Alberta Tomassini

Exploring human breast milk composition by NMR-based metabolomics

Breast milk is a complex fluid evolutionarily adapted to satisfy the nutritional requirements of growing infants. In addition, milk biochemical and immunological components protect newborns against infective agents in the new environment. Human milk oligosaccharides, the third most abundant component of breast milk, are believed to modulate the microbiota composition, thus influencing a wide range of physiological processes of the infant. Human milk also contains a number of other bioactive compounds, the functional role of which has not yet been clearly elucidated. In this scenario, NMR-based metabolic profiling can provide a rapid characterisation of breast milk composition, thus allowing a better understanding of its nutritional properties.

Effects of resveratrol on HepG2 cells as revealed by 1H-NMR based metabolic profiling

BACKGROUND Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis. METHODS We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a (1)H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways. RESULTS Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation. CONCLUSIONS Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation. GENERAL SIGNIFICANCE NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.

Monitoring of metabolic profiling and water status of Hayward kiwifruits by nuclear magnetic resonance

The metabolic profiling of kiwifruit (Actinidia deliciosa, Hayward cultivar) aqueous extracts and the water status of entire kiwifruits were monitored over the season (June-December) using nuclear magnetic resonance (NMR) methodologies. The metabolic profiling of aqueous kiwifruit extracts was investigated by means of high field NMR spectroscopy. A large number of water-soluble metabolites were assigned by means of 1D and 2D NMR experiments. The change in the metabolic profiles monitored over the season allowed the kiwifruit development to be investigated. Specific temporal trends of aminoacids, sugars, organic acids and other metabolites were observed. The water status of kiwifruits was monitored directly on the intact fruit measuring the T(2) spin-spin relaxation time by means of a portable unilateral NMR instrument, fully non-invasive. Again, clear trends of the relaxation time were observed during the monitoring period. The results show that the monitoring of the metabolic profiling and the monitoring of the water status are two complementary means suitable to have a complete view of the investigated fruit.

Peach Fruit: Metabolic Comparative Analysis of Two Varieties with Different Resistances to Insect Attacks by NMR Spectroscopy

The metabolite profile of aqueous extracts of two peach varieties, Percoca Romagnola 7 and Flaminia, with different susceptibilities to Ceratitis capitata attack was investigated by means of 1D and 2D high-field NMR spectroscopy. Water-soluble metabolites belonging to different classes such as organic acids (citric, fumaric, malic, quinic, shikimic, and succinic acids), sugars (fucose, fructose, fructose-6-phosphate, glucose, glucose-6-phosphate, rhamnose, sucrose, and xylose), amino acids (alanine, asparagine, isoleucine, threonine, and valine) and other metabolites such as myo-inositol, choline, trigonelline, catechin, chlorogenic and neochlorogenic acids, orthophosphate, and α-l-glycerophosphorylcholine were identified. The metabolite profile together with a suitable statistical analysis was used to make a comparison between the two varieties. The levels of glucose, xylose, myo-inositol, choline, isoleucine, and valine were found to be higher in Flaminia than in Percoca Romagnola 7 samples, whereas the levels of fumaric acid, alanine, quinic acid, sucrose, fucose, and chlorogenic and neochlorogenic acid were found to be higher in Percoca Romagnola 7 than in Flaminia samples.

Fecal and urinary NMR-based metabolomics unveil an aging signature in mice

BACKGROUND Aging is characterized by derangements in multiple metabolic pathways that progressively constrict the homeostatic reserve (homeostenosis). The signature of metabolic alterations that accompany aging can be retrieved through the metabolomic profiling of biological fluids. OBJECTIVE To characterize the age-related changes in urinary and fecal metabolic profiles of BALB/c mice through a (1)H nuclear magnetic resonance (NMR)-based metabolomic approach. METHODS Young (n=19) and old (n=13) male BALB/c mice were fed ad libitum standard laboratory chow. Twenty four-hour feces and urine were collected using metabolic cages and analyzed by high-resolution (1)H NMR spectroscopy combined with multivariate statistical analyses. RESULTS An age-related metabolic phenotype was detected both in urine and feces. The metabolic signature of aging consisted of changes in levels of metabolites associated with amino acid metabolism, tricarboxylic acid cycle, tryptophan-nicotinamide adenine dinucleotide pathway, and host-microbiota metabolic axis. CONCLUSIONS Our (1)H NMR-based metabolomic approach was able to characterize the effect of age on urinary and fecal metabotypes. The implementation of this analytical strategy may increase our understanding of the metabolic alterations involved in the aging process and assist in the design of anti-aging interventions.

