Embryonic development of folate binding protein-1 (Folbp1) knockout mice: Effects of the chemical form, dose, and timing of maternal folate supplementation

Abstract

Inactivation of folate binding protein-1 (Folbp1) adversely impacts murine embryonic development, as nullizygous embryos (Folbp1^-/-) die in utero. Administration of folinic acid (N5-formyl-tetrahydrofolate) to Folbp1-deficient dams before and throughout gestation rescues the majority of embryos from premature death; however, a portion of surviving embryos develop structural malformations, including neural tube defects. We examined whether maternal supplementation with L-N5-methyl-tetrahydrofolate (L-5M-THF) has superior protective effects on embryonic development of Folbp1^-/- fetuses compared with L-N5-formyl-tetrahydrofolate (L-5F-THF). We also examined the critical period during gestation when folate supplementation is most beneficial to the developing Folbp1^-/- embryos. Folbp1^-/- pups presented with a range of malformations involving the neural tube, craniofacies, eyes, and abdominal wall. The frequencies of these malformations decreased with increasing folate dose, regardless of the form used. There was no additional benefit provided by L-5M-THF compared with L-5F-THF. Despite rescuing the phenotype in Folbp1^-/- embryos, no significant elevation of Folbp1^-/- maternal folate levels was observed with supplementation. Developmental Dynamics 231:221–231, 2004. © 2004 Wiley-Liss, Inc.

INTRODUCTION

Folic acid deficiency has been associated with the occurrence of birth defects, and several studies in humans have demonstrated the protective role of maternal folic acid supplementation in preventing neural tube, craniofacial and cardiac malformations (McDonald et al., 2003). Periconceptional folic acid supplementation of at least 0.4 mg/day reduces the prevalence of neural tube defects (NTDs) by up to 70% (MRC, 1991; Czeizel and Dudas, 1992; Berry et al., 1999; Berry and Li, 2002), as well as reducing the occurrence of craniofacial malformations such as cleft lip and cleft palate (Shaw et al., 1995; Czeizel et al., 1999; Loffredo et al., 2001). The mechanism by which this protection is achieved remains unknown.

Folates enter cells by way of the folate receptors (FRs in humans), also known as folate binding proteins (Folbps) in mice. Most FRs/Folbps isoforms are externally bound to the plasma membrane by glycosyl-phosphatidylinositol anchors and have tissue-specific and cell-specific expression patterns (Antony, 1992; Ross et al., 1994; Antony, 1996). The human FR-α and its murine homologue Folbp1 are primarily found in placenta, choroid plexus, and the brush border membrane in kidney (Selhub and Franklin, 1984; Elwood, 1989; Holm et al., 1991; Antony, 1992; Prasad et al., 1994). In the developing mouse embryo, Folbp1 is highly expressed in yolk sac, the neural folds, and neural tube (Barber et al., 1999; Saitsu et al., 2003). In a previously published study, the gene coding for Folbp1 has been functionally inactivated (Piedrahita et al., 1999). The embryonic development of nullizygous Folbp1 (Folbp1^-/-) mice is severely retarded; they fail to complete anterior neural tube closure and eventually die by gestational day 10. Daily oral supplementation of dams with the racemic mixture of N5-formyl-tetrahydrofolate (5F-THF, folinic acid; Leucovorin) before and throughout gestation reduces embryonic mortality and ameliorates a significant portion of the adverse developmental effects in Folbp1 nullizygotes (Piedrahita et al., 1999; Finnell et al., 2002). Although complete reversal of abnormal embryonic development is not achieved with folinic acid supplementation, a significant portion of the surviving Folbp1^-/- pups have seemingly normal postnatal development and are able to reproduce (Finnell et al., 2002).

Pteroylmonoglutamates, better known as folates, are essential water-soluble B vitamins that participate in the transfer of one-carbon units in two key metabolic pathways: biosynthesis of DNA and methylation. Folate is the commonly used generic term that refers to various chemical forms. All folates consist of a pteridine moiety attached at the C-6 position to p-aminobenzoyl glutamate (Shane, 1995). The most widely recognized chemical form is folic acid, which has an oxidized pteridine group. Folic acid is the common form used in vitamin supplements and food fortification due to its relatively low synthetic cost, chemical stability, and bioavailability. Folic acid per se does not have any known biological activity; however, it provides the fundamental backbone structure for the in vivo formation of biologically active folate coenzymes. Biologically active folates have the L optical configuration, reduced pteridine moieties, and are usually referred to as tetrahydrofolates (THF). The predominant biologically relevant folate circulating in blood and present intracellularly is L-N5-methyl-tetrahydrofolate (L-5M-THF). Unlike folic acid or naturally occurring polyglutamated folates, L-5M-THF requires no in vivo biochemical modifications such as reduction of the pteridine moiety, methylation, or hydrolysis of glutamate residues (Shane, 1995). Thus L-5M-THF would be expected to have superior biological activity compared with other folates and recently has been patented and marketed in a pure crystalline form for human use (Metafolin; http://www.metafolin.com).

