Is the Antioxidant Capacity of Stored Human Milk Preserved?

Background: In the neonatal intensive care unit (NICU) expressed mothers' milk usually is stored frozen until used. We found that when human milk was stored at -20°C for up to 9 months there were reduced bacterial counts and pH, increased free fatty acids, but unchanged immune proteins. Antioxidant protection is an important benefit of human milk. Few studies have evaluated long-term effects of cold storage on the antioxidant capacity of human milk. We hypothesized that the antioxidant capacity of human milk is affected adversely by long-term storage at -20°C. Objective: To study the impact of long-term cold storage on the oxidative capacity of human milk and the biological impact of these changes on macromolecular constituents of human milk. Methods: Freshly expressed milk was obtained from mothers in the NICU, stored at -20°C for 6 months, and compared with the baseline. Paired samples were analyzed for glutathione, hydrogen peroxide (H2O2), 8-isoprostane, catalase, and superoxide dismutase. Results: There was no change in H2O2 concentration between baseline and 6 months. Significant reductions from baseline in both catalase and superoxide dismutase concentrations and activities, total glutathione, oxidized glutathione, reduced glutathione, and the ratio of reduced to oxidized glutathione were observed (p < 0.05). There was a significant increase in 8-isoprostane concentrations (p < 0.001). Conclusion: These data indicate significant changes in antioxidant capacity of human milk, including oxidation of macromolecules, after storage at -20°C for 6 months. The clinical implication of these findings may explain the nonuniform protection against oxidant disease in preterm infants fed human milk.

Bacterial stability with freezer storage of human milk.

OBJECTIVES: Human milk supports the development of a beneficial newborn intestinal microflora. We have shown previously that human milk had reduced bacteria but unchanged nutrient composition when stored at -20 °C for up to nine months. We suspected declining bacterial colony counts were manifestations of bacterial dormancy and not failure of survival. We investigated differences in selected bacterial colony counts (lactobacillus, bifidobacteria, staphylococcus, streptococcus and enterococcus) in human milk stored for 2 and 12 weeks at -20 °C in either manual or automatic defrost freezers and whether reduced bacterial counts at 12 weeks were the result of dormancy or failure of survival. METHODS: Freshly expressed milk was obtained from mothers in the NICU, divided into aliquots and stored for 2 and 12 weeks at -20 °C in either automatic or manual defrost freezers. Subsequently, duplicate aliquots, one thawed and the other thawed and maintained at room temperature for 4 h, were plated to assess bacterial colony counts. RESULTS: Significant declines in bacterial colony counts were seen from 2 to 12 weeks freezer storage for all bacteria. There were no differences in colony counts between freezer types. Once thawed, no further bacterial growth occurred. CONCLUSIONS: Short-term freezer storage for 12 weeks resulted bacterial killing. Type of freezer used for storage did not have an impact on bacterial survival. It is unknown whether the paucity of important probiotic bacteria in stored human milk has adverse effects on infants.

Relationship Between Milk Fat Globule-Epidermal Growth Factor 8 and Intestinal Cytokines in Infants Born Preterm.

OBJECTIVES: To investigate the relationships between dietary intake and fecal concentrations of milk fat globule-epidermal growth factor 8 (MFG-E8), and between fecal concentrations of MFG-E8 and markers of intestinal inflammation in infants born preterm. STUDY DESIGN: Fecal samples were collected daily and enteral feedings were sampled weekly. MFG-E8 in enteral feedings and feces, and cytokine concentrations in feces were quantified by enzyme-linked immunosorbent assay. RESULTS: Milk MFG-E8 concentrations were significantly greater in unfortified mother's own milk (MOM) and MOM with human milk fortifier than either donor human milk or preterm formula. MFG-E8 concentrations in fecal samples were positively correlated with MFG-E8 concentrations in respective milks. High MFG-E8 exposure (≥60 mL/kg/day of feedings that include MOM or MOM with human milk fortifier) was associated with lower concentrations of proinflammatory cytokines (interleukin-8, tumor necrosis factor-α, and monocyte chemoattractant protein-1) and higher concentrations of the anti-inflammatory cytokine interleukin-4 in feces, compared with low MFG-E8 exposure. CONCLUSIONS: Infants born preterm who were fed MOM had greater concentrations of MFG-E8 and lower concentrations of proinflammatory cytokines in fecal samples than other diets or no feedings. These data further support the protective role of MOM, possibly because of MFG-E8, against intestinal inflammation.

