Macronutrients, immunoglobulin A and total antioxidant capacity of human milk during prolonged lactation

Background: A longer duration of breastfeeding of up to two years is encouraged by many health authorities, but there is limited information regarding the composition of the milk after one year postpartum. The aim of the study was to determine the longitudinal changes in human milk macronutrients, immunoglobulin A (IgA) and total antioxidant capacity (TAC) during prolonged lactation. Methods: One hundred eighty-four breastfeeding mothers with full-term healthy children who had been lactating from 1 to 24 months were recruited from January 2019 to April 2019. Human milk was biochemically analyzed for protein and carbohydrate content by colorimetric assays. The fat content was determined by capillary centrifugation, and the energy content was calculated from the results of centrifugation. IgA levels and TAC were determined by ELISA and a Trolox equivalent antioxidant capacity (TEAC) assay, respectively.Pearson correlation analysis was used to determine the association of milk composition with the month of lactation. Results: The concentrations of protein, fat, energy and IgA were positively correlated with the duration of lactation ( r = 0.15, p < 0.05; r = 0.23, p < 0.05; r = 0.23, p < 0.05 and r = 0.29, p < 0.05, respectively). No significant correlations of the carbohydrate concentrations or TAC with the duration of lactation was observed ( r = 0.10, p > 0.05 and r = -0.07, P > 0.05, respectively). Conclusions: We demonstrated that protein, fat, IgA and energy contents increasedduring prolonged lactation lasting up to twoyears postpartum, while carbohydrateconcentrationand TACwerenot related to lactation duration. Based on the results, lactating mothers should be encouraged to continue breastfeeding for at least two years postpartum.


Background
Human milk is widely accepted as an optimal food and provides essential components for the growth and development of infants. Apart from macronutrients and micronutrients, human milk contains various nonnutritive bioactive compounds, including immunologic factors, prebiotics, growth factors, hormones, and antioxidants [1][2][3]. In addition to supporting normal growth and development, breastfeeding offers numerous advantages, including immunological, psychological, economic, and environmental benefits. Recent advances in molecular biological techniques have shown that human milk plays an essential role as an epigenetic modulator of gene expression in milk recipients and may positively impact life-long metabolic programming [4,5].
A longer duration of breastfeeding is encouraged by the World Health Organization (WHO) [6], which recommends exclusive breastfeeding for the first six months, along with continued breastfeeding for at least two years. The American Academy of Pediatrics (AAP) reaffirms the recommendation of exclusive breastfeeding for approximately the first six months, followed by continued breastfeeding as complementary foods are introduced with the continuation of breastfeeding for at least one year of life [7].
It is well established that human milk is a dynamic fluid whose composition continually changes throughout the lactation period. Approximately two weeks after birth, the colostrum and the transitional milk change rapidly, whereas mature milk is relatively similar in its composition with subtle changes up to weaning [8].
Although the composition of the milk produced during the first six months postpartum has been widely reported, information on milk composition during the second year postpartum is limited and inconclusive due to small sample sizes, nonstandardized sample collection protocols, and limitations associated with the study designs. Moreover, immunoglobulin A (IgA), which is the predominant immunoglobulin in human milk, and antioxidant capacity, which supports the immature immune system by neutralizing pathogens and removing free radicals, are rarely reported [9][10][11].
Our study aim was to examine the longitudinal changes in human milk macronutrient compositions, IgA, and total antioxidant capacity (TAC) between one and 24 months of lactation. These data should provide support for the benefits of prolonged breastfeeding.

Study Design
This cross-sectional study included one hundred eighty-four breastfeeding mothers with full-term healthy children who had no underlying medical conditions and had been lactating for 1 to 24 months. Participants were recruited from January 2019 to April 2019 through study posters posted in the well-baby clinic and lactation room of 4 hospitals in Chiang Mai City. Participants were also recruited from a Facebook parenting group. Before providing information and breast milk samples, all participants signed informed consent forms.

Sample Collection
Participants were required to collect milk samples in the lactation room of Maharaj Nakorn Chiang Mai Hospital, Nakornping Hospital, Health Promotion Hospital Region one and Lampang Hospital. To minimize possible circadian influences [9] and to ensure uniformity of the samples, all breast milk samples were expressed between 8:00 AM and 12:00 PM using a Lactina Electric Selection pump (Medela®, Switzerland  µL of the diluted samples and the IgA standard were loaded into each well of the ELISA plate. Incubation was performed at 37 °C for two hours, and the solution in each well was replaced by 100 µL of biotinylated IgA detector antibody. Incubation was performed at 37 °C for an hour, and the solution in each well was discarded and washed. The avidin-HRP conjugate mixture was added at 100 µL into each well, and incubation was performed at 37 °C for another hour. Next, the solution in the well was discarded, and the plate was washed. Then, 90 µL of TMB substrate was added to each well, and the plate was incubated in the dark at 37 °C for 15 minutes. The reaction was allowed to run for six minutes, and the absorbance at 734 nm was read with Genesys™ 20 (Thermo Scientific, USA). The TAC in each human milk sample was calculated using a Trolox (Sigma, 238813) standard curve with a concentration range of 0-5 mM, and the TAC was reported as mM Trolox equivalence.

