Scientia et Arte

If science were art and art were science, then the howling black wolf has probably swallowed some N-(4-hydroxyphenyl)ethanamide.

Case Study 1: Tryptophan and Blue Diaper Syndrome (with some postscripts)

Urine color could tell many things. Of course, the normal color is yellow, due to the pigment urochrome. A cause for alarm is when one’s urine changes color, from varying degrees of yellow, to disturbing ones such as red, green, or blue. The normal yellow color in urine varies depending on one’s fluid intake. The latter ones are really disturbing, and might need medical intervention. The diagnoses might be related to the amount and extent of absorption of certain compounds into the bloodstream and urine. It is thus important to characterize the components of urine, their possible sources (dyes, food, medications, toxins) and/or underlying and consequential medical conditions. Treatment would not tackle the discoloration per se, but the underlying disease or syndrome.

Here is an interesting case from Richard Ludueña’s Learning Biochemistry: 100 Case-Oriented Problems (1995):

Your patient is a 10-month-old boy whose urine is blue. You find that the blue color follows the administration of oral tryptophan. His serum levels of tryptohan are very low. His urinary levels of tryptophan are very low. [When analyzed, the blue pigment in his urine turned out to be indigo blue.]

Tryptophan (Trp) is an essential amino acid (also hydrophobic and aromatic), hence the human body needs to acquire it from food. Plants and microorganisms are the most possible sources of such compounds, since they can produce the enzymes necessary for synthesis. Tryptophan has an indole group. This fact will be helpful in this discussion. L-Trp is the most common stereoisomer.

Structure of Tryptophan

Tryptophan is introduced into the body via oral administration (i.e. as a drug).  However, as mentioned in the case, the tryptophan levels in the boy’s serum is low. Here are three possibilities which could explain the scenario:

1. Tryptophan is being poorly absorbed in the bloodstream.

2. There is increased catabolism for tryptophan.

3. The excretion of tryptophan is higher than normal.

The second clue is that his urine’s trytophan level is quite low. Hence possibility 3, which dwells on a possible kidney defect, can be discarded.

The second possibility gives a more complicated issue. Catabolites of tryptophan, especially those made by mammalian enzymes, are not blue. Thus, possibility 2 would also not be correct, since our enzyme activity for tryptophan metabolism would not account for the color.

We are left with the first option: tryptophan is poorly absorbed. It has been known that bacteria could metabolize tryptophan into products that are indigo blue. Resident bacteria in the intestine could do this. Hence, tryptophan is most probably being left in the intestine instead of being absorbed into the bloodstream. What will instead be absorbed and carried out into urine is the indigo blue bacteria catabolite of tryptophan.

The picture below demonstrates a pathway for the conversion of tryptophan to indigo blue:

Conversion of tryptophan to indigo blue (lifted from Ludueña, 1995)

This condition is known as Blue Diaper Syndrome (BDS). From the name itself, and the age of the patient, this disease affects newborn infants. Children with the condition leave blue-stained diapers. BDS is a metabolic disorder (an “inborn error”) and could be inherited (through autosomal recessivity). Amino acid metabolism (in this case, tryptophan) is defective since the compound in question does not enter substantially into the bloodstream.

The problem lies might lie in defects in the protein responsible for transporting tryptophan in the intestine. Research has been done on the mutations in the genes LAT2 and TAT1, which encodes for solute carrier proteins, including the protein (an enzyme) for the transport and absorption of tryptophan. Mutations could also come from base substitutions or other factors which might impede the transcription-translation process. The said protein might not be produced and present after all.

BDS has a more technical name: familial hypercalcemia with nephrocalcinosis and indicanuria. This is the name given by the classic and most referred paper on BDS: a study of Drummond, et al., entitled “The Blue Diaper Syndrome: Familial hypercalcemia with nephrocalcinosis and indicanuria; a new familial disease, with definition of the metabolic abnormality”, published in December 1964 in the American Journal of Medicine. [Unfortunately, I was not able to have a copy of the paper as of this post.] Consequently, BDS has been also known as Drummond’s Syndrome.

Let us digest the technical term one by one.

– BDS is familial hypercalcemia. Familial could also be synonymous with being hereditary or the disease related to members of the family. This is already mentioned.

The blue tint is also a symptom of hypercalcemia, or high levels of calcium. This is why BDS is linked to and is a possible cause for hypercalcemia. Hypercalcemia could also lead to kidney problems. The general method for treating hypercalcemia is looking at its underlying cause.

