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PubMed2000

A new HG/LT-GC/ICP-MS multi-element speciation technique for real samples in different matrices.

An improved speciation technique is presented for metal(loid)organic compounds, enabling identification and quantification of species from twelve elements: germanium, arsenic, selenium, molybdenum, tin, antimony, tellurium, iodine, tungsten, mercury, lead and bismuth. At this time it is possible to identify 29 species with boiling points between -88.5 degrees C and 250 C in gaseous, liquid and solid samples in a few minutes. This study shows as an example results from measurements of soil samples from municipal waste deposits. The HG/LT-GC/ICP-MS-(hydride generation/low temperature-gas chromatography/inductively coupled plasma-mass spectrometry) apparatus contains a home-built gas chromatograph that enables satisfactory separation of various species with a boiling point difference of > or = 14 degrees C. The absolute detection limits for the elements mentioned above were below 0.7 pg.

Chromatography, Gas↗

PubMed1993

[Quantitative 201Tl-scintigraphy of the lower limb in arterial occlusive disease].

201Tl scintigraphy is useful in evaluating the hemodynamic consequences of arterial stenoses in arterial occlusive disease. The aim of the present study was to determine normal values for absolute Tl uptake in the lower leg, for the quotient between left and right lower leg uptake as well as for the redistribution pattern after bicycle exercise. We examined as reference 49 patients without clinical evidence of peripheral or coronary artery disease: absolute Tl uptake in the lower leg in anterior scintigraphy (acquisition time: 120 sec) was 5.02 +/- 1.70 counts/pixel (left) and 5.21 +/- 1.53 counts/pixel (right), the quotient between right and left lower leg was 0.85 +/- 0.09 and the redistribution pattern (quotient of the mean activity exercise/rest) 0.82 +/- 0.17 (left) and 0.83 +/- 0.15 (right). The arithmetic mean minus 2 standard deviations was regarded as the lower limit of normal. In 8 patients with mechanical compression of the left femoral artery before injection at least two scintigraphic parameters were abnormal, in 7 patients with angiographically proven peripheral artery disease at least one scintigraphic parameter was below normal. Quantification of Tl redistribution offers diagnostic advantages in bilateral peripheral artery disease.

Adult↗

PubMed2000

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2'-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection.

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2'-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml(-1) (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml(-1) urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg(-1) creatinine (range 0.17-5.9 ng mg(-1) creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1996

Quantification and pharmacokinetics of blood-brain barrier disruption in humans.

Hyperosmolar blood-brain barrier disruption (HBBBD), produced by infusion of mannitol into the cerebral arteries, has been used in the treatment of brain tumors to increase drug delivery to tumor and adjacent brain. However, the efficacy of HBBBD in brain tumor therapy has been controversial. The goal of this study was to measure changes in vascular permeability after HBBBD in patients with malignant brain tumors. The permeability (K1) of tumor and normal brain blood vessels was measured using rubidium-82 and positron emission tomography before and repeatedly at 8- to 15-minute intervals after HBBBD. Eighteen studies were performed in 13 patients, eight with glioblastoma multiforme and five with anaplastic astrocytoma. The HBBBD increased K1 in all patients. Baseline K1 values were 2.1 +/- 1.4 and 34.1 +/- 22.1 microl/minute/ml (+/- standard deviation) for brain and tumor, respectively. The peak absolute increases in K1 following HBBBD were 20.8 +/- 11.7 and 19.7 +/- 10.7 microl/minute/ml for brain and tumor, corresponding to percentage increases of approximately 1000% in brain and approximately 60% in tumor. The halftimes for return of K1 to near baseline for brain and tumor were 8.1 +/- 3.8 and 4.2 +/- 1.2 minutes, respectively. Simulations of the effects of HBBBD made using a very simple model with intraarterial methotrexate, which is exemplary of drugs with low permeability, indicate that 1) total exposure of the brain and tumor to methotrexate, as measured by the methotrexate concentration-time integral (or area under the curve), would increase with decreasing infusion duration and would be enhanced by 130% to 200% and by 7% to 16%, respectively, compared to intraarterial infusion of methotrexate alone; and 2) exposure time at concentrations above 1 microM, the minimal concentration required for the effects of methotrexate, would not be enhanced in tumor and would be enhanced by only 10% in brain. Hyperosmolar blood-brain barrier disruption transiently increases delivery of water-soluble compounds to normal brain and brain tumors. Most of the enhancement of exposure results from trapping the drug within the blood-brain barrier, an effect of the very transient alteration of the blood-brain barrier by HBBBD. Delivery is most effective when a drug is administered within 5 to 10 minutes after disruption. However, the increased exposure and exposure time that occur with methotrexate, the permeability of which is among the lowest of the agents currently used clinically, are limited and the disproportionate increase in brain exposure, compared to tumor exposure, may alter the therapeutic index of many drugs.

