Wesam Elremaly

Oxygen and parenteral nutrition two main oxidants for extremely preterm infants: ‘It all adds up’

OBJECTIVES To assess the effect of early exposure to O2 and parenteral nutrition (PN) on oxidative stress at 36 weeks post-menstrual age (PMA) and on bronchopulmonary dysplasia (BPD) in extremely preterm infants. STUDY DESIGN A prospective observational study including 116 infants <29 weeks of gestation. Baseline clinical characteristics, FiO2 on day 7, duration of PN and clinical outcomes data were collected. In 39 infants, whole blood glutathione (GSH) and oxidized glutathione (GSSG) at 36 weeks PMA were measured and the redox potential was calculated using Nernst equation. Students t-test, Chi-square, Spearman correlation, ANOVA, and logistic regression analyses were used as appropriate. P <  0.05 was considered significant. RESULTS FiO2 ≥25% was associated with higher level of GSSG (0.29 ± 0.04 versus 0.18 ± 0.02 nmol/mg of protein), a more oxidized redox potential (-191 ± 2 versus -198 ± 2 mV) and more BPD (90% versus 45%). PN duration >14 days was also associated with higher level of GSSG (0.26 ± 0.03 versus 0.13 ± 0.02 nmol/mg of protein), a more oxidized redox potential (-193 ± 5 versus -203 ± 2 mV) and more BPD (89% versus 24%). In logistic regression model, each 1% increase in FiO2 and each day increase in PN duration resulted in an increase in the OR for BPD by 1.57 (1.09 -2.28) and 1.17 (1.03 -1.33) respectively. CONCLUSION Early O2 supplement and PN have additive effects that were associated with prolonged oxidative stress and increased risk of BPD. Strategies targeting judicious use of O2 and decreasing the duration or developing a safer formulation of PN can be targeted to decrease BPD.

Adding glutathione to parenteral nutrition prevents alveolar loss in newborn Guinea pig

UNLABELLED Bronchopulmonary dysplasia, a main complication of prematurity, is characterized by an alveolar hypoplasia. Oxidative stress is suspected to be a trigger event in this population who has a low level of glutathione, a main endogenous antioxidant, and who receives high oxidative load, particularly ascorbylperoxide from their parenteral nutrition. HYPOTHESIS the addition of glutathione (GSSG) in parenteral nutrition improves detoxification of ascorbylperoxide by glutathione peroxidase and therefore prevents exaggerated apoptosis and loss of alveoli. METHODS Ascorbylperoxide is assessed as substrate for glutathione peroxidase in Michaelis-Menten kinetics. Three-days old guinea pig pups were divided in 6 groups to receive, through a catheter in jugular vein, the following solutions: 1) Sham (no infusion); 2) PN(-L): parenteral nutrition protected against light (low ascorbylperoxide); 3) PN(+L): PN without photo-protection (high ascorbylperoxide); 4) 180 μM ascorbylperoxide; 5) PN(+L)+10 μM GSSG; 6) ascorbylperoxyde+10 μM GSSG. After 4 days, lungs were sampled and prepared for histology and biochemical determinations. Data were analysed by ANOVA, p < 0.05 RESULTS: The Km of ascorbylperoxide for glutathione peroxidase was 126 ± 6 μM and Vmax was 38.4 ± 2.5 nmol/min/ U. The presence of GSSG in intravenous solution has prevented the high GSSG, oxidized redox potential of glutathione, activation of caspase-3 (apoptosis marker) and loss of alveoli induced by PN(+L) or ascorbylperoxide. CONCLUSION A correction of the low glutathione levels observed in newborn animal on parenteral nutrition, protects lungs from toxic effect of ascorbylperoxide. Premature infants having a low level of glutathione, this finding is of high importance because it provides hope in a possible prevention of bronchopulmonary dysplasia.

