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PubMed1999

Quantitative analysis of regular and irregular astigmatism induced by pterygium.

PURPOSE: To quantitatively evaluate the influence of pterygium and its removal surgery on both regular and irregular corneal astigmatism. METHODS: In 19 eyes of 19 patients undergoing pterygium surgery, videokeratographic measurements were taken before and 1 month after surgery. Using Fourier harmonic analysis, dioptric data on mire rings were decomposed into spherical, regular astigmatism, and irregular astigmatism (decentration and higher order irregularity) components. The distance between the line of sight and the advancing edge of pterygium was measured, and the eyes were classified into two groups: large pterygium group (the distance <2.0 mm, n = 7) and small pterygium group (> or =2.0 mm, n = 12). Fifteen eyes of 15 subjects served as age-matched normal control eyes. RESULTS: Before surgery, the magnitudes of regular astigmatism and higher order irregular astigmatism showed significant correlation with the size of pterygium. Regular astigmatism, asymmetry, and higher order irregularity in the large pterygium group were significantly greater than those of normal control eyes. The pterygium removal surgery significantly improved these changes, but regular astigmatism and higher order irregularity in the large pterygium group still remained significantly greater than those in the normal control eyes. CONCLUSION: Pterygium induces a significant amount of regular and irregular astigmatism in proportion to its size. The removal surgery can improve these changes, but corneal distortion does not normalize completely in eyes with advanced pterygium.

Aged↗
PubMed2000

Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.

Leptin, the protein hormone product of the obese (ob) gene, functions in the regulation of appetite, energy expenditure, and reproduction in animals and humans. Since changes in the level of circulating leptin can have marked physiological consequences, it is important to be able to accurately quantify leptin gene expression. Toward this goal, we have constructed a chicken leptin RNA competitor and successfully employed it as an internal standard in the development of a quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR) assay for leptin mRNA. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was utilized for the separation and analysis of chicken leptin target (261 bp) and competitor (234 bp) dsDNA products from QC-RT-PCR assay samples. Leptin amplicons were separated using a DB-1 coated capillary (27 cm x 100 microm ID) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 x TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microg/mL EnhanCE fluorescent intercalating dye. Samples were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. QC-RT-PCR/CE-LIF was used to quantify leptin mRNA in liver and adipose tissue from 8-week-old male and female broiler chickens. This study is the first report of quantitative analysis of leptin gene expression using QC-RT-PCR/CE-LIF.

Adipose Tissue↗
PubMed1986

Quantitative analysis of sex-chromosome mosaicism with X-Y DNA probes.

Sex-chromosome mosaicism was quantitatively analyzed in two patients using DNA probes specific for human X and Y chromosomes. Both patients were female with stigmata of the Turner syndrome, and both had a 45,X cell line and a 46,XY cell line. One of the patients had a morphologically abnormal, nonfluorescent Y chromosome, dic(Y)(q11). Hybridization of DNA from this patient with two repetitive DNA sequences specific for the heterochromatic region of the Y chromosome indicated that most of the Y-heterochromatic sequences were deleted. DNA from both patients was hybridized with a probe for the DXYS1 locus and found to have the X- and Y-linked loci. Densitometric measurements of the relative intensities of the X- and Y-linked bands were used to calculate the degree of mosaicism in each case. The percentages of 45,X cells obtained by DNA analysis agreed with those obtained by chromosome analysis. DNA analysis provides a way to quantitate mosaicism at the DNA level and in nondividing tissue.

Adolescent↗
PubMed1986

Quantitative analysis of individual motor unit potentials: a proposition for standardized terminology and criteria for measurement.

The physiology of the motor unit potential (MUP) is reviewed. The aim is to identify the electrophysiological events in the motor unit that generate the individual parts of the MUP. This is based on insight gained from new experimental techniques, such as single-fiber electromyography (EMG), scanning EMG, and simulation studies of the MUP. A terminology for the different parts of the MUP is also suggested, and nine parameters used to describe different features of the MUP are delineated: duration, spike duration, amplitude, area, spike area, phases, turns, satellites, and variability. Technical aspects, such as electrode type, filtering, and sampling rate of the computers, are discussed as well. In Appendix A, different manual and computer-aided methods for quantitative MUP analysis are described. Despite minor systematic differences between the methods, MUP durations measured by different methods correlate highly with each other (Appendix B). The manual and computer-aided methods have comparable variability between repeated measurements.

