Perchloric Acid Extracts Research Articles

Assessment of the intrinsic radiosensitivity of glioma cells and monitoring of metabolite ratio changes after irradiation by 14.7-T high-resolution1H MRS

Gliomas are the most common type of primary brain tumor. Radiation therapy (RT) is the primary adjuvant treatment to eliminate residual tumor tissue after surgery. However, the current RT guided by conventional imaging is unsatisfactory. A fundamental question is whether it is possible to further enhance the effectiveness and efficiency of RT based on individual radiosensitivity. In this research, to probe the correlation between radiosensitivity and the metabolite characteristics of glioma cells in vitro, a perchloric acid (PCA) extracting method was used to obtain water-soluble metabolites [such as N-acetylaspartate (NAA), choline (Cho), creatine (Cr) and succinate (Suc)]. Spectral patterns from these processed water-soluble metabolite samples were acquired by in vitro 14.7-T high-resolution ¹H MRS. Survival fraction analysis was performed to test the intrinsic radiosensitivity of glioma cell lines. Good ¹H MRS of PCA extracts from glioma cells was obtained. The radiosensitivity of glioma cells correlated positively with the Cho/Cr and Cho/NAA ratios, but negatively with the Suc/Cr ratio. Irradiation of the C6 cell line at different X-ray dosages led to changes in metabolite ratios and apoptotic rates. A plateau phase of metabolite ratio change and a decrease in apoptotic rate were found in the C6 cell line. We conclude that in vitro high-resolution ¹H MRS possesses the sensitivity required to detect subtle biochemical changes at the cellular level. ¹H MRS may aid in the assessment of the individual radiosensitivity of brain tumors, which is pivotal in the identification of the biological target volume.

Recovery correction technique for NMR spectroscopy of perchloric acid extracts using DL-valine-2,3-d2: validation and application to 5-fluorouracil-induced brain damage.

We explored a recovery correction technique that can correct metabolite loss during perchloric acid (PCA) extraction and minimize inter-assay variance in quantitative (1)H nuclear magnetic resonance (NMR) spectroscopy of the brain and evaluated its efficacy in 5-fluorouracil (5-FU)- and saline-administered rats. We measured the recovery of creatine and dl-valine-2,3-d2 from PCA extract containing both compounds (0.5 to 8 mM). We intravenously administered either 5-FU for 4 days (total, 100 mg/kg body weight) or saline into 2 groups of 11 rats each. We subsequently performed PCA extraction of the whole brain on Day 9, externally adding 7 µmol of dl-valine-2,3-d2. We estimated metabolite concentrations using an NMR spectrometer with recovery correction, correcting metabolite concentrations based on the recovery factor of dl-valine-2,3-d2. For each metabolite concentration, we calculated the coefficient of variation (CEV) and compared differences between the 2 groups using unpaired t-test. Equivalent recoveries of dl-valine-2,3-d2 (89.4 ± 3.9%) and creatine (89.7 ± 3.9%) in the PCA extract of the mixed solution indicated the suitability of dl-valine-2,3-d2 as an internal reference. In the rat study, recovery of dl-valine-2,3-d2 was 90.6 ± 9.2%. Nine major metabolite concentrations adjusted by recovery of dl-valine-2,3-d2 in saline-administered rats were comparable to data in the literature. CEVs of these metabolites were reduced from 10 to 17% before to 7 to 16% after correction. The significance of differences in alanine and taurine between the 5-FU- and saline-administered groups was determined only after recovery correction (0.75 ± 0.12 versus 0.86 ± 0.07 for alanine; 5.17 ± 0.59 versus 5.66 ± 0.42 for taurine [µmol/g brain tissue]; P < 0.05). A new recovery correction technique corrected metabolite loss during PCA extraction, minimized inter-assay variance in quantitative (1)H NMR spectroscopy of brain tissue, and effectively detected inter-group differences in concentrations of brain metabolites between 5-FU- and saline-administered rats.

