Cysteine Supplementation in Critically Ill Neonates

Specific Aims:

Critically ill neonates have demonstrated low concentrations of the antioxidant glutathione and a high incidence of disease associated with oxidative injury compared to healthy neonates. Cysteine is considered to be a conditionally essential amino acid for neonates and is the rate limiting substrate for the synthesis of glutathione. We hypothesize that parenterally-fed, critically ill neonates administered cysteine supplementation will have higher concentrations of total glutathione, lower ratios of oxidized to reduced glutathione, higher glutathione synthetic rates, lower levels of inflammatory cytokine production and lipid peroxidation, and decreased severity of disease associated with oxidative injury compared to similarly ill parenterally fed neonates without cysteine supplementation. To test this hypothesis, critically ill, parenterally fed neonates assigned randomly to receive a cysteine or an isonitrogenous cysteine-free supplement to their TPN (total parenteral nutrition) regimen will be prospectively studied in a double-blind fashion according to the following specific aims:

1. to measure total concentrations of erythrocyte glutathione, oxidized to reduced (GSSG:GSH) erythrocyte glutathione ratios, and the in vivo fractional and absolute synthetic rates of erythrocyte glutathione utilizing a [13C]-glycine tracer,
2. to measure plasma interleukin-6 (IL-6), tumor necrosis factor (TNF-a), and malondialdehyde concentrations as determinants of illness severity and degree of oxidative injury reflected by lipid peroxidation,
3. and, to measure duration of mechanical ventilation, duration of supplemental oxygen, and duration of hospitalization as the primary clinical outcomes of disease severity.

By demonstrating whether cysteine supplementation increases the synthesis and concentration of glutathione along with the subsequent decrease in oxidative injury and associated disease, the widespread morbidity and mortality for vulnerable, critically ill neonates may be improved.

Background:

Critically ill neonates treated in the Neonatal Intensive Care Unit (NICU), whether premature or full term, experience a high incidence of diseases that may be secondary to or exacerbated by oxidative injury. These include a variety of multi-factorial disease processes such as respiratory distress syndrome (RDS), persistent pulmonary hypertension (PPHN), chronic lung disease (CLD), sepsis, meconium aspiration, retinopathy of prematurity (ROP), and necrotizing enterocolitis (NEC). Within NICUs across the U.S. per year, Neonatologists and Pediatric Surgeons care for an estimated 100,000 critically ill neonates, of which approximately 35% are full-term and up to 20% will ultimately expire of their illnesses despite major advances in neonatal care. Some neonatal diseases are well known to have a varying prevalence according to neonatal maturity. Premature neonates more commonly suffer from RDS, CLD, ROP and NEC, with the most susceptible being the extremely premature. However, 10-15% of neonates who develop NEC are full-term and have a resultant mortality rate of up to 35%. Furthermore, other disease processes causing significant respiratory distress (e.g., PPHN, pneumonia, and meconium aspiration) more commonly occur in full-term neonates.

The high morbidity of these diseases has a significant impact for the neonate, their parents, and society in general. Neonatal survivors of critical illness who are without obvious neurologic injury have been found later to have lower IQ's, increased behavioral problems, decreased ability to perform normal activities of daily living, decreased scholastic abilities, and decreased health-related quality of life compared to healthy children. The economic burden to the parents and society from the care of these neonatal illnesses can be astounding. For example, the economic costs of neonatal healthcare alone can increase 32-71% when a critically ill neonate has NEC compared to appropriately matched controls without NEC. Adjusted for inflation to the year 2005, an additional expense of approximately $281,666 is expected for every NEC survivor prior to discharge from the NICU (equivalent to $8.45 million per year for a single large healthcare center). Similarly, the continued healthcare costs of critically ill neonates even after discharge can be insurmountable. Children who were survivors of a critical illness as a premature neonate have been shown to require increased utilization of Pediatric Intensive Care Unit (PICU) resources (e.g., mechanical ventilation), increased length of ICU care, and more frequent readmission to the PICU compared to other PICU patients.

Although cysteine is a non-essential amino acid made from methionine via cystathionine in children and adults, most premature and term neonates have a decreased capacity to synthesize cysteine due to their low expression of the rate limiting enzyme cystathionase. As a result, plasma cysteine and cystine concentrations in critically ill neonates are low compared to healthy infants. Unfortunately, plasma cysteine concentrations of neonates receiving cysteine-free TPN remain low despite generous methionine intakes and relatively high plasma methionine concentrations in TPN fed neonates. This is particularly relevant since cysteine is the limiting substrate for the synthesis of the principal intracellular antioxidant in the body - glutathione.

