PEDIATRICOBESITY ORIGINALRESEARCH
Breast milk leptin and adiponectin in relation to infant body composition up to 2 years S. Brunner1, D. Schmid1, K. Zang1, D. Much1, B. Knoeferl2, J. Kratzsch3, U. Amann-Gassner1, B. L. Bader4 and H. Hauner1,4 1 Else Kröner-Fresenius-Center for Nutritional Medicine, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 2Institute for Medical Statistics and Epidemiology, Klinikum rechts der Isar, Technische, Universität München, Munich, Germany; 3Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; 4ZIEL – Research Center for Nutrition and Food Sciences, Nutritional Medicine Unit, Technische Universität München, Freising, Germany
Received 16 September 2013; revised 20 December 2013; accepted 17 February 2014
Summary Background: Adipokines in breast milk have been associated with infant growth trajectories. Objective: We aimed to explore the relationship of leptin and adiponectin in breast milk with infant weight gain and body composition up to the age of 2 years. Methods: Breast milk samples were collected from exclusively or partially breastfeeding mothers at 6 weeks (n = 152) and 4 months (n = 120) post-partum. Leptin and adiponectin were determined in skim breast milk and related to infant growth and fat mass assessed by skin-fold thickness measurements. A total of 118 infants were examined at 2 years. Results: The levels of both milk adipokines were slightly lower at 4 months compared with 6 weeks post-partum. Breast milk leptin was largely unrelated to infant anthropometric measures up to 2 years. Milk adiponectin tended to be inversely related to early infant anthropometry up to 4 months, but beyond was positively associated with weight gain and the sum of skin-folds up to 2 years. Conclusions: Our results suggest that higher adiponectin levels in breast milk might be associated with greater weight gain and higher fat mass in the offspring up to 2 years. Keywords: Adipokines, adiponectin, breast milk, body composition, leptin, weight gain.
Introduction The perinatal nutritional environment is suggested to have a programming effect on an individual's susceptibility to develop obesity (1). Breastfeeding is suggested to be protective against later obesity (2), which could partly be attributable to bioactive compounds present in breast milk, but absent in infant formula (3). However, the specific components of breast milk accounting for these effects have not yet been identified. Since the discovery of the adipocytesecreted factors leptin and adiponectin in breast milk (4,5), both adipokines have been proposed as strong candidates (6). Leptin is mainly produced in
adipose tissue, but also by other tissues including the mammary gland (7). It is released from adipose tissue in amounts proportionate to the body fat depots and plays a fundamental role in the hypothalamic regulation of energy homeostasis by decreasing food intake and increasing energy expenditure (8). In contrast, adiponectin is down-regulated in obesity and exerts insulin-sensitizing and antiinflammatory effects (9). Several animal and human studies have suggested a role of oral leptin administration or the amount of adipokines in breast milk in the regulation of early weight gain or growth trajectory (10–17). However, the majority of available human studies merely
Address for correspondence: Professor H Hauner, Else Kröner-Fresenius-Center for Nutritional Medicine, Klinikum rechts der Isar, Technische Universität München, Uptown München Campus D, Georg-Brauchle-Ring 60/62, München D-80992, Germany. E-mail: [email protected] Trial registration: clinicaltrials.gov, NCT00362089.
© 2014 The Authors Pediatric Obesity © 2014 International Association for the Study of Obesity. Pediatric Obesity ••, ••–••
ORIGINALRESEARCH
doi:10.1111/j.2047-6310.2014.222.x
ORIGINALRESEARCH
2
|
S. Brunner et al.
focused on rather global measures of infant overweight such as body mass index (BMI) or simple weight development and did not include more specific measurements of fat mass. We therefore aimed to explore the relationship of the adipokines leptin and adiponectin in breast milk with infant weight gain and body composition assessed by skin-fold thickness measurements.
Methods Data came from the INFAT study, a randomized controlled trial originally designed to examine the effect of reducing the maternal dietary n-6/n-3 fatty acid ratio during pregnancy and lactation on infant fat mass. The study design and the clinical results on infant body composition over the first year of life were previously published (18,19). Briefly, a total of 208 healthy pregnant women with a pre-pregnancy BMI between 18 and 30 kg m−2 were randomly assigned to either an intervention or a control group from the 15th week of gestation until 4 months post-partum. The dietary intervention consisted of a dietary supplementation with 1200 mg n-3 long-chain polyunsaturated fatty acids (LCPUFA) per day and a nutritional counselling to normalize the consumption of arachidonic acid to a moderate level of intake (∼90 mg per day). In contrast, women of the control group were advised to keep a healthy diet according to current recommendations. The study protocol was approved by the ethical committee of the Technische Universität München and all participants gave written informed consent.
