Studies on the influence of folic acid and riboflavin on nitric oxide production in cultured murine macrophage cells
Creators: name: Rittmann, Marijke
Publisher: name: No Publisher Supplied
Year: 2018

description: Methylenetetrahydrofolate reductase (MTHFR) is a flavin adenine dinucleotide (FAD) - dependent, folate-metabolizing enzyme. The main product of MTHFR is methylenetetrahydrofolate, an important substrate for homocysteine metabolism and synthesis of methionine and the universal methyl donor, S-adenosylmethionine. MTHFR also catalyzes the reduction of dihydrobiopterin to tetrahydrobiopterin (BH4). BH4 is an essential cofactor for nitric oxide synthase (NOS) that produces nitric oxide (NO) 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 and that deficiency in either folic acid or riboflavin will result in decreased NO output.  We investigated this hypothesis in vitro in stimulated murine macrophage RAW cells, which express the inducible form of NOS (iNOS). The cells were exposed to standard and reduced levels of folic acid and riboflavin. They were then stimulated with LPS. The 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 iNOS and arginase. 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 ≤ 0.001). Expression of iNOS after LPS induction increased in all three media conditions.  Based on the findings above we further hypothesized that the decreased NO output from the B vitamin-deficient cells was due to insufficient BH4 availability to the cells. To test this hypothesis, a precursor of BH4, sepiapterin, was provided to the LowFA and LowB2 cells before stimulating NO production with LPS. Cells were treated with the same level of deficiency in folic acid and riboflavin as above, but were supplemented with 10 umol/L of sepiapterin at 0 hour and at the time of stimulation with 100 ng/mL LPS. Cells receiving sepiapterin before and during LPS exposure did not increase NO output when compared to those exposed only to LPS (p&gt;0.8).  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. They also show that providing substrate for BH4 production is not sufficient to overcome the decreased NO output caused by deficiency.