A new statistical workflow (R-packages based) to investigate associations between one variable of interest and the metabolome.

INTRODUCTION: In metabolomics, the investigation of associations between the metabolome and one trait of interest is a key research question. However, statistical analyses of such associations are often challenging. Statistical tools enabling resilient verification and clear presentation are therefore highly desired. OBJECTIVES: Our aim is to provide a contribution for statistical analysis of metabolomics data, offering a widely applicable open-source statistical workflow, which considers the intrinsic complexity of metabolomics data. METHODS: We combined selected R packages tailored for all properties of heterogeneous metabolomics datasets, where metabolite parameters typically (i) are analyzed in different matrices, (ii) are measured on different analytical platforms with different precision, (iii) are analyzed by targeted as well as non-targeted methods, (iv) are scaled variously, (v) reveal heterogeneous variances, (vi) may be correlated, (vii) may have only few values or values below a detection limit, or (viii) may be incomplete. RESULTS: The code is shared entirely and freely available. The workflow output is a table of metabolites associated with a trait of interest and a compact plot for high-quality results visualization. The workflow output and its utility are presented by applying it to two previously published datasets: one dataset from our own lab and another dataset taken from the repository MetaboLights. CONCLUSION: Robustness and benefits of the statistical workflow were clearly demonstrated, and everyone can directly re-use it for analysis of own data.

Roux-En-Y Gastric Bypass (RYGB) Surgery during High Liquid Sucrose Diet Leads to Gut Microbiota-Related Systematic Alterations.

Roux-en-Y gastric bypass (RYGB) surgery has been proven successful in weight loss and improvement of co-morbidities associated with obesity. Chronic complications such as malabsorption of micronutrients in up to 50% of patients underline the need for additional therapeutic approaches. We investigated systemic RYGB surgery effects in a liquid sucrose diet-induced rat obesity model. After consuming a diet supplemented with high liquid sucrose for eight weeks, rats underwent RYGB or control sham surgery. RYGB, sham pair-fed, and sham ad libitum-fed groups further continued on the diet after recovery. Notable alterations were revealed in microbiota composition, inflammatory markers, feces, liver, and plasma metabolites, as well as in brain neuronal activity post-surgery. Higher fecal 4-aminobutyrate (GABA) correlated with higher Bacteroidota and Enterococcus abundances in RYGB animals, pointing towards the altered enteric nervous system (ENS) and gut signaling. Favorable C-reactive protein (CRP), serine, glycine, and 3-hydroxybutyrate plasma profiles in RYGB rats were suggestive of reverted obesity risk. The impact of liquid sucrose diet and caloric restriction mainly manifested in fatty acid changes in the liver. Our multi-modal approach reveals complex systemic changes after RYGB surgery and points towards potential therapeutic targets in the gut-brain system to mimic the surgery mode of action.

Targeted ultra-performance liquid chromatography/tandem mass spectrometric quantification of methylated amines and selected amino acids in biofluids.

RATIONALE: Methylated amino compounds and basic amino acids are important analyte classes with high relevance in nutrition, physical activity and physiology. Reliable and easy quantification methods covering a variety of metabolites in body fluids are a prerequisite for efficient investigations in the field of food and nutrition. METHODS: Targeted ultra-performance liquid chromatography/tandem mass spectrometric (UHPLC/MS) analysis was performed using HILIC separation and timed ESI-MRM detection, combined with a short sample preparation. Calibration in urine and blood plasma was achieved by matrix-matched standards, isotope-labelled internal standards and standard addition. The method was fully validated and the performance was evaluated using a subset from the Karlsruhe Metabolomics and Nutrition (KarMeN) study. RESULTS: Within this method, a total of 30 compounds could be quantified simultaneously in a short run of 9 min in both body fluids. This covers a variety of free amino compounds which are present in very different concentrations. The method is easy, precise and robust, and has a broad working range. As a proof of principle, literature-based associations of certain metabolites with dietary intake of respective foods were clearly confirmed in the KarMeN subset. CONCLUSIONS: Overall, the method turned out to be well suited for application in nutrition studies, as shown for the example of food intake biomarkers in KarMeN. Application to a variety of questions such as food-related effects or physical activity will support future studies in the context of nutrition and health.

