1. European Journal of Clinical Nutrition (2006) 60, 120–128
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ORIGINAL ARTICLE
Folate and vitamin B12 in relation to lactation:
a 9-month postpartum follow-up study
CH Ramlau-Hansen1,2, UK Møller1,3, TB Henriksen4,5, E Nexø6 and J Møller7
1 ˚
Institute of Public Health, Department of Health Science, Aarhus University, Arhus, Denmark; 2Department of Occupational Medicine,
Aarhus University Hospital, A ˚ rhus, Denmark; 3Department of Endocrinology and Metabolism, Aarhus University Hospital, Arhus,
˚
4
Denmark; The Perinatal Epidemiology Research Unit, Aarhus University, A ˚ rhus, Denmark; 5Departments of Obstetrics and
˚
Paediatrics, SKS, Aarhus, University Hospital, Arhus, Denmark; 6Departments of Clinical Biochemistry, NBG, Aarhus University
˚ ˚
Hospital, Arhus, Denmark and 7Departments of Clinical Biochemistry, SKS, Aarhus University Hospital, Arhus, Denmark
Objective: To investigate the relation between lactation and markers of folate and vitamin B12 (B12) deficiency in women
with and without vitamin supplementation.
Design: A 9-month follow-up study.
Subjects and methods: Blood samples from 91 women, who gave birth to a single healthy child, were collected 3 weeks, 4 and
9 months postpartum and analysed for circulating level of homocysteine (tHcy), methylmalonic acid (MMA), folate and B12.
The participants were categorized as exclusively, partly or not breast-feeding dependent on the degree of lactation 4 months
postpartum. During follow-up, lifestyle factors were recorded by structured interviews.
Results: Among 72 exclusively breast-feeding women, the median (10–90% percentile) tHcy was 5.8 (3.1–8.3) mmol/l 3 weeks
postpartum, 6.1 (4.1–10.3) mmol/l 4 months postpartum and 5.3 (3.6–8.7) mmol/l 9 months postpartum. At 9 months
postpartum, none of the women breast-fed exclusively. No significant change occurred in the concentration of B12 and folate.
Exclusively breast-feeding women without vitamin supplementation had higher median tHcy than supplemented exclusively
breast-feeding women 4 and 9 months postpartum (7.0 vs 5.4 mmol/l (Po0.001) and 5.8 vs 4.5 mmol/l (P ¼ 0.003),
respectively). Six women had increased (415 mmol/l) tHcy; four of these were unsupplemented and exclusively breast-feeding.
Conclusion: We found no overall indication of depletion of the folate and B12 stores during the lactation period in this
population. However, folate-supplemented women had lower tHcy and higher folate levels, suggesting a beneficial effect of
supplementation with folate throughout lactation.
Sponsorship: The Biomedical Laboratory Scientist Education and Research Fund and LEO Pharma Research Foundation
supported this study. DPC Scandinavia, Denmark donated reagents for the folate and tHcy analysis.
European Journal of Clinical Nutrition (2006) 60, 120–128. doi:10.1038/sj.ejcn.1602275; published online 28 September 2005
Keywords: breast-feeding; serum folate; serum B12; plasma homocysteine; serum methylmalonic acid
Introduction two vitamins (Allen, 1994; O’Connor, 1994; O’Connor et al.,
1997; Bjorke Monsen and Ueland, 2003). B12 insufficiency
Folate and vitamin B12 (B12) is released into human milk, during lactation may cause anaemia and neurological damage
hence the nursing mother has an increased demand for the in both the mother and the breast-fed child, as has been
reported in vegetarians (Metz, 1970; Michaud et al., 1992; Weiss
˚
Correspondence: CH Ramlau-Hansen, Arbejdsmedicinsk klinik, Arhus sygehus, et al., 2004). If maternal folate stores are insufficient prior to a
˚
Nørrebrogade 44, bygn. 2C, DK 8000 Arhus C, Denmark. subsequent conception, the risk of adverse pregnancy outcome
E-mail: craha@as.aaa.dk such as preterm delivery and birth defects in the following
Guarantor: CH Ramlau-Hansen.
pregnancy is increased (Smits and Essed, 2001).
