Measurement Of Metabolite Concentrations Research Articles

PSIX-4 A Veterinary Gastrointestinal Diet Affects Fecal Characteristics, Metabolites, Bile Acids, and Microbiota Concentrations of Antibiotic-Treated Cats

Abstract Antibiotics are often used to treat animals with gastrointestinal diseases or infections, but their use is known to have negative effects on the intestinal microbiota community. Identifying dietary strategies that may aid in the recovery from antibiotic use is of interest. The objective of this study was to determine how a veterinary gastrointestinal diet affected the fecal characteristics and metabolite, bile acid (BA), and microbiota concentrations of cats recovering from metronidazole administration. Twenty-four healthy adult cats were used in an 8-wk completely randomized design study. The study began with a 2-wk baseline phase where all cats consumed a leading grocery brand dry diet (GBD; wk 0 to wk 2). Over the next 2 wk, cats continued to consume GBD and received metronidazole (20 mg/kg BW; wk 2 to wk 4). At wk 4, cats were randomly allotted to one of two treatments [GBD; Blue Buffalo Gastrointestinal Support (BB)] for the remaining 4 wk (wk 5 to wk 8). Fecal scores were recorded daily, and fresh fecal samples were collected at wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter, and metabolite and BA concentrations. Fecal microbiota were analyzed by 16S rRNA sequencing and qPCR, which was used to calculate dysbiosis index. All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, with treatment treated as a fixed effect, cat as a random effect, and significance set at P< 0.05 and trends at P < 0.10. Metronidazole administration had dramatic effects on the outcomes measured, including increased fecal scores (looser stools), reduced fecal pH and dry matter %, reduced fecal short-chain fatty acid (SCFA), branched-chain fatty acid, ammonia, phenol, and indole concentrations, and altered fecal BA concentrations (increased primary BA; reduced secondary BA). Furthermore, fecal bacterial alpha diversity was reduced, dysbiosis index was increased, and the abundance of several individual bacterial taxa were altered. Fecal outcome variables partially recovered over the next 4 wk, with some being impacted by diet. The change in fecal acetate concentrations were greater and the change in total SCFA concentrations tended to be greater in cats fed BB. Dysbiosis index and alpha diversity measures slowly recovered over 4 wk, without differences due to diet. Recovery of about 20 bacterial genera were impacted by diet. Primary and secondary BA concentrations and ratios demonstrated a prolonged impairment of primary to secondary BA conversion. Most of these changes were an effect of time, but the change of fecal cholic acid concentrations were less in cats fed BB. In conclusion, our data demonstrate that metronidazole is a powerful antibiotic that has long-lasting effects on the fecal microbiota and metabolites of cats. The veterinary gastrointestinal diet tested aided in the recovery of some variables measured.

PSIX-3 A Veterinary Gastrointestinal Low-Fat Diet Affects Fecal Characteristics, Metabolites, Bile Acids, and Microbiota Concentrations of Antibiotic-Treated Dogs