Does Human Milk Fortifier Affect Intestinal Inflammation in Preterm Infants?

Background: Fecal calprotectin, a recognized marker of intestinal inflammation, is derived from neutrophil migration to a site of inflammation. Introduction of bovine-based human milk fortifier containing intact protein in preterm infants is associated with an increase in fecal calprotectin suggestive of intestinal inflammation. Newer fortifiers contain protein hydrolysates in place of intact protein. Objective: To measure fecal calprotectin in human milk-fed preterm infants before and after human milk fortification using a fortifier containing hydrolyzed protein. Methods: Serial stool samples were collected from 24 infants beginning at the first week to 60 days postnatal age. To compare the effect of human milk fortification, samples collected before and after fortification were compared. Infant demographics, diet, postnatal morbidities, and maternal characteristics were recorded. Results: A total of 401 stool samples were collected from 24 study infants who had a birth weight of 993 ± 277 g (mean ± standard deviation), gestational age 27.5 ± 2.8 weeks, and fortifier initiation at 14 days. Median fecal calprotectin before and after fortification were similar. Calprotectin levels were not correlated with birth weight or gestational age but were inversely correlated with postnatal age (p = 0.005), use of fortifier (p < 0.001), receipt of antibiotics antenatally (p = 0.007) and postnatally (p = 0.008). After adjusting for postnatal age, calprotectin levels were significantly lower following receipt of fortifier (p < 0.001) and postnatal antibiotics (p < 0.001). Conclusions: The feeding of protein hydrolysate-containing human milk fortifiers does not appear to be associated with increases in a marker of intestinal inflammation.

Effects of human milk fortifier properties on intrinsic probiotic bacteria.

Background To meet the nutritional needs of preterm infants, multicomponent nutrient fortifiers are added to human milk. The fortified human milk (FHM) product changes the physical and biochemical characteristics of the milk. We questioned whether such physical-chemical changes in the milk would alter intrinsic probiotic bacterial activity. The objective of the study was to evaluate the effect of osmolality and pH on the growth of probiotic bacterial species intrinsic to human milk. Methods Human milk samples (n = 26) were collected from mothers in the neonatal intensive care unit (NICU) and stored at -20°C until analyzed. The samples were thawed and divided into three portions. Human milk fortifiers (HMFs) were added to two portions to prepare concentrations of FHM. The remaining portion was the unfortified control sample. Each sample was then divided into two parts. One part (baseline) was used to measure the osmolality and pH and plated on selective agar to enumerate the growth of lactobacilli and bifidobacteria species. The remaining part was incubated at 37°C for 24 h to further test bacterial integrity (post-incubation) and then the same measurements were made (osmolality, pH, bacterial colony counts). Results When compared with unfortified milk at baseline, osmolality increased and pH decreased significantly after the addition of HMFs. Lactobacilli and bifidobacteria colony counts did not differ among the groups pre-incubation. Post-incubation lactobacilli and bifidobacteria increased in all the groups. Conclusion The appropriate addition of HMFs differentially affected the osmolality and pH of the milk. These physical changes did not affect the growth of probiotic bacterial species.

Pasteurization Preserves IL-8 in Human Milk.

