Male and Female Mitochondria Respond Differently after Exercising in Acute Hypoxia.

The use of hypoxic devices among athletes who train in normobaric hypoxia has become increasingly popular; however, the acute effects on heart and brain metabolism are not yet fully understood. This study aimed to investigate the mitochondrial bioenergetics in trained male and female Wistar rats after acute hypoxia training. The experimental plan included exercising for 30 min on a treadmill in a Plexiglas cage connected to a hypoxic generator set at 12.5% O2 or in normoxia. After the exercise, the rats were sacrificed, and their mitochondria were isolated from their brains and hearts. The bioenergetics for each complex of the electron transport chain was tested using a Clark-type electrode. The results showed that following hypoxia training, females experienced impaired oxidative phosphorylation through complex II in heart subsarcolemmal mitochondria, while males had an altered ADP/O in heart interfibrillar mitochondria, without any change in oxidative capacity. No differences from controls were evident in the brain, but an increased electron transport system efficiency was observed with complex I and IV substrates in males. Therefore, the study's findings suggest that hypoxia training affects the heart mitochondria of females more than males. This raises a cautionary flag for female athletes who use hypoxic devices.

Sardinian Infants of Diabetic Mothers: A Metabolomics Observational Study.

Gestational diabetes mellitus (GDM) is a condition characterized by glucose intolerance, with hyperglycemia of varying severity with onset during pregnancy. An uncontrolled GDM can lead to an increased risk of morbidity in the fetus and newborn, and an increased risk of obesity or developing type 2 diabetes, hypertension or neurocognitive developmental impairment in adulthood. In this study, we used nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GS-MS) to analyze the urinary metabolomic profile of newborns of diabetic mothers (NDMs) with the aim of identifying biomarkers useful for the monitoring of NDMs and for early diagnosis of predisposition to develop related chronic diseases. A total of 26 newborns were recruited: 21 children of diabetic mothers, comprising 13 in diet therapy (NDM-diet) and 8 in insulin therapy (NDM-insulin), and 5 control children of non-diabetic mothers (CTR). Urine samples were collected at five time points: at birth (T1), on the third day of life (T2), one week (T3), one month (T4) and six months postpartum (T5). At T1, variations were observed in the levels of seven potential biomarkers (acetate, lactate, glycylproline/proline, isocitrate, N,N-dimethylglycine, N-acetylglucosamine and N-carbamoyl-aspartate) in NMD-insulin infants compared to NDM-diet and CTR infants. In particular, the altered metabolites were found to be involved in several metabolic pathways such as citrate metabolism, glycine, serine and threonine metabolism, arginine and proline metabolism, amino sugar and nucleotide sugar metabolism, and pyruvate metabolism. In contrast, these changes were not visible at subsequent sampling times. The impact of early nutrition (maternal and formula milk) on the metabolomic profile was considered as a potential contributing factor to this finding.

Halogen-Bonding-Mediated Radical Reactions: The Unexpected Behavior of Piperazine-Based Dithiooxamide Ligands in the Presence of Diiodine.

N,N'-Dialkylpiperazine-2,3-dithiones (R2pipdt) were recognized as a class of hexa-atomic cyclic dithiooxamide ligands with peculiar charge-transfer donor properties toward soft electron-acceptors such as noble metal cations and diiodine. The latter interaction is nowadays better described as halogen bonding. In the reaction with diiodine, R2pipdt unexpectedly provides the corresponding triiodide salts, differently from the other dithiooxamides, which instead typically achieve ligand·nI2 halogen-bonded adducts. In this paper, we report a combined experimental and theoretical study that allows elucidation of the nature of the cited products and the reasons behind the unpredictable behavior of these ligands. Specifically, low-temperature single-crystal X-ray diffraction measurements on a series of synthetically obtained R2pipdt (R = Me, iPr, Bz)/I3 salts, complemented by neutron diffraction experiments, were able to experimentally highlight the formation of [R2pipdtH]+ cations with a -S-H bond on the dithionic moiety. Differently, with R = Ph, a benzothiazolylium cation, resulting from an intramolecular condensation reaction of the ligand, is obtained. Based on density functional theory (DFT) calculations, a reasonable reaction mechanism where diiodine plays the fundamental role of promoting a halogen-bonding-mediated radical reaction has been proposed. In addition, the comparison of combined experimental and computational results with the corresponding reactions of N,N'-dialkylperhydrodiazepine-2,3-dithione (R2dazdt, a hepta-atomic cyclic dithiooxamide), which provide neutral halogen-bonded adducts, pointed out that the difference in the torsion angle of the free ligands represents the structural key factor in determining the different reactivities of the two systems.

