Effects of dietary branched-chain amino acid supplementation on serum and milk metabolome profiles in dairy cows during early lactation.

The 3 branched-chain AA (BCAA), Val, Leu, and Ile, are essential AA used by tissues as substrates for protein synthesis and energy generation. In addition, BCAA are also involved in modulating cell signaling pathways, such as nutrient sensing and insulin signaling. In our previous study, dietary BCAA supplementation was shown to improve protein synthesis and glucose homeostasis in transition cows. However, a more detailed understanding of the changes in metabolic pathways associated with an increased BCAA availability is desired to fine-tune nutritional supplementation strategies. Multiparous Holstein cows (n = 20) were enrolled 28 d before expected calving and assigned to either the BCAA treatment (n = 10) or the control group (n = 10). Cows assigned to BCAA were fed 550 g/d of rumen-protected BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, whereas the cows assigned to the control were fed only 200 g/d of dry molasses. Serum samples were collected on d 10 before expected calving, as well as on d 4 and d 21 postpartum. Milk samples were collected on d 14 postpartum. From a larger cohort, we selected 20 BCAA-supplemented cows with the greatest plasma urea nitrogen concentration, as an indicator for greater BCAA availability, for the metabolomics analysis herein. Serum and milk samples were subjected to a liquid chromatography-mass spectrometry-based assay, detecting and measuring the abundance of 241 serum and 211 milk metabolic features, respectively. Multivariable statistical analyses revealed that BCAA supplementation altered the metabolome profiles of both serum and milk samples. Increased abundance of serum phosphocholine and glutathione and of milk Val, Ile, and Leu, and decreased abundance of milk acyl-carnitines were associated with BCAA supplementation. Altered phosphocholine and glutathione abundances point to altered hepatic choline metabolism and antioxidant balance, respectively. Altered milk acyl-carnitine abundances suggest changes in mammary fatty acid metabolism. Dietary BCAA supplementation was associated with a range of alterations in serum and milk metabolome profiles, adding to our understanding of the role of BCAA availability in modulating dairy cow protein, lipid, and energy metabolism on a whole-body level and how it affects milk composition.

Mechanism of antioxidant action of natural phenolics on scallop (Argopecten irradians) adductor muscle during drying process.

Lipid hydrolysis and oxidation occurred in Argopecten irradians adductor muscle during hot air drying. Using an in vivo imaging system, we found that antioxidants of bamboo leaves (AOB) could diffuse into the adductor muscle upon marinating. Both tea polyphenols (TP) and AOB efficiently retarded lipid oxidation but had a slight effect on lipid hydrolysis during drying process. The in situ antioxidant mechanisms of AOB as well as TP were revealed, including quenching of free radicals detected by electron spin resonance, chelating metal ions determined by confocal laser scanning microscopy and inhibiting lipoxygenase. Less than 8% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in AOB and TP marinated adductor muscle were decreased compared to more than 28% decrease in control adductor muscle during the drying process. Overall, these natural antioxidants, TP and AOB, efficiently maintained high nutritive value of adductor muscle, especially, their lipid quality.

In vivo Metabolic Profiles as Determined by 31P and short TE 1H MR-Spectroscopy : No Difference Between Patients with IDH Wildtype and IDH Mutant Gliomas.

PURPOSE: Previous ex vivo spectroscopic data from tissue samples revealed differences in phospholipid metabolites between isocitrate dehydrogenase mutated (IDHmut) and IDH wildtype (IDHwt) gliomas. We investigated whether these changes can be found in vivo using 1H-decoupled 31P magnetic resonance spectroscopic imaging (MRSI) with 3D chemical shift imaging (CSI) at 3 T in patients with low and high-grade gliomas. METHODS: The study included 33 prospectively enrolled, mostly untreated patients who met spectral quality criteria according to the World Health Organization (WHO II n = 7, WHO III n = 17, WHO IV n = 9; 25 patients IDHmut, 8 patients IDHwt). The MRSI protocol included 1H decoupled 31P MRSI with 3D CSI (3D 31P CSI), 2D 1H CSI and a 1H single voxel spectroscopy sequence (TE 30 ms) from the tumor area. For 1H MRS, absolute metabolite concentration values were calculated (phantom replacement method). For 31P MRS, metabolite intensity ratios were calculated for the choline (C) and ethanolamine (E)-containing metabolites. RESULTS: In our patient cohort we did not find significant differences for the ratio of phosphocholine (PC) and phosphoethanolamine (PE), PC/PE, (p = 0.24) for IDHmut compared to IDHwt gliomas. Furthermore, we found no elevated ratios of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), GPC/GPE, (p = 0.68) or GPC/PE (p = 0.12) for IDHmut gliomas. Even the ratio (PC+GPC)/(PE+GPE) showed no significant differences with respect to mutation status (p = 0.16). Nonetheless, changes related to tumor grade regarding intracellular pH (pHi) and phospholipid metabolism as well as absolute metabolite concentrations of co-registered 2D 1H CSI data for tumor and control tissue showed the anticipated results. CONCLUSION: Using 3D-CSI data acquisition, in vivo 31P MR spectroscopic measurement of phospholipid metabolites could not distinguish between IDHmut and IDHwt.

