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Rapid and sensitive quantitative analysis of alkyl phosphates in urine after organophosphate poisoning.

Organophosphate compounds are widely used as pesticides. After ingestion by humans, organophosphates decompose into alkyl phosphates. Decomposition continues postmortem. We developed a rapid (< 3 h), quantitative, and sensitive analysis of the human organosphosphate metabolites O,O-dimethylphosphate (DMP), O,O-diethylphosphate (DEP), O,O-dimethylthiophosphate (DMTP), O,O-diethylthiophosphate (DETP), O,O-dimethyldithiophosphate (DMDTP), and O,O-diethyldithiophosphate (DEDTP). Urine is dried under azeotropic conditions with isopropanol and nitrogen. All metabolites are converted into their corresponding benzyl esters reacting with benzyl bromide and diazotoluene. The protocol prevents the isomerization of DMTP and DETP occurring when diazo compounds are used exclusively. The benzyl ester derivatives are purified on solid-phase extraction silica columns. The quantitative analysis is performed by gas chromatography-mass spectrometry. All metabolites can be identified by the parent molecular ions. Urine samples from eight cases of fatal suicidal poisoning dialkyl phosphates were quantitated. The limits of detection ranged from 3 to 6 ng/mL. Hence, this protocol is sufficiently sensitive to detect and quantitate organophosphate metabolites beyond cases of fatal poisoning, in the clinical setting, and even following average environmental exposure.

Forensic Medicine↗
PubMed2003

Quantitative analysis of dinuclear manganese(II) EPR spectra.

A quantitative method for the analysis of EPR spectra from dinuclear Mn(II) complexes is presented. The complex [(Me(3)TACN)(2)Mn(II)(2)(mu-OAc)(3)]BPh(4) (1) (Me(3)TACN=N, N('),N(")-trimethyl-1,4,7-triazacyclononane; OAc=acetate(1-); BPh(4)=tetraphenylborate(1-)) was studied with EPR spectroscopy at X- and Q-band frequencies, for both perpendicular and parallel polarizations of the microwave field, and with variable temperature (2-50K). Complex 1 is an antiferromagnetically coupled dimer which shows signals from all excited spin manifolds, S=1 to 5. The spectra were simulated with diagonalization of the full spin Hamiltonian which includes the Zeeman and zero-field splittings of the individual manganese sites within the dimer, the exchange and dipolar coupling between the two manganese sites of the dimer, and the nuclear hyperfine coupling for each manganese ion. All possible transitions for all spin manifolds were simulated, with the intensities determined from the calculated probability of each transition. In addition, the non-uniform broadening of all resonances was quantitatively predicted using a lineshape model based on D- and r-strain. As the temperature is increased from 2K, an 11-line hyperfine pattern characteristic of dinuclear Mn(II) is first observed from the S=3 manifold. D- and r-strain are the dominate broadening effects that determine where the hyperfine pattern will be resolved. A single unique parameter set was found to simulate all spectra arising for all temperatures, microwave frequencies, and microwave modes. The simulations are quantitative, allowing for the first time the determination of species concentrations directly from EPR spectra. Thus, this work describes the first method for the quantitative characterization of EPR spectra of dinuclear manganese centers in model complexes and proteins. The exchange coupling parameter J for complex 1 was determined (J=-1.5+/-0.3 cm(-1); H(ex)=-2JS(1).S(2)) and found to be in agreement with a previous determination from magnetization. The phenomenon of exchange striction was found to be insignificant for 1.

Algorithms↗
PubMed2002

Qualitative and quantitative analysis of agricultural dust in working environment.

