Perchloric Extracts Research Articles

1 H NMR metabolomic profiling elucidated attenuation of neurometabolic alterations by lercanidipine in MCAo model in rats.

Stroke is a leading cause of death and disability worldwide with limited therapeutic interventions. The current study explored proton nuclear magnetic resonance spectroscopy (1 H NMR)-based metabolomic approach to elucidate the effect of lercanidipine on neurometabolic alterations in transient model of ischaemic stroke in rats. In the present investigation, male Wistar rats were subjected to middle cerebral artery occlusion (MCAo) for 2h followed by reperfusion using intraluminal filament method. Rats were randomly divided into three groups as vehicle-treated sham control, vehicle-treated MCAo control and lercanidipine-treated MCAo. Vehicle or lercanidipine (0.5mg/kg, i.p.) was administered 120min post-reperfusion. The rat brain cortex tissues were isolated 24h post-MCAo and were investigated by 1 H NMR spectroscopy through perchloric extraction method. A total of 23 metabolites were altered significantly after cerebral ischaemic-reperfusion injury in MCAo control as compared to sham control rats. Lercanidipine significantly reduced the levels of valine, alanine, lactate, acetate and tyrosine, while N-acetylaspartate, glutamate, glutamine, aspartate, creatine/phosphocreatine, choline, glycerophosphorylcholine, taurine, myo-inositol and adenosine di-phosphate were elevated as compared to MCAo control. Present study illustrates effect of lercanidipine on neurometabolic alterations which might be mediated through its antioxidant, anti-inflammatory, vasodilatory and anti-apoptotic property in MCAo model of stroke.

Suitability of flavan‐3‐ol analysis to differentiate grapes from Vranac, Kratošija and Cabernet Sauvignon ( Vitis vinifera L.) grown in Montenegro

Background and Aims Interest in the production of wines from indigenous cultivars such as Vranac and Kratošija is steadily increasing, since they offer distinct characteristics. In contrast to popular cultivars, little is known about their chemical composition, especially in Montenegro. Many important characteristics of red wines are related to phenolic substances. Therefore, it is imperative to determine the phenolic composition of these cultivars. Methods and Results Flavan-3-ol monomers of seeds were analysed by reversed phase HPLC after thioacidolysis. In the perchloric extract of skins, flavan-3-ol monomers and dimers were determined by HPLC. Catechin represented the most abundant flavan-3-ol in seeds, with a high concentration of epicatechin-3-O-gallate also found in Vranac. In the skins, procyanidin B1 and catechin were the most abundant phenolic substances. Using multivariate statistics, a unique profile of the ratio of flavan-3-ols from each cultivar was found. Conclusions Cultivar and geographical origin considerably influenced the flavan-3-ol composition of the three cultivars. The flavan-3-ol concentration of berries exhibited characteristic varietal differences, with the order of concentration being Cabernet Sauvignon > Kratošija > Vranac. The indigenous Montenegrin cultivars had a significantly lower proportion of seed and skin mass than that of Cabernet Sauvignon, which resulted in a lower flavan-3-ol concentration in the berries. Significance of the Study The flavan-3-ol concentration of grapes from Vranac, Kratošija and Cabernet Sauvignon cultivars grown in Montenegro was determined for the first time. These results further broaden knowledge relating to characterisation of wine, optimisation of its production and authenticity control.

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism.

