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Zinc concentrations in human milk and infant serum during the first six months of lactation
Accepted Manuscript Title: Zinc concentrations in human milk and infant serum during the first six months of lactation Authors: Dijana Djurovi´c, Branka Milisavljevi´c, Boban – Mileti´c, Sneˇzana Spasi´c, Mugoˇsa, Nikoleta Lugonja, Srdan Miroslav Vrvi´c PII: DOI: Reference:
S0946-672X(16)30333-9 http://dx.doi.org/doi:10.1016/j.jtemb.2017.02.012 JTEMB 25897
To appear in: Received date: Revised date: Accepted date:
25-10-2016 15-2-2017 17-2-2017
Please cite this article as: Djurovi´c Dijana, Milisavljevi´c Branka, Mugoˇsa Boban, – Spasi´c Sneˇzana, Vrvi´c Miroslav.Zinc concentrations Lugonja Nikoleta, Mileti´c Srdan, in human milk and infant serum during the first six months of lactation.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2017.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Zinc concentrations in human milk and infant serum during the first six months of lactation
Dijana Djurović,1 Branka Milisavljević,2 Boban Mugoša,1 Nikoleta Lugonja,3 Srđan Miletić,3Snežana Spasić,3 Miroslav Vrvić 4
1
Institute of Public Health of Montenegro, Džona Džeksona bb, Podgorica, Montenegro
2
Department of Neonatology, Subotica Hospital, Izvorska 3, Subotica, Serbia
3
Department of Chemistry of the Institute of Chemistry, Technology and Metallurgy, University
of Belgrade, Njegoševa 1, Belgrade, Serbia 4
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia
*Corresponding author: Srđan Miletić, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, email: [email protected], Telephone: +381 60 3514360
Short title: Zinc in human milk
1
Abstract
Normal supply of zinc to the newborn via milk is essential for normal development. Using ICPOES, we analyzed changes in the level of Zn in milk and infant serum in the neonatal period (Day 1 and Day 28 post partum) and at 6 months after delivery, in the cohort of 60 mothers and exclusively breastfed babies. Zn level in the serum showed increase (significant at 6 months) during this period (mg/mL): Day 1: 0.52 ± 0.12; Day 28: 0.59 ± 0.19; 6 months: 0.68 ± 0.28. The concentration of Zn in the milk showed an opposite (decreasing) trend during the follow up: Day 1: 4.70 ± 1.74 mg/L; Day 28: 2.65 ± 1.06; 6 months: 0.46 ± 0.36. A significant negative correlation was established between serum and milk [Zn] at day 28 (R = -0.338; p = 0.008), whereas a positive correlation was found at 6 months between these parameters (R = 0.306; p = 0.018). There was no significant correlation between [Zn] in the milk and serum and infants' body mass, mothers' age and mass at delivery. The level of Zn in the milk at 6 months of lactation is not sufficient to meet the recommended values. This implies that in Serbian population, Zn supplementation might be needed in the later phase of lactation.
Keywords: zinc; human milk; serum; lactation; ICP-OES; neonate
2
Introduction Human milk is considered to be the best source of nutrition for the newborn infants. WHO recommends exclusive breastfeeding up to 6 months of age [1]. Human milk contains almost all essential components as protective elements against infections, allergies and many different chronic-degenerative diseases [2]. In the early childhood, especially in the first few months of life, human milk enables immune system protection and health development [3]. Zinc plays a crucial role in metabolism of proteins, carbohydrates, and lipids, cell differentiation and body growth. It is also very important in gene transcription and hormonal structure and represents a constituent of almost 300 enzymes [4]. During lactation, Zn concentration in human milk decreases much faster compare to other minerals [5,6]. The highest level is reached in the early period of lactation and strongly declines during the first three months. The most reliable biochemical indicator for the assessment of zinc status in healthy adult population is the measurement of the concentration of zinc in serum or plasma [8]. Only a few studies addressed in parallel Zn levels in milk and in serum of breastfed infants [9-12], but to the best of our knowledge only one study estimated correlation between these parameters [12]. The aim of this study was to investigate the relationship between the levels of zinc in mother's milk and infant serum on the first, the twenty eighth day, and after 6 months of lactation. The main objective was to evaluate the changes in zinc concentrations and to detect potential correlation between a set of parameters during the lactation period. This kind of study was performed for the first time in Serbia and obtained data will be very useful to define zinc status of lactating mothers and its influence on infant zinc serum status.
