Miori Tanaka

Background/Objectives: Obesity is associated with increased bone marrow fat content and saturation, which contribute to enhanced osteoclastogenesis and bone loss. Although saturated, monounsaturated, and polyunsaturated fatty acids (FAs) have been reported to differentially affect bone metabolism, their comparative effects and the underlying mechanisms remain unclear. This study compared the effects of palmitic acid (PA), oleic acid (OA), α-linolenic acid (ALA), and linoleic acid (LA) on osteoclastogenesis in vitro and examined the effects of PA-, OA-, and LA-enriched diets on bone metabolism. Methods: Receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-stimulated RAW264.7 macrophages were treated with PA, OA, ALA, or LA. The number of osteoclasts, tartrate-resistant acid phosphatase (TRAP) activity, osteoclastic gene and protein expression, NF-κB signaling, reactive oxygen species (ROS)-related fluorescence, and antioxidant-related gene expression were evaluated. The involvement of toll-like receptor 4 (TLR4) signaling was examined using TAK-242. Male C57BL/6J mice were fed high-fat high-sucrose diets enriched with PA, OA, or LA for 8 weeks, and bone mineral density (BMD), plasma bone turnover markers, and osteoclast-related gene expression were assessed. Results: PA enhanced RANKL-induced osteoclastogenesis and NF-κB transcriptional activity, which were partially attenuated by TAK-242. ALA and LA attenuated PA-enhanced osteoclastogenesis and oxidative stress-related responses. Mice fed a PA-enriched diet exhibited lower BMD and higher plasma free fatty acid levels and bone turnover markers. Conclusions: PA promotes RANKL-induced osteoclastogenesis, which can be alleviated by ALA and LA in vitro. These findings suggest that different FAs exert distinct effects on osteoclastogenesis and that dietary FA composition may influence bone metabolism.

Mineral composition of donor human milk (DHM) is important for the nutritional management of preterm infants, but data remain limited, particularly in Asian populations. This study determined calcium (Ca), inorganic phosphorus (IP), and zinc (Zn) concentrations in DHM from a Japanese human milk bank and their associations with maternal and infant factors. We analyzed 457 DHM samples from 331 donors, including 106 preterm and 351 term samples. Mineral concentrations were measured by a colorimetric assay, and associations with maternal and infant factors were evaluated using linear mixed-effects models accounting for repeated samples. Zn concentrations were higher in preterm DHM than in term DHM in the unadjusted comparison (141 [100–183] µg/dL vs. 113 [72–147] µg/dL, p < 0.001), but the difference was not significant after adjustment for postpartum week. Ca and IP concentrations did not differ between groups. Postpartum week was negatively associated with all minerals (all p < 0.001). Maternal age was positively associated with IP, while parity 2 was associated with lower Ca than parity 1. These findings identify postpartum week as a major source of variation in DHM mineral composition and may inform future approaches to DHM compositional assessment and nutritional management of preterm infants.

Background: Secondhand exposure to heated tobacco products (HTPs) is increasingly common, but its impact on human milk composition is unclear. We investigated whether secondhand exposure to HTP aerosols affects major human milk components and cotinine concentrations in lactating women. Methods: This observational study included 15 lactating women whose household members used HTPs (secondhand HTP exposure group) and 33 lactating women who did not live with any smokers (non-exposed group). Human milk was analyzed for macronutrients, total solids, energy, lactoferrin, secretory immunoglobulin A (sIgA), calcium, inorganic phosphorus, zinc, and cotinine. Cotinine was measured in all women in the secondhand HTP exposure group and in three women in the non-exposed group. Results: Background characteristics did not differ significantly between groups. No significant differences were observed in lipid, protein, total solids, energy, true protein, lactoferrin, calcium, inorganic phosphorus, or zinc. Carbohydrate concentration differed significantly between the non-exposed and secondhand HTP exposure groups (non-exposed vs. secondhand HTP exposure: 8.20 vs. 8.10 g/dL, p = 0.032), although the absolute difference was small. sIgA tended to be higher in the secondhand HTP exposure group (non-exposed vs. secondhand HTP exposure: 1244 vs. 1706 μg/mL, p = 0.072). Cotinine concentrations did not differ significantly between groups; qualitative cotinine tests were negative in all samples. Conclusions: Secondhand exposure to HTPs was not associated with clear differences in major human milk components or cotinine concentrations. However, the findings should be interpreted cautiously because of the small sample size and limited cotinine assessment. Larger studies with objective exposure assessment and infant follow-up are needed.

