Modulation of the crystallization of rapeseed oil using lipases and the impact on ice cream properties.

We investigated the possibility to use rapeseed as a main oil in ice cream formulations by changing its functionality when using different kinds of lipases. Through a 24 h-emulsification and a centrifugation, the modified oils were further used as functional ingredients. All lipolysis was first assessed as a function of time by 13C NMR, where triglycerides consumption and the formation of low-molecular polar lipids (LMPL: monoacylglycerol and free fatty acids, FFAs) were selectively identified and compared. The more the FFAs, the sooner the crystallization (from -55 to -10 °C) and the later the melting temperatures (from -17 to 6 °C) measured by differential scanning calorimetry. These modifications were exploited in ice cream formulations with a significant impact on overall hardness (range of 60-216 N) and flowing during defrosting (from 1.29 to 0.35g/min). The global behavior of products can be controlled by the composition of LMPL within oil.

Study of rapeseed oil gelation induced by commercial monoglycerides using a chemometric approach.

Commercial oleogelators rich in monoglycerides (MGs) are complex mixtures of acylglycerides with variable gelling properties, depending on the oil used and their concentration. In this study we developed a chemometric approach to identify the key parameters involved in gelling process. Analytical parameters have been defined, using GC and NMR analysis to identify fatty acids and acylglycerides composing the mixtures. Specific acylglyceride families and compound ratios were calculated to streamline the analytical results. To determine the key analytical parameters, artificial neural networks were used in a QSPR study related to the gelling properties measured by rheology through oscillatory experiments. At low oleogelator concentrations, the MGs especially rich in C16:0 and the ratio of specific isomers both have a positive influence on G'. For high oleogelator concentrations, C18:0-rich acylglycerides and unsaturated/saturated fatty acid ratios have a positive influence on G'. Conversely, at low concentrations, C18:0-rich acylglycerides show a lesser effect on G'.

Hepatocyte nuclear factor-1ß shapes the energetic homeostasis of kidney tubule cells.

Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1ß (HNF-1ß) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1ß deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined 1 H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b-/- ), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1ß in metabolism. Hnf1b-/- cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b-/- cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b-/- cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1ß. HNF-1ß shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI.

Synthetic cannabinoids in e-liquids: A proton and fluorine NMR analysis from a conventional spectrometer to a compact one.

The 1H NMR profiles of 13 samples of e-liquids supplied by French customs were obtained with high-field and low-field NMR. The high-field 1H NMR spectra allowed the detection of matrix signals, synthetic cannabinoids, and flavouring compounds. Quantitative results were obtained for the five synthetic cannabinoids detected: JWH-210, 5F-MDMB-PICA, 5F-ADB, 5F-AKB48, and ADB-FUBINACA. Conventional GC-MS analysis was used to confirm compound identification. Fluorine-19 NMR was proposed for the quantification of fluorinated synthetic cannabinoids and was successfully implemented on both 400 MHz and 60 MHz NMR spectrometers. This study based on few examples explored the potentiality of low-field NMR for quantitative and quantitative analysis of synthetic cannabinoids in e-liquids.

Urinary metabolomic signature of muscle-invasive bladder cancer: A multiplatform approach.

Bladder cancer (BCa) is ninth amongst the most common types of cancer in the human population worldwide. The statistics of incidence and mortality of BCa are alarming and the currently applied diagnostic methods are still not sensitive enough. This leads to a large number of undiagnosed BCa cases, usually among patients in the early stages of the disease. Despite the fact that many risk factors of BCa have been recognized, the pathomechanism of development of bladder cancer has not been fully explained yet. Therefore, in the present study, multiplatform urinary metabolomics has been implemented in order to scrutinize potential diagnostic indicators of BCa that might help to explain its pathomechanism and be potentially useful in diagnosis and determination of stage of the disease. Urine samples collected from muscle-invasive high grade BCa patients (n = 24) and healthy volunteers (n = 24) were matched in terms of most common BCa risk factors i.e. gender, age, BMI and smoking status. They were analyzed by high performance liquid chromatography coupled with time of flight mass spectrometry detection (HPLC-TOF/MS) using RP and HILIC chromatography, gas chromatography hyphenated with triple quadruple mass spectrometry detection (GC-QqQ/MS) in scan mode, and proton nuclear magnetic resonance (1H NMR). The six datasets obtained were submitted to univariate and multivariate statistical analyses. 17 metabolites significantly discriminated urinary profiles of BCa patients from urinary profiles of healthy volunteers. These metabolites are mainly involved in amino acid metabolism, pyrimidine and purine metabolism, as well as energy metabolism and might play a crucial role in the pathogenesis of BCa.

