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1H-NMR Identifies Unique Metabolic Signature of Prematurity

Confounding Factors Critical When Comparing Newborn Urine Metabolomes

Premature birth is linked to a host of challenges for a child. According to the World Health Organization, complications of preterm birth are the leading cause of death for children under age five and lead to a million deaths worldwide each year. In all, about 15 million babies a year are born three or more weeks early.

The statistics underscore the gravity of the situation and the importance of researching prematurity. Although blood tests have gone a long way toward providing key information about newborns, testing urine would make it easier to gather samples and, ideally, increase testing. Ultimately, better information might indicate which interventions would most improve outcomes. 1H-NMR is well-suited to analyzing metabolomes and providing actionable intelligence.

A group of researchers from Portugal set out to answer a number of questions: What does the metabolome of a newborn tell us about prematurity? What confounding factors distort results? Is it possible to find a specific metabolic “signature” in urine collected from premature babies that is different from that of babies born with other issues?

Answers to these questions appear in, Diaz, Sílvia O., et al. “Newborn Urinary Metabolic Signatures of Prematurity and Other Disorders: A Case Control Study,” published January 2016 in the Journal of Proteome Research.

Confounding variables

Scientists from two universities in Portugal, the University of Aveiro and the University of Coimbra, assessed information from medical records and questionnaires on 46 babies in the control group. All were healthy, full-term babies born to healthy mothers after normal pregnancies. The researchers considered a number of confounding variables, including gender, delivery mode (Caesarian vs. vaginal) gestational age and what day of life the urine was collected.

After preparing the samples, they researchers recorded the spectra on a Bruker Avance DRX 500 spectrometer at 300 K with the help of Bruker BioSpin Germany. Peak assignments were carried out using the Bruker Bbiorefcode spectral database and the human metabolome database, HMDB33.

The researchers measured 56 metabolites including, for the first time, phenylacetylglutamine (PAG) and indoxyl sulfate (IS) in newborn urine.

The results were clear: Gender and method of birth significantly influence urine composition, and the metabolic differences were easy to see in the 1H-NMR spectra. The urine of newborn, healthy control group females had higher levels of fumarate, glucose, lactose, taurine and threonine than males. It had lower amounts of acetone, creatine and formate than males.

The researchers went on to evaluate the spectra to see if there was any impact on the urine metabolome linked to the method of delivery. They noted distinct differences. Babies born via Caesarean section had increases in 1-methylhistidine, 2-KG, acetone, betaine, DMA, ethanolamine, lactose, and decreases in 4-deoxyerythronic acid (4-DEA), 4-hydroxyphenylacetate (4- HPA), cis-aconitate, IS, myo-inositol, PAG, trigonelline and tyrosine.

The researchers did not detect differences in urine composition related to gestational age or day of sample collection.

The results demonstrate that gender and delivery method should be considered in newborn research when comparing control groups to disease groups.

Premature birth or respiratory depression?

Next, the researchers evaluated the metabolic signatures of urine samples from premature newborns and newborns with other disorders to see if they could spot distinct spectra specific to premature birth. They evaluated urine from several groups of newborns: late premature newborns (birth ranging from 33 to 36 gestational weeks), newborns who experienced respiratory depression, newborns whose birth weight was above the 90th percentile and newborns with malformations.

The researchers identified clear differences in the spectra between groups. They identified what they term a specific “metabolic signature of prematurity” that includes 25 known metabolites and several at unassigned resonances. These metabolic changes indicate disturbances in nucleotide metabolism, lung surfactants biosynthesis, and renal function, along with enhancement of tricarboxylic acid cycle activity, fatty acids oxidation and oxidative stress.

In addition to identifying a metabolic signature of prematurity, the researchers also found distinct signature changes to the spectra for each group of newborns, those with respiratory depression, those who were large for their gestational age and those with malformations.

This research proves that 1H-NMR of newborn urine can accurately and precisely distinguish different conditions, which may prove useful in understanding the health of newborns. Future research might help assess the consequences of prematurity in specific cases or provide the basis for diagnostic or prognostic markers.

For more information:

World Health Organization Preterm Fact Sheet, November 2015 http://www.who.int/mediacentre/factsheets/fs363/en/

Mussap, M.; Antonucci, R.; Noto, A.; Fanos, V. The role of metabolomics in neonatal and pediatric laboratory medicine. Clin. Chim. Acta 2013, 426, 127−138.

The Human Metabolome Database (HMDB) is a freely available electronic database containing detailed information about small molecule metabolites found in the human body. http://www.hmdb.ca

Lamarre, S. G.; Morrow, G.; et al. Formate: an essential metabolite, a biomarker, or more? Clin. Chem. Lab. Med. 2013, 51, 571−578.  DOI: 10.1515/cclm-2012-0552

Diaz, S. O.; Barros, A. S.; et al. Following healthy pregnancy by nuclear magnetic resonance (NMR) metabolic profiling of human urine. J. Proteome Res. 2013, 12, 969− 979. DOI: 10.1021/pr301022e