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PubMed1984

Quantification of alprazolam in serum or plasma by liquid chromatography.

A reversed-phase "high-performance" liquid-chromatographic assay for the quantification of alprazolam in serum or plasma is described. Serum or plasma is extracted with toluene/isoamyl alcohol (99/1 by vol), evaporated, and reconstituted in the mobile phase. The latter is washed with hexane, then subjected to reversed-phase liquid chromatography and ultraviolet detection at 202 nm. Either U-31485, an alprazolam analog, or lorazepam, a 3-hydroxybenzodiazepine, is satisfactory as internal standards. Major alprazolam metabolites and various other commonly used drugs do not interfere. The useful lower limit of sensitivity for quantification is 2.5 micrograms/L. Peak height and alprazolam concentration are linearly related from 2.5 to 100 micrograms/L. For 10 and 20 micrograms/L concentrations, within-run CVs were 1.4% and 0.9% and the between-runs CVs 4.8% and 3.2%. Steady-state serum concentrations ranged from 25 to 55 micrograms/L in patients taking 1.5 to 6.0 mg per day, orally. Preliminary data suggest the method is also suitable for analysis of the structurally similar triazolobenzodiazepine, triazolam.

Alprazolam↗
PubMed1984

Quantification of proteins in tracheobronchoalveolar lavage fluid by isoelectric focusing and scanning densitometry. A methodological study on rats.

The aim of this study was to evaluate whether isoelectric focusing is a technique that is capable of quantifying a number of proteins simultaneously, in a single analysis of tracheobronchoalveolar lavage fluid. The aim included presentation of an animal model for such analysis after exposure to a pulmonary irritant. The studies were performed on tracheobroncoalveolar lavage fluid from rats. One-millimetre-thick agarose gels were used. The gel plates were stained with Coomassie Brilliant Blue R 250, which primarily stains proteins. Quantification of different protein bands was performed by laser scanning densitometry and computerized processing of the results. The effects of different steps in the analysis were examined. Recommendations for application of the method are proposed. An investigation was also made of the variance for the entire method, the variance attributable to isoelectric focusing and individual variation, for different protein bands. A light-microscopic examination for mechanical injury caused by tracheobronchoalveolar lavage was performed. The effects of perfusion of the pulmonary circulation with 0.9% sodium chloride solution during lavage was investigated. Application of the method in an experiment in which rats exposed to tobacco smoke were compared with rats not thus exposed showed that the method is powerful enough to show significances between these two groups. Corresponding results were obtained when rats, the pulmonary circulation of which was perfused as stated above, were compared with non-perfused rats. The experiments showed that isoelectric focusing combined with laser scanning as applied in this study, can be used for quantification of a system of proteins in tracheobronchoalveolar lavage fluid when different groups are to be compared.

Animals↗
PubMed1983

Noninvasive radioisotopic technique for detection of platelet deposition on bovine pericardial mitral-valve prosthesis and in vitro quantification of visceral microembolism in dogs.

Platelet deposition on bovine pericardial-tissue mitral-valve prostheses in dogs was observed noninvasively by use of 111In-labeled platelets and quantified after sacrifice at one, 14 and 30 days postimplantation (300-400 microCi of labeled platelets having been injected 24 hrs previously). Thrombosis on the sewing ring and pericardial leaflets at one and 14 days and on the leaflets at 30 days was delineated in scintiphotos. In vitro quantification (% injected dose) indicated that the sewing ring and perivalvular tissue retained 0.75% of labeled platelets at one day postimplantation, 0.084% at 14 days, and 0.0042% at 30 days. Platelet survival was reduced to 38 hrs at 21 days postimplantation but returned toward the normal (50 hrs) with endothelial covering of the sewing ring. Microemboli in lung and kidney, as measured by tissue/blood radioactivity ratio, decreased significantly at 30 days. 111In-labeled platelets thus provide a sensitive marker for noninvasive imaging and in vitro quantification of platelet deposition on valvular prostheses and microemboli trapped in viscera.

Animals↗
PubMed1983

[Reaction time and eye movements in the recognition task of hand-written Katakana-letters: an experimental verification of the discriminant analysis of letter recognition by Hayashi's Quantification].

