Urinary Metabolic Distinction of Niemann-Pick Class 1 Disease through the Use of Subgroup Discovery.

In this investigation, we outline the applications of a data mining technique known as Subgroup Discovery (SD) to the analysis of a sample size-limited metabolomics-based dataset. The SD technique utilized a supervised learning strategy, which lies midway between classificational and descriptive criteria, in which given the descriptive property of a dataset (i.e., the response target variable of interest), the primary objective was to discover subgroups with behaviours that are distinguishable from those of the complete set (albeit with a differential statistical distribution). These approaches have, for the first time, been successfully employed for the analysis of aromatic metabolite patterns within an NMR-based urinary dataset collected from a small cohort of patients with the lysosomal storage disorder Niemann-Pick class 1 (NPC1) disease (n = 12) and utilized to distinguish these from a larger number of heterozygous (parental) control participants. These subgroup discovery strategies discovered two different NPC1 disease-specific metabolically sequential rules which permitted the reliable identification of NPC1 patients; the first of these involved 'normal' (intermediate) urinary concentrations of xanthurenate, 4-aminobenzoate, hippurate and quinaldate, and disease-downregulated levels of nicotinate and trigonelline, whereas the second comprised 'normal' 4-aminobenzoate, indoxyl sulphate, hippurate, 3-methylhistidine and quinaldate concentrations, and again downregulated nicotinate and trigonelline levels. Correspondingly, a series of five subgroup rules were generated for the heterozygous carrier control group, and 'biomarkers' featured in these included low histidine, 1-methylnicotinamide and 4-aminobenzoate concentrations, together with 'normal' levels of hippurate, hypoxanthine, quinolinate and hypoxanthine. These significant disease group-specific rules were consistent with imbalances in the combined tryptophan-nicotinamide, tryptophan, kynurenine and tyrosine metabolic pathways, along with dysregulations in those featuring histidine, 3-methylhistidine and 4-hydroxybenzoate. In principle, the novel subgroup discovery approach employed here should also be readily applicable to solving metabolomics-type problems of this nature which feature rare disease classification groupings with only limited patient participant and sample sizes available.

Metabolic biomarkers of radiotherapy response in plasma and tissue of an IDH1 mutant astrocytoma mouse model.

Astrocytomas are the most common subtype of brain tumors and no curative treatment exist. Longitudinal assessment of patients, usually via Magnetic Resonance Imaging (MRI), is crucial since tumor progression may occur earlier than clinical progression. MRI usually provides a means for monitoring the disease, but it only informs about the structural changes of the tumor, while molecular changes can occur as a treatment response without any MRI-visible change. Radiotherapy (RT) is routinely performed following surgery as part of the standard of care in astrocytomas, that can also include chemotherapy involving temozolomide. Monitoring the response to RT is a key factor for the management of patients. Herein, we provide plasma and tissue metabolic biomarkers of treatment response in a mouse model of astrocytoma that was subjected to radiotherapy. Plasma metabolic profiles acquired over time by Liquid Chromatography Mass Spectrometry (LC/MS) were subjected to multivariate empirical Bayes time-series analysis (MEBA) and Receiver Operating Characteristic (ROC) assessment including Random Forest as the classification strategy. These analyses revealed a variation of the plasma metabolome in those mice that underwent radiotherapy compared to controls; specifically, fumarate was the best discriminatory feature. Additionally, Nuclear Magnetic Resonance (NMR)-based 13C-tracing experiments were performed at end-point utilizing [U-13C]-Glutamine to investigate its fate in the tumor and contralateral tissues. Irradiated mice displayed lower levels of glycolytic metabolites (e.g. phosphoenolpyruvate) in tumor tissue, and a higher flux of glutamine towards succinate was observed in the radiation cohort. The plasma biomarkers provided herein could be validated in the clinic, thereby improving the assessment of brain tumor patients throughout radiotherapy. Moreover, the metabolic rewiring associated to radiotherapy in tumor tissue could lead to potential metabolic imaging approaches for monitoring treatment using blood draws.