Background: Pasteurized donor human milk is an alternative feeding when mothers' own milk is not available for premature infants. The effects of pasteurization on the host defense properties of human milk are unclear. We investigated the effects of Holder pasteurization on concentrations of anti-inflammatory and pro-inflammatory cytokines in human milk. Objective: To compare concentrations of anti-inflammatory and pro-inflammatory cytokines before and after pasteurization of donor human milk. Study Design: A single milk sample was obtained from each of 24 mothers of premature infants in the neonatal intensive care unit by electric breast pump and was stored at -80°C. At the time of pasteurization, milk samples were thawed and divided into two aliquots. The first aliquot was re-stored at -80°C and the second aliquot was heat-treated at 62.5°C for 30 min and then re-stored at -80°C. At the time of batch cytokine analyses samples were thawed rapidly. Results: Most cytokine concentrations declined following pasteurization. The most prevalent cytokine, IL-8, was preserved (89%) following pasteurization. There were no relationships between gestational age, postnatal age of milk collection, duration of milk storage, and the concentrations cytokines. Conclusion: In contrast to most cytokines after pasteurization, IL-8 is preserved or liberated from another compartment. The maintenance of IL-8 in human milk after pasteurization and the loss of anti-inflammatory cytokines following pasteurization, suggests that the effects of inflammatory activity in pasteurized human milk should be evaluated. These data may account, in part, for the lesser protective effect on the host of pasteurized donor human milk compared with mother's own milk.

Effects of Extended Freezer Storage on the Integrity of Human Milk.

OBJECTIVE: To examine the integrity (pH, bacterial counts, host defense factors, nutrient contents, and osmolality) of freshly expressed and previously refrigerated human milk subjected to long-term freezer storage. STUDY DESIGN: Mothers donated 100 mL of freshly expressed milk. Samples were divided into baseline, storage at -20°C (fresh frozen) for 1, 3, 6, and 9 months, and prior storage at +4°C for 72 hours (refrigerated frozen) before storage at -20°C for 1 to 9 months. Samples were analyzed for pH, total bacterial colony count, gram-positive and gram-negative colony counts, and concentrations of total protein, fat, nonesterified fatty acids, lactoferrin, secretory IgA, and osmolality. RESULTS: Milk pH, total bacterial colony count, and Gram-positive colony counts decreased significantly with freezer storage (P < .001); bacterial counts decreased most rapidly in the refrigerated frozen group. The gram-negative colony count decreased significantly over time (P < .001). Nonesterified fatty acid concentrations increased significantly with time in storage (P < .001). Freezing for up to 9 months did not affect total protein, fat, lactoferrin, secretory IgA, or osmolality in either group. CONCLUSIONS: Freezer storage of human milk for 9 months at -20°C is associated with decreasing pH and bacterial counts, but preservation of key macronutrients and immunoactive components, with or without prior refrigeration for 72 hours. These data support current guidelines for freezer storage of human milk for up to 9 months for both freshly expressed and refrigerated milk.

Synergistic protection against hyperoxia-induced lung injury by neutrophils blockade and EC-SOD overexpression.

BACKGROUND: Oxygen may damage the lung directly via generation of reactive oxygen species (ROS) or indirectly via the recruitment of inflammatory cells, especially neutrophils. Overexpression of extracellular superoxide dismutase (EC-SOD) has been shown to protect the lung against hyperoxia in the newborn mouse model. The CXC-chemokine receptor antagonist (Antileukinate) successfully inhibits neutrophil influx into the lung following a variety of pulmonary insults. In this study, we tested the hypothesis that the combined strategy of overexpression of EC-SOD and inhibiting neutrophil influx would reduce the inflammatory response and oxidative stress in the lung after acute hyperoxic exposure more efficiently than either single intervention. METHODS: Neonate transgenic (Tg) (with an extra copy of hEC-SOD) and wild type (WT) were exposed to acute hyperoxia (95% FiO2 for 7 days) and compared to matched room air groups. Inflammatory markers (myeloperoxidase, albumin, number of inflammatory cells), oxidative markers (8-isoprostane, ratio of reduced/oxidized glutathione), and histopathology were examined in groups exposed to room air or hyperoxia. During the exposure, some mice received a daily intraperitoneal injection of Antileukinate. RESULTS: Antileukinate-treated Tg mice had significantly decreased pulmonary inflammation and oxidative stress compared to Antileukinate-treated WT mice (p < 0.05) or Antileukinate-non-treated Tg mice (p < 0.05). CONCLUSION: Combined strategy of EC-SOD and neutrophil influx blockade may have a therapeutic benefit in protecting the lung against acute hyperoxic injury.

