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Importanza del supporto di acido folico durante l'allattamento


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Importanza del supporto di acido folico durante l'allattamento

  1. 1. European Journal of Clinical Nutrition (2006) 60, 120–128 & 2006 Nature Publishing Group All rights reserved 0954-3007/06 $30.00 ARTICLEFolate and vitamin B12 in relation to lactation:a 9-month postpartum follow-up studyCH Ramlau-Hansen1,2, UK Møller1,3, TB Henriksen4,5, E Nexø6 and J Møller71 ˚ 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, ˚ 4Denmark; 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, DenmarkObjective: To investigate the relation between lactation and markers of folate and vitamin B12 (B12) deficiency in womenwith 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 and9 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 monthspostpartum. 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 weekspostpartum, 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 monthspostpartum, 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 exclusivelybreast-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 thispopulation. However, folate-supplemented women had lower tHcy and higher folate levels, suggesting a beneficial effect ofsupplementation with folate throughout lactation.Sponsorship: The Biomedical Laboratory Scientist Education and Research Fund and LEO Pharma Research Foundationsupported 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 2005Keywords: breast-feeding; serum folate; serum B12; plasma homocysteine; serum methylmalonic acidIntroduction two vitamins (Allen, 1994; O’Connor, 1994; O’Connor et al., 1997; Bjorke Monsen and Ueland, 2003). B12 insufficiencyFolate and vitamin B12 (B12) is released into human milk, during lactation may cause anaemia and neurological damagehence 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 outcomeE-mail: such as preterm delivery and birth defects in the followingGuarantor: CH Ramlau-Hansen. pregnancy is increased (Smits and Essed, 2001).Contributors: CHR, JM, UKM and TBH designed the study. CHR and UKMcollected the samples. JM and EN were responsible for the biochemical Data concerning folate- and B12 status during lactation isanalyses; CHR performed the statistical analyses, wrote the original manuscript conflicting. Several investigations show an increased risk ofand edited all subsequent versions. JM, EN and TBH contributed to the suboptimal folate status (Matoth et al., 1965; Donangelointerpretation 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 storespublished online 28 September 2005 during the lactation period (Shapiro et al., 1965; Tamura
  2. 2. Folate and B12 in lactation CH Ramlau-Hansen et al 121et al., 1980; Butte et al., 1981; Sneed et al., 1981; Smith et al., Women were invited to participate provided they met the1983; 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 bornPicciano, 1999) in women with no folic acid supplementa- 38–42 completed weeks of gestation with a birth weighttion and with a diet insufficient in folate. Others have shown above 2500 g, blood loss less than 500 ml during labour, lessthat lactation does not affect the level of folate (Cole et al., than 14 drinks of alcohol per week (the upper limit of1974; Thomas et al., 1980; Ek, 1983). A possible B12 alcohol consumption for women recommended by thedepletion 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 bypotential deficiency with folate in whole blood reflecting the telephone. The study was approved by the local ethicsuptake of folate during erythropoiesis, thus giving a measure committee (Project no. 20030093) and was monitored by theof the long-term average folate intake and folate in plasma local Good Clinical Practice (GCP) Unit. Written informedreflecting the intake in recent days. Also, metabolites consent was obtained from all the participants.accumulating upon vitamin depletion can be used asmarkers of the vitamin status. The concentration ofcirculating homocysteine (tHcy) is elevated in both folate Study designand B12 deficiency (Refsum et al., 1998; Stabler and Allen, Data were collected approximately 3 weeks (baseline), 42004), 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 UniversityKlee, 2000; Stabler and Allen, 2004). In healthy young Hospital, Denmark during May 2003–April 2004. It wasadults, folate status is the main determinant of the tHcy, and assumed that if lactation had any influence on markers oftHcy 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 regardingduring 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? Howdelivery (Andersson et al., 1992). The decrease in tHcy during many cigarettes per day?’), intake of coffee (‘How many cupspregnancy is suggested to be caused by an endocrine-based of coffee do you drink per day?’) and alcohol (How manymechanism (Murphy et al., 2002) or a change in renal drinks of alcohol do you drink per week?’), physical exercisehaemodynamics during pregnancy (Brattstrom, 2003). A (‘Do you exercise (defined as minimum 30 min of walkingdecrease 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 their2002). 