Patients with RA having RFC 80A and GGH-401T alleles were less responsive to MTX than those with RFC 80A and without GGH-401T alleles. Thus, this data may be useful for guiding treatment of RA patients with MTX.

Rheumatoid arthritis (RA) is a chronic disease characterized by the painful joints, inflammation, uncontrolled proliferation of synovial tissue and multisystem comorbidities. Weekly low-dose methotrexate (MTX) has been established as effective treatment in RA patients. MTX is converted to γ -glutamyl polyglutamates, an active form of MTX, through the action of folylpolyglutamate synthetase in the cells. MTX-polyglutamates (MTX-PGs) in red blood cells (RBCs) may be useful as a therapeutic marker of RA. However, the previously reported methods for the quantification of MTX and MTX-PGs in RBCs are impractical for clinical use due to time-consuming, laborious and high cost. We attempted to apply a method with the commercially available fluorescence polarization immunoassay (FPIA) kit. We found that anti-MTX monoclonal antibody showed the reactivity to 4-amino-10-methylpteroylheptaglutamic acid (MTX-PG 7 ) as equal to MTX. Good agreement was observed in the concentration-response curves between MTX and MTX-PG 7 spiked samples. Accordingly, the anti-MTX monoclonal antibody for FPIA appeared to show the equal reactivity to MTX and MTX-PGs. The recoveries of MTX and MTX-PG 7 from RBCs were 99.0% and 94.1%, respectively. Furthermore, we determined total MTX-PGs concentrations in RBCs of 71 patients with RA treated with weekly pulse MTX. Total MTX-PGs concentrations in 70% of the patients were found to be more than 50 nM that is the lower limit of MTX-PGs concentration in RBCs for expected therapeutic outcome. The routine measurement of total MTX-PGs concentration in RBCs might be useful for prediction about therapeutic outcome of MTX in RA patients.methotrexate; methotrexate-polyglutamate; rheumatoid arthritis; therapeutic drug monitoring; fluorescence polarization immunoassay.Tohoku

Hyperhomocysteinemia is a known risk factor of cardiovascular disease. Homocysteine has been also linked to inflammation in rheumatoid arthritis (RA). In this study, we investigated the relationship between plasma homocysteine levels and single nucleotide polymorphism (SNP) of the gene coding for methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the biosynthesis of homocysteine, and the correlation between the plasma homocysteine levels and generally used inflammatory markers (C-reactive protein, erythrocyte sedimentation rate and matrix metalloproteinase-3) in 96 Japanese patients with RA. Plasma homocysteine levels in patients with the MTHFR 677TT genotype were significantly higher than in those with the 677CC genotype (p < 0.05). In addition, plasma homocysteine levels were increased along with the elevation of general inflammatory markers. Therefore, we conclude that homocysteine might affect the inflammatory status of patients, and the MTHFR 677C>T SNP could be a predictive factor of hyperhomocysteinemia in patients with RA.

We investigated the genotype distribution and allele frequency of C452T polymorphism of g g-glutamyl hydrolase (GGH) gene, which causes the decreased enzymatic activity affecting the efficacy of methotrexate (MTX), in a Japanese population. The polymerase chain reaction-restriction fragment length polymorphism assay was applied to determine the genotype of C452T polymorphism in 269 Japanese healthy individuals. The genotype distribution was as follows: C/C, 89.2% (n‫;)042؍‬ C/T, 10.4% (n‫;)82؍‬ T/T, 0.4% (n‫.)1؍‬ The frequency of C and T allele was 0.944 and 0.056, respectively. The obtained genotype distribution was well agreed with those expected by Hardy-Weinberg equilibrium. The genotype distribution and allele frequency in a Japanese population were found to be similar to those of African-Americans but significantly different from Caucasians. Although the frequency of variant T allele in a Japanese population is not so high as compared to Caucasians, determination of C452T polymorphism of GGH may be useful for monitoring of efficacy and side-effects of MTX for treatment of diseases such as rheumatoid arthritis or childhood acute leukemia. To our knowledge, this is the first report about the examination of C452T polymorphism of GGH in a Japanese population.