Intracellular glutathione serves many roles in the cell including protection against oxidative damage caused by free radicals through glutathione peroxidase and detoxification of electrophilic metabolites through multiple glutathione transferases. Thereby, the level of glutathione in a specific organ or tissue is thought to be a major determinant of the vulnerability of that organ or tissue to oxidative stress. Although the majority of whole body glutathione is produced in hepatocytes, most cells, including the erythrocyte, have the equivalent capacity to synthesize glutathione de novo from the 3 amino acids cysteine, glutamate, and glycine utilizing (-glutamylcysteine and glutathione synthetases. The active form of glutathione (reduced or GSH) is also regenerated from recycling of oxidized glutathione (glutathione disulfide or GSSG) through glutathione reductase. However, when the use of reduced glutathione exceeds the capacity of regeneration, the GSSG is expelled from the cell and intracellular glutathione stores are depleted.

Regardless of the degree of stress or neonatal weight, critically ill neonates have activity levels of these glutathione synthetic enzymes comparable to those of adults. Furthermore, these neonates demonstrate unimpeded ability to transport amino acids, including cysteine derivatives, across cellular membranes. Hence, neonates demonstrate the capacity to synthesize glutathione given the appropriate substrates are available. Despite the potential ability to synthesize glutathione, low glutathione concentrations and high ratios of GSSG:GSH have been demonstrated from the blood, lung, and intestine of critically ill neonates in multiple studies. Consequently, a likely limiting factor in the production of glutathione for critically ill neonates is the availability of cysteine in their diet.

Most critically ill neonates receive their nutrition solely through the parenteral route during the acute duration of their illness, from a few days to weeks of post-natal life. Unfortunately, there is no parenteral amino acid formulation commercially available in the U.S. which includes cysteine as a significant nutrient. This is because cysteine is relatively unstable and cystine (i.e., cysteine dimer) is insoluble in aqueous solution. However, cysteine supplementation to TPN is possible in the form of cysteine-HCl if given within 24 hours prior to infusion. Subsequent provision of cysteine in this manner results in higher plasma cysteine concentrations in critically ill neonates. Although cysteine-HCl supplementation to TPN has been shown to be possible and safe, it is not routine practice in current neonatal care. Nevertheless, cysteine-HCl supplementation is standard practice in some major neonatal centers in the U.S. in order to augment the calcium and phosphorus solubility in TPN, which further benefits growing infants.

Several in vitro and animal studies demonstrate cysteine supplementation increases glutathione concentrations in the blood, liver, and intestines. Cysteine supplementation to premature neonatal erythrocytes in cell cultures have demonstrated increased glutathione concentrations compared to non-cysteine supplemented controls. Malloy et. al. have shown cysteine-HCl supplemented TPN-fed beagle pups have higher concentrations of plasma cysteine and hepatic glutathione than unsupplemented TPN-fed pups. Similarly, Pollack et. al. have demonstrated intestinal glutathione concentrations significantly increase in premature newborn rabbits administered cysteine-HCl supplementation compared to non-supplemented controls. Moreover, the colonic glutathione concentrations were found to be similar to those of maternally reared, term newborn rabbits.

As a clinical corollary, human studies in stressed adults with acute respiratory distress syndrome have demonstrated elevated concentrations of glutathione and improvements in recovery after cysteine supplementation. Septic and malnourished children supplemented with enteral cysteine have demonstrated increased glutathione concentrations and in vivo glutathione synthetic rates measured utilizing a novel non-radioactive, stable isotope methodology. Furthermore, the septic children supplemented with cysteine resolved their illnesses quicker than their unsupplemented counterparts. These studies suggest that cysteine-HCl supplementation may improve glutathione production and decrease oxidative stress in critically ill neonates.

There has yet to be an investigation confirming that improved glutathione concentrations and in vivo glutathione synthetic rates occur in critically ill neonates supplemented with cysteine-HCl and that these perturbations lead to a decrease in the amount of oxidative tissue injury that these patients endure. This gap in studies serves as a perfect setting for our proposed randomized, blinded, placebo-controlled trial that is adequately powered to detect a pre-determined difference between the two groups.