Validation of the custo screen pediatric blood pressure monitor according to the European Society of Hypertension International Protocol revision 2010.

The purpose of the study was to validate the ambulatory blood pressure monitoring (ABPM) device custo screen pediatric in children aged 3 to 12 years according to the International Protocol of the European Society of Hypertension (ESH-IP revision 2010). Thirty-three children were included and systolic and diastolic blood pressure measurements were performed according to the ESH-IP. The protocol was modified for children considering data from the German Health Interview and Examination Survey for Children and Adolescents (KIGGS). The custo screen pediatric met all the requirements of the ESH-IP. The mean difference between the test device and the reference was -1.4 ± 3.0 mmHg for systolic blood pressure (SBP) and -0.7 ± 3.2 mmHg for diastolic blood pressure (DBP). For SBP and DBP, all 99 measurements were within the absolute difference of 10 mmHg between the test device and the reference. As to part 2 of the protocol, for DBP in all subjects, two out of three measurements were within 5 mmHg between the device and the standard, whereas for SBP in 32 of 33 subjects, two out of three measurements were within this range. CONCLUSION: The custo screen pediatric met all criteria of the ESH-IP review 2010, modified for children from 3 to about 12 years, and can be recommended for ABPM in children. What is Known: • Validation of blood pressure measuring devices is essential to provide patients with an accurate blood pressure measuring device. • The majority of devices has not been validated in children. What is New: • Prior to the present validation, study protocol adjustments of ESH-IP review 2010 for children were defined according to German Health Interview and Examination Survey for Children and Adolescents 2013 (KIGGS). • The custo screen pediatric test device met all criteria of ESH-IP revision 2010, modified for children, and can be recommended for ABPM in children aged 3 to about 12 years.

Dietary essential α-linolenic acid and linoleic acid differentially modulate TNFα-induced NFκB activity in FADS2-deficient HEK-293 cells.

The pro- or anti-inflammatory bioactivity of dietary essential linoleic acid (LA) and alpha-linolenic acid (ALA) is mainly attributed to rate-limiting delta-6 desaturase (D6D) activity. The aim of this study was to analyze mechanisms of D6D-substrates ALA, LA and D6D-product gamma-linolenic acid (GLA) under D6D-deficient conditions. Fatty acid profiles (GC-MS), D6D gene expression (real-time RT-PCR) and NFκB activity (luciferase assay) were assessed in HEK293 cells. FADS2 gene expression was approved being marginal. Incubation with ALA or LA did not increase D6D products but their elongase products C20:3n-3 and C20:2n-6. Bypassing the D6D, GLA elevated C20:3n-6 and C20:4n-6. LA significantly increased (+18% at 60 µM; p < .001), ALA reduced (-32% at 100 µM; p < .001) and GLA did not specifically change NFκB activity. Our data indicate that D6D might not be essential for the distinct effects of LA and ALA on NFκB activity.

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.

Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men-A multi-platform metabolomics approach.

Introduction: Endurance exercise alters whole-body as well as skeletal muscle metabolism and physiology, leading to improvements in performance and health. However, biological mechanisms underlying the body's adaptations to different endurance exercise protocols are not entirely understood. Methods: We applied a multi-platform metabolomics approach to identify urinary metabolites and associated metabolic pathways that distinguish the acute metabolic response to two endurance exercise interventions at distinct intensities. In our randomized crossover study, 16 healthy, young, and physically active men performed 30 min of continuous moderate exercise (CME) and continuous vigorous exercise (CVE). Urine was collected during three post-exercise sampling phases (U01/U02/U03: until 45/105/195 min post-exercise), providing detailed temporal information on the response of the urinary metabolome to CME and CVE. Also, fasting spot urine samples were collected pre-exercise (U00) and on the following day (U04). While untargeted two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) led to the detection of 608 spectral features, 44 metabolites were identified and quantified by targeted nuclear magnetic resonance (NMR) spectroscopy or liquid chromatography-mass spectrometry (LC-MS). Results: 104 urinary metabolites showed at least one significant difference for selected comparisons of sampling time points within or between exercise trials as well as a relevant median fold change >1.5 or <0. 6 ¯ (NMR, LC-MS) or >2.0 or <0.5 (GC×GC-MS), being classified as either exercise-responsive or intensity-dependent. Our findings indicate that CVE induced more profound alterations in the urinary metabolome than CME, especially at U01, returning to baseline within 24 h after U00. Most differences between exercise trials are likely to reflect higher energy requirements during CVE, as demonstrated by greater shifts in metabolites related to glycolysis (e.g., lactate, pyruvate), tricarboxylic acid cycle (e.g., cis-aconitate, malate), purine nucleotide breakdown (e.g., hypoxanthine), and amino acid mobilization (e.g., alanine) or degradation (e.g., 4-hydroxyphenylacetate). Discussion: To conclude, this study provided first evidence of specific urinary metabolites as potential metabolic markers of endurance exercise intensity. Future studies are needed to validate our results and to examine whether acute metabolite changes in urine might also be partly reflective of mechanisms underlying the health- or performance-enhancing effects of endurance exercise, particularly if performed at high intensities.

Validation of the Microlife BP B3 AFIB upper arm blood pressure monitor in adults and adolescents according to the ANSI/AAMI/ISO 81060-2:2019 protocol.

OBJECTIVE: Aim of this study was to validate the Microlife BP B3 AFIB/enterprise resource planning (ERP) No: BP3KT1-3 N blood pressure (BP) monitor according to the American National Standards Institute (ANSI)/Association for the Advancement of Medical Instrumentation (AAMI)/International Organization for Standardization (ISO) 81060-2:2019 in adolescents and adults from a general population. METHODS: BP measurements on the upper arm were performed in 85 subjects (age range 12-88 years), using the Microlife BP B3 AFIB and a standard mercury reference sphygmomanometer. RESULTS: A total of 255 valid BP comparisons were performed for the present validation analysis. The mean ± SD difference between the test and the reference device was 0.70 ± 7.05 mmHg for SBP (pass criterion ≤5 mmHg) and -0.85 ± 4.70 mmHg for DBP (pass criterion ≤5 mmHg) with the SD below the required value of ≤8 mmHg. The mean ± SD of the intraindividual differences between the test and the reference device was 0.70 ± 5.87 mmHg for SBP (pass criterion for the SD ≤6.90 mmHg) and -0.85 ± 4.19 mmHg for DBP (pass criterion for the SD ≤6.88 mmHg). CONCLUSION: The Microlife BP B3 AFIB/ERP No: BP3KT1-3 N has passed the criteria of the ANSI/AAMI/ISO 81060-2:2019 protocol and can be recommended for home BP measurements in adolescents and adults.

Validation of the blood pressure measurement device Beurer BM 28 according to the European Society of Hypertension International Protocol revision 2010.

OBJECTIVE: The aim of the present study was to validate the blood pressure (BP) monitor Beurer BM 28 according to the International Protocol of the European Society of Hypertension (ESH-IP) revision 2010. METHODS: In 33 subjects of age 27-81 years, BP measurements were performed according to the ESH-IP protocol, which alternates reference mercury sphygmomanometer and device-under-test (Beurer BM 28) measurements, resulting in a total of 99 comparisons. RESULTS: As to part 1 of the protocol, an absolute difference within 5 mmHg between the Beurer BM 28 and the test device was found in 83 out of 99 comparisons for the SBP and 82 out of 99 comparisons for the DBP. In 95 out of 99 SBP comparisons and 96 out of 99 DBP comparisons, the difference was found to be within 10 mmHg, whereas only one outlier was noted with an SBP difference higher than 15 mmHg. Mean difference between the test device and the reference was 0.4 ± 4.4 mmHg for SBP, and 0.5 ± 4.3 mmHg for DBP. According to part 2 of the protocol, 30 out of 33 subjects for SBP, and 28 out of 33 for DBP had a minimum of two out of three comparisons staying within the range of 5 mmHg. In none of the subjects, all three comparisons stayed outside the 5 mmHg absolute difference, while in three subjects this was the case for the DBP. CONCLUSION: The Beurer BM 28 met all requirements of the ESH-IP revision 2010 and can be recommended for BP measurements in the study population under investigation.

