1. Precursor preference in surfactant synthesis of newborns Aaron Hamvas, MD Kimberly Spence, MD Division of Newborn Medicine Washington University / St. Louis Children’s Hospital St. Louis, MO

2. stable isotope methodology summary of experience to date protocol specifics Overview

3. Stable isotope tracers Tracer - a substance added to a system to interrogate one or more metabolic pathways - indistinguishable from the naturally occurring molecule of interest Stable isotopes - non-radioactive (one extra neutron) - mass difference detectable with mass spectrometry - naturally occurring - safe - used extensively to study carbohydrate, protein, fat, and energy metabolism in infants, children and adults

4. Natural isotopic abundances

5. Palmitate O-O-O-O-C C C C C C C C C C C CC CC C =O 1.1% chance for each carbon to be 13 C 1.1% chance for each carbon to be 13 C x 16 carbons x 16 carbons → 17.6 % of all palmitate has at least one 13 C ubiquitous, abundant fatty acid ubiquitous, abundant fatty acid 60% of the fatty acids in surfactant 60% of the fatty acids in surfactant

6. Surfactantpalmitate acetyl-CoA acetate Plasma Type II Alveolar cell Cellular lipids glucose, fatty acids, amino acids palmitate de novo palmitate 13 C 4 -palmitate 13 C 1 -acetate

7. = O-O-O-O- O C-C * [1- 13 C]acetate C PC synthesis from labeled acetate or palmitate C DPPC C-C N(CH 3 ) 3 O C C C C C C C C C C C C C C C C C C C CC O C C C C C C C C C C C C C C C C C C C C C C C C O C = OOP = O = O * (*)(*) * O-O-O-O- C C C C C C C C C C C C C C C C = O (*)(*) labeled palmitate [m+1 (m+2)] [m+1 (m+2)] * * * * O-O-O-O- C C C C C C C C C C C C C C C C = O [1,2,3,4- 13 C 4 ] palmitate * * * * [m+4]

8. Methods 24-hour tracer infusion:24-hour tracer infusion: –[1- 13 C 1 ] acetate –[1,2,3,4- 13 C 4 ] palmitate Sequential blood samples in conjunction with clinically indicated blood sampling (up to 2.5 ml in 5 aliquots over 27 hours)Sequential blood samples in conjunction with clinically indicated blood sampling (up to 2.5 ml in 5 aliquots over 27 hours) Sequential tracheal aspirate samples in conjunction with routine airway suctioning, otherwise discarded (2-4x daily for up to 2 weeks)Sequential tracheal aspirate samples in conjunction with routine airway suctioning, otherwise discarded (2-4x daily for up to 2 weeks) Surfactant phospholipid extractedSurfactant phospholipid extracted Enrichment of 13 C measured with mass spectrometryEnrichment of 13 C measured with mass spectrometry

9. Incorporation of [1- 13 C 1 ]acetate into surfactant Bohlin K, et al. Pediatr Res. 2003; 54:185 FCR = 76+21% FCR = 18+6% Enrichment (%) 18 19 20 21 23 22

10. Conclusions surfactant synthesis is slower in preterm and term newborns with RDS surfactant synthesis is slower in preterm and term newborns with RDS

11. Safety monitoring (2001-2005) Study group (n=53) Comparison group (n=57) Electrolyte disturbance Bloodstream infection within 1 mo Death within 1 month

12. Safety monitoring (2001-2005) Study group (n=53) Comparison group (n=57) P-value Electrolyte disturbance 11 (21%)24 (42%)0.02 Bloodstream infection within 1 mo 11 (21%)19 (33%)0.14 Death within 1 month 2 (4%)8 (14%)0.06

13. no SAEs identified no SAEs identified no increased risk of electrolyte imbalance, infection, or death from participation in study no increased risk of electrolyte imbalance, infection, or death from participation in study Summary to date

14. Background Chronic lung disease continues to be a significant cause of morbidity and mortality in premature infants. Surfactant composition and function are abnormal Nothing is known about surfactant metabolism in premature newborns who develop chronic lung disease. Pigs fed diets low in palmitate had lower levels of surfactant DPPC and lung compliance.

15. Objective To determine the rate of and contribution to surfactant production from palmitate and from acetate in preterm infants < 28 weeks gestational age at: - birth - 2 weeks - 4 weeks

16. Inclusion criteria Preterm infants with RDSPreterm infants with RDS Mechanical ventilation anticipated for 5 daysMechanical ventilation anticipated for 5 days Intravenous line in placeIntravenous line in place Blood drawing for clinical purposes at least twice a dayBlood drawing for clinical purposes at least twice a day Clinical care team assentClinical care team assent

17. Exclusion criteria Need for escalating interventionNeed for escalating intervention Anticipated deathAnticipated death Active infectionActive infection Chromosomal abnormalities or multiple congenital anomaliesChromosomal abnormalities or multiple congenital anomalies Fluid sensitivity / electrolyte imbalanceFluid sensitivity / electrolyte imbalance

18. Consent Family approached after discussion with clinical care teamFamily approached after discussion with clinical care team Study describedStudy described Family given consent form and pamphletFamily given consent form and pamphlet Revisited 24 hours laterRevisited 24 hours later

19. Tracer preparation Must be prepared extemporaneouslyMust be prepared extemporaneously Commercially available powder - clinical gradeCommercially available powder - clinical grade –stored under vacuum in dessicator Vehicle: albumin / 5% glucose water (D5W)Vehicle: albumin / 5% glucose water (D5W) Sterile preparation PharmD in the clinical pharmacySterile preparation PharmD in the clinical pharmacy –laminar flow hood –weigh isotope powder –dissolve in warm D5W –filter through 0.22 micron filter into albumin / syringe Handled and administered like other infusatesHandled and administered like other infusates –24 hour hang-time Infusate saved and frozen at conclusion of infusionInfusate saved and frozen at conclusion of infusion

