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Table of Contents    
ORIGINAL ARTICLE
Year : 2018  |  Volume : 66  |  Issue : 4  |  Page : 1016-1019

Homocysteine and vitamin B12: Other causes of neural tube defects in Eastern Uttar Pradesh and Western Bihar population


1 Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Neurosurgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of Neurology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Department of Pediatric Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Web Publication18-Jul-2018

Correspondence Address:
Dr. Royana Singh
Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.236968

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 » Abstract 


Background: Neural tube defects (NTDs) are congenital malformations with an incidence of 1–10/1000 live births. Homocysteine and vitamin B12 metabolism have been shown to be associated with NTDs.
Aim: To investigate the status of maternal and neonate's folic acid, homocysteine, and vitamin B12 levels and their association with the risk of development of NTDs in the population of Eastern Uttar Pradeshand Western Bihar, India.
Materials and Methods: This study is a cross-sectional, retrospective study where 96 mothers who either had a first NTD child or had a history of NTD child in the family and 126 neonates with spina bifida were recruited during the period 2012-2015. Eighty-four control mothers whose previous and current pregnancies were normal, and 87 control neonates who had no defects and were within the same age range as the NTD affected neonates, recruited from the department of pediatric surgery, were enrolled in the study. Plasma concentrations of folic acid, vitamin B12, and homocysteine were compared between cases and controls.
Results: The folic acid level in the mothers and neonates was within the normal limit. A significant increase in the level of homocysteine in mothers with affected pregnancy and in neonate cases in comparison to control mothers was obseved. Further, a significant decrease in the level of vitamin B12 in mothers with NTD neonates and in the affected neonates was noted. A negative correlation was found between homocysteine and vitamin B12 levels in case and control mothers.
Conclusion: A correlation of an increase in serum homocysteine with a decrease in vitamin B12 was seen in mothers of neonates with NTD. A similar observation as made in the neonates with NTDs. It may be suggested that maternal decrease in vitamin B12, in mothers who have normal folic acid may be associated with NTD in their children.


Keywords: Homocysteine, neural tube defect, vitamin B12
Key Message: In mothers of neonates exhibiting neural tube defects, a significant increase in the serum level of homocysteine with decrease in vitamin B12 level was observed even when their folic acid levels were normal. Thus, maternal decrease in vitamin B12 in mothers who have normal folic acid may be associated with NTD in their children.


How to cite this article:
Gupta R, Kumari P, Pandey S, Joshi D, Sharma SP, Rai SK, Singh R. Homocysteine and vitamin B12: Other causes of neural tube defects in Eastern Uttar Pradesh and Western Bihar population. Neurol India 2018;66:1016-9

How to cite this URL:
Gupta R, Kumari P, Pandey S, Joshi D, Sharma SP, Rai SK, Singh R. Homocysteine and vitamin B12: Other causes of neural tube defects in Eastern Uttar Pradesh and Western Bihar population. Neurol India [serial online] 2018 [cited 2018 Aug 17];66:1016-9. Available from: http://www.neurologyindia.com/text.asp?2018/66/4/1016/236968




Among several birth defects known till date, neural tube defects (NTDs) are congenital malformations with an incidence ranging from 1–10/1000 live births, depending on the ethnic group and geographical region studied. In India, a population-based study done by Cherian et al., in 2005, elaborated a higher incidence of 8.21/1000 live birth.[1] A hospital-based study done in 2012 to determine the incidence of NTD in the Eastern Uttar Pradesh revealed a much higher incidence of 21.1/1000 live births.[2]

NTDs occur due to the improper closure of neural tube during the first month after conception, in which spina bifida and anencephaly are the two most recurring forms. Previous studies have reported that maternal supplementation of folic acid during the periconceptional period is the factor that has been responsible for reducing the occurrence and recurrence of this defect.[3] However, not only folate but also vitamin B12 has been known to show prevention against NTDs.[4] Methionine synthase requires vitamin B12 for the formation of methionine. A study by Afman et al., in 2002, and Refsum, in 2001, showed that a low level of vitamin B12 leads to an increase in homocysteine level, which often manifests as NTDs.[5],[6]

Several studies have shown that the abnormal serum homocysteine and vitamin B12 levels may be associated with NTD. Vitamin B12 is involved in the formation of methyltetrahydrofolate synthase in a one-carbon metabolism, which affects folic acid metabolism and the formation of homocysteine to methionine during the methylation cycle. If the level of Vitamin B12 is decreased, it leads to hyperhomocysteinemia. Therefore, this study was undertaken with the purpose of investigating the status of maternal and neonatal serum levels of folic acid, homocysteine, and vitamin B12.