Glucose metabolism and NRF2 coordinate the antioxidant response in melanoma resistant to MAPK inhibitors.

Targeted therapies as BRAF and MEK inhibitor combination have been approved as first-line treatment for BRAF-mutant melanoma. However, disease progression occurs in most of the patients within few months of therapy. Metabolic adaptations have been described in the context of acquired resistance to BRAF inhibitors (BRAFi). BRAFi-resistant melanomas are characterized by an increase of mitochondrial oxidative phosphorylation and are more prone to cell death induced by mitochondrial-targeting drugs. BRAFi-resistant melanomas also exhibit an enhancement of oxidative stress due to mitochondrial oxygen consumption increase. To understand the mechanisms responsible for survival of BRAFi-resistant melanoma cells in the context of oxidative stress, we have established a preclinical murine model that accurately recapitulates in vivo the acquisition of resistance to MAPK inhibitors including several BRAF or MEK inhibitors alone and in combination. Using mice model and melanoma cell lines generated from mice tumors, we have confirmed that the acquisition of resistance is associated with an increase in mitochondrial oxidative phosphorylation as well as the importance of glutamine metabolism. Moreover, we have demonstrated that BRAFi-resistant melanoma can adapt mitochondrial metabolism to support glucose-derived glutamate synthesis leading to increase in glutathione content. Besides, BRAFi-resistant melanoma exhibits a strong activation of NRF-2 pathway leading to increase in the pentose phosphate pathway, which is involved in the regeneration of reduced glutathione, and to increase in xCT expression, a component of the xc-amino acid transporter essential for the uptake of cystine required for intracellular glutathione synthesis. All these metabolic modifications sustain glutathione level and contribute to the intracellular redox balance to allow survival of BRAFi-resistant melanoma cells.

Metabolic rewiring in cancer cells overexpressing the glucocorticoid-induced leucine zipper protein (GILZ): Activation of mitochondrial oxidative phosphorylation and sensitization to oxidative cell death induced by mitochondrial targeted drugs.

Cancer cell metabolism is largely controlled by oncogenic signals and nutrient availability. Here, we highlighted that the glucocorticoid-induced leucine zipper (GILZ), an intracellular protein influencing many signaling pathways, reprograms cancer cell metabolism to promote proliferation. We provided evidence that GILZ overexpression induced a significant increase of mitochondrial oxidative phosphorylation as evidenced by the augmentation in basal respiration, ATP-linked respiration as well as respiratory capacity. Pharmacological inhibition of glucose, glutamine and fatty acid oxidation reduced the activation of GILZ-induced mitochondrial oxidative phosphorylation. At glycolysis level, GILZ-overexpressing cells enhanced the expression of glucose transporters in their plasmatic membrane and showed higher glycolytic reserve. 1H NMR metabolites quantification showed an up-regulation of amino acid biosynthesis. The GILZ-induced metabolic reprograming is present in various cancer cell lines regardless of their driver mutations status and is associated with higher proliferation rates persisting under metabolic stress conditions. Interestingly, high levels of OXPHOS made GILZ-overexpressing cells vulnerable to cell death induced by mitochondrial pro-oxidants. Altogether, these data indicate that GILZ reprograms cancer metabolism towards mitochondrial OXPHOS and sensitizes cancer cells to mitochondria-targeted drugs with pro-oxidant activities.

Identification of altered brain metabolites associated with TNAP activity in a mouse model of hypophosphatasia using untargeted NMR-based metabolomics analysis.

Tissue non-specific alkaline phosphatase (TNAP) is a key player of bone mineralization and TNAP gene (ALPL) mutations in human are responsible for hypophosphatasia (HPP), a rare heritable disease affecting the mineralization of bones and teeth. Moreover, TNAP is also expressed by brain cells and the severe forms of HPP are associated with neurological disorders, including epilepsy and brain morphological anomalies. However, TNAP's role in the nervous system remains poorly understood. To investigate its neuronal functions, we aimed to identify without any a priori the metabolites regulated by TNAP in the nervous tissue. For this purpose we used 1 H- and 31 P NMR to analyze the brain metabolome of Alpl (Akp2) mice null for TNAP function, a well-described model of infantile HPP. Among 39 metabolites identified in brain extracts of 1-week-old animals, eight displayed significantly different concentration in Akp2-/- compared to Akp2+/+ and Akp2+/- mice: cystathionine, adenosine, GABA, methionine, histidine, 3-methylhistidine, N-acetylaspartate (NAA), and N-acetyl-aspartyl-glutamate, with cystathionine and adenosine levels displaying the strongest alteration. These metabolites identify several biochemical processes that directly or indirectly involve TNAP function, in particular through the regulation of ecto-nucleotide levels and of pyridoxal phosphate-dependent enzymes. Some of these metabolites are involved in neurotransmission (GABA, adenosine), in myelin synthesis (NAA, NAAG), and in the methionine cycle and transsulfuration pathway (cystathionine, methionine). Their disturbances may contribute to the neurodevelopmental and neurological phenotype of HPP.

