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Leptin determination in colostrum and early human milk from mothers of preterm and term infants
Early Human Development 87 (2011) 415–419
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Early Human Development j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e a r l h u m d ev
Leptin determination in colostrum and early human milk from mothers of preterm and term infants Elisabeth Eilers a,1, Thomas Ziska b,1, Thomas Harder b, Andreas Plagemann b, Michael Obladen c, Andrea Loui c,⁎ a b c
Department of Pediatrics, Neonatal Intensive Care Unit, Vivantes Medical Center Berlin, Neukölln, Germany Department of Obstetrics, Division of Experimental Obstetrics, Charité University Medicine Berlin, Germany Department of Neonatology, Charité University Medicine Berlin, Germany
a r t i c l e
i n f o
Article history: Received 2 November 2010 Received in revised form 28 February 2011 Accepted 8 March 2011 Keywords: Body mass index Breast feeding Human milk Leptin Preterm infant
a b s t r a c t Background: Leptin is involved in the regulation of food intake and energy expenditure and is therefore important for growth and brain development. Analytical methods used for leptin measurement in human milk differ widely in the literature and yield varying results. Aims: To compare different preparation methods for the analysis of leptin in human milk and to investigate the leptin levels in colostrum and mature human milk from mothers of preterm or term infants. Methods: Mothers delivering a preterm (n = 37) or a term infant (n = 40) were recruited for a prospective study and were ask to collect breast milk on the 3rd and 28th day of lactation. Leptin, protein and fat concentrations were analysed. Clinical data of mother and child were recorded prospectively. Results: Skim milk was most appropriate for leptin analysis. Human milk leptin concentrations did not differ between preterm and term human milk. In term milk, leptin concentration on day 28 was lower than on day 3 (p b 0.05). Milk leptin levels on the 3rd and 28th day were positively correlated with mothers' body mass index, but not with fat content in milk. Conclusion: Skim milk was the most stabile preparation for leptin analysis. Preterm and term human milk contain leptin in equal concentrations. Human milk leptin depends on mothers' body mass index. © 2011 Published by Elsevier Ireland Ltd.
1. Introduction Human milk contains a number of bioactive factors and hormones which may be relevant for adequate nutrition, regulation of metabolic pathways and development of the newborn [1]. Leptin, the 167 amino acid polypeptide hormone product of the ob gene, is involved in the regulation of food intake and energy expenditure and therefore of body weight [2]. Developmentally, leptin is important in angiogenesis, bone metabolism, haematological differentiation, brain development, and growth [3]. Leptin contributes to the peripheral insulin resistance by affecting insulin secretion and attenuating insulin action in various insulin-responsive cell types [4,5]. Leptin is produced and secreted from white adipocytes into the blood and is transported to the brain via a saturable system [6]. Leptin is also produced by mammary glands and is secreted by epithelial cells [7]. In breast milk, leptin may be important for the postnatal growth and development of different organs of the newborn. Humans with leptin deficiency due to genetic
⁎ Corresponding author at: Department of Neonatology, Charité University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 450566122; fax: +49 30 450566922. E-mail address: [email protected] (A. Loui). 1 These authors contributed equally to this publication. 0378-3782/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.earlhumdev.2011.03.004
mutations are affected by congenital obesity and endocrine abnormalities. Administration of exogenous leptin to a leptin-deficient child resulted in a decreased energy intake and a dramatic loss of fat mass, whereas the lean body mass was unaffected [8]. In animal experiments reduced inhibition of neurons of the arcuate hypothalamic nucleus by leptin was observed in neonatally overfed rats compared with normally fed rats. This might indicate a hypothalamic leptin resistance contributing to persistent hyperphagia and overweight [9]. In rats, exogenous induction of hyperleptinaemia during the first 10 days of life leads to leptin resistance in adulthood [10]. Hormones and bioactive factors in human milk play an important role in nutrition. Leptin may prime the endocrine system at a different homeostatic energy regulation balance. The incidence of obesity has increased to epidemic proportions in recent decades and evidence is accumulating that this can be attributed in part to perinatal nutrition. Paediatricians are now searching for strategies to prevent later complications in metabolic programming such as hypertension, cardiovascular disease, diabetes and obesity. Markedly suppressed circulating leptin levels were reported in extremely premature infants [11]. The goal in feeding preterm infants is to prevent undernutrition and obesity. Different studies [12,13] showed that leptin concentration varies widely and changes during lactation. But the numbers of lactating women included were generally small and the methods used
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to measure leptin levels differed. Some studies measured leptin in whole milk [14–19] whereas others used skim milk and sonication [12,13,20–23]. Because the reported leptin levels in human milk are inconsistent, we performed a pilot study with the following aims: - To compare different methods of preparing human milk for leptin measurement in order to establish the most appropriate method. - To assess leptin in colostrum and mature human milk of mothers from preterm and term infants to search for differences between their milk leptin levels at two defined time points in lactation. 2. Materials and Methods 2.1. Subjects This study was approved by the local ethics committee and a written informed consent was obtained from the parents. A power calculation based on the literature data suggested that a comparison of two groups with at least 20 samples would show a difference of 0.5 ng/mL in leptin concentrations to be statistically significant. Milk samples were collected from 37 mothers who delivered preterm between 24 and 36 weeks of gestation and 40 mothers who delivered at term between 37 and 41 weeks of gestation. The mothers were questioned about height and weight before pregnancy. Medical data from mother and child were collected prospectively. Small for gestational age was defined according to Fenton [24]. Mothers with twins and triplets, vegetarian diet, diseases with malabsorption, gestational diabetes or pre-eclampsia were excluded from the study. 2.2. Sample Collection In both groups milk samples were collected on days 3 and 28 of lactation. Sometimes there was a delay in the onset of lactation after birth so that the newborn's day of life and lactation day differed and were recorded. To standardise the collection the milk samples were obtained by pumping electrically for ten minutes between 4 p.m. and 8 p.m. A sample of the milk volume from this one pumping procedure was taken. The mothers were asked not to have a big meal in the twohour period prior to pumping. The milk samples were frozen immediately and stored at −20 °C until analysis. 2.3. Analytical Procedures 2.3.1. Methodical Study: Preparation of Milk Used for the Investigation of the Effect of Milk Dilution and Recovery of Leptin Milk samples from 22 mothers (14 samples from mothers of term infants, 8 samples from mothers of preterm infants) collected 2–6 days postpartum were used for this part of the study and stored at − 20 °C until use. Milk samples were thawed and vortexed vigorously before pipetting. We compared the following five different milk preparation regimens, most often used regimens to prepare milk samples for leptin measurement: a) whole milk without further preparation, b) centrifugation at 40,000 g for 1 h at 4 °C; the fat layer was discarded (skim milk), c) sonication 2 x 10 bursts of 0.3 s (Sonoplus ultrasonic homogeniser, Bandelin, Germany) at 4 °C (type 1), d) sonication 2 x 10 bursts of 0.3 s at 4 °C (type 1) and centrifugation at 40 000 g for 1 h at 4 °C; the fat layer was discarded, e) sonication 2 x 20 bursts of 0.5 s at 4 °C (type 2) and centrifugation at 40 000 g for 1 h at 4 °C; the fat layer was discarded. 5 and 10 ng/ml leptin were added to one sample from each preparation to study the recovery of leptin. All preparations were diluted 1:2 and 1:4 with an RIA buffer to test the effect of milk dilution.