Role of EC-SOD overexpression in preserving pulmonary angiogenesis inhibited by oxidative stress.

UNLABELLED: Angiogenesis is one of the most important processes for normal lung development. Oxidative stress can impair the pulmonary angiogenesis, leading to chronic lung disease or Bronchopulmonary dysplasia (BPD). OBJECTIVE: To investigate the protective effects of EC-SOD overexpression on pulmonary angiogenesis on neonates following exposure to acute hyperoxia. DESIGN/METHODS: Transgenic (TG) and wild-type (WT) neonatal mice (10 mice per group) were exposed either to air (control group) or 95% O(2) for 7 days starting at birth. After exposure, all animals were sacrificed. ROS concentration was measured in lung homogenates using OxiSelect ROS assay kit. Mean vascular density (MVD) was measured using anti CD34 staining. RNA was extracted and the angiogenesis markers, VEGF, VEGFR1 and VEGFR2 and PECAM-1 were analyzed by RT-q PCR. VGEF protein was measured using Western blots. Endothelial progenitor cells (EPCs) was assayed by flow cytometer. RESULTS: There was a significant reduction of ROS in TG hyperoxic neonate group (156±14.2) compared to WT hyperoxic animals (255±35.1). Evaluation of MVD, using anti-CD34, showed marked significant increase of MVD in the TG group following hyperoxic exposure (85±12) in comparison to the WT hyperoxic group (62±8.4), (P<0.05). Among the hyperoxic groups, both RNA and protein of VEGF expression were significantly reduced in the WT animals compared to the TG group (P<0.05). The same trend was found in VEGFR 1 and 2 which were significantly reduced in WT group compared to the TG group (P<0.05). There was no significant difference between hyperoxia TG and control group (P>0.05). PECAM expression was significantly reduced in both hyperoxic compared to normoxic groups (P<0.05). EPC's showed significant reduction in WT hyperoxic group compared to others (P>0.05). CONCLUSIONS: EC-SOD plays a key role in preserving angiogenesis by scavenging free radicals which has an inhibitory effect on angiogenesis process in neonatal mice lung following exposure to hyperoxia.

Refrigerator storage of expressed human milk in the neonatal intensive care unit.

OBJECTIVE: To provide recommendations for refrigerator storage of human milk, the overall integrity (bacterial growth, cell counts, and component concentrations) of milk was examined during 96 hours of storage at 4 degrees C. STUDY DESIGN: Fresh milk samples (n = 36) were divided and stored at 4 degrees C for 0, 24, 48, 72, and 96 hours. At each time, pH, white cell count, and osmolality were measured and additional samples were stored at -80 degrees C until analyzed for bacteria and concentrations of lactoferrin, secretory (s)IgA, fat, fatty acids, and protein. RESULTS: There were no significant changes for osmolality, total and Gram-negative bacterial colony counts or concentrations of sIgA, lactoferrin, and fat. Gram-positive colony counts (2.9 to 1.6 x 10(5) colony-forming units per mL), pH (7.21 to 6.68), white blood cell counts (2.31 to 1.85 x 10(6) cells per mL), and total protein (17.5 to 16.7 g/L) declined, and free fatty acid concentrations increased (0.35 to 1.28 g/L) as storage duration increased, P < .001. CONCLUSIONS: Changes were minimal and the overall integrity of milk during refrigerator storage was preserved. Fresh mother's milk may be stored at refrigerator temperature for as long as 96 hours.

