Biochemical Investigations in the Rare Disease Alkaptonuria: Studies on the Metabolome and the Nature of Ochronotic Pigment

Norman, Brendan ORCID: 0000-0001-9293-4852
(2019) Biochemical Investigations in the Rare Disease Alkaptonuria: Studies on the Metabolome and the Nature of Ochronotic Pigment. PhD thesis, University of Liverpool.

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Alkaptonuria (AKU) is a rare inherited disorder of tyrosine metabolism caused by lack of the enzyme homogentisate 1,2-dioxygenase (HGD). The primary biochemical consequence of HGD deficiency is increased circulating concentration of homogentisic acid (HGA); this is the central cause of the devastating multi-systemic damage observed in AKU. One of the most striking pathophysiological features of AKU is accumulation of ochronotic pigment derived from HGA in tissues throughout the body. HGA has an affinity for cartilaginous tissue. Presence of ochronotic pigment in cartilage of load-bearing joints causes severe early-onset osteoarthropathy; the greatest cause of morbidity in AKU. This thesis addresses two major unanswered questions in AKU. First is the wider metabolic consequences both of HGD deficiency and those observed following treatment with the promising HGA-lowering agent nitisinone. A metabolomics approach was employed to address these questions, with the aim of discovering biomarkers with potential clinical value in monitoring disease in AKU and in assessing the wider metabolic consequences of nitisinone treatment. Second, this thesis explores the nature of ochronotic pigment derived from HGA. The chemical structure of ochronotic pigment is not known, nor are the mechanisms by which HGA interacts with and binds into the cartilage extracellular matrix. Various analytical chemistry techniques were employed to study the chemical properties of ochronotic pigment and pigmented cartilage in patients and mice with AKU. The data from chemical analyses presented in this thesis provide new insights into the nature of ochronotic pigment. The chemical structure of ochronotic pigment remains unknown, but the pigment had long been widely cited as a polymer in the literature. First, these data fundamentally challenge the idea that ochronotic pigment is a polymeric species. Through study of ochronotic tissue samples, the data also indicate for the first time a specific alteration to the cartilage matrix that enables binding of HGA-derived species. An LC-QTOF-MS metabolic phenotyping strategy was developed, enabling identification of hundreds of metabolites simultaneously, based on chemical properties of accurate mass and chromatographic retention time. Application of this technique to AKU showed that the biochemical alterations following a) targeted deletion of the HGD gene in mice to cause AKU, and b) nitisinone treatment, reflect a complex, interconnected network of metabolite changes. The data not only stimulate myriad further lines of research into AKU pathophysiology but also reveal previously uncharacterised association between metabolites at a network level. These studies provide a prime example of how rare diseases such as inborn errors of metabolism can offer unique windows into metabolism and human physiology more generally.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences
Depositing User: Symplectic Admin
Date Deposited: 08 Apr 2020 14:31
Last Modified: 19 Jan 2023 00:03
DOI: 10.17638/03074243
  • Gallagher, James A
  • Ranganath, Lakshminarayan R
  • Roberts, Norman B