Contributors: CHR, JM, UKM and TBH designed the study. CHR and UKM
collected the samples. JM and EN were responsible for the biochemical Data concerning folate- and B12 status during lactation is
analyses; CHR performed the statistical analyses, wrote the original manuscript conflicting. Several investigations show an increased risk of
and edited all subsequent versions. JM, EN and TBH contributed to the suboptimal folate status (Matoth et al., 1965; Donangelo
interpretation of data and commented on the manuscript. All authors
et al., 1989; Lehti, 1989; Ramlau-Hansen et al., 2003;
approved the final version of the manuscript.
Received 21 March 2005; revised 27 June 2005; accepted 12 July 2005; Villalpando et al., 2003) and a reduction in folate stores
published online 28 September 2005 during the lactation period (Shapiro et al., 1965; Tamura

2. Folate and B12 in lactation
CH Ramlau-Hansen et al
121
et al., 1980; Butte et al., 1981; Sneed et al., 1981; Smith et al., Women were invited to participate provided they met the
1983; Dostalova, 1984; Bruinse et al., 1985; Bates et al., 1986; following criteria: healthy, no medication, no drugs of abuse,
Salmenpera et al., 1986; Keizer et al., 1995; Mackey and no pregnancy complications, a single healthy infant born
Picciano, 1999) in women with no folic acid supplementa- 38–42 completed weeks of gestation with a birth weight
tion and with a diet insufficient in folate. Others have shown above 2500 g, blood loss less than 500 ml during labour, less
that lactation does not affect the level of folate (Cole et al., than 14 drinks of alcohol per week (the upper limit of
1974; Thomas et al., 1980; Ek, 1983). A possible B12 alcohol consumption for women recommended by the
depletion during lactation is apparently less common than Danish National Board of Health (Gronbaeck et al., 1997))
folate depletion (Shapiro et al., 1965; Sneed et al., 1981; and ability to understand Danish in writing and speaking.
Dostalova, 1984; Donangelo et al., 1989; Keizer et al., 1995). Potential participants were contacted by mail 1 week
Measurement of the vitamins in serum is used to identify postpartum and those who replied were contacted by
potential deficiency with folate in whole blood reflecting the telephone. The study was approved by the local ethics
uptake of folate during erythropoiesis, thus giving a measure committee (Project no. 20030093) and was monitored by the
of the long-term average folate intake and folate in plasma local Good Clinical Practice (GCP) Unit. Written informed
reflecting the intake in recent days. Also, metabolites consent was obtained from all the participants.
accumulating upon vitamin depletion can be used as
markers of the vitamin status. The concentration of
circulating homocysteine (tHcy) is elevated in both folate Study design
and B12 deficiency (Refsum et al., 1998; Stabler and Allen, Data were collected approximately 3 weeks (baseline), 4
2004), whereas an elevated concentration of methylmalonic months (1. follow-up) and 9 months (2. follow-up) post-
acid (MMA) is specific to B12 deficiency (Allen et al., 1990; partum at The Osteoporosis Clinic, Aarhus University
Klee, 2000; Stabler and Allen, 2004). In healthy young Hospital, Denmark during May 2003–April 2004. It was
adults, folate status is the main determinant of the tHcy, and assumed that if lactation had any influence on markers of
tHcy increases early during development of folate deficiency folate and B12 status, this effect would be detectable after 4
(Refsum et al., 1998). months of breast-feeding.
Several authors have observed a 20–50% decrease in tHcy Upon each visit, participants were interviewed regarding
during pregnancy (Kang et al., 1986; Andersson et al., 1992; lactation and current use of vitamin supplements. Informa-
Murphy et al., 2002), which normalize very fast following tion about smoking habits (‘Do you smoke cigarettes? How
delivery (Andersson et al., 1992). The decrease in tHcy during many cigarettes per day?’), intake of coffee (‘How many cups
pregnancy is suggested to be caused by an endocrine-based of coffee do you drink per day?’) and alcohol (How many
mechanism (Murphy et al., 2002) or a change in renal drinks of alcohol do you drink per week?’), physical exercise
haemodynamics during pregnancy (Brattstrom, 2003). A (‘Do you exercise (defined as minimum 30 min of walking
decrease in folate and B12 during pregnancy has also been every day)?’, Yes/No), medication, illnesses and diet (vege-
observed (Bruinse et al., 1985; Cikot et al., 2001; Chery et al., tarian or meat eater) were also recorded because of their
2002). These seem to normalize in a matter of weeks or potential influence on especially the tHcy concentration.
months postpartum depending on the nutritional status and Participants who used vitamin supplementation were ques-
lactation (Smith et al., 1983; Dostalova, 1984; Bruinse et al., tioned in detail about the brand and type of supplements.
