What Is Known
- Osteopontin is a multifunctional protein present in most tissues and body fluids where it is involved in, for example, immune maturation and regulation.
- The osteopontin concentration in human milk is much higher than in bovine milk and infant formulas.
- Current knowledge on the osteopontin level in human milk is based on a small study comprising few Danish mothers.
What Is New
- Multicenter study comprising 629 mothers from 4 countries in Asia and Europe.
- Osteopontin concentration differed significantly between countries, also when adjusting for infant age.
- Osteopontin concentration in human milk decreased with infant age.
Human milk provides an unsurpassed nutritional support for the development of infants. It is an optimal source of energy, essential amino acids and lipids, as well as specific oligosaccharides.
There is increased awareness regarding the content of bioactive proteins in human milk, which may deliver important signals or actions in the growth and development of the infant. These include lactoferrin, α-lactalbumin and caseins, which upon digestion can release peptides that have antibacterial and opioid agonist effects as well as immune stimulating and mineral binding activities (1–3). Many of the bioactive proteins are present in significantly lower concentrations in bovine milk compared to human milk, and therefore also lower in infant formulas based on bovine milk (4). This could contribute to the differences observed between breastfed and formula fed infants in both development and well-being.
Osteopontin (OPN) is another bioactive protein present in higher concentration in human milk than in bovine milk (4–6). OPN is a multifunctional protein expressed in most cells and tissues (7). OPN is present in all physiological fluids, for example, plasma (approx. 35 μg/L) (4), urine (approx. 4 mg/L) (8) and with the by far highest concentration in human milk (approx. 138 mg/L) (4). The function of OPN in milk remains unclear; however, the high concentration of OPN in human milk, cord blood (263 μg/L) and infant plasma (342 μg/L) (4) suggests a role in infant growth and development. OPN can activate cellular signaling cascades by interaction with cellular receptors via its integrin binding sequences and has been shown to be a key cytokine in the regulation of the Th1/Th2 balanced immune response (9). Likewise, OPN is shown to activate macrophages and induce their phagocytosis of bacteria (10), suggesting that OPN could also play a more direct role in the immune response by interacting directly with invading pathogens.
Bovine milk contains much less OPN than human milk (18 and 138 mg/L, respectively) (4). Bovine and human milk OPN are structurally very similar (11–13) and it has been suggested to use bovine OPN in infant formulas to close the concentration gap to human milk. In vitro experiments have shown that bovine OPN is partly resistant to proteolysis by neonatal gastric juice (14). Furthermore, studies have shown that large OPN fragments can be found in the plasma of animals ingesting bovine milk OPN (15–17). This makes OPN a bioactive component of milk and infant formulas.
Studies have shown that bovine OPN have beneficial effects on formula fed animal and human infants. In a study with rhesus monkeys, addition of OPN (125 mg/L) to the formula reduced the number of genes expressed differently between the breastfed and formula fed infants from 1017 to 217 (18), suggesting that bovine milk OPN shifts intestinal gene expression in a way that reduces the difference between formula fed and breastfed infants. In a randomized double-blind controlled human trial, mothers either breast- or formula fed their infants from 1 to 6 months of age. Formula was standard or with 65 or 130 mg/L bovine OPN added. The addition of OPN changed the plasma levels of several amino acids and cytokines to be more similar to breastfed infants. Interestingly, addition of OPN lowered the levels of the pro-inflammatory cytokine TNF-α and increased the levels of interleukin-2 that plays a key role in oral tolerance (19). Furthermore, the OPN groups had fewer days with fever compared to the group receiving standard infant formula (19). Supplementing infant formula with OPN also shifted the gene expression of peripheral blood mononuclear cells to be more similar to breastfed infants and increased the proportion of circulating T-cells (20,21).
Overall, studies indicate that OPN in milk plays an important role in infant immune development. Information about the level of OPN in human milk is important both in understanding the physiological role of the protein in milk and for use as a guideline in the production of infant formulas containing OPN. Currently, knowledge about the level and variation of OPN in human milk is based on a single study including only 29 Danish mothers (4). In this explorative, multicenter study we therefore aimed to investigate the OPN content in a large number of human milk samples from 4 different countries; China, Denmark, Japan and Republic of Korea.