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.

Impact of glucuronide interferences on therapeutic drug monitoring of posaconazole by tandem mass spectrometry.

BACKGROUND: Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. METHOD: A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time <3 min was developed and compared to a LC-MS/MS method and HPLC method with fluorescence detection. RESULTS: During evaluation of UPLC-MS/MS, two earlier eluting peaks were observed in the MRM trace of posaconazole. This was only seen in patient samples, but not in spiked calibrator samples. Comparison with LC-MS/MS disclosed a significant bias with higher concentrations measured by LC-MS/MS, while UPLC-MS/MS showed excellent agreement with the commercially available HPLC method. In the LC-MS/MS procedure, comparably wide and left side shifted peaks were noticed. This could be ascribed to in-source fragmentation of conjugate metabolites during electrospray ionisation. Precursor and product ion scans confirmed the assumption that the additional compounds are posaconazole glucuronides. Reducing the cone voltage led to disappearance of the glucuronide peaks. Slight modification of the LC-MS/MS method enabled separation of the main interference, leading to significantly reduced deviation. CONCLUSIONS: These results highlight the necessity to reliably eliminate interference from labile drug metabolites for correct TDM results, either by sufficient separation or selective MS conditions. The presented UPLC-MS/MS method provides a reliable and fast assay for TDM of posaconazole.

Determination of globotriaosylceramide in plasma and urine by mass spectrometry.

BACKGROUND: Fabry disease is an X-chromosomally inherited lysosomal storage disorder leading to accumulation of glycosphingolipids, mainly globotriaosylceramide (ceramide-trihexoside, Gb3). Concentrations of Gb3 in plasma and urine have been used to diagnose Fabry disease and to monitor enzyme replacement therapy with recombinant alpha-galactosidase. METHODS: Gb3 was purified from plasma or urine by combined liquid extraction/protein precipitation and solid-phase extraction, and was detected by flow-injection analysis electrospray mass spectrometry (MS) using multi-reaction-monitoring. Calibration was performed via standard addition using C17-Gb3 as internal standard. The most abundant isoforms were monitored for calculation of total Gb3. RESULTS: A MS-based assay for quantification of Gb3 in plasma and urine was established and validated. Intra- and inter-assay coefficient of variation (CV) of the method were

Determination of naltrexone and 6beta-naltrexol in human blood: comparison of high-performance liquid chromatography with spectrophotometric and tandem-mass-spectrometric detection.

We present data for a comparison of a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and a high-performance liquid-chromatographic method with column switching and UV spectrophotometric detection. The two methods were developed for determination of naltrexone and 6beta-naltrexol in blood serum or plasma aiming to be used for therapeutic drug monitoring to guide the treatment of patients with naltrexone. For the high-performance liquid chromatography (HPLC)/UV detection, online sample cleanup was conducted on Perfect Bond C(18) material with 2% (vol/vol) acetonitrile in deionized water. Drugs were separated on a C(18) column using 11.5% (vol/vol) acetonitrile and 0.4% (vol/vol) N,N,N,N-tetramethylethylenediamine within 20 min. LC-MS/MS used naltrexone-d (3) and 6beta-naltrexol-d (4) as internal standards. After protein precipitation, the chromatographic separation was performed on a C(18) column by applying a methanol gradient (5-100%, vol/vol) with 0.1% formic acid over 9.5 min. The HPLC/UV method was found to be linear for concentrations ranging from 2 to 100 ng/ml, with a regression correlation coefficient of r (2) > 0.998 for naltrexone and 6beta-naltrexol. The limit of quantification was 2 ng/ml for naltrexone and 6beta-naltrexol. For the LC-MS/MS method the calibration curves were linear (r(2) > 0.999) from 0.5 to 200 ng/ml for both substances, and the limit of quantification was 0.5 ng/ml. The concentrations measured by the two methods correlated significantly for both substances (r(2) > 0.967; p < 0.001). Both methods could be used for therapeutic drug monitoring. The HPLC/UV method was advantageous regarding automatization and costs, whereas LC-MS/MS was superior with regard to sensitivity.