20. Testing preparation procedure Every 3 months:Every 3 months: –culture in microbiology lab With each new bottle of isotope:With each new bottle of isotope: –particulate matter and pyrogenicity –culture Annually:Annually: –assay for constituent concentration

21. Infusion details Does not interfere with other intravenous solutionsDoes not interfere with other intravenous solutions No disruption in fluid, electrolyte, or nutrient intake.No disruption in fluid, electrolyte, or nutrient intake. Infusion (/kg/24h) Usual intake (/kg/d) Sodium (mmol)3.63-5 Acetate (mmol)3.61-8 Potassium58 μmol2-4 mmol Palmitate (μmol)58~1500 Albumin (g)10-2 Fluid (ml)24100-150

22. New surfactant synthesis in ventilated preterm newborns (Spence et al, J Pediatr, in press) Surfactant turnover increases in evolving chronic lung diseaseSurfactant turnover increases in evolving chronic lung disease Plasma derived palmitate is the predominant precursor for surfactant synthesisPlasma derived palmitate is the predominant precursor for surfactant synthesis Proportion of new synthesis coming from sources other than acetate / palmitate decreases (recycling?)Proportion of new synthesis coming from sources other than acetate / palmitate decreases (recycling?) birth2 wks4 wks Gestational age at birth: 26 + 2 wks

23. Surfactantpalmitate acetyl-CoA 13 C-acetate Plasma Type II Alveolar cell Cellular lipids glucose, fatty acids, amino acids 13 C 4 -palmitate de novo palmitate

24. Surfactantpalmitate acetyl-CoA 13 C-acetate Plasma Type II Alveolar cell Cellular lipids glucose, fatty acids, amino acids 13 C 4 -palmitate de novo palmitate recycling

25. New surfactant synthesis in ventilated preterm newborns Are these changes a function of development, disease, or both?Are these changes a function of development, disease, or both? Recycling estimates - animals Term rabbits – 90%Term rabbits – 90% Adult rabbits – 50%Adult rabbits – 50% Lambs with RDS – 30-40%Lambs with RDS – 30-40%

26. Hypothesis The decrease in surfactant recycling over time is associated with the evolution of chronic lung disease rather than normal developmentin premature infants. The decrease in surfactant recycling over time is associated with the evolution of chronic lung disease rather than normal development in premature infants.

27. Comparison group Ideal: –gestational and chronological age matched –“normal lungs” –require mechanical ventilation extremely rare

28. Comparison group Realistic: -condition that requires intensive care with mechanical ventilation and intravascular catheters as part of routine clinical care -normal chest X-rays and gas exchange Near-term or term infants abdominal wall defects, neurological defect, craniofacial abnormalities, congenital heart disease

29. Chest X-ray of RDS Endotracheal tube Hazy lungs

30. Case 3 Chest X-ray of gastroschisis Endotracheal tube Clear lungs Intestines Umbilical artery catheter

31. Interventions specific to the research protocol Stable isotope infusionStable isotope infusion up to 2.5 ml of additional blood drawnup to 2.5 ml of additional blood drawn

32. Risks From standard clinical care: –blood drawing, airway manipulations, infection, –adverse outcome from underlying condition From research protocol: –2.5 ml additional blood (coordinated with clinical samples) –infection –transient electrolyte imbalance

33. Benefits Information about “normal” surfactant metabolism in newbornsInformation about “normal” surfactant metabolism in newborns Context for interpreting data from preterm newborns with evolving chronic lung diseaseContext for interpreting data from preterm newborns with evolving chronic lung disease Establish new paradigms in thinking about the role of surfactant in newborn lung diseaseEstablish new paradigms in thinking about the role of surfactant in newborn lung disease “opportunity to understand, prevent, or alleviate a serious problem affecting the health or welfare of children”

36. Surfactant phospholipid synthesis DPPC glucose acetate fatty acid PCPSPE choline PG PI CDP-diacylglycerol glycerol phosphatidic acid

37. Natural abundance isotopomer distribution We can calculate the isotopomer distribution for any molecular formula taking into account natural abundance of 13 C, 2 H, 17 O, 18 O, etc.: methyl palmitate C 17 H 34 O 2 0.0001 274 (m+4) 0.00170.0014273 (m+3) 0.02110.0174272 (m+2) 0.18970.1564271 (m+1) 1.00000.8246270 (m+0) Relative abundance Fractional abundance Mass

38. Surfactant metabolism in evolving chronic lung disease PopulationFSR acetate %/day FSR palmitate %/day P-value*FCR acetate %/day NEWBORN (n=16) 4.05+2.17.6+4.60.00625.3+7.0 INTERMEDIATE (n=7) 8.0+5.321.3+15.70.0243.6+14.0 BPD (n=9) 14.9+6.830.5+16.30.0153.8+14.4 P-values (ANOVA between groups) <0.001 N/A0.001 FSR increases with time FSR palmitate > FSR acetate FCR increases With time

39. Stable isotope enrichment in surfactant m+1 (from [1- 13 C 1 ]acetate) m+4 (from [1,2,3,4-13C4]palmitate m+4 (from [1,2,3,4- 13 C 4 ]palmitate) FSR = upslope/precursor enrichment (% per day) FCR = downslope (% of the pool per day) FSR/FCR = contribution of individual substrate to total surfactant production