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PubMed1996

Direct quantitative analysis of peptides using matrix assisted laser desorption ionization.

The protocol and various matrices were examined for quantification of biomolecules in both the low ca. 1200 amu and mid mass 6000-12000 amu ranges using an internal standard. Comparative studies of different matrices of MALDI quantitative analysis showed that the best accuracy and standard curve linearity were obtained for two matrices: (a) 2,5-dihydroxybenzoic acid (DHB) combined with a comatrix of fucose and 5-methoxysalicylic acid (MSA) and (b) ferulic acid/fucose. In the low mass range, the quantitative limit was in the 30 fmol range and in the mid mass range the quantitative limit was in the 250 fmol range. Linear response was observed over 2-3 decades of analyte concentration. The relative error of the standard curve slope was 1.3-1.8% with correlation coefficients of 0.996-0.998.The main problem for quantitative measurement was suppression of the signal of the less concentrated component (analyte or internal standard) by the more concentrated component. The effect was identified with saturation of the matrix by the analyte. The threshold of matrix saturation was found to be in the range of 1/(3000-5000) analyte/matrix molar ratio. To avoid matrix saturation the (analyte+internal standard) to matrix molar ratio should be below this threshold. Thus the internal standard concentration should be as low as possible.DHB/MSA/fucose and ferulic acid/fucose matrices demonstrated good accuracy and linearity for standard curves even when the internal standard had chemical properties different from the analyte. However, use of an internal standard with different chemical properties requires highly stable instrumental parameters as well as constant (analyte+internal standard)/matrix molar ratio for all samples.

Journal Article↗

PubMed1983

Quantitative analysis of glycosaminoglycans in urothelium and bladder wall of calf.

Glycosaminoglycans (GAG) were described by histochemical staining procedures in the normal urothelium of the urinary bladder; they are supposed to be involved in the antibacterial defense mechanism. Our quantitative analysis, however, demonstrated only heparan sulfate in the normal calf urothelium (less than 600 nmol/Gm d.wt.); only trace amounts of other GAG were to be analyzed. High concentrations of GAG were found in the submucosa and muscle layers as to be expected in mesoderm originating tissues. According to these results there were no GAG with the exception of heparan sulfate at the surface of the normal urothelium; therefore, glycoproteins detected in mumol/Gm d.wt. ranges are more likely to be involved in the antibacterial defense mechanism.

Animals↗

PubMed1992

Quantitative analysis of DNA-sequencing electrophoresis.

At the heart of the DNA-sequencing process is a remarkably selective electrophoretic separation of up to 1000 oligonucleotide fragments, each differing in size by only a single nucleotide unit. A quantitative analysis of this separation is performed in terms of both selectivity and efficiency. It is shown that both the Ogston sieving and reptation migration mechanisms are operative. It is demonstrated that, under the conditions used in traditional sequencing electrophoresis, Joule heating does not significantly contribute to band broadening, and that diffusion is the primary contributor to plate height. An analytic expression is derived relating the peak width for each fragment to its molecular size. Calculations are presented showing that, when longer sequences are required, the maximum electrical field strength will be limited by the influence of biased reptation on the separation selectivity. Finally, it is shown that, when short sequences are required, the electrical field strength is limited by the ability to dissipate Joule heat, and that in these cases a tube format will be approximately 50% faster than a slab having a thickness equivalent to the tube diameter.

DNA↗

PubMed1997

Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

ADP-ribosyl Cyclase↗

PubMed1980

The application of high-speed cinematography for the quantitative analysis of equine locomotion.

Locomotive disorders constitute a serious problem in horse racing which will only be rectified by a better understanding of the causative factors associated with disturbances of gait. This study describes a system for the quantitative analysis of the locomotion of horses at speed. The method is based on high-speed cinematography with a semi-automatic system of analysis of the films. The recordings are made with a 16 mm high-speed camera run at 500 frames per second (fps) and the films are analysed by special film-reading equipment and a mini-computer. The time and linear gait variables are presented in tabular form and the angles and trajectories of the joints and body segments are presented graphically.

Animals↗

PubMed1990

[Experimental analytical electron microscopic studies on the quantitative analysis of elemental concentrations in biological thin specimens and its application to dental science].

