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Quantitative analysis of cannabinoids from Cannabis sativa using 1H-NMR.

A (1)H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of delta 4.0-7.0 in the (1)H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.

Cannabinoid Receptor Modulators↗

Foqus: a FORTRAN program for the quantitative analysis of x-ray spectra from thin biological specimens.

An online FORTRAN program for the quantitative analysis of energy dispersive X-ray spectra from thin biological specimens is presented. The methods of background suppression by digital filtering and peak deconvolution by linear least-squares fitting with measured peak profiles are used. The continuum quantitation method for spectra from thin biological sections as proposed by Hall is applied. The performance of the computer program, utilizing the facilities of a disk operating system, is demonstrated. The routines were optimized for speed, resulting in a run-time of less than 5 seconds on a 16 bit minicomputer for a full quantitation for 7 elements of an energy dispersive thin section X-ray spectrum, including an optional absorption correction. Since no assembly language subroutines are implemented, the restrictions for the use of the program with different computer systems are minimized.

Calibration↗

Quantitative analysis of histamine in biological samples by gas chromatography--mass spectrometry.

A mass fragmentographic method for the quantitative analysis of histamine in the supernatant from antigen-challenged leukocytes, whole blood, and urine is described. Histamine labeled with two 15N atoms was synthesized and added to the sample as an internal standard. N alpha-Heptafluorobutyryl-N tau-ethoxycarbonylhistamine was prepared for mass fragmentographic analysis and the molecular ions at m/z 379 and 381 were used for monitoring histamine and 15N2-labeled histamine, respectively. The quantitation limit of histamine was 2 ng by this method. The experimental error of the method was less than 7% at the level of 5 ng in the supernatant from antigen-challenged leukocytes. The value obtained by this method correlated well with that from radioisotopic enzymatic assay (r=0.990).

Gas Chromatography-Mass Spectrometry↗

Ultrastructural quantitative analysis of glutamatergic and GABAergic synaptic terminals in the phrenic nucleus after spinal cord injury.

Quantitative analysis of electron microscopic postembedding immunochemically stained material indicates that 48% of all terminals in the rat phrenic nucleus are glutamatergic and 33% are gamma-aminobutyric acid (GABA)ergic. Three distinct types of glutamatergic terminals were observed in the rat phrenic nucleus: terminals characterized by large, loosely arranged spherical synaptic vesicles (SI) or small, compact spherical synaptic vesicles (Ss) and elongated terminals containing spherical synaptic vesicles with neurofilaments (NFs). All three types of glutamatergic terminals display asymmetrical synaptic membrane densities with postsynaptic dense bodies being present in some of the S-type terminals. The GABAergic immunoreactive terminals in the phrenic nucleus most closely resemble F-type terminals. They are characterized by flattened or pleomorphic synaptic vesicles and symmetric synaptic membrane densities. Among the 48% glutamatergic terminals, 27% are SI, 65% are Ss, and 8% are NFs, respectively. Significantly fewer glutamate, GABA, and unlabeled terminals per unit area are present in the phrenic nucleus 30 days after a C2 spinal cord hemisection as compared to nonhemisected controls. The average number of active zones per terminal, however, is greater in the hemisection group (1.45 +/- 0.03) than in the control group (1.34 +/- 0.03), with the active zones in the glutamate terminals mainly accounting for this difference. Moreover, the length of the active zones in the glutamate terminals was significantly longer in the hemisection group (0.37 +/- 0.013 microns) as compared to the controls (0.24 +/- 0.008 microns). In addition, the mean length of synaptic active zones in GABAergic terminals was also found to be longer in the hemisection group (0.36 +/- 0.022 microns) as compared to controls (0.28 +/- 0.014 microns). Finally, there is also a significantly higher ratio of synaptic active zones to the total number of glutamate-labeled terminals after injury (1.73 +/- 0.08) as compared to controls (1.41 +/- 0.04). The number of double/multiple synapses, the percentages of Sl, Ss, and NFs-type terminals, and the percentages of synaptic active zones contacting either distal dendrites or proximal dendrites/somata do not change significantly 30 days after injury. These results are important for a more complete understanding of the synaptic plasticity that occurs in the phrenic nucleus after spinal cord injury and to show how the plasticity may relate to the unmasking of latent bulbospinal respiratory connections which restore function to the hemidiaphragm paralyzed by an ipsilateral spinal cord hemisection.

