Macronutrient, immunoglobulin A and total antioxidant capacity proles of human milk from 1 to 24 months postpartum

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 milk after one year postpartum. The goal of this study was to determine the changes in human milk macronutrient, immunoglobulin A (IgA), and total antioxidant capacity (TAC) proles during extended 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 assays. IgA levels and TAC were determined by ELISA and a Trolox equivalent antioxidant capacity (TEAC) assay, respectively. Pearson’s correlation coecient and Spearman’s rank correlation coecient were used to determine the association of milk composition with month of lactation. Results: The fat, energy and IgA contents were positively correlated with the duration of lactation (r = 0.229, p = 0.002 ; r = 0.229, p =0.002 and r = 0.304, p < 0.001, respectively). No signicant correlations between protein, carbohydrate concentrations and TAC with the duration of lactation were observed (r = 0.106, p = 0.15; r = -0.032, p = 0.67; r = -0.056, p = 0.45, respectively). Conclusions: We demonstrated that fat, energy and IgA contents increased during extended lactation lasting up to two years postpartum, while protein and carbohydrate concentrations and TAC were not related to lactation duration. Based on these results, lactating mothers should be encouraged to continue breastfeeding for at least two years postpartum.

mother was under 18 years old or above 40 years old, (c) the mother was illiterate for Thai, and (d) the mother could not travel to our lactation room by themselves. Paper-based questionnaires were used for data collection using the Thai language. All participants completed a self-report questionnaire on baseline information that included maternal age, education level, rst antenatal care (ANC), gestational age, birth order, parental status and breastfeeding frequency. The weight and height of each participant was measured before their milk samples were collected. All eligible participants were then asked to make an appointment for milk collection. Before providing information and breast milk samples, all participants signed informed consent forms. The participants received no payments.

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 in uences [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). The pump was left on for approximately 15 minutes or until there was no further milk that could be expressed for at least ve minutes. For storage, the samples were aliquoted into 1.5 ml microcentrifuge tubes and frozen at -80℃ until further analysis. Samples collected for antioxidant activity measurements were stored at 0℃ and analyzed within 72 hours to preserve the antioxidant activity.

Carbohydrate Content
The total carbohydrate content in human milk was estimated using a 3,5-dinitrosalicylic acid (DNS) solution prepared by solubilizing one gram of DNS (Sigma, 128848) in a 2 M NaOH (VWR Chemicals, 28244.295) solution containing 30 g Na-K tartrate (VWR Chemicals, 27068.233), after which DI H 2 O was added to reach a total volume of 100 mL; this solution is referred to as the working DNS solution. The milk samples were diluted 25× with DI H 2 O, and 500 μL of each diluted sample was mixed with 500 μL of working DNS solution. The mixture was then boiled for ve minutes and cooled down in running tap water. Then, 4 mL of DI H 2 O was added to each reaction, and the absorbance was read at 540 nm with a Synergy H4 Hybrid Reader (BioTek ® , USA). The concentration of carbohydrates in the milk was calculated from a D-lactose (Sigma, 61345) standard curve with a concentration range of 0-100 mg/mL.

Protein Content
The total protein content in human milk was determined by Lowry's method using Folin-Ciocalteu solution (VWR Chemicals, 31360.264). The milk samples were diluted 100× with DI H 2 O, and 500 μL of each diluted sample was mixed with 2.5 mL of an alkaline solution and 250 μL of the Folin-Ciocalteu solution. The mixture was incubated at room temperature (RT) for ten minutes, and the absorbance was read at 650 nm with a Synergy H4 Hybrid Reader (BioTek ® , USA). The concentration of protein in the milk was calculated from a bovine serum albumin (GE Healthcare, K41-001) standard curve with a concentration range of 0-100 mg/mL.

Creamatocrit, Lipid Content, and Energy Conversion
The percentage of cream (creamatocrit) in the human milk was examined by capillary centrifugation followed by calculating the lipid content and energy yield. The milk samples were individually loaded into each capillary tube to 4/5 of the tube capacity, and the lled tube was capped with clay. Then, the tubes were microcentrifuged (Hettich Haematokrit, Germany) for 15 minutes. The thickness of the cream (A) and the total solution heights (B) were measured. Creamatocrit was calculated as 100 (A÷B), lipid content (g/L) was calculated as (creamatocrit × 5.57) -3.08, and energy (kcal/100) was calculated as (creamatocrit × 5.57) + 45.13.

