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1.  NMR-Based Metabolomic Profiling of Overweight Adolescents: An Elucidation of the Effects of Inter-/Intraindividual Differences, Gender, and Pubertal Development 
BioMed Research International  2014;2014:537157.
The plasma and urine metabolome of 192 overweight 12–15-year-old adolescents (BMI of 25.4 ± 2.3 kg/m2) were examined in order to elucidate gender, pubertal development measured as Tanner stage, physical activity measured as number of steps taken daily, and intra-/interindividual differences affecting the metabolome detected by proton NMR spectroscopy. Higher urinary excretion of citrate, creatinine, hippurate, and phenylacetylglutamine and higher plasma level of phosphatidylcholine and unsaturated lipid were found for girls compared with boys. The results suggest that gender differences in the metabolome are being commenced already in childhood. The relationship between Tanner stage and the metabolome showed that pubertal development stage was positively related to urinary creatinine excretion and negatively related to urinary citrate content. No relations between physical activity and the metabolome could be identified. The present study for the first time provides comprehensive information about associations between the metabolome and gender, pubertal development, and physical activity in overweight adolescents, which is an important subject group to approach in the prevention of obesity and life-style related diseases. While this study is preliminary, these results may have the potential to translate into clinical applicability upon further investigations; if biomarkers for Tanner stage can be established, these might be used for identification of individuals susceptible to an early pubertal development.
doi:10.1155/2014/537157
PMCID: PMC3985195  PMID: 24800239
2.  Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds 
Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain and exerting anti-pathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds; Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contain significantly higher levels of fucosylated oligosaccharides than Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid) suggesting it may therefore offer advantages in term of a wider array of bioactivities.
doi:10.1021/jf300015j
PMCID: PMC3386800  PMID: 22632419
oligosaccharides; dairy cow; sialyllactose; fucosylation; mass spectrometry
3.  NMR-Based Milk Metabolomics 
Metabolites  2013;3(2):204-222.
Milk is a key component in infant nutrition worldwide and, in the Western parts of the world, also in adult nutrition. Milk of bovine origin is both consumed fresh and processed into a variety of dairy products including cheese, fermented milk products, and infant formula. The nutritional quality and processing capabilities of bovine milk is closely associated to milk composition. Metabolomics is ideal in the study of the low-molecular-weight compounds in milk, and this review focuses on the recent nuclear magnetic resonance (NMR)-based metabolomics trends in milk research, including applications linking the milk metabolite profiling with nutritional aspects, and applications which aim to link the milk metabolite profile to various technological qualities of milk. The metabolite profiling studies encompass the identification of novel metabolites, which potentially can be used as biomarkers or as bioactive compounds. Furthermore, metabolomics applications elucidating how the differential regulated genes affects milk composition are also reported. This review will highlight the recent advances in NMR-based metabolomics on milk, as well as give a brief summary of when NMR spectroscopy can be useful for gaining a better understanding of how milk composition is linked to nutritional or quality traits.
doi:10.3390/metabo3020204
PMCID: PMC3901264  PMID: 24957988
milk; NMR spectroscopy; metabolomics; metabolites
4.  The Serum Metabolite Response to Diet Intervention with Probiotic Acidified Milk in Irritable Bowel Syndrome Patients Is Indistinguishable from that of Non-Probiotic Acidified Milk by 1H NMR-Based Metabonomic Analysis  
Nutrients  2010;2(11):1141-1155.
The effects of a probiotic acidified milk product on the blood serum metabolite profile of patients suffering from Irritable Bowel Syndrome (IBS) compared to a non-probiotic acidified milk product was investigated using 1H NMR metabonomics. For eight weeks, IBS patients consumed 0.4 L per day of a probiotic fermented milk product or non-probiotic acidified milk. Both diets resulted in elevated levels of blood serum L-lactate and 3-hydroxybutyrate. Our results showed identical effects of acidified milk consumption independent of probiotic addition. A similar result was previously obtained in a questionnaire-based evaluation of symptom relief. A specific probiotic effect is thus absent both in the patient subjective symptom evaluations and at the blood serum metabolite level. However, there was no correspondence between symptom relief and metabolite response on the patient level.
doi:10.3390/nu2111141
PMCID: PMC3257620  PMID: 22254002
metabonomics; 1H-NMR spectroscopy; Irritable Bowel Syndrome; acidified milk products; multivariate data analysis; lactate; probiotics
5.  Metabolomic phenotyping of a cloned pig model 
BMC Physiology  2011;11:14.
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.
doi:10.1186/1472-6793-11-14
PMCID: PMC3174869  PMID: 21859467
6.  Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics 
Background
Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach.
Methods
Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH.
Results
The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH2 oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium.
Conclusions
The explorative approach of this study combined with the determination of a decreased intracellular DCFH2 oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.
doi:10.1186/1550-2783-7-9
PMCID: PMC2822831  PMID: 20205771

Results 1-6 (6)