Folate and vitamin B₁₂ status of women in Newfoundland at their first prenatal visit

James D. House,* Sandra B. March,† Sam Ratnam,† Elizabeth Ives,‡ John T. Brosnan,§ James K. Friel§

CMAJ 2000;162(11):1557-9

• Abstract
• Introduction
• Methods
• Results
• Interpretation
• References

Background: Newfoundland has one of the highest rates of neural tube defects in North America. Given the association between low maternal folic acid levels and neural tube defects, a cross-sectional study was conducted to obtain baseline data on the folate and vitamin B₁₂ status of a sample of women in Newfoundland who were pregnant.

Methods: Blood samples were collected between August 1996 and July 1997 from 1424 pregnant women in Newfoundland during the first prenatal visit (at approximately 16 weeks' gestation); this represented approximately 25% of the women in Newfoundland who were pregnant during this period. The samples were analysed for serum folate, vitamin B₁₂, red blood cell folate and homocysteine.

Results: Median values for serum folate, red blood cell folate and serum vitamin B₁₂ were 25 nmol/L, 650 nmol/L and 180 pmol/L, respectively. On the basis of the interpretive criteria used for red blood cell folate status, 157 (11.0%) of the 1424 women were deficient (< 340 nmol/L) and a further 180 (12.6%) were classified as indeterminate (340–420 nmol/L). Serum homocysteine levels, measured in subsets of the red blood cell folate status groups, supported the inadequate folate status. Serum vitamin B₁₂ levels of 621 (43.6%) women were classified as deficient or marginal; however, the validity of the interpretive criteria for pregnant women is questionable.

Interpretation: A large proportion of pregnant women surveyed in Newfoundland in 1997 had low red blood cell folate levels.

[Contents]

Maternal folate status and folic acid intake at the time of conception are strongly implicated in the etiology of neural tube defects. Several case-control studies documented significantly decreased relative risks of occurrence^2,3 and recurrence⁴ of neural tube defect births for women who consumed folic acid supplements during the periconceptional period. Kirke and colleagues⁵ showed that plasma and red blood cell folate levels were significantly lower in women who gave birth to a child with a neural tube defect. In Newfoundland 64% of a sample of mothers who gave birth to children with neural tube defects had folate intakes below the national recommended levels (168 µg/d),⁶ compared with 27% of mothers who gave birth to healthy babies.⁷ These findings suggest that poor maternal folate status may be associated with the high incidence of neural tube defects in Newfoundland.

We assessed the folate levels of women in Newfoundland at their first prenatal visit to establish baseline data prior to the implementation of cereal grain fortification with folic acid. In addition, given the association between low serum vitamin B₁₂ and increased risk for neural tube defects,⁵ we also assessed serum B₁₂ concentrations.

[Contents]

An anonymous cross-sectional survey of patients was conducted from August 1996 to July 1997, and blood samples were collected from 1424 women in Newfoundland (25% of all pregnancies) at their first prenatal visit. For each subject blood was collected and frozen, and samples (linked by identification numbers) were shipped to the Public Health Laboratory in St. John's, Nfld., for the preparation of sera and red blood cell lysates. Folate concentrations in sera and red blood cell lysates were determined using the IMx Folate System (Abbott Laboratories, Abbott Park, Ill.), and vitamin B₁₂ concentrations in sera were determined using the IMx B12 System (Abbott Laboratories). Samples were classified with respect to folate and vitamin B₁₂ status as deficient (serum folate < 6 nmol/L, red blood cell folate < 340 nmol/L, serum vitamin B₁₂ < 130 pmol/L); indeterminate (6–7 nmol/L, 340–420 nmol/L, 130–160 pmol/L, respectively); or normal (> 7 nmol/L, > 420 nmol/L, > 160 pmol/L, respectively) based on criteria established by the manufacturer. Serum homocysteine concentrations, a further biochemical indicator of cellular folate status, were evaluated for a subset of 370 samples, comprising equal numbers of samples from the red blood cell folate status groups, using reverse-phase high-performance liquid chromatography with fluorescence detection.⁸ Significant differences (p < 0.05) between log-transformed variables were determined by 1-way ANOVA, and differences between groups were assessed by the Neuman–Keuls procedure.^9,10

Ethics approval for the study was obtained from the Human Investigation Committee, Memorial University of Newfoundland.

