scholarly journals Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels

2004 ◽  
Vol 380 (2) ◽  
pp. 449-454 ◽  
Author(s):  
Dev CHURAMANI ◽  
Elizabeth A. CARREY ◽  
George D. DICKINSON ◽  
Sandip PATEL
Keyword(s):  
Nicotinic Acid ◽  
Radioligand Binding ◽  
Rat Hepatocytes ◽  
Experimental Conditions ◽  
Ligand Interaction ◽  
Human Red Blood Cells ◽  
Enhanced Sensitivity ◽  
Sea Urchin Egg ◽  
Low Concentrations

Nicotinic acid–adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725–729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor–ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.

scholarly journals Unique kinetics of nicotinic acid–adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand

2000 ◽  
Vol 352 (3) ◽  
pp. 725-729 ◽  
Author(s):  
Sandip PATEL ◽  
Grant C. CHURCHILL ◽  
Antony GALIONE
Keyword(s):  
Nicotinic Acid ◽  
Sea Urchin ◽  
Binding Sites ◽  
Cell Types ◽  
Unique Property ◽  
Single Class ◽  
High Affinity ◽  
Sea Urchin Egg ◽  
Low Concentrations ◽  
Kinetics Of

Nicotinic acidŐadenine dinucleotide phosphate (NAADP) is a novel and potent Ca2+-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [32P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca2+. Binding of [32P]NAADP was lost in preparations that did not mobilize Ca2+ in response to NAADP, indicating that [32P]NAADP probably binds to a receptor mediating Ca2+ mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [32P]NAADP binding, did not result in displacement of bound [32P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels.

scholarly journals A Square-Wave Adsorptive Stripping Voltammetric Method for Determination of Fast Green Dye

2009 ◽  
Vol 92 (6) ◽  
pp. 1714-1719
Author(s):  
Ali F Al-Ghamdi
Keyword(s):  
Soft Drink ◽  
Supporting Electrolyte ◽  
Pulse Amplitude ◽  
Fast Green ◽  
Experimental Conditions ◽  
Analytical Methodology ◽  
Square Wave ◽  
Carbonate Buffer ◽  
Enhanced Sensitivity

Abstract Square-wave adsorptive stripping voltammetric (SW-AdSV) determinations of trace concentrations of the coloring agent fast green were described. The analytical methodology used was based on the adsorptive preconcentration of the dye on the hanging mercury drop electrode, and then a negative sweep was initiated. In pH 10 carbonate supporting electrolyte, fast green gave a well-defined and sensitive SW-AdSV peak at 1220 mV. The electroanalytical determination of this dye was found to be optimized in carbonate buffer (pH 10) with the following experimental conditions: accumulation time (120 s); accumulation potential (0.8 V); scan rate (800 mV/s); pulse amplitude (90 mV); frequency (90 Hz); surface area of the working electrode (0.6 mm2); and the convection rate (2000 rpm). Under these optimized conditions, the AdSV peak current was proportional over the concentration range 2 1086 107 M (r = 0.999), with an LOD of 1.63 1010 M (0.132 ppb). This analytical approach possessed more enhanced sensitivity than conventional chromatography or spectrophotometry, and was simple and quick. The precision of the method in terms of RSD was 0.17, whereas the accuracy was evaluated via the mean recovery of 99.6. Possible interferences by several substances usually present as food additive azo dyes (E110, E102, E123, and E129), natural and artificial sweeteners, and antioxidants were also investigated. Applicability of the developed electroanalysis method was illustrated via the determination of fast green in ice cream and soft drink samples.

scholarly journals Use of a Mixed Cationic-Reverse Phase Column for Analyzing Small Highly Polar Metabolic Markers in Biological Fluids for Multiclass LC-HRMS Method

2020 ◽  
Vol 10 (20) ◽  
pp. 7137
Author(s):  
Marco Roverso ◽  
Iole Maria Di Gangi ◽  
Gabriella Favaro ◽  
Paolo Pastore ◽  
Sara Bogialli
Keyword(s):  
High Selectivity ◽  
Biological Fluids ◽  
Polar Compounds ◽  
Reverse Phase ◽  
Experimental Conditions ◽  
Metabolic Markers ◽  
Low Concentrations ◽  
Polar Metabolites ◽  
Highly Polar Compounds

