Early diagnosis of rheumatoid arthritis (RA) is hampered by suboptimal accuracy of currently available serological biomarkers. Recent advancements in metabolomic profiling include dansylation liquid chromatography mass spectrometry (LC-MS), resulting in 1000-fold increase in detection sensitivity of amine/phenol-containing metabolites, and universal metabolome-standard (UMS) methodology in conjunction with differential chemical isotope labeling (CIL LC−MS), to provide long-term analytical reproducibility and facilitate metabolome comparisons among different data sets. CIL LC-MS uses different labeling reagents to target chemical group-based submetabolomes to provide in-depth metabolomic analysis. We aimed to identify a metabolite signature with consistently high accuracy for RA.

12C-dansylation and acid labeling of individual serological samples and 13C-dansylation and acid labeling of pooled samples from 2 RA cohorts was undertaken. Cohort A samples were from 50 RA patients, 39 female (mean age 49.9), 11 male (mean age 47.8), symptom duration <3 years, DAS >3.7, naïve to b-DMARD, and 50 age and sex-matched healthy controls. Cohort B samples were from 50 RA patients, 40 female (mean age 53.4), 10 male (mean age 57.2), symptom duration <5 years, samples from both pre- and post- (3 months) treatment with TNFi and a second set of 50 age and sex-matched healthy controls.

A total of 3415 amine/phenol metabolites were commonly detected in 80% of the samples, of which 116 metabolites were positively identified using dansyl standard library search. Orthogonal partial least squares discriminant analysis showed a clear separation of the groups for each cohort (R2=0.98/Q2=0.92 for cohort A and R2=0.93/Q2=0.79 for separation of controls, pre- and post-treatment cohort B). 18 positively identified metabolites from cohort A and 23 from cohort B were identified as potential RA biomarkers with ROC AUC(95%CI) of 0.99(0.96-0.99) and 0.98(0.93-0.99) for the top 10 metabolites, respectively. L-Cystine, O-Phosphoethanolamine, Gamma Glutamylglutamic acid, Glycyl-Valine, and 3 unidentified metabolites were significant in both cohorts, with the combination ROC AUC(95%CI) of 1.0(1.0-1.0) and 1.0(0.99-1.0) for cohorts A and B, respectively. For the 4 positively identified metabolites, sensitivity/specificity plus AUC(95%CI) were 89%/88% plus 0.95(0.87-0.99) and 91%/91% plus 0.97(0.92-1.0) for cohorts A and B, respectively. None of these biomarkers correlated with age, gender, or symptom duration.

7 metabolites, 4 of which are positively identified, have consistently high discriminatory capacity for RA.