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Modeling aging and age-related pathologies presents a substantial analytical challenge given the complexity of gene–environment influences and interactions operating on an individual. A top-down systems approach is used to model the effects of lifelong caloric restriction, which is known to extend life span in several animal models. The metabolic phenotypes of caloric-restricted (CR; n= 24) and pair-housed control-fed (CF; n= 24) Labrador Retriever dogs were investigated by use of orthogonal projection to latent structures discriminant analysis (OPLS-DA) to model both generic and age-specific responses to caloric restriction from the 1H NMR blood serum profiles of young and older dogs. Three aging metabolic phenotypes were resolved: (i) an aging metabolic phenotype independent of diet, characterized by high levels of glutamine, creatinine, methylamine, dimethylamine, trimethylamine N-oxide, and glycerophosphocholine and decreasing levels of glycine, aspartate, creatine and citrate indicative of metabolic changes associated largely with muscle mass; (ii) an aging metabolic phenotype specific to CR dogs that consisted of relatively lower levels of glucose, acetate, choline, and tyrosine and relatively higher serum levels of phosphocholine with increased age in the CR population; (iii) an aging metabolic phenotype specific to CF dogs including lower levels of liproprotein fatty acyl groups and allantoin and relatively higher levels of formate with increased age in the CF population. There was no diet metabotype that consistently differentiated the CF and CR dogs irrespective of age. Glucose consistently discriminated between feeding regimes in dogs (≥312 weeks), being relatively lower in the CR group. However, it was observed that creatine and amino acids (valine, leucine, isoleucine, lysine, and phenylalanine) were lower in the CR dogs (<312 weeks), suggestive of differences in energy source utilization. 1H NMR spectroscopic analysis of longitudinal serum profiles enabled an unbiased evaluation of the metabolic markers modulated by a lifetime of caloric restriction and showed differences in the metabolic phenotype of aging due to caloric restriction, which contributes to longevity studies in caloric-restricted animals. Furthermore, OPLS-DA provided a framework such that significant metabolites relating to life extension could be differentiated and integrated with aging processes.

Personalized nutrition has been traditionally based on the adjustment of food and diet according to individual needs and preferences. At present, this concept is being reinforced through the application of state-of-the-art high-throughput technologies to help understand the molecular mechanisms underlying a healthy state. This knowledge could enable the adjustment of general dietary recommendations to match the needs of specific population groups based on their molecular profiles. The optimal development of evidence-based nutritional guidance to promote health requires an adequate assessment of nutrient bioavailability, bioactivity, and bioefficacy. To achieve this, reliable information about exposure to nutrients, their intake, and functional effects is required; thus, the identification of valid biomarkers using standardized analytical procedures is necessary. Although some nutritional biomarkers are now successfully used (eg, serum retinol, zinc, ferritin, and folate), a comprehensive set to assess the nutritional status and metabolic conditions of nutritional relevance is not yet available. Also, there is very limited knowledge on how the extensive human genetic variability influences the interpretation of these biomarkers. In this context, nutrigenomics seems to be a promising approach to identify new biomarkers in nutrition, through an integrative application of transcriptomics, proteomics, metabolomics, epigenomics, and nutrigenetics in human nutritional research.

The feline odontoclastic resorptive lesion (FORL) is a common oral problem in cats. The disease has increased steadily since the domestication of cats and etiology of this disease has not been fully determined although several theories have been proposed. Feeding practices, vaccination, and neutering programs have all been suspected to be associated with FORL. The aim of the current study is to assess the feasibility of metabonomics to detect at an early stage the onset of the disease. The diagnostic biomarkers could then be used as “efficacy markers” for nutritional intervention in preventing and/or slowing the progression of FORL. 1H-NMR- and LC/MS-based metabonomic analysis of saliva samples obtained from a group of 21 cats (11 healthy and 10 FORL diseased) showed clear differences in the metabolic composition of saliva from healthy and FORL-diseased cats. To identify biomarkers, the spectroscopic data was processed using partial least-squares discriminant analysis (PLS-DA) and validated by leave-one-subject-out cross validation. The PLS-DA model predicted FORL- diseased cats with over 60% accuracy. The maximum value of Q2of the random permutation sets was less than 0.3. The diseased cats showed increased levels of many organic and amino acids, such as acetate, lactate, propionate, isovalerate, tryptamine, and phenylalanine suggesting changes in oral microflora in the disease situation. This study is preliminary and a larger study with more samples to further validate the biomarker profile predictive of an early FORL pathophysiological status is in progress.