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 mealand no study on the relationship between lactation and (no eggs, less than 150 g of meat and less than 2 dl of milk orMMA 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 smallinfluence of lactation on markers of folate and B12 status in meal will have no influence (Refsum et al., 2004). Bloodwomen 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 wereSubjects 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 wereAll 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 within2003 were considered for participation. The study was part of 30 min of sampling (Rasmussen and Moller, 2000). Plasmaan investigation also on the effect of lactation and bone and serum were stored at À201C until analysed. Blooddensity (Moller, 2003). samples for haemoglobin (Hgb) and MCV were analysed on European Journal of Clinical Nutrition
  3. 3. Folate and B12 in lactation CH Ramlau-Hansen et al122 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.European Journal of Clinical Nutrition
  4. 4. Folate and B12 in lactation CH Ramlau-Hansen et al 123Table 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-valueAge (years)a 31.9 (20–42) 29.9 (23–36) 0.1bPrimiparaus, n (%) 29 (40) 12 (71) 0.02cNumber of childrend 2 (1–4) 1 (1–3) 0.05eSmoking, n (%) 4 (6) 2 (12) 0.3fCoffee, n (%) 39 (54) 7 (41) 0.3cAlcohol, n (%) 38 (53) 7 (41) 0.4cExercise, n (%) 48 (67) 10 (59) 0.5cFolic acid during pregnancy, n (%) 70 (97) 17 (100) 1.0cFolic acid and B12 during the study period, n (%) 45 (63) 10 (59) 0.8cHgb (mmol/l)Mean (10 and 90 percentiles) 8.4 (7.5,9.2) 8.3 (7.7,9.0) 0.6ba 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 womenDanish diet including meat. The vegetarian did not differ The median of tHcy and MMA and mean of folate and B12 atfrom 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 inally declined from 86 at 3 weeks to 72 at 4 months mean folate and B12 across the three time periods among thepostpartum. 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 monthsextent 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 concentrationbetween duration of the lactation period and tHcy at 9 decreased statistically significant throughout the studymonths 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 andmonths postpartum (Spearman’s rank-correlation: rs ¼ 0.29, not breast-feeding women is shown in Table 4. These twoP 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 oftHcy for all the participants at the three visits are presented smokers, coffee and alcohol drinkers or number of womenin Table 2. There was a statistically significant inverse who exercised on a daily basis. These 17 women wererelation between tHcy and folate (Spearman’s r À0.63, therefore combined into one reference group. There was noPo0.001). change in mean folate and B12 or median tHcy throughout European Journal of Clinical Nutrition
  5. 5. Folate and B12 in lactation CH Ramlau-Hansen et al124 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-European Journal of Clinical Nutrition
  6. 6. Folate and B12 in lactation CH Ramlau-Hansen et al 125 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 VisitFigure 2 Concentrations of vitamins and metabolites in serum and plasma in the lactation period. THcy, MMA, folate and B12 in breast-feedingwomen (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 vertical lines.tration of tHcy had decreased to below 10 mmol/l at the 9 Discussionmonths visit, exhibiting a significant decrease nearly to butnot 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 3for the six women with hyperhomocysteinaemia as com- weeks to 4 months postpartum and to return to normal afterpared to the remaining group (Wilcoxon’s rank-sum test: cessation of lactation. Considering the presumed inverseP ¼ 0.03). All six hyperhomocysteinaemic women drank relation between folate and tHcy and B12 and MMA, wecoffee daily (1–5 cups/day) compared to 47 (57%) among would also expect an initial increase in tHcy