Immunoglobulin A (IgA) Determination
IgA levels in human milk were determined using a commercial ELISA kit (Aviva System Biology, OKEH00516) according to the manufacturer's protocol. Brie y, the human milk samples were diluted 200,000× in water as well as assay diluent buffer. Then, 100 μL of the diluted samples and the IgA standard were loaded into each well of the ELISA plate. The samples were incubated at 37°C for two hours, after which the solution in each well was replaced with 100 μL of biotinylated IgA detector antibody. The samples were incubated 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. Finally, 50 μL of the stop solution was added to each well, and the plate was read at an absorbance of 450 nm with a Synergy H4 Hybrid Reader (BioTek ® , USA). The concentration of IgA in the milk was calculated from an IgA standard curve with a concentration range of 0-4000 pg/mL.

Total Antioxidant Capacity (TAC)
The TAC of human milk was determined as the Trolox equivalence antioxidant capacity (TEAC) using ABTS solution, which was prepared by mixing two equal volumes of 0.768 g% of ABTS® (AppliChem, A1088,0005) and 0.132 g% of K 2 S 2 O 8 (VWR Chemical, 26915.291). The mixture was incubated at RT for 12 hours, and the working ABTS was made by diluting the stock solution 50× in DI H 2 O. Twenty microliters of human milk sample was mixed with 2 mL of the diluted ABTS solution. The reaction was allowed to run for six minutes, and then the absorbance at 734 nm was read with a Genesys TM 20 instrument (Thermo Scienti c, 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 millimolar Trolox equivalence.

Statistical Analysis
This study was a cross-sectional study. The data are presented as descriptive statistics, including the mean, standard deviation (SD), frequency (n), percentage (%), median, interquartile range, and range. Kruskal-Wallis and Mann-Whitney tests were used to test the differences in macronutrient and energy contents in breast milk by month of lactation, whereas one-way ANOVA post hoc tests was used to test the differences in IgA levels and TAC in breast milk by month of lactation. Pearson's correlation coe cient and Spearman's rank correlation coe cient were used to determine associations between milk composition with month of lactation, and multiple regression analysis was used to assess the association between maternal age, maternal body mass index (BMI), and breastfeeding frequency with milk composition. Differences were considered signi cant at p < 0.05.

Results
The participants were divided into four groups based on breastfeeding periods: 1-6 months (n = 43), 6-12 months (n = 47), 12-18 months (n = 50) and 18-24 months (n = 44). There were no signi cant differences between the groups with respect to demographics or baseline characteristics (Table 1).  3 Fisher's exact test were used for statistical calculations, and a p-value lower than 0.05 was regarded as significant.
BMI, body mass index; ANC, antenatal care; SD, standard deviation; P, percentiles All the participants were Thais.

Macronutrients
The fat and energy contents in human milk expressed by mothers who had been lactating from 1-24 months showed a positive correlation with the duration of lactation (r = 0.229, p = 0.002 and r = 0.229, p = 0.002, respectively) ( Figure 1b,1c.). There were no signi cant correlations between protein and carbohydrate concentrations with the length of lactation (r = 0.106, p = 0.15; r = -0.032, p = 0.67, respectively) (Figure 1a,1d).

Factors Affecting Human Milk Composition
Correlations among maternal age, maternal BMI, breastfeeding frequency, and milk composition were tested using Spearman's Rank correlation coe cient and Pearson's correlation coe cient (Table 3). Maternal BMI was positively correlated with the fat and energy contents in human milk (r = 0.233, p = 0.001 and r = 0.233, p = 0.001, respectively) and negatively correlated with carbohydrate content (r = -0.193, p = 0.01). Maternal age was positively associated with changes in the carbohydrate concentration (r = 0.148, p = 0.04), while breastfeeding frequency was negative associated with the carbohydrate content of human milk (r = -0.182, p = 0.01) ( Table 3). In addition, the multiple regression analysis results indicated that maternal BMI was positively associated with fat and energy contents in human milk (p < 0.001) and negatively associated with carbohydrate content (p < 0.05) ( Table 4).

