HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hobbs, C. A Articles by James, S J. Search for Related Content PubMed PubMed Citation Articles by Hobbs, C. A Articles by James, S J. Agricola Articles by Hobbs, C. A Articles by James, S J.

Congenital heart defects and abnormal maternal biomarkers of methionine and homocysteine metabolism^1,2,3,4

¹ From the Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences and Arkansas Children's Hospital Research Institute, Little Rock

² The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the CDC or the NIH.

³ Supported by Cooperative Agreement no. U50/CCU613236-06 from the Centers for Disease Control and Prevention and by grants from the National Institute of Child Health and Human Development (5R01 HD39054-03) and the National Center for Research Resources (1C06 RR16517-01 and 3C06 RR16517-01S1).

⁴ Reprints not available. Address correspondence to CA Hobbs, Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, 11219 Financial Centre Parkway, Suite 250, Little Rock, AR 72211. E-mail: hobbscharlotte{at}uams.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: It is well established that folic acid prevents neural tube defects. Although the mechanisms remain unclear, multivitamins containing folic acid may also protect against other birth defects, including congenital heart defects.

Objective: Our goal was to establish a maternal metabolic risk profile for nonsyndromic congenital heart defects that would enhance current preventive strategies.

Design: Using a case-control design, we measured biomarkers of the folate-dependent methionine and homocysteine pathway among a population-based sample of women whose pregnancies were affected by congenital heart defects (224 case subjects) or unaffected by any birth defect (90 control subjects). Plasma concentrations of folic acid, homocysteine, methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), vitamin B-12, and adenosine were compared, with control for lifestyle and sociodemographic variables.

Results: After covariate adjustment, case subjects had higher mean concentrations of homocysteine (P < 0.001) and SAH (P < 0.001) and lower mean concentrations of methionine (P = 0.019) and SAM (P = 0.014) than did control subjects. Vitamin B-12, folic acid, and adenosine concentrations did not differ significantly between case and control subjects. Homocysteine, SAH, and methionine were identified as the most important biomarkers predictive of case or control status.

Conclusions: The basis for the observed abnormal metabolic profile among women whose pregnancies were affected by congenital heart defects cannot be defined without further analysis of relevant genetic and environmental factors. Nevertheless, a metabolic profile that is predictive of congenital heart defect risk would help to refine current nutritional intervention strategies to reduce risk and may provide mechanistic clues for further experimental studies.

Key Words: Birth defects • congenital heart defects • risk • methionine • homocysteine • folate

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Congenital heart defects occur among 11 per 1000 live births in the United States (1) and are substantially more prevalent among stillbirths and miscarriages (2). Annually, heart defects account for 6000 total deaths and one-tenth of infant deaths (1). Only 15% of heart defects can be attributed to a known cause (1). The remaining cases are thought to result from complex interactions involving environmental exposures, maternal lifestyle factors, and genetic susceptibilities.

Folic acid is well established in preventing neural tube defects (NTDs). Evidence also suggests that multivitamins containing folic acid may protect against congenital heart defects (3-5) However, the mechanism or mechanisms underlying this effect remain unidentified. Several studies measured plasma biomarkers of folate metabolism among women with pregnancies affected by NTDs and orofacial clefts (6-10). In general, those studies demonstrated that increased plasma homocysteine and decreased folic acid are detrimental to the developing embryo.

Only 2 studies reported altered concentrations of biomarkers associated with homocysteine metabolism among women with pregnancies affected by heart defects. In one study, median fasting plasma homocysteine concentrations among 27 women who had affected pregnancies were significantly higher than 56 women with unaffected pregnancies (11). In the other study, fetal cells recovered from amniotic fluid of pregnancies affected by heart defects had elevated intracellular homocysteine relative to fetal cells of unaffected pregnancies (12).

