol/h, and esophageal dilatations are performed every 2 to 3 months for recurrent strictures. Case Report 2 A 51-year-old man who had a 6-month history of epigastric pain, diarrhea, and 15-kg weight loss was hospitalized for a severe episode of abdominal pain, vomiting, diarrhea, and dehydration. He rapidly improved with intravenous liquids and pantoprazole. Computed tomodensitometry (CT scan) showed inflammatory changes of the third segment of the duodenum and enlarged lymph nodes in the periduodenal area, while liver and pancreas were normal. Gastrointestinal endoscopy showed severe esophagitis and 3 ulcers in the third segment of the duodenum. Gastrin level in serum was 88 pmol/l (Dia- Sorin kit). The slight increase in gastrin level was attributed to long-term intake of a proton pump inhibitor and acute renal failure due to dehydration. Two days after stopping treatment with pantoprazole, the severe digestive symptoms relapsed and resolved when pantoprazole was readministered. Endoscopic ultrasonography suggested enlarged gastric folds and showed 2-cm lymph nodes where fine needle aspiration indicated neuroendocrine tumor. Octreoscan showed increased uptake corresponding to the lymph nodes found on CT scan and endoscopic ultrasonography. A second gastrin measurement with the DiaSorin kit was still normal (32 pmol/l). A secretin stimulation test failed to increase serum gastrin concentrations. Basal gastric acid output was 14.5 meq/h (normal, 5 meq/h) while treatment continued with oral pantoprazole 80 mg twice daily. Following these results, the diagnosis of ZES was confirmed, and the dosage of pantoprazole was increased to 120 mg twice daily. At laparotomy, 2 lymph nodes containing metastasis of a neuroendocrine tumor were removed near the pancreas head, but no primary tumor was identified. Six years after surgery, gastric acid hypersecretion is still controlled by esomeprazole 20 mg twice daily. Diagnostic Sensitivity of the Gastrin Kits Table 2 shows the results of the measurements in plasma from 40 fasting patients with ZES using the commercial immunoassay kits. Defined by the reference assay, the first group of 14 patients had concentrations less than 100 pmol/l (Table 2, group A). The patients Table 3. Gastrin Concentrations in 0 Plasma to Which Synthetic Human Peptides and an Extract of Human Antral Mucosa Have Been Added Commercial gastrin kits Added gastrin peptide Reference assay Biohit Correlate DiaSorin DRG Diagnostics Euro- Diagnostica Immulite MP Biomed Peninsula Phoenix EIA Phoenix Siemens US Biological 0 plasma Gastrin-14 (ns) Gastrin-14 (s) Gastrin-52 (ns) CCK-8 (s) ( pmol/l) Antral extract Upper concentration limit for normal subjects (see also Table 1) NOTE. Values are expressed as pmol/l. ns, nonsulfated; s, sulfated.

6 May 2011 GASTRIN MISMEASUREMENT IN PATIENTS WITH GASTRINOMA 1449 with the lowest concentrations were those with controlled disease after surgical removal of gastrinomas (nos. 1 5, 8, and 11 in Table 2 [group A]). Five of the patients (nos. 6, 7, 12, 13, and 14 [Table 2, group A]) still had active disease and 2 (nos. 10 and 14 [Table 2, group A]) were strongly suspected of having ZES yet without histochemical verification of gastrinoma. In group A, the Biohit and US Biological kits measured false-low concentrations in 5 and 4 patients, respectively. The second group of 14 patients with gastrinoma were not surgically treated but on long-term medication with proton pump inhibitors. The medication was stopped for 5 days before blood was drawn for measurement of gastrin until the diagnosis was established. According to the reference assay, they had concentrations ranging between 100 and 400 pmol/l (Table 2, group B). The results show that the Biohit ELISA, US Biological ELISA, and Phoenix failed to provide adequate diagnostic measurements in 35%, 43%, and 77% of patients, respectively. In addition, the DiaSorin and the Phoenix ELISA kits measured too low concentrations in 2 and 3 cases, respectively. The last 12 patients had concentrations greater than 400 pmol/l (Table 2, group C). Despite a considerable interkit variation, the concentrations in this group are so high that they support a diagnosis of gastrinoma anyway. The only exception was a patient in whose plasma the US Biological kit measured a concentration of 80 pmol/l. In all groups of