1985; Allen, 1994). Measures of the participants’ heights and weights were
To our knowledge, very few investigations on the relation- obtained.
ship between lactation and tHcy have been conducted The evening and morning prior to blood sampling, the
(Mackey and Picciano, 1999; Ramlau-Hansen et al., 2003) women were asked to consume a low protein content meal
and no study on the relationship between lactation and (no eggs, less than 150 g of meat and less than 2 dl of milk or
MMA exists. other milk products) because a large protein-rich meal may
The main objective of our work was to investigate the increase the tHcy by about 10% after 6–8 h, whereas a small
influence of lactation on markers of folate and B12 status in meal will have no influence (Refsum et al., 2004). Blood
women with and without vitamin supplementation. samples were collected by vein puncture with participants
sitting upright after at least 10 min rest using a minimum of
tourniquet application (Rasmussen et al., 1999). Blood were
Subjects and methods collected into EDTA-treated evacuated tubes for whole blood
and into untreated evacuated tubes for serum preparation.
Participants Blood samples for tHcy, folate, MMA and B12 were
All women who gave birth at Aarhus University Hospital, refrigerated immediately, and plasma and serum was sepa-
Denmark, during a period of 3 months in spring and summer rated by centrifugation at 1500 g for 15 min at 41C within
2003 were considered for participation. The study was part of 30 min of sampling (Rasmussen and Moller, 2000). Plasma
an investigation also on the effect of lactation and bone and serum were stored at À201C until analysed. Blood
density (Moller, 2003). samples for haemoglobin (Hgb) and MCV were analysed on
European Journal of Clinical Nutrition

3. Folate and B12 in lactation
CH Ramlau-Hansen et al
122
the day of collection by ADVIA 120 (Bayer, Copenhagen, Index population
Denmark). The tHcy and folate concentrations were deter- 1215 women giving birth
mined using IMMULITE 2000 (DPC, Koege, Denmark), and between March and July 2003
B12 was measured by Bayer Centauer (Bayer, Copenhagen,
Denmark). MMA were analysed by GS-MS (Rasmussen et al.,
1996). The relevant reference intervals are as follows: tHcy,
778 women (64%)a fulfilled the 437 women (36%)a did not
o8.1 mmol/l; MMA, o0.28 mmol/l; B12, 200–600 pmol/l; and eligibility criteria and were fulfill the eligibility criteria and
folate, 46 nmol/l. The CVs for all assays were o10%. potential participants. A letter were not contacted
of invitation was sent to 746
The participants were categorized into two groups with women (61%)b
regard to the degree of lactation 4 months postpartum: (1)
exclusively breast-feeding women (n ¼ 72), and (2) partly
(n ¼ 8) or not (n ¼ 9) breast-feeding women, who substituted
from one to all daily meals of human milk with formula or 116 women replied in writing 630 women (52%)a did not
and were contacted by reply in writing and were not
solid food. The participants were further categorized accord- telephone contacted further
ing to use of vitamin supplementation.
Statistical analyses
Study population 25 women (2%)a were not
All statistical analyses were performed using the NCSS/PASS 91 women of whom 88 women included after the telephonic
2000 Dawson Edition. completed all three visits interview (2 were unwilling, 14
were non-eligible and 9 were
All variables that were not normally distributed were not included because the
natural log-transformed. Normally distributed data were inclusion period had ended)
described with the mean and 10 and 90 percentiles. Relative
Figure 1 Study design. aPercentage of the index population. bIn all,
(%) changes in body weight were calculated by use of
32 women (3%) were not invited due to lack of capacity.
naturally log-transformed data. The distributions of tHcy
and MMA were skewed even after log-transformation.