– Nephrocalcinosis happens when calcium salts are deposited into the kidneys. This could be a consequence of hypercalcemia.

– Bacteria in the gut can transform tryptophan into indigo blue. This condition is that of indicanuria because of excessive production and excretion of indican, an immediate precursor of indigo blue via oxidation. Interestingly, the blue tint can be termed as indoluria, or excretion of indole. Indican is an indole derivative.

The most obvious symptom and sign of BDS is the blue-stained diapers. Since BDS is connected to hypercalcemia, symptoms include digestive disturbances, fever, irritability and visual difficulties. Infants with the condition might develop kidney disease. BDS is not harmful to newborns, but could be disturbing. Well, blue urine should be disturbing.

There are possible treatments for BDS. In the case of dietary restrictions, diet should be low in calcium and tryptophan (food such as turkey and warm milk). Antiobiotics could decrease bacterial activity and infection. Genetic counselling could also be an option since this is an inherited disease.

Nevertheless, this disease is rare and uncommon. A full profile and description of BDS is incomplete and undergoing research. The explanations provided above are mostly hypotheses due to limited information. Hypercalcemia, indicanuria, and bacterial infection and pigmentation, are also possible explanation for blue urine in general, which may not necessarily be BDS.

Since BDS could be ascertained from urine, serum and urinary tests could be used for analysis. There has been a study by Chen, et al. entitled “The ocular abnormalities of blue diaper syndrome” published in 1991 in the journal Metabolic Pediatric and System Ophthalmology. Key findings are restated here. Thus, one could see that BDS manifests itself in very physical signs. There is a postscript below on a qualitative test used to ascertain the presence of indican in urine.

To summarize, Blue Diaper Syndrome:

– [from a review article on hypercalcemia

“Blue diaper syndrome is an uncommon metabolic disorder that is due to a defect in tryptophan metabolism. The block in tryptophan metabolism leads to urinary excretion of excessive amounts of indole derivatives, including a derivative called “indican” that gives the urine-soaked diaper a blue tint. Affected infants have hypercalcemia, hypercalciuria and nephrocalcinosis, but the mechanism is unknown.”

– is a rare, inherited, metabolic disorder possibly caused by mutations in the genes that code for tryptophan transport proteins; and 

– has blue stains in an infant’s diaper as a primary symptom, caused by excessive bacterial breakdown of unabsorbed tryptophan into indigo blue.

 

Postcript 1: Obermeyer Test for Indican

Indican

Structure of Indican

The Obermeyer Test determines the presence of indican in urine, which could be a sign of overgrowth of bacteria in the intestine, or as an affirmation for BDS. As explained by Dr. Jeremy Kaslow, indican is found in urine only in small amounts (the majority is in the bowel). A high protein diet and/or inefficient protein digestion might yield to a greater indican amount. In the case of BDS, there is inefficient or incomplete tryptophan digestion since the amino acid is not completely absorbed into the bloodstream.

As mentioned above, oxidation of indican leads to indigo blue. The test consists of mixing a sample of urine with Obermayer’s reagent (ferric chloride in HCl) and chloroform. The resulting color of the resulting chloroform layer (due to the absorption of indigo blue) is compared against a chart.

Indigo Blue

Structure of Indigo Blue

Indican Color Test

Indican Color Chart

A positive result could range from #1 (slightly blue) to #4 (jet black).

Postcript 2: The urine of King George III

King George III, known for losing the Americas, and for being mad.

George III (1738-1820) is the King of Great Britain and Ireland from 1760 to 1801. He is widely remembered for losing the American colonies. In his older years, he suffered from recurring mental instability, baffling the doctors during his time. One curiosity that challenged experts (for instance, the king’s attending physician, Sir Henry Halford) was the appearance of a “blue ring” in his urine, consequently examined just at the outset of those troubled years of his life. This, based from the discussion above, could be characteristic of indigo blue.

First, there is the need to explain the connection of blue urine with indigo. This comes from the ingenuity of Edward Schunck, a noted 19th century German-British chemist who specialized in natural coloring materials (i.e. dyes). His investigated purple urine, and devised an experiment to determine the presence of indigo and indirubin, colored blue and red, respectively. Next, he created a test to determine the presence of indigo in individuals. He tested the urine of 40 individuals, in which only one (a publican by trade), resulted positive for his test. My reference for this postscript (see link below) contains more description of these experiments of Schunck. The present test for blue urine is the Obermayer Test, mentioned in Postscript 1. It is faster and uses only few reagents.

