A severe sunburn-like rash and unusual urinary metabolites

These are all neutral amino acids, with an uncharged side-chain. We know that they are all transported by the same neutral amino acid transporter. There are separate transporters for acidic, basic and neutral amino acids.

The same amino acid group transporters are found in:

the intestinal mucosa, permitting absorption of amino acids from the gut

the kidney tubules, permitting reabsorption of amino acids that have been filtered in the glomerulus

at the surface of other cells, permitting the uptake of amino acids from the bloodstream

As well as abnormally large amounts of amino acids, his urine also contained a number of indole derivatives, including indolepyruvate, indolelactate, indoleacetate, indoxyl sulphate and indican (indoxyl glucoside), which are normally excreted in only very small amounts.

The most likely metabolic precursor of these indoles in the amino acid tryptophan. Indole pyruvate is the transamination product of tryptophan. Indolelactate can be formed by reduction of indolepyruvate, and indoleacetate by oxidation.

Cleavage of the side-chain of tryptophan yields indole, which can be metabolised further to the sulphate and glucoside, which are water-soluble compounds that can be excreted in the urine.

The results to date suggest that Eddie has an abnormality of tryptophan metabolism. He was therefore given a test dose of 0.5 mmol of tryptophan per kg body weight by mouth, and his plasma tryptophan was measured at intervals over the next 8 hours. The results are shown below:

His plasma tryptophan does not increase significantly in response to the oral dose. THis suggests that he does not absorb tryptophan for the gut.

His urine was collected over 10 hours after the oral dose of tryptophan, and his excretion of kynurenine (an intermediate in the pathway of tryptophan metabolism shown above) was measured:

Eddie excreted 457 µmol of kynurenine over 10 hours after a tryptophan load

A control subject excreted 1018 µmol of kynurenine over 10 hours after a tryptophan load

This result confirms the lack of tryptophan absorption as a possible basis of his problem. After a tryptophan load the flux through the pathway is greater than the capacity of kynurenine hydroxylase and kynureninase, and kynurenine accumulates and is excreted in the urine.

He was then given an intravenous infusion of 0.5 mmol of tryptophan /kg body weight. The results are shown on the right.

Again his excretion of kynurenine was measured over 10 hours:

Eddie excreted 1020 µmol of kynurenine over 10 hours after the intravenous infusion of tryptophan

A control subject excreted 1030 µmol of of kynurenine over 10 hours after the intravenous infusion of tryptophan.

Eddie's plasma tryptophan increases normally after the intravenous infusion, and his excretion of kynurenine is also normal. This confirms that his problem was in the absorption of tryptophan from the gut, and not a defect in the pathway of tryptophan metabolism.

Pellagra is due to deficiency of niacin to form NAD, and much (indeed, most) NAD synthesis is normally from tryptophan rather than preformed dietary niacin. If he does not absorb tryptophan from the gut, he will be deficient in tryptophan, and therefore have reduced NAD synthesis.

There are two possible explanations here:

He is obviously deficient in tryptophan, and this means that he is likely to have impaired synthesis of the neurotransmitter serotonin (5-hydroxytryptophan). An argument against this is the fact that patients with pellagra due to a lack of dietary tryptophan and niacin do not show the same neurological signs, although their synthesis of serotonin must be similarly impaired. (Patients with pellagra do develop a depressive psychosis, which can indeed be attributed to reduced serotonin synthesis). However, if Eddie has a defect in the transport protein for neutral amino acids, then it is likely that he will have greatly impaired uptake of these amino acids across the blood-brain barrier, so that his central nervous system will be more severely tryptophan deprived than is the case in dietary deficiency of tryptophan.

He is excreting a variety of unusual indole compounds. It is possible that some of these are neurotoxic.

His previous neurological attacks have been in winter, associated with common winter-time infections. It is likely that under normal conditions he can synthesise just about enough NAD to meet his needs, but the added stress of infection has precipitated an attack. The skin lesions of pellagra are only seen in areas of the skin exposed to sunlight - the rash is indeed a severe form of sunburn. He is most unlikely to be exposed to enough sunlight to cause sunburn in winter.

The attack that caused his hospital admission occurred in summer, so he would have been exposed to enough sunlight to cause sunburn.

The most likely reason why he had not suffered attacks in summer before is that he is now starting his adolescent growth spurt - like infection, this produces a significant metabolic stress, and also requires more use of tryptophan for net new protein synthesis, so that less will be available for NAD synthesis, so precipitating pellagra.

There must be a way of absorbing tryptophan and other amino acids other than as the free amino acids. It is known that even relatively large peptides can cross from the intestinal lumen into the bloodstream by paracellular transport (transport through the gaps between mucosal cells - this is the basis of allergic reactions to foods.

We also know that the pancreatic proteolytic enzymes do not act on dipeptides and tripeptides, so that the end-result of protein digestion in the small intestine is a mixture of free amino acids and dipeptides and tripeptides. It is possible that these are absorbed into mucosal cells intact, rather than undergoing hydrolysis in the intestinal lumen.