Advances in measuring cancer cell metabolism with subcellular resolution.

Characterizing metabolism in cancer is crucial for understanding tumor biology and for developing potential therapies. Although most metabolic investigations analyze averaged metabolite levels from all cell compartments, subcellular metabolomics can provide more detailed insight into the biochemical processes associated with the disease. Methodological limitations have historically prevented the wider application of subcellular metabolomics in cancer research. Recently, however, ways to distinguish and identify metabolic pathways within organelles have been developed, including state-of-the-art methods to monitor metabolism in situ (such as mass spectrometry-based imaging, Raman spectroscopy and fluorescence microscopy), to isolate key organelles via new approaches and to use tailored isotope-tracing strategies. Herein, we examine the advantages and limitations of these developments and look to the future of this field of research.

Cysteine is a limiting factor for glioma proliferation and survival.

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. However, little is known about the mechanisms that make cancer cells require certain nutrients from the microenvironment. Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13 C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to support glutathione synthesis through the transsulfuration pathway, which allows methionine to be converted to cysteine in cysteine/cystine-deprived conditions. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, although this increase did not attain statistical significance, with concomitant reductions in plasma glutathione and cysteine levels. At the end point, however, tumors displayed the ability to synthesize glutathione, even though higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolite levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

Gut microbiome and metabolome in a non-human primate model of chronic excessive alcohol drinking.

A relationship between the gut microbiome and alcohol use disorder has been suggested. Excessive alcohol use produces changes in the fecal microbiome and metabolome in both rodents and humans. Yet, these changes can be observed only in a subgroup of the studied populations, and reversal does not always occur after abstinence. We aimed to analyze fecal microbial composition and function in a translationally relevant baboon model of chronic heavy drinking that also meets binge criteria (drinking too much, too fast, and too often), i.e., alcohol ~1 g/kg and blood alcohol levels (BALs) ≥ 0.08 g/dL in a 2-hour period, daily, for years. We compared three groups of male baboons (Papio anubis): L = Long-term alcohol drinking group (12.1 years); S = Short-term alcohol drinking group (2.7 years); and C = Control group, drinking a non-alcoholic reinforcer (Tang®) (8.2 years). Fecal collection took place during 3 days of Drinking (D), followed by a short period (3 days) of Abstinence (A). Fecal microbial alpha- and beta-diversity were significantly lower in L vs. S and C (p's < 0.05). Members of the commensal families Lachnospiraceae and Prevotellaceae showed a relative decrease, whereas the opportunistic pathogen Streptococcus genus showed a relative increase in L vs. S and C (p's < 0.05). Microbiota-related metabolites of aromatic amino acids, tricarboxylic acid cycle, and pentose increased in L vs. S and C (FDR-corrected p < 0.01), with the latter two suggesting high energy metabolism and enhanced glycolysis in the gut lumen in response to alcohol. Consistent with the long-term alcohol exposure, mucosal damage and oxidative stress markers (N-acetylated amino acids, 2-hydroxybutyrate, and metabolites of the methionine cycle) increased in L vs. S and C (FDR-corrected p < 0.01). Overall, S showed few differences vs. C, possibly due to the long-term, chronic alcohol exposure needed to alter the normal gut microbiota. In the three groups, the fecal microbiome barely differed between conditions D and A, whereas the metabolome shifted in the transition from condition D to A. In conclusion, changes in the fecal microbiome and metabolome occur after significant long-term excessive drinking and are only partially affected by acute forced abstinence from alcohol. These results provide novel information on the relationship between the fecal microbiome and metabolome in a controlled experimental setting and using a unique non-human primate model of chronic excessive alcohol drinking.

Magnetic resonance spectroscopy for the study of cns malignancies.

