Metabolic maturation in the infant urine during the first 3 months of life.

The infant urine metabolome provides a body metabolic snapshot, and the sample collection can be done without stressing the fragile infant. 424 infant urine samples from 157 infants were sampled longitudinally at 1-, 2-, and 3 months of age. 49 metabolites were detected using proton nuclear magnetic resonance spectroscopy. Data were analyzed with multi- and univariate statistical methods to detect differences related to infant age-stage, gestational age, mother's pre-pregnancy BMI, C-section, infant birth weight, and infant sex. Significant differences were identified between age-stage (pbonferoni < 0.05) in 30% (15/49) of the detected metabolites. Urine creatinine increased significantly from 1 to 3 months. In addition, myo-inositol, taurine, methionine, and glucose seem to have conserved levels within the individual over time. We calculated a urine metabolic maturation age and found that the metabolic age at 3 months is negatively correlated to weight at 1 year. These results demonstrate that the metabolic maturation can be observed in urine metabolome with implications on infant growth and specifically suggesting that the systematic age effect on creatinine promotes caution in using this as normalization of other urine metabolites.

Thermal degradation of metabolites in urine using multiple isotope-labelled internal standards for off-line GC metabolomics - effects of injector and oven temperatures.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). An optimized derivatization protocol has been successfully applied using multiple isotope labelled analytical internal standards of selected deuterated and 13C selected compounds, covering a range of different groups of metabolites for non-automated GC metabolomics (off-line). Moreover, the study was also realized in a pooled urine sample, following metabolic profiling. A study of thermal degradation of metabolites due to GC inlet and oven programs (fast, slow) was performed, where the results indicated that both GC oven programs (fast and slow) negatively affected the thermal stability of the metabolites, while the fast-ramp GC program also suppressed MS signals. However, the use of multiple internal standards can overcome this drawback. The application of extended temperature ramp GC program presented identical behaviour on metabolite stability and better chromatographic separation combined with much lower signal suppression, compared to a short temperature ramp program. No effects were observed for organic acids, fatty acids, sugars and sugar alcohols, while significant differences were observed for amino acids. GC metabolomics is a strong tool that can facilitate analysis, but special attention is required for sampling handling and heating, before and during the GC analysis. The use and application of multiple multi-group internal standards is highly recommended.

Metabolic Effects of Bovine Milk Oligosaccharides on Selected Commensals of the Infant Microbiome-Commensalism and Postbiotic Effects.

Oligosaccharides from human or bovine milk selectively stimulate growth or metabolism of bacteria associated with the lower gastrointestinal tract of infants. Results from complex infant-type co-cultures point toward a possible synergistic effect of combining bovine milk oligosaccharides (BMO) and lactose (LAC) on enhancing the metabolism of Bifidobacterium longum subsp. longum and inhibition of Clostridium perfringens. We examine the interaction between B. longum subsp. longum and the commensal Parabacteroides distasonis, by culturing them in mono- and co-culture with different carbohydrates available. To understand the interaction between BMO and lactose on B. longum subsp. longum and test the potential postbiotic effect on C. perfringens growth and/or metabolic activity, we inoculated C. perfringens into fresh media and compared the metabolic changes to C. perfringens in cell-free supernatant from B. longum subsp. longum fermented media. In co-culture, B. longum subsp. longum benefits from P. distasonis (commensalism), especially in a lactose-rich environment. Furthermore, B. longum subsp. longum fermentation of BMO + LAC impaired C. perfringens' ability to utilize BMO as a carbon source (potential postbiotic effect).

Predictive ability of host genetics and rumen microbiome for subclinical ketosis.