Abstract In a recent dog study, the antibiotic metronidazole proved to be an effective disruptor of the fecal microbiota and resulted in looser stools and altered fecal bile acid (BA) profiles. Recovery took several weeks and was aided by a dietary supplement. The objective of this study was to determine how a veterinary gastrointestinal low-fat diet affected the fecal characteristics and metabolite, BA, and microbiota concentrations of dogs recovering from metronidazole administration. Twenty-four healthy adult dogs were used in an 8-wk completely randomized design study. The study began with a 2-wk baseline phase where all dogs consumed a leading grocery brand dry kibble diet (GBD; wk 0 to wk 2). Over the next 2 wk, dogs continued to consume GBD and received metronidazole (20 mg/kg BW; wk 2 to wk 4). At wk 4, dogs were randomly allotted to one of two treatments [GBD; Blue Buffalo Gastrointestinal Support Low-Fat (BB)] for the remaining 4 wk (wk 5 to wk 8). Fecal scores were recorded daily, and fresh fecal samples were collected at wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter, and metabolite and bile acid concentrations. Fecal microbiota populations were also analyzed by 16S rRNA sequencing and qPCR, which was used to calculate dysbiosis index. All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, with treatment treated as a fixed effect, dog as a random effect, and significance set at P < 0.05. As anticipated, metronidazole administration increased (P < 0.0001) fecal scores (looser stools) and drastically altered (P < 0.05) fecal metabolite concentrations, reducing fecal short-chain fatty acid (SCFA), branched-chain fatty acid (BCFA), phenol, and indole concentrations, increasing primary BA concentrations, and decreasing secondary bile acid concentrations. Metronidazole dramatically impacted fecal microbiota populations by reducing alpha diversity, altering the abundance of multiple bacterial taxa, and increasing dysbiosis index. Fecal outcome variables recovered over the next 4 wk, with some being impacted by diet. The change in fecal SCFA and immunoglobulin A concentrations were greater (P < 0.01), while the change in fecal BCFA, phenol, and indole concentrations were decreased (P < 0.05) in dogs fed BB. Dysbiosis index and alpha diversity measures recovered quickly for all dogs and without dietary effects. Recovery of over 25 bacterial genera were impacted by diet (P < 0.05). While many bacterial taxa returned to baseline levels after 4 wk, others had still not fully recovered. Fecal BA concentrations recovered quickly for all dogs. The change in lithocholic acid concentration was less (P < 0.0001) in dogs fed BB, but other BA concentrations were not affected by diet. In conclusion, metronidazole drastically alters the fecal microbiota and metabolites of dogs. While most variables return to normal over time, a veterinary gastrointestinal diet may aid in the recovery.

The effect of teff (Eragrostis tef) hay inclusion on feed intake, digestibility, and milk production in dairy cows

Teff (Eragrostis tef) is a drought-tolerant, multi-harvest, high-quality summer forage crop. We conducted a study aiming at assessing the effect of replacing wheat hay with teff hay in diets on the feed intake, digestibility, and lactational performance of dairy cows. Thirty-four multiparous (≥3rd parity) Israeli Holstein Friesian dairy cows averaging (± SD) 182 days in milk ± 8 days in milk, 45 kg/d ± 4.8 kg/d of milk yield, and a body weight of 647.1 kg ± 51 kg at the beginning of the study were recruited to a 6-week feeding trial. Cows were randomly divided into two balanced groups based on parity, days in milk, and milk yield. Cows were subjected to two low-roughage dietary treatments (~30% roughage): a control wheat hay-based diet and a teff hay-based diet. Production performances, dry matter intake, and nutrient digestibility were measured. Milk samples were analyzed for their composition and fatty acids profile. Blood samples were used to measure metabolite concentrations. The statistical model included fixed effects of dietary treatments, time, and random effects of cows nested in treatment. Production data and feed intake were analyzed as repeated measures using a covariance structure. Dietary treatments did not affect dry matter intake (26 kg/d). However, the teff-fed cows demonstrated higher crude protein digestibility than control cows (61.9% vs. 59.2%). Dietary teff inclusion increased milk yield by 1.5 kg/d. Polyunsaturated fatty acids and omega-3 fatty acids profiles in milk were greater in the teff cows than in the control cows (4.77 g/100 g vs. 4.36 g/100 g and 3.71 g/100 g vs. 3.43 g/100 g, respectively). Non-esterified fatty acids, beta-hydroxybutyrate, and blood urea nitrogen concentrations in circulation were higher in the control group than in the teff group. The acetic-to-propionic-acid ratio in the rumen fluid was higher in control cows than in teff cows (2.90 vs. 2.43). However, the ruminal ammonia-N concentration was higher in the teff cows than in the control cows (18.5 mg/dL vs. 15.8 mg/dL). In conclusion, teff hay inclusion in the rations of high-producing dairy cows increased milk yield, which could be attributed to improved crude protein digestibility and energy partition to production.