Despite intensive research, brain tumors are amongst the malignancies with the worst prognosis; therefore, a prompt diagnosis and thoughtful assessment of the disease is required. The resistance of brain tumors to most forms of conventional therapy has led researchers to explore the underlying biology in search of new vulnerabilities and biomarkers. The unique metabolism of brain tumors represents one potential vulnerability and the basis for a system of classification. Profiling this aberrant metabolism requires a method to accurately measure and report differences in metabolite concentrations. Magnetic resonance-based techniques provide a framework for examining tumor tissue and the evolution of disease. Nuclear Magnetic Resonance (NMR) analysis of biofluids collected from patients suffering from brain cancer can provide biological information about disease status. In particular, urine and plasma can serve to monitor the evolution of disease through the changes observed in the metabolic profiles. Moreover, cerebrospinal fluid can be utilized as a direct reporter of cerebral activity since it carries the chemicals exchanged with the brain tissue and the tumor mass. Metabolic reprogramming has recently been included as one of the hallmarks of cancer. Accordingly, the metabolic rewiring experienced by these tumors to sustain rapid growth and proliferation can also serve as a potential therapeutic target. The combination of 13C tracing approaches with the utilization of different NMR spectral modalities has allowed investigations of the upregulation of glycolysis in the aggressive forms of brain tumors, including glioblastomas, and the discovery of the utilization of acetate as an alternative cellular fuel in brain metastasis and gliomas. One of the major contributions of magnetic resonance to the assessment of brain tumors has been the non-invasive determination of 2-hydroxyglutarate (2HG) in tumors harboring a mutation in isocitrate dehydrogenase 1 (IDH1). The mutational status of this enzyme already serves as a key feature in the clinical classification of brain neoplasia in routine clinical practice and pilot studies have established the use of in vivo magnetic resonance spectroscopy (MRS) for monitoring disease progression and treatment response in IDH mutant gliomas. However, the development of bespoke methods for 2HG detection by MRS has been required, and this has prevented the wider implementation of MRS methodology into the clinic. One of the main challenges for improving the management of the disease is to obtain an accurate insight into the response to treatment, so that the patient can be promptly diverted into a new therapy if resistant or maintained on the original therapy if responsive. The implementation of 13C hyperpolarized magnetic resonance spectroscopic imaging (MRSI) has allowed detection of changes in tumor metabolism associated with a treatment, and as such has been revealed as a remarkable tool for monitoring response to therapeutic strategies. In summary, the application of magnetic resonance-based methodologies to the diagnosis and management of brain tumor patients, in addition to its utilization in the investigation of its tumor-associated metabolic rewiring, is helping to unravel the biological basis of malignancies of the central nervous system.

IDH1 mutations induce organelle defects via dysregulated phospholipids.

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.

Metabolic plasticity of IDH1-mutant glioma cell lines is responsible for low sensitivity to glutaminase inhibition.

BACKGROUND: Targeting glutamine metabolism in cancer has become an increasingly vibrant area of research. Mutant IDH1 (IDH1 mut ) gliomas are considered good candidates for targeting this pathway because of the contribution of glutamine to their newly acquired function: synthesis of 2-hydroxyglutarate (2HG). METHODS: We have employed a combination of 13C tracers including glutamine and glucose for investigating the metabolism of patient-derived IDH1 mut glioma cell lines through NMR and LC/MS. Additionally, genetic loss-of-function (in vitro and in vivo) approaches were performed to unravel the adaptability of these cell lines to the inhibition of glutaminase activity. RESULTS: We report the adaptability of IDH1 mut cells' metabolism to the inhibition of glutamine/glutamate pathway. The glutaminase inhibitor CB839 generated a decrease in the production of the downstream metabolites of glutamate, including those involved in the TCA cycle and 2HG. However, this effect on metabolism was not extended to viability; rather, our patient-derived IDH1 mut cell lines display a metabolic plasticity that allows them to overcome glutaminase inhibition. CONCLUSIONS: Major metabolic adaptations involved pathways that can generate glutamate by using alternative substrates from glutamine, such as alanine or aspartate. Indeed, asparagine synthetase was upregulated both in vivo and in vitro revealing a new potential therapeutic target for a combinatory approach with CB839 against IDH1 mut gliomas.