Adult↗

PubMed2005

Usage of human reliability quantification methods.

Human reliability quantification (HRQ) methods are becoming increasingly important in risk and accident assessment in systems these terms are usually related to (hi-tech industrial systems, including nuclear and chemical plants). These methods began to intensively develop after numerous accidents caused by human error or inadequate activity of people who controlled and managed complex technological processes. For already existing systems, but also for new ones, it is important to assess the possibility of an accident. Determination of possible preventive activities, which include the influence of human error on the safety of a system, is also required. These are the main goals of the HRQ method. Using Absolute Probability Judgment (APJ) and Success Likelihood Index Methods (SLIM) HRQ techniques in control and management centers in electro-power systems in Belgrade and railway traffic in Nis (both in Serbia and Montenegro) are shown in this paper.

Accidents↗

PubMed1994

A new immunohistochemical method for quantification of fast-myosin in frozen histologic sections of the rat skeletal muscles.

An immunohistochemical micromethod for quantification of fast-myosin in frozen histologic sections of rat skeletal muscles was developed. The principle of this method was enzyme-linked immunosorbent assay (ELISA). We used frozen tissue sections as models of the antigen-coated wells of ELISA plate. The intensity of immunoreactivity of the frozen section to an anti-fast-myosin monoclonal antibody was quantified directly from the color developed with the second antibody coupled with peroxidase using phenol-4-aminoantipyrine as a substrate. Then, the same section was incubated in 0.01 M acetic acid solution to cleave antigen-antibody complexes, followed by colorimetric measurement to obtain the absolute value of total protein per section. Fast-myosin content in the frozen tissue section was expressed as mg of fast-myosin per g of total protein. In this micromethod, the minimum area and the optimum thickness of the section were 5 mm2 and 10 microns, respectively. Fast-myosin contents in the extensor digitorum longus and soleus muscles were 185.0 +/- 6.1 and 17.5 +/- 2.4 mg/g, respectively. The results obtained by this micromethod were in good agreement with those obtained by two conventional methods, immunohistochemical morphometry and biochemical determination. This micromethod is useful for a quantitative evaluation of the contractile function of the mammalian skeletal muscles.

Actins↗

PubMed2003

Developmental and regional changes in the neurochemical profile of the rat brain determined by in vivo 1H NMR spectroscopy.

Sixteen metabolites were quantified from 11-24 micro l volumes in three different brain regions (hippocampus, striatum, and cerebral cortex) during postnatal development. Rat pups from the same litter were repeatedly measured on postnatal days 7, 10, 14, 21, and 28 using a completely noninvasive and longitudinal study design. Metabolite quantification was based on ultra-short echo-time (1)H NMR spectroscopy at 9.4 T and LCModel processing. Most of the brain metabolites were quantified with Cramer-Rao lower bounds (CRLB) less than 20%, which corresponded to an estimated concentration error <0.2 micro mol/g. Taurine and total creatine were quantified with CRLB < or = 5% from all 114 processed spectra. The resulting high reliability and reproducibility revealed significant regional and age-related changes in metabolite concentrations. The most sensitive markers for developmental and regional variations between hippocampus, striatum, and cerebral cortex were N-acetylaspartate, myo-inositol, taurine, glutamate, and choline compounds. Absolute values of metabolite concentrations were in very good agreement with previously published in vitro results based on chromatographic measurements of brain extracts. The current data may serve as a reference for studies focused on developmental defects and pathologies using neonatal rat models.

