CHAPTER 12

Clinical biochemistry of the gastrointestinal tract

Ingvar T. Bjarnason; Roy A. Sherwood

CHAPTER OUTLINE

INTRODUCTION

Gastrointestinal complaints continue to account for a sizeable proportion of medical consultations. This is best illustrated by the fact that irritable bowel syndrome (IBS), the most commonly encountered gastrointestinal disorder, is estimated to have a prevalence of 20% in the UK. About 30% of these people seek medical attention, which in turn accounts for as many as 20–50% of patients attending general gastroenterology outpatient clinics in the UK. Furthermore, the prevalence of reflux oesophagitis and non-specific dyspepsia is increasing, and with the advent of safe and effective drugs to inhibit gastric acid secretion, it is estimated that the cost of treatment for these disorders may grow to account for as much as 20% of drug expenditure in primary care.

The development of endoscopic techniques has revolutionized the investigation of gastrointestinal disorders. The combination of upper gastrointestinal endoscopy, colonoscopy and optic and/or wireless capsule enteroscopy now allows visualization of the whole of the gastrointestinal tract and biopsies can be obtained from all parts of the bowel. This has resulted in loss of demand for many of the classic biochemical investigations (such as gastric acid secretion) and, as some of these methods are now obsolete, we have omitted them from this chapter. Endoscopy and biochemical investigations, however, provide different kinds of information that are, in many respects, complementary. Endoscopy provides a static morphological picture that by itself, or with biopsy, has the potential to provide a diagnosis that automatically translates to treatment. Biochemical methods, on the other hand, provide a wider range of information. These include the following.

• Providing a diagnosis. This is a frequently desired purpose of clinical biochemistry, but is rarely achieved. Nevertheless, the indirect documentation of the presence of Helicobacter pylori is often the only test required before treatment, and non-invasive tests for small bowel bacterial overgrowth may be the only tests required in order to treat diabetic patients with diarrhoea.

• Clinical screening tests prior to invasive investigation. Examples of these kinds of investigations are serum transglutaminase antibody measurements and tests of intestinal permeability, which, if normal, may avoid the need for more invasive tests and, if positive, are an indication for jejunal or duodenal biopsy.

• The detection and assessment of the severity of intestinal dysfunction. These tests are called upon in order to provide an explanation for clinical signs (such as weight loss), for monitoring response to therapy and for confirmation of diagnosis (e.g. measurement of intestinal permeability following gluten withdrawal and challenge in coeliac disease, although this is rarely necessary in practice).

• Providing prognostic information. Biochemical tests are uniquely suited to assess functional changes that may herald a drastic change in the activity of the disease. This is best exemplified by increased concentrations of inflammatory markers in the faeces of patients with clinically quiescent inflammatory bowel disease (IBD, i.e. ulcerative colitis and Crohn disease), as these predict imminent clinical relapse.

• Investigating the impact of non-intestinal factors on intestinal function, whether biochemical or physiological. These may be exogenous, for example related to radiotherapy, drugs, alcohol, dietary or environmental factors, or endogenous, for example due to malnutrition, reduced blood flow, anaemia etc. Non-steroidal anti-inflammatory drug (NSAID)-induced enteropathy is an example of gastrointestinal disease caused by an exogenous factor.

Our intention in this chapter is to review new and established laboratory-based investigations of the gastrointestinal tract that are useful clinically or for research purposes. We will not always provide the reference ranges for test results, as these may differ depending on environmental, geographical and racial factors, as well as differences in methodology.

MOUTH AND OESOPHAGUS

Clinical biochemistry has not had a major impact in the investigation of oral or oesophageal diseases. The reason is obvious in that the main functions of these organs are physical, namely grinding of food and transport to the stomach. Nevertheless, the buccal mucosa is a common site for obtaining material for genetic analysis and saliva can be assayed for a number of antibodies (although this is rarely done in clinical practice). Although the salivary glands and parotid secretions contain amylase (and initiate digestion of complex carbohydrates), peptides and growth factors (that may confer a degree of protection to the stomach and accelerate healing of gastric lesions), measurements of these are not helpful clinically and are used only in research.

