Enteral L Citrulline Supplementation in Preterm Infants - Safety, Efficacy and Dosing

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in preterm infants
(PI). Preterm birth causes disruption in pulmonary vascular growth that leads to decreased
vascular surface area that increases pulmonary vascular resistance (PVR). Increased PVR leads
to altered vasoreactivity and structural remodeling with intimal hyperplasia and increased
muscularization of the small pulmonary arteries. There is no definite treatment for BPD_PH.

Nitric Oxide: Nitric Oxide (NO) is a potent pulmonary vasodilator. Endothelial Nitric oxide
synthase (eNOS) mediates production of NO from L-Arginine. L-citrulline is a precursor for
L-arginine. L-Arginine is a precursor of nitric oxide (NO). In infants with BPD_PH, there are
decreased levels of L-arginine & L-citrulline with decreased production of NO (measured by
urinary nitrates & nitrites) leading to increased PVR. Several studies have shown the benefit
of oral L-citrulline supplementation in increasing serum citrulline levels, increasing NO
production and reducing pulmonary hypertension. Oral L-arginine was not effective in
increasing NO production in previous studies and it was due to increased break down of oral
L-arginine by intestinal arginases.

Source of L-arginine in preterm infants: Routinely, extremely premature infants receive
nutrition as total parental nutrition (TPN i.e. infants get infusion of protein, fat and
carbohydrate via central venous line) that contains L-arginine (approximately 1mg/1mL) to
metabolize ammonia via urea cycle. PIs receive adequate amount of intra venous arginine from
TPN. Routinely, PIs are started with small volumes of enteral feeds which are increased
slowly overtime. TPN is slowly decreased as enteral feeds are increasing. As the TPN is going
down, intra venous L-arginine intake also drops down and ultimately when the PI are off TPN,
they don't get any IV supplemental L-arginine.

Why oral citrulline: Enteral feeds (formula as well as breast milk) is poor source of
arginine. Once PIs are on full enteral feed, an enteral feed is the only source of arginine.
Interestingly, 40% of enteral arginine gets metabolized by arginase enzyme present in
intestine. We speculate that plasma levels of arginine drop once TPN is discontinued and
infants are on full feeds. Oral L-arginine has poor bio-availability that is why oral
L-arginine supplementation does not increase blood levels of arginine. Since oral citrulline
has high bioavailability, the best way to increase serum arginine levels is by oral
citrulline supplementation. Oral supplementation of L-citrulline in preterm infants once they
are off TPN will likely to increase arginine levels and NO production.

Safety of oral citrulline: L-citrulline has been safely used for decades in patients with
urea cycle defects. It has been used in pediatric patients with sickle cell disease and in
infants undergoing cardiac surgery. No side effects were reported in these studies. In a
study in newborn rats exposed to hyperoxia, L-citrulline caused a marked increase in
arginase-2 expression in the lungs and this could have an impact on lung development and
remodeling. However, this is only a theoretical risk.

Inclusion Criteria:

- Infants less than or equal to 30 weeks' gestational age born at UTMB, Galveston.

- Parents have provided informed consent/assent in a manner that is approved by the IRB

Exclusion Criteria:

- Known congenital or chromosomal anomalies.

- Congenital heart disease affecting cardio-respiratory system (other than PDA, PFO or
ASD)

- Necrotizing enterocolitis, sepsis, or any condition requiring surgery prior to
recruitment