More than 80 mutations in the HGD gene have been identified in people with the disease. Many of these mutations lead to changes in single amino acids in the homogentisate oxidase protein. A substitution of the amino acid valine for methionine at position 368 is the most common HGD mutation in European populations. Mutations in the HGD gene probably deactivate the enzyme by changing its structure.
In the body of an AKU patient, HGA accumulates at more than 2,000 times the normal rate. Some of this is excreted in the urine, but a significant quantity remains. HGA turns black on oxidation, leading to black urine and occasional black sweat which can be indicative of alkaptonuria. A small proportion of patients, however, will not develop these symptoms.
Much of the research into AKU has focused on the urine. It was discovered that the darkening was delayed in an acidic solution and accelerated in alkaline conditions. This affinity for alkali led Boedeker, in 1859, to describe the urine as containing an alkapton, later leading to the name alkaptonuria.
Through a simple test with Benedict’s sugar reagent, it was noted that the urine was a powerful reducing agent. This not only reduces the copper reagent to an orange precipitate, but also darkens the solution due to its alkalinity. The net effect is orange particles suspended in a muddy brown solution.