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Keywords:

  • H2-receptor antagonist;
  • Zollinger-Ellison syndrome;
  • Gastrinoma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References

Background

In people, serum gastrin concentrations increase in response to administration of H2 receptor antagonists, but the effect of famotidine administration on serum gastrin concentrations has not been evaluated in dogs.

Objectives

To determine if serum gastrin concentrations increase in response to 14 days of famotidine treatment and the time needed to return to baseline after discontinuation of famotidine; define stability of gastrin in samples held at room temperature.

Animals

Eleven healthy dogs were included in part A (famotidine treatment) and 7 healthy dogs in Part B (serum gastrin stability). In part A, famotidine (0.5 mg/kg PO q12h) was administered for 14 days. Fasting blood samples were collected on days 0, 3, 7, 11, 14, 16, 18, 20, and 22. In part B, blood was collected after a 12-hour fast. Gastrin concentrations in serum samples held at room temperature for ≤30 minutes after sampling were compared to concentrations in samples held at room temperature for 150 minutes after sampling.

Results

Serum gastrin concentrations increased by day 3 of famotidine administration and returned to baseline concentrations in all dogs by day 14 despite continued famotidine administration. Serum gastrin concentrations were lower (20% mean decrease; P = .0005) in samples held at room temperature for 150 minutes.

Conclusions and Clinical Importance

After 14 days of famotidine administration, clinically healthy dogs have normal serum gastrin concentrations. In a dog with clinical features consistent with gastrinoma, chronic famotidine administration is unlikely to contribute to increases in serum gastrin concentrations.


Abbreviations
Ach

acetylcholine

GI

gastrointestinal

H2RA

H2-receptor antagonists

PPI

proton pump inhibitor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References

Gastric parietal cells, also known as oxyntic cells, secrete hydrochloric acid in response to stimulation by acetylcholine (Ach) released from the vagus nerve, gastrin produced and secreted by gastric G cells, and histamine produced and secreted by enterochromaffin-like cells (ECL).[1]

Gastrin is a heterogeneous hormone consisting primarily of 2 molecular forms differing in size and biological activity but both having a similar carboxy-terminal pentapeptide amide sequence. Stimulation of gastrin secretion is mediated by gastrin releasing peptide (GRP).[1, 2] Gastric antral distention and intragastric amino acids, amines, and calcium stimulate the release of GRP. Inhibition of hydrochloric acid secretion is mediated mainly by somatostatin (SST). Somatostatin is secreted by gastric D cells in response to increased gastric concentrations of HCl[3] and suppresses gastrin secretion from gastric G cells, acid secretion from parietal cells, and histamine release by ECL cells.[4-6] In dogs, clearance of exogenously administered human gastrin by the kidney[7, 8] and lung[9] has been demonstrated with an approximate half-life of 5 minutes.[10] Increased serum gastrin concentrations can result from chronic pharmacologic suppression [H2-receptor antagonists (H2RA) and proton pump inhibitors (PPIs)] of gastric acid secretion in people and rats,[3, 11, 12] gastrinoma, Helicobacter pylori infection in humans,[5] hepatic ischemia[7], and renal failure in humans[13] and cats.[14] Although Hpylori can stimulate gastrin secretion in canine cell culture systems,[15] it does not appear to affect serum gastrin concentrations.[16]

Gastrinomas are neuroendocrine tumors that secrete gastrin. Gastrinomas usually are located in the pancreas, but a gastrinoma has been documented in the duodenum of a dog.[17] Dogs with gastrinomas typically develop vomiting, weight loss, anorexia, lethargy, diarrhea, polydipsia, and melena.[18-21] The presence of a gastrinoma may be suspected after documenting increased serum gastrin concentrations in a patient with compatible clinical signs in the absence of other conditions that increase serum gastrin concentrations. Serum gastrin concentrations in patients with gastrinoma may be 3–100 fold higher than normal,[22] but in affected humans, two-thirds of patients have fasting gastrin concentrations <10 times normal.[23]

Famotidine is an H2RA commonly used for symptomatic treatment of dogs exhibiting gastrointestinal (GI) signs. Famotidine suppresses gastric acid production by competitive inhibition of the histamine receptor on gastric parietal cells. The use of H2RAs and PPI in humans interferes with the diagnosis of gastrinoma by masking clinical signs.[23-25] Current recommendations for measuring serum gastrin concentration in human patients are to discontinue PPIs 7 days, and H2RAs for 48 hours, before blood sampling.[25]