Action Potentials↗
PubMed2000

Quantitative analysis of cytokeratin 20 gene expression using RT-PCR and capillary electrophoresis with fluorescent DNA detection.

OBJECTIVE: We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue. DESIGN AND METHODS: Our method incorporates a calibrated PCR with an internal competitor and an external standard. RESULTS: The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples. CONCLUSION: To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

Biomarkers, Tumor↗
PubMed1980

Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols.

The effect of different fixation solutions on the denaturation of membrane-associated antigens in murine lymphoid cells was determined quantitatively using microfluorometric analysis and a radioimmunoassay. Paraformaldehyde and periodate-lysine-paraformaldehyde solutions preserved the antigenicity of cell surface-associated immunoglobulin (S-Ig) antigens when used in concentrations ranging from 0.01 to 4%. However, glutaraldehyde destroyed the antigenicity of S-Ig and Thy 1.2 molecules at concentrations higher than 0.1%. Electron microscopic analysis of the different fixed cell suspensions, after labelling of the cells with a rabbit anti-mouse immunoglobulin-horseradish peroxidase conjugate (RaM-Ig-HRP) showed that prefixation of the sample with 0.1% glutaraldehyde was optimal for immunoelectron microscopical studies, since this concentration preserved both the antigenicity of membrane-associated antigens as well as the ultrastructure of the cells under study. Prolonged fixation periods affected antibody binding. However, S-Ig molecules denatured at a slower rate than Thy 1.2 molecules. A preparation method for the immunoelectron microscopical localization of lymphoid and non-lymphoid cell types in lymphoid organs is reported.

Animals↗
PubMed1999

Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS.

Electrospray mass spectrometry (ES-MS) is a powerful tool for analysis of carcinogen-adducted DNA. In this study, we developed a quantitative isotope dilution method for analysis of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP), the principal nucleoside adduct derived from enzymatic hydrolysis of 4-aminobiphenyl (4-ABP)-modified DNA. The method used column switching valves to perform on-line sample concentration and cleanup, which permitted direct analysis of enzymatic DNA hydrolysates using narrow-bore liquid chromatography (LC). ES-MS detection was performed using a single quadrupole instrument by monitoring M+H+ and two fragment ions characteristic for dG-C8-4-ABP, along with M+H+ and a fragment ion for the deuterated internal standard. The detection limit for dG-C8-4-ABP in DNA hydrolysates was approximately 10 pg on-column, equivalent to 0.7 dG-C8-4-ABP adducts in 10(7) normal nucleotides for a sample containing 100 microg DNA. The method was applied to the analysis of calf thymus DNA modified in vitro through reaction with N-hydroxy-4-ABP and of hepatic DNA isolated from mice treated in vivo with two dose levels of 4-ABP.

Aminobiphenyl Compounds↗
PubMed2005

Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.

Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the sphingolipids in a biological system are bioactive and are often closely related structurally and metabolically (for example, complex sphingolipids<-->ceramide<-->sphingosine<-->sphingosine 1-phosphate), to understand the role(s) of sphingolipids in a given context one must conduct a "sphingolipidomic" analysis-i.e., a structure-specific and quantitative measurement of all of these compounds, or at least all members of a critical subset. Liquid chromatography tandem mass spectrometry (LC MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples ( approximately 10(6) cells). This review describes a series of protocols that have been developed for the relatively rapid analysis of all of the molecular species from 3-ketosphinganines through sphingomyelins and some glycosphingolipids (including all the compounds that are presently regarded as sphingolipid "second messengers") using normal- and reverse-phase LC to separate isometric and isobaric species (such as glucosylceramides and galactosylceramides) in combination with triple quadrupole (for MS/MS) and hybrid quadrupole-ion trap (for MS3) mass spectrometry. Also discussed are some of the issues remaining to be resolved in the analysis of the full sphingolipidome.

Animals↗
PubMed1998

Quantitative analysis of sirolimus (Rapamycin) in blood by high-performance liquid chromatography-electrospray tandem mass spectrometry.