Irritable Bowel Syndrome (IBS) is a common multifactorial intestinal disorder for which the aetiology remains largely undefined. Here, we have used a Trichinella spiralis(T. spiralis)-induced model of post-infective IBS, and the effects of probiotic bacteria on gut dysfunction have been investigated using a metabonomic strategy. A total of 44 mice were divided into four groups:  an uninfected control group and three T. spiralis-infected groups, one as infected control and the two other groups subsequently treated with either Lactobacillus paracasei(L. paracasei) NCC2461 in spent culture medium (SCM) or with L. paracasei-free SCM. Plasma, jejunal wall and longitudinal myenteric muscle samples were collected at day 21 post-infection. An NMR-based metabonomic approach characterized that the plasma metabolic profile of T. spiralis-infected mice showed an increased energy metabolism (lactate, citrate, alanine), fat mobilization (acetoacetate, 3-D-hydroxybutyrate, lipoproteins) and a disruption of amino acid metabolism due to increased protein breakdown, which were related to the intestinal hypercontractility. Increased levels of taurine, creatine and glycerophosphorylcholine in the jejunal muscles were associated with the muscular hypertrophy and disrupted jejunal functions. L. paracasei treatment normalized the muscular activity and the disturbed energy metabolism as evidenced by decreased glycogenesis and elevated lipid breakdown in comparison with untreated T. spiralis-infected mice. Changes in the levels of plasma metabolites (glutamine, lysine, methionine) that might relate to a modulation of immunological responses were also observed in the presence of the probiotic treatment. The work presented here suggests that probiotics may be beneficial in patients with IBS.

Stress in the form of moderate periods of maternal separation of newborn rats has been postulated to cause permanent changes in the central nervous system and diseases in later life. It is also considered that dietary supplementation with long chain polyunsaturated fatty acids (LC-PUFAs) can potentially ameliorate the effects of stress. The metabolic consequences of early life maternal separation stress were investigated in rats (2−14 days after birth), either alone or in combination with secondary acute water avoidance stress at 3−4 months of age. The effect of a LC-PUFA-enriched dietary intervention in stressed animals was also assessed. Systematic changes in metabolic biochemistry were evaluated using 1H nuclear magnetic resonance spectroscopy of blood plasma and multivariate pattern recognition techniques. The biochemical response to stress was characterized by decreased levels of total lipoproteins and increased levels of amino acids, glucose, lactate, creatine, and citrate. Secondary acute water avoidance stress also caused elevated levels of O-acetyl glycoproteins in blood plasma. LC-PUFAs dietary enrichment did not alter the metabolic response to stress, but did result in a modified lipoprotein profile. This work indicates that the different stressor types resulted in some common systemic metabolic responses that involve changes in energy and muscle metabolism, but that they are not reversible by dietary intervention.

Abstract: Statistical model validation tools such as cross-validation, jack-knifing model parameters and permutation tests are meant to obtain an objective assessment of the performance and stability of a statistical model. However, little is known about the performance of these tools for megavariate data sets, having, for instance, a number of variables larger than 10 times the number of subjects. The performance is assessed for megavariate metabolomics data, but the conclusions also carry over to proteomics, transcriptomics and many other research areas. Partial least squares discriminant analyses models were built for several LC-MS lipidomic training data sets of various numbers of lean and obese subjects. The training data sets were compared on their modelling performance and their predictability using a 10-fold cross-validation, a permutation test, and test data sets. A wide range of cross-validation error rates was found (from 7.5% to 16.3% for the largest trainings set and from 0% to 60% for the smallest training set) and the error rate increased when the number of subjects decreased. The test error rates varied from 5% to 50%. The smaller the number of subjects compared to the number of variables, the less the outcome of validation tools such as cross-validation, jack-knifing model parameters and permutation tests can be trusted. The result depends crucially on the specific sample of subjects that is used for modelling. The validation tools cannot be used as warning mechanism for problems due to sample size or to representativity of the sampling.

