This chapter explains normal gastrointestinal function and provides an overview of inflammatory bowel disease and neuroendocrine tumors. Disorders reviewed include: irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, colorectal cancer, and neuroendocrine tumors. For each analyte used in the gastrointestinal field the biological function is explained (if applicable), with the clinical applications of the test and its limitations. Typical assay technology is also described. The type of sample and frequency of use are included, with an example reference interval (for background information only). The analytes included are: calprotectin, chromogranin A, chromogranin B, cocaine- and amphetamine-regulated transcript, pancreatic polypeptide, gastrin, somatostatin, vasoactive intestinal peptide, and glucagon.

Pancreatic polypeptide (PP) is predominantly expressed in the pancreas, with very low concentrations being detected in the endocrine cells of the small and large intestine (1). PP concentrations rise sharply postprandially and take approximately 6 hours to return to fasting levels. In obese subjects, lower basal and postprandial circulating concentrations of PP have been measured compared to normal weight subjects (2). Following weight loss, PP concentrations generally return to levels observed in lean subjects (3). Plasma PP concentrations are significantly higher in type 2 diabetic patients compared to non-diabetic subjects. Recently it has been demonstrated that in mammalian somatostatin-containing cells PP, possibly via the Y4 receptor, inhibits somatostatin secretion (4). This reduction in somatostatin secretin is thought to allow the necessary postprandial rise in gastrointestinal endocrine hormones.

In the circulation, PP has a comparatively long half-life compared to other satiety hormones (2). PP has been found to be extremely stable. No significant differences in PP concentrations were measured when samples were collected using lithium heparin, lithium heparin tubes containing aprotinin (Trasylol), ethylenediaminetetraacetic acid (EDTA), plain and Serum Separation tubes, or when the time between blood collection and plasma and serum separation (up to 4 and 5 hours respectively) and freeze-thaw cycle number (up to 4) was investigated (data awaiting publication).

The proglucagon (PG) peptide consists of 158 amino acids and is differentially processed to produce the biologically active peptides, glucagon, oxyntomodulin, glicentin, GLP-1 and GLP-2. Glucagon corresponds to PG residues 33–61 and is formed by the action of prohormone convertase (PC) 2 in the pancreas. Oxyntomodulin contains the full glucagon amino acid sequence and is C-terminally extended with IP1. Glicentin, which also contains the full glucagon and IP1 sequence, is N-terminally extended with GLRP. Glicentin corresponds to PG residues 1–69 and is formed by the action of PC1/3 in the gut (Figure 1). Glicentin can be further processed to produce oxyntomodulin which corresponds to PG residues 33–69. As glicentin and oxyntomodulin are expressed in the intestinal they have been termed “enteroglucagon”. The remaining part of PG is processed to produce GLP-1 and GLP-2 in the intestinal L-cells.

N-terminal

C-terminal

1

158

GLRP

Glucagon

IP1

GLP-1

IP2

GLP-2

33

Glucagon

.61

Pancreas

33

Oxyntomodulin

69

}

1

Glicentin

69

Gut

Figure 1: Proglucagon peptide is processed to produce glucagon in the pancreas, oxyntomodulin and glicentin in the gut. GLP-1 and GLP-2 are also products of proglucagon. GLRP – glicentin related peptide, IP1 – intervening peptide 1, IP2 – intervening peptide 2, GLP-1 – glucagon like peptide 1, GLP-2 – glucagon like peptide 2.

Due to the way PG is processed the development of specific antibodies for glucagon needs to be C-terminally directed. Glucagon antibodies directed to the mid-region of the molecule also detect glicentin and oxyntomodulin; while N-terminally directed antibodies detect glucagon and oxyntomodulin. To further complicate antibody development for glucagon assays, it is the mid-region which is more antigenic. In addition, glucagon circulates in low concentrations so the antibody needs to be sensitive. These requirements increase the likelihood that more animals will need to be immunized in order to produce a specific and sensitive antibody for glucagon.