These experiments were undertaken to (1) determine whether L-5M-THF is superior to L-5F-THF (levoleucovorin) in preventing the occurrence of gross congenital malformations in Folbp1^-/- mice at various dosages; (2) examine the critical periods during gestation when folate supplementation is most effective for survival and proper development of the Folbp1-deficient embryos; and (3) examine adult Folbp1^-/- mice for the presence of growth disturbances and structural defects.

RESULTS

Continuous Folate Supplementation

In total, 421 Folbp1^-/- newborn pups from 77 litters, and 67 Folbp1 wild-type pups from 9 litters were collected (Table 1). Of the total 488 pups collected, 16 were disfigured by the dam, leaving 472 pups (96.7%) that were examined for external malformations, including defects of the neural tube, lip and palate, ventral body wall, limbs and digits, and eyes (Figs. 1–4). No internal organs such as the heart were examined for congenital malformations in this series of experiments. The proportion of Folbp1^-/- pups with any malformation, ranged from 19% to 72%. The specific malformations observed among the abnormal Folbp1^-/- pups and their frequencies in the various experimental groups are summarized in Table 2. No malformations were observed among the 65 Folbp1 wild-type control pups that were examined.

Table 1. Summary of Litters From Control and Folbp1^−/− Dams Supplemented With LOW (10.75 or 21.5 μmol folate/kg/day) and HIGH (43 μmol folate/kg/day) Doses of L-N5-Formyl-tetrahydrofolate (L-5F-THF) and L-N5-Methyl-tetrahydrofolate (L-5M-THF)a

Characteristics of litters and pups	Control dams	Folbp1^−/− dams
		L-5F-THF			L-5M-THF
		LOW dose		HIGH dose	LOW dose		HIGH dose
		10.75	21.5	43	10.75	21.5	43
a Control dams are wildtype mice (Folbp1^+/+). b Significantly lower than the mean litter size of the control dams: adjusted P = 0.0055. c Malformed fetuses are defined as those fetuses having at least one malformation.
Number of litters	9	14	13	10	15	10	15
Number of pups	67	69	77	37	103	46	89
Number of pups examined	65	69	76	36	101	46	79
Mean litter size (±SD)	7.44 ± 1.94	4.93 ± 2.62	5.92 ± 1.80	3.70 ± 2.63b	6.87 ± 2.59	4.60 ± 2.46	5.93 ± 2.46
Number of malformed pups (%)c	0	46 (66.7)	50 (65.8)	12 (33.3)	73 (72.3)	26 (56.5)	15 (19.0)
Mean number of malformations/Malformed pup (± SD)	0	2.17 ± 1.23	1.98 ± 1.00	1.75 ± 1.14	1.95 ± 0.86	1.88 ± 1.03	2.00 ± 0.76

Figure 1. Neural tube, abdominal wall, and eye defects seen in Folbp1^-/- newborn pups. A: Encephalocele and cleft lip. B: Craniorrhachischisis. C: Exencephaly.

Figure 2. Multiple defects seen in individual Folbp1^-/- newborn pups. A: Exencephaly and abdominal wall defect. B: Exencephaly and agnathia. C: Encephalocele, midline cleft lip, and cleft palate. D: Midline cleft lip, polydactyly, and open eyelids.

Figure 3. Ocular defects seen in Folbp1^-/- adult mice. A: Anophthalmia. B: Bilateral anophthalmia.

Figure 4. Craniofacial defects seen in Folbp1^-/- newborn pups. A: Unilateral cleft lip. B: Bilateral cleft lip. C: Midline cleft lip. D: Midline cleft lip and cleft palate. E: Bilateral cleft lip, cleft palate, and micrognathia. F: Midline cleft lip, cleft palate, and encephalocele. G: Control fetus. H: Micrognathia. I: Agnathia.

Table 2. Summary of Structural Malformations Detected in the Pups of Folbp1^−/− Dams Supplemented With LOW (10.75 or 21.5 μmol folate/kg/day) and HIGH (43 μmol folate/kg/day) Doses of L-N5-Formyl-tetrahydrofolate (L-5F-THF) and L-N5-Methyl-tetrahydrofolate (L-5M-THF)a