Animals↗

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films.

A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100 degrees C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the alpha-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O-H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility.

Animals↗

Quantitative analysis of short echo time (1)H-MRSI of cerebral gray and white matter.

Quantitative analysis of (1)H-magnetic resonance spectroscopic imaging (MRSI) data was developed using the user-independent spectral analysis routine LCModel. Tissue segmentation was performed using statistical parametric mapping software (SPM 96), and the results were used to correct for cerebrospinal fluid contamination. A correction was developed for the imperfections in the spectroscopic excitation profile in order to improve the uniformity of metabolite images. After validation in phantoms, these techniques were applied to study differences in metabolite concentrations between gray and white matter in normal volunteers (n = 13). A positive correlation was found between concentration and gray matter content for most metabolites studied. The estimated ratios of metabolite concentration in gray vs. white matter were: N-acetyl aspartate + N-acetyl aspartyl glutamate (NAc) = 1.16+/- 0.11; creatine = 1.7+/-0.3; glutamate + glutamine = 2.4+/-0.5; myo-inositol = 1.6+/-0.3; choline = 0.9+/-0.2. The ratio of NAc/Cr was negatively correlated with gray matter content: gray/white = 0.69 +/-0.08. These methods will be useful in the evaluation of metabolite concentrations in MRSI voxels with mixed tissue composition in patient groups.

Adult↗

Quantitative analysis of two-dimensional electrophoretograms using shape-fitting.

This paper describes an automatic quantitative analysis of protein using two-dimensional electrophoresis. We made 2-D electrophoretograms with index spots of some proteins whose mass is known. First, we cut out the protein spots from the background. Then we detected connected regions which may correspond to spot regions and measured their mass as the sum of the gray levels in the region. For confluent spots, we propose a method of separating them by shape-fitting, and examine this method by comparing it with the spot-dividing method.

Biometry↗

EndoCyte, an interactive computer program for quantitative analysis of receptor-mediated endocytosis.

We present EndoCyte, a user friendly interactive program for quantitative analysis of receptor-mediated endocytosis. The data, comprised of time-dependent concentrations of the ligand at the cell surface and the ligand internalized by cells, are analyzed by the application of a set of nested mathematical models of endocytosis. EndoCyte reduces data to parameters conventional in description of receptor-mediated endocytosis and a parameter which describes the non-linear effects. The performance of EndoCyte is documented by the analysis of applications to synthetic data.

Computer Simulation↗

Comparison of flow cytometry and image morphometry in the quantitative analysis of cell population markers in the lymph node of sheep.

Two approaches to the quantitative analysis of cell population markers in tissues are flow cytometry and image morphometry. To compare these methods, sheep lymph nodes were collected and analysed for CD8+ and CD21+ cell populations, which were selected to represent dispersed and concentrated cell populations, respectively. These two populations were measured as a percentage of total cell count (flow) or total tissue area (morphometry). The two populations were also measured as a percentage of respective base populations (CD2+ cells for CD8 and MHC II+ cells for CD21). A simple linear regression analysis showed that when the cell populations were assessed as a percentage of total cell count or total area, measurements obtained with flow and morphometry only correlated significantly with the dispersed CD8+ population and not with the highly concentrated CD21+ population. However, when the cell populations were assessed as a percentage of their base population, measurements obtained with flow and morphometry showed a significant correlation for both the dispersed and concentrated cell populations. This study demonstrates that measurements of lymph node cell populations obtained with the two methods are comparable, but that tissue distribution of cell populations should be considered, when the unit of measurement is chosen.

Animals↗

Quantitative analysis of catechol and 4-methylcatechol in human urine.

