Gastrointestinal and pancreatic function

14.1 Laboratory diagnosis of gastric disease

Barbara Braden, Bernhard Lembcke, Lothar Thomas

The stomach comprises three anatomical and functionally distinct regions:

The cardia that contains mucus-secreting cells
The corpus, which constitutes 80–90% of the stomach and contains parietal cells secreting HCl and intrinsic factor and the pepsinogen secreting chief cells
The antrum, which contains the gastrin-secreting G-cells.

14.1.1 Regulation of gastric acid secretion

The central nervous system regulates the vagal activity of gastric secretion (smell, sight, taste and chewing of food). The vagal stimulation initiates the secretion of H⁺ and the G-cells to release the hormone gastrin, which enters the venous effluent and circulates to the parietal cells, inducing them to secrete H⁺.

HCl and pepsinogen are the main secretory products of the gastric mucosa. The mucosal epithelium of the stomach resists the destructive action of HCl and pepsin and forms a barrier between the lumen and the interstitial space. H⁺ are secreted by the parietal cells into the lumen at concentrations as high as 160 mmol/L. The H⁺ secretion is controlled by the carbonic anhydrase system of the parietal cell which regulates the conversion of carbon dioxide to carbonic acid. The carbonic acid then dissociates to H⁺ and HCO₃^–. H⁺ is released into the gastric juice and HCO₃^– enters via the interstitial fluid the circulation. Basal acid secretion is below 5 mmol/L, but under appropriate stimulation H⁺ secretion can rise to 5–20 mmol/L.

14.1.1.1 Functional heartburn

Functional heartburn consists of retrosternal burning similar to that experienced in patients with proven gastrooesophageal reflux disease (GERD), but without abnormal oesophageal acid exposure or oesophageal mucosal pathology such as erosive gastritis, Barrett's oesophagus, or eosinophilic oesophagitis. Functional heartburn is important to diagnose because without investigation, this nonerosive reflux disease might be considered equivalent to GERD and treated with acid suppressive therapy. A flow diagram demonstrating evaluation of persisting heartburn symptoms is shown in Ref.  /3/.

14.1.1.2 Peptic ulcer disease

The term peptic ulcer disease designs ulcerations of the mucous membrane of the oesophagus, stomach or duodenum caused by acid and pepsin in the gastric juice. Patients with peptic ulcer disease make up a substantial proportion of the overall gastroenterological patient population. Diagnostic laboratory examinations are, however, of only limited relevance in the diagnostics, course and therapeutic assessment of diseases of the upper gastrointestinal tract. It is mainly the diagnosis of Helicobacter pylori (H. pylori) infection, the main cause of gastritis in Western populations.

If the gastric mucosa is inflamed, the pattern of secretory products changes substantially. In H. pylori infection moderate hypergastrinemia and hyperacidity are found. Colonization of the antral mucus layer with H. pylori is associated with structural alterations of the gastric mucus and antral gastritis. H. pylori gastritis is believed to be caused by:

The urease that is produced by H. pylori, which leads to the formation of ammonia that directly stimulates the G- cells
The formation of pro-inflammatory cytokines such as IL-2, IL-8 and TNF-α, which are stimulatory to the G-cells.

The diagnosis of H. pylori infection is made /1/:

Invasively, by the detection of H. pylori or by determination of the urease activity in the antral gastric mucosal biopsy
Non-invasively, by the measurement of serum antibodies directed against H. pylori and antigen determination in stool.

The invasive procedures imply endoscopy with specimen collection. Whenever endoscopy is performed, the combination of a urease rapid test and two biopsies for histology is advantageous. The culture of H. pylori is complex and the biopsies must be dispatched in a special culture medium. Cultures are, therefore, usually employed for resistance testing. The reliability of the detection procedures is shown in Tab. 14.1-1 – Accuracy of procedures for the detection of H. pylori.

14.1.1.3 Drug-related damage of the gastrointestinal tract

Adverse drug events are a frequent cause of upper gastrointestinal tract bleeding, particularly in elderly individuals. If the drug history is ambiguous, the drugs must be determined in serum or urine. The most widely prescribed medications related to adverse events are non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin. The relative risk of upper gastrointestinal tract bleeding due to NSAIDs is shown in Tab. 14.1-2 – NSAIDs as triggers of gastrointestinal bleeding. In elderly individuals, the odds ratios for bleeding caused by aspirin at daily doses of 75 mg, 150 mg and 300 mg are, respectively, 2.3, 3.2 and 3.9.

The odds ratios for ticlopidine and clopidogrel are in the range of those for 100 mg of aspirin, namely, 2.7 /2/.

14.1.1.4 Reflux esophagitis

Gastroesophageal reflux disease (GERD) is a chronic disorder that is caused by abnormal reflux of acid, pepsin, bile and pancreatic enzymes. GERD is a result of an imbalance between the aggressive forces within the refluxate and defensive mechanism of the esophagus. About 65–70% of patients with GERD have normal endoscopy, and these patients are considered to have Non-erosive reflux disease (NERD) /3/. The presence of typical symptoms (heartburn and regurgitation) and a good response to proton pump inhibitors (PPIs) to typical symptoms, which is called the PPI-test is a sensitive test in diagnosing gastro-oesophageal erosive reflux. The cumulative sensitivity of the PPI test was 82.3% with a specificity of 51.5%, the positive predictive value was 79% and the negative predictive value was 56.9% /3/.

References

1. Fischbach W, Malfertheiner P, Hoffmann JC, Bolten W, Kist W, Koletzko S. Helicobacter pylori und gastroduodenale Ulkuskrankheit. Dtsch Ärztebl 2009; 106: 801–8.

2. Sostres C, Gargallo C, Lanas A. Drug-related damage of the ageing gastrointestinal tract. Best Practice & Research in Clinical Gastroenterology 2009; 23: 849–60.

3. Fass R, Zerbib F, Gyawali CP. Aga clinical practice update of functional heartburn: expert review. Gastroenterology 2020; 158 (8): 2286–93.

14.1.2 Helicobacter pylori infection

Helicobacter pylori (H. pylori) is a gram-negative bacterium involved in the pathogenesis of peptic ulcer disease, gastric carcinoma, and lymphoma. The ecological niche of H. pylori is the human stomach, where the organism establishes long-term colonization of the gastric mucosa.

14.1.2.1 Indication

Diagnosis of H. pylori infection and evidence for eradication.

14.1.2.2 Method of determination

A distinction is made as follows:

When patients are examined by upper endoscopy, gastric biopsies can be obtained and used for diagnosis of H. pylori
Determination of H. pylori antigen in the stool
Serological diagnosis of H. pylori infection is a major non-invasive diagnostic tool in epidemiologic studies.

The exclusive implementation of non-invasive procedures (serological H. pylori test, ¹³C urea breath test, or antigen detection in the stool) for the diagnosis of H. pylori infection does not make possible the recognition of the type of injury that is associated with the bacterium (e.g., gastritis, peptic ulcer, MALT lymphoma, carcinoma) /1/.

14.1.2.2.1 Biopsy-dependent procedures

Two biopsy specimens are taken from the antrum and two from the corpus of the stomach.

Urease test

Principle: biopsy specimens from the antrum and the corpus are incubated with urea and a pH indicator dye (e.g., phenolphthalein). H. pylori forms urease which catalyzes the transformation of urea into ammonia and bicarbonate and the pH indicator turns red within 1 to 2 hours.

NH₂-CO-NH₂ + H₂O + H⁺ → 2 NH₄⁺ + HCO₃^–

14.1.2.2.2 Histological examination of pathogens

Biopsy specimens from the antrum and the corpus are embedded in paraffin wax, and stained with hematoxylin and eosin, and with cresylviolet. Gastritis is scored on the basis of depth of inflammation as superficial and pan mucosal. In H. pylori positive patients, the intragastric bacterial load is scored.

14.1.2.2.3 Culture

An antral biopsy is inoculated in a transport medium and incubated in a culture medium under microaerophilic conditions at 37 °C for 5 days. The identification of H. pylori is based upon the biochemical demonstration of urease, oxidase, catalase and gamma-glutamyl transferase, as well as morphological characteristics.

14.1.2.2.4 Serologic tests

All patients colonized with H. pylori elicit a local antibody response against antigens covering the surface and flagella of the bacterium. In the majority of cases the antibody response is also detectable in serum. Currently, the majority of routine serodiagnostic tests for H. pylori measure IgG antibodies. Locally the antibodies are mostly of the IgA class, but the circulating antibodies are primarily of the IgG class, usually the IgG₁, IgG₂, and IgG₄ subclasses. The ELISA is the method of choice /2/.

14.1.2.2.5 ¹³C urea breath test

Principle: the non-radioactive isotope ¹³C is a natural constituent of the organism, making up approximately 1% of the total carbon content. Orally ingested ¹³C-labeled urea is metabolized by H. pylori urease in the stomach to ¹³CO₂, resulting in an increase of the ¹³CO₂/¹²CO₂ ratio in the exhaled air, which is quantifiable with isotope-ratio mass spectrometry (IRMS) or with non-dispersive isotope selective infrared spectroscopy (NDIRS) /2/.

Procedure /3/: after an overnight fast, each individual receives 200 mL of full-cream cows milk to delay gastric emptying. After 5 min., individuals drink a 50 mL aqueous solution containing 75 mg of ¹³C urea. Breath samples are taken before the meal and every 15 min. for 1 h after ingestion of the urea solution. At each sampling time, patients blew directly into 10 mL tubes by means of a straw. The analytical data are expressed as percentages of ¹³CO₂ recovery per hour.

14.1.2.2.6 Antigen determination in the stool

Principle: demonstration of H. pylori antigens using enzyme immunoassay.

Procedure: the micro titer plate of the test kit is coated with mono- or polyclonal antibodies against H. pylori antigens. The supernatant of a stool suspension is incubated in the wells of the micro titer plate. H. pylori antigens in the stool suspension bind to antibodies of the wells. In a second step peroxidase-linked antibodies against the bound antigens are added, forming a sandwich complex. Following washing steps for the removal of unbound antibodies the peroxidase mediated enzyme reaction is measured spectrophotometrically.

14.1.2.3 Specimen

Biopsy-dependent procedures: antral and corpus biopsy specimens
H. pylori serology: serum 1 mL
¹³C urea breath test: end-expiratory breath
Antigen determination in the stool: approximately 0.1 g or 100 μL of a random stool sample.

14.1.2.4 Reference interval

Biopsy-dependent procedures: negative urease test or absence of H. pylori
H. pylori serology: H. pylori-negative individuals have IgG antibody concentrations ≤ 10 U/mL
¹³C urea breath test: a rise in the difference of the ¹³CO₂/¹²CO₂ ratio of ≤ 0.5% in comparison with the basal value rules out an H. pylori infection
H. pylori antigen determination in the stool: in the enzyme immunoassay, an optical density of up to 0.15 at 450 nm rules out an H. pylori infection.

14.1.2.5 Clinical significance

H. pylori is considered to be the causal factor in chronic type B-gastritis and non-drug dependent peptic duodenal ulcer and gastric ulcer, an etiological stimulus of gastric MALT lymphoma, and a pathogen involved in the development of stomach cancer. The prevalence of H. pylori infection in the industrialized nations of the Western countries is characterized by a linear increase with age, while in developing countries the spread of the infection is already very high in children and adolescents /4/.

Patients with H. pylori infection suffer from dyspepsia.The term dyspepsia is used for a spectrum of symptoms localized by the patient to the epigastric region (between the navel and the xiphoid process) and the flanks. The clarification of dyspeptic complaints is based upon the endoscopic examination, which includes the bioptic evidence of H. pylori infection /5/.

Asymptomatic individuals should undergo testing only in the case of a clear family history of gastric cancer or a request on the part of the individual provided that the intention is to subsequently treat the infection.

14.1.2.5.1 Gastric cancer

Gastric cancer is the fifth most common malignancy and the forth cancer related death worldwide. Geographic and temporal variation in genotypes of H. pylori in relation to gastric patients are observed. In African population with gastric adenocarcinoma the H. pylori VacA s1m1 genotype was associated with adenocarcinoma (Odds ratio = 2.68) whilst VacAs2m2 was associated with a reduced probability of gastric adenocarcinoma (Odds ratio = 0.23). No association between cytotoxin associated gene A (CgA) and gastric adenocarcinoma was observed /9/.

Germline pathogenic variants in nine genes (APC, ATM, BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, and PALB2) were associated with the risk of gastric cancer. H. pylori infection modified the risk of gastric cancer associated with germline pathogenic variants in homologous-recombination genes /10/. The authors suggest that in persons known to carry a pathogenic variant in a homologous-recombination gene, evaluation and eradication of H. pylori infection may be particularly important.

14.1.2.5.2 Biopsy-dependent procedures

The histological examination of biopsy material from the gastric mucosa provides evidence of H. pylori infection or monitors the success of antibiotic treatment. It is recommended that following eradication therapy, diagnostic confirmation of success should be made at the earliest 4 weeks after completion of treatment. If a gastroduodenal ulcer was present but has not healed, a further biopsy of the remaining ulcer (from the margin and the base of the ulcer) or the ulcer scar must be taken. A follow-up endoscopy is not necessary if an duodenal ulcer was present /3/.

In suspicion of antibiotic resistance, the biopsy enables culture systems for the determination of resistance.

14.1.2.5.3 H. pylori serology

H. pylori serology is suitable for the screening of asymptomatic individuals and possibly as a contact investigation in the families of individuals with the infection. The examination only indicates that a confrontation with the pathogen has taken place; it does not, however, permit a statement to be made as to whether or not an active H. pylori infection is still present. Rapid tests based upon simplified latex agglutination or solid phase ELISA tests demonstrate the presence of H. pylori antibodies qualitatively, from a drop of whole blood (lancet puncture) and within a few minutes, by means of a simple color reaction. The extent of H. pylori colonization of the gastric mucosa does not influence the circulating antibody titers in a significant manner. The IgG and IgA antibody determination is useful for confirming eradication of H. pylory infection. A decline of IgG antibodies ≥ 25% after 6 months indicates eradication. IgA antibodies may drop sooner in response to treatment.

14.1.2.5.4 ¹³C urea breath test

The quantitative result of the ¹³C urea breath test correlates with the urease activity in the stomach or with the H. pylori density and thereby, indirectly, with the extent of the H. pylori-induced gastritis as well.

Following antibiotic therapy or the ingestion of a proton pump inhibitor, the test can provide a false negative result, in spite of the fact that the H. pylori infection is still present. The reason is a temporary suppression of the pathogen. By definition the pathogen has only been eradicated if, following 4 weeks of therapy, no positive evidence of H. pylori can be provided. Accordingly, the ¹³C urea breath test for the monitoring of the therapeutic success is performed 4 weeks following the end of treatment.

14.1.2.5.5 H. pylori antigen determination in the stool

The stool antigen test does not allow a quantitative statement to be made regarding the assessment of the colonization density of H. pylori infection in the stomach. However, in contrast to serological antibody detection, the stool test, like the ¹³C urea breath test, provides an indication of the current H. pylori infection /6/.

14.1.2.5.6 Comparison of tests

Diagnostic sensitivity, specificity and accuracy of the tests for the diagnosis of H. pylori infection are shown in Tab. 14.1-3 – Sensitivity, specificity and accuracy of tests for the diagnosis of H. pylori infection.

14.1.2.6 Comments and problems

Urease test

A test which is positive only after 24 hours should not be evaluated.

Culture

Storage and transport may cause false negative results if special transport media are not used. The cooling of the sample during the transport increases the survival of H. pylori.

H. pylori serology

ELISAs using complex antigens detect about 85–95% of those patients with H. pylori infection detectable by culture and/or histology. The diagnostic specificity of these tests is ≥ 95%.

The serological tests are not recommended for use in children or adolescents, neither for diagnostic purposes nor for the monitoring of therapeutic success. The reason is that the diagnostic sensitivity and specificity of serological tests vary considerably in children /7/. A positive IgG result can still occur months or years following an infection.

¹³C urea breath test

Proton pump inhibitors have to be interrupted 4 weeks before the test, otherwise false positive results may be obtained. The Maastricht Consensus Conference proposed the non-invasive ¹³C urea breath test or the stool antigen test for the monitoring of antibiotic therapy with the goal of eradication, preferably 4 weeks following the completion of treatment /8/. Accordingly, not every eradication treatment requires a mandatory follow-up; nonetheless, in cases of a high clinical need for clarification (e.g., following ulcer bleeding, persistence of symptoms following therapy in non-ulcer dyspepsia) one should not dispense with the documentation on the antibiotic eradication.

If the test conditions are not adhered to concurrent intake of natural ¹³C enriched substrate, such as caramel products, false positive results are to be expected.

Antigen determination in the stool

The advantages of the stool test consist of the simple sample collection and the availability of the analytical methodology in every laboratory. The stool test is suitable for the monitoring of therapy as early as 2–4 weeks following the completion of the treatment. Stool tests that use monoclonal antibodies have a higher accuracy than those that employ polyclonal antibodies /7/.

Point of care tests determine H. pylori antigens within minutes, using a simplified chromatographic technique.

14.1.3 Autoimmune atrophic gastritis

Helicobacter pylori infection and autoimmune atrophic gastritis are the two major reasons in which corpus atrophic gastritis, a precancerous condition, occurs /11/.

Clinical findings: autoimmune atrophic gastritis may manifest in a clinical spectrum of diseases ranging from neurological, hematological, and gastrointestinal manifestations. Other important autoimmune disorders are e.g., thyroid disorders, vitiligo, type 1 diabetes. Autoimmune atrophic gastritis causes serious iron and cobalamin malabsorption.

Laboratory findings:

anti-parietal cell antibodies (anti-PCA). The main antigen in autoimmune atrophic gastritis is the gastric proton pump H⁺-K⁺ ATPase localized on parietal cells. An anti-PCA-ELISA has 81% sensitivity and 90% specificity /12/.
Anti-intrinsic factor antibodies (IFA). IFA are considered to have a low sensitivity but high specificity.
Serum gastrin concentrations are elevated. The principle role of gastrin is to control parietal cells action in gastric acid secretion. However, gastrin release is stimulated also by alcohol, hypoglycemia, caffeine, increased calcium concentration, and secretory drugs such as proton pump inhibitors.
Pepsinogens (PGs) are proenzymes for pepsin and are classified in PGI and PGII. PGI is produced by the chief cells and neck cells of the gastric corpus. PGII is produced by the chief cells and neck cells of the gastric corpus, in the pyloric glands of the antrum, and in the Brunner’s glands of the proximal duodenum. The ratio between PGI/PGII is helpful to distinguish corpus involvement from antral H. pylori gastritis and to overcome false interpretations of the PGI result as the sole marker /11/. The PGI concentration in serum is correlated to the corpus chief cell mass.

References

1. Caspary WF, Rösch W. Diagnostik und Therapie der Helicobacter pylori-Infektion. Dt. Ärztebl 1996; 93: B 2094–7.

2. Chen TS, Chang FY, Lee SD. Serodiagnosis of Helicobacter pylori infection: comparison and correlation between enzyme-linked immunosorbent assay and rapid serological test results. J Clin Microbiol 1997; 35: 184–6.

3. Perri F, Clemente R, Pastore M, Quitadamo M, Festa V, Bisceglia M, et al. The ¹³C-urea breath test as a predictor of intragastric bacterial load and severity of Helicobacter pylori gastritis. Scand J Clin Lab Invest 1998; 58: 19–28.

4. DGVS-Leitlinien. I. Diagnostik und Therapie der Helicobacter pylori-Infektion. Z Gastroenterol 1996; 34: 302–401.

5. Madisch A, Andresen V, Enck P, Labenz J, Frieling T, Schemann M. The diagnosis and treatment of functional dyspepsia. Dtsch Arztebl Int 2018; 115: 222–32.

6. Braden B, Teuber G, Dietrich CF, Lembcke B, Caspary WF. Comparison of a new faecal antigen test with ¹³C-urea breath test for detecting Helicobacter pylori infection and monitoring eradication treatment: prospective clinical evaluation. Br Med J, 2000; 320: 148–149.

7. Guarner J, Kalach N, Elitsur Y, Koletzko S. Helicobacter pylori diagnostic tests in children: review of the literature from 1999 to 2009. Eur J Pediatr 2010; 169: 15–25.

8. Malfertheiner P, Megraud F, O’Morain C, Hungin AP, Jones R, Axon A, Graham DY, Tytgat G; European Helicobacter Pylori Study Group (EHPSG). Current concepts in the management of Helicobacter pylori infection – the Maastricht 2-2000 Consensus Report. Aliment Pharmacol Ther 2002; 16: 167–180.

9. Njenga P, Njau A, Moloo Z, Revathi G, Yamaoka Y. Pattern and trends of Helicobacter genotypes in gastric cancer: A Kenyan 8-year study. Front Med 2023; 17 February 2023.

10. Usui Y, Taniyama Y, Endo M, Koyanaki Y, Kasugai Y, Oze I, et al. Helicobacter pylori, homologous-recombination genes, and gastric cancer. N Engl J Med 2023; 388 (13): 1181–90.

11. Dottori L, Pivetta G, Annibale B,Lahner E. Update on serum biomarkers in autoimmune atrophic gastritis. Clin Chem 2023; 69 (10): 1114–23.

12. Strickland RG, Mackay IR. A reappraisal of the nature and significance of chronic atrophic gastritis. Am J Dig Dis 1973; 18: 426–40.

14.2 Diseases of the pancreas

Paul Lankisch, Bernhard Lembcke, Lothar Thomas

14.2.1 Acute pancreatitis

Acute pancreatitis, as defined by the Marseilles-Rome classification, is an inflammatory disorder of the pancreas in which normal pancreas function will be restored once the primary cause of the acute event is relived /1/. Most cases of acute pancreatitis have a favorable outcome, but 20–25% of affected patients have a severe form of progression which is associated with the development of the systemic inflammatory response syndrome (SIRS), multiple organ failure and death.

14.2.1.1 Common causes of acute pancreatitis

Common causes are gallstones (50–60%) and alcohol abuse (30–40%). Other causes include medications, types I, IV and V hyperlipidemia, viral infection (mumps), post-ERCP, post operative, post traumatic, and hereditary factors /2/.

In acute pancreatitis without identifiable disease causes, hereditary pancreatitis should be considered /3/. This is frequently based upon a mutation of the cationic trypsinogen gene (PRSS1), which is located on the long arm of chromosome 7. The inheritance is autosomal dominant, with a penetration for the phenotype of up to 80%.

14.2.1.2 Mechanism of cellular injury

Pancreatic duct obstruction , irrespective of the mechanism, leads to upstream blockage of pancreatic secretion, which in turn impedes exocytosis of zymogen granules (containing digestive enzymes) from acinar cells. Consequently, the zymogen granules coalescence with intracellular lysosomes to form condensing or autophagic vacuoles containing an admixture of digestive and lysosomal enzymes. The lysosomal enzyme cathepsin B can activate the conversion of trypsinogen to trypsin. The resulting accumulation of active trypsin within the vacuoles can activate a cascade of digestive enzymes leading to auto digestive injury /1/.

A block in the healthy apical exocytosis of zymogen granules can cause basolateral exocytosis in the acinar cell, releasing active zymogens into the interstitial space (rather than the acinar lumen), with subsequent protease-induced injury to the cell membrane /1/.

14.2.1.3 Definition of severity in acute pancreatitis

The Atlanta classification is the standard classification of the severity of acute pancreatitis /2/. Clinical severity of acute pancreatitis is stratified into three categories /1, 2/: mild, moderately severe, and severe.

Mild acute pancreatitis. No organ failure or systemic or local complications. Patients do not need pancreatic imaging and are frequently discharged within 3–7 days of onset of illness.
Moderately severe acute pancreatitis. The pancreatitis is characterized by one or more transient organ failure (defined as organ failure lasting < 48 h), systemic complications, or local complications. Organ failure includes respiratory, cardiovascular and renal failure. Systemic complications are defined as exacerbations of pre-existing co morbidities, including congestive heart failure, chronic liver disease, and chronic lung disease. Local complications include interstitial pancreatitis (peripancreatic fluid collections, pancreatic pseudo cysts), and necrotizing pancreatitis.
Severe pancreatitis is characterized by the presence of persistent single-organ or multi organ failure (defined by organ failure that is present ≥ 48 h). Most patients who have persistent organ failure have pancreatic necrosis and mortality of at least 30%. Radiological severity of acute pancreatitis includes acute peripancreatic fluid collections within the first several days of interstitial pancreatitis. An acute peripancreatic fluid collection that does not resolve can develop into a pseudocyst, which contains a well defined inflammatory wall. There is very little, if any, solid material within the fluid of a pseudocyst

14.2.1.4 Clinical significance

The cardinal symptom of acute pancreatitis is mid gastric pain, which within minutes or hours usually radiates directly through the back. About 80% of patients experience nausea and vomiting when the pain is at its maximum /3/. Prognostically meaningful is information on previous episodes of acute pancreatitis, since recurrent episodes are rarely severe or necrotizing in comparison with the initial episode /3/. In the severe necrotizing pancreatitis an early episode (first 2 weeks), associated with mortality, and a late episode, are differentiated. The mortality in patients with acute pancreatitis is about 1%. However, with the development of severe systemic complications (SIRS) or local complications (infection of pancreatic necrosis) the mortality rises steeply. Complicating sepsis usually leads to multiple organ failure and death in the second episode. Factors that determine the course of acute pancreatitis are age, obesity and a history of chronic alcoholism.

14.2.1.5 Biomarkers in acute pancreatitis

Laboratory tests are important for the diagnosis of acute pancreatitis, for the evaluation of the cause, and for the assessment of the prognosis (Tab. 14.2-1 – Laboratory tests for the diagnosis and monitoring of acute pancreatitis) /4/.

14.2.1.5.1 Laboratory diagnosis of acute pancreatitis

The hallmark of acute pancreatitis is an increased serum lipase level or an increased serum and urine α-amylase level. With most procedures, a lipase level of ≥ 3 fold than the upper reference interval value confirms the diagnosis with a diagnostic sensitivity and specificity of over 90% (see also Tab. 1.12-1 – Lipase reference intervals). The accuracy of the pancreas-specific amylase is comparable.

14.2.1.5.2 Evaluation of the cause of acute pancreatitis

Biliary etiology

The early diagnosis of a biliary etiology is important. For this purpose the cholestasis markers bilirubin, GGT and ALP in relation to ALT are determined. ALT values ≥ 3-fold the upper reference interval value and an even more marked increase in ALP and GGT are indicative of biliary etiology, with a positive predictive value of some 95% Refer to:

Drugs

Drugs for which a definite association with acute pancreatitis has been reported /1/:

acetaminophen, asparaginase, azathioprine, bortezomib, capecitabine, carbamazepine, cimetidine, cisplatin, cytarabine, didanosine, enalapril, erythromycin, estrogens, furosemide, hydrochlorothiazide, interferon alpha, itraconazole, lamivudine, mercaptopurine, mesalazine, olsalazine, methyldopa, metronidazole, octreotide, olanzapine, opiates, oxyphenbutazone, pentamidine, pentavalent antimony compounds, penformin, simvastatin, steroids, sulfasalazine, co-trimoxazole

Hypertriglyceridemia

The hazard ratio for acute pancreatitis associated with severe hypertriglyceridemia (≥ 500 mg/dL) is 3.20 (1.95–5.16). The risk of incident acute pancreatitis increases by 4% by every 100 mg/dL increase in triglyceride concentration (after adjustment for covariates and removal of patients hospitalized for gall stones, chronic pancreatitis, alcohol related morbitidies, renal failure and other biliary diseases) /22/.

14.2.1.5.3 Prognosis of acute pancreatitis

Important is the early differentiation of the presence of mild edematous pancreatitis or a severe necrotic form. There exists no single marker that reliably answers this question /5/. Of the serum tests, the best clues are provided by CRP and IL-6. Pancreas-specific scores are usually utilized for prognostic assessment.

C-reactive protein (CRP)

CRP reaches its peak value 48–72 hours following the onset of the acute symptoms; these are higher in necrotizing pancreatitis than in the edematous form. Thus, patients whose degree of severity was classified based upon scores had the CRP values that are listed in Tab. 14.2-2 – Severity of acute pancreatitis and CRP values. According to a consensus /6/, a CRP serum level of above 150 mg/L within the first 48 hours following the occurrence of clinical symptoms indicates, with a diagnostic sensitivity and specificity of over 80% and an accuracy of 86%, necrotizing acute pancreatitis.

Interleukin-6 (IL-6)

Already upon admission to the clinic, the necrotizing form of acute pancreatitis manifests substantially higher values than the edematous form. IL-6 is thereby helpful early on in the stratification of the patients. Peak values are recorded on the third day following the occurrence of symptoms. The diagnostic sensitivity is 69–100% with a specificity of 70–86%. Thus, the IL-6 values in one study /7/ were 91 ± 71 ng/L within the first 72 hours in mild pancreatitis, and 146 ± 53 ng/L in severe disease. Patients who developed organ failure had concentrations of 162 ± 53 ng/L; by comparison, the values in patients without organ failure were 88 ± 66 ng/L.

Lymphopenia

In severe pancreatitis, lymphopenia develops in addition to SIRS and sepsis.

Importance of scores

For early prediction of risk with regard to the degree of severity of acute pancreatitis, risk scores such as the Ranson criteria, the Apache II scoring system and the Imrie-Glasgow score are used. The Ranson criteria are listed in Tab. 14.2-3 – Ranson criteria for the assessment of severity of acute pancreatitis. This score is calculated from parameters obtained at admission and 48 hours later. Each criterion receives a point. A score of 3 points indicates the presence of severe pancreatitis /8/.

14.2.2 Chronic pancreatitis

Chronic pancreatitis is defined as a chronic inflammatory state that results in irreversible damage to pancreatic structure and function /9, 10, 11/. Due to recurrent inflammatory episodes, the pancreatic parenchyma is replaced by fibrotic tissue. The result of the connective tissue remodeling is an increasing loss of exocrine and endocrine pancreatic function. The major causes of chronic pancreatitis are smoking, alcohol ingestion, malnutrition, and certain gene disorders (Tab. 14.2-4 – Causes of chronic pancreatitis). Complications of the exocrine insufficiency, which develops in 30–60% of patients, are maldigestion, formation of pancreatic duct stenosis, pseudo cysts, duodenal stenosis, compression of the biliary tract, vascular complications, and the development of pancreatic carcinoma. The typical clinical symptoms of maldigestion (disturbance of food digestion) are abdominal complaints, steatorrhea, and signs of malnourishment. Steatorrhea only occurs with lipase secretion of ≤ 5–10% of normal.

When the diagnosis of chronic pancreatitis is made, pancreatic insufficiency should, in principle, be expected generally, however, only some 10 years after the onset of the clinical symptoms. The risk of vitamin D deficiency, along with the risk of osteoporosis and fractures as well as of vitamin E deficiency, exists even in mild or subclinical exocrine insufficiency.

In Western Europe, the incidence is approximately 8 per 100,000 people per year; in some regions it is as high as 20. The disease can occur at any age; the frequency peaks during the decade 5–6 of life. Approximately 70–75% of the cases are associated with alcohol, while in 20–25% the causes are recurrent cholelithiasis, metabolic endocrine diseases, hemochromatosis, and the like. The classification of the degree of severity of pancreatic insufficiency is made according to the criteria shown in Tab. 14.2-5 – Classification of the severity of chronic pancreatitis.

For the individual patient with chronic pancreatitis, the course of progression and the pain is not predictable. Nonetheless, according to the burnout theory, the pain should diminish following an average duration of 10 years after recognition of the insufficiency.

An important pathophysiological mechanism of chronic pancreatitis is believed to be based upon the pancreatic stellate cells (PSCs). They are localized in the periacinar space and, morphologically, are comparable with the Kupffer stellate cells of the liver. The PSCs are activated in chronic pancreatitis, and they are transformed to myofibroblast-like cells. The conversion is triggered by alcohol and inflammatory cytokines. The continual activation of the PSCs leads to degradation of the parenchymal cells and to fibrosis of the pancreas /10/.

Transition of acute pancreatitis to chronic pancreatitis develops in alcoholics. In a German study /12/ over a period of almost 8 years, only alcoholics developed chronic pancreatitis, independently of both severity of first acute pancreatitis and dis continuation of alcohol and nicotine. The cumulative risk of the development of chronic pancreatitis was 13% within 10 years and 16% within 20 years The risk of chronic pancreatitis in those who survived the second episode of acute pancreatitis was 38% within 2 years. Laboratory investigations for impairment of pancreas function and monitoring of chronic pancreatitis are shown in (Tab. 14.2-11 – Laboratory testing and monitoring of chronic pancreatitis).

14.2.2.1 Pancreatic function tests

In the symptom-free interval of chronic pancreatitis α-amylase, and lipase indicate no increase in activity, whereas pancreatic function tests show pathologic results when chronic pancreatitis is established.

Pancreas function can be evaluated in different way

By measuring pancreatic enzyme secretions after stimulation by hormones or a test meal
By measuring metabolites of ingested substances, which provides indirect estimate of pancreatic enzyme secretion
By measuring pancreatic enzymes in serum or stool.

The tests are categorized into:

Invasive tests which require placement of duodenal tubes to aspirate pancreatic secretion after the pancreas has been stimulated by secretin cholecystokinin (secretin-pancreozymin test) or a test meal (Lundh test)
Noninvasive direct tests which measure enzyme activity in stool (elastase-1, chymotrypsin activity, stool fat).
Noninvasive indirect tests which measure serum or urine levels of metabolites of ingested substances (¹³C breath test, pancreolauryl test, NBT-PABA).

The testing for endocrine pancreatic insufficiency is performed with the determination of HbA_1c or the oral glucose tolerance test /7/.

Because chronic pancreatitis increases the mortality by 38.4% after 20 years, annual noninvasive tests, and the determination of CRP, ALP and HbA_1c are recommended for monitoring the course of the disease. Tumor markers such as CA 19-9 or CEA are not recommended.

14.2.2.1.1 Indication

Suspicion of chronic pancreatitis and confirmation of its course
Following acute pancreatitis, to clarify if the acute inflammation has healed without sequelae, if damage persists post-healing, or if there has been a transition to chronic pancreatitis.

14.2.2.1.2 Clinical significance

The sensitivity of non-invasive pancreatic function tests is not sufficient for the diagnosis of mild to moderate exocrine pancreatic insufficiency /13/. The causes of chronic pancreatitis are shown in Tab. 14.2-4 – Causes of chronic pancreatitis.

Direct invasive tests

With the use of these tests, the pancreatic secretions (volume, bicarbonate, enzymes) are recorded quantitatively following the placement of an duodenal tube and stimulation of pancreatic secretions /14/. The secretin-pancreozymin test has a diagnostic sensitivity of 92% with a specificity of 94% /15/. The Lundh test is comparable. The direct invasive tests are considered to be the gold standards; they are the best procedures for the demonstration or the exclusion of exocrine pancreatic insufficiency. These tests also reliably capture mild to moderate forms of chronic pancreatitis, but they are expensive and time-consuming, and they represent a burden to the patient.

Non-invasive direct tests

The diagnostic sensitivity and specificity of these tests is shown in Tab. 14.2-6 – Sensitivity and specificity of noninvasive tests in comparison with the secretin-caerulein test in chronic pancreatitis.

¹³C breath test

Patients are administered ¹³C-labeled, non-absorbable substrates (triglycerides), which are cleaved by pancreatic enzymes in the small intestine. Conclusions can be drawn about the secreted enzyme activity following reabsorption and metabolism of the cleavage products based upon the measurement of the ¹³C/¹²C ratio in the exhaled air. Triglycerides (hydrolysis with lipase), cholesterol esters (cholinesterase) and starches (hydrolysis with α-amylase) are administered with a stimulating test meal.

14.2.3 Pancreatic cancer

Exokrine pancreatic cancer is an extraordinarily deadly disease and is diagnosed in more than 60,000 people in the United states annually and represents the fourth leading cause of cancer death. The majority of cases are classified as ductal adenocarcinoma. The disease most likely in adults who are 60 to 70 years and is rare in adults below 40 years /23/. In chronic pancreatitis, the relative risk of pancreatic carcinoma is increased 16-fold to 13.1% (in smokers 25-fold). The majority of patients live for a maximum of 1 year following diagnosis and the 5-year survival rate is less than 5%. The poor prognosis is due to the late detection of the carcinoma; most patients are only diagnosed when curative surgery is no longer possible. The symptoms of pancreas carcinoma are uncharacteristic and include weight loss, fatigue, abdominal pain, newly diagnosed diabetes mellitus, nausea and jaundice.

Hereditary pancreatic cancer

The relative risk of pancreatic carcinoma in hereditary chronic pancreatitis is 69%. Pancreatic carcinoma occurs with increased frequency in families with a history of adenocarcinoma. Familial predispositions are observed in 5–10% of the cases of pancreatic carcinoma and a hereditary component is believed to play a role in 10–20% of these carcinomas /16/. Diseases that are associated with an elevated risk of pancreas carcinoma are shown in Tab. 14.2-7 – Cancer syndromes associated with increased pancreatic cancer risk.

14.2.3.1 Laboratory diagnosis of pancreatic cancer

In comparison with imaging procedures with high diagnostic accuracy /17/, the determination of the tumor markers CA19-9 and CEA is only meaningful (see Section 28 – Tumor markers):

For estimating the prognosis
For the assessment of the therapeutic efficiency
In the postoperative disease course assessment, for the detection of a relapse.

CA 19-9

In the diagnostic investigation of pancreatic cancer, the diagnostic sensitivity of CA19-9 is 67–92%, with a specificity of 68–92%. Only 50% of the carcinomas with a diameter of less than 2 cm have elevated CA19-9, and Lewis a negative patients (4–15%) do not generate CA19-9. The tumor marker is also elevated in non-tumor associated diseases like cholestasis or chronic pancreatitis.

CEA

The diagnostic sensitivity for pancreatic cancer is 48–55%, with a specificity of 87–90%. This tumor marker is, however, also over expressed in colon carcinoma and other gastrointestinal tumors, in inflammatory diseases of the intestine, and in smokers.

Genetic testing

Investigation for germline mutation is recommended in patients with pancreatic cancer. The percentage of patients with exocrine pancreatic cancer is 4–20%. Mutations in BRCA1 and BRCA2 are most common /18/.

References

1. Lankisch PG, Apte M, Banks PA. Acute pancreatitis. Lancet 2015; 386: 85–96.

2. Banks PA, Bollen TL, Dervenis C, et al., and the Acute Pancreatitis Working Group. Classification of acute pancreatitis – 2012: revision of the Atlanta classification and definition by international consensus. Gut 2013; 62: 102–12.

3. Huber W, Schmid RM. Akute Pankreatitis: Evidenzbasierte Diagnostik und Therapie. Dtsch Ärztebl 2007; 104: B1615–B1623.

4. Harper SJF, Cheslyn-Curtis S. Acute pancreatitis. Ann Clin Biochem 2011; 48: 23–37.

5. Rau B, Schilling MK, Berger HG. Laboratory markers of severe acute pancreatitis. Dig Dis 2004; 22: 247–57.

6. Dambrauskas Z, Gulbinas A, Pundzius J, Baraukas G. Value of the different prognostic systems and biological markers for predicting severity and progression of acute pancreatitis. Scand J Gastroenterol 2010; 45: 959–70.

7. Sathyanarayan G, Garg PK, Prasad H, Tandon RK. Elevated level of interleukin-6 predicts organ failure and severe disease in patients with acute pancreatitis. J Gastroenterol Hepatol 2007; 22: 550–4.

8. Ranson JH, Rifkind KM, Roses DF, Fink SD, Eng K, Spencer FC. Prognostic signs and the role of operative management in acute pancreatitis. Surg Gynecol Obstet 1974; 139: 69–81.

9. Ansari D, Andersson E, Andersson B, Andersson R. Chronic pancreatitis: potential future interventions. Scand J Gastroenterol 2010; 45: 1022–8.

10. Andersson R, Tingstedt B, Xia J. Pathogenesis of chronic pancreatitis: a comprehensive update and a look into future. Scand J Gastroenterol 2009; 44: 661–3.

11. Vege SS, Chari ST. Chronic pancreatitis. N Engl J Med 2022; 386 (9): 869–78..

12. Lankisch PG, Breuer N, Bruns A, Weber-Dany B, Löwenfels AB, Maisonneuve P. Natural history of acute pancreatitis: a long term population based study. Am J Gastroenterol 2009; 104: 2797–805.

13. Sigmund E, Löhr JM, Schuff-Werner P. Die diagnostische Validität nichtinvasiver Pankreasfunktionstests – Eine Metaanalyse. Z Gastroenterol 2004; 42: 1117–28.

14. Lankisch PG, Schmidt I. Exocrine pancreatic function tests, 1999. Is the best we have good enough? Scand J Gastroenterol 1999; 34: 945–7.

15. Otte M. Pankreasfunktionsdiagnostik. Internist 1979; 20: 331–40.

16. Greer JB, Lynch HT, Brand ER. Hereditary pancreatic cancer: a clinical perspective. Best Practice and Research Clinical Gastroenterology 2009; 23: 159–70.

17. Michl P, Pauls S, Gress TM. Evidence-based diagnosis and staging of pancreatic cancer. Best Practice and Research Clinical Gastroenterology 2006; 20: 227–51.

18. Simon B, Hlouschek V, Domschke W, Lerch MM. Hereditäre Pankreatitis. Internist Prax 2003; 43: 499–508.

19. Finkelberg DL, Sahani D, Deshpande V, Brugge WR. Autoimmune pancreatitis. N Engl J Med 2006; 355: 2670–6.

20. Ghazale A, Chari ST, Smyrk TC, Levy MJ, Topazian MD, et al. Value of IgG₄ in the diagnosis auf autoimmune pancreatitis and in distinguishing it from pancreas cancer. Am J Gastroenterol 2007, 102: 1646–53.

21. Detlefsen S, Drews AM. Autoimmune pancreatitis. Scand J Gastroenterol 2009; 44: 1391–1407.

22. Murphy MJ, Sheng X, MacDonald TM, Wei L. Hypertriglyceridemia and acute pancreatitis. JAMA intern Med 2013; 173: 162–4.

23. DuBois JS, Kambadakone A, Wo JY, Zhang L. Case19-2022: A 29-year-old woman with jaundice and chronic diarrhea. N Engl J Med 2022; 386 (25): 2413–23.

14.2.4 Fecal elastase-1

Human pancreatic elastase-1 (EC 3.4.21.36) belongs to the family of serine proteases together with digestive enzymes such as chymotrypsin and trypsin, as well as some proteases of the cascade of blood coagulation and the complement system. These proteases share more than 40% of homology for primary and tertiary structure. Elastase is a carboxy endopeptidase, which catalyzes the hydrolysis of elastin but not of collagen and keratin. Elastase-1 is synthesized by the acinar cells of the exocrine pancreas, has a molecular weight of 26 kDa and is composed of 240 amino acids. The enzyme concentration in pancreatic juice is 170–360 mg/L /1/.

Elastase-1 is released with other digestive enzymes from the acinar cells and shows a linear correlation to lipase, amylase and trypsin; furthermore, duodenal elastase secretion correlates linearly with fecal elastase-1 concentrations. Unlike other pancreatic enzymes, elastase-1 is not significantly degraded during intestinal transit where it is mainly bound to bile salts. In contrast to chymotrypsin, the fecal content which is only some 0.5% of the pancreatic-duodenal juice, the fecal concentration of elastase-1 is 5–6-fold increased. The elastase-1 concentration in feces reflects the secretory capacity of the pancreas. The secretion of elastase-1 is reduced in exocrine pancreatic insufficiency, resulting in decreased concentrations of the enzyme in the feces /2/.

Refer to Tab. 28.2-3 – Oncogenes, oncogene products and tumor suppressor genes in cancer patients.

14.2.4.1 Indication

Suspected maldigestion in pancreatic insufficiency.

14.2.4.2 Method of determination

Enzyme immunoassay (EIA) using monoclonal or polyclonal antibodies against human pancreatic elastase-1 /3/. For the EIA determination, the stool samples are processed as follows: 100 mg of stool are homogenized with 10 mL of extraction buffer (10 mg stool/mL) and subsequently diluted 1 : 500 or, if necessary, higher, and then pipetted into the micro titer plate wells.

14.2.4.3 Specimen

Stool sample: approximately 1 mL

14.2.4.4 Reference interval

175–2,500 μg elastase-1/g stool /4/

Values for adults and children older than 3 months. Values are 2.5 and 97.5 percentiles

14.2.4.5 Clinical significance

Patients with exocrine pancreatic insufficiency have diminished elastase-1 excretion. In one study /4/, with cutoff of 175 μg/g stool, the diagnostic sensitivity of elastase-1 was 93% with a diagnostic specificity of 94%. If the patients who had pathological secretin-caerulein test results were subdivided into groups with steatorrhea and without (moderate pancreatic insufficiency), the diagnostic sensitivity in the group with steatorrhea increased to 96%, while that in the group with moderate pancreatic insufficiency decreased to 88%.

