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Food intake triggers extensive changes in the blood metabolome. The kinetics of these changes depend on meal composition and on intrinsic, health-related characteristics of each individual, making the assessment of changes in the postprandial metabolome an opportunity to assess someone's metabolic status. To enable the usage of dietary challenges as diagnostic tools, profound knowledge about changes that occur in the postprandial period in healthy individuals is needed. In this study, we characterize the time-resolved changes in plasma levels of 634 metabolites in response to an oral glucose tolerance test (OGTT), an oral lipid tolerance test (OLTT), and a mixed meal (SLD) in healthy young males (n = 15). Metabolite levels for samples taken at different time points (20 per individual) during the challenges were available from targeted (132 metabolites) and non-targeted (502 metabolites) metabolomics. Almost half of the profiled metabolites (n = 308) showed a significant change in at least one challenge, thereof 111 metabolites responded exclusively to one particular challenge. Examples include azelate, which is linked to ω-oxidation and increased only in OLTT, and a fibrinogen cleavage peptide that has been linked to a higher risk of cardiovascular events in diabetes patients and increased only in OGTT, making its postprandial dynamics a potential target for risk management. A pool of 89 metabolites changed their plasma levels during all three challenges and represents the core postprandial response to food intake regardless of macronutrient composition. We used fuzzy c-means clustering to group these metabolites into eight clusters based on commonalities of their dynamic response patterns, with each cluster following one of four primary response patterns: (i) “decrease-increase” (valley-like) with fatty acids and acylcarnitines indicating the suppression of lipolysis, (ii) “increase-decrease” (mountain-like) including a cluster of conjugated bile acids and the glucose/insulin cluster, (iii) “steady decrease” with metabolites reflecting a carryover from meals prior to the study, and (iv) “mixed” decreasing after the glucose challenge and increasing otherwise. Despite the small number of subjects, the diversity of the challenges and the wealth of metabolomic data make this study an important step toward the characterization of postprandial responses and the identification of markers of metabolic processes regulated by food intake.

Cardiorespiratory fitness (CRF) represents a strong predictor of all-cause mortality and is strongly influenced by regular physical activity (PA). However, the biological mechanisms involved in the body’s adaptation to PA remain to be fully elucidated. The aim of this study was to systematically examine the relationship between CRF and plasma metabolite patterns in 252 healthy adults from the cross-sectional Karlsruhe Metabolomics and Nutrition (KarMeN) study. CRF was determined by measuring the peak oxygen uptake during incremental exercise. Fasting plasma samples were analyzed by nuclear magnetic resonance spectroscopy and mass spectrometry coupled to one- or two-dimensional gas chromatography or liquid chromatography. Based on this multi-platform metabolomics approach, 427 plasma analytes were detected. Bi- and multivariate association analyses, adjusted for age and menopausal status, showed that CRF was linked to specific sets of metabolites primarily indicative of lipid metabolism. However, CRF-related metabolite patterns largely differed between sexes. While several phosphatidylcholines were linked to CRF in females, single lyso-phosphatidylcholines and sphingomyelins were associated with CRF in males. When controlling for further assessed clinical and phenotypical parameters, sex-specific CRF tended to be correlated with a smaller number of metabolites linked to lipid, amino acid, or xenobiotics-related metabolism. Interestingly, sex-specific CRF explanation models could be improved when including selected plasma analytes in addition to clinical and phenotypical variables. In summary, this study revealed sex-related differences in CRF-associated plasma metabolite patterns and proved known associations between CRF and risk factors for cardiometabolic diseases such as fat mass, visceral adipose tissue mass, or blood triglycerides in metabolically healthy individuals. Our findings indicate that covariates like sex and, especially, body composition have to be considered when studying blood metabolic markers related to CRF.