UK) at the 15th and 32nd week of gestation. The mean of triplicate measurements per site was used for analysis. Maternal body fat percentage was calculated according to Durnin and Wormersley (20) and van Raaij et al (21). Infants were examined at birth (n = 188), at 6 weeks (n = 180), 4 months (n = 174), 1 year (n = 170) and 2 years (n = 118) post-partum. Weight, length and skin-fold thicknesses at four body sites were measured as previously described (19). Percentage body fat was calculated according to Weststrate and Deurenberg (22). Infant weight gain between the time point of milk collection and later time points of follow-up was also calculated. If infants could not be clinically examined at 2 years, parents were asked to provide weight and height as assessed in the routine pediatric ‘well-child check-up visit’ at 24 months (n = 52). Infant feeding practices (fully breastfed, partially breastfed, formula) were documented at each visit over the first year of life. The individuals lost to follow-up did not differ from those remaining in the study with regard to the major sociodemographic and clinical parameters, such as maternal age, education, parity, prepregnancy BMI or infant feeding practices (data not shown). The infants of the intervention group (treatment with n-3 LCPUFA) did not differ from the control group in growth or body composition, neither over the first year of life, except higher birth weight and ponderal index due to prolonged pregnancy duration (19), nor at 2 years of age (data not shown). For the present analysis, only the mother–infant pairs with available breast milk sample at 6 weeks or 4 months post-partum were included.
Collection of biosamples Breast milk samples of the lactating mothers were collected with an electric breast pump after an overnight fast at 6 weeks (n = 152) and 4 months (n = 120) post-partum. Approximately, 20 mL of a complete breast expression were kept, aliquoted and stored at −80°C until analysis. Maternal EDTA plasma samples were taken after an overnight fast at the same time points. Plasma was separated from red blood cells by centrifugation at 2000 × g at 4°C for 10 min, aliquoted and stored at −80°C until analysis.
Maternal and infant characteristics Maternal height, pre-pregnancy weight and weight development over the course of pregnancy were retrieved from the maternity records issued to the women by their gynecologists. Maternal skin-fold thickness measurements were performed at four sites (biceps, triceps, subscapular and suprailiacal) by a Holtain Caliper (Holtain Ltd, Croswell, Crymych,
Laboratory analyses For the analysis of leptin and total adiponectin, milk samples were pre-treated as follows: milk samples were thawed at room temperature and vortexed. The whole milk was sonicated three times for a 5-s burst by using an ultrasound stick (ultrasonic processor UP100H, 100 W, 30 kHz, 100% amplitude; Hielscher Ultrasonics GmbH, Teltow, Germany). Skim milk was prepared by centrifugation of whole milk (14000 rpm, 30 min). Leptin and adiponectin were detected in skim milk by commercially available radioimmunoassays (leptin: Mediagnost, Reutlingen, Germany; adiponectin: Millipore, St. Charles, MO, USA). Leptin and high molecular weight (HMW) adiponectin in maternal plasma were quantified using enzyme-linked immunosorbent assay (Leptin: Mediagnost, Reutlingen, Germany; HMW adiponectin: ALPCO Diagnostics, Salem, MA, USA) in a subsample of n = 82 women.
© 2014 The Authors Pediatric Obesity © 2014 International Association for the Study of Obesity. Pediatric Obesity ••, ••–••
|
3
Table 1 Concentrations of the adipokines, leptin and adiponectin in breast milk at 6 weeks and 4 months post-partum
Milk leptin (ng mL−1) Plasma leptin (ng mL−1) Milk total adiponectin (ng mL−1) Plasma HMW adiponectin (μg mL−1)
6 weeks post-partum
4 months post-partum
Median (IQR*)
n
Median (IQR*)
n
0.11 (0.19) 9.52 (8.56) 10.93 (8.34) 2.13 (1.43)
152 82 151 82
0.09 (0.18) 8.30 (10.64) 10.36 (9.40) 2.03 (1.32)
120 82 120 82
P (over time)†
P‡
0.65 0.29 0.03 0.01
P < 0.001 P < 0.001
*IQR, interquartile range. †Change over time by Wilcoxon test. ‡Comparison of the respective adipokine levels in breast milk vs. plasma (Wilcoxon test).