Because dogs with gastrinoma can exhibit gastrointestinal signs, some patients are treated with H2RAs before measurement of serum gastrin concentrations. Dogs admitted to our hospital often have been treated, sometimes for prolonged periods of time, with H2RAs such as famotidine. H2RAs, by virtue of their ability to suppress gastric acid secretion, may remove the negative feedback on gastrin secretion caused by luminal acid. They are thus likely to enhance gastrin secretion, which would falsely increase serum concentrations of gastrin. Although studies in human patients have confirmed H2RA-induced hypergastrinemia, this phenomenon has not been documented in dogs chronically treated with H2RAs. Knowing if hypergastrinemia is a consequence of chronic H2RA therapy in dogs has substantial clinical relevance in patients suspected to have gastrinoma. Whether or not H2RAs should be discontinued or whether or not there is a need for a “washout period” before measuring serum gastrin concentration in dogs has not been established. The purpose of this study was to determine the effect of long-term (14 days) famotidine treatment and its subsequent withdrawal on serum gastrin concentrations in dogs. An additional goal was to assess the stability of gastrin concentrations in serum samples held at room temperature before freezing. We hypothesized that famotidine treatment would increase serum gastrin concentrations in dogs and that discontinuation of famotidine would result in return of serum gastrin concentrations to baseline within 7 days. In addition, we hypothesized that room temperature storage of serum samples for 150 minutes would significantly decrease measured serum gastrin concentrations as compared to samples held at room temperature for <30 minutes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References

Animals

Part A

Dogs belonging to employees or students of the Washington State University College of Veterinary Medicine who consented to enroll their dogs in the study were included. The study was approved by the Institutional Animal Care and Use Committee at Washington State University. Subjects were required to be healthy based on physical examination and could not be receiving any medications. Patients were screened for underlying metabolic disorders with a CBC, serum biochemical profile, and urinalysis. They had not shown signs of anorexia, vomiting, or diarrhea in the past month. Because gastrin clearance is delayed in patients with decreased glomerular filtration rates, dogs with a serum creatinine concentration >1.4 mg/dL and concurrent urine specific gravity <1.030 were ineligible for this study. Eleven dogs enrolled and completed the study.

Part B

Blood samples from 1 dog that participated in Part A of the study and 6 additional dogs were used to determine the stability of gastrin stored at room temperature. The additional dogs also belonged to employees or students of the Washington State University Veterinary College of Veterinary Medicine and were enrolled with the consent of the owners. Inclusion and exclusion criteria were the same as for part A.

Procedures

Part A

Famotidine (0.5 mg/kg PO q12h) was administered for 14 days. Venous blood was collected before the 1st dose (day 0), and on days 3, 7, 11, 14, 16, 18, 20, and 22 after the first dose. Patients were fasted for a minimum of 12 hours before blood collection for all samples. Blood samples were collected and centrifuged in nonserum separator evacuated glass tubes without anticoagulant. Serum was decanted into a plastic aliquot tube and frozen at −80°F within 30 minutes of venipuncture. All serum samples were shipped as a batch overnight on dry ice to the Michigan State University Endocrinology Laboratory for measurement of serum gastrin concentration.

Serum gastrin concentrations were measured using a commercially available radioimmunoassay1 that recognizes the major circulating forms of gastrin. This assay, which uses rabbit anti-human gastrin antibodies as the primary antibody, and goat anti-rabbit antibodies as the precipitating antibody, has been previously validated for dogs using linearity and dilutional parallelism.2

Part B

Subjects were fasted for 12 hours before blood collection. Venous blood was collected and prepared as described for Part A. Then serum was decanted into 4 plastic aliquot tubes. Two of the 4 samples were frozen within 30 minutes of collection. The other 2 samples were kept at room temperature for 150 minutes and then frozen at −80°F. Samples were labeled so as to not reveal their storage conditions and all samples were shipped as a batch to the Michigan State University Endocrinology Laboratory for measurement of gastrin concentration as for Part A.

Statistical Analysis

For the data of Part A, the D'Agostino test showed that all data were normally distributed except those at day 20. We thus analyzed the time course of serum gastrin concentration changes by repeated measures analysis of variance, followed by a one-sided Dunnett's test for statistical differences between day 0 and all subsequent days (nonparametric Friedman's test followed by pairwise Wilcoxon tests led to the same statistical conclusions as reported below [data not shown]). For the data in Part B, the D'Agostino test showed that all data were normally distributed. Accordingly, a paired t-test was used to compare the difference in gastrin concentrations in samples held at room temperature for 30 and 150 minutes post sampling. Statistical significance was set at a value ≤.05. Intra-assay variability in Part B was calculated as the difference in measured gastrin concentration between the paired samples divided by the mean of the 2 samples.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References