NMR Metabonomic Profile of Preterm Human Milk in the First Month of Lactation: From Extreme to Moderate Prematurity.

Understanding the composition of human milk (HM) can provide important insights into the links between infant nutrition, health, and development. In the present work, we have longitudinally investigated the metabolome of milk from 36 women delivering preterm at different gestational ages (GA): extremely (<28 weeks GA), very (29-31 weeks GA) or moderate (32-34 weeks GA) premature. Milk samples were collected at three lactation stages: colostrum (3-6 days post-partum), transitional milk (7-15 days post-partum) and mature milk (16-26 days post-partum). Multivariate and univariate statistical data analyses were performed on the 1H NMR metabolic profiles of specimens in relation to the degree of prematurity and lactation stage. We observed a high impact of both the mother's phenotype and lactation time on HM metabolome composition. Furthermore, statistically significant differences, although weak, were observed in terms of GA when comparing extremely and moderately preterm milk. Overall, our study provides new insights into preterm HM metabolome composition that may help to optimize feeding of preterm newborns, and thus improve the postnatal growth and later health outcomes of these fragile patients.

A Comparison of Mother's Milk and the Neonatal Urine Metabolome: A Unique Fingerprinting for Different Nutritional Phenotypes.

The ability of metabolomics to provide a snapshot of an individual's metabolic state makes it a very useful technique in neonatology for investigating the complex relationship between nutrition and the state of health of the newborn. Through an 1H-NMR metabolomics analysis, we aimed to investigate the metabolic profile of newborns by analyzing both urine and milk samples in relation to the birth weight of neonates classified as AGA (adequate for the gestational age, n = 51), IUGR (intrauterine growth restriction, n = 14), and LGA (large for gestational age, n = 15). Samples were collected at 7 ± 2 days after delivery. Of these infants, 42 were exclusively breastfed, while 38 received mixed feeding with a variable amount of commercial infant formula (less than 40%) in addition to breast milk. We observed a urinary spectral pattern for oligosaccharides very close to that of the corresponding mother's milk in the case of exclusively breastfed infants, thus mirroring the maternal phenotype. The absence of this good match between the infant urine and human milk spectra in the case of mixed-fed infants could be reasonably ascribed to the use of a variable amount of commercial infant formulas (under 40%) added to breast milk. Furthermore, our findings did not evidence any significant differences in the spectral profiles in terms of the neonatal customize centile, i.e., AGA (adequate for gestational age), LGA (large for gestational age), or IGUR (intrauterine growth restriction). It is reasonable to assume that maternal human milk oligosaccharide (HMO) production is not or is only minimally influenced by the fetal growth conditions for unknown reasons. This hypothesis may be supported by our metabolomics-based results, confirming once again the importance of this approach in the neonatal field.

Unraveling the Microbiome of Necrotizing Enterocolitis: Insights in Novel Microbial and Metabolomic Biomarkers.

Necrotizing enterocolitis (NEC) is among the most relevant gastrointestinal diseases affecting mostly prematurely born infants with low birth weight. While intestinal dysbiosis has been proposed as one of the possible factors involved in NEC pathogenesis, the role of the gut microbiota remains poorly understood. In this study, the gut microbiota of preterm infants was explored to highlight differences in the composition between infants affected by NEC and infants prior to NEC development. A large-scale gut microbiome analysis was performed, including 47 shotgun sequencing data sets generated in the framework of this study, along with 124 retrieved from publicly available repositories. Meta-analysis led to the identification of preterm community state types (PT-CSTs), which recur in healthy controls and NEC infants. Such analyses revealed an overgrowth of a range of opportunistic microbial species accompanying the loss of gut microbial biodiversity in NEC subjects. Moreover, longitudinal insights into preterm infants prior to NEC development indicated Clostridium neonatale and Clostridium perfringens species as potential biomarkers for predictive early diagnosis of this disease. Furthermore, functional investigation of the enzymatic reaction profiles associated with pre-NEC condition suggested DL-lactate as a putative metabolic biomarker for early detection of NEC onset. IMPORTANCE Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease occurring predominantly in premature infants whose etiology is still not fully understood. In this study, the analysis of infant fecal samples through shotgun metagenomics approaches revealed a marked reduction of the intestinal (bio)diversity and an overgrowth of (opportunistic) pathogens associated with the NEC development. In particular, dissection of the infant's gut microbiome before NEC diagnosis highlighted the potential involvement of Clostridium genus members in the progression of NEC. Remarkably, our analyses highlighted a gastrointestinal DL-lactate accumulation among NEC patients that might represent a novel potential functional biomarker for the early diagnosis of NEC.

