The study of neural tube defects after the Human Genome Project and folic acid fortification of foods.

The implementation of folic acid fortification will eliminate a proportion of neural tube defects (NTD). As a result, the etiologic and clinical profiles of the developmental disorder may both change. In the assessment of NTD as it evolves, the bioinformatics structure and content of the Human Genome Project will find vital application. One important development will be an enhanced understanding of the role of folic acid in global regulation of gene expression through epigenetic processes. In addition, bioinformatics will facilitate coordination of research in the basic sciences with clinical investigations to better define remaining etiologic factors.

Altered antioxidant enzyme activities in children with a serious adverse experience related to valproic acid therapy.

Specific oxidative metabolites of valproic acid (VPA) have been associated with the clinically defined toxicity of the drug. To investigate the role of enzymatic detoxification in clinical toxicity, we compared activities of five antioxidant enzymes in 15 patients with a serious adverse experience (SAE) related to VPA therapy, to enzyme activities measured in 35 patients with good clinical tolerance of VPA, and 50 healthy, age-matched subjects. These enzymes included glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione transferase, superoxide dismutase, and catalase in erythrocytes; and GSH-Px in plasma. We also determined levels of Se, Cu, and Zn, trace elemental cofactors for these enzymes, in plasma from each individual. In patients with a VPA-associated SAE, GSH-Px was significantly depressed and GSSG-R was significantly elevated relative to values for the other groups. Selenium and zinc concentrations were lower in SAE patients than in controls. These findings may indicate a role for selenium dependent antioxidant activity in individual susceptibility to an SAE related to VPA therapy.

Ahomocysteinemia in molybdenum cofactor deficiency.

We report an infant with molybdenum cofactor deficiency (MCD) and a unique clinical presentation of hemiplegia, hypotonia, dystonia, and bilateral basal ganglia changes. Biochemistry revealed absent serum homocysteine, low concentrations of plasma cystine, high levels of urinary S-sulfocysteine and sulfite, and high levels of oxypurines in serum and urine. The depletion of cysteine and cystine through reaction with sulfite suggests that other thiols and thiol-dependent proteins may be similarly depleted. Ahomocysteinemia may be a clue to the mechanism of cytotoxicity in MCD.

Cell cycle properties in lymphocytes from children with myelomeningocele.

Children with spina bifida display a constellation of clinical features which include growth retardation, latex allergies, and recurrent infections. Clinical and epidemiological findings support the view that principle components of this congenital syndrome originate in an inherent susceptibility, and not necessarily as a secondary complication of the early central nervous system malformation. Critical requirements for normal neurulation include folic acid and a fully functional methylation cycle, both of which also promote cell proliferation. This suggests that elements of susceptibility may emerge as cells from an individual with myelomeningocele are induced to synthesize DNA and divide. As an in vitro correlate of proliferative activity, we compared cell cycle properties of peripheral blood lymphocytes (PBL) from children with spina bifida to those in age-matched healthy controls. The four patients selected for study all have typical lumbar level myelomeningocele with the Chiari II malformation as well as clinical features which we commonly observe. We exposed PBL to phytohemagglutinin in the presence of the thymidine analog bromodeoxyuridine. Using bivariate flow cytometric analysis of Hoechst 33258- and ethidium bromide-induced fluorescence, we measured percentages of cells which responded to the mitogen, and relative rates at which case and control cells traversed the first three cell cycles. In three of the four experiments a greater percentage of PBL from the patient than from the control responded to mitogen. Cells from these children also appeared to progress more rapidly into second and third cell cycles. In the fourth patient, an unusually high percentage of cells failed to respond to mitogen and the remainder progressed more slowly into later cycles. The biological functions of folic acid and methionine led us to expect that in the cells from children with spina bifida, DNA synthesis would be retarded, S phase prolonged and transitions between cycles delayed. These preliminary results contrast with original expectations for fractions of mitogen-responsive cells and rates of traversal. Further experiments with PBL and other cell types will be required to confirm the differences we observed and establish their biological significance. An association between abnormal proliferative capacity and NTD, whether positive or negative, will create a biological and experimental context in which to define metabolic factors in this condition.