pH detection in biological samples by 1D and 2D 1H-31P NMR.

The chemical shifts of several important endogenous phosphorus compounds under different pH conditions were explored, including adenosine-5'-triphosphate, adenosine-5'-diphosphate, adenosine-5'-monophosphate, phosphorylcholine and phosphorylethanolamine. Their 31P NMR and 1H NMR chemical shifts were all pH-sensitive in the similar pH range. Two dimensional (2D) 1H-31P NMR spectra were found helpful to identify these endogenous phosphorus markers in biological samples from rather complicated NMR spectra. Herein, for the first time, a pH sensor based on 2D 1H-31P NMR was established and applied to biological samples analysis with pH values determined in good agreement with those by potentiometric method. Apart from being simple, green, rapid and less sample-consuming, information concerning both the endogenous phosphorus markers and pH status could be attained in a single NMR run, which demonstrated the great potential of this method in rare sample analysis and even disease diagnosis.

EVALUATION OF ANTIVIRAL THERAPY TREATMENT FOR LIVER CIRRHOSIS CAUSED BY CHRONIC HEPATITIS C AND HEPATITIS C BY 31P-MRS, BASED ON METABOLITE DETECTION.

This study discusses the application of magnetic resonance spectrum (MRS) to evaluate the efficacy of antiviral therapy in the treatment of liver cirrhosis caused by chronic hepatitis C and hepatitis C, based on metabolite detection. A total of 54 patients with liver cirrhosis caused by chronic hepatitis C and hepatitis C were selected and divided into treatment group and control group. 31P-MRS imaging was carried out on patients in the two groups both before receiving antiviral treatment and 6 months after treatment to compare the change of metabolite ratio (PE+PC)/(GPE+GPC). It was revealed that no statistically significant difference was found in the comparison of (PC+PE)/(GPC+GPE) ratio in the two groups before treatment, but the difference was found 6 months after treatment; ratio of (PC+PE)/ (GPC+GPE) in the treatment group distinctly decreased 6 months after treatment compared to before treatment, with a statistically significant difference, while the control group had no remarkable change or statistical significance. Moreover, 32 patients were found with sustained virus response to antiviral therapy. Of these, 25 patients possessed a decreased ratio of (PC+PE)/ (GPC+GPE), 4 remained without change and 3 had a slightly increased ratio after antiviral treatment. Of 12 patients with no response, 1 had a decreased ratio of (PC+PE)/ (GPC+GPE), 2 remained without change and 9 had a slightly increased ratio. The differences were all statistically significant in comparison of the two groups. 31P-MRS is thought to be effective for evaluating the efficacy of antiviral therapy through non-invasive detection of liver energy metabolism.

Bacterial colonization due to increased nurse workload in an intensive care unit / Colonização bacteriana por causa do aumento da carga de trabalho da equipe de enfermagem em unidade de terapia intensiva / Colonización bacteriana debido al aumento de la carga de trabajo del equipo de enfermería en una unidad de cuidados intensivos