The common oesophageal disturbances such as reflux and dysmotility (oesophageal spasms, neuromuscular incoordination, achalasia etc.) are investigated and diagnosed by imaging techniques (endoscopy, radiology), high-resolution manometry and pH recording, rather than by biochemical methods. Oesophageal cancer, infections (cytomegalovirus, Candida albicans and herpes simplex) and drug- and radiation-induced diseases are similarly diagnosed largely from the clinical history and by endoscopy and biopsy.

STOMACH

The stomach acts as a reservoir for ingested food, where it is mixed with acid, mucus and pepsin and then released at a controlled rate into the duodenum. The mucosal surface of the stomach is lined with mucus-secreting columnar epithelial cells, interrupted by gastric pits containing acid-secreting parietal (or oxyntic) cells and pepsinogen-secreting chief cells. The secreted hydrochloric acid kills many ingested bacteria, provides the acid pH necessary for pepsin (from pepsinogen) to begin digesting proteins and stimulates bile secretion. Mucus secretion is necessary to protect the mucosal surface of the stomach wall from its acidic contents. The stomach also secretes intrinsic factor, which binds vitamin B₁₂ and allows it to be absorbed in the terminal ileum. The muscular contractions of the stomach wall have the mechanical effect of macerating food.

Helicobacter pylori

Helicobacter pylori is now accepted to be the main cause of gastric and duodenal ulcers; other causes include NSAIDs and, very rarely, the Zollinger–Ellison syndrome. Helicobacter pylori may play an important role in gastric cancers (adenocarcinomas and mucosal–associated lymphoid tissue lymphomas) and is undoubtedly the main cause of chronic gastritis, though in most patients this is asymptomatic. Many experts also believe H. pylori to be a major cause of non ulcer-related dyspepsia, and recent recommendations suggest that anyone who so wishes should be tested for H. pylori and treated if found to be positive.

The mode of transmission of H. pylori is uncertain. It is thought to be an infection acquired in childhood, presumably by the faeco–oral route, which would explain the higher prevalence in developing countries and the progressive decline in prevalence in developed countries, with better hygiene, preservation of food and smaller family sizes. Whatever the mode of transmission, H. pylori elicits an inflammatory response that is usually asymptomatic. This takes the form of chronic gastritis, with an acute inflammatory cell infiltrate of variable severity. Nine out of ten people infected by H. pylori do not develop ulcers. The variable development of clinically significant disease relates to the site of infection, virulence factors (e.g. phospholipases, vacuolating cytotoxins (VAC), CagA protein) and poorly defined host factors that include blood flow, mucus secretion and pepsinogen stimulation. There are three recognized patterns of infection by H. pylori in the stomach. The most common type is that of low-grade inflammation of the mid-body of the stomach. This occurs in people with a high threshold for immune response and H. pylori that has a low expression of CagA and VAC. Gastric acid secretion is reduced and there are usually no significant clinical consequences. If the microbe is predominantly in the antrum, the inflammation leads to dysfunction of the antral G-cells that become hyper-reactive and secrete disproportionate amounts of gastrin in response to food and gastric distension. Gastrin has a trophic effect on the gastric parietal cells, increasing their number and stimulating them directly, via cell surface receptors, to produce more acid, resulting in the hyper-acidic state characteristic of patients with duodenal ulceration.

The third pattern, a pangastric infection with H. pylori, is characteristic of those who develop gastric ulcers and those at risk of developing gastric cancer. These individuals tend to have normal or reduced gastric acid secretion. In theory, it would be possible to perform an endoscopy on every patient with indigestion and document the pattern of infection. However, this would be prohibitively expensive owing to the large number of symptomatic patients. A less expensive option would be to assess a panel of hormones that are secreted from defined anatomical locations in the stomach (for instance pepsinogen 1 and 2 and gastrin-17), but further work is required before this approach becomes widely accepted. A more pragmatic approach has, therefore, been adopted in adult patients under 50–55 years of age, namely to test and treat for H. pylori infection, irrespective of the precise diagnosis (ulcer, gastritis etc.), unless ‘red flag’ indicators suggestive of malignancy (e.g. weight loss, dysphagia) are present.