Eddie was then given an oral dose of 0.5 mmol of the dipeptide tryptophanyl-glycine /kg body weight, and again his plasma tryptophan was measured at intervals over 8 hours. The results are shown on the right.

His plasma tryptophan rises normally after the oral dose of tryptophanyl-glycine.This suggests that he can indeed absorb dipeptides into intestinal mucosal cells, where they are hydrolysed to free amino acids.

The easiest experiment here would be to use everted gut sacs form a rat or mouse.

You prepare them by removing the small intestine, washing it through with buffered saline solution to remove the gut contents, then turning it inside out by sliding it carefully over a glass rod, tying down one end and pulling it so that the mucosal side is now outside and the serosal side inside.

You can now cut the everted gut into pieces 2 - 3 cm long, tie off one end of each piece, fill it with buffer, and tie off the other end. These everted gut sacs are then incubated in buffer containing glucose and free amino acid, dipeptide or both together. Transport from the mucosal side (now outside) to the serosal side (now inside) will occur, at at the end of the incubation you can withdraw to contents of the sac using a needle.

If transport of the free amino acid and dipeptide occur by the same transporter then once the concentration of either is high enough to saturate the transporter, adding the other will have no additional effect on the amount of amino acid + dipeptide found inside the sac at the end of the incubation.

However, if there are separate transporters for free amino acids and dipeptides, then once one transporter is saturated, adding more of the other substrate will lead to an increase in the amount of amino acid + dipeptide found inside the sac at the end of the incubation. The two processes will be additive.

If Eddie is not absorbing free tryptophan from the gut, then there will be a considerable amount in the intestinal lumen, and this will provide a substrate for bacterial metabolism. It is known that intestinal bacteria can ferment tryptophan; indeed, much of the odour of faeces is due to indole and skatole (methylindole) formed by bacterial metabolism of tryptophan.

The simplest experiment would be to treat him with a broad spectrum antibiotic such as neomycin, which is not absorbed from the gut. Neomycin is commonly used to treat persistent intestinal bacterial infections. It does not sterilise the gut, since it does not kill yeasts and fungi, but it does kill more or less all the bacterial microflora.

When Eddie was treated with neomycin for three days, chromatography of his urine showed none of the abnormal indoles, although his urinary excretion of tryptophan (and other neutral amino acids) was still very much higher than normal.

The disappearance of the abnormal indoles after antibiotic treatment provides good evidence that he is not synthesising them himself, but that they are the result of bacterial fermentation of unabsorbed tryptophan in the intestinal lumen.

He seems to be able to maintain reasonable health under most conditions, but to prevent the development of pellagra it would be sensible to prescribe him supplements of nicotinamide to permit adequate synthesis of NAD. A relatively high protein diet will ensure that he has enough tryptophan available from the absorption of dipeptides and tripeptides.

Key points from this exercise:

• The amino acid transporters in cell membranes are group specific. Rather than having a separate transporter for each amino acids, they are transported in groups depending on the chemical nature of the side-chain: acidic, basic or neutral.
• The same amino acid transporters are involved in absorption of amino acids from the small intestine, re-absorption in the kidney of amino acids filtered at the glomerulus, and in transport of amino acids across the blood-brain barrier.
• Proteolytic enzymes secreted into the intestinal lumen do not hydrolyse dipeptides and tripeptides.
• Dipeptides and tripeptides resulting from hydrolysis of dietary proteins in the small intestine are absorbed intact into the intestinal mucosal cells, then hydrolysed to their constituent amino acids.
• The transport of dipeptides and tripeptides is by transporters that are separate from those for amino acids.
• In addition to its role in protein synthesis and the synthesis of the neurotransmitter serotonin (5-hydroxytryptamine), tryptophan provides much, or even most, of the nicotinamide required for synthesis of the coenzymes NAD and NADP.
• Unabsorbed amino acids in the intestinal lumen are substrates for intestinal bacterial fermentation. The products of bacterial metabolism can be absorbed and excreted unchanged or after onward metabolism (e.g. conjugation with sulphate, glucose of glucuronic acid) in the urine. Some of the products of tryptophan metabolism by bacteria may be neurotoxic.

This case was originally described in 1956 by Baron DN, Dent CE, Harris H et al. Hereditary pellagra-like skin rash with temporary cerebellar ataxia, constant renal amino-aciduria, and other bizarre biochemical features. Lancet 271, 421-428. The patient was called Eddie Hartnup, and the condition is now known as Hartnup disease or Hartnup syndrome. Study of this patient provided the early evidence that amino acids were transported by proteins that have specificity for groups of among acids (acidic, basic, neutral), as well as early evidence that dipeptides are absorbed from the intestinal lumen by a separate mechanism, and undergo hydrolysis to the free amino acids intracellularly.

For more on Hartnup disease click here