 » Materials and Methods Top


Recruitment of subjects

This study was a cross-sectional, retrospective study where 96 mothers who either had a previous history of a NTD child or had a family history of a NTD, and 126 neonates with spina bifida were recruited from the Department of Pediatrics Surgery, Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, after taking an informed consent. Blood samples of control neonates (n = 87) within the same age range as that of the cases, and control mothers (n = 84) were also taken. The personal and family history of all the recruited families were taken considering the following parameters: (1) consanguinity, (2) any previous case of spina bifida in the family, (3) supplementation of folic acid during pregnancy, (4) supplementation of vitamin B12 during pregnancy, (5) food habit, i.e., vegetarian or non-vegetarian, (6) maternal diabetes and hyperthermia, and, (7) alcohol and tobacco intake. The study protocol was approved by the Institutional Ethical Committee of Sir Sunderlal Hospital, Banaras Hindu University, Varanasi.

Collection of the blood sample and assay

Venous blood of both neonates and mothers was drawn on the day of presentation of the neonates (ranging from 1 day to 4 weeks) into ethylenediaminetetraacetic acid coated vacutainers for serum folic acid, homocysteine, and vitamin B12 estimation. The vacutainers were immediately placed in ice. Serum was collected after centrifugation at 1000 rpm for 5 min. Long-term storage of serum was done at −20°C. Levels of folic acid, vitamin B12, and homocysteine were determined using Abbott Architect automated assay.

Statistical analysis

Statistical analysis was done using GraphPad Prism version 6.[7] Data analysis for mean, and standard deviation (SD) or counts with percentage was done. Biochemical levels of folic acid, homocysteine, and B12 were expressed as mean ± SD. A two-tailed unpaired t-test was used to check the significance of the study parameters between the cases and the control subjecs, as deemed appropriate.


 » Results Top


From March 2012 until January 2015, different cases of NTD were recruited from different districts in and around Eastern Uttar Pradesh. These patients were not only from Varanasi but also other nearby districts; Chandauli, Jaunpur, Sant Ravidas Nagar, Ghazipur, Mirzapur, Sonbhadra, Pratapgarh, Sultanpur, Allahabad, Basti, Ambedkar Nagar, Azamgarh, Ballia, Deoria, Gorakhpur, and Faizabad districts of Uttar Pradesh.

Patients also hailed from other states such as Bihar, Madhya Pradesh, and Jharkhand. The maximum number of patients were from the Western part of Bihar district including Paschim Champaran, Purvi Champaran, Gopalganj, Rohtas, Kaimur (Bhabhua), Aurangabad, and Gaya. Patients from Jharkhand were mostly from district Palamu, and those from Madhya Pradesh were from the district Singrauli.

The mean age of the NTD patients was 0.80 ± 0.69 months for male patients, and 0.84 ± 0.57 months for the female ones. The mean age for the healthy control subjects was 0.64 ± 0.23 months for the male ones, and 0.83 ± 0.34 months for the female ones. The ratio of male-to-female patients was 2.32, and the ratio in the case of healthy controls was 2.41.

During the study, it was observed that meningomyelocele was the most prevalent NTD, i.e., with an incidence of 77.78% among the different types of NTDs. While a few of NTD neonates presented with no complications, some others had involvement of the bladder and bowel (47.22%); other neurologic deficits included weakness in the lower extremity, sensory loss in the same distribution (7.64%), paraplegia (39.58%), hydrocephalus (15.28%),  Chiari malformation More Details (0.69%), patulous anus (0.68%), clubfoot (8.33%), tethered cord (1.39%), and congenital talipes equinovarus (0.69%). Some neonates of anencephaly also had associated polydactyly (12.5%), cleft palate (12.5%), syndactyly (12.5%), polycystic kidney (12.5%), and myelomeningocele [MMC] (12.5%).

Several factors known to be associated with mother lead to the development of NTD. In this context, the maternal age, the dietary intake of iron, folic acid or vitamins, and the prenatal exposure to passive smoking were given special consideration.

The mothers aged below 18 years and above 35 years exhibited 0.51% of NTD offsprings. Mothers within an age range of 18–25 years had 58.16% of the affected pregnancies, and mothers within the range of 26–35 years had 40.81% of the affected pregnancies.

The mothers who were on periconceptional folic acid supplementation constituted 16.67% of the cohort. The intake of iron-vitamin supplementation during the first trimester was 19.44%; and, the intake of iron-vitamin supplementation after the first trimester was 53.47%. Maternal education was present in 17.86% of the subjects. Epilepsy was seen in 1.39%, and exposure to passive smoking was seen in 20.14% of the mothers.

The folic acid level was within the normal range in the control mothers and neonates, with their mean levels being 11.83 ± 3.91ng/ml and 12.96 ± 4.47ng/ml, respectively. The mean levels of folic acid were 10.95 ± 4.31ng/ml and 11.90 ± 5.48ng/ml in the affected mothers and neonates.