Having established the presence of H. pylori (see below), there are a number of eradication regimens available, and the eradication failure rate, after trying at least three different regimens, should be < 5–10%. If H. pylori is successfully eradicated, reinfection rates are extremely low (< 1%).

Diagnosis of H. pylori infection

There are various methods for the diagnosis of H. pylori infection. At endoscopy, it is possible to take biopsies from which the organism can be visualized histologically or cultured, the latter being performed only if there are repeated treatment failures. Alternatively, the biopsy can be tested directly using commercially available kits incorporating a gel containing urea and an indicator that changes colour at an alkaline pH. In the presence of H. pylori (which contains urease), the urea is broken down to carbon dioxide and ammonia. The latter increases the pH of the gel and a colour change takes place. Less invasive methods involve measurement of H. pylori-specific antibodies (IgG or IgA). These are eminently suitable for screening (sensitivity 92%, specificity 83%), but cannot be used to document the success of treatment as high antibody titres can persist despite successful eradication.

The H. pylori breath test is currently the most widely used method for non-invasive diagnosis. It uses the same principle as the biopsy-based test and comes at a fraction of the cost of endoscopy and biopsy. Patients are given isotopically labelled urea (¹³C or ¹⁴C) to drink. If there is no urease present, the urea is absorbed intact and excreted in the urine. If H. pylori is present, labelled carbon dioxide is absorbed into the circulation and exhaled in the breath. Breath samples are obtained before, and 45–60 min after, drinking the labelled urea. The detection of labelled carbon is most commonly performed by mass spectrophotometry. The breath test is widely used and can also be used to assess the success of the treatment (sensitivity 95%, specificity 96%). Lastly, there are commercial kits that use the polymerase chain reaction to amplify nuclear sequences specific for H. pylori from saliva or faeces (sensitivity 95%, specificity 94%). The stool tests are increasingly being used for the detection and confirmation of successful eradication of H. pylori rather than the breath test.

Gastric acid secretion

Before the discovery of the role of H. pylori in peptic ulcer disease, it was common practice to investigate gastric acid secretion in patients with duodenal ulcers, who tend to be hyper-secretors, and those with gastric ulcers, who tend to have normal or low secretion rates. Gastric cancers are often associated with hypochlorhydria, and achlorhydria is common in pernicious anaemia and in gastric cancer.

Acid secretion tests are now only rarely used in the research setting and are no longer available in the vast majority of clinical biochemistry departments: they have become obsolete as the test results do not alter clinical practice or management.

Gastrin

In < 0.5% of patients with gastroduodenal ulcers, the cause is unregulated gastrin release from an endocrine tumour termed a gastrinoma, which can lead to the Zollinger–Ellison syndrome characterized by multiple gastroduodenal ulcers. The persistently high plasma gastrin concentrations not only lead to marked hypersecretion of gastric acid, but also, because the hormone is trophic for parietal cells, to increased parietal cell mass. Patients may present with symptoms that are indistinguishable from H. pylori-associated peptic ulcer disease. However, coexisting diarrhoea (due to acidic inactivation of pancreatic enzymes), multiple ulcers involving the second part of the duodenum, recurrent ulceration and ulcers refractory to conventional treatment should always arouse suspicion. Gastrinomas are either sporadic (the more common form) or associated with multiple endocrine neoplasia type 1 (MEN 1, Wermer syndrome), a syndrome characterized by the presence of two or more of pituitary, pancreatic islet and parathyroid tumours. Multiple endocrine neoplasia type 1 is also associated with an increased prevalence of carcinoid, adrenocortical and thyroid tumours.

