Lipid peroxidation products as predictors of oxidant-mediated disease in preterm infants.

BACKGROUND: Preterm infants are susceptible to "oxygen radical diseases" (ORD). 8-isoprostane (8-IP) is a bioactive eicosanoid generated by reactive oxygen species-catalyzed peroxidation of arachidonic acid. Malondialdehyde (MDA) is generated by the decomposition of oxidant-induced lipid hydroperoxides. We hypothesize that the development of ORD is associated with elevated plasma 8-IP on day 0-1, and increasing urine levels of MDA in the first month. METHODS: Preterm (<32 weeks, n = 39) and term (n = 39) infants were recruited at birth. Plasma 8-IP was quantified by ELISA on day 0-1, and urine MDA by colorimetric assay of thiobarbituric acid reactive substances (TBARS) on days 0-1, 7, 14, 21, and 28. ORD was defined as retinopathy of prematurity ≥ stage 1, pneumatosis, or oxygen requirement at 36 weeks corrected gestational age. RESULTS: Plasma 8-IP was higher on day 0-1 in preterm infants who developed ORD compared to term infants. Urine TBARS levels increased in preterm infants from day 0-1 to day 28 but were not different in infants with or without ORD. Preterm infants who developed ORD demonstrated a significant rise in urine TBARS levels from day 1 to 14. CONCLUSIONS: Elevated plasma 8-IP on day 1 is associated with ORD in preterm infants. If validated as a biomarker for ORD, it may be useful in directing antioxidant therapies to the most susceptible infants. Urine TBARS during the first month are not significantly different in term infants, preterm infants with ORD, and preterm infants without ORD, but rapid rise of TBARS in the first 2 weeks may be associated with ORD.

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.

Determinants of the lung microbiome in intubated premature infants at risk for bronchopulmonary dysplasia.

BACKGROUND: Airway dysbiosis in premature infants may be associated with bronchopulmonary dysplasia (BPD). Early oropharyngeal colostrum (OPC) administration alters the oral microbiome, which may impact the lung microbiome. We aim to compare the oral and tracheal microbiota during the first week of life, and to determine whether early OPC administration affects microbial diversity or leukocyte inflammatory activity in the lung. METHODS: Intubated premature infants (n = 42) were evaluated. The oral microbiome was characterized on day of life (DOL) 3, and the tracheal microbiome on DOL 3 and DOL 7, using 16S ribosomal DNA sequencing. Gene expression for inflammatory markers was quantified in airway leukocytes by real-time q-PCR. RESULTS: The oral and tracheal microbiota were significantly different on DOL 3, but the tracheal microbiome on DOL 7 was more similar to the oral from DOL 3. Tracheal bacterial diversity decreased from DOL 3 to DOL 7. Longer time to first OPC administration tended to be associated with lower bacterial diversity in the airways. CONCLUSIONS: The tracheal microbiome in intubated premature infants in the first week is likely determined, in part, by the composition of the oral microbiome. Bacterial diversity in intubated babies decreases during the first week of life, a pattern that could be consistent with risk for BPD. Decreased bacterial diversity and increased inflammatory activity in the lung may also be associated with delayed administration of OPC.

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.

Early oral colostrum administration in preterm infants.

BACKGROUND: Early administration of colostrum may provide preterm infants with immune components. Previous studies illustrating the effects of oral colostrum (OC) have been confounded by the coincidence of enteral feedings. OBJECTIVE: To quantify OC absorption, as measured by urinary sIgA and lactoferrin, in preterm infants prior to enteral feedings. MATERIALS AND METHODS: Colostrum was obtained from mothers delivering infants ≤32 weeks and ≤1500 g. sIgA and lactoferrin were measured in infant urine, and microflora in saliva and tracheal aspirates were characterized. RESULTS: Urinary sIgA and lactoferrin were significantly greater in infants receiving OC by syringe compared to swab (p < 0.002). Urinary sIgA correlated with the total number of doses in 72 h (R2 = 43%, p < 0.01). CONCLUSIONS: Administration of OC by syringe and higher cumulative dose are associated with increased absorption of sIgA and lactoferrin, and early dosing may contribute to a more diverse tracheal microbiome.

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.

Effects of Nutrition Supplementation on Osmolality of Expressed Human Milk.