Another study /5/ classified the pancreatic insufficiency as shown in Tab. 14.2-8 – Assessment of elastase-1 and provided the following diagnostic sensitivities with a cutoff below 200 μg/g stool:

In mild pancreatic insufficiency, 63%.
In severe and moderately severe pancreatic insufficiency, 100%, with a specificity of 93%.
For all patients with exocrine pancreatic insufficiency, diagnostic sensitivity of 93%.

In children with cystic fibrosis and a cutoff below 200 μg/g, the diagnostic sensitivity for the investigation of exocrine pancreatic insufficiency was 91.1%, with a specificity of 95.8% /6/. The adult reference value for elastase-1 can be applied to infants older than 2 weeks, independent of gestation age, birth weight, and the type of nutrition /7/.

Watery stools lead to low and therefore erroneously pathological results /8/.

In contrast to fecal chymotrypsin, the immunoreactive elastase-1 test is uneffected by pancreatic enzyme replacement therapy /4/.

14.2.4.6 Comments and problems

Method of determination

In contrast to the elastase-1 test with monoclonal antibodies, a commercial test that utilizes polyclonal antibodies detects not only elastase-1, and in consequence manifests lower diagnostic specificity /9/. In another study /10/, this assay permitted an interpretation that was 91% identical to that provided by the monoclonal assay; the remaining patients varied by ± 1 classification level with reference to the classification described in Tab. 14.2.8 – Assessment of elastase-1.

Stability

In stool samples at 20 °C, 3 days. At 22 °C, decrease of around 8% per week. High temperatures (56 °C) lead to inactivation within minutes.

Pancreatic enzyme preparations

Medicines such as porcine pancreatin do not interfere, since only human elastase-1 is detected. Consequently, it is possible to monitor exocrine pancreatic function without interrupting treatment.

Refer to Tab. 28.2-3 – Oncogenes, oncogene products and tumor suppressor genes in cancer patients.

References

1. Dominici R, Franzini C. Fecal elastase-1 as a test for pancreatic function: a review. Clin Chem Lab Med 2002; 40: 325–32.

2. Sziegoleit A, Krause E, Klör HU, Kanacher L, Lindner D. Elastase and chymotrypsin B in pancreatic juice and feces. Clin Biochem 1989; 22: 85–9.

3. Scheefers-Borchel U, Scheefers H, Arnold R, Fischer P, Sziegoleit A. Pancreatic elastase 1: parameter for the diagnosis of chronic and acute pancreatitis. Lab Med 1992; 16: 427–32.

4. Stein J, Jung M, Sziegoleit A, Zeuzem S, Caspary WF, Lembcke B. Immunoreactive elastase 1: clinical evaluation of a new noninvasive test of pancreatic function. Clin Chem 1996; 42: 222–6.

5. Löser C, Mölgaard A, Fölsch UR. Fecal elastase 1: a novel, highly sensitive, and specific tubeless pancreatic function test. Gut 1996; 39: 580–6.

6. Walkowiak J, Lisowska A, Przyslaski J, Grzymislawski M, Krawcynski M, Herzig KH. Faecal elastase-1 test is superior to faecal lipase test in the assessment of exocrine pancreatic function in cystic fibrosis. Acta Paediatr 2004; 93: 1042–5.

7. Nissler K, von Katte I, Huebner A, Henker J. Pancreatic elastase 1 in feces of preterm and term infants. J Pediatr Gastroenerol Nutr 2001; 33: 28–31.

8. Brydon WG, Kingstone K, Ghosh S. Limitations of faecal elastase-1 and chymotrypsin as tests of exocrine pancreatic disease in adults. Ann Clin Biochem 2004; 41: 78–81.

9. Schneider A, Funk B, Caspary W, Stein J. Monoclonal versus polyclonal ELISA for assessment of fecal elastase concentration: pitfalls of a new assay. Clin Chem 2005; 51: 1052–4.

10. Erickson JA, Aldeen WE, Grenache DG, Ashwood ER. Evaluation of fecal pancreatic elastase-1 enzyme-linked immunosorbent assay: assessment versus an established assay and implication in classifying pancreatic function. Clin Chim Acta 2008; 397: 87–91.

14.2.5 Fecal fat

Fecal fat analysis is the standard test for diagnosing and quantifying fat malabsorption in chronic pancreatitis, even though it is both insensitive and nonspecific in the diagnosis of chronic pancreatitis.

14.2.5.1 Indication

Suspicion of exogenous pancreatic insufficiency.

14.2.5.2 Method of determination

Van de Kamer method

This method quantitatively determines free fatty acids as well as fatty esters (triglycerides) /1/.

Principle: ethanolic KOH is added to a weighed amount of stool; after 20 min. heating period, including 25% HCl, free fatty acids are released. The addition of ethanol and benzine results in the separation of the fatty acids in the benzine layer. These fatty acids are subsequently titrated with NaOH and thymol blue as the indicator. The daily fecal fat content is calculated taking into account the amount of NaOH consumed as well as the daily stool amount.

It is important to use appropriate extraction reagents. Thus, polar reagents also extract water soluble short chain fatty acids that arise during the metabolism of carbohydrates. This is not the case with the use of non-polar extraction reagents. A method such as this is described in Ref. /2/.

Test performance: stool is collected in three 24-hour fractions over a period of 72 hours. During the collection period, a minimal quantity of fat of some 80 g per day should be ingested with the food /3/.

Preparations of pancreatic enzymes should be discontinued at least 72 hours prior to the collection.

Assessment criteria: the average amount of fat daily excreted with the feces.

Near infrared reflectance analysis (NIRA)

Principle: the spectrum of the light in the infrared range (700–2500 nm) that is reflected from the surface of a stool sample is characteristically determined by the composition of the sample. Important determinants of the reflection spectrum are the absorption bands due to specific functional groups such as CH, NH, OH as well as the matrix that surrounds them. The method is, however, not applicable if the water content of the stool is above 75%. A new procedure which combines the CEM SmartTrac technology (microwave drying technology) with NIRA makes possible the determination of fecal fat within minutes /3/.

Assessment criteria: fecal fat concentration. Conversion to 24-hour stool fat excretion.

14.2.5.3 Specimen

Stool collected during 72 hours in three 24-hour fractions.

14.2.5.4 Reference interval

Stool fat concentration /4, 5/	0.32–13.4 g/100 g wet weight
Stool fat excretion /1/	≤ 7.0 g/24 h

14.2.5.5 Clinical significance

The quantitative stool fat analysis is an important screening test if there is suspicion of maldigestion or malabsorption. The excretion of fecal fat is elevated in both disorders if severe digestive symptoms are present. Causes of maldigestion are exocrine pancreatic insufficiency or bile acid deficiency. Malabsorption is based upon either a reduction in the small intestinal surface area due to atrophy of the villi (e.g. in celiac disease) or a fat transport deficiency due to (e.g., intestinal lymphangiectasia). Further tests such as the D-xylose absorption test (intestinal absorption function) and stool elastase-1 determination (pancreatic enzyme digestive function) are necessary for the differentiation of malabsorption and maldigestion (Fig. 14.2-1 – Algorithm for the differentiation of malabsorption syndrome) /6/.

Steatorrhea in a normal D-xylose test is suggestive of pancreas dependent maldigestion (severe pancreatic insufficiency). In such a case the determination of elastase-1 or the secretin-caerulein test should be performed as pancreatic function tests.

In the presence of steatorrhea and a pathological D-xylose test, malabsorption due to intestine-specific affections such as extensive Crohn’s disease, celiac disease or tropical sprue must be considered. Diseases that cause elevated stool fat excretion are listed in Tab. 14.2-9 – Diseases associated with increased fecal fat excretion.

In healthy individuals, daily stool fat excretion is quite constant, independent of the quantity of dietary fat, and even with complete deprivation of dietary fat some 3 g of fat per day are found in the stools, due to intestinal bacterial decomposition and cell regeneration. Due to lipase deficiency in exocrine pancreatic insufficiency, dietary fats can be hydrolyzed only to an inadequate degree and, in consequence, they end up in the stool in large quantities. The fecal fat excretion occurs in the non-cleaved form or, as the case may be, following cleavage via bacterial lipases.

Bile acid deficiency occurs in bacterial overgrowth of the small intestine, in the afferent loop syndrome, in the blind loop syndrome, and in strictures of the small intestine. In these cases the conjugated bile acids are hydrolyzed by bacteria, especially Bacteroides species. In most cases ileum dysfunction reduces the reabsorption of bile acids in the terminal ileum. The bile acid deficiency that results in both cases leads to an insufficient emulsification of dietary fats and, in consequence, to steatorrhea.

In malabsorption, steatorrhea occurs due to impairment of the absorption capability of the small intestine. Causes are diseases of the small intestine with atrophy of the villi.

14.2.5.6 Comments and problems

Method of determination

The van de Kamer procedure detects only 60 to 70% of the total fat content of the stool. NIRA correlates well with the van de Kamer method.

Influence factors

Collection errors and watery stools cause false low concentrations of fecal fat excretion. If a minimal dietary fat content of 70 g per day is not achieved, false negative results may be obtained, even in severe pancreatic insufficiency. Inflammatory stools with mucous and blood, or special dietary conditions such as nursing of infants, lead to false negative results with both methods of determination.

References

1. Van de Kamer JH, ten Bokkel Heunik H, Weyers HA. Rapid method for the determination of fat in faeces. J Biol Chem 1949; 177: 347–55.

2. Berstad A, Erchinger F, Hjartholm AS. Fecal fat determination with a modified titration method. Scand J Gastroenterol 2010; 45: 603–7.

3. Korpi-Steiner NL, Ward JN, Kumar V, McConnell JP. Comparative analysis of fecal fat quantitation via nuclear magnetic resonance spectroscopy (¹H NMR) and gravimetry. Clin Chim Acta 2009; 400: 33–6.

4. Stein J, Purschian B, Bieniek U, Caspary WF, Lembcke B. Near infrared reflectance analysis (NIRA): a new dimension in the investigation of malabsorption syndromes. Eur J Gastroenterol Hepatol 1994; 6: 889–94.

5. Benini L, Caliari S, Guidi GC, Brentegani MT, Castellani G, Sembrenini C, et al. Near infrared spectroscopy for fecal fat measurement: comparison with conventional gravimetric and titrimetric methods. Gut 1989; 30: 1165–76.

6. Lembcke B, Grimm K, Lankisch PG. Raised fecal fat concentration does not differentiate pancreatic from other steatorrheas. Am J Gastroenterol 1987; 82: 526–31.

14.2.6 Secretin-caerulein test

14.2.6.1 Indication

Suspected exocrine pancreatic insufficiency.

14.2.6.2 Test performance

Principle: the exocrine pancreas is stimulated by the intravenous injection of the hormones secretin and caerulein; subsequently the secretions are collected via a duodenal tube and analyzed. Caerulein is a synthetic polypeptide hormone which occurs naturally in the skin of frog. Its C-terminal amino acid sequence is similar to that of cholecystokinin/pancreozymin and is produced synthetically.

Secretin increases not only the volume of secretion but also the bicarbonate secretion (hydrokinetic function). The increase in enzyme activity observed under the influence of secretin can be traced back to a rising effect of the ductal system whereby the enzyme concentration drops in comparison to the resting secretion. Because this enzyme secretion is unpredictable and only reflects the changing enzyme content of the ductal system, subsequent stimulation of the enzyme secretion (ecbolic function) with caerulein is necessary for the assessment of pancreatic function.

Protocol: after discontinuing any pro digestive medications 72 hours prior to the performance of the test and following a 12 hours fasting period, a duodenal tube with a double lumen is introduced into the duodenum under X-ray guidance, in order to obtain a quantitative collection of pancreatic secretions. To prevent losses in the collection of pancreatic secretion, the patient should rest in right lateral position. If alkaline, bile-colored duodenal juice flows out of the duodenal tube and acidic gastric juice from the gastric tube, it is possible to proceed with the test. There are essentially two types of methods /1, 2/.

Two-step stimulation: after a 15-min. period of spontaneous secretion, secretin (1 CU = clinical unit/kg/h) is administered via a syringe pump for 1 hour, and the duodenal juice is collected in 15-min. periods. After 30 minutes, caerulein (30 ng/kg/h) is administered in addition during the last half hour via the syringe pump. The duodenal secretions are collected, cooled on ice, after each hormonal administration for 2 × 15 minutes; the secreted volumes are measured, and the bicarbonate concentration as well as the enzyme activities (amylase, trypsin, lipase) are determined.

Continuous stimulation: after intravenous administration of secretin (1 CU/kg body weight), the duodenal secretions are collected in multiple fractions over a period of 1 hour. During the second hour, secretin and caerulein (1 CU or 75 ng/kg body weight/hour) are infused continuously. The duodenal secretions are again collected in multiple fractions.

Criteria for assessing the secretory capacity of the pancreas

Secretin phase: volume secretion in mL/min., maximal fractional bicarbonate concentration in mmol/L, bicarbonate secretory capacity in mmoL/min. or in mmoL/30 min.

Caerulein phase: enzyme secretion of α-amylase /3/, lipase /4/, trypsin /5/, possibly also chymotrypsin, in U/min. or U/30 minutes.

To retroactively correct possible volume losses by means of mathematical calculation, a defined amount of ⁵⁸Co-labeled vitamin B₁₂ or polyethylene glycol is continuously instilled into the duodenum during the collection period and aspirated again with the pancreatic secretions. According to corresponding examinations using polyethylene glycol as the labeling substance, the detection rate is so high that no significant improvement in the overall diagnostic evaluation can be expected based on the correction of possible volume losses. Therefore, it is not absolutely necessary to routinely employ this correction technique in clinical diagnostic investigations /6, 7/.

14.2.6.3 Reference interval

Refer to Tab. 14.2-10 – Reference intervals for biomarkers of the secretin-caerulein test.

14.2.6.4 Clinical significance

Like every pancreatic function test, the secretin-caerulein test only allows an assessment of the functional state of the pancreas. In the presence of exocrine pancreatic insufficiency, the test cannot differentiate the underlying cause (i.e., it cannot determine whether the insufficiency is a result of chronic pancreatitis or of pancreatic cancer) /2, 8/.

For the comparability of test results, exocrine pancreatic insufficiency should be classified according to severity. For this purpose a classification which is based upon the results of the secretin-caerulein test and the fecal fat analysis was provided, and has proven to be valuable (Tab. 14.2-11 – Severity of exocrine pancreatic insufficiency) /8, 10/.

This classification has also therapeutic implications. In the case of mild to moderate exocrine pancreatic insufficiency, compensated functional impairment is present (i.e., replacement therapy with pancreatic enzymes is not required). In severe functional impairment, decompensated pancreatic insufficiency is present (i.e., a condition usually necessitating pancreatic enzyme substitution).

The results of the secretin-caerulein test have been repeatedly compared to those obtained by endoscopic retrograde cholangiopancreatography (ERCP). When both examinations were classified according to stages of severity, concordance between both examination methods was found in only half to two thirds of all patients examined /11/. Follow-up examinations have shown that in patients with an initially abnormal secretin-caerulein test result but a normal ductal system shown by ERCP, chronic pancreatitis developed later on, whereas this is rarely the case in patients with initially abnormal ERCP findings but normal secretin-caerulein test /11/. This is possibly due to the fact that following acute pancreatitis, ductal alterations often remain consisting of residual scarring which is falsely interpreted as a sign of chronic pancreatitis /12/.

14.2.6.5 Comments and problems

Reference interval

The secretin-caerulein test is not standardized. Therefore, each laboratory should determine its own reference intervals.

Test performance

Pancreatic secretions must be collected in ice-cooled graduated cylinders. In this way, enzyme activities remain almost stable for up to 8 hours.

Analytical and pre analytical factors

Incomplete collection of secretions
Reflux of duodenal juice into the stomach
Influx of gastric secretions, thus stimulating reduced secretion of both bicarbonate and enzymes
Increased influx of bicarbonate containing bile
Use of insufficiently purified secretin
Loss of enzyme activity due to thawing and freezing if the enzyme activities cannot be determined immediately after the examination
In order to prevent a loss of enzyme activity freezing is recommended using glycerol (87%) as an additive in equal parts
Falsely high amylase activities can occur as a result of an admixture of salivary amylase since this enzyme is not always inactivated by the gastric secretions /13/. If the amylase values seem to be too high in comparison to the lipase or trypsin, a determination of the pancreatic isoamylase is recommended /14/.

References

1. Schmidt H, Lankisch PG. Funktionsdiagnostik des exokrinen Pankreas. Med Klin 1975; 70: 1227–36.

2. Lankisch PG. Progress report: exocrine pancreatic function tests. Gut 1982; 23: 777–98.

3. Rick W, Stegbauer HP. α-Amylase. Messung der reduzierenden Gruppen. In: Bergmeyer HU (ed). Methoden der enzymatischen Analyse, Band I. 3rd ed. Weinheim: Verlag Chemie, 1974: 848–53.

4. Rick W. Kinetischer Test zur Bestimmung der Serumlipaseaktivität. Z Klin Chem Klin Biochem 1969; 7: 530–9.

5. Erlanger BF, Kokowsky N, Cohen W. The preparations and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961; 95: 271–8.

6. Staud R, von Kleist D, Stopik D, Hampel KE. Bedeu-tung der Volumenverlustkorrektur für die Ergebnisse des Sekretin-Pankreozymin-Tests. Z Gastroenterol 1980; 18: 474–7.

7. Lankisch PG, Creutzfeldt W. Effect of synthetic and natural secretin on the function of the exocrine pancreas in man. Digestion 1981; 22: 61–5.

8. Lankisch PG. Function tests in the diagnosis of chronic pancreatitis. Critical evaluation. Int J Pancreatol 1993; 14: 9–20.

9. Lankisch PG, Schreiber A, Otto J. Pancreolauryl test. Evaluation of a tubeless pancreatic function test in comparison with other indirect and direct tests for exocrine pancreatic function. Dig Dis Sci 1983; 28: 490–3.

10. Lankisch PG, Andrén-Sandberg Ĺ. Standards for the diagnosis of chronic pancreatitis and for the evaluation of treatment. Int J Pancreatol 1993; 14: 205–12.

11. Lankisch PG, Seidensticker F, Otto J, et al. Secretinpancreozymin test (SPT) and endoscopic retrograde cholangiopancreatography (ERCP): both are necessary for diagnosing or excluding chronic pancreatitis. Pancreas 1996; 12: 149–52.

12. Seidensticker F, Otto J, Lankisch PG. Recovery of the pancreas after acute pancreatitis is not necessarily complete. Int J Pancreatol 1995; 17: 225–9.

13. Lankisch PG, Otto J. Salivary isoamylase in duodenal aspirates. Dig Dis Sci 1986; 31: 1299–1302.

14. Junge W, Troge B, Klein G, Poppe W, Gerber M. Evaluation of a new assay for pancreatic amylase: performance characteristics and estimation of reference intervals. Clin Biochem 1989; 22: 109–14.

14.2.7 Pancreolauryl test

14.2.7.1 Indication

Suspected exocrine pancreatic insufficiency.

14.2.7.2 Test performance

The fluorescein product that is formed in the pancreolauryl test can be determined in urine or serum.

Urine test

Principle: hydrolytic cleavage of the synthetic substance fluorescein-dilaurate (dilauric acid ester of fluorescein) by the cholesterol ester hydrolase of the pancreatic secretions into lauric acid and free, water-soluble fluorescein /1/. This dye is then absorbed, partially metabolized in the liver, and excreted via the kidneys. The amount of dye excreted in the urine is used to assess exocrine pancreatic function /2/.

Protocol /3/: the fasting patient receives 0.5 liter of tea without sugar and cream at 6:30 a.m., at 7:00 a.m. 20 g of butter on a roll and the two blue test capsules (0.5 mmol of fluorescein dilaurate) which are taken in unchewed with a portion of the chewed roll mid-way through the breakfast, one additional cup of tea is served with breakfast. The urine collection starts as of 7:00 a.m.

No further food intake until 10:00 a.m.
At 10:00 a.m. 1 liter of tea which should be drunk within a 2-h period; after that, normal food intake is resumed
At 5:00 p.m., urine collection is terminated after emptying the bladder
After at least a one-day break, the examination is performed under the same conditions using a control substance (0.5 mmol of fluorescein sodium), administered in the form of a red capsule.

Laboratory measurement of the dye

The 10-h urine collection is mixed, the volume precisely determined, and 4.5 mL of 0.1 mol/L of NaOH added to a 0.5 mL sample of urine. In order to measure the total proportion of fluorescein, the sample is placed in a water boiler at a temperature of 65–70 °C for a period of 10 min., followed by centrifugation after cooling. This results in the hydrolysis of fluorescein glucuronides. The optically homogeneous mixture is spectrophotometrically analyzed at 492 nm against water.

Calculation of the excretion of the dye

Absorption × urine volume (mL)/35 = dye excretion (% of administered dose). After the dye excretion has been calculated on the test (T) day and a control (C) day, the T/C ratio is ascertained.

Serum test

No standardized test sequence /4/.

14.2.7.3 Reference interval

T/C higher than 30 /3/

14.2.7.4 Clinical significance

According to data provided by the manufacturer, a T/C ratio < 20 indicates the presence of exocrine pancreatic insufficiency with or without steatorrhea. Given a ratio between 20 and 30, repeat testing is suggested; if a ratio of < 30 is confirmed, exocrine pancreatic insufficiency is presumably present /5/.

According to the authors findings, the presence of steatorrhea must be assumed if the T/C ratio is < 10.

A normal pancreolauryl test rules out the presence of moderate or severe exocrine pancreatic insufficiency /3, 5/.

In the event of mild exocrine insufficiency, the test may produce falsely-normal results. An abnormal pancreolauryl test is usually proof of exocrine pancreatic insufficiency. Falsely abnormal test results have been described after gastric resections (postprandial asynchronism?), in conjunction with biliary diseases (inadequate ester hydrolysis?), and with inflammatory bowel diseases /6/.

As is the case with any pancreatic function test, the pancreolauryl test only allows the assessment of the functional status of the pancreas. It cannot aid in differentiating the underlying cause (i.e., it cannot determine whether this exocrine pancreatic insufficiency is a result of chronic pancreatitis or of pancreatic cancer).

14.2.7.5 Comments and problems

Falsely low fluorescein excretion rates can be expected if the urine collection is incomplete or if the intake of the breakfast and the capsules was inadequate.

False-negative test results have to be assumed if pancreatic enzyme substitution therapy is continued throughout the test. Such preparations must be discontinued 3 days prior to the start of the test.

High-dose vitamin B₂ administration also causes interference with the test since riboflavin is measured at 492 nm as well. The administration of azulfidine apparently also interferes with the method.

Since accurate urine collections can be difficult to achieve in elderly, severely ill or ambulatory patients, attempts were undertaken to measure fluorescein in the serum /4, 7/. According to own experiences, the serum test is of equivalent value to the urine test. The best distinction between patients with normal and abnormal pancreatic function was possible after 210 min. /7/.

An optimized pancreolauryl test method, which is considered to be more sensitive than the one described herein, has been published /8/.

References

1. Meyer JG. Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase. Biochim Biophys Acta 1989; 1002: 89–92.

2. Kaffarnik H, Meyer-Bertenrath JG. Zur Methodik und klinischen Bedeutung eines neuen Pankreaslipase-Tests mit Fluoresceindilaurinsäureester. Klin Wschr 1969; 47: 221–3.

3. Ventrucci M, Gullo L, Daniele C, Priori P, Labň G. Pancreolauryl test for pancreatic exocrine insufficiency. Am J Gastroenterol 1983; 78: 806–9.

4. Dominguez-Muńoz JE, Pieramico O, Büchler M, Malfertheiner P. Clinical utility of the serum pancreolauryl test in diagnosis and staging of chronic pancreatitis. Am J Gastroenterol 1993; 88: 1237–41.

5. Stock K-P, Schenk J, Schmack B, Domschke W. Funktions-Screening des exokrinen Pankreas. FDL-, N-BT-PABA-Test, Stuhl-Chymotrypsinbestimmung im Vergleich mit dem Sekretin-Pankreozymin-Test. Dtsch Med Wschr 1981; 106: 983–7.

6. Freise J, Ranft U, Fricke K, Schmidt FW. Chronic pancreatitis: Sensitivität, Spezifität und prädiktiver Wert des Pankreolauryltests. Z Gastroenterol 1984; 22: 705–12.

7. Lankisch PG, Brauneis J, Otto J, Göke B. Pancreolauryl and NBT-PABA tests. Are serum tests more practicable alternatives to urine tests in the diagnosis of exocrine pancreatic insufficiency? Gastroenterology 1986; 90: 350–4.

8. Dominguez-Muńoz JE, Malfertheiner P. Optimized serum pacreolauryl test for differentiating patients with and without chronic pancreatitis. Clin Chem 1998; 44: 869–75.

14.3 Diseases producing malabsorption

Lothar Thomas

14.3.1 Conceptual explanation

Malassimilation

Malassimilation is a decreased ability of the gastrointestinal tract to incorporate nutrients into the body. Malassimilation causes nutritional deficiency states and are either due to maldigestion or malabsorption /1/.

Maldigestion

Maldigestion is caused by deficiency of the following pancreatobiliary secretions:

Pancreatic digestive enzymes (α-amylase, lipase, trypsin, elastase) and bicarbonate
Bile acids.

Malabsorption

Malabsorption is attributable to a generalized severe flattening of the mucosa of the small intestine. Features and laboratory tests to differentiate malabsorption from maldigestion are shown in Tab. 14.3-1 – Features and diagnostic tests to differentiate malabsorption from maldigestion.

14.3.2 Malabsorption syndrome

Cardinal symptoms of diseases of the small intestine are non-specific abdominal pain, postprandial bloating, meteorism, chronic diarrhea and loss of weight. In Western industrial nations the prevalence of chronic diarrhea is 4–5% and the most common reason for hospitalization in a gastroenterologic clinic. Diarrhea is the condition of having at least three loose bowel movements each day, and a stool weight above 200 g. In chronic disease diarrhea lasts more than 4 weeks.

Numerous diseases of the small intestine are associated with malabsorption. Causes are /2/:

Defects in the absorption of specific carbohydrates such as lactose or sucrose. In these cases the transport mechanisms across the luminal cell membrane of the enterocytes are disturbed, with no morphological change in the mucosa (primary malabsorption).
The reduction of the absorptive epithelium, with a simultaneous morphological change in the mucosa or an impaired movement of food due to a disturbance in lymphatic transport (secondary malabsorption). Chronic inflammatory diseases of the intestine, such as Crohn’s and ulcerative colitis, are the most important examples of diseases of decreased absorptive epithelium.

A distinction is made between global and partial or isolated malabsorption. A malabsorption syndrome is present in some 50% of patients with weight loss and watery, voluminous stools without blood, who have neither fever nor pain. Diseases of the small intestine that are associated with malabsorption are listed in Tab. 14.3-2 – Diseases of the small intestine with global and partial malabsorption.

14.3.2.1 Diseases producing malabsorption

Diseases producing malabsorption are short-bowel syndrome after small bowel resection, celiac disease, Crohn’s disease, ulcerative colitis, amyloidosis of the small intestine, Whipple’s disease, radiation enteritis, and lactose and fructose malabsorption.

The idiopathic inflammatory bowel diseases comprise two types of chronic intestinal disorders: Crohn’s disease and ulcerative colitis /4/. There is some evidence that inflammatory bowel disease results from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. The peak of onset is in individuals 15–30 years of age. Diseases producing malabsorption are listed in (Tab. 14.3-3 – Diseases producing malabsorption).

14.3.2.2 Laboratory tests in patients with malabsorption

In patients with diseases of the small bowel associated with malassimilation routine laboratory tests usually do not indicate malabsorption. Besides routine laboratory tests specific tests of nutriment assimilation are important to differentiate malabsorption and to define the specific site of the defect /3/. Important screening tests for the general investigation on malabsorption are the D-xylose test and the determination of fat excretion in the stool. For further information refer to

Tab. 14.3-4 – Routine laboratory test results in malabsorption
Tab. 14.3-5 – Tests of digestive-absorptive function.

Special examinations

Examinations for the differentiation between Crohn’s disease and ulcerative colitis are /5/:

Anti-neutrophil cytoplasmic antibodies (ANCA): often positive in colitis, seldom positive in Crohn’s disease Anti-Saccharomyces cerevisiae antibodies (ASCA): often positive in Crohn’s disease, seldom positive in colitis
Molecular biological testing of genes that are involved in the expression of Crohn’s disease and ulcerative colitis.

Disease activity in Chrohn’s disease

Mild: patient is able to walk, tolerates oral intake of food, loss of weight > 10%, slightly elevated CRP.

Moderate: loss of weight > 10%, intermittent vomiting, lack of responsiveness to mild Crohn’s disease drug therapy, moderately elevated CRP (< 50 mg/L).

Severe: cachexia with body mass index < 18 or ileus or abscess. Persistent symptoms in spite of intensive treatment, elevated CRP.

Remission: No elevation of CRP.

Disease activity in ulcerative colitis

Mild (S1): up to 4 stools in 24 hours, possibly bloody, normal pulse, temperature and erythrocyte sedimentation rate (ESR), no anemia.

Moderate (S2): 4–6 daily stools, and no signs of systemic involvement.

Severe (S3): more than 6 bloody stools and signs of systemic involvement, temperature above 37.5 °C, heart rate over 90/min., Hb levels below 105 g/L, erythrocyte sedimentation rate > 30 mm in the first hour.

References

1. Caspary WF. Dünndarmkrankheiten. Dtsch Ärztebl 1995; 92: B-2177–84.

2. Rubenstein E, Federman E. Scientific American Medicine. New York, Scientific American 1995.

3. Duncan A. A review of the quality of gastrointestinal investigations performed in UK laboratories. Ann Clin Biochem 2007; 44: 145–58.

4. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med 2009; 361: 2066–78.

5. Baumgart DC. Diagnostik und Therapie von Morbus Crohn und Colitis ulcerosa. Dtsch Ärztebl 2009; 106: 123–33.

6. Kaukinen K, Lindfors K, Collin P, Koskinen O, Mäki M. Coeliac disease – a diagnostic and therapeutic challenge. Clin Chem Lab. Med 2010; 48: 1205–16.

7. Bergamachi G, Markopoulos K, Albertini R, Di Sabatino A, Biagi F, Ciccocioppo R, et al. Anemia of chronic disease and defective erythropoietin production in patients with celiac disease. Haematologica 2008; 93: 1785–91.

8. Husby S, Murray JA, Kastzka D. AGA clinical practice update on diagnosis and monitoring of celiac disease–changing utility of serology and histologic measures: expert review. Gastroenterology 2019; 156 (4): 885–9.

9. Enattah NS, Sahi T, Savilathi E, Terwilliger JD, Peltonen L, Jarvela I. Identification of a variant associated with adult-type hypolactasia. Nat Genet 2002; 30: 233–7.

10. Raithel M, Weidenhiller M, Hagel AFK, Hetterich U, Neurath M, Konturek PC. Kohlenhydratmalassimilation häufig vorkommender Mono- und Disaccharide. Dtsch Arztebl Int 2013; 110: 775–82.

11. Obermayer-Pietsch BM, Bonelli CM, Walter DE, Kuhn RJ, Fahrleitner-Pammer A, Berghold A, et al. Genetic predisposition for adult lactose intolerance and relation to diet, bone density, and bone fractures. J Bone Miner Res 2004; 19: 42–7.

12. Noomen CG, Hommes DW, Fidder HH. Update on genetics in inflammatory disease. Best Practice and Research Clinical Gastroenterology 2009; 23: 233–43.

13. Yazdanyar S, Weischer M, Nordestgaard BG. Genotyping for NOD2 genetic variants and Crohn disease: a metaanalysis. Clin Chem 2009; 55: 1950–7.

14. Hellström PM. Pathophysiology of the irritable bowel syndrome– Reflections of today. Best Practice & Research Clinical Gastroenterology 2019; 40–41: 101620.

14.3.3 D-xylose test

D-xylose is a pentose found naturally in plants. Its incomplete absorption allows it to be used as an absorptive test. D-xylose allows assessment of small intestine function.

14.3.3.1 Indication

Suspected malabsorption syndrome
Suspected disorders of the functional integrity of the proximal small intestine.

14.3.3.2 Test performance

Principle: perorally administered D-xylose is actively absorbed within the proximal small bowel; while a portion is subject to the intermediary metabolism, approximately one half of that absorbed is excreted renally. The D-xylose, which is detectable in a 5-h urine collection, depends on the intestinal absorption capacity for carbohydrates. The D-xylose test evaluates the functional integrity of the duodenum and the jejunum. Diseases which reduce the absorptive intestinal surface are associated with abnormally low D-xylose excretion in the urine as well as with decreased serum D-xylose concentrations.

Protocol in adults: after bladder emptying, the fasting patient drinks 25 g of D-xylose in 300 mL of water or weak tea. Another 300 mL of water or tea are drunk to ensure adequate diuresis. The 5-h urine is collected and stored; venous blood samples (3 mL) are collected after 15 min., 1 h, and 2 h.

Protocol in children: see comments and problems.

The total volume of the 5-h urine collection must be measured; a 1 : 10 dilution with distilled water is used. The D-xylose concentration is determined by means of the spectrophotometric p-bromine-aniline method /1/. Serum is deproteinized for the analysis by using trichloroacetic acid (final concentration in the test mixture approximately 0.1 mol/L), followed by filtration. Alternatively, the determination can also be conducted using high-pressure liquid chromatography (HPLC).

Assessment criteria: quantitative D-xylose excretion in a 5-h urine collection, D-xylose serum concentration /2, 3/.

14.3.3.3 Specimen

5-hour urine collected over a 5-hour period, addition of 5 mL of 10% thymol in isopropanol. The total volume of the urine collection is to be delivered to the laboratory.

Serum: 1 mL

14.3.3.4 Reference interval

Refer to Ref. /4/ and Tab. 14.3-6 – Reference intervals of D-xylose test

14.3.3.5 Clinical significance

The D-xylose test is an established test for detecting disorders within the proximal small intestine in the absorption of carbohydrates. The test checks the absorption capacity of the small intestine for monosaccharides (Tab. 14.3-7 – Gastrointestinal diseases with reduced D-xylose excretion).

A decreased urinary excretion rate of D-xylose and an insufficient increase of D-xylose in serum suggest the presence of a disease of the duodenum or jejunum. The urinary D-xylose excretion does not allow a more definitive distinction to be made than the serum D-xylose determination. The 1-hour serum level provides information on the rate constant for intestinal absorption (proximal small intestinal function), whereas the 5-hour urine content (assuming normal renal function) correlates with overall bio availability (a function of the rate constant for absorption and the inverse of the rate constant for non absorptive losses, such as from small intestinal bacterial overgrowth and intestinal hurry) /9/.

The D-xylose test, employed in parallel to tests of the pancreatic function, a small intestine biopsy and quantitative fecal fat determination, is the essential tool allowing the distinction to be made between enteral malabsorption and pancreatic impaired digestion.

The D-xylose test does not detect isolated reductions of individual disaccharidase activities in the brush border (primary forms of carbohydrate intolerance) such as a deficiency in lactase or in sucrase-isomaltase. Special types of malabsorption (e.g., involving vitamin B₁₂ or bile acids) as a result of pathological changes within the ileum cannot be excluded based on a normal D-xylose test. The test is normal in chronic pancreatitis. Bacterial colonization of the small intestine should be taken into consideration if the fecal fat excretion is abnormal in conjunction with an abnormal D-xylose test and normal morphology of the brush border villi.

A normal D-xylose test does not rule out an intestinal cause of malabsorption. However, given such a test, the disease is most likely to be caused by changes in the distal small intestine (e.g., by ileal dysfunction as seen in Crohn’s disease or by radiation enteritis).

If steatorrhea is present, a normal D-xylose test in conjunction with unremarkable findings during the gastrointestinal passage should induce a diagnostic investigation of pancreatic function.

D-xylose test for monitoring

In celiac disease the excretion of D-xylose increases under therapy with a gluten-free diet, however, complete normalization is not always achieved in adults. In patients with tropical sprue normalization of the D-xylose test often does not occur even after many years of treatment and despite improvements in the clinical symptoms.

14.3.3.6 Comments and problems

Test protocol

Possible side effects of the 25 g D-xylose test include symptoms of carbohydrate intolerance such as intestinal distention, diarrhea, and flatulence as well as nausea. Therefore, some authors prefer to use the 5-g D-xylose test. However, using the 5 g dose, the test does not reach an optimal carbohydrate load thus resulting in a correspondingly lower clinical sensitivity. Furthermore, a higher susceptibility to interferences must be recognized with underlying bacterial colonization of the small intestine and disturbances in gastric emptying are present.

The use of the hydrogen breath test does not result in any diagnostic improvement in comparison to the classic D-xylose test /4/.

Age dependency

The results concerning a decrease of the D-xylose excretion with advancing age are controversial. A decreased urinary excretion of D-xylose in elderly individuals in conjunction with a normal increase in serum D-xylose after 1 to 2 hours usually reflects a decrease in renal function.

D-xylose test in children

In pediatrics, serum D-xylose determination is preferred because of the difficulties in obtaining adequate urine collections. Numerous modifications of the test exist.

According to reference /10/ in children with a body weight of 4–30 kg: 5 g of D-xylose as a standard dose in 100–200 mL of water, venous blood sampling (1 mL) after 1 h, a serum D-xylose concentration of > 20 mg/dL (1.33 mmol/L) is considered to be the reference value.

According to reference /9/, 14.5 g of D-xylose per m² of body surface are administered as a 10% solution. A D-xylose concentration of > 25 mg/dL (1.67 mmol/L) after 1 h is considered to be the reference value.

Pre analytical factors resulting in reduced D-xylose excretion rate are incomplete urine collection (very common), incomplete bladder emptying (residual urine), vomiting, food intake during the test period.

Biological influence factors

Causes of reduced D-xylose excretion:

Renal insufficiency, ascites, inadequate hydration, reduced effective circulatory volume, aspirin and aspirin-like medications, hypothyroidism, pernicious anemia, bacterial overgrowth of the small intestine, dumping, extreme delays in gastric emptying /11/
An abnormal urinary D-xylose excretion under the above mentioned circumstances is not indicative of an absorption disorder if the serum D-xylose concentration is normal /12/
In the case of bacterial overgrowth of the small intestine, the D-xylose test may produce abnormal results. The normalization of an abnormal D-xylose test after antibiotic therapy serves as an indirect indicator of bacterial overgrowth /12/ in situations when clinically more useful tests are not available.
In chronic alcoholics acute alcohol consumption will decrease the urinary D-xylose excretion while chronic alcohol intake in conjunction with adequate nutrition will lead to an increase in the absorption of D-xylose
Cholestasis leads to reduced urinary and serum D-xylose values in urine and serum
Phenformin and indomethacin inhibit the intestinal absorption of D-xylose /11/.

Causes of increased D-xylose excretion:

With adequate nutrition, chronic alcohol intake leads to an increase in D-xylose absorption
Liver cirrhosis leads to an abnormal D-xylose excretion only in conjunction with ascites (a rise after the mobilization of the ascitic fluid); following shunt surgery due to portal hypertension, a rise in the urinary D-xylose excretion is also observed.

Stability

Urinary D-xylose: at 4 °C without additives for 24 h, at room temperature with thymol-isopropanol as an additive for 48 h.

Serum D-xylose: at 4 °C for 3 days.

Storage of the samples is possible for weeks at –16 °C.

14.3.3.7 Pathophysiology

Studies of the vesicles of the microvilli show that passive absorption is the predominant mechanism of D-xylose absorption /13/. In healthy people only 58% of a 25 g dose of perorally administered D-xylose is enterally absorbed; of this portion 90% are absorbed within the proximally 100 cm of the small intestine. Approximately 42–60% of enterally absorbed D-xylose appear unchanged in a 24-h urine collection, corresponding to 5.5–7.8 g /14/. The large majority of this portion can already be found in the urine after a period of 5 h. The highest serum D-xylose concentration is measured after 1–2 h.

The D-xylose test is a sensitive functional test of the proximal small bowel; the practical-diagnostic consequence of an abnormal D-xylose test is a small bowel biopsy (morphology/enzyme analysis) which, in some of the cases, contributes to the etiological differentiation of the underlying malabsorption syndrome.

References

1. Roe JH, Rice EW. A photometric method for the determination of free pentoses in animal tissues. J Biol Chem 1948; 173: 507–12.

2. Benson JA, Culver PJ, Ragland S, Jones CM, Drummey ED, Bougas E. The D-Xylose absorption test in malabsorption syndromes. N Engl J Med 1957; 256: 335–9.

3. Finlay JM, Hogarth J, Wightman KJR. A clinical evaluation of the D-Xylose tolerance test. Ann Intern Med 1964; 61: 411–22.

4. Lembcke B, Bornholdt C, Kirchhoff S, Lankisch PG. Clinical evaluation of a 25 g D-Xylose hydrogen (H₂) breath test. Z Gastroenterol 1990; 28: 555–60.

5. Caspary WF. On the mechanism of D-Xylose absorption from the intestine. Gastroenterology 1972; 63: 531–5.

6. Fordtran JS, Soergel KH, Ingelfinger FJ. Intestinal absorption of D-Xylose in man. N Engl J Med 1962; 267: 274–9.

7. Wilson FA, Dietschy JM. Differential diagnostic approach to clinical problems of malabsorption. Gastroenterology 1971; 61: 911–31.

8. Ehrenpreis ED, Ganger DR, Kochvar GT, Patterson BK, Craig RM. D-xylose malabsorption: characterisic finding in patients with AIDS wasting syndrome and chronic diarrhea. J Acquir Immune Defic Syndr 1992; 5: 1047–50.

9. Craig RM, Ehrenpreis ED. D-xylose-testing. J Clin Gastroenterol 1999; 29: 143–50.

10. Rolles CJ, Kendall MJ, Nutter S, Anderson CM. One-hour blood-xylose screening-test for coeliac disease in infants and young children. Lancet 1973; 2: 1043–5.

11. Kendall MJ, Nutter S, Hawkins CF. Xylose test: effect of aspirin and indomethacin. Brit Med J 1971; 1: 533–6.

12. Goldstein F, Karacadac S, Wirts CW, Kowlessar OD. Intraluminal small-intestinal utilisation of D-Xylose by bacteria. Gastroenterology 1970; 59: 380–6.

13. Rolston DDK, Manthan VI. Xylose transport in the human jejunum. Dig Dis Sci 1989; 34: 553–8.

14. Wyngaarden JB, Segal S, Foley JB. Physiological disposition and metabolic fate of infused pentoses in man. J Clin Invest 1957; 36: 1395–407.

14.3.4 Carbohydrate malassimilation

Adverse food reactions are common in the population and are claimed by up to 67% of individuals with functional gastrointestinal disorders (FGIDs). The FGIDs include a number of separate idiopathic disorders which affect different parts of the gastrointestinal tract. The Rome meetings have proposed a consensual classification system and terminology of FGIDs.

A food is defined as any substance intended for human consumption, including drinks, food additives, and dietary supplements. Food allergy and intolerance describe a wide range of reactions to foods. The expert panel of the US National Institute of Allergy and Infectious Diseases /1/ proposes that all adverse food reactions be classified as

either immune-mediated (food allergy and celiac disease)
or non immune mediated (formerly known as food intolerances), which are themselves subdivided into four categories.

The categories of immune mediated adverse food reactions are /2/: IgE-mediated, non-IgE-mediated, mixed IgE-mediated, and cell-mediated.

Non immune-mediated conditions (food intolerance) include /2/:

Metabolic (carbohydrate malabsorption)
Pharmacological (vasoactive amines, salicylates, caffeine, theobromines)
Toxic (scombroid poisoning)
Other, idiopathic (food additive hypersensitivity).

Carbohydrate intolerances, defined as symptoms associated with their ingestion, are one important cause of FIGDs. The most important carbohydrates that routinely cause clinical abdominal complaints are lactose, fructose, and the sugar alcohol sorbitol.

14.3.4.1 Indication

Suspicion of carbohydrate malabsorption, a non immune mediated metabolic condition (food intolerance). Symptoms are in the range from a feeling of mild bloating to severe diarrhea.

14.3.4.2 Examinations

The examinations in carbohydrate malabsorption include:

Food and medication history
Hydrogen exhalation test (hydrogen breath test)
Lactose tolerance test
Sonography
Endoscopy and enzyme determination (for example disaccharidases) in the aspired small intestinal mucosa biopsy material.

