Gastrointestinal and pancreatic function
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.
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.
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. .
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.
- 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 .
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 . 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.
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) . 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% .
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.
Diagnosis of H. pylori infection and evidence for eradication.
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) .
Two biopsy specimens are taken from the antrum and two from the corpus of the stomach.
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.
22.214.171.124.2 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.
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.
126.96.36.199.4 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 .
188.8.131.52.5 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) .
Procedure : 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.
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.
- 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.
- 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.
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 .
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 .
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.
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 .
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 . 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.
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 .
In suspicion of antibiotic resistance, the biopsy enables culture systems for the determination of resistance.
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.
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.
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 .
A test which is positive only after 24 hours should not be evaluated.
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 . 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 . 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 .
Point of care tests determine H. pylori antigens within minutes, using a simplified chromatographic technique.
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.
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.
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.
Paul Lankisch, Bernhard Lembcke, Lothar Thomas
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 . 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.
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 .
In acute pancreatitis without identifiable disease causes, hereditary pancreatitis should be considered . 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%.
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 .
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 .
The Atlanta classification is the standard classification of the severity of acute pancreatitis . Clinical severity of acute pancreatitis is stratified into three categories /, /: 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
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 . Prognostically meaningful is information on previous episodes of acute pancreatitis, since recurrent episodes are rarely severe or necrotizing in comparison with the initial episode . 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.
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 ). The accuracy of the pancreas-specific amylase is comparable.
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:
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
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) .
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 . 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 . According to a consensus , 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.
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 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.
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 . 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 .
Chronic pancreatitis is defined as a chronic inflammatory state that results in irreversible damage to pancreatic structure and function /, , /. 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 (). 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 .
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 .
Transition of acute pancreatitis to chronic pancreatitis develops in alcoholics. In a German study 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 ().
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).
Because chronic pancreatitis increases the mortality by 38.4% after 20 years, annual noninvasive tests, and the determination of CRP, ALP and HbA1c are recommended for monitoring the course of the disease. Tumor markers such as CA 19-9 or CEA are not recommended.
- 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.
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 . The secretin-pancreozymin test has a diagnostic sensitivity of 92% with a specificity of 94% . 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
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.
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 . 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 . Diseases that are associated with an elevated risk of pancreas carcinoma are shown in .
- For estimating the prognosis
- For the assessment of the therapeutic efficiency
- In the postoperative disease course assessment, for the detection of a relapse.
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.
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.
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 .
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.
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.
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.
Human pancreatic elastase-1 (EC 184.108.40.206) 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 .
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 .
Suspected maldigestion in pancreatic insufficiency.
Enzyme immunoassay (EIA) using monoclonal or polyclonal antibodies against human pancreatic elastase-1 . 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.
Stool sample: approximately 1 mL
Values for adults and children older than 3 months. Values are 2.5 and 97.5 percentiles
Patients with exocrine pancreatic insufficiency have diminished elastase-1 excretion. In one study , 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%.
- 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% . 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 .
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 . In another study , 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 .
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.
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.
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.
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.
Suspicion of exogenous pancreatic insufficiency.
Van de Kamer method
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. .
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 .
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 .
Assessment criteria: fecal fat concentration. Conversion to 24-hour stool fat excretion.
Stool collected during 72 hours in three 24-hour fractions.
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 () .
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 .
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.
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.
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.
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.
Suspected exocrine pancreatic insufficiency.
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 /, /.
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.
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 /, /.
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) /, /.
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 () /, /.
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 . 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 . 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 .
The secretin-caerulein test is not standardized. Therefore, each laboratory should determine its own reference intervals.
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 . If the amylase values seem to be too high in comparison to the lipase or trypsin, a determination of the pancreatic isoamylase is recommended .
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.
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.
Suspected exocrine pancreatic insufficiency.
The fluorescein product that is formed in the pancreolauryl test can be determined in urine or serum.
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 . 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 .