Three measurements were performed in each preparation and after adding 5 and 10 ng/ml leptin. 2.3.2. Preparation of Milk Samples from Mothers Who Delivered a Preterm or Term Infant Based on the results of the pre-study (see Results 3.2.), we decided to use regimen b) (skim milk) for all analyses in the main study. Milk samples were thawed and vortexed vigorously before pipetting. Whole milk was centrifuged at 40,000 g for 1 h at 4 °C and the fat layer was discarded. The resulting skim milk was used to determine leptin concentrations. 2.3.3. Quantitative Measurement of Leptin in Human Milk Leptin concentration was measured using the sensitive human leptin RIA kit (Cat. # SHL-81 K, Linco Research, St. Charles, MO, USA) according to the manufacturer's instructions. Recombinant human leptin (Linco Research, St. Charles, MO, USA) served as the standard preparation in a concentration range of 0.05–10.0 ng/ml. The intraassay variation was 3.7–7.5% and the inter-assay variation was 3.2– 8.9%. The recovery of leptin was determined using the human leptin RIA kit (Cat # HL-81 K, Linco Research, St. Charles, MO, USA). The recombinant human leptin served as the standard preparation in a concentration range of 0.5–100.0 ng/ml. The intra-assay variation was 3.4 −8.3% and the inter-assay variation was 3.0–6.2%. Since we expected relatively low concentrations of leptin in human milk, we used a RIA kit during the methodological part of the study which has a high sensitivity in the lower concentration range of leptin (SHL-81 K). However, for the recovery rate study, the addition of 5 and 10 ng/ml leptin would have exceeded the upper limit of the concentration range of this assay. Therefore, we had to switch to another Kit which has a higher upper concentration range (HL-81 K). The first one of these assays was also used for the main study. 2.3.4. Quantitative Measurement of Total Protein in Human Milk Total milk protein was measured by the bicinchoninic acid method, a well-established protein quantification assay (BCA™ Protein Assay, Thermo Scientific, Meridian Rd., Rockford, USA) based on the photometric determination of a coloured copper complex, the formation of which is dependent on the amount of protein present [25]. 2.3.5. Quantitative Measurement of Total Fat in Human Milk Milk samples in a haematocrit capillary tube were centrifuged in a haematocrit centrifuge TH12 (MLW, Germany). The percentage of cream was read from the haematocrit capillary tube and was linearly related to the fat content [26]. 2.4. Statistical Analysis Statistical analysis was carried out with SPSS software 12.0 (SPSS Inc., Chicago, USA). Data are expressed as mean ± SD or n (%). Group differences were identified with the Mann–Whitney test and the Chisquare test; all tests were two-sided and non-parametric. Spearman rank correlation coefficient was used to relate leptin, fat and protein concentrations and clinical data. Significance was assumed for p b 0.05 in all statistical tests used. 3. Results 3.1. Clinical Data The clinical data of women included in the study are shown in Table 1. All mothers were in good clinical and nutritional condition and consumed a balanced diet. BMI was higher in mothers of term infants (p b 0.01), while mean values for this parameter were found in both groups to be in the upper normal range for women (18.5–24.9 kg/m2)
E. Eilers et al. / Early Human Development 87 (2011) 415–419 Table 1 Maternal clinical data.
Maternal age (years) Multipara (n, %) BMI before pregnancy (kg/m2) Prenatal steroids (n, %) Lactation day of sample 1 Lactation day of sample 2
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Table 3 Effect of preparation and dilution on leptin concentration in human milk. Preterm birth (n = 37)
Term birth (n = 40)
P value*
31 ± 5 15 (40) 22 ± 4 16 (43) 3±1 28 ± 1
30 ± 5 15 (38) 24 ± 3 1 (3) 3±0 28 ± 2
ns ns b 0.01 b 0.001 ns ns
All data as mean ± SD or n (%). *Chi-square test, Mann–Whitney test. BMI, body mass index, defined as weight-for-high index. ns, not significant at p b 0.05.
[27]. Taking both groups together, a BMI of 25–29 was found in 5 mothers and a BMI of N30 in 17 mothers. 43% of the mothers who had given birth to preterm infants had been treated with prenatal steroids (p b 0.001) as a routine treatment to induce lung maturation because of suspected premature labour. Maternal age and the lactation days on which samples were collected did not differ between the groups (Table 1). Table 2 shows the newborns' clinical data. As expected, gestational age, birth weight, body length, head circumference (p b 0.001) and ponderal index (p b 0.05) at birth differed between the groups. In both groups, the day of life on which the first sample was collected ranged from the 2nd to 10th day of life while the second sample was collected between the 28th and the 35th day of life (Table 2).
