An unconscious child with hyperammonaemia and keto-acidosis

The standard treatment for acidosis is intravenous bicarbonate to permit respiratory compensation for the acidosis by shifting the bicarbonate / carbon dioxide equilibrium to the left

Intravenous glucose.

A rectal infusion of lactulose.

See the exercise on Hyperammonaemic coma due to liver failure for discussion of how intestinal bacterial fermentation of lactulose acidifies the intestinal contents and permits lowering of plasma ammonia.

Click here for a summary of the ammonia-lowering action of the products of lactulose fermentation.

A glucose tolerance test gave normal results, and she showed a normal increase in insulin secretion in response to the glucose load.

One possible cause of hypoglycaemia and keto-acidosis is diabetes mellitus. The normal glucose tolerance test and insulin secretion eliminate this as a likely diagnosis.

Defects of any of the the enzymes of urea synthesis lead to hyperammonaemia after a moderately protein-rich meal. Therefore a liver biopsy sample was taken, and the following enzyme activities were determined. The control samples were from six infants of about the same age who were being investigated for conditions that did not lead to hyperammonaemia. The results are shown in the table below:

The results suggest that she has a partial defect of carbamoyl phosphate synthetase, which would explain the hyperammonaemia after a moderately protein-rich meal. Note that the activities of the other enzymes of urea synthesis are moderately elevated, as might be expected in response to hyperammonaemia, when there is induction of these enzymes.

Lack of carbamoyl phosphate synthetase would not explain either the severe keto-acidosis and hypoglycaemia, or her poor muscle tone and muscle weakness.

She remained well on a high-carbohydrate, very low-protein feed for several days, although the poor muscle tone and muscle weakness persisted. A second liver biopsy sample was taken after four days when her plasma ammonia was well within the normal range. The results are shown in the table below:

The activity of carbamoyl phosphate synthetase is now normal, thus suggesting that the initial diagnosis of a urea cycle enzyme defect was incorrect. The activities of the other enzymes of urea synthesis have all fallen somewhat. This is as would be expected since they will no longer be being induced in response to hyperammonaemia.

Before the results from this second biopsy sample were available, the keto-acids of several of the essential amino acids (threonine, methionine, leucine, isoleucine and valine) were introduced into her feed. This is standard practice in cases of urea cycle defects where it is desirable to maintain the nitrogen intake as low as possible to prevent hyperammonaemia, yet ensure an adequate supply of essential amino acids for growth.

This precipitated another attack of ketosis and metabolic acidosis, although her plasma ammonia was normal this time.

This suggests that the underlying problem is in the metabolism of the carbon skeletons of one or more of these essential amino acids.

In order to investigate this further, a plasma sample and a urine sample were analysed by high pressure liquid chromatography. This revealed a number of abnormalities, as shown in the table below:

She obviously has a problem metabolising propionic acid; her plasma concentration is abnormally high and she is excreting a great deal of propionic acid as propionyl carnitine.

Propionyl CoA is the homologue of acetyl CoA, and can be expected to act as a weak substrate for, and therefore competitive inhibitor of, enzymes that utilise acetyl CoA. It is formed in the metabolism of the carbon skeletons of leucine, valine, methionine and threonine - those keto-acids she was given in an attempt to ensure an adequate supply of essential amino acids.

Therefore, assuming that her condition is due to accumulation of propionyl CoA that cannot be metabolised onwards to methylmalonyl CoA and then the citric acid cycle intermediate succinyl CoA, we can explain her adverse reaction to milk. It is likely that the especially severe reaction that led to her admission was the effect of protein intake at a time of fever, when she would anyway have been metabolising a significant amount of tissue protein.

CoA is derived from the vitamin pantothenic acid, any unmetabolised propionyl CoA is likely to undergo hydrolysis to free propionic acid, or acyl transfer to carnitine, which is not normally a dietary essential, in order to spare CoA and its vitamin precursor.

Propionyl CoA is the homologue of acetyl CoA, and will be a weak substrate for, and therefore a competitive inhibitor of, citrate synthase. Methylcitrate cannot be metabolised further in the citric acid cycle.

Forming methylcitrate that cannot be metabolised, but is excreted, will deplete her tissue pool of oxaloacetate, leading to reduced citric acid cycle activity. She will therefore be more than usually reliant on glucose metabolism in the fasting state, since she will be unable to metabolise much of the acetyl CoA arising from fatty acids and ketone bodies. However, in response to glucagon released as a result of hypoglycaemia, she will continue to release fatty acids form adipose tissue and synthesise ketone bodies in the liver. The unmetabolised ketone bodies are the cause of keto-acidosis.

She is excreting a large amount of carnitine, as propionyl carnitine.

The main function of carnitine is in the uptake of fatty acids into the mitochondrial matrix for beta-oxidation. Fatty acyl CoA cannot cross the mitochondrial membrane. At the outer face of the outer mitochondrial membrane the fatty acyl group is transferred onto carnitine, catalysed by carnitine acyltransferase 1 (CAT 1). Acyl carnitine is transported across the membranes in exchange for free carnitine.and at the inner face of the inner mitochondrial membrane the acyl group is transferred onto CoA, catalysed by carnitine acyltransferase 2.

Carnitine was measured in the first liver biopsy sample taken from Angela, as well as in a muscle biopsy sample. The results are shown in the table below:

She is obviously carnitine depleted - total and free carnitine in muscle and liver are well below the reference range; her muscle short-chain acyl carnitine (mainly propionyl carnitine) is high, and we know that she is excreting a large amount of short-chain acyl carnitine in her urine. Obviously, her excretion of acyl carnitine is outstripping her capacity to synthesise carnitine.

