WORK SESSION 4: Malabsorption/Ion Channels

AIM

To enable an understanding of the biochemical/physiological basis of some malabsorption syndromes.

OBJECTIVES

By the end of this Unit you will:

• have an understanding of the general principles of nutrient absorption in the gut, with particular emphasis on the small intestine.
• appreciate the principles of electrolyte, sugar and water transport in the small intestine, and electrolyte transport in the pancreas.
• have an understanding of a number of malabsorption syndromes, their relation to normal absorption processes, and their underlying biochemistry/physiology. In particular, sugar intolerance, cholera toxin, cystic fibrosis and coeliac disease.
• appreciate malabsorption events in the context of diarrhoeal symptoms.

Medical lecture notes; appropriate textbook (e.g. Kumar and Clark). Complete questions 1-4 before the class.

A. ILEAL ELECTROLYTE/WATER TRANSPORT, INCLUDING SUGARS

[Kumar and Clark p.689 (6th Edition), and lecture notes]

Virtually all nutrients are absorbed into the blood across the mucosa of the small intestine. In addition the intestine absorbs water and electrolytes, thus playing a critical role in maintenance of body water and acid-base balance. The single most important process that makes such absorption possible is the establishment of a Na⁺ ion electrochemical gradient across the epithelial cell boundary. All the cells in the body are required to maintain a low intracellular sodium concentration to establish the electrochemical gradient and membrane potential. Low intracellular sodium is maintained by Na⁺/K⁺ ATPases, so called sodium pumps, that are embedded in the basolateral membrane Each pump exports 3 sodium ions in exchange for 2 potassium ions, thus establishing both a charge and concentration gradient across the basolateral membrane.

The Na+/K+ ATPase is a highly conserved integral membrane protein that is expressed in virtually all cells. It has been estimated that about 25% of cellular ATP is hydrolysed by sodium pumps. Depending on cell type, there may be 800,000 to 30 million pumps on the surface of a cell! They may be distributed evenly on the cell surface, or clustered in certain membrane domains, as in the basolateral membranes of polarised epithelial cells in the kidney and intestine.

Absorption of water and electrolytes

An average sized individual takes in 1-2 litres of dietary fluid each day; another 6-7 litres is received by the small intestine as secretions from salivary glands, stomach, pancreas and the small intestine itself. By the time ingesta reaches the large intestine, so 80% of this fluid has been (re) absorbed.

• Net movement of water across cell membranes always occurs by osmosis
• Absorption of water is absolutely dependent on absorption of solutes, particularly sodium

The process would occur as follows:

QUESTION 1

Use the information above to complete Figure 1. Include the ion or solute being transported (A-D), indicate the direction of the osmotic gradient and the major route of water absorption.

Figure 1

QUESTION 2

Using Figure 1, give examples of an antiport transporter, a symporter and an active transport system; what is the source of energy for the active transport system?

INFORMATION

Bicarbonate excretion is now known to occur in the stomach, the small and large intestine, and is a necessary component in the maintenance of the "microclimate" near the apical membranes of gastric and intestinal surface cells. Duodenal bicarbonate excretion is thought to be mediated in part by electroneutral Cl^--HCO₃^- exchange (as shown in Figure 1), in part by an electrogenic secretory pathway, and in part by paracellular diffusion [electrogenic = movement of an ion, without an accompanying counterion resulting in an endergonic separation of a positive and negative charge; paracellular = through the cell-cell tight junctions]. Duodenal electrogenic bicarbonate secretion is proportionally greater than electroneutral bicarbonate, and is the secretory pathway activated by most secretagogues. Its molecular nature is unclear; possibilities are an anion channel with high bicarbonate permeability, or a Cl^- channel functionally coupled to an anion exchanger through which the secreted Cl^- is recycled. The cystic fibrosis transmembrane regulator (CFTR) channel is abundantly expressed in all parts of the small intestine and is permeable to bicarbonate ion, albeit much less than to Cl^- (not shown in Figure1).

Secretion of water

It is important to realise that the epithelial cells lining the small intastine are responsible foe both absorption and secretion of water. Large quantities of water are secreted intl the lumen of the small intestin during the the digestive process. this is essential to disperse the chyme, maximise contact with the epithelium, and allow efficient enzymatic digestion ot occur. Regardless of whether it is being secreted or absorbed, water flows across the mucosa in response to an osmotic gradient.

There are 2 distinct processes that establish an osmotic gradient that pulls water into the lumen of the intestine

1. Increase in luminal osmotic pressure resulting from influx and digestion of foodstuffs.

QUESTION 3

Why would the osmotic pressure of the gut lumen increase as digestion of foodstuffs proceeds?