Protocol : 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.
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 .
According to the authors findings, the presence of steatorrhea must be assumed if the T/C ratio is < 10.
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 .
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).
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 /, /. 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. .
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.
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.
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 .
Maldigestion is caused by deficiency of the following pancreatobiliary secretions:
- Pancreatic digestive enzymes (α-amylase, lipase, trypsin, elastase) and bicarbonate
- Bile acids.
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.
- 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 .
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 . 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 ().
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 . 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
- 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.
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.
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.
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.
- Suspected malabsorption syndrome
- Suspected disorders of the functional integrity of the proximal small intestine.
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 . 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).
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
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 ().
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) .
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.
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 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 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 , 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
- 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
- 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 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 .
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.
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.
Studies of the vesicles of the microvilli show that passive absorption is the predominant mechanism of D-xylose absorption . 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 . 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.
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.
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 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.
- 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.
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.
The examinations in carbohydrate malabsorption include:
- Food and medication history
- Hydrogen exhalation test (hydrogen breath test)
- Lactose tolerance test
- Endoscopy and enzyme determination (for example disaccharidases) in the aspired small intestinal mucosa biopsy material.
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 . 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 : 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 : 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 : 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.
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).
Lactose tolerance test: venous or capillary blood: 0.1–1 mL.
The term carbohydrate malabsorption describes conditions of incomplete absorption of carbohydrates in the small intestine and reach the colon . This can be physiological due to the ingestion of carbohydrates for which the healthy gastrointestinal tract has a limited digestive or absorptive capacity (. 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 .
- 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.
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 (). 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.
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 . Diseases involving carbohydrate malabsorption are listed in .
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 .
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)
- 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.
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.
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.
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.
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.
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 . 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.
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.
Stool sample: 1 spatula filling, approximately 1–2 g
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 .
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.
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) . 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 . The prevalence of IBD is 0.6–1%.
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 :
- 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 .
The determination of f-CP is a useful screening test in adults and children for identifying those patients who are most likely to need endoscopy for IBD . 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.
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.
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 . 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 .
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 . The comparison of 6 assays showed similar results. Assay specific cutoffs were recommended .
F-CP and lactoferrin 1 week.
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.
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.
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.
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.
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. . 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 .
- 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 .
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.
Suspicion for advanced adenoma and colorectal cancer.
- 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)
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 . The detection limit of the iFOBTs is in the range of 2–17 μg hemoglobin/g feces .
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.
Randomized trials have shown that annual or biannual screening with gFOBTs could reduce the CRC mortality by up to 30%.
CRC is the third most cancer worldwide . 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 . 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 . 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 .
About 25% of the population have pre-malignant polyps by the age of 50 years and the prevalence increases with age . 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% .
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%.
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 :
- 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.
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.
According to a study 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.
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 .
In a study 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).
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 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 . 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.
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.
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.
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.
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.
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.
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.
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 . 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 .
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 (). The WHO included histopathologic and functional parameters in a classification.
- 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) .
- 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 .
- 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 .
- 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 .
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 .
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) . 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 . 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 . MENs are characterized by high penetration in many neuroendocrine tissues.
- 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.
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 . 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% .
Hormonally active tumor cells manifest characteristics that are similar to those of neuroendocrine cells . 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 ().
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 . 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 .
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 /, /.
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 ). 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 ).
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 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 . 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 (). 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.
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.
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.
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.
- In patients with irritable bowel symptoms (IBS)
- Suspicion of gastropancreatic neuroendocrine tumors (NETs) and monitoring their course
- Suspected pheochromocytoma.
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 or using monoclonal antibodies against the central domain 145–245 of the molecules . Competitive radio immunoassays use a polyclonal antibody against the 116-439 amino acid sequence of CgA . Intact CgA as well as fragments are bound. IRMA use monoclonal antibodies . They detect the central 125–245 domain of the CgA molecule.
