An anatomical investigation of alkaptonuria: Novel insights into ochronosis of cartilage and bone
Authors: Hughes Juliette H. , Charlesworth Gemma , Prior Amanda , Tierney Claire M. , Rothwell Paul D. , Thomas Neil P. , Ranganath Lakshminarayan R. , Gallagher James A. , Bond Alistair P. 
Published: December 2024

Ochronotic pigmentation of connective tissue is the central pathological process in the rare metabolic disease alkaptonuria (AKU). Tissue pigmentation in AKU occurs due to unmetabolised homogentisic acid (HGA) in the circulation, caused by an enzyme deficiency in the liver. Ochronotic pigmentation, derived from HGA, has previously been reported and described in large joints obtained from arthroplasty surgeries, which typically have advanced disease. Many tissues that are affected by ochronosis are not accessible for study during life, including tissues subjected to early and mid‐stage disease. Here, the opportunity arose to anatomically examine a 60‐year‐old AKU female body donor, allowing the investigation of previously understudied tissue, including those undergoing early‐stage pathological changes. Dissection of fresh‐frozen tissue was carried out and harvested tissues were fixed and examined histologically using H&E and Schmorl's stains to aid identification of ochronotic pigment. This work focusses on osteochondral tissues including extra‐skeletal cartilage, viscera and eyes. Gross and histological images demonstrating pigmentation in the cartilage and perichondrium of the ear ossicles, tympanic membrane and the pubic symphysis fibrocartilaginous disc are described for the first time here. We also show the first examination of the temporomandibular joint, which macroscopically appeared unpigmented, with histological analysis of the fibrocartilaginous disc showing no pigmentation. Pigmentation of non‐articular hyaline cartilage was observed in the respiratory tract, in both the hyaline cartilage and perichondrium, confirming previous findings. Within smaller joints, pigmentation of chondrons and the surrounding territorial matrix was observed, but was confined to calcified articular cartilage, and was not generally found in the hyaline articular cartilage. Dark pigmentation of the perichondrium adjacent to the articular surface was observed in numerous small joints. The calcified bone matrix was not pigmented but ochronosis was identified in a small fraction of trabecular osteocytes in the capitate and radius, with substantially more pigmented osteocytes observed in bone of the ear ossicles. Viscera examined were unpigmented. This anatomical examination of tissues from an AKU individual highlights that most osteochondral tissues are susceptible to HGA‐derived pigmentation, including the ear ossicles which are the smallest bones in the body. Within joints, calcified cartilage and perichondrium appear to be the earliest affected tissues, but why this is the case is not understood. Furthermore, why the TMJ disc was unaffected by pigmentation is intriguing. The heterogenous appearance of pigmentation both within and between different tissues indicates that factors other than tissue type (i.e. cartilage, perichondrium) and matrix composition (i.e. collagen‐rich, calcified) may affect the process of ochronosis, such as oxygen tension, loading patterns and tissue turnover. The effect of nitisinone treatment on the ochronotic disease state is considered, in this case 7 years of treatment, however comparisons could not be made to other cases due to inter‐individual variability.