Mitochondrial oxidative stress is the Achille's heel of melanoma cells resistant to Braf-mutant inhibitor.

Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition.

Inactivation of the HIF-1α/PDK3 signaling axis drives melanoma toward mitochondrial oxidative metabolism and potentiates the therapeutic activity of pro-oxidants.

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Synthesis of lipophilic dimeric C-7/C-7-linked ciprofloxacin and C-6/C-6-linked levofloxacin derivatives. Versatile in vitro biological evaluations of monomeric and dimeric fluoroquinolone derivatives as potential antitumor, antibacterial or antimycobacterial agents.

The synthesis of C-7/C-7-linked ciprofloxacin (CP) and C-6/C-6-linked levofloxacin (LV) derivatives with modulated lipophilicity is described herein. The synthesized compounds, along with the monomeric analogs described previously, were evaluated in vitro for (i) their growth inhibitory effect against five human cancer cell lines, (ii) their antibacterial activity against Gram-positive Staphylococcus aureus and Enterococcus hirae and Gram-negative Escherichia coli and Pseudomonas aeruginosa strains and (iii) their antimycobacterial activity. The most efficient derivatives as antiproliferative agents (C-7/C-7-linked CP 7e and C-6/C-6-linked LV 11f) displayed IC(50) values in the 0.1-8.7 and 0.2-0.7 µM ranges respectively while IC(50) values for parent CP and LV ranged from 89 to 476 µM and from 67 to 622 µM respectively depending on the cell line. A specific antibacterial activity against S. aureus was found for the monomeric and dimeric derivatives of CP. The most efficient derivative against S. aureus (monomeric oxoethyloctanoate CP derivative 3d) displayed MIC <1 nM. Monomeric alkanoyloxymethyl LV esters (9a,c,e,f) and C-6/C-6-linked LV derivatives (11f-h) were the most efficient derivatives as antimycobacterial agents with MIC and IC(50) values in the 2.5-5 µM and 1.3-≤ 2.5 µM ranges respectively while MIC and IC(50) values for parent LV were 2.5 and 0.8 µM, respectively.

Exploiting mitochondrial dysfunction for effective elimination of imatinib-resistant leukemic cells.

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.

Novel lipophilic 7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid derivatives as potential antitumor agents: improved synthesis and in vitro evaluation.

A convenient route for the synthesis of some acyloxymethyl esters and carboxamides of levofloxacin (LV) with modulated lipophilicity is described. The synthesized compounds were evaluated in vitro for their growth inhibitory effect in five human cancer cell lines. The most efficient LV derivatives (ester 2e and amide 4d) displayed IC(50) values in the 0.2-2.2 µM range, while IC(50) values for parent LV ranged between 70 and 622 µM depending on the cell line. The esters displayed no in vivo toxicity up to 80 mg/kg when administered intraperitoneally. This study thus shows that LV analogs displayed antitumor efficacy, at least in vitro, a feature that appeared to be independent from the lipophilicity of the grafted substituent.

7-((4-Substituted)piperazin-1-yl) derivatives of ciprofloxacin: synthesis and in vitro biological evaluation as potential antitumor agents.

Ciprofloxacin (CP), an antibiotic has been shown to have antiproliferative and apoptotic activities in several cancer cell lines. Moreover, several reports have highlighted the interest of increasing the lipophilicity to improve the antitumor efficacy. These studies have led us to synthesize new CP derivatives of various lipophilicities and to evaluate their activity in five human cancer cell lines. With an easy and cost-efficient procedure, 31 7-((4-substituted)piperazin-1-yl) derivatives of CP were prepared that displayed IC(50) values ranging from microM to mM concentrations and are non-toxic in vivo in healthy mice as shown by their maximal tolerated dose (MTD) indices >80 mg/kg. Several derivatives displayed higher in vitro antitumor activity than parent CP however this was not dependent on the lipophilicity of the substituent. Among all synthesized derivatives, the most potent were 2 and 6h whose IC(50) values were 10 microM in three (derivative 2) or four (derivative 6h) cancer cell lines.

Two-dimensional DOSY experiment with Excitation Sculpting water suppression for the analysis of natural and biological media.