INTRODUCTION: The rates of multiresistant bacteria colonization or infection (MRB+) development in intensive care units are very high. The aim of this study was to determine the possible association between the risk of development of nosocomial infections and increased daily nurse workload due to understaffing in intensive care unit. METHODS: We included 168 patients. Intensity of workload and applied procedures to patients were scored with the Project de Recherché en Nursing and the Omega scores, respectively. The criteria used for infections were those defined by the Centers for Disease Control. RESULTS: Of the 168 patients, 91 (54.2%) were female and 77 (45.8%) were male patients. The mean age of female and male was 64.9 ± 6.2 years and 63.1 ± 11.9 years, respectively. The mean duration of hospitalization in intensive care unit was 18.4 ± 6.1 days. Multiresistant bacteria were isolated from cultures of 39 (23.2%) patients. The development of MRB+ infection was correlated with length of stay, Omega 1, Omega 2, Omega 3, Total Omega, daily PRN, and Total PRN (p < 0.05). There was no correlation between development of MRB+ infection with gender, age and APACHE-II scores (p > 0.05). CONCLUSION: The risk of nosocomial infection development in an intensive care unit is directly correlated with increased nurse workload, applied intervention, and length of stay. Understaffing in the intensive care unit is an important health problem that especially affects care-needing patients. Nosocomial infection development has laid a heavy burden on the economy of many countries. To control nosocomial infection development in the intensive care unit, nurse workload, staffing level, and working conditions must be arranged. .

INTRODUÇÃO: As taxas de desenvolvimento de infecção ou colonização por bactérias multirresistentes (BMR+) em unidades de terapia intensiva são muito elevadas. O objetivo deste estudo foi determinar a possível associação entre o risco de desenvolvimento de infecções hospitalares e o aumento da carga de trabalho diária da equipe de enfermagem devido à insuficiência de pessoal em unidade de terapia intensiva. MÉTODOS: Cento e sessenta e oito pacientes foram incluídos. O volume da carga de trabalho e os procedimentos realizados em pacientes foram avaliados com o uso de instrumentos de medidas como o Projeto de Pesquisa em Enfermagem (Project de Recherché en Nursing) e o Omega, respectivamente. Os critérios usados para definir infecções foram os definidos pelos Centros de Controle de Doenças. RESULTADOS: Dos 168 pacientes, 91 (54,2%) eram do sexo feminino e 77 (45,8%) do sexo masculino. As médias das idades de mulheres e homens foram 64,9 ± 6,2 e 63,1 ± 11,9 anos, respectivamente. A média do tempo de internação em unidade de terapia intensiva foi de 18,4 ± 6,1 dias. As bactérias multirresistentes foram isoladas a partir de culturas de 39 (23,2%) pacientes. O desenvolvimento de infecção por BMR+ foi correlacionado com tempo de internação, Omega 1, Omega 2, Omega 3, Omega total, PPE diário e PPE total (p < 0,05). Não houve correlação entre desenvolvimento de infecção por BMR+ e gênero, idade e escores no APACHE-II (p > 0,05). CONCLUSÃO: O risco de desenvolvimento de infecção hospitalar em unidade de terapia intensiva está diretamente relacionado com o aumento da carga de trabalho de enfermagem, as intervenções praticadas e o tempo de internação. A falta de pessoal em unidade de terapia intensiva é um problema de saúde importante que afeta principalmente os pacientes que requerem cuidados. A infecção hospitalar colocou um fardo pesado sobre a economia de muitos países. Para controlar o desenvolvimento de infecção hospitalar em UTI, a carga ...

INTRODUÇÃO: as taxas de desenvolvimento de infecção ou colonização por bactérias multirresistentes [BMR (+)] em unidades de terapia intensiva são muito elevadas. O objetivo deste estudo foi determinar a possível associação entre o risco de desenvolvimento de infecções hospitalares e o aumento da carga de trabalho diária da equipe de enfermagem por causa da insuficiência de pessoal em unidade de terapia intensiva. MÉTODOS: foram incluídos 168 pacientes. O volume da carga de trabalho e os procedimentos feitos em pacientes foram avaliados com o uso de instrumentos de medidas como o Projeto de Pesquisa em Enfermagem (Project de Recherché en Nursing) e o Omega, respectivamente. Os critérios usados para definir infecções foram os estabelecidos pelos Centros de Controle de Doenças. RESULTADOS: dos 168 pacientes, 91 (54,2%) eram do sexo feminino e 77 (45,8%) do masculino. As médias das idades de mulheres e homens foram 64,9 ± 6,2 e 63,1 ± 11,9 anos, respectivamente. A média do tempo de internação em unidade de terapia intensiva foi de 18,4 ± 6,1 dias. As bactérias multirresistentes foram isoladas a partir de culturas de 39 (23,2%) pacientes. O desenvolvimento de infecção por BMR (+) foi correlacionado com tempo de internação, Omega 1, Omega 2, Omega 3, Omega total, PPE diário e PPE total (p < 0,05). Não houve correlação entre desenvolvimento de infecção por BMR (+) e gênero, idade e escores no Apache-II (p > 0,05). CONCLUSÃO: o risco de desenvolvimento de infecção hospitalar em unidade de terapia intensiva está diretamente relacionado com o aumento da carga de trabalho de enfermagem, as intervenções praticadas e o tempo de internação. A falta de pessoal em unidade de terapia intensiva é um problema de saúde importante que afeta principalmente os pacientes que requerem cuidados. A infecção hospitalar colocou um fardo pesado sobre a economia de muitos países. Para controlar o desenvolvimento de infecção hospitalar em UTI, a carga de trabalho ...