The aim of this study is to employ an energy dispersive X-ray spectrometer (EDS) in developing a computer software system for the quantitative analysis of elemental concentrations in biological specimens. The methods and the software were applied to the examination of the coronal dentin of human deciduous and permanent teeth. Results 1. Examination methods. Chemical compounds known for their calcium (Ca) and phosphorus (P) contents were used to determine optimum conditions for analysis. The following were the best analytical conditions: 100 kV accelerating voltage, 2 x 10(-10) A probe current, 10 ekV energy scale, 100 sec. counting time, and 100-150 nm section thickness. Under these conditions, it is possible to obtain statistically sufficient integral spectra values. By calculating with the computer software(t-factor) developed in this study, it was possible to arrive at analytical calcium and phosphorus concentration values that are very close to theoretical values. 2. Application to human dentin. Deciduous intertubular dentin contained 24.9% (w/w) Ca and 12.1% (w/w) P; peritubular dentin in the same teeth contained 30.7% (w/w) Ca and 15.3% (w/w) P. Permanent intertubular dentin contained 25.5% (w/w) Ca and 12.5% (w/w) P; peritubular dentin in the same teeth contained 34.5% (w/w) Ca and 16.9% (w/w) P. These results show that, in both permanent and deciduous teeth, concentrations of Ca and P are higher in peritubular than in intertubular dentin. Concentrations Ca and P in both peritubular and intertubular dentin are lower in deciduous than in permanent teeth. The computer software developed for this study differs from chemical analysis and may prove very useful in microanalysis of mineralized tissues on the basis of their ultrastructures.

Autoanalysis↗

PubMed2006

Quantitative analysis of enzymatic assays using indoxyl-based substrates.

Hydrolysis of indoxyl-based substrates by hydrolytic enzymes is a commonly used semiquantitative detection system that generates a water-insoluble indigo dye which is difficult to quantify. This work describes the quantitative analysis and enzyme kinetics for alkaline phosphatase (AP) and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) in solution obtained by applying known solubilization methodology from the textiles industry to the enzymatic product. This proposal is based on the reduction of the tetrahalo-indigo blue dye in a basic medium with the aim of generating its aqueous-soluble parent compound termed indigo white, which gives a rich yellow color in solution and is fluorescent. A quantitative ELISA (where a soluble end product is required) is accomplished for first time using BCIP as substrate.

Alkaline Phosphatase↗

PubMed1987

Quantitative analysis of the interaction between immune complex and C1q complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of C1q to immune precipitates.

A novel method was developed for the analysis of the interaction of large multivalent ligands with surfaces (matrices) to analyse the binding of complement subcomponent C1q to immune precipitates. Our new evaluation method provides quantitative data characteristic of the C1q-immune-complex interaction and of the structure of the immune complex as well. To reveal the functional role of domain-domain interactions in the Fc part of IgG the binding of C1q to different anti-ovalbumin IgG-ovalbumin immune complexes was studied. Immune-complex precipitates composed of rabbit IgG in which the non-covalent or covalent bonds between the heavy chains had been eliminated were used. Non-covalent bonds were abolished by splitting off the CH3 domains, i.e. by using Facb fragments, and the covalent contact was broken by reduction and alkylation of the single inter-heavy-chain disulphide bond. The quantitative analysis of the binding curves provides a dissociation constant (K) of 200 nM for the interaction between C1q and immune precipitate formed from native IgG. Surprisingly, for immune precipitates composed of Facb fragments or IgG in which the inter-heavy-chain disulphide bond had been selectively reduced and alkylated, stronger binding (K = 30 nM) was observed. In this case, however, changes in the structure of the immune-complex matrix were also detected. These structural changes may account for the strengthening of the C1q-immune-complex interaction, which can be strongly influenced by the flexibility and the binding-site pattern of the immune-complex precipitates. These results suggest that domain-domain interactions in the Fc part of IgG affect the segmental mobility of IgG molecules and the spatial arrangement of the immune-complex matrix rather than the affinity of individual C1q-binding sites on IgG.

Animals↗

PubMed2004

Quantitative analysis and classification of AFM images of human hair.