Structural defect	Folbp1^−/− dams
	L-5F-THF		L-5M-THF
	LOW dose (n = 145)b	HIGH dose (n = 36)	LOW dose (n = 147)	HIGH dose (n = 79)
a Neural tube defects include exencephaly, encephalocele, or craniorachischisis; craniofacial defects include unilateral cleft lip, bilateral cleft lip, midline cleft lip, agnathia, or micrognathia; abdominal wall defects include omphalocele or gastroschisis; and ocular defects include unilateral or bilateral anophthalmia or microphthalmia. b n = number of pups that were scored for all of the malformations in Table 2.
Neural tube defects	51 (35.2)	6 (16.7)	50 (34.0)	9 (11.4)
Exencephaly	43 (29.7)	5 (13.9)	39 (26.5)	4 (5.1)
Encephalocele	7 (4.8)	1 (2.8)	11 (7.5)	5 (6.3)
Craniorachischisis	1 (0.7)	0 (0)	0 (0)	0 (0)
Craniofacial defects	67 (46.2)	7 (19.4)	74 (50.3)	12 (15.2)
Cleft lip - unilateral or bilateral	52 (35.9)	4 (11.1)	64 (43.5)	4 (5.1)
Cleft lip - unilateral	33 (22.8)	2 (5.6)	36 (24.5)	4 (5.1)
Cleft lip - bilateral	19 (13.1)	2 (5.6)	28 (19.1)	0 (0)
Cleft lip - midline	10 (6.9)	2 (5.6)	8 (5.4)	7 (8.9)
Agnathia or micrognathia	11 (7.6)	1 (2.8)	5 (3.4)	2 (2.5)
Abdominal wall defects	31 (21.4)	2 (5.6)	23 (15.7)	2 (2.5)
Ocular defects	21 (14.5)	1 (2.8)	36 (24.5)	6 (7.6)
Club foot	11 (7.6)	3 (8.3)	4 (2.7)	0 (0)
Polydactyly	12 (8.3)	2 (5.6)	1 (0.7)	0 (0)

Litter Size

The mean litter size (including disfigured pups) for each treatment/dose group was smaller than the mean litter size for the wild-type controls (Table 1). However, only the mean litter size for the L-5F-THF group, supplemented at 43 μmol/kg per day, was significantly lower than the mean litter size for controls (Bonferroni adjusted P = 0.0055). The mean litter sizes for L-5M-THF and L-5F-THF treatment groups were not significantly different at equimolar doses, and the mean litter sizes for the LOW vs. HIGH dose groups did not differ significantly within treatment group.

Malformation Frequencies in Folbp1-Deficient Mice

The mean number of malformations per abnormal Folbp1^-/- pup ranged from 1.8 to 2.2 (Table 1). There were no significant differences in the number of malformations per abnormal pup either between folate forms at the same dose or between doses of the same folate form (P > 0.05).

Among the abnormal pups, there was evidence that the various malformations did not occur independently (Table 3). NTDs were observed to be strongly associated (P ≤ 0.0006) with both craniofacial and abdominal wall defects (AWDs) at LOW doses of both folate forms. Except for the association between NTDs and clefts in embryos supplemented with L-5F-THF, these associations remained significant in the HIGH treatment groups. Craniofacial anomalies were also significantly associated with ocular anomalies in the L-5M-THF group, and this association was present both at LOW and HIGH dosages. In addition, craniofacial anomalies were significantly associated with AWDs, but only in the LOW dosage levels of both folate forms.

Table 3. Statistical Significance (P values) for the Pair-wise Associations Between Malformation Categories in the Affected Pups of Folbp1^−/− Dams Supplemented With LOW (10.75 or 21.5 μmol folate/kg/day) and HIGH (43 μmol folate/kg/day) Doses of L-N5-Formyl- tetrahydrofolate (L-5F-THF) and L-N5-Methyl-tetrahydrofolate (L-5M-THF)a

Folate form	Folate dose	Defect #2	Defect #1
			Neural tube defects	Facial clefts	Abdominal wall defects
a Facial clefts include unilateral cleft lip, bilateral cleft lip, or midline cleft lip; neural tube defects include exencephaly, encephaocele, or craniorachischisis; abdominal wall defects include omphalocele or gastroschisis; and ocular defects include unilateral or bilateral anophthalmia or microphthalmia.
L-5F-THF	LOW	Facial clefts	<0.0001	-	-
		Abdominal wall defects	0.0001	0.02	-
		Ocular defects	0.09	0.42	0.05
L-5M-THF	LOW	Facial clefts	0.0006	-	-
		Abdominal wall defects	<0.0001	0.01	-
		Ocular defects	0.92	0.01	0.47
L-5F-THF	HIGH	Facial clefts	0.07	-	-
		Abdominal wall defects	0.02	0.36	-
		Ocular defects	1.00	1.00	1.00
L-5M-THF	HIGH	Facial clefts	<0.0001	-	-
		Abdominal wall defects	0.01	0.28	-
		Ocular defects	0.14	<0.0001	1.00

Analysis of the distribution of malformations (using the broadest definition of malformation) indicated that malformations were not randomly distributed across pups, but rather tended to cluster within litters (χ²₁ = 43.0; P < 0.001). Hence, all subsequent analyses account for the clustered nature of these data.

Generalized estimating equations (GEE) were used to assess the association between folate form and malformation frequency, while accounting for the clustered nature of the data and controlling for the effects of dose. Folate form was not significantly associated with malformation frequency at either HIGH or LOW doses (P > 0.05; Table 4). GEE were also used to assess the association between dose (LOW vs. HIGH) and malformation frequency, while accounting for the clustered nature of the data and controlling for the effects of folate form. Dose was significantly and inversely related to the frequency of each malformation category in both folate forms (P < 0.05; Table 4).

