AKU Society

An investigation of the nature of ochronotic pigment and its interaction with tissues in order to explore treatment avenues

 
Objective: To understand the nature of the interaction of the pigment produced in alkaptonuria (AKU) by in vivo and in vitro studies and assess possible strategies to minimise the damage to tissues, especially to joint cartilage, in AKU.
 
AKU pigment: Accumulation of homogentisic acid (HGA) leads to the formation of a bezoquinone polymer catalysed by the enzyme polyphenol oxidase. The exact nature of this pigment is incompletely studied and factors affecting this process remain to be fully understood. ‘Alkapton’ refers to the oxidizability of the pigment under alkaline conditions. HGA has a very low renal threshold and is rapidly excreted through the kidneys. The low concentration of HGA in body fluids and tissues is converted to the polymeric BQA that binds to tissues and leads to tissue damage. Although HGA has been shown to have a direct toxic effect, it is believed that the toxicity is mainly due to the BQA pigment. The exact nature of polyphenol oxidase, also called phenolase or tyrosinase or catechol oxidase, is uncertain; tyrosinase is a polyphenol oxidase and is present in more than one form. It is debatable whether there are other forms of polpyphenol oxidase(s).
 
Post-mortem study of AKU: A post-mortem study on a 74 year old woman with AKU in our department, funded by the AKU Society, revealed the extent of deposition of ochronotic pigment not just in cartilage but also in virtually all tissues of the body. The pigmentation was intense in cartilage and other connective tissue, especially in the joints (synovial and fibro-cartilaginous), kidneys, and circulatory system. Pigmentation of the musculoskelatal system – the following features were noted:

• Tendons and ligaments showed little pigmentation.
  
• Bone: Mild pigmentation of periosteal collagen was present and no pigment was seen in bone.
  
• Cartilage: The elastic cartilages of the airways showed intense pigmentation, most prominently in the extracellular matrix of the perichondrial region.
  
• Synovial joints: Marked pigmentation was noted, diffusely distributed in the cartilage matrix, without prominent intracellular or perilacunar accentuation. Granular pigment was present in the superficial articular cartilage and in fibrocartilaginous areas at the edge of joints. Focal chondrocyte necrosis was present
• Fibrocartilaginous joints: The fibrocartilage of the costal cartilages, sacro-iliac joints, pubic symphysis and intervertebral discs was intensely pigmented.

 
The deposition of pigment in Alkaptonuria is partly related to the type of collagen present in connective tissue, with a preference for type II articular cartilage and to type I collagen in fibrocartilage and elastic cartilages. Chondrocyte necrosis was a novel observation and may help to explain the premature degeneration of articular cartilages. It is tempting to speculate that HGA or the BQA may be toxic to the cartilage cells and induce apoptosis. The exact nature of the affinity of the BQA or HGA to tissue is unknown. Pigmentation of the circulatory system – the following features were noted:

• The dark grey pigmentation of the intima was patchy and more prominent around the branches and bifurcations of the major arteries.
  
• The adventitia of the aorta and carotid arteries were also pigmented to varying degree but not the media.
  
• Prominent pigmentation of the aortic valve cusps and valve ring, and of the anterior mitral valve cusp and mitral valve ring was noted.
  
• Very little pigmentation was observed in the right heart and pulmonary artery.
  
• The venous system appeared normal. The deposition of ochronotic pigment in the cardiovascular system following life-long exposure was not uniform. The reason(s) for the patchy pigmentation of the tissues of the cardiovascular system are unknown.
  

 
Hypothesis: Factors influencing pathogenicity in AKU may be loosely termed SEED, SOIL and WATER elements. Disease severity in AKU depends upon generation of the toxic compound (SEED) (factors influencing this may be degree of HGO deficiency, dietary stress, catabolic stress, pro- and anti-oxidant defences, acid-base status, levels of polyphenol oxidase activity), affinity of tissue for the toxic compound (SOIL) (cartilage, other connective tissue elements, damaged or undamaged tissue, elastic or other connective tissue elements, types of collagen, polyanionic ground substance), and factors affecting the interaction of the toxic compound with the tissue (WATER) (pressure, blood pressure, turbulence, temperature, acid-base status). The pathogenetic mechanisms described in the previous section relating to the seed, soil and water factors may be tested by considering the following analytical techniques as well as subject and samples.
 
Potential mechanisms                        Techniques                                         
Oxidation                                                 Oxid- LDL; MDA/F2-Isoprostanes; In vitro oxidising system; Polyphenol oxidase assay    
Acid-Base                                               Cell-culture studies; Human acidification or alkalinisation
Polymerisation to BQA                         HPLC/Tandem MS; Cellular or Extracellular