Introduction/objectives <p>The aim of the present study was to identify key amino acid (AA) pathways in osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA), as AAs have emerged as potential biomarkers for the detection of degenerative joint diseases. It was hypothesized that we would detect distinct metabolic pathways activated in OA and RA due to different degrees of inflammation.</p> Method <p>Samples of synovial fluid (SF) and infrapatellar Hoffaʼs fat pad (IFP) were collected from end-stage knee OA (<i>n</i> = 10) and RA patients (<i>n</i> = 10), and from non-inflammatory controls (<i>n</i> = 5). Metabolites were analyzed utilizing a liquid chromatography high-resolution mass spectrometry approach, followed by univariate and multivariate statistical testing and pathway analysis by MetaboAnalyst. Receiver operating characteristic analysis was used to examine diagnostic values.</p> Results <p>SF results identified o-tyrosine as a promising biomarker for distinguishing RA patients from OA patients and controls, and cystine, cysteine, and methionine separating OA patients from controls. Regarding IFPs, <i>β</i>-alanine could have diagnostic value to discriminate RA and OA. The present data indicate alterations in metabolic pathways, such as cysteine and methionine metabolism in RA and OA SFs compared to control SF, selenocompound metabolism in RA <i>vs.</i> OA SFs, and pyrimidine metabolism in RA <i>vs.</i> OA IFPs.</p> Conclusions <p>The identified nitrogen compounds, particularly o-tyrosine, and AA metabolism pathways have potential as novel diagnostic and therapeutic targets for degenerative joint diseases.</p> <p><Table Float="No" ID="Taba"> <tgroup cols="2"> <colspec align="left" colname="c1" colnum="1" /> <colspec align="left" colname="c2" colnum="2" /> <tbody> <row> <entry align="left" nameend="c2" namest="c1"> <p><b>Key Points</b></p> <p>•<i> Synovial fluid o-tyrosine can distinguish rheumatoid arthritis from osteoarthritis and control.</i></p> <p>• <i>Synovial fluid cystine, cysteine, and methionine separate osteoarthritis from control.</i></p> <p>• <i>β-Alanine in intra-articular fat has diagnostic value between rheumatoid arthritis and osteoarthritis.</i></p> <p>• <i>A routine measurement of o-tyrosine would be useful in the future as an indicator of rheumatoid arthritis.</i></p> </entry> </row> </tbody> </tgroup> </Table></p>