2. Crypt cells actively secrete electrolytes, leading to water secretion. The apical or lunimal membranes of crypt epithelial cells contain an ion channel of immense medical significance, a cyclic AMP-dependent chloride channel also known as the cystic fibrosis transmembrane conductance regulator or CFTR

The two fundamental structures or the small intestinal mucosa are shown in the diaram below. The villi are covered predominantly by mature, absorptive enterocytes along with occasional mucus-secreting goblet cells. Crypts (of Lieberkuhn) are lined with younger secreting cells. Stem cells in the crypts give rise to enterocytes, enterendocrine cells, goblet cells or Paneth cells. Enterocytes begin life as secreting cells in the crypt, migrate up the walls of the crypt, and end life as absorptive cells on the villi

The process of water secretion across crypt clls is explained as follows:

• Elevated intracellular concentrations of cAMP in crypt cells activate the CFTR chloride channel. The channel has a high specificity for chloride ions which move in one direction only through this channel, in this case from within to the outside of the cell (into the crypt).
• Accumulation of chloride ions in the crypt creates an electrical potential that draws sodium ions across the epithelium, principally through the tight junctions. The net result is secretion of NaCl.
• An osmotic gradient is created across the tight junction and water is drawn into the lumen.
• A symporter on the basolateral cell surface allows sodium and chloride to enter the cell to maintain intracellular chloride concentration.
• Sodium/potassium pumps on the basolateral surfaces remove sodium from the cells, providing a source of sodium in the intracellular space, and maintaining the sodium gradient.

  [The description above is fairly simplistic, but valid. The movement of electrolytes across epithelial cell layers is medicated by a complicated array of different channels. For exapmle, while transport of chloride by CFTR is very important, there are other chloride channels. Furthermore, the distribution and relative importance of these processes varies considerably in different areas of the gut.]

  QUESTION 4

  Using the above information on water secretion, identify A-E in Figure 2 below.

  Figure 2: electrolyte movement in secretory crypt cells of the small intestine.

  

  B. MALABSORPTION

  QUESTION 5 Define the term "malabsorption".

  QUESTION 6 List the most common causes of malabsorption.

  C. DIARRHOEA

  [Kumar and Clark p331]

  QUESTION 7 List the four most common classifications of diarrhoea.

  D. CASE STUDIES

  The following case studies (glucose-galactose intolerance, cystic fibrosis, cholera toxin and coeliac disease) will be used to exemplify different aspects of malabsorption, effects on electrolyte balance, and resulting symptoms of diarrhoea.

  CASE STUDY 1: GLUCOSE-GALACTOSE INTOLERANCE

  [There is very little information in Kumar and Clark on this topic (p.298); the following passage will provide you with relevant information].

  INFORMATION

  The sugars lactose, sucrose and maltose are broken down by the enzymes lactase, sucrose and maltase respectively, which are located in the lining of the small intestine. Normally the enzymes break these sugars into simple sugars such as glucose, which are then absorbed into the blood through the intestinal wall. If the necessary enzyme is lacking, the sugars are not digested, and so can't be absorbed. Thus, they remain in the small intestine. The resulting high concentration of sugar draws fluid into the small intestine, causing diarrhoea. The unabsorbed sugar is then fermented by bacteria in the large intestine, producing acidic stool and flatulence. Enzyme deficiency occurs in coeliac disease, tropical sprue, and infections of the intestine. Enzyme deficiency can also be caused by antibiotics, especially neomycin, or it can be congenital. In the inherited disorder, there is a complete loss in the ability to absorb the sugar, and inevitably diarrhoea will result. If untreated, a new-born baby will die from dehydration within a couple of days.

  An different example of congenital disorder resides in the Na⁺/glucose transporter itself. Because the Na⁺/glucose co-transporter has been cloned (SGLT1), it has been possible to screen for mutations in the gene sequence in individuals with this condition. Every affected individual studied has a missense mutation in the gene encoding SGLT1. Some of these mutations result in improper trafficking of the protein whilst others result in reduced transport ability. Either way, glucose and galactose transport across the small intestinal epithelium is significantly impaired.

  Lactose intolerance

  Some degree of lactose intolerance occurs in about 75% of adults. It affects fewer than 20% of adults of northwestern European origin, but 90% of Asians. Lactose intolerance is common among people from the Mediterranean area. About 75% of non-white North Americans gradually develop lactose intolerance between ages 10 and 20. In such cases, the extent of synthesis of the enzyme lactase is reduced with age, and is a normal occurrence in such races.