Modulation of rat intestinal nuclear factor NF-kappaB by gum arabic.

The objective of this study was to test the hypothesis that in an animal model of cathartic-induce intestinal dysfunction the proabsorptive effects of gum arabic (GA) could be associated with modulation of nuclear factor-kappaB (NF-kappaB) and with reduction of the inflammatory response caused by cathartics, as evidenced by intestinal mucosa cytokine production and gene expression. Juvenile male rats were given a phenolphthalein-magnesium citrate solution for 6 days, by itself or supplemented with either 10 or 20 g L(-1) GA, as a sole source of fluid. The controls given were tap water alone or with added 20 g L(-1) GA. The animals were euthanized and small-intestinal mucosa nuclear fractions and RNA were isolated. NF-kappaB p65 activity was highest after administration of cathartics, lowest in controls, and intermediate in GA-treated rats. Mucosal IL-1beta was overexpressed in tissues from cathartic-treated rats and from rats given high-GA solutions. Gene-array analysis revealed a complex pattern of gene regulation by cathartics which selectively upregulated several subfamilies of cytochrome P-450 family 2 genes. Co-administration of GA did not block this effect. These findings suggest that local anti-inflammatory effects on the small intestine could be obtained by administration of a nonabsorbable proteoglycan such as GA.

Enhancement of absorption by gum arabic in a model of gastrointestinal dysfunction.

OBJECTIVE: Diarrhea is a common and deadly threat to millions of infants and children. Similarly, malabsorption can aggravate the health status of the chronically sick and especially the elderly. Prompt recovery from intestinal dysfunction may have a substantial impact on many populations. The aim of this study was to test the hypothesis that, in an animal model of cathartic-induce diarrhea, the previously shown proabsorptive effects of gum arabic (GA) could directly reduce and ameliorate intestinal dysfunction. METHODS: Young male rats were offered a standard solid feed and as a sole source of fluid a phenolphthalein-magnesium citrate solution for 3 or 6 days (PC), or the same plus either 10 (GA1) or 20 (GA2) g/L of GA. Other groups had tap water without (CTL) or with 20 g/L GA (CTL + GA), after which the animals were jejunally perfused under anesthesia to test their absorptive capacity. Similarly treated rats were killed and the small intestinal mucosa scraped and processed for nitric oxide synthase (NOS) determination. RESULTS: In 6-day studies addition of GA to the cathartic solution led to increases in net water, sodium and glucose absorption with the higher GA2, relatively to the PC rats. For water (means +/- SEM): PC = 42.4 +/- 3.6; GA2 = 57.9 +/- 3.9 nmol/g.min, p < 0.05. For sodium: PC = 2,139 +/- 334; GA2 = 4,465 +/- 444 nmol/g.min, p < 0.05. After only 3-day exposure, effects were less marked. Total NOS activity was increased in the PC, GA1 and GA2 groups (333 +/- 26; 334 +/- 27; 336 +/- 23 nmol/h.g) compared to CTL (233 +/- 27 nmol/h.g, p < 0.05), while CTL + GA showed a further reduction of activity (190 +/- 18 nmol/h.g, p < 0.05 vs. CTL). CONCLUSIONS: These findings substantiate earlier physiologic and biochemical effects of GA on the gastrointestinal tract, presently conducted in a model of gastrointestinal dysfunction. The data further suggest that a natural proteoglycan such as GA can reduce secretory effects induced by cathartics and, hence, are predictive of potential effectiveness in the context of diarrhea or malabsorption by infectious or functional causes.

Effect of human milk fortifiers on bacterial growth in human milk.

