Biochemical mechanisms involved in pulmonary hypo-alveolarization induced by peroxides contaminating parenteral nutrition in newborn guinea pig

Bronchopulmonary dysplasia (BPD) is a major complication of preterm newborns, affecting nearly 50% of infants born before 29 weeks of gestation. BPD is characterized by an arrest in alveolar development. The onset of BPD is related to oxidative stress. Research has shown that parenteral nutrition (PN), which is given to preterm newborns to bypass an immature gastrointestinal system, is a major source of oxidative stress. Indeed, PN is contaminated with peroxides, including ascorbylperoxide, an oxidized form of dehydroascorbic acid. Ambient light is a catalyst for the generation of peroxides. Photo-protection of PN, although difficult to apply in the clinical situation, is associated with a lower incidence of BPD in premature infants and with better alveolar outcomes in animal models of neonatal PN. We hypothesized that the ascorbylperoxide in PN disrupts alveolar development. The main mechanism of action is an inhibition of the transformation of methionine into cysteine in the liver, leading to a lower glutathione synthesis in the liver as well as in peripheral tissues such as lung. Lower glutathione (GSH) concentrations favour a shift of redox potential to a more oxidized state and consequently, to exaggerated apoptosis. If our hypothesis is correct, the addition of glutathione to PN would help detoxify ascorbylperoxide through the action of glutathione peroxidase and prevent the deleterious impact of PN. Objectives: The aims of my research project were to investigate the biochemical mechanisms linking PN to the development of BPD in premature newborns and to propose a nutritional alternative that would prevent the occurrence of this frequently observed complication. Specific objectives were: 1) to assess the effect of intravenously infused ascorbylperoxide on the metabolic axis redox potential of glutathione in the lung; specifically, apoptosis and the alveolarization index; 2) to study the impact of ascorbylperoxide and the redox potential on the activity of methionine adenosyltransferase (MAT) in the liver; methionine adenosyltransferase is the first enzyme in the metabolic cascade from methionine to cysteine; and 3) to try to prevent the deleterious impact of PN or ascorbylperoxide infusions on the lung by improving glutathione status.  Methods: Through a catheter in the jugular vein, 3-day-old guinea pigs (n = 8 per group) received continuous infusions of PN or a simple solution (dextrose + NaCl) enriched with different molecules for testing. The first objective was achieved by enriching the basic solution with ascorbylperoxide at concentrations of 0, 20, 60 and 180 M. To mimic clinical conditions, these solutions contained, or not, 350 M H2O2. The second objective was achieved by investigating the mechanisms of MAT inhibition in animals infused, or not, with solutions consisting of the basic solution, peroxides, glutathione, and PN (dextrose + amino acids + multivitamins + lipids). The third objective was achieved by adding, or not, 10 M of glutathione (GSSG) to the ascorbylperoxide or PN solution until a normal plasma concentration of glutathione was obtained. After 4 days, the lungs were removed. GSH and GSSG levels in the lungs were determined by capillary electrophoresis. The redox potential was calculated using the Nernst equation. The activation and the concentration of active caspase-3 (marker of apoptosis) were determined by Western blot, and the alveolarization index quantified by the number of intercepts between histological structures and a calibrated straight line. Data were compared by ANOVA; effects were considered significant if p was less than 0.05. Results: The infusion of ascorbylperoxide, independently of H2O2, induced hypoalveolarization, activation of caspase-3, and oxidation of the redox potential, in a dose-dependent manner. These effects were prevented by the addition of GSSG to the ascorbylperoxide (180 M) or PN solutions. Ascorbylperoxide and H2O2 inhibited MAT activity in the liver. Hepatic MAT activity was linearly modulated by the value of the redox potential. Conclusion: Our results suggest that ascorbylperoxide is the active ingredient in PN that leads to the development of BPD. Correcting the low glutathione levels induced by peroxides in PN solutions would promote the detoxification of peroxides and re-establish proper pulmonary redox potentials. Glutathione correction further protects the lungs from the deleterious effects of PN by bypassing hepatic MAT inhibition. This result is of great importance because it gives hope for the possible prevention of BPD.

Keyphrases: Bronchopulmonary dysplasia, Preterm newborns, Oxidative stress, Alveolarization, Parenteral nutrition and ascorbylperoxide