Quantification of valproic acid and its metabolite 2-propyl-4-pentenoic acid in human plasma using HPLC-MS/MS

A specific and sensitive HPLC-MS/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid in human plasma has been developed, using VPA-d15 as the internal standard. The method was based on pre-column derivatization using 4-dimethylaminobenzylamine dihydrochloride. The derivatives were separated with a gradient elution and quantified by positive electrospray ionization with multiple reaction monitoring. The assay provides routine quantification limits of 200 ng/mL for VPA and 20 ng/mL for 4-ene VPA with within- and between-day coefficients of variation of <10%. This method has been applied to the analysis of plasma samples obtained from patients treated with this drug.     

Inhibition of medium-chain fatty acid beta-oxidation in vitro by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid

Valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid, were found to inhibit strongly the metabolism of decanoic acid in homogenates of rat liver. Reductions in decanoate consumption in response to inhibitors were paralleled by decreases in the formation of octanoic and hexanoic acids, two products of decanoate beta-oxidation. In contrast, 4-pentenoic acid, an established inhibitor of long-chain fatty acid beta-oxidation, had little effect on the metabolism of decanoate. It is concluded that the title compounds are potent, broad-spectrum inhibitors of fatty acid beta-oxidation, a property which may be of key toxicological importance in the pathology of valproate-induced liver injury.     

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.     

Valproic acid-induced neural tube defects: reduction by folinic acid in the mouse

Neural tube defects were induced dose-dependently by single injections of the anticonvulsant drug valproic acid (VPA) as sodium salt in mice on gestational day 8. Folinic acid (5-CHO-THF) coadministration by i.p. injection or by a constant rate infusion via osmotic minipumps, implanted s.c., significantly reduced the exencephaly rates using a randomized double-blind experimental procedure. 5-CHO-THF supplementation cut the exencephaly rates into half even at high maternal plasma levels of VPA (p less than 0.005, chi 2-test); resorption rates were not affected. The VPA plasma kinetics were not changed by any of the application regimens of 5-CHO-THF. The investigation of the folate metabolite pattern (determined by HPLC) showed that 5-CHO-THF and 5-methyl-tetrahydrofolic acid (5-CH3-THF) were the main metabolites in untreated mice. After supplementation with 5-CHO-THF, only the concentrations of this folate vitamer were increased in the plasma from 0.3 microgram/ml (normal) to 0.6 or 1.9 micrograms/ml (after injection of 3 x 1 mg/kg or 3 X 4 mg/kg) and to 4.2 micrograms/ml (after infusion via osmotic minipumps). Our results indicate that VPA-induced exencephaly in mice combined with the investigation of the plasma levels of VPA and the different folate metabolites could be an appropriate animal model to study protective effects of folates on the occurrence of neural tube defects.     

Early morphological abnormalities in splotch mouse embryos and predisposition to gene- and retinoic acid-induced neural tube defects

Genetic and environmental factors contribute to an individual's neural tube defect liability. In the mouse, the gene mutation Splotch (Sp) causes a pigmentation defect in heterozygotes while homozygotes have spina bifida +/- exencephaly. Splotch homozygotes, heterozygotes, and wild-type embryos were examined for somite number, anterior neuropore closure, and posterior neuropore length. The aim was to distinguish potentially affected homozygotes early in pathogenesis and find a morphological basis for increased teratogen susceptibility in heterozygotes. Posterior neuropore closure as well as anterior neuropore closure was significantly delayed in potentially affected Sp as compared to wild-type litter embryos exceeding the incidence found in day-10-diagnosed homozygotes. Part of this excess was attributed to a transient delay in heterozygotes which in turn might predispose to retinoic acid-induced neural tube defects. This idea was supported by an outcross of Sp heterozygote males by inbred SWV females and wild-type males by SWV where a significant increase in retinoic acid-induced neural tube defects was found in Sp carrier litters.     

Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice

BACKGROUND: Valproic acid (VPA) causes the failure of neural tube closure in newborn mice. However, the molecular mechanism of its teratogenesis is unknown. This study was conducted to investigate the genomewide effects of VPA disruption of normal neural tube development in mice. METHODS: Microarray analysis was performed on the head part of NMRI mouse embryos treated for 1 hr with VPA on gestational day (GD) 8. Subsequently, we attempted to isolate genes that changed in correlation with the teratogenic action of VPA by employing reduced teratogenic VPA analogs, valpromide (VPD) and valnoctamide (VCD), in a real-time PCR study. RESULTS: Microarray results demonstrated that during neurulation, many genes, some of whose functions are known and some unknown, were either increased or decreased after VPA injection. Some genes were affected by VPD or VCD in the same way as VPA, but others were not changed by the analogs. In this way, our system identified 11 increased and 20 decreased genes. Annotation analysis revealed that the increased genes included gadd45b, ier5, per1, phfl3, pou3f1, and sox4, and the decreased genes included ccne2, ccnl, gas5, egr2, sirt1, and zfp105. CONCLUSIONS: These findings demonstrate that expression changes in genes having roles in the cell cycle and apoptosis pathways of neural tube cells were strongly expected to relate to the teratogenic, but not antiepileptic, activity of VPA. Our approach has allowed the expansion of the catalog of molecules immediately affected by VPA in the developing neural tube.     

Metabolism of 4-pentenoic acid and inhibition of thiolase by metabolites of 4-pentenoic acid

The metabolism of 4-pentenoic acid, a hypoglycemic agent and inhibitor of fatty acid oxidation, has been studied in rat heart mitochondria. Confirmed was the conversion of 4-pentenoic acid to 2,4-pentadienoyl coenzyme A (CoA), which either is directly degraded via beta-oxidation or is first reduced in a NADPH-dependent reaction before it is further degraded by beta-oxidation. At pH 6.9, the NADPH-dependent reduction of 2,4-pentadienoyl-CoA proceeds 10 times faster than its degradation by beta-oxidation. At pH 7.8, this ratio is only 2 to 1. The direct beta-oxidation of 2,4-pentadienoyl-CoA leads to the formation of 3-keto-4-pentenoyl-CoA, which is highly reactive and spontaneously converts to another 3-ketoacyl-CoA derivative (compound X). 3-Keto-4-pentenoyl-CoA is a poor substrate of 3-ketoacyl-CoA thiolase (EC 2.3..1.16) whereas compound X is not measurably acted upon by this enzyme. The effects of several metabolites of 4-pentenoic acid on the activity of 3-ketoacyl-CoA thiolase were studied. 3,4-Pentadienoyl-CoA is a weak inhibitor of this enzyme that is protected against the inhibition by acetoacetyl-CoA. The most effective inhibitor of 3-ketoacyl-CoA thiolase was found to be 3-keto-4-pentenoyl-CoA, which inhibits the enzyme in both a reversible and irreversible manner. The reversible inhibition is possibly a consequence of the inhibitor being a poor substrate of 3-ketoacyl-CoA thiolase. It is concluded that 4-pentenoic acid is metabolized in mitochondria by two pathways. The minor yields 3-keto-4-pentenoyl-CoA, which acts both as a reversible and as a irreversible inhibitor of 3-ketoacyl-CoA thiolase and consequently of fatty acid oxidation.     