The blue ring might have been indigo. However, the next step is to ascertain the source of the indigo. As mentioned above, indigo blue could be produced from tryptophan by intestinal bacteria. Professor Wilfrid Arnold, in his 1996 article in The Lancet, reported that:

Because the patient was constipated, putrefaction during stasis (sluggish performance) of the bowel led to indole formation from dietary tryptophan by the normal intestinal flora. Some indole was absorbed into the blood stream and converted in the liver first to indoxyl and then to the sulfate ester. The colourless, water-soluble indoxyl sulfate (metabolic indican in the older published research papers) was excreted in the urine. In the presence of a sulfatase of bacterial origin, either from urinary tract infection or as an environmental contamination of the chamber pot, indoxyl was slowly released by hydrolysis and oxidatively dimerised to indigo, which precipitated on the porcelain”.

Perhaps, what differs this case from BDS is that George’s urine might have the blue ring (oxidized form of indoxyl sulfate) only after it has already been excreted by the body, with the blue ring happening from the oxidation of indoxyl sulfate. Babies who have BDS have blue urine the moment they urinate in their diapers.

Conversion of indoxyl sulfate to indigo (from the Royal Society of Chemistry)

Could his blue urine be correlated with his mental illness? It was widely accepted that George III suffered from porphyria. However, the link between indigo and mental instability is still not clearly drawn, or might not be present at all. Porphyria itself is not a disease easy to diagnose. King George was only known for blood-stained urine. This could be related to porphyria since the disease has defective or enzymes for the production of porphyria and heme. Nevertheless, indigo and indirubin could be attributed to some illnesses such as Crohn’s disease.

My reference also contains an experiment for testing one’s urine for indigo blue. The procedure makes use of the Obermayer Test mentioned above.

The University of Missouri in Kansan City has a video recording of a presentation made on the topic of King George III’s illness. It could be found here.

Postcript 3: Biotech Blue

Blue jeans could be getter greener.Since the underlying cause of blue-stained urine is the bacterial production of indigo blue, why not use bacteria as alternative dye manufacturers? This research opportunity is explored in an article from the Journal of Industrial Microbiology and Biotechnology, volume 28 (2002), entitled “Application of metabolic engineering to imrpove both the production and use of biotech indigo” (abstract here and the review article linked below).  The bacteria used was Escherichia coli. As with some generic aims of biotechnology, the use of bacteria presents environment-friendly alternatives to the present dye and denim industries. Such organisms naturally produce indigo blue as part of their metabolism.

I like one of the article’s caption: “Blue jeans could be getting greener”.

Other review articles, aside from my main reference, could be found here and here.

Main References:

1. Ludueña, R. F. Learning Biochemistry: 100 Case-Oriented Problems, Problem 4, Wiley-Liss, Inc. (1995).

2. Articles on Blue Diaper Syndrome:

3. Dr. Jeremy Kaslow on the Obermayer Test: http://www.drkaslow.com/html/urine_indican.html

3. Cooksey, C. and A. Dronsfield. George III, indigo and the blue ring test. Education in Chemistry. Royal Society of Chemistry, March 2008. Available at: http://www.rsc.org/Education/EiC/issues/2008Mar/GeorgeIIIindigoBlueRingTest.asp

4. Review article of Application of metabolic engineering to imrpove both the production and use of biotech indigo in Nature, 27 March 2012: http://www.nature.com/news/2002/020327/full/news020325-4.html

Image Sources:

http://www.orato.com/files/main//5/4/38343645d5b22f6d0.jpg

http://1.bp.blogspot.com/_qMxRPn4iW3k/S4my1hNnq3I/AAAAAAAAAcA/N7bJDjHcvOs/s320/diaper.jpg

http://www.daviddarling.info/images/tryptophan.jpg

http://i130.photobucket.com/albums/p257/a_series_of_blurs/miscellany/Indican.png

http://www.worlddyevariety.com/wp-content/uploads/2012/08/Vat-Blue-1.gif

http://sutherlandshirenaturopathicclinic.files.wordpress.com/2010/09/indican.jpg?w=600

http://upload.wikimedia.org/wikipedia/commons/5/54/George_III_in_Coronation_edit.jpg

http://www.rsc.org/images/cooksey%20scheme%203_tcm18-113563.jpg

[post by Ajep Perez]

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