No abstract

Objective: Alkaptonuria (AKU) is a rare, inherited disorder of tyrosine metabolism, where patients are unable to breakdown homogentisic acid (HGA), which increases systemically over time. It presents with a clinical triad of features; HGA in urine, ochronosis of collagenous tissues, and the subsequent ochronotic arthritis of these tissues. In recent years the advance in the understanding of the disease and the potential treatment of the disorder looks promising with the data on the efficacy of nitisinone. However, there are limited methods for the detection and monitoring of ochronosis in vivo, or for treatment monitoring. The study aim was to test the hypothesis that Raman spectra would identify a distinct chemical fingerprint for the non-ochronotic, compared to ochronotic cartilage. Design: Ochronotic and non-ochronotic cartilage from human hips and ears were analysed using Raman spectroscopy. Results: Non-ochronotic cartilage spectra were similar and reproducible and typical of normal articular cartilage. Conversely, the ochronotic cartilage samples were highly fluorescent and displayed limited or no discernible Raman peaks in the spectra, in stark contrast to their non-ochronotic pairs. Interestingly, a novel peak was observed associated with the polymer of HGA in the ochronotic cartilage that was confirmed by analysis of pigment derived from synthetic HGA. Conclusion: This technique reveals novel data on the chemical differences in ochronotic compared with non-ochronotic cartilage, these differences are detectable by a technique that is already generating in vivo data and demonstrates the first possible procedure to monitor the progression of ochronosis in tissues of patients with AKU.

Purpose: Alkaptonuria is a rare genetically inherited form of OA which is characterised by urine which darkens on standing, pigmentation of articular cartilages and early-onset, rapidly progressing osteoarthropathy of multiple weight bearing joints. The pigmentation is caused by presence of a tyrosine metabolite; homogentisic acid (HGA), which polymerises over time to for a pigment in the articular cartilages. The presence of pigment changes the biochemical and biomechanical properties of the cartilage causing anatomical and physiological dysfunction. To date there is no evidence to suggest the exact time when ochronosis and therefore pathological change in tissues commences. Our study will examine ochronotic and non-ochronotic cartilage to determine if Raman spectroscopy can detect pigmentation in macroscopically non-ochronotic cartilage. It will also compare the spectra of ochronotic and non-ochronotic cartilage to normal cartilage spectra in the current literature. Methods: Cartilage samples were obtained as surgical waste with informed patient consent following ethical approval. The samples were dissected immediately following surgery into ochronotic and nonochronotic pairs and stored unfixed at -80 C. Individual cartilage samples were placed on to calcium fluoride discs for Raman spectral analysis. Spectra were acquired using an InVia Raman microspectrometer (Renishaw plc, UK) with an 785 nm laser. Spectra were collected using 20 s and 3 accumulations, at 100 % power (~10mW at sample) from the non-ochronotic tissue. However, applying the same settings to the ochronotic tissue resulted in detector saturation. Therefore spectra were collected using 1 s and 10 accumulations, at 100 % power. Data were baseline corrected, using polynomial subtraction, to remove fluorescence and normalised. Results: Macroscopically non-ochronotic cartilage displayed spectra typical of normal articular cartilage with peaks corresponding to all major functional groups and amino acids: proline, hydroxyproline, phenylalanine, amide I (carbonyl group) and amide III (C-N and N-H). Cartilage which showed observable macroscopic ochronotic pigmentation and those which were completely ochronotic displayed an absence of a recognisable spectra with no discernible peaks corresponding to functional groups seen in the non-ochronotic sample, but were highly fluorescent. The macroscopically pigmented sample showed two peaks, previously described in the literature, corresponding to aromatic C-C twisting and phosphatidylinositol (lipid). Conclusions: Our data demonstrates that the semi-pigmented and pigmented samples of cartilage were in stark contrast to the non-pigmented samples. While it was relatively straightforward to collect spectra from the non-ochronotic cartilage, which produced 'typical' cartilage spectra, the ochronotic cartilage was very fluorescent, which masked the Raman signal. Therefore, there were very few identifiable peaks; two were identified at 626 and 775 cm -1 , but were not present in the non-pigmented spectra. The presence ...