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

Importance of Contaminated Soils in Supplying Bioaccessible Fluoride to Grazing Animals From the Historic Metalliferous Mining Areas of the UK

Soil-plant-animal and soil-animal pathways are the principal routes through which trace element e.g fluorine (F) enters the animal body systems. It is believed that soils and herbage contaminated with such trace elements may, eventually, reflect in the bones and other animal tissues. However, the correlationship between soil F and Bone F among grazing animals has not been substantially, established. This study aimed at investigating the association between F concentration in soil to those found in the bones of sheep and cattle reared in metalliferous mining areas of the United Kingdom. The study area included Derbyshire, a site of fluorite (CaF2) mineralization; Ceredigion and Mendips, sites of mostly galena (PbS) mineralization, the latter two sites used as control sites for this study. The analytical approach involved alkali fusion, perchloric acid digestion and sequential extraction procedures in determining total soil F, total bone F and soil bioavailable F, respectively. The spectrophotometric technique was then used to determine soil F from solution extracts. The results showed mean total soil F concentrations of 302.3 mg/kg, 175.4 mg/kg and 70.8 mg/kg in Derbyshire, Mendips and Ceredigion respectively. The same order was observed for bone F with as high as 218.3 mg/kg, 118.1 mg/kg and 88.9 mg/kg found in Derbsyhire, Mendips and Ceredigion respectively. Analysis of Spearman rank coefficients established that there is a moderate association between soil bioavailable F and bone F (rs=0.571), significant at p < 0.1; a conclusion suggesting possible high risk from F on animals grazing within heavily contaminated areas affected by historical F mining.

Quantification of methylone and metabolites in rat and human plasma by liquid chromatography-tandem mass spectrometry

Methylone is a commonly abused synthetic cathinone derivative marketed as a “legal” alternative to “ecstasy” or cocaine. Previous studies examined the metabolism of methylone in vitro and in vivo; 4-hydroxy-3-methoxymethcathinone (HMMC) was identified as the primary metabolite, with other reported minor metabolites, 3,4-methylenedioxycathinone (MDC) and 3,4-dihydroxymethcathinone (HHMC). However, limited information is known about methylone and its metabolites’ pharmacokinetics. We developed and fully validated a method for the simultaneous quantification of methylone, HMMC, MDC and HHMC by liquid chromatography-tandem mass spectrometry in 100 µl rat and human plasma. β-Glucuronidase was utilized for plasma hydrolysis, followed by perchloric acid protein precipitation and solid-phase extraction utilizing cation exchange columns. Chromatographic separation was performed with a Synergi Polar column in gradient mode, and analytes were determined by two multiple reaction monitoring (MRM) transitions. Linear ranges of 0.5–1,000 µg/l (methylone, HMMC and MDC) and 10–1,000 µg/l (HHMC) were achieved. Bias and imprecision were generally acceptable, although quantification of HHMC exhibited variability (16.2–37 %). Extraction efficiencies and ion suppression were 89.9–104 % (for HHMC, 15.9–16.2 %) and < 11.4 %, respectively. Methylone and metabolites were stable in plasma for 24 h at room temperature, 72 h at 4 °C, and after three freeze–thaw cycles (except for a 60 % HMMC increase). Human and rat plasma were cross-validated, documenting that rat plasma quality control samples were accurately quantified against a human plasma calibration curve (−23.8 to 12 % bias). As proof of method, rat plasma specimens were analyzed pre-injection and after subcutaneous administration of methylone at 6 mg/kg from 15 to 480 min post-dosing. Methylone, HMMC, MDC and HHMC concentrations ranged from 1.1 to 1,310, 11.2 to 194, 1.9 to 152 and 24.7 to 188 µg/l, respectively.