BACKGROUND: Provision of human milk to premature infants optimizes outcomes, but it must be supplemented to meet their nutrient and caloric requirements for growth. Our objective was to quantify the osmolality of human milk mixed with commercially available human milk fortifiers (HMF) and powdered infant formula, as currently fed to premature infants, simulating standard neonatal intensive care unit feeding practices for mixing and refrigerator storage. METHODS: Expressed human milk (EHM) samples obtained from mothers of premature infants (≤32 weeks gestation) were mixed with standard commercial products, and osmolalities were quantified. RESULTS: HMF significantly increased the micronutrient content and osmolality of EHM. Osmolalities were 291 ± 6 mOsm/kg (mean ± SD) for unsupplemented milk, and 505 ± 5 and 315 ± 19 mOsm/kg after supplementation to 24 kcal/oz using 2 current U.S. Liquid EHM fortifiers. When using powdered infant formulas to further increase the caloric content of fortified EHM >24 kcal/oz, osmolalities increased by 10.5-23.0 mOsm/kg for each additional kcal/oz. The use of powdered formulas alone (without HMF) increased osmolality without comparable increases in nutrient content. Refrigeration for 24 hours did not affect osmolalites. CONCLUSION: Our finding that several common feeding formulations exceed 450 mOsm/kg, and the lack of evidence of adverse effect, raise the question of whether current maximum osmolality guidelines should be reevaluated to enable optimal nutrition for infants in neonatal intensive care.

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.

Overexpression of extracellular superoxide dismutase protects against brain injury induced by chronic hypoxia.

Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of 18FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.

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.

Overexpression of extracellular superoxide dismutase has a protective role against hyperoxia-induced brain injury in neonatal mice.

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P< 0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P < 0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.

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.

Extracellular superoxide dismutase overexpression can reverse the course of hypoxia-induced pulmonary hypertension.

Hypoxia leads to free radical production, which has a pivotal role in the pathophysiology of pulmonary hypertension (PH). We hypothesized that treatment with extracellular superoxide dismutase (EC-SOD) could ameliorate the development of PH induced by hypoxia. In vitro studies using pulmonary microvascular endothelial cells showed that cells transfected with EC-SOD had significantly less accumulation of xanthine oxidase and reactive oxygen species than nontransfected cells after hypoxia exposure for 24 h. To study the prophylactic role of EC-SOD, adult male wild-type (WT) and transgenic (TG) mice, with lung-specific overexpression of human EC-SOD (hEC-SOD), were exposed to fraction of inspired oxygen (FiO(2)) 10% for 10 d. After exposure, right ventricular systolic pressure (RVSP), right ventricular mass (RV/S + LV), pulmonary vascular wall thickness (PVWT) and pulmonary artery contraction/relaxation were assessed. TG mice were protected against PH compared with WT mice with significantly lower RVSP (23.9 ± 1.24 versus 47.2 ± 3.4), RV/S + LV (0.287 ± 0.015 versus 0.335 ± 0.022) and vascular remodeling, indicated by PVWT (14.324 ± 1.107 versus 18.885 ± 1.529). Functional studies using pulmonary arteries isolated from mice indicated that EC-SOD prevents hypoxia-mediated attenuation of nitric oxide-induced relaxation. Therapeutic potential was assessed by exposing WT mice to FiO(2) 10% for 10 d. Half of the group was transfected with plasmid containing cDNA encoding human EC-SOD. The remaining animals were transfected with empty vector. Both groups were exposed to FiO(2) 10% for a further 10 d. Transfected mice had significantly reduced RVSP (18.97 ± 1.12 versus 41.3 ± 1.5), RV/S + LV (0.293 ± 0.012 versus 0.372 ± 0.014) and PVWT (12.51 ± 0.72 versus 18.98 ± 1.24). On the basis of these findings, we concluded that overexpression of EC-SOD prevents the development of PH and ameliorates established PH.

The protective effect of overexpression of extracellular superoxide dismutase on nitric oxide bioavailability in the lung after exposure to hyperoxia stress.

The objective of this study was to determine whether overexpression of human extracellular superoxide dismutase (hEC-SOD) can preserve nitric oxide (NO) bioavailability. In vitro studies examined the transient expression of hEC-SOD in mouse epithelial (C10) cells and its effect on extracellular accumulation of NO, intracellular cyclic guanosine monophosphate (cGMP), and nuclear factor kappa B (NF-κB) activation under normal and oxidative stress conditions. In vivo, newborn rabbits were treated with a plasmid containing hEC-SOD cDNA or vehicle plasmid alone, followed by exposure to hyperoxia (Fio2 = 95% for 7 days). A third group was raised under normoxic conditions. cGMP and NF-κB activation were studied. There was significantly higher NO accumulation in cells expressing hEC-SOD exposed to oxidative stress compared with nontransfected cells. Accumulation of cGMP was significantly higher in cells expressing hEC-SOD. Oxidative stress induced NF-κB activation, which was abrogated by hEC-SOD expression. In vivo, there was significantly higher cGMP accumulation in transfected neonatal rabbit lung tissue at 3 and 7 days of hyperoxic exposure. Immunostaining for NF-κB, showed a marked increase in NF-κB concentration in nontreated neonatal rabbit lung tissue compared to transfected neonatal lung with hEC-SOD and the control air group. These results show that transient EC-SOD overexpression maintains NO bioavailability, which directly leads to maintenance of cGMP activity and reduction of NF-κB activation under oxidative stress.

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.