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state‐of‐the‐art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

Purpose Differences in resting energy expenditure (REE) between men and women mainly result from sex-related differences in lean body mass (LBM). So far, a little is known about whether REE and LBM are reflected by a distinct human metabolite profile. Therefore, we aimed to identify plasma and urine metabolite patterns that are associated with REE and LBM of healthy subjects. Methods We investigated 301 healthy male and female subjects (18–80 years) under standardized conditions in the cross-sectional KarMeN (Karlsruhe Metabolomics and Nutrition) study. REE was determined by indirect calorimetry and LBM by dual X-ray absorptiometry. Fasting blood and 24 h urine samples were analyzed by targeted and non-targeted metabolomics methods using GC × GC–MS, GC–MS, LC–MS, and NMR. Data were evaluated by predictive modeling of combined data using different machine learning algorithms, namely SVM, glmnet, and PLS. Results When evaluating data of men and women combined, we were able to predict REE and LBM with high accuracy (> 90%). This, however, was a clear effect of sex, which is supported by the high degree of overlap in identified important metabolites for LBM, REE, and sex, respectively. The applied machine learning algorithms did not reveal a metabolite pattern predictive of REE or LBM, when analyzing data for men and women, separately. Conclusions We could not identify a sex independent predictive metabolite pattern for REE or LBM. REE and LBM have no impact on plasma and urine metabolite profiles in the KarMeN Study participants. Studies applying metabolomics in healthy humans need to consider sex specific data evaluation. Electronic supplementary material The online version of this article (10.1007/s00394-018-1767-1) contains supplementary material, which is available to authorized users.

Background: Casein is considered a slowly digestible protein compared with whey protein, and this may cause differences in hormone responses and the kinetics of delivering amino acids into the circulation. Objective: We investigated whether postprandial plasma hormone and metabolite responses were different when bovine casein or whey protein was co-administered with carbohydrates in healthy and prediabetic adults. Methods: White healthy male adults (n = 15) and white, well-defined male and female prediabetic adults (n = 15) received test drinks randomly on 3 different occasions at least 2 d apart which contained 50 g of maltodextrin19 (MD19) alone or in combination with 50 g of whey protein isolate (WPI) or 50 g of sodium caseinate (SC). Blood samples were collected over a 240-min time period and were analyzed for hormone profiles and defined metabolites. Results: No evidence was found that gastric emptying was different between the 2 protein drinks. Both proteins increased peak plasma insulin concentrations in prediabetic persons by 96% compared with MD19 (each, P < 0.05), which was accompanied by a reduction of peak venous blood glucose by 21% (each, P < 0.0001) without a difference between the 2 proteins. Peak plasma glucagon concentrations increased by 101% in both groups after the protein drinks (P < 0.05). The WPI drink also increased peak plasma glucose-dependent insulinotropic polypeptide concentrations in healthy volunteers by 56% (P < 0.01). Differences in plasma metabolite concentrations in volunteers could be attributed exclusively to the differences in the amino acid composition of the 2 proteins ingested. Conclusion: The WPI and the SC drinks similarly reduced postprandial glucose excursions when ingested together with carbohydrates in healthy and prediabetic volunteers. Under our experimental conditions, however, no evidence was found that gastrointestinal processing of the 2 protein varieties differed substantially. This trial was registered at clinicaltrials.gov as DRKS00005682. J Nutr doi: 10.3945/jn.114.199190.

Whey protein was shown to reduce blood glucose responses in humans and various other positive effects have been attributed to this protein. In contrast, studies using glycomacropeptide (GMP) as part of the whey fraction of bovine milk are rare. We, therefore, studied the postprandial responses to GMP administration in humans with impaired glucose tolerance compared to the effects of pure whey protein in a random design. Fifteen prediabetic volunteers received on different occasions one of three test drinks containing 50 g of maltodextrin19 (MD19) alone or in combination with either 50 g GMP or 50 g whey protein isolate (WPI). Blood was collected over 4 h with analysis of blood glucose and hormones, gastric emptying rate as well as plasma amino- and fatty acids, β-hydroxybutyrate and acylcarnitines. The WPI drink reduced the AUC of venous blood glucose compared to the MD19 drink in the prediabetic group by 11% (p = 0.0018) whereas GMP reduced the AUC by 18% (p