We report here a quantitative method for the analysis of sirolimus in blood using solid-phase sample preparation and HPLC-electrospray-tandem mass spectrometry detection. Blood samples (500 microl) were prepared by pre-treatment with acetonitrile: 15 mM zinc sulphate (70:30, v/v), containing 32-demethoxysirolimus (internal standard) and C18 solid-phase extraction. The electrospray conditions were chosen to enhance the [M+NH4]+ species at the expense of other species. Detection was by multiple reactant monitoring with the mass transitions m/z 931.8-->864.6 and m/z 901.8-->834.4 employed for sirolimus and the internal standard, respectively. The method was linear over the range 0.2 to 100.0 microg l(-1). The accuracy and inter-day precision, over this concentration range, was 94.4% to 104.4% and 1.4% to 5.0%, respectively. The accuracy and total precision at the limit of quantitation (0.2 microg l(-1)) was 103.0% and 10.8%, respectively. The mean absolute recovery of sirolimus and the internal standard were 80.5% and 81.3%, respectively. The sensitivity and analytical concentration range of the method make it suitable for therapeutic drug monitoring and pharmacokinetic studies. Further, the ability of the method to measure parent drug specifically will facilitate the evaluation of immunoassays for sirolimus.

Anti-Bacterial Agents↗
PubMed2005

An investigation of microleakage from root-end fillings in ultrasonic retrograde cavities with or without finishing: a quantitative analysis.

OBJECTIVE: The aim of this study was to make a quantitative assessment of the sealing ability of Super-EBA, IRM, and Pro Root MTA root-end fillings subjected to 3 different finishing techniques. STUDY DESIGN: Eighty-one ultrasonically prepared root-end cavities in human canines were separated randomly into 3 test groups of 27 roots each. The cavities were filled with Super-EBA, IRM, or Pro Root MTA and finished by ball burnishing. Sequentially, 18 roots from each group received a final smoothing with either a 30-fluted tungsten carbide finishing bur or a Zekrya carbide 28-mm bur. Samples were prepared and immersed in 2% methylene blue dye neutral solution for 12 hours. Roots were ground into a powder and prepared for analysis in an absorbency spectrophotometer. RESULTS: The results revealed that Pro Root MTA displayed significantly less mean dye microleakage ( P < .05) than Super-EBA and IRM root-end fillings. The Super-EBA root-end fillings, although presenting a greater mean dye microleakage, did not differ significantly from IRM ( P > .05). The finishing technique did not significantly ( P > .05) affect the incidence of microleakage among the materials tested. CONCLUSIONS: The favorable results obtained with MTA in leakage studies may be related to its good marginal adaptation. Spectrophotometric analysis may provide valuable information about the sealing capacity of root-end fillings. None of the procedures tested were able to avoid leakage, a finding that stresses the importance of the eradication of irritants within the root canal system.

Aluminum Compounds↗
PubMed1981

Quantitative analysis of microvascular structure and function in the amelanotic melanoma A-Mel-3.

Blood cell velocity, capillary diameter, and capillary length were determined in the microcirculation of the amelanotic hamster melanoma A-Mel-3 as well as in s.c. tissue of tumor-free animals. Studies were carried out using a dorsal skin flap chamber, intravital microscopy, and television techniques after transplantation of a 0.5-cu mm piece of tumor tissue. The tumor revealed a special microvascular configuration of short, thin-walled, sometimes dilated capillaries running around the edge of the tumor. Large avascular areas appeared in the center part approximately 5 days after tumor transplantation. Although mean capillary blood cell velocity was not different in tumor-containing and tumor-free preparations, localized irregularities of blood flow were observed close to points of endothelial sacculations. Huge platelet conglomerates were consistently noted in capillaries of the tumor, blocking the blood stream temporarily. Due to discrepancies in microvascular morphology and lack of visible vascularization, large parts of this tumor seem to be inaccessible to tumor treatment. This implies that better vascularization of these regions might enhance the efficiency of cancer treatment. The chamber technique, intravital microscopy, and television methods combined with the subsequent, quantitative microvascular analysis may provide a unique means for direct evaluation of local therapy, particularly during early melanoma growth.

