Low Concentrations Of Metabolites Research Articles

A unified design allows fine-tuning of biosensor parameters and application across bacterial species

In recent years, transcriptional biosensors have become valuable tools in metabolic engineering as they allow semiquantitative determination of metabolites in single cells. Although being perfectly suitable tools for high-throughput screenings, application of transcriptional biosensors is often limited by the intrinsic characteristics of the individual sensor components and their interplay. In addition, biosensors often fail to work properly in heterologous host systems due to signal saturation at low intracellular metabolite concentrations, which typically limits their use in high-level producer strains at advanced engineering stages.We here introduce a biosensor design, which allows fine-tuning of important sensor parameters and restores the sensor response in a heterologous expression host. As a key feature of our design, the regulator activity is controlled through the expression level of the respective gene by different (synthetic) constitutive promoters selected for the used expression host. In this context, we constructed biosensors responding to basic amino acids or ring-hydroxylated phenylpropanoids for applications in Corynebacterium glutamicum and Escherichia coli. Detailed characterization of these biosensors in liquid cultures and during single-cell analysis using flow cytometry showed that the presented sensor design enables customization of important biosensor parameters as well as application of these sensors in relevant heterologous hosts.

Trends in the Use of Tissue Culture, Applications and Future Aspects

Plant tissue culture has developed widely incorporated into biotechnology, the agricultural systems being a key factor to support many pharmaceutical and industrial outcomes. Since 1902 there is vast progress in plant culture and its application has emerged having great diversity in the science filed. Due to development and desire to grow on high scale production in the past few decades, tissue culture techniques were manipulated for improvement of plant growth, biological activities, transformation, and secondary metabolites production. A significant advance in techniques has been sought to deal with problems of low concentrations of secondary metabolites in whole plants. The augmented use of plant culture is due to a superior perceptive of plant oriented compounds and secondary metabolites from economically important plants. Due to development in modern techniques, several particular protocols have been developed for the production of a wide array of secondary metabolites of plants on a commercial scale. Plant tissue culture has to lead to significant contributions in recent times and today they constitute an indispensable tool in the advancement of agricultural sciences and modern agriculture. This review would enable us to have an analysis of plant tissue culture development for agriculture, human health and wellbeing in general.

Spironolactone metabolite concentrations in decompensated heart failure: insights from the ATHENA-HF trial.

In Aldosterone Targeted Neurohormonal Combined with Natriuresis Therapy in Heart Failure (ATHENA-HF), high-dose spironolactone (100 mg daily) did not improve efficacy endpoints over usual care [placebo or continued low-dose spironolactone (25 mg daily) in patients already receiving spironolactone] in the treatment of acute heart failure (HF). We hypothesized that low concentrations of the long-acting active metabolites of spironolactone [canrenone and 7α-thiomethylspironolactone (7α-TMS)] in the high-dose group could have contributed to these neutral results. In patients randomized to high-dose spironolactone not previously treated with spironolactone (high-dose-naïve, n = 112), concentrations of canrenone and 7α-TMS increased at 48 and 96 h compared to baseline, and between 48 and 96 h (all P < 0.005), indicating that steady-state concentrations had not been reached by 48 h. In patients previously on low-dose, high-dose spironolactone (high-dose-previous, n = 37), concentrations of canrenone increased at 48 and 96 h compared to baseline (both P < 0.0005), with a marginal increase between 48 and 96 h (P = 0.0507). At 48 h, both high-dose groups had higher concentrations of both metabolites than the low-dose spironolactone group (P < 0.0001). Moreover, concentrations of both metabolites were higher in high-dose-previous vs. high-dose-naïve patients (P < 0.01), indicating that previous spironolactone use was significant, and that steady-state has not been reached in high-dose-naïve patients at 48 h. We found limited and inconsistent evidence of correlation between metabolite concentrations and endpoints. Lower-than-anticipated concentrations of spironolactone active metabolites were observed for at least 48 h in the high-dose spironolactone group and may have contributed to the absence of pharmacological effects of spironolactone in the ATHENA-HF trial.