Subclinical metabolic disorders such as ketosis cause substantial economic losses for dairy farmers in addition to the serious welfare issues they pose for dairy cows. Major hurdles in genetic improvement against metabolic disorders such as ketosis include difficulties in large-scale phenotype recording and low heritability of traits. Milk concentrations of ketone bodies, such as acetone and ß-hydroxybutyric acid (BHB), might be useful indicators to select cows for low susceptibility to ketosis. However, heritability estimates reported for milk BHB and acetone in several dairy cattle breeds were low. The rumen microbial community has been reported to play a significant role in host energy homeostasis and metabolic and physiologic adaptations. The current study aims at investigating the effects of cows' genome and rumen microbial composition on concentrations of acetone and BHB in milk, and identifying specific rumen microbial taxa associated with variation in milk acetone and BHB concentrations. We determined the concentrations of acetone and BHB in milk using nuclear magnetic resonance spectroscopy on morning milk samples collected from 277 Danish Holstein cows. Imputed high-density genotype data were available for these cows. Using genomic and microbial prediction models with a 10-fold resampling strategy, we found that rumen microbial composition explains a larger proportion of the variation in milk concentrations of acetone and BHB than do host genetics. Moreover, we identified associations between milk acetone and BHB with some specific bacterial and archaeal operational taxonomic units previously reported to have low to moderate heritability, presenting an opportunity for genetic improvement. However, higher covariation between specific microbial taxa and milk acetone and BHB concentrations might not necessarily indicate a causal relationship; therefore further validation is needed before considering implementation in selection programs.

An Integrated Multi-Omics Analysis Defines Key Pathway Alterations in a Diet-Induced Obesity Mouse Model.

Obesity is a multifactorial disease with many complications and related diseases and has become a global epidemic. To thoroughly understand the impact of obesity on whole organism homeostasis, it is helpful to utilize a systems biological approach combining gene expression and metabolomics across tissues and biofluids together with metagenomics of gut microbial diversity. Here, we present a multi-omics study on liver, muscle, adipose tissue, urine, plasma, and feces on mice fed a high-fat diet (HFD). Gene expression analyses showed alterations in genes related to lipid and energy metabolism and inflammation in liver and adipose tissue. The integration of metabolomics data across tissues and biofluids identified major differences in liver TCA cycle, where malate, succinate and oxaloacetate were found to be increased in HFD mice. This finding was supported by gene expression analysis of TCA-related enzymes in liver, where expression of malate dehydrogenase was found to be decreased. Investigations of the microbiome showed enrichment of Lachnospiraceae, Ruminococcaceae, Streptococcaceae and Lactobacillaceae in the HFD group. Our findings help elucidate how the whole organism metabolome and transcriptome are integrated and regulated during obesity.

Background Diet Influences TMAO Concentrations Associated with Red Meat Intake without Influencing Apparent Hepatic TMAO-Related Activity in a Porcine Model.

Red meat has been associated with an increased cardiovascular disease (CVD) risk, possibly through gut microbial-derived trimethylamine-N-oxide (TMAO). However, previous reports are conflicting, and influences from the background diet may modulate the impact of meat consumption. This study investigated the effect of red and white meat intake combined with two different background diets on urinary TMAO concentration and its association with the colon microbiome in addition to apparent hepatic TMAO-related activity. For 4 weeks, 32 pigs were fed chicken or red and processed meat combined with a prudent or western background diet. 1H NMR-based metabolomics analysis was conducted on urine samples and hepatic mRNA expression of TMAO-related genes determined. Lower urinary TMAO concentrations were observed after intake of red and processed meat when consumed with a prudent compared to a western background diet. In addition, correlation analyses between urinary TMAO concentrations and relative abundance of colon bacterial groups suggested an association between TMAO and specific bacterial taxa. Diet did not affect the hepatic mRNA expression of genes related to TMAO formation. The results suggest that meat-induced TMAO formation is regulated by mechanisms other than alterations at the hepatic gene expression level, possibly involving modulations of the gut microbiota.

NMR-Based Metabolomics of Food.

