A Longitudinal Study of Serum Cobalamins and Its Binding Proteins in Lactating Women

ORIGINAL ARTICLE A longitudinal study of serum cobalamins and its binding proteins in lactating women

AL Mørkbak1*, CH Ramlau-Hansen2, UK Møller3, TB Henriksen4, J Møller5 and E Nexø1

1Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus Sygehus, Aarhus, Denmark; 2Department of Occupational Medicine, Aarhus University Hospital, Aarhus Sygehus, Aarhus, Denmark; 3Department of Endorinology, Aarhus University Hospital, Aarhus Sygehus, Aarhus, Denmark; 4Perinatal Epidemiology Research Unit, Department of Obstetrics and Paediatrics, Aarhus University Hospital, Skejby Sygehus, Aarhus, Denmark and 5Department of Clinical Biochemistry, Aarhus University Hospital, Skejby Sygehus, Aarhus, Denmark

Objective: To examine longitudinal changes in serum cobalamins, transcobalamin (TC) and haptocorrin (HC) during lactation and to investigate the influence of vitamin B12 supplementation on these parameters. Design: A 9-month follow-up study. Subjects and methods: Lactating mothers (N ¼ 89) including 23 supplemented with vitamin B12 (1–18 mg/daily), 41 partly supplemented and 25 not supplemented. Blood samples collected 3 weeks (baseline) and 4 and 9 months post-partum were analysed for cobalamins, TC and HC. Both the total concentration and the cobalamin-saturated form (holo) of TC and HC were analysed. Results: No significant differences were observed in serum cobalamins or its binding proteins related to supplementation with vitamin B12 or the duration of lactation. Serum cobalamins remained unchanged from 3 weeks to 9 months post-partum. Total TC (holoTC) (median7s.e. pmol/l) decreased between 3 weeks (710723 (85712)) and 9 months (602721 (76711)) (Po0.0001 (P ¼ 0.0002)), whereas total HC (holoHC) increased from (422711 (30079)) at 4 months to (455713 (317710)) to 9 months post-partum (Po0.0001 (Po0.0001)). Conclusion: We report a decrease in TC and an increase in HC during a 9-month period post-partum. No differences were observed between the vitamin B12-supplemented and the unsupplemented groups. Thus, supplementation with vitamin B12 has no impact on the circulating level of serum cobalamins or its binding proteins in a Danish population of lactating mothers. European Journal of Clinical Nutrition (2007) 61, 184–189. doi:10.1038/sj.ejcn.1602502; published online 9 August 2006

Keywords: transcobalamin; haptocorrin; holoTC; holoHC; cobalamins

Introduction in breast milk (Baker et al., 1962; Specker et al., 1990; Black

et al., 1994) and with the vitamin B12 status of the baby The requirements for many micronutrients such as vitamin (Specker et al., 1988), indicating that the amount of vitamin

B12 are higher during pregnancy and lactation owing to B12 released into the breast milk and thereby available to the transfer to the baby (Allen, 1994; Mackey and Picciano, baby depends on the vitamin B12 status of the mother. 1999). A few studies have shown that the vitamin B12 status Many babies have low serum levels of cobalamins (Specker of the mother is associated with the content of vitamin B12 et al., 1990; Minet et al., 2000; Monsen et al., 2001). In most