and MMAthe 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-nourishedcentrations of MMA, decreasing from 1.40 mmol/l 3 weeks population of Danish women, but we did observe changespostpartum to 0.53 mmol/l 9 months postpartum (reference suggesting that folate supplementation may be warrantedinterval 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 inverseB12 (41000 pmol/l). She took vitamin supplements at the relation between tHcy and folate, also found in ourfirst two visits. In addition, three exclusively breast-feeding preliminary cross-sectional investigation (Ramlau-Hansenwomen had MMA above the reference limit (0.28 mmol/l) 4 et al., 2003) and in agreement with results from themonths postpartum. At the 9 months visit, the MMA had double-blind, randomized, longitudinal supplementationdeclined 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 significantlyand tHcy. higher folate and lower tHcy than unsupplemented breast- European Journal of Clinical Nutrition
  7. 7. Folate and B12 in lactation CH Ramlau-Hansen et al126 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-European Journal of Clinical Nutrition
  8. 8. Folate and B12 in lactation CH Ramlau-Hansen et al 127tion of eligible women, Gurli Tanderup and DPC Scandinavia Klee GG (2000). Cobalamin and folate evaluation: measurement offor providing reagents for the tHcy and folate analyses and methylmalonic acid and homocysteine vs vitamin B(12) and folate. Clin Chem 46, 1277–1283.finally The Biomedical Laboratory Scientist Education and Lehti KK (1989). Iron, folic acid and zinc intakes and status of lowResearch Fund and LEO Pharma Research Foundation for socio-economic pregnant and lactating Amazonian women. Eur Jtheir financial support. Clin Nutr 43, 505–513. Mackey AD, Picciano MF (1999). Maternal folate status during extended lactation and the effect of supplemental folic acid. Am J Clin Nutr 69, 285–292. Matoth Y, Pinkas A, Sroka C (1965). Studies on folic acid in infancy.References 3. Folates in breast fed infants and their mothers. Am J Clin Nutr 16, 356–359.Allen LH (1994). Vitamin B12 metabolism and status Metz J (1970). Folate deficiency conditioned by lactation. Am J Clin during pregnancy, lactation and infancy. Adv Exp Med Biol 352, Nutr 23, 843–847. 173–186. Michaud JL, Lemieux B, Ogier H, Lambert MA (1992). NutritionalAllen RH, Stabler SP, Savage DG, Lindenbaum J (1990). Diagnosis of vitamin B12 deficiency: two cases detected by routine newborn cobalamin deficiency I: usefulness of serum methylmalonic acid urinary screening. Eur J Pediatr 151, 218–220. and total homocysteine concentrations. Am J Hematol 34, 90–98. Moller UK (2003). Calcium homostase og knogleomsætning underAndersson A, Hultberg B, Brattstrom L, Isaksson A (1992). Decreased og efter amning. Thesis, Protokol i forbindelse med speciale pa ˚ serum homocysteine in pregnancy. Eur J Clin Chem Clin Biochem Aarhus Universitet. 30, 377–379. Murphy MM, Scott JM, McPartlin JM, Fernandez-Ballart JD (2002).Bates CJ, Fuller NJ, Prentice AM (1986). Folate status during The pregnancy-related decrease in fasting plasma homocysteine is pregnancy and lactation in a West African rural community. not explained by folic acid supplementation, hemodilution, or a Hum Nutr Clin Nutr 40, 3–13. decrease in albumin in a longitudinal study. Am J Clin Nutr 76,Bjorke Monsen AL, Ueland PM (2003). Homocysteine and methyl- 614–619. malonic acid in diagnosis and risk assessment from infancy to O’Connor DL (1994). Folate status during pregnancy and lactation. adolescence. Am J Clin Nutr 78, 7–21. Adv Exp Med Biol 352, 157–172.Brattstrom L (2003). Pregnancy-related decrease in total plasma O’Connor DL, Green T, Picciano MF (1997). Maternal folate status homocysteine. Am J Clin Nutr 77, 993–994. and lactation. J Mammary Gland Biol Neoplasia 2, 279–289.Bruinse HW, van der BH, Haspels AA (1985). Maternal serum folacin Ramlau-Hansen CH, Moller UK, Moller J, Thulstrup AM (2003). levels during and after normal pregnancy. Eur J Obstet Gynecol Lactation – a risk factor for elevated plasma homocysteine? Ugeskr Reprod Biol 20, 153–158. Laeger 165, 2819–2823.Butte NF, Calloway DH, Van Duzen JL (1981). Nutritional assessment Rasmussen K, Moller J (2000). Total homocysteine measurement in of pregnant and lactating Navajo women. Am J Clin Nutr 34, 2216– clinical practice. Ann Clin Biochem 37, 627–648. 