Teores de minerais em polpas e cascas de frutos de cultivares de bananeira

O objetivo deste trabalho foi determinar a percentagem de matéria seca, o teor de minerais e a capacidade de fornecimento de minerais, com base na ingestão dietética de referência na polpa e na casca de frutos verdes e maduros de 15 cultivares de bananeira. Utilizaram-se quatro cachos por cultivar e seis frutos por unidade amostral. Foram coletadas amostras frescas da polpa e da casca, para digestões sulfúricas (0,2 g) e nitroperclóricas (0,5 g). Foram determinados: no extrato sulfúrico, o N total pelo método Kjeldahl; e, no extrato nitroperclórico, P, K, Ca, Mg, Cu, Mn, Fe, Zn e Se. O P foi determinado por colorimetria, e os demais nutrientes, por espectrofotometria de absorção atômica. O teor de minerais na polpa e na casca foi expresso com base na média dos dois estádios. Há diferenças entre cultivares de bananeira quanto à concentração de macro e micronutrientes na polpa e na casca, mas não entre frutos verdes e maduros. A cultivar Terrinha apresentou a maior percentagem de matéria seca na polpa, e as cultivares Marmelo e Maçã, na casca. Para N, P, Fe, Zn e Cu, a casca apresentou duas vezes o teor da polpa. O teor de K e Mn na casca foi de aproximadamente quatro vezes o da polpa. A polpa da cultivar Caipira (AAA) e a casca das cultivares Prata e Pacovan (AAB) apresentam os maiores teores de minerais.

INFLUENCE OF TEMPERATURE ON PRODUCTION OF BIOGENIC AMINES IN PORK, BEEF, AND POULTRY AND THEIR HPLC DETERMINATION AFTER POSTCOLUMN DERIVATIZATION

The main biogenic amines in foods are histamine, tryptamin, cadaverin, putrescin, spermin, and spermidin. The samples of pork, beef, and poultry were extracted with 0.6 mol · l−1 perchloric acid and extract was filtered. Histamine, cadaverine, putrescine, and tyramine were determined by reverse-phase HPLC using a C18 stationary phase column. Acetate buffer with water (pH 4.5) and acetate buffer with acetonitrile (45/55 v/v) was used as a mobile phase, the flow-rate was 1 mL · min−1. The post column derivatization technique with OPT buffer (phthaldialdehyde buffer) as derivatization agent and the fluorescence detector at 330 nm and 465 nm as excitation and emission wavelengths was used. Limit of detection for biogenic amines was 0.2 mg · kg−1 and limit of quantification was 0.5 mg · kg−1. The method was useful over a wide concentration range of biogenic amines. The concentrations from 0.5 to 1 000 mg · kg−1 of histamine, tyramine, putrescine, and cadaverine in pork, beef, and poultry could be detected easily without any noticed disadvantage. Concentrations of biogenic amines were determined in pork, beef, and poultry and concentrations were increased depending on temperature and time. Concentrations of cadaverine and putrescine were highest for ground meat stored at 24°C after 10 days. Highest influence of temperature on production of biogenic amines was found in poultry samples. The concentrations of tyramine, putrescine and histamine were higher than 100 mg · kg−1 and the concentration of cadaverine was more than 400 mg · kg−1.

S-Adenosylmethionine-dependent Protein Methylation Is Required for Expression of Selenoprotein P and Gluconeogenic Enzymes in HepG2 Human Hepatocytes

Cellular methylation processes enable expression of gluconeogenic enzymes and metabolism of the nutrient selenium. Selenium status has been proposed to relate to type II diabetes risk, and plasma levels of selenoprotein P (SEPP1) have been positively correlated with insulin resistance. Increased expression of gluconeogenic enzymes glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) has negative consequences for blood glucose management in type II diabetics. Transcriptional regulation of SEPP1 is directed by the same transcription factors that control the expression of G6PC and PCK1, and these factors are activated by methylation of arginine residues. We sought to determine whether expression of SEPP1 and the aforementioned glucoconeogenic enzymes are regulated by protein methylation, the levels of which are reliant upon adequate S-adenosylmethionine (SAM) and inhibited by S-adenosylhomocysteine (SAH). We treated a human hepatocyte cell line, HepG2, with inhibitors of adenosylhomocysteine hydrolase (AHCY) known to increase concentration of SAH before analysis of G6PC, PCK1, and SEPP1 expression. Increasing SAH decreased 1) the SAM/SAH ratio, 2) protein-arginine methylation, and 3) expression of SEPP1, G6PC, and PCK1 transcripts. Furthermore, hormone-dependent induction of gluconeogenic enzymes was reduced by inhibition of protein methylation. When protein-arginine methyltransferase 1 expression was reduced by siRNA treatment, G6PC expression was inhibited. These findings demonstrate that hepatocellular SAM-dependent protein methylation is required for both SEPP1 and gluconeogenic enzyme expression and that inhibition of protein arginine methylation might provide a route to therapeutic interventions in type II diabetes.