[Contents]

Median values for serum folate, red blood cell folate and serum vitamin B₁₂ were 25 nmol/L, 650 nmol/L and 180 pmol/L, respectively (Table 1). On the basis of the interpretive criteria, 157 (11.0%) of the 1424 women were deficient (< 340 nmol/L) in red blood cell folate, and a further 180 (12.6 %) were classified as indeterminate (340–420 nmol/L) (Table 2). Serum homocysteine levels measured in subsets of the red blood cell folate status groups supported the inadequate folate status; homocysteine levels for the red blood cell folate-deficient group were 11.9 ± 0.5 µM, the indeterminate group, 10.3 ± 0.7 µM, and for the normal group, 8.2 ± 0.6 µM.

[Contents]

On the basis of serum folate levels less than 4% of the women in our sample were deficient or at risk for becoming deficient in folate. Using the less conservative criteria of Bailey and colleagues¹¹ (i.e., < 7 nmol/L = deficient, 7–13 nmol/L = marginal), 2.4% and 23.3% of the women sampled would be categorized as deficient or of marginal status, respectively. Despite the lack of consensus concerning the limits for normal and deficient levels for serum folate during pregnancy,^12–15 it is generally agreed that serum folate is more reflective of recent folate intake and that levels normally decline with advancing pregnancy.^16,17 However, red blood cell folate concentration is independent of parity and plasma-volume expansion and is therefore more stable throughout pregnancy.¹⁶ Given the long half-life of red blood cells, red blood cell folate concentration at the first prenatal clinic (16 weeks' gestation) should better reflect the folate status of pregnant women at the time of neural tube closure (21 days' gestation); using criteria similar to those used by other groups^11,13 27% of the women in our sample had either deficient or marginal systemic folate stores. Serum homocysteine concentrations, which were inversely correlated with the folate concentrations, confirmed the depleted systemic folate stores.

According to category limits (i.e., deficient, indeterminate and normal) that were based on samples from women who were not pregnant, 43.6% of the women in our study were of deficient or indeterminate status for serum vitamin B₁₂. Although serum vitamin B₁₂ levels are known to decline with advancing pregnancy,^18–20 there may not be any other reliable marker for B₁₂ deficiency.²¹ Benjamin and colleagues²² set their limit for deficiency at 100 pg/mL (approx. 70 pmol/L), which would result in less than 5% of the women in our study classified as deficient in vitamin B₁₂. Clearly, further work is required to define appropriate interpretive criteria for serum vitamin B₁₂ status in pregnant subjects, perhaps by linking serum B₁₂ levels to other indices of B₁₂ status, such as methylmalonic acid.²³ Although serum homocysteine concentration has also been used as an index of vitamin B₁₂ status,^23,24 our regression analysis yielded no significant relationship between serum B₁₂ status and serum homocysteine concentrations.

Unfortunately, we were unable to correlate the low folate levels with other relevant patient variables (e.g., dietary preferences, multivitamin usage, socioeconomic status or gravidity) because blood samples were given anonymously. Nevertheless, the fact that 27% of the pregnant women presented with deficient or indeterminate red blood cell folate levels is a serious public health concern that should be addressed.

We thank all participating hospitals and the women who took part in the study. We also thank Claude Mercer and Allison MacDonald for data analysis and Marian Crowley for collecting rates of neural tube defect occurrence.

This study was supported by the Janeway Research Foundation, the Medical Research Council of Canada and the Canadian Diabetic Association.

Competing interests: None declared.