Aging↗

PubMed2004

Quantitation of simulated short echo time 1H human brain spectra by LCModel and AMARES.

LCModel and AMARES, two widely used quantitation tools for magnetic resonance spectroscopy (MRS) data, were employed to analyze simulated spectra similar to those typically obtained at short echo times (TEs) in the human brain at 1.5 T. The study focused mainly on the influence of signal-to-noise ratios (SNRs) and different linewidths on the accuracy and precision of the quantification results, and their effectiveness in accounting for the broad signal contribution of macromolecules and lipids (often called the baseline in in vivo MRS). When applied in their standard configuration (i.e., fitting a spline as a baseline for LCModel, and weighting the first data points for AMARES), both methods performed comparably but with their own characteristics. LCModel and AMARES quantitation benefited considerably from the incorporation of baseline information into the prior knowledge. However, the more accurate quantitation of the sum of glutamate and glutamine (Glx) favored the use of LCModel. Metabolite-to-creatine ratios estimated by LCModel with extended prior knowledge are more accurate than absolute concentrations, and are nearly independent of SNR and line broadening.

Brain Chemistry↗

PubMed1990

Quantitative evaluation of benzodiazepine receptors in live Papio papio baboons using positron emission tomography.

The binding of the 11C-labeled benzodiazepine antagonist Ro 15-1788 (flumazenil) was measured in the neocortex of live Papio papio baboons by positron emission tomography. This allowed us to calculate in vivo (i.e., at physiological temperature, neurotransmitters concentrations, and ionic environment) the apparent density of available benzodiazepine receptors (B'max) and the dissociation constant of Ro 15-1788 (Kd). By coadministering increasing doses of unlabeled Ro 15-1788 with [11C]Ro 15-1788 and assuming that nonsaturable radioactivity indicated the free ligand concentration, we were able to obtain saturation isotherms. We showed that a state of quasiequilibrium was reached 50 min after the administration of the radioligand. Linear Scatchard plots allowed us to calculate B'max at 78 and 50 pmol/ml of cerebral tissue in the occipital and frontal cortices, respectively. In both these areas, Kd is on the order of 6 nM, with a Hill number very close to unity. This indicates that Ro 15-1788 binds in vivo with high affinity to an homogeneous population of saturable sites. A similar measurement was carried out on a naturally photosensitive P. papio baboon. Absolute values of B'max, Kd, and Hill number were similar to those of the control baboons. Although results concerning this baboon can only be considered as a case report, this similarity may suggest that its epileptic syndrome is not related to a large change in B'max or Kd, at least in occipital and frontal cortices. Our results showed that quantitative estimation by positron emission tomography of some characteristics of benzodiazepine receptors is possible in live baboons and may represent a supplementary tool for investigating further the molecular mechanisms of benzodiazepine receptor function in physiological and physiopathological conditions. We suggest that a similar method of quantification of classic in vivo [3H]Ro 15-1788 binding could be usefully adapted when studying rodent models of epilepsy, stress, and other neuropsychological disorders. On the other hand, the similarity between the B'max and Kd values we obtained in baboons and those recently reported in humans using similar methods emphasizes that most of the in vivo characteristics of the benzodiazepine receptors of baboons are very close to those of human benzodiazepine receptors. This confirms that P. papio baboons are a suitable animal model for studying the pharmacology of benzodiazepine receptor ligands before clinical applications in humans.

Animals↗

PubMed1999

Identification and rapid quantification of early- and late-lytic human herpesvirus 8 infection in single cells by flow cytometric analysis: characterization of antiherpesvirus agents.

