Following Healthy Pregnancy by NMR Metabolomics of Plasma and Correlation to Urine

This work presents the first NMR metabolomics study of maternal plasma during pregnancy, including correlation between plasma and urine metabolites. The expected decrease in circulating amino acids early in pregnancy was confirmed with six amino acids being identified as required by the fetus in larger extents. Newly observed changes in citrate, lactate, and dimethyl sulfone suggested early adjustments in energy and gut microflora metabolisms. Alterations in creatine levels were also noted, in addition to creatinine variations reflecting alterations in glomerular filtration rate. Regarding plasma macromolecules, HDL and LDL+VLDL levels were confirmed to increase throughout pregnancy, although at different rates and accompanied by increases in fatty acid chain length and degree of unsaturation. Correlation studies suggested (a) an inverse relationship between lipoproteins (HDL and LDL+VLDL) and albumin, with a possible direct correlation to excreted (unassigned) pregnancy markers resonating at δ 0.55 and δ 0.63, (b) a direct link between LDL+VLDL and <i>N</i>-acetyl-glycoproteins, together with excreted marker at δ 0.55, and (c) correlation of plasma albumin with particular circulating and excreted metabolites. These results have unveiled specific lipoprotein/protein metabolic aspects of pregnancy with impact on the excreted metabolome and, therefore, provide an interesting lead for the further understanding of pregnancy metabolism

Prediction of Gestational Diabetes through NMR Metabolomics of Maternal Blood

Metabolic biomarkers of pre- and postdiagnosis gestational diabetes mellitus (GDM) were sought, using nuclear magnetic resonance (NMR) metabolomics of maternal plasma and corresponding lipid extracts. Metabolite differences between controls and disease were identified through multivariate analysis of variable selected ¹H NMR spectra. For postdiagnosis GDM, partial least squares regression identified metabolites with higher dependence on normal gestational age evolution. Variable selection of NMR spectra produced good classification models for both pre- and postdiagnostic GDM. Prediagnosis GDM was accompanied by cholesterol increase and minor increases in lipoproteins (plasma), fatty acids, and triglycerides (extracts). Small metabolite changes comprised variations in glucose (up regulated), amino acids, betaine, urea, creatine, and metabolites related to gut microflora. Most changes were enhanced upon GDM diagnosis, in addition to newly observed changes in low-<i>M</i>_w compounds. GDM prediction seems possible exploiting multivariate profile changes rather than a set of univariate changes. Postdiagnosis GDM is successfully classified using a 26-resonance plasma biomarker. Plasma and extracts display comparable classification performance, the former enabling direct and more rapid analysis. Results and putative biochemical hypotheses require further confirmation in larger cohorts of distinct ethnicities

Second Trimester Maternal Urine for the Diagnosis of Trisomy 21 and Prediction of Poor Pregnancy Outcomes

Given the recognized lack of prenatal clinical methods for the early diagnosis of preterm delivery, intrauterine growth restriction, preeclampsia and gestational diabetes mellitus, and the continuing need for optimized diagnosis methods for specific chromosomal disorders (e.g., trisomy 21) and fetal malformations, this work sought specific metabolic signatures of these conditions in second trimester maternal urine, using ¹H Nuclear Magnetic Resonance (¹H NMR) metabolomics. Several variable importance to the projection (VIP)- and b-coefficient-based variable selection methods were tested, both individually and through their intersection, and the resulting data sets were analyzed by partial least-squares discriminant analysis (PLS-DA) and submitted to Monte Carlo cross validation (MCCV) and permutation tests to evaluate model predictive power. The NMR data subsets produced significantly improved PLS-DA models for all conditions except for pre-premature rupture of membranes. Specific urinary metabolic signatures were unveiled for central nervous system malformations, trisomy 21, preterm delivery, gestational diabetes, intrauterine growth restriction and preeclampsia, and biochemical interpretations were proposed. This work demonstrated, for the first time, the value of maternal urine profiling as a complementary means of prenatal diagnostics and early prediction of several poor pregnancy outcomes

Representative ¹H NMR spectra of control plasma.

<p>500 MHz ¹H NMR spectra of blood plasma from a control subject: a) standard 1D spectrum; b) CPMG spectrum; c) diffusion-edited spectrum. Signal assignment: 1-lactate; 2-alanine; 3 -glutamine; 4-glucose; 5-isoleucine; 6-leucine; 7-valine; 8-lysine; 9-acetate; 10-pyruvate; 11-citrate; 12-creatine; 13-creatinine; 14-dimethyl sulfone; 15-TMAO, trimethylamine-<i>N</i>-Oxide; 16,proline; 17-methanol; 18-glycine; 19-tyrosine; 20-histidine; 21- phenylalanine; 22-formate; 23-C18H cholesterol; 24-CH₃ lipids; 25-(CH₂)_n lipids; 26-C<u>H</u>₂CH₂CO lipids; 27-C<u>H</u>₂CH₂C = C lipids; 28-C<u>H</u>₂C = C lipids; 29-C<u>H</u>₂CO lipids; 30-C = CC<u>H</u>₂CH = C lipids; 31-albumin lysil groups; 32-N(CH₃)₃ choline; 33-glyceryl C1,3H; 34-glyceryl C1,3H’; 35-glyceryl C2H; 36-HC = CH lipids; 37-NH protein region.</p

Generalized linear regression results.

<p>Generalized linear regression coefficients obtained through modeling of metabolite variations as a function of gender proportion, smoking history, body-mass index (BMI), age and AMD status. F.A.: Fatty acids. Values in bold illustrate the higher contributions of AMD status for each metabolite variation, compared to confounders. Similar metabolite variations in the two cohorts are denoted by underline.</p

Boxplot graphs for metabolites varying in Coimbra cohort.

<p>Coimbra cohort: boxplot representations of the metabolite variations found statistically relevant (* indicates <i>p-</i>value < 0.05) in at least one pairwise PLS-DA model. Compound names in rectangles correspond to compounds differentiating between controls and early AMD patients. C: controls, E: early AMD, I: intermediate AMD, L: late AMD. F.A.: fatty acids.</p

Characterization of the study population.

<p>Characterization of the study populations (Coimbra and Boston cohorts), with corresponding number of subjects (n), age (years), female (F)/male (M) ratio, body mass index (BMI) (kg.m^-2) and smoking history.</p

Variations in plasma metabolites of AMD patients.

<p>Main variations in plasma metabolites across AMD evolution through different severity stages, in Coimbra and Boston cohorts.</p

Effect size plots for CPMG spectra integrals.

<p>Effect size (E.S.) plots for resonances varying in the CPMG NMR spectra across AMD evolution through different severity stages in the a) Coimbra and b) Boston cohorts. Resonances are listed alphabetically within each compound family (amino acids, organic acids, other low-M_w compounds and lipids). The dashed horizontal line refers to null E.S. and the length of the vertical segments corresponds to E.S. range. E.S. segments not intercepting the null E.S. line are considered as relevant variations (shaded rectangles). F.A.: fatty acids.</p

Examples of PLS-DA score plots.

<p>PLS-DA scores scatter plots and MCCV quality parameters (pairwise model Q², Q²_median (obtained through MCCV), % CR, % sens. and % spec.) obtained for variable selected CPMG NMR spectra of late AMD patients <i>vs</i> controls, in the a) Coimbra cohort: late AMD patients (□, n = 32), controls (∎, n = 42) and b) Boston cohort: late AMD patients (◇, n = 38), controls (♦, n = 40).</p