Background One of the most important discoveries in rheumatology is the characterization of citrulline containing auto-antigens [1]. The identification of citrullinated proteins as auto-antigens and the development of new assay based on the detection of anti-citrullinated protein antibodies (ACPAs) become a breakthrough in the diagnosis and treatment of rheumatoid arthritis (RA). Mitsui and coauthors in vitro identified adenosine deaminase (ADA) as an ACPA antigen [2]. Earlier we have reported the enhance of ADA activity in synovial fluids (SFs) of RA patients [3].This increasing was in correlation with the ratio of small isoenzyme (SADA) to the large (LADA) [4].The comparison of citrullination states of SADA and LADA can be a clue for understanding the mechanism of SADA/LADA increase in RA, which is important both in diagnosis and treatment of the disease.

Objectives The objectives of this study were a) to separate SADA and LADA isoforms from SFs of RA patients and b) to compare their citrullination degree.

Methods The SADA and LADA isoforms from SFs of RA patients were separated and purified using gel-filtration and ion-exchange chromatography [4]. The citrullination degree of the isoforms was measured in the colorimetric assay with diacetylmonoxime (DAMO) [6] using the absorbance at 530 nm and free L-citrulline as a standard.

Results The SF samples of 20 RA patients with initial ADA activity in the range 35–190 IU/L were used. Earlier we demonstrated a negligible level of SADA at initial ADA lower of this interval. In the samples with medium initial activity (35–55 IU/L), SADA/LADA ratio was $≈ $ 1/2. In those with high ADA activity (100–190 IU/L) this ratio was $≈ $ 4. The separation and purification of LADA from 10 SFs revealed that it is not citrullinated in any case. In SADA from SFs with medium ADA activity, we failed to register the citrullination. In SADA from SFs with the initial ADA activity ≥100 IU/L, the significant citrullination ($≈ $ 0.6 μmol/mg of protein) was registered.

Conclusions The obtained results evidence that SADA from SFs with high ADA activity can serve as new citrulline containing ACPA antigen. This finding can be a base of developing new strategy (e.g. seeking specific inhibitors) for treatment of RA patients because citrullination enhances the ADA activity [2], hampering the increase of adenosine by methotrexate.

References

1. vanVenrooij WJ, van Beers JJ, Pruijn GJ. Anti-CCP Antibody, a Marker for the Early Detection of Rheumatoid Arthritis. Ann NY Acad Sci.1143:268–85, 2008.

2. Mitsui H, Arito M, Sato T, Yokoyama MK, Suematsu N, et al. Novel anti-citrullinated peptide autoantibodies identified by proteomics with in vitro citrullinated proteins in patients with rheumatoid arthritis. Inflammation and Regeneration 33:121–30, 2013.

3. Antonyan A, Sharoyan S, Haroyan A, Harutyunyan R, Mardanyan S. Adenosine Deaminase activity in synovial fluid at arthritis. Proceedings of YSU. Chemistry and Biology 3:28–32, 2013.

4. Antonyan A, Sharoyan S, Haroyan A, Harutyunyan R, Mardanyan S. Adenosine Deaminase Isoforms in synovial fluid at Rheumatoid arthritis. Proceedings of YSU. Chemistry and Biology 2:54–57, 2014.

5. Boyde TR, Rahmatullah M. Optimization of conditions for the colorimetric determination of citrulline, using diacetylmonoxime. Anal Biochem 107:424–431, 1980.

References

Disclosure of Interest None declared