14.3.4.2.1 Hydrogen breath test

The technique of hydrogen breath tests is based on the principle, that there is no human hydrogen gas production, but hydrogen is produced by intestinal bacteria when ingested carbohydrates escape complete absorption in the small intestine /3/. Usually, hydrogen producing bacteria only colonise the colon. A fixed fraction of this colonic hydrogen diffuses into the blood stream and is exhaled by the lungs where it can be analyzed in breath. Hydrogen concentrations in end expiratory breath samples are measured using gas chromatography or electrochemical cells.

Lactose hydrogen breath test protocol /3/: for testing for lactose malabsorption, 50 g lactose are dissolved in 100–500 mL of water and orally ingested. Hydrogen concentrations in end expiratory breath are analyzed before and at 15–30 min. intervals for 4 h after intake. A rise of more than 10–20 ppm over the basal hydrogen value (detected in at least two breath samples) indicates lactose malabsorption.

Fructose hydrogen breath test protocol /3/: for testing for fructose malabsorption, 25–50 g of fructose are dissolved in 200–400 mL of water and orally ingested. Hydrogen concentrations in end expiratory breath are analyzed before and at 15 min. intervals for 3 h after intake. A rise of more than 20 ppm over the basal hydrogen value (detected in at least two breath samples) indicates fructose malabsorption.

Sorbitol hydrogen breath test protocol /3/: for testing for sorbitol malabsorption, 5–10 g sorbitol are dissolved in 200–500 mL of water and orally ingested. Hydrogen concentrations in end expiratory breath are analyzed before and at 15 min. intervals for 3 h after intake. A rise of more than 10–20 ppm over the basal hydrogen value (detected in at least two breath samples) and the occurrence of typical symptoms provoked by the sorbitol load indicates sorbitol malabsorption.

14.3.4.2.2 Lactose tolerance test

Principle: the lactase of the mucosa of the small intestine is a β-galactosidase located in the brush border which causes the cleavage of lactose into the monomers glucose and galactose. The monosaccharides are actively absorbed and lead to a rise in the blood glucose concentration. The rate limiting step of lactose assimilation is the hydrolytic cleavage of the disaccharides. In the event of decreased lactase activity, the rise in blood glucose therefore is absent.

Protocol: the fasting patient drinks 50 g of lactose in 400 mL of water, venous or capillary blood samples are obtained (0, 30, 60, 90, 120 min.) and the blood glucose level is determined. Children receive 2 g of lactose/kg of body weight, up to maximally 50 g.

Assessment criteria: the rise in blood glucose in comparison to the basal level; the clinical symptomatology during the course of an 8-hour period following the start of the test (abdominal distention, abdominal cramping, bloating, diarrhea).

14.3.4.3 Specimen

Lactose tolerance test: venous or capillary blood: 0.1–1 mL.

14.3.4.4 Reference interval

Lactose tolerance test /8/: rise in whole blood or serum glucose of > 20 mg/dL (1.11 mmol/L), or in capillary blood glucose of > 25 mg/dL (1.39 mmol/L)
Lactose hydrogen breath test /3/: ≤ 20 parts per million (ppm) during 240 minutes
No gastrointestinal symptoms

14.3.4.5 Clinical significance

Carbohydrates are classified according to their structure based on the number of basic sugar or saccharide units they contain. Important disaccharide sugars present in diet are shown in Tab. 14.3-8 – Disaccharides present in the diet.

The term carbohydrate malabsorption describes conditions of incomplete absorption of carbohydrates in the small intestine and reach the colon /4/. This can be physiological due to the ingestion of carbohydrates for which the healthy gastrointestinal tract has a limited digestive or absorptive capacity (Tab. 14.3-9 – Food that can cause malassimilation of monosaccharides and disaccharides). Carbohydrate malabsorption may result in bloating, passing of gas, flatulence and cramping. Fructose and lactose intolerances are common in patients with FGIDs. Intolerance prevalence across all FGIDs was 60% to fructose, 51% to lactose and 33% to both. Malabsorption, using hydrogen breath testing, occurred in 45%, 32% and 16%, respectively /5/.

Malabsorption may result from /6/:

Congenital defects of a single transport system (primary malabsorption)
From impairment of the epithelial surface of the small intestine (secondary malabsorption), due to general intestinal diseases such as celiac disease or Crohn’s disease, which may inhibit the absorption of all carbohydrates.

14.3.4.5.1 Primary carbohydrate malabsorption

The primary forms of carbohydrate malabsorption are the result of congenital deficiencies of enzymes and proteins which are responsible for the digestion and transport of sugars (Tab. 14.3-8 – Disaccharides present in the diet). The physiological loss of enzymes such as lactase may also be involved. As a rule, patients with primary carbohydrate malabsorption manifest symptoms that are the result of the primary disorder, (e.g., lactase deficiency). Lactose, fructose and sorbitol intolerances are 7–20%, 15–25% and 8–12% respectively.

Tab. 14.3-9 – Food that can cause malassimilation of monosaccharides and disaccharides shows food products that contain these carbohydrates. The severity of the symptoms depends on the extent of the enzyme deficiency.

14.3.4.5.2 Secondary carbohydrate malabsorption

This form is caused by malabsorption of carbohydrates, associated with damage involving loss of the brush border of the small intestine. In cancer patients, treatment with radiotherapy or chemotherapy may affect the cells in the intestine that normally secrete the digestive enzymes, leading to malabsorption. In such cases and in inflammatory intestinal disease, the malabsorption involves, as a rule, a number of carbohydrates. Diseases are, for example, duodenal ulcer, gastric resection, ulcerative colitis, Crohn’s disease, infectious and nonspecific diarrhea in adults, giardiasis, cystic fibrosis and acute viral hepatitis /7/. Diseases involving carbohydrate malabsorption are listed in Tab. 14.3-10 – Primary and secondary carbohydrate malabsorption.

14.3.4.5.3 Differential diagnosis

Difficulties can occur in the differentiation of primary carbohydrate malabsorption from irritable bowel syndrome, bacterial overgrowth of the small intestine, irritable stomach and food allergy. The symptoms that can occur with lactose, fructose and sorbitol provocation are indistinguishable from those of inflammatory bowel syndrome. Therefore, in suspicion of irritable bowel, carbohydrate malabsorption should be ruled out, since its prevalence in patients with irritable bowel corresponds roughly to that in the normal population /8/.

14.3.4.6 Comments and problems

Hydrogen breath test

All measures that lead to a reduction of the bacterial flora of the colon (e.g., lavage, laxatives prior to endoscopy and radiography, broad antibiotic therapy) result in a false normal test outcome. The test is not meaningful in patients with ileostomy.

Due to abnormal intestinal flora, 10–15% of the European population cannot form hydrogen gas; thus, in their exhaled air, the hydrogen fraction is ≤ 20 ppm, and this represents a false normal finding. In order to establish if patients with a normal finding have a normal bacterial flora, the hydrogen breath test is repeated using lactulose. Lactulose is a disaccharide that is not cleaved in the human small intestine; it reaches the colon and is cleaved by bacteria. A hydrogen formation of > 20 ppm demonstrates that a normal finding is valid.

Lactose tolerance test (LTT)

Influence factors: in LTT a flattened blood glucose profile is seen following oral lactose loading in 25% of the cases, in spite of normal lactase activity /9/.

Causes are:

Motility factors such as delayed emptying of the stomach, inappropriately rapid intestinal passage with maximal blood glucose values within as little as 15 minutes (e.g., following surgical gastric resection).
Augmented glucose uptake by tissues and absorptive function disorders (deficient monosaccharide transport) of the small intestine.

Monitoring of monosaccharide absorption: a lactose absorption disorder is demonstrable by repetition of the LTT on the following day with oral administration of 25 g D-glucose + 25 g D-galactose in 400 mL of water. In lactose intolerance, the ratio of the blood glucose rise 50 g lactose/(25 g D-glucose + 25 g D-galactose) is < 0,4. In pathological glucose tolerance and manifest diabetes mellitus, the outcome of the LTT may be false-negative.

References

1. Boyce JA, Assa’ad A, Burks WA, Jones SM, Sampson HA, Wood RA, et al. Guidelines for the diagnosis and management of food allergy in the United States: Summary of the the NIAID-sponsored expert panel report. J Allergy Clin Immunol 2010; 126 (suppl): S1–S58.

2. Skypala I. Adverse food reactions – An emerging issue for adults. J Amer Dietetic Assoc 2011; 111: 1877–91.

3. Braden B. Methods and functions: Breath tests. Best Practice&Research Clinical Gastroenterology 2009; 23: 337–52.

4. Hammer HF, Hammer J. Diarrhea caused by carbohydrate malabsorption. Gastroenterol Clin North Am 2012; 41: 611–27.

5. Wilder-Smith CH, Materna A, Wermelinger C, Schuler J. Fructose and lactose intolerance and malabsorption testing: the relationship with symptoms in functional gastrointestinal disorders. Aliment Pharmacol Ther 2013; 37: 1074–83.

6. Born P. Carbohydrate malabsorption in patients with non-specific abdominal complaints. World J Gastroenterol 2007; 21: 5687–91.

7. Plotkin, GR, Isselbacher KJ. Secondary disaccharidase deficiency in adult coeliac disease (non tropical sprue) and other malabsorption states. N Engl J Med 1964; 271: 1033–7.

8. Shaw AD, Davies GJ. Lactose tolerance. Problems and treatment. J Clin Gastroenterol 1999; 28: 208–16.

9. Arola H. Diagnosis of hypolactasia and lactose malabsorption. Scand J Gastroenterol 1994; 29, Suppl 202: 26–35.

10. Suarez FL, Savaiano DA, Levitt MD. A comparison of symptoms after the consumption of milk or lactose-hydrolyzed milk by people with self-reported severe lactose intolerance. N Engl J Med 1995; 333: 1–4.

11. Enattah NS, Sahi T, Savilathi E, Terwilliger JD, Peltonen L, Jarvela I. Identification of a variant associated with adult-type hypolactasia. Nat Genet 2002; 30: 233–7.

12. Obermayer-Pietsch B, Bonelli CM, Walter DE, Kuhn RJ, Fahrleitner-Pammer A, Berghold A, et al. Genetic predisposition for adult lactose intolerance and relation to diet, bone density, and bone fractures. J Bone Miner Res 2004; 19: 42–7.

13. Newcomer AD, McGill DB, Thomas PJ, Hofmann AF. Prospective comparison of indirect methods for detecting lactase deficiency. N Engl J Med 1975; 293: 1232–5.

14. Hüppe D, Tromm A, Langhorst H, May B. Lactoseintoleranz bei chronisch entzündlicher Darmerkrankung. Dtsch Med Wschr 1992; 117: 1550–5.

15. Kyaw MH, Mayberry JF. Fructose malabsorption: true condition or a variance from normality. J Clin Gastroenterol 2011; 45: 16–21.

16. Bauditz J, Norman K, Biering H, Lochs H, Pirlich M. Severe weight loss caused by chewing gum. Br Med J 2008; 336: 96–7.

17. Born P, Zeck J, Stark M, Claaasen M, Lorenz R. Zuckeraustauschstoffe: Vergleichende Untersuchung zur intestinalen Resorption von Fructose, Sorbit und Xylit. Med Klinik 1994; 89: 575–8.

14.3.5 Fecal calprotectin (f-CP)

Calprotectin has been also known as L1 protein, MRP-8/14, calgranulin and cystic fibrosis antigen. It is a 36 kDa calcium- and zinc-binding protein, belongs to the S100 protein family and is predominantly expressed in neutrophils, in which it constitutes up to 60% of the total cytosolic protein. Calprotectin has antimicrobial and anti proliferative properties and plays a regulatory role in inflammatory processes /1/. Calprotectin is excreted in feces (f-CP) and concentrations are increased in patients with intestinal mucosal inflammation. In these patients the f-CP concentration correlates well with the fecal excretion of ¹¹¹Indium-labeled leukocytes, the gold standard for quantifying the severity of gastrointestinal inflammation.

14.3.5.1 Indication

The determination of fecal calprotectin (f-Cp) is indicated:

As a non invasive marker for distinguishing inflammatory bowel disease (ulcerative colitis, Crohn’s disease) from inflammatory bowel syndrome prior to endoscopy
To assess disease activity in inflammatory bowel disease
To predict relapse in patients with clinical remission from inflammatory bowel syndrome.

14.3.5.2 Specimen

Stool sample: 1 spatula filling, approximately 1–2 g

14.3.5.3 Method of determination

Principle: some 100 mg of feces are homogenized in 5 mL of extraction buffer, 1 mL of suspension is centrifuged, and the supernatant is taken for the determination of f-CP. The determination is performed with a two-step immunoassay using two monoclonal antibodies. In the first step f-CP is bound to antibodies on the micro titer plate well forming an immune complex. Superfluous stool material is removed by washing. In the second step the immune complex is labeled with an enzyme-marked antibody. The enzyme activity bound to a well is determined by adding a corresponding substrate. The development of a product is measured spectrophotometrically and is proportional to the f-CP concentration. The clinical and analytical verification of an automated immunoassay with extraction device is published /16/.

14.3.5.4 Reference interval

Cutoff for exclusion of intestinal inflammatory activity /2/: ≤ 50 μg/g wet feces (some authors recommend ≤ 100 μg/g stool for adults).

14.3.5.5 Clinical significance

Cp represents over 60% of the cytosolic proteins in the neutrophil cytoplasm. Consequently, f-Cp concentration may be related to inflammation of the bowel mucosa in inflammatory bowel disease.

14.3.5.5.1 Diagnosis of inflammatory bowel disease

Inflammatory bowel disease (IBD), mainly ulcerative colitis (UC) and Crohn’s disease (CD) are a chronic inflammatory condition of the gastrointestinal tract. These conditions are indicated by persistent mucosal inflammation caused by the adaptive and innate immune system.

UC presents with widespread inflammation and ulcers that can extend along a varying distance from the caecum to the rectum.
CD is mainly characterized by transmural inflammation occurring at any location in the gastrointestinal tract, with the terminal ileum and colon being the most affected areas.

The IL-1 cytokines and their receptors may be involved as a promoter of inflammation in patients with IBD due to their accepted involvement in the development of inflammatory disorders /17/.

Gastroenterologists are often faced with the diagnostic difficulty of differentiating patients with irritable bowel syndrome (IBS) from those with intestinal pathology, in particular inflammatory bowel disease (IBD) /3/. IBS and IBD have many symptoms in common including abdominal pain, bloating, excessive flatus and altered bowel habit. Features like diarrhea or rectal bleeding are, rather, suggestive of IBD. Because the differentiation remains problematic, gastroenterologists order invasive endoscopic and radiographic imaging in patients in the IBS category to make a diagnosis of exclusion. In the USA and Great Britain, the prevalence of IBS is 14–19% in men, and 14–24% in women /4/. The prevalence of IBD is 0.6–1%.

Since there is no biological marker for the diagnosis of IBS, the Rom-I criteria for IBS and other functional intestinal diseases were defined at a consensus conference (Tab. 14.3-11 – The Rome I diagnostic criteria for irritable bowel syndrome).

The most striking difference between IBS and IBD is that the former is noninflammatory in nature. In patients with suspicion of IBS based upon positive Rom I criteria, the following laboratory tests, as surrogate markers for the differentiation of IBS and IBD, were additionally recommended /5/:

Thyroid function tests for the diagnosis of hyperthyroidism-associated IBS
Investigation of helminth eggs and parasites in the stool to rule out IBS.

Inflammatory markers (erythrocyte sedimentation rate and CRP); these markers are disappointing, may be they lack sensitivity and specificity.

In a relative large proportion of individuals with suspected IBD the results of endoscopy are negative. A third of adults with bleeding related symptoms have no abnormalities on endoscopy, and this proportion increases to half with non-bleeding symptoms such as diarrhea, abdominal pain and weight loss. The identification of patients with a sufficiently low likelihood of inflammatory bowel disease would reduce the number of unnecessary endoscopic procedures /6/.

14.3.5.5.2 Fecal calprotectin for screening of inflammatory bowel disease

The determination of fecal calprotectin (f-CP) is a useful screening test in adults and children for identifying those patients who are most likely to need endoscopy for IBD /6/. An elevated value may indicate an urgent need for endoscopy whereas normal levels are less likely associated with intestinal inflammation. The exception to this rule is persistent rectal bleeding. F-CP provides better information regarding intestinal inflammation than systemic inflammatory markers. The concentration of f-CP correlates well with the histological findings of inflammatory intestinal activity.

Refer to:

Tab. 14.3-12 – Fecal calprotectin in healthy controls and in disease groups
Tab. 14.3-13 – Fecal calprotectin in functional and inflammatory intestinal diseases.

14.3.5.5.3 Causes of abnormal results for fecal calprotectin other than inflammatory bowel disease /6/

Infections: Giardia lamblia, bacterial dysentery, Helicobacter pylori gastritis.

Malignancies: colorectal carcinoma, gastric carcinoma, intestinal lymphoma.

Medications: NSAID, proton pump inhibitors, food allergy (untreated).

Miscellaneous: gastroesophageal reflux, cystic fibrosis, celiac disease (untreated), diverticulitis, protein losing enteropathy, colorectal adenoma, juvenile polyposis, autoimmune enteropathy, microscopic colitis, liver cirrhosis, children under the age of 5 years.

Lactoferrin

Like f-CP, lactoferrin is present in specific granules of neutrophil granulocytes and is delivered to the feces if granulocytes are released during inflammatory mucosal reactions. In patients with IBD, the diagnostic accuracy of lactoferrin is similar to that of f-CP, and will, therefore, not be dealt with in a separate section /7/. In a study the fecal lactoferrin in normal individuals was 0.75 ± 0.83 μg/g feces, in ulcerative colitis 307 ± 234 μg/g feces and in Crohn’s disease was 197 ± 231 μg/g feces /8/.

14.3.5.6 Comments and problems

The f-CP immunoassays are not standardized. Depending on the procedure, extraction leads to an underestimation of 7.8–28.1%. In three commercial tests that were evaluated, the difference in the values was as high as a factor of 3.8, although all manufacturers use the same cutoff /9/. The comparison of 6 assays showed similar results. Assay specific cutoffs were recommended /15/.

Fecal rapid calprotectin test: the diagnostic sensitivity and negative predictive value were both 100%, whereas they were only 78% and 95% for a fecal rapid lactoferrin test /13/.

Stability

F-CP and lactoferrin 1 week.

Influence factors

The f-CP values in active-drinking alcoholics are not significantly different, compared with healthy controls /14/.

References

1. Aadland E, Fagerhol MK. Faecal calprotectin: a marker of inflammation throughout the intestinal tract. Eur J Gastroenterol Hepatol 2002; 14: 823–5.

2. von Roon AC, Karamountzos L, Purkayastha S, Reese GE, Darzi AW, Teare JP, et al. Diagnostic precision of fecal calprotectin for inflammatory bowel disease and colorectal malignancy. Am J Gastroenterol 2007; 102: 803–13.

3. Hellström PM, Benno P. The Rome IV: Irritable bowel syndrome– a functional disorder. Best Practice & Research Clinical Gastroenterology 40-41 (2019) 101634.

4. Alibrahim B, Aljasser MI, Dalh B. Fecal calprotectin use in inflammatory bowel disease and beyond: a minireview. Can J Gastroenterol Hepatol 2015; 29: 157–163.

5. Tolliber BA, Herrera JL, DiPalma JA. Evaluation of patients who meet clinical criteria for irritable bowel syndrome. Am J Gastroenterol 1994; 89: 176–8.

6. Van Rheenen PF. Faecal calprotectin for screening of patients with suspected inflammatory bowel disease: diagnostic meta-analysis. BMJ 2010; 341: c3369. doi: 10.1136/bmj.c3369.

7. Kane SV, Sandborn WJ, Rufo PA, Zholudev A, Boone J, Lyerly D, et al. Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J. Gastroenterol 2003; 98: 1309–14.

8. Uchida K, Matsuse R, Tomita S, Sugi K, Saitoh O, Ohshiba S. Immunochemical detection of human lactoferrin in feces as a new marker for inflammatory gastrointestinal disorders and colon cancer. Clin Biochem 1994; 27: 259–64.

9. Whitehead SJ, French J, Brookes MJ, Ford C, Gama R. Between-assay variability of faecal calprotectin enzyme-linked immunosorbent assay kits. Ann Clin Biochem 2013; 50: 53–61.

10. Keohane J, O’Mahony C, O’Mahony L, O’Mahony S, Quigley EM, Shanahan F. Irritable bowel syndrome-type symptoms in patients with inflammatory bowel disease: a real association or reflection of occult inflammation? Am J Gastroenterol 2010; 105: 1789–94.

11. Sipponen T, Kolho K-L. Faecal calprotectin in children with clinically quiescent inflammatory bowel disease. Scand J Gastroenterol 2010; 45: 872–7.

12. Turner D, Leach ST, Mack D, Uusoue K, McLernon R, Hyams J, et al. Faecal calprotectin, lactoferrin, M2-pyruvate kinase and S100A12 in severe ulcerative colitis: a prospective multicentre comparison of predicting outcomes and monitoring response. Gut 2010; 59: 1207–12.

13. Otten CMT, Kok L, Witteman BJM, Baumgarten R, Kampman E, Moons KGM, et al. Diagnostic performance of rapid tests for detection of fecal calprotectin and lactoferrin and their ability to dicriminate inflammatory from irritable bowel syndrome. Clin Chem Lab Med 2008; 46: 1275–80.

14. Montalto M, Gallo A, Ferrulli A, Visca D, Campobasso E, Cardone S, et al. Fecal calprotectin concentrations in alcoholic patients: a longitudinal study. Eur J Gastroenterol Hepatol 2011; 23: 76–80.

15. Oyaert M, Boel A, Jacobs J, van den Brent S, de Slovere M, Vanpoucke H, et al. Analytical performance and diagnostic accuracy of sic different faecal calprotectin assays in inflammatory bowel disease. Clin Chem Lab med 2017; 55: 1564–73.

16. Wyness SP, Lin L, Jensen R, Bird J, Norgyal T, Jensen G, et al. Clinical and analytical verification of an automated fecal calprotectin immunoassay with extraction device. JALM 2021; July: 931–41.

17. Aggeletopoulou SP, Kalafateli M, Tsounis EP, Triantos C. Exploring the role of IL-1β in inflammatory bowel disease. Frontiers in Medicine 2024. doi: 10.3389/fmed.2024.1307394.

14.4 Colorectal cancer screening

For colorectal cancer (CRC) screening and screening for colorectal adenoma, which is a common precursor of CRC, fecal tests for blood are recommended. Bleeding is an important symptom of carcinoma and adenoma of the colon, but it is visible with the naked eye only when substantial quantities of blood are present.

Stool tests for CRC screening are based on:

Detection of blood. The tests exist for the detection of invisible (occult) blood and fecal occult blood testing (FOBT) has proven to be successful. Thus, with the regular use of such tests, CRC mortality has been significantly reduced in a cost-effective manner.
Tumor methylation analysis for leading markers such as NDRG4 and SDC2 that are integral part of the test. Stool based DNA testing is increasingly important for noninvasive detection of CRC. For further information refer to Ref. /15/. Reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using supernatant from the homogenized stool was chosen for analysis for genomic DNA /16/.

German recommendations proceed as follows /1/:

1 × FOBT every year, beginning at 50 years of age
2 × colonoscopy at least 10 years apart or 2 × FOBT every 2 years beginning at 55 years
In persons with positive family history if first degree relative colonoscopy 10 years before colorectal cancer diagnosis of the index patient.
Fecal occult blood must be determined using a quantitative immunologic assay. The analysis must be carried out in a specialist medical laboratory.

The U.S. Preventive Services Task Force (USPSTF) recommends screening for colorectal cancer in adults using FOBT, sigmoidoscopy, or colonoscopy, beginning at 50 years of age and continuing until 75 years of age /2/.

Intervals for recommended screening strategies:

Annual screening with high-sensitivity FOBT
Sigmoidoscopy every 5 years, with high-sensitivity FOBT every thee years
Screening colonoscopy every 10 years.

14.4.1 Indication

Suspicion for advanced adenoma and colorectal cancer.

14.4.2 Method of determination

The following stool test procedures are employed /3/:

Fecal occult blood testing (FOBT)
Fecal immunological testing for blood (FIT)

Guaiac-based FOBT (gFOBT)

Principle: hemoglobin peroxidase activity in the fecal sample is determined. A filter paper impregnated with guaiac resin has to be coated with a sample of feces. For development, an alcoholic solution of hydrogen peroxide is added as oxygen donor. In the presence of peroxidases, such as hemoglobin, guaiac is oxidized and turns blue. The detection limit is in the range of 0.3–0.6 mg hemoglobin/g feces.

Fecal immunochemical tests (iFOBT)

Fecal immunochemical tests of occult blood use antibodies directed against the globin part of hemoglobin /4/. Quantitative and qualitative fecal immunochemical tests are used.

Principle: an ELISA was conducted as follows; some 100 mg of feces are homogenized in extraction buffer, 1 mL of suspension is centrifuged, and the supernatant is removed for the determination of Hb. The determination is performed with a two-step immunoassay using two monoclonal antibodies. In the first step Hb is bound to antibodies on the micro titer plate wells forming an immune complex. Superfluous stool material is removed by washing. In the second step the immune complex is labeled with an enzyme-labeled antibody. The enzyme activity bound to a well is determined by adding a corresponding substrate. The development of a product is measured spectrophotometrically and is proportional to the fecal Hb content. The iFOBT detects only human blood and does not tend to provide false positive results as does the gFOBT because of non-human Hb, peroxidases of plant origin, vitamin C and aspirin /3/. The detection limit of the iFOBTs is in the range of 2–17 μg hemoglobin/g feces /4/.

14.4.3 Specimen

The following specimens are recommended:

iFOBT; a serratic plastic stick for stool collection is stabbed into 3 different parts of the stool sample and inserted into a buffer containing vial
gFOBT; collection of 2 samples from each of 3 consecutive bowel movements. Altogether, two samples will be smeared on each of 3 cards.

14.4.4 Reference interval

Guaiac-based FOBT (gFOBTs):

Qualitative; positive or negative

Immunological assays (iFOBTs):

Quantitative; the preset manufacturer’s thresholds differentiate between positive and negative

14.4.5 Clinical significance

Randomized trials have shown that annual or biannual screening with gFOBTs could reduce the CRC mortality by up to 30%.

14.4.5.1 The clinical problem

CRC is the third most cancer worldwide /5/. The yearly new diagnosed cases in Germany are 40,000, with 17,000 deaths; in the USA the corresponding numbers are 147,000 and 50,000. The age-adjusted incidence of CRC in the USA is 61.2 per 100,000 population among men and 44.8 per 100,000 population among women /5/. In the UK CRC is the second most cancer with over 30,000 new cases and almost 20,000 deaths per year, 93% of them in the over 55 years age group /6/. In general, in individuals over the age of 50 years, the incidence and mortality of CRC double every 10 years.

The prognosis of CRC is good if it is detected early. This is due to the slow growth and the low penetration to malignant transformation of polyps to adenoma and to carcinoma. The transformation from polyps to advanced adenomas is a process that can last for decades. Early detection of polyps and advanced adenomas offers a good opportunity for preventing the development of CRC. Thus, the US National Polyp Study demonstrated that colonoscopic polypectomy resulted in a 90% reduction in CRC incidence. Seventy to 90% of CRC develop from adenomatous polyps. The most CRC screening studies evaluate the detection rate of invasive CRCs as well as advanced adenomas, which conventionally are defined as polyps ≥ 10 mm or histologically having high-grade dysplasia or significant villous components /7/.

About 25% of the population have pre-malignant polyps by the age of 50 years and the prevalence increases with age /6/. However, only a small fraction of the polyps develop into CRC (2.5 polyps per 1,000 patient years). Villous adenomas are much more likely to develop into CRC than tubular adenoma. It takes an average 10 years for a polyp with a diameter of less that 1 cm to develop into an invasive carcinoma. Progressive growth of polyps is more likely to lead to malignancy. The chance of a very small polyp being cancerous may be 1 in 500; in polyps with a diameter of 1 cm it is some 10%; and in those of 2 cm diameter, it is 50% /6/.

The 5-year survival rates in CRC are dependent upon the stage; they are:

Dukes A (tumor limited to the mucosa) 83%
Dukes B and C (mucosal margin exceeded) 64%
Dukes D (metastasized) 5%.

14.4.5.2 Colorectal cancer risk (CRC) factors

The most common indicator of high risk is a first-degree relative with CRC before 50 years of age. In such a case the individual of risk should undergo investigation for hereditary syndromes such as /5/:

Familial adenomatous polyposis
Hereditary non-polyposis colorectal cancer syndrome (HNPCC)
Mut Y homolog (MUTYH) polyposis.

If a first-degree relative had CRC at ≥ 50 years of age, the lifetime risk for CRC nearly doubles among the family members.

Patients with Crohn’s disease and ulcerative colitis are at increased risk of CRC; they should undergo surveillance with colonoscopy, starting 8–10 years after diagnosis.

Additional factors associated with elevated CRC risk are a low-fiber diet, lack of movement, physical inactivity, alcoholism, smoking and obesity.

14.4.5.3 Diagnosis of patients with colorectal cancer (CRC) risk

Procedures for the diagnostic investigation of CRC are the endoscopic examination of the colon and the sigmoid, and the investigation of fecal blood with a gFOBT or iFOBT. All of the procedures are effective for the early detection of CRC and for reducing CRC mortality. The diagnostic sensitivity and specificity of iFOTBs is better than of gFOBTs.

14.4.5.3.1 Endoscopy

According to a study /8/ the hazard ratio for CRC in patients who underwent endoscopy, in comparison with patients who did not undergo the procedure, was 0.57 after polypectomy, 0.60 after negative sigmoidoscopy, and 0.44 after negative coloscopy. Multivariate hazard ratios for death from CRC were 0.59 after screening sigmoidoscopy and 0.32 after screening coloscopy.

14.4.5.3.2 FOBT

It is assumed that colorectal loss of 2–3 mL of blood, representing 0.3 mg Hb/g stool, is the lower limit of blood loss that is associated with pathological events in the colon (CRC or advanced adenoma).

The gFOBT with a detection limit of 0.3–0.6 mg Hb/g stool have, in dependence of the number of stool samples tested, a diagnostic sensitivity for CRC and advanced adenoma of 33% and 8.6%, respectively.
The iFOBT with a detection limit of 2–17 μg/Hb/g stool have a diagnostic sensitivity for CRC and advanced adenoma of 73.3% and 25.7%, respectively
The accuracy of FOBTs to detect CRC and advanced adenoma is shown in Tab. 14.4-1 – FOBT for the diagnosis of colorectal cancer and advanced adenoma detection of CRC and of adenoma.

In a study /4/ comparative evaluations of diagnostic performance for advanced adenoma were made at preset manufacturers’ thresholds (9 assays, range 2–17 μg Hb/g feces) at a uniform threshold (15 μg Hb/g feces) and at adjusted thresholds yielding defined levels of specificity (99%, 97%, and 93%). Sensitivities and specificities for advanced adenoma varied widely when the preset thresholds were used. Using the uniform threshold and adjusting thresholds to yield a specificity of 99%, 97% or 93% resulted in almost equal sensitivities for detection of advanced adenoma (14.4–18.5%, 21.3–23.6%, and 30.1–32.5% respectively) and almost equal positivity rates (2.8–3.4%, 5.8–6.1%, and 10.1–10.9%, respectively).

14.4.6 Comments and problems

The fecal immunological test (iFOBT) has proven utility for CRC detection in symptomatic patients. However, most patients with increased fecal Hb do not have CRC found at colonoscopy. In a study /17/ an elevated free Hb in iFOBT was independently associated with older age, deprivation, anticoagulants, rectal bleeding, advanced adenoma, non-advanced polyps and inflammatory bowel disease in symptomatic patients. Deprivation was associated with an increased free hemoglobin in the absence of pathology. This must be considered when utilising iFOBT in symptomatic patients.

Biological influence factors

gFOBT: false-positive results may be based upon non-human peroxidase activity, due to nonobservance of dietaries. During the last 3 days prior to the test, the following should be avoided: raw meat, horse radish, broccoli, cauliflower, liver, radish, small radish, bananas, cherries, iron and iodine-containing medicines. A range of medicines such as acetylsalicylic acid, glucocorticoids, nonsteroidal anti-inflammatory drugs, or coumarin derivatives can lead to gastrointestinal bleeding. Vitamin C leads to false-negative results.

iFOBT: with immunological assays it is not necessary to adhere to a special diet because the antibodies of test are directed against human hemoglobin. Because of the heterogeneity of study designs, study populations, pre-analytical sample handling, and positivity thresholds the comparative evaluation of the diagnostic performance of different iFOBT brands is difficult. Thus, five tests were compared, and out of 71 stool samples, 31 (43.7%) showed a significant difference /9/. Furthermore, the authors recommend that in in-patients, CRC screening with FITs should not be performed, due to the likelihood of numerous false positive results because of the high sensitivity of the tests.

There is little evidence on replicate and repeat iFOBTs because significant gaps surrounding application exist /18/.

Stability

gFOBT: stool samples can be stored at room temperature for 2 days. Freezing of the sample at –20 °C can prevent hemolytic degradation of hemoglobin.

iFOBT: stability of hemoglobin in the buffer containing sample tube at 2–8 °C for 2 weeks and at 15–30 °C for 1 week.

References

1. Richtlinien des Bundesausschusses der Ärzte und Krankenkassen über die Früherkennung von Krebserkrankungen 2017

2. U.S. Preventive Services Task Force. Screening for colorectal cancer. JAMA 2016; 315: 2564–575.

3. Loitsch SM, Shastri Y, Stein J. Stool test for colorectal cancer screening – it’s time to move. Clin Lab 2008; 54: 473–84.

4. Gies A, Cuk K, Schrotz-King P, Brenner H. Dirct comparison of diagnostic performance of 9 quatitative fecal immunochemical tests for colorectal cancer screening. Gastroenterology 2018; 154: 93–104.

5. Lieberman DA. Screening for colorectal cancer. N Engl J Med 2009; 361: 1179–87.

6. Starkey BJ. Screening for colorectal cancer. Ann Clin Biochem 2002; 39: 351–65.

7. Levin B, Lieberman DA, McFarland B, Andrews KS, Brooks D, Bond J, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American cancer society, the US multi-society task force on colorectal cancer, and the American college of radiology. Gastroenterology 2008; 134: 1570–95.

8. Nishihara R, Wu K, Lochhead P, Morikawa T, Liao X Quian ZR, et al. Long-term colorectal-cancer incidence and mortality after lower endoscopy. N Engl J Med 2013; 369: 1095–1105.

9. Tannous B, Lee-Lewandowski E, Sharples C, Brugge W, Bigatello L, Thompson T, et al. Comparison of conventional guaiac to four immunochemical methods for fecal occult blood testing: implications for clinical practice in hospital outpatient settings. Clin Chim Acta 2009; 400: 120–2.

10. Hardcastle JD, Chamberlain JO, Robinson MHE, Moss SM, Amar SS, Balfour TW, et al. Randomized controlled trial of faecal occult blood screening for colorectal cancer. Lancet 1996; 348: 1472–7.

11. Kronborg O, Fenger C, Olsen J, Jorgensen OD, Sandergaard O. Randomized study of screening for colorectal cancer with faecal occult blood test. Lancet 1996; 348: 1467–71.

12. Shaukat A, Mongin SJ, Geisser MS, Lederle FA, Bond JH, Mandel JS, et al. Long-term mortality after screening for colorectal cancer. N Engl J Med 2013; 369: 1106–14.

13. Brenner H, Tao S. Superior diagnostic performance of faecal immunochemical tests for haemoglobin in head-to-head comparison with guaiac bases faecal occult blood test among 2235 participants of screening colonoscopy. Eur J Cancer 2013; 49: 3049–54.

14. Jensen CD, Corley DA, Quinn VP, Doubeni CA, Zauber AG, Lee JK, et al..Fecal immunochemical test program performance over 4 rounds of annual screening. Ann Intern Med 2016; 164: 456–63.

15. Ahlquist DA. Molecular detection of colorectal neoplasia. Gastroenterology 2010; 138: 2127–39.

16. Jin S, Ye Q, Hong Y, Dai W, Zhang C, Liu W, et al. A systematic evaluation of stool preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers. Clin Chem Lab Med 2021; 59 (1): 91–9.

17. Johnstone MS, Miller G, Pang G, Burton P, Kourounis G, Winter J, et al. Alternative diagnoses and demographics associated with a raised quantitative fecal immunochemical test in symptomatic patients. Ann Clin Biochem 2022; 37 (2): 457–66.

18. Pin-Vieito N, Puga M, Fernandez-de-Castro D, Cubiella J.Faecal immunochemical test otsise colorectal cancer screening? World J Gastroenterol 2021; 27 (38): 6415–29.

14.5 Gastrointestinal neuroendocrine tumors

14.5.1 Neuroendocrine tumors (NETs)

Neuroendocrine cells share a number of antigens with nerve elements (neuron-specific enolase, chromogranin, synaptophysin). The major function of neuroendocrine cells is to produce, store and release small peptides and biogenic amines.

Traditionally, this classification has tended to exclude pituitary and parathyroid tissue /1/. NETs constitute a heterogeneous group of neoplasms that originate from a common neuroendocrine precursor cell population. The neuroendocrine system includes glands like the pituitary, the parathyroid glands, the neuroendocrine adrenal as well as endocrine islets within the pancreas and the thyroid, and cells dispersed between exocrine cells, such as the endocrine cells of the respiratory and gastrointestinal tracts /1/.

NETs associated with hyper functional syndromes are defined as functioning, whereas NETs exhibiting immune positivity for endocrine markers and/or elevated serum markers but not associated with distinct clinical syndrome are called non-functioning tumors (Tab. 14.5-1 – Tumor markers and distribution of somatostatin receptors in patients with gastroenteropancreatic tumors, chromaffin cell tumors, and medullary thyroid carcinoma). The WHO included histopathologic and functional parameters in a classification.

The following types of NETs have been recognized /2/:

Type 1: well-differentiated endocrine tumor. Most NETs are well-differentiated tumors that are characterized by a solid trabecular or glandular structure; tumor cell monomorphism with absent or low cytologic atypia; and a low mitotic (≤ 2 mitosis/mm²) and proliferative status (≤ 2% Ki-67-positive cells) /3/.
Type 2: well-differentiated neuroendocrine carcinoma (low malignancy). Such tumors are slow-growing but can occasionally exhibit more aggressive behavior (> 2 mitosis/mm² or proliferative index > 2% Ki-67-positive cells). Only in the presence of metastases or invasiveness a tumor is defined as well-differentiated neuroendocrine carcinoma /3/.
Type 3: poorly differentiated neuroendocrine carcinoma (highly malignant). Poorly differentiated NETs are invariably malignant tumors. They are characterized by a predominantly solid structure with abundant necrosis; cellular atypia with a high mitotic index (> 10 mitosis/mm²), a proliferative status > 15% Ki-67-positive cells; diffuse reactivity for cytosolic markers or neurosecretory products /3/.
Type 4: mixed exocrine-endocrine carcinoma: these carcinoma are epithelial tumors with a predominant exocrine component admixed with an endocrine component comprising at least on third of the entire tumor cell population. Their biologic behavior is essentially dictated by the exocrine component, which may be of acinar or ductal type /3/.

The majority of NET-predisposing diseases is related to tumor suppressor genes; exceptions are MEN II and the inherited form of medullary thyroid carcinoma, which are based on dominant activation of the RET protooncogene. The latter encodes a transmembrane tyrosine kinase receptor, that causes cellular proliferation, differentiation, and increased cell motility /1/.

14.5.1.1 Gastropancreatic neuroendocrine tumors (GEP-NETs)

Tumors arising from gut endocrine cells have been classified as GEP-NETs. These tumors are usually divided between carcinoids and endocrine tumors of the pancreas. The tumors are generally clinically differentiated between those producing hormonal or hormone-related symptoms/syndromes and non-functioning tumors (not presenting with any hormonal symptoms) /4/. Most GEP-NETs are well-differentiated, with a solid or glandular structure. There is tumor cell monomorphism without, or with only a few, atypical cells. Such tumors generally grow slowly, but they maintain their multi potent development potential. In this way, they can express a number of metabolically active substances and cell membrane receptors such as somatostatin receptors /1/. A small proportion of the GEP-NETs manifests invasive and metastatic growth.

GEP-NETs occur sporadically or in a familial context of autosomal dominant inherited syndromes such as multiple endocrine neoplasia (MEN). Four MEN syndromes (MEN I, MEN II, von Hippel-Lindau (VHL) disease, and the Carney complex) are the most common inherited NETs /1/. MENs are characterized by high penetration in many neuroendocrine tissues.

14.5.1.2 Pancreatic neuroendocrine tumors (PETs)

Functioning islet cell tumors are traditionally named according to the predominant hormone(s) that are released. A distinction is made between three groups of tumors /5/:

Small tumors, which synthesize relatively large amounts of active peptide, such as insulinomas, gastrinomas and VIPomas
Tumors with relatively few symptoms, or with late symptoms that first appear when the tumor becomes large (e.g., glucagonomas)
Tumors that are first noticed when, due to their size, they lead to a functional impairment of other organs.

PETs often synthesize more than one single peptide and these often in different molecular forms. With the exception of insulinomas, the majority of the tumors are malignant or can degenerate to form malignancies.

14.5.1.3 Gut neuroendocrine tumors

Endocrine tumors of the gut comprise about two thirds of the gastroenteropancreatic endocrine tract. They most commonly occur in the midgut; one third of this group arise in the appendix /5/. Small intestinal neuroendocrine tumors (SINETs) make up 21% of the GEP-NETs, and 38% of the active endocrine small intestinal tumors. SINETs are derived mainly from enterochromaffin cells, and they produce multiple hormonally active substances, such as serotonin, bradykinin and tachykinin. They are responsible for the carcinoid syndrome, and are considered to be an aggressive type of cancer, since their 5-year survival rate is only 56–79% /6/.

14.5.1.4 Markers of neuroendocrine tumors

Hormonally active tumor cells manifest characteristics that are similar to those of neuroendocrine cells /1/. There are a few general markers that can be determined for the diagnosis of hormonally active tumors, and specific markers for certain GEP-NET tumors and chromaffin cell tumors (Tab. 14.5-1 – Tumor markers and distribution of somatostatin receptors in patients with gastroenteropancreatic tumors, chromaffin cell tumors, and medullary thyroid carcinoma).

Specific tumor markers

Peptide hormones which are processed in a sequence- and tissue-specific manner yield biologically active peptides, however their fine-tuning is usually deficient in NET cells /1/. Measurement of the peptide hormones or their precursors establishes the tumor diagnosis. Specific markers are insulin from an insulin-producing tumor, gastrin from a gastrinoma, and glucagon from glucagonoma /7/.

Nonspecific tumor markers

Nonspecific tumor markers are general tumor markers, the most interesting in the biochemical diagnosis of NETs are chromogranin A, neuron-specific enolase (NSE), the subunits of human chorionic gonadotrophin (hCG) and pancreatic polypeptide /1, 7/.

Chromogranins: this is a group of acidic monomeric soluble proteins that are localized within secretory granules in which they are co stored and co secreted with the locally present peptides. Chromogranin A (CgA) is the granin mostly determined in clinical practice (Fig. 14.5-1 – Chromogranin A (CgA) as a marker of NETs). Plasma CgA may be elevated in a variety of NETs, including pheochromocytomas, paragangliomas, carcinoid and pancreatic islet cell tumors, medullary thyroid carcinoma, parathyroid and pituitary adenomas.

NSE: the hormone is distributed diffusely in the cytoplasm of neuroendocrine cells. NSE is only present in neurons and NE cells and can serve as a circulating marker for NETs. Most frequently NSE is elevated in patients with small cell lung cancer (74%), but is also detected in 30–50% of patients with carcinoid, medullary thyroid carcinoma, islet cell tumors and pheochromocytoma. See Section 28.18 – Neuron specific enolase (NSE, γ-enolase).

hCG: the subunits of hCG are markers of non-functioning GEP-NETs, well as of medullary thyroid carcinoma and small-cell lung cancer. About 30% of patients with GEP-NETs have high concentrations of hCG subunits.

It must be taken into consideration that in active neuroendocrine tumor cells the fine tuning of a peptide can be disturbed, so that with immunoassay methods, due to a lack of antibody specificity, the recognition may be only partial, or even absent.

The principal GEP-NETs and their diagnostic procedures are listed in Tab. 14.5-2 – Neuroendocrine tumors (NETs) of the gastroenteropancreatic system.