Hyperthermia and neural tube defects of the curly-tail mouse

The mutant gene curly-tail produces neural tube defects (NTD) in 60% of mice, predominantly at the caudal end of the neural tube. Only 1% of individuals have exencephaly. Pregnant curly-tail mice and C57BL mice which are not genetically pre-disposed to NTD, were subjected to various regimes of hyperthermia on day 8 or on day 9 or on day 10 of gestation. Normal body temperature was around 36.8 degrees C, but it was found to be extremely labile in response to heat exposure. It was significantly raised for 15 min of a 20-min exposure period, and, after removal from the heat, it dropped rapidly. In C57BL mice, heat treatment produced exencephaly alone and in only 3% of mice. In curly-tail mice, none of the heat-treatment regimes had any consistent effect on the incidence of posterior NTD but produced specifically exencephaly. The incidence was increased slightly at an environmental temperature of 37 degrees C when the body temperature was 4.01 degrees C; at an ambient temperature of 43 degrees C and a body temperature of 42 degrees C, the incidence of exencephaly was 20%. Exencephaly was produced by two periods of 20 min heat exposures 7 hr apart or a single exposure of 1 hr, especially on day 8 of gestation, but not by a single 20 min exposure. It is concluded that these experiments, performed in a mutant predisposed to lesions especially at the caudal end of the neural tube, demonstrate the specificity of hyperthermia for affecting closure of the cranial neural folds.     

Interaction between the splotch mutation and retinoic acid in mouse neural tube defects in vitro

The interaction between the splotch gene (Sp) and all-trans retinoic acid (RA) was investigated using cytogenetically marked Sp/+ and +/+ mouse embryos cultured in the presence of RA. Retinoic acid retarded the development of and had a teratogenic effect on mouse embryos in culture. In particular, RA had seemingly opposite effects on the posterior neural tube, inducing abnormally early fusion in some embryos and causing a dose-dependent delay in others. When the effects of RA on identified Sp/+ and +/+ embryos were compared, the only observed difference in their responses was in the degree of the delay in posterior neuropore (PNP) closure. At the end of the culture period, among the untreated control embryos, the Sp heterozygotes showed retardation of PNP closure compared to +/+ embryos. In addition, the RA treatment was found to have induced a greater delay in posterior neural tube closure in Sp/+ than in +/+ embryos. The basis for this difference in response to RA is presumed to be the retardation of PNP closure that is caused by the Sp gene in heterozygous form. The effects of the gene and the teratogen are additive and the gene carriers thus have greater mean PNP lengths at the end of culture. Since the length of the PNP is an indication of an embryo's likelihood of developing spina bifida, this provides an explanation for the observation that Sp/+ embryos are more sensitive to the spina bifida-causing effects of RA than are +/+ embryos.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

Failure of homocysteine to induce neural tube defects in a mouse model

BACKGROUND: Folate deficiencies have been associated with many adverse congenital abnormalities. It is not clear, however, whether these defects are due to a folate deficiency or to an increase in homocysteine. Homocysteine has been shown to be teratogenic in the chicken-embryo model and it has been suggested that homocysteine-induced defects are mediated by inhibiting the N-methyl-D-aspartate (NMDA) receptor on neural crest cells. The majority of the teratology studies have been carried out using the chicken embryo model. In an effort to develop a murine model of homocysteine-induced neural tube defects, several inbred mouse strains were treated with homocysteine or the NMDA inhibitor MK801 and the fetuses examined for any induced-NTD. METHODS: Several in-bred mouse strains were administered homocysteine once on gestational day (GD) E8.5 or once daily on GD 6.5-10.5. Additionally, because homocysteine was been reported to mediate its effects through the NMDA receptor, the effect of MK801, an antagonist of this receptor, was also investigated. RESULTS: Regardless of the mouse treatment time, homocysteine failed to induce neural tube defects in our in-bred mouse strains. Homocysteine also failed to increase the number of neural tube defects in the splotch strain, regardless of the genotype. CONCLUSIONS: Irrespective of the mouse strain or treatment, homocysteine failed to induce neural tube defects in our mouse models, which is in contrast to what has been reported in the chicken embryo models.     

Anencephaly: its spectrum and relationship to neural tube defects

Anencephaly patients are of renewed interest because they are regarded as a potential source of organ donation. While there has been a longstanding scientific curiosity on this subject, studies have frequently included such cases as part of the larger spectrum of neural tube defects (NTDs). This paper will discuss some unusual features of anencephaly. Following a review of classification and pathogenesis, associated malformations, growth parameters (organ size and anthropometric measurements), and associations with other entities are discussed. Finally, the relationship of anencephaly to NTDs is presented.     

