Hyperammonaemic coma due to liver failure

Jaundice is due to accumulation of bilirubin the blood plasma, giving it and his skin a yellow colour.

Jaundice can develop for two reasons:

• when bilirubin arising from the catabolism of haem in old red blood cells is not conjugated in the liver
• when the bile duct is obstructed (e.g. with gall stones) so that bilirubin cannot be excreted in the bile

The GP suspects that Mr ABV's problem is viral hepatitis, but also thinks that it may be due to alcoholic liver damage, because she can smell alcohol on his breath, and on abdominal examination his liver is palpable and firm. She takes blood samples for both standard liver function tests (measurement of liver enzymes in the blood plasma) and also immunology to test for viral hepatitis.

On questioning, Mr ABV says he only drinks a glass or two of whisky each evening, and the occasional glass of wine.

The immunology results come back negative. The liver function test results are shown in the table below.

They have been released from dead and dying liver cells. They indicate the extent of liver damage, but have no function in the bloodstream.

The amount of an enzyme in plasma is measured by measuring its catalytic activity.

This means that the limiting factor in the assay must be the amount of enzyme that is present, and not the amount of substrate that is available for the enzyme to act on.

In other words, the enzyme should be more or less saturated with substrate, so that it is acting at its maximum rate (Vmax).

Saturation of the enzyme is achieved when the concentration of substrate is about 10 - 20x greater than the Km.

At lower concentrations of substrate the rate of product formation will depend on both the amount of enzyme present and the amount of substrate available, and the results will be meaningless.

In non-alcoholic liver disease the ratio of aspartate transaminase : alanine transaminase activities in plasma is < 1. In alcoholic liver disease it is > 2.

No. The ratio of aspartate transaminase : alanine transaminase activities in his plasma is 2.5, which is strongly indicative of alcoholic liver disease, suggesting that he must be drinking considerably more than a glass or two of whisky each evening, and the occasional glass of wine

This is the result of zonation in the liver.

Hepatocytes in the region around the hepatic portal vein (periportal hepatocytes) have a different complement of enzymes from those around the central vein (perivenous hepatocytes), and notably have a higher content of aspartate transaminase than of alanine transaminase.

Alcohol reaches the liver through the hepatic portal vein, which drains the gut, and therefore will affect periportal hepatocytes more than those further away from the hepatic portal vein.

In non-alcoholic liver disease, cells throughout the liver, and not just those adjacent to the hepatic portal vein, are affected. Perivenous hepatocytes make up a considerably greater proportion of total hepatocytes than do periportal cells.

Bilirubin metabolism is summarised below:

We now suspect that Mr ABV is a heavy drinker, and his jaundice is due to alcoholic liver disease. He has an enlarged liver with signs of cirrhosis.

The picture above on the left shows the surface anatomy of a cirrhotic liver, and below a cross section.

The picture on the right shows the histology of a cirrhotic liver, with regenerating hepatocytes surrounded by fibrous connective tissue.

It will be unconjugated bilirubin - his is obviously in liver failure and his liver cannot conjugate the bilirubin that is formed by macrophages.

You would see conjugated bilirubin when jaundice is due to obstruction of the bile duct (e.g. by gall stones) because although the liver can conjugate bilirubin, it cannot be adequately excreted in the bile.

Mr ABV was admitted to his local hospital, and one morning some days later (when it was assumed that he had not been drinking alcohol) he was found in a coma. His blood ammonia concentration was found to be 200 µmol /L, compared with a reference value of < 60 µmol /L.

There is equilibrium between ammonia and ammonium ions in plasma:

Almost all will be present as ammonium ions (NH4+), with only a very small amount as ammonia (NH3)

Both acidosis and alkalosis can lead to loss of consciousness.

No. There is a considerable buffer capacity in plasma (as well as haemoglobin in red blood cells), and an 240 µmol /L of ammonia is not enough to have any effect on plasma pH.

The equilibrium of glutamate dehydrogenase is finely poised, and the direction of reaction (deamination of glutamate or synthesis of glutamate from alpha-ketoglutarate (2-oxoglutarate) depends on the relative concentrations of the substrates, and especially on the concentration of ammonium.