A method was developed for the quantitative analysis of catechol and 4-methylcatechol in human urine. [U-14C]Catechol was used as in internal standard. Urine was treated with beta-glucuronidase and sulphatase, acidified and extracted with ether. The ether extract was silylated and analysed by glass capillary gas chromatography. Catechol and 4-methylcatechol occurred in urine primarily as conjugates. Levels of catechol and 4-methylcatechol in the urine of nonsmokers on unrestricted diets were 10 +/- 7.3 (mean +/- 1 SD) and 3.4 +/- 2.3 mg/24 hr, respectively. Nonsmokers on uniform restricted diets, in which the intake of plant-derived products was limited, excreted 4.4 +/- 1.2 mg catechol and 8.1 +/- 1.7 mg 4-methylcatechol/24 hr. Smokers on the same restricted diet excreted 6.8 +/- 3.0 mg catechol and 6.1 +/- 2.6 mg 4-methylcatechol/24 hr. These results indicate that diet is a major factor in determining urinary catechol levels and that the contribution of smoking is comparatively small. Catechol and 4-methylcatechol appear to have different dietary precursors.

Adult↗

Automated evaluation of chemical shift perturbation spectra: New approaches to quantitative analysis of receptor-ligand interaction NMR spectra.

This paper presents new methods designed for quantitative analysis of chemical shift perturbation NMR spectra. The methods automatically trace the displacements of cross peaks between a perturbed test spectrum and the reference spectrum (or among a series of titration spectra), and measure the changes of chemical shifts, heights, and widths of the altered peaks. The methods are primary aimed at the (1)H-(15)N HSQC spectra of relatively small proteins (<15 kDa) assuming fast exchange between free and ligand-bound states on the chemical shift time scale, or for comparing spectra of free and fully bound states in the slow exchange situation. Using the (1)H-(15)N HSQC spectra from a titration experiment of the 74-residue Pex13p SH3 domain with a Pex14p peptide ligand (14 residues, K (d)= approximately 40 microM), we demonstrate the scope and limits of our automatic peak tracing (APET) algorithm for efficient scoring of high-throughput SAR by NMR type HSQC spectra, and progressive peak tracing (PROPET) algorithm for detailed analysis of ligand titration spectra. Simulated spectra with low signal-to-noise ratios (S/N ranged from 20 to 1) were used to demonstrate the reliability and reproducibility of the results when dealing with poor quality spectra. These algorithms have been implemented in a new software module, FELIX-Autoscreen, for streamlined processing, analysis and visualization of SAR by NMR and other high-throughput receptor/ligand interaction experiments.

Algorithms↗

Quantitative analysis of strychnine and Brucine in Strychnos nux-vomica using 1H-NMR.

A quantitative analysis using (1)H-NMR (Q-NMR) has been developed for the determination of strychnine and brucine in Strychnos nux-vomica seeds and stems. The advantages of the method are that no reference alkaloids are needed for calibration curves, the quantification could be directly realized on a crude extract, strychnine and brucine could easily be distinguished, an overall profile of the preparation (including non alkaloid compounds) could be directly obtained, and a very significant time-gain could be achieved, in comparison to conventional HPLC methods, for instance.

Chromatography, High Pressure Liquid↗

A quantitative analysis of agonistic behavior in juvenile American lobsters (Homarus americanus L.).

In these studies a quantitative analysis of agonistic (fighting) behavior in lobsters in presented as a first step in our attempt to relate patterns of behavior to underlying neurobiological mechanisms. The agonistic behavior of juvenile American lobsters (Homarus americanus L.) was studied in laboratory tanks at the New England Aquarium. Using video analyses and statistical techniques: (1) an ethogram of agonistic behavior was constructed; and (2) the temporal structure of the behavior was identified. We demonstrated that fighting in juvenile lobsters proceeds according to strict rules of conduct. All animals exhibit six common behavioral patterns in a stereotypical manner. A temporal sequence of these patterns was evident, representing an increase in intensity during confrontations. The typical scenario of an encounter begins with extensive threat displays upon first contact, continues with periods of ritualized aggression and restrained use of the claws, and terminates in a brief session of unrestrained combat. Predictions of game theory (i.e. assessment strategies) provide a useful framework for the understanding of fighting in lobsters. The presence of a highly structured behavioral system may reduce the potential for damage in fights among conspecifics, and may prove useful in attempts to study the neurobiological causes of complex behavioral patterns such as aggression.

Aggression↗

Quantitative analysis of hypothalamic-hypophyseal-testicular system: why testosterone can act under negative feedback control.