Material and Methods Subjects Our study included 60 healthy non-smoker mothers and their infants at the Department of Neonatology, Subotica Hospital in the period from January to June 2013. The demographic characteristics of the cohort are presented in Table 1. Mothers were invited to participate in the study with the following criteria: individual consent to participate, the ability to supply at least 10 mL of milk in 6 months period, and to exclusively breastfeed their infants during that period (unless advised otherwise by neonatologist/pediatrician). All mothers gave written consent for blood sampling from their infants and for the collection of their blood at Day 1. For mothers 3
below the age of 18, additional inform consents were obtained from parents. All mothers and infants were assessed healthy on days of sample collection by an experienced clinician, except for Day 1 where 1/3 of neonatal cohort suffered from jaundice. Ethical Committee of Medical Faculty in Belgrade approved research, No.01-434/4, dated 22/05/2012. The study protocol was in accordance with the guidelines of the Ethical Committee, Faculty of Medicine.
Table 1. Baseline characteristics Parameter
Value
Maternal age [years]
28.6 ± 4.3 (range: 16–36)
Maternal mass at delivery [kg]
82.0 ± 10.8
Number of parities
1.7 ± 0.9 (range: 1–5)
Gestational age [weeks]
39.4 ± 1.2 (range: 38–42)
Neonatal mass at birth [g]
3424 ± 381 (range: 2440–4100)
Sex [male/female]
32/28
Number of neonates with IUGRa
3/60
Number of neonates with neonatal 20/60 jaundice a
IUGR (intrauterine growth restriction) was defined as estimated fetal and neonatal birth weight
beyond 10th percentile for the gestational age. Data are presented as mean ± standard deviation.
Sample collection and analysis Milk samples (10–20 mL) were collected on Day 1 (within 24 h after delivery), Day 28,and after 6 months of lactation. Every mother has previously cleansed breast and nipples with Mili-Q water using protective gloves. Milk was collected using a manual breast milk pump and/or a passive breast milk sampler. Mothers were instructed to sample milk both at the beginning and at the end of the breast feeding session. Milk was stored at -20ºC before analysis. Blood was 4
collected from the infants by venous puncture on the dorsum of the hand into Vacutainer tubes with K3EDTA (BD, Franklin Lakes, NJ, USA). To obtain serum samples, blood was collected using Vacutainer tubes with no anticoagulant (BD). After centrifugation, the serum was separated and kept at -20ºC until further analysis. The collection of milk, blood and sera was conducted in the morning, before the first meal/breast feeding session. Milk and serum (1 mL) were diluted to 10 mL with deionized Mili-Q water. Zn content was determined by Spectro Arcos ICP-OES analyzer (Spectro Analytical Instruments GmbH, Kleve, Germany). All used chemical reagents were of analytical grade. Zn working analytical solutions were prepared after serial dilution of stock reference solution containing 1000 mg/L of element (LGC-ICP-OES stock solution) in a gradient as needed. Reference material National Institute of Standards and Technology (NIST) standard reference material infant/adult nutritional formula SRM-1849 was used for determination of accuracy and precision of this study.
Statistical methods Statistical analyses were conducted using STATISTICA 8.0 (StatSoft Inc., Tulsa, OK, USA). Results are presented as box plots. Boxes represent the median and the 25th and 75th percentiles; whiskers represent the non-outlier range. Outliers and extremes are defined as data point values that are more than 1.5× and 3× interquartile range (IQR) outside the box. p-Values were determined using 2-way analysis of variance with post hoc Duncan test or using nonparametric 2-tailed Mann-Whitney U test. Correlation was established via Pearson coefficient (R). Results were considered to be statistically significant if p < 0.05.
