INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse 
the posttranslational conversion of peptidylarginine to peptidylcitrulline. 
Citrullination of proteins is well described in rheumatoid arthritis (RA), and 
hypercitrullination of proteins may be related to inflammation in general. PAD 
activity has been demonstrated in various cell lysates, but so far not in 
synovial fluid. We aimed to develop an assay for detection of PAD activity, if 
any, in synovial fluid from RA patients.
METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the 
immobilized substrate for citrullination and anti-citrullinated fibrinogen 
antibody as the detecting agent were used for measurement of PAD activity in 
synovial fluid samples from five RA patients. The concentrations of PAD2 and 
calcium were also determined.
RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) 
than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was 
detected in four of five synovial fluid samples from RA patients and correlated 
with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium 
requirement for half-maximal activities of PAD2 and PAD4 were found in a range 
from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium 
levels for the citrullination process to occur.
CONCLUSIONS: We present an assay with high specificity for PAD2 activity and 
show that citrullination of fibrinogen can occur in cell-free synovial fluid 
from RA patients.