The Buffer Capacity and Calcium Concentration of Water Influence the Microbial Species Diversity, Grain Growth, and Metabolite Production During Water Kefir Fermentation.

Eight water kefir fermentation series differing in buffer capacity and calcium concentration of the water used for fermentation were studied during eight backslopping steps. High buffer capacities resulted in high pH values and high calcium concentrations resulted in low pH values at the end of each backslopping step. When the water buffer capacity and/or calcium concentration were below certain minima, the water kefir grain growth decreased gradually over multiple backsloppings. High water buffer capacities resulted in high concentrations of residual total carbohydrate concentrations and low metabolite concentrations. Further, high water buffer capacities resulted in high ratios of lactic acid bacteria to yeasts, which was reflected in high molar ratios of the concentrations of lactic acid to ethanol and acetic acid to ethanol. The most prevalent microorganisms of the water kefir grain inoculum and grains of all fermentation series at the end of the eighth backslopping step were Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus paracasei, Bifidobacterium aquikefiri, Saccharomyces cerevisiae, and Dekkera bruxellensis. These microbial communities were influenced by the water buffer capacity and had an impact on the substrate consumption and metabolite production during water kefir fermentation.

Profiling glucosinolate metabolites in human urine and plasma after broccoli consumption using non-targeted and targeted metabolomic analyses

Broccoli is a popular brassica vegetable and its consumption may decrease the occurrence of cancer in certain populations. To gain insight into the metabolites that may induce physiological responses to broccoli intake, a non-targeted metabolomic approach and a targeted approach for analysis of glucosinolate metabolites were developed using high resolution accurate mass spectrometry. A human study was conducted in which 6 subjects consumed a single meal of 200 g of uncooked broccoli florets. The metabolomic analysis revealed changes in endogenous metabolites and a decrease in hippuric acid after broccoli consumption. Targeted analysis using high-resolution, accurate mass-mass spectrometry (HRAM-MS) enabled detection of low concentrations (nM) of glucosinolate metabolites in human urine and plasma. Glucosinolate metabolites were found in human urine (13) and plasma (8), respectively. Metabolites from methoxyl-indole glucosinolates, arising from broccoli consumption, are reported for the first time. Most glucosinolate metabolites reached their peak concentration in urine 2–4 h after consumption while, in plasma, peak maxima were achieved 2 h after intake. The results suggest that glucoraphanin metabolites (sulforaphane, sulforaphane cysteine, sulforaphane N-acetyl cysteine) and indole metabolites (ascorbigen and methoxyl ascorbigen from indole glucosinolates) may serve as marker compounds for the intake of broccoli.

Pharmacodynamic Effects, Pharmacokinetics, and Metabolism of the Synthetic Cannabinoid AM-2201 in Male Rats

Novel synthetic cannabinoids are appearing in recreational drug markets worldwide. Pharmacological characterization of these new drugs is needed to inform clinicians, toxicologists, and policy makers who monitor public health. [1-(5-Fluoropentyl)-1H-indol-3-yl](1-naphthyl)methanone (AM-2201) is an abused synthetic cannabinoid that was initially created as a research tool for investigating the endocannabinoid system. Here we measured the pharmacodynamic effects of AM-2201 in rats, and simultaneously determined plasma pharmacokinetics for the parent drug and its metabolites. Male Sprague-Dawley rats were fitted with surgically implanted temperature transponders and indwelling jugular catheters under pentobarbital anesthesia. One week later, rats received subcutaneous injection of AM-2201 (0.1, 0.3, and 1.0 mg/kg) or its vehicle, and serial blood specimens were withdrawn via catheters. Core temperatures and catalepsy were measured just prior to each blood withdrawal, and plasma was assayed for drug and metabolites using liquid chromatography-tandem mass spectrometry. We found that AM-2201 produced dose-related hypothermia and catalepsy that peaked at 2 hours and lasted up to 8 hours. AM-2201 plasma concentrations rose linearly with increasing dose and ranged from 0.14 to 67.9 µg/l. Concentrations of three metabolites, AM-2201 N-(4-hydroxypentyl) (≤0.17 µg/l), naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) N-(5-hydroxypentyl) (≤1.14 µg/l), and JWH-018 N-pentanoic acid (≤0.88 µg/l) were detectable but much lower. Peak AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations occurred at 1.3, 2.4, and 6.5 hours, respectively. Concentrations of AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid were negatively correlated with body temperature, but, given the low concentrations of metabolites detected, AM-2201 is likely the major contributor to pharmacodynamic effects under our experimental conditions.

Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolic activation of primaquine, an antimalarial drug. CYP2D6 is genetically polymorphic, and these polymorphisms are associated with interindividual variations observed in the therapeutic efficacy of primaquine. To further understand this association, we performed in vitro enzymatic analyses of the wild-type CYP2D6.1 and 49 CYP2D6 allelic variants, which were expressed in 293FT cells, using primaquine as a substrate. The concentrations of CYP2D6 variant holoenzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild type and 27 variants showed a peak at 450 nm. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of primaquine 5-hydroxylation were characterized. The kinetic parameters of the wild type and 16 variants were measured, but the values for the remaining 33 variants could not be determined because of low metabolite concentrations. Among the variants, six (i.e., CYP2D6.17, .18, .35, .39, .53, and .70) showed significantly reduced intrinsic clearance compared with that of CYP2D6.1. Three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2D6 variants. Our findings provide insights into the allele-specific activity of CYP2D6 for primaquine, which could be clinically useful for malaria treatment and eradication efforts.

The potential effect of low cell osmolarity on cell function through decreased concentration of enzyme substrates.

Some freshwater algae have lower (<130 osmol m-3) intracellular osmolarities than most others (>180 osmol m-3). Low osmolarities are related to the presence of flagella and the low energy cost of active water efflux following downhill water influx unconstrained by cell walls covering the plasmalemma, and the low resource cost of cell wall synthesis with the same mechanical degree of safety. One consequence of low intracellular osmolarity is limitation on the concentration of metabolites, that is, substrates and products of enzyme activity. Models of the flux through metabolic pathways, and hence the specific growth rate, using steady-state concentrations of enzymes and metabolites have involved organisms with intracellular metabolite osmolarities >280 osmol m-3, where the metabolite concentrations are much greater than the total osmolarity of some freshwater algae. Since the protein concentration (mol m-3) in the cells and the specific growth rates of freshwater cells with low and with higher intracellular osmolarity are highly similar, the models of trade-offs between enzyme and metabolite concentrations for cells with high intracellular osmolarity need modification for cells with low intracellular osmolarity. The soluble free-radical scavenger ascorbate can constitute as little as 0.2% of the low intracellular metabolite concentration (mol m-3) of low-intracellular-osmolarity cells.

The Complement-1q-Like-3 Inhibits Insulin Secretion by an Adhesion G-Protein Coupled Receptor, BAI3 in Pancreatic ß Cells

Secreted proteins are important metabolic regulators and our objective was to identify proteins that are important regulators of β-cell function. Microarray data from multiple tissues including liver, adipose, gastrocnemius, islets from lean and obese mice were analyzed to identify candidate regulators. For this, we developed a data filtering approach to identify candidate secreted protein regulators. Next, cross-tissue correlation network and gene enrichment based analyses were performed to determine cellular pathways affected by each candidate secreted protein in the target tissue. By using this methodology, complement-1q-like-3 (C1ql3) was identified to affect ’secretory processes’ in islets. Preliminary data showed that the expression of C1ql3 was increased by 32-fold in islets of obese mice. Incubating islets with C1ql3 containing culture media inhibited high glucose, GLP-1 receptor agonist (exendin-4), cAMP, and potassium-induced insulin secretion in mouse islets. Interestingly, C1ql3 did not affect insulin secretion in response to low and submaximal concentrations of glucose, fatty acids, amino acids, and mitochondrial metabolites. We further observed that the expression of BAI3, a receptor for C1ql3, is elevated in islets of obese compared to lean mice. Knockdown of BAI3 in β-cells increased glucose-stimulated insulin secretion by 2-fold and soluble C1ql3-binding fragment of BAI3 completely blocked the inhibitory effects of C1ql3 on cAMP-stimulated insulin secretion. These findings report the identification of a novel pathway in C1ql3/BAI3 that antagonizes GLP-1/cAMP signaling to inhibit insulin secretion from β-cells. We propose that the activation of C1ql3/BAI3 signaling contributes to type 2 diabetes and may offer therapeutic alternatives to treat type 2 diabetes. Disclosure R. Gupta: None. M. Schaid: None. M.E. Kimple: None. J.E. Koltes: None. S. Bhatnagar: None.

