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PubMed2001

Imaging and quantitative analysis of tritium-labelled cells in lymphocyte proliferation assays using microchannel plate detectors originally developed for X-ray astronomy.

Microchannel plate detectors have been used in many astronomical X-ray telescopes. Recently we have begun to use similar detectors to image electron emission from radiolabelled biological assays. Here we show how a microchannel plate (MCP) detector can be used to image tritium uptake in T lymphocyte proliferation assays. Quantitative analysis using the MCP detector has the same sensitivity and speed as conventional liquid scintillation counter (LSC) analysis whilst obviating the need for scintillation fluid. In addition the system permits the imaging of whole plate harvests from a range of plate sizes. Here we present data obtained with 96-well plates and Terasaki plates.

Cell Division↗
PubMed2006

Quantitative analysis of N-phenylpropenoyl-L-amino acids in roasted coffee and cocoa powder by means of a stable isotope dilution assay.

Since recent reports on the role of N-phenylpropenoyl-L-amino acids as powerful antioxidants and key contributors to the astringent taste of cocoa nibs, there is an increasing interest in the concentrations of these phytochemicals in plant-derived foods. A versatile analytical method for the accurate quantitative analysis of N-phenylpropenoyl-L-amino acids in plant-derived foods by means of HPLC-MS/MS and synthetic stable isotope labeled N-phenylpropenoyl-L-amino acids as internal standards was developed. By means of the developed stable isotope dilution assay (SIDA), showing recovery rates of 95-102%, 14 N-phenylpropenoyl-L-amino acids were quantified for the first time in cocoa and coffee samples. On the basis of the results of LC-MS/MS experiments as well as cochromatography with the synthetic reference compounds N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4'-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine, respectively, were detected for the first time in cocoa powder, and (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4'-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tryptophan, respectively, were detected for the first time in coffee beverages.

Amino Acids↗
PubMed1994

Rate modulated pacing based on right ventricular dP/dt: quantitative analysis of chronotropic response.

Right ventricular contractility increases in response to catecholamine stimulation and greater ventricular preload, factors that increase with exercise workload. Thus, the maximum systolic dP/dt may be a potentially useful sensor to control the pacing rate of a permanent pacing system. The present study was designed to test the long-term performance of a permanent pacemaker that modulates pacing rate based on right ventricular dP/dt and to quantitatively analyze the chronotropic response characteristics of this sensor in a group of patients with widely varying structural heart diseases and degrees of hemodynamic impairment. A permanent pacing system incorporating a high fidelity pressure sensor in the lead for measurement of right ventricular dP/dt was implanted in 13 patients with atrial arrhythmias and AV block, including individuals with coronary artery disease, hypertension, severe obstructive pulmonary disease with prior pneumonectomy, atrial septal defect, dilated cardiomyopathy, restrictive cardiomyopathy, and mitral stenosis. Patients underwent paired treadmill exercise testing in the VVI and VVIR pacing modes with measurement of expired gas exchange and quantitative analysis of chronotropic response using the concept of metabolic reserve. The peak right ventricular dP/dt ranged from 238-891 mmHg/sec with a pulse pressure that ranged from 19-41 mmHg. There was a positive correlation between the right ventricular dP/dt and pulse pressure (r = 0.70, P = 0.012). The maximum pacing rate and VO2max were 72 +/- 6 beats/min and 12.61 +/- 4.0 cc O2/kg per minute during VVI pacing and increased to 124 +/- 18 beats/min and 15.89 +/- 5.9 cc O2/kg per minute in the VVIR pacing mode (P < 0.0003 and P < 0.002, respectively). The integrated area under the normalized rate response curve was 96.7 +/- 45.7% of expected during exercise and 100.1 +/- 43.4% of expected during recovery. One patient demonstrated an anomalous increase in pacing rate in response to a change in posture to the left lateral decubitus position. Thus, the peak positive right ventricular dP/dt is an effective rate control parameter for permanent pacing systems. The chronotropic response was proportional to metabolic workload during treadmill exercise in this study population with widely varying forms of structural heart disease.

Adult↗
PubMed2003

The quantitative analysis of the vascularization following two basic auditory canal skin incisions.