Results and Discussion Fig 1A. shows changes in the Zn concentration in infant serum at birth (Day 1), at the end of neonatal period (Day 28), and 6 months post partum. It can be observed that Zn level in serum rises with age, and that at 6 months shows significantly higher values compared to two other time points. It is worth mentioning that 38% of infants did not experience a rise in serum [Zn] between Day 28 and 6 months. This might be related to the lower [Zn] in breast milk in this subcohort (0.38 ± 0.24 mg/L) compared to the milk that was fed to the babies showing an increase in serum [Zn] during the first 6 months (0.50 ± 0.38 mg/L); although the difference was not statistically significant. Further, a significant percentage of infants showed Zn levels that were 5
below the lower reference limit (0.6 mg/L): 61% at Day 1; 53% at Day 28; and 40% at 6 months. It is important to note that Zn levels in serums of newborns with (n = 20) and without (n = 40) neonatal jaundice did not differ. In addition, there was no statistically significant difference in [Zn] in serum from infants of different sex. We also compared [Zn] in serum samples obtained from mothers and newborns at Day 1. Mean values were 0.540 ± 0.179 and 0.520 ± 0.116 mg/L, respectively, and there was no correlation between these two parameters (R = 0.047; p > 0.05). Zn concentration in the milk showed an opposite trend compared to infant serum levels i.e. it gradually decreased with time (Fig. 1B). This is in line with previous reports [13–16]. There was no significant correlation between serum and milk [Zn] (at all three time points) and mothers' age and weight, number of parities, or leukocytes count in neonates' blood at Day 1 (13.5 ± 4.4 × 109 L-1) (Table 2). Serum [Zn] is known to be the most reliable marker of Zn status in healthy adults. Whether or not it is an appropriate marker in newborns is unclear so far. Nevertheless, as the lack of correlation between leukocytes count and [Zn] in serum observed here speaks in favor of applicability of this parameter in assessing Zn status in the neonates.
Table 2. Correlation coefficients (R) showing dependency between Zn level in milk and sera and maternal age and mass at delivery, number of parities, and leukocytes count in the blood of neonates at Day 1. Maternal age
Maternal mass Number parities
of Leukocytes count
in
neonates
at
Day 1 [Zn] in serum at Day 1
[Zn] in serum at Day 28
[Zn] in serum at 6 months
[Zn] in milk at Day 1
R = -0.048
R = -0.156
R = -0.053
R = -0.184
p = 0.716
p = 0.233
p = 0.687
p = 0.158
R = -0.014
R = -0.203
R = 0.031
R = -0.094
p = 0.917
p = 0.120
p = 0.870
p = 0.478
R = 0.091
R = -0.005
R = 0.115
R = 0.041
p = 0.488
p = 0.968
p = 0.380
p = 0.756
R = 0.052
R = -0.100
R = 0.019
R = -0.037
p = 0.690
p = 0.954
p = 0.886
p = 0.780
6
[Zn] in milk at Day 28
[Zn] in milk at 6 months
R = -0.022
R = -0.106
R = -0.070
R = 0.230
p = 0.866
p = 0.418
p = 0.606
p = 0.077
R = 0.048
R = 0.010
R = 0.237
R = 0.135
p = 0.720
p = 0.938
p = 0.068
p = 0.303
The obtained values for [Zn] in full-term milk are in accordance with previous population-based and methodological studies [13-17]. Of note, Zn deficiency of milk has been found in specific local populations [18-20]. In the case of exclusively breastfed babies, the quantity of Zn of 2 mg/day (according to Dietary Reference Intake), must be contained in the daily volume of human milk ingested by the infants [21]. Lactating woman in good health can produce 640 to 820 mL of milk per day [22]. If we take into account the mean volume of milk per day (780 mL) and the measured concentrations of Zn in the examined cohort, about 50% of infants were provided with less than 2 mg/day. At 6 months post partum, the concentration of Zn in the milk showed a drastic drop (from 4.70 ± 1.74 mg/L at Day 28 to 0.46 ± 0.36 mg/L). This implies that Zn supplementation should be advised to mitigate Zn deficiency during the later period of lactation at least. Colostrum (i.e. milk at Day 1) showed the highest Zn concentration. Pertinent to this, it is known that adequate supply of Zn is essential to the newborn particularly during the first days of life. This is implied by the early death of pups from Zn deficiency due to mutation in Zn transporter ZnT-4 that suppresses the transfer of Zn into the milk in mammary glands [23]. High level of Zn adds to some other supreme features of colostrum compared to mature milk, such as high antioxidative activity [24] and sufficient levels of iron and copper [25]. As expected, infant mass increased with life time (Fig. 1C). Next, Pearson correlation coefficients were calculated to determine dependency between concentrations of Zn in the serum and in the milk. Significant negative correlation was established between serum Zn level and milk Zn concentration at Day 28 (R = -0.338; p = 0.008). In addition, a significant positive correlation was found for serum and milk [Zn] at 6 months (R = 0.306; p = 0.018). A previous study has reported positive correlation between Zn level in mother's milk and in serum of infants 4–6 months old [12]. Correlations and trends observed here imply that concentration of Zn in milk changes to accommodate the needs of the infant. Level of Zn in milk decreases as the concentration of Zn in infant serum increases to reach the homeostasis. It appears that following 6 months, milk serves to maintain rather than to raise infant Zn level. There was no significant 7
correlation between body mass and the level of Zn in the milk and serum. Available data on this are ambiguous [26].
