Trial Information

Effect of Cysteine Supplementation on Glutathione Production 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.