Animals↗
PubMed2006

Quantitative analysis of SNRPN(correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are 2 distinct neurodevelopmental disorders caused primarily by deficiency of specific parental contributions at an imprinted domain within the chromosomal region 15q11.2-13. In most cases, lack of paternal contribution leads to PWS either by paternal deletion (approximately 70%) or maternal uniparental disomy (UPD; approximately 30%). Most cases of AS result from the lack of a maternal contribution from this same region by maternal deletion (approximately 70%) or by paternal UPD (approximately 5%). Analysis of allelic methylation differences at the small nuclear ribonucleoprotein polypeptide N (SNRPN) locus can differentiate the maternally and paternally inherited chromosome 15 and can be used as a diagnostic test for AS and PWS. METHODS: Sodium bisulfite-treated genomic DNA was PCR-amplified for the SNRPN gene. We used pyrosequencing to individually quantify the resulting artificial C/T sequence variation at CpG sites. Anonymized DNA samples from PWS patients (n = 40), AS patients (n = 31), and controls (n = 81) were analyzed in a blinded fashion with 2 PCR and 3 pyrosequencing reactions. We compared results from the pyrosequencing assays with those obtained with a commonly used methylation-specific PCR (MS-PCR) diagnostic protocol. RESULTS: The pyrosequencing assays had a sensitivity and specificity of 100% and provided quantification of methylation at 12 CpG sites within the SNRPN locus. The resulting diagnoses were 100% concordant with those obtained from the MS-PCR protocol. CONCLUSIONS: Pyrosequencing is a rapid and robust method for quantitative methylation analysis of the SNRPN locus and can be used as a diagnostic test for PWS and AS.

Angelman Syndrome↗
PubMedDate not supplied

[Development of rapid methods for quantitative analysis of proteolytic reactions].

The approaches for development of express methods for quantitative control of proteolytic reactions are discussed. Recently, these reactions have taken on special significance for revealing many important problems of theoretical and practical medicine and biology as well as for technological, pharmacological and ecological monitoring. Traditional methods can be improved both by use of immobilized enzymes and substrates, and on the basis of combination of various classic biochemical and immunological approaches. The synthesis of substrates with specified properties allows new methods to be realized for the study of the proteinase activity and kinetic characteristics of the corresponding reactions both in vitro and in vivo. An application of biosensor technology is promising trend since it allows the analysis time and cost to be saved, the direct interaction between enzymes and their inhibitors and activators to be studied in a real time scale, the quantitative measurements to be performed both in liquids and in the air. Besides, biosensor technique is well compatible with computer data processing.

Animals↗
PubMed1986

[Quantitative analysis of capillary and neuron images under normal conditions and in dementia].

Stereological investigations (Leitz-Classimat) of the capillary net of young (19-44 yrs), old (85-95 yrs) and age-matched demented patients with Alzheimer's disease show a condensation (40%) of the capillary volume in the cerebral cortex of the Alzheimer group (n-3) compared with the age-matched controls (n-7), without change of the capillary diameter. These results represent gross atrophy of the frontal brain in senile dementia of Alzheimer type (SDAT). No changes of this kind can be observed between young individuals (n-6) and normal aged group. The behaviour of the capillary net in the putamen is different from that of the cortex. Already during normal aging a 80% condensation of the capillary volume is observed (capillary volume and length per unit increase, intercapillary distances decrease). A comparison between the aged group and the Alzheimer patients exhibits neither additional alterations of capillary parameters nor decreased volume of the putamen. In all anatomical layers of the frontal cortex a significant atrophy (27-36%) of neuronal perikarya (size and shape measurements with the TAS of Leitz) occurs in the Alzheimer group, compared with the normal aged ones. In the same way, neuronal surface area decreases by 30% in the putamen. Significant changes of perikaryal shape in both brain regions confirm marked neuronal atrophy in Alzheimer's disease. During normal aging only 85-95 years old group shows significantly smaller (15-35%) neurons in comparison to young individuals. Quantitative image analysis facilitates considerably evaluating new morphometrical data of the aging process in the human brain, which are important for a pharmacological concept of treating cerebral insufficiency symptoms.

Adult↗
PubMedDate not supplied

Quantitative analysis of glucose loss during acute therapy for hyperglycemic hyperosmolar syndrome.

Four patients with severe hyperglycemia and hyperosmolality were studied to quantitate the major mechanisms responsible for the fall in blood glucose concentration. Insulin was not administered to any of these patients during the first 15 h of therapy. In each case, there was a fall in glucose concentration due to dilution; this was quantitated by chloride space analysis and accounted for 24-34% of the fall in concentration. The size of the glucose pool decreased for two reasons. Glucosuria accounted for the majority of the reduction in the size of the glucose pool in the patients with the smallest decrease in extracellular fluid (ECF) volume [and hence the best preserved glomerular filtration rate (GFR)]. In contrast, glucosuria was a less important factor in causing glucose loss in the patients with very low GFR values. The size of the glucose pool also decreased due to glucose metabolism that did not require exogenous insulin. Thus the fall in glucose concentration in the initial therapy in patients with the hyperglycemic hyperosmolar syndrome is multifactorial and is not absolutely dependent on exogenous insulin. Furthermore, the patients grouped in this diagnostic category represent a heterogeneous population with the common features of severe hyperglycemia, hyperosmolality, and a negative or weakly reactive test for serum ketones.