Hepatocytes Entrapped in Alginate Gel Beads and Cultured in Bioreactor: Rapid Repolarization and Reconstitution of Adhesion Areas

The maintenance of the differentiated hepatocyte phenotype and its specific physiological properties is known to depend on several factors, such as chemical signals, cell-cell and extracellular matrix molecular interactions, as well as the use of three-dimensional matrices. The entrapment of hepatocytes within Ca-alginate at high cell density and the culture under continuous flow favour the development of three-dimensional organization and promote expression of the differentiated hepatic phenotype. This system could represent an improvement in hepatocyte cultivation for basic studies of liver physiology and metabolism; it could also be applicable in toxicology, hepatocyte transplantation or development of bioartificial organs. This report describes the effect of alginate entrapment and culture in a bioreactor on hepatocyte aggregate formation, with particular attention to the re-establishment of cell polarity, cell junctions and three-dimensional re-organization of the cytoskeleton. Oxygen supply and cell oxygen consumption rate were monitored in order to evaluate possible changes in hepatocyte energy requirement. Our data show that after only 6 h of perfusion in the bioreactor, actin and cytokeratin localize along the adhesion areas of the plasma membrane, in which reconstituted bile canaliculi were also observed. Moreover, the presence of connexin at the level of joined membranes of neighbouring cells suggests the establishment of gap junctions between hepatocytes. After the first 30 min of perfusion the oxygen consumption rate remained constant throughout the experimental period.

Metabolic profiling and outer pericarp water state in Zespri, CI.GI, and Hayward kiwifruits.

The metabolic profiling of aqueous extracts of Zespri Gold ( Actinidia chinensis ) and CI.GI (a controlled crossbreed from different species of Actinidia deliciosa ) kiwifruits and the water state of the outer pericarp of entire fruits were monitored over the season by means of high-field NMR spectroscopy and T(2) relaxation time measurements, respectively, and compared with the corresponding ones of Hayward kiwifruits previously investigated. A more complete assignment of the (1)H spectrum with respect to that obtained previously was reported: histidine, phenylalanine, quercetin 3-rhamnoside, and epicatechin were identified. Metabolic profiling confirmed Zespris earlier maturation compared with the two other varieties. The water state of entire kiwifruits was measured nondestructively on fruits attached to the plants or detached from the plants. T(2) relaxation times were found to be sensitive to the kiwifruit developmental stage.

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 Induce Different Age-Related Metabolic Profiles Revealed by 1H-NMR Spectroscopy in Urine and Feces of Mice

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 × 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age × treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction.

Assessment of Fecal Microbiota and Fecal Metabolome in Symptomatic Uncomplicated Diverticular Disease of the Colon.

Goal: The aim of this study was to assess fecal microbiota and metabolome in a population with symptomatic uncomplicated diverticular disease (SUDD). Background: Whether intestinal microbiota and metabolic profiling may be altered in patients with SUDD is unknown. Patients and Methods: Stool samples from 44 consecutive women [15 patients with SUDD, 13 with asymptomatic diverticulosis (AD), and 16 healthy controls (HCs)] were analyzed. Real-time polymerase chain reaction was used to quantify targeted microorganisms. High-resolution proton nuclear magnetic resonance spectroscopy associated with multivariate analysis with partial least-square discriminant analysis (PLS-DA) was applied on the metabolite data set. Results: The overall bacterial quantity did not differ among the 3 groups (P=0.449), with no difference in Bacteroides/Prevotella, Clostridium coccoides, Bifidobacterium, Lactobacillus, and Escherichia coli subgroups. The amount of Akkermansia muciniphila species was significantly different between HC, AD, and SUDD subjects (P=0.017). PLS-DA analysis of nuclear magnetic resonance -based metabolomics associated with microbiological data showed significant discrimination between HCs and AD patients (R2=0.733; Q2=0.383; P<0.05, LV=2). PLS analysis showed lower N-acetyl compound and isovalerate levels in AD, associated with higher levels of A. municiphila, as compared with the HC group. PLS-DA applied on AD and SUDD samples showed a good discrimination between these 2 groups (R2=0.69; Q2=0.35; LV=2). SUDD patients were characterized by low levels of valerate, butyrate, and choline and by high levels of N-acetyl derivatives and U1. Conclusions: SUDD and AD do not show colonic bacterial overgrowth, but a significant difference in the levels of fecal A. muciniphila was observed. Moreover, increasing expression of some metabolites as expression of different AD and SUDD metabolic activity was found.

Energy metabolism and re-establishment of intercellularadhesion complexes of gel entrapped hepatocytes

We studied the effect of continuous medium flow on the viabilityand structural organization of hepatocytes high density entrapped inalginate gel beads in the first few hours after isolation.The metabolic energy status of the entrapped cells, monitored invivo by 31P NMR spectroscopy, was stable during theexperimental time and a physiological redox ratio was reachedafter the first three hours of culture. The morphologicalanalysis revealed that the entrapped hepatocytes placed in a fixed-bed bioreactor under continuous flow showed a polyhedricalshape with numerous microvilli on cell surface and reconstitutedtight junctions as well as bile canalicular structures, closelyresembling those present in the liver.These results suggest that continuous flow allows the culture ofhepatocytes at very high cell density within a matrix withoutloss of viability and accelerates cellular tissue reconstructionat very short times after isolation. This type of culture couldrepresent a very useful model for physiological andtoxicological studies as well as a promising approach toward thedevelopment of a bioartificial hybrid support device in acuteliver failure.