Metabolic challenge protocols, such as the oral glucose tolerance test, can uncover early alterations in metabolism preceding chronic diseases. Nevertheless, most metabolomics data accessible today reflect the fasting state. To analyze the dynamics of the human metabolome in response to environmental stimuli, we submitted 15 young healthy male volunteers to a highly controlled 4 d challenge protocol, including 36 h fasting, oral glucose and lipid tests, liquid test meals, physical exercise, and cold stress. Blood, urine, exhaled air, and breath condensate samples were analyzed on up to 56 time points by MS- and NMR-based methods, yielding 275 metabolic traits with a focus on lipids and amino acids. Here, we show that physiological challenges increased interindividual variation even in phenotypically similar volunteers, revealing metabotypes not observable in baseline metabolite profiles; volunteer-specific metabolite concentrations were consistently reflected in various biofluids; and readouts from a systematic model of β-oxidation (e.g., acetylcarnitine/palmitylcarnitine ratio) showed significant and stronger associations with physiological parameters (e.g., fat mass) than absolute metabolite concentrations, indicating that systematic models may aid in understanding individual challenge responses. Due to the multitude of analytical methods, challenges and sample types, our freely available metabolomics data set provides a unique reference for future metabolomics studies and for verification of systems biology models.

Der zunehmende Einsatz von Hochdurchsatz-Profilierungstechniken in der Ernahrungsforschung dient der Identifizierung metabolischer Phanotypen als Auspragung der genetischen Heterogenitat sowie dem Nachweis von biofunktionalen Wirkungen ausgewahlter Nahrungsinhaltsstoffe.

Branched-chain amino acids (BCAA) in plasma are discussed as risk factors for the onset of several diseases. Information about the contribution of the overall diet to plasma BCAA concentrations is controversial. Our objective was to investigate which dietary pattern is associated with plasma BCAA concentrations and whether other additional nutrients besides BCAA further characterize this dietary pattern. Based on the cross-sectional KarMeN study, fasting plasma amino acid (AA) concentrations, as well as current and habitual dietary intake were assessed in 298 healthy individuals. Using reduced rank regression, we derived a habitual dietary pattern that explained 32.5% of plasma BCAA variation. This pattern was high in meat, sausages, sauces, eggs, and ice cream but low in nuts, cereals, mushrooms, and pulses. The age, sex, and energy intake adjusted dietary pattern score was associated with an increase in animal-based protein together with a decrease in plant-based protein, dietary fiber, and an unfavorable fatty acid composition. Besides BCAA, alanine, lysine and the aromatic AA were positively associated with the dietary pattern score as well. All of these factors were reported to be associated with risk of type 2 diabetes and cardiovascular diseases before. Our data suggest that rather than the dietary intake of BCAA, the overall dietary pattern that contributes to high BCAA plasma concentrations may modulate chronic diseases risk.

Bile acids (BA) play an important role in lipid metabolism. They facilitate intestinal lipid absorption, and BA synthesis is the main catabolic pathway for cholesterol. The objective of this study was to investigate associations of age, sex, diet (fat intake) and parameters of lipid metabolism (triglycerides, LDL, HDL, body fat content) with fasting plasma BA concentration of healthy individuals. Fasting plasma samples from a cross-sectional study were used to determine the concentrations of 14 BA using an LC-MS stable isotope dilution assay. Triglycerides, LDL and HDL were analyzed by standard clinical chemistry methods and body fat content was measured with a DXA instrument. The dietary fat intake of the 24 h period prior to the sampling was assessed on the basis of a 24 h recall. Subsequent statistical data processing was done by means of a median regression model. Results revealed large inter-individual variations. Overall, higher median plasma concentrations of BA were observed in men than in women. Quantile regression showed significant interactions of selected BA with age and sex, affecting primarily chenodeoxycholic acid and its conjugates. No associations were found for LDL and the amount of fat intake (based on the percentage of energy intake from dietary fat as well as total fat intake). Additional associations regarding body fat content, HDL and triglycerides were found for some secondary BA plasma concentrations. We conclude that age and sex are associated with the fasting plasma concentrations. Those associations are significant and need to be considered in studies investigating the role of BA in the human metabolism.