NMR spectroscopy is one of the major analytical techniques used in the metabolomics studies of food. There are many applications of metabolomics on food-related topics and on the food itself. Here, we describe protocols for performing NMR-based metabolomics of foods ranging from simple beverages to solid foods and semisolid foods. Beverages can be analyzed either directly or after sample preprocessing to remove interfering macromolecules, muscle-based foods can be analyzed after extraction, and semisolid foods can be analyzed directly using high-resolution magic-angle spinning (HR-MAS) NMR. Finally, we discuss metabolomic data analysis as well as different procedures and strategies for targeted and untargeted approaches.

Lactose and Bovine Milk Oligosaccharides Synergistically Stimulate B. longum subsp. longum Growth in a Simplified Model of the Infant Gut Microbiome.

Increasing awareness of the importance of a healthy Bifidobacterium-rich microbiome has led to a need for more knowledge on how different prebiotic carbohydrates specifically impact the infant microbiome, especially as a community instead of single bacterial targets. In this study, we combined proton nuclear magnetic resonance (1H NMR) metabolomics and molecular biology methods for quantification of bacteria to compare the prebiotic effect of bovine milk oligosaccharides (BMO) and synthetic galacto oligosaccharides (GOS) using mono- and cocultures of eight major bacteria related to a healthy infant microbiome. The results revealed that BMO treatments supported growth of Bifidobacterium longum subsp. longum and Parabacteroides distasonis, while at the same time growth of Clostridium perfringens and Escherichia coli was inhibited. In addition, there was a synergistic effect of combining lactose and BMO in regards to reducing C. perfringens, maintaining stable numbers of P. distasonis and simultaneously increasing numbers of the beneficial B. longum subsp. longum. These results indicate that the oligosaccharide composition plays a vital role in shaping the developing microbiota.

Influence of herd diet on the metabolome of Maasdam cheeses.

The untargeted metabolic profiles of ripened Maasdam cheese samples prepared from milk derived from three herd groups, fed: (1) indoors on total mixed ration (TMR), or outdoors on (2) grass only pasture (GRA) or (3) grass and white clover pasture (CLO) were studied using high resolution nuclear magnetic resonance (1H NMR), high resolution magic angle spinning nuclear magnetic resonance (1H HRMAS NMR) and headspace (HS) gas chromatography mass spectrometry (GC-MS). A total of 31 compounds were identified using 1H NMR and 32 volatile compounds including 7 acids, 5 esters, 4 alcohols, 4 ketones, 4 sulfur compounds, 2 aldehydes, 3 hydrocarbons, 2 terpenes and a lactone were identified using GC-MS in Maasdam cheeses ripened for 97-d. On comparing the 1H NMR metabolic profiles, TMR-derived cheese had higher levels of citrate compared to GRA-derived cheese. The toluene content of cheese was significantly higher in GRA or CLO compared to TMR cheeses and dimethyl sulfide was identified only in CLO-derived cheese samples as detected using HS GC-MS. These compounds are proposed as indicator compounds for Maasdam cheese derived from pasture-fed milk. Clear differences between outdoor or indoor feeding systems in terms of cheese metabolites were detected in the lipid phase, as indicated by principal component analysis (PCA) from 1H HRMAS NMR spectra, although differences based on PCA of all 1H NMR spectra and HS-GC-MS were less clear. Overall, this study presented the metabolite profile and identified specific compounds which may be useful for discriminating between ripened Maasdam cheese and related cheese varieties manufactured from indoor or outdoor herd-feeding systems.

Salmon in Combination with High Glycemic Index Carbohydrates Increases Diet-Induced Thermogenesis Compared with Salmon with Low Glycemic Index Carbohydrates⁻An Acute Randomized Cross-Over Meal Test Study.