Hayashi's Quantification, model II, is a multivariate discriminant analysis for qualitative data. This method was applied to the quantitative indices about confusability of recognition of handwritten Japanese Katakana-letters. The experimental results of reaction time and eye movements in the recognition task corresponded well with indices computed by this statistical procedure. (a) The labels, given by the subjects to the letters, were in line with the prediction by the discriminant, (b) the reaction time and the number of fixations were bigger for highly confusable letters, and (c) those features, which were important according to the discriminant, were fixated more frequently. Thus, Hayashi's quantification procedure is valid for psychological experiments.

Adolescent↗
PubMed1982

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA). Quantification of antigen by diffusion over an antibody-coated surface.

The diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) has previously been used for quantification of antibodies. In the present study, using bovine serum albumin as antigen and a corresponding rabbit antiserum as antibody source, the DIG-ELISA principle was used for quantification of antigen. Antibodies were immobilized on a plastic surface, over which the antigen was allowed to diffuse in an agarose gel. The antigen-antibody reaction zones were then detected by binding of an enzyme-labelled antibody directed against the antigen. The enzyme-substrate reaction, yielding a coloured reaction product, was visualized in a substrate-containing gel. The density and quality of immobilized antibody on the surface was found to be critical. Reproducible results were obtained only when antibodies purified with affinity chromatography on immobilized antigen were used. After 46 h of diffusion the diameter of the reaction zones was directly proportional to the logarithm of the antigen concentration in the range 0.4 mg/1-1 g/1. The variability of the method (CV) was 3%.

Animals↗
PubMed1982

Immunonephelometric quantification of serum vitamin D-binding globulin.

Rapid quantification of serum vitamin D-binding globulin (VDBG) is described. An immunonephelometric method was developed and compared with the conventional immunodiffusion method for VDBG. A correlation coefficient of 0.896 (P less than 0.001) was found with a regression line y = 0.965x + 0.011 (n = 23). The within-assay and day-to-day coefficient of variation of the immunonephelometric method was 6 and 11% at 0.150 g/l (low VDBG concentration), 5 and 7% at 0.230 g/l (normal), and 4 and 8% at 0.570 g/l (high). Levels of VDBG and total 25-hydroxyvitamin D (25(OH)D, 25(OH)D2 + 25(OH)D3) were analysed from 33 serum samples obtained in April-May 1980 from 14-month-old children undergoing vitamin D substitution. 25(OH)D was extracted from 0.1-0.5 ml serum followed by semipreparative silica column chromatography and final quantification by a competitive protein-binding method. The VDBG mean value was 0.308 +/- 0.068 g/l (SD) and 25(OH)D had a mean value of 121 +/- 70 nmol/l (SD). There was no significant correlation between the levels of VDBG and 25(OH)D. The results of this study in general agreed well with other findings.

25-Hydroxyvitamin D 2↗
PubMed1983

Liquid-chromatographic quantification of piracetam.

Piracetam, an analog of gamma-aminobutyric acid, absorbs maximally at 197 nm. Its molar absorptivity at 208 nm and pH 4.5 is 3576 (SD 251) L . mol-1 cm-1, approximately 45% of its absorptivity at 197 nm. Direct quantification of piracetam at 197 nm in biological extracts is complicated by the fact that many other compounds absorb between 190 and 220 nm due to carbon-nitrogen bonding. Chromatography of methanol extracts of serum and aqueous humor on a reversed-phase C-18 column developed isocratically with KH2PO4 (0.1 mol/L, pH 4.8) allows detection and quantification of 0.2 mmol of piracetam per liter. Under these conditions the retention time of piracetam is about 5 min. The detector response is linear for quantities between 5 and 15 nmol. The method is rapid, inexpensive, and convenient for the clinical laboratory.

Animals↗
PubMed1982

Lipoprotein quantification: an electrophoretic method compared with the Lipid Research Clinics method.

