Analysis of Melanin-like Pigment Synthesized from Homogentisic Acid, with or without Tyrosine, and Its Implications in Alkaptonuria

Abstract

Alkaptonuria is an iconic disease used by Archibald Garrod to demonstrate the theory of “inborn errors of metabolism”. AKU knowledge has advanced in recent years: development of an in vitro model, discovery of murine models and advances in understanding bone and cartilage phenotypes and arthropathy in AKU. These discoveries have aided in a new clinical trial into nitisinone. However, there are still knowledge gaps surrounding the pigment in AKU and the pigmentation process. We demonstrate an advance in the understanding in the kinetics and chemistry of the polymerisation of homogentisic acid (HGA) into its pigment using size-exclusion chromatography and IR spectroscopy. We compared the properties of HGA-based pigments that were freshly prepared to those stored in solution for 2 years. Our results demonstrate the importance of pH in the polymerisation process and that colour change seen in solution (analogous to AKU patient urine) is not initially due to presence of ochronotic pigment but the quinone intermediary. In addition, we observed that pigment formation from HGA can occur in the presence of tyrosine, without the inclusion of this tyrosine into the pigment. These observations have positive implications for patients with alkaptonuria; an increased understanding of the pigment polymer chemistry, the presence of an intermediary and their kinetics present more therapeutic opportunities for treating the condition, including preventing the pigment from forming, binding or reversing established pigmentation. AKU patients treated with nitisinone show elevated tyrosine levels causing side effects such as corneal opacities; our data demonstrates that elevated tyrosine levels should not contribute or add to the ochronotic pigment burden in these patients.

Appendices

“Take-Home” Message

Our data shows initial colour change in samples of HGA solution is due to the formation of intermediaries which are not polymerised pigment. C–C and C–O bonds are involved in linking aromatic units in the formation of ochronotic pigment. Tyrosine is not incorporated into ochronotic pigment meaning that patients with elevated plasma tyrosine will not have elevated plasma tyrosine adding or contributing to the pigment burden.

Contribution

Drs. Taylor and Vercruysse have been involved in conception, design, analysis and interpretation of data. Both authors drafted the chapter and revised it critically for important intellectual content.