Fructose–Maltodextrin Ratio Governs Exogenous and Other CHO Oxidation and Performance

Fructose coingested with glucose in carbohydrate (CHO) drinks increases exogenous-CHO oxidation, gut comfort, and physical performance. This study aimed to determine the effect of different fructose-maltodextrin-glucose ratios on CHO oxidation and fluid absorption while controlling for osmolality and caloricity. In a crossover design, 12 male cyclists rode 2 h at 57% peak power then performed 10 sprints while ingesting artificially sweetened water or three equiosmotic 11.25% CHO-salt drinks at 200 mL·15 min, comprising weighed fructose and maltodextrin-glucose in ratios of 0.5:1 (0.5 ratio), 0.8:1 (0.8 ratio), and 1.25:1 (1.25 ratio). Fluid absorption was traced with D2O, whereas C-fructose and C-maltodextrin-glucose permitted fructose and glucose oxidation rate evaluation. The mean exogenous-fructose and exogenous-glucose oxidation rates were 0.27, 0.39, and 0.46 g·min and 0.65, 0.71, and 0.58 g·min in 0.5, 0.8, and 1.25 ratio drinks, representing mean oxidation efficiencies of 54%, 59%, and 55% and 65%, 85%, and 86% for fructose and glucose, respectively. With the 0.8 ratio drink, total exogenous-CHO oxidation rate was 18% (90% confidence interval, ±5%) and 5.2% (±4.6%) higher relative to 0.5 and 1.25 ratios, respectively, whereas respective differences in total exogenous-CHO oxidation efficiency were 17% (±5%) and 5.3% (±4.8%), associated with 8.6% and 7.8% (±4.2%) higher fructose oxidation efficiency. The effects of CHO ratio on water absorption were inconclusive. Mean sprint power with the 0.8 ratio drink was moderately higher than that with the 0.5 ratio (2.9%; 99% confidence interval, ±2.8%) and 1.25 ratio (3.1%; ±2.7%) drinks, with total- and endogenous-CHO oxidation rate, abdominal cramps, and drink sweetness qualifying as explanatory mechanisms. Enhanced high-intensity endurance performance with a 0.8 ratio fructose-maltodextrin-glucose drink is characterized by higher exogenous-CHO oxidation efficiency and reduced endogenous-CHO oxidation. The gut-hepatic or other physiological site responsible requires further research.

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

Elaeis guineensis: Adenosine phosphates and nicotinamides involvement in fatty acid biosynthesis

Adenosine phosphates (ATP, ADP, AMP) and nicotinamides (NAD, NADH, NADP and NADPH) are nucleotides that are involved in various plant biosynthetic pathways such as photosynthesis, nitrogen uptake, purine metabolism and lipid biosynthesis. In oil palm fruit formation, kernel and mesocarp are separated by a shell and differ mainly by lipid composition. Oil palm mesocarp and kernel tissues were extracted using a modified perchloric acid extraction and separated via HPLC to quantify the accumulation of these seven nucleotides in relation to lipid composition. Principal component analysis on palm mesocarp and kernel samples displayed clustering and indicated that palm mesocarp contained higher ATP and NAD+ than palm kernel. The higher levels of ATP and NADH may be attributable to the higher content of unsaturated and long chain fatty acids in found palm mesocarp.

Point-of-Care Glucose Analysis in Neonates Using Modified Quinoprotein Glucose Dehydrogenase