3.2. Analysis of Leptin in Human Milk 3.2.1. 3.2.1. Methodical Study: Comparison of Milk Preparation Regimens Sonication of milk samples did not lead to an increase in measured leptin concentrations (Table 3, c–e). Both skim milk (Table 3b) and milk centrifuged and sonicated according to regimens d and e (Table 3d, e) showed good dilution characteristics. By contrast, the dilution of whole milk (regimen a) and of whole milk after sonication (regimen c) did not lead to a linear decrease of leptin concentrations in the samples. Recovery rates of 5 ng and 10 ng of leptin in undiluted whole milk were only 64.7 and 55.9%, respectively. In undiluted, sonicated whole milk the respective values were 63.4% and 81.3%. Only at a dilution of 1:4 was an acceptable recovery rate observed (Table 4). An addition of 5 ng and 10 ng leptin to skim milk led to recovery rates of 94.6% and 94.0%, respectively. Again, dilution characteristics of skim milk were excellent (Table 4). Table 2 Neonatal clinical data.
Male gender (n, %) Gestational age (weeks) Umbilical arterial pH Apgar score at 5 min Birth weight (g) Head circumference at birth (cm) Body length at birth (cm) Ponderal index at birth (g/cm3 * 100) SGA (n, %) Day of life of sample 1 Day of life of sample 2
Preterm infants (n = 37)
Term infants (n = 40)
P value*
20 (54) 32 + 1 ± 3 + 5 7.30 ± 0.05 8±2 1863 ± 663 30 ± 4 43 (39/46) 2.35 ± 0.33 6 (16) 5±1 29 ± 2
22 (55) 39 + 4 ± 1+4 7.26 ± 0.09 9±1 3437 ± 669 35 ± 2 52 (50/53) 2.51 ± 0.25 5 (13) 4±1 29 ± 1
ns b 0.001 b 0.05 b 0.05 b 0.001 b 0.001 b 0.001 b 0.05 ns ns ns
All data as mean ± SD or n (%). *Mann–Whitney test, Chi-square test. SGA, small for gestational age. Ponderal index, weight-for-length index, defined as g/cm3 *100. ns, not significant at p b 0.05.
Dilution
Observed [ng/ml]
Expected [ng/ml]
Expected [%]
a) Whole milk 1:1 1:2 1:4
1.34 ± 1.08 0.97 ± 1.08 0.36 ± 0.23
– 0.67 0.34
– 144.8 107.5
b) Whole milk, centrifuged (skim milk) 1:1 1.05 ± 0.98 1:2 0.55 ± 0.47 1:4 0.30 ± 0.22
– 0.53 0.26
– 104.8 114.3
c) Whole milk, sonicated type 1 1:1 1.34 ± 1.08 1:2 1.08 ± 0.92 1:4 0.67 ± 1.34
– 0.67 0.34
– 161.2 200.0
d) Whole milk, sonicated type 1 and centrifuged 1:1 1.12 ± 1.06 – 1:2 0.57 ± 0.49 0.56 1:4 0.30 ± 0.22 0.28
– 101.8 107.1
e) Whole milk, sonicated type 2 and centrifuged 1:1 1.04 ± 0.85 – 1:2 0.50 ± 0.40 0.52 1:4 0.28 ± 0.22 0.26
– 96.2 107.7
Data are mean ± SD, n = 22.
3.2.2. Comparison of Samples from Mothers of Preterm and Term Infants On lactation days 3 and 28 leptin levels in human milk were not different in mothers of preterm and term infants (Table 5). Milk leptin declined during the first 4 weeks of lactation in THM and PHM, but significantly only in THM (p b 0.05). Taking all infants together leptin concentrations from the 1st and 2nd sample correlated with each other (r = 0.504, p b 0.001). At both time points, leptin in human milk weakly correlated with mother's BMI before pregnancy (r = 0.28, p b 0.01 on day 3, r = 0.45, p b 0.001 on day 28). This correlation was stronger in PHM (r = 0.40, p = 0.016 on day 3, r = 0.56, and p b 0.001 on day 28, Fig. 1). In mothers with a BMI higher than 25 we found higher milk leptin concentrations on the 28th day of lactation than in mothers with a BMI lower than 25 (p b 0.01). No correlations of leptin with milk fat content were found (data not shown). Prenatal steroid treatment in the preterm group did not influence leptin levels in PHM on lactation days 3 and 28 (data not shown). 3.2.3. Comparison of Protein and Fat in Milk of Mothers of Preterm and Term Infants Protein concentrations on lactation days 3 and 28 did not differ between the groups, but were found to decrease up to week 4 (p b 0.001) in THM and PHM. The milk fat content at 28th day was lower in THM (p b 0.05) than in PHM (Table 5). In both groups the milk fat concentration did not change during the first 4 weeks of life. No significant correlations were found between protein and fat content in mother's own milk and mother's BMI before pregnancy. 4. Discussion The results of our methodological study show that skim milk is most suitable for the measurement of leptin in human milk. The recovery of leptin is very good for skim milk, but unsatisfactory for other milk preparations. Our results are in agreement with the results of Lönnerdal and Havel [28]. These authors showed that milk fat interacts substantially with the assay method. In our study the nonlinear dilution behaviour of whole milk (without or with sonication) supports this idea. Concentrations of leptin are higher (although not significant) in whole milk because of the perturbing effects of fat in the leptin RIA. In fact, we did not perform a combination of sonication and centrifugation in the recovery study (Table 4). However, this was
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Table 4 Recovery of leptin in human whole und skim milk. Dilution
Initial concentration Observed [ng/ml]
Leptin added 5 ng/ml observed [ng/ml]
Expected [ng/ml]
% recovery
10 ng/ml observed [ng/ml]
Expected [ng/ml]
% recovery
a) Whole milk 1:1 10.27 ± 0.02 1:2 0.54 ± 0.09 1:4 0.15 ± 0.02
9.88 ± 0.72 2.90 ± 0.02 1.61 ± 0.18
15.27 3.04 1.40
64.7 95.6 115.3
11.32 ± 0,23 7.07 ± 0.11 2.78 ± 0.04
20.27 5.54 2.65
55.9 127.7 105.0
b) Whole milk, centrifuged (skim milk) 1:1 0.58 ± 0.03 1:2 0.30 ± 0.02 1:4 0.19 ± 0.01
5.28 ± 0.08 2.87 ± 0.05 1.50 ± 0.07
5.58 2.80 1.44
94.6 102.4 104.6
9.94 ± 0.95 5.27 ± 0.45 2.77 ± 0.01
10.58 5.30 2.69
94.0 99.3 103.1
17.11 ± 0.92 9.68 ± 0.94 1.65 ± 0.02
26.96 5.09 1.51
63.4 190.3 109.2
25.99 ± 0.40 21.37 ± 0.54 3.17 ± 0.14
31.96 7.59 2.76
81.3 281.7 114.8
c) Whole milk, sonicated type 1 1:1 21.96 ± 0.70 1:2 2.59 ± 0.07 1:4 0.26 ± 0.01
Measurements in duplicate, data are mean ± SD. Recovery was calculated on observed vs expected values.