BACKGROUND: As a component in human milk fortifiers (HMF), iron may equilibrate with human milk for as long as 24 hours, bind important bacteriostatic proteins, and potentially affect the host defense properties of human milk. OBJECTIVE: We compared bacterial growth in human milk prepared with each of two HMF differing in their content of iron. STUDY DESIGN: Samples of human milk obtained from mothers of premature infants were divided and mixed with one of two HMF and maintained at refrigerator temperature. Refrigerated milk samples were removed at 0, 24, and 72 hours for determination of total bacterial colony counts (TBCC). RESULTS: TBCC did not differ between groups but declined from 0 to 72 hours, p<0.001. CONCLUSION: These data suggest that differences in iron content, or other nutrients in HMF, do not affect bacterial growth in human milk. Storage of fortified human milk at refrigerator temperature for 72 hours results in decreased bacterial growth. As a component in human milk fortifiers (HMF), iron may equilibrate with human milk for as long as 24 hours, bind important bacteriostatic proteins, and potentially affect the host defense properties of human milk. We compared bacterial growth in human milk prepared with each of two HMF differing in their content of iron. Samples of human milk obtained from mothers of premature infants were divided and mixed with one of two HMF and maintained at refrigerator temperature. Refrigerated milk samples were removed at 0, 24, and 72 hours for determination of total bacterial colony counts (TBCC).

Proabsorptive action of gum arabic in isotonic solutions orally administered to rats. II. Effects on solutes under normal and secretory conditions.

Gum arabic (GA) is a natural proteoglycan with proabsorptive capacity attributable to its physico-chemical properties. Previous experiments showed that in rats oral administration of GA in an isotonic solution had a generally positive effect. This study extends the investigation to include acetaminophen and to evaluate whether GA could also act under secretory conditions induced by theophylline. Test solutions were orally administered to rats under CO2 anesthesia and blood concentrations followed for 3 hr. The secretory effects of theophylline were clearly observed for sodium and zinc. Addition of GA resulted in a more rapid rate of glutamate absorption, under normal physiologic conditions, as indicated by the higher area under the curve (AUC). There were no differences in the presence of theophylline. Acetaminophen blood concentrations peaked about 30 min after administration, and the AUC in rats that received GA was higher than in those that got the solution without GA. AUCs for total body water distribution with time and those for glucose concentrations were indistinguishable whether the solutions contained or did not contain either GA or theophylline. The results confirm that oral administration of GA can accelerate absorption of some solutes, including pharmacologic agents.

Modulation of small intestinal nitric oxide synthase by gum arabic.

Preceding studies have revealed that gum arabic (GA), a natural proteoglycan (>/= 250,000 Da), has proabsorptive properties-as shown by increased sodium and water absorption-in normal rats, and especially in two animal models of diarrhea. Because nitric oxide (NO) metabolism is linked to gastrointestinal physiology, the goals of this study were to determine whether GA modulated NO and to determine intestinal function in vivo when NO production was enhanced by l-arginine (Arg), added at either 1 or 20 mM. Mechanistically, the goal was also to determine whether GA was a NO scavenger and a small intestinal NO synthase (NOS) inhibitor. Using a glucose-electrolyte solution in rat jejunal perfusions we found that GA at +/-10 microM (2.5 g/l) decreased nitrite and nitrate formation, tending to normalize water, sodium, and glucose absorption when modified by Arg addition. In vitro tests, with oxyhemoglobin as a marker, showed that GA at >/= 5 microM scavenged NO. For GA effects on NOS, small intestinal homogenate supernatants (10,000 g) from frozen tissues of either adult or 2-day-old rats were incubated for 1 hour at 37 degrees C in the presence of 2 mM Arg and increasing GA concentrations (0-100 microM). GA produced a concentration-dependent inhibition of NOS, reaching approximately 31% inhibition with 5 microM GA and up to 51% with 50 microM GA. GA at 100 microM produced no further inhibition. The data indicate that GA, in addition to its ability to remove NO diffused into the intestinal lumen, may also partially inhibit intestinal NOS and thus modulate intestinal absorption through these mechanisms. Use of GA as a food additive may help in restoring or improving small intestinal function in conditions where functional damage has occurred.