1H/13C NMR metabolomics in a neonatal rat brain slice model of early and late mild hypothermia treatments of asphyxia

Clinical trials with neurological outcomes have led to using mild therapeutic hypothermia (≈4°C decrease) in early treatments of newborns with hypoxic‐ischemic encephalopathy from birth asphyxia, which we simulated by oxygen‐glucose deprivation (OGD) in superfused brain slices. Temperature differently modulates metabolic fluxes and concentrations. Although 4°C changes are small, three hypothermia treatment groups (nothing, immediate, and delayed) were distinctly separated by PLS‐DA multivariate analyses in 14.1 T (Tesla) and 21.15 T NMR metabolic profiling of perchloric acid brain slice extracts. Changes in tricarboxylic acid cycle (TCA) fluxes and neuron‐glial metabolic differences were measured after simultaneous administration of [1‐13C]glucose and [1,2‐13C]acetate. High energy phosphates, quantified by 31P NMR at 14.1 T, were quantified by 1H NMR at 21.15 T (900 MHz). Mild hypothermia's influence on neuron/glial metabolic differences was measured via two ratios: acetate/glucose utilization, which had significant changes; and (pyruvate carboxylase)/(pyruvate dehydrogenase) activity, which had minimal changes. Combining 13C data with 1H data further sharpened metabolomic distinctions. Support: NIH GM34767

Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis

Despite the crucial roles of flavin cofactors in metabolism, we know little about the enzymes responsible for the turnover of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and their subcellular localization. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation, because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial function in both bioenergetics and cellular regulation. In this context, the capability of yeast mitochondria to metabolize externally added and endogenous FAD and FMN was investigated and use was made of purified and bioenergetically active mitochondria prepared starting from the Saccharomyces cerevisiae cell. To determine whether flavin metabolism can occur, the amounts of flavins in aliquots of neutralized perchloric extracts of both spheroplasts and mitochondria were measured by HPLC, and the competence of S. cerevisiae mitochondria to metabolize FAD and FMN was investigated both spectroscopically and via HPLC. FAD deadenylation and FMN dephosphorylation were studied with respect to dependence on substrate concentration, pH profile and inhibitor sensitivity. The existence of two novel mitochondrial FAD pyrophosphatase (diphosphatase) (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) activities, which catalyse the reactions FAD + H₂O → FMN + AMP and FMN + H₂O → riboflavin + Pi respectively, is here shown by fractionation studies. Considering cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial contents via HPLC, probably mitochondria play a major role in regulating the flavin pool in yeast and in relation to flavin homeostasis.

SU‐GG‐J‐135: Exploring the Neuroprotective Effects of Bee Venom in a Mouse Parkinson Model with Immunohistochemistry, Magnetization Transfer Ratio Imaging and Spectroscopy