Inhibition of hepatic methionine adenosyltransferase by peroxides contaminating parenteral nutrition leads to a lower level of glutathione in newborn Guinea pigs

Premature newborn infants on total parenteral nutrition (TPN) are at risk of oxidative stress because of peroxides contaminating TPN and low glutathione level. Low cysteine availability limits glutathione synthesis. In this population, the main source of cysteine derives from the hepatic conversion of methionine. The first enzyme of this conversion, methionine adenosyltransferase (MAT), contains redox-sensitive cysteinyl residues. We hypothesize that inhibition of MAT by peroxides contaminating TPN leads to a lower availability of cysteine for glutathione synthesis. At 3 days of life, animals were fitted with a jugular catheter for intravenous infusion. Four groups were compared by ANOVA (P<0.05): (1) Control, without surgery, fed regular chow; (2) Sham, fitted with an obstructed catheter, fed orally regular chow; (3) TPN, fed exclusively TPN (dextrose, amino acids, fat, vitamins) containing 350 μM peroxides; (4) H2O2, fed regular chow orally and infused with 350 μM H2O2. Four days later, MAT activity and glutathione in liver and blood were lower in TPN and H2O2 groups. The redox potential was more oxidized in blood and liver of the TPN group. In conclusion, peroxides generated in TPN inhibit methionine adenosyltransferase activity with, among consequences, a low level of glutathione and a more oxidized redox potential.

Ascorbylperoxide from parenteral nutrition induces an increase of redox potential of glutathione and loss of alveoli in newborn guinea pig lungs

Background Bronchopulmonary dysplasia is one of the main complications associated with extreme prematurity. Oxidative stress is suspected to be a trigger event of this lung disease, which is characterized by impaired alveolar development. Peroxides, mainly ascorbylperoxide and H2O2, are known contaminant of parenteral nutrition. We hypothesize that these oxidant molecules induce bronchopulmonary dysplasia development. The aim was to determine if the infusion of ascorbylperoxide, whether in presence or absence of H2O2, is associated with oxidative stress, apoptosis and loss of alveoli in the lungs of newborn guinea pigs. Method Three-day-old guinea pigs received parenteral solutions containing 0, 20, 60 or 180 µM ascorbylperoxide in the presence or not of 350 µM H2O2 (concentrations similar to those measured in parenteral nutrition). After 4 days, the lungs were collected for determination of glutathiones redox potential, caspase-3 activation (an apoptosis marker), alveolarization index (by histology), activation of Nrf2 and NF?B (biological markers of oxidative stress), and IL-6 and PGJ2 levels (markers of NF?B activation). Groups were compared by ANOVA, p < 0.05. Results Loss of alveoli was associated with ascorbylperoxide in a dose-dependent manner, without an influence of H2O2. The dose-dependent activation of caspase-3 by ascorbylperoxide was lower in the presence of H2O2. Ascorbylperoxide induced an increase of redox potential in a dose-dependent manner, which reached a plateau in presence of H2O2. Nrf2 and NF?B were activated by H2O2 but not by ascorbylperoxide. Conclusion Results suggest that ascorbylperoxide, generated in parenteral nutrition, is involved in the development of bronchopulmonary dysplasia, independently of the increase of the redox potential. This study underlines the importance of developing a safer formulation of parenteral nutrition.

Ascorbylperoxide Contaminating Parenteral Nutrition Is Associated With Bronchopulmonary Dysplasia or Death in Extremely Preterm Infants.

Background: Ascorbylperoxide (AscOOH) is a hydrogen peroxide–dependent by-product of ascorbic acid that contaminates parenteral nutrition. In a guinea pig model, it caused oxidized redox potential, increased apoptosis, and decreased alveolarization. AscOOH detoxification is carried out by glutathione peroxidase (GPX). We hypothesize that extremely preterm infants have limited capacity for AscOOH detoxification. Our objective was to determine if there is an association between an early level of urinary AscOOH and later development of bronchopulmonary dysplasia (BPD) or death. Materials and Methods: This prospective cohort study included 51 infants at <29 weeks of gestation. Baseline clinical characteristics and clinical outcomes data were collected. Urine samples were collected on days 3, 5, and 7 of life for urinary AscOOH. Blood samples on day 7 were collected for total plasma glutathione, GPX, and glutathione reductase. &khgr;2, Student’s t test, Spearman correlation (r), linear regression (adjusted r2), and repeated-measure analysis of variance were used as appropriate. P < .05 was considered significant. Results: Urinary AscOOH increased over time (P = .001) and was higher in infants who later developed BPD or died (P = .037). Compared with adults and full-term infants, total plasma glutathione concentration was low (median, 1.02 µmol/L; 25th–75th percentiles, 0.49–1.76 µmol/L), whereas GPX and glutathione reductase activities were sufficient (3.98 ± 1.25 and 0.36 ± 0.01 nmol/min/mg of protein, respectively). Conclusion: Extremely preterm infants have low glutathione levels, which limit their capacity to detoxify AscOOH. Higher first-week urinary AscOOH levels are associated with an increased incidence of BPD or death.

