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2.2. AUCs for (6S)-5-Methyl-THF-Ca Versus Folic Acid and Methods of Indirect Comparisons with the AUCs for (6S)-5-Methyl-THF-Na

Prinz-Langenohl et al. (2009) compared the AUC0–8 h of plasma total folate between (6S)-5-Methyl-THF-Ca and folic acid in 24 young women []. shows a brief summary of the study design compared to the present study. Equimolar oral doses of (6S)-5-Methyl-THF-Na (436 µg, present study) and (6S)-5-Methyl-THF-Ca (451 µg, in Prinz-Langenohl et al.) have been tested against the same reference substance, folic acid (400 µg). Plasma concentrations of (6S)-5-Methyl-THF were selectively measured in both studies. In the study by Prinz-Langenohl et al., 2009 [], the measurements of plasma (6S)-5-Methyl-THF were conducted at VU University Medical Centre, Department of Clinical Chemistry, Amsterdam (The Netherlands), by using liquid chromatography tandem mass-spectrometer (LC-MS-MS system) using 13C5-(6S)-5-Methyl-THF as internal standard []. We here show novel data on the AUC0–8 h of plasma (6S)-5-Methyl-THF from the original study [].

Abstract

[6S]-5-methyltetrahydrofolic acid (MTHF) is a proposed replacement for folic acid (FA) in diets and prenatal supplements. This study compared the effects of these two forms on maternal metabolism and hypothalamic gene expression. Pregnant Wistar rats received an AIN-93G diet with recommended FA (1X, 2 mg/kg, control), 5X-FA or equimolar levels of MTHF. During lactation they received the control diet and then a high fat diet for 19-weeks post-weaning. Body weight, adiposity, food intake, energy expenditure, plasma hormones, folate, and 1-carbon metabolites were measured. RNA-sequencing of the hypothalamus was conducted at parturition. Weight-loss from weaning to 1-week post-weaning was less in dams fed either form of the 5X vs. 1X folate diets, but final weight-gain was higher in 5X-MTHF vs. 5X-FA dams. Both doses of the MTHF diets led to 8% higher food intake and associated with lower plasma leptin at parturition, but higher leptin at 19-weeks and insulin resistance at 1-week post-weaning. RNA-sequencing revealed 279 differentially expressed genes in the hypothalamus in 5X-MTHF vs. 5X-FA dams. These findings indicate that MTHF and FA differ in their programing effects on maternal phenotype, and a potential adverse role of either form when given at the higher doses.

2.1. Animals and Diets

All animal work was approved by the University of Toronto Animal Care Committee (#20011892). First-time pregnant Wistar rats (2–3 days, 170–250 g) were purchased from Charles River Farms (Quebec, Canada) and individually housed upon arrival. Animals were maintained on a reverse 12-h dark-light cycle (lights on at 07:00 a.m. at 22 ± 1 °C) and ad libitum food and water were provided throughout the study. Dams were randomly allocated to one of four dietary interventions that were provided contemporaneously and only during pregnancy until term (3 weeks). Pregnant dams (n = 16–18/group) received an isocaloric AIN-93G diet (energy density 4.0 kcal/g) [] containing either the recommended (1X, 2 mg/kg diet, control) or 5X-quantity of FA for rodents (1X-FA or 5X-FA) or equimolar recommended levels of [6S]-5-methyltetrahydrofolic acid, calcium salt (1X, 2.1 mg/kg diet, Metafolin®, Merck & Cie, Schaffhausen, Switzerland) or 5X-quantity of MTHF (1X-MTHF or 5X-MTHF). FA and MTHF diets at equimolar doses have been used in clinical trials assessing folate status and related-health effects [,,]. The recommended (1X) dose was chosen based on the FA requirements for rodents for adequate growth rate [] and reflects the FDA requirements for non-pregnant women (400 µg/day) []. The 5X dose was chosen to reflect FA intakes commonly consumed in North America above the basal dietary requirements for humans [,]. Diets were custom made by Research Diets Inc. (New Brunswick, NJ, USA) () and stored at 4 °C and in the dark until provided fresh weekly. The additions of the MTHF form of folate into foods has been shown to be stable at both 4 °C and room temperature for up to 24 months []. Analytical testing of the high folate diets following a 36-month storage at −80 °C showed >80% retention of folates (), further confirming their long-term stability.