We compared a turbidimetric electrophoretic method (Lipidophor) for lipoprotein quantification with the standardized Lipid Research Clinics (LRC) method. In the Lipidophor procedure, major lipoproteins are separated by electrophoresis on agarose gels, precipitated on the gels with phosphotungstate-Mg2+ reagent, and the resulting turbidity is measured densitometrically. Measurements of relative turbidity were converted into lipoprotein cholesterol values by the use of numeric constants provided by the manufacturer. Among-day CVs (n = 46) for the Lipidophor method were 6.0%, 3.6%, and 9.9% for cholesterol in the alpha-, beta-, and pre-beta lipoproteins, respectively. The Lipidophor alpha-cholesterol was significantly lower (n = 171 specimens) than the LRC high-density lipoprotein (HDL) cholesterol (514 vs 586 mg/L), and beta-cholesterol was significantly higher than the corresponding LRC low-density lipoprotein (LDL) cholesterol values (1505 vs 1409 mg/L). The linear relation between the two methods for lipoprotein cholesterol quantification is as follows: Lipidophor alpha = 0.77 LRC HDL + 63 mg/L with correlation coefficient (r) of 0.87; Lipidophor beta = 0.95 LRC LDL + 166 mg/L (r = 0.96); Lipidophor pre-beta = 0.57 LRC very-low-density lipoprotein + 39 mg/L (r = 0.82). We derived a revised algorithm for estimating lipoprotein cholesterol from turbidity measurements. Lipoprotein cholesterol values by the Lipidophor method agree well with those obtained by the LRC method when these constants are used.

Adult↗
PubMed1982

Drool quantification: noninvasive technique.

Drooling can lead to physical and psychologic problems for the 10-13% of all persons with cerebral palsy who exhibit this symptom. Different therapeutic attempts have been made to eliminate drooling, ranging from speech therapy to radical surgery. However, the effectiveness of all previous approaches is open to question since no satisfactory drool quantification technique has been devised. This paper describes a new inexpensive drool quantification system that has been tested with a number of cerebral palsy subjects. The system provides measures of drooled saliva to within 0.2ml, and allows for the possibility of more objective studies related to natural variations in drooling (for example, diurnal rhythms or activity levels) and clinical outcome studies of therapeutic interventions.

Adolescent↗
PubMed1981

Diagnosis of lactose intolerance through the quantification of hydrogen in exhaled air.

Considerations are made on diarrhea secondary to intestinal malabsorption of carbohydrates and on the diagnostic means available in our environment, among which are the reactive strip, the clinitest tablet, tolerance curves and research of intestinal enzymes. The quantification of exhaled hydrogen is mentioned as a diagnostic method, used by several other authors, and a review of the literature on the subject is made. Considerations are made on a hydrogen quantification in the breath of patients with a transitory lactose intolerance. 16 patients were studied, 15 of whom showed an increase in the exhaled H2. 32 children with diarrhea, free from lactose intolerance, were studied. The method used did not show a significant increase in the exhaled hydrogen. 27 patients showed no H2 in their breath. In 4 there was elimination of H2 (false positives). 17 diarrhea-free children were studied. A significant difference was found when comparing the children with diarrhea and lactose intolerance to the other two groups who were free from lactose-intolerance. It is considered that this method can be useful in the diagnosis of intolerance to lactose and other sugars.

Chromatography, Gas↗
PubMed1980

Immunological quantification by high-affinity antibodies of O6-ethyldeoxyguanosine in DNA exposed to N-ethyl-N-nitrosourea.

Three immunological methods [radioimmunoassay (RIA), enzyme-linked immunosorbent assay, and radioimmunosorbent technique] were established for quantification of the potentially mutagenic O6-ethyldeoxyguanosine (O6-EtdGuo) in DNA treated with the carcinogen ethylnitrosourea in vivo or in vitro. To obtain high-affinity antibodies for specific detection of low levels of O6-EtdGuo in small amounts of DNA (cells), different schemes were applied for immunization of rabbits with the hapten O6-ethylguanosine coupled to various carrier proteins (rat serum albumin, bovine serum albumin, keyhold limpet hemocyanin). Low-dose immunization with the hapten-keyhold limpet hemocyanin conjugate resulted in antibodies with an affinity constant of 1 to 2 X 10(10) liters/mol and very low levels of cross-reactivity with normal as well as other alkylated DNA components. The RIA (the most sensitive of the three assays) detects 0.05 pmol of O6-EtdGuo at 50% inhibition of tracer (O6-ethyl[8,5'-3H]-3'-deoxyguanosine)-antibody binding. This permits quantification by RIA of O6-EtdGuo at an O6-EtdGuo:2'-deoxyguanosine molar ratio of approximately 3 X 10(-7) in a hydrolysate of 100 micrograms of ethylated DNA. By chromatographic separation of O6-EtdGuo prior to the RIA, this value can be lowered to less than 5 X 10(-8).