Consequently, they were characterized by medians, 10 and of the 89 participants, who completed at least two visits, are
90 percentiles, and nonparametric statistics were used as presented in Table 1. Women who were not from Scandina-
described below. Folate, B12, tHcy and MMA concentrations via (5%) did not differ from Scandinavians concerning MMA
were analysed both cross-sectionally and longitudinally. and tHcy and no further analysis according to ethnicity were
Five folate concentrations exceeded the upper limit of the performed. At baseline, 46 of the women consumed alcohol
IMMULITE 2000 method (454.4 nmol/l). The concentration weekly (median 2 (range 1–7) drinks/week). At 9 months
of folate in these five samples was arbitrarily set to 10% over postpartum, the number of alcohol consumers was 55
the upper analytical limit (59.8 nmol/l) before statistics were (median 2 (range 1–10) drinks/week). At 3 weeks and 9
performed. months postpartum, respectively, six and 13 women smoked
One exclusively breast-feeding woman had high concen- cigarettes daily (median 3 (range 2–10) cigarettes/day and
trations of MMA. Her MMA and B12 results are not included median 4 (range 1–12) cigarettes/day). A total of 47 women
in the statistics and figures. had a daily coffee intake (median 2 (range 1–5) cups/day) 3
Wilcoxon’s rank-sum test was used to compare contin- weeks postpartum; 9 months postpartum, 53 drank coffee
uous, not normally distributed data (tHcy and MMA daily (median 3 (range 1–8) cups/day). At 3 weeks and 9
concentrations) between supplemented and unsupplemen- months postpartum, 60 and 68 women, respectively, ex-
ted or exclusively breast-feeding and partly and not breast- ercised daily. None of the women became pregnant during
feeding women. Two-sample t-test was used, when data were the study period. At 3 weeks, 4 and 9 months postpartum,
normally distributed (folate and B12 concentrations). Fish- respectively, one, seven and 18 women used oral contra-
er’s exact test or w2 test was used to compare proportions in ceptives. The above lifestyle factors did not correlate to tHcy
two groups. (data not shown); only the median MMA was statistically
A two-tailed probability level of 0.05 was chosen as the higher in oral contraceptive users 4 months postpartum
level of statistical significance. compared to nonusers (Wilcoxon’s rank-sum test: P ¼ 0.002).
The women had between zero and four previous children.
No association was observed between the concentration of
Results tHcy or MMA and the number of previous children born to
the mother (data not shown). All remained apparently
Participants healthy throughout the study apart from one participant,
In all, 91 women aged mean 31.5 years (range 20–42 years) who was diagnosed with thyroid disease but was normalized
were included, 89 completed the first two visits and 88 at the visit 9 months postpartum. She did not differ from the
completed all three visits (Figure 1). Baseline characteristics rest concerning tHcy and MMA.
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Table 1 Selected characteristics at 3 weeks postpartum (baseline) of participants divided according to the degree of lactation 4 months postpartum
(1. follow-up)
Exclusively breast-feeding (n ¼ 72) Partly and not breast-feeding (n ¼ 17) P-value
Age (years)a 31.9 (20–42) 29.9 (23–36) 0.1b
Primiparaus, n (%) 29 (40) 12 (71) 0.02c
Number of childrend 2 (1–4) 1 (1–3) 0.05e
Smoking, n (%) 4 (6) 2 (12) 0.3f
Coffee, n (%) 39 (54) 7 (41) 0.3c
Alcohol, n (%) 38 (53) 7 (41) 0.4c
Exercise, n (%) 48 (67) 10 (59) 0.5c
Folic acid during pregnancy, n (%) 70 (97) 17 (100) 1.0c
Folic acid and B12 during the study period, n (%) 45 (63) 10 (59) 0.8c
Hgb (mmol/l)
Mean (10 and 90 percentiles) 8.4 (7.5,9.2) 8.3 (7.7,9.0) 0.6b
a
Mean (range).
b
P-value tested with two-sample t-test.
c
P-value tested with w2 test.
d
Median (range).
e
P-value tested with Wilcoxon’s rank-sum test.
f
P-value tested with Fisher’s exact test.