Statistical analyses Statistical analyses were performed with the R software package (version 2.8.1; R Foundation for Statistical Computing, http://www.r-project.org) and PASW software (version 20.0; SPSS Inc, Chicago, IL, USA). Changes of parameters over time within the groups were assessed by Wilcoxon tests. Mann Whitney-U tests were performed to explore differences in the parameters between the control and the intervention group at the individual time points. Correlations between breast milk and plasma adipokines as well as with maternal anthropometric variables were explored by Spearman correlation coefficients. To analyze associations of breast milk adipokines with infant growth and body composition up to 2 years of age, multiple regression models adjusting for maternal pre-pregnancy BMI, gestational weight gain, pregnancy duration, sex, ponderal index at birth, group and degree of breastfeeding (full vs. partial) were performed. A two-sided P-value ≤ 0.05 was considered statistically significant.
Results Adipokines in breast milk and maternal plasma As no differences in maternal or milk adipokines between the original intervention and control group were found at any time point (data not shown), pooled data from both groups are presented. Median concentrations of leptin and total adiponectin in breast milk at 6 weeks post-partum were 0.11 (interquartile range: 0.19) ng mL−1 and 10.93 (8.34) ng mL−1, respectively (Table 1). Both leptin and adiponectin concentrations in breast milk were slightly lower at 4 months compared with 6 weeks, with a significant difference over time only for total adiponectin. Median plasma levels of leptin and HMW adiponectin showed the same pattern (Table 1). Maternal plasma leptin levels over the course of pregnancy and lactation were previously reported (23). Concentrations of both adipokines were substantially lower in breast milk
Table 2 Correlations of breast milk leptin with maternal anthropometry Anthropometric variable
n
R*
P
Pre-pregnancy BMI BMI (15th wk gest) Sum 4 SFTs (15th wk gest) Percentage body fat (15th wk gest) BMI (32nd wk gest) Sum 4 SFTs (32nd wk gest) Percentage body fat (32nd wk gest)
152 152 98 115 149 108 117
0.49 0.52 0.57 0.54 0.57 0.61 0.61
Breast milk leptin and adiponectin in relation to infant body composition up to 2 years S. Brunner1, D. Schmid1, K. Zang1, D. Much1, B. Knoeferl2, J. Kratzsch3, U. Amann-Gassner1, B. L. Bader4 and H. Hauner1,4 1 Else Kröner-Fresenius-Center for Nutritional Medicine, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 2Institute for Medical Statistics and Epidemiology, Klinikum rechts der Isar, Technische, Universität München, Munich, Germany; 3Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; 4ZIEL – Research Center for Nutrition and Food Sciences, Nutritional Medicine Unit, Technische Universität München, Freising, Germany
Received 16 September 2013; revised 20 December 2013; accepted 17 February 2014
Summary Background: Adipokines in breast milk have been associated with infant growth trajectories. Objective: We aimed to explore the relationship of leptin and adiponectin in breast milk with infant weight gain and body composition up to the age of 2 years. Methods: Breast milk samples were collected from exclusively or partially breastfeeding mothers at 6 weeks (n = 152) and 4 months (n = 120) post-partum. Leptin and adiponectin were determined in skim breast milk and related to infant growth and fat mass assessed by skin-fold thickness measurements. A total of 118 infants were examined at 2 years. Results: The levels of both milk adipokines were slightly lower at 4 months compared with 6 weeks post-partum. Breast milk leptin was largely unrelated to infant anthropometric measures up to 2 years. Milk adiponectin tended to be inversely related to early infant anthropometry up to 4 months, but beyond was positively associated with weight gain and the sum of skin-folds up to 2 years. Conclusions: Our results suggest that higher adiponectin levels in breast milk might be associated with greater weight gain and higher fat mass in the offspring up to 2 years. Keywords: Adipokines, adiponectin, breast milk, body composition, leptin, weight gain.