Animals↗
PubMed1994

[Use of the AgNOR method for the differential diagnosis of tumors and for the quantification of non-neoplastic epidermal hyperproliferation in dermatohistology].

The AgNOR number (NN) and the AgNOR quotient (NQ) of 228 melanocytic, epidermal and fibrohistiocytic lesions were investigated by image analysis. In melanocytic nevi, dysplastic nevi and Spitz nevi, significantly less AgNORs were seen than in malignant melanomas and melanomas in situ. Significant differences in AgNOR expression were detected between the subtypes of melanoma, too. Epidermal carcinomas showed a significantly higher AgNOR expression than keratoacanthomas and other benign tumors. Between the G1 carcinomas and the G2 and G3 groups, an increase of the NN and NQ was found. Actinic keratosis demonstrated a significantly lower AgNOR expression than carcinomas and--like M. Bowen--higher NN and NQ than the benign lesions. The lowest AgNOR results of fibrohistiocytic lesions were registered in regressive palmar fibromatosis and in scars, the highest in malignant fibrous histiocytomas. Between dermato-fibrosarcoma protuberans, atypical fibroxanthoma and malignant fibrous histiocytomas, significant differences were found to exist. Furthermore we examined the suitability of the AgNOR technique for quantification of epidermal cell kinetics in non-neoplastic epidermal hyperproliferations. Especially in the grading of psoriasis this technique is helpful. First convincing results could be achieved for the use of the AgNOR method in the verification of antiproliferative effects of new substances in an experimental mouse tail test. This study demonstrated that the AgNOR technique in addition to the dermatohistological differential diagnosis of tumors is useful for the quantification of antiproliferative effects of different treatments in hyperproliferative epidermal dermatosis.

Cell Division↗
PubMed1994

Quantification of area percentage of immunohistochemical staining by true color image analysis with application of fixed thresholds.

Most image analysis systems (IAS) use black-and-white cameras. However, true color IASs are considered to be useful for quantification of immunohistologically stained structures. Using a true color IAS, we evaluated two methods of segmentation for quantification of area percentage of staining: one using fixed, preset thresholds and one using thresholds interactively set per image. Furthermore, the effect of shading correction was evaluated, and measurements in both color and black-and-white mode were compared. The results of segmentation with fixed thresholds did not differ significantly from those of control percentages, established by interactive morphometry using a grid, which served as reference. Interactive segmentation was significantly different from the reference (t test, P = .0001). The effect of shading correction was negligible. Measurements with and without this procedure correlated highly (r = .99, P < .001). Comparison of the results obtained in color and black-and-white mode showed a significant difference in the latter from the reference (t test, P = .005). We conclude that it is possible to quantify, in a reliable way, area percentage of positive staining using a true color IAS with application of a segmentation method with fixed thresholds.

Breast Neoplasms↗
PubMed1994

[Quantification of messenger RNA expression in tumors: which standard should be used for best RNA normalization?].

Alterations in gene expression are frequently encountered in malignant tissues. Quantification of the often quite subtle changes in mRNA content is routinely performed by northern blot hybridization. Practical experience shows that the methodology is subject to considerable error, necessitating the use of an internal mRNA standard for quantification. Many different control mRNAs have been employed over the years, but no systematic study has proven their applicability to comparisons made between tumor cells and normal tissues. The validity of these control RNAs, usually housekeeping genes, must be established to exclude the possibility that they themselves are expressed at different levels in tumor and non-tumor cells. Our experience has revealed that serious discrepancies may arise when incorrect standard genes are used to compare tumor and normal tissues.

Actins↗
PubMed1995

New method for stem cell quantification: applications to the management of peripheral blood stem cell transplantation.