INTRODUCCIÓN: Las tasas de desarrollo de infección o colonización por bacterias multirresistentes en unidades de cuidados intensivos son muy elevadas. El objetivo de este estudio fue determinar la posible asociación entre el riesgo de desarrollo de infecciones hospitalarias y el aumento de la carga de trabajo diaria del equipo de enfermería debido a la falta de personal en la unidad de cuidados intensivos. MÉTODOS: Ciento sesenta y ocho pacientes fueron incluidos. El volumen de la carga de trabajo y los procedimientos realizados en pacientes fueron evaluados con el uso de instrumentos de medidas como el Proyecto de Investigación en Enfermería (Project de Recherché en Nursing) y el Omega, respectivamente. Los criterios usados para definir infecciones fueron los definidos por los Centros de Control de Enfermedades. RESULTADOS: De los 168 pacientes, 91 (54,2%) eran del sexo femenino y 77 (45,8%) del sexo masculino. La edad media de las mujeres y de los hombres fueron 64,9 ± 6,2 y 63,1 ± 11,9 años, respectivamente. El tiempo medio de ingreso en la unidad de cuidados intensivos fue de 18,4 ± 6,1 días. Las bacterias multirresistentes fueron aisladas a partir de cultivos de 39 (23,2%) pacientes. El desarrollo de infección por bacterias multirresistentes fue correlacionado con el tiempo de ingreso, Omega 1, Omega 2, Omega 3, Omega total, PPE diario y PPE total (p < 0,05). No hubo correlación entre el desarrollo de la infección por bacterias multirresistentes y el sexo, la edad y las puntuaciones en el APACHE-II (p > 0,05). CONCLUSIÓN: El riesgo de desarrollo de infección hospitalaria en una unidad de cuidados intensivos está directamente relacionado con el aumento de la carga de trabajo de enfermería, las intervenciones practicadas y el tiempo de ingreso. La falta de personal en la unidad de cuidados intensivos es un problema de sanidad importante que afecta principalmente a los pacientes que necesitan esos cuidados. La infección hospitalaria ...

MR-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition.

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF-7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS-detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF-7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized (13) C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3-kinase pathway and AMP-activated protein kinase. Our findings demonstrate the value of translatable non-invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition.

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania.

Miltefosine (MT) (hexadecylphosphocholine) was implemented to cope with resistance against antimonials, the classical treatment in Leishmaniasis. Given the scarcity of anti- Leishmania (L) drugs and the increasing appearance of resistance, there is an obvious need for understanding the mechanism of action and development of such resistance. Metabolomics is an increasingly popular tool in the life sciences due to it being a relatively fast and accurate technique that can be applied either with a particular focus or in a global manner to reveal new knowledge about biological systems. Three analytical platforms, gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) have been coupled to mass spectrometry (MS) to obtain a broad picture of metabolic changes in the parasite. Impairment of the polyamine metabolism from arginine (Arg) to trypanothione in susceptible parasites treated with MT was in some way expected, considering the reactive oxygen species (ROS) production described for MT. Importantly, in resistant parasites an increase in the levels of amino acids was the most outstanding feature, probably related to the adaptation of the resistant strain for its survival inside the parasitophorous vacuole.

Influence of calcium on choline measurements by 1H MR spectroscopy of thigh muscles.