In the presence of moderately high concentrations of ammonium, the reaction will proceed in the direction of glutamate formation.

Alpha-ketoglutarate is a key intermediate in the citric acid cycle, which is the main pathway for ATP formation in the cell. If there is excessive withdrawal of alpha-ketoglutarate to form glutamate, then the citric acid cycle will slow down, leading to reduced formation of ATP.

This is especially important in the brain, which cannot oxidise fatty acids by beta-oxidation, and so is wholly reliant on glycolysis and citric acid cycle activity.

A reduction in the rate of ATP formation in the brain will lead to reduced ion transport, and hence loss of the normal transmembrane ion gradients, and loss of consciousness.

The nurses call a physician, who gives Mr ABV a rectal infusion of lactulose, and he recovers consciousness. A blood sample now shows that his plasma ammonia has fallen to below 50 µmol /L.

Lactulose is a disaccharide that is not normally found in foods, although small amounts are formed in heated milk. It is not hydrolysed by intestinal enzymes, but is a substrate for fermentation by intestinal bacteria to yield 4 mol of lactate and 4 hydrogen ions.

Although most plasma ammonia is present as ammonium (NH4+), this is in equilibrium with a very small amount of ammonia (NH3). Ammonia is lipid soluble, and can diffuse across cell membranes, from the bloodstream into the intestinal lumen. The fermentation of lactulose acidifies the intestinal contents, and as ammonia diffuses into the gut, it is trapped as ammonium, which cannot cross cell membranes. This means that more ammonia can diffuse into the gut lumen, down its concentration gradient, so lowering the plasma concentration of ammonia, which is replaced from the larger pool of ammonium.

Within a short time, the plasma concentration of (ammonia + ammonium) will fall to the within the normal range, and the reaction of glutamate dehydrogenase will proceed in the direction of formation of alpha-ketoglutarate, restoring citric acid cycle activity and ATP formation in the brain.

Key points from this exercise:

• Dead and dying cells release enzymes into the bloodstream, and measurement of these enzymes in plasma can be useful diagnostically, although they have no function in the bloodstream.
• In order to measure the activity of an enzyme in the bloodstream, the limiting factor in the formation of product must be the amount of enzyme, not the amount of substrate. In order to ensure that the enzyme is saturated with substrate and acting at Vmax, the concentration of substrate needs to be 10 - 20x higher than the Km of the enzyme
• Bilirubin is synthesised in macrophages by oxidation of the haem from old red blood cells, and is conjugated with glucuronic acid in the liver, then excreted in the bile. In obstructive jaundice, when the bile duct is blocked, liver function is normal, and conjugated bilirubin accumulates in the bloodstream. In liver failure, bilirubin cannot be conjugated adequately, and unconjugated bilirubin accumulates in the bloodstream.
• Hepatocytes in the periportal region of the liver have a different complement of enzymes from those in the periportal region. In alcoholic liver disease it is mainly cells around the hepatic portal vein that are damaged, while in non-alcoholic liver disease cells are damaged in both regions.
• The direction of the reaction of glutamate dehydrogenase is dependent on the amount of ammonium available; even a moderate increase in [plasma and tissue ammonium will lead to formation of glutamate from alpha-ketoglutarate. This depletes the tissue pool of alpha-ketoglutarate and slows the rate of citric acid cycle activity, leading to impaired formation of ATP. In the brain this leads to failure of ion transport across membranes, and loss of consciousness.
• Although most ammonia in the bloodstream is as ammonium ions (NH4+), this is in equilibrium with a small amount of ammonia (NH3). Ammonia is lipid soluble, and can diffuse across cell membranes, whereas ammonium cannot. Lactulose is fermented by intestinal bacteria to form lactate and hydrogen ions. This acidifies the intestinal contents and traps ammonia that diffuses into the gut as ammonium. Ammonia continues to diffuse into the gut down a concentration gradient, so lowering blood ammonia + ammonium.