Sphingolipid Pathway as a Source of Vulnerability in IDH1mut Glioma.

In addition to providing integrity to cellular structure, the various classes of lipids participate in a multitude of functions including secondary messengers, receptor stimulation, lymphocyte trafficking, inflammation, angiogenesis, cell migration, proliferation, necrosis and apoptosis, thus highlighting the importance of understanding their role in the tumor phenotype. In the context of IDH1mut glioma, investigations focused on metabolic alterations involving lipidomics' present potential to uncover novel vulnerabilities. Herein, a detailed lipidomic analysis of the sphingolipid metabolism was conducted in patient-derived IDH1mut glioma cell lines, as well as model systems, with the of identifying points of metabolic vulnerability. We probed the effect of decreasing D-2HG levels on the sphingolipid pathway, by treating these cell lines with an IDH1mut inhibitor, AGI5198. The results revealed that N,N-dimethylsphingosine (NDMS), sphingosine C17 and sphinganine C18 were significantly downregulated, while sphingosine-1-phosphate (S1P) was significantly upregulated in glioma cultures following suppression of IDH1mut activity. We exploited the pathway using a small-scale, rational drug screen and identified a combination that was lethal to IDHmut cells. Our work revealed that further addition of N,N-dimethylsphingosine in combination with sphingosine C17 triggered a dose-dependent biostatic and apoptotic response in a panel of IDH1mut glioma cell lines specifically, while it had little effect on the IDHWT cells probed here. To our knowledge, this is the first study that shows how altering the sphingolipid pathway in IDH1mut gliomas elucidates susceptibility that can arrest proliferation and initiate subsequent cellular death.

Metabolic Landscape of a Genetically Engineered Mouse Model of IDH1 Mutant Glioma.

Understanding the metabolic reprogramming of aggressive brain tumors has potential applications for therapeutics as well as imaging biomarkers. However, little is known about the nutrient requirements of isocitrate dehydrogenase 1 (IDH1) mutant gliomas. The IDH1 mutation involves the acquisition of a neomorphic enzymatic activity which generates D-2-hydroxyglutarate from α-ketoglutarate. In order to gain insight into the metabolism of these malignant brain tumors, we conducted metabolic profiling of the orthotopic tumor and the contralateral regions for the mouse model of IDH1 mutant glioma; as well as to examine the utilization of glucose and glutamine in supplying major metabolic pathways such as glycolysis and tricarboxylic acid (TCA). We also revealed that the main substrate of 2-hydroxyglutarate is glutamine in this model, and how this re-routing impairs its utilization in the TCA. Our 13C tracing analysis, along with hyperpolarized magnetic resonance experiments, revealed an active glycolytic pathway similar in both regions (tumor and contralateral) of the brain. Therefore, we describe the reprogramming of the central carbon metabolism associated with the IDH1 mutation in a genetically engineered mouse model which reflects the tumor biology encountered in glioma patients.

Triptolide suppresses IDH1-mutated malignancy via Nrf2-driven glutathione metabolism.

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, served as a potent Nrf2 inhibitor, which exhibited selective cytotoxicity to patient-derived IDH1-mutated glioma cells in vitro and in vivo. Mechanistically, triptolide compromised the expression of GCLC, GCLM, and SLC7A11, which disrupted glutathione metabolism and established synthetic lethality with reactive oxygen species derived from IDH1 mutant neomorphic activity. Our findings highlight triptolide as a valuable therapeutic approach for IDH1-mutated malignancies by targeting the Nrf2-driven glutathione synthesis pathway.

Metabolic reprogramming associated with aggressiveness occurs in the G-CIMP-high molecular subtypes of IDH1mut lower grade gliomas.