A targeted strategy to identify untargeted metabolites from in vitro to in vivo: Rapid and sensitive metabolites profiling of licorice in rats using ultra-high performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry

It is challenging to conduct in vivo metabolic study for traditional Chinese medicines (TCMs) because of complex components, unpredictable metabolic pathways and low metabolite concentrations. Herein, we proposed a sensitive strategy to characterize TCM metabolites in vivo at an orally clinical dose using ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS). Firstly, the metabolism of individual compounds in rat liver microsomes was studied to obtain the metabolic pathways and fragmentation patterns. The untargeted metabolites in vitro were detected by multiple ion monitoring-enhanced product ion (EPI) and neutral loss-EPI scans. Subsequently, a sensitive multiple reaction monitoring-EPI method was developed according to the in vitro results and predicted metabolites to profile the in vivo metabolites. Licorice as a model herb was used to evaluate and validate our strategy. A clinical dose of licorice water extract was orally administered to rats, then a total of 45 metabolites in urine, 21 metabolites in feces and 35 metabolites in plasma were detected. Among them, 18 minor metabolites have not been reported previously and 6 minor metabolites were first detected in vivo. Several isomeric metabolites were well separated and differentiated in our strategy. These results suggested that this new strategy could be widely used for the detection and characterization of in vivo metabolites of TCMs.

Real-Time Visualization of Cysteine Metabolism in Living Cells with Ratiometric Fluorescence Probes.

Sulfite from cysteine metabolism in living cells plays a crucial role in improving the water solubility of metabolic xenobiotics for their easier excretion in urine or bile. However, an imbalance of sulfite in vivo would lead to oxidative stress or age-related diseases, and an effective strategy for real-time imaging of cysteine metabolism in living cells is still lacking due to its low metabolite concentration and rapid reaction kinetics. Herein, a cyanine moiety based ratiometric fluorescence probe was developed for highly selective and sensitive detection of sulfite in aqueous solution and living cells. The free probe exhibited an orange emission color, and the fluorescence color would gradually change to blue once sulfite anions selectively reacted with the unsaturated carbon double bonds in the probe molecule. This ratiometric fluorescence manner endowed the probe excellent sensitivity with a detection limit of 0.78 nM, which was then explored to image the kinetic process of sulfite release in hepatic BRL cells after incubating with an excess amount of cysteine. This strategy opens new opportunities for revealing thiol-containing species metabolism and even quantitatively tracking their distributions in live cells or organelles.

Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.

Primordial Krebs-cycle-like non-enzymatic reactions detected by mass spectrometry and nuclear magnetic resonance