Table 4. Statistical Significance (P values) for the Likelihood Ratio Tests Evaluating the Relationship Between Treatment Group (L- 5M-THF versus L-5F-THF) and Folate Dose (LOW versus HIGH) and the Risk of Structural Malformations in the Fetuses of Folbp1^−/− Damsa

Malformation	L-5M-THF versus L-5F-THF		LOW versus HIGH dose
	HIGH dose	LOW dose	L-5M-THF	L-5F-THF
a The comparison group for all of these analyses was restricted to those pups in which no malformations were observed. L-5F-THF, L-N5-formyl-tetrahydrofolate; L-5M-THF, L-N5-methyl-tetrahydrofolate (L-5M-THF). LOW, 10.75 or 21.5 μmol folate/kg/day; HIGH, 43 μmol folate/kg/day group. Neural tube defects include exencephaly, encephaocele, or craniorachischisis; craniofacial defects include unilateral cleft lip, bilateral cleft lip, midline cleft lip, agnathia, or micrognathia; abdominal wall defects include omphalocele or gastroschisis, and ocular defects include unilateral or bilateral anophthalmia or microphthalmia.
Any malformation	0.59	0.96	0.002	0.02
Neural tube defects	0.91	0.73	0.004	0.026
Exencephaly	0.79	0.89	0.015	0.04
Craniofacial defects	0.41	0.98	0.004	0.03
Cleft lip - unilateral or bilateral	0.76	0.89	0.012	0.04
Abdominal wall defects	0.79	0.56	0.002	0.02
Ocular defects	0.35	0.07	0.006	0.03

Folate Supplementation During Different Periconceptional Windows

Developmental outcome of Folbp1^+/- dams that were mated to Folbp1^-/- males and supplemented with L-5M-THF 20 mg/kg per day during various times of gestation is presented in Table 5. Compared with the nonsupplemented dams, a lower proportion of resorbed embryos were observed in dams treated daily before and throughout gestation (−14-E18) and dams treated on embryonic day (E) 1→9 (P < 0.05). Compared with the −14-E18 treated group, all other groups had higher rates of resorptions; however, this difference was only significant for dams treated on E4–6.

Table 5. Summary of Embryonic Survival and Malformations Among the Offspring of Folbp1^+/− Dams Supplemented With 20 mg/kg/day of L-5M-THF During Various Periconceptional Periodsa

Periconceptional period of supplementation	Number of litters	Number of implants	Mean number of implants/litter (SD)	Number (%) of embryonic resorptions	Number of pups examined	Number of Folbp1^−/− pups (%)	Number of malformed Folbp1^−/− pups (%)
a Percent Folbp1^−/− pups calculated from the number of pups examined. Percent malformed Folbp1^−/− pups calculated from the number of Folbp1^−/− pups. b The proportion of embryos that were resorbed was significantly greater for dams treated on gestational days 4 → 6 than for dams treated 2 weeks preconception through gestational day 19 (P < 0.05). c The proportion of embryos that were resorbed was significantly different from nonsupplemented dams (P < 0.05).
Not supplemented	11	97	8.82 ± 1.54	48 (49.5)	49	0	-
E1-3	6	41	6.83 ± 2.14	29 (70.7)bc	12	0	-
E4-6	7	58	8.28 ± 3.73	41 (70.7)bc	17	0	-
E7-9	10	80	8.0 ± 3.09	32 (40.0)	48	14 (29.2)	2 (14.3)
E1-9	8	55	6.88 ± 3.23	15 (27.3)c	40	18 (45.0)	2 (11.1)
E1-18	5	40	8.0 ± 3.16	14 (35.0)	26	14 (53.8)	4 (28.6)
-14-E18	7	62	8.86 ± 1.07	13 (21.0)c	49	21 (42.9)	6 (28.6)

Among surviving liveborn pups, those treated on E1–3 and E4–6 appeared to fare no better than the nonsupplemented group with respect to survival of null pups, i.e., all Folbp1^-/- embryos died and were resorbed during early gestation. On the other hand, liveborn Folbp1^-/- fetuses were present in dams of all experimental groups that were supplemented on E7–E9 as a minimum, i.e., E7–9, E1–9, E1–18 and −14→E18. In all the aforementioned experimental groups where Folbp1^-/- fetuses were born, a certain proportion (11–29%) developed abnormally and presented with neural tube, craniofacial, and ocular defects. However, the relatively small number of embryos for which these data are available precludes meaningful statistical analyses of these relationships.