Three groups of nine patients each were analyzed. The first two groups consisted of those that underwent tympanoplastic due to chronic inflammation of middle ear. Two different standard auditory canal skin incisions were applied, i.e. tympanomeatal flap (TMF) or vascular strip (VS). The third control group consisted of non-operated patients. All the operated patients were subjected to a quantitative analysis of the auditory canal revascularization by means of the Weibel stereological test method, i.e. the B 100 double network system. The density of capillaries, arterioles, venulolymphatic spaces and a total volume density of all vascular elements of the auditory canal skin were measured. The obtained results of vascularization were compared with those of the target control group. It was found out that there were no significant differences in vascularization of auditory canal skin between TMF and VS patients from one side and the control group on the other side.

Adult↗
PubMed2002

Quantitative analysis of the novel depsipeptide anticancer drug Kahalalide F in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray ionization tandem mass spectrometry.

Kahalalide F (KF) is a novel cyclic depsipeptide anticancer drug, which has shown anticancer activity both in vitro and in vivo especially against human prostate cancer cell lines. To characterize the pharmacokinetics of KF during a phase I clinical trial in patients with androgen refractory prostate cancer, a method was developed and validated for the quantitative analysis of KF in human plasma using high-performance liquid chromatography (HPLC) coupled to positive electrospray ionization tandem mass spectrometry (ESI-MS/MS). Microbore reversed-phase liquid chromatography (LC) performed with mobile phases containing trifluoroacetic acid, an additive commonly used for separating peptides, resulted in substantial suppression of the signal for KF on ESI-MS/MS. An alternative approach employing a basic mobile phase provided an excellent response for KF when detected in the positive ion mode. Plasma samples were prepared for LC MS/MS by solid-phase extraction on C(18) cartridges. The LC separation was performed on a Zorbax Extend C(18) column (150 x 2.1 mm i.d., particle size 5 micro m) with acetonitrile -10 mM aqueous ammonia (85 : 15, v/v) as the mobile phase, at a flow-rate of 0.20 ml min(-1). A butyric acid analogue of KF was used as the internal standard. The lower limit of quantitation (LLQ) using a 500 micro l sample volume was 1 ng ml(-1) and the linear dynamic range extended to 1000 ng ml(-1). The inter-assay accuracy of the assay was -15.1% at the LLQ and between -2.68 and -9.05% for quality control solutions ranging in concentration from 2.24 to 715 ng ml(-1). The inter-assay precision was 9.91% or better at these concentrations. The analyte was stable in plasma under all relevant conditions evaluated and for a period of 16 h after reconstituting plasma extracts for LC analysis at ambient temperature.

Antineoplastic Agents↗
PubMed1988

Quantitative analysis of aldosterone's role in potassium regulation.

Aldosterone is part of a complex system that regulates plasma potassium concentration by affecting the renal excretion of the ion as well as its distribution within the body. Because there are other components of the system, it has been difficult to determine the physiological significance of aldosterone in potassium regulation by attempting to study the hormone's effects in isolation. In this presentation a quantitative analysis of the potassium control system is used to provide information concerning the physiological role of aldosterone in potassium regulation under normal and pathological conditions, as well as during pharmacological interventions.

Aldosterone↗
PubMed2003

Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): detection of HTLV-I tax-induced CTGF from a human carcinoma cell line.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

Antibodies, Monoclonal↗
PubMed2006

Quantitative analysis of chimerism using a short tandem repeat method on a fluorescent automated DNA sequencer.

Stem cell transplantation (SCT) is the treatment of choice for a number of malignant and nonmalignant diseases. Monitoring of SC engraftment or microchimerism (MC) is important for diagnosis of relapse, rejection or graft vs. host disease (GVHD). The goal of this study was to develop a sensitive and relatively simple method for MC lineage analysis using the Visible Genetics fluorescence automated sequencer. Sensitivity of the method was studied by polymerase chain reaction (PCR) amplification of informative short tandem repeats (STR) using donor/recipient DNA mixtures as the templates and DNA extracted from donor and recipient CD3+, CD19+ and CD15+ cells mixed at various ratios. Semi-quantitative analysis was performed using the Visible Genetics software and percent of donor specific signal was calculated. The sensitivity of this method varied from 0.8% to 6.2% for both DNA and cellular MC in CD3+, CD19+ and CD15+ subsets. Regression analysis revealed linearity (r = 0.94) between the number of donor cells in the mixture and intensity of MC fluorescent signal. These data indicate that the Visible Genetics polyacrylamide gel sequencer can be successfully used for MC analysis in SC recipients providing a relatively high level of sensitivity.

Antigens, CD↗
PubMed1989

Quantitative analysis of labelled inner retinal proteins in experimental optic neuritis.

