title: Red blood cell folate concentrations and polyglutamate distribution in juvenile arthritis:  predictors of folate variability.
abstract: OBJECTIVE
  Methotrexate (MTX) has several enzymatic targets in the folate pathway. To better understand the variability in response to MTX, we characterized the interindividual variability of intracellular folate pools in children with juvenile arthritis (JA) and determined clinical and genetic contributors to this variability.
  
  
  METHODS
  This exploratory single-center cross-sectional study evaluated 93 patients with JA not currently receiving MTX. Whole blood, plasma, and erythrocyte folate concentrations were determined after deconjugation and analyzed through reversed-phase separation and stable isotope dilution tandem mass spectrometry. Folate polyglutamates were measured in red blood cell lysates using an ion-pair reversed phase chromatography tandem mass spectrometry method.
  
  
  RESULTS
  Intracellular concentrations of 5-methyl-tetrahydrofolate (5-CH3-THF) and 5,10-methenyl-tetrahydrofolate varied approximately 20-fold and 80-fold, respectively. The polyglutamated forms of 5-CH3-THF as a percentage of total 5-CH3-THF (5-CH3-THFGlun) were also measured. Hierarchical clustering of 5-CH3-THFGlun revealed two groups, each with two distinct clusters. There was an inverse relationship between 5-CH3-THFGlun chain length and plasma 5-CH3-THF concentrations. A subgroup of patients with a historical intolerance to MTX had significantly lower cellular folate concentrations (P<0.0001). In univariate analyses, clinical variables including sex, age, and folate supplementation in addition to variations in MTHFR, MTR, and SLC25A32 were associated with differential intracellular folate redox concentrations. Multivariate analysis further supported the association of single nucleotide polymorphisms in SLC25A32, MTHFR, and MTR with variability in intracellular 5-CH3-THF and 5,10-methenyl-tetrahydrofolate concentrations, respectively.
  
  
  CONCLUSIONS
  Measurement of intracellular folate isoforms may contribute toward a better understanding of individual MTX effects in JA. Clinical variables in addition to genotypic differences beyond MTHFR may additionally explain differential intracellular folate concentrations and variable responses to MTX.
authors.affiliation: Division of Clinical Pharmacology and Medical Toxicology, Children's Mercy Hospitals and Clinics, Kansas City, Missouri, USA. mlbecker@cmh.edu
authors.forename: Mara L
authors.initials: ML
authors.lastname: Becker
authors.forename: Leon
authors.initials: L
authors.lastname: van Haandel
authors.forename: Roger
authors.initials: R
authors.lastname: Gaedigk
authors.forename: Bradley
authors.initials: B
authors.lastname: Thomas
authors.forename: Mark F
authors.initials: MF
authors.lastname: Hoeltzel
authors.forename: Andrew
authors.initials: A
authors.lastname: Lasky
authors.forename: Hongying
authors.initials: H
authors.lastname: Dai
authors.forename: John
authors.initials: J
authors.lastname: Stobaugh
authors.forename: James S
authors.initials: JS
authors.lastname: Leeder
mesh_terms: D000293:Adolescent; D001171:Arthritis, Juvenile / Q000097:blood* / Q000188:drug therapy* / Q000235:genetics; D002648:Child; D005260:Female; D005287:Ferredoxin-NADP Reductase / Q000235:genetics; D005493:Folic Acid Antagonists / Q000009:adverse effects* / Q000493:pharmacokinetics / Q000627:therapeutic use; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008727:Methotrexate / Q000009:adverse effects* / Q000493:pharmacokinetics / Q000627:therapeutic use; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D010084:Oxidation-Reduction; D020641:Polymorphism, Single Nucleotide; D011624:Pteroylpolyglutamic Acids / Q000097:blood*; D013763:Tetrahydrofolates / Q000097:blood*
chemical_list: D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D011624:Pteroylpolyglutamic Acids; C417506:SLC25A32 protein, human; D013763:Tetrahydrofolates; C018092:5,10-methenyltetrahydrofolate; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase; D042965:Methylenetetrahydrofolate Reductase (NADPH2); C005984:5-methyltetrahydrofolate; D008727:Methotrexate