The study investigated the acute effects of meals containing either salmon or veal in combination with carbohydrates with high or low glycemic index (GI) on diet-induced thermogenesis (DIT) (primary endpoint), appetite sensations, and energy intake (EI). Twenty-five overweight men and women ingested four iso-caloric test meals: salmon with mashed potatoes (high GI) (SM), salmon with wholegrain pasta (low GI) (SP), veal with mashed potatoes (VM) and veal with wholegrain pasta (VP). Energy expenditure was measured in the fasting state and six times postprandially for 25 min with 5-min breaks between each measurement. Appetite sensations were measured every 30 min. Blood samples, from arterialized venous blood, were drawn every 20 min until an ad libitum buffet-style lunch was served 3.5 h later. DIT was 40% higher after the SM meal compared to the SP meal (p = 0.002). Prospective food consumption was lower after the SM meal compared with the VP meal (p = 0.01). There were no differences in satiety, hunger, fullness, or ad libitum EI between the test meals (all p > 0.05). In conclusion, salmon with high GI carbohydrates increased DIT compared to salmon with low GI carbohydrates. This indicates that DIT is sensitive to the GI of the carbohydrates after intake of salmon but not veal.

Quantification of Human Milk Phospholipids: the Effect of Gestational and Lactational Age on Phospholipid Composition.

Human milk (HM) provides infants with macro- and micronutrients needed for growth and development. Milk phospholipids are important sources of bioactive components, such as long-chain polyunsaturated fatty acids (LC-PUFA) and choline, crucial for neural and visual development. Milk from mothers who have delivered prematurely (<37 weeks) might not meet the nutritional requirements for optimal development and growth. Using liquid chromatography tandem-mass spectrometry, 31 phospholipid (PL) species were quantified for colostrum (<5 days postpartum), transitional (≥5 days and ≤2 weeks) and mature milk (>2 weeks and ≤15 weeks) samples from mothers who had delivered preterm (n = 57) and term infants (n = 22), respectively. Both gestational age and age postpartum affected the PL composition of HM. Significantly higher concentrations (p < 0.05) of phosphatidylcholine (PC), sphingomyelin (SM) and total PL were found in preterm milk throughout lactation, as well as significantly higher concentrations (p < 0.002) of several phosphatidylethanolamine (PE), PC and SM species. Multivariate analysis revealed that PLs containing LC-PUFA contributed highly to the differences in the PL composition of preterm and term colostrum. Differences related to gestation decreased as the milk matured. Thus, gestational age may impact the PL content of colostrum, however this effect of gestation might subside in mature milk.

The effect of casein, hydrolyzed casein, and whey proteins on urinary and postprandial plasma metabolites in overweight and moderately obese human subjects.

BACKGROUND: Casein and whey proteins differ in amino acid composition and absorption rate; however, the absorption rate of casein can be increased to mimic that of whey proteins by exogenous hydrolysis. In view of these compositional differences, we studied the metabolic responses to intake of casein, hydrolyzed casein, and whey proteins in overweight and moderately obese men and women by investigating select urinary and blood plasma metabolites. RESULTS: A total of 21 urinary and 23 plasma metabolites were identified by nuclear magnetic resonance spectroscopy. The postprandial plasma metabolites revealed a significant diet-time interaction for isoleucine (P = 0.001) and tyrosine (P = 0.001). The level of isoleucine and tyrosine peaked 90 min postprandially with a 1.4-fold difference following intake of whey proteins compared with either casein or hydrolyzed casein. A 1.2-fold higher urinary level of lactate was observed after intake of whey proteins compared with intake of intact casein (P < 0.01). CONCLUSION: The plasma metabolites revealed different amino acid profiles reflecting the amino acid composition of casein and whey proteins. Furthermore, the results support that casein hydrolysates neither affect the postprandial amino acid absorption rate nor the amino acid level compared with that of intact casein. The urinary lactate increases following whey protein intake might indicate a higher metabolism of glucogenic amino acids. © 2018 Society of Chemical Industry.

A Comprehensive Approach to Assess Feathermeal as an Alternative Protein Source in Aquafeed.