A dramatic increase in peripheral blood stem cells (PBSC) is observed after high-dose chemotherapy followed by haematopoietic growth factors. The degree of mobilisation of PBSC is quantified by the level of clonogenic cells detected by CFU assays (CFU-GM or CFU-GEMM) or CD34+ cell determination. Working under the hypothesis that, in peripheral blood, mononuclear cells in DNA synthesis (MCDS) are proliferating stem cells, we decided to detect these cells by flow cytometric measurement of their DNA content. The relations between the number of MCDS and well-known haematopoietic progenitor indicators such as CFU-GM or CD34+ cells were analysed. We studied the kinetics of recruitment of PBSC in cancer patients, treated with rmeHuG-CSF following VP-16 cytoxan chemotherapy, until the first day of leukapheresis. For the 31 patients studied the individual curves of peripheral MCDS and CFU-GM reconstitutions showed identical profiles and a good correlation was noted between the numbers of peripheral MCDS and CFU-GM (r = 0.73). In the leukapheresis product, the predictive value of MCDS was equivalent to CFU-GM for PBSC quantification (r = 0.70). In conclusion, MCDS analysis by flow cytometry provides reliable results and appears to be an alternative to CFU-GM assay or CD34+ cell determination for PBSC quantification.

Adult↗
PubMedDate not supplied

[Quantification of pulmonary emphysema with computerized tomography. Comparison with various methods].

Computed Tomography (CT) has been proved to be the most accurate imaging modality to diagnose emphysema in vivo. Our study was aimed at comparing different CT methods for pulmonary emphysema quantification in patients with severe chronic obstructive pulmonary disease (COPD). Forty-six consecutive inpatients affected with COPD underwent high resolution CT (HRCT). Three scans were acquired at 3 preselected anatomic levels at both full inspiration and expiration. Three different observers were asked to subjectively evaluate, under blind conditions, the extent alone and both the severity and the extent of emphysema on the 6 scans. HRCT findings were also analyzed quantitatively by measuring the mean CT number in Hounsfield Units (HU) and the % of lung area with CT numbers < -900 HU (pixel index). Quantitative CT data were compared with reference values obtained in 7 normal nonsmokers. The CT visual score of emphysema exhibited medium-high interobserver reproducibility with correlation coefficients ranging from 0.80 to 0.96 and a good correlation with pulmonary function tests, particularly relative to the assessment of the extent of emphysema alone as expressed by one observer. CT quantification demonstrated an excellent correlation with functional indices of expiratory airflow, lung volumes and diffusion coefficients (p < 0.001). The expiratory measurements were better than the inspiratory ones while the analysis of both CT number and pixel index gave comparable results. Only the CT expiratory quantitative data allowed to differentiate the patients affected with COPD from the controls. In conclusion, the severity of emphysema as expressed by CT correctly reflects the functional impairment of patients with severe COPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Quantification of apolipoproteins B-100, B-48, and E in human triglyceride-rich lipoproteins.

We have developed a procedure to quantify apolipoprotein (apo) B-100, apoB-48, and apoE in human triglyceride-rich lipoproteins. This procedure permits delipidation of small amounts of triglyceride-rich lipoproteins without appreciable losses, and quantification of these apolipoproteins in samples containing as little as 10 micrograms of protein. Delipidated triglyceride-rich lipoproteins are subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis, and the mass of apolipoproteins is estimated after densitometric scanning and volume integration of Coomassie blue-stained bands. The chromogenicities of apoB-100 and apoB-48 are virtually identical, and twofold lower than that of apoE. The standard curve for each apolipoprotein follows a power function over a wide protein range, permitting quantification of as little as 0.2 microgram of apoB-48 and as much as 30 micrograms of apoB-100 from a single application of triglyceride-rich lipoproteins to the gels. This method is suitable for routine use in studies of the intestinal and hepatic contributions to triglyceride-rich lipoproteins and their responses to postprandial lipemia.

Apolipoprotein B-100↗
PubMed1995

[A trial for quantification of proton MR spectroscopy: relaxation times of each metabolite and concentration using water signal as an internal standard].

We conducted quantification of proton MR spectroscopy (1H-MRS) using water signal as an internal standard. The 1H-MR spectrum was measured with and without water suppression pulses. For calculation of relaxation times, measurement conditions of the PRESS sequence were as follows: TR = 1500, 3000, 5000 ms, TE = 135,270 ms. The T1 relaxation and T2 relaxation of each metabolite were calculated by fitting to the curve of the equation of the spin echo sequence. The signal intensities of each metabolite were corrected using relaxation times and such corrected intensities were used for the quantitative calculation. The concentration of each metabolite was obtained from ratios of the corrected intensities of water and metabolites. The value of each concentration was coincident with that reported in the previous literature. We considered that this quantification using water signal as an internal standard would be very useful when the proton-weighted image does not show remarkable change.

Adult↗