Table 2 Folate, tHcy, Hgb and body weight at 3 weeks, 4 and 9 months postpartum in all participants
3 weeks (n ¼ 91) P-valuea 4 months (n ¼ 89) P-valueb 9 months (n ¼ 88)
Folatec (nmol/l) 18.5 (8.6, 37.0) 0.1d 15.4 (6.3, 36.4) 0.4d 16.5 (8.6, 30.8)
THcye (mmol/l) 5.8 (3.2, 8.3) 0.1f 6.1 (3.8, 10.3) 0.04f 5.5 (3.6, 9.0)
Hgbc (mmol/l) 8.4 (7.6, 9.2) 0.04d 8.2 (7.6, 8.8) 0.7d 8.2 (7.6, 8.9)
Body weightc (kg) 69.3 (59.1, 83.0) 0.1d 66.9 (55.4, 82.4) 0.3d 65.5 (55.2, 81.0)
a
Comparing values at 3 weeks to values at 4 months postpartum.
b
Comparing values at 4 months to values at 9 months postpartum.
c
Mean (10 and 90 percentiles).
d
P-value tested with two-sample t-test.
e
Median (10 and 90 percentiles).
f
P-value tested with Wilcoxon’s rank-sum test.
All but one (a vegetarian) reported eating an average Breast-feeding as compared to partly or not breast-feeding women
Danish diet including meat. The vegetarian did not differ The median of tHcy and MMA and mean of folate and B12 at
from the rest concerning tHcy and MMA. the three visits according to lactation at 4 months post-
The number of exclusively breast-feeding women gradu- partum are presented in Table 3. There was no change in
ally declined from 86 at 3 weeks to 72 at 4 months mean folate and B12 across the three time periods among the
postpartum. At 9 months postpartum, none of the women women who were exclusively breast-feeding. The concentra-
breast-fed exclusively. In total, 44 breast-fed to a very limited tion of tHcy did not change from 3 weeks to 4 months
extent and 44 did not breast-feed. The median duration of postpartum (Wilcoxon’s rank-sum test: P ¼ 0.1), but de-
the lactation period for those 44 not-breast-feeding women creased from 4 to 9 months postpartum (Wilcoxon’s rank-
was 28 weeks (range 0–40 weeks). There was no association sum test: P ¼ 0.01). The median MMA concentration
between duration of the lactation period and tHcy at 9 decreased statistically significant throughout the study
months postpartum. However, there was a positive correla- (Wilcoxon’s rank-sum test: Po0.001).
tion between duration of the lactation period and MMA at 9 The median tHcy and MMA at the three visits in partly and
months postpartum (Spearman’s rank-correlation: rs ¼ 0.29, not breast-feeding women is shown in Table 4. These two
P ¼ 0.007). groups were similar with respect to MMA and tHcy, and the
The mean body weight, mean folate and Hgb and median groups did not differ with respect to the percentages of
tHcy for all the participants at the three visits are presented smokers, coffee and alcohol drinkers or number of women
in Table 2. There was a statistically significant inverse who exercised on a daily basis. These 17 women were
relation between tHcy and folate (Spearman’s r À0.63, therefore combined into one reference group. There was no
Po0.001). change in mean folate and B12 or median tHcy throughout
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Table 3 Indicators of folate and B12 status at 3 weeks, 4 and 9 months postpartum in exclusively breast-feeding (both combined and separated in
supplemented and unsupplemented) and partly and not breast-feeding women
Exclusively breast- Supplemented Unsupplemented P-valuec Partly and not P-valued