Evidence demonstrates that homocysteine is elevated among women with a history of adverse pregnancy outcomes, including preeclampsia (13), recurrent early pregnancy losses (14), NTDs (6), orofacial clefts (15), and congenital heart defects (11). Elevated homocysteine is also associated with neurodegenerative disorders, cardiovascular disease, and cancer (16-19). Homocysteine is the product of the intracellular methionine cycle, in which methionine is initially activated by ATP to S-adenosylmethionine (SAM), the primary methyl donor for essential methyltransferase reactions (Figure 1). After methyl transfer, SAM is converted to S-adenosylhomocysteine (SAH). The sole source of homocysteine in the body is the hydrolysis of SAH. Interestingly, the equilibrium dynamics favor the reverse reaction, the synthesis rather than the hydrolysis of SAH. Thus, elevated homocysteine concentrations cause SAH to accumulate (20). Increased SAH is a potent product inhibitor of cellular methyltransferases, which during organogenesis can alter gene expression, cell differentiation, and apoptosis (21, 22).

View larger version (14K): [in this window] [in a new window]   FIGURE 1.. The folate-dependent methionine and homocysteine metabolic pathway. Biomarkers indicated by bold type were measured in the present study. B12, vitamin B-12 (cobalamin); MAT, methionine adenosyltransferase; mRNA, messenger RNA; MS, methionine synthase; MT, methyltransferase; SAH, S-adenosylhomocysteine; SAHH, SAH hydrolase; SAM, S-adenosylmethionine; THF, tetrahydrofolate; tRNA, transfer RNA.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The protocol and provisions for informed consent were reviewed and approved by the Institutional Review Board at the University of Arkansas for Medical Sciences. Case subjects were identified and ascertained through the Arkansas Reproductive Health Monitoring System, a statewide birth defects registry. Inclusion criteria for the case subjects were as follows: 1) resident of Arkansas at the time of the completion of the index pregnancy and at the time of enrollment in the study; 2) pregnancy outcome was a liveborn, stillborn, or elective termination; 3) pregnancy ended between February 1998 and September 2003; 4) a physician diagnosis of a nonsyndromic septal, conotruncal, or right- or left-sided obstructive heart defect that was confirmed by prenatal or postnatal echocardiogram, surgical, or autopsy report; 5) English or Spanish speaking; and 6) the case and control subjects had completed participation in the National Birth Defects Prevention Study (NBDPS) (23). Case subjects for whom the pregnancy was also affected by a known single gene disorder, chromosomal abnormality, or syndrome were excluded; only nonsyndromic congenital heart defects were included in this study.

Control subjects were Arkansas residents who had live births that were unaffected by any birth defect and were English or Spanish speaking. They were randomly chosen from all birth certificates registered at the Arkansas Department of Health, with birth dates between June 1998 and February 2003. Case or control subjects who were pregnant at the time of the blood draw or on antiepileptic medications were not eligible for the study. After determining eligibility for case or control subjects, a research nurse contacted the subjects by mail and telephone, describing this local study. During scheduled home visits, the nurse obtained written informed consent and a blood sample by routine venipuncture.

Covariates
The maternal educational level and household income were reported during a structured computer-assisted telephone interview that was specifically designed for an ongoing multisite case-control study, the NBDPS). Further details about the NBDPS were published previously (23). At the time of the home visit, a Block Abbreviated Food-Frequency Questionnaire (24) was administered, and information about the current use of multivitamins, cigarettes, and alcohol and about caffeine intake was obtained.

Sample preparation and biomarker measurement
Blood samples were collected into evacuated tubes containing EDTA, immediately chilled on ice, and centrifuged at 4000 x g for 10 min at 4 °C. Aliquots of the plasma layer were transferred into cryostat tubes and stored at –80 °C until analysis. Aliquots were thawed for extraction and HPLC quantification. Plasma folic acid and vitamin B-12 concentrations were measured by using Quantaphase II radioimmunoassay kit from Bio-Rad Laboratories (Hercules, CA).

