ARTHRITIS & RHEUMATISM Vol. 50, No. 10, October 2004, pp 3104–3111 DOI 10.1002/art.20516 © 2004, American College of Rheumatology The Effect of Folic Acid and Folinic Acid Supplements on Purine Metabolism in Methotrexate-Treated Rheumatoid Arthritis Sarah L. Morgan, Robert A. Oster, Jeannette Y. Lee, Graciela S. Alarco ´n, and Joseph E. Baggott Objective. To determine if folinic acid supplemen- tation during methotrexate (MTX) therapy for rheuma- toid arthritis (RA) reduces both urinary 5-amino- imidazole-4-carboxamide (AICA) and urinary adenosine excretion more than does folic acid supple- mentation. AICA and adenosine are markers for MTX interference with purine metabolism. Methods. Forty patients with RA who received MTX for 6 weeks were randomized to receive either daily folic acid or folinic acid supplements during an additional week of MTX therapy. Colorimetric and radio- immunocompetition assays were used to measure 24-hour urinary AICA and adenosine excretion levels, respectively. Results. At the end of 6 weeks, 24-hour urinary levels of AICA, but not adenosine, were elevated as compared with baseline levels (i.e., prior to MTX ther- apy). Folinic acid, but not folic acid, supplementation normalized urinary AICA levels during MTX therapy. Relatively high urinary levels of AICA were correlated with reduced disease activity. No similar correlations were seen with urinary adenosine levels. Conclusion. The blockade of purine nucleotide biosynthesis by MTX at the AICA ribonucleotide transformylase–catalyzed step may be related to the efficacy of MTX, and this blockade is effectively relieved by folinic acid, but not by folic acid, supplementation. Methotrexate (MTX) is an antifolate with a chemical structure similar to that of folic acid and folinic acid (5-formyl-tetrahydrofolic acid) (Figure 1). Admin- istered in low dosages (7.5–25 mg/week), MTX inhibits a number of folate-dependent metabolic steps, including a very potent inhibition of dihydrofolate reductase (DHFR), which reduces folic acid to dihydrofolic acid and to tetrahydrofolate. Once absorbed, MTX is metab- olized to polyglutamate derivatives, which have a greater ability to inhibit other folate-dependent enzymes, such as thymidylate synthetase, and 5-aminoimidazole-4- carboxamide ribonucleotide (AICAR) transformylase, as compared with MTX (1). The mechanism of action of low-dose MTX in the treatment of rheumatoid arthritis (RA) is unclear. Pro- posed mechanisms of action of MTX in RA have centered on adenosine-mediated antiinflammatory ef- fects (2–8). Inhibition of AICAR transformylase causes increased levels of AICAR, with subsequent inhibition of AMP deaminase and adenosine deaminase (ADA) (1). MTX therapy then results in an increase in extra- cellular adenosine concentration. Adenosine binds to many transmembrane-spanning adenosine surface re- ceptors (e.g., A 1 ,A 2 ,A 2 , and A 3 ) (3). As a result of binding to A 2 and A 3 receptors, lymphocyte prolifera- tion is inhibited and a cytokine environment favorable for lowering inflammation is fostered (3). However, the importance of adenosine-mediated mechanisms has re- cently been questioned in rat adjuvant-induced arthritis, where there was no attenuation of antiarthritic effect when 3 adenosine antagonists were combined with MTX (9). We hypothesized that the effect of MTX in the treatment of RA is due to the inhibition of AICAR Supported by a grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Office of Dietary Supplements (1R29-AR-42674), and in part by grants from the De- partment of Research Resources Clinical Research Center (M01-RR- 00032) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (P60-AR-40895), NIH. Sarah L. Morgan, MD, RD, Robert A. Oster, PhD, Jeannette Y. Lee, PhD, Graciela S. Alarco ´n, MD, MPH, Joseph E. Baggott, PhD: University of Alabama at Birmingham. Address correspondence and reprint requests to Sarah L. Morgan, MD, RD, University of Alabama at Birmingham, 354A Learning Resource Center, 1714 9th Avenue South, Birmingham, AL 35294-1270. E-mail: slmorgan@uab.edu. Submitted for publication March 1, 2004; accepted in revised form June 15, 2004. 3104