OBJECTIVE: To study the effects of calcium on the choline peak measurements with 1H MR spectroscopy. MATERIAL AND METHODS: The thigh muscles of two cadaveric specimens were prospectively evaluated on a 3 T MR unit before and after the injection of calcium carbonate (up to 0.4322 g). The choline peaks of 147 spectra from 10 different anatomic locations were quantitatively evaluated. The influence of the calcium concentration and its disposition with respect to the main magnetic field were considered. B0 phase maps were used to evaluate field inhomogeneities. RESULTS: The presence of calcium led to a 43 % underestimation of the choline peak and the choline concentration (p = 0.0002 and 0.0036). The mean choline concentrations before and after CaCO3 injection were 3.53 ± 1.72 mmol/l and 1.58 ± 0.63 mmol/l. The influence of calcium carbonate on the choline peak estimations was proportional to the calcium concentration. There was a significant position-dependent difference in the estimation of the choline peak amplitude (p < 0.0154). Calcium injection led to a measurable increase in field inhomogeneities. CONCLUSION: There was a significant underestimation of the choline peak amplitude and concentration in the presence of calcium, which might cause misinterpretations of MR spectra. KEY POINTS: The presence of calcium led to significant underestimation of choline measurements. The influence of calcium is dependent on its concentration and distribution. Quantitative MR spectroscopy of calcified tumours should be interpreted with caution.

Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry.

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many "targeted" approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Thalamic metabolic abnormalities in patients with Huntington's disease measured by magnetic resonance spectroscopy

Huntington's disease (HD) is a neurologic disorder that is not completely understood; its fundamental physiological mechanisms and chemical effects remain somewhat unclear. Among these uncertainties, we can highlight information about the concentrations of brain metabolites, which have been widely discussed. Concentration differences in affected, compared to healthy, individuals could lead to the development of useful tools for evaluating the progression of disease, or to the advance of investigations of different/alternative treatments. The aim of this study was to compare the thalamic concentration of metabolites in HD patients and healthy individuals using magnetic resonance spectroscopy. We used a 2.0-Tesla magnetic field, repetition time of 1500 ms, and echo time of 135 ms. Spectra from 40 adult HD patients and 26 control subjects were compared. Quantitative analysis was performed using the LCModel method. There were statistically significant differences between HD patients and controls in the concentrations of N-acetylaspartate+N-acetylaspartylglutamate (NAA+NAAG; t-test, P<0.001), and glycerophosphocholine+phosphocholine (GPC+PCh; t-test, P=0.001) relative to creatine+phosphocreatine (Cr+PCr). The NAA+NAAG/Cr+PCr ratio was decreased by 9% and GPC+PCh/Cr+PCr increased by 17% in patients compared with controls. There were no correlations between the concentration ratios and clinical features. Although these results could be caused by T1 and T2 changes, rather than variations in metabolite concentrations given the short repetition time and long echo time values used, our findings point to thalamic dysfunction, corroborating prior evidence.

Direct electrospray ionization mass spectrometric profiling of real-world samples via a solid sampling probe.

This study presents a novel direct analysis strategy for rapid mass spectrometric profiling of biochemicals in real-world samples via a direct sampling probe (DSP) without sample pretreatments. Chemical modification is applied to a disposable stainless steel acupuncture needle to enhance its surface area and hydrophilicity. After insertion into real-world samples, biofluid can be attached on the DSP surface. With the presence of a high DC voltage and solvent vapor condensing on the tip of the DSP, analyte can be dissolved and electrosprayed. The simplicity in design, versatility in application aspects, and other advantages such as low cost and disposability make this new method a competitive tool for direct analysis of real-world samples.

Iron supplementation dose for perinatal iron deficiency differentially alters the neurochemistry of the frontal cortex and hippocampus in adult rats.

BACKGROUND: Long-term prefrontal cortex (PFC)- and hippocampus-based cognitive deficits are the sequelae of perinatal iron deficiency, despite iron supplementation starting in the newborn period. Whether high-dose iron supplementation prevents these deficits is yet to be determined. METHODS: Perinatal iron deficiency was induced in rat pups using a low-iron (3 mg/kg diet) diet during gestation until postnatal day (P)8. Iron was supplemented using a standard (40 mg/kg diet) or a 10-fold higher (400 mg/kg diet) iron-containing diet until P21. PFC and hippocampal neurochemistry was determined using in vivo (1)H nuclear magnetic resonance (NMR) spectroscopy at 9.4 Tesla on P90. RESULTS: Both standard and 10-fold higher iron supplementation doses corrected anemia and brain iron deficiency by P21. The neurochemical profile of the PFC in both supplementation groups was comparable with the control group. In the hippocampus, standard-dose iron supplementation resulted in lower concentrations of N-acetylaspartate (NAA) and phosphoethanolamine (PE) and higher concentrations of N-acetylaspartylglutamate (NAAG) and glycerophosphocholine + phosphocholine (GPC + PC). High-dose iron supplementation resulted in lower PE and higher GPC + PC concentrations. CONCLUSION: The iron supplementation dose for perinatal iron deficiency differentially alters the neurochemical profile of the PFC and hippocampus in adults. The neurochemical changes suggest altered glutamatergic neurotransmission, hypomyelination, and abnormal phospholipid metabolism in the formerly iron-deficient (FID) hippocampus.