Discussion
The goal of this study was to assess the changes in the patterns of human milk macronutrients, IgA, and TAC during extended lactation. A slight but signi cant increase in fat, energy, and IgA contents was observed during extended lactation lasting up to two years postpartum, while protein and carbohydrate concentrations and TAC were not related to lactation duration. Comparisons of milk components with the duration of lactation were made using four groups (1-6, 6-12, 12-18, and 18-24 months). In the subsequent lactation period, the IgA concentration was signi cantly lower from 1-6 months than that observed in the other groups (6-12, 12-18, and 18-24 months). The primary factor that exhibiting an association with human milk composition in our study was maternal BMI.
The demographics or baseline characteristics of our participants did not show signi cant differences between the groups. However, the participants had a higher than average education level among individuals in Thailand, as the recruitment yers were distributed in the hospital's lactation room in an urban area, and those who were interested in participating would need to travel to our lactation room by themselves. Breastfeeding frequency was not observed to be signi cantly different across two years of breastfeeding. Our results are consistent with those of other studies [12,13] that reported no signi cant differences in the mean of numbers of breastfeeding per day.
Mandel et al. [12] reported that the feeding frequency of a short-duration group (6-12 months) and a long-duration group (12-39 months) was 7.1 and 5.9 feed/day, respectively. Shehadeh et al. [13] reported mean lactation frequencies for participants whose breastfeeding duration was under one year (approximately 3 months postpartum) and longer (approximately 14 months postpartum) of 7.1 and 6.9 feed/day, respectively. These results could explained by the need to maintain frequent nursing throughout the lactation phases to maintain milk supply.

Macronutrients
In our study, we observed that protein concentration was not related to lactation duration.
In contrast, two recent studies [14,15]  They also compared the protein content in human milk in the second year postpartum with unpasteurized milk samples from 51 approved donors less than one year postpartum from milk banks. The mean protein content for participants whose breastfeeding duration was 11-17 months postpartum was signi cantly higher than the protein content of milk from a milk bank(average time of lactation was 4.8 + 3.3 months) [15]. We reported that the fat and energy contents positively correlated with the duration of lactation. Similarly, Mandel et al. [12] demonstrated that human milk expressed by mothers who had been lactating over one year (12-39 months) had a signi cant increase in fat content compared to that expressed by mothers who had been lactating for shorter periods (6-12 months). Czosnykowska-Łukacka et al. [14] showed that the fat content signi cantly increased in human milk expressed by mothers lactating beyond 18 months postpartum. However, Shehadem et al. [13] and Perrin et al. [15] concluded that fat concentration was not related to lactation duration. No signi cant correlations between carbohydrate concentrations and the duration of lactation were observed in this study. The few studies that have examined the carbohydrate concentration in human milk beyond the rst year of lactation have produced con icting results. Czosnykowska-Łukacka et al. [14] showed that carbohydrate content decreased signi cantly in a group of women 12 to 18 months of lactation compared to that observed in women lactating between 1 and 12 months, while no change was observed in our study or others [13,15].
We assessed the macronutrient composition of human milk and changes in concentrations of the components among four periods of time assayed. We observed that the protein concentration in human milk after 18 months postpartum signi cantly increased compared with human milk collected from 6-12 and 12-18 months postpartum. The fat and energy contents were higher in human milk after 18 months than those observed in the other groups (1-6 and 12-18 months of lactation). This variation may be due to decreases in volume and mammary gland involution during the weaning process, which regularly occur in longitudinal breastfeeding. Decreasing volume and mammary gland involution during the weaning process have been correlated with human milk composition. Garze et al. [28] reported that protein and fat concentrations increased during weaning. Neville et al. reported a signi cant increase in protein but a decrease in lactose concentration was only observed during gradual weaning when the milk volume was below 400 mL/day [29].
Immunoglobulin A (IgA) A signi cant increase in IgA contents was observed during extended lactation lasting up to two years postpartum. Similar to our results, Perrin et al. [15] observed that the IgA concentration gradually increased (P<0.05) over a study period of 11-17 months postpartum. Prentice et al., 1984 [16] measured the concentration of IgA in the mature breast milk of 153 rural Gambia mothers who lactation on 14 days to 26 months postpartum. In contrast to our study, they observed that IgA concentrations decreased signi cantly (p<0.001) during the rst year of lactation. The measurement of milk volume in their study was made on or close to the day of milk sampling for 95 milk samples from mothers who lactated from infant to 18 months postpartum. Furthermore, the breast milk volume of the women in their study peaked at 2-3 months postpartum and decreased in the rst 12 months of lactation, and although they discussed the relationship between IgA and milk volume, the relationship was not analyzed. In contrast with our results, Hennart et al., 1991 [17] studied 127 Zairean mothers (54 urban and 73 rural mothers) who lactated between the rst week to 18 months postpartum. They observed that the concentration of IgA remained stable throughout the 18 months of lactation and reported that the IgA concentration in milk was signi cantly higher in rural mothers than in urban mother (p<0.05). The urban mothers had much higher milk yields (612 +27 ml/day) than did the rural mothers (307 + 16 ml/day), and the mean breastfeeding frequency was signi cantly higher (p<0.05) in the urban mothers (10.1 times/day) than for the rural mothers (6.8 times/day). However, they did not report the association between IgA levels, breastfeeding frequency and milk output per day. With respect to lactation period, we observed that the mean IgA concentration was signi cantly lower from 1-6 months than that observed in the longer duration groups. We also observed a non-signi cantly decreasing in the mean IgA concentration from 12-18 months. This variation may due to our samples having been collected from different women, and we did not control for factors that potential in uenced of IgA level, such as breastfeeding frequency, milk output per day, geographical region (rural or urban area), maternal nutritional status, and the stage of lactogenesis (weaning and non-weaning) [16][17][29][30].