Impact of glutathione supplementation of parenteral nutrition on hepatic methionine adenosyltransferase activity.

Background The oxidation of the methionine adenosyltransferase (MAT) by the combined impact of peroxides contaminating parenteral nutrition (PN) and oxidized redox potential of glutathione is suspected to explain its inhibition observed in animals. A modification of MAT activity is suspected to be at origin of the PN-associated liver disease as observed in newborns. We hypothesized that the correction of redox potential of glutathione by adding glutathione in PN protects the MAT activity. Aim To investigate whether the addition of glutathione to PN can reverse the inhibition of MAT observed in animal on PN. Methods Three days old guinea pigs received through a jugular vein catheter 2 series of solutions. First with methionine supplement, (1) Sham (no infusion); (2) PN: amino acids, dextrose, lipids and vitamins; (3) PN-GSSG: PN+10 μM GSSG. Second without methionine, (4) D: dextrose; (5) D+180 μM ascorbylperoxide; (6) D+350 μM H2O2. Four days later, liver was sampled for determination of redox potential of glutathione and MAT activity in the presence or absence of 1 mM DTT. Data were compared by ANOVA, p<0.05. Results MAT activity was 45±4% lower in animal infused with PN and 23±7% with peroxides generated in PN. The inhibition by peroxides was associated with oxidized redox potential and was reversible by DTT. Correction of redox potential (PN+GSSG) or DTT was without effect on the inhibition of MAT by PN. The slope of the linear relation between MAT activity and redox potential was two fold lower in animal infused with PN than in others groups. Conclusion The present study suggests that prevention of peroxide generation in PN and/or correction of the redox potential by adding glutathione in PN are not sufficient, at least in newborn guinea pigs, to restore normal MAT activity.

Total parenteral nutrition induces sustained hypomethylation of DNA in newborn guinea pigs

Background:Neonatal total parenteral nutrition (TPN) is associated with animals with low glucose tolerance, body weight, and physical activity at adulthood. The early life origin of adult metabolic perturbations suggests a reprogramming of metabolism following epigenetic modifications induced by a change in the pattern of DNA expression. We hypothesized that peroxides contaminating TPN inhibit the activity of DNA methyltransferase (DNMT), leading to a modified DNA methylation state.Methods:Three groups of 3-d-old guinea pigs with catheters in their jugular veins were compared: (i) control: enterally fed with regular chow; (ii) TPN: fed exclusively with TPN (dextrose, amino acids, lipids, multivitamins, contaminated with 350 ± 29 μmol/l peroxides); (iii) H2O2: control + 350 μmol/l H2O2 intravenously. After 4 d, infusions were stopped and animals enterally fed. Half the animals were killed immediately after treatments and half were killed 8 wk later (n = 4–6 per group) for hepatic determination of DNMT activities and of 5′-methyl-2′-deoxycytidine (5MedCyd) levels, a marker of DNA methylation.Results:At 1 wk, DNMT and 5MedCyd were lower in the TPN and H2O2 groups as compared with controls. At 9 wk, DNMT remained lower in the TPN group, whereas 5MedCyd was lower in the TPN and H2O2 groups.Conclusion:Administration of TPN or H2O2 early in life in guinea pigs induces a sustained hypomethylation of DNA following inhibition of DNMT activity.

