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.

Ochronotic pigmentation is caused by homogentisic acid and is the key event in alkaptonuria leading to the destructive consequences of the disease—A review

“…The absorbance spectra showed a broad band at wavelengths 280 to 350 nm, with increased general absorbance across the UV‐visual range without distinct peaks. Similar absorbance properties have been reported previously for OP and also melanin (eumelanin derived from 5,6,‐dihydroxyindole‐2‐carboxylic acid), and are thought to reflect a chemically heterogeneous structure of oligomers formed by a range of different bonding mechanisms, also referred to as “chemical disorder” . These absorbance properties might also account for the physical appearance of some pigmented substances, which in the case of melanin is thought to provide its physiologically important optical characteristics, that is, its ability to absorb UV light .…”

“…More recent analysis of synthetically derived OP solutions using size exclusion chromatography suggest that OP has greater molecular weight than HGA, as indicated by a peak at shorter retention time. However, visually pigmented solutions could be formed from HGA (over a shorter period of time; 10 days as opposed to 2 years) without evidence of the peak corresponding to OP and no observed decrease in the HGA peak . This suggests that the visual darkening of the solution due to increased pH can be due to presence of the low molecular weight oxidized form of HGA over a relatively shorter period.…”

Fe2+/H2O2-mediated oxidation of homogentisic acid indicates the production of ochronotic and non-ochronotic pigments. Implications in Alkaptonuria and beyond

“…[6] Using air-oxidation, HGA is readily converted into a darkly-colored, melanin (MN)-like pigment, but strong alkaline conditions appear to be required to initiate the process. [7] In vivo such conditions are unlikely to be the norm, however some cartilage samples display a neutral or alkaline pH. [8] A more likely means of oxygen-mediated oxidation and polymerization of HGA would require the presence of a catalyst; enzyme or other.…”

Dark- or Light-Colored Melanins: Generating Pigments Using Fe2+ and H2O2

“…[9][10][11][12][13]26 In SEC, molecules are separated on the basis of differences in their hydrodynamic volume (often related to the molecular mass of the molecule): the lower the retention time, the higher the hydrodynamic volume of the analyte. In our SEC analyses, the lower limit of exclusion, as determined by the injection of water, was about 15 minutes.…”

“…Some of these compounds have been the subject of previous studies on the formation of MN-like pigments performed in our laboratories. [9][10][11][12][13] Fig. 1: Chemical structures of compounds used in this study: catechol (1), pyrogallol (2), dopamine (3), norepinephrine (4), epinephrine (5), tyrosine (6), serotonin (7) and homogentisic acid (8).…”