The Bipolar Pulse Pair Stimulated Echo NMR pulse sequence was modified to blend the original Excitation Sculpting water signal suppression. The sequence is a powerful tool to generate rapidly, with a good spectrum quality, bidimensional DOSY experiments without solvent signal, thus allowing the analysis of complex mixtures such as plant extracts or biofluids. The sequence has also been successfully implemented for a protein at very-low concentration in interaction with a small ligand, namely the salivary IB5 protein binding the polyphenol epigallocatechine gallate. The artifacts created by this sequence can be observed, checked and removed thanks to NPK and NMRnotebook softwares to give a perfect bidimensional DOSY spectrum.

Interest of fluorine-19 nuclear magnetic resonance spectroscopy in the detection, identification and quantification of metabolites of anticancer and antifungal fluoropyrimidine drugs in human biofluids.

BACKGROUND: The metabolism of fluorouracil and fluorocytosine, two 5-fluoropyrimidine drugs in clinical use, was investigated. METHODS: (19)F nuclear magnetic resonance (NMR) spectroscopy was used as an analytical technique for the detection, identification and quantification of fluorinated metabolites of these drugs in intact human biofluids as well as fluorinated degradation compounds of fluorouracil in commercial vials. RESULTS: (19)F NMR provides a highly specific tool for the detection and absolute quantification, in a single run, of all the fluorinated species, including unexpected substances, present in biofluids of patients treated with fluorouracil or fluorocytosine. Besides the parent drug and the already known fluorinated metabolites, nine new metabolites were identified for the first time with (19)F NMR in human biofluids. Six of them can only be observed with this technique: fluoride ion, N-carboxy-alpha-fluoro-beta-alanine, alpha-fluoro-beta-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O(2)-beta-glucuronide of fluorocytosine. CONCLUSION: (19)F NMR studies of biological fluids of patients treated with anticancer fluorouracil or antifungal fluorocytosine have furthered the understanding of their catabolic pathways.

Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs.

Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy provides a highly specific tool for the detection, identification and quantification of fluorine-containing drugs and their metabolites in biofluids. The value and difficulties encountered in investigations on drug metabolism are first discussed. Then the metabolism of three fluoropyrimidines in clinical use, 5-fluorouracil, 5-fluorocytosine and capecitabine are reported. Besides the parent drug and the already known fluorinated metabolites, 12 new metabolites were identified for the first time with 19F NMR in human biofluids. Nine of them can only be observed with this technique: fluoride ion, N-carboxy-alpha-fluoro-beta-alanine, alpha-fluoro-beta-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O2-beta-glucuronide of fluorocytosine, fluoroacetaldehyde hydrate and its adduct with urea, fluoromalonic acid semi-aldehyde adducts with urea. This emphasizes the high analytical potential of 19F NMR for the furtherance in the understanding of fluoropyrimidine catabolic pathways. 19F NMR should also play a role in the therapeutic monitoring of FU and its prodrugs in specific groups of patients, e.g. hemodialyzed patients or patients with deficiency in FU catabolic enzymes.

Usefulness of in vitro 1H magnetic resonance spectroscopy for the characterization of primary brain tumors: report of two cases.

Pathological diagnosis of brain tumors provides an index of brain disease severity and guides clinical practice in their treatment. Diagnoses are often made from biopsy material obtained using stereotactic techniques with the difficulty of making a histological diagnosis in small samples. In our experience, the estimation of the degree of malignancy on the basis of in vitro 1H magnetic resonance spectroscopy (1H MRS) appears to well correlate with histology and clinical evolution. We report two cases with a discordance between the diagnoses on the basis of histology examination and in vitro 1H MRS whose evolution seems to correlate better with the data of 1H MRS.

Isolation of an unknown metabolite of capecitabine, an oral 5-fluorouracil prodrug, and its identification by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry as a glucuroconjugate of 5'-deoxy-5-fluorocytidine, namely 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine.

A new metabolite of capecitabine, a prodrug of 5-fluorouracil, was detected by (19)F NMR in bile and liver of rats treated with this anticancer drug. Crude bile and perchloric acid extract of liver was subjected to liquid-liquid separation followed by a pre-purification step on a preparative octadecyl silane column (C(18)). The compound was purified by HPLC optimised to allow the detection of the unknown metabolite and its assumed precursor 5'-deoxy-5-fluorocytidine (5'-DFCR). Treatment with beta-glucuronidase from three sources showed that it was a glucuroconjugate of 5'-DFCR. HPLC-TIS-MS-MS and (1)H NMR allowed identification of the unknown metabolite as 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine.