A molecular spectroscopy approach for the investigation of early phase ochronotic pigment development in Alkaptonuria

Andrea Atrei,Fabrizio Manetti,Elena Petricci,Annalisa Santucci,Andrea Bernini,Maria Camilla Baratto

doi:10.1038/s41598-021-01670-z

Abstract

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.

Highlights

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene
Alkaptonuria development is connected to the accumulation of HGA and subsequent deposition of ochronotic pigment in joints and cartilages
The transformation from the metabolite into the polymeric pigment has been supposed to rely on benzoquinone acetic acid (BQA) as intermediate at basic pH mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the para-benzoquinone

Summary

Introduction

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction Such behaviour can be observed in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. HGA belongs to the metabolic pathway of phenylalanine and tyrosine It is a precursor of plastoquinone and tocopherols in plants and is involved in the machinery designated to protect chlorophyll from sunlight degradation. Electrochemical studies demonstrated that a twoproton-two-electron reaction[18] is responsible for the transformation of HGA (0.8 mM in 0.1 M phosphate buffer)

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Conclusion