Abstract

Supplementation with [6S]-5-methyltetrahydrofolic acid (MTHF) is recommended as an alternative to folic acid (FA) in prenatal supplements. This study compared equimolar gestational FA and MTHF diets on energy regulation of female offspring. Wistar rats were fed an AIN-93G diet with recommended (2 mg/kg diet) or 5-fold (5X) intakes of MTHF or FA. At weaning, female offspring were fed a 45% fat diet until 19 weeks. The 5X-MTHF offspring had higher body weight (>15%), food intake (8%), light-cycle energy expenditure, and lower activity compared to 5X-FA offspring (p < 0.05). Both the 5X offspring had higher plasma levels of the anorectic hormone leptin at birth (60%) and at 19 weeks (40%), and lower liver weight and total liver lipids compared to the 1X offspring (p < 0.05). Hypothalamic mRNA expression of leptin receptor (ObRb) was lower, and of suppressor of cytokine signaling-3 (Socs3) was higher in the 5X-MTHF offspring (p < 0.05), suggesting central leptin dysregulation. In contrast, the 5X-FA offspring had higher expression of genes encoding for dopamine and GABA- neurotransmitter receptors (p < 0.01), consistent with their phenotype and reduced food intake. When fed folate diets at the requirement level, no differences were found due to form in the offspring. We conclude that MTHF compared to FA consumed at high levels in the gestational diets program central and peripheral mechanisms to favour increased weight gain in the offspring. These pre-clinical findings caution against high gestational intakes of folates of either form and encourage clinical trials examining their long-term health effects when consumed during pregnancy.

2.1. Animals and Diets

First-time pregnant (2–3 days) Wistar rats weighing 170–250 g (Charles River Farms, QC, Canada) were singly housed in a 12 h dark-light cycle (lights on at 0700 hrs at 22 ± 1 °C) and provided ad libitum food and water throughout the study. The protocol (# 20011892) was approved by the University of Toronto Animal Care Committee. Upon arrival, dams (n = 16–18/group) were randomly allocated to one of four custom-made dietary interventions (Research Diets Inc., New Brunswick, NJ, USA) that were provided only during their 3-week pregnancy: an AIN-93G diet (energy density 4.0 kcal/g) containing FA at either recommended (1X-FA, 2 mg/kg diet, control) or high (5X-FA, 10 mg/kg diet) quantities for rodents or [6S]-5-methyltetrahydrofolic acid calcium salt (Metafolin®, Merck & Cie, Schaffhausen, Switzerland) at equimolar recommended (1X-MTHF, 2.1 mg/kg diet) or high quantities (5X-MTHF, 10.4 mg/kg diet). Diets were kept in the dark at 4 °C until freshly provided to the animals weekly. Diets were analytically tested for long-term stability []. FA and MTHF were provided at equimolar levels in the diet due to a suggested comparable bioavailability of MTHF at equimolar levels to FA. Equimolar levels are used in clinical trials comparing folate status and homocysteine level maintenance [,]. The FA form was used as a reference to calculate the MTHF equimolar dose equivalent as only the synthetic folate form contains a tolerable upper level (1000 µg/day). The recommended (1X) FA dose provides 2 mg/kg of FA (4000 kcal) and represents the basal requirement for rodents for adequate growth rate []. This dose reflects recommendations of 0.4 mg of FA/day for women at low risk of neural-tube defects planning a pregnancy []. The supplemented FA diet contains 5X the basal requirement for rodents or 10 mg/kg of FA (4000 kcal). This dose reflects FA intakes commonly consumed by pregnant women in North America during the post-fortification era at >2-fold above basal dietary requirements for humans [,]. As MTHF is incorporated at the equivalent dose as FA in supplements, intakes may soon increase beyond recommendations [].

3.1. X-MTHF Offspring Gained more Weight and Ate more Food Than 5X-FA Offspring

A Dose×Form interaction effect was also found on measures of cumulative food intake up to 19 weeks post-weaning (Dose p = 0.7, Form p = 0.05, Dose×Form p = 0.02, B). The 5X-MTHF female offspring consumed ~8% more food compared to the 5X-FA offspring, but food intakes of the 1X folate offspring were similar to both 5X groups. Cumulative food intake and total weight gain of the offspring were also strongly and positively correlated within each group (1X-FA r = 0.89, p < 0.001, 1X-MTHF r = 0.90, p < 0.001, 5X-FA r = 0.71, p < 0.01; 5X-MTHF r = 0.72, p < 0.01).

5. Conclusions

MTHF compared to FA consumed at high levels in the gestational diet program central and peripheral mechanisms to favour increased weight gain in the female offspring. These pre-clinical findings caution against high gestational intakes of folates of either form, and encourage clinical trials examining their long-term health effects when consumed during pregnancy.