Children with fatty diarrhoea

Although LD has some chylomicrons in his circulation, there is no increase after a meal, suggesting either that he cannot absorb fat from the meal or that he has an impaired ability to form chylomicrons.

In the next experiment LD was given an intraduodenal infusion of highly emulsified fat, with droplets of the order of 4 - 6 nm in diameter. The results are shown on the right:

It seems that LD can absorb fat and form chylomicrons if he is provided with highly emulsified fat. This suggests that his problem lies in the emulsification of fat into small enough droplets to be absorbed.

We now need to consider what factors are involved in the emulsification of dietary fat into droplets small enough to be absorbed.

In the following experiment volunteers were fed a meal containing two synthetic triacylglycerols:

• One consisted of C18:0 esterified to carbons-1 and -3, and C18:2 n-6 esterified to carbon-2. Both the glycerol and the C18:2 n-6 in this triacylglycerol were labelled with 13C.
• The other consisted of C16:0 esterified to carbons-1 and -3, and C18:3 n-3 esterified to carbon-2. This triacylglycerol was unlabelled.

Three hours after the meal blood samples were taken and chylomicrons were isolated. Their triacylglycerol was analysed by gas chromatography and mass spectrometry, when the results shown in the right were obtained. Red shows the 13C labelled compounds.

There has obviously been exchange of fatty acids between carbons-1 and -3 of the two triacylglycerols, since both C16:0 and C18:0 occur in both the labelled and unlabelled triacylglycerols.

There has not been any significant exchange of the fatty acid at carbon-2 of either triacylglycerol, since [13C]C18:2 n-6 is still only associated with the [13C]glycerol, and unlabelled C18:3 n-3 is still only associated with the unlabelled glycerol.

This suggests that the dietary triacylglycerol has undergone partial hydrolysis to remove the fatty acids esterified at carbons-1 and -3, leaving the 2-monoacylglycerols and non-esterified fatty acids.

You could follow the digestion of triacylglycerol by gas chromatography, measuring the disappearance of triacylglycerol and the appearance of non-esterified fatty acids.

As triacylglycerol disappears, the resulting non-esterified fatty acids, diacylglycerol and monoacylglycerol, as well as phospholipids present in the dietary lipid and bile salts secreted by the gall bladder increasingly emulsify the lipid into smaller and smaller droplets.

What non-esterified fatty acids, diacylglycerol and monoacylglycerol, phospholipids and bile salts have in common is that all have a hydrophobic region that can dissolve in triacylglycerol, and a hydrophobic group that can interact with water. As these amphiphilic compounds increase in concentration, so they permit the emulsification of triacylglycerol into micelles - droplets 4 - 6 nm in diameter, as shown on the right - that are small enough to be taken up into intestinal mucosal cells.

The hydrophobic core of the micelles contains vitamins A, D, E and K, as well as carotenes and cholesterol.

It is apparent from the results above that the 2-monoacylglycerol is re-esterified in the intestinal mucosal cell to form triacylglycerol that is packaged into chylomicrons.

Since LD is able to absorb highly emulsified triacylglycerol and package it into chylomicrons, it is most likely that either he lacks pancreatic lipase or has a defect in bile salt synthesis or secretion. It would be easy to determine which by analysing the jejunal contents for lipase activity and the presence of bile salts. In either case there would be little or no hydrolysis of triacylglycerol and little or no emulsification of lipid into small droplets.

There are two approaches to treatment:

• administration of pancreatin, a preparation of pancreatic enzymes in enteric coated capsules (i.e. capsules that remain intact in the acid conditions of the stomach, and dissolve in the alkaline conditions of the duodenum and jejunum) taken with each meal or snack;
• control over his fat intake.

The graph on the right shows the effect of giving LD pancreatin capsules with a meal. There is now significant formation of chylomicrons, but not as much as usual.

The original case report states that "he is now 13½ years old and is very healthy and intelligent...In spite of persistent therapy with pancreatin ... he occasionally soils his clothes with liquid oil unless his dietary fat is carefully controlled. His mother has learned just how much fat she can allow him. He invariably soils if a dose of pancreatin is omitted."