Background: Metabolism is the process of nutrient uptake and conversion, and executed by the metabolic network. Its evolutionary precursors most likely originated in non-enzymatic chemistry. To be exploitable in a Darwinian process that forms a metabolic pathway, non-enzymatic reactions need to form a chemical network that produces advantage-providing metabolites in a single, life compatible condition. In a hypothesis-generating, large-scale experiment, we recently screened iron and sulfur-rich solutions, and report that upon the formation of sulfate radicals, Krebs cycle intermediates establish metabolism-like non-enzymatic reactivity. A challenge to our results claims that the results obtained by liquid chromatography-selective reaction monitoring (LC-SRM) would not be reproducible by nuclear magnetic resonance spectroscopy (1H-NMR). Methods: This study compared the application of the two techniques to the relevant samples. Results: We detect hundred- to thousand-fold differences in the specific limits of detection between LC-SRM and 1H-NMR to detect Krebs cycle intermediates. Further, the use of 1H-NMR was found generally problematic to characterize early metabolic reactions, as Archean-sediment typical iron concentrations cause paramagnetic signal suppression. Consequently, we selected non-enzymatic Krebs cycle reactions that fall within the determined technical limits. We confirm that these proceed unequivocally as evidenced by both LC-SRM and 1H-NMR. Conclusions: These results strengthen our previous conclusions about the existence of unifying reaction conditions that enables a series of co-occurring metabolism-like non-enzymatic Krebs cycle reactions. We further discuss why constraints applying to metabolism disentangle concentration from importance of any reaction intermediates, and why evolutionary precursors to metabolic pathways must have had much lower metabolite concentrations compared to modern metabolic networks. Research into the chemical origins of life will hence miss out on the chemistry relevant for metabolism if its focus is restricted solely to highly abundant and unreactive metabolites, including when it ignores life-compatibility of the reaction conditions as an essential constraint in enzyme evolution.

Abstract 1485: Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination

Abstract Cancer deaths, including breast cancer, are caused by metastasis of the malignant tumors to distant locations. However, current methods of detection cannot distinguish pre-invasive breast cancer from noninvasive breast tumor or benign breast disease. Population-wide mammographic screenings have led to increased detection of ductal carcinoma in situ or DCIS, noninvasive, proliferative cells contained by the basement membrane of the terminal ductal lobular unit. DCIS is usually not associated with metastasis and/or cancer death. Each year, in the US alone, about 1.5 million of women who have been diagnosed with DCIS or a suspicious lump/lesion by mammography will require resection or breast biopsy after diagnosis for further pathologic analysis. However, ~80-85% of biopsies result in noninvasive breast disease or benign findings. As a result, a considerable number of patients suffer from side effects caused by breast biopsy and/or overtreatment. Therefore, there is an urgent need to find a biomarker for pre-invasive breast cancer. The ability of cancer cells to produce lactate through aerobic glycolysis (the Warburg effect) is a consistent hallmark of cancer, including breast cancer. Recent advancements in liquid chromatography-mass spectrometry (LC-MS) technology have significantly improved the sensitivity of this method compared to traditional NMR or GC-MS-based technologies, which make it feasible to detect very low concentrations of small molecules or metabolites. We have recently established a positional isotopic labeling and LC-MS-based targeted metabolomics method that can directly measure the conversion from [1-13C]glucose to [3-13C]lactate through glycolysis. Our results show that metastatic breast cancer cells exhibit a dramatically increased production of [3-13C]lactate from [1-13C]glucose even under aerobic conditions as compared to low- or noninvasive breast cancer cell lines. We found that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We have also observed significantly elevated production of [3-13C]lactate in serum samples of early stage metastatic mammary tumors developed in mice. Since elevated levels of lactate are closely correlated to increased tumor aggressiveness, these results suggest that monitoring of lactate production from glycolysis by targeted metabolomics may provide a biomarker for pre-invasive breast cancer. These results will pave the way for further exploration of the elevated production of lactate as a promising biomarker for pre-invasive breast cancer and for assessment of therapeutic response in clinical trials. Citation Format: Da-Qing Yang, Margot Cleary. Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1485. doi:10.1158/1538-7445.AM2017-1485

Effects of CO2 pretreatment on the volatile compounds of dried Chinese jujube (Zizyphus jujuba Miller)

Abstract The aim of this study was to investigate whether anaerobic metabolites could induce volatile compounds and improve aroma of dried jujube (Ziziphus jujuba Miller). Jujube fruits were incubated in a polyvinyl chloride bag containing 5% CO2 and 95% N2 for up to 168 h at 25 °C and 3 samples were randomly removed every 6 h and oven dried to a moisture content of ≅ 20%. The volatile compounds of control and 5% CO2-pretreated Chinese jujube fruits were extracted by simultaneous distillation extraction and identified by gas chromatography-mass spectrometry(GC-MS). The acetaldehyde and ethanol contents were determined by gas chromatography (GC). The results indicated that a large accumulation of acetaldehyde and ethanol caused changes in aroma composition of dried jujube products and 5% CO2 pretreatment led to an increase in the levels of some compounds, particularly esters, acetaldehydes, and ethanol, whereas the amount of acids were decreased significantly. Principal component analysis showed that integrative scores of 5% CO2 pretreatment at 120 h were the highest, and aroma quality was better than that of the control. Relatively low concentrations of anaerobic respiration metabolites are good for jujube fruit aroma composition.

First report on organochlorine pesticides in water in a highly productive agro-industrial basin of the Central Valley, Chile

The Ñuble River flows through an agricultural area in the central zone of Chile in which different types of fruits are grown. The objective of this study was to identify the levels and source of organochlorine pesticides (OCPs) in the surface waters of the Ñuble River. Surface water samples were collected at 10 sampling points and analyzed for 19 organochlorine pesticides during the dry (spring-summer) and wet (autumn-winter) periods of 2013 and 2014. The results revealed that concentrations of total OCPs in surface waters ranged from 0.12 to 26.28 ng/l. Endosulfan and lindane were the main OCPs in the water. The maximum OCP levels were found in the dry period, while significantly lower concentrations were recorded in the wet period. This indicates that patterns of OCPs have varied in the last 10 years as this study found low concentrations of DDT and metabolites but significantly increased concentrations of α-endosulfan. Given the fact that OCPs were withdrawn from agricultural used many years ago, their presence indicates that they may be still in use clandestinely. Findings of significant concentrations of endosulfan and lindane in this study lead to the conclusion that the chemical fallow practices contribute to runoff into the river and surface water infiltration into groundwater. Because no studies have previously measured OCP levels in agricultural basins in Chile, this study is an important contribution to the knowledge of organochlorine contaminants present in surface water in Chile.

In vivo online magnetic resonance quantification of absolute metabolite concentrations in microdialysate.

In order to study metabolic processes in animal models of diseases and in patients, microdialysis probes have evolved as powerful tools that are minimally invasive. However, analyses of microdialysate, performed remotely, do not provide real-time monitoring of microdialysate composition. Microdialysate solutions can theoretically be analyzed online inside a preclicinal or clinical MRI scanner using MRS techniques. Due to low NMR sensitivity, acquisitions of real-time NMR spectra on very small solution volumes (μL) with low metabolite concentrations (mM range) represent a major issue. To address this challenge we introduce the approach of combining a microdialysis probe with a custom-built magnetic resonance microprobe that allows for online metabolic analysis (1H and 13C) with high sensitivity under continuous flow conditions. This system is mounted inside an MRI scanner and allows performing simultaneously MRI experiments and rapid MRS metabolic analysis of the microdialysate. The feasibility of this approach is demonstrated by analyzing extracellular brain cancer cells (glioma) in vitro and brain metabolites in an animal model in vivo. We expect that our approach is readily translatable into clinical settings and can be used for a better and precise understanding of diseases linked to metabolic dysfunction.

Maternal Buprenorphine Maintenance and Lactation.

In addition to the well-known benefits of human milk and breastfeeding for the mother and infant, breastfeeding may mitigate neonatal abstinence syndrome severity in prenatally opioid-exposed infants. However, lack of conclusive data regarding the extent of the presence of buprenorphine and active metabolites in human milk makes the recommendation of breastfeeding for buprenorphine-maintained women difficult for many providers. This study seeks to determine the concentrations of buprenorphine and its active metabolites (norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide) in human milk, maternal plasma, and infant plasma of buprenorphine-maintained women and their infants. Up to 10 buprenorphine-maintained women provided paired breast milk and plasma samples at 2, 3, 4, 14, and 30 days postdelivery, and 9 infants provided plasma samples on day 14 of life. All samples were analyzed via liquid chromatography tandem mass spectrometry to determine concentrations of buprenorphine, norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide by a fully validated method. Concentrations of buprenorphine and metabolites are low in human milk and maternal plasma. Breastfed infant plasma concentrations of buprenorphine were low or undetectable and metabolite concentrations undetectable at 14 days of infant age. There were significant correlations between maternal buprenorphine dose and maternal plasma and human milk buprenorphine concentrations. These data find low concentrations of buprenorphine and metabolites in human milk and lend support to the recommendation for lactation among stable buprenorphine-maintained women. However, the correlation between maternal dose and maternal plasma and human milk buprenorphine concentrations bears further study.

Climatic conditions driving a part of changes in the biochemical composition in land snails: Insights from the endangered Codringtonia (Gastropoda: Pulmonata)

AbstractMost land snails in order to ensure their survival under adverse environmental conditions interrupt their activity entering a state of dormancy. The transition from activity to dormancy is accompanied by several changes in the animals’ physiology. To identify these changes we examined the seasonal patterns in the biochemical composition and the LDH activity of three different tissues in four endangered land snail species. Additionally, within a phylogenetic framework we investigated the correlation of the measured parameters with the spatial and climatic variables of the sampling sites. Our results indicate that there are both differences and similarities in the pattern exhibited by the four species, depending on the physiological parameter investigated and the climatic conditions of the sampling sites, which sometimes have a significant effect on the seasonal pattern exhibited. Snails can successfully deal with the winter dormancy by maintaining high metabolite concentrations and stable water content whereas there is no indication of anaerobic pathways’ activation. At the same time, they deal successfully with the low humidity and high temperatures during the summer, but they are forced to maintain low metabolite concentrations and seem to activate anaerobic pathways to meet their energy demands. Therefore, from a biochemical perspective, it seems that winter is a less stressful period for snails compared to summer. According to the prevailing climate change scenarios, the Mediterranean region is going to exhibit a sudden transition towards a dryer and longer summer. This transition will exert a very high adaptation pressure on the already vulnerable

Specific Metabolic Markers Are Associated with Future Waist-Gaining Phenotype in Women.

ObjectiveOur study aims to identify metabolic markers associated with either a gain in abdominal (measured by waist circumference) or peripheral (measured by hip circumference) body fat mass.MethodsData of 4 126 weight-gaining adults (18–75 years) from three population-based, prospective German cohort studies (EPIC, KORA, DEGS) were analysed regarding a waist-gaining (WG) or hip-gaining phenotype (HG). The phenotypes were obtained by calculating the differences of annual changes in waist minus hip circumference. The difference was displayed for all cohorts. The highest 10% of this difference were defined as WG whereas the lowest 10% were defined as HG. A total of 121 concordant metabolite measurements were conducted using Biocrates AbsoluteIDQ® kits in EPIC and KORA. Sex-specific associations with metabolite concentration as independent and phenotype as the dependent variable adjusted for confounders were calculated. The Benjamini-Hochberg method was used to correct for multiple testing.ResultsAcross studies both sexes gained on average more waist than hip circumference. We could identify 12 metabolites as being associated with the WG (n = 8) or HG (n = 4) in men, but none were significant after correction for multiple testing; 45 metabolites were associated with the WG (n = 41) or HG (n = 4) in women. For WG, n = 21 metabolites remained significant after correction for multiple testing. Respective odds ratios (OR) ranged from 0.66 to 0.73 for tryptophan, the diacyl-phosphatidylcholines (PC) C32:3, C36:0, C38:0, C38:1, C42:2, C42:5, the acyl-alkyl-PCs C32:2, C34:0, C36:0, C36:1, C36:2, C38:0, C38:2, C40:1, C40:2, C40:5, C40:6, 42:2, C42:3 and lyso-PC C17:0.ConclusionBoth weight-gaining men and women showed a clear tendency to gain more abdominal than peripheral fat. Gain of abdominal fat seems to be related to an initial metabolic state reflected by low concentrations of specific metabolites, at least in women. Thus, higher levels of specific PCs may play a protective role in gaining waist circumference.