Raman Spectroscopy identifies differences in ochronotic and non-ochronotic cartilage; a potential novel technique for monitoring ochronosis

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.

Ochronotic pigmentation is caused by homogentisic acid and is the key event in alkaptonuria leading to the destructive consequences of the disease—A review

“…Ochronotic samples were highly fluorescent and, unlike non‐ochronotic samples, provided limited to no discernible Raman spectral peaks. A novel peak was obtained from the ochronotic sample that was also observed in the spectra from pigment derived from synthetic HGA . The ability of Raman spectroscopy to clearly distinguish between ochronotic and non‐ochronotic tissue warrants further study, as the technique appears to have potential to provide fundamental information on the chemical nature of OP.…”

“…The ability of Raman spectroscopy to clearly distinguish between ochronotic and non‐ochronotic tissue warrants further study, as the technique appears to have potential to provide fundamental information on the chemical nature of OP. Furthermore, there is potential for the technique to be employed as an in vivo tool for measuring and monitoring ochronosis progression in a clinical setting …”

“…However, direct detection of OP in vivo by Raman spectroscopy is possible. Such a technique has been validated in ex vivo tissue and is being adapted as an in vivo technique using ear cartilage and Achilles tendon, both tissues with little subcutaneous tissue …”

“Lessons from Rare Forms of Osteoarthritis”

“…Spectra provide the overall chemical measurement of a given sample, based on the energy exchange between the incident laser and energy used when the molecular bonds in a sample vibrate. In a study comparing non-ochronotic and ochronotic cartilage from hips and knees two main differences were found [ 120 ]. Specifically, non-ochronotic cartilage resulted in spectra comparable with that in the cartilage literature [ 121 , 122 ], whereas the pigmented cartilage was highly fluorescent.…”

The potential of nitisinone for the treatment of alkaptonuria

“…Recently it has been shown that Raman spectroscopy may be a useful tool for monitoring ochronosis in vivo. The ex vivo analysis of cartilage samples from the hips and ears of individuals with AKU have shown unique cartilage spectra from those with the presence of ochronotic pigment, whilst cartilages without ochronosis demonstrated a spectra that was consistent with normal cartilages [73]. Raman has the potential to be used in vivo via transdermal analysis of cartilage, it has been used on analysing cartilage and bone samples from patients with Osetogenesis Imperfecta (OI).…”