Statistical Analysis
This study was a cross-sectional study. Data are presented as descriptive statistics, including the mean, standard deviation (SD), frequency (n), and percentage (%).
One-way ANOVA was used to test the differences in macronutrients, energy content, IgA and antioxidant capacity in breast milk by month of lactation. Pearson correlation analysis was used to determine the association of milk composition with month of lactation and to determine the association of maternal body mass index (BMI), maternal age and breastfeeding frequency with milk composition. Statistics were considered significant at P < 0.05.

Macronutrients
The concentrations of protein, fat and energy in human milk expressed by mothers who had been lactating from 1-24 months showed a positive correlation with the duration of lactation (r = 0.15, p < 0.05; r = 0.23, p < 0.05 and r = 0.23, p < 0.05, respectively) ( Fig. 1a-c).
In the subsequent lactation period ( correlation between the concentration of carbohydrates and the length of lactation (r = 0.10, p > 0.05) (Fig. 1d).   Immunoglobulin A (IgA) The concentration of IgA in human milk showed a positive correlation with lactation duration (r = 0.29, p < 0.05) (Fig. 1e)

Factors Affecting Human Milk Composition
Correlations among maternal BMI, maternal age, breastfeeding frequency, and milk composition were tested using Pearson's correlation coefficient (Table 3)  Pearson correlation analysis was used for statistical calculations, and a p-value lower than 0.05 was regarded as significant. * p-value < 0.05, ** p-value < 0.01 Pearson correlation analysis was used for statistical calculations, and a p-value lower than 0.05 was regarded as significant. * p-value < 0.05, ** p-value < 0.01

Macronutrients
Our study and two recent studies [12,13]  Mandel et al. [14] demonstrated that human milk expressed by mothers who had been lactating over one year had a significant increase in fat content compared to that expressed by mothers who had been lactating for shorter periods. This finding was consistent with our study and with Czosnykowska-Łukacka et al. [12] who showed that the fat content significantly increased in human milk expressed by mothers lactating beyond 18 months postpartum. However, Shehadem et al. [15] and Perrin et al. [13] concluded that fat concentration was not related to the duration of lactation.
Limited studies examining carbohydrate concentration in human milk beyond the first year of lactation have shown equivocal results. Czosnykowska-Łukacka et al. [12] showed that carbohydrate content decreased significantly in the group of 12 to 18 months of lactation compared to that in those lactating between 1 and 12 months, while no change was observed in our study and others [13,15].

Immunoglobulin A (IgA)
Prior research on the concentration of IgA in the context of prolonged lactation has reported inconclusive results. Perrin et al. [13] and our study demonstrated that the IgA concentration of human milk increased in the second year postpartum.
Conversely, other studies [16,17] showed that the IgA concentration was stable Zarban et al. [18] measured TAC at five different times from 115 healthy mothers of full-term infants. Their final samples measured colostrum at 2 ± 1 day after birth (n = 115), transitional milk at 7 ± 3 (n = 97) and 30 ± 3 days after birth (n = 102), mature milk at 90 ± 7 days (n = 100) and 180 ± 10 days after birth (n = 91). They reported that the TAC in milk was significantly higher in colostrum than in transitional and mature milk [18]. The same pattern of TAC was reported by Quiles et al. [19], who evaluated the changes in TAC in human milk during the first month of lactation. Based on limited research, it can be concluded that antioxidant components and TAC were at their highest levels in colostrum and declined during early lactation [18][19][20]. Decreasing volume and mammary gland involution during the weaning process have been correlated with human milk composition. These factors may affect human milk components, especially in longitudinal studies. Garze et al. [27] documented that protein and fat concentrations increased during weaning. A significant increase in protein, but a decrease in the lactose concentration, was observed during gradual weaning only when milk volume was below 400 mL/day, as reported by Neville et al. [28]. The concentration of IgA was also affected by maternal nutritional status and the stage of lactogenesis (weaning and nonweaning) [29,30]. Furthermore, both genetic variation [8,31] and environmental factors such as ethnicity [32] and geographical region [33], respectively, have been shown to influence human milk composition, and these factors were uncontrollable and beyond the limitations of this study. Future research should include a prospective cohort study to reduce individual bias at each time point with careful adjustments for the potential effects.

Conclusions
We demonstrated that the concentration of protein, fat, energy and IgA significantly increased (p < 0.05), while no change was observed in carbohydrate or antioxidant capacity over prolonged lactation of up to two years postpartum. Based on these results, lactating mothers should be encouraged and supported to continue breastfeeding for at least two years postpartum.

Consent for Publication
Not applicable.

Availability of Data and Materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests
The authors declare that they have no competing interests.

Funding
The study was financially supported by the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (grant number 017/2562). The funding bodies had no role in the design of the study; the collection, analysis, and interpretation of data; or the writing of the manuscript.

Authors' Contributions
OK designed the study, managed the study approval, drafted the initial manuscript and revised the manuscript. RJ supervised the sample collection and the sample analysis and revised the manuscript. OK, SP (1) and MR participated in fieldwork management, sample collection, and analysis. SR and AP analyzed the data. KK and SP (2) critically reviewed the manuscript. All authors read and approved the final manuscript. Figure 1 a-f) The Correlation of Macronutrients, IgA and TAC of Human Milk with Month of Lactation.