HPLC and mass spectrometry analysis of the free radical degradation of chondroitin sulfate and its implications in osteoarthritis
Chondroitin sulfate (CS) is vital component of bone and cartilage; it is widely used as a daily supplement in the management of arthritis. Reactive oxygen species (ROS) are involved in vast array of biological processes ranging from regulatory functions to damaging effects in disease pathogenesis. Mass spectrometry is a major tool in deciphering unknown chemical structures, particularly tandem MS. In this study mass spectrometry coupled with High performance liquid chromatography (HPLC) gives some insight into the potential mechanisms involved when ROS are made to attack CS. HPLC and coupled mass spectrometry showed that the free radical depolymerisation of CS yielded an N-acetylgalactosamine (GalNAc) and a uronic acid with varied sulfation. This led us to investigate the possibility of other means of free radical generation with the potential to degrade CS. Alkaptonuria an ultra-rare (1:100000-1:250000) in born error of metabolism resulting in the accumulation of homogentisic acid (HGA) due to a deficiency in homogentisate1,2-dioxygenase. Polymerised HGA is excreted in the urine and deposited as an ochronotic pigment in cartilage. The mechanism of HGAs polymerisation is yet to be concluded due to the complexity of its reactions. This study aims to breakdown individual steps in HGAs biochemistry using mass spectrometry. This will provide further insight into the potential reactions HGA can undergo in the body and propose some mechanisms on how it may polymerise. Using mass spectrometry it became apparent that HGA polymerised when in the presence of copper and whilst HGA in water also yielded a dimer, the structure of which was proposed computationally. Little is known on the mechanisms that underpin the displayed symptoms of AKU, it is known however that the polymerisation of HGA does play a role and is responsible for the ochronotic pigmentation in AKU patients. HPLC and ESI-MS was used to investigate the potential interactions between HGA and CS and propose that HGA is able to degrade CS disaccharides when in the presence of a copper catalyst. And that this interaction will play a role in the early onset of arthritis in AKU patients.
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