Participants and Sample Deliveries
Participating mothers were recruited from well-developed areas with comparable high-level environmental and sanitary conditions and economic wealth. In China, mothers were from the city of Changsha. In Korea, mothers were from postpartum care centers located in the center of the city in the provinces of Seoul, Gyeonggi-do, Gangwon-do, Sejong-si and Chungcheongbuk-do. In Denmark, mothers were from the municipality of Odense. In Japan, mothers were from the prefectures of Hokkaido, Aomori, Niigata, Ibaraki, Saitama, Tokyo, Chiba, Kanagawa, Aichi, Shiga, Osaka, Wakayama, Hyogo, Okayama, Tottori, Fukuoka, Oita, Saga, Nagasaki, and Kagoshima. At each study site, mothers were invited to deliver a milk sample with no requirements regarding the sample being fore- or hind milk. In China and Japan, mothers delivered a single sample at up to 3 different time points (China: day 30, 60, and 90 postpartum (pp); Japan: approx. 8, 16, and 24 weeks pp). Japanese samples were collected once a day for 7 consecutive days and were individually pooled and mixed afterward. In Republic of Korea and Denmark, mothers delivered a single sample (Korea: approx. 5 weeks pp; Denmark: approx. 3 months pp). After delivery, samples were mixed and stored at −80 °C until analysis. Due to ethical and legal conditions, transport of milk samples between the 4 study sites was not possible. Therefore, OPN and protein analyses were performed at each of the 4 study sites, whereas data management and statistical analyses were conducted at 1 site. Information about the milk sample collection is included as Supplementary material (Supplemental Digital Content 1, http://links.lww.com/MPG/B367).
At all sites, OPN was measured using the Quantikine Human Osteopontin ELISA (R&D Systems, Minneapolis, MN), which is validated for quantitative determination of OPN in human milk. Before analysis, samples were thawed and centrifuged to remove residual cream or precipitate as described (4). The samples were diluted 40,000-fold after the following scheme: 20 μL milk + 980 μL H2O (50×) from which 20 μL was diluted in 780 μL H2O (40×) from which 10 μL was diluted in 190 μL of the RD5-24 diluent buffer (20×) supplied with the ELISA kit. Milk samples and dilutions were kept on ice until analysis. All samples were analyzed in duplicates, and doublets that deviated more than 10% (Japan: 20%) from each other were re-analyzed. Sample concentrations were calculated using logistic curve fitting. To ensure comparability of the ELISA measurements, all standard curves from the 4 study sites were inspected and compared. Parameters used for the logistic fit standard curves from the 4 sites are included as Supplementary material (Supplemental Digital Content 2, http://links.lww.com/MPG/B368).
Before analysis, samples were thawed at 5 °C overnight, placed in a 37 °C to 40 °C water bath and homogenized. Protein analysis was performed using either a mid-infrared transmission spectroscopy device (Miris HMA, Uppsala, Sweden) or a Fourier transformation infrared spectroscopy device (MilkoScan FT2, Foss Analytical, Hillerød, Denmark), both developed for in-hospital analysis of human milk. In China, Denmark, and Japan, the Miris HMA was used, and the samples were tested once (China) or twice (Denmark and Japan). In Republic of Korea, the MilkoScan FT2 was used, and each sample was tested once. Since the MilkoScan FT2 output was total/crude and not true protein, a total of 10 samples was re-analyzed using a Miris HMA. These results were used to calculate a conversion factor (0.802926), by which the MilkoScan FT2 output was converted for comparison of protein concentrations across sites.
Study data were collected and managed using REDCap electronic data capture tool hosted at University of Southern Denmark, Odense, Denmark (22). Descriptive statistics were performed to describe the participating mothers and their infants. Due to non-normal distribution (tested graphically and by Shapiro-Wilk test), OPN and protein concentrations, OPN as percentage of true protein (OPN/protein%) and infant age are presented as medians and interquartile ranges (IQRs), whereas maternal age is presented as mean ± standard deviation (SD). The median OPN concentration and OPN/protein% were calculated based on the first milk sample provided, that is, mothers delivering more than 1 sample were included only once. Due to unequal variances, differences between sites were investigated using linear regression, both unadjusted and adjusted for infant and maternal age.
Based on samples from mothers delivering more than 1 sample, multilevel (mixed model) linear regression analysis was performed to investigate any correlation between the dependent variable OPN concentration and the independent variables infant and maternal age and sample age when OPN analysis was performed. The multilevel approach takes into account the longitudinal aspect of data, that is, the correlation between OPN concentrations within each mother over time. Statistics were performed using STATA 15.0/IC (College Station, Texas) and P values <0.05 were considered statistically significant.