Asymptomatic hypoglycemia in neonates may contribute to neurologic deficits during development. Whole-blood glucose sensors are often imprecise and inaccurate at the low glucose concentrations found in neonates. In this study, a glucose sensor using a mutated glucose dehydrogenase that does not cross-react significantly with maltose was evaluated at three pediatric centers. Blood samples (n=575) from infants less than 30 days of age (hematocrit 23-70%) were analyzed using six reagent lots on three ACCU-CHEK(®) meters (Roche Diagnostics, Indianapolis, IN): the Inform II, Performa, and Aviva. Reference glucose level was determined in duplicate in perchloric acid extracts using a coupled hexokinase procedure. Imprecision of glucose measurement using stable control materials ranged from 2.0% to 3.1% (coefficient of variation) using the glucose meters and from 0.8% to 5.3% (coefficient of variation) in perchloric acid-treated controls. The difference between meter glucose values and reference values showed a slight dependence on hematocrit from 23% to 70% (r=-0.391, P<0.001) but not in the typical range of neonatal hematocrit from 45% to 70% (r=-0.036, P=0.239). Linear regression of the aggregated results yielded the following relationship: Meter glucose=0.99×Reference Glucose+0.04; r(2)=0.976; Syx=0.249. Receiver-operator characteristic analysis of the data using 2.2 mmol/L as the reference threshold for hypoglycemia yielded an area under the curve value of 0.993. All infants with a glucose level of <2.2 mmol/L were detected (100% sensitivity) when the meter glucose value was below 2.8 mmol/L. These data indicate that the modified ACCU-CHEK chemistry may be used effectively in neonatal settings to detect clinically significant hypoglycemia.

Effects of lovastatin treatment on the metabolic distributions in the Han:SPRD rat model of polycystic kidney disease

BackgroundWe previously demonstrated that lovastatin decreases cyst volume and improves kidney function in the Han:SPRD (Cy/+) rat model of ADPKD. Since endothelial dysfunction and inflammatory activity are evident in patients with ADPKD, we investigated whether lovastatin reduces the inflammation and vascular dysfunction and improves kidney cell energy metabolism of Cy/+ rats.MethodsCy/+ and normal littermate control animals (+/+) were treated with either lovastatin (4 mg/kg/day) or vehicle (ethanol) from 3–8 weeks of age. 1H-NMR analysis was performed on water-soluble and lipid kidney fractions following perchloric acid extraction. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess endothelial dysfunction, oxidative stress and inflammation markers in plasma and kidney tissue extracts.ResultsCy/+ rats showed perturbations in fatty acid metabolism and increased synthesis of pro-inflammatory lipoxygenases-produced bioactive lipids was observed. Lovastatin decreased inflammatory markers, specifically 13-HODE, 12-HETE and leukotriene B4. In Cy/+ rats, lovastatin reduced the elevated homocysteine and allantoin plasma levels and increased arginine, that is known to positively affect NO production.In terms of kidney cell metabolism, Cy/+ rats showed reduced Krebs cycle activity. Treatment with lovastatin increased the Krebs cycle activity as well as the glycolytical lactate production, thus improving the overall energy state of the cystic kidney.ConclusionAs previously described, lovastatin was able to decrease kidney weight and cyst volume density in Cy/+ rats. The decrease in cyst volume was accompanied by a reduction in arachidonic acid-mediated inflammation markers, the normalization of metabolism of NO precursors and the improvement of kidney energy cell metabolism.

Validation of an HPLC Methodology for the Identification and Quantification of Biogenic Amines in Chicken Meat

This study validated a high performance liquid chromatography (HPLC) method to determine biogenic amines in chicken meat. For the identification of biogenic amines, an isocratic elution system coupled with a UV detector (254 nm) was used after a perchloric acid (5 %) extraction and benzoyl chloride derivatization of the samples. The standards of tyramine, putrescine, cadaverine, spermidine, and spermine were used for the following validation parameters: selectivity, linearity, accuracy, recovery, limit of detection and quantification, and robustness. Finally, chicken meat commercialized in two types of packaging was evaluated. The results showed an excellent selectivity and separation of all amines, r 2 > 0.99, relative standard deviation <5 %, recovery between 64 and 112 %, limits of detection and quantification between 0.03–1.25 and 0.15–5.00 μg L−1, respectively, and appropriate robustness for the proposed methodology. Moreover, both chicken meat commercial packages had similar values for all amines; only tyramine was significantly different (P ≤ 0.05). The proposed method was suitable to detection and quantification of simultaneous five biogenic amines in chicken meat.