BACKGROUND: Early detection of increased aggressiveness of brain tumors is a major challenge in the field of neuro-oncology because of the inability of traditional imaging to uncover it. Isocitrate dehydrogenase (IDH)-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with tumorigenicity because they appear indolent and non-glycolytic initially, but eventually a subset progresses toward secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood. METHODS: We employed model systems for IDH1 mutant (IDH1mut) gliomas with different growth and proliferation rates in vivo and in vitro. We described the metabolome, transcriptome, and epigenome of these models in order to understand the link between their metabolism and the tumor biology. To verify whether this metabolic reprogramming occurs in the clinic, we analyzed data from The Cancer Genome Atlas. RESULTS: We reveal that the aggressive glioma models have lost DNA methylation in the promoters of glycolytic enzymes, especially lactate dehydrogenase A (LDHA), and have increased mRNA and metabolite levels compared with the indolent model. We find that the acquisition of the high glycolytic phenotype occurs at the glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP)-high molecular subtype in patients and is associated with the worst outcome. CONCLUSION: We propose very early monitoring of lactate levels as a biomarker of metabolic reprogramming and tumor aggressiveness.

Targeting Glycolysis through Inhibition of Lactate Dehydrogenase Impairs Tumor Growth in Preclinical Models of Ewing Sarcoma.

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.

Methylphenidate alters monoaminergic and metabolic pathways in the cerebellum of adolescent rats.

Abnormalities in the cerebellar circuitry have been suggested to contribute to some of the symptoms associated with attention deficit hyperactivity disorder (ADHD). The psychostimulant methylphenidate (MPH) is the major drug for treating this condition. Here, the effects of acute (2.0 mg/kg and 5.0 mg/kg) and chronic (2.0 mg/kg, twice daily for 15 days) MPH treatments were investigated in adolescent (35-40 days old) rats on monoaminergic and metabolic markers in the cerebellum. Data acquired indicates that acute MPH treatment (2.0 mg/kg) decreased cerebellar vesicular monoamine transporter (VMAT2) density, while chronic treatment caused an increase. In contrast, protein levels of tyrosine hydroxylase (TH) and the dopamine D1 receptor were not significantly altered by neither acute nor chronic MPH treatment. In addition, while chronic but not acute MPH treatment significantly enhanced dopamine turnover (DOPAC/dopamine) in the cerebellum, levels of dopamine and homovanillic acid (HVA) were not altered. Acute MPH (5.0 mg/kg) significantly modified levels of a range of cerebellar metabolites with similar trends also detected for the lower dose (2.0 mg/kg). In this regard, acute MPH tended to decrease cerebellar metabolites associated with energy consumption and excitatory neurotransmission including glutamate, glutamine, N-acetyl aspartate, and inosine. Conversely, levels of some metabolites associated with inhibitory neurotransmission, including GABA and glycine were reduced by acute (5.0 mg/kg) MPH, together with acetate, aspartate and hypoxanthine. In conclusion, this study demonstrated that MPH alters cerebellar biochemistry, and that this effect depends on both dose and duration of treatment. The therapeutic significance of these results requires further investigation.

Detection of Metabolic Changes Induced via Drug Treatments in Live Cancer Cells and Tissue Using Raman Imaging Microscopy.

Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NAD⁺) salvage pathway, FK866, and observed decreased viability in these cells. To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined analyses point to a decrease in cell division due to loss of lipid content that contributes to membrane formation in the in vitro setting. However, the FK866 drug did not have the same potency in vivo. The use of Raman imaging microscopy indicated an opposite trend of lipid distribution in the tissue collected from treated versus untreated mice when compared with the cells. These results demonstrate the role of Raman imaging microscopy to identify and quantify metabolic changes in cancer cells and tissue.

NMR analysis reveals significant differences in the plasma metabolic profiles of Niemann Pick C1 patients, heterozygous carriers, and healthy controls.