Purpose: Neuroprotective therapeutics stop or slowdown the degeneration process in animal models of Parkinson's disease (PD). In the present study, we investigated the anti‐inflammatory effect of bee venom (BV) on lipopolysaccharide (LPS)‐stimulated microglia. Method and Materials: To investigate whether MR techniques have added value in neuroprotection research in the MPTP mouse model, both MRI and MRS were applied to investigate the neuroprotective effects of BV. BV is suggested to stimulate anti‐inflammatory processing indirectly via DA‐dependent mechanisms or γ‐aminobutyric acid (GABA) and glutamate release. Magnetization transfer ratio (MTR) and 1H‐MRS studies were performed on control mice, MPTP‐intoxicated mice and BV treatment mice. The successfully induced lesions were verified by tyrosine hydroxylase immunolabelling on the substantia nigra (SN) and striatum (ST) performed on perchloric extracts of mice brains, after the control mice and the MPTP‐intoxicated mice were killed. All the MTR and 1H‐MRS experiments were performed on a 9.4 T MRI/MRS system using a standard head coil. Outer volume suppression combined with the ultra‐short echo‐time stimulated echo acquisition mode was used for the localized 1H‐MRS. The quantitative analysis of metabolites was determined using jMRUI from the 1H spectra obtained in vivo on the striatum. Results: The peak height of the MTR histograms in the PD model group was significantly lower than that in the BV treatment group (P&lt;0.05). The present study revealed that the peak height of the MTR histogram was significantly decreased in the ST and SN, and a significant increase of the Glx/Cr ratio was found in the striatum of the PD group, as compared with that of the BV treatment group. Conclusion: These results demonstrate that BV possess a potent suppressive effect on proinflammatory responses of microglia and suggest that these compounds may offer substantial therapeutic potential for treatment of neurodegenerative diseases that are accompanied by microglial activation.

Metabolic profiling and aquaculture differentiation of gilthead sea bream by 1H NMR metabonomics

Abstract This work describes a metabolic profiling study of gilthead sea bream, from three different aquaculture systems, using 1 H NMR and chemometrics. A total of 54 samples under two different storage regimens were analysed. The assignment of all major NMR signals of the perchloric extracts was performed. A comprehensive multivariate data analysis proved able to distinguish the fish metabolism amongst the different aquaculture systems and to determine whether a fish was stored or not. The state of energy metabolism of inosine proved a robust biomarker for evaluating storage time. A new multivariate classification tool, i ECVA, revealed several metabolites which are important biomarkers for characterising the three different aquaculture systems: glycogen (stress indicator), histidine, alanine and especially glycine for long storage times and mainly betaine for fresh samples. The findings represent a step forward in understanding how in vivo and postmortem processes affect the total quality of the final product.

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

Fructose-1,6-bisphosphate does not preserve ATP in hypoxic–ischemic neonatal cerebrocortical slices

Fructose-1,6-bisphosphate (FBP), an endogenous intracellular metabolite in glycolysis, was found in many preclinical studies to be neuroprotective during hypoxia–ischemia (HI) when administered exogenously. We looked for HI neuroprotection from FBP in a neonatal rat brain slice model, using 14.1 T 1H/ 31P/ 13C NMR spectroscopy of perchloric acid slice extracts to ask: 1) if FBP preserves high energy phosphates during HI; and 2) if exogenous [1- 13C]FBP enters cells and is glycolytically metabolized to [3- 13C]lactate. We also asked: 3) if substantial superoxide production occurs during and after HI, thinking such might be treatable by exogenous FBP's antioxidant effects. Superfused P7 rat cerebrocortical slices (350 μm) were treated with 2 mM FBP before and during 30 min of HI, and then given 4 h of recovery with an FBP-free oxygenated superfusate. Slices were removed before HI, at the end of HI, and at 1 and 4 h after HI. FBP did not improve high energy phosphate levels or change 1H metabolite profiles. Large increases in [3- 13C]lactate were seen with 13C NMR, but the lactate fractional enrichment was always (1.1 ± 0.5)%, implying that all of lactate's 13C was natural abundance 13C, that none was from metabolism of 13C-FBP. FBP had no effect on the fluorescence of ethidium produced from superoxide oxidation of hydroethidine. Compared to control slices, ethidium fluorescence was 25% higher during HI and 50% higher at the end of recovery. Exogenous FBP did not provide protection or enter glycolysis. Its use as an antioxidant might be worth studying at higher FBP concentrations.