Plasma homocysteine and methionine concentrations in children with neural tube defects.

Mild to moderate homocysteinemia in women has been associated with an increased frequency of pregnancies with neural tube defects (NTD). Homocysteinemia is also an independent risk factor for premature vascular disease. In addition to folic acid, supplemental Vitamin B12, Vitamin B6 and betaine may normalize homocysteine metabolism, decrease the risk for NTD formation, and correct related metabolic imbalances in children with NTD. By means of automated amino acid analysis, we assessed total non-fasting homocysteine and methionine in plasma from 24 children with myelomeningocele. This study group (mean age 10.5 +/- 4.9 years) included 12 girls and 12 boys randomly selected from our Birth Defects Clinic. Homocysteine concentrations in our patients (4.7 +/- 1.8 mumol/L) did not differ from those of 20 randomly selected child controls (5.1 +/- 2.6 mumol/L). The mean homocysteine concentration for 36 adult controls (9.3 +/- 3.0 mumol/L) was significantly higher than the mean for either group of children (p < 0.0001). Linear regression analysis revealed negative correlation of total plasma homocysteine with serum folate (r = -0.53; p = 0.01), but not of homocysteine with either methionine or B12. Plasma methionine concentrations from our patients did not differ from adult reference values. Elevated homocysteine in some mothers of children with NTD has been attributed to defective methylation of homocysteine. These preliminary results do not indicate such a defect in the children themselves. A more comprehensive study of homocysteine, methionine and related metabolites in children with NTD and age-matched controls will be required to determine the clinical significance of these findings.

Comparison of erythrocyte antioxidant enzyme activities and embryologic level of neural tube defects.

Increased exposure to oxidant-derived free radicals or inadequate systems for antioxidant defense could alter cellular response at critical points in development. We measured 5 antioxidant enzymes, glutathione peroxidase (GSH-Px), glutathione reductase, glutathione-S-transferase, catalase and superoxide dismutase in erythrocytes and their plasma cofactor trace elements (Se, Zn, Cu) in 37 children with myelomeningocele and in 37 age-matched controls. We placed the patients into 3 groups according to motor level of the lesion at birth. We found significantly lower GSH-Px activities (p = 0.007) in children with myelomeningocele. For paired comparisons among the 3 patient groups and controls, there were significant differences (p < 0.05) between controls and both high (thoracic) and raid (lumbar) level embryologic lesions. The finding of antioxidant enzyme variations in our patients with myelomeningocele may indicate a role for abnormal oxidative metabolism in the development of this defect. The contribution of oxidative stress to human birth defects warrants investigation. We discuss potential relationships between oxidative stress and energy metabolism during primary neurulation.

Purification, properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

S-adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis(guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32,000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [3H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme synthesis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic activity. There was no further increase in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occurring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.

Regulation of the level of S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

Bovine lymphocytes were mitogenically activated by the lectin concanavalin A and the activity of the polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SDC), was followed (R. H. Fillingame and D. R. Morris, Biochem. Biophys. Res. Commun. 52: 1020-1025, 1973). Enzyme activity began to increase at approximately 3 hours after cellular activation, reaching a peak at 9-12 hours. A second elevation of SDC activity coincided with the entry of the cells into S phase at 24 hours. The early and late increases in SDC activity were regulated by different mechanisms. The initial elevation of activity was due to approximately a 10-fold enhancement of the rate of enzyme synthesis; the enhanced rate of synthesis was maintained throughout the remainder of the culture period. The second increase in SDC activity was due to a lengthening of the intracellular half-life of the enzyme from 80 to 170 min. These changes in the rates of SDC synthesis and degradation fully account for the observed biphasic enzyme induction curve. In vitro translation of the SDC messenger RNA has been achieved and this will be used in further studies of the mechanism of enhanced enzyme synthesis.