Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies: an underlying mechanism to reduce lipid accretion by whey protein ingestion?

Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by (1)H NMR and LC-MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC-MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC-MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice.

Metabolomic phenotyping of a cloned pig model.

BACKGROUND: Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. RESULTS: The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. CONCLUSIONS: From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.

Metabolic Effects of a 24-Week Energy-Restricted Intervention Combined with Low or High Dairy Intake in Overweight Women: An NMR-Based Metabolomics Investigation.

We investigated the effect of a 24-week energy-restricted intervention with low or high dairy intake (LD or HD) on the metabolic profiles of urine, blood and feces in overweight/obese women by NMR spectroscopy combined with ANOVA-simultaneous component analysis (ASCA). A significant effect of dairy intake was found on the urine metabolome. HD intake increased urinary citrate, creatinine and urea excretion, and decreased urinary excretion of trimethylamine-N-oxide (TMAO) and hippurate relative to the LD intake, suggesting that HD intake was associated with alterations in protein catabolism, energy metabolism and gut microbial activity. In addition, a significant time effect on the blood metabolome was attributed to a decrease in blood lipid and lipoprotein levels due to the energy restriction. For the fecal metabolome, a trend for a diet effect was found and a series of metabolites, such as acetate, butyrate, propionate, malonate, cholesterol and glycerol tended to be affected. Overall, even though these effects were not accompanied by a higher weight loss, the present metabolomics data reveal that a high dairy intake is associated with endogenous metabolic effects and effects on gut microbial activity that potentially impact body weight regulation and health. Moreover, ASCA has a great potential for exploring the effect of intervention factors and identifying altered metabolites in a multi-factorial metabolomic study.

Metabolomics to Explore Impact of Dairy Intake.

Dairy products are an important component in the Western diet and represent a valuable source of nutrients for humans. However, a reliable dairy intake assessment in nutrition research is crucial to correctly elucidate the link between dairy intake and human health. Metabolomics is considered a potential tool for assessment of dietary intake instead of traditional methods, such as food frequency questionnaires, food records, and 24-h recalls. Metabolomics has been successfully applied to discriminate between consumption of different dairy products under different experimental conditions. Moreover, potential metabolites related to dairy intake were identified, although these metabolites need to be further validated in other intervention studies before they can be used as valid biomarkers of dairy consumption. Therefore, this review provides an overview of metabolomics for assessment of dairy intake in order to better clarify the role of dairy products in human nutrition and health.

Metabolomics investigation to shed light on cheese as a possible piece in the French paradox puzzle.

An NMR-based metabolomics approach was used to investigate the differentiation between subjects consuming cheese or milk and to elucidate the potential link to an effect on blood cholesterol level. Fifteen healthy young men participated in a full crossover study during which they consumed three isocaloric diets with similar fat contents that were either (i) high in milk, (ii) high in cheese with equal amounts of dairy calcium, or (iii) a control diet for 14 days. Urine and feces samples were collected and analyzed by NMR-based metabolomics. Cheese and milk consumption decreased urinary choline and TMAO levels and increased fecal excretion of acetate, propionate, and lipid. Compared with milk intake, cheese consumption significantly reduced urinary citrate, creatine, and creatinine levels and significantly increased the microbiota-related metabolites butyrate, hippurate, and malonate. Correlation analyses indicated that microbial and lipid metabolism could be involved in the dairy-induced effects on blood cholesterol level.

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.

Lactose-hydrolyzed milk is more prone to chemical changes during storage than conventional ultra-high-temperature (UHT) milk.

The enzymatic hydrolysis of lactose to glucose and galactose gives rise to reactions that change the chemistry and quality of ambient-stored lactose-hydrolyzed ultra-high-temperature (UHT) milk. The aim of the present study was to investigate and compare chemical changes in lactose-hydrolyzed and conventional UHT milk during a 9 month ambient storage period. Several complementary analyses of volatiles, free amino acids, acetate, furosine, and level of free amino terminals were concluded. The analyses revealed an increased level of free amino acids and an increased formation rate of specific compounds such as furosine and 2-methylbutanal in lactose-hydrolyzed UHT milk compared to conventional UHT milk during storage. These observations indicate more favorable conditions for Maillard and subsequent reactions in lactose-hydrolyzed milk compared to conventional UHT milk stored at ambient temperature. Furthermore, it is postulated that proteolytic activity from the lactase-enzyme preparation may be responsible for the observed higher levels of free amino acids in lactose-hydrolyzed UHT milk.

Enzymatic browning and after-cooking darkening of Jerusalem artichoke tubers (Helianthus tuberosus L.).

Jerusalem artichoke tubers (Helianthus tuberosus L.) undergo enzymatic browning when peeled or cut, and turn grey after boiling, due to after-cooking darkening reactions between iron and phenolic acids. In an attempt to reveal the components responsible for these discolouration reactions, sensory evaluation and instrumental colour measurements were related to contents of total phenolics, phenolic acids, organic acids and iron in three varieties of raw and boiled Jerusalem artichoke tubers harvested in the autumn and the spring. No differences were found between varieties in sensory evaluated enzymatic browning, but Rema and Draga had higher scores than Mari in after-cooking darkening. Jerusalem artichoke tubers had higher contents of total phenolics, phenolic acids and citric acid in the autumn and low contents in the spring, while it was the opposite for malic acid. None of the chemical parameters investigated could explain the discolouration of the Jerusalem artichoke tubers.