Part A

Study participants ranged in age from 2 to 11 years (mean, 5.9 years) and ranged in weight from 9.75 to 35 kg (mean, 20 kg). Breeds included in the study were Greyhound (1), English Cocker Spaniel (1), Labrador Retriever (1), Jack Russell Terrier (1), Golden Retriever (1), mixed breed (1), Border Collie (2) and Australian Cattle Dog (3). All subjects completed the study. No adverse effects related to the treatment were observed. The mean famotidine dose was 0.585 mg/kg (range, 0.5–0.66 mg/kg). Gross lipemia or hemolysis was not noted in any serum samples. Baseline (day 0) serum gastrin concentration ranged from 22 to 84 ng/L (mean, 42.4 ng/L; median, 45 ng/L). Six of the 11 subjects in Part A and 3 of 7 subjects in Part B had fasting serum gastrin concentration above the reported reference interval of 10–40 ng/L. Mean serum gastrin on days 3 (116 ng/L; range, 38–314), 7 (106.9 ng/L; range, 22–233), and 11 (86.3 ng/L; range, 19–206) were significantly higher than on day 0. There was no significant difference in mean serum gastrin concentration between day 0 and days 14 through 22 (Fig 1). Although all gastrin concentrations had returned to baseline by day 14, there was variability among the individual dogs as to when each gastrin concentration returned to baseline, with the range being 3–14 days.

image

Figure 1. Mean serum gastrin concentration over time in 11 dogs treated with famotidine 0.5 mg/kg PO q12h for 14 days. Serum gastrin was measured before administration of famotidine (day 0), and on days 3, 7, 11, 14, 16, 18, 20 and 22. Points on the line reflect the mean (±SD) serum gastrin concentration for all dogs at each point. Compared to baseline at day 0, mean serum gastrin concentration was significantly different (P < .05) at days 3, 7 and 11 (denoted by asterisks).

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Part B

Serum samples from 7 dogs ranging in weight from 30 to 54 kg (median, 40 kg) were obtained for measurement of serum gastrin concentration in samples maintained at room temperature for 30 and 150 minutes. Dogs ranged in age from 2 to 6 years (median, 4 years). Breeds included Great Dane (1), Golden Retriever (1), German Shepherd (1), Saint Bernard (1), and Labrador Retriever (2). Gross lipemia or hemolysis was not noted in any serum samples. Serum gastrin concentrations (mean of each paired aliquot) in the 30-minute samples ranged from 28 to 76 ng/L. There was a significant decrease (Fig 2) in mean serum gastrin concentrations (43.9 ng/L for all dogs) between samples held at room temperature for 30 minutes post-sampling compared to those held at room temperature for 150 minutes (mean, 34.7 ng/L for all dogs; P = .0005); the range of the mean of each paired aliquot at 150 minutes was 22–52 ng/L. Serum gastrin concentrations in individual paired samples held for 150 minutes decreased by a mean of 20% of their 30-minute concentrations (range, 13–36.3%). For the paired gastrin samples, there was an intra-assay variability of 14.3% in the mean gastrin concentration of the pair.

image

Figure 2. Serum gastrin concentration decreases in samples held at room temperature for 150 minutes compared to serum gastrin concentration measured after storage at room temperature for 30 minutes. Serum was obtained from 6 fasted dogs and each sample divided into 4 aliquots, 2 of which were frozen within 30 minutes of collection, and 2 of which were held at room temperature for 150 minutes after collection before freezing. The figure depicts the mean gastrin concentration of each pair of samples at 30 minutes and the mean gastrin concentration of each pair of samples at 150 minutes; each pair of dots and its connecting line represent data from a single dog. The mean gastrin concentration for all dogs at 30 minutes was 43.9 ng/mL and 34.7 ng/mL at 150 minutes. The means for all dogs at 30 and 150 minutes were significantly different (P = .0005).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References

As hypothesized, mean serum gastrin concentrations peaked by day 3 of famotidine administration, but instead of remaining above basal concentrations, serum gastrin concentration had returned to baseline by day 14, the last day of famotidine administration, in all dogs. In a study of dogs treated with ranitidine for 7 days, basal serum gastrin concentrations peaked at day 4 after starting treatment, comparable to our results, and remained increased through day 7.[26] In this study, fasting gastrin concentrations had returned to baseline 8 days after ranitidine was stopped, but there were no measurements of serum gastrin concentrations between days 7 and 15 so the time at which dogs returned to baseline could not be determined. Results of studies in humans of the effect of H2RAs on serum gastrin concentrations have been comparable to, or different from, our results. Earlier investigations showed administration of H2RAs for 8 days, or up to 29 days of continual daily dosing, resulted in consistently increased gastrin concentrations.[27, 28] In a more recent study, however, serum gastrin concentrations returned to baseline by day 14 of continual daily famotidine administration to human patients without evidence of H. pylori infection.[29] In the earlier studies, the presence of H. pylori was not assessed. The interaction between H. pylori infection and H2RA administration has not been evaluated in dogs but gastrin concentrations were unaltered in dogs with Helicobacter infections.[16]

Return of serum gastrin concentrations to baseline in people is thought to occur from tolerance to H2RAs. The mechanism of tolerance is not known, but is speculated to be a consequence of gastrin's trophic properties on gastric mucosa. Hypergastrinemia initially results in hypertrophy of ECL cells (days) and then hyperplasia (weeks), increasing histamine production.[30] Increased numbers of histamine molecules then are able to competitively combine with receptors to stimulate acid production. Other suggested mechanisms of tolerance to H2RAs include upregulation of parietal cell receptors for other mediators, sensitization of H2 receptors, or alteration in H2 receptor turnover after chronic competitive inhibition. Consistent with the hypothesis of tolerance, abrupt discontinuation of HR2As causes rebound acid hypersecretion in humans as a result of the trophic properties of gastrin on ECL cells.[29] The phenomenon of tolerance has not been investigated in veterinary patients.

