Identifying the Safe and Adequate Folic Acid Intake Range Using Biomarkers in 2 Mouse Models

High intakes of folic acid (FA), the synthetic form of folate added to fortified foods and supplements, have been associated with adverse effects in studies in animals and humans, albeit inconsistently. Animal models are integral to investigating these relationships; however, it is unclear how commonly used FA doses in animal feeding studies relate to intakes in humans.

To identify adequate and excess FA intakes in mice based on biomarkers of inadequate [plasma homocysteine (Hcy)] and excessive [plasma unmetabolized FA (UMFA)] FA intakes.

Four-week-old male and female BALB/c and C57BL/6 mice were fed diets containing 0, 0.2, 0.5, 1, 2, 5, 20, or 40 mg/kg FA for 12 wk. Total folate in red blood cells (RBC) and plasma was measured biweekly. Plasma Hcy, plasma and liver folate vitamers including UMFA, total blood counts, and kidney pathology were assessed at necropsy.

Sex and strain effects were evident for nearly all endpoints. Plasma Hcy reached baseline in mice fed FA as low as 0.2 mg/kg. Plasma and RBC total folate were dose responsive and took 2-4 and 10+ wk to reach homeostasis in plasma and RBC, respectively. Plasma UMFA was only higher in mice fed the 20 and 40 mg/kg FA; UMFA represented ∼50% of total folate in these mice. UMFA did not accumulate in liver in any mice. Other endpoints were not FA dependent.

The AIN-93 diet, the most commonly used control diet, contains 2 mg FA/kg diet, which is higher than adequate. On the basis of increased UMFA, commonly used supplemental FA doses associated with adverse effects in mice are supraphysiological and likely do not reflect human exposures. Our findings suggest caution is warranted when interpreting findings in mice fed these very high FA doses.