The presented quantitative and qualitative analysis of dust in agricultural working environment is a continuation of the process of recognizing the exposure to dust among private farmers. The study covers the following: determination of respirable fraction of dust in the respiratory zone (on the background of total dust) while performing individual farming activities which constitute an annual work cycle, organic and mineral components of settled dust for basic groups of farming activities, and the main mineral pathogenic component - free silica in airborne and settled dust. The study was conducted on 5 farms specialising in: cultivation of cereals, root plants, vegetables, dairy cattle and swine breeding and mixed production. The analysis of settled dust covered 17 types of dust accompanying field work and farm/indoor activities. Studies of airborne dust were conducted on farmers while performing 40 main work activities which contributed to the annual work cycle. Results of the study confirmed the following: agricultural work activities are accompanied by a high level of dustiness and showed the presence of a respirable fraction in airborne dust of up to 25%, a higher level of pathogenic free silica SiO(2) in settled dust samples in the working environment of a farmer, compared to dust in respiratory zone, a comparable level of SiO(2) in total and respirable airborne dust, and a high level of organic component in settled dust at work activities with plant material. These results indicated that the evaluation of farmers' exposure to dust should be based on the examination of samples taken in the respiratory zone while performing individual work activities

Adult↗
PubMed1996

Nonlinear dynamics and quantitative EEG analysis.

Quantitative, computerized electroencephalogram (EEG) analysis appears to be based on a phenomenological approach to EEG interpretation, and is primarily rooted in linear systems theory. A fundamentally different approach to computerized EEG analysis, however, is making its way into the laboratories. The basic idea, inspired by recent advances in the area of nonlinear dynamics and chaos theory, is to view an EEG as the output of a deterministic system of relatively simple complexity, but containing nonlinearities. This suggests that studying the geometrical dynamics of EEGs, and the development of neurophysiologically realistic models of EEG generation may produce more successful automated EEG analysis techniques than the classical, stochastic methods. A review of the fundamentals of chaos theory is provided. Evidence supporting the nonlinear dynamics paradigm to EEG interpretation is presented, and the kind of new information that can be extracted from the EEG is discussed. A case is made that a nonlinear dynamic systems viewpoint to EEG generation will profoundly affect the way EEG interpretation is currently done.

Brain↗
PubMed1994

Purification and immunoelectron microscopic localization of cellular glutathione peroxidase in rat hepatocytes: quantitative analysis by postembedding method.

To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/microns 2) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.

Animals↗
PubMed1987

Human mumps virus-specific cytotoxic T lymphocytes: quantitative analysis of HLA restriction.

To obtain quantitative information about the use of HLA antigens as restriction element by antiviral cytotoxic T lymphocytes (CTL), we have analyzed precursors of human mumps virus-specific CTL by limiting dilution. CTL generated by restimulation of peripheral blood T lymphocytes with autologous mumps virus (MV)-infected stimulator cells were restricted by autologous HLA class I antigens, and derived from the T4-8+ population. They were specific for MV and did not lyse autologous target cells infected with other viruses. Frequencies of MV-specific CTL precursors ranged from 1/500 to 1/8000. HLA restriction was analyzed by split-well analysis of individual CTL colonies. CTL recognizing HLA-A or B antigens were unequally distributed: HLA-B7, -B13, and -B27 were found to function as predominant, in some cases as exclusive, restriction elements, whereas other antigens such as HLA-A24 were never or rarely used. In several combinations, there was no evidence for antigenic variants of HLA molecules as reason for the failure to be recognized. The proportion of CTL precursors recognizing HLA-A2 and -B8 seemed to be dependent on the presence or absence of "dominant" restriction elements. We conclude that CTL precursors recognizing certain virus-HLA combinations are preferentially expanded during an infection, but that low responsiveness to a given combination is not necessarily absolute.

Antigens, Viral↗
PubMed1987

Problems of quantitative urine analysis.

Quantitative determinations of various analytes in urine represent only a small part of all analyses performed in the routine clinical chemistry laboratory. Consequently, analytical as well as pre-analytical problems are less often considered in urine analysis. In the first part of this paper, pre-analytical factors affecting quantitative determinations are discussed, e.g., physical activity, posture, circadian rhythms, different solubilities of the various substances, errors in urine collecting, or drug effects. Subsequently, aspects of linearity and specificity of the routine determinations will be illustrated with special consideration given to protein, calcium, and enzyme determinations as well as some specialized measurements, e.g., porphyrins, catecholamines and their metabolites, or 5-hydroxyindole-acetic acid. Effects of pre-analytical and analytical factors on intra-assay and between-assay variations found in urine analysis are discussed. Using the results of surveys, it is shown that the standardization of methods as well as the use of suitable standards are mandatory for an effective amelioration of the present quality of quantitative urine analysis.