Impact of SMOFLipid on Pulmonary Alveolar Development in Newborn Guinea Pigs

BACKGROUND Parenteral nutrition (PN) is associated with bronchopulmonary dysplasia in premature infants. In animals, PN leads to alveolar loss following stimulation of apoptosis by oxidative stress (oxidized redox potential). Peroxides and aldehydes generated in PN can induce hypo-alveolarization. The implication of peroxides, which is reduced by light protection, is demonstrated. The implication of aldehydes from omega-6 fatty acids oxidation is expected. The hypothesis is that composition and light exposure of PN influences bronchopulmonary dysplasia development. Since SMOFLipid (SMOF) contains a lower amount of omega-6 fatty acids than Intralipid (IL), the aim was to compare, the impacts of PN compounded with SMOF or IL, photo-protected or not, on alveolar development. MATERIALS AND METHODS Three-day-old Guinea pigs received PN, photo-protected or not, made with SMOF or IL through a jugular vein catheter. After 4 days, lungs were sampled for determinations of redox potential of glutathione, apoptosis (caspase-3, caspase-8, and caspase-9) and alveolarization index (histology: number of intercepts/mm). RESULTS Compared with IL, SMOF induces a greater oxidation of redox potential (-200 ± 1 versus [vs] -205 ± 1 mV), apoptosis (caspase-3: 0.27 ± 0.04 vs 0.16 ± 0.02; caspase-9: 0.47 ± 0.03 vs 0.30 ± 0.03), and a lower alveolarization index (27.2 ± 0.8 vs 30.0 ± 0.9). Photo-protection prevented activation of caspase-9 and was statistically without effect on redox potential, caspase-3, and alveolarization index. CONCLUSION In our model, SMOF is pro-oxidant and induces hypo-alveolarization following exaggerated apoptosis. These results highlight the need for further studies before introducing SMOFLipid in standard neonatal care.

PS-279 Prolonged Oxidative Stress And Increased Incidence Of Neonatal Morbidities After Early Postnatal Exposure To Oxidants In Infants Less Than 29 Weeks Gestational Age

Background The antioxidant defenses are poorly developed in preterm infants. Oxygen and parenteral nutrition (PN) which is contaminated with peroxides are two major sources of oxidants. Objective To assess the effect of early oxygen (on day 7 and 28) and the PN duration on oxidative stress markers at 36 weeks post menstrual age (PMA) and on the incidence of neonatal morbidities. Design/methods A prospective observational study including 120 infants less than 29 weeks gestational age without major congenital anomalies. Consent for blood sample at 36 weeks PMA was obtained for 51 infants. GSH and GSSG (nmol/mg protein) were measured by capillary electrophoresis and were used for redox potential (mV) calculation using Nernst equation, and expressed as mean (± sem). BPD was defined as the need of O2 supplement at 36 weeks PMA. ROP that required either laser or anti-VGF treatment and NEC grade 2 or higher according to Bell’s criteria were included. Student’s t test or Chi squared were used as appropriate, * = p < 0.05, **= p < 0.01. Results FiO2 ≥ 25% on day 7 and 28 of life and PN duration > 14 days resulted in higher GSSG concentration, more oxidised redox potential at 36 weeks PMA and increased the incidence of BPD, ROP and NEC Conclusions Early life exposure to oxidants is associated with prolonged oxidative stress and higher incidence of neonatal morbidities. These results suggest that strategies targeting judicious O2 use and either decreasing the duration or using safer formulation PN will help decreasing the incidence of BPD, ROP and NEC. Abstract PS-279 Table 1 GSH GSSG Redox potenial BPD or Death ROP NEC FiO2 < 25% on day 7 7.6 (0.5) 0.18 (0.02) -198 (2) 26/54 2/56 7/56 FiO2 ≥25% on day 7 7.4 (0.6) 0.29 (0.04) -191 (2) 46/50 6/50 17/50 P NS * * ** NS ** FiO2 < 25% on day 28 8.3 (0.8) 0.17 (0.02) -201 (4) 9/36 0/37 4/37 FiO2 ≥25% on day 28 7.3 (0.5) 0.26 (0.03) -193 (2) 55/60 8/60 17/60 P NS NS * ** * * PN ≤14 days 7.5 (1.2) 0.13 (0.02) -203 (5) 16/42 0/44 2/44 PN >14 days 7.5 (0.4) 0.26 (0.03) -193 (2) 58/64 8/65 22/65 P NS * * ** * **