Application of NMR-based metabolomics to the study of marine bivalves

This paper reviews the results of three studies on the application of high-field proton Nuclear Magnetic Resonance (1 H NMR) spectroscopy and metabolomics to the assessment of different issues related to marine bivalves. The outcomes of such applications yielded insights into multiple aspects concerning the impact of cold storage on shellfish quality and the effect of natural and anthropogenic stressors on bivalve metabolic profiles. In the first work, the effect of different cold storage conditions on the Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819) was investigated, revealing substantial time-related changes both in the metabolic profiles and microbial loads of stored mussels. In the second study, the seasonal change and reproductive cycle were demonstrated to be the leading causes behind the fluctuation in the amino acid and carbohydrate contents in the European clam Ruditapes decussatus (Linnaeus, 1758). Finally, NMR metabolomics was applied for assessing the effect of lead and zinc short-term exposure on the metabolic profiles of two clam species, Ruditapes decussatus and Ruditapes philippinarum (Adams & Reeve, 1850). The results evidenced a sensible metabolic response upon metal pollution, pointing out a main variability in the content of amino acids and organic osmolytes in relation to both metal nature and bivalve species

Clinical metabolomics and nutrition: the new frontier in neonatology and pediatrics.

In the pediatric clinic, nutritional research is focusing more and more on preventing the development of long-term diseases as well as supporting the repair processes important in the therapy of already fully developed diseases. Most children who are hospitalized or affected by chronic diseases could benefit from specific and careful attention to nutrition. Indeed, the state of nutrition modulates all body functions, including the different metabolic processes which, all together, have a profound effect on the development of the health and future of all individuals. Inappropriate food, even in the first periods of life, can accelerate the development of chronic metabolic diseases, especially in the pediatric age. To gain further insights into metabolic cycles and how they are connected with diet and health, nutrition and metabolomics interact to develop and apply modern technologies for metabolic assessment. In particular, nutritionists are evaluating the metabolomic approach to establish the single nutritional phenotypes, that is, the way in which diet interacts with individuals' metabolisms. This strategy offers the possibility of providing a complete definition of the individual's nutritional and health status, predict the risk of disease, and create metabolomic databases supporting the development of "personalized nutrition," in which diet is attuned to the nutritional needs of individual patients

Studio dell'impronta isotopica del deuterio di oli alimentari mediante la spettroscopia NMR del 2H e 1H

A report is given on 2H and 1H NMR investigations of a number of triacylglycerol mixtures of edible oils and squalene samples extracted from extra virgin olive oil, shark liver and synthetic. The average numbers of hydrogen and deuterium for the olefinic, methylene and methyl sites were determined, and some parameters describing the different fractionation of deuterium in the various sites were obtained. The results have been discussed in terms of the biosynthesis steps involved in the elongation and desaturation processes of the fatty acids, and in the condensation and reduction processes of the squalene. From a principal components analysis distinct domains have been found for the squalene samples from shark liver, olive oil and synthetic

Exploring the effect of vitamin D and DHA supplementation on the urine metabolome of preterm infants by 1H NMR-based metabolomics

Background and objectives: Vitamin D and docosahexaenoic acid (DHA) insufficiency and deficiency could potentially have a great impact on health outcomes in preterm infant. Due to the importance of early nutrition intervention in this population and given the lack of metabolomic studies concerning the supplementations effect on the metabolome of preterm infants, 44 premature infants were studied, divided in two groups, one receiving only vitamin D (DS) and the second both vitamin D and DHA (D-DHAS) supplementation. Two were the main objectives of the study: 1) to look at changes over time in the urinary metabolic profiles of infants before and over two months of supplementation; 2) to compare the urinary metabolome of the two groups after supplementation. Methods: 1H NMR-based metabolomics approach was used to analyze urine samples obtained from preterm newborns at three different time points: at the time of hospital discharge and before supplementation (T0), 1 month (T1) and 2 months (T2) after the beginning of supplementation. Results: A clear temporal dynamics of the urinary metabolic profiles of preterm infants was highlighted by OPLS analysis. Both groups were characterized by growing levels of betaine, N,N-dimethylglycine, creatinine, creatine and guanidinoacetate and diminishing levels of myo-inositol and hydroxyproline with increasing postmenstrual age (PMA). Additionally, for D-DHAS citrate and dimethylamine increased, while lactate decreased over time. OPLS-DA clearly discriminated the two groups after two months of supplementation. Compared to DS, D-DHAS group was characterized by higher levels of betaine, N,N-dimethylglycine, creatinine and dimethylamine and lower amounts of lactate and myo-inositol. Conclusions: Metabolomic analysis of urine from the neonatal period could be a useful tool to understand metabolic processes linked to early nutrition and supplementation. According to our results, vitamin D supplementation exerts in preterm newborns positive effects evaluated with urinary metabolomics. Moreover, it seems that the supplementation with vitamin D and DHA exerts a higher antioxidant and protective action on newborns, and it could also positively affect the body fat composition