To determine total homocysteine and methionine, 50 µL freshly prepared 1.43 mol/L sodium borohydride solution containing 1.5 µmol/L EDTA, 66 mmol/L NaOH, and 10 µL iso-amyl alcohol was added to 200 µL plasma to reduce sulfhydryl bonds. The samples were incubated at 40 °C on a shaker for 30 min. To precipitate proteins, 250 µL ice-cold 10% meta-phosphoric acid was added and mixed well, and the sample was incubated for 10 min on ice. After centrifugation at 18 000g for 15 min at 4 °C, the supernatant fluid was filtered through a 0.2-µm nylon membrane filter (PGC Scientific, Frederic, MD) and a 20-µL aliquot was injected into the HPLC system.

For determination of SAM, SAH, and adenosine, 100 µL 10% meta-phosphoric acid was added to 200 µL plasma to precipitate protein; the solution was mixed well and incubated on ice for 30 min. After centrifugation for 15 min at 18 000 x g at 4 °C, supernatant fluids containing SAM and SAH were passed through a 0.2-µm nylon membrane filter, and 20 µL was injected into the HPLC system. The methodologic details for metabolite elution and electrochemical detection were described previously (25, 26).

Statistical analysis
Sociodemographic and lifestyle characteristics of case and control subjects were compared with the Fisher exact test for categorical variables or with the Mann-Whitney U or Wilcoxon rank-sum test for continuous variables. All plasma biomarkers exhibited positively skewed distributions; therefore, to achieve normality, biomarker data were log-transformed (natural log) before analysis. Normality of the transformed data was verified by using the Anderson-Darling test (27). Outlier analysis was performed for each biomarker, stratified by case-control status. Among the case subjects, 4 questionable values that fell beyond 3 SDs from the mean (in both the log-transformed and untransformed data) were considered to be outliers. There were no outliers identified among the control subjects.

Mean log-transformed biomarker concentrations of case and control subjects were compared by using a Student t test, whereas linear regression was used to adjust these comparisons for age, race, educational level, household income, multivitamin supplement intake, number of cigarettes smoked daily, alcohol consumption, caffeine intake, and interval between the end of pregnancy and the blood draw. Crude and adjusted odds ratios and corresponding 95% CIs for the association between plasma biomarkers and case-control status were computed by using logistic regression. The most important predictors were identified by using a "best subset" approach (28). Analyses were performed with use of the SAS statistical package version 8.2 (SAS Institute, Cary, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study population
Two hundred fifty-six case subjects were contacted to participate in the study; 32 (12.5%) of the case subjects refused to participate. Similarly, one hundred nine control subjects were contacted to participate in the study; 19 (17.4%) of them refused to participate. Two case subjects had pregnancies that resulted in a stillbirth; the remaining women had affected pregnancies that ended in a live birth. Selected characteristics of the 224 case subjects and 90 control subjects that participated in this study are presented in Table 1. The sample consisted primarily of white women. At the time of the blood draw, 62.5% of the case subjects were younger than 30 y, and less than one-half reported alcohol consumption (45.1%). No significant difference was observed between the number of case subjects who reported regular multivitamin use (45.1%) and the number of control subjects who reported regular use (46.7%). Smoking was more prevalent among case subjects (28.1%) than control subjects (16.7%; P = 0.043). The mean caffeine intake did not vary significantly between case subjects (44.59 mg/d) and control subjects (44.16 mg/d; P = 0.628). The median time between the end of pregnancy and the blood draw was significantly longer for control subjects (24.5 mo; range = 4.8–49.3 mo) than for case subjects (14.9 mo; range = 0.1–52.2 mo; P < 0.01). Eighty-nine (98.9%) of the 90 control subjects and 183 (81.7%) of the 224 case subjects participated 6 mo after the end of pregnancy (data not shown).