Liquid chromatography-tandem mass spectrometry based simultaneous quantification of tryptophan, serotonin and kynurenine pathway metabolites in tissues and cell culture systems

BackgroundKynurenine and respective metabolites exhibit bioactivity as well as tryptophan, an essential amino acid, and the neurotransmitter serotonin. Dysregulations in the kynurenine pathway are involved in neurodegenerative/neuropsychiatric disorders and diabetes mellitus type 2 but also in cancer. Therefore, measurements of kynurenine-related metabolites will improve the general understanding for kynurenine pathway relevance in disease pathogenesis. MethodsTryptophan, serotonin, picolinic acid, quinolinic acid, 3–OH-kynurenine, kynurenine, 3-OH-anthranilic acid, kynurenic acid, anthranilic acid as well as nicotinic acid and the redox cofactor NAD+ were analyzed in heterogeneous matrices by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After validation, the described method was applied for measurements of native metabolite concentrations in murine tissues and cellular systems including pathway-shift monitoring after treatment with the tryptophan-2,3-dioxygenase-inhibitor 680C91. In addition, the method was evaluated for its ability for integration into multi-omics approaches using a single sample metabolite extraction procedure. ResultsA simple and sensitive UPLC-MS/MS method for simultaneous quantification of up to 10 kynurenine-related metabolites in four biological matrices was developed. Within a run time of 6.5 min, chromatographic separation of kynurenine-related metabolites, including the isomers nicotinic acid and picolinic acid, was achieved without derivatization. Validation parameters, including interday precision (<14.8%), mean accuracy (102.4% ± 12.9%) and linear detection ranges of more than three orders of magnitude, indicate method reliability. Depending the investigated sample matrix, the majority of metabolites were successfully detected and quantified in native murine and cell culture derived sample materials. Furthermore, the method allowed to monitor the impact of a tryptophan-2,3-dioxygenase-inhibitor on kynurenine pathway in a cellular system and is suitable for multi-assay analyses using aliquots from the same cell extract. ConclusionThe described UPLC-MS/MS method provides a simple tool for the simultaneous quantification of kynurenine pathway metabolites. Due to its suitability for many physiological matrices, the method provides wide application for disease-related experimental settings.

Closed Bipolar Electrode-Enabled Electrochromic Sensing of Multiple Metabolites in Whole Blood.

We report a closed bipolar electrode (CBE)-based sensing platform for the detection of diagnostic metabolites in undiluted whole human blood. The sensor is enabled by electrode chemistry based on: (1) a mixed layer of blood-compatible adsorption-resistant phosphorylcholine (PPC) and phenylbutyric acid (PBA), (2) ferrocene (Fc) redox mediators, and (3) immobilized redox-active enzymes. This scheme is designed to overcome nonspecific protein adsorption and amplify sensing currents in whole human fluids. The scheme also incorporates a diffusing mediator to increase electronic communication between the immobilized redox enzyme and the working electrode. The use of both bound and freely diffusing mediators is synergistic in producing the electrochemical response. The sensor is realized by linking the analyte cell, containing the specific electrode surface architecture, through a CBE to a reporter cell containing the electrochromic reporter, methyl viologen (MV). The colorless-to-purple color change accompanying the 1e- reduction of MV2+ is captured using a smartphone camera. Subsequent red-green-blue analysis is performed on the acquired images to determine cholesterol, glucose, and lactate concentrations in whole blood. The CBE blood metabolite sensor produces a linear color change at clinically relevant concentration ranges for all metabolites with good reproducibility (∼5% or better) and with limits of detection of 79 μM for cholesterol, 59 μM for glucose, and 86 μM for lactate. Finally, metabolite concentration measurements from the CBE blood metabolite sensor are compared with results from commercially available FDA-approved blood cholesterol, glucose, and lactate meters, with an average difference of ∼3.5% across all three metabolites in the ranges studied.

Quantitative measurement of peritumoral concentrations of glutamate, N-acetyl aspartate, and lactate on magnetic resonance spectroscopy predicts glioblastoma-related refractory epilepsy.