The effect of partially replacing fishmeal in aquafeed with feathermeal (FTH) at three levels (0%: FTH0, 8%: FTH8, 24%: FTH24) and two extrusion temperatures (100 and 130 °C) was evaluated in rainbow trout (Oncorhynchus mykiss) with respect to growth performance, metabolism response, and oxidative status of the feed proteins. Multivariate data analyses revealed that FTH24 correlated positively with high levels of oxidation products, amino acids (AA) racemization, glucogenic AAs level in liver, feed intake (FI), specific growth rate (SGR), and feed conversion ratio (FCR); and low AAs digestibility. Both FI and SGR were significantly increased when 8 and 24% feathermeal was included in the feed extruded at 100 °C, while there was a negative effect on FCR in fish fed FTH24. In conclusion, higher oxidation levels in FTH24 may give rise to metabolic alterations while lower levels of FTH may be considered as fishmeal substitute in aquafeed for rainbow trout.

High resolution magic angle spinning NMR spectroscopy reveals that pectoralis muscle dystrophy in chicken is associated with reduced muscle content of anserine and carnosine.

Increased incidences of pectoralis muscle dystrophy are observed in commercial chicken products, but the muscle physiological causes for the condition remain to be identified. In the present study a high-resolution magic angle spinning (HR-MAS) proton ((1)H) NMR spectroscopic examination of intact pectoralis muscle samples (n=77) were conducted to explore metabolite perturbations associated with the muscle dystrophy condition for the very first time. Both in chicken with an age of 21 and 31days, respectively, pectoralis muscle dystrophy was associated with a significantly lower content of anserine (p=0.034), carnosine (p=0.019) and creatine (p=0.049). These findings must be considered intriguing as they corroborate that characteristic muscle di-peptides composed of ß-alanine and histidine derivatives such as anserine are extremely important in homeostasis of contractile muscles as a results of their role as buffering, anti-oxidative, and anti-glycation capacities.

1H-13C NMR-Based Profiling of Biotechnological Starch Utilization.

Starch is used in food- and nonfood applications as a renewable and degradable source of carbon and energy. Insight into the chemical detail of starch degradation remains challenging as the starch constituents amylose and amylopectin are homopolymers. We show that considerable molecular detail of starch fragmentation can be obtained from multivariate analysis of spectral features in optimized 1H-13C NMR spectroscopy of starch fragments to identify relevant features that distinguish processes in starch utilization. As a case study, we compare the profiles of starch fragments in commercial beer samples. Spectroscopic profiles of homooligomeric starch fragments can be excellent indicators of process conditions. In addition, differences in the structure and composition of starch fragments have predictive value for downstream process output such as ethanol production from starch. Thus, high-resolution 1H-13C NMR spectroscopic profiles of homooligomeric fragment mixtures in conjunction with chemometric methods provide a useful addition to the analytical chemistry toolbox of biotechnological starch utilization.

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

The Effect of Gestational and Lactational Age on the Human Milk Metabolome.

Human milk is the ideal nutrition source for healthy infants during the first six months of life and a detailed characterisation of the composition of milk from mothers that deliver prematurely (<37 weeks gestation), and of how human milk changes during lactation, would benefit our understanding of the nutritional requirements of premature infants. Individual milk samples from mothers delivering prematurely and at term were collected. The human milk metabolome, established by nuclear magnetic resonance (NMR) spectroscopy, was influenced by gestational and lactation age. Metabolite profiling identified that levels of valine, leucine, betaine, and creatinine were increased in colostrum from term mothers compared with mature milk, while those of glutamate, caprylate, and caprate were increased in mature term milk compared with colostrum. Levels of oligosaccharides, citrate, and creatinine were increased in pre-term colostrum, while those of caprylate, caprate, valine, leucine, glutamate, and pantothenate increased with time postpartum. There were differences between pre-term and full-term milk in the levels of carnitine, caprylate, caprate, pantothenate, urea, lactose, oligosaccharides, citrate, phosphocholine, choline, and formate. These findings suggest that the metabolome of pre-term milk changes within 5-7 weeks postpartum to resemble that of term milk, independent of time of gestation at pre-mature delivery.

