## 3 Results
### MTHF Concentration in Non-Pregnant and Pregnant Rat Tissues Post Folate Form Administration  
In the pregnant rat on normal folate replete chow (Lab Diet 5001, St. Louis, MO, USA), the baseline MTHF level is highest in the liver and was similar to that in the non-pregnant and young rats [36; 37]. The ovary contained the next highest level of MTHF with lower  
Figure 1: Serum concentration of folate forms following oral administration of PGA (**A**); L-5-methylfolate (**B**); Fusilev (**C**); Leucovorin (**D**); determined at 30 min, 60 min, 120 min, and 240 min post oral gavage. Values shown are ng/mL serum for all folate forms; Two rats were used for each folate form administered at each time point. levels in the brain. The placentas and the embryos contained substantially low levels of MTHF in comparison (Figure 3). When analyzing non-pregnant rats 24 h post folate administration, we observed a slight increase in liver MTHF compared to the baseline value. Liver MTHF was similar with all four forms administered. The MTHF accumulation in the kidneys, ovary, and  
Figure 3: Endogenous tissue MTHF in a GD 14 pregnant dam (\(n=2\)). Figure 2: Absorbed unconverted folate form in blood. The values are expressed as a percentage of total folate in the serum. Levofolinate and d,l-folinic acid identified as unconverted are deduced from the total concentration in the serum. Therefore, the MTHF formed is likely to include THF and formyl-THF formed, which are minor components of the unconverted folate forms. MTHF administered appears to be absorbed as such without further conversion. brain was also similar with all four forms of folate administered (Figure 4A) with values comparable to baseline values in control dams shown in Figure 3.  
In GD 14 dams, tissue distribution of MTHF concentration 24 h post dosing was somewhat higher compared to non-pregnant rats. Moreover, substantially higher amount of MTHF was observed in the placentas and embryos with all forms of folate administered (Figures 3 and 4b for comparison) (placenta: F(4, 14.1) = 623, \(p<0.001\), embryo: F(4, 15.4) = 146, \(p<0.001\)). When comparing the MTHF concentration in placenta among the administered folate forms via Gammell-Howell test, levofoliate had higher MTHF levels than folic acid (\(t\)-value = \(-\)6.35, df = 12.61, \(p<0.001\)) and 5 MTHF (\(t\)-value = \(-\)7.35, df = 10.34, \(p<0.001\)). Placental MTHF concentration with d,l-folinic acid was higher than with folic acid (\(t\)-value = \(-\)4.11, df = 24.9, \(p=0.003\)) and 5 MTHF (\(t\)-value = \(-\)5.4, df = 21, \(p<0.001\)) but not when compared to levofoliate (\(t\)-value = 2.86, df = 18.1, \(p=0.069\)). MTHF uptake in embryonic tissue was also increased when comparing levofoliate to folic acid (\(t\)-value = \(-\)9.34, df = 41, \(p<0.001\)) and 5 MTHF (\(t\)-value = \(-\)5.11 df = 41, \(p<0.001\)) via  
Figure 4: MTHF concentration in tissues of non-pregnant rats (**A**) and in GD 14 pregnant rats (**B**) 24 h post oral administration of various folate forms. (Sample sizes non-pregnant (**A**): Folic acid (\(n=2\)), 5 MTHF (\(n=2\)), Levofoliate (\(n=2\)), d,l-folinic acid (\(n=2\)); Sample sizes pregnant (**B**): Folic acid (\(n=2\)), 5 MTHF (\(n=2\)), Levofoliate (\(n=3\)), d,l-folinic acid (\(n=3\))). Asterisks indicate the following: **\(p<0.01\), ***\(p<0.001\). post-hoc Tukey's test. Moreover, d,l-folinic acid had higher concentration levels than folic acid (\(t\)-value = \(-8.3\), df = 41, \(p<0.001\)) and 5 MTHF (\(t\)-value = \(-3.79\), df = 41, \(p=0.004\)), but no difference in concentration when compared to levofolinate (\(t\)-value = 2.4, df = 41, \(p=0.137\)).