not necessary because in the dilution study (Table 3), after having centrifuged the samples, we did not observe a further improvement of the dilution characteristics when these probes were additionally sonicated (see Table 3, d and e). Moreover, as shown in Table 4, regimen b) (centrifugation only) had a very good recovery rate which was judged by us to be suitable for the purpose of our main study. The methodological study indicated that we obtained the best dilution characteristics and recovery rates when the samples were centrifuged only (regimen b on page 7). Moreover, some of the data from the sonicated samples (Table 3, regimen c and Table 4, regimen c) indicate that sonication might lead to higher than expected leptin values and non-linear dilution characteristics. Therefore, one might speculate that under certain circumstances sonication of milk samples might even disturb leptin measurements. Therefore, we used the regimen which showed the most stable and reliable results. Leptin concentrations in term and preterm human milk were not different and correlated with the mother's BMI. During the first 4 weeks of life leptin levels within the groups decreased significantly in term but not in preterm human milk. Our literature search for studies on leptin measurements in human milk showed that various preparation methods were used. Whereas some authors used skim milk [12,13,20–23] others analysed whole milk partly treated with sonication [14–19]. Leptin levels were found in whole milk in a higher concentration than in skim milk probably because of the association of leptin with milk fat globules [29]. Therefore, in the following we compare our results with studies which used skim milk for analysis, as we did. Our leptin concentrations in human milk are comparable with those found by Uysal et al. [21] and Weyermann et al. [13]. Uysal examined the milk of 50 obese and non-obese mothers by using RIA, whereas Weyermann analysed 766 mothers 6 weeks postnatally by
ELISA. At 4 weeks of lactation Resto et al. found 10 times higher leptin levels in PHM than we did [12]. Other authors described leptin concentrations 3 times higher in colostrum of obese mothers [20] or 7 times higher in mature milk at 4 weeks of lactation [22]. Interindividual variations in milk leptin concentrations between the mothers may be one cause for different results in the above mentioned studies. Beside this the milk leptin level is also influenced by adiposity and the plasma leptin level of the mother. Lean mothers with very low plasma leptin levels produce milk with little or even no detectable leptin [12]. Our study confirmed the results of Houseknecht et al. who found a correlation of milk leptin with mother's BMI [18]. Accordingly, our mothers with a BMI above 25 had significantly higher milk leptin levels at 4 weeks of lactation. Studies showed that human placenta synthesises quantitative amounts of leptin, which explain the elevated plasma levels of the mother during pregnancy [30]. During lactation plasma leptin concentration of the mother returns to prepartum levels [31]. These levels are higher in obese women. Casabiell et al. investigated the human milk of 44 obese mothers 5 days after delivery and found higher leptin levels than we did [20]. Doneray et al. excluded only mothers with BMI N 35 from their study [22]. In both studies mothers' adiposity may have increased the leptin in milk. Some studies [12,20] which used skim milk for analyses differed in some important preparation and analytical steps from ours. Resto
Table 5 Leptin, protein and fat concentrations in human milk#. Preterm infants (n = 37) Term infants (n = 40) P value* 3 rd lactation day Leptin (ng/mL) Fat (vol%) Total protein (g/dl)
0.70 ± 0.79 5.0 ± 2.2 2.39 ± 1.60
0.65 ± 0.67 4.5 ± 2.4 2.22 ± 0.87
ns ns ns
28th lactation day Leptin (ng/mL) 0.50 ± 0.40 Fat (vol%) 5.5 ± 1.7 Total protein (mg/dl) 1.51 ± 2.67
0.50 ± 0.50 4.6 ± 1.6 1.58 ± 5.53
ns b0.05 ns
All data as mean ± SD. *Mann- Whitney test. ns, not significant at p b 0.05. # Leptin measured in skim milk.
Fig. 1. Correlation of human milk leptin on lactation day 28 with the body mass index from 37 healthy mothers who delivered preterm (b 37 weeks' gestation). Spearman rank correlation (p b 0.001, r = 0.558).
E. Eilers et al. / Early Human Development 87 (2011) 415–419
et al. used pancreatic lipase digestion, instead of centrifugation, to remove fat from the milk samples. Casabiell et al. used dialysis against 10 potassium chloride/tris puffer (KT puffer) to purify the leptin from milk. Such differences might, of course, lead to differences in the measured leptin concentrations. This is the first study which investigated leptin in skim milk of mothers who had given birth to preterm and term infants and did not find a difference in leptin levels of their milk on days 3 and 28 of lactation. This confirms the data from Resto et al.[12] and might suggest that leptin can regulate nutrient intake and energy balance equally in preterm and term infants. In our study, leptin concentration in THM decreased over 4 weeks of lactation as already decribed by others [12], whereas a number of other studies found constant [23] or even increased levels of human milk leptin over the same time course [22]. Beside differences in the analytical method used or adiposity of the mother this may also point to widely varying leptin levels in human milk. 5. Conclusions Skim milk was shown to be the most suitable preparation for leptin analysis. Preterm and term human milk contain leptin, which is important for the regulation of food energy intake and therefore of body weight, in equal concentrations. Human milk leptin depends on the mother's BMI. Funding Supported by the German Research Foundation (DFG; PL 241/5–1). The study sponsor had no involvement in the study design, collection, analysis and interpretation of the data, in writing the manuscript or in the decision to submit the manuscript for publication. Conflict of interest statement All authors confirm that they do not have any financial or personal relationships with other people or organisations that could inappropriately influence their work. Acknowledgements We thank all parents of the newborn infants who enabled this study. We thank Boris Metze for assistance with data processing and Anne Carney for editorial assistance. References [1] Kobata R, Tsukahara H, Ohshima Y, Ohta N, Tokuriki S, Tamura S, Mayumi M. High levels of growth factors in human breast milk. Early Hum Dev 2008;84:67–9. [2] Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin: a review. J Anim Sci 1998;76:1405–20. [3] Locke R. Preventing obesity: the breast milk–leptin connection. Acta Paediatr 2002;91:891–6.