cases, an apparent vitamin B12 deficiency may be attributed to a low vitamin B12 status in the mother (Specker et al., *Correspondence: Dr AL Mørkbak, Department of Clinical Biochemistry, 1988, 1990; Casterline et al., 1997; Ueland and Monsen Aarhus University Hospital, Aarhus Sygehus, Nørrebrogade 44, DK 8000 2003). These observations suggest that lactating mother do Aarhus, Denmark. E-mail: [email protected] Guarantor: AL Mørkbak. not have a sufficient intake of vitamin B12 to ensure the Contributors: EN and ALM were responsible for analyses and wrote the original needs of both the mother and the baby and emphasize the manuscript. CHR, JM, UKM and TBH designed the study. CHR and UKM need to examine the status of cobalamin and its binding collected samples. CHR, JM, UKM and TBH commented on the manuscript. All proteins in lactating mothers. authors approved the final version of the manuscript. Received 20 July 2005; revised 27 April 2006; accepted 14 June 2006; In healthy non-pregnant adults, most of the circulating published online 9 August 2006 cobalamins are bound to haptocorrin (HC), and this protein Serum cobalamins and its binding proteins in lactating women AL Mørkbak et al 185 is almost fully saturated with vitamin B12 (holoHC). Less immunosorbent assay (ELISA) (Nexø et al., 2000; Mørkbak than one-third of the circulating cobalamins are bound to et al., 2005). Total coefficient of variation (CV) was less than transcobalamin (TC), and most of this protein circulates 10%. unsaturated with cobalamins. HoloTC is the metabolically Serum holoHC (holoTC) was quantified by measurement active form responsible for the transport of cobalamin to of the amount of HC (TC) not removed by treating the the cells and thereby also for the delivery of cobalamin to samples with vitamin B12-covered beads following the the mammary glands (Nexo and Hollenberg, 1980; Willis protocol developed for measurement of holoTC (Nexø et al., 1989). et al., 2002). In brief, 90 ml of serum was pretreated with Longitudinal data on the level of the cobalamin-binding neuraminidase and peptide N-glycosidase F dissolved in proteins and cobalamins during lactation are limited phosphate buffer, 0.1 mol/l pH 7.4, as described by Mørkbak (Fernandes-Costa and Metz, 1982; Dosta´lova´, 1984; Donan- et al (Mørkbak et al., 2005). For measurement of holoHC, gelo et al., 1989). Moreover, the available data were reported 100 ml of pretreated serum solution was incubated for 1 h before the development of assays that measure the binding with 10 ml vitamin B12 beads before magnetic separation proteins HC and TC directly (Nexø et al., 2000; Mørkbak of the beads from the supernatant containing the holoHC. et al., 2005). A 290 ml portion of 0.1 M sodium phosphate buffer pH 8.0 The aim of the present study was to examine the containing 0.1% human albumin was added to 50 ml super- longitudinal changes in serum cobalamins and in total and natant, and holoHC was analysed by the same ELISA as used holoTC and holoHC during lactation and to investigate for measurement of total HC. The total CV for the holoHC the influence of vitamin B12 supplementation on these assay was 10% at 157 pmol/l (43 estimates determined in parameters. 43 runs) and 9% at 194 pmol/l (40 estimates determined in 40 runs). TC saturation was calculated as holoTC divided by total Subjects and methods TC; likewise, HC saturation was calculated as holoHC divided by total HC. Study design and populations Prolactin in plasma was measured using the Immuno The study was designed as a follow-up study including 91 Assay system on the Advia Centauer from Bayer A/S, healthy mothers. Two non-lactating individuals were ex- Leverkusen, Germany. cluded. The study population has previously been described in detail (Ramlau-Hansen et al., 2006). Briefly, the partici- Statistical analyses pants were apparently healthy with no use of medication The distribution of weight, cobalamins, prolactin, TC and and no abuse of alcohol, uncomplicated pregnancy and a HC (holo and total) was skewed, and therefore statistical blood loss less than 500 ml during labour. They gave birth to analysis was performed using non-parametric statistics a single child at a gestation age of 38–42 weeks and with a (Mann–Whitney test, Wilcoxon matched pair signed-ranks birth weight of more than 2500 g. Blood samples were drawn test and Friedman’s test (Po0.05)). Associations were at baseline (3 weeks post-partum), first follow-up (4 months analysed using linear regression. All statistics were performed post-partum) and second follow-up (9 months post-partum). in GraphPad Prism version 4.00 for Windows (GraphPad At all three collections, the participants were interviewed Software, San Diego, CA, USA, www.graphpad.com). with respect to degree of lactation and use of vitamin supplements (name of preparation and dose). Furthermore, smoking habits, coffee and alcohol intake, medications, Results exercise level, illnesses and diet (meat eater or vegetarian) were registered. The study was approved by the local ethics The study population was 89 post-partum lactating mothers committee (project nos. 20030093 and 20030094) and with a lactation period ranging from 1.5 to more than monitored by the local unit for Good Clinical Practice. 9 months (51% had not stopped lactating after 9 months). For establishment of a reference interval, plasma samples The median (range) duration of the lactation period for from 71 healthy female donors (age: 23–61 years) were also the 42 women who had stopped lactating at 9 months was analysed. 28 weeks (6–40 weeks). Of the 89 mothers, 23 were supplemented with vitamin