The participation and sample collection at the individual study sites complied with the Declaration of Helsinki II including informed consent. The Chinese study complied with the China Food and Drug Administration's (CFDA) guideline for good clinical practice (GCP), The International Conference on Harmonisation's (ICH) guideline for GCP and were approved by the ethics committee of Changsha Hospital for Maternal and Child Health Care (ref. 20160303). The Danish study was approved by the Danish Data Protection Agency (ref. 12/26892) and the Regional Committees on Health Research Ethics for Southern Denmark (ref. S-20090130, sub protocols 12, 18 and 37). The Japanese study was approved by the internal review board in Fukuda Clinic (ref. IRB20140621-03) and registered in the Japanese Clinical Trials Registry (ref. UMIN000015494). The Korean study was approved by the Institutional Review Boards at the Maeil Asia Human Milk Research Center (ref. 0627-201508-HRBR-002-04).
Across sites, a total of 629 mothers delivered 829 milk samples (521 delivered 1, 16 delivered 2 and 92 delivered 3 samples). An overview of the sample deliveries, including maternal and infant characteristics is shown in Table 1. Infant age, when the sample was provided, ranged from 1 (Republic of Korea) to 30 weeks (Japan, 3rd visit).
Osteopontin Content in Human Milk
OPN analysis was performed on all 829 samples from the 629 mothers. Across sites, the median OPN concentration based on the first sample was 157.0 mg/L (IQR 95.4–229.5, min-max 2.2–474.8). Based on the first sample from 495 mothers with a corresponding protein concentration available, the median OPN/protein% was 1.8% (IQR 1.3–2.6, min-max 0.1–16.5). An overview of the OPN content and the OPN/protein% across sites is shown in Table 1 and Figure 1. Linear regression (with Denmark as baseline) established that the mean OPN concentration differed statistically significant across sites; F (3,625) = 135.1, P < 0.001, R2 and adj. R2 both = 0.39, with coefficients (β) ranging from 77.4 to 154.2 (all P < 0.001). The size of β and the significance remained when adjusting for maternal, infant and sample age; F (6,622) = 67.7, P < 0.001, R2 and adj. R2 both = 0.39, coefficients (β) ranging from 51.2 to 119.5 (all P < 0.05). Except from Japan and Republic of Korea, all sites were pairwise significantly different from each other after adjustment for maternal, infant and sample age.
Protein Content in Human Milk
Protein analysis was performed on 695 of the 829 samples. One Chinese sample was not analyzed due to device breakdown, and 133 of the Danish samples did not have enough material for the protein analysis. The median protein concentration was 1.0 g/100 mL (IQR 0.8–1.1) in the Chinese samples, 0.9 g/100 mL (IQR 0.8–0.9) in the Danish, 0.8 g/100 mL (IQR 0.8–0.9) in the Japanese and 1.3 g/100 mL (IQR 1.2–1.4) in the Korean samples (after conversion).
Osteopontin Content in Relation to Infant Age
An overview of the OPN concentration in relation to infant age across sites is shown in Figure 2. To avoid clustering for mothers delivering more than 1 sample, only first time visit samples are included in the figure.
Based on the 108 mothers delivering more than 1 sample (75 from China and 33 from Japan), the multilevel regression analysis showed a decrease in OPN concentration with infant age; in the Chinese milk with β = (−11.3) (95% CI (−13.9) to (−8.8), P < 0.001), and in the Japanese milk with β = (−2.1) (95% CI (−3.2) to (−0.9), P = 0.001). For both the Chinese and the Japanese milk, the association remained when adjusting for maternal age and sample age at the time of OPN analysis. An overview is shown in Figure 3.
In this explorative multicenter study we aimed to investigate the OPN content in a large number of human milk samples from 4 different countries; China, Denmark, Japan and Republic of Korea. Based on 829 milk samples from 629 mothers, we observed varying levels of OPN, both absolute (mg/L) and relative to the protein concentration (OPN/protein%) across sites and within lactation period.
The highest OPN concentration and OPN/protein% were found in milk from Chinese mothers, followed by slightly lower levels in the Korean and Japanese mothers (Fig. 1), whereas the OPN level in the Danish samples was significantly lower. To examine whether this lower level could be due to longer storage period of the Danish samples, fresh milk samples from 13 Danish mothers were analyzed. The OPN level and OPN/protein% in these 13 fresh milk samples (data not shown) did not significantly differ from the level obtained in the Danish samples in the present study or from the level in samples from 29 Danish mothers previously reported (4). In conclusion, 3 individual sets of Danish human milk samples and analyses all showed significantly lower OPN levels than those observed in milk from Asian mothers.
The reason for the variations in OPN content between the involved countries is not clear. It could be the result of environmental and dietary differences, but it is more likely that the variation is based in genetic differences between the populations. It is known that the composition of human milk varies geographically and between ethnicities. Recently, it was shown that human milk oligosaccharide concentrations and profiles varied extensively in milk samples from 11 international cohorts from 4 different continents (23). Likewise, the concentration of lactoferrin was significantly higher in milk from Ethiopian than Swedish mothers (24) and the levels of both lactoferrin and lactadherin in milk from Indian and South African mothers were significantly higher than in milk from American mothers (25). Finally, levels of transforming growth factor beta (TGF-β), which is involved in immune regulation and mucosal defense, have been shown to be present at significantly higher levels in colostrum from Japanese women compared to Nepalese women (26).