Evaluation of the Antioxidant and Cytotoxic Activities on Cancer Cell Line of Extracts of Parasitic Plants Harvested in Tunisia

Weeds thrive in agricultural environments. However, in certain areas of the world, they are consumed by humans as food, and they can represent a source of valuable active ingredients of ethnomedical interest. In this study, three North African parasitic plants of the Orobanchaceae family, Cistanche violacea, Orobanche crenata and Orobanche lavandulacea, normally considered as weeds, were studied to compare their biological activities. The antioxidant activity of hexane, ethyl acetate, acetone, methanol, and water extracts obtained by sequential maceration was estimated by DPPH, ABTS and FRAP assays. The metabolic profi les of aqueous extracts were studied by 1H NMR spectroscopy. The oils extracted using hexane as solvent were analyzed by GC-FID and HPLC-DAD to characterize the fatty acid profi le. The cytotoxic effect of the oil from the parasitic plants was also studied on B16F10 melanoma cancer cells. All extracts showed antioxidant activity and differences between extracts obtained using different solvents as well as for different plants have been highlighted. The highest total phenolic content was determined in O. crenata acetone extract (3.53±0.20 mmol GAE/g) and in O. lavandulacea methanol extract (2.29±0.02 mmol GAE/g). 1H NMR spectroscopy revealed the presence of amino acids, organic acids and carbohydrates. The major fatty acids in the oils were 18:2 n-6, 16:0, 18:1 n-9, and 18:3 n-3. O. lavandulacea revealed to be a highly cytotoxic species for B16F10 melanoma cells. These data suggest that, despite the extreme climate conditions and the damage that can result, the studied desert parasitic plants can represent a novel and precious source of metabolites, with promising biological activities

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 Clarktype 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

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 H-1 NMR spectroscopy, while the cholesterol, alpha-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 10(8)-10(9) 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

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

Cholinium-Based Ionic Liquids from Hydroxycinnamic Acids as New Promising Bioactive Agents: A Combined Experimental and Theoretical Investigation

Cholinium-based ionic liquids (Cho-ILs) are very attractive compounds for medicinal and pharmaceutical applications as bioavailability enhancers in drug formulations and active components in pharmaceutical ingredients. In this study, we synthesized six Cho-ILs from hydroxycinnamic acids (HCA) ([Cho][HCA] ILs), a group of bioactive compounds with poor water solubility. [Cho][HCA] ILs and their parent acids were evaluated for solubility, thermal stability, and antioxidant activity. Furthermore, [Cho][HCA] ILs were screened for their cytotoxicity. To rationalize the experimental antioxidant activities, density functional theory (DFT) calculations were performed. The theoretical approach allowed for identification of the most likely radical scavenging mechanisms involving HCAs and the corresponding ionic forms under the studied experimental conditions and to rationalize the observed activity differences between salts and acids. Overall, our results revealed a higher solubility and free-radical scavenging activity for [Cho][HCA] ILs than corresponding HCAs, a relatively high thermal stability (melting temperature > 100 °C) and negligible cytotoxicity activity. Furthermore, DFT calculations showed that both the hydrogen atom transfer and the sequential proton loss electron transfer mechanisms are likely to occur in aqueous and ethanolic solutions. The picture emerging from our results supports the increasingly expressed idea that [Cho][HCA]ILs are promising candidates for applications in pharmaceutical formulatio

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 by1H 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