Niemann-Pick type C1 (NPC1) disease is a rare autosomal recessive, neurodegenerative lysosomal storage disorder, which presents with a range of clinical phenotypes and hence diagnosis remains a challenge. In view of these difficulties, the search for a novel, NPC1-specific biomarker (or set of biomarkers) is a topic of much interest. Here we employed high-resolution 1H nuclear magnetic resonance spectroscopy coupled with advanced multivariate analysis techniques in order to explore and seek differences between blood plasma samples acquired from NPC1 (untreated and miglustat treated), heterozygote, and healthy control subjects. Using this approach, we were able to identify NPC1 disease with 91% accuracy confirming that there are significant differences in the NMR plasma metabolic profiles of NPC1 patients when compared to healthy controls. The discrimination between NPC1 (both miglustat treated and untreated) and healthy controls was dominated by lipoprotein triacylglycerol 1H NMR resonances and isoleucine. Heterozygote plasma samples displayed also increases in the intensities of selected lipoprotein triacylglycerol 1H NMR signals over those of healthy controls. The metabolites identified could represent useful biomarkers in the future and provide valuable insight in to the underlying pathology of NPC1 disease.

1H NMR-based metabolomics reveals neurochemical alterations in the brain of adolescent rats following acute methylphenidate administration.

The psychostimulant methylphenidate (MPH) is increasingly used in the treatment of attention deficit hyperactivity disorder (ADHD). While there is little evidence for common brain pathology in ADHD, some studies suggest a right hemisphere dysfunction among people diagnosed with the condition. However, in spite of the high usage of MPH in children and adolescents, its mechanism of action is poorly understood. Given that MPH blocks the neuronal transporters for dopamine and noradrenaline, most research into the effects of MPH on the brain has largely focused on these two monoamine neurotransmitter systems. Interestingly, recent studies have demonstrated metabolic changes in the brain of ADHD patients, but the impact of MPH on endogenous brain metabolites remains unclear. In this study, a proton nuclear magnetic resonance (1H NMR)-based metabolomics approach was employed to investigate the effects of MPH on brain biomolecules. Adolescent male Sprague Dawley rats were injected intraperitoneally with MPH (5.0 mg/kg) or saline (1.0 ml/kg), and cerebral extracts from the left and right hemispheres were analysed. A total of 22 variables (representing 13 distinct metabolites) were significantly increased in the MPH-treated samples relative to the saline-treated controls. The upregulated metabolites included: amino acid neurotransmitters such as GABA, glutamate and aspartate; large neutral amino acids (LNAA), including the aromatic amino acids (AAA) tyrosine and phenylalanine, both of which are involved in the metabolism of dopamine and noradrenaline; and metabolites associated with energy and cell membrane dynamics, such as creatine and myo-inositol. No significant differences in metabolite concentrations were found between the left and right cerebral hemispheres. These findings provide new insights into the mechanisms of action of the anti-ADHD drug MPH.

A Multifactorial Comparison of Ternary Combinations of Essential Oils in Topical Preparations to Current Antibiotic Prescription Therapies for the Control of Acne Vulgaris-Associated Bacteria.

Acne vulgaris, a chronic condition associated with overgrowth of Propionibacterium acnes and Staphylococcus epidermidis, is commonly treated with antibiotics. However, the emergence of antibiotic resistance has resulted in a need for alternative therapies. The aim of this study is to develop a topical preparation incorporating essential oils (EOs) for use against acne-associated bacteria and assess its efficacy against prescription therapies Dalacin T and Stiemycin. Antimicrobial screening of rosewood, clove bud and litsea EOs was conducted before interactions between binary and ternary combinations were determined against P. acnes and S. epidermidis (type and clinical isolates) using minimum inhibitory concentrations and fractional inhibitory concentrations. The EOs were characterised by both gas chromatography-mass spectrometry and nuclear magnetic resonance. A combination of 0.53 mg/mL litsea, 0.11 mg/mL rosewood and 0.11 mg/mL clove bud was formulated into herbal distillates and compared with Dalacin T and Stiemycin against antibiotic sensitive and resistant isolates (erythromycin). The distillate with EO had synergistic activity against P. acnes (7log10 reduction) and indifferent activity against S. epidermidis (6log10 reduction); antimicrobial activity was either significantly (p ≤ 0.05) more antimicrobial or equivalent to that of Dalacin T and Stiemycin. This formulation may serve as a valuable alternative for the control of acne vulgaris-associated bacteria. Copyright © 2017 John Wiley & Sons, Ltd.