Serum, EDTA-plasma, heparin-plasma: 1 mL
Manufacturer’s instruction should be followed.
CgA has been demonstrated to be the most promising marker for the diagnosis of neuroendocrine tumors (NETs).
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 . 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 .
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 . Refer to:
- 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.
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) . 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 . In nonfunctional GEP-NETs with liver metastases, CgA levels did not reflect the tumor burden but rather, tumor progression and response to therapy /, /.
In the assessment of medical therapy efficacy in patients with NET, a reduction of CgA level > 50% should be considered significant . Under therapy with long-acting octreotide, there is an association between the decline in CgA and survival time . 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 .
Method of determination
- 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 :
- DAKO: diagnostic sensitivity 85%, diagnostic specificity 88%
- CIS: diagnostic sensitivity 67%, diagnostic specificity 96%
- Euro Diagnostica: diagnostic sensitivity 93%,diagnostic specificity 88%.
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 .
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 .
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 .
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.
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.
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.
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.
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 . Following carboxyendopeptidase cleavage progastrin G34-Gly is generated which may be converted either to G17-Gly or to G34 amide ().
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 . 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 . 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.
- 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
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.
Serum, heparin or EDTA-plasma: 1 mL
Sampling in the fasting state or within the framework of function tests.
Conversion: ng/L × 0.48 = pmol/L.
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 : 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.
- 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.
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 . 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.
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).
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 .
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.
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.
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.
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 (). 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 .
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 . Three indole markers are used in carcinoid disease:
- 5-HIAA in 24 h collected urine
- Urinary serotonin
- Platelet serotonin content.
- 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.
5-HIAA in urine
Spectrophotometric determination or reversed high-performance liquid chromatography (HPLC) in association with electrochemical detection.
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 . 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.
24-h urine collection
Addition of 10 mL of glacial acetic acid, measurement of the urine volume, dispatching of 20 mL to the laboratory.
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.
Conversion of 5-HIAA: mg/L × 5.23 = μmol/L
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.
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.
- 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.
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 . 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 /, /. 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 .
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 .
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 , and the diagnostic sensitivity of serotonin in platelet-rich plasma is believed to be increased . In midgut carcinoids, LOH is frequently observed at the SDHD gene (succinate-ubiquinone oxidoreductase subunit D) (distal 11q) and distal 18q.
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 . 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 .
- 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:
Preparing the patient
- Bananas, walnuts, tomatoes, pineapple, currants, plums, gooseberries, mirabelle, plums, melon, avocado, eggplant, kiwis.
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.
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.
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.
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.
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.
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.
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 /, /.
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 . It equipotently binds to the VIP-receptor responsible for triggering diarrhea . Its role in the development of flushes in conjunction with the carcinoid syndrome remains to be determined.
- Persistent profuse watery diarrhea (stool quantities greater than 1 liter)
- Severe hypokalemia and hypochlorhydria.
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.
VIP conversion: pg/mL × 0.30 = pmol/L
VIP is quite important in the diagnostic investigation of Verner-Morrison syndrome, as well as the WDHA syndrome or VIPoma /, , /. VIPomas comprise 2–7% of the GEP-NETs. The annual incidence is 1 in 10 million people . 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 . 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.
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 .
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 .
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 .
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.
Only somatostatin and octreotide are known to influence plasma VIP.
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.
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.
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 .
- Suspicion of a neuroendocrine tumor of the gastrointestinal tract (GEP-NET)
- Secretory diarrhea.
- Suspicion of pheochromocytoma, ganglioneuroma, neuroblastoma.
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.
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.
The determination of PP is clinically relevant in the diagnosis of GEP-NET tumors /, /. 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 , diseases associated with elevated PP levels are listed.
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.
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.
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.
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.
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
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.
- 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 ().
- Heme biosynthesis includes 8 individual enzyme catalyzed steps. Metabolic disorders cause the production of porphyrins ().
- 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 .
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) . 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 .