Human herpesvirus 8 (HHV-8) infection is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. In this study, we used monoclonal antibodies (MAbs) directed against HHV-8 lytic cycle-associated proteins encoded by open reading frame (ORF) 59 (nuclear PF-8 protein) and ORF K8.1 (viral envelope glycoprotein K8.1 [gpK8.1]) to investigate HHV-8 lytic infection in single cells. Lytically infected cells were labeled with MAbs, stained with fluorescently conjugated secondary Abs, and analyzed by flow cytometry. A 3-day stimulation of HHV-8-positive PEL cell lines (BCBL-1 and BC-3) with 12-O-tetradecanoylphorbol-13-acetate (30 nM) or n-butyric acid (0.3 mM) maximized the expression of lytic-phase viral proteins and minimized cell toxicity. The absolute number of expressing cells was inducer and cell line dependent. Expression of PF-8 occurred earlier and more frequently (in up to 20% of cells) than did expression of gpK8.1. A subset of PF-8 positive cells (25%) co-expressed gpK8.1, representing the majority of gpK8.1 expressing cells. Acyclovir, foscarnet, cidofovir, and PMEA reduced the number of cells expressing gpK8.1, but not the number expressing the nonstructural early lytic gene product PF-8. By contrast, alpha interferon (IFN-alpha) and IFN-beta reduced expression of both PF-8 and gpK8.1, implying an overall inhibitory effect on viral gene transcription or translation. In summary, we have characterized and quantified HHV-8 lytic infection in single cells by dual measurement of early- and late-lytic-cycle HHV-8 protein expression. This technique should prove useful for screening of possible antiherpesvirus agents and for detailed phenotypic characterization of HHV-8-infected cells in vitro and in patients with HHV-8-associated diseases.

Acyclovir↗

PubMed1985

Routine quantification of rheumatoid factor by rate nephelometry.

In a cross-sectional study of over 3000 consecutive serum specimens the levels of rheumatoid factor (RF) measured by rate nephelometry (Beckman ICS II) were compared with values obtained by the more traditional methods of sheep cell agglutination (Rose-Waaler) and latex agglutination. Similar values for sensitivity and specificity were found for all three methods for rheumatoid arthritis, with nephelometry giving slightly higher levels of sensitivity for other rheumatic disorders. A significant correlation (r = 0.46, p less than 0.01) was found between the nephelometric and Rose-Waaler method for 147 consecutive seropositive specimens. Of interest, however, several disparate results were observed, and explanations for these were sought. Longitudinal studies of RF were performed in 49 seropositive patients over a two-year period. The nephelometric method was considered superior compared with the other techniques because of its ability to detect changes in absolute levels at earlier stages and its low interassay coefficient of variance (11%). We conclude that the nephelometric technique appears suitable for routine diagnostic use, offers several advantages compared with more traditional methods, and is no more expensive per test specimen than the Rose-Waaler technique.

Arthritis, Rheumatoid↗

PubMed1988

Evaluation of marrow cellularity in alcoholism and hepatic cirrhosis, by aspiration, biopsy and histomorphometric.

A study of bone marrow cell content was carried out in 23 non-cirrhotic alcoholics, 20 cirrhotic alcoholics and 19 cirrhotic non-alcoholics compared with a control group of 12 healthy subjects, by means of sternal aspiration and bone marrow biopsy with histomorphometric quantification of fat in the latter. Although in the three pathological groups the mean values of fat area were superior to those of the control group, the only statistical differences found were between non-alcoholic cirrhotic and control subjects. The differences disappeared under covariance analysis with age as the correcting factor, showing a correlation between age and bone marrow fat content. To conclude from the biopsy the bone marrow of the cirrhotic is not hypercellular, contrary to the so far accepted findings obtained through sternal aspiration. Moreover, between the subjective global cellularity evaluation by biopsy and aspiration there was an important disagreement in 23.8% and an absolute agreement only 28.6% of cases. The non-existence of a linear correlation between fat content in bone marrow and liver, suggests that the two phenomena do not develop simultaneously.