Table 2

Table 3

Table 4

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Possible causes for the altered biomarkers include deficiencies in folate or vitamin B-12 or both, genetic polymorphisms encoding enzymes in the methionine cycle, or exogenous factors. Mean plasma folic acid was not significantly lower among case subjects than control subjects, as would be expected if folate deficiency was strongly contributing. Most pregnancies in this study began after January 1, 1998, when folic acid fortification of grain products was mandated (30). The mean folic acid concentration among our control subjects was 11.99 mg/L (31), which is consistent with the mean postfortification concentrations among women of childbearing age in other studies (32).

The similarity in folic acid concentrations between case and control subjects suggest that case subjects were either more genetically susceptible to having increases in homocysteine and SAH or were more likely to have reduced activity of relevant enzymes. For example, an alteration in the function of, or genes encoding, methionine synthase may reduce the conversion of homocysteine to methionine (Figure 1). Multiple case-control studies have evaluated the association between NTDs and common genetic variants in the methionine synthase gene with inconsistent results (33-36). To our knowledge, the association between methionine synthase activity and congenital heart defects is as yet undetermined. Thus, further studies are needed of genetic variants associated with congenital heart defects that would cause elevated homocysteine.

Our findings of elevated homocysteine among case subjects are consistent with other studies comparing vitamin-dependent homocysteine metabolism among mothers of offspring with orofacial clefts (15) and NTDs (6) with those of mothers with nonmalformed offspring (15). Studies have also found elevated homocysteine concentrations in women who have had preeclampsia (13) and recurrent early miscarriages (14). In those studies, maternal vitamin and homocysteine concentrations were measured up to 6 y after the relevant embryonic period (15). Thus, it was not possible to determine whether alterations in biomarkers among nonpregnant women having adverse pregnancy outcomes reflect the biomarker concentrations during organogenesis or early pregnancy loss. However, it is known that temporal changes in homocysteine concentrations are minimal over a 1–2-y time frame (37). Also, we are unable to determine the temporal relation between the alteration in biomarkers and the adverse pregnancy from our findings and those of others. The evidence suggests that these alterations, if not causally related, are associated with an increased risk of having adverse pregnancy outcomes, including congenital heart defects.

Possible mechanisms by which homocysteine may have an embryotoxic effect include oxidative stress and secondary accumulation of SAH, which leads to product inhibition of DNA methyltransferase reactions, DNA hypomethylation, and altered gene expression (38). Lifestyle factors such as alcohol intake and cigarette smoking are associated with increased oxidative stress (39), which can decrease the functional activity of methionine synthase through limiting the availability of reduced vitamin B-12 (35, 40, 41). Of note in our sample is the greater number of smokers among case subjects than among control subjects. When homocysteine concentration was stratified by smoking status among case and control subjects, the differences were higher among smokers than nonsmokers in both the case and the control subjects, but the results were not significantly different (data not shown). Animal studies showed that oxidative stress during fetal organogenesis can result in damage to mitochondrial and nuclear DNA, to protein structure and function, or to membrane lipids and signal transduction pathways. Depending on the timing and duration of exposure, the functional consequences of oxidative injury are embryopathy and dysmorphogenesis. For example, exposure of rats to 20% oxygen during early neurulation significantly increased the incidence of NTDs relative to unexposed embryos (42). Hypomethylation also was shown to lead to embryonic lethality and multiple developmental malformations (43, 44). For example, maternal exposure to valproic acid was shown to induce DNA hypomethylation in NTD-affected pregnancies in mice (45).

Several methodologic limitations of our study should be considered. Despite efforts to enroll participants soon after the index pregnancy, the venipuncture occurred well after the index pregnancies had ended. Thus, our measurements may not represent the biomarker concentrations at the time of organogenesis. However, it is remarkable that, even far after delivery, the biomarkers measured are significantly different between case and control subjects.