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Early Human Development j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e a r l h u m d ev
Leptin determination in colostrum and early human milk from mothers of preterm and term infants Elisabeth Eilers a,1, Thomas Ziska b,1, Thomas Harder b, Andreas Plagemann b, Michael Obladen c, Andrea Loui c,⁎ a b c
Department of Pediatrics, Neonatal Intensive Care Unit, Vivantes Medical Center Berlin, Neukölln, Germany Department of Obstetrics, Division of Experimental Obstetrics, Charité University Medicine Berlin, Germany Department of Neonatology, Charité University Medicine Berlin, Germany
a r t i c l e
i n f o
Article history: Received 2 November 2010 Received in revised form 28 February 2011 Accepted 8 March 2011 Keywords: Body mass index Breast feeding Human milk Leptin Preterm infant
a b s t r a c t Background: Leptin is involved in the regulation of food intake and energy expenditure and is therefore important for growth and brain development. Analytical methods used for leptin measurement in human milk differ widely in the literature and yield varying results. Aims: To compare different preparation methods for the analysis of leptin in human milk and to investigate the leptin levels in colostrum and mature human milk from mothers of preterm or term infants. Methods: Mothers delivering a preterm (n = 37) or a term infant (n = 40) were recruited for a prospective study and were ask to collect breast milk on the 3rd and 28th day of lactation. Leptin, protein and fat concentrations were analysed. Clinical data of mother and child were recorded prospectively. Results: Skim milk was most appropriate for leptin analysis. Human milk leptin concentrations did not differ between preterm and term human milk. In term milk, leptin concentration on day 28 was lower than on day 3 (p b 0.05). Milk leptin levels on the 3rd and 28th day were positively correlated with mothers' body mass index, but not with fat content in milk. Conclusion: Skim milk was the most stabile preparation for leptin analysis. Preterm and term human milk contain leptin in equal concentrations. Human milk leptin depends on mothers' body mass index. © 2011 Published by Elsevier Ireland Ltd.
1. Introduction Human milk contains a number of bioactive factors and hormones which may be relevant for adequate nutrition, regulation of metabolic pathways and development of the newborn [1]. Leptin, the 167 amino acid polypeptide hormone product of the ob gene, is involved in the regulation of food intake and energy expenditure and therefore of body weight [2]. Developmentally, leptin is important in angiogenesis, bone metabolism, haematological differentiation, brain development, and growth [3]. Leptin contributes to the peripheral insulin resistance by affecting insulin secretion and attenuating insulin action in various insulin-responsive cell types [4,5]. Leptin is produced and secreted from white adipocytes into the blood and is transported to the brain via a saturable system [6]. Leptin is also produced by mammary glands and is secreted by epithelial cells [7]. In breast milk, leptin may be important for the postnatal growth and development of different organs of the newborn. Humans with leptin deficiency due to genetic
⁎ Corresponding author at: Department of Neonatology, Charité University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 450566122; fax: +49 30 450566922. E-mail address: [email protected] (A. Loui). 1 These authors contributed equally to this publication. 0378-3782/$ – see front matter © 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.earlhumdev.2011.03.004
mutations are affected by congenital obesity and endocrine abnormalities. Administration of exogenous leptin to a leptin-deficient child resulted in a decreased energy intake and a dramatic loss of fat mass, whereas the lean body mass was unaffected [8]. In animal experiments reduced inhibition of neurons of the arcuate hypothalamic nucleus by leptin was observed in neonatally overfed rats compared with normally fed rats. This might indicate a hypothalamic leptin resistance contributing to persistent hyperphagia and overweight [9]. In rats, exogenous induction of hyperleptinaemia during the first 10 days of life leads to leptin resistance in adulthood [10]. Hormones and bioactive factors in human milk play an important role in nutrition. Leptin may prime the endocrine system at a different homeostatic energy regulation balance. The incidence of obesity has increased to epidemic proportions in recent decades and evidence is accumulating that this can be attributed in part to perinatal nutrition. Paediatricians are now searching for strategies to prevent later complications in metabolic programming such as hypertension, cardiovascular disease, diabetes and obesity. Markedly suppressed circulating leptin levels were reported in extremely premature infants [11]. The goal in feeding preterm infants is to prevent undernutrition and obesity. Different studies [12,13] showed that leptin concentration varies widely and changes during lactation. But the numbers of lactating women included were generally small and the methods used
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to measure leptin levels differed. Some studies measured leptin in whole milk [14–19] whereas others used skim milk and sonication [12,13,20–23]. Because the reported leptin levels in human milk are inconsistent, we performed a pilot study with the following aims: - To compare different methods of preparing human milk for leptin measurement in order to establish the most appropriate method. - To assess leptin in colostrum and mature human milk of mothers from preterm and term infants to search for differences between their milk leptin levels at two defined time points in lactation. 2. Materials and Methods 2.1. Subjects This study was approved by the local ethics committee and a written informed consent was obtained from the parents. A power calculation based on the literature data suggested that a comparison of two groups with at least 20 samples would show a difference of 0.5 ng/mL in leptin concentrations to be statistically significant. Milk samples were collected from 37 mothers who delivered preterm between 24 and 36 weeks of gestation and 40 mothers who delivered at term between 37 and 41 weeks of gestation. The mothers were questioned about height and weight before pregnancy. Medical data from mother and child were collected prospectively. Small for gestational age was defined according to Fenton [24]. Mothers with twins and triplets, vegetarian diet, diseases with malabsorption, gestational diabetes or pre-eclampsia were excluded from the study. 2.2. Sample Collection In both groups milk samples were collected on days 3 and 28 of lactation. Sometimes there was a delay in the onset of lactation after birth so that the newborn's day of life and lactation day differed and were recorded. To standardise the collection the milk samples were obtained by pumping electrically for ten minutes between 4 p.m. and 8 p.m. A sample of the milk volume from this one pumping procedure was taken. The mothers were asked not to have a big meal in the twohour period prior to pumping. The milk samples were frozen immediately and stored at −20 °C until analysis. 2.3. Analytical Procedures 2.3.1. Methodical Study: Preparation of Milk Used for the Investigation of the Effect of Milk Dilution and Recovery of Leptin Milk samples from 22 mothers (14 samples from mothers of term infants, 8 samples from mothers of preterm infants) collected 2–6 days postpartum were used for this part of the study and stored at − 20 °C until use. Milk samples were thawed and vortexed vigorously before pipetting. We compared the following five different milk preparation regimens, most often used regimens to prepare milk samples for leptin measurement: a) whole milk without further preparation, b) centrifugation at 40,000 g for 1 h at 4 °C; the fat layer was discarded (skim milk), c) sonication 2 x 10 bursts of 0.3 s (Sonoplus ultrasonic homogeniser, Bandelin, Germany) at 4 °C (type 1), d) sonication 2 x 10 bursts of 0.3 s at 4 °C (type 1) and centrifugation at 40 000 g for 1 h at 4 °C; the fat layer was discarded, e) sonication 2 x 20 bursts of 0.5 s at 4 °C (type 2) and centrifugation at 40 000 g for 1 h at 4 °C; the fat layer was discarded. 5 and 10 ng/ml leptin were added to one sample from each preparation to study the recovery of leptin. All preparations were diluted 1:2 and 1:4 with an RIA buffer to test the effect of milk dilution.