Increased extracellular glutamate is known to cause epileptic seizures in patients with glioblastoma (GBM). However, predicting whether the seizure will be refractory is difficult. The present study investigated whether evaluation of the levels of various metabolites, including glutamate, can predict the occurrence of refractory seizure in GBM by quantitative measurement of metabolite concentrations on magnetic resonance spectroscopy (MRS). Forty patients were treated according to the same treatment protocol for primary GBM at Ehime University Hospital between April 2017 and July 2021. Of these patients, 23 underwent MRS to determine concentrations of metabolites, including glutamate, N-acetylaspartate, creatine, and lactate, in the tumor periphery by applying LC-Model. The concentration of each metabolite was expressed as a ratio to creatine concentration. Patients were divided into three groups: Type A, patients with no seizures; Type B, patients with seizures that disappeared after treatment; and Type C, patients with seizures that remained unrelieved or appeared after treatment (refractory seizures). Relationships between concentrations of metabolites and seizure types were investigated. In 23 GBMs, seizures were confirmed in 11 patients, including Type B in four and Type C in seven. Patients with epilepsy (Type B or C) showed significantly higher glutamate and N-acetylaspartate values than did non-epilepsy patients (Type A) (p < 0.05). No significant differences in glutamate or N-acetylaspartate levels were seen between Types B and C. Conversely, Type C showed significantly higher concentrations of lactate than did Type B (p = 0.001). Cutoff values of lactate-to-creatine, glutamate-to-creatine, and N-acetylaspartate-to-creatine ratios for refractory seizure were > 1.25, > 1.09, and > 0.88, respectively. Extracellular concentrations of glutamate, N-acetylaspartate, and lactate in the tumor periphery were significantly elevated in patients with GBM with refractory seizures. Measurement of these metabolites on MRS may predict refractory epilepsy in such patients and could be an indicator for continuing the use of antiepileptic drugs.

Longitudinal associations of plasma metabolites with persistent fatigue among colorectal cancer survivors up to 2 years after treatment.

The underlying biological mechanisms causing persistent fatigue complaints after colorectal cancer treatment need further investigation. We investigated longitudinal associations of circulating concentrations of 138 metabolites with total fatigue and subdomains of fatigue between 6 weeks and 2 years after colorectal cancer treatment. Among stage I-III colorectal cancer survivors (n=252), blood samples were obtained at 6 weeks, and 6, 12 and 24 months posttreatment. Total fatigue and fatigue subdomains were measured using a validated questionnaire. Tandem mass spectrometry was applied to measure metabolite concentrations (BIOCRATES AbsoluteIDQp180 kit). Confounder-adjusted longitudinal associations were analyzed using linear mixed models, with false discovery rate (FDR) correction. We assessed interindividual (between-participant differences) and intraindividual longitudinal associations (within-participant changes over time). In the overall longitudinal analysis, statistically significant associations were observed for 12, 32, 17 and three metabolites with total fatigue and the subscales "fatigue severity," "reduced motivation" and "reduced activity," respectively. Specifically, higher concentrations of several amino acids, lysophosphatidylcholines, diacylphosphatidylcholines, acyl-alkylphosphatidylcholines and sphingomyelins were associated with less fatigue, while higher concentrations of acylcarnitines were associated with more fatigue. For "fatigue severity," associations appeared mainly driven by intraindividual associations, while for "reduced motivation" stronger interindividual associations were found. We observed longitudinal associations of several metabolites with total fatigue and fatigue subscales, and that intraindividual changes in metabolites over time were associated with fatigue severity. These findings point toward inflammation and an impaired energy metabolism due to mitochondrial dysfunction as underlying mechanisms. Mechanistic studies are necessary to determine whether these metabolites could be targets for intervention.

Data dimensionality reduction technique for clustering problem of metabolomics data

In metabolomics studies, independent analyses or replicating the metabolite concentration measurements are often performed to anticipate errors. On the other hand, the size of the dataset is increasing. For clustering purposes, obtaining representative information chemically from independent analyses is needed. The objective of this study is to develop a data reduction method such that a dataset that represents chemical information is obtained. Overall a proper data reduction method would simplify the clustering of metabolite data. We propose the modified Weiszfeld algorithm (MWA) to reduce independent analyses. To obtain comprehensive results, we compare MWA with some other well-known reduction methods, including PCA, CMDS, LE, and LLE. Then reduced datasets are clustered using the fuzzy c-means (FCM) algorithm with the Tang Sun Sun (TSS) index and silhouette index as the cluster validity indices. The results show that MWA, together with PCA, present the optimal number of clusters, namely four clusters. This result aligns with the optimal number of clusters before dimensionality reduction. The present results show that MWA is robust to perform dimensionality reduction of independent analyses while maintaining chemical information on the reduced dataset. Therefore, we recommend the reliability of MWA as one of the chemometric techniques, and the present finding has enriched chemometric techniques in metabolomics studies.

Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry for Automated Analysis of Four Sulfated Steroid Metabolites in Saliva Samples.

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL−1 for PREGS, DHEAS, and CRTS and of 0.05–10 ng mL−1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL−1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.

Sex-specific links between the social landscape and faecal glucocorticoid metabolites in semi-captive Asian elephants

Although social behaviour is common in group-living mammals, our understanding of its mechanisms in long-lived animals is largely based on studies in human and non-human primates. There are health and fitness benefits associated with strong social ties, including increased life span, reproductive success, and lower disease risk, which are attributed to the proximate effects of lowered circulating glucocorticoid hormones. However, to deepen our understanding of health-social dynamics, we must explore species beyond the primate order. Here, using Asian elephants as a model species, we combine social data generated from semi-captive timber elephants in Myanmar with measurements of faecal glucocorticoid metabolite (FGM) concentrations. These data enable a “natural experiment” because individuals live in work groups with different demographic compositions. We examine sex-specific FGM concentrations for four different aspects of an individuals’ social world: general sociality, work group size, sex ratio and the presence of immatures (<5 years) within the work group. Males experienced lower FGM concentrations when engaged in more social behaviours and residing in female-biased work groups. Surprisingly, females only exhibited lower FGM concentrations when residing with calves. Together, our findings highlight the importance of sociality on individual physiological function among elephants, which may have broad implications for the benefits of social interactions among mammals.

Reliable Budding Pattern Classification of Yeast Cells with Time-Resolved Measurement of Metabolite Production

Filamentous growth in Saccharomyces cerevisiae is a stress response commonly induced under nutrient deprivation and by certain alcohols. It is a compound phenotype characterized by pseudohyphal growth, invasion and a shift to more polarized budding. Previous methods have not allowed the time-resolved determination of filamentous growth. Here we present a new method for budding pattern characterization that enables the measurement of filamentous growth and metabolite concentration during yeast cell growth at precise time intervals. By combining chemical cell immobilization and single-cell imaging using an oCelloScope™, this method provides more accurate budding pattern classification compared with previous methods. The applications of the method include, for example, investigation of quorum sensing-controlled yeast filamentous growth and metabolism under stress and identification of toxic metabolites.

Advancements in Pulsed Stable Isotope-Resolved Metabolomics.

The understanding of biochemical processes of metabolism is gained through the measurement of the concentration of intermediates and the rate of metabolite conversion. However, the measurement of metabolite concentrations does not give a full representation of this dynamic system. To understand the kinetics of metabolism, the systemmust be described and quantified in terms of metabolite flow as a function of time. In order to measure the metabolite flow, or more precisely the metabolic flux through a biological system, substrates of the cell are labelled with stable isotopes. The usage of these substrates by the cell leads to the incorporation of the isotopes into downstream intermediates.The most important metabolic pathways are encompassed in the central carbon metabolism (CCM). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the central carbon metabolism "is the most basic aspect of life". It includes all metabolites and enzymatic reactions within: glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), amino acids and nucleotide metabolic pathways. Some molecules are at the crossroad of metabolic pathways, interconnecting diverse metabolic and therefore functional outcomes. Labelling these nodal metabolites and analysing their isotopic composition allows the precise determination of the metabolic flow within the biochemical networks that they are in.Application of stable isotope labelled substrates allows the measurement of metabolic flux through a biochemical pathway. The rapid turnover of metabolites in pathways requires pulse-feeding cells with a labelled substrate. This method allows for the determination of different cell states. For example, the action of a drug from immediate impact until the compensatory response of the metabolic system (cell, organs, organisms). Pulsed labelling is an elegant way to analyse the action of small molecules and drugs and enables the analysis of regulatory metabolic processes in short time scales.

Water-suppression cycling 3-T cardiac 1 H-MRS detects altered creatine and choline in patients with aortic or mitral stenosis.