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 . 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 . 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 .
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 .
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 . Every porphyria results in the accumulation of a specific intermediate of the heme biosynthetic pathway. Refer to .
Metabolic intermediates accumulate at the synthesis step that precedes the enzymatic defect; these are the oxidized substrates of the respective defective enzyme . 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.
- Acute porphyrias
- Cutaneous porphyrias
- Recessive porphyrias.
According to clinical complaints porphyrias can be categorized as either acute hepatic (neurovisceral) or cutaneous.
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 .
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 .
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.
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 ().
- Molecular biological testing for gene mutations (mutational analysis).
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 (). 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 /, /.
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 . Lead poisoning and the rare 5-aminolevulinic acid dehydratase porphyria are associated with isolated elevation of 5-aminolevulinic acid.
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 . 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 /, /.
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 :
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.
- 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.
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 a 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 .
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 .
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.
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 .
Mutational analysis is rarely necessary to make a diagnosis of porphyria and may be misleading if a mutation is not found . 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.
Four porphyrias are inherited in an autosomal dominant pattern: acute intermittent porphyria, hereditary coproporphyrinuria, variegate porphyria and porphyria cutanea tarda . 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 . Mutations decrease enzyme activities in all tissues.
- 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 .
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 .
- 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.
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.
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.
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 . 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 . 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.
Important adipokins in obesity medicine are leptin und adiponectin.
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.
Adinonpectin has a regulating effect on glucose metabolism and lipid metabolism and promotes insulin sensitizing, anti atherogenic and anti inflammatoric effects.
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 . Adipogenesis continues throughout life with a median turnover rate of 8% .
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 .
As obesity progresses, preadipocyte differentiation becomes dysfunctional, leading to reduced insulin signaling, glucose uptake, and adiponectin release by the mature adipocytes . 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 :
- 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
Protein-losing enteropathy is an uncompensated loss of plasma proteins in the intestine . 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 /, /.
- Mucosal diseases
- Diseases of the lymphatic return
- Congenital causes.
- Erosive diseases cause a loss of plasma proteins into the gut lumen . 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.
- 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 .
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 . 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 /, /.
Chaple disease results from inherited CD55 mutations.
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)
Protein-losing enteropathy is diagnosed by ruling out other causes of hypoproteinemia and diarrhea or malabsorption.
- 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.
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.
Sens, diagnostic sensitivity; Spec, diagnostic specificity
Values are the 2.5th and 97.5th percentiles
UBT, Urea breath test threshold delta over baseline 0.5%
Acute pancreatitis: diagnosis and monitoring
Examination after 48 h
Each criterion receives a point. A score of 3 points indicates the presence of severe pancreatitis.
Clinical and laboratory findings
* Pancreatic insufficiency; Sens, diagnostic sensitivity; Spec, diagnostic specificity;
Relative risk, Frequency in sporadic cases
AD, autosomal dominant; AR, autosomal recessive; * 80% penetrance
Clinical and laboratory findings
Fecal fat determination
* Classification is based on the results of the secretin-caerulein test and the quantitative stool fat determination
Values from own working group
Clinical and laboratory findings
Test results in malabsorption
* Corresponds to > 16% of the ingested amount.
Conversion: mmol = g × 6.66; mmol/L = mg/dL × 0.0666
Clinical and laboratory findings
Clinical and laboratory findings
Detection rate of SS receptors with 111In-octreotide
CgA, chromogranin A; NSE, neuron specific enolase; * with neuroendocrine differentiation
Clinical and laboratory findings
Gastrointestinal (GI) disease
Non-GI carcinomas/kidney disease
* 5-Hydroxyindole acetic acid in 24-hour urine collection. CgA was determined using the Dako assay.
Clinical and laboratory findings
Clinical and laboratory findings
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
Clinical and laboratory findings
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
* with reference to 1 g dry weight
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
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. . 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. .
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 14.6-3 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. .