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Reprint requests to: Dr. James K. Friel, Department of Biochemistry, Memorial University of Newfoundland, St. John's NF A1B 3X9; fax 709 737-2422; jfriel@morgan.ucs.mun.ca

1. Frecker ME. Neural tube defects in Newfoundland. J Epidemiol Community Health 1994;48:100.
2. Czeizel AE, Dudas I. Prevention of the first occurrence of neural tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327:1832-5. [MEDLINE]
3. Werler MM, Shapiro S, Mitchell AA. Periconceptional folic acid exposure and risk of occurent neural tube defects. JAMA 1993;269:1257-61. [MEDLINE]
4. Medical Research Council Vitamin Study Research Group. Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet 1991;338:131-7. [MEDLINE]
5. Kirke PN, Molloy AN, Daly LE, Burke H, Weir DG, Scott JM. Maternal plasma folate and vitamin B₁₂ and independent risk factors for neural tube defects. Q J Med 1993;86:703-8. [MEDLINE]
6. Health and Welfare Canada. Nutrition recommendations. The Report of the Scientific Review Committee. Ottawa: Minister of Supply and Services; 1990.
7. Friel JK, Frecker M, Fraser FC. Nutritional patterns of mothers of children with neural tube defects in Newfoundland. Am J Med Genet 1995;55:195-9. [MEDLINE]
8. Vester B, Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Eur J Clin Chem Clin Biochem 1991;29:549-54. [MEDLINE]
9. SAS Institute. SAS/STAT user's guide. vol 6. 4th ed. Cary (NC): SAS Institute; 1989.
10. Zar JH. Biostatistical analysis. 2nd ed. Englewood Cliffs (NJ): Prentice-Hall; 1941.
11. Bailey LB, Mahan CS, Dimperio D. Folacin and iron status in low-income pregnant adolescents and mature women. Am J Clin Nutr 1980;33:1997-2001. [MEDLINE]
12. Huber AM, Wallins LL, DeRusso P. Folate nutriture during pregnancy. J Am Diet Assoc 1988;88:791-5. [MEDLINE]
13. Lehti KK. Iron, folic acid, and zinc intakes and status of low socioeconomic pregnant and lactating Amazonian women. Eur J Clin Nutr 1989;43:505-13. [MEDLINE]
14. Nutrition Canada. Nutrition: a national priority; a report by Nutrition Canada to the Department of National Health and Welfare. Ottawa: Information Canada; 1973.
15. O'Connor DL. Folate status during pregnancy and lactation. In: Allen L, King J, Lönnerdal B, editors. Nutrient regulation during pregnancy, lactation, and infant growth. New York: Plenum Press; 1994. p. 157-72.
16. Ek J, Magnus EM. Plasma and red blood cell folate during normal pregnancies. Acta Obstet Gynecol Scand 1981;60:247-51. [MEDLINE]
17. Hall MH, Pirani BB, Campbell D. The cause of the fall in serum folate in normal pregnancy. Br J Obstet Gynaecol 1976;83:132-6. [MEDLINE]
18. Ball EW, Giles C. Folic acid and vitamin B₁₂ levels in pregnancy and their relation to megaloblastic anaemia. J Clin Pathol 1964;17:165-74.
19. Green R, Colman N, Metz J. Comparison of results of micro biologic and radio isotopic assays for serum vitamin B₁₂ during pregnancy. Am J Obstet Gynecol 1975;122:21-4. [MEDLINE]
20. Zamorano AF, Arnalich F, Sanchez Casas E, Sicilia A, Solis C, Vazquez JJ, et al. Levels of iron, vitamin B₁₂, folic acid and their binding proteins during pregnancy. Acta Haematol 1985;74:92-6. [MEDLINE]
21. Metz J, McGrath K, Bennett M, Hyland K, Bottiglieri T. Biochemical indices of vitamin B₁₂ nutrition in pregnant patients with subnormal serum vitamin B₁₂ levels. Am J Hematol 1995;48:251-5. [MEDLINE]
22. Benjamin F, Bassen FA, Meyer LM. Serum levels of folic acid, vitamin B₁₂ and iron in anemia of pregnancy. Am J Obstet Gynecol 1966;96:310-5. [MEDLINE]
23. Pardo J, Peled Y, Bar J, Hod M, Sela BA, Ben Rafael Z, et al. Evaluation of low serum B₁₂ in the nonanaemic pregnant patient. Hum Reprod 2000;15:224-6. [MEDLINE]
24. Stabler SP, Lindenbaum J, Allen RH. The use of homocysteine and other metabolites in the specific diagnosis of vitamin B₁₂ deficiency. J Nutr 1996;126:1266S-72S.

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