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

Abstract

Homogentisic acid (HGA) can be oxidized by a combination of Fe 2+ and H 2 O 2 into a darkly colored high molecular mass pigment. Increasing the concentration of H 2 O 2 can lead to the formation of a high molecular mass material that exhibits less absorbance in the visible range of the electromagnetic spectrum, while maintaining a strong absorbance in the UV range of the electromagnetic spectrum. FT-IR spectroscopy did indicate the presence of a chemical feature in the pigments generated through H 2 O 2 -mediated oxidation that is absent in pigments generated through air-mediated oxidation. Our observations could have implications in the pathophysiology of alkaptonuria. In alkaptonuria, patients suffer from homogentisic aciduria from birth, but develop ochronosis (darkening) of collagenous tissues much later in life due to the formation of a melanin-like pigment. Three major questions regarding ochronosis remain to be answered: 1) how is the pigment formed, 2) why does it appear by the third decade of life and 3) why is it sporadic in appearance? Our results suggest that ochronotic or non-ochronotic pigments can be generated from HGA depending on the oxidation reaction conditions. Thus, the absence of any visible pigment, as for younger alkaptonuria patients, could not necessarily mean the absence of HGA-derived melanin-like molecules. We compare our results and their potential implications for ochronosis to the changes in pigmentation observed in vitiligo or aging (greying) hair.

Light- or Dark-Colored, L-DOPA-Based Melanins

“…27,43 However, these reports involve the treatment of pre-formed MNs with H 2 O 2 , while our work focuses on the use of H 2 O 2 in the synthesis of MNs. In this and our previous reports [14][15][16] we confirmed that the presence of high concentrations of H 2 O 2 does lead to MN-like materials with decreased colored appearance and enhanced fluorescence emission. These physicchemically different MN-like pigments do not appear to undergo any depolymerization as the SEC retention times of all the MN-like materials are very similar; independent of the reaction conditions that generated the pigments.…”

“…However, it is worth pointing out that for greying of hair or vitiligo, conditions that are associated with the loss of pigmentation, the presence of increased levels of H 2 O 2 has been discussed as a potential contributing factor to these changes in pigmentation. [44][45][46] Our current and previous observations [14][15][16] do suggest a hypothesis that the absence of dark colors may not necessarily mean the absence of MN-like molecules, but could mean the presence of a different type of MN-like material. It is worth pointing out that the light-colored MN-like materials generated in the presence of high concentrations of H 2 O 2 do absorb or scatter light in the UV range of the electromagnetic spectrum, although to a less efficient extent than the dark-colored materials.…”

“…Such retention times are in line with those reported for MN-like pigments generated from other phenolic or catecholic compounds. [14][15][16]23 For each of the purified pigment materials a stock solution at 1mg/mL was prepared in distilled water. Figure 1 presents a photograph of these stock solutions.…”

“…Such an absorbance shoulder is weakly present in the spectra of pigment B and absent in the spectra of pigment C. This distinct feature in the FT-IR spectra has been observed for other MN-like pigments generated through Fe 2+ /H 2 O 2 -mediated oxidation. [15][16] An absorbance at that wavenumber can be attributed to the presence of a carbonyl entity (C=O); either from a ketone or a carboxylic acid functionality. 26 This feature in the FT-IR spectra may be due to a chemical reaction whereby a phenolic functional group was converted into a ketone functional group or whereby an aromatic unit was opened and two carboxylic acids were generated into the structure.…”

“…13 In previous reports we have presented our observations regarding the synthesis of MN-like pigments from a multitude of precursors using Fe 2+ and H 2 O 2 as the oxidizing reagents and observed that light-or dark-colored materials could be generated depending on the intensity of the oxidizing reaction conditions. [14][15][16] We have expanded these earlier observations by evaluating the generation of MN-like pigments from L-DOPA using air-oxidation in an alkaline environment and Fe 2+ /H 2 O 2 -mediated oxidation. In addition, we have explored the possibility that the physic-chemically distinct materials generated from L-DOPA could have a different effect on the interleukin release from immune cells.…”