Quantitative analysis of the positive and negative feedback actions of testosterone (T) was carried out using intact rats, and orchidectomized rats implanted with T-filled Silastic capsules. Target organ weights and serum levels of LH and T were examined 7 days later, and response ratios of positive and negative actions were plotted against logarithm of serum T. From x-intercepts and slopes of regression lines, thresholds and responsiveness of reactions were calculated, respectively. Maintenance T levels necessary to maintain onset weights of the organ were also calculated from the regression lines. We compared 5 parameters at various ages, 1) thresholds for lowering serum LH, 2) mean T concentration of intact animals, 3) upper limit of serum T (mean + 2SD), 4) maintenance T levels, and 5) lower 95% limit of the thresholds for target organ response in orchidectomized animals. Though T can act between thresholds for target organ response and upper limit of serum T, the action range of T to induce organ growth over the onset organ weight (growth-inducing range) should be the area between the maintenance T level and the upper limit for serum T. At 3 weeks of age, the threshold for serum LH was very low, which makes the upper limit of serum T lower than maintenance T, allowing no growth-inducing range. From 5 weeks of age, the threshold for serum LH increased, and the serum T level with its upper limit also increased over the maintenance T level, allowing the presence of a growth-inducing range of T to make the growth of target organs possible.

Animals↗

An open environment for quantitative analysis of left ventricular function using ultrasound images.

We have developed an open environment for quantitative analysis of left ventricular function using ultrasound images. The system is primarily intended for experimental purposes, i.e. clinical research and the evaluation of quantification algorithms. The modular design allows easy expansion with new analysis methods. The core of our system is the user-interface and image presentation. Image analysis methods are included as a set of tools available to the operator. Using this approach, the expensive, expert knowledge of the cardiologist is used more efficiently than in most delineation systems. During delineation, images of a complete heart cycle can be simultaneously displayed dynamically ('cine mode') in a separate window. This feature improves visual edge perception and enables accurate determination of the endocardium even in cases of very poor echogenicity. The resulting contour can be corrected locally by interactive modification using 'rubber banding'. After delineation of the images from different echocardiographic views, volume, ejection fraction, and regional wall motion are determined. Studies using contrast-enhanced echocardiography can be quantified by image intensity analysis in a region of interest in order to determine myocardial perfusion. We evaluated our method by quantifying echocardiographic studies from 12 healthy volunteers. We compared the results with magnetic resonance imaging and concluded that there was no significant difference in volume and ejection fraction calculated from both modalities. Moreover, the image display features and contour correction methods were greatly appreciated by the cardiologists who used the system.

Echocardiography↗

[MALD-MS in the quantitative analysis of peptides and proteins].

A modified method of isotope dilution was applied to the quantitative determination of peptides and proteins by MALDI MS at subpicomolar level. The essence of the method consists in the quantitative analysis of the enzymic hydrolysis products rather than the starting compounds. This allows the measurements to be performed at a higher resolution and makes the method independent of the molecular mass of oligopeptides and proteins examined. Fragments obtained by hydrolysis of the same oligopeptide or protein in a known concentration by the same enzyme and labeled with the stable 18O isotope are used as internal standards. The label is introduced by carrying out the hydrolysis in H(2)18O, and the oligopeptide concentration is calculated from the isotope distribution between the labeled and unlabeled hydrolysis products in the mass spectrum. This method was tested in the determination of concentrations of the angiotensinogen (1-14) fragment (oligopeptide), extracellular RNAase from Bacillus amyloliquefaciens (protein) and its protein inhibitor, barstar M. Usefulness of this method in kinetic studies was also demonstrated.

Amino Acid Substitution↗

[Detection of nucleic acid sequences by bioluminescence. Importance of an internal reference system for qualitative and quantitative analysis].

Various luminescent detection systems were used to detect and quantify simultaneously several probes. For quantitative analysis, one probe was used as an internal standard to evaluate the amplification yield and the others to quantify the amplified target. After using hybridization in solution, accurate results were obtained. For a qualitative analysis, amplified sequences were hybridized to specific oligonucleotides which were labelled or immobilized. To compare amplified fragments to products of known sequences, we developed a more efficient technique based on DNA strand exchange occurring during hybridization in solution. This method allowed to detect a single base substitution in DNA fragments of 280 base pairs.

Base Sequence↗