Neonatal Hyperbilirubinemia in a Turkish Cohort: Association of Vitamin B12

Background Deficiency of vitamin B12 (VitB12) causes failure of erytrocyte maturation leading to cell lysis. Red blood cell lysis causes excess heme production that ends with hyperbilirubinemia. In this study, we aimed to evaluate the role of VitB12 in neonatal hyperbilirubinemia (NNH) with prolonged jaundice and to compare patients with control group who did not develop hyperbilirubinemia. Methods A total of 20 patients (M/F = 13/7) with jaundice and 20 healthy controls (M/F = 11/9) were included in the study. Results The mean indirect bilirubin level of patient group was 9.91 ± 1.90 mg/dL (6.71 - 15.2 mg/dL) and control group was 3.18 ± 1.24 mg/dL (1.16 - 4.96 mg/dL). The mean VitB12 level of patient group was 119.9 ± 43.9 ng/L (42.35 - 178 ng/L) and the control group was 286.17 ± 97.43 ng/L (207.90 - 624.10 ng/L). There was a statistically significant difference in terms of VitB12 level (< 0.001) between the study groups. Conclusion To our knowledge, this study is the first study showing that low VitB12 level has been observed as a risk factor in NNH for the first time in the literature. We suggest that prophylactic use of VitB12 by pregnant women so will greatly benefit to prevent VitB12 deficiency and its complications in the first years of life such as NNH.


Introduction
Because of its role in DNA synthesis, vitamin B 12 (VitB 12 ) is essential for cell multiplication during pregnancy [1]. VitB 12 (stored in liver) is crucial for maturation and proliferation of red blood cells (RBCs). Deficiency of VitB 12 causes failure of RBC maturation leading to cell lysis [2]. RBC lysis causes excess heme production that ends with hyperbilirubinemia. Since in newborn the functional capacity of liver is only about 1% of the adult liver [3], we supposed that excess bilirubin due to RBC lysis and low functional capacity of liver would result in inefficient clearance of bilirubin so that indirect hyperbilirubinemia develops. In this study, we aimed to evaluate the role of VitB 12 in neonatal hyperbilirubinemia (NNH) with prolonged jaundice and to compare patients with control group who did not develop hyperbilirubinemia. Prolonged jaundice was defined as total bilirubin level > 5 mg/dL in newborns older than 14 days of age [4,5].

Methods
The present study was conducted on a sample of newborns with confirmed diagnosis of prolonged jaundice and a group of sex-and age-matched healthy subjects as controls. Patients group included a sample of newborn with well-established neonatal jaundice, followed up at the Neonatology Clinic, from December 2013 to May 2014, whose parents gave their consent to participate in the study protocol. In order to define the etiology of jaundice, all patients were submitted to a systematic protocol. Briefly, the protocol included ABO group, Rh group, hematocrit, screening for glucose-6-phosphate dehydrogenase deficiency (G6PD), thyroid function tests and urinary culture. Patients who have ABO, Rh incompatibilty, polycythemia, G6PD disease or thyroid disorder were excluded.

Controls
The control group who had not prolonged jaundice consisted of sex-and age-matched healthy subjects from our Pediatric Clinic. Healthy status was determined through the subjects' medical history and either a parental report or self-report to

Ethical aspects
The Ethics Committee of School of Medicine of the Sutcu Imam University approved the study. Venous blood sample was taken from each newborn patient for determination of serum VitB 12 level. Serum VitB 12 level was determined by using an autoanalyzer instrument (E170 Roche Hitachi). Serum VitB 12 was determined by chemiluminescence method.

Statistical analysis
The Mann-Whitney was used to compare nonparametric continuous variables. The level of significance was set at P < 0.05. All patients informations were anlayzed by SPSS (Statistical Package for Social Science) 16.0 program.

Results
A total of 20 patients (M/F = 13/7) with jaundice and 20 healthy controls (M/F = 11/9) were included in the study. No differences were observed in general clinical characteristics such as gender, age, birth weight, gestational age and hematocrit levels among patient group and healthy controls ( Table 1). The mean age of patient group was 31.95 ± 5.99 days (19 -39 days) and control group was 33.8 ± 4.14 days (27 -42 days). The mean indirect bilirubin level of patient group was 9.91 ± 1.90 mg/dL (6.71 -15.2 mg/dL) and control group was 3.18 ± 1.24 mg/dL (1.16 -4.96 mg/dL). The mean VitB 12 level of patient group was 119.9 ± 43.9 ng/L (42.35 -178 ng/L) and the control group was 286.17 ± 97.43 ng/L (207.90 -624.10 ng/L). There was a statistically significant difference in terms of VitB 12 and indirect bilirubin levels (< 0.001) between the study groups.
In populations in which the incidence of neonatal jaundice or kernicterus is high, pharmacologic treatment may warrant consideration. However, concerns surround the long-term effects of phenobarbital on these children. Because pharmacologic treatment is probably not justified in populations with a low incidence of severe neonatal jaundice as in our center we did not give phenobarbital treatment [4].