Figure 1. concentration of Zn and breastfed infant mass during six months after delivery. Panel A: Concentration of Zn in infant serum; Panel B: Concentration of Zn in milk; Panel C: Infant mass. Boxes represent the median and the 25th and 75th percentiles; whiskers represent the nonoutlier range. Outliers (circles) - data point values that are more than 1.5× IQR outside the box. Extremes (asterisk) - data points more than 3× IQR outside the box. Statistical significance (pvalues) are presented. ns, non-significant (p > 0.05).
Conclusions In this study 60 pairs of mothers and their infants were involved to evaluate changes and correlation between levels of zinc in human milk and infant serum. Levels of Zn in milk and infant serum showed significant correlation and reciprocal trends during first six months post partum. The amount of Zn available in the milk meet the nutritive requirements during the neonatal period, but a drastic drop at six months of lactation imply that exclusively breastfed infants might be exposed to risk of Zn deficiency. Our data should lead to the improvement of the quality of breastfeeding in Serbia. Factors influencing infant and maternal zinc status, especially in exclusively breastfed infants, as well as human milk, blood and serum zinc concentration should be further investigated.
Acknowledgments 8
This study received grants from the Ministry of Education Science of the Republic of Serbia (Grant No 43004)
9
References
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13. M.A. Hanna, N.N. Dogadkin, I.A. Ashur, W.M. Markus, Copper, Selenium and Zinc Concentrations in Human Milk during the First Three Weeks of Lactation, Biol. Trace Elem. Res. 107 (2005)11-23. 14. K.L. Bjorklund, M. Vahter, B. Palm, M. Grandér, S. Lignell, M. Berglund, Metals and Trace Element Concentrations in Breast Milk of First Healthy Mothers: A Biological Monitoring Study, Environ. Health.11 (2012)92-99. 15. J. Mello-Neto, P.H.C. Rondó, M. Oshiiwa, M.A. Morgano, C.Z. Zacari, M.L. Santos, Iron Supplementation in Pregnancy and Breastfeeding and Iron, Copper and Zinc Status of Lactating Women from a Human Milk Bank, J. Trop. Pediatrics59 (2013)140-144. 16. M. Leotsinidis, A. Alexopoulos, E. Kostopoulou-Farri, Toxic and essential trace elements in human milk from Greek lactating women: association with dietary habits and other factors, Chemosphere61 (2005)238–247. 17. S. Fernández-Menéndez, M.L. Fernández-Sánchez, B. Fernández-Colomer, R.R. de la Flor St Remy, G.D. Cotallo, A.S. Freire, B.F. Braz, R.E. Santelli, A. Sanz-Medel, Total zinc quantification by inductively coupled plasma-mass spectrometry and its speciation by size exclusion chromatography-inductively coupled plasma-mass spectrometry in human milk and commercial formulas: Importance in infant nutrition, J. Chromatogr. A 1428 (2016)246254. 18. R. Mahdavi, L. Nikniaz, S.J. Gayemmagami, Association between Zinc, Copper and Iron Concentrations in Breast Milk and Growth of Healthy Infants in Tabriz, Iran, Biol. Trace Elem. Res. 135 (2010)174-181. 19. E. Orun, S.S. Yalçin, O. AIkut, G. Orhan, G.K. Morgil, Zinc and Copper Concentrations in Breastmilk at the Second Month of Lactation. Indian Pediatr. 49 (2012)133-135. 20. A. Winiarska-Mieczan, Cadmiun, Lead, Copper and Zinc in Breast Milk in Poland, Biol. Trace Elem. Res. 157 (2014)36-44. 21. Institute of Medicine – DietaryReference Intakes; the Essential Guide to Nutrient Requirements. National Academy Press, Washington DC; 2006 22. L.A. Del Ciampo, R.G. Ricco, C.A.N. Almeida, Aleitamento Materno. Passagens e Transferências Mãe-Filho. Editora Atheneu, Rio de Janeiro, 2004 23. L. Huang, J. Gitschier, A novel gene involved in zinc transport is deficient in the lethal milk mouse, Nat. Genet. 17 (1997)292–297. 11
