Prominent Metabolite Research Articles

Metabolism and Mass Balance of the Novel Nonsteroidal Androgen Receptor Inhibitor Darolutamide in Humans

The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg ¹⁴C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of (<i>S</i>,<i>R</i>)- and (<i>S</i>,<i>S</i>)-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were <i>O</i>-glucoronide (M-7a/b) and <i>N</i>-glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of (<i>S</i>,<i>S</i>)-darolutamide. <h3>SIGNIFICANCE STATEMENT</h3> The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of (<i>S,S</i>)-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes.

Ataluren metabolism: Ataluren-O-1β-acyl glucuronide is a stable circulating metabolite in mouse, rat, dog and human

Ataluren is an aromatic acid derivative with a 1,2,4-oxodiazole moiety. Ataluren-O-1β-acyl glucuronide is a prominent circulatory metabolite in mice, rats, dogs, and humans following oral administration of ataluren. The objective of this paper was to evaluate the stability in vitro and in vivo of ataluren-O-1β-acyl glucuronide metabolite. Ultrahigh performance liquid chromatography-mass spectrometry methods were developed to separate and monitor ataluren-O-1β-acyl glucuronide and its possible migration isomers. In vitro stability was assessed in phosphate buffered saline as well as in control rat and human plasma. The disappearance of ataluren-O-1β-acyl glucuronide and the formation of migration isomers were monitored by the ultrahigh performance liquid chromatography-mass spectrometry methods. In vitro, ataluren-O-1β-acyl glucuronide underwent isomerization with an estimated half-life of approximately 1 h. However, ataluren-O-1β-acyl glucuronide was stable and was the only detectable acyl glucuronide following oral administration of ataluren in mice, rats, dogs, and humans using the same analytical methods. Ataluren acyl glucuronide in mouse, rat, dog, and human plasma could be hydrolyzed by β-glucuronidase, further confirming the structure of O-1β-acyl glucuronide. These results demonstrated that ataluren-O-1β-acyl glucuronide did not undergo migration in vivo. No clinical safety concern related to ataluren-O-1β-acyl glucuronide migration has been detected.

ω3 fatty acid metabolite, 12-hydroxyeicosapentaenoic acid, alleviates contact hypersensitivity by downregulation of CXCL1 and CXCL2 gene expression in keratinocytes via retinoid X receptor α.

ω3 fatty acids show potent bioactivities via conversion into lipid mediators; therefore, metabolism of dietary lipids is a critical determinant in the properties of ω3 fatty acids in the control of allergic inflammatory diseases. However, metabolic progression of ω3 fatty acids in the skin and their roles in the regulation of skin inflammation remains to be clarified. In this study, we found that 12-hydroxyeicosapentaenoic acid (12-HEPE), which is a 12-lipoxygenase metabolite of eicosapentaenoic acid, was the prominent metabolite accumulated in the skin of mice fed ω3 fatty acid-rich linseed oil. Consistently, the gene expression levels of Alox12 and Alox12b, which encode proteins involved in the generation of 12-HEPE, were much higher in the skin than in the other tissues (eg, gut). We also found that the topical application of 12-HEPE inhibited the inflammation associated with contact hypersensitivity by inhibiting neutrophil infiltration into the skin. In human keratinocytes in vitro, 12-HEPE inhibited the expression of two genes encoding neutrophil chemoattractants, CXCL1 and CXCL2, via retinoid X receptor α. Together, the present results demonstrate that the metabolic progression of dietary ω3 fatty acids differs in different organs, and identify 12-HEPE as the dominant ω3 fatty acid metabolite in the skin.

Untargeted metabolomics of human plasma reveal lipid markers unique to chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

Chronic obstructive pulmonary disease (COPD) is characterised by airway inflammation and progressive airflow limitation, whereas idiopathic pulmonary fibrosis (IPF) is characterised by a restrictive pattern due to fibrosis and impaired gas exchange. We undertook metabolomic analysis of blood samples in IPF, COPD and healthy controls (HC) to determine differences in circulating molecules and identify novel pathogenic pathways. An untargeted metabolomics using an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) was performed to profile plasma of patients with COPD (n=21), and IPF (n=24) in comparison to plasma from healthy controls (HC; n=20). The most significant features were identified using multiple database matching. One-way ANOVA and variable importance in projection (VIP) scores were also used to highlight metabolites that influence the specific disease groups. Non-polar metabolites such as fatty acids (FA) and membrane lipids were well resolved and a total of 4805 features were identified. The most prominent metabolite composition differences in lipid mediators identified at ∼2-3 fold higher in both diseases compared to HC were palmitoleic acid, oleic acid and linoleic acid; and dihydrotestosterone was lower in both diseases. We demonstrated that COPD and IPF were characterised by systemic changes in lipid constituents such as essential FA sampled from circulating plasma.