14.5.1.5 Imaging of tumors with radio nucleotides

The presence of amine uptake mechanisms and a high density of peptide receptors on several NETs can be used for the diagnosis and monitoring these tumors using radioactive techniques /1/. Radioactively labeled amines or peptide analogs are used for the imaging of NETs, based upon their capability of binding to NET ligands. The following is used: ¹²³I-metaiodobenzylguanidine, which is well suited to the imaging of tumors containing chromaffin tissue such as pheochromocytoma, paraganglioma, carcinoid, and medullary thyroid cancer. A further substance is ¹¹¹In-octreotide (Tab. 14.5-1 – Tumor markers and distribution of somatostatin receptors in patients with gastroenteropancreatic tumors, chromaffin cell tumors, and medullary thyroid carcinoma). Octreotide is a cyclic octapeptide, a somatostatin analog that binds to somatostatin receptors. Via its neuroendocrine cell receptors, somatostatin exerts inhibitory actions on neurotransmission, intestinal motility, fluid and nutrient absorption, vascular contraction and cell proliferation.

References

1. Kaltsas GA, Besser GM, Grossman AB. The diagnosis and medical management of advanced neuroendocrine tumors. Endocrine Reviews 2004; 25: 458–511.

2. Solcia E, Kloppel G, Sobin LH. Histological typing of endocrine tumours. 2nd ed. Heidelberg; World Health Organization: 2000.

3. Simon P, Spilcke-Liss, Wallaschofski H. Endocrine tumors of the pancreas. Endocrinol Metab Clin N Am 2006; 35: 431–47.

4. De Herder WW, Lamberts SWJ. Gut endocrine tumours. Best Practice & Research Clinical Endocrinology and Metabolism 2004; 18: 477–95.

5. Ardill JES. Circulating markers for endocrine tumours of the gastroenteropancreatic tract. Ann Clin Biochem 2008; 45: 539–59.

6. Bergestuen DS, Aabakken L, Holm K, Vatn M, Thiis-Evensen E. Small intestinal neuroendocrine tumors: prognostic factors and survival. Scand J Gastroenterol 2009; 44: 1084–91.

7. Öberg K, Eriksson B. Endocrine tumors of the pancreas. Best Practice & Research Clinical Endocrinology and Metabolism 2005; 19: 753–81.

8. Delle Fave G, Capurso G, Milione M, Panzuto F. Endocrine tumours of the stomach. Best Practice & Research Clinical Endocrinology and Metabolism 2005; 19: 659–73.

9. Hoffmann KM, Furukawa M. Duodenal neuroendocrine tumours: classification, functional syndromes, diagnosis and medical treatment. Best Practice & Research Clinical Endocrinology and Metabolism 2005; 19: 675–97.

10. Halfdanarson TR, Rubin J, Farnell MB, Grant CS, Petersen GM. Pancreatic endocrine neoplasms: epidemiology and prognosis of pancreatic endocrine tumors. Endocrine-Related Cancer 2008; 15: 409–27.

11. Panzuto F, Severi C, Cannizzaro R, et al. Utility of combined use of plasma levels of chromogranin A and pancreatic polypeptide in the diagnosis of gastrointestinal and pancreatic endocrine tumors. J Endocrinol Invest 2004; 27: 6–11.

14.5.2 Chromogranin A (CgA)

The CgA protein, a member of the granin family, is an acidic 439 amino acid protein, with a molecular weight of about 70 kDa. All granins are neuroendocrine (NE) cell secretory granule proteins, and are released in a physiological regulatory manner from the NE cells. Granins are localized within secretory granules in which they are co stored and co secreted with the locally present peptides in secretory granulogenesis. Granins are important in the selection of secretory proteins, their maturing and condensation in the granules. CgA is an important tumor marker, because most NE tumors, including silent tumors without secretion of known hormones, express and release CgA.

14.5.2.1 Indication

In patients with irritable bowel symptoms (IBS)
Suspicion of gastropancreatic neuroendocrine tumors (NETs) and monitoring their course
Suspected pheochromocytoma.

14.5.2.2 Method of determination

CgA immunoreactivity mainly consists of high-molecular weight forms and is subject to post-translational processing, which often is specific for the individual tumor. Therefore CgA is found in plasma both as the intact molecule and in the fragmented form. Antibodies of immunoassays for the determination of CgA bind the intact molecule and the fragments with different avidity; the results of commercially available immunoassays are, therefore, not comparable.

The three commercially available methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and immune radio metric assay (IRMA). ELISA CgA is measured with a double antibody sandwich assay which utilizes rabbit antibodies to a 23 kDa C-terminal fragment of CgA /1/ or using monoclonal antibodies against the central domain 145–245 of the molecules /3/. Competitive radio immunoassays use a polyclonal antibody against the 116-439 amino acid sequence of CgA /2/. Intact CgA as well as fragments are bound. IRMA use monoclonal antibodies /3/. They detect the central 125–245 domain of the CgA molecule.

14.5.2.3 Specimen

Serum, EDTA-plasma, heparin-plasma: 1 mL

Manufacturer’s instruction should be followed.

14.5.2.4 Reference interval

EDTA plasma: CgA ELISA (DAKO) /1/ 2.0–41.7 U/L
Heparin plasma: CgA RIA (EuroDiagnostica) /2/ ≤ 4.0 nmol/L
Serum: CgA ELISA (CIS) /3/ 27–94 μg/L
Serum: CgA IRMA (CIS) /3/ 19–98 μg/L
EDTA plasma: CgA IRMA (CIS) /3/ 20–150 μg/L

14.5.2.5 Clinical significance

CgA has been demonstrated to be the most promising marker for the diagnosis of neuroendocrine tumors (NETs).

14.5.2.5.1 CgA in the diagnosis of neuroendokrine tumors

Neuroendocrine tumors, which originate from neuroendocrine cells, are widely distributed in the body. In most patients, the primary tumor is found in the midgut (i.e., jejunum, ileum, appendix, and right-sided colon; 60% to 80%). NETs are a form of cancer that differs from other neoplasia in that they synthesize, store, an release peptides and amines. The incidence of NETs is approximately 5 cases per 100,000 people or 1 case per 1,000 malignancies. NETs are broadly classified into two categories termed functional NETs or non-functional NETs according to whether these tumors give rise to a clinical symptom which are often nonspecific.

The natural history of NETs is attended to a long history of vague abdominal symptoms. The patients complaints are often classified as irritable bowel syndrome. The symptoms persist with a median latency of 9.2 years, by which time the tumor has metastasized, causing symptoms like flushing, diarrhea /4/. A critical issue is that NETs are identified when 60–80% of the tumors are metastasized, and a NET is not considered until indicated by a pathological CgA value. A further diagnostic difficulty is the fact that over 90% of NETs are nonfunctional, that is, they do not secrete hormones that are associated with a clinical syndrome. Even so, a large proportion of these tumors is recognized with the CgA determination /5/.

CgA is a sensitive but nonspecific NET marker. Elevated concentrations are, in principle, associated with tumors such as gastropancreatic NETs (GEP-NETs), bronchopulmonary NETs, pheochromocytoma, neuroblastoma and medullary thyroid cancer /6/. Refer to:

Fig. 14.5-1 – Utility of chromogranin A as a marker of NETs
Tab. 14.5-3 – Non gastrointestinal NET disorders with elevated CgA levels and sensitivity of detection
Tab. 14.5-4 – Diagnostic accuracy of Cg A in comparison with other tumor markers in the detection of gastroenteropancreatic endocrine tumors
Tab. 14.5-5 – CgA in neuroendocrine tumors and other diseases.

A systematic review and meta-analysis points to the diagnostic value of CgA to discriminate NETs from controls /17/:

Diagnostic sensitivity of 73% (71–76%) with a diagnostic specificity of 95% (93–96%)
Odds ratio 56.3 (25.3–125.4)
Positive likely hood ratio 14.6 (6.6–32)
Negative likely hood ratio 0.26 (0.18–0.38).

Hyper gastrinemia and renal insufficiency are the most frequent disorders that lead to increased CgA levels in the absence of NETs.

14.5.2.5.2 CgA and tumor burden

CgA is a valuable marker for the assessment of the tumor burden of NETs. Independent of tumor size, the highest values are found in the carcinoid syndrome (small bowel NETs) /6/. CgA levels are higher with extensive metastases than with localized tumors or limited hepatic involvement. CgA does not only reflect tumor load but correlates with tumor progression. In 238 NET patients, in midgut NETs with multiple liver metastases, CgA concentrations were higher than in patients with only a few liver metastases or with only lymph node metastases /7/. In nonfunctional GEP-NETs with liver metastases, CgA levels did not reflect the tumor burden but rather, tumor progression and response to therapy /8, 9/.

14.5.2.5.3 CgA and tumor therapy

In the assessment of medical therapy efficacy in patients with NET, a reduction of CgA level > 50% should be considered significant /6/. Under therapy with long-acting octreotide, there is an association between the decline in CgA and survival time /6/. CgA reduction of > 80% after cytoreductive surgery of hepatic metastases is predictive of subsequent symptom relief and disease control, and is associated with improved outcome, even after incomplete cytoreduction /6/.

14.5.2.6 Comments and problems

Method of determination

The CgA molecule undergoes post translational processing and results in a series of smaller biologically active peptides such as /6/:

Pancreastatin (corresponding to CgA residues 250–301)
Catestatin (corresponding to CgA residues 352–372)
Vasostatin I and II (corresponding to CgA residues 1–76 and 1–113, respectively).

Since these fragments have different avidities to the CgA antibodies of immunoassays, and also because manufacturers cannot not relate the calibration of the kits to a reference preparation, results obtained with the various assays are very different. Thus, in NET patients, a comparison of kits showed the following diagnostic sensitivities and specificities /14/:

DAKO: diagnostic sensitivity 85%, diagnostic specificity 88%
CIS: diagnostic sensitivity 67%, diagnostic specificity 96%
Euro Diagnostica: diagnostic sensitivity 93%,diagnostic specificity 88%.

Blood sampling

In the morning in fasting patients. Food intake increases CgA concentrations. Thus, 30–60 minutes following food intake increases of 16% and as much as 20–31% were recorded in healthy controls and in MEN I patients, respectively /6/.

Attention must be paid to whether CgA is determined in serum or plasma, because plasma values can be 20–70% higher /3/.

The day to day variation in of CgA in NET patients and healthy controls is approximately 25% /6/.

Stability

Up to 48 hours at room temperature (20 °C) /15/.

14.5.2.7 Pathophysiology

CgA is a member of the granin family (chromogranin or secretogranin), and is present in the granules of many secretory glands. The peptide is composed of 439 amino acids, with an upstream 18-amino acid signal peptide. CgA contains monobasic and dibasic amino acids, which represent targets for proteolytic degradation. CgA mRNA and protein are expressed in all types of neurons, which reflects the extent of dense-core granule formation in various cell types throughout the diffuse NE system. In comparison to the endocrine glands, which comprise localized aggregations of NE cells with well defined functions (adrenal, parathyroid, pituitary), the diffuse NE system is a syncytium integrated throughout the bronchopulmonary and gastrointestinal system /6/.

CgA is the precursor peptide of many biologically active peptides. Included among these are:

Pancreastatin (inhibitor of glucose-induced insulin secretion)
Parastatin, a peptide released by the parathyroid glands (inhibits low Ca²⁺-induced parathyroid hormone secretion)
Catestatin (suppresses catecholamine secretion)
Vasostatin (anti-adrenergic actions).

CgA proteolysis is tissue specific, so that pancreastatin is formed in the α cells of the pancreas, and chromostatin in the β-cells.

The biological functions of GgA are: reduction in the number and size of chromaffin granules; increase in blood pressure; loss of diurnal blood pressure variation; increase in left ventricular mass; and decrease in adrenal catecholamine and neuropeptide Y concentrations /16/.

References

1. Pirker RA, Pont J, Pöhnl R, Schütz W, Griesmacher A, Müller MM. Usefullness of chromogranin A as a marker for detection of relapses of carcinoid tumours. Clin Chem Lab Med 1998; 36: 837–40.

2. Stridsberg M, Öberg K, Li Q, Engström U, Lundquist G. Measurements of chromogranin A, chromogranin B, chromogranin C and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours. J Endocrinol 1995; 144: 49–59.

3. Glinicki P, Kapuscinska R, Jeske W. The differences in chromogranin A (CgA) concentrations measured in serum and in plasma by IRMA and ELISA methods. Polish J Endocrinol 2010; 61: 346–50.

4. Vinik AI, Silva MP, Woltering G, Go VLW, Warner R, Caplin M. Biochemical testing for neuroendocrine tumors. Pancreas 2009; 38: 876–89.

5. Seregni E, Ferrarri L, Bajetta E, Martinetti A, Bombardieri E. Clinical significance of blood chromogranin A measurement in neuroendocrine tumours. Ann Oncol 2001; 12, suppl 2: 69–72.

6. Modlin IM, Gustafsson BI, Moss SF, Pavel M, Tsolakis AV, Kidd M. Chromogranin A – biological function and clinical utility in neuro endocrine tumor disease. Ann Surg Oncol 2010; 17: 2427–43.

7. Campana D, Nori F, Piscitelli L, et al. Chromogranin A: is it a useful marker of neuroendocrine tumors? J Clin Oncol 2007; 25: 25: 1967–73.

8. Nikou GC, Marinou K, Thomakos P, et al. Chromogranin levels in diagnosis, treatment and follow-up of 42 patients with non-functioning pancreatic endocrine tumours. Pancreatology 2008; 8: 510–9.

9. Kaltsas GA, Besser GM, Grossman AB. The diagnosis and medical management of advanced neuroendocrine tumors. Endocrine Reviews 2004; 25: 458–511.

10. Panzuto F, Severi C, Cannizzaro R, et al. Utility of combined use of plasma levels of chromogranin A and pancreatic polypeptide in the diagnosis of gastrointestinal and pancreatic endocrine tumors. J Endocrinol Invest 2004; 27: 6–11.

11. Ceconi C, Ferrari R, Bacchetti T, et al. Chromogranine in heart failure.; a novel neurohumeral factor and a predictor for mortality. Eur Heart J 2002; 23: 967–74.

12. Korse CM, Taal BG, de Groot C, Bakker RH, Bonfrer JMG. Chromogranin-A and N-terminal pro-brain natriuretic peptide: an excellent pair of biomarkers for diagnostics in patients with neuroendocrine tumor. J Clin Oncol 2009; 26: 4293–9.

13. Zhang D, Lavaux T, Voegeli AC, Lavine T, Castelain V, Meyer N, et al. Prognostic value of chromogranin A at admission in critically ill patients: a cohort study in a medical intensive care unit. Clin Chem 2008; 54: 1497–1503.

14. Stridsberg M, Eriksson B, Öberg K, Janson ET. A comparison between three commercial kits for chromogranin A measurements. J Endocrinol 2003; 177: 337–41.

15. Bender H, Maier A, Wiedenmann B, O’Connor DT, Messner K, Schmidt-Gayk H. Immunoluminometric assay of chromogranin A in serum with commercially available reagents. Clin Chem 1992; 38: 2267–72.

16. Bilek R, Safarik L, Ciprova V, Vlcek P, Lisa L. Chromogranin A, a member of neuroendocrine secretory proteins as a selective marker for laboratory diagnosis of pheochromocytoma. Physiol Res 2008; 57, suppl 1, S 171–S179.

17. Yang X, Yang Y, Li Z, Cheng C, Yang T, Wang C, Liu L, Liu S. Diagnostic value of circulating chromogranin A for neuroendocrine tumors: a systematic review and meta-analysis. Plos One 2015. doi: 10.1371/journal.pone.0124884.

14.5.3 Gastrin

Gastrin is released by the G cells of the antrum of the stomach and assists the stimulation of gastric acid secretion. Gastrin and cholecystokinin are the only members of the gastrin family. Both molecules share a common COOH-terminal pentapeptide amide that also includes the sequences essential for biological activity. The gastrin precursor, preprogastrin, is generated in the endoplasmic reticulum, where the N-terminal signal sequence is removed to yield progastrin. In the trans-Golgi network of antral G-cells progastrin is sequestered into storage vesicles of the regulated pathway of exocytosis /1/. Following carboxyendopeptidase cleavage progastrin G34-Gly is generated which may be converted either to G17-Gly or to G34 amide (Fig. 14.5-2 – Structural relationship between different forms of gastrin).

The regulation of gastric acid secretion is mediated by gastrins with a COOH-terminal amide (i.e., G17 and G34). In the plasma of healthy individuals, gastrin-17 (MW 2,098 Da) and gastrin-34 (MW 3,839 Da or 3,919 Da) are the predominant forms /2/. The longer chain gastrin peptides dominate in conditions of elevated gastrin secretion. This is particularly the case with gastrinomas.

In gastrinoma cells, progastrin is less completely processed than in normal G cells, and in consequence progastrin, progastrin intermediates and longer chain forms of gastrin (gastrin-71, -52, and -34) are released into the circulation in increased quantities /3/. Each gastrinoma secretes an individual pattern of gastrins. The plasma half life of gastrin-17 is 4 minutes, while that of gastrin-34 is 40 minutes. Therefore the biological action of gastrin-34 is longer lasting.

14.5.3.1 Indication

Conditions /4/:

Refractory or recurrent peptic ulcer disease
Peptic ulcer disease in unusual locations (e.g., beyond the duodenal bulb)
Peptic ulcer disease with concurrent endocrinopathies
Gastrointestinal reflux disease
Refractory to proton pump inhibitors and/or with distal esophageal stricture
Presence of prominent rugal folds seen on upper endoscopy
Chronic secretory diarrhea
Gastric carcinoids

14.5.3.2 Method of determination

Radioimmunoassay (RIA), enzyme immunoassay /3/.

The use of antibodies against truncated gastrin 2–17 with a free N-terminal NH₂ group is optimal. They are directed against the C-terminal and N-terminal parts of the gastrins. To produce such antibodies, truncated gastrin 2–17, or more commonly these days synthetic peptides conjugated to carrier protein provide the basis for satisfactory immunoassays. Antibodies created in this manner specifically target the C-terminal epitope of gastrin-17, but also the N-terminal epitope of other gastrins.

14.5.3.2.1 Secretin test

The secretin test is a provocation test and a complementary procedure in the differential diagnosis of tumor dependent hyper gastrinemia from other forms of hyper gastrinemia (Tab. 14.5-6 – The secretin test).

14.5.3.2.2 Calcium infusion test

The intravenous calcium infusion test is a complementary test in differential diagnosis for gastrinoma (Tab. 14.5-7 – The calcium infusion test).

14.5.3.3 Specimen

Serum, heparin or EDTA-plasma: 1 mL

Sampling in the fasting state or within the framework of function tests.

14.5.3.4 Reference interval

Gastrin: below 104 ng/L (50 pmol/L) /1/

Conversion: ng/L × 0.48 = pmol/L.

14.5.3.5 Clinical significance

The regulation of gastric acid secretion is mediated by gastrin. Gastrin stimulates acid secretion following food intake via the induction of histamine release, which again activates gastrin secretion. If the pH of the gastric juice falls, gastrin secretion is inhibited via a negative feedback mechanism. If the pH of the gastric juice increases to above 4, gastrin is secreted. Under pathological or pharmacological conditions with reduced acid secretion, the formation of gastrin is continuously stimulated.

Following the ingestion of food, a 2–3-fold rise in gastrin is considered to be physiological. Chronic elevation of plasma gastrin leads to hyperplasia of the histamine-producing enterochromaffin-like (ECL) cells of the gastric corpus. Hyper gastrinemia is defined by a gastrin level greater than 100–150 ng/L. There are different categories of hyper gastrinemia /2/: appropriate gastrin secretion occurs with neutral pH, while unopposed gastrin secretion in the presence of an already acid pH is an inappropriate response as in gastrin-producing tumors or gastrinomas

Hyper gastrinemia with acid hypersecretion. This is the case in Zollinger-Ellison syndrome, H. pylori infection and gastrinoma
Hyper gastrinemia without hypersecretion. Because gastrin secretion maintains gastric pH below a value of 4, there is increased circulating gastrin in patients with reduced or absent acid secretion. This is the case in chronic atrophic gastritis, in some patients on long-term proton pump inhibitors, and in the Ménétrier syndrome. In this case the pH is increased due to exudation of interstitial fluid into the stomach.

Elevated gastrin values may have clinical consequences depending upon their duration /6/:

Short-term effects: the relatively early tolerance in patients treated with H₂ blockers possibly reflects induction of histidine decarboxylase activity by gastrin
Middle-term effects (> 1 day, < 3 months) are based upon stimulation by gastrin of ECL cells. The ECL cell hyperplasia leads to augmented histamine release, which explains tolerance to H₂ blockers
Long-term (over 3 months) effects: every condition with long-term hyper gastrinemia results in ECL cell hyperplasia and predisposes to tumor growth.

The diagram in Fig. 14.5-3 – Differential diagnosis of hyper gastrinemia indicates a diagnostic recommendation for the differentiation of hyper gastrinemia. Diseases with elevated gastrin values are listed in Tab. 14.5-8 – Diseases associated with pathological gastrin levels.

14.5.3.6 Comments and problems

Blood sampling

In the morning following overnight fasting. The sample should be centrifuged within 30 minutes of blood collection, and the serum or plasma is to be frozen /1/.

Method of determination

Out of 12 commercial gastrin kits (7 RIA, 5 ELISA) that were compared with a reference kit, only 4 test kits recorded gastrin concentrations of up to 1247 ng/L (600 pmol/L) in a patient with Zollinger-Ellison syndrome with an accuracy of 90–100%. The others generally determined lower values, because some gastrin forms were not detected /1/. An important reason for this is that these test kits use antibodies that target only a short sequence of the C-terminal portion of gastrin-17. However, some of the gastrin molecules are detected only if the antibodies are directed against both the N-terminal and the C-terminal sequences of gastrin-12.

Most of the tests show a cross-reaction with cholecystokinin; this does not interfere, because the plasma concentration of cholecystokinin is 10–20-fold lower than that of gastrin.

Hemolysis interferes with the gastrin determination.

Reference interval

There are, currently, many gastrin immunoassays without uniform specificity; in consequence, the reference intervals in the various laboratories are different, but the upper reference interval is usually around 40–200 ng/L (20–100 pmol/L).

Drugs

Proton pump inhibitors (PPI) and H₂ blockers must be discontinued at least 1 week prior to blood sampling. Patients with PPI/H₂ therapy were found to have an elevated mean fasting plasma gastrin concentration 22-fold greater than the mean fasting gastrin concentration in the control patients without PPI/H₂ therapy /7/.

Stability

At 4 °C, gastrin loses up to 50% of its activity within 48 hours. Storage for several days at –20 °C, for longer periods of time at –70 °C.

References

1. Dockray GJ. Gastrin. Best Practice & Research Clin Endocrinol Metab 2004; 18: 555–68.

2. Varro A, Ardill JS. Gastrin: an analytical review. Ann Clin Biochem 2003; 40: 472–80.

3. Rehfeld JF, Bardram L, Hilsted L, Poitras P, Goetze JP. Pitfalls in diagnostic gastrin measurements. Clin Chem 2012; 58: 831–6.

4. Dacha S, Razvi M, Massaad J, Cai Q, Wehbi M. Hypergastrinemia. Gastroenterology Report 2015. doi: 10.1093/gastro/gov004.

5. Wada M, Komoto I, Doi R, et al. Intravenous calcium injection test is a novel complementary procedure in differential diagnosis for gastrinoma. World J Surgery 2002; 26: 1291–6.

6. Waldum HL, Fossmark R, Bakke I, Martinsen TC, Qvigstad G. Hypergastrinemia in animals and man: causes and consquences. Scand J Gastroenterol 2004; 6: 505–9.

7. Dhillo WS, Jayasena CN, Lewis CJ, Martin NN, Tang KCN, Meeran K, et al. Plasma gastrin measurement cannot be used to diagnose a gastrinoma in patients on either proton pump inhibitors or histamine type-2 receptor antagonists. Ann Clin Biochem 2006; 43: 153–5.

8. Kaltsas GA, Besser GM, Grossman AB. The diagnosis and medical management of advanced neuroendocrine tumors. Endocrine Reviews 2004; 25: 458–511.

9. Dacha S, Razvi M, Massaad J, Cai Q, Wehbi M. Hypergastrinemia. Gastroenterology Report 2015; 2015: 1–8. doi: 10.1093/gastro/gov004.

10. McColl KEL, Fullarton GM, Chittajalu R, El Nujumi AM, MacDonald AMI, Dahill SW, et al. Plasma gastrin, day-time intragastric pH, and nocturnal acid output before and at 1 and 7 months after eradication of helicobacter pylori in duodenal ulcer subjects. Scand J Gastroenterol 1991; 26: 339–46.

11. Koop H, Eissele R: Gastrale Säurereduktion: Pathophysiologische und klinisch relevante Folgen. Z Gastroenterol 1991; 29: 613–7.

12. Meuwissen SG, Craanen ME, Kuipers EJ. Gastric mucosal morphological consequences of acid suppression: a balanced review. Best Pract Res Clin Gastroenterol 2001; 15: 497–510.

13. Ciccotosto GD, Dawborn JK, Hardy KJ, Shulkes A. Gastrin processing and secretion in patients with endstage renal failure. J clin Endocrinol Metab 1996; 81: 3231–8.

14.5.4 Serotonin, 5-hydroxy indole acetic acid

Serotonin (5-hydroxytryptamine) is an important central nervous system neurotransmitter and neuromodulator. Under normal circumstances, most of the available tryptophan is incorporated into proteins, but 1–3% of the ingested tryptophan is utilized for the synthesis of serotonin (Fig. 14.5-4 – Synthesis and oxidative deamination of serotonin). About 80% of total body serotonin is synthesized in the enterochromaffin-like (ECL) cells of the gastrointestinal tract. The synthesis rate of serotonin depends both on the activity of tryptophan hydroxylase and on the availability of tryptophan in the diet. In the circulation, a substantial portion of serotonin is found in the thrombocytes. The metabolism of serotonin occurs via oxidative deamination by the enzyme monoamine oxidase and further oxidation by the enzyme aldehyde dehydrogenase. Both enzymes are present in the liver, lung and kidney. Oxidation leads to the formation of 5 hydroxy indole acetic acid (5-HIAA), the most quantitatively important metabolite. 5-HIAA is excreted in its free form directly in the urine /1/.

Carcinoids are neuroendocrine tumors derived from ECL cells which produce serotonin as a paracrine hormone. The majority of carcinoids arise in the small intestine and appendix. Markers such as chromogranin A and neuropeptides possess a high sensitivity for neuroendocrine tumors. However, these markers are unable to detect an enhanced serotonin metabolism, which is considered a hallmark of carcinoid tumors /2/. Three indole markers are used in carcinoid disease:

5-HIAA in 24 h collected urine
Urinary serotonin
Platelet serotonin content.

14.5.4.1 Indication

Suspicion of carcinoid tumor (e.g., if the following clinical symptoms occur: flushing, abdominal colic and diarrhea, paroxysmal dyspnea, chronic intermittent incomplete ileus, peptic ulcer disease)
In patients with CgA levels that are suggestive of carcinoid syndrome
Monitoring of carcinoid patients during treatment.

14.5.4.2 Method of determination

5-HIAA in urine

Spectrophotometric determination or reversed high-performance liquid chromatography (HPLC) in association with electrochemical detection.

Serotonin

Immunoassay /3/.

Profiling of indole markers

Using online-solid phase extraction and gradient HPLC with fluorometric detection an automated indole-profiling method that enables the simultaneous analysis of tryptophan, serotonin and 5-HIAA was developed /4/. The determination may be done in plasma, urine and thrombocytes. The methods that are employed for the profiling of serotonin and its principle metabolites have replaced spectrophotometric and fluorometric procedures.

14.5.4.3 Specimen

24-h urine collection

Addition of 10 mL of glacial acetic acid, measurement of the urine volume, dispatching of 20 mL to the laboratory.

Platelet-rich plasma

10 mL of EDTA-plasma are centrifuged immediately following blood sampling for 20 min. at 150 × g; in this manner, platelet-rich plasma (PRP) is obtained. The PRP thrombocytes are counted with a hematology analyzer, and the platelets are then sedimented at 2,000 × g for 15 min. Serotonin in the sediment is determined.

14.5.4.4 Reference interval

5-HIAA in the urine /3/: 2–8 mg (10–42 μmol)/24 h
Serotonin in platelet-rich plasma /2, 4/: 2.5–6.1 nmol/10⁹ thrombocytes

Conversion of 5-HIAA: mg/L × 5.23 = μmol/L

14.5.4.5 Clinical significance

Neuroendocrine tumors (NETs), which originate from ECL cells, are widely distributed in the body. In most patients the primary tumor is found in the midgut (i.e., jejunum, ileum, appendix, and right-sided colon; 60–80%) and less frequently in the lung (20%). At the time of diagnosis 20–30% of patients with NET present with the carcinoid syndrome.

Carcinoid tumors are APUDomas (characterized by amine precursor uptake and decarboxylation) that arise from ECL cells. The tumors have common histological, cytochemical and ultrastructural characteristics. The overall incidence of clinically manifest carcinoid tumors is 1–2 cases per 100,000 of the general population.

14.5.4.5.1 Carcinoid syndrome

The carcinoid syndrome is a pattern of symptoms such as abdominal pain, diarrhea, and flushing, caused by an overproduction of vasoactive peptides. Particularly in midgut tumors, serotonin is the prominent peptide.

Frequency: according to a statistical evaluation /5/:

Out of 8876 patients with GEP-NET 748 (8.4%) had carcinoid syndrome
The GEP-NET patients with carcinoid syndrome tended to be older than those without
In 91.7% of the patients with carcinoid syndrome, the serotonin concentrations were high or very high
In 26.6% of patients without carcinoid syndrome serotonin was elevated
In 73.4% of patients with carcinoid syndrome the tumor was associated with liver or lymph node metastases
The 5-year survival rate following resection of the primary tumor was 67.2% in patients with carcinoid syndrome, while it was 88.7% in NET patients who do not have the syndrome.
Carcinoid crisis: this is a medical emergency that may be caused spontaneously, after palpation of the tumor, during induction of anesthesia or surgery, after administration of chemotherapy or after embolization of a hepatic artery. The symptoms are intense flushing, hypotension or a marked alteration in blood pressure, diarrhea, bronchoconstriction, arrhythmia, hyperthermia, confusion.

14.5.4.5.2 Foregut carcinoid

Foregut carcinoid tumors (bronchopulmonary, thymus, stomach, duodenum, pancreas) secrete directly into the circulation and are, therefore, be associated with carcinoid syndrome. Bronchopulmonary carcinoid makes up 20% of carcinoids /6/. Tumors with a diameter of up to 20 mm account for up to half of these. Diarrhea occurs in 80% of the cases, asthmatic episodes in 10%, and right-sided heart disease in 8%. Approximately half of the patients have increased urinary 5-HIAA excretion, and elevated serotonin concentrations in platelet-rich plasma /2, 7/. Carcinoids in MEN-1 patients are mostly of foregut origin. In MEN-1 patients carcinoids often exhibit loss of heterozygosity (LOH) at 11q13, with deletion of the wild-type MEN-1 gene allele, in accordance with the tumor suppressor nature of the MEN-1 gene product /1/.

In foregut carcinoid patients, platelet serotonin has a higher discriminating power compared with urinary 5-HIAA. Carcinoid tumors of the foregut lack decarboxylase activity. Thus, 5-hydroxy tryptophan (5-HTP) enters the circulation, and blood and urinary concentrations of 5-HTP are increased. However, 5-HTP is partially converted by the kidney into serotonin (5-HT) and than taken up by platelets or excreted directly. Only a small fraction of serotonin is metabolized subsequently to 5-HIAA. In patients with foregut tumors, urinary concentrations of 5-HTP and serotonin may be elevated and 5-HIAA only modestly increased /1/.

14.5.4.5.3 Midgut carcinoid

The carcinoids of the ileum, also known as midgut carcinoid tumors, are found mainly in the 60 cm area of the ileocecal valve. The symptoms that they cause are uncharacteristic. The most common initial symptom is abdominal pain. Only some 25% of the patients with ileum carcinoid tumors have classical carcinoid syndrome. Increased 5-HIAA excretion is observed in 87% of the patients /7/, and the diagnostic sensitivity of serotonin in platelet-rich plasma is believed to be increased /2/. In midgut carcinoids, LOH is frequently observed at the SDHD gene (succinate-ubiquinone oxidoreductase subunit D) (distal 11q) and distal 18q.

In midgut carcinoids all three indole markers (5-HIAA, 5-HTP and platelet serotonin) have a high discriminating capacity /2/.

14.5.4.5.4 Hindgut carcinoid

Carcinoid tumors of the colon and the rectum, also termed hindgut tumors, develop frequently in individuals over the age of 70 years, and particularly in the right part of the colon and the cecum. Clinical symptoms arise in tumors with a diameter of greater than 5 cm, and if distant metastases are present /4/. These tumors are secretory only to a limited extent. Serotonin and increased 5-HIAA excretion are found in only 20% of the patients. These patients then have advanced disease with liver metastases /8/.

14.5.4.5.5 Laboratory findings

The secretory activity of carcinoids is related to the primary site of the tumor /2/:

Forgut carcinoids may produce several substances (histamine, catecholamines) besides serotonin and 5-hydroxy tryptophan (5-HTP). These tumors produce less serotonin than midgut carcinoids. Therefore platelet serotonin is increased and 5-HTP and 5-HIAA in urine may be only modestly increased.
Midgut carcinoids produce serotonin predominantly. All markers (5-HIAA and 5-HTP in urine, platelet serotonin, plama serotonin) have a high diagnostic accuracy.
Hindgut carcinoids secrete limited amounts of serotonin, therefore, except for patients with advanced disease, all markers make a minor contribution to the diagnosis.

The test characteristics of indole markers for carcinoid tumors are described in:

Tab. 14.5-9 – Sensitivity and efficiency of biomarkers for the diagnosis of carcinoids at 97% specificity
Tab. 14.5-10 – Test characteristics of indole markers for the diagnosis of carcinoids with application of high-level cut-off values.

14.5.4.6 Comments and problems

Preparing the patient

5-HIAA excretion: elevations are caused by vitamin-rich foods and certain medications. Therefore, the foods listed below may not be eaten 3–4 days prior to, and during, the urine collection /1/:

Bananas, walnuts, tomatoes, pineapple, currants, plums, gooseberries, mirabelle, plums, melon, avocado, eggplant, kiwis.

Serotonin in platelet-rich plasma: the determination is not affected by amine-rich foodstuffs /1/.

Urine collection

If the collection bottle is kept in the refrigerator during the collecting period, acidification of the urine for the determination of 5-HIAA is not necessary. For dispatching by mail, or collection at room temperature, 20 mL of glacial acetic acid must be added to the collection bottle beforehand.

Stability

5-HIAA: up to 2 weeks at 4 °C in acidified urine at pH 4 /1/.

Serotonin in platelet-rich plasma: the EDTA-blood has to be processed promptly. Following centrifugation of the platelet-rich plasma, the deep frozen thrombocyte pellet is stable over a long period of time.

Influence factors

Falsely high values of 5-HIAA are caused by: paracetamol, cumarin, mephenesin, phenobarbital, acetanilide, ephedrine-HCL, methamphetamine, nicotine, phentolamine, caffeine, phenacetin, methocarbamol.

Falsely low values are caused by: aspirin, levodopa, promethazine, isoniazid, methenamine, streptozocin, chlorpromazine.

References

1. Lips CJM, Lentjes EGWM, Höppener JWM. The spectrum of carcinoid tumours and carcinoid syndromes. Ann Clin Biochem 2003; 40: 612–27.

2. Meijer WG, Kema IP, Volmer M, Willemse PHB, de Vries EGE. Discriminating capacity of indole markers in the diagnosis of carcinoid tumors. Clin Chem 2000; 46: 1588–96.

3. Kluge H, Bolle M, Reuter R, Werner S, Zahlten W, Prudlo J. Serotonin in platelets: comparative analyses using new enzyme immunoassay and HPLC test kits and the traditional fluorometric procedure. J Lab Med 1999; 23; 360–4.

4. Kema IP, Meijer WG, Meiborg G, Ooms B, Willemse PHB, de Vries EGE. Profiling of tryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection. Clin Chem 2001; 47: 1811–20.

5. Soga J, Yakuwa Y, Osaka M. Carcinoid syndrome: a statistical evaluation of 748 reported cases. J Exp Clin Res 1999; 18: 133–41.

6. Soga J, Yakuwa Y. Bronchopulmonary carcinoids: an analysis of 1875 reported cases with special reference to a comparison between typical carcinoids and atypical varieties. Ann Thorac Cardiovasc Surg 1999; 5: 211–9.

7. Feldman JM. Urinary serotonin in the diagnosis of carcinoid tumors. Clin Chem 1986; 32: 840–4.

8. Koura AN, Giacco GG, Curley SA, Skibber JM, Feig BW, Ellis LM. Carcinoid tumors of the rectum: effect of size, histopathology, and surgical treatment on metastasis free survival. Cancer 1997; 79: 1294–8.

9. Carling RS, Degg TJ, Allen KR, Bax NDS, Barth JH. Evaluation of whole blood serotonin and plasma and urine 5-hydroxyindole acetic acid in diagnosis of carcinoid disease. Ann Clin Biochem 2002; 39: 577–82.

14.5.5 VIP and PACAP

Vasoactive intestinal polypeptide (VIP) is a neuropeptide with a molecular mass of 3,326 Da. VIP belongs to the glucagon-secretin peptide family due to its structural similarity. As gut-brain peptide, it occurs jointly with the pituitary adenylate cyclase activating polypeptide (PACAP) in the nervous system where both serve as neurotransmitters and neuromodulators. Within the peptidergic gastrointestinal nervous system both play an important role as regulators of gastrointestinal motility /1, 2/.

Under normal conditions, no significant plasma levels are measurable for either of the two peptides. The clinical relevance of VIP lies in its ectopic overproduction in conjunction with endocrine gastrointestinal tumor syndromes among which a syndrome characterized by profuse, secretory diarrhea is the most prominent. PACAP has also been detected in gastrointestinal endocrine tumors and exerts a potent flush-producing effect /1/. It equipotently binds to the VIP-receptor responsible for triggering diarrhea /3/. Its role in the development of flushes in conjunction with the carcinoid syndrome remains to be determined.

14.5.5.1 Indication

Persistent profuse watery diarrhea (stool quantities greater than 1 liter)
Severe hypokalemia and hypochlorhydria.

14.5.5.2 Method of determination

Radioimmunoassay for VIP and PACAP /4, 5/.

14.5.5.3 Specimen

A 10 mL blood sample is collected with 25 U of heparin/mL of blood and 1,000 KIU of aprotinin/mL of blood. This sample is then immediately cooled in ice and centrifuged using a cool centrifuge. The plasma is frozen and forwarded on dry ice.

14.5.5.4 Reference interval /6, 7/

VIP	Below 65 ng/L (20 pmol/L)
PACAP	Below 10–20 pmol/L

VIP conversion: pg/mL × 0.30 = pmol/L

14.5.5.5 Clinical significance

VIP is quite important in the diagnostic investigation of Verner-Morrison syndrome, as well as the WDHA syndrome or VIPoma /8, 9, 10/. VIPomas comprise 2–7% of the GEP-NETs. The annual incidence is 1 in 10 million people /11/. The mean age is 49 (32–75) years. Approximately 70–80% of the cases involve solitary tumors, localized to the pancreas, with a diameter of 1–7 cm. At diagnosis, metastases in the regional lymph nodes, the liver, the kidneys and the stomach are present in 50–60% of the patients. VIPomas are multi focal; 4–9% are associated with MEN I.

VIP suppresses water and electrolyte transport in the ileum, and reverses net absorption to net secretion /11/. Large quantities of K⁺, Cl^– and bicarbonate are lost in the intestine, since the colon is only capable of reabsorbing some 50%. Since, in addition, up to 9 liters of fluid are released into the colon, and not all of this can be reabsorbed, watery diarrhea results. The tumors that secrete VIP autonomously, and which are often malignant are, in most cases, localized to the pancreas. In addition, there are also VIPoma fractions in tumors of the sympathetic trunk (ganglioneuromas, neuroblastomas) and in pheochromocytomas.

14.5.5.5.1 Clinical presentation

Verner-Morrison syndrome is characterized by watery diarrhea and, in 50% of the cases, by achlorhydria or hypochlorhydria (WDHA syndrome). Further signs are general weakness, seizure-like abdominal pain, exsiccosis, and flush-like symptoms /11/.

14.5.5.5.2 Laboratory findings

Findings are: hypokalemia, hypo- or hyperchloremia, metabolic acidosis, hypomagnesemia, hyperglycemia, hyper reninemic hyperaldosteronism, since VIP stimulates the release of renin, hypercalcemia in some of the cases. Elevated plasma VIP levels are the diagnostic findings; apart from their importance in the diagnostic investigation, they are also relevant with regard to the assessment of disease progression under chemotherapy. In untreated cases, VIP is above 65 ng/L (20 pmol/L). Elevated VIP values normalize following surgical tumor resection in non-metastasizing VIPoma’s, and with successful chemotherapy /11/.

14.5.5.5.3 PACAP

PACAP is a neuropeptide, belonging to the secretin-, glucagon- and VIP-peptide family. It induces the synthesis of VIP in tumor cells. In enterocytes, it stimulates ion transport through interaction with the VIP receptor. VIP-producing tumors of diverse origins also secrete PACAP. This is not the case with tumors that do not release VIP /12/.

14.5.5.6 Comments and problems

Blood sampling

In the morning in fasting patients. Plasma proteases quickly degrade VIP and PACAP, therefore blood sampling with heparin and aprotinin, processing (centrifugation) under continuous cooling, immediate freezing, dispatch on dry ice.

Method of determination

Synthetic, highly purified porcine VIP preparations are used for calibration.

Drugs

Only somatostatin and octreotide are known to influence plasma VIP.

References

1. Brand SJ, Schmidt WE. Gastrointestinal hormones. In: Yamada T, Alpers DH, Powell DW, Owyang C, Silverstein FE (eds). Textbook of gastroenterology, 2nd ed. Philadelphia: Lippincott, 1995: 25–71.

2. Katsoulis S, Schwörer H, Clemens A, Creutzfeldt W, Schmidt WE. Novel brain-gut peptide PACAP induces neurogenic contraction of the isolated guinea-pig ileum. Am J Physiol 1993; 265: G295–305.

3. Schmidt WE, Seebeck J, Höcker M, Schwarzhoff R, Schäfer H, Fornefeld H, et al. PACAP and VIP stimulate enzyme secretion in rat pancreatic acini via interaction with VIP/PACAP-2 receptors: additive augmentation of CCK-/carbachol-induced enzyme release. Pancreas 1993; 8: 476–87.

4. Fahrenkrug J, Schaffalitzky de Muckadell OB. Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. J Lab Clin Med 1977; 89: 1379–88.

5. Bloom SR. Vasoactive intestinal polypeptide, the major mediator of the WDAH (pancreatic cholera) syndrome: value of measurement in diagnosis and treatment. Am J Dig Dis 1978; 23: 373–6.

6. Bloom SR. Vasoactive intestinal polypeptide. In: Jaffe BM, Behrman HR (eds). Methods in hormone radioimmunoassay. New York: Academic Press, 1979: 553–61.

7. Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991; 129: 2787–9.

8. Verner JV, Morrison AB. Endocrine pancreatic islet disease with diarrhoe: report of a case due to diffuse hyperplasia of non-beta islet tissue with a review of 54 additional cases. Arch Intern Med 1974; 133: 492–9.

9. Fahrenkrug J: Vasoactive intestinal polypeptide. J Clin Gastroenterol 1980; 9: 633–43.

10. Öberg K. Endocrine tumours of the pancreas. Best Practice and Research Clin Gastroenterol 2005; 19: 753–81.

11. Park SK, O’Dorisio MS, O’Dorisio TM. Vasoactive intestinal polypeptide-secreting tumours: biology and therapy. Baillieres Clinical Gastroenterology 1996; 10: 673–96.

12. Fahrenkrug J, Buhl K, Hannibal J. PreproPACAP derived peptides occur in VIP-producing tumours and coexist with VIP. Regulatory peptides 1995; 58: 89–98.

14.5.6 PP, PYY and NPY

Pancreatic polypeptide (PP) is secreted by the pancreatic polypeptide cells that are localized mainly in the Langerhans’ islets of the pancreatic head region. Together with peptides YY (PYY) and the neurotransmitter/neuromodulator neuropeptide Y (NPY), which are produced in intestinal endocrine cells, PP forms a peptide family of its own /1/.

14.5.6.1 Indication

PP, PYY

Suspicion of a neuroendocrine tumor of the gastrointestinal tract (GEP-NET)
Secretory diarrhea.

NPY

Suspicion of pheochromocytoma, ganglioneuroma, neuroblastoma.