(From Sheldon W, Arch Dis Childhood 39: 268 1964)

A lipase inhibitor to treat obesity

Orlistat is an inhibitor of pancreatic lipase that is used to treat severe obesity when the patient has made a serious effort to lose weight and has been only partially successful.

If the diet is too high in fat then inhibition of pancreatic lipase will have the same effects as genetic lack of lipase in LD - fatty stools and the possibility of soiling after a high fat meal.

Another child with fatty diarrhoea

CRD is a 10 year old girl. She suffers from fatty stools, but unlike LD she also suffers from diarrhoea and abdominal pain after meals. She is very small for her age, and shows signs of malnutrition (low plasma albumin and low plasma concentrations of vitamins A and E), as well as signs of vitamin A deficiency (impaired dark adaptation and poor ability to see in dim light).

The most likely diagnosis for CRD would be coeliac disease - inflammation and flattening of the intestinal villi as a result of an immune reaction to the gliadin fraction of wheat gluten. This leads to general malabsorption, and hence malnutrition. Therefore, an ileal biopsy sample was taken. It did not show any flattening of the villi, but there was a marked accumulation of droplets of triacylglycerol in the mucosal cells. At endoscopy the surface of her ileal mucosa was covered with a white stippling that resembled frost.

CRD does not suffer from coeliac disease. It seems likely that she suffers from a defect in the transport of absorbed lipid from the intestinal mucosa into the bloodstream, and has a much impaired capacity to form chylomicrons.

She has normal activity of pancreatic lipase and normal secretion of bile salts. She accumulates droplets of triacylglycerol in her intestinal mucosal cells.

Intestinal mucosal cells have a rapid turnover. They proliferate in the crypt, then migrate upwards and undergo apoptosis and are shed into the intestinal lumen about 48 hours after proliferation.

This means that the triacylglycerol they have accumulated is released into the intestinal lumen when the cells are shed.

In the following experiment volunteers were fed a meal containing two synthetic triacylglycerols:

• One consisted of C12:0 esterified to carbons-1 and -3, and C18:2 n-6 esterified to carbon-2.
• The other consisted of C18:0 esterified to carbons-1 and -3, and C18:3 n-3 esterified to carbon-2.

Three hours after the meal blood samples were taken and chylomicrons were isolated. Their triacylglycerol was analysed by gas chromatography and mass spectrometry, when it was found that only two triacylglycerols were present in chylomicrons:

• One consisted of C18:0 esterified to carbons-1 and -3, and C18:2 n-6 esterified to carbon-2.
• The other consisted of C18:0 esterified to carbons-1 and -3, and C18:3 n-3 esterified to carbon-2.

There was no C12:0 in chylomicron triacylglycerol.

Either medium-chain fatty acids like C12:0 are not absorbed, or they are absorbed by some route other than incorporation into chylomicrons.

In the next experiment the hepatic portal vein and thoracic duct of a pig were cannulated. The hepatic portal vein collects blood from the venous drainage of the villi, and delivers it to the liver. The thoracic duct carries lymph from the abdominal lymphatic ducts that arise from the lymphatic drainage of the villi and delivers it into the bloodstream via one of the great veins of the neck.

The pig was fed a meal containing a synthetic triacylglycerol consisting of:

• [14C]C12:0 esterified to carbon-1 of glycerol
• C18:2 n-6 esterified to carbon-2 of glycerol
• [14C]C18:0 esterified to carbon-3 of glycerol

The [14C]C12:0 was found exclusively in the hepatic portal vein, as non-esterified fatty acid.

The [14C]C18:0 was found exclusively in the thoracic duct, as triacylglycerol.

It seems that medium-chain fatty acids are not esterified to form triacylglycerol in the intestinal mucosa, but are transferred into the venous drainage of the villus as non-esterified fatty acids that are taken up by the liver.

By contrast, long-chain fatty acids are esterified in the intestinal mucosa, assembled into chylomicrons and exported into the lymphatic system.