The introduction of molecular tools in food research offers the possibility to the food industry to benefit from the experience gained in the field by pharmaceutical companies. In this work we are showing how in silico virtual screening techniques based on molecular similarity were applied for identifying novel umami-tasting compounds. The results obtained suggest that 5‘-ribonucleotides and monosodium glutamate might elicit the fifth basic taste via the same molecular mechanism. New algorithms were developed and used in this work, such as the dimension reduction of data sets by singular value decomposition and the introduction of the correlation dimension as a natural dimension of a chemical space. It is shown that the representations of molecular data sets in chemical spaces possess self-similar properties, characteristic of fractal objects.

Bacillus amyloliquefaciens α-amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of α-amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.

Modification of liquefying a-amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. The photo-oxidation followed pseudo-first-order kinetics giving maximal value at pH 8.0. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. Diethylpyrocarbonate at low concentration at pH 6.0 and 30 ° C completely inactivated a-amylase. Inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by diethylpyrocarbonate was one, thus indicating modification of a single histidine per mole of the enzyme. Diethylpyrocarbonate-modified enzyme showed increased absorbance at 240 nm which was reversed completely upon treatment with NH2OH at 30 °C for 16 hr. Calculating the histidine residues being modified from the increase in absorbance at 240 nm showed that three residues were ethoxyformylated on treatment with diethylpyrocarbonate, of which only one was found at the active site. Substrate and competitive inhibitor protects the enzyme against both, photo-oxidation, and modification by diethylpyrocarbonate, confirming that histidine plays an essential role at the a-amylase active site.

Bacillus amyloliquefaciens a-amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of a-amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.

An extracellular α-amylase is purified to homogeneity with 62 % recovery of the enzyme activity using heat treatment, ion-exchange and gel filtration chromatographies. The purified enzyme has a molecular weight of 68, 000, isoelectric point 6.25, optimal activity at pH 6 and temperature 65 °C, kest8.8×108s1liquefying amylase units, and Kmfor starch 2.9 mg ml−1. The enzyme is further characterized for its endo action on the starch and related polymers. Calcium stabilizes the active conformation of the enzyme during prolonged exposure to the extremes of pH and temperature. The enzyme retains 100 % activity for 24 h at 65°C and exhibits a half-life of 9, 3, and 0.5 h at 80°, 85° and 90°C, respectively.

ABSTRACTThe gut microbiota enhances the host’s metabolic capacity for processing nutrients and drugs and modulate the activities of multiple pathways in a variety of organ systems. We have probed the systemic metabolic adaptation to gut colonization for 20 days following exposure of axenic mice (n= 35) to a typical environmental microbial background using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy to analyze urine, plasma, liver, kidney, and colon (5 time points) metabolic profiles. Acquisition of the gut microbiota was associated with rapid increase in body weight (4%) over the first 5 days of colonization with parallel changes in multiple pathways in all compartments analyzed. The colonization process stimulated glycogenesis in the liver prior to triggering increases in hepatic triglyceride synthesis. These changes were associated with modifications of hepatic Cyp8b1 expression and the subsequent alteration of bile acid metabolites, including taurocholate and tauromuricholate, which are essential regulators of lipid absorption. Expression and activity of major drug-metabolizing enzymes (Cyp3a11 and Cyp2c29) were also significantly stimulated. Remarkably, statistical modeling of the interactions between hepatic metabolic profiles and microbial composition analyzed by 16S rRNA gene pyrosequencing revealed strong associations of the Coriobacteriaceaefamily with both the hepatic triglyceride, glucose, and glycogen levels and the metabolism of xenobiotics. These data demonstrate the importance of microbial activity in metabolic phenotype development, indicating that microbiota manipulation is a useful tool for beneficially modulating xenobiotic metabolism and pharmacokinetics in personalized health care.IMPORTANCEGut bacteria have been associated with various essential biological functions in humans such as energy harvest and regulation of blood pressure. Furthermore, gut microbial colonization occurs after birth in parallel with other critical processes such as immune and cognitive development. Thus, it is essential to understand the bidirectional interaction between the host metabolism and its symbionts. Here, we describe the first evidence of an in vivoassociation between a family of bacteria and hepatic lipid metabolism. These results provide new insights into the fundamental mechanisms that regulate host-gut microbiota interactions and are thus of wide interest to microbiological, nutrition, metabolic, systems biology, and pharmaceutical research communities. This work will also contribute to developing novel strategies in the alteration of host-gut microbiota relationships which can in turn beneficially modulate the host metabolism.