  Symptoms: People with lactose intolerance usually cannot tolerate milk and other dairy products, which contain lactose. Some people recognize this early in life and consciously avoid dairy products. A child who can't tolerate lactose has diarrhoea and does not gain weight when milk is part of the diet. An adult may have audible bowel sounds (borborygmi), abdominal bloating, flatulence, nausea, an urgent need to defecate, abdominal cramps, and diarrhoea following a meal containing lactose. Severe diarrhoea may prevent proper absorption of nutrients because they are expelled from the gut too quickly. Similar symptoms can be caused by lack of the enzymes sucrase and maltase.

  Diagnosis: A doctor suspects lactose intolerance when a person has symptoms after consuming dairy products. If a person has lactose intolerance, eating a dose of lactose causes diarrhoea, bloating and a feeling of abdominal discomfort in 20 to 30 minutes. Because the test dose is not broken down into glucose, blood glucose levels do not rise. A biopsy of the small intestine may be performed. The specimen of small intestine is viewed under the microscope and tested for lactase or other enzyme activity.

  Treatment: Lactose intolerance can be controlled by avoiding foods containing lactose, primarily dairy products. To prevent a calcium deficiency, calcium supplements should be given. Alternatively, lactase can be added to milk before being consumed.

  Note: Ingestion of large amounts of hexitols (e.g. sorbitol, mannitol), which are used as sugar substitutes, cause diarrhoea as a result of their slow absorption and stimulation of rapid small-bowel motility ("dietetic food" or "chewing gum" diarrhoea). Diarrhoea can result with the use of poorly absorbed salts (Mg sulphate, Na phosphates) as laxatives or antacids.

  QUESTION 8 In your own words, how do you explain the resulting diarrhoea from lactose intolerance?

  QUESTION 9 How would you classify this type of diarrhoea?

  QUESTION 10 What is lactose?

  QUESTION 11 What is the lactose tolerance test?

  QUESTION 12 In a patient suffering lactose intolerance, blood glucose would not be expected to rise following a lactose tolerance test. Why not?

  QUESTION 13 Why is "live" yoghurt usually tolerated in such patients?

  CASE STUDY 2: CYSTIC FIBROSIS

  [Kumar and Clark; p.413, 909]

  QUESTION 14 What is the incidence of cystic fibrosis?

  QUESTION 15 What are the major tissues involved?

  QUESTION 16 The gene responsible for cystic fibrosis is located on chromosome 7. What name has been given to this gene?

  QUESTION 17 The protein product of this gene is a membrane channel with a specific selectivity for one ion. What is the ion?

  INFORMATION:

  By the late 1990s, nearly 45,000 mutant chromosomes had been examined by the Cystic Fibrosis Genetic Analysis Consortium, and more than 900 presumptive mutation sites had been identified. Some of the more common ones are listed in Table 1. The overwhelming majority of CFTR mutations (>80%) are located in the two nucleotide-binding (NB) domains. This group includes the common Phe⁵⁰⁸ deletion (deltaF508) and a number of point mutations (missense) that alter the protein chemistry in regions that are highly conserved among many ATP-binding proteins. Interestingly, CFTR protein is relatively tolerant of missense mutation. Most of the remaining mutations are extremely rare. Mutations that cause CF are either mild or severe, as determined in large part by the level of pancreatic function. The concordance of pancreatic involvement is high among CF patients of the same family, suggesting a genotype-phenotype correlation. About 85% of CF patients are pancreatic insufficient (PI) and are thought to have two severe alleles, either homozygous or compound heterozygous; the other 15% are pancreatic sufficient (PS) and are believed to have at least one mild allele. The common deltaF508 mutation, if homozygous, causes classic severe CF with pancreatic insufficiency (PI) and a high risk of meconium ileus in the infant. Most severe alleles produce an incomplete (or no) CFTR protein, while mild alleles are often missense mutations.

  Table 1. Common CFTR Mutations 1
  Mutation	Type	Frequency (%)
  deltaF508	deletion	28,948 (66.0)
  G542ter	nonsense	1062 (2.4)
  G551D	missense	717 (1.6)
  N1303K	missense	589 (1.3)
  W1282ter	nonsense	536 (1.2)
  R553ter	nonsense	322 (0.7)
  621+1 G->T	splice junction	315 (0.7)
  1717-1 G->A	splice junction	284 (0.6)
  R117H	missense	133 (0.3)
  3849+10kb C->T	alternative splice	104 (0.2)
  1 Data are from the Cystic Fibrosis Genetic Analysis Consortium, based on 43,000 CF chromosomes examined through 1994. Mutations are indicated by amino acid residue or nucleotide position; ter, termination codon.

  QUESTION 18 What do you understand by the terms deletion, missense and nonsense mutation?

  QUESTION 19 What are the major symptoms of cystic fibrosis?

  QUESTION 20 What is Meconium ileus?

  QUESTION 21 Given that the chloride channel in the epithelium of the small intestine is impaired in cystic fibrosis sufferers, how would this explain meconium ileus in the newborn infant?