Fatty acids are a major fuel for muscle contraction; her carnitine deficiency means that muscle contraction and muscle tone will be impaired.

Carnitine supplements. She is excreting more carnitine (as short-chain acyl carnitine) than she can synthesise, so supplements will permit her to continue to excrete propionyl carnitine yet maintain a sufficient amount in muscle.

Carnitine supplements will not prevent the keto-acidosis. She is obviously synthesising ketone bodies, so despite the carnitine depletion she is able to take up fatty acids into liver mitochondria for ketogenesis. It is the depletion of oxaloacetate because of the formation of methylcitrate that accounts for her ketosis - she has limited capacity to oxidise acetyl CoA.

See the exercise on Muscle weakness, heart failure and profound hypoglycaemia in a young girl for more on this.

N-Acetylglutamate is an obligatory activator of carbamoyl phosphate synthetase (see the exercise on Urea synthesis in the liver, and potentially fatal hyperammonaemia in a child)

Propionyl CoA is the homologue of acetyl CoA, and will be a weak substrate for, and therefore a competitive inhibitor of, enzymes that use acetyl CoA including glutamate N-acetyltransferase, the enzyme that catalyses the synthesis of N-acetylglutamate. At the time of her first liver biopsy, during her metabolic and hyperammonaemia crisis, she showed significant propionic acidaemia, and therefore the concentration of propionyl CoA in her liver will have been high. After four days on the low-protein, high-carbohydrate diet the concentration of propionyl CoA will have been considerably lower, so that N-acetylglutamate synthesis will be more or less normal. This will allow normal activity of carbamoyl phosphate synthetase.

The metabolism of a test dose of stable isotopically labelled [13C]propionate given by intravenous infusion was determined in Angela, her parents and a group of control subjects.

Skin fibroblasts were cultured and the activity of propionyl CoA carboxylase was determined in vitro by incubation with propionate, malonate and [14C] sodium bicarbonate, followed by acidification to release carbon dioxide from unmetabolised bicarbonate, and measurement of the radioactivity remaining in the incubation medium.

Malonate is an inhibitor of succinate dehydrogenase (see the exercise on Experiments with isolated liver cells - the citric acid cycle and warming up post-operative patients). If it were not added then the succinyl CoA formed by carboxylation of propionyl CoA would be oxidised in the citric acid cycle, leading to release of [14C]carbon dioxide, and there would be little or no radioactive metabolites to measure.

The results are shown in the table below::

Angela has very low ability to oxidise propionate, and her fibroblasts fix very little [14C]carbon dioxide. This suggests that she has a lack of propionyl CoA carboxylase. Her parents both have about half the normal ability to oxidise propionate, and half the normal activity of propionyl CoA carboxylase. They are both heterozygous for defective propionyl CoA carboxylase (i.e. they have one normal gene and one gene that encodes an enzyme with very low activity). Angela is homozygous for the recessive (inactive) form of the enzyme.

This is the condition of propionic acidaemia.

This is a recessive condition, so there is:

• a 1/4 chance that a child will be homozygous for the recessive allele (aa)
• a 1/4 chance that it will be homozygous for the active (normal) allele (AA)
• a 2/4 chance that it will be heterozygous, and hence a carrier (Aa)

Propionic acidaemia can be diagnosed either by an elevated concentration of methylcitrate in amniotic fluid or by very low activity of propionyl CoA carboxylase in amniocytes. However, contamination by maternal cells can give a more or less normal value propionyl CoA carboxylase activity.

Key points from this exercise:

• Propionyl CoA is normally metabolised by carboxylation to methylmalonyl CoA, followed by isomerisation to succinyl CoA, which is a citric acid cycle intermediate.
• Genetic defects of propionyl CoA carboxylase lead to accumulation of propionyl CoA. In order to conserve CoA, which is formed from the vitamin pantothenic acid, propionyl CoA may be hydrolysed to yield free propionic acid. Alternatively, the propionyl moiety may be transferred onto carnitine; the resultant propionyl carnitine is then excreted in the urine.
• The excretion of propionyl carnitine outstrips the body's capacity for carnitine synthesis, leading to carnitine depletion.
• The major role of carnitine in the body is in the transport of fatty acids across the mitochondrial membrane for beta-oxidation. The depletion of carnitine leads to poor muscle tone and muscle weakness, because of the reliance of muscle on fatty acid metabolism.
• Propionyl CoA is a poor substrate for, and hence a competitive inhibitor of some enzymes that utilise acetyl CoA:
  • citrate synthase, leading to the formation of methylcitrate, which cannot be metabolised further, and hence depletion of oxaloacetate and reduced citric acid cycle activity and reduced ATP formation
  • glutamate N-acetyltransferase, leading to much reduced synthesis of N-acetylglutamate, which is an obligatory activator of carbamoyl phosphate synthetase, the key enzyme for incorporation of ammonium into urea. This leads to potentially life-threatening hyperammonaemia.
• Because of the impaired citric acid cycle activity, tissues are more than usually reliant on glycolysis, leading to hypoglycaemia.
• In response to hypoglycaemia there is increased synthesis of ketone bodies, which cannot be metabolised through the citric acid cycle, leading to keto-acidosis.

See the exercise on Hyperammonaemic coma due to liver failure for a discussion of how intestinal bacterial fermentation of lactulose acidifies the intestinal contents and permits lowering of plasma ammonia.

See the exercise on Muscle weakness, heart failure and profound hypoglycaemia in a young girl for a discussion of the role of carnitine in fatty acid metabolism.

See the exercise on Experiments with isolated liver cells - the citric acid cycle and warming up post-operative patients for a discussion of the citric acid cycle.

For more on propionic acidaemia, see OMIM (On-line Mendelian Inheritance in Man) at http://omim.org/entry/606054)