Metabolic changes detected by proton magnetic resonance spectroscopy in vivo and in vitro in a murin model of Parkinson’s disease, the MPTP‐intoxicated mouse

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta, which project to the striatum. The aim of this study was to analyze in vivo and in vitro consequences of dopamine depletion on amount of metabolites in a mouse model of Parkinson's disease using proton (1)H magnetic resonance spectroscopy (MRS). The study was performed on control mice (n = 7) and MPTP-intoxicated mice (n = 7). All the experiments were performed at 9.4 T. For in vivo MRS acquisitions, mice were anesthetized and carefully placed on an animal handling system with the head centered in birdcage coil used for both excitation and signal reception. Spectra were acquired in a voxel (8 microL) centered in the striatum, applying a point-resolved spectroscopy sequence (TR = 4000 ms, TE = 8.8 ms). After in vivo MRS acquisitions, mice were killed; successful lesion verified by tyrosine hydroxylase immunolabeling on the substantia nigra pars compacta and in vitro MRS acquisitions performed on perchloric extracts of anterior part of mice brains. In vitro spectra were acquired using a standard one-pulse experiment. The absolute concentrations of metabolites were determined using jmrui (Lyon, France) from (1)H spectra obtained in vivo on striatum and in vitro on perchloric extracts. Glutamate (Glu), glutamine (Gln), and GABA concentrations obtained in vivo were significantly increased in striatum of MPTP-lesioned mice (Glu: 15.5 +/- 2.5 vs. 12.9 +/- 1.0 mmol/L, p < 0.05; Gln: 2.3 +/- 0.9 vs. 1.8 +/- 0.6 mmol/L, p < 0.05; GABA: 2.3 +/- 0.9 vs. 1.3 +/- 0.6 mmol/L, p < 0.05). The in vitro results confirmed these results, Glu (10.9 +/- 2.5 vs. 7.9 +/- 1.7 micromol/g, p < 0.05), Gln (6.8 +/- 2.9 vs. 4.3 +/- 1.0 micromol/g, p < 0.05), and GABA (2.9 +/- 0.9 vs. 1.5 +/- 0.4 micromol/g, p < 0.01). The present study strongly supports a hyperactivity of the glutamatergic cortico-striatal pathway hypothesis after dopaminergic denervation in association with an increase of striatal GABA levels. It further shows an increased of striatal Gln concentrations, perhaps as a strategy to protect neurons from Glu excitotoxic injury after striatal dopamine depletion.

Relevance of a perchloric acid extraction scheme to determine mineral and organic phosphorus in swine slurry

To increase the phosphorus recycling potential from swine slurry, mineral phosphorus products which could be used as fertilizers should be obtained and new processes need to be investigated. A routine method is needed to better evaluate the dissolved and solid mineral phosphorus in swine slurry. Cold perchloric acid extraction method previously developed for wastewater or sludge analysis was adapted. Ionic chromatography was used to measure orthophosphate in extracts. Only one extraction step was needed to distinguish between mineral and organic phosphorus in slurry. Reproducibility of the method was high (less than 5% of variation on the measured fractions). Selectivity was assessed by adding several organic and mineral phosphorus sources in the slurry. Cold perchloric extraction followed by ionic chromatography was very selective in quantifying both the mineral and organic forms of phosphorus in swine slurry.

Separation of americium and europium from solutions of nitric and perchloric acid using dipicolinic acid diamides

Extraction of nitric acid, perchloric acid, americium, and europium with dialkyldiarylamides of 2,6-pyridinecarboxylic (dipicolinic) acid in polar fluorinated solvents (diluents) was analysed. Among the extractants studied N, N′-diethyl-N, N′-ditolylamide dipicolinate is characterized by the best extraction properties. On passing from nitric to perchloric acid solutions extraction of metals sharply increases. Extraction of americium from nitric acid solutions is significantly greater as compared with europium (separation coefficient reaches 6), whereas extraction of americium and europium from perchloric acid solutions is nearly similar. The effect of diluent on the extraction was analyzed; for different diluents the maximum of extraction from perchloric acid solutions shifts form 3 to 6 M.