The surface topography of human hair, as defined by the outer layer of cellular sheets, termed cuticles, largely determines the cosmetic properties of the hair. The condition of the cuticles is of great cosmetic importance, but also has the potential to aid diagnosis in the medical and forensic sciences. Atomic force microscopy (AFM) has been demonstrated to offer unique advantages for analysis of the hair surface, mainly due to the high image resolution and the ease of sample preparation. This article presents an algorithm for the automatic analysis of AFM images of human hair. The cuticular structure is characterized using a series of descriptors, such as step height, tilt angle and cuticle density, allowing quantitative analysis and comparison of different images. The usefulness of this approach is demonstrated by a classification study. Thirty-eight AFM images were measured, consisting of hair samples from (a) untreated and bleached hair samples, and (b) the root and distal ends of the hair fibre. The multivariate classification technique partial least squares discriminant analysis is used to test the ability of the algorithm to characterize the images according to the properties of the hair samples. Most of the images (86%) were found to be classified correctly.

Algorithms↗

PubMed2000

Scanning laser polarimetry of the normal human retinal nerve fiber layer: a quantitative analysis.

PURPOSE: To provide a quantitative comparison of retardation values obtained with the scanning laser polarimeter (Nerve Fiber Layer Analyzer; Laser Dignostic Technologies, San Diego, California) in normal subjects, with the known histologic properties of the human retinal nerve fiber layer. METHODS: Scanning laser polarimetry was performed on 48 normal subjects. The retardation values obtained from each eye were processed to remove reflections from the optic nerve head vasculature and to derive peripapillary polar retardation profiles. The location of the maximum and minimum retardation values superior and inferior to the disk was determined. Retardation values were also determined along lines running radially from the center of the optic disk to through the points of maximal retardation. RESULTS: Retardation values were greater in the superior and inferior quadrants of the peripapillary retinal nerve fiber layer compared with the nasal and temporal quadrants. Superiorly, peak retardation values were nasal to the vertical meridian by 22.9 degrees (SD, 17.5) in right eyes and by 15.7 degrees (SD, 17.7) in left eyes. Inferiorly, the peak showed a closer correspondence with the vertical meridian, lying nasal to this by 0.8 degrees (SD, 14.9) in right eyes and temporal to this by 2.8 degrees (SD, 15.4) in left eyes. Radial analysis showed that the peak retardation occurred approximately (0.5 to 0.7 disk diameters from the optic disk margin) at the superior and inferior aspect of the disk. The degree of modulation of retardation around the optic disk was less than would be expected from the variation in peripapillary nerve fiber layer thickness seen histologically. CONCLUSION: The location of the peak retardation values shows reasonable agreement with the angular location of the peak values of nerve fiber layer thickness published for humans. By contrast, the modulation of retardation values around the disk and the change with eccentricity from the optic disk center differ from the anatomic data. The Nerve Fiber Layer Analyzer appears to measure a correlate of nerve fiber layer thickness, but it is important that any discrepancies between the retardation and anatomic data are recognized in the clinical interpretation of polarimetric data.

Adult↗

PubMedDate not supplied

Methods of chromatography and quantitative analysis of galacto- and phospholipids of plant leaves.

Data in the literature on chromatography of glycerolipids of plant leaves on columns of DEAE-cellulose, Sephadex LH-20, silica gel, florisil, in a thin layer of silica gel, paper are generalized. Methods of obtaining chromatographically pure glycerolipids, their subfractionation, and the quantitative analysis of glycerolipids are described. Experimental data are presented on fractionation of lipids of potato leaves on columns of DEAE-cellulose, silica gel, on paper, and the quantitative determination of phospholipids. A method of rapid division of lipids into classes by means of their elution with silica gel KSC by different solvents is described.

Chromatography, DEAE-Cellulose↗

PubMed1996

Quantitative analysis of clustering on biological membranes: methodology and application to ligand-induced asialoglycoprotein receptor redistribution on rat hepatocytes.