Three measurements were performed in each preparation and after adding 5 and 10 ng/ml leptin. 2.3.2. Preparation of Milk Samples from Mothers Who Delivered a Preterm or Term Infant Based on the results of the pre-study (see Results 3.2.), we decided to use regimen b) (skim milk) for all analyses in the main study. Milk samples were thawed and vortexed vigorously before pipetting. Whole milk was centrifuged at 40,000 g for 1 h at 4 °C and the fat layer was discarded. The resulting skim milk was used to determine leptin concentrations. 2.3.3. Quantitative Measurement of Leptin in Human Milk Leptin concentration was measured using the sensitive human leptin RIA kit (Cat. # SHL-81 K, Linco Research, St. Charles, MO, USA) according to the manufacturer's instructions. Recombinant human leptin (Linco Research, St. Charles, MO, USA) served as the standard preparation in a concentration range of 0.05–10.0 ng/ml. The intraassay variation was 3.7–7.5% and the inter-assay variation was 3.2– 8.9%. The recovery of leptin was determined using the human leptin RIA kit (Cat # HL-81 K, Linco Research, St. Charles, MO, USA). The recombinant human leptin served as the standard preparation in a concentration range of 0.5–100.0 ng/ml. The intra-assay variation was 3.4 −8.3% and the inter-assay variation was 3.0–6.2%. Since we expected relatively low concentrations of leptin in human milk, we used a RIA kit during the methodological part of the study which has a high sensitivity in the lower concentration range of leptin (SHL-81 K). However, for the recovery rate study, the addition of 5 and 10 ng/ml leptin would have exceeded the upper limit of the concentration range of this assay. Therefore, we had to switch to another Kit which has a higher upper concentration range (HL-81 K). The first one of these assays was also used for the main study. 2.3.4. Quantitative Measurement of Total Protein in Human Milk Total milk protein was measured by the bicinchoninic acid method, a well-established protein quantification assay (BCA™ Protein Assay, Thermo Scientific, Meridian Rd., Rockford, USA) based on the photometric determination of a coloured copper complex, the formation of which is dependent on the amount of protein present [25]. 2.3.5. Quantitative Measurement of Total Fat in Human Milk Milk samples in a haematocrit capillary tube were centrifuged in a haematocrit centrifuge TH12 (MLW, Germany). The percentage of cream was read from the haematocrit capillary tube and was linearly related to the fat content [26]. 2.4. Statistical Analysis Statistical analysis was carried out with SPSS software 12.0 (SPSS Inc., Chicago, USA). Data are expressed as mean ± SD or n (%). Group differences were identified with the Mann–Whitney test and the Chisquare test; all tests were two-sided and non-parametric. Spearman rank correlation coefficient was used to relate leptin, fat and protein concentrations and clinical data. Significance was assumed for p b 0.05 in all statistical tests used. 3. Results 3.1. Clinical Data The clinical data of women included in the study are shown in Table 1. All mothers were in good clinical and nutritional condition and consumed a balanced diet. BMI was higher in mothers of term infants (p b 0.01), while mean values for this parameter were found in both groups to be in the upper normal range for women (18.5–24.9 kg/m2)
E. Eilers et al. / Early Human Development 87 (2011) 415–419 Table 1 Maternal clinical data.
Maternal age (years) Multipara (n, %) BMI before pregnancy (kg/m2) Prenatal steroids (n, %) Lactation day of sample 1 Lactation day of sample 2
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Table 3 Effect of preparation and dilution on leptin concentration in human milk. Preterm birth (n = 37)
Term birth (n = 40)
P value*
31 ± 5 15 (40) 22 ± 4 16 (43) 3±1 28 ± 1
30 ± 5 15 (38) 24 ± 3 1 (3) 3±0 28 ± 2
ns ns b 0.01 b 0.001 ns ns
All data as mean ± SD or n (%). *Chi-square test, Mann–Whitney test. BMI, body mass index, defined as weight-for-high index. ns, not significant at p b 0.05.
[27]. Taking both groups together, a BMI of 25–29 was found in 5 mothers and a BMI of N30 in 17 mothers. 43% of the mothers who had given birth to preterm infants had been treated with prenatal steroids (p b 0.001) as a routine treatment to induce lung maturation because of suspected premature labour. Maternal age and the lactation days on which samples were collected did not differ between the groups (Table 1). Table 2 shows the newborns' clinical data. As expected, gestational age, birth weight, body length, head circumference (p b 0.001) and ponderal index (p b 0.05) at birth differed between the groups. In both groups, the day of life on which the first sample was collected ranged from the 2nd to 10th day of life while the second sample was collected between the 28th and the 35th day of life (Table 2).
3.2. Analysis of Leptin in Human Milk 3.2.1. 3.2.1. Methodical Study: Comparison of Milk Preparation Regimens Sonication of milk samples did not lead to an increase in measured leptin concentrations (Table 3, c–e). Both skim milk (Table 3b) and milk centrifuged and sonicated according to regimens d and e (Table 3d, e) showed good dilution characteristics. By contrast, the dilution of whole milk (regimen a) and of whole milk after sonication (regimen c) did not lead to a linear decrease of leptin concentrations in the samples. Recovery rates of 5 ng and 10 ng of leptin in undiluted whole milk were only 64.7 and 55.9%, respectively. In undiluted, sonicated whole milk the respective values were 63.4% and 81.3%. Only at a dilution of 1:4 was an acceptable recovery rate observed (Table 4). An addition of 5 ng and 10 ng leptin to skim milk led to recovery rates of 94.6% and 94.0%, respectively. Again, dilution characteristics of skim milk were excellent (Table 4). Table 2 Neonatal clinical data.