Background: Secondhand exposure to heated tobacco products (HTPs) is increasingly common, but its impact on human milk composition is unclear. We investigated whether secondhand exposure to HTP aerosols affects major human milk components and cotinine concentrations in lactating women. Methods: This observational study included 15 lactating women whose household members used HTPs (secondhand HTP exposure group) and 33 lactating women who did not live with any smokers (non-exposed group). Human milk was analyzed for macronutrients, total solids, energy, lactoferrin, secretory immunoglobulin A (sIgA), calcium, inorganic phosphorus, zinc, and cotinine. Cotinine was measured in all exposed women and in three women in the non-exposed group. Results: Background characteristics did not differ significantly between groups. No significant differences were observed in lipid, protein, total solids, energy, true protein, lactoferrin, calcium, inorganic phosphorus, or zinc. Carbohydrate concentration differed significantly between the non-exposed and secondhand exposure groups (non-exposed vs. secondhand HTP exposure: 8.20 vs. 8.10 g/dL, p = 0.032). sIgA tended to be higher in the secondhand HTP exposure group (non-exposed vs. secondhand HTP exposure: 1,244 vs. 1,706 μg/mL, p = 0.072). Cotinine concentrations did not differ significantly between groups; qualitative cotinine tests were negative in all samples. Conclusion: In this study, secondhand exposure to HTPs was not associated with clear changes in major human milk components or cotinine concentrations. While secondhand exposure should be avoided whenever possible, these findings suggest that household members’ use of HTPs does not justify refraining from breastfeeding.

Background: According to the human milk banking guidelines in Japan, issued in 2014, pasteurized donor human milk (PDHM) is discarded 24 hours after thawing. However, previous studies have suggested that PDHM remains safe for administration to preterm infants for up to 48 hours after thawing, potentially reducing unnecessary wastage. Research Aim: This study aimed to assess the microbiological safety and the preservation of nutritional and immunological properties of PDHM stored under refrigeration for up to 48 hours after thawing. Methods: This was a paired-sample experimental study evaluating the safety and composition of PDHM during refrigerated storage after thawing. Individual pasteurized milk samples from 40 donors were thawed overnight in a refrigerator and analyzed at 24 and 48 hours. Samples were cultured on sheep blood agar and incubated for 48 hours at 35 °C to assess bacterial growth. Macronutrients, micronutrients, and immune substances (sIgA and lactoferrin) were measured using specialized analyzers and ELISA kits. The pH of the samples was determined at 24 and 48 hours using a tabletop pH analyzer. Results: Small but statistically significant changes were observed in several components during 48 hours of storage. Fat, carbohydrates, calcium, inorganic phosphorus, sIgA, and lactoferrin showed slight variations, while protein and zinc remained stable. Fat decreased after 24 hours compared with immediately after pasteurization but did not decline further at 48 hours. The mean pH increased slightly from 7.047 to 7.102 ( p = 0.003). No bacterial growth was detected at either 24 or 48 hours. Conclusions: Although minor biochemical changes occurred, these were not clinically significant. Extending PDHM use to 48 hours after thawing maintained its nutritional and microbiological integrity and could inform revisions to current Japanese milk bank guidelines.