Postnatal Growth of Folbp1^-/- Mice

The average weights of 16 Folbp1^+/- females, seven Folbp1^-/- females, 12 Folbp1^+/- males, and three Folbp1^-/- males was measured weekly for 6 weeks (data not shown). Heterozygous mice had consistently higher weights compared with the nullizygous mice across most time points, but these differences were not statistically significant (P > 0.05), most likely due to the relatively small number of nullizygous mice. For example, at 6 weeks of age, Folbp1^+/- females weighed 17.5 ± 1.8 g, whereas Folbp1^-/- females weighed 15.4 ± 2.6 g (P = 0.0554).

Defects in Adult Folbp1^-/- Mice

Three types of defects were observed among adult Folbp1^-/- mice that were born to Folbp1^+/- dams supplemented with various folate regimens and maintained on a standard rodent diet. These defects included brachycephaly (Fig. 5A,B), ocular defects, specifically unilateral and bilateral anophthalmia and microphthalmia (Figs. 3B, 5B), and teeth malocclusion (not presented). No defects were observed in Folbp1^+/- and Folbp1^+/+ littermates.

Figure 5. Developmental defects in Folbp1^-/- adult mice. A: Brachycephaly. B: Adult Folbp1^-/- male (right) with a Folbp1^+/- male littermate. Note that the Folbp1 null mouse is smaller than the heterozygote and has brachycephaly and microphthalmia.

Plasma Folate Levels

Total plasma folate levels of Folbp1^-/- dams supplemented with L-5M-THF and L-5F-THF, as well as of the wild-type controls were determined for the experimental animals. Nonsupplemented Folbp1^-/- mice had folate levels approximately one third as high as those observed in the wild-type controls (12–15 and 40 ng/ml, respectively; P < 0.0001). With all three dosages that were evaluated, plasma folate levels of mice supplemented with L-5M-THF were not significantly different than those supplemented with L-5F-THF (P > 0.05). Of interest, with both folate forms, plasma levels obtained during supplementation of all dosages were not significantly different than levels measured before supplementation (P > 0.05).

DISCUSSION

Folbp1-deficient mice supplemented with L-5F-THF previously have been shown to have abnormal embryonic development that resulted in exencephaly (Piedrahita et al., 1999; Finnell et al., 2002). Recently, these mutant mice were also shown to have abnormal development of craniofacial structures that resulted in midfacial clefts of the lip and palate (Tang and Finnell, 2003). In the current studies, we extend the observation of the diversity of congenital malformations that occur due to complete loss of Folbp1-mediated intracellular folate transport.

Among Folbp1^-/- mice that were supplemented with LOW folate dosages, approximately half had NTDs, predominantly exencephaly. Several fetuses had encephaloceles, while only a single fetus had craniorachischisis, a complete failure of neural fold elevation and fusion (Fig. 1A–C). Overall, craniofacial malformations were the most abundant defect category seen among Folbp1^-/- fetuses, occurring in more than half of the pups in the LOW supplemented groups. The most common craniofacial defects were lateral clefts of the upper lip, which are due to a failure of fusion of the medial nasal and maxillary processes. Additional malformations of the mandible, which have not been previously described in Folbp1-deficient mice, included micrognathia and agnathia (Fig. 4H and 4I, respectively) were observed. In addition, although pups in this study were not systematically examined for the presence of cleft palate, such defects were observed in a significant proportion of Folbp1^-/- pups with cleft lips (Fig. 4D–F). Similarly, the external appearance of several fetuses suggested the existence of holoprosencephaly, a malformation that has been associated with midline clefts (Taub et al., 2003; Konig et al., 2003; Fig. 4C–F). Additional malformations that have not been reported previously in Folbp1^-/- mice involved other external structures: a relatively high percentage of Folbp1^-/- mice on the LOW folate dosages had abdominal wall (16–21%) or ocular defects (14–24%). Whereas newborn pups with gastroschisis or omphalocele (Fig. 2A,B) die immediately after birth, ocular defects such as anophthalmia and microphthalmia (Fig. 3A) are compatible with survival. These ocular defects are also observed in similar frequencies in adult Folbp1-deficient mice (Fig. 3B). Additional defects that were observed to a lesser extent include polydactyly (Fig. 2D) and club foot (Fig. 2A).

The types of malformations observed in this study suggest that cranial neural crest cells (NCC), which may be specifically vulnerable to inadequate folate supplies during embryogenesism, are involved. At LOW levels of supplementation, the incidence of defects is high and appears mostly in structures that involve significant contributions from cranial NCC populations (Le Douarin and Kalcheim, 1999). HIGH levels of supplementation reduce the frequency of malformations but do not eliminate them entirely at the dosages used in this study. Thus, it is hypothesized that NCC, which are highly proliferative during migration to their destination sites, have an elevated demand for folates. If these demands are not met, the cellular integrity of target structures is compromised, leading to their defective development. It is unknown whether folate supplementation at higher doses than those used in this study will completely eliminate defects in Folbp1-deficient mice. In humans, folate supplementation is not 100% effective, as infants continue to be born with nonfolate-responsive NTDs (Berry et al., 1999; Berry and Li, 2002; McDonald et al., 2003).