1H NMR-Linked Metabolomics Analysis of Liver from a Mouse Model of NP-C1 Disease.

Clinical manifestations of Niemann-Pick type C1 (NP-C1) disease include neonatal hepatosplenomegaly and in some patients progressive liver dysfunction and failure. This study involved a 1H NMR-linked metabolomics analysis of liver samples collected from a NP-C1 disease mutant mouse model in order to explore time-dependent imbalances in metabolic pathways associated with NP-C1 liver dysfunction, including fibrosis. NP-C1 mutant (Npc1-/-; NP-C1), control (Npc1+/+; WT), and NP-C1 heterozygous mice (Npc1+/-; HET) were generated from heterozygote matings. Aqueous extracts of these liver samples collected at time points of 3, 6, 9, and 11 weeks were subjected to high-resolution NMR analysis, and multivariate (MV) metabolomics analyses of data sets acquired were performed. A MV random forests (RFs) model effectively discriminated between NP-C1 and a combined WT/HET hepatic NMR profiles with very high predictive accuracy and reliability. Key distinguishing features included significant upregulations in the hepatic concentrations of phenylalanine, tyrosine, glutamate, lysine/ornithine, valine, threonine, and hypotaurine/methionine, and diminished levels of nicotinate/niacinamide, inosine, phosphoenolpyruvate, and 3-hydroxyphenylacetate. Quantitative pathway topological analysis confirmed that imbalances in tyrosine biosynthesis, and hepatic phenylalanine, tyrosine, glutamate/glutamine, and nicotinate/niacinamide metabolism were involved in the pathogenesis of NP-C1 disease-associated liver dysfunction/damage. 1H NMR-linked metabolomics analysis provides valuable biomarker information regarding hepatic dysfunction or damage in NP-C1 disease.

Urinary excretion and metabolism of miglustat and valproate in patients with Niemann-Pick type C1 disease: One- and two-dimensional solution-state (1)H NMR studies.

Niemann-Pick type C1 (NP-C1) disease is a neurodegenerative lysosomal storage disease for which the only approved therapy is miglustat (MGS). In this study we explored the applications and value of both one- and two-dimensional high-resolution NMR analysis strategies to the detection and quantification of MGS and its potential metabolites in urine samples collected from NP-C1 disease patients (n=47), and also applied these techniques to the analysis of the anticonvulsant drug valproate and one of its major metabolites in ca. 30% of these samples (i.e. from those who were also receiving this agent for the control of epileptic seizures). A combination of high-resolution 1D and 2D TOCSY/NOESY techniques confirmed the identity of MGS in the urinary (1)H NMR profiles of NP-C1 patients treated with this agent (n=25), and its quantification was readily achievable via electronic integration of selected 1D resonance intensities. However, this analysis provided little or no evidence for its metabolism in vivo, observations consistent with those acquired in corresponding experiments performed involving an in vitro microsomal system. Contrastingly, the major valproate metabolite 1-O-valproyl-ß-glucuronide was readily detectable and quantifiable in 14/47 of the urine samples investigated, despite some resonance overlap problems (identification of this agent was confirmed by experiments involving equilibration of these samples with ß-glucuronidase, a process liberating free valproate). In order to facilitate and validate the detection of MGS in urine specimens, full assignments of the (1)H NMR spectra of MGS in both buffered aqueous (pH 7.10) and deuterated methanol solvent systems were also made. The pharmacological and bioanalytical significance of data acquired are discussed, with special reference to the advantages offered by high-resolution NMR analysis.