24. V. Marinković, M. Ranković-Janevski, S. Spasić, A. Nikolić-Kokić, N. Lugonja, D. Djurović, S. Miletić, M.M. Vrvić, I. Spasojević, Antioxidative activity of colostrum and human milk: Effects of pasteurization and storage, J. Pediatr. Gastr. Nutr. 62 (2016) 901-906 25. D. Silvestre, C. Martìnez-Costa, M.J. Lagarda, J. Brines, R. Farré, G. Clemente, Copper, iron, and zinc contents in human milk during the first three months of lactation: a longitudinal study, Biol. Trace Elem. Res. 80 (2001)1-11. 26. R.G. Sezer, G. Aydemir, A.B. Akcan, D.S. Bayoglu, T. Guran, A. Bozaykut, Effect of breastfeeding on serum zinc levels and growth in healthy infants, Breastfeed. Med. 8 (2013)159-163.
12
S0946-672X(16)30333-9 http://dx.doi.org/doi:10.1016/j.jtemb.2017.02.012 JTEMB 25897
To appear in: Received date: Revised date: Accepted date:
25-10-2016 15-2-2017 17-2-2017
Please cite this article as: Djurovi´c Dijana, Milisavljevi´c Branka, Mugoˇsa Boban, – Spasi´c Sneˇzana, Vrvi´c Miroslav.Zinc concentrations Lugonja Nikoleta, Mileti´c Srdan, in human milk and infant serum during the first six months of lactation.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2017.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Zinc concentrations in human milk and infant serum during the first six months of lactation
Dijana Djurović,1 Branka Milisavljević,2 Boban Mugoša,1 Nikoleta Lugonja,3 Srđan Miletić,3Snežana Spasić,3 Miroslav Vrvić 4
1
Institute of Public Health of Montenegro, Džona Džeksona bb, Podgorica, Montenegro
2
Department of Neonatology, Subotica Hospital, Izvorska 3, Subotica, Serbia
3
Department of Chemistry of the Institute of Chemistry, Technology and Metallurgy, University
of Belgrade, Njegoševa 1, Belgrade, Serbia 4
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia
*Corresponding author: Srđan Miletić, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, email: [email protected], Telephone: +381 60 3514360
Short title: Zinc in human milk
1
Abstract
Normal supply of zinc to the newborn via milk is essential for normal development. Using ICPOES, we analyzed changes in the level of Zn in milk and infant serum in the neonatal period (Day 1 and Day 28 post partum) and at 6 months after delivery, in the cohort of 60 mothers and exclusively breastfed babies. Zn level in the serum showed increase (significant at 6 months) during this period (mg/mL): Day 1: 0.52 ± 0.12; Day 28: 0.59 ± 0.19; 6 months: 0.68 ± 0.28. The concentration of Zn in the milk showed an opposite (decreasing) trend during the follow up: Day 1: 4.70 ± 1.74 mg/L; Day 28: 2.65 ± 1.06; 6 months: 0.46 ± 0.36. A significant negative correlation was established between serum and milk [Zn] at day 28 (R = -0.338; p = 0.008), whereas a positive correlation was found at 6 months between these parameters (R = 0.306; p = 0.018). There was no significant correlation between [Zn] in the milk and serum and infants' body mass, mothers' age and mass at delivery. The level of Zn in the milk at 6 months of lactation is not sufficient to meet the recommended values. This implies that in Serbian population, Zn supplementation might be needed in the later phase of lactation.