The level and variation of OPN in human milk has only been analyzed in a couple of studies comprising few mothers and milk samples. Based on a total of 97 samples across lactation from 20 Japanese mothers, the median OPN concentration was reported to be 1493.4 mg/L in early milk (72 hours to 7 days pp) declining to 896.3 mg/L (1 month pp), 550.8 mg/L (4–7 months pp) and 412.7 mg/L (11–14 months pp) (27). However, OPN concentrations were determined using an ELISA not validated for use in human milk, and later this assay was shown to overestimate the level of milk OPN up to 10-fold (4). In a study comprising 29 Danish mothers delivering a milk sample between day 6 and 58 pp, the OPN concentration (presented as mean ± SD (min-max)) was reported to be 138 ± 79 mg/L (18–322), corresponding to 2.1 ± 1.4% of the total milk protein concentration (4). In 2015, data were published on term (8 mothers, 32 samples) and preterm (14 mothers, 28 samples) human milk collected from American mothers, 2 to 58 days pp. The study found no significant difference in the OPN levels between the preterm and the term milk measured by peak ion counts in mass spectrometric analyses. The absolute levels of OPN in the milk samples were not reported (28).
In the present study, Chinese and Japanese mothers delivered more milk samples during the course of lactation. In both populations a significant decrease in OPN concentration with infant age was observed. Since OPN has been shown to be involved in immunological processes (9), it could be hypothesized that the presence of OPN is especially important in early milk. A similar scenario exists for other immunologically active milk proteins (29), like lactoferrin which is also known to decrease in concentration over the course of lactation (30).
Comparable protein concentrations were found in Chinese, Danish, and Japanese milk, whereas the protein concentration was significantly higher in Korean milk. The observed Korean protein level is, however, in line with a previous study comprising 2632 Korean mothers (31).
By performing protein and OPN analyzes in 4 different places, a possible source of error is introduced in the comparison of data. The optimal study setup would have been to analyze all samples at 1 time point at the same laboratory or to perform ring analyses of a subset of samples. As described in the Methods section, this was, however, not possible. Instead, actions were taken to minimize potential sources of error and ensure the comparability of the results. As for the OPN ELISA, milk samples had to be diluted 40,000-fold; this step represents the biggest source of experimental error in the process and in order to reduce this, laboratory staff at all sites were instructed to use the same dilution protocol and to follow the ELISA protocol very carefully. Likewise, all data used for generation of the ELISA standard curves obtained at the 4 study sites were inspected and compared to ensure comparability between sites.
In conclusion, this multicenter study investigated the OPN concentration in a large number of human milk samples from 4 different countries; China, Denmark, Japan, and Republic of Korea. Based on the first visit, the median OPN concentration differed statistically significant across sites, from 99.7 mg/L in Danish, 185.0 mg/L in Japanese and 216.2 mg/L in Korean to 266.2 mg/L in Chinese mothers even after adjustment for infant, maternal and sample age. Based on the first sample from all mothers, a decreasing tendency in the OPN concentration with infant age was observed. This was confirmed when performing a multilevel regression analysis on data from mothers delivering more than 1 sample. Knowledge about the level of the bioactive protein OPN in human milk across different countries and within the lactation period provides a better understanding of human milk composition. This knowledge can aid in the development of improved infant formulas based on bovine milk proteins.
Denmark: Mette Vogn Hviid, Hans Christian Andersen Children's Hospital performed the macronutrients analyses. Lise Møller Fogh, Department of Molecular Biology and Genetics, Aarhus University assisted in the analyses of the OPN content. Jan Sørensen, Healthcare Outcomes Research Center, Royal College of Surgeons in Ireland contributed to the statistical analyses.
Japan: Maki Azakami and Tomomi Miyazono collected the human milk samples and participant data in the Japanese Human Milk Study. Toshiya Kobayashi supported the authors as manager of the Research and Development Department of Bean Stalk Snow Co., Ltd.
Republic of Korea: Jia Jung contributed to the setup and management of the Maeil Asia Human Milk Research Center (MAHRC). Byungmoon Jung collected the human milk samples and participant data in the Korean Human Milk Study. Yongki Kim supported the authors as manager of the Nutrition Research and Development Center of Maeil Dairies. Honam Chun supported the authors as head of the Maeil Innovation Center (MIC).
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