2228. Rasmussen K, Moller J, Lyngbak M (1999). Within-person variationChery C, Barbe F, Lequere C, Abdelmouttaleb I, Gerard P, Barbarino P of plasma homocysteine and effects of posture and tourniquet et al. (2002). Hyperhomocysteinemia is related to a decreased application. Clin Chem 45, 1850–1855. blood level of vitamin B12 in the second and third trimester of Rasmussen K, Moller J, Lyngbak M, Pedersen AM, Dybkjaer L (1996). normal pregnancy. Clin Chem Lab Med 40, 1105–1108. Age- and gender-specific reference intervals for total homo-Cikot RJ, Steegers-Theunissen RP, Thomas CM, de Boo TM, Merkus cysteine and methylmalonic acid in plasma before and after HM, Steegers EA (2001). Longitudinal vitamin and homocysteine vitamin supplementation. Clin Chem 42, 630–636. levels in normal pregnancy. Br J Nutr 85, 49–58. Refsum H, Smith AD, Ueland PM, Nexo E, Clarke R, McPartlin JCole JK, Kimber RJ, Kutkaite D (1974). Serum folic acid levels and et al. (2004). Facts and recommendations about total homo- pregnancy in South Australia. A study of the factors associated cysteine determinations: an expert opinion. Clin Chem 50, 3–32. with low serum folic acid levels with particular reference to racial Refsum H, Ueland PM, Nygard O, Vollset SE (1998). Homocysteine groups and lactation. Med J Aust 1, 421–424. and cardiovascular disease. Annu Rev Med 49, 31–62.Danish Veterinary and Food Administration (1997). Folat og Salmenpera L, Perheentupa J, Siimes MA (1986). Folate nutrition is neuralrørsdefekt. Schultz bogtryk: Copenhagen, Denmark. optimal in exclusively breast-fed infants but inadequate in some ofDonangelo CM, Trugo NM, Koury JC, Barreto Silva MI, Freitas LA, their mothers and in formula-fed infants. J Pediatr Gastroenterol Feldheim W et al. (1989). Iron, zinc, folate and vitamin B12 Nutr 5, 283–289. nutritional status and milk composition of low-income Brazilian Shapiro J, Alberts HW, Welch P, Metz J (1965). Folate and vitamin B- mothers. Eur J Clin Nutr 43, 253–266. 12 deficiency associated with lactation. Br J Haematol 11, 498–504.Dostalova L (1984). Vitamin status during puerperium and lactation. ˚ ˚ Skajaa E (1992). Sundhedsprofil af smabørn og smabørnsfamilier i Ann Nutr Metab 28, 385–408. ˚ Arhus – En deskriptiv undersøgelse af 3383 børn i 8-ma ˚nedersEk J (1983). Plasma, red cell, and breast milk folacin concentrations alderen, 1992. Dansk institut for Sundheds – og Sygeplejeforening in lactating women. Am J Clin Nutr 38, 929–935. (report).Gronbaeck MN, Iversen L, Olsen J, Becker PU, Hardt F, Sørensen TI Smith AM, Picciano MF, Deering RH (1983). Folate supplementation (1997). Sensible drinking limits. Ugeskr Laeger 159, 5939–5945. during lactation: maternal folate status, human milk folateHegaard HK, Kjaergaard H, Moller LF, Wachmann H, Ottesen BS content, and their relationship to infant folate status. J Pediatr (2004). The prevalence of passive smoking among pregnant Gastroenterol Nutr 2, 622–628. women in Denmark on their first visit to the midwife. Ugeskr Smits LJ, Essed GG (2001). Short interpregnancy intervals and Laeger 166, 3706–3711. unfavourable pregnancy outcome: role of folate depletion. LancetKang SS, Wong PW, Zhou JM, Cook HY (1986). Total homocyst(e)ine 358, 2074–2077. in plasma and amniotic fluid of pregnant women. Metabolism 35, Sneed SM, Zane C, Thomas MR (1981). The effects of ascorbic acid, 889–891. vitamin B6, vitamin B12, and folic acid supplementation on theKeizer SE, Gibson RS, O’Connor DL (1995). Postpartum folic breast milk and maternal nutritional status of low socioeconomic acid supplementation of adolescents: impact on maternal lactating women. Am J Clin Nutr 34, 1338–1346. folate and zinc status and milk composition. Am J Clin Nutr 62, Stabler SP, Allen RH (2004). Vitamin B12 deficiency as a worldwide 377–384. problem. Annu Rev Nutr 24, 299–326. European Journal of Clinical Nutrition
  9. 9. Folate and B12 in lactation CH Ramlau-Hansen et al128 Tamura T, Yoshimura Y, Arakawa T (1980). Human milk folate and Villalpando S, Latulippe ME, Rosas G, Irurita MJ, Picciano MF, folate status in lactating mothers and their infants. Am J Clin Nutr O’Connor DL (2003). Milk folate but not milk iron concentrations 33, 193–197. may be inadequate for some infants in a rural farming community Thomas MR, Sneed SM, Wei C, Nail PA, Wilson M, Sprinkle III EE in San Mateo, Capulhuac, Mexico. Am J Clin Nutr 78, 782–789. (1980). The effects of vitamin C, vitamin B6, vitamin B12, folic Weiss R, Fogelman Y, Bennett M (2004). Severe vitamin B12 acid, riboflavin, and thiamin on the breast milk and maternal deficiency in an infant associated with a maternal defi- status of well-nourished women at 6 months postpartum. Am J ciency and a strict vegetarian diet. J Pediatr Hematol Oncol 26, Clin Nutr 33, 2151–2156. 270–271.European Journal of Clinical Nutrition