Keywords: zinc; human milk; serum; lactation; ICP-OES; neonate
2
Introduction Human milk is considered to be the best source of nutrition for the newborn infants. WHO recommends exclusive breastfeeding up to 6 months of age [1]. Human milk contains almost all essential components as protective elements against infections, allergies and many different chronic-degenerative diseases [2]. In the early childhood, especially in the first few months of life, human milk enables immune system protection and health development [3]. Zinc plays a crucial role in metabolism of proteins, carbohydrates, and lipids, cell differentiation and body growth. It is also very important in gene transcription and hormonal structure and represents a constituent of almost 300 enzymes [4]. During lactation, Zn concentration in human milk decreases much faster compare to other minerals [5,6]. The highest level is reached in the early period of lactation and strongly declines during the first three months. The most reliable biochemical indicator for the assessment of zinc status in healthy adult population is the measurement of the concentration of zinc in serum or plasma [8]. Only a few studies addressed in parallel Zn levels in milk and in serum of breastfed infants [9-12], but to the best of our knowledge only one study estimated correlation between these parameters [12]. The aim of this study was to investigate the relationship between the levels of zinc in mother's milk and infant serum on the first, the twenty eighth day, and after 6 months of lactation. The main objective was to evaluate the changes in zinc concentrations and to detect potential correlation between a set of parameters during the lactation period. This kind of study was performed for the first time in Serbia and obtained data will be very useful to define zinc status of lactating mothers and its influence on infant zinc serum status.
Material and Methods Subjects Our study included 60 healthy non-smoker mothers and their infants at the Department of Neonatology, Subotica Hospital in the period from January to June 2013. The demographic characteristics of the cohort are presented in Table 1. Mothers were invited to participate in the study with the following criteria: individual consent to participate, the ability to supply at least 10 mL of milk in 6 months period, and to exclusively breastfeed their infants during that period (unless advised otherwise by neonatologist/pediatrician). All mothers gave written consent for blood sampling from their infants and for the collection of their blood at Day 1. For mothers 3
below the age of 18, additional inform consents were obtained from parents. All mothers and infants were assessed healthy on days of sample collection by an experienced clinician, except for Day 1 where 1/3 of neonatal cohort suffered from jaundice. Ethical Committee of Medical Faculty in Belgrade approved research, No.01-434/4, dated 22/05/2012. The study protocol was in accordance with the guidelines of the Ethical Committee, Faculty of Medicine.
Table 1. Baseline characteristics Parameter
Value
Maternal age [years]
28.6 ± 4.3 (range: 16–36)
Maternal mass at delivery [kg]
82.0 ± 10.8
Number of parities
1.7 ± 0.9 (range: 1–5)
Gestational age [weeks]
39.4 ± 1.2 (range: 38–42)
Neonatal mass at birth [g]
3424 ± 381 (range: 2440–4100)
Sex [male/female]
32/28
Number of neonates with IUGRa
3/60
Number of neonates with neonatal 20/60 jaundice a
IUGR (intrauterine growth restriction) was defined as estimated fetal and neonatal birth weight
beyond 10th percentile for the gestational age. Data are presented as mean ± standard deviation.