Calcium↗
PubMed1977

Quantitative analysis of plasma neutral glycosphingolipids by high performance liquid chromatography of their perbenzoyl derivatives.

A quantitative high performance liquid chromatography method for the analysis of neutral glycosylceramides as their perbenzoyl derivatives has been devised. Samples containing more than 2.5 nmol each of mono-, di-, tri-, and tetraglycosylceramide are benzoylated with 10% benzoyl chloride in pyridine at 37degrees C for 16 hr. The products are separated from excess reagents by solvent distribution and injected onto a pellicllar silica gel (Zipax) column (2.1 mm X 50 cm). The derivatives are eluted with a 10 min linear gradient of 2-17% ethyl acetate in hexane at 2 ml/min and absorbance at 280 nm is recorded. The detector response was proportional to the weight of sample used (2-30 nmol) and the lower limit of detection was about 70 pmol. The procedure has been applied to the quantitative analysis of erythrocyte and plasma glycolipids. As little as 0.5 ml of plasma can be used for analysis. The relative standard deviation of repetitive analyses ranged between 2.0% for glucosylceramide to 5.4% for galactosyllactosylceramide.

Benzyl Compounds↗
PubMed1993

Quantitative analysis of aflatoxin-albumin adducts.

Quantitative exposure assessments made using biologically relevant markers will facilitate epidemiological studies of risk from environmental carcinogens. Blood proteins are readily accessible macromolecules that have been shown to be targets for activated chemical carcinogens. Serum albumin is quantitatively the most abundant target for aflatoxin B1 and the measurement of aflatoxin-serum albumin adducts has been used to detect exposed individuals. The goal of these experiments was to devise an analytical procedure that would increase the overall recovery of aflatoxin adducts in serum albumin, and thereby improve the accuracy of exposure monitoring. The method developed consisted of the following procedures. Proteins were precipitated from serum (< or = 100 microliters) with 80% ammonium sulfate, with incubation at 4 degrees C for 2 h. Following dialysis against phosphate-buffered saline (pH 7.0 for 3 h at 4 degrees C), the proteins were digested with protease (Pronase) (1:4.1 w/w enzyme:protein) for 15 h at 37 degrees C with shaking. Enzyme and other undigested proteins were precipitated with acetone (1:2 v/v, 40 min, 4 degrees C). After evaporation of the acetone under vacuum, levels of aflatoxin B1-albumin adducts were determined by radioimmunoassay carried out on 300 microliters fractions. This procedure obviated the isolation of albumin prior to analysis and reduced interference in the radioimmunoassay. High recoveries of aflatoxin B1 adducts were achieved together with a low limit of detection. The applicability of the procedure in epidemiological studies of human aflatoxin exposure was illustrated by results of analysis of aflatoxin-albumin adducts in serum samples from residents of Chongming Island, People's Republic of China.

Aflatoxin B1↗
PubMed1989

A diagnostic programme for quantitative analysis of proteinuria.