Human Milk Oligosaccharides: A Comprehensive Review towards Metabolomics.

Human milk oligosaccharides (HMOs) are the third most represented component in breast milk. They serve not only as prebiotics but they exert a protective role against some significant neonatal pathologies such as necrotizing enterocolitis. Furthermore, they can program the immune system and consequently reduce allergies and autoimmune diseases' incidence. HMOs also play a crucial role in brain development and in the gut barrier's maturation. Moreover, the maternal genetic factors influencing different HMO patterns and their modulation by the interaction and the competition between active enzymes have been widely investigated in the literature, but there are few studies concerning the role of other factors such as maternal health, nutrition, and environmental influence. In this context, metabolomics, one of the newest "omics" sciences that provides a snapshot of the metabolites present in bio-fluids, such as breast milk, could be useful to investigate the HMO content in human milk. The authors performed a review, from 2012 to the beginning of 2021, concerning the application of metabolomics to investigate the HMOs, by using Pubmed, Researchgate and Scopus as source databases. Through this technology, it is possible to know in real-time whether a mother produces a specific oligosaccharide, keeping into consideration that there are other modifiable and unmodifiable factors that influence HMO production from a qualitative and a quantitative point of view. Although further studies are needed to provide clinical substantiation, in the future, thanks to metabolomics, this could be possible by using a dipstick and adding the eventual missing oligosaccharide to the breast milk or formula in order to give the best and the most personalized nutritional regimen for each newborn, adjusting to different necessities.

Urine NMR Metabolomics Profile of Preterm Infants With Necrotizing Enterocolitis Over the First Two Months of Life: A Pilot Longitudinal Case-Control Study.

Objective: To investigate changes in the urine metabolome of very low birth weight preterm newborns with necrotizing enterocolitis (NEC) and feed intolerance, we conducted a longitudinal study over the first 2 months of life. The metabolome of NEC newborns was compared with two control groups that did not develop NEC: the first one included preterm babies with feed intolerance, while the second one preterm babies with good feed tolerance. Methods: Newborns developing NEC within the 3 weeks of life were identified as early onset NEC, while the remaining as late onset NEC. Case-control matching was done according to the gestational age (±1 week), birth weight (± 200 g), and postnatal age. A total of 96 urine samples were collected and analyzed. In newborns with NEC, samples were collected before, during and after the diagnosis over the first 2 months of life, while in controls samples were collected as close as possible to the postnatal age of newborns with NEC. Proton nuclear magnetic resonance (1H NMR) spectroscopy was used for metabolomic analysis. Data were analyzed by univariate and multivariate statistical analysis. Results: In all the preterm newborns, urine levels of betaine, glycine, succinate, and citrate positively correlated with postnatal age. Suberate and lactate correlated with postnatal age in preterms with NEC and in controls with food intolerance, while N,N-dimethylglycine (N,N-DMG) correlated only in controls with good digestive tolerance. Preterm controls with feed intolerance showed a progressive significant decrease of N-methylnicotinamide and carnitine. Lactate, betaine, myo-inositol, urea, creatinine, and N,N-dimethylglycine discriminated late-onset NEC from controls with good feed tolerance. Conclusion: Our findings are discussed in terms of contributions from nutritional and clinical managements of patients and gut microbiota.

Urinary Metabolomics Study of Patients with Bicuspid Aortic Valve Disease.