NMR metabolite profiling analysis reveals changes in phospholipid metabolism associated with the re‐establishment of desiccation tolerance upon osmotic stress in germinated radicles of cucumber

The adaptation of metabolism is thought to play a role in the acquisition of desiccation tolerance (DT). However, the importance of such a role and whether specific regulatory pathways exist remain to be assessed. Using in vitro 31P and 13C nuclear magnetic resonance (NMR) spectroscopy and biochemical assays, we analysed metabolite profiles of perchloric extracts from germinating radicles of cucumber to identify changes in carbon and phosphate metabolism associated with DT. Emerged radicles measuring 2 mm long can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution with a water potential of 1.5 MPa. However, in 4-mm-long emerged radicles, this treatment was ineffective. This manipulable system enabled the discrimination of changes in metabolites associated with DT from those associated with the response to osmotic stress. Independent of radicle length, the PEG treatment resulted in an increase in sucrose (Suc) content, whereas glucose (Glc), fructose (Fru) and the hexose phosphate pool, as well as phosphoenolpyruvate decreased three- to fourfold. In addition, three derivatives arising early during phospholipid catabolism (glycerylphosphorylcholine, glycerylphosphorylethanolamine and glycerylphosphorylinositol) appeared in the PEG-treated radicles. Interestingly, phospholipid degradation was much more pronounced in osmotically challenged radicles that remain sensitive to drying. This was proved by the appearance of catabolites, such as phosphocholine and phosphoethanolamine, solely in 4 mm PEG-treated radicles. Furthermore, glycerol-3-phosphate and its derivative 3-phosphoglycerate increased significantly. Our data suggest that the metabolic response leading to the re-establishment of DT is not entirely identical to that of an osmotic response. It is inferred that membrane remodelling and/or increased phospholipid catabolism is an adaptive response common to osmotic adjustment and DT but is controlled differently in tolerant and sensitive radicles.

Determination of levetiracetam in human plasma with minimal sample pretreatment

We here present a method for the routine quantification of the novel antiepileptic drug levetiracetam in human serum by HPLC–UV. The procedure is very easy, quick, inexpensive and rugged. The sample preparation consists only in the precipitation of serum proteins by perchloric acid and extraction of unpolar components by cyclohexane. The aqueous phase containing the analyte levetiracetam is injected onto a porous graphitic carbon analytical HPLC-column and separated by gradient elution with diluted phosphoric acid/acetonitrile. Detection is carried out at a wavelength of 205 nm. The calibration function is linear in the range of 1–75 μg/ml. The detection limit is 0.1 μg/ml. Using four quality control sample concentrations, the inter-day relative standard deviations (R.S.D.) are lower than 3% and the accuracies are better than 6%. The respective inter-day values are: R.S.D. < 4% and accuracies better than 2%. Frequently co-administered antiepileptic drugs do not interfere with the assay. The method has been successfully applied to patient samples.

Using 31P NMR spectroscopy at 14.1 Tesla to investigate PARP-1 associated energy failure and metabolic rescue in cerebrocortical slices.

PARP-1 activation by H(2)O(2) in an acute preparation of superfused, respiring, neonatal cerebrocortical slices was assessed from PAR-polymer formation detected with immunohistochemistry and Western blotting. (31)P NMR spectroscopy at 14.1 Tesla of perchloric acid slice extracts was used to assess energy failure in a 1-h H(2)O(2) exposure as well as in a subsequent 4-h recovery period where the superfusate had no H(2)O(2) and specifically chosen metabolic substrates. Although more data are needed to fully characterize different bioenergetic responses, a high NMR spectral resolution (PCr full-width at half-max approximately.01 ppm) and narrow widths for most metabolites (<.2 ppm) permitted accurate quantifications of spectrally resolved resonances for ADP, ATP, NAD(+)/NADH, and other high energy phosphates. It appears possible to use brain slices to quantitatively study PARP-related, NAD-associated energy failure, and rescue with TCA metabolites.