Morten Rahr Clausen

Relationship between the metabolite profile and technological properties of bovine milk from two dairy breeds elucidated by NMR-based metabolomics.

The aim of the present study was to investigate the relationship between the metabolite profile of milk and important technological properties by using nuclear magnetic resonance (NMR)-based metabolomics. The metabolomics approach was introduced for the metabolic profiling of a set of milk samples from two dairy breeds representing a wide span in coagulation properties. The milk metabolite profiles obtained by proton and carbon NMR spectroscopy could be correlated to breed and, more interestingly, also with the coagulation profile, as established by traditional methods by using principal component analysis (PCA). The metabolites responsible for the separation into breed could mainly be ascribed to carnitine and lactose, whereas the metabolites varying in the samples with respect to coagulation properties included citrate, choline, carnitine, and lactose. The results found in the present study demonstrated a promising potential of NMR-based metabolomics for a rapid analysis and classification of milk samples, both of which are useful for the dairy industry.

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.

Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH(4)Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10(6) vs 6.47 × 10(6)). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0-4.5, the reducibility of H(+), and the high gas-phase basicity of Ac(-) and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies.

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 1H 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.

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.

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.

Bioactivity and chemical composition of blackcurrant (Ribes nigrum) cultivars with and without pesticide treatment

Eleven blackcurrant cultivars grown with pesticide (PT) and without pesticide treatment (PF) were evaluated to compare the differences in plant growth and physical condition, total anthocyanin content, ascorbic acid content, total antioxidant capacity, effect on prostaglandin E2 (PGE2) production and anticancer cell proliferation activities. Results showed that the yield and growth of PT blackcurrants were higher. However, PF blackcurrants contained a higher amount of ascorbic acid, and displayed an increased inhibition against cancer cells compared to PT blackcurrants, indicating that PF blackcurrants have an increased potential to deliver health-promoting benefit for consumers. Significant differences were observed between blackcurrant cultivars in relation to plant growth and physical condition, total anthocyanin content and PGE2 assay, highlighting the importance of cultivar selection.

Lean‐seafood intake decreases urinary markers of mitochondrial lipid and energy metabolism in healthy subjects: Metabolomics results from a randomized crossover intervention study

SCOPE Proteins constitute an important part of the human diet, but understanding of the effects of different dietary protein sources on human metabolism is sparse. We aimed to elucidate diet-induced metabolic changes through untargeted urinary metabolomics after four weeks of intervention with lean-seafood or nonseafood diets. It is shown that lean-seafood intake reduces urinary excretion of metabolites involved in mitochondrial lipid and energy metabolism possibly facilitating a higher lipid catabolism in healthy subjects. METHODS In a randomized controlled trial with crossover design, 20 healthy subjects consumed two balanced diets that varied in main protein sources for 4 weeks. Morning spot urine samples were collected before and after each intervention period. Untargeted metabolomics based on (1) H NMR spectroscopy and LC-MS analyses were applied to characterize the urinary metabolic response to the interventions. RESULTS The lean-seafood diet period reduced the urinary level of l-carnitine, 2,6-dimethylheptanoylcarnitine, and N-methyl-2-pyridone-5-carboxamide, relative to the nonseafood period. The dietary analysis revealed that the higher urinary level of trimethylamine-N-oxide after the lean-seafood diet period and guanidinoacetate and 3-methylhistidine after the nonseafood diet period was related to the endogenous content of these compounds in the diets. CONCLUSIONS Our data reveal that 4 weeks of lean-seafood intake reduces urinary excretion of metabolites involved in mitochondrial lipid and energy metabolism possibly facilitating a higher lipid catabolism in healthy subjects after the lean-seafood intake.

Time-Saving Design of Experiment Protocol for Optimization of LC-MS Data Processing in Metabolomic Approaches

We describe a time-saving protocol for the processing of LC-MS-based metabolomics data by optimizing parameter settings in XCMS and threshold settings for removing noisy and low-intensity peaks using design of experiment (DoE) approaches including Plackett-Burman design (PBD) for screening and central composite design (CCD) for optimization. A reliability index, which is based on evaluation of the linear response to a dilution series, was used as a parameter for the assessment of data quality. After identifying the significant parameters in the XCMS software by PBD, CCD was applied to determine their values by maximizing the reliability and group indexes. Optimal settings by DoE resulted in improvements of 19.4% and 54.7% in the reliability index for a standard mixture and human urine, respectively, as compared with the default setting, and a total of 38 h was required to complete the optimization. Moreover, threshold settings were optimized by using CCD for further improvement. The approach combining optimal parameter setting and the threshold method improved the reliability index about 9.5 times for a standards mixture and 14.5 times for human urine data, which required a total of 41 h. Validation results also showed improvements in the reliability index of about 5-7 times even for urine samples from different subjects. It is concluded that the proposed methodology can be used as a time-saving approach for improving the processing of LC-MS-based metabolomics data.

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 β-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 β-CN and αs1-CN, which were clearly hydrolyzed after approximately 90 days of storage in the lactose-hydrolyzed milk.