(LB04-01-25) Establishment of Adequate and High Folate Intake Thresholds Based on a Folate Intake-Response Analysis in Male and Female C57Bl6 and BALB/c Mice

Objectives: High intakes of folic acid (FA), the synthetic form of folate added to fortified foods and supplements, may mask B12 deficiency, and have been associated with adverse effects in some studies. Animal models are integral to investigating these relationships, however it is unclear how commonly-used FA doses in animal feeding studies relate to those in humans. The objective was to establish a model of safe and adequate folic acid (FA) intakes in mice that reflects human intakes based on an estimation of adequate and excess FA intakes based on changes in plasma homocysteine (Hcy) and unmetabolized folic acid (UMFA) at low and high intakes, respectively, with consideration of strain and sex.

Methods: Four-week-old male and female Balb/c and C57BL/6 mice were fed AIN-93-based diets containing 0, 0.2, 0.5, 1, 2, 5, 20, or 40 mg/kg folic acid for 12 weeks. Total folate in red blood cells and plasma was measured biweekly and complete blood count, plasma homocysteine and folate vitamers were measured at necropsy. Benchmark Dose Response Modeling (BMDR) was used to identify doses corresponding to adequate and excess FA intake.

Results: Sex and strain effects were evident for nearly all endpoints investigated. The estimated adequate dose of FA as determined by plasma homocysteine was 0.53-0.95 mg/kg diet for BALB/c mice, but could not be calculated for C57BL/6 mice. UMFA increased in plasma at doses above 1.00-1.87 mg/kg depending on strain and sex. UMFA was the predominant folate form in plasma when diets contained >5 mg/kg. Total folate and folate vitamer concentrations in red blood cells and plasma were dose responsive, but hematological parameters did not reflect folate intake.

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

Funding Sources: Health Canada