Background: Pasteurized donor human milk (PDHM) is widely recommended for preterm infants when their mother’s own milk is unavailable. Storage duration of frozen PDHM varies across countries. In Japan, PDHM is stored at −30 °C; however, there is no information on whether long-term storage at −30 °C affects PDHM quality. Research Aims: This study aimed to determine the effects of freezing at −30 °C for 6, 9, and 12 months after expression on bacterial growth and nutrient content of PDHM. Methods: This was an experimental laboratory study of nutrient and bacterial stability over time. A total of 20 milk samples were pasteurized within 3 months after expression and stored at −30 °C. Bacterial counts and levels of macronutrients, minerals, secretory Immunoglobulin A (sIgA), and lactoferrin in PDHM were analyzed after pasteurization and at 6, 9 and 12 months after expression. A Friedman test followed by a Bonferroni-adjusted Wilcoxon signed-rank test was performed. Results: No bacteria were detected in the 20 samples after pasteurization or at any time point. When the concentrations of nutrients were compared to the values immediately after pasteurization, carbohydrates at 9 and 12 months and zinc at 12 months showed slight increases, but the respective increases were minimal (carbohydrate: +3.2% at 9 months and +3.8% at 12 months; zinc: +7.8% at 12 months). The concentrations of protein, fat, calcium, inorganic phosphorus, sIgA, and lactoferrin remained stable at 6, 9, and 12 months. Conclusions: These results suggest that PDHM can be safely stored at −30 °C for 12 months after expression without compromising bacterial safety and nutritional quality.

Background: Calcium (Ca), inorganic phosphorus (IP), and zinc (Zn) are essential minerals in human milk for infant growth and health. Current methods for determining minerals in human milk, including inductively coupled plasma atomic emission spectroscopy (ICP‐AES), are time‐consuming and laborious. This study aimed to measure Ca, IP, and Zn in human milk by a rapid, simple, and accurate method using clinical laboratory reagents based on a colorimetric assay. Methods: 71 pooled donor human milk (DHM) samples were collected after Holder pasteurization. The only pretreatment required was dilution, and samples were diluted 5‐fold. Ca, IP, and Zn concentrations were measured by colorimetric assay using an automated analyzer. The results were validated in terms of repeatability, recovery, and comparison with ICP‐AES. Results: The coefficients of variation for intra‐assay precision were 0.7%–1.9% for Ca, 1.5%–2.1% for IP, and 0.8%–7.4% for Zn. The recovery rates ranged from 104.1% to 104.9% for Ca, 96.3%–103.4% for IP, and 94.5%–103.2% for Zn with high, medium, and low concentrations. There were significant correlations between Ca, IP, and Zn levels determined by colorimetric assay and Ca, total P, and Zn levels determined by ICP‐AES (Ca: r = 0.944, IP: r = 0.463, and Zn: r = 0.949, p < 0.001, n = 58). Conclusion: These results confirm the precision and accuracy of the colorimetric assay using clinical laboratory reagents for determining Ca, IP, and Zn levels in human milk. Its use may contribute to the rapid and easy provision of appropriate DHM to preterm infants.

Holder pasteurization (HoP; 62.5 °C, 30 min) is the method used most frequently in human milk banks to reduce the risk of infection transmission through donor human milk (DHM), but it has the disadvantage of potentially affecting the contents of some bioactive milk components. Dry pasteurizers avoid water handling and are easier to operate than water pasteurizers; however, the effect of dry pasteurization on human milk quality has not been fully elucidated. The aim of this study was to determine the effect of HoP by water or dry pasteurization on the nutrient content of human milk. Milk samples were assigned to 4 groups: No-HoP (untreated milk), Sterifeed (water pasteurizer), Barkey (dry pasteurizer), and Racoon (dry pasteurizer). The processing temperature of each machine was measured over time. Levels of macronutrients, minerals, secretory immunoglobulin A (sIgA), lactoferrin, and human milk oligosaccharides (HMOs) in DHM were analyzed before and after HoP. The mean plateau temperature and the highest temperature of the water pasteurizer was more than 0.5 °C lower and more than 1 °C lower, respectively, than for the dry pasteurizers. Macronutrients, minerals, and HMOs were unaffected or minimally affected by the HoP process in experiments conducted using each type of pasteurizer, whereas sIgA and lactoferrin levels were decreased significantly. The losses of sIgA and lactoferrin through dry pasteurization were more than 10% greater than the losses incurred by water pasteurization. The respective reductions in sIgA, calculated from median values, were 23% for Sterifeed (P < 0.05), 42% for Barkey (P < 0.001), and 44% for Racoon (P < 0.001). The reductions in lactoferrin were 75%, 86%, and 86%, respectively (all P < 0.001). After the cooling process in the two dry pasteurizers, energy decreased by 6% and calcium decreased by 2% in Barkey (all P < 0.05), while lactoferrin increased by 3% in Racoon (P < 0.05). No other components showed significant changes. These results show that dry pasteurization causes a higher reduction in immune components than water pasteurization, suggesting that dry method is not suitable for processing DHM.