Sample collection and analysis Milk samples (10–20 mL) were collected on Day 1 (within 24 h after delivery), Day 28,and after 6 months of lactation. Every mother has previously cleansed breast and nipples with Mili-Q water using protective gloves. Milk was collected using a manual breast milk pump and/or a passive breast milk sampler. Mothers were instructed to sample milk both at the beginning and at the end of the breast feeding session. Milk was stored at -20ºC before analysis. Blood was 4
collected from the infants by venous puncture on the dorsum of the hand into Vacutainer tubes with K3EDTA (BD, Franklin Lakes, NJ, USA). To obtain serum samples, blood was collected using Vacutainer tubes with no anticoagulant (BD). After centrifugation, the serum was separated and kept at -20ºC until further analysis. The collection of milk, blood and sera was conducted in the morning, before the first meal/breast feeding session. Milk and serum (1 mL) were diluted to 10 mL with deionized Mili-Q water. Zn content was determined by Spectro Arcos ICP-OES analyzer (Spectro Analytical Instruments GmbH, Kleve, Germany). All used chemical reagents were of analytical grade. Zn working analytical solutions were prepared after serial dilution of stock reference solution containing 1000 mg/L of element (LGC-ICP-OES stock solution) in a gradient as needed. Reference material National Institute of Standards and Technology (NIST) standard reference material infant/adult nutritional formula SRM-1849 was used for determination of accuracy and precision of this study.
Statistical methods Statistical analyses were conducted using STATISTICA 8.0 (StatSoft Inc., Tulsa, OK, USA). Results are presented as box plots. Boxes represent the median and the 25th and 75th percentiles; whiskers represent the non-outlier range. Outliers and extremes are defined as data point values that are more than 1.5× and 3× interquartile range (IQR) outside the box. p-Values were determined using 2-way analysis of variance with post hoc Duncan test or using nonparametric 2-tailed Mann-Whitney U test. Correlation was established via Pearson coefficient (R). Results were considered to be statistically significant if p < 0.05.
Results and Discussion Fig 1A. shows changes in the Zn concentration in infant serum at birth (Day 1), at the end of neonatal period (Day 28), and 6 months post partum. It can be observed that Zn level in serum rises with age, and that at 6 months shows significantly higher values compared to two other time points. It is worth mentioning that 38% of infants did not experience a rise in serum [Zn] between Day 28 and 6 months. This might be related to the lower [Zn] in breast milk in this subcohort (0.38 ± 0.24 mg/L) compared to the milk that was fed to the babies showing an increase in serum [Zn] during the first 6 months (0.50 ± 0.38 mg/L); although the difference was not statistically significant. Further, a significant percentage of infants showed Zn levels that were 5
below the lower reference limit (0.6 mg/L): 61% at Day 1; 53% at Day 28; and 40% at 6 months. It is important to note that Zn levels in serums of newborns with (n = 20) and without (n = 40) neonatal jaundice did not differ. In addition, there was no statistically significant difference in [Zn] in serum from infants of different sex. We also compared [Zn] in serum samples obtained from mothers and newborns at Day 1. Mean values were 0.540 ± 0.179 and 0.520 ± 0.116 mg/L, respectively, and there was no correlation between these two parameters (R = 0.047; p > 0.05). Zn concentration in the milk showed an opposite trend compared to infant serum levels i.e. it gradually decreased with time (Fig. 1B). This is in line with previous reports [13–16]. There was no significant correlation between serum and milk [Zn] (at all three time points) and mothers' age and weight, number of parities, or leukocytes count in neonates' blood at Day 1 (13.5 ± 4.4 × 109 L-1) (Table 2). Serum [Zn] is known to be the most reliable marker of Zn status in healthy adults. Whether or not it is an appropriate marker in newborns is unclear so far. Nevertheless, as the lack of correlation between leukocytes count and [Zn] in serum observed here speaks in favor of applicability of this parameter in assessing Zn status in the neonates.