Bicuspid aortic valve (BAV) is the most common congenital heart defect responsible for valvular and aortic complications in affected patients. Causes and mechanisms of this pathology are still elusive and thus the lack of early detection biomarkers leads to challenges in its diagnosis and prevention of associated cardiovascular anomalies. The aim of this study was to explore the potential use of urine Nuclear Magnetic Resonance (NMR) metabolomics to evaluate a molecular fingerprint of BAV. Both multivariate and univariate statistical analyses were performed to compare the urinary metabolome of 20 patients with BAV with that of 24 matched controls. Orthogonal partial least squared discriminant analysis (OPLS-DA) showed statistically significant discrimination between cases and controls, suggesting seven metabolites (3-hydroxybutyrate, alanine, betaine, creatine, glycine, hippurate, and taurine) as potential biomarkers. Among these, glycine, hippurate and taurine individually displayed medium sensitivity and specificity by receiver operating characteristic (ROC) analysis. Pathway analysis indicated two metabolic pathways likely perturbed in BAV subjects. Possible contributions of gut microbiota activity and energy imbalance are also discussed. These results constitute encouraging preliminary findings in favor of the use of urine-based metabolomics for early diagnosis of BAV.

Theoretical and Experimental Study of the Excess Thermodynamic Properties of Highly Nonideal Liquid Mixtures of Butanol Isomers + DBE.

Binary alcohol + ether liquid mixtures are of significant importance as potential biofuels or additives for internal combustion engines and attract considerable fundamental interest as model systems containing one strongly H-bonded self-associating component (alcohol) and one that is unable to do so (ether), but that can interact strongly as a H-bond acceptor. In this context, the excess thermodynamic properties of these mixtures, specifically the excess molar enthalpies and volumes (HE and VE), have been extensively measured. Butanol isomer + di-n-butyl ether (DBE) mixtures received significant attention because of interesting differences in their VE, changing from negative (1- and isobutanol) to positive (2- and tert-butanol) with increasing alkyl group branching. With the aim of shedding light on the differences in alcohol self-association and cross-species H-bonding, considered responsible for the observed differences, we studied representative 1- and 2-butanol + DBE mixtures by molecular dynamics simulations and experimental excess property measurements. The simulations reveal marked differences in the self-association of the two isomers and, while supporting the existing interpretations of the HE and VE in a general sense, our results suggest, for the first time, that subtle changes in H-bonded topologies may contribute significantly to the anomalous volumetric properties of these mixtures.

Sportomics in professional soccer players: metabolomics results during preseason.

BACKGROUND: Sportomics is the application of metabolomics to study the metabolism shifts of individuals that practice sports or do physical exercise. This aim was reached by the analysis of low molecular weight metabolites (<1.5 kDa) present in biological fluids such as blood, saliva or urine. METHODS: In this study, authors performed a 1H-NMR analysis of urine from 21 professional soccer players collected at 3 different time points during the preseason preparation period before the beginning of Serie A Championship (first division) in Italy. RESULTS: Urine profile changed during the observational period. In particular, significant variations were observed for trimethylamine-N-oxide, dimethylamine, hippuric acid, hypoxanthine, guanidoacetic acid, 3-hydroxybutyric acid, citric acid and creatine. These modifications could be related to the diet, training and microbiota. For instance, trimethylamine-N-oxide and hippuric acid are both of dietary origins but are also related to the microbiota, while 3-hydroxy-butyric acid is associated with the type of physical exercise. CONCLUSIONS: This is the first sportomics study ever performed on professional soccer players, according to authors' knowledge. In the future, sportomics could be applied in a tailored way to choose the best diet and training program in the single individual to obtain the best possible performances and to prevent injuries of athletes.

Influence of Autochthonous Putative Probiotic Cultures on Microbiota, Lipid Components and Metabolome of Caciotta Cheese.