B12 (1–18 mg, median ¼ 1 mg per day) through out follow-up, Sampling and analytical methods 41 were partly supplemented (1–9 mg, median ¼ 1 mg per day Venous blood was drawn for preparation of serum and indicated at one or two of the visits) and 25 were not plasma at 3 weeks (baseline) and 4 and 9 months post- supplemented at all. Selected baseline characteristics of partum. Samples were stored at À201C until use. Serum the 89 lactating women are listed in Table 1. A statistically cobalamins were measured on the Advia Centauer from significant decrease in the weight of the lactating mothers Bayer A/S, Leverkusen, Germany. Total TC and HC in serum (range 3.5–(À14) kg, median ¼À4.1 kg) was observed from were measured by in-house sandwich enzyme-linked baseline to 9 months post-partum (Friedman’s test,

European Journal of Clinical Nutrition Serum cobalamins and its binding proteins in lactating women AL Mørkbak et al 186 Table 1 Baseline age, lactation, parity, diet, vitamin B12 supplementa- a 900 200 tion and smoking, alcohol and coffee habits of the lactating women (n ¼ 89)

Mean (range) 700 150 [percentage] * Age (years) 31 (20–42) Vitamin B12 supplemented (mg vitamin B12 3 (1–13.5) [63] 500 100 HoloTC, pmol/L HoloTC, per day) TC, pmol/L Total Smoking (cigarettes per day) 4 (2–10) [7] * Alcohol (drinks per day) 2 (1–7) [49] 300 50 Coffee (cups per day) 2 (1–5) [53] 0 2 4 6 8 10 Exclusive lactating [96] Time, months Primiparus [44] Vegetarian diet [1] b 600

500 Po0.0001). This decrease showed a weak but statistically * significant positive association with the increase in holoHC 400 (r2 ¼ 0.06, P ¼ 0.02, n ¼ 86) and cobalamins (r2 ¼ 0.05, P ¼ 0.04, n ¼ 86). No associations were seen with changes in 300 * holoTC, total TC or total HC.

The weight of the baby increased (range 2895–7980 g, pmol/L and holoHC, Total 200 median ¼ 4880 g) from baseline to 9 months post-partum. 0 2 4 6 8 10 The increase was not associated with the change in Time, months cobalamins, TC or HC (holo and total) from baseline to 9 months post-partum. c 600 There were no statistically significant associations between the extent of vitamin B supplementation and levels of 12 500 serum cobalamins, TC and HC (holo and total) at baseline, 4 months or 9 months post-partum. Furthermore, the levels of serum cobalamins, TC and HC (total and holo) after 400 9 months showed no statistical difference between the

