A next-generation sequencing approach for the detection of mixed species in canned tuna.

Tuna cans are relevant seafood products for which mixtures of different tuna species are not allowed according to European regulations. In order to support the prevention of food fraud and mislabelling, a next-generation sequencing methodology based on mitochondrial cytochrome b and control region markers has been tested. Analyses of defined mixtures of DNA, fresh tissue and canned tissue revealed a qualitative and, to some extent, semiquantitative identification of tuna species. While the choice of the bioinformatic pipeline had no influence in the results (p = 0.71), quantitative differences occurred depending on the treatment of the sample, marker, species, and mixture (p < 0.01). The results revealed that matrix-specific calibrators or normalization models should also be used in NGS. The method represents an important step towards a semiquantitative method for routine control of this analytically challenging food matrix. Tests of commercial samples uncovered mixed species in some cans, being not in compliance with EU regulations.

Essential terminology and considerations for validation of non-targeted methods.

Through their suggestive name, non-targeted methods (NTMs) do not aim at a predefined "needle in the haystack." Instead, they exploit all the constituents of the haystack. This new type of analytical method is increasingly finding applications in food and feed testing. However, the concepts, terms, and considerations related to this burgeoning field of analytical testing need to be propagated for the benefit of those associated with academic research, commercial development, or official control. This paper addresses frequently asked questions regarding terminology in connection with NTMs. The widespread development and adoption of these methods also necessitate the need to develop innovative approaches for NTM validation, i.e., evaluating the performance characteristics of a method to determine if it is fit-for-purpose. This work aims to provide a roadmap for approaching NTM validation. In doing so, the paper deliberates on the different considerations that influence the approach to validation and provides suggestions therefor.

Addition of soluble fiber to standard purified diets is important for gut morphology in mice.

Purified diets (PD) increase standardization and repeatability in rodent studies but lead to differences in the phenotype of animals compared to grain-based "chow" diets. PD contain less fiber and are often devoid of soluble fiber, which can impact gut health. Thus, the aim of the present study was to modify the PD AIN93G by addition of soluble fiber, to promote more natural gut development as seen with chow diets. One hundred twenty male C57BL/6J mice were fed over 12 weeks either a chow diet, AIN93G or one of three modified AIN93G with increased fiber content and different ratios of soluble fiber to cellulose. Gut health was assessed through histological and immunohistochemical parameters and gut barrier gene expression. Gut microbiota composition was analyzed and its activity characterized through short chain fatty acid (SCFA) quantification. Feeding AIN93G led to tissue atrophy, a less diverse microbiota and a lower production of SCFA compared to chow diet. The addition of soluble fiber mitigated these effects, leading to intermediate colon and caecum crypt lengths and microbiota composition compared to both control diets. In conclusion, the addition of soluble fibers in PDs seems essential for gut morphology as well as a diverse and functional gut microbiome.

Sex-Specific Relationship between the Cardiorespiratory Fitness and Plasma Metabolite Patterns in Healthy Humans-Results of the KarMeN Study.

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.

An NMR-Based Approach to Identify Urinary Metabolites Associated with Acute Physical Exercise and Cardiorespiratory Fitness in Healthy Humans-Results of the KarMeN Study.

Knowledge on metabolites distinguishing the metabolic response to acute physical exercise between fit and less fit individuals could clarify mechanisms and metabolic pathways contributing to the beneficial adaptations to exercise. By analyzing data from the cross-sectional KarMeN (Karlsruhe Metabolomics and Nutrition) study, we characterized the acute effects of a standardized exercise tolerance test on urinary metabolites of 255 healthy women and men. In a second step, we aimed to detect a urinary metabolite pattern associated with the cardiorespiratory fitness (CRF), which was determined by measuring the peak oxygen uptake (VO2peak) during incremental exercise. Spot urine samples were collected pre- and post-exercise and 47 urinary metabolites were identified by nuclear magnetic resonance (NMR) spectroscopy. While the univariate analysis of pre-to-post-exercise differences revealed significant alterations in 37 urinary metabolites, principal component analysis (PCA) did not show a clear separation of the pre- and post-exercise urine samples. Moreover, both bivariate correlation and multiple linear regression analyses revealed only weak relationships between the VO2peak and single urinary metabolites or urinary metabolic pattern, when adjusting for covariates like age, sex, menopausal status, and lean body mass (LBM). Taken as a whole, our results show that several urinary metabolites (e.g., lactate, pyruvate, alanine, and acetate) reflect acute exercise-induced alterations in the human metabolism. However, as neither pre- and post-exercise levels nor the fold changes of urinary metabolites substantially accounted for the variation of the covariate-adjusted VO2peak, our results furthermore indicate that the urinary metabolites identified in this study do not allow to draw conclusions on the individual's physical fitness status. Studies investigating the relationship between the human metabolome and functional variables like the CRF should adjust for confounders like age, sex, menopausal status, and LBM.

High-Intensity Interval Training Decreases Resting Urinary Hypoxanthine Concentration in Young Active Men-A Metabolomic Approach.

High-intensity interval training (HIIT) is known to improve performance and skeletal muscle energy metabolism. However, whether the body's adaptation to an exhausting short-term HIIT is reflected in the resting human metabolome has not been examined so far. Therefore, a randomized controlled intervention study was performed to investigate the effect of a ten-day HIIT on the resting urinary metabolome of young active men. Fasting spot urine was collected before (-1 day) and after (+1 day; +4 days) the training intervention and 65 urinary metabolites were identified by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite concentrations were normalized to urinary creatinine and subjected to univariate statistical analysis. One day after HIIT, no overall change in resting urinary metabolome, except a significant difference with decreasing means in urinary hypoxanthine concentration, was documented in the experimental group. As hypoxanthine is related to purine degradation, lower resting urinary hypoxanthine levels may indicate a training-induced adaptation in purine nucleotide metabolism.

Specific Wheat Fractions Influence Hepatic Fat Metabolism in Diet-Induced Obese Mice.

Low whole grain consumption is a risk factor for the development of non-communicable diseases such as type 2 diabetes. Dietary fiber and phytochemicals are bioactive grain compounds, which could be involved in mediating these beneficial effects. These compounds are not equally distributed in the wheat grain, but are enriched in the bran and aleurone fractions. As little is known on physiological effects of different wheat fractions, the aim of this study was to investigate this aspect in an obesity model. For twelve weeks, C57BL/6J mice were fed high-fat diets (HFD), supplemented with one of four wheat fractions: whole grain flour, refined white flour, bran, or aleurone. The different diets did not affect body weight, however bran and aleurone decreased liver triglyceride content, and increased hepatic n-3 polyunsaturated fatty acid (PUFA) concentrations. Furthermore, lipidomics analysis revealed increased PUFA concentration in the lipid classes of phosphatidylcholine (PC), PC-ether, and phosphatidylinositol in the plasma of mice fed whole grain, bran, and aleurone supplemented diets, compared to refined white flour. Furthermore, bran, aleurone, and whole grain supplemented diets increased microbial α-diversity, but only bran and aleurone increased the cecal concentrations of short-chain fatty acids. The effects on hepatic lipid metabolism might thus at least partially be mediated by microbiota-dependent mechanisms.