Raman spectroscopy can non-invasively distinguish between ochronotic and non-ochronotic cartilage

Abstract

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 ...

Reversal of ochronotic pigmentation in alkaptonuria following nitisinone therapy: Analysis of data from the United Kingdom National Alkaptonuria Centre

“…The ear cartilage biopsy showed that ochronosis was not uniform throughout the sample and this heterogeneity is a limitation in its usefulness. A non‐invasive analysis is being developed by utilizing Raman Spectroscopy for a future study, 25 in which a much larger area of the ear tissue could be analyzed. The appearance of ear and eye ochronosis coincides with the appearance of low back and knee pain, suggesting a common evolving ochronotic pathogenetic process 2,3 …”