Identification and Functional Characterization of Metabolites for Bone Mass in Peri- and Postmenopausal Chinese Women.

Although metabolic profiles appear to play an important role in menopausal bone loss, the functional mechanisms by which metabolites influence bone mineral density (BMD) during menopause are largely unknown. We aimed to systematically identify metabolites associated with BMD variation and their potential functional mechanisms in peri- and postmenopausal women. We performed serum metabolomic profiling and whole-genome sequencing for 517 perimenopausal (16%) and early postmenopausal (84%) women aged 41 to 64 years in this cross-sectional study. Partial least squares regression and general linear regression analysis were applied to identify BMD-associated metabolites, and weighted gene co-expression network analysis was performed to construct co-functional metabolite modules. Furthermore, we performed Mendelian randomization analysis to identify causal relationships between BMD-associated metabolites and BMD variation. Finally, we explored the effects of a novel prominent BMD-associated metabolite on bone metabolism through both in vivo/in vitro experiments. Twenty metabolites and a co-functional metabolite module (consisting of fatty acids) were significantly associated with BMD variation. We found dodecanoic acid (DA), within the identified module causally decreased total hip BMD. Subsequently, the in vivo experiments might support that dietary supplementation with DA could promote bone loss, as well as increase the osteoblast and osteoclast numbers in normal/ovariectomized mice. Dodecanoic acid treatment differentially promoted osteoblast and osteoclast differentiation, especially for osteoclast differentiation at higher concentrations in vitro (eg,10, 100 μM). This study sheds light on metabolomic profiles associated with postmenopausal osteoporosis risk, highlighting the potential importance of fatty acids, as exemplified by DA, in regulating BMD.

Heuristics in Role of Human Glutathione S-transferase Mu 1 as Nitric Oxide Carrier and its Engineered Variants for Enhanced Activity.

Human glutathione S-transferases (hGSTs) are phase-II detoxification enzymes that catalyze the conjugation of electrophilic compounds and glutathione. Anomalous excess production of NO in the cellular environment under diseased or stressed condition results in lethal effects to the cell. Studies have reported that the evolution of tyrosine-based GSTs as a defense mechanism by the cell to mitigate Nitric Oxide (NO) toxicity. The dual role of hGSTP1 as NO carrier and scavenger is a prelude for the research forthwith. A plausible role of hGSTM1 as NO carrier is considered. Being a prominent cellular messenger and secondary metabolite, excess production of NO is lethal to the cell. Moreover, hGSTM1 polymorphisms lead to diminished catalytic activity that promotes a diseased state. Hence, it is compelling to generate hGSTM1 mutants that have more catalytic efficacy compared to Wild Type (WT). hGSTM1 mutants with enhanced efficiency were generated using in silico and in vitro Site-Directed Mutagenesis (SDM). WT and mutant proteins were overexpressed and purified using affinity chromatography. The catalytic activity and binding efficiency of WT and mutant proteins towards CDNB (1-chloro-2, 4-dinitrobenzene) & NO were determined. NO assay reveals the probable interaction of WT hGSTM1 with NO. In silico, SDM studies provided E129K and Q109K mutants with superior NO binding efficiency as compared to WT. The catalytic activity (GST and NO assays) of the mutants corroborate the in silico results. WT hGSTM1 is recognized as a positive NO carrier. The novel mutant enzymes E129K and Q109K are inferred to possess superior NO carrying capacity.

Resolution of the Hypoxylon fuscum Complex (Hypoxylaceae, Xylariales) and Discovery and Biological Characterization of Two of Its Prominent Secondary Metabolites.