groups of supplemented (supplemented with vitamin B12 at 300 all three visits, N ¼ 25) and unsupplemented (no supplemen- pmol/L Cobalamins, N tation with vitamin B12 at any of the three visits, ¼ 25) 200 lactating women (data not shown). 0 2 4 6 8 10 As expected, the prolactin level decreased (Friedman’s test, Time, months Po0.0001) from baseline (range 0.04–2.4 IU/l, median ¼ Figure 1 Median and interquartile range of total and holoTC (a), 0.6 IU/l) to 9 months (range 0.01–0.8 IU/l, median ¼ 0.15 total and holoHC (b) and total cobalamins (c) by time in lactating mothers. (K) Holoprotein level; (J) the total protein level. IU/l) post-partum. The decrease was not associated with the % Statistically significant difference over time when tested with change in serum cobalamins, TC and HC (holo and total) Friedman’s test (P ¼ 0.0002 for holoTC and p0.0001 for the other from baseline to 9 months post-partum. variables), N ¼ 86. No blood sample was drawn at first and second The levels of serum cobalamins, TC and HC (holo and follow-up for two individuals and at second follow-up for one individual. These individuals were left out in the Friedman’s test. total) after 9 months for mothers having lactated o8 months (N ¼ 30) and mothers having lactated 48 months (N ¼ 56) were compared. No statistically significant differences in levels of these biomarkers in relation to the extent of lacta- and Table 2). A significant decrease was seen for holoTC, tion were observed (Mann–Whitney test, P40.05). There- comparing the level at baseline and at 9 months (Friedman’s fore, the results from all 89 women were analysed together to test, P ¼ 0.0002) (Figure 1a and Table 2). follow changes in serum cobalamins and its binding proteins No statistically significant change in the level of total and post-partum. holoHC was seen when comparing the baseline and the No change was observed in serum cobalamins throughout 4 months data (Wilcoxon matched pair signed-ranks test, the study period, whereas changes in opposite directions P40.05). However, comparing the level at 4 and 9 months, were observed for TC and HC (Figure 1 and Table 2). Total a statistically significant increase was observed (Wilcoxon TC decreased statistically significantly from baseline to 9 matched pair signed-ranks test, Pp0.01) (Figure 1b and months post-partum (Friedman’s test, Po0.0001) (Figure 1a Table 2).

European Journal of Clinical Nutrition Serum cobalamins and its binding proteins in lactating women AL Mørkbak et al 187

Table 2 Median and range of weight of mother and infant, and B12 supplementation vitamin B12-related biomarkers of the lactating mothers

Baseline 4 months 9 months P-value P-value Basline versus 4 months versus 4 months 9 months

Number of women 89 87 86 B12 supplementation (mg per day) 1 (0–13.5) 1 (0–13.5) 0 (0–18.0) Partly lactatinga (%) 3 9 51 Exclusive lactating (%) 97 83 0 Weight mother (kg) 69.0 (54.7–119.5) 66.0 (50.1–116.5) 64.8 (50.4–106.0) o0.0001 o0.0001 Infant weight (g) 4460 (2925–5690) 7110 (4840–9145) 9500 (7290–12950) o0.0001 o0.0001 Cobalamins (pmol/l) 322 (129–1039) 317 (114–1247) 315 (145–1193) NS NS [200–600]b Total TC (pmol/l) 710 (391–1785) 635 (240–1807) 602 (232–1501) o0.0001 0.01 [610–1400]c HoloTC (pmol/l) 85 (30–1068) 87 (20–1020) 76 (30–972) 0.02 0.01 [40–150]d TC saturation 0.1 (0.05–0.6) 0.1 (0.04–0.6) 0.1 (0.05–0.7) 0.0006 NS [0.05–0.20]d Total HC (pmol/l) 413 (180–768) 422 (203–664) 455 (250–844) NS 0.0008 [250–760] HoloHC (pmol/l) 297 (163–613) 300 (142–524) 317 (176–596) NS 0.01 [220–590] HC saturation 0.7 (0.4–0.9) 0.8 (0.5–0.9) 0.7 (0.4–1) NS 0.04 [0.6–1]e

Abbreviations: HC, haptocorrin; NS, nonsignificant; TC, transcobalamin. Number of women lactating are given in percentage. [Reference intervals] for biomarkers are included (where appropriate reference intervals for younger female subjects are given). For statistical analyses Wilcoxon matched pair signed-ranks test is used. aPartly lactating is defined as: lactation continued, but X1 meal is replaced. bReference Nexø, 1983. cReference Nexø et al., 2000. (for female subjects o50 years). dReference Nexø et al., 2002. eFor female subjects p50 years.