Cardiac proton spectroscopy (1H‐MRS) is widely used to quantify lipids. Other metabolites (e.g. creatine and choline) are clinically relevant but more challenging to quantify because of their low concentrations (approximately 10 mmol/L) and because of cardiac motion. To quantify cardiac creatine and choline, we added water‐suppression cycling (WSC) to two single‐voxel spectroscopy sequences (STEAM and PRESS). WSC introduces controlled residual water signals that alternate between positive and negative phases from transient to transient, enabling robust phase and frequency correction. Moreover, a particular weighted sum of transients eliminates residual water signals without baseline distortion. We compared WSC and the vendor's standard ‘WET’ water suppression in phantoms. Next, we tested repeatability in 10 volunteers (seven males, three females; age 29.3 ± 4.0 years; body mass index [BMI] 23.7 ± 4.1 kg/m2). Fat fraction, creatine concentration and choline concentration when quantified by STEAM‐WET were 0.30% ± 0.11%, 29.6 ± 7.0 μmol/g and 7.9 ± 6.7 μmol/g, respectively; and when quantified by PRESS‐WSC they were 0.30% ± 0.15%, 31.5 ± 3.1 μmol/g and 8.3 ± 4.4 μmol/g, respectively. Compared with STEAM‐WET, PRESS‐WSC gave spectra whose fitting quality expressed by Cramér‐Rao lower bounds improved by 26% for creatine and 32% for choline. Repeatability of metabolite concentration measurements improved by 72% for creatine and 40% for choline. We also compared STEAM‐WET and PRESS‐WSC in 13 patients with severe symptomatic aortic or mitral stenosis indicated for valve replacement surgery (10 males, three females; age 75.9 ± 6.3 years; BMI 27.4 ± 4.3 kg/m2). Spectra were of analysable quality in eight patients for STEAM‐WET, and in nine for PRESS‐WSC. We observed comparable lipid concentrations with those in healthy volunteers, significantly reduced creatine concentrations, and a trend towards decreased choline concentrations. We conclude that PRESS‐WSC offers improved performance and reproducibility for the quantification of cardiac lipids, creatine and choline concentrations in healthy volunteers at 3 T. It also offers improved performance compared with STEAM‐WET for detecting altered creatine and choline concentrations in patients with valve disease.

Bilingualism is a long-term cognitively challenging experience that modulates metabolite concentrations in the healthy brain

Cognitively demanding experiences, including complex skill acquisition and processing, have been shown to induce brain adaptations, at least at the macroscopic level, e.g. on brain volume and/or functional connectivity. However, the neurobiological bases of these adaptations, including at the cellular level, are unclear and understudied. Here we use bilingualism as a case study to investigate the metabolic correlates of experience-based brain adaptations. We employ Magnetic Resonance Spectroscopy to measure metabolite concentrations in the basal ganglia, a region critical to language control which is reshaped by bilingualism. Our results show increased myo-Inositol and decreased N-acetyl aspartate concentrations in bilinguals compared to monolinguals. Both metabolites are linked to synaptic pruning, a process underlying experience-based brain restructuring. Interestingly, both concentrations correlate with relative amount of bilingual engagement. This suggests that degree of long-term cognitive experiences matters at the level of metabolic concentrations, which might accompany, if not drive, macroscopic brain adaptations.

Effect of increasing dietary protein with constant lysine:methionine ratio on production and omasal flow of nonammonia nitrogen in lactating dairy cows

