Objective To determine if functional polymorphisms of folate/homocysteine pathway enzymes are associated with homocysteine concentrations and/or coronary artery calcification (CAC) scores in patients with systemic lupus erythematosus (SLE) and controls. Methods We investigated 163 SLE patients and 160 controls. Functional polymorphisms in 6 genes in the folate/homocysteine pathway were genotyped: 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T, MTHFR 1298A>C, cystathionine ß-synthase (CBS) 844ins68, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, thymidylate synthase (TYMS) 1494del6, and dihydrofolate reductase (DHFR) c.86+60_78. Results Homocysteine levels were higher in African American SLE patients than Caucasian patients and African American controls. Genotype distributions were significantly different in African American and Caucasian controls for 6 of the 7 polymorphisms. Genotype distributions for each polymorphism did not differ significantly between SLE patients and controls even after stratification by race. Glomerular filtration rate was strongly negatively correlated to homocysteine levels, and was therefore adjusted for as a covariate in the models of the effects of the polymorphisms on homocysteine levels. In SLE patients none of the 7 polymorphisms was associated with homocysteine concentrations. In Caucasian controls only MTHFR 677C>T and 1298A>C showed effects on homo-cysteine similar to what would be expected from the literature. There were no genotypic associations with median CAC scores in SLE patients or controls with and without stratification by race. Conclusion Polymorphisms in folate/homocysteine metabolizing enzymes do not predict higher homocysteine levels or CAC scores in patients with SLE.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that recruits monocytes into the subendothelial cell layer in atherosclerotic lesions. Elevated homocysteine (hyper-homocysteinemia), which is usually associated with low-folate status, is a known risk factor for many pathologies with inflammatory etiologies. The present study was undertaken to examine whether there are associations between MCP-1 concentrations and folate/Hcy phenotype or methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype in healthy young adults. In females, MCP-1 concentrations were positively correlated with Hcy and negatively correlated with both serum and red blood cell folate; female smokers and MTHFR 677T carriers had particularly elevated MCP-1 concentrations. Similar relationships were not seen in males. These findings may have implications for understanding the female predominance observed for a range of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.

A high homocysteine, low folate phenotype is a feature of many diseases. The effect of the cystathionine b-synthase (CBS) 844ins68 polymorphism on homocysteine and folate concentrations was examined alone and in the context of the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C4T polymorphism in a Northwestern European male population. The MTHFR 677TT genotype is known to be associated with increased homocysteine and decreased folate relative to CT heterozygotes and CC homozygotes in this and other populations. MTHFR 677TT homozygotes who were also CBS 844ins68 carriers had homocysteine and folate concentrations similar to those of individuals with the MTHFR 677CT and CC genotypes. Homocysteine levels in MTHFR 677TT subjects carrying the CBS 844ins68 allele were 24.1% lower than in non-carriers (6.66 vs 8.77 lmol/l, P ¼ 0.045), and serum folate levels were 27.7% higher (11.16 vs 8.74 nmol/l, P ¼ 0.034). These findings suggest that the CBS 844ins68 allele 'normalizes' homocysteine and folate levels in MTHFR 677TT individuals.

Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low (“Lo”) and high (“Hi”) folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency.

EA.hy 926 cells grown under low folate conditions adopt a “pro-atherosclerotic” morphology and biochemical phenotype. Pharmacologically relevant doses of the antifolate drug methotrexate (MTX) were applied to EA.hy 926 cells maintained in normal (Hi) and low (Lo) folate culture media. Under both folate conditions, MTX caused inhibition of cell proliferation without significantly compromising metabolic activity. MTX treated Hi cells were depleted of folate derivatives, which were present in altered proportions relative to untreated cells. Transcript profiling using microarrays indicated that MTX treatment modified the transciptome in similar ways for both Hi and Lo cells. Many inflammation-related genes, most prominently those encoding C3 and IL-8, were up-regulated, whereas many genes involved in cell division were down-regulated. The results for C3 and IL-8 were confirmed by quantitative RT-PCR and ELISA. MTX appears to modify the inflammatory potential of EA.hy 926 cells such that its therapeutic properties may, at least under some conditions, be accompanied by the induction of a subset of gene products that promote and/or maintain comorbid pathologies.