patients, the Siemens Immulite kit measured false-high concentrations due to overreactivity against sulfated gastrins (Table 3 and Figure 2C). Too high concentrations were also measured by the Correlate kit, both in the low and the intermediate ranges (Table 2), apparently due to unspecific interference from plasma proteins (Table 3). Considering patients at risk for an erroneous diagnosis when basal gastrin level is between 50 and 400 pmol/l (20 patients in our series), the kits provided the correct diagnosis in the following percentages of patients: Euro- Diagnostica 100%, MP Biomed and Siemens 95%, DRG and Siemens Immulite 90%, Peninsula and Phoenix EIA 75%, DiaSorin and Biohit 60%, US Biological 55%, and Phoenix 40%. The kit from Correlate measured elevated gastrin concentrations in 100% of patients with 4 Figure 2. Gel chromatography (Sephadex G50 superfine column) of plasma from a patient with ZES. The elution was monitored using 5 gastrin immunoassays that all use synthetic human nonsulfated gastrin-17 as assay calibrator and tracer. (A) The elution monitored by the Biohit ELISA kit. (B) The elution monitored by an specific for the N-terminus (N t )of human gastrin (C) The elution monitored by the Siemens Immulite kit. (D) The elution monitored by an that uses antibody 2605, which binds the amidated C-terminus of bioactive gastrins in nonsulfated form only. 30 (E) The elution monitored by the reference that uses antibody no. 2604, which binds the carboxyamidated C-terminus of all the bioactive gastrins and binds these gastrins with equimolar affinity, irrespective of the degree of tyrosyl sulfation. 26,27

7 1450 REHFELD ET AL GASTROENTEROLOGY Vol. 140, No. 5 Table 4. Deming Regression Analysis Comparing the Results of the Reference Gastrin Assay With Those of Commercial Gastrin Immunoassays From Diagnostic Companies Manufacturer Slope 95% confidence interval Y-intercept when X 0 95% confidence interval P value r (Pearson) Biohit to Correlate to DiaSorin to DRG Diagnostics to Euro-Diagnostica to Siemens Immulite to MP Biomed to Peninsula to Phoenix EIA to Phoenix to Siemens to US Biological to NOTE. Thirty samples were included, defined from a cutoff from the gold standard analysis with plasma gastrin concentration 500 pmol/l (see Table 2). hypergastrinemia, but it also provided false-positive results in 8 of 8 patients with normal gastrin levels. Analytical Specificity of the Gastrin Kits According to the manufacturers, all the immunoassay kits were examined against nonsulfated gastrin-17; 10 kits were also examined against nonsulfated gastrin-34 and 6 kits against sulfated gastrins (Table 1). To extend the specificity examination, we spiked human plasma that had first been charcoal stripped of endogenous gastrins ( 0 plasma) with different synthetic human gastrins. As shown in Table 3, gastrin-14 (sulfated as well as nonsulfated) was not measured by the Biohit, Siemens Immulite, Phoenix ELISA, Phoenix, and US Biological kits. Gastrin-17 (sulfated and nonsulfated) was measured by all kits, but the Biohit, Immulite, and US Biological kits overestimated the sulfated form of gastrin to 7-fold, whereas the Phoenix ELISA kit did not measure sulfated gastrin-17 at all. Human gastrin-34 (nonsulfated) was not measured by the Biohit, Phoenix, and US Biological kits but measured fully by the remaining kits. The same pattern of reactivity was found with synthetic gastrin-52 (nonsulfated). Gel chromatography confirmed that kits that did not bind gastrin-14, gastrin-34, and gastrin-52 in the specificity test recognized only gastrin-17 in plasma from patients with ZES (Figure 2A). The result was confirmed both by our in-house gastrin-17 specific, using antibody no (data not shown), and the specific for the N-terminus of gastrin-17 (Figure 2B). Gel chromatography also confirmed that kits that overestimated sulfated forms of synthetic gastrins also did so in plasma from patients with gastrinoma (Figure 2C). Unspecific plasma interference was assessed using the previously mentioned human 0 plasma stripped free of endogenous gastrins. Significant interference was found in the Correlate and the Phoenix ELISA kits (Table 3). In Deming regression analysis, the correlation between the reference assay and each commercial gastrin kit was found to vary considerably. Thus, the r value (Pearson) varied from 