feeding (n ¼ 72a) exclusively breast- exclusively breast- breast-feeding
feeding (n ¼ 36b) feeding (n ¼ 36) (n ¼ 17)
3 weeks folatee (nmol/l) 18.2 (7.8, 36.9) 22.9 (13.3,46.8) 14.5 (6.8, 35.0) o0.001f 17.6 (8.1, 29.5) 0.2f
3 weeks tHcyg (mmol/l) 5.8 (3.1, 8.3) 5.6 (3.3, 8.5) 6.1 (2.4, 8.8) 0.3h 6.1 (3.7, 10.4) 1.0h
3 weeks B12e (mol/l) 324 (214, 493) 301 (196, 437) 349 (233, 529) 0.04f 278 (170, 392) 0.01f
3 weeks MMAg (mmol/l) 0.17 (0.11, 0.34) 0.17 (0.11, 0.36) 0.17 (0.12, 0.26) 0.7h 0.14 (0.12, 0.25) 0.1h
4 months folatee (nmol/l) 15.5 (6.4, 36.0) 23.2 (14.6, 50.0) 10.4 (5.8, 18.6) o0.001f 15.1 (5.3, 37.5) 0.02f
4 months tHcyg (mmol/l) 6.1 (4.1, 10.3) 5.4 (3.5, 8.8) 7.0 (4.7, 15.3) o0.001h 5.0 (3.0, 14.7) 0.1h
4 months B12e (pmol/l) 320 (228, 491) 310 (227, 448) 330 (231, 495) 0.4f 288 (189, 408) 0.2f
4 months MMAg (mmol/l) 0.16 (0.09, 0.24) 0.15 (0.09, 0.23) 0.16 (0.10, 0.25) 0.3h 0.12 (0.07, 0.19) 0.009h
9 months folatee (nmol/l) 17.1 (9.1, 32.4) 23.0 (13.8, 41.1) 12.8 (7.5, 21.1) o0.001f 14.5 (7.7, 30.0) 0.3f
9 months tHcyg (mmol/l) 5.3 (3.6, 8.7) 4.5 (3.2, 7.3) 5.8 (3.8, 9.5) 0.002h 5.8 (2.8, 16.7) 1.0h
9 months B12e (pmol/l) 339 (237, 519) 333 (237, 517) 346 (233, 535) 0.6f 277 (192, 402) 0.02f
9 months MMAg (mmol/l) 0.14 (0.09, 0.22) 0.13 (0.09, 0.21) 0.15 (0.10, 0.22) 0.2h 0.11 (0.08, 0.19) 0.1h
a
n ¼ 71 concerning the B12 and MMA results.
b
n ¼ 35 concerning the B12 and MMA results.
c
Comparing supplemented and unsupplemented exclusively breast-feeding women.
d
Comparing unsupplemented exclusively breast-feeding women with partly and not breast-feeding women.
e
Mean (10 and 90 percentiles).
f
P-value tested with two-sample t-test.
g
Median (10 and 90 percentiles).
h
P-value tested with Wilcoxon’s rank-sum test.
Table 4 THcy and MMA 3 weeks, 4 and 9 months postpartum in the subgroups of partly breast-feeding and not breast-feeding women
3 weeks tHcya 3 weeks MMAa 4 months tHcya 4 months MMAa 9 months tHcya 9 months MMAa
(mmol/l) (mmol/l) (mmol/l) (mmol/l) (mmol/l) (mmol/l)
Partly breast-feeding, (n ¼ 8) 6.3 (3.8, 16.1) 0.14 (0.12, 0.25) 5.7 (3.8, 12.9) 0.14 (0.10, 0.20) 6.7 (2.8, 13.7) 0.14 (0.08, 0.20)
Not breast-feeding, (n ¼ 9) 5.8 (3.3, 9.0) 0.13 (0.11, 0.26) 5.0 (2.4, 21.8) 0.11 (0.05, 0.18) 5.8 (2.9, 28.7) 0.11 (0.09, 0.19)
P-value 0.7b 1.0b 0.6b 0.1b 0.4b 1.0b
a
Median (10 and 90 percentiles).
b
P-value tested with Wilcoxon’s rank-sum test.
the study among these women. Also, in this group the Also, the unsupplemented women – in contrast to supple-
median MMA decreased throughout the study (Wilcoxon’s mented women – had statistically significantly lower folate
rank-sum test: P ¼ 0.05). concentrations 4 months postpartum compared to 3 weeks
and 9 months postpartum (Figure 2). Only age differed
between the two groups (two-sample t-test: P ¼ 0.04); the
Vitamin supplemented compared to unsupplemented exclusively mean age was 33 (range 25–42) years among supplemented
breast-feeding women exclusively breast-feeding mothers years and 31 (range 20–
Women who breast-fed exclusively 4 months postpartum 41) years among unsupplemented exclusively breast-feeding
were further categorized into those supplemented with mothers at baseline.
vitamins (n ¼ 36) or not (n ¼ 36) at the 4 months visit,
Table 3: 22 took 200 mg/day folic acid and 14 took 400 mg/day.