The determination of small highly polar metabolites at low concentrations is challenging when reverse-phase (RP) chromatography is used for multiclass analysis. A mixed cationic-RP column coupled to high-resolution tandem MS (HR-MS/MS) was tested for highly polar compounds in biological fluids, i.e., trimethylamine N-oxide (TMAO) and the isobaric molecules beta-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). The efficient retention and separation of the above compounds were obtained with common and MS-friendly RP conditions, reaching high selectivity and sensitivity. The method was firstly assessed in plasma and urine, showing good linearity in the range 50–1000 µg/L and 500–10,000 µg/L for TMAO and both BMAA and DAB, respectively. Excellent precision (RDS < 3%) and good accuracies (71–85%) were observed except for BMAA in plasma, whose experimental conditions should be specifically optimized. Preliminary tests performed on compounds with biological relevance and a wider range of polarities proved the effectiveness of this chromatographic solution, allowing the simultaneous analysis of a larger panel of metabolites, from very small and polar compounds, like trimethylamine, to quite lipophilic molecules, such as corticosterone. The proposed LC-HRMS protocol is an excellent alternative to hydrophilic interaction liquid chromatography and ion-pairing RP chromatography, thus providing another friendly analytical tool for metabolomics.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

1996 ◽  
Vol 76 (01) ◽  
pp. 005-008 ◽  
Author(s):  
Jean Claude Lormeau ◽  
Jean Pascal Herault ◽  
Jean Marc Herbert
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Residual Activity ◽  
Coagulation Factors ◽  
Heparin Binding ◽  
Experimental Conditions ◽  
First Order ◽  
Heparin Binding Site ◽  
Coagulant Activity

SummaryWe examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor Vila bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor Vila, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor Vila complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 ± 10,500 min-1 and 112,000 ± 12,000 min-1 (mean ± s.e.m., n = 3)

Determination of alcohols in mixtures by 19F NMR spectroscopy using hexefluoroacetone reagent

1982 ◽  
Vol 47 (7) ◽  
pp. 1973-1978 ◽  
Author(s):  
Jiří Karhan ◽  
Zbyněk Ksandr ◽  
Jiřina Vlková ◽  
Věra Špatná
Keyword(s):  
Standard Deviation ◽  
Nmr Spectroscopy ◽  
Standard Method ◽  
Internal Standard ◽  
Internal Standard Method ◽  
Concentration Region ◽  
Experimental Conditions ◽  
Curve Method ◽  
Sample Preparation Procedure

The determination of alcohols by 19F NMR spectroscopy making use of their reaction with hexafluoroacetone giving rise to hemiacetals was studied on butanols. The calibration curve method and the internal standard method were used and the results were mutually compared. The effects of some experimental conditions, viz. the sample preparation procedure, concentration, spectrometer setting, and electronic integration, were investigated; the conditions, particularly the concentrations, proved to have a statistically significant effect on the results of determination. For the internal standard method, the standard deviation was 0.061 in the concentration region 0.032-0.74 mol l-1. The method was applied to a determination of alcohols in the distillation residue from an oxo synthesis.

scholarly journals Software-guided microscale flow calorimeter for efficient acquisition of thermokinetic data

2021 ◽  
Author(s):  
Timothy Aljoscha Frede ◽  
Marlene Dietz ◽  
Norbert Kockmann
Keyword(s):  
Design Of Experiments ◽  
Process Development ◽  
Reaction Conditions ◽  
Experimental Conditions ◽  
Flow Calorimeter ◽  
Microscale Flow ◽  
Increased Sensitivity ◽  
Important Time ◽  
Optimal Reaction

AbstractFast chemical process development is inevitably linked to an optimized determination of thermokinetic data of chemical reactions. A miniaturized flow calorimeter enables increased sensitivity when examining small amounts of reactants in a short time compared to traditional batch equipment. Therefore, a methodology to determine optimal reaction conditions for calorimetric measurement experiments was developed and is presented in this contribution. Within the methodology, short-cut calculations are supplemented by computational fluid dynamics (CFD) simulations for a better representation of the hydrodynamics within the microreactor. This approach leads to the effective design of experiments. Unfavourable experimental conditions for kinetics experiments are determined in advance and therefore, need not to be considered during design of experiments. The methodology is tested for an instantaneous acid-base reaction. Good agreement of simulations was obtained with experimental data. Thus, the prediction of the hydrodynamics is enabled and the first steps towards a digital twin of the calorimeter are performed. The flow rates proposed by the methodology are tested for the determination of reaction enthalpy and showed that reasonable experimental settings resulted. Graphical abstract A methodology is suggested to evaluate optimal reaction conditions for efficientacquisition of kinetic data. The experimental design space is limited by thestepwise determination of important time scales based on specified input data.

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