Myocardial calcium overload during graded hypothermia and after rewarming in an in vivo rat model

Mechanisms underlying cardiac contractile dysfunction during and after rewarming from hypothermia remain largely unknown. We have previously reported myocardial post-hypothermic calcium overload to be the culprit. The aim of the present study was to measure changes in myocardial [Ca(2+) ](i) during graded hypothermia and after rewarming in an anesthetized, intact rat model, using the (45) Ca(2+) technique. Rats were randomized and cooled to 15 °C. Hearts were excised and perfusion-washed to remove extracellular calcium after 0.5 h of hypothermia (n = 9), 4 h of hypothermia (n = 8), and after 4 h of hypothermia and 2 h rewarming (n = 9). A normothermic group, kept at 37 °C for 5 h, served as control (n = 6). [Ca(2+) ](i) was determined in perchloric acid extracts of heart tissue. Spontaneous cardiac electromechanic work was maintained during hypothermia without cardiac arrest or ischaemia. Between 0.5 and 4 h at 15 °C, a six-fold increase in cardiac [Ca(2+) ](i) was observed (0.55 ± 0.10 vs. 2.93 ± 0.76 μmol (g dry wt)(-1) ). Rewarming resulted in a 33% decline in [Ca(2+) ](i) , but the actual value was significantly above the value measured in control hearts. We show that calcium overload is a characteristic feature of the beating heart during deep hypothermia, which aggravates by increasing duration of exposure. The relatively low decline in [Ca(2+) ](i) during the rewarming period indicates difficulties in recovering calcium homoeostasis, which in turn may explain cardiac contractile dysfunction observed after rewarming.

Lactate detection in inducible and orthotopic Her2/neu mammary gland tumours in mouse models

This study compared the steady state concentration of lactate in an inducible Her2/nue transgenic breast cancer mouse model and in tumours from the same Her2/neu model grown orthotopically. In vivo lactate was detected by MRS using the Hadamard encoded selective multiple quantum coherence pulse sequence (HadSelMQC) recently developed by our laboratory. A lower lactate signal was observed in the inducible tumours compared to orthotopic tumours in vivo, while ex vivo analysis of perchloric acid extracts revealed similar amounts of this metabolite in both models. Histological staining of mammary tumour specimens showed a much higher level of fat tissue in inducible tumours compared to the orthotopic model. Phantom studies with [3-(13) C] lactate indicated that a lipid environment could significantly reduce the T2 of lactate and impede its detection. The transgenic inducible model for breast cancer not only better recapitulated the biological aspects of the human disease but also provided additional characteristics related to in vivo detection of lactate that are not available in orthotopic or xenograft models. This study suggests that the level of lactate measured by the HadSelMQC pulse sequence may be underestimated in human patients in the presence of high lipid levels that are typically encountered in the breast.

Human umbilical mesenchymal stem cell and its adipogenic differentiation: Profiling by nuclear magnetic resonance spectroscopy

To study the metabolic profile of human umbilical mesenchymal stem cells (HUMSC) and adipogenic differentiation by nuclear magnetic resonance (NMR) spectroscopy. HUMSC isolated from human umbilical cord stroma were induced to adipocytes over 2 wk by adding dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin to the culture medium. Adipogenic differentiation was confirmed by Red O staining and transcription-polymerase chain reaction. Perchloric acid extracts of the HUMSCs and adipocytes (about 7 × 10(6)) were characterized for metabolites by using in vitro high resolution 9.4T NMR spectroscopy. Several major metabolites, such as: choline, creatine, glutamate and myo-inositol, acetate, and some fatty acids/triglycerides, were observed in the MR spectroscopic pattern of HUMSCs and their adipogenic differentiation. HUMSCs are characterized by an unusually low number of NMR-detectable metabolites, high choline, acetate, glutamate and creatine content. However, the metabolic profiles of adipogenic differentiation demonstrated considerably higher methionine and fatty acids, and non-detectable creatine. The biomarkers of HUMSCS and adipocytes were obtained and assigned. NMR spectroscopy will be a promising tool for monitoring stem cell differentiation.