14.5.6.2 Method of determination

Radioimmunoassay, peptide-specific /2, 3/.

14.5.6.3 Specimen

Plasma (25 IU heparin/mL blood): 2 mL

Samples need to be cooled in ice immediately after blood collection, plasma is obtained by means of cooled centrifugation and immediately frozen.

14.5.6.4 Reference interval /1, 2, 3/

PP	< 630 pg/mL (150 pmol/L)
PYY	< 100 pmol/L
NPY	< 20–50 pmol/L

Basal plasma concentrations are age dependent; plasma concentrations in the upper normal range may occur particularly in elderly individuals. NPY is normally undetectable in plasma since it is a neuropeptide.

14.5.6.5 Clinical significance

The determination of PP is clinically relevant in the diagnosis of GEP-NET tumors /4, 5/. The clinical symptoms are, as a rule, determined by the peptide that is produced in the tumor (e.g., gastrin, insulin, VIP). Often, several gastrointestinal hormones are synthesized by a tumor and released into the circulation. PP is relatively often co secreted by GEP-NETs along with CgA, although there are also endocrine tumors in which only PP is detectable and which do not secrete other peptides (PPomas). Pancreatic tumors that secrete PP comprise some 20% of all pancreatic GEP-NETs. They are often diagnosed in the 5th and 6th decades of life. These are non-functioning tumors. Causes of this are believed to be hormonal inactivity, the co secretion of inhibitors, or the down regulation of the PP receptors.

NPY is synthesized in organs of the sympathoadrenal system (adrenal medulla, ganglia) and in gastrointestinal neurons. Secretion together with adrenalin and noradrenalin occurs in pheochromocytoma. In Tab. 14.5.-11 – Diseases and conditions with elevated PP concentrations, diseases associated with elevated PP levels are listed.

14.5.6.6 Comments and problems

Blood sampling

To be collected from fasting patients in the morning after a fasting period of at least 8–10 h since postprandial PP plasma levels remain elevated over a prolonged period of time /8/.

Drugs

Therapeutic drugs with an indirect or direct parasympathomimetic effect such as metoclopramide or those with a sympathicolytic effect such as beta-adrenergic blockers must be discontinued within an adequate period of time prior to the test.

Interferences

Patients with insulin-dependent diabetes mellitus of long duration and therapy with insulin preparations prior to the time of chromatographic purification or prior to the era of gene-technological insulin production may have circulating antibodies directed against PP which are the result of PP contamination of insulin used in the past. An exact PP determination is not possible in such patients.

Stability

The stability of plasma PP at room temperature is limited. Therefore, cooling of the blood sample, starting from the time when it is collected, mailed and up to the point of its arrival in the laboratory, is critical.

References

1. Arnold R, McIntosh C, Bothe E, Koop H. Pankreatisches Polypeptid (PP). Z Gastroenterol 1978; 16: 317–24.

2. Adrian T, Ferri G, Bacarese-Hamilton A, Fussel H, Polak JM, Bloom SR. Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 1985; 89: 1070–7.

3. Allen JM, Hughes J, Bloom SR. Presence, distribution and pharmacological effects of neuropeptide Y in mammalian gastrointestinal tract. Dig Dis Sci 1987; 32: 506–12.

4. Floyd JC, Fajans SS, Pek S, Chance RE. A newly recognized pancreatic polypeptide: plasma levels in health and disease. Rec Prog Horm Res 1977; 33: 519–70.

5. Schwartz TW. Pancreatic polypeptide (PP) and endocrine tumours of the pancreas. Scand J Gastroenterol 1979; 14, suppl 53: 93–100.

6. Schwartz TW. Atropine suppression test for pancreatic polypeptide. Lancet 1978; ii: 43–4.

7. Koop H, Eissele R, Moennikes H, Stange EF, Seifert E, Arnold R. Gastrin and pancreatic polypeptide responses to terbutaline and secretin in different states of hypergastrinaemia. Eur J Gastroenterol Hepatol 1990; 2: 291–6.

8. Schwartz TW, Stadil F, Chance RE, Rehfeld JF, Larsson LI, Moon N. Pancreatic polypeptide response to food in duodenal ulcer patients before and after vagotomy. Lancet 1976; i: 1102–5.

14.6 Porphyrias

Lothar Thomas

Porphyria refers to a group of metabolic disorders that involve the heme biosynthetic pathway. Disruptions in the pathway resulting from enzyme deficiencies cause precursors and porphyrins to accumulate.

Accumulation of the porphyrin precursors causes damage of neurons in the gut, brain, and peripheral nervous system.
Accumulation of porphyrins causes photosensitivity and damage of the skin.

Porphyria can be categorized as either acute hepatic or cutaneous.

The acute hepatic porphyrias are:

Acute intermittent porphyria
Variegate porphyria; has both acute neurovisceral features and cutaneous features
Hereditary coproporphyria; has both acute neurovisceral features and cutaneous features
ALA dehydratase-deficiency porphyria.

The cutaneous porphyrias are:

Congenital erythropoietic porphyria
Porphria cutanea tarda
Erythropoietic protoporphyria
X-linked dominant protoporphyria

14.6.1 Heme biosynthetic pathway and porphyrins

Heme, the iron-protoporphyrin complex, is central to biological oxidation reactions. In the bone marrow 80% of heme combines with globin to form hemoglobin. About 20% of the heme biosynthesis is produced in the liver and cells outside the liver to form cytochrome P450 enzymes and hemoproteins. The synthesis of heme involves eight enzymes to catalyze the pathway from glycine and succinyl CoA.The enzyme delta-aminolevulic acid synthase (ALAS) initiates the synthesis pathway.

Heme biosynthesis is regulated by two isoenzymes of ALAS /1/:

In erythroid cells, synthesis of heme is regulated during erythroid differentiation in response to erythropoietin. The ε-ALAS (ALAS2) catalyzes heme synthesis, the rate is limited by iron availability.
In the liver δ-ALAS (ALAS1) is the rate limiting enzyme in the production of heme. About 20% of the organism’s heme is formed. The synthesis of heme is controlled via negative-feedback regulation by the intracellular uncommitted heme pool.

In the heme biosynthetic pathway:

5-aminolevulinic acid is the first specific metabolite of heme biosynthesis (Fig. 14.6-1 – First step of heme synthesis).
Heme biosynthesis includes 8 individual enzyme catalyzed steps. Metabolic disorders cause the production of porphyrins (Fig. 14.6-2 – Heme biosynthetic pathway and porphyrias).
The inhibition of ALAS1 by the intracellular heme pool and the induction of ALAS1 by enzyme deficiencies of the heme biosythetic pathway are shown in Fig. 14.6-3 – Inhibition of hepatic aminolevulinic acid synthase activity.

14.6.2 Porphyrins

In patients with porphyria metabolic intermediates of the heme biosynthetic pathway accumulate at the synthesis step that precedes the enzymatic defect. Porphyrins are the oxidized substrates of the respective defective enzyme.

The name porphyria describes not the diseases but the lustrous purple-red crystalline porphyrins; they are named from the Greek porphuros (purple) /2/. The reason is the structure of the pyrrole ring with its conjugated double bonds. Porphyrins are oxidized products of porphyrinogens, which are the actual substrates of the enzymes catalyzing heme biosynthesis. The porphyrinogens are hexahydroxyporphyrins, in which the four methene bridge carbon atoms and the two pyrrolenine nitrogen atoms are hydroxygenated /3/.

Refer to Fig. 14.6-4 – Oxidation of porphyrinogen to porphyrin).

Porphyrins are tetrapyrroles derived from porphin, a macro cycle, by substituting its eight side hydrogen atoms with characteristic side chains. Most porphyrins have carboxylic acid side chains /3/. Porphyrins with 8, 7, 6, 5, and 4 carboxyl group side chains (uro-, heptacarboxy-, pentacarboxy-, and coproprophyrins) are produced in excess of that required for heme biosynthesis, and are excreted in the urine or the feces. Prophyrins are formed in excess for the most different reasons: hereditary; disturbances of hemoglobin formation; hepatic diseases; in patients on hemodialysis.

Porphyrinogens are reduced porphyrins, and the first of the porphyrinogens synthesized by the heme pathway has eight carboxyl groups. The stepwise decarboxylation of the side chains from eight to two occurs along the pathway, and it confers different physicochemical properties to the subsequent oxidized porphyrins. Thus, porphyrins with higher numbers (8–4) of carboxyl groups are hydrophilic, a quantity that facilitates their excretion in urine; while porphyrins with fewer (2–4) carboxyl moieties as side chains have lipophilic properties and are excreted by the hepatobiliary route /3/. Both types of porphyrins appear in plasma, bound to various proteins and phospholipids.

The porphyrins formed towards the end of the pathway (coproporphyrin, harderoporphyrin and protoporphyrin, with four, three and two carboxyl groups respectively), are found in feces due to their hydrophobicity /3/.

Porphyrin isomers that occur naturally in biological materials are isomers I and III of the polycarboxylated porphyrins, and protoporphyrin IX. Only type III porphyrinogens are physiological precursors of protoporphyrin IX and heme /3/.

14.6.3 Presentation and diagnostics of porphyrias

Hereditary porphyrias are a group of metabolic disorders of the heme biosynthetic pathway. Seven porphyrias are the result of a partial enzyme deficiency, and a gain of function mechanism has been characterized in a new porphyria /1/. Every porphyria results in the accumulation of a specific intermediate of the heme biosynthetic pathway. Refer to Fig. 14.6-3 – Inhibition of hepatic aminolevulinic acid synthase activity.

Metabolic intermediates accumulate at the synthesis step that precedes the enzymatic defect; these are the oxidized substrates of the respective defective enzyme /4/. The metabolic intermediates accumulate in the organism, particularly in the liver, the hematopoietic system and the skin. They can be toxic and can lead to neurovisceral symptoms, skin lesions, or both.

Porphyrias are pan ethnic; their prevalence in the population is between 0.5 and 10 per 100,000 people /5/. Based on clinical presentation porphyrias are divided in /1/:

Acute porphyrias
Cutaneous porphyrias
Recessive porphyrias.

The clinical classification of porphyrias and main clinical presentation is shown in Tab. 14.6-1 – Characterisation of porphyrias.

According to clinical complaints porphyrias can be categorized as either acute hepatic (neurovisceral) or cutaneous.

14.6.3.1 Acute hepatic porphyrias

Autosomal dominant acute porphyrias are:

Acute intermittent porphyria; skin lesions never develop
Variegate porphyria; skin lesions develop in approximately 60% of patients
Hereditary coproporphyria; skin lesions are rare (5%).

Acute porphyrias are due to mutations in the genes that code for enzymes of the heme biosynthetic pathway.

Acute attacks are very rare before puberty and after menopause. The acute attack begins with neurological abnormalities: behavioral changes such as anxiety and restlessness, and then progresses to nausea, vomiting and cramp-like abdominal pain. Tachycardia and hypertension are suggestive of increased sympathetic activity. The pain regresses after one week, but diffuse muscular weakness can last longer /6/.

Although 70% of acute attacks of porphyria are related to ingestion of common drugs and alcohol, the precipitating factors in the residual 30% are unclear, although the roles of infection, dieting or fasting, and endogenous hormones are reasonably assured /1/.

All clinical features of an acute attack can be explained by lesions of the nervous system. The leading hypothesis is that 5-aminolevulinic acid or other metabolites that are overproduced by the liver are neurotoxic. Cutaneous photosensitivity is due to the unique fluorescent properties of the extensive amounts of porphyrins produced. Apart from acute intermittent porphyria, all porphyrias demonstrate more or less photosensitive skin changes.

14.6.3.2 Diagnosis of acute hepatic porphyrias

The diagnostic investigations of porphyrias is based upon:

Acute (neurovisceral) symptoms
Personal and family medical history
Observation of cutaneous changes
First line biochemical tests for diagnosis (Tab. 14.6-2 – First line tests for diagnosis of porphyrias).
Molecular biological testing for gene mutations (mutational analysis).

14.6.3.2.1 Biochemical markers in acute hepatic porphyrias

Examination of urine for excess porphobilinogen is the first-line test for patients with a suspected attack of acute porphyria. Measurement of 5-aminolevulinic acid is not essential /1, 7/.

Porphobilinogen

Increased excretion of porphobilinogen is the most important finding pointing to acute porphyria. Normal excretion is below 10 μmol/L or below 1.5 μmol/mmol creatinine. In the acute phase of porphyria porphobilinogen is usually increased by a factor of 10 above the upper reference limit. With a porphobilinogen over expression above the tenfold limit treatment can be started immediately. Investigations must be ordered for differentiation of acute porphyrias (Tab. 14.6-2 – First line tests for diagnosis of porphyrias). The porphobilinogen excretion decreases with regression of the acute symptoms. In acute intermittent porphyria, porphobilinogen remains elevated over the course of a number of weeks, while in variegate porphyria and hereditary coproporphyria it decreases within the first week /1, 7/.

5-aminolevulinic acid

5-aminolevulinic acid is elevated in all forms of acute porphyria, but not essential to establish the diagnosis. 5-aminolevulinic acid can be helpful for the differentiation of disorder from other metabolic causes of abdominal pain /1/. Lead poisoning and the rare 5-aminolevulinic acid dehydratase porphyria are associated with isolated elevation of 5-aminolevulinic acid.

Total porphyrins

They are of minor relevance since, in acute porphyrias, the increase is due mainly to the condensation of PBG molecules to uroporphyrin. Reference intervals are listed in Tab. 14.6-3 – Reference intervals of urinary porphyrins.

Coproporphyrin

In order to rule out hereditary coproporphyria and variegate porphyria with certainty, the examination of a stool sample for coproporphyrin and protoporphyrin is important, because in both forms of acute porphyria the decrease in porphobilinogen excretion occurs as early as within one week. Reference intervals are listed in Tab. 14.6-4 – Reference intervals of fecal porphyrins. Total fecal porphyrin concentration is increased in variegate porphyria, with protoporphyrin (protoporphyrin IX) levels greater than those for coproporphyrin, whereas it is usually normal in acute intermittent porphyria /1, 7/.

Initial investigations in diagnosis and during remission of porphyria

In patients above the age of 15 years with symptoms of acute porphyria in the recent past or earlier, and in patients with chronic symptoms or uncertain family history, the following examinations are recommended /8/:

1. Urinary porphobilinogen and 5-aminolevulinc excretion in urine

2. Fecal protoporphyrin, coproporphyrin and coproporphyrin III/coproporphyrin I isomer ratio

3. Plasma fluorescence emission spectroscopy

4. Erythrocyte porphobilinogen deaminase activity if routine hematology is normal.

Interpretation and further investigation

If one or more of tests 1–3 are abnormal, porphyria is confirmed
If metabolite tests 1–3 are normal, any current or recent symptoms are not caused by porphyria and an alternative cause of the symptoms should be investigated
If only test 4 is abnormal, mutation analysis of the gene HMBS is required. If a disease specific mutation is identified, acute intermittent porphyria in remission (or latent if asymptomatic with family history) is confirmed. Current data indicates that mutational analysis of the HMBS gene is 95% sensitive and 100% specific.

14.6.3.3 Cutaneous porphyrias

Two types of cutaneous porphyrias are differentiated /1, 9/:

Bullous porphyrias (variegate porphyria, hereditary coproporphyria and porphyria cutanea tarda) which share the same chronic cutaneous photosensitivity.
Acute painful photosensitive porphyrias. Erythropoietic protoporphyria is an inherited disorder that is caused by partial deficiency in mitochondrial ferrochelatase.

Refer to Tab. 14.6-5 – Biochemical investigations and findings in cutaneous porphyrias.

Bullous porphyrias

In bullous porphyrias, large amounts of porphyrins accumulate in the skin. Porphyria cutanea tarda is the most frequent type of porphyria worldwide and presents with skin symptoms only. Porphyria cutanea tarda is caused by a deficiency of uroporphyrinogen decarboxylase (UROD) deficiency. Gene defect of UROD lead to 50% uroporphyrinogen decarboxylase deficiency. Liver dysfunction is common in porphyria cutanea tarda, especially in patients with excessive alcohol intake. Approximately 75% of porphyria cutanea tarda belong to the sporadic subtype and 25% to the familial subtype that has an earlier onset than the sporadic subtype /1/.

Laboratory findings

Differences in the plasma fluorescent spectrum are the best criteria for diagnosis of cutaneous porphyrias, differentiating between porphyria cutanea tarda and variegate porphyria (Tab. 14.6-2 – First line tests for diagnosis of porphyrias).

Acute painful photosensitive porphyrias

In erythropoietic protoporphyria free protoporphyrin accumulates in erythrocytes and other tissues leading to painful photosensitivity and liver disfunction (20–30% of cases). The clinical manifestation is lifelong acute photosensitivity which develops in early childhood, but in rare cases symptoms manifest in adulthood. Clinical symptoms include burning, stinging, and pruritus in sun-exposed skin /1/.

X-linked dominant erythropoietic protoporphyria results from increased activity of ALAS2 attributible to gain-of-function deletions in ALAS2. The ALAS2 gain of function leads to production of protoporphyrin in excess and in quantities causing photosensitivity and liver damage.

Laboratory findings

Excretion of porphyrins does not increase, because protoporphyrin is strictly lyophilic. In symptomatic patients erythrocytes show a strong increase in free protoporphyrin, the plasma porphyrin fluorescence assay shows a characteristic peak at 634 nm. Measured in nucleated cells, ferrochelatase activity is 10–35% of normal value. Screening for mutation and for hypomorphic IVS3-48C/T identifies symptom-free family members and allow definition of mode of inheritance in that family /1/.

14.6.4 Genetic testing of porphyrias

Mutational analysis is rarely necessary to make a diagnosis of porphyria and may be misleading if a mutation is not found /10/. In addition, the exclusion of porphyria is not possible. In addition, low clinical penetration in autosomal dominant porphyrias means that identification of a mutation does not necessarily indicate active porphyria. Nonetheless, molecular genetic testing is now the method of choice in pre-symptomatic diagnosis, family studies and for predictive counselling.

14.6.4.1 Autosomal dominant porphyrias

Four porphyrias are inherited in an autosomal dominant pattern: acute intermittent porphyria, hereditary coproporphyrinuria, variegate porphyria and porphyria cutanea tarda /10/. All four porphyrias show low clinical penetration, a sign that environmental factors and genes from other loci are important in determining their presentation. Approximately 10–20% of the affected individuals in France and the UK develop symptoms but figures as high as 50% have been reported for acute intermittent porphyria from Sweden. There is evidence that mutations for all four disorders are more common in Western European populations than the prevalence of diseases would suggest.

In autosomal dominant acute porphyrias more than 342 mutations have been identified in the HMBS gene in acute intermittent porphyria, about 52 in the CPOX gene in hereditary coproporphyrinuria and more than 150 in the PPOX gene in variegate porphyria. Most are point mutations, but a few large deletions have been detected in the HMBS and CPOX genes /10/. Mutations decrease enzyme activities in all tissues.

Indications for genetic testing are /10/:

In patients with biochemically proven acute intermittent porphyria, hereditary coproporphyrinuria and variegate porphyria is to identify a mutation as an essential preliminary to molecular investigation of that patients family
Presymptomatic diagnosis of affected relatives in the management of patients with acute intermittent porphyria, hereditary coproporphyrinuria or variegate porphyria.

Acute intermittent porphyria

Patients are either hetero allelic or homo allelic for a missense mutation, p.Lys404Glu, in exon 6 of the CPOX gene that impairs the sequential decarboxylation of coproporphyrinogen III, resulting in increased fecal excretion of harderoporphyrin /10/.

Porphyria cutanea tarda

Two main types of porphyria cutanea tarda exist. Most patients have the sporadic form in which URO-D deficiency is restricted to the liver and the UROD gene is normal. About 25% of patients with porphyria cutanea tarda have the autosomal dominant form (familial porphyria cutanea tarda) in which URO-D activity is decreased in all tissues. More than 108 mutations in the UROD gene have been identified /10/.

14.6.4.2 Autosomal recessive porphyrias

The following porphyrias belong to this group /10/:

Congenital erythropoietic porphyria. Affected individuals are homozygous or compound heterozygous for UROS gene mutations, 45 of which are known. Rarely deficient UROS activity is due to mutations in the gene encoding the transcriptional regulator GATA1.
Erythropoietic protoporphyria. Excess accumulation results from the partial deficiency of ferrochelatase (FECH) activity. In the UK, most patients with erythropoietic protoporphyria are compound heterozygotes for a hypomorphic IVS3-48C allele that produces a truncated unstable mRNA, reducing activity by 20–30%.
X-linked dominant protoporphyria (XLDPP). Mutational analysis of ALAS2 gene is essential to confirm the diagnosis of XLDPP and is recommended for all patients with erythropoietic protoporphyria where zinc protoporphyrin comprises 10% or more of the total erythrocyte porphyrin.

A summary about clinics and diagnostics of porphyrias is shown in Tab. 14.6-6 – Typical findings in porphyrias.

References

1. Puy H, Gouya L, Deybach JC. Porphyrias. The Lancet 2010; 375: 924–36.

2. Moore MR. The biochemistry of heme synthesis in porphyria and in the porphyrinurias.Clinics in Dermatology 1998; 16: 203–23.

3. Zaider E, Bickers DR. Clinical laboratory methods for diagnosis of the porphyrias. Clinics in Dermatology 1998; 16: 277–93.

4. Doss MO, Sieg I. Porphyria. In: Thomas L, ed. Clinical Laboratory Diagnostics, 5th ed. Frankfurt, TH-Books 1998.

5. Kauppinen R. Porphyrias. Lancet 2005; 365: 241–52.

6. Lip G, McColl K, Moore MR. The acute porphyrias BJCP 1993; 47: 38–43.

7. Kushner JP. Laboratory diagnosis of the porphyrias. N Engl J Med 1991; 324: 1432–4.

8. European Porphyria Initiative. Porphyrias. www.porphyria-europe.com

9. Murphy GM, The cutaneous porphyrias: a review. Br J Dermatol 1999; 140: 573–81.

10. Whatley SD, Badminton MN. Role of genetic testing in the management of patients with inherited porphyria and their families. Ann Clin Biochem 2013; 50: 204–16.

11. Gross U, Sassa S, Jacob K, Deybach JC, Nordman Y, Frank M, Doss MO. 5-aminolevulinic acid dehydratase deficiency porphyria: a twenty-year clinical and biochemical follow up. Clin Chem 1998; 44: 1892–6.

12. de Matteis F. Porphyria cutanea tarda of the toxic and sporadic varieties. Clinics in Dermatology 1998; 16: 265–73.

13. Poh-Fitzpatrick MB. Clinical features of the porphyrias. Clinics in Dermatology 1998; 16: 251–64.

14.7Adiposity

Lothar Thomas

Adipose tissue consists of brown adipose tissue and white adipose tissue /1/.

14.7.1 Brown adipose tissue (BAT)

BAT arises during the late trimester of pregnancy and protects newborns from cold as they develop the ability to shiver. BAT consumes glucose and triglycerides, generating heat. The distribution of adult BAT is found only in certain anatomical depots in the neck, shoulders, posterior thorax, abdomen, and posterior abdomen. The amount of BAT mass varies according to sex is about 1 kg in adults 20 to 50 years of age and is 0.2 to 3 % of total adipose tissue mass.

14.7.2 White adipose tissue (WAT)

WAT is derived from mesotherm. WAT begins to develop in the second trimester of pregnancy. By birth, both visceral and subcutaneous depots are well established. In lean adults WAT depot is 10–20 kg in men and 20–30 kg in women. WAT stores food calories, creates a layer of thermal insulation and provides mechanical protection. Insulin is the driver of fuel absorption and storage of WAT, with adipose tissue responsible for 5% of glucose uptake in adults who are lean and 20% in those who are obese /2/. WAT is also an endocrine organ. White adipocytes produce adipokines that affect local and distant pathophysiology such as producing proinflammatory proteins (TNFa, monocyte chemotactic protein), and estrogen /1/. The functional status of the adipocyte is communicated at the autocrine, paracrine and endocrine levels. WAT also uses interorgan signaling to coordinate the storage and consumption of nutrients with the liver and skeletal muscle. WAT and BAT are integral and reglatable components of lipoprotein and bile acid metabolism. Chylomikrons from a meal and liver-derived triglyceride-rich lipoproteins deliver their lipids to BAT and WAT.

14.7.3 Adipokins

Important adipokins in obesity medicine are leptin und adiponectin.

14.7.3.1 Leptin

Low concentrations of leptin signal low enegy stores and starvation. People with obesity have leptin resistance. Exogenous administration of leptin does not lead to appetite suppression or weight loss.

14.7.3.2 Adinopectin

Adinonpectin has a regulating effect on glucose metabolism and lipid metabolism and promotes insulin sensitizing, anti atherogenic and anti inflammatoric effects.

14.7.3.3 Estimation of fat mass

Fat mass is estimated using waist circumference, body mass index (BMI) or skinfold clipers. BMI can not provide information about WAT distribution or specific depots. Very precise calculations use computer tomography (CT) or magnetic resonance imaging (MRI). Many features of the adipose-tissue depots (location, size, metabolic bahavior) are influenced by the genetic background and sex /2/. Adipogenesis continues throughout life with a median turnover rate of 8% /3/.

14.7.4 Function and communication of fat cells

Adipocyte precursor cells are present in the stromal vascular fraction and perivascular tissue, which is capable of self-renewal. The growth of adipocytes results both from hypertrophy, enlargement of cell size, hyperplasia, and the increase in cell number /4/.

As obesity progresses, preadipocyte differentiation becomes dysfunctional, leading to reduced insulin signaling, glucose uptake, and adiponectin release by the mature adipocytes /5/. Ultimately hypertrophic WAT growth and expansion restrict the ability of oxygen to diffuse from the capillaries into the adipocytes The hypoxia constitutes a biologic alert for the cells altering the expression of genes and triggering a series of interconnected responses leading to /1/:

insulin and adrenergic signaling
increased inflammation
cellular damage
failure of the adipose tissue to continue expanding leads to overflow and subsequent deposition of triglycerides throughout the body, with ectopic accumulation in the liver and skeletal muscle
immune system dysfunction
increased risk of many types of cancer

References

1. Cypess A. Reassessing human adipose tissue. N Engl J Med 2022; 386: 768–79.

2. Bouchard C. Genetics of obesity: what we have leaned over decades of reseach. Obesity (Silver Spring) 2021; 29: 802–20.

3. Spalting KL, Arner E, Westermark PO, Bernard S, Buchholz BA, Bergmann O, et al. Dynamics of fat cell turnover in humans. Nature 2008; 453: 783–7.

4. Greenhill C. Identification thermogenic adipocyte lineages Nat Rev Endocrinol 2021; 14: 319.

5. Kahn CR, Wang G, Lee KY. Altered adipose tissue and adipocyte function in the pathogenesis of metabolic syndrome. J Clin Invest 201); 129: 3990–4000.

14.8Protein-losing enteropathy

Lothar Thomas

Protein-losing enteropathy is an uncompensated loss of plasma proteins in the intestine /1/. The enteropathy also involves the wasting of all other interstitial fluid components. Epithelial cells connected by tight junctions guard the integrity and composition of the gut interstitium. The syndrome occurs when the interstitial epithelium is not functional and the interstitial fluid leaks into the gut lumen. Protein-losing enteropathy is a nonselective depletion of all plasma proteins /1, 2/.

Three principal mechanisms may be responsible for the pathophysiology of protein-losing enteropathy /2/:

Mucosal diseases
Diseases of the lymphatic return
Congenital causes.

14.8.1 Mucosal diseases

Erosive diseases cause a loss of plasma proteins into the gut lumen /2/. The erosion is caused in inflammatory bowel disease, infections, and carcinoma. Other diseases are ulcerative colitis, Crohn’s disease, pseudomembranous colitis, and Zollinger-Ellison syndrome. Infrequent diseases are sarcoidosis, gastrointestinal sarcoidosis, and graft-versus-host disease.
Non-erosive mucosal diseases alter the permeability of the epithelium in cases of inflammation and infection or genetic abnormality of the mucosa. This is the case in patients with autoimmune diseases e.g., systemic lupus erythematosus, Sjögren’s syndrom, Schönlein-Hennoch purpura, and Pemphigus vulgaris.
Allergic disorders, e.g. eosinophilic gastritis may cause protein-losing enteropathy
Food elimination diets which can reverse the normal process of protein transport out of the gut lumen
Heparan sulfate and surface proteins may disrupt the gut barrier.

The mucosal disorders are generally mild and more amenable to treatment.

14.8.2 Diseases of lymphatic return

Intestinal lymphangiectasia: in these cases diseases block the lymphatic drainage and cause pathological distortion and distention of lymphatics. Proteins of the lymph are massively lost into the lumen causing severe protein-losing enteropathy. Intestinal lymphangiectasia leads to panhypoproteinemia and low concentration of total protein.
Widespread metastatic abdominal cancers can block lyph drainage causing protein-losing enteropathy
Thrombotic disease. Protein losing enteropathy can be both a cause and a consequence of thrombotic disease, e.g., antiphospholipid syndrome, Waldenstroem’s disease
Inflammation or lymphatic damage can result from infection with pathogens, e.g., bacteria, viruses, fungi, parasites
Metastatic abdominal cancers can block lymph drainage
Secondary intestinal lymphangiectasia is caused in constrictive pericarditis and specific surgical interventions. Lymph recirculation requires intact vasculature with a pressure gradient guiding unidirectional flow to the right side of the heart. Heart conditions causing chronically elevated central venous pressure impede lymph return from intestinal lymphatics in secondary intestinal lymphangiectasia /3/.

14.8.3 Congenital causes

Congenital protein-losing enteropathy can result from specific gene mutations causing lymphatic dysplasia and lymphatic malformations that affects multiple organs and is associated by facial dysmorphism and cognitive impairement /2/. The Hennekam lymphangiectasia-lymphedema syndrome (HKLLS) affects multiple organs and is accompanied by facial dysmorphism and cognitive impairement. Deleterious mutations in three genes e.g., CCBE1 (collagen and Ca²⁺ binding EGF domains 1) FAT4 (FAT atypical cadherin 4), and ADAMTS3 (ADAM metallopeptidase with thrombospondin type 1 motif 3) can cause HKLLS types 1, 2, 3, respectively /2, 4/.

Chaple disease results from inherited CD55 mutations.

14.8.4 Clinical findings

Patients present the following features:

Nutritional deficiencies, vomiting
Edema of the face, arms and legs
Gastrointestinal symptoms, e.g. diarrhea, steatorrhea, abdominal pain, effusions (pleural, pericardial, peritoneal)
Infections.

Protein-losing enteropathy is diagnosed by ruling out other causes of hypoproteinemia and diarrhea or malabsorption.

14.8.5 Laboratory findings

Elevated alpha1-antitrypsin in stool. Alpha1-antitrypsin has nearly the same size as albumin and is not actively secreted or absorbed in the bowel
Hypoproteinemia; exclude advanced liver disease or malnutrition
Increased urinary excretion of protein; exclude nephrotic syndrome
Albumin and immunoglobulins are reduced in serum.

Non-selective protein deficiency. Less abundant proteins are reduced e.g., acute phase proteins, transferrin, haptoglobin.

References

1. Greenwald DA. Protein-losing gastroenteropathy. In: Feldman M, Friedman LS Brandt LJ, et al. eds. Sleisinger and Fordtran’s gastrointestinal and liver disease: pathophysiology, diagnosis, management. Philadelphia; Elsevier: 2021, 11th edition, 350–5.

2. Ozen A, Lenardo MJ. Protein-losing enteropathy. N Engl J Med 2023; 389 (8): 733–45.

3. Wilkinson P, Pinto B, Senior JR. Reversible protein-losing enteropathy with intestinal lymphangiectasia secondary to chronic constrictive pericarditis. N Engl J Med 1965; 273: 1178–81.

4. Brouillard P, Dupont L, Helaers R, Coulie GE, Tiller J, Peeden A, et al.Loss of ADAMTS3 activity causes Hennekam lyphangiectasia-lymphedema syndrome 3. Hum Mol Genet 2017; 26 (21): 4095–4104.

Table 14.1-1 Accuracy of tests for the detection of H. pylori /1/

Method	Test	Sens. (%)	Spec. (%)
Invasive	Culture	70–90	100
	Histology	80–98	90–98
	Urease test	90–95	90–95
	PCR	90–95	90–95
Non- invasive	Urea breath test	85–95	85–95
	Stool antigen test	85–95	85–95
	IgG antibodies in serum	70–90	70–90

Sens, diagnostic sensitivity; Spec, diagnostic specificity

Table 14.1-2 NSAIDs as triggers of gastrointestinal bleeding /2/

NSAID	Relative risk
Ibuprofen	2.5 (2–3.1)
Diclofenac	2.1 (1.6–2.7)
Aceclofenac	1.4 (0.9–2.2
Naproxen	4.0 (2.8–5.8)
Piroxicam	7.2 (4.8–10.7)
Indomethacin	3.3 (1.7–6.6)
Meloxicam	3.6 (1.8–7.2)
Ketorolac	8.0 (3.4–18.5)
Lornoxicam	3.5 (1.2–9.8)
Ketoprofen	6.5 (2.3–18.3)
Other	6.7 (2.6–16.9)

Values are the 2.5th and 97.5th percentiles

Table 14.1-3 Sensitivity, specificity and accuracy of tests for the diagnosis of H. pylori infection /3/

	Histo- logy	Culture	Sero- logy	UBT
Sensitivity (%)	99.2	66.6	89.7	90.5
Specificity (%)	100	100	91.3	97.8
Accuracy (%)	99.4	75.6	90.1	92.5

UBT, Urea breath test threshold delta over baseline 0.5%

Table 14.2-1 Laboratory tests for the diagnosis and monitoring of acute pancreatitis /4/

Examination	Acute pancreatitis: diagnosis and monitoring
α-amylase, lipase	Diagnostic sensitivity and specificity refer to Section 1.4 – Amylase and Section 1.12 – Lipase. The level of the enzyme activity does not correlate with the severity of the disease.
ALT	Evaluation of gallstone-induced pancreatitis, values ≥ 3-fold the upper reference interval value are indicative, but in 20% of cases the values are within the reference interval. Values ≥ 120 U/L are prognostically unfavorable.
CRP	Marker of inflammatory activity. Levels ≥ 150 mg/L within 48 hours of the occurrence of acute symptoms are suggestive of necrotic pancreatitis.
Further prognostic markers	Prognostically unfavorable are:
	Initial Blood glucose > 200 mg/dL (11.1 mmol/L) Leukocyte count > 16 × 10⁹/L LD ≥ 350 U/L ALT ≥ 120 U/L Fever ≥ 38.5 °C Age ≥ 55 years Body mass index ≥ 30 kg/m²
	During the course Hematocrit decline ≥ 10% Calcium ≤ 8.0 mg/dL (2.0 mmol/L) Creatinine ≥ 2 mg/dL (177 mol/L) Serum albumin ≤ 32 g/L PO₂ ≤ 60 mmHg Urine flow rate ≤ 50 mL/h Fluid deficit of greater than 6 liters Shock, tachycardia

Table 14.2-2 Severity of acute pancreatitis and CRP values /5/

Acute pancreatitis	CRP (mg/L)	Sensitivity (%)	Specificity (%)
Severe form	> 45	68	65
Necrotic form	> 71	79	71
Infected pancreatitis	> 206	62	62
Death	> 84	77	63

Table 14.2-3 Ranson criteria for the assessment of severity of acute pancreatitis /8/

First examination	Examination after 48 h
Age > 55 years	Volume deficit > 6 liters
Leukocyte count > 16 × 10⁹/L	Rise in BUN > 6 mg/dL (4.3 mmol/L)
AST > 150 U/L	Base deficit > 40 mmol/Ls
LD > 350 U/L	PO₂ fall to ≤ 60 mmHg
Blood glucose > 200 mg/dL (11 mmol/L)	Calcium decrease to < 8 mg/dL (2 mmol/L)

Each criterion receives a point. A score of 3 points indicates the presence of severe pancreatitis.

Table 14.2-4 Causes of chronic pancreatitis (CP)

Clinical and laboratory findings
Alcoholism /9, 10, 11/ Approximately 10% of patients with severe alcohol abuse develop CP. A minimum of 80 g of alcohol per day over the course of 6–12 years is considered to be a risk factor for the development of chronic pancreatitis. Smoking leads to rapid progression. The average age for the onset of chronic alcohol-related pancreatitis is 35–40 years. Alcohol is believed to have a direct toxic effect on the acinar cells and this leads to necrotic inflammation. Pro enzymes such as trypsinogen are activated, whereby lysosomal cathepsin C is, in turn, activated, leading to autodigestion of the acinar cells. Recurrent alcoholic necroinflammatory episodes lead to peri lobular fibrosis, distortion of the pancreatic ducts, and interference with pancreatic duct secretion and stone formation. Gradual fibrosation of the organ with the development of pancreatitis thereby ensue.
Hereditary chronic pancreatitis (HCP) /12/ The prevalence of HCP is 1 in 300,000 in the general population. It is associated with recurrent episodes that begin as early as in childhood. Approximately 66–68% of patients with HCP have a mutation in the PRSS1 gene. This gene codes for the formation of trypsinogen. The mutation leads to the development of HCP via autosomal dominant inheritance with a penetration of 80–93%. Further common risk factors in sporadic HCP are mutations of the genes SPINK1 and CFTR. SPINK1 is a gene that regulates the serine protease inhibitor Kazal type 1 for the pancreatic secretory trypsin inhibitor, which prevents the premature auto-activation of trypsinogen. CFTR is the gene that controls the cystic fibrosis transmembrane conductance regulator. Mutation analysis should be performed: In patients with a positive family history (1 or 2 first-degree relatives with idiopathic CP) In patients with 2 or more episodes of acute pancreatitis prior to the age of 25 In idiopathic CP before the age of 25. The risk of pancreatic carcinoma in HCP is 69%.
Autoimmune pancreatitis (AIP) /13/ AIP is characterized by an autoimmune inflammatory process. Lymphocytic infiltration with fibrosis of the pancreas, leading to functional impairment, occurs. Additional autoimmune diseases are typical in AIP patients. Type 1, which occurs at the age of > 60 years, and type 2, which presents during the 4th and 5th decades of life, are distinguished. AIP accounts for 5–6% of chronic pancreatitis (CP). Major symptoms of AIP are discrete epigastralgia, newly occurring diabetes, or obstructive jaundice that is attributable to pancreatic duct strictures. In about 30% of cases, AIP is associated with other autoimmunopathies such as rheumatoid arthritis, Sjörgren’s syndrome, and inflammatory bowel disease. The enzyme carbo anhydrase is found intracytoplasmatically in most of the organs that are affected in AIP, such as the lungs, the bile ducts and the kidneys. Laboratory findings: in patients with type 1 AIP, IgG₄ concentration is > 2-fold elevated; in type 2 up to 2-fold. In Japan, the prevalence of IgG₄ in AIP is up to 90%, in Spanish studies it is around 50%, and in German studies it is approximately 20% /21/. At a cutoff > 140 mg/dL, the negative predictive value that rules out type 1 is 98%. Patients with IgG₄ levels > 280 mg/dL without AIP were predominantly women /17/. Elevated IgG₄ in AIP is seen in 63% of patients with type 1 AIP, but only in 23% of those with type 2. Lipase values are usually normal or slightly elevated in AIP; diabetes with increased glucose and HbA_1c values may occur in AIP.
Cystic fibrosis (CF) /16/ In Caucasians, CF is an autosomal inheritable disease that is attributable to a mutation in the CFTR gene, which encodes for the transmembrane conductance regulator. This protein is responsible for the chloride channel of the sweat glands, and of the mucus- and gastrointestinal secretion-producing cells. Approximately 2–4% of Caucasians have this gene, and 70% of carriers have the ΔF508 mutation. The clinical manifestation of CF includes obstructive pulmonary disease and pancreatic insufficiency. CF patients are at elevated risk of developing pancreas carcinoma. Laboratory findings: a sweat test should be performed in children with chronic pancreatitis in order to rule out CF.

Clinical and laboratory findings

Alcoholism /9, 10, 11/

Approximately 10% of patients with severe alcohol abuse develop CP. A minimum of 80 g of alcohol per day over the course of 6–12 years is considered to be a risk factor for the development of chronic pancreatitis. Smoking leads to rapid progression. The average age for the onset of chronic alcohol-related pancreatitis is 35–40 years. Alcohol is believed to have a direct toxic effect on the acinar cells and this leads to necrotic inflammation. Pro enzymes such as trypsinogen are activated, whereby lysosomal cathepsin C is, in turn, activated, leading to autodigestion of the acinar cells. Recurrent alcoholic necroinflammatory episodes lead to peri lobular fibrosis, distortion of the pancreatic ducts, and interference with pancreatic duct secretion and stone formation. Gradual fibrosation of the organ with the development of pancreatitis thereby ensue.

Hereditary chronic pancreatitis (HCP) /12/

The prevalence of HCP is 1 in 300,000 in the general population. It is associated with recurrent episodes that begin as early as in childhood. Approximately 66–68% of patients with HCP have a mutation in the PRSS1 gene. This gene codes for the formation of trypsinogen. The mutation leads to the development of HCP via autosomal dominant inheritance with a penetration of 80–93%. Further common risk factors in sporadic HCP are mutations of the genes SPINK1 and CFTR.

SPINK1 is a gene that regulates the serine protease inhibitor Kazal type 1 for the pancreatic secretory trypsin inhibitor, which prevents the premature auto-activation of trypsinogen.

CFTR is the gene that controls the cystic fibrosis transmembrane conductance regulator.

Mutation analysis should be performed:

In patients with a positive family history (1 or 2 first-degree relatives with idiopathic CP)
In patients with 2 or more episodes of acute pancreatitis prior to the age of 25
In idiopathic CP before the age of 25.

The risk of pancreatic carcinoma in HCP is 69%.

Autoimmune pancreatitis (AIP) /13/

AIP is characterized by an autoimmune inflammatory process. Lymphocytic infiltration with fibrosis of the pancreas, leading to functional impairment, occurs. Additional autoimmune diseases are typical in AIP patients. Type 1, which occurs at the age of > 60 years, and type 2, which presents during the 4th and 5th decades of life, are distinguished. AIP accounts for 5–6% of chronic pancreatitis (CP). Major symptoms of AIP are discrete epigastralgia, newly occurring diabetes, or obstructive jaundice that is attributable to pancreatic duct strictures. In about 30% of cases, AIP is associated with other autoimmunopathies such as rheumatoid arthritis, Sjörgren’s syndrome, and inflammatory bowel disease. The enzyme carbo anhydrase is found intracytoplasmatically in most of the organs that are affected in AIP, such as the lungs, the bile ducts and the kidneys.

Laboratory findings: in patients with type 1 AIP, IgG₄ concentration is > 2-fold elevated; in type 2 up to 2-fold. In Japan, the prevalence of IgG₄ in AIP is up to 90%, in Spanish studies it is around 50%, and in German studies it is approximately 20% /21/. At a cutoff > 140 mg/dL, the negative predictive value that rules out type 1 is 98%. Patients with IgG₄ levels > 280 mg/dL without AIP were predominantly women /17/. Elevated IgG₄ in AIP is seen in 63% of patients with type 1 AIP, but only in 23% of those with type 2. Lipase values are usually normal or slightly elevated in AIP; diabetes with increased glucose and HbA_1c values may occur in AIP.

Cystic fibrosis (CF) /16/

In Caucasians, CF is an autosomal inheritable disease that is attributable to a mutation in the CFTR gene, which encodes for the transmembrane conductance regulator. This protein is responsible for the chloride channel of the sweat glands, and of the mucus- and gastrointestinal secretion-producing cells. Approximately 2–4% of Caucasians have this gene, and 70% of carriers have the ΔF508 mutation. The clinical manifestation of CF includes obstructive pulmonary disease and pancreatic insufficiency. CF patients are at elevated risk of developing pancreas carcinoma.

Laboratory findings: a sweat test should be performed in children with chronic pancreatitis in order to rule out CF.

Table 14.2-5 Classification of the severity of pancreatic insufficiency

Insufficiency	Criteria
Mild	Diminished enzyme secretion, normal bicarbonate in the duodenal secretion, normal stool fat.
Moderate	Decrease in enzyme secretion and reduced bicarbonate content in the duodenal secretion, normal stool fat.
Severe	All parameters are pathological, steatorrhea.