Given the relatively rare prevalence of most birth defects, the most common epidemiologic design used to investigate etiologic factors is a case-control study after the completion of pregnancy. Substantially more resources would be required to launch a cohort study to measure biomarkers among women enrolled before conception and followed to the completion of pregnancy. However, confidence in our findings is supported by noting that although folate, vitamin B-12 (46), and homocysteine (47) gradually decrease during pregnancy, at 6 wk after delivery, the concentrations are similar to those before conception or early pregnancy (47). Further, we examined our data to determine whether concentrations of homocysteine and folate correlated with the time interval between the end of pregnancy and the blood draw in case and control subjects. No discernible trend was found. Therefore, it is likely that, on average, the metabolic patterns observed even more than a year after pregnancy reflect stable adult profiles.

We did not examine the relation between cardiac phenotypes and the biomarkers. Particular cardiac phenotypes may be more influenced by altered homocysteine and methionine metabolism by way of different developmental pathways. Another potential source of bias in our study could be the failure to identify cardiac lesions among control pregnancies. Although control subjects were excluded if the medical chart indicated any congenital malformation, echocardiograms were not required to rule out a cardiac lesion.

If further investigations replicate those of this study, this altered metabolic profile might provide a measure of preconceptional risk and, more importantly, new information for the refinement of nutritional intervention strategies designed to reduce risk. Currently, primary prevention efforts to reduce the occurrence of nonsyndromic congenital heart defects are limited to folic acid fortification and supplementation and to avoidance of specific teratogens. Clinical efforts are focused on secondary prevention by using fetal echocardiograms, thereby optimizing postnatal medical and surgical outcomes. Identification of the genetic, epigenetic, and metabolic factors that may increase the likelihood of having a pregnancy affected by congenital heart defects may enhance primary prevention. One might imagine that during routine gynecologic visits women are screened for cancer, sexually transmitted diseases, and an increased risk of adverse pregnancy outcomes. Such screening would only be helpful if effective interventions to modify the risk profile were available. Currently, a national effort is under way to identify biomarkers for cancer risk and prevention (48). Perhaps, it is now time to begin a similar effort for the primary prevention of birth defects.

	ACKNOWLEDGMENTS

 
We appreciate Sadia Ghaffar for her diligent classification of cardiac lesions, and we are indebted to the Arkansas families whose participation made this study possible.