Non-invasive grading of astrocytic tumours from the relative contents of myo-inositol and glycine measured by in vivo mrs

MRI and MRS are established methodologies for evaluating intracranial lesions. One MR spectral feature suggested for in vivo grading of astrocytic tumours is the apparent myo-lnositol (ml) intensity (ca 3.55 ppm) at short echo times, although glycine (gly) may also contribute in vivo to this resonance. The purpose of this study was to quantitatively evaluate the ml + gly contribution to the recorded spectral pattern in vivo and correlate it with in vitro data obtained from perchloric acid extraction of tumour biopsies. Patient spectra (n = 95) at 1.5T at short (20-31 ms) and long (135-136 ms) echo times were obtained from the INTERPRET MRS database (http://gabrmn.uab.eslinterpretvalidateddbl). Phantom spectra were acquired with a comparable protocol. Spectra were automatically processed and the ratios of the (ml + gly) to Cr peak heights ((ml + gly)/Cr) calculated. Perchloric acid extracts of brain tumour biopsies were analysed by high-resolution NMR at 9.4T. The ratio (ml + gly)/Cr decreased significantly with astrocytic grade in vivo between low-grade astrocytoma (A2) and glioblastoma multiforme (GBM). In vitro results displayed a somewhat different tendency, with anaplastic astrocytomas having significantly higher (ml + gly)/Cr than A2 and GBM. The discrepancy between in vivo and in vitro data suggests that the NMR visibility of glycine in glial brain tumours is restricted in vivo.

The X Protein of Hepatitis B Virus Inhibits Apoptosis in Hepatoma Cells through Enhancing the Methionine Adenosyltransferase 2A Gene Expression and Reducing S-Adenosylmethionine Production

The X protein (HBx) of hepatitis B virus (HBV) is involved in the development of hepatocellular carcinoma (HCC), and methionine adenosyltransferase 2A (MAT2A) promotes the growth of liver cancer cells through altering S-adenosylmethionine homeostasis. Thus, we speculated that a link between HBx and MAT2A may contribute to HCC development. In this study, the effects of HBx on MAT2A expression and cell apoptosis were investigated, and the molecular mechanism by which HBx and MAT2A regulate tumorigenesis was evaluated. Results from immunohistochemistry analyses of 37 pairs of HBV-associated liver cancer tissues/corresponding peritumor tissues showed that HBx and MAT2A are highly expressed in most liver tumor tissues. Our in vitro results revealed that HBx activates MAT2A expression in a dose-dependent manner in hepatoma cells, and such regulation requires the cis-regulatory elements NF-κB and CREB on the MAT2A gene promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) further demonstrated that HBx facilitates the binding of NF-κB and CREB to MAT2A gene promoter. In addition, overexpression of HBx or MAT2A inhibits cell apoptosis, whereas knockdown of MAT2A expression stimulates apoptosis in hepatoma cells. Furthermore, we demonstrated that HBx reduces MAT1A expression and AdoMet production but enhances MAT2β expression. Thus, we proposed that HBx activates MAT2A expression through NF-κB and CREB signaling pathways to reduce AdoMet production, inhibit hepatoma cell apoptosis, and perhaps enhance HCC development. These findings should provide new insights into our understanding how the molecular mechanisms underline the effects of HBV infection on the production of MAT2A and the development of HCC.

Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue

Fatigue was studied in intact tibialis anterior muscle of anesthetized mice. The distal tendon was detached and connected to a force transducer while blood flow continued normally. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100-Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5% of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4% after 4 min. Intracellular phosphate concentration ([P(i)]) was measured by (31)P-NMR on perchloric acid extracts of muscles. [P(i)] increased during fatigue from 7.6 ± 1.7 to 16.0 ± 1.6 mmol/kg muscle wet wt and returned to control during recovery. Intracellular Ca(2+) was measured with cameleons whose plasmids had been transfected in the muscle 2 wk before the experiment. Yellow cameleon 2 was used to measure myoplasmic Ca(2+), and D1ER was used to measure sarcoplasmic reticulum (SR) Ca(2+). The myoplasmic Ca(2+) during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR Ca(2+) also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [P(i)] and a reduction in the tetanic myoplasmic Ca(2+). We suggest that both changes contribute to the fatigue. A likely cause of the decline in tetanic myoplasmic Ca(2+) is precipitation of CaP(i) in the SR.

Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Role of Peroxisome Proliferator-activated Receptor δ/β in Hepatic Metabolic Regulation

Pharmacological activation of peroxisome proliferator-activated receptor δ/β (PPARδ/β) improves glucose handling and insulin sensitivity. The target tissues of drug actions remain unclear. We demonstrate here that adenovirus-mediated liver-restricted PPARδ activation reduces fasting glucose levels in chow- and high fat-fed mice. This effect is accompanied by hepatic glycogen and lipid deposition as well as up-regulation of glucose utilization and de novo lipogenesis pathways. Promoter analyses indicate that PPARδ regulates hepatic metabolic programs through both direct and indirect transcriptional mechanisms partly mediated by its co-activator, PPARγ co-activator-1β. Assessment of the lipid composition reveals that PPARδ increases the production of monounsaturated fatty acids, which are PPAR activators, and reduces that of saturated FAs. Despite the increased lipid accumulation, adeno-PPARδ-infected livers exhibit less damage and show a reduction in JNK stress signaling, suggesting that PPARδ-regulated lipogenic program may protect against lipotoxicity. The altered substrate utilization by PPARδ also results in a secondary effect on AMP-activated protein kinase activation, which likely contributes to the glucose-lowering activity. Collectively, our data suggest that PPARδ controls hepatic energy substrate homeostasis by coordinated regulation of glucose and fatty acid metabolism, which provide a molecular basis for developing PPARδ agonists to manage hyperglycemia and insulin resistance.

Metabolomic investigations of American oysters using H-NMR spectroscopy.

The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding 1H NMR peak assignments for future NMR-based metabolomic studies in oysters.

Abstract 1802: Correlating flow-cytometric and 19F-NMR changes in fluorodeoxyglucose signal in response to the Raf-inhibitor RAF265

Abstract FDG-PET (2-Deoxy-2-fluoro-D-glucose-Positron-Emission-Tomography) is an established tool in the management of cancer patients and is increasingly used as an early response marker. Interpreting the cellular basis of the FDG signal however, remains a challenge. The aim of this study was to explore changes in FDG signal by leveraging the quantitative and chemical specificity of 19F-NMR (nuclear magnetic resonance) and correlating these with flow-cytometric (FC) measurement of cell count, apoptosis and cell cycle in A375M melanoma cells treated with the investigational Raf-kinase inhibitor RAF265. Cell culture and flow cytometry: A375M cells (melanoma cell line expressing B-RafV600E) were grown in Eagle's Minimal Essential Media with 10% HI FCS (Hyclone), penicillin 100U/ml and streptomycin 100μg/ml. Cells were incubated in the presence of 1μM RAF265 or a 1:1000 dilution of DMSO as a control. After 24 hours 1.5 mM FDG was added to the culture media followed two hours later by a perchloric acid extraction of trypsinized cells. The neutralized lysate was analyzed via 19F-NMR. FC measurements were performed on trypsinized cells using a B-D Canto II with Diva software and analyzed with FlowJo Analysis software and Dean-Jett-Fox cell cycle modeling software. 19F-NMR: All experiments were performed at room temperature on a Bruker 300 MHz DPX spectrometer equipped with a QNP probe. All samples were doped with D2O and 5-fluorouracil as the internal 19F-reference. A non-1H-decoupled sequence was used to collect and quantitate spectra. Spectral assignments were based on prior reports. The spectra and FC results differed qualitatively and quantitatively between groups. Spectral 19F-NMR resonances consistent with phosphorylated FDG metabolites were identified. The mean quantity of FDG metabolites in the treated group was 113.8 nmol ± 3.06 (SD) and 281.3 nmol ± 1.34 (SD) in the untreated group, representing a significant (p&amp;lt;0.001) 59.5% difference. FC results indicated that RAF265 treatment resulted in a mean increase of 32% in the G0/G1 population and a decrease of 41% and 40% in the percentage of cells in the S and G2/M phases respectively. Correspondingly, there was a 27% decrease in the cell count between the treated and untreated groups. There was a four-fold increase in the proportion of Annexin-V staining cells. In summary, we have identified and quantified FDG metabolites in cell extracts using 19FNMR following treatment with the investigational Raf kinase inhibitor RAF265. There was a qualitative and quantitative drop in FDG metabolites suggesting a modulation in the metabolism of FDG. The corresponding changes in cell cycle and number in the drug-treated samples suggest a less metabolically active profile. By inference, FDG is expected to behave similarly in the clinic and therefore this technique, subject to validation, could provide a cellular basis for the interpretation of response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1802.