A spectrum of quantitative methods was adapted to the Kone Specific Analyser for the purpose of recognizing, quantifying and differentiating various forms of proteinuria. Total protein, IgG, albumin and alpha 1-microglobulin (measured by turbidimetry), N-acetyl-beta-D-glucosaminidase activity and creatinine (measured photometrically), were measured in undiluted urine; in addition alpha 1-microglobulin was measured in serum. Within and between run precision, accuracy and linearity of the turbidimetric methods were in good agreement with nephelometric procedures. All turbidimetric methods exhibited a correlation coefficient r greater than 0.98 when compared with the radial immunodiffusion procedure as reference method. Total protein measured turbidimetrically with the Kone Specific Analyser was in good agreement with the manual biuret procedure. The low detection limits and linearities allowed quantification of urine analytes from the lower range of normals up to ten times the upper limit of normals. The measured analytes exhibited stability in urine at pH 4-8 over at least seven days at 4-6 degrees C and -20 degrees C. Only IgG showed a significant loss (up to 30 percent), when measured after storage at -20 degrees C. Quantities per mol creatinine showed significantly lower intra-individual and inter-individual variability than quantities per liter. In 31 normal persons, the intraindividual variation was lowest for N-acetyl-beta-D-glucosaminidase activity (13%) and highest for total protein (33%), when measured in the second morning urine on 5 consecutive days. When related to creatinine, results obtained in the second morning urine showed no significant differences from those in 24 h urine, except for alpha 1-microglobulin which gave lower values in 24 h urines. The upper normal limits, calculated as the 95% ranges, were determined from 154 urines of 31 individuals. Nearly all analytes showed an asymmetric distribution. Because of a wide tailing of the upper limit, preliminary upper normal limits were set above this range: (table; see text) Application of the newly adapted programme to unselected urines sent for urine analysis revealed a threefold increase in the proportion of results outside the normal ranges, compared with the routinely used protein test strip procedure. All additional positive urines exhibited either signs of glomerular or tubular proteinuria. Determination of albumin or N-acetyl-beta-D-glucosaminidase excretion was sufficient to detect these additional cases.

Diagnosis, Differential↗
PubMed1982

The correlation of histology to needle type in microvascular anastomoses: a quantitative analysis.

This study was undertaken to quantitate the effects of needle trauma to the arterial wall during microvascular anastomoses. End-to-end anastomoses performed on rat femoral arteries were used as a model. Two weeks post-anastomoses, the specimens were processed and measured with a planimeter. The results were analysed statistically to evaluate the effects of different types of needles on the arterial wall. We found significant thickening of the arterial wall (P less than 0.01) but no difference in the amount of thickening between any of the needles evaluated. We found that certain needles were easier to use than others and have applied this observation to clinical work.

Animals↗
PubMed1998

Branched-chain amino acid biosynthesis in Salmonella typhimurium: a quantitative analysis.

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.

Acetolactate Synthase↗
PubMed1996

Adenovirus-mediated gene transfer into injured arteries: quantitative analysis of repeated administration.

We evaluated quantitatively the efficiency and safety of, and influence of either prior arterial injury or immunity against adenovirus on adenovirus-mediated gene transfer into injured arteries of a relatively large animal model, the dog. A replication-defective adenovirus expressing bacterial lacZ was introduced percutaneously into balloon-injured femoral arteries through a double balloon catheter. After a single dose of adenovirus, up to 90% of surface and smooth muscle cells in multiple layers of the media showed transgene expression evaluated by X-Gal histostaining without extralocal expression, as assessed by the polymerase chain reaction. High level expression measured as beta-galactosidase activity peaked at 7 days after transfer and was transient, though retained for a month. Gene transfer performed just after balloon injury induced submaximal gene expression. A second administration of the same adenovirus to the same arterial site enhanced lacZ expression significantly despite the presence of an immune response. Expression of lacZ was also detected in preimmunized dogs, although the expression levels correlated inversely to the titer of neutralizing antibodies in their serum. These data may provide a scientific foundation for the use of adenovirus-mediated arterial gene transfer in future clinical practice.

Adenoviridae↗
PubMed2002

[Relative quantitative analysis of corneal immunogenicity].

OBJECTIVE: To quantitate the relative immunogenicity of three major corneal cell layers (epithelium, stroma, and endothelium) among the whole corneal immunogenicity respectively. METHODS: Cellular immunity: Three porcine major corneal cell layers were heterotopically allografted to subcutaneous layer of 40 BALB-c mice respectively. Twelve days later, the peripheral white blood cells of recipients were double directly stained with anti-mouse IgG-fluorescein conjugate mAb and analyzed by flow cytometry. Humoral immunity: The suspension of porcine cornea or corneal epithelium was used as antigen to produce anti-porcine immune sera in C57BL-6 mice. With the anti-porcine immune sera, enzyme linked immunosorbent assay was performed. RESULTS: On cellular immunity detection, the immunogenicities of intact endothelium, epithelium and stroma, equal in thickness, were 70.75%, 27.63%, and 1.62% respectively. On humoral immunity detection, the immunogenicities were 62.11%, 31.77% and 6.12% in the three respective layers of equal thickness. CONCLUSION: These findings indicate that the immunogenicity of corneal stroma is the lowest among the three corneal cell layers of equal thickness. The preliminary results provide the experimental basis for clinical application of xenogenic graft of corneal stroma.

