Isabel M. Carreira

Metabolic Signatures of Lung Cancer in Biofluids: NMR-Based Metabonomics of Urine

In this study, ¹H NMR-based metabonomics has been applied, for the first time to our knowledge, to investigate lung cancer metabolic signatures in urine, aiming at assessing the diagnostic potential of this approach and gaining novel insights into lung cancer metabolism and systemic effects. Urine samples from lung cancer patients (n = 71) and a control healthy group (n = 54) were analyzed by high resolution ¹H NMR (500 MHz), and their spectral profiles subjected to multivariate statistics, namely, Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Projections to Latent Structures (OPLS)-DA. Very good discrimination between cancer and control groups was achieved by multivariate modeling of urinary profiles. By Monte Carlo Cross Validation, the classification model showed 93% sensitivity, 94% specificity and an overall classification rate of 93.5%. The possible confounding influence of other factors, namely, gender and age, have also been modeled and found to have much lower predictive power than the presence of the disease. Moreover, smoking habits were found not to have a dominating influence over class discrimination. The main metabolites contributing to this discrimination, as highlighted by multivariate analysis and confirmed by spectral integration, were hippurate and trigonelline (reduced in patients), and β-hydroxyisovalerate, α-hydroxyisobutyrate, N-acetylglutamine, and creatinine (elevated in patients relatively to controls). These results show the valuable potential of NMR-based metabonomics for finding putative biomarkers of lung cancer in urine, collected in a minimally invasive way, which may have important diagnostic impact, provided that these metabolites are found to be specifically disease-related.

Metabolic profiling of human lung cancer tissue by 1H high resolution magic angle spinning (HRMAS) NMR spectroscopy.

This work aims at characterizing the metabolic profile of human lung cancer, to gain new insights into tumor metabolism and to identify possible biomarkers with potential diagnostic value in the future. Paired samples of tumor and noninvolved adjacent tissues from 12 lung tumors have been directly analyzed by (1)H HRMAS NMR (500/600 MHz) enabling, for the first time to our knowledge, the identification of over 50 compounds. The effect of temperature on tissue stability during acquisition time has also been investigated, demonstrating that analysis should be performed within less than two hours at low temperature (277 K), to minimize glycerophosphocholine (GPC) and phosphocholine (PC) conversion to choline and reduce variations in some amino acids. The application of Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) to the standard 1D (1)H spectra resulted in good separation between tumor and control samples, showing that inherently different metabolic signatures characterize the two tissue types. On the basis of spectral integration measurements, lactate, PC, and GPC were found to be elevated in tumors, while glucose, myo-inositol, inosine/adenosine, and acetate were reduced. These results show the valuable potential of HRMAS NMR-metabonomics for investigating the metabolic phenotype of lung cancer.

Metabolic Biomarkers of Prenatal Disorders: An Exploratory NMR Metabonomics Study of Second Trimester Maternal Urine and Blood Plasma

This work describes an exploratory NMR metabonomic study of second trimester maternal urine and plasma, in an attempt to characterize the metabolic changes underlying prenatal disorders and identify possible early biomarkers. Fetal malformations have the strongest metabolic impact in both biofluids, suggesting effects due to hypoxia (leading to hypoxanthine increased excretion) and a need for enhanced gluconeogenesis, with higher ketone bodies (acetone and 3-hydroxybutyric acid) production and TCA cycle demand (suggested by glucogenic amino acids and cis-aconitate overproduction). Choline and nucleotide metabolisms also seem affected and a distinct plasma lipids profile is observed for mothers with fetuses affected by central nervous system malformations. Urine from women who subsequently develop gestational diabetes mellitus exhibits higher 3-hydroxyisovalerate and 2-hydroxyisobutyrate levels, probably due to altered biotin status and amino acid and/or gut metabolisms (the latter possibly related to higher BMI values). Other urinary changes suggest choline and nucleotide metabolic alterations, whereas lower plasma betaine and TMAO levels are found. Chromosomal disorders and pre-preterm delivery groups show urinary changes in choline and, in the latter case, in 2-hydroxyisobutyrate. These results show that NMR metabonomics of maternal biofluids enables the noninvasive detection of metabolic changes associated to prenatal disorders, thus unveiling potential disorder biomarkers.

