Metabolite profiling of whole murine embryos reveals metabolic perturbations associated with maternal valproate-induced neural tube closure defects

Abstract

Background: Valproic acid (VPA) is prescribed therapeutically for multiple conditions, including epilepsy. When taken during pregnancy, VPA is teratogenic, increasing the risk of several birth and developmental defects including neural tube defects (NTDs). The mechanism by which VPA causes NTDs remains controversial and how VPA interacts with folic acid (FA), a vitamin commonly recommended for the prevention of NTDs, remains uncertain. We sought to address both questions by applying untargeted metabolite profiling analysis to neural tube closure (NTC) stage mouse embryos.

Methods: Pregnant SWV dams on either a 2 ppm or 10 ppm FA supplemented diet were injected with a single dose of VPA on gestational day E8.5. On day E9.5, the mouse embryos were collected and evaluated for NTC status. Liquid chromatography coupled to mass spectrometry metabolomics analysis was performed to compare metabolite profiles of NTD-affected VPA-exposed whole mouse embryos with profiles from embryos that underwent normal NTC from control dams.

Results: NTDs were observed in all embryos from VPA-treated dams and penetrance was not diminished by dietary FA supplementation. The most profound metabolic perturbations were found in the 10ppm FA VPA-exposed mouse embryos, compared with the other three treatment groups. Affected metabolites included amino acids, nucleobases and related phosphorylated nucleotides, lipids, and carnitines.

Conclusion: Maternal VPA treatment markedly perturbed purine and pyrimidine metabolism in E9.5 embryos. In combination with a high FA diet, VPA treatment resulted in gross metabolic changes, likely caused by a multiplicity of mechanisms, including an apparent disruption of mitochondrial beta-oxidation. Birth Defects Research 109:106-119, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: folic acid; metabolism; metabolomics; neural tube defects; one-carbon metabolism; teratogen; valproic acid.

Figure 1

Compound mass vs. retention time plot for 200 compounds with unique masses and retention times, identified by positive- and negative-ion mode LC/MS. These compounds are annotated by color based on their major metabolic classifications.

Figure 2

Boxplots depicting the somite count of murine embryos in each of the four treatment groups included in the LC/MS metabolomics analysis. All pairwise comparisons are statistically significant with the exception of 2ppm FA Control and 2ppm FA VPA (n = 18 for each group). Exact differences in somite count and p-values can be found in Supplementary Table 1.

Figure 3

Principal component (PC1 and PC2) regression onto somite counts for control and VPA-exposed mouse embryos from dams receiving a diet containing either 2ppm or 10ppm FA. (A) PC1 based on negative mode data (59.9% of all variance explained), no significant regression or meaningful R² for any group. (B) PC2 based on negative mode data (7.6% of all variance explained), 2ppm FA – CNTRL R² = 0.23, no significant regression for the 10ppm FA – CNTRL group, 2ppm FA – VPA R² = 0.24, 10ppm FA – VPA R² = 0.22. (C) PC1 based on positive mode data (49.5% of all variance explained), no significant regression or meaningful R² for any group. (D) PC2 based on positive mode data (13.4% of all variance explained), no significant regression or meaningful R² for any group. (n = 18 embryos for each group.)

Figure 4

Untargeted hierarchical clustering plot demonstrating alterations in the relative abundances of compounds in embryos from VPA-treated vs. control dams receiving a diet containing10ppm FA. Depicted results were represented as a heat map based on compounds identified with negative-ion mode LC/MS analysis. In this data representation, metabolites are clustered in the rows and 10ppm FA control and VPA-exposed embryos are clustered in the columns. Pearson correlation and complete linkage was used to cluster both columns and rows. Compounds in the rows are also marked by major compound classifications. (n = 18 embryos for each group.)

Figure 5

Boxplots describing the relative abundance changes in compounds related to purine and pyrimidine metabolism in embryos from VPA-treated vs. control dams supplemented with 10ppm FA. (A) Aspartic acid: p-val (Tukey HSD) = 7.46 ×10⁻⁵, Neg mode PC1/PC2 = −1.85 (somite-linked). (B) Glutamine: p-val (Tukey HSD) = 0.0016, Neg mode PC1/PC2 = −27.73. (C) Carbamoyl aspartic acid: p-val (Tukey HSD) = 0.0083, Neg mode PC1/PC2 = −2.88. (D) UMP: p-val (Tukey HSD) = 5.01 ×10⁻⁴, Neg mode PC1/PC2 = −2.60. (E) CMP: p-val (Tukey HSD) = 0.0014, Neg mode PC1/PC2 = −3.45. (F) Thymine: p-val (t-test) = 6.87 ×10⁻⁴, Pos mode data. (G) AMP: p-val (Tukey HSD) = 0.0014, Neg mode PC1/PC2 = −3.72. (H) GMP: p-val (Tukey HSD) = 0.0024, Neg mode PC1/PC2 = −3.87. (I) Adenine: p-val (t-test) = 0.0013, Pos mode data. *Indicates a somite-linked compound. (n = 18 embryos for each group).

Figure 6

Boxplots describing the relative abundance changes in compounds related to lipid and carnitine metabolism in embryos from VPA-treated vs. control dams supplemented with 10ppm FA. (A) Palmitic acid: p-val (Tukey HSD) = app. 0, Neg mode PC1/PC2 = −1.01 (somite-linked). (B) Carnitine: p-val (t-test) = 0.0018, Pos mode data. (C) Deoxycarnitine: p-val (t-test) = 0.0013, Pos mode data. *Indicates a somite-linked compound. (n = 18 embryos for each group).