Gastrinomas are commonly situated in the pancreas, but are increasingly recorded arising from the duodenum, stomach and bones. Some 60% are clearly malignant, with multiple metastases at diagnosis. Given a suspicion of the Zollinger–Ellison syndrome, the first step is to measure fasting serum gastrin, as virtually all cases are associated with high concentrations. The differential diagnosis of a mildly elevated gastrin concentration includes hypochlorhydria, long-term proton pump inhibitor drug therapy, pernicious anaemia and antral G-cell hyperplasia. Given a strong suggestion of a gastrinoma, the next step is to localize the tumour, which is best done at a specialist centre. Techniques for localization of the tumour include endoscopic ultrasound, octreotide scanning, magnetic resonance imaging (MRI), positron emission tomography (PET) and computerized tomography (CT) (the latter being particularly useful to detect metastases). Definitive diagnosis is histological. The treatment of gastrinomas usually involves a combination of surgery, chemotherapy and acid suppression for symptomatic relief.

Intrinsic factor

Intrinsic factor is a glycoprotein principally secreted by the parietal cells of the stomach. Its secretion is governed by the same biochemical processes that regulate acid secretion and its action is to assist in the absorption of vitamin B₁₂. Vitamin B₁₂ is released from dietary proteins by the action of pepsin and then combines with R-binders (haptocorrins), glycoproteins secreted by the stomach, that assist in its protection against acid degradation. It is subsequently cleaved from R-binder complexes by pancreatic proteolysis in the duodenum and then binds to intrinsic factor to form a resistant complex that is taken up by ileal cell receptors (cubulin).

The commonest cause of intrinsic factor deficiency is autoimmunity; the reason for requesting intrinsic factor antibody measurement is usually the discovery of a low plasma vitamin B₁₂ concentration – a cause of macrocytic anaemia and various neurological disorders. The diagnosis of pernicious anaemia is based on the finding of a low plasma B₁₂ concentration together with the presence of antiparietal cell antibodies and/or intrinsic factor antibodies (see Chapter 27). Current automated vitamin B₁₂ assays suffer, in differing amounts, from interference from intrinsic factor antibodies; alternative strategies for assessment of B₁₂ status include measurement of serum homocysteine, methylmalonic acid and holotranscobalamin concentrations. Other causes of intrinsic factor deficiency leading to low plasma B₁₂ include gastrectomy, achlorhydria and congenital absence of intrinsic factor, which is rare. Vitamin B₁₂ deficiency can also occur because of deficient intake (e.g. in vegans, starvation or reduced food intake of any cause) or in small bowel disease involving the terminal ileum. Gastric biopsies showing histopathological features of atrophic gastritis may be helpful and there are some indications that patients should undergo surveillance endoscopies once a diagnosis is made because of an increased incidence of gastric cancer. In patients in whom small bowel disease is suspected, the choice is that of wireless capsule enteroscopy or CT/MRI enteroclysis (specialized techniques in which a contrast medium is infused into the small intestine) for diagnosis. Previously, on finding a low vitamin B₁₂ concentration, it was customary to request a Schilling test, which had the potential to help in the differential diagnosis, but this test has become obsolete as imaging techniques have improved. Although proton pump inhibitors induce a state of gastric acid hyposecretion and are widely used, it is exceptionally rare to see vitamin B₁₂ deficiency in these patients.

PANCREAS

The exocrine function of the pancreas includes the production of bicarbonate and enzymes, including amylase, lipase, trypsin, chymotrypsin, esterases and carboxypeptidases. The differential diagnosis of exocrine pancreatic disease in neonates and children is predominantly between cystic fibrosis and pancreatic acinar cell aplasia (Shwachman–Diamond syndrome). The major diseases affecting the pancreas in adults are acute pancreatitis, chronic pancreatitis leading to pancreatic insufficiency and carcinoma of the pancreas. Chronic pancreatitis results in progressive loss of both islet cells and acinar tissue. Presentation is typically with recurrent upper abdominal pain radiating to the back, although malabsorption, for example steatorrhoea, may be the presenting feature. Approximately 90% of the pancreatic acinar tissue must be lost before features of malabsorption become apparent and clinically significant reduction in endocrine function generally occurs late in the disease process.