Monosaccharides and amino acids are absorbed into the venous system of the villus, and go to the liver (via the hepatic portal vein) before they reach peripheral tissues. You have seen in the exercise on Summary - why do we need to eat and how do we survive between meals? that the liver takes up a considerable proportion of the glucose coming in from the small intestine after a meal, so that while the concentration of glucose in the hepatic portal vein may be as high as 20 mmol /L, the concentration in peripheral blood to which extra-hepatic tissues are exposed in normal about 8 mmol /L after a meal. The liver similarly takes up amino acids after a meal, so buffering the amount that extra-hepatic tissues are exposed to.

By contrast, long-chain fatty acids in chylomicrons enter the circulation at the thoracic duct, and all tissues in the body are exposed to chylomicrons before the liver clears the remnants.

Obviously she will require a diet with a strictly limited content of long-chain fatty acids, in order to prevent accumulation of triacylglycerol in intestinal mucosal cells and the resultant steattorhoea. However, because she is completely unable to absorb long-chain fatty acids, she is severely malnourished. One answer would be to provide her with synthetic triacylglycerols consisting mainly of medium-chain fatty acids, which are absorbed into the hepatic portal vein and do not require chylomicron formation.

She will require supplements of vitamins A, D, E and K, as well as essential fatty acids.

A family with hyperchylomicronaemia

Four out of 6 children in the same family all presented in infancy with fat intolerance, episodic abdominal pain and pancreatitis, and eruptive xanthomas. Eruptive xanthomas are clusters of small, red-yellow papules on the skin, especially over the buttocks, shoulders, and the extensor surfaces of the limbs. They result from macrophage accumulation of plasma lipids, associated with severe hyperlipidaemia. Analysis of plasma lipids showed very high concentrations of triacylglycerol, and persistence of chylomicrons in the fasting state (they are normally cleared within a few hours after a meal).

In a study of chylomicron metabolism normal volunteers were given an oral dose of [13C]palmitate (C16:0) with a meal, to label chylomicrons. Blood samples were taken over 3 hours after the test meal, simultaneously from the femoral artery, the superficial epigastric vein (which drains subcutaneous adipose tissue) and the antecubital vein (which drains the forearm, and so represents drainage form. muscle), and [13C]palmitate was measured. The results are shown below as fractional clearance = (arterial concentration - venous concentration) / arterial concentration.

(From data reported by Bickerton Set al., Diabetes 56: 168, 2007; see also Karpe F et al., Biochemical Society Transactions 35: 472, 2007)

Both adipose tissue and muscle contribute to clearance of chylomicrons. Adipose tissue stores triacylglycerol to release non-esterified fatty acids and glycerol into the bloodstream in the fasting state; muscle stores triacylglycerol for its own use, and does not release significant amounts of non-esterified fatty acids for use by other tissues.

The slight rise in adipose tissue clearance of [13C]palmitate after 300 minutes may reflect the uptake by the liver of chylomicron remnants, which is followed by release form the liver of very low density lipoprotein (VLDL), containing both newly synthesised triacylglycerol (which will not be labelled) and triacylglycerol from the chylomicron remnants, which will be labelled. You will investigate VLDL and other plasma lipoproteins in a later exercise.

Isolated adipocytes were incubated with chylomicrons with and without the addition of insulin.

In the absence of insulin there was no detectable hydrolysis of triacylglycerol, and no change in the size of the chylomicrons, as determined by light scattering.

By contrast, when the adipocytes had been incubated with insulin there was a rapid decrease in the size of the chylomicrons, and a reduction in the triacylglycerol concentration. At the same time there was a small increase in the concentration of non-esterified fatty acids in the incubation medium.

Insulin obviously stimulates the synthesis or activation of a lipase that hydrolyses the triacylglycerol in chylomicrons. Since the chylomicrons remain outside the adipocytes, this enzyme must be at the cell surface (or, in vivo, at the epithelial surface of the blood capillaries).

Most of the non-esterified fatty acid released by this enzyme obviously enters the adipocytes (where it is re-esterified to triacylglycerol), but some does not. It remains in the incubation medium. Physiologically it would remain in the bloodstream, and would be taken up by the liver, and re-esterified to triacylglycerol.

