Marginal biotin deficiency is teratogenic in ICR mice

Abstract

The incidence of marginal biotin deficiency in normal human gestation is approximately one in three. In ICR mice, maternal biotin deficiency results in cleft palate, micrognathia, microglossia and limb hypoplasia. However, the relationships among the severity of maternal biotin deficiency, fetal biotin status and malformations have not been reported. This study utilized validated indices of biotin status to investigate the relationships among maternal biotin status, fetal biotin status and the rate of fetal malformations in ICR mice. Biotin status was controlled by feeding diets with varying egg white concentration. In dams and fetuses, biotin status was assessed by hepatic biotin content and hepatic activity of the biotin-dependent enzyme propionyl-CoA carboxylase; in dams, status was also assessed by urinary excretion of biotin and 3-hydroxyisovaleric acid. Malformations were assessed morphologically. Biotin was measured by HPLC/avidin-binding assay. Propionyl-CoA carboxylase (PCC) activity was determined by H(14)CO(3) incorporation. 3-Hydroxyisovaleric acid concentration was determined by GC/MS. Although no overt signs of deficiency appeared, metabolic disturbances caused by biotin deficiency were detectable in dams and fetuses. These disturbances increased with increasing egg white. Fetal biotin status correlated significantly with maternal biotin status (fetal vs. dam hepatic biotin, r = 0.671; fetal vs. dam PCC activity, r = 0.70). The incidences of malformations were strikingly dependent on egg white concentration. We conclude that in ICR mice, marginal maternal biotin deficiency causes fetal biotin deficiency. We speculate that the fetal malformations are primarily the consequence of fetal biotin deficiency. Because murine malformations appeared at degrees of biotin deficiency that are similar to those in human gestation, we speculate that some human fetal malformations may be caused by biotin deficiency.

FIGURE 1

Effect of duration of gestation and increasing concentrations of egg white in the diet on urinary excretion of biotin in pregnant mice. Biotin excretion decreased significantly with duration of gestation (P < 0.001 by repeated-measures ANOVA), but diet had no effect. At baseline, biotin excretion did not differ among the diet groups. By gestational day (GD) 7, biotin excretion was lower (P < 0.05 by Fisher's post-hoc test) for all diet groups consuming egg white. By GD 17, biotin excretion was lower (P < 0.003) for all diet groups, including the control groups. Values are means ± sem, n ≥ 4.

FIGURE 2

Effect of duration of gestation and increasing concentrations of egg white in the diet on urinary excretion of 3-hydroxyisovaleric acid (3HIA) in pregnant mice. Symbols denote values from at least four animals. At gestational day (GD) 0, 3HIA excretion did not differ among the diet groups. Values are means ± sem, n ≥ 4. *Different from the 0% egg white diet group, P < 0.005.

FIGURE 3

Correlation between fetal biotin status and dam biotin status in mice dams and pups when the dams were fed increasing concentrations of egg white in the diet. (A) The hepatic concentration of individual litter pools increased as the hepatic biotin of the corresponding dam increased (r = 0.67, P < 0.0001). (B) Fetal propionyl-CoA carboxylase (PCC) activity increased as the hepatic PCC of the corresponding dam increased (r = 0.70, P < 0.0001).

FIGURE 4

The incidence of cranial malformations increased with increasing concentrations of egg white in the diet. Fetuses whose dams were fed the 25% egg white diet had 100% incidence of clefting. The 50% effective dose (ED₅₀) of clefting was 3% egg white. The ED₅₀ for micrognathia and microglossia were at ∼4% egg white. For fetuses, the mean n for groups was 6; range 4–8.

FIGURE 5

Skeletal malformations in fetuses whose dams were fed increasing concentrations of egg white in the diet. Fetuses whose dams were fed the 25% egg white diet exhibited striking shortening of the long bones of the limbs; control fetuses exhibited normal skeletal anatomy.

FIGURE 6

The incidence of limb hypoplasia in fetuses whose dams were fed increasing concentrations of egg white in the diet. The incidence was 100% in fetuses whose dams were fed the 25% egg white diet; incidence rates showed a dose-dependent increase at egg white concentrations >3%. The 50% effective dose (ED₅₀) for limb hypoplasia was at ∼4% egg white. The n for each diet group was as in Figure 4.