Impact of prenatal disorders on the metabolic profile of second trimester amniotic fluid: a nuclear magnetic resonance metabonomic study.

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 α-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.

1H NMR Based Metabonomics of Human Amniotic Fluid for the Metabolic Characterization of Fetus Malformations

An NMR-metabonomic study of malformed fetuses was carried out through human amniotic fluid (HAF) analysis. Over 70 compounds were detected in control HAF by NMR. Possible confounding variables (fetus gender and gestational and maternal ages) were shown not to induce detectable compositional trends in the control group considered. Malformed fetuses showed variations in glucose, some amino acids and organic acids and proteins. In tandem with enzymatic assays, these NMR results suggest that changes in gycolysis and gluconeogenesis as well as kidney underdevelopment occur in the malformed fetuses studied here.

Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy

The aim of this work was to investigate the effects of cell handling and storage on cell integrity and (1)H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was approximately 70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation- and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The (1)H HRMAS spectra of the three cell types are compared and discussed.

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 (1)H Nuclear Magnetic Resonance ((1)H 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.

Metabolite Profiling of Human Amniotic Fluid by Hyphenated Nuclear Magnetic Resonance Spectroscopy

The metabolic profiling of human amniotic fluid (HAF) is of potential interest for the diagnosis of disorders in the mother or the fetus. In order to build a comprehensive metabolite database for HAF, hyphenated NMR has been used, for the first time, for systematic HAF profiling. Experiments were carried out using reverse-phase (RP) and ion-exchange liquid chromatography (LC), in order to detect less and more polar compounds, respectively. RP-LC conditions achieved good separation of amino acids, some sugars, and xanthines. Subsequent NMR and MS analysis enabled the rapid identification of 30 compounds, including 3-methyl-2-oxovalerate and 4-aminohippurate identified in HAF for the first time, to our knowledge. Under ion-exchange LC conditions, a different set of 30 compounds was detected, including sugars, organic acids, several derivatives of organic acids, and amino acids. In this experiment, five compounds were identified for the first time in HAF: D-xylitol, amino acid derivatives (N-acetylalanine, N-acetylglycine, 2-oxoleucine), and isovalerate. The nonendogenous nature of some metabolites (caffeine, paraxanthine, D-xylitol, sorbitol) is discussed. Hyphenated NMR has allowed the rapid detection of approximately 60 metabolites in HAF, some of which are not detectable by standard NMR due to low abundance (microM) and signal overlap thus enabling an extended metabolite database to be built for HAF.

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 N-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.

Following Healthy Pregnancy by Nuclear Magnetic Resonance (NMR) Metabolic Profiling of Human Urine

In this work, untargeted NMR metabonomics was employed to evaluate the effects of pregnancy on the metabolite composition of maternal urine, thus establishing a control excretory trajectory for healthy pregnancies. Urine was collected for independent groups of healthy nonpregnant and pregnant women (in first, second, third trimesters) and multivariate analysis performed on the corresponding NMR spectra. Models were validated through Monte Carlo Cross Validation and permutation tests and metabolite correlations measured through Statistical Total Correlation Spectroscopy. The levels of 21 metabolites were found to change significantly throughout pregnancy, with variations observed for the first time to our knowledge for choline, creatinine, 4-deoxyerythronic acid, 4-deoxythreonic acid, furoylglycine, guanidoacetate, 3-hydroxybutyrate, and lactate. Results confirmed increased aminoaciduria across pregnancy and suggested (a) a particular involvement of isoleucine and threonine in lipid oxidation/ketone body synthesis, (b) a relation of excreted choline, taurine, and guanidoacetate to methionine metabolism and urea cycle regulation, and (c) a possible relationship of furoylglycine and creatinine to pregnancy, based on a tandem study of nonfasting confounding effects. Results demonstrate the usefulness of untargeted metabonomics in finding biomarker metabolic signatures for healthy pregnancies, against which disease-related deviations may be confronted in future studies, as a base for improved diagnostics and prediction.