Effect of hydroxyurea on neural tube defects in the curly-tail mouse

Around 60% of curly-tail mice spontaneously develop neural tube defects (NTD), that is, exencephaly, and/or spina bifida (open lesions), or a curly tail (closed lesion), due to an incompletely penetrant recessive gene. Various doses of hydroxyurea, a teratogen to the rodent central nervous system, were administered to curly-tail mice on either day 8 or day 9 of pregnancy in an attempt to increase the number of NTD in the embryos. No dose used on either day achieved this. However, on day 8, the proportion of affected mice with open lesions increased from around 30% in control mice to 78% with 400 mg/kg hydroxyurea, and this was accounted for specifically by the production of exencephaly. When administered on day 9, 400, 500, and 600 mg/kg hydroxyurea (the latter two doses being lethal to embryos on day 8) actually reduced the incidence of total NTD, to around 30%. Among these affected mice, even though reduced in number, there was still a slight tendency for an increase in the number of exencephalics. Hydroxyurea also produced gastroschisis in a small percentage of embryos; the greatest incidence was 36% with 400 mg/kg on day 8.     

Expression of the sonic hedgehog gene in human embryos with neural tube defects

BACKGROUND: To estimate the rate of malformations observed during early human development, a series of 38,913 first-trimester abortions were studied. Neural tube defects (NTD) were found in 57 cases. METHODS: A histological study of serial sections performed in 25 embryos revealed a spectrum of axial structure abnormalities. Expression of the SHH gene was studied by in situ hybridization in one case of CRS and in two cases of SB. RESULTS: A cervical notochord duplication was always found in craniorachischisis (CRS, n = 8), but not in spina bifida (SB, n = 10) or diplomyelia (split cord malformation, n = 3). In the embryo with CRS, expression of SHH was found in both domains, corresponding to the duplicated part of the notochord, whereas a single signal was observed in the nonduplicated part. This expression was associated at the cervical level of the open neural tube with a broad SHH expression domain and with two or even three domains in its lumbar region, suggesting multiple functional floor plates. Similarly, in two embryos with SB, two domains of SHH expression were found in the ventral neural tube. CONCLUSIONS: Our findings suggest that notochord splitting in the cervical region might be involved in the pathogenesis of CRS. Interestingly, similar notochord abnormality and altered expression of the shh gene are observed in Lp mice with NTD. This suggests that the Lp gene could be a candidate gene for human CRS. Further studies are needed to establish the primary event responsible for the notochord splitting and for the abnormal expression of the SHH gene in the floor plate in embryos with CRS and SB.     

Changes in the mouse neuroepithelium associated with cadmium-induced neural tube defects

The aim of this study was to investigate the teratogenic action of cadmium (Cd) on the developing mouse CNS. Pregnant mice were injected with 4 mg/kg CdCl2 on day 7, 8, 9, or 10 of gestation. These animals and saline injected controls were sacrificed either on the day before birth or at various times up to 48 hours after injection and the embryos examined grossly and histologically. Exencephaly occurred after Cd treatment on day 7 or 8 and its development was examined in day 8 embryos. Eight hours after Cd injection many cells of the closing neural plate contained dense-staining inclusions, thought to be autophagic vacuoles. After 24 hours this damage had almost disappeared, but the anterior neural folds, although looking histologically normal, were more open than in controls. Forty-eight hours after injection it was apparent that this part of the neural tube was not going to close and would result in exencephaly. Cd exposure on day 9 or 10 did not cause gross CNS defects such as exencephaly. On both days, twelve hours after Cd injection, similar dark-staining inclusions were seen in many cells throughout the CNS. After twenty-four hours there were variable amounts of cell death, resulting in some embryos in breakdown of parts of the wall of the neural tube. Forty-eight hours after treatment all inclusions and cellular debris had disappeared, indicating repair had taken place, but in some embryos, treated on day 9, severe lasting damage was seen as dorsal openings in the previously closed neural tube.     