Pancreatic function tests

Tests of pancreatic function are usually divided into the direct (invasive) tests and indirect tests on blood, urine or faecal samples.

Direct or invasive function tests

The ‘gold standard’ test of pancreatic function is the secretin–pancreozymin test. This test assesses exocrine function by measuring bicarbonate and pancreatic enzyme secretion (amylase and trypsin) in aspirates from a tube sited in the duodenum, usually under fluoroscopic control. Secretin is given to induce fluid secretion, while pancreozymin or its analogue caerulein (secretin–caerulein test), is given to induce enzyme production. This test requires meticulous attention to technique in positioning the tube correctly and maintaining its patency, and is uncomfortable for the patient. This test is now rarely used in routine practice. However, it remains the test that has the highest sensitivity and specificity for the differential diagnosis of pancreatic insufficiency.

Non-invasive pancreatic function testing

Serum enzymes

The measurement of pancreatic enzymes in serum is standard practice in acute pancreatitis, but is seldom useful in the investigation of chronic pancreatitis.

Amylase is the most commonly measured enzyme owing to the availability of cheap, easily automated methods. A disadvantage is the lack of specificity for the pancreas, as amylase in the circulation is derived from both pancreatic and non-pancreatic (mostly salivary) sources in approximately equal amounts. Measurement of the specific pancreatic amylase can be achieved using immunosubtraction techniques. Consideration of the ethnicity of the patient is important when interpreting amylase results, as subjects of African origin have a higher reference range for non-pancreatic amylase. The mechanism for this difference is unknown. An increase in both the pancreatic and non-pancreatic fractions may indicate the presence of macroamylasaemia – immunoglobulin–amylase complexes that have a prolonged half-life in the circulation owing to a reduction in clearance. Non-pancreatic causes of an increased serum amylase are given in Table 12.1. Lipase has greater specificity for pancreatic disease than amylase, but methods available tend to be more complex and expensive. The majority of the lipase in blood is the pancreatic form, although a sublingual lipase is also present. Lipase is not affected by ethnicity. Trypsin is 100% specific to the pancreas and would therefore, theoretically, be the best of the three enzymes to measure. However, there are physiological obstacles that mean that trypsin itself is seldom measured in blood samples. Trypsin is produced and stored in the pancreas as its inactive zymogen form (trypsinogen), and is activated after secretion into the intestinal tract. Active trypsin entering the circulation is bound immediately to the protease inhibitors α₂-macroglobulin and α₁-antitrypsin. Once bound, trypsin is not measurable using standard techniques, but any trypsinogen entering the circulation can be measured as ‘immunoreactive trypsinogen’ (IRT). Immunoreactive trypsinogen can be used as a first-line test for screening for cystic fibrosis using the blood spots taken on Guthrie cards at 7–10 days of age. A national programme of cystic fibrosis screening started in 2006–2007 in the UK; IRT is used for initial screening with subsequent genetic testing for confirmation.

Faecal tests

The measurement of faecal fat excretion as a test of fat malabsorption (and thus indirectly of pancreatic exocrine function), is now regarded as obsolete by most clinical biochemists and gastroenterologists.