Hypoxylon, a large, cosmopolitan genus of Ascomycota is in the focus of our current poly-thetic taxonomic studies, and served as an excellent source for bioactive secondary metabolites at the same time. The present work concerns a survey of the Hypoxylon fuscum species complex based on specimens from Iran and Europe by morphological studies and high performance liquid chromatography coupled to mass spectrometry and diode array detection (HPLC-MS-DAD). Apart from known chemotaxonomic markers like binaphthalene tetrol (BNT) and daldinin F, two unprece-dented molecules were detected and subsequently isolated to purity by semi preparative HPLC. Their structures were established by nuclear-magnetic resonance (NMR) spectroscopy as 3′-malonyl-daldinin F (6) and pseudofuscochalasin A (4). The new daldinin derivative 6 showed weak cytotoxicity towards mammalian cells but bactericidal activity. The new cytochalasin 4 was compared to cytochalasin C in an actin disruption assay using fluorescence microscopy of human osteo-sarcoma U2OS cells, revealing comparable activity towards F-actin but being irreversible compared to cytochalasin C. Concurrently, a multilocus molecular phylogeny based on ribosomal and proteinogenic nucleotide sequences of Hypoxylon species resulted in a well-supported clade for H. fuscum and its allies. From a comparison of morphological, chemotaxonomic and phylogenetic evidence, we introduce the new species H. eurasiaticum and H. pseudofuscum.

Transcriptome analysis to identify key genes involved in terpenoid and rosmarinic acid biosynthesis in lemon balm (Melissa officinalis).

Melissa officinalis (lemon balm) is a well-known pharmaceutical plant in traditional medicine around the world because of the high-value secondary metabolites. Nowadays, advances in computational biology and bioinformatics have opened new avenues to plant-based natural product drug discovery. Despite the pharmacological importance, there is low information about the genes encoding the important biosynthetic pathways related to the secondary metabolite in M. officinalis. In this study, the main genes related to the rosmarinic acid (RA) and terpenoid biosynthesis pathways were detected using transcriptome analysis. Furthermore, we isolated and characterized a novel M. officinalis Hydroxyphenylpyruvate reductase (HPPR) gene involved in RA biosynthesis pathway. An effective pipeline was used to generate 37,055 unigenes by evaluating 42,837,601 Illumina paired-end reads. Functional annotation of the unigenes revealed that 27,363 (73.84%) and 35,822 (96.67%) unigenes had significant similarity to identified proteins in the SwissProt and NR databases, respectively. Also, 10,062 (36.83%) out of 37,055 unigenes were assigned to 399 KEGG pathways. Since terpenes and RA are two prominent metabolites in this plant, the attention of this study has been on the pathways related to them. A total of 149 unigenes were found that are related to the terpenoids biosynthesis, including 75 unigenes involved in the methyl-erythritol phosphate and mevalonate pathway, terpenoid backbone biosynthesis genes, and 74 unigenes related to the terpene synthase. We also identified 144 and 30 unigenes that were associated with the biosynthesis of phenylpropanoid and the rosmarinic acid pathway. Consequently, this investigation can be a comprehensive and accurate transcriptome basis for further investigation in the metabolic engineering and detection of new genes and pathways in M. officinalis.

Potential Metabolic Biomarkers for Early Detection of Oral Lichen Planus, a Precancerous Lesion.

Background: Oral lichen planus (OLP) is a T-cell-mediated chronic inflammatory disorder and precancerous oral lesion with high incidence. The current diagnostic method of OLP is very limited and metabolomics may provide a new approach for quantitative evaluation. Methods: The Ultra-Performance Liquid Chromatography-Quadrupole/Orbitrap High Resolution Mass Spectrometry (UHPLC-Q-Orbitrap HRMS) was applied to analyze the change of metabolites in serum of patients with OLP. A total of 115 OLP patients and 124 healthy controls were assigned to either a training set (n = 160) or a test set (n = 79). The potential biomarkers and the change of serum metabolites were profiled and evaluated by multivariate analysis. Results: Totally, 23 differential metabolites were identified in the training set between OLP group and healthy group. Three prominent metabolites in receiver operating characteristic (ROC) were selected as a panel to distinguish OLP or healthy individuals in the test set, and the diagnostic accuracy was 86.1%. Conclusions: This study established a new method for the early detection of OLP by analyzing serum metabolomics using UHPLC-Q-Orbitrap HRMS, which will help in understanding the pathological processes of OLP and identifying precancerous lesions in oral cavity.