Table 14.2-6 Diagnostic sensitivity (%) and specificity (%) of noninvasive indirect tests in comparison with the secretin-caerulein test in chronic pancreatitis /13/

Test	Mild* Sens (%)	Moderate* Sens (%)	Severe* Sens (%)	Spec. (%)
Elastase-1	54	75	95	85
Stool fat	0	0	78	70
Chymotrypsin in the stool	< 50	~60	80–90	80–90
¹³C breath test	62–100		90–100	80–90

* Pancreatic insufficiency; Sens, diagnostic sensitivity; Spec, diagnostic specificity;

Table 14.2-7 Cancer syndromes associated with increased pancreatic cancer risk /16/

Syndrome	Chromo- some	Gene Gene type	Inheri- tance	Relative risk, Frequency in sporadic cases
Hereditary pancreatitis	7q35	PRSS1 Cationic trypsinogen	AD*	20–75 Unknown
Cystic fibrosis	7q31.2	CFTR Chloride ion channel	AR	Approx. 5 Unknown
Peutz-Jeghers syndrome	19p13.3	STK11/LKB1 Tumor suppressor, serine/threonine kinase	AD	132 4%
Familial atypical multiple melanoma	9p21	p16INK4a/ MTS1 Tumor suppressor	AD	13–22 98%
Hereditary breast and ovarian cancer	17q21–24	BRCA1 Tumor suppressor, linked to RAD5	AD	2.3–3.6 BRCA1 7% for BRCA
Hereditary breast and ovarian cancer	13q12–13	BRCA2 Tumor suppressor, linked to RAD5	AD	3–10 BRCA2 2 N/A for BCRA1
Familial adenomatous polyposis	5q21	APC Tumor suppressor	AD	5–40%
Hereditary nonpolyposis colon cancer	2p22–21 3p21.3	MSH2 and MLH1 Mismatch repair	AD	Unknown 4–11%
Family X: site specific pancreatic cancer	4q32–34	Palladin Cytoskeleton structure	AD	Unknown Unknown

AD, autosomal dominant; AR, autosomal recessive; * 80% penetrance

Table 14.2-8 Assessment of Elastase-1 results /6/

Elastase-1 (μg/g stool)	Assessment
> 175 (200)	Normal
100–175 (200)	Mild to moderate insufficiency
Below 100	Severe exocrine insufficiency

Table 14.2-9 Diseases associated with increased fecal fat excretion

Clinical and laboratory findings
Maldigestion (e.g., in chronic pancreatitis) The increased excretion of fecal fat occurs in the late stages of chronic pancreatitis, and then, only if > 90% of the exocrine secretory functional performance of the organ has failed. With the demonstration of pancreatic calcification, in a high percentage of the cases, pathological fatty acid excretion is associated. In such cases, the secretin caerulein test is always pathological.
Various gastrointestinal diseases Liver parenchymal cell damage, bile duct obstruction, overgrowth of the small intestine with colonic flora, diseases of the ileum with decreased bile acid reabsorption. These diseases lead to reduced concentrations of conjugated bile acids in the lumen of the small intestine. While in severe hepatitides and bile duct obstruction generally only mild steatorrhea (< 20 g fecal fat/24 h) develops, severe forms can occur in overgrowth of the small intestine with bacteria of the Bacteroides species. This is also the case in enteral bile acid loss due to a disease of the ileum (decrease in the bile acid pool).
Acute diarrhea, carcinoid syndrome Due to accelerated intestinal peristalsis, the intraluminal residence time for the digestion and absorption of nutritional components is too short. Steatorrhea normally occurs with less than 20 g of fecal fat/24 hours.
Malabsorption syndrome Among the biochemical methods for the diagnosis of generalized malabsorption fatty acid excretion, as a direct function test, is of the greatest diagnostic value. If the D-xylose test is also pathological, the diagnostic categorization is by and large assured. Celiac disease is the most common cause of generalized malabsorption. Isolated absorption disorders, for (e.g., disaccharidase deficiency), are not associated with pathological fatty acid excretion. Malabsorption occurs in celiac disease and tropical sprue, terminal enteritis, intestinal lymphoma with lymphangiectasia, Whipple’s disease, amyloidosis, scleroderma, dermatitis herpetiformis, and food allergy.

Clinical and laboratory findings

Maldigestion (e.g., in chronic pancreatitis)

The increased excretion of fecal fat occurs in the late stages of chronic pancreatitis, and then, only if > 90% of the exocrine secretory functional performance of the organ has failed. With the demonstration of pancreatic calcification, in a high percentage of the cases, pathological fatty acid excretion is associated. In such cases, the secretin caerulein test is always pathological.

Various gastrointestinal diseases

Liver parenchymal cell damage, bile duct obstruction, overgrowth of the small intestine with colonic flora, diseases of the ileum with decreased bile acid reabsorption. These diseases lead to reduced concentrations of conjugated bile acids in the lumen of the small intestine. While in severe hepatitides and bile duct obstruction generally only mild steatorrhea (< 20 g fecal fat/24 h) develops, severe forms can occur in overgrowth of the small intestine with bacteria of the Bacteroides species. This is also the case in enteral bile acid loss due to a disease of the ileum (decrease in the bile acid pool).

Acute diarrhea, carcinoid syndrome

Due to accelerated intestinal peristalsis, the intraluminal residence time for the digestion and absorption of nutritional components is too short. Steatorrhea normally occurs with less than 20 g of fecal fat/24 hours.

Malabsorption syndrome

Among the biochemical methods for the diagnosis of generalized malabsorption fatty acid excretion, as a direct function test, is of the greatest diagnostic value. If the D-xylose test is also pathological, the diagnostic categorization is by and large assured. Celiac disease is the most common cause of generalized malabsorption. Isolated absorption disorders, for (e.g., disaccharidase deficiency), are not associated with pathological fatty acid excretion. Malabsorption occurs in celiac disease and tropical sprue, terminal enteritis, intestinal lymphoma with lymphangiectasia, Whipple’s disease, amyloidosis, scleroderma, dermatitis herpetiformis, and food allergy.

Table 14.2-11 Severity of exocrine pancreatic insufficiency /8, 10/*

Pancreatic insufficiency	Secretin-caerulein test		Fecal fat determination
Pancreatic insufficiency	Enzyme secretion	Bicarbonate secretion	Fecal fat determination
Mild	Abnormal	Normal	Normal
Moderate	Abnormal	Abnormal	Normal
Severe	Abnormal	Abnormal	Abnormal

* Classification is based on the results of the secretin-caerulein test and the quantitative stool fat determination

Table 14.2-10 Reference intervals for biomarkers of the secretin-cerulein test

30 min. after secretin
Volume	> 67 mL
Bicarbonate concentration	> 70 mmol/L
Bicarbonate secretion	> 6.5 mmol
30 min. following caerulein
Amylase /9/	> 12,000 U/30 min.
Lipase /10/	> 65,000 U/30 min.
Trypsin /11/	> 30 U/30 min.

Values from own working group

Table 14.2-11 Laboratory testing and monitoring of chronic pancreatitis /11/

Laboratory findings
Fat IN STOOL The patient follows a diet containing 100 g of fat daily. Stool collection over a period of 48 to 72 hours. In stage III and IV excretion > 7 g/day. The test should be performed to confirm pancreatic insufficiency if fecal elastase concentrations or vitamin A or E levels are very low in the absence of the classic complex of symptoms of steatorrhea.
Fecal elastase The test is performed annually as a screening test for pancreatic exocrine insufficiency. Concentrations < 200 μg/g stool are abnormal. Concentrations < 50 μg/g stool are predictive of steatorrhea in stage III or IV.
glucose intolerance Loss of islet mass and insulin causes glucose intolerance and eventually diabetes type 3 c.
genetic Mutations Cystic fibrosis transmembrane conductance regulator (CFTR), serine inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC). More than 90% of these cases manifest as apparently sporadic early-onset (< 35 years of age) pancreatitis.

Laboratory findings

Fat IN STOOL

The patient follows a diet containing 100 g of fat daily. Stool collection over a period of 48 to 72 hours. In stage III and IV excretion > 7 g/day. The test should be performed to confirm pancreatic insufficiency if fecal elastase concentrations or vitamin A or E levels are very low in the absence of the classic complex of symptoms of steatorrhea.

Fecal elastase

The test is performed annually as a screening test for pancreatic exocrine insufficiency. Concentrations < 200 μg/g stool are abnormal. Concentrations < 50 μg/g stool are predictive of steatorrhea in stage III or IV.

glucose intolerance

Loss of islet mass and insulin causes glucose intolerance and eventually diabetes type 3 c.

genetic Mutations

Cystic fibrosis transmembrane conductance regulator (CFTR), serine inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC). More than 90% of these cases manifest as apparently sporadic early-onset (< 35 years of age) pancreatitis.

Table 14.3-1 Features and diagnostics tests to differentiate malabsorption and maldigestion Lit. /2/

Features and diagnostic tests	Malabsorption (small intestine)	Maldigestion (pancreas/ biliary tract)
Weight loss	Marked (anorexia)	Mild (low normal weight)
Signs of vitamin deficiency	Smooth tongue margins, cheilitis	Only in malnutrition
Anemia	Frequent, often macrocytic	Not common
Steatorrhea	Moderate (20–35 g)	Marked (40–80 g)
Calcium and magnesium decreased	Common in severe disease	Rare
Vitamin B₁₂ deficiency	In severe ileal disease	Rare
D-xylose test	Abnormal	Normal
NBT-PABA test	Normal	Abnormal
Jejunal mucosa	Abnormal with flattening of villi	Normal

Table 14.3-2 Diseases of the small intestine with global and partial malabsorption /1/

Global malabsorption

Diseases with morphological mucosal changes (Sprue/celiac disease, microvillus inclusion disease)

Diminished absorption surface (short bowel syndrome)

Partial/isolated malabsorption syndrome

Carbohydrate malassimilation
Amino acid absorption disorder
Isolated vitamin B₁₂ absorption disorder
Bile acid loss syndrome
Bacterial overgrowth of the small intestine
Oligosymptomatic sprue
Intestinal protein loss
Steatorrhea in excretory pancreatic insufficiency, cholestasis, bacterial overgrowth, bile acid loss syndrome, gastrinoma, small bowel resection, small intestinal radiation injury, lymph transport disorders

Table 14.3-3 Diseases producing malabsorption

Clinical and laboratory findings
Celiac disease /6/ Celiac disease is an autoimmune-mediated enteropathy that is triggered by the assimilation of an particular dietary factor, namely, gluten from wheat and barley. Immunization occurs via innate and adaptive immune mechanisms. Gluten is not completely cleaved enzymatically in the gastrointestinal tract; rather, peptides like p31–45 mediate contact with the immune system of the intestinal wall, activate it, and lead to a cellular and humoral immune response and inflammation. The consequences are villous atrophy and hyperplasia of the small intestinal mucosa, along with malabsorption; a prompt improvement occurs, however, with gluten-free nutrition. The mucosal changes develop gradually, from mild inflammation (Marsh I) to crypt hyperplasia (Marsh II) and to severe enteropathy (Marsh III). Clinical: the classical diagnostic criteria like chronic diarrhea, severe malabsorption syndrome and childhood development disorders have changed to a considerable extent. Today, oligosymptomatic forms with a more mild constellation, like abdominal symptoms, flatulence, constipation and stomach rumbling, are in prevalent. Anemia and fatigue are also common findings, and isolated iron, folic acid and vitamin B₁₂ malabsorption may also be present without anemia. In addition, extra-intestinal manifestations such as dermatitis herpetiformis, neurological symptoms, articular troubles, gynecological problems, bone symptoms, liver disease and mental disturbances also occur. Of these symptoms, dermatitis herpetiformis can best be assessed objectively. Celiac disease may also occur together with other autoimmune diseases such as autoimmune hepatitis, type 1 diabetes, autoimmune thyroid gland disease, Addison’s disease, selective IgA deficiency and sarcoidosis. Incidence: the prevalence is estimated to be 0.6–1% of the population. Laboratory findings: some 90% of celiac disease patients are HLA-DQ2 positive and the majority of the remaining patients has the HLA-DQ8 haplotype. It should not be forgotten, however, that 30–40% of Europeans have one of these two HLA types. In the diagnostic differentiation of celiac disease from autoimmune enteropathy, giardiasis, Rotavirus-associated gastroenteritis, or a lack of both HLA characteristics rules out celiac disease. Up to 40% of treated patients have a moderate rise in aminotransferases with no significant histological liver findings. In one study /7/, 34% of untreated celiac disease patients manifested anemia which was in 53% of the cases due to a deficiency in iron and/or folic acid or vitamin B₁₂. In suspicion of celiac disease, the determination of anti-trans glutaminase 2 antibodies and of antibodies against endomysium is a meaningful diagnostic approach (see Section 25.10 – Antibodies in gastrointestinal diseases). The diagnostic sensitivity is 90–100%, with a specificity of 100%. Patients with mild celiac disease and antibodies against endomysium in the immunofluorescence test with a titer of ≥ 1 : 5, but a still normal villous structure, already manifest gastrointestinal symptoms, which improve with a gluten-free diet /8/.
Carbohydrate malassimilation /9, 10/ The most important carbohydrates that routinely cause clinical abdominal complaints are lactose, fructose, and the sugar alcohol sorbitol. For further information see Section 14.3.3 – D-xylose test.
Whipple’s disease Whipple’s disease is relatively rare and occurs at any age. The mucosa of the small intestine is infiltrated by polygonal macrophages, so-called SPC cells, which have granular or sickle-shaped inclusions and contain bacteria. Many other organs, apart from the small intestine, are also affected. Chronic diarrhea is a late symptom. The diagnosis is made with a small intestine biopsy.
Bacterial overgrowth of the small intestine /1/ The causes are anatomical changes in the small intestine, such as fistulae, strictures, diverticula, stenoses and motility disorders. The bacteria cause de conjugation and dehydroxylation of the bile acids, and the secondary bile acids that are formed are toxic to the intestinal epithelium. Since the amount of conjugated bile acids is decreased due to the bacterial effect, malabsorption of fats and of the fat soluble vitamins A, D, E and K results. Furthermore, the nutritional carbohydrates are fermented prematurely by the bacteria. Laboratory findings: D-xylose test positive, H₂ breath test following 75 g of glucose positive, ¹⁴C/¹³C-breath test, vitamin B₁₂ test (Schilling test) positive /11/.
Protein loosing enteropathy /1/ Gastrointestinal diseases can lead to loss of protein, exceeding 10–20% of normal albumin turnover. The result is a decrease in plasma proteins with long half-life times, such as albumin and IgG. Clinical symptoms are abdominal pain, diarrhea, peripheral edema and dystrophic yellow nails. Laboratory findings: hypoalbuminemia, reduced total protein, PT, APTT and fibrinogen normal, fecal fat excretion abnormal, α₁-antitrypsin clearance elevated, ⁵¹Cr-albumin test abnormal.
AIDS /1/ Approximately half of AIDS patients have diarrhea during the course of their disease, in most cases the diarrhea is due to infection, while the remaining cases are caused by side effects of medication, or are idiopathic. Common pathogens are Microsporidia species (10–30%), Cryptosporidia species (4–16%), Mycobacterium avium-intracellulare and Lamblia species.
Inflammatory bowel disease – Generally Crohn’s disease and ulcerative colitis are the two idiopathic forms of chronic inflammatory bowel disease (IBD). Patients with IBD are at elevated risk of developing primary sclerosing cholangitis, ankylosing spondylitis, and psoriasis. More than 30 genes that are associated with both diseases have so far been identified /12/. In the USA, 1.4 million residents have IBD. Crohn’s disease and ulcerative colitis can develop extra-intestinal manifestations (musculoskeletal, dermatological, ocular and hepatobiliary) /5/.
– Crohn’s disease (MC) Crohn’s disease generally affects the ileum and the colon, but it can impact all regions of the small intestine intermittently. The inflammation is often transmural and the disease is associated with granuloma, strictures and fistulae. Cigarette smokers have a higher risk of MC than of ulcerative colitis. Affected are mainly Caucasians, in particular in Europe and the USA with a prevalence of 26–199 per 100,000 general population. The first gene that was identified in MC is the NOD2 (nucleotide-binding oligomerization domain containing 2; alias CARD 15) gene. The gene is involved in the innate immune response that protects bacterial cell walls. More than 30 variants of this gene have been identified. The following three variants, which comprise 82% of the variations that are associated with MC, have been identified /13/: A single nucleotide polymorphism, which codes for an Arg702Trp substitution A single nucleotide polymorphism, which codes for a Gly908Arg substitution A frame shift polymorphism Leu1007fsinsC. The product of the NOD2 gene, the NOD protein, is present in many defense cells and is a pattern recognition receptor of muramyl dipeptide (MDP), the bacterial degradation product of peptidoglycans. The perception of MDP stimulates the secretion of antibacterial peptides (defensins) by the paneth cells of the small intestine, and in this way protects the host against bacterial invasion. Mutations of the NOD gene result in diminished secretion of defensins /12/.
– Ulcerative colitis Ulcerative colitis affects the colon and the rectum; the colon may be completely or only partially impacted. The inflammation is limited to the mucosa; neither strictures nor fistulae nor granuloma develop. Ulcerative colitis and MC manifest considerable genetic association; one of the few exceptions is represented by the mutations in the NOD2 gene /12/. Differences are also seen in the presence of ANCA-positivity (ulcerative colitis) and ASCA-positivity (MC).

Clinical and laboratory findings

Celiac disease /6/

Celiac disease is an autoimmune-mediated enteropathy that is triggered by the assimilation of an particular dietary factor, namely, gluten from wheat and barley. Immunization occurs via innate and adaptive immune mechanisms. Gluten is not completely cleaved enzymatically in the gastrointestinal tract; rather, peptides like p31–45 mediate contact with the immune system of the intestinal wall, activate it, and lead to a cellular and humoral immune response and inflammation. The consequences are villous atrophy and hyperplasia of the small intestinal mucosa, along with malabsorption; a prompt improvement occurs, however, with gluten-free nutrition. The mucosal changes develop gradually, from mild inflammation (Marsh I) to crypt hyperplasia (Marsh II) and to severe enteropathy (Marsh III).

Clinical: the classical diagnostic criteria like chronic diarrhea, severe malabsorption syndrome and childhood development disorders have changed to a considerable extent. Today, oligosymptomatic forms with a more mild constellation, like abdominal symptoms, flatulence, constipation and stomach rumbling, are in prevalent. Anemia and fatigue are also common findings, and isolated iron, folic acid and vitamin B₁₂ malabsorption may also be present without anemia. In addition, extra-intestinal manifestations such as dermatitis herpetiformis, neurological symptoms, articular troubles, gynecological problems, bone symptoms, liver disease and mental disturbances also occur. Of these symptoms, dermatitis herpetiformis can best be assessed objectively. Celiac disease may also occur together with other autoimmune diseases such as autoimmune hepatitis, type 1 diabetes, autoimmune thyroid gland disease, Addison’s disease, selective IgA deficiency and sarcoidosis.

Incidence: the prevalence is estimated to be 0.6–1% of the population.

Laboratory findings: some 90% of celiac disease patients are HLA-DQ2 positive and the majority of the remaining patients has the HLA-DQ8 haplotype. It should not be forgotten, however, that 30–40% of Europeans have one of these two HLA types. In the diagnostic differentiation of celiac disease from autoimmune enteropathy, giardiasis, Rotavirus-associated gastroenteritis, or a lack of both HLA characteristics rules out celiac disease. Up to 40% of treated patients have a moderate rise in aminotransferases with no significant histological liver findings. In one study /7/, 34% of untreated celiac disease patients manifested anemia which was in 53% of the cases due to a deficiency in iron and/or folic acid or vitamin B₁₂. In suspicion of celiac disease, the determination of anti-trans glutaminase 2 antibodies and of antibodies against endomysium is a meaningful diagnostic approach (see Section 25.10 – Antibodies in gastrointestinal diseases). The diagnostic sensitivity is 90–100%, with a specificity of 100%. Patients with mild celiac disease and antibodies against endomysium in the immunofluorescence test with a titer of ≥ 1 : 5, but a still normal villous structure, already manifest gastrointestinal symptoms, which improve with a gluten-free diet /8/.

Carbohydrate malassimilation /9, 10/

The most important carbohydrates that routinely cause clinical abdominal complaints are lactose, fructose, and the sugar alcohol sorbitol. For further information see Section 14.3.3 – D-xylose test.

Whipple’s disease

Whipple’s disease is relatively rare and occurs at any age. The mucosa of the small intestine is infiltrated by polygonal macrophages, so-called SPC cells, which have granular or sickle-shaped inclusions and contain bacteria. Many other organs, apart from the small intestine, are also affected. Chronic diarrhea is a late symptom. The diagnosis is made with a small intestine biopsy.

Bacterial overgrowth of the small intestine /1/

The causes are anatomical changes in the small intestine, such as fistulae, strictures, diverticula, stenoses and motility disorders. The bacteria cause de conjugation and dehydroxylation of the bile acids, and the secondary bile acids that are formed are toxic to the intestinal epithelium. Since the amount of conjugated bile acids is decreased due to the bacterial effect, malabsorption of fats and of the fat soluble vitamins A, D, E and K results. Furthermore, the nutritional carbohydrates are fermented prematurely by the bacteria.

Laboratory findings: D-xylose test positive, H₂ breath test following 75 g of glucose positive, ¹⁴C/¹³C-breath test, vitamin B₁₂ test (Schilling test) positive /11/.

Protein loosing enteropathy /1/

Gastrointestinal diseases can lead to loss of protein, exceeding 10–20% of normal albumin turnover. The result is a decrease in plasma proteins with long half-life times, such as albumin and IgG. Clinical symptoms are abdominal pain, diarrhea, peripheral edema and dystrophic yellow nails.

Laboratory findings: hypoalbuminemia, reduced total protein, PT, APTT and fibrinogen normal, fecal fat excretion abnormal, α₁-antitrypsin clearance elevated, ⁵¹Cr-albumin test abnormal.

AIDS /1/

Approximately half of AIDS patients have diarrhea during the course of their disease, in most cases the diarrhea is due to infection, while the remaining cases are caused by side effects of medication, or are idiopathic. Common pathogens are Microsporidia species (10–30%), Cryptosporidia species (4–16%), Mycobacterium avium-intracellulare and Lamblia species.

Inflammatory bowel disease – Generally

Crohn’s disease and ulcerative colitis are the two idiopathic forms of chronic inflammatory bowel disease (IBD). Patients with IBD are at elevated risk of developing primary sclerosing cholangitis, ankylosing spondylitis, and psoriasis. More than 30 genes that are associated with both diseases have so far been identified /12/. In the USA, 1.4 million residents have IBD. Crohn’s disease and ulcerative colitis can develop extra-intestinal manifestations (musculoskeletal, dermatological, ocular and hepatobiliary) /5/.

– Crohn’s disease (MC)

Crohn’s disease generally affects the ileum and the colon, but it can impact all regions of the small intestine intermittently. The inflammation is often transmural and the disease is associated with granuloma, strictures and fistulae. Cigarette smokers have a higher risk of MC than of ulcerative colitis. Affected are mainly Caucasians, in particular in Europe and the USA with a prevalence of 26–199 per 100,000 general population. The first gene that was identified in MC is the NOD2 (nucleotide-binding oligomerization domain containing 2; alias CARD 15) gene. The gene is involved in the innate immune response that protects bacterial cell walls. More than 30 variants of this gene have been identified. The following three variants, which comprise 82% of the variations that are associated with MC, have been identified /13/:

A single nucleotide polymorphism, which codes for an Arg702Trp substitution
A single nucleotide polymorphism, which codes for a Gly908Arg substitution
A frame shift polymorphism Leu1007fsinsC.

The product of the NOD2 gene, the NOD protein, is present in many defense cells and is a pattern recognition receptor of muramyl dipeptide (MDP), the bacterial degradation product of peptidoglycans. The perception of MDP stimulates the secretion of antibacterial peptides (defensins) by the paneth cells of the small intestine, and in this way protects the host against bacterial invasion. Mutations of the NOD gene result in diminished secretion of defensins /12/.

– Ulcerative colitis

Ulcerative colitis affects the colon and the rectum; the colon may be completely or only partially impacted. The inflammation is limited to the mucosa; neither strictures nor fistulae nor granuloma develop. Ulcerative colitis and MC manifest considerable genetic association; one of the few exceptions is represented by the mutations in the NOD2 gene /12/. Differences are also seen in the presence of ANCA-positivity (ulcerative colitis) and ASCA-positivity (MC).

Table 14.3-4 Routine laboratory test results in malabsorption

Examination	Test results in malabsorption
Complete blood count	Frequently moderate microcytic anemia, less frequently anemia of chronic disease (normocytic) or macrocytic anemia.
Ferritin	Reduced in microcytic anemia due to deficient iron absorption via the divalent metal ion transporter.
Folic acid	Often at the lower limit of the reference interval or reduced, particularly in bacterial overgrowth.
Vitamin B₁₂	Often at the lower limit of the reference interval or reduced, particularly in bacterial overgrowth.
Calcium, magnesium	Reduced if total protein is reduced or due to deficient absorption via the divalent metal ion transporter.
Alkaline phosphatase	Elevated due to accumulation of ALP from the small intestine, or with decreased 25(OH)D absorption.
Total protein, albumin	Decreased or at the lower limit of the reference interval.
Prothrombin	Reduced in global malabsorption.
C-reactive protein	Frequently elevated in inflammatory intestinal disease such as Crohn’s disease and ulcerative colitis.

Table 14.3-5 Tests of digestive-absorptive function

Test	Clinical and laboratory findings
Stool weight	Thin or watery stools more than 3 times daily or stool weight of above 200 g/24 h are indicative of diarrhea.
Quantitative fecal fat analysis	Fecal fat analysis is an important test to identify maldigestion or malabsorption.
D-xylose test	Function test indicated whenever quantitative fecal fat analysis is abnormal. The D-xylose test localizes the abnormality to the small intestine.
Hydrogen breath test	Functional test for the assessment of gastrointestinal carbohydrate intolerance.
Lactose tolerance test	The lactose tolerance test is a functional test for the detection of lactose intolerance.
Schilling test	Functional test of ileal disease, and for the diagnostic investigation of bacterial overgrowth.
Bile acid breath test	¹⁴C-glycocholic acid will be de conjugated, metabolized and excreted via the lungs as ¹⁴CO₂. Indicated in patients with steatorrhea caused by suspected bacterial overgrowth
α₁-antitrypsin clearance, ⁵¹Cr-albumin test	Investigations for the detection of intestinal protein loss syndrome and intestinal lymphangiectasia.
Serological tests	Antibodies against endomysium and trans glutaminase 2 are indicative of celiac disease (see Section 25.10 – Antibodies in gastrointestinal diseases).

Table 14.3-6 Reference intervals of D-xylose test /4/

Urine	> 4 g/5 h (26.6 mmol/5 h)*
Serum 15 min.	> 10 mg/dL (0.67 mmol/L)
or 1 h	> 30 mg/dL (2.0 mmol/L)
or 2 h	> 30 mg/dL (2.0 mmol/L)

* Corresponds to > 16% of the ingested amount.

Conversion: mmol = g × 6.66; mmol/L = mg/dL × 0.0666

Table 14.3-7 Gastrointestinal diseases with reduced D-xylose excretion /5, 6, 7/

Clinical and laboratory findings
Non tropical sprue (celiac disease), tropical sprue A reduced excretion of D-xylose in conjunction with steatorrhea indicates underlying enteral causes in the presence of (global) malabsorption syndrome; celiac disease is the most common cause. Furthermore, from a differential diagnostic point of view, the rare diseases mentioned below need to be considered. In some cases it is unknown whether an abnormal D-xylose test should be interpreted as an artifact or as the pathophysiological sequela of a primary disease (e.g., Zollinger-Ellison syndrome or carcinoid syndrome). The diagnostic sensitivity of the D-xylose test is 94% for celiac disease, according to a large-scale statistical review, while tropical sprue is associated with an abnormal D-xylose test in 96% of cases. Rarer diseases associated with an abnormal D-xylose test include: amyloidosis, small bowel resection/bypass surgery, intestinal lymphoma, scleroderma, radiation enteritis, malabsorption in conjunction with mucosal damage due to medications (e.g., neomycin), Whipple’s disease, herpetiform dermatitis, carcinoid syndrome, Zollinger-Ellison syndrome.
Bacterial overgrowth of the small intestine Due to the bacterial effect, the quantity of conjugated bile acids is decreased, resulting in malabsorption of fats and of the fat soluble vitamins A, D, E and K. In addition, D-xylose is fermented by bacteria, leading to a positive test result.
AIDS AIDS patients develop severe chronic diarrhea and suffer from loss of weight. The absorptive defect encompasses the jejunum and the ileum, and this is documented in the form of an absorption disorder of D-xylose and vitamin B₁₂. Patients with weight loss have a severe D-xylose absorption disorder, while those without weight loss have a normal test result /8/.

Clinical and laboratory findings

Non tropical sprue (celiac disease), tropical sprue

A reduced excretion of D-xylose in conjunction with steatorrhea indicates underlying enteral causes in the presence of (global) malabsorption syndrome; celiac disease is the most common cause. Furthermore, from a differential diagnostic point of view, the rare diseases mentioned below need to be considered. In some cases it is unknown whether an abnormal D-xylose test should be interpreted as an artifact or as the pathophysiological sequela of a primary disease (e.g., Zollinger-Ellison syndrome or carcinoid syndrome). The diagnostic sensitivity of the D-xylose test is 94% for celiac disease, according to a large-scale statistical review, while tropical sprue is associated with an abnormal D-xylose test in 96% of cases.

Rarer diseases associated with an abnormal D-xylose test include: amyloidosis, small bowel resection/bypass surgery, intestinal lymphoma, scleroderma, radiation enteritis, malabsorption in conjunction with mucosal damage due to medications (e.g., neomycin), Whipple’s disease, herpetiform dermatitis, carcinoid syndrome, Zollinger-Ellison syndrome.

Bacterial overgrowth of the small intestine

Due to the bacterial effect, the quantity of conjugated bile acids is decreased, resulting in malabsorption of fats and of the fat soluble vitamins A, D, E and K. In addition, D-xylose is fermented by bacteria, leading to a positive test result.

AIDS

AIDS patients develop severe chronic diarrhea and suffer from loss of weight. The absorptive defect encompasses the jejunum and the ileum, and this is documented in the form of an absorption disorder of D-xylose and vitamin B₁₂. Patients with weight loss have a severe D-xylose absorption disorder, while those without weight loss have a normal test result /8/.

Table 14.3-8 Disaccharides present in the diet

Diet	Disacchar- ides	Enzymes	Monosacchar- ides
Starch	Maltose	Maltase	Glucose (2 molecules)
Table sugar	Sucrose	Isomaltase	Glucose + fructose
Milk	Lactose	Lactase	Glucose + galactose

Table 14.3-9 Foods that can cause malassimilation of monosaccharides and disaccharides

Carbohydrates	Foods
Lactose	Dairy products, cheese, cappuccino, condensed milk, chocolate, instant meals
Fructose	Fruit (apple), raisins, honey, potatoes, fruit juices, soft drinks, chocolate, instant meals
Sorbitol, Xylitol	Grapes, pears, plums, peaches, dates (fresh and dried), diabetic food, sugar substitutes, candy, chewing gum

Table 14.3-10 Primary and secondary carbohydrate malabsorption

Clinical and laboratory findings
Primary lactose intolerance /10/ The milk sugar lactose is hydrolyzed to glucose and galactose by the enzyme lactase (β-galactosidase), which is located at the intestinal brush border membrane of the small intestine enterocytes. In the next step the monosaccharides are absorbed. In two thirds of children, lactase activity falls during the second to the fifth year of life to a level that is insufficient for the ingested lactose. This primary lactase deficiency is ethnically determined. It occurs in 70–100% of children in Asia and Africa, in 15–80% in the USA, and in 15–20% in Germany. The inheritance pattern of primary lactase deficiency is autosomal dominant. Following the nursing of the baby the activity of lactase-phlorizin hydrolase (LPH) falls progressively; this process is termed lactose intolerance. The cause is the LCT_{(T/C–13910)} polymorphism of the Lactase gene (LCT) on chromosome 2q21 /11/. Besides lactose intolerance, individuals with the CC genotype have reduced enteral calcium absorption as well as reduced bone density which, especially after menopause, leads to bone fractures /12/. In malabsorption, a proportion of lactose escapes hydrolysis in the small intestine and reaches the colon. Lactose is osmotically active in the colon and undergoes bacterial transformation to CO₂, methane and short chain fatty acids. This leads to flatulence, meteorism, diarrhea and abdominal pain. There is a negative correlation between age of the individual and intestinal mucosal lactase activity, resulting in an increase in the prevalence of lactose malabsorption in elderly people. Adults with primary lactase deficiency do not manifest nutritional deficiencies, so that their clinical symptoms, which may vary markedly in severity, can easily be misinterpreted as a functional gastrointestinal disorder. Laboratory findings: the hydrogen breath test with 50 g of lactose (children 2 g/kg BW) is performed. The diagnostic sensitivity is 90–95%, with a specificity of 95–100%. The diagnostic sensitivity of the lactose tolerance test is maximally 75%, with a diagnostic specificity of 83% /13/. The demonstration of a gene mutation does not explain whether or not, and as of what dose, lactose malabsorption occurs.
Secondary lactose intolerance In lactose intolerance a reduction in other intestinal mucosal enzymes is also often found, but lactase is the most sensitive; lactose malabsorption therefore often accompanies intestinal diseases. The symptoms, which are caused by disaccharide malabsorption, often remain undetected due to the underlying disease. The lactose intolerance is thereby identified through the course of the underlying disease, but it may persist even following mucosal restitution. Secondary lactose intolerance is the consequence of a disturbance of the mucosal integrity for (e.g., in celiac disease, tropical sprue, intestinal lymphoma, Whipple’s disease, genuine intestinal lymphangiectasia, short bowel syndrome, A-β-lipoproteinemia, blind loop syndrome, radiation enteritis, nonspecific infectious diarrhea in children, or with neomycin, colchicine, cytostatic (methotrexate) therapy) /13/. Lactose intolerance is a common problem in active Crohn’s disease; it can be confirmed with the hydrogen breath test. However, the symptomatology is not due solely to a lactase deficiency, which is why a mucosal biopsy is not recommended /14/.
Primary fructose malabsorption /15/ The monosaccharide fructose is taken up in the brush border membrane of the enterocyte by the GLUT-5 transporter and released basolaterally by the GLUT-2 transporter into the blood. Under loading with 30–50 g fructose per hour, the uptake capacity of the GLUT-5 transporter is saturated and fructose reaches the colon. There fructose, independent of the dose, exerts osmotic effects and leads to bacterial overgrowth and the formation of short chain fatty acids, methane and CO₂. The uptake of sorbitol leads to the amplification of the fructose absorption disorder, because sorbitol is transformed to fructose and blocks the GLUT-5 transporter. Fructose malabsorption is not to be mistaken for fructose intolerance, which is a metabolic disease caused by a deficiency in fructose-1-phosphate aldolase. Laboratory findings: the hydrogen breath test with 25 g of fructose is performed. The diagnostic sensitivity and specificity is 80–90% respectively. Important in the assessment is the presence of abdominal discomfort during the test. If there is no abdominal discomfort, asymptomatic fructose malabsorption, in which symptoms only appear with higher doses of fructose, is present.
Secondary fructose malabsorption Apart from fructose malabsorption, intestinal diseases may be important causes of secondary malabsorption. For further disorders see secondary lactose malabsorption.
Sorbitol malabsorption /16/ Sorbitol, a 6-valent sugar alcohol (E 420), is found in fruits. Fructose and sorbitol have become important following recommendations to increase fruit and vegetable consumption, and also as a result of their use as sweeteners in dietary preparations and so-called sugar-free sweets /6/. In addition sorbitol is used in view of its hygroscopic properties, as a humectant. Sorbitol is passively absorbed in the small intestine, and directly inhibits the GLUT-5 transporter. The result is diminished fructose absorption. Sorbitol and fructose malabsorption often occur together, and the clinical symptomatology of both of these disorders is similar. Laboratory findings: the hydrogen breath test with 5–10 g of sorbitol is performed. Important in the assessment is the presence of abdominal discomfort during the test. If 25 g of each of sorbitol, xylitol and fructose are administered, the malabsorption rates of sorbitol, fructose and xylitol in malabsorption patients are 84%, 36% and 12%, respectively /17/.

Clinical and laboratory findings

Primary lactose intolerance /10/

The milk sugar lactose is hydrolyzed to glucose and galactose by the enzyme lactase (β-galactosidase), which is located at the intestinal brush border membrane of the small intestine enterocytes. In the next step the monosaccharides are absorbed. In two thirds of children, lactase activity falls during the second to the fifth year of life to a level that is insufficient for the ingested lactose. This primary lactase deficiency is ethnically determined. It occurs in 70–100% of children in Asia and Africa, in 15–80% in the USA, and in 15–20% in Germany. The inheritance pattern of primary lactase deficiency is autosomal dominant. Following the nursing of the baby the activity of lactase-phlorizin hydrolase (LPH) falls progressively; this process is termed lactose intolerance. The cause is the LCT_{(T/C–13910)} polymorphism of the Lactase gene (LCT) on chromosome 2q21 /11/. Besides lactose intolerance, individuals with the CC genotype have reduced enteral calcium absorption as well as reduced bone density which, especially after menopause, leads to bone fractures /12/.

In malabsorption, a proportion of lactose escapes hydrolysis in the small intestine and reaches the colon. Lactose is osmotically active in the colon and undergoes bacterial transformation to CO₂, methane and short chain fatty acids. This leads to flatulence, meteorism, diarrhea and abdominal pain. There is a negative correlation between age of the individual and intestinal mucosal lactase activity, resulting in an increase in the prevalence of lactose malabsorption in elderly people. Adults with primary lactase deficiency do not manifest nutritional deficiencies, so that their clinical symptoms, which may vary markedly in severity, can easily be misinterpreted as a functional gastrointestinal disorder.

Laboratory findings: the hydrogen breath test with 50 g of lactose (children 2 g/kg BW) is performed. The diagnostic sensitivity is 90–95%, with a specificity of 95–100%. The diagnostic sensitivity of the lactose tolerance test is maximally 75%, with a diagnostic specificity of 83% /13/. The demonstration of a gene mutation does not explain whether or not, and as of what dose, lactose malabsorption occurs.

Secondary lactose intolerance

In lactose intolerance a reduction in other intestinal mucosal enzymes is also often found, but lactase is the most sensitive; lactose malabsorption therefore often accompanies intestinal diseases. The symptoms, which are caused by disaccharide malabsorption, often remain undetected due to the underlying disease. The lactose intolerance is thereby identified through the course of the underlying disease, but it may persist even following mucosal restitution. Secondary lactose intolerance is the consequence of a disturbance of the mucosal integrity for (e.g., in celiac disease, tropical sprue, intestinal lymphoma, Whipple’s disease, genuine intestinal lymphangiectasia, short bowel syndrome, A-β-lipoproteinemia, blind loop syndrome, radiation enteritis, nonspecific infectious diarrhea in children, or with neomycin, colchicine, cytostatic (methotrexate) therapy) /13/. Lactose intolerance is a common problem in active Crohn’s disease; it can be confirmed with the hydrogen breath test. However, the symptomatology is not due solely to a lactase deficiency, which is why a mucosal biopsy is not recommended /14/.

Primary fructose malabsorption /15/

The monosaccharide fructose is taken up in the brush border membrane of the enterocyte by the GLUT-5 transporter and released basolaterally by the GLUT-2 transporter into the blood. Under loading with 30–50 g fructose per hour, the uptake capacity of the GLUT-5 transporter is saturated and fructose reaches the colon. There fructose, independent of the dose, exerts osmotic effects and leads to bacterial overgrowth and the formation of short chain fatty acids, methane and CO₂. The uptake of sorbitol leads to the amplification of the fructose absorption disorder, because sorbitol is transformed to fructose and blocks the GLUT-5 transporter. Fructose malabsorption is not to be mistaken for fructose intolerance, which is a metabolic disease caused by a deficiency in fructose-1-phosphate aldolase.

Laboratory findings: the hydrogen breath test with 25 g of fructose is performed. The diagnostic sensitivity and specificity is 80–90% respectively. Important in the assessment is the presence of abdominal discomfort during the test. If there is no abdominal discomfort, asymptomatic fructose malabsorption, in which symptoms only appear with higher doses of fructose, is present.

Secondary fructose malabsorption

Apart from fructose malabsorption, intestinal diseases may be important causes of secondary malabsorption. For further disorders see secondary lactose malabsorption.

Sorbitol malabsorption /16/

Sorbitol, a 6-valent sugar alcohol (E 420), is found in fruits. Fructose and sorbitol have become important following recommendations to increase fruit and vegetable consumption, and also as a result of their use as sweeteners in dietary preparations and so-called sugar-free sweets /6/. In addition sorbitol is used in view of its hygroscopic properties, as a humectant. Sorbitol is passively absorbed in the small intestine, and directly inhibits the GLUT-5 transporter. The result is diminished fructose absorption. Sorbitol and fructose malabsorption often occur together, and the clinical symptomatology of both of these disorders is similar.

Laboratory findings: the hydrogen breath test with 5–10 g of sorbitol is performed. Important in the assessment is the presence of abdominal discomfort during the test. If 25 g of each of sorbitol, xylitol and fructose are administered, the malabsorption rates of sorbitol, fructose and xylitol in malabsorption patients are 84%, 36% and 12%, respectively /17/.

Table 14.3-11 The Rome IV diagnostic criteria for irritable bowel syndrome /3/

Recurrent abdominal pain, on average, at least 1 day per week in the last 3 months, associated with two or more of the following criteria:

Related to defecation
Associated with a change in frequency of stool
Associated with a change in form (appearance) of stool

Criteria fulfilled for the last 3 months with symptom onset at least 6 months before diagnosis.

Table 14.3-12 Fecal calprotectin (f-Cp) in healthy controls and in disease groups /2/

Patient group comparison of calprotectin	f-Cp difference (μg/g stool)	95% CV (μg/g stool)	p-value
Inflammatory bowel disease vs. normal	219.23	174.49– 263.97	< 0.001
Crohn’s disease vs. normal	170.14	162.06– 178.21	< 0.001
Ulcerative colitis vs. normal	186.19	45.59– 326.80	0.009
Irritable bowel syndrome vs. normal	–4.01	–21.63– 13.61	0.660
Colorectal cancer vs. normal	132.19	–59.18– 323.56	0.180
Crohn’s disease vs. ulcerative colitis	50.73	3.33– 98.14	0.040
Crohn’s disease vs. irritable bowel syndrome	303.67	167.50– 439.83	< 0.001

Table 14.3-13 Fecal calprotectin (f-Cp) in functional and inflammatory intestinal diseases

Clinical and laboratory findings
Screening of patients with suspected inflammatory bowel disease (IBD) /6/ Suspicion of IBD (Crohn’s disease and ulcerative colitis) raises in patients with persistent (≥ 4 weeks) or recurrent periods (≥ 2 episodes in 6 months) of abdominal pain and diarrhea. In addition, rectal bleeding, weight loss or anemia increase the likelihood of IBD. The irritable bowel syndrome (IBS) is characterized by abdominal pain and intestinal discomfort without organic causes. In a meta-analysis /6/ the diagnostic value of f-CP (cutoff 50 μg/g stool) as a screening test, in comparison with endoscopy, was evaluated with the purpose of establishing whether the implementation of the test before endoscopy could reduce the number of endoscopies performed for the diagnosis of IBD. In the adult studies the pooled sensitivity of f-Cp testing was 0.93 (95% confidence interval 0.85 to 0.97) and the pooled specificity was 0.96 (0.79 to 0.99). The corresponding values in the studies of children and teenagers were 0.92 (0.84 to 0.96) and 0.76 (0.62 to 0.86). Because the predictive value of a positive f-CP result corresponds to the prevalence of patients with IBD, the test cannot be recommended in tertiary care facilities. For example /6/, on decreasing the prevalence in adults from 32% to 5%, the positive predictive value of the f-CP test decreases to 55%, whereas the negative predictive value increases above 99.8%. However, at a tertiary care level a diagnostic test with a high positive likelihood ratio is preferred. In children, in whom IBS is much less frequent than in adults, and in whom an IBD prevalence of 61 % can be expected, an abnormal test result for f-CP increases the probability to 86 % whereas a normal test result for f-CP reduces the probability to 15%. A further meta-analysis /2/ showed that f-CP differentiated well patients with IBD, but that a cutoff of 100 μg/g stool distinguished with better accuracy than a cutoff of 50 μg/g stool. F-CP values in the different intestinal diseases are listed in Tab. 14.3-12 – Fecal calprotectin in healthy controls and in disease groups. In a major study /3/, the differentiation of IBS from IBD on the basis of the Rome I criteria, and from CRP and the erythrocyte sedimentation rate (ESR), compared to endoscopy, was investigated. The diagnostic sensitivity of the Rome I criteria for the identification of IBS was 85%, with a specificity of 79%. The f-CP odds ratio (cutoff 50 μg/g stool) for the diagnosis of IBD was 27.8 in comparison to elevated CRP with 4.2 and an elevated ESR with 3.2.
Irritable bowel syndrome (IBS) /2/ In a meta-analysis, there was no difference in f-CP values between healthy individuals and IBS (Tab. 14.3-12 – Fecal calprotectin in healthy controls and in disease groups).
Crohn’s disease, ulcerative colitis /2/ Both IBDs show significantly higher f-CP values than healthy individuals and patients with IBS. However, differentiation of Crohn’s disease from ulcerative colitis is not possible with f-CP.
Inflammatory bowel disease patients in clinical remission /10/ Gastrointestinal symptoms in patients with IBD in apparent remission can reflect the coexistence of IBS or subclinical inflammation. Conventional markers of inflammatory activity, such as CRP, frequently lack sufficient diagnostic sensitivity to detect low-grade inflammation and to help clinicians to differentiate subclinical IBD from coexisting IBS. In a study /8/, the Rome-criteria for IBS were fulfilled in remission in 59.7% and 38.6% of Crohn’s patients and ulcerative colitis patients, respectively. The f-CP concentrations (μg/g) were: In Crohn’s patients in remission with gastrointestinal symptoms 414.7 ± 80.3 and in patients without symptoms 174 ± 49.1. In ulcerative colitis patients in remission with gastrointestinal symptoms 591.1 ± 172.5 and in patients without symptoms 229.8 ± 83.4. The study shows that IBS symptoms are common among IBD patients in apparent remission and such symptoms reflect ongoing IBD activity. When in clinical remission, 35% of children with IBD had normal f-CP (below 100 μg/g) and 13% a very high level (over 1,000 μg/g) without reporting symptoms. A clinical relapse occurred within 12 months in one third of the children. When in clinical remission, the positive predictive value of f-CP for an overt relapse was low ranging from 0.396 to 0.429 for f-CP values > 100 μg/g or > 1,000 μg/g respectively. The negative predictive value was 0.75 for < 100 μg/g /11/. In pediatric IBD, subjective symptoms and clinical assessment associate poorly with the concentration of f-CP.
Steroid refractoriness in severe pediatric ulcerative colitis Four fecal markers were investigated for their ability to predict steroid refractoriness in severe pediatric ulcerative colitis. Median values were very high at baseline for f-CP (4,215 μg/g), lactoferrin, M2 pyruvate kinase (M2-PK) and S100A12. M2-PK was numerically superior to the other three markers and CRP /12/.
Colon carcinoma /2/ F-CP can be elevated in colon carcinoma, but the diagnostic sensitivity is only 36% with a specificity of 71%.
Bacterial diarrhea In patients with diarrhea, a positive f-CP test indicates bacterial etiology with a diagnostic sensitivity of 83% with a specificity of 54%.
Irritable Bowel syndrome Irritable bowel syndrome (IBS) is a chronic gastrointestinal symptom complex defined by abdominal pain and disturbed bowel habits over 3 months within a period of 6 months, in the absence of any identifiable organic pathology. Various functional abnormalities in the gastrointestinal tract and in the brain occur as a consequence of disease development. F-CP is not elevated /14/.