The daily dose of B12 was 1 mg (n ¼ 29), 2–4.5 mg (n ¼ 4) and Women with increased levels of tHcy and/or MMA
more than 5 mg (n ¼ 3). At 9 months postpartum, 25 (69%) Moderate hyperhomocysteinaemia is traditionally defined as
took supplements with at least 200 mg folic acid and 1 mg B12 tHcy between 15 and 30 mmol/l (Refsum et al., 1998) and
on a daily basis. using 15 mmol/l as cutoff value for tHcy; six women had
Unsupplemented mothers had statistically significantly hyperhomocysteinaemia 4 months postpartum. Five of
higher tHcy at 4 and 9 months postpartum and statistically those were exclusively breast-feeding and four of those were
significantly lower folate at all three visits than supplemen- unsupplemented. They all stopped lactation prior to the 9
ted mothers, while no difference was observed for MMA. months follow-up, and in four of the women, the concen-
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tHcy MMA
10.0 0.25
Vitamin supplemented
Vitamin unsupplemented
tHcy, µmol/L
MMA, µmol/L
8.0 0.20
6.0 0.15
4.0 0.10
1 2 3 1 2 3
Visit Visit
Folate B12
30 390
Folate, µmoL/L
B12, pmol/L
20 360
10 330
0 300
1 2 3 1 2 3
Visit Visit
Figure 2 Concentrations of vitamins and metabolites in serum and plasma in the lactation period. THcy, MMA, folate and B12 in breast-feeding
women (n ¼ 72) at 3 weeks, 4 months and 9 months postpartum according to vitamin supplementation. All women were exclusively breast-
feeding at 4 month postpartum and all were giving the child supplementary meals at 9 months postpartum. Mean values are shown with s.e.m.
as vertical lines.
tration of tHcy had decreased to below 10 mmol/l at the 9 Discussion
months visit, exhibiting a significant decrease nearly to but
not within the reference interval. If lactation depleted the women of folate and B12, we would
Concerning lifestyle, only cups of coffee per day differed expect folate and B12 concentrations to decrease from 3
for the six women with hyperhomocysteinaemia as com- weeks to 4 months postpartum and to return to normal after
pared to the remaining group (Wilcoxon’s rank-sum test: cessation of lactation. Considering the presumed inverse
P ¼ 0.03). All six hyperhomocysteinaemic women drank relation between folate and tHcy and B12 and MMA, we
coffee daily (1–5 cups/day) compared to 47 (57%) among would also expect an initial increase in tHcy and MMA
the normohomocysteinaemic women (1–7 cups/day) (data followed by a decrease.
not shown). We found only a modest association between lactation and
One exclusively breast-feeding participant had high con- the indicators of folate and B12 status in this well-nourished
centrations of MMA, decreasing from 1.40 mmol/l 3 weeks population of Danish women, but we did observe changes
postpartum to 0.53 mmol/l 9 months postpartum (reference suggesting that folate supplementation may be warranted
interval 0.08–0.28 mmol/l). She had no other signs or throughout lactation.
symptoms of B12 deficiency and normal tHcy and a high As expected, we found a statistically significant inverse
B12 (41000 pmol/l). She took vitamin supplements at the relation between tHcy and folate, also found in our
first two visits. In addition, three exclusively breast-feeding preliminary cross-sectional investigation (Ramlau-Hansen
women had MMA above the reference limit (0.28 mmol/l) 4 et al., 2003) and in agreement with results from the
months postpartum. At the 9 months visit, the MMA had double-blind, randomized, longitudinal supplementation
declined to below 0.28 mmol/l in two of these women. One trial by Mackey and Picciano (1999).
mother had both increased MMA and tHcy, suggesting At 4 months postpartum, vitamin supplemented exclu-
relative B12 deficiency as the cause of the increased MMA sively breast-feeding women had statistically significantly
and tHcy. higher folate and lower tHcy than unsupplemented breast-
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7. Folate and B12 in lactation
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126
feeding women. This is in accordance with earlier observa- the outcome measures were results from blood samples,
tions by Shapiro et al. (1965), Keizer et al. (1995) and Smith which were analysed by blinded staff. Furthermore, the
et al. (1983). On the other hand, Thomas et al. (1980) were samples were collected according to standardized procedures
unable to detect this difference in folate concentrations and the risk of preanalytic variation was minimized. The
between supplemented and unsupplemented women, prob- relative strict inclusion and exclusion criteria resulted in a
ably explained by the small number of participants in their homogeneous study population and the risk of confounding
cross-sectional study. The vitamin supplemented and the was hereby reduced. In general, the variables that potentially
unsupplemented exclusively breast-feeding women were could give rise to confounding because of their potential
comparable with regard to other factors that can have an effect on tHcy (e.g. smoking, coffee, alcohol, age and daily
impact on the results. We take our results together with exercise (Refsum et al., 1998)) were equally distributed in the
those of others to indicate that folate supplementation may compared groups.