Discussion
NNH is a frequently encountered problem. Although up to 60% of term newborns have clinical jaundice in the first week of life, few have significant underlying disease [6]. Physiologic jaundice in healthy term newborns follows a typical pattern. All etiologies of jaundice beyond physiologic and breastfeeding or breast milk jaundice are considered pathologic [7]. Pathologic jaundice can result from ABO and Rh incompatibiliy, G6PD deficiency, urinary tract infection, and hypothyroidsm [6]. So that in our study, newborns who have an indirect bilirubin level above 5 mg/dL were submitted to a systemic protocol which composed of ABO, Rh groups, screening for G6PD, thyroid function tests, and urinary culture [5]. Newborns who have none of these risk factors were included in the study.
Here in our study, VitB 12 level was analyzed for a risk factor of NNH. We found that low VitB 12 level is a risk factor for developement of NNH. VitB 12 deficiency is a correctable cause for hemolysis. The mechanism of hemolysis in this condition is ineffective erythropoiesis, where in immature erythrocytes are lysed within the bone marrow itself, resulting in the release of excess quantities of biliverdin, which is ultimately converted to indirect bilirubin [8].
VitB 12 (cobalamin) is essential for folate metabolism and DNA synthesis, acting as a cofactor for key enzymatic reactions. Deoxyadenosylcobalamin, one of the coenzyme forms of cobalamin, is a cofactor for methionine synthase, the enzyme which converts homocysteine to methionine [9]. In maternal VitB 12 deficiency, the products homocysteine and methylmalonic acid accumulate and can be transmitted to the fetus. Raised levels of these products have been detected on newborn screening of infants with low VitB 12 stores [10]. Thus, screening for methylmalonic acid cannot be relied upon to detect infants with low VitB 12 stores.
VitB 12 deficiency in newborn, in most cases, results from a maternal deficiency. Maternal causes of infant deficiency can be divided into deficient maternal diet or maternal pernicious anemia in a breastfed infant because infant formulas are supplemented with VitB 12 [11].
In view of maternal diet serum VitB 12 concentrations decrease during pregnancy more than can be accounted for by hemodilution [12]. There is some evidence of increased VitB 12 absorption during pregnancy, with newly absorbed VitB 12 being more important to placental transport than maternal liver stores [13]. So that, lack of VitB rich diet of the mother may cause VitB 12 defiency in newborn.
There is a positive relation between maternal and breastfed infant VitB 12 deficiency linked to low VitB 12 levels in breast milk. According to a study by Specker and colleagues, VitB 12 milk concentrations less than 360 pmol/L, approximately corresponding to a maternal serum VitB 12 of less than 300 pmol/L, could result in an infant who is biochemically deficient in VitB 12 [14]. So that low VitB 12 uptake may cause "not-enough breastmilk-jaundice" that may occur when the baby does not drink enough breast milk. The role of VitB 12 in breast milk in serum level of newborn shows that a normal maternal blood count is not a reliable marker for deficiency in the infant.
A careful review of available medical literature did yield rare previous reports of severe VitB 12 deficiency presenting with isolated indirect hyperbilirubinemia without concomitant significant hematologic or neurologic involvement. There are case reports of infant VitB 12 deficiency being diagnosed on newborn screening. Dasari et al reported a 41-year-old Asian-Indian male who presented with recurrent episodes of jaundice since VitB 12 deficiency [15].
There are shortcomings in our study. The first one was we do not know the VitB 12 level of the mothers of the patients and controls during pregancy so that we could not conclude that the relative low levels of VitB 12 in patients result from the mother's VitB 12 status. The second one is that we measured serum VitB 12 level only once for each patient and number of sample size was relatively small (n = 20) because of financial problems. Briefly, recruitment was conducted to achieve a sample t.
However, statistically with minimum sample sizes (n > 20), it may be obvious that the data are drawn from a normally distributed population [16]. So that further studies are needed to collect the samples at least twice in 3-month intervals and with more number of patients for more accuracy.
It has been shown [17] that neonatal jaundice shows statistically significant association with MTHFR 677T (homocysteine metabolism gene) mutation [18] which result elevated homocysteine. Hypomethylation due to MTHFR 677T and folate deficiency, which are independent risk factors could also be a factor leading to predisposition for NNH. So that the role of VitB 12 in MTHFR 677T mutation related neonatal jaundice need to be functionally established in future prospective studies.
To our knowledge this study is the first study showing that low VitB 12 level has been observed as a risk factor in NNH for the first time in the literature. However we should mention that NNH, or prolonged jaundice of newborns are multifactorial. Breastfeeding is an important factor as well as genetic background.
We suggest that prophylactic use of VitB 12 by pregnant women, similar to iron and folic acid prophylaxis, in addition to nutrition education programs and food enrichment, will greatly benefit to prevent VitB 12 deficiency and its complications in the first years of life such as NNH. This finding in itself emphasizes the need to raise awareness of this presentation among physicians.