Clinical and laboratory findings

Screening of patients with suspected inflammatory bowel disease (IBD) /6/

Suspicion of IBD (Crohn’s disease and ulcerative colitis) raises in patients with persistent (≥ 4 weeks) or recurrent periods (≥ 2 episodes in 6 months) of abdominal pain and diarrhea. In addition, rectal bleeding, weight loss or anemia increase the likelihood of IBD. The irritable bowel syndrome (IBS) is characterized by abdominal pain and intestinal discomfort without organic causes.

In a meta-analysis /6/ the diagnostic value of f-CP (cutoff 50 μg/g stool) as a screening test, in comparison with endoscopy, was evaluated with the purpose of establishing whether the implementation of the test before endoscopy could reduce the number of endoscopies performed for the diagnosis of IBD. In the adult studies the pooled sensitivity of f-Cp testing was 0.93 (95% confidence interval 0.85 to 0.97) and the pooled specificity was 0.96 (0.79 to 0.99). The corresponding values in the studies of children and teenagers were 0.92 (0.84 to 0.96) and 0.76 (0.62 to 0.86).

Because the predictive value of a positive f-CP result corresponds to the prevalence of patients with IBD, the test cannot be recommended in tertiary care facilities. For example /6/, on decreasing the prevalence in adults from 32% to 5%, the positive predictive value of the f-CP test decreases to 55%, whereas the negative predictive value increases above 99.8%. However, at a tertiary care level a diagnostic test with a high positive likelihood ratio is preferred. In children, in whom IBS is much less frequent than in adults, and in whom an IBD prevalence of 61 % can be expected, an abnormal test result for f-CP increases the probability to 86 % whereas a normal test result for f-CP reduces the probability to 15%.

A further meta-analysis /2/ showed that f-CP differentiated well patients with IBD, but that a cutoff of 100 μg/g stool distinguished with better accuracy than a cutoff of 50 μg/g stool. F-CP values in the different intestinal diseases are listed in Tab. 14.3-12 – Fecal calprotectin in healthy controls and in disease groups.

In a major study /3/, the differentiation of IBS from IBD on the basis of the Rome I criteria, and from CRP and the erythrocyte sedimentation rate (ESR), compared to endoscopy, was investigated. The diagnostic sensitivity of the Rome I criteria for the identification of IBS was 85%, with a specificity of 79%. The f-CP odds ratio (cutoff 50 μg/g stool) for the diagnosis of IBD was 27.8 in comparison to elevated CRP with 4.2 and an elevated ESR with 3.2.

Irritable bowel syndrome (IBS) /2/

In a meta-analysis, there was no difference in f-CP values between healthy individuals and IBS (Tab. 14.3-12 – Fecal calprotectin in healthy controls and in disease groups).

Crohn’s disease, ulcerative colitis /2/

Both IBDs show significantly higher f-CP values than healthy individuals and patients with IBS. However, differentiation of Crohn’s disease from ulcerative colitis is not possible with f-CP.

Inflammatory bowel disease patients in clinical remission /10/

Gastrointestinal symptoms in patients with IBD in apparent remission can reflect the coexistence of IBS or subclinical inflammation. Conventional markers of inflammatory activity, such as CRP, frequently lack sufficient diagnostic sensitivity to detect low-grade inflammation and to help clinicians to differentiate subclinical IBD from coexisting IBS. In a study /8/, the Rome-criteria for IBS were fulfilled in remission in 59.7% and 38.6% of Crohn’s patients and ulcerative colitis patients, respectively. The f-CP concentrations (μg/g) were:

In Crohn’s patients in remission with gastrointestinal symptoms 414.7 ± 80.3 and in patients without symptoms 174 ± 49.1.
In ulcerative colitis patients in remission with gastrointestinal symptoms 591.1 ± 172.5 and in patients without symptoms 229.8 ± 83.4.

The study shows that IBS symptoms are common among IBD patients in apparent remission and such symptoms reflect ongoing IBD activity.

When in clinical remission, 35% of children with IBD had normal f-CP (below 100 μg/g) and 13% a very high level (over 1,000 μg/g) without reporting symptoms. A clinical relapse occurred within 12 months in one third of the children. When in clinical remission, the positive predictive value of f-CP for an overt relapse was low ranging from 0.396 to 0.429 for f-CP values > 100 μg/g or > 1,000 μg/g respectively. The negative predictive value was 0.75 for < 100 μg/g /11/. In pediatric IBD, subjective symptoms and clinical assessment associate poorly with the concentration of f-CP.

Steroid refractoriness in severe pediatric ulcerative colitis

Four fecal markers were investigated for their ability to predict steroid refractoriness in severe pediatric ulcerative colitis. Median values were very high at baseline for f-CP (4,215 μg/g), lactoferrin, M2 pyruvate kinase (M2-PK) and S100A12. M2-PK was numerically superior to the other three markers and CRP /12/.

Colon carcinoma /2/

F-CP can be elevated in colon carcinoma, but the diagnostic sensitivity is only 36% with a specificity of 71%.

Bacterial diarrhea

In patients with diarrhea, a positive f-CP test indicates bacterial etiology with a diagnostic sensitivity of 83% with a specificity of 54%.

Irritable Bowel syndrome

Irritable bowel syndrome (IBS) is a chronic gastrointestinal symptom complex defined by abdominal pain and disturbed bowel habits over 3 months within a period of 6 months, in the absence of any identifiable organic pathology. Various functional abnormalities in the gastrointestinal tract and in the brain occur as a consequence of disease development. F-CP is not elevated /14/.

Table 14.4-1 FOBT for the diagnosis of colorectal cancer and of advanced adenoma /13, 14/

Clinical and laboratory findings
Guaiac fecal occult blood tests (gFOBTs) standard tests The diagnostic sensitivities and specificities for the detection of CRC fluctuate /7/: For the standard gFOBTs, such as Hemoccult II, the diagnostic sensitivity is 12.9% with a specificity of 97%. In patients with tumor stages 0, I, II or III, the diagnostic sensitivity was only 14.1%, and in those with high-grade dysplastic adenomas it was only 15% /5/. With comparable sensitivities and specificities, a reduction in CRC mortality of 15% was achieved over 7.8 years in the Nottingham Study /10/, and a reduction of CRC mortality of 18% was seen over 10 years in the Danish Trial /11/. In the Minnesota Cancer Control Study /12/, the Hemoccult II was implemented over a period of 30 years. The mortality risk was reduced by 32% (annual testing) and 22% (testing every 2 years). With a reduction in mortality of 54–58%, men aged 60–69 years and women of > 70 years of age had the most benefit. Women of < 60 years of age had moderate benefit.
Immunologic fecal occult blood tests (iFOBT) The detection limit of iFOBTs is approximately 100 fold lower than of the gFOBT standard tests for the diagnosis of CRC. The diagnostic sensitivity for the detection of CRC of the gFOBT and iFOBT is 33.3% and 73.3%, respectively.
FOBTs and advanced adenomas FOBTs that are employed for detection of advanced adenomas. Among these are polyps with a diameter of 1 cm incorporating, histologically, high-grade dysplasia or, alternatively, significant villous areas. For the detection of advanced adenomas, the diagnostic sensitivities of the standard gFOBT and iFOBT is 8.6% and 25,7%, respectively.

Clinical and laboratory findings

Guaiac fecal occult blood tests (gFOBTs) standard tests

The diagnostic sensitivities and specificities for the detection of CRC fluctuate /7/:

For the standard gFOBTs, such as Hemoccult II, the diagnostic sensitivity is 12.9% with a specificity of 97%. In patients with tumor stages 0, I, II or III, the diagnostic sensitivity was only 14.1%, and in those with high-grade dysplastic adenomas it was only 15% /5/. With comparable sensitivities and specificities, a reduction in CRC mortality of 15% was achieved over 7.8 years in the Nottingham Study /10/, and a reduction of CRC mortality of 18% was seen over 10 years in the Danish Trial /11/.
In the Minnesota Cancer Control Study /12/, the Hemoccult II was implemented over a period of 30 years. The mortality risk was reduced by 32% (annual testing) and 22% (testing every 2 years). With a reduction in mortality of 54–58%, men aged 60–69 years and women of > 70 years of age had the most benefit. Women of < 60 years of age had moderate benefit.

Immunologic fecal occult blood tests (iFOBT)

The detection limit of iFOBTs is approximately 100 fold lower than of the gFOBT standard tests for the diagnosis of CRC. The diagnostic sensitivity for the detection of CRC of the gFOBT and iFOBT is 33.3% and 73.3%, respectively.

FOBTs and advanced adenomas

FOBTs that are employed for detection of advanced adenomas. Among these are polyps with a diameter of 1 cm incorporating, histologically, high-grade dysplasia or, alternatively, significant villous areas. For the detection of advanced adenomas, the diagnostic sensitivities of the standard gFOBT and iFOBT is 8.6% and 25,7%, respectively.

Table 14.5-1 Tumor markers and somatostatin (SS) receptors in patients with gastroenteropancreatic tumors, chromaffin cell tumors, and medullary thyroid carcinoma /1/

NET tumor type	Specific serum tumor marker	Not specific serum marker	Detection rate of SS receptors with ¹¹¹In-octreotide
Thymus	Somatostatin (SS), serotonin	CgA, NSE	50–80%
C-thyroid cells	Calcitonin, calcitonin gene-related peptide (CGRP), ACTH, SS, serotonin	CgA, CEA	70–75%
Lung	Gastrin-releasing peptide (GRP), calcitonin, SS, proopiomelanocortin (POMC), ACTH, antidiuretic hormone (ADH), serotonin, β-hCG	CgA, NSE	80%
Gastro- intestinal tract	Gastrin, cholecystokinin (CCK), gastrointestinal peptide (GIP), vasoactive intestinal peptide (VIP), motilin, glucagon, GRP, pancreatic peptide (PP), GHRH, POMC, ACTH, serotonin	CgA, NSE, hCG	80–90%
Pancreatic islet cells	Insulin, gastrin, VIP, glucagon, SS, serotonin	CgA, NSE, hCG	60–95%
Ovary	Serotonin, β-hCG, parathyroid hormone-related peptide (PTHrP), POMC, CGRP	CgA, NSE
Chromaffin cells	Noradrenaline, adrenaline, dopamine, POMC, calcitonin, neuropeptide Y, neurotensin, SS	CgA, NSE	85–95%
Adeno- carcinoma*	POMC, CGRP	CgA, NSE	20–35%

CgA, chromogranin A; NSE, neuron specific enolase; * with neuroendocrine differentiation

Table 14.5-2 Neuroendocrine tumors (NETs) of the gastroenteropancreatic system

Clinical and laboratory findings
NETs of the stomach /1, 5/ The incidence of endocrine tumors of the stomach, or gastric carcinoids, is 1–2 cases per 100,000 general population per year; they comprise 8.7% of all GEP-NETs. All endocrine tumors of the stomach are enterochromaffin neoplasias; they are infrequently G-cell tumors or enterochromaffin serotonin-producing tumors /8/. Three groups are distinguished /4, 8, 9/: Type 1 tumors, which arise in association with chronic atrophic gastritis (CAG) and are associated with secondary hyper gastrinemia. These tumors contain enterochromaffin-like (ECL) cells. CAG tumors develop in up to 1% of patients with atrophic gastritis and represent 70–80% of the GEP-NETs of the stomach. The tumors usually have a diameter of less than 1 cm and are multi focal. Since, not infrequently, the patients are clinically asymptomatic, this type of tumor is classified together with the non-functional GEP-NETs. The mortality rate is low. The patients manifest hypochlorhydria, hyper gastrinemia and in about 50% of the cases pernicious anemia. Type 2 tumors may develop in the course of hyper gastrinemia due to Zollinger-Ellison syndrome, associated with multiple endocrine neoplasia type 1 (MEN-1; OMIN 131100). This type represents up 5–10% of the gastric NETs, the patients are asymptomatic, and the diagnosis is often made fortuitously on gastroscopy. It is believed that these tumors develop from ECL cell hyperplasia. The mortality rate is low. Type 3 tumors are sporadically occurring gastric carcinoids that occasionally secrete histamine. They usually are single and large (diameter of over 2 cm) and grow from the gastric body/fundus in the context of a normal surrounding mucosa and in the absence of circulating hyper gastrinemia. The tumors account for the remaining 15–20% of the gastric NETs. Type 3 tumors have often already metastasized to the lymph nodes and the liver at diagnosis. Average survival time following diagnosis is 7 months. Laboratory findings: hypergastrinemia is found with tumor types 1 and 2, and type 3 tumors may be associated with elevated histamine values, particularly if flush symptomatology is present.
Duodenal NETs /9/ Duodenal NETs comprise 2–3% of all gastrointestinal endocrine tumors. Duodenal NETs include five types of tumors (i.e., gastrinoma, somatostatinoma, non-functional NET, gangliocytic paraganglioma, and poorly differentiated or undifferentiated neuroendocrine carcinomas). The term carcinoid is synonymous with the term "well differentiated NET". Globally, 50–75% of the duodenal NETs are well differentiated tumors (carcinoids), 20–25% are well differentiated neuroendocrine carcinomas, and 0–3% are undifferentiated or poorly differentiated NETs. The duodenal NETs comprise about 10% of the duodenal tumors in Japan. The average age at clinical presentation is the 6th decade of life, but generally between the ages of 15–91 years. If there is no secretion of hormones, the major clinical symptoms are: abdominal pain (37%), upper gastrointestinal bleeding (21%), icterus (18%), anemia (21%), lower gastrointestinal bleeding (4%), diarrhea (4%). Gastrinomas: duodenal gastrinomas comprise up to 66% of duodenal NETs; they occur sporadically (75–80%) or are associated with MEN I. They can arise together with Zollinger-Ellison syndrome; this was the case earlier in 30–50% of the cases, but today it occurs to a lesser extent. In sporadic cases duodenal gastrinomas are usually a solitary lesion, whereas in MEN 1 they are almost invariably multiple. These are small, and in 40–60% of the cases are associated with regional lymph node swelling, if the tumor has invaded the muscularis. Somatostatinomas: they comprise 15–20% of duodenal NETs and show preference for the periampullary region, particularly in patients with neurofibromatosis I. Somatostatinomas are differentiated from other tumors histologically, based upon the glandular pattern with the presence of psammoma bodies. Unlike pancreatic somatostatinomas, duodenal somatostatinomas do not lead to the somatostatinoma syndrome (cholelithiasis, diabetes mellitus, weight loss, diarrhea). Non-functional NETs: they comprise up 19% of duodenal NETs. Gastrin, serotonin, calcitonin and somatostatin are detectable immune cytologically, but the patients do not produce clinical symptoms that are relatable to these hormones. Gangliocytic paragangliomas: these tumors, generally occur in the periampullary region, make up only a small fraction of NETs. They are large, benign tumors that invade the muscularis. Histologically, they manifest epithelial cells (somatostatin and pancreatic peptide are detectable immune histochemically) and gangliocytic differentiation with the demonstration of NSE and S100 protein. Poorly differentiated neuroendocrine carcinomas: these are hormonally inactive tumors, located in the ampullary region, and generally with a diameter of greater than 2 cm. They grow in the muscularis and metastasize to the regional lymph nodes and the liver. Immunohistologically, there is strong labelling of synaptophysin, while CgA is negative or only weakly positive. Laboratory findings Gastrinoma: the following peptides are secreted: CgA (59–100%), NSE (38–100%), pancreatic peptide (10–62%), motilin (29%), neurotensin (0–20%). Some of the patients have normal gastrin values. In these cases, a secretin stimulation test should be performed (see section 14.4.2). More than 90% of the patients in whom Zollinger-Ellison syndrome (ZES) is present have elevated fasting gastrin values. A value ≥ 1,000 ng/L, or ≥ 500 ng/L along with a basal acid output of 39 ± 3 mmol/L are confirmatory. Duodenal ZES is not distinguished from pancreatic ZES. H₂ antagonists and proton pump inhibitors must be discontinued 1–3 weeks prior to sampling for the gastrin determination. Somatostatinoma: these tumors do not lead to elevated plasma somatostatin values.
Non-functional NETs and poorly differentiated carcinomas: the most important marker is CgA.
NETs of the pancreas (PETs) – Generally /7, 10/ Endocrine pancreatic tumors are uncommon tumors occurring in approximately 1 in 100,000 people. In autopsy studies the incidence is 0.8–10%, and this signifies that these tumors often go undetected. PETs account for less than 3% of pancreatic neoplasms. PETs are generally more indolent than adenocarcinoma of the pancreas, and the prognosis is also substantially better. With regard to their functional status, PETs are differentiated into: Functional tumors; these manifest clinical symptoms that are due to hormone or peptide formation. Insulinomas, gastrinomas, VIPomas and glucagonomas are the functional PETs. Non-functional tumors manifest no hormone-dependent clinical symptoms, but nonetheless express hormones or react immunologically to hormones. These are tumors whose cells produce pancreatic polypeptide, neurotensin or somatostatin. Many somatostatin-producing tumors are inapparent, since this hormone does not trigger a hormonal syndrome. Laboratory findings: the diagnostic investigation is based upon the hormones and amines that are secreted by the PETs (e.g., gastrin from gastrinomas, glucagon from glucagonomas). There are, however, important general markers such as CgA, pancreatic polypeptide (PP) and the α-subunit of hCG. Thus, in non-functional PETs, the diagnostic sensitivity of CgA is 84%, and this value increases to 96% with the additional determination of PP, while alone PP has a diagnostic sensitivity of only 30% /11/. In functional PETs, the diagnostic sensitivity of CgA is increased by the additional determination of PP from 74% to 94% /11/. The diagnostic sensitivity of the α-subunit of hCG is 30%. Patients with MEN I have elevated CgA values.
– Insulinoma Insulinomas account for 60% of islet cell tumors. These are hyper vascular solitary tumors, 90% of which have a diameter of less than 2 cm. Approximately 6–13% of the insulinomas are multiple, and 4–6% are associated with MEN 1. Their incidence is 2–4 per 1 million people per year. Further information can be found in Section 3.7 – Insulin, C-peptide, proinsulin.
– Gastrinoma Most gastrinomas are localized close to the head of the pancreas. After insulinomas, they are the second most common PETs; 25% of gastrinomas occur in association with MEN I. Most gastrinomas become malignant and, at diagnosis, 70–80% of the patients have lymph node or liver metastases and 12% have bone metastases. Gastrin-producing tumors lead to the development of Zollinger-Ellison syndrome. Laboratory findings: see duodenal NETs in this table. See also Section 14.5.3 – Gastrin.
– VIPoma /1/ VIPoma is caused by hypersecretion of vasoactive intestinal peptide (VIP). It occurs sporadically and is localized in the pancreas in 70–80% of the cases. Other locations are the adrenals, the retroperitoneal space, the mediastinal space, the lungs and the jejunum. Severe watery diarrhea is the cardinal symptom. Patients produce more than 3 liters of watery stool daily. Laboratory findings: hypokalemia, often below 2.5 mmol/L, due to loss of over 400 mmol potassium per day. Paradoxically, the hypokalemia is associated with low bicarbonate. Severe hypochloremic acidosis, hypophosphatemia and hypomagnesemia. Plasma concentrations of VIP are greater than 60 pmol/L.
– Somatostatinoma /1/ These are rare tumors of the pancreas and the upper small intestine. More than 60% are large tumors (diameter greater than 5 cm), found in the head and the body of the pancreas. Most of the tumors are located close to the papilla vateri, and can therefore be associated with bile duct occlusion, pancreatitis and gastrointestinal bleeding. The incidence of somatostatinomas is 1 in 40 million people per year. Somatostatin hypersecretion is inhibitory to hormonal secretion and intestinal functions such as absorption, motility and fluid transport. Clinical symptoms are hyperglycemia (95%), cholelithiasis (68%), diarrhea (60%), steatorrhea (47%) and hypochlorhydria (26%). Laboratory findings: hyperglycemia, icterus, increased fasting plasma somatostatin levels.
– Glucagonoma /1, 7/ These are glucagon-secreting α-cell tumors of the pancreas. They represent 5% of the clinically relevant PETs, and 8–13% of the functional PETs. Most glucagonomas occur sporadically, and 5–17% are associated with MEN I. The tumors commonly occur in the tail of the pancreas with a diameter of up to 6 cm and are highly malignant. At the time of diagnosis, more than 80% of the patients have liver metastases. Clinical symptoms are weight loss (70–80%), diabetes (75%), cheilosis or stomatitis (30–40%), and necrotic migratory erythema that begins in the groin and the perineum and migrates to the lower extremities. The patients tend to develop deep vein thrombosis. Laboratory findings: Hyperglycemia (50% of the cases), normocytic anemia (up to 30%), plasma glucagon values above 50 pmol/L; one third of the patients have increased fasting gastrin values. Furthermore, hypoalbuminemia and hypocholesterolemia may be present.
– Carcinoid tumors These well differentiated NETs of the pancreas arise from neoplastic proliferation of enterochromaffin cells. See also Section 14.5.4.5.1 – Carcinoid syndrome.
– Functionally inactive pancreatic tumors /7/ Endocrine tumors of the pancreas are clinically classified as non-functional or non-functioning when they are not related to any definite clinical syndrome. Usually diagnosed in the 5th and 6th decades of life, they comprise 30–50% of tumors of the pancreas. There are many reasons for the absence of clinical hormonal symptoms with these tumors. Many of the tumors produce peptides that do not induce symptoms, for (e.g., pancreatic polypeptide, CgA, the α-subunit of hCG, neurotensin, or ghrelin). Other causes are low secretions of hormones, biologically inactive forms, no release of the synthesized peptide, simultaneous synthesis of an inhibitory peptide like somatostatin, or non-functional receptors. Laboratory findings: the patients have elevated CgA levels; pancreatic polypeptide, hCG-α and hCG-β are increased in 58%, 40% and 20% of cases, respectively /11/.
– MEN 1 syndrome /1/ Pancreatic endocrine tumors can be associated with the MEN 1 syndrome. MEN 1 is an autosomal dominant disease with a prevalence of 1: 20,000 to 1 : 40,000. The endocrine tumors that are most commonly described in MEN I are adenomas of the parathyroid and the pituitary glands, and pancreatic and duodenal NETs. The hormonally active MEN 1 tumors of the pancreas are principally gastrinomas (20–40%) and insulinomas (5–10%). Glucagonomas and VIPoma occur only in less than 5% of MEN 1 patients. Laboratory findings: MEN I tumors form many peptides which can change over the course of time. Important markers are gastrin, insulin, CgA, pancreatic polypeptide and glucagon. Taken together, these markers are believed to have a diagnostic sensitivity of 70%.

Clinical and laboratory findings

NETs of the stomach /1, 5/

The incidence of endocrine tumors of the stomach, or gastric carcinoids, is 1–2 cases per 100,000 general population per year; they comprise 8.7% of all GEP-NETs. All endocrine tumors of the stomach are enterochromaffin neoplasias; they are infrequently G-cell tumors or enterochromaffin serotonin-producing tumors /8/.

Three groups are distinguished /4, 8, 9/:

Type 1 tumors, which arise in association with chronic atrophic gastritis (CAG) and are associated with secondary hyper gastrinemia. These tumors contain enterochromaffin-like (ECL) cells. CAG tumors develop in up to 1% of patients with atrophic gastritis and represent 70–80% of the GEP-NETs of the stomach. The tumors usually have a diameter of less than 1 cm and are multi focal. Since, not infrequently, the patients are clinically asymptomatic, this type of tumor is classified together with the non-functional GEP-NETs. The mortality rate is low. The patients manifest hypochlorhydria, hyper gastrinemia and in about 50% of the cases pernicious anemia.
Type 2 tumors may develop in the course of hyper gastrinemia due to Zollinger-Ellison syndrome, associated with multiple endocrine neoplasia type 1 (MEN-1; OMIN 131100). This type represents up 5–10% of the gastric NETs, the patients are asymptomatic, and the diagnosis is often made fortuitously on gastroscopy. It is believed that these tumors develop from ECL cell hyperplasia. The mortality rate is low.
Type 3 tumors are sporadically occurring gastric carcinoids that occasionally secrete histamine. They usually are single and large (diameter of over 2 cm) and grow from the gastric body/fundus in the context of a normal surrounding mucosa and in the absence of circulating hyper gastrinemia. The tumors account for the remaining 15–20% of the gastric NETs. Type 3 tumors have often already metastasized to the lymph nodes and the liver at diagnosis. Average survival time following diagnosis is 7 months.

Laboratory findings: hypergastrinemia is found with tumor types 1 and 2, and type 3 tumors may be associated with elevated histamine values, particularly if flush symptomatology is present.

Duodenal NETs /9/

Duodenal NETs comprise 2–3% of all gastrointestinal endocrine tumors. Duodenal NETs include five types of tumors (i.e., gastrinoma, somatostatinoma, non-functional NET, gangliocytic paraganglioma, and poorly differentiated or undifferentiated neuroendocrine carcinomas). The term carcinoid is synonymous with the term "well differentiated NET".

Globally, 50–75% of the duodenal NETs are well differentiated tumors (carcinoids), 20–25% are well differentiated neuroendocrine carcinomas, and 0–3% are undifferentiated or poorly differentiated NETs. The duodenal NETs comprise about 10% of the duodenal tumors in Japan.

The average age at clinical presentation is the 6th decade of life, but generally between the ages of 15–91 years. If there is no secretion of hormones, the major clinical symptoms are: abdominal pain (37%), upper gastrointestinal bleeding (21%), icterus (18%), anemia (21%), lower gastrointestinal bleeding (4%), diarrhea (4%).

Gastrinomas: duodenal gastrinomas comprise up to 66% of duodenal NETs; they occur sporadically (75–80%) or are associated with MEN I. They can arise together with Zollinger-Ellison syndrome; this was the case earlier in 30–50% of the cases, but today it occurs to a lesser extent. In sporadic cases duodenal gastrinomas are usually a solitary lesion, whereas in MEN 1 they are almost invariably multiple. These are small, and in 40–60% of the cases are associated with regional lymph node swelling, if the tumor has invaded the muscularis.

Somatostatinomas: they comprise 15–20% of duodenal NETs and show preference for the periampullary region, particularly in patients with neurofibromatosis I. Somatostatinomas are differentiated from other tumors histologically, based upon the glandular pattern with the presence of psammoma bodies. Unlike pancreatic somatostatinomas, duodenal somatostatinomas do not lead to the somatostatinoma syndrome (cholelithiasis, diabetes mellitus, weight loss, diarrhea).

Non-functional NETs: they comprise up 19% of duodenal NETs. Gastrin, serotonin, calcitonin and somatostatin are detectable immune cytologically, but the patients do not produce clinical symptoms that are relatable to these hormones.

Gangliocytic paragangliomas: these tumors, generally occur in the periampullary region, make up only a small fraction of NETs. They are large, benign tumors that invade the muscularis. Histologically, they manifest epithelial cells (somatostatin and pancreatic peptide are detectable immune histochemically) and gangliocytic differentiation with the demonstration of NSE and S100 protein.

Poorly differentiated neuroendocrine carcinomas: these are hormonally inactive tumors, located in the ampullary region, and generally with a diameter of greater than 2 cm. They grow in the muscularis and metastasize to the regional lymph nodes and the liver. Immunohistologically, there is strong labelling of synaptophysin, while CgA is negative or only weakly positive.

Laboratory findings

Gastrinoma: the following peptides are secreted: CgA (59–100%), NSE (38–100%), pancreatic peptide (10–62%), motilin (29%), neurotensin (0–20%). Some of the patients have normal gastrin values. In these cases, a secretin stimulation test should be performed (see section 14.4.2). More than 90% of the patients in whom Zollinger-Ellison syndrome (ZES) is present have elevated fasting gastrin values. A value ≥ 1,000 ng/L, or ≥ 500 ng/L along with a basal acid output of 39 ± 3 mmol/L are confirmatory. Duodenal ZES is not distinguished from pancreatic ZES. H₂ antagonists and proton pump inhibitors must be discontinued 1–3 weeks prior to sampling for the gastrin determination.

Somatostatinoma: these tumors do not lead to elevated plasma somatostatin values.

Non-functional NETs and poorly differentiated carcinomas: the most important marker is CgA.

NETs of the pancreas (PETs) – Generally /7, 10/

Endocrine pancreatic tumors are uncommon tumors occurring in approximately 1 in 100,000 people. In autopsy studies the incidence is 0.8–10%, and this signifies that these tumors often go undetected. PETs account for less than 3% of pancreatic neoplasms. PETs are generally more indolent than adenocarcinoma of the pancreas, and the prognosis is also substantially better. With regard to their functional status, PETs are differentiated into:

Functional tumors; these manifest clinical symptoms that are due to hormone or peptide formation. Insulinomas, gastrinomas, VIPomas and glucagonomas are the functional PETs.
Non-functional tumors manifest no hormone-dependent clinical symptoms, but nonetheless express hormones or react immunologically to hormones. These are tumors whose cells produce pancreatic polypeptide, neurotensin or somatostatin. Many somatostatin-producing tumors are inapparent, since this hormone does not trigger a hormonal syndrome.

Laboratory findings: the diagnostic investigation is based upon the hormones and amines that are secreted by the PETs (e.g., gastrin from gastrinomas, glucagon from glucagonomas). There are, however, important general markers such as CgA, pancreatic polypeptide (PP) and the α-subunit of hCG. Thus, in non-functional PETs, the diagnostic sensitivity of CgA is 84%, and this value increases to 96% with the additional determination of PP, while alone PP has a diagnostic sensitivity of only 30% /11/. In functional PETs, the diagnostic sensitivity of CgA is increased by the additional determination of PP from 74% to 94% /11/. The diagnostic sensitivity of the α-subunit of hCG is 30%. Patients with MEN I have elevated CgA values.

– Insulinoma

Insulinomas account for 60% of islet cell tumors. These are hyper vascular solitary tumors, 90% of which have a diameter of less than 2 cm. Approximately 6–13% of the insulinomas are multiple, and 4–6% are associated with MEN 1. Their incidence is 2–4 per 1 million people per year. Further information can be found in Section 3.7 – Insulin, C-peptide, proinsulin.

– Gastrinoma

Most gastrinomas are localized close to the head of the pancreas. After insulinomas, they are the second most common PETs; 25% of gastrinomas occur in association with MEN I. Most gastrinomas become malignant and, at diagnosis, 70–80% of the patients have lymph node or liver metastases and 12% have bone metastases. Gastrin-producing tumors lead to the development of Zollinger-Ellison syndrome.

Laboratory findings: see duodenal NETs in this table. See also Section 14.5.3 – Gastrin.

– VIPoma /1/

VIPoma is caused by hypersecretion of vasoactive intestinal peptide (VIP). It occurs sporadically and is localized in the pancreas in 70–80% of the cases. Other locations are the adrenals, the retroperitoneal space, the mediastinal space, the lungs and the jejunum. Severe watery diarrhea is the cardinal symptom. Patients produce more than 3 liters of watery stool daily.

Laboratory findings: hypokalemia, often below 2.5 mmol/L, due to loss of over 400 mmol potassium per day. Paradoxically, the hypokalemia is associated with low bicarbonate. Severe hypochloremic acidosis, hypophosphatemia and hypomagnesemia. Plasma concentrations of VIP are greater than 60 pmol/L.

– Somatostatinoma /1/

These are rare tumors of the pancreas and the upper small intestine. More than 60% are large tumors (diameter greater than 5 cm), found in the head and the body of the pancreas. Most of the tumors are located close to the papilla vateri, and can therefore be associated with bile duct occlusion, pancreatitis and gastrointestinal bleeding.

The incidence of somatostatinomas is 1 in 40 million people per year. Somatostatin hypersecretion is inhibitory to hormonal secretion and intestinal functions such as absorption, motility and fluid transport. Clinical symptoms are hyperglycemia (95%), cholelithiasis (68%), diarrhea (60%), steatorrhea (47%) and hypochlorhydria (26%).

Laboratory findings: hyperglycemia, icterus, increased fasting plasma somatostatin levels.

– Glucagonoma /1, 7/

These are glucagon-secreting α-cell tumors of the pancreas. They represent 5% of the clinically relevant PETs, and 8–13% of the functional PETs. Most glucagonomas occur sporadically, and 5–17% are associated with MEN I. The tumors commonly occur in the tail of the pancreas with a diameter of up to 6 cm and are highly malignant. At the time of diagnosis, more than 80% of the patients have liver metastases. Clinical symptoms are weight loss (70–80%), diabetes (75%), cheilosis or stomatitis (30–40%), and necrotic migratory erythema that begins in the groin and the perineum and migrates to the lower extremities. The patients tend to develop deep vein thrombosis.

Laboratory findings: Hyperglycemia (50% of the cases), normocytic anemia (up to 30%), plasma glucagon values above 50 pmol/L; one third of the patients have increased fasting gastrin values. Furthermore, hypoalbuminemia and hypocholesterolemia may be present.

– Carcinoid tumors

These well differentiated NETs of the pancreas arise from neoplastic proliferation of enterochromaffin cells. See also Section 14.5.4.5.1 – Carcinoid syndrome.

– Functionally inactive pancreatic tumors /7/

Endocrine tumors of the pancreas are clinically classified as non-functional or non-functioning when they are not related to any definite clinical syndrome. Usually diagnosed in the 5th and 6th decades of life, they comprise 30–50% of tumors of the pancreas. There are many reasons for the absence of clinical hormonal symptoms with these tumors. Many of the tumors produce peptides that do not induce symptoms, for (e.g., pancreatic polypeptide, CgA, the α-subunit of hCG, neurotensin, or ghrelin). Other causes are low secretions of hormones, biologically inactive forms, no release of the synthesized peptide, simultaneous synthesis of an inhibitory peptide like somatostatin, or non-functional receptors.

Laboratory findings: the patients have elevated CgA levels; pancreatic polypeptide, hCG-α and hCG-β are increased in 58%, 40% and 20% of cases, respectively /11/.

– MEN 1 syndrome /1/

Pancreatic endocrine tumors can be associated with the MEN 1 syndrome. MEN 1 is an autosomal dominant disease with a prevalence of 1: 20,000 to 1 : 40,000. The endocrine tumors that are most commonly described in MEN I are adenomas of the parathyroid and the pituitary glands, and pancreatic and duodenal NETs. The hormonally active MEN 1 tumors of the pancreas are principally gastrinomas (20–40%) and insulinomas (5–10%). Glucagonomas and VIPoma occur only in less than 5% of MEN 1 patients.

Laboratory findings: MEN I tumors form many peptides which can change over the course of time. Important markers are gastrin, insulin, CgA, pancreatic polypeptide and glucagon. Taken together, these markers are believed to have a diagnostic sensitivity of 70%.

Table 14.5-3 Non gastrointestinal NET disorders with elevated CgA and sensitivity of endocrine disease detection /6/

Gastrointestinal (GI) disease
Chronic atrophic gastritis (78–100%) Pancreatitis (23%) Inflammatory bowel syndrome (28–55%) Irritable bowel syndrome (20–31%) Liver cirrhosis (19–48%) Chronic hepatitis (20%) Colon cancer (1–20%) Hepatocellular carcinoma (70–83%) Pancreatic cancer (43–83%)
Non-GI carcinomas/kidney disease
Small cell lung cancer (53–72%) Prostate carcinoma (52–88%) Breast cancer (25%) Ovary cancer (no data) Renal insufficiency (92%)
Endocrine disease
Pheochromocytoma (71–100%) Hyperparathyroidism (27%) Pituitary tumors (22%) Medullary thyroid carcinoma (27–100%) Hyperthyreosis (68%)
Inflammatory disease
Rheumatoid arthritis (100%) SIRS (no data) Chronic bronchitis (no data) Airway obstruction in smokers (no data
Cardiovascular disease/drugs
Arterial hypertension (18–40%) Cardiac insufficiency (100%) Acute coronary syndrome (no data) Giant cell arteritis (27%) Proton pump inhibitors (100%) H₂-Blockers (0–8%)

Table 14.5-4 Diagnostic sensitivity and specificity of Cg A in comparison with other tumor markers in the detection of gastroenteropancreatic endocrine tumors /5/

Marker	Cutoff	Sensitivity (%)	Specificity (%)
CgA	34 U/L	68	86
NSE	12.5 μg/L	33	100
CEA	5 μg/L	15	91
5-HIAA*	10 mg/L	35	100

* 5-Hydroxyindole acetic acid in 24-hour urine collection. CgA was determined using the Dako assay.

Table 14.5-5 CgA in neuroendocrine tumors and other diseases

Clinical and laboratory findings
GEP-NETs CgA is a sensitive marker for the detection of foregut and midgut NETs, and is more suitable than 5-hydroxy indoleacetic acid (5-HIAA). In 127 patients with diverse NETs, the diagnostic sensitivity of CgA was 68%, with a specificity of 86%. In comparison, 5-HIAA and neuron-specific enolase (NSE) had specificities of 100%, but their respective diagnostic sensitivities were only 35% and 33% (Tab. 14.5-4 – Diagnostic accuracy of Cg A in comparison with other tumor markers in the detection of gastroenteropancreatic endocrine tumors) /6/. In symptomatic patients with disseminated NETs, the diagnostic sensitivity of 5-HIAA is higher in comparison to CgA. The highest CgA levels (up to 200-fold the upper reference interval value) are found in NETs of the ileum, and in MEN I (up to 150-fold). For GEP-NETs, the combination of CgA and pancreatic polypeptide had a diagnostic sensitivity of ≥ 95% /10/. Pancreatic NETs and type 2 and type 3 NETs of the stomach (see Tab. 14.5-1 – Tumor markers and distribution of somatostatin receptors in patients with gastroenteropancreatic tumors, chromaffin cell tumors, and medullary thyroid carcinoma), as well as Zollinger-Ellison syndrome, have intermediate values (80–100-fold), while type 1 gastric NETs manifest only slight elevations (2–4-fold) /6/. Well-differentiated NETs secrete more CgA than poorly-differentiated NE carcinomas, because the cells of the latter have hardly any CgA-secreting dense granules. In these cases, serum CgA levels may be normal.
Tumor of the pituitary and the parathyroid gland CgA concentrations are elevated 2–4-fold. The respective diagnostic sensitivities for pituitary and parathyroid gland tumors are 22% and 27%. CgA can also be of value in the differentiation of the etiology of Cushing’s syndrome (hypophyseal, adrenocortical or ectopic). CgA is elevated in NETs that produce ectopic ACTH or corticotrophin-releasing hormone (CRH) /6/.
Non-NE tumor – Generalized /6/ Some non-NE tumors may be associated with elevated CgA concentrations.
– Prostate carcinoma The incidence of NE cells in prostatic adenocarcinomas is 10–100%, and a positive correlation between the fraction of NE cells in the tumor and the CgA level exists. Elevated CgA values seem to be associated with a poorer prognosis.
– Small-cell lung carcinoma The CgA value in small-cell lung carcinoma is higher than in patients with non-small-cell lung carcinoma. In extensive disease CgA values are more frequently elevated, and to a greater extent. In a comparative evaluation of CgA with NSE the diagnostic sensitivity for small-cell lung carcinoma was 61% vs. 57%.
– Hepatocellular carcinoma Values higher than the upper reference interval value were found in 83% of patients with HCC as well as in liver cirrhosis (48%) and chronic hepatitis (20%).
– Colon carcinoma NE differentiation is relative common in these carcinomas (34%); the extent to which they lead to elevated CgA is unknown.
Non-neoplastic disease – Generalized /6/ CgA elevations can be caused by gastropancreatic and cardiovascular diseases, and by medication that inhibits the secretion of the gastric juice.
– Renal insufficiency CgA is increased in proportion to the decrease of the glomerular filtration rate.
– Autoimmune chronic gastritis The ECL cells are stimulated to produce gastrin and CgA. Approximately 78–100% of the patients have increased values, but these are not more than double the upper reference interval value.
– Chronic heart disease (CHD) The CgA levels correlate with the extent of essential hypertension; they are also elevated in CHD. Enhanced sympathoadrenal activity is believed to be a cause of the rise in CgA. The CgA concentration correlates with the severity of the CHD and with the mortality risk /11/. Due to thickening of the tricuspid valve and endocardial fibrotic plaques, 20–70% of carcinoid patients with liver metastases develop CHD with right atrial or right ventricular enlargement. Patients with CgA values above 784 μg/L (cutoff 120 μg/L) had CHD more frequently /12/. NT-ProBNP and CgA were very important markers in the diagnosis of CHD in patients with NET. Furthermore, patients with elevated NT-proBNP in addition to elevated CgA levels showed worse overall survival than patients with elevated CgA alone.
– Critically ill patients /13/ At admission to the intensive care unit, critically ill non-surgical patients show a positive correlation between their CgA values and inflammatory markers such as CRP, procalcitonin (PCT) and creatinine. The CgA level is a prognostic indicator of survival. Thus, the key laboratory results were: Survivors: CRP 74 (24–151) mg/L, PCT 1 (0–6) μg/L, CgA 86 (53–175) μg/L, creatinine 131 (86–220) μmol/L Non-survivors: CRP 72 (18–145) mg/L, PCT 7 (2–23) μg/L, CgA 293 (163–699) μg/L, creatinine 186 (112–329) μmol/L.
Proton pump inhibitors, histamine type 2 receptor antagonists CgA serum level can be elevated by a factor of 10 and more. The rise begins 6 days post-medication and increases further to high values following 1 year of medication. The values normalize 1–2 weeks following the dis continuation of medication. Proton pump inhibitors result in higher CgA values than histamine type 2 receptor antagonists.