Non-neuroendocrine tumour causes of plasma elevation

Glucagon concentrations rise moderately in response to a meal, to low plasma glucose concentrations and by certain amino acids. Therefore, secretion is stimulated by protein-rich meals, but inhibited by carbohydrate-rich meals. Patients with type 2 diabetes have higher fasting concentrations of glucagon than healthy subjects. 

High levels of plasma glucagon may be measured in patients with end-stage renal function, however, it has been proposed that the high concentrations are due to N-terminally extended forms of glucagon that can be detected with assays directs to the C-terminal of glucagon and not with assays that employ two antibodies to detect intact glucagon (5).

Sample collection

The recommended collection method for glucagon samples has been to add the protease inhibitor, aprotinin. A recent study demonstrates that this is not necessary as no significant difference in glucagon concentrations was found in samples collected from healthy subjects or patients with gastrointestinal diseases (6).

Proton pump inhibitors (PPIs) increase circulating CgA concentrations; the mean increase is approximately 2.3-3 times. The magnitude of increase is dependent on the duration and dose of treatment. Short term PPI treatment of 30 mg/day for 7 days results in a significant mean increase of 2.5 fold in either plasma or serum CgA concentrations (11). The increase in short term CgA elevation is significantly higher with daily PPI doses of 40 mg compared to 20 mg (7). Medium term use of 40 mg/day also resulted in a mean increase of serum CgA concentration of 2.5 times (12), and an increase of 3 times with long term PPI administration for 6 to more than 12 months was measured 154 patients (13).  Discontinuation of PPI resulted in significant decreases of serum CgA after 5 days (7), and continues to decline with a half-life of 4-5 days (11). However, PPIs need to be discontinued for 2 weeks before concentrations return to pre-treatment levels. However, there is considerable inter-individual variation in the rise of circulating CgA concentrations in response to PPIs, with some patients experiencing no change and others up to a 10-fold increase.

It has been recommended that H­­2 receptor antagonists be used instead as circulating CgA concentrations are not elevated when used (10;13).

1. Cox, H.M. Neuropeptide Y receptors; antisecretory control of intestinal epithelial function. Auton. Neurosci. 133 (1), 76−85 (2007).
2. Batterham, R.L., Le Roux, C.W., Cohen, M.A. et al. Pancreatic polypeptide reduces appetite and food intake in humans. J. Clin. Endocrinol. Metab. 88 (8), 3989−3992 (2003).
3. Reinehr, T., Roth, C.L., Enriori, P.J., Masur, K. Changes of dipeptidyl peptidase IV (DPP-IV) in obese children with weight loss: relationships to peptide YY, pancreatic peptide, and insulin sensitivity. J. Pediatr. Endocrinol. Metab. 23 (1−2), 101−108 (2010).
4. Kim, W., Fiori, J.L., Shin, Y.K. et al. Pancreatic polypeptide inhibits somatostatin secretion. FEBS Lett. 588 (17), 3233−3239 (2014).
5. Wewer Albrechtsen, N.J., Hartmann, B., Veedfald, S. et al. Hyperglucagonaemia analysed by glucagon sandwich ELISA: nonspecific interference or truly elevated levels? Diabetologia 57 (9), 1919−1926 (2014).
6. Bak, M.J., Albrechtsen, N.W., Hartmann, B. et al. No Effect of Aprotinin (Trasylol™) on Degradation of Exogenous and Endogenous Glucagon in Human, Mouse and Rat Plasma. J. Endocrinol. & Diabetes 1 (1), 5 (2014).
7. Pregun, I., Herszenyi, L., Juhasz, M. et al. Effect of proton-pump inhibitor therapy on serum chromogranin a level. Digestion 84 (1), 22−28 (2011).
8. Fossmark, R., Johnsen, G., Johanessen, E., Waldum, H.L. Rebound acid hypersecretion after long-term inhibition of gastric acid secretion. Aliment. Pharmacol. Ther. 21 (2), 149−154 (2005).
9. Jianu, C.S., Fossmark, R., Viset, T. et al. Gastric carcinoids after long-term use of a proton pump inhibitor. Aliment. Pharmacol. Ther. 36 (7), 644−649 (2012).
10. Korse, C.M., Muller, M., Taal, B.G. Discontinuation of proton pump inhibitors during assessment of chromogranin A levels in patients with neuroendocrine tumours. Br. J. Cancer 105 (8), 1173−1175 (2011).
11. Mosli, H.H., Dennis, A., Kocha, W., Asher, L.J., Van Uum, S.H. Effect of short-term proton pump inhibitor treatment and its discontinuation on chromogranin A in healthy subjects. J. Clin. Endocrinol. Metab. 97 (9), E1731−E1735 (2012).
12. Waldum, H.L., Arnestad, J.S., Brenna, E., Eide, I., Syversen, U., Sandvik, A.K. Marked increase in gastric acid secretory capacity after omeprazole treatment. Gut 39 (5), 649−653 (1996).
13. Sanduleanu, S., De, B.A., Stridsberg, M. et al. Serum chromogranin A as a screening test for gastric enterochromaffin-like cell hyperplasia during acid-suppressive therapy. Eur. J. Clin. Invest. 31 (9), 802−811 (2001).