Total Antioxidant Capacity (TAC)
The results of our study showed that TAC was not related to lactation duration. A few studies have focused on the relationship between TAC in breast milk and postnatal age. In 2009, Zarban et al. [18] measured TAC at ve different times from 115 healthy mothers of full-term infants for colostrum at 2+1 days after birth (n=115), transitional milk at 7+3 days (n=97) and 30+3 days (n=102), mature milk at 90+7 days (n=100) and 180+10 days after birth (n=91). They reported the TAC in milk was signi cantly higher in colostrum than in transitional and mature milk [18]. The same TAC pattern was reported by Quiles et al. [19], who evaluated the changes in TAC in human milk during the rst month of lactation. Based on limited research, it can be concluded that the highest levels of antioxidant components and TAC were observed in colostrum and decreased during early lactation [18][19][20]. To the best of our knowledge, this is the rst study to describe a longitudinal change in TAC in breast milk where there was no observed change in antioxidant capacity in the second year postpartum.

Factors Affecting Milk Composition
We investigated the potential in uence of factors on human milk composition, including maternal age, maternal BMI, and breastfeeding frequency. The primary factor showing an association with human milk composition in our study was maternal BMI, which was signi cantly and positively correlated (p<0.001) with the fat concentration and energy content in human milk and negatively correlated (p<0.05) with carbohydrate concentration in the multiple regression analysis. This nding is consistent with those of previous studies [21][22][23][24]. Bzikowska et.al [21] measured body composition and analyzed the data at three-time points: during the rst (n=40), third (n=22), and sixth (n=15) month of lactation, observing a positive correlation between human milk fat content and maternal BMI in the rst month postpartum (r=0.33; p=0.032). Hahn et.al [23] evaluated the association between the fat concentration in human milk and interactions between maternal age and BMI and energy in a study where the participants were subgrouped by age and BMI. They observed that both maternal age and BMI signi cantly affected the fat, energy, and carbohydrate contents, while the protein content in human milk was only affected by maternal BMI. A positive correlation between fat content and maternal BMI has been repeatedly reported, while there has been limited and inconclusive information on the association between maternal BMI with protein and carbohydrate contents and inconclusive. In contrast with our results, Chang et al. [24] measured the concentrations of macronutrients from 2632 mature breastmilk samples (1-8 months postpartum) and observed that maternal BMI was negatively associated with lactose content. Human milk is a dynamic uid that can vary in composition according to maternal diet. Elucidating the reason for the association between maternal BMI and human milk macronutrient contents is di cult, because diet type and eating behavior may differ for each group (normal or abnormal BMI). However, the majority of studies have reported that maternal diet has a slight effect on the contents of many nutrients in human milk [22,[25][26].
A limitation of this study was that it was not possible to infer causality because of the study design. The average education level of our participants was higher than that of the general population, and implementing the ndings of this study in the general population would require further investigation. Furthermore, both genetic variation [8,31] and environmental factors such as dietary intake, time since last feed, and ethnicity [32,33] have been shown to in uence 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 their potential effects.

Conclusions
In this study, we demonstrated that over extended lactation of up to two years postpartum, the fat, energy and IgA contents of human milk signi cantly increased (p < 0.05), whereas no change was observed in carbohydrate levels or antioxidant capacity. 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 nancially 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.
Author 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 eldwork 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 nal manuscript. (a-f) The Correlations Between Macronutrients, IgA and TAC of Human Milk with Month of Lactation. Protein, fat, energy, and carbohydrate content data were analyzed using Spearman's rank correlation coe cient, while IgA and TAC data were analyzed using Pearson's correlation coe cient, * p < 0.05, ** p < 0.01