The wealth of genomic information and high-throughput profiling technologies are now being exploited by scientists in the disciplines of nutrition and food science. Diet and food components are prime environmental factors that affect the genome, transcriptome, proteome and metabolome, and this life-long interaction defines the health or disease state of an individual. For the first time the interaction of foods, and individual food constituents, with the biological systems can be defined on a molecular basis. Profiling technologies are used in basic-science applications for identifying the mode of action of foods or particular ingredients, and are similarly taken into the science-driven development of foods with a defined biofunctionality. Biomarker profiles and patterns derived from genomics applications in humans should guide nutrition and food science in developing evidence-based dietary recommendations and health-promoting foods.

A range of 1-ethynylpyrene-modified oligonucleotides (Py−≡−dU) has been prepared by a special semi-automated strategy using Sonogashira-type cross-coupling conditions in the solid phase. The absorption and fluorescence properties of the Py−≡−dU-modified single strands and their corresponding double-stranded duplexes have been characterized. The presented spectra show that the Py−≡−dU emission is quenched in DNA duplexes bearing a thymine or cytosine adjacent to the Py−≡−dU group. This shows that the Py−≡−dU moiety is able to inject an electron into the base stack while simultaneously acting as a spectroscopic label to enable the observation of these processes. Hence, Py−≡−dU-modified oligonucleotides are promising donor systems in order to study electron injection and electron transport in DNA spectroscopically. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

cope : Knowledge on the influence of current diet on TMAO levels in humans is still inconsistent. Thus, we aimed to investigate associations of current diet with urine and plasma TMAO levels and to determine the effect of different foods on TMAO variation. Methods and results : TMAO concentrations of 297 healthy individuals were assessed using 1H-NMR spectroscopy for 24h urine collection and spot urine, and LC-MS for plasma. Of 35 assessed food groups, those with a correlation of ρ >|0.15| with plasma or urine TMAO levels were further investigated in multivariate linear regression models showing current fish and (red) meat consumption as plausible dietary sources of TMAO. Overall, explained variance of TMAO levels by current diet and co-variates (age, sex, lean body mass, glomerular filtration rate) was small. Associations with urine and plasma concentrations differed depending on the TMAO source. Fish consumption was associated with urine and plasma TMAO concentrations, whereas meat consumption was only associated with TMAO concentrations in plasma. Furthermore, associations of plasma TMAO concentration with fish consumption were two times stronger than with meat consumption. Conclusions : Meat and fish consumption differentially affects TMAO concentrations in body fluids. Only a small fraction of variance is explained by current diet. This article is protected by copyright. All rights reserved

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction. Formation of loop-loop kissing complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5-10-fold fluorescence emission decrease (F(max) = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average K(D) = 2.6 +/- 1.7 nM) and affinity for Rom (average K(D) = 60 +/- 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins. Using stopped-flow experiments, it was found that the 2-AP probes experienced at least three different microenvironments during association of the RNA complex, thus suggesting a kinetic intermediate in the kissing pathway. In contrast, dissociation of the complex was found to fit a single exponential decay (average k(off) = 8.9 x 10(-5) s(-1)). Consistent with these observations, a two-step mechanism for RNA loop-loop complex association is proposed in which the complementary loops of R1inv and R2inv first base pair to form the loop-loop helix (average k(1) = 0.13 microM(-1)s(-1)) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k(2) = 0.09 s(-1)), where the helical stacking around the junctions is optimized.