When the experiment with insulin was repeated in the presence of cycloheximide (an inhibitor or protein synthesis) there was no detectable hydrolysis of triacylglycerol, and no change in the size of the chylomicrons.

Insulin obviously stimulates the expression of the lipase, and does not activate a pre-existing enzyme.

The gene for this lipase in adipose tissue has been cloned. It encodes a protein of 475 amino acids that becomes a mature protein of 448 residues after cleavage of a signal peptide.

The presence of a signal peptide means that this is a protein that is destined for export from the cell. In the case of this lipoprotein lipase, it does indeed leave the adipocyte, and is anchored to the luminal surface of epithelial cells lining the blood capillaries.

The enzyme has a rapid turnover, with a half-life of 1 hour.

Lipoprotein lipase is synthesised in response to insulin in the fed state, but in the fasting state its activity must fall, so that adipose tissue can switch from taking up fatty acids and synthesising triacylglycerol to its fasting state role of hydrolysing its stores of triacylglycerol and exporting non-esterified fatty acids and glycerol.

The most likely cause is a genetic defect of lipoprotein lipase, so that it is either:

• not expressed at all
• expressed but inactive or with very low activity
• expressed, with normal activity, but with a defect in the post-synthetic modification to form the region that anchors it in the cell membrane of capillary endothelial cells.

Both parents of these children have normal plasma triacylglycerol after an overnight fast, but show an exaggerated post-prandial lipaemia, with chylomicrons persisting for 7 - 8 hours after a meal, compared with a normal clearance within 4 - 5 hours.

The affected children must be homozygous for the defective lipoprotein lipase, and their parents are both heterozygotes. Obviously, half the normal amount of active lipoprotein lipase is not adequate for normal clearance of chylomicrons, but does permit gradual clearance.

As for CRD discussed above, these children will require a diet that is low in long-chain fatty acids, so as to minimise the formation of chylomicrons, and supplements of triacylglycerol containing medium-chain fatty acids that are absorbed in to the hepatic portal vein and do not form chylomicrons.

Key points from this exercise:

• After a moderately fatty meal the plasma appears milky, due to the presence of chylomicrons. Chylomicrons are relatively large plasma lipoproteins - droplets of triacylglycerol emulsified by surface proteins, unesterified cholesterol and phospholipids. They have a diameter of 0.1 - 1 µm, which is large enough to scatter light.
• Over a period of 2 - 3 hours the plasma becomes clear again, as chylomicrons are cleared from the circulation.
• Pancreatic lipase hydrolyses the fatty acids esterified to carbons 1 and 3 of glycerol in triacylglycerols, resulting in the formation of non-esterified fatty acids and 2-monoacylglycerol.
• Non-esterified fatty acids, monoacylglycerol, phospholipids and bile salts emulsify the dietary lipid into micelles - droplets 4 - 6 nm in diameter that are small enough to be taken up into intestinal mucosal cells.
• In the intestinal mucosal cell monoacylglycerol is re-esterified with long-chain fatty acids to form triacylglycerol, which is packaged into chylomicrons that enter the lymphatic circulation, and then enter the bloodstream at the thoracic duct.
• This means that the products of lipid digestion and absorption are available to all tissues in the body before the liver clears the remnants. This is in contrast to the water-soluble products of digestion (monosaccharides and amino acids), which are absorbed through the hepatic portal vein, so that the liver controls what enters the circulation.
• Medium-chain fatty acids are not esterified in the intestinal mucosa, but are absorbed into the hepatic portal vein.
• A rare genetic condition results in impairment of chylomicron assembly, and accumulation of lipid droplets in the intestinal mucosal cells. This leads to fatty diarrhoea (steattorhoea) when the mucosal cells are shed into the intestinal lumen. Intestinal mucosal cells proliferate in the crypt, migrate up the villus, and undergo apoptosis and are shed into the intestinal lumen about 48 hours after proliferation.
• Chylomicrons are cleared by the action of lipoprotein lipase in adipose tissue and muscle. This is an extracellular enzyme with a short half-life.Its synthesis is upregulated by insulin.