The neural crest cell involvement hypothesis is strengthened by the observation that Folbp1 nullizygotes are rescued by folate supplementation during E7–E9, a time in which NCC migrate from the dorsal aspects of the neural folds to their destination tissues. Supplementation at narrow gestational windows that do not include E7–E9, results in litters completely devoid of Folbp1^-/- mice. On the other hand, enhanced success of Folbp1^-/- rescue by folate acid is achieved when the supplementation is provided from conception to neurulation, as in group E1–9. We were unable to achieve greater protection against the development of congenital defects by initiating supplementation before conception and continuing beyond neurulation, as in the −14-E18 group.

Supplementing dams with the HIGH folate dosage provided significantly more protection against abnormal development in Folbp1^-/- mice, compared with LOW concentrations. This dose–response effect was observed for all individual and classes of malformations that were analyzed (Table 4). Our results are in agreement with human epidemiological data, which supports a dose–response effect of folate supplementation in preventing congenital malformations.

The Folbp1^-/- fetuses in this study were not examined for the presence of cardiac defects. However, these defects have been described recently in folate supplemented Folbp1 nullizygous fetuses (Tang et al., in press). One would expect such defects to occur in our mouse model, based upon the contribution of NCC to the conotruncal portion of the developing heart (Le Douarin and Kalcheim, 1999). More recent work by Boot and colleagues (2003, 2004) demonstrated that abnormalities in conotruncal heart development could be traced back to hyperhomocysteinemia-induced alterations in NCC differentiation. Specifically, explanted chick embryos cultured under folate deplete conditions and provided exogenous homocysteine had expanded domains of largely undifferentiated neural crest cells. The extent to which these crest cells differentiated was directly related to the concentration of homocysteine in the culture (Boot et al., 2003). Failure to properly differentiate into the smooth muscle cellular components of the conotruncus could lead to the observed malformations in the chick embryos (Boot et al., 2004) and are typical of the Folbp1 heart defects reported elsewhere (Tang et al., in press).

Despite the theoretical advantages of L-5M-THF as a folate supplement (http://www.metafolin.com), it failed to provide additional protection compared with L-5F-THF in this model system. Structurally, these two naturally occurring folates differ only in the oxidation state of the transferable one-carbon group. Both compounds are biologically active, have comparable affinity to the reduced folate carrier and, thus, are absorbed to a similar extent (Assaraf and Goldman, 1997; Said et al., 2000; Matherly and Goldman, 2003). Biochemically, L-5F-THF does not participate directly in homocysteine remethylation, the predominant biochemical pathway that generates SAM, the universal methyl donor. L-5F-THF serves as the precursor of 5,10-methylene-THF, which is converted by the enzyme 5,10-methylenetetrahydrofolate reductase to 5-methyl-tetrahydrofolate, the form of folate that has the greatest binding affinity to the folate binding protein 1. Supplementation with these two folate forms generated comparable total folate plasma levels in Folbp1^-/- mice. Collectively, it can be concluded that both folate forms contribute similarly to the total folate pool in the dam and that available to the developing embryo, which seems to be the important factor in prevention of malformations in Folbp1^-/- mice, rather than the specific folate form that is provided.

It is important to note that the theoretical advantages of L-5M-THF should be evident when they are compared with folic acid, the nonbioactive folate form (Shane, 1995), which was not tested in this study. Folic acid is the form used in most multivitamin tablets due to its chemical stability and simple synthetic process compared with the laborious selective crystallization process required for L-5M-THF synthesis. Thus, folic acid requires several biochemical steps for bioactivation, including the reduction of the pteridine moiety to the THF form, and the addition of a methyl group. In a recent study, Venn and colleagues (2003) compared the ability of L-5M-THF and folic acid to elevate folate levels and lower plasma homocysteine levels in healthy human volunteers. Folate levels in both plasma and red blood cells were elevated to similar extents under the two regimens, and only a marginal statistical improvement in lowering homocysteine concentrations were observed with L-5M-THF. Similar results with L-5M-THF indicated that it was not superior to folic acid in elevating folate levels among women of childbearing age (Venn et al., 2002).

In the present study, we confirmed our previous findings (Spiegelstein et al., 2003a) where an approximate threefold difference in plasma folate levels was detected between Folbp1^-/- and wild-type Folbp1^+/+ mice. However, all folate supplementation regimens failed to significantly elevate steady-state plasma levels in the mutant Folbp1^-/- mice. This finding is intriguing, given that a highly significant dose response was observed with regard to the prevention of birth defects in Folbp1^-/- mice with increasing levels of supplementation.