A 12-hour fast before blood sampling was chosen due to constraints imposed by the blood sampling schedule after famotidine discontinuation, namely that dogs would have had to be fasted for 24 hours every other day for the last 8 days of the study period. The laboratory's reference interval for fasting serum gastrin concentration has been established for dogs fasted for >24 hours. The fact that dogs in our study were fasted for only 12 hours rather than 24 may have contributed to our observation of basal serum gastrin concentrations greater than the laboratory's reported reference interval in 9 of 18 dogs.

One important consideration when interpreting results of this study is that in people, gastrin secretion can fluctuate during the course of a day, and only a single sample was used to determine serum gastrin concentrations on each day of the study.[23] Fluctuations in serum gastrin concentration also may have been a contributing factor in the increased basal serum gastrin concentration in 50% of our study animals. Perhaps a more accurate assessment of serum gastrin concentrations would have been to measure them in blood samples obtained hourly for 24 hours as has been performed in studies of humans. Despite this potential limitation, however, our findings of increased serum gastrin concentration in response to H2RA administration followed by decreased serum gastrin concentration in the face of continued administration are consistent with results of studies in humans and rats.[31]

We were not able to establish a washout period of famotidine before measuring serum gastrin concentrations because gastrin concentrations returned to baseline while dogs were still receiving famotidine. Determining a washout period for an individual patient may be difficult for a number of reasons. First, owner compliance with oral medication administration is variable[32] and can be poor, making it difficult to predict when famotidine actually was administered and withdrawn from a patient. Second, although mean serum gastrin concentration at 14 days was not significantly different from baseline, there was variability in the time at which serum gastrin concentration returned to baseline among individual dogs. Finally, owners also may administer other over-the-counter medications, such as antacids, that might also alter gastric acid-gastrin physiology. Although it might be ideal to measure serum gastrin concentrations in the absence of famotidine treatment, our data suggest that famotidine therapy is unlikely to confuse interpretation because none of our study patients developed serum gastrin concentrations typical of dogs with gastrinoma, and dogs on famotidine for over 11 days actually had serum gastrin concentrations within reference ranges.

Our finding that serum gastrin was not stable when serum samples were stored at room temperature for 150 minutes as compared to 30 minutes after blood sampling is consistent with results of studies of gastrin stability in people. Recommendations made by the authors of one study were that serum should be frozen at −70°C as soon as possible before analysis.[31] Serum gastrin concentrations in samples stored at room temperature for 24 hours decreased by almost 50%. Surprisingly, gastrin concentrations in samples stored for 24 hours at 4 and −6°C declined by >10%. The average decrease in serum gastrin concentration in our study samples maintained at room temperature for over 2 hours before freezing was 20%. Dogs in which serum gastrin concentrations are only marginally increased, or at the lower end of the spectrum for a gastrinoma, may appear to have normal or only slightly increased gastrin concentrations if the sample is inappropriately handled. This could result in an inappropriate dismissal of gastrinoma as a differential diagnosis for a particular patient. Based on our findings, it is recommended that serum samples be frozen as quickly as possible after collection.

Finally, the results of our study raise an important and clinically relevant question, which is whether or not administration of famotidine for more than 14 days is effective in suppressing gastric acid production. Although Komazawa et al[29] documented a return of serum gastrin concentrations to normal in humans treated with famotidine for 14 days, there appeared to be some continued suppression of acid secretion. We did not measure gastric acid secretion or intraluminal pH in our study subjects and thus cannot make a definitive statement as to the efficacy of long-term famotidine treatment in suppression of acid secretion in dogs. Based on results of a recent study in dogs in which intragastric pH was >3 for only a short percentage of time during a 7-day famotidine treatment,[33] the utility of famotidine for long-term acid suppression would seem to warrant investigation.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References

The authors thank Ms Peggy Bryan and Dr Bryan Slinker for their assistance with statistical analysis.

Footnotes
  1. 1

    Gastrin 125I Radioimmunoassay Kit, MP Biomedicals, Orangeburg, NY

  2. 2

    personal communication, Dr Patricia Schenck, Michigan State University

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of Interest
  8. Acknowledgment
  9. References