In order to determine if axonal transport changes in chronic experimental allergic encephalomyelitis (EAE) were due to blockade or increased discharge of fast transported proteins from the inner retina, we examined the presence of pulse labeled proteins in autoradiograms of the optic nerve head, retinal ganglion cell and nerve fiber layers of juvenile strain-13 guinea pigs with chronic EAE and normal controls. Quantitative analysis of silver grains, performed six and twenty-four hours following the intravitreal injection of tritiated leucine, showed a decrease in inner retinal radioactivity in those with EAE, whereas no difference was detected between the two groups after three days. Grain counts within the optic nerve heads of guinea pigs with EAE were reduced at all time intervals studied. These results are consistent with an increase in discharge of fast transported proteins from retinal ganglion cells into optic nerve axons and support our previous observations of increased radioactivity at the foci of optic nerve demyelination.

Animals↗
PubMed2000

Quantitative analysis, by ultrastructural in situ hybridization, of mitochondrial genomes and their expression in mid-gut and ovarian cells of a mutant strain of Drosophila subobscura.

In the mitochondrial deletion mutant strain studied here, two types of DNA coexist (heteroplasmy): intact mtDNA (15.9 kb) and mutant mtDNA (10.9 kb), which represents about 80% of the mitochondrial genomes in somatic tissues. The heteroplasmy level is lower in ovary (63%). Mutation is transmitted unchanged through generations. Quantitative analysis of in situ DNA hybridization demonstrated that for the 12SrDNA probe, of a gene outside the deletion, the mitochondrial DNA cellular content in the studied cells of the mutant strain is 1.5 times higher than in the wild-type strain. For the probe encoding Cyto b, a mitochondrial gene affected by the mutation, the ratios (mutant versus wild-type content) differ according to cell type: close to 0.4 in MGE cells and 0.7 in ovary cells. These values indicate heteroplasmic levels of about 72% in MGE cells and 50% in stage 10 oocytes, which is lower than that previously reported for stage 14 oocytes (60%) and embryos (69%). Analysis of in situ RNA hybridization showed that for the 12SrDNA probe, the transcript concentrations do not differ significantly between MGE cells and cells of germinal origin from the two strains. For the Cyto b probe, the mutant RNA/wild-type RNA ratios are lower in somatic cells than in stage 10 nurse cells and oocytes, but in each case less than expected. These studies indicate that the progressive heteroplasmy increase may be related to intense phases of mitochondria biogenesis and that different compensatory phenomena may exist.

Animals↗
PubMed1976

Anticonvulsant osteomalacia determined by quantitative analysis of bone changes. Population study and possible risk factors.

Material has been obtained by biopsy from the right iliac crest of 60 adult epileptic out-patients receiving chronic anticonvulsant therapy with diphenylhydantoin (DPH), either in single-drug or combined-drug regime, and of 16 controls with the same distribution by sex and age. Four (7%) of the epileptics were hypocalcemic and 25 (42%) had elevated serum alkaline phosphatase values. A quantitative analysis of the morphological bone changes was performed on decalcified and undecalcified bone, using integrating filters and the point count principle. An increased amount of unmineralized bone was found in 32 (53%) of the epileptics. The trabecular osteoclastic resorption surfaces and the mean volume of periosteocytic lacunae were increased in 36 (69%) and 45 (75%) patients, respectively. The calcification rate was decreased in relation to what is referred to elsewhere as normal. The bone changes suggest a mineralization defect analogous to osetomalacia with secondary hyperparathyroidism. An increased osteoid volume or thickness and decreased calcification rate were correlated to low dietary vitamin D intake, low exposure to sunlight, high hepatic clearance rate of DPH, combined-drug treatment and the male sex. These parameters should be considered risk factors of anticonvulsant osteomalacia.

Adolescent↗
PubMed1996

Quantitative analysis of glycogen content in hepatocytes of portal and central lobule zones of normal human liver and in patients with chronic hepatitis of different etiology.