It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at −20 °C, on dry ice, at −80 °C or in liquid nitrogen and then stored at −20 °C, −80 °C or in liquid nitrogen vapor phase for 1–5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at −20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.

Adverse levels of lipoproteins are highly heritable and constitute risk factors for cardiovascular outcomes. Hitherto, genome-wide association studies revealed 95 lipid-associated loci. However, due to the small effect sizes of these associations large sample numbers (>100 000 samples) were needed. Here we show that analyzing more refined lipid phenotypes, namely lipoprotein subfractions, can increase the number of significantly associated loci compared with bulk high-density lipoprotein and low-density lipoprotein analysis in a study with identical sample numbers. Moreover, lipoprotein subfractions provide novel insight into the human lipid metabolism. We measured 15 lipoprotein subfractions (L1-L15) in 1791 samples using (1)H-NMR (nuclear magnetic resonance) spectroscopy. Using cluster analyses, we quantified inter-relationships among lipoprotein subfractions. Additionally, we analyzed associations with subfractions at known lipid loci. We identified five distinct groups of subfractions: one (L1) was only marginally captured by serum lipids and therefore extends our knowledge of lipoprotein biochemistry. During a lipid-tolerance test, L1 lost its special position. In the association analysis, we found that eight loci (LIPC, CETP, PLTP, FADS1-2-3, SORT1, GCKR, APOB, APOA1) were associated with the subfractions, whereas only four loci (CETP, SORT1, GCKR, APOA1) were associated with serum lipids. For LIPC, we observed a 10-fold increase in the variance explained by our regression models. In conclusion, NMR-based fine mapping of lipoprotein subfractions provides novel information on their biological nature and strengthens the associations with genetic loci. Future clinical studies are now needed to investigate their biomedical relevance.

Scope: The adipose tissue is a major site of insulin action and contributes substantially to energy homeostasis. Insulin increases the extraction of glucose from circulation into adipose tissue by recruiting the glucose transporter GLUT4 to the plasma membrane. It has been proposed that dietary flavonoids may interfere with glucose transport processes. Methods and results: We have used murine 3T3-L1 adipocytes and isolated mature human adipocytes to assess the interaction of selected flavonoids with glucose uptake, both in the basal state and after insulin stimulation. Kinetic characterization of 2-deoxyglucose uptake in the basal state revealed in both cell types an apparent Km of around 8 mM with no change in affinity but a significant increase in maximal influx in the presence of insulin. A screening of representative flavonoids of different structural classes revealed the flavanone naringenin and the isoflavone daidzein to affect glucose transport significantly with half-maximal inhibition at concentrations of around 60–80 μM for basal and 70–110 μM for insulin-stimulated glucose uptake in both 3T3-L1 adipocytes and mature human adipocytes. Conclusion: Considering attainable plasma concentrations of flavonoids in vivo, we assume that even under physiological conditions naringenin and daidzein could impair glucose removal from plasma, which may pose a risk to patients with diabetes mellitus.

Ultrafast structural dynamics concomitant to excitation energy transfer in DNA has been studied using a pair of pyrene-labeled DNA bases. The temporal evolution of the femtosecond pump−probe spectra reveals the existence of two electronic coupling pathways, through-base stack and through-space, which lead to excitation energy transfer and excimer formation even when the labeled DNA bases are separated by one AT base pair. The electronic coupling which mediates through-base stack energy transfer is so strong that a new absorption band arises in the excited-state absorption spectrum within 300 fs. From the analysis of time-dependent spectral shifts due to through-space excimer formation, the local structural dynamics and flexibility of DNA are characterized on the picosecond and nanosecond time scale.

1-ethynylpyrene shows remarkable absorption changes upon DNA hybridization when it is covalently attached to the 8-position of guanine. An absorption band at approximately 420 nm is only present in the duplex, exhibits thermal melting behaviour and provides the basis for a molecular beacon together with 1-ethynylpyrene-modified cytosine.

Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.