In contrast to Folbp1^-/- mice, the plasma folate levels of Folbp2^-/- females supplemented with L-5M-THF (20 mg/kg per day) are elevated approximately threefold compared with their baseline pretreatment levels (Spiegelstein et al., 2003b). This discrepancy in response to folate supplementation between the two mutant mice strains likely reflects the difference in tissue expression and the physiological roles of these two folate receptors. Whereas the Folbp2 gene is expressed at low to moderate levels in many tissues and has no vital physiological and developmental role (Ross et al., 1999; Piedrahita et al., 1999), Folbp1 is highly expressed only in select tissues. Most notably, Folbp1 is highly expressed in the brush border membranes of the kidney proximal tubules, where it is hypothesized to enable folate reabsorption, serving as a folate sparing mechanism for maintaining folate homeostasis (Selhub and Franklin, 1984; Elwood, 1989; Birn et al., 1993). Thus, Folbp2^-/- mice that have no apparent alteration of folate homeostasis and folate reabsorption are able to retain a portion of the folate administered. On the other hand, Folbp1-deficient mice are likely to be unable to reabsorb folates from their urine; thus, their plasma folate levels do not elevate significantly when provided with folate supplementation, even at the highest dosage.

We hypothesize that, when supplemented with pharmacological doses of folate, Folbp1^-/- embryos are able to harvest and use sufficient folate cofactors for their survival and development in a dose-dependent manner during the peak plasma folate levels achieved after supplemental folate administration. Although folate supplementation is highly effective in preventing many congenital anomalies in the Folbp1-deficient mouse model, maternal plasma folate cannot serve as an indicator of the folate status of the embryo, its environment, or ultimately, the pregnancy outcome. Further support for this hypothesis requires that folate levels be measured in embryonic tissues during the critical time of development. Such experiments are currently in progress in our laboratory.

EXPERIMENTAL PROCEDURES

Folate Solutions

For the continuous supplementation study, the highest concentrations (4.3 μmol/ml) of L-5M-THF and L-5F-THF were prepared by dissolving 500 mg of L-N5-methyl-tetrahydrofolate calcium salt (Metafolin) and 512 mg of L-N5-formyl-tetrahydrofolate calcium salt (kindly provided by Merck Eprova AG, Schaffhausen, Switzerland), respectively, in 200 ml of 1% sodium ascorbate (Sigma, St. Louis, MO) aqueous solution. The intermediate concentration (2.15 μmol/ml) was prepared by mixing equal volumes of the 4.3 μmol/ml solution and 1% ascorbate. The low concentration solutions (1.075 μmol/ml) were prepared similarly.

For the intermittent supplementation study, as well as for supplementation of the breeding colony mice, a 2.0 mg/ml L-5M-THF solution was prepared by dissolving 400 mg of L-5M-THF calcium salt in 200 ml of 1% sodium ascorbate solution. The 5F-THF and L-5M-THF solutions were prepared similarly by using 500 mg of folinic acid calcium salt (Acros Organics, Fairlawn, NJ) and 250 mg of L-5M-THF (Merck Eprova AG), respectively. All solutions were filtered through a 0.2-μm Nalgene syringe filter (Nalge, Rochester, NY) and aliquoted into 1.5-ml vials. Each vial was briefly flushed with argon, sealed, and stored protected from light at 20°C until used.

Experimental Animals and Treatments

Continuous folate supplementation.

Folbp1-deficient dams were randomly divided into one of six experimental groups: L-5M-THF or L-5F-THF at 10.75, 21.5, or 43.0 μmol/kg per day. An additional group of wild-type Folbp1 (Folbp1^+/+) dams that did not receive any treatment and were mated to wild-type males was used as a control group. Each group consisted of three to seven females receiving a supplementation regimen that was initiated 2 weeks before the first attempted mating, and continued throughout the duration of the study. Supplementation was administered daily by oral gavage (10 μl/g body weight) using a mouse-feeding needle (Fine Science Tools, Foster City, CA). Folbp1-deficient dams were mated overnight to Folbp1^-/- males, and the pregnancy was determined by the presence of a vaginal plug observed the following morning. On E18, pregnant females were placed in a metabolic cage (Nalge Nunc International, Rochester, NY) equipped with a rat wire for collection of newborn pups. Liveborn pups were killed by hypothermia (4°C for several hours) and fixed in 4% formaldehyde solution (Fisher Scientific, Fair Lawn, NJ). All pups were examined for the presence of gross external malformations such as neural tube, craniofacial, abdominal wall, ocular, and digital defects. Images of selected fetuses were taken by using a Leica MZ95 microscope (Heerbrug, Switzerland) and a Magnafire digital camera (Optronics, Goleta, CA). Mice were housed in clear polycarbonate microisolator cages, were allowed free access to water and food (Harlan Teklad, Madison, WI; Rodent Diet #8604; ∼2.7 mg folic acid/kg diet), and were maintained on a 12-hr light/dark cycle in the Vivarium at the Institute of Biosciences and Technology (Houston, TX). The study was approved by the institutional animal care and use committee of the Institute of Biosciences and Technology.

Folate supplementation during different periconceptional windows.