be warranted throughout the lactating period. Our results were obtained on a somewhat selected group of
In order to judge whether the changes in the vitamin- women. The participants were generally healthier than the
related parameters is caused by the pregnancy or by the background population, as illustrated by the relatively small
lactation per se, one would have liked to compare results number of smoking participants (7% at 3 weeks postpartum)
obtained on mothers who never breast-fed with those breast- compared to the number of Danish women smoking during
feeding for a prolonged period of time. Owing to the high pregnancy (25%) (Hegaard et al., 2004). This may also be the
frequency of breast-feeding, we were unable to study a group reason why a larger than expected proportion of the women
of mothers who did not breast-feed. However, due to the breast-fed their children 4 months postpartum (Skajaa,
similarity, we chose to combine the partly and not breast- 1992). When extrapolating our findings to women with a
feeding women into one reference group. Comparing this less optimal diet (e.g. women in developing countries), one
group to the one at the highest risk for developing vitamin would expect that lactation would pose a greater risk of
depletion, the unsupplemented exclusively breast-feeding developing suboptimal folate status, as previously found in,
women revealed a minor but interesting difference. Folate for example, low-income Brazilian mothers (Donangelo
was lower 4 months postpartum in the ‘high-risk’ group of et al., 1989; Lehti, 1989).
women as compared to the reference group. This supports a Six women had hyperhomocysteinaemia (P-tHcy
negative folate balance in the unsupplemented breast- 415 mmol/l) 4 months postpartum. Four of these reported
feeding women, unexplained by the pregnancy period but to be unsupplemented and exclusively breast-feeding, one to
due to breast-feeding, although no difference in tHcy was be supplemented and exclusively breast-feeding and one to
observed between the groups. We chose the measurement of be unsupplemented and not breast-feeding. The reason for
folate in serum rather than in whole blood for practical the high level of tHcy in the latter two women is not known.
reasons and in order to get a more timely measure of the One possibility could be a genetic predisposition (e.g. the
possible fluctuations in folate intake, by supplementation or MTHFR polymorphism (Refsum et al., 1998)). A total of nine
by food. (10%) of the women had clearly elevated metabolites,
We observed a minor decrease (5.8%) in the mean body suggesting cobalamin deficiency, folate deficiency or both.
weight for all the participants from 3 weeks to 9 months Possible consequences for the development of the child
postpartum. However, it seems unlikely that this decrease in taken into account, and awareness of the vitamin status in
body weight has influenced the results, since neither Hgb the lactation period are important according to these results.
nor tHcy or folate increased during the study period. In conclusion, we report no overall indication of depletion
The participants were asked if they were vegetarians or of the folate and B12 stores during the lactation period in
consumed an average Danish diet including meat, but no this well-nourished Danish population of lactating women.
detailed information about their diet was recorded. Danish However, the folate-supplemented women had lower tHcy
fertile women in general are estimated to consume 248 mg and higher folate levels, suggesting a beneficial effect of
(10 and 90 percentiles: 159 and 347 mg) folate/day (Danish supplementation with folate throughout lactation.
Veterinary and Food Administration, 1997) and we do not
expect that the participants consumed less folate than the
general population of fertile women in Denmark. Acknowledgements
The exposure status (lactation and vitamin supplementa-
tion) was based on self-reported information. Misclassifica- We thank the women who participated in the study, the staff
tion regarding lactation status seems unlikely. However, at The Osteoporosis Clinic, Aarhus University Hospital,
information on vitamin supplement is probably more Denmark for collecting the blood samples, Lene Damm
critical. It is well known that participants often over-report Christensen and the other staff at Department of Clinical
in the most socially acceptable way, and maybe some women Biochemistry, Aarhus University Hospital, Skejby, Denmark
overstated their use of vitamins. The result of this potential for analysing the blood samples, the secretaries at Depart-
over-report would be that our results are underestimated. ment of Obstetrics and Gynaecology, Aarhus University
The risk of information bias in the study was minimal, since Hospital, Skejby, Denmark for assisting during the identifica-
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8. Folate and B12 in lactation
CH Ramlau-Hansen et al
127
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