The present study was undertaken to produce probiotic Caciotta cheeses from pasteurized ewes' milk by using different combinations of autochthonous microbial cultures, containing putative probiotic strains, and evaluate their influence on gross composition, lipid components, sensory properties and microbiological and metabolite profiles of the cheeses throughout ripening process. A control cheese was produced using commercial starter cultures. The hydrophilic molecular pools (mainly composed by amino acids, organic acids, and carbohydrates) were characterized by means of 1H NMR spectroscopy, while the cholesterol, α-tocopherol and fatty acid composition by HPLC-DAD/ELSD techniques. Conventional culturing and a PCR-DGGE approach using total cheese DNA extracts were used to analyze cheese microbiota and monitor the presence and viability of starters and probiotic strains. Our findings showed no marked differences for gross composition, total lipids, total cholesterol, and fatty acid levels among all cheeses during ripening. Differently, the multivariate statistical analysis of NMR data highlighted significant variations in the cheese' profiles both in terms of maturation time and strains combination. The use of autochthonous cultures and adjunct probiotic strains did not adversely affect acceptability of the cheeses. Higher levels of lactobacilli (viability of 108-109 cfu/g of cheese) were detected in cheeses made with the addition of probiotic autochthonous strains with respect to control cheese during the whole ripening period, suggesting the adequacy of Caciotta cheese as a carrier for probiotic bacteria delivery.

Urinary Metabolomic Profile of Preterm Infants Receiving Human Milk with Either Bovine or Donkey Milk-Based Fortifiers.

Fortification of human milk (HM) for preterm and very low-birth weight (VLBW) infants is a standard practice in most neonatal intensive care units. The optimal fortification strategy and the most suitable protein source for achieving better tolerance and growth rates for fortified infants are still being investigated. In a previous clinical trial, preterm and VLBW infants receiving supplementation of HM with experimental donkey milk-based fortifiers (D-HMF) showed decreased signs of feeding intolerance, including feeding interruptions, bilious gastric residuals and vomiting, with respect to infants receiving bovine milk-based fortifiers (B-HMF). In the present ancillary study, the urinary metabolome of infants fed B-HMF (n = 27) and D-HMF (n = 27) for 21 days was analyzed by 1H NMR spectroscopy at the beginning (T0) and at the end (T1) of the observation period. Results showed that most temporal changes in the metabolic responses were common in the two groups, providing indications of postnatal adaptation. The significantly higher excretion of galactose in D-HMF and of carnitine, choline, lysine and leucine in B-HMF at T1 were likely due to different formulations. In conclusion, isocaloric and isoproteic HM fortification may result in different metabolic patterns, as a consequence of the different quality of the nutrients provided by the fortifiers.

A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

Special Issue on "NMR-Based Metabolomics and Its Applications Volume 2".

Over the last decade, the number of scientific publications in the metabolomics area has increased exponentially. The literature includes ~29,000 contributions (articles and reviews) during the period of 2009-2019, revealing metabolomics applications in a wide range of fields, including medical, plant, animal, and food sciences (this bibliographic data were retrieved from the SCOPUS database, searching "metabolomics" in keywords). The high applicability of this approach is due to its ability to qualitatively and quantitatively characterize the chemical profile of all the low molecular weight metabolites (metabolome) present in cells, tissues, organs, and biological fluids as end products of the cellular regulatory pathways. Thus, providing a snapshot of the phenotype of a biological system, metabolomics offers useful contributions to a comprehensive insight into the functional status of human, animal, plant, and microbe organisms. The contributions collected in this Special Issue (12 articles, one review and one technical report) report on the recent technical advances and practical applications of NMR spectroscopy to metabolomics analyses.

Metabolomics of Breast Milk: The Importance of Phenotypes.

Breast milk is the gold standard of nutrition for newborns. Its composition is tailored to the nutritional needs of the infant and varies between mothers. In recent years, several bioactive molecules have been discovered in addition to the main nutrients, such as multipotent stem cells, hormones, immunoglobulins, and bacteria. Furthermore, the human milk oligosaccharides (HMOs) seem to exert several important protective biological functions. According to the HMOs' composition, breast milk can be classified as a secretory or non-secretory phenotype. In our study, we investigated the metabolome of milk collected from 58 mothers that delivered neonates at term, that were appropriate, small or large for gestational age, by performing nuclear magnetic resonance spectroscopy (¹H-NMR). From the data analysis, two groups were distinguished based on their different types of oligosaccharides, and classified according the mother phenotype: secretory and non-secretory. This information is of major importance given the different biological function of the different HMOs, such as immune-modulation and protection against disease. This would allow us to predict whether the neonate would be, for instance, more prone to developing certain diseases, and to tailor her or his nutrition to fit their needs perfectly and pave the way to a personalized nutrition.

The Effect of Season on the Metabolic Profile of the European Clam Ruditapes decussatus as Studied by ¹H-NMR Spectroscopy.