0.34 to 0.96 (Table 4). The variation is further illustrated for 3 kits: one that is gastrin-17 specific with frequent false-low measurement (Biohit, Figure 3A and B); one that overreacts with sulfated gastrin, which causes false-positive measurements (Siemens Immulite, Figure 3C and D), and one with acceptable correlation (Siemens, Figure 3E and F). Discussion The patient reports show that failure to measure gastrin levels in plasma correctly can have deleterious consequences. The ensuing assay examination showed that the failures are due to antibodies that do not appropriately recognize the molecular forms of gastrin secreted by gastrinomas. Gastrinomas are well known for their short-term and long-term complications. Short-term complications are related to gastric acid hypersecretion and aggressive peptic disease, often with ulcer perforation. Fortunately, peptic complications can now be treated effectively with proton pump inhibitors. Modern management of patients with ZES can therefore focus on long-term complications of tumor growth. The consequences of missing a diagnosis of ZES are serious. Our first patient developed esophagitis and peptic stenosis that prohibited oral alimentation for weeks and forced serial dilatations of the esophagus for years. The second patient had repeated episodes of dehydration and severe ulcerations with microperforation of the distal duodenum. In these situations, the clinician disregarded the diagnosis of ZES because of low gastrin concentrations and therefore failed to prescribe the antisecretory regimen that would have been appropriate.

8 May 2011 GASTRIN MISMEASUREMENT IN PATIENTS WITH GASTRINOMA 1451 Figure 3. Deming regression analyses (left panels) and the corresponding bias plots (right panels) comparing 3 commercial gastrin kits (A and B, C and D, and E and F, respectively) with the reference assay. Only samples with a gastrin concentration 500 pmol/l defined by the reference assay were included (n 30). The dashed lines indicate the 95% confidence interval. Note that the Biohit kit measures false-negative low concentrations (bias, 67%; SD, 78%) and the Siemens Immulite kit measures false-high concentrations (bias, 28%; SD, 54%). Accurate measurement of gastrin in plasma together with assessment of gastric acid secretion are decisive for diagnosis of ZES. Gastrin, however, circulates in multiple forms, both in healthy subjects and in patients with ZES (Figure 1) Moreover, the peptide pattern and the concentration vary considerably, not only between

9 1452 REHFELD ET AL GASTROENTEROLOGY Vol. 140, No. 5 healthy subjects and patients with ZES as such (Figure 1B and C), but also among the individual patients with gastrinoma. 23,24,30 To ensure a high diagnostic sensitivity, the first-line assays should therefore measure all the bioactive (ie, amidated) gastrin peptides, irrespective of size and modification such as tyrosyl sulfation. 23,24 A few patients with gastrinoma can have truly normal concentrations of amidated gastrin in plasma For these patients, assays that measure progastrin or the total progastrin product may be necessary in a second line of measurement. During the past decades, commercial immunoassay kits have, to a large extent, been implemented in routine diagnostic measurements. 13,25 An open question is to what extent these kits meet the pathognomonic specificity. Prompted by missed diagnoses of ZES, we have in the present study compared the measurements of available gastrin kits with those of a fully characterized reference assay (Tables 2 4). The study shows considerable variation of the gastrin concentration in the same sample as measured by different kits. The 40 plasma samples were randomly collected among patients known to have, known to have had, or strongly suspected to have ZES. False-low concentrations were frequently encountered using 4 of the kits. The diagnostic significance of false-low concentrations is minor for the group of patients who have plasma concentrations greater than 400 pmol/l. The major problem is the false-low concentrations measured in patients with moderately but diagnostically significant increased plasma gastrin concentrations (50 or pmol/l [Table 2, groups A and B]). In this category, false-low measurements may be responsible for failure to establish the correct diagnosis, putting the patients at risk for severe, even lethal complications. Our study also uncovers the causes of the deviating results in precise molecular terms. Hence, false-low results are due to antibodies that bind only gastrin-17, but neither shorter nor longer forms (Figures 1 and 2A). The results, however, depend not only on the length of the gastrin peptide. The antibody binding is also heavily influenced by modifications such as tyrosine sulfation. Thus, a study of different gastrin antisera showed a 100-fold variation in reactivity with sulfated gastrins. 