A randomised, controlled, crossover study of the effect of diet on angiopoietin-like protein 4 (ANGPTL4) through modification of the gut microbiome.

Angiopoietin-like protein 4 (ANGPTL4) is a lipoprotein lipase inhibitor that is involved in lipid metabolism and angiogenesis. Animal studies have suggested that the ANGPTL4 protein is modulated by the gut microbiota, possibly through increased concentrations of SCFA, such as C4, found in whole-fat milk or as a result of fermentation of inulin. This study investigated whether a standardised diet either high in fat content or supplemented with inulin powder would increase plasma ANGPTL4 in overweight men and whether this increase was mediated through a compositional change of the gut microbiota. The study had a crossover design with three arms, where participants were given a standardised isoenergetic diet supplemented with inulin powder, whole-fat milk or water (control). Plasma and urine samples were collected before and after each intervention period. Faecal samples and adipose tissue biopsies were collected after each intervention period. The study included twenty-one participants of whom eighteen completed the study. The dietary interventions did not change ANGPTL4 plasma concentration, nor was plasma ANGPTL4 associated with plasma lipids, TAG or NEFA concentration. The relative abundance of bifidobacteria following the inulin diet was higher, compared with the control diet. However, the changes in microbiota were not associated with plasma ANGPTL4 and the overall composition of the microbiota did not change between the dietary periods. Although weight was maintained throughout the dietary periods, weight was negatively associated with plasma ANGPTL4 concentration. In the adipose tissue, ANGPTL4 expression was correlated with leptin expression, but not with hypoxia-inducible factor 1α (HIF-1α) expression.

Chemical and proteolysis-derived changes during long-term storage of lactose-hydrolyzed ultrahigh-temperature (UHT) milk.

Proteolytic activity in milk may release bitter-tasting peptides and generate free amino terminals that react with carbohydrates, which initiate Maillard reaction. Ultrahigh temperature (UHT) heat treatment inactivates the majority of proteolytic enzymes in milk. In lactose-hydrolyzed milk a ß-galactosidase preparation is applied to the milk after heat treatment, which has proteolytic side activities that may induce quality deterioration of long-term-stored milk. In the present study proteolysis, glycation, and volatile compound formation were investigated in conventional (100% lactose), filtered (60% lactose), and lactose-hydrolyzed (<1% lactose) UHT milk using reverse phase high-pressure liquid chromatography-mass spectrometry, proton nuclear magnetic resonance, and gas chromatography-mass spectrometry. Proteolysis was observed in all milk types. However, the degree of proteolysis was significantly higher in the lactose-hydrolyzed milk compared to the conventional and filtered milk. The proteins most prone to proteolysis were ß-CN and αs1-CN, which were clearly hydrolyzed after approximately 90 days of storage in the lactose-hydrolyzed milk.

Association between the bovine milk metabolome and rennet-induced coagulation properties of milk.

The milk metabolomes of 407 individual Swedish Red dairy cows were analyzed by nuclear magnetic resonance spectroscopy as part of the Danish-Swedish Milk Genomics Initiative. By relating these metabolite profiles to total milk protein concentration and rheological measurements of rennet-induced milk coagulation together using multivariate data analysis techniques, we were able to identify several different associations of the milk metabolome to technological properties of milk. Several novel correlations of milk metabolites to protein content and rennet-induced coagulation properties were demonstrated. Metabolites associated with the prediction of total protein content included choline, N-acetyl hexosamines, creatinine, glycerophosphocholine, glutamate, glucose 1-phosphate, galactose 1-phosphate, and orotate. In addition, levels of lactate, acetate, glutamate, creatinine, choline, carnitine, galactose 1-phosphate, and glycerophosphocholine were significantly different when comparing noncoagulating and well-coagulating milks. These findings suggest that the mentioned metabolites are associated with milk protein content and rennet-induced coagulation properties and may act as quality markers for cheese milk.