In this study, the metabolome of Ruditapes decussatus, an economically and ecologically important marine bivalve species widely distributed in the Mediterranean region, was characterized by using proton Nuclear Magnetic Resonance (¹H-NMR) spectroscopy. Significant seasonal variations in the content of carbohydrates and free amino acids were observed. The relative amounts of alanine and glycine were found to exhibit the same seasonal pattern as the temperature and salinity at the harvesting site. Several putative sex-specific biomarkers were also discovered. Substantial differences were found for alanine and glycine, whose relative amounts were higher in males, while acetoacetate, choline and phosphocholine were more abundant in female clams. These findings reveal novel insights into the baseline metabolism of the European clam and represent a step forward towards a comprehensive metabolic characterization of the species. Besides providing a holistic view on the prominent nutritional components, the characterization of the metabolome of this bivalve represents an important prerequisite for elucidating the underlying metabolic pathways behind the environment-organism interactions.

Impact of Early Postnatal Nutrition on the NMR Urinary Metabolic Profile of Infant.

NMR-based metabolomics was used to compare the metabolic urinary profiles of exclusively breast-fed term infants (n = 11) with those of a double-blinded controlled trial with 49 formula-fed term newborns randomized to receive either an infant formula enriched by functional ingredients (n = 24) or a standard formula (n = 25). Anthropometric measurements and urine samples were taken at enrollment (within the first month of life), at around 60 days of life, and at the end of study period (average age of 130 days). The metabolic profiles were examined in relation to time and diet strategy. A common age-dependent modification of the urine metabolome was observed for the three types of nutrition, mainly characterized by similar temporal trends of choline, betaine, myoinositol, taurine, and citrate. Contrariwise, differences in the metabolic profiles were identified according to the type of diet (human versus formula milk), while no significant difference was observed between the two formulas. These modifications are discussed mainly in terms of the different milk compositions. Despite the low number of enrolled infants (n = 60), these findings pointed out the potential of the metabolomics approach for neonatal nutritional science, in particular to provide important contributions to the optimization of formula milk.

Metabolomics in necrotizing enterocolitis: the state of the art.

INTRODUCTION: Necrotizing enterocolitis (NEC) is a serious inflammation of the bowel that most often affects premature infants. The exact cause of NEC is still poorly understood but investigators believe that these different factors can play an important role: prematurity and immaturity of the intestine of the preterm newborns, differences in the intestinal bacterial colonization and an abnormal immune response. Since the early symptoms of NEC are often rather non-specific, the diagnosis of NEC can be difficult. Therefore, the search for diagnostic biomarkers for NEC remains warranted. This review focuses on the results of recent metabolomics investigations on NEC, providing important contributions to the understanding of the biochemical process characterizing this disease. Areas covered: Only a few metabolomics applications of NEC are reported in the literature. The metabolic pathway principally altered in NEC patients is suggested to be linked to the carbohydrates metabolism and the role of gluconate as a predictive marker for this pathology is investigated. Expert commentary: Biomarkers are crucial as a confident diagnostic tool to make NEC therapy more efficient. Although only a few metabolomics studies have been performed in NEC research, this technique proved to be helpful to understand the pathophysiological mechanisms of this disease.

Metabolic responses of clams, Ruditapes decussatus and Ruditapes philippinarum, to short-term exposure to lead and zinc.

This study investigated the effects of 48h heavy metal exposure upon the metabolic profiles of Ruditapes decussatus and Ruditapes philippinarum using (1)H NMR metabolomics. Both species were exposed to increasing concentrations of lead nitrate (10, 40, 60 and 100µg/L) and zinc chloride (20, 50, 100 and 150µg/L), under laboratory conditions. ICP-OES analysis was further performed on the clams' samples in order to verify the occurrence of heavy metal bioaccumulation. With respect to the controls, the metabolic profiles of treated R. decussatus exhibited higher levels of organic osmolytes and lower contents of free amino acids. An opposite behavior was shown by R. philippinarum. In terms of heavy metal, the exposure effects were more evident in the case of Pb rather than Zn. These findings show that NMR-based metabolomics has the required sensitivity and specificity for the identification of metabolites that can act as sensitive indicators of contaminant-induced stress.