Histone deacetylase inhibition increases levels of choline kinase α and phosphocholine facilitating noninvasive imaging in human cancers.

Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.

1H magnetic resonance spectroscopy metabolites as biomarkers for cell cycle arrest and cell death in rat glioma cells.

BACKGROUND: Improved non-invasive imaging biomarkers of treatment response contribute to optimising cancer management and metabolites detected by proton magnetic resonance spectroscopy ((1)H MRS) show promise in this area. Understanding (1)H MRS changes occurring in cells during cell stress and cell death in vitro should aid the selection of pertinent biomarkers for clinical use. METHODS: BT4C glioma cells in culture were exposed to either 50 µM cis-dichlorodiammineplatinum II (cisplatin) or starvation by culture in phosphate buffered saline. High resolution magic angle spinning (1)H MRS was performed on cells using a Varian 600 MHz nanoprobe and metabolites were quantified by a time domain fitting method. Cell viability was assessed by trypan blue, H&E, 4',6-diamino-2-phenylindole (DAPI), DNA laddering and annexin V-FITC labelled flow cytometry; propidium iodide flow cytometry was used to assess the cell cycle phase. RESULTS: With cisplatin exposure, cells initially accumulated in the G1 stage of the cell cycle with low numbers of apoptotic and necrotic cells and this was associated with decreases in phosphocholine, succinate, alanine, taurine, glycine and glutamate and increases in lactate and glycerophosphocholine (GPC). Starvation, leading to necrotic cell death within 6-18 h, caused decreases in succinate, alanine, glycine, and glutamate and increases in GPC. Principal component analysis revealed two patterns of metabolite changes, one common to both types of cell stress and another specific for necrosis secondary to cell starvation. CONCLUSIONS: (1)H MRS reveals alterations in multiple metabolites during cell cycle arrest and cell death which may provide early biomarker profiles of treatment efficacy in vivo.

Multimodal mass spectrometric imaging of small molecules reveals distinct spatio-molecular signatures in differentially metastatic breast tumor models.

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.

Mouse brain plasmalogens are targets for hypochlorous acid-mediated modification in vitro and in vivo.

Plasmalogens, 1-O-alk-1'-enyl-2-acyl-sn-glycerophospholipids, are significant constituents of cellular membranes and are essential for normal brain development. Plasmalogens, which contain a vinyl ether bond at the sn-1 position, are preferential targets for hypochlorous acid (HOCl), generated by myeloperoxidase (MPO) from H(2)O(2) and chloride ions. Because MPO is implicated in neurodegeneration, this study pursued two aims: (i) to investigate the reactivity of mouse brain plasmalogens toward HOCl in vitro and (ii) to obtain in vivo evidence for MPO-mediated brain plasmalogen modification. Liquid chromatography coupled to hybrid linear ion trap-Fourier transform-ion cyclotron resonance mass spectrometry revealed plasmalogen modification in mouse brain lipid extracts at lower HOCl concentrations as observed for diacylphospholipids, resulting in the generation of 2-chloro fatty aldehydes and lysophospholipids. Lysophosphatidylethanolamine accumulation was transient, whereas lysophosphatidylcholine species containing saturated acyl residues remained stable. In vivo, a single, systemic endotoxin injection resulted in upregulation of cerebral MPO mRNA levels to a range comparable to that observed for tumor necrosis factor-α and cyclooxygenase-2. This inflammatory response was accompanied by a significant decrease in several brain plasmalogen species and concomitant in vivo generation of 2-chlorohexadecanal. The present findings demonstrate that activation of the MPO-H(2)O(2)-chloride system under neuroinflammatory conditions results in oxidative attack of the total cerebral plasmalogen pool. As this lipid class is indispensable for normal neuronal function, HOCl-mediated plasmalogen modification is likely to compromise normal synaptic transmission.