Untargeted Metabolomic Fingerprinting As a Potential Tool in the Diagnostic Evaluation of Diamond Blackfan Anemia

Background: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) marked by erythroid hypoplasia, reticulocytopenia and macrocytosis, and associated with congenital malformations and an increased risk of developing malignancies. From a clinical and molecular perspective this disease is highly heterogeneous, and no clear genotype-phenotype correlations can be found. Since the majority of molecular defects have been found in ribosomal protein (RP) genes, DBA is regarded a "ribosomopathy". While the molecular basis has been studied intensively, the pathophysiology of DBA is still not fully understood. One of the major unresolved issues is how RP gene mutations result in the specific erythroid defect seen in DBA, with macrocytic erythrocytes and increased adenosine deaminase activity. In addition, the highly heterogeneous and variable clinical presentation and disease course, even in patients with similar molecular defects, remains enigmatic. Hence, in order to investigate the cellular defect, and increase our understanding of disease pathophysiology and clinical heterogeneity, novel tools are needed. In this study, we explore the potential of untargeted metabolomics on dried blood spots and report for the first time a metabolic fingerprint for DBA. Aims: Defining a metabolic signature for DBA in order to: 1. Increase our understanding of cellular determinants of impaired ribosome biogenesis, and 2. Extend the toolbox for diagnostic evaluation. Methods: Untargeted metabolic profiling was performed on DBS samples obtained from 18 DBA patients and 45 healthy controls using direct infusion high resolution mass spectrometry following a previously established approach1. Statistical analysis was performed in MetaboAnalyst and predictive modeling was executed within R-software. Results: In total, 1917 unique metabolite features were identified in DBS samples from patients and controls. Multivariate analysis yielded distinct metabolic profiles, reflected by natural separation detected by principal component analysis (Figure 1A), and emphasized by clear distinction with partial least square discriminant analysis (Figure 1B). This 'metabolic fingerprint' was incorporated into a machine learning algorithm, and subsequently a binary classification (or prediction) model was constructed by randomly dividing patient and controls into 'training' (32 HC, 13 DBA) or 'test' set (13 HC, 5 DBA). Accurate class assignment was achieved for all patients and controls in the training set (Figure 1C). Prominent metabolites in the fingerprint and classification algorithm were a.o. menadione (a vitamin K precursor), 4-hydroxyproline (collagen component) and methylmalonylcarnitine (an acylcarnitine). In addition a large number of interesting metabolites were identified that could provide novel starting points for studying/understanding downstream effects of RP defects in DBA and therapeutic mechanisms (Figure 1D). Conclusion: In this study we performed untargeted metabolomics on dried blood spots from a substantial cohort of DBA patients and report for the first time a metabolic fingerprint of this disease. By incorporating this fingerprint in a machine learning algorithm we underlined the diagnostic potential of this approach. Moreover, the metabolites identified in this fingerprint, provide promising starting points for further studies to increase our insights in disease pathophysiology, including the mechanism involved in elevated eADA activity, as well as the development of new therapeutic strategies.

3-Hydroxytyrosol regulates biofilm growth in Cunninghamella elegans

In contrast to yeast biofilms, those of filamentous fungi are relatively poorly understood, in particular with respect to their regulation. Cunninghamella elegans is a filamentous fungus that is of biotechnological interest as it catabolises drugs and other xenobiotics in an analogous manner to animals; furthermore, it can grow as a biofilm enabling repeated batch biotransformations. Precisely how the fungus switches from planktonic to biofilm growth is unknown and the aim of this study was to shed light on the possible mechanism of biofilm regulation. In dimorphic yeasts, alcohols such as tyrosol and 2-phenylethanol are known to control the yeast-to-hypha switch, and a similar molecule might be involved in regulating biofilm in C. elegans. Gas chromatography-mass spectrometry analysis of crude ethyl acetate extracts from supernatants of 72 h planktonic and biofilm cultures revealed 3-hydroxytyrosol as a prominent metabolite. Further quantification revealed that the amounts of the compound in planktonic cultures were substantially higher (>10-fold) than in biofilm cultures. In the presence of exogenous 3-hydroxytyrosol the growth of aerial mycelium was inhibited, and there was selective inhibition of biofilm when it was added to culture medium. There was no biotransformation of the compound when it was added to 72 h-old cultures, in contrast to the related compounds tyrosol and 2-phenylethanol, which were oxidised to a number of products. Therefore, we propose that 3-hydroxytyrosol is a new signalling molecule in fungi, which regulates biofilm growth.

Targeted LC–MS/MS analysis of steroid glucuronides in human urine

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1−3 G), 17β-estradiol 17-glucuronide (E2−17 G), 17β-estradiol 3-glucuronide (E2−3 G), estriol 16-glucuronide (E3−16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F–G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F–G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC–MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC–MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC–MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15−20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0−1 month of age: 184.5 nmol/L; 15−20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.

Secondary Metabolites From Halotolerant Plant Growth Promoting Rhizobacteria for Ameliorating Salinity Stress in Plants.

Soil salinization has emerged as one of the prime environmental constraints endangering soil quality and agricultural productivity. Anthropogenic activities coupled with rapid pace of climate change are the key drivers of soil salinity resulting in degradation of agricultural lands. Increasing levels of salt not only impair structure of soil and its microbial activity but also restrict plant growth by causing harmful imbalance and metabolic disorders. Potential of secondary metabolites synthesized by halotolerant plant growth promoting rhizobacteria (HT-PGPR) in the management of salinity stress in crops is gaining importance. A wide array of secondary metabolites such as osmoprotectants/compatible solutes, exopolysaccharides (EPS) and volatile organic compounds (VOCs) from HT-PGPR have been reported to play crucial roles in ameliorating salinity stress in plants and their symbiotic partners. In addition, HT-PGPR and their metabolites also help in prompt buffering of the salt stress and act as biological engineers enhancing the quality and productivity of saline soils. The review documents prominent secondary metabolites from HT-PGPR and their role in modulating responses of plants to salinity stress. The review also highlights the mechanisms involved in the production of secondary metabolites by HT-PGPR in saline conditions. Utilizing the HT-PGPR and their secondary metabolites for the development of novel bioinoculants for the management of saline agro-ecosystems can be an important strategy in the future.

Glucosinolates: Natural Occurrence, Biosynthesis, Accessibility, Isolation, Structures, and Biological Activities.

Glucosinolates (GSLs) are secondary plant metabolites abundantly found in plant order Brassicales. GSLs are constituted by an S-β-d-glucopyrano unit anomerically connected to O-sulfated (Z)-thiohydroximate moiety. The side-chain of the O-sulfate thiohydroximate moiety, which is derived from a different amino acid, contributes to the diversity of natural GSL, with more than 130 structures identified and validated to this day. Both the structural diversity of GSL and their biological implication in plants have been biochemically studied. Although chemical syntheses of GSL have been devised to give access to these secondary metabolites, direct extraction from biomass remains the conventional method to isolate natural GSL. While intact GSLs are biologically inactive, various products, including isothiocyanates, nitriles, epithionitriles, and cyanides obtained through their hydrolysis of GSLs, exhibit many different biological activities, among which several therapeutic benefits have been suggested. This article reviews natural occurrence, accessibility via chemical, synthetic biochemical pathways of GSL, and the current methodology of extraction, purification, and characterization. Structural information, including the most recent classification of GSL, and their stability and storage conditions will also be discussed. The biological perspective will also be explored to demonstrate the importance of these prominent metabolites.

Omega-3 lipid metabolites as mediators of metformin’s anti-proliferative effect in ovarian cancer

Objective: Metformin is being repurposed for treatment of gynecologic malignancies and other cancers. It is known to alter the cancer cell metabolism, primarily the energy metabolism. Our aim was to identify and test the preclinical efficacy of the prominent metabolite changes occurring in response to metformin treatment in ovarian cancer cell lines.

Proton nuclear magnetic resonance J-spectroscopy of phantoms containing brain metabolites on a portable 0.05 T MRI scanner

We examined approaches for obtaining 1H NMR spectra of brain metabolites on a low-field (B0 = 0.05 T) portable MRI scanner, which was developed in our laboratory with the aim of bringing cost-effective radiological services to populations in underserved, remote regions. The low static magnetic field B0 dictates low signal to noise ratio for metabolites in the mM concentration range, and results in an overall spectral region for the 1H resonances of these metabolites narrower than the linewidth obtainable in our scanner. The narrow spectral range also precludes the possibility of suppressing the large contribution of the water resonance at the acquisition stage.We used a spectroscopic Carr-Purcell-Meiboom-Gill (CPMG) sequence to acquire multiecho data from solutions of J-coupled brain metabolites, focusing on lactic acid, a metabolite whose concentration is negligible in the healthy brain and increases significantly in several disease conditions. The J spectra we obtained for lactate from the Fourier transformation of the multiecho data are spectrally well-resolved for a range of echo spacing values. We show that the J spectra at different echo spacings fit well with simulations of the evolution of echo train signal of the lactate under the same conditions. Applying a J-refocused variant of the CPMG sequence, the J modulation of the echo decay is removed, providing a way for subtracting the large contribution of the non-modulated component in the J spectrum in conditions where notching it using post-processing methods is impossible. We also demonstrate by means of experimental data and simulations that in our experimental conditions, J-spectra of other prominent brain metabolites, such as the neurotransmitter glutamate, do not yield discernible peaks and only contribute to a broad peak at zero frequency.

Application of lignosulfonate as the growth promotor for freshwater microalga Euglena gracilis to increase productivity of biomass and lipids

Biomass derived supplements have been recognized as environment-friendly and cost-effective sources of stimulants which can promote growth and valuable metabolites biosynthesis of microalgae. In this research, effect of lignosulfonates (LIGNs) on the growth, cell morphological changes, and valuable products accumulation of microalga Euglena gracilis was explored. At the optimal concentration of 5000 mg·L−1, LIGNs could promote the growth of E. gracilis up to 1.95 folds, and increase the yield of chlorophyll a, chlorophyll b and carotenoids by 3.50, 1.59 and 3.48 times, respectively. Cell morphological changes in aspect ratio and size caused by LIGNs were also observed. Fourier transform infrared spectroscopy measurements followed by multivariate analysis revealed difference in metabolic patterns and prominent metabolites with LIGNs treatment. LIGNs improved the lipid yield since both cell density and lipid content increased, which could contribute to the biofuel production in the future.

Water Pollution and Environmental Concerns in Anesthesiology.

Medications administered by anesthesia health care providers and subsequently excreted into the water supply system have the potential to affect ecological systems. Presently, there is a lack of literature examining which medications or metabolites enter the waste stream. Further, their potential environmental impacts are often unknown or simply not considered as an externality of medical practice. Recent work examining the practice of anesthesiology has explored the solid waste stream, and the global warming potential of anesthetic gases, however the potential aquatic impacts remain unexplored. To address the potential for waterborne pollution and environmental toxicity, we extracted the total intravenous medications (by mass) administered by anesthesiologists in 2017 at The University of Vermont Medical Center (UVMMC), a mid-size regional Level 1 trauma center in Burlington, VT. The most commonly administered medications were: cefazolin, propofol, acetaminophen, sugammadex and lidocaine. To estimate the amount of each medication that entered the wastewater stream, we used published metabolism profiles to adjust from the total amount administered to the amount excreted unchanged or as prominent metabolites. For each medication we reviewed existing literature concerning their environmental fate and impacts in water. Due to the constraints of current knowledge, it is not possible to determine the exact fate and impacts of these drugs. Some medications, like propofol, have the potential for significant bioaccumulation and persistence. Others, such as lidocaine and acetaminophen, have short half-lives in the environment but their constant delivery and excretion result in pseudo-persistence. The current literature mostly assesses acute exposure at doses higher than could be expected in the environment on select species. While significant toxicities across a variety of species have been found repeatedly, chronic low dose exposures require further study for all the medications discussed. Finally, multi-drug impacts are likely to be more impactful than single-drug toxicities. While we cannot state definitive impacts, the pharmaceuticals most used in anesthesiology have a clear toxic potential and future studies should more closely examine the relative contribution of anesthesia to pharmaceutical pollution, as well as points of intervention for minimizing these unintended consequences of healthcare delivery.

The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.

Increasing Doses of Blueberry Polyphenols Alters Colonic Metabolism and Calcium Absorption in Ovariectomized Rats.

Blueberries are rich sources of bioactive polyphenols that may provide health benefits when consumed regularly, leading to their increased marketing as dietary supplements. However, the metabolic changes associated with consuming concentrated doses of purified polyphenols, as may be present in dietary supplements, are unknown, especially when considering the colonic metabolites formed. This study aimed to evaluate the pharmacokinetics of high doses of purified blueberry polyphenols. 5-month old, ovariectomized Sprague-Dawley rats are acutely dosed with purified blueberry polyphenols (0, 75, 350, and 1000mg total polyphenols per kg body weight (bw)) and 45 Ca to measure calcium absorption. Blood and urine are collected for 48 h after dosing and phenolic metabolites measured via ultra high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The most prominent metabolites are colonically generated cinnamic and hippuric acids. Smaller amounts of other phenolic acids, flavonols, and anthocyanins are also detected. Most metabolites follow a dose-response relationship, though several show saturated absorption. Maximal metabolite concentrations are reached within 12 h for a majority of compounds measured, while some (e.g., hippuric acid) peaked up to 24 h post-dosing. Calcium absorption is significantly increased in the highest dose group (p = 0.03). These results indicate that increased doses of blueberry polyphenols induce changes in intestinal phenolic metabolism and increase calcium absorption.