Metabolomics reveals drastic compositional changes during overwintering of Jerusalem artichoke (Helianthus tuberosus L.) tubers.

Metabolic changes were investigated in overwintering Jerusalem artichoke (Helianthus tuberosus L.) tubers using proton nuclear magnetic resonance ((1)H NMR) metabolomics. Three varieties were studied; as a result of overwintering, the amount of inulin was found to decrease in Jerusalem artichoke tubers. This was mainly due to its conversion to sucrose and, at the same time, formation of inulin with a lower degree of polymerization. Major effects on the concentration of citric acid, malic acid, γ-aminobutyric acid (GABA), and adenosine were also found. Intriguingly, malic acid concentration increased and citric acid concentration decreased. These changes, together with an increase in sucrose and GABA concentrations, were ascribed to mobilization of nutrients prior to sprouting, suggesting that malic acid and GABA serve as carbon and nitrogen sources during sprouting of Jerusalem artichokes.

Quality of sour cherry juice of different clones and cultivars (Prunus cerasus L.) determined by a combined sensory and NMR spectroscopic approach.

Juice was manufactured from seven different sour cherry clones/cultivars and evaluated by quantitative descriptive sensory analysis and (1)H NMR spectroscopy. The sensory evaluation showed a large variation in several sensory attributes between the sour cherry clones/cultivars, which could be divided into two groups on the basis of both the sensory data and the NMR spectroscopic data. These groups were closely related to the genetic background of the clones. Kelleris clones were distinctly different from Stevnsberry and Fanal clones. Hence, (1)H NMR spectroscopic data seem to correlate with sensory quality of different sour cherry clones. In addition, malic acid was the most important metabolite for modeling the two highly correlated sensory attributes sweetness and sourness, whereas the glucose content had a slight effect and the fructose content had no impact on sweetness/sourness. Other metabolites (ethyl acetate, asparagine, ethanol) could be correlated with sensory attributes; however, a direct causal connection could not be established.

Characterization of peroxides formed by riboflavin and light exposure of milk. Detection of urate hydroperoxide as a novel oxidation product.

Characterization of peroxides by size exclusion chromatography (SEC) of milk following exposure to riboflavin and light showed that hydrogen peroxide was the most abundant peroxide formed since it could be removed by catalase. Formation of peroxides after separation by SEC showed that hydrogen peroxide formation was primarily increased in the presence of caseins and ascorbate, although whey proteins also were found to contribute. Caseins and beta-lactoglobulin also formed catalase-resistant peroxides, presumably protein hydroperoxides. A catalase-resistant and unstable peroxide was observed in fractions containing urate. Experiments performed with pure urate suggested that urate radicals reacted further with superoxide leading to a urate hydroperoxide. Electron paramagnetic resonance spectroscopy using spin-traps showed that the presence of oxygen was required for urate radical formation, which could be assigned as nitrogen-centered radicals. These results suggest a new route during light-induced oxidation sensitized by flavins, in effect making urate pro-oxidative.

Characterization of major radical scavenger species in bovine milk through size exclusion chromatography and functional assays.

Radical scavenging activities of bovine milk components were quantified following size exclusion chromatography (SEC) with postcolumn characterization of fractions using the scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS*(+)) in the Trolox equivalent antioxidant capacity (TEAC) assay and peroxyl radicals formed from cleavage of 2,2'-azobis(2-amidinopropane) (AAPH) in the oxygen radical absorbance capacity (ORAC) fluorometric assay. Caseins were quantitatively the major radical scavenger species in both assays, whereas beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) were much less active and only in the peroxyl radical assay. The radical scavenging activity of the caseins could be quantitatively accounted for by their constituent amino acids, as there were no effects of denaturing agents or complete digestion with proteases. In contrast, the activities of the whey proteins were dependent on denaturation or partial hydrolysis and dominated by the free thiol in beta-lg. A component in milk serum with a molecular mass of approximately 100 kDa contributed significantly to both ABTS*(+) and peroxyl radical scavenging but was absent in whey. This radical scavenger was identified as beta-casein. The only significant low molecular weight radical scavenger species were identified as ascorbate and urate in both assays.

Inhibition of lactoperoxidase-catalyzed 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and tyrosine oxidation by tyrosine-containing random amino acid copolymers.

Oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by lactoperoxidase was found to be inhibited by tyrosine-containing random amino acid copolymers but not by tyrosine. Both electrostatic effects and polymer size were found to be important by comparison of negatively and positively charged copolymers of varying lengths, with poly(Glu, Tyr)4:1 ([E 4Y 1] approximately 40) as the strongest competitive inhibitor (EC 50 approximately 20 nM). This polymer did not form dityrosine in the presence of lactoperoxidase (LPO) and peroxide. Furthermore, incubation with tert-butyl hydroperoxide, as opposed to hydrogen peroxide, resulted in a peculiar long lag phase of the reaction between the redox intermediate compound II and [E 4Y 1] approximately 40, indicating a very tight association between enzyme and inhibitor. We propose that interactions between multiple positively charged areas on the surface of LPO and the polymer are required for optimal inhibition.