Author contributions were as follows: CAH (experimental design and analysis, manuscript writing), MAC (statistical data analysis design and manuscript writing), SM (laboratory biomarker analyses), WZ (statistical analyses), and SJJ (experimental analysis, manuscript writing). There were no potential conflicts of interests for the authors of this manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Botto LD, Correa A. Decreasing the burden of congenital heart anomalies: an epidemiologic evaluation of risk factors and survival. Prog Pediatr Cardiol 2003;18:111–21.
2. Tennstedt C, Chaoui R, Korner H, Dietel M. Spectrum of congenital heart defects and extracardiac malformations associated with chromosomal abnormalities: results of a seven year necropsy study. Heart 1999;82:34–9.[Abstract/Free Full Text]
3. Botto LD, Mulinare J, Erickson JD. Do multivitamin or folic acid supplements reduce the risk for congenital heart defects? Evidence and gaps. Am J Med Genet 2003;121A:95–101.
4. Shaw GM, O'Malley CD, Wasserman CR, Tolarova MM, Lammer EJ. Maternal periconceptional use of multivitamins and reduce risk for conotruncal heart defects and limb deficiencies among off spring. Am J Med Genet 1995;59:536–45.[Medline]
5. Czeizel AE. Periconceptional folic acid containing multivitamin supplementation. Eur J Obstet Gynecol Reprod Biol 1998;78:151–61.[Medline]
6. van der Put NM, van Straaten HW, Trijbels FJ, Blom HJ. Folate, homocysteine and neural tube defects: an overview. Exp Biol Med (Maywood) 2001;226:243–70.[Abstract/Free Full Text]
7. Rosenquist TH, Finnell RH. Genes, folate and homocysteine in embryonic development. Proc Nutr Soc 2001;60:53–61.[Medline]
8. van der Put NM, Blom HJ. Neural tube defects and a disturbed folate dependent homocysteine metabolism. Eur J Obstet Gynecol Reprod Biol 2000;92:57–61.[Medline]
9. Eskes TK. Open or closed? A world of difference: a history of homocysteine research. Nutr Rev 1998;56:236–44.[Medline]
10. van Rooij IA, Swinkels DW, Blom HJ, Merkus HM, Steegers-Theunissen RP. Vitamin and homocysteine status of mothers and infants and the risk of nonsyndromic orofacial clefts. Am J Obstet Gynecol 2003;189:1155–60.[Medline]
11. Kapusta L, Haagmans ML, Steegers EA, Cuypers MH, Blom HJ, Eskes TK. Congenital heart defects and maternal derangement of homocysteine metabolism. J Pediatr 1999;135:773–4.[Medline]
12. Wenstrom KD, Johanning GL, Johnston KE, DuBard M. Association of the C677T methylenetetrahydrofolate reductase mutation and elevated homocysteine levels with congenital cardiac malformations. Am J Obstet Gynecol 2001;184:806–17.[Medline]
13. Raijmakers MT, Roes EM, Zusterzeel PL, Steegers EA, Peters WH. Thiol status and antioxidant capacity in women with a history of severe pre-eclampsia. Br J Obstet Gynaecol 2004;111:207–12.
14. Nelen WL, Blom HJ, Steegers EA, den Heijer M, Thomas CM, Eskes TK. Homocysteine and folate levels as risk factors for recurrent early pregnancy loss. Obstet Gynecol 2000;95:519–24.[Medline]
15. Wong WY, Eskes TK, Kuijpers-Jagtman AM, et al. Nonsyndromic orofacial clefts: association with maternal hyperhomocysteinemia. Teratology 1999;60:253–7.[Medline]
16. Mattson MP, Shea TB. Folate and homocysteine metabolism in neural plasticity and neurodegenerative disorders. Trends Neurosci 2003;26:137–46.[Medline]
17. Maxwell SR. Coronary artery disease–free radical damage, antioxidant protection and the role of homocysteine. Basic Res Cardiol 2000;95:I65–71.
18. Cavalca V, Cighetti G, Bamonti F, et al. Oxidative stress and homocysteine in coronary artery disease. Clin Chem 2001;47:887–92.[Abstract/Free Full Text]
19. Wu LL, Wu JT. Hyperhomocysteinemia is a risk factor for cancer and a new potential tumor marker. Clin Chim Acta 2002;322:21–8.[Medline]
20. Yi P, Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ. Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J Biol Chem 2000;275:29318–23.[Abstract/Free Full Text]
21. Ehrlich M. Expression of various genes is controlled by DNA methylation during mammalian development. J Cell Biochem 2003;88:899–910.[Medline]
22. Finnell RH, Spiegelstein O, Wlodarczyk B, et al. DNA methylation in Folbp1 knockout mice supplemented with folic acid during gestation. J Nutr 2002;132:2457S–61S.[Abstract/Free Full Text]