Adult↗

PubMed2004

Quantification of venlafaxine and O-desmethylvenlafaxine in human serum using HPLC analysis.

We describe an isocratic reversed-phase liquid chromatographic method for the determination of venlafaxine (VLX) and its main active metabolite O-desmethylvenlafaxine (ODV) in serum, using haloperidol as internal standard and liquid/liquid extraction for sample preparation. VLX and ODV were separated on a C18 column with a mobile phase of acetonitrile/buffer (30/70, v:v) at 60 degrees C and a flow rate of 1.5 ml/min. The measurement of the native fluorescence signals of the eluted compounds were carried out at 227/300 nm (excitation/emission) without interference from endogenous components in serum. High linearities for VLX and ODV for concentrations between 20 and 500 microg/l were obtained (r = 0.9997). A large spectrum of routinely prescribed drugs did not interfere in the assay. The coefficients of variation for repeatability varied between 5.40% and 5.99% and for reproducibility between 9.43% and 21.63%. Absolute recoveries were more than 52% for both substances.

Antidepressive Agents, Second-Generation↗

PubMed1996

Quantification of estrogen receptor messenger RNA by quantitative polymerase chain reaction using internal standard fragment.

A simple and reliable polymerase chain reaction-based method for quantifying human and rat estrogen receptor (ER)-mRNA is described. This method involves co-amplification of cDNA transcribed from sample RNA with an internal standard to reduce tube to tube amplification variations. Human and rat internal standards were synthesized by insertion of a DNA fragment near the midportion of human and rat target ER cDNA molecules. After co-amplification, both products of target ER cDNA and internal standard were separated on agarose gel by electrophoresis and transferred onto a membrane filter. The radioactivity hybridized with 32P-labeled ER cDNA was counted. The ER mRNA content was calculated by linear regression analysis after obtaining a logarithm of ratios between the sample and the internal standard radioactivity. The coefficient of variation of this assay was 18.8%. An absolute amount of ER mRNA in less than 10(4) cultured cells could be measured.

Animals↗

PubMed2005

Determination of flavonoids from Orthosiphon stamineus in plasma using a simple HPLC method with ultraviolet detection.

A simple liquid chromatographic method was developed for the simultaneous determination of flavonoids from Orthosiphon stamineus Benth, namely sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, in plasma. Prior to analysis, the flavonoids and the internal standard (naproxen) were extracted from plasma samples using a 1:1 mixture of ethyl acetate and chloroform. The detection and quantification limits for the three flavonoids were similar being 3 and 5 ng/ml, respectively. The within-day and between-day accuracy values, expressed as percentage of true values, for the three flavonoids were between 95 and 107%, while the corresponding precision, expressed as coefficients of variation, for the three flavonoids were less than 14%. In addition, the mean recovery values of the extraction procedure for all the flavonoids were between 92 and 114%. The calibration curves were linear over a concentration range of 5-4000 ng/ml. The present method was applied to analyse plasma samples obtained from a pilot study using rats in which the mean absolute oral bioavailability values for sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone was 9.4, 1.0 and 1.5%, respectively.

Animals↗

PubMed2000

The contributions of emissions and spatial microenvironments to exposure to indoor air pollution from biomass combustion in Kenya.

