Homocysteine and vitamin B12: Other causes of neural tube defects in Eastern Uttar Pradesh and Western Bihar population
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.236968
Source of Support: None, Conflict of Interest: None
Keywords: Homocysteine, neural tube defect, vitamin B12
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. 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.
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. However, not only folate but also vitamin B12 has been known to show prevention against NTDs. 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.,
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.
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 was done using GraphPad Prism version 6. 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.
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].
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.
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. 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, 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.
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. 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.
Mills et al., in 1995, 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.
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. However, in a study done by Gaber et al., in 2007, 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.,, 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.
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.