Male gender (n, %) Gestational age (weeks) Umbilical arterial pH Apgar score at 5 min Birth weight (g) Head circumference at birth (cm) Body length at birth (cm) Ponderal index at birth (g/cm3 * 100) SGA (n, %) Day of life of sample 1 Day of life of sample 2
Preterm infants (n = 37)
Term infants (n = 40)
P value*
20 (54) 32 + 1 ± 3 + 5 7.30 ± 0.05 8±2 1863 ± 663 30 ± 4 43 (39/46) 2.35 ± 0.33 6 (16) 5±1 29 ± 2
22 (55) 39 + 4 ± 1+4 7.26 ± 0.09 9±1 3437 ± 669 35 ± 2 52 (50/53) 2.51 ± 0.25 5 (13) 4±1 29 ± 1
ns b 0.001 b 0.05 b 0.05 b 0.001 b 0.001 b 0.001 b 0.05 ns ns ns
All data as mean ± SD or n (%). *Mann–Whitney test, Chi-square test. SGA, small for gestational age. Ponderal index, weight-for-length index, defined as g/cm3 *100. ns, not significant at p b 0.05.
Dilution
Observed [ng/ml]
Expected [ng/ml]
Expected [%]
a) Whole milk 1:1 1:2 1:4
1.34 ± 1.08 0.97 ± 1.08 0.36 ± 0.23
– 0.67 0.34
– 144.8 107.5
b) Whole milk, centrifuged (skim milk) 1:1 1.05 ± 0.98 1:2 0.55 ± 0.47 1:4 0.30 ± 0.22
– 0.53 0.26
– 104.8 114.3
c) Whole milk, sonicated type 1 1:1 1.34 ± 1.08 1:2 1.08 ± 0.92 1:4 0.67 ± 1.34
– 0.67 0.34
– 161.2 200.0
d) Whole milk, sonicated type 1 and centrifuged 1:1 1.12 ± 1.06 – 1:2 0.57 ± 0.49 0.56 1:4 0.30 ± 0.22 0.28
– 101.8 107.1
e) Whole milk, sonicated type 2 and centrifuged 1:1 1.04 ± 0.85 – 1:2 0.50 ± 0.40 0.52 1:4 0.28 ± 0.22 0.26
– 96.2 107.7
Data are mean ± SD, n = 22.
3.2.2. Comparison of Samples from Mothers of Preterm and Term Infants On lactation days 3 and 28 leptin levels in human milk were not different in mothers of preterm and term infants (Table 5). Milk leptin declined during the first 4 weeks of lactation in THM and PHM, but significantly only in THM (p b 0.05). Taking all infants together leptin concentrations from the 1st and 2nd sample correlated with each other (r = 0.504, p b 0.001). At both time points, leptin in human milk weakly correlated with mother's BMI before pregnancy (r = 0.28, p b 0.01 on day 3, r = 0.45, p b 0.001 on day 28). This correlation was stronger in PHM (r = 0.40, p = 0.016 on day 3, r = 0.56, and p b 0.001 on day 28, Fig. 1). In mothers with a BMI higher than 25 we found higher milk leptin concentrations on the 28th day of lactation than in mothers with a BMI lower than 25 (p b 0.01). No correlations of leptin with milk fat content were found (data not shown). Prenatal steroid treatment in the preterm group did not influence leptin levels in PHM on lactation days 3 and 28 (data not shown). 3.2.3. Comparison of Protein and Fat in Milk of Mothers of Preterm and Term Infants Protein concentrations on lactation days 3 and 28 did not differ between the groups, but were found to decrease up to week 4 (p b 0.001) in THM and PHM. The milk fat content at 28th day was lower in THM (p b 0.05) than in PHM (Table 5). In both groups the milk fat concentration did not change during the first 4 weeks of life. No significant correlations were found between protein and fat content in mother's own milk and mother's BMI before pregnancy. 4. Discussion The results of our methodological study show that skim milk is most suitable for the measurement of leptin in human milk. The recovery of leptin is very good for skim milk, but unsatisfactory for other milk preparations. Our results are in agreement with the results of Lönnerdal and Havel [28]. These authors showed that milk fat interacts substantially with the assay method. In our study the nonlinear dilution behaviour of whole milk (without or with sonication) supports this idea. Concentrations of leptin are higher (although not significant) in whole milk because of the perturbing effects of fat in the leptin RIA. In fact, we did not perform a combination of sonication and centrifugation in the recovery study (Table 4). However, this was
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Table 4 Recovery of leptin in human whole und skim milk. Dilution
Initial concentration Observed [ng/ml]
Leptin added 5 ng/ml observed [ng/ml]
Expected [ng/ml]
% recovery
10 ng/ml observed [ng/ml]
Expected [ng/ml]
% recovery
a) Whole milk 1:1 10.27 ± 0.02 1:2 0.54 ± 0.09 1:4 0.15 ± 0.02
9.88 ± 0.72 2.90 ± 0.02 1.61 ± 0.18
15.27 3.04 1.40
64.7 95.6 115.3
11.32 ± 0,23 7.07 ± 0.11 2.78 ± 0.04
20.27 5.54 2.65
55.9 127.7 105.0
b) Whole milk, centrifuged (skim milk) 1:1 0.58 ± 0.03 1:2 0.30 ± 0.02 1:4 0.19 ± 0.01
5.28 ± 0.08 2.87 ± 0.05 1.50 ± 0.07
5.58 2.80 1.44
94.6 102.4 104.6
9.94 ± 0.95 5.27 ± 0.45 2.77 ± 0.01
10.58 5.30 2.69
94.0 99.3 103.1
17.11 ± 0.92 9.68 ± 0.94 1.65 ± 0.02
26.96 5.09 1.51
63.4 190.3 109.2
25.99 ± 0.40 21.37 ± 0.54 3.17 ± 0.14
31.96 7.59 2.76
81.3 281.7 114.8
c) Whole milk, sonicated type 1 1:1 21.96 ± 0.70 1:2 2.59 ± 0.07 1:4 0.26 ± 0.01
Measurements in duplicate, data are mean ± SD. Recovery was calculated on observed vs expected values.