The Folbp1^+/- females were mated to Folbp1^-/- males and randomly assigned to one of the following experimental groups: supplementation of L-5M-THF 20 mg/kg per day on E1–3, E4–6; E7–9; E1–9; E1–18; and starting 2 weeks before and throughout gestation (−14→E18), or they received no supplementation at all. Pregnant dams were killed on E18; the uterine contents removed and examined for the presence of resorptions and gross external malformations. Fetuses were genotyped according to previously published methods (Piedrahita et al., 1999).

Postnatal growth of Folbp1^-/- mice.

Postnatal growth was assessed weekly starting at 7 days of age and continuing for 6 weeks by weighing Folbp1^+/- and Folbp1^-/- littermates that were born to Folbp1^+/- dams supplemented daily with L-5M-THF 20 mg/kg per day.

Defects in adult Folbp1^-/- mice.

Adult Folbp1^-/- mice from the breeding colony and the progeny of Folbp1^+/- dams that were supplemented with various regimens such as 5F-THF 25 mg/kg per day, L-5F-THF 12.5 mg/kg per day, and L-5M-THF 20 mg/kg per day were examined for the presence of external structural defects.

Plasma Folate Concentrations

Plasma folate concentrations were determined for mice used in the continuous folate supplementation study. Twelve untreated nulliparous Folbp1^-/- female mice, 2–3 months of age, were randomly divided into two groups containing six animals each. After collection of the baseline (pretreatment) blood sample, mice received daily oral supplementation with either L-5M-THF or L-5F-THF 10.75 μmol/kg per day. After 2 weeks, a blood sample was collected 24 hr after the last 10.75 μmol/kg per day dose, which was subsequently increased to 21.5 μmol/kg per day and administered for 2 weeks. Another blood sample was collected, and the dose was further increased to 43 μmol/kg per day for an additional 2 weeks, after which the final blood sample was obtained. All blood samples (∼250 μl) were used to determine the total plasma folate concentrations. The blood was obtained from the retro-orbital sinus using heparinized capillary tubes and centrifuged for 5 min at 5,000 × g, and the plasma was separated and stored at −80°C until analyzed. The Lactobacillus casei microbiological assay was used for the analysis of plasma folate levels (Spiegelstein et al., 2003a).

Statistical Methods

The t-test was used to assess pairwise differences in mean litter size between controls and treatment groups at a given dose and between the doses of any given folate form. The t-test was also used to assess pairwise differences in the mean number of malformations per abnormal pup between treatment groups at a given dose and between doses of a given treatment. Chi-squared analysis or Fisher's exact test were used to determine whether, within treatment group, these malformation categories occurred independently or whether certain types of malformations tended to co-occur within the same fetus.

Analyses of fetal malformations used a general definition of malformation, which included the presence of at least one of the structural defects listed in Table 2. When appropriate and numbers permitted, subgroups defined by the presence of a specific defect (e.g., exencephaly) or defect category (e.g., neural tube defects) were also evaluated.

The likelihood ratio test (LRT) was used to determine whether malformations (defined broadly to include any malformation or group of malformations) were randomly distributed across fetuses or clustered within litters. Two logistic regression models, one with and the other without a random effect for litter, were used to obtain the LRT statistic. The distribution of this statistic approximately follows a chi-squared distribution, with degrees of freedom equal to the difference in the number of parameters estimated by the two models.

The effects of treatment group and dose on the risk of malformation were assessed by using GEE, to account for the correlations between embryos within litters. These analyses used a logit link and binomial error structure, as in logistic regression. The effects of treatment and dose were assessed using the LRT comparing the model that included both treatment and dose, with the model that included only treatment or dose. In these analyses, fetuses with no abnormalities were considered as the comparison group.

For all analyses related to dose, the litters/fetuses treated at 10.75 and 21.5 μmol/kg per day were combined and are referred to as the LOW dose. This pooling strategy was motivated by the small cell sizes available for some of the analyses and was guided by the similarities observed upon visual inspection of the data in Table 1. Therefore, the LOW dose group is compared with litters/fetuses treated at 43 μmol/kg per day, which is referred to as the HIGH dose.

Differences in the frequency of embryonic resorptions for dams supplemented with folate during different periconceptional windows were assessed by chi-squared analyses. Differences in postnatal body weights between Folbp1^-/- and Folbp1^+/- pups, and in plasma folate levels between the supplementation regimens were assessed by using the Mann–Whitney test. All statistical analyses were performed using SAS version 8.2. When appropriate, the Bonferroni approach was used to adjust for multiple comparisons. Test statistics with P values < 0.05 (adjusted for multiple comparisons when appropriate) were considered to be significant.

Acknowledgements

The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. The authors thank Ms. Melissa Scott and Ms. Marlene Tsie from the Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A&M University System Health Science Center for their technical assistance in performing these studies, including the maintenance of the mouse colony. The generous contribution of L-N5-methyl-tetrahydrofolate (Metafolin) and L-N5-formyl-tetrahydrofolate from Merck Eprova AG (Schaffhausen, Switzerland) is greatly appreciated. R.H.F. was funded by grants from the March of Dimes, and L.E.M. was funded by the NIH.