Pancreatic enzymes that have been measured in faeces include chymotrypsin and elastase. Stool chymotrypsin measurements have suffered from a lack of standardization in the techniques used, making it difficult to compare results obtained from the various groups who have used the test. Measurement of faecal pancreatic elastase-1 is now recommended as the marker of choice for detecting pancreatic insufficiency. Elastase is an endopeptidase and sterol binding protein. As with chymotrypsin, elastase is not degraded during transit through the intestinal tract and is stable in faecal samples in vitro. Two commercially available enzyme linked immunosorbent assays (ELISAs) using antibodies specific for pancreatic elastase have been studied in patients with pancreatic insufficiency. Sensitivities of 60–100% for moderate to severe pancreatic disease, using a cut-off of 200 μg/g wet weight, have been reported for faecal elastase. Discrimination between diarrhoea of pancreatic and non-pancreatic origins has been reported to be good, with better specificity than chymotrypsin. Faecal elastase measurements may also be useful in determining the amount of pancreatic enzyme replacement therapy required in patients with cystic fibrosis or chronic pancreatic insufficiency (chymotrypsin cannot be used for this purpose). While biochemical tests attempt to give a functional diagnosis in chronic pancreatitis, imaging techniques provide further information that may disclose the cause. Plain abdominal radiography, endoscopic ultrasound, CT and magnetic resonance cholangiopancreatography (MRCP) are all useful. Endoscopic retrograde cholangiopancreatography (ERCP) with, or more commonly without, secretin stimulation is widely used, but carries a 10% risk of significant complications.

SMALL BOWEL BACTERIAL OVERGROWTH

With the discovery of microbes in the 19th century, theories were formulated linking the intestine with the development of systemic disease as a result of an unfavourable interaction between intestinal luminal microbes and the body. These ideas are equally evident today in the mass media, whereby producers of live yoghurts (containing viable bacteria) seek to equate ingestion of their products (probiotics) with health, vitality and happiness.

The normal intestinal microflora

Despite the perceived importance of intestinal bacteria, the reality is that we are still at the descriptive stages of assessing the normal intestinal flora and do not fully understand its function and impact on our well-being. Though the bulk of intestinal microbes inhabit the lower bowel, even the stomach is not usually sterile with bacterial population counts (predominantly Gram-positive aerobes) of up to 10³ per gram of luminal content. The jejunum has a similarly Gram-positive flora, with a higher population of up to 10⁴ per gram of luminal contents. The ileum contains a more varied flora that includes both aerobes and anaerobes, with populations in the region of 10⁵–10⁸ per gram. In the large intestine, Gram-negative anaerobes become the predominant species, overall bacterial populations rising significantly to 10¹⁰–10¹² per gram of caecal content. Similar counts are present in the distal colon where the bacterial flora corresponds to that seen on faecal analyses.

The above data have been obtained using invasive techniques not commonly used in clinical practice. However, intestinal bacterial populations and species differ between individuals and relate in a complex way to age, diet, geographical and racial factors, antimicrobial treatment and intrinsic gut diseases.

Definition, causes and symptoms of small bowel bacterial overgrowth

The relatively small numbers of bacteria normally present in the small intestine are probably of little significance. However, problems can arise when the bacterial population of the small intestine is increased – bacterial overgrowth. An essential consideration of this definition is that bacterial overgrowth is based on quantitative and qualitative estimates of coliform bacteria in small bowel aspirates, something which is not possible in routine clinical practice. There are a number of diseases and conditions associated with small bowel bacterial overgrowth, a combination of factors predisposing to overgrowth in any given condition. Gastric hypochlorhydria of any cause may contribute to small bowel bacterial overgrowth. Other factors include: alterations in systemic immunology (e.g. isolated immunoglobin A deficiency, hypogammaglobulinaemia, combined immune deficiency, infection with human immunodeficiency virus); impaired motility (e.g. advanced age, autonomic neuropathy in diabetes, fibrosis in scleroderma); pancreatic insufficiency; oral antibiotics; intrinsic small bowel disease (e.g. jejunal diverticulosis, coeliac disease, small bowel Crohn disease), and surgery (removal of the ileocaecal junction, surgically induced blind loops and, increasingly, bariatric surgery).

Small bowel bacterial overgrowth can be asymptomatic or be associated with non-specific symptoms, such as abdominal distension (bloating), eructation, flatulence, borborygmi and diarrhoea. The classic clinical presentation includes vitamin B₁₂

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