Animals↗
PubMed2004

Paradoxes of the first-night effect: a quantitative analysis of antero-posterior EEG topography.

OBJECTIVE: The first-night effect (FNE) is a common issue in sleep research. Being considered fragmented and poorly efficient, the adaptation night is discarded for data analysis. The present study aims to provide a quantitative and topographical EEG analysis of this phenomenon. METHODS: Eight healthy subjects slept for two consecutive nights (adaptation (AD) and baseline (BSL)), and their polysomnography was visually scored and then submitted to spectral power analysis. RESULTS: The results showed a decreased quality and quantity of first-night sleep as indicated by more stage 1 and intrasleep wake, paralleled by a reduced sleep efficiency and a longer sleep onset latency. On the other hand, EEG quantitative data showed a more complex and apparently paradoxical picture. An increase in delta power was observed, particularly over the central areas during the first night, paralleled by an increased power in beta bin frequencies solely at posterior scalp locations. CONCLUSIONS: These results have been interpreted as caused by, respectively, a reduced total sleep time during the adaptation night and a cortical hyperactivity, typical of psychophysiological insomnia. The present results confirm the need to exclude the laboratory sleep adaptation night from data analysis since it is not a reliable index of sleep on subsequent nights as regards both visual scoring and quantitative EEG analysis. Finally, regional differences between REM and NREM sleep have been confirmed. SIGNIFICANCE: This is the first attempt to evaluate the FNE with a quantitative approach to the antero-posterior EEG topography, providing both a Hz-by-Hz and a classical EEG band-based analysis.

Adaptation, Physiological↗
PubMed1993

Quantitative analysis of Her-2/neu (ERBB2) gene expression using reverse transcriptase polymerase chain reaction.

Inappropriate expression of Her-2/neu (ERBB2) gene has been associated with impaired breast cancer prognosis, suggesting a functional role in tumor progression. Herein we describe a quantitative method for analysis of Her-2/neu gene messenger RNA (mRNA), which employs reverse transcriptase polymerase chain reaction (RT-PCR) on a 10-microns cryostat section. The technique combines modified RNA extraction with complementary DNA (cDNA) synthesis to achieve a high level of sensitivity. Utilizing this PCR-based gene expression assay, we were able to quantitate variable amounts of Her-2/neu mRNA in cell lines with established levels of gene expression and in clinical human breast cancer specimens. In clinical samples, mRNA levels correlated with intensity of immunoperoxidase staining for corresponding oncoprotein. We conclude that PCR-based mRNA quantitation can be applied to quantitative analysis of Her-2/neu gene expression, and potentially many other genes, in samples of limited size.

Base Sequence↗
PubMed1997

Cytoplasmic microtubules in two different mouse melanoma cell lines: a qualitative and quantitative analysis using confocal laser scanning microscopy and computer-assisted image analysis.

The microtubular system as one part of the cellular cytoskeleton is not only necessary for mitotic activity of malignant cells but also for invading neighboring tissues and for the formation of distant metastases. In the present study, the amount and distribution of tubulin in two murine melanoma cell lines (K1735-M2: high metastatic clone; K1735-c116: low metastatic clone) were determined quantitatively using an indirect immunofluorescence technique, confocal laser scanning microscopy (CLSM) and computer-assisted image analysis. Additionally, qualitative and quantitative changes after application of the microtubule-inhibitor nocodazole were investigated. Quantitative analysis showed a significant difference between the high and low metastatic cell line for the parameter TEXTURE, indicating a finer structured network within the high metastatic cells. After treatment with nocodazole the parameters TEXTURE and DENSITY were reduced, suggesting a decrease of assembled tubulin and a less delicate structure of the remaining microtubules. Our study shows that CLSM combined with computer-assisted image analysis provides a new method to examine quantitative variations of the cytoskeleton possibly related to cell function.

Animals↗