36 Moreover, while approximately half of each gastrin form is sulfated in normal human tissue and plasma, 24,30 the pattern changes in a highly individual manner in patients with gastrinoma, where sulfation has been found to vary between 11% and 82%. 24 The variation is further complicated by the observation that some antibodies of the gastrin-17 specific kits also underreact or overreact with sulfated gastrins. Hence, if plasma contains a large fraction of sulfated gastrin-17, the gastrin-17 specific assays measure false-high gastrin concentrations, as seen for the Biohit and US Biological kits. Accordingly, the gastrin-17 specific Phoenix kit measures more consistently false-low concentrations (Table 2) because the antibody reacts poorly with sulfated gastrin. These results emphasize the necessity of evaluating not only peptide length but also amino acid derivatizations. The study also reveals examples of false-high gastrin concentrations. The explanation seems 2-fold. Two kits are sensitive to unspecific plasma effects (Table 3). The other factor is tyrosine sulfation, as discussed previously. This seems true also for a kit that is not gastrin-17 specific such as the Siemens Immulite kit, where the false-high results (Table 2) are caused by overreaction with sulfated gastrins (Figure 2C). The implications of false-high concentrations may be less severe than falselow concentrations because the tumor diagnosis is not missed. On the other hand, false-high concentrations are a cause of anxiety, and patients have to undergo unnecessary and costly investigations. At its extreme, they can lead to unnecessary surgery and inappropriate, lifelong treatment. A broader perspective of the present study is that neuroendocrine tumors in general release peptide hormones and chromogranins that, like gastrin, circulate in different molecular forms. An obvious question is, therefore, whether the immunoassay kits used for the other neuroendocrine tumors have the necessary diagnostic sensitivity and specificity. The perspective becomes even wider when diagnostic use of immunoassays as such is challenged. Do antibodies in diagnostic assays today thus cover the complexity of substances unveiled by modern biochemistry in normal and diseased tissue and plasma? References 1. Gregory RA, Grossman MI, Tracy HJ, et al. Nature of the gastric secretagogue in Zollinger-Ellison tumours. Lancet 1967;2: Gregory RA, Tracy HJ, Agarwal KL, et al. Aminoacid constitution of two gastrins isolated from Zollinger-Ellison tumour tissue. Gut 1969;10: Zollinger RM, Ellison EH. Primary peptic ulcerations of the jejunum associated with islet cell tumors of the pancreas. Ann Surg 1955;142: Creutzfeldt W, Arnold R, Creutzfeldt C, et al. Pathomorphologic, biochemical, and diagnostic aspects of gastrinomas (Zollinger- Ellison syndrome). Hum Pathol 1975;6: Wolfe MM, Jensen RT. Zollinger-Ellison syndrome. Current concepts in diagnosis and management. N Engl J Med 1987;317: Metz DC, Jensen RT. Gastrointestinal neuroendocrine tumors: pancreatic endocrine tumors. Gastroenterology 2008;135: Walsh JH, Grossman MI. Gastrin. N Engl J Med 1975;292: Malagelada JR, Davis CS, O Fallon WM, et al. Laboratory diagnosis of gastrinoma. I. A prospective evaluation of gastric analysis and fasting serum gastrin levels. Mayo Clin Proc 1982;57: Jacobsen O, Bardram L, Rehfeld JF. The requirement for gastrin measurements. Scand J Clin Lab Invest 1986;46: Rehfeld JF, van Solinge WW. The tumor biology of gastrin and cholecystokinin. Adv Cancer Res 1994;63: Varro A, Ardill JE. Gastrin: an analytical review. Ann Clin Biochem 2003;40:

10 May 2011 GASTRIN MISMEASUREMENT IN PATIENTS WITH GASTRINOMA Berna MJ, Hoffmann KM, Serrano J, et al. Serum gastrin in Zollinger-Ellison syndrome: I. Prospective study of fasting serum gastrin in 309 patients from the National Institutes of Health and comparison with 2229 cases from the literature. Medicine (Baltimore) 2006;85: Rehfeld JF. The art of measuring gastrin in plasma: a dwindling diagnostic discipline? Scand J Clin Lab Invest 2008;68: Wolfe MM, Jain DK, Edgerton JR. Zollinger-Ellison syndrome associated with persistently normal fasting serum gastrin concentrations. Ann Intern Med 1985;103: Zimmer T, Stolzel U, Bader M, et al. Brief report: a duodenal gastrinoma in a patient with diarrhea and normal serum gastrin concentrations. N Engl J Med 1995;333: Jais P, Mignon M. Normal serum gastrin concentration in gastrinoma. Lancet 1995;2: Dubois A, Eerdewegh PV, Gardner JD. Gastric emptying and secretion in Zollinger-Ellison syndrome. J Clin Invest 1977;59: Song Y, Chey WY, Chang TM, et al. Mechanism of gastric acid hypersecretion in patients with islet cell tumor without hypergastrinemia: studies in rats. Gastroenterology 1997;113: Gregory RA, Tracy HJ. Isolation of two big gastrins from Zollinger-Ellison tumour tissue. Lancet 1972;2: Gregory RA, Tracy HJ, Harris JI, et al. Minigastrin; corrected structure and synthesis. Hoppe Seylers Z Physiol Chem 1979; 360: Rehfeld JF, Johnsen AH. Identification of gastrin component I as gastrin-71. The largest possible bioactive progastrin product. Eur J Biochem 1994;223: Rehfeld JF, Stadil F, Vikelsoe J. Immunoreactive gastrin components in human serum. Gut 1974;15: Rehfeld JF, Stadil F. Gel filtration studies on immunoreactive gastrin in serum from Zollinger-Ellison patients. Gut 1973;14: Andersen BN, Petersen B, Borch K, et al. Variations in the sulfation of circulating gastrins in gastrointestinal diseases. Scand J Gastroenterol 1983;18: Wolfe MM, Paquet RJ, Reel GM. Specificity of commercially available antibodies used for gastrin measurement. J Lab Clin Med 1985;105: Rehfeld JF, Stadil F, Rubin B. Production and evaluation of antibodies for the radioimmunoassay of gastrin. Scand J Clin Lab Invest 1972;30: Stadil F, Rehfeld JF. Determination of gastrin in serum. An evaluation of the reliability of a radioimmunoassay. Scand J Gastroenterol 1973;8: Stadil F, Rehfeld JF. Preparation of 125 I-labelled synthetic human gastrin I for radioimmunoanalysis. Scand J Clin Lab Invest 1972;30: Bardram L, Rehfeld JF. Production and evaluation of monospecific antibodies for a processing-independent sequence of human progastrin. Scand J Clin Lab Invest 1989;49: Andersen BN, De Magistris L, Rehfeld JF. Radioimmunochemical quantitation of sulfated and non-sulfated gastrins in serum. Clin Chim Acta 1983;127: Bardram L, Rehfeld JF. Processing-independent radioimmunoanalysis: a general analytical principle applied to progastrin and its products. Anal Biochem 1988;175: Bardram L. Progastrin in serum from Zollinger-Ellison patients. An indicator of malignancy? Gastroenterology 1990;98: Jais P, Mignon M, Rehfeld JF. Processing-independent assay of serum gastrin for diagnosis of liver metastases in the Zollinger- Ellison syndrome. Int J Cancer 1997;71: Jorgensen NR, Rehfeld JF, Bardram L, et al. Processing-independent analysis in the diagnosis of gastrinomas. Scand J Gastroenterol 1998;33: Rehfeld JF. Processing of precursors of gastroenteropancreatic hormones: diagnostic significance. J Mol Med 1998;76: Rehfeld JF, De Magistris L, Andersen BN. Sulfation of gastrin: effect on immunoreactivity. Regul Peptides 1981;2: Rehfeld JF, Zhu X, Norrbom C, et al. Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17. Biochem J 2008; 415: Stadil F, Rehfeld JF, Christiansen L, et al. Patterns of gastrin components in serum during feeding in normal subjects and duodenal ulcer patients. Scand J Gastroenterol 1975;10: Received November 11, Accepted January 24, Reprint requests Address requests for reprints to: Professor Jens F. Rehfeld, MD, DSc, Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark. jens.f.rehfeld@rh.regionh.dk; fax: (45) Acknowledgments The authors thank Alice von der Lieth for skillful technical assistance and Diana Skovgaard for skillful secretarial assistance. Conflicts of interest The authors disclose no conflicts. Funding The earlier gastrin assay studies upon which the present study is based have been supported by grants from the Danish Medical Research Council, the Danish Cancer Union, and the Lundbeck and the Novo Nordisk Foundations.