Introduction The perinatal nutritional environment is suggested to have a programming effect on an individual's susceptibility to develop obesity (1). Breastfeeding is suggested to be protective against later obesity (2), which could partly be attributable to bioactive compounds present in breast milk, but absent in infant formula (3). However, the specific components of breast milk accounting for these effects have not yet been identified. Since the discovery of the adipocytesecreted factors leptin and adiponectin in breast milk (4,5), both adipokines have been proposed as strong candidates (6). Leptin is mainly produced in
adipose tissue, but also by other tissues including the mammary gland (7). It is released from adipose tissue in amounts proportionate to the body fat depots and plays a fundamental role in the hypothalamic regulation of energy homeostasis by decreasing food intake and increasing energy expenditure (8). In contrast, adiponectin is down-regulated in obesity and exerts insulin-sensitizing and antiinflammatory effects (9). Several animal and human studies have suggested a role of oral leptin administration or the amount of adipokines in breast milk in the regulation of early weight gain or growth trajectory (10–17). However, the majority of available human studies merely
Address for correspondence: Professor H Hauner, Else Kröner-Fresenius-Center for Nutritional Medicine, Klinikum rechts der Isar, Technische Universität München, Uptown München Campus D, Georg-Brauchle-Ring 60/62, München D-80992, Germany. E-mail: [email protected] Trial registration: clinicaltrials.gov, NCT00362089.
© 2014 The Authors Pediatric Obesity © 2014 International Association for the Study of Obesity. Pediatric Obesity ••, ••–••
ORIGINALRESEARCH
doi:10.1111/j.2047-6310.2014.222.x
ORIGINALRESEARCH
2
|
S. Brunner et al.
focused on rather global measures of infant overweight such as body mass index (BMI) or simple weight development and did not include more specific measurements of fat mass. We therefore aimed to explore the relationship of the adipokines leptin and adiponectin in breast milk with infant weight gain and body composition assessed by skin-fold thickness measurements.
Methods Data came from the INFAT study, a randomized controlled trial originally designed to examine the effect of reducing the maternal dietary n-6/n-3 fatty acid ratio during pregnancy and lactation on infant fat mass. The study design and the clinical results on infant body composition over the first year of life were previously published (18,19). Briefly, a total of 208 healthy pregnant women with a pre-pregnancy BMI between 18 and 30 kg m−2 were randomly assigned to either an intervention or a control group from the 15th week of gestation until 4 months post-partum. The dietary intervention consisted of a dietary supplementation with 1200 mg n-3 long-chain polyunsaturated fatty acids (LCPUFA) per day and a nutritional counselling to normalize the consumption of arachidonic acid to a moderate level of intake (∼90 mg per day). In contrast, women of the control group were advised to keep a healthy diet according to current recommendations. The study protocol was approved by the ethical committee of the Technische Universität München and all participants gave written informed consent.
UK) at the 15th and 32nd week of gestation. The mean of triplicate measurements per site was used for analysis. Maternal body fat percentage was calculated according to Durnin and Wormersley (20) and van Raaij et al (21). Infants were examined at birth (n = 188), at 6 weeks (n = 180), 4 months (n = 174), 1 year (n = 170) and 2 years (n = 118) post-partum. Weight, length and skin-fold thicknesses at four body sites were measured as previously described (19). Percentage body fat was calculated according to Weststrate and Deurenberg (22). Infant weight gain between the time point of milk collection and later time points of follow-up was also calculated. If infants could not be clinically examined at 2 years, parents were asked to provide weight and height as assessed in the routine pediatric ‘well-child check-up visit’ at 24 months (n = 52). Infant feeding practices (fully breastfed, partially breastfed, formula) were documented at each visit over the first year of life. The individuals lost to follow-up did not differ from those remaining in the study with regard to the major sociodemographic and clinical parameters, such as maternal age, education, parity, prepregnancy BMI or infant feeding practices (data not shown). The infants of the intervention group (treatment with n-3 LCPUFA) did not differ from the control group in growth or body composition, neither over the first year of life, except higher birth weight and ponderal index due to prolonged pregnancy duration (19), nor at 2 years of age (data not shown). For the present analysis, only the mother–infant pairs with available breast milk sample at 6 weeks or 4 months post-partum were included.
Collection of biosamples Breast milk samples of the lactating mothers were collected with an electric breast pump after an overnight fast at 6 weeks (n = 152) and 4 months (n = 120) post-partum. Approximately, 20 mL of a complete breast expression were kept, aliquoted and stored at −80°C until analysis. Maternal EDTA plasma samples were taken after an overnight fast at the same time points. Plasma was separated from red blood cells by centrifugation at 2000 × g at 4°C for 10 min, aliquoted and stored at −80°C until analysis.