Adult↗
PubMed1997

Quantitative analysis of spiral ganglion projections to the cat cochlear nucleus.

A quantitative examination of the tonotopic organization of primary afferent projections to the cochlear nucleus (CN) in adult cats was conducted by using focal extracellular injections of Neurobiotin (NB) into the spiral ganglion of the basal cochlea. One to three injections separated by intervals of at least 2 mm were positioned along the basal one-third of the cochlea. Each injection produced discrete projection laminae that appeared as parallel horizontal sheets of labeled axons terminals distributed sequentially dorsally to ventrally across each major CN subdivision: the anteroventral, posteroventral, and dorsal cochlear nucleus, (AVCN, PVCN, and DCN, respectively). The length (rostrocaudal dimension), width (mediolateral dimension), thickness (dorsoventral dimension), and relative placement of 18 "frequency-band" laminae were measured in 10 adult cochlear nuclei. The average AVCN projection thickness was approximately twice that of the PVCN and DCN projections. In double injection cases, the center-to-center separation between AVCN laminae was also approximately twice that in the PVCN and equal to that in the DCN. Lamina thickness did not differ significantly as a function of frequency representation. However, in both width and length, mid-frequency laminae were up to two times larger than high-frequency laminae. Thus, the results indicate that DCN projections are the most discrete (i.e., are the thinnest and have the least overlap between adjacent frequency projections), whereas the AVCN projections are the largest but are as discrete as PVCN projections. In addition, high-frequency projections are smaller and more discrete than mild-frequency projections, which are larger and have greater overlap with adjacent frequency projections.

Animals↗
PubMed2001

Quantitative analysis of the dendrites of sacral preganglionic neurons in the cat.

Quantitative analyses were performed on the dendrites and somata of 25 electrophysiologically identified preganglionic neurons (PGN) obtained from the sacral spinal cord of the cat by intracellular injection of Neurobiotin or horseradish peroxidase. Total dendritic length and surface area were measured for each dendrite. The sizes of the stem dendrites measured at their base were positively correlated with the sizes of the entire tree and numbers of end branches. Total surface area of somata and dendrites averaged 39,138 microm(2); 90.7% of that was from the dendrites. To obtain measurements of the relative contributions of PGN dendrites to specific regions of the spinal cord, the percentage of each dendrite occupying eight spinal cord regions was recorded. Sixty-three percent of the dendrites projected dorsal to their somata, whereas an average of 33.3% of dendrites were located in the white matter, most of them in the lateral and dorsolateral funiculi. The neurons within this sample formed a continuum with some neurons having a large percentage of dendrites in lamina I but little in the white matter, whereas at the other end of the continuum were cells with the reverse configuration. The intermediate neurons had dendrites in both locations. Taken together, these data indicate a heterogeneous population of PGN in the lateral band of the sacral parasympathetic nucleus.

Animals↗
PubMed2004

Quantitative analysis of synaptic distribution along thalamocortical axons in adult mouse barrels.

Quantitative data on thalamocortical synapses in adult mouse barrels have been obtained largely by using lesion-nduced degeneration to label thalamic afferents. By the time degenerating axons can be identified with the electron microscope, they have broken up into many separate pieces, making it impossible to assess the distribution of synapses along unbroken lengths of afferent. Here, this deficiency is rectified by examining intact lengths of axon labeled by the injection of biotinylated dextran amine into ipsilateral thalamus. Serial thin section reconstructions were analyzed to determine the numbers of synapses per axon length made with dendritic spines vs. shafts and the locations of synapses with respect to axonal varicosities. Results for seven axonal segments from six mice showed an average of 0.2 synapses/microm; 80% were made with spines and 20% with dendritic shafts. Just over two-thirds of axonal varicosities formed one synapse; most of the remainder formed two and rarely three, whereas 8% formed none. Although most synapses occurred at varicosities (88%), more than 12% were made at cylindrically shaped regions of the reconstructed axonal segments. These results serve as a caveat for the use of light microscopy to quantify synapses, wherein the usual approach is to equate one varicosity with one synapse. For thalamocortical afferents to mouse barrels, equating one varicosity with one synapse would prove to be incorrect more than 30% of the time and would exclude the roughly 12% of synaptic connections made at cylindrical regions of thalamocortical afferents.

Animals↗