Arne Røseth MD, PhD, started his medical training in San Diego, USA and graduated from University of Oslo in 1985. He is a board-certified gastroenterologist in both the USA and Norway and is currently working as a gastroenterology consultant in Oslo, Norway. He is also working as a medical consultant for Bühlmann laboratories in Switzerland. He developed as part of his PhD project the Calprotectin assay and has more than 20 years of clinical experience using this biomarker in his clinical practice. Furthermore, he has been involved in many clinical trials around the world using this assay as a part of the study protocol.

Richard Chapman was formerly Lead Consultant Clinical Scientist at Imperial College Healthcare NHS Trust, Honorary Senior Lecturer at Imperial College Faculty of Medicine and Director of the Supra-regional Assay Service for endocrinology and gut hormones. His major research interests were in immunoassay and antibody production and development, particularly monoclonal antibodies. His career began at the Middlesex Hospital Medical School then at the Scottish Antibody Production Unit and the Royal Infirmary in Glasgow before returning to London at the Royal Postgraduate Medical School and Hammersmith Hospital. Here, he managed a programme of modernisation for clinical biochemistry through the development of a four-site "teaching hospital" network with a fully automated laboratory, community and specialist services centralised at the Charing Cross hospital. He continues as an enthusiast for teaching and training in immunoassay and antibody technology and acted as an overseas “expert” for the International Atomic Energy Agency.

Radha Ramachandran is NIHR Doctoral Research Fellow, Division of Diabetes, Endocrinology and Metabolism, Imperial College, London, UK (Dr. Ramachandran is currently funded by an NIHR Doctoral Research Fellowship).

Chris Sheehan is an experienced and effective Healthcare Commercial Coach/Consultant/Interim, with a strong Sales and Marketing track record in large corporate, international, diagnostics and capital markets. He is expert in all aspects of sales processes and CRM implementation and has coached high growth companies in planning for and achieving strong sales in business-to-business markets. Has worked with pre-start and SMEs (small to medium sized enterprises) in healthcare and has carried out several assignments at incubators internationally mentoring start-ups, creating business and marketing plans and project managing commercialization. Chris enjoys motivating and persuading people, whether in international project teams, as line manager or in customer and opinion leader consultative selling settings to achieve business results and people development. Previously with Johnson and Johnson, Chris works with Oxford Innovation as well as leading a breast cancer start-up and work for Fleet Bioprocessing.

Paul Bech, PhD, runs an specialist assay service for the Department of Diabetes, Endocrinology and Metabolism, Imperial College, London UK. The service measures hormones used for the diagnosis, treatment follow-up and disease recurrence of neuroendocrine tumors. Paul Bech is also involved with NET biomarker research and assay development.

Gastrointestinal tract, stomach, intestines, irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, colorectal cancer, neuroendocrine tumor, neuroendocrine neoplasm, calprotectin, chromogranin A, chromogranin B, cocaine- and amphetamine-regulated transcript, pancreatic polypeptide, gastrin, somatostatin, vasoactive intestinal peptide, glucagon.