The homocysteine level in the control mothers was 4.424 ± 1.067 micomoles/l and in the control neonates was 5.225 ± 0.2349 micromoles/l; whereas, the corresponding levels in mothers having neonates with NTD was 14.57 ± 0.2602 micromoles/l and in the affected neonates with NTS was was 15.32 ± 0.6631 micromoles/l. This difference in the homocysteine levels was significantly different (P< 0.0001). The level of vitamin B12 in the control mothers and neonates was 478.0 ± 105.0 pg/ml and 293.4 ± 12.60 pg/ml, respectively, whereas in mothers with NTD neonates, the corresponding level was 191.8 ± 6.053 pg/ml and the affected neonates had a level of 156.8 ± 9.597 pg/ml, which was significantly less in both mothers as well as affected neonates (P< 0.0001) [Table 1].
Table 1: Mean±standard deviation of serum homocysteine and vitamin B12 levels in neonates and mothers with neural tube defect as well as in control mothers and children

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The Pearson correlation coefficient between homocysteine and vitamin B12 levels was −0.177 in the control mothers as compared to 0.055 in mothers having the affected pregnancy. The Pearson correlation coefficient between homocysteine and vitamin B12 was −0.214 in control neonates as compared to 0.600 in case neonates. The inverse correlation indicated that homocysteine levels increased with a decrease in the vitamin B12 levels in mothers having affected pregnancy.


 » Discussion Top


In this study, a male preponderance was seen, with the male-to-female ratio being 2.32. However, Seller et al., in 1987, have reported amongst patients with NTDs, a male: female ratio of 0.73, with a female preponderance.[8] The preponderance of males having a NTD may be due to an underestimation of the true situation, which can be attributed to fact that this is a hospital-based study where a girl neonate with NTD may not have been brought for consultation. The true preponderance of male patients with regard to their propensity to developing a NTD can only be determined when a population study investigating the incidence of NTDs is carried out.

MMC (77.78%) was the most commonly occurring type of NTD in our study. While Feuchtbaum et al., in 1999, reported the occurrence of MMC to be 42% among the NTDs seen in California during the period 1990–1994,[9] a study done in the Asir Region of Saudi Arabia by Asindi and Al-Shehri in 2001, reported that 70% of patients with a NTD had MMC.[10]

The maternal age seems to have no correlation with the occurrence of NTD. A high percentage of women (83.33%) did not have periconceptional folic acid supplementation, and 80.56% of the women did not have iron-vitamin intake during the first trimester, although according to the ongoing national program, folic acid and iron-vitamin supplementation are mandatory for all mothers. The folic acid level was, however, within the normal limits, which can be attributed to the vegetarian diet consisting of green vegetables, the staple diet prevalent in this part of the country.

On the basis of the observations, it may be suggested that the serum vitamin B12 and homocysteine levels in mothers and neonates are strongly associated with an increased risk of NTD. Previous studies have elaborated that periconceptional vitamin B12 supplementation prevents hyperhomocysteinemia and NTDs.[11] A significant decrease in the level of vitamin B12 in mothers with NTD babies and affected neonates has also been seen in this study, which is similar to the previous findings that had shown an increase in the level of homocysteine during pregnancy and a decrease in the level of vitamin B12 in mothers having NTD babies.[12]

Mills et al., in 1995,[13] confirmed the finding that mothers of children with NTD has hyperhomocysteinemia; however, in this study, hyperhomocysteinemia was observed in neonates also. Molloy et al., 2009, suggested that a mother who has a vitamin B12 concentration below 300 pg/ml is at an increased risk of NTD-affected pregnancy.[14]

According to Ceyhan et al., in a study conducted in 2010, vitamin B12 does not play a causative role in the development of fetal NTDs and suggested the importance of homocysteine level in the genesis of fetal NTDs.[15] However, in a study done by Gaber et al., in 2007,[16] low serum vitamin B12 level emerged as the leading cause of NTD.

The negative correlation between the homocysteine and vitamin B12 levels in the mothers and neonates in the case as well as control groups indicates that serum homocysteine levels increase with a decrease in vitamin B12 levels in mothers. Vitamin B12 functions as a coenzyme in the one-carbon metabolism pathway for the conversion of homocysteine to methionine. Further, vitamin B12 is involved in the formation of methylenetetrahydrofolate which is known to play a significant role in the conversion of 5-methyltetrahydrofolate to tetrahydrofolate for the synthesis of folic acid.[4],[5],[6] A decrease in the serum vitamin B12 (<300 pg/ml) level leads to hyperhomocysteinemia in mothers during pregnancy. Furthermore, this decrease in the vitamin B12 level impairs the pathway leading to the synthesis of folate and methionine, and subsequently results in the genesis of NTDs.