Background/Objectives: Sodium (Na) concentration and the sodium-to-potassium (Na/K) ratio in human milk reflect epithelial tight junction integrity and have been proposed as non-invasive biomarkers of lactational dysfunction, including subclinical mastitis and ductal obstruction. However, their discriminative performance across varied mammary duct conditions, as well as their relevance to milk quality and nutritional integrity, remain underexplored. This study aimed to evaluate the ability of Na, K and the Na/K ratio to discriminate ductal obstruction from non-obstructed lactation—including normal, mixed, and donor milk—and to assess their applicability as nutritional and clinical screening biomarkers. Methods: The study analyzed 635 human milk samples from four groups: obstructed ducts (n = 94), mixed ducts (n = 39), normal ducts (n = 102), and donor milk (n = 400). Na and K concentrations were measured using validated handheld ion-selective electrode analyzers. Statistical analyses included Quade’s ANCOVA and receiver operating characteristic curve analysis, adjusting for infant age, gestational age, birth body weight, maternal age and storage duration. Results: Na concentrations were highest in obstructed ducts (Group A: median 810 ppm, IQR 368–1725) compared with normal ducts (Group C: 220 ppm, IQR 140–283) and donor milk (Group D: 98 ppm, IQR 80–130) (p < 0.001). A similar pattern was observed for the Na/K ratio (Group A: 1.5, IQR 0.6–3.1 vs. Group C: 0.3, IQR 0.2–0.5; Group D: 0.3, IQR 0.2–0.3). After adjusting, both Na and the Na/K ratio remained significantly elevated in milk from obstructed ducts compared to non-obstructed samples (p < 0.001). Donor milk exhibited the lowest and most stable electrolyte levels. Na demonstrated excellent discriminative performance (area under the curve = 0.96), slightly outperforming the Na/K ratio (area under the curve = 0.92). Conclusions: Na concentration and the Na/K ratio in human milk are sensitive and practical biomarkers of mammary gland integrity. Given that Na alone can be measured without additional calculations, its simplicity and strong performance support its application as a potential biomarker for ductal obstruction, with implications for both lactation support and nutritional science.

Background: Freezing is commonly used to preserve human milk; however, microwave thawing is not recommended due to nutritional loss and creation of hotspots in the milk. Data on compositional changes after microwave thawing and uneven temperature distribution are scarce. Research Aim: To investigate the association between microwave heating and the composition and temperature distribution of human milk. Methods: In this laboratory-based cross-sectional experimental study, 35 milk samples were divided into six groups based on preheating operations and milk bag material (35 samples each). After thawing at 600 watts for 30 seconds, temperature was immediately measured using thermography. Uneven temperature distribution was evaluated by the difference between maximum and minimum temperatures. Subsequently, it was mixed by inverting, and the temperature was measured again. The secretory Immunoglobulin A and lactoferrin concentrations were analyzed using enzyme-linked immunosorbent assays. Macronutrients were analyzed using mid-infrared transmission spectroscopy. Results were compared with thawing in running water to explore the feasibility of microwave thawing. Results: The median temperature in the sonicated group (33.6 °C) was significantly decreased compared to that in the untreated group (54.9 °C). The median temperature in the polyethylene bag group (42.0 °C) was also significantly decreased compared to that in the polypropylene bag group (53.2 °C). The temperature after inversion mixing was close to the recommended temperature for feeding. The median concentration significantly decreased for secretory Immunoglobulin A (0.9–16.6%) and lactoferrin (21.3–29.1%) after microwaving. Conclusion: Component losses caused by microwave thawing were not clinically problematic compared to the standard value and could be minimized. Microwave heating may be an option for thawing human milk.