Ligand-induced receptor clustering is the first step in receptor mediated endocytosis of asialoglycoproteins by rat hepatocytes. This well-characterized receptor was used as a model system to set up a general method for the quantitative analysis and the visualization of molecular clustering on surface replicas, using a two-step approach. In the first step, aiming at the quantitation of clustering, gold-labeled asialoglycoprotein receptors on the cell surface were assumed to reflect two populations, one of clustered and one of not-clustered receptors. The experimental distribution of nearest neighbor distances of labeled receptors was adjusted by least square fitting to the sum of two functions, each corresponding to the theoretical nearest neighbor distance distribution of randomly distributed points, corresponding to non-clustered and clustered particle concentrations, respectively. The resolution of the experimental nearest neighbor distribution into these two components yielded an objective estimate of the number of labeled receptors in clusters and of the total surface of clusters. The second step, aiming at the visualization of clusters, rested on the fact that the distance of a point to its neighbors is shorter in a cluster than outside the cluster. Accordingly, each particle was ordered according to the sum of the distances to its nearest neighbors. Clustered particles with the lowest cumulative distances were extracted, in proportion of the extent of clustering determined in the first step, and displayed on the computer screen. This translated the two components into the two corresponding populations of identified particles. This method demonstrated that, if rat hepatocytes were incubated at 4 degrees C with either asialofetuin-gold complexes before fixation, or with asialofetuin prior to fixation and immunogold labeling of the ASGP-R, up to 65% of the receptors became clustered on the dorsal cell surface in areas where receptors could be concentrated up to 20-fold, as compared with randomly distributed molecules. In ultrathin sections, clusters essentially corresponded to clathrin-coated pits. In principle, this method is generic and can be applied to other biological systems, especially when statistical analysis is needed for studying the dynamics of the clustering process.

Animals↗

PubMed2004

Development and validation of an isotope-dilution electrospray ionization tandem mass spectrometry method with an on-line sample clean-up device for the quantitative analysis of the benzene exposure biomarker S-phenylmercapturic acid in human urine.

An isotope-dilution electrospray ionization tandem mass spectrometry (ESI-MS/MS) method with an on-line sample clean-up device, for the quantitative analysis of human urine for the benzene exposure biomarker S-phenylmercapturic acid (SPMA), was developed and validated. The sample clean-up system was constructed from an autosampler, a reversed-phase C18 trap cartridge, a two-position switching valve, and controlling computer software and hardware. The sample clean-up system was interfaced via 1/20 splitting to the ESI source of a triple-quadrupole mass spectrometer using negative ion mode and multiple reaction monitoring for SPMA and the isotope-labeled internal standard. A strategy was adopted to acquire pooled blank urine matrix and quality control samples spiked with standards. Validated procedures and data on method specificity, detection limits, standard curves, precision and recovery, sample storage stability, and inter-laboratory comparison are presented. The analytical system was fully automated. No tedious manual sample clean-up procedures are required. With the selectivity and the sensitivity provided by ESI-MS/MS detection, the analytical system can be used for high-throughput and accurate determination of SPMA levels in human urine samples, as a biomarker for environmental as well as occupational benzene exposure.

Acetylcysteine↗

PubMed1983

Quantitative analysis of changes in cell shape of Amoeba proteus during locomotion and upon responses to salt stimuli.

A new parameter expressing the complexity of cell shape defined as (periphery)2/(area) in 2D projection was found useful for a quantitative analysis of changes in the cell shape of Amoeba proteus and potentially of any amoeboid cells. During locomotion the complexity and the motive force of the protoplasmic streaming in amoeba varied periodically, and the Fourier analysis of the two showed a similar pattern in the power spectrum, giving a rather broad peak at about 2.5 X 10(-3) Hz. The complexity increased mainly due to elongation of the cell as external Ca2+ increased. This effect was blocked by La3+, half the inhibition being attained at 1/200 amount of the coexisting Ca2+. On the other hand, the complexity decreased due to rounding up of the cell as the concentration of other cations, such as Sr2+, Mg2+, Co2+, Ni2+, Na+, K+ etc., increased. Irrespective of the opposite effects of Ca2+ and other cations on the cell shape, the ATP concentration in amoeba decreased in both cases with increase of all these cations. The irregularity in amoeboid motility is discussed in terms of a dynamic system theory.