 » Conclusion Top


Our study confirms the fact that a low vitamin B12 and a higher homocysteine level increases the chance of NTDs in neonates. Therefore, periconceptional vitamin B12 supplementation to mothers during the antenatal period or a little before conceiving may be a good adjuvant therapy. Vitamin B12 is available from food materials like milk, meat, and fish; vitamin B12 tablets may also be given to decrease NTD in neonates. A prospective study to evaluate the role of vitamin B12 in increasing the risk of the NTD needs to be conducted.

Financial support and sponsorship

This work was supported by the Institute of Medical Sciences University, Grant Commission (UGC), New Delhi, India, in the form of Junior and Senior research fellowships to Dr. Rashmi Gupta.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

1.
Cherian A, Seena S, Bullock RK, Antony AC. Incidence of neural tube defects in the least-developed area of India: A population-based study. Lancet 2005;366:930-1.  Back to cited text no. 1
    
2.
Singh R, Gupta R, Johnson AA, Ojha U, Gangopadhyay AN. Incidence of neural tube defect: A hospital based study. Q J Surg Sci 2012;1:38-44.  Back to cited text no. 2
    
3.
MRC Vitamin Study Research Group. Prevention of neural tube defects: Results of the medical research council vitamin study. Lancet 1991;338:131-7.  Back to cited text no. 3
    
4.
Kirke PN, Molloy AM, Daly LE, Burke H, Weir DG, Scott JM. Maternal plasma folate and vitamin B12 are independent risk factors for neural tube defects. Q J Med 1993;86:703-8.  Back to cited text no. 4
    
5.
Afman LA, Lievers KJ, van der Put NM, Trijbels FJ, Blom HJ. Single nucleotide polymorphisms in the transcobalamin gene: Relationship with transcobalamin concentrations and risk for neural tube defects. Eur J Hum Genet 2002;10:433-8.  Back to cited text no. 5
    
6.
Refsum H. Folate, Vitamin B12 and homocysteine in relation to birth defects and pregnancy outcome. Br J Nutr 2001;85 Suppl 2:S109-13.  Back to cited text no. 6
    
7.
A two tailed unpaired t-test, mean and standard deviation, Pearson Correlation were performed using GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA. Available from: https://www.graphpad.com/scientific-software/prism/. [Last accesed on 2018 Jul 07].   Back to cited text no. 7
    
8.
Seller MJ, Opitz JM, Reynolds JF. Neural tube defects and sex ratios. Am J Med Genet 1987;26:699-707.  Back to cited text no. 8
    
9.
Feuchtbaum LB, Currier RJ, Riggle S, Roberson M, Lorey FW, Cunningham GC. Neural tube defect prevalence in California (1990-1994): Eliciting patterns by type of defect and maternal race/ethnicity. Genet Test 1999;3:265-72.  Back to cited text no. 9
    
10.
Asindi A, Al-Shehri A. Neural tube defects in the Asir Region of Saudi Arabia. Ann Saudi Med 2001;21:26-9.  Back to cited text no. 10
    
11.
Menkes JH, Sarnat HB, Flores-Sarnat L. Malformations of the central nervous system. In: Menkes JH, Sarnat HB, Maria BL, editors. Child Neurology. 7th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. p.172-74   Back to cited text no. 11
    
12.
Haslam RHA. Congenital anomalies of the central nervous system. In: Behrman RE, Kliegman RM, Jenson HB, editors. Nelson Textbook of Pediatrics. 16th ed. Philadelphia: WB Saunders Co., 2000; 1803-10.  Back to cited text no. 12
    
13.
Mills JL, McPartlin JM, Kirke PN, Lee YJ, Conley MR, Weir DG, et al. Homocysteine metabolism in pregnancies complicated by neural-tube defects. Lancet 1995;345:149-51.  Back to cited text no. 13
    
14.
Molloy AM, Kirke PN, Troendle JF, Burke H, Sutton M, Brody LC, et al. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics 2009;123:917-23.  Back to cited text no. 14
    
15.
Ceyhan ST, Beyan C, Atay V, Yaman H, Alanbay I, Kaptan K, et al. Serum vitamin B12 and homocysteine levels in pregnant women with neural tube defect. Gynecol Endocrinol 2010;26:578-81.  Back to cited text no. 15
    
16.
Gaber KR, Farag MK, Soliman SE, El-Bassyouni HT, El-Kamah G. Maternal vitamin B12 and the risk of fetal neural tube defects in Egyptian patients. Clin Lab 2007;53:69-75.  Back to cited text no. 16
    



 
 
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