Clinical and laboratory findings

GEP-NETs

CgA is a sensitive marker for the detection of foregut and midgut NETs, and is more suitable than 5-hydroxy indoleacetic acid (5-HIAA). In 127 patients with diverse NETs, the diagnostic sensitivity of CgA was 68%, with a specificity of 86%. In comparison, 5-HIAA and neuron-specific enolase (NSE) had specificities of 100%, but their respective diagnostic sensitivities were only 35% and 33% (Tab. 14.5-4 – Diagnostic accuracy of Cg A in comparison with other tumor markers in the detection of gastroenteropancreatic endocrine tumors) /6/. In symptomatic patients with disseminated NETs, the diagnostic sensitivity of 5-HIAA is higher in comparison to CgA. The highest CgA levels (up to 200-fold the upper reference interval value) are found in NETs of the ileum, and in MEN I (up to 150-fold). For GEP-NETs, the combination of CgA and pancreatic polypeptide had a diagnostic sensitivity of ≥ 95% /10/.

Pancreatic NETs and type 2 and type 3 NETs of the stomach (see Tab. 14.5-1 – Tumor markers and distribution of somatostatin receptors in patients with gastroenteropancreatic tumors, chromaffin cell tumors, and medullary thyroid carcinoma), as well as Zollinger-Ellison syndrome, have intermediate values (80–100-fold), while type 1 gastric NETs manifest only slight elevations (2–4-fold) /6/.

Well-differentiated NETs secrete more CgA than poorly-differentiated NE carcinomas, because the cells of the latter have hardly any CgA-secreting dense granules. In these cases, serum CgA levels may be normal.

Tumor of the pituitary and the parathyroid gland

CgA concentrations are elevated 2–4-fold. The respective diagnostic sensitivities for pituitary and parathyroid gland tumors are 22% and 27%. CgA can also be of value in the differentiation of the etiology of Cushing’s syndrome (hypophyseal, adrenocortical or ectopic). CgA is elevated in NETs that produce ectopic ACTH or corticotrophin-releasing hormone (CRH) /6/.

Non-NE tumor – Generalized /6/

Some non-NE tumors may be associated with elevated CgA concentrations.

– Prostate carcinoma

The incidence of NE cells in prostatic adenocarcinomas is 10–100%, and a positive correlation between the fraction of NE cells in the tumor and the CgA level exists. Elevated CgA values seem to be associated with a poorer prognosis.

– Small-cell lung carcinoma

The CgA value in small-cell lung carcinoma is higher than in patients with non-small-cell lung carcinoma. In extensive disease CgA values are more frequently elevated, and to a greater extent. In a comparative evaluation of CgA with NSE the diagnostic sensitivity for small-cell lung carcinoma was 61% vs. 57%.

– Hepatocellular carcinoma

Values higher than the upper reference interval value were found in 83% of patients with HCC as well as in liver cirrhosis (48%) and chronic hepatitis (20%).

– Colon carcinoma

NE differentiation is relative common in these carcinomas (34%); the extent to which they lead to elevated CgA is unknown.

Non-neoplastic disease – Generalized /6/

CgA elevations can be caused by gastropancreatic and cardiovascular diseases, and by medication that inhibits the secretion of the gastric juice.

– Renal insufficiency

CgA is increased in proportion to the decrease of the glomerular filtration rate.

– Autoimmune chronic gastritis

The ECL cells are stimulated to produce gastrin and CgA. Approximately 78–100% of the patients have increased values, but these are not more than double the upper reference interval value.

– Chronic heart disease (CHD)

The CgA levels correlate with the extent of essential hypertension; they are also elevated in CHD. Enhanced sympathoadrenal activity is believed to be a cause of the rise in CgA. The CgA concentration correlates with the severity of the CHD and with the mortality risk /11/. Due to thickening of the tricuspid valve and endocardial fibrotic plaques, 20–70% of carcinoid patients with liver metastases develop CHD with right atrial or right ventricular enlargement. Patients with CgA values above 784 μg/L (cutoff 120 μg/L) had CHD more frequently /12/. NT-ProBNP and CgA were very important markers in the diagnosis of CHD in patients with NET. Furthermore, patients with elevated NT-proBNP in addition to elevated CgA levels showed worse overall survival than patients with elevated CgA alone.

– Critically ill patients /13/

At admission to the intensive care unit, critically ill non-surgical patients show a positive correlation between their CgA values and inflammatory markers such as CRP, procalcitonin (PCT) and creatinine. The CgA level is a prognostic indicator of survival. Thus, the key laboratory results were:

Survivors: CRP 74 (24–151) mg/L, PCT 1 (0–6) μg/L, CgA 86 (53–175) μg/L, creatinine 131 (86–220) μmol/L
Non-survivors: CRP 72 (18–145) mg/L, PCT 7 (2–23) μg/L, CgA 293 (163–699) μg/L, creatinine 186 (112–329) μmol/L.

Proton pump inhibitors, histamine type 2 receptor antagonists

CgA serum level can be elevated by a factor of 10 and more. The rise begins 6 days post-medication and increases further to high values following 1 year of medication. The values normalize 1–2 weeks following the dis continuation of medication. Proton pump inhibitors result in higher CgA values than histamine type 2 receptor antagonists.

Table 14.5-6 The secretin test

Indication: the secretin test is a provocation test. It serves to distinguish between tumor-induced hyper gastrinemia (e.g., gastrinoma) and other types of elevated gastrin levels.

Protocol: secretin is administered to the fasting patient by bolus injection within 30 sec. at a dose of 2 KU/kg of body weight, after two basal blood samples have been obtained at a time interval of 15 min.; some authors only use 1 KU/kg of body weight. Further blood samples are collected after 2, 5, 10, 15 and 30 minutes.

Assessment: the test increases plasma gastrin concentration over 50% within 2–5 min. in gastrinoma patients. In non gastrinoma patients, the increase is in many cases inhibited, but in some individuals there is a very modest rise immediately after secretin administration /2/. According to other data, a rise of ≥ 100 pmol/L within 15 minutes of the secretin injection is considered to indicate the presence of gastrinoma /1/.

The secretin test is reported to be a valuable predictor of recurrence after surgery with curative intent. Secretion of gastrin from antral G-cells can be demonstrated using a standard meal test (two eggs, toast); plasma gastrin typically increases two- to threefold in non gastrinoma patients, with a peak at about 30 min. Gastrinoma patients exhibit little or no increase in plasma gastrin with feeding /2/.

Table 14.5-7 The calcium infusion test

Indication: complementary procedure in patients with suspected gastrinoma but with circulating gastrin below 50% in the secretin test.

Protocol: 54 mg calcium/kg body weight/hour are infused as a 10% calcium gluconate solution to the fasting patient, over 3 hours, after two basal blood samples have been obtained at a time interval of 15 min. Further blood samples are collected every 30 minutes.

Assessment: the test is performed in patients with suspicion of gastrinoma with gastrin values of below 500 pmol/L, if their secretin test is negative. A rise of greater than 200 pmol/L relative to the basal values is indicative of a gastrinoma. In one study /5/, all gastrinomas were detected with the combined use of the secretin and the calcium infusion tests. Neither the secretin test nor the calcium infusion test distinguishes gastrinoma with and without MEN-I.

Table 14.5-8 Diseases associated with pathological serum gastrin levels

Clinical and laboratory findings

Zollinger-Ellison Syndrome (ZES) /8/

In ZES, the clinical picture is characterized by massive hydrochloric acid production, peptic ulcer, severe reflex esophagitis and diarrhea. Tumors of the pancreas, the duodenum or the stomach, autonomously secreting gastrin and its precursor (pro gastrin), occur most commonly. Fasting gastrin values are generally above 1,000 ng/L (500 pmol/L). The basal acid output is over 15 mmol/h. High serum pro gastrin values can confirm the diagnosis.

Gastrinoma /8/

In patients with gastrinomas gastrin levels are, as a rule, 100–20,000 ng/L (50–10,000 pmol/L). Some 10% of the patients have gastrin concentrations of below 100 ng/L (50 pmol/L) /8/. In these patients, and in those with concentrations below 1,000 ng/L (500 pmol/L), a secretin test should be performed (Tab. 14.5-6 – The secretin test). In approximately 10% of gastrinomas the secretin test is, nonetheless, negative, and in these cases a complementary calcium infusion test can follow (Tab. 14.5-7 – The calcium infusion test). Post-operatively, normalization of the gastrin value indicates complete removal of the gastrinoma. Since, in gastrinoma patients, gastrin manifests considerable molecular heterogeneity, its determination should be performed with a test that detects all amidated forms of the molecule. Thus, assays that solely determine gastrin-17 recognize only 10% of the gastrins /4/. Gastrinoma patients under therapy with proton pump inhibitors and H2 blockers had higher gastrin values (298 ± 33 pmol/L) than those not receiving such medication (204 ± 30 pmol/L) /7/. Dis continuation of therapy can lead to life threatening situations (bleeding of the ulcer), which is why the patient must be closely monitored.

Gastrinoma in multiple endocrine neoplasia (MEN type I) /1/

MEN type 1 is found in 20–38% of gastrinoma, The gastrin levels are > 2,000 ng/l.The clinical picture corresponds to that of gastrinoma, as described above, but in addition, further endocrine tumors with typical symptomatology (e.g., hyperparathyroidism with adenoma of the parathyroid glands, insulinoma, glucagonoma, gastric carcinoids) may be present. It is important that in every case of diagnostically demonstrated primary hyperparathyroidism, which is often the cardinal clinical symptom, the possible presence of MEN type I (or type IIa) be investigated.

Antral G-cell hyper function /9/

The rare clinical picture of antral G-cell hyper function is not generally accepted as a distinct disease. It is characterized by moderate hyper gastrinemia which, as in Zollinger-Ellison syndrome, is associated with elevated, but rarely excessive, basal acid output and a tendency for recurrent ulcers. The secretin test is negative, but food-stimulated gastrin secretion is markedly increased with elevations of more than 100% in spite of raised basal gastrin values. Gastrin values normalize following resection of the antrum.

Helicobacter pylori gastritis /10/

In Helicobacter pylori gastritis, inflammation of the gastric mucosa is associated with a decreased gastric acid secretion. Circulating gastrin concentrations increase as a consequence, although pro inflammatory cytokines in the vicinity of G-cells also stimulate gastrin release /2/. Serum gastrin levels are usually modest (100 ng/L, 50 pmol/L), especially if the patients are not fasting. Basal as well as postprandial gastrin values are usually elevated.

Autoimmune atrophic gastritis /2/

In these patients, unlike gastrinoma patients, acid secretion is absent. Gastrin values are elevated, since the acid inhibition of gastrin formation is canceled. The consequence is pernicious anemia in which achlorhydria, over the course of many years, can lead to gastrin values of over 1,000–3,000 ng/L (500–1,500 pmol/L). Infrequently, values of up to 30,000 pmol/L are seen. In order to confirm the diagnosis, the determination of parietal cell antibodies should be performed.

Retained antrum syndrome /11/

Anastomotic ulcerations and peptic ulcers of the jejunum in patients with Billroth II gastric surgery may indicate the presence of retained antrum if the gastrin level is increased. Differential diagnostic distinction from gastrinoma: after a provocation test with secretin, there is no detectable in crease in serum gastrin.

Vagotomy /11/

Following vagotomy, the gastrin level is normal or slightly elevated; rarely more than 2–3-fold the upper reference interval value and about 200 ng/L. In the case of recurrent ulcers after vagotomy with hyper gastrinemia and normal acid secretion, the performance of the secretin test allows the differential diagnostic distinction from a possible Zollinger Ellison syndrome.

Anti-secretory therapy (proton pump inhibitors, histamine H-2 receptor antagonists) /12/

Drug treatment for the inhibition of acid secretion leads to elevated gastrin levels due to the loss of negative feedback. Values reach twice the upper reference interval value, but gastrin falls substantially within 3 days of dis continuation. This is not the case in patients who have been taking these medications for a long period of time; rather, in these patients, it takes a number of months for gastrin to decrease. The level of gastrin correlates with the gastrin concentration prior to treatment. If mild hyper gastrinemia is already present before administration of the anti-secretory therapy gastrin may, in isolated cases, rise markedly under proton pump inhibition; in such cases, differentiation with respect to gastrinoma can be difficult. Under these conditions, the secretin test is negative. Proton pump inhibitors are better inhibitors of gastric acid secretion than H2 receptor blockers and are therefore associated with higher gastrin levels. Gastrin concentrations are in the range of 200–400 ng/l.

Pheochromocytoma

Gastrin secretion is stimulated by circulating catecholamines and patients with pheochromocytoma may have elevated plasma gastrin concentrations /2/.

Renal failure

In end stage renal disease patients have higher than normal levels of gastrin, probably due to renal clearance of gastrin, increased gastric G cell density, and decreased inhibition secondary to diminished somatostatin levels /13/.

Table 14.5-9 Sensitivity and efficiency of biomarkers for the diagnosis of carcinoid tumor at 97% specificity /9/

Marker	Sensitivity (%)	Efficiency (%)	Cutoff
Serotonin (blood)	77	88	6.1 nmol/10⁹ thrombocytes
5-HIAA (plasma)	89	63	116 nmol/L
5-HIAA (urine)	77	88	56 μmol/24 h

Table 14.5-10 Test characteristics of indole markers for carcinoid tumors with application of high-level cutoff values /2/

Marker	Cutoff value	Sens. (%)	Spec. (%)	PPV (%)	NPV (%)
5-HIAA (urine)	6.7	52	98	87	90
Serotonin (urine)	99	46	93	89	60
Serotonin (platelets)	9.3	63	99	89	93

Urinary 5-HIAA expressed in mmol/mol creatinine; urinary serotonin in μmol/mol creatinine; platelet serotonin in nmol/10⁹ thrombocytes; Sens, diagnostic sensitivity; Spec, diagnostic specificity

Table 14.5-11 Diseases and conditions with elevated pancreatic polypeptide (PP) levels

Clinical and laboratory findings
Normal individual In some clinically healthy people, mostly in elderly persons, abnormally high serum PP concentrations can be measured. From a differential diagnostic point of view, the atropine test can help to differentiate such a situation from autonomous hypersecretion of PP /6/. In healthy people elevated PP levels can be decreased by > 50%, in comparison to the basal level, 30 or 60 min. after the intravenous injection of 1 mg of atropine (positive test result).
PPoma (PP producing endocrine tumor) Because of the absence of hormone-associated clinical features, PPomas which are mainly located within the pancreas, are commonly not diagnosed until local complications arise such as invasive growth into adjacent organs or general signs of tumorous disease are noted such as weight loss, fatigue, and metastases. Only a few cases have been described to date. The atropine test /6/ is negative in these cases; the administration of atropine does not suppress the abnormally high PP concentrations. It remains to be determined whether hypersecretion of PP alone may be responsible for secretory diarrhea.
VIPoma (Verner-Morrison syndrome) The clinical picture is characterized by profuse watery diarrhea and is due to increased VIP secretion. In approximately 2/3 of the cases elevated PP levels should be anticipated as well. The atropine test is abnormally negative in the presence of elevated basal concentrations. However, normal PP levels do not rule out an underlying VIPoma. For further details regarding the clinical picture, refer to Reference /7/.
Zollinger-Ellison syndrome, Gastrinoma, Insulinoma These slightly more common endocrine tumors of the gastrointestinal tract are associated with elevated PP levels in approximately 20% of cases (refer to Section 14.5.2). The atropine test is negative in the case of autonomous PP secretion by the tumor (no decline in the PP concentration). From a differential diagnostic point of view, the rise in the PP concentration cannot be used in the secretin test /7/.
Visceral neuropathy Basal levels of PP do not differ from the normal range. As a result of the decline in blood glucose, insulin-induced hypoglycemia (0.2 U/kg of body weight, i.v.) in healthy people leads to the activation of both cholinergic (vagal) as well as adrenergic portions of the autonomous nervous system and thus to a rise in the plasma PP concentration. This rise does not occur or is already limited prior to the onset of definite clinical neuropathic signs in patients with visceral neuropathy (e.g., as observed in conjunction with diabetes mellitus, or in those with idiopathic visceral neuropathy).
Chronic pancreatitis Meal-stimulated increases in the PP level are not reduced until far advanced stages of exocrine pancreatic insufficiency are present, usually in combination with steatorrhea, therefore, the determination of the PP is not relevant as a diagnostic test of pancreas function.

Clinical and laboratory findings

Normal individual

In some clinically healthy people, mostly in elderly persons, abnormally high serum PP concentrations can be measured. From a differential diagnostic point of view, the atropine test can help to differentiate such a situation from autonomous hypersecretion of PP /6/. In healthy people elevated PP levels can be decreased by > 50%, in comparison to the basal level, 30 or 60 min. after the intravenous injection of 1 mg of atropine (positive test result).

PPoma (PP producing endocrine tumor)

Because of the absence of hormone-associated clinical features, PPomas which are mainly located within the pancreas, are commonly not diagnosed until local complications arise such as invasive growth into adjacent organs or general signs of tumorous disease are noted such as weight loss, fatigue, and metastases. Only a few cases have been described to date. The atropine test /6/ is negative in these cases; the administration of atropine does not suppress the abnormally high PP concentrations. It remains to be determined whether hypersecretion of PP alone may be responsible for secretory diarrhea.

VIPoma (Verner-Morrison syndrome)

The clinical picture is characterized by profuse watery diarrhea and is due to increased VIP secretion. In approximately 2/3 of the cases elevated PP levels should be anticipated as well. The atropine test is abnormally negative in the presence of elevated basal concentrations. However, normal PP levels do not rule out an underlying VIPoma. For further details regarding the clinical picture, refer to Reference /7/.

Zollinger-Ellison syndrome, Gastrinoma, Insulinoma

These slightly more common endocrine tumors of the gastrointestinal tract are associated with elevated PP levels in approximately 20% of cases (refer to Section 14.5.2). The atropine test is negative in the case of autonomous PP secretion by the tumor (no decline in the PP concentration). From a differential diagnostic point of view, the rise in the PP concentration cannot be used in the secretin test /7/.

Visceral neuropathy

Basal levels of PP do not differ from the normal range. As a result of the decline in blood glucose, insulin-induced hypoglycemia (0.2 U/kg of body weight, i.v.) in healthy people leads to the activation of both cholinergic (vagal) as well as adrenergic portions of the autonomous nervous system and thus to a rise in the plasma PP concentration. This rise does not occur or is already limited prior to the onset of definite clinical neuropathic signs in patients with visceral neuropathy (e.g., as observed in conjunction with diabetes mellitus, or in those with idiopathic visceral neuropathy).

Chronic pancreatitis

Meal-stimulated increases in the PP level are not reduced until far advanced stages of exocrine pancreatic insufficiency are present, usually in combination with steatorrhea, therefore, the determination of the PP is not relevant as a diagnostic test of pancreas function.

Table 14.6-1 Characterisation of porphyrias /1, 8, 10/

Disease (OMIM, heredity)	Enzyme defect, Prevalence	Enzyme activity	Screening test	Clinical presentation
Acute porphyria
Acute intermittent porphyria (176000, AD)	HMBS 1 : 100,000	50%	Urine PBG	Acute attack
Acute cutaneous porphyrias
Hereditary coproporphyria (121300, AD)	Coproporphyrin oxidase 1 : 1 × 10⁶	50% ⁽¹	Urine PBG	Acute attack Skin blisters
Variegate porphyria (176200, AD)	Protoporphyrin oxidase 1 : 25,000	50% ⁽¹	Urine PBG	Acute attack Skin blisters
Cutaneous porphyria
Porphyria cutanea tarda (176090, AD), (176100 sporadic)	Uoporphyrinogen decarboxylase 1 : 25,000	50% ⁽¹	Plasma 618–620 nm	Acute attack Skin blisters
Acute painful photosensitive porphyrias
Erythropoietic protoporphyria (177000, AR)	Ferrochelatase 1 : 140,000	5–30%	Plasma 630–634 nm	Burning sensation after sun exposure
X-linked dominant protoporphyria (300752, X-linked)	ALA synthase 1 : 6 million	Elevated	Plasma 630–634 nm	Photosensitive pain
Rare recessive porphyrias
ALA-dehydratase deficiency porphyria (125270, AR)	ALA dehydratase Unknown	< 5%		Acute and chronic neuropathy
Congenital erythropoietic porphyria (606398 AR) Günther disease	Uroporphyrin- ogen-III-synthase 1 : 330,000	2–30%	Plasma 615–618 nm	Severe photo- sensitivity
Hepatoerythropoietic porphyria (176100)	Uroporphyrin- ogen decarboxylase		Plasma 615–618 nm	Severe photo- sensitivity

ALA dehydratase, 5-aminolevulinic acid dehydratase; HMBS, hydroxymethylbilan synthase (porphobilinogen deaminase); decarboxylase; ALA synthase, 5-aminolevulinic acid synthase 2; AD, autosomal dominant; AR, autosomal recessive; ¹⁾ Enzyme activity in lymphocytes, other activities in erythrocytes; prevalence data from Great Britain

Table 14.6-2 First-line tests for diagnosis of porphyrias /1/

Clinical presentation Examination	Result
Akute attacks (unexplained abdominal pain, nausea, vomiting, constipation, uropsychiatric symptoms) Porphobilinogen and (5-aminolevulinic acid) in urine	Normal: no acute porphyria. Elevated: excess of porphobilinogen is the first-line test in all three acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria). For diagnosis of the type of porphyria plasma fluorescence emission spectroscopy is the first-line test. However, it does not distinguish acute intermittent porphyria from hereditary coproporphyria. Total fecal porphyrin concentration is increased in variegate porphyria, with protoporphyrin (protoporphyrin IX) levels greater than those for coproporphyrin, whereas it is usually normal in acute intermittent porphyria.
Erosive photodermatosis (blisters, skin fragility, hypertrichosis) Plasma fluorescence emission spectroscopy	Normal: porphyria is ruled out. Plasma fluorescence emission spectroscopy is the best initial test for cutaneous porphyrias differntiating variegate porphyria (plasma peak 624–627 nm) and porphyria cutanea tarda (plasma peak 618–620 nm).
Acute painful photosensitivity (burning sensation after sun exposure) Protoporphyrin IX in erythrocytes	Normal: porphyria is ruled out. Elevated: porphyria confirmed. Further investigation: FECH gene sequenching including detection of week IVS3-48C allel, low FECH activity in lymphocytes
Neonatal porphyrias (neonatal icterus, hemolytic anemia, bullae, severe neurological defects Porphobilinogen, 5-aminolevulinic acid and total porphyrins in urine

Clinical presentation
Examination

Result

Akute attacks (unexplained abdominal pain, nausea, vomiting, constipation, uropsychiatric symptoms)

Porphobilinogen
and (5-aminolevulinic acid) in urine

Normal: no acute porphyria.

Elevated: excess of porphobilinogen is the first-line test in all three acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria). For diagnosis of the type of porphyria plasma fluorescence emission spectroscopy is the first-line test. However, it does not distinguish acute intermittent porphyria from hereditary coproporphyria. Total fecal porphyrin concentration is increased in variegate porphyria, with protoporphyrin (protoporphyrin IX) levels greater than those for coproporphyrin, whereas it is usually normal in acute intermittent porphyria.

Erosive
photodermatosis
(blisters, skin fragility, hypertrichosis)

Plasma fluorescence emission spectroscopy

Normal: porphyria is ruled out.

Plasma fluorescence emission spectroscopy is the best initial test for cutaneous porphyrias differntiating variegate porphyria (plasma peak 624–627 nm) and porphyria cutanea tarda (plasma peak 618–620 nm).

Acute painful photosensitivity (burning sensation after sun exposure)

Protoporphyrin IX in erythrocytes

Normal: porphyria is ruled out.

Elevated: porphyria confirmed. Further investigation: FECH gene sequenching including detection of week IVS3-48C allel, low FECH activity in lymphocytes

Neonatal porphyrias (neonatal icterus, hemolytic anemia, bullae, severe neurological defects

Porphobilinogen, 5-aminolevulinic acid and total porphyrins in urine

Table 14.6-3 Reference intervals of urinary porphyrins /4/

Porphyrins	μg/24 h	nmol/24 h
Total porphyrins	< 100	< 120
Uroporphyrin	3–24	4–29
Heptacarboxyporphyrin	0–3	0–4
Hexacarboxyporphyrin	0–2	0–3
Pentacarboxyporphyrin	0–4	0–6
Coproporphyrin	14–78	21–119
Tricarboxyporphyrin	0–2	0–2
Dicarboxyporphyrin(s)	0–1	0–1
Coproporphyrin isomer I fraction		17–31%
Coproporphyrin isomer III fraction		69–83%

Table 14.6-4 Reference intervals of fecal porphyrins /4/

Porphyrins	μg/g*	nmol/g*
X-porphyrins	0–2	0–3
Uroporphyrin	1–3	1–4
Heptacarboxyporphyrin	0–3	0–4
Hexacarboxyporphyrin	0–1	0–1
Pentacarboxyporphyrin	1–4	1–5
Isocoproporphyrins	0	0
Coproporphyrin	3–24	5–37
Tricarboxyporphyrin	0–6	0–8
Protoporphyrin	12–85	21–151

* with reference to 1 g dry weight

Table 14.6-5 Biochemical investigations and findings in cutaneous porphyrias /8, 10/

Disease	Urine	Stool	Red cells	Plasma fluorescence*
Variegate porphyria	ALA, PBG, Copro III	Proto> Copro III	NE	624–627 nm
Congenital erythropoietic porphyria (CEP)	Uro I, Copro I	Copro I	ZnPP + free Proto, Copro I, Uro I	615–620
Porphyria cutanea tarda (PCT)	Uro I+III, hepta	Isocopro, hepta	NE	615–620
Erythropoietic protoporphyria (EPP)	NE	Proto	Free proto	626–634
X-linked dominant protoporphyria (XLDPP)	NE	Proto	ZnPP + free proto	626–634

ALA, 5-aminolevulinic acid; hepta, heptacarboxyporphyrin; isocopro, isocoproporphyrin; copro, coproporphyrin; NE, not elevated; PBG, porphobilinogen; proto, protoporphyrin; uro, uroporphyrin; ZnPP, zinc protoporphyrin, * Plasma fluorescence emission spectroscopy

Table 14.6-6 Typical findings in porphyrias

Clinical and laboratory findings

Acute intermittent porphyria (AIP) /6/

AIP is the most common and severe of the acute porphyrias with reduction of the activity of porphobilinogen (PBG) deaminase (or uroporphyrinogen I synthase). The prevalence of AIP is 1–2/100,000 in the general population. Men become symptomatic in their fourth decade of life, women in their third. The clinical symptoms begin with colicky abdominal pain, hypertension and tachycardia, often preceded by nausea, vomiting or constipation. One third of the patients develop confusion, anxiety and depression. A neuropathy occurs in two-thirds of patients with symmetrical weakness mainly involving limb and girdle muscles. An associated hyponatremia (Na⁺ < 120 mmol/L), may indicate the syndrome of inappropriate ADH secretion (SIADH). The acute attacks are the result of neuropathy, which is due to heme accumulation in neurons of the autonomic nervous system. Skin changes do not occur.

Laboratory findings: hyponatremia, light elevations in liver enzyme activities. The key investigation is the examination of urine for excess porphyrin precursors (PBG and ALA), total porphyrin and uroporphyrin I are moderate elevated. Stool porphyrins are increased in only 20% of the patients. In an acute attack, urine when passed becomes dark on standing, due to PBG polymerizing to uroporphyrin and a brownish-red pigment, porphobilin. This process can be retarded by means of the cooling of urine at 4 °C, alkaline pH (8–9), and protection from light. In remission, urinary PBG excretion falls, but rarely to normal. Latent patients rarely have normal urinary ALA or PBG excretion. The estimation of erythrocyte PBG deaminase activity may be useful in the diagnosis of latent cases.

Hereditary Coproporphyria /6/

Acute porphyria, which is caused by reduced coproporphyrinogen oxidase activity leading to excess coproporphyrin in the urine and elevated ALA synthase. Autosomal dominant inheritance. Coproporphyrinogen oxidase catalyzes the conversion of coproporphyrin III to protoporphyrinogen. Homozygous trait carriers have a decrease in enzyme activity of up to 2%. The determination of the activity is performed, if necessary, on lymphocytes. HC is often clinically latent but may present with symptoms similar to AIP, and with cutaneous photosensitivity. The most prominent and distressing presenting feature is abdominal pain (80%). As with AIP, there are also symptom-free gene carriers. Cutaneous photosensitivity is seen in up to 30% of the patients, particularly in body parts that are exposed to light, like the face and the hands.

Laboratory findings: the principle biochemical abnormalities in acute attack are increased urinary PBG, ALA and coproporphyrin III excretion. In clinical remission these markers normalize. Fecal porphyrin excretion is > 200 nmol/g of dry stool, whereby coproporphyrin III is the main component, and the coproporphyrin III/I ration is above 2 /8/. Patients with latent HC excrete mainly coproporphyrin III in the stool, and less in the urine. Some patients have a plasma fluorescence emission peak at 615–620 nm.

Variegate porphyria (VP) /4/

VP is an acute porphyria with variable clinical symptomatology; this is the reason for the term variegate. VP is an autosomal dominant disorder associated with deficiency of the enzyme protoporphyrinogen oxidase (PPOX), the penultimate enzyme of the heme synthetic pathway. Because of PPOX deficiency protoporphyrinogen is not converted to protoporphyrin. Most of the patients are heterozygous carriers with PPOX activity decreased by some 50%. In many heterozygous gene carriers, the disease is not manifested. Clinical features are similar to HC, except for the more severe skin photosensitivity features with scarring. The patients are also at risk for acute attacks of porphyria, characterized by episodes of severe abdominal pain, autonomic disturbance, and a motor neuropathy. Homozygous carriers of the genetic defect are rare. In these, the clinical symptomatology begin as early as in childhood with neurological symptoms, marked photosensitivity and growth retardation.

Laboratory findings: in attacks the urine develops a red (port wine) discoloration on exposure to light. Urinary PBG, ALA and coproporphyrin III are elevated during acute attacks; they normalize almost completely in the remission phase, within one week. Plasma X-porphyrins are specific to VP. These are porphyrin-peptide complexes which are detectable with fluorescence emission at 624–627 nm. A fluorescence emission peak at this wavelength differentiates VP from all other porphyrias /8/. Fecal protoporphyrin concentrations are at least two-fold higher than those of coproporphyrin.

ALA dehydratase deficiency porphyria (ADP)

ADP (Doss porphyria) is a very rare autosomal recessive hereditary disease. The disease can occur for the first time in adolescence and resembles, clinically, AIP.

Laboratory findings: urinary ALA elevated to above 72 μmol/mmol creatinine, and urinary coproporphyrin III elevated to above 250 nmol/mmol creatinine, PBG slightly elevated; erythrocyte zinc protoporphyrin elevated. Erythrocyte ALA dehydratase activity reduced to below 5% and cannot be reactivated /11/.

Porphyria cutanea tarda (PCT) /3/

The term PCT refers to a common cutaneous form of porphyria that includes both inherited and acquired forms of non-acute porphyria. In both forms, there is a block in heme biosynthesis at the stage of decarboxylation of uroporphyrinogen III. A deficient activity of the relevant enzyme uroporphyrinogen decarboxylase (URO-D) is demonstrated in the liver. The reduced URO-D activity causes the accumulation of uroporphyrin and additional highly carboxylated porphyrins. The enzyme defect of URO-D is accompanied by a compensatory stimulation of 5-aminolevulinic acid synthetase, and in this way the accumulation of uroporphyrin is readily accounted for by overproduction as well as under utilization of uroporphyrinogen III. This situation leads to accumulation of uroporphyrin III in the liver, from where it is distributed throughout the organism, and activates cutaneous oxidative reactions resulting in tissue necrosis during light exposure. Main precipitating factors in PCT are mutations in the HFE-Locus (Cys282Tyr and His63Asp), polymorphisms in the cytochrome gene (CYP1A2) and the transferrin receptor 1 (TFR1) gene, viral infections (hepatitis C and HIV), estrogens, chronic alcohol consumption.

Two types of PCT are distinguished:

Type 1: this type is acquired and the enzyme defect is limited to hepatic tissue. This form is the most frequent cause of human porphyrias; its prevalence is 10–100/100,000 people. The cause is believed to be oxidative stress, due to changes in iron metabolism. Triggering factors are chronic alcoholism, poisons such as hexachlorobenzene-treated wheat, polybrominated biphenyls, viral infections like HBV and HCV /12/. Type 1 accounts for up to 80% of the PCT cases. Sporadic PCT normally does not occur before the last third of life; if it is observed in younger individuals, an association with HCV or HBV disease should be taken into consideration.
Type 2: this familial type is an autosomal dominant hereditary form. URO-D is deficient in both hepatocytes and erythrocytes. In another familial form of PCT (type 3), erythrocyte URO-S is normal, but hepatic URO-D activity is decreased. Activity is found in the erythrocytes and the liver.

In both PCT types, light sensitivity with cutaneous symptoms and chronic hepatopathy are the main clinical findings. Light exposed skin parts manifest blisters and scar formation, the hands are easily vulnerable and hypertrichosis is seen in the temporal and zygomatic bone areas.

Laboratory findings: the analysis for PCT include plasma porphyrin assessment, erythrocyte porphyrin determination, and urinary and fecal porphyrin fractionation. The urinary porphyrin profile shows normal PBG and ALA and increased uroporphyrin and heptacarboxyl porphyrin. Fecal porphyrin fractionation in PCT reveals the largest fraction to be isocoproporphyrin, followed by heptacarboxyl porphyrin, hexacarboxyl porphyrin and pentacarboxyl porphyrin. PCT must be differentiated from VP, since PBG and ALA are not sufficiently reliable to differentiate VP. VP can be ruled out in a satisfactory manner using plasma fluorescence scan (PV emission peak at 624–627 nm, as the unique porphyria) or with the determination of stool porphyrins (isocopro- and heptacarboxyporphyrins elevated in PCT). The liver enzymes are usually elevated, transferrin saturation and ferritin concentrations are often elevated as a sign of increased iron storage. With ferritin values of over 800 μg/L, iron stores are mainly in the liver. In Great Britain, approximately 20% of the PCT patients are homozygous for the C282Y mutation.

Congenital erythropoietic porphyria (CEP) /13/

This non-acute porphyria is also termed Morbus Günther. In the rare autosomal recessive disorder of CEP, defective activity of uroporphyrinogen III synthase leads to overproduction of the isomer I porphyrinogens in the bone marrow erythrocyte precursors. Isomer I porphyrinogens cannot be decarboxylated by enzymes of the heme pathway further than to coproporphyrinogen I and they accumulate as their corresponding oxidized photo active porphyrin by-products. The accumulated porphyrins (chiefly uroporphyrin I and coproporphyrin I) are released from the erythroid cells into plasma by hemolysis or diffusion, and they are excreted in urine and feces. CEP is the most severe form of cutaneous porphyria. The diagnosis of this extremely rare disorder is often suspected in the neonatal period by the pink staining of diapers by reddish porphyrin pigments that discolor the urine. The pigments are also deposited in tissues, bones and teeth, where they produce a red to brown discoloration. Unprotected exposure to sunlight leads to progressive skin necrosis with the formation of blisters, ulcerations and, over the years, to marked changes in appearance. Splenomegaly is found, and hemolysis occurs in phases; anemia, however, does not develop. Generally, the clinical picture can range from hydrops fetalis to mild cutaneous changes in the adult years.

Laboratory findings: the biochemical hallmark is accumulation of uroporphyrin I in erythrocytes, plasma and urine. Urinary porphyrins consist mainly of uroporphyrin I and to a lesser extent of coproporphyrin I, but PBG and ALA are normal. Increased protoporphyrin in plasma, in feces protoporphyrin > coproporphyrin /3/.

Erythropoietic protoporphyria (EPP) /13/

This autosomal dominant non-acute porphyria has a prevalence of 1 : 140,000 people and is more prevalent than CEP. Decreased mitochondrial ferrochelatase activity leads to accumulation of large excess of protoporphyrin, a hydrophobic photo active molecule, in erythrocytes, liver, plasma, bile and feces. Ferrochelatase catalyzes the conversion of protoporphyrin to heme by insertion of ferrous iron into the porphyrin molecule. The activity of this enzyme in bone marrow, reticulocytes, lymphocytes, liver and skin fibroblasts ranges from 10–40% of normal. Because protoporphyrin is lipophilic, it reaches the intestine via the bile and is excreted in the stool. The predominant organ source of the excess protoporphyrin is the bone marrow. Deposition of protoporphyrin in liver is associated with toxic effects that may be manifested by cholestasis, cirrhosis, progressive decompensation and death in 20–30% of the patients. The initial presentation of EPP occurs most often in childhood, with complaints of cutaneous itching, or burning during or shortly after sunlight exposure.

Laboratory findings: laboratory diagnosis of EPP is based on demonstration of increased protoporphyrin in erythrocytes, plasma, and feces. With normal urinary porphyrin excretion, stool protoporphyrin can be elevated with protoporphyrin > coproporphyrin. Since the remaining ferrochelatase activity is sufficient for the synthesis of heme, no induced rise in ALA synthetase occurs and, therefore, the porphyrin precursors PBG and ALA are not elevated in the urine. An early sign of impeding liver dysfunction can be sought by measurement of urinary porphyrin levels, which are normal in EPP, unless hepatic function becomes compromised. Asymptomatic patients can often be identified only through the determination of reduced ferrochelatase activity.

Pseudoporphyria

PCT has to be differentiated from pseudoporphyrias. These are clinical pictures in which skin changes like those occurring in PCT are seen, but are not associated with abnormal porphyrin biosynthesis. The cause can be non-porphyrin photo toxic substances, such as tetracycline, furosemide or nalidixic acid. Patients with liver cirrhosis or chronic kidney disease can also manifest skin symptoms that are comparable with those of photosensitive cutaneous porphyria.

Lead poisoning

The increased ALA excretion in lead poisoning is due to the direct inhibition of ALA dehydratase (PBG synthetase) activity caused by lead. An increase in and persistence of the ALA dehydratase inhibition can lead to an extreme rise in ALA excretion, resulting from the synergistic effect of PBG synthase inhibition and ALA synthase induction from a hepatic reaction. The induction of ALA synthase in lead intoxication is due to the down regulation of the negative feedback with the decrease in heme biosynthesis.

Laboratory findings: urinary ALA and coproporphyrin III markedly elevated, PBG slightly elevated; erythrocyte zinc protoporphyrin elevated. Erythrocyte ALA dehydratase activity markedly reduced to below 10% of values in healthy individuals, can, however, be reactivated with zinc and thiols.

Figure 14.2-1 Algorithm for the differentiation of malabsorption syndrome.

Figure 14.5-1 Chromogranin A (CgA) as a marker of NETs. The diagnostic sensitivity (y-axis) vs. the maximum CgA increase (normalized to the upper limit of normal is shown). With kind regards of Ref. /6/. Normal =1, x-axis; ECL-1, gastrinoma type I; ECL-II, gastrinoma type II; ECL-III, gastrinoma type III; ECL-IV, gastrinoma type IV; MCC, Merkel cell carcinoma; MTC, medullary thyroid carcinoma; EPT, enteropancreatic tumor; ZES, Zollinger-Ellison syndrome; MEN, multiple endocrine neoplasia.

Figure 14.5-2 Structural relationship between different forms of gastrin. Preprogastrin, initially secreted, is rapidly converted to progastrin, which may be phosphorylated (P) at serine 96. Cleavage at pairs of arginine residues, followed by carboxypeptidase activity, generates G34-Gly, which may be converted either to G17-Gly by cleavage at lysine (K) residues or to G34 amide (NH₂) by the action of petidyl-α-amidating monoxygenase (which may be in turn be cleaved to yield G17). Examples of antibodies reacting with the COOH-terminus of progastrin (L289), COOH-terminus of gly-gastrin (Mab 109-21), COOH-terminus of amidated gastrins (L2), NH₂ terminus of G17 (1295), and with intact G17 (L6) are shown. The dark regions show the amino acid sequences that are shared with cholecystokinin. The insert shows the shared sequence at the COOH-terminus of gastrin and cholecystokinin. With kind regards from Ref. /2/.

Figure 14.5-3 Differential diagnosis of hyper gastrinemia; BAO, basal acid output.

Figure 14.5-4 Synthesis and oxidative deamination of serotonin.

Figure 14.6-1 First step of heme synthesis. Catalyzed by aminolevulinic acid synthase (ALAS), succhinyl-CoA and glycine are converted to aminolevulinic acid (ALA). In the next step, aminolevulinic acid dehydrase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to porphobilinogen (PBG).

Figure 14.6-2 Heme biosynthetic pathway and porphyrias /4/.

Figure 14.6-3 Inhibition of hepatic aminolevulinic acid synthase (ALA synthase) by heme via negative feedback regulation and induction of the enzyme in disorders of heme synthesis. The disorders result from partial enzyme deficiencies in acute intermittent porphyria (AIP), hereditary coproporphyria (HC), variegate porphyria (VP) and lead intoxication. ALA, 5-aminolevulinic acid; PBG, porphobilinogen; URO, uroporphyrinogen; COPRO, coproporphyrinogen; PROTO, protoporphyrinogen; ALA dehydratase = PBG synthase.

Figure 14.6-4 Oxidation of porphyrinogen to porphyrin. The porphyrinogens are classified according to their substituents at the peripheral ring positions. The numbering of the substituents runs from 1–8. The substituents are vinyl, ethyl, methyl, acetic acid and propionic acid groups. With kind permission from Ref. /3/.

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Gastrointestinal and pancreatic function

14.1 Laboratory diagnosis of gastric disease

14.1.1 Regulation of gastric acid secretion

14.1.1.1 Functional heartburn

14.1.1.2 Peptic ulcer disease

14.1.1.3 Drug-related damage of the gastrointestinal tract

14.1.1.4 Reflux esophagitis

14.1.2 Helicobacter pylori infection

14.1.2.1 Indication

14.1.2.2 Method of determination

14.1.2.2.1 Biopsy-dependent procedures

14.1.2.2.2 Histological examination of pathogens

14.1.2.2.3 Culture

14.1.2.2.4 Serologic tests

14.1.2.2.5 13C urea breath test

14.1.2.2.6 Antigen determination in the stool

14.1.2.3 Specimen

14.1.2.4 Reference interval

14.1.2.5 Clinical significance

14.1.2.5.1 Gastric cancer

14.1.2.5.2 Biopsy-dependent procedures

14.1.2.5.3 H. pylori serology

14.1.2.5.4 13C urea breath test

14.1.2.5.5 H. pylori antigen determination in the stool

14.1.2.5.6 Comparison of tests

14.1.2.6 Comments and problems

14.1.3 Autoimmune atrophic gastritis

14.2 Diseases of the pancreas

14.2.1 Acute pancreatitis

14.2.1.1 Common causes of acute pancreatitis

14.2.1.2 Mechanism of cellular injury

14.2.1.3 Definition of severity in acute pancreatitis

14.2.1.4 Clinical significance

14.2.1.5 Biomarkers in acute pancreatitis

14.2.1.5.1 Laboratory diagnosis of acute pancreatitis

14.2.1.5.2 Evaluation of the cause of acute pancreatitis

14.2.1.5.3 Prognosis of acute pancreatitis

14.2.2 Chronic pancreatitis

14.2.2.1 Pancreatic function tests

14.2.2.1.1 Indication

14.2.2.1.2 Clinical significance

14.2.3 Pancreatic cancer

14.2.3.1 Laboratory diagnosis of pancreatic cancer

14.2.4 Fecal elastase-1

14.2.4.1 Indication

14.2.4.2 Method of determination

14.2.4.3 Specimen

14.2.4.4 Reference interval

14.2.4.5 Clinical significance

14.2.4.6 Comments and problems

14.2.5 Fecal fat

14.2.5.1 Indication

14.2.5.2 Method of determination

14.2.5.3 Specimen

14.2.5.4 Reference interval

14.2.5.5 Clinical significance

14.2.5.6 Comments and problems

14.2.6 Secretin-caerulein test

14.2.6.1 Indication

14.2.6.2 Test performance

14.2.6.3 Reference interval

14.2.6.4 Clinical significance

14.2.6.5 Comments and problems

14.2.7 Pancreolauryl test

14.2.7.1 Indication

14.2.7.2 Test performance

14.2.7.3 Reference interval

14.2.7.4 Clinical significance

14.2.7.5 Comments and problems

14.3 Diseases producing malabsorption

14.3.1 Conceptual explanation

14.3.2 Malabsorption syndrome

14.3.2.1 Diseases producing malabsorption

14.3.2.2 Laboratory tests in patients with malabsorption

14.3.3 D-xylose test

14.3.3.1 Indication

14.3.3.2 Test performance

14.3.3.3 Specimen

14.3.3.4 Reference interval

14.3.3.5 Clinical significance

14.1.2.2.5 ¹³C urea breath test

14.1.2.5.4 ¹³C urea breath test