Acute and chronic respiratory diseases, which are causally linked to exposure to indoor air pollution in developing countries, are the leading cause of global morbidity and mortality. Efforts to develop effective intervention strategies and detailed quantification of the exposure-response relationship for indoor particulate matter require accurate estimates of exposure. We used continuous monitoring of indoor air pollution and individual time-activity budget data to construct detailed profiles of exposure for 345 individuals in 55 households in rural Kenya. Data for analysis were from two hundred ten 14-hour days of continuous real-time monitoring of concentrations of particulate matter [less than/equal to] 10 microm in aerodynamic diameter and the location and activities of household members. These data were supplemented by data on the spatial dispersion of pollution and from interviews. Young and adult women had not only the highest absolute exposure to particulate matter (2, 795 and 4,898 microg/m(3) average daily exposure concentrations, respectively) but also the largest exposure relative to that of males in the same age group (2.5 and 4.8 times, respectively). Exposure during brief high-intensity emission episodes accounts for 31-61% of the total exposure of household members who take part in cooking and 0-11% for those who do not. Simple models that neglect the spatial distribution of pollution within the home, intense emission episodes, and activity patterns underestimate exposure by 3-71% for different demographic subgroups, resulting in inaccurate and biased estimations. Health and intervention impact studies should therefore consider in detail the critical role of exposure patterns, including the short periods of intense emission, to avoid spurious assessments of risks and benefits.

Adolescent↗

PubMed1998

Experience-induced neurogenesis in the senescent dentate gyrus.

We demonstrate here that under physiological conditions neurogenesis continues to occur in the dentate gyrus of senescent mice and can be stimulated by living in an enriched environment. Neurogenesis was investigated by confocal microscopy of three-channel immunofluorescent staining for the proliferation marker bromodeoxyuridine (BrdU) and neuronal and glial markers. Quantification was performed with unbiased stereological counting techniques. Neurogenesis decreased with increasing age. Stimulation of adult and aged mice by switching from standard housing to an enriched environment with opportunities for social interaction, exploration, and physical activity for 68 d resulted in an increased survival of labeled cells. Phenotypic analysis revealed that, in enriched living animals, relatively more cells differentiated into neurons, resulting in a threefold net increase of BrdU-labeled neurons in 20-month-old mice (105 vs 32 cells) and a more than twofold increase in 8-month-old mice (684 vs 285 cells) compared with littermates living under standard laboratory conditions. Corresponding absolute numbers of BrdU-positive astrocytes and BrdU-positive cells that did not show colabeling for neuronal or glial markers were not influenced. The effect on the relative distribution of phenotypes can be interpreted as a survival-promoting effect that is selective for neurons. Proliferation of progenitor cells appeared unaffected by environmental stimulation.

Aging↗

PubMed1994

Flow cytometric reticulocyte quantification in the evaluation of hematologic recovery. Spanish Multicentric Study Group for Hematopoietic Recovery.

The role of flow cytometric reticulocyte (RET) counting and the immature RET fractions (IRF) in the evaluation of hematopoietic recovery following chemoradiotherapy-induced aplasia was studied. RET counts and IRF were studied using an automated flow cytometric reticulocyte counter (Sysmex R-2000) in three groups of patients: 58 patients undergoing an autologous bone marrow transplantation (ABMT group), 28 of whom received granulocyte colony-stimulating factor (G-CSF); 28 patients undergoing an allogeneic bone marrow transplantation (BMT group); and 28 patients receiving remission-induction chemotherapy for acute leukemia (CHEMO group). To evaluate the IRF the percentages of RET fractions with middle and high fluorescence reticulocyte (MFR and HFR, respectively) were used. A rising IRF (expressed as the percentage of MFR + HFR) was the first sign of hematopoietic recovery (ABMT group, IRF 9 days versus 18 days for the absolute neutrophil count (ANC); BMT group, 15 versus 18 days; CHEMO group, 9 versus 11 days). When recovery of the ANC (> 0.5 x 10(9)/l) was compared with that of the iRF (MFR + HFR > 5%), statistically significant differences were found in all three groups. Additionally, 93.1% of the ABMT, 92% of the BMT and 91.2% of the CHEMO recovered the IRF before the ANC. In conclusion, an elevation in the percentage of IRF is the first sign of hematologic recovery in the majority of patients receiving remission-induction chemotherapy and the first sign of engraftment in those submitted to ABMT or BMT. Serial automated flow cytometric quantitative reticulocyte counting provides a useful and early measure of erythropoiesis indicative of hematopoietic reconstitution or successful bone marrow engraftment following marrow transplantation.

Adolescent↗