# Presentation: 910494

## 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**

## Overview 

** ****summary of experience to date**
** ****protocol specifics**

**Overview **

## 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**

## Natural isotopic abundances 

## Palmitate

** ****1.1% chance for each carbon to be ****13****C**

** ****x 16 carbons**

**→ ****17.6 % of all palmitate has at least one ****13****C **

** ****ubiquitous, abundant fatty acid**

** ****60% of the fatty acids in surfactant**

## Plasma

**Surfactant**

**palmitate**

**acetyl-CoA**

**acetate**

**Plasma**

**Type II Alveolar cell**

**Cellular lipids**

**glucose, fatty acids, **
**amino acids**

**palmitate**

***de novo***** palmitate**

## PC synthesis from labeled acetate or palmitate

**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**

**C**

**C**

**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**

**=**

**O**

**O**

**P**

**=**

**O**

**=**

**O**

## Methods

**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 tracheal aspirate samples in conjunction with routine airway suctioning, otherwise discarded (2-4x daily for up to 2 weeks) **
**Surfactant phospholipid extracted **
**Enrichment of ****13****C measured with mass spectrometry**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Incorporation of [1-13C1]acetate 

**into surfactant**

*Bohlin K, et al. Pediatr Res. 2003; 54:185*

## Conclusions

**Conclusions**

** ****surfactant synthesis is slower in preterm and term newborns with RDS**

## Safety monitoring

**(2001-2005)**

|  | Study group
(n=53) | Comparison  group (n=57) |
| --- | --- | --- |
| Electrolyte disturbance |  |  |
| Bloodstream infection within 1 mo |  |  |
| Death within 1 month
 |  |  |

## 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
 |

## Summary to date

** ****no increased risk of electrolyte imbalance, infection, or death from participation in study **

**Summary to date**

##    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**.

## 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**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Inclusion criteria

**Preterm infants with RDS**
**Mechanical ventilation anticipated for 5 days**
**Intravenous line in place **
**Blood drawing for clinical purposes at least twice a day  **
**Clinical care team assent**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Exclusion criteria

**Need for escalating intervention**
**Anticipated death**
**Active infection **
**Chromosomal abnormalities or multiple congenital anomalies**
**Fluid sensitivity / electrolyte imbalance**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Consent

**Family approached after discussion with clinical care team**
**Study described**
**Family given consent form and pamphlet**
**Revisited 24 hours later**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Tracer preparation

**Must be prepared extemporaneously**
**Commercially available powder - clinical grade **
**stored under vacuum in dessicator**
**Vehicle: albumin / 5% glucose water (D5W)**
**Sterile 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 infusates**
**24 hour hang-time **
**Infusate saved and frozen at conclusion of infusion**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Testing preparation procedure

**Every 3 months: **
**culture in microbiology lab**
**With each new bottle of isotope: **
**particulate matter and pyrogenicity**
**culture**
**Annually: **
**assay for constituent concentration**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Infusion details

**Does not interfere with other intravenous solutions**
**No disruption in fluid, electrolyte, or nutrient intake. **

|  | Infusion
(/kg/24h)  | Usual intake
(/kg/d) |
| --- | --- | --- |
| Sodium (mmol) | 3.6 | 3-5  |
| Acetate (mmol) | 3.6 | 1-8 |
| Potassium | 58 μmol | 2-4 mmol |
| Palmitate (μmol) | 58 | ~1500 |
| Albumin (g) | 1 | 0-2 |
| Fluid (ml) | 24 | 100-150 |

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## New surfactant synthesis in ventilated preterm newborns 

*(Spence et al, J Pediatr, in press)*

**Surfactant turnover increases in evolving chronic lung disease **
**Plasma derived palmitate is the predominant precursor for surfactant synthesis**
**Proportion of new synthesis coming from sources other than acetate / palmitate decreases  (recycling?)**

**birth**

**2 wks**

**4 wks**

**Gestational age at birth: 26 **_**+**_** 2 wks**

## Plasma

**Surfactant**

**palmitate**

**acetyl-CoA**

**13****C-acetate**

**Plasma**

**Type II Alveolar cell**

**Cellular lipids**

**glucose, fatty acids, **
**amino acids**

**13****C****4****-palmitate**

***de novo***** palmitate**

## Plasma

**Surfactant**

**palmitate**

**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**

## New surfactant synthesis in ventilated preterm newborns 

**Are these changes a function of development, disease, or both? **

_**Recycling estimates - animals **_
**Term rabbits – 90%**
**Adult rabbits – 50%**
**Lambs with RDS – 30-40% **

## Hypothesis

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

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Comparison group

**Ideal: **
**gestational and chronological age matched **
**“****normal lungs” **
**require mechanical ventilation**

**extremely rare**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## 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**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Chest X-ray of RDS

**	**

**Chest X-ray of RDS**

**Endotracheal tube**

**Hazy lungs**

## Chest X-ray of gastroschisis

**Case 3**

**Chest X-ray of gastroschisis**

**Endotracheal tube**

**Clear lungs**

**Intestines**

**Umbilical artery **
**catheter**

## Interventions specific to the research protocol

**Stable isotope infusion**
**up to 2.5 ml of additional blood drawn  **

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## 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**
**	**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Benefits

**Information about “normal” surfactant metabolism in newborns**
**Context for interpreting data from preterm newborns with evolving chronic lung disease**
**Establish 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”**

**Notes:**
Stable isotope methodology relies on the administration of naturally occurring, non-radioactive, stable isotopes of carbon (13C). 

For the study I’m presenting, we used glucose uniformly labeled with carbon 13 as a metabolic precursors of surfactant phospholipids synthesis and than we measured their appearance in tracheal effluent with gas chromatography-combustion interface isotope ratio mass spectrometry (GC-IRMS)

## Slide 34

## Slide 35

## Surfactant phospholipid synthesis

**DPPC**

**glucose**

**PC**

## Natural abundance isotopomer distribution

- We can calculate the isotopomer distribution for any molecular formula taking into account natural abundance of 13C, 2H, 17O, 18O, etc.:
- methyl palmitate C17H34O2

## Surfactant metabolism in evolving chronic lung disease

| Population | FSRacetate
%/day | FSRpalmitate
%/day | P-value* | FCR acetate
     %/day |
| --- | --- | --- | --- | --- |
| NEWBORN
   (n=16) | 4.05+2.1 | 7.6+4.6 | 0.006 | 25.3+7.0 |
| INTERMEDIATE
   (n=7) | 8.0+5.3 | 21.3+15.7 | 0.02 | 43.6+14.0 |
| BPD
   (n=9) | 14.9+6.8 | 30.5+16.3 | 0.01 | 53.8+14.4 |
| P-values (ANOVA between groups) | <0.001 | <0.001 | N/A | 0.001 |

## 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**