not necessary because in the dilution study (Table 3), after having centrifuged the samples, we did not observe a further improvement of the dilution characteristics when these probes were additionally sonicated (see Table 3, d and e). Moreover, as shown in Table 4, regimen b) (centrifugation only) had a very good recovery rate which was judged by us to be suitable for the purpose of our main study. The methodological study indicated that we obtained the best dilution characteristics and recovery rates when the samples were centrifuged only (regimen b on page 7). Moreover, some of the data from the sonicated samples (Table 3, regimen c and Table 4, regimen c) indicate that sonication might lead to higher than expected leptin values and non-linear dilution characteristics. Therefore, one might speculate that under certain circumstances sonication of milk samples might even disturb leptin measurements. Therefore, we used the regimen which showed the most stable and reliable results. Leptin concentrations in term and preterm human milk were not different and correlated with the mother's BMI. During the first 4 weeks of life leptin levels within the groups decreased significantly in term but not in preterm human milk. Our literature search for studies on leptin measurements in human milk showed that various preparation methods were used. Whereas some authors used skim milk [12,13,20–23] others analysed whole milk partly treated with sonication [14–19]. Leptin levels were found in whole milk in a higher concentration than in skim milk probably because of the association of leptin with milk fat globules [29]. Therefore, in the following we compare our results with studies which used skim milk for analysis, as we did. Our leptin concentrations in human milk are comparable with those found by Uysal et al. [21] and Weyermann et al. [13]. Uysal examined the milk of 50 obese and non-obese mothers by using RIA, whereas Weyermann analysed 766 mothers 6 weeks postnatally by
ELISA. At 4 weeks of lactation Resto et al. found 10 times higher leptin levels in PHM than we did [12]. Other authors described leptin concentrations 3 times higher in colostrum of obese mothers [20] or 7 times higher in mature milk at 4 weeks of lactation [22]. Interindividual variations in milk leptin concentrations between the mothers may be one cause for different results in the above mentioned studies. Beside this the milk leptin level is also influenced by adiposity and the plasma leptin level of the mother. Lean mothers with very low plasma leptin levels produce milk with little or even no detectable leptin [12]. Our study confirmed the results of Houseknecht et al. who found a correlation of milk leptin with mother's BMI [18]. Accordingly, our mothers with a BMI above 25 had significantly higher milk leptin levels at 4 weeks of lactation. Studies showed that human placenta synthesises quantitative amounts of leptin, which explain the elevated plasma levels of the mother during pregnancy [30]. During lactation plasma leptin concentration of the mother returns to prepartum levels [31]. These levels are higher in obese women. Casabiell et al. investigated the human milk of 44 obese mothers 5 days after delivery and found higher leptin levels than we did [20]. Doneray et al. excluded only mothers with BMI N 35 from their study [22]. In both studies mothers' adiposity may have increased the leptin in milk. Some studies [12,20] which used skim milk for analyses differed in some important preparation and analytical steps from ours. Resto
Table 5 Leptin, protein and fat concentrations in human milk#. Preterm infants (n = 37) Term infants (n = 40) P value* 3 rd lactation day Leptin (ng/mL) Fat (vol%) Total protein (g/dl)
0.70 ± 0.79 5.0 ± 2.2 2.39 ± 1.60
0.65 ± 0.67 4.5 ± 2.4 2.22 ± 0.87
ns ns ns
28th lactation day Leptin (ng/mL) 0.50 ± 0.40 Fat (vol%) 5.5 ± 1.7 Total protein (mg/dl) 1.51 ± 2.67
0.50 ± 0.50 4.6 ± 1.6 1.58 ± 5.53
ns b0.05 ns
All data as mean ± SD. *Mann- Whitney test. ns, not significant at p b 0.05. # Leptin measured in skim milk.
Fig. 1. Correlation of human milk leptin on lactation day 28 with the body mass index from 37 healthy mothers who delivered preterm (b 37 weeks' gestation). Spearman rank correlation (p b 0.001, r = 0.558).
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et al. used pancreatic lipase digestion, instead of centrifugation, to remove fat from the milk samples. Casabiell et al. used dialysis against 10 potassium chloride/tris puffer (KT puffer) to purify the leptin from milk. Such differences might, of course, lead to differences in the measured leptin concentrations. This is the first study which investigated leptin in skim milk of mothers who had given birth to preterm and term infants and did not find a difference in leptin levels of their milk on days 3 and 28 of lactation. This confirms the data from Resto et al.[12] and might suggest that leptin can regulate nutrient intake and energy balance equally in preterm and term infants. In our study, leptin concentration in THM decreased over 4 weeks of lactation as already decribed by others [12], whereas a number of other studies found constant [23] or even increased levels of human milk leptin over the same time course [22]. Beside differences in the analytical method used or adiposity of the mother this may also point to widely varying leptin levels in human milk. 5. Conclusions Skim milk was shown to be the most suitable preparation for leptin analysis. Preterm and term human milk contain leptin, which is important for the regulation of food energy intake and therefore of body weight, in equal concentrations. Human milk leptin depends on the mother's BMI. Funding Supported by the German Research Foundation (DFG; PL 241/5–1). The study sponsor had no involvement in the study design, collection, analysis and interpretation of the data, in writing the manuscript or in the decision to submit the manuscript for publication. Conflict of interest statement All authors confirm that they do not have any financial or personal relationships with other people or organisations that could inappropriately influence their work. Acknowledgements We thank all parents of the newborn infants who enabled this study. We thank Boris Metze for assistance with data processing and Anne Carney for editorial assistance. References [1] Kobata R, Tsukahara H, Ohshima Y, Ohta N, Tokuriki S, Tamura S, Mayumi M. High levels of growth factors in human breast milk. Early Hum Dev 2008;84:67–9. [2] Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin: a review. J Anim Sci 1998;76:1405–20. [3] Locke R. Preventing obesity: the breast milk–leptin connection. Acta Paediatr 2002;91:891–6.
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