Table 2. Correlation coefficients (R) showing dependency between Zn level in milk and sera and maternal age and mass at delivery, number of parities, and leukocytes count in the blood of neonates at Day 1. Maternal age
Maternal mass Number parities
of Leukocytes count
in
neonates
at
Day 1 [Zn] in serum at Day 1
[Zn] in serum at Day 28
[Zn] in serum at 6 months
[Zn] in milk at Day 1
R = -0.048
R = -0.156
R = -0.053
R = -0.184
p = 0.716
p = 0.233
p = 0.687
p = 0.158
R = -0.014
R = -0.203
R = 0.031
R = -0.094
p = 0.917
p = 0.120
p = 0.870
p = 0.478
R = 0.091
R = -0.005
R = 0.115
R = 0.041
p = 0.488
p = 0.968
p = 0.380
p = 0.756
R = 0.052
R = -0.100
R = 0.019
R = -0.037
p = 0.690
p = 0.954
p = 0.886
p = 0.780
6
[Zn] in milk at Day 28
[Zn] in milk at 6 months
R = -0.022
R = -0.106
R = -0.070
R = 0.230
p = 0.866
p = 0.418
p = 0.606
p = 0.077
R = 0.048
R = 0.010
R = 0.237
R = 0.135
p = 0.720
p = 0.938
p = 0.068
p = 0.303
The obtained values for [Zn] in full-term milk are in accordance with previous population-based and methodological studies [13-17]. Of note, Zn deficiency of milk has been found in specific local populations [18-20]. In the case of exclusively breastfed babies, the quantity of Zn of 2 mg/day (according to Dietary Reference Intake), must be contained in the daily volume of human milk ingested by the infants [21]. Lactating woman in good health can produce 640 to 820 mL of milk per day [22]. If we take into account the mean volume of milk per day (780 mL) and the measured concentrations of Zn in the examined cohort, about 50% of infants were provided with less than 2 mg/day. At 6 months post partum, the concentration of Zn in the milk showed a drastic drop (from 4.70 ± 1.74 mg/L at Day 28 to 0.46 ± 0.36 mg/L). This implies that Zn supplementation should be advised to mitigate Zn deficiency during the later period of lactation at least. Colostrum (i.e. milk at Day 1) showed the highest Zn concentration. Pertinent to this, it is known that adequate supply of Zn is essential to the newborn particularly during the first days of life. This is implied by the early death of pups from Zn deficiency due to mutation in Zn transporter ZnT-4 that suppresses the transfer of Zn into the milk in mammary glands [23]. High level of Zn adds to some other supreme features of colostrum compared to mature milk, such as high antioxidative activity [24] and sufficient levels of iron and copper [25]. As expected, infant mass increased with life time (Fig. 1C). Next, Pearson correlation coefficients were calculated to determine dependency between concentrations of Zn in the serum and in the milk. Significant negative correlation was established between serum Zn level and milk Zn concentration at Day 28 (R = -0.338; p = 0.008). In addition, a significant positive correlation was found for serum and milk [Zn] at 6 months (R = 0.306; p = 0.018). A previous study has reported positive correlation between Zn level in mother's milk and in serum of infants 4–6 months old [12]. Correlations and trends observed here imply that concentration of Zn in milk changes to accommodate the needs of the infant. Level of Zn in milk decreases as the concentration of Zn in infant serum increases to reach the homeostasis. It appears that following 6 months, milk serves to maintain rather than to raise infant Zn level. There was no significant 7
correlation between body mass and the level of Zn in the milk and serum. Available data on this are ambiguous [26].
Figure 1. concentration of Zn and breastfed infant mass during six months after delivery. Panel A: Concentration of Zn in infant serum; Panel B: Concentration of Zn in milk; Panel C: Infant mass. Boxes represent the median and the 25th and 75th percentiles; whiskers represent the nonoutlier range. Outliers (circles) - data point values that are more than 1.5× IQR outside the box. Extremes (asterisk) - data points more than 3× IQR outside the box. Statistical significance (pvalues) are presented. ns, non-significant (p > 0.05).
Conclusions In this study 60 pairs of mothers and their infants were involved to evaluate changes and correlation between levels of zinc in human milk and infant serum. Levels of Zn in milk and infant serum showed significant correlation and reciprocal trends during first six months post partum. The amount of Zn available in the milk meet the nutritive requirements during the neonatal period, but a drastic drop at six months of lactation imply that exclusively breastfed infants might be exposed to risk of Zn deficiency. Our data should lead to the improvement of the quality of breastfeeding in Serbia. Factors influencing infant and maternal zinc status, especially in exclusively breastfed infants, as well as human milk, blood and serum zinc concentration should be further investigated.
Acknowledgments 8
This study received grants from the Ministry of Education Science of the Republic of Serbia (Grant No 43004)
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