Evolutionary change from induced to constitutive expression of an indirect plant resistance.

Induced plant resistance traits are expressed in response to attack and occur throughout the plant kingdom. Despite their general occurrence, the evolution of such resistances has rarely been investigated. Here we report that extrafloral nectar, a usually inducible trait, is constitutively secreted by Central American Acacia species that are obligately inhabited by ants. Extrafloral nectar is secreted as an indirect resistance, attracting ants that defend plants against herbivores. Leaf damage induces extrafloral nectar secretion in several plant species; among these are various Acacia species and other Fabaceae investigated here. In contrast, Acacia species obligately inhabited by symbiotic ants nourish these ants by secreting extrafloral nectar constitutively at high rates that are not affected by leaf damage. The phylogeny of the genus Acacia and closely related genera indicate that the inducibility of extrafloral nectar is the plesiomorphic or 'original' state, whereas the constitutive extrafloral nectar flow is derived within Acacia. A constitutive resistance trait has evolved from an inducible one, obviously in response to particular functional demands.

Trimethylamine-N-Oxide Postprandial Response in Plasma and Urine Is Lower After Fermented Compared to Non-Fermented Dairy Consumption in Healthy Adults.

Trimethylamine-N-oxide (TMAO) can be produced by the gut microbiota from dietary substrates and is associated with cardiovascular disease. While dairy products contain TMAO precursors, the effect of fermented dairy on TMAO metabolism remains unclear. We used plasma and urine samples collected for two randomised cross-over studies to evaluate the effects of fermented dairy consumption on TMAO metabolism. In Study 1, thirteen healthy young men tested a yogurt and an acidified milk during postprandial tests and a two-week daily intervention. In Study 2, ten healthy adults tested milk and cheese during postprandial tests. TMAO and five related metabolites were measured in plasma and urine by LC-MS/MS and NMR. Faecal microbiota composition was assessed in Study 1 (16S rRNA metagenomics sequencing). Fermented milk products were associated with lower postprandial TMAO responses than non-fermented milks in urine (Study 1, p = 0.01; Study 2, p = 0.02) and in plasma, comparing yogurt and acidified milk (Study 1, p = 0.04). Daily consumption of dairy products did not differentially affect fasting TMAO metabolites. Significant correlations were observed between microbiota taxa and circulating or urinary TMAO concentrations. Fermentation of dairy products appear, at least transiently, to affect associations between dairy products and circulating TMAO levels.

Quantification of protein phosphorylation by microLC-ICP-MS.

Determination of the protein amount and of the extent of protein phosphorylation is crucial for a variety of research fields, but is not always straightforward. We describe the application of capillary LC-ICP-MS (liquid chromatography-inductively coupled plasma-mass spectrometry) for quantification of phospho-proteins and their phosphorylation degree. Element mass spectrometry is ideally suited for monitor ing and quantification of compounds with heteroelements such as phosphorus and sulphur, particularly because the ICP-MS response is virtually independent from the chemical form of the element. Determination of the phosphorylation stoichiometry, i.e. the relative abundance of the phosphorylated isoforms, can be assessed by the relative abundance of phosphorus compared with sulphur as a marker for the protein amount. Moreover, isotope dilution analysis by post-column addition of a 34S-Spike provides absolute protein quantification with exceptionally high accuracy. Phosphoprotein analysis by capillary LC-ICP-MS may be applied to isolated proteins or protein digests and may include separation of impurities by 1D-SDS-PAGE followed by enzymatic digestion. Alternatively, digestion of complex protein mixtures such as cellular protein extracts allows determination of global, tissue-specific phosphorylation degrees.

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.

µLC coupled to ICP-SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification.

Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (µLC) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (α-fetoprotein). For CRM 393 excellent agreement (37.0 ± 1.4 µmol L(-1)) was obtained with the certificate (37.7 ± 1.8 µmol L(-1)). However, the recovery rate for α-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation.