Folic Acid Impairs the Uptake of 5-Methyltetrahydrofolate in Human Umbilical Vascular Endothelial Cells

Abstract

Background: Adequate folate status supports endothelial structure and function. Folic acid (FA), an oxidized synthetic folate, which is present in the plasma of patients consuming fortified food or FA supplements, may impair cellular uptake of physiological, reduced folates. We studied the effect of FA on uptake of the dominant circulatory folate, 5-methyltetrahydrofolate (5MTHF) in endothelial cells.

Methods and results: For short-term effects of FA, primary human umbilical vein endothelial cells (HUVECs) were maintained in growth medium containing 200 nM 5MTHF and preincubated with 20 nM FA 10 minutes before the 5MTHF uptake assessment. For long-term effects, HUVECs were cultured for 3 passages in growth medium containing either 200 nM 5MTHF, or a combination of 100 nM 5MTHF and 100 nM FA. 5MTHF uptake was assessed after exposing cells to 200 nM [C5]-5MTHF, after which intracellular [C5]-5MTHF was quantified using liquid chromatography/tandem mass spectrometry. Acute FA exposure caused a 57% reduction in 5MTHF uptake compared with control conditions (51 ± 12 vs. 22 ± 7 fmol·min·mg protein; P = 0.01). Long-term exposure to FA reduced 5MTHF uptake by 41% (51 ± 12 vs. 30 ± 11 fmol·min·mg protein; P = 0.05) and reduced total cellular 5MTHF levels by 47 ± 21% in HUVEC (P = 0.02).

Conclusion: Unmetabolized FA, which appears in the plasma after consumption of fortified food or FA supplements, may impair uptake of 5MTHF, the dominant bioactive form of folate, in HUVEC.

FIGURE 1.

Timecurve 5MTHF Uptake in HUVECs.

FIGURE 2.

Effect of long- and short-term FA exposure on the uptake of 5MTHF in primary HUVECs. I: control, II: 10 minutes of FA exposure, III: long-term FA exposure. Bars represent mean ± SD for 6 HUVECs isolates.

FIGURE 3.

Effect of long-term FA exposure on intracellular 5MTHF concentration in HUVECs. Results depict 6 HUVECs isolates: left is cultured on 200 nM 5MTHF for 3 passages, and right on a combination of 100 nM 5MTHF and 100 nM FA.

FIGURE 4.

Relative gene expression of folate transporters in primary HUVECs. Results depict the mean relative gene expression levels normalized to βs2M (2-ΔCt ± SD) of 3 HUVEC isolates. n.d., not detectable.