Changes in the NMR metabolic profile of human microglial cells exposed to lipopolysaccharide or morphine.

Microglial cells play a major role in host defense of the central nervous system. Once activated, several functional properties are up-regulated including migration, phagocytosis, and secretion of inflammatory mediators such as cytokines and chemokines. Little, if anything, is known about the metabolic changes that occur during the activation process. High-resolution (1)H nuclear magnetic resonance spectra obtained from perchloric acid extracts of human microglial cell cultures exposed to lipopolysaccharide (LPS) or morphine were used to both identify and quantify the metabolites. We found that human microglia exposed to LPS had increased concentrations of glutamate and lactate, whereas the cells exposed to morphine had decreased concentrations in creatinine, taurine, and thymine. Glutamate and creatinine were the key metabolites differentiating between the two stimuli. These results are discussed in terms of activation and differences in the inflammatory response of human microglial cells to LPS and morphine.

Increased vigabatrin entry into the brain by polysorbate 80 and sodium caprate.

The effects of a non-ionic surfactant, polysorbate 80, and the sodium salt of the saturated fatty acid, sodium caprate (C10), as potential brain absorption enhancers for vigabatrin were studied. Vigabatrin is an enzyme-activated irreversible inhibitor of gamma-aminobutyric acid (GABA) transaminase that increases brain and cerebrospinal GABA concentrations in animals and man. Before intravenous administration, a range of concentrations of the surfactants were tested using erythrocyte lysis or the red blood cell lysis test to establish the non-toxic concentration range. Vigabatrin was dissolved in 0.1% polysorbate 80 and 0.1% sodium caprate and administered intravenously in doses of 4 mL kg(-1) to male Wistar rats (230-250 g; n = 3). Rats were killed 2 h after drug and surfactant administration and the brains were immediately removed and homogenized in 0.4 M perchloric acid. Selected ion monitoring electrospray mass spectrometry was used to determine the concentration of vigabatrin and GABA directly from the perchloric acid extract of the rat brain. This method was developed to increase the speed and efficiency of the analysis by removing the need for complex extraction and derivatization procedures while retaining the specificity of the mass spectrometer as a detector. The stability of both vigabatrin and GABA in perchloric acid was established by monitoring their pseudo molecular ions in standard solutions at timed intervals over 24 h. Although the detection level for vigabatrin and GABA was at least 50 pg, only GABA was detected in rat brain. Vigabatrin caused a small increase in whole brain GABA. However, GABA levels were higher in the samples with vigabatrin + enhancer than in the samples where vigabatrin alone was administered. One-way analysis of variance indicated a significant effect of the surfactants on GABA levels (F (5,17) = 11.86, P < 0.01) and vigabatrin absorption was presumed. The rectal temperature of the rats is lowered by the presence of vigabatrin in the brain. Vigabatrin alone decreased rectal temperature by 6%. When given with either polysorbate 80 or sodium caprate, the extent of temperature lowering was significantly greater (P < 0.001). There was no significant difference after 2 h between polysorbate 80 + vigabatrin, and sodium caprate + vigabatrin.