Premium
Folic Acid and Riboflavin Deficiencies in Murine Macrophage RAW Cells Reduce LPS‐induced Nitric Oxide Production
Author(s) -
Rittmann Marijke,
Miller Joshua W,
Gow Andrew
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.802.22
Subject(s) - nitric oxide , riboflavin , nitric oxide synthase , chemistry , biochemistry , methylenetetrahydrofolate reductase , flavin adenine dinucleotide , enzyme , cofactor , organic chemistry , genotype , gene
Methylenetetrahydrofolate reductase (MTHFR) is a flavin adenine dinucleotide (FAD)‐dependent folate metabolizing enzyme that catalyzes the conversion of dihydrobiopterin to tetrahydrobiopterin (BH 4 ). BH 4 is an essential cofactor for nitric oxide synthase (NOS) that produces nitric oxide for multiple functions including vasodilation and blood pressure (BP) regulation. Genome‐wide association studies have linked a common polymorphism, C677T, in MTHFR with BP, and riboflavin supplements have been shown to reduce BP in individuals homozygous for the 677TT variant form of the enzyme. We hypothesize that this relationship between MTHFR, riboflavin, and BP is mediated through nitric oxide synthesis. We investigated this hypothesis in vitro in stimulated murine macrophage RAW cells, which express the inflammatory form of NOS, exposed to standard and reduced levels of folic acid and riboflavin. RAW cells were grown in medium with 0.4 mg/L riboflavin and 4.0 mg/L folic acid (control), 0.04 mg/L riboflavin (lowB2), or 0.4 mg/L folic acid (lowFA) for 48 hours, and then exposed to 100 ng/ml or 1000 ng/ml lipopolysaccharide (LPS) for 24 hours. Media was then analyzed for nitric oxide production by chemiluminescence assay using a Nitric Oxide Analyzer. Quantitative PCR was used to analyze gene expression of inducible NOS (iNOS). In all three media conditions, no differences in RAW cell proliferation rates were observed over 48 hours. After LPS exposure, nitric oxide production in the lowB2 and lowFA cells was 30–35% and 35–40% of the control cells, respectively (p ≤ 0.02). Expression of iNOS after LPS induction increased in all three media conditions. LPS‐induced nitric oxide production is reduced in RAW cells grown in either riboflavin or folic acid deficient media independent of iNOS expression. These results demonstrate the importance of the folate cycle in maintaining NOS function, and indicate a potential mechanism for the effects of MTHFR polymorphisms on BP. Support or Funding Information Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