Maternal and infant characteristics Maternal height, pre-pregnancy weight and weight development over the course of pregnancy were retrieved from the maternity records issued to the women by their gynecologists. Maternal skin-fold thickness measurements were performed at four sites (biceps, triceps, subscapular and suprailiacal) by a Holtain Caliper (Holtain Ltd, Croswell, Crymych,
Laboratory analyses For the analysis of leptin and total adiponectin, milk samples were pre-treated as follows: milk samples were thawed at room temperature and vortexed. The whole milk was sonicated three times for a 5-s burst by using an ultrasound stick (ultrasonic processor UP100H, 100 W, 30 kHz, 100% amplitude; Hielscher Ultrasonics GmbH, Teltow, Germany). Skim milk was prepared by centrifugation of whole milk (14000 rpm, 30 min). Leptin and adiponectin were detected in skim milk by commercially available radioimmunoassays (leptin: Mediagnost, Reutlingen, Germany; adiponectin: Millipore, St. Charles, MO, USA). Leptin and high molecular weight (HMW) adiponectin in maternal plasma were quantified using enzyme-linked immunosorbent assay (Leptin: Mediagnost, Reutlingen, Germany; HMW adiponectin: ALPCO Diagnostics, Salem, MA, USA) in a subsample of n = 82 women.
© 2014 The Authors Pediatric Obesity © 2014 International Association for the Study of Obesity. Pediatric Obesity ••, ••–••
|
3
Table 1 Concentrations of the adipokines, leptin and adiponectin in breast milk at 6 weeks and 4 months post-partum
Milk leptin (ng mL−1) Plasma leptin (ng mL−1) Milk total adiponectin (ng mL−1) Plasma HMW adiponectin (μg mL−1)
6 weeks post-partum
4 months post-partum
Median (IQR*)
n
Median (IQR*)
n
0.11 (0.19) 9.52 (8.56) 10.93 (8.34) 2.13 (1.43)
152 82 151 82
0.09 (0.18) 8.30 (10.64) 10.36 (9.40) 2.03 (1.32)
120 82 120 82
P (over time)†
P‡
0.65 0.29 0.03 0.01
P < 0.001 P < 0.001
*IQR, interquartile range. †Change over time by Wilcoxon test. ‡Comparison of the respective adipokine levels in breast milk vs. plasma (Wilcoxon test).
Statistical analyses Statistical analyses were performed with the R software package (version 2.8.1; R Foundation for Statistical Computing, http://www.r-project.org) and PASW software (version 20.0; SPSS Inc, Chicago, IL, USA). Changes of parameters over time within the groups were assessed by Wilcoxon tests. Mann Whitney-U tests were performed to explore differences in the parameters between the control and the intervention group at the individual time points. Correlations between breast milk and plasma adipokines as well as with maternal anthropometric variables were explored by Spearman correlation coefficients. To analyze associations of breast milk adipokines with infant growth and body composition up to 2 years of age, multiple regression models adjusting for maternal pre-pregnancy BMI, gestational weight gain, pregnancy duration, sex, ponderal index at birth, group and degree of breastfeeding (full vs. partial) were performed. A two-sided P-value ≤ 0.05 was considered statistically significant.
Results Adipokines in breast milk and maternal plasma As no differences in maternal or milk adipokines between the original intervention and control group were found at any time point (data not shown), pooled data from both groups are presented. Median concentrations of leptin and total adiponectin in breast milk at 6 weeks post-partum were 0.11 (interquartile range: 0.19) ng mL−1 and 10.93 (8.34) ng mL−1, respectively (Table 1). Both leptin and adiponectin concentrations in breast milk were slightly lower at 4 months compared with 6 weeks, with a significant difference over time only for total adiponectin. Median plasma levels of leptin and HMW adiponectin showed the same pattern (Table 1). Maternal plasma leptin levels over the course of pregnancy and lactation were previously reported (23). Concentrations of both adipokines were substantially lower in breast milk
Table 2 Correlations of breast milk leptin with maternal anthropometry Anthropometric variable
n
R*
P
Pre-pregnancy BMI BMI (15th wk gest) Sum 4 SFTs (15th wk gest) Percentage body fat (15th wk gest) BMI (32nd wk gest) Sum 4 SFTs (32nd wk gest) Percentage body fat (32nd wk gest)
152 152 98 115 149 108 117
0.49 0.52 0.57 0.54 0.57 0.61 0.61