Adenosine Triphosphate↗

PubMed1980

Quantitative analysis for innervation of smooth muscle cells in the wall of the urinary bladder.

The distribution of nerve terminals to different parts of the urinary bladder was studied by electron microscopy with the aid of 6-hydroxydopamine treatment. A quantitative analysis was made for each region. In the sphincter and trigone areas nerve terminals were found to be much more numerous (3 times more) than in sections from the body and the apex of the bladder. In much of the bladder there were 6--7 smooth muscle cells for each nerve fiber, however in the trigone area 1--2 muscle cells were supplied by terminal nerve fiber. The narrowest gap between nerve terminals and smooth muscle cells was found to be 150--250 nm. It was commonly seen that Desmosome-like attachments, called nexuses, connected adjacent smooth muscle cells. After 6-hydroxydopamine treatment degenerated terminals could be found mostly in the fine periarterial plexuses but some were also observed that were not related to blood vessels, especially in the trigone area. Summation of our figures and results for terminal density suggests to us that contractions of the urinary bladder are initiated or controlled in the trigone region and that other parts of the wall are activated by local nerve processes and the numerous nexal contacts.

Animals↗

PubMed2001

Electroencephalography in juvenile neuronal ceroid lipofuscinosis: visual and quantitative analysis.

Fourteen patients with a confirmed diagnosis of juvenile neuronal ceroid lipofuscinosis (JNCL) (aged 6-12.5 years at the beginning of the study) were prospectively followed for 5 years. An electroencephalogram (EEG) was recorded and analysed both visually and quantitatively and a neuropsychological examination was performed once a year. In addition, a cross-sectional EEG study of 32 patients aged 5-27 years was performed. The EEG was often normal before the age of 9 years, and thereafter a progressive background abnormality and increase in paroxysmal activity took place. The EEGs were significantly slower than those of the controls, and the speed of slowing of EEG correlated to the decrease in intelligence quotients (IQ). Quantitative analysis was superior to visual analysis in detecting the deterioration of the background activity. The best parameter describing this was the fast/slow ratio. Peak frequency, percentage of theta and the fast/slow ratio correlated with IQ.

Adolescent↗

PubMed1999

Effects of scatter and attenuation correction on quantitative analysis of beta-CIT brain SPET.

The aim of this study was to evaluate the effects of scatter correction (SC) and attenuation correction (AC) on the quantification of dopamine transporters using 123I-beta-CIT brain SPET images. Quantitative analysis was carried out using static SPET images obtained 23 h after injection. We calculated V3" [(striatal-occipital)/occipital ratio] values from images without correction, with AC, and with SC and AC. Two types of regions of interest (ROI) were placed on the striatum: a small square ROI and a larger ROI containing most of the striatum. After validating the correction method in a phantom experiment, a human study was carried out involving eight normal volunteers and 15 patients. The larger ROI yielded smaller V3" values. The effect of attenuation correction was modest, whereas that of scatter correction was marked. It was shown that beta-CIT SPET quantification was affected by the size of the ROI, photon scattering and attenuation, and that scatter and attenuation correction improved the accuracy of the quantification. Methodological standardization in image processing and the type of ROI should be considered when a multi-centre trial is planned.

Aged↗

PubMed1989

[Aldehydes and ketones in silage: quantitative analysis by high performance liquid chromatography].

Carbonyl compound toxicity is known in several species but no study has been carried out with ruminants. Such volatile compounds exist in silages. After condensation of aldehydes and ketones with 2,4-dinitrophenylhydrazine, quantitative analysis was performed with 37 silages. It was found that quantities of carbonyl compounds varied from 36 mg/kg of dry matter (DM) to 1,535 mg/kg DM with a mean value of 642 mg/kg DM. Ethanal was 63% of the total amount of carbonyl compounds (mol/kg DM). Other molecules were propanal, propanone, butanal (n- and iso-), butanone and n- and iso- pentanal. The total amount of carbonyl compounds correlated positively with the dry matter percentage and negatively with the pH, crude fiber, ash content and volatile fatty acids. Thus, carbonyl compounds seem to be dependent up on silage storage conditions. Lactic flora could be involved in the synthesis of these compounds.

Aldehydes↗