Impact of Prenatal Disorders on the Metabolic Profile of Second Trimester Amniotic Fluid: A Nuclear Magnetic Resonance Metabonomic Study Gonc¸alo Grac¸a,† Iola F. Duarte,† Anto ´ nio S. Barros,‡ Brian J. Goodfellow,† Sı´lvia O. Diaz,† † § Joana Pinto, Isabel M. Carreira, Eula´lia Galhano,| Cristina Pita,| and Ana M. Gil*,† CICECO-Department of Chemistry, Campus Universita´rio de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, QOPNA Research Unit, Department of Chemistry, Campus Universita´rio de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, Po´lo III, University of Coimbra, 3000-354 Coimbra, Portugal, and Maternidade Bissaya Barreto, Centro Hospitalar de Coimbra, 3000 Coimbra, Portugal Received August 9, 2010
This paper describes a metabonomic study of prenatal disorders using nuclear magnetic resonance (NMR) spectroscopy of amniotic fluid (AF) collected in the second trimester of pregnancy, to search for metabolite markers of fetal malformations, prediagnostic gestational diabetes (GD), preterm delivery (PTD), early rupture of membranes (PROM), and chromossomopathies. Fetal malformations were found to have the highest impact on AF metabolite composition, enabling statistical validation to be achieved by several multivariate analytical tools. Results confirmed previous indications that malformed fetuses seem to suffer altered energy metabolism and kidney underdevelopment. Newly found changes (namely in R-oxoisovalerate, ascorbate, creatinine, isoleucine, serine, threonine) suggest possible additional effects on protein and nucleotide sugar biosynthesis. Prediagnostic GD subjects showed an average increase in glucose and small decreases in several amino acids along with acetate, formate, creatinine, and glycerophosphocholine. Small metabolite changes were also observed in the AF of subjects eventually undergoing PTD and PROM, whereas no relevant changes were found for chromossomopathies (for which a low number of samples was considered). The potential value of these results for biochemical insight and prediction of prenatal disorders is discussed, as well as their limitations regarding number of samples and overlap of different disorders. Keywords: Amniotic fluid • prenatal disorders • fetal malformations • gestational diabetes • preterm • NMR spectroscopy • metabonomics
Introduction Human amniotic fluid (AF) results from the diffusion of metabolites and water from fetal plasma through fetal skin, which acts as a permeable barrier. Throughout gestation, there is an increased contribution of fetal urine to amniotic fluid, and the concentration of many of its constituents is determined by the synergistic equilibrium between mother and fetus. As AF composition reflects the metabolic status of both mother and fetus during pregnancy,1-4 this biofluid is an important target for metabolic studies to monitor the mother’s and fetus’s health. AF is routinely collected through amniocentesis, in the second trimester, mainly for risk pregnancies such as advanced maternal age, detection of fetal malformations by ultrasound, * Corresponding author. Tel.: +351 234 370707. Fax: +351 234 370084. E-mail:
[email protected]. † CICECO-Department of Chemistry, Universidade de Aveiro. ‡ QOPNA Research Unit, Department of Chemistry, Universidade de Aveiro. § University of Coimbra. | Centro Hospitalar de Coimbra.
6016 Journal of Proteome Research 2010, 9, 6016–6024 Published on Web 09/17/2010
or prior history of genetic abnormalities. This biofluid is used mainly to obtain amniocyte cells to be tested for chromosome abnormalities such as aneuploidies, mainly involving the 21,18,13, X and Y chromosomes, and certain fetal infections, e.g., cytomegalovirus (CVM). AF is also analyzed for R-fetoprotein and choline-esterase to aid detection of open-tube defects, for certain digestive enzymes for the diagnosis of cystic fibrosis, and for surfactant phospholipids to assess fetal lung maturity in cases of preterm delivery.5 Several studies have shown possible correlations between AF metabolite composition and the occurrence of different types of prenatal diseases. For instance, changes in the concentrations of choline and several amino and organic acids have been suggested in women diagnosed with preeclampsia3 and spina bifida3,6 as well as other cases of suspected fetal abnormalities.7 In addition, several organic acids and glycine conjugates seem to be associated with the occurrence of metabolic diseases (e.g., type I tyrosinaemia, propionic acidaemia, and organic acidurias),8 and specific protein changes seem to be related to preterm birth,9 preeclampsia,10 Down and Turner syndromes, and premature rupture of membranes 10.1021/pr100815q
2010 American Chemical Society
Metabolic Study of Prenatal Disorders by NMR-Metabonomics 11-13
(PROM). To eventually obtain disease biomarkers, it is necessary to consider large numbers of subjects to build sturdy statistical models and to seek validation of the compositional variations related to particular diseases. This may be difficult in practice, due to lower incidences of some disorders (e.g.,
