Folate Depletion and Increased Glutamation in Juvenile Idiopathic Arthritis Patients Treated With Methotrexate

Abstract

Objective Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of MTX activity. This study evaluates circulating folate content and folate polyglutamate distribution in Juvenile Idiopathic Arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply. Methods Blood, plasma and red blood cell (RBC) measurements of MTX and folates were obtained from previously published data sets and additional sample analysis for JIA patients receiving (n=98) and not receiving (n=78) MTX therapy. Erythroblastoid cells maintained in culture were exposed to MTX and grown under varying levels of folic acid supplementation. Samples were analyzed for cellular folate and MTX content. Results Circulating folate levels were lower in JIA patients receiving MTX, with reduced levels of blood, plasma and RBC 5-methyl-tetrahydrofolate (5mTHF) (p<0.0001). Average polyglutamate chain-length (Gluavg) of RBC 5mTHF was elevated in JIA patients receiving MTX (5.63±0.15 vs. 5.54±0.11, p<0.0001) and correlated with both RBC MTX accumulation (p=0.02) and reduced plasma 5mTHF levels (p=0.008). MTX exposure and folate deprivation in erythroblastoid cells resulted in a depletion of bioactive folate species that was associated with a shift to higher Gluavg values for several species, most notably tetrahydrofolate (THF) and 5,10-methylenetetrahydrofolate (CH2-THF). Increased Gluavg resulted from the depletion of short-chain and the accumulation of long-chain glutamate species. Conclusion Folate content and polyglutamate distribution are responsive markers of MTX activity and folate supply in vivo and in vitro, and may provide novel clinical markers of pharmacologic activity of MTX.

Metabolomic Profiling to Identify Molecular Biomarkers of Cellular Response to Methotrexate In Vitro

“…In particular, several markers of pyrimidine and purine biosynthesis, including deoxyuridine monophosphate, 2‐deoxycytidine, 2‐deoxyuridine, and IMP may represent highly sensitive markers of MTX activity. These findings are consistent with previous studies that have found MTX treatment is associated with systemic depletion of circulating folates and corresponding dysregulation of intermediates of nucleotide metabolism …”

“…In this study, K562 cells under normal growth conditions were either left untreated (i.e., controls) or treated with MTX at a concentration of 1,000 nM for 24 hours. The concentration of 1,000 nM was chosen based on MTX peak concentrations in the treatment of RA, and to allow analysis utilizing a complimentary data set including targeted metabolic profiling of folates in K562 cells treated with MTX . Under these conditions, K562 cells are efficient at the uptake and polyglutamation of MTX, which is believed to be important in its pharmacological activity .…”

“…Following statistical analysis for individual analytes measured by the untargeted metabolomic analysis, chemometric and metabolic pathway analysis techniques were utilized to detect specific chemical classes and metabolic pathways associated with the observed pharmacological response to MTX. In addition to the metabolomic data collected in this study, targeted analysis of folates and select markers of purine and pyrimidine biosynthesis that were previously collected were included in the analysis . Initial chemometric analysis was conducted using lipophilicity (i.e., xlogP) as a defining chemical descriptor to stratify the individual metabolites and plotted as a function of fold‐change with node size representing the negative logarithmic transformation of the FDR adjusted P value for each metabolite ( Figure a ).…”

“…Previous efforts to identify biomarkers of MTX response in autoimmune arthritis have primarily focused on the targeted analyses of MTX and its metabolites, or specific analytes hypothesized to be foundational to its impact on inflammation and immunity . A major limitation to these studies has been the biased investigative approach that largely focuses on a handful of metabolites or metabolic pathways, primarily revolving around the folate pathway and adenosine formation . In fact, despite over 70 years of research on MTX, the biochemical basis through which MTX mediates its effects in autoimmune arthritis remains controversial and, therefore, has been a potential obstacle in the search for biochemical markers of efficacy .…”

“…14 A major limitation to these studies has been the biased investigative approach that largely focuses on a handful of metabolites or metabolic pathways, primarily revolving around the folate pathway and adenosine formation. [15][16][17][18] In fact, despite over 70 years of research on MTX, the biochemical basis through which MTX mediates its effects in autoimmune arthritis remains controversial and, therefore, has been a potential obstacle in the search for biochemical markers of efficacy. 19 As an -omics approach, metabolomics offers a unique opportunity to apply a largely unbiased and untargeted approach to investigate potential biochemical markers of MTX response and to unravel the underlying biochemical basis of MTX efficacy.…”