The baseline levels were significantly associated with the weeks to 9 months post-partum. Also, total TC decreased and levels at 9 months post-partum for total TC (total HC) (r2 ¼ total HC increased during the same period. 0.51 (r2 ¼ 0.50)), holoTC (holo HC) (r2 ¼ 0.93, (r2 ¼ 0.68)) Information concerning the physiological changes in and TC saturation (HC saturation) (r2 ¼ 0.66 (r2 ¼ 0.27)) serum cobalamins and the cobalamin-binding proteins (Po0.0001). during pregnancy and lactation is limited. Serum cobalamins The 95% reference interval for holoHC, total HC and HC decrease during pregnancy (Bartels et al., 1989; Willis saturation determined on plasma from 71 healthy female et al., 1989; Koebnick et al., 2002), but based on the results donors was 220–590 pmol/l (median ¼ 340 pmol/l), 250– from our study and a previous study (Dosta´lova´, 1984), 760 pmol/l (median ¼ 408 pmol/l) and 0.6–1 pmol/l (med- it normalizes within a few weeks post-partum. These ian ¼ 0.9 pmol/l), respectively. The HC saturation was observations are confirmed by comparing the level of significantly higher for women p50 years (95% reference serum cobalamins just before birth measured in a previous interval ¼ 0.6–1 pmol/l, median ¼ 0.9, n ¼ 36) compared with study (Obeid et al., 2005) with the level 3 weeks post- women 450 years (95% reference interval ¼ 0.7–0.9 pmol/l, partum obtained in our study. Between 3 weeks and 9 median ¼ 0.8, n ¼ 35) (Mann–Whitney test, P ¼ 0.0004). No months post-partum, we found no alteration in serum statistically significant association with age was seen for holo cobalamins, and the levels obtained were well in accord- and total HC. ance with established reference intervals for non-lactating women, Table 2. Interestingly, the subfractions of serum cobalamins, Discussion holoTC and holoHC changed with time post-partum as did total TC and HC. These changes could be explained by the The aim of this study was to examine serum cobalamins, HC influence of several factors, such as change in weight of and TC (holo and total) in lactating women. We found no the mother (blood volume), vitamin B12 supplementation, change in the level of serum cobalamins, whereas the two the extent of lactation and/or hormonal changes during compartments of serum cobalamins changed in lactating lactation. Surprisingly, our results indicate that the weight women. HoloTC decreased and holoHC increased from 3 decrease of the mother only influences the level of holoHC

European Journal of Clinical Nutrition Serum cobalamins and its binding proteins in lactating women AL Mørkbak et al 188 and cobalamins, whereas holoTC, total TC or total HC were Foley, 1971; Ford et al., 1975; Trugo et al., 1985; Adkins and not influenced by the weight change. Lonnerdal, 2002). However, the relationship between HC in We did not find any difference in cobalamins or its bind- human serum and milk is unknown.

ing proteins between the groups of vitamin B12-supplemen- Our data indicate that measurement of total serum ted and unsupplemented lactating mothers. Furthermore, cobalamins in lactating women will be of limited interest if

the extent of vitamin B12 supplementation was not signi- one wants to evaluate the amount of cobalamin available for ficantly associated with the levels of serum cobalamins, TC the cells (i.e. the holoTC). This is in excellent agreement or HC (holo and total). Therefore, supplementation with with recent data, suggesting that holoTC rather than cobal-

vitamin B12 does not seem to influence the levels of amins is a sensitive marker of vitamin B12 status (Herbert cobalamins, TC and HC (holo and total). However, no et al., 1990; Nexo et al., 2002; Herrmann et al., 2003; Hvas conclusion can be drawn concerning the influence of and Nexo, 2003).