Glycogen content was determined in hepatocytes of different lobule zones of the normal human liver (23 patients without any liver pathology) and the liver of patients with chronic viral B hepatitis (30 patients) and chronic alcohol hepatitis (28 patients). All the patients were males and aged between 17-50 years. Quantitative analysis of the glycogen content in hepatocytes of portal and central lobule zones was carried out in sections of the human liver (material of functional biopsies) stained with PAS-reaction. The measurements were carried out using an image analyser 'Magiscan' which allows combined cytophotometric analysis of a substance in cells and determination of the cell localization in tissue. The results showed significant differences of the glycogen content in different lobule zones in the normal liver and in the liver in chronic viral and alcohol hepatitis. Ratios of glycogen content in hepatocytes of the portal and the central zones of liver lobule were 1.128 +/- 0.004 and 1.061 +/- 0.003 in normal human liver, and liver of patients with chronic viral hepatitis respectively, i.e. the glycogen content in hepatocytes of the portal lobule zone was much higher than in the central lobule zone in the normal liver and in the liver of patients with chronic viral B hepatitis. The ratio in patients with chronic alcohol hepatitis was less than 1.0 (0.930 +/- 0.003), i.e. a significantly higher glycogen content was found in hepatocytes of the central liver lobule zone. Possible mechanisms of this phenomenon are discussed. Thus, the pattern of the glycogen content in hepatocytes of different lobule zones can be used as an indicator of etiology of chronic hepatitis.

Adolescent↗
PubMed1997

[Quantitative analysis of radiologic progression in rheumatoid arthritis: controversies and perspectives].

Rheumatoid arthritis (RA) is a chronic inflammatory disease with symmetrical polyarthritis as the major feature. Persistent inflammation leads to largely irreversible joint damage which can be seen radiographically. Radiographs depict the progression of joint damage and alterations, which are one of the major parameters of RA evolution. Delayed radiologic progression is a good indicator of the success/failure of long-term drug treatment, but the quantitative analysis of changes over time and the reliability of scoring systems remain difficult steps. This paper focuses of the main current scoring systems, with an emphasis on the following four: Larsen method and its modification by Kayle, Sharp method and its modification by van der Heijde. The scoring method--be it grading, counting, or weighted counting--did not appear to influence reliability or repeatability, while the radiologist's training and the film reading technique were critical to identify disease progression accurately. Our data consistently suggest the paired reading method as the most suitable for radiologic progression assessment in RA. The radiologic studies of 62 patients with early RA after 7 years' follow-up showed joint damage in 82% of patients. The average annual progression rate of the total radiologic score obtained with Sharp method, summing erosions and joint space abnormalities, was faster in the earlier years of the disease (8.8 units/year) than later on (4.9 units/year) (p < 0.01). From the trialist's point of view, these results imply that disease duration is a critical feature for RA treatment outcome.

Adult↗
PubMed1989

Immobilized sulfatase:beta-glucuronidase enzymes for the qualitative and quantitative analysis of drug conjugates.

The enzymes sulfatase and beta-glucuronidase from Helix pomatia were simultaneously immobilized on aminopropyl control pore glass. Once immobilized, these enzymes retained activity under varied conditions of pH, organic solvent, and temperature. To hydrolyze the sulfate and glucuronide conjugates of xenobiotics, the immobilized enzymes were either added directly to incubation mixtures for qualitative in vitro studies or packed in a short stainless steel column and placed in an HPLC system for quantitative studies. By incorporating specific inhibitors (D-saccharic acid-1,4-lactone to inhibit beta-glucuronidase or phosphate ions to inhibit sulfatase) into the incubation mixture or into the HPLC mobile phases, selective hydrolysis of either sulfate or glucuronide conjugates was achieved. Upon removal of the inhibitors from the incubation mixtures or from the mobile phases, original enzyme activity was restored. The utility of immobilized enzymes was demonstrated for quantitative analysis of sulfate and glucuronide conjugates of fenoldopam, where the liberation of the catechol aglycone moiety was necessary for electrochemical detection.

Benzazepines↗
PubMed2006

Quantitative analysis of multi-component spherical virus assembly: scaffolding protein contributes to the global stability of phage P22 procapsids.

Assembly of the hundreds of subunits required to form an icosahedral virus must proceed with exquisite fidelity, and is a paradigm for the self-organization of complex macromolecular structures. However, the mechanism for capsid assembly is not completely understood for any virus. Here we have investigated the in vitro assembly of phage P22 procapsids using a quantitative model specifically developed to analyze assembly of spherical viruses. Phage P22 procapsids are the product of the co-assembly of 420 molecules of coat protein and approximately 100-300 molecules of scaffolding protein. Scaffolding protein serves as an assembly chaperone and is not part of the final mature capsid, but is essential for proper procapsid assembly. Here we show that scaffolding protein also affects the thermodynamics of assembly, and for the first time this quantitative analysis has been performed on a virus composed of more than one type of protein subunit. Purified coat and scaffolding proteins were mixed in varying ratios in vitro to form procapsids. The reactions were allowed to reach equilibrium and the proportion of the input protein assembled into procapsids or remaining as free subunits was determined by size exclusion chromatography and SDS-PAGE. The results were used to calculate the free energy contributions for individual coat and scaffolding proteins. Each coat protein subunit was found to contribute -7.2(+/-0.1)kcal/mol and each scaffolding protein -6.1(+/-0.2)kcal/mol to the stability of the procapsid. Because each protein interacts with two or more neighbors, the pair-wise energies are even less. The weak protein interactions observed in the assembly of procapsids are likely important in the control of nucleation, since an increase in affinity between coat and scaffolding proteins can lead to kinetic traps caused by the formation of too many nuclei. In addition, we find that adjusting the molar ratio of scaffolding to coat protein can alter the assembly product. When the scaffolding protein concentration is low relative to coat protein, there is a correspondingly low yield of proper procapsids. When the relative concentration is very high, too many nuclei form, leading to kinetically trapped assembly intermediates.