  INFORMATION

  Meconium. Definition: The infant's first stools, it is composed of amniotic fluid, mucous, lanugo (the fine hair that covers the baby's body), bile, and cells that have been shed from the skin and the intestinal tract. Meconium is thick, greenish black, and sticky. During pregnancy the baby floats in amniotic fluid inside the mother's uterus. This fluid protects the baby while it grows and develops. The baby swallows the amniotic fluid which contains all the other constituents mentioned above.

  QUESTION 22 Symptomatic steatorrhoea is a common clinical feature in cystic fibrosis. What is steatorrhoea, what is its biochemical basis, and how may it be treated?

  The role of the CFTR in sodium chloride transport across epithelia has been a puzzling issue for many years. The airways and pancreas, colon and sweat ducts all express CFTR and the epithelial sodium channel (ENaC) in their apical membranes. However, these epithelia use strikingly different mechanisms to transport NaCl.
  
  Pancreas and airways
  In the epithelia of pancreas and lungs, Na is absorbed transcellularly through the ENaC channel, whereas Cl enters through the tight junctions; this scenario would constitute the absorptive mode. The osmotic potential generated on the basolateral surface would draw water through from the lumen.
  When raised levels of cAMP activate the CFTR channel (which in turn inhibits the ENaC channel, chloride ions pass into the lumen, sodium ions follow through the tight junctions, and the resulting osmotic gradient secretes water into the lumen.

  Figure 3: Electrolyte movement in pancreas and lung epithelial cells

  
  

  

  QUESTION 23 Annotate Figure 3 with replacements for A-F, assuming the cell to be in the secretory mode

  QUESTION 24 In your own words, explain what might be the underlying cause of pancreatic dysfunction in cystic fibrosis patients.

  Some related reading, for interest - a recent article in New Scientist investigates the success of the Cystic Fibrosis screening programme in the USA

  Sweat ducts
  In contrast, in the sweat ducts cells both chloride and sodium ions are absorbed transcellularly from the duct, following the activation in parallel of the CFTR and ENaC by cAMP. Chloride transport through the CFTR is into the cell. A chloride ion channel on the basolateral surface membrane allows for the exit of CL^- to retain electrical balance. Tight junctions between these epithelial cells are particularly impermeable to water.

  The sweat gland defect in cystic fibrosis has been known for many years and this fact is still used today to diagnose F with the sweat test.

  QUESTION 25 Annotate Figure 4 with replacementd for A-E

  Figure 4: Electrolyte movement in sweat Duct Cells

  QUESTION 26 In your own words, explain why cystic fibrosis sufferers often have salty sweat.

  
  

  

  CASE STUDY 3: CHOLERA TOXIN

  [Kumar and Clark; p.84]

  QUESTION 27 What is the causative organism of cholera?

  QUESTION 28 What are the principal symptoms of cholera?

  QUESTION 29 What are the molecular events that produce the symptoms? Use Figure 5 below and fill in the legends.

Figure 5

Step 1:	Step 2:
Step 3:	Step 4:

QUESTION 30 What form of maintenance therapy is used to treat cholera victims?

Figure 6 below explains the basis of action of the sodium/glucose cotransport system.

Figure 6

The Na + glucose cotransport protein binds Na and glucose in the lumen. Both substances must be present in the lumen at the same time.	The cotransport protein changes in conformation and releases Na and glucose into the intestinal cells. This raises the osmotic pressure within the cell and sucks water back into the body by osmosis.

QUESTION 31 How does this Figure form the basis of oral rehydration therapy?

QUESTION 32 Enterotoxigenic E.Coli produces a diarrhoeal illness that may be indistinguishable from severe cholera. What are the biochemical similarities/differences in the mode of action of E.coli ST-toxin and cholera toxin?

QUESTION 33 It has been suggested that there is a selective advantage to individuals carrying the CFTR mutation, that might explain the high incidence of this mutation. Can you offer an explanation for this in view of the symptoms of cholera?

CASE STUDY 4: COELIAC DISEASE

[Kumar and Clark; p.301]

Coeliac disease has a high incidence (approximately 1:100 adults) and is commonly underdiagnosed. It leads to an atrophy of the villi in the small intestines, as exemplified in the photographs below, and a consequent reduction in surface area for absorption.

QUESTION 34 Is coeliac disease likely to be associated with symptoms of diarrhoea? Explain your reasoning.

SUMMARY OF CASES

Complete the following table.

Disorder	Glucose-Galactose Intolerance	Cholera	Cystic Fibrosis	Coeliac Disease
Is there a Genetic basis to this disorder?
What are the major tissue involvements?
Associated with diarrhoea? Secretory or Osmotic?




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