Pigmentation Chemistry and Radical‐Based Collagen Degradation in Alkaptonuria and Osteoarthritic Cartilage

Abstract

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA‐derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone‐resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.

Anatomical Distribution of Ochronotic Pigment in Alkaptonuric Mice is Associated with Calcified Cartilage Chondrocytes at Osteochondral Interfaces

“…Changes to the ECM of the ACC appear to occur early in AKU mice allowing pigmentation, so it may be possible that these potential ECM changes in the ACC also occur in normal ageing that predisposes to conditions such as OA. The intrastrand disruption of the collagen triple helix by alterations in hydrogen bonding reported in pigmented AKU cartilage was also reported in non-AKU OA cartilage, suggesting a common change in the ECM that may be related to loading and ageing, predisposing to pigmentation and OA pathology [24].…”

“…A modified version of Schmorl's stain, usually used to identify melanin pigments, has been used both in cells incubated with HGA (in vitro) [4] and in human AKU tissue sections [21,22] to enhance ochronotic pigment from a yellow-brown to an intense bluegreen colour, by the reduction of ferricyanide to ferrocyanide by ochronotic pigment [23]. A recent analysis of human AKU pigmented cartilage using NMR indicated the presence of hydroquinones that are known for their redox activity, and also identified radicals via EPR (electron paramagnetic resonance) spectroscopy of synthetic pigment [24]. Changes in glycine signals were observed in the pigmented cartilage samples, most likely indicating intrastrand disruption of the collagen triple helix due to a disruption in hydrogen bonds involving glycine, that probably changes the mechanical properties of the cartilage [24].…”

“…A recent analysis of human AKU pigmented cartilage using NMR indicated the presence of hydroquinones that are known for their redox activity, and also identified radicals via EPR (electron paramagnetic resonance) spectroscopy of synthetic pigment [24]. Changes in glycine signals were observed in the pigmented cartilage samples, most likely indicating intrastrand disruption of the collagen triple helix due to a disruption in hydrogen bonds involving glycine, that probably changes the mechanical properties of the cartilage [24]. The authors propose that formation of glycyl radicals, enabled by HGA radical formation, can disrupt hydrogen bonding within the collagen triple helix and oxidise nearby HGA molecules, amplifying the process of HGA oxidation, increasing pigment formation and leading to further collagen damage.…”

Homogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural Characterization and Antioxidant Activity

“…These mimics show physico-chemical characteristics similar to the insoluble eumelanin-like materials. Recently a homogentisic acid derived pigment has been proposed as a biocompatible label for optoacoustic imaging of macrophages and it is used as a reference to obtain more insights on composition and collagen disruption in alkaptonuria cartilage [5,12]. Laccases are oxidative enzymes able to oxidize a wide variety of molecules comprising polyphenols, aromatic amines etc.…”

“…In humans, the genetic disorder alkaptonuria is related to a deficiency of the enzyme homogentisate 1,2 dioxygenase. This leads to the accumulation of HGA monomer which, then, for spontaneous autoxidation to 1,4-benzoquinone-2-acetic acid, polymerizes into the characteristic ochronotic pigment with not yet clearly identified structure [4,5]. All melanins exhibit interesting physico-chemical properties associated with their broad band visible light absorption, a persistent paramagnetism related to their free radical properties, antioxidant activity, and conduction properties [6].…”