Bacteriophage P22↗
PubMed1978

Quantitative analysis of activation and inactivation of asymmetry currents in biological membranes, based on a conformational transition model.

A basic voltage-dependent conformational transition mechanism is proposed. It comprises one relatively fast conversion between two individual states which are comparatively slowly coupled with a third state. Having introduced voltage as an additional parameter of state, standard methods of thermodynamics and rate theory are employed to describe the equilibrium and kinetic behavior of the system. In particular, a quantitative discussion is given regarding the asymmetrical displacement currents generated by switching on and off a voltage pulse. Effects of temperature, pulse duration, and application of a conditioning prepulse are examined. The results provide a comprehensive basis for a quantitative analysis of pertinent experimental work. The so far presented measuring data can indeed by very well described along these lines.

Animals↗
PubMedDate not supplied

Quantitative analysis of sulpiride and impurities of 2-aminomethyl-1-ethylpyrrolidine and methyl-5-sulphamoyl-2-methoxybenzoate in pharmaceuticals by high-performance thin-layer chromatography and scanning densitometry.

A simple and reliable thin-layer chromatographic method for determining sulpiride and impurities of 2-aminomethyl-1-ethylpyrrolidine and methyl-5-sulphamoyl-2-methoxybenzoate was developed and validated. A methylene chloride-methanol-ammonia solution (25%; 18 + 2.8 + 0.4, v/v) solvent system is used for separation and quantitative evaluation of chromatograms. The chromatographic plate is first scanned at 240 nm to locate chromatographic zones corresponding to sulpiride and methyl-5-sulphamoyl-2-methoxybenzoate. Then 2-aminomethyl-1-ethylpyrrolidine is derivatized in situ with ninhydrin, and resulting colored spots are measured at 500 nm. The method is reproducible and convenient for quantitative analysis and purity control of sulpiride in its raw material and in its dosage forms.

Benzoates↗
PubMed1997

[Comparison of computerized tomography and magnetic resonance with fast sequences with and without paramagnetic contrast media in the assessment of liver metastasis: qualitative and quantitative analysis].

We compared unenhanced and contrast-enhanced fast MRI and CT in the detection of liver metastases. Eleven patients with single or multiple hepatic lesions (42 in all) were submitted to CT and MR studies; T1- and T2-weighted TSE, T2-weighted TSE with fat suppression, unenhanced breath-hold TFE and early or delayed enhanced breath-hold TFE images were acquired with a 1.5 T super-conductive magnet (Philips NT). The quantitative analysis of all MR images was performed for contrast/noise ratio (CNR) and number of detected lesions; MR and CT images were also compared qualitatively for lesion conspicuity, anatomical structure identification and artifacts. The results were compared with Student's t test. Early enhanced breath-hold TFE was statistically superior to T1-weighted TSE (p = .0009), T2-weighted TSE (p = .01) and CT (p = .0004) for lesion conspicuity and to T1-weighted TSE, T2-weighted TSE, unenhanced TFE (p = .0001) and CT (p = .01) for anatomical structure identification. CT was superior to T1- and T2-weighted TSE (p = .0001) and unenhanced TFE (p = .004) for the lack of artifacts. Fat-suppressed T2-weighted TSE images had a statistically higher CNR than T2-weighted TSE (p = .02), T1-weighted TSE (p = .0006) and unenhanced and delayed TFE sequences (p = .007; p = .0001, respectively). To conclude, MRI appears superior to CT in the detection of liver metastases; the examination should include early enhanced breath-hold T1-weighted TFE and T2-weighted fat-suppressed TSE images.

Adult↗