the vitamin B12 from the diet. Our data indicated that levels of TC and HC (holo and We observed a decrease in TC and an increase in HC from total) were independent of the extent of vitamin B12 3 weeks to 9 months post-partum, changes that are unlikely supplementation and lactation. These results may indicate to be related to the extent of lactation or hormonal changes that Danish women are supplied with sufficient amounts of

(prolactin) because no statistically significant association nutritional vitamin B12 to fulfil needs even during prolonged was observed between these parameters. lactation. Our data do not allow us to conclude whether the

Comparing the levels of TC and HC obtained 3 weeks post- baby gets a sufficient supply of vitamin B12, but judged from partum with the levels obtained just before birth using the the results obtained for the mothers, there seems to be no

same ELISA methods (Obeid et al., 2005), the levels of total need for supplementation with vitamin B12 during lactation proteins (TC and HC) seem to decrease significantly at or just in a Danish population of healthy mothers. after birth, whereas the levels of holoproteins (TC and HC) In conclusion, we report a decrease in total and holoTC seem to increase, significantly. In order to explore this obser- and an increase in total and holoHC in lactating mothers vation, a longitudinal study on pregnant women including during a 9-month period post-partum. No differences were samples drawn just before and after birth is needed. observed between mothers who were supplemented or not

Total and holoTC were relatively high 3 weeks and 4 with vitamin B12, independent of the length of the lactation months post-partum, a time during which the flux of period. Thus, supplementation with vitamin B12 has no cobalamin through the circulation is expected to be impact on the circulating level of serum cobalamins or its increased because cobalamin has to be delivered to both binding proteins in a Danish population of lactating the mother and through the milk to the baby. The amount of mothers. total and holoTC present in the circulation is the sum of the influx minus the efflux. As the efflux of holoTC is likely to be increased during lactation, the relatively high level of holoTC indicates that also the influx is increased. The Acknowledgements increased influx could be explained by an increased absorption of cobalamin through the intestine or mobilization of We thank the women who participated in the study; Anna cobalamin stores from the body. The relatively high level of Lisa Christensen and Jette Fisker Petersen at the Department total TC early post-partum combined with the expected of Clinical Biochemistry, Aarhus University Hospital, increased efflux of the protein suggests an increased syn- Denmark, for technical assistance; the staff at The Osteo- thesis of the protein during lactation. The decrease in total porosis Clinic, Aarhus University Hospital, Denmark, for TC can be explained by the increased efflux of holoTC collecting the blood samples; and the secretaries at the during lactation and/or a decreased synthesis of TC. Department of Obstetrics and Gynaecology, Aarhus Uni- Whereas total and holoTC decrease post-partum, the versity Hospital, Denmark, for assisting in the identification opposite is true for HC. Total HC increases steadily from of eligible women. This study was supported by the 3 weeks through 4 and 9 months, whereas holoHC increases Biomedical Laboratory Scientist Education Research Fund, from 4 to 9 months post-partum when the mothers the LEO Pharma Research Foundation and by the EU decreased their lactation activities. HoloHC levels decrease Demonstration Project on the diagnostic utility of holoTC during pregnancy, whereas total HC, calculated as apo-HC (QLK3-CT-2002-01775). plus holoHC, increases slightly from the second to third trimester (Koebnick et al., 2002). Our data suggest that the increase in total HC continues, whereas holoHC starts to References increase post-partum. Adkins Y, Lonnerdal B (2002). Mechanisms of vitamin B(12) HC is the major vitamin B12-binding protein in human J Pediatr Gastroenterol Nutr milk. Previous animal and human studies indicate that HC absorption in breast-fed infants. 35, 192–198. in milk mediates vitamin B12 absorption in infants during Allen LH (1994). Vitamin B12 metabolism and status during the neonatal period (Boass and Wilson, 1963; Gallagher and pregnancy, lactation and infancy. Adv Exp Med Biol 352, 173–186.

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