Ultrastructural changes in the neural tube of 10-day-old mouse embryos exposed to colchicine and hydroxyurea

The ultrastructural changes in the neural tube of 10-day-old mouse embryos were investigated between 1.5 hr and 4 hr after application of either 1 mg/kg colchicine (Col) or 500 mg/kg hydroxyurea (HU) or simultaneous application of both substances. During the investigated period, the shape of the nuclei of the neuroepithelial cells had changed from elongated to round after Col application. The chromatin in the nuclei was condensed and arranged in clusters. A breakdown of polysomes into ribosomes and an enlargement of the rough ER was observed in the cytoplasm. At the luminal surface, bleb-like cytoplasmic processes of the neuroepithelial cells containing monoribosomes protruded into the lumen. No cell necroses were visible in the neural tube after Col application. A condensation of chromatin in the nuclei of some neuroepithelial cells was visible 1.5 hr after HU application. Shortly thereafter, cell necroses appeared in the neural tube and 4 hr after HU application the entire spinal cord was strongly damaged. After simultaneous application of Col and HU, the ultrastructural changes in the neuroepithelial cells of the neural tube did not differ from the results obtained after Col application alone. In contrast to the results obtained after HU application alone, no necroses occurred after simultaneous application of Col and HU.     

Abnormalities of neural tube formation in pre-spina bifida splotch-delayed mouse embryos

The splotch-delayed homozygous mutant (Spd/Spd) develops spina bifida with or without exencephaly, has spinal ganglia abnormalities, and delays in posterior neuropore closure and neural crest cell emigration. The heterozygote (Spd/+) has a pigmentation defect, and occasionally neural tube defects. To investigate the underlying mechanisms, we compared the neuroepithelium in the posterior neuropore region of cytogenetically identified 15-18 somite pair Spd/Spd, Spd/+, and +/+ mouse embryos by transmission electron and light microscopy. The notochordal area and cell number in the non-fused neuroepithelium region of Spd/Spd and Spd/+ embryos were significantly reduced compared to those of normal (+/+) embryos, which suggests an abnormality in notochord elongation. In the mesoderm, the mean cell number and mean ratio of cell number to area in the non-fused region were significantly lower in the Spd/Spd compared with +/+ embryos. The distance of exposed neuroepithelium above the mesoderm in the just-fused region was significantly lower in the Spd/Spd versus +/+ embryos, which may indicate an insufficient force exerted by the mesoderm during neural tube closure. Within the neuroepithelium, significantly more intercellular space was found in Spd/Spd than in +/+ embryos indicating disorganization. The basal lamina was poorly organized and the formation delayed around the neural tube in Spd/Spd and Spd/+ embryos. All together, these results suggest an early abnormality in interactions among the neuroepithelium, mesoderm, and notochord, which may lead to the delay or inhibition of neural tube closure observed in Spd/Spd mutants.     

Reduction in the frequency of neural tube defects in splotch mice by retinoic acid

In the homozygous state, the splotch (Sp) gene causes spina bifida and exencephaly. Close to 25% of the embryos from Sp/ + X Sp/+ litters are affected. The frequency of these defects is significantly reduced by maternal treatment with 5 mg/kg retinoic acid on day 9 of gestation. There is no significant increase in the resorption frequency with this treatment, indicating that the fall in the frequency of neural tube defects is not due to differential mortality of the affected fetuses. The effects of retinoic acid are time specific, with treatment at different times on day 9 having the greatest influence on either the anterior or posterior neuropore. Treatment on day 8 with the same dose of retinoic acid causes an increase in both resorptions and neural tube defects, although only the increase in the former was significant.