Choline intake and genetic polymorphisms influence choline metabolite concentrations in human breast milk and plasma.

BACKGROUND: Choline is essential for infant nutrition, and breast milk is a rich source of this nutrient. Common single nucleotide polymorphisms (SNPs) change dietary requirements for choline intake. OBJECTIVE: The aim of this study was to determine whether total choline intake and/or SNPs influence concentrations of choline and its metabolites in human breast milk and plasma. DESIGN: We gave a total of 103 pregnant women supplemental choline or a placebo from 18 wk gestation to 45 d postpartum and genotyped the women for 370 common SNPs. At 45 d postpartum, we measured choline metabolite concentrations in breast milk and plasma and assessed the dietary intake of choline by using a 3-d food record. RESULTS: On average, lactating women in our study ate two-thirds of the recommended intake for choline (Adequate Intake = 550 mg choline/d). Dietary choline intake (no supplement) correlated with breast-milk phosphatidylcholine and plasma choline concentrations. A supplement further increased breast-milk choline, betaine, and phosphocholine concentrations and increased plasma choline and betaine concentrations. We identified 5 SNPs in MTHFR that altered the slope of the intake-metabolite concentration relations, and we identified 2 SNPs in PEMT that shifted these curves upward. Individuals who shared sets of common SNPs were outliers in plots of intake-metabolite concentration curves; we suggest that these SNPs should be further investigated to determine how they alter choline metabolism. CONCLUSION: Total intake of choline and genotype can influence the concentrations of choline and its metabolites in the breast milk and blood of lactating women and thereby affect the amount of choline available to the developing infant. This study was registered at clinicaltrials.gov as NCT00678925.

Analytical approaches toward successful human cell metabolome studies by NMR spectroscopy.

The aim of this work was to investigate the effects of cell handling and storage on cell integrity and (1)H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was approximately 70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation- and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The (1)H HRMAS spectra of the three cell types are compared and discussed.

Quantification of choline- and ethanolamine-containing metabolites in human prostate tissues using 1H HR-MAS total correlation spectroscopy.

A fast and quantitative 2D high-resolution magic angle spinning (HR-MAS) total correlation spectroscopy (TOCSY) experiment was developed to resolve and quantify the choline- and ethanolamine-containing metabolites in human prostate tissues in approximately 1 hr prior to pathologic analysis. At a 40-ms mixing time, magnetization transfer efficiency constants were empirically determined in solution and used to calculate metabolite concentrations in tissue. Phosphocholine (PC) was observed in 11/15 (73%) cancer tissues but only 6/32 (19%) benign tissues. PC was significantly higher (0.39 +/- 0.40 mmol/kg vs. 0.02 +/- 0.07 mmol/kg, z = 3.5), while ethanolamine (Eth) was significantly lower in cancer versus benign prostate tissues (1.0 +/- 0.8 mmol/kg vs. 2.3 +/- 1.9 mmol/kg, z = 3.3). Glycerophosphocholine (GPC) (0.57 +/- 0.87 mmol/kg vs. 0.29 +/- 0.26 mmol/kg, z = 1.2), phosphoethanolamine (PE) (4.4 +/- 2.2 mmol/kg vs. 3.4 +/- 2.6 mmol/kg, z = 1.4), and glycerophosphoethanolamine (GPE) (0.54 +/- 0.82 mmol/kg vs. 0.15 +/- 0.15 mmol/kg, z = 1.8) were higher in cancer versus benign prostate tissues. The ratios of PC/GPC (3.5 +/- 4.5 vs. 0.32 +/- 1.4, z = 2.6), PC/PE (0.08 +/- 0.08 vs. 0.01 +/- 0.03, z = 3.5), PE/Eth (16 +/- 22 vs. 2.2 +/- 2.0, z = 2.4), and GPE/Eth (0.41 +/- 0.51 vs. 0.06 +/- 0.06, z = 2.6) were also significantly higher in cancer versus benign tissues. All samples were pathologically interpretable following HR-MAS analysis; however, degradation experiments showed that PC, GPC, PE, and GPE decreased 7.7 +/- 2.2%, while Cho+mI and Eth increased 18% in 1 hr at 1 degrees C and a 2250 Hz spin rate.