Eight lactating cows were fed 4 diets in which dietary crude protein (CP) was increased in steps of approximately 2 percentage units from 11 to 17% of DM by replacing high-moisture corn with soybean meal supplemented with rumen-protected Met to maintain a Lys:Met ratio of 3:1 in metabolizable protein. Trial design was a replicated 4 × 4 Latin square; experimental periods lasted 28 d, with data and sample collection being performed during wk 3 and 4 of each period. Digesta samples were collected from the rumen as well as the omasum to measure metabolite concentrations and ruminal outflow of N fractions using infusion of 15N-enriched ammonia to quantify microbial nonammonia N (NAN) and nonmicrobial NAN. Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc.). There were linear increases in the yields of milk and true protein and concentration of milk urea N, and a linear decrease in N efficiency, with increasing dietary CP. Apparent ruminal and total-tract N digestibility increased linearly with increasing dietary CP, but estimated true total-tract N digestibility was not affected. Apparent digestibility of the other macronutrients was not influenced by diet. Ruminal ammonia, total AA and peptides, and branched-chain VFA also increased linearly with dietary CP. The 15N enrichment of liquid- and particle-associated microbes linearly declined with increasing dietary CP due to decreasing 15N enrichment of the ammonia pool. Although no effect of dietary CP on nonmicrobial NAN flow was detected, total NAN flow increased linearly from 525 g/d at 11% CP to 637 g/d at 17% CP due to the linear increase in microbial NAN flow from 406 g/d at 11% CP to 482 g/d at 17% CP. Under the conditions of this study, when dietary CP was increased by adding soybean meal supplemented with rumen-protected Met, improved milk and protein yields were driven not by RUP supply but by increased ruminal outflow of microbial protein.

The interplay between social rank, physiological constraints and investment in courtship in male spotted hyenas

Abstract In many animal societies, dominant males have a higher reproductive success than subordinate males. The proximate mechanisms by which social rank influences reproductive success are poorly understood. One prominent hypothesis posits that rank‐related male attributes of attractiveness and fighting ability are the main mediators of reproductive skew. Yet, empirical evidence in support of this hypothesis is limited. An alternative hypothesis emphasises the inherent social dimension of dominance relationships and posits that the relationship between male social rank and reproductive success is mediated by the physiological costs of male–male competition. This has not been tested in systems in which the two hypotheses can be disentangled. We investigated the interplay between male social rank, physiological costs and male investment in social and sexual activities. We used measurements of faecal glucocorticoid metabolite concentrations (fGMC) as biomarkers of physiological costs and long‐term behavioural data of 319 males in free‐ranging spotted hyenas Crocuta crocuta, a species in which male access to females and reproductive success do not depend on physical attributes. When males courted females and interacted with male competitors, low‐ranking males had higher fGMC than high‐ranking males. In contrast, fGMC did not vary with social rank when males were alone or when they courted females and competitors were absent. Low‐ranking males minimised their exposure to physiologically costly intrasexual competition; they spent more time alone and less time engaging in social and sexual activities than did high‐ranking males. They also invested less than high‐ranking males in courting the most contested and highest‐quality females. Our findings demonstrate that the physiological costs of intrasexual competition in male spotted hyenas vary with social rank and shape behavioural trade‐offs between the allocation of time and physiological resources to social integration, reproduction and self‐maintenance. Our study suggests that physiological and social constraints play a pivotal role in the emergence of rank‐related male reproductive success. The study provides insights into how the social organisation and breeding system shape physiological constraints and patterns of reproductive skew within and between species. A free Plain Language Summary can be found within the Supporting Information of this article.

Magnetic resonance spectroscopy in the rodent brain: Experts' consensus recommendations.

In vivo MRS is a non-invasive measurement technique used not only in humans, but also in animal models using high-field magnets. MRS enables the measurement of metabolite concentrations as well as metabolic rates and their modifications in healthy animals and disease models. Such data open the way to a deeper understanding of the underlying biochemistry, related disturbances and mechanisms taking place during or prior to symptoms and tissue changes. In this work, we focus on the main preclinical 1H, 31P and 13C MRS approaches to study brain metabolism in rodent models, with the aim of providing general experts' consensus recommendations (animal models, anesthesia, data acquisition protocols). An overview of the main practical differences in preclinical compared with clinical MRS studies is presented, as well as the additional biochemical information that can be obtained in animal models in terms of metabolite concentrations and metabolic flux measurements. The properties of high-field preclinical MRS and the technical limitations are also described.

Urinary Concentrations of Phthalate Metabolite Mixtures in Relation to Serum Biomarkers of Thyroid Function and Autoimmunity among Women from a Fertility Center.

Background:Although previous epidemiological studies have explored associations of phthalate metabolites with thyroid function, no studies to date have assessed associations of mixtures with thyroid function and autoimmunity among potentially susceptible subgroups such as subfertile women.Objective:We aimed to explore associations of mixtures of urinary phthalate metabolites with serum markers of thyroid function and autoimmunity.Methods:This cross-sectional study included 558 women attending a fertility center who provided one spot urine and one blood sample at enrollment (2005–2015). We quantified urinary concentrations of eight phthalate metabolites using mass spectrometry, and biomarkers of thyroid function [thyroid-stimulating hormone (TSH), free and total thyroxine (, ) and triiodothyronine (, ), and autoimmunity [thyroid peroxidase and thyroglobulin antibodies (TPOAb and TgAb, respectively)] in serum using electrochemiluminescence assays. We applied principal component analysis (PCA) and Bayesian kernel machine regression (BKMR) to identify the main patterns of urinary phthalate metabolites. We used linear mixed models to assess the association between PCA-derived factor scores in quintiles and serum thyroid function and autoimmunity, adjusting for age, body mass index (BMI), specific gravity (SG), and, for the PCA, other factor scores.Results:We observed two factors using PCA, one representing the di(2-ethylhexyl) (DEHP) and another non-DEHP metabolites. Compared to women in the lowest quintile of the DEHP factor scores, women in the highest quintile had significantly lower serum concentrations of , , , and [absolute difference: ; 95% confidence interval (CI): , ; ; absolute difference: ; 95% CI: , ; ; absolute difference: ; 95% CI: 0.54, ; ; and absolute difference: ; 95% CI: , ; , respectively]. Using BKMR, we observed that mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was the primary contributor to these negative associations. DEHP and non-DEHP factor scores were not associated with serum TSH, TgAb, or TPOAb.Conclusions:Mixtures of urinary DEHP metabolites were inversely associated with serum biomarkers of thyroid function but not with autoimmunity, which were within normal ranges for healthy adult women. https://doi.org/10.1289/EHP6740

Simultaneous Measurement of Metabolite Concentration and Isotope Incorporation by Mass Spectrometry.

Studies of the topology, functioning, and regulation of metabolic systems are based on two main types of information that can be measured by mass spectrometry: the (absolute or relative) concentration of metabolites and their isotope incorporation in 13C-labeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two sets of information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of 2 both the number of cultivations to perform and the number of samples to collect, prepare, and analyze. The proposed approach is based on an IS labeled with other isotope(s) that can be resolved from the 13C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.

Assessment of Uterine Cervical Cancer by Means of MR Spectroscopy

Abstract Background Proton magnetic resonance spectroscopy has been shown to have clinical value in managing cancers of the brain, breast, cervix and prostate, it is useful for diagnosing and monitoring treatment of cervical cancer by analyzing the metabolite composition of cervical tumors, providing details of tumors metabolism that might assist tumor grading and leading to a better understanding of the biochemical pathways found within the lesion, serving as a noninvasive biomarker of metabolism in tumors. 1H-MRS has achieved great strides as a molecular imaging technique since its introduction, and its scope in many clinical scenarios and research settings is rising. Objective In this study, MRS was performed in all cases using single voxel spectroscopy (SVS) and patient spectra were interpreted qualitatively by inspection of the peaks of lipid and choline. Aim and Patients and Methods This study was carried out during the period between December 2017 and September 2018. Twenty three patients with cancer cervix diagnosed clinically and/or pathologically proved cancer were recruited from (Oncological department) in Ain Shams University Hospitals. Results Our study revealed high lipid level in 65% of cervical cancer patients which has 100% sensitivity and 74% specificity in detecting cervical cancer, choline level which considered most consistent difference between majority of normal tissue and tumors shows high level in measured 69.2% of the patients. Conclusion Abnormal metabolism is a key tumor hallmark. Proton magnetic resonance spectroscopy (1H-MRS) allows measurement of metabolite concentration that can be utilized to characterize tumor metabolic changes. 1H-MRS measurements of specific metabolites have been implemented in the clinic. This study interpret ate the role of 1H-MRS for cancer evaluation, evaluates its strengths and limitations, and correlates metabolite peaks at 1H-MRS with diagnostic and prognostic parameters of cervical cancer.