23. Yoon PW, Rasmussen SA, Lynberg MC, et al. The National Birth Defects Prevention Study. Public Health Rep 2001;116:32–40.[Medline]
24. Block G, Hartman AM, Naughton D. A reduced dietary questionnaire: development and validation. Epidemiology 1990;1:58–64.[Medline]
25. Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ. A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulemetric electrochemical detection. J Nutr Biochem 1999;10:490–7.[Medline]
26. Melnyk S, Pogribna M, Pogribny IP, Yi P, James SJ. Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5-phosphate concentrations. Clin Chem 2000;46:265–72.[Abstract/Free Full Text]
27. Anderson TW, Darling DA. A test of goodness of fit. J Am Stat Assoc 1954;49:765–9.
28. Hosmer DW, Lemeshow S. Applied logistic regression. New York: Wiley, 2001.
29. Selhub J, Jacques PF, Rosenberg IH, et al. Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991–1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med 1999;131:331–9.[Abstract/Free Full Text]
30. Food and Drug Administration. Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid. Fed Reg 1996;61:8781–97.
31. Centers for Disease Control and Prevention. Folate status in women of childbearing age–United States, 1999. MMWR Morb Mortal Wkly Rep 2000;49:962–5.[Medline]
32. Than LC, Watkins M, Daniel KL. Serum folate levels among women attending family planning clinics–Georgia, 2000. MMWR Morb Mortal Wkly Rep 2002;51:4–8.
33. Gueant-Rodriguez RM, Rendeli C, Namour B, et al. Transcobalamin and methionine synthase reductase mutated polymorphisms aggravate the risk of neural tube defects in humans. Neurosci Lett 2003;344:189–92.[Medline]
34. De Marco P, Calevo MG, Moroni A, et al. Study of MTHFR and MS polymorphisms as risk factors for NTD in the Italian population. J Hum Genet 2002;47:319–24.[Medline]
35. Wilson A, Platt R, Wu Q, et al. A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida. Mol Genet Metab 1999;67:317–23.[Medline]
36. Christensen B, Arbour L, Tran P, et al. Genetic polymorphisms in meth-ylenetetrahydrofolate reductase and methionine synthase, folate levels in red blood cells, and risk of neural tube defects. Am J Med Genet 1999;84:151–7.[Medline]
37. Nurk E, Tell GS, Vollset SE, et al. Changes in lifestyle and plasma total homocysteine: the Hordaland Homocysteine Study. Am J Clin Nutr 2004;79:812–9.[Abstract/Free Full Text]
38. Perna AF, Ingrosso D, Lombardi C, et al. Possible mechanisms of homocysteine toxicity. Kidney Int Suppl 2003;S137–40.
39. Moller P, Wallin H, Knudsen LE. Oxidative stress associated with exercise, psychological stress and life-style factors. Chem Biol Interact 1996;102:17–36.[Medline]
40. McCaddon A, Regland B, Hudson P, Davies G. Functional vitamin B(12) deficiency and Alzheimer disease. Neurology 2002;58:1395–9.[Abstract/Free Full Text]
41. Ma J, Stampfer MJ, Christensen B, et al. A polymorphism of the methionine synthase gene: association with plasma folate, vitamin B12, homocyst(e)ine, and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev 1999;8:825–9.[Abstract/Free Full Text]
42. Ishibashi M, Akazawa S, Sakamaki H, et al. Oxygen-induced embryopathy and the significance of glutathione-dependent antioxidant system in the rat embryo during early organogenesis. Free Radic Biol Med 1997;22:447–54.[Medline]
43. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992;69:915–26.[Medline]
44. Martin CC, Laforest L, Akimenko MA, Ekker M. A role for DNA methylation in gastrulation and somite patterning. Dev Biol 1999;206:189–205.[Medline]
45. Alonzo-Aperte E, Ubeda N, Achon M, Perez-Miguelsanz J, Varela-Moreias G. Impaired methionine synthesis and hypomethylation in rats exposed to valproate during gestation. Neurology 1999;52:750–6.[Abstract/Free Full Text]
46. Cikot RJ, Steegers-Theunissen RP, Thomas CM, de Boo TM, Merkus HM, Steegers EA. Longitudinal vitamin and homocysteine levels in normal pregnancy. Br J Nutr 2001;85:49–58.[Medline]
47. Walker MC, Smith GN, Perkins SL, Keely EJ, Garner PR. Changes in homocysteine levels during normal pregnancy. Am J Obstet Gynecol 1999;180:660–4.[Medline]
48. Srivastava S, Gopal-Srivastava R. Biomarkers in cancer screening: a public health perspective. J Nutr 2002;132:2471S–5S.[Abstract/Free Full Text]

This article has been cited by other articles:

				L. M.J.W. van Driel, H. P.M. Smedts, W. A. Helbing, A. Isaacs, J. Lindemans, A. G. Uitterlinden, C. M. van Duijn, J. H.M. de Vries, E. A.P. Steegers, and R. P.M. Steegers-Theunissen Eight-fold increased risk for congenital heart defects in children carrying the nicotinamide N-methyltransferase polymorphism and exposed to medicines and low nicotinamide Eur. Heart J., June 1, 2008; 29(11): 1424 - 1431. [Abstract] [Full Text] [PDF]

				S. Malik, M. A. Cleves, M. A. Honein, P. A. Romitti, L. D. Botto, S. Yang, C. A. Hobbs, and and the National Birth Defects Prevention Study Maternal Smoking and Congenital Heart Defects Pediatrics, April 1, 2008; 121(4): e810 - e816. [Abstract] [Full Text] [PDF]

				J. Yang, S. L. Carmichael, M. Canfield, J. Song, G. M. Shaw, and the National Birth Defects Prevention Study Socioeconomic Status in Relation to Selected Birth Defects in a Large Multicentered US Case-Control Study Am. J. Epidemiol., January 15, 2008; 167(2): 145 - 154. [Abstract] [Full Text] [PDF]

				I.M. van Beynum, M. den Heijer, H.J. Blom, and L. Kapusta The MTHFR 677C->T polymorphism and the risk of congenital heart defects: a literature review and meta-analysis QJM, December 1, 2007; 100(12): 743 - 753. [Abstract] [Full Text] [PDF]

				S. Malik, M. A. Cleves, W. Zhao, A. Correa, C. A. Hobbs, and and the National Birth Defects Prevention Study Association Between Congenital Heart Defects and Small for Gestational Age Pediatrics, April 1, 2007; 119(4): e976 - e982. [Abstract] [Full Text] [PDF]

				M. A. Riedijk, B. Stoll, S. Chacko, H. Schierbeek, A. L. Sunehag, J. B. van Goudoever, and D. G. Burrin Methionine transmethylation and transsulfuration in the piglet gastrointestinal tract PNAS, February 27, 2007; 104(9): 3408 - 3413. [Abstract] [Full Text] [PDF]

				J. M. Robbins, J. M. Tilford, T.M. Bird, M. A. Cleves, J. A. Reading, and C. A. Hobbs Hospitalizations of Newborns With Folate-Sensitive Birth Defects Before and After Fortification of Foods With Folic Acid Pediatrics, September 1, 2006; 118(3): 906 - 915. [Abstract] [Full Text] [PDF]

				T. Tamura and M. F. Picciano Folate and human reproduction Am. J. Clinical Nutrition, May 1, 2006; 83(5): 993 - 1016. [Abstract] [Full Text] [PDF]

				I. M. van Beynum, L. Kapusta, M. den Heijer, S. H.H.M. Vermeulen, M. Kouwenberg, O. Daniels, and H. J. Blom Maternal MTHFR 677C>T is a risk factor for congenital heart defects: effect modification by periconceptional folate supplementation Eur. Heart J., April 2, 2006; 27(8): 981 - 987. [Abstract] [Full Text] [PDF]

				C. A. Hobbs, S. Malik, W. Zhao, S. J. James, S. Melnyk, and M. A. Cleves Maternal Homocysteine and Congenital Heart Defects J. Am. Coll. Cardiol., February 7, 2006; 47(3): 683 - 685. [Full Text] [PDF]

				C. A Hobbs, M. A Cleves, W. Zhao, S. Melnyk, and S J. James Congenital heart defects and maternal biomarkers of oxidative stress Am. J. Clinical Nutrition, September 1, 2005; 82(3): 598 - 604. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hobbs, C. A Articles by James, S J. Search for Related Content PubMed PubMed Citation Articles by Hobbs, C. A Articles by James, S J. Agricola Articles by Hobbs, C. A Articles by James, S J.